CN117535156B - 一种篮状菌cft-1及其应用 - Google Patents
一种篮状菌cft-1及其应用 Download PDFInfo
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Abstract
本发明涉及一种篮状菌（Talaromyces sp.）CFT‑1，保藏编号为CGMCC No.40342。其在PDA培养基上，菌落初期呈白色，菌丝有横隔，分生孢子梗，孢子囊呈扫帚状，孢子形态为圆形或椭圆形，成熟的孢子呈青绿。该菌株具有较高的释硅、溶磷、解钾、产嗜铁素和分泌IAA的能力。该菌株还具有较强的酸碱、盐耐受特性。本发明同时涉及了该菌株在促进植物生长、土壤质量提升等方面的应用。以该菌株为有效成分可制成生物有机肥，减少化学硅、磷、钾肥的施用；同时还可以利用其强抗逆性，用于盐碱地、酸性或碱性土壤改良，具有广阔的推广应用前景。
Description
技术领域
本发明属于环境微生物技术领域，具体涉及一种篮状菌CFT-1及其应用。
背景技术
当前，硅虽然不是植物生长发育所必需的营养元素，但其是氮、磷、钾之外植物健康生长所必须的第四大元素，在农业生产中作用显著。硅的缺乏在一定程度上影响着作物的产量和品质，同时也影响作物与环境及作物与其他生物间的生态关系。由于磷、钾元素被土壤大量固定，土壤中95%以上是难溶性磷和矿物钾，很难被植物利用，存在土壤微生态失衡，作物抗逆性能低等问题。
释硅、溶磷、解钾功能微生物能够利用自身代谢产物或者通过与其他生物的协同作用，将土壤或矿物中难溶的硅、磷、钾转化为植物生长可利用的有效态养分，对提高作物产量和品质，增强作物抗病、抗逆性，改善土壤质量，提高肥料利用率，开发新的功能性微生物产品具有重要作用。
土壤中释硅、溶磷和解钾微生物主要包括细菌、真菌和放线菌。目前研究较多且较为深入的主要是单一功能菌种，极少报道同时具有释硅、溶磷和解钾功能的微生物，因此，筛选同时具有释硅、溶磷和解钾功能微生物，对研制新型微生物有机肥、改善土壤质量，以及尾矿区生态复垦有重大意义。
发明内容
本发明的目的是提供一种既可高效释硅，又具有良好解磷、解钾能力的耐酸碱篮状菌CFT-1，利用其同时进行释硅和解磷、解钾、生产嗜铁素和IAA等功能，可在土壤质量提升，生物有机肥生产过程中进行应用。
本发明采用如下技术方案：
一种篮状菌（Talaromyces sp.）CFT-1，保藏于中国微生物菌种保藏管理委员会普通微生物中心，地址为北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，保藏编号为CGMCC No. 40342，保藏日期为2022年10月21日。
所述的篮状菌CFT-1在PDA培养基上生长良好，在PDA培养基上，菌落初期呈白色，菌丝有横隔，分生孢子梗，孢子囊呈扫帚状，孢子形态为圆形或椭圆形，成熟的孢子呈青绿。
所述篮状菌CFT-1，具有释硅能力。
所述篮状菌CFT-1，具有溶磷、解钾能力。
所述篮状菌CFT-1，具有产嗜铁素和分泌IAA能力。
所述篮状菌CFT-1，具有酸碱耐受能力，在初始培养基pH为3~12范围内均能很好生长，最适生长pH为4~8。
所述篮状菌CFT-1，具有盐耐受特性，在初始培养基NaCl浓度0~15%范围内均能很好生长。
所述篮状菌CFT-1，其兼具释硅能力、解磷解钾和耐酸碱能力。
所述篮状菌CFT-1，可耐受生长温度为20~40℃，最适生长温度为25~30℃。
一种上述篮状菌CFT-1在植物促生和/或土壤修复中的应用。
一种包含上述篮状菌CFT-1的生物硅肥/磷肥/钾肥和/或土壤改良剂。
在生物硅肥/磷肥/钾肥和/或土壤改良剂中，所述篮状菌CFT-1的孢子含量不低于106个/mL。
本发明的有益效果在于：
（1）本发明的篮状菌CFT-1不仅有释硅作用，还能溶磷、解钾、产嗜铁素和IAA。
