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. BANDANI1, 4, B. P. S. KHAMBAY1, J. L. FAULL2, R. NEWTON3, M. DEADMAN4 and T. M. BUTT1, 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 In spite of their insecticidal and putative antifungal properties

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.
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 mC 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 23m
and for 5 d except for P. infestans, which was kept at 18m 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 30m.
Production of efrapeptins by Tolypocladium species 538

Table 1. Origin of the strains of Tolypocladium.

Origin Host

ARSEF 616 T. niveum Czechoslovakia Unknown

ARSEF 3321 T. niveum Canada Choristoneura fumiferana (Lepidoptera : Torticidae)
ARSEF 3394 T. niveum Canada Root of black spruce, clay soil
ARSEF 3398 T. niveum USA Maple\beech\birch forest floor
ARSEF 3434 T. nubicola Canada Dryas site, Warcup soil plate, alpine meadow
ARSEF 3436 T. parasiticum Canada Bdelloid rotifers in farm soil
ARSEF 3399 T. tundrense Canada Raised beach soil
ARSEF 3401 T. tundrense United Kingdom Festuca grasslands, moorland soil
**IMI 292558 T. cylindrosporum United Kingdom Mosquito larvae (Diptera : Bibionidae)
UAMH 4561 T. cylindrosporum USA Mosquito larvae (Aedes sierrensis)
UAMH 4562 T. cylindrosporum New Zealand Mosquito larvae (A. australis)
UAMH 4563 T. cylindrosporum Czechoslovakia Sewage
UAMH 5002 T. cylindrosporum USA Mosquito larvae (A. sierrensis)

* 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 Last larval 15.4 % dried skimmed milk, 30.8 % wheat germ, 30.8 % baby IACR-Rothamsted
(Lepidoptera : Pyralidae) rice, 11.5 % honey, 11.5 % glycerine at 30m in the dark
Manduca sexta Last larval According to method of Samuels et al. 1988 Bath University*
(Lepidoptera : Sphingidae)
Calliphora vomitoria Last larval Mammalian blood Bangor University**
(Diptera : Calliphoridae)
Tenebrio molitor Last larval Wheat bran Bangor University**
(Coleoptera : Tenebrioidae)
Periplaneta americana Adults Dry bread, cheese Bangor University**
(Dictyoptera : Blattidae)
Myzus persicae Adults Chinese cabbage at 20m, 16 : 8 hr light : dark photoperiod IACR-Rothamsted
(Homoptera : Aphidae)

* Kindly provided by Dr Keith Charnley.

** Kindly provided by Dr David Shaw.

Table 3. Fungi and bacteria against which efrapeptins were assayed, and haemocytometer and the suspension was diluted to provide
their source. the desired conidial concentration using 0.03 % aqueous
Species Source Tween 80. All Tolypocladium strains assayed (Table 1) were
grown in 100 ml of Czapek Dox (Oxoid) liquid medium
Phytophthora infestans Dr David Shaw, Bangor University supplemented with 0.5 % w\v Bactopeptone (Oxoid) in
Metarhizium anisopliae (V245) IACR-Rothamsted
Fusarium moniliforme (C2804)
250 ml Erlenmeyer flasks. The flasks were inoculated with
F. oxysporum (C2799) 1 ml of conidial suspension containing 10) conidia ml−" and
F. proliferatum (C2172) incubated at 23m in a cooled orbital incubator (Gallenkamp) at
Cladosporium sp (C1089) 150 rpm for 14 d in the dark. The cultures were harvested by
Aspergillus ochraceus filtering the mycelium through four layers of cheesecloth. The
Mucor sp. IACR-Rothamsted
Serpula lacrymans (FPRL 12C) Dr John Palfreyman, University of
culture filtrate was filtered through a Buchner funnel lined
Sporobolomyces roseus Abertay Dundee with Whatman filter paper No. 1 to ensure complete removal
Coniophora puteana (BAM 515) of conidia and hyphal debris. The weight of mycelium residue
Coriolus versicolor (FPRL 28G) was recorded at the end of the incubation period for all
Poria placenta (FPRL 280) experiments. The mycelium was freeze-dried before weighing.
Micrococcus luteus Dr Jane Faull, London University
Bacillus cereus (B34) Prof. Norman Ratcliffe, University
Unless indicated otherwise there were three replicates per
Escherichia coli (D31) of Wales Swansea 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
Toxin production in vitro
determine pH, biomass, crude extract, and efrapeptin profiles.
Conidia were harvested from 14 d old sporulating cultures by The last samples were taken 30 d after inoculation.
scraping the surface with a spatula and suspending the conidia Culture filtrates were extracted as described by (Gupta et al.
in sterile 0.03 % v\v aqueous Tween 80 (BDH). The number 1992). This entailed extraction of culture filtrate with analar
of conidia was determined using an improved Neubauer 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 40m. The
residue was weighed and stored at 4m.
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 4m.
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,
25i0.46 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 1i10(
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 23m. 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 80m
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 (40m) 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 30m, 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 2i2 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 30m for 24 h before recording the RF 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 540

