Tram. Br. my co!. Soc. 84 (3), 519-526 (1985)

[ 519 ]

Primed i l l Great Britain

SELECTIVE MEDIUM FOR ISOLATION OF TERMITOMYCESFROM TERMITE NESTS

By REBECCA THOMAS

Tr opical Development and R esearch Institute, College H ouse, W rights Lane, London W 8

A selective medium for the isolation of Termitomyces, a fungus symbiotic with termites ofthe sub-family Macroterrnitinae, was developed. Candidate subs tances for the selectivemedium were tested for the ir effect on growth and germination of Termitomyces and severalcommon contaminant fung i. Of the 38 substances tested eight were included in the selectivemedium. The radial growth of Termitomy ces cultures associated with different termite specieswas measured on the selective medium and was not significantly different from growth onth e cont ro l medium.

Termites in the sub-family Macrotermitinae havea symbiotic relationship with th e basidiomycetegenus Termitomyces Heim. The fungus inhabitsstructures, built from the termites faecal pellets,called fungus combs. Fungus combs are foundwith in the termite nest system and may be larg ecentralized masses, as in Macrotermes bellicosus(Smeathrnan) , or structures up to 5 cm diam,enclosed in small chambers in th e soil, as inMicrotermes. Termitomy ces is found as mycelium inthe fungus comb, and as round white asexualreproductive struc tures on the comb surface. Thesehave been called mycotetes (H eirn, 1977), synnern-ata (Rohrmann , 1978) or br ornar ia (D ixon , 1983).Termitomy ces is found only on th e fungus comb,in th e gut of termites, and, in some species, asbasid iocarps which develop from th e fungus comb.

T ermitomy ces is very slow-growing in agarculture and may be rapidly overgrown by otherfung i. This makes it very difficult to isolate from thenatural habitat where many other fungus propagulesare present. Many authors have found it difficult orimpossible to isolate Termitomy ces (Perch , 1906;Abo-Khatwa, 1977; Zoberi, 1979), although Swift(1976, pers . comm.) had some success using acellulose based medium. This paper report s on thedevelopment ofa selective medium for the isolationof Termitomyces.

METHOD

S trategy and choice of test substances

The development of a selective med ium can becarried out in two ways, i.e. by inclus ion ofsubstances wh ich inhibit the growth of un wantedorgan isms , or by including energy sources or othernutrients whi ch are available on ly to a restri ctedrange of micro-organisms including the preferredone (T sao, 1970). Both approach es were tri ed here .

Substances for inclusion in the selective growthmedium were screened by the standard method ofmeasuring radial growth of Termitomyces in theirpresence and ab sence (Vaartaja, 1960; Papavizas,1967 ; U scuplic & Pawsey, 1970). Substances whichdid not inhibit the growth of Termitomy ces werefurther tested for their effects on common con-taminant fungi. Those substances which inhibitedthe growth of the common contaminant fungiwere tested for their effects on the germination ofspores of Termitomy ces and contaminant fungi .

Five ant ibacterial substances, penicillin, rosebengal, novobiocin, chloramphenicol and strepto-mycin sulphate, were tested. The broad spectrumantibiotic, str eptomy cin sulphate, had been used ,together with penicillin, to isolate Termitomyces byBatra & Batra (1979) . It has been used to preventthe growth of bacter ia in media in concentrationsranging from 0 '03 to 6 g I-I (Papavizas & Davey,1959 ; Uscup lic & Pawsey, 1970). Martin (1950)found it had an inhibitory effect on only 2 out of117 fungal isolates.

Five fungicides were tested. Benomyl is asystemic fungicide of the benzimidazole groupwhich controls a wide range of diseases. It isespecially effect ive against Penicillium and someother Hyphomycetes. Maloy (1974) found that at0'001 g 1- 1 it completely inhibited Penicillium sp .and Phialophora f 'astigiata and greatly reduced thegrowth of Tr ichoderma. Dinocap, a dinitrophenylderivative, is a contact fungicide. Thiophanatemethyl is one of the thi ophanate group of systemicfungicides based on thi ourea. It is simi lar in itsanti-fungal properties to benomyl and is charac-terized by br oad-spectrum effects . The otherfungicides tested were pentachloronitrobenzene(PCN B), a narrow spectrum soil fungic ide whichinhibits rapidly growing saprophytic fungi, anddithane, a dithiocarbamate fungicide .

