The ASH1-PEX16 regulatory pathway controls peroxisome biogenesis for appressorium-mediated insect infection by a fungal pathogen.

Entomopathogenic fungi infect insects by penetrating through the cuticle into the host body. To breach the host cuticle, some fungal pathogens produce specialized infection cells called appressoria, which develop enormous turgor pressure to allow cuticle penetration. However, regulatory mechanisms underlying appressorium turgor generation are poorly understood. Here, we show that the histone lysine methyltransferase ASH1 in the insecticidal fungus Metarhizium robertsii, which is strongly induced during infection of the mosquito cuticle, regulates appressorium turgor generation and cuticle penetration by activating the peroxin gene Mrpex16 via H3K36 dimethylation. MrPEX16 is required for the biogenesis of peroxisomes that participate in lipid catabolism and further promotes the hydrolysis of triacylglycerols stored in lipid droplets to produce glycerol for turgor generation, facilitating appressorium-mediated insect infection. Together, the ASH1-PEX16 pathway plays a pivotal role in regulating peroxisome biogenesis to promote lipolysis for appressorium turgor generation, providing insights into the molecular mechanisms underlying fungal pathogenesis.

Utilization of plant-derived sugars and lipids are coupled during colonization of rhizoplane and rhizosphere by the fungus Metarhizium robertsii.

Plant-derived sugars and lipids are key nutritional sources for plant associated fungi. However, the relationship between utilization of host-derived sugars and lipids during development of the symbiotic association remains unknown. Here we show that the fungus Metarhizium robertsii also needs plant-derived lipids to develop symbiotic relationship with plants. The fatty acid binding proteins FABP1 and FABP2 are important for utilization of plant-derived lipids as the deletion of Fabp1 and Fabp2 significantly reduced the ability of M. robertsii to colonize rhizoplane and rhizosphere of maize and Arabidopsis thaliana. Deleting Fabp1 and Fabp2 increased sugar utilization by upregulating six sugar transporters, and this explains why deleting the monosaccharide transporter gene Mst1, which plays an important role in utilization of plant-derived sugars, had no impact on the ability of the double-gene deletion mutant ΔFabp1::ΔFabp2 to colonize plant roots. FABP1 and FABP2 were also found in other plant-associated Metarhizium species, and they were highly expressed in the medium using the tomato root exudate as the sole carbon and nitrogen source, suggesting that they could be also important for these species to develop symbiotic relationship with plants. In conclusion, we discovered that utilization of plant-derived sugars and lipids are coupled during colonization of rhizoplane and rhizosphere by M. robertsii.

The composition and function of bacterial communities in Bombyx mori (Lepidoptera: Bombycidae) changed dramatically with infected fungi: A new potential to culture Cordyceps cicadae.

Cordyceps cicadae (Hypocreales: Cordycipitaceae) is a renowned entomopathogenic fungus used as herbal medicine in China. However, wild C. cicadae resources have been threatened by heavy harvesting. We hypothesised that Bombyx mori L. (Lepidoptera: Bombycidae) could be a new alternative to cultivate C. cicadae due to the low cost of rearing. Bacterial communities are crucial for the formation of Cordyceps and for promoting the production of metabolites. To better understand the bacterial community structure associated with Cordyceps, three Claviciptaceae fungi were used to explore the pathogenicity of the silkworms. Here, fifth-instar silkworms were infected with C. cicadae, Cordyceps cateniannulata (Hypocreales: Cordycipitaceae) and Beauveria bassiana (Hypocreales: Cordycipitaceae). Subsequently, we applied high-throughput sequencing to explore the composition of bacterial communities in silkworms. Our results showed that all three fungi were highly pathogenic to silkworms, which suggests that silkworms have the potential to cultivate Cordyceps. After fungal infection, the diversity of bacterial communities in silkworms decreased significantly, and the abundance of Staphylococcus increased in mummified larvae, which may play a role in the death process when the host suffers infection by entomopathogenic fungi. Furthermore, there were high similarities in the bacterial community composition and function in the C. cicadae and C. cateniannulata infected samples, and the phylogenetic analysis suggested that these similarities may be related to the fungal phylogenetic relationship. Our findings reveal that infection with different entomopathogenic fungi affects the composition and function of bacterial communities in silkworms and that the bacterial species associated with Cordyceps are primarily host dependent, while fungal infection affects bacterial abundance.

