Isolation And Identification Of Endophytic Bacteria Resistant To Nickel And Manganese In Wetland Plants In The Yellow River Delta And Preliminary Study On Their Resistance To Heavy Metals

The Yellow River Delta Wetland is a national nature reserve.It is located at the junction of land and sea,and carries the material exchange between land and sea.In recent years,heavy industry of China has developed rapidly and a large amount of industrial wastewater has been discharged.The content of heavy metals carried by the Yellow River sand,especially manganese,has increased by 53.7%compared with ten years ago.This has caused the accumulation of heavy metals in the wetland soils of the Yellow River Delta to increase,which is potentially toxic to the ecology.In the remediation of heavy metal pollution,the bioremediation method has the advantages of low cost,high efficiency,wide application range and not causing secondary pollution.Heavy metal wetland phytoremediation technology refers to planting wetland plants grown naturally or cultivated by genetic engineering in heavy metal contaminated soil or water bodies.It uses the symbiotic system of wetland plants or plants and microorganisms to achieve the purpose of repairing heavy metal pollution.Wetland plants can use the root system to change the environment of the root zone and provide a place for microorganisms to attach and grow.And heavy metal ions can be absorbed into the plant to reduce the concentration of heavy metal ions in the soil environment.Plant endophytes are bacteria that are isolated from healthy plant tissues and symbiotic with plants.Plant endophytes promote plant growth by secreting phytohormones.Plant endophytes inhibit the infestation of plants by pathogenic bacteria by secreting siderophore.Plant endophytes enable plants to survive in stressful environments such as soil contaminated by heavy metals through the adsorption of heavy metal ions.In recent years,the use of endophytic bacteria to strengthen plant pollution repair has become a hot research topic.In this project,through the identification of heavy metal-resistant nickel and manganese endophytic bacteria in dominant plants of Tamarix chinensis and Suaeda salsa in the Yellow River Delta,several potential nickel-resistant endophytic bacteria and manganese-resistant endophytic bacteria were discovered.Preliminary studies have been conducted on the mechanism of resistance to heavy metals.The research content of this topic is as follows:(1)Isolation and identification of a variety of nickel-resistant endophytic bacteria and manganese-resistant endophytic bacteria:Through isolation and identification,three types of nickel-resistant endophytic bacteria were obtained,which belonged to the genus Stenotrophomonas,Pseudomonas,Sphingobium respectively.Ten kinds of manganese-resistant endophytic bacteria were obtained.Through the measurement of manganese-oxidation activity,it was found that all manganese-resistant endophytic bacteria have manganese oxidation activity.(2)Three nickel-resistant endophytic bacteria were tested for nickel removal rate and seed germination experiments:All of three endophytic bacteria had a nickel-removing effect,the Sphingobium sp.S42 was found to have the strongest nickel-removing ability.The nickel removal rate reached 33.68%after 36 hours.For the root length of plants in presence of 200mg/L Ni,the plants inoculated with isolate S42 showed the most extended length,4.3 fold higher compared with the un-inoculated plants.(3)Determination of physicochemical properties and growth promotion potential of manganese-oxidizing endophytes of the Salinicola genus:It was found that the salt tolerance range of the Salinicola genus is generally 3%-15%.The survival rate was higher than 8.2%under 2 mmol/L H₂O₂conditions.They could produce indole-3-acetic acid(IAA),siderophore and ACC(1-aminocyclopropane-1-carboxylic acid)deaminase.This lays a foundation for further exploring the molecular mechanism of manganese-oxidizing endogenous bacteria.(4)Investigating the effects of two manganese oxidizing endophytic bacteria Pantoea eucrina SS01 and Pseudomonas composti SS02 on plant growth promotion and stress tolerance:It was found that the maximum manganese-tolerant concentrations of the two manganese oxidizing endophytes were 30 m M and 20 m M.They could secrete IAA above 12.08 mg/L,and can promote the increase in plant height and root length of Suaeda salsa by colonization.X-ray fluorescence spectrometry(XRF)was used to detect manganese and iron in the black matter of the root of Suaeda salsa.It proved that SS01 and SS02 can significantly promote the formation of manganese oxide film on the root of Suaeda salsa,which can be used for plant-endophytic bacteria repair.(5)A new type of manganese catalase Mn Cat was discovered,and its molecular mechanism in the stress tolerance of manganese-oxidizing endogenous bacteria was studied through in vivo and in vitro experiments:Through whole-genome analysis,it was found that Salinicola genus contains a new type of manganese catalase.We could predict that the protein has a molecular weight of about 27.56 k Da and contains six trivalent manganese ions according to the bioinformatics method.Through RT-q PCR and gene knockout,it was found that when the external Na Cl concentration is too low(less than 3%)and the hydrogen peroxide concentration is increased,the transcription level of the gene is increased.It is speculated that this protein plays a role in the antistress response of Salinicola bacteria.Through exogenous expression and non-denaturing polyacrylamide gel electrophoresis analysis of Mn Cat protein,it was found to have catalase activity.The inclusion body protein was purified by nickel column.In summary,this subject has discovered a variety of nickel-resistant and manganese-resistant endophytic bacteria,which have different characteristics such as reducing the content of nickel ions in the environment,oxidizing manganese ions,generating plant growth hormones,and promoting the formation of manganese oxide films.This may give them the ability to promote the growth of plants in soils contaminated by heavy metals nickel and manganese.It was found that the endogenous bacteria of the Salinicola genus have better stress tolerance characteristics,growth promotion potential and manganese-oxidation activity,and they may have a certain promotion effect on the growth of plants in the environment of heavy metal manganese.It was found that the manganese catalase Mn Cat in the Salinicola genus may play a role in anti-stress response to bacteria,especially salt-resistance and anti-oxidation.This subject has certain application value for the joint plant-endophytic bacteria repairing heavy metal soil,and lays a good foundation for the application of plant-endophyte environmental management.