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。     

Rep-PCR技术对中国水稻条斑病菌的遗传多样性初析

 采用Rep-PCR技术,对30个水稻细菌性条斑病菌株(Xanthomonas oryzae pv.oryzicola)进行遗传多样性分析,同时对李氏禾条斑病菌等其它10个参试菌株也进行了比较。Rep-PCR是利用一些基于细菌的短的重复序列引物(ERIC和BOX)的DNA扩增特性,2种引物组合的电泳图谱结合并分析,以水稻细菌性条斑病菌各自的指纹谱型在相似率80%时可分为6簇,初步表明我国水稻细菌性条斑病菌群体的遗传分化明显;发现自然界存在的弱或无毒性菌株与毒性菌株的Rep-PCR指纹图谱差异很大;毒性菌株的遗传分簇与其致病性具有一定的相关性。用ERIC扩增水稻条斑病菌基因组DNA的指纹比BOX更为多样,两者对菌株的分辨率不同。因此,Rep-PCR技术可有效地用于监测水稻细菌性条斑病菌的遗传变异,还可应用于菌株的鉴定和分类学研究。     

四川省籼稻区水稻稻曲病菌遗传多样性分析

应用ERIC-PCR指纹技术,对采自四川省6个籼稻自然生态区的60个稻曲病菌菌株进行了DNA分子水平上的遗传多样性研究,并进行UPGMA聚类分析和相似性分析.结果表明,所采用的ERIC引物在不同供试菌株中分别扩增出5～20条不等的带谱;在0.75相似水平上,供试菌株被划分为11个遗传型群,其中L1、L4、L5为优势群,并存在次要小型群和特异性型群;来自同一地区的稻曲病菌菌株具有较高的遗传相似性,而不同地区的稻曲病菌则表现出程度不同的变异,且寄主品种与稻曲病菌遗传差异之间的相关性较小.     

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。     

广东水稻白叶枯病菌遗传多样性和小种分化研究

 通过IS-PCR和rep-PCR指纹技术,分析了广东水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae)群体遗传多样性。用2对特异性引物J3和ERIC对114个菌株基因组DNA进行了PCR扩增,分别呈现89和40种谱型,以彼此间的带位相似率达70%为界,J3扩增的谱型被分为11簇,ERIC的谱型被分为8簇。J3的簇1包含89个菌株,占总数的78.07%,ERIC的簇1包含52个菌株,占总数的45.61%,均为优势簇群。群体遗传多样性值J3为0.8919,ERIC为0.8278。上述结果表明,广东水稻白叶枯病菌的遗传多样性较高。全部参试菌株接种于含有不同抗性基因近等基因系及高感品种金刚30共6个鉴别品种,被划分为6个小种(X-gd1,X-gd2,X-gd3,X-gd4,X-gd5和X-gd6),X-gd4出现频率最高,为广东省的优势小种。     

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。     

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。     

甘肃省大麦条纹病菌遗传多样性及致病力差异

为了解甘肃省大麦条纹病病原菌Pyrenophora graminea的遗传多样性及致病力差异,运用RAPD分子标记技术对大麦条纹病菌不同菌株进行遗传多样性分析,并采用三明治法进行菌株致病力差异研究。结果表明:17个RAPD标记从45个菌株中扩增出126条带,平均每个标记7.41条带,遗传相似系数范围为0.468 3～0.984 1,平均值为0.830 8,当遗传相似系数为0.723 6时,可将供试菌株划分为4个类群,分别包含41、2、1和1个菌株;致病力测定结果显示菌株QWC较菌株QQ致病力强,两菌株除在品种‘甘啤2号’和‘GP-3’上无致病力外,在其他供试品种上致病力均存在差异。表明大麦条纹病菌不同菌株间存在遗传差异,且菌株QWC和菌株QQ存在致病力差异。     

