The regulation of BbLaeA on the production of beauvericin and bassiatin in Beauveria bassiana.

Beauvericin and bassiatin are two valuable compounds with various bioactivities biosynthesized by the supposedly same nonribosomal peptide synthetase BbBEAS in entomopathogenic fungus Beauveria bassiana. To evaluate the regulatory effect of global regulator LaeA on their production, we constructed BbLaeA gene deletion and overexpression mutants, respectively. Deletion of BbLaeA resulted in a decrease of the beauvericin titer, while overexpression of BbLaeA increased its production by 1-2.26 times. No bassiatin could be detected in ΔBbLaeA and wild type strain of B. bassiana, but 4.26-5.10 µg/mL bassiatin was produced in OE::BbLaeA. Furthermore, additional metabolites with increased production in OE::BbLaeA were isolated and identified as primary metabolites. Among them, 4-hydroxyphenylacetic acid showed antibacterial bioactivity against Ralstonia solanacearum. These results indicated that BbLaeA positively regulates the production of beauvericin, bassiatin and various bioactive primary metabolites.

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.

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.

The Drosophila Baramicin polypeptide gene protects against fungal infection.

The fruit fly Drosophila melanogaster combats microbial infection by producing a battery of effector peptides that are secreted into the haemolymph. Technical difficulties prevented the investigation of these short effector genes until the recent advent of the CRISPR/CAS era. As a consequence, many putative immune effectors remain to be formally described, and exactly how each of these effectors contribute to survival is not well characterized. Here we describe a novel Drosophila antifungal peptide gene that we name Baramicin A. We show that BaraA encodes a precursor protein cleaved into multiple peptides via furin cleavage sites. BaraA is strongly immune-induced in the fat body downstream of the Toll pathway, but also exhibits expression in other tissues. Importantly, we show that flies lacking BaraA are viable but susceptible to the entomopathogenic fungus Beauveria bassiana. Consistent with BaraA being directly antimicrobial, overexpression of BaraA promotes resistance to fungi and the IM10-like peptides produced by BaraA synergistically inhibit growth of fungi in vitro when combined with a membrane-disrupting antifungal. Surprisingly, BaraA mutant males but not females display an erect wing phenotype upon infection. Here, we characterize a new antifungal immune effector downstream of Toll signalling, and show it is a key contributor to the Drosophila antimicrobial response.

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.

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.

Dominant egg surface bacteria of Holotrichia oblita (Coleoptera: Scarabaeidae) inhibit the multiplication of Bacillus thuringiensis and Beauveria bassiana.

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 as the microbial frontline against the alien Eucalyptus pest Gonipterus platensis in Brazil.

The eucalyptus snout beetle (ESB), Gonipterus platensis, is endemic to Australia but has become a major invasive, destructive pest of Brazilian eucalyptus plantations. Efforts to develop insecticides based on entomopathogenic fungi against ESB are limited by the lack of known virulent strains. We therefore explored the virulence of indigenous Brazilian strains of major entomopathogenic fungi-Beauveria spp. and Metarhizium anisopliae-against ESB adults. We found widely varying virulence and later capacities for conidial production on infected adult cadavers. Two strains stood out, B. bassiana IBCB-240 and M. anisopliae IBCB-364, as especially lethal for ESB adults under laboratory conditions, sporulated abundantly on infected insects, and also outperformed comparable strains used in commercial mycoinsecticides. Notably, B. bassiana IBCB-240 exhibited lower LT50 values at low inoculum levels (≤ 107 conidia mL-1) and smaller LC50 values than M. anisopliae IBCB-364. Taken together, this study emphasizes natural variation in virulence among indigenous Beauveria and Metarhizium strains against ESB adults and identifies fungal strains with superior lethality to existing commercialized strains for managing this eucalyptus pest in Brazil.

Molecular insight into the endophytic growth of Beauveria bassiana within Phaseolus vulgaris in the presence or absence of Tetranychus urticae.

Entomopathogenic fungi are an important factor in the natural regulation of arthropod populations. Moreover, some can exist as an endophyte in many plant species and establish a mutualistic relationship. In this study, we have investigated the endophytic growth of Beauveria bassiana within different tissues of Phaseolus vulgaris in the presence and absence of Tetranuychus urticae. After the colonization of the B. bassiana within the internal tissues of P. vulgaris. The susceptibility of T. urticae appeared to depend on the life stage where high, moderate, and low mortalities were recorded among adults, nymphs, and eggs, respectively. In addition, this study provided, for the first time, molecular insight into the endophytic growth of B. bassiana by analyzing the expression of several genes involved in the development of the entomopathogenic fungi at 0-, 2-, and 7- days post-inoculation. B. bassiana displayed preferential tissue colonization within P. vulgaris that can be put into the following order based on the detection rate: leaf > stem > root. After analyzing the development-implicated genes (degrading enzymes, sugar transporter, hydrophobins, cell wall synthesis, secondary metabolites, stress management), the most remarkable finding is the detection of behavioral change between parasitic and endophytic Beauveria during post-penetration events. This study elucidates the tri-trophic interaction between fungus-plant-herbivore.

