Virulence and proteomic responses of Metarhizium anisopliae against Aedes albopictus larvae.

The tropical climate in Malaysia provides an ideal environment for the rapid proliferation of Aedes mosquitoes, notably Aedes aegypti and Aedes albopictus, prominent vectors of dengue fever. Alarmingly, these species are increasingly developing resistance to conventional pesticides. This study aimed to evaluate the efficacy of Metarhizium anisopliae isolate HSAH5 spores, specifically on conidia (CO) and blastospores (BL), against Ae. albopictus larvae. The study centered on evaluating their pathogenic effects and the resultant changes in protein expression. Spore suspensions with varying concentrations were prepared for larvicidal bioassays, and protein expressions were analysed using liquid chromatography-mass spectrometry. Subsequently, protein annotation and network analysis were conducted to elucidate infection mechanisms and the proteomic response. Based on the lethal concentrations and time frames, CO exhibited faster larval mortality than BL at lower concentrations. Despite this, both spore types demonstrated comparable overall pathogenic effects. Results from the proteomic profiling revealed 150 proteins with varied expressions following exposure to Ae. albopictus extract, shedding light on distinct infection strategies between the spores. Gene Ontology enrichment and network analysis illustrated the diverse metabolic adaptations of M. anisopliae and interactions with mosquito larvae. This highlighted the complexity of host-pathogen dynamics and the significance of biosynthetic processes, energy storage, and cellular interaction pathways in disease progression. The BL network, consisting 80 proteins and 74 connections, demonstrates the intricate fungal mechanisms triggered by host stimuli. Conversely, the CO network, though smaller, displayed notable interconnectivity and concentrated involvement at the cell periphery, suggesting a deliberate strategy for initial host contact. This study offers valuable insights into proteome dynamics of M. anisopliae's BL and CO for managing mosquito populations and combating disease transmission, thereby significantly advancing public health and environmental conservation efforts.

N-acetylglucosamine kinase (BbHxk1) has pleiotropic effects on vegetative growth, cell wall integrity, morphological transition, cuticle infection, and metabolic modulation in the biological pesticide Beauveria bassiana.

Beauveria bassiana is a popular and eco-friendly biopesticide. During its pathogen-pest interaction, both N-acetylglucosamine (GlcNAc) catabolism and anabolism are crucial for nutrient supply and cell-wall construction. The initiation of GlcNAc metabolism relies on the catalysis of GlcNAc kinase, which has been extensively studied in the human pathogen Candida albicans. However, the physiological function of GlcNAc kinase remains poorly understood in entomopathogenic fungi. In the present study, a GlcNAc kinase homolog was identified and designated as BbHxk1 in B. bassiana. Deletion of BbHxk1 resulted in viable but reduced vegetative growth on various carbon sources. ΔBbHxk1 mutants displayed severe defects in cell wall integrity, making them more susceptible to cell wall stress cues. Furthermore, the absence of BbHxk1 resulted in an increase in conidial yield and blastospore production, and a faster rate of germination and filamentation, potentially attributed to higher intracellular ATP levels. BbHxk1 deficiency led to a reduction in the activities of cuticle-degrading enzymes, which might contribute to the attenuated pathogenicity specifically through cuticle penetration rather than hemocoel infection towards Galleria mellonella larvae. Being different from C. albicans Hxk1, which facultatively acts as a catalyzing enzyme and transcriptional regulator, BbHxk1 primarily acts as a catalyzing enzyme and metabolic regulator. The altered metabolomic profiling correlated with the phenotypic defects in ΔBbHxk1 mutants, further implicating a potential metabolism-dependent mechanism of BbHxk1 in mediating physiologies of B. bassiana. These findings not only unveil a novel role for GlcNAc kinase in B. bassiana, but also provide a solid theoretical basis to guide metabolic reprogramming in order to maintain or even enhance the efficiency of fungi for practical applications.

Functional characterization of BbEaf6 in Beauveria bassiana: Implications for fungal virulence and stress response.

