Plant tissue localization of the endophytic insect pathogenic fungi Metarhizium and Beauveria

Endophytic fungi may display preferential tissue colonization within their plant hosts. Here we tested if the endophytic, insect pathogenic fungi (EIPF) Metarhizium and Beauveria showed preferential localization within plant tissues, in the field and under laboratory conditions. In the field, plants were sampled from three separate sites (Brock University, St. Catharines, Ontario; Pelham, Ontario; and Torngat Mountains National Park, Newfoundland, Canada) and EIPF were isolated from plant roots, the hypocotyl, and stem and leaves. Two genera of EIPF, Metarhizium spp. and Beauveria bassiana, were isolated from plants sampled, as well as the nematophagous fungus, Pochonia chlamydosporium. Metarhizium spp. were almost exclusively found in roots, whereas B. bassiana and P. chlamydosporium were found throughout the plant. The Metarhizium species were identified by RFLP and 95 % were Metarhizium robertsii, 4.3 % were M. brunneum, and 0.7 % were M. guizhouense. Lab studies with M. robertsii and B. bassiana reflected observations found in the field, that is, Metarhizium was restricted to the roots of plants while B. bassiana was found throughout the plant. Insect infection by these EIPF is preferential with respect to above and below ground insects, and the present study correlates above and below ground insect infections with endophytic colonization by these EIPF.     

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.     

Ubiquity of Insect-Derived Nitrogen Transfer to Plants by Endophytic Insect-Pathogenic Fungi: an Additional Branch of the Soil Nitrogen Cycle

The study of symbiotic nitrogen transfer in soil has largely focused on nitrogen-fixing bacteria. Vascular plants can lose a substantial amount of their nitrogen through insect herbivory. Previously, we showed that plants were able to reacquire nitrogen from insects through a partnership with the endophytic, insect-pathogenic fungus Metarhizium robertsii. That is, the endophytic capability and insect pathogenicity of M. robertsii are coupled so that the fungus acts as a conduit to provide insect-derived nitrogen to plant hosts. Here, we assess the ubiquity of this nitrogen transfer in five Metarhizium species representing those with broad (M. robertsii, M. brunneum, and M. guizhouense) and narrower insect host ranges (M. acridum and M. flavoviride), as well as the insect-pathogenic fungi Beauveria bassiana and Lecanicillium lecanii. Insects were injected with ¹⁵N-labeled nitrogen, and we tracked the incorporation of ¹⁵N into two dicots, haricot bean (Phaseolus vulgaris) and soybean (Glycine max), and two monocots, switchgrass (Panicum virgatum) and wheat (Triticum aestivum), in the presence of these fungi in soil microcosms. All Metarhizium species and B. bassiana but not L. lecanii showed the capacity to transfer nitrogen to plants, although to various degrees. Endophytic association by these fungi increased overall plant productivity. We also showed that in the field, where microbial competition is potentially high, M. robertsii was able to transfer insect-derived nitrogen to plants. Metarhizium spp. and B. bassiana have a worldwide distribution with high soil abundance and may play an important role in the ecological cycling of insect nitrogen back to plant communities.     

Effect of Norwegian entomopathogenic fungal isolates against Otiorhynchus sulcatus larvae at low temperatures and persistence in strawberry rhizospheres

Biological control of belowground stages of the black vine weevil Otiorhynchus sulcatus F. (Coleoptera: Curculionidae) in strawberries in cool temperate regions using entomopathogens is challenged by low temperatures during the periods when larvae are vulnerable to infections. In a laboratory study we tested six indigenous Norwegian isolates of entomopathogenic fungi (one Beauveria bassiana, three Beauveria pseudobassiana, and two Metarhizium brunneum; Ascomycota: Hypocreales) for their efficacy against O. sulcatus larvae at 6, 12, and 18 °C. At the lowest temperature only Beauveria spp. affected survival of O. sulcatus while all three fungal species reduced larval survival compared to the control treatment at 12 and 18 °C. Two of the Norwegian isolates, one B. pseudobassiana and one M. brunneum, were then evaluated for long-term persistence (>1 year) in the bulk soil and the rhizosphere soil of strawberries in a semi-field experiment. An exotic isolate of M. brunneum sharing origin with a widespread commercial biocontrol agent (F52/Met52 (Novozymes)) was included for comparison. All three isolates showed significantly higher abundances in the rhizosphere soil compared to bulk soil at 153, 366, and 471 days after inoculation, thus indicating rhizosphere competence for B. pseudobassiana. Notably, CFU levels for both Norwegian isolates were much higher than for the exotic M. brunneum isolate. Selection of locally adapted isolates may therefore be of importance when considering biocontrol strategies of belowground pests in strawberry production.     

