玉米丛枝菌根真菌根外菌丝表面定殖细菌解磷功能鉴定

柴小粉， 张 林， 田芷源， 王 菲， 冯 固

(中国农业大学资源与环境学院， 北京 100193)



玉米丛枝菌根真菌根外菌丝表面定殖细菌解磷功能鉴定

柴小粉， 张 林， 田芷源， 王 菲， 冯 固*

(中国农业大学资源与环境学院， 北京 100193)

玉米; AM真菌; 植酸钙; 解磷细菌; 菌丝际

丛枝菌根(arbuscular mycorrhizal, AM)真菌是一类重要的土壤微生物，能够与80%以上的陆生植物形成共生关系[1]，产生大量的根外菌丝来增强植物对移动性差的土壤养分(如磷、 锌)的吸收[2]，提高植物对干旱和重金属等非生物胁迫以及病原微生物等生物胁迫的抗性，促进土壤团聚体的形成[3]。此外，AM真菌的根外菌丝能够分泌质子、 糖、 有机酸阴离子[4]，改变菌丝周围土壤的理化性质，形成“菌丝际(hyphosphere)”[5]，为土壤微生物提供定殖场所[6-9]。解磷细菌也是土壤中一类重要的功能微生物，数量可以占到可培养细菌的40%[10-11]，通过释放质子、 有机酸和磷酸酶等代谢产物来活化土壤难溶解的有机态或无机态的磷酸盐。许多研究已经证明，定殖于作物根际的解磷细菌能够帮助植物吸收难溶性磷，改善植物的磷营养[12]。

AM真菌与解磷细菌的互作一直是生态学和植物营养学的研究热点。大量研究工作表明，在盆栽条件下同时接种AM真菌和解磷细菌能够显著提高植物对磷的吸收，促进植物生长[13-14]。但这些研究都是在根际范围内进行，未排除根系对两种微生物的影响。利用两室分隔的根盒装置，在不受根系影响的菌丝室接种解磷细菌的研究发现AM真菌Rhizophagusirregularis(原名Glomusintraradices)与解磷细菌互作可以提高土壤中磷酸酶的活性、 促进土壤中植酸磷的矿化[15-16]。而Tisserant等[17]通过对AM真菌模式菌株Rhizophagusirregularis(DAOM 197198)的转录组分析发现AM真菌不含编码植酸酶蛋白的基因，这就意味着AM真菌不具备直接矿化植酸磷的能力，之前报道的AM真菌促进有机磷矿化是通过AM真菌菌丝刺激解磷细菌的活性实现的[18-19]。然而，AM真菌与解磷细菌互作的研究大多是在室内模拟条件下进行的，在真实的生态系统(如农田生态系统)中的研究尚不多见。

本试验的目的是研究在田间原位条件下AM真菌根外菌丝上是否有活化有机磷的细菌定殖。然后分离纯化出这些解磷细菌，对它们的种类和解磷能力进行鉴定，从而揭示菌丝表面解磷细菌的定殖过程和细菌种类，对于理解菌丝际微域土壤难溶性磷的周转和生物地球化学循环过程提供借鉴。

1　材料和方法

1.1菌丝收集

图1　田间埋放尼龙袋示意图Fig.1　Schematic diagram of burying nylon bags in the field

1.2菌丝表面可培养解磷细菌的分离与纯化

1.3菌株鉴定

对矿化植酸钙的细菌进行16S rDNA测序分析，来确定这些菌株的种类。首先使用细菌基因组DNA提取试剂盒(TIANGEN DP302)提取细菌DNA，方法为在LB培养液中活化细菌，吸取菌液进行离心(11500×g，1 min)，取下部沉淀，按试剂盒说明提取细菌DNA，所得DNA用NanoDrop 2000检测其浓度及纯度，-20℃保存。其次PCR扩增采用细菌16S rDNA通用引物(27F: 5′-AGA GTT TGA TCM TGG CTC AG-3′; 1492R : 5′-TAC GGY TAC CTT GTT ACG ACT T-3′; M 为A 或C )，PCR反应在Biomatro T3扩增仪上进行，反应体系为50 μL。PCR 原液组成为: 10 ×PCR扩增缓冲液5 μL，dNTPs 4 μL(dATP、 dGTP、 dCTP、 dTTP均为25 mmol/L)，正反向引物各1 μL(6 μmol/L)，Taq聚合酶1 μL，以及1 μL DNA模板，37 μL超纯水。PCR反应程序为94℃预变性5 min; 94℃变性1 min，58℃退火1 min，72℃延伸90 s，循环30次; 最后72℃延伸7 min。然后将PCR产物交由华大基因进行测序分析，所得序列提交到Genbank中利用Blast程序(www.ncbi.nlm.nih.gov/BLAST/)进行相似性比对，确定细菌的种类，最后用MEGA 软件(版本4.0)构建系统进化树。

