Oxaloacetate hydrolase gene links the cytoplasmic route of oxalate formation to differentiation and virulence of entomopathogenic fungus Beauveria bassiana

Oxalic acid is used as a functional molecule by most filamentous fungi, and produced via cytoplasmic pathway and mitochondrial pathway. The cytoplasmic pathway of oxalate production from oxaloacetate is a one-step reaction catalyzed by oxaloacetate hydrolase. The entomopathogenic fungus Beauveria bassiana contains a unique oxaloacetate hydrolase gene (BbOAH). The role of cytoplasmic pathway of oxalate production in B. bassiana development and virulence was studied via construction of a targeted gene disruption mutant of BbOAH. Disruption of BbOAH resulted in a slight decrease (~ 30%) in oxalate production, but has no significant influence on fungal growth. The mutant strain displayed a significant delay at early stage of conidial development, and a significant defect in dimorphic transition. Additionally, bioassay using the greater waxmoth as host indicated a slight (~ 20%) decrease in mortality caused by the gene disruption strain. The phenotypic defects of the ΔBbOAH strain could be restored by ectopic integration of BbOAH. Our findings indicate that BbOAH gene connects the cytoplasmic route of oxalate production to fungal development and virulence, although the cytoplasmic route is not indispensable for oxalate production in B. bassiana.     

Role of cuticle-degrading enzymes of Beauveria bassiana and Metarhizium anisopliae in virulence on Plodia interpunctella (Lepidoptera,Pyralidae) larvae

Entomopathogenic fungi (EPF) are important biological control agents in pest management programs in agroecosystems against insect pests. EPF such as Beauveria bassiana (Bals.) Vuillemin and Metarhizium anisopliae (Metchn.) Sorokin produce a wide range of extracellular enzymes involved in disturbance of the first barrier in the insect cuticle comprising proteins, chitin, and lipids. Realizing relationships between the expression of these enzymes and fungal virulence might aid in development of effective mycoinsecticides. The virulence of B. bassiana (isolates TV and OZ1) and M. anisopliae (isolate CS1) were investigated on Plodia interpunctella (Hübner) larvae in this study. The third instar larvae were immersed in a suspension containing 1 × 10⁸ conidia mL^?1 of fungal conidial inoculum. The results revealed that all three fungal isolates caused mortality in larvae, but there was a considerable variation in their virulence. Total proteinase, exochitinase and lipase assays were done for these isolates. The TV isolate with the highest mortality with 41.7%, had the highest level of specific activity of exochitinase, protease, and lipase with 0.148, 0.654, and 0.190 U. mg^?1 protein, respectively. In the current study, a positive correlation was determined between the virulence of fungal isolates and the activities of protease and lipase, but this link was not significant for exochitinase. Our results demonstrated that extracellular enzymes, particularly protease and lipase, may play a crucial role in the virulence of these fungal isolates against P. interpunctella larvae.     

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.     

Pathogenicity of Beauveria bassiana ANU1 to the red imported fire ant,Solenopsis invicta workers in Korea

The red imported fire ant (RIFA), Solenopsis invicta, is one of the globalized invasive pests. This study focused on pathogenicity and virulence of entomopathogenic fungi as one of the biological control agents to RIFA workers under different temperatures. The fungal pathogen, Beauveria bassiana ANU1 was isolated from Korea in 2015 and showed the pathogenicity to RIFA workers. A conidial suspension (1 × 10⁷ conidia/ml) induced a low mortality from day 2 after treatment and reached to 100% mortality at day 7 and day 8 after treatment for major and minor workers, respectively. The median lethal concentrations of B. bassiana ANU1 were calculated as 3.9 × 10³ for major and 4.6 × 10³ for minor workers at day 7 after treatment. Low temperatures decreased a virulence of B. bassiana ANU1 (1 × 10⁷ conidia/ml) to RIFA and showed mortality of 26.6% for major and 20% for minor workers. Based on bioassay results, this study provides one of possibilities of effective and successful strategy for controlling RIFA by entomopathogenic fungi.     

