西藏阿里高寒草原四种牧草根际促生菌资源筛选及促生机理研究
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摘要
西藏是我国传统的五大牧区之一，是“世界屋脊”，阿里地区是世界屋脊的屋脊，主要以高原寒带草原为主，生态环境极其脆弱，对全球变化和人类活动的响应高度敏感，生态系统与环境的关系以其自身的临界性和生物独特的适应机理为基本特征，分布着特有、珍稀的生物资源。特殊的地域特点和丰富的自然资源成为了许多学者关注的热点地域，收集、研究特殊地域的微生物资源为我国生物资源的保护和开发利用有着重要的意义。
本研究以西藏阿里地区高寒草原四种牧草（披碱草Elymus dahuricus、燕麦Avenasterilis、紫花苜蓿Medicago sativa和草地早熟禾Poa pretensis）为研究对象，采用常规分析法测定了植物根际土壤的理化性质，用选择性培养基分离了植物根际不同部位（根系较远的土壤、根表土壤、根系表面和根内）细菌和PGPR菌株的数量和分布，分析了根际养分含量与细菌数量和分布的相关性；研究PGPR菌株的固氮、溶磷和分泌IAA等促生特性，筛选优良菌株并进行了鉴定，同时测定了优良菌株在披碱草根际的定殖能力及对生长的影响，并研究了溶磷菌的溶磷机理，主要研究结果如下：
1．不同植物根际土壤理化性质、细菌和PGPR菌株数量及分布差异显著
四种植物根际土壤养分含量较低，土壤总体有效养分的供给能力较弱，并且不同植物根际养分含量、分布细菌的数量差异显著，植物根际土壤的理化性质和养分含量与细菌的数量间存在一定的相关性。四种供试植物根际分离到PGPR菌株222株，其中，固氮菌71株、溶解无机磷菌株73株和溶解有机磷菌株78株。PGPR菌株的数量表现出“根表>根表土>远根土>根内”的分布趋势，表现出强烈的根际效应。
2．PGPR菌株促生特性各异
采用乙炔还原法（ARA）对分离获得的71株固氮菌的固氮能力进行了测定，采用平板溶磷圈法和液培钼锑抗比色法、显色法和Salkowaki比色法对分离到的222株菌株的溶磷和分泌IAA能力进行了测定、筛选，结果表明：28株具有良好的固氮酶活性，固氮酶活性在1.08~446.63nmol C-12H4·hmL~(-1)；15株具有良好的溶解无机磷的能力，D/d值在1.12~1.69，溶磷量在1.66~40.89μg/mL；32株具有良好的溶解有机磷的能力，D/d值在1.23~3.47，溶磷量在11.05~26.03μg/mL；38株具有分泌IAA的能力，分泌量在7.63~26.20μg/mL。
3．优良PGPR菌株以假单胞属和杆菌属为主
优良PGPR菌株16S rDNA鉴定结果表明：菌株PWXZ10、PYXP1、NXZ8、NXZ17、003PWXZ3、003NXZ4、003NXZ6、003NXZ9与Pseudomonas sp.具有很高的同源性，因此鉴定属假单胞菌属；菌株PYXZ1、PWXZ6、NXZ4与Pseudomonas fluorescens具有很高的同源性，因此鉴定为荧光假单胞菌；菌株PWXZ23、NXP17与Bacterium具有很高的同源性，因此鉴定属杆菌属。
4．优良PGPR菌株可在植物根际成功定殖，对植物促生效果显著
选择优良PGPR菌株26株（溶磷菌17株，固氮菌9株）制备接菌剂，进行了接种试验，结果表明：PGPR接菌处理后披碱草的的株高、地上和地下植物干重显著高于对照，各菌株接菌处理后对披碱草根系性状的影响差异显著（P<0.05），其中，菌株003PWXZ6、NXP17处理后对植株的株高的促进作用最为明显；菌株PYXP1、 NXP17处理对植株的地上、地下植物干重及总干重增加最显著；菌株PWXZ10、003NXZ5处理的根冠比最高；菌株PYXZ7、NXZ9处理对根系平均直径的影响最大；菌株003NXZ5接菌处理后，植株的根总长、根表面积、根体积均最大。各菌株处理对披碱草根系不同直径段植株根总长、根表面积、根体积及其分布的影响也各有不同，菌株PYXZ19处理根系直径大于0.45mm和菌株NXZ16处理根系直径大于0.15mm的根长、根表面积、根体积所占比例最高。供试菌株能在披碱草根际成功定殖，对披碱草地上、地下部分和整株磷、氮含量和植株地上部分粗蛋白含量的影响差异显著（P<0.05），菌株003PWXZ6处理植株地上、地下及部分及整株的含磷量最高，菌株NXP17处理植株地上、地下部分及整株的氮含量和植株地上部分粗蛋白含量最高。综合分析接菌处理对各指标的影响，菌株003PWXZ6、NXP17、PYXP1、PWXZ10、003NXZ5、PYXZ7、NXZ9等菌株具有研制微生物肥料的潜质。
