来源于同一穗不同稻曲球的稻曲病菌的致病性及遗传多样性

 本研究从源于6穗稻曲病穗的48个稻曲球中分离获得稻曲病菌（Ustilaginoidea virens）48株,从3个稻曲球的不同部位分离获得稻曲病菌23株。用注射接种法将菌株分别接种到水稻品种两优培九（感病品种）、淮稻5号（中抗品种）和武育粳3号（抗病品种）上,结果显示分离的菌株致病力分化较大,而菌株在水稻品种上的致病力强弱与已知水稻品种对稻曲病菌的感、抗性趋势基本一致。相同孢子量接种水稻,不同分离菌株之间仍有致病力分化,生长速率测定也发现菌株之间可能存在差异。利用REP PCR (repetitive extragenic palindromic sequence PCR)技术进行菌株遗传多样性分析表明,同穗不同稻曲球分离的菌株中,1号穗分离的4个菌株聚在同一簇群,其余5穗的菌株分别聚在3～5个簇群;同一稻曲球不同部位分离的菌株中,一个稻曲球分离的8个病菌聚在同一簇群,而其余2个稻曲球分离的病菌则分别聚在2～3个簇群。由此推测同一稻穗上不同稻曲球可能是由来源不同的稻曲病菌侵染所形成;而一个稻曲球可以由同一稻曲病菌引起,也存在多个侵染源共同侵染的可能。     

辽宁省稻曲病菌遗传多样性和致病力分析

为有效防治辽宁省稻曲病菌Ustilaginoidea virens,利用重复序列PCR(repetitive elementbased PCR,rep-PCR)分子指纹技术,对2017年自辽宁省8个市8个主产稻区采集的51株稻曲病菌菌株进行遗传多样性和致病力分析。结果显示,在3对引物中,以BOX1/BOX2和ERIC1/ERIC2为引物扩增的DNA指纹图谱的遗传多样性值分别为0.764、0.707,均大于0.7,故选择这2种引物扩增的DNA指纹图谱进行遗传多样性分析;当DNA指纹相似系数为0.78时,以BOX1/BOX2为引物和以ERIC1/ERIC2为引物扩增的DNA指纹图谱分别将供试菌株划分为12个和10个遗传类群;供试菌株致病力可划分为弱致病型、中等致病型和强致病型3个致病型,所占比例分别为33.33%、58.82%和7.85%,强致病型菌株仅在沈阳市、鞍山市和大连市出现;所有优势类群均包含3种致病型菌株。表明辽宁省稻曲病菌遗传结构复杂,不同地理来源的稻曲病菌菌株致病力存在一定差异,相同致病型的稻曲病菌菌株分属于不同的遗传类群,同一遗传类群中包含不同的致病型菌株。     

稻曲病菌遗传多样性与群体结构的初步分析

 利用随机扩增多态性DNA (random amplified polymorphic DNA,RAPD)初步分析了稻曲病菌(Ustilaginoidea virens)的群体遗传结构。从1 60个随机引物中筛选32个扩增带型清晰、重复性好的引物,对不同年份采自辽宁、云南、湖北和浙江等水稻种植区的5 6个菌株进行扩增。32个引物扩增出2 2 3条带,绝大多数引物对不同年度采自不同稻区的菌株扩增的DNA谱型相同,大多数菌株间相似性系数达0.80以上。根据扩增DNA片段的多态性,从空间分布来看,来源于北方、长江流域和南方的菌株难以划分出明显的地理宗谱;不同年度的菌株DNA多态性也无明显的差异。上述结果初步表明稻曲病菌遗传稳定,寄主选择作用(寄主的基因型及其时空分布)对稻曲病菌变异的影响较小。但是尚需采用其它的分子技术测试更多的菌系,才能较系统地分析我国稻曲病菌系的遗传变异及群体结构特点。     