Analysis of Whitefly Transcriptional Responses to Beauveria bassiana Infection Reveals New Insights into Insect-Fungus Interactions

Background

The fungal pathogen, Beauveria bassiana, is an efficient biocontrol agent against a variety of agricultural pests. A thorough understanding of the basic principles of insect-fungus interactions may enable the genetic modification of Beauveria bassiana to enhance its virulence. However, the molecular mechanism of insect response to Beauveria bassiana infection is poorly understood, let alone the identification of fungal virulent factors involved in pathogenesis.

Methodology/Principal Findings

Here, next generation sequencing technology was applied to examine the expression of whitefly (Bemisia tabaci) genes in response to the infection of Beauveria bassiana. Results showed that, compared to control, 654 and 1,681genes were differentially expressed at 48 hours and 72 hours post-infected whiteflies, respectively. Functional and enrichment analyses indicated that the DNA damage stimulus response and drug metabolism were important anti-fungi strategies of the whitefly. Mitogen-activated protein kinase (MAPK) pathway was also likely involved in the whitefly defense responses. Furthermore, the notable suppression of general metabolism and ion transport genes observed in 72 hours post-infected B. tabaci might be manipulated by fungal secreted effectors. By mapping the sequencing tags to B. bassiana genome, we also identified a number of differentially expressed fungal genes between the early and late infection stages. These genes are generally associated with fungal cell wall synthesis and energy metabolism. The expression of fungal cell wall protein genes might play an important role in fungal pathogenesis and the dramatically up-regulated enzymes of carbon metabolism indicate the increasing usage of energy during the fungal infection.

Conclusions/Significance

To our knowledge, this is the first report on the molecular mechanism of fungus-whitefly interactions. Our results provide a road map for future investigations on insect-pathogen interactions and genetically modifying the fungus to enhance its efficiency in whitefly control.     

Microsatellite markers to monitor a commercialized isolate of the entomopathogenic fungus Beauveria bassiana in different environments: Technical validation and first applications

Here, we report on the application of five previously developed microsatellite markers (simple sequence repeats, SSRs) to monitor an isolate of the entomopathogenic fungus Beauveria bassiana (Bals.) Vuill. in different environments. Discriminatory power of these SSR markers was assessed in two commercialized B. bassiana isolates as well as in 16 B. bassiana isolates from a world-wide collection, and three of the five SSR markers were estimated to allow a confident discrimination among the given isolates. Sensitivity thresholds of 0.1 pg DNA were subsequently determined for all SSR markers in case pure genomic fungal B. bassiana DNA was used as a template for PCR assays, but threshold levels varied depending on the environment (soil, plant) of the PCR assay. Furthermore, presence of a commercialized B. bassiana isolate was monitored via these SSR markers in three different types of potting substrates over a period of 14 weeks. With two SSR markers, strain-specific products were detected up to 14 weeks after application of B. bassiana to the substrate. Infectivity of B. bassiana conidia in the respective soil samples was confirmed by the Galleria baiting technique. Together these results indicate that molecular markers like SSRs specific for commercialized strains of entomopathogenic fungi are important tools to monitor a particular fungal strain in complex environmental samples such as bulk soil or plant DNA.     

Transcriptomic analysis of entomopathogenic fungus Beauveria bassiana infected by a hypervirulent polymycovirus BbPmV-4

Polymycoviridae is a recently established family of mycoviruses. Beauveria bassiana polymycovirus 4 (BbPmV-4) was previously reported. However, the effect of the virus on host fungus B. bassiana was not clarified. Here, a comparison between virus-free and virus-infected isogenic lines of B. bassiana revealed that BbPmV-4 infection of B. bassiana changes morphology and could lead to decreases in conidiation and increases in virulence against Ostrinia furnacalis larvae. The differential expression of genes between virus-free and virus-infected strains was compared by RNA-Seq and was consistent with the phenotype of B. bassiana. The enhanced pathogenicity may be related to the significant up-regulation of genes encoding mitogen activated protein kinase, cytochrome P450, and polyketide synthase. The results enable studies of the mechanism of interaction between BbPmV-4 and B. bassiana.     

