【作者】 易艳梅；

【导师】 黄为一；

【作者基本信息】 南京农业大学 ， 微生物学， 2007， 博士

【摘要】 溶磷微生物是农业生态系统中一类重要的土壤微生物,在农业土壤和自然土壤中广泛存在。由于不同溶磷微生物对某一特定的环境适应存在差异,导致它在不同条件下的溶磷功能发挥显著不同,因而有针对性地掌握不同生态环境中溶磷微生物的分布特征,不同类型溶磷微生物的溶磷特性,是溶磷微生物走向田间应用的前提。本文通过调查不同盐渍土区、不同重金属污染区以及不同磷矿种类成矿区的溶磷微生物资源,研究了不同生态区土壤中溶磷微生物分布的特殊性以及盐分、重金属对溶磷微生物数量分布和种群结构的影响。在此基础上,开展了溶磷细菌对磷矿粉中部分伴生性重金属元素的耐受性与其溶解磷矿粉能力的关系研究;产多糖溶磷细菌对土壤难溶性固定磷的溶磷特性研究;胞外多糖在产多糖溶磷细菌溶磷过程中的作用机理探讨以及盐渍土中接种溶磷细菌EnHy-401对小麦盐胁迫的缓解效应及缓解机理研究。具体结果如下:分别在新疆、山东、江苏的盐渍土区,湖南、江苏、安徽的重金属污染区,湖南中方、石门、浏阳的磷矿区采集土样,从中分离出12个属的溶磷微生物,它们分属于细菌、真菌和放线菌。对溶磷细菌进行分离纯化,得到18个具有溶磷能力的纯菌株。分析不同土壤条件下溶磷微生物的多样性特点及其影响因素发现,重金属污染土壤和盐渍土中均以假单胞菌属（Pseudomonas）、芽孢杆菌属（Bacillus）的出现频度最高,盐渍土中有机质含量对溶磷细菌种群丰度的影响程度大于对溶磷细菌数量丰度的影响程度。重金属污染土壤中,综合污染指数和有机质含量是影响溶磷细菌数量的主要因素。单一重金属严重污染下的土壤,其种群丰度小于多金属复合下的轻度污染。磷矿区土壤中的溶磷细菌分布受磷矿石速效磷含量、土壤有机质水平以及土壤重金属本底值的复合影响,具有适度的速效磷水平和一定的有机质含量的磷矿区,其溶磷细菌数量多,种群丰富,反之,则数量少,种群简单。因此,一个生态区溶磷微生物种群多样性可在一定程度上反映该生态区的生态环境条件。以在NBRI-溴苯酚蓝（BPB）液体培养条件下BPB的褪色程度和滤液在680 nm处的吸光度作为筛选高效溶磷菌株的主要标准,在所分离纯化的18个菌株中,选出HH-1、HH-2、EnHy-401、ArHy-505和AzHy-510等高效溶磷菌株,根据其生理生化特性和16SrDNA的序列分析,将HH-1、HH-2、EnHy-401、ArHy-505和AzHy-510初步鉴定为肠杆菌属（Enterobacter）、成团泛菌（Pantoea agglomerans）、球状节杆菌（Arthobacter globiformis）、肠杆菌属（Enterobacter）和德克斯氏固氮菌（Dexiragumosa）,均分离于湖南中方磷矿区土壤。此结果显示,从缺磷环境下分离到的溶磷微生物并不一定具备高溶磷活性,溶磷微生物并不局限于在缺磷土壤中存在。通过对溶磷细菌溶解不同磷矿粉以及连续发酵磷矿粉的溶磷效果研究,发现,磷矿粉中的部分伴生性金属元素会随着磷的释放而溶出,,其溶出的难易程度随伴生性金属元素在随磷矿粉中的位置而不同。在测定的5种伴生性金属元素中,Zn是与磷的释放联系最密切的金属元素,随磷释放的Zn量最多可占该磷矿粉中总Zn量的90%以上,其溶出率可间接反映磷矿粉磷的释放程度。而Pb是不易随磷溶出的伴生性金属元素。这些伴生性金属的溶出对溶磷细菌的生长和溶磷相关代谢产生抑制作用,从而影响菌株溶磷作用的发挥。在一般情况下,菌株在同一磷矿粉的第二次发酵的溶磷能力要大于对该磷矿粉第一次发酵的溶磷能力。在低磷条件下不耐锌的溶磷菌,其在发酵不同批次磷矿粉间的溶磷能力差异越大;伴生性锌较稳定的磷矿粉,其在菌株不同次发酵中的溶磷效果差异较小。因此,在选用溶磷菌提高磷矿粉生物有效性时,应兼顾其溶磷能力与低磷下的抗锌能力。通过比较不同类型溶磷细菌对土壤不同难溶性磷酸盐的活化效果发现,不同类型的溶磷细菌对不同形式固定态磷的活化有不同的针对性。产多糖溶磷细菌溶解Al-P和Fe-P的能力较弱,但在促进Ca-P型固定磷的溶解能力上具有明显优势。将各产多糖溶磷菌株磷酸钙发酵液中的速效磷分别与发酵液中的总多糖与胞外多糖做相关性分析,发现,产多糖溶磷细菌的溶磷能力与胞外多糖高度相关。根据胞外多糖中磷与胞外多糖提取前后上清液Pi之差的高度相关性（r=0.97,p＜0.05）,可以推测胞外多糖具有吸持溶液中可溶性磷的能力,此持磷能力的大小预示着胞外多糖对有机酸溶磷协同效应的强弱。除此之外,胞外多糖与有机酸溶磷的协同效应还受到胞外多糖浓度、有机酸种类的影响。有机酸溶磷动力曲线与有机酸-胞外多糖复合溶磷动力曲线的差异表明,多糖对有机酸的协同溶磷效应主要是改变了有机酸的溶磷动力学特征,在有机酸单独溶解磷酸三钙时,16 h时磷酸钙的溶解趋于平衡。在有多糖存在的条件下,磷酸钙的溶解一直可以缓慢持续到40 h,溶解平衡时间的延迟,表明在此之前的溶解平衡被打破,并向磷释放的方向发生了移动,使溶磷量提高11.6%。难溶性磷酸钙盐的溶解动态平衡与三个因素有关:环境中的酸度;溶液中的钙离子;溶液中的H₂PO₄^-或HPO₄^2-量。由于多糖具有一定的持磷作用,因此,多糖对有机酸溶磷动态平衡的改变可以理解为多糖通过对有机酸溶下的速效磷的即时吸持,从而影响了了单纯有机酸溶磷的平衡,并推动了平衡向磷溶解方向移动,促进了磷酸钙盐的持续溶解,但这种促进作用受到多糖持磷量的限制。环境中的C/N、N源、可溶磷水平分别作用于产多糖溶磷菌EnHy-401的溶磷相关因子（有机酸、多糖）,溶磷相关因子与溶磷的相关程度决定了产多糖溶磷细菌EnHy-401的溶磷作用受营养条件中C/N、氮源以及可溶性磷酸盐水平等不同变量的影响程度,该菌株只有在适合于产溶磷高效有机酸的前提下,才有较强的溶磷作用,如果又有持磷多糖的分泌,其溶磷作用会更强。盆栽试验结果显示,产多糖溶磷细菌EnHy-401可以在滨海盐渍土中很好地定殖,并在一定程度上提高盐渍土中的有效磷水平,改变了盆栽条件下小麦根系形态的分布特证。与对照相比,生长在接种处理的小麦,其根系表现出根轴数量增多而长度减少,侧根密度降低而长度增加的现象,该现象既是小麦磷素营养得到改善的结果,又进一步加强了小麦对磷、钾、钙以及镁等矿质营养元素的吸收,从而对小麦盐胁迫产生缓解效应,与对照相比,接菌处理的小麦植株中对磷、钙、钾和镁的吸收率分别比对照提高了34.4%、36.3%、31.5%、6.3%。该菌株所表现出来的对盐渍土壤中磷素有效性的提高、对小麦吸收K⁺、Ca²⁺、Mg²⁺、P等矿质营养的促进作用以及小麦生长与矿质营养元素吸收之间高度的相关性表明,溶磷菌株Enterobacteria sp.EnHy-401对小麦盐胁迫的缓解效应主要是通过改善盐渍土中部分矿质营养的供应,增强植株对P、Ca,Mg、K等营养元素的选择性吸收以及多糖调渗作用而实现,该缓解效应受到土壤盐分和营养基质的影响,在贫瘠的高盐分盐渍土中,添加适量的生物有机肥可以提高土壤有机质含量,降低盐胁迫对溶磷菌株溶磷活性的抑制作用,提高菌株对植株盐胁迫的缓解作用。更多还原

