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Reactive oxygen species (ROS), synthesized by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) complex, are vital molecules in biological cells, influencing various physiological processes such as fungal growth, development, and virulence. Beauveria bassiana , an entomopathogenic fungus, is a promising biopesticide for agricultural, forestry, and urban pest control. This study focuses on the characterization of NADPH oxidases (Noxs) in B. bassiana . Gene expression profiles of Noxs in B. bassiana (BbNoxs) were analysed using RT-qPCR. Knockout strains of single BbNoxA, BbNoxB, BbNoxR, and double BbNoxA and BbNoxB were constructed via homologous recombination, and their phenotypic characteristics were examined. Fungal virulence was evaluated using Galleria mellonella larvae, and infection structures formation and penetration ability were assessed on cicada wings. ROS production and actin assembly during fungal growth and infection were detected using staining and marker methods. Expression analysis revealed significant upregulation of BbNoxs during fungal growth and infection. Compared to the wild-type strain, single knockouts (ΔBbNoxA/B/R) and double knockout (ΔBbNoxAB) of BbNoxs exhibited reduced conidial yields, accelerated conidial germination rates. Deletion of BbNoxB or BbNoxR decreased fungal virulence compared to the WT strain in topical inoculation experiments. Additionally, loss of BbNoxB or BbNoxR impaired infection structures formation, penetration ability, ROS production, and actin aggregation during fungal infection. BbNoxs are crucial for fungal growth, development, and virulence in B. bassiana , playing essential roles in infection structures formation, penetration, ROS production, and actin assembly. Understanding their functions provides insights into B. bassiana 's pathogenic mechanisms.

Mycoviruses can alter the biological characteristics of host fungi, including change virulence or pathogenicity of phytopathogens and entomopathogenic fungi (EPF). However, most studies on the mycoviruses found in EPF have focused on the effects of the viruses on the virulence of host fungi towards insect pests, with relatively few reports on the effects to the host fungi with regard to plant disease resistance in hosts. The present study investigated the effects of the mycovirus Beauveria bassiana chrysovirus 2 (BbCV2) virus infection on host biological characteristics, evaluated antagonistic activity of BbCV2 against two phytopathogenic fungi ( Sclerotinia sclerotiorum and Botrytis cinerea ), and transcriptome analysis was used to reveal the interactions between viruses and hosts. Our results showed that BbCV2 virus infection increased B. bassiana 's growth rate, spore production, and biomass, it also enhanced the capacity of host fungi and their metabolic products to inhibit phytopathogenic fungi. BbCV2 virus infection reduced the contents of the two pathogens in tomato plants significantly, and transcriptome analysis revealed that the genes related to competition for ecological niches and nutrition, mycoparasitism and secondary metabolites in B. bassiana were significantly up-regulated after viral infection. These findings indicated that the mycovirus infection is an important factor to enhance the ability of B. bassiana against plant disease after endophytic colonization. We suggest that mycovirus infection causes a positive effect on B. bassiana against phytopathogens, which should be considered as a potential strategy to promote the plant disease resistance of EPF.

Rad6 functions as a ubiquitin-conjugating protein that regulates cellular processes in many fungal species. However, its role in filamentous entomopathogenic fungi remains poorly understood. This study characterizes Rad6 in Beauveria bassiana , a filamentous fungus widely employed as a critical fungicide globally. The results demonstrate a significant association between Rad6 and conidial properties, heat shock response, and UV-B tolerance. Concurrently, the mutant strain exhibited heightened sensitivity to oxidative stress, cell wall interfering agents, DNA damage stress, and prolonged heat shock. Furthermore, the absence of Rad6 significantly extended the median lethal time (LT 50 ) of Galleria mellonella infected by B. bassiana . This delay could be attributed to reduced Pr1 proteases and extracellular cuticle-degrading enzymes, diminished dimorphic transition rates, and dysregulated antioxidant enzymes. Additionally, the absence of Rad6 had a more pronounced effect on genetic information processing, metabolism, and cellular processes under normal conditions. However, its impact was limited to metabolism in oxidative stress. This study offers a comprehensive understanding of the pivotal roles of Rad6 in conidial and hyphal stress tolerance, environmental adaptation, and the pathogenesis of Beauveria bassiana .

The Eaf6 protein, a conserved component of the NuA4 and NuA3 complexes in yeast and MOZ/MORF complexes in humans, plays crucial roles in transcriptional activation, gene regulation, and cell cycle control. Despite its significance in other organisms, the functional role of Eaf6 in entomopathogenic fungi (EPF) remained unexplored. Here, we investigate the function of BbEaf6, the Eaf6 homolog in the entomopathogenic fungus Beauveria bassiana . We demonstrate that BbEaf6 is predominantly localized in nuclei, similar to its counterpart in other fungi. Deletion of BbEaf6 resulted in delayed conidiation, reduced conidial yield, and altered conidial properties. Transcriptomic analysis revealed dysregulation of the genes involved in asexual development and cell cycle progression in the Δ BbEaf6 mutant. Furthermore, the Δ BbEaf6 mutant exhibited decreased tolerance to various stresses, including ionic stress, cell wall perturbation, and DNA damage stress. Notably, the Δ BbEaf6 mutant displayed attenuated virulence in insect bioassays, accompanied by dysregulation of genes associated with cuticle penetration and haemocoel infection. Overall, our study elucidates the multifaceted role of BbEaf6 in stress response, development, and virulence in B. bassiana , providing valuable insights into the molecular mechanisms governing fungal pathogenesis and potential targets for pest management strategies.

Beauveria bassiana has potential for Aedes aegypti biological control. However, its efficacy depends on the strain's geographic location, host susceptibility, and virulence. The present study aimed to evaluate the effectiveness of B. bassiana strain BBPTG4 conidia in controlling Ae. aegypti adults and its detection via introns profile on exposed mosquito corpses. Morphologic characteristics among strains were highly similar. Comprehensive testing of these strains demonstrated that BBPT4 exhibited the ideal biological activity for Ae. aegypti control, with a median lethal time (TL 50 ) of 7.5 d compared to ~3 d and ~10 d for BB01 and BB37 strains, respectively. Infected mosquitoes died after GHA and BBPTG4 exposure, and corpses were analyzed for infecting strains detection. Differences among the seven evaluated strains were determined, assessing five different insertion group I intron profiles in BBTG4, BB01, GHA, BB37, and BB02 strains. Mosquitoes infected by BBPTG4 and non-exposed (negative control) intron profiles were obtained. We detected the presence of introns in the BBPTG4 strain, which were not present in non-exposed mosquitoes. In conclusion, B. bassiana strains showed similarities in terms of their cultural and microscopic morphological characteristics and biologicals virulence level, but different intron profiles. BBPTG4 strain-infected Ae. aegypti adult corpses, showing specific amplicons, enabled us to identify B. bassiana at the strain level among infected mosquitoes. However, monitoring and detection of field-infected insects is essential for further verification.

Basic leucine zipper domain transcription factors (TFs), of which yeast activator protein (Yap) is a significant class, are crucial for the development of sclerotia, the stress response, vegetative growth, and spore adhesion. Nevertheless, nothing is known about how Yap TFs contribute to the pathogenicity of entomopathogenic fungus. In this work, Beauveria bassiana was used to identify and knock out the yeast gene BbYap1 , which is similar to Yap. The BbYap1 gene deletion has an impact on lipid homeostasis of B. bassiana ; oleic acid, for example, dropped by 95.69%. The BbYap1 mutant exhibited much less virulence and vegetative development in comparison to the wild strain, while demonstrating a greater sensitivity to chemical stress. It is noteworthy that the physiological abnormalities brought on by BbYap1 deletion were largely repaired by the addition of exogenous oleic acid, as seen by the notable increase in insect survival in the blood cavity injection group. Following infection with the BbYap1 mutant, the host exhibits a considerable down-regulation of the expression of β-1,3-glucan recognition protein, gallerimycin, gloverin, and moricin-like protein genes. Likewise, the introduction of exogenous oleic acid markedly increased the host's expression of the aforementioned genes. In summary, BbYap1 regulates cellular enzyme lipid homeostasis and fungal virulence by eluding host humoral defense, which contributes to fungal chemical stress and vegetative development., Importance: Entomopathogenic fungi (EPF) offer an effective and eco-friendly alternative to curb insect populations in biocontrol strategy. When EPF enter the hemolymph of their host, they encounter a variety of stress reactions, such as immunological and oxidative stress. Basic leucine zipper domain transcription factors, of which yeast activator protein (Yap) is a significant class, have diverse biological functions related to metabolism, development, reproduction, conidiation, stress responses, and pathogenicity. This study demonstrates that BbYap1 of Beauveria bassiana regulates cellular enzyme lipid homeostasis and fungal virulence by eluding host humoral defense, which contributes to fungal chemical stress and vegetative development. These findings offer fresh perspectives for comprehending molecular roles of YAP in EPF., Competing Interests: The authors declare no conflict of interest.

Background: Enhancing crops' drought resilience is necessary to maintain productivity levels. Plants interact synergistically with microorganisms like Beauveria bassiana to improve drought tolerance. Therefore, the current study investigates the effects of biopriming with B. bassiana on drought tolerance in Malva parviflora plants grown under regular irrigation (90% water holding capacity (WHC)), mild (60% WHC), and severe drought stress (30% WHC)., Results: The results showed that drought stress reduced the growth and physiological attributes of M. parviflora. However, those bioprimed with B. bassiana showed higher drought tolerance and enhanced growth, physiological, and biochemical parameters: drought stress enriched malondialdehyde and H 2 O 2 contents. Conversely, exposure to B. bassiana reduced stress markers and significantly increased proline and ascorbic acid content under severe drought stress; it enhanced gibberellic acid and reduced ethylene. Bioprimed M. parviflora, under drought conditions, improved antioxidant enzymatic activity and the plant's nutritional status. Besides, ten Inter-Simple Sequence Repeat primers detected a 25% genetic variation between treatments. Genomic DNA template stability (GTS) decreased slightly and was more noticeable in response to drought stress; however, for drought-stressed plants, biopriming with B. bassiana retained the GTS., Conclusion: Under drought conditions, biopriming with B. bassiana enhanced Malva's growth and nutritional value. This could attenuate photosynthetic alterations, up-regulate secondary metabolites, activate the antioxidant system, and maintain genome integrity., (© 2024. The Author(s).)

