论文标题:海洋微生物及其活性物质防治植物病害的研究
Study on Marine Microorganism and Its Antifungal Substances to Control Plant Disease
论文作者
论文导师 陈志谊,论文学位 硕士,论文专业 植物病理
论文单位 南京农业大学,点击次数 284,论文页数 96页File Size6088K
2007-06-01论文网 http://www.lw23.com/lunwen_209689397/
Marine microorganism;; Antifungal strains;; Plant pathogenic fungi;; Crude antifungal proteins;; Chitinase;; 16S rDNA
从连云港海域的海水和海泥中分离到了644株海洋细菌，以水稻纹枯病菌为指示菌，经过初筛、纯化、定量复筛获得11株抑菌效果较好的海洋细菌。室内抑菌谱测定结果表明，海洋细菌Ns-sw-6、Ps-sw-20和Ps-sw-21对8种植物病原真菌有很好的抑制作用，分别是白菜黑斑病菌(Alternaria brassicae)、稻瘟病菌(Pyricularia grisea)、番茄灰霉病菌(Botrytis cinerea)、水稻纹枯病菌(Rhizoctonia solani)、油菜菌核病菌(Sclerotinia sclerotiorum)、辣椒炭疽病菌(Colletotrichum capsici)、水稻恶苗病菌(Fusarium moniliforme)和辣椒疫霉(Phytopthora capsici)。菌株MG-sm-3，PY-sw-1，PY-sw-9对7种病原真菌有较好的抑制效果，而对辣椒疫霉(Phytopthora capsici)的抑菌活性很弱。产几丁质酶测定结果表明，在11株海洋源拮抗细菌中，菌株Ps-sw-20产几丁质酶。 盆栽和田间小区试验测定海洋源拮抗细菌对水稻纹枯病的控病能力，探索海洋源拮抗细菌在生物防治中的应用前景。试验结果表明，海洋细菌PY-sw-1及其胞外物质在盆栽和田间对水稻纹枯病的防治效果最好，盆栽试验7天和15天后调查的菌体防效分别为63.8％和61.1％，分别比陆源生防菌Bs-916的防效提高了13.73％和25.46％，其胞外物质的防效分别为61.2％和55.5％，比陆源生防菌在Bs-916的防效分别提高了25.41％和24.16％；两次调查菌株PY-sw-1菌体在田间的防效分别为62.2％和58.3％，比陆源生防菌Bs-916的防效提高了22.96％和41.16％，其胞外物质的防效分别为53.9％和50.8％，比陆源生防菌Bs-916的防效提高了28.64％和35.83％。盆栽和田间试验结果表明，海洋源拮抗菌对植物病害具有一定的防治效果，在植物病害生物防治方面具有潜在的应用价值。 用硫酸铵沉淀法提取11株海洋源拮抗细菌的胞外粗蛋白质，抑菌活性结果表明，菌株PY-sw-1的胞外蛋白提取物对7种植物病原真菌具有较强的抑制作用，它们分别是白菜黑斑病菌(A.brassicae)、稻瘟病菌(P. grisea)、番茄灰霉病菌(B.cinerea)、水稻纹枯病菌(R.solani)、油菜菌核病菌(S. sclerotiorum)、辣椒炭疽病菌(C.capsici)和水稻恶苗病菌(F. moniliforme)，对辣椒疫霉(P. capsici)没有抑制作用。菌株Ls-sm-1、Ps-sm-9、Ns-sm-1、Ps-sm-7、MG-sm-2和MG-sm-3的硫酸铵提取物对A. brassicae有较弱的活性，而对4种病原真菌C. capsici, P. capsici, F. monilloforme和P. grisea没有抑菌活性；菌株Ns-sw-6、Ps-sw-20、Ps-sw-21和PY-sw-9的硫酸铵提取物对8种病原真菌均无抑菌活性。考马斯亮兰G250测定各菌株胞外蛋白浓度的结果表明，菌株Ps-sw-20和Ps-sw-21的胞外蛋白浓度最高，分别为2.509 g／L和2.203 g／L，它们对水稻纹枯病菌没有抑制作用；菌株PY-sw-1产生的胞外蛋白质浓度为1.255 g／L，抑菌活性结果表明，其对7种植物病原真菌有较强烈的抑制作用。海洋细菌PY-sw-1产生的粗抗菌蛋白对植物病原真菌的抑制作用主要表现在菌丝生长受抑制或萎缩、菌丝扭曲、顶端膨大，原生质外泄等。 海洋细菌PY-sw-1最佳生长NaCl浓度为3％左右，能够耐受最高NaCl浓度为14％；其最佳生长pH为6.75；其最适生长温度范围为25～35℃，28℃时抗菌物质的产量最高。用硫酸铵沉淀法提取获得的该菌株的胞外粗蛋白在37℃、经蛋白酶K处理30min后，其活性下降了64.49％，木瓜蛋白酶、胃蛋白酶和胰蛋白酶处理后仍具有部分抑菌活性，初步推断海洋细菌PY-sw-1产生的抗菌物质可能包含蛋白类。温度高于50℃时，该粗抗菌蛋白活性下降，高温高压(120℃，0.1～0.5mpa)处理20 min后其活性下降了71.97％；在pH 7.0时其抑菌活性最强，pH 2和12时其抑菌活性几乎丧失。这些结果表明海洋细菌PY-sw-1产生的粗抗菌蛋白抑菌活性稳定、适应范围广，其在防治植物病害方面将具有重要的作用。 通过比较分析16S rDNA基因序列，海洋细菌PY-sw-1被鉴定为Bacillus sp.，与B. subtilis最为接近，有99.9％的相似性；产几丁质酶菌株Ps-sw-20被鉴定为Pseudoalteromonas sp.，与P. flavipulchra有99.6％的相似性。分类鉴定结果为深入研究海洋细菌及其抗菌物质防治植物病害奠定了基础。
The 644 strains of marine bacteria were isolated from sea mud and water collectedfrom Lian Yungang sea area, and 11 strains with antifungal activity were screened by thefirst and second screening, purifying and fix quantify screening. The antifungal spectrumsof 11 strains against 8 plant pathogenic fungi were tested and the results showed that thestrains Ns-sw-6, Ps-sw-20 and Ps-sw-21 were wide antifungal spectrum and had strongantifungal activity against 8 plant pathogenic fungi, including Alternaria brassicae,Pyricularia grisea, Botrytis cinerea, Rhizoctonia solani, Sclerotinia sclerotiorum,Colletotrichum capsici, Fusarium moniliform and Phytopthora capsici. The strainsMG-sm-3, PY-sw-9 and PY-sw-1 also had high antifungal activity against 7 plantpathogenic fungi except P. capsici. Among 11 antifungal marine bacteria, only the strainPs-sw-20 can produce chitinase. In order to know whether the marine-derived antagonistic bacteria could be applied