Identification of Cyt2Ba from a New Strain of Bacillus thuringiensis and Its Toxicity in Bradysia difformis
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Identification of Cyt2Ba from a New Strain of Bacillus thuringiensis and Its Toxicity in Bradysia difformis Fan‑Fan Wang1 · Shao‑Xuan Qu1,2 · Jin‑Sheng Lin1 · Hui‑Ping Li1 · Li‑Juan Hou1 · Ning Jiang1 · Xin Luo1 · Lin Ma1 Received: 20 November 2019 / Accepted: 7 May 2020 © The Author(s) 2020
Abstract Bradysia difformis is one of the most damaging pests in mushroom production in China. In this study, eight Bacillus thuringiensis strains were analyzed for insecticidal activity in B. difformis. The strain JW-1 showed the highest insecticidal activity against B. difformis larvae, but did not inhibit the mycelial growth of Pleurotus ostreatus and P. geesteranus. The 16S rRNA gene (1397 bp) and cyt2 gene (792 bp) were obtained from strain JW-1. The phylogenetic tree based on 16S rRNA gene and Cyt2 toxin showed that strain JW-1 was a member of B. thuringiensis and Cyt2 toxin belonged to Cyt2Ba toxin cluster. The Cyt2Ba toxin from strain JW-1 was overexpressed in E. coli as a fusion protein and the fusion protein (70 kDa) was purified by Ni-IDA affinity chromatography. The purified Cyt2Ba fusion protein was toxic to B. difformis larvae (LC50 was 2.25 ng/ mL). The identification of Cyt2Ba from strain JW-1 and confirmation of the insecticidal activity of Cyt2Ba in B. difformis provided a new means of biological control of the important pest in mushroom production.
Introduction Bradysia difformis, belonging to the Sciaridae family, is one of the most destructive greenhouse pests, primarily affecting edible mushroom cultivation [1]. Adult female flies lay eggs on the surface of the growing mushrooms, and the larvae mostly eat hyphae and fruiting bodies, directly damaging the mushroom produce [2]. Adult flies also spread fungal spores and mites when flying over mushrooms, which indirectly leads to lower yields [3]. Bradysia spp. can be controlled by traditional chemical agents because of their simplicity, effectiveness, and speed [4]. However, resistance and residual insecticide pose major threats to the mushroom industry [5, 6]. The current trend is to shift to integrated and eco-friendly pest-management, involving microorganisms with insecticidal activity, such as Bacillus thuringiensis [7, 8]. Bacillus thuringiensis (Bt) is a gram-positive, endosporeforming, ubiquitous soil bacterium that produces parasporal crystals during the sporulation phase of its growth cycle * Lin Ma [email protected] 1
Vegetable Research Institute, Jiangsu Key Laboratory for Horticultural Crop Genetic Improvement, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China
Institute of Life Science, Jiangsu University, Zhenjiang 210023, China
2
[9, 10]. These parasporal crystals contain different types of proteins with insecticidal activity, the insecticidal crystal proteins (ICP) [11]. Different ICPs possess highly specific insecticidal activities against numerous target insects, such as Lepidoptera, Diptera, Coleoptera, Hymenoptera, Homoptera, as well as some invertebrates such as Nemathelminthes and Platyhelminthes [12–14]. The
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