5．溶磷菌株溶磷机理存在多样性
选择6株溶磷菌株进行液培处理，分别在第2、4、6、8、10d，采用钼锑抗比色法、pH计法、3,5-二硝基水杨酸法、磷酸苯二钠比色法和高效液相色谱法，动态测定了各处理培养液的有效磷含量、pH的变化、蔗糖酶的活性、磷酸酶的活性、有机酸的种类和含量，并分析有效磷的含量和其他各指标间的相关性，结果表明：培养第10d时PKO和蒙金娜培养液有效磷含量与pH值间均呈现线性相关(P<0.01)。在各处理培养10d内，蒙金娜培养液各处理的蔗糖酶活性较在PKO培养液高，菌株PWXZ6在PKO培养液和菌株PYXZ23在蒙金娜培养液有效磷含量与蔗糖酶活性间存在一定的正相关性（P<0.05）。培养液有效磷含量与磷酸酶活性间存在正相关关系，但各处理有效磷含量与磷酸酶活性间的相关性差异显著。各菌株在培养期间均能分泌一定量的乳酸、甲酸、乙酸、柠檬酸、酒石酸、草酸、苹果酸、富马酸、丁二酸等，但菌株不同，分泌的有机酸的种类和数量差异较大。在培养期间各菌株分泌有机酸总量动态变化呈先增加后减少，再增加再减少的变化趋势。
6．溶磷菌株溶磷途径存在多样性
本研究供试菌株中，菌株PYXZ1在溶解无机磷时的主要途径是溶液的pH的降低和磷酸酶的作用，菌株PWXZ10的主要溶磷途径是溶液的pH的降低的作用，菌株PWXZ6的主要途径是溶液的pH的降低、蔗糖酶、磷酸酶和有机酸的共同作用，菌株PYXZ3的主要途径是溶液的蔗糖酶、磷酸酶和有机酸的共同作用。
Tibet, one of the five major traditional pastoral areas of China, is named by "the roofof the world". However, Ali region is the roof of the roof of the world with vast grassland,used as the main substance basis of grassland animal husbandry development Ali region.Mainly plateau frigid desert grassland and ecological environment is fragile, is highlysensitive to the response of the global change and human activities, ecosystem and theenvironment relations to its criticality and the unique biological adaptation mechanism asthe basic characteristics, there are unique, rare biological resources. There has become afocus of many scholars because of its special geographical features and abundant naturalresources. Research on special geographical microbial resources laid the foundation forresources protection and resources application.
