四倍体西瓜抗枯萎病菌生理小种1的机理

doi:10.3864/j.issn.0578-1752.2018.19.012

摘要/Abstract

摘要： 【目的】近年来由于西瓜重茬种植面积的不断增加及连作障碍的发生导致枯萎病在西瓜上迅速蔓延，该病害已经成为限制西瓜生产的主要因素之一。田间观察发现四倍体西瓜比其同源二倍体西瓜抗枯萎病。本研究通过对比四倍体与二倍体抗性差异，揭示四倍体西瓜抗枯萎病的机理，为西瓜多倍体育种和抗病育种提供理论依据。【方法】以同基因型的郑州3号二倍体和四倍体西瓜幼苗为材料，在一叶一心时期接种枯萎病菌生理小种1（Fusarium oxysporum f. sp. niveum race 1，Fon 1），比较不同倍性西瓜苗期枯萎病抗性差异；运用绿色荧光蛋白标记技术观察枯萎病菌的侵染过程；并分析过氧化物酶（POD）、苯丙氨酸解氨酶（PAL）、丙二醛（MDA）、总酚及类黄酮等与抗病相关代谢物质活性/含量差异，及PR3、MPK7、PAL、MYB基因表达变化。【结果】西瓜苗期接种枯萎病菌后，二倍体西瓜幼苗4 d发病，10 d时大部分枯死；四倍体西瓜幼苗7 d开始发病，13 d时大部分枯死，四倍体相对其同源二倍体延后3 d发病。枯萎病菌侵染过程对比发现，病菌在二倍体和四倍体西瓜中的侵染路径相同；此外不同倍性西瓜根系分生孢子的萌发率无明显差别，但是随着病菌的侵染，枯萎病菌在四倍体幼苗中的侵染速度明显较慢，细胞间菌丝较少；枯萎病菌在四倍体西瓜幼苗维管束中的定殖率比其同源二倍体低，且在幼苗根部差异显著、叶柄部差异极显著，病菌在四倍体植株茎部及叶部的扩展得到了一定的限制；枯萎病菌在四倍体西瓜中的侵染过程明显滞后，与枯萎病的发病症状相吻合。接种枯萎病菌后，四倍体西瓜幼苗根系POD、PAL活性均呈增加的趋势，且四倍体幼苗根系POD活性增加的幅度比二倍体大，病菌侵染后四倍体根系生成了更多的PAL和POD以增强植物抗病能力，保护细胞免受伤害；四倍体西瓜根系中总酚、类黄酮含量的增加量明显高于同源二倍体，这些次生代谢物质含量的优势，使得四倍体植株更易于抵御枯萎病菌的入侵；另外四倍体西瓜根系MDA含量明显比同时期的二倍体低，表明接种枯萎病菌后四倍体西瓜幼苗根系细胞膜损伤程度更小。抗病基因的表达分析发现，接种枯萎病菌后四倍体PR3的表达量不断增加，在接种后10 d表达量达到最大，是二倍体同时期表达量的10倍；接种后期四倍体根系能表达更多的MPK7，传递抗病信号，促进抗病基因的表达，从而降低枯萎病菌对西瓜幼苗的伤害；PAL的表达量呈现先增高后降低的趋势，表达量均高于同时期的二倍体幼苗，最大表达量是二倍体PAL表达量的6倍；四倍体MYB的表达量明显高于其同源二倍体，接种后7、10 d四倍体根系的表达量分别是二倍体的80、35倍。【结论】通过苗期枯萎病菌接种鉴定、病菌侵染过程观察、代谢物质含量及基因表达水平变化等多方面的研究，表明四倍体西瓜幼苗比同源二倍体抗枯萎病。

