四川籼稻区稻瘟病菌群体遗传结构

应用rep-PCR分子指纹技术对2000～2002年采自四川6个籼稻自然生态区的137个稻瘟病菌菌株进行了DNA分子指纹扩增和聚类分析,共获得73个不同的DNA指纹图谱(单元型)和62条分子量不等的DNA带型.结果显示,无论以何种遗传相似水平划分,四川稻瘟病菌的群体结构都表现很突出的优势宗谱,又存在着具有较多遗传多样性的次要小宗谱和特异性宗谱,蕴含着极其丰富的遗传信息;在0.19遗传相似水平,所有供试菌株可以划分成37个遗传宗谱,层次较为丰富.四川稻瘟病菌群体结构具有明显的时空特点,不同年度间稻瘟病菌群体存在一定的亲缘关系,又各自拥有当年的特异性宗谱;在空间上,不同稻作区表现出从复杂到简单的病菌群体变化特点.稻瘟病菌的遗传宗谱与生理小种致病型不存在一一对应的关系,作者认为将二者横向比较没有可比性.     

江西省稻区稻瘟病菌遗传宗谱与致病型的关系

为了探寻稻瘟病菌无性世代DNA水平的变异,明确江西省稻区稻瘟病菌遗传宗谱与致病型之间的对应关系,利用rep-PCR(repetitive element-based PCR)分子指纹分析技术,对稻区稻瘟病菌的群体结构和遗传多样性进行分析,并用41株代表性菌株对35个水稻品种进行了致病性测定。结果表明,以相似度75%为界,可以将不同稻区采集的99个菌株划分为14个遗传宗谱,其中,宗谱4、1和10为优势宗谱,分别包含37、18和12个菌株,占总数的37.37%、18.18%和12.12%;稻瘟病菌遗传宗谱与致病型间存在复杂的关系,同一宗谱的菌株对应多个致病型,而同一致病型的菌株,分属于不同的遗传宗谱,两者之间不存在简单的对应关系。     

福建病圃与大田稻瘟菌群体遗传谱系和毒性类型的比较

用rep-PCR方法分析了病圃和大田的稻瘟菌的遗传谱系组成,并在CO39NILs6个近等基因系品种上进行毒性类型分析。结果表明,不同群体之间的遗传谱系和毒性类型均不完全相同,福建稻瘟菌群体在年度间存在明显的优势谱系,1999、2000年的优势谱系均为CFL03,2001年为CFL07;两个季节中的谱系类型组成差异小,早季病圃和大田及晚季大田优势谱系均为CFL07。年度间病圃与大田的毒性类型组成和优势类型都有很大的变化,1999年病圃的优势毒性类型为I20.1,而2000年则为I24.1和I34.1两个类型;毒性类型I1.1、I5.1、I11.1、I26.1和I35.0只在1999年出现,而毒性类型I4.1和I14.1只在2000年出现。两个季节中病圃的毒性类型组成有所差异,早季有1个毒性类型(I35.1)在晚季未出现,晚季有4个毒性类型(I10.1、I31.1、I32.1、I21.1)在早季中未出现,优势毒性类型早季的I14.1变为晚季的I20.1,毒性类型组成也有很大差异,且晚季的比早季的丰富。     

