Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/31751
標題: 十字花科硫配醣體之揮發性分解物防治蔬菜苗土媒病害
Volatile compounds Released from Cruciferous Glucosinolates Degradation to Control Soilborne Diseases of Vegetable Seedlings
作者: 鍾文全
Chung, Wen-Chuan
關鍵字: 猝倒病
damping-off
立枯絲核病菌
包心芥菜種子粉末
異硫氰化物
十字花科蔬菜
MBF混合物
硫配醣體
PBGG 粒劑
Rhizoctonia solani AG-4
Brassica spp
ground mustard seed meal -Biolan peat B3 (MBF) mixture
allyl isothiocyanate
Pseudomonas boreopolis
myrosinase genes TGG1 and TGG2
3-butenyl isothiocyanate
PBGG
出版社: 植物病理學系
摘要: 土壤燻蒸劑-溴化甲烷,在二十世紀被廣泛用於農地的殺菌與滅菌工作,效果雖佳,惟這種燻蒸劑卻有破壞臭氧層及造成環保污染之虞;同時也會導致土壤微生物相失衡,破壞土壤物理和化學結構;此外,若施用不當,尚會危害人體健康。致使許多國家已開始研擬禁用此種藥劑的計畫,尤其在歐美先進國家將於公元2005年全面禁用溴化甲烷。因此開發天然生物性燻蒸劑,藉以替代溴化甲烷,是當前嘗試努力的重要課題。 十字花科蔬菜植體或種子含有硫配糖體,經芥子酵素作用後,可釋放出異硫氰化物,具有類似溴化甲烷的殺菌作用,因此,本研究第一部分試驗是嘗試分析十字花科蔬菜種子粉中具有殺菌及抑菌作用的物質及研製天然異硫氰化物種子粉衣劑防治蔬菜立枯病的效果評估,第二部分試驗即以菜籽粕作為主體,再搭配具有分泌芥子酵素的拮抗微生物或轉殖芥子酵素基因的拮抗微生物,藉以研製天然異硫氰化物燻蒸粒劑,運用於防治作物土媒病原菌,建立安全蔬菜生產體系,不僅減緩溴化甲烷對環境的衝擊外,尚符合有機與永續農業經營的精神。 評估10種十字花科蔬菜種子粉水解後,釋放之揮發性物質對立枯絲核病菌(Rhizoctonia solani AG-4) 之抑制效果,結果發現包心芥菜種子粉釋放之揮發性物質的抑菌效果最佳。各種子粉釋放的主要揮發性物質是 allyl isothiocyanate (ITC),其中以包心芥菜種子粉的含量最高。在九種種子粉衣劑添加物的篩選試驗中,發現包心芥菜種子粉與 Biolan peat B3 (MBF) 調製成的混合物,粉衣於甘藍種子上,可以有效防治甘藍立枯絲核病的發生。在 MBF 混合物中,包心芥菜種子粉與Biolan peat B3兩者按2比3體積比混拌的組成,具有最佳防治甘藍立枯絲核病的效果外,亦不會影響甘藍種子的發芽百分率。在農委會種苗改良繁殖場自動化環控溫室進行大面積試驗,發現甘藍種子粉衣 MBF 混合物後,能有效降低立枯絲核病菌的為害及提高甘藍苗之鮮重與乾重。顯然應用MBF混合物處理甘藍種子,藉以提高種苗的生產,確具商品化潛力。 評估57微生物菌株分解 sinigrin 的能力,發現僅有26支菌株具有分解 sinigrin 的能力,其中以 Pseudomonas boreopolis 分解 sinigrin 的能力最強。菜籽粕經 P. boreopolis 分解後所釋放的氣體,主要是 allyl ITC 與 3-butenyl ITC 兩類。菜籽粕接種P. boreopolis 製成的 BPP 產品,除對Fusarium oxysporum f. sp. lactucum 之生長無抑制效果外,對 R. solani AG-4、 Sclerotium rolfsii、 Sclerotinia sclerotiorum、 Pythium aphanidermatum 及 Phytophthora capsici 等五種土媒植物病原菌,均具有顯著的抑菌功效,其抑菌率高達94.5%以上。應用 BPP 產品進行西瓜猝倒病的防治試驗,發現 BPP 產品不僅能有效降低 P. aphanidermatum 游走子與厚膜胞子的存活,且提高健康西瓜苗的數目。 自阿拉伯芥植株分離的芥子酵素 (myrosinase) 基因 TGG1 及 TGG2 的 cDNAs,分別構築於 Escherichia coli 和 Pichia pastoris 兩種微生物,發現兩基因可在 E. coli 和 P. pastoris 兩種微生物中表現酵素活性,且以在 P. pastoris 表現的酵素活性最佳。比較 TGG1 及 TGG2 兩基因在兩種微生物中所產生芥子酵素對硫配醣體 (glucosinolate) 的專一性,結果發現 TGG1 和 TGG2 芥子酵素對 sinigrin, gluconapin, glucobrassicanapin 及 glucoraphanin 均具有相當高的專一性。硫配醣體 sinigrin 經 TGG1 和 TGG2 芥子酵素分解後,釋放之 allyl ITC 揮發性物質對六種土媒植物病原菌之抑制評估,結果發現 R. solani AG-4、S. rolfsii 和 P. aphanidermatum 等菌的生長,明顯受抑制;顯然利用微生物系統表現的芥子酵素,再配合硫配醣體可有效防治土媒植物病原菌。 將菜籽粕、甘油、 P. boreopolis 、藻酸鈉及氯化鈣調製成 PBGG 粒劑產品後,以不同濃度 PBGG 粒劑施用至土壤中時,結果發現 1% (w/w) PBGG粒劑不但可顯著降低 R. solani AG-4 在土壤中存活時間,且可提高放線菌族群的增殖。比較 Streptomyces spp. 不同菌株間對 R. solani 的拮抗能力,發現 S. padanus 和 S. xantholiticus 兩菌株展現最佳的拮抗能力。當 1% (w/w) PBGG 添加至土壤中時,除可有效抑制 R. solani 的存活外,尚可大量促進 S. padanus 菌株的增殖。進一步,利用1% (w/w) PBGG 配合 0.5% (w/w) SS-07 添加物 (每克介質含有108 cfu S. padanus菌株) 在網室與田間大面積試驗,結果發現白菜立枯病發生率顯著的降低, 且不影響種子發芽時間,發芽率與植株鮮重。
