海洋微生物抗稻瘟菌（Pyricularia grisea）活性菌株的筛选
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摘要
水稻稻瘟病是全世界稻区危害最严重的水稻病害之一，本文报道从海洋取样，进行微生物分离，采用一种以稻瘟菌孢子生长异常为指标的筛选模型，筛选抗稻瘟菌的活性菌株的研究结果。
通过采用高氏一号、伊莫逊氏、土壤浸汁培养基、M1培养基、YE琼脂、V8培养基、淀粉培养基、马丁培养基等不同的培养基，采用热处理、SDS处理、苯酚处理、几丁质富集培养、氯霉素富集培养等几种预处理方法，并在分离培养基中加入相应的抑制剂，对采自南海、西沙、北部湾、海南岛近海的71份样品进行菌株分离，共得到646株海洋微生物菌株。其中细菌297株，丝状真菌111株，放线菌238株。
包括本实验室其他合作者筛选得到的菌株共1593株作为筛选抗稻瘟菌活性菌株的待筛菌株，采用96-孔板法和琼脂挖块法，初筛共获得对稻瘟菌有不同程度活性的微生物共468株，活性菌株比率达29.4％。经SAS 8.2统计，分析了样品类型等七种因素对初筛活性菌株比率的影响，结果表明，样品类型、琼脂挖块法中的对峙时间、菌株分离时对样品的预处理、微生物种类的影响达极显著，采样地点的影响达显著，菌株分离时的培养基、测活方法的影响不显著。
在初筛468株活性菌株的基础上，第一次复筛选出240株，第二次复筛选出84株仍然表现对稻瘟菌的抑制活性；第三次复筛选出高活性菌株14株(包括特别有潜力的7株，其中，菌株031057热稳定性和遗传稳定性均表现较好。)
活性菌株筛选中，使用的两种测活方法均适合作为初筛，其中琼脂挖块法的假阴性率较高，96-孔板法的假阳性率稍高。
抗稻瘟菌的高活性菌株031057是从南海海沙中分离到的一株属于G+、内生芽孢、不运动的杆状细菌。
菌株031057所产抗生素粗品的效价：相当于潮霉素B的100mg／mL；经
海洋微生物抗稻瘟菌（卸ricularia grLSea）活性菌株的筛选
24 h发酵，菌体生长达最大密度，其最大生长密度为 3．68 X 10’Cfu／InL，而其
产生活性物质是在sd后达到最大值。
不同通气量对活性菌株03 057发酵产生活性物质的影响不大，它可耐受
5人2％的盐，但它产生活性物质并不依赖于盐的存在，且以不添加盐的蒸馏水
配制的黄豆粉培养基进行发酵，效价最高。
经5种培养基发酵的定性试验，03 1057在黄豆粉培养基中生长最好，产
活最高。
菌株031057所产活性物质热稳定性、干燥加工稳定性、常温贮藏稳定性
均较好，但酸碱稳定性较差。菌株031057主要产胞外水溶性活性物质，这种
物质可以被无水乙醇和丙酮沉淀，但无水乙醇使之变性，丙因使之部分变性；
另外，有机溶剂可以导致该物质失活，初步推测该物质可能为杂蛋白类物质。
菌株 03 1057的抗菌谱较窄，其所产抗生素粗品 500倍液能有效防治盆栽
水稻稻瘟病，且对水稻不会造成药害。
Rice blast is one of the most severe disease of the rice. This thesis reported screening on marine microorganisms antagonized Pyricularia grisea, using a screening model based on observing inhibiton of the co'udiospore of P. grisea.
Seventy-one marine samples were collected from South Sea, Xisha, North Bay and near shore coast of Hainan Island. During the isolation procedure, some effective media, such as Cause's Synthetic Medium, Emerson's Medium, Soil Extract Agar, Ml Medium, YE Agar, V8 Vegetable Juice Agar, Starch Agar Medium, Martin Medium and so on, and pretreatments, as heat, SDS, phenol, chitin enrichment, chlormycetin enrichment were conducted and some inhibition agents were added into the media. Six hundred and forty-six strains were isolated, including 297 of bacteria, 111 of filamentous fungi and 238 of actinomycetes.
