Responses Of Rhizobia And Arbuscular Mycorrhizal Fungi To Cadmium And Nitrogen Stress In Robinia Pseudoacacia

As one of the most important soil heavy metal contaminants,the heavy metal cadmium（Cd）is characterised by high toxicity,high transport capacity,inability to be degraded by soil microorganisms and slow natural purification.Exposure of plants to Cd contamination can cause a strong toxic reaction.Plants can reduce the level of heavy metals in the environment by absorbing and immobilising them.Studies have found that symbiosis between microorganisms and plants can improve the inter-root microenvironment of plants and increase their ability to withstand heavy metal stress.Rhizobia and mycorrhizal fungi（AMF）are widely distributed in the environment,and nitrogen fixation by rhizobia in symbiosis with plants can promote plant growth and heavy metal uptake by plants;AMF build a large mycelial network through external mycelium,providing a channel for plants to take up essential nutrients.However,the mechanisms by which the symbiosis of rhizobia,AMF and host plants respond to Cd stress under N deficiency and the changes in the inter-root microbial community are not yet clear.Therefore,in this study,we investigated in detail the effects of different inoculation treatments on the growth and physiological response of acacias under N deficiency and Cd stress and the mechanisms of the symbiotic microbial response to acacia nutrient uptake and its role in the mitigation of Cd stress,as well as the response of the inter-rhizosphere bacterial community.Specific findings were as follows:（1）Mycorrhizal colonisation was not inhibited by cadmium stress,but rather the rate of infestation was increased,and the rate of mycorrhizal infestation was higher compared to normal nitrogen conditions under low nitrogen conditions,with the highest rate of infestation（56.2%）achieved when rhizobia and AMF were co-inoculated.The inoculation of rhizobia and AMF effectively alleviated the inhibition of cadmium stress on acacia growth,where the overall biomass of acacia was at a higher level under normal nitrogen supply than under low nitrogen.The effect of nitrogen on acacia growth was significantly reduced under symbiotic conditions.Under R conditions,the normal N treatment increased acacia root and above-ground biomass by 65%and 90%,respectively,compared to the low N treatment,but the inoculation treatment effectively reduced the difference between the two,especially at the RRM treatment where the increases were 19%and 45%.The proportion of acacia root biomass under Cd stress was also significantly higher.Cd stress significantly reduced the chlorophyll content of all treated acacias.Inoculation alleviated the effects of Cd stress on acacia chlorophyll and significantly increased chlorophyll content,with the best improvement occurring when co-inoculated.The net photosynthetic rate（P_n）,stomatal conductance（g_s）and transpiration rate（E）of acacia plants were significantly reduced under Cd stress,while intercellular CO₂concentration（C_i）did not change significantly,while net photosynthetic rate（P_n）,stomatal conductance（g_s）and transpiration rate（E）of acacia plants were significantly increased under co-inoculation with Rhizobium and AMF,and the inhibition of chlorophyll content and photosynthesis by nitrogen stress under co-inoculation The inhibition of chlorophyll content and photosynthesis by nitrogen stress under co-inoculation was significantly reduced.（2）The concentration of Cd in acacia decreases sequentially from the root to the stem and leaves,and the greater biomass under Cd stress is allocated to the roots,which directly leads to the accumulation of Cd in the roots of acacia and significantly reduces the proportion of Cd transported to the ground while ensuring normal growth and development of the above ground,all inoculation treatments can reduce the proportion of Cd transported to the ground,especially the most significant reduction in the RRM treatment,compared with the R treatment.The percentage of Cd translocation to the ground was reduced by 13%and25%under low and normal N conditions,respectively,in addition to the lower percentage of Cd translocation to the ground under normal N conditions compared to low N.The MDA and H₂O₂contents of acacia leaves were significantly increased under Cd stress,and inoculation treatments reduced MDA and H₂O₂contents in all treatments,with the greatest reduction in the RRM treatment,which greatly reduced the effect of N deficiency on MDA and H₂O₂.The addition of rhizobia and AMF significantly increased the antioxidant capacity of acacia leaves,with significantly higher CAT,POD and SOD activities in all treatments compared to the non-inoculated treatments,and the highest antioxidant enzyme activities were found in the rhizobia and AMF co-inoculation,in addition to higher enzyme activities in acacia leaves at normal nitrogen levels.