苹果茎沟病毒微滴数字RT-PCR检测方法的建立和应用

我国是第一大苹果生产国。苹果茎沟病毒(apple stem grooving virus, ASGV)在我国各苹果产区分布广泛,侵染果树导致树势变弱,生长量减少,果实品质下降,严重威胁我国苹果产业的可持续发展。病毒检测是果树病毒病防控的重要前提。本研究根据苹果茎沟病毒外壳蛋白基因保守序列,设计了引物和探针,建立了微滴数字PCR (droplet digital PCR, ddPCR)检测方法。所建立的检测方法能特异性检出苹果茎沟病毒,检测重复性好,灵敏度比qRT-PCR法高10倍,可适用于田间样品及组培苗检测。该方法的建立为苹果茎沟病毒的检测提供了重要的技术手段。     

我国北方部分苹果主产区病毒病的发生与检测

苹果病毒病在我国广泛发生,已成为限制我国苹果优质高产的关键因素。20世纪80年代曾对其做过详细的调查和研究,但近年来由于各地农业产业结构调整等因素,苹果病毒病的发生特点有了不同程度的变化。为了解目前我国苹果病毒病的发生情况,在我国北方苹果主产区山东、陕西、山西、辽宁、北京和黑龙江6个省市的部分地区采集苹果样品共计267份,经RT-PCR检测和扩增产物的克隆与测序分析表明在上述地区采集的样品中,苹果褪绿叶斑病毒(ACLSV)、苹果茎沟病毒(ASGV)、苹果茎痘病毒(ASPV)、苹果锈果类病毒(ASSVd)的发生率分别为66.7%~100.0%、38.1%~94.1%、4.8%~85.7%和4.8%~48.6%;苹果凹果类病毒(ADFVd)仅在山东的两个果园零星发生;6个省市样品中病毒复合侵染率分别为67.1%、92.1%、75.0%、88.2%、94.1%和76.2%。     

北京地区番茄黄化曲叶病毒病的鉴定及防治对策

番茄黄化曲叶病毒病是一种由烟粉虱传播的病毒病,给番茄生产造成严重威胁。2009年在北京郊区调查时发现部分保护地种植的番茄植株表现典型黄化曲叶症状。通过提取典型症状样品总DNA利用粉虱传双生病毒检测简并引物PA/PB,进行PCR扩增到541bp的特异条带。通过测序和核苷酸序列比对表明该序列与番茄黄化曲叶病毒序列相似性最高为99%。分子检测结果表明北京郊区部分保护地种植的番茄已被烟粉虱传播的番茄黄化曲叶病毒侵染危害。     

基于PCR的植物病毒检测技术研究进展

植物病毒是引起植物重大病害的病原之一.植物病毒病危害大、防治难度高,一旦感染病毒终生携带,而早期的检测成为病毒预防及治理的先决条件.基于基因扩增技术的病毒检测方法,由于检测迅速、敏感性高、成本低等优点而被广泛应用于各种病毒的检测.本文对常用的基于PCR的病毒检测方法进行了综述,并对其优缺点进行了比较,以期为选择合适的植...     

河北省番茄黄化曲叶病毒和番茄褪绿病毒复合侵染及分布

 番茄黄化曲叶病毒(Tomato yellow leaf curl virus, TYLCV) 及番茄褪绿病毒(Tomato chlorosis virus, ToCV)是危害番茄的主要病毒。2016~2017年,在河北省18个番茄主产区调查番茄病毒病危害情况,采集262份植株矮化、叶片黄化、褪绿、卷曲的番茄样品,利用分子生物学技术对病原进行鉴定。结果表明:2016~2017年河北省番茄病毒病发生普遍,所检样品中TYLCV的侵染率为68.66%,其中与ToCV复合侵染率为19.5%;除栾城、滦南、乐亭、永年4地样品暂未检测到ToCV外,其他14个采样点的样品均检测到ToCV,侵染率为19.5%,且全部表现为与TYLCV复合侵染,河北省番茄主产区暂未发现ToCV单独侵染的样品。     

苹果褪绿叶斑病毒双引物对PCR检测体系的建立及应用

<正>病毒病在我国苹果树上普遍发生,其中苹果褪绿叶斑病毒(apple chlorotic leaf spot virus,ACLSV)、苹果茎沟病毒(apple stem grooving virus,ASGV)和苹果茎痘病毒(apple stem pitting virus,ASPV)最常见,常混合侵染,引起树体衰弱,苹果产量和品质下降,经济损失严重(CieniewiczFuchs,2016)。因此,建立快速、准确的检测技术对病毒病防控具有重要意义。Hu et al.(2015)认为在病毒检测过程中,需     

