本文選題：梨輪紋病 + 梨干腐病��； 參考：《華中農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：輪紋病和干腐病是梨產(chǎn)區(qū)常年發(fā)生且為害嚴(yán)重的病害,對(duì)我國(guó)梨產(chǎn)業(yè)造成了嚴(yán)重的威脅。長(zhǎng)久以來,梨輪紋病和干腐病被認(rèn)為是由葡萄座腔菌屬(Botryosphaeria)真菌引起的兩種不同的病害,而對(duì)于我國(guó)梨輪紋病和干腐病病原菌的種類、分布、致病性以及其所攜帶真菌病毒的情況研究甚少。本研究對(duì)來源于我國(guó)不同省市梨產(chǎn)區(qū)分離得到的梨輪紋、干腐病菌進(jìn)行了種類鑒定、致病性測(cè)定,根據(jù)其菌落特征及所含有dsRNA的情況篩選到不同的真菌病毒,并對(duì)真菌病毒的基因組結(jié)構(gòu)、病毒粒子形態(tài)、對(duì)寄主致病力的影響進(jìn)行了詳細(xì)的研究,為進(jìn)一步研究梨樹輪紋病、干腐病發(fā)生規(guī)律及有效利用真菌病毒進(jìn)行防治提供了參考依據(jù),取得的研究結(jié)果如下:從我國(guó)20個(gè)省、市梨主栽區(qū)采集表現(xiàn)輪紋或干腐癥狀的病斑組織,通過組織分離法共得到243份原始菌株,將其中129份經(jīng)過單菌絲純化后得到131株純化菌株。根據(jù)菌株的形態(tài)學(xué)特征并結(jié)合其ITS、β-tubulin和EF1-α序列分析,可將上述純化得到的葡萄座腔菌屬真菌分為Botryosphaeria dothidea、B.rhodina、B.parva和B.obtusa四類。通過分析菌株的種類來源與其分布的關(guān)系,發(fā)現(xiàn)除來源于新疆和甘肅的樣品上沒有分離得到葡萄座腔菌屬真菌外,其它地區(qū)的樣品均分離得到了該屬真菌,但數(shù)量和分類上存在差異,其中B.dothidea為梨上的優(yōu)勢(shì)種類,共計(jì)116株,而且北至吉林,南至云南均有分布,顯示出其對(duì)不同地理環(huán)境的適應(yīng)性。另外B.rhodina、B.parva和B.obtusa菌株數(shù)量分別為2、7和6株。而且菌株的分類與其癥狀之間也存在相關(guān)性,在表現(xiàn)輪紋癥狀的病斑上只分離得到了B.dothidea,而在表現(xiàn)干腐癥狀的病斑上卻分離得到了B.dothidea、B.rhodina、B.parva和B.obtusa。選取B.dothidea、B.rhodina、B.parva和B.obtusa的代表菌株分別接種梨1年生枝條,結(jié)果表明,接種B.dothidea菌株的枝條表現(xiàn)干腐癥狀和疣狀突起癥狀,而接種其它3個(gè)種菌株的枝條只表現(xiàn)干腐癥狀,這些不同種類菌株對(duì)梨枝條的侵染條件也存在差異,其中,B.dothidea和B.parva菌株無傷接種和有傷接種均能導(dǎo)致接種部位產(chǎn)生癥狀,而B.rhodina和B.obtusa菌株只能通過傷口侵染使接種部位表現(xiàn)癥狀。當(dāng)上述菌株接種離體梨成熟果實(shí)時(shí),B.parva、B.rhodina和B.obtusa菌株所造成的病斑顯著大于B.dothidea菌株所產(chǎn)生的病斑。因此,葡萄座腔屬菌的侵染途徑及不同種病菌對(duì)梨的危害可能存在差異。綜上所述,b.dothidea為梨輪紋病和干腐病常見的病原,而b.parva、b.rhodina和b.obtusa只引起梨干腐病。該結(jié)果系統(tǒng)研究了我國(guó)梨上的botryosphaeria病原菌種類的多樣性及其接種后所表現(xiàn)癥狀間的相關(guān)性。通過觀察菌株在pda平板上的形態(tài)特征發(fā)現(xiàn),243株原始菌株中有7株菌株生長(zhǎng)異常,其中4株菌株生長(zhǎng)很慢、氣生菌絲弱且易產(chǎn)生黑色色素;2株菌株的中央菌絲氣生性較弱、邊緣菌絲稀薄,且在邊緣易產(chǎn)生扇變;1株菌株的菌絲擴(kuò)展快、但氣生菌絲消失、且易產(chǎn)生墨綠色的色素。提取了所有原始菌株的dna及dsrna,發(fā)現(xiàn)均沒有dna病毒的侵染,而在表型異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。