• Scopus收录期刊
  • CSCD(核心库)来源期刊
  • 中文核心期刊
  • 中国科技核心期刊
  • F5000顶尖学术来源期刊
  • RCCSE中国核心学术期刊
高级检索

杀线虫真菌Sr18发酵液对松材线虫超微结构的影响

王修清, 王倩, 王亚萍, 冯欣, 孙建华, 孟庆恒

王修清, 王倩, 王亚萍, 冯欣, 孙建华, 孟庆恒. 杀线虫真菌Sr18发酵液对松材线虫超微结构的影响[J]. 北京林业大学学报, 2017, 39(7): 69-75. DOI: 10.13332/j.1000-1522.20170048
引用本文: 王修清, 王倩, 王亚萍, 冯欣, 孙建华, 孟庆恒. 杀线虫真菌Sr18发酵液对松材线虫超微结构的影响[J]. 北京林业大学学报, 2017, 39(7): 69-75. DOI: 10.13332/j.1000-1522.20170048
WANG Xiu-qing, WANG Qian, WANG Ya-ping, FENG Xin, SUN Jian-hua, MENG Qing-heng. Effects of nematicidal filamentous fungus Sr18 metabolites on the ultrastructure of Bursaphelenchus xylophilus[J]. Journal of Beijing Forestry University, 2017, 39(7): 69-75. DOI: 10.13332/j.1000-1522.20170048
Citation: WANG Xiu-qing, WANG Qian, WANG Ya-ping, FENG Xin, SUN Jian-hua, MENG Qing-heng. Effects of nematicidal filamentous fungus Sr18 metabolites on the ultrastructure of Bursaphelenchus xylophilus[J]. Journal of Beijing Forestry University, 2017, 39(7): 69-75. DOI: 10.13332/j.1000-1522.20170048

杀线虫真菌Sr18发酵液对松材线虫超微结构的影响

基金项目: 

国家自然科学基金项目 31272019

详细信息
    作者简介:

    王修清。主要研究方向:植物线虫的生物防治。Email:clearmoon2008@126.com  地址:300387  天津市西青区宾水西道延长线393号天津师范大学主校区生命科学学院

    责任作者:

    孟庆恒,副教授。主要研究方向:应用微生物。Email:jbv3139@163.com  地址:同上

  • 中图分类号: S763.3; S432.4+5

Effects of nematicidal filamentous fungus Sr18 metabolites on the ultrastructure of Bursaphelenchus xylophilus

