| Large-scale coal mining not only has a serious impact on the ecological environment,but also on the land resources in the mining area.Reclamation is the main way to protect the cultivated land,alleviate the human-land conflict and maintain food security.How to rapidly improve the fertility of reclaimed soil is the main task of researchers.Previous studies showed that after engineering flattening,the nutrients in the surface soil were deficient,especially the bioavailability of phosphorus was low,and phosphorus nutrient was the limiting factor affecting the recovery of the reclaimed soil.Phosphate solubilizing bacteria could activate the insoluble phosphorus in the soil,promote the utilization of nitrogen and phosphorus by the plant,which was of positive significance to the reclamation of the mining area.Thus,this study focused on two strains of phosphate solubilizing bacteria(w134 and w137,both of which are Pseudomonas fluorescens)isolated by our laboratory,and carried out a systematic study on their mechanism of phosphate solubilizing,their ability of colonization in reclaimed soil and their effect on corn growth.Furthermore,we applied them in field experiments of mining area reclamation,to investigate the ability of phosphate solubilizing in reclamation soil in mining subsidence area.Meanwhile,we also studied the effects of different fertilizers and different reclamation times on soil properties and soil microorganisms.The main results are as follows:(1)The traditional phosphate solubilizing mechanism of w134 and w137: HPLC showed that w134 mainly secreted 6 kinds of organic acids: Citric acid,Tartaric acid,Oxalic acid,Formic acid,Acetic acid and Succinic acid;w137 mainly secreted 5 kinds of organic acids: Citric acid,Tartaric acid,Oxalic acid,Acetic acid and Succinic acid.The total amount of organic acids secreted by w137 is more than w134,and the ability of phosphorus dissolving is higher than that of w134.The test of pure organic acid solubilizing phosphate showed that the contribution of organic acid to phosphate solubilizing of the two strains were 72.64% and80.13% respectively,and the main way of phosphate solubilizing was to secrete organic acid.The p H value of NBRIP culture medium was adjusted by hydrochloric acid to the lowest p H value when it was inoculated.The contribution of p H to phosphate solubilizing of the two strains were 26.58% and 40.23% respectively,which indicated that the contribution of H+ to phosphate solubilizing was limited.The decrease of p H value in NBRIP medium just should be the concomitant phenomenon when bacteria secreted organic acids.The activity of phosphatase secreted by the two strains was the highest on the first day in the phosphate solubilizing experiment,and then decreased gradually.There was a negative correlation between the activity of phosphatase and the content of available phosphorus in NBRIP medium.(2)Both w134 and w137 belong to Pseudomonas fluorescens.w137 strain with better phosphate solubilizing effect was selected for transcriptome sequencing.,further explaining the mechanism phosphate solubilizing of w137 at gene level.Under different phosphorus source conditions,the transcriptome sequencing results of w137 bacteria showed that,compared with the soluble phosphorus group,the GO function of up-regulated genes of w137 under insoluble phosphorus condition was mainly focused on Carbohydrate metabolism,Fatty acid metabolism and Cell sugar metabolism.At the same time,the results of KEGG enrichment also showed that the genes of Glycometabolism pathway closely related to organic acid production were significantly upregulated.HPLC showed that the content of organic acids increased in the insoluble phosphorus culture medium and non-phosphorus culture medium,which further confirmed that the up-regulated genes should be related to the secretion of organic acids by bacteria.In addition,compared with the soluble phosphorus group,the genes related to phosphorus metabolism of w137 in the insoluble phosphate and non-phosphate groups were significantly down-regulated,which indicated that phosphorus deficiency reduced the ability of w137 to phosphorus transport,absorb and assimilate.(3)The pTRGFP plasmid was transferred into w134 and w137 respectively by electric shock transformation.Under the condition of no selective pressure,the green fluorescence can still be observed under fluorescence microscope after w134 GFP and w137 GFP have been passed on for 15 times continuously,and the plasmid can be extracted,which showed that GFP plasmid can be inherited stably.Moreover,the GFP plasmid had no significant effect on growth curve and phosphate solubilizing capacity of w134 and w137.In the experiment of colonization in reclaimed soil,the numbers of GFP-labeled bacteria reached the maximum after 7 days of inoculation,among which,w134 GFP was 7.6 × 108 CFU/g fresh soil,and w137 GFP was 1.5 × 1010 CFU/g fresh soil.After that,the number of w134 GFP and w137 GFP decreased gradually with the increase of time.The number decreased to 104 CFU/g soil in 35 days,and maintained at about 3 × 103CFU/g in 60 days,and reached the low limit of 20 CFU/g at three months.