【作者】 刘涛；

【导师】 蒋士君；

【作者基本信息】 河南农业大学 ， 植物病理学， 2009， 硕士

【摘要】 烟草病毒病是一类在烟草上普遍发生且危害严重的侵染性病害,也是世界范围内的重要病害之一。促成烟草病毒病发生的因素较多,防治困难较大,防治研究进展较慢,被称为“烟草癌症”,目前还没有很好的防治措施。近年来,烟草根际微生物研究越来越受到人们的重视。通过改善烟草根际微生态环境来促进烟草生长以及从根际微环境中筛选具有良好促生和抗菌作用的有益菌群,日益为人们所重视。很多土壤细菌都能通过非核糖体途径合成并分泌一种或几种铁载体。铁载体(siderophores)是一类具有很强的特异螯合Fe3＋的小分子化合物,它的产生是地球上的微生物适应地球地壳高氧低铁环境的结果。细菌利用合成的铁载体攫取外界游离Fe3+来维持正常的生理活动。环境中游离的铁载体也可以为其他细菌所用,细菌在环境中的生存地位也可以通过其对环境中铁源的争夺能力而反映出来。因此对自然条件下产铁载体菌的研究能够帮助我们解决许多因细菌引起的植物病害问题。本试验拟以烟草根围产铁载体细菌为研究对象,采用生物测定法结合分子生物学方法,从烟草根围分离并鉴定产铁载体细菌；通过对产铁载体细菌进行培养纯化,然后接菌至无菌苗根部及盆栽烟草根部,一方面研究产铁载体细菌对烟草生长的影响,另一方面研究产铁载体细菌对提高烟草抗病毒病能力的积极作用。从而为找到防治烟草病毒病新途径奠定基础。主要结果包括：1)在平顶山郏县的烟田土样中,分离到19种不同类型的细菌分离物,经过鉴定,分离物JX001、JX002、JX003、JX004、JX005、JX006、JX008、JX0011为产铁载体细菌。其中分离物JX003、JX004、JX006、JX008经16s rDNA鉴定,JX004为绿真假单孢菌,JX003和JX006属于假单胞菌属的待定种,而JX008未鉴定出属种,属于未知菌。此8株产铁载体细菌均来自病毒病轻微发生的烟株根围土样,而非根围土样和病毒病严重发生的烟株根围土样中未分离出产铁载体细菌。多次分离结果初步表明,烟草根围土壤中的产铁载体细菌与烟株抗病毒病之间有一定的关系。2)产铁载体细菌能够显著促进烟草幼苗的生长。供试的4株产铁载体细菌(JX003、JX004、JX006、JX008)均能不同程度促进烟草幼苗的生长发育。经产铁载体细菌处理过的烟草幼苗在培养皿中生长健壮,生长势好,成活率高,明显好于对照。3)产铁载体细菌能促进烟草幼苗对铁营养的吸收利用。高价铁的高度不溶解性使铁很难被植物吸收利用,铁的可给性非常低,而Fe2+更容易被植物吸收利用,因此,在高价铁的环境中,烟草幼苗很难生长存活。试验结果表明,经产铁载体细菌处理过的高价铁的MS培养基中的烟草幼苗仍能健康生长,而且生长好于对照。这说明产铁载体细菌能将环境中的高价铁转化为可供烟草育苗吸收利用的铁的形式,能促进烟草幼苗对铁营养的吸收利用。4)产铁载体细菌对烟草地上部分和根系的生长发育具有显著的促进作用。经产铁载体细菌JX003处理的烟株生长一个月的平均株高最高达8.35cm,而不经产铁载体细菌处理的烟株平均株高最高为5.85cm；经产铁载体细菌JX004处理的烟株生长一个月的平均株高最高达12.13cm,而不经产铁载体细菌处理的烟株平均株高最高为9.48cm；经产铁载体细菌JX006处理的烟株生长一个月的平均株高最高达11.33cm,而不经产铁载体细菌处理的烟株平均株高最高为8.7cm；经产铁载体细菌JX008处理的烟株生长一个月的平均株高最高达12.1cm,而不经产铁载体细菌处理的烟株平均株高最高为8.8cm。经产铁载体细菌处理的烟株均具有发达的根系。5)产铁载体细菌能显著抑制烟草病毒病的发生。试验结果表明,经产铁载体细菌处理的烟株在接种后18d才开始发病,比正常处理的烟株发病晚7d左右,病情指数5-13.8。与不经产铁载体细菌处理的烟株的病情指数(13.9-27.8)之间存在极显著差异,且经产铁载体细菌处理的烟株其病毒病症状在生长后期有进一步减轻的趋势。总之,产铁载体细菌能促进烟草对铁营养的吸收,从而促进烟草幼苗的生长发育；还能促进盆栽烟草地上部分的生长和根系发育,并能显著提高烟株的抗病毒病能力。产铁载体细菌的这些生物活性及其作用机制值得进一步研究。更多还原