  • 摘要: Sr18真菌是一株自主分离获得的具有广谱杀线虫活性的生防菌。为探明该菌的杀线虫作用机理,以松材线虫为靶标,借助场发射高分辨扫描电镜及透射电镜,对1/2浓度的Sr18发酵液小分子活性组分处理后的线虫及未处理的正常线虫进行了超微观察。高分辨率扫描电镜观察结果表明,小分子物质对松材线虫头部、体壁和尾部均有损伤,并观察到内容物外溢的现象。透射电镜观察结果显示,线虫表皮和体腔分离,细胞核受损严重,形状不规则,粗面内质网核糖体聚集,线粒体空泡样变,肌纤维受损。这些现象从形态结构角度证实了该发酵液小分子活性成分对线虫的损伤是整体性的毒杀作用,排除了表面触杀的机制。这为进一步揭示Sr18的杀线虫机理以及线虫生防制剂的开发提供了重要理论依据。
    Abstract: Filamentous fungus Sr18 is a documented bio-control fungus against nematodes with broad-spectrum nematocidal activity. In present research, high resolution field emission scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to observe the micro-structure surface as well as internal cellular ultrastructure of Bursaphelenchus xylophilus. The results of SEM indicated that small molecular active products damaged the B. xylophilus' heads, body wall and tail. The dissolved substance inside the cell flowed out was observed by SEM. The results of TEM indicated that nematodes' cuticle and coelom were separated, and cell nucleus were seriously damaged, and became irregular. The ribosomes on rough endoplasmic reticulum became aggregated. The mitochondria appeared vacuolated. The muscle cells were also damaged. The above phenomena demonstrated that the nematocidal mechanism of small molecular active products was poisoned holistically rather than surface contact action. The research provides scientific evidence for further revealing the nematocidal mechanism of Sr18 as well as the development of biological pesticide controlling nematodes.
  • 植物寄生线虫对农林业发展构成严重的威胁[1-2],为了防治线虫病害,并避免传统化学防治带来的环境污染[3],开发利用杀线虫生防制剂已成为当今的研究热点[4-5]。真菌次生代谢产物具有药效高、环境相容性好、不容易造成二次污染等优点已被越来越多的人所关注[6-7]。丝状真菌Sr18(Syncephalastrum racemosum)(简称Sr18真菌),是一株以松材线虫(Bursaphelenchus xylophilus)为靶标,自主分离获得的具有广谱杀线虫活性的丝状真菌,其代谢物对大豆孢囊线虫、甘薯茎线虫、南方根结线虫等都具有良好的防治作用,用1/2浓度的Sr18发酵液处理松材线虫、南方根结线虫、大豆孢囊线虫等不同线虫24 h,校正死亡率均能达到100%[8-9]。前期研究通过实验已证实发酵液中杀线虫活性组分主要为小分子物质[10],其小分子活性组分具有水溶性好且耐热的优势。进一步的机理研究表明:小分子活性组分对线虫体内的超氧化物岐化酶(Superoxide dismutase, SOD)、过氧化物酶(Peroxidase, POD)、过氧化氢酶(Catalase, CAT)等抗氧化保护酶系,神经递质相关的总胆碱酯酶(Total Cholinesterase, TChE),以及解毒酶谷胱甘肽转硫酶(Glutathione S-transferases, GST)均有明显的抑制作用[11]。同时还发现小分子活性组分对线虫卵的孵化也有很强的抑制作用,原始浓度发酵液处理南方根结线虫的虫卵48 h,孵化抑制率能达到85%以上[9]。在此基础上,实验借助场发射扫描电镜和高分辨透射电镜的手段,对1/2浓度Sr18发酵液小分子活性组分处理后的松材线虫进行了体表和内部结构观察[12-13],旨在为进一步揭示Sr18发酵液小分子活性组分毒杀线虫的机制提供依据。

    松材线虫,中国农业科学院植物保护研究所提供。

    Sr18真菌,由本研究室筛选及保藏。

    灰葡萄孢霉(Batrytis cinerea)(简称BC菌),由天津师范大学提供。

    在工厂用5t发酵罐采取优化放大工艺[14](发酵条件:罐压0.1 mPa;排气1;温度27 ℃;转速120 r/min,变频器324.7;发酵时间40 h)制得Sr18发酵液[10]

    取原浓度的Sr18发酵液,加入等量无菌水稀释成1/2浓度的Sr18发酵液,经微孔滤膜(0.22 μm)过滤除菌后,采用截流分子量为6 000 da的UEOS-503型中空纤维膜组件对发酵液进行超滤分级[15],除去大分子物质后所得到的小分子收集物即为实验所用的1/2浓度的Sr18发酵液小分子活性组分。

    在无菌操作条件下,将灰葡萄孢霉接种于察氏培养基上,21 ℃下避光培养7 d[16],待菌丝长满培养皿后,将松材线虫接种于培养皿进行培养繁殖,26 ℃下避光培养5~7 d。

    培养好的线虫采用贝尔曼(Baermann)漏斗法[17]进行收集,用无菌水离心洗涤3次(2 000 r/min,3 min),制备成约15 000条/mL的悬液,供试。