52 0 Isolation of Termitomyces

Table 1. Growth of Termitomyces and common contaminating fungi on media containing substances forinclusion in selective medium

Conen Da ysgl-' incubation Acremonium WPP P. variocii Penicillium A . niger

Control 2 4 2 NO 2 2

Benomyl 0 -01 2 4 1 NO 1* 1 *

Thi ophanate methyl 0 '01 2 4 1 NO 2 1*

Control 5 4 NO 3 3 3Benomyl, thiophanate 0 -01 5 4 NO 2 2* 1*

methyl 0-01

Benomyl, thiophanate 0 '0 1 4 NO 1* 1*

methyl and 0 '01

cellophane

Control 5 4 NO 3 4 4Dinocap 0 '002 5 3 NO 2 4 3Control 6 4 NO 3 4 4Ox bile 5 '0 6 1 NO 1 4 2

Control 7 4 NO 2 4 3Gallic acid 0 '01 7 4 NO 2* 4 4

Scale: radial growth from plug 1 = 0-0'49 em, 2 = 0' 5-<J -99 em, 3 = 1'0-1 '49 em, 4 = > 1 '49 em, NO = nodetermination.

* Substance prevents production of satellite colonies.

Different carbon sources tested for their selec-tivity were glucose, cellobiose, cellophane, man-nitol and peptone.

Other substances were selected on the basis oftheir p revious use in selective media. T heseincl uded a phenolic compound, gallic acid . F lowers& Hendrix (1969) found that at 0 '025 g I- I itprevented development of non-pythiaceous fungi .Hintikka (1971) found that some basidiomycetescou ld grow in it up to 15 g I-I but in general itreduced their growth . Another substance testedwas ox bile which has been used in many selectivemedia as it suppresses the growth of many fungi . Itwas used by Papavizas (1967) at 0'5 g I- I in aselective medium to isolate Fusarium . At higherconcentrations it also suppresses bacteria ; Baker-spig el & Miller (1953) found that agar containing1 ':' 0 ox bile suppressed bacterial colonies byone-fifth to one -tenth.

Sui table ingredients when used in combinationmay cause problems, and synergism betweeninhibitors may antagonise the desired isolate .Various permutations and combinations of likelyingredients were therefore tested in order todevelop the appropriate selective medium .

Test fungi

All Termitomy ces cultures were obtained frommycotetes in termite nests in the vic in ity of Mokwain the Southern Guinea savanna vegetation zone of

Nigeria, They were isolated on a general soil fungusmed ium (G M ) containing in 1 I di stilled H p ;10'0 g glucose, a's g (NH4)2S0 ,,, 0'5 g KCI , 1'0 gKH2PO~, 0'2 g MgSO I.7H20, 0 '1 g CaCI2 , 0 '5 gyeast extract, 20'0 g agar, 0 '1 g novobiocin disso lvedin 10 ern" distilled H 20 and added prior toautoclaving and maintained at 29 °C on thismedium without nov ob iocin (BM) . The T ermite-my ces cu ltures used were associated with Ancistro-termes cavithorax (S iostcd t) , Macrotermes bellicosus(Smeathrnan), Macrotermes subhyalinus (Rambu r),Microtermes aluco Siostedt (sp . D ), A-ficrotermesgra ssei Ghidini (sp. B), M icrotermes lepidu s Siostedt(sp . A), Microtermes subhy alinus Silve stri (sp . G .from Mokwa, and sp. R [ = M. subhyalinus?] fromRabba, a riparian fore st near Mokwa), Microtermessp. nov . (sp . C), Microtermes sp. Z, Odontotermessmeathmant (Fuller) and Odontotermes sp.Holmgren.

The common contaminating fungi investigatedwere A spergillus niger van Tiegh., A cremonium sp.,Paecilomy ces variotii Bainier, Penicillium sp . anda sterile mycelial isolate WPP. These were allobtained from dilution plates from M. bellicosusfungus comb and maintained on BM at 29°.

