水稻内生成团泛菌HAUM1对宿主的定殖及促生作用的研究
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摘要
随着世界人口的迅速增长,粮食危机逐渐成为人们关注的焦点问题。国际水稻研究所指出,提高水稻的产量是解决此危机的关键。水稻是我国主要粮食作物,目前,实现水稻高产增收的主要途径是大量施用工业化肥和含重金属物质的农药,而这样会造成土壤原有的理化性状改变,反而导致农作物的产量逐渐降低,甚至影响农产品的质量安全,危害到人类身体健康。近年来人们开始致力于寻找替代型的生物资源,阻止这一现象的恶化。有报道证实部分内生菌具有实现这一环保生态型农业的潜在价值。本研究以揭示一株此类水稻内生菌的定殖规律及促生作用为实验目的,进行了以下实验。
从表面灭菌的野生水稻的根、茎、叶组织,初步分离得到固氮细菌18株、解磷细菌11株,经由回接复筛实验,生理生化试验以及16SrDNA测序分析,确定了三株为内生菌,分别为具有固氮能力的克雷伯氏菌HAUM3,阴沟肠杆菌HAUM12和解磷能力的成团泛菌HAUM1。通过纤维素及果胶降解试验,发现HAUM1对宿主的侵染能显著高于另外两株菌；盆栽回接实验,证明HAUM1接种的水稻在15天后株高方面显著高于其他处理。综合以上结果,挑选了水稻内生优势成团泛菌HAUM1为研究菌株。
通过接合实验,将菌株HAUM1进行gfp基因标记,体外传代15次后,挑选出了保持抗性及荧光强度最稳定的接合子,利用根据Roseateles depolymeran光捕捉器操纵子上的一段特异序列设计的特异性引物,对该接合子进行gfp基因扩增,挑选出了结果为阳性的进行下一步研究。
将此菌株回接入无菌试管水稻苗中,结合激光共聚焦扫描技术与平板计数法,研究其接种后3、5、7、15天在宿主中的定殖及分布规律,结果说明接种三天后菌体主要以特殊的symplasmata结构由侧根部侵入宿主,并开始向上迁移,接种五天后菌体逐渐分散定殖于根部皮层的各细胞及细胞间隙中,接种7天后菌体扩散到茎组织中,并在第15天大量定殖于叶组织及细胞中。
在对HAUM1促生作用的研究中,将HAUM1与另两株解磷菌PA10(Agrobacterium sp.)、P11(Pantoea agglomerans),同时进行盆栽水稻的接种处理,结果证明前者在提高宿主水稻的株高、鲜重及干重方面均有显著优势,且其接种后的植株叶绿素(a+b)及磷含量,分别达到了未进行接种处理的空白对照组水稻的1.76、1.22倍。此外液相色谱分析证明,在产植物生长激素能力方面,HAUM1的吲哚乙酸,脱落酸分泌量高达23.9mg/L和38.5mg/L。说明HAUM1与宿主植物建立优于普通根际菌的和谐联合关系,而其对宿主植物的影响,不仅在于根部解磷作用,而且与其对光合作用的促进及植物激素的调节都有关系。
综上所述,菌株HAUM1与宿主水稻建立了有效的和谐联合关系,是一株值得更深入研究的水稻内生促生菌。
With the rapid growth of world population, the food crisis has become the focus of attention.International Rice Research Institute pointed out that increase of rice yeild is the key to solve this impending crisis, as for now, China where rice is the staple crops, Application of industrial chemical fertilizers and pesticides containing heavy materials had been considered as the main way to achieve the goal, which caused changes in the physical and chemical properties of soil, and led to decrease in crop production, and even affected the crop quality and safety, finally did harm to human health. The endophytic bacteria were gradually proved to be an alternative bio-ecological resources to prevent the deterioration of this phenomenon, For the purpose of revealing the colonization and growth promotion properties by this endophytic bacteria in rice, the following experiments were conducted.
18 nitrogen-fixing bacterial strains and 11 phosphate-solubilizing bacterial strains were isolated from surface sterilized roots, stems and leaves of wild rice.On the basis of rescreening experiments、physiological and biochemical tests and 16S rDNA sequence analysis,three endphytic bacteria were confirmed which were Klebsiella strain HAUM12、Enterobacter strain HAUM3 and Pantoea agglomerans strain HAUM1. By cellulose and pectin degradation test, HAUM1 showed better of infection capability than the other two strains. Results of pot experiment indicated that the rice inoculated with HAUM1 were significantly higher than the other treatments at 15 day after inoculation.In conclusion, HAUM1 was chosen as the reseach strain.
