本文選題：獼猴桃軟腐病 + Botryosphaeriaceae科�。� 參考：《四川農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：四川是獼猴桃種植大省,種植歷史悠久,2011年年產(chǎn)量約為13萬(wàn)噸。獼猴桃軟腐病是貯藏期危害最為嚴(yán)重的病害,發(fā)病迅速且難于防治。有關(guān)獼猴桃軟腐病的研究尚處于初始階段,其病原種類、致病機(jī)制和發(fā)病果實(shí)的生理物質(zhì)變化等報(bào)道甚少�；谝陨显�,本論文對(duì)該病病原物進(jìn)行了系統(tǒng)研究并獲得以下結(jié)果：從四川省蒼溪、都江堰、名山、彭州、邛崍和雙流等地的發(fā)病獼猴桃果實(shí)上共分離到135株Botryosphaeriaceae科真菌。根據(jù)病原菌的形態(tài)特征以及內(nèi)轉(zhuǎn)錄間隔區(qū)(internal transcribed space,ITS)、翻譯延長(zhǎng)因子(transcription elongation factor 1-a, TEF)和p-微管蛋白(β-tubulin, BT)序列將病原菌鑒定為Botryosphaeria dothidea、Lasiodiplodia theobromae和Neofusicoccum parvum,其中L. theobromae和N. parvum是第一次報(bào)道為該病的病原物。研究也發(fā)現(xiàn)B. dothidea能以分生孢子器和假囊殼越冬,越冬的分生孢子和子囊孢子與從果實(shí)上分離的3種病原菌(B. dothidea、L. theobromae和N. parvum)一樣,均能危害果實(shí)、葉片和枝條,表明越冬的B. dothidea分生孢子和子囊孢子為獼猴桃軟腐病的初侵染源。病原菌生物學(xué)特性研究結(jié)果表明,3種病原菌(Botryosphaeria dothidea、Lasiodiplodia theobromae和Neofusicoccum parvum)的菌絲生長(zhǎng)和孢子萌發(fā)的最適溫度為25-30℃。菌絲生長(zhǎng)最適pH值為5-8,而孢子萌發(fā)最適pH值為6-8。不同病原菌對(duì)碳源和氮源的利用程度不盡相同。Neofusicoccum parvum的最適碳源是木糖,Botryosphaeria dothidea和Lasiodiplodia theobromae的最適碳源為葡萄糖。氮源利用試驗(yàn)表明,NH4Cl為N. parvum和B. dothidea的最適氮源,而L. theobromae的最適氮源為谷氨酸。對(duì)37株Botryosphaeria dothidea,2株Lasiodiplodia theobromae和13株Neofusicoccum parvum,共52株分離物進(jìn)行致病力測(cè)定和遺傳多樣性分析。結(jié)果表明它們均能造成果實(shí)發(fā)病,但致病力有明顯差異。對(duì)于B. dothidea種類來(lái)說(shuō),弱致病力菌株占29.73%(11株),中致病力菌株占43.24%(16株),強(qiáng)致病力菌株占27.03%(10株)。同樣,中致病力菌株在N. parvum類群中所占比例也最高(53.85%),弱致病力菌株占7.69%(1株),強(qiáng)致病力菌株占38.46%(5株)。2株L. theobromae均為強(qiáng)致病力菌株。利用簡(jiǎn)單重復(fù)序列區(qū)間擴(kuò)增多態(tài)性標(biāo)記(inter-simple sequence repeats, ISSR)和相關(guān)序列擴(kuò)增多態(tài)性標(biāo)記(Sequence Related Amplified Polymorphism, SRAP)分析這52株病原菌的多樣性。9個(gè)ISSR引物共擴(kuò)增出128條譜帶,其中多態(tài)性條帶為86條,占總條帶數(shù)的67.19%,每對(duì)引物平均獲得9.56條。8對(duì)SRAP引物組合共擴(kuò)增出114條譜帶,其中多態(tài)性條帶為78條,占總條帶數(shù)的68.42%,每對(duì)引物平均獲得9.75條。在遺傳距離約0.645處,9個(gè)ISSR分子標(biāo)記可將獼猴桃軟腐病菌劃分為3個(gè)大類：類群Ⅰ所有菌株為B. dothidea(37個(gè)菌株)；類群Ⅱ所有菌株均為N. parvum(括13個(gè)菌株)；而類群Ⅲ只有2個(gè)菌株,它們均為L(zhǎng). theobromae。在0.798處類群Ⅰ分為5個(gè)組。在0.708處類群Ⅱ分為3個(gè)組。在0.996處類群Ⅲ各分為兩個(gè)菌株,均為L(zhǎng). theobromae。8對(duì)SRAP分子標(biāo)記在遺傳距離約0.608處,可將獼猴桃軟腐病菌劃分為2個(gè)大類：類群Ⅰ所有菌株為B. dothidea(37個(gè)菌株)；類群Ⅱ包括13個(gè)N. parvum菌株和2個(gè)L.theobromae菌株。