【作者】 李红梅；

【导师】 王树桐； 曹克强；

【作者基本信息】 河北农业大学 ， 植物病理学， 2009， 硕士

【摘要】 近年来,板蓝根根腐病在一些主产区发生逐年加重,成为板蓝根生产的限制因素之一。本研究试图从从多种作物的根际土壤和中草药粗提液中筛选到能够用于防治板蓝根根腐病的有益生防物质,为板蓝根根腐病的有效控制提供新的线索。以板蓝根根腐病的主要致病菌-茄腐镰刀菌为指示菌,对从全国各地采集的137个土壤样品进行了拮抗微生物的分离筛选,获得了201株拮抗微生物,其中拮抗细菌172株,真菌29株。经过平板对峙培养,获得了对茄腐镰刀菌的抑制效果最好的3个菌株Bs-28,Bs-84和B1-105。采用常规方法结合分子生物学方法对Bs-28、Bs-84和Bl-105菌株进行了鉴定。根据Bs-28、Bs-84和Bl-105的16S rDNA序列与芽孢杆菌的同源性,初步将菌株Bs-28、Bs-84和Bl-105鉴定为芽孢杆菌。又根据菌株Bs-28、Bs-84和Bl-105的形态特征、培养性状以及生理生化反应,根据伯杰氏细菌鉴定手册（第八版）,最终将Bs-28和Bs-84鉴定为枯草芽孢杆菌（Bacillus subtilis）的两个菌株,将菌株Bl-105鉴定为地衣芽孢杆菌（Bacillus licheniformis）。对菌株Bs-28、Bs-84和Bl-105的培养条件进行了优化。①Bs-28的优化培养条件为:碳源为2%的玉米粉,氮源为2%的酵母粉,无机盐为0.2%磷酸二氢钠+磷酸氢二钠;最适装液量为30 mL/300 mL,最适初始接种量为1 mL,最适初始pH值为7.5;②Bs-84的优化培养条件为:碳源为2%的玉米粉,氮源为2%的酵母粉,不添加无机盐,最适装液量为10 mL/300 mL,最适初始接种量为1.5 mL,最适初始pH值为8.0;③Bl-105的优化培养条件为:碳源为0.5%的玉米粉,氮源为1%的酵母粉,无机盐0.4%MgSO₄,最适装液量为10 mL/300 mL,最适初始接种量为1.5 mL,最适初始pH值为6.0。以茄腐镰刀菌为指示菌,测试了123种中草药粗提液对菌丝生长及分生孢子萌发的抑制作用。结果表明:粗提液浓度为0.01 g/mL时,有99种粗提液对菌落生长具有显著的抑制作用,其中北沙参的抑制率达到了100%;苦楝皮和威灵仙的抑制率大于50%,有112种粗提液对分生孢子萌发具有显著的抑制作用,其中11种粗提液的抑制率在50%以上,而且北沙参和牛膝的抑制率达到100%。将抑制率大于50%的11种粗提液稀释为0.005g/mL,进行二次筛选,发现北沙参对菌落生长抑制率仍为100%;0.005 g/mL北沙参粗提液对分生孢子萌发抑制率为74.50%,苦楝皮的次之为59.39%。北沙参和苦楝皮对菌丝生长的抑制中浓度(EC₅₀)为3.62 mg/mL和6.63mg/mL。将拮抗微生物与植物提取液混合发酵,分别测定了混合和单一的拮抗菌发酵液对茄腐镰刀菌的菌丝生长的抑制作用。结果显示,混合发酵处理“Bs-28+Bs-84+Bl-105+北沙参”效果最优。2008年在河北唐山进行的田间试验测试了3株拮抗微生物和2种植物提取物对板蓝根根腐病的防治效果。结果表明:拮抗细菌和中草药粗提物处理对板蓝根根腐病均具有显著防治效果,而拮抗菌株Bs-28和Bs-84还具有显著的增产作用。其中Bs-28的防效最好为49.76%,增产47.03%。更多还原

【Abstract】 Isatis indigotica root rot disease is found to be more and more serious in production areas of China recent years.In the present study,we tried to screen plant extract and effective antagonistic microorganisms from soil samples collected from different crop rhizosphere.The aim is to provide a new clue for controllingⅠ.indigotica root rot disease effectively.Fusarium solani was confirmed as the main causal agent ofⅠ.indigotica root rot disease in previous study.One hundred and seventy two out of the tested 201 isolates are bacteria isolates,the other 29 are fungal isolates.All the selected isolates were tested against F.solani for their antagonistic effect.Bacteria isolates named as Bs-28,Bs-84 and B1-105 showed strong antagonistic effect against F.solani.Antagonistic isolates Bs-28、Bs-84 and Bl-105 were identified by traditional and molecular biology methods.Bs-28、Bs-84 and Bl-105 strains were identified as Bacillus spp.by 16S rDNA sequence analysis.Furthermore,Bs-28 and Bs-84 were finally identified as Bacillus subtilis,Bl-105 was identified as Bacillus licheniformis based on the morphological characterization,cultural characters,physiology and biochemical characteristics according to Bergey’s Manual of Determinative Bacteriology（Eighth Edition）.The selected three isolates were fermentated.The optimized fermentation condition for Bs-28 was 2%corn meal,2%yeast extract,0.2%sodium dihydrogen phosphate （NaH₂PO₄） and disodium hydrogen phosphate（Na₂HPO₄）,broth content 10 ml/300 ml flask,initial inoculum 1 ml and initial pH 7.5;The optimized fermentation condition for Bs-84 was 2%corn meal,2%yeast extract,broth content 10 ml/300 ml flask,initial inoculum 1.5 ml and initial pH 8.0.The optimized fermentation condition for Bl-105 was 0.5%corn meal,1%yeast extract,0.4%magnesium sulfate（MgSO₄）,broth content 10 ml/300 ml flask,initial inoculum 1.5 ml and initial pH 6.0.The antifungal activity of ethanol extract of 123 species of Chinese herbs against F. solani was tested on in vitro.Results indicated that ninety nine species of Chinese herbs had significant inhibitory effect against colony growth at the concentration 0.01 g/mL.The inhibition ratio of Glehnia littoralis extract reached 100%,and that of Cortex meliae and Clematis chinensis were over 50%.One hundred and twelve species of Chinese herbs had significant inhibitory effect against conidial germination,inhibition rates of eleven extracts were over 50%,the inhibition rates of Glehnia littoralis and Radix Achyranthis bidentatae were 100%.Those extracts with inhibition rates over 50%were chosen for the secondary screening tests at the concentration of 0.005 g/mL.G.littoralis had 100%inhibitory effect in the second screening tests against colony development,while the the inhibition rate of G. littoralis against conidial germination was 74.50%.The corresponding inhibition rates of Cortex meliae were 59.59%and 53.46%.EC₅₀ of G.littoralis and Cortex meliae were 3.621 mg/mL and 6.631 mg/mL seperately.Three tested antagonistic microorganism isolates were fermentated together with G. littoralis and/or Cortex meliae extracts.The inhibition of the mixtures and single fermentation were tested against the mycelium growth of F.solani.The optimized fermentation was the combination Bs-28+Bs-84+Bl-105+G.littoralis.In field trial of 2008,the selected three isolates and two plant extracts were tested to controlⅠ.indigotica root rot disease in Tangshan of Hebei province.All the tested isolates and plant extracts showed control efficacy againstⅠ.indigotica root rot disease, antagonistic isolates Bs-28 and Bs-84 improvedⅠ.indigotica yield significantly. Antagonistic isolate Bs-28 gave the best control effect 49.67%and 47.03%yield increasing.更多还原