ISSN 2079?0597, Russian Journal of Genetics: Applied Research, 2015, Vol. 5, No. 2, pp. 102–109. © Pleiades Publishing, Ltd., 2015.Original Russian Text © D.V. Krutylo, V.S. Zotov, 2013, published in Ekologicheskaya Genetika, 2013, Vol. 11, No. 4, pp. 86–95.102INTRODUCTIONNodule bacteria are known to play an integral partin the process of molecular nitrogen fixation from theatmosphere as they are able to initiate the formation ofnitrogen?fixing nodules on legume roots. Due to thisability rhizobia are considered an important geneticresource for the creation of microbial preparation.Considerable attention has been recently paidtowards the study of natural populations of nodulebacteria—microsymbionts of traditional and rarelegumes. One of the most topical problems of contem?porary soil microbiology is to describe genetic varietyof specific nodule bacteria (Spaink et al., 2002).Considering the importance of soybean we got ori?ented towards the study of populations of itsmicrosymbionts (nodule bacteria) in soils of differentregions of Ukraine.We know that soybean can make symbiosis withsome nodule bacteria—with Bradyrhizobium japoni?cum, B. elkanii, and B. liaoningense (Spaink et al.,2002), which have slow growth rates, and withSinorhizobium fredii, S. xinjiangensis (Chen et al.,1988), and Mesorhizobium thianshanense, which haveintensive growth rates (Chen et al., 1995).We admit that there are no aboriginal nodule bac?teria of soybean in soils of Ukraine, as wild soya andother legumes belonging to cross?infected plants (cowpea, mung bean) in natural phytocenosises are notfound (Tolkachov, 1997). The formation of soil popu?lations of specific soybean rhizobia has startedrecently, from the moment of the intensive introduc?tion of this culture into crop rotation. According to theliterature, bacteria B. japonicum with a slow growthrate are considered to be typical microsymbionts ofcultured soybean in soils of Ukraine and they wereintroduced into agrocenosis in commercial biologicalpreparations (Rhizotorfin of Russian and Ukrainianmanufacture) (Babich, 1993; Patyka et al., 2003; Tol?kachov, 1990). Thus, populations of soybean rhizobiaare a convenient object to study the processes ofmicroevolutions resulting in the divergence of soybeanmicrosymbionts connected with their adaptation todifferent plant genotypes, as well as the new soil andclimatic conditions.For some years (2001–2004) we studied the spreadof nodule bacteria of soybean in agrocenosises withdifferent growth rate intensities of this culture (soy?bean in a crop rotation for 10–30 years). The resultsshow that in the soils of the country a different numberlocal populations of nodule bacteria of soybean areformed and function. Despite the homogeneity of theintroduced strains?inoculants, soil populations arequite heterogeneous. As a result of the analysis of themorphological, cultural, physiological, and biochem?ical properties of 180 strains of soybean rhizobia iso?lated from soils of different regions of Ukraine, weGenotypic Analysis of Nodule Bacteria Nodulating Soybeanin Soils of UkraineD. V. Krutyloa and V. S. ZotovbaInstitute of Agricultural Microbiology and Agro?Industrial Manufacture, National Academy of Agrarian Sciences of Ukraine, ul. Shevchenka 97, Chernihiv, 14027 Ukrainee?mail: krutilod@mail.rubBach Institute of Biochemistry, Russian Academy of Sciences, pr. Leninskiy 33, Moscow, 119071 Russiae?mail: adni83@yandex.ruReceived April 5, 2013; in final form, November 1, 2013Abstract—We have conducted the analysis of sequences of the 16S rRNA gene and intergenic 16S?23S rRNAregion (ITS) of nodule bacteria of soybean which differ in growth speed and are isolated from soils of Ukrainewith different growth rate intensities. As a result we have shown the similarity of strains with an intense growthrate to soybean rhizobia of group USDA 123. The studied strains, as other representatives of this group, pos?sess increased saprophytic competence. The restriction analysis divided all sequences of the intergenic ITSregion of soybean rhizobia into two ITS types: the first ITS?type includes strains with an intensive growth rateand the second ITS ?type contains strains with a slow grow rate. These types also coincide with their physio?logical groups.Keywords: Bradyrhizobium japonicum, 16S rRNA, 16S?23S rRNA (ITS), RFLP?analysis, DNA sequencing,Glycine maxDOI: 10.1134/S2079059715020057
