Delineation of the composition of an indigenous soil population of Rhizobium meliloti in root nodules of uninoculated field grown Medicago sativa L. Public Deposited

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  • Agronomic and microbiological studies were conducted on "Anchor" alfalfa (Medicago sativa L.) established from uninoculated seed on three Aridisols in central Oregon. Significant responses to 230 Kg N per ha (NH₄NO₃), which was applied in mid-April and after each first and second harvests, were observed in yield parameters of the third harvest in five of seven post-establishment cuttings. Of the isolates of Rhizobium meliloti obtained from nodules of ten-week old plants at one of the sites 20% were ineffective, 16% were of intermediate effectiveness and 64% were highly effective. In the second post-establishment season no ineffective nodule isolates were recovered, but nodule numbers declined from 66 to 11 per plant between the first and third harvests. All isolates recovered from plants at the third harvest were of intermediate effectiveness. Microbiological studies were focused on the development of methods to discriminate between and identify R. meliloti isolates occupying nodules of alfalfa at one of these field sites. A collection of 300 field isolates of R. meliloti were subdivided into seven groups based on intrinsic antibiotic resistance characteristics. Two hundred and four and 55 isolates were placed into two groups, C and F, respectively. Group C isolates dominated the root nodule population in all but one of nine quadrants analyzed. Antiserum raised to a group C isolate, #31, cross agglutinated with 46 of 55 group C isolates. There were no cross reactions between isolates from any of the other six groups. Thirty three of 35 cross agglutinating field isolates from group C had the same sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) protein profile pattern as isolate #31 whereas non-agglutinating isolates had different protein profiles from the latter. Further analysis of 32 isolates obtained from ten-week old plants in one replicate plot by SDS-PAGE showed that 12 and 6 isolates were represented by two protein profiles, gel types A and B, respectively. The remaining 14 isolates were represented by unique gel types. Antiserum raised to #31, a gel type A isolate, cross agglutinated with the 12 gel type A isolates and only with two of the other 20 isolates, each of which was represented by a distinct gel type. Gel-immune-diffusion analysis showed that 9 of the 12 gel type A isolates were antigenically identical to #31. Analysis of 79 nodule isolates obtained from a systematic sampling of plants from the same replicate plot during the second postestablishment season showed that 53% of these isolates were identical to #31 and were distributed in nodules of plants growing throughout the plot. Isolates identical to #3] by serological and SDS-PAGE methodologies could still be subdivided further into two symbiotic effectiveness classes. The data suggest the need for complementary methods to identify unequivocally field isolates of R. meliloti, that some members of the diverse indigenous population are more dominant nodule occupants than others, and that turnover and change of nodule occupants may be a factor in limiting symbiotic N₂ fixation, and in the yield potential of alfalfa in the latter part of the growing season in this region of Oregon.
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