Emulsifiable oil-formulated Beauveria bassiana competes with imidacloprid for seasonal control of cereal aphids in Zhejiang, China.

BACKGROUND: Alternatives to neonicotinoids against cereal aphids are needed to mitigate aphid resistance and non-target effects. The emulsifiable oil formulations of two Beauveria bassiana strains, namely Bb registered as a mycoinsecticide and TBb overexpressing an endogenous virulence factor, were tested for seasonal control of cereal aphids at the elongating (April 7) to milk ripening (May 12) stages of winter wheat crop in Yuhang, Zhejiang. Each of three field trials consisted of blank control and the treatments (three randomized 100-m2 plots per capita) of each fungal strain sprayed biweekly at rates of 1.0 × 1013 and 1.5 × 1013 conidia ha-1 and 10% imidacloprid WP sprayed biweekly at a label rate. RESULTS: Tiller infestation percentage and aphid density in the 5-week field trials after the first spray were reduced to 18.7-22.4% and 9.1-12.4 aphids per tiller in the fungal treatments, and 12.8-25.3% and 2.8-20.9 aphids per tiller in the chemical treatment, contrasting with 49.2-60.3% and 37.1-108.5 aphids per tiller in the control. Percent control efficacies (±SD) computed with weekly aphid densities over the period averaged 84.0 ± 1.6 and 85.3 ± 1.8 versus 78.0 ± 4.0 and 79.9 ± 3.2 in the high-rate versus low-rate treatments of Bb and TBb, respectively, and 84.5 ± 7.8 in the chemical treatment. Imidacloprid showed faster kill action but more variable efficacy than the fungal treatments throughout the trials. CONCLUSION: Either Bb or TBb formulation competes with imidacloprid in reducing percent infestation and aphid density. The overall efficacy was significantly higher in the treatments of TBb than of Bb. © 2024 Society of Chemical Industry.

Timing of Fungal Insecticide Application to Avoid Solar Ultraviolet Irradiation Enhances Field Control of Rice Planthoppers.

Thechemical control of rice planthoppers (RPH)is prohibited in annual rice-shrimp rotation paddy fields. Here, the fungal insecticides Beauveria bassiana ZJU435 and Metarizhium anisoplae CQ421 were tested for control of RPH populations dominated by Nilaparvata lugens in three field trials. During four-week field trials initiated from the harsh weather of high temperatures and strong sunlight, the rice crop at the stages from tillering to flowering was effectively protected by fungal sprays applied at 14-day intervals. The sprays of either fungal insecticide after 5:00 p.m. (solar UV avoidance) suppressed the RPH population better than those before 10 a.m. The ZJU435 and CQ421 sprays for UV avoidance versus UV exposure resulted in mean control efficacies of 60% and 56% versus 41% and 45% on day 7, 77% and 78% versus 63% and 67% on day 14, 84% and 82% versus 80% and 79% on day 21, and 84% and 81% versus 79% and 75 on day 28, respectively. These results indicate that fungal insecticides can control RPH in the rice-shrimp rotation fields and offer a novel insight into the significance of solar-UV-avoiding fungal application for improved pest control during sunny summers.

Optional strategies for low-risk and non-risk applications of fungal pesticides to avoid solar ultraviolet damage.

BACKGROUND: Solar ultraviolet (UV) irradiation is harmful to formulated conidia as active ingredients of fungal pesticides and hence restrains their field application in sunny days of summer, a season requiring frequent pest controls. This conflict makes it necessary to explore optimal strategies for the application of fungal pesticides to suppress pest populations but avoid solar UV damage during summer. RESULTS: The conidia of Beauveria bassiana, a wide-spectrum fungal pesticide, were tolerable to UVB (major solar UV wavelengths) damage of ≤0.5 J cm-2 . The damage of this upper limit caused a loss of conidial viability and infectivity if not photoreactivated by light exposure after irradiation. Intriguingly, the light exposure resulted in a high photoreactivation rate of UVB-inactivated conidia and an insignificant or marginal difference in insecticidal activity between normal conidia and those photoreactivated. Modeling analysis of solar UVB intensity recorded hourly over the daylight of five sunny summer days from 5:00 am to 7:00 pm at 30° 17'57'' N and 120°5'7'' E revealed a variation of daily accumulated UVB dose from 2.07 to 2.78 J cm-2 , which was far beyond the upper limit. A more tolerable dose of ~0.2 J cm-2 appeared between 3:00 pm and 5:00 pm, and no harmful dose accumulated between 5:00 pm and 7:00 pm. CONCLUSION: Fungal UVB tolerance, fungal photoreactivation capability and the daily accumulation pattern of solar UV irradiation are based to propose an optional strategy for low-risk or non-risk application of fungal pesticides after 3:00 or 5:00 pm during summer. © 2022 Society of Chemical Industry.

