CN106222096B - 一株塔宾曲霉菌ct1及其在盐碱地解磷方面的应用 - Google Patents
一株塔宾曲霉菌ct1及其在盐碱地解磷方面的应用 Download PDFInfo
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Abstract
本发明涉及一株塔宾曲霉菌CT1及其在盐碱地解磷方面的应用，塔宾曲霉菌CT1的保藏编号为：CGMCC No.12770；保藏单位为：中国微生物菌种保藏管理委员会普通微生物中心；保藏日期为：2016年7月20日；保藏地址为：北京市朝阳区北辰西路1号院3号。本发明可以有效提高土壤磷的利用率，进而促进作物增产。
Description
技术领域
本发明属于生物技术和农业增产技术领域，具体地说，它涉及一种塔宾曲霉菌CT1(Aspergillus tubingensis)及其在盐碱地解磷方面的应用。
背景技术
盐碱地在我国分布广泛，尤其在黄河三角洲地区分布有大面积的盐碱地，使得农业效益低下，作物产量低。磷是植物的必需营养元素之一，但土壤中的磷大多以植物难以利用的无机态或者有机态存在，难以利用的磷占土壤总磷的95％，大大降低了植物对磷的利用效率。
解磷菌可以提高土壤磷的利用，解磷菌在代谢过程中产生各种酶类如植酸酶、核酸酶、磷脂酶和有机酸类，并通过酸化、螯合和离子交换等过程将难溶磷变为可溶磷供植物吸收利用；施加解磷菌能促进土壤有效磷转化，提高作物产量。盐渍化土壤作为一种特殊的盐生环境，分布着各种耐盐微生物。
发明内容
本发明针对盐碱地农田作物产量低、磷利用效率低的问题，提供了一株塔宾曲霉菌CT1(Aspergillus tubingensis)及其在盐碱地解磷方面的应用。本发明采集黄河三角洲，经分离纯化后获得塔宾曲霉菌CT1，该菌的保藏编号为：CGMCC No.12770；保藏单位为：中国微生物菌种保藏管理委员会普通微生物中心；保藏日期为：2016年7月20日；保藏地址为：北京市朝阳区北辰西路1号院3号。
本发明提供的曲霉菌CT1(Aspergillus tubingensis)的菌落特征：PDA固体培养基中菌落菌丝成白色，菌落内圈成黑色或深黄绿色，25℃培养7天菌落直径61-69mm。在无机磷液体培养时，发酵液第四天已经澄清，呈现淡黄色或黄绿色，有轻微的粘稠感，菌丝球呈现不规则的团状。曲霉菌CT1的分类命名为塔宾曲霉Aspergillus tubingensis。
塔宾曲霉菌CT1(Aspergillus tubingensis)的细胞形态特征：菌丝细长，枝多，顶囊为球状，老时黄色或黄褐色。
塔宾曲霉菌CT1(Aspergillus tubingensis)的生理生化特征：生长温度20℃～35℃，pH的大小不影响其生长，NaCl耐受性1％～10％。NaNO3、KNO3、尿素、(NH4)2SO4、葡萄糖、蔗糖、麦芽糖、乳糖下均能正常生长。
塔宾曲霉菌CT1(Aspergillus tubingensis)ITS序列分析：利用PDA固体培养基培养真菌，刮取平皿表面的菌丝体放入研钵中用液氮研磨。研磨好的菌丝体采用真菌总DNA提取试剂盒提取其基因组DNA。利用真菌ITS序列的PCR通用引物(上游引物为ITS1：5′-TCCGTAGGTGAACCTGCGG-3′；下游引物为ITS4：5′-TCCTCCGCTTATTGATATGC-3′)进行PCR扩增。采用25μl PCR反应体系：2.5μmol L-1dNTPs、10μmol L-1上游引物、10μmol L-1下游引物、3U Taq plus DNA聚合酶、1×PCR反应缓冲液。PCR扩增程序为：94℃5min；94℃1min，57℃45s，72℃45s，共35个循环；72℃10min。用1.5％琼脂糖凝胶电泳对PCR扩增产物进行检测，PCR产物经试剂盒纯化后送至北京三博远志生物科技有限公司测序后，将获得的DNA序列，输入GenBank，用Blast程序与数据库中的序列进行比较分析，利用MEGA4.1软件进行系统发育树的构建。分析发现与Aspergillus tubingensis(KP725001)相似度100％，属于曲霉菌属。
该曲霉菌能够很好的分解土壤中的无机磷并对作物产量(小麦和玉米)都具有明显的增产作用。
本发明的优点和积极效果：
本发明可以有效提高土壤磷的利用率，进而促进作物增产。本发明菌株在盐浓度为0.03％时解磷能力最强，可达523.5mg/L。在0.03％～6％时，菌株CT1的解磷能力随发酵液盐浓度升高降低，但下降不太大。
附图说明
图1是塔宾曲霉菌CT1(Aspergillus tubingensis)解无机磷的效果；
图2是塔宾曲霉菌CT1(Aspergillus tubingensis)的菌落形态；
图3是塔宾曲霉菌CT1(Aspergillus tubingensis)的细胞形态；
