Molecular phylogenetic,morphological, and pathogenic analyses reveal a single clonal population of Septoria lycopersici with a narrower host range in Brazil

Septoria leaf spot, caused by Septoria lycopersici, is considered one of the most important diseases of tomato in Brazil. Despite its importance, the disease agent is still poorly studied. Septoria isolates collected from different production regions of Brazil were characterized by molecular, morphological, and pathogenic methods. A set of 104 isolates was sequenced for the DNA Tub, Cal, and EF1-α loci. Ten isolates were selected, according to geographical region of origin and type of leaf lesion (typical or atypical), for morphological characterization and for evaluation of aggressiveness on tomato cultivar Santa Clara. To evaluate the pathogen host range, cultivated and wild Solanaceae plants were inoculated with four selected isolates. The results showed that all isolates grouped with the type isolate of S. lycopersici in maximum likelihood and Bayesian inference trees. The isolates were morphologically similar. All isolates selected for pathogenicity testing on tomato were able to induce typical symptoms of the disease, but differed in their aggressiveness. A total of eight species of Solanaceae were also identified as potential alternative hosts for S. lycopersici. This information will provide a more accurate assessment of the risks involved with the introduction of new crops, especially of the genus Solanum, in areas where the species is already present. In addition, it will provide the basis for the establishment of more efficient methods in the management of Septoria leaf spot of tomatoes in natural conditions and in the different production systems.     

Phylogenetic and serological analyses reveal genetic diversity of Agrobacterium vitis strains in Japan

Twenty-eight strains of Agrobacterium vitis , including both tumorigenic and nonpathogenic phenotypes involving 26 isolated in Japan and strains NCPPB 3554^T and NCPPB 2562 isolated in Australia and Greece, respectively, were characterized by means of a slide agglutination test (SAT) using antisera prepared against somatic antigens. Phylogenetic analyses were also carried out, using the results of repetitive sequence-based polymerase chain reaction and the partial nucleotide sequences of pyrG , recA and rpoD . The A. vitis strains separated into four serogroups (A to D) based on the SAT reactivity. The phylogenetic analyses showed A. vitis strains separated into four tumorigenic groups (A to D) and one nonpathogenic group (E). Serogroups A to C corresponded exactly to genetic groups A to C, respectively, whereas serogroup D further divided into two distinct genetic groups, D and E. Genetic group E was isolated in Okayama Prefecture, Japan, and all strains of it have been found to coexist with tumorigenic strains belonging to the other groups within the same galled grapevine tissues. These results suggest that A. vitis strains are genetically heterogeneous and can be separated into several genetic groups. The differences between the nucleotide sequences of pyrG , recA and rpoD of the genetic groups will enable the development of a simple and convenient monitoring method, which will increase understanding of the dynamics of each genetic group of A. vitis in the natural environment.     

臭矢菜丛枝病植原体的分子鉴定研究

 本实验采用DAPI荧光显微镜、PCR、克隆和测序等技术,对海南臭矢菜丛枝病样进行了检测和鉴定。以染病臭矢菜总DNA为模板应用3对植原体特异性引物进行PCR扩增,获得PCR产物为16S rDNA(1 430 bp)、16S-23S rDNA(358bp)、rp DNA(1 294 bp)。应用DNA回收试剂盒获得了3个PCR扩增片断的纯化产物,并克隆到DH5α大肠杆菌中测序。应用DNAMAN和MEGA软件对获得的序列与NCBI数据库中植原体序列进行同源性分析和构建系统发育树。结果显示臭矢菜丛枝病植原体与花生丛枝病植原体序列同源性最高,16S rDNA的序列同源性为99.9%,16S-23S rDNA高达100%,rp为99.7%,因而将臭矢菜丛枝病植原体归为花生丛枝组(16SrⅡ),根据16S rDNA的RFLP分析,将其归为16SrⅡ-A亚组。     

三叶草绿变病植原体的分子鉴定

三叶草(Trifolium pratense Linn.)为车轴草属,蝶形花科多年生草本植物,原产亚洲南部和欧洲东南部,是一种世界性分布与栽培的优良牧草.因其花叶兼优、草姿美、绿期长而具有较高的观赏价值,近几年作为草坪用草被广泛种植.在自然条件下,三叶草很容易受到不同种植原体的侵染,国外已报道的侵染三叶草的植原体有:三叶草绿变植原体( Clover phyllody phytoplasma,CPh)和三叶草增殖植原体(Clover proliferation phytoplasma,CP)等,这些植原体分别属于16SrI组和16SrⅥ组[1,2].     

