CN113337445B - 一种地单胞菌rf4及其应用 - Google Patents
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Abstract
本发明公开了一种地单胞菌(Geomonas niftrogeniifigens)RF4及其应用。属于微生物技术领域。地单胞菌RF4为严格厌氧、革兰氏阴性、运动、周身鞭毛。菌落为红色、圆形。接触酶和氧化酶反应为阴性。菌株RF4具有产碱性磷酸盐酶、酸性磷酸酶和萘酚‑AS‑BI‑磷酸水解酶的能力。首次发现地单胞菌同时具有铁还原和固氮菌功能。在农业生产中驱动水稻促长、降低氮肥施用、减少环境污染，具有良好的应用前景。
Description
技术领域
本发明涉及微生物技术领域，更具体的说是涉及一种地单胞菌RF4及其应用。
背景技术
水稻是我国的第一大粮食作物，氮是水稻产量的主要限制因子。生物固氮和化学氮肥是水稻生长主要氮源，但化学氮肥的大量施用会带来一定程度的环境污染。生物固氮是水稻田的一种重要过程，能维持水稻田淹水状态下氮素的平衡，减少化学肥料施用，降低环境污染。
长期化学施肥，致使水稻土壤微生物群落结构发生改变，从而降低了水稻田固氮菌多样性。此外，还发现关键固氮微生物菌群在长期不施肥的环境下呈系统发育聚集状态，而施肥条件下则呈现随机性状态，导致其生物固氮功能逐渐退化。
研究表明，淹水稻田优势固氮细菌为具有铁还原作用的地杆菌科(Geobacteraceae)成员，如地杆菌属(Geobacter)和厌氧粘细菌属(Anaeromyxobacter)，而且发现铁还原反应与固氮呈正相关。增加水稻田铁还原能力，可提高固氮菌的固氮活性。由此可知，铁还原固氮菌对淹水稻田的生物固氮起着重要作用。
地单胞菌属(Geomonas)是地杆菌科新发现的一类严格厌氧、革兰氏阴性细菌。目前，地单胞菌属仅包含9个有效种，关于地单胞菌属资源研究报道甚少。全球可培养微生物约为1％，而厌氧微生物纯培养物占全球可培养微生物不到0.1％，大部分厌氧微生物处于未培养状态。
因此，挖掘具有铁还原和固氮菌功能的地单胞菌新种是本领域技术人员亟需解决的问题。
发明内容
有鉴于此，本发明提供了一种地单胞菌RF4及其应用。从未施肥的水稻土壤中分离获得了1株地单胞菌RF4，经多相分类(生理生化、化学和基因型特性)分析证明菌株RF4为地单胞菌属的新种，命名为固氮地单胞菌(Geomonas niftrogeniifigens)，首次发现该菌株具有较好的固氮特性。
为了实现上述目的，本发明采用如下技术方案：
分类学名称：Geomonas niftrogeniifigens；保藏中心：广东省微生物菌种保藏中心，地址：广州市先烈中路100号大院59号楼5楼，保藏时间2021年06月28日，保藏编号为GDMCC No：61761。
一种地单胞菌(Geomonas niftrogeniifigens)RF4，保藏编号为GDMCC No：61761。
本发明还提供了一种地单胞菌RF4在铁还原和固氮中的应用，保藏编号为GDMCCNo：61761。
本发明还提供了上述的地单胞菌RF4在农业生产中的应用。
本发明还提供了上述的地单胞菌RF4在制备菌肥中的应用。
本发明还提供了上述的地单胞菌RF4在促进水稻增产中的应用。
本发明还提供了上述的地单胞菌RF4在制备碱性磷酸盐酶、酸性磷酸酶和萘酚-AS-BI-磷酸水解酶中的应用。
本发明还提供了一种微生物菌剂，包括固氮地单胞菌RF4，保藏编号为GDMCC No：61761。