    参照日本Kawazu等[18]处理线虫的实验方法(即1 mL杀线虫药剂+1 500头松材线虫),取24孔板,每孔加200 μL线虫悬液,实验组加入2 mL 1/2浓度的Sr18发酵液小分子活性组分,对照组则加入等量的无菌水,于26 ℃培养箱中处理1~3 d,每24 h取样观察1次。

    将处理的线虫按照预定时间(24、48、72 h)取样,离心后用无菌水洗4次,加入用磷酸缓冲液配制的2.5%戊二醛,4 ℃过夜后,再用0.1 M PBS漂洗3次,每次15 min;然后用磷酸缓冲液配制的1%锇酸室温下固定2 h,PBS漂洗3次,每次15 min;分别用30%、50%、70%、80%、90%、100%I、100%Ⅱ梯度乙醇溶液脱水,每次10 min;六甲基二硅胺烷法干燥;离子溅射镀膜仪镀膜[19];场发射扫描电镜(型号:Nova NanoSEM 230)观察,拍照记录,每24 h取样观察1次。

    按照电镜样品制备的常规方法[20],用PBS反复清洗包含有线虫的小琼脂块,2.5%戊二醛和1%锇酸双重固定(37 ℃,12 h;45 ℃,12 h;60 ℃,24 h),分别用30%、50%、70%、80%、90%、100%I、100%Ⅱ梯度乙醇溶液脱水,每次10 min;环氧树脂梯度浸透,包埋,LEICA EMUC6超薄切片机进行超薄切片(70~80 nm),铜网收集,醋酸双氧铀-柠檬酸铅双重染色[21],Hitachi H-600透射电子显微镜观察并拍照,每24 h取样观察1次。

    正常线虫及小分子活性组分处理后的线虫SEM观察结果如图 1所示。观察发现,对照组线虫虫体丰满,呈自然弯曲状态(图 1A),线虫头部与虫体分界明显,体壁环纹清晰,尾部表面光滑,雄虫可见交合刺外凸。

    图  1  线虫虫体低倍观察结果
    A.对照(Bar=100 μm);B.处理3天后的线虫(Bar=200 μm)。
    Figure  1.  Ultrastructure observation of Bursaphelenchus xylophilus at low magnification
    A, normal Bursaphelenchus xylophilus(Bar = 100 μm); B, 3 days treated Bursaphelenchus xylophilus by Sr18(Bar = 200 μm).

    与对照组相比,小分子活性组分处理后的线虫虫体皱缩并扭曲(图 1B),体壁环纹模糊,表面凹凸不平,附着的细菌消失,表皮呈片状脱落,体壁出现明显的局部凹陷,放大后可见体表内陷形成的孔洞(图 2A2B),并可见内容物从体壁破损的孔洞溢出(图 2C)。

    图  2  处理后线虫体表SEM观察结果
    A.处理1天后线虫体表出现孔洞(Bar=4 μm);B.放大后的孔洞(Bar=1 μm);C.处理3天后内溶物由孔洞溢出(←)(Bar=1 μm)。
    Figure  2.  Ultrastructure observation of the Bursaphelenchus xylophilu's body surface at high magnification
    A, holes on body surface of nematode treated 1 day by Sr18(Bar=4 μm); B, magnifying hole on body surface(Bar=1 μm); C, dissolved substance inside the cell flew out from the holes after 3 days treated(Bar=1 μm).

    图 3A图 3B可以看出,正常线虫的头部与虫体界限分明, 连接处环形平台清晰,头部六片唇瓣之间有凹陷间隔(图 3B),并可观察到唇片上存在乳状突起,唇片上角质环纹明晰, 6个唇片在口周形成角质口环, 环中可见口针外凸。此外,还发现线虫体表局部有携带细菌[22]的存在(图 3A)。与之相对应,小分子组分处理过的线虫头部和虫体皱缩严重,连接处环形平台缢缩;唇瓣之间有凹陷间隔消失,难以分辨出6片唇,角质口环和横纹消失、口针完全被破坏(图 3C)。

    图  3  线虫头部SEM观察结果
    A.对照线虫头部及体壁的细菌(Bar=20 μm);B.对照线虫头部(Bar=3 μm);C.处理3天后萎缩的线虫头部(Bar=1 μm)。
    Figure  3.  Ultrastructure observation of the Bursaphelenchus xylophilu's head at high magnification
    A, head and bacteria on body wall of normal nematode(Bar=20 μm); B, head of normal nematode(Bar=3 μm); C, shrinking head of nematode treated 3 days by Sr18(Bar=1 μm).

