【作者】 陶冶；

【导师】 廖金铃；

【作者基本信息】 华南农业大学 ， 植物病理学， 2018， 博士

【摘要】 根结线虫(Meloidogyne spp.)是一种植物固着性内寄生线虫,种类多、寄主范围广、致病性强且在全球分布广泛。了解根结线虫的种类、分布及寄主是制定防治策略的基础。本研究对我国南方7个省市部分地区的果蔬作物上的根结线虫进行了鉴定。在此基础上,对鉴定到的四种根结线虫进行线粒体基因组全长测序,并结合其他线虫的线粒体基因组信息构建了系统发育树,为根结线虫的分类和系统进化研究提供依据。另外,基于线粒体基因组及其他一些序列的信息,构建了奇异根结线虫(M.aberrans)和象耳豆根结线虫(M.enterolobii)的分子快速检测系统。主要研究成果如下:1.结合形态学、同工酶电泳技术和分子生物学的方法,对89个种群的根结线虫进行鉴定,其中62个种群为南方根结线虫(M.incognita),占总样本数的70.8%;12个种群为象耳豆根结线虫,占总样本数的13.5%;11个种群为爪哇根结线虫(M.javanica)占总样本数的11.2%;4个种群为本研究发现的一个新种——奇异根结线虫(Meloidogyne aberrans n.sp.),占总样本数的4.5%。研究表明南方根结线虫是我国南方果蔬作物上的优势种。杨桃和葡萄是象耳豆根结线虫的寄主新纪录。2.描述了根结线虫新种——奇异根结线虫,其形态鉴别特征如下:雌虫会阴部凸起,颈部位于身体侧面,会阴花纹很弱、圆,口针中等长度(13.6-15.5μm);雄虫口针长(18.2-19.6μm),交合刺长(22.7-36.8μm),侧线11-15条;二龄幼虫唇区光滑,侧面观凹陷,口针长(15.9-16.8μm),侧线4条,透明尾极短(2.2-5.5μm)。其与一户町根结线虫(M.ichinohei)形态最相似,但与一户町根结线虫相比,奇异根结线虫的雄虫、雌虫和二龄幼虫口针均更长;雄虫体长更长、尾更短、DGO更小、侧线更多;二龄幼虫侧线更少。奇异根结线虫的酯酶表型为罕见的S2型,其两条带的相对迁移率(Rm)分别是40.5%和44.5%,而苹果酸脱氢酶的表型为常见的N1型。同时,本研究获得了奇异根结线虫的SSU、LSU D2D3、ITS和cox2-16S rRNA的序列并构建了系统发育树。序列比对及系统发育树均表明该线虫与已知的根结线虫分子特征不一致。SSU和LSU的系统发育树显示奇异根结线虫和一户町根结线虫亲缘关系最近,而在ITS和cox2-16S rRNA的系统发育树上,和奇异根结线虫亲缘关系最近的分别是巨大根结线虫(M.megadora)和山茶根结线虫(M.camelliae)。另外,组织病理学研究表明奇异根结线虫诱导取食位点形成4-6个多核的巨细胞。3.利用长片段PCR技术对奇异根结线虫、象耳豆根结线虫、南方根结线虫和爪哇根结线虫的线粒体基因组全长进行测序,四种根结线虫的线粒体全长分别为:17,004bp、17,494 bp、17,543 bp和18,392 bp;四种根结线虫线粒体基因组的22个tRNA均为非典型的三叶草结构,并且都是单拷贝;12个蛋白编码基因的排列方式也均相同,但tRNA的排列方式具有一定的变化;所有基因具有相同的转录方向;此外,非编码区的数量及长度也有一定的变化,其中象耳豆根结线虫具有3个大的非编码区,其余三种根结线虫只有2个大的非编码区;另外,基于12个蛋白编码基因,采用贝叶斯法(Bayesian inference,BI)和最大似然法(Maximum-likehood,ML)构建了线虫系统进化树,结果显示:所有的根结线虫聚在同一支,其中南方根结线虫,爪哇根结线虫,花生根结线虫和象耳豆根结线虫聚在同一分支上,拟禾本科根结线虫和哥伦比亚根结线虫聚在另一分支上,而奇异根结线虫则单独落在基部的分支上,与其他几种根结线虫形成姐妹支的关系;整个根结线虫的分支和伤残短体线虫亲缘关系最接近,而与孢囊线虫的亲缘关系相对较远。4.基于线粒体基因组设计了检测根结线虫的通用引物对Mt-RKN-F/Mt-RKN-R、检测奇异根结线虫的特异引物对Mab-Mt-F/Mab-Mt-R和象耳豆根结线虫的特异引物对Me-Mt-F/Me-Mt-R,基于这些引物构建了检测奇异根结线虫和象耳豆根结线虫的双重PCR体系,该体系检测灵敏度可达到单条线虫。此外,在奇异根结线虫rDNA-ITS区分别设计了实时荧光定量PCR特异引物对Mab-qF/Mab-qR和探针Mab-Probe;在象耳豆根结线虫特异性扩增区(Sequence characterized amplified region,SCAR)分别设计了实时荧光定量PCR特异引物对Me-qF/Me-qR和探针Me-Probe,利用这些引物和探针构建了检测奇异根结线虫和象耳豆根结线虫的的实时荧光定量PCR体系,该体系对单条线虫DNA稀释1000倍后的模板仍可灵敏检测。将两种方法用于不同土壤样本的检测,结果表明:实时荧光定量PCR可以100%检测到靶标线虫,即奇异根结线虫和象耳豆根结线虫,但双重PCR无法检测到某些靶标线虫含量较低的土壤样本。更多还原

