梨轮纹病与干腐病的病原关系及轮纹病菌携带真菌病毒多样性研究

发布时间：2018-07-11 17:24

  本文选题：梨轮纹病 + 梨干腐病　； 参考：《华中农业大学》2016年博士论文

【摘要】：轮纹病和干腐病是梨产区常年发生且为害严重的病害,对我国梨产业造成了严重的威胁。长久以来,梨轮纹病和干腐病被认为是由葡萄座腔菌属(Botryosphaeria)真菌引起的两种不同的病害,而对于我国梨轮纹病和干腐病病原菌的种类、分布、致病性以及其所携带真菌病毒的情况研究甚少。本研究对来源于我国不同省市梨产区分离得到的梨轮纹、干腐病菌进行了种类鉴定、致病性测定,根据其菌落特征及所含有dsRNA的情况筛选到不同的真菌病毒,并对真菌病毒的基因组结构、病毒粒子形态、对寄主致病力的影响进行了详细的研究,为进一步研究梨树轮纹病、干腐病发生规律及有效利用真菌病毒进行防治提供了参考依据,取得的研究结果如下:从我国20个省、市梨主栽区采集表现轮纹或干腐症状的病斑组织,通过组织分离法共得到243份原始菌株,将其中129份经过单菌丝纯化后得到131株纯化菌株。根据菌株的形态学特征并结合其ITS、β-tubulin和EF1-α序列分析,可将上述纯化得到的葡萄座腔菌属真菌分为Botryosphaeria dothidea、B.rhodina、B.parva和B.obtusa四类。通过分析菌株的种类来源与其分布的关系,发现除来源于新疆和甘肃的样品上没有分离得到葡萄座腔菌属真菌外,其它地区的样品均分离得到了该属真菌,但数量和分类上存在差异,其中B.dothidea为梨上的优势种类,共计116株,而且北至吉林,南至云南均有分布,显示出其对不同地理环境的适应性。另外B.rhodina、B.parva和B.obtusa菌株数量分别为2、7和6株。而且菌株的分类与其症状之间也存在相关性,在表现轮纹症状的病斑上只分离得到了B.dothidea,而在表现干腐症状的病斑上却分离得到了B.dothidea、B.rhodina、B.parva和B.obtusa。选取B.dothidea、B.rhodina、B.parva和B.obtusa的代表菌株分别接种梨1年生枝条,结果表明,接种B.dothidea菌株的枝条表现干腐症状和疣状突起症状,而接种其它3个种菌株的枝条只表现干腐症状,这些不同种类菌株对梨枝条的侵染条件也存在差异,其中,B.dothidea和B.parva菌株无伤接种和有伤接种均能导致接种部位产生症状,而B.rhodina和B.obtusa菌株只能通过伤口侵染使接种部位表现症状。当上述菌株接种离体梨成熟果实时,B.parva、B.rhodina和B.obtusa菌株所造成的病斑显著大于B.dothidea菌株所产生的病斑。因此,葡萄座腔属菌的侵染途径及不同种病菌对梨的危害可能存在差异。综上所述,b.dothidea为梨轮纹病和干腐病常见的病原,而b.parva、b.rhodina和b.obtusa只引起梨干腐病。该结果系统研究了我国梨上的botryosphaeria病原菌种类的多样性及其接种后所表现症状间的相关性。通过观察菌株在pda平板上的形态特征发现,243株原始菌株中有7株菌株生长异常,其中4株菌株生长很慢、气生菌丝弱且易产生黑色色素;2株菌株的中央菌丝气生性较弱、边缘菌丝稀薄,且在边缘易产生扇变;1株菌株的菌丝扩展快、但气生菌丝消失、且易产生墨绿色的色素。提取了所有原始菌株的dna及dsrna,发现均没有dna病毒的侵染,而在表型异常的菌株中均提取到了dsrna,在表型正常的菌株中有95株b.dothidea提取到了dsrna,其余菌株均未提取到dsrna。从分离于河南郑州的一株气生菌丝稀少、致病力丧失的菌株yzn115中发现了一种新的dsrna病毒,将其命名为botryosphaeriadothidearnavirus1(bdrv1/yzn115)。bdrv1/yzn115的基因组包含5条dsrna,大小分别为2397、2184、1967、1131和1060bp,每条dsrna包含1个开放阅读框。bdrv1/yzn115和aspergillusfumigatustetramycovirus-1(afutmv-1)有较近的亲缘关系,其中,bdrv1/yzn115的dsrna1-4编码的蛋白氨基酸序列分别和afutmv-1编码的相应蛋白具有55%、47%、43%和53%的相似性。bdrv1/yzn115的dsrna1、3和4分别编码依赖rna的rna聚合酶(rdrp),甲基转移酶(methyltransferase)和p-a-s-rich类似蛋白(pasrp-like),而dsrna2和dsrna5编码的蛋白功能未知。经超速离心纯化后,bdrv1/yzn115的核酸和蛋白能从产物中获得,但免疫电镜结果表明bdrv1/yzn115没有形成病毒粒子。bdrv1/yzn115可以通过分生孢子和菌丝进行接触传播,得到的传毒衍生菌株也表现出生长异常及致病力丧失的特性。从分离于湖北武汉的一株气生菌丝稀少、致病力下降的菌株ew220中发现了一种双节段dsrna病毒,将其命名为botryosphaeriadothideabotybirnavirus1(bdbrv1)。其中,dsrna1大小为6434bp,gc含量为48.1%,该dsrna只包含1个开放阅读框(orf1,nt546-6323),编码1925个氨基酸,包含1个rdrp保守结构域;dsrna2大小为5986bp,gc含量为57.1%,包含1个开放阅读框(orf2,nt568-5671),编码1767个氨基酸。dsrna1和dsrna2的5′-末端非翻译区长度分别为545和567 nt,且这部分序列高度相似,3′-末端非翻译区长度分别为111和115nt,其中有63个核苷酸序列一致。BdBRV1的dsRNA1和dsRNA2编码的蛋白序列和Sclerotinia sclerotiorum botybirnavirus 1(SsBRV1)所编码的蛋白相似性为82%和74%,但相似序列的分布不均匀,其中一些区域的相似性达到了90%,而最低区域只有39%。BdBRV1的病毒粒子为球形,直径约为37 nm,结构蛋白分子量大小约为100 kDa、90 kDa和80 kDa。BdBRV1能够降低B.dothidea菌株的生长速率,且使其致病力下降。从分离于江西鹰潭的一株表现正常的菌株GY25中分离得到了一种dsRNA病毒,将其命名为Botryosphaeria dothidea victorivirus 1(BdV1)。BdV1基因组大小为5322 bp,包含两个重叠的开放阅读框(ORF),其中,ORF1编码743个氨基酸(nt512-2743),ORF2编码840个氨基酸(nt 2743-5265)。ORF1编码的氨基酸序列和Rosellinia necatrix victorivirus 1(RnV1)编码的衣壳蛋白相似性最高,为37%(E value=8e-101),ORF2编码的氨基酸序列和Beauveria bassiana victorivirus NZL/1980(BbV-NZL/1980)编码的RNA-directed RNA聚合酶相似性最高,为44%(E value=0)。BdV1在翻译链的5′-端存在一个小ORF,但所预测的氨基酸序列在NCBI网站上没有比对到任何具有同源性的已知蛋白。
