CN108504583B - 一种冠突散囊菌fdwt1及其应用 - Google Patents
一种冠突散囊菌fdwt1及其应用 Download PDFInfo
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Abstract
本发明提供一种冠突散囊菌FDWT1及其应用,通过对菌株的18S rDNA序列进行分子鉴定,发现其属于冠突散囊菌属(Eurotium cristatum),是一种冠突散囊菌属真菌。该菌不仅具有利用难溶性无机磷酸盐的能力,还具有分泌酸性磷酸酶、降解有机磷除草剂草甘膦的能力。本发明提供一种具有能够分泌酸性磷酸酶,溶解难溶性无机磷酸盐,降解茶叶上草甘膦农残的真菌菌株,为茶叶高效、安全生产提供候选的功能微生物。
Description
技术领域
本发明属于微生物领域,涉及一种冠突散囊菌FDWT1及其应用。
背景技术
冠突散囊菌(Eurotium cristatum)——俗称“金花”:属于散囊菌目发菌科散囊菌属的一种真菌,可生长在土壤、茯砖茶、冬虫夏草、中药片、沉香、木屑等基物上。
有机磷农药是一类有机磷酯类化合物。1944年德国拜耳公司制造出世界上第一种有机磷杀虫剂“1605 对硫磷”,距今有机磷农药的生产和应用己有近70年的历史。20 世纪60年代以来,许多国家开始禁止或限制使用有机氯农药,使得有机磷农药逐步发展成为国内外广泛生产和使用的农药产品,其产品已达百种以上。随着有机磷农药的大规模使用,80%~90%的有机磷农药进入环境,它对土壤、水体、大气环境造成了严重污染,危害了人类的生存安全。有机磷农药能够自然降解,但时间长,其残留就给人类带来了不可估量的危害,因此研究人工降解方法迫在眉睫。现有的机磷农药降解菌对有机磷农药,如氧乐果的降解率仅为 60%左右,不能快速高效的降解有机磷。
发明内容
本发明的目的在于提供一种冠突散囊菌FDWT1及其应用,寻找具有解磷能力和降解茶叶上农残能力的真菌。本发明从“金花”白茶上分离到了一株属于冠突散囊菌属的真菌,研究发现其能够在磷源只含有磷酸钙的培养基上正常生长,具有溶磷功能,能够提高难溶性磷的可利用性;具有分泌酸性磷酸酶的能力,并且具有能够降解有机磷除草剂草甘膦的能力。
为实现上述目的,本发明采用如下技术方案:
一种冠突散囊菌(Eurotium cristatum)FDWT1(即白茶金花菌1号),于2017年12月04号保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO:M 2017755,地址为武汉大学。
不同生长阶段观察白茶上分离的冠突散囊菌菌落的外观:
①在PDA培养基上“白茶金花菌1号”在2d时出现,直径约为4mm,到5d时达到14~16mm,8d时到30~32mm,出现金黄色晕圈,并出现大量黑褐色分泌物。
②“白茶金花菌1号”菌落在察氏培养基上生长较PDA培养基明显减慢,28℃培养箱内培养5d,菌落直径可至20~23 mm,培养14 d菌落直径至32~35mm,菌落整体呈较规则圆形,质地较PDA上显松散,有黄色晕圈,边缘淡黄色,向内近于金黄色,中央部分颜色较深近于褐色,呈现大量黄色具饰菌丝;菌落背面在早期有清晰的黄色晕圈,后期整个菌落背面呈黑褐色。
进一步,表明分离到的“白茶金花菌1号”具有分泌酸性磷酸酶的能力,能够利用难溶性无机磷酸盐,具有溶磷的作用;研究还发现了该“白茶金花菌1号”菌对草甘膦除草剂也有明显的降解作用,在农残降解方面有应用潜力。
本发明的优点在于:利用茶叶表面优势菌株自身的有益功能与生理特性,对磷元素的利用和降解能力,可有效改变白茶的风味与品质,已证明冠突散囊菌属有一定保健功用,利用本菌可提高白茶的保健功能。同时利用该菌不仅能够降解难溶无机磷、提高土壤磷有效性供茶树使用,还能降解有机磷、降低有机农残对茶叶危害,将对茶叶的高效、安全生产起到十分重要的作用。
附图说明
图1 茶叶上“金花菌”的菌落外观。
图2 “白茶金花菌1号”菌在PDA(左)和察氏培养基(右)上的菌落。
图3 体视镜观察“白茶金花菌1号”菌菌落。
图4 “白茶金花菌1号”菌的物种进化树。
图5 “白茶金花菌1号”溶解难溶性无机磷酸盐的能力分析。
图6 “白茶金花菌1号”酸性磷酸酶活性测定。
图7 “白茶金花菌1号”降解草甘膦农药能力测定。
具体实施方式
实施例1白茶“白茶金花菌1号”株系FDWT1的分离
1)取成品白茶的叶片,用无菌毛刷刷下茶叶上的微生物到无菌水中,混匀为菌液。
2)吸取500µl收集的菌液用无菌棒涂布到PDA培养基,倒扣放于28℃恒温培养箱2-3天;
3)待培养基上长出单菌落,挑去单菌落上的菌到新的无菌水中,通过划线方法划到新的固体PDA培养基上,28℃恒温培养。
4)根据划线平板的上长出的菌落外观是否一致判断所分离的菌是否已经是单一的菌株。
表1 马铃薯葡萄糖琼脂培养基PDA培养基配方(L-1)
表2 察氏琼脂(CZG)琼脂培养基配方(L-1)
