甘薯病毒病害（SPVD）病原的基因组测序、检测及种传效率研究
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摘要
甘薯病毒病害(Sweet potato virus disease, SPVD)是由甘薯褪绿矮化病毒(Sweet potato chlorotic stunt virus, SPCSV)和甘薯羽状斑驳病毒(Sweet potato feathery mottle virus, SPFMV)协生共侵染甘薯引起的病毒病害。SPVD对甘薯产量影响极大,严重时可造成90%以上的产量损失,甚至绝收,是甘薯上最严重的病毒病害之一。我国于2010年首次发现了SPVD的发生,目前主要分布在广东、江苏、四川、安徽和福建等地。为了加强对SPVD的预警和控制,防止该病的进一步蔓延危害,有必要对该病害的两个病原SPCSV和SPFMV进行深入研究。
SPCSV可划分为东非(EA)和西非(WA)两个株系,目前侵染我国甘薯的主要是WA株系(SPCSV-WA).本文以SPVD的两个主要病原SPCSV-WA和SPFMV为研究对象,完成了SPCSV-WA中国分离物的基因组全序列测定；分析了我国甘薯上SPCSV-WA的分子变异情况；建立了SPCSV-WA和SPFMV实时荧光定量PCR检测技术；对SPCSV-WA的寄主范围以及SPCSV和SPFMV的种传特性进行了初步研究,以期为SPVD的防控提供技术手段和理论依据。论文主要结果如下：
1、利用RT-PCR结合3'-RACE和5'-RACE方法,成功地从传毒介体烟粉虱中克隆了SPCSV-WA江苏分离物(SPCSV-WA-JS)基因组RNA1和RNA2全长序列(GenBank登录号：KC146840和KC146841)。序列分析表明,SPCSV-WA江苏分离物RNA1全长8637nt,包含89nt的5’端非翻译区(UTR)和193nt的3'-UTR。RNA2全长8107nt,包含191nt的5'-UTR和192nt的3'-UTR。RNA1包含4个开放阅读编码框(ORF)。其中,ORFla位于90-6053nt,ORF1b位于6052-7569nt,ORF2位于7583-8272nt, ORF3位于8277-8444nt,分别编码polyprotein蛋白、RdRp. RNase3蛋白、p7蛋白。RNA2包含9个ORF,其中,ORF4位于192-329nt, ORF5位于333-467nt, ORF6位于406-534nt, ORF7位于879-2543nt, ORF8位于2565-4121nt, ORF9位于4103-4324nt, ORF10位于4352-5125nt, ORF11位于5128-7182nt,ORF12位于7187-7915nt。分别编码p5.2蛋白、p5蛋白、p5.1蛋白、Hsp70, p60蛋白、p8蛋白、CP蛋白、mCP蛋白、p28蛋白。将SPCSV-WA-JS与西班牙Can181-9分离物进行比对,RNA1和RNA2的相似性分别为98.90%和98.68%,表明SPCSV-WA的基因组序列高度保守,分子变异较小。
2、利用RT-PCR获得了重庆(SPCSV-WA-CQ)和四川(SPCSV-WA-SC-8、SPCSV-WA-SC-12)等地区3个分离物的近全长序列(GenBank登录号分别为：KC888966和KC888963、KC888964和KC888961、KC888965和KC888962)。对我国不同地区SPCSV-WA分离物的基因组序列进行相似性分析,结果表明4个分离物的核苷酸序列相似性达到98%以上,核苷酸序列呈现高度保守性,分子变异较小
3、依据GenBank中登录的甘薯羽状斑驳病毒(SPFMV)外壳蛋白(CP)基因的保守区设计引物和TaqMan探针,通过对反应体系和反应条件的优化,建立了特异、灵敏、高效的SPFMV实时荧光定量PCR检测方法。结果表明,该方法只能检测到目的病毒,最低可检测到约5.46个拷贝·μL-1的阳性质粒,灵敏度比常规PCR高2个数量级。本研究建立的实时荧光定量PCR方法可用于田间样品的检测。
4、依据GenBank中登录的SPCSV-WA核苷酸序列,分别设计两对特异性引物和一条TaqMan探针。以SPCSV-WA CP基因的重组质粒为阳性标准质粒绘制标准曲线,通过优化反应体系和反应条件,建立了SPCSV-WA的实时荧光定量PCR检测方法。试验结果表明,该方法只能检测到目的病毒,最低可检测到约3.31个拷贝·μL-1的阳性质粒,灵敏度比常规PCR高1000倍。本研究建立的SPCSV实时荧光定量PCR方法可用于田间样品的检测。
