Effects Of Plant Growth Promoting Bacteria From Sedum Alfredii On Plant Growth,Cd Uptake And Accumulation Of Brassica Juncea L.and Their Underlying Mechanisms

In recent years,with the development of global Microbiome Project,more and more microorganisms have been used to assist phytoremediation of heavy metal contaminated soils.Numerous plant growth promoting bacteria(PGPB)isolated from hyperaccumulators,such as Sedum alfredii Hance and Pteris vittata,have been verified to play a beneficial role in promoting plant growth and facilitating heavy metal uptake of their host plants,which consequently improved phytoremediation efficiency.However,little is known about the functional mechanisms and application effects of these PGPB on the non-host plants with promising remediating potential.Therefore,a genotype of Cd-accumulating Brassica juncea was selected in this study to conduct hydroponic experiments,pot experiments as well as field experiments.The effects of PGPB isolated from Sedum alfredii on B.juncea growth,Cd uptake and accumulation,antioxidative response,photosynthetic response and rhizospheric bacterial community were investigated with in situ fluorescence analysis,molecular biology,transcriptome and microbiome techniques.Furthermore,field experiment was conducted to illustrate the the effects of single bacteria inoculants and combined bacteria consortium inoculants.The main results were listed below:(1)Hydroponic and pot experiments combined with GFP-labeling were conducted to elucidate the colonization pattern of Sphingomonas Sa MR12 in B.juncea.The results by a confocal laser scanning microscope showed that Sa MR12 entered the roots through the conjunction of taproot and lateral roots.In addition,fluorescence was also detected in stems and leaves of B.juncea.(2)A hydroponic experiment was conducted to investigate the effects of 12different PGPB strains isolated from S.alfredii Hance on plant growth,Cd uptake and accumulation of B.juncea.The results showed that inoculation of most PGPB strains promoted plant growth,boosted root development and improved chlorophyll content in the absence of Cd.Inoculation of PGPB strains promoted plant growth up to 111%in the shoots and 358%in the roots when treated with 2?M Cd.In addition,PGPB inoculation of Sa CR1,Sa MR10,Sa MR12,Sasm05 and Sa ZS4 under 2?M Cd treatment not only ameliorated plant root morphology including total root length,total surface area,total root volume and number of root tips,but also facilitated photosynthesis of B.juncea.While most strains were beneficial for Cd uptake,inoculation of Sa MR10,Sa MR12,Sa PS17,Sasm03,Sasm05 and Sa ZS4 facilitated Cd transport.Among all 12 PGPB strains,Burkholdria Sa MR10 and Sphingomonas Sa MR12 were identified as the promising microbes for B.juncea to improve phytoremediation efficiency of Cd contaminated soils.(3)A hydroponic experiment was conducted to study the effects of Sphingomonas Sa MR12 on antioxidative responses of its non-host plant B.juncea under four different Cd treatments.The results showed that Cd exposure resulted into the antioxidant system damage,which induced the production of hydrogen peroxide(H₂O₂),increased the content of malondialdehyde(MDA)and proline accumulation.Inoculation of Sa MR12not only reduced H₂O₂ content and MDA content by 11%-38%and 21%-68%respectively,but also decreased proline content by 7-30%in the shoots and 7-32%compared to non-inoculated plants.Additionally,Sa MR12 inoculation promoted the activities of superoxide dismutase,peroxidase,catalase,and ascorbate peroxidase and facilitated the relative gene expression levels of dehydroascorbate reductase and glutathione reductase involved in the glutathione(GSH)-ascorbic acid(As A)cycle.The results demonstrated that,under Cd stress,Sa MR12 inoculation could alleviate the Cd-induced damage of B.juncea by decreasing the concentrations of H₂O₂,MDA and proline,increasing the activities of antioxidative enzymes,and regulating the GSH-As A cycle.Through these ways,Sa MR12 inoculation facilitated plant growth and Cd accumulation of B.juncea under Cd stress,which further contributed to the elevated phytoextracton efficiency.