经由上述的技术方案可知，与现有技术相比，本发明公开提供了一种同时具有铁还原和固氮菌功能的地单胞菌RF4，取得的技术效果为地单胞菌RF4为严格厌氧、革兰氏阴性、运动、周身鞭毛。菌落为红色、圆形。接触酶和氧化酶反应为阴性。生长条件为10～40℃(最佳30～33℃)、pH 5.5～8.5(最佳pH 6.0～7.0)，0～0.7％(w/v)NaCl(最佳0.2～0.4％)。Fe(III)–NTA为电子受体，乙酸钠、甲苯、丁醇、甲醇、乳酸钠、苹果酸钠、丙酸钠、琥珀酸盐和富马酸钠能作为电子供体，但苯酚和苯甲醛不能作为电子供体。乙酸钠作为电子供体时，柠檬酸铁、Fe(III)-NTA、水铁矿、富马酸钠和硝酸钠可以作为电子受体，而苹果酸钠、氧气、硫、MnO2不能。在缺乏电子受体时，生长缓慢，富马酸钠、乙醇、甘油和乳酸钠作为底物时可观测微弱生长，而苹果酸盐和丙酮酸钠不行。菌株RF4具有产碱性磷酸盐酶、酸性磷酸酶和萘芬-AS-BI-磷酸水解酶的能力。乙酸钠作为电子供体，水铁矿作为电子受体，大量的Fe(III)可被还原为Fe(II)。
RF4与最相近模式菌株Geomonas terrae Red111T的16S rRNA基因相似性为98.5％，与最相近模式菌株的ANI和dDDH分别为81.4％和25.0％，主要脂肪酸为C14:0，C15:1ω6c，C16:0，iso-C15:0，和Summed Feature 3，主要呼吸醌组分为MK-8。基因组DNA G+C含量为61.7％。表明菌株RF4为地单胞菌属的新种。基因组含有固氮酶核心基因元件nifHDK，PCR扩增获得了nifH基因片段，乙炔还原法测定表明具有较高的固氮酶活性，将氮气还原为作物可利用的氮肥。在农业生产中驱动水稻促长，减少化学氮肥施用、降低环境污染等方面具有良好的应用前景。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据提供的附图获得其他的附图。
图1附图为本发明提供的地单胞菌RF4生长曲线示意图。
图2附图为本发明提供的基于16S rRNA基因的系统发育树图。
图3附图为本发明提供的铁还原能力示意图。
图4附图为本发明提供的细胞色素光谱图。
图5附图为本发明提供的PCR扩增结果图。
图6附图为本发明提供的固氮酶活性示意图。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
本发明实施例公开了一种地单胞菌RF4及其应用。
实施例1
样本富集培养
称取10g水稻土至90mL无菌MFM液体培养基，N2：CO2(V/V，80：20)除氧0.5h形成厌氧环境，铝盖封口后30℃静止富集培养2周。
MFM培养基(L-1)：2.0g KHCO3，0.02g MgSO4·7H2O，0.3g KH2PO4，1.0g NH4Cl，0.1gMgCl2·6H2O，0.08g CaCl2·2H2O，0.6g NaCl，9.52g HEPES，10mL矿物质溶液[(L-1)：NTATrisodium Salt(Free acid)1.50g，MgSO43.00g，MnSO4·H2O 0.50g，NaCl 1.00g，FeSO4·7H2O 0.10g，CaCl2·2H2O 0.10g，CoCl2·6H2O 0.10g，ZnCl20.13 g，CuSO4·5H2O 0.01g，AlK(SO4)2·12H2O 0.01g，H3BO30.01 g，NaMoO4·2H2O 0.09g]，10mL维生素混合液[(L-1)：生物素0.002g，泛酸0.005g，维生素B12 0.0001g，对氨基苯甲酸0.005g，硫辛酸(α-)0.005g，烟酸0.005g，硫胺素0.005g，核黄素0.005g，盐酸吡哆醇0.01g，叶酸0.002g]，pH 6.8。
菌株的分离纯化
取100μL梯度稀释的新鲜土壤悬浮液和富集液，涂布于改良的R2A(添加20mM富马酸钠)平板上，30℃严格厌氧培养10d。挑取平板上红色菌落，采用连续划线法进行纯化，直至获得单个纯培养物。所有纯化的菌株采用10％DMSO保菌液于-80℃保藏。所有操作均是在厌氧手套箱工作台中进行，以保证厌氧环境。在改良R2A平板上培养3天后。菌株RF4的菌落形状为圆形，边缘光滑，表面光滑，菌落红色，菌落直径在0.5～1mm。
