樱桃带化病组织中植物菌原体16SrDNAPCR扩增及其产物RFLP分析

樱桃带化病是从以色列引种樱桃园中发现的新病害。本文报道了按照检测植物菌原体（ｐｈｙｔｏｐｌａｓｍａ）的方法提取ＤＮＡ，扩增患病植株中植物菌原体的１６ＳｒＤＮＡ片段，证明樱桃带化病中有植物菌原体存在，并对此扩增片段进行限制性酶切片段长度多态性（ＲＦＬＰ）分析。根据ＲＦＬＰ分析结果，参照Ｌｅｅ，Ｉ．Ｍ．等的文献资料，对本次樱桃带化病病原进行鉴定与分类，初步定为Ⅶ类。     

西种竹子的RAPD指纹图谱的初步研究

本实验以ＲＡＰＤ技术对考顺竹，凤尾竹，绿竹，白绿竹四个品种用２０种已知序列的１０ｎｔ引物进行ＰＣＲ反应扩增，其中有１７种可扩增出ＤＮＡ条带，构成ＲＡＰＤ指纹图谱，各中引物ＰＣＲ扩增的ＤＮＡ条带数目在０－６条之间，大小在０．３－２．０ｋｂ之间，各种竹子的１７种引物扩增的ＤＮＡ条带总数在２３－４４条之间，四种竹子两两之间的相似系数在３６－７３％之间。     

AFLP分子标记技术及其在林木遗传育种上的应用

ＡＦＬＰ是以ＰＣＲ扩增为基础的，是扩增片段长度多态性的简称。它是ＲＦＬＰ和ＰＣＲ相结合的一种标记技术，其基本原理是对组ＤＮＡ限制性酶切片断的选择性扩增。ＡＦＬＰ的基本反应过程包括模板ＤＮＡ制备、酶切片段的扩增和扩增产物的凝胶电泳分析３个步聚。ＡＦＬＰ是一种新颖和强大的ＤＮＡ指纹技术，结合了ＲＦＬＰ和ＲＡＰＤ的优点，具有高效、快捷、稳定、可靠的特点。本文将ＡＦＬＰ和ＲＦＬＰ、ＲＡＰＤ、ＳＳＲ等分子标     

寡核苷酸管芯片技术检测和鉴别我国不同组植原体

[目的]不同组植原体检测和鉴别的特异性探针已有报道,为了筛选出适合于我国不同组植原体检测和鉴别的特异性探针,建立管芯片检测和鉴别植原体技术,并对我国发生的疑似植原体病害进行鉴别。[方法]通过PCR扩增结合管芯片杂交技术,对收集到的15种植原体侵染的植物样品及其健康对照进行检测和鉴别。[结果]建立了管芯片检测和鉴别植原体技术体系。15种病害样品中,13种获得显著的阳性杂交信号,并且所有的健康对照都呈现为阴性。13种植原体病害依16Sr DNA直接测序可分为16SrⅠ、Ⅱ、Ⅴ、XIX四组植原体。在所有探针中,植原体的通用探针(Pp-502)可以检测到所有确定的植原体样品。16SrⅠ组特异性探针(PpⅠ-465)可以确定16SrⅠ组的泡桐丛枝、苦楝丛枝、桑树萎缩和莴苣黄化4种植原体样品。16Sr II组特异性探针(PpⅡ-629)仅可以确定16Sr II组的花生丛枝、甘薯丛枝和臭矢菜丛枝3种植原体样品。但16Sr V组的枣疯病、樱桃致死黄化和重阳木丛枝及16Sr XIX组的板栗黄化皱缩植原体与其他组专化性探针皆有明显的交叉杂交信号。相比于PCR扩增的凝胶电泳检测,管芯片检测的灵敏度提高了1 000倍。对疑似植原体病害的诊断结果显示河南濮阳的红花槐丛枝的病原应为16Sr V组植原体,福建福州的长春花黄化丛枝应为16SrⅠ组植原体;而北京戒台寺牡丹黄化皱叶和内蒙古包头柳树丛枝未出现任何植原体专化的杂交信号。[结论]管芯片杂交技术作为一种检测和鉴别植原体的方法,可应用于我国植原体病害调查和诊断,并为植原体的鉴别和分类提供可靠的依据。     