The entomopathogenic fungus Beauveria bassiana provides an eco-friendly substitute to chemical insecticides for mosquito control. Nevertheless, its widespread application has been hindered by its comparatively slow efficacy in eliminating mosquitoes. To augment the potency of B. bassiana against Aedes mosquitoes, a novel recombinant strain, Bb -Cyt1Aa, was developed by incorporating the Bacillus thuringiensis toxin gene Cyt1Aa into B. bassiana . The virulence of Bb -Cyt1Aa was evaluated against Aedes aegypti and Aedes albopictus using insect bioassays. Compared to the wild-type (WT) strain, the median lethal time (LT 50 ) for A. aegypti larvae infected with Bb -Cyt1Aa decreased by 33.3% at a concentration of 1 × 10 8 conidia/mL and by 22.2% at 1 × 10 7 conidia/mL. The LT 50 for A. aegypti adults infected with Bb -Cyt1Aa through conidia ingestion was reduced by 37.5% at 1 × 10 8 conidia/mL and by 33.3% at 1 × 10 7 conidia/mL. Likewise, the LT 50 for A. aegypti adults infected with Bb -Cyt1Aa through cuticle contact decreased by 33.3% and 30.8% at the same concentrations, respectively. Furthermore, the Bb -Cyt1Aa strain also demonstrated increased toxicity against both larval and adult A. albopictus , when compared to the WT strain. In conclusion, our study demonstrated that the expression of B. thuringiensis toxin Cyt1Aa in B. bassiana enhanced its virulence against Aedes mosquitoes. This suggests that B. bassiana expressing Cyt1Aa has potential value for use in mosquito control., Importance: Beauveria bassiana is a naturally occurring fungus that can be utilized as a bioinsecticide against mosquitoes. Cyt1Aa is a delta-endotoxin protein produced by Bacillus thuringiensis that exhibits specific and potent insecticidal activity against mosquitoes. In our study, the expression of this toxin Cyt1Aa in B. bassiana enhances the virulence of B. bassiana against Aedes aegypti and Aedes albopictus , thereby increasing their effectiveness in killing mosquitoes. This novel strain can be used alongside chemical insecticides to reduce dependence on harmful chemicals, thereby minimizing negative impacts on the environment and human health. Additionally, the potential resistance of B. bassiana against mosquitoes in the future could be overcome by acquiring novel combinations of exogenous toxin genes. The presence of B. bassiana that expresses Cyt1Aa is of significant importance in mosquito control as it enhances genetic diversity, creates novel virulent strains, and contributes to the development of safer and more sustainable methods of mosquito control., Competing Interests: The authors declare no conflict of interest.

Successful host tissue colonization is crucial for fungal pathogens to cause mycosis and complete the infection cycle, in which fungal cells undergo a series of morphological transition-included cellular events to combat with hosts. However, many transcription factors (TFs) and their mediated networks regulating fungal pathogen colonization of host tissue are not well characterized. Here, a TF (BbHCR1)-mediated regulatory network was identified in an insect pathogenic fungus, Beauveria bassiana , that controlled insect hemocoel colonization. BbHCR1 was highly expressed in fungal cells after reaching insect hemocoel and controlled the yeast ( in vivo blastospores)-to-hyphal morphological switch, evasion of immune defense response, and fungal virulence. Comparative analysis of RNA sequencing and chromatin immunoprecipitation sequencing identified a core set of BbHCR1 target genes during hemocoel colonization, in which abaA and brlA were targeted to limit the rapid switch from blastospores to hyphae and fungal virulence. Two targets encoding hypothetical proteins, HP1 and HP2, were activated and repressed by BbHCR1, respectively, which acted as a virulence factor and repressor, respectively, suggesting that BbHCR1 activated virulence factors but repressed virulence repressors during the colonization of insect hemocoel. BbHCR1 tuned the expression of two dominant hemocoel colonization-involved metabolite biosynthetic gene clusters, which linked its regulatory role in evasion of immune response. Those functions of BbHCR1 were found to be collaboratively regulated by Fus3- and Hog1-MAP kinases via phosphorylation. These findings have drawn a regulatory network in which Fus3- and Hog1-MAP kinases phosphorylate BbHCR1, which in turn controls the colonization of insect body cavities by regulating fungal morphological transition and virulence-implicated genes.IMPORTANCEFungal pathogens adopt a series of tactics for successful colonization in host tissues, which include morphological transition and the generation of toxic and immunosuppressive molecules. However, many transcription factors (TFs) and their linked pathways that regulate tissue colonization are not well characterized. Here, we identified a TF (BbHCR1)-mediated regulatory network that controls the insect fungal pathogen, Beauveria bassiana , colonization of insect hemocoel. During these processes, BbHCR1 targeted the fungal central development pathway for the control of yeast (blastospores)-to-hyphae morphological transition, activated virulence factors, repressed virulence repressors, and tuned the expression of two dominant hemocoel colonization-involved immunosuppressive and immunostimulatory metabolite biosynthetic gene clusters. The BbHCR1 regulatory function was governed by Fus3- and Hog1-MAP kinases. These findings led to a new regulatory network composed of Fus3- and Hog1-MAP kinases and BbHCR1 that control insect body cavity colonization by regulating fungal morphological transition and virulence-implicated genes., Competing Interests: The authors declare no conflict of interest.

Cla4, an orthologous p21-activated kinase crucial for non-entomopathogenic fungal lifestyles, has two paralogs (Cla4A/B) functionally unknown in hypocrealean entomopathogens. Here, we report a regulatory role of Cla4A in gene expression networks of Beauveria bassiana required for asexual and entomopathogenic lifecycles while Cla4B is functionally redundant. The deletion of cla4A resulted in severe growth defects, reduced stress tolerance, delayed conidiation, altered conidiation mode, impaired conidial quality, and abolished pathogenicity through cuticular penetration, contrasting with no phenotype affected by cla4B deletion. In ∆ cla4A , 5288 dysregulated genes were associated with phenotypic defects, which were restored by targeted gene complementation. Among those, 3699 genes were downregulated, including more than 1300 abolished at the transcriptomic level. Hundreds of those downregulated genes were involved in the regulation of transcription, translation, and post-translational modifications and the organization and function of the nuclear chromosome, chromatin, and protein-DNA complex. DNA-binding elements in promoter regions of 130 dysregulated genes were predicted to be targeted by Cla4A domains. Samples of purified Cla4A extract were proven to bind promoter DNAs of 12 predicted genes involved in multiple stress-responsive pathways. Therefore, Cla4A acts as a novel regulator of genomic expression and stability and mediates gene expression networks required for insect-pathogenic fungal adaptations to the host and environment.

Asexual development is the main propagation and transmission mode of Beauveria bassiana and the basis of its pathogenicity. The regulation mechanism of conidiation and the key gene resources for utilization are key links to improving the conidia yield and quality of Beauveria bassiana . Their clarification may promote the industrialization of fungal pesticides. Here, we compared the regulation of morphology, resistance to external stress, virulence, and nutrient utilization capacity between the upstream developmental regulatory gene fluG and the key genes brlA , abaA , and wetA in the central growth and development pathway. The results showed that the Δ brlA and Δ abaA mutants completely lost the capacity to conidiate and that the Δ wetA mutant had seriously reduced conidiation capacity. Although the deletion of fluG did not reduce the conidiation ability as much as deletions of brlA , abaA , and wetA , it significantly reduced the fungal response to external stress, virulence, and nutrient utilization, while the deletion of the three other genes had little effect. Via transcriptome analysis and screening the yeast nuclear system library, we found that the differentially expressed genes in the Δ fluG mutants were concentrated in the signaling pathways of ABC transporters, propionate metabolism, tryptophan metabolism, DNA replication, mismatch repair, and fatty acid metabolism. FluG directly acted on 40 proteins that were involved in various signaling pathways such as metabolism, oxidative stress, and cell homeostasis. The analysis indicated that the regulatory function of fluG was mainly involved in DNA replication, cell homeostasis, fungal growth and metabolism, and the response to external stress. Our results revealed the biological function of fluG in asexual development and the responses to several environmental stresses as well as its influence on the asexual development regulatory network in B. bassiana .

Beauveria bassiana is an entomopathognic fungus, which is widely employed in the biological control of pests. Gene disruption is a common method for studying the functions of genes involved in fungal development or its interactions with hosts. However, generating gene deletion mutants was a time-consuming work. The transcriptional factor OpS3 has been identified as a positive regulator of a red secondary metabolite oosporein in B. bassiana. In this study, we have designed a new screening system by integrating a constitutive OpS3 expression cassette outside one of the homologous arms of target gene. Ectopic transformants predominantly exhibit a red colour with oosporein production, while knockout mutants appear as white colonies due to the loss of the OpS3 expression cassette caused by recombinant events. This screening strategy was used to obtain the deletion mutants of both tenS and NRPS genes. Correct mutants were obtained by screening fewer than 10 mutants with a positive efficiency ranging from 50% to 75%. This system significantly reduces the workload associated with DNA extraction and PCR amplification, thereby enhancing the efficiency of obtaining correct transformants in B. bassiana., (© 2024 The Author(s). Microbial Biotechnology published by John Wiley & Sons Ltd.)

Introduction: In yeast, the cytoplasm-to-vacuole targeting (Cvt) pathway acts as a biosynthetic autophagy-related process, in which vacuolar targeting of hydrolase is mediated by the machineries involved in the selective autophagy. However, the mechanistic insights into vacuolar targeting of hydrolases through the selective autophagy pathway still remain enigmatic in filamentous fungi., Objectives: Our study aims to investigate the mechanisms involved in vacuolar targeting of hydrolases in filamentous fungi., Methods: The filamentous entomopathogenic fungus Beauveria bassiana was used as a representative of filamentous fungi. We identified the homologs of yeast aminopeptidase I (Ape1) in B. bassiana by bioinformatic analyses and characterized their physiological roles by gene function analyses. Pathways for vacuolar targeting of hydrolases were investigated via molecular trafficking analyses., Results: B. bassiana has two homologs of yeast aminopeptidase I (Ape1) which are designated as BbApe1A and BbApe1B. The two homologs of yeast Ape1 contribute to starvation tolerance, development, and virulence in B. bassiana. Significantly, BbNbr1 acts as a selective autophagy receptor to mediate the vacuolar targeting of the two Ape1 proteins, in which BbApe1B interacts with BbNbr1 also directly interacting with BbAtg8, and BbApe1A has an additional requirement of the scaffold protein BbAtg11 that interacts with BbNbr1 and BbAtg8. Protein processing occurs at both terminuses of BbApe1A and only at carboxyl terminus of BbApe1B, which is also dependent on the autophagy-related proteins. Together, the functions and translocation processes of the two Ape1 proteins are associated with autophagy in fungal lifecycle., Conclusion: This study reveals the functions and translocation processes for vacuolar hydrolases in the insect-pathogenic fungi and improves our understandings of the Nbr1-mediated vacuolar targeting pathway in the filamentous fungi., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Production and hosting by Elsevier B.V.)

