Autecology in Rhizospheres and Nodulating Behavior of Indigenous Rhizobium trifolii

Abstract

Indigenous serotype 1-01 of Rhizobium trifolii occupied significantly fewer nodules (6%) on plants of soil-grown noninoculated subterranean clover (Trifolium subterraneum L.) cv. Woogenellup than on cv. Mt. Barker (36%) sampled at the flowering stage of growth. Occupancy by indigenous serotype 2-01, was not significantly different on the two cultivars (16 and 26%). Serotype-specific, fluorescent-antibody conjugates were synthesized and used to enumerate the indigenous serotypes in host (clovers) and nonhost (annual rye-grass, Lolium multiflorum L.) rhizospheres and in nonplanted soil. The form and concentration of Ca2+ in the flocculating mixture and the presence of phosphate anions in the extracting solution were both critical for enumerating R. trifolii in Whobrey soil. The two serotypes were present in similar numbers in nonplanted soil (ca. 106 per g of soil) and each represented ca. 10% of the total R. trifolii population. Although host rhizospheres did not preferentially stimulate either serotype, the mean population densities of serotype 2-01 were significantly greater (P = 0.05) than those of serotype 1-01 in clover rhizospheres on 8 of 14 samplings made between the time of seeding and the appearance of nodules (day 12). In this experiment, and in contrast to our earlier findings, serotype 1-01 occupied significantly fewer (P c 0.05) of the nodules (7 to 16%) on both cultivars than serotype 2-01 (51 %) when sampled at 4 weeks. Differences between cultivars became apparent as the plants matured. There was a threefold increase (7 to 21 %) in nodules occupied by serotype 1-01 on cv. Mt. Barker between 4 and 16 weeks. This was accompanied by increases in nodules coinhabited by both nonidentifiable occupants and either serotype 1-01 (0 to 20%) or 2-01 (11 to 51%). No increases in either of these parameters were observed on cv. Woogenellup.

Determination of viability within serotypes of a soil population of Rhizobium leguminosarum bv. trifolii

“…(v) Immunofluorescence analysis. Preparation of fluorescein-labeled immunoglobulin conjugates and the methods used for enumerating rhizobia have been described elsewhere (12).…”

“…A portion (0.2 ml) of the nalidixic acid stock solution was immediately added to each growth tube to achieve a final concentration of 10 mg/liter. Over a period of 24 h, replicate tubes were sacrificed at 4-h intervals by adding Formalin (final concentration, 2% [wt/ vol]) and were then cooled to 4°C and processed for microscopy as described elsewhere (7,12). Included in every experiment were extra tubes containing filtered soil suspension supplemented with yeast extract and incubated in the presence or absence of nalidixic acid.…”

Population Size and Distribution of Rhizobium leguminosarum bv. trifolii in Relation to Total Soil Bacteria and Soil Depth

“…All previous immunofluorescence studies of soilborne rhizobia have involved the use of 0.4-p.m-pore-size filters to collect the bacterial cells (16,29). Our initial discovery of small biovar trifolii organisms led us to suggest that 0.2-pLm-pore-size filters, commonly used by aquatic microbial ecologists, might be necessary for accurate population determinations (2).…”

“…The filters were destained sequentially with 0.1 M citrate buffers at pHs 6.6, 5.5, and 4.0 and then with distilled water. Each filter was air dried and mounted on Cargille type B immersion oil (Cargille Laboratories, Inc., Cedar Frame, N.J.) on a microscope slide, oil was added to the upper surface of the filter, a cover slip was applied, and bacteria were enumerated by epifluorescence as described previously (16) Efficiency of bacterial recovery from soil. Soil was extracted and flocculated, and a 2-ml portion of this supernatant was inoculated into a dilute medium composed of yeast extract and Bacto-Peptone (0.1 g of each liter-').…”

Plasmids and saprophytic growth of Rhizobium leguminosarum bv. trifolii W14-2 in soil

“…Rhizobia are saprophytic microorganisms [ 1,2] and there is a wide range of organic substrates present in soil [3,4] that can be utilized [51. Most organic substrates are found at population-limiting concentrations in soil away from the rhizosphere [3,4,6] and addition of organic substrates increases their populations .…”