PCR检测树木菌原体的研究

按莱氏无胆甾原体和ＭＬＯ的核糖体蛋白基因序列的保守区域，合成一对寡核苷酸引物，用ＣＴＡＢ法直接提取田间发病的桑萎缩和泡桐丛枝病树皮层的ＤＮＡ，以健康树作平行对照。结果用ＰＣＲ成功地从病树ＤＮＡ模板中扩增出１．２６ｋｂ左右的产物，而对照样品均未出带，表明此项技术可以检测ＭＬＯ，并用于研究其分子系统学关系。     

PCR检测树木菌原体的研究

按菜氏无胆甾原体和ＭＬＯ的核糖体蛋白基因序列的保守区域，合成一对寡核苷酸引物，用ＣＴＡＢ法直接提取田间发病的桑萎缩和泡桐丛枝病树皮层的ＤＮＡ，以健康树作平行对照，结果用ＰＣＲ成功地从病树ＤＮＡ模板中扩增出１．２６ｋｂ左右的产物，而对照样品均未出带，表明此项技术可以检测ＭＬＯ，并用于研究其分子系统学关系。     

中国松树枯梢病菌遗传多态性的RAPD分析

运用随机增增多态ＤＮＡ（ＲＡＰＤ）技术对发生于我国１３省区１６种松树和其它２种针叶树上的松枯梢病菌（Ｓｐｈａｅｒｏｐｓｉｓ ｓａｐｉｎｅａ）的５５个菌株进行基因组ＤＮＡ多态性分析。用１７个随机引物经ＰＣＲ扩增共得到２００个ＲＡＰＤ标记,其中９８．５％具有多态性。ＵＰＧＭＡ聚类分析确定了供试菌株间的亲缘关系,将５５个菌株分为３个类群。各菌株间的差异与其寄主种类无明显关系,与其地理来源在某些类群间有一定联系,但在大多数菌株间相关趋势不明显。     

绣线菊丛枝病病原的分子鉴定

用植原体16S rRNA基因的通用引物,对表现黄化、丛枝、顶枯等症状的绣线菊DNA进行PCR扩增,得到了约1.2kb的特异性片段.测序结果证明其病原为植原体.RFLP和序列分析表明:该分离物属于翠菊黄化组的16SrⅠ -B亚组.与GenBank中其他植原体分离物序列比较,发现该分离物与16SrⅠ -B亚组中的西方翠菊黄化植原体(SAY)同源性高达99.6%.     

PCR在真菌与植物病理学中的应用现状

介绍ＰＣＲ的原理、操作和影响反应的条件。论述了ＰＣＲ应用于克隆抗病虫目的基因、鉴定转基因植物中的靶基因的方法和策略。综述了几年来用于研究真菌系统进化和分子分类的现状，包括真菌能用引物的位置。反映了ＰＣＲ研究类菌原体的最新进展，对其在根结线虫、类病毒等方面的应用也作一简述。同时介绍近一年来应用较多的ＲＡＰＤ进行遗传标记和种以下水平的分类的情况。     

猪屎豆丛枝病植原体的分子检测与鉴定

植原体(phytoplasma),原称类菌原体(MLO),在植物和昆虫中广泛分布,为无细胞壁原核微生物,尚不能在人工培养基上离体培养.迄今,世界各地已统计有1000多种植物自然感染植原体病害(Seemüller et al.,1998),我国也报道了100多种植物植原体病害(赖帆等,2008).     