In present paper, the physicochemical properties of plant rhizosphere soil from fourdominant forages (Elymus dahuricus, Avena sterilis, Medicago sativa and Poa pretensis) inAli region, Tibet, was measured using routine analysis method. The number anddistribution of bacteria and PGPR (plant growth promoting rhizobacteria) was determinedand analyze the correlation between the nutrient content of rhizosphere and number anddistribution of bacteria. The PGPR strains were selected and identified based on themeasurement of nitrogen fixation capacity, phosphate-solubilization capacity and IAAsecretion capacity. The potential PGPR were used for in vitro inoculation experiment andpot inoculation experiment, respectively in order to study the effects of PGPR on thegrowth parameters of Elymus and colonization activity in the rhizosphere of Elymus, andexplore the phosphorus-solubilizing mechanisms of several strains. The results wereobtained as follows：
1. Different plant rhizosphere soil physical and chemical properties, the number ofbacteria and PGPR strains and significant distribution differences
The nutrient content of four plant rhizosphere soils was low, the supply ability of totaleffective soil nutrient was weak, and significant difference was detected for nutrientcontent and the number of distribution in the rhizosphere of different plants, there was acorrelation between soil physical and chemical property of rhizosphere, nutrient contentand the number of bacteria. There were222strains of PGPR were isolated from therhizosphere of four tested plants, consist of71strains nitrogen fixing bacteria,73strains soluble inorganic phosphorus strains and78strains dissolving organic phosphorus strains.The number of bacteria was presented as RS (root soil)> NRS (soil away from roots)> RP(rhizoplan or surface of roots)≧HP (histoplan or interior of roots), having an obviousrhizosphere effect.
2. PGPR strain grows characteristic difference was different
The nitrogen fixation ability of71azotobacter strains were determined by using theacetylene reduction assay method (ARA) while the phosphorus-solubilizing activity andIAA secretion capacity of222strains were measured and screened base onphosphate-solubilizing ring method, molybdenum blue method, Salkowski colorimetrymethod. The results indicated that28strains had good nitrogenase activity, which variedfrom1.08to446.63nmol C2H4·h-1mL-1;15strains have good ability of dissolvinginorganic phosphorus, the values of D/d were between1.12and1.69and the amount ofdissolved phosphorus in1.66-40.89μg/mL while32strains can dissolve organicphosphorus, the values of D/d were between1.23and3.47and the amount of dissolvedphosphorus in11.05-26.03μg/mL;38strains have the ability to secreting IAA, itssecretion was in7.63-26.20μg/mL.
3. Good PGPR strain mainly were the bacteria belong to the pseudomonas andbacillus genus
The potential PGPR were identified based on16s rDNA, the results showed that eightstrains (PWXZ10, PYXP1, NXZ8, NXZ17,003PWXZ3,003NXZ4,003NXZ6,003NXZ9),having high homology with Pseudomonas sp., were identified as Pseudomonas genus;Strain PYXZ1, PWXZ6, NXZ4have high homology with Pseudomonas fluorescens,therefore identified them as Pseudomonas fluoresceut while Strain PWXZ23, NXP17wereidentified as bacillus genus for having a very high homology with Bacterium.
4. Good PGPR strains in plant rhizosphere colonization, the success of plant growseffect were remarkable
Inoculants, composed of17stains soluble phosphorus bacteria and9strainsazotobacter were used for inoculation experiment. The results indicated that the height,aboveground biomass and underground biomass of Elymus dahuricus treated by PGPRinoculants were significantly higher than that of control (CK), different inoculants hadsignificant effect on the root parameters of Elymus dahuricus (P<0.05). Strains003PWXZ6, NXP17had the most obvious promoting effect on plant height while PYXP1 and NXP17had positive effect on aboveground biomass, underground biomass and totalweight. The root shoot ratio of PWXZ10and003NXZ5were the highest while StrainPYXZ7and NXZ9had the maximum root volume. Root length, root surface area, rootvolume of plant treated by PYXZ19were maximum. The effects of different stains on totalroot length, root surface area, root volume and their distribution were different, there is thehighest ratio among plant root length, root surface area, root volume between strainPYXZ19treatment root diameter greater than0.45mm and strain NXZ16treatment rootdiameter greater than0.15mm. Strains can be successful colonization on the rhizosphereof Elymus dahuricus, and had a significant impact on the phosphorus, nitrogen content ofaboveground, underground part and the whole plant and protein content of abovegroundpart (P<0.05), the phosphorus content of aboveground, underground part and the wholeplant after inoculating003PWXZ6was the highest while NXP17had the highest nitrogencontent of aboveground, underground part and the whole plant and protein of abovegroundpart. Therefore, the comprehensive analysis of all parameters confirmed that these strains(003PWXZ6、NXP17、PYXP1、PWXZ10、003NXZ5、PYXZ7、NXZ9) had the potentialto produce biofertilizer.
