CN116640670B - 一株有促生作用的紫花风铃木内生真菌及其应用 - Google Patents
一株有促生作用的紫花风铃木内生真菌及其应用 Download PDFInfo
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Abstract
本发明公开了一株有促生作用的紫花风铃木内生真菌及其应用。菌株Myrmaeciumsp.FLM1,该菌株于2023年4月6日保藏于广东省微生物菌种保藏中心(GDMCC),地址:广东省广州市越秀区先烈中路100号大院,邮编:510070,保藏编号为GDMCC No.63329。本发明从野生紫花风铃木成年大树枝条中分离得到内生真菌功能菌株Myrmaecium sp.FLM1,将其接种至紫花风铃木栽培品种‘紫绣球’种子苗后,能侵染紫花风铃木根系,并主要侵入根表皮和皮层细胞内,为较典型的内生真菌定殖方式。此外,接种试验显示,接种菌株Myrmaeciumsp.FLM1显著促进了‘紫绣球’栽培品种新生根发生和地上部生长,再者其能纯培养,且产孢量大,易于工业化生产,具有良好的应用前景。
Description
技术领域
本发明属于微生物技术应用领域,更具体地,涉及一株有促生作用的紫花风铃木内生真菌及其应用。
背景技术
紫花风铃木(Handroanthus impetiginosus)是紫葳科(Bignoniaceae)风铃木属(Handroanthus)的落叶乔木。原产于巴西、巴拉圭、危地马拉、阿根廷等国家和地区。自20世纪70年代引入我国,目前在我国华南地区和西南热带地区的广东、海南、广西、福建等地均有种植。栽培品种‘紫绣球’具有紫红色风铃状花朵,圆锥花序,很容易构成一片亮丽的花海,因其具有良好的观赏效果,广泛应用为行道树、庭院树和公园景观树种等,是我国非常重要的热带亚热带景观树种。目前紫花风铃木的繁殖仍以播种和嫁接为主,但是由于每年种子产量不稳定,且种子在田间条件下3个月后就丧失发芽能力,或种子苗初期生长缓慢,所以优质苗木仍然供不应求。
大量研究表明,有些互利共生关系的有益真菌可在侵染植物根系后,提高植物对基质中营养的吸收,尤其是提高植物对基质中氮磷营养的利用,提高植物幼苗的生长能力和质量。因此,高效功能性有益真菌的筛选与应用对于紫花风铃木栽培发展具有重要意义。一方面,能提高紫花风铃木的引种和栽培效率;另一方面,有益真菌可与适宜有机肥复配,形成功能性的生物有机肥,能减少化学肥料的施用量,从而降低园林植物栽培养护成本,保护城市生态环境。
发明内容
本发明目的在于提供一株紫花风铃木内生真菌功能菌株Myrmaecium sp.FLM1及其应用,该菌是一种能够促进紫花风铃木植株生长的菌株。
为实现上述目的,本发明提供的技术方案如下:
本发明的第一个目的是提供一株紫花风铃木内生真菌功能菌株Myrmaeciumsp.FLM1,该菌株于2023年4月6日保藏于广东省微生物菌种保藏中心(GDMCC),地址:广东省广州市越秀区先烈中路100号大院,邮编:510070,保藏编号为GDMCC No.63329。
本发明的第二个目的是提供一种菌剂,其含有上述Myrmaecium sp.FLM1作为活性成分。
本发明的第三个目的是提供上述紫花风铃木内生真菌功能菌株Myrmaeciumsp.FLM1或其菌剂在促进紫花风铃木生长方面的应用。
优选,是在制备促进紫花风铃木生长的肥料中的应用。
优选,所述的Myrmaecium sp.FLM1菌剂是将Myrmaecium sp.FLM1菌株接种于PDA培养基上进行培养获得。进一步优选,所述的培养是28℃恒温暗培养。
本发明的第四个目的是提供一种促进紫花风铃木生长的方法,其是将Myrmaeciumsp.FLM1接种到紫花风铃木旁边或上面,使其定殖于紫花风铃木根部,促进紫花风铃木生长。