The mitochondrial genome contribution to the phylogeny and identification of Metarhizium species and strains

The genus Metarhizium is composed of entomopathogenic fungal biological control agents (BCAs) used for invertebrate pest control. The phylogenetic relationships of species within this genus are still under scrutiny as several cryptic species can be found. In this work, the mitochondrial (mt) genome of Metarhizium brunneum ARSEF 4556 was fully sequenced and a comparative genome analysis was conducted with 7 other available mt genomes, belonging to 5 Metarhizium species: M. anisopliae, M. brunneum, M. robertsii, M. guizhouense and M. majus. Results showed that Metarhizium demonstrates greater conserved stability than other fungal mt genomes. Furthermore, this analysis located 7 diverse regions in both intergenic domains and gene fragments which were ideal for species/strain discrimination. The sequencing of these regions revealed several SNPs among 38 strains tested, 11 of which were uncharacterized. Single gene phylogenies presented variable results which may be used further for intra-species discrimination. Phylogenetic trees based on the concatenation of mt domains and the nuclear ITS1-5.8S-ITS2 region showed discrimination of the species studied and allowed the identification of uncharacterized strains. These were mostly placed within species M. anisopliae and M. brunneum. Five strains clustered together in a clade related to M. brunneum, suggesting that they comprise a cryptic species.     

Species of the Metarhizium anisopliae complex with diverse ecological niches display different susceptibilities to antifungal agents

Species of the Metarhizium anisopliae complex are globally ubiquitous soil-inhabiting and predominantly insect-pathogenic fungi. The Metarhizium genus contains species ranging from specialists, such as Metarhizium acridum that only infects acridids, to generalists, such as M. anisopliae, Metarhizium brunneum, and Metarhizium robertsii that infect a broad range of insects and can also colonize plant roots. There is little information available about the susceptibility of Metarhizium species to clinical and non-clinical antifungal agents. We determined the susceptibility of 16 isolates comprising four Metarhizium species with different ecological niches to seven clinical (amphotericin B, ciclopirox olamine, fluconazole, griseofulvin, itraconazole, tebinafine, and voriconazole) and one non-clinical (benomyl) antifungal agents. All isolates of the specialist M. acridum were clearly more susceptible to most antifungals than the isolates of the generalists M. anisopliae sensu lato, M. brunneum, and M. robertsii. All isolates of M. anisopliae, M. brunneum, and M. robertsii were resistant to fluconazole and some were also resistant to amphotericin B. The marked differences in susceptibility between the specialist M. acridum and the generalist Metarhizium species suggest that this characteristic is associated with their different ecological niches, and may assist in devising rational antifungal treatments for the rare cases of mycoses caused by Metarhizium species.     

Exploring virulence of new and less studied species of <Emphasis Type="Italic">Metarhizium</Emphasis> spp. from Brazil for two-spotted spider mite control

