玉米叢枝菌根真菌根外菌絲表面定殖細菌解磷功能鑒定

柴小粉， 張 林， 田芷源， 王 菲， 馮 固

(中國農業大學資源與環境學院， 北京 100193)

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玉米叢枝菌根真菌根外菌絲表面定殖細菌解磷功能鑒定

柴小粉， 張 林， 田芷源， 王 菲， 馮 固*

(中國農業大學資源與環境學院， 北京 100193)

玉米; AM真菌; 植酸鈣; 解磷細菌; 菌絲際

叢枝菌根(arbuscular mycorrhizal, AM)真菌是一類重要的土壤微生物，能夠與80%以上的陸生植物形成共生關系[1]，產生大量的根外菌絲來增強植物對移動性差的土壤養分(如磷、 鋅)的吸收[2]，提高植物對干旱和重金屬等非生物脅迫以及病原微生物等生物脅迫的抗性，促進土壤團聚體的形成[3]。此外，AM真菌的根外菌絲能夠分泌質子、 糖、 有機酸陰離子[4]，改變菌絲周圍土壤的理化性質，形成“菌絲際(hyphosphere)”[5]，為土壤微生物提供定殖場所[6-9]。解磷細菌也是土壤中一類重要的功能微生物，數量可以占到可培養細菌的40%[10-11]，通過釋放質子、 有機酸和磷酸酶等代謝產物來活化土壤難溶解的有機態或無機態的磷酸鹽。許多研究已經證明，定殖于作物根際的解磷細菌能夠幫助植物吸收難溶性磷，改善植物的磷營養[12]。

AM真菌與解磷細菌的互作一直是生態學和植物營養學的研究熱點。大量研究工作表明，在盆栽條件下同時接種AM真菌和解磷細菌能夠顯著提高植物對磷的吸收，促進植物生長[13-14]。但這些研究都是在根際范圍內進行，未排除根系對兩種微生物的影響。利用兩室分隔的根盒裝置，在不受根系影響的菌絲室接種解磷細菌的研究發現AM真菌Rhizophagusirregularis(原名Glomusintraradices)與解磷細菌互作可以提高土壤中磷酸酶的活性、 促進土壤中植酸磷的礦化[15-16]。而Tisserant等[17]通過對AM真菌模式菌株Rhizophagusirregularis(DAOM 197198)的轉錄組分析發現AM真菌不含編碼植酸酶蛋白的基因，這就意味著AM真菌不具備直接礦化植酸磷的能力，之前報道的AM真菌促進有機磷礦化是通過AM真菌菌絲刺激解磷細菌的活性實現的[18-19]。然而，AM真菌與解磷細菌互作的研究大多是在室內模擬條件下進行的，在真實的生態系統(如農田生態系統)中的研究尚不多見。

本試驗的目的是研究在田間原位條件下AM真菌根外菌絲上是否有活化有機磷的細菌定殖。然后分離純化出這些解磷細菌，對它們的種類和解磷能力進行鑒定，從而揭示菌絲表面解磷細菌的定殖過程和細菌種類，對于理解菌絲際微域土壤難溶性磷的周轉和生物地球化學循環過程提供借鑒。

1　材料和方法

1.1菌絲收集

圖1　田間埋放尼龍袋示意圖Fig.1　Schematic diagram of burying nylon bags in the field

1.2菌絲表面可培養解磷細菌的分離與純化

1.3菌株鑒定

對礦化植酸鈣的細菌進行16S rDNA測序分析，來確定這些菌株的種類。首先使用細菌基因組DNA提取試劑盒(TIANGEN DP302)提取細菌DNA，方法為在LB培養液中活化細菌，吸取菌液進行離心(11500×g，1 min)，取下部沉淀，按試劑盒說明提取細菌DNA，所得DNA用NanoDrop 2000檢測其濃度及純度，-20℃保存。其次PCR擴增采用細菌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 為A 或C )，PCR反應在Biomatro T3擴增儀上進行，反應體系為50 μL。PCR 原液組成為: 10 ×PCR擴增緩沖液5 μL，dNTPs 4 μL(dATP、 dGTP、 dCTP、 dTTP均為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超純水。PCR反應程序為94℃預變性5 min; 94℃變性1 min，58℃退火1 min，72℃延伸90 s，循環30次; 最后72℃延伸7 min。然后將PCR產物交由華大基因進行測序分析，所得序列提交到Genbank中利用Blast程序(www.ncbi.nlm.nih.gov/BLAST/)進行相似性比對，確定細菌的種類，最后用MEGA 軟件(版本4.0)構建系統進化樹。

