Isolation and identification of tomato bacterial spot pathogen in Ningxia and screening of identification methods for disease resistance

doi:10.3785/j.issn.1008-9209.2022.07.042

Journal of Zhejiang University (Agriculture and Life Sciences)

2023, Vol. 49

Issue (3): 349-357 DOI: 10.3785/j.issn.1008-9209.2022.07.042

Plant protection

Isolation and identification of tomato bacterial spot pathogen in Ningxia and screening of identification methods for disease resistance

Xiaohong LI¹(

),Xiaomin WANG^1,^2,³(

),Wenxia DENG¹,Kaibin WANG¹,Meng GUO^1,^2,³,Guoxin CHENG^1,^2,³

^1.School of Agriculture, Ningxia University, Yinchuan 750021, Ningxia, China
^2.Ningxia Modern Facility Horticulture Engineering Technology Research Center, Yinchuan 750021, Ningxia, China
^3.Key Laboratory of Modern Molecular Breeding for Dominant and Special Crops in Ningxia, Yinchuan 750021, Ningxia, China

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Abstract

In order to identify the pathogens causing tomato bacterial spot disease in Ningxia, tomato leaves infected with bacterial spot disease from Zhongwei and Wuzhong cities of Ningxia were used as the materials, and the strains were isolated using the conventional tissue isolation method, and their morphology, molecular biology and pathogenicity were identified. The four-factor and three-level orthogonal design of experiment [L₉(3⁴)] was conducted to screen the indoor resistance identification method at the seedling stage. The results showed that the colony morphologies of strain 1 isolated from Zhongwei City and strain 2 isolated from Wuzhong City were all milky white, full edge, opaque, smooth surface, producing green fluorescence, rod-shaped cells, and Gram staining was negative. The 16S rDNA sequences of both strain 1 and strain 2 were 99.93% similar to that of Pseudomonas syringae pv. tomato pathogenic variant KT783475.1. After reinoculation, brown lesions with yellow halos were presented on the leaves of the tomato, which was consistent with the natural symptoms. So, it was confirmed that the pathogen causing tomato bacterial spot disease in Ningxia was P. syringae pv. tomato. Under the treatment combination of inoculating seedlings at the four-leaf stage, the bacterial suspension concentration of 1.00×10⁷ CFU/mL, with stem inoculation method and 96 h moisture, the disease index was 72.22%, which was significantly higher than that in other treatments, and thus it was considered to be the best indoor resistance identification method at the seedling stage for tomato bacterial spot disease. The above results will provide a theoretical basis for the prevention and control of tomato bacterial spot disease and resistance breeding in Ningxia.

Key words： tomato bacterial spot disease morphological identification molecular biological identification

Received: 04 July 2022 Published: 25 June 2023

CLC:

S436.412.19

Corresponding Authors: Xiaomin WANG E-mail: lxh0895@163.com;wangxiaomin_1981@163.com

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Cite this article:

Xiaohong LI,Xiaomin WANG,Wenxia DENG,Kaibin WANG,Meng GUO,Guoxin CHENG. Isolation and identification of tomato bacterial spot pathogen in Ningxia and screening of identification methods for disease resistance. Journal of Zhejiang University (Agriculture and Life Sciences), 2023, 49(3): 349-357.

URL:

https://www.zjujournals.com/agr/10.3785/j.issn.1008-9209.2022.07.042 OR https://www.zjujournals.com/agr/Y2023/V49/I3/349

宁夏地区番茄细菌性斑点病病原菌的分离鉴定及其抗病性鉴定方法的筛选

为明确引起宁夏地区番茄细菌性斑点病的病原菌，以宁夏中卫市和吴忠市感染细菌性斑点病的番茄病叶为材料，通过常规组织分离法分离菌株，对其形态学、分子生物学、致病性进行鉴定，并采用四因素三水平正交试验设计[L₉（3⁴）]筛选室内苗期抗病性鉴定方法。结果表明：菌株1（分离自宁夏中卫市的病原菌）、菌株2（分离自宁夏吴忠市的病原菌）的菌落形态均呈乳白色，全缘，不透明，表面光滑，产生绿色荧光，菌体杆状，革兰氏染色呈阴性；菌株1、菌株2的16S rDNA序列与丁香假单胞菌番茄致病变种KT783475.1的相似度为99.93%；回接后叶片产生褐色病斑，伴有黄色晕圈，与自然发病植株的症状一致。因此，确定引起宁夏地区番茄细菌性斑点病的病原菌是丁香假单胞菌番茄致病变种（Pseudomonas syringae pv. tomato）。在接种苗期为四叶期、菌悬液浓度为1.00×10⁷ CFU/mL、接种方法为茎秆接种法、保湿时间为96 h的条件下，病情指数为72.22%，显著高于其他处理，是番茄细菌性斑点病室内苗期抗病性鉴定的最佳方法。本研究为宁夏地区番茄细菌性斑点病的防治及抗病育种提供了一定的理论依据。

关键词： 番茄, 细菌性斑点病, 形态学鉴定, 分子生物学鉴定

Table 1 Factors and levels of resistance identification of tomato bacterial spot disease

Table 2 Orthogonal design table for resistance identification of tomato bacterial spot disease at the seedling stage

Fig. 1 Symptoms of tomato disease samples collected in the field in NingxiaThe front (A) and back (B) tomato leaves infected with bacterial spot disease in Wuzhong City of Ningxia; and the stems (C) and fruits (D) of tomato infected with bacterial spot disease in Zhongwei City of Ningxia.

Fig. 2 Morphological characteristics of strain 1 and strain 2A. Strain 1 colony morphology; B. Strain 1 fluorescent reaction; C. Strain 2 colony morphology; D. Strain 2 fluorescent reaction; E. Strain 1 Gram staining reaction; F. Strain 2 Gram staining reaction.

Fig. 3 16S rDNA amplification (A) and specific amplification (B) of strain 1 and strain 2M: DNA marker; 1-2: PCR products of strain 1; 3-4: PCR products of strain 2.

Fig. 4 Partial multi-sequence alignment of Pseudomonas syringae pv. tomatoPst-1: Strain 1; Pst-2: Strain 2. The same as below.

Fig. 5 Phylogenetic tree of Pseudomonas syringae pv. tomato

Strains isolated in this study.

Fig. 6 Pathogenicity identification results of strain 1 and strain 2A-B. Strain 1; C. Strain 2. S: Lesion.

Table 3 Analysis of incidence and disease index in patho-genicity identification of strain 1 and strain 2

Table 4 Comparisons of indoor resistance identification methods of tomato bacterial spot disease at the seedling stage


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