基于毒性相关基因序列的稻瘟病菌群体遗传多样性分析

 选用16对毒性相关基因特异性引物对四川和重庆9个县(市)分离到的200个稻瘟病菌单孢菌株进行PCR扩增,并采用最长距离法进行聚类分析,结果显示各引物均能扩增出其目的条带,多态位点百分率(P)高达93.75%,扩增频率差异较大;200个菌株可归为70个不同的单元型,其中单元型SCH13为优势单元型;在0.86遗传相似水平上,200个菌株可划分为27个遗传宗谱,包括1个优势宗谱,3个亚优势宗谱,14个次要宗谱,9个小宗谱,层次丰富;在群体平均水平上,病菌群体具有丰富的遗传多样性(H=0.324 4,I=0.484 2),且群体间差异较大;9个种群在遗传距离为0.05水平上可分为4个类群,种群遗传谱系与地理区域分布呈一定相关性。同时,该地区的群体存在一定的遗传分化(HT=0.320 0),群体内多样性大于群体间多样性(Hs=0.179 6,Dst=0.140 4),总遗传变异的56.13%存在于群体内(Gst=0.438 7),群体间基因流动性较小(Nm=0.639 6)。本研究揭示了四川和重庆部分区域稻瘟病菌群体遗传结构、遗传多样性及其与地理分布之间的关系,为抗病育种和品种布局奠定了基础。     

黑龙江省稻瘟病菌育性及其交配型分析

为明确黑龙江省采集自不同年份、不同地区的稻瘟病菌Magnaporthe oryzae的育性能力和交配型分布,采用2株标准菌株GUY11(MAT1-2)和KA3(MAT1-1)对2016—2017年黑龙江省西部、东部、中部3个地区经单孢分离的241株稻瘟病菌进行育性测定,并利用PCR技术对其交配型进行检测。结果表明,黑龙江省西部、东部、中部的241株稻瘟病菌中可育性菌株比例为11.62%,其中雌性菌株、雄性菌株、两性菌株分别占1.66%、4.56%和1.25%,不能判断其性别的未知菌株占4.15%。采集自不同地区、不同年份的稻瘟病菌可育性差异均较大,西部、东部、中部地区可育性菌株出现频率分别为13.25%、7.27%和12.62%;2016年采集的稻瘟病菌可育性较高,可育性菌株出现频率为25.30%。黑龙江省稻瘟病菌群体中同时存在MAT1-1和MAT1-2两种交配型,主要以交配型MAT1-1占优势,出现频率为58.92%,交配型为MAT1-2的菌株出现频率为8.30%。不同地区稻瘟病菌的交配型亦有差异,交配型为MAT1-1的菌株在黑龙江省东部地区出现频率最高,为72.73%,在中部、西部地区的出现频率次之,分别为61.17%和46.99%。表明黑龙江省水稻种植区的稻瘟病菌同时存在2种交配型菌株,其交配型存在丰富的多态性,但其可育性及交配型分布不均衡。     

基于SSR序列的江西省不同生态地区稻瘟病菌遗传多样性分析

为明确江西省稻瘟病菌Magnaporthe oryzae群体遗传结构及其多样性水平,选用13对SSR引物对分离自5个不同生态地理县(市)水稻穗颈瘟标样的稻瘟病菌单孢菌株的全基因组进行PCR扩增,利用最长距离法和POPGENE 32生物学软件对其进行聚类分析和群体遗传多样性分析。结果显示,共分离获得189株稻瘟病菌菌株,13对SSR引物对其均能扩增出1条大小相同且清晰的条带,多态性位点百分率高达100.00%。供试189株稻瘟病菌菌株在相似系数为0.74时可划分为15个遗传宗谱,其中宗谱JXL01包含71株菌株,占总菌株数的37.57%,为优势宗谱;宗谱JXL02、JXL14为亚优势宗谱,分别包含31、26株菌株,占总菌株数的16.40%和13.76%;宗谱JXL03、JXL08、JXL10为次要宗谱,包含10～17株菌株;其它9个宗谱为小宗谱,包含菌株都在5株以下。在群体水平上,来源于不同生态型地区的5个稻瘟病菌群体的Nei’s基因多样性指数为0.375,Shannon信息指数为0.558,具有丰富的遗传多样性,且群体间差异较大;这5个种群基于非加权配对平均法大多聚为一类,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0.373,存在一定的遗传分化,且群体内多样性大于群体间多样性,总遗传变异的64.56%存在于群体内。表明江西省稻瘟病菌群体结构既包含明显的优势宗谱,又存在复杂多变的特异性小宗谱,遗传多样性丰富,且与地理分布有一定的相关性。     

