乌头产吲哚乙酸内生细菌遗传多样性、抗逆性及其对水稻幼苗生长的影响

Genetic diversity,stress resistance,and effect on rice seedling growth of indoleacetic acid-producing endophytic bacteria isolated from Aconitum carmichaelii Debeaux

【目的】探究乌头产吲哚乙酸(IAA)内生细菌的遗传多样性、溶磷解钾能力、抗逆能力及其对水稻幼苗生长的影响,为道地产区乌头产业可持续发展提供科技支撑。【方法】从健康乌头植株分离可培养内生细菌,采用Salkowski比色法测定内生细菌产IAA能力,16SrDNA限制性片段长度多样性(16SrDNA-RFLP)及16SrDNA全长序列研究其遗传多样性及系统发育地位,平板培养法测定产IAA内生细菌溶磷解钾及其对pH、温度、盐、抗生素等的耐受能力,平板法测定菌悬液浸泡后水稻种子萌发率和室内水培法探究产IAA内生细菌对水稻幼苗生长的影响。【结果】从健康乌头植株分离获得24株高产IAA的内生细菌,其分泌IAA的值为21.39–84.43 mg/L。16S rDNA-RFLP分析表明,24株产IAA的内生细菌可分为12个类群,代表菌株16S rRNA基因系统发育结果显示这些菌分属于肠杆菌属(Enterobacter)、克雷伯氏杆菌属(Klebsiella)、泛菌属(Pantoea)、假单胞菌属(Pseudomonas)、微杆菌属(Microbacterium)、芽孢杆菌属(Bacillus)、鞘氨醇杆菌属(Sphingobacterium)和金黄杆菌属(Chryseobacterium),表现出较丰富的遗传多样性。另外,24株菌中有12株菌具有较强的溶钾能力,大部分供试菌株可耐受较广范围的pH、温度和盐浓度,但对抗生素耐受性不强;10株内生细菌可显著提高水稻种子的萌发率,16株内生细菌可显著促进水稻幼苗的生长,综合筛选出3株内生细菌(JY1-1S、JY3-6S和JY10-1L)既能提高水稻种子发芽率,又能促进水稻幼苗生长。【结论】乌头植株含有较丰富的高产IAA的内生细菌资源,且其遗传多样性丰富;本研究筛选出3株对水稻种子萌发和幼苗生长具显著促进作用的内生细菌,可为后续生物肥料的开发提供优质菌种。

[Objective]To explore the genetic diversity,phosphorus-and potassium-solubilizing ability,stress resistance,and effect on rice seedling growth of indoleacetic acid(IAA)-producing endophytic bacteria isolated from Aconitum carmichaelii Debeaux and to provide scientific support for the sustainable development of A.carmichaelii industry.[Methods]Culturable endophytic bacteria were isolated from surface sterilized healthy plants of A.carmichaelii,and their IAA-producing capacities were measured by Salkowski spectrophotometry.The genetic diversity and phylogenetic status of these bacteria were studied via 16S rDNA-restriction fragment length polymorphism(16S rDNA-RFLP)and 16S rDNA full-length sequence.The phosphorus-and potassium-solubilizing abilities and stress resistance of them were studied by plate culture method.The effects of IAA-producing endophytic bacteria on the seed germination rate of rice were tested by semi-solid culture method after the seeds were immersed in bacterial liquids for 30 min.The effects of the endophytic bacteria on rice seedling growth were investigated by hydroponic experiment in our laboratory.[Results]Twenty-four endophytic bacterial strains were isolated from healthy A.carmichaelii,with the IAA-producing capacities ranging from 21.39 mg/L to 84.43 mg/L.The 16S rDNA-RFLP analysis indicated that these 24 strains were classified into 12 groups.The phylogenetic tree constructed based on 16S rRNA gene sequences showed that the representative strains belonged to Enterobacter,Klebsiella,Pantoea,Pseudomonas,Microbacterium,Bacillus,Sphingobacterium,and Chryseobacterium,which demonstrated high genetic diversity.Twelve strains showed strong potassium-solubilizing capacities.Most of the strains could grow normally within wide ranges of pH,temperature,and NaCl concentration,while they showed little resistance to different antibiotics.Ten strains significantly improved the germination rate of rice seeds,and 16 strains significantly promoted the growth of rice seedlings.We screened out 3 strains JY1-1S,JY3-6S,and JY10-1L that could not only improve the seed germination rate but also promote the seedling growth of rice.[Conclusion]A.carmichaelii harbored rich and diverse IAA-producing endophytic bacteria.We screened out 3endophytic bacterial strains that could promote the seed germination and seedling growth of rice.This study laid a foundation for the development of plant growth-promoting biological fertilizer in the future.