番茄抗青枯病种质TK083的筛选和鉴定

doi:10.16420/j.issn.0513-353x.2024-0084

摘要/Abstract

摘要：

在江西省北部地区对130份番茄种质进行青枯病抗性田间自然筛选，并验证了这些种质对当地优势青枯病菌菌株RS100的苗期抗性，发现TK083为优异的番茄抗青枯病种质。接种青枯病菌后，TK083木质部汁液中的青枯病菌数远低于易感型番茄‘Moneymaker’；TK083叶片中SOD和POD活性明显上升，MDA含量在接种后初期小幅上升；几丁质酶和β-1,3-GA活性分别在接种前期和后期上升，表明它们参与不同阶段的免疫反应；定量PCR结果显示参与青枯病免疫调控的SlNAP1、SlERF2a和SlERF3的表达量在TK083根中显著被青枯病菌诱导上调。以上结果表明TK083可能通过多个途径协同应对青枯病菌对番茄的侵染。

关键词: 番茄, 青枯病, 青枯雷尔氏菌, 种质, 抗性, 鉴定

Abstract:

In the northern part of Jiangxi Province，130 tomato accessions were naturally screened for bacterial wilt resistance，and the seedling resistance of these accessions to the local dominant Ralstonia solanacearum strain RS100 was verified，and TK083 was found to be an excellent tomato resistant to bacterial wilt accessions. After inoculation with Ralstonia solanacearum，the concentration of the pathogen in the xylem sap of TK083 was much lower than that of susceptible tomato‘Moneymaker’. The activities of SOD and POD in the leaves of TK083 increased markedly，while the MDA content showed a slight increase in the early stages post-inoculation. The activity of chitinase and β-1，3-GA increased in the early and late stages of inoculation respectively，indicating that they were involved in different stages of immune response. RT-qPCR results revealed that the expression levels of SlNAP1，SlERF2a and SlERF3，which are involved in the immune regulation of bacterial wilt，were significantly up-regulated by Ralstonia solanacearum in TK083 roots. These results suggested that TK083 likely employs multiple pathways to coordinately combat the infection of Ralstonia solanacearum.

Key words: tomato, bacterial wilt, Ralstonia solanacearum, accession, resistance, identification

方俊仪, 巫伟峰, 陆乔, 凌宏清, 孔丹宇. 番茄抗青枯病种质TK083的筛选和鉴定[J]. 园艺学报, 2025, 52(6): 1477-1487.

FANG Junyi, WU Weifeng, LU Qiao, LING Hongqing, and KONG Danyu. Screening and Identification for Bacterial Wilt Resistance Accession TK083 in Tomato[J]. Acta Horticulturae Sinica, 2025, 52(6): 1477-1487.

图/表 7

图1 130份番茄种质青枯病的田间自然发病情况 I类：发病率 ≤ 5%；Ⅱ类：5% < 发病率 ≤ 30%；Ⅲ类：30% < 发病率 ≤ 75%；Ⅳ类：75% < 发病率。SLL：普通栽培番茄；SLC：樱桃番茄；SP：醋栗番茄

Fig. 1 The natural incidence of bacterial wilt in 130 tomato accessions under field conditions ClassI：Incidence ≤ 5%；ClassⅡ：5% < incidence ≤ 30%；ClassⅢ：30% < incidence ≤ 75%；ClassⅣ：75% < Incidence. SLL：Solanum lycopersicum var. lycopersicum；SLC：S. lycopersicum var. cerasiforme；SP：S. pimpinellifolium

表1 qRT-PCR 所用引物

Table 1 Primers for qRT-PCR

引物名称 Primer name	正向引物（5′-3′） Forward primer	反向引物（5′-3′） Reverse primer	片段/bp Fragment length	参考文献 Reference
qSlNAP1	CCAAATAGAGCAGCTGTGTCA	TTGGTGGTTTGCCTTTGTAA	121	Wang et al.,2020
qSlERF2a	TATGCACAATTACTTCGCGATG	GCTTGTTGTTGTTGTTGTTG	155	Ke et al.,2023
qSlERF2b	CGACGATACAGAGGAGTTAGAC	AAAAGTACCTAACCAAACACGC	96	Ke et al.,2023
qSlERF3	GGTGCAGTTACAGCTATGGC	CTAGCAGCGTTATCATAAGC	191	Pan et al.,2010
qSlWRKY33	AAGGCAACAACAGACCACTG	CCATCTTCCGCCTTCTGTTC	162	Ke et al.,2023
qSlUBI	TCGTAAGGAGTGCCCTAATGCTGA	CAATCGCCTCCAGCCTTGTTGTAA	119	姜静等,2017