固体R2A培养基成分(g/L)：0.5g酵母浸出粉，0.5g蛋白胨，0.5g酪蛋白水解物，0.5g葡萄糖，0.5g可溶性淀粉，0.3g磷酸二氢钾，0.024g无水硫酸镁，0.3g丙酮酸钠，15.0g琼脂，蒸馏水1000mL，pH7.0。
生物学特性：
生长曲线
用注射器准确吸取2mL(接菌量1％)的Geomonas菌液注入装有100mL R2A+20mM富马酸钠液体培养基的500mL厌氧瓶中，置于30℃摇床振荡培养。当菌液注入到厌氧瓶中记为0时，于未接菌、0、12、18、20、24、26、28、30、32、34、36、38、40、42、44、48、52h时取样2mL，将取出的样品测定OD600值。设置三个重复。生长量的测定：将未接菌取样倒入比色皿中，选用600nm波长分光光度计调0，作为空白对照，对不同时间取出的样品依次进行测定，结果参见图1。由图1知，菌株RF4培养33～34h达到对数期。
革兰氏染色：于载玻片上滴加3％的KOH溶液一滴，用接菌环挑取一环待测菌落，于该玻片3％的KOH溶液混匀，经过30～60s，细菌在KOH溶液中的混悬物变得粘稠以至形成胶冻状，此为革兰氏阴性菌，如果呈现出均匀混悬，不出现胶状物者为革兰氏阳性菌。
菌株RF4为革兰氏阴性细菌。
生理生化特征
温度适应性：配制改良R2A培养基(1.5％琼脂)平板，用接菌环蘸取菌液，用连续划线法接种与平板上，放置于6、10、13、16、20、25、30、33、37、40和42℃，每个温度设置3个平行，一周后，测量菌落的直径。
pH适应性：配置pH(5.0～9.0，梯度为0.5)改良R2A培养基+1.5％琼脂平板，用接菌环蘸取菌液，用连续划线法划线与平板上，每个pH做3个平行，一周后，测量菌落的直径。
NaCl浓度适应性：配置NaCl浓度(0～5％，[w/v]，梯度为0.5％)改良R2A培养基+1.5％琼脂平板，用接菌环蘸取菌液，用连续划线法划线与平板上，每个盐浓度做3个平行，一周后，测量菌落的直径。
新菌株RF4生长温度范围10～40℃(最佳30～33℃)，pH 5.5～8.5(最佳pH 6.0～7.0)，0～0.7％(w/v)NaCl(最佳0.2～0.4％)。
需氧性：将菌株划线于改良R2A平板上，接种的R2A平板置于30℃恒温培养箱好氧培养两周。观察发现菌株RF4未在平板上生长形成菌落，表明氧气条件下菌株RF4不能进行生长。
运动能力：配置改良R2A半固体培养基(0.5％的琼脂)，用接菌环刮取平板上的菌落，插入到半固体培养基内，三天后观测菌株的生长状况及运动能力。观察发现接种的穿刺线上生长外，在穿刺线的两侧均可见羽毛状或云雾状浑浊生长表明菌株RF4具有一定的运动能力。
抗生素敏感性：吸取200μL对数期菌液在改良R2A培养基上进行涂布，夹取抗生素药物药敏纸片放置于平板上，30℃培养3d，观察抑菌圈有无判断菌株对抗生素敏感性。
结果判定：菌株RF4对头孢唑林(30μg/片)、青霉素(10μg/片)、氨苄西林(10μg/片)、苯唑西林(1μg/片)、新霉素(30μg/片)、庆大霉素(10μg/片)、链霉素(10μg/片)、万古霉素(30μg/片)和阿奇霉素(15μg/片)不敏感，对克林霉素(2μg/片)、氯霉素(30μg/片)、丁胺卡那(30μg/片)、卡那霉素(30μg/片)、利福平(5μg/片)、多粘菌素B(30μg/片)、多西环素(30μg/片)、四环素(30μg/片)和红霉素(15μg/片)敏感。
利用API ZYM试剂条检测新种菌株RF4的产酶特性(操作步骤见说明书即可)。
结果表明，菌株RF4具有产碱性磷酸盐酶、酸性磷酸酶和萘酚-AS-BI-磷酸水解酶的能力，不产酶脂(C4)、类脂酯酶(C8)、类脂酶(C14)、白氨酸芳胺酶、缬氨酸芳胺酶、胱氨酸芳胺酶、胰蛋白酶、胰凝乳蛋白酶、α-半乳糖苷酶、β-半乳糖苷酶、β-糖醛酸苷酶、α-葡萄糖苷酶、β-葡萄糖苷酶、N-乙酰-葡萄糖胺酶、α-甘露糖苷酶和β-岩藻糖苷酶。