在0.779處類群Ⅰ分為7個(gè)組,在0.77處類群Ⅱ分為6個(gè)組,其中1至5個(gè)組均由N. parvum菌株構(gòu)成,第6個(gè)組由兩株L. theobromae菌株構(gòu)成。綜合結(jié)果表明,來(lái)源于四川省6個(gè)地區(qū)的獼猴桃軟腐病菌的遺傳多樣性與致病性強(qiáng)弱及地理來(lái)源并無(wú)明顯相關(guān)關(guān)系。對(duì)獼猴桃軟腐菌致病因子研究結(jié)果表明,B. dothidea能在PDA、Fries與Czapek培養(yǎng)液中產(chǎn)生毒素,但在Fries與Czapek培養(yǎng)液中產(chǎn)生的毒素活性更強(qiáng)。在光暗交替振蕩條件下病原菌產(chǎn)生的毒素活性明顯強(qiáng)于黑暗振蕩培養(yǎng)中產(chǎn)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。致病性測(cè)定結(jié)果表明,B. dothidea、 Lasiodiplodia theobromae和Neofusicoccum parvum產(chǎn)生的毒素、果膠酶及纖維素酶能對(duì)獼猴桃果實(shí)和葉片產(chǎn)生傷害。根據(jù)Neofusicoccum parvum的目的纖維素酶基因和看家基因序列,設(shè)計(jì)了ubiquitin conjugating enzyme (ubcB)、Beta-tubulin (β-tub)和RNA polymerase iii transcription factor (TFC1)3個(gè)看家基因的特異性引物,和6對(duì)跨內(nèi)含子和1對(duì)不跨內(nèi)含子(基因無(wú)內(nèi)含子)的纖維素酶基因的特異性引物。結(jié)果表明,由這3個(gè)看家基因作為參照分別計(jì)算出來(lái)的7個(gè)纖維素酶基因的表達(dá)量趨勢(shì)高度一致,但是由TFC1作為參照計(jì)算出來(lái)的7個(gè)纖維素酶基因表達(dá)量均高于其它兩個(gè)看家基因作為參照時(shí)的表達(dá)量。在6d的侵染過程中,UCRNP2_2427和UCRNP2_7122纖維素酶基因的表達(dá)量為先下降后上升再下降又再上升的W型趨勢(shì),UCRNP2_2356、UCRNP2_5067和UCRNP2_7847纖維素酶基因的表達(dá)量趨勢(shì)為先下降后上升,呈U型趨勢(shì),UCRNP2_3883的表達(dá)量趨勢(shì)為先上升后下降的倒V型,而UCRNP2_8897的表達(dá)量趨勢(shì)則為先下降再上升然后再下降。對(duì)于7個(gè)基因來(lái)說(shuō),UCRNP2_2427基因的表達(dá)量在第1d最低,而UCRNP2_2356最高；UCRNP2_5067的表達(dá)量在第2、3和4d最低,而UCRNP2_3883表達(dá)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表達(dá)量一樣,均為最低,而在同一天UCRNP2_2356的表達(dá)量最高；在第6d時(shí),UCRNP2_2356表達(dá)量最高,而UCRNP2_3883表達(dá)量最低。根據(jù)B. dothidea的ITS序列設(shè)計(jì)了一對(duì)用于特異擴(kuò)增該病原菌的引物BZY-1/BZY-2。除B. dothidea能擴(kuò)增出目標(biāo)片段外,該對(duì)引物對(duì)參試的其它25屬真菌均不能擴(kuò)增出相應(yīng)的條帶。該引物不僅能從接種B. dothidea的果實(shí)里擴(kuò)增出特異性條帶,并且也能從自然發(fā)病的果實(shí)中擴(kuò)增出B. dothidea特有的條帶,而健康果實(shí)則未有任何條帶。對(duì)水楊酸和生防菌Lecythophora luteoviridis發(fā)酵液誘導(dǎo)獼猴桃抗軟腐病的研究結(jié)果表明,水楊酸和L. luteoviridis的無(wú)菌發(fā)酵液浸泡獼猴桃果實(shí)后,能使果實(shí)發(fā)病時(shí)間延遲。利用水楊酸和L. luteoviridis發(fā)酵液誘導(dǎo)果實(shí)后,在6d內(nèi)連續(xù)測(cè)量獼猴桃果實(shí)超氧化物歧化酶(SOD)、過氧化物酶(POD)、過氧化氫酶(CAT)、脯氨酸(Pro)、丙二醛(MDA)的變化結(jié)果表明,這5種物質(zhì)在不同處理中有不同的變化趨勢(shì)。但是大多數(shù)情況下接種病原菌的果實(shí)CAT、POD、SOD、Pro和MDA含量均比不接種病原菌的高。蒸餾水浸泡果實(shí)后接種病原菌的果實(shí)CAT、POD、SOD、Pro和MDA在大多數(shù)情況下均比其它處理的高。
[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.
【學(xué)位授予單位】：四川農(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)】：S436.634.1

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