图2 青枯病菌菌株RS100与其他Ralstonia代表序列的系统进化树

Fig. 2 Evolutionary tree of Ralstonia solanacearum strain RS100 with other Ralstonia representative sequences

图3 番茄种质青枯病菌抗性筛选 A：幼苗接菌发病率；B：田间自然筛选与幼苗接菌筛选青枯发病率

Fig. 3 Screening of tomato accession for resistance to Ralstonia solanacearum A：Incidence of seedling inoculation；B：Comparison of bacterial wilt incidence between natural screening in the field and seedling inoculation screening

图4 抗病种质TK083和感病种质Moneymaker（Mm）幼苗在接种青枯病菌5 d后的表型（A）、木质部细菌数（B）和24 d内的发病率（C） t检验，** 表示在P < 0.01水平上差异显著

Fig. 4 Phenotype（A），number of xylem bacteria（B）and incidence rate（C）within 24 days of inoculation of resistant accession TK083 and susceptible accession Moneymaker（Mm）seedlings 5 days after inoculation with Ralstonia solanacearum t-Test，** indicates significantly difference at P < 0.01 level

图5 抗病种质TK083和感病种质Moneymaker（Mm）幼苗在接种青枯病菌后的生理指标变化差异显著性分析采用Duncan’s检验，不同小写字母表示同一种质不同时间差异显著（P < 0.05）

Fig. 5 Changes of physiological indexes in seedlings of resistant accession TK083 and susceptible accession Moneymaker（Mm）after inoculation with Ralstonia solanacearum Significant difference analysis used Duncan’s test，different lowercase letters indicate significant differences in the same accession at different times（P < 0.05）

图6 抗病种质TK083和感病种质Moneymaker（Mm）幼苗在接种青枯病菌5 d后相关免疫应答基因的表达模式 t检验，* 表示在P < 0.05水平上差异显著，** 表示在P < 0.01水平上差异显著，ns表示差异不显著

Fig. 6 Expression patterns of related immune response genes in resistant accession TK083 and susceptible accession Moneymaker（Mm）seedlings 5 days after inoculation with Ralstonia solanacearum t-Test，* indicates significantly difference at P < 0.05 level，** indicates significantly difference at P < 0.01 level，ns indicates no significant difference

