Beauveria bassiana JEF-007 with strong virulence against Riptortus pedestiris was selected for the Agrobacerium tumefaciens-mediated transformation(AtMT). AtMT generated two transformants, B1-06 and C1-49, showed significantly reduced virulence against R. pedestris. To identify the virulence-related genes, thermal asymmetric interlaced(TAIL) PCR and flanking region analysis were performed. From the analysis, two genes, Complex I intermediated-associated protein 30(CIA30) and Autophagy protein 22(Atg22), possibly related virulence in B. bassiana JEF-007. For the analysis of two putative virulence-related genes in JEF-007, hairpin RNA interference (hpRNAi) is under consideration. This work can provide the functional roles of the virulence-related genes in B. bassiana JEF-007.

Myeloid differentiation factor 88 (MyD88) is an intracellular adaptor protein involved in Toll pathway. In this study, we monitored the response of 4 key genes of the insect immune system against Beauveria bassiana JEF-007 in Tenebrio molitor using RT-PCR. To better understand the roles of Toll pathway in mealworm immune system, TmGPR and TmMyD88 was knocked down by RNAi silencing. Target gene expressions were decreased at 6 days post-dsRNA injection. Therefore, mealworms were compromised by B. bassiana JEF-007 at 6 days post-dsRNA injection. Silencing of the TmMyD88 and TmGPR resulted in reducing the resistance of the host to fungal infection. However, only dsTmMyD88 showed significant difference with dsEGFP by statistical analysis, which may be due to partial gene knock down of dsGPR. These results indicate that TmMyD88 is required in mealworms for survival against B. bassiana JEF-007.

A viral genome was assembled de novo from next-generation sequencing (NGS) data from bean bugs, Riptortus pedestris, infected with an entomopathogenic fungus, Beauveria bassiana (Bb), and was further confirmed via the RACE method. Based on the phylogenetic analysis of the RdRp sequences, RiPV-1 was clustered in the unassigned insect RNA viruses with two other viruses, APV and KFV. These three viruses were suggested to constitute a new group of insect RNA viruses. Interestingly, RiPV-1 replication was increased dramatically in bean bugs 2 to 6 days after fungal infection. In conclusion, a novel insect RNA virus was found by NGS data assembly. This virus can provide further insight into the interaction between virus, fungus and the host.

A novel insect-infecting positive sense single-stranded RNA virus, Riptortus pedestris virus-1 (RiPV-1), was found in the Riptortus pedestris transcriptome data by de novo assembly and further confirmed by RACE method. The genome of RiPV-1 consists of 10,554 nucleotides (nt) excluding the poly(A) tail and contains a single large open reading frame (ORF) of 10,371 nt encoding a 3,456 aa polyprotein and flanked by 71 and 112 nt 5' and 3' noncoding regions, respectively. RiPV-1 genome contains the consensus genome organization of picorna-like RNA helicase, cysteine protease, and RNA-dependent RNA polymerase (RdRp) array in that order from the 5' to the 3' end. From the phylogenetic analysis, RiPV-1 was clustered with unassigned insect RNA viruses, APV and KFV, which suggests that these three insect picorna-like viruses might constitute a novel group of insect-infecting RNA viruses. Tissue tropism analysis revealed that RiPV-1 was relatively abundant in the thorax, abdomen, midgut and fat body. Interestingly, RiPV-1 replication was enhanced by Beauveria bassiana JEF-007 infection that was quantified using qRT-PCR. This study identified a novel insect-infecting virus and provided further insight into the relationship between virus, fungus and host.

Beauveria bassiana (Bb) is an entomopathogenic fungus with a wide host range, and is commonly used as an environment-friendly biopesticide. However, the molecular mechanisms of Bb-host interactions are not well understood. Here, RNA isolated from a highly virulent strain of B. bassiana (Bb JEF-007) and Riptortus pedestris (Hemiptera: Alydidae) (bean bug) infected with this strain were subjected to high throughput next generation sequencing (NGS) to analyze and compare transcriptomes. Differentially expressed gene (DEG) analysis showed that 2,381 genes were up-regulated and 2,303 genes were down-regulated upon infection. Most DEGs were classified into the categories of single-organism, cellular and metabolism processes by gene ontology (GO) analysis. Carbon metabolism-related enzymes in the glyoxylate cycle were significantly up-regulated, suggesting a possible role for them in Bb growth in the host. This work provides insight into how entomopathogenic B. bassiana occupies agriculturally harmful bean bug at the late stage, which might be essential during fungal infection.

Bean bug, Riptortus pedestris (Hemiptera: Alydidae) is an agriculturally serious pest in East Asian countries. Chemical pesticides have been contributed to the management of the pest, but nowadays insect resistance limits the use of chemical pesticides, thus alternatively new pesticides with different mode of actions such as entomopathogenic fungi are considered. Herein entomopathogenic Beauveria bassiana JEF isolates were collected, identified and assayed against bean bugs in laboratory conditions. Some isolates showed >80% virulence by contact-exposure and spray methods. The Agrobacterium tumefaciens-mediated transformation of B. bassiana JEF-007 generated random transformants and some mutants showed reduced virulence against Tenebrio molitor (Coleoptera: Tenebrionidae) larvae and R. pedestris nymph. Compared to the wild-type, the two transformants showed remarkably different morphology, conidial production, and thermotolerance. To figure out pathogenicity-related genes, thermal asymmetric interlaced (TAIL) PCR of the random transformants was performed and possibly some virulence-related genes were predicted. This work can be a strong platform for the functional genetics of bean bug-pathogenic B. bassiana.