5、采用烟粉虱传毒和实时荧光定量PCR检测的方法测定了SPCSV-WA的寄主范围。共测定4科8种植物。结果表明,经烟粉虱传毒后,胡萝卜、雍菜表现为轻微花叶,巴西牵牛表现为叶片皱缩,番茄表现为轻微花叶和叶片皱缩,普通烟、本氏烟、白菜和萝卜均不表现明显症状。实时荧光定量PCR检测结果表明,8种供试植物均呈SPCSV-WA阳性,说明SPCSV-WA能侵染所有供试的8种植物。其中,胡萝卜、雍菜、萝卜、白菜、普通烟和番茄6种植物为首次报道。对胡萝卜进行了回传实验,接种烟粉虱2周后,健康的胡萝卜上出现轻微花叶症状,实时荧光定量PCR方法检测呈SPCSV-WA阳性。
6、通过烟粉虱传毒获得感染SPCSV-WA的烟草植株,以感病烟草植株上收获的种子为材料,利用NCM-ELISA检测种子实生苗的带毒率,共检测了760株烟草,SPCSV-WA的种传率为5.39%。应用实时荧光定量PCR方法检测SPCSV-WA在烟草种子和花器上的分布情况,结果表明烟草种子、花冠、雄蕊、雌蕊、花萼均携带SPCSV。
7、将SPVD甘薯植株的茎蔓作为接穗嫁接到供试甘薯品种上,待其发病表现典型SPVD症状后,将其移栽至海南试验田进行种子繁育。以SPVD甘薯植株上收获的种子为材料,应用NCM-ELISA和实时荧光定量PCR方法检测种子实生苗的传毒率、SPVD甘薯植株的花器及种子不同部位带毒情况。NCM-ELISA方法共检测了1311株实生苗,2012年和2013年SPCSV的种传率分别为4.56%和0.90%,SPFMV的种传率分别为7.49%和2.59%；实时荧光定量PCR检测了55份甘薯实生苗,SPCSV和SPFMV的种传率均为100%。应用实时荧光定量PCR方法检测SPVD甘薯植株的花器及种子不同部位带毒情况,结果表明种皮、胚乳、胚、雄蕊、雌蕊、花萼均携带SPCSV和SPFMV;花冠携带SPFMV,不携带SPCSV.
Sweet potato virus disease (SPVD), which is caused by the synergistic interaction of sweet potato chlorotic stunt virus (SPCSV) and sweet potato feathery mottle virus (SPFMV), is the most serious viral disease of sweet potato (Ipomoea batatas). SPVD causes severe yield loss of about90%, sometimes reaching100%. In China, SPVD is mainly distributed in Guangdong, Jiangsu, Sichuan and Anhui provinces currently since its first report in2010. Furher study on the two pathogens, SPCSV and SPFMV, is necessary to control the rapid spread of SPVD.
SPCSV can be differentiated into two distantly related strains, East African (SPCSV-EA) and West African (SPCSV-WA), The SPCSV-WA strain has a wider geographic distribution in China than the SPCSV-EA strain. Focusing on SPCSV-WA and SPFMV in this study, we cloned and sequenced the genome of Chinese SPCSV-WA isolates, analyzed molecular variation of the Chinese SPCSV-WA isolates, developed two real-time fluorescent quantitative PCR assays to detect SPCSV-WA and SPFMV, studied the host range of SPCSV-WA and seed transmission of SPCSV-WA and SPFMV using real-time PCR and other detection methods. The results showed (summarized below) are an attempt to determine the mechanism of the wide spread of SPVD, which will help to develop disease control strategies.
