CN117417840B - 一种外生菌根真菌云南硬皮马勃zss01及其应用 - Google Patents
一种外生菌根真菌云南硬皮马勃zss01及其应用 Download PDFInfo
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Abstract
本发明涉及农业种植技术领域,尤其公开一种外生菌根真菌云南硬皮马勃ZSS01及其应用,外生菌根真菌云南硬皮马勃ZSS01菌株,保藏于中国典型培养物保藏中心,保藏地址是中国武汉武汉大学;保藏编号为CCTCC NO:M 2023594,本发明筛选获得的云南硬皮马勃ZSS01菌株,菌根侵染率可达到90%以上,同时可提高松树幼苗的抗病性和生长,为实现该野生菌的人工栽培提供了种苗保障。
Description
技术领域
本发明涉及农业种植技术领域,尤其涉及一种外生菌根真菌云南硬皮马勃ZSS01及其应用。
背景技术
外生菌根(Ectomycorrhiza, ECM)在森林生态系统中具有举足轻重的作用。外生菌根真菌和树木在长期的共同进化过程中,形成共生关系,帮助宿主植物扩大根部的吸收面积,提高对水分和矿物质的吸收,同时菌根还能提高土壤的生物活性,改良土壤的理化性质,进而增强宿主植物的抗氧化性、抗逆性和抗病性,提高造林成活率,促进林木茁壮成长,对林业的发展起着十分重要的作用。
松树是北半球温带森林的主要建群树种,也是我国重要的造林和用材树种。其中,作为我国西南特别是云南境内主要的建群树种云南松和思茅松,在造林过程中,存在幼苗成活率、人工林的品质、树林的稳定性低等问题,云南硬皮马勃(Scleroderma yunnanense)作为国内唯一可食用的硬皮马勃种,生长于针叶林和混交林内,是硬皮马勃属中的一个特有种。虽然现有技术CN201410425245.9中公开了用获得的硬皮马勃孢子接种宿主后,获得了较高侵染率的菌根苗,但是该专利存在以下问题,首先,并未交代具体是哪个硬皮马勃的种,硬皮马勃属(Scleroderma)共25个种,分布在世界各地,从北到南森林、公园和农地均有分布,种类繁多包括大孢硬皮马勃、光硬皮马勃、多根硬皮马勃等,不同种硬皮马勃作为ECM的效果无法预期;其次,孢子接种虽然接种周期短、成本低,但野外采集的硬皮马勃成熟度、孢子活性等难以保障,接种成功率的稳定性不高;最后,大多数外生菌根真菌传代数次后就会老化或退化,需要重新从野生硬皮马勃中分离,而新鲜的硬皮马勃于云南只在固定的几个月生长,该方法下,存储和运输困难,不利于工业化的推广和应用。
发明内容
本发明的目的在提供一种外生菌根真菌原种的制备方法及其应用,以提高幼苗成活率,提高人工林的品质,增强树林的稳定性着手,达到增加木材储量、发挥生态效益、创建优美环境。
为了实现上述发明目的,本发明提供以下技术方案:
云南硬皮马勃ZSS01菌株,保藏于中国典型培养物保藏中心,保藏地址是中国武汉武汉大学;保藏编号为 CCTCC NO:M 2023594,保藏日期为2023年4月23日,分类命名为scleroderma yunnanense ZSS01。
一种菌剂,含有云南硬皮马勃ZSS01菌株培养后获得的菌丝体发酵液。
进一步的,本发明提供了菌剂制备方法,包括以下步骤:
(1)制备液体种子:将云南硬皮马勃ZSS01菌株在无菌环境条件下转接到改良PDA固体培养基上,在25℃培养箱内培养活化,用打孔器在活化的菌落上打孔,转至灭菌后的改良PDA液体培养基内,25℃条件培养20 d,得到菌丝体种子,备用;
(2)制备菌丝体:取步骤(1)制备的菌丝体种子在无菌环境下接种到改良PDA液体培养基中,扩大培养,获得菌丝体发酵液并收集。
进一步的,所述改良PDA固体培养基配方为:马铃薯200g,琼脂18.0 g,葡萄糖20.0g,KH2PO43.0 g,MgSO4·7H2O 1.5 g,维生素B1 10 mg,NH4NO3 0.25 g,pH5.7±0.1,蒸馏水1000 mL,应当能理解的是,改良PDA液体培养基的配置即在改良PDA固体培养基基础上去除琼脂18.0 g即可。
