【作者】 都业娟；

【导师】 向本春； 黄家风；

【作者基本信息】 石河子大学 ， 作物栽培学与耕作学， 2013， 博士

【摘要】 本文对危害新疆番茄的病毒病及植原体病害进行了研究：通过田间调查，明确新疆加工番茄病毒病的危害症状可分为花叶、蕨叶和坏死条斑三种类型，其中坏死条斑病毒病发病晚，但危害最严重。针对该病害，2007-2009年对石河子地区不同播期的加工番茄(里格87-5)进行发病率和病情指数调查，结果显示：晚播(5月20日)的发病率(67.94%)和病情指数(43.47)，明显高于早播(4月5日)和适播(4月25日)，三个播期间病情指数差异显著；对15个加工番茄品种(系)发病情况调查分析显示，15个品种(系)对病毒病均无显著抗性，其中‘里格87-5’、‘石番15’、‘屯河34’、‘石番18’4个品种(系)田间发病较重(病情指数为64.03～65.77)；‘新番9’、‘新番4’、‘Q027’、‘石番3’4个品种(系)田间发病相对较轻(病情指数为44.02～45.47)。建立烟草花叶病毒（TMV）、番茄花叶病毒（ToMV）、黄瓜花叶病毒（CMV）、马铃薯Y病毒（PVY）及蚕豆萎蔫病毒（BBWV）五种病毒的有效检测体系。对15个加工番茄品种(系)的种子进行检测结果显示：11个品种(系)可检测到ToMV，说明种子带毒是ToMV田间发病的初侵染来源之一；2009-2012对400份田间病样进行5种病毒的分子检测，显示ToMV的侵染率(88.75%)远高于TMV的侵染率(2.50%)；田间发病主要以ToMV与另2种病毒(CMV+PVY、CMV+BBWV、PVY+BBWV)的复合侵染为主，侵染率分别为28.25%、20.75%和15.25%。对表现坏死条斑的病株通过生物学分离得到ToMV分离物XJT-1、BBWV分离物XJ14-3和CMV分离物SFQT1-2，并对其进行了全长克隆和基因组序列分析。结果显示：XJT-1基因组全长6383个核苷酸(FN985165)，与Genebank中已登录的ToMV序列相似性高达98.7%～99.3%。XJ14-3基因组RNA1全长5955个核苷酸(FN985164)，编码一个1870个氨基酸的多聚蛋白，与Genebank中已登录的BBWV2基因组RNA1序列相似性高达78.6%～92.4%；RNA2全长3635个核苷酸(HQ283389)，编码一个1064个氨基酸的多聚蛋白，与Genebank中已登录的BBWV2基因组RNA2序列相似性高达79.9%～97.4%。CMV分离物SFQT1-2基因组包括三条正单链RNA，基因组RNA1全长为3356个核苷酸(HQ283392)，编码1a蛋白，与Genebank中已登录的CMV ⅠA亚组代表株系基因组RNA1序列相似性最高；基因组RNA2全长3042个核苷酸(HQ283391)，编码2a和2b蛋白；基因组RNA3全长2219个核苷酸(HQ283393)，编码移动蛋白（MP）和外壳蛋白（CP），SFQT1-2基因组RNA2和RNA3与CMV亚组ⅠB代表株系的序列相似性高。CP基因核酸序列系统发生树分析表明，SFQT1-2属于CMV亚组ⅠB。为明确危害新疆喀什温室番茄的病毒种类，利用双生病毒兼并引物对表现黄化曲叶症状的番茄植株进行PCR检测，8株番茄样品均扩增到约500bp的特异片段，从中选取分离物KS2-5克隆测序。结果显示该片段由543个核苷酸组成，与番茄黄化曲叶病毒(TYLCV) DNA-A相似性达99.5%。对KS2-5进行全基因组DNA-A的克隆和测序，KS2-5DNA-A全长为2781个核苷酸(JQ807735)，具有典型的双生病毒基因组特征，与TYLCV各分离物同源性达98.6%～99.5%，而与其他双生病毒的序列相似性却低于75.4%。由此表明，新疆喀什温室番茄黄化曲叶病由TYLCV引起。为明确表现丛枝、小叶、花器变形的加工番茄病原种类，利用植原体通用引物R16mF2/R16mR1对2株显症番茄植株(SYC-1，ZYT-3)的16S rRNA基因进行PCR扩增，均扩增出约1400bp的目标条带，测序结果表明，二者序列相似性达99.8%，均与三叶草丛簇组(16SrⅥ)成员相似性最高(高于98.4%)；利用iPhyClassifier在线分析表明，番茄植原体16S rRNA基因的RFLP图谱与16SrⅥ-A亚组代表株AY39021完全相同。基于16S rRNA基因的核酸序列系统发生树分析表明，二者均与16SrⅥ-A亚组分离物位于同一亚分枝上。这说明侵染加工番茄的植原体属于三叶草丛簇组(16SrⅥ)A亚组。更多还原

