ECOLOGICAL STUDIES ON TILE SYMBIOSIS OF TER ITOMYCES ...

ECOLOGICAL STUDIES ON TILE SYMBIOSIS OF 

TER ITOMYCES HEIM WITH NIGERIAN 

MACROTERMITINAE 

by 

REBECCA JANE THOMAS B. Sc. 

A thesis submitted for the degree of Doctor 

of Philosophy of the University of London 

AUGUST 1981 Dept. of Plant Biology and Microbiology, 

Queen Mary College, 

Mile Eid Road, 

London.

ABSTRACT 

The relationship between termites of the sub-family Macro- 

termitinae and the symbiotic Basidiomycete fungus Termitomyces was 

investigated. 

Fieldwork was carried out on several termite species at Mokwa 

in the Southern Guinea savanna vegetation zone of Nigeria. 

Termitomyces in nature and in culture is described. 

A selective medium was developed to facilitate isolation of 

Tenaitanyces. Optimum conditions of temperature and p11 for 

Termitomyces cultures associated with different termite species were 

found to be 29°C and 5.2. 

The appearance, production of cultural mycotetes, blastospore 

size and germination of Termitomyces in culture was investigated. 

The cultures from different termite species could not be separated 

by these criteria. 

Methods were established for investigating the microbial ecology 

of Macrotermitinae nests, The fungal population and numbers and 

location of TemiteMces, was, detemined for Nacrotermes bellicosuss 

Macrotermes subhyalinusq Hicratemes species and for adjacent soilse 

Termitomyces was only found In the fungus combo Other fungi were 

present there only as sporest but when the comb was removed from 

the nest these grew rapidlys A substance was found in extracts of 

foodstore and termites preventing gemination of contaminant fungi. 

Termitomycea was present in the digestive tract of Microtermes 

foragers but only in very few Macrotermes bellicosus and Macroternnes 

subhyalinus 

foragers. 

2.

The establishment of Tennisces in new nests occurred by 

carriage of spores in the slate guts in Macrotermes bellicosus and 

Microtermes and probably from basidiospores in tho non-carriers. 

Termitomyces cultures produced polyphenol oxidases and cellulases 

but Temitamyces was unable to utilize starcho chitin and pectic 

CM12 My 0 

substancese The nitrogen content of fOOdi fUnWs cb and cot""tes 

was o. 289 Ma and 6.68% respectively* Termites consume the fungus 

comb and wycotAtes and therefore Teruit2=ces'degrades nitrogen-poor 

food and provides the termites with a relatively nitrogen-rich diet* 

3"

CONTENTS 

ABSTRACT 2 

CONTENTS 

LIST OF FIGURES 9 

LIST OF TABLES 11 

PAGE 

LIST OF PLATES 18 

GENERAL INTRODUCTION 20 

CHAPTER ONEs A HISTORY OF THE STUDY OF TERMITOMYCES 26 

CHAPTER TWOs G04EIIAL DESCRIPTIONS OF SITES AND ORGIANISMS 3 Ik 

2el DESCRIPTION OF STUDY AREAS 35 

2*1*1 Vegetation 35 

2*1,2 Soils 39 

2e2 CLIMATE 41 

2*2*1 lRainfall 41 

2*2o2 Temperature 43 

2-3 TM-1ITE SPECIES STUDIED 46 

2.3.1 General biology 46 

2.3e2 Species of termites studied 50 

2A TEPMXTOMYCES 62 

204"1 Termitawycei In nature 62 

2.4.2 Termitomyces In culture 62 

CHAPTER TUREE, 

i DETERMINATION OF ISOLATION MMIA AND OPTIMUM 

CONDITIONS FOR GROWTH 

OF TEIMITOHYCES 64 

3.1 DEVB LOPMM OF A SELECTIVE MM)IUM 65 

3elel Introduction 65 

3-2o2 Methods of measuring Growth of Termitomyces 65 

3-1-3 Candidate antibacterial,, antifungal and 

growth-promoting substances 67 

3-1-3-1 nesults 67 

3--l-3,. 2 Conclusions 82 

391A Effects of compounds favouring growth of 

Termitomyces, on common contaminating fungi 82 

3a. 4. i Methods 82 

3.1.4.2 Results 82 

3*1*4*3 Conclusions 92 

4 

4.

3*1,5 The effect on spore gemination of 

compounds appearing suitable for a 

selective medium 93 

30105.1 Methods 93 

3*lo5*2 Results 94 

3*1493 Discussion 97 

391o6 Conclusions - the selective medito 97 

3*2 ISOIATION OF TERMITOMYCES 

FROM TEM11TE GUTS 99 

3.2.1 Surface sterilizationo Introduction 99 

3.2.11. Methods 99 

3*20 Results and discussion 100 

3-3 DETERWHATION OF THE OPTIMUM TEMPMUTURE FOR GROM 102 

3.3.1 Introduction 102 

3o3*2 Methods 102 

3.3.3 Results 102 

3*3.4 Discussion 116 

3.4 DErERMINATION OF THE OPTIMM pit FX)R GRMTH 121 

3.4.1 Introduction 121 

3.4.2 methods 121 

3-4-3 Results 121 

3-4.4 Discussion 129 

3-5 GROWTH 

OF DIFFýý TMMITOMYCES ON THE SELF=IVF, 

HMIUM 132 

3*5*1 Introduction 132 

3-5.2 Methods 132 

3-5-3 Results 132 

34.4 Discussion 140 

CILAPTER FOURs TERMITOMYCES 

IN CULTURE 143 

4.1 =4RACTERISTICS OF TERMITOMYCES 

IN CULTURE 144 

4.1.1 Introduction 141* 

4. io2 Methods 144 

4.1.3 Results 144 

4.1*4 Discussion 156 

4.2 SpOrm FROM CULTLMES AND MYCOTETE-S 159 

4.2.1 Methods 159 

4.12.2 Resultsý 159 

4.2-3 Discussion 166 

5.

4.3 BLASTOSPORE 

GEW11KATION 168 

4-3-1 Methods 168 

4.3**2 Results 168 

4-3.3 Discussion 172 

CHAPTEn FI VEs THE MICMBIAL ECOLOGY OF MACROTEMITINAE NESTS 173 

501 M=1ODS 174 

501*1 Introduction 174 

5*1*2 Methods. Establishment of methods to be 

used in the investigation 175 

5-1-3 Itesults and discussion 176 

5.204 Conclusiont methods to be used In the 

Investigation 181 

5*2 HACnOTEPMES BE, LLICOSUS 183 

5*2.1 Macrotermes bellicosus nest system 183 

5*2*2 General fungal flora of the Macrotermes 

bellicosiLs nest system 191 

5*2*3 Location of Termitomyces In the Macrotermes 

belliconus nest system 199 

5.2.4 Fungi found In the gut of Macrotermes 

belliconus foragers and adult and young 

major worker termites In the nest 204 

5.3 HAMOTERMES SUMALINUS 210 

5-3.1 Introduction and methods 210 

5ý3-2 Results 210 

5*393 Discussion 214 

5.4 MICTZOTMMES 

, SPECIES 215 

5*4.1 Introduction and methods 215 

5.4*2 Results 216 

5.4*3 Discussion 222 

505 OVER TERMITE SPECIES AND GENERAL DISCUSSION 225 

5*5*1 Introduction and methods 225 

5-5.2 Results 225 

50503 General dinewslon 227 

5.6 GWMI OF SPEClES OTIUn THAN TEIRMITOMYCES 230 

5.6'. 1 Incubation experiments 230 

. 5.6.12 Organisms associated with nests of funWa- 

growing termites 251 

6.

5-7 PRI-: VMTION OF SPORE CM1111NATION IN THE, NEST SYSTEM 

5-7-1 Effect of zaliva in soil on spore germination 255 

5-7-2 Inhibition of spore germination by extracts 

255 

of foodstore, fungus comb and termites 258 

508 WASHING TECHNIQUES 268 

50601 Introduction 268 

598e2 Methods 268 

5.0.3 Results 269 

50004 Discussion 273 

CHJU)TER SIXi T11E MICROBIAL ECOLOGY OUTSIDE TIM NEST SYSTEM 275 

6.1 FUNGI IN SOILS 276 

6.1.1 Fungal POPUlAtiOn. 3 of different soils 

6.1.2 Fungal populations at different depths in 

the soil 

ESTADLIS1114ENT OF THE TERMITOMYCES cumunc. IN NEW NESTS 287 

6,1-2-01 Carriage of Termitomyces and other fungi by 

alates 

6.2.2 DazLdiocarp production in Termitomyces 294 

CHAJ'y= SEVINS THE PHYSIOLOGICAL BASIS OF THE ASSOCIATION ULTWEEN 

7, -1 

TERNITOMYCES AND THE TERMITES 310 

tl)ISTURE CONTENTS 311 

7-1-1 Methods 311 

7*1*2 Results 311 

7.1.3 Discussion 315 

7-12' P11 318 

7-2-1 Methods 318 

7*2*2 results 318 


7*3 LIGNIN DEGRADATION 322 

7-3*1 Introduction 322 

7-3.2 HathodLa 326 

7.3.3 Results 326 

7-3-4 Discussion 329 

7-4 CELLULOSE DEGRADATION 332 

7, -4*1 

Introduction 332 

7-4*2 Methods 333 

7.4.3 Results 334 

7-4-4 Discussion 338 

7. 

276 

282 

287

7-5 UTILIZATION OF STARCH9 CHITIN AND PECTIC SUBSTANCES 342 

7-5.1 Introduction 342 

7.5o2 Methods 343 

7.5.3 Results 343 


7.6 MINERAL CONTENT OF DIFFERENT COMPONENTS OF THE 

TERMITE-FUNGUS SYSTEM 353 

7.6.1 Introduction and methods 353 

7o6*2 Results 353 

7o6*3 Discussion 358 

CHAPTER EIGHT$ GENERAL DISCUSSION 363 

8.1 TERMITOMYCES IN CULTURE 364 

8*2 TERMITOMYCES IN THE WILD 365 

8.3 ENZYMES, DECOMPOSITION AND NUTRIENT CYCLING 367 

8*4 SY11BIOSIS BErWEEN INSECTS AND FUNGI 370 

8.5 EVOUJTION OF THE SYMBIOSIS 372 

ACKNOWLEDGEMENTS 

APPENDIX 376 

REFERENCES 394 

8. 

375

LIST OF FIGURES 

Legends have been abbreviated 

in many cases. 

10.0101 The vegetation zones of Nigeria 37 

2010,21 Fieldwork sites 

2*2*1 Climate diagram for Mokwa 45 

293-1 Vertical section of a Macrotermes bellicosus nest 53 

30101 Position of inocula of fungL on petri dish containing 

agar medium incorporating the antifunCal substance to 

be tested 

30*1 Growth of lermit2MLces cultures at different 

PAGE 

temperaturese First experiment 108 

3-3*2 Growth of Termitomyces cultures at different 

temperatures. Second experiment 109 

3-3-3 Mean diameter of Termit2Mces. cultures Crown over a 

range of temperatures* Day 7- 110 

3-3-4 Mean diameter of Termitannrces cultures grown over a 

range of temperatures. Day 14. First experiment 

30*5 Hean diameter of Temitomyces. cultures grown over a 

range of temperatures* Day 14., Second experiment 112 

3-3., 6 Growth during second week of TermItomyces cultures 113 

3-3-7 Relative growth ratesof cultures of Termitomyces grown 

at different temperatures 114 

3.4'. 1 Growth of Termlt2wýces cultures at different pH values 126 

3*4*2 Mean diameter of Termit==ces cultures Crown over a 

range of TAI'se Day 4 127 

3.4-3 Mean diameter of Termitomyces cultures grown over a 

range of pIlOse Day 11 128 

305.1 Growth of Termitomyces cultures on two media 134-5 

3.5.2 Regression of growth of the Termlt2=ces cultures on 

the amount of cellulose decomposition 141 

4.201 Length of Termitomyces spores 164 

4*2*2 Width of Termitomyces spores 165 

4-3-1 Progression of germination with time 170-1 

5*6*1 Conidia bearing stroma of Xylaria 237 

Pod-shaped stroma from Macrotermes bellicosus fungus 

comb 

38 

89 

9. 

237

5.6*3 Fungi doveloping on incubated Macrotermes belliconus 

fungus comb 

5.6A Fungi developing on Incubated Macrotermes bellicosus 

fungus comb 

5&6*5 Growth oflXXIaria after 122 days incubation on SP medium 

Plates 

5-AX Growth of X-Y-laria, after 5 days incubation on SF medium 

plates 

5*8*1 Washing apparatus 270 

5.8.2 Mean number of fungi frora wa3lnrater from the foodstore 271 

5.0.3 Mean number of fungi from washwater from fungus comb 272 

7-6.1 Amount of nitrogen In different components of the 

10. 

Macrotermes bellicosua nest system 355 

Thirty figures in all, 

238 

238 

239 

240

LIST OF TABLES 

1, egends have been abbreviated In many cases., 

2*lol Chemical and particle size analyses of soils from 

the fieldworic sites 40 

2*2el Monthly rainfall figures for Mokwa 42 

2*24 Monthly temperature figures for Mokwa 44 

3-1-1 Growth and appearance of Termitomyces cultures on 

PAGE 

media containing antibiotics 75 

3ole2 Antifungal substances preventing the growth of 

TermitomXces 76 

31ol*3 Diameter and appearance oflermlt2MLces colonies 

on media containing antifungal substances 77 

361.4 Growth of TermitgMees and contaminating fungi on 

media contaWng antLfungal wid growth promoting 

substances 

39105 Number of fungal colonies developing from spores 

on plates of different media 95 

3e2el Comparison of mycotOtes washed In Hiltons Fluid and 

unwashed mycotfites 100 

3-3-1 Analysis of variance on the diameters of Termitomyces 

cultures on Day 7- First experiment 103 

3-3-2 Analysis of variance on the diameters of Termitonffces 

cultures on Day 14., First experiment 103 

3-3-3 Analysis of variance on the diameters of Termitomyces 

cultures on Day 15o Second experiment 104 

34-4 Analysis of variance of growth of Termitowyces 

cultures during the 2nd veek 104 

3*3-5 Analysis of variance of relative growth rates of 

Termitomyces cultures during the 2nd week 107 

3*3.6 lUblished. values for the temperature of Macrotermitinae 

nests 115 

3.4.1 Analysis of variance of the diameters of Termitomyces 

cultures on day 11 122 

3.4.2 Initial and final pH of the medium 129 

3.5.1 Analysis of variance on the diameters of Termitomyces 

cultures on day 14. First experiment 133 

8 1* 

11.

3e5e2 Analysis of variance on the dimmeters of Termitomyces 

cultures on day 14. Second earperiment 137 

3.5.3 Growth of Termitomyces cultures on selective mediuml 

and cellulose decomposition 139 

3*594 Regression of growth of Termitonvyces cultures on the 

selective medium against amount of cellulose decomposi- 

tion 

4.1*1 Appearance of Termitomyces_, cultures after 2 vee)us 

growth at 290C* 

4.1.2 Production of cultural mycotetes on different media 154 

4.1.3 Production of cultural mycotetes at different pil 

values 

4a. 4 Production of cultuml mycotetes at different 

temperatures 155 

4.1-5 Production of mycotOtes with increasing number of 

subcultures 

4.1 Appearance of spores of Termitgar Ices 

160 

4.2.2 Spore sizes of Termitomyces 161 

4.2*3 Other reported TermitoWces spore sizes 163 

4.3.1 Mode of gemination of TermitMces, blastospores 168 

4-3-2 Events during course of germination and growth of 

Termitomycem blastospores In culture 169 

5.1.1 Numbers of fungal colonies obtained on soil dilution 

plates on two media at two teMeratures 179 

5.1.2 Number of fungal colonies on soil dilution plates 

after 29 5 and 7 days Incubation 180 

5-1-3 Number of fungal colonies obtained by 2 methods of 

Isolation from mycot8tes, 18o 

5*2el Dimensions of various parts of a Macrotemes bellicopus 

nest eystem 

5*2,, 2 Extension of the food store down a Macrotermes bellicoaus 

hive 188 

5*2*3 Lengths of the different zones of Macrotemes bellicosus 

fungus c(xab 189 

5.2.4 Dry weights of mycotetes fron Macrotennes bellicosus 

139 

155 

156 

fungus comb 189 

5.2.5 Density of Macroterm s bellicosus fungus comb and food 

store 190 

12. 

188

5.2.6 Fungi cultured by direct isolation from parts of the 

Macrotermes bellicosus nest system 192 

5*297 Mean number of fungal colonies per g dry weight of 

different parts of tho 11acrotermes bellicosus nest 

system 

5.2.8 Analysis of variance on fungl found in different parts 

of the Macrotemes bellicosuS nest system 194 

5*2*9 Fungi isolated from different parts of the Macrotermes 

bellicosuE nest system 195 

5.2.10 Similarities in fungal species composition between 

different parts of the Macroternes bellicosus nest 

system 

5*2911 Isolation of Termitomycen, by the direct isolation 

method from parts of the Macrotermes bellicosus nest 

system 

54*12 Mean number of Termitom yces colonies per U dry weight 

of different parts of the Macrotermes bellicosus nest 

system 

5*2*13 Analysis of variance on the numbers of colonies of 

Termitomyces found in different parts of the Macroter-mes 

bellicosUB nest system 202 

5e2*14 Presence of bolus in crop of major workers of Macrotermes 

bellicosus 205 

5.2-15 Growth of Termitoaces from the Cut of Macrotermes 

bellicosuB major workers 205 

5.2.16 Fungi isolated from Macroterues bellicosus major worker 

cuts 

5-3-1 Mean number of fungal colonies per a dry weight of 

different parts of the Macrotermes subhyalinus, nest 

system 

5-3-2 Analysis of variance on the numbers of fungal colonies 

found In different parts of the Macrotermes subhy; Llinus 

nest system 211 

5-3-3 Fungi isolated from the different parts of the Macrotermen 

subhyalinux nest system 212 

5-3.4 Presence of bolus In crop of major workers of Macrotermen 

subhyalinus 

13. 

193 

196 

200 

201 

207 

211 

212

5-7.2 Analysis of variance on the nu: mbers of fungal colonies 

developing from the different soils 

5-7-3 Hean mzmber of fungi per g dry weight Isolated from 

soil freshly worked by termitesq older worked soil 

and unworked soil 

5-7.4 The numbers of fungi developing on soil d1lution platen 

after treatment with extracts of foodstore and fungus 

comb 

5.7.5 The numbers of fungi developing on soil dilution plates 

after treatment with extracts of whole termitess termite 

guts and termite head and thorax 

5-8*1 Ilean number of fungi per g dry weight from washed and 

unwashed foodstore and fungus ccub 

6aa The mean number of fungi per C dry weight of soil in 7 

Boils 

6.1.2 Analysis of variance on the numbers of fungal colonies 

developing from the different soils 

6.1.3 Mean number of fungi per g dry weight isolated from 

different soils 

0.1.4 Similarities In fungal species composition between the 

different soils 

6.1.5 Hean number of fungal propagules per g dry weight of 

*oil at different depths 

6.1.6 Analysis of variance of the numbers of fungal colonies 

at different depths In the soil 

6-1-7 Fungi isolated from different depths of soil 

6*2*1 Thq presence of a bolus in the crop of alates of several 

species of Macrotemitinae 

6*2o2 Growth of Termlt22Xcen from the guts of alates of several 

species of Macrotermitinae 

6.24 Association between the carriage of Termitowces in the 

guts of alates and the ability to establish viable 

fungus comb in laboratory cultures 

6., 2.4 Species of fungi carried by alates of Macrotermitinae 

species 

6.2.5 The association between the carriage of Ternitotnyces 

spores by alatca, and the development of basidiocarps 

from the fungus comb 

25 6 

257 

260 

261 

269 

277 

277 

279 

15. 

280 

283 

283 

285 

288 

289 

289 

290 

296

6.21.6 The first flight and appearance of Termit=ces basidio- 

carps for some species of Macrotermitinae in 1978 at 

S=aru in the Northern Guinea savanna zone of Nigeria 297 

6.2-7 The association of termites with TermiteUces basidio- 

carps 

7-1-1 Moisture content of Macrotermes bellicosus foodstore, 311 

7-192 Moisture content of Hacrotemes bellicosus fungus comb 312 

7ol*3 Moisture content of Kicrotermes bellicosus nest 

structure 

7-1-4 Moisture content of Hacrotermes bellicoaul% food (wood 

litter) 

7914 Moisture content of the fungus combs of other species 

of Hacrotermitinne 

7.1-. 6 Moisture contents of myr-ot4tes and pseudorhiza of 

TemitoMces 

7.1.7 Other reported values for the moisture content of fungus 

combs within the Macrotermitinae 314 

7*1-8 Moisture content of the soil in the Mokwa area N4 

7*2*1 p1l of foodstore and fungus comb of Hacrotervwx ballicorAts 318 

7*2,2 pit of fungus comb of other species of termite 319 

7-2-3 Reported values for the pit of fungus comb of tormite 

species 

7-3.1 Some organic components of plant material 325 

7-3. *2 Reaction of Termit2=ces cultures an red cabbage extract 327 

7-34 Reaction of Termitomyces cultures an gallic and tannic 

acid media 328 

7-3.4 Reactions of guts of Macrotermes bellicosus and Hicro- 

termes , species on Gallic and tannic acid plates 329 

7.4.1 The amount of cel%ulose removed by Termlt2nces cultures 

after two weeks growth on cellophane strips 334 

7.4.2 Some values for lIgnLn and cellulose content of fungus 

comb and food of different termite species 335 

7-5.1 Utilization of starch by Terwitomyces cultures from 

different termite species 344 

7*5.2 Utilization of chitin by TermitomXces cultures from 

different termite species 345 

7-5-3 Enzymes found in different termite species 346 

298 

16. 

313 

313 

313 

314 

319

7.6.1 laneral content of fungus comb* foodstore, food, 

mycotetes and TeMit2Tyces. associated with different 

termite species 354 

7.6.2 Published values for the nutrient content of fungus 

combs and Termit M ces mycotetes 356 

One hundred and thirteen tables in all* 

17.

LIST OP PLATES 

Leoends have been abbreviated In many casese 

2*3el 1-ticrotermes belliconus plate and hive showing food- 

PAGE 

store and fungus comb 49 

2.93e2 Ancistrotermes cavithorax fungus comb showing old 

and new areas 

2*3.3 Macrotermen bellicosus mound 54 

203.4 Macratermes bellicosus hive showing position of queen 

cell 

2.3.5 K-1croterries bellicoatis fungus c=b In position In 

the nest 

2*3.6 Hacrotemes gubbyallnus mound 56 

2-3-7 Racrotermes suhhyalinus fungus comb 56 

2*3.8 Microtermcs funaus comb In a chamber in the soil 57 

2*309 tticroter! ýes fungus combs 6o 

4.1.1 TemitS! Mces cultures from Ancistroternres cavithorax 148 

4.1.2 TermtVýces cultures from Macrotermes belliconus 148 

4.1.3 Ternit2=ea cultures from MAcrotermax subhyalinus 149 

4.1.4 Termit2=ces cultures from Hicrotermes op. A 149 

4.1-5 Termitomycen cultures from M crotermes ape D 150 

4.1.6 Termit2=ces cultures from Hicrotermes spo C 150 

4.1.7 Termit=es cultures frcxzi Hicrotennes an* D 151 

4a. 8 Ternitomyces cultures from Mcriftermen eve G 151 

4. le9 Temitarnyces cultures from Hicrotermes ape R 152 

4.1*10 Termitomyces cultures from Hicrotermes ape Z 152 

4.1*11 Termitmyces cultures from Odontotermes smeathmant 153 

4.1*12 TermitmnycesIcultures from Odontoternes op. (OD54) 153 

5*2*1 Macrotermes bellicorus fungus comb showing zonos 186 

5-6.1 Incubated Macrotermes bellicostsov fungus ccxnb showing 

rhizomorphs of alarin 241 

5.6.2 Incubated K-tcrotermes bellicosus, fungus comb showing 

conidia-bearing stroma of XXIaria 241 

5-6-3 Incubated Macrotemes bellicosus fungus comb showing 

pod-shaped stroma 242 

5-6-4 Blackened rtilzomorphs of Xylaria 242 

54 

55 

55 

18.

5*64 Soil plastered over Incubated 1-facrotermes belliconus 

fungus comb 

7.4.1 Termit=ces culture clearing Remazol Blue dyed 

cellophane overlay 337 

28 plates in all* 

243 

19.

GENMAL INTRODUCTION 

Termites are Insects In the order Isoptera, They occur In 

tropicals sub-tropical,, semi-arid and to some extent warm temperate 

regions as far as 450 north and southe This results In approximately 

two-thirds of the land surface coming within the limits of their 

distribution (wood 1976, Wood and Sands 1978)* Mmy are important 

and often dominant members of the soil faunaq feeding on plant 

tinsuo which may be living$ dead or decaying, and playing a 

significant role In the cycling of nutrients by affecting all 

stages of decomposition* They are social Insects, with different 

morphological forms or castesq living in highly organized 

communities with each caste performing different functions. They 

differ from hymenopteran social insects in that they are homi- 

metabolous 9 the castes are usually bisexual and there are no known 

subsocial groups (Krishna 1969)o They usually live in nests of 

their own constructiong the nest system often being very extensive. 

Their burrowing and nest buildingg together with their feeding 

habitaq renults In them having a large effect on soil physical and 

chemical properties as well as vegetation, agriculture and wooden 

structures of man. 

There are approximately 11900 living and fossil species 

(Krishna 1969)s of which approximately 1400 are living (CM-Aton 

1961), The fossils are closely related to the living species, 

little change having occurred over millions of yearn (Wood and 

Sands 1978). In tho 5 families of lower termites and the family, 

TermLtidae, the higher termitesq there are 168 Genera, The 

TermLtidao Includes approximately 75% of knawn species (Lee and 

20.

Wood 19709 mid four-fifths of all known Nigerian species (Johnson 

et ale 198O)e The lower termites havo symbiotic protozoa In the 

hind gut which digest cellulose., The hLgher termites lack these 

protozoa. 

The Temitidae is the most advanced and diverse family 

exhibiting a wide range of social specializations 

(Krishna 1969)9 

It has 4 sub-f=ilies of which one, the Macrotemitinaev has a 

symbiotic relationship with Basidiomyceto fungi of the genu3 

t 

TemitMcen, which lives on fungus combz composed of the termites 

faccal pellets within the nest systems One of the general 

Sphaerotermess builds fungus combs which do not have Termitemces 

associated with them. In this subfamily all the faecal material 

goes Lnto the fungus combg and so the nest structure and covered 

runways are made only from soLle Another distinguLahLng characterLstLc 

of the Macrotermitinne In thaLr prImLtLve gut anatomy, The 13 genera 

of the HacrotermitLnae are CLven belows 

GENMIA OF ZOOGMGMUMICAL NUMUM OF SPECIES 

MACIZOTEMITINAE REGION FOUND IN NIGERIA(l) 

Acanthotermas Ethiopian I 

AllodonteMes,. Ethiopian 0 

Ancistrotermes Ethiopian, Oriental 3 

Euscaiotermex Oriental 

Hypotermes Oriental 

Macrotemes Ethiopian, Oriental 5 

HegaLrotemes Ethiopian I 

111crotermes Ethiopian, Malagasyl Oriental >9(2) 

Cdontotemes Ethiopian, Oriental >3 

Protermes Ethiopian 3 

- 

21.

Popudacanthotermes Ethiopian 2 

Sphafbrotermes Ethiopian I 

S= canthotermes Ethiopian 0 

(1) Johnson et al* 1980* 

(2) Wood (pers. comm. ) 

Ancistrotem. es has previously only been recorded as occurring in the 

Ethiopian region but Hicrotermes Insperatus (Kemner 1934) from Java 

appears to be an Ancistrotermes (Wood perse comm. ). Akhtar and 

Ilussain (1930) also found Microtermes 22kistanicus from Bangladesh 

to be an Ancistrotermes based on its enteric valve armature and 

other featurese 

The Macrotermitinae originated in the Ethiopian region during 

the Tertiary period, the fauna of this region Including the most 

primitive genera Acanthotermes and Poeudacanthotennes which are 

endemic to Africa (Krishm 1970), The most advanced genera 

Hacrotermes, p Microtermps and Odontoterman reached the Oriental region 

in the Hlocene period where Odontoteýs gave rise to Itypotermes and 

EUscalotermem. Macrotermitinae did not reach the Australian and 

Papuan regions and do not occur in the Neotropical region where 

fungus growing ants have evolved a similar type of system* The 

occurrence of Micratermes In the Malagasy region Is considered an 

anomalous distribution as the Microtermes are considered to have 

originated after the separation of Hadagascar from Africa (Krishna 

1970)- There are various theories as to how they reached Madagascar 

(Thakur and Sen-Sarma 1978) but none are very satisfactory* 

The Basidiomycoto fungi found growing on the fungus combs of the 

Macrotermitinao vere brought together in the gems Tormit2M. ces by 

lleLm; (1940) because of their combination of characters* lie considered 

22.

they constituted a systematic groupt links vith other agarics being 

vague* Heim (1942b) placed Termit2=ces in tho family Amanitaceae 

(Order AgaricaleA where the tribe Termitomycateae formed the bridge 

between the Lepioteae and the Collybieaoe Thin genus is found only in 

association with termites. Termit2Mcesl grotm an the fungus coWW 

of members of the Ilacrotermitinae where it is present as Mycelium 

and round white bodiesj called mycotetool which are the asexual 

reproductive structureal having within them couidia, of blastosporic 

ontogenys 

There are two sub-genera* (1) PraetermitcnTees which includes 

Termitomyces microcarrain and other small species which grow from 

fungus combs scattered on the soil surface by the termitese The cap 

diameter Is less than or equal to 3 cm and does not have a pileic 

perforatorium and no, or very reduced% pseudorhiza 

(11eim 1958)0 Heim 

(1942a) considered T& microca! Ms formed a kind of link between the 

cavernicolous Termlt2=ces with pseudorhLza and non-termitophLIous 

epigean agaricsq ancestral to the genuno (2) Eutermitomyces which 

Includes the larger species with a cap diameter greater or equal to 

3*5 cm, joined to the fungus comb by means of a long pseudorhLza and 

with a perforatorium (Heim 1958)o 

There are probably over 30 different species of Ternitomyces, 

Heim (1952a) recognized 12 species and 5 varieties or forms of African 

Termit=Cea and '2 Asiatic species, Singer (1962) recorded 10 species. 

Other species have been recorded and described by Alasoadura 19661, 

19671 Heim 19771 Natarajan 1975,1979 and Otieno 19649 1968. 

Termit=ces. in highly prized an food In many parts of the world 

and In considered by soma to be superior In taste to all other mushrooms, 

The places it Is eaten include India Matra and Batra. 1979)% Thailand 

r3"

(Bela perse comj, China (Cheo, 1948), Uganda (Makilbi 1973)9 za ia 

(Piearce pers. com. ) and Nigeria (Zoberi 1973)- Its crude protein, 

carbohydrate and fat content makes its food quality comparable to 

that of many horticultural crops 

often been mentioned in folklore and proyerbs 

pers* cam. ). 

(Mukiibi 1973). Termitom. ces has 

(Cheo, 1948, Piearce 

The role of the fungus comb and Termit2=cen in the life of the 

temite, how Termit2=ces reaches newly founded colonies and the 

degree of specificity between termites and Termitomyces are some of 

the questions which will be dealt with In this thesiso 

Certain termite species cause damage to crops, trees Olarrig 19699 

sands 1973) and pastures (Wood And Ohiagu 1976), but the majority of 

species are not pests, For example of the 120 or so known species In 

Nigeria only about 20 damage crops or buildings (Johnson and Wood 

1979)* The major pests are In general members of the Macrotermitinae 

(Datra 

and Batra 19799 Coaton 19619 Johnson and Wood 1979) with 

Hicratermes species being the most important termite crop peat In 

Nigeria 

(Johnson 

and Wood 19799 Woods Johnson and Ohiagu 1977)e The 

obligate association of these crop pests with Termit2=cea suggests a 

novel way of controlling them by killing the fungus* To develop this 

Idea much more had to be known about details of the association 

between Termitomyces and termites* An alternative control method 

such an this could become increasingly Important If Insecticides used 

in termite controls such as aldring are banned In countries where 

termites are a problem. 

The work described here was carried out during the tenure of nn 

N=- research studentship based at Queen Hary College, Londom Field 

21k.

work was carried out at the Agricultural ]Research Station, Hokwal of 

the Institute for Agricultural Researchl Ahmadu Bello University, 

Zariag Nigeria. The field work van organized and supervised by the 

Centre for Overseas Pest Rosearchs Overseas Development Aduinistrationg 

1, ondone 

2g.

alAPrER 

ONP 

A HISTORY OF THE STUDY OF 

TL'RNITOMYCES 

2G.

Ono of the earliest records of tho fungi and fungus combs 

associated with the toruite subfamily Macrotermitinae was by the 

I 

German naturalist Klonigs, (1779) in the East Indies. lie considered 

that the fungi actod as a food supply for the young termites (cited 

by Heim 1942ag Sands 1969). 

In IL781 an Englishmant Henry Smeathman, 

published an account 

of his Investigations of Macrotermes bellicosus nests in West Africae 

He called the fungus combs nurseries and described an them small white 

globules (later called the mycoteten)l which he took to be mushrooms* 

The first record of the basidiocarps was In 11347 when Berkeley, 

an English cryptogamic botanistj n=ed a specimen he had received 

from Gardener in Ceylonj Lentinux cartilagineus. This was recorded 

an being found 4 feet (1,2 m) below the surface of the earth growing 

on the combs of termites, The specimen was damaged and he considered 

it possible more specimens might justify a new Venuse According to 

Petch (1913b) other collectionB of the same species were also named 

Lepicta albuminosa and Armillarin eurhiza by Berkeley* In 1869 

Berkeley received basidlocarps developed from Indian termite nests. 

Ile called these Agaricus termitlgena, q but Petch (1913b) had no doubt 

they were Identical with L. cartilagineus, Thvaites. sent more 

basidiocarps, associated with termites to Berkeley including those 

collected by Gardenere This time the basidLocarp was named Collybbia 

sparxibarbial but Berkeley noted its similarity to A. eurhiza but it 

lacked a PSeudorhizal this having been cut off (Patch 1913b). 

In 1850 Savage examined the mycotkas from Went African nests and 

thouGht the fungus van a Trichia,, He stated that Kirby and Spence 

considered it a Mucor* Gibbon (1874) was the first person to realise 

27"

there van a connection between the mycotetes and'the basidiocarpe 

In 1882 Berkeley named the mycotetes as Ae2erita cluthiei. Laterv in 

1935, Ciferri transferred It to a new genusq Termitomphaern, and named 

it To duthiei, However, the fungus he studied could not have been the 

asexual stage of Tem. 1t2nffSpj; as it was fram a Nnautiternes nest in 

South America. 11oltermann (1900) also described the mycotCtess which 

he thought formed a laroe part of the food of the termites, but he 

did not associate these with the basidiocarp. Jumelle and Perrier do 

la Bathie (1907) considered the mycotOtesq which they found in termite 

nests in Madagascar, to be a conidial stages or form of myceliuml of 

Xylarin. TrUghRrd (1904) described tho structure of the mycot9tes from 

the Sudan on the combs of Odontotermes vulaaris (cited by Wheeler 1907)e 

In 1879 Cesati published figures and descriptions of fungi 

collected in Borneog one of which he called Tricholm. a segambosums 

considered by Petch (1913b) to be the termite agarico Holtermann 

named the basidiocarp, he found in Java Pluteus rajal! 

(Itoltormann 1900)0 

Hennings and Nyman redescribed it as. Phollota janseanA and later as 

Flnmmula Janseana* They also recorded It as Pluteus bogoripnxing 

Pluteus treubianum and Flammula filtREndula, believing those to be 3 

different species (cited by Petch 1906l 1913b)e Patouillard named 

the basidlocarp from Javan termite mounds Collybia radicata (cited 

by Petch 1913b)o 

In IL906 the eminent English mycologistq Petch (1906), who for a 

large part of his career was director of the Royal Botanic Gardenag 

PcradeniYat Ceylon, published a cajor work on the fungi associated 

with Odontotemen redemanni and Cdontotemes obscUrice2j3, lie 

described fungus vxnbs mycellumi Mycotaton and also the basidiocarpl 

which he named Volvaria eurhizat recording 7 oynonyma an detailed 

28.

above. He suspected the nature of the relationship of the mycotiate to 

the basidiocarp but could not prove it* lie thought the fungus was 

eaten by the termites but was not enough to supply food for the larvae 

unless the fungal growth was abnormally rapid, lie also considered the 

position of Entolomn microcArpum 

(n Termitomyces mAcrocaMs). This 

develops from pieces of fungus comb ejected from nests by termiteaq 

but its connection with termites had not previously been suspectede 

Doflein (1906) also described ftmgus combs of Odontotermes 

obscuriceps and proved that larvae and nymphs ate the mycotetese 

E3cherich (1911) was one of the first to consider the metabolic heat 

created by the ftmguj3 gardens 

(cited by Datra and Batra 1966)e 

In 1913 Patch again discussed and described Entolon. a microcarpum 

(Patch 1913a)* lie considered the fungus "spheres" resembled those of 

, Aenerita duthiet but there were differences In the degree of differentia- 

tion. Ile still had no proof for the association he suspected between 

B. microcarTxim and mycotAtes from the combl his opinion being that after 

a period In the termite neat the fungus lost its vigour and required 

rejuvenating so the termites carried the mycotOteis to the surface, 

where they produced basidiocarps, providing spores which the termites 

took back to the nests 

Patch (1913b) also collected together all the obaervations of 

fungal species associated with termite nests@, Those developing on the 

fungus comI within the nest while the nest was inhabited by termites 

he divided into 2 groupaq 

(1) the vhite conidial. spheres (2) A2arlcus 

opp. lie collected 16 synonyms for the agaric under the name Collybia 

etlburninoja.. The earliest name was LeRiota albuminosa but the general 

-opinion was that it was a Collybia. 

to have been fruitless, was to establish the link 

'His aim, which he considered 

29"

etween A. duthipi and the basidiocarp. In 191*2 Cheo finally 

established its link with Collybiaalbuminosas 

Bottomley and Fuller (1921) discussed Petch's conviction of the 

association between E. microcaMm and termites. They observed 

Cdontotermes badiuS bringing fUngus comb up to the surface and 

spreading it arounde Fungi developed from these which resembled 

Petch's E. microca! 3=, # pointing to the truth in the connection 

between it and the termitese They also observed immature termites 

feeding on the MYCOtA etes, which they thought grew rapidly to replace 

those being eaten, 

Bathellier (1927) confirmed that the mycelium which covered the 

fungus combs of certain "Termes" in Indo-China was part of the cycle 

of the Basidiomycetes as well as the mycotates. Ile was also one of 

the first to suggest that the comb consisted of masticated woody 

materials incompletely triturateds then regurgitated by the worker 

termitenj rather than of excreta, as had previously been believed (13oze 

19239 Dottomley and Fuller 19211, Dofloin 1906, Petch 1906)o Ile was 

also suspicious of the belief that the culture was controlled by the 

Insects themselves for nutritional purposes, but believed combs to be 

nurseries. 

Ilia method of comb construction was further considered by Ca-asse 

(1937)s who stated that the normal fneces of 14acrotermLtinae were dark 

brown and semi-liquid, and agreed with Bathelliers viewe Over a 

number of years Grasse and co-workers continued to be of the opinion 

that the fungus combs were of macerated and undigested plant tissueg 

chewed for a long time by the woricers and shaped Into small balls by 

rolling in the mouthparts, from which the fungus comb was constructed 

(Grasse 1949, Grasse and Heim 1950, Grass; O and Noirot 1961), Although 

30.

Sands (1960) observed and photographed the building up of fungus comb 

from faecal pellets other authors continued to believe Grassele view 

(Cmelik and Douglas 19709 Heim 19779 Rohrmann 1978), while others 

agreed with the earliest views of a faecal pellet origin (Abo-Khatwa 

1977, Datra and Batra 19799 Choo 1948, Coaton 1961, Josens 19719 Wood 

and Johnson 1978). Grasse (1973) finally agreed that the material 

from which the fungus combs wero constructed had passed through the 

termites Cut and called these mylosphereso 

Grass6's (1937) view on the role of the fungus was that it and 

the comb played a small nutritional rolep but he did not realise the 

dynamic nature of the combs until latere 

A different type of function of the fungus comb was proposed by 

Ghidini (1938)t that of humidity controle Iles and later authors 

Olarris 2951, Hesse 1957)s thought that the large convoluted surface 

area of tho comb could function to control humidity by taking up 

excess moisture or releasing it whcn the atmosphere was dry* 

The major taxonomic work on the fungi associated with termites 

was carried out over a number of years by the French mycologist$ Ileimq 

whog (Ileim 1940)9 placed all agarics that grew from the fungus combs 

of the Macrotermitinae In the genus Termitomyces because of their 

combination of characterse lie also cultured the asexual stage of 

Termitcqnycen, In the laboratorys Ile wrota many papers over a number 

of years on theSiology and taxonmy of Termit9mycess most of which 

was collected together and published in 2 papers (Ileim 1942b, 1958) 

and In a book (Ileim 1977). tie believed Oletm 1942a) that the comb was 

a normal part of the nest architecture and Termit2=cen only Orew 

there an it was a suitable medium and was tolerated by the termites. 

He rej, ýcted the classical theory that Temit ces1was cultivated 

31.

Intentionally by the termites and regarded them as commensals which 

are tolerated by the termitest with the mycot'etes occasionally being 

eaten by the adults to limit the growth of the funguso Heimts theory 

was that the quality, nature and traits of Tormitomycenlresulted. from 

exceptional adaptation to the conditions of life on the comb and that 

there was no symbiotic relationship between the termites and fungi 

(Heim 1952a)o Later, (Helm 1977)9 he amended his view and considered 

it a symbiotic association, 

Grassii (1949) showed that the fungus was eaten by finding 

remains of mycotAtes in the alimentary canal of workers and same 

larvae* lie considered it might be the source of some nutritional 

requirement such as vitamins or growth substancese 

it 

Uischer (1951a, 1961) revived the microclimato function theory to 

include the maintenance of a more constant nest temperature by the 

metabolic heat of the fungi, This heat production caused convection 

currents which circulated air and allowed for Can exchange,, Other 

proponents of this role havo included Cheem& at alo 1962 and Harris 

1951- Collins (1977) and Rohrmann (1977) measured fungus comb 

respiration and calculated metabolic heat and water production showing 

a high respiration rate for fresh combs* 

Sands (1956) established the vital role of the fungus in nutrLtion 

of the termites,, Without the fungus CMontotermes badius. survived no 

longer In captivity than when starved, and he concluded their relation- 

ship was a symbiotic ones This was In contrast to the previously 

mentioned view of Helm (1952&),, Ausat et ale (1962) also found the 

fungus essential in the diet of Cdontotermen obeaus. 

32,

Grass6 and Noirot (1957,1958) rediscovered the dynamic nature 

and eating of the fungus comb by the termitesj and confirmed the 

symbiosis between TermitS! Mces and termites* They proposed that the 

fungus attacked the lignin-cellulose complexes thought to be 

inaccessible to the termites, decomposed the lignin and thus made 

available the cellulose. The ter-mites then ato the fungus com. b, 

especially when food vas In short supplyl and ate fungal tissue at 

the same time. They considered the comb to be part of the normal food 

cycle of the fungus., Josens (1971) established a2 month renewal time 

for Ancistrotennes cavithorax combs* 

In 1966 Datra and Batra suggested that yet another function of the 

fungus might be that of conserving nitrogen. Later analyses on the 

mycot6tes showed that the fungus was a concentrated source of nitrogen 

and also of phosphorus (Abo-hlmtwa 1977, Matsumoto 1976, Rohrmann 1978). 

It han also been suggested that, a3 well as decomposing plant 

material within the fungus comb, the fungus may act an a source of 

cellulose decomposing enzymes within the termite gut* Abo-Khatwa 

(1978) and Martin and Martin (197119 1979) suggested that Cl enzymes 

and perhaps some Cx and P-glucosidases were obtained by Ingestion of 

mycoteteso 

Since Ifelm other taxonomic worit. on Temit2aces. has been carried 

out by Alasoadura 1966,19679 NataraJAn 19759 1979, Otleno, 19649 19681, 

Peoler 1969,1977, Peolor and Rayner 1969 and nayner 1969. 

33.

MAKER 

" WO 

GENERAL DESCRIPTIONS OF SITES AND 

ORGANISM 

34.

2*1,1 VEGETATION 

2.1 DESCRIPTION OF STUDY AREAS 

The fieldwork associated with this project was carried out in and 

around Holwa Agricultural Research Stations in Niger State, Nigeria at 

9018IN9 50046E. Mokwa lies close to the boundary of the Southern 

Guinea savanna vegetation zone of West Africa (Figure 2.1.1). 

The sites in which work wan carried out include 

(1) Prim. -x= savanna woodland. This was mainly at zugurmal 15 km 

northwest of Ik)kwa (Figure 2olo2), and as far as could be determined 

had never been cultivated, The natural vegetation was closed savanna 

woodland of semi-deciduous trees 12-15 m high with tall perennial 

grasses uP to 2-3 m high (Wood and Johnson 1978)e The most common 

trees were 3 Caesalpiniaceous legumes, Afzelia africana Smo, Burkea 

africana. Iloo1c. and Detarlum microcaum Guille and Per. (Collins 1977)e 

The grasses were composed mainly of Andron2gon nayanus Kunth. 

Eragrostis trenula Olochat 

ex Steud) and Ilyparrhenia dissoluta Noes. 

ex Steud (Wood et al. 1977)9 Annual fires in the dry season burned 

most of the grass, leaf litter and killed leaves-on trees, 

(2) Seconda! Z woodlands Theno areas around tho Agricultural 

lResearch'Station vere cleared during the 1949-1953 Colonial Development 

Corporations Groundnut Scheme (Baldwin 1957). Piegrowth was prevented 

by cutting until 1959 after which time regrowth was unchecked* The 

species present were those In (1) but without largo trees. 

(3) Experimental plots. * This area (Figure Zel,, Z) had been 

cleared and regenerated as Wo In 1973 it was cleared again and 12 

plots established in the 1974 growing season 

(Wood ot ^1,1980). Two 

j 

35.

plots remained as woodland, 2 were cleared and maintained as grazed 

pasture and the others were planted with various crops. In 1979 

these were left fallowe 

(4) Local farmers fields. These were cleared and cultivated 

using traditional methods* Crops were usually maize and yam. The 

previous history was urdmowne 

(5) Rile I gardens and tjolf course* This was the area around 

the housing built for the Colonial Development Corporation Schemeo 

Both garden3 and golf course were mainly rm)wn grass 

(Figure 2.1*2). 

(6) Riparian forest at Rnbba, 90121N, 50041E, 12 km south of 

Mokwa (Figure 2.1.2). This was a narrow belt of forest fringing the 

flood plains of the River Niger's northern banks existing due to the 

much hiGher moisture content of the soile The savanna forest boundary 

was extremely sharpq with the forest having a maximum width of 0*5 I=* 

The dominant vegetation was Elneis nuineensts Jacq and Cola nitida 

(Vente) Schott and Endle with a few jainya sene2alensis 

(Wood et ale in press), 

(Desv. ) Juss. 

36.

42- 

o 

8 

0 

km 

200 

FIGURE 2olel. The vegetation zones of Nigeria, 

(after Keay 1959). 

37"

p5 

4 

Experimentý! _ý, 

Plots 

ß. % 

10 km 

Zugurma 

2* 

I 

1 

Mile 5 

0 Cattle Ranch 

Rabba., 

ýf 

Mile 1 

5m/,, 

Mokwa 

FIGURE 2.1.2. Fieldwork Citelle (1) primary woodland, 

ain 

secondary woodlands (3) experimental 

38. 

plots. (4) local farmers fields, (5) Hilo is 

(6) riparian forest at Rabba, 

T

2el*2 SOILS 

The experimental sites around tlolcwa were at elevations between 

180-230 m. The general area van gently undulating with broad inter- 

fluvess and was occupied by soils of the Kulfo Association which are 

developed on coarse Nupe Sandstone (Valette 1973)o The association 

Is dominated by very deep, reddish soils with a sandy clay loam to 

clay texture in the subsoil. Moat of the soils are ferrisolsl with 

a sandy surface layer with low water-retention capacity. The soils 

in the experimental sites were a complex mosaic of the Kulfo Series 

(very deep, red sandy clay D horizon between 2511 and 6011) and the 

Dangappo Series (very deep red clay D horizon within 25") (Valette 

1979- 

At nabba the soils on the northern boundary of the forest were 

of the Rabba. Complex Association on an old alluvial sandy terrace* 

The main characteristics were deep to very deep brownish sands (Valette 

1973)e The forest was situated where the terrace descended to the 

floodplaine The forest *oil was hydromorphics developed on alluviuml 

with a dark Creyl weak crumb-structured topsoil (0-4ý5 cm) overlying a 

laroely structurelesel pale yellow-grey subsoile In the wet season 

the water table was at 50-150 cm (Wood et ale In press)* 

Analyzes of reprosentative profiles are given in Table 2,1,1, 

39.

TAMZ 2olol ChemIc&I and particle size analyses of soils from the 

fLeldwork, sites 

U)CATION 

(Wood et al. 1977, Wood et al,. in press). 

IDEPTH 

PARTICLE SIZE W 

cm ORGANIC CIP N 

SAND SILT CLAY 

Primary 0-5 72 15 13 0090 0-131 

woodland 5-25 72 13 15 0-50 0.065 

(Site 1) 25-50 65 9 26 0.50 0.075 

50-75 47 10 1*3 o. 48 O.. C88 

75-100 39 

1 10 51 0-35 0.075 

Secondary 0-5 74 17 9 0.80 OX75 

woodland 5-25 73 16 11 0-30 0-075 

(Site 2) 25-50 66 11 27 3 0.20 o. o63 

50-75 51 U". 37 0020 o. o6g 

75-100 45 12 43 O-ZO 0.057 

Rabba 0-25 60 15 25 lk*71 0.316 

forest 25-50 73 11 16 '. 1.80 0413 

(Site 6) 50-75 74 10 16 2*02 0.146 

75-100 86 5 9 1.59 0.149 

Cultivated 0-5 67 17 16 0.50 01098 

soil 5-25 49 13 38 0-30 0.0138 

(site 3) 25-50 47 10 43 Oo25 0-075 

First year of 50-75 45 a 47 0.40 0.031 

cultivation '75-100 45 12 1*3 Oa2O 0-057 

% 

40.

2.2.1 TZAINrALL 

.. 2 2 CLIIIATE 

The rainfall for the 3 years during which this work was carried 

out in given in Table 2*2*lo A climate diagram showing rainfall and 

temperature is given in Figure 2o2,1. 

The mean annual rainfall at Wkva is 1,175 mm (GhiaQu and Wood 

1976)e The annual rainfall for 1977 (I(Y*O. o mm) and 1978 (995*2 WM) 

were both below this figure. Two-thirds of the rain falls in Mays 

Junel July and Septemberg with 2 years In 3 having a double peak rain- 

fall regime with a drop in rainfall In August (Valette 1973)e This 

2-peak pattern was followed In 1977 and 1973, with the rains in 1973 

building up carliere The start of 1979 appeared different with rains 

in April followed by a reduction in May (Fig* 1_e2*l)9 

There Is a pronounced wet and dry season with the dry season 

beginning October/November and ending Harch/Aprile 

41.

TABLE 2.2.1 Monthly rainfall figures for Mokwag 19779 

1973 and part of 1979 (m)- 

kIDNTII 

MONTHLY WNFALL r. iGuREs 

1977 197.8 1979 

January 0*00 0100 0.00 

February 0.00 0100 0000 

March 2.54 61-97 6.10 

April 2-03 131.83 102962 

May 206. '-14 168.66 65-522 

June 218-43 156.70 216.4o 

July 174.49 147-57 - 

August 75-06 98-56 - 

September 268.48 190-2-5 - 

October 924,72 39.6,3 - 

November 0.00 0*00 - 

December 0*00 0000 

TOTAL 1039-99 995-17 

42.

2.2.2 TDIPLIIATLrZE 

The temperature of noil and air for the 3 years during which 

this work was carried out in given In Table 

The lower night temperatures from December to February are caused 

by dust-laden winds (harmattan) blowing from the north, 

tja

ff, 

tn 

II c14 21 CII 

ID 

Ct 8 U b- 4 %D to t, ) 1 j %D i'n la 

w u u w u 

l e g 1 4 1 

%D 

89- CO \0 %0 -4 --2 

\A 8 w 

b- 

P. 2 ta N ti ua 

to 

ei 1 

i 

0 4 

, ý% 

0 

0 0 0 

ei 

u 

t13 

-4 

ti 

' ta ch ei (> u w fli 0 

ra 

cl, 

b. 4 

t83 ti w ri 

0 ý- 

m ND m 

0 0 8 0 

ra 

\A 

ta 

c> 4 

0 

0 %D 

Z - jn 

-x 5 

G 1 b zn , ý', ýn G , ým :n G u ci U" 

8 p ei N 

3 b- >O %0 C% %D 

-4 %0 8 Vi to %0 

ra 

c% 

%0 W 1 3 

%0 8 ta 0 . 

%0 %D %0 %0 1 0 u u C) 3 

8 8 8 8 1 >O b" b" 1 to 1.4 (» - 

ý t5 

H 

t3 

co 

0 a 

pý 

Ir 

:Z 

tA. 

in 

p 

R 

%0 

44.

FIGURE 2,2.1, Climate diagram for Mokwas 1977,1978 and part of 1979- 

45,

-ff 

C: 

) 

0 

CO 

0 

(0 

6060,0', 6::.. 

0.0 

6........ 66.. 00.: 6 

6000000- 

**ob*** 

............ 

060.000460000006000004 

..................... 

00000000000060 

xt 

00.000004000 

00000*0*000 

0004 

*0000**400 00.0000.. 

..................... 

.................. 

00,0.0,0.000.00 

............ 

............... 

0»0*060 00000- 

.......... 

LL 

LL 

0 

2 

00 

a) 

cr)

2.3.1 GMMIAL BIOLOGY 

22-3 TERMITE SPECIES STUDIM 

Termites of the sub-family Macrotermitinae live in highly 

organised communitien which may be composed of hundreds of thousands 

of individuals. The West African savannas aro dominated by the 

Macrotermitinao (Ulood and Sands 1978)o 

There are several morphologically distinct castes which have 

, Avision of labourf performing different biological functions* There 

are 3 larval instarn in the Macrotermitinno and newly hatched larvae 

are capable of developing Into any caste. This is determined before 

their first moult and in probably controlled by pheromones* The 

proportions of different castes are maintained further by selective 

cannibalism of soldiers or reproductives by the workers (1, ee and 

Wood 1971), 

The workers, which may be dimorphict are sterile, and are the 

most numerous castee For exa plot in Kicrotermen bellicosun, the 

mean canto proportions are 0,7% major soldiers, 1.0%, minor soldiersq 

10,, I% major workers, 2995% minor workers and 58.8% larvae (Collins 

1977), The workers consist of "etable" Individuals which have 

achieved their final development and also other individuals which 

are capable of further moultingg either whilst remaining an workers, 

or by transformation to soldiers* Workers do all the foraging for 

foodq care for the eggag laryno and reproductives Including feeding 

the larvaos soldiers and reproductives, They also maintain the 

fungus combs. In H. ballicoaux and Macrotermen subhyalinuq the 

foraging major workers were distinguished from "nurne" workers which 

were found attending the fungus comb and larvae* 

46.