中国部分地区稻曲病菌培养特性及其遗传多样性分析

采用生物学方法和RAPD-PCR技术,对来自11个省(市)84个菌株的菌丝生长速率、分生孢子数量、孢子萌发率及其遗传多样性进行分析,以明确中国部分地区稻曲病菌株的培养特性和遗传多样性。依据菌丝生长速率,菌株可被划分为快和慢2种类型,分别占58.33%和41.67%。依据产孢力和分生孢子萌发力,菌株可划分为强、中和弱3种类型。采用12条RAPD引物共扩增出323条带,多态性条带比率为98.14%,遗传距离变化范围为0.02～1.00。在遗传距离0.725水平上,所有菌株被划分成7个遗传聚类组,聚类组R4和R5为优势聚类组,并存在一些亚组。不同地区之间和同一地区内的菌株表现出不同程度的变异,内陆地区的菌株群体变异程度明显高于沿海地区。从采用相同接种体接种的水稻品种上分离的稻曲病菌具有紧密的亲缘关系。     

辽宁稻区稻曲病菌生物学特性及遗传多样性分析

为明确采自辽宁省12个稻区稻曲病菌Ustilaginoidea virens的生物学特性、群体遗传多样性与地理区域的关系。本研究采用生物学方法测定稻曲病菌菌株的生长速率和产孢能力,并采用SPSS 20.0分析软件对稻曲病菌菌株的菌丝生长速率和产孢能力进行相关性分析。提取稻曲病菌基因组DNA,采用特异性引物US、交配型引物MAT、遗传多样性引物ERIC对其进行PCR扩增,通过聚类分析进行群体遗传多样性研究。结果显示:157个稻曲病菌菌株的菌丝生长速率与产孢能力相关系数为0.19。采用稻曲病菌特异性引物US进行扩增,157个菌株均为稻曲病菌株;采用交配型引物MAT进行扩增,53个菌株为MATⅠ型,104个菌株为MATⅡ型。采用ERIC引物可扩增出2~7条不等的条带,157个稻曲病菌株被划分为10个基因类群,其中第1类群为优势类型,有44个菌株,占总数的28.0%;第2类群有20个菌株,占总数的12.7%;第3类群1个菌株,占总数的0.6%;第4类群1个菌株,占总数的0.6%;第5类群有21个菌株,占总数的13.4%;第6类群有17个菌株,占总数的10.8%;第7类群有2个菌株,占总数的1.2%;第8类群有5个菌株,占总数的3.2%;第9类群有30个菌株,占总数的19.1%;第10类群有16个菌株,占总数的10.2%。来自辽宁省12个稻区的157个稻曲病菌株菌丝生长速率与菌株产孢量之间没有相关性,产孢量与地域之间有相关性。基于ERIC-PCR扩增的稻曲病菌株基因组DNA指纹图谱的多态性进行划分的基因类群与地理区域之间有相关性,基因类群与生物学特性之间没有相关性。     

水稻上三种条斑病细菌DNA的多态性初析

 试用40个引物对我国水稻条斑病菌、"稻短条斑病菌"和李氏禾条斑病菌等14个代表菌株进行RAPD分析,其中11个引物的扩增产物表现明显的多态性,共扩增出158条谱带,多态性为89.74%。聚类分析结果显示14个菌株可区分为3个类群,第1群包括LLS2、LLS3、LLS4、RS05、R1008、TAS和TAX;来自不同稻区水稻条斑病菌的群体结构差异明显,分属于第2群(如RS-Hai等)和第3群(如RS105等)。菌株DNA-RAPD指纹分析和致病性测定结果证明:李氏禾条斑病菌、"稻短条斑病菌"在水稻和李氏禾上不仅可相互侵染,而且遗传背景的相似性较高,确认是同一种病菌,其与小麦黑颖病菌亲缘关系较近,但与水稻细菌性条斑病菌具有明显差异。     

采用AFLP技术分析稻曲病菌的遗传多样性Ⅰ:同一块稻田中稻曲病菌的遗传结构

 采用AFLP (Amplified fragment length polymorphism)技术分析了来自北京昌平同一块稻田中不同水稻品种和育种中间材料上稻曲病菌(Ustilaginoidea virens)的遗传多样性。从256对EcoRⅠ和MseⅠ引物中选择30对扩增40个菌株。结果表明,同一块稻田中稻曲病菌菌株间的相似性系数达0.72以上,来自同一小区的多数菌株能聚成亚类;发现从同一水稻品种分离的菌株没有特异性的AFLP谱带。初步推断稻曲病菌与水稻品种不存在明显的专化性互作。     

