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Investigations on the destruxin production of the entomopathogenic fungus Metarhizium anisopliae
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INVERTEBRATE PATHOLOGY Journal of Invertebrate Pathology 85 (2004) 168–174 www.elsevier.com/locate/yjipa
Investigations on the destruxin production of 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 by Metarhizium 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 postinoculation 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. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Metarhizium anisopliae; Destruxin; Carbon/nitrogen ratio; Toxin production
1. Introduction The entomopathogenic fungus Metarhizium anisopliae 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), produced 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 Kodaira (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: [email protected] (C. Wang).
0022-2011/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2004.02.008
2000). Destruxins A and E were proven to be more insecticidal than others (Dumas et al., 1994). The mechanism of dtx biosynthesis in M. anisopliae has not been well documented. The multifunctional nonribosomal peptide synthetases (PES) have been supposed 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 different 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; Kershaw 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. anisopliae strains V245 and V275 were monitored both on solid and in liquid media to meet the requirements for developing these strains as potential commercial
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mycoinsecticides. The influence of the culture medium on 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 additionally provide useful information to assess the potential risks of fungal toxic metabolites during the mass production of M. anisopliae.
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tracted three times for 30 min in 100 ml ethyl acetate:dichloromethane (1:1, v/v) in a sonicator. After extraction, the mixture was filtered through WhatmanÕs No. 1 filter paper, the organic phases were pooled and washed with 300 ml deionised water and the crude extracts were harvested by evaporating the solvents in vacuo. Two independent assessments with three replicates each were conducted. 2.3. Submerged cultivation
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 °C when fully covered with spores, usually after 14 days. 2.2. Solid cultivation Petri dishes (£ 9 cm) were charged with 20 ml autoclaved Czapek Dox (CD) medium (K2 HPO4 , 1 g/L; MgSO4 H2 O, 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 (0.5 ml, 108 conidia/ml) was inoculated and spread evenly. After incubation at 25 °C in the dark, two plates were taken out every three days and the microfibre filter papers with fungal propagules were carefully peeled off. The agar medium was sliced and soaked in 100 ml dichloromethane:ethyl acetate (1:1, v/v) in a 250 ml conical flask over night. Crude extracts were harvested by evaporating the organic solvent and analysed by HPLC to determine the production of toxic metabolites. 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 50 ml deionised water in a 500 ml conical flask. The autoclaved rice was inoculated with 1 ml 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 suspended in 1000 ml 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-
2.3.1. Toxin production in large-scale fermentation Metabolite production in fermenter cultures was assessed for both strains. The fungi were pre-cultured in 1000 ml conical flasks with 250 ml sabouraud dextrose broth (SDB, Difco) for five days at 23 °C and 150 rpm and then 100 ml were transferred to a 10 L fermenter with 9000 ml SDB, growing for seven days at pH 6, 500 rpm, 23 °C and 150 L/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 extracted twice with 300 ml dichloromethane:ethyl acetate (1:1, v/v) for 6 h. Extracts were diluted in methanol:acetonitrile (1:1, v/v) and analysed by HPLC described below. 2.3.2. Influence of incubation time on toxin production Conical flasks (250 ml) were charged with 100 ml CD liquid medium containing 5% mycological peptone. Each flask was inoculated with 0.5 ml spore suspension (108 conidia/ml). The flasks were incubated in dark at 24 °C and 110 rpm on a rotary shaker (Sanyo Gallenkamp). Every three days, the cultures from the duplicated flasks were taken out and filtered through two layers of Kimwipes and then through WhatmanÕs 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 determined. Correlations between the production of different dtxs as well as pH and biomass were analysed by calculating PearsonÕs correlation coefficient with the program 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 30 min at )80 °C and homogenised under liquid nitrogen. The homogenates were dried in oven under 60 °C for 48 h. Additions of
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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
10 20 30
V275
Dtx A
Dtx B
Dtx E
Dtx A
Dtx B
Dtx E
5.4 1.0 17.6 0.44 28.6 3.8
5.8 0.8 7.6 1.0 14.2 2.4
n.d. n.d. n.d.
