侵染小苹果的苹果锈果类病毒的检测和全序列分析

小苹果是抗寒海棠类与大苹果的杂交后代,因耐寒能力强,口感好,被广泛用于嫁接与育种材料,是我国寒凉苹果产区宝贵的种质资源。苹果锈果类病毒(Apple scar skin viroid,ASSVd)侵染苹果后引起果实表面产生锈状斑、花脸、畸形等症状,严重降低果实品质和市场价值。2013年在调查苹果病毒病时发现北京市延庆县种植的小苹果表现出典型畸形症状,应用RT-PCR方法对其进行检测后经测序和序列分析表明,小苹果被苹果锈果类病毒侵染。克隆此分离物(ASSVd-YQ)全基因组序列,分析后得到其基因组全长为329个核苷酸,系统发育分析表明该分离物与来自不同国家和地区的ASSVd分离物间的进化关系极近。二级结构预测表明,该分离物的中央保守区与末端保守区与ASSVd参考序列相同。这是对侵染小苹果的ASSVd的首次检测和鉴定。     

NaCl胁迫对八棱海棠和山定子光合特性的影响

以盆栽一年生苹果砧木山定子和八棱海棠为实验材料,研究不同浓度(0、0.1%、0.2%、0.3%、0.4%、0.5%)NaCl处理对山定子和八棱海棠光合特性的影响。结果表明:不同浓度NaCl处理后,山定子胞间CO2(Ci)浓度呈现高于对照的趋势;蒸腾速率(E)和气孔导度(Gs),净光合速率(Pn)均表现下降的趋势,并且低于对照;八棱海棠在NaCl浓度高于0.2%,Ci浓度才高于对照,NaCl浓度高于0.1%,蒸腾速率(E)和气孔导度(Gs),净光合速率(Pn)才低于对照。另外,山定子的盐害指数和盐害率均高于八棱海棠,八棱海棠对NaCl胁迫的耐受性较强。     

多重PCR检测籽用西葫芦种子带毒及热处理脱毒效果分析

为了明确籽用西葫芦种子携带病毒的情况,以籽用西葫芦种子为材料,采用多重PCR技术检测种子带毒率。同时为了筛选出有效脱除病毒的方法,通过相对定量法分析了不同温度湿热和干热处理对种子携带病毒的钝化效果。结果表明,从7个籽用西葫芦品种种子幼苗中均可检测到CMV、ZYMV及WMV,其检出率分别为67.1%、50.0%和72.3%,多个病毒的复合检出率为66%。不同品种种子幼苗的病毒检出率有明显差异,其中‘粒丰9号’种子幼苗的病毒检出率最低,为60%,而‘京丰9号’,‘瑞丰9号’,‘绿丰9号’及‘金葫360’的病毒检出率均为100%。8种处理均能不同程度降低籽用西葫芦种子幼苗的带毒率,其中干热60、70、75℃,湿热75℃处理同时脱除WMV、CMV和ZYMV的效果最好。     

侵染‘舞美’苹果的苹果锈果类病毒检测与全序列分析

2017年9月,在山东胶州调查苹果病毒病时发现‘舞美’果实表现出明显的花脸症状,取发病树体芽组织嫁接于健康‘富士’苹果幼树上,采用RT-PCR技术对其是否含有苹果锈果类病毒进行了检测,并利用DNAMAN、MEGA 5.1等软件对其全序列进行了分析。结果表明,显症‘舞美’果实、发病树体枝条及嫁接‘富士’枝条中均可检测出ASSVd,无症状‘舞美’果实和相应树体枝条中均未检测到ASSVd。‘舞美’分离物基因组主流序列为333 nt (登录号MG745387),与GenBank中已报道的ASSVd序列一致性为92%～99%。序列多重比对及系统进化树分析发现,该分离物的末端保守区和中央保守区与ASSVd参考序列一致,且与不同来源的ASSVd分离物亲缘关系较近。这是首次在‘舞美’苹果上发现和鉴定出苹果锈果类病毒。     

我国花生品种资源种子带病毒检测

在防虫网室内,对1245份入库花生品种资源材料带病毒情况进行检测,其中707份材料(占56.8%)种子传带花生条纹病毒(PStV),平均种传率5%以上。带毒材料主要来源于北方PStV病毒流行省(市);63份材料(占3.4%)种子传带黄瓜花叶病毒(CMV),分别来源于辽宁大连、河北唐山和山西汾阳。辽宁大连带CMV病毒材料比例最高占(47.6%),平均种传率达3.6%。     