The Eaf6 protein, a conserved component of the NuA4 and NuA3 complexes in yeast and MOZ/MORF complexes in humans, plays crucial roles in transcriptional activation, gene regulation, and cell cycle control. Despite its significance in other organisms, the functional role of Eaf6 in entomopathogenic fungi (EPF) remained unexplored. Here, we investigate the function of BbEaf6, the Eaf6 homolog in the entomopathogenic fungus Beauveria bassiana. We demonstrate that BbEaf6 is predominantly localized in nuclei, similar to its counterpart in other fungi. Deletion of BbEaf6 resulted in delayed conidiation, reduced conidial yield, and altered conidial properties. Transcriptomic analysis revealed dysregulation of the genes involved in asexual development and cell cycle progression in the ΔBbEaf6 mutant. Furthermore, the ΔBbEaf6 mutant exhibited decreased tolerance to various stresses, including ionic stress, cell wall perturbation, and DNA damage stress. Notably, the ΔBbEaf6 mutant displayed attenuated virulence in insect bioassays, accompanied by dysregulation of genes associated with cuticle penetration and haemocoel infection. Overall, our study elucidates the multifaceted role of BbEaf6 in stress response, development, and virulence in B. bassiana, providing valuable insights into the molecular mechanisms governing fungal pathogenesis and potential targets for pest management strategies.

Phenotypic and genotypic characterization of fifty strains of Beauveria spp. (Ascomycota, Cordycipitaceae) fungal entomopathogens from diverse geographic origins against the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae).

BACKGROUND: The diamondback moth (DBM) (Plutella xylostella) causes large losses to global crop production. Conventional insecticides are losing effectiveness due to resistance. Consequently, there is a growing interest in sustainable control methods like entomopathogenic fungi (EPF) in Integrated Pest Management. However, the field efficacy of fungi varies due to environmental influences. In this study, a group of 50 Beauveria strains sourced from different locations were characterized by genotype and phenotype with respect to their conidial production, temperature and UV-B radiation tolerance, and virulence against DBM. RESULTS: Phylogenetic analysis revealed two distinct species: Beauveria bassiana (84%) and B. pseudobassiana (16%). Most strains showed optimal growth between 25 °C and 28 °C, with germination severely affected at 10 °C and 33 °C. Notably, 44% displayed high resistance to UV-B radiation (5.94 kJ m-2), with germination rates between 60.9% and 88.1%. Geographical origin showed no correlation with temperature or UV radiation tolerance. In virulence experiments, 52% of strains caused mortality rates exceeding 80% in DBM second instars at 7 days after exposure to a 4 mL conidial suspension (107 conidia/mL). CONCLUSION: Survival under environmental conditions is crucial for EPF-based commercial products against DBM. Results suggest strain tolerance to environmental stressors is more tied to specific micro-climatic factors than geographical origin. Each strain exhibited unique characteristics; for example, the most virulent strain (#29) was highly UV-sensitive. Therefore, characterizing diverse strains provides essential genotypic and phenotypic insights, which are fundamental for understanding their role as biocontrol agents while facilitating efficient biopesticide product development and uptake. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

The fungal protease BbAorsin contributes to growth, conidiation, germination, virulence, and antiphytopathogenic activities in Beauveria bassiana (Hypocreales: Cordycipitaceae).

The fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae), is one of the most destructive agricultural pests. The entomopathogenic fungus Beauveria bassiana (Hypocreales: Clavicipitaceae) is a biopesticide widely used for biocontrol of various pests. Secreted fungal proteases are critical for insect cuticle destruction and successful infection. We have previously shown that the serine protease BbAorsin in B. bassiana has entomopathogenic and antiphytopathogenic activities. However, the contribution of BbAorsin to fungal growth, conidiation, germination, virulence and antiphytopathogenic activities remains unclear. In this study, the deletion (ΔBbAorsin), complementation (Comp), and overexpression (BbAorsinOE) strains of B. bassiana were generated for comparative studies. The results showed that ΔBbAorsin exhibited slower growth, reduced conidiation, lower germination rate, and longer germination time compared to WT and Comp. In contrast, BbAorsinOE showed higher growth rate, increased conidiation, higher germination rate and shorter germination time. Injection of BbAorsinOE showed the highest virulence against S. frugiperda larvae, while injection of ΔBbAorsin showed the lowest virulence. Feeding BbAorsinOE resulted in lower pupation and adult eclosion rates and malformed adults. 16S rRNA sequencing revealed no changes in the gut microbiota after feeding either WT or BbAorsinOE. However, BbAorsinOE caused a disrupted midgut, leakage of gut microbiota into the hemolymph, and upregulation of apoptosis and immunity-related genes. BbAorsin can disrupt the cell wall of the phytopathogen Fusarium graminearum and alleviate symptoms in wheat seedlings and cherry tomatoes infected with F. graminearum. These results highlight the importance of BbAorsin for B. bassiana and its potential as a multifunctional biopesticide.

Transfection of entomopathogenic Metarhizium species with a mycovirus confers hypervirulence against two lepidopteran pests.

Although most known viruses infecting fungi pathogenic to higher eukaryotes are asymptomatic or reduce the virulence of their host fungi, those that confer hypervirulence to entomopathogenic fungus still need to be explored. Here, we identified and studied a novel mycovirus in Metarhizium flavoviride, isolated from small brown planthopper (Laodelphax striatellus). Based on molecular analysis, we tentatively designated the mycovirus as Metarhizium flavoviride partitivirus 1 (MfPV1), a species in genus Gammapartitivirus, family Partitiviridae. MfPV1 has two double-stranded RNAs as its genome, 1,775 and 1,575 bp in size respectively, encapsidated in isometric particles. When we transfected commercial strains of Metarhizium anisopliae and Metarhizium pingshaense with MfPV1, conidiation was significantly enhanced (t test; P-value < 0. 01), and the significantly higher mortality rates of the larvae of diamondback moth (Plutella xylostella) and fall armyworm (Spodoptera frugiperda), two important lepidopteran pests were found in virus-transfected strains (ANOVA; P-value < 0.05). Transcriptomic analysis showed that transcript levels of pathogenesis-related genes in MfPV1-infected M. anisopliae were obviously altered, suggesting increased production of metarhizium adhesin-like protein, hydrolyzed protein, and destruxin synthetase. Further studies are required to elucidate the mechanism whereby MfPV1 enhances the expression of pathogenesis-related genes and virulence of Metarhizium to lepidopteran pests. This study presents experimental evidence that the transfection of other entomopathogenic fungal species with a mycovirus can confer significant hypervirulence and provides a good example that mycoviruses could be used as a synergistic agent to enhance the biocontrol activity of entomopathogenic fungi.

Purpureocillium lilacinum SBF054: Endophytic in Phaseolus vulgaris, Glycine max, and Helianthus annuus; Antagonistic to Rhizoctonia solani; and Virulent to Euschistus heros.

Microorganisms with multiple ecological functions can be a useful biotechnological resource in integrated pest- and disease-management programs. This work aimed to investigate the potential endophytic and virulent effects of a strain of Purpureocillium lilacinum on organic cultivation in Brazil. Specifically, the strain's ability to establish itself as an endophyte in common bean, soybean, and sunflower plants when inoculated via seed was evaluated. Furthermore, its antifungal activity against phytopathogens and its pathogenicity and virulence against insects of the order Lepidoptera, Coleoptera, and Hemiptera were evaluated. Furthermore, the strain was evaluated for its biochemical and physiological characteristics. For virulence bioassays, the experiments were conducted under a factorial scheme (2 × 3), with the following factors: (a) fungal inoculation and control without inoculum and (b) types of inocula (blastospores, aerial conidia, and metabolites). The treatments were sprayed on insect species at different stages of development. In summary, it was found that the SBF054 strain endophytically colonized the common bean, with partial recovery from the root tissues of soybean and sunflower plants, 30 days after inoculation; suppressed 86% of Rhizoctonia solani mycelial growth in an in vitro assay; and controlled eggs, nymphs, and Euschistus heros adults. These multifunctional abilities are mainly attributed to the strain's mechanisms of producing metabolites, such as organic acids, soluble nutrients, and hydrolytic enzymes.