FluG and FluG-like FlrA Coregulate Manifold Gene Sets Vital for Fungal Insect-Pathogenic Lifestyle but Not Involved in Asexual Development.

The central developmental pathway (CDP) activator gene brlA is activated by the upstream genes fluG and flbA-flbE in Aspergillus nidulans. Increasing evidences of fungal genome divergence make it necessary to clarify whether such genetic principles fit Pezizomycotina. Previously, fluG disruption resulted in limited conidiation defect and little effect on the expression of brlA and flbA-flbE in Beauveria bassiana possessing the other FluG-like regulator FlrA. Here, single-disruption (SD) mutants of flrA and double-disruption (DD) mutants of flrA and fluG were analyzed to clarify whether FlrA and FluG are upstream regulators of key CDP genes. Despite similar subcellular localization, no protein-protein interaction was detected between FlrA and FluG, suggesting mutual independence. Three flrA SD mutants showed phenotypes similar to those previously described for ΔfluG, including limited conidiation defect, facilitated blastospore production, impaired spore quality, blocked host infection, delayed proliferation in vivo, attenuated virulence, and increased sensitivities to multiple stresses. Three DD mutants resembled the SD mutants in all phenotypes except more compromised pathogenicity and tolerance to heat shock- or calcofluor white-induced stress. No CDP gene appeared in 1,622 and 2,234 genes dysregulated in the ΔflrA and ΔfluG mutants, respectively. The majority (up/down ratio: 540:875) of those dysregulated genes were co-upregulated or co-downregulated at similar levels in the two mutants. These findings unravel novel roles for flrA and fluG in coregulating manifold gene sets vital for fungal adaptation to insect-pathogenic lifestyle and environment but not involved in CDP activation. IMPORTANCE FluG is a core regulator upstream of central developmental pathway (CDP) in Aspergillus nidulans but multiple FluG-like regulators (FLRs) remain functionally uncharacterized in ascomycetes. Our previous study revealed no role for FluG in the CDP activation and an existence of sole FLR (FlrA) in an insect-pathogenic fungus. This study reveals a similarity of FlrA to FluG in domain architecture and subcellular localization. Experimental data from analyses of targeted single- and double-gene knockout mutants demonstrate similar roles of FrlA and FluG in stress tolerance and infection cycle but no role of either in CDP activation. Transcriptomic analyses reveal that FlrA and FluG coregulate a large number of same genes at similar levels. However, the regulated genes include no key CDP gene. These findings uncover that FlrA and FluG play similar roles in the fungal adaptation to insect-pathogenic lifestyle and environment but no role in the activation of CDP.

Fungal insecticidal activity elevated by non-risky markerless overexpression of an endogenous cysteine-free protein gene in Beauveria bassiana.

BACKGROUND: Fungal insecticides are notorious for slow kill action, an intrinsic trait that can be improved by the genetic engineering of an exogenous or endogenous virulence factor. However, transgenic insecticides expressing exogenous toxin and herbicide-resistant marker genes may cause unexpected ecological risks and are hardly permitted for field release due to strict regulatory hurdles. It is necessary to improve biotechnology that can speed up fungal insect-killing action and exclude ecological risk source. RESULTS: A markerless transformation system of Beauveria bassiana, a main source of wide-spectrum fungal insecticides, was reconstructed based on the fungal uridine auxotrophy (Δura3). The system was applied for overexpression of the small cysteine-free protein (120 amino acids) gene cfp previously characterized as a regulator of the fungal virulence and gene expression. Three cfp-overexpressed strains showed much faster kill action to Galleria mellonella larvae than the parental wild-type via normal cuticle infection but no change in vegetative growth and aerial condition. The faster kill action was achieved due to not only significant increases in conidial adherence to insect cuticle and total activity of secreted cuticle-degrading Pr1 proteases and of antioxidant enzymes crucial for collapse of insect immune defense but acceleration of hemocoel localization, proliferation in vivo and host death from mummification. CONCLUSION: The markerless system is free of any foreign DNA fragment as a source of ecologic risk and provides a novel biotechnological approach to enhancing fungal insecticidal activity with non-risky endogenous genes and striding over the regulatory hurdles. © 2022 Society of Chemical Industry.

