Differential expression of genes involved in entomopathogenicity of the fungi Metarhizium anisopliae var. anisopliae and M. anisopliae var. acridum (Clavicipitaceae)

Expression analysis of the genes involved in germination, conidiogenisis and pathogenesis of Metarhizium anisopliae during its saprophytic and pathogenic life stages can help plan strategies to increase its efficacy as a biological control agent. We quantified relative expression levels of the nitrogen response regulator gene (nrr1) and a G-protein regulator of genes involved in conidiogenesis (cag8), using an RT-qPCR assay. Comparisons were made between M. anisopliae var. anisopliae and M. anisopliae var. acridum during germination and conidiogenesis and at different stages of pathogenesis. The cag8 gene was repressed during germination and induced during conidial development and the pathogenic phase, and the nrr1 gene was induced during germination, conidiogenesis and the pathogenic phase. Both genes were more expressed in M. anisopliae var. anisopliae, demonstrating that different varieties of M. anisopliae differ in activation of genes linked to virulence for certain environments and hosts. This suggests that differences among these varieties in the ability to adapt could be attributed not only to specific genomic regions and genes, but also to differential gene expression in this fungus, modulating its ability to respond to environmental stimuli.     

Dry mycelium preparations of entomopathogenic fungi,Metarhizium anisopliae and Beauveria bassiana

Several different harvesting procedures were used to obtain dry mycelium preparations of the entomopathogenic fungi, Metarhizium anisopliae and Beauveria bassiana. The effects of these procedures on the survival of the fungal preparations and on their conidiation after short periods of storage at room temperature and at 4°C were examined. Harvesting procedures consisted of filtering the mycelium produced in airlift containers from the culture medium, washing with deionized water, spraying with a sugar solution, and incubating for 18 hr at 4°C before drying. Conidial production of treated mycelia stored 1.5 and 4.5 months at 4°C was not significantly different for and procedure. For dry mycelium of M. anisopliae stored 1.5 months at 4°C and then at room temperature for 3 months, maltose- and sucrose-treated preparations produced more conidia than preparations sprayed with dextrose solution, with water only, or not sprayed. B. bassiana preparations dried soon after mat formation were superior to those incubated at 4°C, and maltose-and dextrose-treated mycelia were superior to other treatments when stored at room temperature.     

Development of a simple and rapid Agrobacterium tumefaciens-mediated transformation system for the entomopathogenic fungus Metarhizium anisopliae var. acridum

AIMS: To examine the ability of Agrobacterium to attach to Metarhizium anisopliae var. acridum strain CG423 under co-cultivation and to develop an Agrobacterium-mediated method of gene delivery into strain CG423, a promising agent for biological control of grasshoppers. METHODS AND RESULTS: The co-cultivation of Agrobacterium tumefaciens and M. anisopliae var. acridum was analysed under scanning electron microscopy. We observed that Agrobacterium attached to and formed aggregates around Metarhizium conidia and germ tubes. We also observed the occurrence of fibril-like structures connecting neighbouring bacterial-fungal cells. The Agrobacterium-mediated transformation was applied using two binary vectors carrying a benomyl resistance gene as a selection marker. The efficiency of transformation was up to 53 transformants per 10(5) target conidia. High mitotic stability of the transformants (89-97%) was demonstrated after five successive transfers on non-selective media. Molecular analysis revealed the occurrence of high frequency of gene conversion. CONCLUSIONS: In our study, we report that A. tumefaciens strain AGL-1 attaches to and genetically transforms the entomopathogenic fungus Metarhizium anisopliae var. acridum. SIGNIFICANCE AND IMPACT OF THE STUDY: We report for the first time, the attachment of Agrobacterium to fungal cells opening new avenues for the study of this essential step of the T-DNA transfer process. Considering the efficiency of the transformation protocol herein described, this is a useful tool for gene disruption in M. anisopliae var. acridum.     

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.     