5. Mechanism of dissolving phosphorus strains were diverse
Six solubilizing phosphorus bacteria were cultivated in liquid LB medium, theavailable phosphorus content, change of pH and sucrase activity, phosphatase activity, thetype and content of organic acid of culture solution were detected in different time (2,4,6,8,10d) by using molybdenum antimony colorimetric method, pH meter method,3,5-dinitrosalicylic acid method, pyrocatechol phosphate disodium colorimetric methodand high performance liquid chromatography (HPLC) method, respectively, and analyzedthe correlation of available phosphorus content and other index. The results indicated thatthere had a linear correlation (P<0.01) between available phosphorus content and pH valuefor the10d PKO and Mengjinna liquid medium. All treatments training within10d, thesucrase activity of PKO were superior to that of Mengjinna, strain PWXZ6in PKO brothand strain PYXZ23in Mengjinna broth had certain positive correlation between availablephosphorus content and sucrase activity(P<0.05). Meanwhile, there had a significantlypositive correlation relationship between available phosphorus content and phosphataseactivity of broth. All the strainscultured in broth can secrete a certain amount of lactic acid,formic acid, acetic acid, citric acid, tartaric acid, oxalic acid, malic acid, fumaric acid, succinic acid, etc., however, the type and amount of organic acid depended on strain.During cultivation period, the dynamic change of total organic acid secreted by strains wasbimodal curve.
6. Soluble phosphorus strains of dissolving phosphorus approaches were diverse
In this study, strains PYXZ1when dissolved inorganic phosphorus is the main way toreduce the pH of solution and the role of phosphatase, strain PWXZ10the main way ofdissolving phosphorus was to reduce the pH of solution, strain PWXZ6the main way is toreduce the pH of solution, the common role of sucrase, phosphatase, and organic acids,strain PYXZ3solution was the main way of the combination of sucrase, phosphatase, andorganic acids.
本研究以西藏阿里地区高寒草原四种牧草（披碱草Elymus dahuricus、燕麦Avenasterilis、紫花苜蓿Medicago sativa和草地早熟禾Poa pretensis）为研究对象，采用常规分析法测定了植物根际土壤的理化性质，用选择性培养基分离了植物根际不同部位（根系较远的土壤、根表土壤、根系表面和根内）细菌和PGPR菌株的数量和分布，分析了根际养分含量与细菌数量和分布的相关性；研究PGPR菌株的固氮、溶磷和分泌IAA等促生特性，筛选优良菌株并进行了鉴定，同时测定了优良菌株在披碱草根际的定殖能力及对生长的影响，并研究了溶磷菌的溶磷机理，主要研究结果如下：
1．不同植物根际土壤理化性质、细菌和PGPR菌株数量及分布差异显著
四种植物根际土壤养分含量较低，土壤总体有效养分的供给能力较弱，并且不同植物根际养分含量、分布细菌的数量差异显著，植物根际土壤的理化性质和养分含量与细菌的数量间存在一定的相关性。四种供试植物根际分离到PGPR菌株222株，其中，固氮菌71株、溶解无机磷菌株73株和溶解有机磷菌株78株。PGPR菌株的数量表现出“根表>根表土>远根土>根内”的分布趋势，表现出强烈的根际效应。
2．PGPR菌株促生特性各异