Molecular identification of a phytoplasma associated with Elm witches’‐broom in China

Elm samples with and without witches’‐broom symptoms (EWB) were collected from Tai’an and Zhaoyuan, Shandong Province, China. Phytoplasmal cells were observed in the phloem cells of symptomatic plants under electron microscope. Specific fragments of about 1.2 kb in length were amplified with nested‐PCR from symptomatic samples, while no fragment was obtained from healthy plants. The 16S rRNA gene sequences of the phytoplasmas associated with elm witches’‐broom in Tai’an (EWB‐TA) and Zhaoyuan (EWB‐ZHY) had high similarities, and formed a sublineage in phylogenetic tree, with members of subgroup B or D of aster yellows group (16SrI). Computer simulated restriction fragment length polymorphism analysis of 16S rRNA gene revealed that EWB‐TA and EWB‐ZHY patterns had similarity coefficients of 1.00 with the pattern from the representative strains of subgroup 16SrI‐B, and had a similarity coefficient of lower than 0.97 with representatives of other subgroups. These results indicated that the phytoplasma strain associated with elm witches’‐broom in China was very closely related to ‘Candidatus Phytoplasma asteris’ OAY, belonging to subgroup‐B of aster yellows group (16SrI‐B). This is the first report of a phytoplasma associated with elm witches’‐broom disease in China.     

Salix tetradenia Hand.‐Mazz: a new natural plant host of ‘Candidatus phytoplasma’

This article reports Salix tetradenia Hand.‐Mazz as a new host of Candidatus phytoplasma and demonstrates its association with witches' broom disease on S. tetradenia plants. Plants exhibited typical visual symptoms of phytoplasma with virescence, abnormality of flowers and witches' broom, and phytoplasma bodies were observed by transmission electron microscopy. Products of 1.2 kb were amplified by nested PCR using phytoplasma universal primer pairs R16F2n/R16R2, but no amplification products were obtained from symptomless plants. The sequence analysis of three 16S rDNA isolates showed 99.84%, 99.68% and 99.76% identify, respectively, with the homologous gene (nc_005303) of member of ‘Candidatus phytoplasma asteris’ (16SrI) group. Phylogenetic and virtual computer‐simulated restriction fragment length polymorphism analysis of the 16S rRNA, tuf and rp gene sequences confirmed that this phytoplasma clustered in the 16SrI‐B subgroup. These results indicated that the diseased S. tetradenia plants were infected by a phytoplasma of the 16SrI group. This is the first report on the occurrence of phytoplasma disease on S. tetradenia worldwide.     

枣疯病和泡桐丛枝病原植原体分离物的组织培养保藏和嫁接传染研究

分别从河北唐县赞皇大枣、辽宁凌源梨枣和河南濮阳扁核酸3个品种的枣疯病和来自山东、江西和北京的不同无性系的泡桐丛枝病树上采集丛枝枝条作为组织培养材料,获得的枣疯病和泡桐丛枝病组培苗皆表现典型的丛枝症状。其中感染植原体的赞皇大枣组培苗(Ft)和扁核酸组培苗(HPD)在未加任何激素的MS培养基和其它培养基交替继代培养1a以上仍能维持丛枝苗生长;而发病梨枣(LD)除产生丛枝外,还出现叶片黄化和植株矮化、枯梢等衰退症状。泡桐丛枝病植原体可在不同无性系组培苗上通过各种培养基交替和单纯的MS培养基继代培养,并已在实验室内连续保藏达10a,其引致丛枝症状的能力无明显的改变。用枣树Ft染病材料作接穗,以健康冬枣(DJ)和抗病婆枣(W14)砧木进行组培苗间嫁接传病试验,可使部分DJ和W14发病;而嫁接未发病的砧木多数像健苗一样正常生长。用感染山东泡桐丛枝病植原体ZD株系丛枝组培苗为接穗,嫁接健康泡桐无性系组培苗致使无性系MB33、TY2T和C125发病。用植原体16SrDNA通用引物进行PCR,确证了泡桐和枣树发病苗和嫁接发病组培苗体内存在植原体。用DAPI荧光显微镜技术比较组培苗韧皮部筛管中的植原体浓度,结果显示,Ft和嫁接发病冬枣(DJ-Ft)筛管中植原体浓度相对较高,但仍低于各泡桐无性系染病丛枝组培苗;HPD和LD浓度中等,而发病的W14砧木含有植原体的筛管数量较少、且浓度很低。在嫁接不成功或未发病的DJ和W14砧木组织及健康对照组织中皆检测不到植原体荧光。     