图4是塔宾曲霉菌CT1(Aspergillus tubingensis)的发酵液形态；
图5是不同盐度处理情况下塔宾曲霉菌CT1(Aspergillus tubingensis)的溶磷量；
图6是塔宾曲霉菌CT1(Aspergillus tubingensis)菌剂处理前后小麦的生物量；
图7为本发明塔宾曲霉菌CT1(Aspergillus tubingensis)对小麦茎鲜重的影响；
图8为本发明塔宾曲霉菌CT1(Aspergillus tubingensis)对小麦根长的影响。
具体实施方式
下面结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。但实施例仅是范例性的，并不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本发明的保护范围内。
实施例1菌株筛选
采集黄河三角洲将土样按马丁氏培养基的培养方法进行菌株的初筛，挑选具有透明圈较大且透明度高，生长旺盛的菌落，进行分离筛选和纯化，直至分离出单个菌落后接入察氏培养基斜面上，4℃冰箱保藏。通过初筛我们得到了14株解磷菌。
再对这14株具有解磷菌利用无机磷培养基进行复筛，挑选出解磷能力最强的解磷菌，进行有机磷解磷能力的测定。最终获得解磷菌塔宾曲霉菌CT1，该菌株具有较高的解磷能力，可达523.5mg/L。结果如图1所示。
塔宾曲霉菌CT1，该菌的保藏编号为：CGMCC No.12770；保藏单位为：中国微生物菌种保藏管理委员会普通微生物中心；保藏日期为：2016年7月20日；保藏地址为：北京市朝阳区北辰西路1号院3号。
实施例2：塔宾曲霉菌CT1(Aspergillus tubingensis)菌株的形态特征
在培养皿底放一张滤纸片，滤纸上放一载玻片架，在载玻片上放一块载玻片和两块盖玻片，盖上皿盖后外用用报纸后密封，经高压蒸汽灭菌锅0.101MPa、121℃灭菌20min，置于烘箱中低温烘干。用灭菌的滴管吸取少量融化的査氏培养基，滴加1/2滴到载玻片的接种处，接着用接种针挑取少量待观察的解磷菌菌丝至湿室内的载玻片上。在培养基未彻底凝固前，盖上盖玻片，使盖玻片和载玻片间的距离相当接近但不能接触。最后在皿中倒适量20％的事先无菌好的甘油使滤纸完全润湿，置28℃恒温培养4d。每天取出观察菌丝生长情况。
结果表明，塔宾曲霉菌CT1在无机磷固体培养基上生长时产生明显的解磷圈，如图2所示，透明圈的产生可能是因为菌落在生长过程中，产生的代谢产物与磷酸钙发生反应的结果，我们可以通过解磷圈与菌落直径的比值来初步判断解磷菌的解磷能力。塔宾曲霉菌CT1菌落菌丝成白色，菌落内圈成黑色或深黄绿色。
用湿室培养法培养塔宾曲霉菌CT1菌丝，如图3所示，图中为第三天时的菌丝长势，顶囊为球状。
实施例3塔宾曲霉菌CT1(Aspergillus tubingensis)菌株的序列测定和鉴定
ITS序列分析利用PDA固体培养基培养真菌，刮取平皿表面的菌丝体放入研钵中用液氮研磨。研磨好的菌丝体采用真菌总DNA提取试剂盒提取其基因组DNA。利用真菌ITS序列的PCR通用引物(上游引物为ITS1：5′-TCCGTAGGTGAACCTGCGG-3′；下游引物为ITS4：5′-TCCTCCGCTTATTGATATGC-3′)进行PCR扩增。采用25μl PCR反应体系：2.5μmol L-1dNTPs、10μmol L-1上游引物、10μmol L-1下游引物、3U Taq plus DNA聚合酶、1×PCR反应缓冲液。PCR扩增程序为：94℃5min；94℃1min，57℃45s，72℃45s，共35个循环；72℃10min。
用质量分数为1.5％的琼脂糖凝胶电泳对PCR扩增产物进行检测，PCR产物经试剂盒纯化后送至北京三博远志生物科技有限公司测序后，将获得的DNA序列，输入GenBank，用Blast程序与数据库中的序列进行比较分析，利用MEGA4.1软件进行系统发育树的构建，如图4所示，与Aspergillus tubingensis(KP725001)相似性为100％。序列如SEQ ID No.1所示。
实施例4：塔宾曲霉菌CT1的发酵培养
采用液体发酵培养法，将塔宾曲霉菌CT1在难溶性磷液体培养基上培养(培养基组成：葡萄糖10g；(NH4)2·SO4 0.5g；MgSO4·7H2O 0.3g；NaCl 0.3g；FeSO4·7H2O 0.03g；KCl0.3g；MnSO4·H2O 0.03g；Ca3(PO4)2 5g；琼脂18g；去离子水1000mL；pH 7.0-7.5)，按照配方配制液体培养基进行高压灭菌，在超净工作台上，按照无菌接种法，接种塔宾曲霉菌CT1后，将培养基放置在摇床中，设置30℃，120r/min转速培养7d，每天观察发酵液的变化，并记录菌丝球的出现日期，菌丝球颜色，目测其大小。