樱桃花变绿病植原体的分子鉴定

 植原体(phytoplasma)是一类没有细胞壁,不能人工培养,存在于植物筛管细胞中的类似植物病原细菌的原核生物。迄今为止,世界各地报道的1 000余种植物病害与植原体有关,引起的症状主要包括丛枝、黄化、花变绿、花变叶、花器退化等。     

金莲花绿变植原体的分子鉴定

本研究对河北省大面积发生的金莲花绿变病的病原进行检测和鉴定。以金莲花叶片的总DNA为模板,使用植原体16S rDNA和核糖体蛋白(ribosomal protein)基因rp的特异性引物进行PCR扩增,在感病金莲花样品中扩增到植原体的16S rDNA(1 432 bp)片段和rp基因(1 240 bp)片段。序列分析发现,获得的16S rDNA和rp基因片段与洋葱黄化植原体Onion yellows phytoplasma(GenBank登录号:AP006628)的相似度最高,分别为99.9%和99.3%,确定金莲花绿变病的病原为植原体,暂命名为金莲花绿变植原体Trollius chinensis virescence phytoplasma。对金莲花绿变植原体的16S rDNA进行虚拟RFLP分析,发现其酶切图谱与16SrⅠ-B亚组的洋葱黄化植原体的参照图谱完全一致,相似系数1.00。16S rDNA和rp基因的系统发育进化树显示,金莲花绿变植原体与16SrⅠ-B亚组的植原体聚为一支,属于植原体16S rⅠ-B亚组。     

紫花苜蓿丛枝病植原体的分子检测及鉴定

 利用植原体16S rRNA基因通用引物对云南昆明发生的苜蓿丛枝病感病植株总DNA进行巢式PCR扩增,得到1.2kb的特异片段,从分子水平证实了苜蓿丛枝病的病原是植原体。从PCR产物的RFLP酶切图谱可看出,该植原体株系的酶切图谱与马里兰翠菊黄化植原体(AY1)相同。对扩增片段进行克隆及序列测定后,利用最小进化法做Bootstrap验证的系统进化树,表明苜蓿丛枝病植原体为Candidatus Phytoplasma asteris成员之一,与植原体16SrI-B亚组成员关系密切。     

丁香卷叶病植原体的分子鉴定

 通过透射电子显微镜,在表现卷叶、褪绿症状的丁香(Syringa oblata)样品的叶脉韧皮部筛管细胞内观察到大量植原体粒子。应用植原体16S rRNA基因通用引物对P1/P7和R16F2n/R16R2对表症丁香植株总DNA进行巢式PCR扩增,得到了约1.2 kb的目标片段,通过对扩增片段进行测序、系统发育分析和同源性分析,结果表明,该片段长度为1 246 bp,在系统发育进化树上与翠菊黄化组（Candidatus Phytoplasma asteris）成员是聚集在一起的,与该组成员同源性均在98%以上。用16Sr RNAⅠ组和Ⅴ组特异引物确定了该病害非混合侵染所致,相似性系数和RFLP分析表明该植原体属于16SrⅠ B亚组。这是国内关于翠菊黄化组植原体在丁香上感染的首次报道。     

滇朴丛芽病植原体的分子鉴定

滇朴Celtis kunmingensis Cheng et Hong是云南的乡土树种,适宜全国大部分地区种植,极具观赏价值,是近年来最热门的绿化首选树种—绿化行道树,云南部分地区滇朴近年常表现丛芽的症状.本研究采用形态学与分子生物学结合的方法,对染病的幼嫩枝条进行扫描电镜(SEM)观察;利用16S rDNA植原体通用...     