    松材线虫的尾部为生殖孔所在部位,其结构的完好与否与繁殖密切相关。观察发现,正常线虫尾部因雌雄而异,雄虫侧面观察呈尖形(图 1A),可以观察到交合刺,尾尖片状翼膜清晰(图 4A);小分子活性组分处理后,尾部与虫体变化相似,表面凹凸不平,严重皱缩,交合刺消失,但尾尖片状翼膜变化不大(图 4B4C)。

    图  4  线虫尾部SEM观察结果
    A.对照线虫尾部及交合刺(Bar=20 μm);B.处理2天后线虫尾部缢缩,肿胀物形成(Bar=20 μm);C.肿胀物(Bar=5 μm)。
    Figure  4.  Ultrastructure observation of the Bursaphelenchus xylophilu's tail at high magnification
    A, tail and spicule of normal nematode(Bar=20 μm); B, shrinking tail and swellings of nematode treated 2 days by Sr18(Bar=20 μm); C, magnifying swelling on body surface(Bar=5 μm).

    实验对小分子活性组分处理后引起的的松材线虫内部结构变化进行了TEM超微观察。观察发现,对照组线虫表皮、体壁等结构完整,其3层结构(角质层、下皮层、肌肉层)完整;体腔内的细胞界限清晰,细胞核核膜完整,核糖体丰富,分布均匀;线粒体形态完好、且多聚集于肌纤维[20]附近,内质网等内膜结构清楚(图 5A5B)。

    图  5  线虫内部结构透射电镜(TEM)观察结果
    A.对照线虫肌纤维(M)及线粒体(MI)(Bar=500 nm);B.正常的细胞核(n)(Bar=500 nm);C.处理1天后的细胞核(n)及内质网(ER)(Bar=500 nm);D.处理2天后的体壁(↘)及细胞损伤(Bar=500 nm);E.处理3天后体壁分离(Bar=2 μm);F.处理3天后体壁分离后组织细胞结构消失(Bar=2 μm)。
    Figure  5.  Ultrastructure observation of the Bursaphelenchus xylophilu's internal structure at high magnification
    A, muscle fiber and mitochondria of normal nematode(Bar=500 nm); B, cell nucleus of normal nematode(Bar=500 nm); C, damaged cell nucleus and endoplasmic reticulum of nematode treated 1 day by Sr18(Bar=500 nm); D, swellings on body wall and cellular damage of nematode treated 2 days by Sr18(Bar=500 nm); E, separation of cuticle and coelom of nematode treated 3 days by Sr18 (Bar=2 μm); F, disappearance of tissue and cell structure after separation of nematode treated 3 days by Sr18 (Bar=2 μm).

    经小分子活性组分处理后,体腔内部分细胞受损明显,细胞核形状不规则,核膜受损,染色质分散,内质网出现断裂,核糖体集聚,线粒体明显固缩(图 5C5D),局部体壁和体腔分离,肌纤维严重受损,已看不清体壁层次,并形成空腔,体腔内细胞间界限勉强可见;最终体内细胞界限消失,失去细胞功能(图 5E5F)。

