甘蔗黄叶病毒外壳蛋白基因克隆及其实时荧光RT-PCR检测

 甘蔗黄叶病毒（Sugarcane yellow leaf virus, SCYLV）引起的甘蔗黄叶病是一种新的全球性病毒病害。本文以YLSCPF1和YLSCPR591为引物,采用RT-PCR方法克隆了甘蔗黄叶病毒福建分离物（CHN-FJ1）外壳蛋白（CP）基因,编码196个氨基酸。分析不同地理来源的SCYLV病毒分离物cp基因核苷酸及其推导编码的氨基酸序列,同源性达95%以上。根据cp基因的保守序列,设计1对特异性引物和TaqMan探针,建立了SCYLV的TaqMan实时荧光RT-PCR方法。结果表明,检测下限为初始质粒模板DNA 1 000拷贝/μL（约3.61 fg/μL）,比常规PCR方法的灵敏度提高100倍。检测甘蔗花叶病毒、宿根矮化病菌和黑穗病菌,没有典型的扩增曲线和无Ct值。应用实时荧光RT-PCR、常规RT-PCR和组织印迹免疫杂交（TBIA）对田间甘蔗叶片样品进行检测,阳性检出率分别为100%、61.5%和69.2%,表明该方法比常规RT-PCR和TBIA具有更高的灵敏度,适合于对SCYLV的检测。     

果蔗脱毒种苗甘蔗花叶病、黄叶病和宿根矮化病分子检测

为监测2016-2017年种植的果蔗脱毒种苗脱毒效果,分别采集广州市南沙区和增城区、湛江市麻章区及华南农业大学甘蔗育种基地共83份果蔗脱毒种苗样本,进行甘蔗花叶病毒(SCMV)、高粱花叶病毒(SrMV)和甘蔗黄叶病毒(SCYLV)RT-PCR检测。结果表明SCMV的阳性样本数为3个,阳性检出率3.61%;SrMV的阳性样本数为0;SCYLV的阳性样本数为78个,阳性检出率93.98%。采用常规PCR和巢式PCR技术对采集于广州市增城区和华南农业大学甘蔗育种基地的30份果蔗脱毒种苗样本进行宿根矮化病菌(Lxx)检测,常规PCR检测阳性样本数为0,巢式PCR检测疑似阳性样本数为8,疑似阳性检出率26.67%。本研究采用茎尖组织培养脱毒技术培育的果蔗脱毒种苗能有效脱除果蔗种苗内的SCMV、SrMV和Lxx,但SCYLV的脱除效果有待进一步研究。     

甘蔗宿根矮化病菌巢式PCR检测

为了探索甘蔗宿根矮化病快速检测技术,以细菌16S-23S rDNA内转录间隔区(ITS)通用引物ITS1/ITS2为第1轮引物,以甘蔗宿根矮化病菌特异引物Lxx1/Lxx2为第2轮引物,建立甘蔗宿根矮化病菌巢式PCR检测技术。巢式PCR能扩增出438 bp的目的条带,其核苷酸序列与巴西、澳大利亚及美国甘蔗宿根矮化病菌分离物核苷酸序列同源率为100%或99.8%;其检测灵敏度为10fg甘蔗宿根矮化病菌基因组DNA,较常规PCR提高100倍;样品检测结果也表明巢式PCR检测灵敏度明显优于常规PCR,可用于+1片嫩叶和心叶等微量病菌样品的检测。     

