Rhizobium leguminosarum bv. trifolii forms nitrogen-fixing root nodules on the pasture legume Trifolium repens, and T. repens seed is often coated with a compatible R. leguminosarum bv. trifolii strain prior to sowing. However, significant losses in bacterial viability occur during the seed-coating process and during storage of the coated seeds, most likely due to desiccation stress. The disaccharide trehalose is known to function as an osmoprotectant, and trehalose accumulation due to de novo biosynthesis is a common response to desiccation stress in bacteria. In this study we investigated the role of endogenous trehalose synthesis in desiccation tolerance in R. leguminosarum bv. trifolii strain NZP561. Strain NZP561 accumulated trehalose as it entered the stationary phase due to the combined actions of the TreYZ and OtsAB pathways. Mutants deficient in either pathway showed near-wild-type levels of trehalose accumulation, but double otsA treY mutants failed to accumulate any trehalose. The double mutants were more sensitive to the effects of drying, and their survival was impaired compared to that of the wild type when glass beads were coated with the organisms and stored at relative humidities of 5 and 32%. The otsA treY mutants were also less competitive for nodule occupancy. Gene expression studies showed that the otsA and treY genes were expressed constitutively and that expression was not influenced by the growth phase, suggesting that trehalose accumulation is controlled at the posttranscriptional level or by control of trehalose breakdown rates. Our results indicate that accumulated trehalose plays an important role in protecting R. leguminosarum bv. trifolii cells against desiccation stress and against stress encountered during nodulation.Rhizobium leguminosarum bv. trifolii forms nitrogen-fixing root nodules on the pasture legume Trifolium repens (white clover). Inoculation of legumes is an efficient and convenient way of introducing rhizobia into soil and subsequently into the rhizosphere of legumes. The seeds of many clover species (including white clover) are coated with compatible R. leguminosarum bv. trifolii strains prior to sowing. One of the main reasons for the ineffectiveness of legume inoculants in the field is the rapid death of rhizobia due to desiccation (12, 55). Significant losses in inoculant viability occur during the seedcoating process, which involves a drying stage, during storage of the coated seeds and after introduction into the field environment. These losses in bacterial viability are likely to be due mainly to desiccation stress.Bacteria accumulate osmoprotective compounds, referred to as compatible solutes or osmolytes, in response to osmotic or desiccation stress. Osmolytes can be obtained by uptake from the environment (exogenous) or through de novo biosynthesis (endogenous). De novo biosynthesis of trehalose, a nonreducing (␣,␣-1,1)-glucose disaccharide, is a common response to desiccation and osmotic stress in many bacteria (11,35,57). Trehalose has been shown to protec...

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Caucasian clover (Trifolium ambiguum) is being commercially released for use in New Zealand agriculture. Seed must be inoculated at sowing, as caucasian clover forms a nitrogen-fixing symbiosis only with specific rhizobia that are not native to New Zealand. These rhizobia have the potentially undesirable property of readily forming nodules on white clover that do not fix nitrogen. Caucasian clover inoculant strains available for use in New Zealand were found to be genetically unstable in the laboratory, and must be frequently monitored for their symbiotic properties. A strain of caucasian clover rhizobia that gives better establishment and growth of caucasian clover under oversowing conditions was identified. This strain maintained a higher level of viability under stress conditions in the laboratory and showed enhanced survival on seed in the environment. Growth of caucasian clover results in large populations of caucasian clover rhizobia in the soil. These formed nodules on white clover in the same field environment, although white clover rhizobia dominated nodule formation. Nevertheless it is possible that present caucasian clover inoculants may over time reduce nitrogen fixation and hence the production and persistence of white clover. Further research is required to develop a caucasian clover strain that does not form ineffective nodules on white clover. Keywords: caucasian clover, inoculation, nodulation, rhizobia, Trifolium ambiguum, Trifolium repens, white clover

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