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Mycol. Res. 104 (5) : 537–544 (May 2000).

Printed in the United Kingdom.

537

Production of efrapeptins by Tolypocladium species and evaluation of their insecticidal and antimicrobial properties

A. R. BANDANI 1, 4 , B. P. S. KHAMBAY 1 , J. L. FAULL 2 , R. NEWTON 3 , M. DEADMAN 4 and T. M. BUTT 1, 3

IACR-Rothamsted, Harpenden, Hertfordshire AL5 2JQ, UK. Department of Biological Sciences, Birkbeck College, University of London, Malet Street W4E 7HX, UK. School of Biological Sciences, University of Wales Swansea, Singleton Park, Swansea SA2 8PP, UK. Department of Agriculture, University of Reading, Earley Gate, Reading, RG6 2AT, UK. E-mail: T.Butt Swansea.ac.uk

Received 7 December 1998 ; accepted 12 August 1999.

This study shows for the first time that Tolypocladium species produce efrapeptins, a group of toxic peptides, in vivo but the quantities are too small to account for insect death, suggesting that these insecticidal compounds work in concert with other pathogenicity determinants. There is inter- and intraspecific variation in efrapeptin production in vitro by Tolypocladium species. T. parasiticum produced only efrapeptin E, in small quantities. Efrapeptins were detectable 48 h after inoculation and increased with biomass. The relative amounts of individual efrapeptins (C, D, E, F, G) produced by T. niveum in vitro were D E F C G but in vivo they were D F C E G. Efrapeptins were toxic to a wide range of insects when injected into the haemocoel. Mortality was dose-related. Efrapeptins also exhibited limited antifungal and antibacterial activity. Micrococcus luteus was considered an excellent indicator of efrapeptin presence in culture filtrate extracts because of its extreme sensitivity to these compounds.

INTRODUCTION

Tolypocladium species are widespread, soil-borne fungi, some of which are pathogenic towards insects while others exist as saprotrophs (Bissett 1983, Samson & Soares 1984). Some strains of T. cylindrosporum are considered potential agents for the control of mosquitoes and other insect pests (Samson & Soares 1984, Lam, Geottel & Soares 1988). Tolypocladium species produce a wide range of metabolites including cyclosporins, efrapeptins, elvapeptins and the antibiotic LP237-F8 (Dreyfuss et al. 1976, Jackson et al. 1979, Bullough et al. 1982, Krasnoff et al. 1991, Gupta et al. 1992, Rehacek 1995, Tsantrizos et al. 1996). Weiser & Matha (1988) showed that extracts of T. niveum and T. cylindrosporum were toxic to Galleria mellonella, Drosophila melanogaster and Culex pipiens. They referred to the toxin as tolypin but Krasnoff et al. (1991) showed that the active compounds were efrapeptins. The five efrapeptins (C, D, E, F, G) identified were shown to be useful taxonomic markers (Krasnoff & Gupta 1992, Hodge et al. 1996). Krasnoff et al. (1991) showed that efrapeptins had antifungal and insecticidal properties but their studies were limited to foliar assays, studies on mitochondrial ATPases from a house fly, and Metarhizium anisopliae and T. niveum. Crude efrapeptin extracts sprayed onto leaves were toxic to Colorado potato beetle (CPB), Southern armyworm, tobacco budworm and the two-spotted spider mite, with CPB being the most sensitive.

In spite of their insecticidal and putative antifungal properties very little is known about the production of these compounds in vitro and their ecological significance, i.e. role in fungal survival in the field and pathogenicity towards insect hosts. The aim of this study was to examine the production of efrapeptins by different Tolypocladium species and to determine their activity against selected bacteria, fungi and insect species.

MATERIALS AND METHODS

Maintenance of fungal, bacterial and insect cultures

Origins of the strains of Tolypocladium used in this study are listed in Table 1. All cultures were grown on Sabouraud dextrose agar (Oxoid) supplemented with 1% w v yeast extract (Fluka) at 23 C and 16 :8 light :dark photoperiod. The insects used and details of their source and maintenance are given in Table 2. The source of the fungi and bacteria against which efrapeptins were tested are given in Table 3. All the fungi were grown on potato dextrose agar (PDA, Oxoid) except for P. infestans, which was maintained on rye agar (Caten & Jinks 1968). Fungal cultures were incubated at 23 and for 5 d except for P. infestans, which was kept at 18 for

7 d. All fungal cultures were exposed to 16 :8 h light :dark photoperiod. Bacteria were grown on tryptone soya agar (TSA, Oxoid) at 30 .