Metabolomic profiling of virulent and non-virulent Beauveria bassiana strains: insights into the pathogenicity of Tetranychus truncatus.

In this study, the pathogenicity of local Beauveria bassiana strains was elucidated using molecular and metabolomics methodologies. Molecular verification of the B. bassiana-specific chitinase gene was achieved via phylogenetic analysis of the Bbchit1 region. Subsequent metabolomic analyses employing UPLC-Q-TOF-MS revealed a different number of non-volatile metabolite profiles among the six B. bassiana strains. Bb6 produced the most non-volatile compounds (17) out of a total of 18, followed by Bb15 (16) and Bb12 (15). Similarly, Bb5, Bb8, and Bb21, three non-virulent B. bassiana strains, produced 13, 14, and 14 metabolites, respectively. But unique secondary metabolites like bassianolide and beauvericin, pivotal for virulence and mite management, were exclusively found in the virulent strains (Bb6, Bb12, and Bb15) of B. bassiana. The distinctive non-volatile metabolomic profiles of these strains underscore their pathogenicity against Tetranychus truncatus, suggesting their promise in bio-control applications.

The homeobox transcription factor MrHOX7 contributes to stress tolerance and virulence in the entomopathogenic fungus Metarhizium robertsii.

Entomopathogenic fungi, including Metarhizium species, represent promising environmentally friendly biopesticides. Understanding the molecular mechanisms governing their infection processes is vital for enhancing their effectiveness. Transcription factors (TFs) play critical roles in gene regulation, yet the functions of many TFs in M. robertsii remain unknown. Homeobox transcription factors, implicated in diverse cellular processes, have received limited attention in entomopathogenic fungi. Here, we identify and characterize, a homeobox TF, MrHOX7, in the model entomopathogenic fungus M. robertsii. Subcellular localization and transcriptional profiling revealed MrHOX7's nuclear localization and high expression during conidia and appressoria formation. Deletion of Mrhox7 (ΔMrhox7) enhanced conidial tolerance to heat and UV-B stress, accompanying with upregulated stress-related gene expression. Intriguingly, ΔMrhox7 exhibits inhibited virulence exclusively through topical inoculation. Further investigations unveiled reduced conidial adhesion and appressorium formation, with downregulation of the adhesion gene Mad1 and appressorium-related genes, as the underlying causes of the reduced fungal virulence. Our findings illuminate the role of MrHOX7 in stress tolerance and virulence, providing insights into the molecular basis of fungal biopesticides.

Genetic diversity of the entomopathogenic fungus Metarhizium rileyi based on de novo microsatellite markers.

Epizootics of the entomopathogenic fungus Metarhizium rileyi regulate lepidopteran populations in soybean, cotton, and peanut agroecosystems to the point that insecticide applications could be unnecessary. However, the contribution and how different strains operate during the epizootic are unknown. Several unanswered questions remain: 1. How many genotypes of M. rileyi are present during an epizootic? 2. Which genotype is the most common among them? 3. Are the genotypes involved in annual epizootics at the same location the same? Therefore, the development of molecular markers to accurately identify these genotypes is very important to answer these questions. SSR primers were designed by prospecting in silico to discriminate genotypes and infer the genetic diversity of M. rileyi isolates from the collection kept at Embrapa Soybean. We tested 13 SSR markers on 136 isolates to identify 43 clones and 12 different genetic clusters, with genetic diversity ranging from Hs = 0.15 (cluster I) to Hs = 0.41 (cluster IV) and an average diversity of 0.24. No clusters were categorically distinguished based on hosts or geographical origin using Bayesian clustering analysis. Nonetheless, some clusters comprised most of the isolates with a common geographic origin; for example, cluster VIII was mainly composed of isolates from Central-western Brazil, cluster II from Southern Brazil, and cluster XII from Quincy, Northern Florida, in the United States. Underrepresented regions (few isolates) from Pacific Island nations of Japan, the Philippines, and Indonesia (specifically from Java) were placed into clusters IX and X. Although the analyzed isolates displayed evidence of clonal structure, the genetic diversity indices suggest a potential for the species to adapt to different environmental conditions.

Thermal ecology shapes disease outcomes of entomopathogenic fungi infecting warm-adapted insects.

