| Phytoremediation is considered as a cost-effective and environment-friendly technology for remediation of heavy metal-contaminated soils. A large number of active rhizosphere microorganisms affect growth and heavy metal accumulation of plant in heavy metal-polluted environment, and then change phytoremediation efficiency. In this paper, Pb&Cd-resistant and maize-growth promoting bacterial strains were screened out by their lead and cadmium resistance, ACC deaminase activity and growth promotion to corn seedlings. The effects of bacteria on plant growth and Pb and Cd accumulation under heavy metals stress and mechanism of microbes improving heavy metal tolerance of maize were studied. The results may further clarify mechanism of plant's heavy metal tolerance and offer the theoretical and experimental basis for application of plant-microbe remediation. Major conclusions were summarized as follows:1. Bacterial strains J62, LR113, JYC17, G16, J17-2a and Y3-9 were screened out and respectively identified as Burkholderia cepacia J62, Rahnella sp. LR113, Microbacterium oxydans JYC17, Microbacterium foliorum G16, Enterobacter agglomerans J17-2a and Pseudomonas thivervalensis Y3-9. All bacteria did not cause disease to crops.2. The plant growth-promoting characteristics of isolated bacteria were studied in this paper. The results showed that test strains expected LR113 had the ACC deaminase activity. Strain J62, JYC17, J17-2a and Y3-9 had the arginine decarboxylase activity. All the strains could dissolve calcium phosphate and produce siderophores and indole acetic acid (IAA). The phosphate solubilization of strain LR113 and J62, siderophores secretion of strain J62 and LR113 and IAA production capacity of strain G16 and LR113 were better than that of other strains.3. The heavy metals characteristics of isolated bacteria were assessed. Strains J62, LR113, J17-2a and Y3-9 could grow in media with Pbl000 mg.L-1 or Cd50 mg.L-1 and tolerated Cu, Zn and Ni as well. All test bacteria could promote PbCO3 and CdCO3 solubilization. The bacteria enhanced Pb2+ contents by 3.45 to 39.3 times, compared to non-inoculated control. Cd2+contents in media were increased from 1.03 mg.L-1 to 1.23～127.53 mg.L-1. The lead carbonate activation of strain J62 and JYC17 and cadmium carbonate activation of strains J62 and J17-2a were significantly better than that of other strains. Strain J62 and LR113 were chosen to investigate release and absorption dynamic and mechanism of lead and cadmium. The data revealed that pH descent arose by strains growth increased water-soluble Pb2+or Cd2+concentration in liquid media. The insoluble Pb and Cd solubilization ability of strain J62 was higher than that of LR113. The organic acid species and concentrations produced by bacteria were determined by HPLC. In the media inoculated strain J62, Pb release had no significant effect on organic acids production and Cd release reduced organic acids contents. Carbonate lead and cadmium solubilization promoted organic acids secretion by strain LR113. The organic acids types produced by these two strains were quietly different. Lead and cadmium adsorbed by bacterial cell were measured through the desorption ability of NH4OAc, HCl and EDTA. NH4OAc extracted more Pb (6.17 mg.L-1) on J62 cells. HCl had the best desoiption capability to Cd (77.41mg.L-1) on strain J62 mycelium and Pb (33.65mg.L-1) or Cd (91.92mg.L-1) adsorbed by strain LR113. The Pb and Cd absorption capacity of strain LR113 were better than that of strain J62. The results of bacteria impact on insoluble lead and cadmium in soils demonstrated that strains J62 and LR113 could colonize in the soils; strain J62 had stronger lead and cadmium activation ability than strain LR113 and significantly increased water-soluble Pb and Cd in soils.4. Strain J62, JYC17 and Y3-9 were selected as test bacteria to research the effect of bacterial inoculation on maize growth and Pb and Cd accumulation in aqueous culture with heavy metal stress. The experiments clearly showed that all strains promoted maize growth. Strain J62 and JYC17 significantly promoted the growth of corn in nutrient solution without heavy metal ions. In nutrient solution with Pb2+50 or 100 mg.L-1, dry weights of corn inoculated with strains were higher (25%～94%) than that of non-inoculated control. In nutrient solution with Cd+2 or 4 mg.L-1, all bacteria enhanced root weight of maize by 8%～102%; the growth-promoting role of strain JYC17 was better than J62 and Y3-9. The enhancement of heavy metals stress reduced the growth-promoting capability of bacteria. Bacterial inoculation could improve lead and cadmium concentrations by 17%to 182%in com roots, whereas had little effect on lead and cadmium uptake in corn shoots. Inoculation with strain J62, JYC17 and Y3-9 all increased total lead