Screening And Identification Of Manganese Tolerant Endophytic Bacteria And Their Effect On Manganese Responsive In Wheat

Endophytic bacteria live in symbiosis with their host plants for a long time,they can improve the plant's ability to resist biotic and abiotic stresses.Manganese is an important trace element for plant growth and development,but high concentration of manganese will cause serious toxicity to plants.Therefore,screening and inoculation of Manganese tolerant endophytes in plants is an effective means to improve plant Manganese tolerance.In this study,endophytes with manganese tolerance were isolated and identified from plants such as Solanum solanum,and molecular markers were developed for rapid identification based on lo op-mediated isothermal amplification(LAMP).Then,biological characteristics such as the maximum tolerance concentration and manganese ion removal rate were studied,and finally,the response of endophytes with manganese tolerance to manganese stress in wheat was explored.The main research conclusions are as follows:(1)The plate scribing method was used to preliminarily screen 365 bacteria stored in the laboratory.Among them,165 endophytic bacteria have tolerance to manganese.After identification,the 165 bacteria belong to 4 genera,including Serratia genera(Serratia liquefaciens,MTEB-1);Bacillus genera(Bacillus aerius,MTEB-2);Alcaligenes genera(Alcaligenes faecalis,MTEB-3)and Enterococcus genera(Enterococcus gallinarum,MTEB-4).Four high manganese tolerant endophytes still have good tolerance to manganese under the stress of manganese ion concentration up to 2000 mg/L.The strain MTEB-1 with the strongest tolerance can grow in the environment of manganese ion concentration of 5000 mg/L.Furthermore,an experiment was set to determine the manganese ion removal ability of the four strains with high tolerance under the concentration of 2000 mg/L manganese stress.The results showed that the manganese ion removal rate of the four strains ranged from10.64%to 11.46%at 72h.Among them,MTEB-1 has the highest manganese ion removal rate and the maximum improvement of manganese removal capacity between48 h and 72 h is 5.85%.LAMP markers were developed for rapid detection,and the results showed that the designed primers could quickly and specifically identify MTEB-1.(2)Colonization experiments showed that the EGFP-labeled strain MTEB-1 emitted bright green fluorescence after blue light excitation,and the fluorescence could be clearly observed in wheat,indicating that the strain MTEB-1 could survive and colonize in wheat.The effects of MTEB-1 broth soaking on germination rate,root length,bud length,biomass and morphology of wheat seeds in different concentrations of Mn were analyzed.The results showed that MTEB-1 broth soaking had a certain promoting effect on germination rate,root length,bud length,biomass and morphology of wheat seeds.Especially under the Mn stress concentration of 0 and700 mg/L,the germination rate increased from 95%to 98%,and the yellowing phenomenon of wheat under Mn stress was alleviated after seed soaking treatment.(3)Hydroponic experiments were carried out to investigate the effects of different manganese concentration gradients on the activities of antioxidant enzymes,the content of osmotic regulatory substances,the content of H₂O₂,the content of chlorophyll and the absorption of manganese.The results showed that MTEB-1inoculation could promote the activities of antioxidant enzymes,the contents of osmotic regulatory substances,the contents of H₂O₂,chlorophyll and manganese absorption in wheat seeds.Endophytic bacteria MTEB-1 under different treatment times and different concentrations of manganese stress have a certain influence on antioxidant enzyme activity in wheat leaves,and SOD,POD and CAT activities are significantly increased in seed soaking treatment group compared with the control group;The contents of MDA and proline in seed soaking treatments and control wheat leaves increase with the increase of manganese ion concentration.After seed soaking,the contents of MDA and proline of MTEB-1 are reduced to a certain extent,but with the increase of treatment time and concentration,the damage degree of wheat is aggravated,and the contents of MDA and proline reach the highest content.MTEB-1can promote the increase of chlorophyll content in wheat leaves after treatment under different concentrations of manganese stress.DAB staining was used to observe the changes of H₂O₂ content in wheat.Under manganese stress,H₂O₂ content in both treatment group and control group increased with the increase of manganese concentration,and the higher the manganese concentration,the higher the H₂O₂content was.Under different concentrations of manganese stress,the manganese content of aboveground and underground parts of wheat increases with the increase of manganese concentration.After seed soaking treatment,the manganese content of aboveground and underground parts of wheat increases,indicating that MTEB-1 can promote the absorption of manganese in wheat.In this study,Mn-tolerant endophytic bacteria can significantly improve the Mn absorption capacity of wheat,providing a new idea for improving the Mn content of wheat.Endophytic microorganisms were used to promote the growth of wheat in Mn-contaminated soil,so that it could accumulate heavy metals while growing normally,and realize the production and remediation at the same time,which provided an effective and feasible alternative green remediation method for heavy metal remediation.