Reclassification of aster yellows group phytoplasmas in Korea

Aster yellows group phytoplasmas were reclassified by analysis of the 16S rRNA gene sequence, their phylogeny and the presence of interoperon heterogeneity. Nine phytoplasmas were classified into subgroups 16SrI-B and 16SrI-D using the 16S rRNA gene sequence. Then, based on the presence of interoperon heterogeneity, subgroup 16SrI-B phytoplasmas were differentiated into three subunits as 16SrI-B(a): mulberry dwarf, sumac witches’ broom and porcelain vine witches’ broom; 16SrI-B(b): angustata ash witches’ broom and Japanese spurge yellows; and 16SrI-B(c): onion yellow dwarf, water dropwort witches’ broom and hare’s ear yellow dwarf phytoplasma.     

Distinctions between phytoplasmas at the subgroup level detected by heteroduplex mobility assay

A total of 62 phytoplasma isolates were collected from North America, Europe and Asia and analysed by heteroduplex mobility assay (HMA) of the 16/23S spacer region amplified by the polymerase chain reaction. The results revealed wide genetic diversity among the phytoplasmas studied and a number of new phytoplasma strains were identified from known or new plant hosts in Alberta, Canada. Two distinctive subgroups were revealed by HMA in phytoplasmas associated with canola yellows, Chinese aster yellows, dandelion yellows and monarda yellows. In Alberta, two subgroups of the aster yellows group of phytoplasmas, I-A and I-B, were prevalent in naturally infected field crops and ornamentals in open gardens. The results indicated that HMA is a simple, but rapid and accurate, alternative method for the detection and estimation of genetic divergence of phytoplasmas when finer molecular characterization of phytoplasmas is required at the subgroup level.     

Comparison of phytoplasmas infecting winter oilseed rape in the Czech Republic with Italian Brassica phytoplasmas and their relationship to the aster yellows group

Winter oilseed rape grown in several areas in South Bohemia showed symptoms of stunting, leaf reddening and extensive malformation of floral parts. Phytoplasmas were consistently observed by using electron microscopy only in phloem tissue of symptomatic plants. DNA isolated from infected and healthy control plants was used in PCR experiments. Primer pairs R16F2/R2, P1/P7 and rpF2/R2, amplifying, respectively, 16S rDNA, 16S rDNA plus spacer region and the beginning of the 23S and ribosomal protein gene L22 specific for phytoplasmas, were used. According to RFLP and sequence analyses of PCR products, the phytoplasma from rape was classified in the aster yellows phytoplasma group, subgroup 16SrI-B. The PCR products from the Czech phytoplasma-infected rape also had RFLP profiles identical to those of phytoplasma strains from Italian Brassica . This first molecular characterization of phytoplasmas infecting rape compared with strains from Brassica does not, however, clearly indicate differences among isolates of the same 16SrI-B subgroup. Further studies on other chromosomal DNA portions could help the research on host specificity or on geographical distribution of these phytoplasmas.     

3种检疫性葡萄黄化植原体的快速检测

 葡萄上的植原体病害由于引起叶片黄化而被称为葡萄黄化病。由于这一病害极为严重,葡萄黄化植原体被列为我国的植物检疫对象。其中,葡萄金黄化植原体(16SrV)、维吉尼亚葡萄黄化植原体(16Sr芋) 和澳大利亚葡萄黄化植原体 (16Sr狱) 是引起葡萄黄化病的主要3 个株系,它们导致的病害症状相似,难以区分。本文进行了3 个株系16S rRNA 基因 DNA 序列比对,而后根据同源性相对低的序列设计了43 条特异性引物、103 对引物对组合,对葡萄黄化植原体3 个株系各自的DNA 及混合DNA 进行PCR 扩增,从中筛选出来特异性较强的8 个引物对组合。这些引物对组合,能够同步、特异、快速地检测3 种葡萄黄化植原体。     

Detection and differentiation of grapevine yellows phytoplasmas belonging to the elm yellows group and to the stolbur subgroup by PCR amplification of non-ribosomal DNA

