| Iron is an essential micronutrient element in plants,which participates in many important metabolic pathways in plants,such as chlorophyll synthesis,photosynthesis and respiration.Although the content of total iron is rich in most soils,the content of soluble iron that can be absorbed and utilized by plants is very low in alkaline or calcareous soils.Cinnamomum camphora is an important greening tree species in China.However,with the rapid development of urban and rural greening construction,the phenomenon of iron deficiency and yellowing of camphor trees in many areas is becoming more and more serious,which greatly affects its landscape value and ecological function.Rhizobacteria have been proved to play an important role in improving iron nutrition of plants.However,no attempt has been made to use microorganisms to control chlorosis of camphor trees.For this reason,this study first investigated the characteristics of iron ore solubilization of rhizosphere growth-promoting bacteria Rahnella aquatilis JZ-GX1 in vitro,further analyzed the types of siderophores by genome-wide prediction and ultra-high performance liquid chromatography-mass spectrometry（UPLC-MS）,and explored the ability of model plants to resist iron deficiency stress induced by volatile organic compounds（VOCs）produced by JZ-GX1 strain.The effects of R.aquatilis JZ-GX1 on the growth and physiological metabolism of C.camphora seedlings growing in alkaline soil,as well as on the physical and chemical properties of rhizosphere soil,microbial community structure and soil metabolism were studied systematically,in order to clarify the green mechanism of JZ-GX1 on iron-deficient C.camphora.The main results are as follows:1.Analysis of the characteristics of biodegradation of insoluble iron by R.aquatilis JZ-GX1Firstly,the iron dissolving ability of four bacteria strains from forest rhizosphere was determined.It was found that the four tested bacteria had the ability to dissolve Fe2O3,but the solubility and characteristics were different.Among them,the strongest solubilizing ability to Fe2O3 was R.aquatilis JZ-GX1,followed by Bacillus pumilus HR10,Pseudomonas aurantiaca ST-TJ4 and Burkholderia multivorans WS-FJ9.Among them,two strains with strong iron solubilizing ability had the ability to reduce the p H of culture medium.However,the production capacity of siderophores and Fe3+reductase is moderate,indicating that acidified environment is one of the most important characteristics of microbial dissolution of iron.Furthermore,the organic acids of JZ-GX1 strain fermented continuously for 5 days were quantitatively analyzed by high performance liquid chromatography（HPLC）.It was found that JZ-GX1 strain could produce ten kinds of organic acids,including oxalic acid,malic acid,citric acid,gluconic acid,formic acid,acetic acid,α-ketoglutaric acid,lactic acid,fumaric acid and succinic acid,among which formic acid,acetic acid and gluconic acid were the highest.2.Effects of R.aquatilis JZ-GX1 on the growth and physiological characteristics of C.camphoraTo explore the benefits of iron nutrition and application of JZ-GX1 strain,the control effect of potted seedling was evaluated by inoculation or Fe SO4 treatment.The results showed that in the first 20 days of treatment,the green effect of Fe SO4 treatment was obvious,and then the leaves treated with JZ-GX1were greener and glossy,and the growth rate of chlorophyll reached263.97%after 60 days.Compared with CK and Fe SO4 treatments,the shoot growth and specific leaf weight of camphor trees inoculated with JZ-GX1 strain for 2 months were the highest.It is worth noting that the number of lateral roots and dry matter accumulation increased significantly.Transmission electron microscopy（TEM）was used to observe the ultrastructure in three treatments.It was found that the chloroplasts of control plants grown in alkaline soil showed typical characteristics of matrix lamellar disorder induced by Fe deficiency,and the increase of osmiophilic particles was observed.In contrast,plants from Fe SO4 treatment and inoculation showed more normal thylakoid accumulation than that of CK.In addition,the presence of inoculum significantly increased the volume of starch grains.Compared with non-bacterial treatment,inoculation of JZ-GX1 significantly increased the transfer of active Fe from roots to leaves in C.camphora,and increased the activities of peroxidase（POD）,catalase（CAT）and aseorbateperoxidase（APX）enzyme in leaves,thus reducing the membrane damage of iron-deficient camphor caused by reactive oxygen species（ROS）.3.The species analysis of specific siderophores of R.aquatilis JZ-GX1According to the sequencing of the whole genome,the secondary metabolites were predicted and analyzed by anti SMASH website.The results showed that JZ-GX1 strain could synthesize and secrete siderophores.After comparison,it was found that the gene cluster of siderophores synthesized by JZ-GX1 had 100%homology with the known deferoxamine E synthesis gene cluster dfo JACS of Pantoea agglomerans.In addition,the supernatant of siderophores fermentation of JZ-GX1 was analyzed by UPLC-MS.It was found that JZ-GX1 could synthesize deferoxamine.In the tetrazolium salt experiment,the color of the solution can be changed to be dark red immediately,indicating that JZ-GX1 can produce hydroxamic acid type siderophores,and deferoxamine belongs to hydroxamic acid type siderophores.Therefore,based on the color reaction of siderophores type,genome prediction and UPLC-MS results,it was confirmed that the siderophores secreted by R.aquatilis JZ-GX1 was deferoxamine（DFO）.4.Analysis of transcriptional regulation of biological siderophore-deferriamine on iron absorption of C.camphoraTo further explore the molecular mechanism of microbial siderophores,DFO,promoting iron absorption in plants.Firstly,the concentration of deferoxamine which is the most suitable to promote the absorption and utilization