Application of population profiling and detection of Ralstonia solanacearum on integrated management of tomato bacterial wilt

由土壤傳播的植物病原細菌Ralstonia solanacearum所引起的青枯病是全球性的重要病害。在台灣已記載被本病為害的寄主計有28種植物和數種雜草，青枯病菌是一種在表現型和基因型上均有顯著差異的複合種類，在台灣已知的青枯病菌皆屬於第一生理小種，第三或第四生物型，但是在不同植物種類上的毒力卻有很大的差異，近來則在馬鈴薯上出現第三生理小種第二生物型的菌株。本研究目的在於（一）以新的演化型分類系統決定台灣青枯病菌菌株的演化型，並利用egl和mutS基因的部分序列分析台灣各地區不同來源菌株間的親緣關係，再探討生物型、毒力、菌株來源及地域來源與前述分類結果的相關性；（二）利用半選擇性培養基MSM-1和青枯病菌專一性的引子對AU759/760發展簡易的BIO-PCR方法，評估此方法對田間樣品的偵測效果；（三）發展初步篩選系統，以選擇適合當地應用於綜合防治番茄青枯病的抗病根砧及土壤添加物。首先，評估不同來源的157個台灣菌株在生物型、毒力及親源關係的差異性。除了分離自馬鈴薯的2個菌株屬於第二生物型外，其餘供試菌株皆為第三或第四生物型，其中第三生物型菌株為優勢族群。以演化型專一性的多引子PCR進行檢定，除了2個第二生物型的馬鈴薯菌株屬於第二演化型外，其餘供試菌株皆為第一演化型。從157個菌株中選出58個供試菌株，接種在三種具有不同抗病性的番茄品種上，可區分為7個毒力群。分析供試菌株的egl和mutS基因的部分序列皆發現第一演化型呈現分歧，這個發現首次證明在亞洲的第一演化型菌株可能存在著演化分歧，可區分為2個亞群。根據卡方檢定，發現egl和mutS基因的部分序列分析所獲得的2個亞群，其菌株分佈與生物型及菌株來源有關。至於毒力的表現型，僅與egl基因演化群的菌株分佈有關，與mutS基因演化群的菌株分佈無關。在病菌檢測方面，建立BIO-PCR方法偵測田間土壤、雜草及灌溉水中的青枯病菌，此方法是先以液態的MSM-1培養基在30oC培養田間樣品三天，再利用專一性引子對AU759/760直接與培養液進行PCR檢測。於台灣主要番茄產區的8個具有不同青枯病發病史及耕作制度的田區進行採樣，共收集320個土壤、91個雜草、85個灌溉水樣品進行病原菌偵測。不論是土壤、雜草或灌溉水樣品，以BIO-PCR方法所偵測到帶菌樣品的比例皆高於單獨使用選擇性培養基的稀釋平板法。結果並顯示若要追蹤田間青枯病菌，以雜草根圈土樣品可能是最理想的標的物。以BIO-PCR方法證明了病原菌在田間呈現不規則分佈。若每5.5 m2面積採集1個土壤次樣品，將36個土壤次樣品混合成1個土壤樣品，則每1,000 m2田區只需採集5個土壤樣品，即可得知青枯病菌的存在與否。在綜合防治策略上，發展初步篩選系統，以增加防治策略應用在各地區的成功機率。經初步篩選試驗，結果顯示尿素與石灰混合物對三種供試土壤具有最佳的抑菌效果。根據苗期對代表性菌株的抗病篩選結果，選出具穩定抗病性的茄子品種EG203及番茄品種Hawaii 7996做為根砧。田間試驗結果僅一個田區顯示綜合使用Hawaii 7996根砧和土壤添加物比單獨使用Hawaii 7996根砧具有顯著的防治效果；以EG203做為根砧時，土壤添加物則無法提供顯著的防治效果。了解台灣青枯病菌菌株的差異性、應用BIO-PCR偵測方法追蹤病原菌在田間的分佈及應用初步篩選系統擬定防治策略，將有助於番茄青枯病的永續管理。Bacterial wilt caused by the soil-borne plant pathogenic bacterium, Ralstonia solanacearum, is a globally important disease. The disease has been recorded on 28 plant species as well as several weed species in Taiwan. This pathogen is a species complex with significant phenotypic and genetic diversity. In Taiwan, all reported strains of R. solanacearum are race 1 and biovar 3 or 4, but they are highly variable in virulence on different plant species including tomatoes. Recently, race 3 biovar 2 strains were detected from infected potatoes. The purposes of this study were to (i) determine the phylotype of Taiwanese strains using the new phylotyping classification scheme, determine the phylogenic origins of strains collected from different locations and sources in Taiwan using endoglucanase (egl) and mutase (mutS) partial gene sequences, and examine associations of the classification results with biovar, virulence, strain origins, and geographical origins; (ii) develop a simple BIO-PCR protocol using the semi-selective medium, MSM-1, and species-specific primer pairs, AU759/760, and evaluate the effectiveness of this protocol with field samples; and (iii) develop a preliminary screen to select locally adapted resistant rootstock and soil amendment for integrated management of tomato bacterial wilt in the field. First, the diversity among 157 strains of R. solanacearum isolated from 22 host, 11 weed, 2 water, and 4 soil origins in Taiwan was evaluated on their biovar, virulence, and phylogenic origin. All strains tested were biovar 3 or 4 except for two strains isolated from potato were biovar 2. The biovar 3 strains are predominant. The phylotype-specific multiplex PCR identified all tested strains as phylotype I, except the two biovar 2 potato strains as phylotype II. The 58 out of 157 strains were selected and inoculated on three tomato varieties with different resistance to bacterial wilt. Seven groups with various profiles of virulence were detected. Phylogenetic analysis of partial egl and mutS gene sequences of the same 157 strains revealed a major branch within phylotype I. This is the first evidence to imply the possible existence of an evolution divergence