苹果炭疽病病原菌ISSR-PCR反应体系的优化及遗传多样性分析

为了从分子水平探讨苹果炭疽病病原菌的群体遗传多样性,采用L16 (45)正交设计对ISSRPCR反应体系中的Mg2浓度、dNTP浓度、Taq DNA聚合酶用量、引物浓度和DNA含量进行优化,并利用优化的体系进行引物筛选及苹果炭疽病菌遗传多样性分析.确立最优反应体系为2.0mmol/L Mg2+、0.2 mmol/L dNTP、2UTaqDNA聚合酶、1μmol/L引物和DNA 100 ng.筛选获得的10条引物对供试菌株共扩增出42条谱带,均为多态性条带.供试菌株在相似系数0.50处分为2个类群,分别与根据形态学鉴定的胶孢刺盘孢Colletotrichum gloeosporioides和尖孢刺盘孢C.acutatum 2个类群相一致,在相似系数0.74处,分为4个亚群,表明苹果炭疽病菌存在明显的种间及种内遗传分化.     

苦瓜枯萎病菌的鉴定及其遗传多样性分析

 对分离获得的32株苦瓜枯萎病菌菌株进行形态学特征和寄主专化型测定, 结果表明, 测试的苦瓜枯萎病菌株均为尖孢镰刀菌苦瓜专化型 (Fusarium oxysporum f. sp. momordicae), 这些菌株可以侵染苦瓜和瓠瓜幼苗, 但不侵染其他葫芦科瓜类作物。对苦瓜枯萎病菌菌株的rDNA-ITS区 (ITS1、5.8S和ITS2)序列进行扩增测序, 结果显示其序列长度均为456 bp;聚类分析表明测序菌株与镰刀菌属中尖孢镰刀菌不同专化型的菌株聚为一群。利用RAPD标记技术分析苦瓜枯萎病菌的遗传多样性, 结果显示苦瓜枯萎病菌株与其他葫芦科瓜类作物枯萎病菌株间的遗传相似系数范围为0.59~0.99, 当遗传相似系数为0.85时, 供试的48个菌株分成10个类群 (G_1~10)。在RAPD聚类树中所有苦瓜枯萎病菌株聚在一个分支上 (G₁群), 菌株间的遗传相似系数范围为0.92~1.00, 具有较高的遗传相似性, 且菌株的聚群与地理来源存在一定的相关性。     

甘薯茎腐病菌的遗传多样性及致病力差异分析

为了解不同地区甘薯茎腐病菌Dickeya dadantii种群遗传多样性水平及致病力差异,采用重复序列PCR基因指纹(repetitive element palindromic PCR,REP-PCR)技术和薯片接种方法,对采自广东省、广西壮族自治区和重庆市的6个市区县的59株菌株进行分析。结果表明,5对引物对59株菌株扩增出41个清晰的条带,其中36个为多态性条带,每对引物的扩增条带数在4～10之间,平均为7.2。在物种水平上,有效等位基因数、Nei’s基因多样性指数和Shannon信息指数分别为1.4768、0.2801和0.4186,其中湛江种群多样性最高,南宁种群多样性最低;当遗传相似系数为0.79时,59株菌株可被划分为5个类群,类群划分与菌株来源地间有一定的相关性。此外,不同地区病菌种群间存在明显的致病力差异,其中合浦种群与湛江种群致病力最强,万州种群致病力较弱。表明甘薯茎腐病菌种群具有丰富的遗传多样性,不同地区的病菌种群存在明显的遗传多样性与致病力差异。     

水稻品种构成对稻曲病菌遗传结构影响的初步研究

由稻曲病菌（Ustilaginoidea virens）引起的病害是当前限制我国水稻优质高产的重要病害之一。家畜食用被稻曲病菌厚垣孢子污染的谷物可导致中毒。近20年来，稻曲病防治主要以喷施化学农药为主。研究品种布局对稻曲病菌群体结构的影响，有助于了解病害的发生规律及提出有效的病害防治策略。本文初步分析了稻曲病菌群体结构与水稻品种构成的关系。     

Pathotypic and genetic diversity in the population of Rhizoctonia solani AG1‐IA causing rice sheath blight in China