1. The complete genome sequence of RNA1and RNA2of SPCSV-WA isolated from Jiangsu Province (SPCSV-WA-JS) was cloned using reverse transcriptase PCR (RT-PCR) and rapid amplification of cDNA ends to capture the terminal ends of the viral genomes. The viral RNA was extracted from infected whiteflies and the sequences were deposited in GenBank with accession numbers KC146840and KC146841. Sequence analysis showed that RNA1from the SPCSV-WA-JS isolate was8,637nucleotides (nt) long, including an89-nt5' untranslated region (5'-UTR), a193-nt3'-UTR and four open reading frames (ORFs). The four ORFs in the RNA1at positions90to6053(ORFla),6052to7569(ORFlb),7583to8272(ORF2) and8277to8444t (ORF3), encode the polyprotein, RNA-dependent RNA polymerase (RdRP), RNase3and p7, respectively. RNA2was8,107nt long, including a191-nt5'-UTR, a192-nt3'-UTR, and nine ORFs:nucleotides192to329(p5.2),333to467 (p5),406to534(p5.1),879to2543(heat shock protein70h),2565to4121(p60),4103to4324(p8),4352to5125(major coat protein),5128to7182(minor coat protein) and7187to7915(p28), respectively. Comparing the genome sequence of SPCSV-WA-JS with that of the Can181-9isolate from Spain, RNA1and RNA2showed98.90%and98.68%sequence identity, respectively. The results revealed that the genomic sequences of SPCSV-WA are highly conserved.
2. RT-PCR was used to obtain the nearly full-length sequences of three isolates from Chongqing (SPCSV-WA-CQ) and Sichuan (SPCSV-WA-SC-8, SPCSV-WA-SC-12). The sequences were deposited in GenBank with accession numbers KC888966, KC888963, KC888964, KC888961, KC888965and KC888962. Similarity analysis was performed for the genomic sequences of SPCSV-WA isolates from different regions of China. The analysis showed that the four genomic sequences of SPCSV-WA from Sichuan, Jiangsu and Chongqing shared more than98%nucleotide identity, indicating that SPCSV-WA isolates from China have highly conserved genomic sequences and little molecular variation.
3. Primers and TaqMan probes were designed according to the conserved regions of the coat protein (CP) gene of SPFMV from GenBank. By optimizing the reaction conditions, a specific, sensitive and efficient real-time RT-PCR assay was established to detect SPFMV. The results showed that this assay was specific for detecting the targeted virus:the detection limit was about5.46copies/μL of positive plasmids. The assay was up to two-order more sensitive than conventional PCR. The real-time PCR assay established in this study could be suitable for detecting field samples.
4. Two specific primers and one TaqMan probe were designed according to the nucleotide sequence of SPCSV-WA from GenBank. A recombinant plasmid containing the SPCSV-WA CP gene was used to establish a standard curve. By optimizing the reaction conditions, a real-time RT-PCR assay was established to detect SPCSV-WA. The results showed that this assay was specific for detecting the targeted virus:its detection limit was about3.31copies/μL of the positive plasmid. The sensitivity of the assay was1000times higher than that of conventional PCR. The real-time PCR assay for detecting SPCSV established in this study could be suitable for detecting field samples.
