Factors affecting occurrence of oilseed rape clubroot caused by Plasmodiophora brassicae

油菜係世界上重要的四大油料作物之一，近年來，根瘤病在全球各油菜產區陸續有油菜根部遭受根瘤病菌 (Plasmodiophora brassicae Wornin)為害的報導，且造成頗重大的經濟損失。西元 2013 年 11 月在台灣台南市將軍區亦於採種田發現油菜發生根瘤病的情形。本文旨在於探討寄主植物品種與品系、土壤性質、土壤添加物及微生物對於油菜根瘤病發生的影響，祈有助於油菜根瘤病的防治工作。 本研究係以根毛感染率作為調查與評估病原菌為害油菜的指標，發現種子直播於病菌土中，第 5 天為調查根毛受感染率的基準時間點。試驗統計分析接種濃度、根毛感染率及植株罹病度間的相關性後，發現接種濃度與感染率 (Y = 1.6109－1.0222X + 1.1929X2, r2 = 0.9892)，接種濃度與罹病度 (Y=－0.8754 +23.3430X－1.2666X2, r2 = 0.9612)間皆具有高度的正相關性；至於感染率與罹病度 (Y =4.2267 + 3.4614X－0.0287X2, r2 = 0.9818) 間亦具有頗高的正相關。利用十種不同油菜品種與品系分別播種於 P. brassicae 的病菌土 (107 spores/g soil)中 結果發現，F-99-268 品系的罹病度約 52%，具有耐病性，其次為 F-97-210 品系，其餘秀珍、阿茵、阿麗、油菜心、農友晚生油菜心、F-97-194、F-99-250 及 F-100-324 等均呈現極感病。將南投縣仁愛鄉翠巒(A)、華岡(B)及翠巒(C)等三區罹病田所採得的田土與台中市霧峰區興大農場的 D 田土，分別加入病原菌後，隨即直播油菜 (cv.油菜心)種子後，發現在 A、B、C 及 D 土壤中種植之植株的罹病度依序為 0%、11%、44%及 100%，顯示 A、B 及 C 三種田土均具有抑病功效。進一步，分析各田土的理化性質，發現 A、B 及 C 抑病土 (Suppressive soil)的 pH 值、有效磷、交換性鈣及交換性鉀的濃度均顯著高於 D 導病土 (Conducive soil)。將 0.25%(w/w)蓖麻粕、菜仔粕、魚鱗粉、蝦蟹殼粉、羽毛粉及幾丁聚醣等分別添加於病菌土中，7 天後播種油菜種子，於 24 ℃下 5 天後，各處理之植株根部遭受感染 的百分率皆顯著低於對照組，其中以處理過蝦蟹殼粉之植株，其受感染率較對照組降低 34%最為顯著 此外 將油菜種子粉衣處理 10 種不同拮抗微生物菌株後，播植於病菌土中，隨後每星期澆灌一次各供試拮抗菌的 100 倍稀釋菌液，發現Bacillus mycoides Wt16 菌株可使罹病度顯著下降約 44.4%。進一步，以種子處理方式評估非生物因子與生物因子對油菜根毛受根瘤病菌感染的影響，採用 100mg/L 的硫酸銅、硫酸鋅、二氧化氯、矽酸鈉、氯化鈣及磷酸氫二鉀等無機物及B. mycoides BM02、Wt16 及 RA08 菌株 (108 cfu/ ml)分別處理催過芽的油菜種子 30 及 60 分鐘，結果發現二氧化氯與磷酸氫二鉀分別可使根毛感染率較對照組顯著降低 11%及 15%；至於處理過 BM02、Wt16 及 RA08 菌株分別使根毛感染率顯著降低 19%、23%及 29%。此外，油菜種子分別處理硫酸銨、幾丁聚醣和磷酸氫二鉀以及 B. mycoides Wt16 懸浮液 3 小時亦皆可使根毛感染率較對照組顯著降低，其中以磷酸氫二鉀之處理組可降低 28.3%最為顯著，然拮抗菌 Wt16 與各化學品間並不具協力加成作用。至於土壤添加物搭配種子處理拮抗菌 B. mycoides Wt16 之試驗研究方面，發現蝦蟹殼粉與幾丁聚醣搭配生石灰處理病菌土後皆可使油菜的罹病度顯著降低 44.4% - 55.6%；其中 F-99-268 耐病品系以幾丁聚醣搭配生石灰添加的處理與對照組差異最顯著，其植株罹病度約可減少 55.6%，惟種子搭配處理 Wt16 菌株後，其罹病度卻僅減少 44.4%；至於油菜心感病品種以蝦蟹殼粉與幾丁聚醣搭配生石灰處理後其罹病度則可減少 41.7%與 44.4%，種子再以 Wt16 菌株處理後，其罹病度則可減少 55.6%。Clubroot (Plasmodiophora brassicae Wornin) is one of the most economically important diseases of cruciferous crops. Outbreaks of clubroot in recent years have been reported to be of particular concern in the oilseed rape-growing districts around the world. In Taiwan, the clubroot disease of oilseed rape was found at Jiangjun district, Tainan city in November, 2013. The purpose of this study was to evaluate the effects of host cultivars, soil properties, soil amendments, and microbes on oilseed rape clubroot disease. The establishment of inoculation platform based on root hair infection by the pathogen was used for evaluating the disease development of oilseed rape. The optimal time point for infection analysis of oilseed rape was at the 5th day after inoculation. Multiple regression equations indicated that the relationships among inoculum density, root hair infection percentage, and disease severity were significantly positive correlation. Ten cultivars/ lines of oilseed rape were chosen to analyze their susceptibility to P. brassicae. Among ten cultivars/ lines of oilseed rape tested, F-99-268 was the most tolerant line, followed by F-97-210, and all others were susceptible cultivars. Soils A, B, and C, respectively, obtained from three infested fields of Renai were significantly effective in suppressing clubroot disease of oilseed rape; those soils had higher pH value, organic matter, available phosphate , and exchangeable calcium and potassium ions as compared to conducive soil D obtained from non-infested field of Wufeng. Moreover, 0.25% (w/w) shrimp and crab shell powder mixed with infested soil was effective in reducing 34% of root infection of oilseed rape (cv. Yu-Tsai-Sum). Bacillus mycoides Wt16 could significantly reduce 44.4% of disease severity by seed coating method and drenching the bacterial suspension (100X) once per week for three times. Furthermore,germinated seeds of oilseed rape treated respectively with K2HPO4, antagonistic bacterium isolate RA08, and B. mycoides Wt16 were able to reduce 15%, 21%, and 26% of root infection significantly. Combination of chemicals and B. mycoides Wt16 had no synergistic effects on reducing root infection percentage. Amendments of infested soil with shrimp and crab shell powder and chitosan combined respectively with CaO were effective in reducing disease severity of oilseed rape ranged from 44.4% - 55.6%.