The immune response of the desert locust Schistocerca gregaria during mycosis of the entomopathogenic fungus,Metarhizium anisopliae var acridum

Topical application of Metarhizium anisopliae var acridum to the desert locust Schistocerca gregaria resulted in changes in the biochemistry and antimicrobial defenses of the haemolymph. M. anisopliae var acridum colonized the host haemolymph from day two post application. The haemocytes did not attach to, phagocytose or nodulate elements of the fungus. However, the presence of the fungus appeared to stimulate hemocyte aggregation over the first few days of mycosis though the number of aggregates declined subsequently. The total hemocyte count increased two days after application, indicating an overall stimulation of the immune system, but declined to a value below that for uninoculated controls by day four. The differential haemocyte count showed that the initial increase in total haemocyte count was primarily due to a larger number of coagulocytes. After day two consistent declines in cell number were observed for all haemocyte classes in mycosed insects. The activity of the enzyme, phenoloxidase, decreased during the course of infection. However, the converse was true for prophenoloxidase. Lysozyme levels were significantly smaller in infected than control locusts. There was a significant correlation between lysozyme and PO activities when data from mycosed and control insects were combined. The total protein content of the haemolymph decreased during the course of infection.     

Review on safety of the entomopathogenic fungus Metarhizium anisopliae

The entomopathogenic fungus Metarhizium anisopliae (Metschn.) Sorokin is widely used for biocontrol of pest insects, and many commercial products are on the market or under development. The aim of this review is to summarise all relevant safety data of this fungus, which are necessary for the commercialisation and registration process. The review contains the following sections: (1) identity, (2) biological properties (history, natural occurrence and geographical distribution, host range, mode of action, production of metabolites/toxins, effect of environmental factors), (3) methods to determine and quantify residues, (4) fate and behaviour in the environment (mobility and persistence in air, water and soil), (5) effects on non-target organisms (microorganisms, plants, soil organisms, aquatic organisms, predators, parasitoids, honey bees, earth worms, etc.), (6) effects on vertebrates (fish, amphibia, reptiles, and birds), and (7) effects on mammals and human health (allergy, pathogenicity/toxicity). On the basis of the presented knowledge, M. anisopliae is considered to be safe with minimal risks to vertebrates, humans and the environment.     

Diversity of entomopathogenic fungi near leaf-cutting ant nests in a neotropical forest, with particular reference to Metarhizium anisopliae var. anisopliae

We investigated the prevalence of entomopathogenic fungi associated with leaf-cutting ant colonies in a small area of tropical forest in Panama. There was a high abundance of Metarhizium anisopliae var. anisopliae near the colonies. Beauveria bassiana was also detected in the soil, Aspergillus flavus in dump material, and six Camponotus atriceps ants were found infected with Cordyceps sp. Based on a partial sequence of the IGS region, almost all of the M. anisopliae var. anisopliae isolates fell within one of the three main clades of M. anisopliae var. anisopliae, but with there still being considerable diversity within this clade. The vast majority of leaf-cutting ants collected were not infected by any entomopathogenic fungi. While leaf-cutting ants at this site must, therefore, regularly come into contact with a diversity of entomopathogenic fungi, they do not appear to be normally infected by them.     

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.     

Efficacy of entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae against Thaumetopoea pityocampa Shiff. (Lepidoptera: Thaumatopoeidae)

Abstract

In this study, Metarhizium anisopliae TR 106 and Beauveria bassiana TR 217 was tested against fourth instar larvae of Thaumetopoea pityocampa. The LT₅₀ and LT₉₀ of 1?×?10⁶ concentration of M. anisopliae against T. pityocampa were 3.60 and 4.11 for direct application, while these were 2.87 and 3.60?days, respectively in leaves application. The LT₅₀ and LT₉₀ of the 1?×?10⁸ concentration of the same isolate were 2.50 and 2.95?days for direct application, and 2.98 and 3.74?days for leaves application. The LT₅₀ of insect and leaves application for 1?×?10⁶ of B. bassiana were 3.75 and 3.49?days, respectively. The LT₉₀ of same concentration for insect application was 4.48?days, while LT₉₀ for leaves application was 4.63?days. Similarly, LT₅₀ of insect and leaves application for 1?×?10⁸ of B. bassiana were 3.03 and 3.31?days, while LT₉₀ were 3.68 and 4.29?days, respectively. Approximate 100% mycosis was observed in all treatments.     

