桑叶穿孔病叶面生物种群的鉴定及致病性研究

发布时间：2018-01-14 15:19

本文关键词：桑叶穿孔病叶面生物种群的鉴定及致病性研究　出处：《西南大学》2017年硕士论文　论文类型：学位论文

【摘要】：桑树是一种重要的经济作物,其根、茎、叶、果实均具有较大的经济价值,且桑叶是家蚕最好的天然饲料,桑树为蚕桑业带来了丰厚的收入。桑树病害的发生严重制约了蚕桑业的发展。桑穿孔病是近些年来才受到关注的一种桑病,该病害的发生严重影响了桑叶产量和品质以及桑树的生长。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现为叶片穿孔、脱落,同时对枝条和果实也有一定的影响。在2008年有研究报道了桑穿孔病的发生,但桑穿孔病的病因及病原体迄今尚不清楚。2015年8月四川省绵阳市三台县的桑园发生了较大规模的桑树穿孔病,同时还伴有大量微型害虫的危害,这一复杂的生态现象给穿孔病发生的原因的准确诊断增加了难度。本课题组成员于2015年三台县桑园爆发穿孔病时,前往该桑园进行了现场观察和病叶样本的采集,并进一步对受害叶面的微生物进行了分离、纯化和鉴定,对叶面上分布的微型害虫进行了调查和初步分类;根据科赫法则将分离得到的5种疑似病原菌Pantoea agglomerans MB01、Pantoea ananatis MB02、Nigrospora sphaerica MF03、Phomopsis sp.MF05和Epicoccum nigrum MF06回接到健康的桑叶上,以检测疑似病原菌的致病性,将产生穿孔症状的桑叶带回实验室进行病原菌的再分离,并将再分离菌株与3种接种菌株对比分析,确认为桑树穿孔病病原菌。从已验证的3株桑树穿孔病病原菌中选择致病性最强的菌株Nigrospora sphaerica MF03,通过观察其生长周期和温度、pH值、光照条件这三种因素对菌丝生长的影响以及测试菌丝的致死温度来研究菌株的生物学特性。本文的主要研究内容和实验结果如下:1病叶表面微生物的分离鉴定及微型害虫调查根据植物病原微生物的常规分离方法,从桑叶病斑组织分离到了若干菌株,再根据16S或者ITS鉴定为5株细菌,6株真菌。通过对16S rDNA或ITS rDNA基因片段的扩增与测序并构建系统发育树,同时结合形态特征将分离到的细菌菌株分别鉴定并命名为:Pantoea agglomerans MB01、Pantoea ananatis MB02、Pseudomonas oryzihabitans MB03、Pseudomonas argentinensis MB04以及Acinetobacter calcoaceticus MB05;分离到的真菌菌株分别鉴定并命名为:Nigrospora sp.MF01、Nigrospore oryzae MF02、Nigrospora sphaerica MF03、Phanerochaete sordid MF04、Phomopsis sp.MF05和Epicoccum nigrum MF06。此外,本实验中还观察到在病桑叶表面分布着大量的微型害虫,经过统计、调查,可将它们初步鉴定为桑蓟马、桑粉虱、叶螨和叶甲。2疑似病原菌的致病性试验结合文献资料,本研究从分离得到的菌株中选择2株疑似植物致病性细菌:Pantoea agglomerans MB01、Pantoea ananatis MB02以及3株真菌:Nigrospora sphaerica MF03、Phomopsis sp.MF05和Epicoccum nigrum MF06进行科赫试验,将疑似病原菌回接到健康的桑叶表面以检测分析。结果显示,细菌分离株没有引起桑叶发病;而真菌试验分为桑叶创伤和无创伤两种处理,3株真菌接种无创伤处理的桑叶后,虽能低频率地感染桑叶使其产生病斑但均不产生穿孔症状;3株真菌接种创伤处理的桑叶后均能引起桑叶产生典型的穿孔症状,且以菌株Nigrospora sphaerica MF03的致病性最高,在夏秋两季接种桑叶都能引起非常高的穿孔率。另外两株真菌Phomopsis sp.MF05和Epicoccum nigrum MF06在夏季接种桑叶能引发非常高的穿孔率,而在秋季接种桑叶仅能引起桑叶发生病斑却不能出现穿孔现象。同时对接种后产生典型穿孔症状的桑叶进行病原菌的再分离,并将再分离的菌株的ITS rDNA序列、形态特征分别与原接种菌株对比,比较分析结果表明再分离菌株与原接种菌株一致。因此综合以上研究结果可确定这3株真菌Nigrospora sphaerica MF03、Phomopsis sp.MF05和Epicoccum nigrum MF06均能引起桑叶穿孔。3致病性菌株Nigrospora sphaerica MF03的生物学特性研究基于致病性试验结果,选择致病性最高的真菌菌株Nigrospora sphaerica MF03为目的菌株并进一步研究该菌株的生物学特性,以期为桑叶穿孔病的防治提供基础数据与参考。通过检测菌株的生长周期和温度、pH值、光照条件对菌丝生长的影响以及测定菌丝的致死温度,研究结果显示在实验室条件下未观察到菌株Nigrospora sphaerica MF03的有性生殖周期,初步确定该菌通过孢子进行繁殖的。菌丝对生长温度、pH值表现出较大的耐受范围,但培养温度高于35℃或pH值小于2.0时菌丝不能生长;且菌丝生长不依赖光照,其致死温度为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.

【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S888.71

【参考文献】