水稻品种抗病性变化与稻瘟病菌致病性变异的相关效应研究

 根据1979~1992年对四川省稻瘟病菌致病性与水稻品种抗病性的监测结果表明:稻瘟病菌种群变异与水稻品种抗病性变化密切相关。潜在致病小种随水稻种植面积比率的扩大而增殖,最后成为优势小种。     

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。     

河南商丘地区棉花黄萎病菌分离鉴定和致病力分析

为探讨河南商丘地区棉花黄萎病菌的致病型群体变异,对该地区棉花上分离的8株单孢菌株的菌落形态、显微结构、致病力、ITS序列、系统进化及菌体蛋白等方面进行了研究。结果表明:这些菌株均属于棉花黄萎病菌Verticilliumdahliae;系统进化树显示8株菌株并没有聚在同一进化枝上;8株黄萎菌菌株存在致病力差异,SQ4菌株致病力最强,属于落叶型,而其它致病力较弱的7个菌株属于非落叶型;不同致病力的菌株间蛋白谱带存在差异。     

江西省稻瘟病菌的致病性分化

应用30个水稻抗稻瘟病近等基因系或单基因系及7个中国鉴别寄主,于水稻苗期接种,测定2006-2008年从江西省水稻产区分离的195个稻瘟病单孢菌株的致病性.结果表明,江西省稻瘟病菌以广谱致病性的菌株为主,将致病率按PF<30%、30%≤PF<50%、50%≤PF<70%和PF≥70%4区段划分,各区段菌株所占的比例分别为0、8.72%、31.28%和60.00%;病菌群体对所测定的30个抗瘟基因均表现出毒性,毒力频率为17.95%～100%,表现出较低毒力频率的水稻抗瘟基因是pi-z~t、PI-1(1)、PI-z~5和Pi-k,其毒力频率分别为17.95%、26.67%、27.46%和29.23%.此外,江西省稻瘟病菌含6群40个生理小种,在稻瘟病流行的2006年,主要的优势小种为ZB_(13)和ZA_1;在稻瘟病发生较轻的2007年和2008年,优势小种均为ZB_(15)和ZB_(13).进一步分析表明,同一优势小种内的不同菌株存在明显的致病性分化,28个ZB_(13)菌株对30个抗瘟基因的致病率为50.00%～96.67%,26个ZB_(15)菌株的致病率为46.67%-86.67%.对ZB_(13)与ZB_(15)菌株的聚类分析发现,毒力频率相近的菌株致病相似性差异很大.     

云南元阳哈尼梯田稻瘟病菌遗传多样性分析

 利用12对SSR引物,对来自云南元阳哈尼梯田不同海拔高度、不同水稻品种上的稻瘟病菌菌株进行遗传多样性分析,并运用日本清泽建立的鉴别品种对其中62个菌株进行生理小种鉴定。结果显示:稻瘟病菌总体遗传多样性水平较高(He=0.57±0.27,I=1.17±0.60)。聚类分析表明,在80%相似水平下菌株可划分为9个多样性群体。供试菌株划分为16个生理小种,其中002、000和006为优势小种。分离的不同海拔稻瘟病菌生理小种和遗传多样性存在差异。海拔1600~1700m生理小种最丰富,达9个生理小种;海拔1500~1600m、1400~1500m和1700~1800m,生理小种分别为6、5和4个;遗传多样性分析表明,从海拔1400~1500m分离的菌株遗传多样性水平最高(He=0.57±0.24),并划分为6个群体。综合分析表明,在元阳哈尼梯田系统中,栽培传统品种遗传多样性越丰富的区域,其稻瘟病菌群体多样性和生理小种也越丰富。     

Evidence of parasexual exchange of DNA in the rice blast fungus challenges its exclusive clonality

