| Plant growth-promoting microbes(PGPMs)are a group of plant-beneficial microbes which can effectively colonize in plant roots,and then stimulate plant growth,improve stress resistance and protect plants against foliar pathogens by a series of signaling network responses from molecular recognition between the two partners.The rational application of biofertilizers is an alternative method for reducing the chemical fertilizer input,sustaining high production crop and promoting the sustainable development of ecological agriculture.Trichoderma guizhouense NJAU 4742 was originally isolated from rhizosphere soil of cucumber root,which was widely available for commercialization(solid fermentation,microbial fertilizer)by this research group.Moreover,as revealed by research in recent years,NJAU 4742 strains can directly interact with roots and then increased a plurality of crops growth potential.In order to address more about the plant growth-promoting of NJAU 4742,the present work mainly focused on understanding the comprehensive molecular mechanisms.First of all,bio-organic fertilizer was prepared by developing the solid-state fermentation technology of NJAU 4742 to improve the ability of sporulation production.Then the NJAU 4742 bioorganic fertilizer was applied to study its effect on pepper plant growth(Capsicum annuum L)and the possible role of increasing nutrients availabilities.Moreover,the signal response of molecular dialogue to different proteins and metabolic pathways from NJAU 4742 and cucumber were identified during coculturing by methods the proteomics,transcriptomics and biochemical metabolism.In addition,a characteristic indole acetic acid(IAA)related phenotype in root were observed and IAA biosynthesis were proved as an important signal substance to regulate root morphological in Trichoderma spp.by insitu analysis.Therefore,the mutant deficient in IAA biosynthesis were further constructed to elaborate the molecular mechanism of NJAU 4742 regulating root morphology.The main results of this study are summarized as follows:1.Exogenous addition of amino acid hydrolysate(containing 6.5%free amino acid content)in rice straw can significantly increase the number of NJAU 4742 sporulation.The optimum fermentation parameters for spores production was initial pH 3.5,moisture content 75%,10%corn powder,under which the production of spores can reach to 2.40×1 010 spore·g-1.2.The pot experiment showed that NJAU 4742 bio-organic fertilizer can significantly promote tomato and pepper growth.These fertilization treatments presented a obvious differences in soil enzyme activity,in which NJAU 4742 bio-organic fertilizer(AT6)significantly improved soil invertase,urease and peroxidase activities comparing to chemical fertilizer(CK),inoculation NJAU 4742(T6)and amino acid organic fertilizer(AA).Moreover,NJAU 4742 bio-organic fertilizer can significantly increase the content of soil available phosphorus(AP),available potassium(AK),total nitrogen(TN)and soil organic carbon(SOC).3.Differentially enriched proteins and metabolic pathways in NJAU 4742 and cucumber,which play an important role in regulating cucumber root morphology during co-cultivating.The total proteins of NJAU 4742 strain and the extracellular proteins of cucumber were extracted for proteomic analysis after co-cultivating for 25 days.The results showed that 510 in 1059 intracellular quantified proteins from NJAU 4742 were expressed with significantly difference,and 62 of 24 extracellular proteins were expressed differently.Meanwhile,36 of 11 extracellular proteins secreted were differently regulated in cucumber.Based on the analysis of KEGG pathway and PPI,the intracellular proteins of NJAU 4742 with high abundance were mainly involved in amino acid metabolism,carbon metabolism,energy production,shikimate metabolism(tryptophan,tyrosine,and phenylalanine metabolism)and IAA biosynthesis.In addition,NJAU 4742 could significantly increase the IAA content of cucumber roots with increasing time,especially in 30th day,the IAA content was increased by 1.15 folds more than control.Furthermore,NJAU 4742 was revealed a strong ability of converting glutamate,aspartic acid,arginine,nitrate,nitrile and amines into ammonia by method of proteomics,transcriptomics and isotope tracing.4.The IAA produced by NJAU 4742 plays a major role in regulating arabidopsis root morphology.Inoculation of NJAU 4742 on plant roots could inhibit the elongation of primary root and significantly increase the number of lateral roots.Meanwhile,an increasing DR5::GFP signal was occurrence on root tips after internacting with roots.In Arabidopsis arj7/19 and shy2 mutants,inoculation with Trichoderma harzianum NJAU 4742 had a significantly changed in root architecture,which indicates that the IAA from NJAU 4742 could sufficiently regulate root morphology of Arabidopsis as a main signal than other secreted compounds.5.Based on the results of proteomics and transcriptome,the nits,iaaH and ald genes were selected to further study.Five of sixteen genes were detected to be significantly induced with the addition of tryptophan:Acetamide dehydrogenase gene in acetamide pathway(iaaH),nitrilase gene in indole acetonitrile pathway(nit1 and nit2)and indole acetaldehyde dehydrogenase gene in indole pyruvate and tryptamine pathway(aldl and ald2)were up-regulated by 43.7,7.4,46.0,40.4 and 27.1 folds compared to the without tryptophan treatment.Considering the proposed IAA biosynthesis pathways in microbes and the identified proteins related to IAA synthesis of NJAU 4742,the candidate IAA synthesis genes were screened,which might potentially involved in the IAA biosynthesis.6.Effects of different mutants on IAA production.Five single gene knock-out mutants and four multiple gene knock-out mutants were obtained by using a mutipile genetic manipulation.The results showed that the IAA content of Δald2 mutant strain and Δnit2 mutant strain were significantly decreased by 40.0%and 27.9%compared to wild type strain,while other mutants did not change significantly.Comparing to wild-type strain,the IAA content of ΔiaaH&ald1&ald2&nit1 mutant and ΔiaaH&ald1&ald2&nit1&nit2 mutant were significantly decreased by 66.8%and 72.5%,respectively.Therefore,it is suggested that ald gend was involved in encoding indolealdehyde dehydrogenase gene and nit gene involved in encoding nitrilase,which play an important role in IAA production.7.Effects of different mutants on Arabidopsis root growth and development.To study the effect of different IAA production strains of NJAU 4742 on the growth promoting ability,strains that showed variation in IAA production were applied to this part.The compatible ΔiaaH,Δaldl and Δnit1 strains were not significantly decreased in the capacity of regulating Arabidopsis lateral under co-culture conditions,however the Δald2,Δnit2,ΔiaaH&ald1&ald2&nit1 mutant and ΔiaaH&ald1&ald2&nit1&nit2 mutants performed an obviously decreasing the number of root tips compared with the wild-type strain.The results showed that the IAA synthesis of NJAU 4742 played an important role in promoting the occurrence of lateral roots and regulating root morphology. |
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