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。     

越冬病枝水培与PCR结合检测枣疯病植原体的存活动态

以感染枣疯病的婆枣为材料进行了以下两方面的研究：其一,采集越冬期和越冬前后的枣疯病枝条并对越冬期枝条进行水培,通过萌芽的症状观察和PCR检测,发现病枝条韧皮部和萌芽内均可扩增出1.5 Kb的枣疯病植原体特异性条带,此结果表明,枣疯病植原体可以在-10℃左右的地区在枣树枝条内安全越冬;其二,利用PCR技术检测了冬季轻病树的病枝、根部、主干及树冠内未表现症状的枝条韧皮部,结果只在病枝内检测到植原体的存在,而在其它部位都未发现植原体.     

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%.     

车桑子丛枝病的病原分子检测鉴定及风险分析

在攀枝花市米易县车桑子Dodonaea viscosa种植区调查丛枝病的发生情况。应用植原体16S rDNA基因的通用引物R16m F2/R16mRl和R16F2/R16R2对具有典型植原体侵染症状和无症车桑子进行检测,巢式PCR得到约1.2 kb的特异性片段,经过测序并在NCBI数据库进行比对,获得1条植原体特定的16S rDNA基因序列(DvWB-PZHMY1);将测得的序列与已报道的植原体序列进行同源性比对,并构建系统进化树,结果显示获得的植原体序列均聚类到16S rI-B亚组中。风险评估分析表明,车桑子丛枝病达到中度风险级别,应给予重视。     

以tuf基因为靶标的5种16SrⅠ组植原体环介导恒温扩增技术

【目的】建立一种能够特异性检测和鉴定16SrⅠ组植原体的环介导恒温扩增技术,实现16SrⅠ组植原体的快速检测。【方法】以植原体tuf基因作为靶标,通过序列比对,设计多组具有特异性的LAMP引物组,筛选出一套适用于16SrⅠ组植原体的特异性检测体系。以16SrⅡ组、16SrⅤ组、16SrⅩⅨ组植原体样品按照此检测体系进行反应,来验证其特异性;同时将稀释后的感病组织样品同步进行PCR和LAMP检测,以确定其检测灵敏度。对来自不同省市的6份田间样品以及9份组培样品进行检测,以验证其检测的稳定性。【结果】16SrⅠ-LAMP在恒温63℃条件下40 min内完成扩增,能检测5种分别引起泡桐丛枝、苦楝丛枝、桑树萎缩、莴苣黄化和长春花绿变病的16SrⅠ组植原体,不能检测其他组植原体包括16SrⅡ组的花生丛枝、甘薯丛枝、臭矢菜丛枝、16SrⅤ组的枣疯病、红花槐丛枝、樱桃致死黄化和16SrⅩⅨ组的板栗黄化皱缩植原体。通过在反应液中加入钙黄绿素肉眼判断绿色为阳性结果,褐色为阴性结果,与用荧光定量设备进行判读扩增曲线的结果一致。相比于PCR检测,16SrⅠ-LAMP检测的灵敏度提高了8倍;16SrⅠ-LAMP能够快速检测出来自不同区域的田间泡桐发病样品、感病泡桐组培苗和嫁接传病脱毒苗。【结论】以tuf基因作为靶标,建立能同时检测5种16SrⅠ组植原体的环介导恒温扩增技术,具有高效、特异、操作简便、检测时间短及成本较低等优点,适合于基层和现场检测。     

灰叶丛枝病病原的分子鉴定及系统发育分析

实验应用PCR扩增、iPhyClassifer分析、序列同源性及系统发育分析等方法对海南省灰叶丛枝病病害进行了分子检测及其病原的鉴定、系统进化研究.iPhyClassifer结果显示:灰叶丛枝病植原体为花生丛枝组(16SrⅡ组)16SrⅡ-A成员.序列同源性分析结果表明:灰叶丛枝病植原体与16SrⅡ、16SrⅡ-A成员...     