5)将分离到的菌接种到察氏琼脂(CZG)和PDA琼脂培养基上,不同生长阶段观察白茶上分离的冠突散囊菌菌落的外观:
①在PDA培养基上“白茶金花菌1号”在2d时出现,直径约为4mm,到5d时达到14-16mm,8d时到30-32mm,出现金黄色晕圈,并出现大量黑褐色分泌物。
②“白茶金花菌1号”菌落在察氏培养基上生长较PDA培养基明显减慢,28℃下培养箱内培养5d,菌落直径可至20~23 mm,培养14 d菌落直径至32-35mm,菌落整体呈较规则圆形(图1),质地较PDA上显松散,有黄色晕圈,边缘淡黄色,向内近于金黄色,中央部分颜色较深近于褐色(图1-3),呈现大量黄色具饰菌丝;菌落背面在早期有清晰的黄色晕圈(图2),后期整个菌落背面呈黑褐色 (图3)。
实施例2 白茶“白茶金花菌1号”株系FDWT1的鉴定
为了鉴定所分离到的“白茶金花菌1号”的系统发育地位,对所分离到的“白茶金花菌1号”首先提取其基因组的DNA,利用18S rDNA序列设计引物ITS1(5’-TCCGTAGGTGAACCTGCGG-3’)和ITS4(5’-TCCTCCGCTTATTGATATGC-3’) 然后进行PCR扩增,产物在1.0%的琼脂糖凝胶上检测后用于测序,测序的结果如SEQ ID NO.1所示。
将获得的DNA序列提交到NCBI(https://blast.ncbi.nlm.nih.gov/Blast.cgi)进行BLAST比对分析。从GenBank数据库获取了6个参比,应用MEGA5.0构建系统发育树,如图4所示。
白茶上“金花菌”的外观如附图1所示,在PDA培养基上“金花菌”菌落整体呈较规则圆形,有黄色晕圈,边缘淡黄色,向内近于金黄色,中央部分颜色较深近于褐色,呈现大量黄色具饰菌丝(图1),不同阶段的菌落外观如图2所示。图3展示了“金花菌”菌落在体式显微镜下的外观。
实施例3 “金花菌”溶磷能力分析
为了进一步研究“白茶金花菌1号”菌是否具溶解难溶性无机磷酸盐的能力,我们将“白茶金花菌1号”接种到磷源只含有难溶性Ca-P的液体培养基中,发现“白茶金花菌1号”能够溶解难溶性无机磷提高培养基中可溶性磷的含量,如图5所示。并且随着培养天数的增加,培养基中可溶性磷的含量也随着增加,表明“白茶金花菌1号”具有溶磷功能。
为了进一步探究“白茶金花菌1号”是否具有分泌酸性磷酸酶的能力,我们进一步测定了培养基中酸性磷酸酶的活性。发现随着培养天数的增加,分泌的酸性磷酸酶活性也随之增加(图6)。由此可见,白茶金花菌1号”具有通过分泌酸性磷酸酶,利用培养基中难溶性磷酸盐的能力。
实施例4 “白茶金花菌1号”菌对草甘膦的分解作用进一步探讨具有酸性磷酸酶分泌能的“白茶金花菌1号”菌是否能够降解有机磷,以草甘膦为唯一磷源,加入“金花”菌菌液,液体中的浓度加入1000mg/L草甘膦标准物质溶液1ml,当天取样并在30℃下培养,在菌球出现明显时(7d)和菌球明显成型时(12d)各取样一次。各取1ml培养液经抽滤后上机测得指标。结果见图(图7)。结果显示随着培养天数的增加,以不接种菌的液体培养基为对照,液体培养基中接入本菌,草甘膦降解率随着培养天数明显增加,表明了“白茶金花菌1号”菌对草甘膦农药有明显的分解作用。
本发明通过对“金花”白茶上的优势微生物进行分离、纯化培养分离得到了一株菌落外观呈金黄色的真菌,经18S rDNA 测序鉴定为冠突散囊菌属真菌,并且命名为“白茶金花菌1号”,通过构建进化树,发现新纯化的“白茶金花菌1号”与已经报道的冠突散囊菌属的其他真菌属于同一分支,但是在保守DNA序列上不完全相同。进一步研究表明分离到的“白茶金花菌1号”具有分泌酸性磷酸酶的能力,能够利用难溶性无机磷酸盐,具有溶磷的作用;进一步的研究还发现了该“白茶金花菌1号”菌对除草剂中的草甘膦有机磷也有明显的降解作用,培养7天时达到约50%,12天时60%以上,在农残降解方面有应用潜力。
以上所述仅为本发明的较佳实施例,凡依本发明申请专利范围所做的均等变化与修饰,皆应属本发明的涵盖范围。
SEQUENCE LISTING
<110> 福建农林大学
<120> 一种冠突散囊菌FDWT1及其应用
<130> 3
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 1125
<212> DNA
<213> 人工序列
<400> 1
gctcgtagtt gaccttgggt ctggctggcc ggtccgcctc accgcgagta ctggtccggc 60
tggacctttc cttctgggga acctcatggc cttcactggc tgtgggggga accaggactt 120
ttactgtgaa aaaattagag tgttcaaagc aggcctttgc tcgaatacat tagcatggaa 180
taatagaata ggacgtgcgg ttctattttg ttggtttcta ggaccgccgt aatgattaat 240