Primer pairs were designed from a cloned DNA probe of a strain of flavescence dorée (FD) phytoplasma and from a cloned DNA probe of a strain of stolbur phytoplasma. Among an array of reference phytoplasma strains maintained in periwinkle, pair FD9f/r amplified a 1.3 kb DNA fragment only with phytoplasma strains of elm yellows (EY) group, i.e. two strains of FD and two strains of EY. Tru9I restriction analysis of the fragment amplified by FD9f/r revealed a diversity among EY-group phytoplasmas. The FD strains differed from the strains isolated from elm. The profile of the phytoplasmas infecting the grapevine samples from Catalonia and most of the samples from Northern Italy were identical to that of a FD strain. Three other profiles were detected in grapevine from Palatinate, in Germany.The two primer pairs derived from a stolbur strain, STOL4f/r and STOL11f2/r1, specifically amplified a 1.7 kb and a 0.9 kb DNA fragment, respectively, with all strains in the stolbur subgroup. However, the pair STOL4f/r did not recognise strain MOL. Both pairs allowed to detect phytoplasmas in diseased grapevines from France, Italy, Spain and Israel. Attempts to differentiate between phytoplasmas in the stolbur subgroup by restriction analyses failed. The pairs FD9f/r and STOL11f2/r1 could be used in the same reaction (multiplex PCR) to detect EY-group phytoplasmas, stolbur-subgroup phytoplasmas or both phytoplasmas simultaneously when template DNAs were mixed.     

Ecological implications from a molecular analysis of phytoplasmas involved in an aster yellows epidemic in various crops in Texas

ABSTRACT In the spring of 2000, an aster yellows (AY) epidemic occurred in carrot crops in the Winter Garden region of southwestern Texas. A survey revealed that vegetable crops, including cabbage, onion, parsley, and dill, and some weeds also were infected by AY phytoplasmas. Nested polymerase chain reaction (PCR) and restriction fragment length polymorphism analysis of PCR-amplified phytoplasma 16S rDNA were employed for the detection and identification of phytoplasmas associated with these crops and weeds. Phytoplasmas belonging to two subgroups, 16SrI-A and 16SrI-B, in the AY group (16SrI), were predominantly detected in infected plants. Carrot, parsley, and dill were infected with both subgroups. Onion and three species of weeds (prickly lettuce, lazy daisy, and false ragweed) were predominantly or exclusively infected by subgroup 16SrI-A phytoplasma strains, while cabbage was infected by subgroup 16SrI-B phytoplasmas. Both types of phytoplasmas were detected in three leafhopper species, Macrosteles fascifrons, Scaphytopius irroratus, and Ceratagallia abrupta, commonly present in this region during the period of the epidemic. Mixed infections were very common in individual carrot, parsley, and dill plants and in individual leafhoppers. Sequence and phylogenetic analyses of 16S rDNA and ribosomal protein (rp) gene sequences indicated that phytoplasma strains within subgroup 16SrI-A or subgroup 16SrI-B, detected in various plant species and putative insect vectors, were highly homogeneous. However, based on rp sequences, two rpI subgroups were identified within the subgroup 16SrI-A strain cluster. The majority of subgroup 16SrI-A phytoplasma strains were classified as rp subgroup rpI-A, but phytoplasma strains detected in one onion sample and two leafhoppers (M. fascifrons and C. abrupta) were different and classified as a new rp subgroup, rpI-N. The degree of genetic homogeneity of the phytoplasmas involved in the epidemic suggested that the phytoplasmas came from the same pool and that all three leafhopper species may have been involved in the epidemic. The different phytoplasma population profiles present in various crops may be attributed to the ecological constraints as a result of the vector-phytoplasma-plant three-way interaction.     

Note: A comparison of molecular diagnostic procedures for the detection of aster yellows phytoplasmas (16Sr-I) in leafhopper vectors

Different molecular procedures were compared for the detection of aster yellows phytoplasmas (AYP) in the leafhopper vectorsMacrosteles quadripunctulatus (Kirschbaum),Euscelidius variegatus (Kirschbaum) andEuscelis incisus (Kirschbaum). Polymerase chain reaction (PCR) with universal and group-specific primers designed on the 16S-rDNA sequence was most sensitive in nested assays. A dot-blot procedure with an oligoprobe designed on the 16S-rDNA was less sensitive and consistent to detect phytoplasmas in total insect DNA, but consistently detected amplicons from direct PCR. The dot-blot assay with a probe based on a phytoplasma plasmid sequence detected AYP in most vector specimens and did not react with DNAs from leafhoppers infected by flavescence dorée and psyllids infected by apple proliferation phytoplasmas. This last assay is almost devoid of contamination risks, faster and cheaper compared to PCR, therefore it has to be preferred for field-scale analysis of leafhopper populations. http://www.phytoparasitica.org posting Feb. 24, 2004.     