（2）本发明的篮状菌CFT-1可以为释硅、解磷解钾功能微生物的开发提供优质菌种资源，为开发新的生物有机肥和微生物菌剂奠定基础。
（3）利用本发明的篮状菌CFT-1应用于土壤，不仅可以提高土壤中硅、磷、钾含量，而且显著促进植物生长。
（4）利用本发明的篮状菌CFT-1，可以用于尾矿区土壤生态复垦、缺硅磷钾土壤改良等。
（5）本发明的篮状菌CFT-1，在酸性或碱性土壤耐受性方面优势显著，定殖能力强，可用于土壤改良及质量提升，有广阔的推广应用前景。
附图说明
图1为篮状菌CFT-1的系统发育树。
具体实施方式
下面结合实施例和附图对本发明做进一步的说明。本发明保护范围不限于实施例，本领域技术人员在权利要求限定的范围内做出任何改动也属于本发明保护的范围。
实施例1篮状菌CFT-1分离和保藏
篮状菌CFT-1是从中国河北省承德铁矿尾矿砂样品中，通过梯度稀释平板法初筛、生物浸矿复筛等方法分离获得。
具体方法是：将采集回的铁矿尾矿砂样品研磨过200目筛制备成10-3、10-4、10-5的3个稀释度菌悬液，均匀涂布于亚历山大罗夫培养基上，于30℃恒温培养箱中培养2~3天，挑取不同形态单菌落进一步分离纯化，直至获得单菌落，根据菌落形态初步判断为真菌，因此转接到PDA培养基斜面，4℃冰箱保存备用。以铁尾矿砂为底物，以发酵液中有效硅含量的增加为标准，对初筛得到的单菌落接种至浸矿脱硅培养基内进行释硅能力复筛。发酵第5 d时进行取样，发酵液离心取上清，用硅钼蓝比色法测定有效硅含量，有效硅含量达212.5 mg/L。
所用培养基配方：
亚历山大罗夫培养基（g/L）：蔗糖5.0 g、Na2HPO4 2.0 g、MgSO4·7H2O 0.5 g、FeCl30.005 g、CaCO3 0.1 g、Mg2O8Si3 2.5 g、琼脂15-20 g，pH为7.0~7.4。
浸矿脱硅培养基（g/L）：葡萄糖10 g、KH2PO4 0.2 g、MgSO4·7H2O 0.2 g、NaCl 0.2g、CaCl2·2H2O 0.2 g、CaCO3 5 g、铁尾矿砂1.0 g，pH为7.0~7.2。
PDA培养基（g/L）： 马铃薯 200 g、葡萄糖 20 g、琼脂 15~20g、蒸馏水1 L，pH 自然、固体培养基需加琼脂 15~20 g，pH自然。
菌株CFT-1在PDA培养基上生长良好，菌落初期呈白色，菌丝有横隔，分生孢子梗，孢子囊呈扫帚状，孢子形态为圆形或椭圆形，成熟的孢子呈青绿。
取培养72 h的菌丝体，利用真菌基因组DNA提取试剂盒提取菌株CFT-1基因组DNA，以其DNA为模板， ITS1（上游引物）5’-TCCGTAGGTGAACCFTGCGC-3’，ITS4（下游引物）5’-TCCFTCCGCFTTATTGATATGC-3’ 为引物，进行PCR扩增。经上海生工测序得基因序列。将其与GenBank数据库中的序列进行BLAST比对，并用MEGA 5.0做系统发育树（如图1所示）。
结果表明，该菌株与Talaromycesfuniculosus的同源性最高。结合形态学等特征，将其鉴定为篮状菌Talaromyces sp.。该菌株于2022年10月21日在中国微生物菌种保藏管理委员会普通微生物中心进行了保藏，地址为北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，保藏编号为CGMCC No. 40342。
实施例2 篮状菌CFT-1溶磷、解钾能力测定
制备菌株CFT-1种子平板，点接于NBRIP溶磷和解钾检测培养基上，于30℃恒温培养箱中培养3 d后，观察其生长情况及是否有溶解圈。计算方法为：溶解能力＝溶解圈直径D/菌落直径d。
NBRIP培养基：葡萄糖 10g、Ca3(PO4)2 5 g、(NH4)2SO4 0.5 g、MgSO4·7H2O 0.25 g、KCl 0.2 g、MgCl2·6H2O 5 g、琼脂15 g、0.4%溴酚蓝 6 mL、超纯水1 L，pH 7.0~7.2。
