猕猴桃软腐病病原学研究

发布时间：2018-06-13 00:10

本文选题：猕猴桃软腐病 + Botryosphaeriaceae科　；参考：《四川农业大学》2016年博士论文

【摘要】：四川是猕猴桃种植大省,种植历史悠久,2011年年产量约为13万吨。猕猴桃软腐病是贮藏期危害最为严重的病害,发病迅速且难于防治。有关猕猴桃软腐病的研究尚处于初始阶段,其病原种类、致病机制和发病果实的生理物质变化等报道甚少。基于以上原因,本论文对该病病原物进行了系统研究并获得以下结果：从四川省苍溪、都江堰、名山、彭州、邛崃和双流等地的发病猕猴桃果实上共分离到135株Botryosphaeriaceae科真菌。根据病原菌的形态特征以及内转录间隔区(internal transcribed space,ITS)、翻译延长因子(transcription elongation factor 1-a, TEF)和p-微管蛋白(β-tubulin, BT)序列将病原菌鉴定为Botryosphaeria dothidea、Lasiodiplodia theobromae和Neofusicoccum parvum,其中L. theobromae和N. parvum是第一次报道为该病的病原物。研究也发现B. dothidea能以分生孢子器和假囊壳越冬,越冬的分生孢子和子囊孢子与从果实上分离的3种病原菌(B. dothidea、L. theobromae和N. parvum)一样,均能危害果实、叶片和枝条,表明越冬的B. dothidea分生孢子和子囊孢子为猕猴桃软腐病的初侵染源。病原菌生物学特性研究结果表明,3种病原菌(Botryosphaeria dothidea、Lasiodiplodia theobromae和Neofusicoccum parvum)的菌丝生长和孢子萌发的最适温度为25-30℃。菌丝生长最适pH值为5-8,而孢子萌发最适pH值为6-8。不同病原菌对碳源和氮源的利用程度不尽相同。Neofusicoccum parvum的最适碳源是木糖,Botryosphaeria dothidea和Lasiodiplodia theobromae的最适碳源为葡萄糖。氮源利用试验表明,NH4Cl为N. parvum和B. dothidea的最适氮源,而L. theobromae的最适氮源为谷氨酸。对37株Botryosphaeria dothidea,2株Lasiodiplodia theobromae和13株Neofusicoccum parvum,共52株分离物进行致病力测定和遗传多样性分析。结果表明它们均能造成果实发病,但致病力有明显差异。对于B. dothidea种类来说,弱致病力菌株占29.73%(11株),中致病力菌株占43.24%(16株),强致病力菌株占27.03%(10株)。同样,中致病力菌株在N. parvum类群中所占比例也最高(53.85%),弱致病力菌株占7.69%(1株),强致病力菌株占38.46%(5株)。2株L. theobromae均为强致病力菌株。利用简单重复序列区间扩增多态性标记(inter-simple sequence repeats, ISSR)和相关序列扩增多态性标记(Sequence Related Amplified Polymorphism, SRAP)分析这52株病原菌的多样性。9个ISSR引物共扩增出128条谱带,其中多态性条带为86条,占总条带数的67.19%,每对引物平均获得9.56条。8对SRAP引物组合共扩增出114条谱带,其中多态性条带为78条,占总条带数的68.42%,每对引物平均获得9.75条。在遗传距离约0.645处,9个ISSR分子标记可将猕猴桃软腐病菌划分为3个大类：类群Ⅰ所有菌株为B. dothidea(37个菌株)；类群Ⅱ所有菌株均为N. parvum(括13个菌株)；而类群Ⅲ只有2个菌株,它们均为L. theobromae。在0.798处类群Ⅰ分为5个组。在0.708处类群Ⅱ分为3个组。在0.996处类群Ⅲ各分为两个菌株,均为L. theobromae。8对SRAP分子标记在遗传距离约0.608处,可将猕猴桃软腐病菌划分为2个大类：类群Ⅰ所有菌株为B. dothidea(37个菌株)；类群Ⅱ包括13个N. parvum菌株和2个L.theobromae菌株。在0.779处类群Ⅰ分为7个组,在0.77处类群Ⅱ分为6个组,其中1至5个组均由N. parvum菌株构成,第6个组由两株L. theobromae菌株构成。综合结果表明,来源于四川省6个地区的猕猴桃软腐病菌的遗传多样性与致病性强弱及地理来源并无明显相关关系。对猕猴桃软腐菌致病因子研究结果表明,B. dothidea能在PDA、Fries与Czapek培养液中产生毒素,但在Fries与Czapek培养液中产生的毒素活性更强。在光暗交替振荡条件下病原菌产生的毒素活性明显强于黑暗振荡培养中产生的毒素活性。利用丙酮提取的毒素活性高于用甲醇+氯仿提取的活性。致病性测定结果表明,B. dothidea、 Lasiodiplodia theobromae和Neofusicoccum parvum产生的毒素、果胶酶及纤维素酶能对猕猴桃果实和叶片产生伤害。根据Neofusicoccum parvum的目的纤维素酶基因和看家基因序列,设计了ubiquitin conjugating enzyme (ubcB)、Beta-tubulin (β-tub)和RNA polymerase iii transcription factor (TFC1)3个看家基因的特异性引物,和6对跨内含子和1对不跨内含子(基因无内含子)的纤维素酶基因的特异性引物。结果表明,由这3个看家基因作为参照分别计算出来的7个纤维素酶基因的表达量趋势高度一致,但是由TFC1作为参照计算出来的7个纤维素酶基因表达量均高于其它两个看家基因作为参照时的表达量。在6d的侵染过程中,UCRNP2_2427和UCRNP2_7122纤维素酶基因的表达量为先下降后上升再下降又再上升的W型趋势,UCRNP2_2356、UCRNP2_5067和UCRNP2_7847纤维素酶基因的表达量趋势为先下降后上升,呈U型趋势,UCRNP2_3883的表达量趋势为先上升后下降的倒V型,而UCRNP2_8897的表达量趋势则为先下降再上升然后再下降。对于7个基因来说,UCRNP2_2427基因的表达量在第1d最低,而UCRNP2_2356最高；UCRNP2_5067的表达量在第2、3和4d最低,而UCRNP2_3883表达量在第2d最高,但是UCRNP2_2356在第3和4d最高；UCRNP2_2427和UCRNP2_5067在第5d表达量一样,均为最低,而在同一天UCRNP2_2356的表达量最高；在第6d时,UCRNP2_2356表达量最高,而UCRNP2_3883表达量最低。根据B. dothidea的ITS序列设计了一对用于特异扩增该病原菌的引物BZY-1/BZY-2。除B. dothidea能扩增出目标片段外,该对引物对参试的其它25属真菌均不能扩增出相应的条带。该引物不仅能从接种B. dothidea的果实里扩增出特异性条带,并且也能从自然发病的果实中扩增出B. dothidea特有的条带,而健康果实则未有任何条带。对水杨酸和生防菌Lecythophora luteoviridis发酵液诱导猕猴桃抗软腐病的研究结果表明,水杨酸和L. luteoviridis的无菌发酵液浸泡猕猴桃果实后,能使果实发病时间延迟。利用水杨酸和L. luteoviridis发酵液诱导果实后,在6d内连续测量猕猴桃果实超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)、脯氨酸(Pro)、丙二醛(MDA)的变化结果表明,这5种物质在不同处理中有不同的变化趋势。但是大多数情况下接种病原菌的果实CAT、POD、SOD、Pro和MDA含量均比不接种病原菌的高。蒸馏水浸泡果实后接种病原菌的果实CAT、POD、SOD、Pro和MDA在大多数情况下均比其它处理的高。