The two-spotted spider mite Tetranychus urticae is an important pest of strawberry crops in Brazil and many other countries. Focus for biocontrol studies involving entomopathogenic fungi has been on three species from the genus Metarhizium: M. anisopliae sensu stricto (s.s.), M. brunneum and M. robertsii. Also, the species Beauveria bassiana has been studied for spider mite control and one isolate (ESALQPL63) is commercially available in Brazil. New and undescribed Metarhizium species have been found recently in Brazil and provide a pool of isolates with potential for biocontrol in Brazil and probably also elsewhere. The mortality of adult females of T. urticae when exposed to four new Brazilian species of Metarhizium was compared to the mortality when exposed to M. anisopliae s.s., M. brunneum, M. pingshaense, M. robertsii and Beauveria bassiana ESALQPL63. Fungal suspensions were sprayed onto mites at 10⁷ conidia/mL with 0.05% Tween 80 in laboratory bio-assays. We measured total mortality and percentage sporulating cadavers 10 days after exposure and calculated median lethal time (LT₅₀). The lowest LT₅₀ (4.0 ± 0.17) was observed for mites treated with Metarhizium sp. Indet. 1 (ESALQ1638), which also performed well with respect to mortality after 10 days and capacity to sporulate from cadavers. Among the other little studied species tested, M. pingshaense (ESALQ3069 and ESALQ3222) and Metarhizium Indet. 2 (ESALQ1476) performed well and were comparable to B. bassiana (ESALQPL63). The new Metarhizium isolates and species thus showed potential for biological control.     

The plant beneficial effects of Metarhizium species correlate with their association with roots

Metarhizium species have recently been found to be plant rhizosphere associates as well as insect pathogens. Because of their abundance, rhizospheric Metarhizium could have enormous environmental impact, with co-evolutionary implications. Here, we tested the hypothesis that some Metarhizium spp. are multifactorial plant growth promoters. In two consecutive years, corn seeds were treated with entomopathogenic Metarhizium spp. and field tested at the Beltsville Facility in Maryland. Seed treatments included application of green fluorescent protein (GFP)-tagged strains of Metarhizium brunneum, Metarhizium anisopliae, Metarhizium robertsii, and M. robertsii gene disruption mutants that were either avirulent (Δmcl1), unable to adhere to plant roots (Δmad2), or poorly utilized root exudates (Δmrt). Relative to seeds treated with heat-killed conidia, M. brunneum, M. anisopliae, and M. robertsii significantly increased leaf collar formation (by 15, 14, and 13 %), stalk length (by 16, 10, and 10 %), average ear biomass (by 61, 56, and 36 %), and average stalk and foliage biomass (by 46, 36, and 33 %). Their major impact on corn yield was during early vegetative growth by allowing the plants to establish earlier and thereby potentially outpacing ambient biotic and abiotic stressors. Δmcl1 colonized roots and promoted plant growth to a similar extent as the parent wild type, showing that Metarhizium populations are plant growth promoters irrespective of their role as insect pathogens. In contrast, rhizospheric populations and growth promotion by Δmrt were significantly reduced, and Δmad2 failed to colonize roots or impact plant growth, suggesting that colonization of the root is a prerequisite for most, if not all, of the beneficial effects of Metarhizium.     

Diversity and Genetic Population Structure of Fungal Pathogens Infecting White Grub Larvae in Agricultural Soils

White grub larvae are important soil-dwelling pests in many regions of Mexico as they attack many important crops such as maize. The use of synthetic chemicals is currently the main control strategy, but they are not always effective; thus, other alternatives are needed. Microbial control using entomopathogenic fungi represents an important alternative strategy, and species within the genera Beauveria and Metarhizium are considered amongst the most promising candidates. Seventeen Beauveria spp. and two Metarhizium spp. isolates were obtained in surveys of white grub larvae from different regions of Guanajuato, Mexico. All isolates were capable of infecting healthy larvae of the white grub Phyllophaga polyphilla in laboratory assays, but mortality never exceeded 50 %. Isolates were identified using morphological and molecular methods. Based on elongation factor1-α and ITS partial gene sequence data, all Beauveria isolates were identified as Beauveria pseudobassiana. Elongation factor1-α and β-tubulin sequence data identified the Metarhizium isolates to be Metarhizium pingshaense. In contrast, three additional Metarhizium isolates obtained the previous year in the same region were identified as M. pingshaense, Metarhizium anisopliae and Metarhizium robertsii. Microsatellite genotyping showed that all B. pseudobassiana isolates were the same haplotype. Enterobacterial Repetitive Intergenic Consensus fingerprinting information confirmed no significant variation amongst the B. pseudobassiana isolates. The ecological role of these isolates and their impact on white grub larvae populations are discussed.     

