Investigations on the destruxin production of the entomopathogenic fungus Metarhizium anisopliae

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 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.     

Production of cyclodepsipeptides destruxin A and B from Metarhizium anisopliae

Maltose and peptone were the best carbon and nitrogen sources for the production of destruxins from Metarhizium anisopliae. With the addition of 0.1% (w/v) beta-alanine to the basal medium, the yields of cyclodepsipeptides DA and DB were 7.2 and 279 mg/L, respectively, which was 2-fold higher than that of control experiment. Response surface methodology (RSM) was applied to optimize the compositions of maltose, peptone, beta-alanine, and glucose used in a shaker-flask cultivation of M. anisopliae for the production of DA and DB. Estimated optimal compositions for the DA production were maltose 2.58%, peptone 0.72%, beta-alanine 0.02%, and glucose 0.55%. The predicted DA yield was 18.5 mg/L. On the other hand, the optimal compositions for DB production were maltose 2.51%, peptone 0.75%, beta-alanine 0.02%, and glucose 0.43%. A maximum DB yield of 232 mg/L was predicted. These were confirmed by cultivation experiments conducted at the optimized conditions for maximum destruxins production in a shaker-flask. Furthermore, a modest high level of DA (49 mg/L) and DB (268 mg/L) yields were obtained by employing the response surface methodology optimized DB production medium in a no-baffle, stirred-tank fermentor.     

Chitooligosaccharides enzymatic production by Metarhizium anisopliae

The products of chitosan hydrolysis are chitooligosaccharides and are used mainly for medical applications due to their specific biological activities. The objective of this study was to detect and identify the products of enzymatic hydrolysis of chitosan (dimers to hexamers) using a crude extract of chitosanolytic enzymes produced by the fungus Metarhizium anisopliae. These fungus was able to produce, during 48 h cultivation in a medium containing chitosan, chitooligosaccharides ranging from dimers, trimers, tetramers and pentamers at concentrations 0.2, 0.19, 0.06, 0.04 mg/mL, respectively, and the enzymatic activity was 2.5 U/L. Using the crude enzyme extract for chitosan hydrolysis, we detected the presence of dimers to hexamers at hydrolysis times of 10, 20, 30, 40, 50 and 60 min of enzymatic reaction, but the yields were higher at 10 min (54%). The hexamers was obtained only with 30 min of reaction with concentration of 0.004 mg/mL.     

Swainsonine production in fed-batch fermentations of Metarhizium anisopliae

Summary Extended and enhanced production of swainsonine was achieved from fed-batch fermentations of the fungus Metarhizium anisopliae. A complex medium based on oatmeal extract was intermittently fed with D-glucose and/or lysine. Swainsonine titres were improved eleven-fold and the duration of production extended was from 240 to 550 hours compared with a batch culture under the same conditions.     

Circular dichroism analysis of destruxins from Metarhizium anisopliae

Destruxins are secondary metabolites secreted by Metarhizium anisopliae [Y. Kodaira, Toxic substances to insects, produced by Aspergillus ochraceus and Oopsra destructor, Agric. Biol. Chem., 25 (1961) 261-262. D.W. Roberts, Toxins from the entomogenous fungus Metarhizium anisoplaie: Isolation from submerged cultures, J. Invertebr. Pathol., 14 (1969) 82-88. D.W. Roberts, Toxins from the entomogenic fungi in microbial control of pest and plant disease, Academic press, New York, 1981, pp441-464.]. In recent research, other than being used as insecticides, destruxins exhibited great potential in therapeutical applications such as antitumor, antivirus, and animal cell immunization effectiveness, etc. In this study, the conformations purified destruxins were determined by circular dichroism (CD). The results indicated that these cyclic peptides have the type I beta-turn conformation. In addition, different types of destruxins exhibited different CD spectra in acetonitrile. Therefore, these characters can be used as fingerprints to identify each type of destruxin. To further investigate the interactions among destruxins, various combinations of destruxins in 10 mM phosphate-buffered saline (PBS) were also studied by CD. The results strongly suggested that destruxins might work independently in vivo. To our knowledge, this is the first report presenting the CD analysis of purified destruxins.     