The thermal environment is a critical determinant of outcomes in host-pathogen interactions, yet the complexities of this relationship remain underexplored in many ecological systems. We examined the Thermal Mismatch Hypothesis (TMH) by measuring phenotypic variation in individual thermal performance profiles using a model system of two species of entomopathogenic fungi (EPF) that differ in their ecological niche, Metarhizium brunneum and M. flavoviride, and a warm-adapted model host, the mealworm Tenebrio molitor. We conducted experiments across ecologically relevant temperatures to determine the thermal performance curves for growth and virulence, measured as % survival, identify critical thresholds for these measures, and elucidate interactive host-pathogen effects. Both EPF species and the host exhibited a shared growth optima at 28 °C, while the host's growth response was moderated in sublethal pathogen infections that depended on fungus identity and temperature. However, variances in virulence patterns were different between pathogens. The fungus M. brunneum exhibited a broader optimal temperature range (23-28 °C) for virulence than M. flavoviride, which displayed a multiphasic virulence-temperature relationship with distinct peaks at 18 and 28 °C. Contrary to predictions of the TMH, both EPF displayed peak virulence at the host's optimal temperature (28 °C). The thermal profile for M. brunneum aligned more closely with that of T. molitor than that for M. flavoviride. Moreover, the individual thermal profile of M. flavoviride closely paralleled its virulence thermal profile, whereas the virulence thermal profile of M. brunneum did not track with its individual thermal performance. This suggests an indirect, midrange (23 °C) effect, where M. brunneum virulence exceeded growth. These findings suggest that the evolutionary histories and ecological adaptations of these EPF species have produced distinct thermal niches during the host interaction. This study contributes to our understanding of thermal ecology in host-pathogen interactions, underpinning the ecological and evolutionary factors that shape infection outcomes in entomopathogenic fungi. The study has ecological implications for insect population dynamics in the face of a changing climate, as well as practically for the use of these organisms in biological control.

Metarhizium pingshaense photolyase expression and virulence to Rhipicephalus microplus after UV-B exposure.

The current study examined the impact of ultraviolet (UV)-B radiation in Metarhizium pingshaense blastospores' photolyase expression and their virulence against Rhipicephalus microplus. Blastospores were exposed to UV under laboratory and field conditions. Ticks were treated topically with fungal suspension and exposed to UV-B in the laboratory for three consecutive days. The expression of cyclobutane pyrimidine dimmers (CPDs)-photolyase gene maphr1-2 in blastospores after UV exposure followed by white light exposure was accessed after 0, 8, 12, 24, 36, and 48 h. Average relative germination of blastospores 24 h after in vitro UV exposure was 8.4% lower than 48 h. Despite this, the relative germination of blastospores exposed to UV in the field 18 h (95.7 ± 0.3%) and 28 h (97.3 ± 0.8%) after exposure were not different (p > 0.05). Ticks treated with fungus and not exposed to UV exhibited 0% survival 10 days after the treatment, while fungus-treated ticks exposed to UV exhibited 50 ± 11.2% survival. Expression levels of maphr1-2 8, 12, and 24 h after UV-B exposure were not different from time zero. Maphr1-2 expression peak in M. pingshaense blastospores occurred 36 h after UV-B exposure, in the proposed conditions and times analyzed, suggesting repair mechanisms other than CPD-mediated-photoreactivation might be leading blastospores' germination from 0 to 24 h.

Novel report on soil infection with Metarhizium rileyi against soil-dwelling life stages of insect pests.