电子供体与电子受体
所有的电子受体试验中，都以10mM的乙酸钠作电子供体；所有的电子供体试验中，都以10mM的Fe(III)-NTA作电子受体。菌株RF4接种至无氧的无菌MFM培养基30℃下培养，测定生物量和培养基颜色变化进行判断电子受体和供体谱。
Fe(III)–NTA为电子受体，乙酸钠、甲苯、丁醇、甲醇、乳酸钠、苹果酸钠、丙酸钠、琥珀酸盐和富马酸钠能作为电子供体，但苯酚和苯甲醛不能作为电子供体。乙酸钠作为电子供体时，柠檬酸铁、Fe(III)-NTA、水铁矿、富马酸钠和硝酸钠可以作为电子受体，而苹果酸钠、氧气、硫、MnO2不能。
发酵生长
不加电子受体的情况下，检测富马酸钠、乳酸钠、甘油、丙酮酸、苹果酸和乙醇发酵生长情况。
在缺乏电子受体时，生长缓慢，富马酸钠、乙醇、甘油和乳酸钠作为底物时可观测微弱生长，而苹果酸盐和丙酮酸钠不行。
实施例2
16S rRNA基因鉴定
通过细菌16S rRNA基因通用引物27F(5′-GAG TTTGAT CCT GGC TCA G-3′)和1492R(5′-ACG GCT ACCTTG TTA CGA CTT-3′)对分离菌株进行扩增。
PCR反应程序：94℃预变性5min；94℃变性30s，55℃退火60s，72℃延伸90s，共30个循环；最后72℃延伸10min。取5μL PCR产物，点样于1％的琼脂糖凝胶中，以100bp Marker作为标准分子量，100V电压，电泳30min，用凝胶成像系统观察电泳结果。检测出有条带的菌株PCR产物送至生工生物工程(上海)股份有限公司进行测序。采用ContigExpress软件对测序获得16S rRNA基因序列进行校对，去除头尾两端杂乱碱基，将获得的有效序列提交至EZBioCloud(https：//www.ezbiocloud.net/)和NCBI(https：//www.ncbi.nlm.nih.gov/)上进行序列比对分析。若比对结果表明16S rRNA基因相似性高于98.65％，则初步判断认定其与最相近菌株处于同一种水平分类地位；反之，则判断为潜在新分类单元。
结果表明：RF4与最相近模式菌株Geomonas terrae Red111T的16S rRNA基因相似性为98.5％，低于原核生物种界定阈值98.65％，为地单胞菌属的1个潜在新种。
系统发育分析
基于EZBioCloud和NCBI数据库比对结果，下载数据库与分离菌株相近的模式菌株的16S rRNA基因序列，利用MEGA X软件，采用Kimura 2-parameter法计算进化距离、构建最大自然法(Maximum Likelihood)系统进化树(参见图2)，进行Bootstrap值为1000次重复验证。
基因组ANI和dDDH分析
使用GGDC在线计算软件(Genome-to-Genome Distance Calculator)估算了数字DNA-DNA杂交(dDDH)，利用ANI Calculator计算细菌与其模式菌株间的平均核苷酸一致性(ANI)。
RF4与最相近模式菌株Geomonas terrae Red111TANI和dDDH分别为81.4％和25.0％，低于原核生物种界定阈值96％和70％。因此，表明RF4是地单胞菌属的1个新种。RF4的基因组大小为4829642bp，DNA G+C含量为61.7％。
基因组功能分析，发现RF4具有固氮酶核心基因nifHDK，推测该菌株具有固氮功能。
保藏信息：分类学名称：Geomonas niftrogeniifigens；保藏中心：广东省微生物菌种保藏中心，地址：广州市先烈中路100号大院59号楼5楼；保藏时间2021年06月28日，保藏编号为GDMCC No：61761。
实施例3
脂肪酸检测