Molecular basis and regulatory mechanisms underlying fungal insecticides' resistance to solar ultraviolet irradiation.

Resistance to solar ultraviolet (UV) irradiation is crucial for field-persistent control efficacies of fungal formulations against arthropod pests, because their active ingredients are formulated conidia very sensitive to solar UV wavelengths. This review seeks to summarize advances in studies aiming to quantify, understand and improve conidial UV resistance. One focus of studies has been on the many sets of genes that have been revealed in the postgenomic era to contribute to or mediate UV resistance in the insect pathogens serving as main sources of fungal insecticides. Such genetic studies have unveiled the broad basis of UV-resistant molecules including cytosolic solutes, cell wall components, various antioxidant enzymes, and numerous effectors and signaling proteins, that function in developmental, biosynthetic and stress-responsive pathways. Another focus has been on the molecular basis and regulatory mechanisms underlying photorepair of UV-induced DNA lesions and photoreactivation of UV-impaired conidia. Studies have shed light upon a photoprotective mechanism depending on not only one or two photorepair-required photolyases, but also two white collar proteins and other partners that play similar or more important roles in photorepair via interactions with photolyases. Research hotspots are suggested to explore a regulatory network of fungal photoprotection and to improve the development and application strategies of UV-resistant fungal insecticides. © 2021 Society of Chemical Industry.

Essential Role of COP9 Signalosome Subunit 5 (Csn5) in Insect Pathogenicity and Asexual Development of Beauveria bassiana.

Csn5 is a subunit ofthe COP9/signalosome complex in model fungi. Here, we report heavier accumulation of orthologous Csn5 in the nucleus than in the cytoplasm and its indispensability to insect pathogenicity and virulence-related cellular events of Beauveria bassiana. Deletion of csn5 led to a 68% increase in intracellular ubiquitin accumulation and the dysregulation of 18 genes encoding ubiquitin-activating (E1), -conjugating (E2), and -ligating (E3) enzymes and ubiquitin-specific proteases, suggesting the role of Csn5 in balanced ubiquitination/deubiquitination. Consequently, the deletion mutant displayed abolished insect pathogenicity, marked reductions in conidial hydrophobicity and adherence to the insect cuticle, the abolished secretion of cuticle penetration-required enzymes, blocked haemocoel colonisation, and reduced conidiation capacity despite unaffected biomass accumulation. These phenotypes correlated well with sharply repressed or abolished expressions of key hydrophobin genes required for hydrophobin biosynthesis/assembly and of developmental activator genes essential for aerial conidiation and submerged blastospore production. In the mutant, increased sensitivities to heat shock and oxidative stress also correlated with reduced expression levels of several heat-responsive genes and decreased activities of antioxidant enzymes. Altogether, Csn5-reliant ubiquitination/deubiquitination balance coordinates the expression of those crucial genes and the quality control of functionally important enzymes, which are collectively essential for fungal pathogenicity, virulence-related cellular events, and asexual development.

SET1/KMT2-governed histone H3K4 methylation coordinates the lifecycle in vivo and in vitro of the fungal insect pathogen Beauveria bassiana.