甘蔗赤条病菌巢式PCR检测

甘蔗赤条病是由燕麦食酸菌燕麦亚种(Acidovorax avenae subsp.avenae,Aaa)引起的一种世界性甘蔗细菌病害。为建立Aaa快速、灵敏的检测技术,根据该病菌16S~23S核糖体基因及其转录间隔区ITS分别设计2对特异性引物,建立Aaa巢式PCR检测方法。结果表明,建立的巢式PCR方法对Aaa标准菌株、水稻食酸菌A.oryzae具有特异性,可扩增出454 bp目的条带,对近缘种德氏食酸菌A.delafieldii及其它科属的红色雷夫松氏菌Leifsonia rubra和甘蔗宿根矮化病菌L.xyli subsp.xyli未扩增出任何条带。以感染Aaa的甘蔗叶片总DNA、含ITS靶标片段的质粒DNA标准品及Aaa标准菌液为模板,巢式PCR灵敏度最低检测限分别为10 fg/μL、10拷贝/μL和36 CFU/mL,是常规PCR灵敏度的1 000倍。应用巢式PCR和常规PCR对14份有赤条病症状的田间甘蔗叶片样品进行平行检测,阳性检出率分别为100.0%和28.6%,表明巢式PCR比常规PCR检测方法具有更高的灵敏度。本研究建立的巢式PCR方法适合于田间甘蔗赤条病害的分子检测与鉴定。     

甘蔗绵蚜为害损失研究

甘蔗绵蚜Ceratovacuna lanigera Zehntner是现今广泛分布于我国各甘蔗种植区,严重影响甘蔗产量和品质的叶部害虫。为探明现有生产水平条件下甘蔗绵蚜对甘蔗实测产量及糖分的为害损失和对新植宿根出苗的影响情况,给甘蔗绵蚜的科学有效防控提供理论依据和翔实的实测数据。本研究于2014—2016年,选择主栽品种,同田设立为害区和未为害区,调查评估绵蚜为害对新植宿根出苗的影响,甘蔗成熟期分别收砍称量和测定分析甘蔗产量及糖分含量,并计算甘蔗实测产量及糖分损失。研究结果显示,甘蔗实测产量减少2 503~4 123kg/667m~2,平均3 079kg/667m~2;产量损失率为28.5%~45.7%,平均35.9%;出汁率减少2.4%~4.13%,平均3.01%;甘蔗糖分降低5.48%~8.16%,平均6.38%;蔗汁锤度降低6.95~9.05°BX,平均7.66°BX;蔗汁重力纯度降低8.43%~19.97%,平均12.35%;而蔗汁还原糖分则增加1.01%~1.3%,平均1.21%;新植出苗率降低24.7%~27.3%,平均26.0%;宿根出苗数减少3 829~5 083株/667m~2,平均4 456株/667m~2,相对出苗损失率为57.6%~58.0%,平均57.8%。可见,目前云南蔗区甘蔗绵蚜为害造成的甘蔗产量及糖分损失十分严重,甘蔗绵蚜为害已成为现阶段严重影响甘蔗产量和品质的主要挑战之一。研究结果对加强甘蔗绵蚜的科学有效防控和支持中国蔗糖产业的可持续发展具有重要意义。     

甘蔗黄叶病及其病原分子生物学研究进展

甘蔗黄叶病是由甘蔗黄叶病毒(Sugarcane yellow leaf virus,SCYLV)引起的一种病毒病害,在全球主要甘蔗种植国家或地区普遍发生,危害日益严重。SCYLV经甘蔗蚜虫以持久性方式传播,在寄主植株内主要侵染韧皮部组织。该病毒起源于黄症病毒科属间基因重组,被国际病毒分类命名委员会确认为黄症病毒科马铃薯卷叶病毒属成员。文章综述了甘蔗黄叶病毒生物学特征、病害发生和危害、病原鉴定和检测、分子进化和遗传多样性、基因组结构和基因功能以及抗病转基因等方面研究进展,并对甘蔗黄叶病抗病育种和防治措施作了讨论。     

我国甘蔗主要杂交亲本黄叶病病原鉴定及田间发病率

甘蔗黄叶病是一种发生普遍、危害严重的病毒病害,选育抗病品种是控制该病发生与蔓延的有效措施.利用逆转录-聚合酶链反应(RT-PCR)、组织印迹杂交免疫测定(TBIA)和抗原直接包被酶联免疫吸附(DAC-ELISA)方法对我国43份甘蔗主要杂交亲本黄叶病病原(Sugarcane yellow leaf virus,SCYLV)进行鉴定,并调查SCYLV田间自然侵染条件下甘蔗黄叶病发病率.已明确36份亲本材料感染或未感染SCYLV,其中32份鉴定结果为阳性,品种(系)感病率达88.9％.甘蔗植株田间自然发病率可分为无发病、发病率低、中等和高4个等级,方差分析表明,各等级间的植株发病率差异达极显著水平(P＜0.01).从美国引进的多数CP和HoCP系列亲本发病率较高,而多数崖城和新台糖系列亲本材料的发病率较低,可作为甘蔗抗黄叶病杂交亲本.     

