【作者】 薛磊；

【导师】 薛泉宏；

【作者基本信息】 西北农林科技大学 ， 微生物学， 2013， 博士

【摘要】 棉花黄萎病是由大丽轮枝菌Verticillium dahliae Kleb.引起的土传性病害，是我国棉花生产的主要限制因素，寻找合适有效的防治途径是目前解决棉花产业可持续发展的首要任务。国内外关于棉花黄萎病防治采取的措施主要有抗性品种选育、改善栽培措施、化学农药等。由于抗病育种无重大进展，其他措施效果不佳，探索新的防治途径已成为目前亟待解决的问题。对土传根系真菌病害而言，生物防治具有化学农药所不具备的优点。在生防微生物中，放线菌可产多种抗生素、胞外酶等活性物质，且孢子抗逆性强，在土传病害防治方面具有较好的应用前景，但在防治棉花黄萎病上缺乏系统研究。本研究从土壤微生态修复需要出发，对拮抗大丽轮枝菌的放线菌进行筛选和鉴定，系统研究放线菌对棉花黄萎病的防病促生效果、生防机理，以及生防放线菌与大丽轮枝菌、棉花植株的互作机理，为棉花黄萎病防治提供高效多功能生防放线菌菌株，并为后期生防放线菌活菌制剂的实际应用提供理论依据。论文主要研究结果如下：1.从本研究室保藏的分离自我国新疆、青海、陕西、西藏和黑龙江作物根区土壤中的712株拮抗放线菌中，通过琼脂块法拮抗性和发酵液抑菌活性逐级筛选，得到11株对棉花黄萎病原大丽轮枝菌均有较强抑菌作用的放线菌；入选放线菌及其发酵液可有效的抑制大丽轮枝菌生长、孢子形成和萌发；并能利用酚酸类棉花自毒物质（对羟基苯甲酸、阿魏酸和没食子酸）作为唯一碳源生长。通过形态学特征、生理生化特征、聚类分析及16S rRNA序列分析对其进行分类鉴定，11株放线菌属于链霉菌属（Streptomyces）的蓝微褐链霉菌（S. cyaneofuscatus）、卡那霉素链霉菌（S. kanamyceticus）、娄彻氏链霉菌（S. rochei）、黄三素链霉菌（S. flavotricini）和弗氏链霉菌（S. fradiae）。2.供试11株链霉菌对NaCl具有一定的耐受性，在培养基中NaCl含量在15g·L^-1以下时，11株链霉菌均可以生长，含量在7g·L^-1以下时，对菌株正常菌落形态及拮抗活性影响较小，表明供试链霉菌可用于新疆等含盐量较高的土壤中施用。在NaCl含量为3g·L^-1的高氏1号培养基上，供试链霉菌垂直传代15代后，其菌丝生长速度、产孢量及抑菌活性均优于普通高氏1号培养基。3.以大丽轮枝菌菌体为唯一碳能源时，可诱导供试生防链霉菌同步合成几丁质酶、β^-1,3-葡聚糖酶、β-葡萄糖苷酶、纤维素酶、多酚氧化酶及蛋白酶6种细胞壁溶解酶；当菌体添加量为10g·L^-1，28℃培养7d时，上述诱导酶活性可达峰值；链霉菌粗酶液对大丽轮枝菌菌丝有溶解作用；供试生防链霉菌菌丝均可缠绕在大丽轮枝菌菌丝上，并在缠绕处使病原菌菌丝溶解；以大丽轮枝菌菌体为碳氮能源时供试链霉菌能产生活性较强的抑菌物质；当菌体添加量为20g·L^-1，发酵液稀释5倍时菌株B49所产生抑菌活性物质的最大抑菌率达95.7%。4.采用菌丝生长速率法研究了培养基pH、盐分浓度与种类及链霉菌发酵液对病原菌生长及微菌核形成的影响。结果表明：供试大丽轮枝菌菌落生长最适pH为7.0，偏酸或偏碱会抑制菌落生长。但培养基偏碱可显著促进微菌核形成，当pH值为8.0时，大丽轮枝菌菌落生长受抑制较小，同时微菌核区面积较pH为7.0时增加22.6%。盐分浓度影响大丽轮枝菌菌丝生长及微菌核形成。随培养基NaCl浓度增加，供试大丽轮枝菌生长受到抑制，菌落面积和菌丝面积均逐渐减小，但微菌核形成量却显著增加；当NaCl浓度为10g·L^-1时，微菌核区面积较对照（NaCl浓度为0）增加40.7%。不同种类盐分对供试大丽轮枝菌生长均有影响。氯化物（NaCl和KCl）和硫酸盐（Na₂SO₄和MgSO₄）均随盐分浓度增加而促进大丽轮枝菌微菌核形成；而CaCl2则显著促进菌丝生长并在浓度大于7g·L^-1时抑制微菌核形成。11株链霉菌的无菌发酵滤液对大丽轮枝菌菌落生长、菌核形成和微菌核萌发有明显抑制作用。菌株B49和D184的5倍稀释发酵液对微菌核形成的抑制率达100%。并且，将经链霉菌发酵液处理后丧失形成微菌核能力的大丽轮枝菌菌株，转接至不含发酵液的PDA培养基，连续传代至第5代，其仍然不能恢复形成微菌核的能力。微菌核在含有菌株D1845倍稀释发酵液培养基上培养168h时，微菌核萌发率仅为38.3%。5.播种时向灭菌土壤中接种生防链霉菌，棉花幼苗对大丽轮枝菌毒素危害的防御反应能力及抗逆性大幅度提高。主要表现在：棉花叶片、根系的防御酶活性，二元酚及木质素含量在温室培养6周后显著提高。在毒素处理后，棉苗的保护性反应在24h内即可迅速响应。大丽轮枝菌毒素的毒害作用减轻；其中丙二醛（MDA）在棉苗叶片及根系中的积累减少，棉苗根系活力、叶绿素含量及叶片含水量的下降幅度减缓。最终，接种链霉菌减弱了毒素在棉苗上的毒害作用及致萎作用，72h时生防效率最高，为68.2%。6.生防链霉菌单独接种或与病原菌混合接种时，供试链霉菌均可有效提高棉花植株不同生长时期的诱导抗病性。生防链霉菌可显著提高棉花叶片、根系的过氧化物酶（POD）、多酚氧化酶（PPO）和苯丙氨酸解氨酶（PAL）活性，增加棉花叶片内单宁、二元酚、木质素及游离脯氨酸含量，减少棉花叶片中MDA积累，减缓根系活力和叶片含水量的下降幅度。7.生防链霉菌可制成活菌粉状制剂，并可采用种子包衣或土壤接种的方法施用链霉菌菌剂。在苗期、盆栽和田间病圃试验中，生防链霉菌菌剂对棉花黄萎病均表现出良好、稳定的抗病效果，有效降低了黄萎病病情指数及病情进展曲线下面积（AUDPC）。其中链霉菌X4对棉花黄萎病的生防效果总体表现最好，苗期、盆栽和田间病圃中的最高生防效率分别达到59.3%、59.2%及51.4%。8.生防链霉菌具有产吲哚乙酸（IAA）的能力，培养17d时，无菌发酵滤液中IAA含量可达47.56⁵6.19μg·mL^-1；用发酵滤液浸种可促进棉花种子发芽和棉苗胚轴及胚根的生长。在盆栽、田间试验中，生防链霉菌菌剂接种可显著提高棉花植株高度和根茎直径，促进总生物量、地上部分、根系和棉铃重量的增加，并对叶片绿色度和光合作用有促进作用。其中链霉菌X4对植株高度、总鲜重、根鲜重、棉铃鲜重及净光合速率的促进作用总体表现最好，最高增幅可分别达24.4%、62.4%、107.7%、45.1%及31.9%。9.生防链霉菌可在棉花根区、根表土壤中有效定殖，其中链霉菌X4在盆栽和田间病圃根区土壤中的最高定殖量分别为2.85×10⁶和2.44×10⁶CFU g^-1干土。生防链霉菌可有效改善棉花根区、根表土壤的微生物生态，降低真菌总数量和比例，增加土壤中细菌、放线菌总数量和比例，促使棉花根域土壤微生物由“真菌型”向“细菌型”转变。当生防链霉菌与病原菌混合接种时，链霉菌X4对棉花根区土壤中细菌、放线菌总数量的增幅分别为40.6%、32.1%，而真菌总数量则降低64.6%。更多还原

