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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.

Chemical control is widely used to control the Japanese pine sawyer beetle, Monochamus alternatus, but strong chemical regulations require an environmentally sound management strategy. In this work, we investigated the use of entomopathogenic fungi and their application as a means of practical pest management. Thirty-two diverse species of fungal isolates were assayed against adult pine sawyer beetles using a contact method under laboratory conditions, and four isolates showed over 70% virulence consequently. These isolates, two each of Beauveria bassiana and Metarhizium anisopliae were sprayed on the adult beetles at 1 × 107 conidia/ml in plastic containers, respectively. The M. anisopliae-treated adult beetles showed 67% mortality. M. anisopliae isolates JEF-197 and JEF-279 demonstrated dosage-dependent insecticidal activity. Following the laboratory experiments, semi-field trials were conducted in young pine trees under high (RH 94%) and low (RH 35%) humidity conditions. In the high humidity conditions, most of the adult beetles stayed on the top of the branches. When the two M. anisopliae isolates were sprayed on the beetles, they showed ca. 50–70% insecticidal activity 11 days after application. In contrast, in low humidity conditions, the adult beetles tried to move off the branches and onto the soil. When the beetles reached the JEF-197 and JEF-279-treated soil, we measured >90% insecticidal activity. This work suggests that M. anisopliae was the most virulent entomopathogenic fungus against adult Japanese pine sawyer beetles, and this forest insect could be ecologically controlled by the spray and soil application of the M. anisopliae isolates.

Root-feeding Scarabaeidae, particularly white grubs are considered among the most harmful coleopteran insect pests in turfgrass. In this work, sixteen entomopathogenic fungal species were assayed against flower chafer beetle, Protaetia brevitarsis (Coleoptera: Scarabaeidae) and Metarhizium anisopliae JEF-314 showed high virulence. The control ability of the isolate JEF-314 has been in detail tested for a model insect flower chafer beetle. Further analyses showed insect stage-dependent virulence where the fungal virulence was the highest against smaller instar larvae. Additionally, we confirmed that millet-based solid cultured granule was effective against the soil-dwelling larval stage. The isolate also showed a similar ability for a representative pest (Popillia spp.) in laboratory conditions. Our results clearly suggest a high potential of M. anisopliae JEF-314 to control the flower chafer beetle, possibly resulting in controlling of root-feeding white grubs in turfgrass. Based on the insect life cycle and susceptibility to the fungus, late spring and summer time would be the optimum time to apply JEF-314 granules for an effective control. Further characterization of the efficacy of the fungus under field conditions against the Scarabaeidae beetles might provide an efficient tool to control this beetle in an environment-friendly way.

We investigated the colonization potential of five Metarhizium anisopliae isolates on pine tree surfaces under laboratory conditions, determined the influence of the pine bark extract on fungal growth and evaluated the insecticidal activity following colonization on the Japanese pine sawyer. Finally, the effect of colonization on adults pine sawyer was evaluated using the top three performing isolates (JEF-197, JEF-271 and JEF-279) under laboratory and field conditions. As a result, isolate JEF-197 showed the highest conidial production on the pine surfaces, and five isolates, including JEF-197, showed higher hyphal growth on autoclaved pine bark extract agar, compared to a water agar. Pine bark treated with the isolates showed 40–70 % mortality of adults pine sawyer. Under mimicked overwintering conditions, in the JEF-197 treatment group, 40 % of the inserted larvae became adults and all were dead after 59 d. In a field test, colonized isolate JEF-197 also showed 37 % insecticidal activity against emerged adults from the pine logs as overwintering sites. This work suggests that M. anisopliae isolate JEF-197 possibly colonized the pine surface and application of a conidial suspension on the pine logs as overwintering sites could be an effective strategy to control the pine sawyer.

Beauveria bassiana (Balsamo) Vuillemin (Hypocreales: Cordycipitaceae) ERL836 has been commercialized under the name ChongchaeSak to control an agricultural insect pest, the western flower thrips Frankliniella occidentalis Pergande (Thysanoptera: Thripidae), in the Republic of Korea. As soon as it was launched in 2017, it became a popular product and has received a positive response. However, study of the storage stability of the fungus ERL836 has yet to be investigated. To determine the optimum conditions for long-term storage, we assessed conidial viability and insecticidal activity of B. bassiana ERL836 according to storage temperature and culture substrate. Viability of B. bassiana ERL836 conidia from mycotized grains (millet and rice) stored at low (4 °C) and moderate (25 and 30 °C) temperatures was maintained at >85% for 24 and 18 months, respectively, along with insecticidal activity. In contrast, the samples stored at 37 °C showed low germination rate (about 80% germination rate for only 5 months). This result suggests that low and moderate temperatures (4 to 30 °C) conserve B. bassiana ERL836 viability and virulence.