The soldiers may also be dimorphic, with trimorphic soldiers In 

Pseudacanthotermeso They are also sterile and have strongly chitinised 

heads with greatly modified mouthparts for defence. They are a 

"stable" caste, incapable of moulting and are always preceded by a 

"white soldier" stage which may develop from a larva or worker in the 

Flacrotermitinae (Lee and Wood 1971). Their function is to defend the 

nest, and the workers when foraging away from the nest. They may 

also produce defence secretions. 

The primary reproductivos consist of a king and queen, and when 

the neat to in the right physiological state it contains secondary 

reproductives, 

("alates"), which have no function within that nest 

but leave it in synchronised flights after the start of the rains to 

found new colonies. Following post-flight courtship the newly- 

established pairs burrow into the soil and construct a copulariumo 

Copulation and oviposition occur and, in the case of Mcrotermes spe A, 

the first larvae appear after 28 days (John3on 1980)s The Icing does 

not change much during the course of his lifetimos his only function 

being to fertilize the queen* In tho Macrotermitinae the queen is 

rhysogastric, more or less immobile, produces eggs almost continuously 

and lives in a specially von3tructed royal chamber with the kings 

The nests may be either hypogeal or epLgeal and monocalic or 

polycalice Unlike other termites the nests, mounds and runways are 

made solely from soil* The nesto serve to house and protect the 

colony, store food and togather with the fungus comb maintain an 

optimum environment. which together with the social behaviour of the 

termites produces a condition of homeostasis. There is a contimious 

exchange of sub3tancess called trophallaxLs, within the colony which 

guides the behaviour of Individuals* It includes the exchange of 

47.

nutrients, colony-specific compounds which pe-m. it recognition between 

individuals in the same colony and the distribution of pheromones 

involved in caste differontiation and caste elimination 

cannibalism) 

(Lee and Wood 1971). 

The termites feed an dead wood and other plant detritus rich In 

lignin and cellulose which the workers bring back to the nest* They 

feed either within the food, or covered and protected by soil sheeting* 

In some cases the food may then go into a foodstore (e. g. Mkicrotermes 

bellicosuS), which consists of finely divided comminuted material, 

like damp sawdust in appearance, sitting above and slightly around 

the fungus comb (Plate 29391), H. bellicosus foodstoro contained 

nematodes, collembola. and diptera larvae. Usually there is no food- 

store and the food in eaten by the termites which build convoluted 

structures, brain- or sponge-liko in appearance, called fungus combs, 

from their faccal pollets consisting of only poorly digested food. 

It is on the fungus combs that the symbiotic fungus Termit2=ces grown* 

The termites Pat Termitomyces. and re-eat the old portions of the 

fungus comb on which Termitomyces has acted* The action of the 

termites eating the old portions of the fungus combl while building 

it up with faecal pellets results in a recycling of their food through 

the termito-fungus system thus leading to its complete or almost 

complete degradation* The fungus combs may be concentrated In one 

central chamber as in the case of M. belliconuS, or scattered In 

individual chambers through the soil. 

The major losses of organic matter and nutrientis from the termite- 

fungus system are the death, or eating by predators, of alatesq together 

vith the losses of termites from established colonies to predators 

especially antse 

(by 

480

PLATE 2.3.1. 

Macrotermes bellicosus (young colony) plate and hive with some 

nest structure removed to show fungus comb and foodstore'ý 

49.

ý, ft . 

S 

S 

.ä

2.3-2 SPECIES OF TMtITES STUDIED 

Ancistrotermes cavithorax 

The ne., As of this species aro subterranean and polycalic with 

fungus combs in individual chambers. In Babba the nests w(, re some- 

times associated with old Cubitermes mounds. 

The fungus combs are fairly small 

(3-5 cm long)l and are eaten 

away at the bottom which is the oldest partl and are built up on the 

top and sideso They are Oencrally round in shape with a domed surface 

and flattened base, They sit in the chambers on projections of the 

fungus comb. They are fissured, brain-like structures, some of the 

rounded fissures penetrating through the whole comb. The mycotZtes 

sit within the fissures. The newer parts of the comb are lighter in 

colour than the older (Plate 2.3.2). 

A. cavithorax has both dimorphic soldiers nnd workers* They are 

a small specics, the major soldiers head width being around I mm 

Ofarris 1966). 

They are ca=xm in the forest at Rabba and in primary savanna 

woodlande Continuous cultivation results in their loss (Wood,, Johnson 

and Chiagu 1977). 

They feed on dead wood and plant litter and the outer bark of 

living trees and forage in rumays and under shoots of sub-soil* It 

forages most during the wet season but has more dry season activity 

than Microtermns (Collins 1977). 

The alates fly around 7*30 pm. the day after raing on several 

Occagions early in the rainy season. 

50.

Macrotermes bellicosus 

This species builds very large conical opigeal wounds which may 

be 0-7 m high with a base diameter of 3-4 m (Collins 1979) (Plate 

2-3-3)- Other types of viound are built in other places but around 

Mokwa the habitacle in entirely below ground levele There are 

wounds where the fungus comb sits on a bass plate with spiral vanes 

on Its undersides and wounds without a base plates but only the 

former were found during the course of this work. The main spires are 

hollows connecting with the space around the hive, 

The fungus combs are all concentrated in a hive around which 

there In a thin layer of earth sheeting. Earthen supports also ramify 

through the fungus combo This earth worked by the termites was termed 

the nest structure, The foodstore sits on top and slightly down the 

sides of the fungus comb (%2*lj Plate 2-3-It Fig* 293*0e The queen 

cells which Is a thLekwalledl hardl clay structure sits within the 

: fungus comb above the larval galleries which are at the top of the 

base plate (Plate 2-3-49 Fig@ 2-3-I)a 

The fungus combs are larget convoluted structures with a brain- 

like appearance (Plate 2*3,, 5)9 The fresh edge of the comb which is 

towards the outside In darker In colour than the rest* 

7he soldiers and vorkers are diworphice It In a large speciesl 

the head width of the major soldiers being around 4 mm. 

It was found in the primary and secondary woodland but was absent 

from the forest at Rabbae In order to mechanically cultivate land 

the mounds have to be destroyede 

It is primarily a wood-feeding species., but also takes plant litter, 

with bark in the dry season and some leaflitter early In the rainy 

510

season (Collins 1977)o It forages under runways and sheeting mostly 

of brown topsoil. Foraging occurs all year round with a suggestion 

of slightly greater activity In Mays July and November (Collins 1977)o 

Macrotermen bellicopus alates fly between 7 and 10 P-m- the night 

after raing very early in the rainy seasons It generally flies after 

the first heavy raing with only 2 or 3 flights per seasons 

Macrotermes subhyalinue 

In the area where this woric was carried out M- subhyalinus built 

low multi-domed mounds around the gardens and golf-course at Mlo I 

(Plate 2-3.6), large domed mounds up to 4m high in the forest at 

Rabbaq and had entirely subterranean nests in the primary savanna 

woodlande 

Unlike M. bellicosus, it has no foodstore and no central hiveo 

The fungus combs are found In chambers closely associated with each 

othero The fungus combs are largel convoluteds Irregularly shaped 

structures with newly deposited material being darker in colour. The 

combs sit in the chambers on downward projections of the combo The 

large faecal pellet composition of the comb Is obvious (Plate 2-3-7)- 

It has dimorphic soldiers and workers and in an even larger 

species than Macrotermes bellicosus, the head width of the major 

soldiers being about 5 am. 

Mechanical cultivation destroys the vxxinds,, It feeds on grass 

and plant debris and way also feed on leavenj dung and vood littero 

It feeds under runways and sheeting of mainly sub-solle It forages 

all year round vith a peak'In. the dry season (Collins 1977). 

The slates fly In the evening throughout most of the rainy season, 

starting later in the season than M. bellicosum. 

520

FIGURE 2,3ol,, Vertical Bection of a Macrotermes bellicosus nest 

with spiral plate$ 

2 

3 

4 

5 

6 

7 

53. 

(; Lfter Collins 1979). 0) dentral 

chimneys (2) food store, (3) nest structure, (4) queen 

cells 

plixtee 

(5) fungus comb, (6) larval galleries, (7) base 

11

PLATE 2.3.2 

Ancistrotermes cavithorax fungus combs showing old and new areas 

PLATE 2.1.3. 

Hacrotermes bellicosus mound 

54.

WdvAm- 

Rocivreonafts 

c, av%. T 

A146VTROUSMS 

cr"v%-r"D"x

PIATE 2.3.4 

Macrotermes bellicosus hive with some nest structure and 

fungus comb removed to show position of queen cell. 

II 

PLATE 

-. 2.5 

Macrotermes bellicosus fungus comb in position in the nest 

55o

PLATE 2,2.6* 

Macrotermes subhy4inus, mound 

PLATE 2.3.7. 

Macrotermes subhyllinus fungus comb removed from nest 

56.

. OWIA 

fr/ 

o"m law 

pp 

,. 1, ýP, 

--Alm 

,I 

-S 

loop-

'A 

PLATE 2*1.8o 

Microtermes fungus comb in a chamber in the soil. 

- 57o

,.; 

ý; I.. 

4, 

.. 

. 4, 

WL 

r7.. 

I It* 

III(.

Mcrotermes aDecies 

There was a complex of 7 sympatric Microtermes species occurring 

in the savanna areaq for which the taxonomy has still to be clarifiedo 

There were also 2, or 3 species in the Rabba forest area* The different 

species were given different letters of the alphabet to distinguish 

them., 

They have diffuse subterranean nests consisting of single fungus 

combs in chambers UP to 3-5 cm diametert the chambers being linked by 

narrow galleries 

(Plate 20*09 The nests probably extend below 1*5 m, 

with the queen cell being 50-100 cm; deep (Wood and Johnson 1978)o The 

termites move deeper Into the soil in the&7 season, the majority of 

fungus combs being found below 50 cme Fungus combs are also common in 

abandoned mounds of M, bellicosus and In the walls of living utounds, 

Hicrotermes op. * D was found asnociated with mounds of TrInerviterrms. 

The fungus combs are small, of 5 cmmaximum diameter and an 

average weight of Ig (Wood and Johnson 1978), The fresh material 113 

lighter In colour than the older which in daric grey* The faecal pellet 

structure in obviotLs In the freish materials but not In the older* 

Mycelial projection3 stretch from the comb to the sides of tho chn era. 

The workers are dimorphic but there is OnlY One tYPe of soldier* 

These are small species, the head width of the soldier being 0.6-0.8 V=. 

They were coemmon in primary savanna woodland and forest, and some 

species increase in abundance under cultivation as they exploit the 

crop residues when potential competing species are rcmoved, 

They feed on all: brms of vood and plant debrial subterranean rootg 

and on some living plant tissues making them the major termite crop pent. 

58e

They forage an the surface in the wet sea on, but this Is negligible in 

the dry season when they feed on subterranean roots and the fungus 

combs* They enter their food from the soil and park the eaten parts 

with soil. 

The different species have characteristic flight timese Hicrotermes 

ape A flies from small turrets at around '"' po=4, the day after rain* 

1,11crotermes ape B flies 2 days after rain at the same time as spo A* 

Merotermes ape C flies the day after rain in the early evening at 

6 p. m. Hicrotermes species D flies around 7.30 p. m. on the day after 

rain and sometimes the second and third day* The male bites the females 

abdomene Microtermes ape G files the day after rain around 6o3o p. m. 

The males attach themselves to the feewles in flighto Mcrotermes ap. 

Z, a rare speciess flies 3 or 4 days after rain, In the early evening 

7 to 8 P-m- 

Odontotermes smeathmani and Odontotermes op. 

(OIY4) 

,j 

The nests are entirely subterraneang consisting of fungus combs 

in separate chambers In the soil. The newly deposited portions of 

the comb are darkest In colour, 

0. menthmani was found in the primary savanna woodland and 

around Mile 19 Odontotermen op. was found in the forest at Rabba, 

Continuous mechanical cultivation results in the loss or odontotermes 

speciese 

There Is only one form of soldier but dimorphic workers. They 

are of medium isizee 

They feed largely on wood-litter and also on bark and grass littere 

Odonto-termes spe also feeds on leaves and 0. smeathmni, takes leaves 

early in the rainy season. 

590

? LATE 2.3.9. 

Microternes fungus combs showing fresh and old zones 

6o.

MIC. 1 

toots 

CAL& 

Ail

They feed under soil runways and sheetinge 

0. ameathmani fileis around 2 pemo 2 days after raing early In the 

rainy season. There are several flights during the season. 

61.

2.4.1 TMMXTOMYCES 

IN NATURE 

2*4 TMMITOWCES 

emit=ces In a Basidlomycote fungus which grows on the fungus 

combs of termites of the sub-family Hacrotermitinse, withwhich it has 

a symbiotic association. It can be seen on the fungus combs as 

mycelium and also as round white bodies called mycot4tes (Heim 190). 

These have variously been called spherules (Datra, and Datr, % 1979)s 

nodules and synnemata, (Mgkrtin and Martin 1978)1, synnemata (Rohrman 

1978)9 conidial spheres (Coaton 1961) and conidia (Abo-, Qmtwa 1977)- 

The mycotOte consists of spherocysts bearing conidia of blastosporic 

ontogeny and is the asemal reproductive structure of Temitomyces, 

They are easily detached from the fungtw comb, 

The sexual reproductive stage of Termitomyce. 9% the basLdiocarps 

was only rarely encountered during the course of this worke It 

consists of a cap with a prominent umbo or porforatorium, and a stipe 

which in continued below the ground as a paeudorhiza which connects 

It to the fungus comb. Keys to Identify the species of Tensit=ces 

from the basidlocarp have been produced by Heim (1951,1958)9 Otieno 

(1964)9 Pegler (1977) and Singer (1949). 

The c=ct location of Termitomyces, in the nest systemg in termites 

and outside the nest system were investigated, as was the establishment 

of Temitomycess in new coloniesq and tho3e will be discussed latero 

2.4.2 TM11TOMYCES IN CULTURE 

Cultures of Termit ces associated with the termite species 

mentioned in 2*3*2 vere made, ThO IBOIAtlon of Terinitomyces required 

the development of a selective medium which WL11 be discussed In 

Chapter 39 as will the incubation conditions. Isolations were made 

6,.,.

from mycot8tesq fungus combs termLtes and basidlocarpso 

The method used to name cultures Is by a code vhIch Identifies 

the species of termite it In associated with, has a mimber Identifying 

that particular culture, a letter indicating vhere tho culture was 

from (oogo mycot0tog termite) and the sub-culture number* 

eege for the culture named MAC 36 A (5) 

MAC a associated with Hacrotemes belliCOSU3 (others are SUB 

Racrotermea subhyallnuall AN - Ancistrotermes cavithoraxl OD - 

Odontotermes and HIC u Microtermes). 

36 dintLnguishes thLa from other cultures from M. bellicosus. 

Al Indicates the culture was from an alate (others are T- worker 

termiteag Cm conidia in mycotftesq F= : fUngull, comb). 

(5) Indicates this culture has been sub-cultured 5 timese 

Details of the Orowth and enzymatic capabilities of Ter*ItcwXces 

In culture will bo discussed In Chapters 3 and 7- 

63o

CHAPTEn WEE 

DETERMINATION OF ISOIATION MWIA AND OPTIMUM 

CONDITIONS FOR GROWTH OF TMMITOMYCES 

64.

3.1,1 INTRODUCTION 

3.1 DEVELOPMENT 01" A Sr-LECTIYE MDIUH 

Ternitanycel Is very slow growing In agar culture and may be 

rapidly overgrown by other fungi. This made It very difficult to 

isolate Termitomyges from wycotates and almost Impossible to isolate 

from situations like termite Cuts and fungus comb whore many other 

fungus propagules were presents Many authors have found It difficult 

or impossible to Isolate Termit2Mcf%s (Abo-Matwa. 1977,1 Pet-ch 1906 

and Zoberl 1979)* Swift (1976 Peroe c, ý-. ) had some success with 

mycotete isolations using a cellulose based medium, 

This problem was approached by the development of a selective 

medium which would Inhibit the growth of contaminating fungi but 

maintain that of TermiteMees near to its optimum. 

of medium 

Radial Crowthmeasurements of the fungus in questions on plates 

incorporating the substances to be tested In a common 

method of screening substances for selective media 

(Papavizas 19679 

Uscuplic and Pavsey 19709 Vaartaja 1960). This method was adopted 

hereq and those substances appearing suitable were further tested 

for their effects on the growth of common contaminating fungig and 

on tho germination of TormitMceso 

Selective media can Include substances preventing the growth 

of contaminating fungi as well as substances which enhance the growth 

of the fungus being growne Both were tested by the same Mothode 

3*1*"- 1W'=DS OF MEASURING GIZOW211 

OF TM111rOMYCr!, 

-- 

The substances to be tested vero Incorporated into the basic 

medium (Appendix Do The volume of medium Used and therefore the 

650

depth was the same for all plates as there Is controversy as to the 

Influence of the depth of the medium on radial growth (Brancato and 

Golding 19539 Chaudhuri 1923 and Trinci 1969)e Plugs of Termitomycess 

approximately 5m In diameter, were cut from just Inside the edge of 

actively growing cultures with a sterile cork borer, and placed 

centrally on the plates of medium* Two measurements of the diameter 

of the colonies were then taken at right angles to each otherg and 

this was repeated on several occasions over the 2 week course of the 

experiment. After 2 weeks the final measurements were taken and the 

form of growth of TeraitgýLcea noted* Incubation was at 290C* 

In addition to fungal contamination, bacterial contamination of 

isolations was also a problem, Four antibacterial antibiotics and 

rose bengal were tested for their effect on the radial growth of 

, Tqrmit2=ces@ Novoblocin was dissolved In sterile water at the rate 

of 0.1 g In 10 ml and then added to the medium prior to autoclavinge 

chloramphenicol was dissolved in 10 ml of 95% alcohol and added to 

the medium prior to autoclaving, Streptomycin sulphate and penicillin 

sodium salt were added to the medium after autoclaving a! s those lose 

their activity If autoclaved, 

To prevent growth of and contamination by other fungi a total of 

33 different substances were tested* Substances which Inhibit the 

growth of undesired fungi were tested as well as substances which 

selectively enhanced the medium isee provided in the medium an energy 

source or other nutrients which are assimilabla only by a limited 

number of micro-organisms. The term selective medium Is here meant 

to Include any medium which achieves selective ex lusion of undesired 

organim=9 whether by selective Inhibition or selective enhancement 

(Tsao 1970). 

660

3-1-3 CANDIDATE ANTIBACTERIAL, ANTIFIJNGAL AND GROWTH-PMOTING 

SUBSTANCES 

3-1-3*1 Results 

The results are given in Tables 3-1-1 to 3-1*3* 

(1) Antibiotics 

(a) Chloramphonicol 

Chloramphenicol acts by Inhibiting the incorporation of amino 

acids into ribosomal proteins (Lukerm 1971). At the 

-dose 

by the C-Hel, of 0*05 91-1 the growth of Termitomyces was Greatly 

reduced (Table 3ol-1) and so this substance was rejected* 

(b) Novobiocin 

Novobiocin In an antibiotic which prevents synthesis of call 

recommended 

wall materials In bacterial and in an Inhibitor of protein synthesis* 

It also affects bacteria In a variety of other ways Including acting 

on the cell membrane (Lukens 1971), It is active against both gram + 

and gram - bacterial and also Inhibits the growth of P. Xthium and 

? hytophthorae Butler and [line (1958) found that it retarded the 

radial growth of fungal colonies and here it reduced the growth of 

TemitqmYce-s bY 35% at 0.1 gl-1 (Table 3ol. l)- Duo to this it was 

rejected for the selective medium but It was decided to use It In 

the general soil fungus medium (sr. ) (Appendix 1) to prevent bacterial 

growth* It has the useful advantage that It can be autoclavod., 

(c) Penicillin 

Penicillin acts In a similar way to novobiocing preventing 

synthesis of cell wall materials In bacteria and inhibiting protein 

synthesis 

O. c)5 gl-1 

(Inkens 1971)o It has been used at 0,0050 0*015# OeO3 and 

(Bakerapioel 

and Miller 1953), OeO3 gl-l (c. ý1.1. jL968) and 

67o

0,05 ol-l 

(Eckert et al. 1961)- As penicillin at OoO5 glýl did not 

reduce the growth of Termitomypes, by much (Table 3*1*1)t and had no 

effect on the growth form It was decided to incorporate it Into the 

selective medlun but at the lower rate, of 0*02 ()1-1. This had later 

to be increazed to 0*05 91-1 due to Inadequate control of contaminating 

bacteria* 

(d) Rose bengal 

11ose bengal has been used to control bacterial growth In selective 

media at various concentrations including 0*0005 (Flowers and Hendrix 

1969), o. 03 (Vaartaja 1960)l OeO6 (Flowers and Hendrix 1969)9 0.067 

(Bakerepigel and Miller 19-')3)9 0.1 (Papavizas 1967) and 0,42 01-1 

(Papavizas 1964)e It has also been found to reduce the rate of growth 

o: r spreading : fungi (]3akersrAocl and Miller 19539 Singh and Mitchell 

1961, and Vaartaja 1960)'though Papavizas (1967) found that even at 

0,1 91-1 it did not reduce the colony size of some of the rapid 

Crowerm. It also inhibits the growth of actinomycetes (Bakerspigel 

and Mller 19539 Vaartaja 1960). Some bacteria can still grow In the 

presence of rose bengall and In many cases it lowers the fungal count 

Wartin l%O)* Taking these considerations into account and especially 

In tho light of the laree reduction in the growth of Torraltomyces It 

caused (Table 3*1-1)9 rose bengal was rejectede 

(0) Streptomycin sulphate 

Streptomycin jiulphate in a broad spectrum antibiotic which 

inhibits a single enzym system and Interferes SPOCifiCally with 

electron transport in the cytochromo system. It has been Used At 

concentrations of 0*006 91-1 by Batra and Batra (1979) for the 

Isolatlon of Tpmit2=eex Other concentrations at which It has 

been used In media Include 0,019 OeQ39 0-059 0*19 0--JL3* 0.3 and 6 

68.

(Bakerapigel and Miller 1953, j C*M*19 19639 Kuhlman and Hendrix 1962, 

Nadakavu1taren and Horner 19699 Papavizas and Davey 19599 Papavizan 

1967 and Uscuplic and Pawsey 1970* Martin (1950) found it had an 

inhibitory effect an only two out of 117 cultures, At 0,02 gl-l 

streptomycin aulphate did not adversely effect either the rate or 

form of growth of Termitomyces and it was decided to incorporate 

this Into the selective medium. 

Penicillin and atreptomycin are often used torjetherl and Datra, 

and Batra (1979) used them to isolate Termit=cem 

bellicomiso 

The Termitonyces cultures tested were all from Macrotemps 

(2) Antifungal and_11rowth I! romtina substances 

(a) Denlate 

Benlaia fungicides or benomylq in a systemic fungicide of the 

benzimidazole group$ which controls a wide range of diseases* It 

functions by the inhibition 

of DNA blosynthesis (Cremlyn 1978)o 

Benomyl is especially effective against Penicillium and some other 

llyphomycotes* MaloY (1974) found that at 0*001 01-1 It completely 

Inhibited Penicillium sp. and Phialophora fastialata, and greatly 

reduced the growth of Trichodermai. 

(b) Borax 

Borax - disodium tetraboratee 

(c) Cellobiose 

This was used In placo of glucose in the basic modium. 

(d) Cellophane 

This was used ast a cellophane overlay In place of glucose In the 

basic medium* 

690

(e) Cornmeal agar 

CUS04- 51lqO@ 

(g) Cyclaheximide 

(Appendix 09 

Cycloheximide Is an inhibitor of nuclelc acid and protein 

synthesis, causing thickening of the cell valls of hyphal tips, It 

also attacks the synthesis of DNA to a lesser extents Its effect 

has been tested at 09004,0*029 Oel and 0,5 gI-I by VaartaJa (1960) 

and has been used at 0925 Ul-I by Schrothl Thompson and HLIdebrand 

1965. 

(h) Czapek agar 

(Appendix 1) 

This was used by Heim In his Initial attempts to obtain 

artificial cultures from orfcotttes (Ileim ]L977). 

Dithane 

A dithlocarbamate fungicide, 

Fungus comb 

Fungus comb of Macrotermen bellicosus was Incorporated Into the 

basic medium in placo of glucose and also tested with : fungus; comb and 

agar only, 

Gallic acid 

Gallic acid In a phenolic compound that has been used In selective 

media. Flowers and Hendrix (1969) found that the Incorporation of 

Callic acid at a rate of O, W-5 ol-1 prevented developnent of non- 

pythiaceous fungi@ 11intikka (1971) found that Bome Dasidiomycetes 

could grow In up to 15 CI-1 but In general It reduced their growth,, 

raodiume 

(1) Glucose 

This vas tested at a range ofdLfferent concentrations In the 

70-

(m) Icarathane 

Karathanes cr dinocap,, Is a dinitrophanyl derivative, It is a 

contact fungicide and interferes with respiration through uncoupling 

oxidative phosphorylation in mitochondriae 

Ibmitol 

This was tested at a range of different concentrations in place 

of glucose in the basic medium* 

(o) Murphy fungicide 

Murphy fungicidel or thiophanate methyll is ono of tho thiophanate 

group of systemic fungicides based on thiourea* It Is similar in its 

antifungal properties to beriamylo 

(p) Nystatin 

Nystatin is an antifungal polyane antibiotic which destroyn the 

selective permeability of the cell membrane. Concentrations used in 

selective media Include 0.03, (). 05 and 0.5 gl-1 (Papavizas 1964)9 and 

are of the same order as the concentrations tested here of 0.1 and 

0.01 Ul"I 

(table 3-1-3)- 

(q) Oatmeal agar (Appendix 1) 

Batra and Batra (1979) obtained growth frov: mycatOtes of 

Termitponyces albumin-oaus- on oatmeal agar* In his Initial attempts 

to obtain artificial cultures of Termitg=ces Heitz (1977) Included 

oatmeal agar* 

(r) Ortho-phonylphenol (OPP) 

This was used by Russel (1956) In a Gel6ctiVo medium for the 

isolation of Mmidimycetes. At 0,06 gl-l q the concentration used 

here, it restricts colony growth of fungi such as Alte qnsria, Cladosporium 

71*

and Penicilliun. Several Banidlomycetes will grow slowly In media 

containing up to Oel gl-l. 

(s) Ox bile 

Ox bile has been used In many selective media* It supresses the 

Crowth of saprophytic fungle It was used by Papavizas (1967) at 0.5 

Cl-1 in a selective medium to isolate Fusarium,, and at higher concentra- 

tions it also supresses, bacteria* Bakerspigel and Miller (1953) found 

that agar containing 1% ox bile supressed bacterial colonies by one 

fifth to one tenth* It restricts the growth of Penicilliumg Rhizorus 

and Trichodermal as well as other spreading fungi. 

(t) Para-phenylphenol (PPP) 

(u) Pentachloronitrobenzene (PCN13) 

rCJM is a narrow spectrum soil fungicide which inhibits rapidly 

growing saprophytic fungig slowing the growth of Trichoderma'l and 

oupposedly limiting that of Anperpillus 

and Penicillivm* Kuhlman and 

Hendrix (196") USOd it at 0919 gl-1 and found it prevented growth of 

the Macorales and Greatly restricted the Growth of bacterias Singh 

and tUtchell (1961) found PCNB Inhibited the growth of many soil fungi 

such as j1hiKoctonia, Scleratinin and Sclerotium but not gythium, 

PhXto]Lhthorq or]Fxisarium. Farley and Lockwood (1969) found fungal 

numbers were reduced by 27-40% with concentrations of PCNU ranging 

frOM OeOl-0-5 91-10 

(v) Poptone 

The use of peptone as a carbon and nitrogen source slows the 

growth of fungi requiring carbohydrates for growth (ruhlMan wid Hendrix 

1962). It was used here in place Of glucose In thebasic medium. It 

was used by Russel (1956) In his selective Mcdlum for Isolating 

722 

-

Basidlomyceteal at 5 ()1-19 but In general It Is not used in selective 

media an it in especially active In decre"Ing fungitoxicity (Cochrane 

1958)* 

Potato dextrose agar 

(PDA) (Appendix 1) 

PDA is a useful all purpose medium andwas used by Datra and Batra 

(1966) to Isolate Temitomypeag but with the addition of 10 gl-, l of 

yeast extract. 

Richards agar (Appendix 1) 

Heim (1977) used Richards agar in his attempts to establish 

artificial cultures of Termit M ces. 

(y) Sabourauds aW (Appendix 1) 

Sabourauds agar in the classical medium for the culture of 

dermatophyteso This was used by Ifeim (1977) in his initial attempts 

to obtain artificial cultures frOM mYcotOtes of Termit=cas. Datra 

and Datra (1966v 1979) alsO used it in culturing Tarmitomyroa 

(z) Salicylanilide 

Salicylanilide luts been found to be active against Asnerpillus 

niner. WW Penicillium e2M 

by Vaartaja 

Sodium pentachlorphenoxide. 

(A) Sodium proplonate 

Sodium propionate has been screened for use In selective media 

(19GO) at O, W4* 0.00.9 0.1 and 095 91-19 and by 

Papavlzas (1967),, It does not inhibit the growth of actir=yccteo 

(Corice and Chaae 1956). 

Vg) Sodium taurocholate 

73a

(e) Sorbic acid 

Sorble acid at 0.05 to 1*5 91-1 in active against Apper2illus 

and Ponicillium species* 

(6) Tannic acid 

Swo Ilymenomycetes have been found to be tolerant of UP to 7--5 

gl-l of tannic acid (11intikka, 1971). The concentration (5 gl-l) 

used hero was used in a selective medium by Vaartaja (1968)o Tarmic 

acid is also useful In that it Inhibits the growth of most bacteria 

(Henderson and Farmer (1955), The tannic acid was substituted for 

glucose In the basic medium at 10 gl-l and added to the basic medium 

at 5 01-19 

(-"-) Thiamine hydrothloride 

Thiamine hydrochloride (Vitamin DI) was added to the medium to 

see if an increased amount of this vitamin vould enhance the growth 

of Terrnit=ces. 

CA) Vanillin 

Vanillin was added to the basic medium In place of GlUC030- 

74-

RI 0 

t#l Z 97 9 

e f+ 

2 

0 

0 

c 0 

In 

O< 0 1 

Ili- 9 - 

e 10 rr Irl to: tr 5 n 

re uZ0 0. - 0 1Z7 9 

? 

O= 411161 

9 e_ 

C 

ý < )- *i te 

b- w - 

9 

fD r, 3 9) 0 :Z ba :3 ts 0 c 

8 

000 

l' l' 0# 

ti N- 9 

0 

;a 

02 % 

2 2, 

". o 

b-i 

0 0 

& 

0 

& 

bi 

00 

.. P, ce- 

#.. d ra 9 

a 

4 

b V. 

C 

bw 

. ýa 

t4 3 w ri 

86t+ 

00 0 ow 0 0 0 00b-0 

% 

Za Z4 

w m 

0* 

1. 

-0 

(D 

e8 

to 0. fl> 

0 

IK 

9; 0e0 

0e 

1 1 

vi u 

00 

Zn 

0 0d - 

ca 0% c%, o 

- %§ :a 1: 4 i. i- ü§ -0 

u 0 0 

L 

1 

b- en 

:ya 

l eI l 

be ep m & cr, 

60" 8 4 1 

n 

0 So 

b 

0 

:3 

1.6. 

0 

2 

- 

p. 5 

:t 

2ä" 

0 

u 

l', ' 1 94 

da ý 

Z 2 

"0 

m 

be 0 fß 1--4 

' 

b- 

014 - 

P 

0+ 

0 %W Z 

ti 

04 

06 

fo 0 

(D 

01 

0 

m 

0 

0 

ell 

cr 

19 1. " 

0 

75. 

f-6 

n to, 

0. " 

el 

7-p

TABLE 3*lo2 Antifungal substances preventing the growth of 

Temitarnrces, whcn incorporated In the medium. 14 days incubation 

at 290C. 

CULTURE ANTIFUNGAL SUBSTANCZ 

CONCENTRATION 

HAC 12c (4) Borax 70-00 

AN 33c ( 2) Cycloheximide 0106 

HIC llc (3) Cycloheximide o. o6 

MAC 12c (7) Dithane 0*10 

HAC 12C (4) OPP o. o6 

tfic lie (3) OPP o. 06 

tac 12c Ox bile 5.00 

MAC 12C PPP 0.20 

HAC 12C (9) 13DA see Appendix I 

MAC nc (4) Salicylanilide 0.20 

MAC 1, 

"Ic (3) Sodium pentachlorphenoxide 01016 

HIC lic (3) Sodium pentachlorphenoxide o. o16 

MC 12C (7) Sorbic acid 0.10 

MAC 12c (5) Tantilc acid 10000 

MAC 12c (7) Torsnic acid 5"00 

IMC I 2C (4) Vanillin 10000 

MAC = jermilomycos, cultures : from Macrotermes bellicosus, 

HIC = Termijam cps cultures from Hicrotervwn species* 

ELC I 

AN alermitg!! yces cultures from Ancistratemes ca-vithorax, Wý- 

gl-l 

76.

do% 

w 

0 

%., 1-a %w W %. 

W do% in 0-4 0 4- %» #-% 0 a. % 

g 

o 0 0 

0 0 c 0 

00 

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a 

n ta 

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CO C* 0 -, 1 

1. 

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98 

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8.. a 

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4- 0 %0 c% 

Z-Z3 

r 2u t; 

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N. WU 

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m 11 

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mu m4 

c152 % 0 " d ug 

00 

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nnmn 

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c za !. ýh ý, ýb 't"3 , 88111t01t 11 

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+ 0+ 1+1+1+ 1+ 1+I+I+ 1* 1+I+ # +1 + I+ 1+ 1+ 1+1 + 

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%0 

1-d I-d 

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NO %0 " \-q 1-- -4 

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to to 0 

1+ 0 a5 1* m 

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to 2 r+ 12 

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rg 

IA 0 (D 0 

to 

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R 

n 0"w 95 

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d 

n '' 0" "08 

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ýW I I 

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Go*

m 

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3-1.3-',, ', ConclusiOns 

(1) Substances rejected for use In the nelective medium 

The substances which allowed no (Table 3*1,, _2) or very reduced 

(under 50% of control growth 

(Table 3-1-3) wero rejected. These were 

Dithane, Karathane (at 0,, 0201-1)9 nystatin, OPP,, tannic acIdj 

salic7ylanilides sorbic acid* P*L)oA*t Richards arjar and Oatmeal agar. 

Zoberi (1979) vas unable to get. (; rowth of mycotCtes on P*D*A* but 

13atra and Batra (1979) obtained growth from mycotates of Termitgamces 

albuminopus on it. Batra and Batra (1966) isolated Termitamycen-on 

PDA but with the addition of 10 gl-l of yeast extract which presumably 

supplied the Ingredients missing from PDA necessary for the growth of 

Termito=cene Although cycloheximide allowed the growth of 

Ancistrotermen cavithorax and Hicrotermes associated cultures at 

0.003 01-1 (Table 3-1-3). the culture from Microternes was reduced 

bY 34%. It also affected the form of growth of Tormit=ce-9 and so 

was rejected* Sabourauds agarp Czapek agar and Cornmeal agar were 

not tested further as a great deal of contamination occurred an tlie 

plates* 

(1-7) Substances selected for further testing 

Sub: 3tances which did noteffect the form of growth of Ter-mitomyces 

(those mariced ' in Table 3,1,3) were tested further to assess their 

effect on cont=inating fungi. 

3.1.4 Er-FECTS OF CDIUX«DS FAVOURING GROWIII OF TERMITOMYCES UN 

COI-MN COWAMINATING FUNGI 

3.1.4.1 

Methods 

Those substances not adversely affecting the growth of ýTLCrmitqmYqpI3 

(Table 3.1.3) were screened for their effect on 4 ccx=on contaminating 

fungi - 

82.

Inocula of the fungi were placed on plates of the medium, 

incorporating the substance to be tested, as shown in Fig. 3-1-1-- 

The fungi used were An=. rpillus ni2er, CophalosL2rium sp. ', 

Penicillium, spo and a sterile mycelial isolate WPP, In the course 

of testing the substances it was reallsed that WIT would not pose 

a great problem due to its slow growth and Paecilomyces variottl 

was substituted for it* 

The plates wero Incubated at 29'OC and the growth of the fungi 

recordod after 24 wW 43 hours., If t1le plates ware not overgrown by 

this time the experiment was continued for 5 days* 

The scale used to record the growth of the fungi from the 

inoculum plug towards TrrmitomXces was 

GROWTH (cm) SCALE 

0 0 

0-00-)0.49 1 

0.5040.99 2 

1-0041.49 3 

> x. 49 4 

One of the problems in developing selective media In that 

substances appearing suitable when incorporated in the medium on 

thoir own may be unsuitable when used in combinations This may be 

due to antagonism between thmt reducing their affectivenesal or 

synergism resulting in an increased inhibition of the fungus being 

isolated (Tsao 1970). For this reason various combinations of likely 

ingredients were tested an the selectivo medium was developed, 

3-1.4.2 Results 

The results are given in Table 3.1-4. 

83.

Q2 

bw- 0 

). d 

5. 

8 (+ 

0 c (2 c: :1 0 r. \il o 

W 

8 b- 

(0 n 

w0 

b" 

8 

t- t + 80 1. -e 

b-to 

9 

1 

-n 

4 C) 

t* b- Da w to b" 0 6- ri b- to N- tli w %A ra )-b w b- 

00 b-b-0 00 

ri b- w b" ti 8- CJ )- ti 1. 

-4 V b- W 0" b3 8- w to 4-a 

9- p- t) ime ta b- ra 0- %A) bm r13 NW 19- ei ta w b- op, w n 

W cn (n cn cn mw NW 

te 

» 

,Z 

ic 

88 

0. 

94. 

4 

0. 

-

ýP 

Z. 

OP s- 

W 

0 

in 

n 0 -e 

2 

1.44 

F-- tr 

8 cr 

l> 

er p g, W 

0Z 

F - 

4- (A 

R IM & (D bý 

Z i> :ý s 

5 

e c) NW 

o ý'n 1 

0 

0 

- 9 

ti %D 

42 W. 

12 0 N- f+ 

u 

- 

0 0 

r 1 

0 4 

ID 

ri ta b- to ta 0- ta b- ti 9. 

- ta b- 

00 00 e. 0 NM 9.0 be 0" w b" 6- ). w 

4- Co 

, 

)- )" bo 0.. & t13 1. -4 1. -8 km 

0-% «% 

tn 

ok- to O> u 4- fe ä> " c- 

f to 4- b- 4- ei 

16. 

a b- ti b- ta b- t13 0- ra 6- t, 3 b- ti 0- 

4-% 

NW %. - %., 

tn 

0-% &% 

cn 

" $- ri " ti 9. -0 m p- ba NO to 1. d w f- U b- 

rA cn rn cn cn 

%W Nbo 

-- 

w )- A- b- 

4-. 

En 

%. # 

0-% 

ti b- 

#-% 

. 

9 

1. 

)i- 

85. 

Z 

n 

4m. 

tn Ln Nd 

%. 0 %. & Po 

W. 

Ln cn 

.. ........ ........................... 

.. .................... 

2

0 0o1: 

1, 

.n 

R a I- ? in p pp 6- 0 0-0 

0- 0m 

000 ts 

on 

IW 

0n 

:3 t- 8 w pi 

1., 0 H. 0 

to 0V 

I 

o 

ft 

10,1 10" 

r+ V 

p 06. 

ID Od. 

J.. 

0 0 -W R 

.00 

0 

i 

0 r) 

10 W ý. q w 

I 

ol 

A 

0-1 pw. 

ra ow f-, P- ta I-d %. 4 6- W t3 0.4 0- 1.0 1.0 b) 1- 06- 

00 w 00 

0-k 

00 1. - 63 to W 0- " 

EA cn 

C) 

1ý 

".. 0-ý 

10 C4 

tT 

0 

Vý CC c 

0 

0 

ta 

F 

En 

0 

1 

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10 

un 

I. " 

0-k 0.4 

* 

(n 1.4 

86.

f-" 

> 

a b 

- : 31 0 Wd 0 

>- ýD p 0 1 >- 

:i 0 

tD 0 e b" 0 

0 

t 2 

9 

1-4 

0 

Z. 

ý i 2- 

i 

b- p 

e 

p 

MT 9, 10-0 i 0 i 1 em 

ý n 0 $ - 0 

=0 

1.4 

lý b cl 

0 

t 

b- 

N. 

4 b.. 

d 0 

- 't . Q2 - 2- ). 

f 41 

W 

a 

(Z fD 0w 

b.. b 

&0 

W4 Co c+ 

>- bý Md >" bý bý b- b" W 6.0 00 

4- to 0, t- 4- ro 

+ 

Z) 

). 0 f) 

:S 

" 

00 

). -40 

(D b- 

> 1 >. O 

ha 

bw 8 

1-. 

s- s- W >d bd. 

t> t> t> 01 

et- 0. 

- 19- te b> t3 4-s-s > 

i 

NM 

tA 

Q 

87* 

C) 

1-4 b- to s- 0 b- u 

6- M UW %» >w 

m WO- u NM ei b- Z 

Nd 

0-. 

0-, 

0 

tn tn tn f+ 

0-1 

m ?h tn b* 

4- b- e- W b" b. 

-6 f" t- >O >d lp, m %A b" %i b- U im b- >O

'"l r 

8 (A 

fl- 

bo. 

'2 

lot. 

a -4 

1- -j 1- b. A 0% I-d 

ta 0 ta O-d W ta 6- 

tn tA 

% op %0 

Ch 0. - Q1 0- %. n I" IV 

, ý- 0- 4'- 1. - 191. $- W 6- fs: - 0-4 op- I. - IF- I-" 

ul to tn 00 tA w 

to 

tn 

%. 0 1 - %- a 

10 

3 

"I 

88.

Penicillium SP. 

TermitomYc 

FIGUO 3*1.1. Position of iziocula of funj; i on petri dish 

containina azar medium 

incorporatins the 

antif=gal subatance to be tested* 

89* 

Cephalosporium sp. 

Aspergillus niger 

sterile mycelium 

or 

PaecilomYces variodi

(1) Substances appearino to be unsuitable 

Mannitol at 59 101 30 and 50 01-1 Increased the radial growth 

of Termit2aces-compared to the control 

(Table 3--l-3)o The growth 

was less dense with sparser mycellum and iso the total production of 

mycellum on mannitol, was probably not increasede Abo-Khatwa (1977) 

found that only agar media containing 30 Ul"I of D-mannitol, was able 

to support monocultures of Termitomyces* He found that Inclusion of 

other sugars In the agar media resulted In the growth of a variety of 

contaminating fungiq whereas the inclusion of mannitol, prevented these 

growing& This result was not borne out here where mannitol at all the 

concentrations tested did not inhibit the growth of any of the 

contaminating fungi. In fact in many cases the presence of mannitol 

stimulated the growth of Aspergillus niner and the Penicillium species* 

Mannitol was therefore considered to be no Improvement on glucose an a 

carbon source and was rejected* 

Other fungi do not grow on the undisturbed fungus comb In the 

nest (5.6), but the incorpomtion of fungus comb into the medium In 

place of glucoses or simply added to agarg did not1revent or reduce 

the growth of any of the contaminating fungis Whatever prevents the 

growth of the contaminating fungi Is destroyed by the action of auto- 

claving or just simply the rmoval of the comb from the nest system* 

Datra and Batra (1979) showed that growth of Termitmyces albuminosus 

was reduced on the fungus garden filtrate and residue compared to 

their basal medium and vitamin mixturee Petch (1906) 

obtained 

germination of mycotle'tes In comb extract than In water but he wan 

unable to extend the growth beyond 3 to 4 days due to bacterial 

contamination- 

better 

900

The addition of Vitamin Bl (thiamine hydrochloride) did not 

enhance the growth of Termit2=cea (Table 391*3), Yea t extract 

contains a large number of organic supplements such as vitamins and 

growth factors and it would appear that it supplies sufficient 

Vitamin DI, but In the absence of yeast extract growth Is reduced 

compared to the control 

(Table 3.1.3), showing that the yeast In 

providing Termit2=cas with other necessary vitamins. 

(2) SubstanceSaRpearingto be suitable 

The addition of both Denlate and Murphy to the basal medium did 

not reduce the growth of Termit = cps much but they both reduced the 

growth and gemination of Penicillium op, and Asperaillus niver, and 

also had a small effect on the sterile mycelium. The substitution of 

cellophane for olucose, with the fungicideng did not decrease growth 

of Tertnitowncesl In fact there was a slight Increaset and the growth 

of Aspergillusniner and Penicillium op. was still prevented* It was 

decided that cellophane would be the best carbon source as an organism 

requires the full cOmPlment of enzymes needed for the digestion of 

native cellulose to decompose It and It would therefore be selective* 

The addition of CUS04* 5H20 reduced the growth of TermIt2!! Ycmm 

considerably, but it also reduced the growth of Cephalosporium as 

well as the sterile myceliap Am"rnillus niger and Penicillium, ap. 

The fungicides Plus CUW4- 51120 with Glucose as carbon source did not 

reduce the growth of Ceghaloa22rlum but reduced the growthof other 

fungi. Unfortunately it also reduced the growth of TormitomYces, by 

over 40%. 

Karathane at 09002 iWl did not reduce the growth of Termit2mcels 

very much and did not affect the form of the growth* It also had a 

91.

slight controlling Offect on the 

_Paecil2Mces; variotii* 

growth of Cephalos]22rium, and 

Cellophanes novoblocin and the fungicides slightly Increased 

the growth of Termit2=ces, and prevented or greatly reduced the 

growth of Penicillium sp,, A. niger and prevented germination of 

their spores. The growth of sterile imycelium was slightly reduced$ 

but there was no effect on Cephalosl! 2rium. 

Ox bile inhibited the growth of P. variotii, A. nin and 

Penicillium spog but also reduced the growth of TermitpmXSexp Gallic 

acid slightly reduced the growth of CepjMlosg2rium. 

The most suitable medium at this stage would appear to be that 

containing the fungicides Benlatel Murphy and Itarathanal plus ox bilet 

Gallic acid, C'011OPhanOt CuW4.5H,,, O and antibiotics, 11ovever its 

effect on spore germination needed to be tested* 

3*1.4*3 

Concluaions 

Substances a2l! earing to be unsultablo 

Substances rejected due to their non-inhibitory effect on the 

growth of the contaminating fungi and non-stimulatory effect on 

Termit=vs, were PMj sodium propionatel mannitolv peptoneq fungus 

comb and thiamine hydrochloride. 

(2) Subfitances- 2MearInjL to be suitable 

Those substances appearing suitable vere Benlatel ýýhyj 

cellobiose or cellophane as a more selective carbon source than 

olucoses KarathAne at 0-0012 91-1, ox bile at I glýlq gallic acid 

md 

CuSO4* 511n 

100 

92o

3*le5 THE WECT ON SPORE GMINATION Or, COMUNDS APPEARING 

SUITABIZ FM A SELECTIVE MIM 

3*1*5ol Methods 

It Is important that the selective medium does not affect the 

gemination of Ternit=ces spores as vell as not affectina. its 

mycelial growth. Spores and mycelia often have different sensitivities 

to factoras such as Ingredients, in selective media and so high 

concentrations of antimicrobial agents used In telectivo media that aro 

non-inhibitory to mycelial growth might be Inhibitory to spores 

1970), For this reason the spore gemination of Temitomyces was 

tested on media containing the substances appearing suitable for 

inclusion In the selective mediums 

SWP, ensions of A=rVillus niner and PaecilMXces variotii and 

conidial agglomerations of Term1tq%yqes in sterile distilled waterg 

were pipetted alone and in combination on to plates of basic medium 

W) 

and selective media (see below)* If only one fungus was used 

0,1 ml was pipettedl 2 fungi 0.5 =I each and 3 0.03 ml each* 

The plates were Incubated at 290C and the number of fungi 

developing counted* 

13H (Appendix 1) 

Candidate selective media (CSM) 

CSM (1) = VI4 - glucose 

+ cellophane overlays 0*01 Cl-I 

Denlatet 0.01 Ul'I Murphy, 0.05 gl'I C"5040 5112.09 

Oal al-I novoblocin 

CSIf (21) w BM - alucose 

nannitol 

(10 ol-1) 

(Tsao 

930

CWI (3) 

- MI + Denlate (OoOl gl-Jý)q tUrphy (0*01 gl-l) 

CSH (4) = DII + penicillin sodium salt 

(0,08 ol'I) 9 

streptomycin sulphate (Oo2 91"l) 

CSM (5) = 1314 

- glucose + cellophane 

3,, lo5o2 Results 

+ Benlate (0.01 ol-I)o Murphy (0.01 01-1), 

penicLIlin sodim salt (0-08 gl-l)j streptomycin 

sulphate 

(0*2 gl-l) 

The results am given In Table 3olo5o 

a& 

7"x*

e 

Nd Co 0 

). 0 ý. b b + 

10 

L- & bi 

i 9p 

l mm 0 

+ 

So u 0 

t3 ýi 

8+ 

p 

30 

"0 

0- 0 s" 0 bi 

l»4 Z, zo *0 

dm bo tn 

' 

0 0 -v 0 

1 

m 

C3 

ZD co 

%00 

1+ 

co %0 

;% to, 

3 

1+ 

$- 

co .4 

0- 1- J-d 

I- t'3 %j 

t3 

C% 

14 

%. Ol 0-k 

+ 

ILI 

04 

0- 

S 

I UI 

I. 

" 

S 

0 

R 

Is rA 

v 

I. 

D 

1* 

a 

a. *d. 

m 

'1 

S 

95*

I II It UU 

f) n 

oo 

(D iD 

aa 

bd. 

to 

r 

D 

'1 

bö 

1- ý> 

ow. 

rA 

0 

m 

4 

1+ 

1+ 

W 

0.3 9 

1-4 

%0 C% 0 0W li 

t; Z4 

1+ 

ta 

la I+ I+ 

+ 

o 

0w 

+ 

04 

+ 

&a 

+ 

> 

Ps 

+ 

+ 

a 

s§ 

0.4 

I" 

V g- 

%W 

: + w 

I+ N3 

() 0 

to 0 

%I 

:. 

+ 1+ 1+ 

L 

96. 

WI 

+ P-i -0 

o 

%JI 

0 

t. 2 %A 

+ 

N 

+ 

V 

+ 

to 

w

3-1-5*3 Discussion 

In the initial experiment the combination of ingredients (CSM 

(D) successfully prevented the geminatLon of Aaj! 2rPIllua nivert but 

not Paecil=ps varlotii (Table 3.1.5). Unfortunately It did not 

allow the germination of Termitomycess 

In the second experiment CuSO4- 51120 was emitted from the modiuml 

and Termitoyces, germinated, though In reduced numbers compared to the 

control* It allowed the growth of Termitomyces an the plates with the 

mixture of Tormitomycesl A, -niner 

P. variotli grow (Table 3,1,5), 

and P* variattil though again 

MsmitOl (CSM (2)) had no selective effect on the contaminating 

fungi under these conditions (Table 391*5) with both A. niner and 

P. --variotit, 

developing, and the germination of Termitomyces being 

prevented* Fungicides Denlate and Murphy alone (CSM (3)) prevented 

the germination of A* niner and P. variotil and allowed that of 

Termitomyces When combined with the cellophane overlay the result 

was the samee The antibiotics streptomycin sulphate and pcnicillin 

sodium salts tested separately (CSM (4)), 

allowed the germination of 

Termitomyces when alone in the plateg but did not act on the contaminating 

fungi. rn the final selective medium CSM (5), with all the Ingredients 

combined, the germination of Temitanyces was allowed and Termit2=cos 

could be recovered when added In a mixture of A, _nlaer 

spores, though at slightly reduced levels* 

3.1.6 CONCWSIONS - TIM SLLECTIVE taMIUM 

Tho final selectivo medium vast. 

and Pe variotti 

97-

distilled water 19000 ml 

Kl'. ': IP04 

(? 414), SO4 

KCL 

MgSO4- 71120 

CaC12 

Yeast extract 

Agar 

100 

005 

005 

0,, 2 

0,01 

0*5 g 

20*0 a 

Benlate fungicide 0001 a 

Parphy fungicide 0,001 g 

Karathane fungicide 0.006 g 

Gallic acid 0001 g 

Ox bile loo g 

After autoclaving streptomycin sulphate 

sodium salt 

(0.4 a) and penicillin 

(Ool 9) were added& A sterile cellulose film overlay, 

(cellophanes Dritish Cellophane Co. ), was added as a carbon sourcee 

Although this medium did not stop the growth of all contaminating 

fungi It greatly reduced their numberxe It emblod Temitronycen to 

be isolated from areas where It could not be Isolated on the general 

sF medium, A groat deal of effort was put Into the development of 

the selective medium as many experiments vare dependant on its 

effectiveness 

(Chapters 5 and 6). 

^0 

Wo

3-22 ISOIATION Or, TOMITOMYCES FROM TWMITE GUTS 

3.2.1 SURFACE STMILIZATIONe INTRODIXTION 

The external surfaces of termites are covered with many sporess 

picked up as accidental contaminants from soil and food materials 

Olendee 1933). This presents problems in making isolations from the 

termite gute Bacterial contamination was also a problem. On removing 

fungus combs from soil the comb and mycotibtes were often contaminated 

by soil which meant contaminating fungi would be present leading to 

non-isolation, or lowered isolation frequencies of Termit2acese 

For these reasons it was considered necessary to develop a Means 

of surface sterilizing the termites which did not affect the germina- 

tion of Ter`WitomXces. 

Miltons Fluid (Appendix 2)j used for sterilizing babies bottles, 

fruit and vegetables, was available at Makwas The effects of this 

fluid on contaminating spores, on Termitowyces mycot6tesj and its 

effect on the germination of the mycotetes was tested* 

2a2 MMIODS 

Ten mycotetes were washed In Miltons Fluid by shaking gently for 

one minute and soaking for a further 14 mintstes, Five were plated on 

the general SP mediums 5 an the selective medium and these compared 

with unwashed mycotOtes. The mycotetes were from Macrotermes 

bellicosus, fungus comb* 

Termites were sterilized by soaking for 15 minutes in Miltons 

Fluid,, followed by a washing In sterile distilled water* The guts 

were then dissected out In sterile distilled water, To test the 

effectiveness of this method termites were Durface sterilized and 

plated out whole on SF medium and selective medium, 

990

3*2*3 ]RESULTS AND DISCUSSION 

The results are oiven In Table 3,2.1& 

TABLE 3.291* ComparLson of mycotetes washed Ln Hiltons FluLd and 

unwashed mycotkess germination of mycot6ten and growth of contaminating 

fungi. 5 days. 