甘肃省南瓜及西葫芦小西葫芦黄花叶病毒病鉴定

利用双抗夹心酶联免疫吸附测定(DAS-ELISA)的方法对甘肃出入境南瓜、西葫芦种子及采自河西地区显症病株叶片进行检测,在种子及病叶组织中均检测到ZYMV病毒,其中南瓜种子带毒批次占12.5%,西葫芦种子带毒批次占11.8%。根据已报道的小西葫芦黄花叶病毒(Zucchini yellow mosaic virus)基因组核苷酸序列,设计引物扩增其外壳蛋白(CP)基因,以ELISA阳性种子或病叶组织总RNA为模板,进行RT-PCR扩增,对预期大小的扩增产物进行测序,结果表明扩增获得的核苷酸序列与世界各地的ZYMV分离物CP基因具有高度一致性,综合ELISA检测和RT-PCR的结果,确定南瓜、西葫芦种子可携带ZYMV,且ZYMV是侵染甘肃瓜类作物的重要病毒种类。     

不同品种和植期甘蔗白叶病自然发病率调查

 甘蔗白叶病(sugarcane white leaf, SCWL)是由植原体引起的甘蔗毁灭性病害,对甘蔗生产危害极大。为明确不同品种不同植期SCWL的发病规律,2018年对我国SCWL发病最为严重的耿马芒翁和贺派蔗区进行了SCWL田间发病情况调查和巢式PCR检测分析。田间调查结果表明,不同品种田间自然发病率不同,其中粤糖60号平均发病率最高,为73.50%,柳城05-136平均发病率最低,为13.67%;不同植期田间自然发病率也有差异,新植蔗田间发病率最低,为32.38%,3年宿根蔗的田间发病率最高,为64.33%。病原检测结果表明,所有品种的阳性检出率均在90%以上,其中盈育91-59阳性检出率最低,为90.95%,柳城05-136阳性检出率最高,为96.67%,无白叶症状样品的阳性检出率为81.53%,2年和3年宿根的阳性检出率最高,均为96.67%。本研究结果表明SCWL发病率随宿根年限增加而升高,依据白叶症状进行的田间病害调查不能准确反映SCWL的真实发生情况。     

半巢式RT-PCR检测进口大豆中菜豆荚斑驳病毒的研究

 菜豆荚斑驳病毒(Bean pod mottle virus,BPMV)在美国东部和南部的大豆产区广泛分布。该病毒能够造成3%~52%的产量损失并使大豆品质降低,目前我国未见其分布。针对进口大豆种子的植物病毒检测,本文建立了半巢式RT-PCR技术检测BPMV的方法。该方法采用Trizol快速提取大豆种子病毒总RNA,并根据BPMV的外壳蛋白编码基因设计特异性引物,经第一轮RT-PCR和第二轮半巢式PCR扩增,分别得到275 bp和196 bp大小的特异性基因片段。半巢式RT-PCR扩增产物的测序表明,该产物序列与BPMV的外壳蛋白编码基因存在91%~95%的高度同源性。检测灵敏度比较研究显示,DAS-ELISA与RT-PCR的检测灵敏度相近,半巢式RT-PCR的检测灵敏度比这2种方法高出103倍以上。     

Spread of <Emphasis Type="Italic">Sugarcane yellow leaf virus</Emphasis> in initially disease-free sugarcane is linked to rainfall and host resistance in the humid tropical environment of Guadeloupe