[Abstract]:Sichuan is a big province of gooseberry planting, with a long history of planting, and its annual output is about 130 thousand tons. The soft rot of kiwi fruit is the most serious disease in the storage period. The disease is very fast and difficult to prevent and control. The research on the soft rot of kiwi fruit is still in the initial stage. The species of the pathogen, the pathogenesis and the physiological changes of the fruit are reported very much, and the changes of the physiological substances of the fruit are reported very much. Based on the above reasons, a systematic study of the disease origin was carried out in this paper and the following results were obtained: from Cangxi, Dujiangyan, Dujiangyan, Mingshan, Pengzhou, Qionglai and Shuangliu, 135 Botryosphaeriaceae families were isolated from the fruit of Actinidia. According to the morphological characteristics of the pathogenic bacteria and the internal transcriptional interval (internal TRA) Nscribed space, ITS), the sequence of transcription elongation factor 1-A, TEF and p- microtubule protein (beta -tubulin, BT) identified the pathogenic bacteria as Botryosphaeria, which were first reported as the pathogen of the disease. Dothidea can be overwintered with the conidium and pseudocyst, and the overwintering conidia and osporospore, like the 3 pathogenic bacteria (B. dothidea, L. theobromae and N. parvum) separated from the fruit, can all harm the fruit, leaves and branches, indicating that the overwintered B. dothidea spore and osporospores are the primary infection sources of the soft rot of Actinidia. The results of biological characteristics of the original bacteria showed that the optimum temperature for mycelial growth and spore germination of 3 pathogenic bacteria (Botryosphaeria dothidea, Lasiodiplodia theobromae and Neofusicoccum parvum) was 25-30. The optimum pH value of mycelium growth was 5-8, and the optimum pH value of spores germination was that the use degree of different pathogens to carbon and nitrogen sources was not good. The optimum carbon source for the same.Neofusicoccum parvum is xylose, the optimum carbon source of Botryosphaeria dothidea and Lasiodiplodia theobromae is glucose. The nitrogen source test shows that NH4Cl is the optimum nitrogen source for N. parvum and B. dothidea, while the most suitable nitrogen source for L. is 37. The pathogenicity and genetic diversity of 52 isolates of theobromae and 13 strains of Neofusicoccum parvum were analyzed. The results showed that they all cause fruit onset, but the pathogenicity of B. dothidea