(4)In the pot experiment of maize planted with w134 GFP and w137 GFP,it was found that the GFPlabeled bacteria could still be detected from soil after 35 days of maize growth.The number of two GFPlabeled bacteria in rhizosphere soil was about 104CFU/g,and that in non-rhizosphere soil was about103CFU/g,which was consistent with the results of colonization in reclaimed soil.The application of phosphate solubilizing bacteria increased the content of available phosphorus and the activity of phosphatase in the soil,and increased the plant height,plant diameter,dry and fresh weight of maize and the amount of phosphorus of maize plant.The phosphate solubilizing bacteria can play dual roles of solubilizing phosphate and promoting maize growth in the reclaimed soil.(5)Applying w134 and w137 to the reclaimed fields in coal mining subsidence area,we found that the nutrient content of reclamation soil accumulated with the increase of reclamation time,and the nutrient content of soil reclaimed for10 years approached or exceeded the surrounding mature soil.The application of organic manure combined with phosphate solubilizing bacteria can not only significantly improve the available phosphorus content,but also increase the alkaline nitrogen content and the yield of maize in the reclaimed soil,and slow down the decline of soil p H caused by long-term fertilization.(6)The effects of w134 and w137 on bacteria in reclaimed soil were as follows: the diversity index of bacteria in reclaimed soil increased with the reclamation time,and the succession of bacterial community structure showed obvious time sequence.The bacterial community structure of the 10 years reclaimed soil was the closest to that of the mature soil.There was a good correlation between bacterial diversity and soil nutrients.SOM,TN and AN were the main environmental factors that affect the change of bacterial community.Proteobacteria,Acidobacteria,Actinobacteria,Planctomycetes are the dominant phyla in the reclaimed soil,accounting for more than 68% of the bacterial community.The application of phosphate solubilizing bacteria(w134,w137)can promote the recovery of bacterial diversity in reclaimed soil,but has no significant effect on the community structure of bacteria.(7)The effects of w134 and w137 on fungi in reclaimed soil were as follows: the diversity index of soil fungi also increased with the reclamation years.The succession of the fungi community structure of the reclaimed soil also showed a temporal sequence,but the fungal communities of 5 years and 10 years were partially overlapped.The correlation between soil environmental factors and the fungi diversity is not high,and the effects of environmental factors on fungal community structure were not significant.The average abundance of Ascomycetes was 76.97%,which was the dominant phylum of fungi,followed by Chytridiomycota,Mortierellomycota,Basidiomycota,Glomeromycota,and the unclassified fungi accounted for 11.98%.Application of phosphate solubilizing bacteria(w134,w137)was beneficial to Ascomycetes,but had no significant effect on fungal diversity and community structure in reclaimed soil.(8)In the process of reclamation,reclamation time had more significant effects on soil properties and microbial diversity than fertilization,and reclamation time was the main driving force for soil properties and soil microbial recovery in coal mining subsidence area.In the same reclamation time,organic fertilizer was slightly better than chemical fertilizer,although there were no significant differences in soil nutrient contents and microbial diversity between them.Thus,the two fertilizers could be used for reclaimed farmland in coal mining subsidence area.But from the perspective of phosphate solubilizing bacteria(w134,w137),organic fertilizer + phosphate solubilizing bacteria can better play the function of phosphate solubilizing bacteria.To sum up,w134 and w137 mainly secrete organic acids to dissolve phosphorus,and they can effectively colonize in the reclaimed soil,and play a dual role in phosphate solubilizing and promoting corn growth.At the same time,they have no adverse effects on soil microbial communities.In the process of reclamation,reclamation time is the main driving force of soil properties and microbial recovery in mining subsidence area.Fertilization can rapidly improve soil fertility and aid to shorten the recovery time of reclaimed soil. |
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