Journal ofINVERTEBRATE

Journal of Invertebrate Pathology 85 (2004) 168–174

PATHOLOGY

www.elsevier.com/locate/yjipa

Investigations on the destruxin productionof the entomopathogenic fungus Metarhizium anisopliae

Chengshu Wang,* Anke Skrobek, and Tariq M. Butt

School of Biological Sciences, University of Wales Swansea, Singleton Park, Swansea SA2 8PP, UK

Received 23 December 2003; accepted 24 February 2004

Abstract

The dynamics of cyclic peptide destruxins (dtxs) produced byMetarhizium anisopliae strains V245 and V275 were monitored both

on solid and in liquid media. The results showed that both strains did not produce dtxs in large-scale fermenter cultures or solid

Czapek Dox (CD) agar. Production of the major dtxs A and B could be determined in both strains when grown on rice for up to 10–

30 days. The main dtxs A, B, E, and E diol were detected in CD liquid culture filtrate from both strains after three days post-

inoculation on. Parallel decrease of dtx E and increase of E diol in the culture medium were found, indicating that the latter is the

hydrolytic product from the former. Production of dtxs A and B was significantly positively correlated. A negative correlation was

observed between the production of the metabolites and pH value of the medium. The influence of different nutrient sources on dtx

production was evaluated by using media with different carbon and nitrogen ratios as well as with different insect homogenates. The

findings showed that the amount of dtxs A, B, and E increased with the increasing content of peptone in the medium. When insect

homogenate was used as single nutrient source or added to CD medium, no toxins were detected in the culture filtrate. The potential

risk posed by the toxic metabolites during mass production is discussed.

� 2004 Elsevier Inc. All rights reserved.

Keywords: Metarhizium anisopliae; Destruxin; Carbon/nitrogen ratio; Toxin production

1. Introduction

The entomopathogenic fungus Metarhizium anisop-

liae is one of the most studied and applied species

amongst fungal biocontrol agents, several commercial

products have been developed and registered for the

control of different insect pests (Butt et al., 2001). The

insecticidal cyclic depsipeptides, destruxins (dtxs), pro-

duced by M. anisopliae have been suggested to be an

important virulence factors to accelerate the demise of

infected insects (Brousseau et al., 1996; Dumas et al.,1994; Kershaw et al., 1999). Since the first report of the

discovery of dtxs A and B from M. anisopliae by Ko-

daira (1961), a large number of analogues in this family

have been identified of which dtxs A, B, and E are

predominant (>70%) in submerged culture filtrates

(Loutelier et al., 1996; Pedras et al., 2002; Strasser et al.,

* Corresponding author. Fax: +44-1792-295447.

E-mail address: c.s.wang@swansea.ac.uk (C. Wang).

0022-2011/$ - see front matter � 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.jip.2004.02.008

2000). Destruxins A and E were proven to be more in-

secticidal than others (Dumas et al., 1994).

The mechanism of dtx biosynthesis in M. anisopliae

has not been well documented. The multifunctional non-

ribosomal peptide synthetases (PES) have been sup-

posed to function (Bailey et al., 1996; Jegorov et al.,

1993; Marahiel, 1992; Zuber, 1991), however, there is no

confirmative evidence so far. Both in vivo and in vitro

studies showed that the diffusion of dtxs, albeit in dif-

ferent ratios, is a very rapid process from endogenous

mycelia (Amiri-Besheli et al., 2000; Butt et al., 1994;Loutelier et al., 1996). The production of dtxs differed

not only between different strains of M. anisopliae

(Amiri-Besheli et al., 2000; Hsiao and Ko, 2001; Ker-

shaw et al., 1999), but was also highly influenced by the

component type and ratio, usually carbon and nitrogen,

in the culture media (Liu et al., 2000).