within phylotype I strains in Asia and at least two subgroups could be distinguished. Based on the chisquare test, the distribution of the two subgroups of phylotype I defined by egl or mutS gene sequence over biovars or strain origins was not independent. Only distribution of strains of egl phylogenic groups, but not mutS, was associated with virulence phenotypes. For detection of R. solanacearum, a BIO-PCR method was established to detect R. solanacearum from field soil, weed, and water samples. The method involved incubating field samples in liquid MSM-1 medium at 30oC for 3 days and then detecting with species-specific primer pair AU759/760 without DNA purification. Samples of 320 field soil, 91 weed, and 85 water samples were collected from eight fields with different disease histories and cropping systems located in major tomato production areas. The frequency of positive detections by BIO-PCR was higher than that by plating on MSM-1 medium. Results indicated that weed rhizosphere soils could be good sampling targets to monitor the pathogen in the field. Use of the BIO-PCR method confirmed the uneven distribution of the pathogen in the field. Five composite samples per 1,000-m2 field each consisting of 36 sub-samples at a sampling density of one sub-sample per 5.5 m2 were sufficient to determine the presence of R. solanacearum in the field. For integrated control of bacterial wilt, a preliminary screen was developed to increase the chances of identifying successful control measures over locations. Pre-incubation tests indicated that the mixture of urea and slaked lime was most suppressive to R. solanacearum in three tested soils, and was used in subsequent field experiments. Resistant eggplant (EG203) and tomato (Hawaii 7996) rootstocks were selected based on stable resistance against representative strains of R. solanacearum at the seedling stage. Integrated use of Hawaii 7996 as the rootstock and mixture of urea and slaked lime as the soil amendment provided significantly greater control of wilt than use of Hawaii 7996 as rootstock alone in only one of the four locations, whereas the soil amendment did not provide significant control effect when EG203 was used as the rootstock. Understanding strain variations of R. solanacearum in Taiwan, use of the BIO-PCR detection method to monitor pathogen distribution, and application of the preliminary screen scheme to select disease control strategy can contribute greatly to sustainable management of tomato bacterial wilt.中文摘要..................................................i SUMMARY ................................................iii ABBREVIATIONS ............................................v CONTENTS ..............................................viii CHAPTER I Introduction..............................................1 1.1. Importance of tomato.............................1 1.2. Bacterial wilt caused by R. solanacearum.........1 1.3. Infraspecific classification of R. solanacearum..2 1.4. Detection of R. solanacearum.....................3 1.5. Control of bacterial wilt........................5 1.6. Background and goal of this study................5 1.7. Literature cited.................................6 CHAPTER II Genetic Diversity and Virulence of Ralstonia solanacearum Strains Isolated from Different Hosts in Taiwan..........11 2.1. ABSTRACT........................................11 2.2. INTRODUCTION....................................11 2.3. MATERIALS AND METHODS...........................14 2.3.1. Bacterial strains and DNA extraction............14 2.3.2. Phylotype determination.........................14 2.3.3. Virulence analysis..............................15 2.3.4. Biovar determination............................15 