设施农业土壤中聚磷菌的筛选及其生物学特性分析

为在设施农业土壤中筛选获得兼具高效除磷能力和良好环境适应性的聚磷菌(Phosphorus accumulating organisms,PAOs),从设施大棚采集40 cm以上土样,梯度稀释后经PAOs平板初筛、异染粒染色复筛聚磷菌,结合形态学和16S rRNA测序鉴定种属。采用钼锑抗分光光度计法测定聚磷菌的除磷率和菌体磷含量,PCR验证聚磷作用关键基因ppk。采用活菌计数法分析聚磷菌的耐盐和耐高温率,对峙培养法分析对病原菌的抑菌率。本研究共筛选得到7株聚磷菌,经鉴定分别属于节杆菌(Arthrobacter spp.)、微杆菌(Microbacterium spp.)、红球菌(Rhodococcus spp.)和芽孢杆菌(Bacillus spp.)4个菌属。好氧条件下,所有菌株均具有除磷作用,其中A.ureafaciens Fp64的除磷率最高(70.52%),R.rhodochrous Fp31的菌体含磷量最高(18.74%)。在除磷率大于50%的4株聚磷菌中,均可扩增获得ppk基因。所筛选聚磷菌兼具多种其他生物学特性,其中A.ureafaciens Fp64和A.keyseri Fp38具有病原菌拮抗和耐盐性,M.esteraromaticum Np20具有病原菌拮抗和耐高温性,R.rhodochrous Fp31具有极其显著的耐盐性。本研究所筛选的菌株具有较高的除磷能力和菌体含磷量,兼具拮抗病原菌和良好环境适应性等特点,有望用于控制土壤渗漏液磷流失,为进一步研发针对设施农业土壤环境的高效生物除磷技术提供了优良菌种。

Isolation and biological characterization of phosphorus accumulating organisms(PAOs)from greenhouse facility soil

The objective of this study was to isolate efficient phosphorus accumulating organisms (PAOs) with good environment adaptability. Soil samples (0~40 cm depth) were collected from within greenhouses. The PAOs were isolated and screened by serial dilution, PAOplate culturing, and metachromatic granule staining. Taxonomic identification of PAO isolates was based on morphological characters and 16S rRNA sequencing. Phosphorus removal and phosphorus contents of these PAOs were determined by the Mo-Sb colorimetric method. Gene ppk, the key gene in phosphorus-accumulation, was amplified using PCR. Salt and high temperature tolerances were analyzed by counting the number of surviving bacteria following exposure. Antagonistic activity against pathogens was determined by a confrontation test.Seven bacterial PAOs belonging to 4 genera, Arthrobacter, Microbacterium, Rhodococcus, and Bacillus were screened.All strains showed phosphorus-removal effects, of which A. ureafaciens Fp64 showed the largest removal effect (70.52%), and R.rhodochrous Fp31 had the highest phosphorus content (18.74%). The ppk gene was detected in 4 strains with phosphorus removal rate >50%. The PAO strains also showed variable biological characters. A. ureafaciens Fp64 and A.keyseri Fp38 were salt tolerant and antagonistic to pathogens. M.esteraromaticum Np20 showed antagonistic activities and high temperature tolerance. R.rhodochrous Fp31 exhibited prominent salt tolerance. The PAO strains screened in this study had high phosphorus contents and phosphorus removal capacities. Furthermore, they exhibited variable features, including antagonistic activity and environmental adaptability. They are expected to be used to reduce phosphorus loss from soil leaching and act as good agents for biological phosphorus removal technology in the greenhouse industry.