Transformation of the entomopathogenic fungus Metarhizium anisopliae to benomyl resistance

Abstract Protoplasts of the entomopathogenic fungus Metarhizium anisopliae were transformed to benomyl resistance using cosmid pSV50 which harbours a β-tubulin gene cloned from a Neurospora crassa benomyl-resistant mutant. Transformant colonies, which appeared at a frequency of 4 per 50 μg DNA, grew and sporulated on 10 μg/ml benomyl, whereas the wild type was inhibited by 3 μg/ml. Southern blot hybridization of DNA from transformants showed that, in each case, tandem repeats of the cosmid had integrated at several chromosomal loci. The transformants were mitotically stable when subcultured on non-selective agar and retained the ability to infect and kill larvae of Manduca sexta . Two transformants were less virulent than the wild type and one of them showed slower in vitro spore germination. The benomyl-resistant phenotype persisted in reisolates from insect cadavers.     

Transformation of the entomopathogenic fungus, Metarhizium flavoviride strain CG423 to benomyl resistance

Metarhizium flavoviride strain CG423 is being developed as a mycoinsecticide against grasshoppers. This strain has been transformed to resistance to the fungicide benomyl by a polyethylene glycol (PEG)-mediated procedure using a mutant tubulin gene from Neurospora crassa . Transformation frequencies of up to 84 transformants per microgram of transforming DNA were achieved. Benomyl-resistant transformants were obtained that could tolerate greater than 30 μg ml⁻¹ benomyl. Southern blot analysis of genomic DNA reveals that the mechanism of genetic transformation of all transformants was by homologous gene replacement of the β-tubulin allele.     

The role of destruxins in the pathogenicity of 3 strains of Metarhizium anisopliae for the tobacco hornworm Manduca sexta

Three out of 4 isolates of the Deuteromycete Metarhizium anisopliae were pathogenic for larvae of the tobacco hornworm, Manduca sexta. The most virulent isolate (ME1) grew sparsely in the insect prior to death and caused paralysis of its host. The other 2 pathogenic isolates killed Manduca larvae more slowly, grew profusely in the haemolymph and did not induce symptoms of toxicosis. Toxicosis is apparently due to the production by the fungus of several cyclodepsipeptide toxins, destruxins (DTX). ME1 produced large quantities of DTX in vitro, while other isolates produced less. Destruxin A (DTX A) was recovered from the haemolymph of paralysed, diseased insects infected with ME1, but not with other isolates. It is suggested that DTX may have a pathogenic role, when the toxins are active in causing disease, or an aggressive role, when they facilitate the establishment of the pathogen.     

Transformation of Penicillium chysogenum with selection for increased resistance to benomyl

Abstract Incubation of protoplasts of Penicillium chrysogenum with the plasmid pBT-3 permitted selection of transformants on the basis of increased resistance to the anti-fungal agent benomyl. Transformants were obtained at a frequency of 1–2 per μg of DNA. Southern analysis revealed that transformation had occured by integration of vector sequences into the host genome.     

An improved method for Beauveria bassiana transformation using phosphinothricin acetlytransferase and green fluorescent protein fusion gene as a selectable and visible marker

An improved transformation method for the biocontrol agent, Beauveria bassiana, was developed. For convenience of transformation selection and detection, the coding regions of the genes for phosphinothricin acetyltransferase and green fluorescent protein were fused and an expression vector, pBFT, carrying this fusion was constructed. Under optimum conditions, over 60 transformants microg(-1) plasmid DNA were obtained. B. bassiana conidia frozen 1 month at -80 degrees C were fully competent for transformation. The method was significantly less laborious and more rapid than current methods for B. bassiana. The bar::egfp provides a selectable and visible marker which may expedite future genetic engineering of this fungus.     

A technique for accelerating and synchronising germination of conidia of the entomopathogenic fungus Metarhizium anisopliae

The entomopathogenic fungus Metarhizium anisopliae (strain ME1) failed to swell or form germ-tubes in distilled water. However, a period of soaking in distilled water (10–44 h) accelerated the process of germination when a suitable nutrient source was provided. The implications of this novel observation are discussed in terms of mechanisms of germination and the use of parasitic fungi for insect pest control.     