绿僵菌破坏素合成缺失株中分离鉴定pseurotin A

真菌次级代谢产物是医药活性成分的重要来源之一。就真菌基因组编码的次级代谢基因簇数量而言,由于普遍存在的基因沉默现象,常规培养中能够分离鉴定的化合物种类一般很有限。广谱杀虫的罗伯茨绿僵菌在液体培养基中的主要代谢产物为非核糖体环肽类的破坏素,本研究在对破坏素合成缺失突变株ΔdtxS1进行液体培养时获得了新的产物峰,对其中一个产物峰进行分离、纯化及结构鉴定,确定该化合物为螺环类的pseurotin A。结合在烟曲霉菌中解析的pseurotin A合成途径,推测获得了罗伯茨绿僵菌中的潜在合成基因簇,并推测了pseurotin A在绿僵菌中的合成途径。本研究首次在绿僵菌中鉴定获得pseurotin A,并揭示了在真菌中通过对主要次级代谢产物缺失的方法可以鉴定获得新型的化合物。     

Characterisation of optimum cultural environmental conditions for the production of high numbers of Metarhizium anisopliae blastospores with enhanced ecological fitness

The aim of this study was the production of high numbers of M. anisopliae blastospores with enhanced germination efficiency under conditions of water-stress (ecological fitness). Different nitrogen sources and concentrations were screened for their ability to induce blastospore production while keeping pH and water activity (a_w) at fixed levels (6.8, and 0.98 a_w, respectively). After optimum nitrogen status was determined (cornsteep solid (CS) + yeast extract (YE); or cottonseed flour (CF) + YE), the effect of interaction of nitrogen source, pH (3.5, 5, 6.8, 8, 9 and 10) and solutes for a_w adjustment (KCl, NaCl, PEG 200) on blastospore production, endogenous polyol content (glycerol, erythritol, arabitol and mannitol) and total protein, were determined. For both ionic (NaCl, KCl) and non-ionic solutes (PEG 200), optimum blastospore production (between 4×10⁷ and 2×10⁸ blastospores ml^-1) and growth occurred in the pH range 6.8-8, with the CF + YE nitrogen profile giving higher yields than CS + YE. Optimum conditions for high erythritol and total protein endogenous concentrations (40.37-73.44 and 14.33-18.90 mg g^-1 fresh weight, respectively) occurred between pH 6.8 and 8 by ionic a_w modification (KCl, NaCl) and with the CF + YE nitrogen profile. Germination of blastospores produced under these cultural conditions was between 62 and 89% under conditions of water-stress (0.96 a_w). On the other hand, blastospores with lower amounts of erythritol and total protein content had decreased germination (8-67%). These results could have significant implications for developing a liquid fermentation medium for the production of high numbers of fungal propagules with enhanced efficacy under non-optimum environmental conditions.     

Efficacy of Metarhizium anisopliae microsclerotial granules

The entomopathogenic fungus Metarhizium anisopliae has very recently been shown to produce microsclerotia (MS) – compact, heavily melanised, hyphal aggregates – in liquid media. Soil incorporation bioassays of dried MS preparations of three isolates of M. anisopliae were conducted using third instar Tetanops myopaeformis (sugarbeet root maggot) in clay and/or clay loam field soils as a model system to demonstrate efficacy. At rates as low as 23 mg MS granules/100 g dry soil, the biocontrol efficacy of MS granules of M. anisopliae Strain F52 produced in liquid media with a high carbon concentration (36 g/L) and high C:N ratios (30:1, 50:1) were superior to MS preparations produced in low carbon (8 g carbon/L) media and a high carbon medium with a 10:1 C:N ratio. Bioassays using MS formulations of M. anisopliae strains MA1200 and TM109 produced in high carbon and high C:N ratio media were superior in efficacy to the other MS production media tested. MS preparations of M. anisopliae F52 showed superior efficacy against the sugarbeet root maggot in comparison with more conventional, conidia-covered nutritive (corn grit) granules in a clay and clay soil. The MS granules were also highly efficacious against the sugarbeet root maggot at soil moisture levels as low as 0.983 A _w (?2.33 MPa). Granular preparations incorporating Metarhizium MS can serve as a viable formulation for the use of this fungus against soil insects.     

Optimization of agitation for production of swainsonine from Metarhizium anisopliae in stirred tank and airlift reactors

The optimal agitation rate for production of swainsonine from Metarhizium anisopliae grown in batch stirred tank reactors (2 to 20 l) was 400 rpm with a mixed hyphal and pelleted morphology where the specific swainsonine production rate was 9×10^–2 mg g^–1 cell dry wt h from 87 to 142 h. Culture of the fungus in a 6-l airlift reactor produced loose pellets and the production of swainsonine started at least 24 h earlier than in the stirred tank reactor. The final yield (5.9 mg swainsonine g^–1 cell dry wt) after 168 h in the airlift reactor was 18% less than those obtained in the stirred tank reactor with an agitation rate of 400 rpm.     