A new phytoplasma associated with witches’‐broom on Japanese maple in China

In September 2011, five Japanese maple (Acer palmatum Thunb.) trees with symptoms of witches’‐broom were observed growing near each other at a maple grove in Northwest A&F University, Yangling, Shaanxi Province, China. Pleomorphic phytoplasma‐like bodies were observed in the phloem sieve tube elements of symptomatic plants under transmission electron microscope (TEM). The presence of phytoplasma was further confirmed by a nested polymerase chain reaction (PCR), which amplified a 1.2‐kb fragment using universal primer pair R16mF2/R16mR1 followed by further amplification using primer pair R16F2n/R16R2. Phylogenetic analysis and gel‐based restriction fragment length polymorphism (RFLP) analysis demonstrated that the Japanese maple witches’‐broom was associated with phytoplasma belonging to subgroup 16SrI‐D. This is the first report of a phytoplasma disease of Japanese maple.     

根癌农杆菌对感染植原体的泡桐组培苗症状的影响

采用含有激素合成相关基因的根癌农杆菌，伤口接种已感染植原体的泡桐丛植组培苗和健康组培苗，结果发现对丛植苗的致瘤能力明显低于健康对照苗，且被接种病苗的丛枝症状缓解，从健苗获得的T-DNA转化泡桐瘤组织细胞能在无激素培养基上稳定生长和连续继代培养2年以上，说明瘤组织细胞自身已获得了细胞分裂素和生长素合成能力，根据已报道的根癌农杆菌株系pTil5955T-DNA的异戊烯基转移酶基因（ipt)的保守序列，设计了一对引物（CYT和CYT′），用多聚酶链式反应（PCR）扩增了我国杨树致瘤农杆菌ipt基因部分序列（427bp片段），也从遗传转化的两个泡桐无性系瘤组织At-ZH和At-T35扩增出此特异片估，从而进一步肯定了T-DNA已被整合到泡桐的染色体上表明泡桐易于通过Ti质粒载体途径进行基因转移操作，但用此引物未能从泡桐、甘薯健株和感染植原体的组培病苗扩增出相应的427bp特异片段，当用此遗传转化瘤组织嫁接病苗时，可减轻从枝症状的严重度，延长病苗的存活时间和诱导病株生根，这进一步证实了泡桐在与植原体相互作用过程中激素代谢发生了变化。     

Identification and characterization of the phytoplasma associated with elm yellows in southern Italy and its relatedness to other phytoplasmas of the elm yellows group

In the neighbouring regions Basilicata, Campania, and Calabria of southern Italy, diseased trees of European field elm (Ulmus minor) were examined for phytoplasmal infection using polymerase chain reaction (PCR) technology. All affected trees examined tested positively. Using a primer pair specific for the EY phytoplasma group and restriction fragment length polymorphism (RFLP) analysis of PCR-amplified ribosomal DNA, the organism detected was identified as the elm yellows (EY) phytoplasma. RFLP analysis of PCR-amplified ribosomal DNA was also employed to attempt differentiation within the EY group which includes, in addition to the EY agent, phytoplasmas infecting Rubus, alder, eucalypts, Spanish broom, and grapevine. Following separate digestion with AluI, RsaI, Sau3AI, MseI, HhaI, and KpnI, all PCR-products from EY-group phytoplasmas examined had similar RFLP profiles. When the same ribosomal DNA fragments were digested with TaqI restriction endonuclease, three different restriction profiles were detected among the EY-group phytoplasmas. These profiles represented, respectively, (1) the EY phytoplasma (2) the phytoplasmas causing rubus stunt and being associated with alder yellows, spartium witches broom, and eucalyptus little leaf, and (3) the flavescence dorée phytoplasma. RFLP analysis using TaqI endonuclease enabled for the first time the differentiation of the phytoplasmas associated with alder yellows, eucalyptus little leaf, and spartium witches broom from the EY agent.     

