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。     

抗枣疯病枣树品种的DAPI荧光染色检测

该文通过嫁接枣疯病病皮和病枝传病的方法 ,对抗病材料婆枣 (JL)和壶瓶枣 (Hu)共 10棵单株 ,结合感病酸枣 (Suan)品种的健枝和疯枝作对照 ,应用DAPI染色技术对抗病材料体内植原体的侵染情况进行了荧光观察 ,结果表明 :抗病植株体内已感染了植原体病原 ,说明应用嫁接方法进行枣疯病病原接种是可行的 ,但其却不表现丛枝症状 ,证明了抗病材料具有一定的抗病性 ;同时 ,抗病材料中发现的金黄色自发荧光 ,也为其抗病物质的寻找和抗病机理的研究提供了重要线索。     

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.     

枣疯植原体的分布特点及周年消长规律

枣疯病是一种典型的致死性植原体病害,是枣树生产中发生最普遍、危害最严重的毁灭性传染病害,被视为枣树的癌症.     

枣区枣疯病发生情况调查及防治建议

枣疯病是枣树毁灭性病害,由枣疯病植原体引起。针对河南省新郑、濮阳、镇平、灵宝、永城等县(市)枣区的枣疯病发生情况进行调查,发现新郑枣区发病情况较为严重,放任枣园较枣粮间作和集约管理的枣园发病严重。分析发病的原因,主要是疏于管理,导致树体营养失衡,抗性减弱,媒介昆虫增多,加速病原传播,防治措施缺乏,病树得不到处理造成的。建议采取平衡施肥,改善树势,做好枣园植被管理,防治害虫,防止病害传播,及时修剪,消除病原等措施,开展枣疯病综合防治。     

抗枣疯病枣树品种及品系的选择

测定了6个枣树品种的1个酸枣品种对枣疯病的抗性及河北太行山区主栽品种婆枣的45个单株对枣疯病的抗性。利用连续两年嫁接病皮的方法测定了采自山西果树研究所中国枣树基因库的5个枣树品种砘子枣、马牙枣、长红枣、哈蟆枣、壶瓶枣及河北唐县的婆枣和酸枣两个品种的抗病性。通过四年的跟踪观察，以上7种枣树的发病率分别是100％、78．5％、66．67％、0％、0％、100％、80％。结果表明，壶瓶枣和蛤蟆枣对枣疯病具有高度抗性。利用连续3年2次嫁接病皮和1次嫁接病枝的方法测定了采自河北太行山区七县的婆枣45个单株112株根蘖苗的抗病性，经4年观测，结果有3个单株6根蘖苗经过三次测定苗木未疯，表现出了很强的抗性。     

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。     

植原体的系统分类

植原体不能分离培养 ,因此用传统的形态分类方法无法分类。该文简要介绍了应用分子技术研究植原体系统分类的概况和我国的现状 ,概述了所使用的各种分子技术的基本方法和特点     

警惕植原体病害--板栗黄化皱缩病

近日,《京郊日报》刊出一篇文章《密云金丝小枣40年后回“娘家”》,文中写到：时隔40年之后．在密云大地已经绝迹的密云“三宝”之一——金丝小枣,如今重回“娘家”。40年前．在密云县原本颇具规模的金丝小枣产业缘何全军覆没．绝迹多年呢？原因就是植原体病害——枣疯病。     

北京市丰台区主栽枣品种对枣疯病抗性试验

采用以感染枣疯病的枣树作砧木嫁接健康休眠接穗的方式,对丰台区8个主栽枣品种进行了抗病性鉴定,并应用DAPI荧光显微方法和PCR方法对接穗进行了枣疯病植原体检测.结果表明,8个供试品种均能被感染,并表现丛枝症状,对枣疯病植原体均无免疫性,其中金芒果枣和冬枣为易感痛品种,尜尜枣和京枣39为相对抗性品种.     

