柴小粉， 張 林， 田芷源， 王 菲， 馮 固

(中國(guó)農(nóng)業(yè)大學(xué)資源與環(huán)境學(xué)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(xì)菌解磷功能鑒定

柴小粉， 張 林， 田芷源， 王 菲， 馮 固*

(中國(guó)農(nóng)業(yè)大學(xué)資源與環(huán)境學(xué)院， 北京 100193)

玉米; AM真菌; 植酸鈣; 解磷細(xì)菌; 菌絲際

叢枝菌根(arbuscular mycorrhizal, AM)真菌是一類(lèi)重要的土壤微生物，能夠與80%以上的陸生植物形成共生關(guān)系[1]，產(chǎn)生大量的根外菌絲來(lái)增強(qiáng)植物對(duì)移動(dòng)性差的土壤養(yǎng)分(如磷、 鋅)的吸收[2]，提高植物對(duì)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(jìn)土壤團(tuán)聚體的形成[3]。此外，AM真菌的根外菌絲能夠分泌質(zhì)子、 糖、 有機(jī)酸陰離子[4]，改變菌絲周?chē)寥赖睦砘再|(zhì)，形成“菌絲際(hyphosphere)”[5]，為土壤微生物提供定殖場(chǎng)所[6-9]。解磷細(xì)菌也是土壤中一類(lèi)重要的功能微生物，數(shù)量可以占到可培養(yǎng)細(xì)菌的40%[10-11]，通過(guò)釋放質(zhì)子、 有機(jī)酸和磷酸酶等代謝產(chǎn)物來(lái)活化土壤難溶解的有機(jī)態(tài)或無(wú)機(jī)態(tài)的磷酸鹽。許多研究已經(jīng)證明，定殖于作物根際的解磷細(xì)菌能夠幫助植物吸收難溶性磷，改善植物的磷營(yíng)養(yǎng)[12]。

AM真菌與解磷細(xì)菌的互作一直是生態(tài)學(xué)和植物營(yíng)養(yǎng)學(xué)的研究熱點(diǎn)。大量研究工作表明，在盆栽條件下同時(shí)接種AM真菌和解磷細(xì)菌能夠顯著提高植物對(duì)磷的吸收，促進(jìn)植物生長(zhǎng)[13-14]。但這些研究都是在根際范圍內(nèi)進(jìn)行，未排除根系對(duì)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(xì)菌的研究發(fā)現(xiàn)AM真菌Rhizophagusirregularis(原名Glomusintraradices)與解磷細(xì)菌互作可以提高土壤中磷酸酶的活性、 促進(jìn)土壤中植酸磷的礦化[15-16]。而Tisserant等[17]通過(guò)對(duì)AM真菌模式菌株Rhizophagusirregularis(DAOM 197198)的轉(zhuǎn)錄組分析發(fā)現(xiàn)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(bào)道的AM真菌促進(jìn)有機(jī)磷礦化是通過(guò)AM真菌菌絲刺激解磷細(xì)菌的活性實(shí)現(xiàn)的[18-19]。然而，AM真菌與解磷細(xì)菌互作的研究大多是在室內(nèi)模擬條件下進(jìn)行的，在真實(shí)的生態(tài)系統(tǒng)(如農(nóng)田生態(tài)系統(tǒng))中的研究尚不多見(jiàn)。

本試驗(yàn)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(jī)磷的細(xì)菌定殖。然后分離純化出這些解磷細(xì)菌，對(duì)它們的種類(lèi)和解磷能力進(jìn)行鑒定，從而揭示菌絲表面解磷細(xì)菌的定殖過(guò)程和細(xì)菌種類(lèi)，對(duì)于理解菌絲際微域土壤難溶性磷的周轉(zhuǎn)和生物地球化學(xué)循環(huán)過(guò)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(yǎng)解磷細(xì)菌的分離與純化

1.3菌株鑒定

對(duì)礦化植酸鈣的細(xì)菌進(jìn)行16S rDNA測(cè)序分析，來(lái)確定這些菌株的種類(lèi)。首先使用細(xì)菌基因組DNA提取試劑盒(TIANGEN DP302)提取細(xì)菌DNA，方法為在LB培養(yǎng)液中活化細(xì)菌，吸取菌液進(jìn)行離心(11500×g，1 min)，取下部沉淀，按試劑盒說(shuō)明提取細(xì)菌DNA，所得DNA用NanoDrop 2000檢測(cè)其濃度及純度，-20℃保存。其次PCR擴(kuò)增采用細(xì)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(yīng)在Biomatro T3擴(kuò)增儀上進(jìn)行，反應(yīng)體系為50 μL。PCR 原液組成為: 10 ×PCR擴(kuò)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(yīng)程序?yàn)?4℃預(yù)變性5 min; 94℃變性1 min，58℃退火1 min，72℃延伸90 s，循環(huán)30次; 最后72℃延伸7 min。然后將PCR產(chǎn)物交由華大基因進(jìn)行測(cè)序分析，所得序列提交到Genbank中利用Blast程序(www.ncbi.nlm.nih.gov/BLAST/)進(jìn)行相似性比對(duì)，確定細(xì)菌的種類(lèi)，最后用MEGA 軟件(版本4.0)構(gòu)建系統(tǒng)進(jìn)化樹(shù)。