In this study, toxin production dynamics of M. ani-

sopliae strains V245 and V275 were monitored both onsolid and in liquid media to meet the requirements

for developing these strains as potential commercial

C. Wang et al. / Journal of Invertebrate Pathology 85 (2004) 168–174 169

mycoinsecticides. The influence of the culture mediumon dtx secretion was evaluated by using liquid media

with different carbon and nitrogen ratios as well as with

different insect homogenates. The relationships between

the productions of main dtxs, pH, and biomass were

analysed and discussed. The results in this study addi-

tionally provide useful information to assess the poten-

tial risks of fungal toxic metabolites during the mass

production of M. anisopliae.

2. Materials and methods

2.1. Fungal culture and maintenance

The strains V245 and V275 of M. anisopliae were

originally isolated from hay field soil and Carpocapsa

pomonella in Finland and Austria, respectively. The

propagules of strains were preserved in 30% glycerol

under )80 �C for long-term storage and for experiments,

the cultures were grown on potato dextrose agar (PDA,

Difco) at 25 �C in the dark. Plates were kept at 4 �Cwhen fully covered with spores, usually after 14 days.

2.2. Solid cultivation

Petri dishes (£ 9 cm) were charged with 20ml auto-

claved Czapek Dox (CD) medium (K2HPO4, 1 g/L;

MgSO4�H2O, 0.5 g/L; KCl, 0.5 g/L; and FeSO4, 0.01 g/

L) containing 5% mycological peptone and 2% agar and

lined with Whatman No. 1 glass microfibre filter paper

after the medium had solidified. On each plate, conidial

suspension (0.5ml, 108 conidia/ml) was inoculated andspread evenly. After incubation at 25 �C in the dark, two

plates were taken out every three days and the microfi-

bre filter papers with fungal propagules were carefully

peeled off. The agar medium was sliced and soaked in

100ml dichloromethane:ethyl acetate (1:1, v/v) in a

250ml conical flask over night. Crude extracts were

harvested by evaporating the organic solvent and anal-

ysed by HPLC to determine the production of toxicmetabolites. The experiment was repeated twice.

Investigations on toxin production on rice grain were

conducted for comparison. Fifty grams of ‘‘American

Prefluffed Rice’’ (EasyCook, UK) were soaked over

night in 50ml deionised water in a 500ml conical flask.

The autoclaved rice was inoculated with 1ml spore

suspension (107 conidia/ml) and incubated at 25 �C in

the dark. Nine replicates were prepared for both strains.After 10, 20, and 30 days of incubation, the weight of

three batches per strain was determined and each batch

was divided into two equal parts. One part was sus-

pended in 1000ml aq. Tween 80 (0.05 %, v/v) and the

spore concentration was determined with a Rosenthal

haemocytometer. The colonised rice kernels from the

other part were crushed in liquid nitrogen and then ex-

tracted three times for 30min in 100ml ethyl ace-tate:dichloromethane (1:1, v/v) in a sonicator. After

extraction, the mixture was filtered through Whatman�sNo. 1 filter paper, the organic phases were pooled and

washed with 300ml deionised water and the crude ex-

tracts were harvested by evaporating the solvents in

vacuo. Two independent assessments with three repli-

cates each were conducted.

2.3. Submerged cultivation

2.3.1. Toxin production in large-scale fermentation

Metabolite production in fermenter cultures was as-

sessed for both strains. The fungi were pre-cultured in

1000ml conical flasks with 250ml sabouraud dextrose

broth (SDB, Difco) for five days at 23 �C and 150 rpm

and then 100ml were transferred to a 10L fermenterwith 9000ml SDB, growing for seven days at pH 6,

500 rpm, 23 �C and 150L/h air flow. The culture filtrate

was harvested over a CEPA separator with 10,000 rpm

and then filtered. Three replicates of 300 ml were ex-

tracted twice with 300ml dichloromethane:ethyl acetate

(1:1, v/v) for 6 h. Extracts were diluted in metha-

nol:acetonitrile (1:1, v/v) and analysed by HPLC de-

scribed below.

2.3.2. Influence of incubation time on toxin production

Conical flasks (250ml) were charged with 100ml CD

liquid medium containing 5% mycological peptone.

