Study On IAA Production By Tomato Root Endophytic Bacteria And Its Plant Growth Promotion Mechanism

Indoleacetic acid(IAA)synthesis is considered to be one of the most important mechanisms for endophytic bacteria to promote plant growth,and the tryptophan-dependent IAA synthesis pathway of plant bacteria and its promoting effect on plant growth have been studied in detail.However,in previous studies about fermentation experiments in vitro with tryptophan,we found that only a few kinds of bacteria could directly use tryptophan as substrate to synthesize IAA.Therefore,in order to explore comprehensively the growth-promoting activity and mechanism of plant endophytic bacteria,IAA-producing and growth-promoting bacteria were screened from the previously isolated 117 tomato root endophytic bacterial strains.Furthermore,the plant growth promotion effects and IAA biosynthesis pathway were systematically investigated by using genome sequencing analysis and quantitative detection of UHPLC-MS/MS.This stusy aims to define the role of IAA synthesis of different endophytic strains in plant growth and development,and understand growth-promoting mechanism of endophytic bacteria and effects of microbial-plant interaction on IAA production in tomato.The main results were obtained as follows:(1)Among the 117 tomato endophytic bacterial isolates,21 strains(17.95%)significantly promoted the root elongation of wheat,and 28 strains(23.93%)promoted the growth of wheat leave length(p <0.05).In vitro culture,61 endophytic bacteria(52.14%)had the ability to synthesize auxin IAA used tryptophan as substrate.Most of them belonged to Bacillus and Pseudomonas.Among 66 endophytic strains of which could not directly use tryptophan to produce IAA,18 strains showed obvious plant growth promoting activity(p <0.05).Furthermore,these 66 bacterial strains were cultured in the media with the addition of tryptamine and indoleacetamide,the intermediate metabolites of IAA synthesis,respectively.The results demonstrated that strain 165 and 170 could use indoleacetamide to synthesize an appropriate amount of IAA,while strain 121,152 and 165 could use tryptamine to synthesize an appropriate amount of auxin.The results indicated that some endophytic growth-promoting bacteria without ability to synthesize IAA from tryptophan in vitro,could use the intermediate metabolites to produce IAA,indicating that these strains might habor incomplete tryptophan-dependent IAA biosynthesis pathways.(2)To reveal mechanisms for the endophytic strains to biosynthesis IAA using the intermediate metabolites,Strain 121 Bacillus cereus,which can synthesize IAA directly from tryptophan,and strains 127 Bacillus aryabhattai,165 and 170 Bacillus aryabhattai,which cannot use tryptophan but can synthesize IAA from intermediate were selected for Illumnia Miseq genome sequencing.The results for genome sequencng and annotation showed that all the fourbacterial genomes contained genes invovled in auxin synthesis,resistance to antibiotics,and toxic compounds and siderophore secretion etc.By analyzing the tryptophan metabolic pathway in KEGG pathwayfour strains of sequenced genomes Bacillus strain was found to be a complete tryptophan-dependent indoleacetamide pathway for IAA synthesis,while strain 127,165,170 had incomplete tryptophan dependent 3-indoleacetamide pathway,and strain 165 had another incomplete tryptophan dependent 3-indolylpyruvate pathway.(3)High concentration of IAA,and intermediate metabolites from different IAA biosynthesis pathways tryptamine,indole acetamide and indoleglyoxylic acid were detection in plant root tissue extracts by using UHPLC-MS/MS.Furthermore,the fermentation experiments for four strains 121,127,165 and 170,were carried out by adding root tissue extracts.The results showed that IAA were detected in the culturing media of two endophytic strains 121 Bacillus cereus and 165,indicated that some endophytes with incomplete IAA biosynthesis pathway may use plant intermediates to produce auxin to promote plant growth.