两种林木植原体病害的分子鉴定

近年来, 林木植原体病害发生日趋严重, 对我国林业经济和生态造成了很大损失。2021年-2022年, 在山西沙棘主产区的沙棘上和北京冬奥园区的油松上分别出现了典型的植原体病害症状;沙棘叶片皱缩, 呈小叶状;油松过度分枝生长且出现球状结构等。本研究通过荧光显微观察, 16S rRNA和tuf基因序列分析, 并结合虚拟限制性片段长度多态性分析证实了这两种病害均由植原体引起。基于16S rRNA的系统进化分析显示:引起沙棘叶片皱缩的植原体与泡桐丛枝植原体(Paulownia witches’-broom phytoplasma, OP107515.1)的相似性最高(99.92%), 引起油松丛枝的植原体与狗尾草丛枝植原体株系(Setaria viridis witches’-broom phytoplasma, FJ263625.1)的相似性最高(99.52%);基于tuf基因的系统发育树显示, 二者同属于16SrⅠ组D亚组(16SrⅠ-D)。本研究首次明确了沙棘叶片皱缩病和油松丛枝病相关植原体的分类地位, 为这两种植原体病害的准确诊断、快速检测及其防治提供了基础资料。     

Isozyme and RAPD-PCR analyses of Fusarium avenaceum strains from Finland

Differences in isozyme and RAPD-PCR polymorphisms amongst 33 isolates of Fusarium avenaceum were compared using native polyacrylamide gel electrophoresis and agarose gel electrophoresis. The isolates were collected from different regions of Finland. Amongst eight enzymes analysed clear isozyme polymorphism was detected in five enzymes which could be grouped into 20 different electrophoretic phenotypes and three main groups at the similarity level of 70% in unweighted pair group method with arithmetic average (UPGMA) analysis. RAPD-PCR analysis differentiated all F. avenaceum strains from each other. The phenotypes resulting from RAPD-PCR analysis were grouped into five main groups by UPGMA analysis at the similarity level of 55%. These main groups had several similarities with the main groups from isozyme analysis. RAPD-PCR patterns of 16 isolates of Fusarium graminearum F. culmorum F. equiseti F. oxysporum and F. redolens were also studied and strains from each Fusarium species formed individual groups in UPGMA and principal components analyses. Thus, the extent of isozyme and RAPD-PCR polymorphisms found in Fusarium strains potentially provides a method for identifying the fungi both at strain and species level.     

Molecular identification of a new phytoplasma associated with alfalfa witches'-broom in oman

ABSTRACT Alfalfa (Medicago sativa) plants showing witches'-broom symptoms typical of phytoplasmas were observed from Al-Batinah, Al-Sharqiya, Al-Bureimi, and interior regions of the Sultanate of Oman. Phytoplasmas were detected from all symptomatic samples by the specific amplification of their 16S-23S rRNA gene. Polymerase chain reaction (PCR), utilizing phytoplasma-specific universal primer pairs, consistently amplified a product of expected lengths when DNA extract from symptomatic samples was used as template. Asymptomatic plant samples and the negative control yielded no amplification. Restriction fragment length polymorphism profiles of PCR-amplified 16S-23S rDNA of alfalfa using the P1/P7 primer pair identified phytoplasmas belonging to peanut witches'-broom group (16SrII or faba bean phyllody). Restriction enzyme profiles showed that the phytoplasmas detected in all 300 samples belonged to the same ribosomal group. Extensive comparative analyses on P1/P7 amplimers of 20 phytoplasmas with Tru9I, Tsp509I, HpaII, TaqI, and RsaI clearly indicated that this phytoplasma is different from all the other phytoplasmas employed belonging to subgroup 16SrII, except tomato big bud phytoplasma from Australia, and could be therefore classified in subgroup 16SrII-D. The alfalfa witches'-broom (AlfWB) phytoplasma P1/P7 PCR product was sequenced directly after cloning and yielded a 1,690-bp product. The homology search showed 99% similarity (1,667 of 1,690 base identity) with papaya yellow crinkle (PapayaYC) phytoplasma from New Zealand. A phylogenetic tree based on 16S plus spacer regions sequences of 35 phytoplasmas, mainly from the Southern Hemisphere, showed that AlfWB is a new phytoplasma species, with closest relationships to PapayaYC phytoplasmas from New Zealand and Chinese pigeon pea witches'-broom phytoplasmas from Taiwan but distinguishable from them considering the different associated plant hosts and the extreme geographical isolation.     