Myeloid differentiation factor 88 (MyD88) is an intracellular adaptor protein involved in Toll signaling pathway. In this study, we monitored the response of 4 key genes of the insect immune system against Beauveria bassiana JEF-007 in Tenebrio molitor using RT-PCR. TmGPR, antimicrobial peptide Tenecin 1 and Tenecin 2 were up-regulated after fungal infection. To better understand the roles of Toll signaling pathway in mealworm immune system, TmGRP and TmMyD88 was knocked down by RNAi silencing. Target gene expressions were decreased at 2 days post-dsRNA injection, and dramatically reduced at 6 days post-dsRNA injection. Therefore, mealworms were compromised by B. bassiana JEF-007 at 6 days post-dsRNA injection. Silencing of the TmMyD88 and TmGRP resulted in reducing the resistance of the host to fungal infection. However, only dsTmMyD88 showed significant difference with dsEGFP by statistical analysis, which may be due to partial gene knock down of dsGRP. These results indicate that TmMyD88 is required in mealworms for survival against B. bassiana JEF-007.

Western flower thrips, (Pergande) (Thysanoptera: Thripidae) have been recognized as serious pests worldwide. Commercially available chemical pesticides challenges against the resistance of the pest and safety to consumers. Herein we investigated the usability of soil-treatment of entomopathogenic Beauveria bassiana (Bb) ERL isolates in tomatoes. Bb-egfp was highly virulent to western flower thrips in laboratory conditions and colonized in soil, which provided a chance for western flower thrips larvae to contact the colonized fungal mass. In a glasshouse trial, Bb ERL836 and Bb ERL1578 granule (GR) were applied to soil surface and mixed well, and five females were infested to each pot of tomato 3 days after the application. The two treatments significantly reduced thrips population in tomatoes 20 and 40 days post applications, comparable to the control efficacy of clothianidin granule (GR) formulation. In the following field trial, the two ERL fungal treatments showed 69.2% and 61.5% of control efficacy, which were similar to that of clothianidin GR (76.9%). This work suggests that applied B. bassiana colonized in soil well and thrips larvae easily contacted the fungal mass, which possibly enabled the control strategy to be alternative way to the chemical control.

The present issue of insect resistance and environmental toxicity of pesticides is triggering deep discussion about the pest management tactics, in which pest monitoring and control activity are mainly involved. Novel control agents, hopefully overcoming the present issues and problems, should be researched and commercially applied to the farm fields. With the monitoring-based research, additionally we have to focus on the control-based, particularly control agent-based research and application. Entomo- pathogenic fungi can used as one of the possible novel control agents once considerations are given to the control of soil- or water-dwelling pests. In our research group, the entomopathogenic fungal library has been constructed using the mealworm-based isolation system, which showed a variety of opportunities of their use in pest control. Important key production technologies including granular formulation have been developed to increase their industrialization. Some entomopathogenic fungal isolates showed high biological performance in the control of rice weevils, western flower thrips and Japanese bettles in field stands. To elucidate the fungal mode of action, a fungal transformation system using AtMT and gene identification tools were established. Recently a more deep study about the relationship between insect and entomopathogenic fungi is be investigated using RNA seq. We suggest that to make the entomopathogenic fungal products be applied to agricultural farm field, R&D of down-stream process should be seriously considered as the key step.

Bean bug, Riptortus pedestris is an agriculturally serious pest in East Asian countries, reducing the value of crop quality and loss of income in agribusiness. Chemical pesticides have contributed to the management of the pest, but nowadays insect resistance limits the use of chemical pesticides, thus alternatively new pesticides with different mode of actions such as entomopathogenic fungi are considered. Beauveria bassiana and Metarhizium anisopliae JEF isolates were collected, identified and assayed against bean bugs in laboratory conditions. Some isolates showed >80% virulence by spray and contact-exposure methods. Supernatant showed different level of enzyme activity including chitinase, Pr1 protease and lipase. The Agrobacterium tumefaciens-mediated transformation generated random transformants and some mutants had reduced virulence. TAIL-PCR of the random transformants revealed virulence-related genes. This work can be a strong platform for the functional genetics of bean bug-pathogenic B. bassiana.

The ascomycete fungus Beauveria bassiana is a wide host range entomo- pathogenic fungus, which is commonly used as an environmental friendly biopesticide. However, the molecular mechanisms of host-pathogen interaction of B. bassiana are not well understood. Here, the high throughput next generation sequencing was performed to analyze the transcriptome of B. bassiana JEF-007 infected bean bug (Riptorus pedestris). Differentially expressed gene (DEG) analysis results showed that total 4,684 genes including 2,381 up and 2,303 down regulated genes were identified. Most of the DEGs were classified into single- organism, cellular and metabolism processes by gene ontology (GO) analysis. Metabolism pathway was the most abound category of DEGs via KEGG pathway mapping. Several possible candidates of virulence factors were dramatically expressed after infection, such as cytotoxic lectin, bacterial-like toxin, and proteins related to cell wall, hyphal growth, nutrient uptake and halogenated compounds synthesis. Furthermore, we also found the highest expression of a novel small RNA virus in the infected bean bug, but the relationship between fungal virulence and the RNA virus was under determination. The functional roles of these possible virulence factors are remained unclear, but this work provides a new insight for further fungal studies. Our results reflect systemic impacts of fungal pathogenesis and these findings represent a significant advance in the fungal functional genomics.