Ten cultivars of three Brassica species were compared for volatile compounds in hydrated ground seeds that were fungicidal to Rhizoctonia solani AG-4. Among the samples tested, the volatile substances in the ground seed of mustard (Brassica juncea cv. Bau Sin) showed the strongest fungicidal effect. Allyl isothiocyanate was one of the predominant fungicidal compounds detected in the ground seed of all Brassica species tested and the amount was the highest in ground mustard seed meals. Among the nine carriers tested for seed coating with ground mustard seed meal, Biolan peat B3 was the most effective for the seed treatment. Cabbage seeds treated with a mixture of ground mustard seed meal and Biolan peat B3 (MBF mixture) at 2:3 ratio (w/w) resulted in a significant reduction in incidence of Rhizoctonia damping-off with no harmful effects to the germination of treated seeds. Effective control of Rhizoctonia daming-off of cabbage by the seed treatment with the MBF mixture was also obtained in a large-scale experiment conducted in a commercial automated environment-controlled greenhouse. This study suggests that the MBF mixture has potential for use in the commercial seed treatment. A total of 57 isolates of microorganisms (fungi, bacteria and actinomycetes) were screened for degradation of the glucosinolate sinigrin. Pseudomonas boreopolis was the most effective one among 26 isolates showing sinigrin degradation activities. Treatment of ground Brassica seed pomace (BSP) with P. boreopolis resulted in the release of volatile compounds, predominantly allyl isothiocyanate (ITC) and 3-butenyl ITC, which showed strong fungicidal effects on mycelial growth of Rhizoctonia solani AG-4, Sclerotium rolfsii, Sclerotinia sclerotiorum, Pythium aphanidermatum, and Phytophthora capsici. Results of gas chromatographic analyses showed that allyl ITC and 3-butenyl ITC were the major volatile compounds released from enzymatic hydrolyzsis of glucosinolates by P. boreopolis and the compounds were responsible for reducing the survival of zoospores and oospores of P. aphanidermatum. A large scale greenhouse trial showed that amendment of a growth medium (soil and BVB No.4 at 7:3 ratio, w/w) with BPP mixture 【1% (w/w) ground BSP and P. boreopolis (1-1.8 x 108 cfu/mL)】 was effective in controlling Pythium damping-off of watermelon, resulting in increased production of healthy, marketable seedlings. Myrosinases (thioglucoside glucohydrolase, EC 3.2.3.1) are able to hydrolyze glucosinolates in natural plant products. Three different gene families with different tissue-specific expressions and distribution patterns encode myrosinases. cDNAs of myrosinase genes (TGG1 and TGG2) were isolated from Arabidopsis thaliana and constructed