进一步的,扩大培养的条件如下:培养温度25℃,pH 5.7±0.1,150 r·min-1摇床培养14 d。
本发明还提供了一种菌丝体胶丸,所述菌丝体胶丸含有云南硬皮马勃ZSS01菌株的菌丝体。
进一步的,菌丝体胶丸的制备方法,包括以下步骤:
S1:将制备获得的菌丝体发酵液,无菌条件下过滤,获得菌丝体,将菌丝体无菌条件下粉碎,悬浮在吐温-80溶液中,备用;
S2:将S1所得悬浊液加到灭菌后的海藻酸钠溶液中,备用;
S3:将S2所得溶液加入灭菌后的CaCl2溶液中,缓慢搅拌,固化25 min后形成菌丝体胶丸;
S4:将S3中获得的菌丝体胶丸捞出,保存在4℃无菌水中,备用。
优选的,海藻酸钠溶液浓度为2%~4%,悬浊液和海藻酸钠溶液的体积比为1:50,CaCl2溶液的浓度为0.15~0.25 mol/L。
进一步的,本发明还提供了一种外生菌根真菌三级原种的制备方法,包括以下步骤
S1:将菌丝体胶丸在无菌环境条件下接种到装有改良PDA固体培养基的三角瓶1/3处,25℃下培养至菌丝体胶丸上的菌丝长满整个三角瓶培养基表面;
S2:往三角瓶内装入按照体积比1:4混合灭菌后的麦麸和针叶树木屑,至2/3处,25℃下培养至菌丝长满整个三角瓶。
可选的,在实际应用过程中,本发明所提供的云南硬皮马勃ZSS01菌株,以及由包含该菌株或者由该菌株制备的菌剂、菌丝体胶丸、外生菌根真菌三级原种,均可以直接或者间接的接种至松树苗木形成外生菌根,优选的,所述松树为云南松或思茅松。
本发明具有以下有益效果:
首先,本发明对通过组织分离获取纯培养的菌株进行了保藏(CCTCC NO:M2023594),保证侵染根系的为单一菌种且成功率高,可进行大量生产,进而获得高质量的菌根苗,比孢子侵染根系更有效,侵染率达到90%以上,更容易获得高质量的菌根苗。
其次,本发明通过一级平板接种,二级液体发酵培养和制备菌丝体胶丸,三级固体基质扩繁,制备的外生菌根真菌菌剂,操作方便、成本低廉、简单高效,有利于菌根合成及菌根苗的培育;
然后,本发明通过简单的混合方法制备了菌丝体胶丸,菌丝体胶丸直径约2.5 mm,内有大量完整的菌丝,单个菌丝体胶丸就足以形成菌根,最多可保存6个月仍具有活力;
最后,本发明通过此菌剂的接种,可实现云南硬皮马勃的单一侵染,菌根侵染率可达到90%以上,同时可提高松树幼苗的抗病性和生长。
大多数外生菌根真菌传代数次后就会老化或退化,但是通过发酵获得菌丝体,不仅培养时间短,育苗速度快,尤其是本专利涉及的菌丝体胶丸,允许真菌菌丝在这些胶丸内生长,以便在施用前使菌丝片段生长恢复为菌丝体,是更有效的繁殖体,可帮助获得更高质量的菌根苗,同时可提高松树幼苗的抗病性和生长,且利于保存,时效较长,可长达数月,便于工业化的生产、推广和应用。
附图说明
图1 菌种鉴定结果图;
图2 菌丝体接种时间对云南硬皮马勃菌落形成单位数的影响效果图;
图3 接种处理对(A)云南松和(B)思茅松幼苗生长的影响效果图;
图4接种处理对松树幼苗得病率和死亡率的影响效果图;
图5接种后的云南松(a)或思茅松(b)幼苗根系形成的菌根效果图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚,下面对本发明中的技术方案进行清楚、完整地描述,显然,所描述的实施例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作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。
1.1供试材料
云南硬皮马勃子实体采自于云南松或思茅松林下。
云南松或思茅松种子均为良种。种子先经30%过氧化氢(H2O2)表面消毒30分钟,然后在播种前用无菌水洗涤3次,最后将种子浸泡在45℃的无菌水中24小时,待用。在正式实验之前,我们在直径为90mm的培养皿中测试了种子在20℃下的发芽率(每重复5次,每次20颗种子;发芽率接近100%),确定种子是有活力的。
1.2培养基
