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Abstract Background The fall armyworm (FAW), Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae), is an aggressive pest species that causes severe economic losses in outbreak regions. Use of FAW natural enemies, particularly native parasitoids, has been suggested as a promising control strategy. Campoletis chlorideae Uchida (Hymenoptera: Ichneumonidae) is a solitary ichneumonid larval endoparasitoid with a broad host range that includes S. frugiperda. However, its parasitism rate, developmental stages, and population dynamics during parasitization of S. frugiperda remain unclear. A field survey was conducted to determine the emergence rate and sex ratio of C. chlorideae, evaluated the biological control performance, and investigated the production potential of it on the individually reared and group-reared S. frugiperda through age-stage, two-sex life tables, respectively. Results The results showed that C. chlorideae parasitizing individually reared FAW resulted for the parasitoid in a lifetime total fecundity (F) of 301.5 ± 16.4 eggs/female, a net reproduction rate (R 0) of 62.03 ± 9.07 adult females/female, an overall life span of females of 28.3 ± 0.52 days, and an intrinsic rate of increase (r) of 0.1946 ± 0.0076 day−1. In contrast, the parameters of F, R 0, overall life span of females, and r were 87.71 ± 6.32 pupae/female, 6.02 ± 1.61 adult females/female, 25.21 ± 0.79 days, and 0.0918 ± 0.0148 day−1 in group-reared S. frugiperda. Conclusions This study suggests that C. chlorideae has a promising biological control potential against S. frugiperda. Future research should focus on developing methods to increase the production of C. chlorideae.

Abstract Background Response to oxidative stress is universal in almost all organisms and the mitochondrial membrane protein, BbOhmm, negatively affects oxidative stress responses and virulence in the insect fungal pathogen, Beauveria bassiana. Nothing further, however, is known concerning how BbOhmm and this phenomenon is regulated. Results Three oxidative stress response regulating Zn2Cys6 transcription factors (BbOsrR1, 2, and 3) were identified and verified via chromatin immunoprecipitation (ChIP)-qPCR analysis as binding to the BbOhmm promoter region, with BbOsrR2 showing the strongest binding. Targeted gene knockout of BbOsrR1 or BbOsrR3 led to decreased BbOhmm expression and consequently increased tolerances to free radical generating compounds (H2O2 and menadione), whereas the ΔBbOsrR2 strain showed increased BbOhmm expression with concomitant decreased tolerances to these compounds. RNA and ChIP sequencing analysis revealed that BbOsrR1 directly regulated a wide range of antioxidation and transcription-associated genes, negatively affecting the expression of the BbClp1 cyclin and BbOsrR2. BbClp1 was shown to localize to the cell nucleus and negatively mediate oxidative stress responses. BbOsrR2 and BbOsrR3 were shown to feed into the Fus3-MAPK pathway in addition to regulating antioxidation and detoxification genes. Binding motifs for the three transcription factors were found to partially overlap in the promoter region of BbOhmm and other target genes. Whereas BbOsrR1 appeared to function independently, co-immunoprecipitation revealed complex formation between BbClp1, BbOsrR2, and BbOsrR3, with BbClp1 partially regulating BbOsrR2 phosphorylation. Conclusions These findings reveal a regulatory network mediated by BbOsrR1 and the formation of a BbClp1-BbOsrR2-BbOsrR3 complex that orchestrates fungal oxidative stress responses.

The rice planthopper, Sogatella furcifera, is a piercing-sucking insect pest of rice, Oryza sativa. It is responsible for significant crop yield losses, and has developed moderate to high resistance to several commonly used chemical insecticides. We investigated the effects of the insect fungal pathogen Isaria javanica, alone and in combination with the chemical insecticide dinotefuran, on S. furcifera under both laboratory and field conditions. Our results show that I. javanica displays high infection efficiency and mortality for different stages of S. furcifera, reducing adult survival, female oviposition and ovary development. Laboratory bioassays showed that the combined use of I. javanica with a low dose (4–16 mg L–1) of dinotefuran resulted in higher mortality in S. furcifera than the use of I. javanica or dinotefuran alone. The combined treatment also had more significant effects on several host enzymes, including superoxide dismutase, catalase, peroxidase, and prophenol oxidase activities. In field trials, I. javanica effectively suppressed populations of rice planthoppers to low levels (22–64% of the level in untreated plots). Additional field experiments showed synergistic effects, i.e., enhanced efficiency, for the control of S. furcifera populations using the combination of a low dose of I. javanica (1×104 conidia mL–1) and a low dose of dinotefuran (~4.8–19.2% of normal field use levels), with control effects of >90% and a population level under 50 insects per 100 hills at 3–14 days post-treatment. Our findings indicate that the entomogenous fungus I. javanica offers an attractive biological control addition as part of the integrated pest management (IPM) practices for the control of rice plant pests.

Leaves of Camellia sinensis plants are used to produce tea, one of the most consumed beverages worldwide, containing a wide variety of bioactive compounds that help to promote human health. Tea cultivation is economically important, and its sustainable production can have significant consequences in providing agricultural opportunities and lowering extreme poverty. Soil parameters are well known to affect the quality of the resultant leaves and consequently, the understanding of the diversity and functions of soil microorganisms in tea gardens will provide insight to harnessing soil microbial communities to improve tea yield and quality. Current analyses indicate that tea garden soils possess a rich composition of diverse microorganisms (bacteria and fungi) of which the bacterial Proteobacteria, Actinobacteria, Acidobacteria, Firmicutes and Chloroflexi and fungal Ascomycota, Basidiomycota, Glomeromycota are the prominent groups. When optimized, these microbes’ function in keeping garden soil ecosystems balanced by acting on nutrient cycling processes, biofertilizers, biocontrol of pests and pathogens, and bioremediation of persistent organic chemicals. Here, we summarize research on the activities of (tea garden) soil microorganisms as biofertilizers, biological control agents and as bioremediators to improve soil health and consequently, tea yield and quality, focusing mainly on bacterial and fungal members. Recent advances in molecular techniques that characterize the diverse microorganisms in tea gardens are examined. In terms of viruses there is a paucity of information regarding any beneficial functions of soil viruses in tea gardens, although in some instances insect pathogenic viruses have been used to control tea pests. The potential of soil microorganisms is reported here, as well as recent techniques used to study microbial diversity and their genetic manipulation, aimed at improving the yield and quality of tea plants for sustainable production.

