Isolation and Characterization of Endophytic Bacillus methylotrophicus LH-L3 Antagonist of Cotton Verticillium Wilt

The aim of this study is to obtain endophytic Bacillus with control effect on cotton Verticillium wilt. [Method] The cotton tissue grinding liquid was heated at 80 ℃, and spread onto the Luria-Bertani ager medium. The strains with better inhibitory effects on Verticillium dahliae Vd084, a strain with strong pathogenicity, was identified by the morphology, physiological and biochemical traits as well as 16S rRNA, gyrB and rpoB sequences, and its control effect on cotton Verticillium wilt was determined by pot experiments. [Result] 61 similar Bacillus strains were isolated from cotton plants. Among them, 17 strains could inhibit Verticillium dahliae Vd084. A strain named LH-L3 with a relatively higher antagonistic activity and a broad inhibition spectrum was obtained in the second screening. LH-L3 was classified into Bacillus methylotrophicus. Compared with the control, the emergence rate, plant height, root length, fresh weight of aerial part and underground part of cotton with 106 mL^－1 LH-L3 suspension drenching for 3 times increased by 42.85%, 10.24%, 23.83%, 10.05% and 97.62%, respectively. And control effect on Verticillium wilt was 85.24%. [Conclusion] LH-L3 had good growth promotion and Verticillium wilt control effects and showed good potential for biological control.     

Physiological Mechanism of Different Varieties and Potassium Application Amounts on Cotton Resistance to Verticillium Wilt

[Objective] It has theoretical guiding significance for the comprehensive control technology of Verticillium wilt of cotton to explore the effect of potassium nutrition on mechanism of cotton Verticillium wilt resistance under the condition of pathogen stress of Verticillium dahliae. [Method] Three cotton cultivars Jimian 11 (JM-11 as susceptible cultivar), Zhongzhimian 2 (ZZM-2 as control cultivar), Nongda 601 (ND-601 as resistant cultivar) were adopted, and each cultivar had 0, 75, 150, 225, 300 kg· hm－2 potassium levels represented by K0, K75, K150, K225, K300. Two factors split plot experiment was designed under artificial nursery inoculated Verticillium dahlia, to observe disease index of Verticillium wilt, and researched resistance associated substances content and enzyme activity. [Result] (1) The lignin, total phenol contents and POD, PAL activity in leaves or roots of JM-11 were more greatly influenced by Verticillium wilt compared with ZZM-2, ND-601; the crucial disease resistance substances and enzymes were lignin, total phenol and PAL for JM-11, total phenol for ZZM-2 and ND-601. (2) The lignin, total phenol contents and POD, PAL activities of JM-11 were more sensitive to root potassium than ND-601, but weaker than that of ZZM-2. The sensitivity of POD, PAL activities in functional leaves of JM-11 to potassium were more sensitive than ND-601 and ZZM-2. (3) The best effect of potash fertilizer for relieving disease index for JM-11, ZZM-2, ND-601 were 32.5%, 26.4%, 16.5%, and corresponding potassium application amounts were 290.5 kg·hm－2, 253.5 kg·hm－2 and 229.5 kg·hm－2, respectively. [Conclusion] Application of potassium could increase the resistance to Verticillium wilt and reduce the occurrence of cotton Verticillium wilt.     

棉花黄萎病拮抗细菌2-70（Bacillus subtilis）菌株定殖能力的研究

试验通过灭菌土和非灭菌土两种土样,试验前分别对这两种土样进行三种不同的处理,即：用拮抗菌芽孢液浸种,土壤全部混菌,部分混菌进行盆栽试验。主要通过盆栽试验,考察棉花黄萎病拮抗细菌2—70在土壤中的定植情况及在棉花根内和根际的定植情况。结果表明,在实验室条件下,拮抗细菌2．70能够在灭菌土和非灭菌土中定殖,且定殖数量达10^6cfu／g土左右,经过五个月后仍能保持较高的抑菌活性,并且菌株在灭菌土中的定殖数量高于非灭菌土中的数量。通过对棉苗根内细菌的回收,证实拮抗细菌2．70能在棉花根内定殖,数量达到10^3cfu／g。     

Analysis of Cotton Secondary Metabolites under the Interaction between Verticillium dahliae and Upland Cotton