Beauveria bassiana is a widely used entomopathogenic fungus in insect biological control applications. In this study, we investigated the role of two sirtuin homologs, BbHst3 and BbHst4, in the biological activities and pathogenicity of B. bassiana . Our results showed that deletion of BbHst3 and/or BbHst4 led to impaired sporulation, reduced (~50%) conidial production, and decreased tolerance to various stresses, including osmotic, oxidative, and cell wall-disturbing agents. Moreover, BbHst4 plays dominant roles in histone H3-K56 acetylation and DNA damage response, while BbHst3 is more responsible for maintaining cell wall integrity. Transcriptomic analyses revealed significant changes (>1,500 differentially expressed genes) in gene expression patterns in the mutant strains, particularly in genes related to secondary metabolism, detoxification, and transporters. Furthermore, the Δ BbHst3 , Δ BbHst4 , and Δ BbHst3 Δ BbHst4 strains exhibited reduced virulence in insect bioassays, with decreased (~20%) abilities to kill insect hosts through topical application and intra-hemocoel injection. These findings highlight the crucial role of BbHst3 and BbHst4 in sporulation, DNA damage repair, cell wall integrity, and fungal infection in B. bassiana . Our study provides new insights into the regulatory mechanisms underlying the biological activities and pathogenicity of B. bassiana and emphasizes the potential of targeting sirtuins for improving the efficacy of fungal biocontrol agents.IMPORTANCESirtuins, as a class of histone deacetylases, have been shown to play important roles in various cellular processes in fungi, including asexual development, stress response, and pathogenicity. By investigating the functions of BbHst3 and BbHst4, we have uncovered their critical contributions to important phenotypes in Beauveria bassiana . Deletion of these sirtuin homologs led to reduced conidial yield, increased sensitivity to osmotic and oxidative stresses, impaired DNA damage repair processes, and decreased fungal virulence. Transcriptomic analyses showed differential expression of numerous genes involved in secondary metabolism, detoxification, transporters, and virulence-related factors, potentially uncovering new targets for manipulation and optimization of fungal biocontrol agents. Our study also emphasizes the significance of sirtuins as key regulators in fungal biology and highlights their potential as promising targets for the development of novel antifungal strategies., Competing Interests: The authors declare no conflict of interest.

Relative gene expression analysis through RT-qPCR is an important molecular technique that helps understanding different molecular mechanisms, such as the plant defense response to insect pests. However, the use of RT-qPCR for gene expression analysis can be affected by factors that directly affect the reliability of the results. Among these factors, the appropriate choice of reference genes is crucial and can strongly impact RT-qPCR relative gene expression analyses, highlighting the importance in correctly choosing the most suitable genes for the success of the analysis. Thus, this study aimed to select and validate reference genes for relative gene expression studies through RT-qPCR in hybrids of Eucalyptus tereticornis × Eucalyptus camaldulensis (drought tolerant and susceptible to Leptocybe invasa) under conditions of inoculation by the Beauveria bassiana fungus and subsequent infestation by L. invasa. The expression level and stability of eleven candidate genes were evaluated. Stability was analyzed using the RefFinder tool, which integrates the geNorm, NormFinder, BestKeeper, and Delta-Ct algorithms. The selected reference genes were validated through the expression analysis of the transcriptional factor EcDREB2 (dehydration-responsive element-binding protein 2). For all treatments evaluated, EcPTB, EcPP2A-1, and EcEUC12 were the best reference genes. The triplets EcPTB/EcEUC12/EcUBP6, EcPP2A-1/EcEUC12/EcPTB, EcIDH/EcSAND/Ecα-TUB, EcPP2A-1/Ecα-TUB/EcPTB, and EcPP2A-1/EcUPL7/EcSAND were the best reference genes for the control plants, mother plants, plants inoculated with B. bassiana, plants infested with L. invasa, and plants inoculated with B. bassiana and subsequently infested with L. invasa, respectively. The best determined reference genes were used to normalize the RT-qPCR expression data for each experimental condition evaluated. The results emphasize the importance of this type of study to ensure the reliability of relative gene expression analyses. Furthermore, the findings of this study can be used as a basis for future research, comprising gene expression analysis of different eucalyptus metabolic pathways., (© 2024. The Author(s).)

We used Drosophila melanogaster to investigate how differences between Metarhizium species in growth rate and mechanisms of pathogenesis influence the outcome of infection. We found that the most rapid germinators and growers in vitro and on fly cuticle were the fastest killers, suggesting that pre-penetration competence is key to Metarhizium success. Virulent strains also induced the largest immune response, which did not depend on profuse growth within hosts as virulent toxin-producing strains only proliferated post-mortem while slow-killing strains that were specialized to other insects grew profusely pre-mortem. Metarhizium strains have apparently evolved resistance to widely distributed defenses such as the defensin Toll product drosomycin, but they were inhibited by Bomanins only found in Drosophila spp. Disrupting a gene ( Dif ), that mediates Toll immunity has little impact on the lethality of most Metarhizium strains (an exception being the early diverged M. frigidum and another insect pathogen Beauveria bassiana ). However, disrupting the sensor of fungal proteases ( Persephone ) allowed rapid proliferation of strains within hosts (with the exception of M. album ), and flies succumbed rapidly. Persephone also mediates gender differences in immune responses that determine whether male or female flies die sooner. We conclude that some strain differences in growth within hosts depend on immune-mediated interactions but intrinsic differences in pathogenic mechanisms are more important. Thus, Drosophila varies greatly in tolerance to different Metarhizium strains, in part because some of them produce toxins. Our results further develop D. melanogaster as a tractable model system for understanding insect- Metarhizium interactions.

Background: The entomogenous fungus Beauveria bassiana is used as a biological insecticide worldwide, wild B. bassiana strains with high pathogenicity in the field play an important role in controlling insect pests via not only screening of highly virulent strains but also natural infection, but the pathogenicity degeneration of wild strains severely affected aforementioned effects. Previous studies have showed that multiple factors contributed to this phenomenon. It has been extensively proved that the mycovirus infection caused hypovirulence of phytopathogenic fungi, which has been used for plant disease biocontrol. However, it remains unknown whether the mycovirus epidemics is a key factor causing hypovirulence of B. bassiana naturally in the field., Methods: Wild strains of B. bassiana were collected from different geographic locations in Jilin Province, China, to clarify the epidemic and diversity of the mycoviruses. A mycovirus Beauveria bassiana chrysovirus 2 (BbCV2) we have previously identified was employed to clarify its impact on the pathogenicity of host fungi B. bassiana against the larvae of insect pest Ostrinia furnacalis. The serological analysis was conducted by preparing polyclonal antibody against a BbCV2 coat protein, to determine whether it can dissociate outside the host fungal cells and subsequently infect new hosts. Transcriptome analysis was used to reveal the interactions between viruses and hosts., Results: We surprisingly found that the mycovirus BbCV2 was prevalent in the field as a core virus in wild B. bassiana strains, without obvious genetic differentiation, this virus possessed efficient and stable horizontal and vertical transmission capabilities. The serological results showed that the virus could not only replicate within but also dissociate outside the host cells, and the purified virions could infect B. bassiana by co-incubation. The virus infection causes B. bassiana hypovirulence. Transcriptome analysis revealed decreased expression of genes related to insect epidermis penetration, hypha growth and toxin metabolism in B. bassiana caused by mycovirus infection., Conclusion: Beauveria bassiana infected by hypovirulence-associated mycovirus can spread the virus to new host strains after infecting insects, and cause the virus epidemics in the field. The findings confirmed that mycovirus infection may be an important factor affecting the pathogenicity degradation of B. bassiana in the field., (© 2023. The Author(s).)

Cotton mealybug, Phenacoccus solenopsis (Tinsley) and cowpea aphid Aphis craccivora (Koch) are notorious polyphagous, hemipteran sap sucking insect pests. A recombinant toxin gene 'LqqIT1' from the scorpion Leiurus quinquestriatus quinquestriatus (Ehrenberg) was cloned in the pAL1 fungal expression vector and then expressed in the entomopathogenic fungus Beauveria bassiana (Balasmo) using genetic modification techniques. The genetically transformed B. bassiana strain (BbLqqIT1-3) and its un-transformed parent strain (Bb-C) were screened to infect the third instar nymphs of P. solenopsis and first instar nymph of A. craccivora through leaf treatment and topical application (spray) method at 1 * 10 7 spores per ml concentration. The recombinant strain BbLqqIT1-3 was highly pathogenic against A. craccivora but non pathogenic to P. solenopsis . BbLqqIT1-3 induced 72 and 43.33% mortality in A. craccivora nymphs 96 h after leaf treatment and topical application, respectively. The nymphs of A. craccivora infected with BbLqqIT1-3 displayed classical neurotoxic symptoms such as sluggishness, solublize and liquification of the body. Crude soluble toxin protein, BbLqqIT1a-CSE and Bb-WT-CSE was extracted from the BbLqqIT1-3 and Bb-C, respectively using ammonium sulphate precipitation method, and their oral toxicity was analyzed at 5 µg/ml concentration. The survival of the studied insects was negatively affected by the crude soluble toxin extracts. The LT 50 values of BbLqqIT1a-CSE against P. solenopsis and A. craccivora were 22.18 and 17.69 h, respectively. Exposure to crude soluble toxin extracts also accounted for the imbalance of ionic concentrations in the hemolymph of treated insects such as hyperpotassemia (3.53-8.18 meq/ml) in the P. solenopsis and hypopotassemia (7.52-0.47 meq/ml) in A. craccivora . The transformed fungus BbLqqIT1-3 strain exhibited promising results in invitro study., Competing Interests: The authors declare that they have no competing interests., (© 2023 Rajput et al.)