Entomophthoralean and hypocrealean fungal pathogens of the sugarcane aphid, Melanaphis sacchari (Hemiptera: Aphididae), on sorghum in Georgia.

The sugarcane aphid, Melanaphis sacchari, is a widely distributed insect that attacks grasses in different genera including Miscanthus, Saccharum, and Sorghum. The invasive aphid superclone was first discovered in the U.S. attacking grain sorghum in Texas in 2013. Since then, it has been found in at least 25 states including Georgia. We conducted a survey of naturally occurring fungal pathogens of sugarcane aphids on five farms in Georgia, and identified a hypocrealean fungus, Akanthomyces dipterigenus, and two entomophthoralean fungi, Neoconidiobolus spp. From 2018 to 2020, fungal activity differed across farms but at one farm both major fungal species, A. dipterigenus and N. thromboides, were found each of the 3 years infecting sugarcane aphids, attacking adults, both alatae and apterae, and nymphs.

Regulation of morphogenesis and pathogenicity by OsMep2, OsCph1, and OsPes1 in dimorphic entomopathogenic fungus Ophiocordyceps sinensis (Hypocreales: Ophiocordycipitaceae).

Polarized growth plays a key role in all domains of their biology, including morphogenesis and pathogenicity of filamentous fungi. However, little information is available about the determinants of polarized growth. The fungal Mep2, Pes1, and Cph1 proteins were identified to be involved in the dimorphic transition between yeast and hyphal forms in Candida albicans. In this study, evidence that the dimorphic fungal entomopathogen Ophiocordyceps sinensis Mep2, Pes1, and Cph1 proteins are involved in polarized growth is presented. OsMep2 was significantly upregulated at aerial hyphae and conidia germination stages. OsCph1 was significantly upregulated at aerial hyphae, conidia initiation, and conidia germination stages, and OsPes1 was significantly upregulated at the conidia germination stage. Deletions of OsMep2, OsCph1, and OsPes1 provoked defects in the polarized growth. The abilities of hyphal formation and the yields of blastospores and conidia for the ∆ OsMep2, ∆OsCph1, and ∆ OsPes1 mutants were significantly reduced. The conidia yields of the ΔOsMep2, ΔOsCph1, and ΔOsPes1 mutants were decreased by 69.17%, 60.90%, and 75.82%, respectively. Moreover, the pathogenicity of the ∆ OsMep2, ∆OsCph1, and ∆ OsPes1 mutants against Thitarodes xiaojinensis was significantly reduced. The mummification rate caused by wide type and ΔOsMep2, ΔOsCph1, and ΔOsPes1 mutants were 36.98% ± 8.52%, 0.31% ± 0.63%, 1.15% ± 1.57%, and 19.69% ± 5.6%, respectively. These results indicated that OsMep2, OsCph1, and OsPes1 are involved in the regulation of hyphal formation, sporulation, and pathogenicity of O. sinensis. This study provided a basis for the understanding of the fungal dimorphic development and improving the efficiency of artificial cultivation of O. sinensis.

Harmful and Harmless Soil-Dwelling Fungi Indicate Microhabitat Suitability for Off-Host Ixodid Ticks.