我国部分苹果产区苹果锈果类病毒的检测和全序列分析

近年来,由苹果锈果类病毒Apple scar skin viroid(ASSVd)引起的苹果花脸病、锈果病在我国一些苹果产区日趋严重,对我国苹果生产和苹果产业发展造成严重危害。为了解ASSVd在我国一些苹果产区的发生和变异情况,采用RT-PCR对陕西、山东、山西、河北、北京和黑龙江6个苹果产区的35份苹果样品进行检测,并克隆获得30个分离物的基因组全序列,大小为330~333个核苷酸。分析表明,这些分离物的基因组全序列与GenBank中ASSVd基因组核苷酸序列相似度为96%~100%,在苹果锈果类病毒属的末端保守区及中央保守区保守,在致病区和左端区域有突变,一些分离物的突变位点相同。系统发育分析表明分离物因相同的突变位点而聚类,而与地理来源无关。     

黄瓜绿斑驳花叶病毒对西瓜产量、品质及种子带毒的影响

研究了西瓜不同时期接种黄瓜绿斑驳花叶病毒(Cucumber green mottle mosaic virus,CGMMV)后对其生长、产量、品质和种子带毒的影响。西瓜植株接种CGMMV越早其生长受到抑制越明显,砧木期、嫁接后定植前、定植期、压蔓期、授粉期各处理的株高、叶片数、叶片长与对照相比差异极显著(p0.01),而结瓜以后接种CGMMV对西瓜生长状况无显著影响。压蔓期前3个生育期接种该病毒,西瓜产量损失分别为56.49%、55.48%和51.14%。而压蔓期以后接种该病毒,西瓜产量损失与对照相比仍分别降低17%、13.1%和14.35%。关于倒瓤情况,压蔓期前接种该病毒可导致西瓜果实100%倒瓤,压蔓期、授粉期接种处理西瓜倒瓤率分别为48%和34%,即使结瓜后再接种病毒西瓜倒瓤率仍高达18%。血清学检测表明,压蔓期前接种的西瓜其果肉和种子均携带CGM-MV,带毒率达100%;结瓜后再接种CGMMV西瓜种胚和果肉的带毒率仍然高达64%。检测种胚和果肉病毒含量表明,接种时期越早病毒含量越高,但结瓜后接种仍有较高的病毒含量,种胚和果肉的病毒含量分别达5.98 mg/mL和5.64 mg/mL。     

黄瓜绿斑驳花叶病毒在葫芦上的种传规律及热处理效果评价

为明确黄瓜绿斑驳花叶病毒(Cucumber green mottle mosaic virus,CGMMV)在葫芦上的种传规律,以西瓜砧木葫芦种子为材料,采用双抗体夹心酶联免疫吸附测定法(DAS-ELISA)、反转录聚合酶链式反应(RT-PCR)与生物学检测相结合的方法研究葫芦种子的带毒率和传毒率的关系,并评价了干热处理对病毒的钝化效果。结果表明,DAS-ELISA灵敏度检测种子时,在感染种子研磨液∶健康种子研磨液为1∶1 000时,带毒量仍能检测出阳性,RT-PCR和DAS-ELISA两种方法均能准确检测葫芦种子的带毒情况;6个批次的葫芦种子有4个批次呈阳性,带毒率在0~100%之间,贮藏1年后的传毒率在0~5.6%之间;4个为CGMMV阳性的种子批经干热处理后,仅1株实生苗呈阳性。研究表明,CGMMV在葫芦作物上的隐性带毒现象非常普遍;种子的带毒率高而传毒率低,以表面带毒为主,且非常稳定;72℃72 h干热处理葫芦种子能有效地钝化CGMMV。     

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。     

Molecular characterization and variability analysis of <Emphasis Type="Italic">Apple scar skin viroid</Emphasis> in India

Apple scar skin viroid (ASSVd) infection is a major limitation to apple fruit quality and causes huge economic losses. In surveys of apple orchards in the northern Indian state of Himachal Pradesh, fruits with dappling symptoms were noticed. ASSVd was detected from these fruits and molecularly characterized. Ten clones from three isolates were sequenced, of which seven were new sequence variants of ASSVd. The clones had significant sequence variability (94–100%) with each other. Variability was more common in the pathogenic domain of the viroid genome. Four of the clones were 330 nucleotides (nt) long, and the other six had an additional nucleotide. Phylogenetic analysis showed close affinity of the present isolates with some Chinese and Korean isolates. The study reports seven new variants of ASSVd and also provides the first molecular evidence of viroid infection (ASSVd) in apple in India.     

