CN104911112A - 一种切梢小蠹伴生菌野外分离法 - Google Patents
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Abstract
一种切梢小蠹伴生菌野外分离法,该方法先确定林内受害松树上切梢小蠹侵入孔,以侵入孔为起点沿切梢小蠹虫母坑道方向,剥去切梢小蠹虫母坑道表面树皮,露出切梢小蠹蛀食母坑道韧皮组织,若母坑道两侧已形成子坑道,则将其一并露出,检查确证切梢小蠹各蛀食坑道内是否存在切梢小蠹伴生菌孢子、菌丝、子囊壳以及蓝变韧皮组织,根据切梢小蠹伴生菌生长发育进度及蓝变韧皮组织情况,采取对应的切梢小蠹伴生菌分离措施。本发明将野外采样和实验室菌种分离合二为一,步骤简单,操作方便,切梢小蠹伴生菌分离获取成功率高,有效简化了过去通过先将受伴生菌感染木段或带坑道韧皮、蓝变组织带回实验室,再进行分离的诸多环节,节省伴生菌分离时间,精准分离并提高分离效率。
Description
技术领域
本发明涉及切梢小蠹伴生菌分离方法技术领域,属于森林病虫害防治领域。
背景技术
切梢小蠹是以松树为主的针叶树钻蛀性害虫,迄今已经描述的有7种,主要有纵坑切梢小蠹(Tomicus piniperda)、横坑切梢小蠹 (T. minor)、云南切梢小蠹( T. yunnanensis)、松芽切梢小蠹 (T. brevipilosus) 和华山松切梢小蠹 (T. armandii)等。其中,横坑切梢小蠹和华山松切梢小蠹是目前我国主要松树害虫,特别是上世纪80 年代以来,这些切梢小蠹在云南松林区暴发成灾,导致近百万亩云南松林完全毁灭。进入本世纪以来,切梢小蠹灾害仍持续保持高位,在以云南为主的整个西南云南松林区,每年危害面积仍在150万亩以上,给当地林业生产造成严重损失。
切梢小蠹在蛀害松树过程中常常将病原真菌带入树木韧皮部,该病原菌因与切梢小蠹具有较为紧密的生态学联系,且往往由切梢小蠹所携带,故又称之为切梢小蠹伴生菌。其菌丝体在树木韧皮部及木质部内生长,并将受害部位韧皮组织和木质部染成蓝色。研究表明,切梢小蠹伴生菌对于削弱寄主树木抗性、协助切梢小蠹侵害具有积极协同和促进作用。因此,切梢小蠹伴生菌研究,对探索切梢小蠹与其伴生菌的生态学关系,全面阐释切梢小蠹危害成灾机制均具有重要意义。
切梢小蠹在寄主树木内的蛀食活动将在树木韧皮组织内留下蛀食坑道,由切梢小蠹雌虫蛀食所留下坑道称为母坑道,由切梢小蠹幼虫蛀食所留下的坑道称为子坑道。由于切梢小蠹伴生菌是由切梢小蠹所携带,伴随其蛀食过程而传播,因而切梢小蠹伴生菌主要从切梢小蠹母坑道入侵,首先在切梢小蠹母坑道发育形成。为此,切梢小蠹母坑道是确定并分离切梢小蠹伴生菌的理想场所,同时,切梢小蠹伴生菌也会在子坑道中出现,某些情况下母坑道中的伴生菌受其它类型真菌影响不易分离时可从子坑道入手,因此查找伴生菌时需对切梢小蠹各蛀食坑道逐一检查。
切梢小蠹伴生菌分离是切梢小蠹伴生菌研究的基础。在过去,分离切梢小蠹伴生菌的方法大体上是到切梢小蠹发生林区,砍伐被切梢小蠹为害的树木,将受害树木木段或受害韧皮组织、蓝变组织等带回实验室,在实验室内进行伴生菌分离。该分离方法存在木段运输工作量大、采样组织保存时间不长、分离时间周期长、污染率高、分离效率低等不足。
发明内容
本发明的目的是解决现有技术存在采样和分离工作量大、污染率高和分离周期长等问题,提出一种可有效降低污染率和节省分离时间、显著提高小蠹虫伴生菌分离效率的切梢小蠹伴生菌野外分离法。
本发明的目的通过如下技术方案实现:
一种切梢小蠹伴生菌野外分离法,该方法为先确定林内受害松树上切梢小蠹侵入孔,以侵入孔为起点沿切梢小蠹虫母坑道方向,剥去切梢小蠹虫母坑道表面树皮,露出切梢小蠹蛀食母坑道韧皮组织,若母坑道两侧已形成子坑道,则将其一并露出,检查确证切梢小蠹各坑道内是否存在切梢小蠹伴生菌孢子、菌丝、子囊壳以及蓝变韧皮组织,根据切梢小蠹伴生菌生长发育进度及蓝变韧皮组织情况,采取对应的切梢小蠹伴生菌分离措施,具体的对应措施包括有:
1)如果发现切梢小蠹蛀食坑道内黑色成熟子囊壳顶端着生白色或透明、半透明伴生菌孢子,即用灭菌细竹签挑取单个孢子在2%MEA上培养;
2)如果坑道中仅发现深灰色菌丝或不成熟子囊壳,则切割下小薄段带有坑道的韧皮组织于加保湿棉层的自封袋中培养3~7天,待坑道内长出孢子再挑取到2% MEA培养基上;