[Objective] The aim of this study is to investigate the cotton differential metabolites in the interaction between Verticillium dahliae and cotton plant, and to explore new clues for the further study on the defense mechanism of cotton against V. dahliae. [Method] To obtain the cotton root, stem and leaf samples of the pathogen treated and healthy controls, the upland cotton cultivar CCRI 24 was selected as tested cotton material in this paper, and inoculated with V. dahliae conidia or sterile water by injuring root at the 2-true-leaf stage. The 70% (volume fraction) methanol extracts of these samples were separated and detected by ultra-performance liquid chromatography-electrospray ionization-mass spectrometry (UPLC-ESI-MS), and the metabolite data were obtained by the online XCMS software. Through multivariate statistics and student’s t-test, the differential metabolites were investigated. The types of metabolites were putatively identified based on the comparison of the experimental molecular mass and the monoisotopic accurate molecular mass of cotton metabolites. [Result] The efficiency of negative ion mode was higher than that of positive ion detection mode in the cotton metabolites in UPLC-ESI-MS analysis. 576 ions mainly distributed in cotton root were found in the cotton seedling tissues including root, stem and leaf, which were the distinguished differential metabolites between V. dahliae treatment and healthy control. Among them, 77 ions were identified as sesquiterpenoids, diterpenoids, flavonoids, carbohydrates, aliphatics and phenols. In addition to sesquiterpenes, 17 compounds, including caffeic acid, astragalin, isoastragalin, dillenetin, ent-catechin, gossypetin 8-rhamnoside, gossypicyanin, herbacetin 7-glucoside, leucocyanidin, quercetin 3'-glucoside, quercetin 3-glucoside, quercetin 7-glycosides, α,2',3,3',4,4',6-heptahydroxychalcone 2'-glucoside, melibiose, sucrose, sucrose 6-phosphate and 1-tetratriacontanol, had not been reported in the literatures on the interaction between V. dahliae and cotton, which may be the novel pathogenesis-related metabolites on cotton Verticillium wilt. [Conclusion] The putative pathogenesis-related metabolites of cotton Verticillium wilt may play an important role in the interaction of cotton defense against V. dahliae, which provides an important clue for exploring the new resistant mechanism of cotton to Verticillium wilt.     

一株棉花黄萎病拮抗芽孢细菌的分离鉴定及活性检测

 采用改良的琼脂平板扩散法,通过初筛和复筛,得到一株对棉花黄萎病病原菌大丽轮枝菌（Verticillium dahliae）具有较强的抑菌活性的拮抗细菌Z-5菌株。通过对该菌株的形态特征观察、生理生化鉴定和16S rDNA的序列分析,确定拮抗细菌Z-5菌株为Bacillus malacitensis。盆栽实验表明Z-5菌株对大丽轮枝菌的防治效果达到76.05%,对棉花黄萎病具有明显的防治作用。     

陆地棉植株组织结构和生化代谢与黄萎病抗性的关系

通过对8个陆地棉品种的植株组织结构、酶活性和根系分泌物的比较分析,研究陆地棉黄萎病抗性机制。研究结果表明,陆地棉抗病品种根和茎的导管细胞壁厚、直径小、数目多,髓射线数目多、单位面积薄壁细胞数多,有助于抵御棉花黄萎病菌的侵入与扩展。过氧化物酶（POD）活性与棉花抗黄萎病的关系不明显,但苯丙氨酸解氨酶（PAL     

Pathogenicity Differentiation and Pathotype Distribution of Verticillium dahliae from Cotton in Hunan Province

[Objective] The study of pathogenicity differentiation of Verticillium dahliae in the cotton growing areas in Hunan province could provide theories for breeding and promoting resistant varieties as well as integrated control of Verticillium wilt of cotton. [Method] Seventy seven strains of V. dahliae from major cotton growing areas in Hunan province were evaluated their cultural characteristics. The growth rate, sporulation quantity and pathogenicity of 31 strains among them were tested and SAS 9.1.3 statistical analysis software was used to cluster the pathogenicity of the tested strains. The special primer (D-1/D-2 and ND-1/ND-2) for polymerase chain reaction assay was used to test pathotype of 24 strains. [Result] According to their colony morphology on potato dextrose agar media and culture characteristics, the strains were classified into the sclerotium type, the hyphal type and the intermediate type accounted for 14.28%, 42.86% and 42.86%, respectively. The growth rate and sporulation quantity of the strains were 1.22–2.54 mm·d^－1 and 5.3×106–40.6×106 spore·mL^－1, which showed significant difference. In addition, based on the clustering results of average disease index,the strains were clustered into three groups which showed high (Type Ⅰ), low (Type Ⅱ) and moderate (Type Ⅲ) virulence to cotton with the proportion of 3.2%, 12.9% and 83.9%, respectively. [Conclusion] It is conclude that the main culture types of V. dahliae in Hunan province are the hyphal type and the intermediate type, and the strains showed pathogenicity differentiation. The result showed that defoliating pathotype of V. dahliae was dominant in the cotton growing areas of Hunan province．     

