Identification of new isolates of Bacillus thuringiensis using rep-PCR products and delta-endotoxin electron microscopy

Abstract

PCR has been used to analyze the distribution of REP (Repetitive Extragenic Palindromic) and ERIC (Enterobacterial Repetitive Intergenic Consensus) sequences (rep-PCR) found within the genome of the bacterium Bacillus thuringiensis, with the purpose to analyze the genetic similarities among 56 subspecies samples and 95 field isolates. The PCR products were analyzed by EB-AGE (ethidium bromide-agarose electrophoresis) and then submitted to banding comparisons, based on the Phyllip software algorithm. When the banding similarities were considered for comparison purposes among all the strains, the phylogenic tree patterns varied according to the rep-PCR primers considered, but, from a broader point of view, the ERIC sequences produced better results, which, together with electron microscopy analysis of the released parasporal bodies and colony morphology characteristics, allowed to detect two possible new subspecies of B. thuringiensis.

Evidence of Bacillus thuringiensis Intra-Serovar Diversity Revealed by Bacillus cereus Group-Specific Repetitive Extragenic Palindromic Sequence-Based PCR Genomic Fingerprinting

“…B. thuringiensis serovars kenyae , aizawai , galleriae , sooncheon , indiana , tolworthi and japonensis were also included in the same lineage. In disagreement with previous results [Cherif et al, 2007;Joung and Côte, 2001;Lima et al, 2002;Reyes-Ramirez and Ibarra, 2005], strains that belong to serovars galleriae and aizawai clustered together. Lineage 7 encompassed strains, which despite belonging to the same serovar, showed similar but not identical Rep-PCR patterns.…”

“…Lineage 1 encompassed serovar kurstaki HD-1 and HD-73 strains, which despite belonging to the same serovar, showed distinctive Rep-PCR patterns. Similar results were obtained by other authors based on different methodologies [Cherif et al, 2007;Lima et al, 2002]. B. thuringiensis serovars kenyae , aizawai , galleriae , sooncheon , indiana , tolworthi and japonensis were also included in the same lineage.…”

“…Several alternative typing methods for B. thuringiensis strains, mostly based on molecular techniques, have been recently described [Cherif et al, 2007;Gaviria Rivera and Priest, 2003a, b;Joung and Côte, 2001;La Duc et al, 2004;Lima et al, 2002;Manzano et al, 2009;Peruca et al, 2008;Ibarra, 2005, 2008;Xu and Côte, 2006;Yu et al, 2002]. Among them, a B. cereus group-specific repetitive extragenic palindromic (Rep) sequence-based PCR fingerprinting technique was shown to be highly discriminative, fast, easy, and able to identify B. thuringiensis serotypes, including non-flagellar and self-agglutinated strains [Reyes-Ramirez and Ibarra, 2005].…”

Fingerprinting of Bacillus thuringiensis Type Strains and Isolates by Using Bacillus cereus Group-Specific Repetitive Extragenic Palindromic Sequence-Based PCR Analysis

“…It has also been used for strains within the genus Bacillus (including B. thuringiensis), but based on the use of REPs found in other unrelated bacteria, such as the streptococcal BOX (10), the enterobacterial REP (35,46), and the enterobacterial ERIC (35). In fact, a quick search for all these sequences in the six B. cereus group genomes reported showed no significant matches, indicating that actual BOX, REP, or ERIC analyses of these strains may be uncertain and should be reviewed.…”

Characterization of a native Bacillus thuringiensis isolates from Malaysia that produces exosporium-enclosed parasporal inclusion

“…De Respinis et al (2006) mentioned that the colony morphology of B. thuringiensis and B. cereus is indistinguishable and both species show the typical morphology of circular or irregular colonies in white or grey; granular, milky or mat aspect and minimum 5 mm of diameter. In addition to smooth colonies with irregular shape, Lima et al (2002) also characterized a B. thuringiensis isolate that produced dark yellowish colonies. Apart from the characteristics stated, many also reported that B. thuringiensis strains formed "fried-egg" appearance on agar plate (Travers et al, 1987;El-kersh et al, 2012;Mahalakshmi et al, 2012).…”