Bamboo is an economically important crop that has gained prominence as an alternative to wood to reduce deforestation and ecosystem destruction. Diseases of bamboo that typically occur on leaves and stems can cause significant loss, reducing the quality and yield of the bamboo. However, there are few reports identifying the fungal species diversity and potential pathogens of bamboo. Here, we describe four new species of plant fungi from the leaves of bamboo within Fujian provinces, China. Fungi were isolated from diseased leaves collected within Fujian province and identified based on their morphological characteristics and multilocus phylogenies using nucleotide sequences derived from combined datasets of the intervening 5.8S nrRNA gene (ITS), the 28S large subunit of nuclear ribosomal RNA gene (LSU), the large subunit of RNA polymerase I (rpb1), the translation elongation factor 1-α gene (tef1-α), and the partial beta-tubulin gene (tub2). These analyses helped reveal and clarify taxonomic relationships in the family Magnaporthaceae. The new species of bambusicolous fungi identified include two species of Bifusisporella, described as B. fujianensis sp. nov. and B. bambooensis sp. nov., and two species of Apiospora, described as A. fujianensis sp. nov. and A. fuzhouensis sp. nov. This study further expands the characterization and distribution of fungi associated with bamboo.

Members of the fungal order Diaporthales are sac fungi that include plant pathogens (the notorious chestnut blight fungus), as well as saprobes and endophytes, and are capable of colonizing a wide variety of substrates in different ecosystems, habitats, and hosts worldwide. However, many Diaporthales species remain unidentified, and various inconsistencies within its taxonomic category remain to be resolved. Here, we aimed to identify and classify new species of Diaporthales by using combined morphological and molecular characterization and coupling this information to expand our current phylogenetic understanding of this order. Fungal samples were obtained from dead branches and diseasedleaves of Camellia (Theaceae) and Castanopsis (Fagaceae) in Fujian Province, China. Based on morphological characteristics and molecular phylogenetic analyses derived from the combined nucleotide sequences of loci of the internal transcribed spacer regions with the intervening 5.8S nrRNA gene (ITS), the 28S large subunit of nuclear ribosomal RNA gene (LSU), the translation elongation factor 1-α gene (tef1), the partial beta-tubulin gene (tub2), and partial RNA polymerase II second-largest subunit gene (rpb2), three new species of Diaporthales were identified and characterized. They are as follows: Chrysofolia camelliae sp. nov., Dendrostoma castanopsidis sp. nov., and Pseudoplagiostoma wuyishanense sp. nov. They are described and illustrated. This study extends our understanding of species diversity within the Diaporthales.

Introduction: Odorant-binding proteins (OBPs) are a class of small molecular weight soluble proteins that exist as expanded gene families in all insects, acting as ligand carriers mediating olfaction and other physiological processes. During fungal infection, a subset of insect OBPs were shown to be differentially expressed. Objectives: We tested whether the altered expression of insect OBPs during pathogenic infection plays a role in behavioral or immune interactions between insect hosts and their pathogens. Methods: A wide range of techniques including RNAi-directed knockdown, heterologous protein expression, electrophysiological/behavioral analyses, transcriptomics, gut microbiome analyses, coupled with tandem mass spectrometry ion monitoring, were used to characterize the function of a locust OBP in host behavioral and immune responses. Results: The entomopathogenic fungus Metarhizium anisopliae produces the volatile compound phenylethyl alcohol (PEA) that causes behavioral avoidance in locusts. This is mediated by the locust odorant binding protein 11 (LmOBP11). Expression of LmOBP11 is induced by M. anisopliae infection and PEA treatment. LmOBP11 participates in insect detection of the fungal-produced PEA and avoidance of PEA-contaminated food, but the upregulation of LmOBP11 upon M. anisopliae infection negatively affects the insect immune responses to ultimately benefit successful mycosis by the pathogen. RNAi knockdown of LmOBP11 increases the production of antimicrobial peptides and enhances locust resistance to M. anisopliae infection, while reducing host antennal electrophysiological responses to PEA and locust avoidance of PEA treated food. Also, transcriptomic and gut microbiome analyses reveal microbiome dysbiosis and changes in host genes involved in behavior and immunity. These results are consistent with the elevated expression of LmOBP11 leading to enhanced volatile detection and suppression of immune responses. Conclusion: These findings suggest a crosstalk between olfaction and immunity, indicating manipulation of host OBPs as a novel target exploited by fungal pathogens to alter immune activation and thus promote the successful infection of the host.