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.

Development of a fluid-bed coating process for soil-granule-based formulations of Metarhizium brunneum, Cordyceps fumosorosea or Beauveria bassiana.

AIM: Granule-based products of solid state fermented micro-organisms are available for biocontrol. Because liquid fermentation has several advantages, we investigated fluid-bed coating with liquid fermented biomass. METHODS AND RESULTS: Biomass containing mycelium or mycelium and submerged spores of the entomopathogenic fungi Metarhizium brunneum, Cordyceps fumosorosea and Beauveria bassiana were produced in liquid culture, separated and different biomass concentrations were adjusted. Based on the examined thermo-tolerance, we defined fluid-bed coating adjustments and investigated granule colonization and sporulation on granules. Granule colonization depended on the biomass concentration and strain. For C. fumosorosea and B. bassiana, concentrations of 0·003%dry weight resulted in nearly 100% granule colonization, for M. brunneum with concentrations of 0·7%dry weight in only 50%. The conidiation on granules in sterile soil was highly influenced by the moisture content. Because the granule colonization of M. brunneum was unsatisfactory, we pre-coated nutrients followed by coating with biomass, submerged spores or conidia. Malt extract had a positive effect on the granule colonization for biomass and submerged spores. Furthermore, aerial conidia can also be coated. CONCLUSIONS: Fluid-bed coating of fungal biomass is suitable for the development of granules. SIGNIFICANCE AND IMPACT OF THIS STUDY: With this technology, cost-efficient biocontrol products can be developed.

Involvement of BbTpc1, an important Zn(II)2Cys6 transcriptional regulator, in chitin biosynthesis, fungal development and virulence of an insect mycopathogen.

Chitin is one of the major components of the fungal cell wall and contributes to the mechanical strength and shape of the fungal cell. Zn(II)2Cys6 transcription factors are unique to the fungal kingdom and have a variety of functions in some fungi. However, the mechanisms by which Zn(II)2Cys6 proteins affect entomopathogenic fungi are largely unknown. Here, we characterized the Zn(II)2Cys6 transcription factor BbTpc1 in the insect pathogenic fungus Beauveria bassiana. Disruption of BbTpc1 resulted in a distinct changes in vegetative growth and septation patterns, and a significant decrease in conidia and blastospore yield. The ΔBbTpc1 mutant displayed impaired resistance to chemical stresses and heat shock and attenuated virulence in topical and intrahemocoel injection assays. Importantly, the ΔBbTpc1 mutant had an abnormal cell wall with altered wall thickness and chitin synthesis, which were accompanied by transcriptional repression of the chitin synthetase family genes. In addition, comparative transcriptomics revealed that deletion of BbTpc1 altered fungal asexual reproduction via different genetic pathways. These data revealed that BbTpc1 regulates fungal development, chitin synthesis and biological control potential in B. bassiana.

Biotransformation of Methoxyflavones by Selected Entomopathogenic Filamentous Fungi.

The synthesis and biotransformation of five flavones containing methoxy substituents in the B ring: 2'-, 3'-, 4'-methoxyflavones, 2',5'-dimethoxyflavone and 3',4',5'-trimethoxyflavone are described. Strains of entomopathogenic filamentous fungi were used as biocatalysts. Five strains of the species Beauveria bassiana (KCh J1.5, J2.1, J3.2, J1, BBT), two of the species Beauveria caledonica (KCh J3.3, J3.4), one of Isaria fumosorosea (KCh J2) and one of Isaria farinosa (KCh KW 1.1) were investigated. Both the number and the place of attachment of the methoxy groups in the flavonoid structure influenced the biotransformation rate and the amount of nascent products. Based on the structures of products and semi-products, it can be concluded that their formation is the result of a cascading process. As a result of enzymes produced in the cells of the tested strains, the test compounds undergo progressive demethylation and/or hydroxylation and 4-O-methylglucosylation. Thirteen novel flavonoid 4-O-methylglucosides and five hydroxy flavones were isolated and identified.

Three proline rotamases involved in calcium homeostasis play differential roles in stress tolerance, virulence and calcineurin regulation of Beauveria bassiana.