Among 1593 isolates to be screened, including 947 isolates isolated by collaborators in this project, 468 active isolates showed primarily bioactivity against rice blast in the laboratory, based on two screening methods and the percentage of active isolates was 29.4%.
Effects of seven factors on the percentage of active isolates obtained were tested using SAS 8.2 statistic software. Effects of sample types, pretreatments, microorganism types and antagonizing time of agar diffusing method on percentage of active isolates reached the greatly significant level. Sample locations showed significant effects. Media used for isolations and different screening methods showed low effects on percentage of active isolates.
Eighty-four isolates still showed strong bioactivity against rice blast after the third screening. After the fourth screening, 14 isolates still showed high activity, including 7 very high isolates. Among them, strain 031057 showed stable bioactivity.
Both the two screening methods were suitable to be used as the primary screening methods. Between them, Agar Diffusing Methods showed higher false negative rate in detection, and 96-well Plate Method showed higher false positive rate.
Strain 031057 was isolated from sea sand, collected from the South Sea. It was a bacterium with endospore, not movable, belonged to G+.
Relative inhibition concentration of the bioactivity produced by 031057 was 100 mg/mL corresponding to hygromycin B. Its maximum cell density was 3.68+07 CPU /mL after fermentation for 24 h and reached its maximum bioactivity after 5 d.
Bioactivity of 031057 was not affected by aeration. It could stand salinity up to 5.42%, but its bioactivity producing was not dependant salt. It produced maximum bioactivity in Soybean Liquid Mdium without salt.
Among 5 media, strain 031057 produced maximum bioactivity in Soybean Liquid Mdium.
Bioactive substance produced by strain 031057 was stable at heat, preservation under room temperature, dry procession, but not stable at acid and alkali conditions. Strain 031057 produced water-soluble extracellular bioactive substance, which was deposited by ethanol and acetone and denaturalized completely by ethanol and half denaturalized by acetone. Organic agent inactivated it. The active substance was primarily speculated to be a hybri-protein.
Strain 031057 showed a narrow inhibition range and its control on the rice blast in the lab was at 1/500 concentration. It did not cause damage to the rice.
通过采用高氏一号、伊莫逊氏、土壤浸汁培养基、M1培养基、YE琼脂、V8培养基、淀粉培养基、马丁培养基等不同的培养基，采用热处理、SDS处理、苯酚处理、几丁质富集培养、氯霉素富集培养等几种预处理方法，并在分离培养基中加入相应的抑制剂，对采自南海、西沙、北部湾、海南岛近海的71份样品进行菌株分离，共得到646株海洋微生物菌株。其中细菌297株，丝状真菌111株，放线菌238株。
包括本实验室其他合作者筛选得到的菌株共1593株作为筛选抗稻瘟菌活性菌株的待筛菌株，采用96-孔板法和琼脂挖块法，初筛共获得对稻瘟菌有不同程度活性的微生物共468株，活性菌株比率达29.4％。经SAS 8.2统计，分析了样品类型等七种因素对初筛活性菌株比率的影响，结果表明，样品类型、琼脂挖块法中的对峙时间、菌株分离时对样品的预处理、微生物种类的影响达极显著，采样地点的影响达显著，菌株分离时的培养基、测活方法的影响不显著。
在初筛468株活性菌株的基础上，第一次复筛选出240株，第二次复筛选出84株仍然表现对稻瘟菌的抑制活性；第三次复筛选出高活性菌株14株(包括特别有潜力的7株，其中，菌株031057热稳定性和遗传稳定性均表现较好。)
活性菌株筛选中，使用的两种测活方法均适合作为初筛，其中琼脂挖块法的假阴性率较高，96-孔板法的假阳性率稍高。
抗稻瘟菌的高活性菌株031057是从南海海沙中分离到的一株属于G+、内生芽孢、不运动的杆状细菌。
菌株031057所产抗生素粗品的效价：相当于潮霉素B的100mg／mL；经
海洋微生物抗稻瘟菌（卸ricularia grLSea）活性菌株的筛选
24 h发酵，菌体生长达最大密度，其最大生长密度为 3．68 X 10’Cfu／InL，而其
产生活性物质是在sd后达到最大值。
不同通气量对活性菌株03 057发酵产生活性物质的影响不大，它可耐受
5人2％的盐，但它产生活性物质并不依赖于盐的存在，且以不添加盐的蒸馏水
配制的黄豆粉培养基进行发酵，效价最高。
经5种培养基发酵的定性试验，03 1057在黄豆粉培养基中生长最好，产
活最高。
菌株031057所产活性物质热稳定性、干燥加工稳定性、常温贮藏稳定性
均较好，但酸碱稳定性较差。菌株031057主要产胞外水溶性活性物质，这种
物质可以被无水乙醇和丙酮沉淀，但无水乙醇使之变性，丙因使之部分变性；
