| Summary: | 碩士 === 國立屏東科技大學 === 植物保護系所 === 98 === Bacterial crown gall, often found on the root crown, stem, shoot, and pruned parts, is caused by Agrobacterium tumefaciens that can infect more than 1,193 plant species worldwide. In Taiwan, bacterial gall-like symptoms were recently found on the stems and branches of china berry tree (Melia azedarach L.) in Dongshih, ficus (Ficus spp.) in Taipei, peach (Prunus persica L. B.) in Nantou, and rose (Rosa hybrida) in Changhua. The gall tissues were surface-sterilized and ground in SCPAP buffer, and the resultant plant extracts were smeared onto carrot slices to detect if the slices can develop callus. At 21 days post inoculation (dpi), the developed callus was excised for bacterial isolation on nutrient agar (NA) medium, and a gram-negative, aerobic bacterium consistently formed circular, white colonies with convex elevation on NA was isolated. Several strains of the bacterium from different plant species were characterized by biochemical analyses and the Biolog system and confirmed the isolated strains as A. tumefaciens with similarity values of 0.23-0.75. Virulence assays were performed by prick-inoculating bacterial colonies into the stems of greenhouse-grown, disease-free seedlings of tomato (Solanum lycopersicum L. cv. Known-you
301) and sunflower (Helianthus annuus L. cv. Sunbright). At 5 to 7 dpi, the inoculation sites developed small, light green spongy spots that were gradually transformed into prominent galls at 14 dpi, whereas control plants inoculated with sterile water remained symptomless, indicating that the strains of A. tumefaciens originated from different plants are virulent to tomato and sunflower. Koch's postulates were fulfilled by prick-inoculating bacterial colonies into the stems of greenhouse-grown, disease-free, 1-meter-tall seedlings of china berry tree or peach tree, and symptoms were evident at 2 to 3 weeks post inoculation on all bacteria-inoculated plants but not on sterile water-inoculated ones. Elicited gall tissues were extracted and smeared onto carrot slices as described above, and the bacteria isolated from the carrot calli at 21 dpi showed morphological and biochemical similarities to the ones used for inoculation. Based on the results, the causal agent of bacterial galls on china berry tree and peach tree, the two new bacterial gall diseases reported in Taiwan, is confirmed to be A. tumefaciens. In addition, A. tumefaciens strains isolated from china berry tree, peach, ficus and rose were characterized by the physiological and biochemical characteristics, carbon source utilization, and the nucleotide sequences of 6b residing in T-DNA region. The results revealed that the A. tumefaciens strain of china berry tree is biovar 1 harboring a 6b gene whose deduced amino acid sequence is 91% similar to the 6b of octopine-type Ti plasmid found in GenBank database; the A. tumefaciens strain of peach tree belongs to biovar 1 with a 6b protein that shares 98% to 100% similarity to the 6b of nopaline-type Ti plasmid; the strains of rose and ficus belong to biovar 3, and the amino acid sequences of 6b are 98% to 100% similar to the 6b of nopaline-type Ti plasmid. In Taiwan, the A. tumefaciens strains isolated from different woody plants have different biochemical characteristics and 6b sequences, indicating the bacterial gall disease of woody plants might be elicited by different strains of A. tumefaciens, the questions of the disease transmission and the source of the pathogens remain to be elucidated.
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