IntroductionUnderstanding microbial communities in diverse ecosystems is crucial for unraveling the intricate relationships among microorganisms, their environment, and ecosystem processes. In this study, we investigated differences in the fungal community structure and diversity in soils from two contrasting climatic and vegetation conditions: the Xinjiang western China plateau and the Fujian southeastern coastal province.MethodsA total of 36 soil samples collected from two climatic regions were subjected to high-throughput ITS gene sequencing for fungal community analysis. In conjunction soil physicochemical properties were assessed and compared. Analyses included an examination of the relationship of fungal community structure to environmental factors and functional profiling of the community structure was using the FUNGuild pipeline.ResultsOur data revealed rich fungal diversity, with a total of 11 fungal phyla, 31 classes, 86 orders, 200 families, 388 genera, and 515 species identified in the soil samples. Distinct variations in the physicochemical properties of the soil and fungal community structure were seen in relation to climate and surface vegetation. Notably, despite a colder climate, the rhizosphere soil of Xinjiang exhibited higher fungal (α-)diversity compared to the rhizosphere soil of Fujian. β-diversity analyses indicated that soil heterogeneity and differences in fungal community structure were primarily influenced by spatial distance limitations and vegetation type. Furthermore, we identified dominant fungal phyla with significant roles in energy cycling and organic matter degradation, including members of the Sordariomycetes, Leotiomycetes, Archaeosporomycetes, and Agaricomycetes. Functional analyses of soil fungal communities highlighted distinct microbial ecological functions in Xinjiang and Fujian soils. Xinjiang soil was characterized by a focus on wood and plant saprotrophy, and endophytes, whereas in Fujian soil the fungal community was mainly associated with ectomycorrhizal interactions, fungal parasitism, and wood saprotrophy.DiscussionOur findings suggest fungal communities in different climatic conditions adapt along distinct patterns with, plants to cope with environmental stress and contribute significantly to energy metabolism and material cycling within soil-plant systems. This study provides valuable insights into the ecological diversity of fungal communities driven by geological and environmental factors.

Only a handful of microbial mosquito larval pathogens have been described to date. Sampling several natural enzootic infections of mosquito larvae in southwestern Florida indicated the presence of microbial pathogens capable of extensive larval mortality. A microscopic analysis of one sample site revealed extensive apparent growth of a Pythium-like microbe on mosquito larvae, with the highest degree of infection observed in the siphon and head regions. Structures consistent with sporangia were seen on infected insects after lactophenol blue staining, and higher-resolution scanning electron microscopy (SEM) micrographs showed sporangia and encysted zoospores targeting the head and siphon regions. The isolate was single-colony purified, and molecular identification targeting the ITS and COX1 loci coupled to phylogenetic reconstruction indicated that the isolate belonged to the Pythium genus but was distinct from its closest characterized species, P. inflatum. Morphological features were characterized, with the isolate showing rapid growth on all mycological media tested and relatively high thermotolerance, capable of robust growth at 37 °C; hence, it was designated P. thermoculicivorax. Sampling from a second series of natural infections of mosquito larvae resulted in the molecular identification of three Trichoderma isolates, one with high similarity to T. strigosum and the other two clustering closely with T. asperellum. These data highlight the occurrence of natural enzootic infections of mosquito larvae, potentially as a resource for the identification of new mosquito pathogens.

The Asian water plantain, Alisma orientale (Sam.) Juzep, is a traditional Chinese medicinal plant. The dried tubers of the Alisma orientale, commonly referred to as Alismatis rhizome (AR), have long been used in traditional Chinese medicine to treat a variety of diseases. Soil properties and the soil microbial composition are known to affect the quality and bioactivity of plants. Here, we sought to identify variations in soil fungal communities and soil properties to determine which would be optimal for cultivation of A. orietale. Soil properties, heavy metal content, and pesticide residues were determined from soils derived from four different agricultural regions around Shaowu City, Fujian, China, that had previously been cultivated with various crops, namely, Shui Dao Tu (SDT, rice), Guo Shu Tu (GST, pecan), Cha Shu Tu (CST, tea trees), and Sang Shen Tu (SST, mulberry). As fungi can either positively or negatively impact plant growth, the fungal communities in the different soils were characterized using long-read PacBio sequencing. Finally, we examined the quality of A. orientale grown in the different soils. Our results show that fungal community diversity of the GST soil was the highest with saprotrophs the main functional modes in these and SDT soils. Our data show that GST and SDT soils were most suitable for A. orientale growth, with the quality of the AR tubers harvested from GST soil being the highest. These data provide a systematic approach at soil properties of agricultural lands in need of replacement and/or rotating crops. Based on our findings, GST was identified as the optimal soil for planting A. orientale, providing a new resource for local farmers.

The characterization of natural fungal diversity impacts our understanding of ecological and evolutionary processes and can lead to novel bioproduct discovery. Russula and Lactarius, both in the order Russulales, represent two large genera of ectomycorrhizal fungi that include edible as well as toxic varieties. Based on morphological and phylogenetic analyses, including nucleotide sequences of the internal transcribed spacer (ITS), the 28S large subunit of ribosomal RNA (LSU), the second largest subunit of RNA polymerase II (RPB2), the ribosomal mitochondrial small subunit (mtSSU), and the translation elongation factor 1-α (TEF1-α) gene sequences, we here describe and illustrate two new species of Russula and one new species of Lactarius from southern China. These three new species are: R. junzifengensis (R. subsect. Virescentinae), R. zonatus (R. subsect. Crassotunicatae), and L. jianyangensis (L. subsect. Zonarii).