羽脉山黄麻丛枝植原体的分子鉴定及病害调查

【目的】了解羽脉山黄麻丛枝病在云南省玉溪市新平县的发生情况和危害程度,通过分子生物学方法确定病原物及其分类地位,为本地区植原体防控提供参考。【方法】利用普查法获得病害发病率,评估病害危害程度。利用植原体通用引物P1/P7及R16F2n/R16R2对感病植株总DNA进行巢式PCR扩增、克隆和测序,得到16S r DNA序列。再用植原体在线分类鉴定工具i PhyClassifier以及MegaX软件分别进行序列分析和构建基于16S r DNA序列的系统进化树,确定病害病原物及其分类地位,通过在线分析软件MUSCLE比较相关序列的同源性。【结果】根据调查发现羽脉山黄麻丛枝病发生于大约101°35'—101°53'E和23°56'—24°20'N之间,主要是在低海拔400～600 m温度相对较高的干热河谷地区,说明高温有利于植原体病害的发生。该处连续3年发生此病害,2018年7月调查得知其平均发病率为38.9%,属中度危害。PCR扩增获得约1 246 bp的羽脉山黄麻丛枝病植原体16S r DNA序列(株系:TLWBYN01,登录号:MN513329)。分析表明TLWBYN01与翠菊黄化植原体(Candidatus Phytoplasma asteris)的参考菌株(M30790)相似度为99.8%,因此该植原体为‘Candidatus Phytoplasma asteris’相关菌株,属于16S r I组成员。TLWBYN01的F2nR2片段虚拟RFLP模型与16S r I-X亚组的参考模型番木瓜束顶植原体(登录号:JF781308)最为相似,相似系数为0.98,17种限制性内切酶的酶切图谱显示与16S r I-X参考株只有MseI不同,因此该植原体属于16S r I-X亚组的变种。比较得出TLWBYN01与16S r I-X亚组成员番木瓜束顶植原体(JF781308)和印度葫芦植原体(LT594117、LT594118)同源性分别为99.2%和99.6%。【结论】玉溪市新平县发现的羽脉山黄麻丛枝病属局部常发病害,中度危害,但一旦感染,就会严重影响植物生长。羽脉山黄麻丛枝植原体属16 Sr I组成员,也是报道较少的植原体16S r I-X亚组的一个变种,且羽脉山黄麻为新发现的植原体新寄主。     

植原体检测和鉴定技术

植原体是一种广泛传播的植物病害病原,在世界范围内造成了非常大的经济损失。各国的研究人员对植原体造成的病害,一直进行着不懈研究。利用传统的组织和化学检测技术,取得了很多研究成果。随着现代生物技术的飞速发展,人们又建立了许多植原体的鉴定和检测的新方法,为探索植原体的遗传特征和生物学特性,提供了技术基础。本文就植原体被发现以来,对此病害检测和鉴定技术的发展进行论述。     

温度周年变化与桑树植原体消长的关系

通过聚合酶链反应检测了植原体在桑树体内的分布及其相对含量的周年变化。结果显示,茎部中植原体的含量在生长季节逐渐升高,７月达到最大,而冬季检测不到,叶部与茎部相似但植原体的含量在８月达到最大;根部全年均含有植原体,含量较低。     

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。     

Impact of phytoplasma infection of common alder (Alnus glutinosa) depends on strain virulence

In nature, only a minority of alder trees infected by the alder yellows phytoplasma develop symptoms. To elucidate the reason for this phenomenon, healthy alder seedlings were inoculated with scion wood from five infected but differently affected trees. The symptoms developed by the inoculated plants largely corresponded to disease severity of the inoculum sources. Inoculation with tissue from three of four severely affected trees resulted in severe disease of the recipient trees as expressed by reduced vigour, sparse foliage and little leaves. Inoculum from one of the severe sources caused milder symptoms. No symptoms were developed when the inoculum was collected from a vigorous, non-symptomatic tree. From these results it can be concluded that the alder yellows phytoplasma is pathogenic to alder but that avirulent and low virulent strains do occur.     

Phytoplasma associated with Chinese tallow tree yellowing disease in China represents a new 16SrIII subgroup

Recently, samples of the Chinese tallow tree (Sapium sebiferum) displaying yellowing symptoms were collected from a grove in Tai'an, Shandong Province, China. The association of phytoplasma with yellowing disease was ascertained using nested polymerase chain reaction (PCR) of the 16S rRNA gene by using the phytoplasma‐specific universal primer pair P1/P7, followed by R16F2n/R16R2 as nested primers, and rp genes primed using rpL2F3/rp(I)R1A followed by rp(III)‐FN/rp(I)R1A. The sequence and phylogenetic analyses of the 16S rRNA gene and rp genes revealed that the phytoplasma associated with the Chinese tallow tree belonged to the 16SrIII group (the X‐disease group). Computer‐simulated and gel‐based restriction fragment length polymorphism (RFLP) analyses revealed that the RFLP patterns were different from the reference patterns of all previously established 16SrIII subgroups, with the maximum similarity coefficient exceeding the threshold for delineation of a new subgroup RFLP pattern type within the 16SrIII group. Thus, the phytoplasma associated with the Chinese tallow tree yellowing disease, designated as ‘CTTY’, represents a new subgroup (16SrIII‐Y). This study shows the Chinese tallow tree as a new host of phytoplasma belonging to the 16SrIII group in China and worldwide.     