of camphor iron was selected by using SPAD value and Fe2+content as indexes,and then the H+ATPase and Fe3+reductase related to iron absorption and utilization in camphor roots were determined.Finally,the root transcriptome data of camphor trees with and without DFO were compared by RNAseq technique.The results showed that 10μM DFO could significantly increase the SPAD value and Fe2+content of camphor leaves.Compared with CK,H+ATPase activity decreased and Fe3+reductase increased with the increase of DFO concentration.2374 differentially expressed genes were identified by transcriptome analysis,GO and KEGG enrichment analysis showed that the pathways related to redox reaction,iron binding,carotenoid biosynthesis,glutathione metabolism,flavonoids and phenylpropane biosynthesis were activated.Exogenous spraying of reduced glutathione also alleviated the yellowing of C.camphora.In addition,DFO significantly up-regulated the expression of genes related to iron metabolism of C.camphora,including Ccb HLH,Cc FRO6,Cc IRT1,Cc Nramp5Cc OPT3 and Cc VIT4.Our results provided new insights into the mechanism of microbial siderophores promoting iron absorption in plants.5.Response of microbial community structure of C.camphora rhizosphere soil to R.aquatilis JZ-GX1The effects of JZ-GX1 strain on the population diversity and community structure of microorganisms in rhizosphere soil were studied by high-throughput sequencing technique.The results showed that the Shannon index and Chao1 index of bacteria and fungi in rhizosphere soil treated with JZ-GX1 strain decreased,but there was no significant difference,indicating that inoculation treatment did not significantly change theαdiversity of soil fungi and bacteria,but affected the structure and composition of fungal community.It promoted the increase of specific fungal populations such as Glomus,Mortierella,Trichoderma and Penicillium,while decreased the relative abundance of genera in Nectriaceae,Agaricomycetes,Neocosmospora,Paraphoma and Plectosphaerella.In addition,the results of FUNGuild functional analysis showed that JZ-GX1 treatment decreased the abundance of saprophytic fungi,plant and animal pathogens,while increased the proportion of arbuscular mycorrhizal fungi.Through the observation of mycorrhizal infection,it was found that the mycorrhizal infection rate was 270.37%higher than that of the control.Therefore,the application of rhizobacteria JZ-GX1 can alleviate the phenomenon of iron deficiency yellowing of C.camphora through the interaction with local fungal community.6.Regulation of rhizosphere soil metabolism of C.camphora by R.aquatilis JZ-GX1It was found that JZ-GX1 could colonize in the rhizosphere of C.camphora for at least 30days and significantly decreased the p H value of the soil,and the content of available iron and complex iron was higher than that of CK and Fe SO4 treatment,while the content of amorphous iron and free iron was decreased.Furthermore,the effect of JZ-GX1 strain on the soil metabolism was studied by using non-targeted metabolic technique.The results showed that the main differential metabolites of different treatments included phenolic acids,such as coumaric acid and cinnamic acid,antibiotics,such as antimicrobial peptides,myxolone and Tacrolimus,and lipoic acid,arginine and DFO.Among the representative compounds with significant differences,the content of DFO in soil treated with bacteria was 21.53 times higher than that uninoculated.According to the relatively rich changes of metabolites,the metabolic pathways enriched by KEGG mainly include glycolysis/gluconeogenesis,biosynthesis of isoflavones and phenylpropanes,metabolism of porphyrin and chlorophyll,metabolism of tryptophan and biosynthesis of antibiotics.Therefore,the application of bacterial agent affected the availability of iron in the rhizosphere of C.camphora and the accumulation of specific metabolites to alleviate the etiolation of iron deficiency.7.The mechanism of plant resistance to iron deficiency induced by VOCs of R.aquatilis JZ-GX1The effects of VOCs released by JZ-GX1 strain on the growth and root parameters of model plants under iron deficiency were studied by two-part dish culture system,and the genes of iron metabolism and hormone signal pathway in Arabidopsis thaliana were analyzed by RT-q PCR.The results showed that JZ-GX1 could induce the tolerance of A.thaliana to iron deficiency stress and promote the formation of lateral roots and root hairs by producing VOCs.It was found that the iron uptake related genes At AHA2,At FRO2 and At IRT1 exposed to VOCs were up-regulated by 25,1.81 and 1.35 times,respectively.The accumulation of endogenous abscisic acid（ABA）and jasmonic acid（JA）increased,the expression of ABA synthesis gene At NCED3 was up-regulated on the 3rd and 5th day,and the JA synthesis related gene At PDF1 was significantly up-regulated on the 5th day,indicating that VOCs of JZ-GX1 can activate plant response to iron deficiency stress through ABA and JA signal pathways.Further analysis by headspace solid phase microextraction（SPME-GC-MS）showed that 2-Undecanone and 3-Methyl-1-Butanol have ability to promote the growth of A.thaliana and Medicago sativa under iron-limited conditions.The above results showed that the VOCs of R.aquatilis JZ-GX1 also plays an important role in promoting iron absorption in plants.This study revealed that R.aquatilis JZ-GX1 can reduce soil p H by secreting organic acids,directly provide iron source for C.camphora by synthesizing deferrioxamine,and induce iron deficiency resistance of plants by releasing VOCs,and can increase the abundance of soil arbuscular mycorrhizal fungi,and improve the iron deficiency chlorosis of C.camphora in many ways.Our study helped to clarify the mechanism of beneficial microorganisms in promoting iron uptake in plants,and provided a new sustainable bioremediation way for the prevention and treatment of camphor etiolation. |