Biological control potential of insect-pathogenic fungi against pests is an overall output of various cellular processes regulated by signalling and epigenetic networks. In Beauveria bassiana, mono/di/trimethylation of histone H3 Lys 4 (H3K4me1/me2/m3) was abolished by inactivation of the histone lysine methyltransferase SET1/KMT2, leading to marked virulence loss, reductions in conidial hydrophobicity and adherence to insect cuticle, impeded proliferation in vivo, severe defects in growth and conidiation, and increased sensitivities to cell wall perturbation, H2 O2 and heat shock. Such compromised phenotypes correlated well with transcriptional abolishment or repression of carbon catabolite-repressing transcription factor Cre1, classes I and II hydrophobins Hyd1 and Hyd2 required for cell hydrophobicity, key developmental regulators, and stress-responsive enzymes/proteins. Particularly, expression of cre1, which upregulates hyd4 upon activation by KMT2-mediated H3K4me3 in Metarhizium robertsii, was nearly abolished in the Δset1 mutant, leading to abolished expression of hyd1 and hyd2 as homologues of hyd4. These data suggest that the SET1-Cre1-Hyd1/2 pathway function in B. bassiana like the KMT2-Cre1-Hyd4 pathway elucidated to mediate pathogenicity in M. robertsii. Our findings unveil not only a regulatory role for the SET1-cored pathway in fungal virulence but also its novel role in mediating asexual cycle in vitro and stress responses in B. bassiana.

DIM5/KMT1 controls fungal insect pathogenicity and genome stability by methylation of histone H3K4, H3K9 and H3K36.

Mono-, di- and tri-methylation of histone H3 Lys 9, Lys 4, and Lys 36 (H3K_me1/me2/me3) required for mediation of DNA-based cellular events in eukaryotes usually rely upon the activities of histone lysine methyltransferases (KMTs) classified to the KMT1, KMT2, and KMT3 families, respectively. Here, an H3K9-specific DIM5/KMT1 orthologue, which lacks a C-terminal post-SET domain and localizes mainly in nucleus, is reported to have both conserved and noncanonical roles in methylating the H3 core lysines in Beauveria bassiana, an insect-pathogenic fungus serving as a main source of wide-spectrum fungal insecticides. Disruption of dim5 led to abolishment of H3K9me3 and marked attenuation of H3K4me1/me2, H3K9me1/me2 and H3K36me2. Consequently, the Δdim5 mutant lost the whole insect pathogenicity through normal cuticle infection, and was compromised severely in virulence through cuticle-bypassing infection (hemocoel injection) and also in a series of cellular events critical for the fungal virulence and lifecycle in vivo and in vitro, including reduced hyphal growth, blocked conidiation, impeded proliferation in vivo, altered carbohydrate epitopes, disturbed cell cycle, reduced biosynthesis and secretion of cuticle-degrading enzymes, and increased sensitivities to various stresses. Among 1,201 dysregulated genes (up/down ratio: 712:489) associated with those phenotypic changes, 92 (up/down ratio: 59:33) encode transcription factors and proteins or enzymes involved in posttranslational modifications, implying that the DIM5-methylated H3 core lysines could act as preferential marks of those transcription-active genes crucial for global gene regulation. These findings uncover a novel scenario of DIM5 and its indispensability for insect-pathogenic lifestyle and genome stability of B. bassiana.

Distinctive role of fluG in the adaptation of Beauveria bassiana to insect-pathogenic lifecycle and environmental stresses.

The upstream developmental activation (UDA) pathway comprises three fluG-cored cascades (fluG-flbA, fluG-flbE/B/D and fluG-flbC) that activate the key gene brlA of central developmental pathway (CDP) to initiate conidiation in aspergilli. However, the core role of fluG remains poorly understood in other fungi. Here, we report distinctive role of fluG in the insect-pathogenic lifecycle of Beauveria bassiana. Disruption of fluG resulted in limited conidiation defect, which was mitigated with incubation time and associated with time-course up-regulation/down-regulation of all flb and CDP genes and another fluG-like gene (BBA_06309). In ΔfluG, increased sensitivities to various stresses correlated with repression of corresponding stress-responsive genes. Its virulence through normal cuticle infection was attenuated greatly due to blocked secretion of cuticle-degrading enzymes and delayed formation of hyphal bodies (blastospores) to accelerate proliferation in vivo and host death. In submerged ΔfluG cultures mimicking insect haemolymph, largely increased blastospore production concurred with drastic up-regulation of the CDP genes brlA and abaA, which was associated with earlier up-regulation of most flb genes in the cultures. Our results unveil an essentiality of fluG for fungal adaptation to insect-pathogenic lifecycle and suggest the other fluG-like gene to act as an alternative player in the UDA pathway of B. bassiana.