5. To determine the host range, eight test plants of four families were inoculated with SPCSV-WA by whitefly (Bemisia tabaci). SPCSV-WA was detected by symptomatology and real-time PCR. Virus-associated symptoms were induced in four plant species:slight mosaic in Daucus carota and I. aquatica, distorted leaves in I. setosa, slight mosaic and distorted leaves in Solanum lycopersicum. The other four species, Nicotiana tabacum, N. benthamiana, Raphanus sativus and Brassica Chinensis, were symptomless. Using real-time PCR, a positive signal was obtained in all the test plants, revealing that SPCSV-WA could infect all eight species. This is the first report of SPCSV-WA infection in D. carota, I. Aquatica, R. sativus, B. Chinensis, N. tabacum and S. lycopersicum. After SPCSV-WA re-infection of healthy plants with virus-free whitefly, mosaic patterns were observed two weeks later, and SPCSV-WA was also detected in D. carota plants using real-time PCR.
6. SPCSV-WA-infected N. tabacum plants were obtained by whitefly transmission. The seeds, flowers and subsequent seedlings produced from the infected N. tabacum plants were used for virus detection. Nitrocellulose membrane-enzyme linked immunosorbent assay (NCM-ELIS A) was used to detect the virus transmission rate of the seedlings. A total of760N. tabacum seedlings were detected, giving a seed transmission rate of SPCSV-WA of5.39%. Real-time PCR was used to determine the distribution of SPCSV-WA in the seeds and floral organs of N. tabacum. The results showed the seeds, corolla, stamen, pistil and calyx were infected.
7. Plants of several sweet potato cultivars exhibited typical symptoms of SPVD after grafting with the scions of a SPVD-infected sweet potato cultivar. The sweet potato cultivars plants above with typical symptoms of SPVD grew in Hainan Province for mature seed harvesting. Using real-time PCR, SPCSV and SPFMV were both detected in the seed coat, embryo, endosperm, calyx, corolla, stamen and pistil. NCM-ELISA was used to detect the virus transmission rate of the seedlings. A total of1311seedlings were detected, giving seed transmission rates of SPCSV in2012and2013of4.56%and0.90%respectively, and of SPFMV in2012and2013of7.49%and2.59%respectively. Positive signals for SPCSV and SPFMV were detected in55randomly chosen seedlings of sweet potato by real time PCR, suggesting the seed transmission rates of SPCSV and SPFMV were100%. Real-time PCR was used to detect the distribution of SPCSV and SPFMV in the floral organs and different parts of the seeds of sweet potato. The results showed a wide distribution of SPCSV and SPFMV in the episperm, endosperm, stamen, pistil and calyx. SPFMV, but not SPCSV, was distributed in the corolla.