Sugarcane yellow leaf virus, the causal agent of yellow leaf, is transmitted from plant to plant by aphids. Understanding and evaluating the epidemic risks due to spread of yellow leaf by aphids is an important feature for sugarcane production. Four distinct sugarcane trials were set up with disease-free plants to study the relationship between spread of yellow leaf, the vector dynamics and environmental conditions that may favour yellow leaf epidemics. The study was performed by surveys of vector populations and determination of plant infections. Sugarcane cultivar SP71-6163, highly susceptible to yellow leaf, was analyzed spatially at different dates in all four trials and compared to commercial cultivars in two of the four trials. These surveys allowed us to identify a correlation between the aphid dynamics in the field and yellow leaf progress. Additionally, a negative correlation was found between rainfall during the first weeks after transferring sugarcane plants to the field and aphid dispersal within the field. This later result revealed an impact of rainfall on aphid invasion and subsequent plant infection by SCYLV. If aphids are the key factor for disease spread, plant response varied also according to cultivar resistance with high variation depending on rain conditions.     

Elimination of Sugarcane yellow leaf virus from infected sugarcane plants by meristem tip culture visualized by tissue blot immunoassay

Sugarcane yellow leaf virus (SCYLV), a member of the Luteoviridae , is implicated in the sugarcane disease known as yellow leaf syndrome (YLS), which is characterized by yellowing of the leaf midrib followed by leaf necrosis and possible growth suppression. YLS is distributed worldwide and susceptible cultivars are commonly infected with SCYLV. However, not all cultivars infected with SCYLV show symptoms of YLS and some cultivars that show symptoms do so sporadically. Since it is difficult to obtain virus-free plants of susceptible cultivars, it has not been possible to study the factors involved in SCYLV infection nor the effects of infection on plant growth and yield. A tissue blot immunoassay was used to visualize in vivo presence of the virus so that virus-infected and virus-free plants could be distinguished. Meristem tip cultures were used to produce virus-free plantings of six SCYLV-susceptible sugarcane cultivars. Nearly all of the regenerated sugarcane lines remained virus-free over a period of up to 4 years, whether grown in isolated fields or in the glasshouse. Experimental re-infection of the virus-free plants by viruliferous aphids demonstrated that meristem tip culture did not affect susceptibility of sugarcane to SCYLV. Improved diagnosis and production of virus-free plants of SCYLV-susceptible cultivars will facilitate research to quantify the effect of the virus on yield and to analyse the processes involved in disease development.     

Sugarcane yellow leaf virus and sugarcane yellows phytoplasma: elimination by tissue culture

Yellow leaf syndrome (YLS) is a recently reported disease of sugarcane, characterized by yellowing of the leaves. Two pathogens: a virus, Sugarcane yellow leaf virus (SCYLV); and a phytoplasma, sugarcane yellows phytoplasma (SCYP), are associated with the disease. The use of tissue culture was investigated as a means to eliminate both SCYLV and SCYP from exotic varieties undergoing quarantine in Mauritius. Of 43 varieties in quarantine, 28 were infected with SCYLV and 19 with SCYP when checked by RT–PCR and nested PCR, respectively. Seventeen varieties were coinfected with both pathogens. Thirty infected varieties were induced to form callus in vitro using leaf rolls as explants. After two subcultures, 19 varieties were successfully regenerated and tested for SCYLV and SCYP. No pathogen could be detected in any regenerated plantlets. All the regenerated plants remained free from both SCYLV and SCYP over a period of 1 year in the glasshouse, confirming that the pathogens had been eliminated by tissue culture.     

Variation in virus populations and growth characteristics of two sugarcane cultivars naturally infected by Sugarcane yellow leaf virus in different geographical locations

Two sugarcane cultivars (R570 and SP71-6163) naturally infected by Sugarcane yellow leaf virus (SCYLV) were each imported from several geographical locations into a sugarcane yellow leaf-free environment (Montpellier, France). Plants were grown as plant cane for 5–6 months and the experiment was repeated for three consecutive years (2003–2005) in a greenhouse. Several sugarcane-growth and disease characteristics were monitored to identify variation in pathogenicity of SCYLV. Depending on their geographical origin, sugarcane cvs R570 and SP71-6163 were infected by SCYLV genotypes BRA-PER or REU, or a mixture of the two. Severity of symptoms did not vary between plants of cv. R570, but variation in disease severity between plants of cv. SP71-6163 from different geographical locations suggested the occurrence of pathogenic variants of SCYLV. For each sugarcane cultivar, differences in stalk length, number of stalk internodes, virus titre in the top visible dewlap leaf, and percentage of infection of leaf and stalk phloem vessels were also found between plants from different geographical origins. However, these differences were not always reproducible from one year to another, suggesting occurrence of different plant responses to SCYLV isolates under varying environmental conditions.     