塔宾曲霉菌CT1在无机磷液体培养时，如图5所示，发酵液第四天已经澄清，呈现淡黄色或黄绿色，有轻微的粘稠感。菌丝球呈现不规则的团状。
实施例5不同盐度处理情况下塔宾曲霉菌CT1(Aspergillus tubingensis)的溶磷量
采用无机磷液体培养基，培养基的其它成分和用量保持不变，氯化钠的加入量分别为0.03g/L、2g/L、4g/L、6g/L、8g/L、10g/L，将所筛选的解磷菌活化后制成孢子悬浊液，按照1％(v/v)的接种量接入无机磷液体培养基中，每个盐浓度做3个平行，空白组不接菌，于振荡器120rpm、28℃培养5天。测定发酵液pH和吸光度。
结果为，菌株CT1在盐浓度为0.03％时解磷能力最强，可达523.5mg/L。在0.03％～6％时，菌株CT1的解磷能力随发酵液盐浓度升高降低，但下降不太大，发酵液中有效磷浓度可以维持在523.5～338.5mg/L，如图6所示。
实施例6塔宾曲霉菌CT1(Aspergillus tubingensis)菌剂处理前后小麦的生物量
取黄河三角洲某菜地土壤(该土壤为盐碱地土壤)为盆栽用土，以土壤中接入塔宾曲霉菌CT1和未接入塔宾曲霉菌CT1(该处理作为对照)作为对比进行实验。将活化好的菌株配制成孢子悬浊液，并保证菌悬液浓度达106cfu/mL。将小麦种子预先用水浸泡8h，使种子充分吸水，然后用潮纱布包好静置一晚。每个花盆(规格500×210×150mm)中装入100颗预处理后的小麦种子，浇1000mL(接种菌的花盆浇水800mL和菌悬液200mL)；之后整个实验过程中，不再施加任何肥料。两种处理方式各做3组平行，分别在培养的第5、10、15、20天进行各指标的测定。结果如图7、8所示，本发明塔宾曲霉菌CT1逐渐表现出明显的解磷效果，尤其在小麦生长到15天时差距最大，茎鲜重增加了12.35％。在小麦生长到20天时，根长增加了15.65％，说明本发明塔宾曲霉菌CT1有较好的增产效果。
Claims (8)
1.塔宾曲霉菌CT1，其特征在于，塔宾曲霉菌（ Aspergillus tubingensis） CT1的保藏编号为：CGMCC No.12770；保藏单位为：中国微生物菌种保藏管理委员会普通微生物中心；保藏日期为：2016年7月20日；保藏地址为：北京市朝阳区北辰西路1号院3号。
2.如权利要求1所述塔宾曲霉菌CT1在盐碱地解磷方面的应用。
3.如权利要求1所述塔宾曲霉菌CT1的发酵液。
4.含有如权利要求1所述塔宾曲霉菌CT1的制剂。
5.含有如权利要求3所述发酵液的制剂。
6.如权利要求3所述塔宾曲霉菌CT1的发酵液在盐碱地解磷方面的应用。
7.如权利要求4所述含有塔宾曲霉菌CT1的制剂在盐碱地解磷方面的应用。
8.如权要求5所述含有塔宾曲霉菌CT1发酵液的制剂在盐碱地解磷方面的应用。
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Application publication date: 20161214 Assignee: Yangxin Dingzhi Agricultural Development Co.,Ltd. Assignor: Shandong University of Aeronautics and Astronautics Contract record no.: X2024980016249 Denomination of invention: A strain of Aspergillus oryzae CT1 and its application in phosphorus solubilization in saline alkali soil Granted publication date: 20180223 License type: Common License Record date: 20240927 Application publication date: 20161214 Assignee: Binzhou Xinrong bio fertilizer Co.,Ltd. Assignor: Shandong University of Aeronautics and Astronautics Contract record no.: X2024980016245 Denomination of invention: A strain of Aspergillus oryzae CT1 and its application in phosphorus solubilization in saline alkali soil Granted publication date: 20180223 License type: Common License Record date: 20240927 |