Seven strains of slow-growing Rhizobium isolated from different species of Stylosanthes growing in either acid (pH 4.5-5.5) or alkaline soils (pH 8.0-9.0) were characterized with respect to several properties. These included cellular, colonial morphology, motility and growth in media of varying pH. Studies were conducted to investigate their ability to utilize 22 different carbohydrates, 11 nitrogenous compounds as well as their requirements for either biotin, thiamine, calcium pantothenate or combinations of these vitamins. Their optimal temperatures for growth were determined in the range of 10 to 40°C. In one study, the seven strains and 27 other strains of slow-growing Rhizobium isolated from diverse host species growing in soils of various pH were assessed for their effectiveness on Stylosanthes capitata using the Leonard jar technique.
Using 100-125 µg ml-1 streptomycin, spectinomycin, kanamycin, rifampicin or combinations of these antibiotics, attempts were made to obtain doubly- as well as triply-labelled antibiotic resistant mutants from the seven parental strains for use in interstrain competition studies. Before these mutants were used for such purposes, they were assessed in several properties in comparison with their parental strains. These properties included effectiveness, competitiveness using the partially enclosed plant tube technique. They were also compared with their parental strains in the ability to grow in acidified (pH 4.5) and neutral (pH 7.0) liquid defined medium. The results obtained revealed the possible change in effectiveness when kanamycin was used. Differences were also observed in the growth ability and competitiveness of these mutants compared with their parental strains. Difficulties were experienced when the mutants resistant to 125 µg ml-1 of either streptomycin, spectinomycin, kanamycin or combinations of these antibiotics were used under non-sterile conditions. Their recovery was severely affected by the presence of other bacteria having resistance to these antibiotics. Success in recovery of Rhizobium strains from the rhizosphere of the host plants was achieved with the groups of mutants selected for resistance to 100 µg ml-1 rifampicin.
In a series of studies conducted to investigate the competitive ability of several strains of slow-growing Rhizobium, plant solution culture containers with 2.5 1 capacity were used. Strains were paired in various combinations: effective v. effective strains from acid soils, effective v. ineffective strains from acid soils and effective v. effective strains from alkaline soils. Six separate studies were conducted under non-sterile conditions using Stylesanthes hamata cv. Verano exclusively as the host plant. Strains were allowed to compete under acidic (pH 4.7), neutral (pH 7.0) and at root temperatures of 25, 30 and 35°C. For each strain pair, sterile seedlings were inoculated with either pure cultures or five other different inoculum cultures in which the pairs of strains were mixed in various ratios where a root temperature of only 30°C was used. When root temperatures of 25 and 35°C were used, only three mixed-cultures in which viable cell numbers of the competing strains were mixed with the intended ratios of 10:1, 1:1 and 1:10, were used to inoculate the host. The inoculum cultures were diluted with sterile dist. water in :m attempt to establish low cell numbers of the competing strains in the rhizosphere of the host after inoculation. Plants were grown in controlled environment growth cabinets with a constant shoot temperature of 30°C and with the day/night light regime of 14/10 h day-1. The light intensity adopted for all studies was approx. 270 µE m-2s-1. Over a period of 24 days, roots were collected after 0, 5, 10, 18 and 24 days and viable counts were made to determine cell numbers of the competing strains in the rhizosphere of the host plants. Growth of fungi was controlled by use of 5 ml 1-1 of sterile aqueous solution of pimafucin. All fully developed root nodules were collected from each inoculation treatment after 10, 18 and 24 days. The relative proportion of the pair strains in nodule formation was subsequently determined by the fluorescent antibody technique.
It was found that effective strains varied markedly in their competing ability. Conditions of incomplete dominance were detected in another pair of effective strains regarding root surface colonization. The ability of strains to colonize the root surface of the host at different pH and temperatures of the rooting medium was closely related to their ability to grow in culture medium at the same pH and temperature values. Strains responded differently upon exposure to either 25 or 35°C root temperature. In one strain pair, decreases in the rhizosphere populations were detected after 5 days in only one strain at both 25 and 35°C whereas in another case, greater decreases in the rhizosphere populations of the paired strains were detected during the same period only at 35°C and not at 25°C
The effective strain used was found to be superior to the ineffective competitor under all experimental conditions imposed. However, the degree of dominance of the effective strain over the ineffective strain was found to vary with pH and root temperature, Nodulation of the host in all studies related well to the interstrain ratios set at inoculation of the competing strains as well as the initial rhizosphere populations of the paired strains recovered from the seedling root surface after inoculation. The proportions of nodules formed by the competing strains particularly those collected after 18 and 24 days changed according to the relative ability of the two strains to colonize host roots. Small magnitude increases (rarely exceeded 2.0 log units) were consistently detected in the rhizosphere populations of rhizobia. Double infection was found to occur at a low frequency of approx. 1% when a total of 234 nodules were examined. Despite the relatively low numbers of rhizobia, plants bearing effective nodules responded favourably to the effect of inoculation. No adverse effect on plant growth was observed when the plants were inoculated with mixtures of two effective strains. It was also found that plant dry shoot weight, in general, was reduced v/hen greater numbers of ineffective strain were mixed with effective strain in the inoculum cultures. The results obtained from these studies were discussed in comparison with the findings currently reported by various other workers.