我国部分枣树品种(系)的枣疯病抗性鉴定

收集了我国部分地区的不同枣树品种(系)的休眠期健康接穗材料,于2009年和2010年的4-6月嫁接到枣疯病砧木上,定期检查接穗发病状况,并采用PCR技术检测植原体感染.结果显示,不同枣树品种对枣疯病的抗性差异明显,根据接穗发病早晚、症状轻重、发病率和病情指数,将测定品种分为5个类型,其中高度抗病品系2个、中度抗病品系4个、一般抗病品系15个、感病品系7个、高度感病品系6个.高抗品系(交城骏枣、唐县和太原壶瓶枣)嫁接当年萌生枝条枣疯病无明显症状或嫁接早期无症状,后期症状较轻.嫁接到病树遮阴部位的接穗发病程度要重于向阳部位;存放期长和嫁接时间早的接穗,易诱发抗性品系发病.巢式PCR能检测到无症抗病接穗内低浓度植原体侵染.与以往所采用的昆虫自然传染和病皮或病枝嫁接到待鉴定的砧木品系上鉴定抗性技术相比,该方法具有接种强度大、诱导发病快、测定方法简便等优点,但对一般抗性、整株或成年株抗性、抗介体传毒等类型的抗性鉴定有局限性.     

Identification of a phytoplasma associated with Syringa reticulata witches' broom disease in China

During the summer of 2018, one Syringa reticulata plant showing witches' broom and small leaves was observed in Beijing, China. Molecular diagnostic tools and electron microscopic cell observation were used to detect the possible pathogen of this disease. As a result, the phytoplasma in the symptomatic S. reticulata tree was confirmed by amplifying the 16S rRNA gene using the phytoplasma‐specific universal primer pair R16mF2/R16mR1 and observation with transmission electron microscopy. The rp and tuf genes of the phytoplasma were also cloned and sequenced as the 16S rRNA gene. Sequence and phylogenetic analyses of the 16S rRNA, rp and tuf genes indicated that the phytoplasma associated with S. reticulata witches' broom (SrWB) disease belonged to the 16SrV‐B subgroup, and it was closely related to the 16SrV‐B subgroup phytoplasma strain jujube witches' broom, which causes serious disease of jujube fruit trees in China. This study shows the S. reticulata tree as a new host of a phytoplasma belonging to the 16SrV‐B subgroup in China.     

枣疯病的综合治理措施

枣疯病是由植原体引起的枣树生产中发生最普遍、危害最严重的传染性检疫病害。主要从科学建园、病树治疗与康复及其他技术措施等方面对枣疯病的综合防治措施进行了概述。     

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.     

Relationship between witches’ broom protein and dynamic of some amino acids inPaulownia tree leaves

This paper dealt with the SDS polyacrymide gel electrophoresis of proteins and analysis of the amino acids in the diseased and healthy leaves with the same strain respectively, which were at the same side and height, ofPaulownia catalpifolia, P. elongata, P. albiphloea andP. kauakamii respectively. The results indicated that the leaves of 4 species ofPaulownia trees with witches’ broom had one protein band, of which molecular weight was 12 kD, which did not appear in the healthy leaves free of phytoplasmas. Moreover, the protein quantity in the affected leaves was more than that in the healthy leaves free of phytoplasmas; At the same time, there was significant difference on the amino acids between the healthy and diseased leaves ofP. catalpifolia andP. kawakamii. The amount of cystine in the affected leaves was higher than that in the healthy leaves, but the change of amount of phenylalanine in the affected and healthy leaves was contrary. These changes of proteins and amino acids in the leaves might be related to the witches’ broom of theParlounia trees. (Responsible Editor: Dai Fangtian)     

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.     

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.     

新疆红枣PGPR解钾效果及其与有机酸的相关性研究

通过分析高效解钾菌对含钾矿物的解钾效果及其与有机酸的相关性,以探究解钾菌解钾作用机制。采用摇瓶实验,将经过筛选出的4株解钾菌K3、K7、K11、K24在液体培养条件下发酵培养,然后测定发酵液中的有机酸含量、速效钾含量及pH值。利用通径分析法,研究草酸、酒石酸、丙酮酸、乙酸、柠檬酸5种有机酸对解钾效果的直接与间接影响。结果表明,4株解钾菌发酵液中的速效钾含量比对照(CK)增加了674.88%,且速效钾的含量与有机酸的含量之间呈现出显著相关(P0.05),故5种有机酸均对速效钾的含量变化有影响,其中丙酮酸对钾长石溶解的直接作用影响最大,其直接通径系数为1.689,酒石酸对速效钾的间接作用影响最大,其间接通径系数为0.205。有机酸与含钾矿物解钾量呈正相关关系,速效钾的含量随着有机酸浓度的提高而增加。