Niche separation of species of entomopathogenic fungi within the genera Metarhizium and Beauveria in different cropping systems in Mexico

Entomopathogenic fungi from the genera Beauveria and Metarhizium, were isolated from soil using the Galleria mellonella baiting method, and from infected white grub larvae from a diversity of cropping systems in Puebla and Guanajuato, Mexico. Isolates were identified to species level using Bloc and Elongation Factor 1-α sequence information. Although widespread, Beauveria bassiana (41 isolates) was only isolated from soil and not from infected white grubs. In contrast, Beauveria pseudobassiana (six isolates) was predominantly isolated from white grub larvae (only one isolate from soil). Haplotype analysis of B. bassiana Bloc sequences identified 25 haplotypes indicating substantial genetic diversity; neither geographical origin nor crop type explained this genetic variation. Metarhizium brunneum (three isolates) and Metarhizium robertsii (17 isolates) were also only isolated from soil, while Metarhizium anisopliae (six isolates) and Metarhizium pingshaense (four isolates) were only isolated from white grub larvae. M. anisopliae was only found infecting Paranomala species while M. pingshaense was only found infecting Phyllophaga species. Species diversity in Metarhizium was influenced by crop type. Our results showed that entomopathogenic fungi species could co-exist in the same soil ecosystem but in separate niches. The potential ecological roles of these species are discussed.     

A PCR-based tool for cultivation-independent detection and quantification of Metarhizium clade 1

The entomopathogenic fungus Metarhizium anisopliae and sister species are some of the most widely used biological control agents for insects. Availability of specific monitoring and quantification tools are essential for the investigation of environmental factors influencing their environmental distribution. Naturally occurring as well as released Metarhizium strains in the environment traditionally are monitored with cultivation-dependent techniques. However, specific detection and quantification may be limited due to the lack of a defined and reliable detection range of such methods. Cultivation-independent PCR-based detection and quantification tools offer high throughput analyses of target taxa in various environments. In this study a cultivation-independent PCR-based method was developed, which allows for specific detection and quantification of the defined Metarhizium clade 1, which is formed by the species Metarhizium majus, Metarhizium guizhouense, Metarhizium pingshaense, Metarhizium anisopliae, Metarhizium robertsii and Metarhiziumbrunneum, formerly included in the M. anisopliae cryptic species complex. This method is based on the use of clade-specific primers, i.e. Ma 1763 and Ma 2097, that are positioned within the internal transcribed spacer regions 1 and 2 of the nuclear ribosomal RNA gene cluster, respectively. BLAST similarity searches and empirical specificity tests performed on target and non-target species, as well as on bulk soil DNA samples, demonstrated specificity of this diagnostic tool for the targeted Metarhizium clade 1. Testing of the primer pair in qPCR assays validated the diagnostic method for specific quantification of Metarhizium clade 1 in complex bulk soil DNA samples that significantly correlated with cultivation-dependent quantification. The new tool will allow for highly specific and rapid detection and quantification of the targeted Metarhizium clade 1 in the environment. Habitat with high Metarhizium clade 1 densities can then be analyzed for habitat preferences in greater detail using cultivation-dependent techniques and genetic typing of isolates.     

Conidiation under illumination enhances conidial tolerance of insect-pathogenic fungi to environmental stresses