Following blood meals or questing bouts, hard ticks (Ixodidae) must locate moist off-host microhabitats as refuge. Soil-dwelling fungi, including entomopathogenic Beauveria bassiana (Bb), thrive in moist microhabitats. Working with six species of ixodid ticks in olfactometer bioassays, we tested the hypothesis that ticks avoid Bb. Contrary to our prediction, nearly all ticks sought, rather than avoided, Bb-inoculated substrates. In further bioassays with female black-legged ticks, Ixodes scapularis, ticks oriented towards both harmful Bb and harmless soil-dwelling fungi, implying that fungi-regardless of their pathogenicity-signal habitat suitability to ticks. Only accessible Bb-inoculated substrate appealed to ticks, indicating that they sense Bb or its metabolites by contact chemoreception. Bb-inoculated substrate required ≥24 h of incubation before it appealed to ticks, suggesting that they respond to Bb metabolites rather than to Bb itself. Similarly, ticks responded to Bb-inoculated and incubated cellulose but not to sterile cellulose, indicating that Bb detection by ticks hinges on the Bb metabolism of cellulose. 2-Methylisoborneol-a common fungal metabolite with elevated presence in disturbed soils-strongly deterred ticks. Off-host ticks that avoid disturbed soil may lower their risk of physical injury. Synthetic 2-methylisoborneol could become a commercial tick repellent, provided its repellency extends to ticks in diverse taxa.

Essential roles of ferric reductase-like proteins in growth, development, stress response, and virulence of the filamentous entomopathogenic fungus Beauveria bassiana.

In yeasts, ferric reductase catalyzes reduction of ferric ion to ferrous form, which is essential for the reductive iron assimilation system. However, the physiological roles of ferric reductases remain largely unknown in the filamentous fungi. In this study, genome-wide annotation revealed thirteen ferric reductase-like (Fre) proteins in the filamentous insect pathogenic fungus Beauveria bassiana, and all their functions were genetically characterized. Ferric reductase family proteins exhibit different sub-cellular distributions (e.g., cell periphery and vacuole), which was due to divergent domain architectures. Fre proteins had a synergistic effect on fungal virulence, which was ascribed to their distinct functions in different physiologies. Ten Fre proteins were not involved in reduction of ferric ion in submerged mycelia, but most proteins contributed to blastospore development. Only two Fre proteins significantly contributed to B. bassiana vegetative growth under the chemical-induced iron starvation, but most Fre proteins were involved in resistance to osmotic and oxidative stresses. Notably, a bZIP-type transcription factor HapX bound to the promoter regions of all FRE genes in B. bassiana, and displayed varying roles in the transcription activation of these genes. This study reveals the important role of BbFre family proteins in development, stress response, and insect pathogenicity, as well as their distinctive role in the absorption of ferric iron from the environment.

Morphological and phylogenetic resolution of Conoideocrella luteorostrata (Hypocreales: Clavicipitaceae), a potential biocontrol fungus for Fiorinia externa in United States Christmas tree production areas.

The entomopathogenic fungus Conoideocrella luteorostrata has recently been implicated in natural epizootics among exotic elongate hemlock scale (EHS) insects in Christmas tree farms in the eastern United States. Since 1913, C. luteorostrata has been reported from various plant feeding Hemiptera in the southeastern United States, but comprehensive morphological and phylogenetic studies of U.S. populations are lacking. The recovery of multiple strains of C. luteorostrata from mycosed EHS in North Carolina provided an opportunity to conduct pathogenicity assays and morphological and phylogenetic studies to investigate genus- and species-level boundaries among the Clavicipitaceae. Pathogenicity assays confirmed that C. luteorostrata causes mortality of EHS crawlers, an essential first step in developing this fungus as a biocontrol. Morphological studies revealed that conidia aligned with previous measurements of the Paecilomyces-like asexual state of C. luteorostrata, with conidiophore morphology consistent with historical observations. Additionally, a Hirsutella-like synanamorph was observed in select C. luteorostrata strains. In both a four-locus, 54-taxon Clavicipitaceae-wide phylogenetic analysis including D1-D2 domains of the nuclear 28S rRNA region (28S), elongation factor 1 alpha (EF1-α), DNA-directed RNA polymerase II subunit 1 (RPB1), and DNA-directed RNA polymerase II subunit 2 (RPB2) and a two-locus, 38-taxon (28S and EF1-α) phylogenetic analysis, all three Conoideocrella species were resolved as strongly supported monophyletic lineages across all loci and both methods (maximum likelihood and Bayesian inference) of phylogenetic inference except for 28S for C. tenuis. Despite the strong support for individual Conoideocrella species, none of the analyses supported the monophyly of Conoideocrella with the inclusion of Dussiella. Due to the paucity of RPB1 and RPB2 sequence data, EF1-α provided superior delimitation of intraspecies groupings for Conoideocrella and should be used in future studies. Further development of C. luteorostrata as a biocontrol against EHS will require additional surveys across diverse Hemiptera and expanded pathogenicity testing to clarify host range and efficacy of this fungus.

