Phosphorus Accumulating Characteristics And Mechanisms Of The Mining Ecotype Of Polygonum Hydropiper

The reliance on high rates of phosphorus（P）-fertilizer application in modern agriculture leads not only to the gradual depletion of finite rock phosphate reserves,but also to excess P in soils and water pollution by agricultural P-runoff.Usage of P-accumulating plants is an efficient way to extract excess P from P-polluted environments.Harvest of the P-accumulating plants can be used as green fertilizer,to decrease the environmental risk caused by application of chemical fertilizers and avoid the waste of resources.However,systematic studies on P-accumulating characteristics and mechanisms is relatively limited.Thus,pot and plot experiments were carried out to investigate P-accumulating characteristics and mechanisms in the P-accumulating plant,the mining ecotype（ME）of Polygonum hydropiper.A summary of the primary results are as follows:1.Root H₂PO₄^-absorption rate increased with the increasing P concentration for the ME,but reached the peak at 2 mmol P L^-1 for the non-mining ecotype（NME）.The ME showed a higher capability of P absorption,with the maximum P absorption rate being 0.023 mmol g^-1 h^-1.Significantly higher total root length,surface area,volume and specific root length in the ME of P.hydropiper at high P treatments compared with the control;however,the root morphological parameters of the NME were inhibited by high P treatments.At 4 mmol L^-1 and 8 mmol L^-1,the ME displayed significantly greater length,surface area and volume of roots with diameter≤0.45 mm than the NME,indicating that more fine roots of the ME may be the basis for its efficient P uptake.The bleeding sap amount in the ME increased with the increasing inorganic P（Pi）treatments,but that in the NME decreased;it reached the peak for both ecotypes at 4 mmol L^-1 of phytate.The bleeding sap amount and P concentration of the ME was significantly higher than that of the NME,indicating that the ME have higher root activity and higher P transfer capability than the NME.2.The ME showed a greater ability to tolerate high P than the NME,as shown by its superior growth at both 3 weeks and 5 weeks with 1.1-2.3 times and 1.0-2.7 times higher shoot biomass and root biomass,respectively.At 8 mmol L^-1 of Pi treatment,the NME showed partial collapsed and damaged epidermal cells with larger size;however,the ME displayed intact leaf epidermal cells with smaller size and with appropriate stoma distributed.The ME maintained an integrated anatomical structure of leaves;however,high P treatments caused the palisade tissues and spongy tissues out of shape and random arrangement in leaves of the NME.Shoot P accumulation was significantly higher in the ME than in the NME,with maximum value of 16.4 mg plant^-1 for the ME at 8 mmol L^-1.P in leaves and roots of P.hydropiper was allocated in cell wall and soluble fraction（vacuoles）,which reduced excess P entering into the cytoplasm and avoided P toxicity at organelle fraction.High P treatments significantly increased all P forms,particularly Pi form.Compared with the NME,lower Pi concentration and higher nucleic and ester P concentration in the leaves of the ME was good for maintaining the range of metabolic P concentration to keep its function;higher Pi concentration in the roots of the ME was good for P transfer to shoots.3.Shoot biomass of the ME and NME increased by increasing Pi concentration,but decreased at 1600 mg kg^-1 and 800 mg kg^-1 of phytate,respectively.Shoot P accumulation of the ME was significantly higher than that of the NME,reaching the peak value of 76.1 mg plant^-1 at 8 mmol L^-1of Pi treatments.Compared with bulk soil,pH decreased in the rhizosphere,with the ME rhizosphere decreasing 0.15-0.45 and the NME rhizosphere decreasing 0.04-0.14.As detected by the solution nuclear magnetic resonance technique,orthophosphate was the most abundant form.Concentrations of soil pyrophosphate and diester-P were very low with the values ranging from 5.5to 14.0 mg kg^-1 and from 6.1 to 22.1 mg kg^-1,respectively.The concentration of orthophosphate,monoester-P and diester-P depleted greatly in the rhizosphere,with higher effect by the ME than by the NME.Glycerophosphate and inositol hexakisphosphate concentrations increased in the rhizosphere under high phytate treatments with higher effect by the ME than by the NME.Soil acid phosphomonoesterase,alkaline phosphomonoesterase,phosphodiesterase and phytase activities of both ecotypes were higher in high P treatments than the control,except for acid phosphomonoesterase at 1600 mg kg^-1 of Pi treatment.Significantly higher enzyme activities were observed in the rhizosphere soil of the ME than the NME.Probably,the ME might obtain higher shoot P than the NME from P-enriched soils through changes in rhizosphere properties.4.Bacterial community diversity were independent with