参考文献 41

[1]	Allen C. 2007. Strategies for managing bacterial wilt diseases of tomato,potato,and export ornamentals. Phytopathology, 97 (7):148-149.
[2]	Benhamou N, Joosten M H, de Wit P J. 1990. Subcellular localization of chitinase and of its potential substrate in tomato root tissues infected by Fusarium Oxysporum f. Sp.Radicis-Lycopersici. Plant Physiology, 92 (4):1108-1120.
[3]	Cai H Y, Yang S, Yan Y, Xiao Z L, Cheng J B, Wu J, Qiu A L, Lai Y, Mou S L, Guan D Y, Huang R H, He S L. 2015. CaWRKY 6 transcriptionally activates CaWRKY40,regulates Ralstonia solanacearum resistance,and confers high-temperature and high-humidity tolerance in pepper. Journal of Experimental Botany, 66 (11):3163-3174.
[4]	Dang F F, Wang Y N, Yu L, Eulgem T, Lai Y, Liu Z Q, Wang X, Qiu A L, Zhang T X, Lin J, Chen Y S, Guan D Y, Cai H Y, Mou S L, He S L. 2013. CaWRKY40,a WRKY protein of pepper,plays an important role in the regulation of tolerance to heat stress and resistance to Ralstonia solanacearum infection. Plant Cell Environment, 36 (4):757-774.
[5]	Denny T P, Hayward A C. 2001. Gram-negative bacteria:Ralstonia.// Schaad N W,Jones J B,Chun W.Laboratory guide for identification of plant pathogenic bacteria,third edition. USA: American Phytopathological Society Press:151-174.
[6]	Dong H H, Xu X, Gao R X, Li Y Q, Li A Z, Yao Q, Zhu H H. 2022. Myxococcus xanthus R31 suppresses tomato bacterial wilt by inhibiting the pathogen Ralstonia Solanacearum with secreted proteins. Frontiers in Microbiology, 12 (2):801091.
[7]	Fang Shumin, Gu Gang, Chen Yusen, Huang Chunmei, Chen Shunhui. 2013. Colonization and infection of Ralstonia Solanacearum in weed roots. Acta Tabacaria Sinica, 19 (5):72-81,88. (in Chinese)
	方树民, 顾钢, 陈玉森, 黄春梅, 陈顺辉. 2013. 烟草青枯菌在杂草根部的定殖和传病作用. 中国烟草学报, 19 (5):72-81,88.
[8]	Grimault V, Prior P, Anaïs G. 1995. A monogenic dominant resistance of tomato to bacterial wilt in Hawaii 7996 is associated with plant colonization by Pseudomonas Solanacearum. Journal of Phytopathology, 143 (6):349-352.
[9]	Habe I, Miyatake K, Nunome T, Yamasaki M, Hayashi T. 2019. QTL analysis of resistance to bacterial wilt caused by Ralstonia solanacearum in potato. Breeding Science, 69 (4):592-600.
[10]	Hayward A C. 1991. Biology and epidemiology of bacterial wilt caused by Pseudomonas Solanacearum. Annual Review of Phytopathology,29:65-87.
[11]	Huet G. 2014. Breeding for resistances to Ralstonia Solanacearum. Frontiers in Plant Science, 5 (12):715.
[12]	Ifnan Khan M, Zhang Y W, Liu Z Q, Hu J, Liu C, Yang S, Hussain A, Furqan Ashraf M, Noman A, Shen L, Xia X Q, Yang F, Guan D Y, He S L. 2018. CaWRKY40b in pepper acts as a negative regulator in response to Ralstonia solanacearum by directly modulating defense genes including CaWRKY40. International Journal of Molecular Sciences, 19 (5):1403.
[13]	Jiang G F, Wei Z, Xu J, Chen H L, Zhang Y, She X M, Macho A P, Ding W, Liao B S. 2017. Bacterial wilt in China:history,current status,and future perspectives. Frontiers in Plant Science, 8 (9):1549.
[14]	Jiang Jing, Wang Yinlei, Zhao Liping, Zhou Rong, Li Yaru, Zhao Tongmin, Yu Wengui. 2017. Selection of tomato reference genes for qRT-PCR. Jiangsu Journal of Agricultural Sciences, 33 (2):389-396. (in Chinese)
	姜静, 王银磊, 赵丽萍, 周蓉, 李亚茹, 赵统敏, 余文贵. 2017. 番茄qRT-PCR内参基因的筛选. 江苏农业学报, 33 (2):389-396.
[15]	Kadota Y, Shirasu K, Zipfel C. 2015. Regulation of the NADPH Oxidase RBOHD during plant immunity. Plant Cell Physiology, 56 (8):1472-1480.
