Structural Analysis Of The Rhizosphere Microbial Community Of Malus Sieversii(Ldb.) Roem.and Discovery Of Soil Nitrogen,Phosphorus And Potassium Activating Bacteria

The Xinjiang wild apple（Malus sieversii（Ldb.）Roem.）is an important germplasm resource in China,and its main distribution is the Tianshan Mountains of Xinjiang.The rhizosphere microorganisms can reflect the inter-root environment and the growth of plants.In the unique natural environment,the inter-roots of Xinjiang wild apple breed a large number of microorganisms.The discovery of beneficial microorganisms is essential for achieving sustainable agriculture.In this study,16S/18S r DNA gene sequencing,functional prediction and functional strain screening of microorganisms from wild apple rhizosphere soils in Xinjiang located at eight sites in Ily were conducted.The effects of human activities and geographic changes on the rhizosphere microorganisms of wild apple were investigated through analysis of community structure and diversity,from which highly active nitrogen,phosphorus and potassium-activating bacteria were identified.In addition,the effects of the selected strains on the Red Fuji apple seedlings were clarified through pot trials and the optimum combination and proportion of functional strains to promote their growth was obtained.The main findings of this study are as follows:（1）There were significant differences in the diversity and community structure of rhizosphere bacteria from Xinjiang wild apple across the 8 sampling sites.The highest number of operational taxonomic units（OTUs）was 3,361 at site C（Wild-fruit forest of Xinyuan County）,and the ACE,Chao,Shannon and Simpson indices were significantly higher than those of the other sampling sites.The number of OTUs for the remaining sampling sites was,in descending order,sites D（Daimolhu in Gongliu County）,H（Guozigou in Laofengkou of Tori County）,G（Wild-fruit forest of Emin County）,B（Resource Nursery）,F（Naze Work Team in Gongliu County）,A（Daxigou of Huocheng County）and E（Xiaomolhu in Gongliu County）.Firmicutes,Proteobacteria and Actinobacteriota were most abundant at the phylum level.Firmicutes had the highest relative abundance in sites A（56.47%）,C（24.46%）,D（32.45%）,E（57.58%）and F（56.43%）.The relative abundance of Proteobacteria was highest in sites B（40.82%）,G（54.70%）and H（46.23%）.The dominant taxa at the genus level were Bifidobacterium,Burkholderia-Caballeronia-Paraburkholderia and Methylotenera.Representational difference analysis（RDA）and correlation analysis showed that soil effective phosphorus,effective potassium,alkaline decomposition nitrogen,p H,water content,catalase activity,altitude,temperature and relative humidity influenced the rhizosphere bacterial community.（2）The diversity and community structure of rhizosphere eukaryotes in Xinjiang wild apple from 8 sites differed significantly.Site A had a maximum number of 2,578 OTUs.The number of OTUs at the remaining sampling sites was,from largest to smallest,sites B,F,E,C,G,D,and H.The ACE,Chao,Shannon and Simpson indices were all significantly higher at site B than at other locations.The number of OTUs,α-diversity,and species evenness of eukaryotes at the four sites（sites C,D,G and H）were lower than that of bacteria.However,the opposite results were observed for the remaining four sites（sites A,B,E and F）.The main taxa of eukaryotes were Ascomycota,Phragmoplastophyta and Basidiomycota.At the genus level,the predominant taxa were Trichoderma,Mortierella and Geminibasidium.RDA and correlation analyses showed that soil effective phosphorus,effective potassium,alkaline decomposition nitrogen,p H,water content,catalase activity,altitude,temperature and relative humidity influenced the rhizosphere eukaryotic community.The results of the Mantel test revealed that soil microbialβ-diversity was correlated with geographic distance,climatic distance and soil p H.Moreover,geographic and climatic differences were found to be critical predictors of microbialβ-diversity.