| Toxic metal pollution has received considerable attention over the years as aresult of increased industrial activities. Heavy metals can be accumulated inorganisms and finally threaten human health through food chains. Therefore, aneffective and affordable solution is urgently needed to remove toxic metals fromenvironment. Established technologies to remediate metal contaminated soils arefrequently expensive and environmentally invasive. In situ phytoremediation has beenproposed as a low-cost, environmentally friendly and effective method to removetoxicant from contaminated soils. However, phytoremediation of heavy metal still hasto deal with some important shortcomings such as phytotoxicity, slower thanmechanical methods, and a limited contaminant uptake. Some phytoremediationenhancing strategies, such as transgenics and chelating agent, are comparativelyavailable but usually expensive and posing potential risks to environment. Therefore,it is important to search a microbe-strengthened phytoremediation approach toenhance plant remediation economically.In the present study, a number of endophytes were isolated from Cdhyperaccumulator plant Solanum nigrum L.. An excellent strain Serratianematodiphila LRE07was selected to study the feasibility of phytoremediationenhancement using plant-endophyte symbiotic system. After inoculating with S.nematodiphila LRE07, plant Solanum nigrum L. was grown in hydroponics withincreasing Cd concentrations. The growth, physiological parameters, photosyntheticpigments, and Cd accumulation characteristics were investigated to study the effect ofendophyte inoculation on plant stress resistance. Further, the nutrient uptake, lipidperoxidation levels, and activated oxygen metabolism were determined order to knowthe possible mechanisms of endophyte in phytotoxicity reduction and growthpromotion involved in phytoremediation. Moreover, the stability of S. nematodiphilaLRE07and the effects of endophyte inoculation on non-host plant were investigated.The main results were indicated as follows:①Isolation and selection of endophytic bacteria from Solanum nigrum L.97endophytes were isolated from cadmium hyperaccumulator Solanum nigrum L.which collected at the sewage discharge canal bank of Zhuzhou Smeltery (27°52′N,113°05′E). Most of them were bacterial endophytes. All endophytes showed different degrees of heavy metals resistance by minimal inhibitory concentration test,suggesting that endophytes of hyperaccumulators are a main source which heavymetals resistant bacteria derived from. A carmine-pigmented bacterium, named asstrain LRE07, was selected from the root of Solanum nigrum L.. The isolated strainwas identified as Serratia nematodiphila by using16S rDNA analysis. S.nematodiphila LRE07showed a high degree of resistance to heavy metals, especiallyto Cu, Cd and Cr. Meanwhile, it had the ability to produce indole acetic acid andsiderophore and could solubilize mineral phosphate. The characters mentioned abovesuggested that endophytic bacterium LRE07was capable of facilitating the plantgrowth and the biomass yield. Finally, the strain LRE07was determined for the nextstep of experimental strain.②Effect of endophyte inoculation on growth and cadmium uptake of Solanumnigrum L.Physiological responses of S. nigrum seedings to Cd stress and the effect ofendophyte inoculation on plant stress resistance were investigated by nutrient solutionculture method. After21and70d of growth, plant samples were harvested todetermine the inoculation efficiency. Endophytic bacteria were isolated from rootsand shoots separately and plated on LB medium. The results showed that S.nematodiphila LRE07was inoculated into plant successfully. The number of CFU thatcould be re-isolated showed a time-dependent growth in roots and shoots of their hostplants and it was not inhibited by the presence of Cd. The number of re-isolated CFUtended to increase with time, indicating that S. nematodiphila LRE07could liveconcordantly with its host plants.The increasing concentration of Cd produced growth inhibition of S. nigrum L.,measured as tillers, FW and shoot height. At0and10μM Cd, the ratio increased overtime for both infected and non-infected plants. However, at50and100μM Cd theratio rapidly reached constant values and even decreased at the end of the experiment.The higher the Cd concentration applied to the nutrient solution, the weaker the ratioof the tillers was obtained. It can be seen that the infected plants showed higher ratiovalues than noninfected controls in an equal Cd concentration as shown in Table2(P<0.05, MANOVAs), that is, greater number of leaves were obtained in infected plants.Similar beneficial effects of endophytic bacterium LRE07on plant growth wereobserved in FW, shoot height, and root weight. At10μM Cd, no visible growthinhibition was observed for infected plants and the FW of these