WASHED WCOTILTES UNWASHED MYCOTBITES 

SP SEI=IVE sp SEL=IVE 

HEDIUM MEDIUM HODIUM MMIUM 

Number germinating 5 5 5 5 

Number contaminated 4 0 3 0 

Washed termites 

On the selective medium Growth occurred from one termite only. 

On the general SP medium growth of contaminating fungi occurred but 

this mainly originated from the protruding rectum. The unwashed 

controls were covered In fungal growth, 

One of the main effects of the WIton appeared to be the removal 

from the termites of adhering soll and this would result In the removal 

of large numbers of contaminating fungi. 

As soaking in Milton did not adversely affect the gemination of 

Termit2=qes (Table 3.2.1)9 or those fungi in the outs it was decided 

to use this method to surface sterilize the termites before dissection, 

Due to the effectiveness of the selective t*dium It was not necessary 

to use Hilton to Isolate Termitomyces. from the mycotetes., 

Other methods for surface sterilizing termites include Immersion 

In iodine followed by rinsing In phymfological Salt sOlUtion Olendee 

1000

1933), and Immersion In 119C12 (Dickman 19319 RaJgopalt Rao and Varna 

1979)* Neither method was infallible but Hendee considered the most 

effective method to be the one with the highest degree of wetting* 

Other authors have only washed the gut In sterile distilled water (Das 

et, al* 1962). 

1010

3.3,1 

3-3 DETEMINATION OF THE OPTIK314 TEMPUZATURE FOR GROWTH 

INTRODUCTION 

In order to get the maximum amount of c0owth of Tormit2aces, it 

is necessary to know the optimum temperature for incubation. As the 

temperature optima of species within a genus way vary widely 

1958) the experiment was carried out on cultures associated with 

(Cochrane 

different termite species to see if any differences could be found* 

The radial growth of Termitomyces on agar plates is considered a 

reliable method for determining the optimum temperature for growth 

(Brancato and Golding 1953, Cochrane 19-58 and Trinci 1969), and there 

in excellent correlation of radial growth with dry weight in temperature 

studies (Chaudhurl 1923). 

3.3.2 KMIODS 

The optimum temperature for Growth was determined by measuring the 

radial growth of Termitmyet-F, culturex on plates of SP medium Incubated 

at 20t 24,299 32 and 350C. Five plates were act up at each temperature 

for each Termitomyc= culture* The plates contained 25.5 ml of agar 

vhich was therefore of a standard depth* The Innocula of 

Tem. itanyce-8 

vero cut with a cork borer and placed upside down centrally on the agar 

plate* 

In the first experiment the cultures of Termita"yces used were 

from Ancistrotermex cavithorax, Macrotermes bellicogun, Macrotomes 

subhnlinual Odontotermes ameathmant and Odqntotem. eýk op. In the 

second experiment cultures from 4 Micratemes species were used, 

3.3.3 MULTS 

393.6. 

The results are given in Figs* 3-3-1 - 3.3.7 and Tables 3-3.1 - 

1020

TADIX, 3.3.1 Analysis of variance on the diameters 

cultures on DaY 7- First experiment. 

SOURCE OF VARIATION 

Between Termitomyc P, --a 

DEGREES OF 

FREEDOM 

SMIS or 

SCUARES 

of the Termit2aces 

MEAN 

SQUARES 

1030 

VARIANCE 

RATIO 

8 8 NSD 

0 

cultures 4 12-359 995 

3-0 -73 

Between temperatures 4 520.1554 130-03885 30.69*** 

Interaction 16 67. "7 4.23779375 2000* 

Residual 100 211.8671 2-118671 

TOTAL 124 812.187 

There in Interaction between Termitomyces culture and temperature* 

NSD no significant difference 

0 significant difference (5% level) 

000 nigniticant ditterence (Ool% level) 

TABLZ 3,3e2 Analysis of variance on the diameters of the Termitommcems 

cultures on Day 14o First experiment, 


Between Terwitomyces 

cultures 

DEGREES or 

FRL=o1t 

sums OF 

SQUARES 

MAN 

SQUARLS 

VARIANCE 

RATIO 

4 190-64774 47.66194 1*22NSD 

Between temperatures 4 1535oll95 383-77988 9.81, 

Interacticm 16 62597,202B 39-1075' 2. 17.47*** 

Residual 99 221I. Q952 2*23853 

TOTAL 1223 2573-10704 

There In interaction between Usperature and Termit=cen culture., 

NSD no significant 

difference 

*** significant difference (0.1%) 

0 Significant difference (5%)

TABLE, 3.3.3 Analysis of variance on the diameters of the TermitamXces 

cultures on Day 15* Second experiment. 


Between Termitomyces 

DEGRE M, OF 

FREJMOM 

SUYIS OF 

SQUARES 

Ym, AN 

SQUARES 

cultures 3 99-47115 33-15705 2. 

104. 

VARIANCE 

RATIO 

Between temperatures 4 2373.80869 593.45217225 38-2.3' 

Interaction 12 186. "Pa594 15-52161667 7.33' 

Residual 79 167-30084 2.117732152 

TOTAL 98 2826.84ooS 

There Is interaction between temperature and Termitomyces culture* 

TABLE 3-3.4 Analysis of variance of growth of TermiteMpen cultures 

during the 2nd wec4c, First experiment. 

SOURCE OF VARIATIM DEMEZS OF 

Between TermttomXceo 

FULMOM 

sums or 

SQUARES 

IMAN 

SQUARES 

14NSD 

VAMANCE 

RATIO 

cultures 4 108-379405 27-0905125 1.40SD 

Between temperatures 4 328-97768.5 82*24442125 00.16*10 

Intemction 16 301-124859 18-82030369 18.34*4* 

Residual 99 101.569r. 1*02.5958788 

TOTAL 1223 84o. o5i869 

There is interaction between temperature and Termito=ces- culture. 

The levels of significant difference between the meana At the different 

temperatures are given below.

woc 

240C 

2ee 

.:. )OOC 240C 29C 32c)c 

320C NSD 

35cle NSD *0* *ov 

*** significant difference (Ool% level) 

NSD no significant difference 

Interaction 

NAC 36 2(rc 240c 291)C 32'DC 35'C 

2. Ooc 

240C 

290c 

3'-'C NSD 

. 350c **# 0** *0* 

105*

SLn3 21 MOC 240C 290C 320C 350C 

20K 

240C 

. 

f)goc NSD 

320C NSD NSD 

350C NSD 

OD 42 

200C 

240C 

290C 

320f, 

200C 240C 1.191DC 320C 350C 

350C NSD 

OD 54 200c 240c 290c 3', loc 350c 

200c 

240c 

NSD 

2 oir **6 **t 

320c 

350c NSD 

- 

io6.

AN 53 200c 240c 290C : 32oc 350c 

2o(ýC 

240C 

290c 

320c 

UM 

350C NSD *0* 


difference 

** significant difference (1% level) 

*** significant differonce (0,1% level) 

1 

*0* 1- 

TAME 3-3.5 Analysis of variance of relative (; rowth rates of 

TermltomXcea cultures during the second weelce Firat experiment* 

SOUME OF VARIATION 

Between TPrnitomyces 

DMREES. OF 

FTWMOM 

sums (. W 

SQUARES 

MEAN 

SQUARES 

107- 

VAMANCE 

RATIO 

cultures 4 2*09936CO 0*5249652 1.56NSD 

Between temperatures 4 2,1005328 0-5251332 1,56N-', "'D 

Interaction 16 5138831W 0*3368012 18.24**# 

Residual 99 i. 828o44 0.0184650%* 

TOTAL 123 11-4172568 

There to Interaction betveen temperature and Termit2=cos culture. 


difference 

0*0 significant difference (Ool% level)

Mean 

diameter 

of 

colony 

(m m) 

16-1 

0' 2579 

16- 

12- 

2 

8 

o" 

c OD 42 

MAC 36 

0,,,,. - C3 

5 

0 

E3 

0 

13 

A 

14 0 

Days 

A 

0 

D 

149 0 

/ (Days 

Days 

257 

C-C3 

0-0 

0-0 

A-A 

A. -A 

Ä==- 

ez 

A mcc=::: Z: 

t, 

Aa -=X- 

20 Oc 

24 0 

29 0c 

32 0c 

35 0c 

FIGURE 3-3-1* (A) (b) (c) (d) and (0) 

Growth of Termito=Xces cultures at different 

tomperatureso First experimanto 

(a) Y-Acrotermen bellico4mn_, (b) Macrotermem 

, nubhZalin,!. ý!. 3, (a) Odontotermas Amenthmani 

(d) Ldontotermen inp., (e) Ancistrotermes 

cavi=horax, 

SUB 21 

d OD 54 

7 

los* 

14 

0

I. 

Mean 

diameter 

of 

Fojýny 

M IC 61 

a 

Days 

1-2 zo 267 

Days 

hm1 r- än 

d MIC 37 

109. 

, FIGURE 3-392a (a) (b) (c) and (d) Growth of Ter-Atomyces, cultures at different 

temperatures, Second experiment. Key as Figure 3.3-1 

(a) Hicrotermes op. Al (b) Microtermes op. D, (c) Hicrotermes 6p, R9 

(d) MicrotermOIS op. Ze

FIGURE 3.3-3. Mean diameter of Termitomycen cultures from 

different termite species# grown over a range 

of temperatures, DaY 

110,

Mean 

diameter 

10 

f 

colony 

(mm) i 

11-11 

A-A 

0-0 

0-0 

A-A 

20 

1 A-A Microtermes sp. A 

0-9 

0-0 

A-A 

a Firstexperiment 


M. subhyalinus 

Odontotermes smeathmani 

M. sp. D 

M. sp. R 

M- sp. Z 

i4 

b Second experiment 

2,9 32 

Temperature (IC 

25 29 32 35 

Temperature (OC) 

i5

Mean 

diameter 

of colony 

(M m) 

16 

14 

1 

8 

6 


M. subhyalinus 

0-0 Odontotermes smeathmani 

0-0 0. sp. 

A-& Ancistrotermes cavithorax 

ill, 

N. 

6 

20 24 29 32 34 

Temperature (OC) 

FIGM 3-3.4. Mean diameter of Termito! Xces , cultures from different 

termite speciesq grown over a range of temperatures. 

Day 14, First experiment. 

C3 

0

Mean 

diarnei 

of 

colony 

(mm) 

112* 

Temperature (OC ) 

FIGURE 3-3-59 Mean diameter of Termitomyces cultures from different 

termite species, grown over a range of temperatures. 

Day 14 Second experiment,

Change 

in 

mean 

diametE 

(mm) 

1 

day 14- 

day 7 

1 

a First experiment 


FIGURE 3-3.6, arvAh darbw second week of Termitom ce, 3 

cultures from different termite species. 

113. 

20 24 29 32 35 

Temperature (OC ) 

21 25 29 U 35 

Temperature flIC )

114. 

FIGURE 3-3-7. Relative growth rate of cultures of Termitomyces grown 

at different temperatures. Relative growth rate is 

Mean diameter day 14 - Mean diameter day 

Mean diameter daY 7

[3-[3 Macrotermes bellicosus 

A-A M. subhyalinus 

a First experiment 

1.0- 0-0 Odontotermes smeathmani 

0-0 0. Sp. 

Relative 

. growth 

A-& Ancistrotermes cavithorax 

rate 

0-8,1 

0-6- 

0-4- 

0- 2 

0 

A: 

it 

0'8- A-AMicrotermes sp. A 

0-6 

0-4 

0-2 

0-40 M. sp -D 

0-0 M. sp. R 

A-A M. sp. Z 

ol IIIII- 

210214 


Ir 

21 - 25 29 32 35 

13 

15 

29 32 3 

Temperature (0 C

TABLE 3-3.6 Published values for the temperature of Macrotermitinae 

nests* 

TDIMITE SPECIES TOtPEnATLME OC AUTHaIITY 

thcrotermes annand. -Alet 28*8 Matsumoto (1977) 

M. bellicosus 30.67-31*122 Collins (1979) 

M. bellicosus 30 lZzcher (1951a) 

M. carbonarius 27-52 Matsumoto (1977) 

H. malaccenals 27.6 Matsumoto (1977) 

M. ukuzit 28 Rohrmann (1977) 

Odontotermes redemanni 2B95 Petcl, (19W) 

0. obesus 18,27 Batra, and Datra (1977) 

vu=cr 28-32 Checma et al. (1962) 

winter 

18-30 

1150

3-3-4 DISCUSSION 

The growth curves in Fig* 3,, 3,, l Indicated that there was an 

Interaction between temperature and day for some species with a 

change In growth rate during the second week at some temperatures. 

It therefore secmed reasonable to suppose most of the information 

could be extracted by considering the whole period and the first and 

second weeks separatelyo 

For the whole period and for the first week it was sufficient 

to look at the size on day 14 and the size on day 7 respectively* 

This was because there was so little variability between the size 

of colonies on day 0.11oweverl since by daY 7 colonies were of 

different sizes this was not true for week- 2. Hence, (size on day 

14 - size on day 7) was looked at to got an absolute measure of 

change In diameterl and ((size on day 14 - size on day 7) .6 size on 

day 7) to get a measure of the relative growth rateo 

Macrotermes bellicosus MAC 36 

The optimum temperature for growth under the conditions of this 

experiment was 290C (Flo* 3*3*1(a))* The temperature growth curve its 

116. 

asymmetrical (Pigs, 3,30(09 3-3.4)9 with the colony diameter decreasing 

rapidly above the optimm at 290C9 very little growth occurring at 350C* 

In general HAC 36 was more tolerant of temperatures below the optim= 

than those above its The growth during the second week given the same 

picturol the relative growth rates being very similar from 20-320C, 

but very MUCh lower at 351DC* 

The opti== for growth In very close to the Mean nest temperature 

of 30.67-31.220C recorded for M, bellicosus (CollIns 1979). Other nest 

temperature values for Pacrotem-ex sPecics Ore also shown in Table 3,3,6.

Macrotemes subhXalinus 

SUB 21 

The optimum temperature for growth under the conditions of this 

experiment was 290C (Fig* 3.3.1(b))* The temperature growth curve is 

more symmetrical than for the culture from Macrotermc-st bellicosus 

(Figs* 3-3-3(a)t 30,09 that from Macrotercies subhy4llnus being more 

tolerant of higher temperatures* At 290C and below the cultures from 

M. bellicosus are larger, but at 320C and 35'OC those from M. subhyalinus 

are larger. There appears to be some adaptation to higher tomperatures 

117o 

during the second weekq the relative growth rate being greatest at 320Ce 

These differences betveen cultures from 14. bellicosus and 

ýj. suhUalinut may reflect the differing regulation of the nest 

temperature. M, bellicosus with Its centralised fungus comb and 

efficient themo-regulation Is able to maintain a more or less constant 

nest temperatures often below that of the airo In contrast K, subhyallnus 

with its more scattered fungus comb may experience higher temperatures 

within 

the nest* 

Odontotemes smeathmani OD 42 

The culture from 0. ameathmani had its optimum at 290C (Fig- 3-3-1 

(0). During the first week, it has a syT=etricAl temperature growth 

curve about the optimum 

(Fig- 3-3.3(a)) but ovor the course of the 

experiment growth in better at temperatures higher than the optimum 

than at those belowe 

The maximum amount of growth occurs at 320C during the second 

week showing that the culturo is beginning to adapt to the higher 

temperaturesq although the total size of the culture in still greatest 

at 290C. The relative growth rate in in fact greatest at 350C Mg* 

3.3-7),, and decreases vith a drop in tomperatura.

Tbis species in not mound building and so has no chi=cy systam 

for regulating nest temperature and the Tern. it2Mces culture Is 

therefore likely to experience higher temperatures than those of mound 

building species. It appears able to adapt and grow well at these 

higher temperaturese 

Odontotermes, ap. CD 54 

The culture OD 54 had its optimum temperature for growth at 290C 

under the conditions of this experimento Its temperature growth curve 

is asymmetrical and similar to that of MC 36 although the diameter in 

less at all temperatures (Figs- 3-30(09 3-3.4)o The growth during 

the second week is greatest at 290C (Fig- 3o3.6)9 with very little 

growth occurring at the higher temperatureso No adaptation occurred 

to these higher temperatures, unlike OD 42o 

OD 54 was obtained from Rabba, where the soil temperature is 

unlikely to rise &3 high as at Mokwa where OD 42 was obtaLnede At 

Rabba the soil Is more moist than at MWcwa and Is In constant shadeo 

Anclatrotermes cavithorax AN 58 

The optimum temperature for the growth of the Termitomces 

culture from A* cavithorax was 2()OC under the conditions of this 

experiment (Fig. 3-3-1(0)). AN 58 produced slightly more growth at 

lower temperatures than at those higher than the optimum during the 

first week 

(Fige 3*3*3(a)), but the temperature growth curve was more 

symmetrical over the whole 14 days of the experiment (Fig. 3.3.4). 

Tho growth during the second week was greatest at qg(>c (rig, 

3.3.6(a)), but the relative growth rate wax higher at 320Ct showing 

AN 58 wax adapting to thimi higher tempcratures This culture was 

obtained from Rabbal but A. cavithorax In also : found in the sayarum 

118*

at tk)l-. wa where higher isoil temperatures would be experienced. This 

ability of Termitomyepa, to, adapt to the higher temperature conditions 

can be compared to the non-adaptability of OD 54 whoso associated 

termite is only found at Rebba. 

In general there is considerable Interaction between temperature 

and species, Ioee the response to temperature Is not the same for each 

species. The only consistent results are that the cultures are all 

largest at 29OCo 

1190 

There in no significant difference between the mean colony diameters 

of different species at DaYs 7 and 14 (Tables 3.3-1 and 3*3*2)9 and no 

significant difference between the growth ofs and relative growth rate 

of, the different cultures during the second week (Tables 3*3-4 and 

3-3-5). 

In the second experiment the cultures from the different Hicrotermes 

species considered as a whole appear to be more tolerant of temperatures 

slightly higher than the optimim than those in the first experiment, 

but the growth falls extremely rapidlys 35'C being too high for growth 

in most cases* 

The culture associated with Hicratermes op. A has an optimum for 

growth at 320C, the only TermitcM. ces culture to have this (Flo* 3*3*2 

(a)), All the others have an optimum of 290C over the 14 days of the 

experiment although the growth at 320C is very close and the true 

optimum may fall between these two values* The temperature-growth 

curves are all very similar (Fig, 30*3(b) and Fig, 3*3*5) in shape 

although the culture associated with species A has much more growth 

at 290C and 320C than the others, with R having the next greatest 

amount of growtho

3.4.1 INTRODUCTION 

3*4 MMIMNATION OF THE OPTIMN 21 FOR (ZZOM 

In order to get the maxic= amount of growth from TennitqMces it 

is necessary to know the optimum pit of the Medi= for growth* This 

was doterminedl for cultures asnociated uith different termite spocieso 

by measuring the radial ()rowth of Termit=ces, on agar plates. 

3.4.,. ) H=ICDS 

Measurements were made of the radial growth of Termit=ccs 

cultures on plates of S17 medium with the p1l adjusted by the addition 

of RCI and NaOlle The pit values used were 4.0,, 4-3* 4.6,4.9,5.2 and 

5.5 which covers the range of p1l values of the fungus comb (4.1 to 4.6, 

Tables 7-2.1 and 7*2*2)* Below pit 390 agarx donIt gel* Five plates 

were set up at each pit value for each Termitomyces culturee The plates 

contained 25-5 MI of agar which was therefore of a standard depth* The 

Inocula of Temitor3yees were cut with a cork borer and placed upside 

down centrally on the agar plate* The platex were incubated at 290C 

for 11 days when the final pli of the medium was determinede Cultures 

of Termitomyces from Ancistroternex cavithorax. Macrotermes bellicosung 

Odontotermes zmeathmani and Odontotermen spo were used. 

3.4.3 FIESULTS 

3A. ".. 

The results are given In Pigs* 3.4-1-3-4-3 and Tablw 3.4-1- 

121. 

I

TABLE 3.4.1 Analysis of variance on the diameters of the Termitomyces 

cultures on day 11. 


Between Termitomycer; 


F'REMOM 

SUILS OF 

SQUARES 

MAN 

SQUAPrZ 

VARIANCE 

RATIO 

cultures 3 244-761 81-587 6.55** 

Between pH's 5 805.134 161.0268 12, lr-*** 

Interaction 15 186-917 12.461133 13.26**4 

Residual 93 87.427 0.9400753 

TOTAL 116 1324.239 

NSD = no significant 

difference 

significant difference (5% level) 

significant difference (1% level) 

*** - sioniticant difference (0*1% level) 

There is interaction between pil and Termtt=cea cultural, i. e. the 

response to pH is not the s=e for each species. 

There to a significant difference between the Termit = ces cultures* 

There in a significant difference between the pit values* 

Betwoen species 

MAC 36 

OD 42 

OD 54 

AN 58 

HAC 36 0D 4'", OD 54 AN 58 

NSD 

122.

Between rIlls 

Interaction 

4. o - 

4-3 

4.6 

4.0 4-3 4.6 4.9 5.2 5.5 

4.9 #* - 

59Z 

5-5 

pil 4.0 MAC36 OWAI, oiD54 AN58 

MAC 36 

OD42 

OD54 NSD 

AN58 

PH 4-6 14AC36 OD42 OD54 AN58 

MAC 36 

OD42 

OD54 NSD 

AN511 NSD 1 .6 

123. 

PH 4-3 RAC36 oD42 OVA AN58 

MAC 36 

OD42 

OD54 NSD 

AN53 

Of 4.9 MAC36 OD412 oD54 AN58 

MC 36 

OD43 NSD 

01154 $0 

Arl, 58 HSD NSD

F 0,11 5.2 mAC36 OD4?. OVA AN58 

MAC 

36 

CV42 

OD54 

AN58 

P11 5-5 MAC36 OD42 OD54 AN58 

MAC 36 

oD42 NSD 

OD54 

AN58 

IIAC 36 4.0 4.3 4.6 4.9 5.. -. 

4.0 - 

4.3 

4.6 NSD 

4.9 NSD NSD 

5-2 NSD 

1 505 0e 

11 1 

*o* 

1 *** 

1 *t 

1 NSD 

0D4,. 3 4.0 4-3 4.6 4.9 5 5-5 

4. o 

4.3 

4.6 

4.9 40* 

502 0*0 

0 NSD 

- 

1- 

14-04.

OD 154 4. o 4.3 4.6 4.9 5. " 5.5 

4. o 

4.3 

4.6 NSD 

4.9 NSD 

5.2 NSD 

1 5-5 NSD NSD 

I NSD 

AN 58 4.0 4-3 4.6 4.9 5o2 5.5 

4. o - 

4-3 

4.6 

4.9 NSD 

5.2 

5 -5 

1250

FIG = 3.4.1. (a)$ (b), (c) and (d) Growth of Termitomyces 

cultures at different pH values of the medium 

126.

Mean 

15 I diameter 

13 

I 

9 

1 

7 

5 

of 

colony 

(mm) 

0247 

11-0 4-0 

Z ------ a 4.3 

A-A 4-6 

o-o4-9 

&-, ä 5-2 

6-05-5 

MAC 36 

ýýo Wl" 

-aýý ___. __-__Q_. -_--__----__ 

A 

79 110 24 

Days 

0 

11 0 

Days 

b OD 42 

0' Z 

0Z 

Z 

4/, 

'0/ 

0 C 

9 11 

/

8 

Mean 

diameter 

of 

colony 

(mm) 

9 ] A-A Macrofermes bellicosus 

7 


0-0 a. sp. 

Ancistrotermes cavithorax 

4-0 4.2 4-6 4-9 5*3 5-5 

Initial PH 

FIGME 3.4,, 2* Mean diameter of , TermitomXces cultures from different 

127 

termite species,, grown over a r6mge of pHIs, Day 4.

Mean 

diameter 

of colony 

(M m) 

20 -t 

12 

I 

6 

4 i 

_6, ý 

0 4-0 

(3-c Macrotermes bellicosus 


0-0 0. sp. 

, &-& Ancistrotermes cavithorax 

13'------ 

ED 

c)"----- 

4-8 5-2 

Initial pH 

]FIGURE 3.4.3. Mean diameter of Termitomyces, culturels from different 

termite speciesq grown over a range of pH's. Day 11. 

128o 

-0 

5-6

TA13IZ 3.4.2 Initial and final p1l of the medium 

MAC 36 

OD42 

01) 54 

AN 53 

. 3.4.4 DISCUSSION 

4. o 4.3 4.6 4.9 502 5-5 

4. o 

4. o 

3-8-3*9 

4-3 

4.2 

4.,., 

3.6-3*7 3*8-3*9 

FINAL p1l OF MEDIUM 

4.6 4.8-4.9 4.8-4.9 5-0 

4-3 4.4 4.6 4-7-4*8 

4.4A. 5 4.5 4*5-4.6 4.8-4.9 

4. o 4.4 4-5 4.6 

The mean diameters of the cultures on Day 11 were compared as there 

was so little variability between the size of colonies on Day 0. On 

comparing Piose 3-4-2 and 3.4.3 the pattern of response of the cultures 

to the initial PH value appeared to be the same of Uays 1* and Ile 

kfacrotermes belliconum (HAC 36) 

The optimum pit of the medium for growth under the conditions of 

this experiment could not be determined an maximum growth occurred at 

the highest pit (5,, 5)9 and the optimum may have been above this value* 

Growth at Of 4.0 was significantly lower than at all the other pills,, 

but above 4.0 there was little difference between colony sizes* Colony 

size at pit 5*5 was significantly greater than at all other VAIU03 

except 5*2* In general the picture In of a broad tolerance to the pit 

valuen of 4.3 and above. This high optimum is surprising considering 

the pH of the fungus comb ranges from 4.1-4.6 in this opecies (Table 

7o2,1), This culture had the broadest tolerance of pit. 

Odontotermes smeathmani 

(OD 42) 

Tho optimm p1l for growth under the conditions of thip expcriment 

was between P11 5**2 and 5.5 The culture from this species was far loss 

129.

tolerant of low 01 values than those from Macrotermos bellicos"s and 

Ancistrotermes cavithoraxe There Is a significant difference between 

the size or the culture at all pil values except 5*2 and 5*5 (Table 

3.4.1). 

Odontotermes spe (OD 54) 

This species showed a similar picture to that of Odontotermes 

eneathn, kini, with an optimum P11 of the medium of 592 under the 

conditions of this experiment. It was more tolerant of lower P11 

values than U. smeathmni but less tolerant than H. bellicostagiand 

A. cavithorax, q and showed the least growth of all the cultures at the 

higher values 

Wig- 3-4-3)- 

Ancistroternes cavithorax (AN 58) 

The culture from this species had an --ptimm pit value of the 

medium for growth of 5.2, under the conditiona of this experiment* 

The optim= was far more pronounced in this species, Growth at this 

p1l value being significantly different (0*1% level) from growth at all 

other p1l valuese 

Under the conditions of this experiment the most suitable initial 

pH of the medium would appear to be 5*2,, Many factors such as 

temperature and nitrogen source will change the shape of the pH curve 

(Coclurane 1958). 

In most cases the pH was altered during growth by the motabolic 

activities of Tormit=ces-, (Table 3,,, 492)9 Uywering of the pill as 

occurred here. is due to the formation of orCanic acids or absorption 

of cations,, and the broad optima may be a reflection of the ability of 

the fungus to alter the pil of the Medi=* Generally acidic plies favour 

Basidiomycetos, alkaline plies causing inhibition (Sharp 

130- 

and Eggins jLq7O)q

and larger Basidicarfcetes are often unable to grow In culture above 

an initial P11 of 7.0 (Cochrane 1958). Chandra and Purkayastha 

(1977) obtained an optinm of pit 4.0 for the growth of 

Tem. Itmcon 

eurhizus but this was In liquid culture with a different mediume 

131o

3.5 GROWTH OF DIFFERWT TLP-MITOMYCES ON THE SELECTIVE MEDIUM 

3*591 INTRODUCTION 

The growth of 12 cultures pf Termlt2acpa from different terzdto 

species waro tested on the selective medium (3.1.6) 

at a y9I of 5,2 

and incubated at 2290C to ensure, that the growth was satisfactory. 

3.5.2 MMODS 

The radial growth of TermitMMces cultures on the selective 

medium was compared with the growth of Termitomyres cultures on 

plates of SF medium an control. 5 plates were set up on each medium 

for each Termitomyces. culture. The plates contained 25.5 =1 of agar 

which was therefore of a standard depth. The inocula of Termit=cos 

were cut with a coric barer and placed upside down centrally on the 

agar plate. 

In the first experiment the cultures of Termitomyces were from 

Anclstrotprmem cavithorax. Kicrotermes bellicosus. Macrotermes 

subhyalinuag Odontotermes ameathmant and Pdontotormos sp. In the 

second experiment cultures from 7 Micratermes species woro used. 

3-5-3 RLSULTS 

The results are Civen In Tables 3.5.1-3-5.4 and Figo. 

Photographs of the cultures on SP medium and tho nelective inedium are 

given In Plates 4.1.1-4olel2o 

132.

TABLE 3.5.1 Analysis of variance on the diameters of the Termit = ces 

cultures on day 14. First experiment. 

SOUPCE OF VARIATION 

DMIRLES OF 

Fn MOM 

SUMS OF 

SQUARES 

tMAN 

SQUARES 

VARlANCL, 

RATIO 

Between species 4 1301-21885 75-3047125 : "-7!; NSD 

Between media 1 47-35591 47-35591 1-73NSD 

Interaction lk 109-35037 '27-3375925 8*520** 

Pesidual W 128.1-822 3.20-e(r)5 

TOTAL 49 586-ZO733 


difference 

= significant difference (5% level) 

** = significant difference (1% level) 

*** = significant difference (0.1% level) 

There is interaction between the media and the TerMit2MXceS speciess 

i. e. the response to the media In not the same for each species. 

There is no significant difference between the Termitomyc 

There is no significant difference between the media, 

,, Ps cultures. 

133o

FIGURE 3-5-1- (a), (b)v (c), (d), (e), Mv (g)j (h)v (i)j (J)$ (k) 

and (1). 

Growth of Termitomyces cultures on two media. 

(a) Macrotermes bellicosus, (b) H. Bubhyalinus, 

(c) Odontotermes smeathmani (d) Odontotermes op*$ 

(e) Ancistrotermes cavithoraxi (f) Microtermes sp* A, 

(g) Hicratermes op. Z$ (h) Microtermes op. Ds 

(i) Microtermes op. B$ (j) Microtermes op. Gs 

(k) Microtermes op, R and (1) Microtermes sp. C. 

'134-5-

Mean 

diameter 

of , 

colony 

(mm) 

Days 1ý 1'4 

Al

16- Me an 

1 

0 

diameter 

of 

colony 

(m m) 

gMIC 

37 

MIC 61 

Days I 14 

a 

A---& SF medium

Interaction 

SF 

Wzium 

MAC 36 

SU13 62 NSD 

MAC 36 SUB 62 OD 42 OD r)4 AN 53 

OD'42 woo 

OD 54 NSD NSD 

Al", 53 0*10 

S=CrIVE 

MEDIUM 

MC 36 

SUB 62 NSD 

1. 

'' 

I 

TISD 

MAC 36 SLI3 62 OD 42 OD 54 AN 53 

OID 42 NSD NSD 

01) 54 *40 

AN 53 NSD NSD NSD 

Growth on the 2 media 

MC 36 NSD 

SUD 62 

01) 4" 

OD 54 

AN 53 

1 

136.

TABLE 3-5.12 Analysis of variance on the diameters of the Tprmitomyces 

cultures from Ificrotermes species on day 14. Second experiment* 



FlWMDOM 

SLU"A OF 

SQUAF%LS 

MAN 

SQUATZLS 

VARI4NCE 

TATIO 

Between species 6 3,29-94288 54.9901*3 It. 61 

Between m. 

edia 1 3-90344 3-90344 0-33NSD 

Interaction 6 71-52585 11-920975 2*9G* 

Residual 55 21.21.84184 4-033488 

TOTAL 68 627,21401 

There is Interaction between the media and the Temitomyces cultures 

from different Microtermes species, lee. the response to the media in 

not the same for each culture. There Is a significant difference 

between the Tormitanyces cultures from different h1crotormes species 

(5%) level). 

There is no significant difference between the media. 

Detwoen species 

A c 

NSD 

c NSD NSD 

D v*O o*» *v* 

G NSD NSD 

R 

Z NSD N. SD 

137*

Interaction 

SP 

MUDIUM 

A 

D NSD 

c NSD NSD 

L) 

G NSD NSD IISD 

rz 

c D 

z NSD 14SD NSD 

SELECTIVR 

MEDIUM 

A 

B NSD 

c NSD 

INSD 

A c D G n z 

D NSD 

G NSD NSD 

r NSD NSD *, A* 

z 

j 

--I 

NSD 

NSD 

Growth on tho 2 media 

A 4 

B NSD 

C # 

D NSD 

G 

R NSD 

Z NSD 

-I 

NSD 

1313-

TABLZ 3.5.3 The growth of Termitomyces cultures on the selective 

medium, and the amount of cellulose decomposition by these cultures 

(14 days growth) 

ASSOCIATED 

TERMITE 

S13ECIIZ 

GrZOWTH ON 

SELECTIVE tMDIUM 

(expressed as % of 

growth on SF 

medium control) 

139- 

JUK)UNT OV dFJ-LULOSE 

DECOMPOSITION 

(see Table 7e4ol) 

(%CRLWL0SI,; 

LF110VEM mm, 

Ancistrotermes caviihorax 75-21 0 

Macrotermen bellicosus 102.52 1.58 

Microtermes sp. A 118.63 1-113 

Mi croterm! ý, g sp. B 104.88 1.95 

Hicrotermes sp. C 85-57 1.07 

Hicroterings, sp. D 102.80 1.47 

Microtermts sp. R 96.63 1.61 

_ýUcroterrt2s sp. Z 91e65 1.35 

Odontotermes smenthmini 86.8o 0-57 

TABLE 3-5.4 Regression of growth of Termit2ýi=es cultures on the 

nelective medium against the amount Of cellulose decomposition by 

these cultures. 

Slope - 16-72 

Intersection - 75-62 

Regression line pointx a (1*239 96*19) and (0-5,83-98) 

. 78 


DEGREE. 33, OF 

FREEDOM 


SQUARES 

Pegression 1 802,45 

Mr. " 

SQUARLS 

Residual 514.212) 73.46 

TMAL 1316 67 

VARIANCE 

IrNATIO 

I 

Go-2-45 10.9,2) 

*

,.. e 

3-5-4 DISCUSSION 

First exLerimont. In this experiment there was no significant 

difference in the growth of the different Termitomyceg cultures, and 

there was no significant difference in growth on the 2 media (Table 

395*1)o There was a significant Interaction between species and 

media showing that the response to the two media was not the same 

for each species* 

The cultures from the 2 Kicrotermes, species both grew slightly 

larger on the selective mediumq whereas the cultures from the 12 

OdontotermesIspecies and Anciatrotermes cavithorax all produced most 

growth on the SF medium* OD 42 and AN 53 produced significantly 

greater growth on the SF medium than cultures fren the other species& 

On the selective medium the culture from OD 54 was significantly 

smaller than all the other cultures. 

Second exl2eriment. In this experiment there was no significant 

difference between the growth an the 2 media, but there van a 

significant difference between the growth of cultures from different 

Hicrotermen species at the 5% level of significance (Table 3*5.2). 

There was a significant Interaction between species and media showing 

that the response to the 2 media was not the same for each species. 

The culture from Mcrotermes bps P grow significantly bigger than all 

tho others, and that from op. D was significantly smaller than all 

except that from Hicrot_crmes, spo Zo Three out of the 7 cultures (from 

Microterme'S app# As L) and D) were bigger on the selective medium* 

Initially growth was greatest on the SP medium but after the first 

week the growth increased at a faster rate on the selective medium* 

on tjie 

103F medium the culture from spe R was significantly bigger than 

all the rests with that from D significantly smaller than the rest 

except for the culture from op* Zo 

140.

FIGURE 3.5.2. Regression of growth of the Termitomlces cultures 

Growth 

on the 

selective 

medium 

120n 

1 

on the amount Of cellulose decomposition (Growth 

on the selective medium expressed as % of growth 

of the control at day 14). 

141. 

0.5 1.0 1.5 2-0 

Amount of cellulose 

decomposition

Growth of the cultures on the selective medium could be divided 

into two categoriess the growth of members in each category not being 

significantly different from each other. Growth of the cultures from 

111croternes spp. Ct D, G and Z was significantly smaller than growth 

of those from Hicrotemps spp. A, 13 and Re 

There was a significant correlation (r 

," level 

- e78") at the TO 

betwoen the growth on the selective medium and the amount of cellulose 

decomposition produced by the different cultures, showing that the 

differing abilities of the cultures to grow on the selective medium 

may be largely explained by their differing abilities to utilize the 

cellophane overlay (Table 3-5-4). 

In general satisfactory growth of all cultures occurred on the 

selective medium. 

14, 

-, .

CHAPTEn FOUR 

TERHITOWCES IN CULTURE 

143.

4a CHARACTEMSTICS OF TEP14ITOMYCES IN CULTWE 


In culture Termit2gces prgWuces colonies with thin-walled septate 

hyphaes for instance Termitg!! Xces albuminosus was described as having 

sparsely septate hyphae 3-5(-B), um wido (Batra and Batra 1979)% 1-4(-6) 

Iva vido (Bakshi 195I)e Ilyphae of the genus Termit2myces In naturo do 

not possess clamp connections 

(Rayner 1969, p Singer 19499 1962) and 

Bakshi (1951) describes Termit=ces. in culture as only rarely having 

theme 

If grown under suitable conditions cultural equivalents of the 

natural mycotletes are produced (13atra and Batra 1979). These consist 

of globular Internal elemental grouped In chainsl which are not capable 

of germinating 

(apherocysts)o 

144a 

and ovoid conidiag In dichotomous brancheaq 

which geminate readily (blastospores)o These blastospores are 

binucleate with the cytoplasm and Its greasy Inclusions being grouped 

in the centre round the nuclei, with vacuoles in the extreme parts 

containing relatively numerous and voluminous metachrvmatic precipita- 

tions Ofeim 19401 1942a)* 

4.1*2 HMIODS 

Cultures of Termitomyces associated with different termite species 

were grown for 2 weeks on SF and selective media (Appendix 111 3.1.6), 

at 290C* The cultures were examined with the naked eye and low power 

binocular Microscope and described. 

The appearance of cultural tmycote^tes vas noted In these cultures 

and in cultures grown for ather o(periments. 

4.1-3 RESULTS 

The results are given In Tables 4.1.1-4.1. s and Plates 4-1.1-4.1.12.

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PiATE 4.1.1 

Termitomyces cultures from Ancintrotermes 

cavithorax on St and selective modia. 

(For Plates 4.1-1 to 4-1-12 TD, = SF 

medium and ++ = selective medium) 

PLkTt 4eI*2* 

Termitomyces cultures from Macratermes bellicosus 

on SF and selective media, 

148. 

It I

AN 93

PLATE 4.1.2. 

Termitomyces cultures from 14acrotermes subkyellinus 

, on SF and selective 

media. 

PLATE 4.1.4. 

Termitomyces cultures from Microtermen op. A 

on SF and selective media. 

I -Z -II 

149.

IS 

5' 

SI 

PLATE 

Termitomyces cultures from Hicrotermen up. B* 

on 'F and selective. media, 

PLATE 4.1.64o 

F sp* Be 

Termitomyces cultures from Microtermes, sp. C 

on BF and selective media. - 

; 

I 

. 

. 1, ' 

i'.. '- 

- 

. 5.5 

: '--- 

. 

150* 

I'1

PLATE 4.1.7. 

,, Termitomlces cultures from Microtermes sp. D 

on SF and eel-ective media. 

PLATE 4.1.8. 

Termitomyces cultures from Microtermes sp. 0---- 

on BF and selective media. ' 

- 

V 

151.

PLATE 4.1.2. 

TermitomIces cultures from Hicrotermes sp. R 

on SF and selective 

m'edia 

PLATE 4.1.1o 

Termitomyces cultures from Microtermes, sp. Z 

on SF and selective media. 

"4 

52.

PLATE 4.1.11. 

Termitomyces cultures from Odontotermes 

smeathmani on SF and selective media. 

RLATE 4.1.12* 

Termitopyces cultu'res from Odontotermes I sp, 

(OD 54) on SF and selective media. 

153.

TA= 4,1,3 production of cultural mycotetes on SF medium at 

different p1l values (2900 

ASSOCIATED =MITZ 

SPECIES 

CULTURE 

Ancletrotermes cavithomx AN 58 - 

TIM FOR > 5099 OF CULTURES TO 

PRODUCE MYCOTt=S (DAYS) 

1550 

4.0 4.3 4.6 4.9 5.2 5-5 

Macrotermes bellicoaus MAC 36 9 9 14 9 9 7 

Macrotemeis subhyAlinus SUB '21 - 7 7 4 7 7 

0dontotemes emeathusani OD 4.1 

Odontoterines SiDe OD 54 

- - - 

- indicates cultural mycotgtes were not prcWuced within 14 days* 

TABLE 4.1.4 Production of cultural mycotates on SF medium at 

different temperatures 

ASSOCIATED TETMTE 

SPECIES 

CULTURE 

Ancistrotermes cavitharax AN 53 

TIM FOR 'ý. 50% OF CULTURES To 

M0DtJCE MYCOl(UES (DAYS) 

200C 240C 290C 320C 350C 

- - 

Macrotermes bellicasus HAC 36 14 7 7 - 

Hncrotermos subhyalinus StM 21 7 14 5 14 16 

Odontotermes ameathmani OD 42 

0dontotemes sDo OD 54 9 14 

- - - - - 

- Indicates cultural =ycotgtes were not produced within 16 days,

TABLE 4.1,21 Production of cultural mycotetes on different media 

at 290ce 

ASSOCIATED TERMITE 

SPECIES 

CULTURE 

Ancistrotermes c"ithorax AN 53 

TIME, FOR > 50% OF CULTURES TO 

PRODUCE MYCOTril-TES J (DAYS) 

SF MEDIUM SELECTIVE MMIUM 

Macrotermes bellicosust MAC 36 8 

Macrotermeis aubhyalinuz SUB 62 5 a 

Hicrotermes one A MIC 30 - 14 

Hicrotermes op. n MIC 43 

Hicrotermes op* C HIC 41 

Hicroterin2s ape D filic 40 

Mlcrotemes op G MIC 68 

Microtermes spo R HIC 61 

- - 

- 

14 

- - 

- 

12 

- - 

Hicrotermt-9- op* Z HIC 37 9 

Odontotermes ameathmani OD 42 12 

Odontotermes op. (Rabba) OD 54 14 

- Indicates cultural mycotlkee were not Produced within 14 dayse 

154.

TABLE 4.1.5 Production of mycotUtes with increasing number of 

subcultures of TemAt2w 

ASSOCIATED 

TEMITE SPECIES 

. ýcqa 

CULTURE 

SUBCULTURE 

NW93ER 

TIM FUR > 50% Or' CULTURES 

TO PRODUCE MYCOT'L4TES (DAYS) 

Macrotermes MAC 12c 3 7 

bellicosum 4 a 

4.1.4 DISCUSSION 

4 

6 

4 6 

5 7 

6 8 

8 

7 

8 6 

9 

Visually it was only possible to dixtinguish 2 Croups (Table 

4.1.2) in culture. 

(1) The Ifacrotermes bellicosus and MacrotOrmes xu! jýallnus 

group which has the most aerial mycelium and very definite obvious 

cultural mycotCtea on both media* These Mycotetes are the closest 

In culture to natural myr-otetes In term of general appearance. 

(2) The, other OrOUP which includox the MIcrotermes, 

Ancistrotermes cavithorax and Odontotermem associated cultures, 

The cultures can be quite variable In appearance and at one end of 

the range can resemble the white felty Cdontotermes associated 

cultureal sometimes waxyt and at the other the more Irregular growth, 

sometimes fawn In colours of the Anclatrotermen cavithorax associated 

cultures. 

7 

5 

7 

5 

6 

156.

The appearances of these cultures agree In general with those 

described by other autherse Helm (1952b) described Terwitomyces 

mammiformis eA being a flaky culture, with a white circumferencet very 

pleated and cream ochre In the centret and described the culture frx= 

Macrotemes natalensis as being tuberculousj offlorescentl either 

wrinkled or cerebroidg cream with relatively big mycotete3 sitting 

on a thin veil of mycelium* 

On PDA (Appendix 1) Batra and Batra (1979) obtained white, some 

pinkish or pale cream cultureal effuse and often furfuraceous with 

aggreuateig of sprout callse 

Holm (1940,1958 and 1977) defined 2 types of Termit2aces 

developing In artificial cultures (1) Yeast-like with opalescent 

mycot"Itese (2) becomes "dusty-scaly4i with less individualized 

mycot: tese 

157o 

The form taken depends on the cultural conditions such as temperatUrO 

and humiditye Heim (1977) found that the cultures from all species of 

Termitomyces examined* both from Africa and Indial were practically 

Identical* 

It appears from Table 4.1.2 that mycotetes are produced more 

readily on the selective medium, This may be duo to the carbon source 

being cellulose rather than glucose* In many cases It was difficult 

to determine whon the flocculose aggregates of mycellum formed actual 

cultural mycoecteso In scuie cases spores could be found on microscopic 

examination of the cultures when cultural =Ycat'etes were not obvious. 

From Table 4.. 1.3 no pattern In the effect Of P11 on =Ycotete production 

could be observede The temperature producing mycat9tes the fastest 

was 290C for SUD 21 and 24-, '&,. 90C for MAC 36. Lower temperatures appeared

more favourable for their production than higher (Table 4.1-4). 

Helm (191*0) found the optimum for the production of opalescent 

mycotie'tes was 2Ge5-28,50C. * Above 301C he found temperatures more 

favourable for the development of filamentou3 elements at the 

expense of the mycot8teme At low temperatures mycotetes wero not 

formed until the temperature approached 240C (Heim 1940)o The number 

of subcultures made does not affect mycotete production (Table 4.1.5). 

1580

4o2el MHODS 

4.2 sPonEs FM,, M CULTMEs AM IlYclyr M-Es 

Spores ftom 2 week old Termitomyces cultures acsociated with 

different termite species were measured., The size of the longest 

and shortest axes of 30 sporesj where possible$ were recorded. 

Measurements were mado on cultures from both SP and selectivo media* 

Me form of the spores and hyphas were recorded* Measurements 

were also made of spores from mycotOtes from the coiub. 

4.2.2 RESULTS 

The results are given in Tables 4.2.1-4.243 and Fig. 4.22.1-4.2.2. 

1590

#W. 

FA 40 

60 

0 

P. * 

n 

ic 3 

32 

f+ 

ti 

9 

rA 

90 u u 

ý2 n ý5 n 

et» g+ b- 

a 

0 

0 e2 

. fß 2 8 1- 

cr 

3 

ti 

a 

eb 

c: 5' clDY 

e 2 el 

:r b. -6 n Z 

)" 

bo. 

). t Z 2 0, X bw 

e CL 

,0 

6 rA 3 0 

ný a0 -i .e :r '0,1 Z 

(A e :s 

,'1: 

(4 

0 

). d Co 

ni 

C) 0 

Nd fa 

1 

0) 

ci 

1 

0 

rA 

c9 

to t* 

0 w. 0 

0.0 n bm 

R k- m c6 

1>. 1% a - 

R 

f+ ý 

bd- C) 

b- b- 

b- D) 0 

e+ 14 

ýO, 

A. t4 

tr re- 

ý 

f+ 

ro 

0 ic 

1,4b W. 

s. 4b e+ N*ä 

Co 0 =3 :r0 

k 

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to & .,. 

to 

8 

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(to 

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2 ci, b- 8 00 

9 

eW t* 

lw- *o 

b. 4 :3" 

f» w. 01 

9 0 

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91 

9.0 

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i 30: 

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0 

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(5 00 01 

tt 

n 

3 

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5 0 m. 

:i 

(D s. 44 

Nd 

Nd 90 

8 9 s * er, 

99 

fA 

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l» 10 

6. 

d n fA th 

rk 

ei 

19 

2 4 C) 

m 

:r 

Z. 2: a 

a 

bm w 

:x ta 

C) 

1 

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tn ý 

Im 

CA 

w4 

9 

9 

2 

0-% 

ta 

11 

ra 

41+ 

rlj 

'10 

16o. 

I 

0 

Co 

wo 

rr

I 

rr . 90 m 1-3 

0m 

V+ e+ P 

1 1 

ft MJý 0 C) M. % 0) 

"I C+ 

a 0. 

II 

0 

-j Z5 

%. 4 

CN 

7 

0' ý- ý 

" C+ 

15 0 

ý 1 

to- 9 C+ 

0 cr to 

cn > 

x 10 Ili 

" elk 

tA CA IAI W- tn 

W 0- 

16 61.1 

-4 C% 

1+1+1+ 1+, 1*1+1+1+1+ 0+ 

0000 00000, 

Ca v: 

n 

W %0 %D %0 C0 C% co ti 

42% C% 0" 6%j M-,, j tj Cý 

01 

0 a 0'a 00 0 

, 

" 1- P. - 6 - 

"zn 

, ý-; " * -, 

CA' R"t 

0 

II 

'-4 

0 

. 

I f* 

6. % 

0 

m0 

R 

Ell 

- 

VI 

to 

0 

8 

co : I 

aE6 

4+ 

f* 0-0 

93 , 

V 

- 

CA 

4 0 

m 

1 

161.

": 

I 

p 

I 

a *m. 

". 4 

S 

0 

(+ 

11 3 

-%A 

I 

g -0 s? A. ad 10 F. 

V 

-V 19- 

00 d- W ta 

Co -3 

tv 

a 9 9 9 

0 on 

I 

to 

I 

r+ I 

4 4 , 

- 

00 

eel f+ 

I 

I 

to 

1 

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I (A 0 

.. 8 6 

1. 

'Os ýý ,9 

- $- b" co W 9 

0-% #, % 

(A tq 

#-- 

Ot- %03 0ý C% 

(7A %A 0 - 

% 

6.0 4ý 

pV --j %A %A %a %. n co 

: 6 Zo to o 

4 

co wl co 

ý) ;3:. 

0% 

;. ;a 

1+14 , 

-iD 

1+1+ 1+ 14 1+ 1+1+ 1* 1+1+1+1+ 1414 1+ 

00 

0 

00 

0 0 0 00 0 0000 00 CA 

ta o* L w Ip * 1- - 6 L *14 zm :. L *tj iL ', a ýL 

Co I- 

1 1- ' -14 7 CIO ? Ir 

) la o - 1"3 1. - t3 to 0.0 0" 6" 

6 to (ý% ý% ý% 

OW I- S- 

M 

0.1.6 

C* 

l? '? TI 

C3 

%D 

cs cc) (7, 

Nd 1- 0- 

1+1+ 4+1+ 1+8+ 1+ 1+1+0+1+ý, 

01, 

;N ýl 6 I 

0 00 

;" ýý 

%a 

'1- 

, 

%A 

"1 

; 

1 

ig 

2 

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1+1+ 1+ 

21 

162o

51 9 112 

öl AN- Z- 0 RE ? m bi 

I 

0 

ro, iä 

offl 

te 

(1) 

d-4 

VOM ei 

mv8. d 41 

8 

1 

9 

6-4 

0) 

tr x4 rA 

ic 

03 

99E g 

9 

n 0. d w b" 0 

9 

ce 

te a 

1 

uvwu 0 

p 

re- et, 1 

, 

m 

-1 (D 0 

0 %0 (b 

to ti 

9) 

f- ? %A 7' 

bl. %n %A il 

W CK) 

1 ta 8 

bd. 

a 

C% vi 

er 0 

9 

n 

0 to x8 M 

0=e 

(D CL 

le 

to 03 0u0 

0 

5 

-5 ag -g 

pýs, - 

.. M 

(D 

-1 

1 

ID 

1 

1 

w- K- B 

to 9 

rA 

ý. u s. b-. 

0p 2 

to 0 Z 

t ,- ,r t-g 

o g to o 

1rn 

(D in 

in 

2 r. 

ec 2r-. 

fi 

' 2 

rA 0 

to u 

.i %A 

Co 

8 

lb 

c3 

8 

v4 

r3 > 

-4 

'? j8 

cl 

d-4 

el 

0 

- 

r> 

e 

61 

vr 

1.4 

lip 

1-0 

Ir 

be 

bd :1 

e Co :i 

1 

r4 

1 

1630 

Zo 

2 

zr 

re 

b. 4 

Me

1649 

FIGURZ 4*2*1* Lengtb, of TermitomIces spores (with 95% confidence limits)

N) PO 

cil Ul 0 Cn 

K 

0 

Cf) 

-Ti 

9 

CD 

a 

c 

3

FIGURE 4.2*2. Width of Termitomyces spores (with 95% confidence limits). 

I- I 

165.

. '. 

'; 

Th 

E 

-> 

a) 

E 

E 

D 

. 

a) 

Li a) 

Cl) u 

EE 

00 

d) 

r 

:i C2 

CC) 11 

U 

I., 

Q) 

0 

.2 

Z 

Q) 

_Q 

Cý 

CZL 

E

4.2.3 -PISCUSSION 

Spores were not produced by all the cultures within Zweeks on 

both media. The medium on which spores were not produced variodl no 

Oporq. 4ýpalng produced on SF medi= by cultures from Hicrotermen 

species-, Ai D and Z, and no xpores-being produced on solective, madium 

by cultures from Hicrotemen species Dj Gj R and Zo The cultures 

from G only produced spores after 5 weeks growthl and 2 weeks may have 

been too short for their production In other cultures cog. AN 531 and 

cultures from Hicrotermes app. A and R on Sr- medium, 

.. The spores from the Ancistroternwx cavithorax cultures vere vider 

Man those frcm the natural--mycotetes but of tho same lengthl tihereas 

those from - Macrotermeg bellicoauq cultures vore very much longer than 

In the natural mYcotetes. - Helm (1942a) stated that the only difference 

between cultural and natural mycotetes was that in culture the blasto- 

spores are more distinctly yeast-like andmore easily crushed than the 

natur&19 but there Is no visible anatomical differences Batra and 

Datra 1979 found that the conidia-bearing aggregates In culture 

rescmbled mycotOtes on combs but vera less compactly arranged and 

this would agree with the results here* Bakshl (1951) 

also considered 

that the cultural mycotetes were essentially tho same as found In 

naturee 

The sizes of spores produced on the 2 media are In general little 

differente In terms of the length the spores fall Into 3 groupas 

zlx)ros from Macrotermos bollicosus, Macrotermen subhyalinux and 

Odontotermes spo (OD 54) being much longer than those from Ancintra. - 

terven eavithorax, all the Microtemes species and Odontoternes 

166.

smeathm. -int 

(Table 4-2.2, Figure 4.. 2.1). Batra and Datra (1979) 

stated that except for minor variation in size the microtmorphology 

of the mycot*jte, spherocynts and conidia in all species of 

Termit2=ces from Inding Pakistan and 1halland were similar. Heim 

(1977) 

said that cultures of different species yielded practically 

identical results, 

Nola (1077) found that blastospores in'culture were very 

variable "d Irregular In for= and dimensionne Ile concluded that 

L 

tI 

the diverse calls which constitute the adult artificial Mycoteteg 

sometimes oval in chains, sometimes terminal sporeal sometimes larget 

unequal and Irregular forming the flesh of an opalescent mycot0to 

and sometimes elongate spores on powdery colonies, are all of the 

same nature loo. blastospores. 