Molecular biological study on the survival strategy of phytoplasma

Journal of General Plant Pathology - Phytoplasmas (genus ‘Candidatus Phytoplasma’) are plant pathogenic bacteria that reside intracellularly within the plant phloem. They infect...     

广东桉树黄化(丛枝)病植原体分子鉴定与检测

从表现黄化(丛枝)症状的桉树上采集病叶,抽提主脉总DNA,采用植原体通用引物与巢式引物进行PCR和巢式PCR扩增,对扩增产物进行克隆和序列测定,获得了植原体的近全长16S rRNA基因及部分16～23S rRNA基因间隔区序列.序列分析揭示,所获得的序列与已知植原体基因组相应区段的序列高度同源,与柳叶菜变叶植原体(epilobium phyllody)和白腊树丛枝植原体(ash witches'-broom)相应序列(GenBank登录号:AY101386和AY566302)同源率为99.9%,与白腊树黄化植原体(aster yellows BD2)相应序列和番茄巨芽植原体(tomato big bud)相应序列同源率分别为99.6%和99.3%.该序列构建的系统进化树表明,引起我国广州地区桉树黄化(丛枝)病的植原体属于16SrI组(即翠菊黄化组),将其暂命名为桉树黄化(丛枝)植原体广东株系(Eucalyp-tus yellowing and witches'-broom phytoplasma strain Guangdong,EYWB-Gd).建立了桉树植原体巢式PCR检测方法,对疑似病样及桉树组培苗进行了检测,多数疑似病样检测结果为阳性,供试的10株组培苗未发现阳性样品.     

Molecular analyses of colletotrichum species from almond and other fruits

ABSTRACT Isolates of Colletotrichum spp. from almond, avocado, and strawberry from Israel and isolates of the pink subpopulation from almond from the United States were characterized by various molecular methods and compared with morphological identification. Taxon-specific primer analysis grouped the avocado isolates within the species C. gloeosporioides and the U.S. almond and Israeli strawberry isolates within the species C. acutatum. However, the Israeli almond isolates, previously identified morphologically as C. gloeosporioides, reacted with C. acutatum-specific primers. Arbitrarily primed polymerase chain reaction and A+T-rich DNA analyses determined that each population from almond and strawberry was distinct and clonal. Sequence analysis of the complete internal transcribed spacer (ITS) region (ITS 1-5.8S-ITS 2) revealed a similarity of between 97.03 and 98.72% among almond isolates from Israel, C. acutatum almond isolates from the United States, and C. acutatum strawberry isolates from Israel. Similarity of the above populations to that of C. gloeosporioides of avocado was between 92.42 and 92.86%. DNA sequence analysis of the entire ITS region supported the phylogeny inferred from the ITS 1 tree of 14 different Colletotrichum species. Although morphological criteria indicated that the Israeli isolates from almond are unique, this population was grouped within the C. acutatum species according to molecular analyses.     

Molecular characterization of a phytoplasma causing Phyllody in Clover and other herbaceous hosts in Northern Italy

Red clover (Trifolium pratense) and Ladino clover (Trifolium repens) plants showing phytoplasma-associated symptoms (yellowing/reddening, virescence and phyllody) have been recovered in Friuli-Venezia Giulia, Italy. Using AluI RFLP analysis of PCR amplified 16S rDNA we showed that the disease can be caused independently by two phylogenetically distinct phytoplasmas. One of them showed the very typical 16S rDNA RFLP pattern of the agent of Clover Phyllody in Canada (CCPh). The 16S rDNA of the other phytoplasma (Italian Clover Phyllody phytoplasma, ICPhp) has been PCR amplified, cloned and sequenced. The sequence revealed high similarity (>98%) with phytoplasmas belonging to the X disease cluster, which includes organisms not reported to cause phyllody on their hosts. The analysis by AluI RFLP of the PCR amplified pathogen 16S rDNA from other herbaceous plants (Crepis biennis, Taraxacum officinale, Leucanthemum vulgare) collected nearby with phytoplasma-associated symptoms showed similar patterns. Southern blot hybridization of their EcoRI digested total DNA revealed identical RFLP patterns, suggesting that the causative agent may be the same organism.Abbreviations PCR Polymerase Chain Reaction - rDNA gene for the small subunit ribosomal RNA - RFLP Restriction Fragment Length Polymorphism     