Diverse β-carboline (βC) alkaloids are produced by microbes, plants, and animals with myriad bioactivities and drug potentials. However, the biosynthetic mechanism of βCs remains largely elusive, especially regarding the hydroxyl and glucosyl modifications of βCs. Here, we report the presence of the bacterial-like Pictet-Spenglerase gene Fcs1 in the entomopathogenic Beauveria fungi that can catalyze the biosynthesis of the βC skeleton. The overexpression of Fcs1 in Beauveria bassiana led to the identification of six βC methyl glycosides, termed bassicarbosides (BCSs) A-F. We verified that the cytochrome P450 (CYP) genes adjacent to Fcs1 cannot oxidize βCs. Alternatively, the separated CYP684B2 family gene Fcs2 was identified to catalyze βC hydroxylation together with its cofactor gene Fcs3 . The functional homologue of Fcs2 is only present in the Fcs1- containing fungi and highly similar to the Fcs1- connected yet nonfunctional CYP. Both evolved quicker than those from fungi without Fcs1 homologues. Finally, the paired methyl/glucosyl transferase genes were verified to mediate the production of BCSs from hydroxy-βCs. All these functionally verified genes are located on different chromosomes of Beauveria , which is in contrast to the typical content-clustered feature of fungal biosynthetic gene clusters (BGCs). We also found that the production of BCSs selectively contributed to fungal infection of different insect species. Our findings shed light on the biosynthetic mechanism of βC glycosides, including the identification of a βC hydroxylase. The results of this study also propose an evolving process of fungal BGC formation following the horizontal transfer of a bacterial gene to fungi.

Background: The mountain pine beetle, Dendroctonus ponderosae, is an irruptive bark beetle that causes extensive mortality to many pine species within the forests of western North America. Driven by climate change and wildfire suppression, a recent mountain pine beetle (MPB) outbreak has spread across more than 18 million hectares, including areas to the east of the Rocky Mountains that comprise populations and species of pines not previously affected. Despite its impacts, there are few tactics available to control MPB populations. Beauveria bassiana is an entomopathogenic fungus used as a biological agent in agriculture and forestry and has potential as a management tactic for the mountain pine beetle population. This work investigates the phenotypic and genomic variation between B. bassiana strains to identify optimal strains against a specific insect., Results: Using comparative genome and transcriptome analyses of eight B. bassiana isolates, we have identified the genetic basis of virulence, which includes oosporein production. Genes unique to the more virulent strains included functions in biosynthesis of mycotoxins, membrane transporters, and transcription factors. Significant differential expression of genes related to virulence, transmembrane transport, and stress response was identified between the different strains, as well as up to nine-fold upregulation of genes involved in the biosynthesis of oosporein. Differential correlation analysis revealed transcription factors that may be involved in regulating oosporein production., Conclusion: This study provides a foundation for the selection and/or engineering of the most effective strain of B. bassiana for the biological control of mountain pine beetle and other insect pests populations., (© 2023. The Author(s).)

Many filamentous fungi develop a conidiation process as an essential mechanism for their dispersal and survival in natural ecosystems. However, the mechanisms underlying conidial persistence in environments are still not fully understood. Here, we report that autophagy is crucial for conidial lifespans (i.e., viability) and vitality (e.g., stress responses and virulence) in the filamentous mycopathogen Beauveria bassiana. Specifically, Atg11-mediated selective autophagy played an important, but not dominant, role in the total autophagic flux. Furthermore, the aspartyl aminopeptidase Ape4 was found to be involved in conidial vitality during dormancy. Notably, the vacuolar translocation of Ape4 was dependent on its physical interaction with autophagy-related protein 8 (Atg8) and associated with the autophagic role of Atg8, as determined through a truncation assay of a critical carboxyl-tripeptide. These observations revealed that autophagy acted as a subcellular mechanism for conidial recovery during dormancy in environments. In addition, a novel Atg8-dependent targeting route for vacuolar hydrolase was identified, which is essential for conidial exit from a long-term dormancy. These new insights improved our understanding of the roles of autophagy in the physiological ecology of filamentous fungi as well as the molecular mechanisms involved in selective autophagy. IMPORTANCE Conidial environmental persistence is essential for fungal dispersal in ecosystems while also serving as a determinant for the biocontrol efficacy of entomopathogenic fungi during integrated pest management. This study identified autophagy as a mechanism to safeguard conidial lifespans and vitality postmaturation. In this mechanism, the aspartyl aminopeptidase Ape4 translocates into vacuoles via its physical interaction with autophagy-related protein 8 (Atg8) and is involved in conidial vitality during survival. The study revealed that autophagy acted as a subcellular mechanism for maintaining conidial persistence during dormancy, while also documenting an Atg8-dependent targeting route for vacuolar hydrolase during conidial recovery from dormancy. Thus, these observations provided new insight into the roles of autophagy in the physiological ecology of filamentous fungi and documented novel molecular mechanisms involved in selective autophagy.

The Aedes aegypti mosquito transmits devastating flaviviruses, such as Zika, dengue, and yellow fever viruses. For more effective control of the vector, the pathogenicity of Beauveria bassiana, a fungus commonly used for biological control of pest insects, may be enhanced based on in-depth knowledge of molecular interactions between the pathogen and its host. Here, we identified a mechanism employed by B. bassiana, which efficiently blocks the Ae. aegypti antifungal immune response by a protease that contains an ovarian tumor (OTU) domain. RNA-sequencing analysis showed that the depletion of OTU7B significantly upregulates the mRNA level of immunity-related genes after a challenge of the fungus. CRISPR-Cas9 knockout of OTU7B conferred a higher resistance of mosquitoes to the fungus B. bassiana. OTU7B suppressed activation of the immune response by preventing nuclear translocation of the NF-κB transcription factor Rel1, a mosquito orthologue of Drosophila Dorsal. Further studies identified tumor necrosis factor receptor-associated factor 4 (TRAF4) as an interacting protein of OTU7B. TRAF4-deficient mosquitoes were more sensitive to fungal infection, indicating TRAF4 to be the adaptor protein that activates the Toll pathway. TRAF4 is K63-link polyubiquitinated at K338 residue upon immune challenge. However, OTU7B inhibited the immune signaling by enzymatically removing the polyubiquitin chains of mosquito TRAF4. Thus, this study has uncovered a novel mechanism of fungal action against the host innate immunity, providing a platform for further improvement of fungal pathogen effectiveness. IMPORTANCE Insects use innate immunity to defend against microbial infection. The Toll pathway is a major immune signaling pathway that is associated with the antifungal immune response in mosquitoes. Our study identified a fungal-induced deubiquitinase, OTU7B, which, when knocked out, promotes the translocation of the NF-κB factor Rel1 into the nucleus and confers enhanced resistance to fungal infection. We further found the counterpart of OTU7B, TRAF4, which is a component of the Toll pathway and acts as an adaptor protein. OTU7B enzymatically removes K63-linked polyubiquitin chains from TRAF4. The immune response is suppressed, and mosquitoes become much more sensitive to the Beauveria bassiana infection. Our findings reveal a novel mechanism of fungal action against the host innate immunity.

In yeasts, bcs1 is a mitochondrial AAA protein (ATPase associated with diverse cellular activities) and required for biogenesis of the complex III in mitochondrial electron transfer chain. However, the presence and biological roles of bcs1 remain largely unknown in the filamentous fungi. In present study, genome-wide identification revealed that there were six BCS1-domain containing proteins (Bbbcs1a through f) in the filamentous insect pathogenic fungus Beauveria bassiana, five of which (except for Bbbcs1f) were functionally analyzed. Phenotypic evaluation revealed that only Bbbcs1b and Bbbcs1c contributed to fungal physiologies, and they localized to nuclei and mitochondria, respectively. Hence, Bbbcs1c is considered as the ortholog of yeast bcs1 in B. bassiana. Ablation of Bbbcs1c did not affect biogenesis of mitochondria, but its loss significantly attenuated mitochondrial functionality (e.g., ATP synthesis and mitochondrial targeting of proteins) significantly. ΔBbbcs1c mutant displayed the impaired phenotypes in vegetative growth, stress response, development, and virulence. Notably, ΔBbbcs1c mutant displayed the increased sensitivity to linoleic acid (LA) stress and lost the intracellular fatty acid homeostasis. The Bbbcs1c loss compromised the mitochondrial membrane potential, and LA stress exacerbated this damage. These findings indicate that Bbbcs1c is a functional homolog of yeast bcs1 in B. bassiana and links mitochondrial functionality to unique lifestyle in the entomopathogenic fungi., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

The entomopathogenic fungus Beauveria bassiana can produce the secondary metabolite oosporein under alkaline conditions or in fungus-killed cadavers. However, the regulatory mechanism of oosporein synthesis is not fully understood. In thisstudy, we found that the pH signaling transcription factor BbPacC is involved in the regulation of oosporein production. Overexpression of BbPacC promotes oosporein production in B. bassiana at pH 6.0 or under alkaline conditions (pH 8.0), but deletion of this gene abolished oosporein production. Under acidic conditions (pH 4.0), no oosporein production was observed in the wild-type and BbPacC overexpression strains. Yeast one-hybrid assays and electrophoretic mobility shift assay (EMSA) confirmed the binding ability of BbPacC with 4 putative PacC-binding sites in the promoter region of BbOpS3 , a transcription factor located in the oosporein synthetic gene cluster regulating the expression of oosporein synthetic genes. Overexpression of Bbmsn2 , a previously reported negative regulator of oosporein synthesis, in OEPacC or wild-type strains abolished oosporein production in all tested conditions. However, deletion of Bbmsn2 in the BbPacC overexpression strain significantly improved oosporein production even at pH 4.0. These results indicated that BbPacC is a positive regulator of oosporein production and functions jointly with Bbmsn2 to regulate oosporein production in different environments and particularly under alkaline conditions. IMPORTANCE B. bassiana produces the red dibenzoquinone pigment oosporein under certain specific conditions, such as alkaline conditions and fungus-killed cadavers. Ooporein possesses antibiotic and insect immune inhibition activities and plays multiple roles during the infection process of B. bassiana against insect hosts. Several negative regulators involved in oosporein synthesis have been reported; however, we know little about the positive regulators outside the biosynthetic gene cluster. Here, we found that the pH signaling transcription factor BbPacC positively regulates oosporein production by binding to several PacC-binding sites. In addition, our results also indicate that BbPacC jointly acts with the negative regulator Bbmsn2 to regulate oosporein synthesis. Our results provide insight into understanding the regulatory mechanism of oosporein production as well as targets to engineer B. bassiana strains producing high levels of oosporein.

Mycoviruses can spread interspecifically and intraspecifically in plant pathogenic fungi, as well as spreading intraspecifically in entomogenous fungi, especially Beauveria bassiana. However, whether mycoviruses are common in Beauveria spp. and can spread interspecifically between Beauveria species are unclear. Herein, four Beauveria species, but not B. bassiana, were randomly selected for double stranded RNA (dsRNA) detection. Furthermore, two previously reported dsRNA mycoviruses from B. bassiana, BbCV-2 and BbPmV-4, were used to study the interspecific transmission among B. bassiana, B. amorpha, and B. aranearum, using hyphal anastomosis and a novel insect coinfection transmission method. The results showed that dsRNA mycoviruses exist universally in Beauveria spp. and could spread interspecifically between different Beauveria species. The transmission efficiency from B. bassiana to the other two Beauveria species was significantly higher than that of the reverse transmission. Both viruses could stably and vertically spread in B. amorpha and B. aranearum, which affected their growth rate and colony morphology., Competing Interests: Conflict of interest The authors declare that they have no conflict., (Copyright © 2022. Published by Elsevier B.V.)

