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Beauveria bassiana is one of the commercially available entomopathogenic fungi (EPF), and a number of isolates with high virulence and broad host spectrum have been used to control agricultural and forest pests. Although the functional importance of genes in EPFs' pathogenesis have been extensively studied, the precise ultrastructural mechanism of the fungal infection, particularly penetration of the host insect cuticles, is not well understood.In this study, we investigated the morphology and ultrastructure of the larval cuticle of the red flour beetle, Tribolium castaneum, after treatment with B. bassiana ERL1170 expressing an enhanced green fluorescent protein (Bb-eGFP). The Bb-eGFP showed high virulence against the larvae, with approximately 90% mortality at 48 h after treatment (HAT) and 100% at 72 HAT under our infection conditions. In these larvae, the regions of the body wall with flexible cuticles, such as the ventral and ventrolateral thorax and abdomen, became darkly melanized, but there was little to no melanization in the rigid dorsal cuticular structures. Confocal microscopy and transmission electron microscopy (TEM) indicated that germinated conidia on the surface of the larval cuticle were evident at 6 HAT, which formed penetration pegs and began to penetrate the several cuticle layers/laminae by 12 HAT. The penetration pegs then developed invading hyphae, some of which passed through the cuticle and reached the epidermal cells by 24 HAT. The larval cuticle was aggressively and extensively disrupted by 48 HAT, and a number of outgrowing hyphae were observed at 72 HAT.Our results indicate that Bb-eGFP is capable of infection and penetrating T. castaneum larvae shortly after inoculation (~24 HAT) at the body regions with apparently flexible and membranous cuticles, such as the ventral intersegmental regions and the ventrolateral pleura. This study provides details on the histopathogenesis of the host cuticle by infection and penetration of EPFs, which can facilitate the management of insect pests. © 2022 Society of Chemical Industry.

The species of Beauveria bassiana is widely used for the management of agricultural insect pests. In this study, we integrated egfp-double-stranded RNA (dsRNA) to a previously generated egfp-expressing B. bassiana transformant (Bb-egfp#3) using a protoplast integration method. The Bb-egfp#3 protoplast was mixed with the dsRNA under PEG/CaCl2 conditions and liquid-cultured in Sabouraud dextrose broth for 5 days. A control culture followed the same procedure without dsRNA. Bb-egfp#3/egfp-dsRNA cultures showed very low fungal growth (OD630 = 0.2) compared to the control culture, Bb-egfp#3 only (OD630 = 1.1). Screening of possible transformants on Sabouraud dextrose agar revealed a transformant T3, without egfp signal. T3 was confirmed as B. bassiana through sequencing of conserved genes and insect bioassays. Interestingly, the genomic egfp fragment of T3 was disrupted, and the egfp signal was not detected over four subcultures, which was also confirmed by RNA-seq of Bb-egfp#3 and T3. This study provides an interesting observation that protoplast integration with dsRNA could possibly generate significantly reduced gene expression in B. bassiana and it is stable across several generations.

All living things on Earth experience various diseases such as those caused by viruses, bacteria, and fungi. Insects are no exception to this rule, and fungi that cause disease in insects are called entomopathogenic fungi. These fungi have been developed as microbial insecticides and are used to control various pests. Generally, the mode of action of entomopathogenic fungi is divided into the attachment of conidia, germination, penetration, growth, and generation of secondary infectious conidia. In each of these steps, that entomopathogenic fungi use genes in a complex manner (specific or diverse) has been shown by gene knock-out and RNA-sequencing analysis. In this review, the information mechanism of entomopathogenic fungi was divided into six steps: (1) attachment of conidia to host, (2) germination and appressorium, (3) penetration, (4) fungal growth in hemolymph, (5) conidia production on host, and (6) transmission and dispersal. The strategy used by the fungi in each step was described at the genetic level. In addition, an approach for studying the mode of action of the fungi is presented.

Background Entomopathogenic Beauveria bassiana has been used as a biocontrol agent for insect pests, but its effect at the molecular level on the hosts has not been studied in detail. Herein, we performed transcriptome analysis of bean bug, Riptortus pedestris (Hemiptera: Alydidae) in response to infection with a highly virulent strain of B. bassiana JEF-007 (Bb JEF-007). Results Based on RNA-seq data from R. pedestris infected with Bb JEF-007 compared with non-infected bean bugs, infection was assumed to strongly activate (i) the energy production pathway by expressing dehydrogenases, (ii) metabolic pathways by expressing secreted proteins, GTPase, MBF2 transcription factor family, pigment-dispersing factor, antioxidants, and cuticle proteins, and (iii) the immune response pathway by expressing serine-threonine kinase in Toll pathway of bean bug. Conclusion We have established the platform for functional studies of the genes required for an immune response against entomopathogenic fungi like B. bassiana in the bean bug, R. pedestris. Moreover, this study also paves the way for genetic modification of B. bassiana to combat with the defense mechanism of R. pedestris. © 2018 Society of Chemical Industry.