Morphological Alterations of Metarhizium anisopliae During Penetration of Boophilus microplus Ticks

Chronological histological alterations of Metarhizium anisopliae during interaction with the cattle tick Boophilus microplus were investigated by light and scanning electron microscopy. M. anisopliae invades B. microplus by a process which involves adhesion of conidia to the cuticle, conidia germination, formation of appressoria and penetration through the cuticle. Twenty-four hours post-infection conidia are adhered and germination starts on the surface of the tick. At this time, the conidia differentiate to form appressoria exerting mechanical pressure and trigger hydrolytic enzyme secretion leading to penetration. Massive penetration is observed 72 h post-inoculation, and after 96 h, the hyphae start to emerge from the cuticle surface to form conidia. The intense invasion of adjacent tissues by hyphae was observed by light microscopy, confirming the ability of M. anisopliae to produce significant morphological alterations in the cuticle, and its infective effectiveness in B. microplus.     

Metarhizium anisopliae, a potential agent for the control of grape phylloxera

This investigation deals with the control effects of the insect pathogenic fungus Metarhizium anisopliae var. anisopliae on Daktulosphaira vitifoliae. In pot experiments, the soil surrounding phylloxera-infected grapes was inoculated with barley colonised with M. anisopliae. After thirty-two days, ineight of ten Metarhizium-applied pots nofresh phylloxera infections could be observed.In two of ten plants, a few fresh nodositiessingly occupied with phylloxera or phylloxeraeggs could be found. In all untreated plants,fresh nodosities with either single (two of sixplants) or multiple (four of six plants)occupation with phylloxera could be observed.M. anisopliae could be re-isolated in aconcentration of <1 × 10³ CFU g^$minus;1 soil dry weight from those pots with phylloxera-infected plants that had been treated with the fungus. The potential role of M. anisopliae in grape phylloxera management is discussed.     

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.     

A Mutant of Metarhizium anisopliae var. acridum with Enhanced Submerged Conidiation

Summary An insertional mutant of Metarhizium anisopliae is described with enhanced submerged conidiation. In a 500 ml submerged culture, this mutant produces a mean of 4.05 × 10⁸ propagules ml⁻¹ from an inoculum of 1 × 10⁶ conidia, where the parental strain accumulates only 3.75 × 10⁴ propagules ml⁻¹.     

Differentially-expressed glycoproteins in Locusta migratoria hemolymph infected with Metarhizium anisopliae

Glycoproteins play important roles in insect physiology. Infection with pathogen always results in the differential expression of some glycoproteins, which may be involved in host-pathogen interactions. In this report, differentially-expressed glycoproteins from the hemolymph of locusts infected with Metarhizium anisopliae were analyzed by two-dimensional electrophoresis (2-DE) and PDQuest software. The results showed that 13 spots were differentially expressed, of which nine spots were upregulated and four were downregulated. Using MS/MS with de novo sequencing and NCBI database searches, three upregulated proteins were identified as locust transferrin, apolipoprotein precursor, and hexameric storage protein 3. These proteins have been reported to be involved in the insect innate immune response to microbial challenge. Due to the limited available genome information and protein sequences of locusts, the possible functions of the other 10 differentially-expressed spots remain unknown.     

Invasion of the pathogenic fungus Metarhizium anisopliae through the guts of germfree desert locusts,Schistocerca gregaria

Less than 1% of an ingested inoculum of the pathogenic fungus Metarhizium anisopliae was retained for long enough (ca. 24 h) in the gut of the desert locust, Schistocerca gregaria, for germination and penetration to have occurred. The residual inoculum did not initiate an infection in guts of fed conventional or axenic locusts. However, symptoms of mycosis (hyphal bodies in the haemolymph, fungal penetration of the hindgut intima and epithelium, tetanic paralysis) were consistently observed in axenic but not conventional locusts which were starved post-inoculation.It is concluded that the antifungal toxin produced by the gut bacteria defends the desert locust against gut invasion by Metarhizium anisopliae during periods of starvation when the physical defences, prominent in fed insects, are less apparent.     