1.4解磷細菌解磷能力的測定

將上述步驟篩選的各株解磷細菌分別接入LB培養液中，37℃培養16 h后，用分光光度計調整細菌濃度至OD值(λ=600 nm)為0.6，然后向無菌的蒙金娜液體有機磷培養基(30 mL)中加入2 mL細菌懸液，以不接菌液的處理作為對照，從而確定每株解磷細菌礦化有機磷的能力，每個菌株重復3次，37℃振蕩(180 r/min)培養48 h后，取2 mL培養液進行離心(4℃，12000 r/min，5 min)，吸取1 mL上清液，采用鉬銻抗比色法測定無機磷的濃度[20]，并用pH計(DENVER, UB-7)測定培養液pH值。

1.5菌絲際解磷細菌回接試驗

供試土壤是2010年5月取自山東省泰安市低磷農田的棕壤，土壤基本理化性狀: 速效磷(Olsen-P)3.30 mg/kg、 有效鉀(NH4Cl 提取)97.6 mg/kg、 有機質7.27 g/kg、 有效氮7.20 mg/kg、 pH 6.40，過2 mm篩后，送至北京鴻儀四方輻射技術有限公司(Beijing Hongyisifang Radiation Technology Co, Ltd)進行γ射線(輻照強度: 10 kGy60Co γ-ray)滅菌。供試裝置為隔網分室根盒培養裝置(圖2)，根室種植玉米，設置接種AM真菌與不接種AM真菌(Rhizophagusirregularis)兩個處理，每個處理重復三次。播種一個月后，在菌絲室中接入篩選出的解磷細菌的混合菌懸液，繼續培養一個月后收獲，收集菌絲室的土壤，在體式顯微鏡下挑取菌絲放入有機磷選擇性培養基上進行篩選。后續的分離和鑒定過程同1.2和1.3。

圖2　兩室培養系統示意圖Fig.2　Schematic diagram of two compartments cultivation system

1.6數據處理

試驗數據均采用Excel進行整理，利用SPSS軟件(16.0版本，SPSS Inc.)進行數據分析。對無機磷濃度數據先進行方差同質性檢驗(Levene′s檢驗)，再進行單因素方差分析和LSD法多重比較(P< 0.05)。對無機磷濃度和培養液pH數據進行相關性分析。利用MEGA 軟件(版本4.0)構建系統進化樹。

2　結果與分析

2.1解磷細菌的鑒定

本研究對分離篩選出的29株解磷細菌進行了16S rRNA基因系統發育樹的構建(圖3)。結果表明29株解磷細菌分別屬于芽胞桿菌屬(Bacillus)、 假單胞菌屬(Pseudomonas)、 沙雷氏菌屬(Serratia)、 葡萄球菌屬(Staphylococcus)和腸桿菌屬(Enterobacter)，其中，芽胞桿菌屬(15株，53.3%)和假單胞菌屬(10株，33.3%)為優勢菌群，其他依次為沙雷氏菌屬(2株，6.7%)、 葡萄球菌屬(1株，3.3%)和腸桿菌屬(1株，3.3%)。

2.2菌絲表面解磷細菌的分離和解磷能力的測定

2.3菌絲際解磷細菌回接分析

圖3　菌絲際分離篩選出的解磷細菌16S rRNA基因系統發育樹Fig.3　Phylogenetic relationship of bacterial 16S rRNA gene sequences of phytate mineralizing bacteria isolated from AM fungal hyphae in maize field[注(Note): 粗體標注為本研究中獲得的序列The sequences obtained in this study are shown in bold，HM+數字為解磷細菌的命名，其中HM表示hyphae associated with maize, HM+number represents the name of phytate mineralizing bacteria.]

利用分室根盒培養系統進行解磷細菌回接試驗。從菌絲際的土壤中挑取菌絲，通過選擇性培養基篩選后得到47株解磷細菌。將這些細菌提取DNA進行16S rDNA測序分析，所得序列在NCBI數據庫上進行比對，發現這47株解磷細菌分別屬于以下5個類群: 芽胞桿菌屬(Bacillus)、 沙雷氏菌屬(Serratia)、 葡萄球菌屬(Staphylococcus)、 腸桿菌屬(Enterobacter)和貪銅菌屬(Cupriavidus)。其中，Bacillus(76.6%)占優勢，其他依次為Serratia(14.9%)、Enterobacter(4.3%)、Staphylococcus(2.1%)和Cupriavidus(2.1%)，表明這些解磷細菌能夠在Rhizophagusirregularis的根外菌絲上定殖。