Entomopathogenic fungi are the most effective control remedy against a wide range of medical and agricultural important pests. The present study aimed to isolate, identify, and assess the virulence of Metarhizium rileyi against Spodoptera litura and Spodoptera frugiperda pupae under soil conditions. The biotechnological methods were used to identify the isolate as M. rileyi. The fungal conidial pathogenicity (2.0 × 107, 2.0 × 108, 2.0 × 109, 2.0 × 1010, and 2.0 × 1011 conidia/mL-1) was tested against prepupae of S. litura and S. frugiperda at 3, 6, 9, and 12 days after treatments. Additionally, the artificial soil-conidial assay was performed on a nontarget species earthworm Eudrilus eugeniae, using M. rileyi conidia. The present results showed that the M. rileyi caused significant mortality rates in S. litura pupae (61-90%), and S. litura pupae were more susceptible than S. frugiperda pupae (46%-73%) at 12 day posttreatment. The LC50 and LC90 of M. rileyi against S. litura, were 3.4 × 1014-9.9 × 1017 conidia/mL-1 and 6.6 × 105-4.6 × 1014 conidia/mL-1 in S. frugiperda, respectively. The conidia of M. rileyi did not exhibit any sublethal effect on the adult stage of E. eugeniae, and Artemia salina following a 12-day treatment period. Moreover, in the histopathological evaluation no discernible harm was observed in the gut tissues of E. eugeniae, including the lumen and epithelial cells, as well as the muscles, setae, nucleus, mitochondria, and coelom. The present findings provide clear evidence that M. rileyi fungal conidia can be used as the foundation for the development of effective bio-insecticides to combat the pupae of S. litura and S. frugiperda agricultural pests.

Pathogenicity of Metarhizium rileyi (Hypocreales: Clavicipitaceae) against Tenebrio molitor (Coleoptera: Tenebrionidae).

Tenebrio molitor L., also known as the mealworm, is a polyphagous insect pest that infests various stored grains worldwide. Both the adult and larval stages can cause significant damage to stored grains. The present study focused on isolating entomopathogenic fungi from an infected larval cadaver under environmental conditions. Fungal pathogenicity was tested on T. molitor larvae and pupae for 12 days. Entomopathogenic fungi were identified using biotechnological methods based on their morphology and the sequence of their nuclear ribosomal internal transcribed spacer (ITS). The results of the insecticidal activity indicate that the virulence of fungi varies between the larval and pupal stages. In comparison to the larval stage, the pupal stage is highly susceptible to Metarhizium rileyi, exhibiting 100% mortality rates after 12 days (lethal concentration 50 [LC50] = 7.8 × 106 and lethal concentration 90 (LC90) = 2.1 × 1013 conidia/mL), whereas larvae showed 92% mortality rates at 12 days posttreatment (LC50 = 1.0 × 106 and LC90 = 3.0 × 109 conidia/mL). The enzymatic analyses revealed a significant increase in the levels of the insect enzymes superoxide dismutase (4.76-10.5 mg-1) and glutathione S-transferase (0.46-6.53 mg-1) 3 days after exposure to M. rileyi conidia (1.5 × 105 conidia/mL) compared to the control group. The findings clearly show that M. rileyi is an environmentally friendly and effective microbial agent for controlling the larvae and pupae of T. molitor.

Hypothetical protein MAA_07646 is required for stress resistance and pathogenicity in Metarhizium robertsii.

Metarhizium robertsii, a vital entomopathogenic fungus for pest management, relies on various virulence-related proteins for infection. Identifying these proteins, especially those with unknown functions, can illuminate the fungus's virulence mechanisms. Through RNA-seq, we discovered that the hypothetical protein MAA_07646 was significantly upregulated during appressorium formation in M. robertsii. In this study, we characterized MAA_07646, finding its presence in both the nucleus and cytoplasm. Surprisingly, it did not affect vegetative growth, conidiation, or chemical tolerance. However, it played a role in heat and UV radiation sensitivity. Notably, ΔMAA_07646 exhibited reduced virulence in Galleria mellonella larvae due to impaired appressorium formation and decreased expression of virulence-related genes. In conclusion, MAA_07646 contributes to thermotolerance, UV resistance, and virulence in M. robertsii. Understanding its function sheds light on the insecticidal potential of M. robertsii's hypothetical proteins.

Isolation and characterization of a native strain of the entomopathogenic fungus Beauveria bassiana for the control of the palm weevil Dynamis borassi (Coleoptera: Curculionidae) in the neotropics.