另外，有机溶剂可以导致该物质失活，初步推测该物质可能为杂蛋白类物质。
菌株 03 1057的抗菌谱较窄，其所产抗生素粗品 500倍液能有效防治盆栽
水稻稻瘟病，且对水稻不会造成药害。
Rice blast is one of the most severe disease of the rice. This thesis reported screening on marine microorganisms antagonized Pyricularia grisea, using a screening model based on observing inhibiton of the co'udiospore of P. grisea.
Seventy-one marine samples were collected from South Sea, Xisha, North Bay and near shore coast of Hainan Island. During the isolation procedure, some effective media, such as Cause's Synthetic Medium, Emerson's Medium, Soil Extract Agar, Ml Medium, YE Agar, V8 Vegetable Juice Agar, Starch Agar Medium, Martin Medium and so on, and pretreatments, as heat, SDS, phenol, chitin enrichment, chlormycetin enrichment were conducted and some inhibition agents were added into the media. Six hundred and forty-six strains were isolated, including 297 of bacteria, 111 of filamentous fungi and 238 of actinomycetes.
Among 1593 isolates to be screened, including 947 isolates isolated by collaborators in this project, 468 active isolates showed primarily bioactivity against rice blast in the laboratory, based on two screening methods and the percentage of active isolates was 29.4%.
Effects of seven factors on the percentage of active isolates obtained were tested using SAS 8.2 statistic software. Effects of sample types, pretreatments, microorganism types and antagonizing time of agar diffusing method on percentage of active isolates reached the greatly significant level. Sample locations showed significant effects. Media used for isolations and different screening methods showed low effects on percentage of active isolates.
Eighty-four isolates still showed strong bioactivity against rice blast after the third screening. After the fourth screening, 14 isolates still showed high activity, including 7 very high isolates. Among them, strain 031057 showed stable bioactivity.
Both the two screening methods were suitable to be used as the primary screening methods. Between them, Agar Diffusing Methods showed higher false negative rate in detection, and 96-well Plate Method showed higher false positive rate.
Strain 031057 was isolated from sea sand, collected from the South Sea. It was a bacterium with endospore, not movable, belonged to G+.
Relative inhibition concentration of the bioactivity produced by 031057 was 100 mg/mL corresponding to hygromycin B. Its maximum cell density was 3.68+07 CPU /mL after fermentation for 24 h and reached its maximum bioactivity after 5 d.
Bioactivity of 031057 was not affected by aeration. It could stand salinity up to 5.42%, but its bioactivity producing was not dependant salt. It produced maximum bioactivity in Soybean Liquid Mdium without salt.
Among 5 media, strain 031057 produced maximum bioactivity in Soybean Liquid Mdium.
Bioactive substance produced by strain 031057 was stable at heat, preservation under room temperature, dry procession, but not stable at acid and alkali conditions. Strain 031057 produced water-soluble extracellular bioactive substance, which was deposited by ethanol and acetone and denaturalized completely by ethanol and half denaturalized by acetone. Organic agent inactivated it. The active substance was primarily speculated to be a hybri-protein.
Strain 031057 showed a narrow inhibition range and its control on the rice blast in the lab was at 1/500 concentration. It did not cause damage to the rice.
引文
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