Suppression of the insect cuticular microbiomes by a fungal defensin to facilitate parasite infection.

Insects can assemble defensive microbiomes on their body surfaces to defend against fungal parasitic infections. The strategies employed by fungal pathogens to combat host cuticular microbiotas remains unclear. Here, we report the identification and functional characterization of the defensin-like antimicrobial gene BbAMP1 encoded by the entomopathogenic fungus Beauveria bassiana. The mature peptide of BbAMP1 can coat fungal spores and can be secreted by the fungus to target and damage Gram-positive bacterial cells. Significant differences in insect survival were observed between the wild-type and BbAMP1 mutant strains during topical infection but not during injection assays that bypassed insect cuticles. Thus, BbAMP1 deletion considerably reduced fungal virulence while gene overexpression accelerated the fungal colonization of insects compared with the wild-type strain in natural infections. Topical infection of axenic Drosophila adults evidenced no difference in fly survivals between strains. However, the gnotobiotic infections with the addition of Gram-positive but not Gram-negative bacterial cells in fungal spore suspensions substantially increased the survival of the flies treated with ∆BbAMP1 compared to those infected by the wild-type and gene-overexpression strains. Bacterial colony counts and microbiome analysis confirmed that BbAMP1 could assist the fungus to manipulate insect surface bacterial loads. This study reveals that fungal defensin can suppress the host surface defensive microbiomes, which underscores the importance to extend the research scope of fungus-host interactions.

OTU7B Modulates the Mosquito Immune Response to Beauveria bassiana Infection via Deubiquitination of the Toll Adaptor TRAF4.

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.

Late effects of Beauveria bassiana on larval stages of Aedes aegypti Linneo, 1762 (Diptera: Culicidae) / Efeitos tardios de Beauveria bassiana nos estágios larvais de Aedes aegypti Linneo, 1762 (Diptera: Culicidae)

Aedes aegypti is a culicide that has gained relevance over the years due to its ability to transmit various viruses that cause diseases in humans that all the years cause high mortality rates in the world population. The main problem is that Ae. aegypti has managed to establish and maintain a close relationship with humans and their habitat, which is why the search for alternatives to control vector populations becomes imperative. The objective of the present work was to study the effects of two Beauveria bassiana strains on Aedes aegypti. Third instar larvae of Ae. aegypti in 250 mL plastic containers were inoculated with the GHA and NB3 strains at different concentrations (1.5 × 104, 1.5× 105, 1.5 × 106 and 1.5 × 107 conidia/mL). The NB3 strain presented highest mortality values with 63% in the highest concentration i.e., 1.5 × 107, while for the GHA strain the highest mortality value was 30.7% at the same concentration. The results showed significant difference in mortality with respect to the strain and days post treatment (P = 0.0001), but not with respect to the conidial concentration (P = 0.634). The average mortality of larvae per day for the NB3 for different concentrations ranged from 20 to 25 larvae per day, while for the GHA daily mortality ranged from 5 to 12 larvae. In post-treatment mortality, the highest mortality was recorded in the third stage larvae for the NB3, while for GHA the highest percentage mortality was observed in individuals who managed to reach the adult state. The findings of the current research depicted the noteworthy role of B. bassiana for the management of an important vector of human disease.

O Aedes aegypti é um culicida que vem ganhando relevância ao longo dos anos devido à sua capacidade de transmitir diversos vírus causadores de doenças em humanos que ao longo dos anos ocasionam altas taxas de mortalidade na população mundial. O principal problema é que Ae. aegypti tem conseguido estabelecer e manter uma relação próxima com o homem e seu habitat, por isso a busca por alternativas para o controle das populações de vetores torna-se imperativa. O objetivo do presente trabalho foi estudar os efeitos de duas cepas de Beauveria bassiana sobre Ae. aegypti. Larvas de terceiro instar de Ae. aegypti em recipientes plásticos de 250 mL foram inoculados com as cepas GHA e NB3 em diferentes concentrações (1.5 × 104, 1.5 × 105, 1.5 × 106 e 1.5 × 107 conídios/mL). A cepa NB3 apresentou os maiores valores de mortalidade com 63% na concentração mais alta, ou seja, 1.5 × 107, enquanto para a cepa GHA o maior valor de mortalidade foi 30.7% na mesma concentração. Os resultados mostraram diferença significativa na mortalidade com relação à cepa e dias pós-tratamento (P = 0.0001), mas não com relação à concentração de conídios (P = 0.634). A mortalidade média de larvas por dia para o NB3 para diferentes concentrações variou de 20 a 25 larvas por dia, enquanto para o GHA a mortalidade diária variou de 5 a 12 larvas. Na mortalidade pós-tratamento, a maior mortalidade foi registrada nas larvas de terceiro estágio para o NB3, enquanto para o GHA o maior percentual de mortalidade foi observado em indivíduos que conseguiram atingir o estado adulto. Os resultados da pesquisa atual retratam o papel notável de B. bassiana no manejo de um importante vetor de doenças humanas.

