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Screening of high toxic Metarhizium strain against Plutella xylostella and its marking with green fluorescent protein

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Abstract

Entomopathogenic fungus is proposed to be one of the best biocontrol agents against the destructive insect pest Plutella xylostella. In this study, we tested the virulence of 11 Metarhizium strain isolates against P. xylostella using a leaf dipping method, and found one strain, named 609, which had displayed the highest pathogenicity. Bioassay results showed that the accumulated corrected mortality rate was 86.7 % on the eighth day after inoculation with a spore concentration 1 × 108 conidia/mL, and that the time to 50 % lethality was 5.7-day. The strain was identified as Metarhizium anisopliae var. acridum by internal transcribed spacer (ITS) region sequencing. A green fluorescent protein (GFP) marker containing vector, camben-gfp, was constructed and delivered into strain 609 by Agrobacterium tumefaciens-mediated transformation. Six positive clones expressing GFP were selected and tested for toxicity against P. xylostella, all of which displayed the same toxicity as the parental wild type strain. The survival rate of transformant T1 was investigated by monitoring GFP levels at 4-day intervals after inoculation into soil. We found that the concentration of Metarhizium spores decreased sharply from 1 × 107 conidia/g to 1 × 106 conidia/g in the first 5 days after inoculation. The decreasing trend then stabilized and the spore count declined to approximately 1 × 104–105 conidia/g after 1 month. The results of this study indicate that the expression of gfp gene in strain 609 does not alter the virulence capability of Metarhizium. This strain will therefore be useful for the control of P. xylostella and as a tool to study molecular biology properties and monitor colonization of M. anisopliae in the field.

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Acknowledgments

We thank Jianxiu Yao from Texas A&M University for her assistance in reviewing the manuscript and Zhihong Lang from Biotechnology Research Institute, CAAS for supplying reagents. This work was supported by the earmarked fund for China Agriculture Research System (CARS-35-07) and the Special Fund for Agro-scientific Research in the Public Interest (201003079).

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Authors and Affiliations

  1. State Key Laboratory for Biology of Plant Diseases and Insect Pests/Institute of Plant Protection, Chinese Academy of Agricultural Sciences (CAAS), No. 12, Zhongguancun South Street, Beijing, 100081, China

    Qianqian Cui, Yi Zhang, Yanchao Zang, Xiangqun Nong, Guangjun Wang & Zehua Zhang

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  1. Qianqian Cui
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  2. Yi Zhang
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  3. Yanchao Zang
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  4. Xiangqun Nong
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  5. Guangjun Wang
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  6. Zehua Zhang
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Correspondence to Guangjun Wang.

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Cui, Q., Zhang, Y., Zang, Y. et al. Screening of high toxic Metarhizium strain against Plutella xylostella and its marking with green fluorescent protein. World J Microbiol Biotechnol 30, 2767–2773 (2014). https://doi.org/10.1007/s11274-014-1700-6

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  • DOI: https://doi.org/10.1007/s11274-014-1700-6

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