167.

4.3.1 MMUDS 

4.3 BLASTMPME, GMMINATION 

A spore suspension was prepared in distilled water using 

cultural mycotetes from a Termitomycon culture associated with 

tfacrotemes bellicosus. 0.1 ml of the sporo suspension was pipetted 

cnto-CA plates (Appendix 1) and incubated at 300C. The gemination 

and development of the spores and mycellum was observed at Oo 69 24 

and 48 hours and at 41 7,10,14 and 17 days after inoculation. 

4.3,2 RESULTS 

4.3.1. 

The results are given In Tables 4.3.1 and 4.3.2 and Figure 

TABLE 40.1 Hode o f germination of Termlt2=ps blastospores. 

POSITION OF G1201 TU132 NU1,23M % 

Prom one end only 48-3 

From both enos UI 

From both ends-. (2 t, l) 

'irom both ends 

From both endB 

From one end 

I) Q 34*2 

10 8-3 

(3 1 1) 2 1*7 

(2 1 2) 

4 3-3 

(2 1 0) 2 1-7 

Prom side 3 

TOTAL 120 100.0 

168. 

"05

TABIZ, 4.3.2 Events during course of germination and growth of 

Te-rmitowyces blantospores In culture. 

TIME 

6 hours ru) germination. 

22 hours I nomo spores have geminatedo 

(CA medium. 3000 

24 hours I 

geraLnaticm and growth of many JIPOrcze SOMO still 

ungeminated* 

48 houris many side branches have developed. 

4,1days aide branches have been produced to fill In the centre 

of the colony* Anastomoses have been formed* Dominance 

of leading hyphae, 

7 days ac - cumulations of hyphae have occurred to form cultural 

Mycot'hese 

10 daya I some of the Initial colonies have disappeared and very 

little growth has occurred In some of the others. 

14 days 10 the actively growing colonies have reached approximatelY 

cm diameter* 

169. 

17 days some colonies have stopped growing and appear to be under- 

going autolysis, In all cases there in a larger actively 

growing colony nearby which suggests competition and 

local nutrient exhaustion. Those actively growing have 

assumed organized form with dominance of leading hyphae, 

anastocioses and nite; =te braxwhinge 

/2 

7 

/ 

/

a0 

FiGuRE 4.3-1- (a)q (b), (c), Progression of germination with 

hours 

b' 24-hours 

time (CA medium, 360C)* 

/// 

Z. 

170,

c 

48 hours 

00, 

171.

4.3.3 DISCUSSION 

The blastospores germinated readily, the majority with one germ 

tube, or one gem tube from each end of the spore (Table 4.3*1). 

Other authors have germinated blastospores but found the spherocysts 

did not germinate% or only rarely, being very poor in cytoplasm and 

having a purely vegetative role Olelm 191*0, o 1942a, 1977; Petch 19Mt 

191309 

Helm (1940ý 1977) found that the bla2tospores could put out one 

to four Mycelial filamental, usuallyat the extremities, rarely starting 

from the middle of the spore. Due to there being never more than two 

nuclei the filaments without one aborted* Petch (1906) 

reported 

hyphae were usually produced at both ends-of tho blastospore, with 

perhaps ono or two additional hyphae arising on either side later* 

Datra and Datra (1979) reported that ellipsoid-allantoid conidia did 

not Geminate but that other blastic cells did so readily, usually 

with a Polar germ tubeq but often with twoq t1iree, or more tubes from 

various altese 

that 

172-

. 

------- 

- 

- 

-- 

---a, 

e ; --. 

-- 

-- 

4; S 

-- 

-- 

--- 

alATvrM FIVE 

THE HICIOBIAL EMLOGY OF 

RAMOTIMMITINAE NESTS 

-- ---. --- 

- --- 

-__-__-( 

--- 

-y-. 

a*____ --- 

--- 

- 

173.

5-1-1 INTPODUCTION 

5.1 HMIMS 

In order to investigate the microbial ecology of termite nestal 

both quantitatively and qualitatively* suitable methods had to be 

found which were both applicable to the problems set by the nature 

of the system being inventioatedl and which could be carried out 

Oiven the tacilities available at Mokwa. 

All methods for Isolating the fungi present in materials such as 

soil are selective. The use of different nutrientain tile agar media, 

different incubation tenperatures and varying p1l of the media will 

favour those fungi whose germination and growth optima are closest to 

the experimental conditions, No technique can yet give the total 

microflora of the system under Investigation* While this is the case 

the dilution plate method will probably continue to be useds whicho 

provided its limitations are realized, in a useful tool, 

Major criticism Of theo dilution plate method arcs 

(1), it gives a large advantaqfx to abundantly sporing species* 

Warcup (1955a) demonstrated that the vast majority of colonies on 

dilution platess arise from spores. The mycellum often rccains 

embedded In organic material or soil fragments* and so tho numbers 

of a particular species on a plate may not bear any resemblance to 

its mycelial. development In the material being te3tede Both dilution 

plates, and zoil; -plates give little Information on species present In 

an active,, hyphal state at the time the Isolations are madeq although 

Inferencen may be made regarding apedes, Which had previously been 

active 

(PArkinsan 1973)-ý Montegut (1960)9 

174. 

althougluagreeing that, If, 

given- an advantage to1zeavily-sporulating species, considers it may

not distort-the numbers as much as It might first appear tot as a 

large number of species do not exhibit a frequency in the field 

proportional to their sporulative capacity In artificial media, 

cog. Trichoderma. - Thus the majority of fungi found on the ordinary 

dilution plates out be judged to originate from spores, 

An advantage Is given to rapidly growing fungif and slow 

starting and slow growing fungi are likely to be suppressed 

1951)e Warcup (1955a) 

showed that nasidiomycates are particularly 

(Garrott 

selected against* The fast growing fungi are also favoured in the 

&oil plate method, Garrett (1951) 

considered this need not be too 

serious provided final dilutions are chosen so that the number of 

fungi/plate are not too highl thus reducing the Interference between 

developing colonies through competition and antagonism. The ideal 

number of colonies per plate to variou3ly given an 30-100 (Waksman 

1944), 35-45 (Brierley, Jewson and Brierley 19,27). an average of 25 

(Disby, James and Timonin 1933, Garrett 1951), this final figure being 

found MtatiBt: iCallY Permissible by Jamex and Sutherland (1939)9 

Howevers even wLth low numbers suppression does, occure 

Those ftmgi whose propagules are attached to coarso particles 

175- 

that undergo a rapid sedimontation-or flotAtLon. will not-be Included In 

the dilution, sarless but Ifornby (1969) found that. the contribution of 

any-fungal material attachod to soil particles was Insignificant. 

5,1,2 lXnICDS. r=ABLLSIUZVT OF MMODS TO DE U. 

SED IN TIM 

INVE STIGATION 

There are a large. raimber of different, mothod, 0-- of Asolating micro- 

erganis=,: rrom soll, and otlier, materials. ä. 4,. -, 

Curl 19729 Park1noon 1973 and Warcup 19GO)o

The Isolation of Termft2=ces, posiDs different problem to that 

of Isolating the general range of fungi* 

' This means that methods 

for Isolating Ternitomyces and also methods for looking at the wa 1 11- 

number of fungal species had to be e8tablifihede Qualitative methods 

for Isolation only were compared-to quantitative nothodise 

Initial trial experiments were carried out to determinel 

(1) the type of isolation method to be used. 

(a) direct Isolation from the mycoteten of Termitomyces 

(both Intact and dispersed In sterile water), from particles of 

food store and from small pieces of fungus comb. 

W the dilution plate mothod*(Jensen 1968). 

(C) the wall plate method (Warcup 1950)o 

(2) the isolation media, The Isolation media used were the 

general soil fungus medium (SF)s 

and cellulose agar medium (CA)s 

Identical except for the carbon source which was a cellophane film 

overlay Instead of glucose (Appendix I)* 

(3) the ter4mrAture of incubations Two incubation temperatures 

were uBed, ambient 3500 and 200C- 

ý (4) the number of days Incubation. Tho prepared plates wore 

Inspected after 2,5 and Tdays IncubatLon'and the number of fungal 

colonies counted, 

5-1-3 RESULTS AM) DISCUSSION 

On the direct Isolation and dilution plates a large ninaber of 

diff, ar, ont fungal species were' isolateds BY Comparison theýWarcup, 

soll plate mathod IW38 MOM 20le-ct1v* with Pal*clla-mycem 

176. 

varioiji being

IsOlated frica all the matbrial plated out, so that no adviintage tould 

be seen In using this method as leeii species were IsolateA. An' 

adýantage of the direct Isolation from fragments of material to thiit 

it in applicable to a wide range of materials under study, Including 

tho digestive tracts of termites* 

on comparing the numbera of fungal colonies obtained on the two 

different media OF and CA) 9 slightly more fungi were obtained on the 

Sr, 'r4edium, althbugh tho numbers were not significantly different 

(Table 5.1.2)* Cellulose in a inore selective carbon source than 

glucoae and therefore tho general soil fungus medium (SP) was used 

In the dilution plate technique to give the general range of fungi 

present as spores in the material being tested* Using SF medium the 

problem in Isolailng 13ýasidiomycetos by the dilution plate technique 

was seen an Termitonr yces was only isolated on those plates containing 

a lower number of other fungi. This was overcome in the main 

experiments by the use of the selective medium for the Isolation of 

Termit2=cps (Chapter 3). 

There was no significant difference in the number of fungal 

colonies developing at the two different temperatures (Table 5*I,, l)* 

177* 

For the main wcpariments a temperature between these twol of 20-30OC9 

was selectedg an this In the optLcntc3 for growth of Tormitamycas and 

close to the mean temperature of 30&9'OC maintained in the habitacle 

of Kicrotermas ballicosus, mounds (Collins 1977), 

Few extra fungi develop on the dilution plates after day 5 (TablV 

5*1*2). It in also difficult to distinguish Individual colonies duo 

to the covering of the plate3 by the growth of the fungi* Therefore 

5 days incubation was chosen As the standard for comparing fungi

developing on the plates. %- The number, of fungi growing an the plates 

were recorded after 2--days In came any fast spreading fungi developed* 

At day 5 any a-ttm fungi appearing on the plates were recorded and 

the fungi Identified, 

'Comparison of the tvo methods of plating mycotetes showed that 

More -ieraitm-yces, 

' colonies were obtained by breaking up mycotAtes -in 

sterile water* than by placing whole ones on the plate 

(Table %10), 

This may Indicate som Inhibition of germination due to the close 

proximity of spores in the Intact mycotaten* Although the spread 

t3ethod. gives a higher number of colonies than the whole mycot8te 

methodl tlie latter, modified slightly by breaking up the mycotete on 

the surface of the plate to spread the spores$ vax adoptedt due to 

there being lower contamination with this-mothod, 

178.

TABLE 5.1*1 Nwbers of fxmgal colonies obtained on noll dilution 

- 

platen on two different media and at two different temperatures* 

ISOLATION 

MEDIUM DIUM 

(nT7, V -) 

dilution* 

TEMPERNTURE 

TEEMPERATI 

AMIENT 350C 

NO* COLONIES/PLATE MW 

INCUBATED 200C 

179- 

NO., COLONIES/PLAT2 MAN 

Cellulose aaar 10 10 6 8.4 5 13 10 10 

(l(r3) 7 9 

12 10 

C*A,, /A 0 7 4 3 11.11 

(JLO-4) 6 3 2 1 

1 

2 2' 

soil fungus 9 15 210 13.6 5 11 21 10*6 

medium 10 14 3 13 

(10-3) 

S*Fo 4 5 2 7.4 5 4 fk 1-4.4 

6 20 

t values 

CoA. vs. S. F* Table value (5% level of oiWdficance) 

10-3 t- 1L. 5!;, 2.31 

lor*, 4 

Ambient vas Incubated 

I NSD a no significant 

1 

NSD 

t= O"og '2.31 NSD 

CA 10-3 t. 1.00 2-31 NSD 

SF io-3 t-0.96 2-31 NSD 

differenco.

TABLE 5*14 Number of fungal colonieg on soil dilution ýIates 

after 2,5 and 7 days incubation. OF medium) 

DILUTION 

NO* OF 

DAYS 

INCUBATION 

NUMERS OF FUNGAL COLONIES 

1806 

PLATE I PLATE 2 PLATE 3 PLATE 4 'PLATE 5 

2 is 50 19 ". W 

10-3 5 20 53 24 23 19 

7 20 53 24 23 19 

2 1 4 1 2 

jL(r4 5 1 5 2 3 

7- 3 

TABLE 5ol. 3 Number of Amgal colonies obtained by 2 different 

methods of Isolation : fn= Termitg=ces raycotates (SIP medi=)& 

MMIOD OF ISOLATION 

NU123En OF PLATES WITH 

Zo. 

COLONIES 

_rmJLtomyce9 

19 

TOTAL NMMER OF 

FUNGAL COLONIES 

Intact mycotates, 

(30 plateso one mycot0to 77 

per plate) 

crushed mycot0tom 

(30 mycotteltes broken up In 

20 t2l aterLIe 112ol 0.1 mi 13 105 

pipetted, onto each of 30 

plates)

5.1.4 

CONCLUSIONS1 MTHODS To BE USM) IN ME INVESTIGATION 

rrom the results of the initial experiments the methods selected 

for investigating the microbial ecology of the next systems weres 

direct isolation (i) onto SF ciedium to isolatetho General 

range of fungi present. 

I 

(it) onto selectivo modLum to isolato, 

TermltMErces. 

This involved using a sterile Innoculating needle to place sm. 11 

pieces of foodstore, fungus comb or whole mycot'oten onto agar plates* 

(2) the dilution plate technique,, using both media an above* 

The importance of standardizing thin method has been frequently 

e=phasized (Jensen 1968) 9 and the some procedures were used throughout. 

The fcmdatore, funguis comb and nest structure were collected In sterile 

containers* The materials were plated out oti the day of collection to 

avoid problems arising from atomCe, This created a limit to the 

number of plates that could be prepared In any one experiments 

Approximately Ia of material, weighed accuratelys was Placed in 

loo ul of sterile water In a 250 ml flask* In the case of fungus comb 

and nest structure the material was first broken up Into small 

fragments. The suspension was prepared by stirring with a magnetic 

stirrer for 30 minutest and the successive dilutions prepared by 

pipetting I yal aliquota Into 9 ml of sterile water* Standard 

pipetting technique: s were used throughout& Prior to pipetting the 

solutims were shaken by h=d* 0*1 ml aliquotb of the required 

dilutions were plpottOd onto each plate and spread over the plate 

surfaco with a flAmed glass spreadere The plates were incubated at 

28-30)C and the fungi developing counted after 2 days, and again after 

181.

5 days when the fungi were assigned code letters and isolations made 

for identification Purposes* 

'Two, dilutions were used for each dilution plate experiment* 

These dilutigns were, dete. ruined by preliminary experiments* The 

moisture content of the materials used was determined by drying over- 

night at I(r>OC* 

is.,,.

5.2 MCROTSIMS BELLICOSM3 

5,2*1 HACROTIT-NES DELLICOSUS WST SYSTEM 

Introduction 

The structure of t1le 14, bellicosus nest system was described 

in Chapter 2 (2-3-29 PLO- 2.3-1)- 

Vixually the fungus combs of 11. bellicosus appear to be divided 

Into three distinct zones (Plate 5,2*1)o These arei 

(1) Fresh combi the freshly deposited edge of the fumaus combo 

This is darker In colour 

than the rest of the comb and the faecal 

pellet structure In more obvious. This will be termed fresh comb. 

(2) Middle combs the middle zone of the comb,, which is 

characterized by having the mycotetes of Termit on its surface* 

This will be called the Middle comb, 

Old combs the older part of the combg which has red/13rown 

superficial patches of soil Present On ito This Will be called the 

old combe 

In order to givo an idea of the relative sizes of tile parts of 

the nest measurements were made on one H. bellicosus mounds 

Nothods 

A small nest was excavated and the sizes of the various parts. of 

the mound recardede These were% 

height of mound 

circumference at base of wound 

height of habitaclp 

circumference of plate 

JL83.

circumferences of queen cell 

(Plate 2.3.4) 

curved distance from top of hive to base (Plate 2.3-1) 

extension of food store down habitacle (Plate 1, *3*1) 

The size of the zones of the fungus comb were measured by means 

of cotton-thread transectse The comb as found in position in the n03t 

consists of Obr4in-likel fissured masses with the fresh edge of the 

comb to the outside so the measurements often involved breaking the 

mass of comb open. The numbers of mycotates occurring in 0,25 cm 

aided squares in the middle zone of the comb wore counted, and the 

density of the mycot8tes on the comb surface calculatedo The mean 

dry weights of the mycotOtes was determined. The density of the comb 

and foodstore was measured by weighing the material in a container of 

known weight and voltimo, 

flesults, and Discussion 

The dimensions of the variou3 part3 of the 11. belliconxis nest 

measured are given in Table 5*2, lo From the work of Collinx (1977) 

on M. bellicosus m9undp In this area a height of 0,9 a indicates the 

colony to be between 3 and 4 years old* The largest mounds can reach 

6-7 in height and 3-4 m video Collins found that smound height 

wi(; th were linearly related by the'equaiiýn 

Uning ifib 

Height 

'f4442'. iridih 

- Oe-5781- 

-i&bOve"'f'0r1muift one would expect a wound of this height to 

have a width of 0-85 mg rather than the 0., 75 found, HaXJLM= building 

activity of tilese teriniten occurs at the end Of the dry season in 

March/April which Is when theBe measurements were made% perhaps 

accounting 

for the slight difference* The diameter of the base plate 

ILD the same dus the mound width@ 

184.

It can be seen 

(Table 5-2e2) that the foodstore extends 

approximately halfway down the hives It sits above and covering a 

laroe part of the fungus comb. It is therefore well situated to 

absorb water lost from the fungus combs by respiration and metabolic 

activity of Tormitomycesq and also water lost by the termites In the 

combo This, together with the fungus combt probably acts as a 

reservoir of water helping to maintain the humidity within the nest 

(see Chapter 7)- 

The sizes of the zones of the fungus comb (Table 5.24) indicate 

that the middle zone Is the largestq occupying almost half the total 

areas with the fresh zone being very much smaller than the other two* 

TemitýMces mycotaten can thus be seen to be prosent on almost halt 

of the comb area. The red-brown patches present In the older zone of 

the comb were only superficiall, and were the colour of the moil. They 

way be due to the termites plastering soil on the comb. One of the 

effects of this may be the Inhibition of the growth of other fungi 

(see incubation exWriments, Inhibition experiments Chapter 5)9 or 

they may be the products of the decomposition of the fungus c=b by 

TermitomLces 

(see 

analysess Chapter 7)- 

The mean number of mycotOtes In the middle zone of the cmb was 

142 =-2, Petch (1906) obtained a value of up to I'M cd-2 for 

Odontotermes rademinni. 

185.

PLATZ- 5.2.1, 

Fungus comb removed from Macrotermes bellicosus nest 

showing 3 zones. 

A 

186

The I numbors ranged from 4-18 per 0.25 cm2. The. mycotates 

appeared to occur In clumps rather than evenly spread over the comb 

surface. The variance'(13.19') is larger than the mean (8.90) 

Indicating that the distribution Is contagious and the population 

clumped or aggregated (Southwood 1966). (Appendix 5)e 

The mean dry weight of the mycotetes was 13.17 x l(r59 ! 0.85 

(Table 5.244). 

The densities of the different'zones of the fungus comb and food- 

store are shown In Table 5.2*5. There is an increase In density in tho 

material when it passes from the foodatore to the fungus comb., There 

was no significant difference In the densities obtained for the 

different mounds,, but there was a significant increase in density from 

the foodntore to all the zones of the fungus combo There was no 

difference In the density of the different r. ones of the comb, 

187-

TABLE 5*3*1 The dimensionx of various parts of al4acrotermes 

bellicosus nest system 

(MAC 49)- 

)MASURMOIT 

Height of mound 0.90 

Circumference of mound base 2.35 

calculated mound width 0-75 

11abitacle heioht 0050 

Circumference of plate 2-35 

calculAted diameter of plate 0.75 

Queen cell O. "A 

circumferences 0-30 

TABLE 5*2o2 The extension of the food store down a lHacratermea 

belliconut hive OAC 49). 

MAN ! SEM 

CURVED DISTANCE 

or - 

IIIVE A (cm) 

LXTENSIGN OF 

FOODSTORS IDOWN 

HIVE B (cm) 

Iss" 

FRACTION OF HIVE 

COVERED By 

F=DSTORE (B/A) 

63 32-3 0-51 

67 32.9 0.49 

6.,,., 36.2 0.58 

64 29,5 6.46 

62 3018 0,48 

62 26.: l 0.42 

59 35410 0.59 

61' 30.2 0-50 

62.5010*82 31-64 : 1.23 0.50 1 0.022

TABLE 5.2*3 The lengths of the different zones of Macrotermer; 

bellicosyS fungus corabl OUC 49) (cm). 

na 50 

All values = FMAN * S. E. M. 

MESH 001-91 

Mm Sul 

length, cm 0*55 0003 

fraction of total length 0-15 0s0I 

HIDDLC ýOOM3 

length cm 1-72 ! 0-03 

fraction of total length 0-47 ! 0.02 

Olz COND 

length cm 1-39 1 0-09 

fraction of total length 0.37 1 0.02 

TOTAL C0183 

length cm 3.66 o. A 

TABLE 5o2o4 Dry weights of vorcataten of Termlt2nMcp_u frou Ilacroter"Pa 

b_qlllcq9u!. fungus comb (MAC 50). 

11 

HAC ! iO 

MEAN MY WEIGHT ! SWI 

(o m 10-5) 

13-1710-85 

-1 --- 

n 

21 

1 

i8q.

TA13LE 5,2.5 Density of Macrotermes bellicosus funmis comb and food 

store (g crr3). 

FIREMI COM Ftimiz- COM OLE) COND FOODSTORE 

HAC 63 o. 44 0.40 0-39 0.213 

IIAC 65 0.1ko 0.28, 0-33 0019 

MAC F UNG 0.31 0.31 0.40 0*20 

Analysis of variance on the density of the fungus c(xmbo 

SOUTICL Or, VARIATION 

DOGIMLS 

OF 

ME=N 


SQUATIM 

190. 

IC AN VARIANCE 

SQUARES WNTIO 

between termite mounds 2 0*01505 0-007525 4.83 NSD 

between different zones of 

comb and foodstore 3 0.048 Z- 0.016075 10-324* 

Interaction 6 0-00935 0-0015583 

TOTAL 11 0.0r.. 625 

There Is no significant difference between the density of fungus comb 

and foqdstore from different termite mounda, Thereja a aignificant 

difference between the 4iffe, rent parts O: f the nest (1% level). 

Between different parts of tl4e pento 

F'OODST6RE 

FRESH CUý3 

MIDDLE CW8 NSD 

FR=l com HIDDIM Colm OLD COIB 

OLD C0113 NSD NSD

Table 5.2.5 continued 

There Is no significant difference between the different zones of 

the fungus cemb, but there Is between all zones of the fungus comb 

and the foodstores 

NSD - no sionificant 

difference 

= sionificant difference (5% level) 

*-significant difference (1% level) 

= significant difference (0*1% level) 

5*2*2 GIXERAL FUNGAL FLOIZA OF THE K. %CIZOTERMr. 

-P Dr-11JECOSUS NEST SYSTEM 

Methods 

Two different methods were uxod to determine the general fungal 

flora of the Macroterpma ballicosys neat system* These were the 

direct Isolation method and the dilution plate method (see 5.1-4)., 

In the direct Isolation method small plecea of tho material were 

plated out on SF medium, Isolations were made from 7 moundat the total 

number of Isolations made being shown in Table 50206. 

In the dilution Plate method three replicate dilution series were 

set up from material frcm one mound on SF medium Plates, 5 plates were 

prepared for each material and replication* (For more details of the 

dilution plate method see 5.1.4. ) Results are shown In Table 5.2-7. 

Results 

The results are given in Tables 5*2.6 

1 

191.

J*- i 0--k M0 

'0 0 b-0 

101 

1.0 V 

42%0% b- 

a 

0 0v to 

to a a 

to 

1.0 0, 

ca -f 

10 

0- 

? 

S-1, 

*r, 

b4 

Pý 

1. - 6- b. Ow 0.0 be tj %,. 

* ýj M. 0- 6- be 0.0 

" 

2,0 

26 

P- b- '. 

I - 1 

n el cU tr in 

0 

0- 6ý a *-ý. 

OA 

2 1 -- 

is 

b. 

5m 

D. 60 05 

it T - loft j 

ce 1 41 . 

6ý 

1.0 6.0 f3 a- 

a- co W, :4 

;jao go 

*0 

0.0 6. * too 

i 

IS 

1A 

to 4 64 

1 w0 

R 

S; 

9, " 

tj 

Sft 12 a 64 94 

ý - 

n 

192* 

tA 

C' 

0 

IN- 

Z 

b% 

90 

beb 

0* 

i 

a 

717

: I- 

flo, 

IK 

7 

sp, 

4< 

CD 

Pd. 

e-9- 

CL 

p 

C. 

C-1 

I. - ýr 

0+ 

r- to 

to 

W ow VII 0 0,4 

Co C% GN 

co 

k 

%0 

to 

"'o 

CID 

1+ 4+ 

I I I 

a 

co "0 

ik 

1 

1 

4- 

1+ 

:i 

cr 

lb 

w 

(D 

l' 

A 

1.0 

0 

Alý lu 

03 

0 ('26 g! '0' 

0 Q o--ý ed. 3m a 

9) 

9 so -2 

0 V. - 

bý :r3 

e. v 

v 

>o 

#w- 

%_o 

0 3 

Irl 

re, 

be 

p ;1 

)4 

1 

0 

0 

ý0 

>4 

ý3 

ü 

193*

i 

TABLE 5*2.8 Analysis of variance on fungi found in different parts 

of the Macroternex bellippaus nest system. 

SOURCE OF 

VARIATION 

Between different 

parts of the nest 

system 

DEGRMS 


rn M. CH 

SM5 OF 

SQUARES 

tw VARIANCE 

SQUARES ]RATIO (F) 

4 527*190-53 13lt797-63 ios8.96*** 

Between rpplIcate 

dilution series 2 794-71 397-35 

IL940 

Interaction a '. 

2.55.28 2010NSD 

Residual (Arror) P6 6,777.89 121*03 

TOTAL 70 536, W5.4i 

There is no Interaction between replicates and the different materials 

of the nest system, 

The're Is a very highly significant difference between the number of 

funci. found In the different materials of the nest system (0*1% leveDo 

I 

There to a significant difference between the replicate dilution series. 

7ho levels of sionificant difference between the different parts of the 

aystem are giVen below, 

FOOLISTO= 

MIS11 COM 

FOODSTORS FRESH Com MIDDLE COM 

111MIS Colo NSD 

OLD COM NSD NSD 

NEST STRUCTURE NSD NSD 

'OLD COMO 

NUST 

STRUCrURE

MI'LICATF, D11=101i 

SERIES 

A 

9 

c 

significant difference (0.1% level) 

= sionificant difference (5% level) 

NSD a no significant 

difference 

A u c 

TABU 5o2,9 Fungi isolated fro= the different parts of the Macrotermes 

bellicasu nest system. 

flaan*number of Mgt per 9 dry weight of mteriale All numbers x 103. 

NSD 

NEST 

STRUCTWE FOODSTOn- Piz WIC I 

Colin 

RIDDLE 

Colin, 

OLD 

195. 

COI13 

AspeMillus ni ger 

3.18 134.. = 2 JL. 94 4., 0., 45 

Pnecll=es varlotit 1.53 1.08 0,47 01.10 

-kenicillilLm - 

Opp. 1023 7.84 1.60 5.43 2.80 

terile white 

CladoeMrium 

mycellum 

appe 

I- 3 

-21 

0: 1)0 

0.22 

Oe22 

0.94 

0.82 

21 76 

- 

0,20 

2.88 

ýItcor 

up. 0.66 

Cunninnhamella opp* 0-58 0-34 

-Aitpprgillum flavus 0.51 1.82 

sterile white myceli= (WR) 0e51 0-77 0.13 0.95 

cerimlompqrium app. 0-37 6.83 0.12 0-31 0.45 

'kusarium app. 0015 0*12 

Trichoderm app, 0*15 0.. 27 0*10 

sterile mycellum (WC) 04,15 

Absidia Spa 0.07 12-33 

EOFLllus terricola 0.07 

A* zulj! ý. ureus 

1 

18,01 

TOTAL NO* OF SPLCIES 15 10 10 

E7 

5

1 

TABLE !;, 2*10 Similarities In fungal species cor4oosition between 

different parts of the Macrotemkm bellicagun nest system, 

Uses 50rensenOs Index of Similarity 2x (Southvood 1966) 

M+n 

where xa the number of species the two materials have in common. 

FOODSTORE 

in a the number of species in material I& 

na the'ýnmber of specleis In material 21* 

FRESH COMB 0-70 

FnEsu 

COM 

HIDDLE COM 0.71 Oe82 

HIMLE 

Collm 

OID Oe53 0,67 0-83 

NEST STRUCrURB 

-1 

- 

OID COMB 

0-721 0-72 ol 64 ' 1 

0-50 

NEST 

1964o 

STRUCTURE

Discussion 

Two different methods of Isolation were used to Olve a wider range 

of fungi isolated. All methods are selective and so different fungi 

may be obtained by the two methods* For example Termitomycen, is the 

most comron fungus Isolated from the fungus comb by the direct Isolation 

method, whereas it does not appear in the dilution plate method 

5,2*9)o TermitpMycpq Is present as wycelium: actively growing In the 

comb and so when small pieces of comb are placed on the plate the 

(Table 

mycelium can actively grow out from the innoculation, The fungi growing 

on dilution plates generally arise from spores, the mycelial fragwnto 

197- 

tending to adhere to lumps of material and to float or sediment out more 

rapidly depending on the material* This could account for the non- 

isolation of Temit=yceq and also Xylaria, by the dilution plate Methods 

an XXIarin has been reported as actively growing in the fungus combl its 

cjycelium being mixed with that of TprmitomXces (Grasso' 1937)- XYIftria 

was isolated by the direct Isolation method from the older coob material, * 

TermitcoXces would also be selected against in the dilution plate method 

as It to slow growing and would be rapidly covered by other fungi present 

on the plate. 

The fungi obtained by plating ou t the foodstore (Table 5*2,6) are 

species commonly isolated from the soil In the Mokwa region (14CDonald 

19681,1970), Trichodema shows a higher frequency than Its Presence in 

the local soils might lead one to expectj it being of very infrequent 

, Occurrence In the soil 

(IScDonald 1970). These fungi are probably 

brought Into the foodstoro as spores by the termites, both mixed with 

the collected food that will constitute the foodatorot and also adhering 

to the terziten bodies,, having been picked up from the 3011 and vegetation 

while the termites were foraging&

After approximately I week In the next (Colling, 1977) the food- 

store in Ingested by the te=itess, passes. through the Cut and. the 

1980 

faecal pellets are deposited to form the fresh edge of the fungus combe 

The fungi isolated from the comb are again those-commonly found In the 

soils except for Termit2=ces which is the =oat coc=n species Isolated 

from the fresh comb by the direct isolation method* 

The mean number of fungal propagules present in the foodstore in 

very much greater than that occurring in either the comb or nest structure 

(Table 5.2.7). There is approximately 230 x 103 g-l dry"weight foodstoreq 

compared to 10*5 x 103 U-1 dry weight fungus comb and 16*5 x 103 gýl dry 

weight nest structures This large drop from foodstore to fungus comb 

indicates that passage through the termite gut must have greatly reduced 

the viability and number of the spores, as all the material present *a 

foodstore Iz. deposjte4 on the rpngua comb after passage through the t; ute 

There is no significant, difference between the. numbers of-the fungi 

(excluding Ternit2MLcps) In the different zones of the co, -vb, 

Table 5*2.9 oives the species of fungl'that were'found on the 

dilution plates* It is not a complete species list but those not 

included were found very Infrequently* 7h, e species isolated are ones 

that have been found as, common soil fungi in the kkkwA soils (McDonald 

1968,1970)e The changes in species diversity of the different parts 

of the system can be seen. in comparing the total number of species 

found In each of the different materialso 

The most diverse population In the nest structure vhich as It 

consists of soil to probably to be expected* The foodstore and fresh 

Comb cc)mo next with ix reduction In species n=bcr from freshq to middle 

to old comb indicating a _'progresaive 

reduction In sPecies number with

Discussion 

The bolus-carried by the foraging workerx was dark In colour 

and was probably composed of soil and/or the foraged materialo The 

foragers are carryino a far higher tu=ber of fUngi within their gut 

than those termites in the neatip there being a reduction In rxL=ber 

from foragers to nurse workers to the young, The foragers would 

also be contminated by spores on the outside of their body and In 

these two ways the spores would be easily Introduced Into the food- 

store-accounting for the high numberx of spores-found there. The 

species vf4ungi found In the guts (Table 5.2*16), are all comnonly 

found in the Mokwa soils (McDonald-1968)9 and many occur In the- 

foodstore (Table 5*2*9)9 

Dy contrast the bolung when presentl In nurne workers and young 

was much lighter In colour. This-probably Indicates that In1he 

young It consists of Term1t9=ceso, Tervdtom ! ycee,, being isolated with 

such high frequency from their out (Table !;, 2*15), The gut of the 

nurse workers way contain fungus combo the Isolation of Termitomyces 

being lower than In the young. 

These results probably, reflect,, difterences In-diets the-older 

workers feeding on funUtts comb with Termitamypes forming, &n important 

part, In the diet -of 

the young terldtose Vary taw Ispecios of fUng, 

apart frcm Termit2. n yces, were found In the 710=9 which would further 

indicate that they are feeding on Termitomwess alone, It they were 

feeding on comb the fungal mpecies present In It would be Introduced 

into the gut and be Isolated As In the nurse w()rkerm. 

only & very small proportion of the foragers carry jerwita"ye.. 

(Table 5*2*15)o Thus when the food to plaCed In the foodstore it jLs 

203. 

--

time In the foodstore-lUngua comb syntem. ThIx may indicate a decreased 

viability of the spores due to their time In the fungus comb, 

Using Sýrcnscnls index of Similarity (Table 5.2.10) the similarities 

between the different materials were compared. The most similar were the 

middle and old comb (0,, 83)9 followed by the middle and fresh comb (0.82)o 

199. 

5*2e3 LOCATION OF TMNIII)HYCES IN THB RACROTEMMS BELLICOSUS NEST SYSTEM 

Introduction and tletluAs 

In order to establish the locations of Temit=ces in the Micro- 

temes bellicosus nest system the selective mediums (Chapter 3), was 

used. The selective medium prevents many of the other fungi presents 

which grow on SF medium, from growing and Increases the chances of 

Isolating TermitomX2ox. 

Direct Isolation from particles of the foodstore and fungus comb 

from 7 mounds were placed on plates of selective Mediume The total 

M=ber of isolations made is shown In Table 5.2&11* 

The dilution Plate Method was also used on the various parts of 

one Macrotermon bellicopus mound In order to Isolate Termito=ces, 

Three replicate dilution series were act up on selective medium plates* 

Five plates were prepared for each material and replication (for more 

dotaile of the dilution PlAte method see 5*1*4). 

lResults 

The results are oiven In Tables 5*2.11 - 5,2.13,

a0 

0 

0 

o 

01" " 

-OW E: 

0-4 et 

Ch 

\. n o o 

0- 0 

W o 

110 

3 140 W 6.3 

C* 

%0 

ILI 

so 

I'll 

+ 

9t 

V) 

C- 

5 1 

2 

9 

C) 

P ol %W 

ý; 

El 

ta 

Er "o 

0 

loo. 

9 

1 

40 

0 

opt 

tr 

M 

0-d 

1-4 

oft 

0 

0 

200.

TABLE 5., 2*12 Mean number of Termlt2! Mcex colonie3 per g dry weight of 

different parts of the Kicrotermes bellicosus, nest systme Results of 

the dilution plato experiment on selective medium, (All numbers x 104)0 

S. E. H. ) 

REPLICATE 

SAMPLES A D C mms 

Foodistore 0 0 0 0 

Fresh fungus comb 0-74 ! 0-45 3-85 ! 1-50 1-36 1-36 1.90 

lUddle fungus ceab 24-75 ! 2-77 49-38 ! 3-20 58-38 3.45 ý44.17 

Old fungus comb i. oB ! o. 44 4-75 ! 1-36 2-13 1 0.66 2,65_ 

Nest structure 0 0 0 0 

A two-way analysis of variance was caxried out on the results of the 

individual plates* 

201*

TAMIS So'-'*13 Analysis of variance on the numbers of colonies of 

Tem. itp. ycos found In different parts of the Kicraternes bellicosus 

T 

nest systeme 



Between different 

parts of the nest 

DMREES 


FREEDOM 


SQUAPMS 

MiAN 

SQUARES 

202o 

VARIANCE 

RATIO (F) 

system 2 17,511975 89755-875 i8.96*s 

Dotween replicate 

samples 2 It-2150.45 625.225 NSD 

1-35 

Interaction 4 1,047*60 461.900 78 

Razidual Urror) 36 730.16 20"280 

TOTAL 64 21039-96 

There its a very highly significant difference between the number of 

Temitomnes colonies found 

Systea 

samples* 

- In the different materials of the nest 

(1% level)* There Is, no significant difference between the 

The levels of significant difference between the different parts 

of the system are given below. 

RUSH CDIM 1. IN, 

MIDDIZ colm 

OID COM 

Mmi com MIDDLE COM OID com 

- 0aw 

KSD 

isignificant differenve, (O-ý% level) 

significant 

NSD . no significant 

difference 

difference* 

ý

Discussion 

In comparing Tables 5-2*6 and 5*2*11 It can be seen ttiat the 

selective medium has greatly increased the probability of isolating 

Tormitonnes. Proin fresh comb the isolatien index increases from 39 

to 72, and from older comb from 41 to 89. The contamination from other 

fungi and bacteria Is very low, and plates were obtained on which nothing 

Crew from the material placed on them. This indicates that the presence 

of Tormit = cen in material placed on this medium would not be masked 

by the growth of other organisms. The conclusion may be drawn therefore 

that Termit =. cealis not prevent in the foodstore. On both media the 

isolation Index of Termitomyces from freshly deposited comb is lower 

than from older comb indicating that the Termitomyces may be colonizing 

the freshly deposited material by growing In from the older combo 

The results of the dilution plate experiment again show that 

Termitomycog is not present In the foodstore or in the nest structures 

(the earthen supports which ramify between and around the outside of 

the fungus comb)j and to confined to the fungus comb within the nest 

system& The numbers of TerMtjg=e9 colonies obtained from the three 

zones of the fungus comb confirm Its original visual divisions LOW 

numbers of Ternit2Liyces colonies were obtained from the fresh edge$ and 

also the older comb* Separating these two zones in the middle zone of 

the comb where TemitooXcen. can be seen as being present due to the 

large numbers of mycotetes which are found In this zone. 

203. 

This experiment was also carried out at 10-3 dilution and again no 

TermitomXces was obtained from the foodstore, and nest structures 

Although the selective modium greatly reduces the numbers of fungis 

other than Termitomyces, Isolateds some other specien were obtained on

the direct isolation and dilution plates, These were Aspergillus 

carnpus, Asrergillus violacelial Curvularin sp, j 1-baarium appe, 

rhielavia op., Phom op*, Xylaria spog Cunninnhamella, sp*1 and an 

unknown Hasidlomycetee The fact that some fungi are found on this 

medium that were not Isolated on the SF medium reflects the different 

pictures of a fungal flora that are obtained with different media$ each 

medium, even general ones, being selective for particular fungi (see 

5.2.4 FUNGI FOUND IN THE GUT Or, HAMOTMWIS DELLICOsUs MRAGE3zs ANý 

ADULT AND YOUNG M&JQP WOMM TERMITES IN TIM NEST 

Introduction and Hethods 

Foraging major workers 

(foragers) collectod from baits in the 

vicinity of a M. bellicosus moundl nurse major workers from the comb 

and young major workern were surface sterilized in lUlton's fluid,, their 

digestive tract dissected (mt in sterile water* and placed on selective 

medium plates to culture any Tormit=ces present, and on SF medium 

platesto deterMine the general range of fungi, being carried in the 

gut (Table 5.2.16), The young major workers ware distinguished. frM 

larvae by the presence of darkened heads which are absent from larvae* 

Results 

The pre3ence or absence of a bolus in the crop was recorded* 

The results are given in Tablen 5.2.14 - 5.2.16. 

2(Y*.

TABLE 5.2olt& Presence of bolus In crop of major workers of 

Flacrotermes bellicotus (1978) 

with 

without 

bolus 

bolus 

FOPAGW-, S NOISE WORKMIS YOUNG 

41 59% 

29 

41% 

14 (21) , o4 

56 (119) 80% (85%) 

TOTAL 70 70 (140) 70 

)m results obtained in 1979, 

2 

XI a 22*26 table value 0*1% df (2) 

205, 

13 19% 

23 workers In each category vould be expected to have a bolus if there 

was no difference in their feeding habits, 

There in a h1ohly significant difference between the mmbers of major 

workers (foragersl nurse workers and YOUID) with a bolus in the crope 

(0.1% level) 

TA13LE 5*2ol! i Growth of Termitomyces from the gut of Macroternes 

bellicosui major workers (on selective medLum)o (Gut 

including bolus) 

57 

- whole Cut 

81% 

FWAGMS, NmSu WOMUIS YOUNG 

No* of Termitomyces colonies 1 7 10 

Total noo of guts plated 30 30 15 

Percantage of guts plated from 

which Term tomyces, obtained 3% 21 3% rof 

X12 - '. 

'Oe43* Table valuo Ool% df a2a 13.81. 

Therefore there is a highly significant difference (0.1% level) between 

the number of Temitomyces colonies obtained from the different types 

of t=jor workers*

The results, shown In Tables 5.. *2.14 and 5*2ol5 indicate that boll 

occur In foragers more frequently than in nurse and youna workerso 

However the frequency of Isolation of Termitomyces was in the order 

young workers, nurse worker3, foragers, 

2069

m 

10 0 Md 

re m 

La 

0 

> 

r 

1 

eg 

o 

V3 (+ m0 0-2 age 

W. n 

pp cr M 

M 

f)). -O 

O-z crb 1: 1 1. 0a, TA 0 

ba. 

C+ 14 m to 9) .1', a9 

1 

- 

1g I 

1. -# 

ý 

tr 0m tý $I. MW 

It 

4 

pd. 

:: v9 

4m to 0- 1 

cm 

m ctl 12 4 to 

I 04 V 

t o 1 I zi I c 

11 M0 tA 0 

I- 

w 

Cý 

%D 

W 

w 

0 

CN OP- 

9 0.0 J. -d I- 

0OW b., 0% 0 ta 0Wm 0- w" 00 0- 0 

Cý \Jq 

00 0W -4 w0 -4 0 -3 -4 kli 0 \A a0% 

- 

j0 

0 1- 6- OPI 0 C* -Q 4- ra 0 C% 1., 0- 1- 1.0., 0 

I- 6ý 

co Ow V fo P- 0 %a br, U) 

0 

%., 1 0 -, j ý.. a w -4 000 

W000000 1- 00 CO 000000 

\Jn 

z 

C 

b"i 

1-4 

c 

Z9 

Er 

c 

on 

14 

6 ý: 

WO OOOO -30O W 0000-40-400 1-4 

Ss 

2 

" 

5 

tn 

0 

; 

E 

. 

207* 

to 

(3 

bm

not mixed with Termit2Mcex and this agrees with the non-Isolation of 

Teraltomyces from this matertale The mixing of Termll: gMes with the 

food material saint either occur In the transfer of the food material 

from the, foodatore through the gut before deposition of the fresh 

edge of the combs or also by the Termit2=es colonizing the froshly 

deposited material by growing in fromj4e middle zone of the combo 

2099

5-3-1 IIMZODUCTICV AND MVIODS 

5-3 MACROTEnMES StMMALINUS 

At Wma Macrotermem subhyallnum builds a more diffuse* less 

structured nest than I M. I bellicomis (Chapter 2)e There'le no food 

store and only two zones can be distinguished in the fungus combo 

There In a narrow band of darker freshly deposited comb material on 

the edge, vith. the main body of the comb con3inting of lighter 

coloured older material on which the mycotetes can be seen 

(Plate 

The general fungal flom of the Macrotermessubhyalinus nest 

system was Investigated by the dilution plate method, using the SF 

modium (5.1.4). Three samples were taken from each of the two zones 

of vomb and the nest structure* 5 replicates of each dil'uiion series 

being set up for each material tested. 

The fungi carried In the Cut of M. subhyallnus major workers 

were Isolated by washing forager& and nurse workers In Milton, 

dissecting the guta In sterile water and placing then on -SF medium 

and selective mediumplates. 30 Cuts were used for each category of 

worker on each medium. 

5.3.2 rtEstiLTS 

The rezultxýars given In Tables 5.3.1 - 5-3.6. 

,4 

210*

TADIZ 5*3,, 1 Hean twaber of fungal colonies per g dry weight of 

different parts of the Wcrotermes subhyallnus nest system* Ul Res ts 

of the dilution plate experiment on SP medium (All mimbers x 103) 

Q S*E*Mo) n 

A C MEAN 

Fresh comb 7-58 ! 1-93 25.52 1 3.58 1548 ! 2920 1,2080 

Old comb - 1-38 10-65 2-76 ! 1.03 6-54 ! 1-36 3.56 

Nest structure 10.21 ! 0.61 ý 25*00 ! 'q,., 38_ 21*2119 : 3-79 18-83 

A-two-way analysis of variance was carried out on the results of the 

Individual platen* 

TABLE 5*3*2 Analysis of variance'on the mobers of fungal colonies- 

found In different parts of the Macrotermessubh3 ralirms nest system* 

SOURCE or 

VARIATIM 

DEM 


FTtE=M 

SLIKS OF 

SQUARES 

MAN 

SQUARES 

VARIANCE 

RATIO (F) 

botween, parts, of nest system 2 1775-2.2 887.61 3!; *41*** 

between samples 2 64o. 99 320.495 JL2-79*** 

Interacti-on 4 226-07 56.518 2.25NSD 

Residual 36 902-35 25.065 

1 

L 

TOTAL 44 3544. ý3_ 

no levels of significant difference, between the different parts of 

the xyst" are given below* 

FRESH COM 

OLD COM 

NEST STRt=URE 

MESII, COM3 OID _ Col Mo NEST SMtCTURS 

21le

D 

0*0 ft 

*** a siqnlficaný dIfference (0.1% level) 

*4 m significant difference (1% level) 

NSD a no significant 

difference 

TABLE 5-3-3 - FungL isolated from the different parts of the Macrotermes 

gunZalinus nest systeC3. Now number of fungL 'per g dry weight of 

material* All numbers x 103a 

Aboldla ape o. 6.5 

NEST STRI=URE FRESH CUMB OLD COM 

A812e!: gillus flavus 0-72' 0.166 

A. nicer : 3.98 5.41 0.48 

A. ochraceus 0.56 0-23 

A, aull)huretin 0.43 L 

G 23 

A, terricoln 097JL 0: 0. 0*12 

CetWosMrlum up* 0-57 0-23 0-34 

Ciadoon2rttm spa 0099 0011 

Cuming1jamella spa 0*72 01.11 

Mucor spa 0.50 0-34 1056 

Penicillium spa 1.135 0059 0.46 

TrIchoderma ove 0059 

sterile Mycolia 0.86 1.66 0*36 

F (unidentified) 0-07 

TOTAL NO* SPP* 13 7 

TABLE. 54*4 Presence of bolus In crop of major workers of Werotemes 

subhyalinus 

With bolus 

Without bolus 

23 

37 

TOTAL 60 

PmGmm HMSE WM= 

(38%) 

(62%) 

24 (40%) 

36 (60%) 

There In no significant difference In the presence of a bo3LUu In the 

crop of foragers and major workers in the neate 

212,

TABLE 5'3-5'"Growth of Termit2m ces from the gut of Macrotermes 

ii 

y. 

I 

subhyallnus major workersq, 

No, of Temit2=es 

colonies 

Total number of 

(on selective inedlum) 

VCMGERS NURSE WOMMS 

guts plated 30 30 

Isolation index 0 13 

Cri's 13% 

X12 - 4. Table value X2 (O'leirel of significance) a 3,84 

They are significantly different at the 5% level- 

TABIZ 5*3*6 Fungi isolated from I&crotermfxis munyallnus major worker 

Cuts (Daý !S SP Imedlum) 

=C193 MRAGMS, NURSE WOMMS 

0 

NO* ISOLATION INDEX NO. ISOLATION INDEX 

A92=111us flavus 11 37 3 10 

X. nicer 26 87 69 230 

feusarium solani 27 

Mucor racemsus 2 7 

Penicillim funiculosum 18 60 

- 

Po MMirogenum 3 

1zhizopus o! =ae,, 29 

1 97 15 

50 

IISOrdariall bosensix -' 1 ' 3 

spo 

3 

. 

10 

1. 3 

WPP (sterile mycellum) 2 1 3 

elastrum racomogum 

' frichoderma. 

nothina 

TOTAL NOa FUNGI ISOLATED. 80 110 

TOTAL NO* GUTS 

I PLATEP : 10 30 

Isolation Index a rAmbee'of Isolations of 'fungus xx, 

0 

total noe of guts plated 

I- 

100 

213s

5,3*3 DISCUSSION 

There In a significant difference between the numberls of fUngi 

found in the different parts of the Pacrotermew RubhXalinum nest 

system 

(Tables 5.3-1 and 5,3,2), The nest structuro'has the largest 

number of fungi present and in also the most diverse with'13 species 

present (Tablii'5-3-3)- Thoý numbers of 'fungi and tho mmbcrs'of 

different species decline from the'-fresh comb to the"old c an 11% 

Racratemen bellicosus. Tho species found are all commonly salated 

frow Wcwa 

soils (FAcDonald 1968)o 

In contrast to M. bellicomus there in no difference In the 

presence of a bolus In the crop of the workers (Table 5-3.4). The 

bolus of the nurse workers In again light In colour and appears to 

be fungus ccob., When p lated out on selective, medium no Termit=as 

was obtained from the gut of the forageraq whereas 13% of the nurse 

workers had Lervit2Mces Isolated from their gut (Table Thlsý 

issimilar to that Of ! LzbOllic0sus And again indicates that the trish 

comb must bee innOcUlated with Termlip mycei, when it Is witlAn the' 

nest systemo This In borne out by direct Isolations fa the comb 

material where the Isolation frequency of Terwitmlyces from older comb 

is much greater, than. from the freshly deposited comb* 

The species of fungi Isolated fr6wAhe Cuts (Table 5.3.6) are 

found in the Mokwa soils (McDonald 1968)e Ruyooka (1978) found a 

range ofIrungi Imperfecti carried by all castes except minor soldiers* 

214.

5o4*1 INMODUCTION AND ICnIMS 

s. 4 mza=ma= !; Pncxr. 

-, v ý 

The fungtw combs of Hicrotermen species are very small 

(mean 

weight 0 1*0 09 Wood and Johnson 1978). Theold and newly deposited 

portions can again be dlstingulshedý theýnewly deposited comb being 

lighter In colour 

(contrast Macrotermes bellicosus and m. subbMlinus), 

with an obvious faccal pellet structure, The older material, found In 

the middle of the base of the comb Is darker In colour and the 

Individual faecal pellets cannot be distinoulished (Plate 2.3*9), Due 

to the small amount of material avallable it was not possible to carr7 

out separate d1lution plate experiments on fr03h and old comb materials 

215* 

Although groat care was talcon It to hard to avoid soco cont=ination of 

Meroternpa fungus combs when digging them out of tho soil., 

Direct Isolation experiments were carried out on !; combs (4 specleg) 

onto both stelective and SF me(Ijum plates (5 Isolations per medlumq per 

comb), and on 4 combs from the frosh and old zones onto tho two media 

(TAble 5.4.1). 

WeroterMOS 

Dilution plate experiments were carried out on_, whole.., combs for"'3 

species, onto o5f,. medium and selective medium. plates* Due 

to the limited material no replicate dilution series could be made 

(Table 1;. 4.2)o 

Microtermei major worXerforagers can be divided into two groupal 

those with dark gutgo'containing soil aM those with lighter coloured 

guts which are carrying food* 

, Aome intermediate* -are also, found. The 

nurse woricers were those major workersý, Uken from the fungus comb, if 

isqueczed the termite ejects a faecal Dell ot'vhl ch- was picked upwith 

sterile needle and spread on plates or SP medium and selective medium,

"'"30 plates of "ch medium were set up tar soil carrierst food carriers 

and nurse workerq, of spo Go 15 plates were also sot up from nurse 

workers of a different species 

5.4.2 RESULTS 

(spe n). (Tables 5-4-7 and 5-4-8)- 

The results are given in Tables 5.4.1 to 5.4.8. 

TABIZ 5'*4.1 Fungi cultured by airect'isolation. from ccuba of 

Hicratemes species 

(onto BF and selective media) 

MICWTEMIES WHOLE FUNGUS COM 

216. 

SPECIES Sp XMIUM SE=IVE I=Ium 

NO* OF ISOLATIONS ISOU16 NO. OF ISOLATIONS =LN0 

OF FUNGUS 

INDIM OF FUNGUS INDEX 

MIC-4118p. C) 5 Cunninghamqliý, sp* 100. -0 5 ermit=ces 100*0 

MIC'43 (sPe B) 2 Termit2nees 40., 0 1 Termit! xMem 20*0 

2 Trichoderma Op. 40.0 4 U0.0 

I bacteria 20.0 

MC 44 (sp. A) I Tprm1t2=Ceq 2000 3 Termitomyces 60.0 

4 80.0 2 40.0, 

MIC 45 (sp,, G) 3 Lerpdt! ýMes 60., 0 4 Termit2nces 80.0 

-4o. o, I '2OeO 

MIC 46 (upw D) 3, Termit2Mcex 60.0 4 Termitomycen 80.0 

2 Cunninghni-ella op. 40.0 1 2090 

MIC 61 (op. R) 3 Termit=ces 50*0 2 Temltg! Mc= 33*0 

Fresh comb %ý&=rglllurj flavus 16-7 4 67--0 

6 Isolations I Clados22rium 

cladosgg; 

1 unidentified 

loiýes, 

M S - D) 

16-7 

16-7 

141C 61 (ap. R) 1 Tervdt2=ps 16-7 1 Tormitomyces 16-7 

Old colab I Axn=tllus flavus 16-7 1 Curvularia 

6 Isolations I A* nicer 16-7 lunata 16*7 

1 Axj!!, Mtllus tip. (T) 16-7 4 67*0 

1 unidentifMI (WS13) 16o7 

I unidentified (WRA) 16-7 

MIC 67 (8p. G) I Fusarium soleni 2000 2 Tprmit=ce s 4ox 

Fresh comb I sterile mycellum _ 

3 6o. o 

5 Isolations (wil) 2010 

3 

6o. o

MICROTIMMS, 

WHOLE FUNGUSý OOMB 

SPECIES Sp HEDIUM SELECTIVE 14=1 

MT1, I 

217. 

