甜叶菊褐斑病的病原菌鉴定及MeJA的抗病作用

doi:10.3864/j.issn.0578-1752.2018.18.008

摘要/Abstract

摘要：

【目的】明确引起甜叶菊褐斑病的病原菌种类,分析MeJA在甜叶菊响应链格孢菌过程中的作用,为甜叶菊褐斑病的防治及抗病育种提供依据。【方法】对取自江苏省东台市富安镇甜叶菊生产基地的发病甜叶菊植株的叶片进行病原菌分离、纯化培养,观察菌落及分生孢子的形态、大小和病原菌的致病性。采用离体叶片接种的方法进行致病性测定。利用真菌通用引物ITS1和ITS4对7个致病菌株ST1-ST7的rDNA-ITS区进行扩增,对扩增产物进行回收和测序,并利用MEGA 7软件的邻接法（neighbor-joining,NJ）构建基于病原菌的rDNA-ITS序列和GenBank中相关链格孢菌序列的系统发育树,确定病原菌的种类。利用台盼蓝染色、光学显微镜观察分析链格孢菌分生孢子在甜叶菊叶片上的萌发状态及侵入叶片的方式。通过向马铃薯葡萄糖琼脂培养基（potato dextrose agar,PDA）上外源添加MeJA分析其对链格孢菌菌丝生长的影响;对甜叶菊离体叶片饲喂MeJA并接种链格孢菌,观察叶片对链格孢菌的抗性;采用qPCR方法分析JA通路相关基因在甜叶菊叶片接种链格孢菌前后的表达情况。【结果】从甜叶菊发病叶片上共分离到7个菌株,所有菌株在PDA培养基上呈近圆形等径辐射生长,气生菌丝较为发达,初期为白色,后期逐渐变为不同程度的灰黑色,分生孢子单生或成链,多为近球形、倒棒状或倒梨形,大小为（20.5—45.5）×（6.5—16.0）μm。将所分离得到的菌株接种于甜叶菊离体叶片上,发现7个菌株对甜叶菊叶片致病程度存在一定差异,ST2、ST3和ST7 3个菌株侵染叶片后病斑扩展速度快,致病力较强。3个致病力较强的菌株的rDNA-ITS序列长度分别为569、570和570 bp,通过系统进化树分析,ST2、ST3与菌株KY814634.1、DQ491089.1等（Alternaria alternata、Alternaria sp.,链格孢）的相似度达到99%—100%,ST7与菌株HQ402558.1（Alternaria tenuissima,细极链格孢）的相似度达到99%。对接种细极链格孢ST7分生孢子的甜叶菊叶片台盼蓝染色后的观察结果发现,分生孢子可以从孢子的头部、侧面、尾部多个位置萌发,从叶片的气孔和表皮细胞间隙侵入叶片表皮细胞内。外源施加浓度高于200 μmol·L^-1的MeJA能有效抑制细极链格孢菌丝的生长;甜叶菊离体叶片饲喂100 μmol·L^-1的MeJA后接种细极链格孢,病斑面积明显小于对照,表明MeJA可增强甜叶菊对细极链格孢的抗性;JA通路相关基因LOX3和JAR1在甜叶菊接种细极链格孢后上调表达,JAZ1和JAZ4反之下调表达,表明JA通路参与甜叶菊对细极链格孢的响应。【结论】江苏省东台市富安镇甜叶菊生产基地甜叶菊褐斑病的致病菌为链格孢菌。细极链格孢菌丝可以从叶片的气孔以及表皮细胞间隙侵入表皮细胞。外源施加MeJA能够有效增强甜叶菊叶片对细极链格孢的抗性,在甜叶菊褐斑病的防治中具有很好的应用前景。

关键词: 甜叶菊, 褐斑病, 链格孢菌, 病原菌鉴定, MeJA, 信号通路

Abstract:

