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.     

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.     

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.     

Diversity of rhizosphere associated entomopathogenic fungi of perennial herbs, shrubs and coniferous trees

Understanding habitat selection of fungal entomopathogens is critical to improve the efficacy, persistence and cost of these fungi as microbial insecticides. This study sought to determine the prevalence of Metarhizium and Beauveria spp. isolated from the rhizosphere of strawberry, blueberry, grape and Christmas tree crops in the Willamette Valley of Oregon. Entomopathogenic fungi were assigned to thirteen species based on molecular phylogenetic criteria. Four species of Metarhizium were isolated including Metarhizium brunneum, Metarhizium guizhouense, Metarhizium robertsii, and Metarhizium flavoviride var. pemphigi. Nine Beauveria species were isolated including, Beauveria brongniartii, an undescribed species referred to as Clade C and seven phylogenetic species of Beauveria bassiana. Strawberries and blueberries were significantly associated with M. brunneum and Christmas trees with M. guizhouense and M. robertsii. Grapes were significantly associated with B. bassiana phylogenetic species Bbas-16. All of the Metarhizium isolates screened were pathogenic to Otiorhynchus sulcatus larvae in laboratory bioassays but only M. brunneum and M. robertsii caused significant levels of infection. The study results suggest that certain species of Metarhizium and Beauveria are significantly associated with the strawberry, blueberry and Christmas tree rhizosphere and could potentially provide better control of O. sulcatus.     

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.     

Comparative gene expression and genomics reflect geographical divergence in the plant symbiotic and entomopathogenic fungal genus Metarhizium

Several species within the fungal genus Metarhizium can both infect insects and colonize plant roots. In Brazil, a specific subgroup within Metarhizium anisopliae s.str. named “subclade Mani 2” is frequently observed infecting above-ground insects, whereas sympatric M. robertsii and M. brunneum predominantly occur in the soil environment. Genotypic variability within the genus may be linked to adaptations to these different habitats. We present a comparative analysis of the complete genomes and the adhesin genes Mad1 and Mad2 of 14 Metarhizium isolates representing M. anisopliae Mani 2 (n = 6), M. robertsii (n = 5) and M. brunneum (n = 3). In addition, the relative gene expression of six selected target genes was compared in root exudate solution and insect cuticle suspension. We hypothesized that M. anisopliae Mani 2 is adapted to insect-pathogenicity in the above-ground environment, reflected by higher relative expression of pathogenicity-related genes. In contrast, M. robertsii and M. brunneum are adapted to the soil environment, hence hypothesized to have a higher expression of genes related to plant associations. Phylogenomic and adhesin phylogenetic trees revealed species differences but also intraspecific variability associated with the geographic origin of isolates. Differences in relative gene expression were observed, with one pathogenicity-related gene (Pr1) being higher expressed in M. anisopliae. The insect adhesion Mad1 gene was more conserved than the plant adhesion Mad2 and similarly expressed in exudate solution, while Mad2 was highly expressed by all Brazilian isolates in both exudate and cuticle conditions. The variabilities observed correlated with different habitats and lifestyles, demonstrating the importance of selecting a diverse collection of isolates in genomic and gene expression studies.     

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.     

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.     

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.     

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.     

Genetic diversity of the Metarhizium anisopliae complex in Colima,Mexico, using microsatellites

Metarhizium anisopliae is a complex of cryptic species with wide geographical distribution and versatile lifestyles. In this study, 45 isolates of the Metarhizium genus harbored in the “Colección de Hongos Entomopatógenos” of the “Centro Nacional de Referencia de Control Biológico” from different substrates, insect-host, and localities from Colima, Mexico, were phylogenetically identified using the 5′end of translation elongation factor 1-α (5′TEF) and intergenic nuclear region MzFG543igs. Seven species were recognized, M. acridum (n = 26), M. pemphigi (n = 1), and within the PARB and MGT clades: M. anisopliae (N = 7; sensu stricto: n = 2; sensu lato: n = 5), M. brunneum (n = 2), M. guizhouense (n = 2), M. pingshaense (n = 2), and M. robertsii (n = 5). Twenty-nine SSR markers were developed for M. acridum; according to the analysis of 12 polymorphic SSR loci, M. acridum showed low genetic diversity, revealing five genotypes with a dominant one (n = 21). Based on the analysis of 13 specific SSR loci, 14 genotypes were identified within the PARB and MGT clades. This study contributes to generating valuable information about the community structure and genotypic diversity of Metharhizum species in the state of Colima, Mexico.     