plants increased by58%compared with non-infected ones. With increasing Cd concentration there was a net decrease in pigment contentfrom day0to the end of the experiment whether the plants were infected or not.Endophyte-infection positively influenced the photosynthetic pigment contents of itshost plant. It can be seen that for all Cd concentrations (except for day3at100μM),chlorophyll a, chlorophyll b, and carotenoid of infected plants leaves were higher thannon-infected ones (P <0.05, MANOVAs). In addition, the decreases of photosyntheticpigment caused by Cd exposure in infected plants were much less than that of inendophyte-free plants.Increasing concentrations of Cd in nutrient solutions led to an increase of Cdcontents in shoots and roots. Nevertheless, no significant differences were observedbetween infected and non-infected plants (except in stem: Cd10and100μM). On adry-weight basis, the plants infected and non-infected with S. nematodiphila LRE07took up approximately the same amount of cadmium. Compared with noninoculatedcontrols, endophyte inoculation did not greatly influence the cadmium concentrationsin plant tissues, but achieved a higher biomass production, thus resulting in more totalCd-uptake per plant (72±5%increase at10μM), reflecting the promotion ofphytoextraction efficiency.③Possible mechanisms of endophyte bacterium in Cd phytotoxicity reduction andplant growth promotion.Cd produced increase in the uptake of Fe in tissues, while the uptake of Mn andZn were decreased except no significant changes of Zn in the root. These resultsindicated that Cd induced alteration on translocation of other heavy metals in planttissues. The inoculation of endophyte S. nematodiphila LRE07alleviated thisCd-induced changes on metal contents. Mn content in infected plant tissues (roots,leaves and stems) was higher, either at10,50or100μM, than that of in non-infectedcontrols. Compared with non-symbiotic controls, symbiotic plants assimilated moreFe and Zn in leaves and stems.The MDA content showed a linear enhancement with time and with the increasein concentration of Cd whether the plants infected or not. However, it can be seen thatthe MDA contents in infected plants were lower than that of in the noninfected plants,although differences were not statistically significant. In addition, the increment valueof MDA content with time was smaller in infected plants than those in noninfectedplants.Activities of enzymes (SOD, GPX, CAT and APX) detoxifying the cells fromactive oxygen species were measured as representative enzymes involves in antioxidant metabolism upon Cd exposure. Cd induced obvious increase in the contentof SOD, and SOD activity is higher in infected plant tissues compared to non-infectedplants for Cd concentration of10and50μM. CAT activity in leaves showed nosignificant differences between both types of plants. Leaves of infected plants showedhigher activity of GPX and APX compared with non-symbiotic plants at10and50μM.In roots, increasing cadmium concentrations caused a linear enhancement of GPX inboth infected and non-infected plants, while for50and100μM Cd the GPX activityof infected plants were significantly higher compared with non-infected ones. Theseresults suggested that the presence of the endophytic bacterium S. nematodiphilaLRE07improved the antioxidative capability of its host plant and reduced ROS injurycaused by Cd exposure.④Stability of endophyte in plant generation and effect of endophyte inoculationon growth and Cd resisitanse of non-host plantStability of endophyte in host plant generation was investigated by comparingthe endophytic bacterial conoly and plant growth condition of two different treatedplant generations. The results showed that there were no significant differencesbetween CFUs of objective strain in two plant generations. As a result of DGGEanalysis, the endophytic bacteria community composition was relatively highconsistency between the generations. Meanwhile, endophytic bacteria remained thepositive effect on plant growth, resulting in no significant differences in growthparameters between the generations. These results suggested that endophytic bacteriacould survive in plant steadily and remain its activity in plant offspring seeds.Effect of endophyte inoculation on growth and Cd resisitanse of non-host plantwas investigated by inoculating S. nematodiphila LRE07into pea (non-host plant of S.nematodiphila LRE07) with Cd explosure. The results showed that Cd inhibited thegermination of pea seed. The endophyte inoculation could not alleviate the stresseffect in pea germination caused by Cd explosure. The inoculation of S.nematodiphila LRE07could promote plant growth under some Cd concentrations. Buton the whole, the endophyte inoculation could not significantly improve the growth ofits non-host plant. |
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