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.     

Establishment and persistence of the entomopathogenic fungus Metarhizium anisopliae in maize fields

The entomopathogenic fungus Metarhizium anisopliae (Metsch.) Sorokin (Hypocreales: Clavicipitaceae) was applied in maize fields to control the Western Corn Rootworm Diabrotica virgifera virgifera Le Conte (Coleoptera: Chrysomelidae). Establishment and persistence of two strains of M. anisopliae were investigated after application as ‘fungal colonized barley kernels’ (FCBK) into the soil and as a spore suspension on maize leaves and on the soil surface in 2006 and 2007 at two locations in Hungary. The applied fungal strains were able to establish at both locations and a long‐term persistence of at least 15 months could be recorded in the soil. A positive correlation between density of colony forming units (CFU) in the soil and the soil inhabiting stages of the host insect D. v. virgifera could be found. M. anisopliae spores applied on maize leaves were able to survive for no longer than 3 days after application, whereas on the soil surface a noticeably increase of fungus densities were found after treatments. Molecular markers were used to identify the applied M. anisopliae strains before and after application of FCBK into the soil of the maize field.     

Isolation and characterization of microsatellite markers from the entomopathogenic fungus Metarhizium anisopliae

Fourteen polymorphic microsatellite markers were isolated from the entomopathogenic fungus, Metarhizium anisopliae, based on enriched genomic libraries. In order to assess allelic variability, the microsatellite loci were analysed in a collection of 34 isolates sampled from across Switzerland. The number of detected alleles in 14 loci ranged from two to eight and expected heterozygosity from 0.265 to 0.808. Because of the high expected heterozygosity, the 14 microsatellite loci are very useful for ecological studies and analysis of population diversity, and to identifying, monitoring, and tracking M. anisopliae strains applied as biological control agents.     

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.     

Cellular and humoral immune defenses of Oxya japonica (Orthoptera: Acrididae) to entomopathogenic fungi Metarhizium anisopliae

One Indonesian isolate of the fungus Metarhizium anisopliae, named Majalengka strain, was evaluated not only for its virulence but also for the immune response of rice grasshopper Oxya japonica (Orthoptera: Acrididae) as a target organism. Five aqueous suspensions with different conidia concentrations in logarithmic series were prepared. The fungus showed high virulence as it caused 100% mortality at low conidia concentration (1.5 × 10² conidia/mL). Remarkable changes in the cellular and humoral responses were also observed when adult grasshoppers were infected with the fungus. The number of hemocytes decreased significantly within 12 h after infection. In addition, the total number of granulocytes increased rapidly in the first 12 h then gradually decreased 24 and 48 h after infections, while the number of coagulocytes fluctuated over time. The infection influenced the humoral response by increasing the phenoloxidase activity.     

CTC medium: A novel dodine-free selective medium for isolating entomopathogenic fungi,especially Metarhizium acridum,from soil

The selective media most commonly used for isolating hyphomycetous species of entomopathogenic fungi from non-sterile substrates rely on N-dodecylguanidine monoacetate (dodine) as the selective fungicide. Although these media are effective for isolating many species of Metarhizium and Beauveria from soil, they are inefficient media for isolation of an important Metarhizium species, Metarhizium acridum, from non-sterile soil. Our current study was directed to formulating a dodine-free selective medium that is efficient for isolating naturally occurring Beauveria spp. and Metarhizium spp., especially M. acridum, from soil. The selective medium (designated CTC medium) consists of potato dextrose agar plus yeast extract (PDAY) supplemented with chloramphenicol, thiabendazole and cycloheximide. In comparisons with selective media previously reported in the literature, the CTC medium afforded colonies that were larger and had both earlier and more abundant conidiation of entomopathogenic fungi, features which greatly facilitated identification of the emerging entomopathogenic fungi. In addition to efficient re-isolation of M. acridum, this medium also is an effective tool for selective isolation of Metarhizium brunneum, Metarhizium robertsii, Beauveria bassiana and Beauveria brongniartii from non-sterile field-collected soil samples inoculated (spiked) with fresh conidia in the laboratory.     