Each flask was inoculated with 0.5ml spore suspension

(108 conidia/ml). The flasks were incubated in dark at

24 �C and 110 rpm on a rotary shaker (Sanyo Gallenk-

amp). Every three days, the cultures from the duplicatedflasks were taken out and filtered through two layers of

Kimwipes and then through Whatman�s No. 1 filter

paper and the filtrate was extracted to determine the

dynamics of toxin production. The variations in pH and

biomass throughout the incubation time were deter-

mined. Correlations between the production of different

dtxs as well as pH and biomass were analysed by cal-

culating Pearson�s correlation coefficient with the pro-gram SPSS 11.0.0 (SPSS Inc.).

2.3.3. Influence of different carbon and nitrogen ratios and

insect homogenates on toxin production

The impact of different nutrient components on the

level of toxic metabolite secretion was evaluated by

growing fungal cultures in media with different ratios of

glucose and peptone as carbon and nitrogen sources(Table 3). Different amounts of insect homogenates

from Tenebrio molitor adults and from the cockroach

Blaberus discoidalis were also used as nutrient sources

for evaluations (Table 3). The adults of Tenebrio and

cockroach were frozen to death for 30min at )80 �C and

homogenised under liquid nitrogen. The homogenates

were dried in oven under 60 �C for 48 h. Additions of

Table 1

Destruxin productions in rice cultures from M. anisopliae strains V245 and V275 over time

Days post-inoculation Dtx yield in mg per 100 g rice (dry weight)

V245 V275

Dtx A Dtx B Dtx E Dtx A Dtx B Dtx E

10 5.4� 1.0 5.8� 0.8 n.d. 8.2� 2.2 0.8� 0.16 n.d.

20 17.6� 0.44 7.6� 1.0 n.d. 9.6� 2.0 6.4� 2.4 n.d.

30 28.6� 3.8 14.2� 2.4 n.d. 1.8� 1.8 34.0� 4.2 n.d.

Mean� SE; n.d., not detected.

170 C. Wang et al. / Journal of Invertebrate Pathology 85 (2004) 168–174

both insect homogenates to CD CN3 (C:N¼ 50:50)

were included to compare the induction effect on toxinproduction. Culture filtrates were harvested after incu-

bating for eight days. The putative toxins were extracted

as described above. Each treatment had two replicates.

2.4. Analytical analysis

The chromatographic profiles of above crude extract

samples were detected with a Dionex HPLC system asdescribed before (Wang et al., 2003). Briefly, the mobile

phase was a linear gradient of double-deionised water

and acetonitrile at a flow rate of 1ml/min. Twenty-five

microlitres of each sample (50 ll in case of extracts from

rice cultures) were injected and monitored at 215 nm.

Dtxs were identified by comparison with standard

chromatograms and by mass spectrometry. Electrospray

ionisation (ESI) was carried out on a MicromassQuattro II triple quadrupole instrument by loop injec-

tion using a Hewlett-Packard 1050 LC autosampler into

a methanol:water (1:1, v/v) stream. Low-resolution

electronic ionisation (EI)/chemical ionisation (CI) mass

measurements were carried out on the same instrument

using a Fisons AS 200 autosampler injecting onto a

dedicated automated roboprobe system into a stream of

methanol:water (1:1, v/v).

Fig. 1. Toxin production dynamics of strains V245 (A) and V275 (B) in

Czapek Dox liquid medium.

3. Results

3.1. Toxin production in solid cultivation

No cyclic peptide metabolites were detected in CD

solid medium from both strains after different incuba-tion periods (data not shown) by HPLC analysis. Con-

idia and dtx production of strains V245 and V275 grown

on rice were compared over time. The spore yield from

strain V275 was lower compared to that of strain V245

at 30 days post-inoculation (dpi). The production of

dtxs A and B could be determined in rice cultures from

both strains in an increasing trend with the elongated

incubation time, while no dtx E was detected from bothcultures up to 30 dpi (Table 1). Interestingly, strain V275

produced approx. 9mg per 100 g rice dtx A at 10 and

20 dpi but after 30 days, only approx. 2mg of this

compound could be detected. Dtx B production was

very low at 10 dpi but increased to 34mg at 30 dpi(Table 1).

