Beruflich Dokumente
Kultur Dokumente
537
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.
Origin Host
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)
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 539
through Whatman No. 1PS (phase separator) filter paper to 10 ml distilled water and the toxins extracted with dichloro-
remove any aqueous residue, then removal of solvent on a methane as described earlier.
rotary evaporator (Gallenkamp). The residue (crude extract) Toxin residues (10 µl of 1 mg ml−") from the haemolymph
was dissolved in methanol, filtered through a cotton plug, and were applied to a Whatman TLC plate (silica particles 150A,
concentrated under a stream of dry nitrogen at 40m. The gel thickness 250 µm) and developed with a dichloro-
residue was weighed and stored at 4m. methane : methanol (93 : 7) solvent mixture. The separation
Freeze-dried mycelium was ground to a fine powder, was monitored by visualisation with uv 254. Efrapeptin bands
dissolved in absolute methanol (4 g 100 ml−") then filtered scraped from the TLC plate were placed in Eppendorf tubes
through Whatman No. 1 filter paper. The solvent was and dissolved in 500 µl methanol and the silica\debris
evaporated in vacuo and the residue weighed and stored at 4m. 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.
Purification of efrapeptins Residues from the insect homogenate were further purified
by a combination of flash chromatography and HPLC (see
The crude extract was dissolved in dichloromethane and
sections above) and the identity of individual efrapeptins
subjected to flash chromatography as described by Still et al.
verified by mass spectroscopy.
(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 Antimicrobial assay
225 nm. The identity of each compound was verified by fast
The anti-microbial activity of efrapeptins was determined
atom bombardment mass spectrometry (Krasnoff & Gupta
using the paper disc diffusion assay and TLC overlay assay
1991).
(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
Toxin production in vivo
culture filtrate or mycelium) or 2 mg ml−" purified efrapeptin
Conidia harvested from axenic cultures of T. niveum 616 were mixture. Three treated and one control (solvent only) discs
suspended in sterile 0.03 % v\v aqueous Tween 80, sieved were placed perpendicular to each other, on freshly inoculated
through cheesecloth to remove hyphal fragments then pelleted bacterial and fungal cultures in 9 cm diam. Petri dishes. To
by centrifugation (3000 g for 5 min). The conidia were inoculate with bacteria, 100 µl of 10* bacteria ml−" were
washed 3 times with 0.03 % aqueous Tween 80 with spread over the medium and incubated at 30m, resulting in a
intervening centrifugation steps, and diluted to 1i10( bacterial lawn within 24 h. The presence of a zone of
conidia ml−" using distilled water. Two hundred final instar inhibition in the bacterial lawn was interpreted as positive
Galleria larvae were injected with the conidial suspension (5 µl activity. A plug of fungal mycelium (ca 2i2 mm) was taken
per insect) or saline only (control) through the first proleg from the edge of an actively growing colony to inoculate the
using a 1 ml Burkard syringe and microinjector (Burkard, U.K.) centre of the culture medium. The treated and control filter
with 30 gauge sterile disposable needle. Insects were kept in discs were placed halfway between the plug and the edge of
batches of ten in 9 cm diam. Petri dishes containing the the Petri dish. Inhibition of fungal growth was interpreted as
appropriate diet (Table 2) at 23m. Haemolymph was collected positive activity. Each test had two replicates and the
from mycosed Galleria larvae at the paralysis stage (i.e. larvae experiment was repeated twice.
were alive but when placed on their backs were unable to right In the TLC overlay assay, 10 µl of 1 mg ml−" crude extract
themselves, although they were able to move their legs). was applied to a Whatman TLC plate (silica particles 150A ;
Larvae were pierced at the base of the proleg with a sterile 30 gel thickness 250 µm) together with Cyclosporin A and
gauge needle and haemolymph collected in a sterile glass efrapeptin standards and developed as described before. The
capillary tube (50 µl per larva). The haemolymph from five TLC plate, after air drying at room temperature, was placed
insects was pooled in a glass test tube and heat fixed at 80m flat on a sheet of 1 % w\v water agar (Fisons) which not only
for 3 min. The sample was dissolved in 5 ml of distilled water stabilised the plate but also promoted a humid environment.
and centrifuged at 3000 g for 5 min. The toxin mixture was The TLC plate was then overlaid with 50 ml TSA and
extracted from the supernatant with dichloromethane as inoculated with 1 ml of Micrococcus luteus (10* bacteria ml−")
described before. The experiment was repeated twice. and incubated at 30m for 24 h before recording the RF values
In another experiment, two hundred and fifty additional of the inhibition zones.