IntroductionTea is one of the most widely consumed beverages around the world. Larvae of the moth, Ectropis obliqua Prout (Geometridae, Lepidoptera), are one of the most destructive insect pests of tea in China. E. obliqua is a polyphagus insect that is of increasing concern due to the development of populations resistant to certain chemical insecticides. Microbial biological control agents offer an environmentally friendly and effective means for insect control that can be compatible with “green” and organic farming practices.MethodsTo identify novel E. obliqua biological control agents, soil and inset cadaver samples were collected from tea growing regions in the Fujian province, China. Isolates were analyzed morphologically and via molecular characterization to identity them at the species level. Laboratory and greenhouse insect bioassays were used to determine the effectiveness of the isolates for E. obliqua control.ResultsEleven isolates corresponding to ten different species of Metarhizium were identified according to morphological and molecular analyses from soil and/or insect cadavers found on tea plants and/or in the surrounding soil sampled from eight different regions within the Fujian province, China. Four species of Metarhizium including M. clavatum, M. indigoticum, M. pemphigi, and M. phasmatodeae were documented for the first time in China, and the other species were identified as M. anisopliae, M. brunneum, M. lepidiotae, M. majus, M. pinghaense, and M. robertsii. Insect bioassays of the eleven isolates of Metarhizium revealed significant variation in the efficacy of each isolate to infect and kill E. obliqua. Metarhizium pingshaense (MaFZ-13) showed the highest virulence reaching a host target mortality rate of 93% in laboratory bioassays. The median lethal concentration (LC50) and median lethal time (LT50) values of M. pingshaense MaFZ-13 were 9.6 × 104 conidia/mL and 4.8 days, respectively. Greenhouse experiments and a time-dose-mortality (TDM) models were used to further evaluate and confirm the fungal pathogenic potential of M. pingshaense MaFZ-13 against E. obliqua larvae.DiscussionIsolation of indigenous microbial biological control agents targeting specific pests is an effective approach for collecting resources that can be exploited for pest control with lowered obstacles to approval and commercialization. Our data show the presence of four different previously unreported Metarhizium species in China. Bioassays of the eleven different Metarhizium strains isolated revealed that each could infect and kill E. obliqua to different degrees with the newly isolated M. pingshaense MaFZ-13 strain representing a particularly highly virulent isolate potentially applicable for the control of E. obliqua larvae.

Little is known concerning terpenoids produced by members of the fungal order Ophiostomales, with the member Harringtonia lauricola having the unique lifestyle of being a beetle symbiont but potentially devastating tree pathogen. Nine known terpenoids, including six labdane diterpenoids (1–6) and three hopane triterpenes (7–9), were isolated from H. lauricola ethyl acetate (EtOAc) extracts for the first time. All compounds were tested for various in vitro bioactivities. Six compounds, 2, 4, 5, 6, 7, and 9, are described functionally. Compounds 2, 4, 5, and 9 expressed potent antiproliferative activity against the MCF-7, HepG2 and A549 cancer cell lines, with half-maximal inhibitory concentrations (IC50s) ~12.54–26.06 μM. Antimicrobial activity bioassays revealed that compounds 4, 5, and 9 exhibited substantial effects against Gram-negative bacteria (Escherichia coli and Ralstonia solanacearum) with minimum inhibitory concentration (MIC) values between 3.13 and 12.50 μg/mL. Little activity was seen towards Gram-positive bacteria for any of the compounds, whereas compounds 2, 4, 7, and 9 expressed antifungal activities (Fusarium oxysporum) with MIC values ranging from 6.25 to 25.00 μg/mL. Compounds 4, 5, and 9 also displayed free radical scavenging abilities towards 2,2-diphenyl-1-picrylhydrazyl (DPPH) and superoxide (O2−), with IC50 values of compounds 2, 4, and 6 ~3.45–14.04 μg/mL and 22.87–53.31 μg/mL towards DPPH and O2−, respectively. These data provide an insight into the biopharmaceutical potential of terpenoids from this group of fungal insect symbionts and plant pathogens.

The insect pathogenic fungus, Ascosphaera apis, is the causative agent of honeybee chalk brood disease. Amylases are secreted by many plant pathogenic fungi to access host nutrients through the metabolism of starch, and the identification of new amylases can have important biotechnological applications. Production of amylase by A. apis in submerged culture was optimized using the response surface method (RSM). Media composition was modeled using Box–Behnken design (BBD) at three levels of three variables, and the model was experimentally validated to predict amylase activity (R2 = 0.9528). Amylase activity was highest (45.28 ± 1.16 U/mL, mean ± SE) in media composed of 46 g/L maltose and1.51 g/L CaCl2 at a pH of 6.6, where total activity was ~11-fold greater as compared to standard basal media. The enzyme was purified to homogeneity with a 2.5% yield and 14-fold purification. The purified enzyme had a molecular weight of 75 kDa and was thermostable and active in a broad pH range (> 80% activity at a pH range of 7–10), with optimal activity at 55 °C and pH = 7.5. Kinetic analyses revealed a Km of 6.22 mmol/L and a Vmax of 4.21 μmol/mL·min using soluble starch as the substrate. Activity was significantly stimulated by Fe2+ and completely inhibited by Cu2+, Mn2+, and Ba2+ (10 mM). Ethanol and chloroform (10% v/v) also caused significant levels of inhibition. The purified amylase essentially exhibited activity only on hydrolyzed soluble starch, producing mainly glucose and maltose, indicating that it is an endo-amylase (α-amylase). Amylase activity peaked at 99.38 U/mL fermented in a 3.7 L-bioreactor (2.15-fold greater than what was observed in flask cultures). These data provide a strategy for optimizing the production of enzymes from fungi and provide insight into the α-amylase of A. apis.

ABSTRACT Metarhizium anisopliae is a commercialized entomopathogenic fungus widely used for the control of insect pests. Significant efforts have been expended to screen and/or select for isolates that display increased virulence toward target insect hosts. UV-induced mutagenesis has resulted in the isolation of a number of hypervirulent M. anisopliae mutants; however, the underlying mechanisms that have led to the desired phenotype have yet to be characterized. Here, we performed a comparative proteomic analysis of an M. anisopliae UV-induced hypervirulent mutant (MaUV-HV) and its wild-type parent using tandem mass tag (TMT)-based quantitative proteomics. A total of 842 differentially abundant proteins were identified, with 360 being more abundant in the hypervirulent mutant and 482 in the wild-type parent. In terms of differential abundance, the critical pathways affected included those involved in secondary metabolite production, virulence, and stress response. In addition, a number of genes involved in terpenoid biosynthesis pathways were identified as significantly mutated in the MaUV-HV strain. In particular, mutations in the farnesyl pyrophosphate synthase (FPPS1) and geranylgeranyl diphosphate synthase (GGPPS5) genes were seen. The effects of the FPPS1 mutation were confirmed via the construction and characterization of a targeted gene knockout strain (ΔMaFPPS1). The overall effects of the mutations were increased resistance to UV stress, faster growth, and increased virulence. These results provide mechanistic insights and new avenues for modulating fungal virulence in efforts to increase the biological control potential of insect-pathogenic fungi. IMPORTANCE The mechanisms that underlie and contribute to microbial (fungal) virulence are known to be varied; however, the identification of contributing pathways beyond known virulence factors remains difficult. Using TMT-based proteomic analyses, changes in the proteomes of an M. anisopliae hypervirulent mutant and its wild-type parent were determined. These data revealed alterations in pathogenicity, stress, and growth/developmental pathways, as well as pathways not previously known to affect virulence. These include terpenoid pathways that can be manipulated to increase the efficacy of fungal insect biological control agents for increased sustainable pest control.

