The Effects Of Endophytic Bacteria Isolated From Metal Tolerant-plants On The Tolerances And Accumulation Of Heavy Metals By Brassica Napus

Phytoremediation is an emerging in-situ remediation technology due to its less cost, convenient operation and friendly-environment for remediation of heavy metal-contaminated soils. A large number of microorganisms which colonized plant tissue interior can affect growth and heavy metal accumulation of plants in heavy metal-polluted environment by many approaches. Therefore, endophytic bacteria play an important role in bacteria-assisted phytoremediation of heavy metal-contaminated soils. By isolating plant growth promoting endophytic bacteria and utilization for remediation, we can not only improve the remediation efficiency and enrich microbial resource librarys, but also elucidate certain mechanisms on how plant growth promoting endophyte improve the biomass of plant and uptake of metals in heavy metal-polluted environments.Twenty1-aminocyclopropane-l-carboxylic acid (ACC) deaminase-producing strains were isolated from chrgsanthemum indicum, bidens pilosa and chenopodium ambrosioides heavy metal-tolerant plant species in mining area and characterized with respect to metal resistance, production of ACC deaminase, indole-3-acetic acid (IAA) and siderophores and mineral phosphate solubilization. Eight strains were isolated from Cu-tolerant plants in Tangshan Cu mine wasteland and the other twelve strains were isolated from qixiashan Lead-Zinc mine tailing. All twenty strains have different heavy metal (Cu, Cd, Pb, Ni) resistance, of which six strains (Y1-3-9、Jp3-3、J1-22-2、Q2BJ2、Q2EJ2、Q2BG1) could produce high level of ACC deaminase (120.3-369.6μM h-1mg-1). The twenty strains were identified as Pantoea%Ralstonia、Pseudomonas、Bacillus、Acinetobacter and Agrobacterium based on16S rRNA gene sequencesanalyses.We selected Brassica napus for phytoremediation in pot experiment to investigate the effect of ACC deaminase-producing endophytic bacteria on the growth and Cu/Pb accumulation of the plant in quartz sand with copper/lead stress. The results indicated that all six strains with high level of ACC deaminase activity have ability to increase the biomass of Brassica napus in the absence of heavy metal stress, and strains Y1-3-9, Jp3-3and J1-22-2could increase the dry weight (24.3-166.8%), Cu content (86.4-192.6%) and total Cu uptake (43.3-124.4%) of Brassica napus in the presence of2.5and5mg kg-1of Cu2+. In the presence of50mg L"1lead, the test strains did not influence the Brassica napus biomass significantly, however, the strains Q2BJ2, Q2EJ2and Q2BG1increased the Pb contents by34.6%to175%and the total Pb uptake from84.0%to147%. In experiments involving Brassica napus grown in quartz sand containing100mg kg-1of Pb, inoculation with the isolates (Q2BJ2, Q2EJ2and Q2BG1) resulted in the increased dry weights (ranging from34.5%to72.2%), lead contents (ranging from156%to310%) and total lead uptake (ranging from28.6%to325%) of Brassica napus compared to the uninoculated control. Strains Q2EJ2and Q2BJ2increased the translocation factor of Pb (1.399and1.049respectively) in the Brassica napus compared with the uninoculation control (0.575), suggesting that bacterial inoculation may influence the translocation factor and metal hyperaccumulating ability of Brassica napus.The mechanism and effect of the growth-promoting endophytic bacteria on microbial-assisted phytoremediation of heavy metal polluted-soil were investigated in pot experiment using in-situ polluted soil collected from Tangshan Cu mine and Qixiashan Pb/Zn mine. Two strains (Y1-3-9and Jp3-3) with high-level of ACC deaminase activity and other two strains (Q2EY2and Q1BY6) that can produce arginine decarboxylase were seleced for pot experiments.Pot experiment showed that inoculation with the strains (Y1-3-9, Jp3-3, Q2EY2and Q1BY6) could increase the biomass of rape by24.4-124%, the Cu/Pb contents by55.8-685%and total Cu/Pb uptake by46-494%compare to the controls. In the experiment involving Brassica napus grown in in-situ heavy metal polluted soil, the analyses of PCR-DGGE revealed that the endophytic bacterial community in the roots of Brassica napus was influenced more obviously by the test strains compared with the rhizosphere bacterial community of Brassica napus.Inoculation with strain Q1BY6resulted in increased relative chlorophyll cotent in leaves in Brassica napus. The content of acetic acid in shoots of Brassica napus was increased by inoculating strains Y1-3-9, Jp3-3and Q1BY6compared with controls and the content of citric acid was significantly increased by16.4%by inoculation with strain Q1BY6. The strains Y1-3-9, Jp3-3and Q1BY6induced root tissues of Brassica napus to produce succinic acid. Inoculation with strains Y1-3-9, Q2EY2and Q1BY6can reduce free putrescine contents of rape to1.7-398.9%compared to controls and inoculation with the strains Yl-3-9and Q2EY2increased value of (Spd+Spm)/Put. The content of phytochelatin was increased by22.5%-256%by inoculation with strains Y1-3-9and Jp3-3and indicated that srains can alleviate the toxicity of heavy metals to Brassica napus.