1.4解磷细菌解磷能力的测定

将上述步骤筛选的各株解磷细菌分别接入LB培养液中，37℃培养16 h后，用分光光度计调整细菌浓度至OD值(λ=600 nm)为0.6，然后向无菌的蒙金娜液体有机磷培养基(30 mL)中加入2 mL细菌悬液，以不接菌液的处理作为对照，从而确定每株解磷细菌矿化有机磷的能力，每个菌株重复3次，37℃振荡(180 r/min)培养48 h后，取2 mL培养液进行离心(4℃，12000 r/min，5 min)，吸取1 mL上清液，采用钼锑抗比色法测定无机磷的浓度[20]，并用pH计(DENVER, UB-7)测定培养液pH值。

1.5菌丝际解磷细菌回接试验

供试土壤是2010年5月取自山东省泰安市低磷农田的棕壤，土壤基本理化性状: 速效磷(Olsen-P)3.30 mg/kg、 有效钾(NH4Cl 提取)97.6 mg/kg、 有机质7.27 g/kg、 有效氮7.20 mg/kg、 pH 6.40，过2 mm筛后，送至北京鸿仪四方辐射技术有限公司(Beijing Hongyisifang Radiation Technology Co, Ltd)进行γ射线(辐照强度: 10 kGy60Co γ-ray)灭菌。供试装置为隔网分室根盒培养装置(图2)，根室种植玉米，设置接种AM真菌与不接种AM真菌(Rhizophagusirregularis)两个处理，每个处理重复三次。播种一个月后，在菌丝室中接入筛选出的解磷细菌的混合菌悬液，继续培养一个月后收获，收集菌丝室的土壤，在体式显微镜下挑取菌丝放入有机磷选择性培养基上进行筛选。后续的分离和鉴定过程同1.2和1.3。

图2　两室培养系统示意图Fig.2　Schematic diagram of two compartments cultivation system

1.6数据处理

试验数据均采用Excel进行整理，利用SPSS软件(16.0版本，SPSS Inc.)进行数据分析。对无机磷浓度数据先进行方差同质性检验(Levene′s检验)，再进行单因素方差分析和LSD法多重比较(P< 0.05)。对无机磷浓度和培养液pH数据进行相关性分析。利用MEGA 软件(版本4.0)构建系统进化树。

2　结果与分析

2.1解磷细菌的鉴定

本研究对分离筛选出的29株解磷细菌进行了16S rRNA基因系统发育树的构建(图3)。结果表明29株解磷细菌分别属于芽胞杆菌属(Bacillus)、 假单胞菌属(Pseudomonas)、 沙雷氏菌属(Serratia)、 葡萄球菌属(Staphylococcus)和肠杆菌属(Enterobacter)，其中，芽胞杆菌属(15株，53.3%)和假单胞菌属(10株，33.3%)为优势菌群，其他依次为沙雷氏菌属(2株，6.7%)、 葡萄球菌属(1株，3.3%)和肠杆菌属(1株，3.3%)。

2.2菌丝表面解磷细菌的分离和解磷能力的测定

2.3菌丝际解磷细菌回接分析

图3　菌丝际分离筛选出的解磷细菌16S rRNA基因系统发育树Fig.3　Phylogenetic relationship of bacterial 16S rRNA gene sequences of phytate mineralizing bacteria isolated from AM fungal hyphae in maize field[注(Note): 粗体标注为本研究中获得的序列The sequences obtained in this study are shown in bold，HM+数字为解磷细菌的命名，其中HM表示hyphae associated with maize, HM+number represents the name of phytate mineralizing bacteria.]

利用分室根盒培养系统进行解磷细菌回接试验。从菌丝际的土壤中挑取菌丝，通过选择性培养基筛选后得到47株解磷细菌。将这些细菌提取DNA进行16S rDNA测序分析，所得序列在NCBI数据库上进行比对，发现这47株解磷细菌分别属于以下5个类群: 芽胞杆菌属(Bacillus)、 沙雷氏菌属(Serratia)、 葡萄球菌属(Staphylococcus)、 肠杆菌属(Enterobacter)和贪铜菌属(Cupriavidus)。其中，Bacillus(76.6%)占优势，其他依次为Serratia(14.9%)、Enterobacter(4.3%)、Staphylococcus(2.1%)和Cupriavidus(2.1%)，表明这些解磷细菌能够在Rhizophagusirregularis的根外菌丝上定殖。