采用乙炔还原法（ARA）对分离获得的71株固氮菌的固氮能力进行了测定，采用平板溶磷圈法和液培钼锑抗比色法、显色法和Salkowaki比色法对分离到的222株菌株的溶磷和分泌IAA能力进行了测定、筛选，结果表明：28株具有良好的固氮酶活性，固氮酶活性在1.08~446.63nmol C-12H4·hmL~(-1)；15株具有良好的溶解无机磷的能力，D/d值在1.12~1.69，溶磷量在1.66~40.89μg/mL；32株具有良好的溶解有机磷的能力，D/d值在1.23~3.47，溶磷量在11.05~26.03μg/mL；38株具有分泌IAA的能力，分泌量在7.63~26.20μg/mL。
3．优良PGPR菌株以假单胞属和杆菌属为主
优良PGPR菌株16S rDNA鉴定结果表明：菌株PWXZ10、PYXP1、NXZ8、NXZ17、003PWXZ3、003NXZ4、003NXZ6、003NXZ9与Pseudomonas sp.具有很高的同源性，因此鉴定属假单胞菌属；菌株PYXZ1、PWXZ6、NXZ4与Pseudomonas fluorescens具有很高的同源性，因此鉴定为荧光假单胞菌；菌株PWXZ23、NXP17与Bacterium具有很高的同源性，因此鉴定属杆菌属。
4．优良PGPR菌株可在植物根际成功定殖，对植物促生效果显著
选择优良PGPR菌株26株（溶磷菌17株，固氮菌9株）制备接菌剂，进行了接种试验，结果表明：PGPR接菌处理后披碱草的的株高、地上和地下植物干重显著高于对照，各菌株接菌处理后对披碱草根系性状的影响差异显著（P<0.05），其中，菌株003PWXZ6、NXP17处理后对植株的株高的促进作用最为明显；菌株PYXP1、 NXP17处理对植株的地上、地下植物干重及总干重增加最显著；菌株PWXZ10、003NXZ5处理的根冠比最高；菌株PYXZ7、NXZ9处理对根系平均直径的影响最大；菌株003NXZ5接菌处理后，植株的根总长、根表面积、根体积均最大。各菌株处理对披碱草根系不同直径段植株根总长、根表面积、根体积及其分布的影响也各有不同，菌株PYXZ19处理根系直径大于0.45mm和菌株NXZ16处理根系直径大于0.15mm的根长、根表面积、根体积所占比例最高。供试菌株能在披碱草根际成功定殖，对披碱草地上、地下部分和整株磷、氮含量和植株地上部分粗蛋白含量的影响差异显著（P<0.05），菌株003PWXZ6处理植株地上、地下及部分及整株的含磷量最高，菌株NXP17处理植株地上、地下部分及整株的氮含量和植株地上部分粗蛋白含量最高。综合分析接菌处理对各指标的影响，菌株003PWXZ6、NXP17、PYXP1、PWXZ10、003NXZ5、PYXZ7、NXZ9等菌株具有研制微生物肥料的潜质。
5．溶磷菌株溶磷机理存在多样性
选择6株溶磷菌株进行液培处理，分别在第2、4、6、8、10d，采用钼锑抗比色法、pH计法、3,5-二硝基水杨酸法、磷酸苯二钠比色法和高效液相色谱法，动态测定了各处理培养液的有效磷含量、pH的变化、蔗糖酶的活性、磷酸酶的活性、有机酸的种类和含量，并分析有效磷的含量和其他各指标间的相关性，结果表明：培养第10d时PKO和蒙金娜培养液有效磷含量与pH值间均呈现线性相关(P<0.01)。在各处理培养10d内，蒙金娜培养液各处理的蔗糖酶活性较在PKO培养液高，菌株PWXZ6在PKO培养液和菌株PYXZ23在蒙金娜培养液有效磷含量与蔗糖酶活性间存在一定的正相关性（P<0.05）。培养液有效磷含量与磷酸酶活性间存在正相关关系，但各处理有效磷含量与磷酸酶活性间的相关性差异显著。各菌株在培养期间均能分泌一定量的乳酸、甲酸、乙酸、柠檬酸、酒石酸、草酸、苹果酸、富马酸、丁二酸等，但菌株不同，分泌的有机酸的种类和数量差异较大。在培养期间各菌株分泌有机酸总量动态变化呈先增加后减少，再增加再减少的变化趋势。
6．溶磷菌株溶磷途径存在多样性
本研究供试菌株中，菌株PYXZ1在溶解无机磷时的主要途径是溶液的pH的降低和磷酸酶的作用，菌株PWXZ10的主要溶磷途径是溶液的pH的降低的作用，菌株PWXZ6的主要途径是溶液的pH的降低、蔗糖酶、磷酸酶和有机酸的共同作用，菌株PYXZ3的主要途径是溶液的蔗糖酶、磷酸酶和有机酸的共同作用。
Tibet, one of the five major traditional pastoral areas of China, is named by "the roofof the world". However, Ali region is the roof of the roof of the world with vast grassland,used as the main substance basis of grassland animal husbandry development Ali region.Mainly plateau frigid desert grassland and ecological environment is fragile, is highlysensitive to the response of the global change and human activities, ecosystem and theenvironment relations to its criticality and the unique biological adaptation mechanism asthe basic characteristics, there are unique, rare biological resources. There has become afocus of many scholars because of its special geographical features and abundant naturalresources. Research on special geographical microbial resources laid the foundation forresources protection and resources application.