【Abstract】 Verticillium wilt of cotton, caused by Verticillium dahliae Kleb., is a serious soil-bornedisease that limits cotton production in China. Finding effective disease control approaches isthe first priority to solve the problem of sustainable cotton production. Conventionalmanagement options for Verticillium wilt of cotton include the development of partiallyresistant cultivars, tillage management, and the application of chemical fungicides. However,there was no significant progress in breeding for disease resistance, and other managementoptions are ineffective for controlling Verticillium wilt of cotton. Exploring new ways ofcontrolling this disease has become the current problems to be solved. Microbial biocontrolagents have been proposed as a more effective and environmentally friendly means ofcontrolling soil-borne diseases. Actinomycetes are widely used in control of soil-bornediseases for their production of various antibiotics, extracellular enzymes and survivablespores. However, few specialized studies have been conducted about the biocontrolmechanism and efficacy of actinomycetes against Verticillium wilt of cotton. This study wasconducted from the perspective of soil microbial ecological restoration. The authorsinvestigated the occurrence of cotton Verticillium wilt, screened and identified antagonisticactinomycetes against V. dahliae, evaluated biocontrol and growth promoting effects ofselected actinomycete strains, and finally explored mechanisms of interaction amongpathogenic fungi, biocontrol actinomycetes and cotton plants. Biocontrol actinomycetes withhigh efficacy and stability were obtained for control of cotton Verticillium wilt andmanagement cotton continuous cropping obstacles. Results provided a scientific basis forfuture commercial production and practical application of biocontrol actinomycete agents.The results and conclusions are listed below:1. A total of712antagonistic actinomycete were previously isolated from croprhizosphere in Xinjiang, Qinghai, Shaanxi, Tibet and Heilongjiang, primarily in northwest ofChina. Of these,11actinomycete isolates with high inhibitory activity against V. dahliaewere selected by stepwise screening using the agar diffusion method and mycelium growthrate measurement. The selected actinomycete isolates efficiently inhibited V. dahliae growth, conidia production, and germination, and utilized cotton self-toxic phenolic acids （i.e.,p-hydroxybenzoic acid, ferulic acid and gallic acid） as the sole carbon source. Morphologicaland physiological tests, cluster analysis, combined with16S rRNA-based molecularidentification indicate that these11isolates are members of the genus Streptomycescyaneofuscatus, S. kanamyceticu, S. rochei, S. flavotricini and S. fradiae, respectively.2. The11Streptomyces isolates showed certain tolerance to NaCl and survived in thepresence of <15g·L^-1content. There were few changes in colony morphology andantagonistic activity of these Streptomyces isolates with <7g·L^-1NaCl. This result indicatesthat the tested Streptomyces can be used in the salt soil of Xinjiang. The mycelia growth rate,spores production and antagonistic activity of the11Streptomyces isolate grown with3g·L^-1NaCl were better than that with0.5g·L^-1NaCl Gause No.1agar medium.3. With V. dahliae mycelium as the sole carbon source, the11biocontrol Streptomycesproduced6extracellular fungal cell-wall degrading