A desirable substitute for chemical pesticides is mycopesticides. In the current investigation, rDNA-ITS (Internal transcribed spacer) and TEF (Transcriptional Elongation Factor) sequencing were used for molecular identification of six Beauveria bassiana strains. Both, leaf discs and potted plant bioassaye were carried out to study their pathogenicity against the cassava mite, Tetranychus truncatus. LC50 and LC90 values of potential B. bassiana strains were estimated. We also discovered a correlation between intraspecific B. bassiana strains pathogenicity and comprehensive metabolome profiles. Bb5, Bb6, Bb8, Bb12, Bb15, and Bb21 strains were identified as B. bassiana by sequencing of rDNA-ITS and TEF segments and sequence comparison to NCBI (National Center for Biotechnology Information) GenBank. Out of the six strains tested for pathogenicity, Bb6, Bb12, and Bb15 strains outperformed against T. truncatus with LC50 values 1.4×106, 1.7×106, and 1.4×106 and with a LC90 values 7.3×107, 1.4×108, and 4.2×108 conidia/ml, respectively, at 3 days after inoculation and were considered as potential strains for effective mite control. Later, Gas Chromatography-Mass Spectrometry (GC-MS) analysis of the above six B. bassiana strains was done on secondary metabolites extracted with ethyl acetate revealed that the potential B. bassiana strains (Bb6, Bb12, and Bb15) have higher levels of acaricidal such as Bis(dimethylethyl)-phenol: Bb6 (5.79%), Bb12 (6.15%), and Bb15 (4.69%). Besides, insecticidal (n-Hexadecanoic acid), and insect innate immunity overcoming compound (Nonadecene) were also identified; therefore, the synergistic effect of these compounds might lead toa higher pathogenicity of B. bassiana against T. truncatus. Further, these compounds also exhibited two clusters, which separate the potential and non-potential strains in the dendrogram of Thin Layer Chromatography. These results clearly demonstrated the potentiality of the B. bassiana strains against T. truncatus due to the occurrence of their bioactive volatile metabolome., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Chaithra et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Succinate dehydrogenase (SDH), also known as respiratory chain complex II, plays a crucial role in energy production in which SdhC functions as an anchored subunit in the inner membrane of mitochondria. In this study, domain annotation analyses revealed that two SdhC domain-containing proteins were present in the filamentous insect-pathogenic fungus Beauveria bassiana, and they were named BbSdhC1 and BbSdhC2, respectively. Only BbSdhC1 localized to mitochondria; hence, this protein is considered the ortholog of SdhC in B. bassiana. Ablation of BbSdhC1 led to significantly reduced vegetative growth on various nutrients. The Δ Bbsdhc1 mutant displayed the significantly reduced ATP synthesis and abnormal differentiation under aerial and submerged conditions. Notably, the BbSdhC1 loss resulted in enhanced intracellular levels of reactive oxygen species (ROS) and impaired growth of mycelia under oxidative stress. Finally, insect bioassays (via cuticle and intrahemocoel injection infection) revealed that disruption of BbSdhC1 significantly attenuated fungal virulence against the insect hosts. These findings indicate that BbSdhC1 contributes to vegetative growth, resistance to oxidative stress, differentiation, and virulence of B. bassiana due to its roles in energy generation and maintaining the homeostasis of the intracellular ROS levels. IMPORTANCE The electron transport chain (ETC) is critical for energy supply by mediating the electron flow along the mitochondrial membrane. Succinate dehydrogenase (SDH) is also known as complex II in the ETC, in which SdhC is a subunit anchored in mitochondrial membrane. However, the physiological roles of SdhC remain enigmatic in filamentous fungi. In filamentous insect-pathogenic fungus B. bassiana, SdhC is required for maintaining mitochondrial functionality, which is critical for fungal stress response, development, and pathogenicity. These findings improve our understanding of physiological mechanisms of ETC components involved in pathogenicity of the entomopathogenic fungi.

The growing threat of insecticide resistance prompts the urgent need to develop additional tools for mosquito control. Entomopathogenic fungi provide an eco-friendly alternative to chemical insecticides. One limitation to the use of mycoinsecticides is their relatively low virulence. Here, we report an approach for suppressing mosquito immunity and increasing fungal virulence. We engineered Beauveria bassiana to express Aedes immunosuppressive microRNAs (miRNAs) to induce host RNA interference (RNAi) immune responses. We show that engineered strains can produce and deliver the miRNAs into host cells to activate cross-kingdom RNAi during infection and suppress mosquito immunity by targeting multiple host genes, thereby dramatically increasing fungal virulence against Aedes aegypti and Galleria mellonella larvae. Importantly, expressing host miRNAs also significantly increases fungal virulence against insecticide-resistant mosquitoes, creating potential for insecticide-resistance management. This pathogen-mediated RNAi (pmRNAi)-based approach provides an innovative strategy to enhance the efficacy of fungal insecticides and eliminate the likelihood of resistance development., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Despite the significant progress that has been made in the genome sequencing of Beauveria species, mitochondrial genome (mitogenome) used to examine genetic diversity within fungal populations. Complete mitogenomes of Beauveria species can be easily sequenced and assembled using various sequencing techniques. However, since mitogenome annotations are mainly derived from similar species comparison and software prediction, and are not supported by RNA-seq transcripts data, it leads to problems with the accuracy of mitochondrial annotations and the inability to understand RNA processing. In this study, we assembled and annotated the mitogenome of eight Beauveria strains using Illumina DNA and RNA sequencing data. The circular mitogenome of eight Beauveria strains ranged from 26,850 bp ( B. caledonica strain ATCC 64970) to 35,999 bp ( B. brongniartii strain GYU-BMZ03), with the intronic insertions accounting for most of the size variation, thus contributing to a total mitochondrial genome (mitogenome) size of 7.01% and 28.95%, respectively. Intron number variations were not directly related to the evolutionary relationship distance. Besides ribosomal protein S3 ( rps3 ), most introns are lost too quickly and lack the stability of protein-coding genes. The short RNA-seq reads from next-generation sequencing can improve the mitochondrial annotation accuracy and help study polycistronic transcripts and RNA processing. The transcription initiation sites may be located in the control region. Most introns do not serve as taxonomic markers and also lack open reading frames (ORFs). We assumed that the poly A tail was added to the polycistronic transcript before splicing and one polycistronic transcript ( trnM (1) - trnL (1) - trnA - trnF - trnK - trnL (2) - trnQ - trnH - trnM (2) - nad2 - nad3 - atp9 - cox2 - trnR (1) - nad4L - nad5 - cob - trnC - cox1 - trnR (2) - nad1 - nad4 - atp8 - atp6 - rns - trnY - trnD - trnS - trnN - cox3 - trnG - nad6 - trnV - trnI - trnS - trnW - trnP - rnl ( rps3 )- trnT - trnE - trnM (3) ) was first processed from the mitogenome and was subsequently processed into smaller mono-, di-, or tricistronic RNAs., Competing Interests: The authors declare there are no competing interests., (©2022 Bai et al.)

Entomopathogenic fungi (EF) provide a potent biocontrol tool; also, their endophytic behavior has broadened their contribution to integrated pest management (IPM) and crop production. In this work, Beauveria bassiana and Metarhizium brunneum were applied to bread wheat (Triticum aestivum) seedlings to elucidate how fungal colonization influences plant growth and the relative expression of 24 genes involved in hormonal syntheses and plant immune mechanisms. A preliminary assay was used to determine the time needed for fungal colonization and assess its effect on wheat growth. Then, plant material collected at various times after inoculation ( viz. , 2, 8, 20, and 36 h and 9 and 15 days) was used to investigate gene expression by quantitative reverse transcription PCR (RT-qPCR). During the colonization time, B. bassiana and M. brunneum caused strong downregulation of most genes associated with plant immunity and the synthesis of hormones like auxin, cytokinin, and gibberellin. This effect was concomitant with a slowdown of endophytic-colonization-related plant growth until 19 days postinoculation (dpi). However, the wheat started to recover at 15 dpi, simultaneously with upregulation of auxin- and gibberellin-related genes. The results suggest that the EF trigger induced systemic resistance rather than acquired systemic resistance during early plant-microbe cross talk in wheat. Also, they confirm that the hormone and immune responses of wheat triggered by EF inoculation influenced plant growth, which can be useful with a view to optimizing management of these microorganisms for sustainable agriculture. IMPORTANCE Microbial control of insect and mite pests is a key tool to develop integrated pest management (IPM) and sustainable agriculture. Entomopathogenic fungi (EF) may have associations with the plants, playing additional ecological roles in the rhizosphere, in the phylloplane, and as plant endophytes. Beauveria bassiana 04/01TIP and Metarhizium brunneum 01/58Su are two strains that showed very good results either in pest control or plant growth promotion and would be good candidates to develop mycoinsecticides as an alternative to pesticides. However, deep knowledge about their interaction with the plant would let farmers optimize their use and understand the plant response, enhancing and promoting their broader contribution to IPM and crop production.

The entomopathogenic fungus Beauveria bassiana is a typical filamentous fungus and has been used for pest biocontrol. Conidia are the main active agents of fungal pesticides; however, we know little about conidial developmental mechanisms and less about maturation mechanisms. We found that a Zn 2 Cys 6 transcription factor of B. bassiana (named BbCmr1) was mainly expressed in late-stage conidia and was involved in conidium maturation regulation. Deletion of Bbcmr1 impaired the conidial cell wall and resulted in a lower conidial germination rate under UV (UV), heat shock, H 2 O 2 , Congo red (CR) and SDS stresses compared to the wild type. Transcription levels of the genes associated with conidial wall components and trehalose synthase were significantly reduced in the ΔBbcmr1 mutant. Further analysis found that BbCmr1 functions by upregulating BbWetA, a well-known transcription factor in the central development of BrlA-AbaA-WetA. The expression of Bbcmr1 was positively regulated by BbBrlA. These results indicated that BbCmr1 played important roles in conidium maturation by interacting with the central development pathway, which provided insight into the conidial development networks in B. bassiana. IMPORTANCE Conidium maturation is a pivotal event in conidial development and affects fungal survival ability under various biotic/abiotic stresses. Although many transcription factors have been reported to regulate conidial development, we know little about the molecular mechanism of conidium maturation. Here, we demonstrated that the transcription factor BbCmr1 of B. bassiana was involved in conidium maturation, regulating cell wall structure, the expression of cell wall-related proteins, and trehalose synthesis. BbCmr1 orchestrated conidium maturation by interplaying with the central development pathway BrlA-AbaA-WetA. BbBrlA positively regulated the expression of Bbcmr1 , and the latter positively regulated BbwetA expression, which forms a regulatory network mediating conidial development. This finding was critical to understand the molecular regulatory networks of conidial development in B. bassiana and provided avenues to engineer insect fungal pathogens with high-quality conidia.