ecotypes and soil components;rather it positively correlated with soil organic matter concentration.There were 49 phyla in soils,with Proteobacteria being the most dominated phylum.At the phylum,the ME showed higher relative abundance of Proteobacteria,Bacteroidetes and Firmicutes in rhizospheric microzone soils than the NME.Rhizoplane and rhizosphere bacterial communities showed more similarity compared with those in bulk soil.At the phylum level,the relative abundance of Proteobacteria,Bacteroidetes and Firmicutes was high in the rhizospheric microzone soils of the ME,indicating these bacteria may play an important role in adapting the phosphate-mine environment.At the genus level,the Halomonas,Flavisolibacter,Blastocatella,Thiobacillus,Sphingomonas and Bacillus in rhizospheric microzone soils of the ME showed higher relative abundance than in that of the NME,positively with soil AP concentration,suggesting these genera might be important in mobilizing soil mineral-P and in accumulating P for P.hydropiper.Soil pH,available P,available nitrogen,available potassium and distance from root surface played major roles in shaping the bacterial community compositions.5.Thirty-five endophytic bacteria from P.hydropiper were tolerant of a high P concentration(800 mg L^-1)and classified into 10 taxonomic groups through 16S rDNA sequencing.Eight isolates belonged to the genus Bacillus,nine to the genus Pseudomonas,four each to the genera Arthrobacter and Staphylococcus,three each to the genera Pantoea and Micrococcus,and one each to the genera Acinetobacter,Rahnella,Brevibacterium and Kocuria.30 isolates were indole-3-acetic acid positive,and 11 endophytes exhibited siderophores activities and phosphate-solubilizing capability on an agar medium supplied either with tricalcium phosphate（TCP）or phytin.All strains exhibited the capability of solubilizing TCP and phytin in a liquid medium,with P concentration ranging from 11.2 to 166.2μg mL^-1 and 9.7 to 141.6μg mL^-1,respectively.Pseudomonas putida SLr12 showed a greatest TCP-solubilizing capability and Pseudomonas plecoglossicida SLr02 showed a greatest phytin-solubilizing capability.Phosphate-solubilizing activity of these strains from TCP was associated with a decrease in pH and production of organic acids,while that from phytin was associated with a decrease of pH but increases of acid and alkaline phosphatase activities.Six high-P-tolerant endophytes showed better combination of all PGP characteristics,offered the most potential as inoculants.6.At high P treatment,inoculation of Pantoea hericii SLr10,Kocuria sp.SLr15,Arthrobacter koreensis SLs08 and Bacillus subtilis SLs13 significantly increased root biomass compared with the no-inoculation control.Inoculation of Kocuria sp.SLr15,B.subtilis SLs13,Arthrobacter koreensis SLs18 and Pseudomonas plecoglossicida SLr02 significantly increased the stem and leaf biomass.Inoculation of P.plecoglossicida SLr02 just significantly improved stem P accumulation and decreased bioaccumulation coefficient and translocation factor of P.hydropiper.Inoculation of Kocuria sp.SLr15,B.subtilis SLs13 and Arthrobacter sp.SLs18 significantly enhanced stem and leaf P accumulation,and they could promote root growth and development,as shown by the significant increases in total root length and root surface area.Thus,strains of Kocuria sp.SLr15,Bacillus subtilis SLs13 and Arthrobacter sp.SLs18 could be used as efficient inoculants for assisting P.hydropiper in P-phytoextraction studies.7.After continuous planting for 3 years,dry weight,shoot P concentration and shoot P accumulation of the ME varied from 14.7 to 25.8 Mg ha^-1,4.1 to 5.6 g kg^-1 and 62.7 to 150.3 kg ha^-1,respectively.Dry weight and P accumulation of the ME grown in swine manure-and dairy manure-deposited soils were greater than in poultry manure-deposited soil and the cultivable soil.Soil water-soluble P concentration increased in animal manure-deposited soils after planting P.hydropiper in first year,but decreased in the second and third year.After planting 3 years,available P concentration in surface soil（0-10 cm）greatly decreased,with a decrease of 15.9%-26.5%in the cultivable soil,10.9%-23.8%in swine manure-deposited soil,13.2%-25.2%in dairy manure-deposited soil and 1.6%-9.2%in poultry manure-deposited soil.The ME showed relatively low shoot efficiency number in swine manue-and dairy manure-deposited soils,with values of2.5-3.2.P removal amounts and shoot P extraction rates of the ME from topsoil（0-20 cm）in swine manure-and dairy manure-deposited soils were higher than from that in poultry manure-deposited soil,with P removal amounts ranging from 42.7 to 56.3 mg kg^-1 and P extraction rates ranging from3.6%to 15.6%.Planting the ME of P.hydropiper could efficiently extract excess P from animal manure-deposited soils and decrease the concentration of soil soluble P to reduce P runoff risk.