Production of efrapeptins by Tolypocladium species

Table 1. Origin of the strains of Tolypocladium.

538

 

Origin

Host

ARSEF 616

T. niveum

Czechoslovakia

Unknown

ARSEF 3321

T. niveum

Canada

Choristoneura fumiferana (Lepidoptera : Torticidae) Root of black spruce, clay soil Maple beech birch forest floor Dryas site, Warcup soil plate, alpine meadow Bdelloid rotifers in farm soil Raised beach soil Festuca grasslands, moorland soil

ARSEF 3394

T. niveum

Canada

ARSEF 3398

T. niveum

USA

ARSEF 3434

T. nubicola

Canada

ARSEF 3436

T. parasiticum

Canada

ARSEF 3399

T. tundrense

Canada

ARSEF 3401

T. tundrense

United Kingdom

**IMI 292558

T.

cylindrosporum

United

Kingdom

Mosquito

larvae

(Diptera : Bibionidae)

UAMH 4561

T. cylindrosporum

USA

Mosquito larvae (Aedes sierrensis) Mosquito larvae (A. australis) Sewage Mosquito larvae (A. sierrensis)

UAMH 4562

T. cylindrosporum

New Zealand

UAMH 4563

T. cylindrosporum

Czechoslovakia

UAMH 5002

T. cylindrosporum

USA

* ARSEF strains kindly provided by Dr Richard Humber, USDA-ARS, Ithaca, NY. ** IMI from the collection of CABI Bioscience, Egham, Surrey.

Table 2. Insect species including, their source and diet.

 

Stage

Diet

Source

Galleria mellonella (Lepidoptera : Pyralidae) Manduca sexta (Lepidoptera : Sphingidae) Calliphora vomitoria (Diptera : Calliphoridae) Tenebrio molitor (Coleoptera : Tenebrioidae) Periplaneta americana (Dictyoptera : Blattidae) Myzus persicae (Homoptera : Aphidae)

Last larval

15.4% dried skimmed milk, 30.8% wheat germ, 30.8% baby rice, 11.5% honey, 11.5% glycerine at 30 in the dark According to method of Samuels et al. 1988

IACR-Rothamsted

Last larval

Bath University*

Last larval

Mammalian blood

Bangor University**

Last larval

Wheat bran

Bangor University**

Adults

Dry bread, cheese

Bangor University**

Adults

Chinese cabbage at 20 , 16 :8 hr light :dark photoperiod

IACR-Rothamsted

* Kindly provided by Dr Keith Charnley. ** Kindly provided by Dr David Shaw.

Table 3. Fungi and bacteria against which efrapeptins were assayed, and their source.

Species

Source

Phytophthora infestans Dr David Shaw, Bangor University Metarhizium anisopliae (V245) IACR-Rothamsted Fusarium moniliforme (C2804)

F.

oxysporum (C2799)

F.

proliferatum (C2172)

Cladosporium sp (C1089) Aspergillus ochraceus Mucor sp. Serpula lacrymans (FPRL 12C) Sporobolomyces roseus Coniophora puteana (BAM 515) Coriolus versicolor (FPRL 28G) Poria placenta (FPRL 280)

Micrococcus luteus Dr Jane Faull, London University

Bacillus cereus (B34) Escherichia coli (D31)

IACR-Rothamsted Dr John Palfreyman, University of Abertay Dundee

Prof. Norman Ratcliffe, University of Wales Swansea

Toxin production in vitro

Conidia were harvested from 14 d old sporulating cultures by scraping the surface with a spatula and suspending the conidia in sterile 0.03% v v aqueous Tween 80 (BDH). The number of conidia was determined using an improved Neubauer