Diverse 2-pyridone alkaloids have been identified with an array of biological and pharmaceutical activities, including the development of drugs. However, the biosynthetic regulation and chemical ecology of 2-pyridones remain largely elusive. Here, we report the inductive activation of the silent polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) ( tenS ) gene cluster for the biosynthesis of the tenellin-type 2-pyridones in the insect-pathogenic fungus Beauveria bassiana when cocultured with its natural competitor fungus Metarhizium robertsii. A pathway-specific transcription factor, tenR , was identified, and the overexpression of tenR well expanded the biosynthetic mechanism of 15-hydroxytenellin (15-HT) and its derivatives. In particular, a tandemly linked glycosyltransferase-methyltransferase gene pair located outside the tenS gene cluster was verified to mediate the rare and site-specific methylglucosylation of 15-HT at its N-OH residue. It was evident that both tenellin and 15-HT can chelate iron, which could benefit B. bassiana to outcompete M. robertsii in cocultures and to adapt to iron-replete and -depleted conditions. Relative to the wild-type strain, the deletion of tenS had no obvious negative effect on fungal virulence, but the overexpression of tenR could substantially increase fungal pathogenicity toward insect hosts. The results of this study well advance the understanding of the biosynthetic machinery and chemical ecology of 2-pyridones. IMPORTANCE Different 2-pyridones have been identified, with multiple biological activities but unclear chemical ecology. We found that the silent tenS gene cluster was activated in the insect pathogen Beauveria bassiana when the fungus was cocultured with its natural competitor Metarhizium robertsii. It was established that the gene cluster is regulated by a pathway-specific regulator, tenR , and the overexpression of this transcription factor expanded the biosynthetic machinery of the tenellin 2-pyridones. It was also found that the paired genes located outside the tenS cluster contribute to the site-specific methylglucosylation of the main compound 15-hydroxytenellin. Both tenellin and 15-hydroxytenellin can chelate and sequester iron to benefit the producing fungus to compete for different niches. This study well advances the biosynthetic mechanism and chemical ecology of 2-pyridones.

Fungi are the most common pathogens of insects and thus important regulators of their populations. Lipid-binding aegerolysin proteins, which are commonly found in the fungal kingdom, may be involved in several biologically relevant processes including attack and defense against other organisms. Aegerolysins act alone or together with membrane-attack-complex/perforin (MACPF)-like proteins to form transmembrane pores that lead to cell lysis. We performed an in-depth bioinformatics analysis of aegerolysins in entomopathogenic fungi and selected a candidate aegerolysin, beauveriolysin A (BlyA) from Beauveria bassiana . BlyA was expressed as a recombinant protein in Escherichia coli , and purified to further determine its functional and structural properties, including lipid-binding ability. Aegerolysins were found to be encoded in genomes of entomopathogenic fungi, such as Beauveria , Cordyceps , Metarhizium and Ophiocordyceps . Detailed bioinformatics analysis revealed that they are linked to MACPF-like genes in most genomes. We also show that BlyA interacts with an insect-specific membrane lipid. These results were placed in the context of other fungal and bacterial aegerolysins and their partner proteins. We believe that aegerolysins play a role in promoting the entomopathogenic and antagonistic activity of B. bassiana , which is an active ingredient of bioinsecticides.

Morphological transition is an important adaptive mechanism in the host invasion process. Wor1 is a conserved fungal regulatory protein that controls the phenotypic switching and pathogenicity of Candida albicans. By modulating growth conditions, we simulated three models of Beauveria bassiana morphological transitions, including CTH (conidia to hyphae), HTC (hyphae to conidia), and BTB (blastospore to blastospore). Disruption of BbWor1 (an ortholog of Wor1 ) resulted in a distinct reduction in the time required for conidial germination (CTH), a significant increase in hyphal growth, and a decrease in the yield of conidia (HTC), indicating that BbWor1 positively controls conidium production and negatively regulates hyphal growth in conidium-hypha switching. Moreover, Δ BbWor1 prominently decreased blastospore yield, shortened the G 0 /G 1 phase, and prolonged the G 2 /M phase under the BTB model. Importantly, BbWor1 contributed to conidium-hypha switching and blastospore propagation via different genetic pathways, and yeast one-hybrid testing demonstrated the necessity of BbWor1 to control the transcription of an allergen-like protein gene (BBA_02580) and a conidial wall protein gene (BBA_09998). Moreover, the dramatically weakened virulence of Δ BbWor1 was examined by immersion and injection methods. Our findings indicate that BbWor1 is a vital participant in morphological transition and pathogenicity in entomopathogenic fungi. IMPORTANCE As a well-known entomopathogenic fungus, Beauveria bassiana has a complex life cycle and involves transformations among single-cell conidia, blastospores, and filamentous hyphae. This study provides new insight into the regulation of the fungal cell morphological transitions by simulating three models. Our research identified BbWor1 as a core transcription factor of morphological differentiation that positively regulates the production of conidia and blastospores but negatively regulates hyphal growth. More importantly, BbWor1 affects fungal pathogenicity and the global transcription profiles within three models of growth stage transformation. The present study lays a foundation for the exploration of the transition mechanism of entomopathogenic fungi and provides material for the morphological study of fungi.

Beauveria bassiana is an insect pathogenic fungus that serves as a model system for exploring the mechanisms of fungal development and host-pathogen interactions. Clinical and experimental studies have indicated that SND1 is closely correlated with the progression and invasiveness of common cancers as a potential oncogene, but this gene has rarely been studied in fungi. Here, we characterized the contributions of an SND1 ortholog (Tdp1) by constructing a BbTdp1 deletion strain and a complemented strain of B. bassiana. Compared with the wild-type (WT) strain, the Δ BbTdp1 mutant lost conidiation capacity (∼87.7%) and blastospore (∼96.3%) yields, increased sensitivity to chemical stress (4.4 to 54.3%) and heat shock (∼44.2%), and decreased virulence following topical application (∼24.7%) and hemocoel injection (∼40.0%). Flow cytometry readings showed smaller sizes of both conidia and blastospores for Δ BbTdp1 mutants. Transcriptomic data revealed 4,094 differentially expressed genes (|log 2 ratio| > 2 and a q value of <0.05) between Δ BbTdp1 mutants and the WT strain, which accounted for 41.6% of the total genes, indicating that extreme fluctuation in the global gene expression pattern had occurred. Moreover, deletion of BbTdp1 led to an abnormal cell cycle with a longer S phase and shorter G 2 /M and G 0 /G 1 phases of blastospores, and enzyme-linked immunosorbent assay confirmed that the level of phosphorylated cyclin-dependent kinase 1 (Cdk1) in the Δ BbTdp1 strain was ∼31.5% lower than in the WT strain. In summary, our study is the first to report that BbTdp1 plays a vital role in regulating conidia and blastospore yields, fungal morphological changes, and pathogenicity in entomopathogenic fungi. IMPORTANCE In this study, we used Beauveria bassiana as a biological model to report the role of BbTdp1 in entomopathogenic fungi. Our findings indicated that BbTdp1 contributed significantly to cell development, the cell cycle, and virulence in B. bassiana. In addition, deletion of BbTdp1 led to drastic fluctuations in the transcriptional profile. BbTdp1 can be developed as a novel target for B. bassiana development and pathogenicity, which also provides a framework for the study of Tdp1 in other fungi.

Beauveria bassiana holds promise as a feasible biological control agent for tick control. The B. bassiana stress-response transcription factor Msn2 is known to contribute to fungal growth, conidiogenesis, stress-response and virulence towards insects; however, little is known concerning whether Msn2 is involved in infection across Arthropoda classes. We evaluated the effects of Msn2 on B. bassiana virulence against Rhipicephalus microplus (Acari, Ixodidae) using wild-type, targeted gene knockout ( ΔBbmsn2 ) and complemented mutant ( ΔBbmsn2/Bbmsn2 ) strains. Reproductive parameters of R. microplus engorged females treated topically or by an intra-hemocoel injection of conidial suspensions were assessed. Treated cuticles of engorged females were analyzed by microscopy, and proteolytic activity of B. bassiana on cuticles was assessed. Topically treated engorged females showed high mean larval hatching (>84%) in control and ΔBbmsn2 treatments, whereas treatment with the wild-type or ΔBbmsn2/Bbmsn2 strains resulted in significantly decreased (lowered egg viability) larval hatching. Percent control of R. microplus topically treated with ΔBbmsn2 was lower than in the groups treated with wild-type (56.1%) or ΔBbmsn2/Bbmsn2 strains. However, no differences on reproductive parameters were detected when R. microplus were treated by intra-hemocoel injection using low (800 conidia/tick) doses for all strains tested; R. microplus injected with high doses of wild-type or mutant strains (10 6 conidia/tick) died before laying eggs (~48 h after treatment). SEM analyses of B. bassiana infection showed similar conidial germination and formation of pseudo-appressoria on tick cuticle. Histological sections of ticks treated with the wild-type or ΔBbmsn2/Bbmsn2 strains showed fungal penetration through the cuticle, and into the tick interior. Hyphae of ΔBbmsn2 , however, did not appear to penetrate or breach the tick exocuticle 120 h after treatment. Protease activity was lower on tick cuticles treated with ΔBbmsn2 than those treated with the wild-type or ΔBbmsn2/Bbmsn2 strains. These data show that loss of the Msn2 transcription factor reduced B. bassiana virulence against R. microplus , but did not interfere with conidial germination, appressoria formation or sporulation on tick cadavers, and plays only a minimal role once the cuticle is breached. Our results indicate that the BbMsn2 transcription factor acts mainly during the fungal penetration process and that decreased protease production may be one mechanism that contributes to the inability of the mutant strain to breach the tick cuticle., Competing Interests: ÉF is Associate Editor for the section Fungi-Animal Interactions in Frontiers in Fungal Biology, and Guest Associate Editor for the section Invertebrate Physiology, in the research topic: Entomopathogenic Fungi for the Control of Arthropods - Frontiers in Physiology. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Muniz, Ribeiro-Silva, Arruda, Keyhani and Fernandes.)

