中国农业科学 ›› 2018, Vol. 51 ›› Issue (10): 1899-1907.doi: 10.3864/j.issn.0578-1752.2018.10.009

• 植物保护 • 上一篇    下一篇

温度和多胺对柑橘溃疡病发生的影响及作用机制

杨枫1(), 陈传武2(), 范七君2, 石春梅1, 谢宗周1, 郭大勇1, 刘继红1()   

  1. 1华中农业大学园艺林学学院/园艺植物生物学教育部重点实验室,武汉430070
    2广西特色作物研究院/广西柑橘生物学重点实验室, 广西桂林541004
  • 收稿日期:2017-10-16 接受日期:2017-12-06 出版日期:2018-05-16 发布日期:2018-05-16
  • 联系方式: 联系方式:杨枫,E-mail:124272531@qq.com。陈传武,E-mail:jk_ccw@126.com。杨枫和陈传武为同等贡献作者。
  • 基金资助:
    国家公益性行业(农业)科研专项(201003067)、万人计划创新领军人才、湖北省自然科学基金创新群体(2017CFA018)、广西柑橘生物学重点实验室培育基地开放课题(桂柑科201202k003,桂柑科201201z004)

Influence of Temperature and Polyamines on Occurrence of Citrus Canker Disease and Underlying Mechanisms

Feng YANG1(), ChuanWu CHEN2(), QiJun FAN2, ChunMei SHI1, ZongZhou XIE1, DaYong GUO1, JiHong LIU1()   

  1. 1College of Horticulture and Forestry Sciences, Huazhong Agricultural University/Key Laboratory of Horticultural Plant Biology, Ministry of Education, Wuhan 430070
    2Guangxi Academy of Specialty Crops/Guangxi Key Laboratory of Citrus Biology, Guilin 541004, Guangxi
  • Received:2017-10-16 Accepted:2017-12-06 Published:2018-05-16 Online:2018-05-16

摘要:

目的 溃疡病是严重危害柑橘的一种细菌性病害,通常在高温下容易发生。论文旨在阐明高温下柑橘易发生溃疡病的机制,揭示其代谢变化,为利用药剂防治溃疡病提供重要的理论指导。方法 以感病的甜橙(Citrus sinensis)为研究对象,在21℃和30℃下预培养3 d,然后均接种同样浓度(108 cfu/mL)的柑橘溃疡病菌(Xanthomonas citri subsp. citri,Xcc)10 μL,比较两组植株的发病率,采用半定量RT-PCR分析两种温度下4个抗病基因AOSCHIGPXPR4A的表达量,利用HPLC测定预培养3 d后叶片内源多胺(腐胺、亚精胺和精胺)的含量。在此基础上,利用外源亚精胺(0.4 mmol·L-1)处理甜橙植株(以清水处理为对照),比较亚精胺和清水处理的植株接种溃疡病菌后的发病率和病情指数,分析亚精胺处理对内源多胺含量和抗病基因AOSCHIGPXPR4A表达的影响。结果 溃疡病菌接种后观察发现,21℃培养植株溃疡病的发病率在前期低于30℃培养的植株,至第10天时,两个处理组植株的发病率接近;同时HPLC测定发现,21℃培养植株叶片3种自由态多胺(腐胺、亚精胺和精胺)含量高于30℃培养植株;RT-PCR分析表明,CHIGPXPR4A 这3个抗病基因的表达量在21℃培养植株中高于30℃培养植株,而AOS表达水平在两组材料中差异不明显。外源亚精胺处理显著增加了内源腐胺和亚精胺的含量,降低了所处理植株接种溃疡病菌后的发病率和病情指数,接种后14 d发病率比对照降低45%,病情指数比对照降低4.8,而由表型可见对照发病程度重于亚精胺处理材料。此外,亚精胺处理能够增强AOSCHIGPXPR4A 4个抗病基因的表达量。结论 高温下甜橙更易发生溃疡病的可能机制是高温抑制抗病基因的表达和多胺合成。外源多胺处理能够降低甜橙发生溃疡病,可能机制是多胺处理后增强了抗病基因的表达,诱发植株的抗病反应最终表现出抗病。因此,高温是影响溃疡病发生的一个关键环境因子,多胺有助于提高对柑橘溃疡病的抗性。

关键词: 柑橘, 溃疡病, 高温, 多胺, 抗病基因, 抗病性

Abstract:

【Objective】 Canker disease is one of the most devastating diseases that cause serious damages to citrus. It is more likely to occur under high temperature. The objective of this study is to elucidate the mechanism underlying the disease incidence at high temperatures, reveal its metabolic changes, and to provide important theoretical guidance for controlling the disease using certain chemicals.【Method】 Sweet orange (Citrus sinensis), which is sensitive to canker disease, was used as the experimental material. The sweet orange plants were pre-cultured for 3 d at either 21℃ or 30℃ prior to inoculation with Xanthomonas citri subsp. citri (Xcc), followed by evaluation of disease incidence. Expression of four defense-related genes, including AOS (allene oxide synthase), CHI (chitinase), GPX (glutathione peroxidase) and PR4A (pathogenesis-related protein 4A), in the plants pre-cultured at the two temperatures was assessed by semi-quantitative RT-PCR. Meanwhile, endogenous polyamines (putrescine, spermidine and spermine) in the plants pre-cultured at the two temperatures were also analyzed by HPLC. In addition, sweet orange plants were treated with exogenous spermidine (0.4 mmol·L-1), using water treatment as a control, before Xcc inoculation. Disease incidence and index of plants treated with either spermidine or water were compared, while endogenous polyamine contents and expression levels of defense-related genes (AOS, CHI, GPX and PR4A) in response to spermidine or water treatment were assessed. 【Result】 After inoculation with Xcc, it was found that plants pre-cultured at 21℃ exhibited a lower cankder disease incidence at the early stage when compared with the plants pre-cultured at 30℃. On the 10th day, the incidence of the two treatments was similar. HPLC analysis showed that content of the three free polyamines (putrescine, spermidine and spermine) in plants pre-cultured at 21℃ was significantly higher than that in the plants pre-cultured at 30℃. In addition, RT-PCR analysis indicated that the transcript level of three defense-related genes, CHI, GPX and PR4A, in plants kept at 21℃ was higher than that from 30℃, while there was no significant difference in AOS expression between the two groups. Exogenous application of spermidine remarkably enhanced levels of endogenous putresicne and spermidine, reduced disease incidence and index in comparison with water treatment. Spermidine treatment reduced the disease incidence by 45% and in comparison with the control after 14 days of inoculation. In addition, the disease index of the spermidine-treated samples was 4.8 lower than that of the control. Meanwhile, the phenotype indicated that the control displayed more serious symptom than that of spermidine treatment. Moreover, spermidine treatment could up-regualte mRNA abundances of all four defense-realted genes, including AOS, CHI, GPX and PR4A. 【Conclusion】 Sweet orange displayed susceptibility to citrus canker at high temperature, and the potential mechanisms underlying this phenomenon may be ascribed to inhibition of defense-related genes and suppression of polyamine biosynthesis. Exogenous polyamine treatment conferred enhanced tolerance to citrus canker by upregulating defense-related genes and triggering disease resistance response. Taken together, high temperature is one of the environmental factors accounting for outbreak of citrus canker disease, and polyamines are conducive for improving tolerance to citrus canker disease.

Key words: citrus, canker disease, high temperature, polyamines, defense-related genes, disease resistance