A winning formula: sustainable control of three stored-product insects through paired combinations of entomopathogenic fungus, diatomaceous earth, and lambda-cyhalothrin.

This research aimed to assess the effectiveness of Metarhizium robertsii, diatomaceous earth (Protect-It), and lambda-cyhalothrin, for the long-term protection of stored wheat against three destructive grain insect pests, Rhyzopertha dominica, Tribolium castaneum, and Trogoderma granarium. Different treatments were applied, both alone and in paired combinations in laboratory and persistence trials. Single treatments exhibited significantly lower mortality rates in comparison to the paired treatments for all tested insect species. Among the single treatments, lambda-cyhalothrin (Lamb) resulted in significantly higher mortality rates in laboratory trials, followed by diatomaceous earth (DE) and M. robertsii (Mr), with insignificant differences between Mr and DE. Evidently, DE exhibited the highest persistence after 120 days of storage for all insect species and initial exposures, although variations in mortality rates among treatments were mostly insignificant. Overall, the most effective treatment in terms of mortality in laboratory, and persistence trials, and progeny production was DE + Lamb, followed by Mr + Lamb, and Mr + DE for all tested insect species. In general, the most susceptible insect species was R. dominica, followed by T. castaneum and T. granarium. This research highlights the effectiveness of M. robertsii, DE, and lambda-cyhalothrin in providing prolonged protection of stored wheat against all the examined grain insect species.

Suppression of Drosophila antifungal immunity by a parasite effector via blocking GNBP3 and GNBP-like 3, the dual receptors for ß-glucans.

The tactics used by animal pathogens to combat host immunity are largely unclear. Here, we report the depiction of the virulence-required effector Tge1 deployed by the entomopathogen Metarhizium robertsii to suppress Drosophila antifungal immunity. Tge1 can target both GNBP3 and GNBP-like 3 (GL3), and the latter can bind to ß-glucans like GNBP3, whereas the glucan binding by both receptors can be attenuated by Tge1. As opposed to the surveillance GNBP3, GL3 is inducible in Drosophila depending on the Toll pathway via a positive feedback loop mechanism. Losses of GNBP3 and GL3 genes result in the deregulations of protease cascade, Spätzle maturation, and antimicrobial gene expressions in Drosophila upon fungal challenges. Fly survival assays confirm that GL3 plays a more essential role than GNBP3 in combating fungal infections. In addition to evidencing the gene-for-gene interactions between fungi and insects, our data advance insights into Drosophila antifungal immunity.

The immune response mechanism of Nilaparvata lugens against a combined infection of rice ragged stunt virus and Metarhizium anisopliae.

BACKGROUND: Previous studies of brown planthopper (BPH), Nilaparvata lugens, showed that carrying the plant pathogenic virus, rice ragged stunt virus (RRSV), enhanced the lethality of the entomopathogenic fungus, Metarhizium anisopliae (YTTR). The underlying mechanism for this was not established but a serine protease cascade was hypothesized to be involved. RESULTS: Two immune response genes, NlKPI and NlVenomase, were identified and shown to be involved. The synthesized double-strand RNA (dsRNA) techniques used in this study to explore gene function revealed that treatment with dsRNA to silence either gene led to a higher BPH mortality from M. anisopliae infection than the dsRNA control treatment. NlKPI and NlVenomase play vital roles in BPH immunity to defend against alien pathogens. Both genes participate in the immune response process of BPH against co-infection with RRSV and M. anisopliae YTTR by regulating the expression of antimicrobial peptides and phenoloxidase activity. CONCLUSION: Our study provided new targets for BPH biocontrol and laid a solid foundation for further research on the interaction of virus-insect-EPF (entomopathogenic fungus). © 2023 Society of Chemical Industry.