解钾培养基：蔗糖10 g、Na2HPO4 1 g、(NH4)2SO4 0.5 g、MgSO4·7H2O 1 g、酵母粉0.2 g、NaCl 0.1 g、CaCO3 0.1 g、FeCl3 0.005 g、钾长石 5 g、水 1 L。
测定结果表明：在NBRIP溶磷检测培养基和解钾培养基中，菌株CFT-1的菌落周边均出现明显的透明圈，并且D/d值大于2，说明其具有较强的溶磷、解钾能力。
实施例3 篮状菌CFT-1产嗜铁素和IAA测定
嗜铁素产生能力：制备菌株CFT-1种子平板，接种针挑取部分菌落接于CAS检测培养基上，30℃培养3 d，若出现黄绿色晕圈，则说明产嗜铁素。
分泌IAA性能：制备菌株CFT-1种子平板，将部分菌落接种于PDA培养基中（含100mg/L的L-色氨酸），置于30℃、180 r/min的摇床上振荡培养3~5 d，取经过8000 r/min离心后的上清液50μL，加入50μL Salkowski比色液，滴在白瓷板上避光显色30 min后，若出现粉红色则为阳性，表示该菌株能够分泌IAA，颜色越深表示分泌的强度越大，不变色为阴性，表示不能分泌IAA。
实验结果表明，菌株CFT-1具有产IAA、嗜铁素的能力，具有较强的促进植物生长的能力。
实施例4 篮状菌CFT-1的抗逆性实验
制备菌株CFT-1种子平板，挑取部分菌落划线接种于不同初始pH（2、3、4、5、6、7、8、9、10、11、12）、NaCl含量（0、0.5％、2％、4％、6％、8％、10％、12％、15％）的PDA液体培养基内，于25 ℃恒温养 2~3 d。同时将菌株CFT-1菌落接入PDA液体培养基，于不同温度条件下（4℃、10℃、20℃、30℃、40℃、45℃、50℃）培养2~3 d，观察菌落生长情况。
结果表明，该菌株CFT-1可在pH 3~12正常生长。在培养基NaCl含量0~15%均能很好生长，说明其具有很强的耐盐能力。菌株CFT-1在20~30℃均能很好生长，最适生长温度为25℃，说明菌株CFT-1对逆境条件耐受能力强，具有高耐酸碱性和耐盐性。
实施例5 篮状菌CFT-1对土壤及植物生长的影响
制备菌株CFT-1孢子液，将孢子浓度调整为1×108 CFU/mL，按照20 mL·kg-1拌入基质，基质是由部分园林土、铁尾矿砂和蛭石复合而成。对照则在基质中拌入相同体积的培养基，进行玉米盆栽试验。置于25℃温室中，在种子萌发后保留每盆中生长最为良好的三株幼苗，3个月后对土壤和植株（玉米）的地上指标及根系生长状况进行测定。每组实验做3组平行处理。
结果显示：玉米在植株长势及根系方面都要显著优于对照处理，特别是在株高、根长、叶面积等方面差异显著。分析结果见表1和表2所示。同时可以显著提高土壤中有效硅、速效钾和有效磷的含量。
表1 篮状菌CFT-1对玉米植株生长的影响
。
表2 篮状菌CFT-1对土壤肥力的影响
。
根据上述的实施例对本发明作了详细描述。需说明的是，以上的实施例仅仅为了举例说明发明而已。在不偏离本发明的精神和实质的前提下，本领域技术人员可以设计出本发明的多种替换方案和改进方案，其均应被理解为在本发明的保护范围之内。
Claims (6)
1.一种篮状菌（Talaromyces sp.）CFT-1，其特征在于，其保藏编号为CGMCC No.40342。
2.一种如权利要求1所述的篮状菌CFT-1在植物促生和/或土壤修复中的应用。
3.一种包含权利要求1所述的篮状菌CFT-1的生物硅肥。
4.一种包含权利要求1所述的篮状菌CFT-1的生物磷肥。
5.一种包含权利要求1所述的篮状菌CFT-1的生物钾肥。
6.一种包含权利要求1所述的篮状菌CFT-1的土壤改良剂。
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