Seasonal variation of paulownia witches’-broom phytoplasma in paulownia trees and distribution of the disease in the Tohoku district of Japan

Seasonal variation of paulownia witches’-broom (PWB) phytoplasma within different organs (leaves, branch and trunk bark and roots) in paulownia trees was investigated by the amplification of a PWB-specific DNA fragment by the polymerase chain reaction (PCR). In leaf samples, PWB phytoplasma was first detected in June and the incidence gradually increased. On the other hand, the PWB was detected at relatively low incidence in branch bark, trunk bark and roots and the incidence did not change among seasons. A survey of PWB in 27 fields in the Tohoku district of Japan showed that malformed flower buds were observed in paulownia trees in almost all of the fields. PWB-phytoplasma was also detected by PCR from paulownia trees in almost all of the fields in Iwate and Fukushima Prefectures. The frequencies of trees in which phytoplasma was detected by PCR were higher than those in which symptoms were observed. These results indicated that PCR amplification of a PWB-specific DNA fragment is an effective tool for practical diagnose and that PWB is widely distributed in the Tohoku district of Japan.     

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.     

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.     

Molecular identification and characterization of a new phytoplasma strain associated with Chinese chestnut yellow crinkle disease in China

Chinese chestnut trees (Castanea mollissima BL.) planted in a suburb of Beijing, China developed symptoms including yellowing, leaf crinkling, little leaf, shortened internodes, and empty burrs. Transmission electron microscopy revealed presence of phytoplasma cells in phloem sieve elements of the symptomatic chestnut trees. Molecular cloning and sequence analysis of PCR‐amplified near‐full length 16S rRNA gene indicated that the phytoplasma associated with the Chinese chestnut yellow crinkle disease is closely related to Japanese chestnut witches’‐broom phytoplasma. This is the first report of a phytoplasmal disease in Chinese chestnut trees.     

Elm yellows: A phytoplasma disease of concern in forest and landscape ecosystems

Elm yellows (EY) is a lethal or decline phytoplasma disease that affects several Ulmus (elm) species and hybrids, which is widespread in North America and Europe. The symptoms vary among the elm species. In those native to North America, main symptoms include epinasty, chlorosis, premature casting of the leaves, yellow to brown discoloration of the phloem in the roots and stem and tree death that usually occurs within 1 or 2 years from the appearance of foliar symptoms. In contrast, affected trees of European and Asian species are primarily characterized by witches’ broom as a specific symptom, do not show phloem discoloration and are less prone to decline. The disease is caused by a relatively genetically homogeneous phytoplasma, the EY agent “Candidatus Phytoplasma ulmi,” a member of the EY phytoplasma group or 16SrV group, subgroup 16SrV‐A. In nature, this pathogen exhibits a high plant host specificity. The elm leafhopper Scaphoideus luteolus is the only confirmed vector of EY phytoplasma in North America, whereas Macropsis mendax has been reported as a natural vector in Northern Italy. However, other insect vectors are likely to be involved in its natural spread. Phytoplasmas of other taxonomic groups or 16SrV subgroups, which are known to infect a wide range of plant hosts, have been identified in naturally infected elm trees. However, the pathological relevance of these “non‐elm” phytoplasmas needs to be confirmed in many cases. Their detection is mainly based on the highly sensitive nested PCR assays, while pathological data are lacking. This study summarizes, within the framework of a single comprehensive review, the current knowledge of EY. Gaps in knowledge of this disease and prospects for future research are also critically discussed.     

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.     

Molecular characterization of a phytoplasma associated with Euonymus bungeanus witches’ broom in China

Euonymus bungeanus plants exhibiting symptoms of abnormal branches, small leaves and phyllody, which is indicative of E. bungeanus witches’ broom (EbWB) disease, have recently been found in Beijing, China. A phytoplasma from symptomatic E. bungeanus plants was identified by 16S rRNA polymerase chain reaction (PCR) using the phytoplasma‐specific universal primer pair R16mF2/R16mR1. Inoculation of healthy E. bungeanus plants by grafting with diseased scions was also performed. The rp and secY genes of the EbWB phytoplasma were cloned and sequenced as was the 16S rRNA gene. Sequence and phylogenetic analyses of 16S rRNA, rp and secY genes indicated that the phytoplasma associated with E. bungeanus belongs to the 16SrV‐B, rpV‐C and secY‐C subgroup, the same subgroup as the jujube witches’ broom (JWB) phytoplasma that is widely distributed among jujube trees in China. Comparative analyses based on virtual restriction fragment length polymorphism (RFLP) showed that the EbWB phytoplasma is more closely related to another 16SrV‐B subgroup strain: RPWB (Robinia pseudoacacia witches’ broom). To the best of our knowledge, this is the first report of a witches’ broom phytoplasma in E. bungeanus in China, and the findings add a new cultivated plant species to the already broad natural host range of 16SrV‐B subgroup phytoplasmas.