從分離于河南鄭州的一株氣生菌絲稀少、致病力喪失的菌株yzn115中發(fā)現(xiàn)了一種新的dsrna病毒,將其命名為botryosphaeriadothidearnavirus1(bdrv1/yzn115)。bdrv1/yzn115的基因組包含5條dsrna,大小分別為2397、2184、1967、1131和1060bp,每條dsrna包含1個(gè)開放閱讀框。bdrv1/yzn115和aspergillusfumigatustetramycovirus-1(afutmv-1)有較近的親緣關(guān)系,其中,bdrv1/yzn115的dsrna1-4編碼的蛋白氨基酸序列分別和afutmv-1編碼的相應(yīng)蛋白具有55%、47%、43%和53%的相似性。bdrv1/yzn115的dsrna1、3和4分別編碼依賴rna的rna聚合酶(rdrp),甲基轉(zhuǎn)移酶(methyltransferase)和p-a-s-rich類似蛋白(pasrp-like),而dsrna2和dsrna5編碼的蛋白功能未知。經(jīng)超速離心純化后,bdrv1/yzn115的核酸和蛋白能從產(chǎn)物中獲得,但免疫電鏡結(jié)果表明bdrv1/yzn115沒有形成病毒粒子。bdrv1/yzn115可以通過分生孢子和菌絲進(jìn)行接觸傳播,得到的傳毒衍生菌株也表現(xiàn)出生長(zhǎng)異常及致病力喪失的特性。從分離于湖北武漢的一株氣生菌絲稀少、致病力下降的菌株ew220中發(fā)現(xiàn)了一種雙節(jié)段dsrna病毒,將其命名為botryosphaeriadothideabotybirnavirus1(bdbrv1)。其中,dsrna1大小為6434bp,gc含量為48.1%,該dsrna只包含1個(gè)開放閱讀框(orf1,nt546-6323),編碼1925個(gè)氨基酸,包含1個(gè)rdrp保守結(jié)構(gòu)域;dsrna2大小為5986bp,gc含量為57.1%,包含1個(gè)開放閱讀框(orf2,nt568-5671),編碼1767個(gè)氨基酸。dsrna1和dsrna2的5′-末端非翻譯區(qū)長(zhǎng)度分別為545和567 nt,且這部分序列高度相似,3′-末端非翻譯區(qū)長(zhǎng)度分別為111和115nt,其中有63個(gè)核苷酸序列一致。BdBRV1的dsRNA1和dsRNA2編碼的蛋白序列和Sclerotinia sclerotiorum botybirnavirus 1(SsBRV1)所編碼的蛋白相似性為82%和74%,但相似序列的分布不均勻,其中一些區(qū)域的相似性達(dá)到了90%,而最低區(qū)域只有39%。BdBRV1的病毒粒子為球形,直徑約為37 nm,結(jié)構(gòu)蛋白分子量大小約為100 kDa、90 kDa和80 kDa。BdBRV1能夠降低B.dothidea菌株的生長(zhǎng)速率,且使其致病力下降。從分離于江西鷹潭的一株表現(xiàn)正常的菌株GY25中分離得到了一種dsRNA病毒,將其命名為Botryosphaeria dothidea victorivirus 1(BdV1)。BdV1基因組大小為5322 bp,包含兩個(gè)重疊的開放閱讀框(ORF),其中,ORF1編碼743個(gè)氨基酸(nt512-2743),ORF2編碼840個(gè)氨基酸(nt 2743-5265)。ORF1編碼的氨基酸序列和Rosellinia necatrix victorivirus 1(RnV1)編碼的衣殼蛋白相似性最高,為37%(E value=8e-101),ORF2編碼的氨基酸序列和Beauveria bassiana victorivirus NZL/1980(BbV-NZL/1980)編碼的RNA-directed RNA聚合酶相似性最高,為44%(E value=0)。BdV1在翻譯鏈的5′-端存在一個(gè)小ORF,但所預(yù)測(cè)的氨基酸序列在NCBI網(wǎng)站上沒有比對(duì)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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S436.612
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本文編號(hào)：2116014