Molecular identification of a phytoplasma associated with Sophora Root yellows

A phytoplasma infecting Sophora Root (Sophora alopecuroides) was detected and identified in Alar, Xinjiang Uygur Autonomous Region of China. Typical phytoplasma bodies were observed in sieve tubes of the diseased plants by transmission electron microscopy. A partial 16S rRNA gene and ribosomal protein (rp) genes containing rpl22 (rplV) and rps3 (rpsC) were amplified by direct and nested PCR. Based on the sequence similarity of the 16S rRNA and rp genes with accompanying phylogenetic analyses, the phytoplasma associated with Sophora Root yellows belongs to the 16SrI group (aster yellows group). Virtual RFLP analysis of these 16S rRNA and rp gene sequences showed distinct differences from those of reference phytoplasma strains representing previously described subgroups of the 16SrI group. Moreover, the similarity coefficient (0.92) of the RFLP profile of this phytoplasma was less than the threshold similarity coefficient (0.97) required for subgroup classification. Thus, the phytoplasma isolate of Sophora Root plants, designated as ‘SoRY’, represents a new subgroup. Furthermore, this is the first report of phytoplasma disease associated with Sophora Root plants.     

“Candidatus Phytoplasma solani” associated with Eucalyptus witches’ broom in Iran

During summer of 2015, Eucalyptus camaldulensis plants showing witches’ broom, little leaf and general yellowing of the foliage were observed in west of Fars and Khozestan province of Iran. DNA from samples of 22 symptomatic and two asymptomatic trees was extracted and subjected to molecular analyses. Nested‐PCR test using R16F2n/R16R2 primers confirmed phytoplasma presence in 63% of symptomatic Eucalyptus plants. Sequence analysis along with virtual RFLP of the 16S ribosomal DNA allowed to classify three Eucalyptus witches’ broom strains into the “stolbur” (“Candidatus phytoplasma solani”) 16SrXII‐A subgroup. Comparison of the secA and secY gene sequences with sequences deposited in GenBank confirmed the phytoplasma identity. Real and virtual RFLPs of the amplified secY gene using HaeIII, MseI and RsaI restriction enzymes showed profiles indistinguishable from each other. This is the first study reporting E. camaldulensis as a new host species for “Ca. P. solani.”     

Detection of phytoplasma infections in declining Populus nigra ‘Italica’ trees and molecular differentiation of the aster yellows phytoplasmas identified in various Populus species

A disease of Populus nigra‘Italica’ associated with foliar yellowing, sparse foliage, stunting, dieback, and decline was observed in south-western Germany; a witches’ broom disease of Populus alba that is known in other countries was also detected in Hungary and Germany. The aetiology of the diseases was studied by fluorescence microscopy and polymerase chain reaction (PCR) amplification. Using fluorescence microscopy, phytoplasmas could be detected only in P. alba. However, most diseased trees of P. nigra‘Italica’ tested phytoplasma-positive by PCR. In some of the trees the phytoplasma numbers were so low that nested PCR was required to detect the infection. Very low phytoplasma numbers were also observed in diseased Populus tremula. The identity of phytoplasmas from P. nigra‘Italica’ sampled in Germany and France, P. alba and also P. tremula was examined by restriction fragment length polymorphism (RFLP) analysis of PCR-amplified ribosomal DNA. In all poplars, phytoplasmas of the aster yellows group were detected. However, three different RFLP groups were identified that consisted of (1) French strains from P. nigra‘Italica’, (2) German strains from P. nigra‘Italica’ and (3) strains from P. alba and P. tremula. The profile observed in the last group was probably the result of sequence heterogeneity in the two 16S RNA genes.