and cadmium accumulation of maize. Compared to non-inoculated control, strain treatments added 28-126%Pb and 47-130%Cd in maize respectively. Strain J62 and JYC17 had a significant improvement on heavy metals accumulation in maize.5. The mechanism of three bacteria improving maize growth and heavy metals tolerance was further explored. The results displayed that the test strains could colonize in both root and rhizosphere of maize. The bacterial numbers of maize inoculated strains were significantly higher than that of control. Strain J62, JYC17 and Y3-9 inoculation reduced maize peroxidation level under lead or cadmium stress. In the maize inoculated with strains, SOD and POD activity reduced, especially in Pb treatments; the concentration of antioxidants ASA significantly increased by 14%-123%. GSH content increased in maize treated with Pb and slightly dropped in maize treated with Cd, which might a large number of GSH be used for PCs anabolism. The MDA contents obviously reduced in inoculated maize than that in control maize. It was dissimilar effect of different strains on endogenous Pro concentrations in maize. Strain J62 inoculation significantly declined Pro level in maize treated with Pb, but slightly increased Pro concentration in maize treated with Cd. Strain JYC17 and Y3-9 inoculation made Pro concentrations remarkable lower than that of control, in maize treated with high concentrations of Pb or Cd. The data of organic acids determined by HPLC revealed that strains incubation increased significantly malic acid concentrations in maize shoots. Bacterial effects on lead or cadmium accumulation and distribution in corn roots were studied through TEM. The results showed that bacteria significantly changed Pb concentration and distribution in maize root cells. Compared with the non-inoculated control, strain J62 Inoculation significantly increased Pb absorb in corn roots, moreover, inoculation with strain JYC17 caused larger lead particles. Pb in root of control maize mainly distributed in cellular wall and membrane, whereas Pb in maize roots inoculated bacterial strains mainly distributed in external layer of cellular wall and extracellular space. Bacterial inoculation also altered Cd distribution and alleviated Cd phytotoxicity in maize cells. Bacterial cells of test strains were observed in root cells of maize.6. The strain J62 that improved lead and cadmium bioavailability in soils and metal-tolerance of com was selected for pot experiment. The results of pot test one showed that strain J62 could promote growth of maize and tomato in lead and cadmium-contaminated soils. Dry weight of maize and tomato inoculated J62 increased by 54% and 17% in shoots and by 75% and 30% in roots. Strain J62 could improve lead and cadmium uptake in plant roots. The Pb concentrations in Indian mustard, corn and tomato inoculated strain J62 were 27%,59% and 31% respectively more than that in control. The Cd concentrations were 174%,31% and 12% higher than that in control, respectively. In conclusion, lead and cadmium total contents in maize and tomato were significantly increased by inoculation of strain J62. The Pb uptake in maize and tomato inoculated strain J62 were 85% and 38% respectively more than that in control, Cd uptake were 56% and 61% respectively more than that in control. The amount of Pb (200 mg.L-1) and Cd (50 mg.L-1)-resistant bacteria and total bacteria in rhizosphere inoculated with strain J62 were much higher than that in controls. The population of potassium-releasing, phosphorus-soluble and nitrogen-fixing bacteria in rhizosphere was also enhanced by strain inoculation. Strain J62 could colonize in test plants rhizosphere and obtain in heavy metal-resistant plates. Moreover, strain J62 inoculation also significantly improved NH4OAc-extracted Pb and Cd concentration in rhizosphere of maize and tomato. The pH values were decreased in the soils inoculated with strain and dissoluble sugar were increased by 6% to 40%, compared with the uninoculation control. Furthermore, strain J62 inoculation increased ocalic acid concentration in Indian mustard and corn rhizosphere and acetic acid concentration in maize and tomato rhizosphere, stimulated production of succinic acid in Indian mustard and maize rhizosphere and citric acid in tomato rhizosphere. Effects of strain J62 on bacterial number, pH, metal form, organic acid and soluble sugar contents might promote Pb or Cd accumulation of corn and tomato. The results of pot test two were accordant with that of test one. Burkholderia cepacia J62 is a good bioinoculant to strengthen phytoremediation in lead and cadmium contaminated soils.It is potential to apply maize inoculated with strain J62 to remedy metal-polluted soils and produce corn cobs. |
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