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。     

西瓜枯萎病菌遗传多样性的相关序列扩增多态性分析

对30个西瓜枯萎病菌Fusarium oxysporum f.sp.niveum菌株基因组DNA进行相关序列扩增多态性(SRAP)分子标记分析,以探究其遗传多样性与地理来源的关系。采用尖孢镰刀菌西瓜专化型Fusarium oxysporumf.sp.niveum0、1、2号生理小种的基因组DNA为模板,对225对SRAP引物进行筛选,筛选出20对多态性、重复性较好且条带清晰的引物,对30个菌株进行PCR扩增,共扩增出386条带,其中多态性条带有371条,多态性比率为96.11%,平均每对引物扩增出19.3个位点和18.55个多态性位点。UPGMA法聚类分析结果显示,供试菌株两两之间的遗传相似系数范围为0.69~0.90,平均为0.79,说明尖孢镰刀菌西瓜专化型的遗传多样性较为丰富。基于SRAP标记聚类分析表明,30个菌株在遗传相似系数为0.70处被划分为3个类群,I类群包含24个菌株,其中18个来自湖南省,Ⅱ类群只包含1个来自黑龙江省哈尔滨市的菌株,它和另一个来自黑龙江地区的菌株被划分到不同的类群,且遗传距离相对较远;Ⅲ类群包含了5个菌株,其中3个来自海南三亚,其余两个来自湖南省。根据菌株的分布情况来看,菌株的聚集与地理来源没有明显的相关性。     

Characterization,genetic diversity and distribution of Xanthomonas campestris pv. campestris races causing black rot disease in cruciferous crops of India

Black rot, caused by Xanthomonas campestris pv. campestris (Xcc) is a disease of crucifer crops. The objective of this study was to characterize races of Xcc, their distribution and genetic diversity in India. Two hundred and seventeen isolates of bacteria were obtained from 12 different black rot‐infected crucifer crops from 19 states of India; these were identified as Xcc based on morphology, hrpF gene and 16S rRNA gene based molecular markers and pathogenicity tests. Characterization of races was performed by using a set of seven differential crucifer hosts, comprising two cultivars of turnip (Brassica rapa var. rapa) and cultivars of Indian mustard (B. juncea), Ethiopian mustard (B. carinata), rapeseed mustard (B. napus), cauliflower (B. oleracea) and Savoy cabbage (B. oleracea var. sabauda). Races 1, 4 and 6 of Xcc were identified and, among these races, race 1 followed by race 4 dominated most of the states of India. Genetic diversity of the Indian isolates of Xcc was analysed using repetitive sequence‐based PCR (rep‐PCR) including primers for REP (repetitive extragenic palindromic), ERIC (enterobacterial repetitive intergenic consensus) and BOX (amplifying with BOX A1 R primer) repetitive elements. This method of fingerprinting grouped the isolates into 56 different DNA types (clusters) with a 75% similarity coefficient. Among these clusters, DNA types 22 and 53 contained two different races 1 and 4, whereas DNA type 12 contained races 1, 4 and 6. However, no clear relationship was observed between fingerprints and races, hosts or geographical origin.     

Analysis of sources of oxolinic acid-resistant field strains of <Emphasis Type="Italic">Burkholderia glumae</Emphasis> based on rep-PCR analysis and nucleotide sequences of <Emphasis Type="Italic">gyrB</Emphasis> and <Emphasis Type="Italic">rpoD</Emphasis>

Repetitive sequence-based polymerase chain reaction (rep-PCR) analysis using BOX and ERIC as primers showed a highly divergent phylogeny among field strains of Burkholderia glumae. To elucidate the sources of oxolinic acid (OA) resistance in field strains of B. glumae isolated from rice seedlings cultivated in Mie, Toyama, and Iwate prefectures, Japan, the amino acid at position 83 of GyrA (GyrA83), which is involved in OA resistance, and the DNA patterns from the rep-PCR and the partial nucleotide sequences of gyrB and rpoD from various strains were analyzed. The ten Mie strains, in which GyrA83 was isoleucine (Ile), were divided into two groups based on the band patterns in rep-PCR analysis, although the nucleotide sequences of gyrB and rpoD were identical among the strains. Based on the band patterns in the rep-PCR analysis and the gyrB and rpoD sequences, two highly OA-resistant Toyama strains, Pg-13 and Pg-14, for which GyrA83 was serine (Ser) and Ile, respectively, were in the same lineage. This suggests that the bacteria might acquire OA resistance faster than phylogenic diversity as determined with the repetitive sequences BOX and ERIC and with gyrB and rpoD. Furthermore, three Iwate strains (H95, H101, and H104), isolated from seedlings of different cultivars grown in different years and having Ile at GyrA83, are probably in the same lineage, suggesting that OA-resistant bacteria might be transferred among different cultivars.     

