摘要
由真菌、细菌、线虫引起的植物病害的防治一直是农业生产上非常重要的环节。多数病害的防治主要依赖于化学农药,但化学农药的大量使用容易对环境造成污染,危害人畜健康,同时造成病原菌抗性不断上升。而生物防治对环境污染极少,人畜安全,有时对某些有害生物可以达到长期抑制的作用,使用方便,近年来受到了广大的关注。
产酶溶杆菌OH11菌株是一种新型的生防菌,其分类地位划分在黄单胞科,溶杆菌属。该菌富含G+C,有滑行运动,对由真菌、细菌引起的许多病害都有生防作用,应用前景广阔。为了大规模发酵,降低防治成本,本研究对OH11的摇瓶发酵条件进行了优化,并对OH11色素突变体进行了研究。
采用单因素试验法对产酶溶杆菌OH11液体发酵的主要影响因子温度、转速等进行了研究,确定了最佳培养条件:温度为30℃、转速为210 r·min~(-1);通过正交试验以LB为基础培养基对其培养基成分在摇瓶中进行了优化。在优化条件下,发酵培养基中活菌数在72 h时达到8.5×10~9 CFU·mL~(-1),明显高于原基础培养基的结果:72 h时达到1.2×10~9 CFU·mL~(-1);并通过正交试验对摇瓶装量、发酵时间、接种量、初始pH值4种因素进行了优化,优化后的结果为:装液量40 mL/250mL,发酵时间72 h,接种量10~8 CFU,初始pH值7.5。并且测定了产酶溶杆菌OH11在不同条件下的存活情况。
利用mariner转座子对菌株OH11进行转座诱变,成功构建了OH11的转座子插入文库。通过表型筛选,从2000多株突变株中筛选到一株色素突变株OH11-1。在LB固体和液体培养基中,OH11-1能产生黑色素。通过亚克隆的方法,我们鉴定了mariner转座子在染色体上的插入位点为hmgA基因。利用自杀载体pEX18GM,通过单交换的方式,构建了hmgA基因的插入失活突变株OH11-2,菌株OH11-2在表型上与OH11-1相一致。通过基因互补,构建了OH11-2的互补菌株OH11-3,OH11-3在表型上恢复了野生型的性状(即在固体和液体LB中不产生黑色素)。利用高效液相色谱技术(HPLC),鉴定了黑色素为尿黑酸,这一结果与序列分析的结果相一致。对色素突变株OH11-2进行了胞外酶检测,平板抑菌试验和温室试验,结果表明:(1)OH11-2降低了蛋白酶和葡聚糖酶的分泌,但不改变几丁质酶和纤维素酶的外泌;(2)OH11-2在平板上不改变对水稻纹枯病菌(Rhizoctonia solani)的拮抗活性;(3)OH11-2降低了对番茄青枯病菌的生防活性。
The control of plant diseases,caused by fungi,bacteria,and nematodes is a key problem in agricultural production.Till now,many plant diseases were controlled by use of chemical pesticides,which resulted in environmental popullation and doing harm to human beings.Recently,biological control of plant diseases is more and more concerned because of its safety for environment and human beings.In addition, biological control is effective to control some plant diseases for a long time.
Members of the genus of Lysobacter are typically found in soil and water habits and are characterized by a high G+C content,gliding motility,and the ability to lyse other microbes,including other bacteria,fungi,and nematodes.One member of this genus,Lysobacter enzymogenes,characterized by the ability to produce extracellular lytic enzymes,including chitinases,β-1,3-glucanases,and proteases,has been described as potential biological control agents for plant diseases.In this study, fermentation condition optimization and characterization of a pigment mutant of Lysobacter enzymogenes strain OH11 was performed.
The fermentation of Lysobacter enzymogenes strain OH11 was investigated through single-factor test,the main factors include:temperature,rotate speed.The optimal fermentation conditions are:temperature 30℃,rotate speed 150 r/min.The orthogonal experiment was used to optimize the medium,and th number of strain OH11 were improved from 8.5×10~9 CFU·mL~(-1) to 1.2×10~9 CFU·mL~(-1) under optimal condition after 72 h.The fermentation condition of OH11,such as volume, fermentation time,inoculum's size and pH value before sterilized were tested by orthogonal experiment.The optimum fermentation conditions are as follows:volume 40 ml/250 ml,fermentation time 72 h,inoculum size 10~8 CFU,pH7.5 before sterilized, and studied the livability of Lysobacter enzymogenes strain OH11 under different conditions.
The mutant library of Lysobacter enzymogenes strain OH11 was successfully constructed by mating mariner transposon into strain OH11.One pigment mutant OH11-1,which could produce dark-brown pigment on LB plate and in LB broth,was selected by the change of colony morphology.The transposon insertion site was identified as hmgA gene by sub-clone strategy.A hmgA disruption mutant OH11-3, which had the same phenotype as strain OH11-1,was constructed by a plasmid insertion inactivated protocol.The brown-pigment was identified as homogentisate by HPLC method,which was in accordance with that of sequence analysis.The results of detection of lytic enzymes,antifungal activity and greenhouse experiment indicated that:(ⅰ) strain OH11-2 had decreased ability to produce protease andβ-1, 3-glucanases,but retained that to produce chitinase and cellulase;(ⅱ) strain OH11-2 retained the antifungal ability against Rhizoctonia solani compared to wild type strain OH11;(ⅲ) strain OH11-2 displayed significantly decreased effect on biocontrol tomato wilt disease in green house compared to wild type strain OH11.
引文
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