【Abstract】 The root-knot nematode(Meloidogyne spp.)is a kind of sedentary endoparasitic nematode with various species,wide host range,great pathogenicity and wide distribution.Investigation and identification of root-knot nematodes is the basis of prevention and control.Therefore,this study identified root-knot nematodes parasitizing fruit and vegetable crops in seven provinces in the south of China.Meantime,mitochondrial genomes from Meloidogyne spp.identified were sequenced.Based on the mitochondrial genomes,phylogenetic trees were constructed,which provided a basis for the classification and evolution research of root-knot nematodes.In addition,according to the mitochondrial genomes and other sequences,the duplex-PCR and qPCR systems were explored to identify M.aberrans and M.enterolobii.The main results are as follows.1.According to morphology,combining with isozymes and molecular analysis,four species of root-knot nematodes were identified from 89 Meloidogyne populations.Of these,62 populations are M.incognita,12 pupolations are M.enterolobii,11 populations are M.javanica,and 4 populations were identified as a new species named M.aberrans n.sp.The proportion of M.incognita,M.enterolobii,M.javanica and M.aberrans in the total samples is 70.8%,13.5%,11.2% and 4.5% respectively.The results showed that M.incognita is the dominant species and carambola and grape are new hosts of M.enterolobii.2.One new root-knot nematode species named Meloidogyne aberrans n.sp.was described.Meloidogyne aberrans sp.n.can be differed from all other Meloidogyne spp.by morphology,isozymes and molecular characters.It is characterized by prominent posterior protuberance,round and faint perineal pattern and a medium-length stylet(13.6-15.5μm)characterized females.Males with stylet 18.2-19.6 μm long,spicules 22.7-36.8μm long and11-15 lateral lines.J2 s were characterized by a smooth lip region with distinct protruded medial lips and a depression in outline at the oral perture,a relatively long stylet(15.9-16.8μm),four incisures in the lateral field and a very short,even not clearly defined,hyaline tail terminus(2.2-5.5 μm).The new is close to M.ichinohei,but can be differed from M.ichinohei by the longer female,male and J2 stylet,the larger male length,the lower DGO of male,the shorter male tail,more lateral lines in males and fewer incisures in the J2 lateral field.The isozyme electrophoretic analysis of M.aberrans n.sp.showed a rare EST phenotype,S2,two Est bands at Rm = 40.5% and 44.5%.The band of MDH peheotype of M.aberrans n.sp.was N1 phenotype.In this study,the sequences of SSU,LSU D2D3,ITS,and cox2-16 S rRNAs of this new species were also obtained to construct the phylogenetic tree that displayed a closed relationship to M.ichinohei on the SSU and LSU D2D3,to M.megadora on the ITS,and to M.camaelliae on the cox2-16 S rRNAs.Histopathological observations on M.aberrans n.sp.indicated that it can induce the formation of 4-6 large multinucleate feeding cells known as giant cells.3.The complete mitochondrial genomes of M.aberrans,M.enterolobii,M.incognita and M.javanica were sequenced by long PCR amplifications.The full length of mitochondrial genomes were 17,004 bp,17,494 bp,17,543 bp and 18,392 bp for M.aberrans,M.enterolobii,M.incognita and M.javanica,respectively.22 tRNA genes of these four Meloidogyne spp.all encode the typical cloverleaf tRNA secondary structure and are all single copy.The arrangement of twelve protein-coding genes are the same,but the arrangement of tRNA is not always same.All genes in mitochondrial genomes have the same transcription direction.In addition,the number and length of non-coding regions vary slightly,for example,there are three non-coding regions in M.enterolobii,but two in other three Meloidogyne species.Based on twelve protein-coding genes,phylogenetic relationships between nematodes were inferred by Bayesian inference(BI)and Maximum like-hood(ML)methods respectively.The phylogenetic trees showed that all Meloidogyne are positioned in the same clade,in which M.incognita,M.javanica,M.arenaria and M.enterolobii cluster together and M.graminicola and M.chitwoodi are in another clade,and M.aberrans is positioned in the basal clade that sister to other Meloidogyne.The clade comprising Meloidogyne is the closet to the root-lesion nematode,Pratylenchus vulnus,butfar from the cyst nematode.4.Based on the mitochondrial genomes,the conserved primers Mt-RKN-F/Mt-RKN-R for detecting root-knot nematodes,the specific primers Mab-Mt-F/Mab-Mt-R for detecting M.aberrans and the specifc primers Me-Mt-F/Me-Mt-R for the detection of M.enterolobii were developed to construct the duplex-PCR systems,which can detect single nematode.Meanwhile,based on the rDNA-ITS region and SCAR(Sequence characterized amplified region)fragment,the specific primers Mab-qF/Mab-qR and the probe Mab-Probe for detecting M.aberrans and the specific primers Me-qF/Me-qR and the probe Me-Probe for detecting M.enterolobii were designed respectively.Based on these primers and probes,the qPCR systems were developed.The qPCR assays could detect the DNA template 1000-fold diluted of a single nematode.In order to evaluate the application of the duplex and real-time PCR systems as diagnostic tool for the detections of M.aberrans and M.enterolobii,rhizosphere soil samples comprising root-knot nematodes and other nematodes were used.The results displayed that the qPCR system can successfully detect the target nematodes,M.aberrans and M.enterolobii,in all samples,but the duplex PCR can not detect the target nematodes in those samples including the low number of target nematodes.更多还原