HAUM1 was gfp marked by bacterial conjugation experiment. The conjugant which appeared stable in resistance and fluorescence intensity after 15 generation was picked out for a gfp gene amplification,then the positive one was selected for the futher study. Combining the use of LSCM with plating experiment, to examine infection、dissemination and colonization of host rice by gfp-tagged strain HAUM1,Results indicated that the symplasmata structure formed by hundreds of bacterial showed up at the beginning of infection process in roots at 3 day, at 5 day these symplasmatas gradually decentralized and bacteria started to colonize the cells and intercell spaces of cortex tissues, a large number of bacteria migrated into stems at 7 day and then colonized leaves at 15 day.
To reveal the influence by strain HAUM1 on host, the rice seedlings were respectively inoculated with HAUM1 and other two phosphate- solubilizing bacterial PA10(Agrobacterium sp.) and P11(Pantoea agglomerans), HAUM1 was proved possessing the most significant increase almost in all aspects of biomass and accumulating the highest levels of phosphate and cytochrome(a+b). It was also detected to produce indole-3-acetic acid and abscisic acid as high as 23.9mg/L and 38.5mg/L by HPLC test.Results indicated that strain HAUM1 had built an eompatibassociation with host plant, and this growth-promoting influcence was partly due to its phosphate-solubilizing、photosynthesis activity and auxin productivity as well.
Considered collectively,all the findings suggested that this kind of endophytic plant-growth-promoting bacteria are far more inclusive and dynamic than we thought therefore enhancing the study in this field holds a promising potential for a long term of sustainable rural economy.
从表面灭菌的野生水稻的根、茎、叶组织,初步分离得到固氮细菌18株、解磷细菌11株,经由回接复筛实验,生理生化试验以及16SrDNA测序分析,确定了三株为内生菌,分别为具有固氮能力的克雷伯氏菌HAUM3,阴沟肠杆菌HAUM12和解磷能力的成团泛菌HAUM1。通过纤维素及果胶降解试验,发现HAUM1对宿主的侵染能显著高于另外两株菌；盆栽回接实验,证明HAUM1接种的水稻在15天后株高方面显著高于其他处理。综合以上结果,挑选了水稻内生优势成团泛菌HAUM1为研究菌株。
通过接合实验,将菌株HAUM1进行gfp基因标记,体外传代15次后,挑选出了保持抗性及荧光强度最稳定的接合子,利用根据Roseateles depolymeran光捕捉器操纵子上的一段特异序列设计的特异性引物,对该接合子进行gfp基因扩增,挑选出了结果为阳性的进行下一步研究。
将此菌株回接入无菌试管水稻苗中,结合激光共聚焦扫描技术与平板计数法,研究其接种后3、5、7、15天在宿主中的定殖及分布规律,结果说明接种三天后菌体主要以特殊的symplasmata结构由侧根部侵入宿主,并开始向上迁移,接种五天后菌体逐渐分散定殖于根部皮层的各细胞及细胞间隙中,接种7天后菌体扩散到茎组织中,并在第15天大量定殖于叶组织及细胞中。
在对HAUM1促生作用的研究中,将HAUM1与另两株解磷菌PA10(Agrobacterium sp.)、P11(Pantoea agglomerans),同时进行盆栽水稻的接种处理,结果证明前者在提高宿主水稻的株高、鲜重及干重方面均有显著优势,且其接种后的植株叶绿素(a+b)及磷含量,分别达到了未进行接种处理的空白对照组水稻的1.76、1.22倍。此外液相色谱分析证明,在产植物生长激素能力方面,HAUM1的吲哚乙酸,脱落酸分泌量高达23.9mg/L和38.5mg/L。说明HAUM1与宿主植物建立优于普通根际菌的和谐联合关系,而其对宿主植物的影响,不仅在于根部解磷作用,而且与其对光合作用的促进及植物激素的调节都有关系。
综上所述,菌株HAUM1与宿主水稻建立了有效的和谐联合关系,是一株值得更深入研究的水稻内生促生菌。
With the rapid growth of world population, the food crisis has become the focus of attention.International Rice Research Institute pointed out that increase of rice yeild is the key to solve this impending crisis, as for now, China where rice is the staple crops, Application of industrial chemical fertilizers and pesticides containing heavy materials had been considered as the main way to achieve the goal, which caused changes in the physical and chemical properties of soil, and led to decrease in crop production, and even affected the crop quality and safety, finally did harm to human health. The endophytic bacteria were gradually proved to be an alternative bio-ecological resources to prevent the deterioration of this phenomenon, For the purpose of revealing the colonization and growth promotion properties by this endophytic bacteria in rice, the following experiments were conducted.
