Effect Of Soil Bacterial-Feeding Nematodes On The Ability Of Bacteria To Synthesize Indole-3-Acetic Acid(IAA) And Its Mechanism

Bacterial-feeding nematodes are the most abundant nutrient group of soil nemtaodes.The interaction between bacterial-feeding nematodes and soil microbes is of great significance in soil ecosystem.Soil bacterial-feeding nematodes affect the quantities,activity,communities of soil microorganisms through their own metabolic activities and feeding behaviors.Thereby,soil bacterial-feeding nematodes can not only influence soil nutrient transformation and circulation,but also modulate plant growth and development,playing a significant role in soil ecological processes.Many studies have comfirmed that soil bacterialfeeding nematodes could increase the content of indole-3-acetic acid(IAA)in soil by interacting with IAA-producing bacteria,thus promoting the growth of plant roots.However,the interaction between soil bacterial-feeding nematodes and IAA-producing bacteria requires further study.Most organisms can sense surroundings by producing metabolites,so as to regulate physiological metabolism,as well as communicate with other organisms.The metabolites of soil bacterial-feeding nematodes,which not only help to attract soil microorganisms as food,but also modulate the physiological and biochemical behaviors of microorganisms and nematodes,play an important role in soil ecology.The effect and mechanism of soil bacterialfeeding nematodes metabolites on the ability of bacteria to synthesize IAA contribute to reveal the interaction between soil bacterial-feeding nematodes and IAA-producing bacteria.The main research contents of this paper are as follows:(1)the typical model bacterialfeeding nematode Caenorhabditis elegans N2,two soil-dwelling bacterial-feeding nematodes(Mesorhabditis sp.NJ and Acrobeloides sp.NJ)and three IAA-producing bacteria(Bacillus amyloliquefaciens JX1,Arthrobacter pascens ZZ21,A.chlorphenolicus L4)were employed to investigate the effect of bacterial-feeding nematodes and their metabolites on the ability of bacteria to synthesize IAA;(2)according to the results of(1),one of the three IAA-producing bacteria was selected as the material for the following study;based on the reported IAA biosynthesis pathways,a combination of genetic and chemical method was used to analyze the potential IAA biosynthesis pathways in the tested strain;(3)the genomewide map of the tested strain was carried out as a reference genome;the effect of C.elegans metabolites on the gene expressions and metabolism of the tested strain were analyzed by transcriptomes and metabolomics,and the IAA biosynthesis pathways regulated by C.elegans metabolites were screened;(4)classification and polarity of the active ingredients of C.elegans metabolites were estimated by different treatments;the active ingredients were separated and identified by High performance liquid chromatography tandem mass spectrometry,and finally synthesized and verified.The main results were as follows:1.C.elegans and its metabolites could significantly improve the ability of the three bacteria to synthesize IAA:C.elegans increased the IAA production of B.amyloliquefaciens JX1,A.pascens ZZ21 and A.chlorophenolicus L4 by 61%,194%and 129%,respectively.By contrast,C.elegans metabolites increased the IAA production of the three bacteria by 25%,124%and 88%,respectively.The metabolites of the two soil-dwelling bacterialfeeding nematodes,Acrobeloides sp.NJ and Mesorhabditis sp.NJ,could also promote the bacteria to produce IAA.The most sensitive bacteria to bacterial-feeding nematodes and their metabolites for producing IAA was A.pascens ZZ21.2.A.pascens ZZ21 was selected to explore the main IAA biosynthesis pathways.The genome-wide map of A.pascens ZZ21 was sequenced and analyzed by PacBio platform.The genome of A.pascens ZZ21 was 4659169 bp with GC content of 65.67%.It contains 4276 genes,including 6 genes associated with IAA biosynthesis and two tryptophan-dependent IAA biosynthesis pathways(the indole-3-acetamide(IAM)and indole-3-pyruvic acid(IPyA)pathways).IAA production of A.pascens ZZ21 increased 4.5-fold when exogenous tryptophan was supplied.Meanwhile,the expressions of prr and aldH,which were predicted to encode aldehyde dehydrogenases in the IPyA pathway,increased 2.8-fold and 1.8-fold,respectively.IAM,IPyA,indole-3-lactic acid(ILA)and indole-3-ethanol(TOL),the main intermediates in tryptophan-dependent IAA biosynthesis,were detected in A.pascens ZZ21 by metabolomics and HPLC-MS analysis,which demonstrared the existence of the IAM and IPyA pathways.The genome-wide mapping of A.pascens ZZ21 provides a reference for the subsequent transcriptome analysis of the mechanism of effect of bacterial-feeding nematodes metabolites on the ability of bacteria to synthesize IAA.3.Effects of C.elegans metabolites on the transcriptome of A.pascens ZZ21 were analyzed.2168 genes of A.pascens ZZ21 were significantly changed by C.elegans metabolites,and 1055 genes were up regulated while 1113 genes were down regulated.Gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis showed that the biosynthesis,metabolism,translation and some other GO terms of A.pascens ZZ21 were changed by C.elegans metabolites,and the biosynthesis of secondary metabolites,biosynthesis of antibiotics,biosynthesis of amino acid and other pathways were also changed.Morever,the expressions of prr,puuC and aldH,which encoding aldehyde dehydrogenase in the IPyA pathway,were increased by 35%,131%and 93%,respectively,suggesting that C.elegans metabolites were likely to promote the biosynthesis of IAA in A.pascens ZZ21 via the IPyA pathway.4.Effects of C.elegans metabolites on the metanolism of A.pascens ZZ21 were analyzed by metabolomics.Results showed that 29 metabolites of A.pascens ZZ21 were significantly changed by C.elegans metabolites,and 21 metabolites were increased while 8 were reduced.The KEGG analysis of differential metabolites showed the biosynthesis and metabolism pathways in A.pascens ZZ21 were significantly affected.Besides,the KEGG analysis of functional genes and proteins related to differential metabolites showed that the biosynthesis of amino acid,biosynthesis of antibiotic and metabolism were also changed.Furthermore,the content of IPyA and its enzymatic reduction product ILA,which were the characteristic intermadiates in IPyA pathway,increased significantly,indicating that C.elegans metabolites could promote the biosynthesis of IAA in A.pascens ZZ21 through the IPyA pathway.5.The metabolites of C.elegans were treated with boiling,proteinase K,DNase,RNase and five organic solvents of different polarity,respectively.Results showed that the active ingredients were preliminary identified as heat-unstable proteins or peptides.Subsequently,30 peptides were isolated and identified from the metabolites of C.elegans by HPLC-MS.Finally,6 short peptides that could promote the biosynthesis of IAA in A.pascens ZZ21 were obtained by artificial synthesis and activity verification.The amino acid sequences were P1(GNALATR),P9(QHGLPQEV),P19(QPLEVLVPN),P20(PQLEVLVPN),P25(TLVDFLR)and P26(TLVDLFR),respectively.The concentrations of 5 peptides(except P20)in the metabolites of C.elegans were determined by Parallel Reaction Monitoring quantitative analysis.The active short peptides from nematodes metabolites provide an opportunity for further exploration of the ecological functions of the bacterial-feeding nematodes metabolites.