Spodoptera frugiperda is a worldwide generalist pest with remarkable adaptations to environments and stresses, including developmental stage-related behavioral and physiological adaptations, such as diverse feeding preferences, mate seeking, and pesticide resistance. Insects’ odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) are essential for the chemical recognition during behavioral responses or other physiological processes. The genome-wide identification and the gene expression patterns of all these identified OBPs and CSPs across developmental stage-related S. frugiperda have not been reported. Here, we screened for genome-wide SfruOBPs and SfruCSPs, and analyzed the gene expression patterns of SfruOBPs and SfruCSPs repertoires across all developmental stages and sexes. We found 33 OBPs and 22 CSPs in the S. frugiperda genome. The majority of the SfruOBP genes were most highly expressed in the adult male or female stages, while more SfruCSP genes were highly expressed in the larval or egg stages, indicating their function complementation. The gene expression patterns of SfruOBPs and SfruCSPs revealed strong correlations with their respective phylogenic trees, indicating a correlation between function and evolution. In addition, we analyzed the chemical-competitive binding of a widely expressed protein, SfruOBP31, to host plant odorants, sex pheromones, and insecticides. Further ligands binding assay revealed a broad functional related binding spectrum of SfruOBP31 to host plant odorants, sex pheromones, and insecticides, suggesting its potential function in food, mate seeking, and pesticide resistance. These results provide guidance for future research on the development of behavioral regulators of S. frugiperda or other environmentally friendly pest-control strategies.

Beauveria bassiana holds promise as a feasible biological control agent for tick control. The B. bassiana stress–response transcription factor Msn2 is known to contribute to fungal growth, conidiogenesis, stress–response and virulence towards insects; however, little is known concerning whether Msn2 is involved in infection across Arthropoda classes. We evaluated the effects of Msn2 on B. bassiana virulence against Rhipicephalus microplus (Acari, Ixodidae) using wild-type, targeted gene knockout (ΔBbmsn2) and complemented mutant (ΔBbmsn2/Bbmsn2) strains. Reproductive parameters of R. microplus engorged females treated topically or by an intra-hemocoel injection of conidial suspensions were assessed. Treated cuticles of engorged females were analyzed by microscopy, and proteolytic activity of B. bassiana on cuticles was assessed. Topically treated engorged females showed high mean larval hatching (>84%) in control and ΔBbmsn2 treatments, whereas treatment with the wild-type or ΔBbmsn2/Bbmsn2 strains resulted in significantly decreased (lowered egg viability) larval hatching. Percent control of R. microplus topically treated with ΔBbmsn2 was lower than in the groups treated with wild-type (56.1%) or ΔBbmsn2/Bbmsn2 strains. However, no differences on reproductive parameters were detected when R. microplus were treated by intra-hemocoel injection using low (800 conidia/tick) doses for all strains tested; R. microplus injected with high doses of wild-type or mutant strains (106 conidia/tick) died before laying eggs (~48 h after treatment). SEM analyses of B. bassiana infection showed similar conidial germination and formation of pseudo-appressoria on tick cuticle. Histological sections of ticks treated with the wild-type or ΔBbmsn2/Bbmsn2 strains showed fungal penetration through the cuticle, and into the tick interior. Hyphae of ΔBbmsn2, however, did not appear to penetrate or breach the tick exocuticle 120 h after treatment. Protease activity was lower on tick cuticles treated with ΔBbmsn2 than those treated with the wild-type or ΔBbmsn2/Bbmsn2 strains. These data show that loss of the Msn2 transcription factor reduced B. bassiana virulence against R. microplus, but did not interfere with conidial germination, appressoria formation or sporulation on tick cadavers, and plays only a minimal role once the cuticle is breached. Our results indicate that the BbMsn2 transcription factor acts mainly during the fungal penetration process and that decreased protease production may be one mechanism that contributes to the inability of the mutant strain to breach the tick cuticle.

Transcriptional activity is mediated by chromatin remodeling, which in turn is affected by post-translational modifications, including histone acetylation. Histone acetyltransferases (HATs) are capable of promoting euchromatin formation and then activating gene transcription. Here, we characterize the Elp3 GNAT family HAT, which is also a subunit of Elongator complex, in the environmentally and economically important fungal insect pathogen, Beauveria bassiana. BbElp3 showed high localization levels to mitochondria, with some nuclear and cytoplasmic localization also apparent. Targeted gene knockout of BbElp3 resulted in impaired asexual development and morphogenesis, reduced tolerances to multiple stress conditions, reduced the ability of the fungus to utilize various carbon/nitrogen sources, increased susceptibility to rapamycin, and attenuated virulence in bioassays using the greater wax moth, Galleria mellonella. The ΔBbElp3 mutant also showed disrupted cell cycle, abnormal hyphal septation patterns, and enlarged autophagosomes in vegetative hyphae. Transcriptome analyses revealed differential expression of 775 genes (DEGs), including 336 downregulated and 438 upregulated genes in the ΔBbElp3 strain as compared to the wild type. Downregulated genes were mainly enriched in pathways involved in DNA processing and transcription, cell cycle control, cellular transportation, cell defense, and virulence, including hydrophobins, cellular transporters (ABC and MFS multidrug transporters), and insect cuticular degrading enzymes, while upregulated genes were mainly enriched in carbohydrate metabolism and amino acid metabolism. These data indicate pleiotropic effects of BbElp3 in impacting specific cellular processes related to asexual development, cell cycle, autophagy, and virulence.