ABSTRACT We applied DNA markers to determine whether parasexual recombination may contribute to the extreme genetic diversity and variability observed in Magnaporthe grisea, the causal agent of rice blast disease. Dispersed repetitive elements and mapped, low-copy restriction fragment length polymorphism (RFLP) probes were used to detect transfers of DNA between cultured isolates of M. grisea. Low-copy RFLP probes also were used to detect putative recombinants among isolates from well-characterized field populations of the pathogen. Microscopic examination of tufted mycelium between cocultured isolates revealed frequent hyphal fusions. Hyphal tips and conidia were recovered without selection from tufted zones in two separate vegetative pairings involving isolates with dissimilar haplotypes, based on the repetitive element MGR586. Haplotypic changes were observed at a higher frequency in tuft derivatives than in subcultures of each isolate alone. From 136 tuft derivatives analyzed, 5 putative recombinant haplotypes were identified. Introgression was demonstrated with two independent repetitive elements, fosbury and MGR586, as probes on DNA digested with several restriction enzymes. Introgressions were characterized by addition of 1 to 10 MGR586 bands, and 1 to 3 fosbury bands from one parent into the background of the other. Polymorphic single-copy probes were used to analyze putative recombinants. One probe detected an introgression event as predicted by analysis with MGR586. To assess the possible role of parasexual recombination in field populations of the pathogen, isolates in the Philippines previously grouped based on DNA fingerprinting were analyzed with low-copy RFLP markers. Polymorphism in single-copy loci typically was seen between, but not within, putative pathogen lineages. One lineage (designated lineage 4), however, was polymorphic for several probes. For some isolates, alleles at these loci comigrated with alleles characteristic of other lineages, suggesting the transfer of DNA fragments between lineages. One isolate was apparently a merodiploid, carrying an allele typical of lineage 4 plus another allele characteristic of a different lineage. In a survey of isolates from the Indian Himalayas, a merodiploid also was found with single- or low-copy probes. Examination of MGR586 profiles of the putative recombinant and its putative donor strains showed the expected introgression of MGR586 bands. The detection of parasexual DNA exchanges in wild-type strains under unselected conditions and the existence of merodiploids in nature suggest that parasexual recombination occurs in field populations of M. grisea. This raises questions concerning exclusive clonality in the blast fungus.     

Genetic differentiation of Magnaporthe oryzae populations from scouting plots and commercial rice fields in Korea

A previous study of the diversity and population structure of the rice blast fungus, Magnaporthe oryzae, over a 20-year period in Korea, found novel fingerprint haplotypes each year, and the authors hypothesized that populations might experience annual bottlenecks. Based on this model, we predicted that M. oryzae populations would have little or no genetic differentiation among geographic regions because rice blast is commonly found throughout Korea each year and M. oryzae would have to disperse from small populations surviving annually between rice crops. To test this hypothesis, we sampled M. oryzae from rice fields in eight provinces in Korea in a single year (1999). In four provinces, we sampled from a set of rice cultivars commonly grown in commercial fields (group I); because of low disease incidence in four other provinces, we could not sample from commercial fields and instead sampled from scouting plots of different cultivars set up for detecting new pathotypes of M. oryzae (group II). All isolates were genotyped with DNA fingerprint probes MGR586 and MAGGY, a telomere-linked gene family member TLH1, the PWL2 host specificity gene and mating type. Fingerprint haplotypes clustered into two distinct lineages corresponding to the two sets of cultivars (groups I and II), with haplotype similarities of 71% between lineages and >76% within lineages. Isolates from the same cultivar within group I were genetically differentiated among locations, and isolates within the same location were differentiated among cultivars. Differentiation for TLH1 and PWL2 was significant (P < 0.03), but not as strong as for fingerprint markers. Similar analyses were not possible among group II isolates because too few isolates were available from any one cultivar. All isolates were in the same mating type, Mat1-1, ruling out sexual reproduction as a source of novel haplotypes. When the 1999 samples were compared with the historical samples from the previous study, haplotypes of group I formed a separate cluster, while those of group II clustered with haplotypes from the historical sample. Altogether, geographic subdivision, monomorphism of mating type, and correlation of haplotypes to sets of cultivars are not consistent with the hypothesis of repeated turnover of haplotypes. Instead, the previous correlations of haplotypes to year might have been caused by inadequate sampling of haplotypes each year, highlighting the need for studies of population genetics to be conducted with systematic samples collected to address specific questions.     