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 Ca2+ 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+ , Mn2+ , Ca2+ , Cu2+ , metal chelate, heat shock and UVB irradiation than was Δfpr2 while both mutants were equally sensitive to Zn2+ , Mg2+ , Fe2+ , H2 O2 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 Ca2+ 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.

Differential susceptibility of blastospores and aerial conidia of entomopathogenic fungi to heat and UV-B stresses.

We investigated the comparative susceptibility to heat and UV-B radiation of blastospores and aerial conidia of Metarhizium spp. (Metarhizium robertsii IP 146, Metarhizium anisopliae s.l. IP 363 and Metarhizium acridum ARSEF 324) and Beauveria bassiana s.l. (IP 361 and CG 307). Conidia and blastospores were produced in solid or liquid Adámek-modified medium, respectively, and then exposed to heat (45 ± 0.2 °C) in a range of 0 (control) to 360 min; the susceptibility of fungal propagules to heat exposures was assessed to express relative viability. Similarly, both propagules of each isolate were also exposed to a range of 0 (control) to 8.1 kJ m-2 under artificial UV-B radiation. Our results showed that fungal isolates, propagule types and exposure time or dose of the stressor source play critical roles in fungal survival challenged with UV-B and heat. Conidia of ARSEF 324, IP 363, IP 146 and IP 361 exposed to heat survived significantly longer than their blastospores, except for blastospores of CG 307. Conidia and blastospores of IP 146 and IP 363 were equally tolerant to UV-B radiation. We claim that blastospores of certain isolates may be promising candidates to control arthropod pests in regions where heat and UV-B are limiting environmental factors.

Characterization of three FK506-binding proteins in the entomopathogenic fungus Beauveria bassiana.

FK506 binding proteins (FKBPs) participate in regulation of diverse biological processes. However, the role of these proteins in insect-pathogenic fungi is far from well understood. To investigate the functions of FKBPs in Beauveria bassiana, a widely used entomopathogenic fungus for control of insect pests, we identify three putative FKBP genes, Bbfkbp12, Bbfkbp15, and Bbfkbp50, in the fungus. Gene-disruption experiments show that loss of Bbfkbp12 results in a significant increase of resistance of B. bassiana against the immunosuppressive compounds FK506 and rapamycin, while loss of Bbfkbp50 leads to the resistance to the ergosterol synthesis inhibitor lovastatin. Transcription assays of calcineurin (CaN)- and mTOR (mammalian target of rapamycin)-downstream target genes confirm that BbFKBP12 is the target of both FK506 and rapamycin, associated with CaN- and mTOR-signal pathways in B. bassiana. GFP-tagging of the proteins shows that BbFKBP12 and BbFKBP15 localize in cytoplasm while BbFKBP50 in nucleus. Our results provide useful information for the study of functions of CaN- and mTOR-mediated signaling, and ergosterol synthesis in the entomopathogenic fungi.

Participation of a MADS-box transcription factor, Mb1, in regulation of the biocontrol potential in an insect fungal pathogen.

The cell wall is crucial for fungal growth, proliferation and interaction with the environment and host. Understanding the regulatory mechanism of cell wall integrity may help with improvement of fungal biocontrol agents. Here, a putative target of the cell wall integrity pathway-involved Slt2 MAP kinase, Mb1, an orthologue of MADS-box transcription factor Rlm1, was characterized in an economically important insect fungal pathogen, Beauveria bassiana. Mb1 disruption mutant (ΔMb1) displayed reduced growth and increased conidial production on minimal medium but not on rich-nutrient media, which is different from ΔSlt2 to a great extent. Loss of Mb1 resulted in a significant increase in sensitivity to cell wall-perturbing agents (Congo red and calcofluor white), with alteration in cell composition that was inconsistent with ΔSlt2 strain, including increased chitin content and reduced chitin-binding ß-1, 3/1,6-glucan levels in the absence of any stress. Transcription levels of 15 chitin synthesis and metabolism-associated and 17 Pkc1-Slt2 CWI (cell wall integrity) pathway, glucan synthesis, and cell wall remodeling enzyme synthesis-involved genes were significantly increased and repressed in ΔMb1 strain, respectively, some of which were verified to be the targets of Mb1. Insect bioassays revealed decreased virulence for the ΔMb1 strain in both topical and intrahemocoel injection assays. Our results demonstrated that Mb1 control fungal biocontrol potential-associated traits, including growth, conidiation and cell wall integrity, in B. bassiana. The difference of Mb1 and Slt2 in contribution to cell wall integrity is discussed.