Chemical insecticides remain the main strategy to combat mosquito-borne diseases, but the growing threat of insecticide resistance prompts the urgent need to develop alternative, ecofriendly, and sustainable vector control tools. Entomopathogenic fungi can overcome insecticide resistance and represent promising biocontrol tools for the control of mosquitoes. However, insects have evolved robust defense mechanisms against infection. Better understanding of mosquito defenses against fungal infection is critical for improvement of fungal efficacy. Here, we show that as the pathogenic fungus Beauveria bassiana penetrates into the host hemocoel, mosquitoes increase expression of the let-7 and miR-100 microRNAs (miRNAs). Both miRNAs translocate into fungal hyphae to specifically silence the virulence-related genes sec2p and C6TF , encoding a Rab guanine nucleotide exchange factor and a Zn(II) 2 Cys 6 transcription factor, respectively. Inversely, expression of a let-7 sponge (anti-let-7) or a miR-100 sponge (anti-miR-100) in the fungus efficiently sequesters the corresponding translocated host miRNA. Notably, B. bassiana strains expressing anti-let-7 and anti-miR-100 are markedly more virulent to mosquitoes. Our findings reveal an insect defense strategy that employs miRNAs to induce cross-kingdom silencing of pathogen virulence-related genes, conferring resistance to infection., Competing Interests: The authors declare no competing interest.

Holotrichia oblita (Coleoptera: Scarabaeidae) and some other scarab beetles are the main soil-dwelling pests in China. Bacillus thuringiensis (Bt) and Beauveria bassiana (Bb) are entomopathogens that have been used as biocontrol agents of various pests. However, scarab larvae especially H. oblita exhibited strong adaptability to these pathogens. Compared to other scarabs, H. oblita could form a specific soil egg case (SEC) structure surrounding its eggs, and young larvae complete the initial development process inside this structure. In this study, we investigated the role of SEC structure and microorganisms from SEC and egg surface in pathogen adaptability. 16S rRNA gene analysis revealed low bacterial richness and high community unevenness in egg surface, with Proteobacteria, Firmicutes, Bacteroidetes and Fusobacteria dominating. In terms of OTUs composition analysis, the data show that the egg surface contains a large number of unique bacteria, indicating that the egg bacterial community may be derived from maternal transmission. Furthermore, we found that all culturable bacteria isolated from egg surface possessed antimicrobial activity against both Bt and Bb. The Pseudomonas bacteria with a significantly higher abundance in egg surface showed strong Bt- and Bb antagonistic ability. In conclusion, this study demonstrated a unique and antimicrobial bacterial community of H. oblita egg surface, which may contribute to its adaptability. Furthermore, the specific SEC structure surrounding the H. oblita eggs will provide a stable microenvironment for the eggs and egg surface bacteria, which probably provides more advantages for H. oblita adaptation ability.

Entomopathogenic fungi have high potential for controlling insect pests, although the slow killing speed has blocked their widespread application. To increase the virulence of entomopathogenic fungi, genetic modification can be employed. Egf1.0 is an immunosuppressive protein encoded by polydnavirus, carried by parasitoid wasp Microplitis demolitor, which blocks the prophenoloxidase (PPO) activation response of host insects. In this study, we explored the feasibility of genetically modifying entomopathogenic fungi with increased virulence by expressing Egf1.0. In comparison with the wild-type parents, the median lethal concentration (LC 50 ) of Beauveria bassiana expressing Egf1.0 against Helicoverpa armigera was reduced by 2.7-fold, and the median lethal time (LT 50 ) was reduced by 22.8%. In vitro assay showed that recombinant Egf1.0 was able to inhibit the PPO activation response of H. armigera. In vivo assay revealed that the expression of Egf1.0 in B. bassiana caused a higher degree of suppression to PPO activation response of H. armigera. These assays suggested that the increased virulence of the transgenic fungi is due to the increased ability to suppress the host insect's immune response. Moreover, colony growth, conidia yield, and germination assays revealed that the expression of Egf1.0 in B. bassiana had no effect on its growth and development. In conclusion, the expression of Egf1.0 can significantly enhance the pathogenicity of B. bassiana against host insects., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Culturing the entomopathogenic fungus, Beauveria bassiana , under high glucose concentrations coupled with high aeration results in a fungal developmental shift from hyphal growth to mostly blastospores (yeast-like cells). The underlying molecular mechanisms involved in this shift remain elusive. A systematic transcriptome analysis of the differential gene expression was preformed to uncover the fungal transcriptomic response to osmotic and oxidative stresses associated with the resulting high blastospore yield. Differential gene expression was compared under moderate (10% w/v) and high (20% w/v) glucose concentrations daily for three days. The RNAseq-based transcriptomic results depicted a higher proportion of downregulated genes when the fungus was grown under 20% glucose than 10%. Additional experiments explored a broader glucose range (4, 8, 12, 16, 20% w/v) with phenotype assessment and qRT-PCR transcript abundance measurements of selected genes. Antioxidant, calcium transport, conidiation, and osmosensor-related genes were highly upregulated in higher glucose titers (16-20%) compared to growth in lower glucose (4-6%) concentrations. The class 1 hydrophobin gene ( Hyd1 ) was highly expressed throughout the culturing. Hyd1 is known to be involved in spore coat rodlet layer assembly, and indicates that blastospores or another cell type containing hydrophobin 1 is expressed in the haemocoel during the infection process. Furthermore, we found implications of the HOG signaling pathway with upregulation of homologous genes Ssk2 and Hog1 for all fermentation time points under hyperosmotic medium (20% glucose). These findings expand our knowledge of the molecular mechanisms behind blastospore development and may help facilitate large-scale industrial production of B. bassiana blastospores for pest control applications., Competing Interests: Author GMM was employed by Brazilian Agricultural Research Corporation. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Mascarin, Iwanicki, Ramirez, Delalibera and Dunlap.)

Beauveria bassiana is a species complex whose isolates show considerable natural genetic variability. However, little is known about how this genetic diversity affects the fungus performance. Herein, we characterized the diversity of genes involved in various mechanisms of the infective cycle of 42 isolates that have different growth rates, thermotolerance and virulence. The analysed genes showed general genetic diversity measured as non-synonymous changes (NSC) and copy number variation (CNV), with most of them being subjected to positive episodic diversifying selection. Correlation analyses between NSC or CNV and the isolate virulence, thermotolerance and growth rate revealed that various genes shaped the biological features of the fungus. Lectin-like, mucin signalling, Biotrophy associated and chitinase genes NSCs correlated with the three biological features of B. bassiana. In addition, other genes (i.e. DNA photolyase and cyclophilin B) that had relatively conserved sequences, had variable CNs across the isolates which were correlated with the variability of either virulence or thermotolerance of B. bassiana isolates. The data obtained is important for a better understanding of population structure, ecological and potential impact when isolates are used as mycoinsecticides and can justify industrialization of new isolates.

Subtilisin-like Pr1 proteases of insect-pathogenic fungi are a large family of extracellular cuticle-degrading enzymes that presumably determine a capability of hyphal invasion into insect hemocoel through normal cuticle infection, but remain poorly understood although often considered as virulence factors for genetic improvement of fungal potential against pests. Here, we report that not all of 11 Pr1 family members necessarily function in Beauveria bassiana , an ancient wide-spectrum pathogen evolved insect pathogenicity ~200 million years ago. These Pr1 proteases are phylogenetically similar to or distinct from 11 homologues (Pr1A-K) early named in Metarhizium anisopliae complex, a young entomopathogen lineage undergoing molecular evolution toward Pr1 diversification, and hence renamed Pr1A1/A2, Pr1B1-B3, Pr1 C, Pr1F1-F4,4 and Pr1 G, respectively. Multiple analyses of all single gene-deleted and rescued mutants led to the recognition of five conserved members (Pr1C, Pr1G, Pr1A2, Pr1B1, and Pr1B2) contributing significantly to the fungal pathogenicity to insect. The conserved Pr1 proteases were proven to function only in cuticle degradation, individually contribute 19-29% to virulence, but play no role in post-infection cellular events critical for fungal killing action. Six other Pr1 proteases were not functional at all in either cuticle degradation during host infection or virulence-related cellular events post-infection. Therefore, only the five conserved proteases are collectively required for, and hence mark evolution of, insect pathogenicity in B. bassiana . These findings provide the first referable base for insight into the evolution of Pr1 family members in different lineages of fungal insect pathogens.

Entomopathogenic fungi naturally infect insect hosts in environment. Fungal invasion and host immune defense are still in the progress of co-evolution. In this study, entomopathogenic fungus Beauveria bassiana and lepidopteran insect Galleria mellonella were used to investigate host cellular immunity and fungal strategy to evade host defense. First of all, genome-wide expression revealed the transcriptomic responses of hemocytes to insect mycopathogen, which dynamically varied during infection process. Enrichment analysis indicated that differentially expressed genes were primarily involved in metabolism, cellular process and immune system. Notably, cellular response involved a series of hydrolytic enzyme and antimicrobial peptide genes which were sorted together in clustering analysis. In B. bassiana , a cell-wall protein gene ( BbCwp ) contributes to fungal development in host hemocoel and virulence. RT-qPCR analyses indicated that infection by Δ BbCwp mutant strain caused the up-regulated expression of a series of immunity-related genes, including β-1, 3-glucan recognition protein, hydrolytic enzyme and antimicrobial peptide genes. Disruption of BbCwp resulted in a significant change in conidial lectin-binding feature and the enhanced encapsulation by the host hemocytes. After being treated with hydrolytic enzymes, Δ BbCwp mutant displayed a significantly enhanced sensitivity to osmotic and oxidative stresses. In conclusion, fungal invasion initiates comprehensive physiological responses in the host hemocytes. For mycopathogen, cell-wall protein plays an important role in fungal evasion of immunity defense and colonization in host. Our studies provide an initial framework for exploring more mechanistic details about the fungus-host interaction.