8.2 2.2 9.6 2.0 1.8 1.8
0.8 0.16 6.4 2.4 34.0 4.2
n.d. n.d. n.d.
Mean SE; n.d., not detected.
both insect homogenates to CD CN3 (C:N ¼ 50:50) were included to compare the induction effect on toxin production. Culture filtrates were harvested after incubating for eight days. The putative toxins were extracted as described above. Each treatment had two replicates.
compound could be detected. Dtx B production was very low at 10 dpi but increased to 34 mg at 30 dpi (Table 1).
2.4. Analytical analysis
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 culture filtrates from day 3 on (Fig. 1). For strain V245 the production of dtxs A and B reached its maximum at day
The chromatographic profiles of above crude extract samples were detected with a Dionex HPLC system as described before (Wang et al., 2003). Briefly, the mobile phase was a linear gradient of double-deionised water and acetonitrile at a flow rate of 1 ml/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 Micromass Quattro II triple quadrupole instrument by loop injection 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).
3.2. Toxin production in 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 incubation periods (data not shown) by HPLC analysis. Conidia 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 both cultures up to 30 dpi (Table 1). Interestingly, strain V275 produced approx. 9 mg per 100 g rice dtx A at 10 and 20 dpi but after 30 days, only approx. 2 mg of this
Fig. 1. Toxin production dynamics of strains V245 (A) and V275 (B) in Czapek Dox liquid medium.
C. Wang et al. / Journal of Invertebrate Pathology 85 (2004) 168–174
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
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nine days for both V245 and V275 and the production decreased sharply afterwards (Fig. 1). With longer incubation time, the pH value of the culture medium decreased (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 detected 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 0.25 min, 18.85 0.17 min, and 21.09 0.32 min in the HPLC analysis were dtxs E, A, and B, respectively. The compound eluted at 13.93 0.28 min 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 major dtxs A, B, and E, biomass and culture pH were estimated for both M. anisopliae strains (Table 2). The results showed that secretions of dtxs A and B were significantly associated (a ¼ 0:01) and both were significantly 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 associations were observed between the production of dtxs B, E, and biomass (Table 2). 3.3. Effect of different media components on toxin production
Fig. 2. pH and biomass dynamics of strains V245 (A) and V275 (B) in Czapek Dox liquid medium.
Media studies revealed that toxin secretion in liquid medium was highly influenced by different carbon/
Fig. 3. HPLC chromatograph of crude extract from strain V275 after nine days of incubation showing the signals of the main cyclic peptide products and their molecular weight (MW in g/mol) determined by mass spectrometry analysis.
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Fig. 4. E diol production dynamics of strains V245 and V275 in Czapek Dox liquid media. Table 2 Pearson relationships between different parameters of strain V245 (below diagonal) and V275 (above diagonal) Dtx A Dtx A Dtx B Dtx E pH Biomass
Dtx B
Dtx E
0.992 0.926 0.434 )0.741 0.764
0.508 )0.748 0.540
0.226 0.200 )0.221 0.497
pH
Biomass
)0.844 )0.808 )0.318
)0.363
0.661 0.637 0.378 )0.510
*
Significance at 0.05 level. ** Significance at 0.01 level.