【Abstract】 Phosphate-solubilizing microbes are one kind of important soil microbes in agricultural ecosystem,which exist extensively in the agricultural and natural soil. Grasping the microbes’ distribution and their phosphate-solubilizing function characteristics are the preconditions for their application in practice because there is significant difference among different conditions in phosphate-solubilizing function for their different adaptations to certain environment.Their special distribution,the effect of salinity and heavy metals on their quantity and population structure and the relationship between their resistance to heavy metals and p-solubilizing ability,etc.were studied in this study.The results are as follows:12 families of P-solubilizing microbes were isolated from the soil samples collected respectively from the saline area in XinJiang,ShanDong and JiangSu,the polluted area by heavy metal in HuNan,JiangSu,AnHui,the Phosphorus Mineral Region in ZhongFang, ShiMen,LiuYang of HuNan.18 pure strains were isolated from the P-solubilizing bacteria. Analysis of the diversity and the effect of P-solubilizing microbes under different soil conditions showed that Pseudomonas sp.and Bacillus sp.were found high-frequency in the area of heavy metal and salt.The influence degree of organic matter content in the area of salt on popular abundance of P-solubilizing bacteria is lager than number abundance.In the soils polluted by heavy metals,the integrated pollution index and organic matter content are the main influence factors of the number of P-solubilizing bacteria.The popular abundance in the soils heavy polluted by single heavy metal is smaller than in the soils light polluted by combined heavy metals.In the soils of phosphorus mineral region,the distribution of P-solubilizing bacteria was influenced by the content of soil available phosphor,the level of soil organic matter,and the soil background value of heavy metals.There are large quantities of P-solubilizing bacteria and abundant population in the phosphorus mineral region with the proper level of available phosphor and some organic matter content,on the contrary there are few quantities of P-solubilizing bacteria and simple population. Therefore,to a certain extent,the diversity of P-solubilizing microbes can reflect the environmental conditions of this ecological area.The main screening standards of high efficiency P-solubilizing strains were based on the degree of discoloration of BPB cultured in the liquid of NBRI-BPB and the absorbance OD680nm.5 high efficiency P-solubilizing strains named