haemocytometer and the suspension was diluted to provide the desired conidial concentration using 0.03% aqueous Tween 80. All Tolypocladium strains assayed (Table 1) were grown in 100 ml of Czapek Dox (Oxoid) liquid medium supplemented with 0.5% w v Bactopeptone (Oxoid) in 250 ml Erlenmeyer flasks. The flasks were inoculated with 1 ml of conidial suspension containing 10 conidia ml and incubated at 23 in a cooled orbital incubator (Gallenkamp) at 150 rpm for 14 d in the dark. The cultures were harvested by filtering the mycelium through four layers of cheesecloth. The culture filtrate was filtered through a Buchner funnel lined with Whatman filter paper No. 1 to ensure complete removal of conidia and hyphal debris. The weight of mycelium residue was recorded at the end of the incubation period for all experiments. The mycelium was freeze-dried before weighing. Unless indicated otherwise there were three replicates per treatment and all experiments were repeated three times. An additional batch of T. niveum 616 was grown as described above. Five replicate flasks were retrieved from the orbital shaker initially after 48 h then at 3 day intervals to determine pH, biomass, crude extract, and efrapeptin profiles. The last samples were taken 30 d after inoculation. Culture filtrates were extracted as described by (Gupta et al. 1992). This entailed extraction of culture filtrate with analar dichloromethane (Fisons), filtration of the solvent phase

A. R. Bandani and others

through Whatman No. 1PS (phase separator) filter paper to remove any aqueous residue, then removal of solvent on a rotary evaporator (Gallenkamp). The residue (crude extract) was dissolved in methanol, filtered through a cotton plug, and concentrated under a stream of dry nitrogen at 40 . The residue was weighed and stored at 4 . Freeze-dried mycelium was ground to a fine powder, dissolved in absolute methanol (4 g 100 ml ) then filtered through Whatman No. 1 filter paper. The solvent was evaporated in vacuo and the residue weighed and stored at 4 .

Purification of efrapeptins

The crude extract was dissolved in dichloromethane and subjected to flash chromatography as described by Still et al. (1978) and Gupta et al. (1992). Individual efrapeptins were isolated by HPLC on a reverse phase C8 column (Hichrom, 25 0.46 cm, 5 µm particle size) eluted with acetonitrile- ammonium sulphate (65 :35) gradient with uv detection of 225 nm. The identity of each compound was verified by fast atom bombardment mass spectrometry (Krasnoff & Gupta

1991).

Toxin production in vivo

Conidia harvested from axenic cultures of T. niveum 616 were suspended in sterile 0.03% v v aqueous Tween 80, sieved through cheesecloth to remove hyphal fragments then pelleted by centrifugation (3000 g for 5 min). The conidia were washed 3 times with 0.03% aqueous Tween 80 with intervening centrifugation steps, and diluted to 1 10 conidia ml using distilled water. Two hundred final instar Galleria larvae were injected with the conidial suspension (5 µl per insect) or saline only (control) through the first proleg using a 1 ml Burkard syringe and microinjector (Burkard, U.K.) with 30 gauge sterile disposable needle. Insects were kept in batches of ten in 9 cm diam. Petri dishes containing the appropriate diet (Table 2) at 23 . Haemolymph was collected from mycosed Galleria larvae at the paralysis stage (i.e. larvae were alive but when placed on their backs were unable to right themselves, although they were able to move their legs). Larvae were pierced at the base of the proleg with a sterile 30 gauge needle and haemolymph collected in a sterile glass capillary tube (50 µl per larva). The haemolymph from five insects was pooled in a glass test tube and heat fixed at 80 for 3 min. The sample was dissolved in 5 ml of distilled water and centrifuged at 3000 g for 5 min. The toxin mixture was extracted from the supernatant with dichloromethane as described before. The experiment was repeated twice. In another experiment, two hundred and fifty additional Galleria larvae were injected with conidia of T. niveum 616 and the larvae frozen as soon as they died, usually within 3 d of injection. The dead larvae were homogenised in liquid nitrogen using a pestle and mortar. The homogenate was thoroughly dissolved in methanol before the debris was pelleted by centrifugation at 3000 g for 5 min. The supernatant was filtered through Whatman No. 1 filter paper and the solvent evaporated in vacuo. The residue was suspended in

539

10 ml distilled water and the toxins extracted with dichloro- methane as described earlier. Toxin residues (10 µl of 1 mg ml ) from the haemolymph were applied to a Whatman TLC plate (silica particles 150A, gel thickness 250 µm) and developed with a dichloro- methane :methanol (93 :7) solvent mixture. The separation was monitored by visualisation with uv 254. Efrapeptin bands scraped from the TLC plate were placed in Eppendorf tubes and dissolved in 500 µl methanol and the silica debris removed by centrifugation (3000 g for 5 min). The supernatant was concentrated with gentle heating (40 ) under a stream of dry nitrogen before use in bioassays or further purification. Residues from the insect homogenate were further purified by a combination of flash chromatography and HPLC (see sections above) and the identity of individual efrapeptins verified by mass spectroscopy.