【Abstract】 Tobacco viral diseases are important infectious disease in tobacco production. There are many factors to aggravate the viral diseases, called "tobacco cancer", that lead to hard to be controlled and slow progress in disease research.. At present, there are not effective methods to be used. In recent years, the research on tobacco rhizosphere microbiology is becoming more and more attractive. It becomes more and more important that to better tobacco growth by using the beneficial bacteria to improve the eco-environment of tobacco rhizosphere.Many soil bacteria can secret more than one siderophores through non-ribosome pathway. Siderophore is a small molecule which can specifically chelated Fe3+ as a result of bio-adaption to the low ferric environment in the earth. The bacterium can use its siderophore to uptake free Fe3+ in the environment to support its normal life. For a microorganism, its capability to obtain free Fe3+ reflect the capability of competition. As a class of beneficial bacterium, the soderophore-producting rhizobacteria was worth to be researched to help us to control plant diseases.In this study, the main object is the siderophore-producting bacteria living in the tobacco rhizosphere. By biological and molecular biological methods, the siderophore-producting bacteria from the tobacco rhizosphere was isolated and identified. Inoculating their pure culture into tobacco rhizosphore, the development of tobacco seedling, roots and the effect on tobacco resistance to virus disease were investigated, for exploring new pathway to disease control. The main results include:1)19 bacterial isolates were obtained from tobacco field soil samples from Jiaxian, Pingdingshan.8 of them were identified to be siderophore-producting bacteria, their code were JX001, JX002, JX003, JX004, JX005, JX006, JX001, JX0011, respectively. According to the 16S rDNA-based identification, isolate JX004 belongs to Pseudomonas chlorosis, JX003 is Pseudomonas sp., JX006 is Pseudomonas sp. and JX008 is an unknown bacterium. All the 8 isolates come from rhizosphere soil of tobacco with slight mosaic symptom. While no siderophore-producting bacteria be obtained from that of tobacco which infected severe viral disease. The same result reproduced in many repeated testment showed that the siderophore-producting bacteria was related with tobacco health.2) The bioassay suggested that the siderophore-producting bacteria can promote tobacco seedlings growth significantly. All the 4 isolates of siderophore-producting bacteria (JX003, JX004, JX006, JX008) can benefit to seedlings growth and development. Rhizobacteria treated seedlings in the medium plates grew much more vigorous and healthy than the control. 3) The bioassay also showed that siderophore-producting bacteria can improve tobacco to absorb and utilize iron nutrition. Naturally, only the resolvable Fe2+ can be uptake by plants. Seedlings are hard to live when Fe3+ as the sole iron nutrition, because of its very low availability. However, the tobacco seedlings grew vigorously when siderophore-producting bacteria was co-cultured in higher Fe3+ medium. Suggesting that the Fe3+ can be transformed into resolvable by siderophore-producting bacteria, and the necessary iron nutrition for tobacco normal growth and development can be meet.4) The siderophore-producting bacteria can promote tobacco growth and development on aerial parts and root extraordinary. The average height vaccinated siderophore-producting bacteria(JX003) can reach 8.35cm in one month, however the highest average height unvaccinated is 5.85cm;The average height vaccinated siderophore-producting bacteria(JX004) can reach 12.13cm in one month, however the highest average height unvaccinated is 9.48cm; The average height vaccinated siderophore-producting bacteria(JX006) can reach 11.33cm in one month, however the highest average height unvaccinated is 8.7cm; The average height vaccinated siderophore-producting bacteria(JX008) can reach 12.1cm in one month, however the highest average height unvaccinated is 8.8cm. The tobacco plants vaccinated siderophore-producting bacteria all have mature roots.5) The siderophore-producting bacteria can restrain the occurrence of tobacco virus disease significantly. Test results show that the tobacco plants infected siderophore-producting bacteria fall ill in the 18th days after infected, later about 7d than normal process, and disease index is 5-13.8. And the disease index of unvaccinated is 13.9-27.8 which exist significant difference between vaccinated. The vaccinated tobacco plants have a tendency of virus symptom can reduce further in later period of growth..Generally, the siderophore-producting bacteria can improve iron absorption and utilization of tobacco, and then benefit to plant growth and development. And also promote the resistance to virus. Their active substance and action mode were worth to be further investigated.更多还原