【作者】 董国菊；

【导师】 周常勇；

【作者基本信息】 西南大学 ， 植物病理学， 2012， 博士

【摘要】 烟草是一种重要的经济作物,由烟草疫霉Phytophthora nicotianae Breda de Haan引起的烟草黑胫病是烟草生产上的一种毁灭性真菌土传病害。该病原菌破坏性极强,烟株受害后易造成整株死亡,严重影响烟草生产。目前,生产上仍依赖于化学农药防治烟草黑胫病,但长期使用化学农药,容易诱发病原菌产生耐(抗)药性菌株,污染环境和破坏生态系统,同时烟草是叶用经济作物,农药残留会影响烟叶的品质。随着烟草及其制品向更加安全化方向发展的趋势,寻找一种安全、无毒、经济、有效和可持续发展的病害控制途径是当务之急。生物防治由于具有经济有效、且对环境安全友好的特点,被认为是一种最具潜力的、可替代化学农药的方法,现已成为植物病害防治研究的热点。鉴于此,本文以分离自连作烟田健康烟株根际土壤的5个细菌菌株为研究对象,从中筛选出对烟草疫霉具有高效拮抗作用的菌株；进一步围绕该高效菌株对病原菌的抑制机制、其在烟草幼苗根部的定殖规律以及对烟草生长的影响等方面展开系统深入的研究,初步探明该高效菌株的生防机理；在此基础上对该菌株进行鉴定并初步明确其最适的摇瓶培养条件。主要研究结果如下。高效拮抗烟草疫霉根际细菌菌株的筛选5个供试菌株中,除P-70-3菌株外,其余4个菌株对烟草疫霉均有很强的平板拮抗效果,且5个供试菌株的胞外代谢产物对病原菌也表现出不同程度的抑菌作用。其中,P-72-10菌株的拮抗效果和抑菌作用最强,平板对峙培养中抑菌带半径达13.0mm,相对抑制率为68.57%；其胞外代谢产物粗提液对病原菌的抑制作用随浓度的增加而增强,分别为24.92%(10-3)、27.18%(10-2)、39.84%(10-1)和46.03%(原液),且与对照相比差异显著(P<0.05)。P-72-10菌株对烟草黑胫病的温室控病效果该菌株的带菌培养液灌根处理烟草幼苗后,能减轻烟草植株的发病率和降低病情指数；其对抗病和感病品种的控病效果表现出一定的差别,相对防效分别为53.57%和66.37%,与对照处理相比均达到差异显著水平(P<0.05)。P-72-10菌株对烟草疫霉的抑制机制该菌株产生的挥发性代谢产物能抑制烟草疫霉菌丝的生长,相对抑制率达到32.94%。显微观察发现：对照菌丝形态饱满,细胞壁光滑,胞质均匀透明,分支正常；而平板对峙培养中受抑制菌落边缘的菌丝形态发生畸变,其菌丝不正常的分支增多,菌丝顶端细胞畸形生长,并在菌丝的顶端或中间均有泡囊形成；部分菌丝干瘪皱缩,原生质渗漏；还有部分菌丝出现扭曲集结的现象。该菌株的菌体悬浮液和无菌滤液均能诱发病原菌菌丝形态发生类似的畸变现象。该菌株能产生纤维素酶、蛋白酶和嗜铁素等抗菌物质。P-72-10菌株在烟草根部的定殖规律该菌株对抗生素利福平敏感,以利福平为标记抗生素,经不同浓度的利福平逐级选择压力诱导后,最终获得抗利福平(300μg/mL)的突变菌株P-72-10:Rifo该突变菌株的培养性状及对病原菌的拮抗能力与原始菌株无差异。通过种子细菌化,该突变菌株能成功地定殖于烟草幼苗的根部,其在根部的定殖动态呈现为先上升后下降的趋势；种子萌发出苗28天后,在烟草幼苗的根部仍能检测到P-72-10:Rif菌株,其定殖量维持在104cfu/g这一稳定的水平。扫描电镜定性观察发现P-72-10:Rif菌株在烟草幼苗根表不同部位的定殖分布呈现宏观不均匀性,根基部分布的菌体数量多于根中部,而根尖部分的细菌菌体数量最少。P-72-10菌株对烟草的促生效果及作用机制烟草种子经P-72-10菌株不同浓度的培养液浸种处理后,能促进幼根的伸长,其中,以10-1浓度促生效果最好,根长为0.53cm,而对照根长为0.47cm,与对照相比,增加了12.76%,且差异显著(P<0.05)；且种子的可溶性蛋白含量增加,总淀粉酶活性升高。培养液灌根处理烟草幼苗后,也能促进幼苗的生长及对N、P、K的吸收；增强光合效率、提高叶绿素含量；幼苗中丙二醛的积累量降低,与烟草植株抗性相关的防御酶(PPO、POD、PAL、几丁质酶和p-1,3-葡聚糖酶)活性升高,而各种防御酶活性的变化趋势不一致,其活性高峰出现时间与数量也有差异。P-72-10菌株的鉴定该菌株在King’s B培养基上菌落呈乳白色,且能产生水溶性的黄绿色荧光色素；为革兰氏阴性细菌,菌体杆状、大小(8.1-16.2)×(1.8-4.8)μm,单端生鞭毛,不形成芽孢。其最适生长温度为30℃；不耐盐；能利用柠檬酸盐和丙酸盐。接触酶反应阳性,氧化酶反应阳性,精氨酸双水解酶反应阳性,苯丙氨酸脱羧酶反应阴性,脂酶反应阴性,硝酸盐还原反应阴性；水解明胶和酪氨酸,但不水解淀粉；能发酵葡萄糖、木糖和甘露醇。该菌株基因组DNA的(G+C)mol%含量为60.72mol%,16SrDNA基因序列分析显示该菌株与假单胞菌属的荧光假单胞菌多个菌株的序列同源性达到99%,在GenBank上的登陆号为：HQ888871。因此,该菌株鉴定为荧光假单胞菌Pseudomonas fluorescens。P-72-10菌株摇瓶培养条件的优化摇瓶培养条件下,当pH为7.0、培养时间达52h、培养温度为25℃时,P-72-10菌株在标准发酵培养基中能获得最大量的抗菌物质,且其抑菌活性最高；该菌株的抗菌物质不能长时间地耐受高温处理。正交试验表明该菌株在摇瓶培养条件下获得抗菌物质的最佳发酵培养基组分配比为：葡萄糖23g,硫酸铵20g,硝酸钾5.5g,氯化钠0.5g,蒸馏水1000mL,pH为7.0。综上所述,在“根际细菌-植物-病原菌”这一微生态互作系统中,P-72-10菌株对烟草疫霉具有高效的拮抗作用,表现出良好的防病促生功效,且通过种子细菌化能成功地定殖于烟草根部,是一株具有应用潜力的生防荧光假单胞菌菌株。本研究也表明根际细菌在防治植物土传病害具有较强的应用优势和前景。更多还原

【Abstract】 Tobacco is an important cash crop. Tobacco black shank, caused by Phytophthora nicotianae Breda de Haan, is one of the most destructive of soil-borne disease in China and other major tobacco-producing areas throughout of the world. The pathogen caused damage to tobacco at all growth stages, with a range from minor injury to complete destruction on a tobacco plant. Currently, this disease is mainly controlled through the use of the fungicide metalaxyl. Nevertheless, increasing