本文關(guān)鍵詞：桑葉穿孔病葉面生物種群的鑒定及致病性研究　出處：《西南大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 桑樹 穿孔病 微型害蟲 Nigrospora sphaerica 生物多樣性

【摘要】：桑樹是一種重要的經(jīng)濟作物,其根、莖、葉、果實均具有較大的經(jīng)濟價值,且桑葉是家蠶最好的天然飼料,桑樹為蠶桑業(yè)帶來了豐厚的收入。桑樹病害的發(fā)生嚴重制約了蠶桑業(yè)的發(fā)展。桑穿孔病是近些年來才受到關(guān)注的一種桑病,該病害的發(fā)生嚴重影響了桑葉產(chǎn)量和品質(zhì)以及桑樹的生長。植物穿孔病主要由病原微生物引起的,感病植物的典型病征表現(xiàn)為葉片穿孔、脫落,同時對枝條和果實也有一定的影響。在2008年有研究報道了桑穿孔病的發(fā)生,但桑穿孔病的病因及病原體迄今尚不清楚。2015年8月四川省綿陽市三臺縣的桑園發(fā)生了較大規(guī)模的桑樹穿孔病,同時還伴有大量微型害蟲的危害,這一復(fù)雜的生態(tài)現(xiàn)象給穿孔病發(fā)生的原因的準確診斷增加了難度。本課題組成員于2015年三臺縣桑園爆發(fā)穿孔病時,前往該桑園進行了現(xiàn)場觀察和病葉樣本的采集,并進一步對受害葉面的微生物進行了分離、純化和鑒定,對葉面上分布的微型害蟲進行了調(diào)查和初步分類;根據(jù)科赫法則將分離得到的5種疑似病原菌Pantoea agglomerans MB01、Pantoea ananatis MB02、Nigrospora sphaerica MF03、Phomopsis sp.MF05和Epicoccum nigrum MF06回接到健康的桑葉上,以檢測疑似病原菌的致病性,將產(chǎn)生穿孔癥狀的桑葉帶回實驗室進行病原菌的再分離,并將再分離菌株與3種接種菌株對比分析,確認為桑樹穿孔病病原菌。從已驗證的3株桑樹穿孔病病原菌中選擇致病性最強的菌株Nigrospora sphaerica MF03,通過觀察其生長周期和溫度、pH值、光照條件這三種因素對菌絲生長的影響以及測試菌絲的致死溫度來研究菌株的生物學(xué)特性。本文的主要研究內(nèi)容和實驗結(jié)果如下:1病葉表面微生物的分離鑒定及微型害蟲調(diào)查根據(jù)植物病原微生物的常規(guī)分離方法,從桑葉病斑組織分離到了若干菌株,再根據(jù)16S或者ITS鑒定為5株細菌,6株真菌。通過對16S rDNA或ITS rDNA基因片段的擴增與測序并構(gòu)建系統(tǒng)發(fā)育樹,同時結(jié)合形態(tài)特征將分離到的細菌菌株分別鑒定并命名為:Pantoea agglomerans MB01、Pantoea ananatis MB02、Pseudomonas oryzihabitans MB03、Pseudomonas argentinensis MB04以及Acinetobacter calcoaceticus MB05;分離到的真菌菌株分別鑒定并命名為:Nigrospora sp.MF01、Nigrospore oryzae MF02、Nigrospora sphaerica MF03、Phanerochaete sordid MF04、Phomopsis sp.MF05和Epicoccum nigrum MF06。此外,本實驗中還觀察到在病桑葉表面分布著大量的微型害蟲,經(jīng)過統(tǒng)計、調(diào)查,可將它們初步鑒定為桑薊馬、桑粉虱、葉螨和葉甲。2疑似病原菌的致病性試驗結(jié)合文獻資料,本研究從分離得到的菌株中選擇2株疑似植物致病性細菌:Pantoea agglomerans MB01、Pantoea ananatis MB02以及3株真菌:Nigrospora sphaerica MF03、Phomopsis sp.MF05和Epicoccum nigrum MF06進行科赫試驗,將疑似病原菌回接到健康的桑葉表面以檢測分析。結(jié)果顯示,細菌分離株沒有引起桑葉發(fā)病;而真菌試驗分為桑葉創(chuàng)傷和無創(chuàng)傷兩種處理,3株真菌接種無創(chuàng)傷處理的桑葉后,雖能低頻率地感染桑葉使其產(chǎn)生病斑但均不產(chǎn)生穿孔癥狀;3株真菌接種創(chuàng)傷處理的桑葉后均能引起桑葉產(chǎn)生典型的穿孔癥狀,且以菌株Nigrospora sphaerica MF03的致病性最高,在夏秋兩季接種桑葉都能引起非常高的穿孔率。另外兩株真菌Phomopsis sp.MF05和Epicoccum nigrum MF06在夏季接種桑葉能引發(fā)非常高的穿孔率,而在秋季接種桑葉僅能引起桑葉發(fā)生病斑卻不能出現(xiàn)穿孔現(xiàn)象。同時對接種后產(chǎn)生典型穿孔癥狀的桑葉進行病原菌的再分離,并將再分離的菌株的ITS rDNA序列、形態(tài)特征分別與原接種菌株對比,比較分析結(jié)果表明再分離菌株與原接種菌株一致。因此綜合以上研究結(jié)果可確定這3株真菌Nigrospora sphaerica MF03、Phomopsis sp.MF05和Epicoccum nigrum MF06均能引起桑葉穿孔。3致病性菌株Nigrospora sphaerica MF03的生物學(xué)特性研究基于致病性試驗結(jié)果,選擇致病性最高的真菌菌株Nigrospora sphaerica MF03為目的菌株并進一步研究該菌株的生物學(xué)特性,以期為桑葉穿孔病的防治提供基礎(chǔ)數(shù)據(jù)與參考。通過檢測菌株的生長周期和溫度、pH值、光照條件對菌絲生長的影響以及測定菌絲的致死溫度,研究結(jié)果顯示在實驗室條件下未觀察到菌株Nigrospora sphaerica MF03的有性生殖周期,初步確定該菌通過孢子進行繁殖的。菌絲對生長溫度、pH值表現(xiàn)出較大的耐受范圍,但培養(yǎng)溫度高于35℃或pH值小于2.0時菌絲不能生長;且菌絲生長不依賴光照,其致死溫度為50℃。