Phylogenetic relationships among Flavescence dorée strains and related phytoplasmas determined by heteroduplex mobility assay and sequence of ribosomal and Nonribosomal DNA

Heteroduplex mobility assay (HMA) and DNA sequencing were performed on Flavescence dorée (FD) phytoplasma strains and related phytoplasmas belonging to the elm yellows group. Part of the ribosomal RNA gene operon and a nonribosomal DNA region were utilized for phylogenetic analyses. Two FD strains, FD92 and FD-D, detected in France and Italy, respectively, were identical in both DNA fragments, confirming previous results. Other FD strains were all very similar and most closely resembled ALY, an Italian alder phytoplasma. Phytoplasmas associated with German Palatinate grapevine yellows were shown to form a distinct subcluster, also different from the elm yellows phytoplasma subcluster. Strain disparities revealed by HMA and sequence data were mostly in agreement, highlighting the utility of HMA in differentiation and classification of phytoplasmas belonging to the same ribosomal RNA group.     

我国几种植物植原体的快速分子鉴别与鉴定的研究

 选用桑萎缩病(Mulberry dwarf,MD)、枣疯病(Jujube witches'-broom,JWB)、酸枣丛枝病(Wild jujube witches'-broom,WJWB)、泡桐丛枝病(Paulownia witches'-broom,PaWB)和苦楝丛枝病(Chinaberry tree witches'-broom,CWB)5种不同植物植原体和来源于3个不同地区PaWB和JWB材料进行16S rDNA和23S rDNA PCR扩增、异源双链迁移率分析(HMA)、PCR产物的RFLP分析和16S rDNA的克隆和测序等比较研究,建立了一种快速确定未知植原体种类和分类地位的分子鉴别与鉴定优化程序;并可对田间采集的各种植物植原体样品进行快速鉴定和鉴别。16S rDNA PCR产物HMA分析结果显示,JWB与CWB、MD和PaWB皆可形成明显的异源杂交双链;而CWB、MD和PaWB植原体之间未能形成异源双链。JWB和PaWB不同地区样品之间、JWB和WJWB之间也未发现异源杂交双链的形成。而23S rDNA PCR产物HMA分析则可以将MD与PaWB区分开。进一步对未知分类地位的CWB序列测定及与其它植原体16S rDNA的RFLP和同源性比较结果显示,CWB与PaWB同源性为99.5%,其中与MD的同源性高达99.7%,因而应将CWB归为翠菊黄花组16Sr I-B,16Sr I-B (rp-B)。     

Use of terminal restriction fragment length polymorphism (T-RFLP) for identification of phytoplasmas in plants

A terminal restriction fragment analysis (T-RFLP) technique was developed for the simple and rapid detection and diagnosis of phytoplasmas in plants. The selected primers amplified part of the 23S rRNA gene to provide improved resolution between the taxonomic groups compared to conventional restriction enzyme analysis of the 16S rRNA. Using the restriction enzymes Bsh 12361 and Mse I on the PCR products, and fragment analysis in the range 68–640 bp, the technique was tested on 37 isolates from 10 of the 16Sr groups. Distinct and unambiguous T-RFLP profiles were produced for nine of the 10 taxonomic groups, such that almost all isolates within a group shared the same profile and could be distinguished from isolates in other groups. The technique also identified the presence of mixtures of phytoplasmas from different groups in samples. Furthermore, the primers were devised to amplify a terminal restriction fragment (TRF) product of a specific defined size (461 bp) from the host plant chloroplast DNA, so that there was a built-in internal control in the procedure to show that the absence of a phytoplasma peak in a sample was the result of no detectable phytoplasma being present, not the result of PCR inhibition. This method offers the possibility of simultaneously detecting and providing a taxonomic grouping for phytoplasmas in test samples using a single PCR reaction.     

Pcr Assay for specific detection of European stone fruit yellows phytoplasmas and its use for epidemiological studies in France

DNA amplification by polymerase chain reaction was used to specifically detect phytoplasmas associated with severe decline diseases of European stone fruits. PCR primers were designed according to the partial sequence of a nonribosomal genomic fragment of European stone fruit yellows phytoplasmas obtained by direct sequencing of a specific PCR product. A PCR assay was developed which resulted in specific amplification of a 237 bp-DNA fragment from total DNA extracts derived from over 300 stone fruit samples. No PCR product was obtained with DNA from healthy controls or plants diseased with various other phytoplasmas, e.g. the closely related apple proliferation and pear decline phytoplasmas. Phytoplasma infection was checked in all samples by PCR amplification with universal ribosomal primers. Detection rate with specific and universal primers was correlated by 97%. European stone fruit yellows phytoplasmas were detected in samples of 114 out of 139 examined orchards which represent the major stone fruit growing regions of France. Typical symptoms like chlorotic leaf roll in summer and off-season growth in winter were correlated by 95% to the presence of phytoplasmas. However, phytoplasmas were also detected in 51% of samples derived from trees showing non-specific symptoms. A comparison study including 201 samples showed that 81% of the PCR-positive samples were also tested positive using fluorescence microscopy with DAPI staining.     