3.2. Toxin production in liquid medium

In large-scale fermenter cultures, no dtxs could be

detected for either strain after incubation for five days.

However, in lab-scale flask cultures, three cyclic peptide

toxins, dtxs A, B, and E were detected in the culturefiltrates from day 3 on (Fig. 1). For strain V245 the

production of dtxs A and B reached its maximum at day

C. Wang et al. / Journal of Invertebrate Pathology 85 (2004) 168–174 171

six and then decreased slightly (Fig. 1A), while for V275,18 days of incubation gave the highest amount of both

dtxs A and B (Fig. 1B). The highest amounts of dtx E

were produced in liquid cultures after incubation for

Fig. 2. pH and biomass dynamics of strains V245 (A) and V275 (B) in

Czapek Dox liquid medium.

Fig. 3. HPLC chromatograph of crude extract from strain V275 after nine day

and their molecular weight (MW in g/mol) determined by mass spectrometr

nine days for both V245 and V275 and the productiondecreased sharply afterwards (Fig. 1). With longer in-

cubation time, the pH value of the culture medium de-

creased (Fig. 2). At the same time, the fungal biomass

kept increasing up to day 9 and then decreased for V245

(Fig. 2a), while no significant differences could be de-

tected in biomass production of V275 after nine days of

incubation (P > 0:05) (Fig. 2B).Mass spectrometry analysis confirmed that the peaks

at 14.46� 0.25min, 18.85� 0.17min, and 21.09� 0.32

min in the HPLC analysis were dtxs E, A, and B, respec-

tively. The compound eluted at 13.93� 0.28min had a

molecular weight of 611 g/mol and was confirmed to be E

diol (Fig. 3). Secretion of E diol could be detected in liquid

medium from day 3 on and increased gradually for both

strains V245 and V275 (Fig. 4).

Correlations between the production of the majordtxs A, B, and E, biomass and culture pH were esti-

mated for both M. anisopliae strains (Table 2). The re-

sults showed that secretions of dtxs A and B were

significantly associated (a ¼ 0:01) and both were sig-

nificantly negatively correlated with the pH value of the

culture medium (a ¼ 0:05). The production of dtx A was

also found to be significantly associated with biomass

production (a ¼ 0:05), while no significant associationswere observed between the production of dtxs B, E, and

biomass (Table 2).

3.3. Effect of different media components on toxin

production

Media studies revealed that toxin secretion in liquid

medium was highly influenced by different carbon/

s of incubation showing the signals of the main cyclic peptide products

y analysis.

Table 2

Pearson relationships between different parameters of strain V245

(below diagonal) and V275 (above diagonal)

Dtx A Dtx B Dtx E pH Biomass

Dtx A 0.992�� 0.226 )0.844� 0.661�

Dtx B 0.926�� 0.200 )0.808� 0.637

Dtx E 0.434 0.508 )0.318 0.378

pH )0.741� )0.748� )0.221 )0.510Biomass 0.764� 0.540 0.497 )0.363* Significance at 0.05 level.** Significance at 0.01 level.

Table 3

Biomass, pH, and toxin profiles of the strain V245 detected in different nutr

Media Ingredients Conc. (g/L) Biomass (g/100ml)

CN1 Glucose 30 0.73� 0.10

Peptone 0

CN2 Glucose 0 0.58� 0.11

Peptone 30

CN3 Glucose 25 0.64� 0.05

Peptone 5

CN4 Glucose 20 0.52� 0.05

Peptone 10

CN5 Glucose 15 0.46� 0.01

Peptone 15

CN6 Glucose 10 0.51� 0.03

Peptone 20

CN7 Glucose 5 0.39� 0.01

Peptone 25

M1 Mealworm 1 0.44� 0.00

homogenate

M2 Mealworm 3 0.71� 0.02

homogenate

CN3+M1 Mealworm — 0.78� 0.00

homogenate

C1 Cockroach 1 0.34� 0.01

homogenate

C2 Cockroach 3 0.79� 0.05

homogenate

CN3+C1 Cockroach — 0.75� 0.03

homogenate

Fig. 4. E diol production dynamics of strains V245 and V275 in

Czapek Dox liquid media.

