| Summary: | 碩士 === 國立中興大學 === 植物病理學系 === 91 === 柒、英文摘要
The potential of Streptomyces fimbriatus WRS9 as a microbial biofungicide and the development of polymerase chain reaction primer for specific detection of Streptomyces spp.
Chiung-ying Chiu
In the discussed thesis works, the biological characteristics of Streptomyces fimbriatus WRS9 pertaining to its potential application as a biofungicide for plant disease control were evaluated using S. griseobrunneus WRS3, S. lincolnensis WRS7, and S. saraceticus SS31 as compared strains. In a preliminary screening test, WRS9-which was originally isolated from sugarcane rhizosphere, was found superior in strength of chitinolytic activity and antagonisity against Pyricularia oryzae, Pythium aphanidermatum and Phytophthora palmivora, than that of the three compared strains. In a broth culture system under continuous shaking, it produced at least 6 inducible chitinase isozymes with molecular weight ranging from 32 to 60 kDa, in responding to the presence of colloidal chitin or the mycelial suspension of Rhizoctonia solani AG4. The bacterium grows well in 2-3% oat broths, the biomass yield reached 3.19x108 cfu/ml 6 days after inoculation (DAI). As a comparison, the bacterial growth in 2% corn and 2% wheat bran broth were comparatively less well; the biomass yield reached only 2.03-1.99x107 cfu/ml at six DAI. In paralleled to the bacterial growth, the antagonisity against P. aphanidermatum was detected from all oat and corn broth cultures at 1 DAI which then hold throughout the 5th day after inoculation and declined to non-detectable at 6 DAI . In a wheat bran broth culture, however, the antagonisity remained non-detectable throughout the 6 days culture period except that a slight increase of antagonisity was detected at 5 DAI. A total of 7 natural agar media were screened for the performance for supporting the sporulation of the test bacterium. Among them, the 0.2 % maltextract amended potato sucrose agar (PSMe) appeared to be the best, the spore yield reached approximately 4.67x109 cfu/plate at 5 DAI. Other test media with good performance in supporting the sporulation of WRS3 include 2% wheat bran, PSA and chitin agar; the yield of biomass all achieved 2.78-3.36x109 cfu/plate level. When applied as inoculum for liquid culture using oat broth medium, spore samples obtained from all these natural agar media all performed equally well in regarding to the yield of biomass except that from 2% corn agar which tended to have a lower yield. A pilot scale mass production of test bacterium was attempted using 5 to 50 L stirrer type fermentor. By an established protocol developed for SS31 and as well for WRS3, the yield of biomass obtained all remained less than 108 cfu/ml. Although the fine tune of the protocol need to be worked out, the potential application of the broth cultures produced in disease control was attempted using papaya root rot (caused by Phytophthora palmivora) as trial target. In a 7-day old suspension culture of P. palmivora growing in PSB broth, the inoculation by WRS9 at 3x106 cfu/ml caused an increased leakage of electrolytes approximately 24 to 36 hours after inoculation; an antibiotic function on the membrane damage was clearly indicated. A followed examination by light microscopy further revealed that the pathogen mycelia were penetrated and/or tangled up by WRS9 mycelium, lost their cellular contents and were finally totally dismantled or even disintegrated. The mycoparasitic and structural damaging effect on pathogen mycelium seemed to implicate the functioning of lytic enzymes of WRS9. In a greenhouse trial, the drenching of 500 to 1000X diluted WRS9 cultural broth was shown slightly reduced the infection of papaya seedlings by artificially inoculated P. palmivora. The application of WRS9 cultural broth at 100X dilution however, greatly reduced the survival of the seedlings. A plausible reason for the observed deleterious effect on test seedlings appeared to due mainly the antibiotic toxicity on the root system. On papaya fruit artificially inoculated with P. palmivora zoospore suspension, the application of WRS9 at 105-106 cfu/ml in concentration one day before inoculation significantly deterred or even stopped the fruit infection.
Another focus of this thesis work was the development of primer for specific detection and/or identification by polymerase chain reaction the members of Streptomyces spp. The major objective of the devoted efforts was to provide useful tool for monitoring the possible ecological impacts due to repeated application of viable propagules of the antagonistic Streptomyces strains attempted for disease control application. The genomic DNAs were extracted from a total of 9 Streptomyces strains namely WRS2, WRS3, WRS7, WRS9, WRS38, TYS15, TYS17 TYS19 and SS31. And from each of them, the 16S rDNAs were amplified by PCR using universal primer pair UNI16S-L and UNI16S-R and cloned by yT&A vector. All the cloned 16S rDNAs appeared to be approximately 1400 bps in size; the full sequence of that from WRS3, WRS7, WRS9 and SS31 were resolved. The sequence analysis by Pretty software (Seq-Web) using database from NCBI indicated that the cloned sequences from WRS7 had 95% identities comparing to that known for most Streptomyces. The comparative analysis of the 4 resolved sequences further indicated the existence of certain species-specific regions located at 171-181 respectively. And from these data information, WRS3-L, WRS7-L, WRS9-L and SS31-L each respectively as forward primer specific for the denoted strain were designed from 171-181 region; and likewise ScR-1 (679-691) and ScR-2 (951-968) were each designed to serve as the reverse primer. The followed extensive PCR amplification trial using WRS3-L, WRS7-L, WRS9-L each as specific forward primer, 16S-R, ScR-1, and ScR-2 each as reverse primer, and the genomic DNAs obtained from S. griseobrunneus WRS3, S. lincolnensis WRS7, S. fimbriatus WRS9, 5 other Streptomyces spp. including WRS2, WRS38, TYS15, TYS17, and TYS19, and 5 other non-Streptomyces bacteria including Bacillus sp., Pseudomonas sp., Erwinia sp., and Xanthomonas spp. 1 and 2, each respectively, as template DNA. The results obtained indicated the use of WRS3-L/ScR-2 as primer pair detected the predicted 797 bps fragment only from WRS3; that of WRS7-L/ScR-2 detected the 797 bps fragment only from WRS7; whereas that of WRS9-L/ScR-2 detected the 797 bps fragment only from WRS9. All the other tested forward/reverse primer pair combinations, as the contrary, were not successful in terms of specific detection of the attempted bacterial strain. For the specific PCR detection of WRS9, the spore suspension at serial dilution was tested for its efficacy in replacing the template function of genomic DNAs. The application of WRS9-L/UNI 16S-R as primer pair was successful in detecting the predicted 1266 bps fragment; the detection limit went down to about 108 cfu/ml diluted 10-4 level. For the detection/identification of specific Streptomyces species/strains, the detailed protocol of the detection may need further improvement for each specific target strain, the use of species specific 16S rDNA sequence as forward primer appeared to be a valuable tool.
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