Laboratory and semi-field evaluation of Beauveria bassiana (Ascomycota: Hypocreales) against the lettuce aphid,Nasonovia ribisnigri (Hemiptera: Aphididae)

The lettuce aphid, Nasonovia ribisnigri (Mosley), is an economically important pest of lettuce worldwide. The entomopathogenic fungus Beauveria bassiana strain GHA has recently been reported as a potential biocontrol candidate for use against the lettuce aphid. This study provides information on the mortality inflicted by B. bassiana when applied against different life stages of the lettuce aphid under laboratory conditions and how fungus infection affects the aphid fecundity. In addition, temporal changes in persistence of fungus inoculum applied to foliage of young lettuce plants under semi-field conditions was analysed. Immature life stages were generally the least susceptible to fungal infection and the susceptibility of all stages was dose-dependent, with the highest mortality occurring at the highest dose. B. bassiana significantly affected the rate of nymph production by the lettuce aphid, with the highest effect seen when the alatoid fourth instar of N. ribisnigri was inoculated with B. bassiana. The persistence of B. bassiana conidia on lettuce foliage was not influenced by leaf position. Within 5 days, the cumulative percentage decline in the conidial population was 38% which declined further to 92% and 99% on day 11 and 20 post-spraying, respectively. In accordance, the infectivity to second instar lettuce aphid nymphs of B. bassiana conidia deposited on leaves declined according to an exponential decay model predicting an intercept of 0.59 ± 0.03 (S.E), a reduction in aphid mortality at a rate of 11% with each increasing day after fungal application and a fungus half-life of 6.34 ± 0.69 days.     

Interaction between entomopathogenic nematodes and entomopathogenic fungi applied to third instar southern masked chafer white grubs,Cyclocephala lurida (Coleoptera: Scarabaeidae), under laboratory and greenhouse conditions

Four entomopathogenic nematode (EPN) species (Heterorhabditis bacteriophora Poinar, Heterorhabditis megidis Poinar, Jackson & Klein, Steinernema feltiae Filipjev and Steinernema riobrave Cabanillas, Poinar & Raulston) were tested for virulence against 3^rd instar southern masked chafer white grubs, Cyclocephala lurida Bland. H. bacteriophora and H. megidis, being the most virulent, were selected to evaluate the interaction with an entomopathogenic fungus (EPF), Beauveria bassiana (Balsamo) Vuillemin strain GHA or Metarhizium anisopliae (Metsch.) Sorokin strain F-52, under laboratory and greenhouse conditions. Nematodes and fungi were either applied alone or in combination, with nematodes added to fungi at different times. When applied alone, B. bassiana and M. anisopliae did not reduce grub numbers. Under laboratory conditions, additive interactions were found between H. megidis and B. bassiana, and between H. bacteriophora and B. bassiana or M. anisopliae in most combinations against chafer grubs; a few treatments showed synergism or antagonism. The combined effect did not differ significantly for nematode and fungal applications made simultaneously or at different times. Nematode infection and infective juveniles (IJs) production in grub carcasses were not significantly affected by the presence of a fungus. Efficacies of H. bacteriophora and M. anisopliae were affected by temperature, with grub mortality increasing at higher temperatures. Under greenhouse conditions, additive or synergistic interaction was found between H. bacteriophora and B. bassiana or M. anisopliae in different formulations in simultaneous applications or when the nematode was applied 4 weeks after the fungi, except between B. bassiana ES and H. bacteriophora. The impact of H. bacteriophora alone or in combination with M. anisopliae or B. bassiana on 3^rd instar C. lurida was comparable to that of an imidacloprid insecticide used as curative applications. More virulent fungal strains or species may be required to achieve a stronger interaction with nematodes in the management of C. lurida.     

MoGrr1, a novel F-box protein,is involved in conidiogenesis and cell wall integrity and is critical for the full virulence of <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