Light is an important signal for fungi in the environment and induces many genes with roles in stress and virulence responses. Conidia of the entomopathogenic fungi Aschersonia aleyrodis, Beauveria bassiana, Cordyceps fumosorosea, Lecanicillium aphanocladii, Metarhizium anisopliae, Metarhizium brunneum, Metarhizium robertsii, Simplicillium lanosoniveum, Tolypocladium cylindrosporum, and Tolypocladium inflatum were produced on potato dextrose agar (PDA) medium under continuous white light, on PDA medium in the dark, or under nutritional stress (= Czapek medium without sucrose = MM) in the dark. The conidial tolerance of these species produced under these different conditions were evaluated in relation to heat stress, oxidative stress (menadione), osmotic stress (KCl), UV radiation, and genotoxic stress caused by 4-nitroquinoline 1-oxide (4-NQO). Several fungal species demonstrated greater stress tolerance when conidia were produced under white light than in the dark; for instance white light induced higher tolerance of A. aleyrodis to KCl and 4-NQO; B. bassiana to KCl and 4-NQO; C. fumosorosea to UV radiation; M. anisopliae to heat and menadione; M. brunneum to menadione, KCl, UV radiation, and 4-NQO; M. robertsii to heat, menadione, KCl, and UV radiation; and T. cylindrosporum to menadione and KCl. However, conidia of L. aphanocladii, S. lanosoniveum, and T. inflatum produced under white light exhibited similar tolerance as conidia produced in the dark. When conidia were produced on MM, a much stronger stress tolerance was found for B. bassiana to menadione, KCl, UV radiation, and 4-NQO; C. fumosorosea to KCl and 4-NQO; Metarhizium species to heat, menadione, KCl, and UV radiation; T. cylindrosporum to menadione and UV radiation; and T. inflatum to heat and UV radiation. Again, conidia of L. aphanocladii and S. lanosoniveum produced on MM had similar tolerance to conidia produced on PDA medium in the dark. Therefore, white light is an important factor that induces higher stress tolerance in some insect-pathogenic fungi, but growth in nutritional stress always provides in conidia with stronger stress tolerance than conidia produced under white light.     

Improving biocontrol of black vine weevil (<Emphasis Type="Italic">Otiorhynchus sulcatus</Emphasis>) with entomopathogenic fungi in growing media by incorporating spent mushroom compost

Amending a peat-based growing medium with 10% v/v spent mushroom compost, a source of fungal chitin and other nutrients, prolonged the persistence of entomopathogenic fungi (Metarhizium brunneum Petsch and Beauveria bassiana (Balsamo) Vuillemin; Hypocreales: Clavicipitaceae). This resulted in improved efficacy of M. brunneum against black vine weevil, Otiorhynchus sulcatus F. (Coleoptera: Curculionidae) larvae compared with using inoculum without spent mushroom compost. B. bassiana only controlled larvae when used in combination with spent mushroom compost (75?±?7% reduction in live larvae). Mixing entomopathogenic fungal inoculum with spent mushroom compost and growing medium was as effective in controlling black vine weevil larvae as using spent mushroom compost colonised with M. brunneum or B. bassiana in the growing medium (80?±?12% reduction in live larvae). The former method is preferable since it does not require production and storage of colonised spent mushroom compost, or registration of new substrate formulations of M. brunneum or B. bassiana.     

Comparison of the relative efficacy of an insect baiting method and selective media for diversity studies of Metarhizium species in the soil

Metarhizium are a commonly occurring group of entomopathogenic fungi normally found in soil. The most common methods to assess the diversity of Metarhizium species in soil are (i) the use of selective media and (ii) insect baiting using Galleria mellonella larvae. We compared the recovery efficiency from soil of four common species of Metarhizium (Metarhizium anisopliae, Metarhizium pingshaense, Metarhizium brunneum and Metarhizium robertsii) using these two methods. Firstly, we compared the number of colony forming units (CFU) produced in vitro when grown on two selective media, one containing chloramphenicol, thiabendazole and cycloheximidethe (CTC) and one based on the fungicide dodine (n-dodecylguanidine acetate) (DOD). Secondly, we artificially inoculated natural/non-sterile soil with the four fungal species at a rate of 2×10² and 2×10³ conidia g^?1of soil, baited with G. mellonella, and processed for evaluation using the selective media. The in vitro results showed that the greatest number of CFUs were recorded for M. brunneum. In contrast, when inoculated into soil, more G. mellonella larvae became infected by M. anisopliae. Finally, when using selective media, most CFUs recovered were for M. robertsii. The importance of our results in selecting a method to study the natural occurrence of Metarhizium in soil are discussed.     

