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Trichoderma spp. as a Major Biological Factor for The Management Cabbage Seedling Blight and Promotion of Seedling Growth
|摘要:||幾丁質分解酵素為木黴菌防治機制中重要的角色，因此篩選出可產生高量幾丁質分解酵素的培養基質，再以此基質發酵的產物來防治甘藍幼苗立枯病。木黴菌的菌絲、分生孢子與厚膜孢子都可應用於防治病害，但分生孢子的發芽率很低，所以誘導分生孢子發芽，應可以增進防治效果。應用木黴菌防治幼苗病害的方式一般有種子粉衣與基質混拌，比較這兩種方式在不同病原濃度下防治病害能力。又因已可量產厚膜孢子，遂比較與分生孢子防治病害的能力。在燕麥液態基質中木黴菌可以產生較高量的幾丁質分解酵素，且於燕麥稻殼固態發酵中生長及產孢狀況良好，施用於育苗介質中具良好的防治效果，所以燕麥適合作為木黴菌固態量產基質，且燕麥煎汁培養液培養15天時，每毫升有1.25x106個厚膜孢子，且大部分厚膜孢子都已經成熟脫離菌絲。在幾丁質分解酵素活性測定方法中，點漬法可約略比較酵素活性的差異，但其敏感度不足，以螢光基質法則可獲得較精確的定量。添加2 % 蔗糖或Potato sucrose broth（PSB）處理24 hr後可使分生孢子100 %發芽。拮抗菌在不同培養基平板上的抑制效果有顯著差異，可依此作為初步篩選的依據。將不同來源的拮抗菌對病原菌作拮抗測試時，原生長環境與病原菌相同的拮抗菌未有較佳的防治表現。接種源與立枯絲核菌存活及致病的試驗中，立枯絲核菌在混有細沙的介質中存活較佳。防治試驗方面，以YT3與AHS06的固態發酵產物防治甘藍幼苗立枯病可達80與90 %的病害防治率。種子粉衣YT3拮抗菌兩次時，其防治效果較好，可將存活率由25 % 提升到50到60 %，但在高病原濃度時種子粉衣分生孢子或厚膜孢子均無法防治病害，基質混拌可維持一定的防治效果。利用由不同培養基製成之厚膜孢子來防治病害試驗中，G8菌株由NyA或是CH 培養基生產的厚膜孢子與YT3菌株以CH培養基生產的厚膜孢子可防治甘藍幼苗立枯病。木黴菌促進生長機制有，對潛隱性病原的抑制作用、誘導植物產生抗病性、增加或促進根圈與介質營養的利用及幫助植物抵禦環境壓力等，但尚有其他因子尚未被發現，本研究將探討木黴菌促進植物生長的直接因子，將試驗分成兩部分：第一部份針對木黴菌的添加在無其他微生物環境與穴盤中對甘藍幼苗的影響，第二部份針對幾丁質的添加在無其他微生物環境與穴盤中對幼苗的影響，完成上述試驗後將兩者混合添加於穴盤進行試驗，並進行木黴菌菌株幾丁質分解酵素活性與促進生長能力的相關性分析。並添加幾丁質與木黴菌於穴盤進行促進生長與誘導水稻產生PR-protein與PAL的m-RNA試驗。添加木黴菌促進十字花科幼苗生長試驗中，1% YT3固態發酵產物即具有促進生長效果，如結球白菜(Brassica pekinensis, pe-tsai)及青梗白菜(B. chinensis L., pak-choi)幼苗乾重可增加一倍以上。測試YT3 沿根生長的能力時，發現隨著根的延長此菌可在根尖及根冠周遭產孢，而多分佈於根尖端及接近莖的根部，成U型分佈，經觀察並無發現菌絲有侵入根毛的情形。添加木黴菌促使幼苗株高增加以種子粉衣較好。而葉面積方面所有處理皆有增加現象，根長度則以基質混拌處理組最好。經移殖的甘藍施與不施肥，乾重增加以澆灌處理最好而種子粉衣處理組次之。在無其他微生物環境下除YT1、YT2、P1三菌株外，其他菌株對甘藍幼苗皆會抑制其生長。而種植於穴盤的試驗中以G8、T2、P3三株菌在施肥下仍可促進植物生長。種子粉衣過量YT3分生孢子時會抑制甘藍種子發芽與幼苗生長，而以107 conidia/seed的粉衣量最適合。介質中混拌高量幾丁質0.5、1、6 %發酵，可顯著增加甘藍50 % 濕重，混拌低量幾丁質0.025及0.2 %則分別增加178及157 % 的乾重。混拌低量0.8及1 %幾丁質並施肥，其增加乾重只有37及24 %，而基質混拌低量幾丁質發酵除0.1 %幾丁質外，隨著幾丁質含量增加乾重也隨之增加。在無其他微生物系統中幾丁質使植株乾重增加16 %，添加幾丁聚醣可增加50 %乾重。當混合木黴菌和幾丁質時，在所有添加AHS06菌株的處理中，添加幾丁質和幾丁聚醣可分別增加26 及8 %乾重，而對照為39 %。而添加YT3菌株的處理中則是以混拌幾丁聚醣的促進效果最好，約增加22 ％。把具分解幾丁質分解能力的真菌菌株加入介質對甘藍幼苗可增加乾重21 %。消毒介質加幾丁質可使乾重增加100 %，未消毒介質加幾丁質可使乾重增加173 %。介質加幾丁質與拮抗菌發酵後種植可增加乾重27 %，單獨添加幾丁質可增加乾重46 %。|
The chitinase plays an important role in the Trichoderma sp. biocontrol mechanism. The fermentation substrates were screened for the chitinase activity of Trichoderma sp. The selected substrate was used as for fermentation and applied to cabbage seedling blight control. Hyphal fragment, conidia and chlamydospores of Trichoderma sp. were all mixed in commercial product. The conidial germination rate was usually too low for biocontrol, so the nutrition or acid was added to stimulate or trigger germination and therefore increase the biocontrol ability. Two application methods of Trichoderma sp. or Gliocladium i.e., drenched into growth substrate and the seed coating were used under different dose of antagonist or pathogen concentrations. As chlamydospore mass production as possible, the biocontrol ability was compared between the chlamydospore and conidia. The chitinase of Trichoderma sp. could be induced in oat solution. The Trichoderma sp. also grew and sporulated well in oat rice hull solid fermentation and exhibited good disease control ability. The number of chlamydospores in oat decoction liquid at 15th day reached 1.25x106 /ml and most of them were matured and detached from the hyphae. The dot-blotting was one of the detection methods of chitinase, but was not sensitive enough to clearly differentiate the quantity of chitinase. The Florence substrate spectrophotometers method was used to detect the minor differently chitinase activity in antagonists. Using 2 % sucrose or PSB could induce the germination of conidia up to 100 % in 24 hr. The antagonistic ability of antagonists that grown on different media was not significantly different, so it could not be used for