Growth of fungi with test substances

Thirty eight different substances were incorporatedinto BM individually for testing. Plugs of Ter mito-my ces associated with M . bellicosus, approximately

Rebecca Thomas 521

Table 2. Mean number of colonies developing from spores on plates containing test substances ( ± S.E.M.)

Experiment A

Inoculated fungi

BM(control)

CSM 1

Recoveredfungus

T ermitomy cesA , nigerP , variatii

Termitomy cesA , nigerP, uariotii

Days Termit omy ces Termitomy cesincubation Termit omy ces +A . niger +P. variatii

lo'8±I 'l 0 '0 0 '0

S S'O±O'47'O± l'S

O'2±O '2

S

Experiment B

A. niger

0 '0

P. oariotii

0 '0

Termitomy ces+A, niger+P, oariotii

0 '0

3'O±2'4I ' S ±O' S

2'3 ±1 '30 '0

o'S±o'S

Inoculated fungi

Da ys Termitomyces-r A. niger

Recovered fung us incubation Termitomy ces +P . variatii

Control Termitomyces 7 Overgrown 0 '0

A . niger SO'O±3'lP , oariotii 20 'O±5'S

CSM2 Termit omy ces 7 Overgrown OvergrownA . niger 0 '0

P. oariotii 0 '0

CSM 1- Termitomyces 7 Overgrown Overgrownnovobiocin A . niger 0 '0

P. uariotii 0 '0

CSM3 Termitomy ces 7 Overgrown 0 '0

A niger SS'o ±9'7P. variatii SO'7 ±9'2

CSM4 Termuomy ces 7 Overgrown l1 'O±4'9A . niger 0 '0

P. oariotii 0 '0

5 mm diam, were taken from actively growingcultures and placed centrally on 9 em diam Petridishes containing equal volumes of the media to betested . Two measurements of colony diam weretaken at right angles to each other every 3 or 4 days,with the final measurements being made after 2

weeks , whe n the form of growth was also noted,Incubation was at 29°.

Those substances not adversely affecting thegrowth of Termitomyces were screened for theireffect on common contaminating fungi. Plates wereset up as above .

Inocula of A . niger, A cremonium sp., Penicilliumsp , and WPP wer e placed 2'5 em from theTerm itomyces plug, equidistant from each other. Inthe course of testing it was realized that WPP wasslow growing and P , variotii was subst itu ted for it ,P lates were incubated at 29° and the growth of thefungi recorded after 24 and 48 h. If the plates were

not overgrown the experiment was continued for upto 7 days ,

Spore germination tests

Spore suspensions of A . niger, P . variotii andconidial agglomerat ions of Termitomyces in steriledistilled water were pipetted, alone and incombination, onto plates of BM alone andincorporating those test substances appearingsuitable for a selective medi um . Standard inoc ulawere used for each experiment. Plates wereincubated at 29° and the numbers of coloniesdeveloping counted. In experiment A 4 plates wereset up, and in experiment B 3 plates were set up foreach fungus or combination of fungi on eachmedium,

52 2 Isolation of Termitomyces

12 14852

(d) Odontotermes sp.

•(b) Macrot ermes subhyalinus ./

~/A

~-//~

~/

2 5 8 12 14

(f) Microtermes Iepidus . /./.// 0

/ .-:;::::::::::'

852

(e) Ancistrotermes cavithorax

2 5 8 12 IA4

(c) Odontotermes smeathmani /

.>~/

f':/,7/

(a) Macrotermes bellicosus #~

/8//~a~

16

12

12

4

16

o20

o18

E.5 14;>,eo'8...~ 10~.,§:;a 6~

4

4

o 2 5 8 12 14 2

Days5 8 12 14

R ebecca Thomas 523

16

12-

8

c:(g) Microtennes sp. Z /'

,,/ e

.>"/.:;;..~.

~e

(h) Microtermes aluco

4

cE

1412852

(j) Microtermes subhyalinus

2 14o

"0o

o 2 5 8 12 14e

18 ~(il Microtermes grassei / "

./~ 10 / e~ b/ e

i 6/ e

(k) Microtermes sp . R (l) Microtermes sp. C

o22

18

14

10

2 5

I

5

8

I

8I

12

2

2

5

5

8

8

12

12

14

14

Days

Fig. l. G rowth of Termitomy ces cultures associa ted with 12 term ite spp. on f::. - f::. , GM; e-e, FSM.