Development of Strain-specific Primers for a Strain of Gliocladium catenulatum Used in Biological Control

The randomly amplified polymorphic DNA (RAPD) technique was used to develop strain-specific primers for Gliocladium catenulatum strain J1446, which is promising in biological control. One of the primer pairs developed proved to be strain-specific; strain J1446 was differentiated from 16 G. catenulatum strains and six other strains of two Gliocladium species, as well as from Trichoderma virens, and isolates of Nectria spp. and Fusarium spp. Specific primers were also tested with DNA isolated from cucumber leaves, treated or untreated with a solution made from Gliocladium powder. The expected amplification product was produced only from treated leaves. DNA isolated from Gliocladium-treated potato tubers and fungi grown in peat was also used in amplification reactions. Strain-specific primers detected strain J1446 when the amount of DNA was 5pg or more. Some variation between the Gliocladium strains was found by the random amplified microsatellites method (RAMS) and the universally primed polymerase chain reaction method (UP-PCR), but no clear fragments specific to strain J1446 were produced. Cross-blot hybridisation of UP-PCR products differentiated strain J1446 from T. virens, but not from the Gliocladium isolates. The 28S rDNA sequences and -tubulin sequences were identical or very similar in all Gliocladium strains. Thus, it is possible that the Gliocladium strains of the present study are conspecific, which means that a revision in the taxonomy of Gliocladium species may be necessary.     

Genetic fingerprinting of Iranian Xanthomonas campestris pv. campestris strains inducing black rot disease of crucifers

Northern Iran has one of the largest and most diverse populations of cultivated crucifers in Iran. Symptoms of black rot disease were observed in 40 % of fields. To assess the genetic diversity of Xanthomonas campestris pv. campestris (Xcc) strains, associated with black rot disease, 40 strains were isolated from infected samples of crucifers such as cabbage, radish, cauliflower, turnip and kohlrabi, and were collected from different geographic regions of northern Iran including West and East Azarbayjan and Ardabil provinces. Bacterial strains were characterized by their morphological, biochemical and physiological features and pathogenicity tests. Four races were found in northern Iran (1, 4, 5 and 6) and the majority of the tested strains belonged to either race 4 (45 %) or race 6 (20 %). To examine the distribution of dispersed repetitive DNA, Enterobacterial Repetitive Intergenic Consensus (ERIC), BOX, Repetitive Extragenic Palindromic (REP) and random amplified polymorphic DNA (RAPD) sequences in the genome of Xcc using conserved primers. The different markers produced characteristic banding patterns and the similarity matrices from binary banding data was derived with the similarity for qualitative data program (SIMQUAL). On the basis of the fingerprint patterns generated by the combination data set of both rep-PCR and RAPD, the Xcc strains were differentiated into seven clusters (A–G) at 76 % similarity level. The geographical origin of the Iranian strains does not seem to be correlated with the RAPD and rep-PCR clusters. The clusters seem to be more related to the race of the strains. This is the first study on genetic diversity of Xcc strains inducing black rot disease of crucifers in Iran.     

Discrimination of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> isolates from sweet and wild cherry using rep-PCR

Bacterial canker is one of the most important diseases of cherry (Prunus avium). This disease can be caused by two pathovars of Pseudomonas syringae: pv. morsprunorum and pv. syringae. Repetitive DNA polymerase chain reaction-based fingerprinting (rep-PCR) was investigated as a method to distinguish pathovars, races and isolates of P. syringae from sweet and wild cherry. After amplification of total genomic DNA from 87 isolates using the REP (repetitive extragenic palindromic), ERIC (enterobacterial repetitive intergenic consensus) and BOX primers, followed by agarose gel electrophoresis, groups of isolates showed specific patterns of PCR products. Pseudomonas syringae pv. syringae isolates were highly variable. The differences amongst the fingerprints of P. syringae pv. morsprunorum race 1 isolates were small. The patterns of P. syringae pv. morsprunorum race 2 isolates were also very uniform, with one exception, and distinct from the race 1 isolates. rep-PCR is a rapid and simple method to identify isolates of the two races of P. syringae pv. morsprunorum; this method can also assist in the identification of P. syringae pv. syringae isolates, although it cannot replace inoculation on susceptible hosts such as cherry and lilac.     