改良PDA固体培养基(1L):马铃薯200g,琼脂18.0 g,葡萄糖20.0 g,KH2PO43.0 g,MgSO4·7H2O 1.5 g,维生素B1 10 mg,NH4NO3 0.25 g,pH5.7±0.1。
改良PDA液体培养基(1L):马铃薯200g,葡萄糖20.0 g,KH2PO43.0 g,MgSO4·7H2O1.5 g,维生素B1 10 mg,NH4NO3 0.25 g,pH5.7±0.1。
1.3栽培基质
供试栽培基质设置蛭石、珍珠岩和蛭石(体积比1:1)、麦麸和松树木屑混合物(体积比1:4)3种。基质经高压湿热灭菌2次(121℃,0.15 MPa),每次1 h,基质的最终pH为5.7,冷却备用。
实施例1
1菌种制备
1.1菌种分离
采集50%-60%成熟、健康的云南硬皮马勃新鲜子实体,轻拭子实体表面粘连的泥土等杂物,再用75%的无水乙醇对其进行擦拭消毒,然后在无菌操作台上用无菌解剖刀在子实体无感染处切取1 cm×1 cm的组织块,接种至装有固体培养基的平板,于25℃人工培养箱内暗培养30 d,最后转至试管斜面保藏,备用。
2.1.2分子鉴定
(1)总DNA的提取
采用CTAB法提取总DNA。
(2)ITS扩增及产物检测
ITS扩增引物为真菌通用引物:ITS4(5´- TCCTCCGCTTATTGATATGC -3´)和ITS5(5´- GGAAGTAAAAGTCGTAACAAGG -3´)(上海生物工程有限公司合成)。PCR反应体系(25μL):2× PCR Taq MasterMix with Dye 12.5μL,引物ITS 4 为1μL,引物ITS 5为1μL,模板DNA为1.0 μL,ddH2O为9.5 μL。
PCR反应条件:94℃预变性300 s,94℃变性40 s,50℃退火40 s,72℃延伸60 s,共35个循环;72℃延伸420 s。所得产物经1%琼脂糖凝胶电泳检测,后送至北京擎科新业生物技术有限公司进行序列测定。
(3)ITS序列分析与鉴定
对合成的菌根均进行DNA鉴定:首先通过BioEdit Sequence Alignment Editor软件去除两端不可靠峰、钝化现象等引起的杂峰,得到序列的可信区;接着通过CExpress软件进行序列拼接和编辑,将结果进行GenBank (http://www. ncbi. nlm. nih.ov/),运用BLAST在DNA序列数据库中寻找同源DNA序列进行比较分析,根据搜索结果,判断菌株物种或近缘物种,序列相似系数99%以上菌株在分类上被确定为同一种。所测菌株与 DNA 序列数据库中登录序列比较得到所测菌株序列的具体各部分序列组成;最后,使用MEGA 7.0中的Neighbor-joining建系统发育树。
分子鉴定结果
用真菌通用引物ITS4(5´- TCCTCCGCTTATTGATATGC -3´)和ITS5(5´-GGAAGTAAAAGTCGTAACAAGG -3´)扩增真菌rDNA的间隔序列,所得得ITS测序序列如下所示:
AACCAAAGGCCTAGGAGGTCCCCCGCCTCCGAAGCTTCGAACCCTTTGACCCCGGGGGGGCCCCCGCCGAAGGCCCTTCGGAACTAAGGCCTTCTCTTTCAAATCCGATGGCCACCGAAAGGAATCGGGCCCTTCCGGCGACGGGTTAAAGGGCCCCCTTACAATTTTCACAATGGGTTTTCTGGGTTTTCGATTCAAGGAAGGACCCAGGAAATCGCAATAAGAAAGGGGATTGGCAAATTTTCCGGGAATCTTCAATTTTTTAACCGCCCCTGGGGTTCCTGGGATTTCCAAGAAGATGGCTGGTTTAAGGGCCTTCAAACCCCCAACCCCAAGGGGCCCTTCAACCCGCCGAAGTTTGGTTTTCGAACCTGGGAAGATGGGGGGGGGGAACGCCGGTCCCCCTCAAAAGATTTACTGGGGGGGGCCAAGCCTGGCCGAACCCCCGGCCCCGTTCAACGCCGAAACCAATCGCCGGGGCTGGAAGGGGCGGGGGAACGGACTAGCCCCTGGTTTTCAAACCTTTCGGGGACAACCCCGCCCGAACCCAGGGTTGGCCCTAAACTCGGGCGAGATTCCCCC