Two white collar proteins protect fungal cells from solar UV damage by their interactions with two photolyases in Metarhizium robertsii.

The photolyases PHR1 and PHR2 enable photorepair of fungal DNA lesions in the forms of UV-induced cyclobutane pyrimidine dimer (CPD) and (6-4)-pyrimidine-pyrimidone (6-4PP) photoproducts, but their regulation remains mechanistically elusive. Here, we report that the white collar proteins WC1 and WC2 mutually interacting to form a light-responsive transcription factor regulate photolyase expression required for fungal UV resistance in the insect-pathogenic fungus Metharhizum robertsii. Conidial UVB resistance decreased by 54% in Δwc1 and 67% in Δwc2. Five-hour exposure of UVB-inactivated conidia to visible light resulted in photoreactivation rates of 30% and 9% for the Δwc1 and Δwc2 mutants, contrasting to 79%-82% for wild-type and complemented strains. Importantly, abolished transcription of phr1 in Δwc-2 and of phr2 in Δwc1 resulted in incapable photorepair of CDP and 6-4PP DNA lesions in UVB-impaired Δwc2 and Δwc1 cells respectively. Yeast two-hybrid assays revealed interactions of either WC protein with both PHR1 and PHR2. Therefore, the essential roles for WC1 and WC2 in both photorepair of UVB-induced DNA lesions and photoreactivation of UVB-inactivated conidia rely upon their interactions with, and hence transcriptional activation of, PHR1 and PHR2. These findings uncover a novel WC-cored pathway that mediates filamentous fungal response and adaptation to solar UV irradiation.

Essential Roles of Two FRQ Proteins (Frq1 and Frq2) in Beauveria bassiana's Virulence, Infection Cycle, and Calcofluor-Specific Signaling.

Two FRQ proteins (Frq1 and Frq2) distinct in molecular mass and structure coexist in Beauveria bassiana, an asexual insect-pathogenic fungus. Frq1 and Frq2 have been proven to have opposite nuclear rhythms that can persistently activate developmental activator genes and hence orchestrate nonrhythmic conidiation in vitro under light or in darkness. Here, we report the essentiality of either FRQ, but Frq2 being more important than Frq1, for the fungal virulence and infection cycle. The fungal virulence was attenuated significantly more in the absence of frq2 than in the absence of frq1 through either normal cuticle infection or cuticle-bypassing infection by intrahemocoel injection, accompanied by differentially reduced secretion of Pr1 proteases required for the cuticle infection and delayed development of hyphal bodies in vivo, which usually propagate by yeast-like budding in the host hemocoel to accelerate insect death from mycosis. Despite insignificant changes in radial growth under normal, oxidative, and hyperosmotic culture conditions, conidial yields of the Δfrq1 and Δfrq2 mutants on insect cadavers were sharply reduced, and the reduction increased with shortening daylight length on day 9 or 12 after death, indicating that both Frq1 and Frq2 are required for the fungal infection cycle in host habitats. Intriguingly, the Δfrq1 and Δfrq2 mutants showed hypersensitivity and high resistance to cell wall-perturbing calcofluor white, coinciding respectively with the calcofluor-triggered cells' hypo- and hyperphosphorylated signals of Slt2, a mitogen-activated protein kinase (MAPK) required for mediation of cell wall integrity. This finding offers a novel insight into opposite roles of Frq1 and Frq2 in calcofluor-specific signal transduction via the fungal Slt2 cascade.IMPORTANCE Opposite nuclear rhythms of two distinct FRQ proteins (Frq1 and Frq2) coexisting in an asexual fungal insect pathogen have been shown to orchestrate the fungal nonrhythmic conidiation in vitro in a circadian day independent of photoperiod change. This paper reports essential roles of both Frq1 and Frq2, but a greater role for Frq2, in sustaining the fungal virulence and infection cycle since either frq1 or frq2 deletion led to marked delay of lethal action against a model insect and drastic reduction of conidial yield on insect cadavers. Moreover, the frq1 and frq2 mutants display hypersensitivity and high resistance to cell wall perturbation and have hypo- and hyperphosphorylated MAPK/Slt2 in calcofluor white-triggered cells, respectively. These findings uncover a requirement of Frq1 and Frq2 for the fungal infection cycle in host habitats and provide a novel insight into their opposite roles in calcofluor-specific signal transduction through the MAPK/Slt2 cascade.