Antimicrobial assay

The anti-microbial activity of efrapeptins was determined using the paper disc diffusion assay and TLC overlay assay (Homans & Fuchs 1970, Cole 1994). In the disc diffusion assay, 50 mm diam. discs of Whatman No. 1 filter paper were treated with 10 µl of either 10 mg ml crude extract (from culture filtrate or mycelium) or 2 mg ml purified efrapeptin mixture. Three treated and one control (solvent only) discs were placed perpendicular to each other, on freshly inoculated bacterial and fungal cultures in 9 cm diam. Petri dishes. To inoculate with bacteria, 100 µl of 10 bacteria ml were spread over the medium and incubated at 30 , resulting in a bacterial lawn within 24 h. The presence of a zone of inhibition in the bacterial lawn was interpreted as positive activity. A plug of fungal mycelium (ca 2 2 mm) was taken from the edge of an actively growing colony to inoculate the centre of the culture medium. The treated and control filter discs were placed halfway between the plug and the edge of the Petri dish. Inhibition of fungal growth was interpreted as positive activity. Each test had two replicates and the experiment was repeated twice. In the TLC overlay assay, 10 µl of 1 mg ml crude extract was applied to a Whatman TLC plate (silica particles 150A ; gel thickness 250 µm) together with Cyclosporin A and efrapeptin standards and developed as described before. The TLC plate, after air drying at room temperature, was placed flat on a sheet of 1% w v water agar (Fisons) which not only stabilised the plate but also promoted a humid environment. The TLC plate was then overlaid with 50 ml TSA and inoculated with 1 ml of Micrococcus luteus (10 bacteria ml ) and incubated at 30 for 24 h before recording the R F values of the inhibition zones.

Insecticidal activity

Crude extract (2 mg in 20 µl dimethylsulphoxide (DMSO)) of T. niveum 616 was diluted to 0.3 and 1 mg ml with sterile distilled water and 10 µl injected into cohorts of 40 of each of the final instar larvae of Galleria mellonella, Calliphora vomitoria,

Production of efrapeptins by Tolypocladium species

C Ac-PIP-AIB-PIP-AIB-AIB-LEU-b-ALA-GLY-AIB-AIB-PIP-AIB-GLY-LEU-AIB-X (FW = 1606)

D Ac-PIP-AIB-PIP-AIB-AIB-LEU-b-ALA-GLY-AIB-AIB-PIP-AIB-GLY-LEU-IVA-X (FW = 1620)

E Ac-PIP-AIB-PIP-IVA-AIB-LEU-b-ALA-GLY-AIB-AIB-PIP-AIB-GLY-LEU-IVA-X (FW = 1634)

F Ac-PIP-AIB-PIP-AIB-AIB-LEU-b-ALA-GLY-AIB-AIB-PIP-AIB-ALA-LEU-IVA-X (FW = 1634)

G Ac-PIP-AIB-PIP-IVA-AIB-LEU-b-ALA-GLY-AIB-AIB-PIP-AIB-ALA-LEU-IVA-X (FW = 1648)

H X = —NH N ALA = alanine GLY = glycerine LEU = leucine AIB
H
X = —NH
N
ALA = alanine
GLY = glycerine
LEU = leucine
AIB = a-aminoisobutyric acid
b-ALA = b-alanine
PIP = pipecolic acid
+ IVA = isovaline
N AC = acetyl
FW = Formula weight

Fig. 1. Structure of efrapeptins.