1416. OF ISOIATIONS ISOIN. NO . OP ISOIATIONS ISOINe 

OF FUNGUS INDEX OF FUNGUS INDEX 

HIC 67 (xp*G) I Temitamyces 2000 3 Termit2=cpts 60.0 

Old comb I A. n1ger 20*0 2 bacteria 00 

loolationx 1 Fusarium solani 200 

I sterile mycelium 

(WPP) 2000 

1 bacteria 

MIC 68 (op. G) 2 Termitamyres 40*0 1 sterile 

Fresh comb I unidentified (WD) 122000 mycelium (NW) 2000 

5 Isolations 2 40.0 80.0 

MIC 68 (ape G) 2 Termit2nees 40.0 1 Termlt2MýLces 20,0 

20*0 

Old comb As raillus flavus 

I 20*0, 3 sterile 

5 Isolations 

- 

ýI Asn! r1jillus ape (0) 20*0 mycellum (Nw) 1 60.0 

.1 Fusarium ave... 2000 1 20*0 

1 bacteria 20*0 

HIC 71 (up- R) I A. n1ger 2000 2 Termitomyces 40.0 

Fresh ccob 3 A. flavus 60.0 3 60.0 

5 Isol tions 2 Aspernillulm, up* 

IT) 

-a- 

1-40*0 1 

HIC 71 (op, R) 5 A, -nlger., . 10060 1 Termitomyces 2000 

Old comb I A. flavus 20.0 4 80.0 

3 Isolations 

I- I" 

The total numbers of Iso latione of Termit=cps from fresh comb are 

on SP medium, 6 on selective medium. rron old cambi ! 3, on SE medium$ 

6 on selective medium, 

Therefore there Ix no difference in the Isolation of Termitomycex from 

fresh and old Hicrotermes comb'(6oth media). 

"

TADLE 5.4.2 He= number of t=gal tolonien per U-dry wetUht of 

different Hicroteraws species fungus combos Results of the dilution 

plate experiment on SF medium (All numbers z 104). 

ttic 41 UP. C) MIC 43 (nP- D) HIC 44 (sp, A) 

105-52 89.46 77-51 

68.39 36.84 53-04 

115.29 42. io 53.04 

115.29 -47-36 44.88 

65-78 44.68 

)=, N ! SEM 93.09 9. il 56-31 ! 0.62 54-67 1 5-99 

A one vay analysis of variance was carried out on the resultse 

TAULS 5*4*3 Analysis of variance on the ruimbers of fungi obtained 

from the ALnqua comb of different Microtermes species. 

SGURCZ OF DEMEW, sm= OF MEAN VARIANCE 

VARIATION OF SQUARES SQUARES RATIO M 

Fnma)mi 

between 

Microtem2l appo 2 6057-33 30220-66 8.5600 

Error 12 4.,,., 44. oi 353e67 

FTOTAL 14 

J10301-33 

"T 

There Is a highly significant difference at the'l% level between the 

numbers of fungl obtained from the fungus combs of different Mcrotermes 

The levelas of significant difference betvieen the different combs are 

given belcme 

2180

Ilic 41 

MIC 43 

Hic 41 HIC 43 MIC 44 

HIC 1*4 NSD 

** a significant difference at 1% level of significance. 


differencee 

TABLE 5.4*4 Mwn number, of Termitomyces, colonies per g dry weight 

2190 

of different Hicrotemes, species funMw combs* Results of the dilution 

plate experiment on selective medium* (All numbers x, 104) 

HIC 41 Up. C) MIC 43 (spý 13) HIC 44 (up. A) 

12,5ýmO6 18.42 16*32 

54, - 72 31-57 34.68 

87-94 44-73 87-71 

117-25 7-89 IA. -4.8 

91-84 10.152 12 4 

MEAN SEM 95-36 * 12041 22.6) 6.89 33-05 1 14.24 

A one way analysis of variance was carried outs 

TADLE 5-4-5 AMIvals of variance on the ntuabers of TamitcxmLces 

colonies obtained from tho funTa combs of different Mcratermex" 

species. 



between 


EIREEDOM 

sum or MAN VARIANCE 

SQUARES SQUARES RATIO (F) 

Hicrotemas islips 2 15470.64 773502 11*48** 

Error 112 8085-93 673-113 

TOTAL 1 23556-57 

There Is a highly aignificant difference at the 1% level between the 

numbers of TermItMcen SPecles obtained from the fungU3 combs of 

different Hicrotermes gDecles,

The levels of significant difference between the different combs are 

given belowe 

HIC 41 

HIC 43 

HIC 41 HIC 43 MIC 44 

HIC " ** I USD 

"o a significant difference (0.1% level) 

** = significant difference (1% level) 

VSD a no significant 

difference 

TAnIZ 5A. 6 Fungi isolated from the fungus combs of three Microtermes 

species, Mean number of fungi per g dry weight of fung" comb* 

(All numbers x 104), On SF medium. 

2200 

SPECIES MIC 43 (op. 0), HIC 44 (op. A) 

Aal2ergillus flavus 3.16 

A. fumipatus 4*21 

A. niqer 3*16 

CP losR2rlm op, 1.58 '2.1*5 

Claftsporium ap, 0*82 

Curvularia up, 0-53 

Paacilomýcps varlotti 0.41 

_ 

Termitemyces 43.613 50.18 

aterile myceila 0082 

TOTAL NOo FUNGI 107 134 

TOTAL NO. Termitomyces 83 123 

TOTAL NO* OTHER FUNGI 24

TABIj3 5.4-7 Growth of T_ermIt2=cca from the faecal pellet of Hicroter"Ps 

major workers 

(on selective medium)* 

FORAGERS 

(SOIL) 

op. G 

FORAGIMS' 

(FWD) 

sp* G 

NURSU WO=MZ 

up. G 

h=ZB WOM= 

op. 11 

TermitgMLces 27 (90) 27 (93) 24 (83) 5 (71) 

other fungi 2 0 0 0 

bacteria 3 2 1 

nothing 2 3 1 

contaminated plate 0 1 0 

TOTAL NO* OP PIATES- 30 30 -1 30 -1 7 

1 = 29 = 29 

Isolation Index Isolation Index = no. colonies 

total -Termit=eFt 

now of plates 

X12 3.24 5% table valuet df (3) 

- 7.82. 

there is no significant difference In the number of TemitMces 

x 100 

colonies obtalned from the faecal pelletB of the different classes of 

major worker. 

TA13LE 5*4*8 Fungi Istolated from Hicrotertnes major worker faecal 

pellets 

(Day 5, SP medium). 

FORAGMS 

(SOIL) (G) 

NO* 

ISOIN. 

INDEX 

FORAMS 

(FOOD) (G) 

NO, 101 

NUILSE 

'LVIWXM 

(G) 

NO* 101 

WHISE 

woluum 

(R) 

221. 

NO* 1*1 

Termit2=es 13 43-3 18 60.0 14 51.9 JI 73-3 

3312iord Hus flavus 6 20.0 2 6&'7 

A, n1dulans 1 3-3 

4L.. ni&iýr. 2 6-7 

A* nuercinus 1 3.3 

Aspergillu§ sp. (GW) 1 3-3 

Clactomrium 

clados22rioides 

16-7 

Cunnin2hAmella, 

llusarium solani 

ape 

2 

3 '3 

6: 7 2 6-7 

7.4 1 6-7 

Nouroal. nra ape 2 6-7 3*7 

Pa ectiMces varlotil 30-0 6 20.0 

Ponfenlium citrimm 3-3'' 

P. 1 6*7

222o 

NUMB NURSE 

FORAGERS FORAGERS 

WORI =1 WORKERS 

(GOIL(G) (F OOD) (G) ' (G) (R) 

NO* 1010 NO* 161* NO, I010 NO* 101* 

Penicillium am. 22.0 

ýEceE! 

Ialnmtrum 

racemosum 1 6-7 

Trichoderma sp. 1 3-3 

sterile mycelium 

(WPP) 1 3-3 

Basidlomycote (WG) 2 6-7 

unidentified 

(V27) 2 6-7 

unidentified (WOl) 2 6-7 

bacteria 2 6.7 1 3-7 

ru)thing 1 3-3 

11 

3*3 3 

1 1100 2 113-3 

TOTAL NO* OF FUNGI 47 35 24 16 

TOTAL NO. ISOLN5* MADE; 30 30 

TOTAL NO* SPP& ISOIATED ILS a- 

5-4,3 DISCUSSION 

1 

I 

27 15 

The ftmgua combs of Mcrotermes species are covered vith mycelluml 

on both tho fresh and old zones, This observation ia borne out on 

comparing the Isolation Indices of Tem. jtýonnrces on both vaedia whero 

there in no difference in the isolation of Termitomyces from the two 

zonex 

(Table 5.4.1). The other fungi obtained In the Isolations are 

all fungi found in the &oil& and are probably brought In by the 

termites* The greatest number of fungal colonies and species were 

Isolated from comb of Hicrotermes gD,, R-9 which In found In river1ne 

forest at Rabba with vauch more moist Bolls richer in organic matter 

than those of the savannas and so a higher number of soil fungi would 

be expected* 

The use of thO 8016r-tiv4b medium h" again Increased the Isolation 

of Terrdt2=ex above that of the'OrdlMrY SP medium, reducing the 

number of other funCal species obtained from the leolations. Ilany 

Inolatimwo 

-on the selective medium aspeclallys produce no Termitomycog, 

1* 

-1

Indicating that not every part of tho comb contains viable Termit2mcex 

mycellum. Isolations were made'just In from the surface of the comb 

to try and avoid contamination and It may be that the growth of 

TormitoMron is concentrated on the outside of the comb where the 

mycellum and mycottetes are found, especially concentrated In small 

pockets, -on the comb surface* 

On comparing the mean numbers of fungal colonies per g dry weight 

obtained from combs of the three different Hicrotermen species 

significant differences In the numbers were obtained on SP medium 

(Tables 5-4-2 and 5-4-34 MIC 41 (op. 0 (90 x 104/g dry weight) had 

more ftmgal colonies than HIC 43 (op, U) (56 x le"19 dry weight) and 

Hic 44 (op. A) (55 x 104/g dry weight) (0*1% level of significAnce)e 

The combs were dug up In the latter half of AprI19 1977s HIC 41 from a 

Pit on Hilo I Colfcourne (30 cm depth)o HIC 43 from b6neath a' I 

TrInerviternes mound and HIC 44 from a pit on the t*rctite farm. HIC 44 

was very recently constructedl tit 30-40 cm depth* The majorLty of 

fungi obtained an these dilution plates were Termitomycen (owSF 

madium). This Is In contrast to Macrotemes ballicosus and Macrotermen 

subhyalinus uhere Lermltomyc_es was not obtained on the SP medium 

dilution plates* H. belliensun and M. subhyalinux fungus comb are In 

much larger volumes% and In stable cond1tion3 of temperature and 

moisture content which could favour the survival of other fungal 

species In contrast to the small, volumen of Hicraterme-sleomb which 

are subject to the Influence of variations In the soil conditions, 

HIC 41 had a much higher ra=ber of other soil fungi probably due to 

high couttaination as mentioned In the Introduction And'so was omitted 

frced Table 5.4.6. on mic 43 and mIc 44 an xPecles found are common 

In the solls of that area'(McDonald 1968,1970) (Table 5.4.6). 

223.

On the selective medium dilution plates the highest number of 

Tem. lt2=cps colonies. are found on HIC 41 (op. C) (9.5 x 104/g dry 

weight) coMmred to HIC 43 (ape B) (23 x 10ý/g dry weight) and HIC 44 

(ape A) (33 x 104/9 dry weight). During the start of the rainy season 

224. 

the termites begin to establish new fungus cotabs in the 0-50 cm horizon 

of the soil when they start their foraoinge During the dry season the 

termites cease foraging and feed on the fungus combs, mostly being 

found below 50 cm depthl (Vood and Johnson 1978)o Possibly, In HIC 44 

the mycot4tes were not fully developed on this newly constructed comb 

resulting in a lower number of' spores from which colonies In the 

dilution plates could originate* 

In Isolations from the faccal, pellets onto selective medlUm there 

was no significant difference between the species of termites In the 

numbers of Individuals carrying Termtt=cP9* The majority of faecal 

pellets when placed onto the fungus comb are already mixed with 

Tormit2=ces and this agrees with the similarities in Isolation from 

the old and fresh zones of the comb (Table 5*4.1). The presence of 

TermltTý, yces In the gut of those foragers which manipulate the soil 

suggests that all foragers must feed on Temlt2r . Mces beforo leaving 

the combo 

On plating faecal pellets onto SF medium a wide range of fungi 

Including Temitmycps was Isolated (Table 5.4.8). All the species 

obtained are common in Mokwa soils# and aro, similar to those obtained 

an comb dilution plates (Table 5.4.6). 

An expected the total number of fungi and also number of species 

was highest frca those carrying soil In thoir Cut, IDy contrast thq 

number carrying Tormtt2=es was lower than in foragers carrying food

and nurse workers* These factors once again suggesting the presence 

of these other fungi an the comb in as contaminants brought in by the 

termites* The number of species found and the total number of fungi 

decreases again from foragern with food in their Cut to nest workers* 

5*5 OTHER TMHITH SPECIES AND GENERAL DISCUSSIOll 

INTRODUCTION AND HLMIODS 

Isolations were made from the fungus comb of Odontotermes 

ameathr*ni (5 on SF medium$ 5 on selective medium)q and from the 

fungus comb of Ancistratermeos cavithorax (6 per medium from both fresh 

and older cwb) % and frcm the c=b of 11acratermen subhX21 nus per 

mediuM frca both fresh and older comb)* 

Ton isolations from the faccal pellets of foraging Ancistrotermos 

cavithorq2E major workers were made On selective medi=* 

5.5.2 nESULTS 

The results are given in Tableg 5,5,1 and 5*5o2o 

2250

ýo 9 

n er 

e 2: 

le 

0-ý 

n 

9 bm 2ý 03 t> 

c h 0 >O t+ ra 

9 

le ei 

to- q+ 

1 Ja 

4 b 

bi 11 

L2 Z 

bd M ta 

w. bo. 

So 0" 6- 

02 

b- b. 0 t, 3 ). * 

,9 th 1 

> >* 

pn 

i 

0 

g. u 

%0 m 

rA e u 

ID 0 

, W., 

be f+ Z 44, 

, 

ta w %i < 

NM 

25 

0- %n 

b. " 3 

- 4r 

t g 

o 

A fe 1ý f+ 

5 

7ý 

ä 

2 

tn 

, n,. - 6* 

g 

VI 

VI 

I 

R 

fa 

.Z 

Ow 

Cb 

cr 

tli

TADLS 5,5.2 Fungi cultured from the faacal pellets of Ancistrotermea 

cavithorax foracers on selectLvo medi=,, 

5,5,3 GENMAL DISCUSSION 

Noe of 

Termil2mXces 

Other ftngi 0 

Total no* 

IsolatIons made 10 

Those fungi obtained from the fungus combs of the different 

species are &119 with the exception of Xylarla co=wnly found in 

the Wma soils. 

Termit=cen, vas Isolated from both fresh and older Ancistrotermes, 

coch (Tablo 5*5*0% and In carrled*by the foragern (Table SoS*U)e This 

would suggest that when the faccal pellets are placed on the cc=b they 

are already mixed vith Termit2=os, 

The results Insections 5*2 to 5*5 indicate that different termite 

species have different strategies of Incorporating Termitomyces Into 

the food material* In Macrotermen belllcosus only a very stuall 

percentage of foragerm carry TemjtZMLce_js. The foraged food Its then 

placed In the foodutore where there in no Termitomyce_9 present* The 

mixing of TPrm1t=v-iq and the food then either occurs on transfer of 

the food from foodstore to comb* or by growth Into the freshly ,' 

deposited material of Termlt=Xces, myýelium; from the older sections 

of the combo The latter Is the most -likely an Termlt=ces In present 

in far Greater amounts1n. the middle-comb zoneq as shown by Isolations 

ind dilution plate experimentso Grass-b, (1937)'Also 

6 

- found 'only a wall 

2-27* 

ncKxmt 'of debris vhich appeared to'be urcelium or corildia of Termit es 

ý_ - 2M 

r

In the dloostive system of H, bellicosus woricers, 

Macrotermes'subhnllnus would appear to have a similar strategyj 

apart from the absence of foodstorel with very little carriage of 

Termit2=cps by the foragerse Rohrmann (1978) found fresh comb of 

1kcrotermon uk-uxii lacked the hyphae of older co=b ltuUcating a similar 

process here* Grasse (1937) also found no mycellum or spores In the 

intestines of Proternes n1mitus'vorkerso 

In contrast the Hicrotermes species carry Termitozryces In their 

guts and the faecAl pelletal when placod- on tho combs are already 

mixed with Termitopyces. On both selective and SP media there was no 

difference In the 180lAti0n of Termit2MXcpa from the f resh and old comb 

zones* With the Mcrotermes combs being small units In pockets In the 

soil, more isolated from Other COMbs than am those of Macrotermen 

bellicosus with Its centralized fungus comb in the nest, it in 

nec4wsary for TermitEMces to be : introduced Into the food material by 

the termites an It has M POSAIlbilItY of colonining fresh material 

other combs as may occur In M. bellicosus- and other iqacr0t"rn0, g, 

Ancistrotemes cavithorax, appears to be similar to Hicrotermes with 

foragers. carrying Termit2ace3. 

228. 

. from 

An well as tho interspecific differences In carriage of Termit Cos 

there are also differences between castese In K. ýelllcqqux 

the young 

workers have a higher percentage of Termit2=ez in the out than nurse 

workers, perhaps Indicating feeding differences* Other authors have 

also found differences* Doflein (1906) found the crops of all larvae 

and nymphs of 0dontatermas obscurleeps were filled with Temtt=cts, 

but not the workers-* Spores of TemLIMces have bo6n'recorded from 

the Cuts of 0dontoteMes obesus workers Was 2. t al. 1962)j large hymphxt

soldiers iao major and 61nor workers' (Ba"tra''wW Datru 1979). In 

Cklontotern, Ps 'gurdalgrAwensis Tem. it2=ces opores have been found in 

small and large nymphs, and minor woricers, and in Hicratermeal in small 

and large nymphs and minor workers Matra and natra 1979). Batra and 

batra (1979) also found some minorworkers of Odontoternes obeffus with 

only 

; wood and no Ternit=ces In the gut., If these were foragers it 

would Indicate a strategy similar to that of Macrotermes bellicosuse 

Yet elsewhere they said that almost 80%, of actively foraging workers 

had abundantlermtj2Mcea conidia in the guts indicating a strategy 

similar to that of Hicratern". 

* 14)hrmann and Roz=an (1930) found 

conidia in the Cuts of major workers, major and minor soldiers and 

nymphs of ftacroternes Wcuzilo 

These differences Infeeding between castes may relate to'the 

conservation of nutrients in the termite-rungus system, particularly 

nitrogen 

(Chapter 7). 

The results in sections 592 to 5*!; have shown that the temites 

bring into the nest laýce numbers of spores of fungi other than 

Termlt=cese This has also been reported for other termite species, 

Daz et, ale (1963) found Axnernillus opp., 6 Curvularift op.,, Fusaritim spo, 

lzhodotorula op* and Torula op, from workers of 0. obeau!. l 

three being genera commonly obtained from the termites 

the first 

_ investigated 

here* They also obtained many flungl from the combq and FýAch (1906) 

recorded spores a3 being present in the combo of Odontotermes redemanni. 

In 0. obesupo, other species of fungi have been recorded In', the gut of 

workers and soldiers (Bose 1923), j and minor workers (Datra 

and natra 

1979)- RaJgopal ot al. - (1979) obtained Cunninghamelial Penicill1im. 

Fusarium and AstraMillus species from guts of workers of 0, obesuBg 

229, 

,; --- T 

i

and Singh, Ungh and Singh (ITIO) ob. tained WLny species especially 

Axj! e! MiIIu. q frow the exterior of this species and Oe redemannie Othor 

fungi have been rocorded from the gut of Hicrotermes obest (Uose 1923)9 

minor workers of Hicrotermen Watra and Datra, 1979) and from the fungus 

combt nest material and worker Cuts of Odontotermes M! rda9rAirPnsix 

(Datra, 

and Batra 1966). 

5.6 nx OWTII CP SPECI M- WIM ILIL&N TMMTWIYCF. S 

5.6.1 INCUBATION MWEnOWTS 

Introduction 

When the fungus comb is first re=ved from the mound the only 

vImLAI evidence of tungi In that of the mycollum and crycot'Utes of 

Termit=cpsp No fungi can be seen growing In the foodstore or on 

the nest structure* Soon after remval from the*mounds mycelial growth 

occurs and the c(=b to soon enveloped by these other fungi* Termitomyces 

being no longer visible. 

In order to determine which fungi grow an the comb and to follow 

the development and succeasion of these f4hol a series of Incubation 

experiments wero set up. 

Methods 

Plastic-pots* with filter paper fixed to the Inside of a foil Ildl 

were sterilized. Fungus combs were placed In these, and A h1oh h=idity 

maintained by wetting the filter paper, and incubated at 141.90c. The 

fungus combs were checked regularly and cultures made from the : ftmgi 

developing,, 

2-. 

30*

R=rIments I and 2s Combsýfrcc 3 different HlcrotomeS speclex 

and comb frc= !; geparate--Macrotercma bellicosup mounds were Incubated* 

determine$ 

ýE2ericicatjs A separate experimcnt was set up to trY to 

22 31 

(1) the effects of the presence of large numbers of termites on 

the development of fungi on the combo Soldlerst workers and larvae 

were addod to pots ccntaining combo 

(2) the effect of washing the cozab in the comb washing appar-atus 

(5.8) on the development of fungi on the comb. Comb wax broken. up and 

washed for 25 x2 minute washes* 

the Pattern of fungal gro'wt'h -thst might on cur on nest structure 

and foodstore kept under Identical conditims, 

QcMriment 4: Six POU of fOOdstare were placed over water In a 

desiccator and kept for a year. The foodstore was then exAmined and 

5 frag=nts from each pot plated out onto SF medium. 

&j=rlm. cnt-, 5 It has been reported that ZýLlarla mycallum In 

antagonistic to Termlt2aces (116iM 19,52a). In order to Inventloate, 

this further 10 potri dishes of SF medium had plugs of Ularla and 

of Temit2Mces placed on them equidistant from the centre of the 

petri disho 10 potri, dishes had only ? Cylaria and In 10 petri dishes 

the Temi LaM. ces was Cr%wn for 4 weeks and tho, &larin was then placed 

on the plates. The growth of the fungi was recorded after 2 and 4 

days* 

Results 

The results are given In Tables 5.6,1 to

Table 5-6-1 Fungi developing on Incubated Amgus combs of 3 

Mcrotermos sTmciex. Llvperiment 1. 

NUMER 

OF DAYS HICROTMMS spo C MCROTMM sp., B HICHOTMIMS ZD* R 

INCU13ATION 

2-3 daYs 

, 

comb covered In conidial heads ccmb covered in 

sliGht growth of of Aspergillus mycelium 

vhite mycelium V! jLer visible 

5 days an day 2 A* niger still Increase In orowth 

presents of mycelium 

Mycelium an 

present in up* C 

developed 

days 7.14 as day 2 as day 5 day 5 

Fungi koniper A. niger A. nlUer 

cultured Rhizopus spo RhIzolMs mtolonifer 

from C4=b Paecilamyces varlotil Trichoderma spe 

23no

In Im 

Co. 0 

C6 

00. 

0 

. 3 

ti 0 

:r 

0 

,l 

g- 

, 

w 

4 to 

to. 0 

--j 

5 

o. k 4, 

o 

ba 1 9.3 10 

0" 01 

0) 4.4 

60 'M 

R " 1, 

0 i 

to 

a s0 

" 

1111105 

0 00. 10 

Co. 

2 

%A to 

0 0 

a 

W. 

flo- m 0 0.0 f l ow 

vc YA cr so 03 

f+ 

a 

F n 

a 

; 

. 

to 

-a V- 0 

i 

1- 

IA 

4+ 

1 0 

6d. 

I 

0T 

t9 5 a% 

wv 

1 

I' w $1 

loft 

1 

1 8 

. 7 

if 

5 $. -1 4 D ;) *NI 

0. % 0" 

1- w 

1. - :5 Q. 00 

Oft 1.4 

" 14 04 01 00 10 

P#. 

t *9 S 4- 5 

b-4 

0 

0M & i to 19 19 

0ý 

0 to 

i' ' 

1: 6 

CD ,& tr 

I 

F- Q 

L% m 0- a I x 

1 

-- 

$A- 

01 

In .0Wn 

0 

0 

loft 

4 1 ;4 g :3- ' : 0 1 1 11) 

- . 

a 1.6 

. 

1" 0 

cr I 

4 HI 

0 

cr 13 

- 

ca 

m- 

i" 

R 

tm 

IN ' I 

, 

in. 0 1 ", nt0* 0 ". 0" :0 6" Oft I-## 

?A 1 

0-0 04. 

. 

-, 1 

ID C, 

g 

s 

233. 

S 

S 

CO 

$. 

ft 

I 

tr 

04- 

g 

p

4 

cr ic 

%-C " loo 

R. 

V 

Put 

f+ 

0 OV P. 

oft es, 

ti 

0 

K 

04 

--I- -- -- 

q 

*1 

4 

ii 

lod. 

0 

.w 

C6 

I 

rA 

co C3 

C3 C3 

Sp 

rf 

1 4 

. 

n l< 

0- 

f+ 0W 

234.

I 

" 

1.0 

M 

0- O-d ý. n 

ca 0) 1 0 *0 

m 

1-4 : 0. >e 

0, 

. 7%4 0- V :3 

V. 0m olft 

:ý 

ý139 >0 m C) 

14 

t* M 

11350 

V-. 

:$R, M ob. 5 04. 4 9 

0 ý-* " t) I 00 () 1 , 

01 0 

C) 

o W. 

'ri 2 1 ý- , f) B M 

44 06 

1 

7 

lei 1" 

I *a 1-4 a 0- x 

6-d 

n M. 

o 

. 

to a 

re. 

40 a0 

4 

ta 

I 

"D 

9 

a0 

6- t 1. 0 

P-* (0 

1 0.3 

1+ 6. j 0 in 

I& ft 

el. C6 6- 

f+ I ?A 

0 

P- 

ILI* 

2 two 

6 0 

0 

rr 

1 - 

at 

-4 0 1,0 x 

-1 

g0 ; 

t-4 I. - 

&mý. O. 

to 0. 

ý; i , 

O-d 

61. 

# 

:, 

r+ " 1-d F so. 

0 

0 

ell 

1 

C+ 

a s I. I. 

9- 

I'D 

aF9 "1 8 

0 

n t4 

V 

n 090 

t+ 

f+

TABLE 596A Foodstore afterý one years incubation. 

APPEARANCE OF 

FOODSTORD 

NMMM OF POTS 

Wet soil-liko mass 2 

Drier, with more 

obvious foodstore 3 

structure 

As original foodstoro. I 

A little growth of 

web-like mycelium 

TAnLU 5*6o5 Fungi obtained from foodstore Incubated for one year 

(on SF medium)* No* of fragments Plated = 35* 

F=GUS 

140. or, 

CULTU=- 

ISOULTION 

IND= 

Trichodarm virlde 9 2-5-7 

As raillus niller a 22,9 

Fusarim app. 7 '22.000 

Paecilmyces varictii 11.4 

Peniclillms funiculosum 11.4 

AspeE211lus flavus 2 5-7 

nothing 1 2.9 

TOTAL NO. FRArAZMS 

PLATED 

35 

236.

FIGURE 5.6.1. Conidia bearing stroma of Xylaria, sp. from 

Macrotermes bellicosus fungus comb. 

FIGURE 5.. 6.2.. Pod-shaped stroma from Macrotermes belliconus 

fungus combo 

., bd. %. * 

.%, 

p 

AA 

237* 

SCALE X 50 

SCALE X 125

1 

FIGURE 5.6-3. Fungi developing on incubated Macrotermes bellicosus 

fungus comb, Mac 47, daY 5-, (1) Trichoderma up., 

(2) CeRhalosparium ap,, (3) pod-shaped stroma. 

2 

SCALE X3 

SCALE X 1- 5 

FIGURE 5.6.4. Fungi developing on incubated Macrotermes bellicosug fungUS 

comb, mac 48, day 2. (1) White Conidia-bearing stroma of 

Xylarin. (2) mycoteteag (3) fresh edge of funVe comb, (4) 

red/brown soil patch 

3 

3

A 

B 

.. x-0- 

9 

Xylaria 

*)Z* 

., . 

40 

X Xylaria inoculation point 

Termitomyces 

/ 

.x 

I 

. x* 

(. :? 

239s 

FIGURE 5.6*5. Growth of &IsLria spo after 2 daya incubcLtion on SF medium platen 

A, XýftLri_ft only* B* X)ajaaL and 4 wee-c old colony of Termitomyces.

A 

. 

- X. 

'.. 

p 

". 2 

Xylaria 

. x. 

K ". ) 

" 

0: 

X Xylaria inoculation point 

.IX. -0.. 

Termitomyces 

X, 0... 

FIGURE 5,6*6* Growth of Maria, ep* after 5 days incubation on SF cedium 

platens At ILlaria only* B. &ýria and 4 weak old colony 

of Termitncee* 

PAO

PLATE 5ýý6.2. 

Incubated Macrotermes bellicosus fungus comb 

showing conidia-bearing stroma. of Xylaria 

PLATE 5.6.1. 

Incubated Macrotermes bellicosus fungus comb 

showing rhizomorphs of Xylaria Day 2. 

241.

N 

p 

ir

PLATE 

Incubated Macrotermen bellicosus fungus comb showing 

pod-shaped stroma. 

'PtATE 5.6.4. 

Blackened rhizomorphs of Aylaria on incubated 

Macrotermen bellicopus fungus comb. Day 10. 

242.

PLATE 5.6.5. 

Soil plastered over incubated Macrotermes bellicosus 

fungus comb by termites, 

243*

I 1;;; 

I

Discussion 

There was very little fungal growth on tho Mcroterme-a fungus 

combs when compared to the Macrotermes bellicasus funaus combs 

(Tables 5-6-1 and 5*6.2). The main fungi Crowing on the Hicrotarmns 

combs were those coc=nly brought In by the temItes with the foods 

and which do not dovelop until the comb in removed from the natural 

habitat, No growth of, Ularia rhizomorphs or conidia-boaring atroma 

was recorded from any 111crotermas fungus combal or any Xylaria 

cultures obtained from theme One problem with the Mcrotermen fungus 

combs was caused by their small size resulting in them being subject 

to more rapid desiccation than Me bellicosus fungus combs thus 

Inhibiting fungal Growtho 

The general devolopwnt of fungi on M. bellico'nui3 fungus combs 

can be described as follows* 

After "I days the combs are covered by vhite alarla mycellume 

A few loose agglomerations of tho mycellur4 occur whicli fom stnx= 

which develop an columns ft the combs surfaceg reaching approximately 

2 cm In height by day 2, Theso are rhizomorphs (Plate 5.6-I)e 

Approximately half the Incubated combs produce thin upright stroma of 

&Ietrias Initially whitel with a powdery surface caused by the 

production of con1dia'(Figg. 5*6,1,5.6.4 and Plate 5.6e2)* Although 

the mycotetes of Termitcay-cem aire obvious when the comb In placed In 

the pott these do not develop and have disappeared by day 5. This 

has also been reported by Patch (1906) and 11alm (1952a), 

Dy day 5 the rhizomorplis and conidia-boaring sttvm of XXIarin 

are beginnifig to' ýIaclccý 

244. 

: from the base up., Between days 5 and 10 dark 

brown pod-shaped strom may appear on approximately half of thg fungus

cmbs 

(Fig3. %6.2v 5.6.3, Plate SoG-3)- Small yellow patches of 

CeRhAlasga2rimm occurred 

(Fig- 5.6.3), and mycelium and sporing 

i3tructures of Trichoderma apt Aspernillus niner, A* flavus, Penicillium 

sp. and Phizopus stolanifer were seen and isolated* Tho d4rkening of 

the XXIaria rhizomorphis continued* and small dropa of, browny liquid 

appeared an tile old rhizomorphas 

day 10 the rhiz=orphs of Xylaria were almost completely black 

(Plate 5.6.4). There was a brittle black outer layer but the 1pner 

ýty; eliurj rem4ined. white. IMOF rhizOmOrPhs may also develop at this 

stage*, 

By 14 days the fungus comb was fragile and partially collApsOd 

and nolfurther developoent of other fUngi occurred, 

T his development of alaria also occurs 'on Incubatiid Itacrater7nes, 

PublInlinus fungus CCCabs. A similar development of'XXIaria niSrIMs 

wais reported on 6dontotermes 

2AC; 

, 

obesus combs by Doxe (1923), The development 

of XXIaria from abandoned fungus combs or from fungus comb removed frc= 

the nest has bepn reported for many years from mzmerous parts of t" 

world$ ASSOCiAted, with cany different termite species* From Africa$ 

AlAzoAdurct (19GO Coaton '(1961), Domis, (19.58) l lDixon (1965, ),, 

s Ifelm 

(1942av lWat 1977)v Ptleno (1964) and nchrmmm and IROB&'Mn (1980)- 

From Indias Bakahi (1951), Datra and natra (1966), Bose (19, z3) and 

Das ei al. 

(106"Oo From Chinas Cheo (1948)o rrom'Ceyl'ons Berkeley 

and Broome (1875)9 Wflein (190G) and Petch (19069 '1913b)o Pr(= Indo- 

China$ batthelfer 

(19, '. 

)7)* 

Uxny synonyms havo been used but thO OPeCles of Xylarla associated 

with termites appear to be$-

(1) xylaria nIqri]2ex Known ýrm Africaq Indial, Ceylon, 

Indonesia mind the PhL111piness (Dakshl 19519 Dose 19239 Dannja 

1958s Ifelm 1977)9 

Mar authors have also recorded the presence of 2 different 

forms of growth of X. nitirij! ex frcm incubated combs (Patch 1906i 1913b)o 

(a) Loosoly structured sterile rhizomorphso On Isolation the 

cultures also produce such rhizomorphze T'he general process of 

development appears to follow the same pattern wherever recorded* 

The rhizo=rphs develop as loose white colums from the comb after'2 

days incubation. Dy I weelt much of the rhizomorph Is blackened amA 

hard, p and after 3-4 weeks It has diede 

(b) Conidla-bearing stroma. * These are small thin filamental 

which may branchq covered In a white powdery coating of conidial 

gradually blackening from the base up, On Isolation these give a 

slow growing colony which produces small stroma up to 0,5 em In 

height. 

PQtch (. 1906* 1913b) cAmcluded that the production of the different 

f0rW was dePondant on the amount of moisture present In-the combo 

drier combs Producing the 2nd type* In these experiments some fungus 

combs produced both typen, 

Other forms of &Irtria, are produced when the comb In ab=%doned, In 

the nest by the termiteso These are the conidial forms ascosporous 

form and sclerotial form* Due to the saultiplicity of forms of 

X. niorlpen 2 species of Ularia (X,, 

_nlgrlj!! 

L9 Wid X*- furca_ta) were 

sometimea considered to be pregent in one : fungu COMb, (Petch 1906, 

21*6.

1913b)s but 11olm (1977) ronsiders X* furcata to bo a synonym of 

X. njUj: j2ej%. Done (1923) found X. ninripps assumed the form furcata 

(a more branching form of n1gripea)-l It cultivated'a long time In a 

moist-atmosphere., 

(2) XXIarla brasillenniso Known frca Africal and algic froal a 

rsmall area in Brazil (Demis 1956), In Brazil* although recorded an 

associated with temite nests* It ww1d not be found on fungus combs 

as the tiacroten: dtinae are not present In the Americas. 

alarla thXEsus Kwwn from Indial Java and ono specimen - 

from Mauritius (Demis 1958). 

247. 

Xylarin obovata* A rare obligate saprophyto on fungu3 comba 

from the Qmgo Oleim 195.1.1a), 

Other species of fungi have also been recorded developing from 

the fungu3 comb on Its removal from the mound, Patch (1906) found 

the comb surface of Odontotermes redm. anni wan ovor-run by species 

of Mcor and TImmidium,, 

appearing after 5 days& The 

mycelium end an ascocarp of Pezi7. a opipr2rtla eM small Patches Of 

CophaloxMrJun, 

-also -devoloped on the ccakbe after 4 days* flucor, and 

Penicillium, developed on fungus combs Incubated by lioltermann (1900)o 

On Incubating cocibs In the absence of Odontotermes purdaMronsim 

workers and soldiers Batra. and Datra, (1966) obtained Cunn-In2Lmm, 11a, 

Trichodermal and Naoskofitzin as well as Xylarla ManY micr(mycates 

including MUcors Thamnidlum, Cer4mloaMrium and Axpe illus app, were 

found to develop on combs by Heim (1932a). Rohrmann and Rossman 

(1930) obtained -a variety of fungi on Incubating Macratermon ukuzii 

fungus ccmb. These included Penicillium DPP-9 Zr. 1chOderma- vi ride,,

Sordarin f1micala and Epicoccum nip=. s all of which they considered 

to be preamt there accidentally* In ocneral the type of Amoal 

develolment on the combs aareas with that of Macroterries bellicogus 

fungus combo 

two types* 

11-te species developing'an the Incubatod embs appear to be of 

(1) Those carried In by the termites both on their bodiesq and 

in the collected food* e. g. Anl2e. Millus app,, Cunninphanalla, 

Penicillitiml TrIchoder= (Sections 5&2*2 and 5,2,4), These exist an 

ungerminatino spores until the fungus comb is removed from the nest 

or abandoned by the terviltes. 

(2) XXIarla which Is mported to exist as nycellum in the ftmgus 

c=b (Grasse' 19374 but which does not produce spores or stroma until 

the ccmb Is removed from the nest or the nest abandoned* X. niariges 

would appear to be an obligate saprophyto as It has not been recorded 

other than associated with termite nests. The evidence for 2EXIaria 

existing as MYcGl1UM appears scantyo 

The full range or fungal species associated with termites will 

be diE; cusjie4,, later (5.6., ". ). 

The Presence of termites on 'the fungus comb suppressed the growth 

of fungi other than TarmliSMýcea for ever 2 days In conirast to the 

controls 

(Table 5-6-3)- The'tenaites plastered a toil covering over 

part of I 

the fungus cmb (Plate 5.6.5). Batra and Datra USAO also 

found the pre3ence of termites on the Incubated comb retarded the 

dcvelcn=ent of Ularla from I to 5 days as the wOrkers pla3tered the 

'? sprouts" with goil* Thin retarftng of grvwth due to termites was 

248 *

also reported for Odontotermn-s obanus 

(Dose 19,23). As XXIaria 

develops through the soil from abandoned ftnýnw combs it is evidently 

not the presence of soil alone that Inhibits Its growth and a 

contributory factor may be the saliva the termites use to moisten and 

bind the soil. The plAstering of soil to block themselves in In an 

automatic response of the termites when their habitat is disturbed so 

the suppression of the fungi may be an Incidental result. of t4in 

behaviour, although Batra and Datra, (1%6) report, that if a petri. dish 

containing a culture of Cunninahamella is placed in a neat ch=bcr the 

workers plaster it with moist soil killing the fungus* 

When the fungus comb was washed the growth'of fungi was very 

greatly reduced, whereas on just breWdng up the comb the development 

of funot was normal. One of the factors that may be responsible Is 

the high moisture content after washing. Another possibility Is that 

If many of the fungi werx) present as spores their population would be 

highly'reduced by the washing process (!;. 8). 0 

No Growth Of ftnoi was observed on the nest structure and very 

little Growth occurred an the foodstore. The latter Is surprising an 

the foodstore, consists of damp woody material which would be expected 

to be an Ideal zmadium for fungal growth. It also contains a far higher 

number of spores than the fungus comb (Table 5-2-7). This suggests 

that some factor In the termites saliva ci&y ba preventing the growth 

of those other fungi cis the foodstore would have been recently 

manipulated by the termLtest its duration as foodstore being approximately 

1 week (Collins 1977)- 

249o

The appcarance of the foodstore after one years incubation Is 

shown in Table 5&6*4* The fungi obtained from plating out (Table 

5*6,5) are the sametpecies as were originally present (Table 5*2*9) 

and evidently the spores remined viable although them-was little 

sign of fungal growth* 

ý TermitgM2. Fij, when it has grown an the plates appears to-havd 

some retarding effect on the growth of Xylaria (Figs, 5*64 and 5.6.6)., 

Thin could be Interpreted an antagonism or competition for nutrients* 

On the plates on which plugs of Xylarin and TermitgMes wore placed 

at the same times the TerTait2r ow 

.! Xces had no effect on the gr th of 

Xylarias At day 2 there was slight growth of the Termitowyýqenj growth 

of Ularin being as on the XXIaria only plates, By day 4 the Xylaria 

had grown completely over the Termitonneso The presence of actively 

growing Termitomyces in the fungus comb may be one of the factors 

helping to prevent the development of Xyjarin when the fungus comb 

is in an active nest* 

It can be seen that the foodstore and fungus comb contain spores 

and that the fungus comb In potentially able to support the growth of 

these sporestas well an myceliu= of Xylaria Yet no development of 

fungi, other than-Tarmftg2ycesitýcccurz In an active nest. There must 

therefore be some factor* or, fActorsj associated wIt1V the presence of 

the termites In the nest, which prevents1he gratth-of these fungis 

Investigatimw Into this problem are reported in the next, nection 

(5-7)- 

250-

5.6.2 ORQNISHS ASSOCIATED WITH MIE NESTS OF FU14GUS-GMWING TMMTLS 

- Many different types of organisms arefound associated with 

termites, p4including 

the ýhcrotermltinae)j living within their nests 

and in some cases specifically associated with the fungtts comb&'- 

The physical protection and relatively controlled environmental 

conditions found in tho mounds attract many animalst mostly Insects, 

to spend part or all of their lives In association with termites* and 

these are known an termitophiles (14a and Wood 197JL)- nePtilest 

mar=aln and birds also live in termite'tunuids or use them an nest 

siteng and species of parasites (ftmg1t protozoal nematodest mites and 

insects) and predators (especially ants) are also found there., 

At Wwa the foodstores of the Hacrotemes bellicosus mounds 

investioated were found In particular to contain nematodes and larvae 

of the dipteran Tricyclea deepln2i Zx=pt. Mites were present on the 

fungus combso 

The degree of-association between fungi and the Macrotermitinae 

in variable and an attempt Ix made here to classify those relationships$- 

(1) Mutuallstic and obligate symblontse TermltomXcex species 

grow on the fungus combs In InhAbited nests* They may also be 

carried as conidia In the gut of workersand alates In some termite 

species 

(692)9 Fruit bodies develop either by punhing through the 

soil or nest, from the fungus comb to the surface (a, go Ancintrotem, ell 

app)l or by 4developing from fungus comb scattered on the surface by 

the termites (e9go Odontotermes BPPe)- 

(2) Parasites. - Metarhizium ftd! onliae Varo anisonliag Uas found 

on Macratermas- subhyallnus alates. Parasiten 

251, 

are found associated with

all species of termitest and Smay be azoociated with other Insectso 

They may be endoparanites 

or ectoparasites 

1952al and Coroomycetop2is ofadiMs 

(e. go Termitari_aq Dakshi 1962t 1181in 1952a) 

(e. go Antennol! nio on Reticulitermes flavip",, Heim 

Obligate saprophytess 

(Blackwell and Kimbrough 1976)s 

(a) on the fungus combs of the Macroteraitinae. Greatest 

development including fruiting occurs after the nest Is abandoned by 

the termites* 

Ascomycotina Pyrenomycetes Sphaeriales 

conus Ularin, (5.6.1) 

Genus Nvosicotitzia 

Hypocreales 

N. termttum (Datra and Datra 1966, Odontotermes n_prdsisZ! re. nstsjj 

Indial Ifelm 1952aj 1977; Petch 1913b, Odantotermes redem. annt, Ceylon)* 

Genus PP7. iza 

Poerlaj! Artla 

Discomycetes Pezizales 

(Coaton 1.963L, Odontotermem badlu_z, Macrotemes 

subhyallnum, South AfrIcal HoLm 1952a, 1977, Indial Potrlx 1906,1913be 

Odontotermfus redemnnt', Ceylon). 

(b) on the earthen supports of the nest 

Basidiomycotina Gasteromycotes Hymonogastrales 

Genus Podaxon 

Madagascar). 

P. tormitophillial(Heim 195249 1977 Madagascar; Petch 1913b, 

Facultative isaprophytes, 

(a) on the soil lining of the chambers in tho ne3t. 

naeldlomyo-otina Gaisteromyceten llymonogantrale3 

Gems Protubem 

Pe. termitum Oleim 19779 Central Africa). 

252 *

There atre many other fungus species found grclmlng on the casing 

of nVatV, of M=y different species of termites,, which may or may not 

include members of the Macrotermitinae. 

(b) on the earthen supports of the nest, (after Ileim 1977)- 

HymenomYCetes 

iUenUgý Louccmoprinus sp. OICIM 1952a) 

L. - madecassensla 

Genua, Le2iota oppe 

Agaricales 

(Ifelm 1977 Mladagascar) 

grasnei Oleim 1952a, 1977 West Africa) 

. L* ivorlensis (fleim 19779 Ivory Coast) 

L. termitopýila (Ileim 1977, Central Africa) 

4, mip linrnsmius 

ri. palicitinensis Oleim 1952a, 1977,111crocc-zotermes Ivory Coast, 

Conao) 

Genua gMhalia 

Gema Psalliata 

P. cam2estris (Heim 1952a) 

P. termitum Olelm 1977) 

(lielm 1977v Ila"uaacar) 

Genus Doletua a Xerocomus noletochaete 

Ale- luteocyatim (Ilelm 1952ai 1977 Madagascar) 

Genus BOVISta 

L. tormitm 

Genus'G; =ýhraMduii 

G. delilel 

Gasteromycetei3 

(1 1. 

leim 1 

197 1 I 

7, Ce ntral 

tycoperdales 

ifrica) 

Hymenogastrales 

OICIM 1952as 1977 Mdagascar) 

Genus PodAxon I 

P. carclimmlim 

(Berkeleys Australia, cited by Petch 1913bi 

253.

loottbmley mul Fuller 1921, Trinervitemps sp., South Africa% Heim 

1952al 1977 fladagascarl Helm 194D Trlnp. rvitcýrtrzes trinervius). 

P. pistillaris 

(Bottomley and Fuller 1921, Trinervitermes spef, 

South Africa; Heim 1940 9 Trink-rvitermes trinervius; Alazoadura 1906 

i 

Nigeria)* Podaxon species are also found associated with Trinervitermes 

geminatus Coarctotermes, and Tumulitermen in Africa% India, # Ceylon, 

Australia, Mdagascar and Malaysia (Sands 1969)e 

(c) other species are found accidentally growino In woody 

debris included in the earth of nests* 

Basidiomycotina flymenomyceten Aphyllophorales 

Genus Ganoderma (Heim 1952a, Cameroons) 

G. curtinii Oleim 19771 Madagascar) 

Of course many other Imer fungi will be Included in the material 

makinf) UP-the nest Structurot foodatore 6xA f%mgus comb (Tables 5.2*99 

5*3-. 3). These may develop when the nest is abandoned as has been 

previously discussed (cog* Ax22MIllus, 

t Penicillitan and TrichodermaLk 

2540 

Bacteria are also found In ummda of the tiacrotemitinaeo HeLkaljohn 

(1965), Investigating Vacrotermen, mounds In Rhodesia fo und cbllulose 

decomposers to be very much tmore abundant than In tho s6119 with' 

denitrifying bdcteila (Pneudomon., ts., Denitrobacillus) -ýnd'a=Oniflerjs 

also Very abUndants Thiv greater'nbundanco may be due to the use of 

saliva as a cementing m4uUurs thus 'contributing organic matter. Boyer 

(1956) found 20-50% more cellulolytic aerobic flora In the mounds of 

Macrotermen belliconus than In the soile The bacteria are present 

both as inactive spores and actively feeding on the large qUantLtjes 

of organic matter found in the nests* Some bacterial cells are 

probably digested when wound material in re-Ingested during reconstruction

and this may-help to keep down theii growth. This greater abund I 

ance 

of'fungi and'of denitrifying bacteria indicates that the breakdown of 

organic matter proceeds faster in mounds than In the surrounding area 

This contrasts with the carton mounds of Nasutitermes exitlosuFg of 

Australia with an Impoverished flora (Lee and Wood 1971), The 

depression of microbial activity in the wounds of this species In 

dueAo-the carton material* 

5.7 PREVENTION OF SPORE GM'JtINATION IN WE NEST SYSM 

The fungus comb and toodstare contain many spores which are 

potentially capable of aerrdnating 

255. 

(5,, 2*9). Those In the fungus comb 

do not germinato until the comb is removed from the nest system, while,, 

when the foodstoria Is"reinoved, the spores still do not germinate. This 

points to there being factors In the nest system which prevent germina- 

tion and Grawthj and which are still operative when the foodstore to 

removed from the iaound. 

One 'Of thO fACtOrS which appeared to retard the growth of the 

fungi was the presence of the termiteng an the plastering of Boll on 

the comb (5.6.1) 

prevented the Initial development of the funglo 

Experiments to Investigate whether this may be due to tho Incorporation 

of saliva in with the soil are described In 5.7*1. The effect of 

extracts in diffekent solventst of foodstores, fungus comb and termites 

on spore germination are described In !; -7,, 2* 

5-7-1 EFFECT OF SALIVA IN SOIL ON SFO, 'M GMUNATION 

Methods 

Part of a Macrotermox bellicosus Mound Wa3 damaged and the following 

day the freshly worked soil j which the termites had used to repair tho 

damaged SeCtions WaB C011OCtOd In A 6t6r1lQ Pot* Older worked soil

from the wound and unworked soil from the vicinity of the mound were 

also collected, 

A dilution plate e=perim'Onts as described in 5.1.4, was carried, 

out on the three soils to find the numbers of fungi germinating from 

each. There were 10 replicate plates for eachaoile 

Results 

The result3 are ishown In Tablems 5-7-1 - 5*7&3,, 

TAMX 5.. 7.1 Mean number of fungal colonies per 9 dry weight of soil 

freshly worked by termites, older worked coil and unworlsed soil* 

Results of the dilution plate experiment on SF madiumg day 5e (All 

numbers x 103)e 

UNWCMUZ) SOIL FPX-%ILY WOMED OLD W0111= SOIL 

MEAN SEN MMI ! SEM IMAN SEM 

57-45 5.95 

1 --- - --- 

I 

39-44 1 1.51 

17-78 1-99 

A one-way analysis of Variance was carried out on the results of the 

Individual plates. 

TABLZ 5.7.2, Analysis of variance on the numbers of fungal colonies 

devoloping from the different soils, 

SOL =lE Or 


DEGRM- S OF 

MU=)%i 

smr; OF 

SQUARWI 

I 

--- 

MAN 

SQUAnES 

I 

256* 

VARIANCE 

RATIO 

Between solls 2 7889*62 3944-81 28,04rom 

Error 27 3743.24 133,64 

TOTAL 29 11632 

*86

UNWOMM SOIL 

FRMILY Wordcm 

OLD WOMW 

-= significant difference (1% level) 

*'* = Significant difference (Ool% level) 

UNWOMZM SOIL rin MILY WORM OLD WOM= 

TA13LL 5-7-3 Hean number of fungi per g dry weight Isolated from soil 

treshly worked by termitest older worliced soil and unworked soil. Day 

59 SP medium* (All number, 3 x 1()4). 

SPECIES 

MOW= 

WIL 

FRIMILY 

WOMM 

Abaidia cpr3T#ýiferj 0011 

Am=! Millus flavus 0.41 

A. niger 2.65 0011 

A. rk arasiticus 

0*11 

-1 

OIJ)M 

WOMM) 

A. terreus 0*92 0411 0010 

A, violacems 0010 

Cladosporium cladosMrioldes 0*21 0010 

Cunnin0hemella ap, 0020 o. 96 2elO 

Dactylosporitm op. 0010 0.11 0120 

Fusarium appe 0.41 0011 

PapcilomXcem 111acirmis 0*20 

V. vhrtotii 0.10 0011 

Penicillium asperiui3 0.82 o. 96 O. ZO 

Po citrinu-m 0-31 o. 64 0*10 

11. funiculosim 0.21 0020 

MjMronenum 161411. 0.85 0.30 

Rhtz=a-oryzAe 0.10 

"Sordariall bosensim 0.51 0*75 0620 

SynceLhalar-trtm racemostm 0020 

Trichoderma up* 1-33 0*32 

Dasidiomycote (WD) 0*10 Oe'32 0410 

Bamidicxaycete (WG) 0010 0-32 

unidentified 

W 0.10 Oe2l 0610 

unidentified 

(SB) 0.61 0011 

unidentified 

050 0-32 0*20 

(PR) 

unidentified 

sterile mycelium (WPP) 0041 

0-3-2 

0.21 

0030 

tiny unidentified colonies 1-73 o. 64 0.10 

TOTAL NMOM OF 5rMIES 22 23 

257o

Discussion 

The. soil freshly worked by the termites has a significantly lower 

number of spores developing from it than the unworked soil 

(Tables 

5-7-1 and 75-7*n). This could indicate that the presence of naliva 

with the soil is preventing germination of some spores. The older 

worked soil has an even lower ntunber of epores present which could be 

explained by the effects of the saliva on the spores, with a drop in 

the- ntunber of viable spores rerulting from desiccation and ageing due 

to spores having been incorrorated for a long time. The numbers 

obtained from the older worked soil in this experiment are comparable 

to those obtained from the nest structure (Table 502-7)9 i. e. 17*78 x 

103 and 16#46 x 103 colonies per 9 dry weight* 

comparison of the species composition of the 3 soils 

(Table 

5-7-3) shows that the drop in numbers of fungi from unworked soil to 

freshly wart-Led soil in not due to a fall in the number of species 

Isolated. In general the SWNO species are Isolated but in lower 

numbers in the freshly wortiLed soil, Interestingly, Basidiomycotes 

such as WO and WG, and the sterile mycelium VPP, increase in number 

"58 - 

from the unworked to freshly worked soil* This suggests that they are 

not being affected by the saliva to the same extent as the Fungi 

Imperfecti. This might be expected as it is unlikely that the aaliva 

would adversely affect Terml! ýMces. In the older woriced soil both 

number3 and species of fungi have declinadq the Aspergilli mostly having 

disappeared while the Penicillia remain. 

5-7-2 IMUDITION OF SPORE GMU41MTIOII 13Y LXMCTS or, FOIDI)STORr.,, FLINGUS 

C0113 AND TEMIUB. a 

Methoda 

In order to determine whether there Is anything present in the 

foodstoreq : fungus comb or termites preventing Coraination of the sporoa

extracts of these materials in 3 solvents were applied to plates on 

which 0.1 ml of a soil suspension 

(x W03 dilution) had been placed* 

(1) 190 U each of crushed fresh edge of fungus comb and foodstore 

was placed in 10 ml of sterile water, methanol or benzeneg mixed well 

and allowed to settle* 0*1 MI of each of these solutions were then 

spread on freshly inoculated aoil, dilution plates (5 plates per sample). 