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
Insecticidal activity
nitrogen using a pestle and mortar. The homogenate was
thoroughly dissolved in methanol before the debris was Crude extract (2 mg in 20 µl dimethylsulphoxide (DMSO)) of
pelleted by centrifugation at 3000 g for 5 min. The supernatant T. niveum 616 was diluted to 0.3 and 1 mg ml−" with sterile
was filtered through Whatman No. 1 filter paper and the distilled water and 10 µl injected into cohorts of 40 of each of
solvent evaporated in vacuo. The residue was suspended in the final instar larvae of Galleria mellonella, Calliphora vomitoria,
Production of efrapeptins by Tolypocladium species 540
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.
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
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
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
DISCUSSION
The T. niveum 616 crude extract (ca 46 % efrapeptin) was
insecticidal on injection even at 0.3 mg ml−", with Galleria and This study shows intra- and inter-specific variation in
Calliophora being the most sensitive followed by Tenebrio efrapeptin production by Tolypocladium species which confirms
larvae (Table 7). Insects showed no symptoms of immediate the observations of Krasnoff & Gupta (1992). It shows for the
paralysis when using the low dose (0.3 mg ml−") but at first time that T. parasiticum (3436) produces efrapeptin E
1 mg ml−" they became paralysed and died. The speed of whereas earlier studies failed to detect this compound (Krasnoff
paralysis and death was dose-related (data not presented). & Gupta 1992). There is no report of any other fungus
Once paralysed the insects did not recover. Myzus persicae producing these compounds which suggests that efrapeptins
A. R. Bandani and others 543
are limited to Tolypocladium (Krasnoff & Gupta 1992) with toxins to kill their host and to enable them to switch to a
only a few Tolypocladium species producing all five efrapeptins. necrotrophic mode of nutrition. This is often observed in a
Some species of Tolypocladium do not produce these number of plant pathogenic fungi (Buchwaldt & Green 1992).
compounds, e.g. T. nubicola (3434), T. balanoides and T. Paralysis preceded death suggesting that efrapeptins may
extinguense (Krasnoff & Gupta 1992). Based on these immobilise the insect host during very early stages of the
observations, it has been suggested that efrapeptins could be infection process. The insecticidal cyclodepsipeptides secreted
used as chemotaxonomic markers to identify Tolypocladium by M. anisopliae are considered to be immunosuppressants
species and distinguish the various anamorphs from pheno- because they are known to interfere with haemocyte activity
typically similar but unrelated species (Hodge et al. 1996). (Vilcinskas, Matha & Gotz 1997, Huzham, Lackie &
Cordyceps subsessilis was shown to produce efrapeptins, so McCorkindale 1989). This would undoubtedly provide time
Hodge et al. (1996) concluded that this was a teleomorph of for the pathogen to adapt to the new environment before
Tolypocladium. No clear pattern exists between the quantity of extensive colonisation. The role of efrapeptins still remains to
efrapeptin secreted and the ecological or taxonomic status of be elucidated.
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 A C K N O W L E D G E M E N TS
and T. niveum with glycine increased the production of D
The authors wish to thank John Palfreyman, David Shaw, Richard Humber,
relative to F while supplementing with alanine increased the
Norman Ratcliffe, Keith Charnley and the late John Lacey for providing
production of F relative to D. These observations suggest that some of the fungal strains and insects used. The authors also wish to thank
efrapeptin synthesis is influenced by the nutrients available Alan Mudd for providing mass spectrometry data. This project was funded
and the intrinsic physiological attributes of the strain. in part by the Ministry of Agriculture, Fisheries and Food of the UK. IACR-
The fact that efrapeptins were detected in 2 d old cultures Rothamsted receives grant-aided support from the Biotechnology and
Biological Sciences Research Council of the UK. Ali Reza Bandani received
and increased with biomass suggests that their production is
funding from the Government of Iran.
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
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