nitrogen ratios as well as by the addition of insect homogenates (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:glucose ¼ 5:1) and then in medium CN6 (peptone:glucose ¼ 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:N ¼ 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 pH value and biomass production in cultures with both insect 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 production 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
Table 3 Biomass, pH, and toxin profiles of the strain V245 detected in different nutrient media Media
Ingredients
Conc. (g/L)
Biomass (g/100 ml)
pH
Dtx A (mg/l)
Dtx B (mg/l)
CN1
Glucose Peptone Glucose Peptone Glucose Peptone Glucose Peptone Glucose Peptone Glucose Peptone Glucose Peptone Mealworm homogenate Mealworm homogenate Mealworm homogenate Cockroach homogenate Cockroach homogenate Cockroach homogenate
30 0 0 30 25 5 20 10 15 15 10 20 5 25 1
0.73 0.10
4.93 0.01
2.93 0.54
1.59 0.40
2.45 0.42
0.58 0.11
6.63 0.08
18.53 0.70
4.95 0.79
13.13 0.99
0.64 0.05
3.75 0.02
6.91 0.44
2.78 0.17
3.53 0.72
0.52 0.05
3.71 0.11
14.48 0.95
4.55 0.59
3.82 0.53
0.46 0.01
4.07 0.05
6.96 0.82
2.60 0.15
0.46 0.18
0.51 0.03
3.98 0.04
15.19 0.98
4.43 0.25
2.39 0.28
0.39 0.01
5.61 0.01
15.20 0.84
5.33 0.48
10.18 1.06
0.44 0.00
7.83 0.04
0
0
0
3
0.71 0.02
7.60 0.01
0
0
0
—
0.78 0.00
4.97 0.01
0.80 0.17
1.81 0.67
0
1
0.34 0.01
8.18 0.03
0
0
0
3
0.79 0.05
7.79 0.15
0
0
0
—
0.75 0.03
5.77 0.21
0.77 0.06
0
0
CN2 CN3 CN4 CN5 CN6 CN7 M1 M2 CN3 + M1 C1 C2 CN3 + C1
Dtx E (mg/l)
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flask but not in solid CD agar for both strains V245 and V275. 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 mechanism 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 aeration 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 observations 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 detection of E diol, suggesting that the transformation process 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 glutathione dtx E, cysteinyl dtx E, sulphated, and phosphorylated 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 the production 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. Supportively, our recent study showed that a mutant from strain V275 had completely lost the ability to produce dtxs after missing a conditionally dispensable chromosome (Wang et al., 2003). In addition, toxin gene clustering has been extensively reported in plant pathogenic fungi, e.g., fumonisin encoding genes in Fusarium verticillioides (Seo et al., 2001); aflatoxin encoding genes in
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Aspergillus parasiticus (Cary et al., 2002); AK-toxin encoding 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 combinations revealed that higher concentrations of peptone (>60%) in liquid media favours toxin production. Liu et al. (2000) reported that maltose and peptone were the best 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 that the addition of a low concentration (<10 mg/L) of cyclopeptolide 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 infected 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 as single 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, Nutrition and Health, QLK1-2001-01391 (RAFBCA). The authors thank Dr. Alain Vey (INRA, France) for providing samples of pure dtxs A and E as well as Dr. John Davies for his help of the analysis with the mass spectrometry.
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Metarhizium anisopliae for three species of insect. J. Invertebr. Pathol. 74, 213–223. Kodaira, Y., 1961. Biochemical studies on the muscardine fungi in the silkworms, Bombyx mori. J. Fac. Text. Sci. Technol. 5, 1–68. Lange, C., Loutelier, C., Cherton, J.C., Cassier, P., Vey, A., Pais, M., 1992. Desorption of ions from locust tissues. 2. Metabolites of Edestruxin using negative-ion fast-atom-bombardment mass-spectrometry. Rapid Comm. Mass Spectrom. 