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.

Agrobacterium tumefaciens-mediated transformation (AtMT) has been widely used for generation of fungal transformants and recently applied to Beauveria bassiana. In this study to comprehend how the AtMT promoter influences on the expression of selection marker (hygromycin B resistance gene; hph), two different Ti-Plasmids were constructed: pCeg (gpdA promoter-based) and pCambia-egfp (CaMV 35S promoter-based). Putative transformants were subjected to the PCR, RT-PCR and qRT-PCR to inspect the T-DNA insertion rate and gene expression level. In conclusion, more than 80% of the colonies succeeded in AtMT transformation and the hph expression level of AtMT/pCeg colonies was higher than that of AtMT/pCambia-egfp colonies. This result can provide useful information on the AtMT of B. bassiana, especially antibiotics susceptibility and promoter-dependant expression level.

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.

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.

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.

The entomopathogenic fungus, Beauveria bassiana is widely used in integrated pest management (IPM), however its successful application is often limited by the little effort to explore its functions of unknown genes. In this work, egfp-expression cassette was randomly integrated into B. bassiana using Agrobacterium tumefaciensmediated transformation, and the general features of the mutants with unusual characteristics and the localization of the integrated genes were explored. To construct a transformation vector, egfp-expression cassette including gpdA promoter and trpC terminator was cut from pBARKS1-egfp using SacI and HindIII and integrated into pCAMBIA containing hygromycin B resistant hygR gene, designated as pCAMBIA-egfp. Transformed B. bassiana isolates were grown on quarter strength-Sabouraud dextrose agar containing 150 μg hygromycinB ml-1. Expression of egfp was investigated by RT-PCR and a fluorescent microscope (400×). Through the genome walking of the transformants using adaptor primers and gene specific primers, unique bands were detected on the egfp-expressing transformants, which were sequenced to figure out the flanking regions. This work provides a platform of methodology to figure out unknown functional genes of B .bassiana and possibly suggest an improved strategy to use the entomopathogen in IPM.

Enhanced green fluoresce protein gene (egfp) was expressed in Beauveria bassiana ERL836 based on the Agrobacterium tumefaciens-mediated transformation (AtMT) method in this study. The ERL836 transformants were generated with pCambia-egfp binary vector. Ten transformants were randomly selected and analyzed for the T-DNA insertion and gene expression. The results revealed that 60% of the fungal putative transformants were inserted by the T-DNA fragment. Of these transformants, 33.33% (2 transformants) expressed the egfp gene. The egfp transformants showed strong green fluorescence with different expression levels. The results of this study could provide a reference for foreign protein expression in B. bassiana by using the AtMT method.

곤충 병원성 곰팡이 Metharizium anisopilae JEF 003, 004와 Beauveria bassiana JEF 006, 007의 대량생산배지 조건에 따른 열안정성을 평가하였다. 첫 번째로 millet 배지에서 배양된 포자의 열안정성 평가를 위하여 50℃ 조건에 0, 30, 60, 90, 120 min으로 포자 현탁액 상태와 grain상태로 노출한 결과 현탁액 상태에서 포자 의 열안정성이 더 많이 감소하는 것을 확인하였다. 다음으로 각기 다른 고체배지 (조: 1~5.×109 conidia/g, 수수: 1~2×109 conidia/g, 기장: 2~3×109 conidia/g) 조건에 서 생산된 포자의 열안정성 확인을 위하여 배양이 완료된 포자를 현탁액과 grain상 태에서 50℃ 조건에 0, 1, 2, 4, 8 hours동안 노출하여 열안정성을 평가하였다. 실험 결과 현탁액 상태보다 grain상태에서 포자의 열안정성이 더 높은 것을 확인하였으 며, 조 배지 조건에서 포자의 열안정성이 가장 높게 향상된 것을 확인하였다. 마지 막으로 포자의 열안정성의 추가 향상을 위하여 배양이 완료된 고체배지 포자에 cotton seed oil, coconut oil, soybean oil, castor oil, olive oil, mineral oil을 넣고, 5 0℃의 온도에 0, 1, 2, 4, 8 hours으로 열안정성을 평가하였다. 결과적으로 cotton seed oil, soybean oil, castor oil, olive oil을 처리한 포자에서 높은 열안정성을 확인 하였다. 따라서 곤충병원성 곰팡이의 열안정성 실험 결과로 확인 된 조를 이용하여 높은 열안전성을 확보할 수 있을 것으로 판단이 되며, 추가적인 열안정성 확보를 위 하여 식물성 오일을 제제에 이용할 수 있을 것으로 전망된다.