1.4解磷細(xì)菌解磷能力的測(cè)定

將上述步驟篩選的各株解磷細(xì)菌分別接入LB培養(yǎng)液中，37℃培養(yǎng)16 h后，用分光光度計(jì)調(diào)整細(xì)菌濃度至OD值(λ=600 nm)為0.6，然后向無(wú)菌的蒙金娜液體有機(jī)磷培養(yǎng)基(30 mL)中加入2 mL細(xì)菌懸液，以不接菌液的處理作為對(duì)照，從而確定每株解磷細(xì)菌礦化有機(jī)磷的能力，每個(gè)菌株重復(fù)3次，37℃振蕩(180 r/min)培養(yǎng)48 h后，取2 mL培養(yǎng)液進(jìn)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(cè)定無(wú)機(jī)磷的濃度[20]，并用pH計(jì)(DENVER, UB-7)測(cè)定培養(yǎng)液pH值。

1.5菌絲際解磷細(xì)菌回接試驗(yàn)

供試土壤是2010年5月取自山東省泰安市低磷農(nóng)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(jī)質(zhì)7.27 g/kg、 有效氮7.20 mg/kg、 pH 6.40，過(guò)2 mm篩后，送至北京鴻儀四方輻射技術(shù)有限公司(Beijing Hongyisifang Radiation Technology Co, Ltd)進(jìn)行γ射線(xiàn)(輻照強(qiáng)度: 10 kGy60Co γ-ray)滅菌。供試裝置為隔網(wǎng)分室根盒培養(yǎng)裝置(圖2)，根室種植玉米，設(shè)置接種AM真菌與不接種AM真菌(Rhizophagusirregularis)兩個(gè)處理，每個(gè)處理重復(fù)三次。播種一個(gè)月后，在菌絲室中接入篩選出的解磷細(xì)菌的混合菌懸液，繼續(xù)培養(yǎng)一個(gè)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(jī)磷選擇性培養(yǎng)基上進(jìn)行篩選。后續(xù)的分離和鑒定過(guò)程同1.2和1.3。

圖2　兩室培養(yǎng)系統(tǒng)示意圖Fig.2　Schematic diagram of two compartments cultivation system

1.6數(shù)據(jù)處理

試驗(yàn)數(shù)據(jù)均采用Excel進(jìn)行整理，利用SPSS軟件(16.0版本，SPSS Inc.)進(jìn)行數(shù)據(jù)分析。對(duì)無(wú)機(jī)磷濃度數(shù)據(jù)先進(jìn)行方差同質(zhì)性檢驗(yàn)(Levene′s檢驗(yàn))，再進(jìn)行單因素方差分析和LSD法多重比較(P< 0.05)。對(duì)無(wú)機(jī)磷濃度和培養(yǎng)液pH數(shù)據(jù)進(jìn)行相關(guān)性分析。利用MEGA 軟件(版本4.0)構(gòu)建系統(tǒng)進(jìn)化樹(shù)。

2　結(jié)果與分析

2.1解磷細(xì)菌的鑒定

本研究對(duì)分離篩選出的29株解磷細(xì)菌進(jìn)行了16S rRNA基因系統(tǒng)發(fā)育樹(shù)的構(gòu)建(圖3)。結(jié)果表明29株解磷細(xì)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(yōu)勢(shì)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(xì)菌的分離和解磷能力的測(cè)定

2.3菌絲際解磷細(xì)菌回接分析

圖3　菌絲際分離篩選出的解磷細(xì)菌16S rRNA基因系統(tǒng)發(fā)育樹(shù)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(biāo)注為本研究中獲得的序列The sequences obtained in this study are shown in bold，HM+數(shù)字為解磷細(xì)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(yǎng)系統(tǒng)進(jìn)行解磷細(xì)菌回接試驗(yàn)。從菌絲際的土壤中挑取菌絲，通過(guò)選擇性培養(yǎng)基篩選后得到47株解磷細(xì)菌。將這些細(xì)菌提取DNA進(jìn)行16S rDNA測(cè)序分析，所得序列在NCBI數(shù)據(jù)庫(kù)上進(jìn)行比對(duì)，發(fā)現(xiàn)這47株解磷細(xì)菌分別屬于以下5個(gè)類(lèi)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(yōu)勢(shì)，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(xì)菌能夠在Rhizophagusirregularis的根外菌絲上定殖。