Controls containing soil dilution only, and soil dilution plus sterile 

waterv metlutnol or benzene were also set up* 

259. 

(2) Major workers of Macrotemes bellicosus were washed in HLItono 

25 whole termites were crushed In 10 ml, of each. of sterile waterg 

methanol and benzene% and the process carried out an above* Thia was 

also carried out with 25 Cuts and 25 heads plus thoraces* 

After 5 days incubation the numbers of fungi developing an the 

plates were recorded (Table 5-7-4)- 

Results 

TABLE 5-7.4 ThO MnUbers or rungi developing on soil dilution PlateA 

after treatment with extracts of foodstcwo, and fungus combo

P. 

b44 

>d 

4 

* 

9 

6ý 

a 

to 

b-ä- 

bo. 

fleb 

W 

00. 

>t 

H) 11 ' 

0 

+ 

Z7; 

4 2 

6.4 

9 

19KU 

x 

Oak 

IA 

+ 

0 

e+ 

1+ 0+ 1+ 1+ 

v 

C*4 

0-0 

1+ 

ta 

+ 

$- i, 0- 

%Q 

WNS 

& 

+ 

- 

w 

E5 

z0 

aH 

.. % 

X ý 

" 

0 

%-, 69 

E3 

m9 

- ", 9 

260. 

.1 

S 

-4 

S 

S

to 

C 

) 

I. 

d. 

'1 

E - 

4 

tl 

R tj 

9: 

11 

14 

b. 

" 

0.16 

+ + + 9 

v m M 13 to 

0 

I 

0 

O o O 

LN 

0 

t'; 

0 

- 

CN 

0 

I+ 1+ 1+ 1+ 

tz 

Cý 

0 

or- 

0 

ýD 

co 

0 

4ý0 rn 

1+ 14 1+ 

In 

zo 

H. 

x C, 

b. - 

1- 

V+ icz 

%W In 

0 

w ki co 

Zi 

6 [, ý 0-1 

%0; OP* co 

ta 

Ck 

%: 

$ 

+ 

+ + 

(A 

sp* co 

ta 

0- 0 

+ 

to 

ý3 U 

t3 

W 

le 

C) 

g 

N CR 

2 

0 Cl- 0.1 

#-a 

0 

+ 

tj 

le 

%0 0 

11 tm !ý04 

C-1 

261a 

to 

o 

b-d 

a 

I. d. 

f+ 

to. 

Col. 

0 

0 

I- 

tv

Comparing the results. of both sets of controls 

(Table3 5*7*4 

and 5,70) it can be seen thatt although the Addition of sterile Watero 

wthanol an& benzene to the soil dilution plate3 decreases the nL=bDr 

of fungi developingg the only aignificAnt decrease In In the case of 

bcnzme. JDven lEio the extract results were in all cases In the one way 

analysis of variance compared to the relevant solvent control and not 

to the soil control* 

n62, 

Exper, imant (D* Fungux comb extractus- the extract of nmCus comb 

in water slightly Increases the mmber of fungi developing probably due 

to some being put in with the comb materialq but this increase In not 

significante The extract In methanol decreases : Cungal Germination by 

9%9 but this Is not significantly different from the control. The 

benzene extract did not significantly reduce tho germination* Although 

there is a slight reduction in pu; bera of fungi germinating after treat- 

ment with the mothAnol. And benzene extracts the fungub, c=b extracts do, 

not significantly reduce the n=bers. of fungi germinating* 

Foodstore eytractut. an e7. tract of foodstoro in wateir incre"eG 

the number of spores germinating by almo3t 5CXY%* Fro= . the dilution 

plate experiment results on foodstorog (Table 5*2.7)9 this would be 

expected as the foodstore In loaded with 230 x 103 spores c dry weiGhOo 

Lven with the large n=ber of spores being Introduced with the food- 

store the extract In methanol still manages to decrease the number of 

fungi Germinating by almost 50% ft. the methanol control -a 

sicnificant decre"e. (l%, level)* The benzene extract decreasess the 

number. by 11% but this Is not sicinificant,

Experiment (2). Uliole termifte extracts: - although the extract 

of whole termites in water decreases the m=ber of -spores developing 

by- 19% this In not significant 

(Table 5--7-5), # but the decreases of 56% 

and 4TA by the methanol and benzene extracts respectively, are both 

significantly different (0,1% level)* 

Gut extractst- extract of Cuts In water decreasea the germination 

by 13% (not sionificant), but thera In a nignificant differenco of 51% 

and 4(Y4 (Del% level) vith extracts in methanol and benzene re3pectively. 

Ilead and thormx extractal- there in no significant differe=e in 

tho numbers of fungi growing after treatment with any of the extracts* 

In general thexe results indicate there In a substance, or 

substanceal Present in the system preventing germination of tho spores 

Which are Present theree This Substance appears to be most, soluble 

In methanol* The differing solubility of the substances In the solvents 

has not been Investigated. The large effect of tho methanol extract of 

foodstore, agrees with the observAtions on the lack of fungi developing 

from the foodstore even when removed from the nest system 

263* 

(5.6.1).. An 

the foodstore consists solely of freshly macerated food the effectiVO 

substances my be derived from the termites saliVas. IlowevOri methanol 

and benzene extracts of termito3 indicated that effective substances 

were present In the abdominal region and not In the head And thorax 

region where worker salivary glands are situated* 

Utracts of whole termitea In both methanol-and benzene reduce 

germinatl6nj showing there Is acne fActor'-prea4bilt, in the termites 

reducing the germinatLono -, This would OPPear to be present in the Out 

rather than In the bead wW thorax region.

Discussion 

DLfferent ruggostions have been proposed to account for the 

lack of devolopment of fungi other than Tormitmyens vithin-the nest 

systemo Theso 

ýýchwUcal activilZ of the, termites. Petch (1906,1913b) 

described the terviites as I'veeding" the comb to keep dawn alien fungi, 

Cheo (1948) attempted to ishow weeding means eating, but nevor saw It 

happene Heim (IS42*) considered it unlikely the termites could 

Intentionally select a culture of Tormitonyces and keep it pure by 

auppression of other species, and Grasse (1944) stated that termites 

were not known to favour the growth of Termit2gypes by controlling 

others like Xylarin Datra and Datra (1966) 

soil over XXIarin (5.6.1) 

264. 

observed termites plastering 

and in 1979 reported major workers of 

Cdontotem-es obesus biting Zylarla myceliumb removino it from the 

fungus garden and burying it under soil. The offectij; of this 

mechanical activity may In many cases be associated with 

(2) Inhibition by saliva or other termite secretiorm. This In 

borne out here by the results of 5-7--l where spore germination wan decreased 

by saliva in the soil 9 which may also be associated with 

mechanical activity of the termites 

(I)* the 

0 and by the decreaso in gendnation 

after treatment with extract of foodatore In methanol, Activities of 

the tercdtes such an liddno the wycotates (Datra and Batra 1977)l and 

carrying them about the comb (Batra and Datra 1979) would result In the 

presence ot saliva on and around the combo ý Inhibition of Crowth may 

thus be caused partly by the Initial incorporation of saliva with the 

material$ and partly by the continued prexence and activities of t1le 

termites with their anti-fung4l zubstance(s) on the comb. Within the

nest the combs are surrounded by saliva-mistened soil worked 

termitese The re-eating of the comb would also add saliva to the 

by the 

materialo Batra and Datra (19") found soil recently manipulated by 

workers of Odontotemes purdasTairensis Inhibited fungal germin. -ttione 

Sannasi and Sundara RajUlU (1967) found that the e=dAte of queens of 

Odontotermen rf4le. manni arrested the growth of Aspergillux flavits., aP4 

fatty acids extracted from the exudate were active against several 

fungi* The defence secretions of soldiem have also been reported an 

inhibiting germination of alien funai 03atra. and natra 1966)o The 

secretions of Odontotermes badius contain p-benzoquinone 

Truckenbrodt and Heinwald 19'r 

(Wood, 

. 01, those of Odontotemes redemanni and 

Odontotermes 1! raevalens contain toluquinone (von )"chwitz and Tho 

1974) q and those of "me HyMtermes and Macrotermen species contain 

benzoquinoneo Many quinones are toxic to miny fungi (Fawcett and 

Spencer 1969), Batra mid Batra (1979) found XXIaria, cells In tho 

guts of soldiers so they must either attack or are fed XXlar1a 

Petch (1906) SUGgeated, the termites promoted growth of the 

mycotOtez* Datra and Batra (1977) found that in tho laboratory the 

addition of saliva promoted the pzx4uction of mycot3tese ýheO (19,18) 

concluded that an well as this they auppressed Ularia It would 

thereforo Appear there are two actions at least of the paliva. in 

maintaining the state of the combq (a) promotion of TermijZýnexq 

(b) suppression of Xylatia 

Inhibition b4substances In 11je jMt In these experiments 

extracts of tho out In bc=cno and methanol decreased 

cermination, 

Doflain (1906) waa ono of the'earliest authors to sugg'ast that the 

chewed vood: miGht be partially sterilized by the action Of saliva or 

n6! 5

gastric juice so that It became a medium for the exclusive growth of 

Te. 

rmit=ces,, all other fungi being suppressed. Lee and Wood (1971) 

found Inhibition of enzyme activity in Nasutitermos exitiosus by 

compounds in the carton having properties similar to soil humic acidst 

which they considered might contribute to the inhibition of microbial 

activity, Cotq*unds with similar activity may be added to the faccal, 

pellets on their passage through the gut* Termit2=cea conidia 

germination to also enhanced in the out (Batra mid Batra 1979). 

(4) The carbon dioxide content of the viound atmosphere* This 

has not been Investigated in the course of this woric. Nest micro- 

climate has also been suggested as a reason by Heim (1952a) and 

Grange (1944) with Sands (1969) 

suggesting carbon dioxide concentration 

may inhibit spore germination and growth of some fungi, Carbon 

dioxide concentrations found range from 0,3 

carbonarius 

(Matsumoto 1976), 0*6 - 3% (dry 

- 0,6% in Macrotermen 

266, 

season) and I (wet 

season)l with diurnal fluctuationag In ýýcrotermos natalensin (Ruelle 

1964), from 1*2 

up to 2-7% CU2 in Lheroterness 

- 5,2% In 3 Macrotermes 

snecias (Matsumoto 1977) and 

mounds (Wacher 1961)o In all cases 

this is higher than the atmosphere (approximately 0.03% carbon diOxIde)q 

and so may have smae Inhibitory effect, as the amount of C02 present 

may inhibit growth and germination of some fungi (Cochrane 1958, JAlly 

and Barnett 1950* Several Penicillium species are restricted by 

increases in CO, levels$ whereas other speciesq for example Trichoderma 

viridt, even 10% C02 has very little effect (Burges 

and Fenton 1953), b 

Iligh C02 levels generally inhibit the growth of fungi but the level at 

which Inhibition appears In quite variabloe High concentrations aro 

partially or even completely Inhibitory to spore germination (Cochrane 

1958)o Wood decay fungi may be stimulated by C02 IOVOID as high an

0., '. 1 atmospheres 

(Swift, Heal and AxWer3on 1979)- In general wood 

inhabiting Basidiomycetes show a for greater tolerance to car-bon 

dioxide than litter decomposing species* 

Antibiotic rj: 2ductian Termit=ceno Grassle (1944)- 

ouggested this ray affect the Orowth and germination of other fUnals 

And Heim (1952a) mentions antagonism between TermitoeXces and Xylaria 

myceliums. The presence of actively Groving TormitoMIcPA was shown to 

slightly retard the development of XXIaria (5,4,1) so this may be a 

factor in keeping down growth of this fungus in the comb. Howeverj 

Termitomyces has neyer been observed to bavo an antagonistic effect 

on any of the other fungi cocmnly found in the nest, and Is readily 

and rapidly overgrown by them in culture, 

(6) ChkNTi1cAl. S_C=sit1On of the conbe This was suggested by 

Grasse in 19", The chemical asPectis Of the fungus comb will be 

discussed in Ch- 7- 9 but certainly tile pli and etineral content of the 

comb would not change on removal from the nost. The only factors of 

the comb chemistry preventing growth of other fungi could be volatile 

substances related to (2)l inhibitions by termite secretions, and 

(3), Inhibition by substances In the Cutj which would be continually 

replaced by the termites activities on the comb* 

a. 267-

5.8.1 INTRODMTION 

5,11 VAIMING TECHNIQXrZ 

Washing techniques have been used on soil 

(Parkinson and 

Williams 1961, tiering 1966, Bisset and Widden 1972), leAves (SLtzaonds 

1930)9 pine necdles (Kendrick 1958) and roots Olarley and Waid 1955, 

Waid 1957t Warcup 1960)9 in order to wash off contaminating spores and 

to facilitate the isolation of active mycelia. 

A washing apparatus was designed and used an the foodstore and 

fungus comb to see if spores could be washed from these materials, 

thu. % supporting the idea that the fungi other than Termit2Mcos ara 

present as spores and not an actively growing myceliume It was also 

thought. that washing the material might facilitate the isolation of 

Tormitm3ypese 

Me princiTutl aim was to remove most of the fungal spores by 

vigorous serial washings, leaving the mycelium attached to the washed 

material* This allows organiwm present as mycelium to be more easily 

Isolated by reducing the competition from heavily sporing fungi. 

5.0.2 tonIODS 

Apparatuisit- The apparatus used to wash the foodistore and fungus 

comb in shown in Fig. 5o3ol* The urashing in carried out In a 100 =1 

Quickfit dropping funnelq containing a mesh. This is filled from a 

500 ml flask of sterile water* and in emptied into another 500 ml 

flask connected by Quickfit adaptorso The connection to the filter 

pump enables water to be drawn Into the funnel, and air to be drawn 

in to agitate the matorialo All Joint* are sealed with vaseline to 

prevent air leAking In. 

G8.

Washing procedures- The apparatus was first sterilized by 

drawing through alcohol* followed i; rsierile water. The material 

to be washed was placed In the funnels t1up n ciosed and tap A opened* 

269o 

I-.. 11 

The filter pump was t1jenAurned on and wateF was drawn into the funnele 

When the ftu=4ýwas half full tap A was closed and tap B opened. This 

caused air 

jo be drawn in which agitated the water and material In 

the funnel* Eac4 washing was carried out for 2. minutes* The filter 

pump was then turrivd off and the rubber tubing connecting it to the 

funnel, removod to allow air to enter* The water then drained out* 

This process was repeated ! )'0 times for each material. The wash 

water was sampled at wash mnbers (1), (5), (9)9 (13)9 (17)1 (25)v 

(33), (Q) and 

(5o). 0*1 ml per plate of the wash waters 

per x=ple) were plated on SP medium. 

Dilution plate experiments (5.1) were carried 

(3 Plates 

out an the washed 

comb and washed foodstore to compare t1jo nmberg of fungi growing from 

them to those from unwashed material* 

5.8.3 PXSULTS 

and 5*8*39 

The results aro given in Table 5,8,1 and'kqureis 5.8.1,5.8.2 

TABU 5*8.1 Hean num3ber of fungi Ver C dry weight from washed and 

unwashed foodstore and fungus ccqb (Day 5), 

FOMSTORE 

UNWASIM) W"C* LED 

SJSLLCTlvk )CMIUM 14#88 x 1()3 8.49 x 103 

5F IMDIUM 775-03 x IcP 706.44 x lo3 

FUNGUS CMD 

sr. L=IVZI =Iulf 345-39 x Ic>3 154.43 x 103 

SF V=IUH 3200 x 104 126.61 x 164

1 

--) 

D 

.1 

7 

8 

270a 

FIGURE 5.8.1. Washing apparatus* (1) cotton wool, (2) sterile water, 

(3) taP As (4) rubber tubing, (5) to filter pump* (6) 

funnelt 

I 

(7) mesho (8) tap B.

Mean 

number 

of 

fungi 

2710 

lu zu ; ju 4U 

Washwater number 

F7GnE 5.8.2. Mean number of fungi per plate from 0-1 L11 of washwater 

from the foodstore. 

50

mean 

number 

of 

fungi 

25 

2C 

Washwater number 

FIGURE 5-8-3- Mean number of fungi per plate from 0.1 Ml. of washwater 

from fungus comb* 

272. 

10 20 30 40 50

5.8,1* DII. SCUSSION 

one of the original aigm of the washing apparatus had been to 

facilitate the isolation of Termit=ceft from the fungus comb by 

washing away the contaminating spores. Due to the effectiveness of 

the selective medium in isolating Termit2=ps the washing apparatus 

vas not required for this purpose but the results have been included 

here an further proof that the other fungi present in the comb and 

273* 

foodstore are there only as spores and not as actively Growing, mycoliumo 

There is a rapid decrease in the numbers of fungi washed off the 

foodatore Into the wash waters from washes number (1) to (25) (Fig, 

5.8,2), 

From wash 

(25) onwards the number of spores being washed off 

appears to level out* This pattern is common in washing procedures, 

the largest number of spores being removed in the first washes (Parkinson 

and williams 1961, Willianst Parkinson and Durges 1965). Tito number of 

washea needed to achieve the removal of most of the spores varies with 

the material being washed* These resultj3 Indicate that there In a 

large fungal spore population present in t1w foodatoret which are 

easily washed off* 

Although the fungus comb was broken up before washing the pattern 

is not so clear 

(Fig* 5e8e3)9 and indicates the fragmented nature of 

the foodstore in better suited to the washing technique than is the 

fungus ccabo 

The main spore# being washed off the foodatcre wcre initially 

Trichoderma spot Cladong2rium spot Penicillium app. j Paectl=cem 

111acinus and Fusarlum appi, These were followed by As=. rCillus appog 

pMeephalastrum op. and sterile mycolia and finally by Pancilornrqps 

variotiie Heavily oporulatino speCies SUCII als Penicillimm, As=11jus

and TrIchoderma Are usually isolated with high frequency from wash 

waterse 

(Parldnson and Willimm 1961)o 

In the case of the fungus comb Termit2=cen, was washed off in 

large n=bers, probably due to the breaking up of the mycotOtes. Other 

fungi washed off were Ampergillus spp*, Cladosporium spq Penicillium 

&pp., Pusarium op., Paec1l2! =t-, -j variotil and Absidin co! Mbifera* 

In the dilution plate experiments (Table 5*8*1) no Termitomyces 

was obtained an SF medium or selective medium dilution plates of either 

M yces In 

washed or umfushed foodstore, giving further proof that, Teritom 

not present there* The lower-pimbers (? f fungi, obtained on the dilution 

plates from washed foodstore, compare4 to uramshed shows that the fungi 

were present as spores which have been washed off (Table 5.8.1)o 

On the fungus comb 

(selective 

medium) plates there are lower 

fungal counts on the washed than unwashed plates. Theme fungi are 

almost entirely Termit=ces and the reduction in n=ber would be due 

to the large amount oflermitomycog lost during the washing process. 

on the SF medium plates the vashod comb produces far higher fungal 

counts than the unwashed comb* At first might thin may appear strange 

but it in due to the removal of spores of the other fungi allowing the 

appearance and growth of TermAt2! 2Xcr 

-. 

q on the plates from the washed 

combo On the plates from the unwashed comb Termit2ncc%s was only 

picked up on the 10-5 dilution plates, Termitour fcos cooprined 89% 

of the totAl fungi obtained on the Washed comb plates$ while only 35% 

on the unwashed comb plates, 

*' 74 gb

CHAPTEn six 

MICnOBIAL EMLOGY OUTSIDE 

THE NEST SYSTEM 

275m.

6.1 MGZ IN SOILS, 

6.1*1 FLTML POPULATIONS OF DIFFERENT SOILS 

Metýods 

Tho ceneral fungal population of a wmber'of'different soils In 

the Mokwa area was determined* This established a comparative base 

from which to judge whether the fungi found in, the termites digestiv 

_. 

II Ae 

tract may have originated from the soils& The soils were also 

Investigated for the pref. ience of Temitomyces, 

The soLls LnventLgated were: 

U Uncultivated soil In Southern Guinea gavanna primary woodland 

(Site (1) in 2*1*1)* 

C Plot cultivated by local farmeral growing maize, close to the 

primary woodland (site (4) In 2.1.1). 

TW Soil washed from a Macrotermen bellicosus mounds In regenerating 

woodland at site (2) (2, *l-, l)e 

T soil 5m from the base of the above M. bellicosus swundq In an 

5 

am& where foraging was taking place, 

0 Soil around Odontatermes sme . athmant fljýht holeaq In a gardeit at 

mile I '(Site So 201000 

G Soil from golf course Mile I where Hicrotermen slates flews 

2769 

Sampled earlier in th-e. wat season than other sites (Site 5j 2*19I)e 

n Soil from riparian torest at Rabbs, (Site 69,2.1.1). 

TUngi, were isolated and their propagule abundance estimated by 

means of dilution plates for sails at all the above sites* Si medium 

was used to obtain the, general range, of fungi presents and selective 

medium to see if''any termw=es was present 

. in the so , 11. Five plates

of, each -soil on each 0041um were. set upe, . For further details of the 

dilution plate method see 5.1.4. 

]Results - 

The results are shown In Tables 6.1.1 to 6.1.4. 

TABLE 6.1*1 The mean mmber of fUnci per g dry weight of soil in 7 

soils, Results of the dilution plate experiment on SP medium. 

51 all numbers x 103) n= 59 

SOIL 

MAN NUMIM OF FUNGr PER G 

DRY WEIGIT'l Sozemo 

u 23-94 2.59 

c 19.29 1-57 

TW 43-33 3*29 

T5 42.86 0.92 

0 46.19 3. (r. 

G 9o12 1,04 

R 

2206-32 

10,19 

(Day 

A one-way analysis of variance was carried out on the restats, of the 

Individual plates. 

TABLE 6.1*2 Analysis of variance an the numberm of fungal colonies 

developina from the different soils. 



DEGREES 


FRMMOH 

sum OF WAN VARIANCE 

SQUARES SQUARES RATIO 

Between soils 6 137,909.42 229998.24 234-59*** 

Rexidwd 28 2o744-97 98-03 

TOTAL 34 140t734-38 

277o

u 

c NSD 

u T5 n 

TV **o m 

T!; NSD - 

0 NSD NSD 

G NSD 

IR *0* *o4 **$ *0* 

NSD a no signIfIcant 

dLfference 

0a significant. difference (. 5% level) 

00 a significant difference (1% level) 

0" - significant difference (Ool% level) 

278o

I 

I 

C6 of 

a1 

ba. 

0 0. od. CL 

0A 

low 

14* 

aI 

4+ 

a 

:! " 9 

w a 

'a 4 

o 

I-. t1l 

1 

to I I-- $- 

- 

c 

a 

0K 

Ie. I-d 

0 b- 

9 

1- 1aa 0 

go cr #- 

0 f ., 

0 

. 

n 

el 

olbý 

b-A 0 0w 

0% 

- 

0001- 

;3ý 

: z 

woo 

; 

j m 

ON %a 021 .ýk 

0% 

FA 

11 i 6- 1 0) 

g 

ow 

1 , 

0- 6 

0 

Iw 

0 

-00 

n0r. 1. * bu 6" 

Nk 

ow 0 1& Jd 0 

I-d to b- 

t 

Jd 

C 

n 

,. 

lo . I A 

0 

I 

to, 0 

M, 

fol 6 

* 

1 

; 

(I Wd 

bd 

6- 0 :r0 

I 

En '4 

9 2M 

0- j" 

aM 

() to 

O-d m& ý- En 

oft I 

0-d to. 

is 

(a ou m 

0- 

0 

i 

r A 

I 

W4 I? 

oft 

nit 1.4 

0000000000ta 

" f" 

op pp p 

P- 

V. 

s-0 0 

0 '. 4, 

12 m0 

n r- a 

I. " I" ow 

10 0- 

CA 

00 00 0 0V 

000 00 00 0 

Lcs ;a 4 

0 0 

61 

%a 

- 

;3 

0 0 

rn 

0 

I H 

0 

P: - P P'P r 

Blot! B f- S: o 

(7% ý% t; ua " ow 

ta 

C% w 

0 

279. 

g -4 

w %a 

19 

od. 

flat 

i 

fh 

9 

4 

tA 

lz 

0 

ft 

9

TOM 6.1.4 Siodlarities In fungal species composition between the 

different soils* 

2X 

Uses Sgirensents Index of Similarity -- 

M+n 

where x= the number of species two soils have In counon 

u 

ma the t=ber of species In soil I 

n= the tu=ber of species In soil 2 

u c TW T5 ýo G 

c 0055 - 

T 0-55 0-55 

w 

T5 0-55 o. 64 0.55 

0 

0.55 o. 4,5 0.45 0-36 

G 0"29 0.14 019219 0-43 0.14 

plates: - 

o. 44 

A 

04,52 0.44 

I 

I-- 

047 

I-- 

0.44 

0011 

Results of, the dilution plate experiment en. selective mediurs 

No TermitMyces was obtained on any of the plates at any dilution* 

This Includes the moil from r1abba which was, collected 2m from TerultonTeem 

, 

fruit bodies growing from Anclatrotermen Cavithorax combso 

Some fungi were obtained on these plates. These Included 

Asperaillus violacewsl Curvularia op., ftsarium ap, ý Thielavia up.,,,. 

Basidlowycetes and 1Rh1zoM! 

s oEXMe 

-I-I 

280.

Discussion , 

2810 

, The lowest number of fungi were obtained from". the golf-courne soil 

(G) 9 which als* yielded very few species* The next was the cultivated 

, s, oijL (C) whjchý was ve" siodlar In mmiber and in species tO thO PrimaxT 

vood. land soil (U). The highest index of similarity was between 

cultivated soil 

(C) and soil m from the termite mound 

(TS) where 

t- ermites were foraging. The soil washed from the mcAnW (Tw) and TS 

soil were similar in Wth numbers and species composition. The richest 

soil In terms of both m=born and species diversity was the soil from 

the riparian forest at Rabba (R)* This result Is expected due to the 

soil there h4wing a greatnr organic and moisture content (Tables 2olol 

and 7-1*8)9 thun promoting the growth of fungi* which were In evidence* 

The surface vegetation has a considerable effect an the soil 

mycofloms partly due to the accutoulation of organic residues from 

the association of plants and animals q and partly to the development 

of a root-inhabiting flora associated with species of higher plants 

(Orpurt'wW Curtin 19579 Thornton 1960, Waid 1960)* Thexe effects are 

seen when comparing the Rabba, results with the otherse In terms of soil 

and vegetation the other areas are far more similar and the fungal 

populations reflect this* Prior to cultivation soil C had been under 

isavwula V(X)dlaW* and the area around tho termito mound (Tv and T, 5) 

was j3ec4ndary savanna woodlande 

lumbers 

The mobiture content of the moll also has an effect on the fungal 

(Wald 19004 With rain : fungi bacme, activol there is an increase 

In the aMOUnt of viable hyphae and several specle3 sporulates 'thus 

increasing thO. Uxabers of spores to be obtained from the soils This 

is retlected in the low numbers obtained frc= G canWed to Oo uhich

was from a similar area althouoh with slightly more aivojýse vegetation 

cover. G was sampled much earlier ong at the start of the rainy 

season, than when 0 and the other soils were sampled, by which time 

many more fungi may have grown and sporulatede 

In geneml týe-speciss of 1ýmgL obtained - frxxm these soilis are 

those obtained from the termites Outs, foodstore and comb dilution 

plates 

(Tables 5*2e - 90 5*2*16)t showing that the prezence of'these In 

the nest, system can be accounted for by contaminAtion of the termites 

by spores from soil, and by contmination of the food material* Most 

. of these fungi wOxo also obtained by McDonald (19689 1970) from Mokva 

soils* 

6,, l,, 2 FUNGAL POPULATIONS AT DIFFERM DMMUS -IN THE SOIL 

Hathod 

Two pits were dug in prima" Southern Guinea savanna w0odlan4 at 

Zugurva (site As 291ý1)* In each pit 5 s911, samples weretaken by 

means of a cork borer from each of the following depthal 0-5 cmq 5.25 

eml, '25-50 ca and 50-100 cme The number of SUMal propagules per g dry 

weight at each depth was determined bY dilution plate counting on SF 

medium* !; plates were not up for each depth in each dilution series* 

For further details of the dilution plate meth(W see 59104. 

Results 

The soil varied from sandy at 0-5 cm depth to sticky clay at 

50-1009 with, an increasing moisture content with depth (29192, Table 

2#1*1)o 

The results are shown In Tables 6*Ii3-6,, l*7* 

282.,

TABLE 6*14 kkAn mmber of ftmgal propAguleg per g dry weight of 

13011 at different depths, ]Results of dilution plate experiment on 

SP rmWum after 5 days* All numbers x 103o na 59 

i)m, >nt (cm) 

MAN : S. E. H. 

PIT A 

MAN ! SOEOM, 

PIT B 

0-5 28.66 6.56 10*51 1.61 

5-25 5.0r. ) 0.84 8.42 2*35 

1115-50 4*50 1*24 1-08 1 0-34 

50-100_ 9.32 3.34 11*75 ! 4.27 

A two-way AUIAIYXIS of variance wan carried out on the results of the 

Individual plates* 

TABLE 6,, lo6 Analysis oi varlance of the numbers of fungal colonLes at 

different deptha In tho soll* 



DEW= 

or 

FREEDOM 


SQUARES 

ýMAN 

SQLWMS 

B6tween depths 3 1547-44 515,81 

VARIANCE 

RATIO 

. 2602NSD 

Detween pits 1 128.!;! s 128.!; 5 04!; ONSD 

Interaction 3 767-07 255096 4.9300 

nesLdual 

32 1660-03 !; 1.83 

TOTAL 39 4103.89 

There is an interaction betveen the different pits and the depthso 

KSD a m) significant 

difference 

00" a significant difference (0.1% level) 

00 a significant difference (1% level) 

a significant difference (5% level) 

203o

0-5 

". 5-50 

0-5 

Pit A 

5-25 2!; -50 50-100' 

14SD 

50-100 000 NSD NSD 

0-5 

5-25 NSD 

Pit B 

115-50 NSD 

50-100 NSD NSD 

25-50 50-100 

- 

'184.

TABLE 6.1-7 Fungi isolated from different depths of soil* tkmn 

number of fungal propagules per C dry weight of soLle All m=bers 

. 00 x 103. 

FUNGUS S 

SPECIES 

DEPTH (cm) 

0-5 

5-25 25-50 50-100 

Aam! Millua 

Penicillium 

Asp2=11lus 

A9L*rg1lluz 

fumigatum 

app. 

niger 

flavus 

9-90 

3*92 

2*27 

0-72 

2.31 

1-79 

0-53 

00261 

0*97 

ý0-54 

0054 

0*11 

2.44 

1000 

0-67 

0.44 

unidentified species 

Penicillium striatum 

Absid1a corjMbifera 

Clados22rlum __ up* 

sterile white mycelia 

Trichoderms op,, 

. 

Paectl=ces Iflacinus 

0-72 

o. 62 

0*31 

0*21 

0*21 

0.11 

0011 

0-74 

0011, 

0*32 

Oo2l 

0.11 

0*32 

0011 

1 

162"03 

0011 

0011 

1-33 

Mcor op. 

A-sperVillus op* (Y2) 

0011 

0011 

0*21 

Cuminghamella opo 04,21 Oo22 loll 

Cerhalo! =rl spa 1-78 

Nigroargra op. 0*22 

As22raillua terreus, 0* 11 

KmEn op spzcrEs 13 12 

Discussion 

I in both profileD tho fUnGL decrease in numbers from the top 5 cm 

downwardas but Increase again at 50-100 cm depth. Soil fungi usually 

shcow a marked depth diatrIbutiong with decribase In numbers from tho 

iop downwards (Briorleyl' Jewson and Urierley 19279 Griffin 1472 and 

Ste nton 19,1>*3)o This pattern in repeated with the species diversity 

(Vicklow and'VUttingham 

diverse* 

1971*)t the top 5-cm here being the most 

In general them Is a deel ine in fungal species div . ernity 

285o 

during prooressive decom'position so we sho4ld'expect to kiýd sionificant 

differences between numbers and species of fungi found in SUCCeSAVO

horizons of the soil 

(Swift 1976), There In therefore a decline In 

fungal diversity from litter to humus and mineral horizons, butt as 

heret increases In the numbers in the lower depths have been found 

Welter 1973)* Normally the most abundant'sporulations and'therefore 

286* 

the highest numbers of colonies on dilution platest occurs I In the upper 

horizons (Mont6gut 1960). These fungL In the upper horizons are often 

Intolerant of carbon dioxide. Other speciess more tolerant of higher 

carbon dioxide levels,, and better suited by the different-conditions 

of humidity and nutrition found there% may show their maximum frequency 

lower, down, (Durges and Fenton 1953, l Montegut 1960, Waid 1960)o In thin 

soil there are 3 species Ce2mlosporium op., Niarospora op, and 

AsperSillus-terreus vh1ch only occurred at ! 50-100 cm"depth,, 

Cunninghamella op, showed increasing : frequency with depth* 

soil 

Soil animals play an imortant part In the mixing of spores and the 

(vobba and 11inson 1960)e k1ost of the general such an Aspargillues 

Absidia, Cladosn2rium and Penicilliuml which have been Isol#ted here 

exist an passive survival structures requiring carriage In soil water 

or on, or in,, the bodies of soil animals for dispersal (Swift 1976's 

Wald 19W), The vertical distribution may be affocted'by how easily 

the spores can be washed down (Burgen 

and'Fenton 1953)o This carriage 

by'soll animaist as has been shown occurs with termites (Tables 5.2,16, 

3.3,6 and 5.4.8), way account for the increased number of spores at 

. 5o-100 co depth. The experiment was cmvied out at the beginning of 

the rainy season* During the dry season a greater proportion of 

Merotet s are found below 50 cm, than In the wet season 

(Wood and 

Johnson 1978) 0 and so this increase of ftmgL may be as, a result of the 

previous activity of termites and other soil animals at this depth*

6*2 E3TAUTSIMENT OF THS TMMITCWCES CULTME IM NEW NMT-43* 

Introduction 

In the Macrotermitinae, now colonies are established by winoed 

reproductives (alates) which leave established colonies In synchronized 

flights after the start of the rainy seasone The slates undergo post- 

flight sequences of courtship behaviour resulting In the loss of wings 

and the formation of pairs (Stuart 1969)e The pair then enter tj%o 

soils excavate a copularium aW eventually establish a now colony* 

One of the questions concerning the association between termites 

and Tervrdt2=ce, % was how Temit=ces reaches the newly established 

colony, Tvo tiothoda of Inoculation have been suggested In the 

literature and both were Invextigateds 

(1) The carTiage of an Inoculum of Terinilm 7ces1by the alate. 1, 

This was Investigated by wans of gut dissection,, ctilturing of gut 

contents and attempts to establish colonies from pairs of alates In 

sterile xoLl In the laboratorye 

(2) The collection of bazidiospores by the first batch of foragers 

produced by the new colony. This was Investigated by seamhing for 

frultbodibs 

(basidiocarpn) 

and collecting the associated termites, 

6*2*1 CAMZIAGE OF TMMITOMYCES AND OTHM FUNGI DY ALATF. "S 

Hathod3 

Alates of several species of kfacroternitinae were collected'duriho 

flight, and (in the caso of Macraterm2a ballicosus), from the nest prior 

to flight. The alaten were surface sterilized in M111tonts Fluid, their 

digestive tract dissected out In sterile water, and placed on selective 

medium plates to culture any TendLaMLces presents and on SF medium 

287o

plates to'datermine the generaj range of fungi beino carried In the 

gut* 30 Cuts from each sex were placed on each medium for H. bellicosus, 

Hicrot-e-rmos spo A and Hicrotermes up, D, wW 15 from each sex for 

M. subhyallnual Ancistrotemes cavithorax and Odontotermas ameathvianio 

The presence or absence of a bolus In the crop was recorded for 

all &latex disseeted, and was also recorded for the penuitimato nymphal 

Instar of. M. bellicosum, 

Results 

The results are recorded In Tables 6*2*1 ý 6.2*16* 

TABIZ 6.2.1 The presence of -a bolus In the crop of slates, of several 

species of Macrotormitinae. 

=*UTZ SPECIES WITH WITHOM 

13OLUS 13OLUS 

Anclatrotermes cavithoram, 5 

-25 

0 

WITH WITHDUT 

DOWS BOLUS 

(16.7%) (83-3%) (16.7%) (83-3%) 

Macrotermets bellicosus 62 0 78* 15 

U00.0%) (0.0%) (83.9%) (16.1%) 

M, bellicosum 

Mtar) 

nycoial 

(penultimate 0 

(0.0%) 

30 

(100.00,91) 

0 

(0.06A) 

30 

(100.0%) 

Macrotermes subhyalinus 7 28 3 16 

(20*0%) (80*0%) (15.0%) (84,2%) 

Hicrotermes op* A 5 55 81 0 

(8*3%) (91'7%) (1001-01%) 

(0*0%) 

Mcrote rrmx up. D 34* 112 1 

_ (35*8%) (64#2%) (99,1%) (0,9%) 

Microtermes spe G 211 2 7 0 

(50.0%) (50.0%) (100.0%) (0.0%) 

Hicrotermes 

spf, zý 0 41 5 0 

(O-MO (100*0%) (10000%) (0.0%) 

Odontoternes papdalor%no 40 26' V 21 

(13-3%) (86-7%) (12.5%) (874%) 

odontotermes ameathmant 1* 30 n" 29 

1 (3-2%) 1(96.8%) (6.5%) (93.6%) 

* mAterial forming bolus is da* In colour and'appears to be soil 

rather 

than Tormit2Mes, 

25 

283.

TABLE 6.2.2 Grworth of TerwitLanes Vrw the guts of alatos of 

several species of Macrotermftinaeo 

TZRMTZ SPBCIES ne nI 

PLT%=TAGC OF PLATES POICENTAGE OF PLATES 

ON WHICH ON WHICH 

TEMTOMYCES GROWS TFIMITOMYCES GROWS 

Anclatrotermes cavithomx 15 000 15 0*0 

Ifacrotes. a bellicoatts 30 79-2' 30 10-7 

flacratermes subUalinus, 15 000 15 0*0 

Hicrotermes sp. A 30 3-3 30 50.0 

Hicrotermes spo D 30 6*7 30 40.0 

Hicrotermes- spo Z 4 0.10 5 40.0 

Odentotermes smeathmani 15 000 15 O. 'o 

TAM. E 6.2-3 Asx0ciation between the carriage of Termitoomyces, In the 

Cuts of slates and the ability to establixh viablo f=gux comb in 

Ulmratory cultureso 

TEWllTs SpEcirz ESTABLIsimmm 

OF VIAMLE 

nmus com 

SPORE. 

CARRIAGE 

ISOLATION OF 

TEMITOMYCES 

Anclotrotermes; eavithorax o (1) 0 

7. vulneensis 0 (2) oft) 'I' 

Macrotormes bellicosus + + (1) 4. 

liacrotemes subhXalinus 0 (1) o (10 0 

111crotemes zD* A (3) (3) + 

Hicrotemp-s spo B 

kticrotermes aD* D + 

micratermes spo G 

kticrotermen BDO Z 

+ 

+ 

Odontotermes M u]2 erans' 

_ _ 

o,, rmentl=ant 

llseudacanthotermes--spinil 

0 

0 (4) 

oM 

o (I) 

+n positive result (1) Johnson c-t al 

0a nevative result (2) Sands (19W) 

noýýtosted (3) Johnson (1901) 

116cher (1951b) 

(1981 in press) 

289o

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Dizcufý. Asion 

The results show that acne tomito species% such as Hacroterues 

bellicosup and, all Plerptermes speciez inypatigatedt carry a bolus of 

TermiLmqces conidia with them when they leave the pants which in shown 

both by e=Ination of the cropcontents 

(Table 692909 And by the 

culturing of-Term-itomXSes from the gut (T4ble 6*2*2)*, Other speý: ies 

Such as, KAcrotemos subliyallnus, t Ancistroteme3 cavitharax =n4 

Odontotermes smeathmani do. not, carry a bolus of'TermitoUcas with 

them on leaving the neste 

Tho n=bors of TomiLcM. Cces cultures obtained (Table 6.2.2) are 

lwar than tho number. of termites carrying boll of Termit2=es (Table 

6,2*1) due tq Vie fact that, although the selective modiumareatly 

reduces the t=ber of other fungL obtainedg some such, as Asper illus 

vloinceous, CumipjLhamelln spog Curvular-la ap. and ThInjavia sp. still 

gr4ow in some casent vith,, bacterial GrOvth Wng a probleM. 

In the case of Macrotermes bellicagus the Inoc I 

ulum of Termit2=eq 

in given to the termites only In the final Instarj the penultimate 

Instar having a c=plctely mpty crop (Table-6,2.1). 

The results also show differences between the sexes in the species 

carrying Termit=e% The male Is the principal carrier 

M. bellicosuml whereas the female carries the Inoculum In all the 

Hicrotermen species investigated, OccasionAlly the llwron6l' isex carried 

Termlte=. ces 

(Table 64*2)e 

The carring4i. of spores Is associated with, the ability of founding 

pairs to establish viable funmw comb In unfed laboratory cultures In 

in 

292a 

sterile zoLl (Table 6. '. 1*3)9 wherean, aame species (the non-carriers) are 

consistently unable, to establish a vlaýle tungus comb In laboratory 

culturese 

.

In contrast-to-the African Odontaternes species recorded heres 

which do not carry apore3j 0, obesus In India carries spores 

(Batra 

and Batra 1966% 1979) and long lived colonies havorbeen met up in the 

laborAtory (Ausat ot alo 1962)s some with fungus comb although it was 

not recorded whether thin contained Tarmit2=ces Other Indian species 

recorded as carrying Tern-itomyces are Oe obscuriceps (Dorlein 1906), 

0,. M! rdaspurensis and HicrotermeS spo (Batra and Batra 19660 1979). 

Cheo (1948) was una le to establish viable fungus-comb from pairs of 

0, formosanuse- 

A range of fUngi are carried by the alates (Table 6.2.4). In 

contrast to Termif-oWes there do not seem to be any Creat differences 

in the carriage of these fungi between the nexe3. The fungi are In 

general those found in the soil and In the nest system, An exception 

to Hetarhizium anizopline which In a pathogenic fungus which penetrates 

termites and ýCills them within a short tine, IkLny of the Cuts contain 

no fungi,. Trichodermas Penicillium and, Aenergillux species have also 

been obtained from Cdontotermes obeaus guts (Das et al, 1962)o 

The carriage of an 1noculum of T@brmjtor3yc,, e spores represents a 

highly evolved-adaptation to the fungal symbiosis (Johnson 1981)o This 

behayIour appears,... to have doveloped at least twice within the Macro- 

termitinaes once within the primitive genus tkerotemes where carriage 

Is-by male reproductiveal and once within the more advanced genus 

Ricrotermen where. carriage of the spores Is by fmales, This method 

of inoculation would require the conidia to rcuain viable In the Cut of 

the alates for several weeks between flight and the building of the 

fungus combo. All attempts at culturing jermit2=ces from reproductive 

pairs established in soil In the laboratory failed duo to bactorial 

and fungal contamination, and spore survival In the &late out needs 

further Investigation. 

: 930

6*2*2 ''EIASIDICCARP PRODUCTION IN TMMIIN)MYCES 

Introduction and Methods 

-The basidlocarps of TermitomXces have been described and recorded 

from Africa and Asia where they appear during the rainy seasono They 

are often highly prized as food and may be collected and sold in local 

mazitets* They are reported to be widely distributed In Nigeria and 

collectiotw have been made from all vegetation zones from rainforest 

in the south$ to dry savanna grassland in the north (Alasoadura 19669 

1967; 'Ileim 102bl-030 19751 Zobori 1973), Unfortunatelyg rarely have 

the termites been collected and Identified-along with the basidlocarp 

and this has not been done with the species previously collected from 

Nigeria. Twelve species have been collected from Nigeria, six of which 

have been named Wasoadura 1966* 1967)- 

After the start of the rains regular searches were carried out 

for basidiocarps In the tk)kva areaq but between 1976 and 1979 no 

basidiocarps were fountl In the savanna woodlands In this re0ion,. 

Termitýýces In reported to produce baaldlocarps 

the rainy season (Holm 1977, Sands 1969), 

after the start of 

Who start of the rainy xa"on attemptx-vere made to, inittate 

fruiting In two ways* Fungus comb was broken up and scattered on the 

surface of the soil in Imitation of the behaviour of some Cdontotermas 

species prior to Termitomyces microcarpu_s, groving from the fungus comb 

(Coaton 1961)* Fungus comb was removed from nests and buried In 

containers of soil which were kept moiýto Tile fungus combs producing 

basidiocarps are partially or wholly deserted and this was an attempt 

"94. 

to 121imic thOSO c0ndltiOnue This Is also similar to tho, chaff-method of 

BUMUlating, truiting (Warcup 1959)9, whcre the chaff an which the fungus 

in growing wa3 placed in pots covered with soil and kept under humid 

conditions,

Attempts were made to germinate 

baxidlospores from basidiocarps 

on vet and dry plates of selective " SF medium, 4nd on sterile 

maize pith. 

Results 

(1) Initiation of Tjasldloca!: ]2 Production 

No basidiocwnw were produced In either of the two matliods u3cdo 

The ftmgus comb In the soil chambers decouposedo 

Gemination of banidloqL2res 

No geminAtion of bAsidiospores occurred. 

ST%Qcics Of TermitC=YCOS collected 

TermltTv ,., con Zerfomnx associated with Anclatrotermos cavitharaxe 

11abitats from subterranean combo of Anclatrotermes cavithorax1in 

riparian foresýj Rabbas rungus combo often partially or completely 

deserted*. Often at baSe of palm treess occasionally the comb In nests 

of Kicrotormon xubhXallnus or near nests of Cubitermes, 

24.6.78t 26.6.781 7-6-791 12.6-791 18.6*79. 

295, 

Other Termitomyges ispecies found were Tarmit=cen striatux form griseuxt 

from Ancintrotermes crucifer fungus comb In maize field, Samaru (19-9-79)o 

Terml! ýMces aps noveg from Odontoternes Rtupprans fmgus comb, 

Zarin-Kaduna road (IL4.7*78). Thirty to 40 specimens, cap 5-8 cm in 

diameter were -seen coming directly from semi-abandoned comb around 

the base of an abandoned Macrotermes bellicasun mounde and around the 

base of a large tree 10 M from the mounds 

Temit=ces rabuori L-i from 

t a susiDected Ancistrotermom comb wLth 

no termites presents Samaru (4*7*78)*

296. 

Termit2=ps microcarpupq from Cdontotermes pauperans, fUngus comb 

ocattered on the soil surfaces Shika (18-7e73s 20.7*78)o 

Torndtc ! qrmog spe A, from Macrotermes pubhyallnus fungus combs 

Zarin-Kaduna rood-We8s7B)s This was a large-specless estl=ted cap 

di=eter of 12 cm* The basidlocarps were In a poor condition and a 

description was not possible* 

TAI31Z, The association betveen the carriage of Termitc 

_? 

Mr4 qAq spores 

by alates, and the development of basidLocarps frow the fungus coinbe 

TEMITS SPECILS 

Anclatrotermos cavithorax 

A. _cLulneensis 

klacrotermen bolliconus + 

CARMAGE OF 

TMaTOMYCES SPO=, 

H. subhyllinum, + 

Microtemes appe 

odantotermem pauPf-rAnn + 

Odontotermes unwathmani 

GWMI OF 

USIDIOCARPS FROM 

FLINGUS COMBS 

Paeudacanthotermes militarts + (I) 

Poeudacanthotermes sl! intper (2) 

(1) Heim 19409 1942ai 1952, bv 1977- 

(2) L16cher 1951be 

Plearce (pers. comm. ). 

-a negative result 

+w positive result 

(3)

TAmz 6.2.6 The first flight and appearance of Termitomyces 

basidiocArps for stoma "ciao of Racrotermitinae in 1978 at Samru 

in the Northern Guinea savanna zone of Nigeria. 

TE MITS DATE OF TIME (DAYS) AFTER FUNGUS COM BUILDING 

SPECIES IST FLIMIT IST FLIGHT WHEN COMK24CED IN 

Ancistrotermes 

TEM11TOMYCES INCIPIENT COLONIES* 

BASIDIOCARPS SELN IN (DAYS AM-ER JST 

FIELD FLIGHT) 

cavithorax April 10th 77 80 

Macrotermf-mi 

subhnlinus, May 16th 91 95 

Odontotemes 

paun2rans My 14th-- 60 65 

(0 data based on laboratory cultures at Mokwa). 

2979

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Discussion 

(1) Association between Temitomyces onecies and different 

m=ciem of tamiles 

'Observatiom in the Molwa area show that the production of 

Termit = ces basidiocarps does not appear to be such a common occurrence 

as might be thouoht from the literature. This has also been the 

experience of some authors elsewhere eego India (Bakshi 1951) and 

Barkuda (Bose 1923). Temites feed on the cap aml stalk,, which aro 

also eaten by man, buck, beetles (Bose 1923) and diptera larvae* In 

Nigeria many of the basidiocarps, even uhcn very young, were attacked 

by diptera larvae. These were Identified aallemin=ochanta apicifera 

(Curran), 11. unicolor (Bigot) and 11. varia Olough)e Thing together 

with their rapid decompositiong may be a reason they are only rarely 

found in some places* An these termites are widespread in the Nigerian 

savanna the observations indicate a highly restricted production of 

basidlocarpss with some species of termite not having any basidlocarps 

associated with them* Coaton (1961) reported from southern Africa that 

basLdiocarps are not produced by Individual colonies every year. Ilicir 

production may be linked to a certain stage of colony development and 

processes within the termite colony* It In a similar situation to the 

attine ant where sporophores, are extrcmely rare making identification 

difficult (Stradling 1978)o 

Collections from Nigeria (Table 6.2.5) show that,, with the 

exception of Odontotermem smeathman1_0 those species, that are unable to 

inoculate comb in laboratory culturea or have been shown not to carry 

Temlt2Mces spores at fliljhtg have been associated with basidiocarps 

of TermItMelps, growing'from the nests of well-established colonieno 

In contrasts no basidiocarps. have been found associated vith t1wse 

302.

spe, ýles where the alaten carry spores In the Cutl further- evidence Is 

provided by Ruelle (1964) and Van Ryn U974) who never found basidlocarps 

associated, with thcrotermes bellicomus, which In kzxmn -to be -a carrier of 

spores And Coaton (1961), who. nevor found basidlocarps'associated, with 

Ricroterne-9 in southern Africao 

Associations between termites and TqrpltýTayeea recorded, ln'the 

literature are shown In Table 6-2-7 and raises the question of 

specificity between termites And Termit2!! D., cell Grasse (ISY59), and Hein 

(1952a) considered there was a tendency for species of fungi to be - 

associated with Genera of termites* There iscortainly a-degree-of 

specificity on the part of the fungus* Sands, (1960) found that 

Ancistrotermen Mitneensin could feed and survive on fungun comb of. 

A. crucifer, but was unable to inoculate its own sterile TLMgus, comb 

from this sourpe. If supplied with fungus cccib from -their own species 

they could-then Inoculate their own comb, Although 2, species of fungi 

may be associated with the same lezvilte species theyýhave never been 

found together In -the same moun& **go 2: _ 'AicrocaMs and To, n1bum1nonux 

(VatrA and Utra 1979)9 with Cdontatermes obasus taid 0. Ordasipurmsixo 

In order to accurately determine the associationa between the species, 

it is naceggary to follow the pseudorhiza. down to its insertion on the 

funcus comb and collect tervAtes from the funow -comb as termites of a 

different species may be foraging in the vicinity* The fact that 

basidiocarps, occur_cm a wound of a certain ternite-may not mean they, 

are growing from combs of that species as fungus comb of soveral species 

are often closely-associated with mounds* FrultIng was observed 

originating from Ancistrotermes cavithoraX comb in the wall of a mound 

of Macrotermes aluhXallnum and from A. cavithorax comb very near to 

Cubitermes nests* Per these rmson. 9 some of the associations reported 

3030

in Table 6.2.7 miy be Indorrects If we accept this then thera; does 

seem to'be a pattern of speciesof Termit=cps being associated with 

genera of termites* 'The degree of specificity needs to be determined 

Airther by simultaneous collections of'baAldlocarps and the associated 

tormitee 

(2) Collection of ba3ldioaMrom by the first foragipa-r! rties 

of newly!. established colontes 

Evidence from Nigeria concerning the aYnchronisation of the 

occurrence of basidlocarps In the field and the building of the fungus 

comb in now colonies (TAblo 6*2.6) shown that basidiospores were 

3(Yko 

available in the field at the time the first foraging workers were active 

and gathering material to met up the new fungus c(xmb, For other termite 

species the evidence required to correlate fruitingg the flight of 

alates and the appearance of the primordial fungus cozib in not available* 

At Kinshasat In the Republic of Zaires alates of Psoudacanthotermes, 

militarin 

(a non-carrier Table 6*2*5) Usually fly towards the end-of 

April and basidi'dcarps of Tervitom. ycen, associated with- them have been 

observed from the-end of September to the middle of October (Van Ryn 

1 

1074)l an interval of approximately five monthas In Moudacanthotermen 

spjni. Cer (also a non-carrier)'the primordial fungus comb was not 

observed until Moro than 200 days'after colony foundation commencede 

If the two Species have a similar pattern of colony development thils 

would mean basidip3pores would be available when foraging cocmenced. 

There are therefore differences between different specLest the delay 

between flight *nd fruiting being 2 to 3 months In the Nigerian species 

studieds 5 months for P. militaris and I to 2 months for Odontoterme3 

badlus (COatOn 19619 VAn RYn 1974).

. The Inoculation of new combs by basidlosporex has been suggested 

by Itarris (1961) and Sanda (1969) but In Very difficult to prove* An 

no Mcolium of Ternitamyres has been found In the wild Sands auggests 

it would be hard to wcplain the adAptAtion of-Termit2Mcp to soil 

penetration if it was not necessary to produce basidiospores. These 

basidlospores would be of no value unless they promoted the distribution 

of Terrnitg=es, to ncw colonies and It is likely that they constitute a, 

second method by which newly a. %tablished colonies C; ain their inoculum 

of Terptt=rs In general TormitomXces are not the first fungi to 

develop after the start of the rains* Different species fruit at 

different tives Olein 1977) and with different species of termites 

also flying at different times, this further Indicates that fruiting 

and flight times may be linkedo 

(3) Factors stimlating tha LiMguctim of-basidloca=3 

(a) In 411 cases. basidlocarp prDduction Is geared to the rains 

and moisture Ix probably a very Important factor, In China basidlocarp 

production to oomtlnes forced by the use of warm or cold water (Cheo 

1948), ý Specific fungi have specific requirmentgs and In Basidicaycetes 

the formation of swaial structures Proceeds more rapidly at high than 

low moisture levels* The majority of reports an production of 

Termit2=caa basidlocarps come from more inoist forest zones than from 

the drier savatum regions* 

(b) In Dazldiomycetes the production of basidiocArps requires a 

305. 

considerable source or reserve of =terlal to draw on during dovelopmfmt* 

Iniereforo they do not usually arise until a fairly extensive vegetative 

myce. lium has developed* This may be one reason for the lack of 

basidlocarps associated with Hicrotermas species-l the fungus cozbs of

which-aro 2-4 cm in diameter and dispersed through the soil* During 

the dry season the =ount of fungus comb decreases In the top 50 cm 

of soil an the termites consume the comb without replacement 

and Jolumon 19704, At the start of the rains the termites begin 

foraging and building up new fungus comb and there are probably 

(Wood 

insufficient reserves of Mycolium and fungus comb to support basidlocarp 

develo; =ente Alternatively, a reason for the absence of basidiocarps in 

that carrier tesmito species do not need to distribute spores by this 

methott. 