利用Blast软件与NCBI上的物种基因序列进行ITS区域比对,序列相似性99%以上的均为Sclerodema yunnanensis,即云南硬皮马勃。
基于ITS构建了该真菌的系统发育树,综合菌株形态学鉴定和分子生物学鉴定结果(图1),该真菌与云南硬皮马勃(Sclerodema yunnanense)亲缘关系最近,所以鉴定为云南硬皮马勃(Sclerodema yunnanense)。
本发明中分离获得的云南硬皮马勃ZSS01,保藏名称为Scleroderma citrinum ZSS01, 保藏于中国典型培养物保藏中心,保藏地址为中国武汉武汉大学,该培养物于2023年4月23日由保藏中心收到,并登记入册,根据请求,由该日起保存三十年,该培养物的存活性由保藏中心于2023年04月30日检测完毕,结果存续。
实施例2
菌丝体发酵液制备
将实施例1分离获得的云南硬皮马勃ZSS01,直径为5 mm的菌丝块接种改良PDA固体培养基平板,25℃条件下培养活化20 d,用打孔器取5个直径5 mm的菌丝块,接种于装有250mL改良PDA液体培养基的500 mL三角瓶中,为让菌种充分活化,便于菌种扩繁,将三角瓶先置于25℃恒温培养箱中精制培养1 d。第2 d再置于25℃的摇床中以150 r·min-1的转速震荡培养10 d,获得菌丝体种子。
将制备的菌丝体种子500 mL在无菌环境下接种到改良PDA液体培养基(15 L)中,25℃条件下以200 r·min-1的转速液体发酵培养14 d,获得菌丝体发酵液并收集,无菌条件下过滤,获得菌丝体,备用。
实施例3 菌丝体胶丸制备
将实施例2制备的菌丝体无菌条件下粉碎,悬浮在0.05~0.15%的Tween 80溶液中形成悬浮液,然后加到灭菌后的2%~4% 海藻酸钠溶液中(体积比为1:50)混合均匀,接着用26号针头将混合液从10 cm的高度泵入灭菌后的0.15~0.25 mol/L CaCl2溶液中,缓慢搅拌,固化25 min后形成菌丝体胶丸,最后将获得的菌丝体胶丸捞出,保存在4℃无菌水中,备用。
菌丝体胶丸的活力与存储时间:存储会导致菌落形成单位百分比的下降(图2)。本发明制备获得的菌丝体胶丸具有较长的存储时间,云南硬皮马勃菌丝体胶丸在贮藏7个月后,生长活力才逐渐下降。
实施例3
1、外生菌根真菌原种制备
将实施例3制备的菌丝体胶丸1粒到装有改良PDA固体培养基的三角瓶1/3处,25℃下培养至菌丝长满整个三角瓶培养基表面,然后装入灭菌后的麦麸和松树木屑混合物,至2/3处,继续于25℃下暗培养至菌丝长满整个三角瓶,获得外生菌根真菌原种。
2、菌根苗培育及猝倒病防治
轻轻地将30 g菌剂放入用于育苗的栽培基质内,拌匀,每盆播种20粒松树种子,最后加入20 mL 液体培养基,置于室温为25℃左右的人工气候室内培养,每天光照16 h,黑暗8 h。对照不接种菌剂,每个处理10个重复。整个实验期限为12个月,每隔15天浇1次灭菌的液体培养基。试验结束后,查验菌根合成情况、苗木的生长及猝倒病防治情况。
菌根侵染率=(菌根侵染的根段数/检测的根段总数)×100%;
得病率(%) = (发病幼苗/播种数)×100;
死亡率(%)=[(播种数- 成活幼苗)/播种数]×100。
3、菌剂接种处理对松树幼苗菌根合成及其生长的影响
通过表1,可以看出:接种过云南硬皮马勃菌剂的云南松和思茅松幼苗生长指标(侵染率、株高、鲜重和干重)显著高于未接种过云南硬皮马勃菌剂的松树幼苗,其中未接种过云南硬皮马勃菌剂的松树幼苗外生菌根侵染率为0。而且,接种过云南硬皮马勃菌剂的松树幼苗,在体积比为1:4的麦麸和松树木屑混合物育苗基质上,侵染率、株高、鲜重和干重都最高。
表1 不同基质条件下不同处理对云南松和思茅松幼苗生长及根系侵染率的影响