SPCSV可划分为东非(EA)和西非(WA)两个株系,目前侵染我国甘薯的主要是WA株系(SPCSV-WA).本文以SPVD的两个主要病原SPCSV-WA和SPFMV为研究对象,完成了SPCSV-WA中国分离物的基因组全序列测定；分析了我国甘薯上SPCSV-WA的分子变异情况；建立了SPCSV-WA和SPFMV实时荧光定量PCR检测技术；对SPCSV-WA的寄主范围以及SPCSV和SPFMV的种传特性进行了初步研究,以期为SPVD的防控提供技术手段和理论依据。论文主要结果如下：
1、利用RT-PCR结合3'-RACE和5'-RACE方法,成功地从传毒介体烟粉虱中克隆了SPCSV-WA江苏分离物(SPCSV-WA-JS)基因组RNA1和RNA2全长序列(GenBank登录号：KC146840和KC146841)。序列分析表明,SPCSV-WA江苏分离物RNA1全长8637nt,包含89nt的5’端非翻译区(UTR)和193nt的3'-UTR。RNA2全长8107nt,包含191nt的5'-UTR和192nt的3'-UTR。RNA1包含4个开放阅读编码框(ORF)。其中,ORFla位于90-6053nt,ORF1b位于6052-7569nt,ORF2位于7583-8272nt, ORF3位于8277-8444nt,分别编码polyprotein蛋白、RdRp. RNase3蛋白、p7蛋白。RNA2包含9个ORF,其中,ORF4位于192-329nt, ORF5位于333-467nt, ORF6位于406-534nt, ORF7位于879-2543nt, ORF8位于2565-4121nt, ORF9位于4103-4324nt, ORF10位于4352-5125nt, ORF11位于5128-7182nt,ORF12位于7187-7915nt。分别编码p5.2蛋白、p5蛋白、p5.1蛋白、Hsp70, p60蛋白、p8蛋白、CP蛋白、mCP蛋白、p28蛋白。将SPCSV-WA-JS与西班牙Can181-9分离物进行比对,RNA1和RNA2的相似性分别为98.90%和98.68%,表明SPCSV-WA的基因组序列高度保守,分子变异较小。
2、利用RT-PCR获得了重庆(SPCSV-WA-CQ)和四川(SPCSV-WA-SC-8、SPCSV-WA-SC-12)等地区3个分离物的近全长序列(GenBank登录号分别为：KC888966和KC888963、KC888964和KC888961、KC888965和KC888962)。对我国不同地区SPCSV-WA分离物的基因组序列进行相似性分析,结果表明4个分离物的核苷酸序列相似性达到98%以上,核苷酸序列呈现高度保守性,分子变异较小
3、依据GenBank中登录的甘薯羽状斑驳病毒(SPFMV)外壳蛋白(CP)基因的保守区设计引物和TaqMan探针,通过对反应体系和反应条件的优化,建立了特异、灵敏、高效的SPFMV实时荧光定量PCR检测方法。结果表明,该方法只能检测到目的病毒,最低可检测到约5.46个拷贝·μL-1的阳性质粒,灵敏度比常规PCR高2个数量级。本研究建立的实时荧光定量PCR方法可用于田间样品的检测。
4、依据GenBank中登录的SPCSV-WA核苷酸序列,分别设计两对特异性引物和一条TaqMan探针。以SPCSV-WA CP基因的重组质粒为阳性标准质粒绘制标准曲线,通过优化反应体系和反应条件,建立了SPCSV-WA的实时荧光定量PCR检测方法。试验结果表明,该方法只能检测到目的病毒,最低可检测到约3.31个拷贝·μL-1的阳性质粒,灵敏度比常规PCR高1000倍。本研究建立的SPCSV实时荧光定量PCR方法可用于田间样品的检测。
5、采用烟粉虱传毒和实时荧光定量PCR检测的方法测定了SPCSV-WA的寄主范围。共测定4科8种植物。结果表明,经烟粉虱传毒后,胡萝卜、雍菜表现为轻微花叶,巴西牵牛表现为叶片皱缩,番茄表现为轻微花叶和叶片皱缩,普通烟、本氏烟、白菜和萝卜均不表现明显症状。实时荧光定量PCR检测结果表明,8种供试植物均呈SPCSV-WA阳性,说明SPCSV-WA能侵染所有供试的8种植物。其中,胡萝卜、雍菜、萝卜、白菜、普通烟和番茄6种植物为首次报道。对胡萝卜进行了回传实验,接种烟粉虱2周后,健康的胡萝卜上出现轻微花叶症状,实时荧光定量PCR方法检测呈SPCSV-WA阳性。
6、通过烟粉虱传毒获得感染SPCSV-WA的烟草植株,以感病烟草植株上收获的种子为材料,利用NCM-ELISA检测种子实生苗的带毒率,共检测了760株烟草,SPCSV-WA的种传率为5.39%。应用实时荧光定量PCR方法检测SPCSV-WA在烟草种子和花器上的分布情况,结果表明烟草种子、花冠、雄蕊、雌蕊、花萼均携带SPCSV。
7、将SPVD甘薯植株的茎蔓作为接穗嫁接到供试甘薯品种上,待其发病表现典型SPVD症状后,将其移栽至海南试验田进行种子繁育。以SPVD甘薯植株上收获的种子为材料,应用NCM-ELISA和实时荧光定量PCR方法检测种子实生苗的传毒率、SPVD甘薯植株的花器及种子不同部位带毒情况。NCM-ELISA方法共检测了1311株实生苗,2012年和2013年SPCSV的种传率分别为4.56%和0.90%,SPFMV的种传率分别为7.49%和2.59%；实时荧光定量PCR检测了55份甘薯实生苗,SPCSV和SPFMV的种传率均为100%。应用实时荧光定量PCR方法检测SPVD甘薯植株的花器及种子不同部位带毒情况,结果表明种皮、胚乳、胚、雄蕊、雌蕊、花萼均携带SPCSV和SPFMV;花冠携带SPFMV,不携带SPCSV.