172 C. Wang et al. / Journal of Invertebrate Pathology 85 (2004) 168–174

nitrogen ratios as well as by the addition of insect ho-mogenates (Table 3). The amount of dtxs A, B, and E

produced by strain V245 increased with increasing

peptone content in the medium. Comparatively, the

highest amount of toxins was produced in medium with

peptone as single nutrient source (CN2), the second

highest amount in medium CN7 (peptone:glucose¼ 5:1)

and then in medium CN6 (peptone:glucose¼ 2:1) (Table

3). Medium with the same C:N ratio like Czapek Dox(CN3) resulted in a lower amount of toxins, similar to

medium CN5 (C:N¼ 50:50) (Table 3).

Interestingly, no toxins were detected by HPLC when

using insect homogenates in different percentages of

both Tenebrio molitor (M1, 1%; M2, 3%) and Blaberus

discoidalis (C1, 1%; C2, 3%) as single nutrient source.

Media CN3+M1 and CN3+C1 resulted in very low

amounts or not detectable amount of dtxs. The pHvalue and biomass production in cultures with both in-

sect homogenates were usually higher than in the media

with artificial nutrient sources (Table 3). The studies on

strain V275 displayed a similar trend of toxin produc-

tion in different media (data not shown).

4. Discussion

Investigations on dtx production by M. anisopliae in

this study showed that three major dtxs A, B, and E

could be detected in liquid CD medium in a lab-scale

ient media

pH Dtx A (mg/l) Dtx B (mg/l) Dtx E (mg/l)

4.93� 0.01 2.93� 0.54 1.59� 0.40 2.45� 0.42

6.63� 0.08 18.53� 0.70 4.95� 0.79 13.13� 0.99

3.75� 0.02 6.91� 0.44 2.78� 0.17 3.53� 0.72

3.71� 0.11 14.48� 0.95 4.55� 0.59 3.82� 0.53

4.07� 0.05 6.96� 0.82 2.60� 0.15 0.46� 0.18

3.98� 0.04 15.19� 0.98 4.43� 0.25 2.39� 0.28

5.61� 0.01 15.20� 0.84 5.33� 0.48 10.18� 1.06

7.83� 0.04 0 0 0

7.60� 0.01 0 0 0

4.97� 0.01 0.80� 0.17 1.81� 0.67 0

8.18� 0.03 0 0 0

7.79� 0.15 0 0 0

5.77� 0.21 0.77� 0.06 0 0

C. Wang et al. / Journal of Invertebrate Pathology 85 (2004) 168–174 173

flask but not in solid CD agar for both strains V245 andV275. Strain V275 produced more toxic metabolites in

liquid cultures. Interestingly, dtx E was not detected in

rice grains from both strains, the same was reported by

Liu and Tzeng (1999), however, the potential mecha-

nism remains to be elucidated in future studies. No

toxins could be detected in large-scale fermentation,

where the aeration was higher than in the conical flasks

(Patrick et al., 1993). It has been shown that high aer-ation reduces the metabolite production of swainsonine

by M. anisopliae (Patrick et al., 1993). Our data suggest

that the aeration regime also has a significant impact on

dtx production. From a safety point of view, the ob-

servations indicate that the risk assessment of toxic

metabolites produced by fungal biological agents should

be evaluated in association with the methods and media

as well as the strain used in mass production.The results of toxin secretion dynamics of both

strains showed that the concentrations of dtxs increased

up to the 7–9th day of incubation and then this trend

was followed by either a noticeable decrease (dtx E) or

more or less pronounced (dtxs A and B). In contrast, dtx

E diol was detected to increase outstandingly from the

9th day on. The less toxic dtx E diol was previously

described as dtx E (Cherton et al., 1991; Lange et al.,1992) and later confirmed as a new toxin (Jegorov et al.,