Lipid-Binding Aegerolysin from Biocontrol Fungus Beauveria bassiana.

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.

SPINK7 Recognizes Fungi and Initiates Hemocyte-Mediated Immune Defense Against Fungal Infections.

Serine protease inhibitors of Kazal-type (SPINKs) were widely identified in vertebrates and invertebrates, and played regulatory roles in digestion, coagulation, and fibrinolysis. In this study, we reported the important role of SPINK7 in regulating immune defense of silkworm, Bombyx mori. SPINK7 contains three Kazal domains and has 6 conserved cysteine residues in each domain. Quantitative real-time PCR analyses revealed that SPINK7 was exclusively expressed in hemocytes and was upregulated after infection with two fungi, Saccharomyces cerevisiae and Candida albicans. Enzyme activity inhibition test showed that SPINK7 significantly inhibited the activity of proteinase K from C. albicans. Additionally, SPINK7 inhibited the growth of three fungal spores, including S. cerevisiae, C. albicans, and Beauveria bassiana. The pathogen-associated molecular patterns (PAMP) binding assays suggested that SPINK7 could bind to ß-D-glucan and agglutinate B. bassiana and C. albicans. In vitro assays were performed using SPINK7-coated agarose beads, and indicated that SPINK7 promoted encapsulation and melanization of agarose beads by B. mori hemocytes. Furthermore, co-localization studies using immunofluorescence revealed that SPINK7 induced hemocytes to aggregate and entrap the fungi spores of B. bassiana and C. albicans. Our study revealed that SPINK7 could recognize fungal PAMP and induce the aggregation, melanization, and encapsulation of hemocytes, and provided valuable clues for understanding the innate immunity and cellular immunity in insects.

Iron homeostasis in the absence of ferricrocin and its consequences in fungal development and insect virulence in Beauveria bassiana.

The putative ferricrocin synthetase gene ferS in the fungal entomopathogen Beauveria bassiana BCC 2660 was identified and characterized. The 14,445-bp ferS encodes a multimodular nonribosomal siderophore synthetase tightly clustered with Fusarium graminearum ferricrocin synthetase. Functional analysis of this gene was performed by disruption with the bar cassette. ΔferS mutants were verified by Southern and PCR analyses. HPLC and TLC analyses of crude extracts indicated that biosynthesis of ferricrocin was abolished in ΔferS. Insect bioassays surprisingly indicated that ΔferS killed the Spodoptera exigua larvae faster (LT50 59 h) than wild type (66 h). Growth and developmental assays of the mutant and wild type demonstrated that ΔferS had a significant increase in germination under iron depletion and radial growth and a decrease in conidiation. Mitotracker staining showed that the mitochondrial activity was enriched in ΔferS under both iron excess and iron depletion. Comparative transcriptomes between wild type and ΔferS indicated that the mutant was increased in the expression of eight cytochrome P450 genes and those in iron homeostasis, ferroptosis, oxidative stress response, ergosterol biosynthesis, and TCA cycle, compared to wild type. Our data suggested that ΔferS sensed the iron excess and the oxidative stress and, in turn, was up-regulated in the antioxidant-related genes and those in ergosterol biosynthesis and TCA cycle. These increased biological pathways help ΔferS grow and germinate faster than the wild type and caused higher insect mortality than the wild type in the early phase of infection.

Molecular characterization of two dsRNAs that could correspond to the genome of a new mycovirus that infects the entomopathogenic fungus Beauveria bassiana.