马铃薯主产区病毒病发生情况调查分析

在我国马铃薯主产区黑龙江、内蒙古、甘肃、贵州采集了752份具有典型病毒病症状的样品和疑似样品,应用DAS ELISA方法进行检测。主要筛查6种病毒：PVX、PVY、PVS、PLRV、PVM、PVA。结果显示: 270份样品检测到病毒,总体上,PVY的检出率最高,PVS次之,33份样品是由多种病毒混合侵染造成的,田间PVY+PLRV混合侵染率最高,PVS+PVY次之。试管苗PVS病毒发生较多,原原种和大田种薯PVY病毒发生比例最高。     

利用Real-time RT-PCR方法检测苹果中苹果茎沟病毒

以含有苹果茎沟病毒(Apple stem grooving virus,ASGV)的苹果枝条为试材,设计扩增产物片段为176 bp的特异性引物,优化反应条件,构建标准曲线,建立了苹果茎沟病毒的SYBR Green Ⅰ实时荧光定量RT-PCR检测方法.对该方法进行了灵敏度和重复性试验,并且利用建立的方法对受ASGV感染的苹果枝条和树叶进行定量检测.最后,比较了实时荧光定量RT-PCR与常规RT-PCR检测的灵敏度.结果表明:样品的荧光定量PCR有良好的扩增曲线和溶解曲线,该方法的灵敏度可达6.8× 102拷贝/μL,检测其灵敏度是传统RT-PCR的100倍,所建立的荧光定量技术可用于田间样品中此病毒的检测,丰富了ASGV的检测手段,提高了检测灵敏度,具有较好的应用前景.     

我国部分地区梅树病毒病的病原初步检测

梅是一种原产于中国、具有重要经济价值、花果兼用型的树种。在其生长繁育过程中易受病毒等病原侵染,从而可能造成严重的经济损失。目前我国梅树上病毒的发生及分布尚不清楚。为此,对无锡、武汉、上海、杭州、南京的梅园进行了调查,采集了115份样品,其中发现了疑似病毒病害,主要症状表现为花叶、卷叶、叶片皱缩、褪绿、脉间失绿等。RT-PCR检测出了苹果茎沟病毒(ASGV)、樱桃病毒A(CVA)和亚洲李属病毒2(APV 2),检出率分别为30.4%、4.3%和3.5%。此外,克隆了这三种病毒的外壳蛋白基因序列,同GenBank已登录的相应序列相似性为86%~99%(核苷酸水平)和83%~99%(氨基酸水平)。序列比较发现APV2梅分离物与日本梅分离物亲缘关系较近。     

浙江省马铃薯病毒病检测分析

马铃薯病毒病是影响马铃薯产量和品质的主要因素之一,其症状表现为花叶、黄化和卷曲等。2019年4月-5月,在浙江省湖州、杭州、绍兴、宁波、金华、台州和丽水等7个地市的主要马铃薯产区采集了具有典型病毒病症状的马铃薯样品,采用转录组测序和RT-PCR验证的方法进行病毒检测,共筛查并验证出6种马铃薯病毒,包括马铃薯X病毒Potato virus X(PVX)、马铃薯Y病毒Potato virus Y(PVY)、马铃薯S病毒Potato virus S(PVS)、马铃薯H病毒Potato virus H(PVH)、马铃薯M病毒Potato virus M(PVM)以及马铃薯奥古巴花叶病毒Potato aucuba mosaic virus(PAMV)。检测结果表明,在调查的这7个地市中每个地市都具有2种以上的病毒发生,其中PVH作为一种新鉴定的麝香石竹潜隐病毒属成员在除宁波以外的各地区都有发生;此外杭州市、湖州市以及绍兴市均检测出了PAMV。对检测到的PVS、PVH、PVM和PAMV外壳蛋白(coat protein,CP)序列进行系统发育分析表明,这些病毒均存在地域差异性。本研究为浙江省马铃薯病毒的防控奠定了重要基础。     

苹果脱毒技术研究进展

苹果（Malus pumila Mill.）普遍感染病毒。目前, 培育无病毒原种母本树, 建立用于繁殖接穗和营养系砧木的母本园, 栽植无病毒苗木, 是防治病毒病的根本措施。本文针对常见的4 种苹果病毒及1 种类病毒, 综述了茎尖培养、热处理、化学处理、微茎尖嫁接以及低温处理脱除苹果病毒方法的研究进展, 分析了不同方法的应用效果, 及所适合脱除的病毒种类, 以期为我国苹果病毒脱除技术研究提供参考信息。     