1998; Wahlman and Davidson, 1993), a product resulted

from enzymatic hydrolysis of the epoxide function of

dtx E inside the mycelium (Loutelier et al., 1996). The

responsible degradation enzyme(s) has (have) not been

identified, however, interestingly, the injection of dtx E

into the locust haemolymph also resulted in the detec-

tion of E diol, suggesting that the transformation pro-cess had occurred within the insect haemocoel (Cherton

et al., 1991). Nevertheless, it is still unknown whether

the dtx E is detoxified by insect or hydrolysed by M.

anisopliae itself. Additionally, the conjugated glutathi-

one dtx E, cysteinyl dtx E, sulphated, and phosphory-

lated dtx E have been detected in locusts and G.

mellonella (Hubert et al., 1999; Lange et al., 1992).

In this study, we found for the first time that theproduction of dtxs A and B is significantly correlated

(a ¼ 0:01). The overall relationships between dtx A, B,

and dtx E were less positive, but up to the 9th day, these

three toxins had a similar increasing trend and then the

association was not clear due to the degradation of dtx

E into E diol. The observations suggest that the encoded

genes for dtxs A, B, and E are located as a gene cluster

in the genome and up-regulated at the same level. Sup-portively, our recent study showed that a mutant from

strain V275 had completely lost the ability to produce

dtxs after missing a conditionally dispensable chromo-

some (Wang et al., 2003). In addition, toxin gene clus-

tering has been extensively reported in plant pathogenic

fungi, e.g., fumonisin encoding genes in Fusarium verti-

cillioides (Seo et al., 2001); aflatoxin encoding genes in

Aspergillus parasiticus (Cary et al., 2002); AK-toxinencoding genes in Alternaria alternata (Hatta et al.,

2002).

In vitro toxin secretion of M. anisopliae could be

significantly influenced by culture conditions. In this

study, the result of different carbon/nitrogen combina-

tions revealed that higher concentrations of peptone

(>60%) in liquid media favours toxin production. Liu

et al. (2000) reported that maltose and peptone were thebest carbon and nitrogen sources with the addition of

amino acid b-alanine for the production of dtxs by M.

anisopliae. The optimal compositions for the production

of dtxs A and B were different but in contrast to the

observation in this study, usually higher with increased

concentrations of maltose (ca. 70%). It is possibly due to

genetic and physiological differences between different

strains of M. anisopliae. A previous study showed thatthe addition of a low concentration (<10mg/L) of cy-

clopeptolide 90–215, a natural pipecolic acid-containing

cyclopeptolide composed of nine a-amino acid residues,

in the medium could increase the production of dtxs by

1.3- to 12.5-fold associated with different strains of M.

anisopliae (Espada and Dreyfuss, 1997), suggesting that

cyclopeptolide either serves as nitrogen source or an

intermediate product during the synthesis of dtxs.Destruxins have been often implicated as one of the

causes of insect death infected with M. anisopliae (Butt

et al., 1994; Vestergaard et al., 1995; Vey et al., 2001),

and they could be detected in variable amounts in in-

fected larvae of Galleria associated with mycoses by

different M. anisopliae strains (Amiri-Besheli et al.,

2000). However, in this study, it is surprising that no

toxins were detected when using insect homogenates assingle nutrients or included in CN3 liquid medium. The

potential regulation machinery remains to be elucidated

in further studies.

Acknowledgments

This work was supported by the Quality of Life and

Management of Living Resources Programme of the

European Commission, Key Action 1 on Food, Nutri-

tion and Health, QLK1-2001-01391 (RAFBCA). The

authors thank Dr. Alain Vey (INRA, France) for pro-

viding samples of pure dtxs A and E as well as Dr. John

Davies for his help of the analysis with the mass spec-trometry.

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