The Toll signaling pathway is responsible for defense against both Gram-positive bacteria and fungi. Gram-negative binding protein 3 (GNBP3) has a strong affinity for the fungal cell wall component, β-1,3-glucan, which can activate the prophenoloxidase (proPO) cascade and induce the Toll signaling pathway. Myeloid differentiation factor 88 (MyD88) is an intracellular adaptor protein involved in the Toll signaling pathway. In this study, we monitored the response of 5 key genes (TmGNBP3, TmMyD88, and Tenecin 1, 2, and 3) in the Toll pathway of the mealworm Tenebrio molitor immune system against the fungus Beauveria bassiana JEF-007 using RT-PCR. TmGNBP3, Tenecin 1, and Tenecin 2 were significantly upregulated after fungal infection. To better understand the roles of the Toll signaling pathway in the mealworm immune system, TmGNBP3 and TmMyD88 were knocked down by RNAi silencing. Target gene expression levels decreased at 2 d postknockdown and were dramatically reduced at 6 d post-dsRNA injection. Therefore, mealworms were compromised by B. bassiana JEF-007 at 6 d post-dsRNA injection. Silencing of TmMyD88 and TmGNBP3 resulted in reduced resistance of the host to fungal infection. Particularly, reducing TmGNBP3 levels obviously downregulated Tenecin 1 and Tenecin 2 expression levels, whereas silencing TmMyD88 expression resulted in decreased Tenecin 2 expression. These results indicate that TmGNBP3 is essential to induce downstream antifungal peptide Tenecin 1 expression against B. bassiana JEF-007.

Larvae of mealworms Tenebrio molitor L. (Coleoptera: Tenebrionidae) have been used as animal feed, but fungal pathogens rapidly downsize the populations, resulting in economic losses. In this work, we established an effective management strategy for fungal pathogens. An entomopathogenic fungus, Beauveria bassiana, was isolated from mealworm cadavers. The bioassay of some isolates of this species at >90% relative humidity revealed that the ERL1575 isolate had the highest virulence. At 20–30% RH, ERL1575 conidia when ingested produced 80% mortality but when sprayed topically produced only 95%) for 5 days. This experiment produced about 90% mortality except at 35°C where mortality was

BACKGROUND Spraying of entomopathogenic fungi formulated as wettable powders and suspension concentrates based on oils or water has been used for the management of crop pests. However, the use of fungal granules to control paddy field pests has not been fully explored. Herein, several Beauveria bassiana isolates (ERL1170, ERL1578 and ERL836) were produced as granules using a millet-based solid culture. The granules were applied to the rice nursery 3 days before transplanting, and their control efficacy against rice water weevils was determined in paddy fields. RESULTS The solid cultures produced ∼1 × 108 conidia g−1 of millet grains 10 days after the inoculation. The granules were applied to the soil in the rice nursery 3 days before the rice seedlings were transplanted in the paddy fields. Rice in plots with granules of ERL1578 had 17.3% leaf damage (74% control efficacy) 14 days post-application, whereas rice plants in the untreated control had 66.5% damage. Rice plants treated in the nursery with ERL1170 and ERL836 had 52–54% damage. In the rice plots previously treated with ERL1578, the smallest numbers of larvae and adults were observed 38 days post-application. In laboratory conditions, ERL1578-treated larvae turned pink and were covered with mycelial mass. CONCLUSION Applications of millet-based B. bassiana granules on rice nursery soil can be an effective and efficient biological control strategy for the management of rice water weevils. This method can be relatively inexpensive and requires less labor than practices involving the spraying of fungi directly on rice in paddy fields. © 2014 Society of Chemical Industry