concerns about environmental problems caused either directly or indirectly by the use of fungicides and metalaxyl-resistant strains of Phytophthora support the need to find supplemental or alternative control methods such as biological control. Biological control of plant pathogens is an ecologically mind and environmentally safe alternative to chemical control. This study focuses on screening the most effective antagonistic strain against P. nicotianae from five rhizobacteria isolates; the antagonistic mechanisms against pathogen and biocontrol efficiency on black shank disease of this isolate; the colonization dynamics and distribution of this isolate along the roots of tobacco; the influence on the growth of tobacco and its physiological and biochemical mechanisms; the taxonomic position of this isolate and the optimal shake ferment condition of antagonistic compounds produced by this isolate.The detailed results of the study are shown as follows.Screening the most effective antagonistic strain P-72-10Five antagonistic rhizobacteria isolates showed significant suppressive effect on P. nicotianae. Among these isolates, strain P-72-10had the highest suppressive effect, which produced an inhibition zone of13.0mm (radius) and a growth suppression of the fungus at68.57%on the dual culture. It was found in the study that the growth of pathogen’s hypha was also suppressed by the extracellular metabolites of strain P-72-10. With the concentrations of extracellular metabolites increasing from10-3to1×, the inhibitory effect enhanced with a percentage from24.92%to46.03%. As compared with the control, the increasing variance was much significant (P<0.05).Biocontrol effect of strain P-72-10on black shank disease When tested on the tobacco plants grown in green house, strain P-72-10reduced disease incidence and declined the disease index significantly. This strain got a biocontrol effect on tobacco black shank disease infection and the effect on the resistant and susceptible varieties of tobacco were53.57%and66.37%respectively.Antagonistic mechanisms against Phytophthora nicotianae of strain P-72-10The volatile metabolites produced by strain P-72-10suppressed the growth of pathogen at32.94%. Strain P-72-10was observed microscopically to cause excessive branching of the fungal mycelia and malformation of mycelia tips; the hypha of pathogen could not grow normally and they formed chlamydospore-like, thick-walled cells with concentrated plasmas. However, the cell wall of normal hypha was even and smooth, and the form was full with the homogeneous and transparent cytoplasm. On the plate culture, strain P-72-10excreted proteinase, celluase, and siderophore, but not chitinase.Colonization dynamics and distribution along tobacco roots of strain P-72-10To study colonization along tobacco roots, strain P-72-10was tagged with antibiotics rifampicin capable of constitutive expression of antibiotics resistance on the King’s B