[16]	Ke J J, Zhu W T, Yuan Y, Du X Y, Xu A, Zhang D, Cao S, Chen W, Lin Y, Xie J T, Cheng J S, Fu Y P, Jiang D H, Yu X, Li B. 2023. Duality of immune recognition by tomato and virulence activity of the Ralstonia solanacearum exo-polygalacturonase PehC. The Plant Cell, 35 (7):2552-2569.
[17]	Li X Y, Xu B, Xu J Q, Li Z S, Jiang C Q, Zhou Y, Yang Z G, Deng M H, Lv J H, Zhao K. 2023. Tomato-thaumatin-like protein genes and confer resistance to five soil-borne diseases by enhancing β-1,3-glucanase activity. Genes, 14 (8):1622.
[18]	Li Y Y, Feng J, Liu H L, Wang L, Hsiang T, Li X H, Huang J B. 2016. Genetic diversity and pathogenicity of Ralstonia solanacearum causing tobacco bacterial wilt in China. Plant Disease, 100 (7):1288-1296.
[19]	Mandal S, Das R K, Mishra S. 2011. Differential occurrence of oxidative burst and antioxidative mechanism in compatible and incompatible interactions of Solanum Lycopersicum and Ralstonia Solanacearum. Plant Physiology and Biochemistry, 49 (2):117-123.
[20]	Milc J, Bagnaresi P, Aragona M, Valente M T, Biselli C, Infantino A, Francia E, Pecchioni N. 2019. Comparative transcriptome profiling of the response to Pyrenochaeta lycopersici in resistant tomato cultivar Mogeor and its background genotype-susceptible Moneymaker. Functional & Integrative Genomics,19:811-826.
[21]	Ou Xiuling, Geng Yawen, Li Feng, Li Caolong, Zhu Jianbo, Wang Aiying. 2013. The effect of physiological traits of transgenic cotton induced by Verticillium wilt Pathogen. Biotechnology Bulletin,6:94-98. (in Chinese)
	欧秀玲, 耿雅文, 李凤, 李曹龙, 祝建波, 王爱英. 2013. 棉花黄萎病病原菌诱导侵染对转基因抗病棉花生理性状的影响. 生物技术通报,6:94-98.
[22]	Pan I C, Li C W, Su R C, Cheng C P, Lin C S, Chan M T. 2010. Ectopic expression of an EAR motif deletion mutant of SlERF3 enhances tolerance to salt stress and Ralstonia solanacearum in tomato. Planta, 232 (5):1075-1086.
[23]	Rawat S, Ali S, Mittra B, Grover A. 2017. Expression analysis of chitinase upon challenge inoculation to Alternaria wounding and defense inducers in brassica juncea. Biotechnology Reports, 13 (3):72-79.
[24]	Ren Minhua, Zhang Jingyan, Cui Xiaodong, Chen Ronghua, Liu Qiongguang. 2022. Diversity of Ralstonia solanacearum strains from tomato in the south of Jiangxi Province. Journal of South China Agricultural University, 43 (1):67-76. (in Chinese)
	任敏华, 张静燕, 崔晓东, 陈荣华, 刘琼光. 2022. 赣南地区番茄青枯菌菌系多样性分析. 华南农业大学学报, 43 (1):67-76.
[25]	Sequeira L, Rowe P R. 1969. Selection and utilization of Solanum Phureja clones with high resistance to different strains of Pseudomonas Solanacearum. American Potato Journal, 46 (12):451-462.
[26]	Shen Shanna, Hou Xilin. 2009. Changes of protective substances and enzymes in non-heading Chinese cabbage after infection by downy mildew. Journal of Nanjing Agricultural University, 32 (1):23-26. (in Chinese)
	申姗娜, 侯喜林. 2009. 不结球白菜感染霜霉病菌后防御物质及酶的变化. 南京农业大学学报, 32 (1):23-26.
[27]	Wang Guoping, Li Zhenxing, Lin Mingbao. 2003. A new tomato germplasm resistance to Ralstonia solanacearum AS52. Acta Horticulturae Sinica, 30 (6):706. (in Chinese)
	汪国平, 黎振兴, 林明宝. 2003. 番茄抗青枯病新资源材料AS52. 园艺学报, 30 (6):706.
[28]	Wang Guoping, Lin Mingbao, Wu Dinghua. 2004. Classical and molecular genetics of bacterial wilt resistance in tomato. Acta Horticulturae Sinica, 31 (3):403-407. (in Chinese)
	汪国平, 林明宝, 吴定华. 2004. 番茄青枯病抗性遗传研究进展. 园艺学报, 31 (3):403-407.