【Abstract】 The study on tomato disease caused by virus and phytoplawmas had been done:Three types of viral symptoms showing mosaic, fern leaf and necrotic streak were found on processingtomato in Xingjiang through field investigation. Tomato necrotic streak disease causes the most seriousdamage although it occurs at the latest. Effect of sowing date on tomato virus disease was investigated inShihezi in2007-2009. The results showed that there are significant differences among three sowing dates.The incidence rate(67.94%) and disease index(43.47) of processing tomato sowing on May20th(latesowing period)are higher than those of processing tomato sowing on April5th (early sowing period) or onApril25th (proper sowing period).15processing tomato cultivars resistance to virus diseases wereinvestigated. The result showed there existed definite differences in resistance among cultivars, althoughall tested cultivars had no obvious resistance to virus diseases. Four cultivars of ‘Lige-87-5’,‘Shifan15’,‘Tunhe4’ and ‘Shifan18’ showed relatively high disease index (64.03～65.77) in the field while the fourcultivars of ‘Xinfan9’,‘Xinfan4’,‘Q027’ and ‘Shifan3’ showed relatively low disease index (44.02～45.47) in the field.Detection method for Tobacco mosaic virus (TMV), Tomato mosaic virus (ToMV), Cucumber mosaicvirus (CMV), Potato virus Y (PVY) and Broad bean wilt virus (BBWV) was established, respectively.Seeds of15processing tomato cultivares were used to detect for the five viruses. The results showed thatonly ToMV can be detected from11cultivares seeds. It concluded that seed-borne ToMV is one of theprimary infection sources. More than400infected plants collected from fields were detected for species ofvirus during2009-2012. It showed that plants infected by ToMV (88.75%) were more than that infected byTMV (2.50%) and processing tomato virus diseases mainly resulted from the mixed infection of ToMVand other two viruses (CMV+PVY、CMV+BBWV、PVY+BBWV).Isolate XJT-1of ToMV、isolate XJ14-3of BBWV2and isolate SFQT1-2of CMV were obtained bybiological method from processing tomato plant showing necrosis streak symptoms. Genome sequences ofthe three isolates were cloned and sequenced. Genomic complete sequences of isolate XJT-1wascomprised of6383nucleotides (FN915865)and had the highest nucleotide sequence identities(98.7%-99.3%) with sequence of ToMV from Genebank. Genomic RNA1and RNA2of isolate XJ14-3were determined. RNA1consisted of5955nucleotides (FN985164) encoding a polyprotein with1870amino acids residues and shared78.6%-92.4%nucleotide sequence identities with RNA1of other BBWV2.RNA2consisted of3635nucleotides (HQ283389) encoding a polyprotein of1064amino acids residues andshared79.9%～97.4%nucleotide sequence identities with RNA2of other BBWV2. The genome of CMVisolate SFQT1-2contained three positive single stranded RNA. RNA1consisted of3356nucleotides(HQ283392) encoding1a protein and had the highest sequence identities with typical strain which belongsto CMV subgroup Ⅰ A. RNA2consisted of3042nucleotides (HQ283391) encoding2a protein and2bprotein. RNA3contained of2219nucleotides (HQ283393) encoding movement protein (MP) and coat protein (CP). RNA2and RNA3of isolate SFQT1-2shared high sequence identities with typical strainwhich belongs to CMV subgroup Ⅰ B.Phylogenetic analysis of CP revealed that SFQT1-2belongs to CMVsubgroup IB．Tomato plants with yellow leaf curl symptom collected from greenhouses in Kaishi were detected byPCR using universal primers of geminivirus to determine virus species. Amplified products about500bpwere amplified from8tomato samples, and KS2-5was chosen to clone and sequence. The sequencesconsisted of543nucleotides. It shared99.5%nucleotide sequence identities with DNA-A of Tomatoyellow leaf curl virus (TYLCV). Isolate KS2-5was chosen to determine complete sequence of DNA-A, itcomprised2781nucleotides (JQ807735)and had typical genomic organization of begomoviruses.Sequence comparison showed that DNA-A of KS2-5had98.6%-99.5%sequence identities with those ofTYLCV isolates, while less than75.4%identities with other begomoviruses. It was concluded that tomatoyellow leaf curl disease was caused by TYLCV in Xinjiang Kashi.Two tomato plants (SYC-1，ZYT-3) showing arbuscular, little leaf and floral organ deformity weredetedcted for phytoplasmal16S rRNA genes using PCR with universal primers R16mF2/R16mR1. DNAfragments of about1400bp were amplified from the two pants and sequenced. The results showed theyshared99.8%nucleotide sequence identities each other and had the highest nucleotide sequence identities(98.4%) with Clover proliferation group (16SⅥ). The16S rRNA gene of tomato phytoplsama had identicalpatterns with typical strain (AY39021) of subgroup16SⅥ-A with online analysis of iPhyClassifier forgenomic of16S rRNA. Phylogenetic analysis with genomic of16S rRNA gene showed they were locatedthe same branch with subgroup16SⅥ-A. All these results indicated that the phytoplsama associated withtomato belongs to the subgroup A of ‘Clover proliferation group’(16SⅥ).更多还原