(4)A hydroponic experiment combined with RNA-seq technology was conducted to reveal the influences of Sphingomonas Sa MR12 on photosynthetic system of B.juncea under Cd stress.The results showed that Cd stress inhibited the photosynthetic activity of photosystem?,destroyed the photosynthetic electron transport chain of photosystem?,and affected chlorophyll synthesis in leaves,resulting in the decline of photosynthetic capacity of B.juncea.Sa MR12 inoculation significantly increased chlorophyll content,Fv/Fm(16.7%),Y(?)(23%),?_PS?(28%),ETR(28%),and q P(23%),while decreased NPQ(18.8%)under Cd stress.Simoutaneously,149characteristic genes significantly related to photosynthetic parameters were obtained by weighted gene co-expression network analysis(WGCNA),and we also identified some key genes including Bna A07g24880D?Bna C01g18870D?Bna C02g25960D?Bna Cnng21050D?Bna C06g23560D?Bna A05g28580D?Bna A01g02130D?Bna A05g06450D?Bna C04g11150D and Bna A01g21750D.These results indicated that Sa MR12 inoculation could improve the efficiency of light energy trapping and photocatalytic ability of photosystem?,facilitate energy cycle of photosynthetic reaction center and facilitate chlorophyll biosynthesis,thus protecting the photosystem from Cd damage,which further leading to the growth promotion of B.juncea under Cd stress.(5)A pot experiment was conducted here to investigate the effects of different-source PGPB consortium on plant growth,Cd extraction,as well as the rhizospheric bacterial community of B.juncea grown on a Cd-contaminated soil.The results showed that the inoculation of PGPB consortium significantly increased biomass by 6.9%-12.8%in shoots and 10%-22.1%in roots.Moreover,inoculation of both rhizospheric PGPB consortium and endophytic PGPB consortium promoted the Cd phytoextraction efficiency.In addition,inoculation of PGPB consortium increased the abundance of Proteobacteria and Bacteroidota at phylum level;Flavobacterium,Rhodanobacter,Kosakonia,Pseudomonas and Paraburkholderia at genus level.The results showed that inoculation of PGPB consortium could promote plant growth,facilitate Cd uptake and shift the bacterial community as well.However,there was no significant difference between the effects of rhizospheric PGPB consortium and endophytic PGPB consortium.(6)A field experiment was conducted to study the effects of single and combined PGPB agents on rape growth,Cd uptake and accumulation at different growth stages.The results showed that Sa CR1+Sa MR10+Sa MR12 combined agents was more effective for promoting the growth of rape at bud stage,flowering stage,mature stage and seeds yield.Moreover,the single/combined PGPB agents of Sa MR12 and Sa MR10was more beneficial to plant Cd uptake.In addition,Sa MR10+Sa MR12 combined PGPB agents largely facilitated Cd content and accumulation in stems,enhanced the Cd extraction rates by 156%increase.Therefore,the application of single and combined PGPB agents could improve the phytoextraction efficiency of Cd,and combined PGPB agents was more functional than single PGPB agents.In conclusion,we found that the PGPB from hyperaccumulator S.alfredii could colonize and survive in the non-host plant B.juncea.Inoculation of Sa MR12 could promote rape growth and Cd accumulation by facilitating root development,alleviating Cd stress and improving plant photosynthesis.The isolation source of PGPB was not the decisive factor for the functional effects of the PGPB consortium.The results showed that both single and combined PGPB agents could be used as efficient remediation agents for B.juncea,of which combined PGPB agents was more effective.These results will provide scientific basis and technical support for the exploration of the molecular physiological mechanism of PGPB effects on the growth and Cd accumulation of non-host plants,for the establishment of efficient plant-microbe combined phytoremediation,as well as for the augment of phytoextraction efficiency in heavy metal-contaminaed farmlands.