Physiological and molecular detection of crystalliferous Bacillus thuringiensis strains from habitats in the South Central United States

Abstract

Gram-positive, endospore-forming Bacillus thuringiensis-like strains were isolated from 95 of 413 samples collected at the 0-5 cm depth of noncultivated soils and stagnant or dried-up ponds as well as from dust from stored grain products in South Central United States. Based on the production of parasporal crystals, 25 isolates were identified as B. thuringiensis after examining 227 B. thuringiensis-like colonies. The greatest proportion of samples yielding B. thuringiensis were from the dust from grain storage. The sodium acetate selective medium, heat processing, and crystal staining used in the initial screening revealed diverse populations of B. thuringiensis, which were categorized into distinct crystal morphological groups. Sugar fermentation, antibiotic sensitivity, growth characteristics and PCR studies showed diversity among the isolates that were distributed among 25 of the 58 known strains. The most frequently isolated strains were kurstaki, aizawai, morrisoni, thuringiensis, sotto and kenyae that together represented more than 90% of the characterized isolates. PCR analysis using 30 family primer pairs for cry and cyt genes showed that the frequency of the cry1 gene (62%) was predominant followed by the cry2 genes (30%), and the rest (8%) were other cry gene types, such as cry3, cry4, cry10, cry11, cry14, cry15, cry20, cry24 and cry26. Both cyt1 and -2 genes were also detected. Several isolates showed PCR products on the gel that were not consistent with the expected sizes of nucleotides targeted by the primers. These were suggestive of nonspecific amplifications and were not used in the characterization process.

Insecticidal Activity ofBacillus thuringiensisStrains Isolated from Soil and Water

“…Bacillus thuringiensis bacteria are ubiquitous in soil [2, 13, 33, 34], dead larvae [4], sand [5], leaves [3], water [7], or dust from stored grains [6]. Wild strains isolated form environmental samples can synthesize crystals that display higher activity against insect pests in comparison to B. thuringiensis strains already used in pesticide production.…”

“…These genes were also noted as the most frequent in B. thuringiensis strains [2, 3, 5, 6, 33, 34]. All analyzed B. thuringiensis harbored cry1I genes that have been reported as the most abundant in B. thuringiensis isolates [11].…”

“…Primer sequences and steps of PCR for cry9 and the gene subgroups detection were accomplished as proposed by Ben-Dov et al [26]. Identification of cry15, cry16, cry18, cry20, cry22, cry25, cry26, cry28, and cyt2 genes was described by Ejiofor and Johnson [6]. The amplification for cry10, cry17 , cry24, cry27, cry29, cry30, cry32 and cry40 genes was conducted as depicted by Ibarra et al [13].…”

Characterization of Cry Proteins in Native Strains ofBacillus thuringiensisand Activity AgainstAnastrepha ludens1

“…Of the 202 colonies analyzed by PCR, 10 were positive for any of the five genes analyzed by multiplex PCR (cry2, cry4, cry10, cry11, cry19), endpoint PCR for each of the 10 positive samples with primers reported in the literature was used (Carozzi et al 1991, Ejiofor andJohnson 2002), only seven colonies that showed a single band corresponding to the expected size were selected for further analysis, and these were characterized by light microscopy based on the shape of the crystal ( Table 1). The protein crystals were characterized by SDS-PAGE.…”

“…Five pairs of primers (2M, 4M, 10M, 11M, and 19M) were designed for multiplex PCR with the software Mpprimer (Shen et al 2010); primer pair 2M: 5'-ACCCCAGTTCCAGATGCAAGGA/ 5'-TGCTGTGGTCCACTACCACTTGC, product size 369 bp for the gene cry2; 4M: 5'-TGGATGCACGAGTGGCACAAGC/ 5'-GCATTTCCAGTTACATGCCACCCCA, product size 106 bp for the gene cry4; 10M: 5'-CGCAACATAATCTGGGGAGCGGT/ 5'-TCCACCTGTGTGACCAGGACCTT, product size 468 bp for the gene cry10; 11M: 5'-TTTAACTGCGCCAGCACCAGCA/ 5'-ACCCGTATTCCAGCAGGTAAGCGA, product size 588 bp for the gene cry11; 19M: 5'-CGAGGAAGCTTCTTATGCATCTTCAGG/ 5'-CACGCAGCGAGAGCTCGGTTAT, product size 291 bp for the gene cry19, with the corresponding Tm of 59.2°C for all the previous primer pairs, these primers were used for partial classification of genes by multiplex PCR. Subsequent PCR primers reported in the literature (Carozzi et al 1991, Ejiofor andJohnson 2002) were used to confirm the presence of the cry genes by uniplex PCR: primer pair 4U: 5'-CAAGCCGCAAATCTTGTGGA/ 5'-TGGCTTGTTTCGCTACATC, product size 797 bp, for the gene cry4 (Carozzi et al 1991); 10U: 5'-TCGTGGAATGGGCAAAAAC/ 5'-TATCCCCCTTCAACATCCTCA, product size 404 bp for the gene cry10; 11U: 5'-TTTGCACCAGATAATACTAAGGAC/ 5'-AACAACTGCGATAAATACCACTCT, product size 485 bp for the gene cry11; 19U: 5'-AGGGGAGTCCAGGTTATGAGTTAC/ 5'-ATTTCCCTAGTTAGTTCGGTTTTT, product size 355 bp for the gene cry19 (Ejiofor and Johnson 2002), with the corresponding Tm of 55°C for all the previous primer pairs.…”