This study aimed to isolate and characterize a native strain of Beauveria bassiana, coded as Bv065, showcasing its potential as a biological control agent targeting the palm weevil Dynamis borassi. Originating from a naturally infected D. borassi specimen collected in southwestern Colombia, the fungus underwent molecular identification and was identified as B. bassiana, exhibiting high sequence similarity with known reference strains. The physiological characterization revealed that Bv065 thrived within a temperature range of 25 to 30 °C and a pH range of 6 to 9. Moreover, the key carbon sources that allow optimal growth of the strain were identified through metabolic profiling, including sucrose, D-mannose, and γ-amino-butyric acid. These findings offer strategic insights for scalability and formulation methodologies. Additionally, enzymatic analyses unveiled robust protease activity within Bv065, crucial for catalysing insect cuticle degradation and facilitating host penetration, thus accentuating its entomopathogenic potential. Subsequent evaluations exposed Bv065's pathogenicity against D. borassi, causing significant mortality within nine days of exposure, albeit exhibiting limited effectiveness against Rhynchophorus palmarum. This study underscores the importance of understanding optimal growth conditions and metabolic preferences of B. bassiana strains for developing effective biopesticides. The findings suggest Bv065 as a promising candidate for integrated pest management strategies in neotropical regions, particularly for controlling palm weevil infestations in coconut and peach palm cultivation. Future research avenues include refining mass production methodologies, formulating novel delivery systems, and conducting comprehensive field efficacy trials to unlock the full potential of Bv065 in fostering sustainable pest management practices. Overall, this study contributes to the growing body of knowledge on entomopathogenic fungi and their pivotal role in biological control, offering nuanced perspectives on eco-friendly alternatives to conventional insecticidal interventions.

Comparative transcriptome analysis to unveil genes affecting the host cuticle destruction in Metarhizium rileyi.

Insect pathogenic fungi, also known as entomopathogenic fungi, are one of the largest insect pathogenic microorganism communities, represented by Beauveria spp. and Metarhizium spp. Entomopathogenic fungi have been proved to be a great substitute for chemical pesticide in agriculture. In fact, a lot of functional genes were also already characterized in entomopathogenic fungi, but more depth of exploration is still needed to reveal their complicated pathogenic mechanism to insects. Metarhizium rileyi (Nomuraea rileyi) is a great potential biocontrol fungus that can parasitize more than 40 distinct species (mainly Lepidoptera: Noctuidae) to cause large-scale infectious diseases within insect population. In this study, a comparative analysis of transcriptome profile was performed with topical inoculation and hemolymph injection to character the infectious pattern of M. rileyi. Appressorium and multiple hydrolases are indispensable constituents to break the insect host primary cuticle defense in entomopathogenic fungi. Within our transcriptome data, numerous transcripts related to destruction of insect cuticle rather growth regulations were obtained. Most importantly, some unreported ribosomal protein genes and novel unannotated protein (hypothetical protein) genes were proved to participate in the course of pathogenic regulation. Our current data provide a higher efficiency gene library for virulence factors screen in M. rileyi, and this library may be also useful for furnishing valuable information on entomopathogenic fungal pathogenic mechanisms to host.

Comparative transcriptomics of growth metabolism and virulence reveal distinct morphogenic profiles of yeast-like cells and hyphae of the fungus Metarhizium rileyi.

Metarhizium rileyiis an entomopathogenic fungus with a narrow host range which distinguishes it from other Metarhiziumspecies with broad host ranges. This species is also unique because the initial yeast-like growth on solid media is only observed in liquid culture in other Metharizium species. A lack of knowledge about the metabolism and genetic signatures of M. rileyiduring this yeast-like phase on solid and in liquid media is a bottleneck for its large-scale production as a commercial biocontrol agent.In this study wefound that M. rileyiyeast-like cells produced on solid medium infected and killed the important insect pest Spodoptera frugiperda with comparable efficiency as yeast-like cells grown in liquid medium. Secondly, we used comparative transcriptomic analysis to investigate theactive genes and genomic signatures of the M. rileyi yeast-like morphotypes produced on solid and in liquid media. Yeast-like cells grown in liquid medium had upregulated genes relating specifically to signal transduction andparticular membrane transporters. Thirdly, we compared the transcriptomic profiles of yeast-like phases of M. rileyi with those of M. anisopliae. The yeast-like phase of M. rileyi grown on solid medium upregulated unique genes not found in otherMetarhiziumspecies including specific membrane proteins and several virulence factors. Orthologous genes associated with heat shock protein, iron permease, membrane proteins and key virulence traits (e.g. collagen-like protein Mcl1) were upregulated in both species. Comparative transcriptome analyses of gene expression showed more differences than similarities between M. anisopliae and M. rileyi yeast-like cells.

