Research On The Strengthening Effect Of Rhizobia On Leguminous Forage To Repair Soil Heavy Metal Pollutio

Soil heavy metal contamination is an urgent biological and environmental science problem in the world,and is one of the tasks of environmental remediation and treatment faced by Guizhou Province as a result of mining.Among the bioremediation methods for heavy metal contamination of soil,the“legume forage-rhizobium”symbiosis system is currently considered to be the most promising symbiotic remediation system.In this project,nodule samples of wild legume forage were collected from the main heavy metal tailing areas in Guizhou,and the rhizobial strains were isolated,identified and preserved;by means of screening of rhizobial strains resistant to compound heavy metals,screening of suitable forage grasses with excellent heavy metal enrichment in Guizhou,screening of“legume forage-rhizobium”symbiotic dominant combination,combined with the safety evaluation of heavy metal content in the overground parts of legumes using as forage grasses for the symbiotic dominant combination,the following research results were achieved.1.A total of 55 nodule samples of 15 different species of wild leguminous forage grasses were collected from 18 tailings areas,including Bijie Hezhang lead-zinc mining area,Liupanshui Zhongshan lead-zinc mining area and Tongren Songtao manganese mining area in Guizhou.Eighty-three Gram-negative（G^-）strains were isolated and purified,and 71 rhizobial strains were identified by morphological observation and sequence alignment of 16S rRNA gene.The results of floristic analysis showed that 71 rhizobial strains belonged to 28 species in 7 genera,and Rhizobium was the dominant genus,R.indigoferae was the dominant species.Phylogenetic tree analysis showed that the 28 species clustered into five major taxa with eighteen branches,indicating a rich diversity of rhizobial species of natural legume forage in the main tailings areas of Guizhou.2.Based on the selection principle that the strains came from different host herbages of different regions and the colony grew well on YMA plate,the tolerance tests of five common heavy metals(Pb²⁺、Zn²⁺、Cr⁶⁺、Mn²⁺、Cd²⁺)in Guizhou tailings were carried out using 17 strains of different species.The results showed that all the testing strains possessed different degrees of tolerance to two or more kinds of compound heavy metals;according to the tolerant number and concentration of five kinds of heavy metals,the tolerance ability of the 17 strains to the compound heavy metals was ranked in order as follows:ZHQT055-1=HMZM040-1<ZHQT054-1<HXYT049-1=HXYT048-1<HSST046-1=WEMT051-2=HSST044-1<HSST044-2=HMZT039-2=HSSA047-1<HMZM040-3<H XTT050-1<ZHQT052-1=HSST042-1<HSST042-6<HMZT039-6,of which 5 strains,HMZT039-6,HSST042-6,HSST042-1,ZHQT052-1 and HXTT050-1,were able to tolerate all five kinds of heavy metal ions,which belonged to the best resistant rhizobial strains screened.3.Five kinds of suitable leguminous herbage in Guizhou were selected,including Trifolium repens,Medicago truncatula,Medicago sativa,Vicia sepium and Lotus corniculatus,and they were planted in pots loaded with tailings soil substrate.The heavy metal content in aboveground part and underground part were determined after 90 days of growth and development.Based on biological accumulating coefficient（BAC）and biological transfer coefficient（BTC）to determine the uptake and transfer ability of each test forage grass to soil heavy metals,the combined abilities of the five legume forage grasses to remediate soil heavy metal pollution were ranked as follows:Lotus corniculatus=Medicago sativa﹥Medicago truncatula﹥Vicia sepium﹥Trifolium repens.4.Ten rhizobial strains with relatively strong resistance to complex heavy metals were chosen（HMZT039-2,HMZT039-6,HMZM040-3,HSST042-1,HSST042-6,HSST044-1,HSST044-2,HSSA047-1,HXTT050-1,ZHQT052-1）,and according to the pairwise matching method,five kinds of suitable leguminous herbage（Trifolium repens,Medicago truncatula,Medicago sativa,Vicia sepium and Lotus corniculatus）were inoculatted with each testing strain respectively in pots loaded with tailings soil substrate..After 90 days of growth and development,heavy metal content in the aboveground part and underground part of the plants were measured.The symbiotic dominant combinations of legume herbage-rhizobium were selected to repair soil heavy metal pollution.By comparison,15 symbiotic dominant combinations were screened out from 50 groups of symbiotic remediation systems,based on the degree of improvement in the enrichment efficiency of soil heavy metals,which were as follows:HSST042-1-Lotus corniculatus、HSST042-6-Lotus corniculatus、HMZT039-6-Lotus corniculatus、HXTT050-1-Lotus corniculatus、HSST044-2-Lotus corniculatus、HSST044-1-Lotus corniculatus、HMZM040-3-Lotus corniculatus、HMZT039-2-Lotus corniculatus、HSSA047-1-Lotus corniculatus、ZHQT052-1-Lotus corniculatus、HSST042-6-Medicago truncatula、HSST042-6-Vicia sepium、HSST042-1-Trifolium repens、HSST042-6-Trifolium repens and HMZT039-6-Trifolium repens.Of them threr were 10 groups of Lotus corniculatus-rhizobium,3 groups of Trifolium repens-rhizobium,1 group of Medicago truncatula-rhizobium and 1 group of Vicia sepium-rhizobium,the repair efficiency of Lotus corniculatus-rhizobium symbiotic combination was the highest.5.By comparison of the above-ground heavy metal levels measured in each symbiotic association with the national safety standards for each heavy metal in forage,only 20 groups out of the 50 groups of legume forage-rhizobium symbiotic associations were all lower than the the prescribed limits of 5 kinds of metals in above-ground part.Safety evaluation was conducted on 15 screened legume forage-rhizobium symbiotic dominant combination restoration systems,and it was found that the content of one or more heavy metals in 14 dominant combinations exceeded the safety standard,only in the dominant combination of“HSST042-6-Vicia sepium”,the contents of five kinds of heavy metals in the above-ground parts of plants were all below the safety standard stipulated by the state,which could be applied to soil heavy metal pollution remediation and meanwhile be used as forage grass.The results of this study could provide effective symbiotic microbial basis and technical support for the construction of legume forage-rhizobium symbiotic restoration system in Guizhou,and be used as theoretical basis for the safe utilization of leguminous herbage in the symbiotic restoration system.