The production of asexual spores plays a critical role in rice blast disease. However, the mechanisms of the genes involved in the conidiogenesis pathway are not well understood. F-box proteins are specific adaptors to E3 ubiquitin ligases that determine the fate of different substrates in ubiquitin-mediated protein degradation and play diverse roles in fungal growth regulation. Here, we identify a Saccharomyces cerevisiae Grr1 homolog, MoGrr1, in Magnaporthe oryzae. Targeted disruption of Mogrr1 resulted in defects in vegetative growth, melanin pigmentation, conidial production, and resistance to oxidative stress, and these mutants consequently exhibited attenuated virulence to host plants. Microscopy studies revealed that the inability to form conidiophores is responsible for the defect in conidiation. Although the Mogrr1 mutants could develop melanized appressoria from hyphal tips, the appressoria were unable to penetrate into plant tissues due to insufficient turgor pressure within the appressorium, thereby attenuating the virulence of the mutants. Quantitative RT-PCR results revealed significantly decreased expression of chitin synthase-encoding genes, which are involved in fungal cell wall integrity, in the Mogrr1 mutants. The Mogrr1 mutants also displayed reduced expression of central components of the MAP kinase and cAMP signaling pathways, which are required for appressorium differentiation. Furthermore, domain complementation analysis indicated that two putative protein-interacting domains in MoGrr1 play essential roles during fungal development and pathogenicity. Taken together, our results suggest that MoGrr1 plays essential roles in fungal development and is required for the full virulence of M. oryzae.     

Use of Beauveria bassiana in combination with commercial insecticides to manage Phauda flammans (Walker) (Lepidoptera: Phaudidae): Testing for compatibility and synergy

An entomopathogenic fungal strain, Beauveria bassiana PfBb, was identified from Phauda flammans (Lepidoptera: Phaudidae) larvae. The compatibility and synergy of B. bassiana PfBb employed in combination with three concentrations (i.e., recommended concentration, 20% and 10% of the recommended concentration) of five commercial insecticides were determined. Beta cypermethrin at 10% of recommended concentration had the lowest inhibitory effect on the mycelial growth of B. bassiana PfBb compared with other insecticides. Insecticides utilized at recommended concentration had no significant effect on the sporulation of B. bassiana PfBb, while the extent of their effect at 20% and 10% of recommended concentration differed among insecticides. Insecticides at 10% of recommended concentration had the lowest inhibition of sporulation and conidial germination compared with other concentrations. The conidial germination of B. bassiana PfBb was the highest after treatment with beta cypermethrin at 10% of recommended concentration. The cumulative mortality for 1 × 10⁷ spores/mL B. bassiana PfBb combined with each insecticide at 10% of recommended concentration was higher than that observed with the application of insecticides alone. The percent cadavers of Phauda flammans larvae observed after treatment with B. bassiana PfBb combined with beta cypermethrin at 10% of recommended concentration were not significantly different from those observed after infection with B. bassiana PfBb alone. Our findings demonstrate that B. bassiana PfBb combined with beta cypermethrin at 10% of recommended concentration could increase the efficiency of this insecticide.     

A new manganese superoxide dismutase identified from Beauveria bassiana enhances virulence and stress tolerance when overexpressed in the fungal pathogen

A superoxide dismutase (SOD) was characterized from Beauveria bassiana, a fungal entomopathogen widely applied to insect control. This 209-aa enzyme (BbSod2) showed no more than 71% sequence identity to other fungal Mn-SODs, sharing all conserved residues with the Mn-SOD family and lacking a mitochondrial signal. The SOD activity of purified BbSod2 was significantly elevated by Mn²⁺, suppressed by Cu²⁺ and Zn²⁺ but inhibited by Fe³⁺. Overexpressing the enzyme in a BbSod2-absent B. bassiana strain enhanced its SOD activity (107.2 ± 6.1 U mg⁻¹ protein) by 4–10-fold in different transformants analyzed. The best BbSod2-transformed strain with the SOD activity of 1,157.9 ± 74.7 U mg⁻¹ was 93% and 61% more tolerant to superoxide-generating menadione in both colony growth (EC₅₀ = 2.41 ± 0.03 versus 1.25 ± 0.01 mM) and conidial germination (EC₅₀ = 0.89 ± 0.06 versus 0.55 ± 0.07 mM), and 23% more tolerant to UV-B irradiation (LD₅₀ = 0.49 ± 0.02 versus 0.39 ± 0.01 J cm⁻²). Its virulence to Spodoptera litura larvae was enhanced by 26% [LT₅₀ = 4.5 (4.2–4.8) versus 5.7 (5.2–6.4) days]. Our study highlights for the first time that the Mn²⁺-cofactored, cytosolic BbSod2 contributes significantly to the virulence and stress tolerance of B. bassiana and reveals possible means to improving field persistence and efficacy of a fungal formulation by manipulating the antioxidant enzymes of a candidate strain.     