Growth of Termitomyces on the final selectivemedium

Once the final selective medium (FSM) had beendeveloped from the series of tests described above,the radial growths of Termitomyces isolates associa-ted with twelve termite species were compared onFSM and GM. For each isolate five plates were setup on each medium.

Isolation of Termitomyces

CSM 1 = BM-glucose, + cellophane overlay,0'01 g benomyl, 0'01 g thiophanatemethyl, 0'1 g novobiocin.

CSM 2 = BM+0'01 g benomyl, 0'01 g thio-phanate methyl.

CSM 3 = BM+0'08 g penicillin-G sodium salt,0'2 g streptomycin sulphate.

CSM 4 = BM-glucose, + cellophane overlay,0'2 g streptomycin sulphate, 0'08 gpenicillin-G sodium salt, 0'01 gbenomyl, 0'01 g thiophanate methyl.

RESUL TS

Growth offungi with test substances

Of the 38 substances tested 20 were rejected asadversely affecting the growth of Termitomyces.Those remaining were the antibiotics penicillin - Gsodium salt and streptomycin sulphate, the fungi-cides benomyl, dinocap, thiophanate methyl andPCNB, alternative carbon sources cellobiose,cellophane, glucose, mannitol and peptone, andfungus comb extract, gallic acid, CuS04.5H20, oxbile, sodium propionate, sodium taurocholate andthiamine hydrochloride.

A total of8 out of the 18 substances not adverselyaffecting the growth of Termitomyces had sufficientinhibitory effect on the contaminant fungi forfurther testing. Two antibiotics were also retainedfor further testing. Those substances eliminatedwere fungus comb extract, glucose, mannitol,PCNB, peptone, sodium propionate, sodiumtaurocholate and thiamine hydrochloride. Theresults of those included in the final selectivemedium are given in Table 1.

The addition of both benomyl and thiophanatemethyl to BM reduced the growth of A. niger andPenicillium sp., and also had a small effect on WPP.The substitution of cellophane or cellobiose forglucose reduced further the growth of Penicilliumsp. Dinocap at 0'002 g I-I slightly inhibited thegrowth of Acremonium sp. and P. variotii, ox bileinhibited the growth of P. variotii, A. niger andPenicillium sp., gallic acid slightly reduced thegrowth of Acremonium sp., CuSO•.5H20 reducedthe growth of all the contaminant fungi, but whenused in combination with fungicides it also reducedthe growth of Termitomyces and so was rejected.

Spore germination tests

Ten substances remained from which a combinedselective media (CSM) could be formulated to testthe effects of carbon source, fungicides and anti-biotics on spore germination. Spore germinationwas tested in four combinations of test substancesin 1 I BM as follows:

In experiment A (Table 2) CSM 1 slightlyreduced the germination of Termitomyces from thatof the control. It allowed the growth of Termitomyceson plates with a mixture of Termitomyces, A. nigerand P. variotii, though P. variotii also germinatedwhen present in the mixture.

In experiment B the addition of benomyl andthiophanate methyl (CSM 2) to BM prevented thegermination of A. niger and P. variotii and allowedthat of Termitomyces. When combined with acellophane overlay the result was the same.

The antibiotics streptomycin sulphate and peni-cillin-G sodium salt, tested separately (CSM 3),allowed the germination of Termitomyces whenalone in the plate, but did not act on thecontaminating fungi.

Termitomyces germinated in CSM 4, and couldbe recovered when added in a mixture of A. nigerand P. variotii spores, though at slightly reducedlevels.

Field trials with selective medium

The medium, CSM 4 plus dinocap, ox bile andgallic acid, was tested in the field by isolatingTermitomyces from fungus comb and worker guts.This enabled the medium to be refined by trial anderror. After field trials the concentration of someingredients were slightly altered resulting in thefinal selective medium (FSM) being BM - glucose,+ (0'01 g benomyl (Benlate, DuPont), 0'01 gthiophanate methyl (Murphy), 0'006 g dinocap(Karathane, Rohm and Haas), 0'01 g gallic acid and1 g ox bile I-I). After autoclaving streptomycinsulphate (0'4 g 1-1) and penicillin-G sodium salt(0'1 g I-I) were added. A sterile cellulose filmoverlay (Cellophane, British Cellophane Co.) wasadded as a carbon source.