18 nitrogen-fixing bacterial strains and 11 phosphate-solubilizing bacterial strains were isolated from surface sterilized roots, stems and leaves of wild rice.On the basis of rescreening experiments、physiological and biochemical tests and 16S rDNA sequence analysis,three endphytic bacteria were confirmed which were Klebsiella strain HAUM12、Enterobacter strain HAUM3 and Pantoea agglomerans strain HAUM1. By cellulose and pectin degradation test, HAUM1 showed better of infection capability than the other two strains. Results of pot experiment indicated that the rice inoculated with HAUM1 were significantly higher than the other treatments at 15 day after inoculation.In conclusion, HAUM1 was chosen as the reseach strain.
HAUM1 was gfp marked by bacterial conjugation experiment. The conjugant which appeared stable in resistance and fluorescence intensity after 15 generation was picked out for a gfp gene amplification,then the positive one was selected for the futher study. Combining the use of LSCM with plating experiment, to examine infection、dissemination and colonization of host rice by gfp-tagged strain HAUM1,Results indicated that the symplasmata structure formed by hundreds of bacterial showed up at the beginning of infection process in roots at 3 day, at 5 day these symplasmatas gradually decentralized and bacteria started to colonize the cells and intercell spaces of cortex tissues, a large number of bacteria migrated into stems at 7 day and then colonized leaves at 15 day.
To reveal the influence by strain HAUM1 on host, the rice seedlings were respectively inoculated with HAUM1 and other two phosphate- solubilizing bacterial PA10(Agrobacterium sp.) and P11(Pantoea agglomerans), HAUM1 was proved possessing the most significant increase almost in all aspects of biomass and accumulating the highest levels of phosphate and cytochrome(a+b). It was also detected to produce indole-3-acetic acid and abscisic acid as high as 23.9mg/L and 38.5mg/L by HPLC test.Results indicated that strain HAUM1 had built an eompatibassociation with host plant, and this growth-promoting influcence was partly due to its phosphate-solubilizing、photosynthesis activity and auxin productivity as well.
Considered collectively,all the findings suggested that this kind of endophytic plant-growth-promoting bacteria are far more inclusive and dynamic than we thought therefore enhancing the study in this field holds a promising potential for a long term of sustainable rural economy.
引文
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8 黎起秦,谢义灵,林纬等.广西番茄内生细菌的多样性和数量动态.生物多样性,2006,(06)：534-540.
9 龙良鲲,肖崇刚.内生细菌01-144在番茄根茎内定殖的初步研究.微生物学通报,2003,(05)：53-56.
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2 陈振德.不同收获时期对蔬菜硝酸盐含量的影响.中国蔬菜,1989,(03).
3 冯德福.砷污染与防治.沈阳教育学院学报,2000,(02)：110-112.
4 冯永君,宋未.植物内生细菌.自然杂志,2001,(05)：249-252.
5 葛诚,吴薇.我国微生物肥料的生产、应用及问题.中国农学通报,1994,(03)：24-28.
6 郭良栋.内生真菌研究进展.菌物系统,2001,(01)：148-152.
7 何红,邱思鑫,蔡学清等.辣椒内生细菌BS-1和BS-2在植物体内的定殖及鉴定.微生物学报,2004,(01)：13-18.
8 黎起秦,谢义灵,林纬等.广西番茄内生细菌的多样性和数量动态.生物多样性,2006,(06)：534-540.
9 龙良鲲,肖崇刚.内生细菌01-144在番茄根茎内定殖的初步研究.微生物学通报,2003,(05)：53-56.
10 鲁如坤,时正元.退化红壤肥力障碍特征及重建措施Ⅱ.磷、氮、钾库重建措施.土壤,2000,(06)：310-314.
11 鲁素芸,陈延熙.棉花维管系中主要微生物类群初步分析.北京农业大学学报,1989,(03)：326-329.
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