Diversity of Ralstonia solanacearum strains from tomato in the south of Jiangxi Province
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摘要:目的
分离鉴定赣南地区番茄青枯病菌，明确菌系分化，为当地番茄抗青枯病育种和病害防治奠定基础。
方法从江西省赣南地区采集番茄青枯病病株，经选择性平板分离、纯化和分子鉴定，获得不同地理来源的青枯菌Ralstonia solanacearum菌株。通过生理生化测定和接种番茄试验，鉴定青枯菌的生化变种和致病类型。PCR扩增内切葡聚糖酶基因egl序列，明确青枯菌的演化型和序列变种。双层平板培养法测定其对8个不同噬菌体的敏感性。
结果获得了来自赣南地区9个市(县)的番茄青枯菌菌株44个，其中，41个菌株为生化变种Ⅲ，3个菌株为生化变种Ⅳ；致病力测定结果聚为I、II和III类，其致病力分别为强、中和弱，其中，强致病力菌株占65.9%。所有菌株属于亚洲分支演化型(Ⅰ)，并进一步划分为Sequevar13、14、15、17、18、34、44和48等8个序列变种。大部分菌株对供试的8个噬菌体敏感。
结论赣南地区番茄青枯菌以生化变种III和强致病力菌株为主，对噬菌体较敏感，存在8个序列变种，具有明显的菌系分化现象和遗传多样性。
Abstract:ObjectiveIsolating and identifying Ralstonia solanacearum strains from tomato plants in the Southern of Jiangxi Province, and clarifying the bacterial differentiation can lay the foundation for local tomato bacterial wilt resistance breeding and disease control.
MethodThe diseased tomato plants were collected from the south of Jiangxi Province, R. solanacearum strains with different geographical origins were isolated by selective plate, purificated and identificated by PCR. The test of physiology and biochemistry and inoculation on tomato plants were conducted for the determination of biovar and virulence difference. The endoglucanase gene (egl) fragments were amplified by PCR to determine the phylotype and sequevar of R. solanacearum.
ResultA total of 44 R. solanacearum strains were obtained from nine cities (counties) in the south of Jiangxi Province, among which 41 strains were identified as biovar III and three strains were identified as biovar IV. According to the results of virulence difference, 44 strains were clustered into three groups, namely group I (high virulence), group II (moderate virulence) and group III (weak virulence), of which group I (high virulence) strains accounted for 65.9%. All strains were belonged to the phylotype I and further divided into eight sequevars, namely Sequevar 13, 14, 15, 17, 18, 34, 44 and 48 respeclively. Most R. solanacearum strains were sensitive to the eight tested bacteriophages.
ConclusionThe strains of R. solanacearum from tomato in the south of Jiangxi Province are mainly biovar III and high virulence, sensitive to bacteriophages, have eight sequevars, and have obvious differentiation and genetic diversity.
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Keywords:
- Ralstonia solanacearum /
- Tomato bacterial wilt /
- Biovar /
- Virulence /
- Phylotype /
- Sequevar /
- Bacteriophage
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图 1 基于44个青枯菌接种5个番茄品种发病率的聚类分析结果
Figure 1. Clustering results based on the incidence of 44 Ralstonia solanacearum strains inoculated to five tomato cultivars
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图 2 基于青枯菌egl基因序列的系统发育分析
将序列变种相同的菌株置于同一水平分支上，“◆”代表参考菌株
Figure 2. Phylogenetic analysis based on sequence of egl gene of Ralstonia solanacearum