Responsiveness of entomopathogenic fungi to menadione-induced oxidative stress

Entomopathogenic fungi are predisposed to ROS induced by heat and UV–A radiation when outside the insect host. When inside the host, they are subject to phagocytic cells that generate ROS to eliminate invading pathogens. The oxidative stress tolerance of the entomopathogenic fungi Aschersonia aleyrodis (ARSEF 430 and 10276), Aschersonia placenta (ARSEF 7637), Beauveria bassiana (ARSEF 252), Isaria fumosorosea (ARSEF 3889), Lecanicillium aphanocladii (ARSEF 6433), Metarhizium acridum (ARSEF 324), Metarhizium anisopliae (ARSEF 5749), Metarhizium brunneum (ARSEF 1187 and ARSEF 5626), Metarhizium robertsii (ARSEF 2575), Tolypocladium cylindrosporum (ARSEF 3392), Tolypocladium inflatum (ARSEF 4877), and Simplicillium lanosoniveum (ARSEF 6430 and ARSEF 6651) was studied based on conidial germination on a medium supplemented with menadione. Conidial germination was evaluated 24 h after inoculation on potato dextrose agar (PDA) (control) or PDA supplemented with menadione. The two Aschersonia species (ARSEF 430, 7637, and 10276) were the most susceptible fungi, followed by the two Tolypocladium species (ARSEF 3392 and 4877) and the M. acridum (ARSEF 324). Metarhizium brunneum (ARSEF 5626) and M. anisopliae (ARSEF 5749) were the most tolerant isolates with MIC 0.28 mM. All fungal isolates, except ARSEF 5626 and ARSEF 5749, were not able to germinate at 0.20 mM.     

Comparative analysis of immune responses in Colorado potato beetle larvae during development of mycoses caused by <Emphasis Type="Italic">Metarhizium robertsii</Emphasis>, <Emphasis Type="Italic">M. brunneum</Emphasis>, and <Emphasis Type="Italic">M. pemphigi</Emphasis>

Development of mycoses and progress of humoral and cellular immune responses were compared in larvae of the Colorado potato beetle Leptinotarsa decemlineata infected with entomopathogenic fungi Metarhizium robertsii, M. brunneum and M. pemphigi. The larvae were found to be highly susceptible to the strains of M. robertsii and M. brunneum but weakly responsive to M. pemphigi. The extent of susceptibility to the pathogens was not related to the stimulating effect of epicuticular extracts on fungal growth. Metarhizium pemphigi, which is non-specific to the Colorado potato beetle, did not cause any significant changes in the immune response and did not colonize the hemocoel. When infected with M. robertsii and M. brunneum, the larvae exhibited an increase in hemocyte count during the early stage of mycosis (day 2) followed by a drastic decrease on day 3. The immunocompetent cells, plasmatocytes and granulocytes, exhibited the greatest decrease. Elevated phenoloxidase activity was recorded in the hemolymph and cuticle on days 2 and 3 post-infection. These changes in the immune responses correlated with strain-specific virulence. Thus, the immune response in Colorado potato beetle larvae is an important factor, which determines differences in the development of mycoses caused by different Metarhizium species.     

Metarhizium robertsii Produces an Extracellular Invertase (MrINV) That Plays a Pivotal Role in Rhizospheric Interactions and Root Colonization

As well as killing pest insects, the rhizosphere competent insect-pathogenic fungus Metarhizium robertsii also boosts plant growth by providing nitrogenous nutrients and increasing resistance to plant pathogens. Plant roots secrete abundant nutrients but little is known about their utilization by Metarhizium spp. and the mechanistic basis of Metarhizium-plant associations. We report here that M. robertsii produces an extracellular invertase (MrInv) on plant roots. Deletion of MrInv (⊿MrInv) reduced M. robertsii growth on sucrose and rhizospheric exudates but increased colonization of Panicum virgatum and Arabidopsis thaliana roots. This could be accounted for by a reduction in carbon catabolite repression in ⊿MrInv increasing production of plant cell wall-degrading depolymerases. A non-rhizosphere competent scarab beetle specialist Metarhizium majus lacks invertase which suggests that rhizospheric competence may be related to the sugar metabolism of different Metarhizium species.     