[Abstract]:Mulberry is an important economic crop, its root, stem, leaf, fruit has great economic value, and mulberry silkworm is the best natural feed, mulberry sigiificant revenue for sericulture. Occurrence of mulberry disease has seriously hampered the development of sericulture industry. Mulberry perforation disease is a kind of mulberry diseases in recent years to get attention, the occurrence of the disease seriously affects the yield and quality of mulberry and mulberry growth. Plants caverned disease caused by pathogenic microorganism, the typical symptoms of susceptible plants showed that leaf shedding, perforation, branches and fruit at the same time also has a certain influence. In 2008 reported that mulberry perforation of the occurrence of disease, but the etiology and pathogen of mulberry perforation are unclear.2015 years in Mianyang city in August three counties in Sichuan province had larger scale in the mulberry mulberry perforation disease, harm is accompanied by a large number of micro pests, This complex ecological phenomenon to increase the difficulty of accurate diagnosis of disease cause of perforation. The members of the project group in 2015 three county mulberry disease outbreak of perforation to the mulberry, on-site observation and leaf samples collected, and further to the victims in the foliar microbial isolation, purification and identification. The micro distribution of foliar pests were investigated and preliminary classification; according to the 5 suspected pathogen Pantoea MB01 Koch agglomerans law will be isolated, Pantoea ananatis MB02, Nigrospora sphaerica MF03, Phomopsis sp.MF05 and Epicoccum nigrum MF06 get healthy mulberry leaf, to detect pathogenic suspected pathogenic bacteria, then the separation will produce the symptoms of the mulberry leaves perforation back to the laboratory for pathogenic bacteria, and then compared with 3 isolates were inoculated with strain analysis, identified as pathogen. From mulberry perforation has been verified Choose the most pathogenic strain Nigrospora sphaerica MF03 pathogen of perforation in 3 strains of mulberry, by observing the growth cycle and temperature, pH value, the three factors of light condition to study the temperature effect on mycelial growth of lethal strains and the biological characteristics of the mycelium test. The main content of research and experiment results in this paper microbial diseases are as follows: 1 the surface of the leaf and micro pest survey according to conventional separation methods isolation and identification of plant pathogenic microorganisms, isolated from mulberry leaf lesion tissue of some strains, according to the 16S or ITS were identified as 5 strains of bacteria, 6 strains of fungi by amplification of 16S rDNA or ITS rDNA gene fragment and sequencing and system construction at the same time, phylogenetic tree, bacterial strains isolated were identified and named as Pantoea agglomerans MB01 combined with morphological characteristics: Pantoea, ananatis MB02, Pseudomonas oryzihabitans MB03, Pseudom Onas argentinensis MB04 and Acinetobacter calcoaceticus MB05; fungal strains isolated were identified and named as Nigrospora sp.MF01, Nigrospore oryzae MF02, Nigrospora sphaerica MF03, Phanerochaete sordid MF04, Phomopsis sp.MF05 and Epicoccum nigrum MF06. in addition, this study also observed in disease of mulberry leaf surface distribution of a large number of micro pests, through investigation, statistics, they can be identified as Mulberry