Expressing Parasitoid Venom Protein VRF1 in an Entomopathogen Beauveria bassiana Enhances Virulence toward Cotton Bollworm Helicoverpa armigera.

Despite entomopathogenic fungi being used in various insect pest control, it is recognized that they could replace more chemical insecticides if they were more efficient. We have found that cotton bollworm Helicoverpa armigera responded to the infection of entomopathogenic fungus Beauveria bassiana by activating the Toll pathway. Koinobiont wasps also regulate host immunity and development to ensure the survival of their progeny. Previously, venom protein VRF1 was identified in Microplitis mediator. It enters H. armigera hemocytes, suppresses the expression of antimicrobial peptides (AMPs) by inhibiting the Toll pathway, and prevents parasite offspring from being encapsulated. With this in mind, we thought that it might be feasible to increase the virulence of B. bassiana by embedding VRF1 into its genome. Compared with that of wild-type (WT) B. bassiana, the median lethal dose (LD50) of the transformant expressing VRF1 (named BbVRF1) decreased approximately 2.36-fold, and the median time to lethality (LT50) was shortened to 84% when infecting H. armigera (a natural host of M. mediator). The AMP expression level of hemocytes in H. armigera infected with BbVRF1 strain was significantly downregulated compared to that in the control group infected with the WT. In addition, the LD50 of BbVRF1 against the fall armyworm Spodoptera frugiperda (an unnatural host of M. mediator) was decreased 3.45-fold and the LT50 was shortened to 73%, showing a greater virulence. Our research indicated that BbVRF1, an engineered strain of B. bassiana, has greater efficacy against pest insects both within and outside its host range (M. mediator), expanding the utilization of parasitoid wasp virulence effectors. IMPORTANCE Mycoinsecticides are essential for the development of integrated pest management as substitutes to chemical insecticides, but their usage is limited by their inferior virulence. Thus, genetically engineered bioinsecticides, including recombinant entomopathogenic fungi, have been regarded as a breakthrough to rapidly control pests. Deep knowledge of parasitoid wasps allows us to take advantage of this natural enemy of pest insects beyond raising them for field release. Our transformant BbVRF1 (Beauveria bassiana integrated with a venom protein VRF1 from Microplitis mediator) showed a higher virulence in H. armigera and S. frugiperda, demonstrating its potential for managing natural or unnatural hosts of M. mediator. This result provides a new strategy regarding which venom protein of parasitoid wasps can become part of the arsenal with which to equip entomopathogenic fungi. Utilizing parasitoid wasps with this approach could easily overcome the difficulties of artificial culture and enhance the virulence of other biocontrol agents.

Phenotypic variation and genomic variation in insect virulence traits reveal patterns of intraspecific diversity in a locust-specific fungal pathogen.

Intraspecific pathogen diversity is crucial for understanding the evolution and maintenance of adaptation in host-pathogen interactions. Traits associated with virulence are often a significant source of variation directly impacted by local selection pressures. The specialist fungal entomopathogen, Metarhizium acridum, has been widely implemented as a biological control agent of locust pests in tropical regions of the world. However, few studies have accounted for natural intraspecific phenotypic and genetic variation. Here, we examine the diversity of nine isolates of M. acridum spanning the known geographic distribution, in terms of (1) virulence towards two locust species, (2) growth rates on three diverse nutrient sources, and (3) comparative genomics to uncover genomic variability. Significant variability in patterns of virulence and growth was shown among the isolates, suggesting intraspecific ecological specialization. Different patterns of virulence were shown between the two locust species, indicative of potential host preference. Additionally, a high level of diversity among M. acridum isolates was observed, revealing increased variation in subtilisin-like proteases from the Pr1 family. These results culminate in the first in-depth analysis regarding multiple facets of natural variation in M. acridum, offering opportunities to understand critical evolutionary drivers of intraspecific diversity in pathogens.

Virulence of Beauveria sp. and Metarhizium sp. fungi towards fall armyworm (Spodoptera frugiperda).