[Abstract]:Rotten disease and dry rot are perennial and seriously harmful diseases in Pear producing areas, causing serious threat to the pear industry in China. For a long time, pear rotation and dry rot are considered to be two different diseases caused by Botryosphaeria fungi. There are few studies on the pathogenicity of the pathogen and the case of its fungal virus. This study identified the type of pear wheel lines and dry rot pathogens isolated from the pear producing areas of different provinces and cities of China. The pathogenicity of the pathogen was determined by screening the different fungal viruses according to their colony characteristics and the conditions contained in dsRNA, and the genome of the fungal virus. The effects of virus particle morphology on host pathogenicity were studied in detail, which provided reference for further study of pear tree ring disease, the occurrence regularity of dry rot disease and the effective use of fungal virus. The results obtained are as follows: from 20 provinces in China, the disease spot groups that show the symptoms of wheel or dry rot in the main pear planting areas of the city are collected. 243 original strains were obtained by tissue separation method, and 129 of them were purified by single mycelium, and 131 strains were purified. According to the morphological characteristics of the strains and combining with its ITS, beta -tubulin and EF1- alpha sequence analysis, the above purified Staphylococcus fungi can be divided into Botryosphaeria dothidea, B.rhodina, B.parva and B.o. Btusa four. By analyzing the relationship between the species origin and the distribution of strains, it was found that there were no isolates from Xinjiang and Gansu, and the fungi in other regions were isolated from other regions, but the number and classification were different, and the B.dothidea was the dominant species in the pear, a total of 116 strains. The distribution of the north to Jilin and south to Yunnan showed its adaptability to different geographical conditions. In addition, the number of B.rhodina, B.parva and B.obtusa strains were 2,7 and 6, and the classification of the strains was also correlated with their symptoms. Only B.dothidea was isolated on the disease spots showing the symptoms of wheel striae, and it showed dry rot. B.dothidea, B.rhodina, B.parva and B.obtusa. selected the representative strains of B.dothidea, B.rhodina, B.parva and B.obtusa to inoculate the 1 year old branches of pear, respectively. The results showed that the branches of the isolates showed dry rot and wart shape, and the branches inoculated with other 3 species showed only dry rot. There are also differences in the infection conditions of these different strains to the pear branches, among which, both B.dothidea and B.parva strains without injury and inoculation can cause the symptoms of the inoculation site, while the B.rhodina and B.obtusa strains can only show the symptoms of the inoculated sites through the infection of the wound. When the above strain is inoculated to the ripe fruit of the pear, B The disease spots caused by.Parva, B.rhodina and B.obtusa were significantly greater than those produced by the B.dothidea strain. Therefore, there may be differences in the route of infection and the harm of different kinds of pathogens to the pear. To sum up, b.dothidea is a common pathogen of pear Rome and dry rot, and b.parva, b.rhodina and b.obtusa only cause pears. This result systematically studied the variety of Botryosphaeria pathogenic bacteria on the pear and the correlation between the symptoms after inoculation. By observing the morphological characteristics of the strains on the PDA plate, it was found that 7 of the 243 strains of the original strain grew abnormally, of which 4 strains were slow to grow, and the mycelium was weak and easily produced black. Color pigment; the central mycelium of the 2 strains was weak, the marginal mycelium was thin and the fan changed easily on the edge. The mycelium of the 1 strains was fast, but the mycelium disappeared, and the ink green pigment was easily produced. The DNA and