优选,是促进紫花风铃木新根发生和/或地上部生长。
本发明的显著优点在于:
本发明从野生紫花风铃木成年大树枝条中分离得到内生真菌功能菌株Myrmaecium sp.FLM1,将其接种至紫花风铃木栽培品种‘紫绣球’种子苗后,能侵染紫花风铃木根系,并主要侵入根表皮和皮层细胞内,为较典型的内生真菌定殖方式。此外,接种试验显示,接种菌株Myrmaecium sp.FLM1显著促进了‘紫绣球’栽培品种新生根发生和地上部生长,再者其能纯培养,且产孢量大,易于工业化生产,具有良好的应用前景。
保藏信息说明:Myrmaecium sp.FLM1于2023年4月6日保藏于广东省微生物菌种保藏中心(GDMCC),地址:广东省广州市越秀区先烈中路100号大院,邮编:510070,保藏编号为GDMCC No.63329。
附图说明:
图1是Myrmaecium sp.FLM1出现在消毒茎段基部且新芽生长壮实,左1-2,右为对照;
图2是Myrmaecium sp.FLM1形态特征图(a菌丝,b菌落正面,c成熟子囊孢子,d菌落背面);
图3是Myrmaecium sp.FLM1 ITS目的片段扩增结果;
图4是Myrmaecium sp.FLM1系统发育树图谱;
图5是Myrmaecium sp.FLM1侵染紫花风铃木根系表皮和皮层内菌丝分布;
图6是接种Myrmaecium sp.FLM1菌株及不接种对照培养1个月后紫花风铃木新根生长及地上部生长量比较。
具体实施方式:
以下实施例是对本发明的进一步说明,而不是对本发明的限制。
实施例1Myrmaecium sp.FLM1的分离与分子鉴定
供试材料为三年生紫花风铃木‘紫绣球’实生苗。剪取一年生半木质化枝条作为外植体。预处理过程中将采集的枝条叶片剪掉,洗洁精浸泡15min,浸泡过程中毛笔刷洗,洗去表面浮土灰尘,后流水冲洗2小时。常规消毒过程中先将枝条剪成2cm左右带1个腋芽的茎段,浸泡在75%乙醇中(过程中不断摇晃)50s,之后用无菌水冲洗3次,直至冲洗到清澈无泡沫出现。再用0.1%升汞处理10min,无菌水冲洗5次。放置在无菌滤纸上吸干表面的水分后,将茎段两端剪去,接种于MS培养基中。培养一段时间过后,发现组织基部有菌,但新萌芽长势较好(图1)。及时分离纯化并编号记录。
为验证该菌株为紫花风铃木内生真菌,进一步扩大采样单株并采用多种消毒剂和延长消毒时间,从消毒茎段基部可以重复得到该菌株,且新诱导的芽生长不受影响。该菌株分泌红棕色物质到MS培养基中(图1)。PDA平板中纯化培养得到的菌落白色气生菌丝较发达,菌丝分枝少,偶有齿状突起,培养基中有红棕色分泌物,未见孢子产生,由此获得菌株FLM1。
挑取少量菌丝于2mL离心管中,加入600μLDNA裂解液和钢珠,于多样品组织破碎机中粉碎,强度为65Hz,60s后静置10min,后再12000rpm,10min;取上清400μL到新的1.5EP管中,加入无水乙醇800μL,-20℃,静置30min以上;13200rpm,10min,去上清,加入70%乙醇750μL,13200rpm,5min;去上清,倒扣沥乙醇,后加入30mLddH20溶解DNA。以真菌DNA为模板,ITS1(5’-3’:TCCG TAGG TGAACCTG CCG)和ITS4(5’-3’:TCCT CCGC TTAT TGAT ATGC)为引物,对纯化菌株的ITS序列进行扩增。PCR扩增体系25μL,其中Premix Taq12.5μL,ITS1 0.5μL,ITS4 0.5μL,模板DNA0.5μL,ddH2O至25μL。PCR扩增条件:94℃预变性5min,然后94℃变性30s,30s,55℃退火30s,72℃延伸60s,共35个循环;最后72℃充分延伸10min,4℃保存。