The development of effective pest management strategies for Spodoptera frugiperda is a high priority for crop protection across its invasive ranges. Here, we examined six Beauveria and five Metarhizium fungal isolates against this pest. Two Beauveria isolates (B-0571, B-1311) induced high mortality toward 3rd and 6th instar caterpillars and adults. For B-0571 mortality was 82.81 ± 5.75%, 61.46 ± 6.83%, and 93.75 ± 3.61%, and 73.72 ± 2.51%, 71.88 ± 5.41%, and 97.92 ± 2.08% for B-1311, with deaths in caterpillars largely occurring under 24 h (3rd instar control 0.74 ± 0.33%, B-0571 73.96 ± 7.85% and B-1311 62.08 ± 3.67%; 6th instar control 0%, B-0571 66.67% ± 11.02% and B-1311 62.5% ± 9.55%). Infection from both Beauveria isolates fully prevented reproduction in surviving S. frugiperda females. In contrast, all five Metarhizium isolates tested and the remaining four Beauveria isolates exhibited lower virulence. The discovery of two highly virulent Beauveria fungal isolates to S. frugiperda opens avenues to develop novel biological control tools against this highly invasive pest.

Beauveria bassiana submerged spores for control of two-spotted spider mite (Tetranychus urticae Koch (Acari: Tetranychidae)): production, stability, and virulence.

In this study, IBCB 66, IBCB 868, and CBMAI 1306 isolates of Beauveria bassiana were grown in liquid culture for 4 days, leading to elevated submerged spores (SS) levels. The influence of the addition of different glycerol concentrations (0, 3, and 6%) (v/v) in the liquid culture was investigated regarding the stability (at 4 and 27 °C) of dried formulations. The virulence of SS was compared with aerial spores (AS) against Tetranychus urticae (Koch) (Acari: Tetranychidae). The results demonstrate the potential of using SS to control T. urticae. CBMAI 1306 and IBCB 868 isolates caused T. urticae mortality rates of 91.11% and 88.89% 5 days after treatment, respectively, when applied at concentrations of 1 × 108 SS mL-1. The median Lethal Time (LT50) values for these strains were 2.64 and 2.61 days, respectively. The dried formulations showed potential acaricidal activity. Higher glycerol concentrations in the liquid culture medium reduced formulation stability at 27 °C.

Fungal Community Associated with the Leaf-Cutting Ant Acromyrmex crassispinus (Hymenoptera: Formicidae) Colonies: a Search for Potential Biocontrol Agents.

The leaf-cutting ant Acromyrmex crassispinus is considered an important pest in forest plantations in southern Brazil. This work aimed to study the fungal community associated with A. crassispinus colonies, subjected to treatments with subdoses of granulated baits (sulfluramid), which might reduce the ability of the ants to care for their symbiotic fungus and other fungi (maybe biocontrol fungi) would take over, to prospect for potential biological control agents. Samplings of fungus gardens and dead ants allowed the identification of 195 fungal isolates, distributed in 29 families, 36 genera, and 53 species. The most frequent genera were Trichoderma (49.2%), Penicillium (13.8%), Chaetomium (6.2%), and Fusarium (3.6%). This is the first study that conducted a survey of antagonistic and entomopathogenic fungi to A. crassispinus and its symbiotic fungus, reporting for the first time the occurrence of potential biological control agents. Escovopsis weberi, Fusarium oxysporum, Rhizomucor variabilis, Trichoderma atroviride, Trichoderma harzianum, Trichoderma koningiopsis, and Trichoderma spirale are considered some of the potential biocontrol organisms.

Genomic Determinants of Entomopathogenic Fungi and Their Involvement in Pathogenesis.

Entomopathogenic fungi offer an effective and eco-friendly alternative to curb insect populations in biocontrol strategy. The evolutionary history of selected entomopathogenic fungi indicates their ancestral relationship with plant endophytes. During this host shifting, entomopathogenic fungi must have acquired multiple mechanisms, including a combination of various biomolecules that make them distinguishable from other fungi. In this review, we focus on understanding various biochemical and molecular mechanisms involved in entomopathogenesis. In particular, we attempt to explain the indispensable role of enlarged gene families of various virulent factors, viz. chitinases, proteases, lipases, specialized metabolites, and cytochrome P450, in entomopathogenesis. Our analysis suggests that entomopathogenic fungi recruit a different set of gene products during the progression of pathogenesis. Knowledge of these bio-molecular interactions between fungi and insect hosts will allow researchers to execute pointed efforts towards the development of improved entomopathogenic fungal strains.