Isolation of Beauveria bassiana and Metarhizium anisopliae var. anisopliae from Boophilus microplus tick (Canestrini, 1887), in Rio de Janeiro State,Brazil

The objective of this work were to isolate and identify strains of entomopathogenic fungi from ingurgitated female Boophilus microplus ticks, collected from the soil in the municipality of Paracambi, Rio de Janeiro State, Brazil. The ingurgitated females were inoculated in the selective medium oat dodine agar (oda), where 49 colonies of Beauveria bassiana (71%) and 20 of Metarhizium anisopliae var. anisopliae (29%) were isolated. These isolated strains characterize for the first time in Brazil the natural occurrence of these species of fungi in this tick, and will be used to conduct bioassays to evaluate the pathogenicity and virulence of these strains for ticks of the genus Boophilus microplus. This revised version was published online in June 2006 with corrections to the Cover Date.     

Chlorimuron ethyl as a new selectable marker for disrupting genes in the insect-pathogenic fungus Metarhizium robertsii

A lack of selectable markers was a hindrance in investigating gene function in Metarhizium robertsii. A reliable Agrobacterium-mediated transformation system based on the use of chlorimuron ethyl as the selectable marker was developed which could serve as a useful tool to inactivate genes involved in insect pathogenicity.     

Prospects for biological control of the western flower thrips,Frankliniella occidentalis,with the entomopathogenic fungus,Metarhizium anisopliae,on chrysanthemum

The potential of Metarhizium anisopliae (Metsch.) Sorok. for the control of Frankliniella occidentalis (Pergande) on chrysanthemum cuttings was evaluated in greenhouse experiments. The fungus significantly reduced both the adult and larval populations of F. occidentalis, although the level of control of larval populations was much lower than for adults. Combined application of M. anisopliae and Methomyl (Lannate^®), however, resulted in a significant reduction of both the larval and adult stages. The use of both control agents might be helpful in reducing the selection pressure for resistance to chemical insecticides, thereby delaying or preventing the build-up of resistant populations in greenhouses.This revised version was published online in October 2005 with corrections to the Cover Date.     

Development of a whole cell green fluorescent sensor for lysine quantification

As an essential amino acid, lysine is an important component of animal and human diets and its bioavailability can depend on a variety of factors. Therefore, an accurate pre-determination of bioavailable lysine in foods and feeds is important. In this study a whole cell fluorescent biosensor for the quantification of lysine in protein sources was constructed. A gene encoding for green fluorescent protein (GFPmut3) was introduced into an E. coli lysine auxotroph genome as a part of a mini-Tn5-Km transposon. The location of the transposon was determined and the growth kinetics of the newly constructed biosensor were examined. The transposon disrupted the ybhM gene, which encodes for the synthesis of a protein with an unknown function. No effect of the transposon’s location in the genome or the expression of gfp on bacterial growth rates was observed. Based on the fluorescence emitted by GFPmut3, a standard curve after 6-h growth of the strain was generated. A correlation coefficient of 0.95 was observed when the fluorescence method was compared to the conventional optical density (OD) growth-based lysine assay. Using the newly developed lysine fluorescent whole cell sensor we determined the total lysine in casein acid hydrolyzate (7.13 ± 0.34%). When lysine added to 12 μg/ml and 30 μg/ml of casein acid hydrolyzate was quantified, recoveries of 97 ± 1.65% and 103 ± 4.66% respectively were detected. The results suggest that the microbial assay using GFP fluorescence represents a promising alternative method for the potential estimation of lysine in protein sources.     

Isolation of two Locust protein targets of a protein tyrosine phosphatase from Metarhizium anisopliae strain CQMa102

In entomopathogenic fungi, secretory protein phosphatases might function in the utilization of phosphoproteins from the environment. But if secreted into the host, secretory protein phosphatases might play a role in pathogenesis by dephosphorylation of host phosphoproteins. Our group purified a novel phosphatase from entomopathogenic fungi, Metarhizium anisopliae. The substrate specificity and inhibitor sensitivity indicate that the phosphatase is a protein tyrosine phosphatase (PTPase). In order to analyze the targets of the PTPase in Locusta migratoria hemolymph, two-dimensional electrophoresis and mass spectrometry were used. The results indicated that the PTPase could specifically dephosphorylate two phosphoproteins from L. migratoria hemolymph. One phosphoprotein was identified as trans-Golgi p230. Previous studies have shown that trans-Golgi p230 participates in vesicular transport of functional proteins from the distal Golgi compartment. trans-Golgi p230 can be inactivated by dephosphorylation, which implies that M. anisopliae could interfere with the correct transportation of functional proteins by secreting extracellular PTPase into the hemolymph. There are some secretion proteins, such as transferrin, have been thought to participate in the insect innate immune against microbial infection, therefor M. anisopliae could interfere with immune defenses of L. migratoria by secreting extracellular PTPase into the hemolymph.     