In present paper, the physicochemical properties of plant rhizosphere soil from fourdominant forages (Elymus dahuricus, Avena sterilis, Medicago sativa and Poa pretensis) inAli region, Tibet, was measured using routine analysis method. The number anddistribution of bacteria and PGPR (plant growth promoting rhizobacteria) was determinedand analyze the correlation between the nutrient content of rhizosphere and number anddistribution of bacteria. The PGPR strains were selected and identified based on themeasurement of nitrogen fixation capacity, phosphate-solubilization capacity and IAAsecretion capacity. The potential PGPR were used for in vitro inoculation experiment andpot inoculation experiment, respectively in order to study the effects of PGPR on thegrowth parameters of Elymus and colonization activity in the rhizosphere of Elymus, andexplore the phosphorus-solubilizing mechanisms of several strains. The results wereobtained as follows：
1. Different plant rhizosphere soil physical and chemical properties, the number ofbacteria and PGPR strains and significant distribution differences
The nutrient content of four plant rhizosphere soils was low, the supply ability of totaleffective soil nutrient was weak, and significant difference was detected for nutrientcontent and the number of distribution in the rhizosphere of different plants, there was acorrelation between soil physical and chemical property of rhizosphere, nutrient contentand the number of bacteria. There were222strains of PGPR were isolated from therhizosphere of four tested plants, consist of71strains nitrogen fixing bacteria,73strains soluble inorganic phosphorus strains and78strains dissolving organic phosphorus strains.The number of bacteria was presented as RS (root soil)> NRS (soil away from roots)> RP(rhizoplan or surface of roots)≧HP (histoplan or interior of roots), having an obviousrhizosphere effect.
2. PGPR strain grows characteristic difference was different
The nitrogen fixation ability of71azotobacter strains were determined by using theacetylene reduction assay method (ARA) while the phosphorus-solubilizing activity andIAA secretion capacity of222strains were measured and screened base onphosphate-solubilizing ring method, molybdenum blue method, Salkowski colorimetrymethod. The results indicated that28strains had good nitrogenase activity, which variedfrom1.08to446.63nmol C2H4·h-1mL-1;15strains have good ability of dissolvinginorganic phosphorus, the values of D/d were between1.12and1.69and the amount ofdissolved phosphorus in1.66-40.89μg/mL while32strains can dissolve organicphosphorus, the values of D/d were between1.23and3.47and the amount of dissolvedphosphorus in11.05-26.03μg/mL;38strains have the ability to secreting IAA, itssecretion was in7.63-26.20μg/mL.
