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標題: 利用拮抗細菌防治番茄萎凋病
Biological control of Fusarium wilt of tomato by antagonistic bacteria
作者: 賴彥良
Lai, Yahn-Liang
關鍵字: Fusarium wilt of tomato
Biological control
出版社: 植物病理學系
摘要: 本研究目的在探討拮抗菌以胚軸切斷接種法輸入番茄植株體內後,拮抗菌 在植物體內的分布情形以及拮抗菌對番茄萎凋病的防治功效。 自竹仔湖 、信義及官田等地分離到 9 株番茄萎凋病菌分離株,比較各分離株之致 病力強弱後,挑選 Fol-04 分離株做為本研究之病原菌供試菌株。接種源 濃度與發病的關係的試驗中顯示,以濃度 2 × 106 spores/ml 的孢子懸 浮液依剪根接種法接種 28 天後,發病指數可達 8.63, 依土壤混菌接種 法接種時,當土壤中病原菌密度達 3 × 105 propagules /g soil 時發 病度可達 0.62,因此以這兩種濃度分別做為二種病原菌接種法時的接種 源濃度。本研究共自 19 種作物的 36 個根圈土樣中,篩選得到 130 株 拮抗細菌,分別與 Fol-04 菌株在 PDA 平板上對峙培養,有74 株的拮抗 距離在 0.1-0.5 公分之間,45 株拮抗距離在 0.5-1 公分之間,有 11 株拮抗距離在 1 公分以上。再挑選拮抗距離大於 0.7 公分的 32 株拮抗 菌,以胚軸切斷接種法分別輸入番茄植株體內, 並以剪根接種法接種番 茄萎凋病菌,結果有 6 株拮抗菌可使植株存活率提高二倍以上, 挑選其 中編號 008、082 及 085 菌株做為生物防治試驗的供試拮抗菌。 以 Biolog 測定法鑑定此三株拮抗菌, 顯示 008 及 082 菌株為 Bacillusspp. 而 085 菌株為 Pseudomonas sp.。拮抗菌在輸入番茄植株 體內 28 天後,各拮抗菌皆以下胚軸基部的族群量最高,其次是不定根再 其次為下胚軸上半部,而以上胚軸處的族群量最少,同時拮抗菌向上傳佈 的能力,以 082 菌株向上傳佈能力最強,而 085 菌株最弱。觀察植株根 部拮抗菌族群之動態發現,各拮抗菌族群數目皆隨植株培育時間的增加而 遞減, 拮抗菌輸入植株體內 14 週後, 根部拮抗菌族群以 085 菌株最 高, 達 2.18 ×105 cfu/g wet root,其次者為 082 菌株, 達 1.89 × 104 cfu/g wet root。 以 008菌株的族群量最低,僅 4.1 × 103 cfu /g wet root。 以電子顯微鏡觀察也證實,拮抗菌輸入 12 小時後, 在下胚軸維管束組織中可見到菌體的存在, 28 天後,可在皮層組織觀察 到細菌的存在。此三株拮抗菌對番茄產量均無影響。以剪根接種法接種不 同濃度的番茄萎凋病菌孢子懸浮液, 結果顯示 008 及 082 菌株均會有 效抑制病勢的發展, 而 085菌株在兩種接種濃度下,均無明顯防治效果 。將輸入拮抗菌的番茄植株植入番茄萎凋病帶病菌土中,結果顯示,各拮 抗菌均呈現明顯防治效果。
The purpose of this study is to detect the distribution of antagonists withinthe plant and to evaluate the effect of antagonists on control of Fusariumwilt of tomato after introducing antagonists into plant by thehypocotyl- cutting method. Nine isolates of Fusarium oxysporum f.sp.lycopersici (Fol) were collected from Chu Tze Hu, Hsinyi, and Kwantien inTaiwan. Among them, the isolate Fol-04 showed the highest virulence on tomatovareity Know-You 301 in pathogenicity test was used in this study. Based onthe results of evaluating the correlation between the inoculum density ofFol-04 and its disease index with root-prunning and soil infestation method,106 spores / ml and 105 propagules / g soil were selected as inoculumdensities for this study. According to analysis of the antagonistic ability of130 strains against Fol-04 isolates with dural-culture on PDA medium, 74strains expressed the inhibition distance ranging from 0.1~0.5 cm, 45 strainsfrom 0.5 to 1 cm and 11 strains greater than 1 cm. Thirty two strains showingthe inhibition distance greater than 0.7 cm were selected and introduced intotomato plants by the hypocotyl-cutting method to test for the ability ofcontrolling Fusarium wilt of tomato in greenhouse. The results revealed that 6out of 32 strains allowed the survival percentages of plants twice more thannon-treated control. Antagonistic bacterial strains 008, 082, and 085 whichshowed strong ability in disease- controlling were used for furthercharacterization, including their colonization within tomato plants andcontrolling ability. Strains 008 and 082 were Bacillus spp. and 085 strain wasPseudomonas sp. based on the Biolog identification system. The result ofevaluating antagonist populations within the tomato plants showed that thehighest population of these strains were detected in the lower half ofhypocotyl, and consequencely declined in the adventitious roots, the upperhalf of hypocotyl, and epicotyl. It was also found that strain 082 was thebest to spread upward from introducing site, followed by 008 and 085. Thepopulation dynamic of antagonists colonized on roots declined with the timeduring the period of plant growth. Fourteen weeks after introduced antagonistsinto plants, population of 008, 082,and 085 strain on roots remained to be4.1x103 cfu/g wet root, 1.89x104 cfu/g wet root, and 2.18x105 cfu/ g wet root,respectively. Based on the observations under T.E.M., these antagonistslocated in vascular tissues of hypocotyl 12 hours after introduction, and inthe cortex cells 28 days afer introduction. The yield of tomato was notinfluenced after these antagonists were introduced into the plant. Strain 008and 082 reduced significantly tomato Fusarium wilt after inoculation byroot-prunning method, while 085 showed no effectiveness. All theseantagonistic strains significantly reduced the tomato Fusarium wilt afterplants were transplanted in the infested soil.
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