3)如果只发现蓝变的韧皮组织,则用75%酒精在韧皮部进行表面消毒后剥离外层蓝变组织,取小薄片内部蓝变组织插入2% MEA培养基培养。
本发明具有以下优点:
1)直接在林中受害树木上挑取切梢小蠹坑道内着生在韧皮组织表面的伴生菌孢子,简化了过去切梢小蠹伴生菌采集和分离的繁琐步骤,简便易行;
2)当切梢小蠹坑道中只有菌丝或不成熟子囊壳时,将该坑道韧皮组织切成小块,放入加有保湿棉层的塑料自封袋中,在室温条件下培养3~7天,即可获得切梢小蠹伴生菌。免除了过去实验室内采用大量玻璃培养皿等器具,野外伴生菌采样需要携带电锯,以及受害木段运输等大量工作;
3)将蓝变组织用酒精擦拭局部消毒后,剥离外层组织,直接用蓝变内部韧皮组织进行伴生菌培养,可减少蓝变外层韧皮组织上的杂菌污染。
本发明直接在野外对切梢小蠹伴生菌孢子、菌丝或子囊壳或已经蓝变的韧皮组织进行伴生菌采集分离,针对性强,将伴生菌的采集和分离同步进行,可有效降低污染率和节省分离时间。
具体实施方式
一种切梢小蠹伴生菌野外分离法,具体步骤如下:
野外采样前准备一个小型轻质手提箱或背包,准备小砍刀或斧头,便携式100倍带灯放大镜,封口膜,装有酒精棉团的小瓶,若干配有2%麦芽琼脂培养基培养基(即2%MEA培养基,该培养基的百分比为质量百分比,配比为每1000ml蒸馏水加20g麦芽提取粉和20g琼脂)的小型一次性塑料培养皿,建议直径为35mm和直径60mm,装有保湿棉花层的4号自封袋,灭菌手术刀片、小镊子和细竹签,数量可根据具体采样点和采样周期酌情准备。
在云南松、思茅松和华山松等分布密集的林区松树上首先确定切梢小蠹侵入孔,以侵入孔为起点沿切梢小蠹虫母坑道方向,剥去切梢小蠹虫母坑道表面树皮,露出切梢小蠹蛀食母坑道韧皮组织,若母坑道两侧已形成子坑道,则将其一并露出,用便携式100倍放大镜观察各蛀食坑道内是否已有切梢小蠹伴生菌孢子、菌丝或子囊壳。如果发现坑道内黑色成熟子囊壳顶端着生白色或透明、半透明伴生菌孢子,用灭菌细竹签挑取孢子在2% MEA培养基上(培养皿直径可为35mm),并用封口膜封住培养皿边缘;如果坑道中只有深灰色菌丝或不成熟子囊壳,则用手术刀切割一小段长4~5cm、宽2~2.5cm、厚1~2mm的带坑道韧皮组织于加保湿棉层的4号自封袋中培养3-7天,待坑道内长出孢子再挑取到2% MEA培养基上;如果只发现蓝变的韧皮组织,则用75%酒精在韧皮部进行表面消毒后用尖头小镊子剥离外层蓝变组织,取长3~5mm,宽2~3cmm,厚0.5~1mm的内部组织2~3片插于2% MEA培养基上培养(培养皿可为直径60mm),用封口膜封住培养皿边缘。采样后回实验室,将培养皿放置于25℃条件下,7~10天可观察到目标菌落形成,如有污染,则挑取其菌落边缘菌丝于2% MEA培养基上25℃培养10~15天可获得单菌落。
Claims (1)
1. 一种切梢小蠹伴生菌野外分离法,其特征在于,该方法先确定林内受害松树上切梢小蠹侵入孔,以侵入孔为起点沿切梢小蠹虫母坑道方向,剥去切梢小蠹虫母坑道表面树皮,露出切梢小蠹蛀食母坑道韧皮组织,若母坑道两侧已形成子坑道,则将其一并露出,检查确证切梢小蠹各坑道内是否存在切梢小蠹伴生菌孢子、菌丝、子囊壳以及蓝变韧皮组织,根据切梢小蠹伴生菌生长发育进度及蓝变韧皮组织情况,采取对应的切梢小蠹伴生菌分离措施,具体的对应措施包括有:
1)如果发现切梢小蠹蛀食坑道内黑色成熟子囊壳顶端着生白色或透明、半透明伴生菌孢子,即用灭菌细竹签挑取单个孢子在2% MEA上培养;
2)如果坑道中仅发现深灰色菌丝或不成熟子囊壳,则切割下小薄段带有坑道的韧皮组织于加保湿棉层的自封袋中培养3~7天,待坑道内长出孢子再挑取到2% MEA培养基上;
3)如果只发现蓝变的韧皮组织,则用75%酒精在韧皮部进行表面消毒后剥离外层蓝变组织,取小薄片内部蓝变组织插入2% MEA培养基培养。
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CN108260476A (zh) * | 2018-01-18 | 2018-07-10 | 昭通学院 | 一种羊肚菌菌种分离的方法 |
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