Characterization of T-DNA insertion mutants with decreased virulence in the entomopathogenic fungus Beauveria bassiana JEF-007
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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.
KeywordsAgrobacterium tumefaciens-mediated transformation Beauveria bassiana Riptortus pedestris Thermal asymmetric interlaced PCR Virulence
We would like to thank Dr. Teak Soo Shin from the Institute of Dongbu Farm Hannong Agrochemical Co., Korea for providing us with a bean bug colony and Nam Jung Kim from the Rural Development Administration for providing us with a mealworm colony. In addition, we would like to thank Mr. Jong Cheol Kim for his assistance with this study. Lastly, we appreciate Bruce L. Parker, University of Vermont, USA for editing this manuscript.
Compliance with ethical standards
This article does not contain any studies performed on human participants or vertebrate animals.
This work was done with the support of the “Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ0116302015)” Rural Development Administration, Republic of Korea.
Conflict of interest
The authors declare that they have no competing interests.
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