selection of biocontrol antagonists. Using antagonists that had the same ecology niche with pathogen could not get the better biocontrol results. The inoculum of pathogen prepared in artificial disease substrate made by potato or oat survived well if the substrate was mixed with sand. The solid fermentation products of YT3 or AHS06 had good efficiency to control the disease. Seed coated with antagonist twice had a good control the cabbage seedling blight, unless the growth substrate containing too many pathogen propagules. The G8 and YT3 chlamydospores produced from NyA or CH medium had good control ability on cabbage seedlings blight. The mechanisms involved in promoting the plant growth by Trichoderma sp. are inhibition of minor pathogen, inducing plant resistance, improving the nutritional absorption in rhizosphere, and increase defensive ability to environment stress. However there are still some unknown factors to be explored. The objectives of this study are to find the factor involved in Trichoderma sp. that enhances the cabbage seedling growth. These studies were divided into two parts. The first part of studies was the amendment of Trichoderma sp. into substrate to affect the cabbage seedling growth on plug and gnotobiotic system. The second part of studies was the chitin amendment on plug and gnotobiotic system. At last, combining chitin and Trichoderma sp. application into substrate for promoting seedlings growth and the induction of the PR-1 and PAL m-RNA in rice. When 1 % Trichoderma sp. solid fermentation product was amended, the dry weight of Chinese cabbage and Pakchoi increased more than fold. The rhizoplane competence of Trichoderma harzianum YT3 was observed in tube assay method and it was found that the antagonist sporulated in root cap and root base near stem and no hyphae were observed in root hair. Increment of seedling height was better in coating seed, and root length was performed better by amendment in the substrates with antagonist, while all treatments i.e. seed coating, amendment and drenched into substrates are capable of increase leaf area. After seedlings were transplanted either or not with fertilizer applied the drench by conidial suspension treatment had heaviest dry weight and the seed coating treatment ranked second. In the gnotobiotic condition with exception of isolates YT1, YT2 and P1, to other tested isolates could promote the seedlings growth. Isolates G8, T2 and P3 could enhance seedling growth under fertilization condition. The optimum conidial concentration for seed coating is 107 conidia/seed that could increase the germination rate and the seedlings growth, however if concentration higher than that will be resulted in decrease of germination rate. When substrate was amended with high quantity of chitin at 0.5, 1 or 6 % the seedling flesh weight increased about 50 %. If low quantity of chitin at 0.025 or 0.2 % was added the dry weight increased more than 150 %. Under fertilization the substrates amended with 0.8 or 1 % chitin, the seedling dry weight increased only 37 or 24 %, respectively. When the growth substrate containing the low quantity of chitin and fermentation for 3 weeks, the more chitin added in substrate and the more increase of dry weight resulted. In the gnotobiotic condition the chitin and chitosan amendment increased 16 % and 50 % seedlings dry weight, respectively. In the substrate containing isolate AHS06 could increase 39 % dry weight of seedling and in that substrate mixed with chitin or chitosan could increase 26 or 8 % dry weight, respectively. The chitin powder could increase 173 % seedlings growth in unsteriled peatmoss, but only increase 100 % in autoclaved peatmoss.
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