Strains with the same sequevar are settled at the same branch, “◆” indicates reference strain
表 1 参考序列信息
Table 1 Referenced sequence information
参考菌株
Reference strain寄主
Host来源
Origin演化型
Phylotype序列变种
Sequevaregl登录号
Accession numberJT523 马铃薯 Solanum tuberosum 留尼汪岛 Reunion Ⅰ 13 AF295252 PSS8 番茄 S. lycopersicum 中国 China Ⅰ 14 FJ561066 PSS358 番茄 S. lycopersicum 中国 China Ⅰ 15 EU407298 UW151 姜 Zingiber officinale 澳大利亚 Australia Ⅰ 16 AF295254 P11 花生 Arachis hypogaea 中国 China Ⅰ 17 FJ561068 GMI1000 番茄 S. lycopersicum 法国 France Ⅰ 18 AF295251 JT519 天竺葵 Pelargonium hortorum 留尼汪岛 Reunion Ⅰ 31 GU295032 PSS219 番茄 S. lycopersicum 中国 China Ⅰ 34 FJ561167 O3 橄榄树 Olea europaea 中国 China Ⅰ 44 FJ561069 TB28 烟草 Nicotiana tabacum 中国 China Ⅰ 44 FJ561127 Tb43 烟草 N. tabacum 中国 China Ⅰ 44 FJ561129 BdlI 木槿 Hibiscus syriacus 中国 China Ⅰ 44 FJ561098 CIIP365 马铃薯 S. tuberosum 菲律宾 The Philippines Ⅰ 45 GQ907151 MADI7 辣椒 Capsicum annuum 马达加斯加 Madagascar Ⅰ 46 GU295040 GMI8254 番茄 S. lycopersicum 印度尼西亚 Indonesia Ⅰ 47 GU295014 M2 桑树 Morus alba 中国 China Ⅰ 48 FJ561067 CMR87 番茄 S. lycopersicum 喀麦隆 Cameroon Ⅱ 35 EF439727 CMR12 番茄 S. lycopersicum 喀麦隆 Cameroon Ⅱ 52 EF439725 CMR39 番茄 S. lycopersicum 喀麦隆 Cameroon Ⅱ 41 EF439726 CFBP2972 马铃薯 S. tuberosum 马提尼克 Martinique Ⅱ 35 EF371809 UW551 天竺葵 P. hortorum 肯尼亚 Kenya Ⅱ 1 DQ657596 ICMIP7963 马铃薯 S. tuberosum 肯尼亚 Kenya Ⅱ 7 AF295263 
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续表 1 Continued table 1 参考菌株
Reference strain寄主
Host来源
Origin演化型
Phylotype序列变种
Sequevaregl登录号
Accession numberUW162 香蕉 Musa nana 秘鲁 Peru Ⅱ 4 AF295256 MOLK2 香蕉 M. nana 菲律宾 The Philippines Ⅱ 3 EF371841 CMR66 木龙葵 S. scabrum 喀麦隆 Cameroon Ⅲ 49 EF439729 JT525 天竺葵 P. hortorum 留尼汪岛 Reunion Ⅲ 19 AF295272 CFBP3059 茄子 S. melongena 布基纳法索 Burkina Faso Ⅲ 23 AF295270 NCPPB332 马铃薯 S. tuberosum 津巴布韦 Zimbabwe Ⅲ 22 DQ657649 MAFF301558 马铃薯 S. tuberosum 日本 Japan Ⅳ 8 AY465002 Psi 番茄 S. lycopersicum 印度尼西亚 Indonesia Ⅳ 10 EF371804 ACH732 番茄 S. lycopersicum 澳大利亚 Australia Ⅳ 11 GQ907150
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表 2 青枯菌生化变种鉴定1)
Table 2 Biovar identification of Ralstonia solanacearum
来源
Origin菌株编号
No. of strain菌株数/个
Strain quantity麦芽糖
Maltose纤维二糖
Cellobiose乳糖
Lactose甘露醇
Mannitol山梨醇
Sorbitol甜醇
Dulcitol生化变种
Biovar于都县
Yudu CountyTm1901~Tm1908、
Tm1920~Tm19249 + + + + + + Ⅲ 上犹县
Shangyou CountyTm1913~Tm1919 7 + + + + + + Ⅲ 石城县
Shicheng CountyTm1925~Tm1929 4 + + + + + + Ⅲ 瑞金市
Ruijin CityTm1930、Tm1931 2 + + + + + + Ⅲ 大余县
Dayu CountyTm1932~Tm1934 3 − − − + + + Ⅳ 安远县
Anyuan CountyTm1935~Tm1937 3 + + + + + + Ⅲ 会昌县
Huichang CountyTm1938~Tm1943 6 + + + + + + Ⅲ 兴国县
Xingguo CountyTm2046、Tm2047 2 + + + + + + Ⅲ 全南县
Quannan CountyTm1944、Tm1945、
Tm2048~Tm20588 + + + + + + Ⅲ 1)“+”表示被利用，“−”表示不被利用
1)“+”indicates to be used, “−” indicates not to be used
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表 3 44个青枯菌接种5个番茄品种的发病率及聚类分组
