Study on eliminating Xanthomonas campestris pv.campestris from cabbage seeds by high frequency microwave
2019
Hochschulschrift
Zugriff:
107
Black rot of crucifers caused by Xanthomonas campestris pv. campestris (XCC) is one of the most important diseases in the world, and it affects the yield of cabbage especially in Taiwan. The most effective method for prevention and control of black rot disease is the use of healthy pathogen-free seeds to reduce the initial inoculum and thus to decrease the disease incidence in the field. Though many seed disinfection methodshavebeen developed, but most of these methods cannot eliminate pathogenic bacteria completely, affect seed germination, or require long processing time. Therefore, this study intends to use the 24 GHz high-frequency microwave system developed by Department of Physics at National Taiwan University to establish a technology which can quickly and effectively eliminate XCC from vegetable seeds. Therefore, a new seed disinfection option can be provided for seed producers. At first, 1 ml of XCC 1-1 strain suspension was irradiated with 300 W high frequency microwave, and the result showed that XCC 1-1 was completely eliminated in 8.91 seconds, indicating the possibility of seed sterilization by high frequency microwave. The cabbage seeds were immersed in XCC 1-1 suspension, air-dried for 1 day and used as the artificially inoculated seeds, and the average bacterial population per seed was about 60,000 CFU (colony forming unit). While these dry inoculated seeds were directly irradiated with high frequency microwave for 55 seconds and the average seed temperature reach 90℃, XCC 1-1 population decreased to 52%, and seed germination rate was also reduced to 25%. In order to improve the sterilization effect of high frequency microwave, the seeds were misting-treated and then irradiated with high frequency microwave.. The results indicated when the misting-treated seeds were heated to the average temperature of 94℃, bacterial population per seed was reduced to 24%, and germination rate increased to 58%. Because the bacteria elimination of misting-treated seeds were better than dry seeds after irradiation with microwave, indicating that water might be the key factor of improving sterilization effect. To confirm the role of water in sterilization process, bacterial population and germination rate of dry, misting, and water-treated (including water-soaking in inverted tube and in upright tube) seeds were analyzed after irradiated with 200 W high frequency microwave, at about 80℃. The results revealed that the more water seed surface was covered by, the better bactericidal effect it had. In both water-treated seed treatments, XCC 1-1 could be eliminated from seeds completely; but only seeds of water-soaking treatment (water-soaking in upright tube)could maintain 100% germination rate. Consequently, seed with water-soaking treatment and high frequency microwave irradiation is the best disinfection method. In order to further explore the reason seed after water-soaking microwave treatement can still survive at high temperature of 80℃, the cabbage seeds were heated to different temperatures by adjusting the high frequency microwave power and irradiation times, and then tried to find out the temperature range in which XCC 1-1 die but seeds survive. According to the experimental results, it was found that under rapid heating of 600 W microwave irradiation for 0.6-1 seconds, the highest survival temperature of cabbage seeds was 91℃, and the lethal temperature difference between seed and XCC 1-1 reached above 13℃. Whereas under slow heating of 20 W irradiation for 35 seconds, the highest survival temperature of cabbage seed was 63℃, and the lethal temperature between seed and XCC 1-1 was reduced to 11℃. It can be concluded that rapid heating of high power high frequency microwave is the main reason why seed can survive at high temperature, and fast heating rate, is benificial to seed disinfection. Based on the above results, this study successfully developed a standard seed disinfection protocol for high frequency microwave as following water-soaking seed were irradiated with 600 W high frequency microwave for 1 seconds. In addition, our study also demonstrated that high frequency microwave irradiation did not affect seed germination and plant growth, also it could effectively reduce the black rot disease incidence of seedlings from 15% to 0%. So this study became the world’s first successful case of seed disinfection using microwave. Because of the success on artificially inoculated seeds, we further attempted to use the same technology to eliminate XCC inside the seeds. To obtain naturally infected seeds, the XCC 1-1 suspension was sprayed on the rape flowers, and the seed pods were harvested after seed matured. These seeds were surface sterilized, and the internal bacterial population was determined as 3,500-16,000 CFU per seed. Surface-sterilized rape seeds treated with water-soaking microwave irradiation method could eliminate 99% of XCC inside the seed; in advance, it was found that seeds pre-soaking in water for 10 minutes then treated with water-soaking microwave irradiation method could eliminate XCC inside the seed completely, but seed cannot survive. Further more, to understand how XCC was eliminated by high-frequency microwave in a watery environment, XCC 1-1 was collected after water-soaking microwave irradiation treatment, and part of the bacteria were stained with propidium iodide and observed by confocal microscope. Beacuse there had no fluorescence in XCC 1-1 cells, it suggested that bacterial cell membranes were not damaged. Another part of irradiated XCC 1-1 was cultured on medium, but no colony was observed, indicating these bacteria were dead and cell membrane destruction seemed not the causal reason. According to the observations of scanning and transmission electron microscopy, it was found that XCC 1-1 cells became shortend and electron-dense objects appeared in cytoplasm after high frequency microwave irradiation. It is inferred that these effects may be related to the mechanism of XCC 1-1 death, but it still needs to be confirmed.
Titel: |
Study on eliminating Xanthomonas campestris pv.campestris from cabbage seeds by high frequency microwave
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Autor/in / Beteiligte Person: | Liao, Cheng-Hsiang ; 廖證翔 |
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Veröffentlichung: | 2019 |
Medientyp: | Hochschulschrift |
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