Potato common scab caused by Streptomyces scabies in Taiwan - biological characteristics of the pathogen and an attempted biocontrol by antagonistic Bacillus subtilis var. amyloliquefaciens WG6-14

Abstract

民國 95 年冬季在中台灣的台中縣潭子鄉與雲林縣斗南鎮地區所收成之馬鈴薯突見瘡痂病之大發生，由於類似的大規模發生前所未見，且在過去兩年間發病率與嚴重程度均有增加情形，一時成為倍受栽培業者關注之課題。本研究旨在探討最近在中南部地區普遍發生之馬鈴薯瘡痂病病原菌之生物特性，及其可能解決方法。本研究計利用由罹病薯塊樣品所分離到 7 個具典型鏈黴菌菌落形態之分離菌株做為供試病原菌，其中 T1、T2、T3、T4、T5 與 T6 等為本研究由採集自潭子地區罹病薯塊分離獲得，D1 菌株則由行政院農業委員會台南區農業改良場 鄭安秀博士提供，分離自斗南地區所採集樣本。7 個供試病原菌株經於 ISP4 (International Streptomyces Project ) 培養基上培養，所長成菌落俱呈灰色，顯微鏡檢視下可見，其均可產生氣生菌絲並於其上著生螺旋狀之孢子鏈；且均可於 ISP 單一碳素源供給下生長良好；另由tyrosine agar 上黑色素 (melanin) 產生特性及膜脂肪酸含量檢測等，所獲結果顯示此 7 個供試菌株之生理、生物特性均與Streptomyces scabies 相當一致。繼而由選殖、解序各供試菌株之 16S rDNA 序列結果，經GenBank分析比對，進而證實其與同屬於 S. scabies 之已知 4 個菌株相同度 (identity) 高達99％，綜合上述型態特徵、生理與生化特性及16S rDNA 序列分析檢測結果，證實本研究中所應用 7 個供試病原菌株均歸屬於 Streptomyces scabies。由於鏈黴菌屬 (Streptomyces) 成員為土壤中腐生性、並被廣泛應用於對抗土傳性病原真菌的有益微生物，本研究進而比較此些病原菌株與本實驗室經一系列實驗證實對植物性病害具有防治效果的鏈黴菌 S1、S3、S4、B4 與 M6 等 5 個菌株間之差異性，次釐清供生物殺菌劑發展應用鏈黴菌株於作物栽培上廣為應用後可能存在之潛在風險。上述 7 個病原菌株於人工接種下，均可造成馬鈴薯薄片組織之褐化與蘿蔔新生幼苗生長之嚴重受抑制反應，另以 1×108 cfu/ml 濃度孢子懸浮液澆灌盆缽種植馬鈴薯則 7 個菌株皆可在所結成薯塊上造成表皮破裂、龜裂狀、隆起之典型瘡痂病病徵；而相對的 5 個供生物殺菌劑發展應用之鏈黴菌株，於馬鈴薯組織上與薯塊上均未見有典型瘡痂病徵表現。有鑑於 thaxtomin A 產生為本病病原菌感染攸關重要因子，將所自 12 個供試鏈黴菌株經培養，並萃取其毒質成分，所含 thaxtomin A 成分除經內加標品高效液相層析 (HPLC) cochromagraphy 證實存在，並利用串聯式液相層析質譜儀 (LC/MS/MS) 以 MRM 監測模式系統，由 m/z＝437 母離子及m/z＝155、140 與 107 等 3 個子離子斷片之檢出進一步加以證實。綜合檢出結果，已證實 7 個可導致典型嚴重瘡痂病徵的菌株其培養液中 thaxtomin A 產量均可達 mg/ml 以上濃度，相對的 5 個供生物製劑發展應用之鏈黴菌株則培養液中均未檢測到有此毒質之產生。另值得一提的是，7 個供試病原菌株中以 T3 菌株產率最高，其次為 T5、T6、D1、T4、T1，而以 T2 菌株為最低者。，經檢視此 7 個供試菌株於蘿蔔幼苗系統所呈現發病狀況可以明白看出各供試菌株之 thaxtomin A 毒質產生能力與其致病毒力表現有密切關係；另將 7 個供試菌株之基因體 DNA 作為模版，以 txtA 基因之專一性引子對添加後，進而證實均可如預期增幅出大小約為 398bp 之片段，經選殖、解序比對後，發現其與已知 S. acidiscabies 之 txtA 基因相同度 (identity) 高達 99.7％，繼之利用所選殖 txtA 片段所製備核酸探針進行南方雜合反應，証實於上述 5 個供生物性殺菌劑發展應用之鏈黴菌菌株之基因體中均未測到 txtA 基因之存在，此亦顯示其余成為瘡痂病病原菌之風險性應極低。為瞭解本病生物防治應用之可行性，本研究以對峙培養檢測 7 株已知對多數重要病原真菌與細菌具優異抗生活性且產孢性狀良好之枯草桿菌群 (Bacillus subtilis group; BSG) 菌株對 S. scabies T1 菌株之拮抗性，經實驗結果證實以 Bacillus subtilis var. amyloliquefaciens WG6-14 拮抗性最為優異，其次為 TKS1-1、SP4-17與WP8-12。在溫室試驗中，分別以 D1 與 T1 兩供試病原菌株之孢子懸浮液 (菌量約為 1×108 cfu/ml) 澆灌行人工接種，處理六週後採收所產生薯塊，其發病率 (infection rate) 與罹病度 (disease severity) 分別 71％ 與 66.7％ 及 43.8％ 與 44.5％ 左右，相對的接種同時以 WG6-14 澆灌處理組則可使D1 與 T1 接種病害發病率降到只有 35％ 與 28％，罹病度降到只有 11.7％ 與 10.5％，另外接種後一個禮拜以 WG6-14 澆灌處理組則 D1 與 T1 接種下發病率分別降到只有 34％ 與 45％、罹病度分別降到只有 11.2％ 與 16.7％，除了防治效果卓著，另值得注意的是，WG6-14 之澆灌處理對馬鈴薯根系與薯塊生長發育均有顯著促進效果，與未處理之對照組比較，D1 與 T1 菌株所接種植株經 WG6-14 處理後，其植株結薯總數分別多 1.4-2.0 倍與 1.7-2.8 倍。此一結果顯示，WG6-14 之施用可以兼具促進植株生長與保護薯塊使不被病原菌感染之双重效果，其確為馬鈴薯栽培與瘡痂病病害管理上極值得被推薦應用之生物製劑。