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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.

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.

Background In July 2015, the carcasses of 11 cockatiels were submitted for disease diagnosis to the Avian Disease Division of the Animal and Plant Quarantine Agency of Korea. The cockatiels, which appeared dehydrated and underweight, had exhibited severe diarrhea and 22 % mortality over 2 weeks. Traditional diagnosis did not reveal the causes of these symptoms. Methods We conducted metagenomics analysis on intestines and livers from the dead cockatiels using Illumina high-throughput sequencing. To obtain more accurate and longer contigs, which are required for further genetic characterization, we compared the results of three de novo assembly tools (metaSPAdes, MEGAHIT, and IDBA-UD). Results Sequence reads of Campylobacter jejuni (C. jejuni) and Chlamydia psittaci (C. psittaci) were present in most of the cockatiel samples. Either of these bacteria could cause the reported symptoms in psittaciformes. metaSPAdes (ver.3.14.1) identified the 1152 bp flaA gene of C. jejuni and the 1096 bp ompA gene of C. psittaci. Genetic analysis revealed that flaA of C. jejuni was recombinant between C. jejuni and Campylobacter coli, and that ompA of C. psittaci isolated from cockatiel was closely related to strains isolated from humans. Conclusions C. jejuni and C. psittaci were detected in cockatiels in the Republic of Korea using metagenomic analysis. This approach is useful for understanding pathogens of pet birds. Three de novo assemblers were compared to obtain accurate contigs from large quantities of reads, and sequences of C. jejuni and C. psittaci generated by metaSPAdes were analyzed.

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.

We established a fungal production platform by focusing on substrates of solid culture for conidial productivity and thermotolerance, and focusing on surfactants to effectively deliver fungal conid...

The Japanese pine sawyer (JPS) beetle, Monochamus alternatus Hope (Coleoptera: Cerambycidae), damages pine trees and transmits the pine wilt nematode, Bursaphelenchus xylophilus Nickle. Chemical agents have been used to control JPS beetle, but due to various issues, efforts are being made to replace these chemical agents with entomopathogenic fungi. We investigated the expression of immune-related genes in JPS beetle in response to infection with JEF-197, a Metarhizium anisopliae isolate, using RNA-seq. RNA samples were obtained from JEF-197, JPS adults treated with JEF-197, and non-treated JPS adults on the 8th day after fungal treatment, and RNA-seq was performed using Illumina sequencing. JPS beetle transcriptome was assembled de novo and differentially expressed gene (DEG) analysis was performed. There were 719 and 1953 up- and downregulated unigenes upon JEF-197 infection, respectively. Upregulated contigs included genes involved in RNA transport, ribosome biogenesis in eukaryotes, spliceosome-related genes, and genes involved in immune-related signaling pathways such as the Toll and Imd pathways. Forty-two fungal DEGs related to energy and protein metabolism were upregulated, and genes involved in the stress response were also upregulated in the infected JPS beetles. Together, our results indicate that infection of JPS beetles by JEF-197 induces the expression of immune-related genes.

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.

Infectious laryngotracheitis (ILT), fowlpox (FP), and reticuloendotheliosis are important poultry diseases caused by gallid herpesvirus 1 (ILTV), fowlpox virus (FWPV), and reticuloendotheliosis virus (REV), respectively. Coinfections with ILTV and FWPV occur naturally in chickens, and FP in its more virulent wet form is characterized by diphtheritic lesions and easily confused with ILT. Moreover, the insertion of only partial REV-LTR or a nearly full-length REV into the FWPV genome, located between the ORF 201 and ORF 203, has increased recently in wild-type field FWPV isolates. Therefore, it is critical to detect ILTV, FWPV, REV-integrated FWPV, and REV early and accurately. In this study, we successfully developed a multiplex PCR assay for the simultaneous detection of ILTV, FWPV, REV-integrated FWPV, and REV, and the detection limits was 1 × 54 copies/tube. When used to test clinical samples, the results of the multiplex PCR were in 100% agreement with singleplex PCRs and sequencing. This new multiplex PCR is a simple, rapid, sensitive, specific, and cost-effective method for detection of 4 viruses in clinical specimens.

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.

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.