HH-1,HH-2,EnHy-401, ArHy-505 and AzHy-510 were screened from 18 isolated strains.Those bacteria were all isolated from Phosphorus Mineral Region in ZhongFang of HuNan and belong to the Enterobacter,Pantoea agglomerans,Arthobacter globiformis,Enterbacter,and Dexira gumos separately,which were identified primarily according to their physiological and biochemical characteristics and sequence analysis of 16SrDNA.The results indicated that P-solubilizing microbes isolated from low-phosphorus environment may not have high efficiency P-solubilizing activity.Studies on the solubilization of phosphate rock by P-solubilizing bacteria and the effects of soluble phosphate of continuous batch fermentation of phosphate rock show that associated heavy metal in phosphate rock will leach out with the phosphorus release.The degree of difficulty of leach is different according to the association of associated heavy metal in phosphate rock.Test on the 5 associated heavy metals,Zinc is significantly related to the phosphorus release.The amount of released Zinc is about 90%of the total amount of Zinc in phosphate rock.The Leach rate of Zinc could indirectly reflect the degree of the phosphorus release in phosphate rock.Among the associated heavy metals,Pb shows high stability.The leach of those associated heavy metals will inhibit the growth of P-solubilizing bacteria and the metabolism of soluble phosphate,which leads to affect the advantage of phosphate-solubilization of bacteria.Generally,the ability of P-solubilizing of bacteria in the second fermentation is higher than the first fermentation.Under low-phosphorus conditions,the P-solubilizing bacteria is more sensitive to Zinc,the difference of the ability of the phosphate solubilization in different batch fermentation is significantly more;on the contrary,the difference of the ability of the phosphate solubilization in different batch fermentation is less in the phosphate rock with the stable associated Zinc.Therefore,the ability of the P-solubilizing and the ability of the resistance of Zinc in low-phosphorus conditions should be taken full consideration during selection of P-solubilizing bacteria to improve the effect of solubilization of phosphate rock.Results of P-solubilization by different P-solubilizing bacteria on different insoluble phosphates showed that P-solubilizing bacteria producing polysaccharide seem to be more efficient in solubilization of the calcium phosphates,but less effective in solubilizing AlPO₄ and FePO₄ compared to Bacillus megaterium P17.Analyzing the relativity between the amount of soluble phosphate in cultures and the total exopolysaccaride production,and the free water-soluble exopolysaccaride yield,respectively,it was found that the phosphate solubilization of P-solubilizing bacteria