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ISOLATION, CHARACTERIZATION, AND CULTURE OF BACILLUS THURINGIENSIS FROM SOIL AND DUST FROM GRAIN STORAGE BINS AND THEIR TOXICITY FOR MAMESTRA CONFIGURATA (LEPIDOPTERA: NOCTUIDAE)1

Published online by Cambridge University Press:  31 May 2012

O.N. Morris ,
V. Converse ,
P. Kanagaratnam  and
J.-C. Coté
O.N. Morris*
Affiliation:
Cereal Research Centre, Agriculture and Agri-Food Canada, 195 Dafoe Road, Winnipeg, Manitoba, Canada R3T 2M9
V. Converse
Affiliation:
Cereal Research Centre, Agriculture and Agri-Food Canada, 195 Dafoe Road, Winnipeg, Manitoba, Canada R3T 2M9
P. Kanagaratnam
Affiliation:
Cereal Research Centre, Agriculture and Agri-Food Canada, 195 Dafoe Road, Winnipeg, Manitoba, Canada R3T 2M9
J.-C. Coté
Affiliation:
Horticultural Research and Development Centre, Agriculture and Agri-Food Canada, 430 Gouin Boulevard, St-Jean-sur-Richelieu, Quebec, Canada J3B 3E6
*
2 Author to whom all correspondence should be addressed.
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Abstract

One hundred and two Bacillus thuringiensis Berliner strains isolated from six different types of Canadian soil and dust from different grain storage bins were cultured in shake flasks containing Great Northern White Bean (GNWB) protein concentrate (48.6% protein) as the main nitrogen source and dextrose as the main carbohydrate source. The resulting endotoxins were bioassayed against the bertha armyworm, Mamestra configurata Wlk. Thirty-three percent of soil and 66% of grain dust samples were positive for B. thuringiensis. The bacterium was found most frequently in organic-rich soil. The four most toxic soil isolates (which were seven to 15 times more toxic than the international standard, HD1-S-1980), and two nontoxic grain dust isolates were characterized by serological typing, biochemical analysis, carbohydrate utilization, plasmid profile analysis, protein profile analysis using sodium dodecyl sulfate – polyacrylamide gels, and polymerase chain reaction. Four isolates were determined to be subsp. kurstaki containing 130–140 and 63–65 kDa proteins, and two isolates (tested for comparison) were subsp. canadensis containing 31 and 38 kDa proteins. Nonpyramidal-crystal-producing strains did not grow well in culture media containing GNWB, degossypellized cotton seed meal (61% protein), defatted soy flour (55% protein), or peptone as nitrogen sources. Excess of GNWB protein concentrate in shake flask culture media (30 g/L) inhibited bacterial growth and reduced the toxicity of isolate A1.2/72 subsp. kurstaki, which was the most toxic soil isolate. Isolate A1.2/72, which was 15 times more toxic for bertha armyworm larvae than the international standard (HD1-S-1980), contained three cry1A genes (cry1Aa, cry1Ab, and cry1Ac), whereas HD-1 lacked the cry1Ab gene. This strain was synergistic with strain HD-551 subsp. kenyae (cry1A, cry2A, and cry1B genes) but not with HD-133 subsp. aizawai (cry1Ab, cry1B, cry1C, and cry1D genes) when the strains were cultured together in a cotton seed meal medium and fed to M. configurata. The growth rate, economic yield, and toxicity of the new isolate, A1.2/72, produced in a 14-L laboratory fermenter declined when the fermentation ingredients were tripled. We believe that the indigenous strain A1.2/72 warrants further research development for bertha armyworm control.

Résumé

Cent deux souches de Bacillus thuringiensis Berliner isolées de six types différents de sols et de poussières provenant de différentes cellules à grain au Canada ont été cultivées dans des fioles sous agitation renfermant du concentré de protéines de haricot blanc Great Northern (GNWB) (48,6% de proténes) comme source principale d’azote et du dextrose comme source principale de glucide. Les endotoxines obtenues ont été utilisées dans un biodosage chez la légionnaire bertha (Mamestra configurata Wlk.). Trente-trois pour cent des échantillons de sol et 66% des échantillons de poussière de grain renfermaient B. thuringiensis. La bactérie se trouvait le plus souvent dans des sols riches en matières organiques. Les quatre isolats de sol les plus toxiques (qui étaient 7 à 15 fois plus toxiques que l’étalon international HD1-S-1980) et deux isolats de poussière de grain non toxiques ont été charactérisés par un typage sérologique, l’analyse biochimique, l’utilisation des glucides, l’analyse du profil de plasmides, l’analyse du profil des protéines sur des gels de polyacrylamide en présence des SDS et l’analyse par l’amplification par la polymérase. Quatre isolats ont été identifiés comme étant subsp. kurstaki renfermant des protéines de 130–140 kDa et de 63–65 kDa et deux isolats (à des fins de comparaison) ont été identifiés comme étant subsp. canadensis renfermant des protéines de 31 et 38 kDa. Les souches produisant un cristal non pyramidal ne poussaient pas bien dans les milieux de culture renfermant du GNWB, de la farine de graine de coton dégossypolisée (61% de protéines), de la farine de soja dégraissée de GNWB dans les milieux de culture sous agitation (30 g/L) inhibait la croissance bactérienne et réduisait la toxicité de l’isolat A1.2/72, subsp. kurstaki, qui était l’isolat de sol le plus toxique. Cet isolat, qui était 15 fois plus toxique pour les larves de légionnaire bertha que l’étalon international (HD1-S-1980) renfermait trois gènes cry1A (cry1Aa, cry1Ab et cry1Ac) alors que HD-1 était dépourvu du gène cry1Ab. Cette souche agissait en synergie avec la souche HD-551 subsp. kenyae (gènes cry1A, cry1A et cry1B), mais non avec la souche HD-133 subsp. aizawai (gènes cry1Ab, cry1B, cry1C et cry1D) lorsque les souches étaients cultivées ensemble dans un milieu à base de farine de graine de coton et utilisées pour nourrir M. configurata. Le taux de croissance, le rendement économique et la toxicité du nouvel isolât (A1.2/72) produit dans un fermenteur de laboratoire de 14 L diminuaient lorsque les concentrations des ingrédients étaient triplées. Nous estimons que la souche indigène A1.2/72 mérite qu’on y consacre davantage de travaux de recherche et de développement pour la lutte contre la légionnaire bertha.

Type
Articles
Information
The Canadian Entomologist , Volume 130 , Issue 4 , August 1998 , pp. 515 - 537
Copyright
Copyright © Entomological Society of Canada 1998

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Footnotes

1

Contribution No. 1659 of the Cereal Research Centre, Agriculture and Agri-Food Canada, Winnipeg.

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