6, 28–31. Liu, B.L., Tzeng, Y.M., 1999. Water content and water activity for the production of cyclodepsipeptides in solid-state fermentation by Metarhizium anisopliae. Biotechnol. Lett. 21, 657–661. Liu, B.L., Chen, J.W., Tzeng, Y.M., 2000. Production of cyclodepsipeptides destruxin A and B from Metarhizium anisopliae. Biotechnol. Prog. 16, 993–999. Loutelier, C., Cherton, J.-C., Lange, C., Traris, M., Vey, A., 1996. Studies on the dynamics of the production of destruxins by Metarhizium anisopliae: Direct high-performance liquid chromatographic and fast atom bombardment mass spectrometric analysis correlated with biological activity tests. J. Chromatogr. A 738, 181– 189. Marahiel, M.A., 1992. Multidomain enzymes involved in peptide synthesis. FEBS Lett. 307, 40–43. Patrick, M., Adlard, M.W., Keshavarz, T., 1993. Production of an indolizidine alkaloid, swainsonine, by the filamentous fungus Metarhizium anisopliae. Biotechnol. Lett. 15, 997–1000. Pedras, M.S.C., Irina, Z.L., Ward, D.E., 2002. The destruxins: synthesis, biosynthesis, biotransformation, and biological activity. Phytochemistry 59, 579–596. Seo, J.A., Proctor, R.H., Plattner, R.D., 2001. Characterization of four clustered and coregulated genes associated with fumonisin biosynthesis in Fusarium verticillioides. Fungal Genet. Biol. 34, 155–165. Strasser, H., Vey, A., Butt, T.M., 2000. Are there any risks in using entomopathogenic fungi for pest control, with particular reference to the bioactive metabolites of Metarhizium, Tolypocladium and Beauveria species? Biocontrol Sci. Technol. 10, 717–735. Vestergaard, S., Gillespie, A.T., Butt, T.M., Schreiter, G., Eilenberg, J., 1995. Pathogenicity of the hyphomycete fungi Verticillium lecanii and Metarhizium anisopliae to the western flower thrips, Frankliniella occidentalis. Biocontrol Sci. Technol. 5, 185–192. Vey, A., Hoagland, R., Butt, T.M., 2001. Toxic metabolites of fungal biocontrol agents. In: Butt, T.M., Jackson, C.W., Magan, N. (Eds.), Fungi as Biocontrol Agents. CAB International, Wallingford, UK, pp. 311–345. Wahlman, M., Davidson, B.S., 1993. New destruxins from the entomopathogenic fungus Metarhizium anisopliae. J. Nat. Prod. (Lloydia) 56, 643–647. Wang, C.-S., Skrobek, A., Butt, T.M., 2003. Concurrence of losing a chromosome and the ability to produce destruxins in a mutant of Metarhizium anisopliae. FEMS Microbiol. Lett. 226, 373–378. Zuber, P., 1991. Non-ribosomal peptide synthesis. Curr. Opin. Cell Biol. 3, 1046–1050.
INVERTEBRATE PATHOLOGY Journal of Invertebrate Pathology 85 (2004) 168–174 www.elsevier.com/locate/yjipa
Investigations on the destruxin production of 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 by Metarhizium 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 postinoculation 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. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Metarhizium anisopliae; Destruxin; Carbon/nitrogen ratio; Toxin production
1. Introduction The entomopathogenic fungus Metarhizium anisopliae 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), produced 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 Kodaira (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: [email protected] (C. Wang).
0022-2011/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2004.02.008
2000). Destruxins A and E were proven to be more insecticidal than others (Dumas et al., 1994). The mechanism of dtx biosynthesis in M. anisopliae has not been well documented. The multifunctional nonribosomal peptide synthetases (PES) have been supposed 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 different 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; Kershaw 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. anisopliae strains V245 and V275 were monitored both on solid and in liquid media to meet the requirements for developing these strains as potential commercial
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mycoinsecticides. The influence of the culture medium on 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 additionally provide useful information to assess the potential risks of fungal toxic metabolites during the mass production of M. anisopliae.