Sheath blight, caused by Rhizoctonia solani AG1‐IA, is one of the most serious diseases of rice. In this study, a total of 175 isolates of R. solani AG1‐IA were collected from five rice‐growing regions in China. Pathogenicity tests revealed that all isolates were virulent to five cultivars with different levels of resistance at the rice seedling stage in the greenhouse. There was considerable variation in aggressiveness, and the isolates were classified into three pathotypes based on disease severity, with moderately virulent isolates prevalent in the population. Forty‐three haplotypes were identified based on ITS sequencing, and 39 haplotypes were distinct among isolates. There were high levels of haplotype diversity and nucleotide diversity within the populations of R. solani AG1‐IA. High gene flow (Nm = 1·63–5·22) was detected, consistent with relatively low differentiation between pairs of populations. Five populations were divided into two distinct clusters by the unweighted pair group method with arithmetic mean (UPGMA), and no spatial population differentiation was discernible. The majority (97·8%) of genetic diversity was distributed among isolates within populations, with only 2·2% of the genetic diversity attributed to differences among populations. The star‐like shape of the haplotype network provided evidence of signatures of population expansion in recent history. No significant relationships were found between the genetic diversity and aggressiveness or geographic origin among populations of R. solani AG1‐IA. These results highlight that the population characteristics of R. solani AG1‐IA should be taken into account in evaluating the germplasm resistance of rice cultivars to sheath blight.     

Reaction of <Emphasis Type="Italic">Leersia</Emphasis> grasses to <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis> collected from Japan and Asian countries

The present study was conducted to determine if there is specificity in the host-pathogen relationship between the isolates of Xanthomonas oryzae pv. oryzae, the causal bacterium for rice blight and Leersia grasses, the alternative weed hosts of the disease. Plants of three species of Leersia, namely, L. sayanuka, L. oryzoides and L. japonica, were collected from various parts of Japan and were inoculated with the X. oryzae pv. oryzae isolates obtained from various locations in Japan and from 11 Asian countries. Four L. sayanuka plants were found susceptible to all Race II isolates and some Race I isolates, but were resistant to all Race III isolates. Race III is known to have a wider range pathogenicity to rice cultivar groups compared with Race I and II. Although the reactions of two L. oryzoides plants to Race I and II isolates were similar to that of L. sayanuka, the L. oryzoides plant collected from Niigata Prefecture showed a susceptible reaction to some Race III isolates. On the other hand, L. japonica plants gave reactions different those of L. sayanuka and L. oryzoides, with two plants of L. japonica found to be resistant to all test isolates collected from Japan. The Asian isolates exhibited a wide host range against the international differential rice cultivars, but almost all of them were avirulent to Leersia plants. These results indicate that the relationship between the pathogenicity of the causal bacterium and the resistance of host plants is very complex, and suggest that pathogenic diversity of X. oryzae pv. oryzae might be related to the resistance of Leersia spp.     

Population genetic structure of <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> AG1IA from rice field in North India

Rhizoctonia solani AG1IA is an important fungal pathogen causing significant yield and quality losses in rice production. However, little is known about the levels of genetic diversity and structure of this pathogen in North India. Out of 240 samples collected from different rice-growing regions of North India, 112 isolates were identified as R. solani AG1IA subgroups using species-specific primers. All 112 isolates were organized into four groups on the basis of percent disease index (PDI). The majority of the isolates were weakly virulent. Population genetic analysis was performed within and between populations using inter simple sequence repeat (ISSR) markers. A total of 8249 alleles were identified from the 112 isolates of R. solani AG1IA through analysis of the ten inter simple sequence repeat markers. All the ten ISSR markers were polymorphic. The average number of bands per primer was 7.3 which ranged in size from 250 to 1500 bp. Genetic structure of the isolates using inter simple sequence repeat primers showed high degree of polymorphism (PIC ≥0.81). The analysis of molecular variance (AMOVA) indicated that most of the genetic diversity occurred within populations (60%), while the variability among populations and among regions contributed 25 and 15%, respectively. Overall, the present study reveals that a large variation exists among rice-infecting isolates of R. solani AG1IA in North India. Fingerprinting of the isolates using ISSRs along with phenotypic characterization and virulence analysis will help epidemiological studies that can provide new insights into pathogen biology and disease spread.     