Insect-killing fungi have high potential in pest management. A deeper insight into the fungal genes at the whole genome level is necessary to understand the inter-species or intra-species genetic diversity of fungal genes, and to select excellent isolates. In this work, we conducted a whole genome sequencing of Beauveria bassiana (Bb) JEF-007 and characterized pathogenesis-related features and compared with other isolates including Bb ARSEF2860. A large number of Bb JEF-007 genes showed high identity with Bb ARSEF2860, but some genes showed moderate or low identity. The two Bb isolates showed a significant difference in vegetative growth, antibiotic-susceptibility, and virulence against Tenebrio molitor larvae. When highly identical genes between the two Bb isolates were subjected to real-time PCR, their transcription levels were different, particularly in heat shock protein 30 (hsp30) gene which is related to conidial thermotolerance. In several B. bassiana isolates, chitinases and trypsin-like protease genes involved in pathogenesis were highly conserved, but other genes showed noticeable sequence variation within the same species. Given the transcriptional and genetic diversity in B. bassiana, a selection of virulent isolates with industrial advantages is a pre-requisite, and this genetic approach could support the development of excellent biopesticides with intellectual property protection.

Entomopathogenic fungi are soil-dwelling microorganisms that infect host insects, and some have been used as biological control agents. Construction of an entomopathogenic fungal library provides a strong platform for the development of highly effective biopesticides, but the traditional method using antibiotic media has low isolation efficiency. Herein, to increase the efficiency of isolation, a Tenebrio molitor pathogenicity-based fungal collection method was used for the construction of a library. The isolation efficiency using the T. molitor larvae-based pathogenicity assay was 55.4%, which was significantly higher than the method of antibiotic medium with dodine (6.0%). We named it the Jeonbuk (Chonbuk) National University Entomopathogenic Fungi (JEF) library. It consisted of 279 isolates belonging to 12 genera and 29 species. Main features of the species-representative 15 isolates were characterized in terms of morphology, virulence, conidiogenesis, thermotolerance and production of biologically active materials such as virulence-related enzymes. Some of the species-representative isolates showed higher virulence against T. molitor, Riptortus pedestris and Plutella xylostella in laboratory conditions. The production of enzymes such as chitinase, Pr1 protease and lipase all related to pathogenesis in LB medium was higher than in the SDB and PDB media. In the thermotolerance assay, a Purpureocillium lilacinum isolate showed higher thermotolerance than other isolates. This work reports an efficient library method using T. molitor for building a library for development of fungal biopesticides for pest management.

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.

The western flower thrip, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), is recognized as a serious pest worldwide, but overuse of chemical pesticides results in environmental pollution and insect resistance. Herein we report the biocontrol of soil-dwelling stages of western flower thrips using entomopathogenic Beauveria bassiana granules. Using a B. bassiana isolate expressing enhanced green fluorescent protein (Bb-egfp), we confirmed that Bb-egfp was highly virulent to the thrips and colonized the soil, which allowed soil-dwelling pupae to come in contact with the colonized fungal mass. In a glasshouse trial, granules of BbERL836 and BbERL1578 isolates were applied to the soil surface of tomatoes growing in pots and female F. occidentalis were released on tomato plants three days after the fungal application. Populations reductions between 75 and 90% were observed in the treatments of BbERL836 and BbERL1578 at 20 and 40 days after application (chemical control: 85% reduction). In the field trial with cucumbers, the two ERL soil treatments showed ca. 90% control efficacy, similar to that of spinetorm, a semi-synthetic insecticide (85% efficacy). This study shows that using entomopathogenic B. bassiana granules to kill soil-dwelling stages of thrips is an effective strategy to manage F. occidentalis populations in tomato and cucumber.

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.

Most entomopathogenic fungal biopesticides have been produced in wettable powder and liquid concentrate formulations. Separation of fungal conidia from granular substrates to manufacture these formulations is costly and labor-intensive, which has hampered growth of the fungal biopesticide industry. Herein to overcome this challenge, we developed a novel screen bag formulation using mycotized granules produced from solid-substrate cultures of Beauveria bassiana JEF-007 and Metarhizium anisopliae JEF-003. The isolates were solid-cultured on Italian millet grains and packed in screen bags (pore size: ~ 15 μm). The bags containing the mycotized granules were immersed in water and agitated to disperse the conidia, after which the number of conidia released into the water was determined. These results were compared to the number of conidia released after vortexing the granules in water as a control. Various surfactants were added to the water to assess their effect on conidial release. Silwet L-77 yielded the highest conidial release. The insecticidal activity against Riptortus pedestris nymphs of suspensions produced from the screen bag formulation and vortexed conidial suspensions was similar, demonstrating that our novel formulation could be a cost-effective replacement for standard WP and ES formulations. The screen bag formulation has several advantages over other standard fungal-based formulations, such as a shorter and lower-cost production process. This formulation technology could also be applied to other conidial-based biopesticides, such as fungal fungicides, herbicides, and fertilizers.