Tenebrio molitor and adult Periplaneta americana. Control insects were injected with 10 µl 1% v v aqueous DMSO only. Galleria larvae were injected through the first proleg and the other insects were injected through the intersegmental membrane. Batches of five treated or control insects were placed in 9 cm diam. Petri dishes lined with dry filter paper and provided with the appropriate diet (Table 2). Adult Myzus persicae were exposed to leaves of Chinese cabbage sprayed with the toxin or control solution. One ml of crude toxin (0.3 or 1 mg ml ) was applied with a gun sprayer (Humbrol) to each surface of the leaf. The leaves were left on a corrugated sheet of aluminum foil to dry for 1 h at room temperature, then 2.5 cm diam. leaf discs were punched out from treated and control leaves and placed on a thin layer of water agar (1% w v) in 5 cm Petri dishes. There was one disc per Petri dish with ten adult M. persicae placed on each disc. Each treatment was replicated ten times. In another experiment, the pure efrapeptin mixture was dissolved in DMSO and diluted to 300, 100, 50, 25, and 12.5 µg ml with distilled water. The final concentration of DMSO was 1% (v v). Five µl of each dose was injected into 40 cohorts of final instar larvae of G. mellonella and Manduca sexta as described above. In this and above assays, insect mortality was recorded 48 h post-treatment.

540

RESULTS

Toxin production in vitro

Efrapeptins (Fig. 1) were secreted by all Tolypocladium species used in this study, although T. nubicola and T. parasiticum only secreted efrapeptin E (Tables 4–5). Efrapeptins were not detected in extracts of the mycelium. Only strains of T. niveum secreted all five efrapeptins, and no other species secreted efrapeptin C. Efrapeptin D was not secreted by T. tundrense

Bissett and T. cylindrosporum 4563 and 4996.

There was no clear link between efrapeptin production, biomass and the crude extract harvested from culture filtrates (Tables 4–5). Some strains yielded little crude extract (e.g. T. niveum 3396) while others with similar or lower fungal biomass yielded substantially more crude extract (e.g. T. cylindrosporum 2005, 4562). There was also inter- and intra- specific variation in the proportion of efrapeptins in the crude extracts (Tables 4–5). The amounts of efrapeptin (C–G) produced 100 ml culture filtrate ranged between 4.5– 11.6 mg for T. cylindrosporum strains, 2.9–8 mg for T. niveum strains and 0.2 mg for T. parasiticum (Table 5). The quantities of the different efrapeptins produced by T. cylindrosporum were in the order F G E D, for three strains of T. niveum (616, 3396 and 3394) it was D E F C G, while that for T. tundrense was F G E (Tables 4–5). Strain 3321 of T. niveum produced more F than any other efrapeptin (Table 5). T. niveum 616 had a typical growth curve, i.e. biomass (mycelium), crude extract, and efrapeptin production increased rapidly for the first 9 d then slowed down (Table 4). The pH declined gradually from 6.8 to 4.1. The amount of biomass and crude extract in 100 ml of culture filtrate increased 1.7 and 6.7 fold, respectively, between day 2 and 3 while the amount of efrapeptins increased 11 fold between day 2 and 3 (Table

4).

The efrapeptin profile altered slightly during the growth period. Efrapeptin D declined and E increased after 15 d growth. In contrast, efrapeptin C and F did not alter significantly between days 3 and 30 (Table 4). Efrapeptin G constituted 4.9% of the total efrapeptin content on day 2 decreasing to 3.4% by day 30 (Table 4).

Table 4. Efrapeptin production by Tolypocladium niveum (616) over 30 d. CF culture filtrate.

Individual efrapeptins (%)

Days

 

Dry mycelium Crude extract

Efrapeptins (mg 100 ml CF)

 
 

postinoculation

pH of CF

(mg 100 ml CF)

(mg 100 ml CF)

C

D

E

F

G

2

6.8 (0.1)*

120 (1.8)

0.4 (0.1)

0.06 (0.002)

5.2 (0.5)

48.7 (0.2)

24.5 (0.8)

16.6 (0.2)

4.9 (0.5)

3

6.7 (0.1)

205 (2.9)

2.7 (0.3)

0.7 (0.02)

5.1 (0.6)

45.6 (0.7)

22.2 (0.8)

19.2 (0.2)

4.8 (0.7)

6

5.9 (0.1)

242 (2.0)

5.3 (0.6)

2.1 (0.03)

4.6 (0.4)

43.6 (0.8)

28 (0.6)

19 (0.6)

4.8 (0.3)

9

5.5 (0.1)

300 (1.8)

7.8 (0.5)

3.7 (0.03)

4.1 (0.3)

41.4 (0.7)

28.4 (0.6)

21.6 (0.9)

4.6 (0.3)

12

5.0 (0.2)

325 (1.1)

9.3 (0.5)

4.4 (0.06)

4.4 (0.6)

46.4 (0.5)

24.6 (0.6)