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tracted three times for 30 min in 100 ml ethyl acetate:dichloromethane (1:1, v/v) in a sonicator. After extraction, the mixture was filtered through WhatmanÕs No. 1 filter paper, the organic phases were pooled and washed with 300 ml deionised water and the crude extracts were harvested by evaporating the solvents in vacuo. Two independent assessments with three replicates each were conducted. 2.3. Submerged cultivation
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 °C when fully covered with spores, usually after 14 days. 2.2. Solid cultivation Petri dishes (£ 9 cm) were charged with 20 ml autoclaved Czapek Dox (CD) medium (K2 HPO4 , 1 g/L; MgSO4 H2 O, 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 (0.5 ml, 108 conidia/ml) was inoculated and spread evenly. After incubation at 25 °C in the dark, two plates were taken out every three days and the microfibre filter papers with fungal propagules were carefully peeled off. The agar medium was sliced and soaked in 100 ml dichloromethane:ethyl acetate (1:1, v/v) in a 250 ml conical flask over night. Crude extracts were harvested by evaporating the organic solvent and analysed by HPLC to determine the production of toxic metabolites. 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 50 ml deionised water in a 500 ml conical flask. The autoclaved rice was inoculated with 1 ml 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 suspended in 1000 ml 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-
2.3.1. Toxin production in large-scale fermentation Metabolite production in fermenter cultures was assessed for both strains. The fungi were pre-cultured in 1000 ml conical flasks with 250 ml sabouraud dextrose broth (SDB, Difco) for five days at 23 °C and 150 rpm and then 100 ml were transferred to a 10 L fermenter with 9000 ml SDB, growing for seven days at pH 6, 500 rpm, 23 °C and 150 L/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 extracted twice with 300 ml dichloromethane:ethyl acetate (1:1, v/v) for 6 h. Extracts were diluted in methanol:acetonitrile (1:1, v/v) and analysed by HPLC described below. 2.3.2. Influence of incubation time on toxin production Conical flasks (250 ml) were charged with 100 ml CD liquid medium containing 5% mycological peptone. Each flask was inoculated with 0.5 ml spore suspension (108 conidia/ml). The flasks were incubated in dark at 24 °C and 110 rpm on a rotary shaker (Sanyo Gallenkamp). Every three days, the cultures from the duplicated flasks were taken out and filtered through two layers of Kimwipes and then through WhatmanÕs 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 determined. Correlations between the production of different dtxs as well as pH and biomass were analysed by calculating PearsonÕs correlation coefficient with the program 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 30 min at )80 °C and homogenised under liquid nitrogen. The homogenates were dried in oven under 60 °C for 48 h. Additions of
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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
10 20 30
V275
Dtx A
Dtx B
Dtx E
Dtx A
Dtx B
Dtx E
5.4 1.0 17.6 0.44 28.6 3.8
5.8 0.8 7.6 1.0 14.2 2.4
n.d. n.d. n.d.
8.2 2.2 9.6 2.0 1.8 1.8
0.8 0.16 6.4 2.4 34.0 4.2
n.d. n.d. n.d.
Mean SE; n.d., not detected.
both insect homogenates to CD CN3 (C:N ¼ 50:50) were included to compare the induction effect on toxin production. Culture filtrates were harvested after incubating for eight days. The putative toxins were extracted as described above. Each treatment had two replicates.
compound could be detected. Dtx B production was very low at 10 dpi but increased to 34 mg at 30 dpi (Table 1).
2.4. Analytical analysis
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 culture filtrates from day 3 on (Fig. 1). For strain V245 the production of dtxs A and B reached its maximum at day
The chromatographic profiles of above crude extract samples were detected with a Dionex HPLC system as described before (Wang et al., 2003). Briefly, the mobile phase was a linear gradient of double-deionised water and acetonitrile at a flow rate of 1 ml/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 Micromass Quattro II triple quadrupole instrument by loop injection 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).
3.2. Toxin production in 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 incubation periods (data not shown) by HPLC analysis. Conidia 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 both cultures up to 30 dpi (Table 1). Interestingly, strain V275 produced approx. 9 mg per 100 g rice dtx A at 10 and 20 dpi but after 30 days, only approx. 2 mg of this
Fig. 1. Toxin production dynamics of strains V245 (A) and V275 (B) in Czapek Dox liquid medium.
C. Wang et al. / Journal of Invertebrate Pathology 85 (2004) 168–174
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
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nine days for both V245 and V275 and the production decreased sharply afterwards (Fig. 1). With longer incubation time, the pH value of the culture medium decreased (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 detected 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 0.25 min, 18.85 0.17 min, and 21.09 0.32 min in the HPLC analysis were dtxs E, A, and B, respectively. The compound eluted at 13.93 0.28 min 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 major dtxs A, B, and E, biomass and culture pH were estimated for both M. anisopliae strains (Table 2). The results showed that secretions of dtxs A and B were significantly associated (a ¼ 0:01) and both were significantly 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 associations were observed between the production of dtxs B, E, and biomass (Table 2). 3.3. Effect of different media components on toxin production
Fig. 2. pH and biomass dynamics of strains V245 (A) and V275 (B) in Czapek Dox liquid medium.