Compatability of Metarhizium anisopliae var. anisopliae with chemical pesticides

The effects of various insecticides on the mycelial growth, sporulation and conidial germination of Metarhizium anisopliae var. anisopliae isolate E₉ were studied in the laboratory. Chlorpyrifos was the most toxic organophosphate to mycelial growth and sporulation at all concentrations. Temephos, malathion and leptophos were highly toxic to sporulation while malathion was the most inhibitory to germination. The carbamates, carbofuran, methomyl and oxamyl were moderately toxic to mycelial growth and sporulation while oxamyl had an adverse effect on germination. The pyrethroids (pyrethrin, permethrin and resmethrin) and the insect growth regulators (diflubenzuron and methoprene) were not inhibitory to the various developmental stages of isolate E₉. The chlorinated hydrocarbons (chlordane, lindane and toxaphene) were more deleterious than all other insecticide groups tested. Among the fungicides, benomyl and maneb produced the greatest inhibition.     

Variability in esterases of Metarhizium anisopliae

The variability in esterases of the entomogenous fungus Metarhizium anisopliae was determined electrophoretically on 8.5% polyacrylamide gel. Ten isolates from diverse taxonomic groups of insects were analyzed. The electrophoretic analysis showed differences and similarities between these isolates and it was possible to distinguish six different patterns. The results obtained show a great polymorphism for the esterase system of M. anisopliae.     

Phoxim and manure enhanced the population rebound and preservation of Metarhizium anisopliae applied in soil

We investigated the population dynamics of Metarhizium anisopliae during peanut growth and the effect of phoxim and manure on population dynamics. The applied population of M. anisopliae initially decreased rapidly, then slowly, and finally stabilised or revived. The 50% decline in the original three rates of treatment appeared at 41.8–57.6 days; however, the populations decreased towards similar levels at harvest time. Phoxim clearly inhibited the fungal growth and conidiation on the medium. Germination was reduced by half or was completely absent when conidia were inoculated on media containing 25 or 100 ppm phoxim. The colony number decreased by 20–65%, and the colonies grew more slowly, conidiated less and faded in colour on media containing 25–200 ppm phoxim. However, normal growth and enhanced conidiation were restored when they were subsequently subcultured on normal media. Adding phoxim or manure to the fungal treatment in potting soil caused the population to decrease faster within the first 16 days but rebound and level off after 30 days. In the field, the M. anisopliae populations at peanut harvest dropped to approximately 10% of their initial levels, regardless of whether the fungus was applied alone or with phoxim or manure. We speculated that low concentrations of phoxim might stimulate M. anisopliae sporulation and proliferation, and that manure might increase space and nutrient competition between soil microbes and increase M. anisopliae proliferation.     

Genetic diversity of Metarhizium anisopliae var. anisopliae in southwestern British Columbia

The abundance and genetic diversity of the entomopathogenic fungus, Metarhizium anisopliae var. anisopliae, in southwestern British Columbia (BC) and southern Alberta was examined. The fungus was found to be widespread in soil throughout southwestern BC, and was recovered from 56% of 85 sample sites. In contrast to southwestern BC, no M. anisopliae isolates were recovered in southern Alberta. An automated fluorescent amplified fragment length polymorphism (AFLP) method was used to examine genetic diversity. In excess of 200 isolates were characterized. The method identified 211 polymorphic amplicons, ranging in size from ≈92 to 400 base pairs, and it was found to be reproducible with a resolution limit of 86.2% similarity. The AFLP method distinguished Metarhizium from other entomopathogenic fungal genera, and demonstrated considerable genetic diversity (25 genotypes) among the reference strains of M. anisopliae isolates examined (i.e. recovered from various substrates and geographical locations). Although 13 genotypes of M. anisopliae var. anisopliae were recovered from southwestern BC soils, the vast majority of isolates (91%) belonged to one of two closely-related genotypes. Furthermore, these two genotypes predominated in urban, agricultural and forest soils. The reasons for the limited diversity of M. anisopliae var. anisopliae in southwestern BC are uncertain. However, findings of this study are consistent with island biogeography theory, and have significant implications for the development of this fungus for microbial control of pest insects.     

