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Applied and Environmental Microbiology, January 2003, p. 607-615, Vol. 69, No. 1
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.1.607-615.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Bacteria Associated with Cysts of the Soybean Cyst Nematode (Heterodera glycines)

Sarah M. Nour,¹ John R. Lawrence,² Hong Zhu,¹ George D. W. Swerhone,² Martha Welsh,¹ Tom W. Welacky,³ and Edward Topp^1*

Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, London,¹ Greenhouse and Processing Crops Research Centre, Agriculture and Agri-Food Canada, Harrow, Ontario,³ National Water Research Institute, Saskatoon, Saskatchewan, Canada²

Received 7 June 2002/ Accepted 30 September 2002

The soybean cyst nematode (SCN), Heterodera glycines, causes economically significant damage to soybeans (Glycine max) in many parts of the world. The cysts of this nematode can remain quiescent in soils for many years as a reservoir of infection for future crops. To investigate bacterial communities associated with SCN cysts, cysts were obtained from eight SCN-infested farms in southern Ontario, Canada, and analyzed by culture-dependent and -independent means. Confocal laser scanning microscopy observations of cyst contents revealed a microbial flora located on the cyst exterior, within a polymer plug region and within the cyst. Microscopic counts using 5-(4,6-dichlorotriazine-2-yl)aminofluorescein staining and in situ hybridization (EUB 338) indicated that the cysts contained (2.6 ± 0.5) x 10⁵ bacteria (mean ± standard deviation) with various cellular morphologies. Filamentous fungi were also observed. Live-dead staining indicated that the majority of cyst bacteria were viable. The probe Nile red also bound to the interior polymer, indicating that it is lipid rich in nature. Bacterial community profiles determined by denaturing gradient gel electrophoresis analysis were simple in composition. Bands shared by all eight samples included the actinobacterium genera Actinomadura and Streptomyces. A collection of 290 bacteria were obtained by plating macerated surface-sterilized cysts onto nutrient broth yeast extract agar or on actinomycete medium. These were clustered into groups of siblings by repetitive extragenic palindromic PCR fingerprinting, and representative isolates were tentatively identified on the basis of 16S rRNA gene sequence. Thirty phylotypes were detected, with the collection dominated by Lysobacter and Variovorax spp. This study has revealed the cysts of this important plant pathogen to be rich in a variety of bacteria, some of which could presumably play a role in the ecology of SCN or have potential as biocontrol agents.

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* Corresponding author. Mailing address: Agriculture and Agri-Food Canada, Southern Crop Protection and Food Research Centre, 1391 Sandford St., London, Ontario, Canada N5V 4T3. Phone: (519) 457-1470, ext. 235. Fax: (519) 457-3997. E-mail: toppe{at}em.agr.ca.

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Applied and Environmental Microbiology, January 2003, p. 607-615, Vol. 69, No. 1
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.1.607-615.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.