【Objective】The objective of this study is to identify the pathogenic fungus causing brown spot disease on Stevia rebaudiana, and analyze the role of MeJA in response to Alternaria to provide a basis for disease prevention and resistance breeding. 【Method】 The samples of diseased S. rebaudiana leaves were collected from the stevia production base of Fuan Town, Dongtai City, Jiangsu Province. After isolation and purification, the isolates were identified by using the methods of morphological characteristics analysis. Morphology parameters mainly included the colony and conidial morphology, conidial size. The isolates pathogenicity was tested on detached S. rebaudiana leaves. The internal transcribed spacer (ITS) rDNA region of the 7 pathogenic strains was amplified by using the fungal universal primers ITS1 and ITS4, and then the amplified product was recovered and sequenced. MEGA 7 was used to construct phylogenetic tree to confirm the pathogen species based on the rDNA-ITS sequence and the related Alternaria sequences in GenBank. The germination status of A. tenuissima conidial spores and the way they invaded the leaves after stained with trypan blue were analyzed by microscopy. The effect on the growth of A. tenuissima mycelium was analyzed by exogenous application of MeJA to potato dextrose agar (PDA) medium. To test the role of MeJA in the S. rebaudiana resistance to A. tenuissima, detached leaves were fed with MeJA and then used for infection. The qPCR method was performed to analyze the expression of JA pathway-related genes before or after inoculation with A. tenuissima in leaves of S. rebaudiana. 【Result】 Seven Alternaria strains were collected from the diseased S. rebaudiana leaves. All the strains grew on the PDA medium in a nearly circular and equal diameter radiation. The aerial hyphae were relatively developed, with white at the beginning and gradually changed to different degrees of grayish black at the later stage. The conidia are solitary or in a chain, mostly nearly spherical, inverted bar-shaped, or inverted pear-shaped, with a size of (20.5-45.5) × (6.5-16.0) μm. The isolated 7 strains were inoculated on the detached leaves of S. rebaudiana, and the pathogenicity was different. ST2, ST3 and ST7 strains led to the lesions spread rapidly with stronger pathogenicity. The rDNA-ITS sequence length of the ST2, ST3, and ST7 strains was 569, 570, and 570 bp, respectively. Phylogenetic tree analysis indicated that the similarity of ST2 and ST3 with strains KY814634.1 and DQ491089.1 (Alternaria alternata, Alternaria sp.) is between 99%-100%, and similarity between ST7 and strain HQ402558.1 (Alternaria tenuissima) is 99%. The results of trypan blue staining of S. rebaudiana leaves inoculated with A. tenuissima showed that conidia of ST7 could germinate from the head, lateral and caudal of the spores, and the A. tenuissima mycelia could invade into epidermal cells from the stomata or the epidermis intercellular. Exogenous application of MeJA could effectively inhibit the growth of A. tenuissima mycelia when the concentration of MeJA was higher than 200 μmol·L^-1. After inoculated with A. tenuissima, the lesion area of detached leaves that fed with 100 μmol·L^-1 MeJA was significantly smaller than that of the control, indicating that MeJA could enhance the S. rebaudiana resistance to A. tenuissima. JA pathway related genes were involved in the response of S. rebaudiana to A. tenuissima, the expression of LOX3 and JAR1 was up-regulated while the expression of JAZ1 and JAZ4 was down-regulated after S. rebaudiana inoculated with A. tenuissima.【Conclusion】The pathogenic fungus that caused S. rebaudiana leaf brown spot in the stevia production base of Fuan Town, Dongtai City, Jiangsu Province was identified as Alternaria. A. tenuissima mycelia can invade epidermal cells from the stomata or the epidermis intercellular space of the leaves. The resistance of S. rebaudiana leaves to A. tenuissima was enhanced after exogenous application of MeJA, which will become the potential candidates for the control of S. rebaudiana brown spot disease in the field.

Key words: Stevia rebaudiana, brown spot disease, Alternaria, identification of pathogens, methyl jasmonate, signaling pathway

崔晓霞, 束红梅, 蒋璐, 何晓兰, 巩元勇, 倪万潮, 郭书巧. 甜叶菊褐斑病的病原菌鉴定及MeJA的抗病作用[J]. 中国农业科学, 2018, 51(18): 3520-3530.

XiaoXia CUI, HongMei SHU, Lu JIANG, XiaoLan HE, YuanYong GONG, WanChao NI, ShuQiao GUO. Identification of Pathogens Causing Brown Spot and the Role of MeJA in Disease Resistance in Stevia rebaudiana[J]. Scientia Agricultura Sinica, 2018, 51(18): 3520-3530.

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