The entomopathogenic fungus Beauveria bassiana is used worldwide for biological control of insects. Seven dsRNA segments were detected in a single B. bassiana strain, RCEF1446. High-throughput sequencing indicated the presence of three mycoviruses in RCEF1446. Two were identified as the known mycoviruses Beauveria bassiana victorivirus 1 and Beauveria bassiana polymycovirus 1, and the novel mycovirus was designated as "Beauveria bassiana bipartite mycovirus 1" (BbBV1). The complete sequence of the BbBV1 is described here. The mycovirus contains two dsRNA segments. The RNA 1 (dsRNA 4) of BbBV1 is 2,026 bp in length, encoding a RNA-dependent RNA polymerase (RdRp) (68.54 kDa), while the RNA 2 (dsRNA 6) is 1,810 bp in length, encoding a hypothetical protein (35.55 kDa) with unknown function. Moreover, the amino acid sequence of RdRp showed the highest sequence identity of 62.31% to Botryosphaeria dothidea bipartite mycovirus 1. Phylogenetic analysis based on RdRp sequences revealed that BbBV1 represents a distinct lineage of unassigned dsRNA mycoviruses infecting fungi.

Bioassay of Some Indigenous Entomopathogens for Controlling Rhynchophorus ferrugineus, Olivier in Saudi Arabia.

<b>Background and Objective:</b> The red palm weevil is a dangerous date palm pests that cannot be controlled with chemical pesticides only. As a result of the justified concerns of the negative use of synthetic insecticides on human health and the environment. So on, candidate eco-friendly micro-organisms isolated from KSA agri-ecosystems were evaluated in controlling RPW. <b>Materials and Methods:</b> Some indigenous entomopathogenic fungi and bacteria were isolated from naturally infected RPW larvae and adults and evaluated as alternative control methods. <b>Results:</b> The infection of RPW larvae with entomopathogenic fungi and bacteria under natural conditions was higher than in adults. <i>Beauveria bassiana </i>was the most prevalent followed by <i>Aspergillus </i>sp., <i>Metarhizium anisopliae</i>, <i>Mucor</i> sp., <i>Cladosporium chlorocephalum</i>. In contrast, both <i>Bacillus</i> <i>thuringiensis</i> and <i>Bacillus popilliae</i> formed 73.9 and 26.1%, respectively. From the 7th day, mortalities (%) increased gradually and recorded the highest mortalities with 21st days after treatment and recorded 93.33, 66.70, 53.36, 46.69 and 60.00% when treated with <i>B. bassiana</i>, <i>M. anisopliae</i>, <i>C. chlorosphalum</i>, <i>Mucor</i> sp. and <i>Aspergillus</i> sp., respectively. <b>Conclusion:</b> Although there was evidence indicating midgut damage and feeding inhibition among larvae that survived the treatments, instead of lower activity of <i>B. thuringiensis</i> against <i>R. ferrugineus</i> immature stages may refer to that, Both species of <i>Bacillus</i> were more virulent as the days 15-21 post-treatment.

A Short-Type Peptidoglycan Recognition Protein 1 (PGRP1) Is Involved in the Immune Response in Asian Corn Borer, Ostrinia furnacalis (Guenée).

The insect immune response is initiated by the recognition of invading microorganisms. Peptidoglycan recognition proteins (PGRPs) function primarily as pattern recognition receptors by specifically binding to peptidoglycans expressed on microbial surfaces. We cloned a full-length cDNA for a PGRP from the Asian corn borer Ostrinia furnacalis (Guenée) and designated it as PGRP1. PGRP1 mRNA was mainly detected in the fat bodies and hemocytes. Its transcript levels increased significantly upon bacterial and fungal challenges. Purified recombinant PGRP1 exhibited binding activity to the gram-positive Micrococcus luteus, gram-negative Escherichia coli, entomopathogenic fungi Beauveria bassiana, and yeast Pichia pastoris. The binding further induced their agglutination. Additionally, PGRP1 preferred to bind to Lys-type peptidoglycans rather than DAP-type peptidoglycans. The addition of recombinant PGRP1 to O. furnacalis plasma resulted in a significant increase in phenoloxidase activity. The injection of recombinant PGRP1 into larvae led to a significantly increased expression of several antimicrobial peptide genes. Taken together, our results suggest that O. furnacalis PGRP1 potentially recognizes the invading microbes and is involved in the immune response in O. furnacalis.

The Tudor Domain-Containing Protein BbTdp1 Contributes to Fungal Cell Development, the Cell Cycle, Virulence, and Transcriptional Regulation in the Insect Pathogenic Fungus Beauveria bassiana.

