Characterization ofcryGenes in a MexicanBacillus thuringiensisStrain Collection

Abstract

Mexico is located in a transition zone between the Nearctic and Neotropical biogeographical regions and contains a rich and unique biodiversity. A total of 496 Bacillus thuringiensis strains were isolated from 503 soil samples collected from the five macroregions of the country. The characterization of the strain collection provided useful information on the ecological patterns of distribution of B. thuringiensis and opportunities for the selection of strains to develop novel bioinsecticidal products. The analysis of the strains was based on multiplex PCR with novel general and specific primers that could detect the cry1,cry3, cry5, cry7, cry8,cry9, cry11, cry12,cry13, cry14, cry21, andcyt genes. The proteins belonging to the Cry1 and Cry9 groups are toxic for lepidopteran insects. The Cry3, Cry7, and Cry8 proteins are active against coleopteran insects. The Cry5, Cry12, Cry13, and Cry14 proteins are nematocidal. The Cry11, Cry21, and Cyt proteins are toxic for dipteran insects. Six pairs of general primers are used in this method. Strains for which unique PCR product profiles were obtained with the general primers were further characterized by additional PCRs with specific primers. Strains containingcry1 genes were the most abundant in our collection (49.5%). Thirty-three different cry1-type profiles were identified. B. thuringiensis strains harboringcry3 genes represented 21.5% of the strains, and 7.9% of the strains contained cry11 and cyt genes.cry7, cry8, and cry9 genes were found in 0.6, 2.4, and 2.6% of the strains, respectively. No strains carrying cry5, cry12, cry13,cry14, or cry21 genes were found. Finally, 14% of the strains did not give any PCR product and did not react with any polyclonal antisera. Our results indicate the presence of strains that may harbor potentially novel Cry proteins as well as strains with combinations of less frequently observed cry genes.

PCR-based identification of Bacillus thuringiensis pesticidal crystal genes

“…This observation is consistent with the physical location of cry1I genes, which have been reported to be in close vicinity to other genes of the cry1 subfamily [13,27,52,53]. Another frequent combination found almost world-wide is the linkage cry1C^cry1D [15,17,18,51,54]. In fact, only Kim et al [30] reported the common presence of cry1C alone.…”

“…The DNA used as template is obtained either by the typical lysis extraction and puri¢cation methods [14,18,29], or by simple time-saving lysis of a cell suspension using physical methods. In the latter case, DNA is usually released by boiling the suspension for 2^10 min [13,15,30], or by alternate freezing (370 ‡C) and boiling steps [17,24,26]. Template is added to the reaction mixture (¢nal volume 50^100 Wl) and the three-step ampli¢cation cycles are performed 25^35 times.…”

“…The largest collection of B. thuringiensis subjected to PCR screening of cry genes published to date contained isolates from around 500 soil samples [17]. These samples were obtained from ¢ve very di¡erent macroecological regions of Mexico.…”

Diversity of Colombian strains of Bacillus thuringiensis with insecticidal activity against dipteran and lepidopteran insects

“…The 445 isolates of B. thuringiensis were grown for 12 h at 30°C in Petri dishes with GEN nutrient medium (5 g yeast extract, 8AE8 g glucose, 10 g NaCl, 20 g agar in 1000 ml distilled water). A loop of each isolate was transferred to 100 ll sterile distilled water, the mixture was homogenized and stored at )20°C for 3 h, then immediately placed in a water bath at 100°C for 10 min to lyse the cells and finally centrifuged for 10 s at 9280 g. This supernatant was used for PCR as DNA template (Carozzi et al 1991;Ceró n et al 1994Ceró n et al , 1995Bravo et al 1998). General primers were used to characterize cry gene content (cry1, cry2, cry4 and cry11).…”

“…These strains have been isolated from diverse microhabitats such as soil, plant leaves, dead insects and stored grain, as well as aquatic environments such as marine sediments, mangroves and fresh water. Characterization of these collections has revealed the great variability and diversity of B. thuringiensis in nature (Ben-Dov et al 1997;Bravo et al 1998;Ben-Dov et al 1999;Ichimatsu et al 2000;Iriarte et al 2000;Kim 2000;Maeda et al 2001;Mizuki et al 2001;Arango et al 2002;Maduell et al 2002;Uribe et al 2003).…”