取PCR扩增产物用1%琼脂糖凝胶电泳分离检验,扩增得到ITS目的片段长约550-600bp(图3)。将含有目标片段的产物委托宝生物工程公司完成测序,得到的实际有效核苷酸序列如SEQ ID NO.1所示。利用公共数据库NCBI进行BLASTn比对分析,选取具有较高同源性的序列用Clustal X和MEGA软件对需要进行绘制系统发育树的序列进行比对,再采用Neighbor-Joining法构建系统发育树,确定菌株FLM1与Myrmaecium rubricosumstrain2K326101-5(MG708367.1)的亲缘关系最近(图4)。
实施例2Myrmaecium sp.FLM1子囊孢子诱导
Myrmaecium sp.FLM1分离时使用的培养基为植物组培通用MS培养基,纯化菌株所用培养基为PDA培养基,在这两种培养基中可以观察到该菌菌落和菌丝特征,但长时间培养后仍然未观察到任何繁殖体形态。进一步采用改良DE培养基作为FLM1孢子诱导培养基。该培养基成分为:0.4mmol·L-1KH2PO4,1.0mmol·L-1CaCl2,0.5mmol·L-1MgSO4,1.0mmol·L- 1K2SO4,25μmol·L-1H3BO3,33μmol·L-1MnCl2,2.8μmol·L-1ZnSO4,1.0μmol·L-1Na2MoO4,140μmol·L-1Na2-EDTA,100μmol·L-1FeSO4,培养基中添加1g·L-1酵母提取物和9g·L-1可溶性淀粉和6g·L-1琼脂,溶剂为水,其配制方法是将各成分混合均匀,灭菌制得。该培养基中富含有机氮,真菌FLM1在该培养基中诱导15天后即可以观察到大量子囊孢子产生(图2),成熟子囊孢子深褐色,椭圆形,2个细胞,中央部分具加厚和略收缩隔膜,有明显的网状表面纹饰。
结合菌株FLM1的形态特征以及DNA系统发育树中的同源性分析,最终判定菌株FLM1为Myrmaecium属真菌。将菌株FLM1命名为Myrmaecium sp.FLM1,该菌株于2023年4月6日保藏于广东省微生物菌种保藏中心(GDMCC),地址:广东省广州市越秀区先烈中路100号大院,邮编:510070,保藏编号为GDMCC No.63329。
实施例3Myrmaecium sp.FLM1真菌定殖紫花风铃木根系结构观察
本实施例采用瓶内共生培养方式。试验所用植物材料为紫花风铃木品系TZ18于MS培养基中培养20天的无菌种子苗,转入改良DE培养基中。菌种材料的培养过程:从4℃下保存菌株的试管中,挑取菌丝,将菌株接种于PDA培养基上,28℃恒温暗培养,待菌丝长满平板(4-5天)后作为菌种;从PDA平板培养基的菌落边缘取出直径0.5cm菌饼,将菌饼放入待共生的培养瓶中,距种子苗约1.0cm,每瓶1枚菌饼,培养30天,培养条件是:将培养瓶置于26℃、光照强度1200-1500lux、12hrs光照/12hrs黑暗的培养室内培养,然后从共培养的组培苗中取3条根系,用6%的琼脂糖包埋至1.5ml的离心管中,凝固后放置4℃下保存。采用Lecia VT1000S震荡切片机进行切片,切片厚度50μm,用0.05%台盼蓝溶液染色3-5min后放入清水培养皿中,挑取完整的切片放置于载玻片上,盖上盖玻片,于Olympus BX43正置显微镜下观察拍照。
结果如图5所示,在紫花风铃木根系表皮和皮层细胞中都可以观察到Myrmaeciumsp.FLM1真菌菌丝,表明真菌FLM1在紫花风铃木幼苗根部定殖成功。
实施例4Myrmaecium sp.FLM1促进紫花风铃木种子苗生长
本实施例采用瓶内共生培养方式。试验所用植物材料选用紫花风铃木‘紫绣球’生长一致的种子苗,转入改良DE培养基中。菌种材料的培养过程:从4℃下保存菌株的试管中,挑取菌丝,将菌株接种于PDA培养基上,28℃恒温暗培养,待菌丝长满平板(4-5天)后作为菌种;从PDA平板培养基的菌落边缘取出直径0.5cm菌饼,将菌饼放入待共生的培养瓶中,距种子苗约1.0cm,每瓶1枚菌饼,对照为不接菌,每个处理30棵苗。培养条件是:将培养瓶置于26℃、光照强度1200-1500lux、12hrs光照/12hrs黑暗的培养室内培养,培养30天后,分别测量对照和处理的茎段长度、上胚轴长度、下胚轴长度、叶片数以及根数。