Lipid assimilation, storage, and turnover impact growth, development, and virulence in many microbial pathogens including fungi. Perilipins are proteins associated with lipid droplets (LDs) that mediate their assembly and turnover. Here, we characterized the Beauveria bassiana (BbPlin1) perilipin. BbPlin1 expression was higher in minimal media than in rich media, and, using a BbPlin1::eGFP fusion protein, the protein was shown to be co–localized to LDs, with the high expression seen during infection and proliferation within the insect (Galleria mellonella) host that dramatically decreased to almost no expression during fungal outgrowth on cadavers including in conidia, but that BbPlin1 production resumed in the conidia once placed in nutrient–containing media allowing for germination and growth. Characterization of a targeted gene deletion strain (ΔBbPlin1) revealed a dramatic (>30%) reduction in cellular LD content, promotion of aerial hyphal growth, and a small decrease in virulence, with little to no effects on vegetative growth and stress responses. However, in the ΔBbPlin1 strain, expression of the complementary LD–associated caleosin gene, BbCal1, was enhanced under nutrient–poor conditions, although no changes in BbPlin1 expression were seen in a ΔBbCal1 strain and the expression of BbPlin1 in the ΔBbCal1 strain did not change LD patterns in cells. Transcriptome and RT–PCR analyses indicated increased expression of lipid metabolism–related genes, including triacylglyercol lipase 3, enoyl–CoA isomerase, and diacylglycerol–O–acetyl transferase in the BbPlin1 deletion mutant. Lipid profile analyses confirmed that the loss of BbPlin1 significantly reduced the cellular levels of contents of triacylglycerol, diacylglycerol, and phosphatidylethanolamine as compared to the wild–type strain. These results demonstrate the involvement of the B. bassiana perilipin in mediating lipid homeostasis, fungal aerial hyphal growth, and virulence, revealing critical cycling from high expression during nutrient utilization within host cadavers to low expression during growth on the surface of the cadaver during the infection process.

Social insects have evolved acute mechanisms for sensing and mitigating the spread of microbial pathogens within their communities that include complex behaviors such as grooming and sanitation. Chemical sensing involves detection and transport of olfactory and other chemicals that are mediated by at least two distinct classes of small molecular weight soluble proteins known as chemosensory- and odorant binding proteins (CSPs and OBPs, respectively) that exist as protein families in all insects. However, to date, a systematic examination of the expression of these genes involved in olfactory and other pathways to microbial infection has yet to be reported. The red imported fire ant, Solenopsis invicta, is one of the most successful invasive organisms on our planet. Here, we examined the temporal gene expression profiles of a suite of S. invicta CSPs (SiCSPs1-22) and OBPs (SiOBPs1-16) in response to infection by the broad host range fungal insect pathogen, Beauveria bassiana. Our data show that within 24 h post-infection, i.e., before the fungus has penetrated the host cuticle, the expression of SiCSPs and SiOBPs is altered (mainly increased compared to uninfected controls), followed by suppression of SiCSP and select SiOBP expression 48 h post-infection and mixed responses at 72 h post-infection. A smaller group of SiBOPs, however, appeared to respond to fungal infection, with expression of SiOBP15 consistently higher during fungal infection over the time course examined. These data indicate dynamic gene expression responses of CSPs and OBPs to fungal infection that provide clues to mechanisms that might mediate detection of microbial pathogens, triggering grooming, and nest sanitation.

The red imported fire ant, Solenopsis invicta, is a eusocial invasive insect that has spread worldwide. Chemosensory proteins (CSPs) are ligand-binding proteins that participate in a diverse range of physiological processes that include olfaction and chemical transport. Here, we performed a systematic survey of the expression of the 21 gene S. invicta CSP family that includes at least two groups of apparent S. invicta-specific gene expansions. These data revealed caste, tissue, and developmental stage-specific differential expression of the SiCSPs. In general, moderate to high SiCSP expression was seen in worker antennae and abdomen tissues with lower expression in head/thorax regions. Male and female alates showed high antennal expression of fewer SiCSPs, with the female alate thorax showing comparatively high SiCSP expression. SiCSP expression was lower in male alates tissues compared to workers and female alates, albeit with some highly expressed SiCSPs. SiCSP expression was low during development including in eggs, larvae (early and late instars), and pupae. Global analyses revealed examples of conserved, divergent, and convergent SiCSP expression patterns linked to phylogenetic relationships. The developmental and caste-specific variation seen in SiCSP expression patterns suggests specific functional diversification of CSPs that may translate into differential chemical recognition and communication among individuals and/or reflect other cellular roles of CSPs. Our results support a model for CSPs acting as general ligand carriers involved in a wide range of physiological processes beyond olfaction. As compared to the expression patterns of the S. invicta odorant binding proteins (OBPs), an inverse correlation between SiOBP and SiCSP expression was seen, suggesting potential complementary and/or compensatory functions between these two classes of ligand carriers.