Media studies revealed that toxin secretion in liquid medium was highly influenced by different carbon/
Fig. 3. HPLC chromatograph of crude extract from strain V275 after nine days of incubation showing the signals of the main cyclic peptide products and their molecular weight (MW in g/mol) determined by mass spectrometry analysis.
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Fig. 4. E diol production dynamics of strains V245 and V275 in Czapek Dox liquid media. Table 2 Pearson relationships between different parameters of strain V245 (below diagonal) and V275 (above diagonal) Dtx A Dtx A Dtx B Dtx E pH Biomass
Dtx B
Dtx E
0.992 0.926 0.434 )0.741 0.764
0.508 )0.748 0.540
0.226 0.200 )0.221 0.497
pH
Biomass
)0.844 )0.808 )0.318
)0.363
0.661 0.637 0.378 )0.510
*
Significance at 0.05 level. ** Significance at 0.01 level.
nitrogen ratios as well as by the addition of insect homogenates (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:glucose ¼ 5:1) and then in medium CN6 (peptone:glucose ¼ 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:N ¼ 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 pH value and biomass production in cultures with both insect 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 production 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
Table 3 Biomass, pH, and toxin profiles of the strain V245 detected in different nutrient media Media
Ingredients
Conc. (g/L)
Biomass (g/100 ml)
pH
Dtx A (mg/l)
Dtx B (mg/l)
CN1
Glucose Peptone Glucose Peptone Glucose Peptone Glucose Peptone Glucose Peptone Glucose Peptone Glucose Peptone Mealworm homogenate Mealworm homogenate Mealworm homogenate Cockroach homogenate Cockroach homogenate Cockroach homogenate
30 0 0 30 25 5 20 10 15 15 10 20 5 25 1
0.73 0.10
4.93 0.01
2.93 0.54
1.59 0.40
2.45 0.42
0.58 0.11
6.63 0.08
18.53 0.70
4.95 0.79
13.13 0.99
0.64 0.05
3.75 0.02
6.91 0.44
2.78 0.17
3.53 0.72
0.52 0.05
3.71 0.11
14.48 0.95
4.55 0.59
3.82 0.53
0.46 0.01
4.07 0.05
6.96 0.82
2.60 0.15
0.46 0.18
0.51 0.03
3.98 0.04
15.19 0.98
4.43 0.25
2.39 0.28
0.39 0.01
5.61 0.01
15.20 0.84
5.33 0.48
10.18 1.06
0.44 0.00
7.83 0.04
0
0
0
3
0.71 0.02
7.60 0.01
0
0
0
—
0.78 0.00
4.97 0.01
0.80 0.17
1.81 0.67
0
1
0.34 0.01
8.18 0.03
0
0
0
3
0.79 0.05
7.79 0.15
0
0
0
—
0.75 0.03
5.77 0.21
0.77 0.06
0
0
CN2 CN3 CN4 CN5 CN6 CN7 M1 M2 CN3 + M1 C1 C2 CN3 + C1
Dtx E (mg/l)
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flask but not in solid CD agar for both strains V245 and V275. 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 mechanism 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 aeration 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 observations 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 detection of E diol, suggesting that the transformation process 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 glutathione dtx E, cysteinyl dtx E, sulphated, and phosphorylated 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 the production 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. Supportively, our recent study showed that a mutant from strain V275 had completely lost the ability to produce dtxs after missing a conditionally dispensable chromosome (Wang et al., 2003). In addition, toxin gene clustering has been extensively reported in plant pathogenic fungi, e.g., fumonisin encoding genes in Fusarium verticillioides (Seo et al., 2001); aflatoxin encoding genes in
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Aspergillus parasiticus (Cary et al., 2002); AK-toxin encoding 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 combinations revealed that higher concentrations of peptone (>60%) in liquid media favours toxin production. Liu et al. (2000) reported that maltose and peptone were the best 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 that the addition of a low concentration (<10 mg/L) of cyclopeptolide 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 infected 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 as single 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, Nutrition and Health, QLK1-2001-01391 (RAFBCA). The authors thank Dr. Alain Vey (INRA, France) for providing samples of pure dtxs A and E as well as Dr. John Davies for his help of the analysis with the mass spectrometry.
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