Characterisation of Pseudomonas syringae Strains Associated with a Leaf Disease of Leek in Australia

A necrotic leaf disease of leek (Allium ampeloprasum Porrum Group) is reported in Australia for the first time. The fluorescent pseudomonad consistently associated with diseased tissue was identified as Pseudomonas syringae by LOPAT tests (+,−,−,−,+), carbon utilisation, bean and lemon inoculations and fatty acid methyl ester analysis. It was confirmed as P. syringae pv. porri by pathogenicity to leeks, bulb onions, spring onions, shallots and garlic, and by genetic analysis using 16S rDNA PCR, REP, ERIC and BOX PCR, and IS50 PCR. Comparison with reference strains of pv. porri from other countries showed similarity to known strains of pv. porri. The Australian leek strains were generally uniform in their biochemical reactions although three strains tested varied in their pathogenicity to other Allium spp. and varied from published data. All Australian strains shared the same genetic profile with strains from New Zealand, France and California. However, Japanese strains from leek and onion were distinct from the Australian strains and those from New Zealand, France and California. Data strongly support the hypothesis that the pathogen is seed-borne.     

rep-PCR-Mediated Genomic Fingerprinting: A Rapid and Effective Method to Identify Clavibacter michiganensis

ABSTRACT The genomic DNA fingerprinting technique known as repetitive-sequence-based polymerase chain reaction (rep-PCR) was evaluated as a tool to differentiate subspecies of Clavibacter michiganensis, with special emphasis on C. michiganensis subsp. michiganensis, the pathogen responsible for bacterial canker of tomato. DNA primers (REP, ERIC, and BOX), corresponding to conserved repetitive element motifs in the genomes of diverse bacterial species, were used to generate genomic fingerprints of C. michiganensis subsp. michiganensis, C. michiganensis subsp. sepedonicus, C. michiganensis subsp. nebraskensis, C. michiganensis subsp. tessellarius, and C. michiganensis subsp. insidiosum. The rep-PCR-generated patterns of DNA fragments observed after agarose gel electrophoresis support the current division of C. michiganensis into five subspecies. In addition, the rep-PCR fingerprints identified at least four types (A, B, C, and D) within C. michiganensis subsp. michiganensis based on limited DNA polymorphisms; the ability to differentiate individual strains may be of potential use in studies on the epidemiology and host-pathogen interactions of this organism. In addition, we have recovered from diseased tomato plants a relatively large number of naturally occurring avirulent C. michiganensis subsp. michiganensis strains with rep-PCR fingerprints identical to those of virulent C. michiganensis subsp. michiganensis strains.     

Genetic and Pathogenic Diversity of Pseudomonas avellanae Strains Isolated from Corylus avellana Trees in North-west of Italy, and Comparison with Strains from Other Regions

Forty strains of Pseudomonas avellanae isolated from hazelnut (Corylus avellana L.) trees in the Langhe district of Italy were compared with 15 strains collected from various geographic areas. All strains were assessed by rep-PCR genomic fingerprinting using ERIC, REP and BOX primer sets. Cluster analysis was performed by means of UPGMA. To check the possible differential virulence of the strains, pathogenicity tests were carried out by inoculating leaf scars of hazelnut trees in early autumn. Cluster analysis indicated that, during a four-year study, at least five groups of strains were isolated from different hazelnut orchards located in the small district of Langhe. Two groups were isolated from the same twig. Such strains showed around 20% similarity with other P. avellanae strains collected from northern Greece and central Italy. The strains from Langhe were less aggressive to hazelnut than strains from northern Greece and central Italy. These results and previous genomic characterizations indicate a possible correlation between genomic profile type and regional geographic distribution of P. avellanae strains. In addition, the genetic variability found in the strains from Langhe indicates that such populations are older than the more homogenous P. avellanae populations from other regions.