was significantly different. For the B. dothidea species, 29.73% (11 strains) were weak pathogenic strains, 43.24% (16) were pathogenic strains, and 27.03 of strong pathogenic bacteria were found. Also, the proportion of pathogenic strains in N. parvum group was also the highest (53.85%), the weak pathogenic strain accounted for 7.69% (1 strains), the strong pathogenic strain accounted for 38.46% (5 strains).2 strain L. theobromae as the strong pathogenic strain. Using the simple repeat sequence interval amplification polymorphisms markers (inter-simple sequence repeats, ISSR) and the related sequence Sequence Related Amplified Polymorphism (SRAP) was used to analyze the diversity of these 52 strains of pathogenic bacteria, and 128 bands were amplified by.9 ISSR primers, of which 86 bands were polymorphic bands, accounting for 67.19% of the total band number. The average of 9.56.8 for each pair of primers was 9.56 bands, and 114 bands were amplified by the combination of 9.56.8. The 78, accounting for 68.42% of the total number of bands, averaged 9.75 primers per pair. At the genetic distance of about 0.645, 9 ISSR markers could divide the soft rot fungus of kiwi fruit into 3 major categories: all the strains of group I were B. dothidea (37 strains); all of the group II strains were N. parvum (including 13 strains); and group III only 2 strains, it The L. theobromae. was divided into 5 groups in 0.798 groups I divided into 3 groups in 0.708 groups. 0.996 groups were divided into two strains in 0.996 groups, all of which were labeled by L. theobromae.8 at the genetic distance of about 0.608, and could be divided into 2 groups: all the strains of group I were B. dothidea (37 strains). Group II included 13 N. parvum strains and 2 L.theobromae strains. In 0.779 groups I divided into 7 groups, and 0.77 groups were divided into 6 groups, 1 to 5 were composed of N. parvum strains and sixth groups were composed of two L. theobromae strains. The comprehensive results showed that the remains of Actinidia soft rot pathogens from 6 regions of Sichuan Province B. dothidea can produce toxins in PDA, Fries and Czapek culture, but the toxins produced in Fries and Czapek cultures are more active. The toxins produced by pathogenic bacteria under the alternate light and dark oscillations are the results of research on pathogenic factors of Actinidia chinensis soft rot fungi. The toxicity of the toxin produced by the acetone was higher than that extracted by the methanol + chloroform. The pathogenicity test showed that the toxins produced by B. dothidea, Lasiodiplodia theobromae and Neofusicoccum parvum, pectinase and fibrinase could harm the fruit and leaves of kiwi fruit Based on the target cellulase gene of Neofusicoccum parvum and the sequence of the housekeeping gene, the specific primers for ubiquitin conjugating enzyme (ubcB), Beta-tubulin (beta -tub) and RNA polymerase III transcription were designed, and 6 pairs of trans introns and 1 pairs of non introns (genes