BACKGROUND: To date, emphasis has been placed on studying the virulence of Isaria fumosorosea conidia rather than the virulence of their blastospores. In the present study, a comparison was made of the efficacy of blastospores of I. fumosorosea (SFP-198) versus conidia against greenhouse whitefly (GWF) Trialeurodes vaporarioum nymphs and thermotolerance at 50 ◦ C. To improve thermotolerance and potency, the blastospores were suspended in corn oil with a wetting and an insect-filming agent. RESULTS:BlastosporeshadsimilarefficacyagainstGWFnymphstoconidiainglasshouseconditionsbuthadlessthermotolerance than conidia. However, blastospores in corn oil had increased thermotolerance, comparable with that of conidia in corn oil. Application of SFP-198 blastospore oil-based formulation containing isotridecyl alcohol ethoxylated-3EO (TDE-3) and sodium alginate (SA) as adjuvants showed enhanced efficacy (95.7% mortality), compared with the SA-free oil formulation (72.8%) in 10days. CONCLUSION: SFP-198 blastospores can be used as an effective biological control agent, given their virulence against GWF, the enhanced thermotolerance in corn oil and the oil-based formulation studies to enhance their efficacy. Blastospores can be easily produced in liquid cultures in less than 4days, which is significantly shorter than using traditional solid cultures for conidial production. c � 2013 Society of Chemical Industry

Baculovirus chitinase gene (chiA) is a late gene essential for liquefying the host insect at a late stage of infection for its hydrolyzing chitin function. In a previous report, baculovirus ChiA has been shown to offer many interesting new opportunities for pest control. Recently, a putative chiA gene was identified in the Korean isolate of the Spodoptera litura nucleopolyhedorvirus (SpliMNPV-K1) genome. The open reading frame (ORF) contains 1692 nucelotides and encodes a protein of 563 amino acids with a predicted molecular weight of about 62.6 kDa. To study the insecticidal activity of ChiA from SpliMNPV-K1, we constructed a recombinant AcMNPV,Ap-SlChiA, which is designed to express the ChiA under the control of a polyhedrin promoter. Western blot analysis indicated that ChiA was successfully expressed by this recombinant virus. Chitinase assay revealed that the chitobiosidase and endochitinase activity of the recombinant virus was 2.5- and 3.9-flods higher than those of wild-type AcMNPV, respectively. In addition, the recombinant virus showed higher evident insecticidal activity against 3rd instar larvae of Spodotera exigua than that of the AcMNPV. These results suggest that the chiA gene from SpliMNPV-K1 could be successfully applied to improve pathogenicity of baculoviruses.

BACKGROUND: Insecticidal activities of five photoactive dyes against greenhouse whitefly (GWF), Trialeurodes vaporariorum West., in tomatoes were investigated to improve the control efficacy of an entomopathogenic fungal product, Isaria fumosorosea SFP-198 wettable powder (WP). Azorubine, Eosin B, Erythrosine, Brilliant Green and Phloxine B were used in this work, accompanying pyriproxyfen emulsifiable concentrate (EC) as a commercial standard for comparison. RESULTS: Phloxine B had the highest control efficacy in glasshouse conditions. The most suitable dose of Phloxine B was determined as 0.005 g L−1, given the dosage-dependent control efficacy and phytotoxicity of the dye, and its influence on the germination of SFP-198 conidia. In field conditions, SFP-198 WP + Phloxine B (2 + 0.005 g L−1; tank mix) showed 89.1 and 95.3% control efficacy 7 and 14 days post-application, which was significantly higher than the efficacy of SFP-198 WP alone (43.5 and 64.0%), Phloxine B (47.5 and 30.7%) or pyriproxyfen EC (67.7 and 80.2%). CONCLUSION: Phloxine B cooperates with SFP-198 WP complementarily, possibly in the order of killing action (early: Phloxine B; late: SFP-198 WP). The dye can be incorporated into SFP-198 WP to improve its efficacy and applied to other Hypocrelean entomopathogenic fungal products. Copyright © 2010 Society of Chemical Industry

The brown marmorated stink bug. Halyomorpha halys, (BMSB) is an exotic pest causing damage to fruits and vegetables. They also transmit diseases. Integrated pest management strategies are lacking for this invasive. For the first time, information is presented on the virulence of several entomopathogenic fungi against BMSB. Three Beauveria bassiana and two Metarhizium anispoliae isolates were bioassayed against adult BMSB. One B. bassiana isolate was the active ingredient in BotaniGard® which produced 85 and 100% mortality in 9 and 12 days post treatment, respectively. Experimental isolates ERL 1170 and ERL 1540 were also efficacious. Metarhizium anispoliae isolates produced lower mortalities than the B. bassiana isolates. Data presented showed the potential for management of BMSB with entomopathogenic fungi. Copyright © 2012 Society of Chemical Industry