producing polysaccharide was high related to the production of the exopolysaccaride.Basing on the high relativity between the phosphorus content in exopolysaccaride and the dispersion of phosphorus concentration in superuatant between before and after EPS extracted,we could presume that the exopolysaccaride produced by P-solubilizing bacteria had ability for phosphorus-holding,which influenced the synergistic effects of exopolysaccaride and organic acid on Ca₃（PO₄）₂ solubilization. The difference existed in the solubilization kinetics of Ca₃（PO₄）₂ in NBRIP-citric acid medium with or without exopolysaccaride showed that the addition of exopolysaccaride changed the balance in homeostasis of by P-solubilization and resulted in greater phosphorus release from insoluble phosphate.The P-solubilizing activity of EnHy-401 was affected by the rate of carbon,nitrogen resource,and KH₂PO₄ content in culture.The results of spot experiment showed that the strain EnHy-401 had the ability to activate the insoluble accumulated phosphorus in saline soil and enhanced nutrient uptake efficiency by wheat plants,then conferred resistance in wheat plants to salt stress and resulted in a significant growth increase.In saline soil inoculated with Enterobacteria sp EnHy-401,available phosphorus and exchangeable calcium was increased from 6.4 mg kg-1 and 1162 mg kg-1 to 10.3 mg kg-1 and 1214 mg kg-1,respectively.Wheat seedling grown in soil inoculated with the EnHy-401 strain increased shoot weight by 28.1%and root weight by 14.6%compared to the control.Phosphorus,calcium,potassium,and magnesium contents in shoots increased 34.4%,36.3%,31.5%,and 6.3%compared to the control,respectively.In our experiments,the fact that the increases in available P,biomass P, and Ca2+ concentration in saline soil treated with PSB Enterobacter sp EnHy-401 inocula （Table 2）,and high relativity between the P,Ca,K,and Mg content in wheat tissue and dry matter（Table 4） indicated that PSB Enterobacter sp EnHy-401 suppressed the adverse effect of salinity stress in plants through nutrient（P and Ca） supply and nutrient（K and Mg） uptake enhancement.The phosphate solubilizing activity of Enterobacteria sp EnHy-401 and the amelioration of salt stress on wheat plants by the strain varied with the salinity levels and content of organic matter in the saline soil.Inoculation of the strain in low salinity soil had a greater increase in available phosphorus and greatly alleviated the negative effects of salt in wheat plants compared to plants cultivated in high saline soil.Combined inoculation of Enterobacter sp EnHy-401 and organic fertilizer application brought about maximum suppression of salt stress to wheat and resulted in the maximum increase of wheat biomass （65.4%） under high salinity condition.更多还原