The MAP kinase Bbslt2 controls growth, conidiation, cell wall integrity, and virulence in the insect pathogenic fungus Beauveria bassiana

Entomopathogenic fungi, such as Beauveria bassiana, are key environmental pathogens of insects that have been exploited for biological control of insect pests. Mitogen-activated protein (MAP) kinases play crucial roles in regulating fungal development, growth, and pathogenicity, mediating responses to the environment. Bbslt2, encoding for an Slt2 family MAPK, was isolated and characterized from B. bassiana. Gene disruption of Bbslt2 affected growth, caused a significant reduction in conidial production and viability, and increased sensitivity to Congo Red and fungal cell wall degrading enzymes. ΔBbslt2 mutants were altered in cell wall structure and composition, which included temperature dependent chitin accumulation, reductions in conidial and hyphal hydrophobicity, and alterations in cell surface carbohydrate epitopes. The ΔBbslt2 strain also showed hypersensitivity to heat shock and altered trehalose accumulation, which could only be partially attributed to changes in the expression of trehalase (ntl1). Insect bioassays revealed decreased virulence in the ΔBbslt2 strain using both topical and intrahemoceol injection assays. These results indicate that Bbslt2 plays an important role in conidiation, viability, cell wall integrity and virulence in B. bassiana. Our findings are discussed within the context of the two previous MAP kinases characterized from B. bassiana.     

TIL-type protease inhibitors may be used as targeted resistance factors to enhance silkworm defenses against invasive fungi

Entomopathogenic fungi penetrate the insect cuticle using their abundant hydrolases. These hydrolases, which include cuticle-degrading proteases and chitinases, are important virulence factors. Our recent findings suggest that many serine protease inhibitors, especially TIL-type protease inhibitors, are involved in insect resistance to pathogenic microorganisms. To clarify the molecular mechanism underlying this resistance to entomopathogenic fungi and identify novel genes to improve the silkworm antifungal capacity, we conducted an in-depth study of serine protease inhibitors. Here, we cloned and expressed a novel silkworm TIL-type protease inhibitor, BmSPI39. In activity assays, BmSPI39 potently inhibited the virulence protease CDEP-1 of Beauveria bassiana, suggesting that it might suppress the fungal penetration of the silkworm integument by inhibiting the cuticle-degrading proteases secreted by the fungus. Phenol oxidase activation studies showed that melanization is involved in the insect immune response to fungal invasion, and that fungus-induced excessive melanization is suppressed by BmSPI39 by inhibiting the fungal cuticle-degrading proteases. To better understand the mechanism involved in the inhibition of fungal virulence by protease inhibitors, their effects on the germination of B. bassiana conidia was examined. BmSPI38 and BmSPI39 significantly inhibited the germination of B. bassiana conidia. Survival assays showed that BmSPI38 and BmSPI39 markedly improved the survival rates of silkworms, and can therefore be used as targeted resistance proteins in the silkworm. These results provided new insight into the molecular mechanisms whereby insect protease inhibitors confer resistance against entomopathogenic fungi, suggesting their potential application in medicinal or agricultural fields.     

Treatment of millet crop plant (Sorghum bicolor) with the entomopathogenic fungus (Beauveria bassiana) to combat infestation by the stem borer,Chilo partellus Swinhoe (Lepidoptera: Pyralidae)

Experiments were done to test if Beauveria bassiana can become an endophyte in sorghum and confer protection from stem borer. Four-week-old sorghum seedlings were treated with B. bassiana. The plants were examined for endophytic presence of B. bassiana, 30 and 60 days after treatment. Stem cultures from treated plants showed growth of B. bassiana. PCR amplification using fungal specific primers for a conserved region of β tubulin gene yielded identical 360 bp products from both B. bassiana and treated sorghum plants. In a subsequent experiment, B. bassiana treated and untreated (control) sorghum plants were artificially infested with stem borer (Chilo partellus) larvae 15 days post treatment and the extent of damage was compared. About 40% of the control plants developed dead heart while no plant in the B. bassiana treated plot did. In the surviving control plants, stem tunneling by shoot borer was significantly higher compared to B. bassiana treated sorghum plants.