结果如图6所示,接种Myrmaecium sp.FLM1菌株后显著促进了种子苗新根发生以及上胚轴生长。其中菌处理后幼苗新生根数量是对照的2.3倍,处理效果达到极显著水平;菌处理后幼苗上胚轴长度比对照的增加了24%。
ITS序列表
SEQ ID NO.1
GGATTCAAAATGAACTCCCCCCCCCGCGCCTCCCCGGGGGCACATATTTTTTTTAAAAAGATTTGGAAGATTTCCTCTAATTTTTTGCAAATTTCACCATTTACTTTAATCCCCATTTGCGCTGCTTTTTTTGATTCGATGCCAAGAACCCCAAGAGATCCCCTTGGAAAAGTTTGGATTGATTTGTAACGTTTCTTACACGCGAGTTCGACTGGAGCGCCCCCCCGCGAGCGGGGGGAGCGACCGGGTTCGGGTGTGACGATTCAAGGGGTTGGAAGGTGCGCGCGCCCCGTGAGGGGCGGCGCCGATCTCTGTTAAAGACCCTTTCCGGGGGGGGGGCCTCGGAAGGATCATTAACAGAGATCGGCGCCGCCCCTCACCGGGCGCGCGCACCTTCCAACCCCTTGAATCGTCACACCCGACCCGGTCGCTCCCCCCGCTCGCGGGGGGCGCTCCAGTCCAACTCGCGTCTCGAAACGTTGCCGTCTGAGTCGACACGACAAATCAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCCCTGGCAGTCCGGGGGGCACATCTGTTCGAGCGTCATTACAACCCTCAAGCTCTGCTTGGTCTTGGGCGTCCCGTCCCCGCCTCGCGCGGCGGACGCGCCTCAAACTCCTCGGCGGTCCGCACCGGCTTCGAGCGTAGCAATCGCACCTCGCTCACGGAGTCCGGCTCGGGTCCTGCCGCATGACGACACACCGTTTCATTTCGAAAGGTTGACCTCGGATCAGATGGGGATACCCGCTGAACTTAAGCATATCTAAAAACCGGAAAGAACA。
Claims (8)
1.一种紫花风铃木内生真菌Myrmaecium sp.FLM1,保藏编号为GDMCC No.63329。
2.一种菌剂,其特征在于,含有权利要求1所述的Myrmaecium sp.FLM1作为活性成分。
3.权利要求1所述的Myrmaecium sp.FLM1或权利要求2所述的菌剂在促进紫花风铃木生长方面的应用。
4.根据权利要求3所述的应用,其特征在于,所述的应用是在制备促进紫花风铃木生长的肥料中的应用。
5.根据权利要求3所述的应用,其特征在于,所述的Myrmaecium sp.FLM1菌剂是将Myrmaecium sp.FLM1菌株接种于PDA培养基上进行培养获得。
6.根据权利要求5所述的应用,其特征在于,所述的培养是28℃暗培养。
7.一种促进紫花风铃木生长的方法,其特征在于,是将权利要求1所述的Myrmaeciumsp.FLM1或权利要求2所述的菌剂接种到紫花风铃木,使其定殖于紫花风铃木根部,促进紫花风铃木生长。
8.根据权利要求7所述的方法,其特征在于,所述促进紫花风铃木生长是促进紫花风铃木新根发生和/或上胚轴生长。
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