20.4 (0.7)

4.3 (0.5)

15

4.6 (0.1)

336 (1.3)

10.6 (0.4)

4.9 (0.05)

4.9 (0.2)

42.3 (0.5)

27.4 (0.9)

21.3 (0.9)

4.2 (0.5)

18

4.4 (0.1)

342 (1.2)

11.8 (0.7)

5.3 (0.05)

4.0 (0.5)

39.0 (0.8)

34.9 (0.3)

18.0 (0.6)

4.5 (0.4)

21

4.2 (0.2)

367 (1.7)

12.8 (0.5)

5.1 (0.08)

4.5 (0.7)

38.3 (0.7)

34.5 (0.7)

19.2 (0.9)

4.0 (0.5)

24

4.2 (0.2)

391 (2.2)

13.4 (0.4)

5.2 (0.04)

4.5 (0.6)

37.7 (0.3)

33.5 (0.4)

20.0 (0.9)

4.3 (0.5)

27

4.1 (0.1)

404 (1.9)

13.4 (0.5)

5.2 (0.08)

4.4 (0.5)

37.3 (0.7)

34.2 (0.8)

19.5 (0.7)

4.5 (0.6)

30

4.1 (0.2)

406 (1.8)

13.7 (0.4)

5.4 (0.07)

4.5 (0.4)

36.2 (0.5)

34.4 (0.9)

19.5 (0.9)

3.4 (0.7)

A. R. Bandani and others

541

Table 5. Efrapeptin production by isolates of Tolypocladium niveum (TN), T. cylindrosporum (TC), and other Tolypocladium spp., 14 d postinoculation. CF culture filtrate.

Individual Efrapeptins (%)

 

Dry mycelium Crude extract

Efrapeptins (mg 100 ml CF)

 

(mg 100 ml CF)

(mg 100 ml CF)

 

C

D

E

F

G

TN 3396

284.5 (2.3)*

 

9.8 (0.4)

 

2.9 (0.04)

4.7 (0.4)

40 (0.2)

 

28.3 (0.7)

22.0 (0.4)

5.1 (0.4)

TN 3321

385.5 (2.3)

13.7 (0.5)

7.8 (0.1)

2.8 (0.5)

18.8 (0.3)

17.6 (0.4)

40.2 (0.5)

20.8 (0.3)

TN 3394

270 (2.1)

20.9 (0.6)

8.0 (0.1)

6.7 (0.4)

37.6 (0.2)

35.5 (0.4)

14.9 (0.3)

5.4 (0.4)

TC 2005

230.5 (2.6)

42.5 (1.5)

11.6 (0.3)

0.0

2.8 (0.4)

15.1 (0.4)

57.7 (0.5)

24.5 (0.4)

TC 4561

430.5 (1.8)

40 (0.8)

8.4 (0.3)

0.0

5.4 (0.6)

21.0 (0.3)

48.7 (0.4)

25.0 (0.7)

TC 4562

231 (3.3)

45.5 (1.3)

10.3 (0.2)

0.0

14.5 (0.7)

12.45 (0.4)

47.0 (0.4)

26.1 (0.7)

TC 4563

309.5 (2.7)

25.5 (0.2)

4.5 (0.1)

0.0

0.0

14.7 (0.5)

54.8 (0.3)

30.6 (0.4)

TC 4996

91 (2)

38.4 (1.5)

5.5 (0.2)

0.0

0.0

17.8 (0.3)

54.5 (0.4)

27.7 (0.5)

TC 292558

242 (1.7)

 

26.7 (1.3)

6.6 (0.1)

0.0

3.7 (0.3)

 

21.0 (0.3)

54.5 (0.7)

20.8 (0.5)

T.

tundrense 3401

576.5 (4.9)

27.4 (0.6)

7.3 (0.08)

0.0

0.0

6.6 (0.5)

56.7 (0.3)

36.7 (0.3)

T.

parasiticum 3436

116.5 (1.3)

2.8 (0.5)

0.2 (0.01)

0.0

0.0

100.0

0.0

0.0

T.

nubicola 3434

721 (3.4)

36.5 (1.2)

0.00

0.0

0.0

0.0

0.0

0.0

 

* .. in brackets.