P-type Na+/K+ ATPases essential and nonessential for cellular homeostasis and insect pathogenicity of Beauveria bassiana.

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 Δena1a and Δena2b mutants were less tolerant to metal cations (Na+, K+, Li+, Zn2+, Mn2+ and Fe3+), 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.

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.

Phenotypic and molecular insights into heat tolerance of formulated cells as active ingredients of fungal insecticides.

Formulated conidia of insect-pathogenic fungi, such as Beauveria and Metarhizium, serve as the active ingredients of fungal insecticides but are highly sensitive to persistent high temperatures (32-35 °C) that can be beyond their upper thermal limits especially in tropical areas and during summer months. Fungal heat tolerance and inter- or intra-specific variability are critical factors and limitations to field applications of fungal pesticides during seasons favoring outbreaks of pest populations. The past decades have witnessed tremendous advances in improving fungal pesticides through selection of heat-tolerant strains from natural isolates, improvements and innovations in terms of solid-state fermentation technologies for the production of more heat-tolerant conidia, and the use of genetic engineering of candidate strains for enhancing heat tolerance. More recently, with the entry into a post-genomic era, a large number of signaling and effector genes have been characterized as important sustainers of heat tolerance in both Beauveria and Metarhizium, which represent the main species used as fungal pesticides worldwide. This review focuses on recent advances and provides an overview into the broad molecular basis of fungal heat tolerance and its multiple regulatory pathways. Emphases are placed on approaches for screening of heat-tolerant strains, methods for optimizing conidial quality linked to virulence and heat tolerance particularly involving cell wall architecture and optimized trehalose/mannitol contents, and how molecular determinants can be exploited for genetic improvement of heat tolerance and pest-control potential. Examples of fungal pesticides with different host spectra and their appropriateness for use in apiculture are given. KEY POINTS: • Heat tolerance is critical for field stability and efficacy of fungal insecticides. • Inter- and intra-specific variability exists in insect-pathogenic fungi. • Optimized production technology and biotechnology can improve heat tolerance. • Fungal heat tolerance is orchestrated by multiple molecular pathways.

Nuclear Ssr4 Is Required for the In Vitro and In Vivo Asexual Cycles and Global Gene Activity of Beauveria bassiana.

Ssr4 serves as a cosubunit of chromatin-remodeling SWI/SNF and RSC complexes in yeasts but remains functionally uncharacterized due to its essentiality for yeast viability. Here, we report pleiotropic effects of the deletion of the ssr4 ortholog nonessential for cell viability in Beauveria bassiana, an asexual insect mycopathogen. The deletion of ssr4 resulted in severe growth defects on different carbon/nitrogen sources, increased hyphal hydrophilicity, blocked hyphal differentiation, and 98% reduced conidiation capacity compared to a wild-type standard. The limited Δssr4 conidia featured an impaired coat with disordered or obscure hydrophobin rodlet bundles, decreased hydrophobicity, increased size, and lost insect pathogenicity via normal cuticle infection and 90% of virulence via intrahemocoel injection. The expression of genes required for hydrophobin biosynthesis and assembly of the rodlet layer was drastically repressed in more hydrophilic Δssr4 cells. Transcriptomic analysis revealed 2,517 genes differentially expressed in the Δssr4 mutant, including 1,505 downregulated genes and 1,012 upregulated genes. The proteins encoded by hundreds of repressed genes were involved in metabolism and/or transport of carbohydrates, amino acids, and lipids, inorganic ion transport and energy production or conversion, including dozens involved in DNA replication, transcription, translation, and posttranslational modifications. However, purified Ssr4 samples showed no DNA-binding activity, implying that the role of Ssr4 in genome-wide gene regulation could rely upon its acting as a cosubunit of the two complexes. These findings provide the first insight into an essential role of Ssr4 in the asexual cycle in vitro and in vivo of B. bassiana and highlights its importance for the filamentous fungal lifestyle.IMPORTANCE Ssr4 is known to serve as a cosubunit of chromatin-remodeling SWI/SNF and RSC complexes in yeasts but has not been functionally characterized in fungi. This study unveils for the first time the pleiotropic effects caused by deletion of ssr4 and its role in mediating global gene expression in a fungal insect pathogen. Our findings confirm an essential role of Ssr4 in hydrophobin biosynthesis and assembly required for growth, differentiation, and development of aerial hyphae for conidiation and conidial adhesion to insect surface and its essentiality for insect pathogenicity and virulence-related cellular events. Importantly, Ssr4 can regulate nearly one-fourth of all genes in the fungal genome in direct and indirect manners, including dozens involved in gene activity and hundreds involved in metabolism and/or transport of carbohydrates, amino acids, lipids, and/or inorganic ions. These findings highlight a significance of Ssr4 for filamentous fungal lifestyle.