in Escherichia coli and Pichia pastoris, respectively. The enzyme activities of myrosinase TGG1 and TGG2 genes expressed in P. pastoris were higher than those expressed in E. coli. Among six glucosinolates tested for specificity to myrosinases TGG1 and TGG2, the suitable substrates for these two genes expressed in P. pastoris and E. coli were sinigrin, gluconapin, glucobrassicanapin and glucoraphanin. Treatment of sinigrin and myrosinases excreted from reconstructed E. coli and P. pastoris with TGG1 and TGG2 genes resulted in the release of volatile compounds, predominantly allyl isothiocyanate (ITC), which showed strong fungicidal effects on mycelial growth of Rhizoctonia solani AG-4, Sclerotium rolfsii, and Pythium aphanidermatum. This study suggests that the combination of glucosinolate with myrosinases excreted from the reconstructed microbes may be a potential mean to control soilborne disease. Granular formulation of P. boreopolis combined with Brassica seed pomace and glycerin (PBGG) were prepared by using sodium alginate. Using a baiting technique, Rhizoctonia solani AG-4 was significantly suppressed to colonize the cabbage seeds buried in infested soil amended with various concentrations of PBGG. PBGG at a rate of 1% significantly reduced the percentage of seed colonization and enhanced population proliferations of actinomycetes in the soil. Among the predominant populations of Streptomyces spp., Streptomyces padanus and S. xantholiticus were most effective in inhibiting R. solani. Application of 1% (w/w) PBGG was able to significantly increase in population densities of S. padanus and S. xantholiticus. Treatment with either Streptomyces strain alone or Streptomyces strain combined with 1% PBGG resulted in significantly reducing the percentage of seed colonization by R. solani compared to controls. Streptomyces padanus was the most effective in reducing the percentage of seed colonization by the pathogen when the infested soil was amended with 1% PBGG. The disease incidence was significantly reduced, to 6.5-8.6% in the soil amended with S. padanus/PBGG mix, 27.8-31.7% in the soil amended with S. xantholiticus / PBGG-mix, and 36.9-38.6% in the soil amended with PBGG alone, compared to 61.8-64.8% in the control (unamended soil). Results of greenhouse and field tests showed that soil amended with either actinomyces/PBGG mix or PBGG had no negative effects on germination percentage of Chinese cabbage seeds. PBGG combined with S. padanus added to soil before sowing seeds showed a promise result in controlling Rhizoctonia damping-off and increased yield of Chinese cabbage.
URI: http://hdl.handle.net/11455/31751
Appears in Collections:植物病理學系

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