enzymes, i.e., chitinase,β^-1,3-glucosidase, β-glucosidase, cellulose, polyphenol oxidas and protease. Maximumenzyme activities were observed in extracellular hydrolases fermentation of the Streptomycessupplied with10g·L^-1fungi mycelium at28℃for7d. The Streptomyce crude enzymeinduced disintegration of V. dahliae mycelia and hypha. All tested Streptomyces isolates werecapable of twining with V. dahliae hypha, leading to fungal cell wall lysis. The Streptomycesisolates produced antifungal active substances with V. dahliae mycelium as the sole carbonand nitrogen source. When supplied with20g·L^-1fungi mycelium, the5-fold diluted fermentfiltrate of isolate B49showed maximum inhibition rate against V. dahliae,95.7%.4. The influence of salinity （concentration and type） and pH on the microsclerotiaformation in V. dahliae was evaluated by determination of the mycelium growth rate usinglaboratory culture medium. The colony growth of V. dahliae was inhibited at acidic andalkaline pH, with optimal colony growth observed at pH7.0. The microsclerotia formation ofV. dahliae was enhanced at alkaline pH, and the microselerotia area was increased by22.6%at pH8.0compared with that at pH7.0. The mycelium growth and microsclerotia formationof V. dahliae was affected by salinity concentration. With an increasing concentration ofNaCl in the culture medium, the colony growth and mycelium area of V. dahliae wereinhibited and decreased, respectively, whereas the formation of microsclerotia was enhanced.The microselerotia area in the culture medium with10g·L^-1NaCl was increased by40.7%compared with that in the medium without NaCl. The growth of V. dahliae was affected bydifferent salinity types. With an increasing concentration of chloride （i.e., NaCl, KCl） orsulfate （i.e., Na₂SO₄, MgSO₄） salts in the culture medium, the microsclerotia formation of V.dahliae was accelerated, whereas the mycelium growth was enhanced in the presence of CaCl2, and the microsclerotia formation of V. dahlia was inhibited at concentrations wasgreater than7g·L^-1in culture medium. The mycelium growth, microsclerotia formation andgermination of V. dahlia were significantly inhibited by culture filtrates of11Streptomycesisolates.5-fold-diluted ferment filtrates of Streptomyces isolates B49and D184displayedcompletely inhibitory effects on microsclerotia formation of V. dahlia with100%ofinhibitory rate. The mutant strains of V. dahliae was obtained from these3Streptomycesferment filtrate treatments, which produced no microsclerotia. The mutants were not reversedto the wild type after transferred5times in the PDA medium containing no Streptomycesferment filtrate. Its germination rate was38.3%at168h in the PDA medium containing5-fold-diluted ferment filtrate of Streptomyces D184.5. Under greenhouse conditions, the application of a powdered Streptomyces biocontrolagent to soil at the time of sowing considerably enhanced the plant defense-related enzymeactivities as well as the ortho-dihydric phenol and lignin contents in cottons after6-weekcultivation. When treated with V. dahliae phytotoxin, the defense-related responses of cottonwere accelerated and strengthened in