我国玉米灰斑病菌遗传多样性的ISSR分析

为明确我国发生的玉米灰斑病菌地理差异及遗传结构,利用简单序列重复区间(ISSR)对玉米灰斑病菌遗传多样性进行了分析,并利用尾孢菌特异引物对分离自四川、云南、湖北、贵州等西南地区的16个玉米灰斑病菌菌株进行了分子鉴定。结果显示,通过ISSR标记筛选出10个扩增多态性好且稳定的通用引物,共扩增出81条DNA条带,均为多态性条带,扩增片段大小在200~2 000 bp之间,菌株遗传相似系数为0.19~1.00。在遗传相似系数为0.19时,供试菌株被聚为2大类群,来自西南地区和东北地区的菌株各自聚为一组,在DNA水平上表现出明显差异,认为是2类不同的致病类群。分子鉴定结果显示引起西南各地区玉米灰斑病的主要致病菌均为玉米尾孢菌Cercospora zeina。表明我国玉米灰斑病菌存在丰富的遗传多样性,ISSR标记可揭示出玉米灰斑病菌株间的亲缘关系及遗传差异性,可用于其遗传多样性研究。     

Characterization of Pseudoperonospora cubensis isolates from Europe and Asia using ISSR and SRAP molecular markers

Downy mildew caused by Pseudoperonospora cubensis is a major disease of cucurbits worldwide. New genotypes of the pathogen have recently appeared in the USA, EU and Israel causing breakdown of genetic resistance, expansion of host range, and the appearance of a new A2 mating type. Seventy-eight P. cubensis isolates were collected during 1996–2011 from cucurbits fields in different regions of Turkey, Israel and the Czech Republic and genetic diversity was analysed using highly polymorphic ISSR and SRAP molecular markers. The data acquired showed remarkable genetic diversity within and among the isolates. While isolates from Turkey and Czech Republic exhibited uniform genetic background, the isolates from Israel were clearly distinguished from the others. The results may indicate on migration and/or frequent sexual reproduction of the pathogen in Israel. Moreover the selected markers can be suggested for monitoring genetic diversity within P. cubensis isolates in further studies.     

西南地区稻瘟病菌群体遗传多样性分析

为明确西南地区稻瘟病菌Magnaporthe grisea(Hebert)Barr群体遗传结构及其多样性水平,选用13对SSR引物对来自18个县(市)的221个稻瘟病菌单孢菌株进行PCR扩增,利用最长距离法和生物学软件进行聚类分析和群体遗传多样性分析。结果显示,13对SSR引物均能扩增出一条大小相同且清晰的条带,多态位点百分率高达100%。221个菌株在0.16相异水平上可划分为13个遗传宗谱,宗谱SCL01含205个菌株,占总菌株数的92.76%,为优势宗谱;宗谱SCL02~SCL013为劣势宗谱,差异极大。在群体水平上,菌源丰富的8个区域稻瘟病菌群体的Nei’s基因多样性指数为0.2133,Shannon信息指数为0.3588,具有丰富的遗传多样性,且群体间差异较大;这8个种群基于UPGMA法大都聚为一类,种群遗传谱系与地理区域分布呈一定相关性,群体遗传多样性均值为0.2518,存在一定的遗传分化,且群体内多样性大于群体间,总遗传变异的59.37%存在于群体内。总体上,西南地区稻瘟病菌群体结构既有明显的优势宗谱,又存在许多复杂多变的特异性小宗谱,具有丰富的遗传多样性,与地理分布关系较为密切。     

安徽省水稻条斑病菌群体遗传结构分析

 水稻条斑病菌是近年来影响安徽水稻生产的主要有害生物。本研究利用rep-PCR指纹技术分析了来自安徽11个不同县市的水稻条斑病菌群体遗传结构。用引物BOX、REP和ERIC分别对94个菌株的基因组DNA进行了PCR扩增,结果表明3组引物共扩增出了49条指纹条带,且所扩增出的DNA条带均为多态带。在群体平均水平上,安徽省水稻条斑病菌群体Nei’s基因多样性指数(H)为 0.32,Shannon 信息指数(I)为 0.49,表明安徽省水稻条斑病菌的遗传多样性丰富,但病菌的遗传多样性在地区间存在差异。UPGMA聚类分析表明,来自毗邻地区的水稻条斑病菌种群大都聚为一类,水稻条斑病菌种群遗传谱系与地理区域分布呈现一定相关性。同时,安徽省水稻条斑病菌群体存在一定的遗传分化,遗传变异主要来源于群体内部。