本文選題：棉花黃萎病 + 大麗輪枝菌�。� 參考：《新疆師范大學(xué)》2017年碩士論文

【摘要】：棉花黃萎病是由大麗輪枝菌(Verticillium dahliae)引起的土傳性維管束病害,是我國(guó)棉花生產(chǎn)危害最嚴(yán)重的真菌病害。尋找對(duì)其有效的防治方法對(duì)于我國(guó)棉花高產(chǎn)穩(wěn)產(chǎn)具有重大意義。本文從新疆連作棉田棉花根際土壤中分離篩選出大麗輪枝菌拮抗芽孢細(xì)菌,探究拮抗菌的抑菌特性和拮抗機(jī)理,為新疆棉花黃萎病的生物防治提供菌株資源和理論指導(dǎo)。試驗(yàn)共分離到146株芽孢細(xì)菌,29株能拮抗大麗輪枝菌,9株拮抗性極強(qiáng);經(jīng)鑒定,這9株菌中J-02和J-29屬短小芽孢桿菌(Bacillus pumilus),J-15和其他菌株與枯草芽孢桿菌(B.subtilis)相近。J-02熱穩(wěn)定性較好,pH耐受范圍在5-9,培養(yǎng)至衰亡期的抑菌效果最好;J-02能抑制大麗輪枝菌芽管伸長(zhǎng)形成菌絲;在盆栽試驗(yàn)中對(duì)棉花黃萎病也表現(xiàn)出明顯防治作用。J-15在盆栽試驗(yàn)中顯示出對(duì)棉花黃萎病的防治效果。硫酸銨鹽析粗提J-15抑菌物質(zhì)結(jié)果顯示,80%硫酸銨飽和度提取效果最好。J-15對(duì)溫度敏感,100℃可致菌體死亡,但其抑菌物質(zhì)經(jīng)100℃處理1 h后活性僅降低18.3%;J-15經(jīng)pH7-9處理后抑菌性與對(duì)照組無(wú)顯著差異,而其抑菌物質(zhì)能耐受pH 2-10,且pH 2-4能完全沉淀抑菌物質(zhì),卻不影響抑菌活性;J-15培養(yǎng)至穩(wěn)定期抑菌性最強(qiáng),而搖菌12-24 h,OD600=1.7以上為抑菌物質(zhì)最佳提取時(shí)間;此外,J-15抑菌粗提物對(duì)胃蛋白酶、蛋白酶K、紫外照射和丙酮都不敏感。利用pH 2酸沉淀后丙酮抽提J-15抑菌物質(zhì),進(jìn)行SDS-PAGE電泳檢測(cè)和光譜掃描,并利用DEAE-Sepharose Fast Follow陰離子交換層析純化后質(zhì)譜鑒定。結(jié)果顯示,J-15粗提抑菌物質(zhì)經(jīng)丙酮沉淀去除非活性蛋白后,電泳未檢測(cè)到特異蛋白;在210 nm和270 nm處有吸收峰,280 nm處光吸收值較低;采用0.1 mmol/L Tris-HCl緩沖溶液(pH=6.5),0-1 mol NaCl線性洗脫得到較純抑菌物質(zhì),在280 nm不具吸光性;質(zhì)譜鑒定J-15抑菌物質(zhì)分子量可能為828 Da。J-15抑菌物質(zhì)對(duì)大麗輪枝菌孢子萌發(fā)、菌絲生長(zhǎng)和防御酶活力影響的研究結(jié)果表明,J-15抑菌物質(zhì)低濃度抑制孢子萌發(fā),高濃度可致萌發(fā)力喪失;平板試驗(yàn)發(fā)現(xiàn)抑菌物質(zhì)可致菌絲短粗畸形,產(chǎn)孢能力降低;與對(duì)照組相比,J-15抑菌物質(zhì)可抑制大麗輪枝菌SOD、POD和CAT活力,且導(dǎo)致MDA含量較對(duì)照高。表明J-15抑菌物質(zhì)的作用機(jī)制主要表現(xiàn)在,抑制孢子萌發(fā),菌絲伸長(zhǎng),并造成大麗輪枝菌的抗氧化系統(tǒng)和膜系統(tǒng)的損傷。
[Abstract]:Cotton Verticillium wilt is a soil borne vascular disease caused by Verticillium dahliae, which is the most serious fungal disease in China's cotton production. It is of great significance to find the effective control method for the high and stable yield of cotton in our country. This paper isolated and screened the Dali wheel from the cotton rhizosphere soil of Xinjiang continuous cropping cotton field. Mycobacteria antagonized the spore bacteria, explored the antimicrobial properties and antagonistic mechanisms of the antagonistic bacteria, provided strain resources and theoretical guidance for the biological control of Verticillium Wilt in Xinjiang. 146 strains of spores were isolated, 29 strains could antagonize Dali and 9 strains were extremely resistant; the 9 strains of J-02 and J-29 were identified as Bacillus pum Ilus), J-15 and other strains and Bacillus subtilis (B.subtilis) similar to.J-02 thermal stability, pH tolerance range of 5-9, culture to the decay period of the best bacteriostasis effect; J-02 can inhibit the elongation of the bud tube of rhizo Dali to form mycelium; in pot experiment, the cotton Verticillium wilt also showed obvious control effect.J-15 in pot experiment. The results of the prevention and control of Cotton Verticillium wilt. Ammonium sulfate salting out J-15 bacteriostasis results showed that the best extraction effect of 80% ammonium sulfate was.J-15 sensitive to temperature, and 100 C could cause the death of the bacteria, but the activity of the bacteriostat was only 18.3% after 1 h treatment at 100, and there was no significant difference between the J-15 and the control group after pH7-9. Bacteria can tolerate pH 2-10, and pH 2-4 can completely precipitate bacteriostatic substances, but do not affect bacteriostasis activity; J-15 culture to stable period is the most bacteriostasis, while the bacteria 12-24 h, OD600=1.7 above the best extraction time of bacteriostat; in addition, J-15 antibacterial crude extracts are not sensitive to pepsin, egg white enzyme K, ultraviolet radiation and acetone. Use pH 2 acid precipitation J-15 bacteriostat was extracted by acetone, detected by SDS-PAGE electrophoresis and spectrum scanning, and identified by DEAE-Sepharose Fast Follow anion exchange chromatography and purified by mass spectrometry. The results showed that the specific protein was not detected by electrophoresis, and there was a peak of absorption, 280 nm at 210 nm and 270 nm. The light absorption value was low; 0.1 mmol/L Tris-HCl buffer solution (pH=6.5), 0-1 mol NaCl linear elution obtained more pure bacteriostat and no light absorption in 280 nm; mass spectrometry identified that the molecular weight of J-15 bacteriostat may be the effect of 828 Da.J-15 bacteriostat on spore germination, mycelial growth and defense enzyme activity of Dali The low concentration of J-15 inhibited the germination of spores and the loss of germinating force at high concentration; the plate test found that bacteriostatic substances could induce mycelial mycelium deformity and decrease the sporulation ability. Compared with the control group, J-15 inhibitory substances could inhibit the activity of SOD, POD and CAT, and lead to the higher MDA content. The mechanism of J-15 bacteriostasis was shown to be the mechanism of action of J-15 bacteriostat. It is mainly manifested in Inhibiting Germination of spores, elongation of mycelia and damage of antioxidant system and membrane system of Verticillium dahliae.

【學(xué)位授予單位】：新疆師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S435.62;S476.1

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