| White clover(Trifolium repens L.) is a perennial good forage of Fabaceae family, Trifolium genus. It has a lot of advantaged features that can be of great value, such as high quality, good reproducibility and grazing-mowing tolerance, so that it enjoys a fame as the "master" legume forages in South of China. White clover is also of beautiful gesture, full ornamental, and strong ability of nitrogen fixation and good adaptability to environment. It becomes more and more widely used in water and soil conservation, landscaping, and ecological construction. Faced with the severe pressure of environmental deterioration, screening and inoculation of leguminous plants highly effective rhizobia not only can provide nitrogen nutrition, improve the yield and quality, but also decrease chemical fertilizer consumption, reduce the pollution of the environment, which would be of great economic, social and ecological benefits.In this study all the tested rhizobia strains were successfully isolated from more than 80 samples of white clover root nodules obtained from 6 sampling sites of Ya’an, Kangding, Luding, Xichang, Chengdu and Leshan in the wild of Sichuan province. In order to select out superior rhizobia strains matching with white clover symbiotic effect, nodulation experiment was carried out with hydroponics(water culture) and quartz sand culture method after initial inspection of colonial morphology observation and gram staining microscopy on all the separation of strains. At the same time, the genetic diversity and phylogeny of were investigated using recA, atpD, glnll, nodC, nifH sequences analysis of the representative strains selected by 16S rDNA PCR-RFLP molecular methods aimed to verification the taxonomic status of highly effective rhizobia strains that had selected out. The main results concluded as follows:1. The root nodules collected from wild white clover presented various shapes because of different places, such as oval, spherical, cone-shaped and gourd-like and so on, meanwhile most of them had pink appearance. The colony of isolated strains on the YMA plate containing congo red were round, no color but milky, transparent or translucent, sticky, neat, slightly convex surface, full of sheen, gram negative, and in the beef extract peptone media all strains failed to grow except LS1105. All the rest separated strains were of typically individual morphology and colony characteristics of rhizobium, and 72 strains were successfully isolated by using streak plate method in this study.2. The results of hydroponics experiment showed that except 5 strains LS1105, XC1102, XC1103, XC1112, KD1109, white clover plants were all noduled by inoculation with the rest 67 rhizobial strains, and it significantly improved root nodule number, plant height, plant shoot fresh weight and dry weight compared to CK and un-noduled plants. Especially, inoculation effect of 12 strains KD1104, KD1111, KD1117, LD1102, YA1105, YA1103, YA1121, XC1108, XC1109, XC1110, CD1105, LS1104 was relatively the most obvious that white clover plants were tall and strong, full of vitality by inoculation, and be of great potential for screening highly efficient strains using for the subsequent quartz sand experiment.3. The selected 12 strains were further assessed for their effectiveness under quartz sand conditions in paper-cup experiment. The results showed that compared with the non-inoculation, white clover plants were all of nodules, tall and flourishing with dark green, wide and thick leaves by inoculated rhizobium strains. Inoculation with YA1121 and CD 1105 as the screening out effective strains among the twelve not only significantly increased the number of nodules of plant, but also the plant height, plant shoot fresh weight and dry weight, chlorophyll content and total nitrogen content in comparison with the non-inoculation control which were respectively increased by 90.89%,103.09%,187.98%, 51.27%,40.92% and 68.23%,86.32%,114.38%,54.70%,33.22%.4.16S rDNA PCR-RFLP analysis showed that 4 genetic patterns of 16S rDNA were attained and the strains was of relatively abundant genetic diversity. The fact were that I type (AAAA),69 strains, occupied 95.8% of all the tested strains, the remaining were II (BBBB), III(CACA), IV(DCDC) three types each having the sole one strain, which were respectively LS1101, LS1105 and KD1107, and accounted for only 4.2%, demonstrating certain genetic variation and geographical or regional differences.16S rDNA sequences analysis results showed that the tested strains respectively belonged to Rhizobium and Agrobacterium. In addition to LS1105 was the closest relationship with Ag. tumefaciens NCPPB 2437T which shared the similarity of 99.5%, sequences similarity of the majority tested strains with R.1 eguminosarum bv. trifolii ATCC 14480T were above 99.7%.5. The phylogenetic analysis results of housekeeping gene (recA, atpD, glnll) were basically identical with 16S rDNA, the strains could also belonged to genera Rhizobium, Agrobacterium on two system branches. LS1105 also could cluster with Ag. tumefaciens NCPPB 2437T, and the similarity between them was 92.4%. The rest strains were of the closest sequence relationship with R. leguminosarum bv. trifolii ATCC 14480T, and the similarity was between 93.4% and 95.0%. In addition, nodC and nifH gene fragments of strains that belonged to the genus Rhizobium were successfully amplified by PCR, but strains which belonged to the genus Agrobacterium were not the same case. According to the phylogenetic trees constructed from the two genes, it demonstrated that the similarity of the rest strains with R. leguminosarum bv. trifolii ATCC 14480T and R. leguminosarum bv. trifolii 9.2k or R2-1 was between 95.6% and 97.5% except LS1105, therefore they can be determined as white clover rhizobium.In conclusion, strains YA1121 and CD 1105 were the two screened of highly efficient rhizobia isolated from white clover by integrating results of screening and phylogenetic studies, and they could be applied to subsequent field experiments for further research. It was also found that the white clover symbiosis with rhizobia was diversity on the basis of different geographical distribution through the comparison among different sampling sites and it would be of great significance for enriching white clover rhizobium resources, and being beneficial to their exploitation and utilization. |
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