ScxuAl reproduction is most likely to occur when a vigorous mycellum 

starts to exhaust Its nutrients (Cochrane 1958). Nutrients are necessary 

3o6. 

for initiation and probably differentiation of the basidlocarp prLmordiums 

different species of fungi having specific requirements for the nutrient 

concentrations necessary to form fruiting structures* The fomatioh of 

fruiting structures occurs over a narrower range of nutrient concentra- 

tions than does veoetative growth (Cochrane 19:., 

)8). The concentration of 

the carbon sourco in usually Pore important than that of the other 

nutrientsg spore formation usually Occurring when the carbon source is 

becoming exhauatede Therefore the production of frultbodlos to probably 

highly dependant, on the nutritional state of the fungus comb* In 

Ancistrote=es cavithorax those ccmbs producing fruitbodies aro usually 

covered with a greater mycelial growth of Temit, myces, s And no 

mycot-etes arc pre3ento They fall apart more cAsily on handling and 

are lighter In colour than fresh comb. Also the part of tho comb from 

which fruiting originated was abandoned by the tezuLtest although they 

could still be adding fresh material to other parts of the ccmb. In 

general the CO1%1b 

was more or loss Abandoned by the tarmitc3q often 

tOt&llY9 and thCrQ were no larvao, pre-sento ThO termites could still

e active on an adjacent combo This abando=cnt of cctmbj3 supporting 

fruiting has also been reported by Delms (pers* 

and Petch (1906)o 

commo)l Ileim (1977) 

Termites eat the developing primordial as waa also observed by 

Bela (pars. com. ), Ifeim (1977) and Petch (1913b). Ifeim has noted 3 

forms of behaviour of the termitest (1) total tolerance with the 

termites still on the comb, 

(it) the termites eat the developing 

basidlocarpst (iij) the termites cat the developing basidiocarps and 

the fUngus combi but finds that generally the chambern from which the 

basidiocarps grow are abandoned by the termites. 

It is difficult'to determine whether an abandonment of tho fungus 

comb, for whatever roaxong by the termites, leading to an unchecked 

development of mycallum and a consequent exIlaustion of nutrients, leads 

to busidlocarp productlcme or whather the development of basidimarps 

leads to abando=ent of the fungtis cmb, 

307o 

(c) The abandor=ent of the comb by the termites may cause changes 

in other factors affecting basidiocarp production. Tho temp4ýraturq 

around the comb =y drop,, which may also occur with the onset of rains, 

In general the temperature around fungus combs is more constant than 

the surrounding environment from day to day but does vary with the 

seasons 

(Cheema et al* 1962t Collins 1977 and lZacher 1951a)* This In 

more pronounced in m" tJuin In diffuse sýbterranean nests, The 

temperature optimum for fruitbody production may vary from that for 

vegetative growth eoge for fruitbody production of Armillaria nellea 

the temperature required in 100C below the optimtzm for vegetative growth 

(Ingold 1979). The fungus combs of the Macrotermitinne are generally 

maintained around the optimum for vegetative Growth so a drop In

temperature In tile rAiny season rwy, help Initiate frultbody development* 

Wn would cool diffuse nests In the soil more than Mounds. 

(d) Abandonment by the termites may also cause a fall In the 

carbon dioxide content of the atmosphere* In Anariclin and other higher 

fungi carbon dioxide concentrations above a certain level stimulate 

mycelial gtvwth and prevent formation of primordine In general there 

Is a higher oxy0en requirement for fruiting than for mycelial growth* 

Light is unlikely to be an important factor in this case* Other factoraq 

both physical and chemical, are likely to be associated with the presence 

of ternites on the comb and may affect the production of fruitbodies, 

(4) Germination of basidiosporen 

The basidiospores of a great many species are hard to germinate 

and there are still a large number, particularly In such genera an 

Russula and Lnctarliv, 419 where conditions for any gemination have not 

been realized In the laboratory (Ingold 1979). 

Although morphologically normal and apparently fully developedg 

spores often do not Derainate until after a period of timog which may 

be regarded as maturation if ah*rt, or dormancy It weeks or months 

(Cochranes 1958)o There are two categories of dormancyt 

308. 

(a) exogenous, related to the Influence of the extemal environmente 

Tho spores may need specific conditions of moisture, temperaturet pill 

oxygen and carbon dioxide concentrationss and nutrients In order to 

germinate. Complex biological materials Improve germination In many 

species and many funoig especially mycorrhimal fungi, require the 

presence of a stimulatory factor or factors for Germination. 

TemitaDrces is only found growing on fungus comb and it may need a 

combination of the factors there to break dormancyl vh1ch may includo 

substances provided by the termites*

(b) 

constitutive% due to innate properties of the xpore which 

will not germinate even in suitable nutrient and moisture conditions* 

The spores require an acti. vation process such as 

hLQh temperatures 

(50-6000, freozingt or passage through the gut of an animal., It may 

be that the spores require to go through the gut of the termites, but 

in general this type of dormancy is less c(x=on nnd is generally 

associated with thick-walled sporess whereas basLdiospores of 

Term. iton. yces are thin-walled (Pegler 

Chandra 1975)- Patch (1906) 

solutLon, p but Heim (1940) 

giving any details* C4eo (1942) 

sporas of Tem. itMUces 

2% sucroso solutions 

and Rayner 1969, Purkayantha and 

was unable to germinate the spores in any 

reported germinating In the lab-oratory without 

was unable to germinate the basLdio- 

albinAnosus In waterl comb extract solution or 

309-

OLUYrL. 

n SEVEN 

WE MYSTOWGICAL MIS OF TIM ASSOCIATION 

D MMM24 TEMIXTOMCES AND TIM TIMMITES 

310.

7.1.1 1O, 

D3 

7ol MISTMIC CONT114TS 

The moisture content of the fungus combg foodstore and nest 

structure (2-3-2) of 8 MtcroteTnro'-i bellicosun ttounds was determined 

by drying overnight in an even. The moisture content of some Microtermes, 

Ancistroterm-s cavitharax, and Odontotermen smeathr. 1-int fungus combs and 

some paeudorhiza and mycotgte3 of Tervitontypes were also determinedo 

The Initial wolghings were made as soon after collection as possible 

to avoid errors due to material drying out. 

7-1.2 RESULTS 

The results are shcnm In Tables 7.1.1 - 7*1*8. 

TABLE 7*1-1 Moisture content of ýhcrotrrmos bellicosun foodstoro 

(expressed as percentage of fresh weight), 

SAME MAN 1101STMIE CONTENT :! S. E. H. n COLUXTED 

FIAC 36 89.39 ! 1.48 2 APRIL 1978 

I= 47 71,97 1 0*30 9 MAY 1978 

11"'c 43 71-85 ! 0-32 a MAY 1978 

tac 49 71.12 ! 0.76 5 MAY 1978 

MAC 50 75&05 1*11 5 MAY 1978 

MAC 63 70s09 0*32 5 MY 1979 

MAC 65 70.32 1. '. 

'9 5- MAY 1979 

mc 63,50 2*79 3- JUNB 1979 

HEAN 72-91 

RANGE 63e50 89-39 

- 

L L 

311.

i 

w 

Owl' " p 

0 li c 

vi %A 

t" S 9 , 

-n E 

Wn 0.0 

i-D * C3 () 

C % 

1+ 1+ 1+ 1+ 1+ 1+ 1+ 

0 0 0 

9 ,o I" w %0 -4 - to 0 

1-0 

1 

lira 

E3 

" ýC* : 

. b) C2 w 

ý 

n 

1- 

%L 

-3 q 6 

0 1+ 14 ý 

1* 1+ 1+ 1+ 1+ 

o o zi 

o o o o o 

C. - v 

-4 C* 0 

1 1+ 1+ 1+ 1+ 1+ 1+ 14 

0 0 0 0 0 

I 

ý10 

ell 

c* 

o 0 W 0 

61 Itl 

(-, raw Eý 

Z4 '. s 

c- C% 

-3 

Itcoo 

tr. 

IPO 

v 

. Z- 

913 

tr 

13 

Imd 

Nd 

Iti 

312.

TA131Z 7*1,3 Iblature content of Mcrotermes bellicomin nest structure 

(expre3mad as percentage of fresh welght)o 

W-23LU IW HDISTURL CONTMT 

MAC 47 12-30 ! 0.37 a 

H%c 48 14.97 1 Oo23 9 

MAC 49 13.2B ! o. 49 5 

ww 50 16-05 1 0-31 5 

MAC 65 11*76 ! 1.11 5 

13-67 

RANGE 11-76 - 16.05 

TABLE 7-, 1-4, Malsturo content of Macrotem-es bellicosup food (Wood 

litter)'',, (expressed as perccntgge of fresh weicht), 

tT--kN WISTURL, CONTENT 

RANGIC n COLLECTM 

6-30 ! 0-73 2.12 20 MY 1973 

TABLE 7-1-5 Holature content of the fungus combs of othor species of 

klacrotermitinae* (expressed an a percentage of fresh weight). 

TEWIM 51"XIL, S 

1 

313. 

Mý MISTURE CCL%Trm 

+ SX*M4 n COLIXI. CTED 

Ancistraterma cavithorax 43.67 ! 0-90 6 JUNL 1978 

(habba) 

tLIcr, otermes. subhXallmla fresh comb 53*51 0*20 MAY 1979 

older comb 46-07 0.47 5 

Microtermes ape A 39-70 1.22 3 APRIL 1978 

1-acrotermea, apt D lk2.09 1-34 3 AMIL 19-13 

MicroteMICS SP* C 5 0,2,2.1-37 3 APRIL 1973 

Microtermen ape G 48-53 1 AMIL 1978 

ýJjcrnterlrlea ape R 46-01 1.03 4 APRIL 1978 

(Rabba) 

Odontotermen 

- Fyiathnunl 58.47 2.47 3 AM 

I -k 

-L 

19713

TABLS 7-1*6 Hoisture contents of mycotates and pseudorhiza of 

Termitc*aceso (oxpressed as percentage of fresh welg4t)o 

A-S, WCIATED TERMITE 

SPECIES 

tW, 11DISTURE CONTENT 

SoEstle 

Wcatetes I 

K-tcrotermem bellicoqua 55-87 '24,47 

thcrotermes sunyalinus 

41.44 1.1 

Psaudorhiza of Termitonyces 78-58 + 1-40 

2erforens 

ilinclatrotermeo eavithorax 

Comb producing paeudorhiza 52*03 

TABLE 7-1-7 Other reported values for the moisture content of fungus 

ccmabj, within the flacrotemitinao* 

TMMITE 

PERCENTAGE 

MOISTME CONTENT 

AtMIOP 

Vywrotermon natalenst" 8.8-8.9 Zoberi (1979) 

M. subhnlintis 

46-7-47*4 Abo-IU'atwa (1977) 

M, ukuzli 52.8 Rohrmann (1977) 

MLCrote, rmes obesi 45.4t 470 Ilishra and Son-Sarma (1979) 

odontotermem assmithi" 48A Hishra and Son-Sarma (1979) 

cdontoternes 49. 

microdentatus 

Gs 51.2 Mishra and Son-Sarma (1979) 

Cdontotermes obe-sus 47*22 Datra and Datra (1966) 

0. 43.,.,., 

obesum 

-47.2 Batra and Batra (1979) 

0, obesus 

148.51 52.8o 56.6 Hishra and Sen-Sarma (1979) 

TADIZ 7--l-B Moisture content of the sollL in the Ho"a area (expreased 

aa a percentago of fresh weight). 

SOIL 

IMM )JUISTURE CqNT121T 

S. saf. 

314. 

COLLECTED 

Golf course 13-97 0.26 ApRIL 

Near Odontaterr. en. flight holes O. OB JVM 

Wash from flicroternaes ballicosus 

mound 9-47 0.14 JUNE 

5m from Macrotermes bellicosus 

t2ound 8.99 0.09 JUNE 

uncultivated primary wo6dland 6.04 0.21 JUND 

cultivated soil 6.29 0.11 JUND 

Rabba soil 

(forest) 

23.05 0.89 JUNn

7*1-3 'DISCUSSION 

The mointure content of the macrotermex bellicostirs foodstore in 

72,91% (table 7.1-14 When collected by the termites the food in very 

such drier (6-3%t Table 7-1.4) and so must absorb water while in theý 

foodstore. It in well placed to absorb water being above and along- 

side the fungus comb (Table 5e2o2s Plate 2*3*1)e Collins (. 1977) 

suggests almost all the food goes through the foodstorewhero it 

remains from 1-2 vecks. Little decomposition by fungi occurs here 

(5.6.1) though many spores are present, but some bacterial decay may 

occur. This absorption of water by the foodstore may make it more 

palatable 

to the termites* 

The moan moisture content of the fungus comb of H. bellicosus 

in 47-16% (Table 7ole0o In each nest sampled the fresh edge of the 

comb had the hithest moisture contents but there was little difference 

between the zones* The fungus combs. of the other species of termites 

sampled had similar moisture contents (Table 7-1-5). The values are 

similar to those obtained by other authors, except Zobari (1979Y, for 

different termite species (Table 7.1-7). 

Tho fungus combs havo a large, surface turca and would readily be 

able to take up and release water* GhIdIni (1938) was the fLrBt to 

suggest that the fungus comb may help to maintain a suitable humidity 

315. 

In, the nest, -and this has since been considered by Batm and Batm (1966) 

Hesse (1957) and 12scher (1951&# 1961), The humidity In an important 

factor-in the survival Of termites an they aro-susceptiblo to desiccation* 

This would be especially Important for the young termites which-are 

found In the folds of the fungus comb* The relative humidity in the 

fungus combs -In, higher than In the wall of, the mound (316cher 1951&)

cmd In maintained around 93-99% (Ilischer 1961) and never below 96.2% 

in Ricrotermes bellicosust and 85-955 In Odontoterraps, obestin (Cheema 

et al. 1962)o The moisture contents of fungus combs lie within 35-50%9 

the rango most favourable to woodrotting fungi (Cartwright and Findlay 

1958)o The moisture content of the Ancistroterrms cavithorax combs 

producing Termitomyces pseudorhiza in slICjhtly higher than those not 

producing. This increase in moisture centent was one of the factors 

suggented in section 

of cellulose 

I an perhap3 initiating fruitbody production. 

The source of this moisture would be from both the decomposition 

(Lee and Wood 1971) by TernitomXces and by the metabolic 

activity of the termites where the enzymic oxidation of glucoae would 

release water and carbon dioxide 

C6111206 + 602 1 6CO2 + 61120, 

316. 

scmmo of this water would be re-utilized in metabolic proce3sons and there 

is doubt an to whether these two processes alone would provide enough 

water to maintain the relative humidity* 

The moisture content of the nest structure. 14%, (Table 7.1.3), is 

greater than the surrounding soil (Table 7.1.8)t the soil moisture 

content, in the areas where the Macrotermen bellico.., un mounds were found 

usually being equal or less than 12%% even In the rainy season (Collins 

1977). Hesse (1955) found the peftentag(y moisture-of Inhabited Were- 

temea mounds to be -1*21.26.9%, - compared to 44-8% In uninhabited woundse 

Lee and Wood (1971) have reported that termites bring fina wet subsoil 

particles from near the water table for wound construction, and-thin as 

uell an metabolic water would be r*nponsible for the higher moisture 

contento of the internal nest structuro over the adjacent soil. Evapora- 

tion occurs In H. -b-111conus 

also helping to maintain the humidity* 

from the spiral plate (Collins 1977)s thus

An well as supplying moisture the metabolism of the termites 

and fungu3 supplies heatj e*Q* a Continuous production of 3#5 watts 

of heat should result from 10 kg of fungus comb (Rohrmann 1977). The 

temperatures in the mounds around the fungus combs have been shown to 

remain fairly constant at around 300C OZscher 1951a, Checma, et ale 

1962, Collins 1977)e This to close to the optin= Unperature, for 

growth of'Termitomyceis 

(29OC9 Section 3-3), and in tropical te , mites 

the growth and development of-colonies and individuals slows dýwn below 

this temperature (111ficher 1961). Terttit=ces, therefore also plays a 

part in maintaining the temperature of the nest, We to the heat 

production Inside the mound convection currents occur. There is a 

gradient of temperatures with distance from, the fungus comb (Collins 

1977), and there In therefore a movement of heat froa the nest to air 

via the mound wall, * Buring the hottest part of the day the outside air 

may be hotter than inside the nest. Therefore the convection currents 

are not constant and heat loss would probably be due to the cooling 

effect of evaporation and losses to the soil. Weir (1973) 

317. 

showed how 

the airflow through and evaporation in mounds helped to stabilize the 

temperature. These convection currents also enable gas exchange to 

occur through the wall* of the mound thus regulating the Internal 

atmosphere. Batra and'Datra 

(1979) have suggested that this temperature 

gradient in soils may assist tei-mites in bringing up subooll water, 

Collins (1977) showed from the relativq respiration rates of termites 

and fungus comb that it was tho fungus that supplied the =Jority of 

the heat* The termite3 do regulate the mound temperature by their 

buildino activities.

7.2.1 ýMIODS, 

7.2 I! H 

The rAi of the f"stom and three Zones of fungus COMb Of 

flacrotermes bellicosus, and whole fungus comb of Ancintroternpa, 

cavithorax. 

Macrotermes 

-qubhnlinus-. 

Hicrotermta species and 

Odontoterms sneathmani was determined with Merck Spezialindikator 

papers (rango p1l 4.0-7-0). To determine the pit 19 of fungus comb 

was crushed in 6 ml distilled water, and Ig of food3tore in 2 ml 

distilled water. n-1. 

7-: 1-2 RESULTS 

The results are oiven In Tables 7-2.1-7.2-3. 

TABLE 7,2,1 pli of foodstore and fungus ectab of Macrotem. es bellicoaua* 

SAHPLLI; FOOLISTOM 

MESH FUNGUS 

cum 

MIDDLL FUNGUS 

COM 

OLD FUNGUS 

Culm 

MAC 36 5,, 8 4.2-4-3 4.1-4.2 4a-4.2 

MAC 47 5-6 4.2 4.1-4*2 4-3-4.4 

MAC 48 5*5 4*2-4*3 4*2-4.3 

mAc 49 5,4 49,2 4.1-4.2 4.,, 

*, 

MAC 63' 5-3 4*4 4.3-4.4 4.4-4.5 

MAC 65 - 

MAC tF#_ 1 5-6-5*7 

4.4-4.5 4.4-4.5 4.4-4.5 

L-1,05-4.6 L- 

4.4-4.5 4.11k 

318*

TABLE 7-2-2 pli of fungus comb of other species of termitese 

TERMITE SPECIES pil 

Ancistroternes cavithorax 4.4 

Macrotormen subhnlinum 4.4 

Microtermes op* A 4-5-4.6 

Hicrotermes op" D 

Hicrotormes op. C 

Hicrotermes spo G 4.1-4.2 

Microtermes ops R 4.1-4*2 

Odontotermes smmtl-^-tni 4.3-4.4 

TABLE 7-243 t1leported values for ihe pli of f'ungus comb of tertmite 

spocierme 

TERMITE SPJDCIES pli AtMIOR 

Macrotemes bellicosus 4.5 liesse (1957) 

thcroterries lioliath 

4.3 1108SO (1957) 

Macrotermes natalonsis 

4.4 Itessc (1957) 

11. natalensis 

Macrot 

4-5-4-6 Zoberi (1979) 

=rm*,, subhyplirwa 

4.2--4.7 Abo-Yhatwa (1977) 

Microtermes obesi 4.2 4.!; 

j Mil5hra and Sen-Sarma (1979) 

Odontatermem assmuthi 4.6 Mishra and Sen-Somma (1979) 

Otlontotermes gunq2Mrensin 4.. 5-6-85 Datra and Batra (1979) 

Odontoternes 

Odontotemes 

microdentatus 

obesus 

4.4,4.8 

4-5-6-85 

Mishra 

134tra 

and Sen--'Sarma (1979) 

and 134tra (1979) 

Odontotermes rodemanni 3-9-4-3 Wieril and mitra (1949) 

Odont 

otermes obesus 4-71-5-3- Hishra and Sen-Sarma (1979) 

- 

3190

7.2-3 ]DISCUSSION 

Tho fungus combs, of all opm-jes of termites investigated wore 

acidic and ranged frcm P11 4.1 to 4-6 (Tables 7.2.1 and 7-2-2)- This 

agrees with the values obtained by other authors for different termite 

species 

(Table 7-2-3), The food3tore is more alkaline tlmn tho funWS 

comb, varying from Of 5-3-r,, 8, There is tittle difference between'tlie 

zones of the Macroternes bellicosus fungus CoMbo with tile fresh zono 

beino alitihtlY less acidic than the middle zone. Batra and Batra (1979) 

*Amilarly found that the p1l of the upper, younoor parts of the comb was 

higher. This lowering of the pil may be due to the activity of 

Termit2Mcpaj decormosition generally causing a lowering of 01 due to 

selective uptake of cations or the production of oroanic acids and C02 

(Swift, Ileal and 

Anderson 1979)- 

The pit has a definite influence in determining the dominant species 

to 

of fungi (K'aarik 1974). flintikka (1971) found that the acidity of wood 

attacked by wood-rotting fungi varies within fairly wide limits,, pH 2*0- 

1 

4.0 being cocinono while aarik (L974) cites Butcher (1966) as finding 

that p1l values of 4.0-9.0 were tolerated In the wood-inhabiting fungi 

tested by hime The low pil in the fungus corab would tend to prevent 

bacterial developments bacteria being generally less tolerant to low 

p1l than are fungis 

The P11 of the ftmguj% cc=b appears nuitable for tho activity of the 

cellulases of Termito=c =.. Martin and Ilartin (19-t-S) found the major 

activity of the major C, enzyme from TemJt=cp_q, from Itacrotermen 

natalensis was in the p1l range 3*9-4*059 and of the minor enzyme was 

in the range 4-2-4-35. Hisra and lRanganathan (1951, k) found tho optim= 

rJI for the callobinse was pil 49 and of the collulasog 3.8-4.4, from 

Odontotermes abeaus_. These optima aro slightly lower titan reported 

3200 

,"

for collulanes from other sources. - Mandols and reese (1965) found 

fungal cellulases at 300C were activo from P11 3-5-7-0t with an optimum 

at P11 4-5-5. Uesai and Pandey (1971) report that most cellulolytic fungi 

are active at pit 4-6-5. 

It was not Possible todhtermine the pit of the mound material due 

to the colouration of the solutions but In general it in higher than 

the comb and "lightly higher than'the soil it is constructed face (Wow 

and Sands 1978), Variations within the mound may Occur due to mineral 

deposits from evapotranspiration. Zabori (1979) found pli changes with 

the season in the outer layers of the mounds but the comb yAi was stable. 

32-11-

7-3-1 INTRODUCTION 

7.3 LIGNIN DDGRADATION 

The bulk of energy in plant tissues in'tontained vithin a variety 

of polysacchirideng sugars and lignin (SwLtt, ot n1o 1979)o PI&Ut tiesuO 

contains 

herbaceous Material 

'up to 35% lignin, wood generally having higher levels than 

part of the food materials of ternitese 

(Table 7-3-1)-- Lignin therefore comprises a large 

Licnin in a very complex polymer, the chemistry of which in 

incoopletely understood (swift et al. 1979). TAgnins from different 

sources may differ in their properties as units are linked by different 

bondse They are a group of substances based on three-dimensional 

complex aromatic polymers. The basic unit is a phanyl propane skeleton 

with a hydrocarbon chain attached to a phenolic group, The chemistry 

of ligning i*eo the variety of different bonds, the steric interference 

provided by aromatic monomers with a variety of side chains, Its highly 

branched and folded structure and Its hydrophobic riature, hinders 

enzy=tic attacks resulting in lignin being the most slowly decomposed 

of plant cell wall components (Swift ot al. 1979). 

During secondary thickening lignin In laid down in the cell wall 

thereby strengthening it. There is controversy as to whether some 

degree of covalent bonding occurs between lignin and r-oolysaccharides 

(Swift et al. 1979), The association of lignin and cellulose, which 

may be largely physical, forms a mutually interpenetrating systom of 

polymers, with the lignin formino a Icage9 around the carbohydrates,, 

This aasociation protects CeIIUIOSO from attack by collulolytic enzymes 

(La Face and Nutting 1973), and it in thought responsible for the 

resistance of wood to microbial decay, except by fungi that have enzyme 

systms capable of depolymerising lignin an vall an the carbohydrates 

of *ood (Cowling 1961). 

322.

Qnly, a restricted, jrAnge of *rUani8M3 can MQtA1)0l1zQ, t11Q Intact 

lionin molecule (SWift. et al, 1979), Lignin is at least, partially 

degraded by both thl higher and lower termites (La PaGe and Nutting 

323* 

1978)o Some species ot termites are able tog at least partiallyg degrade 

and assimilate lignin units by the, action of symbiotic bacteria and 

protozoa in the Cut (French and Bland 1975, Lee and Wood 1971)o 

Nasutitermen exitioxuas when fed radio-active ligninji produced radio., 

active C021 sho%finq it could demethylate and depolyme-rine natural lignin. 

Butler and Duckertleld (1979) suggested the lignin was partially 

degraded In the out to small units which could pass into protozoan 

cells or through the out epithelium to an aerobic environment where 

oxidation to C02 could take place* Other species, such as the 

Ternopsidae and Rhinotermitidae. usually Infest wood already decayed 

by fungi (Datra and 11atra, 1979), The Macrotermitinao generally feed 

on sound woodt manY species also feeding on plant debrial and socne on 

leavese Their food therefore contains et laroe, aMount of lignin (T. 

'bje 

7*3-I)t andg as they lack symbiotic protozoaq it Is generally considered 

that Termltorycps breaks this down (Grasse land N01rot 1957,19581 Heim 

1977; 14o and Wood 1971 and Sands 1969)9 

Wood inhabitlngýmicroorganisms can be divided into, 

(1), moulds and blue stain funaL 

but do not enzymatically degrade lignified cell walls 

-which exhau3t dead call contents 

(KRIrik 1974)- 

(2) organisms capable of enzymatically degrading lionified call 

walls but with a lWtcd degradation capability 

(a) bacteria* They cause only slow cell wall disintegrationg 

mainly attacldno parenchy= cello of the rays (Kaa"rik 1974). The 

ability of any true bacteria to substantially dec(mpose lignin has 

not been shown (La Page and MuttInO 1978).

(b) soft rot, fungi. Under very moist conditIMIS certain 

Ascomycetes and Fungi Imperfecti can cause substantial destruction 

of wood. They principally attack carbohydratess mainly cellulose of 

the cell wall j hemicellulose and pectic materials$ and lignin may be 

modified or perhaps degraded to a lesser extento They are pioneers an 

newly exposed wood, soft woods being more resistanto They cause slow 

attack inward from the surface 

(I=rik 1974), They generally only show 

a limited capacity, for attack on intact wood cello in pure cultures 

'(Swift 1977). 

(3) Organism with high degradative capability 

(a) brown rot fungis These primarily attack the carbohydrate 

content of wood, but may alter, but not decomposes lignin* 

(b) white rot f=01* These have enzymes capable of degrading 

both lignin and cellulose. These can be further divided into 

simult, aneous rotters which simultanomsly deccmpose all the bubstances 

of the lionified cell wallj and sequential rotters which decompose 

wood successively beginning with lignin and hemicellulose and only 

deteriorating cellulose at a later stagee 

The oxidase or 'Davendamm9 reaction Is used to distinGuish between 

white and brown rot fungi, Certain species of wood decaying fUnoi form 

a darit diffusion zone on agar containing polyphonols (Callic 

acid). This reaction in due to the secretion of an oxtra-collular 

and tAnnic 

polyphenol oxidase of the laccase type (phenolase), An almost total 

correlation has been found between the ability to oxidize gallic acid 

in agar to its brown coloured quinonLc form and possession of lignolytic 

ability by Basidlomycetes (Swift ot al 1979), with approximately 95% of 

white rot fungi giving a positive reaction (OwUng 19619 Davidsong 

324-,

Campbell and Blaisdell 193'3)e Termitomnen_ cultures from different 

termite species were tested, for tho reactions on both gallic and,. 

tannic acid media% and for their effect on rcd_cabbage extract which 

also detects the presence of extracellular polyphenol oxidases 

Ojergen3en 

and Vejlby 1953)o 

TABLE 7.3.1 Some organic components of Plant material. 

LIGNIN CELLULOSE MrXTCMJXLOSE 

PLCTIC 

SUBSTANCES 

Plant tissue 5-30(a) l. 5-6o(a) 3-40(f)

7-3-2- )ODIODS 

(1) Red cabbage extract (JOrgesisen and Vejlby 1953). 

Termitm3T! osa cultures associated with different termite species 

were grown on slopes of Pl)ý. (Appendix 1), When well grown 5 MI of red 

cabbage extract was added* They were left at room temperature and the 

colour of the extract noted after 3 and 7 dayso 

(2) Bavenda= test (Davidson et al. 1938). 

Plates of rýalt agar to which gallic or tannic acid was added were 

inoculated with Termitomyces cultures associated with different tomite 

specieso The cultures used were from botli sr, medium and frcra the 

selective mediumel This waslin case the previous medium had any 

inductive effect. The cultures were incubated at 290C and cbecked 

for the oxidaze, reaction after 2 days* 7 days and 14 days, There was 

generally little change after Tdays* 

Termite outs* 

Thirty outs from Macrotormes bellicosu_9 and 15 Vts from 

Hicroterren or. 0 were placed on PlAtOB Of CPllIc and tannic acid 

media@ The medium contained cycloheximide to prevent fungal growth, 

7-3-3 RESULTS 

The results are given In Tables 7-3-2-7.3-4. 

3z6.

TADLE 7-3-2 Reaction of Termit=. ex, culturen on rod itabbage extractl 

after 

7 daYs- 

ASSOCIATM TMMITE SPECIES I-SOLATIM NMOM =I CTION 

Ancistrotermes cavithorax AN 51 3 

AN 5.3 

3 

AN 5 r) 3 

AN r, 6 3 

AN 57 3 

AN r) 8 

AN 59 

.3 

3 

Hacrotermes bellicosus MAC 12 1 

Mc 36 1 

11AC 47 1 

mc 48 1 

MAC 49 1 

n, %c 50 2 

tlicroternppl spe A mic 38 3 

MIC 44 2 

op. B MIC its 3 

RIC 43 2 

op* C HIC 41 3 

ape D HIC 40 3 

op. G RIC 45 3 

op. IZ RIC 46 3 

op. Z RIC 37 2 

unknoun foraging worker FORAG. 10 3 

Odontotermes smenthmnni OD 42 2 

Cdontotermas op. OD 54 

-1/0 

If tho test in pooitive for polyphenol oxidases the extract goes 

yellow. If the test is negative for polyphenol oxidases the extrnet 

goes red* The Control 

redder than control 

as control 

(PM 

vith no tunql) stays purple* 

I an control at 3 days, slightly paler at 7 days 

2 yellower than control 

Yellow 

Order of production of polyphenoloxidases 

Ancistroterraes c-avithorax > lllcrlOtleMQ3 I3PPo > Odantotemes 

I-lacrotemas bollicosus > ýýontotermcs 

gpe 

32-7*

TABLE 7-3-3 Reaction of TermitVycom cultures on gallic and tannic 

acid media after 14 days# 

ASSOCIATED TLI41ITL SPECIES 

ISOLATION FU=TION ON PEACTION ON 

NUMET, GAMIC ACID TANNIC ACID 

Ancistroterm. oss cavithorAx AN 51 3 4 

AN 53 3 4 

AN 55 3 4 

AN 56 3 4 

AN 57 3 10 

AN 58 3/4 4 

AN 59 

AN 69 

3/4 4 

-3/4 

NOT TESTED 

11-tcratormps bellicosus MAC 12 0/1 0 

I-L%c 

36 0 1 

UU; 47 1 2. 

MAC 48 0 0 

MAC 49 1 1 

MAC 50 1 0 

Mc 65 1 NOT TL3TED 

1-facrotermes subityalinus SU3 21 3/4 NOT TESTED 

sun 6, 

-, 3 NOT TESTED 

Hicroterms op. A HIC 38 3 

mic 44 2 3 

op. 0 HIC I /* 1.1. NOT TLSTZD 

HIC 43 3 3 

op. C 

op. D 

MIC 41 

HIC 

3 3 

40 

3 3 

op. G HIC 4-15 2 1 

ope R 

Mc 68 

HIC 46 

4 

3 

NOT TLSTED 

4 

op. Z tac 37 3 3 

unknown foraging worl-cer FORAG 

* 10 3 

Odontotermes smenthmani OD 42 1/12 

Odontotermem ape OD 54 1 

no discolottration 

I discolouration of agar plug of inoculura 

2 diffusion zone light brown, only visible from underside of pinto 

3 diffusion zone light brown, visible from top of plate extending a 

short 

distance 

diffusion zone daric brown, extending con3lderably 

order oflroduction ot polyphanol oxidases. 

323. 

Anclatroterrips cavithorax > Macrotermen subhyalinjig > tficrotcrm. oppe 

Odontotormes simeathmani > t%crotermes beill COsUs ;;,, Odontotermes up, 

0ý

TABLE 7-3-1k Ile. Ictions of ruts of Macrotermos bellicosus and 

Ydcrotermes species on gallic and tamic acid plater (aftor 24 hourn). 

TEPMITE SPLICUS REACTIM 

NUMV., VS UN 

GALLIC ACID 

NUI-MRS ON 

TANNIC ACID 

Kicrotermes ballicosus 2 20 '23 

1 a 3 

0 

Hicrotermes sp. 2 0 0 

15 soil in gut 1 0 0 

1ý) fo od in gut 0 15 15 

0 no colouration 

1 very faint brown colouration 

2 dartzer brown colouration 

7-3-4 DI6CUSSI014 

These results ahow that Termitmnypen has characteristics of a 

white rot fungus in producing polyphenol oxidases of tho laccase type, 

There is a strong correlation betwecn possession of polyphenol oxidases 

and lignin breakdoum, although the polyphonol oxidases are rarely 

concerned with ring breakdown. The enzymatic breakdown of lignin is 

not well understood but W3 probable a multi-enzyme System Is requira. d 

(Kirk 1971t 5ýllft 1977). Two hypotheaes havo been put forwnrd. The 

first is that tho InItIal depolymerialng steps are by extracellular 

cleavage of the inter-urdt bondse The secand Is that the primary attack 

comes not an Intermonomer bonds but on the aromatic ring. This suogezta 

a single mechanism for the Initial opening up of the polymer rather than 

the multiple enzyme system necessary for Initial depolymerization based 

on cleavage of intermonomer bonds (5witt 

Ot al - 1979). The ccoplote 

process in not yet known'* It to likelY different lignin degrading 

arganis= may have cufferent enzyme syntems. 

32,9 

*

The reactions did not vary In any consistent way from inocula from 

the SF medium or selective medium. Polyphenol oxidases producing these 

changes are often present in other Basidiomycetes only at particular 

times in the culture cycle, especially during autolysis, or only in 

particular morphological structures 

(Cochrane 1958). A similar picture 

was shown in those inocula fron Macrotermes bellicosus which gave a 

colour change. The colour was located in the conidial agglomerations 

which were present on the inoculum. 

No growth of the cultures occurred on gallic or tannic acid media* 

Davidson et al. 

(1938) found that gallic and tannic acid were toxic to 

many fungi, and obtained the most intense colour change of the medium 

from those with no growth on it. Phenolic compounds often exert a toxic 

influence on the growth of Basidiomycete lignin decomposers. The most 

resistant are the primary wood decomposing speciess while those occurring 

at later stages of decay are more sensitive, but there are many 

exceptions 

(Hintikka 1970- 

In general the reactions of the cultures were consistent for 

different isolations from the same species of termite. There was 

agreement between the results obtained with the red cabbage extract, 

and on tannic and gallic acid media. In both cases Termitomyces from 

Ancistrotermes cavithorax was the strongest producer of polyphenol 

oxidases. Termitomyces from Microtermes'was a strong producer, whereas 

that from blacrotermes bellicosus was only a very poor producer of 

polyphenol oxidases. This may have some bearing 

on the fact that 

accumulations of old inactive fungus combs have been reported in the 

centre of some M. bellicos! is nests, whereas in Microtermes the combs 

are completely recycled* 

330-

The reaction of tho Ii. bellicosus out Vouid Indicate the presenco 

of e xtra-cellular Volyphenol oxidases, The intemity of the reaction 

of the conidial agglomerations In culture indicates that these poly- 

phenol oxidases may well be obtained from the mycottten. In Micro- 

termes, guta no polyphenol oxidanes were present, though of course the 

guts are very much smaller than those of M* bellicosus. Vohrwann and 

Possman (1980) found that Termitgaces from Macroternes Wýwzii 

produced lignin decomposing enzymes. 

It therefore appý6'arn that one of the functions of Termitc, ces', Is 

ny _ 

to broik do%M the lignin present In the food of the termites. It 

appears that Torýdtonr fcc s enables the Macroternitinae to utilize 'o a und 

wood'that might'Ahervise be unassimilable, by then and it to interesting 

that, SrhArrotermok which has sterile combs with no Termitomyces feeds 

on r6tttno wood. 

331-


7.4 CELLULOSE DWJUDATION 

Plant tissue contains from 15-64% cellulose (Table 7-3-30- 

Cellulose In wood Its very resistant to bacterial attack largely due to 

the presence of lignin* The cellulose of wood Ix a linear polymer of 

d-glucose units linked by 1.4-jl-glucosidic bondsj In the form of long 

linear molecules In a crystalline lattice bound laterally by hydrogen 

bonds between hydroxyl groups (Cowling 19619 Swift et al- 1979)o 

Cellulose breakdown requires at JeA t three different types of extra 

cellular enzymes (Swift 1977)o Cl acts on crystalline cellulose, 

pushing tho, polymer chains apart physically and creating free chain 

encts,,, C. In a ccuplex of enzymes hydrolysing the P194-olucosidic bonds 

in the cellulose volecule* Those produce celloblose on which colloblase, 

or P-glycosidases acts to produce glucose, Cellujolytic micro-orgmuUM 

which grow on native cellulose produce all the enzymes* 

Cellulases, are produced by symbiotic Intestinal flagellate protozoa 

In lower termiteaq and probably by bacteria In some higher termites 

(Dreznak 1970- Potts and Hewitt (1973) 

showed Trinervitermes 

trinervoldes p roduced Its own callulaneo Some higher termites way eat 

wood which has already undergone decay. Heim (1977) 

considered that 

Terml&ýeg, did not decompose cellulose. The cellulose decomposition 

abilities -of cultures of Tormitgnces associated with different termite 

species were investigated here by the use of cellophan4i films dyed with 

332* 

lkswwl Brilliant Blue R dye* Thin dye Is bound to the cellulose molecule 

and released quantitatively In projx)rtlon to glucose (Moores Banset WO 

Swift 1979)a

7-4-2 

DYIng of cellophane strlpxo 

Approximateir 3a of pro-cut standard non-mistureproof cellophane 

strips (2 x5 cm) were boiled In 2 changes of 500 ml distilled vater to 

remove plasticizers. The hot water was removed and 500 ml of cold 

distilled water added* and heated to 800C while stirring to keep the 

strips 

333- 

I suispendiid le'S'_g of hemazol Brilliant Blue R dri was added., 20 ed 

of sodium sulphatia solution 

(30 a In 100 ml IH20 at 800C) was added at 

2 minute intervals- over a period, of 10 minutes., Then A solution of 

2q64 -g trisodium orthophosphateln'15 ml, distilled- 1120 atý SOOC was 

added* -The solution was-left at 8CPC for 20 minutes and then washed 

with hot water iintil the washings were colourless. The strips were 

then autoclaved In 2 changes of II Hn. 

0 for 15 rdnutes at 15 lbs 

pressure to remove further excess colour. 

Experimental procedure, 

The strips were placed on agar plates of selective medium (without 

the cellophane overlay) and Inoculated with TermitoWcez frM different 

temitO 9POcI099 Five strips were set up for each species, These were 

then Incubated at 2910C for two weeks ww the growth of the cultures an 

the dyed films were measured, 

Utraction of the dyed films* 

After two weeks growth the cultures wero rmoved from the dyed 

strips* The fungus was brushed off the film with a paintbrush dipped 

In distilled water to ensure that no extra ougar, 39 which cause purpling 

of the extracto were presente The films were then autoclavod in 50 ml 

distilled vater to remove unbound dye and any metabolic products that 

may have accimalatedg and then autoclaved in 60 ml of 0,5% solution of 

KC0 to extract the dye rm&Lning In the strip, The optical density of

the solution was read at 595 no against a distilled water blanko 

CZUAJLDSZ REMINING 

IN STRIP 

7.4.3 PMULTS 

OeDs TEST STRIP 

O*Do CONTROL 

The results are given in Table 7-4-1- 

x 100 

TABLE 7*4.1 The amount of cellulose removed by TermitgMces cultures 

after two weeks growth on cellophane strips. 

ASSOCIATED TFMTE SPECIES 

ISOLATION 

MMER 

Coum 

DIAMETM 

(mm) 

% CELLULOSE 

REMOVED 

Ancistratemes c"Ithorax AN 53 17*27 0*33 

% CELLULOSE 

MOVED 

Wcrotermes belliconus MAC 36 15*70 24*76 1.. 513 

Microternes ape A HIC 38 19.56 27-89 1-43 

_ lip. B HIC 43 16&39 31-97 109.5 

ape C MIC 1*1 14.64 15-72 1*07 

lope D mic 40 11-57 17-01 1-47 

ape G MIC 68 18*37 23.61 1*29 

ape R MIC 61 18.98 30.61 1.61 

ape Z HIC 37 14.10 19*05 1-35 

Odontotermes wwathmni OD 42 17-M 9-77 0,57 

=t-l 

334*

ta 1* 

L(y% 

I 

tz 

4 

p OW 

0 

a 

4, 

4 

m 

I 

ý% "4 

6 

cs 

, 

lit 

ol 

0. 

tq 

L--- L-j 

336.

0 :1 

1 m 1 JAN 

5 

k*O 

10 

ýe ý 

tv 

p 

9 

1--- 

6 

e 

:0 

-4 ta %0 

Co 

Mn 

9 00 w 

10 

q 

f> 

3 

ra 

to 

Z R 1 0 

L 

R ? ýE O= F F 50 

9 

p 

0 

la 

0 

pf. 

:r 

Nko 

4 

in 

m 

NN 

ra 

W 

ra c3 

CN 

a 

u %0 

0 

ý3 

0Ze 

3 rem 

:r 

b, 6 8 6 ý, 1 1 

u 

Q2 

t, 3 

ß3 pp F 

4 t* 

Z > 1 ky 0 

O> 

fb jP 

1 

ba 

>% 

m 

ß3 u 

-3 

«2 j!. o los 

6. d 

ý i c 

:2C, -. 0-% 

$*d IP gb 

%W 

- 

42 

W ta 

40 %W %»# 

Cm tA t4 

f 

-] 

-i 

%0- -. W 

0 

6,6, 4, 

% th %4 -j > 9 

CN 

1 

9 

r4 

9 

o 

335o 

S 

s-I 0 

$ 

S 

I 

ca 

1A 

FA 

a

PLATE Z. 4.1. 

Temitomyces culture from Macrotermes bellicosus clearing 

I 

Remazol Brilliant Blue dyed cellophane overlay. 

337*

- «ý 

4e _e 

%, >ýý 

. *- 

A. 0ý I, 

dk '.. 

-* 1* )W- I 

"ý ýrt -, 

Ak 

Owl 

M. 

-

7-4-4 DISC=ICN 

, All the cultures of TermitpMees decomposed cellulose* The 

culture from Ancistrotermen eavithorax was a very poor producer of 

callulasex And decomposition was not evident until 3-4 weeks after 

setting up the experiment. In some cultureng e. g. MAC 369 the 

celloplutne was cleared beyond the edge of the colony 

(Plate 7-4,1), 

this being one of the strongest producers of callulaseso In order to 

decompooe the cellophane the Termitomyces cultures have to produce the 

338- 

full complement of enzymes needed to degrade native calluloac. Variations 

In cellulose production occurred between Irux: uia of the name speciese 

The Macrotermitinae have no symbiotic cellulose digesting protozoa 

=4 there have been different theories as to how cellulose decomposition 

occurs In the sub-family* There In evidence that the enzymes necessary 

for the digestion of native cellulose occur In the Cut of several 

species* e. g. Hkeratermes natalensin (Martin 

Hacrotermes subhn1inus 

and, H&ftIn 1978$ 19799) 

(Abo-Khatwa 1978) and Odantotermes obesus 

(Misra. and nanganathan 1954). The source of these enzymes were 

considered to be bacteria by Misr& and Ranganathan, Rohrmann and 

Rosman (1980) found cellulose decomposing bacteria In the gut of 

Hacrotermes ukuzlie They also found protozoa* In contrast Martin and 

Martin showed that- Cl cam from Termlt2gmyce-s and. the Cý, and P. - 

glucosidase front Ingested fungal material and partly from secretions 

from the midgut epithelium and salivary glandse Abo-Khatwa (1978) 

showed thatj in starved Hacrotemes subhyalinus the level of celloblase 

remained uncluýngad but those of the other cellulolytic enzymes 

decreased$ showing that the latter came from Termitomycese One of 

the problems In using starvation techniques in that the levels of

enz, yme production are affected by the c6acentrations of the substances 

present in the gutj for examples cellulose activity being Induced by 

the presence of cellulose or calloblosea Further evidence that the 

presence of living Termltgpyces to required In cellulose digestion van 

given by Sands (1956) who showed that Odont(itermes badium supplied 

with colltilosic materials only or with sterilized fungus ccmb survived 

tu) " loýnger than when starved, If supplied with fungus comb with li'ving 

A 

mycotetei they survived lonw* In contrast to those ilacrotermltl=6 

with TermitoMmes Sphaerotermem spimerothorax has a cellulolytic flora 

similar to that in the rumen of cattle (La rage' and fluttino 1978)o 

The study of the lIgnin and cellulose content of fungus comb does 

not appear to throw much light an the precise role of Tenm1t=ccjj in 

cellulose decomposition* Apart frca nohrmann (1973) 

339o 

j most authors have 

not distinguished between newly depopited fungus, comb and comb on which 

TermitomXSPshaz been working for Occe times mAking It Impossible to 

assess the changes In composition c6used. by the I activity of Tern'itow-ypen 

(Table 7-4-2)- 14th 'the exceptIca I of Nkcrater, ýV4 

Douglas 1970) the porcentago - of I licnin iii 

- 

Lalfath, (Ck: ellk and' 

t: he"cb6b in lower than that 

of the toruites tiotential foýd ýna In both Wcrotitrmem iýatalensis 

Macroter-mes uýuzilthe perc'eniigo of "119nin'fails frc'm, ý6ish to old 

ýomb (Pohma 

nn 1978) show i ng, the 

s,.. 

actl' on of TermitomXcese 

.I -* wn, "". 

- 

11 11ýýI 

_The altuation, with cellulose In more complex, In the comb It 

appears at the saw levels. as could occur In 

-, woody materials but no 

direct ýýmparirjons are available* The percentage of cellulose appears 

to Increase from fresh to old comb due to the conversion of lignin Into 

fungal, tissue. (]R? hrmann 1978)o The, licnin to cellulose ratio. of newly 

emetructed Nacrotermen ukUzii Cmb im eiwilar to that of tho stma wId 

wd

iiaves 

of crasses on"vhicih they aro foraging Whrmann 1978)o This 

ratio decreases an Vio'comb ages indicating lionin 1ýýeakdown 6y 

Termit2Mcez an d*zcrLbvA above* Grasse6 and Nolrot (1961) considered 

the lignin to cellulose ratios Indicated -relatively undigested plant 

materials were usedo As the majority of cemb analyses are based on 

whole comb on. which_TPrm1t2Mc4-js has accomplished some lignin amd cellulose 

breakdowns little can really be drawn from the Ugnin, to cellulose ratios* 

In contrast Abo-Khatwa, (1977) obtained a very high lignin to cellulose 

ratio for Macrotermes vubhyalimts indicating partially digested plant 

material had been used for Its construction. Datra and Batra (1979) 

found all combs had loss cellulose than the raw materialso This would 

indicate swo prior digestion of cellulose may occur before It reaches 

the c(=bo White rotting fungi have different strategies as to how they 

attack lignin and cellulose* but one of the taost usual In to attack 

lignin firsts followed by cellulose and this probably occurs In the comb. 

It to likely that different termites have different strategies. In 

species where the food in mixed with TemitomXces In the Cut it Is likely 

that some decomposition occurs prior to deposition on the fungus comb, 

340e 

The most likely scheme may be that the zmterial in deposited on the comb 

by the foragers with more or less Intact lignin, with perhaps some 

accessible cellulose having been digested* Comminution of plant material 

by termites will rupture cell walls exposing cell contents to the action 

of gut enzymes or enzymes of Termltggyces In the out. This lit an 

Important difference from "straightl wood decay by fungle Termitgw con 

then breaks down the lignin cellulose complexes, the termites reeat the 

comb with fungal material and cellulose digestion In completed* Cellulo- 

lytic bacteria have been Isolated from the comb (Batra and Datra 1979).

ut In general the ccab in too acidic for the development of bacterlat 

though bacterial action way be Important in the roodstore, of ýkcrotermez 

bellicomis. Rohrmann and lRossiman (1980) 

the main cellulose decomposer in the fungus combo 

considered TemitgMes to be 

341o

7-5 'AVILIZATION OF STAM11, C311TIN AND PECrIC SU13STAYICES 

IMODUCTION 

The major polysaccharide components of wood cello are cellulose* 

homicelluloses and pectic c(=ponents (Swift 1977)o The hemicolluloses 

comprise 3-40% of most'plant coll walls with pectic substances ranging 

from < 1-46% (Mandels andlReene 196!; )* The total carbohydrate fraction 

of wood to known'as holOCellulose wýich to divided Into'two tractlon3s 

tho hemicellulases and ol;. cellulose. 11cmicellulosen consist of two 

fractionag P. ýcclluloze mid 19-colluloseo' The three basic fractions 

differ Maiply in the number and type of sugar units* Tho constituent 

sugars Includo xylose, mannoset galactose and arabinose., In herbaceous 

plants pectic materials are the cementing substances between cells, (in 

woody plants It in lignin) Wirk 1971)s forming the middle lamllag and 

are built up from P 1-4 linked galaciuronic acid unitg (Gray and 

VI'llitims 1971)9 

Starch Is the principle reserve POlYs&ccharide of plants and In an 

excellent carbon source for most fungi (Gray and William 197l)- It lis 

a mixtiire of two'Polymars of glucose, mrflOse mW aMylopectine kkay 

bacterlas actirsomycates and : fungL hydrolyze starch by producing extra- 

cellular camylason. 

342o 

Chitin consists of long straight chain polymers of N-acetylglucoamine 

units joined together by Is4-f-glucosidic bonds* It closely resembles 

calluloneo It occurs In arthropod wwakelatonal call walls of fungi 

other than Domyceten and shells of molluscs. Various fungig and bacteria 

Including actinomycetes can attack chitino

All those substancea are therefore likely'to be present in the 

fUngus combs on which TemitZ=-cps, growss and the ability of TerwltoMcpa 

to decompose these substances vas investigated* 

7-5-2 )=IODS 

(a) Utilization of starcht Inocula of Tamitomycem cultures from 

different termite species were placed on starch agar plates 

343* 

(AppmdLx 09 

Starch hydrolysis was checked for after 29 4 and In soma cases 6 weeks 

by flooding the plates with Iodine which gives a blue-black colour with 

starch. Clearing indicated hydrolysis of the starch had occurred* 

(b) UtilizAtion of chitins PlAtes were Prepared by pouring a thin 

layer of melted chitin agar (Appendix 1) an solid tapwater agar which 

makes decomposition more easily seen (ValdIcamp 195!; ), 

Inocula, ot TermLIMcps trom ditterent termite species were placed 

on the chitin agar platen and incubated for three weeks, Decomposition 

of chLtin could be seen by the presence of a clear zone around or under 

the colonies* 

(C .)0 

ccurrence of pectinase's In Termlt2M cex culturess Two flAsks, 

were set ups with the medium which Induces the production of pectic 

emynes 

(Appendix I) t 

from each Teruita=en culturee They were , 

incubated at room temperaturee The flasks were then filtered and 

assayed for polygalacturonases Filtrate was added to holes cut, in 

pectate gel 

(1% sodium pectate In 12% afuLr)o These were incubated for 

48 hours and then flooded with fiN 1XI., A whiteýhalo indicates that 

hydrolysis of the pectate by the activity of polygalacturomse enzymes 

has occurred* 

7-5-3 RMULTS 

The results am given In Tables 7*5* 1 

1-70*29

FA WM0 ?A ol 

10 

rA 444F. 

C) cl to 

F4 4 ra 4 F, 

n 

Z 4 1 

1 

04 

1 rp, 

Z 

Z4; 0.4 0 

95 35 F. 

, 

0 

ononnonnono 000000 

01 

1" 6.0 I-d ý- 

Vs I 00"%"ftJ00 

5 ý5 95 

k: 

I-A 

9 

0%0 -3 C% W 

ý 1ý- 6-0 "" 

80 ? 

oooo, wn 8 ZA, 

opop 

billp 

0 0 

t; týt; !; t ? 

Zo 

u 

0, 

V) 

W 

ý- V-1 9 

-4 """" 

%0 co -4 CN %A) 

6- 4 4 

-4 C3 W. 3 C* 

000 

I ta 0 \A I %. 

n 1 1W to --j t. ) W, t, ) :3 

? ? ? 

IPP i 

000 0 l I pppooo 

000006600 

0 NO 00111 of III1 -1 

*0 o0 0 1 0 its I 'I 

I 

A 

344. 

S 

C= 

Z 

). h 

: *b 

03 

b% 

t4 

W

I 

RR 

I. 

fD a) 

00 

www 

99 

+ W. m 

4 ý 

0 

r+ f+ 

M 

t, 4 a ti n to >I-. 

f+ l f+ f+ 

ta 0 co 

ti , I 

. 0 

v 

a ra 

V N. 

6.4 

-O 

g 

f 

0 

l 

I 

c ia 0 

69 M44F. 44g; t 

00000 

%A 19- 

6ý1 " L4 

tol, 9 tow Ow %4 v %A CD 

3 n 4.0 ( X )6 - C; N 

0 

H 

113, 

M 

21 

Oft 

f* 

C., 

I 

I, 

rI ei, 

f+ 

tl ts 

I'll 

0&- R 

o* 

6bII 

88 

0M 

I" 

93 

345o

00 0.9 P-d 

2 1-" s10 

a. FH ,H, ;, 

" 

04 

ell 10 

1-k 

z 

a ce 

n 

M 

:19 iL 

oms (Dm, " 

to la 2 0- 

0 

i 

R a 

to 

a 

ril 

rt 0 Ile, 

a 

ýL 

rol 

a 

9 

f) 

to 9 

I'll r 

E 

to 

R F. 