棉花黄萎病拮抗细菌DS45-2菌株在土壤和棉花根内的定殖

通过盆栽试验,对棉花黄萎病拮抗细菌DS45-2菌株在土壤和棉花根际、根内的定殖情况进行了分析。结果表明,在自然条件下,拮抗细菌DS45-2菌株能够在土壤及棉花根际、根内定殖。拮抗菌株DS45-2施到土壤中后的45d内,其定殖数量在开始25d内显著下降,但随后下降缓慢并逐渐趋于稳定,维持在106个·g-1土左右。接菌后45d,DS45-2在棉花根内的定殖菌量达2.35×104个·g-1根,在棉花根际的定殖菌量达97.4×106个·g-1土,且重新分离出的拮抗菌株保持较高的拮抗活性。DS45-2菌株在灭菌土中的定殖数量高于自然土中的数量。随土层深度的增加,定殖菌量呈下降趋势,但变化不明显。     

Proteomics Analysis of Gossypium barbadense Leaves with Resistance to Verticillium dahliae under Infection Stress

[Objective] The purpose of this study is to analyze the resistance mechanism of island cotton under Verticillium wilt stress and to find possible resistance genes. [Method] The cotton resistance mechanism under the stress of Verticillium dahliae was studied by protein two-dimensional electrophoresis and mass spectrometry at the protein level. [Result] In the leaves, eleven proteins were down-regulated and 15 proteins were up-regulated after 2 hours of inoculation with Verticillium dahliae. The down-regulated proteins were mainly related to photosynthesis and carbon assimilation, and then we deduce that Verticillium dahliae is mainly broken cotton photosynthetic system to cause cotton susceptibility. The up-regulated proteins were mainly related to photosynthesis and benzoquinone reductase, beta-D-galactosidase, 14-3-3f protein and other disease-resistant protein. [Conclusion] It is speculated that the defense mechanism of island cotton on Verticillium wilt may occur at two levels: one is passive defense, it is manifested that the isoproteins of the down-regulated proteins such as chloroplast II AB binding protein and ribulose diphosphate carboxylase are highly expressed to maintain the stability of the island cotton photosynthetic system, the histone and 14-3-3f protein are highly expressed to keep the cell stability; the second is active defense, it is manifested that the high expressed β-D-galactosidase and benzoquinone reductase may be involved in the resistance of island cotton to Verticillium wilt.     

棉花黄萎病抗性与过氧化物酶同工酶分析

采用等电聚焦电泳方法 ,对棉花二对抗黄萎病近等基因系以及三对色素腺体近等基因系的子叶进行过氧化物同工酶 (POD)分析。结果表明 ,色素腺体和棉酚含量与棉花抗性没有必然的联系 ;环境条件对棉花抗性表现有一定的影响 ,但对子叶中 POD同工酶谱带无显著的影响 ;4条 POD同工酶酶带 (P4、P6、P7、P10 )的表现与棉花抗黄萎病性有关 ,这些酶带的特殊表现可用于棉花抗病性辅助鉴定和辅助育种     