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.     

Susceptibility of Metarhizium spp. and other entomopathogenic fungi to dodine-based selective media

The fungicide dodine has been widely used in selective media to isolate entomopathogenic fungi (EF) from contaminating microorganisms, primarily bacteria and non-entomopathogenic fungi. In order to isolate the fungus Metarhizium acridum from soil for grasshopper and Mormon cricket control in the western USA, the susceptibility of M. acridum was compared with two Metarhizium spp. and other EF species. The isolates were inoculated onto mycological media with concentrations of dodine ranging from 0.0001 to 0.03% active ingredient (A.I.). In addition, susceptibilities of five Metarhizium spp. isolates to two sources of dodine, Syllit® commercial fungicide (65% A.I.) and Sigma® reagent grade (99% A.I.), were compared using Czapek agar medium. Responses to the two dodine sources were virtually identical. Accordingly, subsequent experiments used the less expensive Syllit dodine. Three media [Czapek, potato dextrose agar plus yeast extract (PDAY) and oatmeal agar] were evaluated for appropriateness as the base in selective media. Germination of all three of the M. acridum isolates tested was almost completely inhibited by dodine concentrations of 0.002% A.I. in Czapek or 0.006% A.I. in PDAY. On the other hand, M. robertsii and M. anisopliae isolates were considerably more tolerant, with germination not being inhibited until 0.010% A.I. in Czapek or 0.030% A.I. in PDAY. The higher vulnerability of the isolates to low concentrations of dodine in Czapek medium suggests that this medium would be less effective than PDAY in a selective medium. Oatmeal agar greatly improved fungal growth, but the levels of inhibition were lower. Therefore, PDAY was selected as the best selective basal medium. The lowest concentration that inhibited a common soil-inhabiting fungus, Aspergillus nidulans, was 0.001% A.I. Dodine tolerances were highest with M. robertsii, M. anisopliae, and Beauveria bassiana, followed by Isaria fumosorosea and Lecanicillium spp. The least tolerant EF isolates were M. acridum.     

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.     

Responses of entomopathogenic fungi to the mutagen 4-nitroquinoline 1-oxide

Survival of entomopathogenic fungi under solar ultraviolet (UV) radiation is paramount to the success of biological control of insect pests and disease vectors. The mutagenic compound 4-nitroquinoline 1-oxide (4-NQO) is often used to mimic the biological effects of UV radiation on organisms. Therefore, we asked whether tolerance to 4-NQO could predict tolerance to UV radiation in thirty isolates of entomopathogenic fungi and one isolate of a xerophilic fungus. A dendrogram obtained from cluster analyses based on the 50 and 90 % inhibitory concentrations (IC₅₀ and IC₉₀, respectively) divided the fungal isolates into six clusters numbered consecutively based on their tolerance to 4-NQO. Cluster 6 contained species with highest tolerance to 4-NQO (IC₅₀ > 4.7 μM), including Mariannaea pruinosa, Lecanicillium aphanocladii, and Torrubiella homopterorum. Cluster 1 contained species least tolerant to 4-NQO (IC₅₀ < 0.2 μM), such as Metarhizium acridum (ARSEF 324), Tolypocladium geodes, and Metarhizium brunneum (ARSEF 7711). With few exceptions, the majority of Metarhizium species showed moderate to low tolerances (IC₅₀ between 0.4 and 0.9 μM) and were placed in cluster 2. Cluster 3 included species with moderate tolerance (IC₅₀ between 1.0 and 1.2 μM). In cluster 4 were species with moderate to high tolerance (IC₅₀ between 1.3 and 1.6 μM). Cluster 5 contained the species with high tolerance (IC₅₀ between 1.9 and 4.0 μM). The most UV tolerant isolate of M. acridum, ARSEF 324, was the least tolerant to 4-NQO. Also, L. aphanocladii, which is very susceptible to UV radiation, showed high tolerance to 4-NQO. Our results indicate that tolerance to 4-NQO does not correlate with tolerance to UV radiation. Therefore this chemical compound is not a predictor of UV tolerance in entomopathogenic fungi.     

