Molecular Detection Of Clavibacter Michiganensis Subsp. Michiganensis Causing Bacterial Canker Of Tomato And Study Of Streptomyces Strain Z-L-22 As A Biocontrol Agent Against The Disease

Bacterial canker of tomato (Lycopersicon esculentum Mill.), caused by Clavibacter michiganensis subsp. michiganensis (Cmm), is one of the most severe diseases on tomato crops. Since its discovery in a Michigan greenhouse of America in 1909, bacterial canker of tomato occurred frequently and spread to all tomato growing regions of the world. Epidemic of the disease is increasing in recent years, thus being considered as a quarantine pathogen in China. Cmm could be disseminated through seeds, transplants, infested debris and soil, thus being the primary inoculum source. No effective ways to control the disease are available currently both in China and abroad. Control methods reported before could only manage the occurence and spread of the disease to some extent. Sensitive and reliable detection techniques and biocontrol of this disease are important measures to reduce the incidence and spread of the disease. A multiplex PCR assay system was developed for the detection of Cmm in this research. Experiments were also carried out on strain Streptomyces setonii Z-L-22 which was used as a biological control agent to bacterial canker of tomato.1. A multiplex PCR assay system was developed for the detection of Cmm, which combined two tests in one reaction mixture. Cmm specific primers, PSA-4/PSA-R, and L. esculentum Mill. specific primers, NS-7-F/NS-8-R (internal PCR control primer) were combined in one PCR reaction mixture as well as Cmm and plant DNA. The primer sets could amplify target sequence successfully. Different combinations and concentrations of primers and annealing temperatures were tested, respectively. The detection level of the optimized multiplex PCR assay was up to 5×102 cfu/mL. To verify the applicability of this system, it was employed to detecting Cmm in tomato seeds and plantlets samples. Seeds mixed with 5×104 cfu/mL Cmm cells and diseased plantlets without symptom (6 d after inoculated by needle penetration) could be detected successfully. The multiplex PCR system would avoid false-negative results and is a reliable method for the detection of Cmm.2. S. setonii Z-L-22 screened and conserved by our laboratory was antagonistic to Cmm, thus being used as a biocontrol agent of bacterial canker of tomato in this experiment. Different treatments were carried out in vivo studies using the strain and the antibiotic it produced. Cmm were inoculated to the soil, and three treatments, including soaking tomato seeds in filtrate of the antagonist prior to sowing, inoculation of the soil with the antagonist 7 days before sowing and coating of tomato seeds with the antagonist before sowing, were used. The results indicated that seed coating, soil pre-inoculation and seed soaking showed a control effect of 70.9%,68.0% and 42.3%, respectively, laying a basis for the field use of the biocontrol agent.3. Bioactive compounds produced by Streptomyces setonii Z-L-22 were analyzed in this part. Thin-layer chromatography, with the solvent system at a ratio of benzene: ethyl acetate: methanol = 19: 19: 4, was used to separate the bioactive compounds extracted from the fermentation broth. Two main bioactive components were collected from the layer with Rf value at 0.64 and 0.56. The paper chromatography in Doskchilova solvents system and confirmation tests were used to identify the antibiotic. The two components were all identified as actinomycin. PCR was carried out using the primers targeted to synthetase of the actinomycin, and a fragment of 770 bp (Accession NO. FJ767838) of actinomycin synthetase gene was cloned. The sequence shows a similarity of 82% to the actinomycin synthetase III gene (acmC, accession NO. AF204401.1). PCR results confirmed the antibiotic type and laid a foundation for the genetic study of the strain.4. Genomic walking strategy was used for extending the sequence of actinomycin synthetase gene got in S. setonii Z-L-22. An improved chromosome walking method in this paper was named as nonspecific primer anchored PCR (NPA-PCR). Nested gene specific primers were designed based on the known region and long random primer with degeneracy oligonucleotide was used for nonspecific anchoring. Annealing temperatures were varied to control the priming. Long random primer was nonspecific primed with template for the purpose of anchoring. Target sequences were gotten by PCR with random primer and gene-specific primer. Nonspecific sequence with long random primers at both ends formed stem loop structure due to inverted terminal repeats. As a result, a fragment of 1474 bp and 701bp were obtained at the up and down stream of known fragment respectively. The walking sequence and the known sequence were located conjointly in the same cluster gene by NCBI Blast analysis. A fragment of 2716 bp was got by assembling the walking sequence and the original sequence. The sequence shows a similarity of 82% to acmC by blast analysis in the Genbank. It showed that the result is correct and this technique is useful for chromosome walking.5. The nucleotide sequence of 2716 bp DNA fragment related to actinomycin synthetase was determined and analysed by frameplot software in NCBI. The result demonstrated that the 2716 bp DNA fragment contained one complete open reading frame (ORF) of 1683 bp, named as Streptomyces setonii actionmycin gene X (ssaX). The putative protein of the frame shows the character of peptide synthetase. Reombination plasmid was constructed by inserting the Kanamycin resistance gene to ssaX and ligated with pKC1139. The ssaX disruption mutant showed the reduced inhibition to Cmm and varied bioactive compounds by TLC analysis with the wild type. It indicated that ssaX is necessary in the normal synthesis of actinomycin in strain S. setonii Z-L-22.