dsRNA of all the original strains were extracted. All of the strains were found to have no DNA virus infection, but all of the strains with abnormal phenotypes were extracted. DsRNA, 95 strains of b.dothidea were extracted from normal strains to dsRNA, and the other strains were not extracted from dsrna. from a strain isolated from Zhengzhou, Henan, and a new dsRNA virus was found in the strain yzn115, which was named botryosphaeriadothidearnavirus1 (bdrv1/yzn115).Bdrv1/yzn115. The genome contains 5 dsRNA, which are 2397218419671131 and 1060bp respectively. Each dsRNA contains 1 open reading frames.Bdrv1/yzn115 and aspergillusfumigatustetramycovirus-1 (afutmv-1), and the dsrna1-4 encoded protein amino acid sequence of bdrv1/yzn115 and the corresponding afutmv-1 encoded protein are 55%, respectively. The dsrna1,3 and 4 of the similarity.Bdrv1/yzn115 of 47%, 43% and 53% encode RNA polymerase (RdRp), methyltransferase (methyltransferase) and p-a-s-rich similar protein (pasrp-like), respectively, while dsrna2 and dsrna5 encoded protein function is unknown. After speeding centrifugation, bdrv1/yzn115 nucleic acid and protein can be obtained from the product, but free from the products. The results of the epidemic electron microscopy showed that bdrv1/yzn115 did not form a viral particle.Bdrv1/yzn115 through the conidium and mycelium, and the virus derived strains also showed abnormal growth and loss of pathogenicity. A strain isolated from Wuhan, Hubei, was found in a strain of ew220, a strain of hyphae and a decrease in pathogenicity. The dsRNA virus is named botryosphaeriadothideabotybirnavirus1 (bdbrv1), in which the size of dsrna1 is 6434bp and the GC content is 48.1%. The dsRNA contains only 1 open reading frames (ORF1, nt546-6323), encoding 1925 amino acids, containing 1 RdRp conserved domains, dsrna2 size is 5986bp, and contains 1 open reading. Frame (ORF2, nt568-5671), the length of the 5 '- terminal untranslated region encoding 1767 amino acids.Dsrna1 and dsrna2 is 545 and 567 NT, respectively, and this part is highly similar, and the length of the 3' - terminal non translation region is 111 and 115nt, respectively, of which 63 nucleotide sequences are consistent with the dsRNA1 and dsRNA2 encoded protein sequences of.BdBRV1 and Sclerotinia sclerotio. The protein similarity encoded by rum botybirnavirus 1 (SsBRV1) was 82% and 74%, but the distribution of similar sequences was uneven, the similarity of some regions reached 90%, while the lowest region only 39%.BdBRV1 virus particles were spherical, the diameter was about 37 nm, the molecular weight of structural proteins was about 100 kDa, 90 kDa and 80 kDa.BdBRV1 could reduce B.dot. The growth rate of hidea strain and its pathogenicity decreased. A dsRNA virus was isolated from a strain GY25 isolated from Yingtan, Jiangxi, which was named Botryosphaeria dothidea victorivirus 1 (BdV1).BdV1 genome size of 5322 BP, including two overlapping open reading frames (ORF), including ORF1 coding 743. The amino acid sequences encoded by 840 amino acids (NT 2743-5265).ORF1 and the Rosellinia necatrix victorivirus 1 (RnV1) encoded capsid protein are the highest, which are 37% (E value=8e-101), ORF2 encoded amino acid sequence and Beauveria encoded.ORF1. The similarity of Ted RNA polymerase is the highest, and 44% (E value=0).BdV1 has a small ORF at the 5 '- end of the translation chain, but the predicted amino acid sequence is not compared to any known homologous protein on the NCBI site.
【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S436.612
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本文编号：2116014