Functional insights of three RING-finger peroxins in the life cycle of the insect pathogenic fungus Beauveria bassiana.

Peroxisomes play important roles in fungal physiological processes. The RING-finger complex consists of peroxins Pex2, Pex10, and Pex12 and is essential for recycling of receptors responsible for peroxisomal targeting of matrix proteins. In this study, these three peroxins were functionally characterized in the entomopathogenic fungus Beauveria bassiana (Bb). These three peroxins are associated with peroxisomes, in which BbPex2 interacted with BbPex10 and BbPex12. Ablation of these peroxins did not completely block the peroxisome biogenesis, but abolish peroxisomal targeting of matrix proteins via both PTS1 and PTS2 pathways. Three disruptants displayed different phenotypic defects in growth on nutrients and under stress conditions, but have similar defects in acetyl-CoA biosynthesis, development, and virulence. Strikingly, BbPex10 played a less important role in fungal growth on tested nutrients than other two peroxins; whereas, BbPex2 performed a less important contribution to fungal growth under stresses. This investigation reinforces the peroxisomal roles in the lifecycle of entomopathogenic fungi and highlights the unequal functions of different peroxins in peroxisomal biology.

Auto-dissemination of Cordyceps fumosorosea amongst adult females of the two-spotted spider mite.

Tetranychus urticae is an important pest worldwide. The auto-dissemination of spores of entomopathogenic fungi from an infected individual to conspecifics may be important for controlling pests that can build high populations. The current study was carried out to determine the auto-dissemination of the entomopathogenic fungus Cordyceps fumosorosea strain PFs-1 (Priority®) between T. urticae females. The study consisted of four experiments. First, the efficacy of entomopathogenic fungus bioassays was assessed in Petri dishes (experiment 1) and on potted bean plants (experiment 2). In the auto-dissemination trials (experiments 3 and 4, in Petri dishes and on potted plants, respectively), contaminated adult females (1-5) were released among uncontaminated females (10 individuals). All experiments were carried out separately, and observations were made on days 3, 5, and 7. In exp. 1, the control was different from Priority on all observation days. In exp. 2, the average number of surviving individuals in the control was significantly higher than in the Priority treatment. In the auto-dissemination experiments, as the number of contaminated individuals increased, the mortality rate of uncontaminated individuals also increased, in exp. 3 (Petri dishes) on all observation days, and in exp. 4 (potted plants) only on days 5 and 7. The median lethal time (LT50) decreased as the number of individuals contaminated with Priority increased in both Petri dish and pot trials. Consequently, the effectiveness of biological control may increase with the occurrence of indirect contamination from infected to uncontaminated individuals.

Transcriptomic Analysis of Metarhizium anisopliae-Induced Immune-Related Long Non-Coding RNAs in Polymorphic Worker Castes of Solenopsis invicta.