3. Good PGPR strain mainly were the bacteria belong to the pseudomonas andbacillus genus
The potential PGPR were identified based on16s rDNA, the results showed that eightstrains (PWXZ10, PYXP1, NXZ8, NXZ17,003PWXZ3,003NXZ4,003NXZ6,003NXZ9),having high homology with Pseudomonas sp., were identified as Pseudomonas genus;Strain PYXZ1, PWXZ6, NXZ4have high homology with Pseudomonas fluorescens,therefore identified them as Pseudomonas fluoresceut while Strain PWXZ23, NXP17wereidentified as bacillus genus for having a very high homology with Bacterium.
4. Good PGPR strains in plant rhizosphere colonization, the success of plant growseffect were remarkable
Inoculants, composed of17stains soluble phosphorus bacteria and9strainsazotobacter were used for inoculation experiment. The results indicated that the height,aboveground biomass and underground biomass of Elymus dahuricus treated by PGPRinoculants were significantly higher than that of control (CK), different inoculants hadsignificant effect on the root parameters of Elymus dahuricus (P<0.05). Strains003PWXZ6, NXP17had the most obvious promoting effect on plant height while PYXP1 and NXP17had positive effect on aboveground biomass, underground biomass and totalweight. The root shoot ratio of PWXZ10and003NXZ5were the highest while StrainPYXZ7and NXZ9had the maximum root volume. Root length, root surface area, rootvolume of plant treated by PYXZ19were maximum. The effects of different stains on totalroot length, root surface area, root volume and their distribution were different, there is thehighest ratio among plant root length, root surface area, root volume between strainPYXZ19treatment root diameter greater than0.45mm and strain NXZ16treatment rootdiameter greater than0.15mm. Strains can be successful colonization on the rhizosphereof Elymus dahuricus, and had a significant impact on the phosphorus, nitrogen content ofaboveground, underground part and the whole plant and protein content of abovegroundpart (P<0.05), the phosphorus content of aboveground, underground part and the wholeplant after inoculating003PWXZ6was the highest while NXP17had the highest nitrogencontent of aboveground, underground part and the whole plant and protein of abovegroundpart. Therefore, the comprehensive analysis of all parameters confirmed that these strains(003PWXZ6、NXP17、PYXP1、PWXZ10、003NXZ5、PYXZ7、NXZ9) had the potentialto produce biofertilizer.
5. Mechanism of dissolving phosphorus strains were diverse
Six solubilizing phosphorus bacteria were cultivated in liquid LB medium, theavailable phosphorus content, change of pH and sucrase activity, phosphatase activity, thetype and content of organic acid of culture solution were detected in different time (2,4,6,8,10d) by using molybdenum antimony colorimetric method, pH meter method,3,5-dinitrosalicylic acid method, pyrocatechol phosphate disodium colorimetric methodand high performance liquid chromatography (HPLC) method, respectively, and analyzedthe correlation of available phosphorus content and other index. The results indicated thatthere had a linear correlation (P<0.01) between available phosphorus content and pH valuefor the10d PKO and Mengjinna liquid medium. All treatments training within10d, thesucrase activity of PKO were superior to that of Mengjinna, strain PWXZ6in PKO brothand strain PYXZ23in Mengjinna broth had certain positive correlation between availablephosphorus content and sucrase activity(P<0.05). Meanwhile, there had a significantlypositive correlation relationship between available phosphorus content and phosphataseactivity of broth. All the strainscultured in broth can secrete a certain amount of lactic acid,formic acid, acetic acid, citric acid, tartaric acid, oxalic acid, malic acid, fumaric acid, succinic acid, etc., however, the type and amount of organic acid depended on strain.During cultivation period, the dynamic change of total organic acid secreted by strains wasbimodal curve.
6. Soluble phosphorus strains of dissolving phosphorus approaches were diverse
In this study, strains PYXZ1when dissolved inorganic phosphorus is the main way toreduce the pH of solution and the role of phosphatase, strain PWXZ10the main way ofdissolving phosphorus was to reduce the pH of solution, strain PWXZ6the main way is toreduce the pH of solution, the common role of sucrase, phosphatase, and organic acids,strain PYXZ3solution was the main way of the combination of sucrase, phosphatase, andorganic acids.
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