tobiocontrol or not, it was tested to control Rice Sheath Blight in pots and field. The resultsdemonstrated that the marine bacterium PY-sw-1 was a good biocontrol agent in controllingRice Sheath Blight in pots and field. The control efficacy of PY-sw-1 were 63.8 % and 61.1%, increased by 13.73 % and 25.46 % over those of Bs-916 which was an effectivebiocontrol agent from the soil after investigating in the pots for 7 and 15 days.Simultaneously, its extracellular substances" were 61.2% and 55.5 %, and increased by25.41% and 24.16 % compared with Bs-916"s. In the field test, the control efficacy of thestrain were 53.9% and 50.8%, and increased by 22.96% and 41.16% compared withBs-916"s. Its extracellular substances" were 53.9 % and 50.8 %, and increased by 28.64 %and 35.83 % respectively. The results in the pots and field indicated that marine-derivedantagonistic bacteria had control efficacy against plant disease and potential usefulness inbiocontrol. The fermentational broth of PY-sw-1 precipitated by Solid Ammonium Sulphate werehighly active against 7 plant pathogenic fungi, involved A. brassicae, P. grisea, B. cinerea, R.solani, S. sclerotiorum, C.capsici and E.moniliforme, but not against P.capsici. Althoughthe extracellular protein concentration of Ps-sw-20 and Ps-sw-21 were higher than others"testing by Coomassie Brilliant Blue G250, they were not against 8 plant pathogenic fungi.Conversely, the extracellular protein concentration of PY-sw-1 was approximately half ofPs-sw-20"s, but had strong inhibited effect against 7 plant pathogenic fungi. The others"were weak or lost their activity against plant pathogenic fungi. The morphological toxicityof the crude antifungal proteins from PY-sw-1 to plant pathogenic fungi was revealed thatthe growth of mycelia was inhibited and atrophic, mycelia apex tumescence, distortion,tumescence and rupture. The optimum salinity and pH value of the growth of PY-sw-1 were about 3% and 7respectively, the range of optimum temperature was 25～35℃and the yield of theantifungal substances was maximal at 28℃. The antifungal activity of crude substanceswas decreased by 64.49% after treated with Protease K for 30min at 37℃, and kept partialactivity after treated with Papain, Pepsin and Trypsin. These results showed that thecomponents of the antifungal substance produced by PY-sw-1 might contain proteins. Theantifungal activity was highest when pH value was 7.0, however, and almostly lost fullantifungal activity when pH value was 2.0 or 12.0. The antifungal activity was decreased by71.97% by treatment with high temperature and pressure for 20 min. These results indicatedthat the antifungal activity of the crude antifungal proteins from the marine bacteriumPY-sw-1 was stable and broad-spectrum antifungal activity and might play an importantrole in biocontrol plant disease. By 16S ribosomal DNA gene sequence analysis, the antifungal strain PY-sw-1 wasindentified as a Bacillus ap., which is close to B.subtilis with 99.9% sequence similarity.The other marine bacterium, Ps-sw-20, which could produce chitinase, was indentified as aPseudoalteromonas sp.. It is close to P.flavipulchra with 99.6% sequence similarity. Theseresults made a foundation for studying marine bacteria and their antifungal substances oncontrolling plant disease further.
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