Sirtuins are a class of histone deacetylases that promote heterochromatin formation to repress transcription. The entomopathogenic fungus Beauveria bassiana contains six sirtuin homologs. The class III histone deacetylase, BbSir2, has been previously shown to affect the regulation of carbon/nitrogen metabolism and asexual development, with only moderate effects on virulence. Here, we examine another class III histone deacetylase (BbSirT2) and show that it contributes to deacetylation of lysine residues on histone H4-K16ac. Directed gene-knockout of BbSirT2 dramatically reduced conidiation, the ability of the fungus to metabolize a range of carbon and nitrogen sources, and tolerances to oxidative, heat, and UV stress and significantly attenuated virulence in both intrahemocoel injection and topical bioassays using the Greater wax moth (Galleria mellonella) as the insect host. ΔBbSirT2 cells showed alterations in cell cycle development and hyphal septation and produced morphologically aberrant conidia. Comparative transcriptomic analyses of wild type versus ΔBbSirT2 cells indicated differential expression of 1148 genes. Differentially expressed genes were enriched in pathways involved in cell cycle and rescue, carbon/nitrogen metabolism, and pathogenesis. These included changes in the expression of polyketide synthases (PKSs) and LysM effector proteins that contribute to degradation of host toxins and target host pathways, respectively. These data indicate contributions of BbSirT2 in helping to mediate fungal stress and development, with the identification of affected gene targets that can help account for the observed reduced virulence phenotype.

Chromatin remodeling is mediated in part by post-translational acetylation/deacetylation modifications of histones. Histone acetyltransferases (HATs), e.g., members of the GNAT/MYST superfamily, activate gene transcription via promotion of euchromatin formation. Here, we characterized a GNAT family HAT, Spt10 (BbSpt10), in the environmentally and economically important fungal insect pathogen, Beauveria bassiana. Targeted gene knockout of BbSpt10 resulted in impaired asexual development and morphogenesis; reduced abilities to utilize various carbon/nitrogen sources; reduced tolerance to heat, fungicides, and DNA damage stress; and attenuated virulence. The ΔBbSpt10 mutant showed disrupted cell cycle development and abnormal hyphal septation patterns. Transcriptome analyses of wild type and ΔBbSpt10 cells revealed the differential expression of 373 genes, including 153 downregulated and 220 upregulated genes. Bioinformatic analyses revealed downregulated genes to be enriched in pathways involved in amino acid metabolism, cellular transportation, cell type differentiation, and virulence, while upregulated genes were enriched in carbon/nitrogen metabolism, lipid metabolism, DNA process, and cell rescue, defense, and virulence. Downregulated virulence genes included hydrophobins, cellular transporters (ABC and MFS multidrug transporters) and cytochrome P450 detoxification genes. These data indicated broad effects of BbSpt10 on fungal development, multi-stress response, and virulence.

The western flower thrips, Frankliniella occidentalis, is an aggressive agricultural insect pest causing significant damage to a wide range of fruit, vegetable, and ornamental crops. Beauveria bassiana is a broad-host-range entomopathogenic fungus capable of infecting and killing F. occidentalis. Infection of thrips by B. bassiana strain BbYT12 using a concentration of 1 × 108 conidia/mL resulted in 81.48% mortality in adults in 6 d (LT50 = 90 ± 15.1 h). Scanning electron microscopy of the infection process revealed preferential adhesion and germination of fungal spores to inter-segmental folds or grooves on the insect body surface with penetrating germlings and extended hyphae visualized during the initial stages of infection (6–24 h). Histological analyses showed the appearance of in vivo hyphal bodies in sagittal sections and the fat body as early as 24 h post-infection. Within 72 h, hyphal bodies and hyphae could be found throughout the infected organism including in the midgut, Malphigian tubules, alimentary canal, ovarioles (in females), and an extended hyphal network could be seen on insect cadavers (>72 h post-infection). Real-time RT-PCR analyses of the expression of select genes implicated in virulence including the Pr1 protease, beauvericin synthase, involved in the production of the secondary metabolite beauvericin, two cytochrome P450 monooxygenases implicated in cuticular hydrocarbon degradation, two multidrug efflux proteins, a perilipin involved in lipid storage, and the Hog1 MAP kinase and protein kinase A signaling factors revealed discrete patterns of infection-time dependent expression. These data provide basic insights into the process of B. bassiana infection of F. occidentalis.

Raffaelea lauricola is the causative agent of laurel wilt, a devastating disease of lauraceous trees. R. lauricola is also an obligate nutritional symbiont of several ambrosia beetle species who act as vectors for the pathogen. Here, we sought to establish the baseline “phenome” of R. lauricola with knowledge concerning its metabolic capability, expanding our understanding of how these processes are impacted by environmental and host nutrients. Phenotypic screening using a microarray of over one thousand compounds was used to generate a detailed profile of R. lauricola substrate utilization and chemical sensitivity. These data revealed (i) relatively restricted carbon utilization, (ii) broad sulfur and phosphate utilization, and (iii) pH and osmotic sensitivities that could be rescued by specific compounds. Additional growth profiling on fatty acids revealed toxicity on C10 substrates and lower, with robust growth on C12–C18 fatty acids. Conditions for lipid droplet (LD) visualization and LD dynamics were examined using a series of lipid dyes. These data provide unique insights regarding R. lauricola metabolism and physiology, and identify distinct patterns of substrate usage and sensitivity which likely reflect important aspects of the host-microbe interface and can be exploited for the development of strategies for mitigating the spread of laurel wilt.

The presence of commensal bacteria enhances both acute and persistent infection of murine noroviruses. For several enteric viral pathogens, mechanisms by which these bacteria enhance infection involve direct interactions between the virus and bacteria. While it has been demonstrated that human noroviruses bind to a variety of commensal bacteria, it is not known if this is also true for murine noroviruses. The goal of this study was to characterize interactions between murine noroviruses and commensal bacteria and determine the impact of bacterial growth conditions, incubation temperature and time, on murine norovirus attachment to microbes that comprise the mammalian microbiome. We show that murine noroviruses bind directly to commensal bacteria and show similar patterns of attachment as human norovirus VLPs examined under the same conditions. Furthermore, while binding levels are not impacted by the growth phase of the bacteria, they do change with time and incubation temperature. We also found that murine norovirus can bind to a commensal fungal species, Candida albicans.