Background Insect-killing fungal species, Beauveria bassiana, is as an environment-friendly pest management tool, and many isolates are on the track of industrialization. However, some of B. bassiana isolates show similar morphology and virulence against insect pests, and so it is hard to differentiate them. Herein we used two patented isolates, ERL836 and JEF-007, and investigated their virulence against western flower thrips, Frankliniella occidentalis, and further analyzed genome structures and transcriptional responses when interacting with cuticles of thrips to see possible differences on the initial step of fungal infection. Results The two isolates showed no significant differences in fungal growth, conidial production, and virulence against thrips, and they were structurally similar in genome. But, in transcription level, ERL836 appeared to infect thrips easily, while JEF-007 appeared to have more difficulty. In the GO analysis of ERL836 DEGs (differentially expressed genes), the number of up-regulated genes was much larger than that of down-regulated genes, when compared to JEF-007 DEGs (more genes down-regulated). Interestingly, in the enrichment analysis using shared DEGs between two infecting isolates, plasma membrane-mediated transporter activity and fatty acid degradation pathway including cytochrome P450 were more active in infecting ERL836. Conclusion The two B. bassiana isolates had similar morphology and virulence as well as genome structure, but in transcription level they differently interacted with the cuticle of western flower thrips. This comparative approach using shared DEG analysis could be easily applied to characterize the difference of the two B. bassiana isolates, JEF-007 and ERL836.

Beauveria bassiana is a species complex whose isolates show considerable natural genetic variability. However, little is known about how this genetic diversity affects the fungus performance. Herein, we characterized the diversity of genes involved in various mechanisms of the infective cycle of 42 isolates that have different growth rates, thermotolerance and virulence. The analysed genes showed general genetic diversity measured as non-synonymous changes (NSC) and copy number variation (CNV), with most of them being subjected to positive episodic diversifying selection. Correlation analyses between NSC or CNV and the isolate virulence, thermotolerance and growth rate revealed that various genes shaped the biological features of the fungus. Lectin-like, mucin signalling, Biotrophy associated and chitinase genes NSCs correlated with the three biological features of B. bassiana. In addition, other genes (i.e. DNA photolyase and cyclophilin B) that had relatively conserved sequences, had variable CNs across the isolates which were correlated with the variability of either virulence or thermotolerance of B. bassiana isolates. The data obtained is important for a better understanding of population structure, ecological and potential impact when isolates are used as mycoinsecticides and can justify industrialization of new isolates.

Background: Insect-killing fungal species, Beauveria bassiana, is as an environment-friendly pest management tool, and many isolates are on the track of industrialization. However, some of B. bassiana isolates show similar morphology and virulence against insect pests, and so it is hard to differentiate them. Herein we used two patented isolates, ERL836 and JEF-007, and investigated their virulence against western flower thrips, Frankliniella occidentalis, and further analyzed genome structures and transcriptional responses when infecting the thrips to see possible differences. Results: The two isolates showed no significant differences in fungal growth, conidial production, and virulence against thrips, and they were structurally similar in genome. But, in transcription level, ERL836 appeared to infect thrips easily, while JEF-007 appeared to have more difficulty. In the GO analysis of ERL836 DEGs (differentially expressed genes), the number of up-regulated genes was much larger than that of down-regulated genes, when compared to JEF-007 DEGs (more genes down-regulated). Interestingly, in the enrichment analysis using shared DEGs between two infecting isolates, plasma membrane-mediated transporter activity and fatty acid degradation pathway including cytochrome P450 were more active in infecting ERL836. Conclusion: The two B. bassiana isolates had similar morphology and virulence as well as genome structure, but in transcription level they differently infected western flower thrips. This comparative approach using shared DEG analysis could be easily applied to characterize the difference of the two B. bassiana isolates, JEF-007 and ERL836.

