Isolation Of Plant Growth Promoting Rhizobacteria And The Mechanism Of Relieving Saline-alkaline Stress On Alfalfa

Soil salinization and alkalization is one of the factors which seriously affect the growth of plants.Saline-alkaline stress could affect the growth of plant by inhibiting seed germination,seedling growth,photosynthesis and respiration.Inoculation of plant growth promoting rhizobacteria?PGPR?could promote the growth of plants,and improve the resistance of plants to stress,such as drought,saline-alkaline,and so on.However,the effect of PGPR in promoting plant growth under saline-alkaline stress and the mechanism are unclear.In the present study,PGPRs containing1-aminocyclopropane-1-carboxylate?ACC?deaminasewereisolatedfrom saline-alkaline soil of the rhizosphere of plant.The growth promoting ability of the strains were evaluated.Then,mechanism of improving the saline-alkaline tolerance of plants and the mechanism of promoting the growth of alfalfa by PGPR were explored.The summary of the main results are as follows:1.Screening and identifying of PGPRNine strains containing ACC deaminase were isolated from rhizosphere of plant under saline-alkaline with ACC as sole nitrogen source.Combined with 16s rDNA sequence analysis and strain physiology and biochemistry,Serratia marcescens L-11and Acinetobacter baumannii L-12 were from Echinochloa crusgalli?L.?Beauv.,Pantoea sp.L-14 and Agrobacterium sp.L-16 were from Cyperus rotundus L.,Agrobacterium sp.L-6 was from Avena sativa L.,Agrobacterium sp.L-17,Klebsiella oxytoca L-18 were from Phragmites communis Trin,Pseudomonas mediterranea M-3and Pseudomonas fluorescens M-5 were from Medicago sativa.2.Promoting characteristic identification of isolatesThe growth promoting characteristics of 9 strains were determined.The highest activity of ACC deaminase was found in L-11,and the order of high to low were L-12,M-3,M-5,L-6,L-16,L-18 and L-17.The lowest was L-14.All 9 strains could synthesize IAA in absence of L-Trp.The amount of IAA synthesis of L-6,L-11,L-12,L-16,M-3 and M-5 increased with the increase of L-Trp concentration.The amount of IAA synthesized by L-11 was the highest when there was no L-Trp,and the amount of IAA was the highest at L-Trp concentration of 50,100,200 and 500?g/m L in L-12.The siderophore production ability of 9 strains from high to low were L-11,L-18,L-14,M-3,L-17,M-5,L-6,L-16 and L-12.The phosphate solubilization from high to low followed by L-12,L-11,L-6,M-5,L-14,L-18,M-3,L-17 and L-16.PGPR strains still had the growth promoting characteristics when the strains were cultured under 4%salt concentration and pH 9.The activity of ACC deaminase of 9strains from high to low were L-11,L-12,M-3,M-5,L-6,L-16,L-17,L-18 and L-14.The amount of IAA synthesis of L-6,L-11,L-12,L-16,M-3 and M-5 increased with the increase of L-Trp concentration.The amount of IAA synthesized by L-11 was the highest when there was no L-Trp,and the amount of IAA was the highest at L-Trp concentration of 50,100,200 and 500?g/mL in L-12.The siderophore production ability of 9 strains from high to low were L-11,L-18,L-14,L-17,M-3,L-6,M-5,L-16and L-12.The phosphate solubilization from high to low followed by M-5,L-12,L-6,L-11,L-14,L-18,M-3,L-16 and L-17.3.Effects of PGPR promote the growth of alfalfa under saline-alkaline stress in potsBased on seed germination test,L-11,L-12,M-3 and M-5 could promote the germination of alfalfa seeds under different saline-alkaline levels.Four PGPR strains could reduce the content of ethylene under saline-alkaline stress.Evaluated the effects of 4 PGPR strains inoculation and co-inoculation on alfalfa in saline-alkaline conditions in pots.It was found that single inoculation and co-inoculation of 4 strains could significantly increase the plant height,root length,fresh weight and dry weight of shoot and root of alfalfa?P<0.05?.Besides,the root vigor,leaf chlorophyll content,soluble protein,free proline,and activity of antioxidant enzymes such like SOD,POD and CAT of alflafa inoculated with PGPR was significantly higher than Control?P<0.05?.The contents of MDA,O^2-·and H₂O₂ of both single and co-inoculation alfalfa were reduced?P<0.05?.The biomass and physiological indexes of co-inoculated with L-11+M-3were significantly higher than Control?P<0.05?.Meanwhile,the biomass and physiological indexes of single inoculation L-11 was also higher than Control?P<0.05?.4.Effects of PGPR promote the growth of alfalfa under saline-alkaline stress in fieldThe effect of PGPR on the yield and quality of alfalfa were analyzed in saline-alkaline field in 2014 and 2015.The heights and yields of alfalfa inoculated with single and co-inoculation were significantly increased?P<0.05?,and L-11+M-3 was highest among co-inoculation,L-11 was highest among single inoculation.The crude protein content of alfalfa inoculated with PGPR were higher than Control?P<0.05?.The crude fiber contents of alfalfa inoculated with PGPR were significantly lower than Control?P<0.05?,and the phosphorus contents of alfalfa were higher than Control?P<0.05?.The biomass and quality of co-inoculated with L-11+M-3 were the highest.5.Effect of genes expression of alfalfa inoculated with PGPR under saline-alkaline stressThe expression levels of ethylene responded genes in alfalfa inoculated with strain L-11 under saline-alkaline stress were analysed by qRT-PCR The genes of ACC oxidase?ACO?,ethylene response factor?ERS1,2?,ethylene receptor?ETR1?,ethylene insensitive?EIN3?,and ethylene response factor?ERF1?were down-regulated expression when alfalfa inoculated with PGPR containing ACC deaminase.The results showed that under saline-alkaline stress,alfalfa inoculated with PGPR containing ACC daminase could reduce the inhibitory effect of ethylene on plant growth by reducing the amount of ethylene.The expression levels of IAA responsed genes in alfalfa inoculated with strain L-11under saline-alkaline stress were analysed by qRT-PCR.The genes involving in SCF^TIR1complex?TIR1 and CUL1?,auxin response factors?ARF1?and genes that affect lateral root growth?IAA3,IAA14,and IAA19?were up-regulated expression in alfalfa when alfalfa inoculated with PGPR synthesizing IAA.The results suggested that inoculation of L-11 could promote root elongation and plant growth.The expressions levels of genes which were related to saline-alkaline tolerance in alfalfa inoculated with strain L-11 under saline-alkaline stress were analyzed by qRT-PCR.Compared with non-inoculated Control,the expression of genes involving in regulate response to stress?NAC6,47?,scavenge the reactive oxide species?GSTU4,10?and gene expression involved in the plant regulation process?WRKY33,75?were up-regulated in alfalfa when alfalfa inoculated with L-11.The results showed that inoculation with PGPR could help the plant to remove harmful substances such as ROS,accumulate the protective substances,and improve the saline-alkaline tolerance of plants.