2.3.5. PCR amplification and DNA sequencing............16 2.3.6. Sequence analysis and association test..........16 2.4. RESULTS.........................................17 2.4.1. Biovar and phylotype determination..............17 2.4.2. Virulence analysis..............................17 2.4.3. Phylogenetic characterization...................18 2.4.4. Associations between phylogenic origins and biovar,virulence,strain origins and geographical origins.........................................19 2.5. DISCUSSION......................................19 2.6. LITERATURE CITED................................22 2.7. TABLES..........................................27 2.8. FIGURES.........................................38 CHAPTER III Detection of Race 1 Strains of Ralstonia solanacearum from Field Samples Using a BIO-PCR Method.....................44 3.1. ABSTRACT........................................44 3.2. INTRODUCTION....................................44 3.3. MATERIALS AND METHODS...........................46 3.3.1. Bacterial culture, bacterial suspension, and soil infestation.....................................46 3.3.2. Bacterial enrichment and PCR....................46 3.3.3. Detection threshold and enrichment period.......47 3.3.4. Field sampling and sample preparation...........47 3.3.5. Determination of sample number for field soils..48 3.4. RESULTS.........................................49 3.4.1. Multiplication of R. solanacearum in MSM-1 broth49 3.4.2. Sensitivity of BIO-PCR method...................49 3.4.3. Detection of R. solanacearum from field soils...49 3.4.4. Detection of R. solanacearum from weed samples..50 3.4.5. Detection of R. solanacearum from water samples.50 3.4.6. Sample number of field soil detection...........51 3.4.7. Distribution of R. solanacearum in the field....51 3.5. DISCUSSION......................................52 3.6. LITERATURE CITED................................55 3.7. TABLES..........................................59 3.8. FIGURES.........................................64 CHAPTER IV Application of a Preliminary Screen to Select Locally Adapted Resistant Rootstock and Soil Amendment for Integrated Management of Tomato Bacterial Wilt in Taiwan.67 4.1. ABSTRACT........................................67 4.2. INTRODUCTION....................................67 4.3. MATERIALS AND METHODS...........................69 4.3.1. Bacterial strains and inoculum preparation......69 4.3.2. Plant materials.................................69 4.3.3. Determination of virulence of local strains.....70 4.3.4. Resistance of tomato and eggplant rootstocks against local strains...........................70 4.3.5. Evaluation of soil amendment....................71 4.3.6. Field experiments...............................72 4.4. RESULTS.........................................73 4.4.1. Virulence of local strains......................73 4.4.2. Reactions of rootstock cultivars and lines against local strains...........................73 4.4.3. Efficacy of soil amendment to suppress pathogen in local soils..................................74 4.4.4. Field experiments...............................75 4.5. DISCUSSION......................................76 4.6. LITERATURE CITED................................79 4.7. TABLES..........................................84 4.8. FIGURES.........................................90 CHAPTER V Conclusions..............................................91 APPENDIX Lin, C.-H., Hsu, S.-T., Tzeng, K.-C., and Wang, J.-F. 2008. Application of a preliminary screen to select locally adapted resistant rootstock and soil amendment for integrated management of tomato bacterial wilt in Taiwan. Plant Disease 92:909-916.................................9