Thrips, mulberry whitefly, mites and pathogenicity tests Chrysomela.2 suspected pathogenic bacteria combined with the literature, this study from the isolated strains in 2 strains of suspected plant pathogenic bacteria: Pantoea agglomerans MB01, Pantoea ananatis MB02 and sphaerica MF03 of 3 strains of fungi: Nigrospora Phomopsis, sp.MF05 Epicoccum and nigrum MF06 of the Koch test, the suspected pathogens were returned to the mulberry leaves to the detection and analysis of surface health . the results showed that the strains did not cause disease of mulberry bacterial isolation; and fungi were divided into invasive and noninvasive mulberry two treatment, mulberry leaf fungal inoculation of 3 strains of non invasive treatment, although the infection rate of the low frequency of mulberry leaves to produce lesions but no perforation symptoms; 3 strains of fungi with trauma treated mulberry after the mulberry leaves can cause typical symptoms of perforation, and the strain Nigrospora sphaerica MF03 pathogenicity highest, can cause the perforation rate very high in the summer two season. The other two strains of fungi inoculated leaves of Phomopsis sp.MF05 and Epicoccum nigrum MF06 can cause the perforation rate very high in the summer with mulberry leaves, mulberry leaf spot caused by the occurrence of but can not appear in the autumn leaves inoculated with perforation phenomenon can only be produced at the same time. Then the typical symptoms of perforation from mulberry leaves of pathogenic bacteria on after inoculation, and then isolates ITS rDNA sequence, Morphological characteristics and primary inoculation respectively compared, comparison analysis showed that isolates and inoculated with strains and consistent. Therefore the above research results can determine which of the 3 strains of fungi Nigrospora sphaerica MF03, Phomopsis sp.MF05 and Epicoccum nigrum MF06 can induce the biological characteristics of the pathogenic strains of Nigrospora.3 leaf perforation sphaerica MF03 pathogenicity test results based on choose the highest, pathogenic fungal strains Nigrospora sphaerica MF03 strain and further investigate the biological characteristics of this strain, in order to provide basic data and reference for the prevention and treatment of perforation mulberry leaves. The measurement of strain growth cycle and temperature, pH value, illumination conditions on the mycelial growth of mycelium and determination of lethal temperature. Research results show that under laboratory conditions was not observed in sphaerica strain Nigrospora MF03 sexual reproduction period It was preliminarily determined that the bacterium was propagated through spores. The hypha showed a larger tolerance to the growth temperature and pH value. However, when the culture temperature was higher than 35 C or the pH value was less than 2, the hypha could not grow, and the mycelial growth did not depend on light. The lethal temperature was 50 degrees.

【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S888.71

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