Sweet potato virus disease (SPVD), which is caused by the synergistic interaction of sweet potato chlorotic stunt virus (SPCSV) and sweet potato feathery mottle virus (SPFMV), is the most serious viral disease of sweet potato (Ipomoea batatas). SPVD causes severe yield loss of about90%, sometimes reaching100%. In China, SPVD is mainly distributed in Guangdong, Jiangsu, Sichuan and Anhui provinces currently since its first report in2010. Furher study on the two pathogens, SPCSV and SPFMV, is necessary to control the rapid spread of SPVD.
SPCSV can be differentiated into two distantly related strains, East African (SPCSV-EA) and West African (SPCSV-WA), The SPCSV-WA strain has a wider geographic distribution in China than the SPCSV-EA strain. Focusing on SPCSV-WA and SPFMV in this study, we cloned and sequenced the genome of Chinese SPCSV-WA isolates, analyzed molecular variation of the Chinese SPCSV-WA isolates, developed two real-time fluorescent quantitative PCR assays to detect SPCSV-WA and SPFMV, studied the host range of SPCSV-WA and seed transmission of SPCSV-WA and SPFMV using real-time PCR and other detection methods. The results showed (summarized below) are an attempt to determine the mechanism of the wide spread of SPVD, which will help to develop disease control strategies.
1. The complete genome sequence of RNA1and RNA2of SPCSV-WA isolated from Jiangsu Province (SPCSV-WA-JS) was cloned using reverse transcriptase PCR (RT-PCR) and rapid amplification of cDNA ends to capture the terminal ends of the viral genomes. The viral RNA was extracted from infected whiteflies and the sequences were deposited in GenBank with accession numbers KC146840and KC146841. Sequence analysis showed that RNA1from the SPCSV-WA-JS isolate was8,637nucleotides (nt) long, including an89-nt5' untranslated region (5'-UTR), a193-nt3'-UTR and four open reading frames (ORFs). The four ORFs in the RNA1at positions90to6053(ORFla),6052to7569(ORFlb),7583to8272(ORF2) and8277to8444t (ORF3), encode the polyprotein, RNA-dependent RNA polymerase (RdRP), RNase3and p7, respectively. RNA2was8,107nt long, including a191-nt5'-UTR, a192-nt3'-UTR, and nine ORFs:nucleotides192to329(p5.2),333to467 (p5),406to534(p5.1),879to2543(heat shock protein70h),2565to4121(p60),4103to4324(p8),4352to5125(major coat protein),5128to7182(minor coat protein) and7187to7915(p28), respectively. Comparing the genome sequence of SPCSV-WA-JS with that of the Can181-9isolate from Spain, RNA1and RNA2showed98.90%and98.68%sequence identity, respectively. The results revealed that the genomic sequences of SPCSV-WA are highly conserved.
2. RT-PCR was used to obtain the nearly full-length sequences of three isolates from Chongqing (SPCSV-WA-CQ) and Sichuan (SPCSV-WA-SC-8, SPCSV-WA-SC-12). The sequences were deposited in GenBank with accession numbers KC888966, KC888963, KC888964, KC888961, KC888965and KC888962. Similarity analysis was performed for the genomic sequences of SPCSV-WA isolates from different regions of China. The analysis showed that the four genomic sequences of SPCSV-WA from Sichuan, Jiangsu and Chongqing shared more than98%nucleotide identity, indicating that SPCSV-WA isolates from China have highly conserved genomic sequences and little molecular variation.