Regulation of production of a trypsin-like protease by the insect pathogenic fungus Metarhizium anisopliae

Abstract The entomopathogenic fungus metarhizium anisopliae produces several cuticle-degrading proteases which may play a role in pathogenesis. The regulation of one of these, a trypsin-like protease PR2, has been investigated using depressed mycelia. Three insoluble protein sources, insect cuticle, elastin and collagen, as well as two soluble proteins, BSA and gelatin, induced PR2. The polymeric carbon sources cellulose and xylan resulted in depressed basal levels but not induced production of PR2. An approximately 15-fold increase in PR2 activity per mg dry weight of mycelium was observed when the fungus was grown in the presence of bovine serum albumin (BSA), as compared with conditions of depression alone. This indicates that PR2 is induced by BSA, and probably by other proteins. Basal levels of PR2 were detected after 8 h when mycelium was starved for both carbon and nitrogen but only after 16 h when starved for either nitrogen or carbon. In the presence of a protein source, nitrogen strongly repressed PR2 whereas carbon had little effect. There was no effect of sulphur on PR2 production.     

Transformation of Metarhizium anisopliae mediated by Agrobacterium tumefaciens

A simple, highly efficient, and reliable Agrobacterium tumefaciens-mediated transformation method was developed for the insect pathogenic fungus Metarhizium anisopliae. Expression of the green fluorescent protein gene, egfp, and the benomyl resistance gene, benA3, were used as markers in transformed M. anisopliae. Transformation efficiencies were dependent on the strain of A. tumefaciens used. With strain AGL-1, 17.0 +/- 1.4 transformants per plate could be obtained using conidial concentrations of 10(6) conidia/mL and a 2 day co-cultivation in the presence of 200 micromol/L acetosyringone. On the other hand, transformations using strain LBA4404 were unsuccessful. Ten transformants were tested by Southern analysis and found to contain a single copy T-DNA. Twenty transformants were subcultured for five generations on nonselective media, and 95% of the transformants were mitotically stable. Agrobacterium tumefaciens-mediated transformation of M. anisopliae can serve as a useful tool to investigate genes involved in insect pathogenicity.     

Transformants of Metarhizium anisopliae sf. anisopliae overexpressing chitinase from Metarhizium anisopliae sf. acridum show early induction of native chitinase but are not altered in pathogenicity to Manduca sexta

Extracellular chitinase activity has been implicated in the pathogenesis of several fungal infections. Following induction with chitin, the insect pathogens Metarhizium anisopliae sf. acridum ARSEF strain 324 and Metarhizium anisopliae sf. anisopliae ARSEF strain 2575 secrete 44-kDa basic and acidic isoforms of endochitinase, respectively. The gene from strain 324 (Chit1) was cloned and inserted into the genome of strain 2575 under the control of Aspergillus regulatory elements such that transgenic 2575 (2575-Chit(+)) expressed CHIT1 in a noninducing medium (i.e., not containing chitin). Isoelectric focusing followed by a zymogram technique revealed that neither wild-type 2575 nor 2575-Chit(+) produced significant amounts of the native 2575 acidic chitinase in a noninducing medium. However, in a chitin-containing medium, 2575-Chit(+) produced the native chitinase earlier than strain 2575, soon after secretion of CHIT1. We hypothesize that this is due to the production of soluble inducers following chitin hydrolysis by CHIT1 and that M. anisopliae uses enzymes expressed at low levels to sense the nature of the polymeric nutrient present in the immediate environment. However, the chitinase overproducers did not show altered virulence to caterpillars (Manduca sexta) compared to the wild-type fungus, suggesting that wild-type levels of chitinase are not limiting for cuticle penetration.     

Metarhizium anisopliae conidia production in packed-bed bioreactor using rice as substrate in successive cultivations

This paper presents a study on scale-up and cost reduction of the production of spores of Metarhizium anisopliae IBCB 425, entomopathogenic fungus used in sugarcane crops. Rice was mixed with sugarcane bagasse (9:1 w/w) for substrate composition, assuring adequate physical structure for cultivation in packed-beds. Spores yield from only rice in bench-scale packed-bed bioreactor was 56 % of the one obtained from the mixture 9:1 w/w. In comparison to plastic packages used in bioindustries, equivalent spores yields per gram of substrate have been achieved in bench and pilot-scale bioreactors built by cylindrical jacketed modules, that provide better control of operational and environmental variables, attested by little variability among replicates. Although non-negligible temperature rise (5 °C above the ideal) occurred within the pilot-scale bioreactor, spores production was not harmed in comparison to bench-scale. By reusing rice up to three successive cultivations, a 2.5-fold increase of spores yields was achieved in comparison to single use. Temperatures and CO₂ profiles corroborates the fungus adapted differently to substrate at each usage. Such results are valuable for industrial producers of commercial formulations of the fungus spores, allowing process modernization by using packed-bed bioreactors and production costs and rice demand reduction by recycling the substrate.