关键词：西瓜；四倍体；枯萎病菌生理小种1（Fon 1）；抗性机理

关键词: 西瓜, 四倍体, 枯萎病菌生理小种1（Fon 1）, 抗性机理

Abstract: 【Objective】The Fusarium wilt disease caused by the continuous cropping obstacles is one of the main factors which limits the production of watermelon in recent years. Field observation shows that tetraploid watermelon is more resistant to Fusarium wilt disease than autodiploid watermelon. The objective of this study is to reveal the mechanism of resistance to Fusarium wilt in tetraploid watermelon by comparing the resistant differences of diploid and tetraploid watermelon, and to provide a theoretical basis for polyploidy breeding and disease resistance breeding of watermelon.【Method】The diploid and autotetraploid watermelon seedlings of Zhengzhou No. 3 were used as materials in this research, the differences of the resistance to Fusarium wilt of different ploidy watermelon seedlings were compared after inoculating with Fusarium oxysporum f. sp. niveum race 1 (Fon 1) at one leaf stage, the Fon 1 with green fluorescent protein (GFP) was used to observe the infection process in different ploidy watermelon seedlings, then the activity/content of metabolites related to disease resistance such as peroxidase (POD), phenylalanine ammonialyase (PAL), malondialdehyde (MDA), total phenols, flavonoid and the expression levels of PR3, MPK7, PAL and MYB at different stages of diploid and tetraploid watermelon roots were determined.【Result】After inoculation with Fon 1, for the diploid watermelon seedlings, the Fusarium wilt was observed at the 4th day, and most of the plants died at the 10th day, while the disease was observed at the 7th day, and most of them died at the 13th day in the tetraploid watermelon seedlings. The wilt symptom delayed 3 days in tetraploid watermelon seedlings, tetraploid watermelon seedlings were more tolerant to Fusarium wilt than diploid ones. The infection pathway of Fon 1 was the same in both diploid and tetraploid watermelons. In addition, there was no significant difference in the germination rate of conidia between diploid and tetraploid watermelon roots. However, with the infection of pathogens, the infection speed of Fon 1 in tetraploid seedlings was obviously slower and the intercellular hyphae were less than those in diploid ones. The colonization rate of Fon 1 in the xylem of tetraploid watermelon seedlings was lower than that in diploid plants, which showed significant differences in the roots and extremely significant differences in the petioles of the seedlings, the expansion of the pathogen in the stems and leaves of tetraploid plants was limited to a certain extent. The infection process of Fon 1 in tetraploid watermelon apparently lagged, which coincided with the disease symptoms. After inoculation with Fon 1, the activity of POD and PAL in tetraploid watermelon seedlings increased, the POD activity of tetraploid watermelon seedlings increased more than that of diploid watermelon seedlings. Tetraploid roots produced more POD and PAL to enhance the plant disease resistance and protect cells from damage. The increasing of total phenols and flavonoid contents in tetraploid watermelon roots were higher than those in diploid ones. The advantages of these secondary metabolites made tetraploid plants more resistant to the invasion of Fon 1. In addition, the content of MDA in the root of tetraploid watermelon was obviously lower than that in diploid at the same time, which indicated that the damage degree of root cell membrane of tetraploid watermelon seedlings was lower after inoculation with Fon 1. Analysis of the expression of resistant genes showed that the expression level of PR3 tetraploid watermelon roots increased continuously after inoculation with Fon 1, and reached the highest level at the 10th day, which was 10 times of that of diploid at the same time. At the late stage of inoculation, tetraploid roots exhibited higher expression of MPK7 to transmit resistance signals, and the expression of disease resistance genes could be promoted, the damage of Fon 1 on watermelon seedlings would be reduced. The expression level of PAL increased firstly and then decreased, and the expression level was higher than that of diploid at the same period, the maximum expression level was 6 times of that of autodiploid. The expressions of MYB in tetraploid watermelon roots was continuously higher than that of autodiploid, and the expression level was 80 and 35 times of that of diploid on the 7th and 10th day after inoculation with Fon 1.【Conclusion】Through inoculation and identification of Fon 1 at seedling stage, observation of pathogen infection process, changes of metabolic substance content and gene expression level research, the results show that tetraploid watermelon seedlings are more resistant to Fusarium wilt than autodiploid watermelon seedlings.

Key words: watermelon, tetraploid, Fusarium oxysporum f. sp. niveum race 1 (Fon 1), resistance mechanism

姬万丽，朱红菊，路绪强，赵胜杰，何楠，耿丽华，刘文革. 四倍体西瓜抗枯萎病菌生理小种1的机理[J]. 中国农业科学, 2018, 51(19): 3750-3765.

JI WanLi, ZHU HongJu, LU XuQiang, ZHAO ShengJie, HE Nan, GENG LiHua, LIU WenGe. The Mechanism of Resistance to Fusarium oxysporum f. sp. niveum Race 1 in Tetraploid Watermelon[J]. Scientia Agricultura Sinica, 2018, 51(19): 3750-3765.

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