3. Primers and TaqMan probes were designed according to the conserved regions of the coat protein (CP) gene of SPFMV from GenBank. By optimizing the reaction conditions, a specific, sensitive and efficient real-time RT-PCR assay was established to detect SPFMV. The results showed that this assay was specific for detecting the targeted virus:the detection limit was about5.46copies/μL of positive plasmids. The assay was up to two-order more sensitive than conventional PCR. The real-time PCR assay established in this study could be suitable for detecting field samples.
4. Two specific primers and one TaqMan probe were designed according to the nucleotide sequence of SPCSV-WA from GenBank. A recombinant plasmid containing the SPCSV-WA CP gene was used to establish a standard curve. By optimizing the reaction conditions, a real-time RT-PCR assay was established to detect SPCSV-WA. The results showed that this assay was specific for detecting the targeted virus:its detection limit was about3.31copies/μL of the positive plasmid. The sensitivity of the assay was1000times higher than that of conventional PCR. The real-time PCR assay for detecting SPCSV established in this study could be suitable for detecting field samples.
5. To determine the host range, eight test plants of four families were inoculated with SPCSV-WA by whitefly (Bemisia tabaci). SPCSV-WA was detected by symptomatology and real-time PCR. Virus-associated symptoms were induced in four plant species:slight mosaic in Daucus carota and I. aquatica, distorted leaves in I. setosa, slight mosaic and distorted leaves in Solanum lycopersicum. The other four species, Nicotiana tabacum, N. benthamiana, Raphanus sativus and Brassica Chinensis, were symptomless. Using real-time PCR, a positive signal was obtained in all the test plants, revealing that SPCSV-WA could infect all eight species. This is the first report of SPCSV-WA infection in D. carota, I. Aquatica, R. sativus, B. Chinensis, N. tabacum and S. lycopersicum. After SPCSV-WA re-infection of healthy plants with virus-free whitefly, mosaic patterns were observed two weeks later, and SPCSV-WA was also detected in D. carota plants using real-time PCR.
6. SPCSV-WA-infected N. tabacum plants were obtained by whitefly transmission. The seeds, flowers and subsequent seedlings produced from the infected N. tabacum plants were used for virus detection. Nitrocellulose membrane-enzyme linked immunosorbent assay (NCM-ELIS A) was used to detect the virus transmission rate of the seedlings. A total of760N. tabacum seedlings were detected, giving a seed transmission rate of SPCSV-WA of5.39%. Real-time PCR was used to determine the distribution of SPCSV-WA in the seeds and floral organs of N. tabacum. The results showed the seeds, corolla, stamen, pistil and calyx were infected.
7. Plants of several sweet potato cultivars exhibited typical symptoms of SPVD after grafting with the scions of a SPVD-infected sweet potato cultivar. The sweet potato cultivars plants above with typical symptoms of SPVD grew in Hainan Province for mature seed harvesting. Using real-time PCR, SPCSV and SPFMV were both detected in the seed coat, embryo, endosperm, calyx, corolla, stamen and pistil. NCM-ELISA was used to detect the virus transmission rate of the seedlings. A total of1311seedlings were detected, giving seed transmission rates of SPCSV in2012and2013of4.56%and0.90%respectively, and of SPFMV in2012and2013of7.49%and2.59%respectively. Positive signals for SPCSV and SPFMV were detected in55randomly chosen seedlings of sweet potato by real time PCR, suggesting the seed transmission rates of SPCSV and SPFMV were100%. Real-time PCR was used to detect the distribution of SPCSV and SPFMV in the floral organs and different parts of the seeds of sweet potato. The results showed a wide distribution of SPCSV and SPFMV in the episperm, endosperm, stamen, pistil and calyx. SPFMV, but not SPCSV, was distributed in the corolla.
引文
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