大丽轮枝菌致萎毒力指示菌的筛选及拮抗作用初探

为了筛选可指示Verticillium dahliae致萎毒素毒力大小的指示菌,并对致萎毒素抑制指示菌生长的机理进行初步探讨,采用指示菌平板打孔法,对黑曲霉(Aspergillus niger)BF-3758、枯草芽孢杆菌(Bacillus subtilis)、金黄色葡萄球菌(Staphylococcus aureus)、啤酒酵母(Saccharomyces cerevisiae)等12种具有微生物种属代表性的菌株进行筛选;采用拮抗试验、致萎试验结合显微观察的方法,初步探索了Verticillium dahliae致萎毒素对指示菌生长的抑制作用。结果表明,Verticillium dahliae粗毒素对Aspergillus niger BF-3758的生长具有较强的抑制作用,且该抑制作用和粗毒素对离体叶片的致萎作用具有一致性,并存在一定的量效关系,Aspergillusniger BF-3758菌株可以作为Verticillium dahliae致萎毒素的毒力指示菌;Verticillium dahliae粗毒素可抑制Aspergillus niger BF-3758的孢子萌发和菌丝生长,对成熟菌丝也有破坏作用。上述研究可为评价Verticillium dahliae的致病力提供一种操作简便、耗时短、费用低的生物学方法。     

拮抗细菌C-02防治棉花黄萎病的机理研究

 从抗生、竞争和诱导系统抗性三个方面对拮抗细菌C-02防治棉花黄萎病的机理展开了研究,结果表明,C-02分泌的抗菌蛋白能溶解病原菌VD-11的细胞壁,抑制其致萎毒素的分泌。与病原菌VD-11相比,C-02利用碳源有明显优势,在棉花根际土壤中具有显著的营养竞争优势和很好的定殖能力。C-02处理棉苗后,棉叶中植株防御性酶系PAL、POD和PPO的活性提高幅度大,维持时间长。C-02防治棉花黄萎病的机理是抗生、溶菌、竞争和诱导棉苗系统抗性的综合表现。     

棉花内生拮抗菌Paenibacillus xylanilyticus YUPP-1抗黄萎病研究

 在棉花现蕾期筛选到一个高抗黄萎病的内生菌菌株,根据16S rDNA序列同源性,将其命名为Paenibacillus xylanilyticus YUPP-1(解木聚糖类芽孢杆菌YUPP-1)。当把该菌标记后,每株棉花施用该菌株达到1×10⁶ cfu时,该菌能顺利定殖于棉花体内。室内盆栽抗病实验表明：当生防菌灌根处理的菌量为每株1×10⁶,1×10⁷,1×10⁸ cfu,其在结铃期的发病株率仅为8%,5%和2%,而此时对照的发病株率高达96%。2008－2009年的小区防治结果表明：当生防菌灌根处理的菌量为每株1×10⁸ cfu时,2008年处理区的棉花黄萎病发病株率和病指为14.8%和8.5,对照分别为53.3%和26.5,2009年处理区的棉花黄萎病发病株率和病指为9.4%和6.5,对照为37.8%和18.8。这些结果表明,解木聚糖类芽孢杆菌YUPP-1对防治棉花黄萎病具有巨大的应用潜力。     

棉花黄萎病菌与抗黄萎病遗传育种研究进展

简要综述了棉花黄萎病菌及抗黄萎病遗传育种的研究进展。研究表明 ,各地的棉花黄萎病菌均存在致病力的分化 ,其致病机理是病菌侵入棉花后菌丝及孢子在导管内大量繁殖 ,同时刺激邻近的薄壁细胞产生胶状物质及侵填体而堵塞导管 ,使水分和养分运输发生困难 ,更重要的是病菌在棉株体内产生的糖蛋白毒素作用的结果。棉花抗黄萎病的遗传方式争论较大 ,但一般在温室由单一菌系接种鉴定时棉花黄萎病抗性表现为单基因遗传 ,而在田间病圃或用多菌系混合鉴定时 ,棉花黄萎病抗性表现为多基因遗传。由于陆地棉内缺乏高抗黄萎病资源 ,给棉花抗黄萎病育种带来一定困难 ,但 90年代以来 ,已育成 86 - 6、川 73 7、川 2 80 2、豫棉 1 9号、豫棉 2 1号等一些抗黄萎病的新品种。上述抗黄萎病品种在棉花黄萎病综合防治中起了重要作用     