3　讨论

AM真菌菌丝表面细菌的定殖是近十年来菌根研究的一个热点方向[7,9,21-22]。Artursson等[6]报道过纯培养条件下Bacilluscereus可以很好的附着在Glomusdussii菌丝表面; 另外菌丝表面还存在可培养的解磷细菌有沙雷氏菌属、 葡萄球菌属和肠杆菌属，其中肠杆菌属已经从菌丝际分离出[23]。但这些研究是在室内的菌丝离体培养条件下进行的，在田间原位条件下筛选AM真菌菌丝表面定殖的解磷细菌种类目前未见报道。本研究从河北曲周长期施用堆肥处理的玉米根系周围收集AM真菌菌丝，筛选得到了定殖于菌丝表面且能够活化植酸磷的解磷细菌，16S rDNA分析表明这些解磷细菌分属五个属，即芽胞杆菌属(Bacillus)、 假单胞菌属(Pseudomonas)、 沙雷氏菌属(Serratia)、 葡萄球菌属(Staphylococcus)和肠杆菌属(Enterobacter)，其中芽胞杆菌和假单胞菌属为优势菌。土壤中99%以上的微生物目前不能被培养技术分离[24]，且解磷细菌菌株在纯化过程中有近50%的解磷菌失去了解磷能力[10]，本研究分离鉴定的解磷细菌只是其中一部分，不能完全反映菌丝表面定殖的解磷细菌群落。

土壤中不同属的解磷细菌拥有不同的解磷能力，如假单胞菌(Pseudomonas)、 芽胞杆菌(Bacillus)和根瘤菌(Rhizobium)是解磷能力较强的细菌类群[25]，它们的解磷功能得到了广泛关注和研究。本研究同样发现假单胞菌属的细菌解磷能力相对较强，活化出的有效磷浓度均在15 mg/L以上(图4)。

表1　平板法测量解磷细菌溶磷圈直径与菌落直径比值

图4　解磷细菌活化出的无机磷浓度Fig.4　Concentrations of inorganic phosphorus mineralized by different phytate mineralizing bacteria strains

图5　培养液中无机磷浓度与pH相关性Fig.5　The correlation between concentrations of inorganic phosphorus mineralized of different phytate mineralizing bacterial strains and pH in culture solution

本研究结果发现有机磷矿化的量与培养基的pH呈显著的负相关性，这说明解磷细菌可通过分泌有机酸等代谢产物提高植酸钙的底物有效性，从而促进其矿化[26]。对于筛选出的可培养解磷细菌的解磷能力的评价是在纯培养条件下进行的，但是在自然生态系统中，植物-AM真菌-细菌三者之间存在着相互作用[27]。研究表明，菌根际中细菌活化土壤养分(尤其是磷)的能力会受到植物根系和AM真菌的影响[4,16]。菌丝际的范围非常微小，很难通过类似根际的取样方法进行研究。Gahan等[22]对菌丝表面、 菌丝际及土体土壤中微生物群落进行研究，发现菌丝表面和菌丝际的可培养细菌的丰富度和群落结构均与土体不同，且菌丝际与菌丝表面的细菌群落不存在显著差异，因此可以收集菌丝，通过测定菌丝表面的微生物群落来反映菌丝际的变化，但这种方法缩小了菌丝际的范围，在未来的研究中需要进一步改进。

Rhizophagusirregularis是一种分布广泛的AM真菌，在我国北方农田土壤中普遍存在[28-29]。本研究将从菌丝表面分离出的解磷细菌接种到Rhizophagusirregularis的菌丝周围，结果发现80%经纯化的解磷细菌可以在其表面定殖，另有20%的细菌在回接试验中未检测到，其可能原因是本回接试验的条件与田间条件存在差异，例如农田中侵染于玉米根系中的AM真菌种类很多[29]，而我们的回接定殖试验只接种了Rhizophagusirregularis一种AM真菌，不同的AM真菌对不同种类细菌的定殖是具有选择性的[30]，上述原因有可能导致只有80%解磷细菌在AM真菌菌丝表面定殖，本试验尚不能肯定其余的20%的解磷细菌是否可以在AM真菌根外菌丝表面定殖，这需要更精细的试验进一步的验证。在回接细菌菌株鉴定时发现贪铜菌属(Cupriavidus)存在，该试验在温室进行，空气中不可避免地存在各种微生物，另外本试验所用的Rhizophagusirregularis菌剂也可能是该菌株的来源。这一结果给我们的启示是当所有细菌菌株接种到AM真菌菌丝周围时，会表现出不同的定殖能力，因此它们参与菌丝际土壤磷的周转和磷在植物-AM真菌系统中的利用效率也会表现出不同的作用。

[1]Smith S E, Smith F A. Roles of arbuscular mycorrhizas in plant nutrition and growth: new paradigms from cellular to ecosystem scales[J]. Annual Review of Plant Biology, 2011, 62: 227-250.