Long non-coding RNAs (lncRNAs) represent a class of RNA molecules that do not encode proteins. Generally studied for their regulatory potential in model insects, relatively little is known about their immunoregulatory functions in different castes of eusocial insects, including Solenopsis invicta, a notoriously invasive insect pest. In the current study, we used Metarhizium anisopliae, an entomopathogenic fungus, to infect the polymorphic worker castes (Major and Minor Workers) and subjected them to RNA sequencing at different intervals (6, 24, and 48 h post-infection (hpi)). Comprehensive bioinformatic analysis identified 5719 (1869 known and 3850 novel) lncRNAs in all libraries. Genomic characteristics analysis showed that S. invicta lncRNAs exhibited structural similarities with lncRNAs from other eusocial insects, including lower exon numbers, shorter intron and exon lengths, and a lower expression profile. A comparison of lncRNAs in major and minor worker ants revealed that several lncRNAs were exclusively expressed in one worker caste and remained absent in the other. LncRNAs such as MSTRG.12029.1, XR_005575440.1 (6 h), MSTRG.16728.1, XR_005575440.1 (24 h), MSTRG.20263.41, and MSTRG.11994.5 (48 h) were only present in major worker ants, while lncRNAs such as MSTRG.8896.1, XR_005574239.1 (6 h), MSTRG.20289.8, XR_005575051.1 (24 h), MSTRG.20289.8, and MSTRG.6682.1 (48 h) were only detected in minor workers. Additionally, we performed real-time quantitative PCR and experimentally validated these findings. Functional annotation of cis-acting lncRNAs in major worker ants showed that lncRNAs targeted genes such as serine protease, trypsin, melanization protease-1, spaetzle-3, etc. In contrast, apoptosis and autophagy-related genes were identified as targets of lncRNAs in minor ants. Lastly, we identified several lncRNAs as precursors of microRNAs (miRNAs), such as miR-8, miR-14, miR-210, miR-6038, etc., indicating a regulatory relationship between lncRNAs, miRNAs, and mRNAs in antifungal immunity. These findings will serve as a genetic resource for lncRNAs in polymorphic eusocial ants and provide a theoretical basis for exploring the function of lncRNAs from a unique and novel perspective.

Post-Application Field Persistence and Efficacy of Cordyceps javanica against Bemisia tabaci.

Previously, Cordyceps javanica Wf GA17, a causing agent of whitefly epizootics in southern Georgia, demonstrated superior temperature tolerance and higher virulence against the whitefly Bemisia tabaci than commercial strains in the laboratory. The post-application persistence and efficacy of this fungus against B. tabaci were compared with that of the commercially available C. javanica Apopka97 strain over a two-year field study in cotton and vegetable crops. When blastospores of both strains were applied alone, whitefly populations were not effectively suppressed. Thus, JMS stylet oil was added to fungal treatments for enhancing efficacy and persistence. For 0-day samples, all fungal treatments caused similar but significant levels of immature mortality regardless of fungal strain, propagule form (conidia vs. blastospores), and application method (alone or mixed with JMS). In follow-up samplings, Wf GA17 blastospores + JMS achieved higher control levels than other treatments in some trials, but the efficacy did not last long. The JMS oil alone caused significant mortality and suppressed whiteflies. Over 90% of spores lost viability 24 h after treatment in all fungal treatments. Across evaluation times, there was no difference between the two fungal strains (conidia or blastospores, alone or combined with JMS), but conidia persisted better than blastospores for both strains. Overall, the field persistence and efficacy of C. javanica did not last long; therefore, improved delivery methods and formulations are needed for enhancement.

Double-strand RNAs targeting MaltRelish and MaltSpz reveals potential targets for pest management of Monochamus alternatus.

Overcoming the innate immunity of insects is a key process to improve the efficiency of biological control. Antimicrobial peptides (AMPs) are important effectors in insect innate immunity, usually mediating resistance to pathogenic microorganisms through Toll and IMD signaling pathways. This study investigated the effect of key genes on upstream immune recognition receptor (GNBP3) and downstream effectors (AMPs) by RNAi technology. The transcriptome KEGG enrichment analysis and differential gene annotation results showed that the immune response genes MaltSpz and MaltRelish are important regulators of Toll and IMD signaling pathways, respectively. Both dsSpz and dsRelish could affect AMP gene expression and increase the expression of the immune recognition receptor MaltGNBP3. Moreover, they significantly reduce the survival rate of Monochamus alternatus and promote hyphal growth after Beauveria bassiana infection. This helps to improve the biological control effect of B. bassiana, control the population of vector insects and cut off the transmission route of pine wood nematode. The combined MaltSpz and MaltRelish knockdown increased the infection rate of M. alternatus larvae from 20.69% to 83.93%, achieving the best efficiency in synergistic B. bassiana infection. Our results showed important roles of MaltRelish- and MaltSpz-mediated regulation of AMP genes function in insect entomopathogenic fungi tolerance and induced significant mortality in larvae. Based on this study, MaltSpz and MaltRelish could represent candidate gene targets for the biological control of M. alternatus by RNAi.