中文摘要................................................................................................. i 英文摘要................................................................................................ iii 目次.................................................................................................... v 表次索引.............................................................................................. viii 圖次索引................................................................................................ ix 前言............................................................................................... 1 材料與方法.................................................................... 5 一、 供試植株與栽培基質 .................................................................. 5 二、 供試病原菌來源與保存 ....................................................................... 5 三、 接種源之製備及接種方法 ....................................................................... 5 四、 調查油菜根瘤病之評估方法 .......................................................................... 5 (一)根毛感染率之調查法................................................................... 5 (二)植株罹病度之調查法................................................................................ 6 (三)觀察根毛感染率之時間點分析................................................................ 6 (四)接種濃度對根毛感染率的影響................................................................ 6 (五)接種濃度對植株罹病度的影響................................................................ 7 (六)根毛感染率與植株罹病度間的相關性.................................................... 7 五、 不同油菜品種與品系對根瘤病菌之抗感性分析 .......................................... 7 六、 不同土壤特性對油菜根瘤病發生的影響 ...................................................... 7 (一)供試土樣來源與理化性質分析................................................................ 7 (二)不同土壤對油菜根瘤病發生的影響..................................................... 8 七、 土壤添加物對根瘤病菌感染油菜的影響 ...................................................... 8 八、 不同拮抗微生物對油菜根瘤病發生的影響 .................................................. 9 (一)供試拮抗菌株來源與保存........................................................................ 9 (二)不同拮抗菌株對油菜根瘤病發生的影響................................................ 9 九、 不同種子處理對於根瘤病菌感染油菜的影響 ............................................ 10 (一)化學品對根瘤病菌感染油菜的影響...................................................... 10 (二)拮抗微生物對根瘤病菌感染油菜的影響.............................................. 10 (三)拮抗微生物與化學品對根瘤病菌感染油菜的影響.............................. 10 十、土壤添加物與種子處理拮抗微生物對油菜根瘤病發生之影響 .................. 11 結果................................................................................................... 12 一、 調查油菜根瘤病之評估方法 ........................................................................ 12 (一)觀察根毛感染率之時間點分析.............................................................. 12 (二)接種濃度對根毛感染率的影響.............................................................. 12 (三)接種濃度對植株罹病度的影響.............................................................. 12 (四)根毛感染率與植株罹病度間的相關性.................................................. 13 二、 不同油菜品種與品系對根瘤病菌之抗感性分析 ........................................ 13 三、 不同土壤特性對油菜根瘤病發生的影響 .................................................... 13 四、 土壤添加物對根瘤病菌感染油菜的影響 .................................................... 14 五、 不同拮抗微生物對油菜根瘤病發生的影響 ................................................ 14 六、 不同種子處理對於根瘤病菌感染油菜的影響 ............................................ 14 (一)化學品對根瘤病菌感染油菜的影響...................................................... 14 (二)拮抗微生物對根瘤病菌感染油菜的影響.............................................. 14 (三)拮抗微生物與化學品對根瘤病菌感染油菜的影響.............................. 15 十、土壤添加物與種子處理拮抗微生物對油菜根瘤病發生之影響 .................. 15 討論.................................................................................................... 17 參考文獻.............................................................................................. 22 附錄........................................................................................................ 5