Mealworm, Tenebrio molitor L. (Coleoptera: Tenebrionidae) has high and safe protein contents, which enables it to be animal feed. However, occurrence of entomopathogenic fungi in mealworms is one of the limitations for mass production. In this work, we investigated relationships between abiotic conditions and occurrence of fungal pathogens and established an effective control method using fungicides. In virulence assay, third instar mealworm larvae were sprayed by six entomopathogenic Beauveria bassiana isolates and kept under high relative humidity; B. bassiana ERL1575 isolate had highest virulence. Under normal humidity, ERL1575 conidia showed different virulence between spray (~0% virulence) and digestion (~80% virulence) method. Furthermore, mealworms, which digested conidia, were exposed to various temperature (20-35°C) and humidity (1-3 ml distilled water spray/35 mm diam. dish) conditions for 5 days. All the treatments showed ~90% virulence except 35°C incubations (~20% virulence), but irrespective to the humidity conditions. Forty chemical fungicides were assayed against conidial germination and hyphal growth of ERL1575. Fluazinam and mancozeb showed strong inhibition of conidial germination at standard application dose (SD), 1/2 SD and 1/5 SD; besides, fluazinam showed strong inhibition of hyphal growth. When fluazinam and mancozeb were applied to the fungal conidia-inoculated wheat bran, most of mealworms were alive after 3 days post application. However, high mortality rate (~100%) were observed in the conidia-inoculated wheat bran without any fungicides. In conclusion, this work suggests that B. bassiana isolates could be pathogens at <30°C when they were digested by mealworms, and fluazinam and mancozeb would be used as effective control agents against the pathogen.

Baculoviral anti-apoptotic genes, p35 and iap (inhibitor of apoptosis), play important roles in the initiation stage of viral infection. However, some iap genes are not involved in the anti-apoptotic activity. To investigate the anti-apoptotic activity of the iap genes of Lymantria xylina multiple nucleopolyhedrovirus (LyxyMNPV), two ly-iap genes (ly-iap2 and ly-iap3) were cloned from LyxyMNPV. From a 5′ RACE analysis, a late promoter motif (TAAG) was found in the upstream (-15 bp) of ly-iap2, but ly-iap3 only posited an enhancer-like element (CGTGC) in the upstream (-22 bp) of 5′ UTR. Gene expression were detected by RT-PCR; the ly-iap2 and ly-iap3 genes began to express in the host cells (IPLB-LD652Y cell line) infected with LyxyMNPV 6 hours post-infection (p.i.) and reached the peak 72 hours p.i., followed by decline 3 to 5 days p.i. Functional assay of the iap genes were performed by an over-expression method in Sf9 cells. Full-length domains of LY-IAP2, LY-IAP3 and LY-IAP2-BIR could differently inhibit the apoptosis which induced by Drosophila RPR protein (DRPR). Interestingly, LY-IAP2-RZF domain was important for LY-IAP2 to rescue apoptosis, but it might be also involved in the ubiquitin activity leading to the degradation of LY-IAP2 protein. LY-IAP3-RZF might be working as a “helper domain” to inhibit DRPR-induced apoptosis. These results can be used to figure out the roles of the ly-iap genes in the apoptosis of host cells.

Bean bug, Riptortus clavatus Thunberg (Heteroptera: Alydidae) causes serious damage to Leguminosae. Herein an entomopathogenic fungal virulence assay system against bean bugs was established to construct a fungal database which can be used in integrated pest management (IPM). First to obtain as many bean bugs as possible at the same stage, host plant-preference and developmental synchronization of bean bugs were investigated. In the preference assay, five pairs of adults were infested in a plastic cage, where a pot of green bean, pea or cowpea was previously placed. The highest fecundity and the fastest development of bean bug was observed in the green bean cage. Secondly, in the synchronization experiment, eggs were collected from the cage of adults in 1, 3, 5 and 7 days after oviposition and transferred to a fresh cage with green beans. From the every 4 days of survey, similar stages of bean bugs were found in the cages with the oviposition for 1 and 3 days, rather than the longer times of oviposition. A fungal bioassay against bean bugs was conducted using the bean bugs from the above insect rearing system. Ten Beauveria bassiana isolates were cultured on quarter-strength Sabouraud dextrose agar (¼SDA) for 7 days at 25°C. Ten 4th instar of nymphs were placed on a cultured plate for 1 hour and tranferred to a fresh moisturized plate with grains of green bean. ERL836 isolate treatment showed the highest virulence and fungal mycosis was observed on the bean bugs. In conclusion, these results can be useful to establish an entomopathogenic fungal database for IPM.