The bean bug, Riptortus pedestris, is a major agricultural pest that reduces crop quality and value. Chemical pesticides have contributed to pest management, but resistance to these chemicals has significantly limited their use. Alternative strategies with different modes of action, such as entomopathogenic fungi, are therefore of great interest. Herein, we explored how entomopathogenic fungi can potentially be used to control the bean bug and focused on identifying virulence-related genes. Beauveria bassiana (JEF isolates) were assayed against bean bugs under laboratory conditions. One isolate, JEF-007, showed >80 % virulence by both spray and contact exposure methods. Agrobacterium tumefaciens-mediated transformation (AtMT) of JEF-007 generated 249 random transformants, two of which (B1-06 and C1-49) showed significantly reduced virulence against Tenebrio molitor and R. pedestris immatures. Both species were used for rapid screening of virulence-reduced mutants. The two transformants had different morphologies, conidial production, and thermotolerance than the wild type. To determine the localization of the randomly inserted T-DNA, thermal asymmetric interlaced (TAIL) PCR was conducted and analysis of the two clones found multiple T-DNA insertions (two in B1-06 and three in C1-49). Genes encoding complex I intermediate-associated protein 30 (CIA30) and the autophagy protein (Atg22) were possibly disrupted by the T-DNA insertion and might be involved in the virulence. This work provides a strong platform for future functional genetic studies of bean bug-pathogenic B. bassiana. The genes putatively involved in fungal virulence should be experimentally validated by knockdown in future studies.

In the present study, an enhanced green fluorescent protein gene (egfp) was expressed in Beauveria bassiana ERL836 based on the Agrobacterium tumefaciens-mediated transformation (AtMT) method. The ERL836 transformants were generated with pCambia-egfp binary vectors. Ten transformants were randomly selected and analyzed for the T-DNA insertion and gene expression. The results revealed that 60% (egfp) of the fungal putative transformants were inserted by the T-DNA fragment. The expressions of egfp in putative transformants were rapidly detected by reverse transcription PCR (RT-PCR) method. Among these transformants, 33.33% (egfp, 2 transformants) expressed the target genes. Two selected egfp transformants showed strong green fluorescence with different expression levels. The results of this study could provide a successful procedure for foreign protein production in B. bassiana by using the AtMT method and accomplished with a rapid selection of fungal transformants using RT-PCR.

Mosquitoes have been becoming serious vectors worldwide thus effective and safe control strategies should be established. Entomopathogenic fungi can be alternative controlling agents by substituting chemical insecticides. Herein we assayed 12 soil-borne entomopathogenic fungi against Asian tiger mosquito (Aedes albopictus) larvae in laboratory conditions and tried to establish an effective application method using millet granular formulation (GR). Twelve fungal isolates which belong to 6 genera (Beauveria, Cordyceps, Metarhizium, Paecilomyces, Purpureocillium and Verticillium) were assayed; M. anisopliae JEF-003 showed the fastest mosquitocidal activity, approximately 73% mortality rate at 2 days post-inoculation (dpi.) and > 90% mortality rate at 5 dpi. Conidia of M. anisopliae JEF-003 also showed a dosage dependent activity at 1 × 105, 1 × 106 and 1 × 107 conidia ml− 1. Hyphal growth of M. anisopliae JEF-003 in the Ae. albopictus larvae was observed by infection of an M. anisopliae JEF-003 EGFP-transformant, which was generated by restriction enzyme-mediated integration (REMI) method. GR of M. anisopliae JEF-003 showed high virulence to Ae. albopictus larvae (> 90% mortality) after 5 days of application. These results suggest that M. anisopliae JEF-003 has a potential to control A. albopictus larvae and GR can be practically used for management of the serious vector in water environment.