agggatagtc gggggcgtca gtattcagct gtcagaggtg aaattcttgg atttgctgaa 300
gactaactac tgcgaaagca ttcgccaagg atgttttcat taatcaggga acgaaagtta 360
ggggatcgaa gacgatcaga taccgtcgta gtcttaacca taaactatgc cgactaggga 420
tcgggcggtg tttctataat gacccgctcg gcaccttacg agaaatcaaa gtttttgggt 480
tctgggggga gtatggtcgc aaggctgaaa cttaaagaaa ttgacggaag ggcaccacaa 540
ggcgtggagc ctgcggctta atttgactca acacggggaa actcaccagg tccagacaaa 600
ataaggattg acagattgag agctctttct tgatcttttg gatggtggtg catggccgtt 660
cttagttggt ggagtgattt gtctgcttaa ttgcgataac gaacgagacc tcggccctta 720
aatagcccgg tccgcatttg cgggccgctg gcttcttagg gggactatcg gctcaagccg 780
atggaagtgc gcggcaataa caggtctgtg atgcccttag atgttctggg ccgcacgcgc 840
gctacactga cagggccagc gagtacatca ccttggccga gaggtccggg taatcttgtt 900
aaaccctgtc gtgctgggga tagagcattg caattattgc tcttcaacga ggaatgccta 960
gtaggcacga gtcatcagct cgtgccgatt acgtccctgc cctttgtaca caccgcccgt 1020
cgctactacc gattgaatgg ctcggtgagg ccttcggact ggctccaggg ggttggcaac 1080
gaccccccag agccggaaag ttggtcaaac ccggtcatta gagaa 1125
<210> 2
<211> 19
<212> DNA
<213> 人工序列
<400> 2
tccgtaggtg aacctgcgg 19
<210> 3
<211> 20
<212> DNA
<213> 人工序列
<400> 3
tcctccgctt attgatatgc 20
Claims (2)
1.一种冠突散囊菌(Eurotiumcristatum)FDWT1,已于2017年12月4日在中国典型培养物保藏中心保藏,保藏编号为CCTCC NO:M2017755。
2.如权利要求1所述的冠突散囊菌在降解有机磷和降解植物农药残留中的应用。
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Citations (3)
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CN103627641A (zh) * | 2013-11-05 | 2014-03-12 | 四川农业大学 | 一株降解氯氰菊酯金花菌的筛选鉴定及其应用 |
CN105861333A (zh) * | 2016-06-16 | 2016-08-17 | 湖南农业大学 | 冠突散囊菌ls1菌株 |
CN106497806A (zh) * | 2017-01-06 | 2017-03-15 | 青岛农业大学 | 一种冠突散囊菌株及其应用 |
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CN103627641A (zh) * | 2013-11-05 | 2014-03-12 | 四川农业大学 | 一株降解氯氰菊酯金花菌的筛选鉴定及其应用 |
CN105861333A (zh) * | 2016-06-16 | 2016-08-17 | 湖南农业大学 | 冠突散囊菌ls1菌株 |
CN106497806A (zh) * | 2017-01-06 | 2017-03-15 | 青岛农业大学 | 一种冠突散囊菌株及其应用 |
Non-Patent Citations (1)
Title |
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冠突散囊菌研究进展;陈桂酶;《西北农林科技大学学报(自然科学版)》;20120331;第40卷(第3期);第179-184页 * |
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