Simulated aerial sprays for field cage evaluation of Beauveria bassiana and Metarhizium brunneum (Ascomycetes: Hypocreales) against Anabrus simplex (Orthoptera: Tettigoniidae) in Montana

Field efficacy of the entomopathogenic Ascomycetes Beauveria bassiana strain GHA and Metarhizium brunneum strain F52 was evaluated against nymphs of the Mormon cricket, Anabrus simplex. Fungi were applied with a new apparatus that allows simulated aerial sprays to 0.1-m² areas in the field. The Mormon crickets were then individually housed in cylindrical, metal hardware cloth cages on treated grass. Both fungi demonstrated only marginal success in reducing immature Mormon cricket survival in the field cages. After 28 days, the field mortality of insects, corrected for control deaths, was 28 and 45% for B. bassiana and M. brunneum, respectively. Field-exposed but laboratory-incubated insects, however, suffered 90–100% mortality within 12 days with initial deaths occurring on Day 6 (Beauveria) or Day 5 (Metarhizium), indicating a lethal rate was applied and acquired by the crickets in the field. Potential daily body temperatures were determined for the entire post-treatment observation period using special thermal surrogates. High ambient temperatures and/or thermoregulation by Mormon crickets, in excess of the upper thermal limits of both fungi, prevented higher mortality from being expressed in the field. Thermal surrogates were used to develop models for predicting onset of mortality from infections. The surrogate data indicated mortality should begin between 8 and 26 days after treatment with M. brunneum and 11 and 33 days after treatment with B. bassiana. The timing of mortality in field cages was consistent with the upper boundaries of the temporal models developed from the thermal surrogates, i.e., at or after 28 days after treatment.     

Diversity of indigenous Beauveria and Metarhizium spp. in a commercial banana field and their virulence toward Cosmopolites sordidus (Coleoptera: Curculionidae)

Metarhizium anisopliae and Beauveria bassiana sensu lato were isolated, from 7 and 41 % of soil samples from a commercial banana field, with average fungal density of 4.3 × 10³ and 8.2 × 10³ CFU g^?1 soil, respectively. Twenty-one morphologically distinct B. bassiana and four M. anisopliae sensu lato isolates from different plots within the field were further characterized. ISSR fingerprinting revealed six different clusters for B. bassiana, whereas gene sequencing revealed three M. anisopliae sensu stricto and one unclassified Metarhizium sp. Bioassays with one or more representative isolates from each Metarhizium species and B. bassiana cluster showed that all indigenous isolates had lower virulence and significantly greater ST₅₀s than reference (exotic) isolates. The data suggest that the low virulence of most indigenous isolates toward Cosmopolites sordidus adults and their relatively low density in soil samples, may help explain the low occurrence of epizootics caused by entomopathogenic fungi in populations of this pest, also known to burrow under the soil surface in banana plantations.     

Differential susceptibility of blastospores and aerial conidia of entomopathogenic fungi to heat and UV-B stresses

We investigated the comparative susceptibility to heat and UV-B radiation of blastospores and aerial conidia of Metarhizium spp. (Metarhizium robertsii IP 146, Metarhizium anisopliae s.l. IP 363 and Metarhizium acridum ARSEF 324) and Beauveria bassiana s.l. (IP 361 and CG 307). Conidia and blastospores were produced in solid or liquid Adámek-modified medium, respectively, and then exposed to heat (45 ± 0.2 °C) in a range of 0 (control) to 360 min; the susceptibility of fungal propagules to heat exposures was assessed to express relative viability. Similarly, both propagules of each isolate were also exposed to a range of 0 (control) to 8.1 kJ m⁻² under artificial UV-B radiation. Our results showed that fungal isolates, propagule types and exposure time or dose of the stressor source play critical roles in fungal survival challenged with UV-B and heat. Conidia of ARSEF 324, IP 363, IP 146 and IP 361 exposed to heat survived significantly longer than their blastospores, except for blastospores of CG 307. Conidia and blastospores of IP 146 and IP 363 were equally tolerant to UV-B radiation. We claim that blastospores of certain isolates may be promising candidates to control arthropod pests in regions where heat and UV-B are limiting environmental factors.