Characterisation of the European pathogen population of Magnaporthe grisea by DNA fingerprinting and pathotype analysis

The genetic variability among 41 isolates of the blast pathogen (Magnaporthe grisea) from five European rice growing countries was studied. The genealogy of the isolates was investigated by DNA fingerprinting and the results compared to the degree of similarity for (a)virulence factors. Fingerprinting grouped the isolates into five discrete lineages, that typically showed less than 65% band similarity. Within each lineage, two or more haplotypes were detected with a band similarity of 80% or higher. Each lineage showed a characteristic virulence pattern. All isolates of lineage E5 belonged to the same pathotype. The other lineages were composed of clusters of closely related pathotypes that showed variation for virulence to cultivars with certain known resistance genes, while remaining invariably (a)virulent to others. In most cases, lineage classification of an isolate could be easily inferred by its pathotype. Certain resistance genes and certain lineage-excluding resistance gene combinations appear to provide protection against all of the virulence factors sampled.     

Genetic structure of Iranian <Emphasis Type="Italic">Pyricularia grisea</Emphasis> populations based on rep-PCR fingerprinting

The population structure of the rice blast fungus Pyricularia grisea was analyzed in two major rice-growing provinces of Iran using rep-PCR DNA fingerprinting. A total of 221 monoconidial isolates of the fungus was collected from 12 cultivars at ten regions during 1997–2000. Long-PCR conditions were used to amplify sequences lying between adjacent Pot2 elements. The frequencies of Pot2 lineages (isolates with 70% amplicon similarity) and haplotypes within lineages were determined. Phenetic analysis differentiated five Pot2 fingerprint lineages, designated A, B, C, D and E. The most common fingerprint group, Lineage E, was recovered from all rice cultivars sampled and was distributed throughout the region. Haplotype E6, the most common haplotype within lineage E, was recovered from almost all regions. Lineage A, the second most common lineage, was found mainly in the western part of the sampled region. Haplotype A1 was found in most sites in the western province. Lineage A occurred at relatively high frequency on the susceptible local cultivar Binam, suggesting that lineage A is specifically adapted to Binam. To test this hypothesis, 193 additional isolates were recovered from four fields at two sites separated by approximately 100 km. This second, field-specific collection of isolates contained lineages A, C, D, and E. Approximately 64% and 29% of the isolates recovered from Binam (the shared cv. at two sites) grouped into lineages A and E, respectively. The other two susceptible cultivars at these sites were infected by lineage E at frequencies of 100% and 71%. Overall, these data indicated a low level of genetic diversity in the Iranian P. grisea population similar to that reported in other countries.     

Monitoring and characterization of Magnaporthe grisea isolates with decreased sensitivity to scytalone dehydratase inhibitors

Rice blast fungus isolates were collected in Kyushu to investigate resistance to scytalone dehydratase inhibitors of melanin biosynthesis (MBI-D). In 2001, failure of control of rice blast was reported in the Saga prefecture, where MBI-Ds have been used since 1998. At that time, the distribution of resistant isolates was mainly limited to that area. However, in 2002, resistant isolates were detected in all prefectures of Kyushu. DNA fingerprinting analysis showed that the mutation causing resistance to MBI-Ds had arisen independently in each area. These data suggest that resistant isolates may occur in any area and become dominant under continuous selection pressure for MBI-Ds. Nevertheless, resistant strains can be controlled by reductase inhibitors of melanin biosynthesis (MBI-R) or commercial rice seed disinfectants.