Mitochondrial fission is necessary for mitophagy, development and virulence of the insect pathogenic fungus Beauveria bassiana.

AIMS: Mitochondrial fission is an essential dynamics that maintains mitochondrial morphology and function. This study seeks to determine the roles of mitochondrial fission in the filamentous entomopathogenic fungus Beauveria bassiana. MATERIAL AND METHODS: Three fission-related genes (BbFis1, BbMdv1 and BbDnm1) were functionally characterized via protein intracellular localization and construction of gene disruption mutants. RESULTS: Mitochondrial localization was only observed for BbFis1 which interacts with BbMdv1, but BbMdv1 did not have interaction with BbDnm1. Single disruption mutant of three genes generated the elongated and enlarged mitochondria which could not be eliminated via the mitophagy. Three mutant strains displayed the reduced ATP synthesis and vegetative growth compared with the wild type. Three genes were involved in the early stage of conidiation and unnecessary for the late stage. However, all three genes significantly contribute to blastospore development under submerged condition, and the loss of BbMdv1 had the greatest effects compared with the losses of BbFis1 or BbDnm1. Finally, disruption of three genes significantly attenuated fungal virulence, but their mutations had different influences. CONCLUSIONS: In addition to their consistent roles in mitochondrial division and mitophagy, three fission-related genes perform divergent roles in the development and virulence of the entomopathogenic fungus B. bassiana. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that mitochondrial fission is associated with lifecycle of B. bassiana. These findings provide information for the manipulation of fungal physiology and facilitate the application of entomopathogenic fungi.

Spores of Beauveria bassiana and Trichoderma lignorum as a bioinsecticide for the control of Atta cephalotes.

BACKGROUND: The leafcutter ant (Atta cephalotes) is associated with losses in the agricultural sector, due to its defoliating activity; for its control, biological, mechanical and chemical methods have been developed, the latter associated with adverse effects on human and environmental health. This research validated in the field for the control of the leafcutter ant (A. cephalotes) using a mixture of Beauveria bassiana and Trichoderma lignorum spores. METHODS: The effectiveness from the combination of spores of B. bassiana and T. lignorum with an initial concentration of 2 × 109 spores/ml, in the following proportions of B. bassiana and T. lignorum, A (1:1), of each fungus. It was evaluated within the university campus, comparing it with two commercial formulations, Mycotrol (B. bassiana) and Mycobac (T. lignorum). Additionally, this formulation was evaluated in 49 nests distributed 16 in 14 locations in Colombia. The formulation application was carried out by direct application, using a pump at a speed of 10 ml/m2. The effectiveness was estimated from the reduction of the flow of ants, evaluating the statistically significant differences using the ANOVA and Tukey-test. RESULTS: Effective control of 90% of the nests was observed in the field phase in 60 days, except in nests with areas > 50 m2 that were located in regions with high rainfall (annual average precipitation above 7000 mm), such as Buenaventura. CONCLUSIONS: In this work, it was demonstrated that the combination of B. bassiana and T. lignorum spores represent a viable alternative for the control of the leafcutter ant, in which the effectiveness is related to several factors, including the size of the nest and the rainfall in the area.

Comparative efficacy of two mycotoxins against Spodoptera litura Fab. And their non-target activity against Eudrilus eugeniae Kinb.

Entomopathogenic fungi are feasible and effective against the agricultural pest. In the current research we investigated the bioactive comparison of two widely accepted entmopathogens (Beauveria bassiana (Bals.) Vuill. and Metarhizium anisopliae, (basionym)) against the Spodoptera litura (Fab.) through the assessment of larval tolerance and regulation of antioxidants and non-target impact on the earth worm, E. eugeniae, along with commercial pesticides. The entomopathogenic fungus exposure resulted in the modification of the levels of detoxification enzymes as well as significant increases in catalase and superoxide dismutase activity after exposure to the entomopathogenic fungus. Bioassay results showed that B. bassiana and M. anisopliae displayed larval mortality against third and fourth instars. Correspondingly, sub-lethal concentrations of B. bassiana showed development impairment as compared to M. anisopliae. Gut-histology revealed that mycotoxins dosage (4 × 105) showed significant changes in the midgut tissues as compared to control larvae. The non-target screening through artificial soil assay on the earth worm E. eugeniae, with mycotoxins B. bassiana (5 × 108 conidia/ml/kg) and M. anisopliae (5 × 108 conidia/ml/kg) showed less toxicity as compared to Monocrotophos (10 ppm/kg). Current results suggest that the fungal mycotoxins of M. anisopliae and B. bassiana significantly reduce the development of lepidopteran pests, while having only lesser impact on beneficial earthworms.