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 ΔBbTdp1 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 ΔBbTdp1 mutants. Transcriptomic data revealed 4,094 differentially expressed genes (|log2 ratio| > 2 and a q value of <0.05) between ΔBbTdp1 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 G2/M and G0/G1 phases of blastospores, and enzyme-linked immunosorbent assay confirmed that the level of phosphorylated cyclin-dependent kinase 1 (Cdk1) in the ΔBbTdp1 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.

BbWor1, a Regulator of Morphological Transition, Is Involved in Conidium-Hypha Switching, Blastospore Propagation, and Virulence in Beauveria bassiana.

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, ΔBbWor1 prominently decreased blastospore yield, shortened the G0/G1 phase, and prolonged the G2/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 ΔBbWor1 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.

Effect of earthworm Eisenia fetida epidermal mucus on the vitality and pathogenicity of Beauveria bassiana.

Beauveria bassiana is one of the most widely studied and used entomopathogenic fungus as biopesticide. In the biological control of pests, B. bassiana will persist in the soil after application, and will inevitably contact with earthworms, especially the epigeic earthworm species. So, what are the effects of earthworm and its epidermal mucus on the activity of B. bassiana? We employed the epigeic earthworm Eisenia fetida, B. bassiana TST05 strain, and the insect Atrijuglans hetaohei mature larvae to study the impact of earthworm epidermal mucus on the vitality and pathogenicity of B. bassiana to insect. Methods included scanning electron microscope observation, detection of spore germination, fungal extracellular enzyme activity, and infection testing to A. hetaohei. The results showed that the B. bassiana spores may attach to the cuticle of E. fetida but they could be covered by the epidermal mucus and became rough and shrunken. After treatment with the epidermal mucus, the spore germination and extracellular enzymes of B. bassiana was significantly inhibited. Inoculation of A. hetaohei larvae with a mixture of B. bassiana and mucus showed that the mucus could reduce the pathogenicity of B. bassiana to the insect, resulting in a slower disease course and lower mortality. It was concluded that the epidermal mucus of the earthworm E. fetida can inhibit the activity of B. bassiana, as well as the infectivity and pathogenicity of fungus to target insects. However, after treatment with epidermal mucus the surviving B. bassiana still had certain infectivity to insects. This is of great significance for the application of B. bassiana in biological control of pests.

Insects defend against fungal infection by employing microRNAs to silence virulence-related genes.

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)2Cys6 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.

Earthworms and their cutaneous excreta can modify the virulence and reproductive capability of entomopathogenic nematodes and fungi.

Earthworms are ecological engineers that can contribute to the displacement of biological control agents such as the entomopathogenic nematodes (EPNs) and fungi (EPF). However, a previous study showed that the presence of cutaneous excreta (CEx) and feeding behavior of the earthworm species Eisenia fetida (Haplotaxida: Lumbricidae) compromise the biocontrol efficacy of certain EPN species by reducing, for example, their reproductive capability. Whether this phenomenon is a general pattern for the interaction of earthworms-entomopathogens is still unknown. We hypothesized that diverse earthworm species might differentially affect EPN and EPF infectivity and reproductive capability. Here we investigated the interaction of different earthworm species (Eisenia fetida, Lumbricus terrestris, and Perionyx excavatus) (Haplotaxida) and EPN species (Steinernema feltiae, S. riojaense, and Heterorhabditis bacteriophora) (Rhabditida) or EPF species (Beauveria bassiana and Metarhizium anisopliae) (Hypocreales), in two independent experiments. First, we evaluated the application of each entomopathogen combined with earthworms or their CEx in autoclaved soil. Hereafter, we studied the impact of the earthworms' CEx on entomopathogens applied at two different concentrations in autoclaved sand. Overall, we found that the effect of earthworms on entomopathogens was species-specific. For example, E. fetida reduced the virulence of S. feltiae, resulted in neutral effects for S. riojaense, and increased H. bacteriophora virulence. However, the earthworm P. excavates increased the virulence of S. feltiae, reduced the activity of H. bacteriophora, at least at specific timings, while S. riojaense remained unaffected. Finally, none of the EPN species were affected by the presence of L. terrestris. Also, the exposure to earthworm CEx resulted in a positive, negative or neutral effect on the virulence and reproduction capability depending on the earthworm-EPN species interaction. Concerning EPF, the impact of earthworms was also differential among species. Thus, E. fetida was detrimental to M. anisopliae and B. bassiana after eight days post-exposure, whereas Lumbricus terrestris resulted only detrimental to B. bassiana. In addition, most of the CEx treatments of both earthworm species decreased B. bassiana virulence and growth. However, the EPF M. anisopliae was unaffected when exposed to L. terrestris CEx, while the exposure to E. fetida CEx produced contrasting results. We conclude that earthworms and their CEx can have positive, deleterious, or neutral impacts on entomopathogens that often coinhabit soils, and that we must consider the species specificity of these interactions for mutual uses in biological control programs. Additional studies are needed to verify these interactions under natural conditions.