Apple necrotic mosaic virus,a novel ilarvirus from mosaic-diseased apple trees in Japan and China

The causal agent of apple mosaic disease has been previously thought to be solely caused by apple mosaic virus (ApMV). In this study, we report that a novel ilarvirus is also associated with apple mosaic disease. Next-generation sequencing analysis of an apple tree showing mosaic symptoms revealed that the tree was infected with three apple latent viruses (apple stem pitting virus, apple stem grooving virus, and apple chlorotic leaf spot virus) and a novel ilarvirus (given the name apple necrotic mosaic virus (ApNMV)) that is closely related to Prunus necrotic ringspot virus (PNRSV) and ApMV. The genome of ApNMV consists of RNA1 (3378 nt), RNA2 (2767 nt), and RNA3 (1956 nt). A phylogenetic analysis based on the coat protein amino acid sequences indicated that the novel virus belongs to the same subgroup 3 of the genus Ilarvirus as PNRSV and ApMV. The presence of mosaic leaves, which tend to be unevenly distributed in diseased apple trees, was correlated with the internal distribution of ApNMV. RT-PCR detection of mosaic-diseased apple trees in Japan indicated that ApNMV was detected in apple trees introduced from China, whereas ApMV was detected from cultivated apple trees in domestic orchards. Consistent with these findings, a survey of mosaic-diseased apple trees in major apple-producing provinces in China revealed that the majority of apple trees showing mosaic symptoms in China are infected with ApNMV.     

戊唑·多菌灵·乙膦铝乳胶液对苹果枝干轮纹病的防治效果

近年,在矮砧密植苹果园发现苗木栽植后幼树主干上的枝干轮纹病发生严重,急需有效的防治方法。针对生产中的需求,本研究对幼树枝干轮纹病的防治方法进行了探索,结果显示:用乳胶液配制的戊唑·多菌灵·乙膦铝混合剂对枝干轮纹病防治效果显著高于同种药剂的水溶液,对剪锯口枝干轮纹病防治效果可达63.7%~100%,是药剂水溶液防效的2~3倍;生长季施用2次,对枝干轮纹病病斑的铲除率为45.2%,是药剂水溶液防效的9.6倍以上。结果表明,戊唑·多菌灵·乙膦铝乳胶液对枝干轮纹病防治效果显著,可以用于苗木剪锯口的防护,以及新建果园中幼树枝干轮纹病的治疗。研究结果为生产中幼树枝干轮纹病的防治提供了有效的方法。     

Effects of fungicides on apple crown rot caused by Phytophthora cactorum

The mycelial growth of Phytophthora cactorum on corn meal agar was completely inhibited by metalaxyl or mancozeb at 50 or 100 μg ml^?1, but not by fosetyl-aluminium at the same concentrations. Soil drenches with metalaxyl, followed by metalaxyl+mancozeb, around the base of naturally infected trees under orchard conditions, prevented the growth of P. cactorum in the infected bark of the tree. Soil drenchs with metalaxyl, followed by metalaxyl+mancozeb, also prevented the infection of apple trees with P. cactorum under orchard conditions. The trunk circumference and spur length were not significantly affected by any of the fungicide treatments in all the trials over 2 years, except in one of the four orchards used in the study. Soil drench with metalaxyl alone, or followed by metalaxyl+mancozeb, may provide an effective control method for crown rot of apple trees.     

2011年烟台苹果产区腐烂病发病情况调查与原因分析

为了解2011年烟台苹果产区腐烂病的发生情况,2011年5月,在腐烂病发病较重的栖霞、海阳等地选择21个农户的果园,对腐烂病的发生情况进行了调查.结果表明,21个果园中具有新病疤的病株率为68.20％,死株率为2.76％,平均受害枝量为23.98％,死枝量10.74％,病株率超过50％的果园占25％～30％,总体发病情况比一般年份严重.调查共发现967块新病疤,平均每株2.32块,其中源自剪锯口的病疤占80.04％,从旧病疤复发的病疤占60.29％.2010年秋季的连续阴雨、冬季低温和2011年春季干旱可能是导致烟台苹果产区2011年春季腐烂病大发生的主要原因.剪锯口是腐烂病菌侵染的主要途径,旧病疤复发是春季腐烂病发病的主体.     