（3）Cd stress inhibited the uptake of N and P nutrients in acacia,and inoculation treatments improved nutrient acquisition well.All inoculated treatments had higher nutrient uptake in acacia than uninoculated treatments,with the highest nitrate N content in acacia roots under co-inoculation conditions and a very high bias towards P uptake in AMF alone.Inoculation under Cd stress increased the uptake of Fe,Mg,Cu and Zn by acacia roots compared to the control.For Fe and Mg uptake,co-inoculation had the most significant promotion effect and increased their translocation to the aboveground,ensuring the stability of the aboveground photosynthetic system,while for Cu and Zn uptake,AMF alone had the most significant promotion effect and ensured the stability of the aboveground Cu and Zn content.（4）Under low nitrogen conditions,both RM and RRM treatments had significantly higher abundance and diversity of bacteria than R treatment,while Rcd had the highest bacterial community fraction and diversity under cadmium treatment conditions,comparing RRcd and RMcd,RRMcd had higher abundance and diversity of bacterial community,and under normal nitrogen supply,R treatment bacterial community had the highest abundance and diversity compared to inoculation treatment.The abundance of bacterial communities in all treatments under Cd stress decreased with inoculation,but the diversity of bacterial communities increased with inoculation,and the highest diversity was found in co-inoculation.The bacterial community composition of the different nitrogen treatments under cadmium stress showed separate clustering,indicating that the bacterial community structure was similar between the same treatments and differed between treatments,and that the cadmium treatment was clearly separated from the treatment without cadmium addition,indicating that cadmium stress had a significant effect on the bacterial community structure.At the phylum level,the dominant phyla in all treatments were Actinobacteria,Proteobacteria and Firmicutes,accounting for more than 80%of all treatments.The abundance of Actinobacteria,Proteobacteria and Firmicutes decreased significantly under Cd stress,while that of Proteobacteria and Firmicutes increased under low N compared to normal N.The changes in the abundance of Proteobacteria,Firmicutes,Chloroflexi,Acidobacteria and Bacillariophyta were more significant under RRMcd treatment.The relative abundance of Phyllostomycetes,Chloroflexi,Acidobacteria and Planctomycetes was higher under low nitrogen conditions than under normal nitrogen treatment,and Phyllostomycetes,Chloroflexi and Planctomycetes,as the core groups of bacteria that dominate the inter-rhizosphere bacterial community of acacia,may be the main reason why acacia copes with nitrogen limitation and cadmium stress during the symbiosis.As the core group of bacteria that dominate the inter-rhizosphere community of Robinia pseudoacacacia,A.deformans,A.greenish curvilinearis and A.thick-walledis,they may be the key phyla recruited by Robinia pseudoacacacacia to enhance nutrient acquisition and relieve heavy metal stress in response to nitrogen limitation and Cd stress during the symbiosis.（5）In this study,the genus level of bacteria was analysed.The results showed that genus Pseudarthrobacter and Bacillus were the dominant genera for all treatments,and for all inoculation treatments,cadmium stress increased the relative abundance of Bacillus and IMCC26256 and decreased the relative abundance of Pseudarthrobacter,Streptomyces and Arthrobacter.This result may be attributed to the fact that Bacillus,Streptomyces and Arthrobacter are more sensitive to heavy metals,while Bacillus and IMCC26256 may be the core genera under inoculation conditions,and Bacillus accounted for a significant proportion of the top 10 relative abundances.Bacillus spp.showed different patterns of variation,A higher relative abundance of Bacillus spp.and a lower abundance of Pseudomonas spp.were also found under different nitrogen treatment patterns compared to the normal nitrogen treatment under low nitrogen conditions.The abundance of Pseudomonas spp.increased with inoculation under low nitrogen conditions and decreased with inoculation under normal nitrogen.In addition RMcd had the highest abundance of Bacillus spp.of all treatments.These may be key species in response to this stress under conditions of low nitrogen availability.In summary,nitrogen and phosphorus nutrient uptake in acacia was significantly improved by rhizobia and AMF inoculation under low N and Cd stress,with more Cd fixed by acacia roots and reduced translocation to the ground,while inoculation promoted the translocation of Fe and Mg to the ground,protecting chlorophyll biosynthesis and the photosynthetic system,a process accompanied by increased antioxidant enzyme activity and reduced malondialdehyde and hydrogen peroxide content,alleviating This process is accompanied by an increase in antioxidant enzyme activity and a reduction in malondialdehyde and hydrogen peroxide content,alleviating the oxidative stress caused by cadmium stress,resulting in higher photosynthetic capacity and resistance,ultimately enabling acacias to resist cadmium stress and increase their biomass.In addition,inoculation increased the abundance and diversity of inter-rhizosphere bacteria in acacia,recruiting bacterial populations that are adapted to cadmium and nitrogen deficiency stress.This will provide theoretical guidance for the phytoremediation of degraded and heavy metal contaminated land.