medium plate. The rifampicin-tagged bacteria strain colonized successfully on the tobacco roots following seeds inoculation with the rifampicin-tagged bacteria strain. The dynamics of colonization increased first, then decreased and also the considerable quantity of the rifampicin-tagged bacteria strain maintained for a while with an amount level of10cfu/g. The observations by scanning electron microscopy Scanning electron microscopy (SEM) showed that colonization pattern of rifampicin-tagged bacteria strain on the tobacco roots was inhomogeneity. Colonization on the basal part of root reached the maximum, the middle root was less, and then the upper root.Physiological and biochemical influence of strain P-72-10on tobacco seeds and seedlings The root elongation of tobacco was significantly increased for the seed with the treatment of inoculation with strain P-72-10(0.53cm vs0.47cm, P<0.05), and increased by12.76%comparing with the treatment of control. The contents of soluble protein and total amylase in seeds were also increased. Strain P-72-10promoted growth, enhanced photosynthetic rate, increased chlorophyll content and nutrient absorption of tobacco seedlings. MDA content of tobacco seedlings was decreased and defense enzymes related to resistance of plant were induced after treatment with root irrigation, such as PPO, POD, PAL, chitinase and beta-1,3-glucanase. The change trend of different enzymes was inconsistent, and also the activities peak and quantity of enzymes were various with the changement of time.Taxonomic position of strain P-72-10The colonies were cream in color on King’s B culture. Cells were gram-negative, rod-shaped [(8.1-16.2)×(1.8-4.8) μm], motile owing to one or several polar flagella and did not form endospores. The growth temperature for strain P-72-10ranged from4to41℃with the optimum at30℃. The results of BIOLOG GN2showed that strain P-72-10belonged to Pseudomonas. The genomic G+C mol content was60.72mol%. Phylogenetic analysis of the16S rDNA gene sequence revealed that P-72-10was the most closely related to Pseudomonas fluorescens, with the sequence similarity of99%. Nucleotide number of the accession was HQ888871. Taxonomically, strain P-72-10was identified as P. fluorescens.Shake flask conditions of strain P-72-10In shake flask conditions, the factors influence strain P-72-10growth, included pH, time and temperature with the optimum pattern at pH7.0,52h and25℃. The culture ingredients were optimized as follows:glucose (23g), ammonium sulphate (20 g), potassium nitrate (5.5g), sodium chloride (0.5g), distilled water (1000mL), pH (7.0).In conclusion, results showed that strain P-72-10had antagonistic activity against P. nicotianae and biological control effect on tobacco black shank as well as plant growth-promoting potential to tobacco and root colonization. Strain P-72-10will also be used as a potential biocontrol agent to reduce the disease incidence and protect the environment.更多还原