Infections mediated by broad host range entomopathogenic fungi represent seminal observations that led to one of the first germ theories of disease and are a classic example of a co-evolutionary arms race between a pathogen and target hosts. These fungi are able to parasitize susceptible hosts via direct penetration of the cuticle with the initial and potentially determining interaction occurring between the fungal spore and the insect epicuticle. Entomogenous fungi have evolved mechanisms for adhesion and recognition of host surface cues that help direct an adaptive response that includes the production of: (a) hydrolytic, assimilatory, and/or detoxifying enzymes including lipase/esterases, catalases, cytochrome P450s, proteases, and chitinases; (b) specialized infectious structures, e.g., appressoria or penetrant tubes; and (c) secondary and other metabolites that facilitate infection. Aside from immune responses, insects have evolved a number of mechanisms to keep pathogens at bay that include: (a) the production of (epi) cuticular antimicrobial lipids, proteins, and metabolites; (b) shedding of the cuticle during development; and (c) behavioral-environmental adaptations such as induced fever, burrowing, and grooming, as well as potentially enlisting the help of other microbes, all intended to stop the pathogen before it can breach the cuticle. Virulence and host-defense can be considered to be under constant reciprocal selective pressure, and the action on the surface likely contributes to phenomena such as strain variation, host range, and the increased virulence often noted once a (low) virulent strain is “passaged” through an insect host. Since the cuticle represents the first point of contact and barrier between the fungus and the insect, the “action on the surface” may represent the defining interactions that ultimately can lead either to successful mycosis by the pathogen or successful defense by the host. Knowledge concerning the molecular mechanisms underlying this interaction can shed light on the ecology and evolution of virulence and can be used for rational design strategies at increasing the effectiveness of entomopathogenic fungi for pest control in field applications.

Reactive carbonyl and oxygen species (RCS/ROS), often generated as metabolic byproducts, particularly under conditions of pathology, can cause direct damage to proteins, lipids, and nucleic acids. Glyoxal oxidases (Gloxs) oxidize aldehydes to carboxylic acids, generating hydrogen peroxide (H2O2). Although best characterized for their roles in lignin degradation, Glox in plant fungal pathogens are known to contribute to virulence, however, the mechanism underlying such effects are unclear. Here, we show that Glox in the insect pathogenic fungus, Metarhizium acridum, is highly expressed in mycelia and during formation of infection structures (appressoria), with the enzyme localizing to the cell membrane. MaGlox targeted gene disruption mutants showed RCS and ROS accumulation, resulting in cell toxicity, induction of apoptosis and increased autophagy, inhibiting normal fungal growth and development. The ability of the MaGlox mutant to scavenge RCS was significantly reduced, and the mutant exhibited increased susceptibility to aldehydes, oxidative and cell wall perturbing agents but not toward osmotic stress, with altered cell wall contents. The ΔMaGlox mutant was impaired in its ability to penetrate the host cuticle and evade host immune defense resulting in attenuated pathogenicity. Overexpression of MaGlox promoted fungal growth and conidial germination, increased tolerance to H2O2, but had little to other phenotypic effects. Transcriptomic analyses revealed downregulation of genes related to cell wall synthesis, conidiation, stress tolerance, and host cuticle penetration in the ΔMaGlox mutant. These findings demonstrate that MaGlox-mediated scavenging of RCS is required for virulence, and contributes to normal fungal growth and development, stress resistance.

Pythium insidiosum ATCC 200269 strain CDC-B5653, an isolate from necrotizing lesions on the mouth and eye of a 2-year-old boy in Memphis, Tennessee, USA, was sequenced using a combination of Illumina MiSeq (300 bp paired-end, 14 millions reads) and PacBio (10 Kb fragment library, 356,001 reads). The sequencing data were assembled using SPAdes version 3.1.0, yielding a total genome size of 45.6 Mb contained in 8992 contigs, N50 of 13 Kb, 57% G + C content, and 17,867 putative protein-coding genes. This Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession JRHR00000000. Keywords: Oomycete, Pythium insidiosum, Pythiosis, Human emerging pathogen, Genome sequencing

Broad host range entomopathogenic fungi such as Beauveria bassiana attack insect hosts via attachment to cuticular substrata and the production of enzymes for the degradation and penetration of insect cuticle. The outermost epicuticular layer consists of a complex mixture of non-polar lipids including hydrocarbons, fatty acids, and wax esters. Long chain hydrocarbons are major components of the outer waxy layer of diverse insect species, where they serve to protect against desiccation and microbial parasites, and as recognition molecules or as a platform for semiochemicals. Insect pathogenic fungi have evolved mechanisms for overcoming this barrier, likely with sets of lipid degrading enzymes with overlapping substrate specificities.. Alkanes and fatty acids are substrates for a specific subset of fungal cytochrome P450 monooxygenases involved in insect hydrocarbon degradation. These enzymes activate alkanes by terminal oxidation to alcohols, which are further oxidized by alcohol and aldehyde dehydrogenases, whose products can enter β-oxidation pathways. B. bassiana contains at least 83 genes coding for cytochrome P450s (CYP), a subset of which are involved in hydrocarbon oxidation, and several of which represent new CYP subfamilies/families. Expression data indicated differential induction by alkanes and insect lipids and 4 CYP proteins have been partially characterized after heterologous expression in yeast. Gene knockouts revealed a phenotype for only one (cyp52X1) out of 6 genes examined to date. CYP52X1 oxidizes long chain fatty acids and participates in the degradation of specific epicuticular lipid components needed for breaching the insect waxy layer. Examining the hydrocarbon oxidizing CYP repertoire of pathogens involved in insect epicuticle degradation can lead to the characterization of enzymes with novel substrate specificities. Pathogen targeting may also represent an important co-evolutionary process regarding insect cuticular hydrocarbon synthesis.

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