without introns) were used for cellulose. The specific primers of the enzyme gene showed that the expression of 7 cellulase genes, calculated by these 3 housekeeping genes as a reference, was highly consistent, but the expression of 7 cellulase genes calculated by TFC1 as a reference were higher than that of the other two housekeeping groups as a reference. The infection in 6D During the process, the expression of UCRNP2_2427 and UCRNP2_7122 cellulase genes decreased first and then decreased and then increased again. The trend of the expression of UCRNP2_2356, UCRNP2_5067 and UCRNP2_7847 genes was decreased first and then increased, showing the trend of U type, and the trend of UCRNP2_3883's apparent amount was the inverted V type which was first increased and then decreased, and UCRNP2_8, and UCRNP2_8, and UCRNP2_8, and UCRNP2_8, and UCRNP2_8, and UCRNP2_8. For the 7 genes, the expression of the UCRNP2_2427 gene was the lowest in 1D and the highest in UCRNP2_2356; the expression of UCRNP2_5067 was the lowest in 2,3 and 4D, while the UCRNP2_3883 expression was highest in 2D, but UCRNP2_2356 was highest in third and 4D; UCRNP2_2427 and UCRNP2_5067 were at the highest level. The expression of 5D was the same as the lowest, and the highest expression of UCRNP2_2356 on the same day; at 6D, the expression of UCRNP2_2356 was the highest and the UCRNP2_3883 expression was the lowest. A pair of primers for specific amplification of the pathogen, BZY-1/BZY-2. except B. dothidea, could be amplified out of the target fragment, and the primers were primed. The other 25 genera fungi of the 25 genera can not amplify the corresponding bands. The primers can not only amplify the specific bands from the fruits of the inoculated B. dothidea, but also amplify the unique bands of B. dothidea from the naturally occurring fruit, while the healthy fruits have no bands. The salicylic acid and the biocontrol bacteria Lecythophora luteoviridis are not found. The results of the fermentation broth induced anti soft rot of kiwi fruit showed that the time of fruit onset was delayed after the aseptic fermentation broth of salicylic acid and L. luteoviridis was soaked in kiwi fruit. After the fruit was induced by salicylic acid and L. luteoviridis fermentation broth, the fruit of actinidia fruit was continuously measured in 6D, and the peroxidase (POD) was measured continuously in the fruit of actinidia fruit. The changes of catalase (CAT), proline (Pro) and malondialdehyde (MDA) showed that the 5 substances had different trends in different treatments. But in most cases, the fruit CAT, POD, SOD, Pro and MDA were higher than those of non inoculated pathogens in the fruits inoculated with pathogens. The fruit inoculated with pathogenic bacteria after distilled water was inoculated with pathogens, CAT, POD, SOD, Pro. And MDA in most cases are higher than others.
【学位授予单位】：四川农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S436.634.1

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