A novel Bacillus thuringiensis isolate toxic to cotton pink bollworm (Pectinophora gossypiella Saunders)

Highlights

• Bacillus thuringiensis isolate T26 showed spores and crystals under microscope and pathogenicity against the pink bollworm.

• SDS-PAGE analysis of the spore-crystal mixture revealed presence of major protein bands with ~ 80, 55 and 40 kDa sizes.

• None of the known lepidopteran active genes could be detected with PCR or with whole genome sequence analysis.

Abstract

In this study, we report a novel indigenous Bacillus thuringiensis (Bt) isolate, T26, which showed spores and crystals under scanning electron microscope and pathogenicity against the pink bollworm (Pectinophora gossypiella Saunders) in artificial diet based bioassay. SDS-PAGE analysis of the spore-crystal mixture of the Bt isolate, T26 revealed presence of three major protein bands of approximate molecular weights of 80, 55 and 40 kDa. The draft genome assembly consists of 56 scaffolds with an entire draft genome size of 5,054,095 bp. NCBI blast analysis revealed that assembled draft genome is spread over in a chromosome (4,818,543 bp) and one plasmid (235,552 bp). NCBI Prokaryotic Genome Annotation Pipeline (PGAP) showed presence of 5033 coding gene sequences and 159 RNAs genes. None of the known lepidopteran active genes (cry1, cry2 and cry9) could be detected with PCR or with whole genome sequence analysis using Bt toxin scanner tool or CryProcessor tool. Thus, presence of protein crystals and toxicity towards cotton pink bollworm and absence of any known cry/vip/cyt type of genes in draft genome indicates it is a novel type of Bt isolate. Further investigation of this genome sequence along with protein sequencing will lead to understand the novel factors responsible for its virulence and could be a useful tool for the insect resistance management in pink bollworm.

Introduction

Bacillus thuringiensis is a Gram positive spore forming bacterium which belongs to Bacillus cereus sensu lato group. This bacterium has been exploited as a biocontrol agent either in the form of formulations or in the development of transgenic plants, resistant to various kinds of insects [[1], [2], [3]] . In the year 2017, biotech crops with the insect resistance characteristics covered an area of 23.3 million hectares [4]. However, some insects started developing resistance against Bt proteins expressed in transgenic crops, in some countries. Insect pest species which have developed field-evolved resistance to a single or more than one Bt proteins in the transgenic crops and reduced crop efficacy have reached 7 in 2019 [5]. Some examples are Busseola fusca (Corn) (Cry1Ab) [6]; Spodoptera frugiperda (Corn) (Cry1F) [7]; Diabrotica virgifera virgifera (Corn) (Cry3Bb) [8]; Helicoverpa zea (Corn) (Cry1Ab) [9]; Pectinophora gossypiella (cotton) (Cry1Ac) [10]. The incidence of pink bollworm, P. gossypiella, has been also observed in Bollgard II versions of Bt cotton in India [11]. Therefore, discovery of novel Bt proteins from new isolates could provide alternatives to counteract the insect resistance issues. Through next generation sequencing (NGS) technologies, genome traits of the organisms can be understood and thus their biocontrol mechanisms can be exploited for further applications. In this way a novel B. thuringiensis isolate with Vip1/Vip2 binary and Cry8 toxins that had high toxicity to Scarabaeoidea larvae was identified [12]. In this study, an indigenous Bt isolate, T26, available at Centre for Plant Molecular Biology and Biotechnology (CPMB&B), Tamil Nadu Agricultural University (TNAU), Coimbatore, India, was explored for its morphological, molecular and toxicity characters, and finally genome-sequencing and bioinformatic analyses were carried out to elucidate virulence factors in it.