浙皖鄂地区水稻纹枯病菌5个种群的遗传结构分析

Genetic structure of five populations of the rice sheath blight pathogen Rhizoctonia solani AG-1 IA from provinces of Zhejiang,Anhui and Hubei

水稻纹枯病是世界性的主要病害之一。目前对该病病原菌种群的遗传多样性研究不多,知之甚少,了解其种群的遗传结构可以增加对其进化历程的了解,以制定科学的防治策略。水稻纹枯病菌通常被认为是以无性克隆繁殖为主,但有研究报道它具有混合繁殖方式。有关我国浙皖鄂地区水稻纹枯病菌种群的遗传多样性研究尚未见报道。为了解该地区水稻纹枯病菌种群的遗传变异、基因流、繁育方式及其遗传背景,采用ITS-5.8SrDNA测序技术,分析了分离自浙江富阳(FY)、安徽绩溪(JX)和巢湖(CH)以及湖北荆州(JZ)和孝感(XG)的5个水稻纹枯病菌种群75个菌株的遗传多样性。RhizoctoniasolaniAG-1IA是采集地区水稻纹枯病菌的优势类群。ITS-5.8SrDNA序列经测定共检测到78个多态位点,碱基A、T、C、G的平均含量分别为25.4%、33.6%、21.0%和20.0%。序列的平均转换与颠换比(Ti/Tv)为1.65,其中密码子第3位点的变异最高。根据序列的核苷酸变异共定义了29种单倍型,其中单倍型H5为5个种群的共享单倍型,占样本数的61.33%。5个种群的单倍型多样性和核苷酸多样性分别为0.627和0.482%,显示水稻纹枯病菌种群具有较高的遗传多样性。种群间固定化指数Fst为-0.0253-0.0170,基因流Nm为5.56-11.12,说明种群间基因交流频繁,基因流抑制了由遗传漂变引起的遗传分化,菌丝或菌核短距离扩散和带菌种子远距离传播增加了种群间的基因交流。AMOVA分析显示,种群间的遗传变异仅占总变异的19.03%,而80.97%的变异存在于种群内部,种群间的遗传分化很低。Mantel检验发现,遗传距离与地理距离无显著相关性(r=-0.241,P=0.499)。采用UPGMA法构建的单倍型间的系统发育树表明,不同地点的单倍型分支混合分布,这进一步验证了Mantel检验的结果。单倍型的网状分析显示,水稻纹枯病菌种群曾经发生过种群暴发而不断扩散,因还未能获得足够的时间建立更加复杂的结构故而呈非典型"星状"。采用中性检验分析了水稻纹枯病菌种群遗传结构,结果表明,种群间存在很强的自然选择作用,群体符合Hardy-Weinberg遗传平衡,说明水稻纹枯病菌群体是一个随机交配群体,具有以担孢子进行有性繁殖和以菌丝或菌核进行无性繁殖的混合繁殖方式。这种生物学特性可能是导致其在较小生态范围内较高的遗传多样性水平和较低的种群遗传分化的原因。另外,水稻纹枯病菌经有性繁殖产生新的基因型,并通过无性繁殖在群体内固定繁殖,这种遗传模式极有可能导致其进化潜能提高,极易对杀菌剂产生抗性。因此,对水稻纹枯病菌的防治,除了施用化学药剂和种植抗性品种外,还需要防治农田灌水引起的病原菌(菌丝和菌核)在地区间的流动传播,减少带菌种子迁移或农用机械的交叉污染,对水稻、大豆和玉米等寄主作物的种子进行播前处理等等,这些对于水稻纹枯病菌的防治也是极为重要的。

Rice sheath blight （RSB） is one of the most devastating rice diseases on a global basis.Genetic diversity of RSB pathogen population is poorly understood.Studying on the genetic structure of the population will help to understand the evolution of the pathogen,and to design better strategies to control the disease.In general,the RSB pathogen is thought to be an asexual fungus,though a mixed reproductive mode has been reported.No information is available about the genetic diversity and population structure of the fungus in provinces of Zhejiang,Anhui and Hubei.To reveal the genetic variation,gene flow,reproductive mode and genetic background of the RSB pathogen in these areas,ITS-5.8S rDNA sequencing approach was used to analysis seventy five isolates from the infected samples collected from Fu Yang （FY） in Zhejiang Province,Ji Xi （JX） and Chao Hu （CH） in Anhui Province,as well as Jing Zhou （JZ） and Xiao Gan （XG） in Hubei Province.The dominant pathogen of RSB in these areas was Rhizoctonia solani anastomosis group AG-1 IA.Seventy eight variable sites were detected from ITS-5.8S rDNA sequences of these seventy five isolates,the content of A,T,C and G nucleotides were 25.4%,33.6%,21.0% and 20.0%,respectively.The transition/transversion （Ti/Tv） ratio was 1.65 and the highest variation position was in the third-codon.Twenty nine haplotypes were identified based on nucleotide variation,and haplotype 5 （H5） was appeared on all 5 area populations with the highest frequency of 61.33%.A high genetic diversity was observed among populations with 0.627 for haplotype diversity and 0.482% for nucleotide diversity.The fixation index （Fst） and gene flow parameter （Nm） among populations were-0.0253 to 0.0170 and 5.56 to 11.12,respectively,indicating that the gene flow were frequent among populations and overcome the impact of genetic drift.Short-distance dispersal of asexual propagules （mycelium or sclerotia）,and long-distance dispersal via contaminated seeds may enhance the gene flow.Analysis of molecular variance （AMOVA） revealed that only 19.03% of genetic variation occurred among populations whereas 80.97% existed within populations,suggesting a very low genetic differentiation among populations.Mantel test showed that no significant correlation was found between genetic distance and geographic distance （r=-0.241,P=0.499）.Phylogenetic tree of haplotypes constructed by UPGMA methods exhibited highly mixed branches among different areas,which was consistent with the results of Mantel test.A ＂star like＂ pattern of interrelationship of haplotypes from R.solani AG-1 IA was found by network analysis,suggesting a bread out of population during its evolution,because of insufficient time for the pathogen to form a more complicated population structure.Neutral test for genetic structure showed that the populations were in Hardy-Weinberg （HW） equilibrium suggesting a high natural selection existed in the populations.These results also showed that a mixed reproductive mode,including asexual and sexual reproduction in the populations of R.solani.The characteristics could explain why a high genetic diversity and low genetic differentiation existed among populations in small areas.Pathogen with a mixed reproductive mode would accelerate its evolution,because more new geontypes could be generated by recombination and kept through asexual reproduction,which would cause high risk of fungicides resistance.Therefore,the disease control measures will include reducing the spread of fungal mycelia and sclerotia via shared irrigation systems or contaminated seed to minimize the gene flow,pretreating the seeds,as well as fungicide and resistance varieties application.