①获菌：挑取约40mg对数期(培养3d)的菌体放入带有螺旋盖的试管中(规格13mm×100mm)。
②皂化：在试管中加入1.0mL的皂化试剂，拧紧盖子，振荡器振荡试管5～10s，沸水浴5min，取出并继续振荡5～10s，再次拧紧盖子，继续水浴25min，移出试管，室温冷却。
③甲基化：往试管中加入2.0mL的甲基化试剂，拧紧盖子，振荡器振荡5～10s，进行80℃水浴10min，移出试管并快速用自来水冲凉冷却至室温。
④萃取：再加入1.25mL的萃取试剂，拧紧盖子，快速振荡10min，打开管盖，用移液管吸出试管的下层似水相并弃之。
⑤洗涤：往试管中加入3.0mL的洗涤试剂，拧紧盖子，快速振荡5min，用注射器吸取出约2/3体积的上层有机体到GC小瓶中，待检。
⑥检测：分析仪器采用美国Agilent 7890N气相色谱系统进行检测。
检测程序：设置汽化室的温度250℃，检测器的温度300℃，载气氢气的流速为2mL·min-1，尾吹气氮气的流速为30mL·min-1，进样分流比100：1，柱前压为68.95kPa；色谱柱采用二阶程序升温，从170℃开始升温，每分钟升温5℃，升到260℃时，再每分钟升温40℃，升到310℃时，保持90s；进样量1μL。
分析软件：美国MIDI公司开发的基于微生物细胞脂肪酸成分鉴定的全自动微生物鉴定系统Sherlock MIS4.5(Microbial Identification System)和LGS4.5(LibraryGeneration Software)。
检测结果表明：RF4的主要脂肪酸C14：0，C15：1ω6c，C16：0，iso-C15：0，和SummedFeature 3，与地单胞菌属的脂肪酸种类一致。
醌组分分析
1)收集菌体
将培养至对数期的菌体，进行离心收集并用蒸馏水洗涤2～3次，离心后进行真空冷冻干燥，备用。
2)提取与纯化醌组分
①称取约100mg冻干的菌体，加入氯仿：甲醇(2:l，v/v)溶液40mL，置于暗处进行磁力搅拌10h左右，并于暗处用滤纸过滤，收集菌体。
②用减压旋转蒸发仪对菌体进行40℃减压蒸馏至干燥。
③再用1～2mL的丙酮重新溶解干燥物，以长条状将样品点于GF 254硅胶板上。
④以甲苯作为展层剂，展层时间约20min，然后取出并进行风干，然后打开紫外灯(254nm)观察。Rf＝0.8，在绿色萤光背景下呈暗褐色的带即为甲基萘醌的位置。
⑤刮下Rf＝0.8的带，用1mL的丙酮溶解，然后用细菌过滤器过滤除去硅胶，收集滤液，即得到甲基萘醌的丙酮溶液，将其置于黑暗处4℃保存。
3)高压液相色谱(HPLC)测定醌组分
采用反相高压液相色谱分析法测定甲基萘醌。高效液相色谱仪为Agilent 1100，反相高压液相柱为十八烷基硅烷，流动相为甲醇(色谱纯)：异丙醇(色谱纯)＝67:33(v/v)，流速为1mL·min-1，柱温为40℃，同时于270nm处进行紫外检测，使用250nm光谱扫描。
主要醌组分为MK-8，与地单胞菌属的醌组分类型相同。
结合实施例2和3，表明菌株RF4为地单胞菌属的一个新种，命名为Geomonasnitrogeniifigens。
实施例4
铁还原能力的测定
标准曲线：制定标准曲线y＝2.0465x-0.0028，R2＝1。
厌氧状态下，将RF4种子发酵液接入装有10mL灭菌的20mM水铁矿培养基的厌氧试管中，设置3个平行，未接菌的作为空白对照。
Fe(Ⅱ)的测定：每隔48h取样0.1mL加入到装有0.9mL的0.5M HCl的离心管中，562nm波长下测定吸光度。
参见图3，RF4具有较高的铁还原能力，将水铁矿中大部分的Fe(III)还原为Fe(II)。
细胞色素测定
分离菌株培养于改良R2A培养基中生长3d。取5mL培养菌液离心后，9mL的20mMPIPES缓冲液(pH 7)与9mL的2mM连二亚硫酸钠的20mM PIPES缓冲液(pH 7)进行悬浮。还原6个小时后，细胞的连二亚硫酸盐还原负空气氧化差异图谱通过UV-2600(SHIMADZU,日本)分光光度计400～800nm波长扫描获得差异图谱。