    扫描电镜及透射电镜的超微结构观察结果显示,Sr18发酵液分离得到的小分子活性组分对松材线虫的杀线作用是“内外兼顾”的,全面影响线虫的结构。就超微观察结果而言,观察到体壁受损,层次消失,肌细胞溶解,线粒体、内质网等细胞器受损。核糖体聚集通常也是细胞损伤的重要指征,从而导致线虫死亡,表明其作用机理应为毒杀,而非触杀[23]。前期酶学研究结果也显示,Sr18发酵液的小分子活性组分对ATP、TChE、GST、以及SOD、POD、CAT等酶均有抑制作用[10]。这与超微观察的结果相对应。失去了这些酶的保护,细胞器和细胞的损伤在所难免,而细胞器或细胞的损伤也必然加剧对酶的影响[24]

    目前,对真菌代谢物对线虫抑制或致死作用相关研究主要集中在这些代谢产物的实验室或大田的时间-剂量效应研究上以及代谢物对J2的致死性,对虫卵孵化的抑制力以及抑制线虫侵染寄主的能力[25];Vos等[26]研究发现丛枝菌根真菌可通过改变其寄主根系分泌物来减少根结线虫的侵染,而且其根系分泌物可在抑制线虫运动方面发挥一定的作用。另有一些则对线虫寄生真菌和食线虫真菌侵染性胞外酶的蛋白分子结构、酶动力学以及其作用于线虫的组织部位进行了相关的分析与研究[27];现在许多微生物的杀线虫代谢产物的真正的作用机制还不清楚,代谢产物可通过不同的作用靶点破坏线虫的正常生活,已有的对微生物杀线代谢产物作用机制的研究多从代谢物对线虫的神经毒作用、体壁破坏作用、与物质代谢及能量代谢相关的酶的变化以及分泌植物生长激素等方面进行[28-33],较深入系统的研究还很缺乏。由于这方面的理论问题没有真正搞清楚,因此大大限制了高效生物杀线虫农药的开发应用,这直接影响对它们更好的利用[29]

    近年来随着系统生物学中代谢组学技术的迅猛发展为在更深层次上揭示活性代谢物的杀线虫机理提供了强有力的手段和工具[34]。未来课题组将在前期研究的基础上,以对农林作物造成严重危害的植物寄生线虫为作用对象,将高分辨电镜、酶学及生理生化等研究手段与系统生物学中新兴的代谢组学的新技术方法相结合,充分利用学科交叉的优势,通过体内、外实验系统深入地研究探讨Sr18生物杀线剂防治植物根结线虫病害的作用机理,寻找生物标记物,确定其作用方式,为今后建立植物寄生线虫代谢组学研究平台及开发对黄瓜、番茄、辣椒等根结线虫病害具有防效高、增产作用明显[29]等特点的线虫生防菌株Sr18新型高效生物杀线剂做有益的探索。

  • 图  1   线虫虫体低倍观察结果

    A.对照(Bar=100 μm);B.处理3天后的线虫(Bar=200 μm)。

    Figure  1.   Ultrastructure observation of Bursaphelenchus xylophilus at low magnification

    A, normal Bursaphelenchus xylophilus(Bar = 100 μm); B, 3 days treated Bursaphelenchus xylophilus by Sr18(Bar = 200 μm).

    图  2   处理后线虫体表SEM观察结果

    A.处理1天后线虫体表出现孔洞(Bar=4 μm);B.放大后的孔洞(Bar=1 μm);C.处理3天后内溶物由孔洞溢出(←)(Bar=1 μm)。

    Figure  2.   Ultrastructure observation of the Bursaphelenchus xylophilu's body surface at high magnification

    A, holes on body surface of nematode treated 1 day by Sr18(Bar=4 μm); B, magnifying hole on body surface(Bar=1 μm); C, dissolved substance inside the cell flew out from the holes after 3 days treated(Bar=1 μm).

    图  3   线虫头部SEM观察结果

    A.对照线虫头部及体壁的细菌(Bar=20 μm);B.对照线虫头部(Bar=3 μm);C.处理3天后萎缩的线虫头部(Bar=1 μm)。

    Figure  3.   Ultrastructure observation of the Bursaphelenchus xylophilu's head at high magnification

    A, head and bacteria on body wall of normal nematode(Bar=20 μm); B, head of normal nematode(Bar=3 μm); C, shrinking head of nematode treated 3 days by Sr18(Bar=1 μm).