榆树黄化病植原体的分子检测与鉴定

 利用植原体16SrRNA基因的通用引物R16rrLF2/R16mR1和R16F2n/R16R2对山东泰山上发生的榆树(Ulmus parvifolia)黄化病感病植株总DNA进行巢式PCR扩增,得到了约1.2kb的特异性片段,从分子水平证实了榆树黄化病的病原(EY-China)为植原体。将扩增到的片段测序,并进行一致性和系统进化树分析。结果表明,该分离物属于植原体榆树黄化组(Candidatus Phytoplasma ulmi),与该组成员16SrRNA序列的一致性均在98.2%以上,其中与16SrV-B亚组中的纸桑丛枝(Paper mulberry wiches'-broom)和枣疯病(Jujube witches'-broom)植原体一致性最高,达到99.4%,在系统进化树中与该亚组成员聚类到同一个分支,说明该分离物属于植原体16SrV-B亚组。本研究首次对在中国引致榆树黄化病的植原体进行了分子检测,并通过核酸序列分析将其鉴定到亚组水平。     

Detection and identification of a phytoplasma from lucerne with Australian lucerne yellows disease

Foliar and root symptoms are described for Australian lucerne yellows (ALuY), a disease common in Australian lucerne seed crops. A phytoplasma was detected in plants exhibiting symptoms, but not in symptomless lucerne plants. Oligonucleotide primers specific to the phytoplasma 16S-23S rRNA intergenic spacer region (SR) were used in polymerase chain reaction (PCR) assays on DNA extracted from lucerne plants with and without symptoms. Identical restriction fragment length polymorphism (RFLP) enzyme profiles were obtained for PCR products amplified from 10 yellows-affected lucerne samples. RFLP profiles obtained for four restriction enzymes were different from those of the tomato big bud (TBB) phytoplasma. ALuY phytoplasma PCR products were sequenced to determine phylogeny and were found to fall within the faba bean phyllody phytoplasma group, or phytoplasma group 16srII. Transmission electron microscopy revealed phytoplasmas in the phloem of yellows-affected plant samples, but not in symptomless plant samples. Fungal, bacterial and viral agents in the aetiology of Australian lucerne yellows were ruled out.     

Phylogenetic relationships and population structure of Ralstonia solanacearum isolated from diverse origins in Taiwan

Bacterial wilt caused by Ralstonia solanacearum is a destructive disease for many crops. The aim of this study was to investigate the phylogenetic relationships and genetic structure of an R. solanacearum population from diverse origins in Taiwan. All 58 tested isolates belonged to phylotype I, except the two biovar 2 isolates from potato. These belonged to phylotype IIB sequevar 1 and were identical to known potato brown rot strains, which were probably introduced. Phylotype I isolates were grouped into 10 sequevars. Sequevar 15 was predominant (34 out of 56 isolates). Its distribution covered the whole island and it was largely associated with solanaceous crops such as tomato, and with tomato field soil. A total of 14 haplotypes were identified based on a partial endoglucanase gene sequence. Parsimony network analysis revealed that haplotype A was the oldest haplotype in the local population. It encompassed all but one of the sequevar 15 isolates. Large variation in virulence on tomato was observed among the 58 isolates, and seven pathotypes were identified. Significant genetic differentiation was detected among pathotypes. Moreover, genetic differentiation was detected between biovar 3 and biovar 4 subgroups and between the strains associated with solanaceous and non‐solanaceous species, but none was detected between strains from different geographic origins. The results suggest that the phylotype I population in Taiwan is homogeneous, while mutation and local adaptation to specific ecological niches keep shaping the population.