3　討論

AM真菌菌絲表面細(xì)菌的定殖是近十年來(lái)菌根研究的一個(gè)熱點(diǎn)方向[7,9,21-22]。Artursson等[6]報(bào)道過(guò)純培養(yǎng)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(yǎng)的解磷細(xì)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(jīng)從菌絲際分離出[23]。但這些研究是在室內(nèi)的菌絲離體培養(yǎng)條件下進(jìn)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(xì)菌種類(lèi)目前未見(jiàn)報(bào)道。本研究從河北曲周長(zhǎng)期施用堆肥處理的玉米根系周?chē)占疉M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(xì)菌，16S rDNA分析表明這些解磷細(xì)菌分屬五個(gè)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(yōu)勢(shì)菌。土壤中99%以上的微生物目前不能被培養(yǎng)技術(shù)分離[24]，且解磷細(xì)菌菌株在純化過(guò)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(xì)菌只是其中一部分，不能完全反映菌絲表面定殖的解磷細(xì)菌群落。

土壤中不同屬的解磷細(xì)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(qiáng)的細(xì)菌類(lèi)群[25]，它們的解磷功能得到了廣泛關(guān)注和研究。本研究同樣發(fā)現(xiàn)假單胞菌屬的細(xì)菌解磷能力相對(duì)較強(qiáng)，活化出的有效磷濃度均在15 mg/L以上(圖4)。

表1　平板法測(cè)量解磷細(xì)菌溶磷圈直徑與菌落直徑比值

圖4　解磷細(xì)菌活化出的無(wú)機(jī)磷濃度Fig.4　Concentrations of inorganic phosphorus mineralized by different phytate mineralizing bacteria strains

圖5　培養(yǎng)液中無(wú)機(jī)磷濃度與pH相關(guān)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(jié)果發(fā)現(xiàn)有機(jī)磷礦化的量與培養(yǎng)基的pH呈顯著的負(fù)相關(guān)性，這說(shuō)明解磷細(xì)菌可通過(guò)分泌有機(jī)酸等代謝產(chǎn)物提高植酸鈣的底物有效性，從而促進(jìn)其礦化[26]。對(duì)于篩選出的可培養(yǎng)解磷細(xì)菌的解磷能力的評(píng)價(jià)是在純培養(yǎng)條件下進(jìn)行的，但是在自然生態(tài)系統(tǒng)中，植物-AM真菌-細(xì)菌三者之間存在著相互作用[27]。研究表明，菌根際中細(xì)菌活化土壤養(yǎng)分(尤其是磷)的能力會(huì)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(guò)類(lèi)似根際的取樣方法進(jìn)行研究。Gahan等[22]對(duì)菌絲表面、 菌絲際及土體土壤中微生物群落進(jìn)行研究，發(fā)現(xiàn)菌絲表面和菌絲際的可培養(yǎng)細(xì)菌的豐富度和群落結(jié)構(gòu)均與土體不同，且菌絲際與菌絲表面的細(xì)菌群落不存在顯著差異，因此可以收集菌絲，通過(guò)測(cè)定菌絲表面的微生物群落來(lái)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(lái)的研究中需要進(jìn)一步改進(jìn)。

Rhizophagusirregularis是一種分布廣泛的AM真菌，在我國(guó)北方農(nóng)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(xì)菌接種到Rhizophagusirregularis的菌絲周?chē)?，結(jié)果發(fā)現(xiàn)80%經(jīng)純化的解磷細(xì)菌可以在其表面定殖，另有20%的細(xì)菌在回接試驗(yàn)中未檢測(cè)到，其可能原因是本回接試驗(yàn)的條件與田間條件存在差異，例如農(nóng)田中侵染于玉米根系中的AM真菌種類(lèi)很多[29]，而我們的回接定殖試驗(yàn)只接種了Rhizophagusirregularis一種AM真菌，不同的AM真菌對(duì)不同種類(lèi)細(xì)菌的定殖是具有選擇性的[30]，上述原因有可能導(dǎo)致只有80%解磷細(xì)菌在AM真菌菌絲表面定殖，本試驗(yàn)尚不能肯定其余的20%的解磷細(xì)菌是否可以在AM真菌根外菌絲表面定殖，這需要更精細(xì)的試驗(yàn)進(jìn)一步的驗(yàn)證。在回接細(xì)菌菌株鑒定時(shí)發(fā)現(xiàn)貪銅菌屬(Cupriavidus)存在，該試驗(yàn)在溫室進(jìn)行，空氣中不可避免地存在各種微生物，另外本試驗(yàn)所用的Rhizophagusirregularis菌劑也可能是該菌株的來(lái)源。這一結(jié)果給我們的啟示是當(dāng)所有細(xì)菌菌株接種到AM真菌菌絲周?chē)鷷r(shí)，會(huì)表現(xiàn)出不同的定殖能力，因此它們參與菌絲際土壤磷的周轉(zhuǎn)和磷在植物-AM真菌系統(tǒng)中的利用效率也會(huì)表現(xiàn)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(guó)家自然科學(xué)基金 (31501831,U1403285)和教育部博士點(diǎn)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(yíng)養(yǎng)與調(diào)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