Factors affecting a bioassay for Agrobacterium tumefaciens in natural soil Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/rn3015067

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  • Tomato seedlings were used to assay the population of A. tumefaciens in artificially contaminated soil. Aqueous soil suspensions were applied directly to the wounded stem. The bioassay was used because not all pathologically important physiological strains of A. tumefaciens would grow on any of the selective media available. This method also eliminated the need to assay each Agrobacterium-like isolate obtained on selective media forpathogenicity. Factors affecting the bioassay such as minimum number of bacteria needed for gall initiation, age of seedling at time of inoculation, treatment of seedlings after inoculation, and effect of soil on bacterial suspension used for inoculation were tested. The infectivity of both the 3-ketoglycoside-negative and positive strains were compared, and both produced similar gall initiation in all tests. A concentration of approximately 10³ bacterial cells per wound was necessary to infect 70% of the plants inoculated. Incubating seedlings at 100% relative humidity for 24 hours after inoculation resulted in a significantly (p = 0.05) higher percentage of plants infected over those left at 45-55% relative humidity immediately after inoculation. Mixing the bacterial suspension with either sterile or natural soil immediately prior to inoculation did not affect the number of cells needed for 70% infection. The sensitivity of this assay is not adequate to detect the numbers of A. tumefaciens in field soil which has been reported in the literature (316/g) (41). In this work a population of 10³ cells /wound was required to infect 7 out of 10 tomato seedlings. However, because only 0.01 ml was applied to each wound, it would require an actual concentration of 10⁵ pathogens/ml of initial soil suspension to achieve 70% infection.
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