玉米丛枝菌根真菌根外菌丝表面定殖细菌解磷功能鉴定

Identification of phytate mineralizing bacteria colonized on the extraradical hyphal surface of arbuscular mycorrhizal fungi in a maize field

【目的】在田间原位条件下研究丛枝菌根(Arbuscular mycorrhizal,AM)真菌根外菌丝表面有无解磷细菌定殖,并对存在的解磷细菌的种类进行鉴定,对其活化有机磷的能力进行检测,从而为更好地认识菌丝际土壤有机磷的周转和磷的生物地球化学循环过程提供依据。【方法】利用河北省曲周县中国农业大学实验站的玉米长期定位试验,采用田间埋膜方式从玉米根系周围收集AM真菌的根外菌丝,用蒙金娜有机磷固体培养基筛选菌丝表面具有矿化植酸钙能力的细菌,对筛选出的细菌进行分离、培养,然后提取细菌DNA,通过16S r DNA测序分析来确定解磷细菌的种类。分离鉴定的菌株先用蒙金娜有机磷固体培养基通过测定菌落直径(d)及溶磷圈直径(D)初步鉴定其活化植酸钙的能力,再用无菌的蒙金娜有机磷液体培养基确定每株解磷细菌矿化植酸磷的能力,并对溶液的p H进行测定,每个菌株重复3次。最后采用两室隔网根盒将分离纯化的解磷细菌回接至AM真菌根外菌丝,鉴定回接成功率,确定分离出的解磷细菌能否成功定殖于菌丝表面。【结果】从AM真菌根外菌丝表面分离得到了29株具有活化有机磷能力的细菌,分属于芽胞杆菌、假单胞菌、沙雷氏菌、葡萄球菌和肠杆菌5个不同的属。通过有机磷液体培养进一步检测这些菌株活化植酸磷的能力,发现它们对植酸磷的矿化率为1.9%21.9%。其中假单胞菌属细菌的解磷能力相对较强,对植酸磷的矿化率达14%以上,液体培养基的p H值下降2 4个单位。将分离纯化的细菌回接至两室隔网根盒的菌丝室,培养30 d后,从菌丝表面再次检测到除假单胞菌属外的芽胞杆菌属(Bacillus)、沙雷氏菌属(Serratia)、葡萄球菌属(Staphylococcus)和肠杆菌属(Enterobacter)细菌,另外还检测到贪铜菌属(Cupriavidus)细菌。【结论】在田间原位条件下,与玉米共生的AM真菌的根外菌丝表面有多种解磷细菌定殖,它们活化有机磷能力存在差异,其中以假单胞菌属细菌的解磷能力相对较强。

【Objectives】The objective of this study was to investigate whether phosphate mineralizing bacteria（ PMB） could colonize on the hyphal surface of arbuscular mycorrhizal（ AM） fungi in maize field and to identify these bacteria and test their abilities in mineralizing organic phosphate. This can help to go to deep into understanding the functions of hyphosphere bacteria in soil phosphorus turnover and biogeochemical cycle.【Methods】We collected the extraradical hyphae of AM fungi associated with maize roots from the field at China Agricultural University long-term experimental station in Quzhou, Hebei province. The PMB attached to AM fungal hyphae were selected using the phytate-selective medium contained phytin as the sole phosphorus source. The DNA of selected bacteria was extracted with TIANamp Bacteria DNA Kit according to the manufacturer s instructions.Then the genera of PMB were identified through bacterial 16 S r DNA sequencing. The abilities of PMB to utilize organic phosphate were determined in solid phytate-selective medium contained phytin by the diameter of bacterialcolony（ d） and phosphorus solubilizing halo（ D）, and by inorganic phosphorus concentration and p H in liquid phytate-selective medium. Each PMB isolated from AM fungal hyphae was analyzed in triplication. PMB strains belonged to different genera were inoculated into the hyphal compartment of a two-compartment microcosm conducted in the greenhouse to test their abilities to re-colonize on the hyphal surface. 【Results 】Twenty-nine strains of PMB were isolated from the surface of AM fungal hyphae and affiliated to Bacillus, Serratia,Pseudomonas, Staphylococcus and Enterobacter, respectively. The abilities of PMB to utilize phytin in the liquid medium showed that the mineralization rates ranged from 1. 9% to 21. 9%. Among them, the genus of Pseudomonas had the highest mineralization rate（ more than 14%）, meanwhile the p H of liquid medium was reduced by 2 to 4 units. All the identified PMB strains were re-inoculated into the hyphal compartment of a twocompartment microcosm in the greenhouse. After 30 days, we found that four genera of the PMB were isolated and identified successfully from the hyphal surface with the exception of the genus of Pseudomonas, interestingly,another PSM strain, Cupriavidus was detected.【Conclusions】The results demonstrated that PMB could attach to the surface of extraradical hyphae of AM fungi associated with maize in the field and 29 bacterial strains belonged to5 genera were identified. They had different ability to mineralize phytin and the genus of Pseudomonas had the highest mineralization rate.