[2]Smith S E, Read D J. Mycorrhizal symbiosis[M]. San Diego, CA: Academic Press, 2008.

[3]冯固, 张福锁, 李晓林．丛枝菌根真菌在农业生产中的作用与调控[J]. 土壤学报, 2010, 47: 995-1004.

Feng G, Zhang F S, Li X L. Functions of arbuscular mycorrhizal fungi in agriculture and their manipulation[J]. Acta Pedologica Sinica, 2010, 47: 995-1004.

[4]Johansson J F, Paul L R, Finlay R D. Microbial interactions in the mycorrhizosphere and their significance for sustainable agriculture[J]. FEMS Microbiology Ecology, 2004, 48: 1-13.

[5]Marschner H. Mineral nutrition of higher plants (2nd Edition)[M]. London: Academic Press, 1995.

[6]Artursson V, Jansson J K. Use of bromodeoxyuridine immunocapture to identify active bacteria associated with arbuscular mycorrhizal hyphae[J]. Applied and Environmental Microbiology, 2003, 69: 6208-6215.

[7]Toljander J F, Lindahl B D, Paul L R,etal. Influence of arbuscular mycorrhizal mycelial exudates on soil bacterial growth and community structure[J]. FEMS Microbiology Ecology, 2007, 61: 295-304.

[8]Warmink J A, Nazir R, Corten B, van Elsas J D. Hitchhikers on the fungal highway: the helper effect for bacterial migration via fungal hyphae[J].Soil Biology and Biochemistry, 2011, 43: 760-765.

[9]Agnolucci M, Battini F, Cristani C, Giovannetti M. Diverse bacterial communities are recruited on spores of different arbuscular mycorrhizal fungal isolates[J]. Biology and Fertility of Soils, 2015, 51: 379-389.

[10]Kucey R. Phosphate-solubilizing bacteria and fungi in various cultivated and virgin Alberta soils[J]. Canadian Journal of Soil Science, 1983, 63: 671-678.

[11]Jorquera M A, Hernandez M T, Rengel Z,etal. Isolation of culturable phosphobacteria with both phytate-mineralization and phosphate-solubilization activity from the rhizosphere of plants grown in a volcanic soil[J]. Biology and Fertility of Soils, 2008, 44: 1025-1034.

[12]Zhang L, Ding X D, Chen S F,etal. Reducing carbon: phosphorus ratio can enhance microbial phytin mineralization and lessen competition with maize for phosphorus[J]. Journal of Plant Interactions, 2014, 9: 850-856.

[13]Vázquez M M, Cesar S, Azcón R, Barea M. Interactions between arbuscular mycorrhizal fungi and other microbial inoculants and their effects on microbial population and enzyme activities in the rhizosphere of maize plants[J]. Applied Soil Ecology, 2000, 15: 261-272.

[14]Sabannavar S J, Lakshman H C. Effect of rock phosphate solubilization using mycorrhizal fungi and phosphobacteria on two high yielding varieties ofSesamumindicumL.[J]. World Journal of Agricultural Sciences, 2009, 5: 470-479.

[15]张林, 丁效东, 冯固, 等. 菌丝室接种解磷细菌BacillusmegateriumC4 对土壤有机磷矿化和植物吸收的影响[J]. 生态学报, 2012, 32: 4079-4086．

Zhang L. Ding X D. Feng G,etal.The effects of inoculation with phosphate solubilizing bacteriaBacillusmegateriumC4 in the AM fungal hyphosphere on soil organic phosphorus mineralization and plant uptake[J]. Acta Ecologica Sinica, 2012, 32: 4079-4086．

[16]Zhang L, Fan J, Ding X,etal. Hyphosphere interactions between an arbuscular mycorrhizal fungus and a phosphate solubilizing bacterium promote phytate mineralization in soil[J]. Soil Biology and Biochemistry, 2014, 74: 177-183.

[17]Tisserant E, Kohler A, Dozolme-Seddas P,etal. The transcriptome of the arbuscular mycorrhizal fungusGlomusintraradices(DAOM 197198) reveals functional tradeoffs in an obligate symbiont[J]. New Phytologist, 2012, 193: 755-769.