The genome sequence of the biocontrol fungus Metarhizium anisopliae and comparative genomics of Metarhizium species

Background

Metarhizium anisopliae is an important fungal biocontrol agent of insect pests of agricultural crops. Genomics can aid the successful commercialization of biopesticides by identification of key genes differentiating closely related species, selection of virulent microbial isolates which are amenable to industrial scale production and formulation and through the reduction of phenotypic variability. The genome of Metarhizium isolate ARSEF23 was recently published as a model for M. anisopliae, however phylogenetic analysis has since re-classified this isolate as M. robertsii. We present a new annotated genome sequence of M. anisopliae (isolate Ma69) and whole genome comparison to M. robertsii (ARSEF23) and M. acridum (CQMa 102).

Results

Whole genome analysis of M. anisopliae indicates significant macrosynteny with M. robertsii but with some large genomic inversions. In comparison to M. acridum, the genome of M. anisopliae shares lower sequence homology. While alignments overall are co-linear, the genome of M. acridum is not contiguous enough to conclusively observe macrosynteny. Mating type gene analysis revealed both MAT1-1 and MAT1-2 genes present in M. anisopliae suggesting putative homothallism, despite having no known teleomorph, in contrast with the putatively heterothallic M. acridum isolate CQMa 102 (MAT1-2) and M. robertsii isolate ARSEF23 (altered MAT1-1). Repetitive DNA and RIP analysis revealed M. acridum to have twice the repetitive content of the other two species and M. anisopliae to be five times more RIP affected than M. robertsii. We also present an initial bioinformatic survey of candidate pathogenicity genes in M. anisopliae.

Conclusions

The annotated genome of M. anisopliae is an important resource for the identification of virulence genes specific to M. anisopliae and development of species- and strain- specific assays. New insight into the possibility of homothallism and RIP affectedness has important implications for the development of M. anisopliae as a biopesticide as it may indicate the potential for greater inherent diversity in this species than the other species. This could present opportunities to select isolates with unique combinations of pathogenicity factors, or it may point to instability in the species, a negative attribute in a biopesticide.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-660) contains supplementary material, which is available to authorized users.     

Hydrated conidia of Metarhizium anisopliae release a family of metalloproteases

Metarhizium anisopliae spores release isoforms of metalloprotease during hydration over a 4-day incubation period. The isoforms were identified and characterized by using one-dimensional native PAGE (1-DE nPAGE) and one-dimensional SDS non-dissociating (1-DE nSDS-PAGE) zymography. The ability of these isozymes to degrade gelatin varied as revealed by 2-D spot densitometry. 1-DE nPAGE zymography revealed five isoforms of gelatinase from Tween wash of conidia. Where as, one to three activities with different intensities appeared on gel from washing of conidia to incubation in water till day 4. The relative migrations of these activities on 1-DE nPAGE zymograms appeared as fast, medium and slow on gel. The 2-D spot densitometry of zymograms indicated isoforms have different proteolytic activity as quantified by pixel intensities. SDS-PAGE zymography indicated the release of two isozymes of Mr 103 and 12 kDa during Tween treatment of conidia. However, during the first washing step with water and incubation of spores at day 2 and 3, respectively, only 12 kDa protein was evident. Majority of these proteases were inhibited by EDTA, but stimulated by CaCl(2), and MgCl(2). The presence of isozymes in conidia and their release during hydration must have functional significance for fungi and in this case it should provide advantages to M. anisopliae in its saprobic or pathogenic modalities. To our knowledge this is the first report describing release of metalloprotease isozymes from conidia.