利用两种方法检测苹果锈果类病毒

以克隆ASSVd的部分序列,通过RT-PCR成功合成了地高辛标记的cDNA探针,提取苹果和梨树枝条的总RNA,用斑点杂交技术对其进行了检测试验,结果表明,探针具有很高的灵敏度和特异性。地高辛标记的cDNA探针不与阴性对照枝条RNA以及感染PBCVd、AFCVd、ADFVd枝条总RNA发生杂交,仅与感染ASSVd样品的总RNA杂交。     

<Emphasis Type="Italic">Citrus exocortis viroid</Emphasis> transmission through commercially-distributed seeds of <Emphasis Type="Italic">Impatiens</Emphasis> and <Emphasis Type="Italic">Verbena</Emphasis> plants

Surveys of Impatiens and Verbena species in local nurseries in Fredericton, Canada and Verbena species in New Delhi, India showed widespread infection of Citrus exocortis viroid (CEVd) in vegetatively-propagated and seed-grown plants. To determine viroid seed transmission, samples of eight varieties of Impatiens and 11 varieties of Verbena were obtained from four commercial sources. All 19 samples collected contained viroid infection irrespective of variety. The presence of viroid in non-germinated seed was 21%, while the transmission rate in seedlings was 66% in Impatiens walleriana in 2006. Following 2 years of seed storage, the respective figures were 6% and 26%. Similarly, in Verbena x hybrida the presence of viroid in seed was 13% in 2006 with a seed-transmission rate in seedlings of 28%, while the respective figures after 2 years of storage were 5% and 45%.     

北京平谷区苹果锈果类病毒检测初报

采集北京市平谷区‘中秋王’、‘寒富’苹果叶片样品共12份,以Northern杂交、RT-PCR方法检测苹果锈果类病毒(Apple scar skin viroid,ASSVd)带毒情况。结果表明,表现明显锈果症状的‘中秋王’样品均检出ASSVd,无明显症状的‘中秋王’样品没有检出ASSVd,而无明显症状的‘寒富’样品全部携带ASSVd。测序发现,所得序列与NCBI中已报道的ASSVd基因组序列相似性为91%~99%,且两品种中ASSVd主流序列完全相同。结果说明‘中秋王’对ASSVd较为敏感,感病后易于表现出明显的锈果症状;‘寒富’对ASSVd耐性较强,感病后不表现出明显症状。     

An outbreak of Potato spindle tuber viroid in tomato is linked to imported seed

In 2011, an outbreak of the quarantine-regulated pathogen Potato spindle tuber viroid (PSTVd) occurred in a commercial glasshouse-grown tomato crop in Queensland, Australia. Phylogenetic studies showed that the genotype of this isolate grouped in a cluster of PSTVd genotypes from tomato and Physalis peruviana, and exhibited an interesting mutation (U₂₅₇→A) that has previously been linked to lethal symptom expression in tomato. Transmission studies showed that the viroid could be mechanically transmitted from crushed fruit sap, but not from undamaged fruits. A low rate of asymptomatic infection was determined for plants in the affected glasshouse, demonstrating the efficacy of using symptoms to detect PSTVd infections in tomato. No PSTVd infections were detected in solanaceous weeds located outside of the infected glasshouse, excluding them from playing a role in the viroid epidemiology. Monitoring and subsequent testing of new tomato crops grown in the facility demonstrated successful eradication of the pathogen. A trace-back analysis linked the outbreak of PSTVd to an infected imported tomato seed-lot, indicating that PSTVd is transmitted internationally through contaminated seed.     

Influence of the amount of blackgram mottle virus in different tissues on transmission through the seeds of Vigna mungo

Blackgram mottle virus (BMoV) was transmitted via up to 16% of seeds of different cultivars of blackgram, as determined by seedling symptom tests. The percentage of seed infection by BMoV as determined by EL1SA was even higher. Seed transmission was highest in cv. PLU-277 (15.9%), followed by cvs T-9 (11.8%), PLU-213 (7.0%) and UH-81-7 (1.3%). Seed transmission was correlated with the amount of virus present in the embryonic axis and later in primary leaves. The presence of virus in the testa alone did not result in its transmission through seeds. Virus concentration in different tissues varied; the mean amount of virus in the three cultivars was found to be 48–1234 ng per embryonic axis, 15–24 ng per cotyledon, and 12–20 ng per testa. The infection of primary leaves through the seed also resulted in systemic infection if the amount of virus in primary leaves exceeded 100 ng/100 mg of tissue. Close agreement was found between the percentage of seedlings with systemic infection and the percentage of seeds and embryonic axes containing more than 100 ng virus. The cultivars that resisted seed transmission contained relatively small amounts of the virus in embryonic axes.