[18]Tarafdar J C, Marschner H. Phosphatase activity in the rhizosphere and hyphosphere of VA mycorrhizal wheat supplied with inorganic and organic phosphorus[J]. Soil Biology and Biochemistry, 1994, 26: 387-395.

[19]Feng G, Song Y C, Li X L, Christie P. Contribution of arbuscular mycorrhizal fungi to utilization of organic sources of phosphorus by red clover in a calcareous soil[J]. Applied Soil Ecology, 2003, 22: 139-148.

[20]Murphy J, Riley J P. A modified single solution method for the determination of phosphate in natural waters[J]. Analytica Chimica Acta, 1962, 27:31-36.

[21]Scheublin T, Sanders I R, Keel C, van der Meer J R. Characteriation of microbial communities colonising the hyphal surfaces of arbuscular mycorrhizal fungi[J]. ISME Journal, 2010, 4: 752-763.

[22]Gahan J, Schmalenberger A. Arbuscular mycorrhizal hyphae in grassland select for a diverse and abundant hyphospheric bacterial community involved in sulfonated sulfurization[J]. Applied Soil Ecology, 2015, 89: 113-121.

[23]Chung H, Park M, Madhaiyan M,etal. Isolation and characterization of phosphate solubilizing bacteria from the rhizosphere of crop plants of Korea[J].Soil Biology and Biochemistry, 2005, 37: 1970-1974.

[24]Pham V H, Kim J. Cultivation of unculturable soil bacteria[J]. Trends in Biotechnology, 2012, 30: 475-484.

[25]林启美, 赵小蓉, 孙焱鑫, 姚军. 四种不同生态系统的土壤解磷细菌数量及种群分布[J]. 土壤与环境, 2000, 9: 34-37.

Lin Q M, Zhao X R, Sun Y X, Yao J. Community characters of soil phosphobacteria in four ecosystems[J]. Soil and Environmental Sciences, 2000, 9: 34-37.

[26]Rodrìguez H, Fraga R. Phosphate solubilizing bacteria and their role in plant growth promotion[J]. Biotechnology Advances, 1999, 17: 319-339.

[27]Reinhardt K, Siva-Jothy M T. Biology of the bed bugs (Cimicidae)[J]. Annual Review of Entomology, 2007, 52: 351-374.

[28]Liu W, Jiang S S, Zhang Y L,etal. Spatiotemporal changes in arbuscular mycorrhizal fungal communities under different nitrogen inputs over a 5-year period in intensive agricultural ecosystems on the North China Plain[J]. FEMS Microbiology Ecology, 2014, 90: 436-453.

[29]Wang X J, Wang X X, Feng G. Optimised nitrogen fertiliser management achieved higher diversity of arbuscular mycorrhiza fungi and high-yielding maize (ZeamaysL.)[J]. Crop and Pasture Science, 2015, 66: 706-714.

[30]Toljander J, Artursson V, Paul L R,etal. Attachment of different soil bacteria to arbuscular mycorrhizal fungal extraradical hyphae is determined by hyphal vitality and fungal species[J]. FEMS Microbiology Letters, 2006, 254: 34-40.

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

CHAI Xiao-fen, ZHANG Lin, TIAN Zhi-yuan, WANG Fei, FENG Gu*

(CollegeofResourcesandEnvironmentalSciences,ChinaAgriculturalUniversity,Beijing100193,China)

【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 16S rDNA sequencing. The abilities of PMB to utilize organic phosphate were determined in solid phytate-selective medium contained phytin by the diameter of bacterial colony (d) and phosphorus solubilizing halo (D), and by inorganic phosphorus concentration and pH 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 toBacillus,Serratia,Pseudomonas,StaphylococcusandEnterobacter, 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 ofPseudomonashad the highest mineralization rate (more than 14%), meanwhile the pH of liquid medium was reduced by 2 to 4 units. All the identified PMB strains were re-inoculated into the hyphal compartment of a two-compartment 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 ofPseudomonas, interestingly, another PSM strain,Cupriaviduswas 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 to 5 genera were identified. They had different ability to mineralize phytin and the genus ofPseudomonashad the highest mineralization rate.

maize; arbuscular mycorrhizal fungi; phytin; phytate mineralizing bacteria; hyphosphere

2015-09-28接受日期: 2015-12-21

国家自然科学基金 (31501831,U1403285)和教育部博士点基金(20120008130001)资助。

柴小粉(1990—)， 女， 河南商丘人， 硕士研究生， 主要从事根际营养与调控研究。 E-mail: xf_chai@cau.edu.cn

Tel: 010-62733885, E-mail: fenggu@cau.edu.cn

S513.01; S144.3

A

1008-505X(2016)04-1031-08