Selection of Entomopathogenic Fungus Isaria javanica FT333 for Dual Control of Thrips and Anthracnose

총채벌레 및 고추탄저병의 동시 방제를 위한 곤충병원성 곰팡이 Isaria javanica FT333 선발

Abstract

Red pepper is seriously damaged by thrips (Thrips palmi) and anthracnose caused by Colletotrichum acutatum throughout its development. Because of biotic constraints, producers often depend on chemicals that are expensive and have adverse effects on the environment, operator, and beneficial insects. In addition, resistance is developed because of the repeated use of chemicals. In recent decades, the use of microorganisms in crop protection has become a credible alternative because it is eco-friendly. In this study, we aimed to select isolates with insecticidal and fungicidal activities against the pathogens that cause anthracnose and thrips. We treated T. palmi adults and juveniles with 13 strains of entomopathogenic fungi (isolated from the soil by using the insect-bait method), and 6 strains showed excellent insecticidal activity (70-100%) 5 days after the treatment. The selected isolates were cultured with C. acutatum to screen for the strain with excellent anti-fungal activities, among which an isolate FT333 showed more than 95% control efficacy against C. acutatum in vitro. The isolate was identified as Isaria javanica through its morphological characteristics and phylogenetic analysis of the ITS and ${\beta}-tubulin$ nucleotide sequences. The Isaria javanica FT333 isolate could be used effectively for dual bio-control of thrips and anthracnose during red pepper cultivation.

고추의 주요 병해충인 총채벌레(Thrips palmi)와 탄저병(Colletotrichum acutatum)을 방제하기 위해 다양한 화학농약이 사용되고 있지만, 농약의 오남용으로 인하여 이에 대한 저항성이 발생하여 방제가 힘들어지고 환경오염을 초래하는 문제가 있다. 그리하여 본 연구에서는 이러한 문제를 극복하고자 총채벌레에 대한 살충효과와 탄저병에 항균력이 우수한 균주를 선발하여 동시 방제의 가능성을 확인하고자 하였다. 토양으로부터 분리한 곤충병원성 곰팡이 13균주($1{\times}10^7conidia/mL$)를 오이총채벌레 성충과 약충에 처리하여 70~100%의 우수한 살충률을 나타내는 6균주를 선발하였다. 선발한 6균주의 항균활성을 검정하기 위하여 고추 탄저병균에 대치 배양한 결과, 항균력이 우수한 FT333 균주를 최종 선발할 수 있었다. 최종 선발된 균주를 형태학적 조사와 internal transcribed spacer, ${\beta}-tubulin$ 영역의 염기서열 분석을 통하여 Isaria javanica로 동정하였고, I. javanica FT333으로 명명하였다. I. javanica FT333 (KACC93316P)의 고추 탄저병균에 대한 방제 효과를 기내에서 검정한 결과($1{\times}10^5$, $1{\times}10^6$, $1{\times}10^7conidia/mL$) 95% 이상의 방제효과를 나타내었다. 이러한 결과들을 바탕으로 I. javanica FT333 균주가 고추 탄저병균과 미소해충인 오이총채벌레를 동시에 방제하기 위한 미생물제제로서 이용될 수 있는 가능성을 제시하였다.

Fig. 1. Control efficacy of first selected isolates against juvenile of thrips. Control was treated with 0.02% Tween 80 (mean ± SE). Spores were observed in the mu㎜ies after bioassay. Data were analyzed using ANOVA (p < 0.001), and the differences were further elucidated using DUNCAN’s multiple range test. Different letters indicate significant differences at p < 0.001 at each time point

Fig. 2. Dual culture assay for in vitro inhibition of mycelial growth of Colletotrichum acutatum by isolates. Isolates were cultured on PDA plates at 25°C for 2 weeks. A, Control; B, FT284; C, FT285; D, FT299; E, FT304; F, FT322; G, FT333; PDA, potato dextrose agar.

Fig. 3. Phylogenetic tree based on the internal transcribed spacer rDNA and partial β-tubulin gene sequences of isolate FT333 and the type strains of Isaria species. The tree was constructed using the neighbor-joining method and Kimuras two-parameter model. The percentage of replicate groupings in which the associated taxa clustered together in the bootstrap test (500 replicates) is shown above the branches. A sequence from Metarhizitum brunneum is used as an outgroup. ARSEF, Agricultural Research Service Collection of Entomopathogenic Fungal Cultures (Ithaca, NY, USA); CBS, Central Bureauvoor Schi㎜elcultures (Utrecht, The Netherlands). Scale bar = 0.05 substitutions per nucleotide position

Fig. 4. Morphology of hyphae, conidiophores, and conidia of Isaria javanica FT333 (scale bar = 100 μm)

Fig. 5. Control effect by Isaria javanica FT333against Colletotrichum acutatum on red pepper at wetting treatment chamber. Anthracnose disease was observed by C. acutatum at 25°C for 2 weeks after inoculation

Table 1. Primers used in this study

Table 2. Cumulative mortality of Thrips palmi treated with entomopathogenic fungi collected from soil insect-bait method

Table 3. The inhibitory effects of 6 isolates against mycelial growth of Colletotrichum acutatum on PDA medium

Table 4. Control effcacy of Isaria javanica FT333 against anthracnose