ENA1 and ENA2 are P-type IID/ENA Na + /K + -ATPases required for cellular homeostasis in yeasts but remain poorly understood in filamentous fungal insect pathogens. Here, we characterized seven genes encoding five ENA1/2 homologues (ENA1a-c and ENA2a/b) and two P-type IIC/NK Na + /K + -ATPases (NK1/2) in Beauveria bassiana , an insect-pathogenic fungus serving as a main source of fungal insecticides worldwide. Most of these genes were highly responsive to alkaline pH and Na + /K + cues at transcription level. Cellular Na + , K + and H + homeostasis was disturbed only in the absence of ena1a or ena2b . The disturbed homeostasis featured acceleration of vacuolar acidification, elevation of cytosolic Na + /K + level at pH 5.0 to 9.0, and stabilization of extracellular H + level to initial pH 7.5 during a 5-day period of submerged incubation. Despite little defect in hyphal growth and asexual development, the Δ ena1a and Δ ena2b mutants were less tolerant to metal cations (Na + , K + , Li + , Zn 2+ , Mn 2+ and Fe 3+ ), cell wall perturbation, oxidation, non-cation hyperosmolarity and UVB irradiation, severely compromised in insect pathogenicity via normal cuticle infection, and attenuated in virulence via hemocoel injection. The deletion mutants of five other ENA and NK genes showed little change in vacuolar pH and all examined phenotypes. Therefore, only ENA1a and ENA2b evidently involved in both transmembrane and vacuolar activities are essential for cellular cation homeostasis, insect pathogenicity and multiple stress tolerance in B. bassiana . These findings provide a novel insight into ENA1a- and ENA2b-dependent vacuolar pH stability, cation-homeostatic process and fungal fitness to host insect and environment.

Background: Insect-killing fungal species, Beauveria bassiana, is as an environment-friendly pest management tool, and many isolates are on the track of industrialization. However, some of B. bassiana isolates show similar morphology and virulence against insect pests, and so it is hard to differentiate them. Herein we used two patented isolates, ERL836 and JEF-007, and investigated their virulence against western flower thrips, Frankliniella occidentalis, and further analyzed genome structures and transcriptional responses when interacting with cuticles of thrips to see possible differences on the initial step of fungal infection., Results: The two isolates showed no significant differences in fungal growth, conidial production, and virulence against thrips, and they were structurally similar in genome. But, in transcription level, ERL836 appeared to infect thrips easily, while JEF-007 appeared to have more difficulty. In the GO analysis of ERL836 DEGs (differentially expressed genes), the number of up-regulated genes was much larger than that of down-regulated genes, when compared to JEF-007 DEGs (more genes down-regulated). Interestingly, in the enrichment analysis using shared DEGs between two infecting isolates, plasma membrane-mediated transporter activity and fatty acid degradation pathway including cytochrome P450 were more active in infecting ERL836., Conclusion: The two B. bassiana isolates had similar morphology and virulence as well as genome structure, but in transcription level they differently interacted with the cuticle of western flower thrips. This comparative approach using shared DEG analysis could be easily applied to characterize the difference of the two B. bassiana isolates, JEF-007 and ERL836.

FK506-sensitive proline rotamases (FPRs), also known as FK506-binding proteins (FKBPs), can mediate immunosuppressive drug resistance in budding yeast but their physiological roles in filamentous fungi remain opaque. Here, we report that three FPRs (cytosolic/nuclear 12.15-kD Fpr1, membrane-associated 14.78-kD Fpr2 and nuclear 50.43-kD Fpr3) are all equally essential for cellular Ca 2+ homeostasis and contribute significantly to calcineurin activity at different levels in the insect-pathogenic fungus Beauveria bassiana although the deletion of fpr1 alone conferred resistance to FK506. Radial growth, conidiation, conidial viability and virulence were less compromised in the absence of fpr1 or fpr2 than in the absence of fpr3, which abolished almost all growth on scant media and reduced growth moderately on rich media. The Δfpr3 mutant was more sensitive to Na + , K + , Mn 2+ , Ca 2+ , Cu 2+ , metal chelate, heat shock and UVB irradiation than was Δfpr2 while both mutants were equally sensitive to Zn 2+ , Mg 2+ , Fe 2+ , H 2 O 2 and cell wall-perturbing agents. In contrast, the Δfpr1 mutant was less sensitive to fewer stress cues. Most of 32 examined genes involved in DNA damage repair, Na + /K + detoxification or osmotolerance and Ca 2+ homeostasis were downregulated sharply in Δfpr2 and Δfpr3 but rarely so affected in Δfpr1, coinciding well with their phenotypic changes. These findings uncover important, but differential, roles of three FPRs in the fungal adaptation to insect host and environment and provide novel insight into their essential roles in calcium signalling pathway., (© 2020 John Wiley & Sons Ltd.)

Background: Beauveria bassiana (B. bassiana) is a famous entomopathogenic fungus that could parasitize on hundreds of insect species, which are being used as an environmentally friendly mycoinsecticide. Nevertheless, the possible effect of genetic diversity of these B. bassiana isolates from different hosts on virulence has not been explored before. In order to explore that issue, we compared the genome sequences among seventeen B. bassiana isolates from 17 different insects using whole genome re-sequencing, with B. bassiana strain ARSEF 2860 as the reference genome., Results: There were a total of 10,098 missense mutated genes, 720 positively selected genes were identified in 17 strains of B. bassiana. Among these, two genes with high frequency mutations encode the toxin-producing non-ribosomal peptide synthase (NRPS) protein. Seven genes undergoing positive selection were enriched in the two-component signaling pathway that is known to regulate the fungal toxicity. In addition, the domain changes of three positively selected genes are also directly related to the virulence plasticity. Besides, the functional categorization of mutated genes showed that most of them involved in the biological functions of toxic proteins involved in., Conclusions: Based on our data, our results indicate that several mutated genes and positively selected genes may underpin virulence of B. bassiana towards hosts during infection process, which provide an insight into the potential effects of natural variation on the virulence of B. bassiana, which will be useful in screening out potential virulence factors in B. bassiana.

Entomopathogenic fungi are one of the key regulators of insect populations in nature. Some species such as Beauveria bassiana with a wide host range have been developed as promising alternatives to chemical insecticides for the biocontrol of insect pests. However, the long-term persistence of the released strains, the effect on non-target hosts and local fungal populations remains elusive, but they are considerable concerns with respect to environmental safety. Here we report the temporal features of the Beauveria population genomics and evolution over 20 years after releasing exotic strains to control pine caterpillar pests. We found that the isolates within the biocontrol site were mostly of clonal origins. The released strains could persist in the environment for a long time but with low recovery rates. Similar to the reoccurrence of host jumping by local isolates, the infection of non-target insects by the released strains was evident to endemically occur in association with host seasonality. No obvious dilution effect on local population structure was evident by the releases. However, the population was largely replaced by genetically divergent isolates once per decade but evolved with a pattern of balancing selection and towards expansion through adaptation, non-random outcrossing and isolate migration. This study not only unveils the real-time features of entomopathogenic fungal population genomics and evolution but also provides added values to alleviate the concerns of environmental safety regarding the biocontrol application of mycoinsecticides.

RAD23 can repair yeast DNA lesions through nucleotide excision repair (NER), a mechanism that is dependent on proteasome activity and ubiquitin chains but different from photolyase-depending photorepair of UV-induced DNA damages. However, this accessory NER protein remains functionally unknown in filamentous fungi. In this study, orthologous RAD23 in Beauveria bassiana , an insect-pathogenic fungus that is a main source of fungal insecticides, was found to interact with the photolyase PHR2, enabling repair of DNA lesions by degradation of UVB-induced cytotoxic (6-4)-pyrimidine-pyrimidine photoproducts under visible light, and it hence plays an essential role in the photoreactivation of UVB-inactivated conidia but no role in reactivation of such conidia through NER in dark conditions. Fluorescence-labeled RAD23 was shown to normally localize in the cytoplasm, to migrate to vacuoles in the absence of carbon, nitrogen, or both, and to enter nuclei under various stresses, which include UVB, a harmful wavelength of sunlight. Deletion of the rad23 gene resulted in an 84% decrease in conidial UVB resistance, a 95% reduction in photoreactivation rate of UVB-inactivated conidia, and a drastic repression of phr2 A yeast two-hybrid assay revealed a positive RAD23-PHR2 interaction. Overexpression of phr2 in the Δ rad23 mutant largely mitigated the severe defect of the Δ rad23 mutant in photoreactivation. Also, the deletion mutant was severely compromised in radial growth, conidiation, conidial quality, virulence, multiple stress tolerance, and transcriptional expression of many phenotype-related genes. These findings unveil not only the pleiotropic effects of RAD23 in B. bassiana but also a novel RAD23-PHR2 interaction that is essential for the photoprotection of filamentous fungal cells from UVB damage. IMPORTANCE RAD23 is able to repair yeast DNA lesions through nucleotide excision in full darkness, a mechanism distinct from photolyase-dependent photorepair of UV-induced DNA damage but functionally unknown in filamentous fungi. Our study unveils that the RAD23 ortholog in a filamentous fungal insect pathogen varies in subcellular localization according to external cues, interacts with a photolyase required for photorepair of cytotoxic (6-4)-pyrimidine-pyrimidine photoproducts in UV-induced DNA lesions, and plays an essential role in conidial UVB resistance and reactivation of UVB-inactivated conidia under visible light rather than in the dark, as required for nucleotide excision repair. Loss-of-function mutations of RAD23 exert pleiotropic effects on radial growth, aerial conidiation, multiple stress responses, virulence, virulence-related cellular events, and phenotype-related gene expression. These findings highlight a novel mechanism underlying the photoreactivation of UVB-impaired fungal cells by RAD23 interacting with the photolyase, as well as its essentiality for filamentous fungal life., (Copyright © 2020 American Society for Microbiology.)

Cereal cyst nematodes cause serious yield losses of wheat in Hunaghuai winter wheat growing region in China. Beauveria bassiana 08F04 isolated from the surface of cysts is a promising biological control agent for cereal cyst nematodes. As the colonization capacity is a crucial criteria to assess biocontrol effectiveness for a microbial agent candidate, we aimed to label B. bassiana 08F04 for efficient monitoring of colonization in the soil. The binary pCAM-gfp plasmid containing sgfp and hph was integrated into B. bassiana 08F04 using the Agrobacterium tumefaciens-mediated transformation. The transformation caused a significant change in mycelial and conidial yields, and in extracellular chitinase activity in some transformants. The cultural filtrates of some transformants also decreased acetylcholinesterase activity and the survival of Heterodera filipjevi second-stage juveniles relative to the wild-type strain. One transformant (G10) had a growth rate and biocontrol efficacy similar to the wild-type strain, so it was used for a pilot study of B. bassiana colonization conducted over 13 weeks. Real-time PCR results and CFU counts revealed that the population of G10 increased quickly over the first 3 weeks, then decreased slowly over the following 4 weeks before stabilizing. In addition, the application of wild-type B. bassiana 08F04 and transformant G10 significantly reduced the number of H. filipjevi females in roots by 64.4% and 60.2%, respectively. The results of this study have practical applications for ecological, biological and functional studies of B. bassiana 08F04 and for bionematicide registration., Competing Interests: The authors have declared that no competing interests exist.