iTRAQ-based quantitative proteomic analysis of silkworm infected with Beauveria bassiana.

Beauveria bassiana is a harmful pathogen to the economically important insect silkworm, always causes serious disease to the silkworm, which results in great losses to the sericulture industry. In order to explore the silkworm (Bombyx mori) response to B. bassiana infection, differential proteomes of the silkworm responsive to B. bassiana infection were identified with isobaric tags for relative and absolute quantitation (iTRAQ) at the different stage of the 3rd instar silkworm larvae. Among the 5040 proteins identified with confidence level of ≥95 %, total 937 proteins were differentially expressed, of which 488 proteins were up-regulated and 449 proteins were down-regulated. 23, 15, 250, 649 differentially expressed proteins (DEPs) were reliably quantified by iTRAQ analysis in the B. bassiana infected larvae at 18, 24, 36, 48 h post infection (hpi) respectively. Based on GO annotations, 6, 4, 128, 316 DEPs were involved in biological processes, 12, 5, 143, 376 DEPs were involved in molecular functions, and 6, 3, 108, 256 DEPs were involved in cell components at 18, 24, 36, 48 hpi respectively. KEGG pathway analysis displayed that 18, 12, 210, 548 DEPs separately participated in 63, 35, 201, 264 signal transduction pathways at different time of infection, and moreover a higher proportion of DEPs involved in metabolic pathways. The cluster analysis on the DEPs of different infection stages distinguished a co-regulated DEP, lysozyme precursor, which was up-regulated at both the mRNA level and the protein level, indicating that the lysozyme protein kept playing an important role in defending the silkworm against B. bassiana infection. This was the first report using an iTRAQ approach to analyze proteomes of the whole silkworm against B. bassiana infection, which contributes to better understanding the defense mechanisms of silkworm to B. bassiana infection and provides important experimental data for the identification of key factors involved in the interaction between the pathogenic fungus and its host.

A p53-like transcription factor, BbTFO1, contributes to virulence and oxidative and thermal stress tolerances in the insect pathogenic fungus, Beauveria bassiana.

The p53-like transcription factor (TF) NDT80 plays a vital role in the regulation of pathogenic mechanisms and meiosis in certain fungi. However, the effects of NDT80 on entomopathogenic fungi are still unknown. In this paper, the NDT80 orthologue BbTFO1 was examined in Beauveria bassiana, a filamentous entomopathogenic fungus, to explore the role of an NDT80-like protein for fungal pest control potential. Disruption of BbTFO1 resulted in impaired resistance to oxidative stress (OS) in a growth assay under OS and a 50% minimum inhibitory concentration experiment. Intriguingly, the oxidation resistance changes were accompanied by transcriptional repression of the two key antioxidant enzyme genes cat2 and cat5. ΔBbTFO1 also displayed defective conidial germination, virulence and heat resistance. The specific supplementation of BbTFO1 reversed these phenotypic changes. As revealed by this work, BbTFO1 can affect the transcription of catalase genes and play vital roles in the maintenance of phenotypes associated with the biological control ability of B. bassiana.

Comparative Virulence of Metarhizium anisopliae and Four Strains of Beauveria bassiana Against House Fly (Diptera: Muscidae) Adults With Attempted Selection for Faster Mortality.