棉花根部拮抗枯萎病菌或黄萎病菌的可培养内生细菌多样性分析

为解析棉花内生细菌的种群结构,更好地开发利用生防内生细菌,以棉花枯萎病菌、黄萎病菌为指示病原真菌,对常抗棉和泗棉3号根部分离获得的内生细菌进行拮抗活性筛选,获得87株至少对其中1个指示病原真菌具有拮抗作用的内生细菌。ARDRA分析(Amplified ribosomal DNA restriction analysis)和16S r DNA序列测定表明棉花根部可培养内生拮抗细菌可分为13个ARDRA类群,分属于芽孢杆菌属(Bacillus)、短芽孢杆菌属(Brevibacillus)、伯克霍尔德氏菌属(Burkholderia)、农杆菌属(Agrobacterium)、中华根瘤菌属(Sinorhizobium)、不动杆菌属(Acinetobacter)和假单孢菌属(Pseudomonas)7个属。其中常抗棉根部内生拮抗细菌分属于芽孢杆菌属、短芽孢杆菌属、伯克霍尔德氏菌属和农杆菌属,而泗棉3号根部内生拮抗细菌则分属于芽孢杆菌属、伯克霍尔德氏菌属、中华根瘤菌属、不动杆菌属和假单孢菌属,二者的优势种群都是芽孢杆菌属细菌。BOX-PCR分析显示这些内生拮抗细菌具有丰富的遗传多样性。通过测定这些内生拮抗细菌对棉花黄萎病菌和棉花枯萎病菌的拮抗力大小,以及是否具有产纤维素酶、几丁质酶、蛋白酶和嗜铁素的活性,表明这些内生拮抗细菌具有不同的生防作用机制。     

棉花黄萎菌间接ELISA方法的建立及其检测棉花体内带茵的应用

利用棉花黄萎病的病原菌的菌丝蛋白作为抗原,制备了该病原菌菌丝蛋白的多克隆抗体,建立了特异性检测棉花黄萎病菌的间接ELISA方法.该方法检测灵敏度达到5 ng/mL棉花黄萎菌菌丝蛋白.应用该方法可定量检测棉花组织及其根部土壤中棉花黄萎菌的含量,同时发现病株的茎、根及根部土壤的带菌量多少与病株发病程度有一定的相关性.本研究说明,该方法可在棉花黄萎病的早期检测和预报上具有重要的应用前景.     

棉花黄萎病抗性遗传和分子生物学研究进展

介绍了棉花黄萎病菌的致病机制 ,棉花黄萎病抗病机制及其遗传以及棉花黄萎病的鉴定方法 ,并对分子生物学在棉花黄萎病研究中的应用状况进展进行了综述。     

Detection of the Colonization Ability of Antagonistic Bacteria Strain Z-5 against Cotton Verticillium Wilt

The first step in the biological control of plant diseases is to determine whether antagonistic microorganisms can colonize successfully the plant rhizosphere soil. Bacillus Z-5 was isolated from cotton plant soil and showed a significant biocontrol effect on cotton Verticillium wilt. The strain Z-5 was marked with resistance to rifampicin and pathogenic fungi. A Z-5 mutant strain that could stably grow in 300 μg·mL^-1 rifampicin NA medium and had wild-type antagonistic activity against pathogen fungi, colony morphology, and cell morphology was selected. The marked strain Z-5 was used to treat cotton seeds by seed soaking, seed dressing, and soil drench. The number of Z-5 cells in cotton plant root soil, roots, stems, and leaves was tested using dilution on rifampicin plate after different periods. The result showed that the number of antagonistic bacteria of soil drench group in cotton plant root soil was 49.72×10⁴ cfu·g^-1, while seed soaking and seed dressing groups yielded about 0.49×10⁴ and 2.07×10⁴ cfu·g^-1. The number of antagonistic bacteria of the soil drench group in cotton plant roots was 4.93×10⁴ cfu·g^-1, while the seed soaking and seed dressing group yielded 4.12×10⁴ and 3.29×10⁴ cfu·g^-1.The number of antagonistic bacteria of the soil drench group in cotton stems was 1.32×10⁴ cfu·g^-1, while the seed soaking and seed dressing group yielded 0.17×10⁴ and 0.74×10⁴ cfu·g^-1. The number of antagonistic bacteria of soil drench group in cotton leaves was 0.27×10⁴ cfu·g^-1, while seed soaking and seed dressing group yielded 0.17×10⁴ and 0.22×10⁴ cfu·g^-1. These results showed that the Z-5 strain could effectively colonize cotton roots soil and that in vivo , the soil drench group showed a better colonization effect by the strain Z-5 than the seed soaking and seed dressing group.