Jong Cheol Kim

Conidiogenesis-related DNA photolyase gene in Beauveria bassiana

Beauveria bassiana is an entomopathogenic fungi used in environmentally mindful pest management. Its main active ingredient, conidia, is commercially available as a fungal biopesticide. Many studies of conidia production have focused on how to optimize culture conditions for maximum productivity and stability against unfavorable abiotic factors. However, understanding of how conidiogenesis-related genes provide improved conidial production remains unclear. In this study, we focus on identifying conidiogenesis-related genes in B. bassiana ERL1170 using a random mutagenesis technique. Transformation of ERL1170 using restriction enzyme-mediated integration generated one morphologically different transformant, ERL1170-pABeG #163. The transformant was confirmed to represent B. bassiana, and the binary vector was successfully integrated into the genome of ERL1170. Compared to the wild type, transformant #163 showed very slow hyphal growth and within 6 days only produced <1 × 106 conidia/0.28 cm2 agar block (wild type: 6.2 × 107 conidia/agar block). Transformant #163 also exhibited different morphology than the wild type, including thicker hyphae with some club-shaped parts. In contrast, the typical morphology of wild type B. bassiana exhibits thread-like hyphae and conidiophore structures and circular conidia. To determine the location of the randomly inserted DNA, we conducted thermal asymmetric interlaced (TAIL) PCR and Escherichia coli cloning to clearly sequence the disrupted region. We identified one colony (colony No. 7) with an insertion site identified as DNA photolyase. This was confirmed through a gene knock-out study. It is possible the gene that encodes for DNA photolyase was disrupted during the insertion process and might be involved in fungal conidiogenesis. This work serves as a platform for exploring the function of a variety of B. bassiana genes involved in pest management and their downstream processing.

Eco-Friendly Organic Pesticides (EFOP)-Mediated Management of Persimmon Pests, Stathmopoda masinissa and Riptortus pedestris

Chemical pesticides have been used to control persimmon pests, however the overuse of the pesticides caused insect resistance, followed by failure in pest management and residual problems. Herein we investigate the potential of eco-friendly organic pesticides (EFOP) on the control persimmon pests, Stathmopoda masinissa (persimmon fruit moth) and Riptortus pedestris (bean bug). Ten commercially available plant-derived organic pesticides and one microbial pesticide were sprayed on the target insects in laboratory conditions. The chemical pesticide, buprofezin+dinotefuran wettable powder served as a positive control. In the first bioassay against persimmon fruit moth, alternatively Plutella xylostella larvae were used due to the lack of persimmon fruit moth population from fields, and three organic pesticides showed high control efficacy, such as pyroligneous liquor (EFOP-1), the mixture of Chinese scholar tree extract, goosefoot and subtripinnata extracts (EFOP-2) and Bacillus thuringiensis subsp. aizawai NT0423 (EFOP-11). When the three selected organic pesticides were treated on the persimmon fruit moths, the EFOP-2 treatment showed the highest control efficacy: 27.7% (5 days), 13.3% (7 days) and 6.7% (10 days) of survival rates. In the bioassay against bean bugs, the mixture of Chinese scholar tree, goosefoot and subtripinnata extracts (EFOP-2 and EFOP-9) and the extracts of sophora and derris (EFOP-10) showed high control efficacy, particularly the highest in the treatment of EFOP-2: 20.0% (5 days) and 16.7% (10 days) of survival rates. These results suggest that the mixture of Chinese scholar tree, goosefoot and subtripinnata extracts (EFOP-2) has high and multiple potential in the management of the persimmon pests.

Genomic Analysis of the Insect-Killing Fungus Beauveria bassiana JEF-007 as a Biopesticide

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.

Transcriptional response of bean bug (Riptortus pedestris) upon infection with entomopathogenic fungus, Beauveria bassiana JEF-007.

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.

Establishment of Optimal Rearing Conditions for the Production of Tenebrio molitor Larvae

Tenebrio molitor larvae contain large amounts of proteins, lipids and other functional materials, enabling this insect to be used as an edible food source in animal feeds and for industrialization. Although many efforts have been made to set up mass rearing systems, few studies have been conducted to establish optimal rearing conditions for the production of high quality T. molitor larvae. Herein we investigated 1) the effects of additional diets on the survival and fecundity of the insect, 2) the relationship between oviposition period and the uniformity of larval size, 3) the effects of rearing density and temperature on insect development, and 4) the storage stability of eggs and pupae at low temperatures given possible temporary production discontinuation. The addition of carrot and zucchini to the traditional wheat bran diet significantly increased the survival and fecundity rate of adult T. molitor. Of the three different oviposition sampling periods (3, 7, and 14 days) used to investigate the uniformity of the hatched larvae in each treatment, the period of 3 and 7 days provided higher uniformity than the 14 days oviposition period. Larval development was faster at 30°C than at 20, 25, and 35°C. Interestingly, oviposition rates were highest at 20°C but showed much slower larval development and lower uniformity at 3 0°C. Regarding the effect of larval rearing densities (1, 10, 20, 30, 40, and 50 larvae per 90 mm diam. dish), larval weight was significantly reduced at higher rearing densities, but larval longevity and length were not influenced by rearing density. The 30 larvae/dish is suggested to be a reasonable density to be applied to mass production systems. When kept at 4°C, T. molitor eggs showed a significant reduction in hatching rate; however, when stored under the same conditions, pupae emergence rates remained high until 10 weeks, suggesting that storage at low temperatures is more suitable for the pupal stage than the egg stage. Our findings suggest that an increase in T. molitor adult survival and fecundity rates and a uniformity of hatched larval development can be achieved with the following recommendations: a combination diet (including wheat bran), a 7-day oviposition period; a larvae-rearing temperature of 30°C, a rearing density of 30 larvae/dish, and the storage of pupal stages at low temperatures in the case of rearing discontinuation. This study serves as a strong foundation for the successful mass production of high quality T. molitor larvae.