（3）There were differences in the number and proportion of organic phosphorus-solubilizing,inorganic phosphorus-solubilizing,nitrogen-fixing and potassium-solubilizing bacteria colonizing the rhizosphere of8 sites for Xinjiang wild apple.Analysis of variance showed that the number of colonized and the total number of strains of the above four bacteria had no significant effect on chlorophyll,osmolytes,superoxide dismutase（SOD）and peroxidase（POD）activities of wild apple leaves.The number of colonized organic phosphorus-solubilizing bacteria and the total number of four functional strains influenced catalase（CAT）activity significantly.RDA analysis showed that the number of colonized rhizosphere organic phosphorus-solubilizing bacteria could reflect the both phosphorus and nitrogen contents of Xinjiang wild apple leaves.The number of colonized potassium-solubilizing bacteria indicated the leaf potassium content Meanwhile,the number of colonized inorganic phosphorus-solubilizing,potassium-solubilizing as well as organic phosphorus-dissolving and nitrogen-fixing bacteria reflected the phosphorus,potassium and nitrogen contents of soil,respectively.The numbers of bacteria were determined to be 7.08×10⁴CFU/g（nitrogen-fixing bacteria）,2.7×10⁷CFU/g（phosphorus-solubilizing bacteria）and 4.98×10⁵CFU/g（potassium-solubilizing bacteria）,respectively,when the nitrogen,phosphorus and potassium contents in the leaves and soil were high and the best nutritional growth was achieved in Xinjiang wild apple.（4）The role of nitrogen,phosphorus and potassium-activating strains of rhizosphere microorganisms from 8 sites of Xinjiang wild apple was evident.A total of 115 nitrogen,phosphorus and potassium-activating strains were isolated.Among them,the strain Pseudomonas migulae P8-3 had the strongest phosphorus-solubilizing capacity of 163.18μg/m L.The strain Pseudomonas poae WP1-1 had the highest phosphorus solubilizing capacity of 72.77μg/m L.The strain Pseudomonas extremaustralis N4-1had the highest nitrogen fixing enzyme activity of 42.71 U/L.The strain Pseudomonas extremaustralis K4-1 had the strongest potassium solubilizing ability,with a relative potassium addition rate of 49.93%,significantly higher than other strains（P<0.05）.（5）The growth of Red Fuji apple seedlings was promoted by dominant bacteria screened from rhizosphere microorganisms of Xinjiang wild apple from eight sites.The effects of concentration,bacterial strain,incubation time and fertilization method on the growth of Red Fuji apple seedlings were investigated by L₁₆（4⁵）orthogonal test.The results showed that treatment 10 had the highest leaf nitrogen（0.198%）,phosphorus（1.95%）and potassium（2.65%）contents,leaf biomass（0.920 g/plant）as well as inter-root soil nitrogen（18.20 mg/kg）,phosphorus（46.40 mg/kg）and potassium（106.24 mg/kg）contents.It also had the highest POD and CAT activities of 3.00 U/g·min and 36.00 U/g·s,respectively.The extreme difference analysis demonstrated that fertilization method had the greatest effect on carotenoid content.P.extremaustralis appeared to have the greatest effect on leaf nitrogen,phosphorus and potassium contents together with leaf biomass.P.poae were found to exhibit the strongest effect on leaves malondialdehyde,SOD activity as well as soil phosphorus and potassium contents.Incubation time showed the largest influence on chlorophyll a,chlorophyll b,proline,soluble sugar,soluble protein,POD,CAT activity and soil nitrogen content.It was found that a mixture of inorganic fertilizer bacterial suspensions gave the biggest boost to the growth of seedlings,significantly higher than that of inorganic fertilizer alone and the control.Combining the results of chlorophyll,osmolytes,enzyme activity along with nitrogen,phosphorus and potassium contents under different treatments,a simultaneous application of inorganic fertilizer and bacterial suspension（1:90 P.migulae and 1:90 P.sextremaustralis）to the roots and leaves was finally determined as the optimum treatment for the growth conditions of the Red Fuji apple seedlings.In summary,the results of this study revealed the diversity and structure of the rhizosphere microbial community from the Xinjiang wild apple,as well as geographic and environmental changes and their effects.The most active nitrogen,phosphorus and potassium activating-bacteria were also identified.Further,the optimal combination of strain,concentration,incubation time and fertilization method for promoting Xinjiang wild apple growth was determined.This research provides a theoretical basis for the restoration and ecological construction of wild apple forests in Xinjiang,and offers excellent strain resources for boosting apple growth and improving the resistance to stress.