Table 3 Incidence of 44 Ralstonia solanacearum strains inoculated to five tomato cultivars and their clustering results
来源
Origin菌株编号
No. of strain发病率/% Incidence rate 聚类分组
Cluster红圣佳2号
Hongshengjia 2金艳
Jinyan多宝
Duobao粉霸
Fenba精棚T红
Jingpeng T red于都县 Yudu County Tm1901 55 80 95 100 95 Ⅱ Tm1902 90 85 75 100 100 Ⅰ Tm1903 85 80 95 100 100 Ⅰ Tm1904 90 95 70 100 100 Ⅰ Tm1907 85 75 80 100 100 Ⅰ Tm1908 37 65 60 72 90 Ⅲ 上犹县 Shangyou County Tm1913 90 70 90 100 90 Ⅰ Tm1914 95 75 85 95 100 Ⅰ Tm1915 85 75 95 90 100 Ⅰ Tm1916 60 75 90 85 95 Ⅱ Tm1917 40 70 85 90 100 Ⅱ Tm1918 50 70 95 90 100 Ⅱ Tm1919 0 5 15 40 65 Ⅲ 于都县 Yudu County Tm1920 80 85 80 90 90 Ⅰ Tm1923 50 80 95 68 95 Ⅱ Tm1924 80 95 85 85 100 Ⅰ 石城县 Shicheng County Tm1925 65 85 85 95 85 Ⅱ Tm1926 95 100 90 100 100 Ⅰ Tm1928 80 75 100 95 100 Ⅰ Tm1929 35 21 40 50 95 Ⅲ 瑞金市 Ruijin City Tm1930 90 95 95 100 100 Ⅰ Tm1931 100 100 100 100 100 Ⅰ 大余县 Dayu County Tm1932 20 35 65 45 80 Ⅲ Tm1933 35 35 55 80 90 Ⅲ Tm1934 20 40 75 60 100 Ⅲ 安远县 Anyuan County Tm1935 40 52 85 95 100 Ⅲ Tm1936 30 35 90 80 89 Ⅲ Tm1937 85 95 90 100 95 Ⅰ 会昌县 Huichang County Tm1938 90 85 95 100 100 Ⅰ Tm1939 80 85 95 100 95 Ⅰ Tm1940 85 90 100 95 100 Ⅰ Tm1941 100 84 100 100 100 Ⅰ Tm1942 95 100 100 100 95 Ⅰ Tm1943 40 85 95 90 100 Ⅱ 全南县 Quannan County Tm1944 100 100 95 100 100 Ⅰ Tm1945 100 90 100 90 100 Ⅰ 兴国县 Xingguo County Tm2046 100 100 100 100 100 Ⅰ Tm2047 100 100 100 100 100 Ⅰ 全南县 Quannan County Tm2048 100 95 100 100 89 Ⅰ Tm2049 100 95 100 100 90 Ⅰ Tm2050 100 95 100 100 100 Ⅰ Tm2054 100 100 100 100 100 Ⅰ Tm2055 100 95 100 100 100 Ⅰ Tm2058 90 90 100 100 100 Ⅰ
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表 4 44个青枯菌对8个噬菌体的敏感性测定
Table 4 Sensitivity determination of 44 Ralstonia solanacearum to eight bacteriophages
来源
Origin菌株编号
No. of strain数量/个
Quantity噬菌体1) Bacteriophage 敏感性2)
SensitivityP1555-L P1555-1 P1555-M P1556-1 P1556-2 P7-1 P574 P1521 于都县
Yudu CountyTm1901~Tm1907 5 – – – + + + – + M Tm1908 1 + + + + + + + + V 上犹县
Shangyou CountyTm1913、Tm1914 2 + + + + – + + + S Tm1915~Tm1919 4 + + + + + + + + V Tm1916 1 + + + + – + + + S 于都县
Yudu CountyTm1920~Tm1924 3 + + + + + + + + V 石城县
Shicheng CountyTm1925~Tm1929 3 + + + + + + + + V Tm1928 1 + + + + – + + + S 瑞金市
Ruijin CityTm1930、Tm1931 2 + + + + + + + + V 大余县
Dayu CountyTm1932~Tm1934 3 + + + + + + + + V 安远县
Anyuan CountyTm1935~Tm1937 3 + + + + + + + + V 会昌县
Huichang CountyTm1938、Tm1939 2 – – – + + + – + M Tm1940~Tm1943 4 + + + + + + + + V 全南县
Quannan CountyTm1944 1 – – – + + + + + M Tm1945 1 – – – – – + + + M 兴国县
Xingguo CountyTm2046 1 + + + + + + + + V Tm2047 1 – – – + – + – – W 全南县
Quannan CountyTm2048、Tm2049 2 – – – + + + + + M Tm2050 1 – – – – + + + + M Tm2054~Tm2058 3 + + + + + + + + V 1)“+”表示产生噬菌斑,“–”表示不产生噬菌斑；2)M:中等，S:强，V:特强，W:弱
1) “+” indicates having plaques，“–” indicates no having plaques；2) M: Moderate, S: Strong, V:Very strong, W: Weak
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