   

10

 

20

 

D

 
   

30

 

40

% Absorbancy (225 nm)

 
 

D

 
   

50

 

E

 
   

60

 

F

 

70

 

C

G

C

F

 

80

 

E

G

 
 
     
   

90

   
 

1234

 

5

12

3

45

 
 

Time (min)

Fig. 2. HPLC chromatogram of Tolypocladium niveum 616 from culture filtrate extraction (left) and from mycosed Galleria mellonella larvae (right).

Production of efrapeptins by Tolypocladium species

Production of efrapeptins by Tolypocladium species Fig. 3. The effect of 10 ml − of crude

Fig. 3. The effect of 10 ml of crude extract of Tolypocladium parasiticum culture filtrate (left) and mycelium (right) on Micrococcus luteus.

Toxin production in vivo

Very little efrapeptin was isolated from infected insects, ca 2 µg was purified from 10 ml of haemolymph derived from 200 Galleria larvae. But 600 µg of efrapeptin was extracted from 250 homogenised Galleria larvae. In both cases, some efrapeptin may have been lost during the purification process. The efrapeptin profile of extracts from haemolymph was D

F

C E G while that from culture filtrate was D E

F

C G (Fig. 2).

Antimicrobial activity

Micrococcus luteus (gram ve) was highly sensitive to efrapeptins compared with E. coli (gram ve) and B. cereus

(gram ve). In the TLC overlay assay the zone of inhibition

of M. luteus corresponded with the efrapeptin band (R F 0.1)

and not with the band for cyclosporin. In the paper disc diffusion assay, M. luteus growth was inhibited by extracts from culture filtrates (not mycelium) of those species of Tolypocladium known to produce efrapeptins (Fig. 3). A pure mixture of efrapeptins was inhibitory to M. luteus at 20 µg per disc (data not presented). In the paper disc diffusion assay, Aspergillus ochraceus was the most sensitive of the fungi at 20 µg of the efrapeptin mixture per disc, but the zone of inhibition was small. Mucor, Fusarium and Cladosporium species were slightly inhibited at 50 µg per disc but none of the basidiomycetes were affected (Table 6).

Insecticidal activity

The T. niveum 616 crude extract (ca 46% efrapeptin) was insecticidal on injection even at 0.3 mg ml , with Galleria and Calliophora being the most sensitive followed by Tenebrio larvae (Table 7). Insects showed no symptoms of immediate paralysis when using the low dose (0.3 mg ml ) but at 1 mg ml they became paralysed and died. The speed of paralysis and death was dose-related (data not presented). Once paralysed the insects did not recover. Myzus persicae

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Table 6. Response of fungi and bacteria to efrapeptins.

Efrapeptins concentration ( µg per disc)

 

20

50

Fungi :

Phytophthora infestans

Metarhizium anisopliae

Fusarium moniliforme

F.

oxysporum

F.

proliferatum

Cladosporium sp.

Aspergillus ochraceus

Mucor sp.

Serpula lacrymans

Sporobolomyces roseus

Poria placenta

Coriolus versicolor

Coniophora puteana

Bacteria :

 

Micrococcus luteus

ND

Bacillus cereus

ND

Escherichia coli

ND

Inhibition, No Inhibition, ND Not determined.

Table 7. Percentage Mortality of different insects when treated with crude efrapeptin extract of Tolypocladium niveum 616.

Insect species

Crude efrapeptin

Concentration (mg ml )

 

0.3

1.0

Galleria mellonella*

72 (0.9)

100

Calliphora sp.*

76 (0.6)

100

Tenebrio molitor*

58 (1.4)

92 (0.5)

Periplaneta americana**

54 (1.2)

82 (0.9)

Myzus persicae**

53 (1.1)

84 (0.8)

* Last instar larvae. ** adults.

.. in brackets.

were killed if they contacted leaf discs treated with the crude extract. Many aphids still died even though they moved away from the discs after a few hours of exposure to the leaves. Mortality was greater at the highest dose (1 mg ml ) with many insects dying within the first 24–36 h. The LD values of the purified efrapeptin mixture for last instar larvae of G. mellonella and M. sexta in injection assays were on average 30 and 47 ng, respectively.