[29]	Wang J, Zheng C F, Shao X Q, Hu Z J, Li J X, Wang P, Wang A R, Yu J Q, Shi K. 2020. Transcriptomic and genetic approaches reveal an essential role of the NAC transcription factor SlNAP 1 in the growth and defense response of tomato. Horticulture Research, 7 (1):209.
[30]	Wang L, Zhang X L, Wang L, Tian Y N, Jia N, Chen S Z, Shi N B, Huang X M, Zhou C, Yu Y W, Zhang Z Q, Pang X Q. 2017. Regulation of ethylene-responsive SlWRKYs involved in color change during tomato fruit ripening. Scientific Reports, 7 (1):16674.
[31]	Wang Q, Huang D, Tu W Y, Ma F W, Liu C H. 2024. Overexpression of auxin/indole-3-acetic acid gene MdIAA24 enhances Glomerella leaf spot resistance in apple(Malus domestica). Horticultural Plant Journal, 10 (1):15-24.
[32]	Wang Rongbo, Wang Haiyun, Zhang Qianrong, Cai Songling, Li Benjin, Zhang Meixiang, Liu Peiqing. 2024. Genetic diversity of Ralstonia solanacearum strains isolated from Fujian Province and evaluation of tomato rootstocks for resistance to bacterial wilt. Acta Horticulturae Sinica, 51 (11):2540-2554. (in Chinese)
	王荣波, 王海云, 张前荣, 蔡松龄, 李本金, 张美祥, 刘裴清. 2024. 福建省番茄青枯病菌遗传多样性分析及其抗性砧木的筛选. 园艺学报, 51 (11):2540-2554.
[33]	Wang X F, Wei Z, Yang K M, Wang J N, Jousset A, Xu Y C, Shen Q R, Friman V P. 2019. Phage combination therapies for bacterial wilt disease in tomato. Nature Biotechnology, 37 (12):1513-1520.
[34]	Wang Y R, Xian L, Yu G, Macho A P. 2021. Tomato stem injection for the precise assessment of Ralstonia solanacearum fitness in planta. Bio-Protocol Journal, 11 (16):e4134.
[35]	Wang Yixi, Yan Shuangshuang, Yu Bingwei, Gan Yuwei, Qiu Zhengkun, Zhu Zhangsheng, Chen Changming, Cao Bihao. 2023. Screening and identification of E 3 ubiquitin ligase genes relate to bacterial wilt resistance in eggplant. Acta Horticulturae Sinica, 50 (10):2271-2287. (in Chinese)
	王亦栖, 颜爽爽, 余炳伟, 甘雨薇, 邱正坤, 朱张生, 陈长明, 曹必好. 2023. 茄子青枯病抗性相关的E3泛素连接酶基因的筛选及鉴定. 园艺学报, 50 (10):2271-2287.
[36]	Xiao Y H, Liu X D, Meng D L, Tao J M, Gu Y B, Yin H Q, Li J. 2018. The role of soil bacterial community during winter fallow period in the incidence of tobacco bacterial wilt disease. Applied Microbiology and Biotechnology, 102 (5):2399-2412.
[37]	Xu Xiuyu, Wang Minghuai, Zhang Weiqiang, Xu Bin. 2017. Effect of Ralstonia solanacearum on activities of protective enzymes and soluble protein content in Casuarina equisetifolia. Journal of Southwest Forestry University, 37 (3):107-112. (in Chinese)
	许秀玉, 王明怀, 张卫强, 徐斌. 2017. 青枯菌对木麻黄防御酶活性及可溶性蛋白含量的影响. 西南林业大学学报(自然科学), 37 (3):107-112.
[38]	Xu J, Xian Q Q, Wang K, Dong J P, Zhang C Y, Du S L, Xu X W, Chen X H. 2022. Screening and identification of candidate Fusarium wilt-resistance genes from pumpkin. Horticultural Plant Journal, 8 (5):583-592.
[39]	Xue H, Lozano-Durán R, Macho A P. 2020. Insights into the root invasion by the plant pathogenic bacterium Ralstonia Solanacearum. Plants, 9 (4):516.
[40]	Yan Xi, He Lei, Yang Hong, Lai Wei, Liu Chongzheng. 2022. Establishment of evacuation model for bacterial wilt resistance based on physiological indexes in Capsicum annuum L. Molecular Plant Breeding, http://kns.cnki.net/kcms/detail/46.1068.S.20220221.1702.011.html. in Chinese)
	严希, 何磊, 杨红, 赖卫, 刘崇政. 2022. 辣椒抗青枯病生理指标评估模型的构建. 分子植物育种, http://kns.cnki.net/kcms/detail/46.1068.S.20220221.1702.011.html.
[41]	Zhang C, Chen H, Cai T C, Deng Y, Zhuang R R, Zhang N, Zeng Y, Zheng Y H, Tang R H, Pan R L, Zhuang W J. 2017. Overexpression of a novel peanut NBS-LRR gene AhRRS5 enhances disease resistance to Ralstonia solanacearum in tobacco. Plant Biotechnology Journal, 15 (1):39-55.