The Xenon Test Chamber Q-SUN® for testing realistic tolerances of fungi exposed to simulated full spectrum solar radiation

The low survival of insect-pathogenic fungi when used for insect control in agriculture is mainly due to the deleterious effects of ultraviolet radiation and heat from solar irradiation. In this study, conidia of 15 species of entomopathogenic fungi were exposed to simulated full-spectrum solar radiation emitted by a Xenon Test Chamber Q-SUN XE-3-HC 340S (Q-LAB^® Corporation, Westlake, OH, USA), which very closely simulates full-spectrum solar radiation. A dendrogram obtained from cluster analyses, based on lethal time 50 % and 90 % calculated by Probit analyses, separated the fungi into three clusters: cluster 3 contains species with highest tolerance to simulated full-spectrum solar radiation, included Metarhizium acridum, Cladosporium herbarum, and Trichothecium roseum with LT₅₀ > 200 min irradiation. Cluster 2 contains eight species with moderate UV tolerance: Aschersonia aleyrodis, Isaria fumosorosea, Mariannaea pruinosa, Metarhizium anisopliae, Metarhizium brunneum, Metarhizium robertsii, Simplicillium lanosoniveum, and Torrubiella homopterorum with LT₅₀ between 120 and 150 min irradiation. The four species in cluster 1 had the lowest UV tolerance: Lecanicillium aphanocladii, Beauveria bassiana, Tolypocladium cylindrosporum, and Tolypocladium inflatum with LT₅₀ < 120 min irradiation. The QSUN Xenon Test Chamber XE3 is often used by the pharmaceutical and automotive industry to test light stability and weathering, respectively, but it was never used to evaluate fungal tolerance to full-spectrum solar radiation before. We conclude that the equipment provided an excellent tool for testing realistic tolerances of fungi to full-spectrum solar radiation of microbial agents for insect biological control in agriculture.     

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.     

Microsclerotia production of Metarhizium spp. for dual role as plant biostimulant and control of Spodoptera frugiperda through corn seed coating

The fungal genus Metarhizium comprises entomopathogenic species capable of producing overwintering structures known as microsclerotia. These structures offer many advantages in pest control due to the formation of infective conidia in situ and their persistence in the environment under adverse conditions. In addition, the in vitro production of Metarhizium microsclerotia under controlled liquid fermentation is faster and with greater process control than the production of aerial conidia. However, the potential of Metarhizium microsclerotia to control pests from the orders Lepidoptera and Hemiptera is unexplored. In this study, we examined the ability of Metarhizium spp. microsclerotia to promote corn growth and to provide plant protection against Spodoptera frugiperda (Lepidoptera: Noctuidae) and Dalbulus maidis (Hemiptera: Cicadellidae), through seed coating using microsclerotial granules. A screening to find higher microsclerotia producers was conducted by culturing 48 native Brazilian isolates of Metarhizium spp. (Metarhizium anisopliae, Metarhizium robertsii, Metarhizium humberi and Metarhizium sp. indeterminate). The best microsclerotia producers, M. anisopliae ESALQ1814, M. robertsii ESALQ2450 and M. humberi ESALQ1638 improved the leaf area, plant height, root length, and dry weight of plants compared to un-inoculated plants. Significant reduction in S. frugiperda survival (mortality > 55% after 7 days) was observed when larvae were fed on corn plants treated with any of the three Metarhizium species. Conversely, survival of D. maidis adults were unaffected by feeding on fungus-inoculated plants. Our results suggest that microsclerotia of Metarhizium spp. may act as biostimulants and to provide protection against S. frugiperda in corn through seed coating, thus adding an innovative strategy into the integrated management of this major worldwide pest.     

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.     

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.