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

RESULTS
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) Toxin production in vitro

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)
ALA = alanine
GLY = glycerine
LEU = leucine
H
AIB = a-aminoisobutyric acid
b-ALA = b-alanine
N
X = —NH 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.
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 l culture filtrate.

Individual efrapeptins (%)

Days Dry mycelium Crude extract Efrapeptins
postinoculation pH of CF (mg 100 ml−" 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)

* .. in brackets.
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
l 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

30

40

% Absorbancy (225 nm)

D 50
E

60

F
70
F
C
C
G 80
E

90

1 2 3 4 5 1 2 3 4 5
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 542

Table 6. Response of fungi and bacteria to efrapeptins.

Efrapeptins concentration
( µg per disc)

20 50

Fungi :
Phytophthora infestans k k
Metarhizium anisopliae k k
Fusarium moniliforme k j
F. oxysporum k j
F. proliferatum k j
Cladosporium sp. k j
Aspergillus ochraceus k k
Fig. 3. The effect of 10 ml−" of crude extract of Tolypocladium Mucor sp. k j
parasiticum culture filtrate (left) and mycelium (right) on Micrococcus Serpula lacrymans k k
Sporobolomyces roseus k k
luteus.
Poria placenta k k
Coriolus versicolor k k
Coniophora puteana k k
Bacteria :
Micrococcus luteus j ND
Toxin production in vivo Bacillus cereus k ND
Escherichia coli k ND
Very little efrapeptin was isolated from infected insects, ca
2 µg was purified from 10 ml of haemolymph derived from j l Inhibition, k l No Inhibition, ND l Not determined.
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.
Table 7. Percentage Mortality of different insects when treated with
The efrapeptin profile of extracts from haemolymph was D
crude efrapeptin extract of Tolypocladium niveum 616.
F
C
E
G while that from culture filtrate was D
E

F
C
G (Fig. 2). Concentration
Insect species Crude efrapeptin (mg ml−")

0.3 1.0
Galleria mellonella* 72 (0.9)† 100
Antimicrobial activity
Calliphora sp.* 76 (0.6) 100
Micrococcus luteus (gramjve) was highly sensitive to Tenebrio molitor* 58 (1.4) 92 (0.5)
Periplaneta americana** 54 (1.2) 82 (0.9)
efrapeptins compared with E. coli (gram kve) and B. cereus
Myzus persicae** 53 (1.1) 84 (0.8)
(gram jve). In the TLC overlay assay the zone of inhibition
of M. luteus corresponded with the efrapeptin band (RF l 0.1) † .. in brackets.
* Last instar larvae.
and not with the band for cyclosporin. In the paper disc
** adults.
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 were killed if they contacted leaf discs treated with the crude
disc (data not presented). In the paper disc diffusion assay, extract. Many aphids still died even though they moved away
Aspergillus ochraceus was the most sensitive of the fungi at from the discs after a few hours of exposure to the leaves.
20 µg of the efrapeptin mixture per disc, but the zone of Mortality was greater at the highest dose (1 mg ml−") with
inhibition was small. Mucor, Fusarium and Cladosporium many insects dying within the first 24–36 h.
species were slightly inhibited at 50 µg per disc but none of The LD values of the purified efrapeptin mixture for last
the basidiomycetes were affected (Table 6). &!
instar larvae of G. mellonella and M. sexta in injection assays
were on average 30 and 47 ng, respectively.

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
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.
A C K N O W L E D G E M E N TS
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