An outbreak of tuber infection showing symptoms closely resembling that of common scab disease was observed during the winter of 2006 on field harvested potatoes in central Taiwan at both Tantzu Taichung and Dounan Yunlin. The disease appeared to be new for potato cultivation in Taiwan, and the increasing incidence and severity of the disease have drawn great attention among growers during the past 2 years. The main objectives of this investigation were to explore the biological and pathological characteristics of the causal agent and the possible resolution to solve the problem. A total of 7 Streptomyces strains isolated from diseased tubers were used for the performed tests. Among them, T1, T2, T3, T4, T5 and T6 were from Tantzu; and D1 (kindly given by Dr. Ann-show Cheng of Tainan DAIS) was from Dounan. Comparative study by use of ISP4 (International Streptomyces Project) medium on the morphological and physiological characteristics of these 7 tested isolates indicated that all of them grew up into grayish colony typical of Streptomyces morphology and produced spores in spiral chains on the aerial mycelia. They all appeared to grow well with the provision of each individual ISP sugar as sole carbon source, produced melanin pigment on tyrosine agar, and consisted in their membrane lipid fatty acid profile similar to that known for Streptomyces scabies. The 16S rDNA of these tested isolates were cloned and sequenced, pair-wise comparison of the sequences with the data provided by GenBank indicated all of them shared a 99% identity with that known for Streptomyces scabies. A phylogenetic tree constructed based on 16S rDNA sequences known for Streptomyces further concluded the 7 tested isolates in the same group as that of S. scabies. The morphological, physiological and molecular characteristics observed indicated that all the 7 tested isolates belong to members of S. scabies. As the majority of Streptomyces spp. are known to be saprophytic and some are important beneficial soil microbial resources which have been widely used as biological control agents, 5 Streptomyces strains S1, S3, S4, B4 and M6 known with potential of biofungicide application were included as compared strains for the pathogenicity study to learn if they may pose potential threat on crop production. Upon artificial inoculation, all the 7 diseased potato tuber derived strains were shown to cause necrotic response on potato tuber slices, reduced stem height and leaf thickening on radish seedlings. An artificial inoculation by drenching application of 1x108 cfu/ml spore suspension on pot grown potato plants (cv. Kennebec) confirmed that all 7 tested isolates were pathogenic and led to development of typical necrotic common scab symptoms. As a comparison, none of the 5 strains with potential of biofungicide application caused observable symptoms on potato tuber slices and pot grown potato plant; although slight inhibition of radish seedling growth was detected. Thaxtomin A is known to be a major determinant of Streptomyces spp. that causing the development of common scab symptoms on potato. The production of thaxtomin A of the tested strains was examined by thin layer chromatography, high performance liquid chromatography (HPLC), and a triple-quad mass detector equipped HPLC system (LC/MS/MS). The identity of thaxtomin A detected was demonstrated by use of internal standard co-chromatography in HPLC, and by LC/MS/MS at multiple reaction monitoring mode