Streptomyces-inoculated cottons relative tonon-inoculated cottons within24h. The changes in the defense-related responses furthersuppressed the accumulation of malondialdehyde （MDA）, and reduced the loss of rootactivity, leaf total chlorophyll, and leaf water contents during V. dahliae phytotoxin treatment.The pathogenic effect of V. dahliae phytotoxin on Streptomyces-inoculated cottons waseventually weakened, and the biocontrol efficacy was68.2%at72h.6. When inoculated individually or mixed with V. dahliae, the biocontrol Streptomyceseffectively improved the induced disease resistance in cotton plants during different growthstages. The Streptomyces treatment considerably enhanced the activities of peroxidase （POD）,polyphenol oxidase （PPO） and phenylalanine ammonialyase （PAL） in cotton leaf and rootwhile increasing the tannin, ortho-dihydric phenol, lignin and free proline contents,decreasing the accumulation of malondialdehyde （MDA） in cotton leaf, and reducing the lossof root activity and leaf water contents.7. The selected Streptomyces isolates were prepared as powdered biocontrol agents forapplication through seed coating or soil inoculation. The ability of the powdered biocontrolStreptomyces agents showed excellent and stable efficacies in control of Verticillium wiltduring the seedling stage in wilt-related pot and field experiments. The disease severity andthe area under the disease progress curve （AUDPC） of cotton Verticillium wilt were bothreduced. Among the tested Streptomyces isolates, X4showed the best biocontrol effect, withbiocontrol efficacies up to59.3%,65.8%and51.4%during the seedling stage in wilt-relatedpot and field experiments, respectively. 8. The selected Streptomyces isolates produced indoleacetic acid （IAA） up to47.56and56.19μg·mL^-1in ferment filtrate at17d. Treatment with Streptomyces ferment filtratepromoted the seed germination and the growth of cotton hypocotyl and radicle. In pot andfield experiments, inoculation of Streptomyces considerably increased plant height, rhizomediameter, total biomass, above-ground weight, root weight, and boll weight of cotton, andprompted the greenness and photosynthesis of cotton leaf. Among the tested Streptomycesisolates, X4showed the most significant growth promoting effect, increasing the plant height,fresh weight of total plant, root and boll, and net photosynthetic rate by24.4%,62.4%,107.7%,45.1%and31.9%, respectively.9. The selected biocontrol Streptomyces survived in the rhizosphere and rhizoplane soilsof cotton. In pot and field experiments, the abundance of the isolate X4in the rhizospheresoil of cotton was up to2.85×10⁶and2.44×10⁶CFU g^-1dry soil, respectively. The biocontrolStreptomyces improved the microbial ecology of cotton rhizosphere and rhizoplane soils bydecreasing soil fungi and increasing soil bacteria and actinomyces, leading to the transition ofmicrobial populations from “fungal-type” to “bacteria-type”. When inoculated by mixingwith V. dahliae, the isolate X4increased the abundance of bacteria and actinomycespopulation in rhizosphere soil of cotton by40.6%and32.1%, respectively, while decreasingthe fungi population by64.6%.更多还原