参见图4，菌株RF4在波长425、523和524nm检测细胞色素吸收峰。
固氮能力：
固氮基因nifH扩增：采用nifH基因Pol-F(5′-TGC GAY CCS AAR GCB GAC TC-3′)和Pol-R(5′-ATS GCC ATC ATY TCR CCG GA-3′)对分离菌株进行PCR扩增，判断是否具有固氮功能。
PCR反应程序：95℃预变性5min；95℃变性30s；60℃退火45s；72℃延伸450s；40个循环，最后保持72℃延仲45s。取5μL PCR产物，点样于1％的琼脂糖凝胶中，以100bp Marker作为标准分子量，100V电压，电泳30min，用凝胶成像系统观察电泳结果。
利用固氮基因nifH对分离菌RF4进行PCR扩增，PCR扩增结果表明，扩增得到长度约为360bp的nifH基因片段(图5)，说明菌株RF4具有固氮能力。
ARA测定固氮酶活性：乙炔还原法(ARA法)来测固氮酶的活性。将RF4菌株用改良的R2A培养基培养至对数生长期时，转移至无菌厌氧离心管中，6000rpm离心10min，去上清液。用30mL改良无氮培养基漂洗后，转移至用He/C2H2(90∶10[vol/vol])交换的厌氧瓶中(先将厌氧瓶与厌氧橡胶塞灭菌，盖上塞子后抽真空，再充入混合气)，同样，用30mL改良无氮培养基漂洗后，转移至用纯He交换的厌氧瓶中做阴性对照。培养3d后，用气象色谱检测还原乙烯的含量。
计数：将在无氮培养基培养的菌液吸取100μL在改良的R2A平板上涂布，记录平板上菌落的个数。
参见图6，乙炔还原法表明，菌株RF4具有较高的固氮酶活性，达(3.2±0.2)×10- 6C2H4/h/cell。
实施例5
菌剂的制备
无菌条件下，菌株RF4的新鲜菌体以1％接种量接种至除氧后无菌的改良R2A液体培养基，30℃静止培养3～5d，即可制备获得固氮地单胞菌RF4的菌剂。
液体R2A培养基成分(g/L)：0.5g酵母浸出粉，0.5g蛋白胨，0.5g酪蛋白水解物，0.5g葡萄糖，0.5g可溶性淀粉，0.3g磷酸二氢钾，0.024g无水硫酸镁，0.3g丙酮酸钠，蒸馏水1000mL，pH7.0。
液体R2A培养基利用混合气N2:CO2(80:20，vol/vol)曝气除氧0.5h形成厌氧环境，铝盖封口后，121℃高压灭菌20min。
本说明书中各个实施例采用递进的方式描述，每个实施例重点说明的都是与其他实施例的不同之处，各个实施例之间相同相似部分互相参见即可。
对所公开的实施例的上述说明，使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下，在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
Claims (4)
1.一种地单胞菌(Geomonas niftrogeniifigens)RF4，其特征在于，保藏编号为
GDMCCNo：61761。
2.一种权利要求1所述的地单胞菌(Geomonas niftrogeniifigens)RF4在铁还原和固氮中的应用，其特征在于，保藏编号为GDMCCNo：61761。
3.一种权利要求1所述的地单胞菌(Geomonas niftrogeniifigens)RF4在制备碱性磷酸盐酶、酸性磷酸酶和萘酚-AS-BI-磷酸水解酶中的应用。
4.一种微生物菌剂，其特征在于，包括权利要求1所述的地单胞菌(Geomonas niftrogeniifigens)RF4，保藏编号为GDMCC No：61761。
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