Additional file 1 : Table S1. de novo assembly of B. bassiana JEF-007 (A) and ERL846 (B) after the sequencing of whole genomes using Pac Bio RSII technology with error correction. Table S2. de novo assembly of B. bassiana RNA-sequencing raw data. Table S3. GO enrichment analysis of differentially expressed and shared genes between cuticle-interacting B. bassiana JEF-007 and ERL836. Table S4. Primers used in qRT-PCR for validation of B. bassiana RNA-sequencing. Figure S1. Validation of RNA-sequencing of B. bassiana ERL836 and JEF-007 using qRT-PCR.

Entomopathogenic fungi have great potential to control agricultural and horticultural insect pests, however optimizing conidial production systems to demonstrate high productivity and stability still needs additional efforts for successful field application and industrialization. Although many virulent entomopathogenic fungal isolates have been viewed as potential candidates in a laboratory environment, very few of the isolates are being used in practice for application in agricultural fields as commercial products. I. javanicus is an entomopathogenic fungus that is parasitic to various diverse coleopteran and lepidopteran insects and thought good candidate as biopesticdes. In this work, the basic characteristics of two entomopathogenic fungi, I. javanica FG340 and Pf04, were investigated in morphological examinations, genetic identification, and virulence against Thrips palmi, and then the feasibility of various grains substrates for conidial production was assessed, particularly focusing on conidial productivity and thermotolerance. Isaria javanica FG340 and Pf04 conidia were solid-cultured on 12 grains for 14 days in a Petri dish. Of the tested Italian millet, perilla seed, millet and barley-based cultures showed high conidial production. The four-grain media yielded >1 × 109 conidia/g of I. javanica FG340 and Pf04. Pf04 strain had enhanced thermotolerance up to 45 °C when cultured on Italian millet. In application, it was easy to make a conidial suspension using the cultured grains, and several surfactants were tested to release the conidia. This work suggests several possible inexpensive grain substrates by which to promote conidial production combined with enhanced stability against exposure to high temperature.

Root-feeding Scarabaeidae, particularly white grubs are considered among the most harmful coleopteran insect pests in turfgrass. In this work, sixteen entomopathogenic fungal species were assayed against flower chafer beetle, Protaetia brevitarsis (Coleoptera: Scarabaeidae) and Metarhizium anisopliae JEF-314 showed high virulence. The control ability of the isolate JEF-314 has been in detail tested for a model insect flower chafer beetle. Further analyses showed insect stage-dependent virulence where the fungal virulence was the highest against smaller instar larvae. Additionally, we confirmed that millet-based solid cultured granule was effective against the soil-dwelling larval stage. The isolate also showed a similar ability for a representative pest (Popillia spp.) in laboratory conditions. Our results clearly suggest a high potential of M. anisopliae JEF-314 to control the flower chafer beetle, possibly resulting in controlling of root-feeding white grubs in turfgrass. Based on the insect life cycle and susceptibility to the fungus, late spring and summer time would be the optimum time to apply JEF-314 granules for an effective control. Further characterization of the efficacy of the fungus under field conditions against the Scarabaeidae beetles might provide an efficient tool to control this beetle in an environment-friendly way.

The longhorned tick (bush tick), Haemaphysalis longicornis (Ixodida: Ixodidae), is a serious pest; it transmits the severe fever with thrombocytopenia syndrome (SFTS) virus to humans and has a wide distribution. The use of chemical control is not favored for environmental and health reasons, so more environmentally sound management methods need to be developed. Herein, we describe the use of an entomopathogenic fungal library to develop a fungus-mediated tick management system. Field-collected nymphs were assayed for their susceptibility to entomopathogenic fungi belonging to genera Beauveria, Metarhizium, Cordyceps, and Akanthomyces. Three M. anisopliae s.l. isolates, JEF-214, -279, and -290 showed high virulence in a dose-dependent manner. One Cordyceps isolate was pathogenic but virulence was much lower than the M. anisopliae isolates. Beauveria isolates were not pathogenic to the tick. Because the longhorned tick dwells on the soil surface except for blood-feeding periods, the soil surface was sprayed with conidial suspensions of the isolates after the release of longhorned ticks. The treatments resulted in 60–90% mortality after 30 days. M. anisopliae s.l. isolates were highly virulent against longhorned tick, and the application of fungus-based biopesticides on the soil surface could be an effective control strategy to reduce the tick population for long-term tick management.