4、菌剂接种处理对松树幼苗猝倒病的影响
由图4可以看出,菌剂接种处理显著降低了松树幼苗的得病率和死亡率,大大提高了对猝倒病的防治率。
图5为通过本发明方法培育的云南松(a)或思茅松(b)幼苗根系形成的菌根,证明本方法可培育大量菌根苗。
最后应说明的是:以上实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围。
Claims (9)
1.一种外生菌根真菌云南硬皮马勃(Scleroderma citrinum)ZSS01菌株,其特征在于,保藏于中国典型培养物保藏中心,保藏地址是中国武汉武汉大学;保藏编号为CCTCC NO:M2023594。
2.一种菌剂,其特征在于,含保藏编号为CCTCC NO:M 2023594的云南硬皮马勃ZSS01菌株液体培养后获得的菌丝体发酵液。
3.一种如权利要求2所述的菌剂的制备方法,其特征在于,所述菌剂的制备方法包括以下步骤:
(1)制备液体种子:将云南硬皮马勃ZSS01菌株在无菌环境条件下转接到改良PDA固体培养基上,在25℃培养箱内培养活化,用打孔器在活化的菌落上打孔,转至灭菌后的改良PDA液体培养基内,25℃条件培养20 d,得到菌丝体种子,备用;
(2)制备菌丝体:取步骤(1)制备的菌丝体种子在无菌环境下接种到装有改良PDA液体培养基的发酵罐中,扩大培养,获得菌丝体发酵液并收集;
所述改良PDA固体培养基1L的配方为:马铃薯200g,琼脂18.0 g,葡萄糖20.0 g,KH2PO43.0 g,MgSO4·7H2O 1.5 g,维生素B1 10 mg,NH4NO3 0.25 g,pH5.7±0.1;
所述改良PDA液体培养基1L的配方为:马铃薯200g,葡萄糖20.0 g,KH2PO4 3.0 g,MgSO4·7H2O 1.5 g,维生素B1 10 mg,NH4NO3 0.25 g,pH5.7±0.1。
4.根据权利要求3所述的菌剂的制备方法,其特征在于,扩大培养的条件如下:培养温度25℃,pH 5.7±0.1,150 r·min-1摇床培养14 d。
5.一种菌丝体胶丸,其特征在于,所述菌丝体胶丸含有权利要求1所述云南硬皮马勃ZSS01菌株的菌丝体。
6.一种如权利要求5所述的菌丝体胶丸的制备方法,其特征在于,包括以下步骤:
S1:将权利要求3-4任一所述的菌剂的制备方法制备获得的菌丝体发酵液,无菌条件下过滤,获得菌丝体,将菌丝体无菌条件下粉碎,悬浮在吐温-80溶液中,备用;
S2:将S1所得悬浊液加到灭菌后的海藻酸钠溶液中,备用;
S3:将S2所得溶液加入灭菌后的CaCl2溶液中,缓慢搅拌,固化25 min后形成菌丝体胶丸;
S4:将S3中获得的菌丝体胶丸捞出,保存在4℃无菌水中,备用。
7.根据权利要求6所述的菌丝体胶丸的制备方法,其特征在于,海藻酸钠溶液浓度为2%~4%,悬浊液和海藻酸钠溶液的体积比为1:50,CaCl2溶液的浓度为0.15~0.25 mol/L。
8.一种外生菌根真菌三级菌种的制备方法,其特征在于,包括以下步骤
S1:权利要求5-7任一所述的菌丝体胶丸在无菌环境条件下接种到装有改良PDA固体培养基的三角瓶内,培养基含量至三角瓶的1/3处,25℃下培养至菌丝体胶丸上的菌丝长满整个三角瓶培养基表面;
S2:往三角瓶内装入按照体积比1:4混合灭菌后的麦麸和针叶树木屑,至2/3处,25℃下培养至菌丝长满整个三角瓶;
所述改良PDA固体培养基1L的配方为:马铃薯200g,琼脂18.0 g,葡萄糖20.0 g,KH2PO43.0 g,MgSO4·7H2O 1.5 g,维生素B1 10 mg,NH4NO3 0.25 g,pH5.7±0.1。
9.权利要求1所述的云南硬皮马勃ZSS01菌株、权利要求2所述的菌剂、权利要求5所述的菌丝体胶丸或权利要求8所述制备方法获得的云南硬皮马勃ZSS01三级菌种接种至松树苗木形成外生菌根的应用。
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