Recovery in apple trees infected with the apple proliferation phytoplasma: an ultrastructural and biochemical study

ABSTRACT Localization of hydrogen peroxide (H(2)O(2)) and the roles of peroxidases, malondialdehyde, and reduced glutathione in three apple cultivars were compared in healthy trees, trees infected with apple proliferation phytoplasma (APP), and trees that had recovered from the infection. In recovered apple trees, symptoms of the disease and the pathogen had disappeared from the canopy, but phytoplasmas remained in the roots. H(2)O(2) was detected cytochemically by its reaction with cerium chloride to produce electron-dense deposits of cerium perhydroxides.H(2)O(2) occurred in the plasmalemma of the phloem of leaves of recovered apple trees, but not in healthy or APP-infected leaves. In all cultivars, the peroxidase activity detected in tissue from APP-diseased trees was greater than or equal to that of tissue from recovered trees, which equaled or exceeded that of tissue from healthy trees, at two sampling times (May and September). In contrast, the glutathione content of leaves decreased in the reverse order. More malondialdehyde was observed in leaves from recovered trees than in leaves from healthy or APP-infected trees in three of six cultivar-date combinations; in the other three combinations, the malondialdehyde contents of leaves from healthy, infected, and recovered trees were not significantly different from one another. The results suggest that some components of the oxidant-scavenging system in recovered leaves are not very active, leading to an overproduction of H(2)O(2) and, possibly, to a membrane lipid peroxidation.The production of H(2)O(2) appears to be involved in counteracting pathogen virulence.     

Flat limb (furede grene) of apple trees

Conclusions The experiments clearly show, that flat limb is a graft-transmissible disease.The experiments also demonstrate, that there is a great difference between the susceptibility of the various apple varieties, and furthermore that some varieties can act as symptomless carriers.As it is rather difficult or impossible to remove all infected trees in the nurseries by inspection and roguing, steps have been taken to establish a nucleus stock of all important apple varieties, from which the nurseries can be supplied with grafting and budding material.Important questions concerning flat limb, which are not yet solved, are a further search for symptomless carriers among the important apple varieties and also among the apple rootstocks.Furthermore it would be of value to know the exact effect of the disease on growth and fruit production of the infected trees. Experiments concerning these problems are now in progress.     

Epidemiology of brown rot (Monilinia fructigena) on apple: infection of fruits by conidia

Effects of fruit maturity, wound age, temperature and the duration of wetness periods on infection of apple fruits by conidia of the brown rot fungus, Monilinia fructigena , were studied. Inoculation of fruits on potted apple trees and harvested mature fruits showed that wounding was essential for infection by M. fructigena . On potted trees, there was a significant difference between the susceptibility of cvs Cox and Gala and this difference depended on wound age. The incidence of brown rot was affected greatly by fruit maturity and wound age. Wounds on younger fruits were more resistant to infection than those on older fruits, whilst the older the wound, the more resistant it was to infection. Furthermore, the degree of wound age-related resistance was greater on younger fruits than on older fruits. These relationships were well described by regression models. The effect of the duration of wetness periods was very small: increasing the duration of wetness periods reduced the incidence of brown rot on older wounds. For detached fruits, all those wounded were rotted after inoculation, except for those in two treatments under 20°C on fruits with wounds which were 8 days old. The incubation period of the fungus was generally very short. Wound age was the single most important factor influencing the length of the incubation period; the incubation period increased as wound age increased.     

三种ELISA方法检测苹果褪绿叶斑病毒和苹果茎沟病毒的比较

苹果褪绿叶斑病毒（ＡＣＬＳＶ）和苹果茎沟病毒（ＡＳＧＶ）是感染苹果和其它一些果树的重要病毒。作者应用ＰＡＳ－ＥＬＩＳＡ法成功地检测了苹果组培苗中的这两种病毒。为了简化操作步骤,试验了ＤＡＳ－ＥＬＩＳＡ和改良ＤＡＳ－ＥＬＩＳＡ法,并与ＰＡＳ－ＥＬＩＳＡ的检测结果比较。试验结果表明,ＤＡＳ－ＥＬＩＳＡ能检测出ＡＳＧＶ,却不能检测出ＡＣＬＳＶ,与此同时,这两种病毒均可用改良ＤＡＳ－ＥＬＩＳＡ检测。ＤＡＳ     

苹果属植物寄主应答病原菌胁迫的蛋白质组学研究进展

苹果基因组研究的不断深入,为苹果属植物蛋白质组学研究奠定了基础。近年来,蛋白质组学分析方法在探讨苹果属植物应答病原菌胁迫时,所启动的抗病相关蛋白的差异表达,以及相关抗病机制方面发挥了重要作用。本文针对近年来蛋白质组学研究技术在果树上的应用,综述了苹果属植物应答火疫病、褐斑病、黑星病等主要病害胁迫的蛋白质组学研究进展,分析了蛋白质组学分析技术在苹果属植物相关研究中存在的问题,以期为进一步探究苹果属植物抗病的分子机制提供参考。