Growth of Termitomyces on the final selectivemedium

Comparing the growth of all the Termitomycesisolates by ANOVA there was no significantdifference between the results on FSM and GM in

R ebecca Thomas

T able 3. Mean diam (m m) of the Termitomycesisola tes growing on GM and F S M fo r 14 days( ± S.E.M.)

utilize the Cellophane ove rlay (T hom as, 1981). Ingeneral , satisfactory growth of all cu ltu res occu rre don F SM.

The final selective medium greatly reduced thenumber s of contamin ant fu ngi but did not sto p thegrowth of all. It enabled T ermitomy ees to be isolatedfrom termite guts where it could not be isolatedwith GM. In general satisfactory gro wth of allTermitomy ees cultures occurred on the select ivemedium. The variab le behaviour of th e d ifferentT erm itomy ees isolates indicates that these may bedi fferent species or d iffer ent physiolog ical races ofthe same species. From identification of theba sid iocarps there appears to be a tendency forspecies of T ermitomy ees to be associat ed wi thd iffere nt genera of termites (Sands, 1969)·

This work was carried out during the cou rse ofa CA SE studentship fu nded by the NaturalEnvironment Research Council and the Centre forOverseas Pest Research. I would like to thank DrT . G . W ood, Profe ssor M . J. Swift and Dr W . A.Sands fo r their ad vice and sug ges tions .

FS M

16'37 ± 0'688'26 ±OA5

14'93±1'5715'86± o'2917'12 ±OAO

19'85 ± 1'3215'47 ± O'2514'70 ± O'31

18'86 ± 1'0213'38± O'34

Experiment D

13'95± 1'00 14-34 ± 0-8417-62±0'89 18A 8 ± o'5417'18± o'51 20'38±O'6518'01 ± 1AO 14'91± 1'9221'69±O'53 20'96 ± O'3216'06±o'32 14'8 8±OA419'06 ± o'91 16'31 ±0-88

A. cavithoraxM. bellicosusMa crotermessubhya linus

O. smeat hmaniOdontotermes sp.

Associated termite species GMExperi ment C

M . alucoM . grasseiM .lepidusM icrotermes subhya linusMicrotermes sp. RMicrotermes sp , ZMicrotermes sp.nov,

two experiments C and D . H owever ther e was noconsistent growth respon se of the T ermitomy eesisolates associa ted with differ ent termite spp . toFSM and GM, wi th some isolates producing mostgrowth on FSM and other s on GM (F ig. 1, T able3). In experim ent C iso lates fro m A . eavithorax andfrom the tw o O don to termes spp. produced moregrowth on GM, whereas M aerotermes subhy alinusisolates grew better on FSM. The M. bell ieosusiso late grew better on GM up to 8 days but finall yproduced most growth on FSM. In exper iment Dthree ou t of the seven isolat es (associated wi th M.aluco, M . grassei and M . lep idus) were b igge r onFSM after 14 da ys (T ab le 3, Fig. 1).

There was a sign ifican t differ ence (P < 0'05)bet ween the growth of iso lates associated withdifferent Mierotermes spp . The results we recompared using the LSD method which showedthat on GM the isolate assoc iat ed with Mierot ermessp . R grew significantly bigger than all the others,and that as sociated with M . alueo was significantlysm aller than all others exc ept that associated withM ierot ermes sp . Z. Ther e we re no sig nificantgr owth differen ces on FSM between iso latesassociated with M. aluco, M ierotermes subhyalin us,Microte rmes sp . nov . and M ierotermes sp. Z, norbet ween M. grassei, M. lepidu s and M icro termessp . R.

There was a sign ificant co rre lation ( r = 0 '78)bet ween the gro wth on FSM and the amoun t ofcellulose decomposition produced by the d ifferentcultures (P < 0 '05), showing that the differingabilities of the cultures to grow on FSM m ay belargely explain ed by their di ffering abilities to

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(Received for publication 12 January 1984)