Detection and molecular characterization of an aster yellows phytoplasma in rhodiola in Alberta,Canada

Journal of Plant Diseases and Protection - Rhodiola plants exhibiting symptoms typical of phytoplasma infection were observed at an experimental farm near Edmonton, Alberta, Canada, in 2007....     

宁夏马铃薯僵顶植原体的分子鉴定

通过透射电子显微镜,在从宁夏回族自治区固原市彭阳县红河镇采集的表现叶片上卷、红叶、气生薯症状的马铃薯样品叶脉韧皮部筛管细胞内观察到大量直径为500~700 nm的球形植原体粒子。以提取的感病和健康马铃薯叶片总DNA为模板,应用植原体16S rRNA基因和rp基因通用引物进行PCR扩增,从感病样品中扩增得到了长度均约为1.2 kb的片段。对获得基因核酸一致性比较分析表明,马铃薯僵顶植原体宁夏株系16S rRNA基因与‘Candidatus Phytoplasma fragariae’槭树株系(MK501642)16S rRNA基因核酸一致性最高,为99.7%,rp基因与‘Ca.P.fragariae’云南马铃薯YN-2G株系(KJ144889)rp基因核酸一致性最高,为100%;基于16S rRNA基因和rp基因构建系统进化树发现,马铃薯僵顶植原体宁夏株系与16SrⅫ-E亚组成员聚在一起。基于透射电镜观察和基因序列比较分析,证明宁夏发生的马铃薯僵顶病与植原体侵染相关,该植原体在分类地位上属于植原体16SrⅫ-E亚组。     

Detection and identification of phytoplasmas in yellows-diseased weeds in Italy

Yellows-diseased plants of Crepis setosa (hawksbeard), Knautia arvensis (field scabious), Convolvulus arvensis (field bindweed), Picris echioides (bristly oxtongue), Echium vulgare (blueweed) and Calendula officinalis (pot marigold) collected in central and southern Italy were examined for phytoplasma infection by means of polymerase chain reaction (PCR) technology using universal phytoplasma primers directed to ribosomal sequences. The detected phytoplasmas were characterized and differentiated using restriction fragment length polymorphism analysis of PCR-amplified DNA. The phytoplasma detected in diseased pot marigold plants was identified as a member of the aster yellows group and proved indistinguishable from a strain of the American aster yellows phytoplasma. The phytoplasma identified in diseased field bindweed plants is a putative new type of the stolbur group that differed from the typical stolbur phytoplasma. Phytoplasmas detected in diseased hawksbeard, blueweed and field scabious plants are all putative new members of the sugarcane white leaf group while the phytoplasma detected in diseased bristly oxtongue plants represents a new member of the faba bean phyllody group. For hawksbeard and field scabious this is the first report on the occurrence of phytoplasma diseases, whereas phytoplasmas infecting bristly oxtongue and blueweed have never been characterized before.     

Genetic comparison of the peach yellow leaf roll agent with European fruit tree phytoplasmas of the apple proliferation group

Restriction fragment length polymorphism (RFLP) analysis of PCR-amplified ribosomal DNA and Southern blot hybridization using cloned chromosomal DNA fragments from the apple proliferation (AP) phytoplasma as probes were used to investigate the genetic relationship of the California peach yellow leaf roll (PYLR) agent with phytoplasmas causing fruit tree diseases in Europe. This comparison showed that the California PYLR phytoplasma is closely related to apple proliferation (AP), pear decline, and European stone fruit yellows phytoplasmas and that it is a member of the phylogenetic AP group. The PYLR agent could clearly be distinguished from the AP and European stone fruit yellows phytoplasmas by Southern blot hybridization with DNA fragments from the AP phytoplasma and by RFLP analysis of ribosomal DNA employing Ssp I, Bsa AI, and Rsa I restriction endonucleases. However, the PYLR phytoplasma was indistinguishable from the pear decline agent by RFLP analysis of PCR-amplified ribosomal DNA.