| Switchgrass,a perennial rhizomatous C4-plant is a promising bioenergy plant due to its high biomass,as well as bioenergy production competency as they can grow in the unproductive marginal lands.Endophytic plant-growth-promoting bacteria(PGPB)may contribute to switchgrass growth and regulate Cd uptake and accumulation under Cd stress.In this study,the physiology and molecular mechanism of two ecotype switchgrass cultivars inoculated with PGPB,lowland type cultivar ’Alamo’ and upland type cultivar ’Cave in Rock(CIR)’,were investigated with hydroponic and soil experiments.This research will provide the theory and practice foundation for efficiently developing bioenergy plant in heavy metal contaminated soils.The main results are as following:1.Four endophytic bacteria viz.,Bc09,So23,E02,and Oj24 were successfully isolated from the interior of the shoots of Brassica campestris L.,Spinacea oleracea L.,an unidentified dicot plant,and Oenanthe javanica Bl.,respectively grown in a Cdcontaminated soil.These bacteria were identified by 16s rRNA sequencing as Pseudomonas grimontii strain Bc09,Pantoea vagans strain So23,Pseudomonas veronii strain E02,and Pseudomonas fluorescens strain Oj24 with the GenBank Accession Number KT326183,KT326184,KT326185,and KT326186,respectively.All the bacteria were gram-negative,aerobic,catalase-positive,and motile.These four bacteria have the excellent criterion of adaptation at high CdCl2 concentrations with high Cd uptake capacity and possessed the plant growth-regulating properties,e.g.,indole acetic acid production(IAA),1aminocyclopropane-1-carboxylic acid deaminase(ACCD)activity,and phosphate solubilization ability.The germinating experiment in plates confirmed the plant growth promotion ability of this four endophytic PGPB because PGPB-inoculated switchgrass has significant(p<0.05)higher root and shoot length compared to the noninoculated control plants under 200 μM CdCl2 stress.The entry of bacteria inside the plant parts(roots and shoots)was also confirmed by monitoring the green fluorescent protein(GFP)-labeled bacteria in ’Alamo’.That proved the endophytic nature of the PGPB.2.A hydroponic experiment was conducted to monitor the influence of four identified PGPB on switchgrass cvs ’Alamo’ and ’Cave in Rock’(’CIR’)grown under 20 μM CdCl2 stress.Growth in terms of height and biomass of both cultivars under Cd stress was increased significantly(p<0.05)in the PGPB-inoculated plants.However,the shoot biomass of PGPBinoculated ’Alamo’ was more than the ’CIR’ under Cd stress.Most importantly,the Cd concentration in PGPB-inoculated plant tissues(roots and shoots)was lower compared to the noninoculated Cd-control plant in both cultivars with some exception.But the Cd amounts was more in the PGPB-inoculated plants,this is because of more biomass content of PGPB compared to the Cd-control plants.However,despite the exception,owing to the beneficial activities of PGPB and decreased levels of Cd concentration in the plant tissues,the PGPBinoculated plant acquired more biomass.In case of biomass,multivariate ANOVA detected significant differences(at p<0.001,p<0.01,and p<0.05 levels)due to PGPB treatments,cultivars,and PGPB × Cultivars interactions.The PGPB treatments and PGPB × Cultivar interactions possessed no significant differences in case of Cd concentration in roots and shoots.However,cultivars have significant differences between them in both case of root and shoot Cd concentrations.In case of the Cd amounts in plants,significant differences were detected in PGPB treatments,cultivars,and PGPB × Cultivars interactions.The influence of single or differentially combined PGPB-inoculated switchgrass cv ’Alamo’ grown in Cdcontaminated soil cultures was also explored.Where it was observed the fresh and dry biomasses of the single PGPB-inoculated and differentially combined PGPB-inoculated plants were significantly higher compared to the noninoculated control plants under Cd stress.Interestingly,in the PGPB inoculated plants(both single PGPB-inoculated and differentially combined PGPB-inoculated plants)the Cd concentration and Cd amounts was less compared to the noninoculated control plants.3.The chlorophyll pigment content in the PGPB-inoculated plants was increased both in Cd-amended hydroponic and soil culture.The root morphological parameters total root length(RL),specific root length(SRL),root surface area(RA),average root diameter(RD),root volume(RV),and number of root tips(RTs)and forks of PGPB-inoculated plants were significantly higher in the plants grown in hydroponic culture.In five classes of root diameter(0.2 mm interval),the RL,RA,RV,and RTs of PGPB-inoculated plants were significantly higher than those of the noninoculated Cd-control plants under Cd stress.Moreover,the multivariate ANOVA detected that the differences due to PGPB,cultivar,and PGPB ×cultivar interactions were highly significant(at p<0.001,p<0.01,and p<0.05 levels).In addition,the principal component analysis(PCA)confirmed the existence of a significant correlation between the root morphology,biomass,chlorophyll content,and Cd concentration.This modified root morphology results in minimizing Cd accumulation in roots.The influence of single or differentially combined PGPB-inoculated switchgrass cv’Alamo’ in soil cultures was explored and lignin content of cell wall residue was determined at a primary level by acetyl bromide procedure.It was found that the noninoculated control plant exhibited more lignin content than the single PGPB-inoculated and differentially combined PGPB-inoculated‘Alamo’cultivated for 116 days and 166 days,respectively.This result of lower lignin content is expected for any bioenergy plants for producing biofuel.Our results suggested that the lignin part of the experiment with the assistance of endophyticPGPB needs more research in depth.4.The gene expression levels of heat shock protein 70(HSP70)and the heavy metal ATPase 3(HMA3)genes in the PGPB-inoculated and noninoculated switchgrass with and without Cd stress have been investigated.The experimental results revealed that HSP70 and HMA3 genes expressed highly in the PGPB-inoculated plants under Cd stress.In addition,the expression of HSP70 and HMA3 genes was considerably higher in the first two days after successive four-day exposure of Cd in plants compared to the last two days of exposure.This up-regulation of the gene expression is one of the reasons for overcoming the adverse effect of stress.The PCA analysis revealed a positive correlation among Cd concentration in roots and shoots,HSP70 and HMA3 genes expression;furthermore,root and shoot biomass(dry weight)were also in a positive correlation.Whereas,Cd concentrations and gene expression were in negative correlation with biomass.Regardless of the mechanisms used,PGPB helps plants to enhance growth and yields;therefore,we obtained higher biomass in all the PGPBinoculated plants compared to the Cd control plants under Cd stress.In this study,four endophytic bacteria(either independently or together)were found to be very efficient and beneficial to assist the switchgrass in a Cd-contaminated area.They could decrease the concentration of Cd inside plant tissue,change root morph-architectural patterns with enhanced biomass,lower lignin content in plant tissues,and modify cellular protein quality control systems.The mentioned endophytic bacteria are suitable agents for sustainable biomass production for bioenergy in a Cd-contaminated environment. |
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