I 

Q 

F9 

0 

to 

FA 

p 

In to 

0a 

t+ ma 

:L 01 10 a ?A . I.. a 

to R, p 

t 

t I- 

ý -- w 

04 

- - 0-1 ta 

0 ;j 

fs 

to, 

M 

0 

C6 0- "d 

LOS 

co 

%00 1 

0 

f+ 

E. 

a 

9 

346* 

IL 

if 

I 

I.

9 

2 3 a W. 

c 5 2 0 

Z

z 

ib 

ýd- 

Z 

si 

!r 

M W. 

IVO 

ýi 

eD 9 %-1- 

fD Za q+ 10 1$ 

a 

1. -0 P" 

' 

9 

9- -1, 1: *16 1a iL 

m n% 9 0 R aa 

6.4 

C lod 

g to 0 a I-- ii 

H 

0 

2 

m m a 

I lb 

in 

130 a 

aI e 

?A 

Id. 0 I a 

0 

i 

to 

ID I 

m *ýv 

2 1 110, c 

6-ft 

0 Q 

?A 

cr 

M 

40 vi 

;r at e 

1 

- 

f 

+ 

I- 

0 

03 

P" 

9 

rA 43 

a0 

goft 

F. 

$- 6.0 9 

1-- 

0 FA 

0 (D 

rl* 4+ fi. Co. rb C+ 

CD 

I ell 

m 

a I 

I 

F 

0 6" 1- 

0 

E 

P" 

9) p 0 X P 81 0 to s' 

1 

404 

-1 

104 0" 

to on 

; of 

CL :3 

& 

n 

cri 4 

%. o 

Ei 

ttv 

1 

3430

l 

I 

r. * 

t+ 

bd. 

n tt 

o 

id. 

1 9 

p 

th :101 

J 

Z Z. 

0 tD 

n 0-& 

es 

0 

IN 

10 cl 

10 0 (0 

b14 2 >d 

1-d 8.4 

0w 

i to, lbd 

b- ei N. 4 ' 

bd. g 9 bd. 0 

1 e g 

1 g3 

93 

fa 

eb 

te 80t a 

o 2 

tr 

, 

, t* re, 

0m lb 0 

u 

23 0 a C) % 

b% 

c 

tr be 

tr 18 01 

'ý4 

349a 

1 

ýO

Presence of polyqalýtcturonase enzymes. 

No polygalacturermse e=yme3 were produced by any of the 

TermitomXcoa zultures fran different termite species. 

7-5*4 DISCUSSION 

Starch is In general an excellent carbon source for most fungig 

although a few are known not to grow with It an tho sole c, arbon sources 

Growth occurred with all the cultures although It was generally quite 

poor. Differences occurred between Termit2 = es cultures from 

different termite species* In all cases where clearing occurred it 

was very little and quite difficult to detect Indicating that 

TermLtcx, vces probably produces little If any extracellular amylaae* 

No utilization of starch could be detected by any of tho Ancistrotermen 

cavithorax cultures, The cultures from Macrotermes ihellicorun ehowed 

slight clearing In about half the culturesq under tho inoculum ordy. 

whereas In Macrotermessubý 

yallnus all the cultUres. showed clearinge 

. 

The Werotermes associated cultures were interesting In that sppo D9 

C, G and 11 did not cleart appe D and Z showed slight clearing In all 

cases and in a few cases sp* showed a(moclearino, Cultures from 

Odc. ntotermes smeathmni showed some clearing and In a few cases those 

from OD 54 cleared the starcho 

Enzymes capable of breaking down stimple polysaccharldesl for 

mcample starchs have been found In several apoc4e3 of termites (Table 

7.5-3), Including a very active mylase in the mid cmd hind out of 

Odontotermes obesus (Singh 1976)* Invertase, which hydrolysen sucroset 

350- 

was also found in the mid_and hind out. It would therefore appear that 

the alto of starch breaMown In likely to bo the termite gut with little 

or none occurring on the fungus comb by the action of Tormit=ce, 

-2,

Temlt2rm7ces, appears to be unusual in that most woodrotting 13asidicl- 

trjcetes are able to utilize starch. It In probable that there in 

little starch In the fungus comb it having been usdd by tho termites 

but there Is little evidence for this* 

None of the TeM. Itoayces cultures were able to utilize chitin 

(Table 7*5*2), only a very little Growth occurred and no clearing 

of chitin was observed. Enzymes capable or degrading chitin hava been 

found In several species of termite (Table 7-5*3)- Chitin-would be, 

present In the temite-fungus system from both the eating'of dead ' 

teruites and from the fungus Itself* Chitin contents from 2,4-26.2% 

of dry weight of fungi have been found (Cochrane 19511). Rohrmann and 

no, qsman (1980) found the mycot'dites of Vkcroterptes ukuxii were 2-1.7% 

cliltint 38% protein, now comb was 0.6% chitin and old ccmb 1%, the 

Increase Indicating Ter7mitoMes, has not broken thin down* It seeem 

likely* therefore, that the site of chitinase production to the termite 

out, This Is probably similar to the fungus growing ants where chitinaso 

has been found in the ants faecal material (Marting Gloselmann and 

Martin 1973)* The production of chitina a would obviously be of great 

significance In termites eating mycotUtese 

No polyoalactunonase enzymes were produced by any of the cultures 

of Tern, 11=valfrom different termite gmcies, An important consequence 

of pectic enzyme, activity Is the exposure of other substances In the 

cell wall to enzyme action, In those species feeding on wood the 

presence of pectinases would be loss Important than In those feeding 

on herbaceous materials. RlcrOtOmOs luktalonsis has pectina3es (Martin 

and Martin 1979)- In general Plant pAthogenLc fungi produce pectinases, 

but many bacteria and saprophytic fungi do also, Production of pectic 

351-

enzymes by Termitomyces Ix likely to be of less Inq*rtance 093 the 

termites have already com inuted. and partially digested the material 

before Tervit2=. cen gets at It* 

Other enzymes found in the--guts of termites Include a&-galactosidase 

In Odontotemes obeaus (Singh 1976)o This bydrolynes raffinose to 

sucrono and galactose, but the pH, was unsuitable for its actLvItye 

Extracts of both Termita=XSex mycotifhes and workers from Macroteraws 

subhyAlinus oxidize vw=wso and ()alactoses but only the mycoCote extract 

oxidizes D-mannitol (Abo-Miatwa 1978)& French (1975) found proteasess 

ureases, phenoloxidasen and nitrogenases in Nasutitermes exitiosms and 

CoRtotermes lacteuso Ono species had bacteria uhich fermented glucoset 

mannosel galactose* arabinoses xylosev sucrose and lactose* Various 

authors have recorded the presence of enzymes capable of breaking down 

hemicelluloses and Kalotermes flavicollis can degrade hemicelluloses 

by 60-70% (Wood 1976). 

352a

7-6 MINERAL CbNTr., 

NT'oF taimmmiT coi-miams 6? nta 

7.6.1 INTIZODUCTION AND ý =, IICDS 

TEM41TE-FtNGUS SYSTUI 

In order to determine the role of the fungus comb and TermitomXces 

In the mineral economy of the termites the following analyzes of 

various rungus combst food material and Termitomcen structures were 

wades total N%t total P%j Ca%g Fig%* total ash % and Kppmo all expressed 

an a percentage of dry weight* 

Analyses were carried out on: Food Material on uhich tkerotempis 

bellicosus was foraging (maluly small pieces of wmd). voodstore and 

3 zones of M. bellicavus fungus comb 15 nozts)o Odontatermes ap. fungus 

comb (OD 54 from Rabba). Ricrotermes ape A fungus combe Ancimitrotemes 

cavithoralfunaus comb from ftbba (4 acti've comba wi - 

th . mycoteites, 

abandoned combs producing pacudorhiza. of Termitotwees I! erforans). 

353* 

4 semi- 

Psoudorhiza and bAsidioca"a of T, - perforans. llýrcotutcjj of Termit2Mcem 

from 14. bellicoaus, - fungus embo - Inidubated fungus comb from H, bellicomus 

on which other fungi had developedo 

Analyses were carried out at the International Institute oj 

Tropical Agricultureq lbadanj using standard UDS*D., 16 techniques* 

7.6,2 RESULTS 

The results are given In Tables 7.6.1 and 7., 6,2.

fl. r6 11- u 1 9 

tr 

0 

0 (+ 

ý8 

li 

1 

ic x 

#. 1. - fb 

ua 2 

90 

m 

99 

tr f+ t- W 

p W 

e+ 

:1 c: 

0 

1. 

-4 1. 

-1 ý> 4- 9.11 6.0 b. 0 

00 

00 0 0 

pp 

-9-9 

0 

CN 

n 

u 

0 

m 

0. 

0 

%n . n %n #" 

0+ l+ 0+ l* f+ 

P 

' 

9 

f+ 

f3 

rA 

gy 

ä > Lo 

to %4 1.11 l', t' 

0 ta u 0 w 0 >. 0 S c» ZO 

f*f+ 6,3 %0 %D -3 , 

B l+ f+ 0+ f+ s+ L 

4a 0 

6 8 0 8. - ok 63 0 >e 

00 0 0 0 o 

0 0 Ze 

Z4 %0 t> oe 

er 

44 l* 

%0 NW z1.191 

C% C% 

%A 

Co e 

, öl 

>A 

ra 

Lo Z, -0 

p 0- 

9 m 

o w 

2 ze ZO 

6 1- > 

w 

354s 

iF I 

1 

ro 

C ., 4 

I+R 

I 

ob" 

1*

FIGURE 7.6.1. Amount of nitrogen in different components of the 

Macrotermen bellicosus nest system, and in 

Termitomyces perforans and Macrotermes ukuzii 

(Rohrmann 1978)o 

355.

-n 

0 

CL 

CL 

Sý 

0 

(D 

-n 

CA 

CL 

Il 

(D 

10 

M 

0 

CD' 

ý D? 

4- (D 

CL 

cx 

I 

b' 0 

CD 

to (D 

-. ß -4 

0 1%1 4>. c6- ro 

CL

0 

:s-w w2 

ooý. 

"I 

0 I 

q+ 

0) 1A r, a 

.., 

0 4+ roll 

0 'T 

f : to f+ 

f+ 

e 4+ 

n0 

03 

cr, 

It* " 

ý -9 

tj 

f+ 

ob 

co i- h ZD C4 

1 1 

92 

m 

ce 

H 40 

FA t+ 

). 0 

f. " 

'a 0 

04 A 

w f+ M) 

c+ 

1 

00 nm 

'I" 

OR: 8 

W Co. 

C4. 

0 

F, 

cc 

P. 

ý 

a 

12 

>o 

0, W. A 

21 th 

r. ) C) 

88 

ý- 000 ýý 4ý, 

0 1- 0 6- 1- 

ýý 6 ;N 

,, %n 

wk Vt 

t) %o co .0 ta t3 

:2 I-i 

L 

a Ca L To 

co I. - cr, 

pdx 

ult a wt 

1 A. 41 9 at 

. ri. 'I %D , ZR q+ P- 

10 %0 

0 

v 

r, 

5 

ýg 

356. 

C' 

I 

t3 

Y 

to 

Vol

m 

Q 

9 CN ý 

cý 0a 

W -i u 

ýo 0 10 

'90 

00 

0 

rr 

Z, 2n0. 

is- 

:i :19 tD 9 

b et, e+ m 

2 

z7 

ffi m tr 3 

0 0- gi. m rA W 

w 

n 

eZeat, 

bd P. 

0 Ir 

:y (D 

0 ). % fle. 

I 

M '> ll re? 90c 

tw* 

L: 

ob 11 

0n le l* 

e+ 

2, 

:r 

>'* 

to to b- u )- 41 tli C% b- 

CD E2 

0- tt- c10 

9 

64 $- 0 

. 0 

Co Na 0%-. 3-4 

3 

19 

if $0 

0 

e 

cý C% 

.o0 

t> 9 

153 

be 

la 

%W 

5 

9 

ý 

357o 

ýi -IN 

',, m 19 ro 

00 Co 1 

0 

M

7-6*3 DISCUSSION 

Nitro2en 

in general the nitrogen contents of the-funguo-combs of different 

species are quite, similar, ranging froto 0*00% - 2sl 7%9 with combs of 

0dontotermes, species, generally being higher than the Macroternes species 

(Tables 7.6ol - 7492). An the food moves through the systea nitrogen 

appears-to be conserveds, increasing from the raw food material to food- 

store to fungus combs -This 

Is due to weight loss in decomposition by 

the removal of carbon compoundse The nitrogen content drops from the 

fresh edge. of the comb to the middle zone due to TerInitomyces utilizing 

the nitrogen* 7he data In Table-7.6-1 Is not statistically significant 

but the trend Is there and It In supported by Rohrmann (1978) and AbO- 

Khatwa (1977) in Table 7-6.2. Some loseea of nitrogen would then occur 

here due to the grazing of the termites on TermitonneS mycotOtes mid 

mycellum, The concentration ot-nitrogen. in the mycotOtes Is very hight 

but similar to values obtained for mycotOtes from other speciess IndIcAt- 

Ing the value of these as a nitrogen source to the tenilites. Itungate 

(1944) concluded that In termites which feed on wood attacked by fungi 

the fungus protoplasm was an Important food and that tile fungi served as 

a relatively rich nitrogenous supplement to the wood dietv which would 

appear to confer similar advantages to those obtained by fungus growing 

termites. The nitrogen value of the mycA)tAten In much higher than that 

of the pseudorhiza and basidiocarps of Ternit=cns 124%rforans which are 

approximately 22.7% nitrouen. The latter are similar to the values In 

various 11asidiomycetes of 1-87% (Stark 1973) for fungus fruit bodies on 

wood, and 1.56% In the wild to 7*6% In cultivated sporophores (Cochrane 

1958). Nitrogen content of nasidiorsycate mycellum varies from 0023 - 

3,27% (Herril and Cowling 1966)o and of rhizomorphs from 0.59 - -4.31% 

358o

(stark 1973)-,, This. show3 what a highly concentrated source of nitrogen 

the myc9tatez are and may explain how the 14acrotermitinae overcome what 

has been considered the twat difficult problem In the metabolism of 

wood-eating Insects IL,, e,, how the deficient nitrogen In obtained. Wood 

in a poor source of nitrogen,, nitrogen values usually ranging from 0*03 

(Ruyooks 1979) to o. 44% (Datra and Batra 1979)- The nitrogen content 

of the termites Is similar but slightly higher than that of the mycot'Otes 

(Rohzvmm 1978)o Nitrogen In esitential for the termites growth and 

development* This may be one of the reasons for the differences In the 

presence of Temitomyces In the out of workers of ýbcrotermes bellicosus 

(Table 5o2*l5),. The YMMO termites had a far higher proportion of 

Termit2nces% probably suggesting they are feeding an mycotAtes, a rich 

source of nitrogen* In comParison the older workers probably eat the 

combs accounting for the lower isolation of Tormitpmyces" There is a 

similar came with XYlebOrus, where developing larvae require a protein 

rich diet while adult workers mainly require carbohydrates as an activity 

fuel (Norris 1972)o 

Non-fungus-grovers may obtain imtra nitrogen trom nltrogen-fWng 

bacteria present in the gut (Denemann 197: 19 13reznak at al., 197,3, French 

et al* 1976), * An additional source of nitrogen could be from cwmiballx= 

(PO-Khatwa 1977)o Dreznak (197.5) considerc4 that these nitrogen-fixing 

bacteria or their metabolic Vro4ucts might serve directly an a nitrogen 

sourceq and might, also provide vitamins and other growth factors* 

Rohrmann and Ropan. an (1930) found no n1trocen fixation In the fungus 

grower htcrotermes ukuzil, 

Nitrogýn in excreted by temItes minly In tho f0M Of Uric acid or 

urates* No insects are known to have tho full c=plcmcnt of enzymes 

359*

needed to degrade uric acid nitrogen to the leVel of ammonia And Urea 

(French 1975)9 and the fungus may Play a Part 110r0o Uric acid was 

present in the comb of RAcratert'08 Pubhyallnus but vas at a lower 

concentration In the parts being eaten by the termites (Abo-Rhatwa 

1977), probably due to the action of the fungus* 

The total nitrogen of the fungus comb Includes both protein and 

non-protein componentnd, In the funaiLs itself 60-70% of the nitrogen 

is protein 

360. 

(Cochrane 1958). Non-protein nitrogen of fungus cells conaLnts 

of chiting nucloic acids% free amino acids and miscellaneous nitrogen 

compounds of low molecular weight* Decker (1976) considered the action 

of fungi on wood would Improve the nutritional value by Increasing the 

protein content and this may also occur here. Rohrmann and Rossman 

(1930) found the mycotetes of Macrotermos uIctizil to be composed of 38% 

protein and concluded from the amino acid analysis that Termliomyces 

was capable of supplying all the necessary amino acids present In 

termite protein. Enzymes capable of brealcing down wood proteins haýe 

been found In several termito'species' (Table 7.5.3). Troielnz In wood 

may be resistant to attack, being associated with't=nins or ligninse 

so the action of the fungus may be necessary before the ter=ites 1. 

proteases can work on the proteins., In attino antsý the fungus lacks 

the ft'll cOmPlement of prote6lytic enzymes necessary to 6ake effoctive 

use of polypeptide nitrogen,, but the facces; of the anta provide a 

proteolytic enzyme supplement (Martin 1970). Nitrogon-fixina, de- 

nitrifying, avraonifers and nitrifying- bacteria have all been Isolated 

from wounds with the fungus combs having a specialized nitrogen-timing 

florA (Boyer 19.560 Moikeljohn 1965).

Other Mneral Elements 

Decomposer fungi and insects require high levels of phosphorus 

an well an nitrogen 

(Swift 1W7)j The lower concentrations occurring 

Inwood way be a factor contributing to Its slow rate of decay* As 

in the case with nitrogen the mycotetes c(mtaln far, greater amounts 

of phosphorus and, potassium than -wood or fungus comb and therefore 

provide a good source for young termitese It Is generally accepted 

that potassium and-phosphorus are both essential for callulolytic 

activity 

Wu 1951). Potassium In successively removed from the food 

an it passes thrcm9h the foodstore and fungus comb systems and 1.9 

concentrated in the fungal tissue. Phosphorus In also present In the 

pseudorhiza and basidiocarp3 in greater amounts than In food or fungus 

comb* 

If : funuL require calcium It in only as a trace element, and In 

the Z010d to funO" c0mb Passa0e the Percentage o: r calcium gradually 

Increases as it In not beino utilized by the funguls, Calcium also tends 

361* 

to accumulate In vmmda and the ftngus combs due to evaporation* It 10 

a puzzlina fAct that Calcium In not -concentrate4f 

In the swcotetes 

7.6a and 7.6.2)4, This-In In contrast to other elementso and to results 

with other'ýfungL (swift 

et'al- 1979)e There in usually extensive 

concentration of calcium in Basidlocyceto mycellum 

and rhizmorphs, but not In basidlocarpse 

(Cromack 

- kUnol also have, a relatively largerequirement for magnesium. 

(Tables 

et al 1975)9 

The principal essential-' function of this In the activation of e=ymen 

necessary to UOMal metabolism and growth* Magnesium Is not concentrated 

here In either mycot0tes or fruit bodies* Thin In in agreement with the 

results of Cromack, Todd and Mon1c (1975) who found It was not concentrated 

In either fungal vegetative or reproductive structures.

on comparing the nutrient content of the Ancistrotermes cavithorax 

fungug comb producing mycotete-ve with that producing paeudorhizas very 

little difference can be seen apart from a decrease in the potassIUV3 

contents The concentration of the carbon source In usually the most 

Important In stimulating fruiting (Cochrane 1958) and it would seem 

that the other minerals do not varyouch from the comb with tricotetes, 

to that producing psoudorhiza* In general the trend Is the same an 

that shown from middle to old fungus comb of 

11acrotermes bellicosuag 

the alight increases in percentage of nitrogen probably being due to 

loss of carbon. 

The ash content of Odontotermes fungus combs are higher than those 

of Ancistrotermess Macrotermen end Weroterzes, speclegg probably 

indicating that more soil Is Incorporated during their construction* 

Comb of Macrotermex falciger also had a high ash content. The ash 

content of fruitbodles in general ranges from 1.00 

- 29-87% with most 

having 5-10% (Cochrane 1958) within which TPrm1tnmXces. =-rfor4ftns falls* 

other ash values for Ternit2=ces are from 6-14% (Mukilbi 1973)- The 

ash content of the fungus combs in higher than the food which indicates 

that not 1rauch of the mineral content could have been utilized on Its 

passage through the termite out* 

Other constituents In the fungus comb includo chlorophylll vitamins 

A and Ct lipidal organic acidal phenolics, tannins and sterolas The 

amounts of fatal oils and tannins are much lower In fungus combs than 

In faecal matter and carton next material indicating Termitomyces, may 

be utilizing some of theneo 

362*

alAPTM LIGHT 

GENFJIAL DISCUSSIOU 

363 *

811 =MITOMYCES IN CULTURE 

Termit=cp. s,. In culture Crew slower than the contamLit4ting Aingi 

and the zelective, medi= was developed to enable It to be isolated 

4nd cultured* 

In order to maxinixe growth the optimum cultural conditions were 

determined, Tho optirsum temperature for growth for all species was 

290C, except for Termit2Ucejs associated with Hterotemes ap. A which 

was, 3,20C. - This In very close to the mean aoil temperature at 30 cm 

(10. aemo) of 29*60C over the 3 yqars of the fieldworke The response 

to tmperature was not the same fqr different speciea pcwhaps reflecting 

the different temperature ranaps likely to be ene-oUntered In the various 

nests end hab#atse 

. For example the culti4re Associated with Kicrotermes 

bo-Mcnaus with a controlled ctound tf-vmperature was rar less tolerant of 

higher temperatures than were cultures ansociAted with Hicrotermos 

species which would experience, the changes In sqij temperature more 

closely* 

'The optimum pH for growth was around 5*2* This in higher than 

the fungus comb pH which, ranges from. 4.1 

- 4.6 (7- 2.2). 

364- 

ý Mýe optima were 

broad and in most cases the pli uras altered during Grow'Ui by the metabolic 

activity of, TermlLorgpesib 

1. 

Termitomycýs in' culture produced white to fawn colonies with a 

felty to efflorescent surface* Cultural equivalents of natural 

crfcot'f; "Ite. s were, formed more obviottaly an those culturex aufjociated with 

the 1-kir. 

raternex species. The cultures of TemitEE.! ycom ., Associated with 

difformt. species Of AprMitea. could, no% ka separate4 oR appearAnco in 

cuiture or apore size*,;, 131ekstosporo, oormLnation occurred readilyl with 

from one to four Oycellal tilaments developing.

892- TMIXTOMYCES IN THE VILD 

A major feature to emerge from thin worliz In the differences 

occurring amongst different sPecies of MAcrotermitinae in ocze 

features of their association with Temit2=. cps, 

An example of this"is the incorporatioii of Ter'nitomyces Into the 

food material, where In some species such as'Mcrotermes the faecal 

pellet Is deposited on the fungus comb mixed with Ternit2=cps. In 

other species, such as flacrotermes bellicosusl the foragers do not 

carry Ternit=ox, and incorporation probably occurs by Growth of 

Temitamyces from the adjacent cocab area. There were also caste 

differences In carriage of Termitomyces with young workers having a 

higher percentage of Termlt2Mcea In the out than nurse woricors and 

foragers, perhaps Indicating feeding differences, Those differences 

in feeding between castes way rolate to the conservation of nutrients 

in the termit"fungus system (8*3), particularly nitrogen, and may help 

to overcome the problem In wood-eating insects of obtaining enough 

nitrogen* 

The termites brought Into the wound large numbers of fungi other 

than Termlt=ces. These spores did not germinate until. the fungus 

cotab was removed from the nestq and It was found that there was & 

substance or substances, preyenting germination present In methanol 

extracts of foodstore and methanol and benzene extracts of termites* 

The establiubment of Termitomyces in now colonies also Illustrated 

the differences In the nature of the termito 

. rmjtOmXc, . e, s association 

Tp 

365- 

between different species of tho Macrotermitinae. In some xpecless such 

M* bellicosus and Hicrotermen the alates carried a bolus of Termit=ces

when they left the-nesto whereas in-all the other species investigated 

Tem, it=ceS was not carried* There were differences in the sex of 

the carrier too* the male carrying in kfacroternes bellicosualand the 

female in the Microtermes species. Evidence suggests that the non- 

carriers may obtain their Termit-anyces inGculum from basidlospores as 

the basidiocarp3 are present at the same time as the first foraging 

workers were active in new nests* The presence of basidiocarps only 

In association with non-carrying species was further evidence. 

366db

Termitan 

8.3 MZYIM 9 DEMMMITION AND NUTRIENT CYCLING 

There were differences in enzyme production between cultures of 

-Ices-associated 

with different termite specieso The cultures 

had characteristics of white rot fungi, with those from Ancistraterries 

cavithorax showing very strong production of polyphenol. oxidases of the 

laccaso type, whereas those from Macrotervies bellicosus were very poor 

produceraq showing differences In their potential for degrading lignino 

All cultures produced collulasess and again there were differences In 

production between species. Littleg if anys extracellular amylasol no 

chitinase and no polygalactouranase enzymes were produced* 

It is likely that the Importance of the different functions of 

Termit2=ces vary betveen termite spoclesl and may Perhaps vary In one 

species with time* For example, In M1crotPrm_es_ there in complete 

recycling of the fungus ccmbs but In M. bellicosus abandoned old combs 

may occur in the nests, perhaps due to the Inability of the termite- 

fungus system to break down some of the toxic wante products that may 

build up during the recycling of the food material. In Ricrotormes 

the most important function of Termit2=es appears to be in nutrition 

whereas in the elaborate central hive type of nest system the metabolic 

heat and water production function of Termitom)Mes'assumes an additional 

important role in the maintenance of nest micro-clim4to. 

Nutrient conservation was shown to occur In the Passage of food 

frcm raw material to fUngal tissue. For eXamplet nitrogen increased 

in concentration from raw food,, to foodstore, to fungus comb and finally 

to mycotOtes* "I 

Nitrog 

en is esocntial for growth and development and the 

young workers h&d a higher percentage of TermitorAycen in the Cut, 

indicating they may have been feeding on mycot8tei3l whereas the older 

367s

workers with a lower nitrogen requirement ate fungua combo OthCr, 

nutrients concentrated In the fungus comb and mycorutes were phosphorus 

and potasniumt but magnesium and calcium were not concentrated in the 

mycot9teso Thisqoncentration and conservation of nutrients in probably 

one of the Important funrtjon: s of Termit=ces and the fungus comb system,, 

Comparisons can be made between normal decomposition processes and 

the termite-fungus system* In normal decomposition of plant material 

It way be very gradually broken down in the s=e spot by a succession 

of different types of organism* This may be a lengthy process, 

(eago 

more than 7 Years for pine needless Gray and William 1971)9 but during 

it soluble compounds would be leached out of the material ard go back 

into the soil quite quicklyo Other litters especially that of deciduous 

forests may be more quickly Incorporated Into the soil 

months for ash leiaves)-q (Gray 

(e,, (), 12 to 18 

3680 

and William 1971). ý! any of the decompost- 

tion products of plant material may bo, Incorporated Into animal and 

microbial cells, but the soil animals will return their faecon to the 

soil and may die there. The normal decomposition processes Would 

Involve a large range of different microbes and animals at different 

stages of the decomposition* 

One Important feature with termites to that food material over the 

foraging area Is brought to and concentrated In one place In the case 

of termites with centrallsed hives such an Macrotermes bellicosuss or 

In several places in species with scattered combse A result of this In 

to remove nutrients from the normal recycling processeal the only losses 

to the system being via animal tissue when alates and foraging workers 

are preyed on, The organic matter and nutrients from a wide Area will 

be concentrated In the nest* and there In no direct return of faeces to

the ecosystem unless the nest in destroyed. idith termites the material 

is quickly removed before other organisms have worked on It and may 

then be kept within the nest systeme All the faecal material of the 

termitasýaoes onto the funaus coimb aW dead termites am recycled 

within the colony* The tcrmitewfungus system isýsppcjez poor In 

relation to normal decomposition* An Important difference when 

comparing It to straight wood decay by fungL is that the material 

Ternit2Mceis to presented with has passed through the termites gute 

Cou: minution of the plant material by the termites will rupture call 

walls exposing the cell contents to the action of gut enzymes or 

.., k-s in enzymes of Termitomyr the Cut* This means that it is likely some 

eeccmposition, probably of the more accessible celluloseq has already 

occurred. 

In both system all the material gets recycled but what may take 

a succession of many organisms a long time in normal decomposition 

takes two speciest the termite and Termit2nMceg-, very little time. 

369.

8*4 SIMMIOSIS BETW M- X? 

4SMTS AND FUNGI 

I 

Insects other than termites also have mutualistic associations 

with certain fungal species, These include the mosquito-liko midges 

of the family Itonididae whose galls contain fungi, scale Insects with 

the fungus 

-Sentoba. 

91dim's wood wasps of the Genera Strex Trcmex and 

. Urocerusq ambrosia beetlest ship timber woms (the Lymexylidae) and 

the fungus-growing ants* 

Tho fungus-grming ants show an e=ple of convergent evolution 

as they occupy a very similar ecological niche In the Nootropical region 

to the flacrotermitinae In the Ethiopian and Oriental regions. The fungue 

Is similar In being very slow growing In culture%, but the presence of 

the antaq like the termites, keeps the cultures viable and free from 

contamination 

(Martin 1970). 

As was suggented with the termites the funguls Is more Important in 

the diet of the larvaes the work-era also taking In plant sap (Quinlan 

and Cherret 1979)o Tho ambrosia beetles are a similar case with the 

adults also eating woodl while In the ship timber worms and woodwasp, 9 

only the larvae eat the fungus,, 

Like Ternit=es the funguz associated vith the funUus-growing 

ants is a Basidiamycete without clamp connections. There to scepticism 

370o 

about fruiting which does not occur until the fungus garden is abandoneds 

and it in considered that these basidlocarps are contaminants, The 

fungus has therefore lost its perfect stage and relies on carriage by 

the queen to reach new nests* showing similarity to some species of the 

tfacrotermitinae (6o2e2)9 Unlike termites the food does not first pass 

through the digestive system of the ants before being; resented to the 

fungus# it to Just cut up and placed on the fungus gardens. The ant-

fungus symbiosis Is not so efficient at utilizing tho foods an with 

termites the food In recycled within the system via the fungus combs 

whereas with the ants spent fungus garden material in du=ped. 

371*

Iý8.5 

EVOLUTION OF ME SYMOSIS 

Tito evolution of the rolationship of the termites and TemitgMcla 

has probably been a very gradual process with mutual adaptations 

causing the relationship to become even closer for the benefit of both 

fungus and termiteg rather than a process striving to rid the termite 

of the fungus- as Heim (1977), proposed, Many termite species star* food 

within their nests and It In likely that a 

372* 

Macrotermltinaeýancestor had 

-gL type of comb foodatore. This van an unexploited niche which was 

-probably 

Invaded by saprophytic fungi a Sphaerotermen 

, which has a 

comb without Termit2Mcps, In a primitive species of Macrotermitinae 

and feeds on rotting wood* The presencq of fungi an the comb which 

could produce cellulases and break down lignin would enable the 

Macrotermitinae to exploit more and more sound wood as the relationship 

evolved. This leads eventually to Mcroterm_qa which have probably the 

greatest nutritional Interdependancej attacking more living plants 

than other speclaxe, An the relationship evolved the carriage of 

Termit=! Yces spores by alates resulted In tho loss of tho perfect 

stage by those species of TernLt! 2=cex* This is an example of co. 

evolution where in the interaction between the termites and the fungus 

reciprocal pressures operate to make the evolution of one depandant on 

the evolution of the other. The greatest development of this relAtion. 

ship can be seen In Hicrotemes where there in no loss of the comb to 

the termites by fruiting,, an there In no baxidiocarp productionj and 

due to the close nutritional relationship coabu are completoly recyclode 

In contM3t to this view lleba (1952A) considered Terml_tomXceft to 

be a camonsal tolamted by the termitcal and that It was thereforo not 

,a symbiotic relationnhip. The evidence points to a complete Inter- 

dependance of thO UrMites and Temitgm ! yces indicating a mutualistic

symbiosis. - The fungus doea not OtILEM outside the environment provided by 

the termites and in dependant on the tormitets to provide food* The 

termLtes cannot establish viable colonies without the presence of 

Termitomnem on tlie fungus ccob. 

Haimls view of the relationshIp led him to propone (11olin 1977) 

that tho progression of the evolution was that the Ditormitomyces. 

which grow through the soil from the underground combs, were followed 

by the Praetermit2=cps which the termites expelled from the nest. The 

next step was the non-basidiocarp forming Termlt2Mces of the Madagascar 

9 

373o 

MicrotormeM., and finally the most advanced being ýýhaeroternen sphaerotharax 

which has got rid of the mycotOten " mycolim an wall, 

There must be both advantages and disadvantages to this relationship 

for the temitcs# with the advantages winning As the Macrotermitinae are 

very successful, for example dominating the termite fauna of the West 

African savannas 

(Wood 

and Sands 1978)e Species such an FIncrotermes 

be. ilicosus are able to produce very large stablo colonies which may 

continue for 20 years* Tho fungus enables them to exploit sound wood 

and crops which may be Inaccessible to other species, resultino In 

Microtermes being one of the major termite crop pests. The fungus 

enables them to utilize the food more efficiently with funguz combs 

being completely recycled* In contrast the carton of other species 

still contains 110nin and collulosoe 

Disadvantages Include the fact that far more food has to bo 

collected than In species without fUngit resulting In an IncrVased drain 

on the Colony's resources by predation on forager** Foundation of new 

colonLoo is made an even more risky business by the necessity of establishing

a viable fungus combo Construction and maintenance of fungus combo 

will also be necessary* 

Overall the advantagea'of'thd symbidals, including providing the 

termites with a nutrient concentrated food source obviously outweighs 

the disadiantages.,; 

374-

ACKNOWLEDGMENTS 

It is a pleasure to thank my supervisors Dr. T. G. Wood and 

Dr. M. J. Swift for their assistance and advice during the course of 

this project. 

I am grateful to N. E. R. C. for awarding me a grant, C. 0, P. R (London) 

for providing funds to carry out work in Nigeria and the Director of 

the institute of Agricultural Research and the Officer-in-Charge, Mokwat 

for providing facilities at the field-station at Mokwa. 

I am indebted to Dr. R. A. Johnson for help and advice, and for his 

hospitality in Nigeria. Ibr valuable discussions and assistance I would 

like to thank Robert Lamb, Lynne Boddy and Lesley Falconer. 

Technical assistance was gratefully received from Beryl Wilson and 

Fred Laws at Queen Mary College# and from the members of the Termite Team 

at Mokwa, without whose assistance the field work would not have been 

possible. Thanks go to Christopher Short for some of the photographs. 

Finally I would like to express my gratitude to my family and friendst 

including many people mentioned above, and especially Nicholette Dunlop 

and Denise Gibbons for the very important encouragement they have given me- 

375.

APPENDICES 

376.

APPENDIX 1. CULTURE MEDIA 

BASIC NEDIUM (EM) 

Ostilled water 11000 mi 

glucose 10.0 g 

(NH4 )2SO4 0.5 g 

KCL 0.5 g 

1.0 

KH PO 

4 

Mg9O 7H 0.2 

4' 20 

CaCl 0.1 g 

yeasi extract 0.5 g 

agar 20.0 g 

pH'adjusted to 5.6 

autoclave at 15 p. s. i. for 15 minutes. 

SOIL FUNGUS MEDIUM (SP) 

basic medium 

novobiOcin 

g 

g 

plus 

0.1 g dissolved in 10ml distilled 

water and added to the basic medium prior to autoclaving. 

CORNMEAL AGAR 

from Oxoid Ltd. Medium cm 103 

CZAPEK AGAR 

distilled water 1,000 ml 

Na NO 

3 

MI P04 

2,0 

1.0 

g 

g 

Mg? 149-, o 

VH20 

KCL 

0.5 

0.5 

g 

g 

FeSO 

4.7H 

sucrose 

20 

Q. 01 g 

3Q. 0 g 

agar 

20.0 g 

autoclave at 15 p. s. i. for 20-minutes. 

OATMM AGAR 

water 

1,000 ml 

powdered oatmeal 30.0 g 

agar 20.6 g 

Add oatmeal to water and gradually heat to boiling in a water- 

bath. Boil for 1 hour. Strain through muslin and make up to 

1,000ml with more water. Add agar and boil until dissolved. 

Autoclave at 15 p. s. i. for 20 minutes. 

377

POTATO DEXTROSE AGAR (PDA) 

water 

11,000 ml 

potato 

200.0 g 

dextrose 2.0.0 g 

agar 

20.0 g 

Scrub the potatoes clean, do not peelp cut into 12mm cubes. 

Weigh out 200go rinse rapidly. 

-in running water, place in 19000ml 

watev and boil until soft (1 hour)p mash, and squeeze as much of 

the pulp as possible through a fine sieve. Add agar and boil until 

dissolved. Add dextrose and stir until dissolved. Make up to 

1,000mi. 

Autoclave at 15 p. s. i. fbr 20 minutes. 

RICHARDS AGAR 

water 

1,000 ml 

IKNO 

ICH 

10.0 g 

k 

5.0 g 

mg9o 7H 

4ý 20 

F e Cl 

i 

pota o starc h 

0.25 

0.02 

10.0 

g 

g 

g 

sucrose 50.0 g 

agar 20.0 g 

SABOURAUDS AGAR 

distilled water 1,000 ml 

glucose 40.0 g 

peptone 1Q. 0 g 

agar 

autoclave 

0 

at 120 C for 

18 

- .0g 

10 minutes, 

CELLULOSE AGAR (CA) 

SF medium - glucose 

STARCH AGAR 

+ cellophane overlay 

distilled water 1'. 000 ml 

soluble starch 20.0 & 

yeast extract 5.0 g 

agar 2Q. 0 g 

autoclave at 15 p. s. i. for 15 minutes, 

378

CRITIN AM 

1% chitin solution 1-, ono TnI 

! ý4 1.0 

miso 

4 

agar 

1.0 g 

20.0 g 

the 12 chitin solution is =ad* by ball-milling chitin at 40C 

for 4 days. The plates were made by pouring a thin layer of 

malted chitin agar on solid tap water agar which makes the 

decomposition more easily seen. 

MEDIUM TO INDUCE PRODUCTION OF PECTIC TWDMS 

distilled vater 

glucose 

sodium polypectate 

asparagine 

KH PO 

Mg? o 

4! 

CACO 

3 

7H20 

1,00oul 

5.0 

10.0 

4.0 

Ito 

0.5 g 

10.0 g (in excess) 

379

APPENDIX 2 MILTONS nUID 

Miltons sterilising fluid contains 1% v/v sodium hypochlorite, and 

8.25% v/v sodium chloride, 

380

APPENDIX 3 SPECIES OF TE1141TES 

it 

Acanthotermes S ostedt 

A. acanthothorax (Sjostedt) 

Allodontermem Silvestri 

Ancistrotermes Silvestri 

A. cavIthorax Mostedt) 

A. crucifer (Sjo"stedt) 

A. guineensis (Silvestri) 

A. latinotus (Holmgren) 

A. 

Coarctotermes _paklstanicus 

(Ahmad) 

I Holmgren 

Coptotermes lacteus (Froggatt) 

C. formosanus 9-h-ir-raki 

Cryptotemes brevis (Walker)- 

Cubitermes Wasmann 

Euscalp-termes Silvestri 

Heterotermes indicola (WasmanO 

Hodotermes mossamb cus (Pagen) 

lUotermes Holmgren 

Kalotermes-. flavicollis (Fabricius) 

K. minor Hagen 

Macrotermes Holmgren 

M. annandalei (Silvestri), 

M. bellicosus (Smeathman) 

M. carbonarius (Hagen) 

M. falciger (Gerstacker) 

M. goliath (Sjostedt) 

M. malaccensis (Haviland) 

M. natalensfa (Haviland) 

M. subhZalinus (Rambur) 

M. ukuzii Fuller 

Mastotermes darwiniensis 

' Froggatt 

Megaprotermes Ruelle 

Microcerotermes Silvestri 

M. endentatus; 

tiasmann 

microtermes Wasmann 

M, insperatus Kemner 

M. obesi Holmgren 

Nanutitermes Dudley 

N. exitiosus (Hill) 

Odontotermes Holmgren 

0. assmuthi Holmgren 

0. badius (Haviland) 

0. formosanus (Shiraki) 

0. Iturdaspurensis Holmgren, K. and N. 

0. horni (Wa; m-ann) 

U. 

-kibarensis (Fuller) 

0. lacustris Harris 

0. latericlus (Haviland) 

0. magdalenae 

0. microdentatus 

0. obesus ( ur) 

Grasse and Noirot 

Roonwal and Sen-Sarma 

381

0. obscuriceRs (Wasmann) 

0. patruus (SJO"stedt) 

0. pauperans (Silvestri) 

0. praevalens (John) 

0. redemanni (Wasmann) 

0. smeathmani (Fuller) 

0. sundaicus Kevarler 

0. transvaalensis (Sjgstedt) 

0. vulpris (Uviiland) 

Protermes Holmgren 

P. minutus (Grassfi 

Pseudacanthotermes Siostedt. 

V. 

militar-ra 

-TH-f! 

gen) 

P. s2iniger (Sjo'stedt) 

Reticulitermes (Holmgren) 

RetIculftermes flaviRes (Kollar) 

S12haerotermes Holmgren 

S. . sphaerothorax (SJO'stedt) 

Synacanthotermes Holmgren 

Trinervitermes Holmgren 

T. Feminams- (Wasmann) 

T. trinervius (Rambur) 

T. trinervoides (Sjo'stedt) 

Tumulitermes Holmgren 

ZootermoRsis Emerson 

Z. angusticollis (Hagen) 

382

APPENDIX 4 SPECIES OF FUNGI 

Absidis van Tiegh 

Vs-17da corymbifera (Cohn)Sacc. and Trott. 

Aegerita duthiei Bark. 

Maricual L. ei-l? r. 0 

AgarIcus termitigena Berk, 

Alternaria Ne; _6 ex Wallr, 

Antennopsis Heim 

Armillaria aurhiza B*rke 

ArtaIllaria mef-lea (Vahl ex Yr. ) Kummer 

Aapargillus Mrch , ex Fr. 

A. carneus (Van Tiegho) Bloch. 

A. Ilavus Link ex Fries 

A. fumintus Pres. 

A. nidulans (Eidam) Winter 

A. er van Tiegh. 

A. och_raceus 

A. ýý (Bain. ) Thom and Church 

A. sulphureus (Fres. ) Thom and Church 

A. terreus Thom 

A. terricola Marchal 

A. violaceous Pannell 'and Raper 

Wilhelm 

A. parasLticus Spear 

Boletochaets Singer 

Boletus Dille ex Pro 

B. luteocystis Haim, 

Boviera Pars 

B. i-er-mitum 

ýeim 

Cephalosporium Corda 

Cladooporium Link ex Pro 

C. cla&sgorioides cl (Fres. ) do Vries 

Coll7bia (rr ) Staude 

(nown. '(Berk. ) Patch 

C. riaicata, Pat. non Rohl. 

C. sparsibarbis Berk. and Broome 

Conialls, HShnel 

Coreomycetopsis oedipus Thaxt. 

Cunniaghat-ella Matr. 

Curvularia Boadijn 

C. lunata' (Wakker) Boadijn 

Cyl ndrocarpon Wolleaw. 

DactylosEorium Harz 

Entoloma tatcrocarpum Berk. and Br. 

Epicoccum nigrum ]link L ex Fr. 

Eutermit=ces Beim 

Flammul&. filipendula Hann and Nym. 

F. janseana Henn. and NYm- 

Fusarium Link ex Yr. 

P. solani (Hart. ) Sacc. 

Canoderms Karst. 

G. curtisif (Berk. ) Hurr. 

Gy; ophragffiittm Mont. 

G. delilei Mont. 

383

Lactarius Pers. ex Gray 

Lentrn-us cartilagineus Berk. 

Leplota Pers. eX Gray 

L. albuminosa (Berk. ) Sacc. 

L. grassei Heim 

L. ivoriensis Heim 

L. termitophila 

Leucocoprinus 

L. madecassensis 

Maras7nius Fr. 

M. pahouTnensis 

Pat, 

Heim 

Heim 

De Seynes 

Metarhizium 

Mucor L. 

anTeopliae- (Metachnikoff) Sorokin 

M. racemQsus Fres. 

Neoskofitzia Schulz. 

N. termItum Hohn. 

Neurospora. Shear and Dodge 

Nýýjr-ospora Zimm. 

Omphalia (Fr. ) Staude 

0. myrmecophila 

Heim 

PaeciloEXces lilacinus (Thom) Samson 

P. variotii Bainier 

Penicillil Link ex Fr. 

P. as2eriu-m (Shear) Raper and Thom 

P. citrinum Thom 

P. expausum Link ex Thom 

P. funiculosum Thom 

P. purýurogenum ' Stoll 

P. striatum Raper and Fennell 

Peziza ep spartia Berk. and Br, 

Phialophora fast Riata (Lagerberg 

Pholiota; janseana_ Henn. and Nym. 

Phoma Fr. 

PhZtophtbo-ra do Bary 

Plutsug 0 Oriensis Henn,, and Nym. 

F. rajap 

Holteiia-u-n 

P. troxiblanum Henn. and Nym 

Podaxon Fr. 

Podaxon carcinomalis Fr. 

P. pleEillaria- - -(L-. --ex Pars. ) Prias 

P. termitoph lug J=. et Perr. do I& Bath. 

Praetermitomyces Heim 

Protubera 1161jer 

P. termT-tum Heim 

Psalliota---(Fr. ) Ku=er 

P. campestris 

P., termitum 

Fr. 

bufour 

PYthLism Pringsh. 

llizoct 

thizopus 

nia DC. ex Yr. 

Ebrenb. 

ex Corda 

R. ory-zae Went and Prinsen Gearligs 

R. stolonifer (Ehrenb. ex Fr. ) 

Rhodotorula Harrison 

Russula (Pers. ex Fr. ) 

and Helin) Conant 

384

Sept6basldiwi Pat. 

Sclerotinia Fuckel 

Scleratium Tode ex Fro 

Sordaria Cos. and de Moto 

So bosensis Das. 

q. f lintenlia (Roberme) Ces. and de Not. 

Syncephalastrum Schrost. 

So racemosum- Cohn ex Schroet. 

Tal! S2USas- Denjanin 

Termit-trin Thazt. 

Termitsues Heim 

To albuminosus (Berk. ) Heim 

To cartilaginaýs 

(Berk. ) Heim 

To clypeatus Haim 

To entolomoides Heim 

To ZB-erko) 

eurhizus 

Heim 

To f us Heim 

To globulun Hai= Ond Coossens 

To heiriii-Natarajan 

To letestut (Pat. ) Heim 

To imrmif ormi a 

To mediua RM 

He In 

and Grasse 

To al-- ý: ýocaýus (Berk. and Br. 

To perforano Heim 

To rabuorii Otleno 

To robustus (Beeli) Heim 

To schim2eri (Pat. ) Heim 

To striatus (Beeli) H*jm 

To striatus var. aurantiacua 

To strIatus form riseus H-eTm 

Termitos]Rhaera C 

To duthiei (Fork. ) Cif. 

Th -idium Tuck. ex Sebwend. 

Th'ielavia Zopf 

Torula sensu Turpin 

Er =ch a Haller em. Rostaf. 

Trrc-hoderma Pers, ex Pro 

To rid-e-Yerso ex Pro 

oms subgambosum Cos. 

a eurhiza (Berk. ) Petch 

ar Hill ex Creve 

grasiliensis (Theiss, ) Lloyd 

ah A. * 

(Klotzsch) Cooke 

Berk. 

(Berk. ) Fr. 

) He im 

Haim 

385

APPENDIX 5. STATISTICAL METHODS 

MEAN, VARIANCE v_ STANDARD DEVTATION AND STANDARD ERROR OF THE 41M. 

For a series of readings xV x2' x3 ` x. 

Mean,, (x 1+x2 **** 

variance 

n 

!x2_ 

Standard Deviation 

n-1 

n 

(Ex) 2 

Spandard Error of the Mean a 

n 

+x n) 

J-Vaýriance 

Standard Deviation 

95% Confidence Limits -+ (1.96 x S. E. M, ) 

,, 

r 

386

01M WAY ANALYSIS OF VARIANCE 

(1) The results recorded would be as follows 

(2) (a) 2A2 .2 n2 : 2: C2 

no. in A no. in B, no. in C 

1 A2 +2B+ 

2 

2 C2 

3 2 (in the above case) 

(b) (TA + 2L B+2 

no* of observations 

(c) ; Eindividual V&lues2 

(3) SS (TOTAL) - (c) -M 

SS (BETWEEN 

TREATMENTS 

A. B and C) m (a) (b) 

(4) A table in prepared 

SOURCE OF 11 

DEGREES OF SUMS OF MW VARIANCE 

VARIATION FREEDOM SQUARES (SS) SQUARES RATIO 

I ; (MS) (VR) 

Between no. of treat- from (a)-(b) 11 SS MS botween 

treatments ments -I above 

treatments 

11 D of F 

A. B - 

end C HS Resf dual 

Residual by sub- by sub- GS 

traction traction 

no. of from (c)-(b) 

TOTAL observations above 

1) 0fI?

significance was tested at p- o, o5,0.01 and 0.001 froin a table of r. 

A significant result was shown if the calculated Variance Ratio > 

tabulated 

F. 

LEAST SIGNIFICANT DIM, PENCE (LSD) METHOD 

This was used to find between which treatments or replicates 

the sigpif Icant dif f erence was. ' It was used af ter ona-way and tvo-vay 

analyses of variance. 

To gee if significant difference is between treatments (1) and (2). 

L. S. D. - treatment (1) Mean - treatment (2) mean, 

d. off. - lRisidual d. of fo 

S. E. (diff) - C5- 

where (:: r, m 

Then if L. S. D. > 

to. In- treatment (1) + no. in treatment (2) 

ReeLdual M. S 

'S. E. (dLff) x to. 05 (d. f. ) 

there is a significant differenca at the 5Z level of significance, 

SWlarly for to. 01 (d. fe) and t(O. 001) 

38a

TWO-WAY ANALYSIS OF VXRIANCE 

(1) The results recorded would be as follows 

TREATMENTS 

SPECIES 

A 

B 

H i TOTALS! 

(2) n TOTA (2) TOT. 2 etc. 

(3) 

-(3) 

27 A 

(2) - TOTA B 

(3) 

C etc. 7- C 

TOTAL zH GT 

(a) I-species totals 2 

no. in ApB or C9 

Y-treatment totals 2YH2+ 

no. in H, I or J-9 

ýA 2+ YB 2+ 

;Ec2 

7-12 + yj2 

(c) Yapecies x treatment tot&182 . TOT. 1 2+ TOT. 2 2+ 

(a) 

no. in each secti 3 

CT 2 

, total no., 0? observation; 

-'Findividual 

(3) SS (TOTAL) - (a) - 

values 2 

SS (BETWEEN SPECIES) - (a) - (d) 

SS (BETWEEN TREATMENTS) - 

(b) - (d) 

SS (SPECIES x TREATMENT TABLE TOTALS) (c) - (d) 

(in the abwve case) 

(in the above case) 

389

(4) 

(5) 

ý6) 

SOURCE OF VARTATION 


FREEDOM 

SUMS OF SQUARES 

Between species no. species 1 from above 

Between treatments no. treatments from above 

Interaction species by subtraction by subtraction 

x treatments 

TOTAL 

(no. species x from above 

(no. treatments)- 

SS (RESIDUAL) - SS (TOTAL) - SS (SPECIES x TREATME14TS TABLE TOTAL) 

SOURCE OF DEGREES OF sums OF HEAN SQUARES VARIANCE 

VARIATION FREEDOM SQUARES (SS) (MS) RATIO (VR);. 

Between species as as ss 

B tween treat- 

m: nts 

interaction 

Residual 

as as (4) 

as (4) 

as (4) 

by subtraction I by subtraction 

Total 

TOTAL observations SS (TOTAL) 

(7) Divide (iii) by (iv) 

D of P see (7) 

ss 

ss 

ss 

D of; F 

D of F 

D of F Uv) 

If this is significant divide (L) by (M) and (ii) by (iii). 

if it is not significant divide M by (iv) and (H) by (iv). 

if not significant then species and treatments do not interact, 

below 

390

REGRESSION AND CORRELATION 

(1) Scatter diagr= va3 plotted. 

(2) For a series of data points xl,, yl; x29 Y2; ,, * x. 9 yu 

:: E x :9 Cv- X) 

15 x22 

Y2 

y 

: -5xy 

were calculated. 

The slope of the regression line b was calculated 

(4) ;, i 

ý5-? 

ýy -- 

(1 x) - (-rz-y) 

X2 , _, x) 

n 

and b were substituted in y- bx +a 

to get a vulue for a (intersection). 

By substituting a value for x* in the equation y* vas obtained. 

2 points were know-n(-xv y-) and W. y*) and the 

I 

regression line could be drawn. 

The correlation coefficient r was calculated 

f zy - 

r . 4-: 1 

(ex) 

n( y) 

2y 

(6) To find the goodness of fit of tho regression line the analysis 

of variance was used. 

391

STUDENTS t-TEIST. COMPARISON OF. IMMS OF SIMLL SAMUS 

(1) The hypothesis was made that there was no significant difference 

between means. 

xynI 

xy n2 

X) 22 

no 2x and ne 2y were calculated 

ns xx(, 

j x) 

2 

C- 2 

S. E. (diff. ) 7 was calculated 

where 

a' 2m 

(5) t was calculated 

txy 

aý 

2 

d. f. -n1+n22 

.1+ 

n, n2 

na 2x+ 

n1+n2-2 

were calculated 

ni (similarly for y) 

ns 2y 

(6) If t> table value then there was a significant difference 

between means and the hypothesis was rejectod. 

392

X2 AND X2 TEST OF HOMOGENEITY 

For X2 the expected value (E) = Total of observed values 

no. of Celle 

The X2 test of homogeneity was used where the uunbers tested weren't 

equal e. g. Table 5.4.7. where different numbers of Hicrotermes workers 

were dissected. 

FORAGERS 

(SOIL) 

+ Termit2Mces A 

FORAGERS 

(FOOD) 

NURSE TOTALS 

WORMS 

Termitcayces D E Y, 

TOTALS z v 

'Expected value (A) -xx: v 

GT 

Expected value (F) wyxv GT etc* 

All expected values were calculated and X2 determined. 

-B 

X2 (Observed value - Expected value) 2 

Expected value 

Degrees of freedom - (rows - 1) x (columns 

and column -1 for test* 

e 

test of homogeneity 

if x2> table value then there is a significant 

Ufference. 

c 

- 1) for 

393

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Q: LEGE

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