Genome Sequencing, Detection Method And Seed Transmission Rate Of Pathogens Responsible For Sweet Potato Virus Disease (SPVD)

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.