Seasonal patterns of persistence and infectivity of Metarhizium anisopliae var. acridum in grasshopper cadavers in the Sahel

Field-based experiments were conducted to evaluate the fate and infectivity of the entomopathogenic fungus Metarhizium anisopliae var. acridum (Deuteromycotina: Hyphomycetes) in grasshopper cadavers in the Sahel. Unlike uninfected cadavers, which were rapidly scavenged, those infected with the fungus persisted in the environment for a number of weeks. The environmental factor most associated with cadaver disappearance was rainfall. The high environmental humidity associated with rainfall was also required for sporulation of the fungus on host cadavers, although the likelihood of sporulation differed between microsites. Characteristics of the infection profile from infective cadavers were investigated by the sequential exposure of uninfected hosts to sporulating cadavers in field cages. This experiment revealed that cadavers remained infective for > 30 days, with the net infectivity changing through time. The most likely explanation for these changes is climatic influences on both the fungus and host. High humidity was not required for infection. A measurement of the transmission coefficient between healthy hosts and sporulating cadavers in the field was obtained at a realistic density of infectious cadavers. This revealed a figure of 0.45 m² day^–1. Overall, these experiments show that following host death, M. anisopliae var. acridum can be persistent in the environment, sporulate on host cadavers and reinfect new hosts at a realistically low field density, although at least in arid or semi-arid areas, rainfall may be critical to the horizontal transmission of this pathogen.     

Genome‐assisted development of nuclear intergenic sequence markers for entomopathogenic fungi of the Metarhizium anisopliae species complex

Entomopathogenic fungi in the genus Metarhizium are useful for biological control programmes against economically important arthropod pests worldwide. However, understanding the true diversity and ecology of these organisms is hampered by convergent morphologies between species. The application of molecular techniques has enabled greater resolution of species than allowed by morphology alone. In particular, the commonly used biocontrol agent M. anisopliae was found to be a species complex composed of nine species. This prior work was conducted with commonly used markers in fungal phylogenetics (BTUB, RPB1, RPB2 and TEF), which likely under‐represent diversity in the M. anisopliae complex. Using sequence data from nuclear genomes of M. acridum and M. robertsii we identified regions of conserved gene synteny and developed primers to amplify intergenic regions of seven loci. Using ex‐type and authenticated tissue specimens for species in the M. anisopliae complex, we demonstrate that sequence data derived from intergenic loci is more variable and phylogenetically informative than previously available markers. These new markers will facilitate investigations at or below the species level for the M. anisopliae complex. The method of marker development employed here should be extendable to any group with sufficiently divergent genome data available.     

Microcycle conidiation and the conidial properties in the entomopathogenic fungus Metarhizium acridum on agar medium

Conidiation of the entomopathogenic fungus Metarhizium acridum on agar media was investigated. M. acridum CQMa102 exhibits two different conidiation patterns on agar media: normal conidiation in which conidia are formed on extended hyphae and microcycle conidiation in which conidiation occurs directly after conidia germination. Microcycle conidiation resulted in a mass of conidia produced via budding by accelerated development at the inoculation site. The mean total conidial yield (conidiation at day 10) was 4–5-fold greater after microcycle conidiation than during normal conidiation. Insect pathology assays indicated that microcycle conidia produced on SYA agar were as effective as normal aerial conidia against the locust. Ultraviolet (UV)-resistance tests showed no significant differences between the two types of cell propagules. However, microcycle conidia were more heat resistant than normal aerial conidia, and accumulated higher levels of trehalose in response to heat induction compared to normal aerial conidia.