the detection of precursor ion at m/z=437 and the major product ions at m/z=150, 140 and 107, respectively. All the 7 diseased tuber-isolated strains were shown to produce中文摘要--------------------------------------------------I 英文摘要--------------------------------------------------IV 目錄----------------------------------------------------VIII 壹、前 言--------------------------------------------------1 貳、前人研究-----------------------------------------------4 一、馬鈴薯之經濟重要性-----------------------------------4 二、馬鈴薯瘡痂病之發生與危害概況-------------------------5 三、植物毒質thaxtomin 研究概況---------------------------7 四、馬鈴薯瘡痂病之防治策略-------------------------------9 五、生物防治 (Biological control)-----------------------10 六、枯草桿菌群 (Bacillus subtilis group) 特性及應用概況-10 七、參考文獻--------------------------------------------13 參、材料與方法--------------------------------------------21 一、供試藥品來源----------------------------------------21 二、供試培養基------------------------------------------21 (一) 幾丁質培養基 (chitin medium)---------------------21 (二) 馬鈴薯蔗糖瓊脂培養基 (potato sucrose agar, PSA)--22 (三) International Streptomyces Project (ISP4) 培養基-22 (四) 燕麥粉頭培養基 (oatmeal medium)------------------22 三、供試菌株來源與培養----------------------------------23 (一) 供試馬鈴薯瘡痂病菌菌株之來源、保存---------------23 (二) 供生物殺菌劑發展應用拮抗性鏈黴菌菌株-------------23 四、接種源之製備----------------------------------------23 五、供試菌形態之光學顯微鏡與掃瞄式電子顯微鏡檢視--------24 六、供試菌株生理生化特性測定----------------------------24 (一) 碳素源利用性-------------------------------------24 (二) 脂肪酸分析 (Fatty acid analysis)-----------------25 七、供試病原菌株 16S rDNA 及 txtA 基因序列之聚合酶連鎖反應增幅、解序---------------26 八、txtA基因片段選殖、質體DNA抽取及南方雜合檢測---------27 (一) txtA基因片段選殖與質體DNA抽取--------------------27 (二) 南方雜合檢測2-1. txt A探針(probe)之製備----------------------28 2-2. 核酸之轉漬-----------------------------------28 2-3. 雜合反應-------------------------------------29 2-4. 洗片與壓片-----------------------------------29 九、所分離病原菌株病原性之生物測定 (Bioassay)-----------30 (一) 蘿蔔幼苗系統-------------------------------------30 (二) 馬鈴薯薄片圓盤法生物測定 (tuber slice assay)-----30 (三) 馬鈴薯植株接種試驗-------------------------------31 十、thaxtomin A 萃取、純化與分析------------------------31 (一) thaxtomin A 萃取流程-----------------------------31 (二) 薄層層析 (TLC；thin-layer chromatography) 分離純-------32 (三) 高效液相層析 (High-Performance Liquid Chromatography, HPLC)---32 (四) 串聯式液相層析質譜儀 ( Liquid Chromatograph/Mass Spectrometer/ Mass Spectrometer)------------------32 十一、供試枯草桿菌群菌株之來源及拮抗性篩選測試----------33 十二、拮抗性枯草桿菌發酵液澆灌處理對瘡痂病菌感染之影響--34 肆、結果-------------------------------------------------35 一、供試病原菌株菌落形態與生理、生化特性----------------35 二、供試病原菌菌株 16S rDNA 序列增幅與同源性分析--------36 三、thaxtomin A synthetase (txtA) 基因片段之偵測--------37 四、供試菌株之病原性測試--------------------------------37 五、thaxtomin A 萃取、純化與分析------------------------39 六、馬鈴薯瘡痂病菌拮抗性枯草桿菌群菌株篩選--------------40 七、Bacillus subtilis var. amyloliquefaciens WG6-14 菌株於 馬鈴薯瘡痂病防治之應用性評估------------------------40 伍、討論-------------------------------------------------43 陸、參考文獻-------------------------------------------50 柒、圖表說明----------------------------------------------56 捌、附錄--------------------------------------------------7