台灣由 Streptomyces scabies 所引起之馬鈴薯瘡痂病－病原菌生物特性及應用拮抗性枯草桿菌於其生物防治之初探

An outbreak of tuber infection showing symptoms closely resembling that of common scab disease was observed during the winter of 2006 on field harvested potatoes in central Taiwan at both Tantzu Taichung and Dounan Yunlin. The disease appeared to be new for potato cultivation in Taiwan, and the increasing incidence and severity of the disease have drawn great attention among growers during the past 2 years. The main objectives of this investigation were to explore the biological and pathological characteristics of the causal agent and the possible resolution to solve the problem. A total of 7 Streptomyces strains isolated from diseased tubers were used for the performed tests. Among them, T1, T2, T3, T4, T5 and T6 were from Tantzu; and D1 (kindly given by Dr. Ann-show Cheng of Tainan DAIS) was from Dounan. Comparative study by use of ISP4 (International Streptomyces Project) medium on the morphological and physiological characteristics of these 7 tested isolates indicated that all of them grew up into grayish colony typical of Streptomyces morphology and produced spores in spiral chains on the aerial mycelia. They all appeared to grow well with the provision of each individual ISP sugar as sole carbon source, produced melanin pigment on tyrosine agar, and consisted in their membrane lipid fatty acid profile similar to that known for Streptomyces scabies. The 16S rDNA of these tested isolates were cloned and sequenced, pair-wise comparison of the sequences with the data provided by GenBank indicated all of them shared a 99% identity with that known for Streptomyces scabies. A phylogenetic tree constructed based on 16S rDNA sequences known for Streptomyces further concluded the 7 tested isolates in the same group as that of S. scabies. The morphological, physiological and molecular characteristics observed indicated that all the 7 tested isolates belong to members of S. scabies. As the majority of Streptomyces spp. are known to be saprophytic and some are important beneficial soil microbial resources which have been widely used as biological control agents, 5 Streptomyces strains S1, S3, S4, B4 and M6 known with potential of biofungicide application were included as compared strains for the pathogenicity study to learn if they may pose potential threat on crop production. Upon artificial inoculation, all the 7 diseased potato tuber derived strains were shown to cause necrotic response on potato tuber slices, reduced stem height and leaf thickening on radish seedlings. An artificial inoculation by drenching application of 1x108 cfu/ml spore suspension on pot grown potato plants (cv. Kennebec) confirmed that all 7 tested isolates were pathogenic and led to development of typical necrotic common scab symptoms. As a comparison, none of the 5 strains with potential of biofungicide application caused observable symptoms on potato tuber slices and pot grown potato plant; although slight inhibition of radish seedling growth was detected. Thaxtomin A is known to be a major determinant of Streptomyces spp. that causing the development of common scab symptoms on potato. The production of thaxtomin A of the tested strains was examined by thin layer chromatography, high performance liquid chromatography (HPLC), and a triple-quad mass detector equipped HPLC system (LC/MS/MS). The identity of thaxtomin A detected was demonstrated by use of internal standard co-chromatography in HPLC, and by LC/MS/MS at multiple reaction monitoring mode the detection of precursor ion at m/z=437 and the major product ions at m/z=150, 140 and 107, respectively. All the 7 diseased tuber-isolated strains were shown to produce