甘蔗病毒病及其防治研究进展

甘蔗是最重要的糖料作物,由于其栽培过程中采用种茎无性繁殖,病毒病发生逐年加重.已知侵染甘蔗的病毒种类有甘蔗花叶病毒(Sugarcane mosaic virus,SCMV)、高粱花叶病毒(Sorghummosaic virus,SrMV)、甘蔗线条花叶病毒(Sugarcane streakmosaic virus,SCSMV)、甘蔗黄叶病毒(Sugarcane yellow leaf virus,SCYLV)、甘蔗斐济病病毒(Sugarcane Fiji disease virus,SFDV)、甘蔗线.条病毒(Sugarcane streak virus,SSV)和甘蔗杆状病毒(Sugarcane bacilliform virus,SCBV).文中简要介绍上述几种病毒的基本特性及其所致病害的发生特点,对目前甘蔗病毒病防治技术进行了评述,提出了我国甘蔗病毒研究中需要关注的若干问题.     

侵染西葫芦的中国南瓜曲叶病毒的检测及序列分析

2019年山东种植的西葫芦上广泛发生病毒病,症状与之前常发症状有差异,发病植株叶片向下卷曲、黄化,植株矮化。为明确引起此次西葫芦病毒病的病原,我们以田间采集的10份西葫芦病叶为材料,进行PCR扩增并测序。测序结果显示扩增片段核苷酸序列与我国广东的中国南瓜曲叶病毒(SLCCNV)南瓜分离物(MW389917.1)一致性最高。根据同源序列设计引物,经PCR扩增获得SLCCNV全长序列,DNA-A 全长为2 730 bp(OM692270.1)、DNA-B 全长为2 711 bp(OM692269.1),经序列比对发现DNA-A序列与已登录的SLCCNV一致性为89.65%~99.42%,其中与我国广东的SLCCNV-GDHY南瓜分离物(MW389917.1)一致性最高,为99.42%;DNA-B序列与已登录的SLCCNV一致性范围为81.82%~97.29%,其中与我国广东的SLCCNV-GDHY南瓜分离物(MW389918.1)一致性最高,为97.29%。因此推测引发山东西葫芦病毒病的病原物是SLCCNV,由于该病毒是在山东西葫芦上首次发现,将其命名为SLCCNV-SD。前人已报道SLCCNV可侵染南瓜、甜瓜、烟草、番茄等作物,但SLCCNV可侵染西葫芦在国内未见报道。     

Impact of <Emphasis Type="Italic">Sugarcane yellow leaf virus</Emphasis> on growth and sugar yield of sugarcane

A survey revealed that Sugarcane yellow leaf virus (SCYLV) is found on all Hawaiian sugarcane plantations including those where no yellow leaf symptoms were observed. In a comparison of growth and yield between SCYLV-infected and SCYLV-free plants of the cultivar H87-4094, germination and early shoot growth of infected plants were retarded. The number of stalks per stool was reduced by 30%, biomass was reduced by 29%, and sugar yield by 26% when plants were harvested after 11 months. Yields did not decrease when plants were harvested after 2 years. Thus, SCYLV could reduce yield, even when the plants were asymptomatic. In a field test of SCYLV-susceptible (infected) and -resistant cultivars to compare growth and yield, 10 commercial cultivars (six susceptible and four resistant to SCYLV) were grown in eight fields with different climates and soils. Primary stalk length, biomass and sugar yield did not differ between susceptible and resistant cultivars under any field conditions. Thus, harmful effects of SCYLV on yield cannot be deduced by comparing different cultivars.