DISCUSSION

This study shows intra- and inter-specific variation in efrapeptin production by Tolypocladium species which confirms the observations of Krasnoff & Gupta (1992). It shows for the first time that T. parasiticum (3436) produces efrapeptin E whereas earlier studies failed to detect this compound (Krasnoff & Gupta 1992). There is no report of any other fungus producing these compounds which suggests that efrapeptins

A. R. Bandani and others

are limited to Tolypocladium (Krasnoff & Gupta 1992) with only a few Tolypocladium species producing all five efrapeptins. Some species of Tolypocladium do not produce these compounds, e.g. T. nubicola (3434), T. balanoides and T. extinguense (Krasnoff & Gupta 1992). Based on these observations, it has been suggested that efrapeptins could be used as chemotaxonomic markers to identify Tolypocladium species and distinguish the various anamorphs from pheno- typically similar but unrelated species (Hodge et al. 1996). Cordyceps subsessilis was shown to produce efrapeptins, so Hodge et al. (1996) concluded that this was a teleomorph of Tolypocladium. No clear pattern exists between the quantity of efrapeptin secreted and the ecological or taxonomic status of the different strains tested. The differences noted may reflect differences in metabolism. Studies by Krasnoff & Gupta (1991) showed that supplementing the medium of T. geodes Games and T. niveum with glycine increased the production of D relative to F while supplementing with alanine increased the production of F relative to D. These observations suggest that efrapeptin synthesis is influenced by the nutrients available and the intrinsic physiological attributes of the strain. The fact that efrapeptins were detected in 2 d old cultures and increased with biomass suggests that their production is constitutive. In ageing cultures, however, the quantities decreased which could be due to either less efrapeptin being secreted or a slow decay of existing compounds. Efrapeptins were only detected in culture filtrates and not in mycelium, suggesting that they are secreted rapidly. This may be a strategy to prevent these compounds from interfering with normal metabolic processes because efrapeptins are known to inhibit the mitochondrial ATPase activity of several organisms including Tolypocladium itself (Krasnoff et al. 1991, Abrahams et al. 1996). Efrapeptins exhibited insecticidal activity with some insects being much more susceptible than others. Myzus persicae were killed on sprayed leaves, suggesting that these compounds have contact toxicity and are readily absorbed through the insect cuticle. This is further supported by the fact that aphids do not ingest leaf material but feed on phloem contents. Efrapeptins also exhibited differential toxicity against several fungi and bacteria. The bacterium M. luteus was extremely sensitive and was shown in this study to be a useful efrapeptin indicator. Because efrapeptins had little or no impact on these microorganisms suggests that in nature their primary role is not in the protection of Tolypocladium against antagonistic soil inhibiting bacteria and fungi. This study shows for the first time that Tolypocladium species do secrete efrapeptins in the insect haemocoel during the infection process. Very few fungal elements were detected in dead insects, suggesting that death was due to toxicosis but the quantities of efrapeptin detected were smaller than those required to cause paralysis and death. Efrapeptins appear to work in concert with other pathogenicity determining factors. Toxins of other entomogenous fungi like Metarhizium anisopliae are often implicated as the cause of death of infected insects (Roberts 1981, Ferron 1985, Samuels et al. 1988, Clarkson & Charnley 1996). Toxins may be important pathogenicity determinants for some strains of entomogenous fungi but not others. Some hemi-biotroph pathogens may use

543

toxins to kill their host and to enable them to switch to a necrotrophic mode of nutrition. This is often observed in a number of plant pathogenic fungi (Buchwaldt & Green 1992). Paralysis preceded death suggesting that efrapeptins may immobilise the insect host during very early stages of the infection process. The insecticidal cyclodepsipeptides secreted by M. anisopliae are considered to be immunosuppressants because they are known to interfere with haemocyte activity (Vilcinskas, Matha & Gotz 1997, Huzham, Lackie & McCorkindale 1989). This would undoubtedly provide time for the pathogen to adapt to the new environment before extensive colonisation. The role of efrapeptins still remains to be elucidated.

ACKNOWLEDGEMENTS

The authors wish to thank John Palfreyman, David Shaw, Richard Humber, Norman Ratcliffe, Keith Charnley and the late John Lacey for providing some of the fungal strains and insects used. The authors also wish to thank Alan Mudd for providing mass spectrometry data. This project was funded in part by the Ministry of Agriculture, Fisheries and Food of the UK. IACR- Rothamsted receives grant-aided support from the Biotechnology and Biological Sciences Research Council of the UK. Ali Reza Bandani received funding from the Government of Iran.

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Corresponding Editor: C. A. Shearer