Entomopathogenic fungi such as Beauveria bassiana (Balsamo) Vuillemin and Metarhizium anisopliae/brunneum (Metchnikoff)/Petch have shown promising results for managing the house fly, Musca domestica L. A primary challenge of using these biological control agents (BCAs) in field situations is the time required to induce high adult house fly mortality, typically 6-7 d post-exposure. In this study, virulence of M. anisopliae (strain F52) and four B. bassiana strains were compared. The B. bassiana strains GHA and HF23 are used in commercial products and those were compared with two strains that were isolated from house flies on dairy farms (NFH10 and L90). Assays were conducted by exposing adult house flies to fungal-treated filter paper disks for 2 h. The lethal time to 50% mortality (LT50) at the high concentration of 1 × 109 conidia ranged from 3.8 to 5.2 d for all five strains. GHA, NFH10, and L90 killed flies faster than M. anisopliae strain F52; HF23 did not differ from either the M. anisopliae or the other B. bassiana strains. Attempts with the NFH10 strain to induce faster fly mortality through selection across 10 fungal to fly passages did not result in shorter time to fly death of the selected strain compared with the unselected strain.

Characterisation of Metarhizium majus (Hypocreales: Clavicipitaceae) isolated from the Western Cape Province, South Africa.

Entomopathogenic fungi (EPF) are important soil-dwelling entomopathogens, which can be used as biological control agents against pest insects. EPF are capable of causing lethal epizootics in pest insect populations in agroecosystems. During a survey of the orchard soil at an organic farm, different EPF species were collected and identified to species level, using both morphological and molecular techniques. The EPF were trapped from soil samples taken from an apricot orchard. The traps, which were baited in the laboratory, used susceptible host insects, including the last-instar larvae of Galleria mellonella (wax moth larvae) and Tenebrio molitor (mealworm larvae). The potential pathogenicity of the local Metarhizium majus isolate was tested and verified using susceptible laboratory-reared last-instar T. molitor larvae. The identification of the M. majus isolated from South African soil was verified using both morphological and molecular techniques. The occurrence of M. majus in the South African soil environment had not previously been reported.

Management alternatives for Carmenta theobromae (Busck, 1910) (Lepidoptera: Sesiidae) and Simplicivalva ampliophilobia (Lepidoptera: Cossidae), limiting pests of guava in Colombia.

The larval stages of Carmenta theobromae Busck (1910) and Simplicivalva ampliophilobia Davis, Gentili-Poole and Mitter (2008) attack the subcortical zone and pith in guava trees, respectively, in the first productive nucleus of fruit trees in Colombia: Hoya del Río Suárez (HRS). The presence of pest insects has been reported in 98% of the farms sampled in HRS (n = 124), with up to 96 and 11 simultaneous larvae per tree, respectively. Although the aspects of the basic biology and life cycle of both pests have been resolved, there are no strategies for managing populations in the field. Therefore, the aim of this study was to evaluate different management alternatives under laboratory and field conditions in HRS. In laboratory conditions, a completely randomized design was used in two separate experiments, each with six treatments: T1: Spinosad (a mixture of Spinosad A and D); T2: S-1,2-di(ethoxycarbonyl) ethyl 0,0-dimethylphosphorodithioate (chemical control); T3: Lecanicillium lecanii; T4: Beauveria bassiana; T5: Mix of B. bassiana and B. brongniartii, and T6: distilled water (control). The number of dead larvae per replicate per treatment was evaluated (DL), with experimental units of five and three larvae, respectively. In the field, to the two best alternatives found for each pest in the laboratory, pruning and keeping the area around the plants free of weeds were added as cultural management, in two separate additional experiments, each with three larvae as experimental unit per treatment. For C. theobromae, the best laboratory alternatives were chemical control (DL: 3.78) and L. lecanii (DL: 2.33), followed without statistical differences by B. bassiana (DL: 1.67). In the field, the virulence of B. bassiana improved (DL: 3), and together with pruning and keeping the area around the plants clear of weeds (DL: 3), they stood out as the best alternatives. For S. ampliophilobia under laboratory conditions, the best alternatives were Spinosad (2.74) and chemical control (DL: 2.66), without significant difference. In the field, there were no statistical differences between the alternatives, except for the control. This statistical parity of cultural practices, and biological and chemical management is an argument in favor of the use of the former to the detriment of the third, especially when the harmful effects of the molecule S-1,2 di (ethoxycarbonyl) ethyl 0, 0-dimethyl phosphorodithioate have been proven in air, water and agricultural soils, in addition to its association with thyroid cancer in humans. This is a strong argument to favor the use of synergies of cultural and biological management methods framed in IPM, as opposed to the use of chemical agents whose harmful effects are strongly documented, and whose use is becoming increasingly prohibited.

Gene diversity explains variation in biological features of insect killing fungus, Beauveria bassiana.

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.