3　討論

AM真菌菌絲表面細菌的定殖是近十年來菌根研究的一個熱點方向[7,9,21-22]。Artursson等[6]報道過純培養條件下Bacilluscereus可以很好的附著在Glomusdussii菌絲表面; 另外菌絲表面還存在可培養的解磷細菌有沙雷氏菌屬、 葡萄球菌屬和腸桿菌屬，其中腸桿菌屬已經從菌絲際分離出[23]。但這些研究是在室內的菌絲離體培養條件下進行的，在田間原位條件下篩選AM真菌菌絲表面定殖的解磷細菌種類目前未見報道。本研究從河北曲周長期施用堆肥處理的玉米根系周圍收集AM真菌菌絲，篩選得到了定殖于菌絲表面且能夠活化植酸磷的解磷細菌，16S rDNA分析表明這些解磷細菌分屬五個屬，即芽胞桿菌屬(Bacillus)、 假單胞菌屬(Pseudomonas)、 沙雷氏菌屬(Serratia)、 葡萄球菌屬(Staphylococcus)和腸桿菌屬(Enterobacter)，其中芽胞桿菌和假單胞菌屬為優勢菌。土壤中99%以上的微生物目前不能被培養技術分離[24]，且解磷細菌菌株在純化過程中有近50%的解磷菌失去了解磷能力[10]，本研究分離鑒定的解磷細菌只是其中一部分，不能完全反映菌絲表面定殖的解磷細菌群落。

土壤中不同屬的解磷細菌擁有不同的解磷能力，如假單胞菌(Pseudomonas)、 芽胞桿菌(Bacillus)和根瘤菌(Rhizobium)是解磷能力較強的細菌類群[25]，它們的解磷功能得到了廣泛關注和研究。本研究同樣發現假單胞菌屬的細菌解磷能力相對較強，活化出的有效磷濃度均在15 mg/L以上(圖4)。

表1　平板法測量解磷細菌溶磷圈直徑與菌落直徑比值

圖4　解磷細菌活化出的無機磷濃度Fig.4　Concentrations of inorganic phosphorus mineralized by different phytate mineralizing bacteria strains

圖5　培養液中無機磷濃度與pH相關性Fig.5　The correlation between concentrations of inorganic phosphorus mineralized of different phytate mineralizing bacterial strains and pH in culture solution

本研究結果發現有機磷礦化的量與培養基的pH呈顯著的負相關性，這說明解磷細菌可通過分泌有機酸等代謝產物提高植酸鈣的底物有效性，從而促進其礦化[26]。對于篩選出的可培養解磷細菌的解磷能力的評價是在純培養條件下進行的，但是在自然生態系統中，植物-AM真菌-細菌三者之間存在著相互作用[27]。研究表明，菌根際中細菌活化土壤養分(尤其是磷)的能力會受到植物根系和AM真菌的影響[4,16]。菌絲際的范圍非常微小，很難通過類似根際的取樣方法進行研究。Gahan等[22]對菌絲表面、 菌絲際及土體土壤中微生物群落進行研究，發現菌絲表面和菌絲際的可培養細菌的豐富度和群落結構均與土體不同，且菌絲際與菌絲表面的細菌群落不存在顯著差異，因此可以收集菌絲，通過測定菌絲表面的微生物群落來反映菌絲際的變化，但這種方法縮小了菌絲際的范圍，在未來的研究中需要進一步改進。

Rhizophagusirregularis是一種分布廣泛的AM真菌，在我國北方農田土壤中普遍存在[28-29]。本研究將從菌絲表面分離出的解磷細菌接種到Rhizophagusirregularis的菌絲周圍，結果發現80%經純化的解磷細菌可以在其表面定殖，另有20%的細菌在回接試驗中未檢測到，其可能原因是本回接試驗的條件與田間條件存在差異，例如農田中侵染于玉米根系中的AM真菌種類很多[29]，而我們的回接定殖試驗只接種了Rhizophagusirregularis一種AM真菌，不同的AM真菌對不同種類細菌的定殖是具有選擇性的[30]，上述原因有可能導致只有80%解磷細菌在AM真菌菌絲表面定殖，本試驗尚不能肯定其余的20%的解磷細菌是否可以在AM真菌根外菌絲表面定殖，這需要更精細的試驗進一步的驗證。在回接細菌菌株鑒定時發現貪銅菌屬(Cupriavidus)存在，該試驗在溫室進行，空氣中不可避免地存在各種微生物，另外本試驗所用的Rhizophagusirregularis菌劑也可能是該菌株的來源。這一結果給我們的啟示是當所有細菌菌株接種到AM真菌菌絲周圍時，會表現出不同的定殖能力，因此它們參與菌絲際土壤磷的周轉和磷在植物-AM真菌系統中的利用效率也會表現出不同的作用。

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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

國家自然科學基金 (31501831,U1403285)和教育部博士點基金(20120008130001)資助。

柴小粉(1990—)， 女， 河南商丘人， 碩士研究生， 主要從事根際營養與調控研究。 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