Photoprotective Role of Photolyase-Interacting RAD23 and Its Pleiotropic Effect on the Insect-Pathogenic Fungus Beauveria bassiana.

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 Δrad23 mutant largely mitigated the severe defect of the Δrad23 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.

Subtilisin-like Pr1 proteases marking the evolution of pathogenicity in a wide-spectrum insect-pathogenic fungus.

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.

Opposite Nuclear Dynamics of Two FRH-Dominated Frequency Proteins Orchestrate Non-Rhythmic Conidiation in Beauveria bassiana.

Non-rhythmic conidiation favors large-scale production of conidia serving as active ingredients of fungal insecticides, but its regulatory mechanism is unknown. Here, we report that two FREQUENCY (FRQ) proteins (Frq1/2) governed by a unique FRQ-interacting RNA helicase (FRH) orchestrate this valuable trait in Beauveria bassiana, an asexual insect-pathogenic fungus. Frq1 (964 aa) and Frq2 (583 aa) exhibited opposite expression dynamics (rhythms) in nucleus and steadily high expression levels in cytoplasm under light or in darkness no matter whether one of them was present or absent. Such opposite nuclear dynamics presented a total FRQ (pooled Frq1/2) level sufficient to persistently activate central developmental pathway in daytime and nighttime and supports continuous (non-rhythmic) conidiation for rapid maximization of conidial production in a fashion independent of photoperiod change. Importantly, both nuclear dynamics and cytoplasmic stability of Frq1 and Frq2 were abolished in the absence of the FRH-coding gene nonessential for the fungal viability, highlighting an indispensability of FRH for the behaviors of Frq1 and Frq2 in both nucleus and cytoplasm. These findings uncover a novel circadian system more complicated than the well-known Neurospora model that controls rhythmic conidiation, and provide a novel insight into molecular control of non-rhythmic conidiation in B. bassiana.

Colony heating protects honey bee populations from a risk of contact with wide-spectrum Beauveria bassiana insecticides applied in the field.

BACKGROUND: The safety of fungal insecticides to apiculture is a public concern but remains poorly understood. This study seeks to evaluate whether, how and why wide-spectrum Beauveria bassiana insecticides are safe to honey bees in a novel assessment system. RESULTS: Mesonotum dipping with a 108 conidia ml-1 suspension and body contact with conidial suspension in sucrose solution caused high mortalities of adult forager bees at 25 °C optimal for conidial germination and hyphal invasion. Intriguingly, colony sizes in the hives contaminated by the forager bees contacting viable and inactivated conidia at two sites (1.2 km in distance), respectively, showed similar increase percentages (31.7% versus 29.2%) during a 4-week summer period of exposure to environment. No sign of fungal infection was found within each of the monitored colonies. Neither was fungal outgrowth observed on surfaces of bee cadavers cleaned from each hive at either site. Hourly counts of cleaned cadavers from videotapes presented no significant difference in colony-cleaning behavior between the two sites. During the period, in-hive temperatures at both sites were persistently stabilized at approximately 35 °C, which abolished conidial germination and were far above the out-hive temperature range. CONCLUSION: It is colony heating that protects honey bee populations from a risk of forager bees' contact with formulated conidia applied for arthropod pest control. No role was detected for colony self-cleaning behavior in protecting the bee colonies from the risk. © 2020 Society of Chemical Industry.