    图  4   线虫尾部SEM观察结果

    A.对照线虫尾部及交合刺(Bar=20 μm);B.处理2天后线虫尾部缢缩,肿胀物形成(Bar=20 μm);C.肿胀物(Bar=5 μm)。

    Figure  4.   Ultrastructure observation of the Bursaphelenchus xylophilu's tail at high magnification

    A, tail and spicule of normal nematode(Bar=20 μm); B, shrinking tail and swellings of nematode treated 2 days by Sr18(Bar=20 μm); C, magnifying swelling on body surface(Bar=5 μm).

    图  5   线虫内部结构透射电镜(TEM)观察结果

    A.对照线虫肌纤维(M)及线粒体(MI)(Bar=500 nm);B.正常的细胞核(n)(Bar=500 nm);C.处理1天后的细胞核(n)及内质网(ER)(Bar=500 nm);D.处理2天后的体壁(↘)及细胞损伤(Bar=500 nm);E.处理3天后体壁分离(Bar=2 μm);F.处理3天后体壁分离后组织细胞结构消失(Bar=2 μm)。

    Figure  5.   Ultrastructure observation of the Bursaphelenchus xylophilu's internal structure at high magnification

    A, muscle fiber and mitochondria of normal nematode(Bar=500 nm); B, cell nucleus of normal nematode(Bar=500 nm); C, damaged cell nucleus and endoplasmic reticulum of nematode treated 1 day by Sr18(Bar=500 nm); D, swellings on body wall and cellular damage of nematode treated 2 days by Sr18(Bar=500 nm); E, separation of cuticle and coelom of nematode treated 3 days by Sr18 (Bar=2 μm); F, disappearance of tissue and cell structure after separation of nematode treated 3 days by Sr18 (Bar=2 μm).

  • [1] 侯金丽.我国植物寄生线虫防治研究进展[J].现代农业科技, 2015(7): 136, 142. http://d.old.wanfangdata.com.cn/Periodical/ahny201507083

    HOU J L. Advances on control of plant-parasitic nematodes in China[J]. Modern Agricultural Science and Technology, 2015(7): 136, 142. http://d.old.wanfangdata.com.cn/Periodical/ahny201507083

    [2] 郑友乐, 王承东, 汪涛.线虫感知行为的研究进展[J].畜牧兽医学报, 2016, 47(5): 857-863. http://d.old.wanfangdata.com.cn/Periodical/xmsyxb201605001

    ZHENG Y L, WANG C D, WANG T. Research advances on chemosensory behaviors of nematodes[J]. Acta Veterinaria et Zootechnica Sinica, 2016, 47(5): 857-863. http://d.old.wanfangdata.com.cn/Periodical/xmsyxb201605001

    [3]

    RAJU J, ADIVAPPAR N, JAYALAKSHMI K, et al. Evaluation of chemicals and bioagents for managing phytophthora root rot and root-knot nematode disease complex in capsicum under naturally ventilated polyhouse[J]. Journal of Pure & Applied Microbiology, 2016, 10(2): 1641-1644. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=557406689f331c0e8e1a46bbb0848836

    [4] 黄金玲, 刘志明, 刘纪霜, 等.植物寄生线虫生防细菌的研究进展[J].广西农业生物科学, 2008, 27(3): 288-293. http://d.old.wanfangdata.com.cn/Periodical/gxnyswkx200803022

    HUANG J L, LIU Z M, LIU J S, et al. Research advances on biological control of plant-parasitic nematode by bacteria[J]. Journal of Guangxi Agricultural and Biological Science, 2008, 27(3): 288-293. http://d.old.wanfangdata.com.cn/Periodical/gxnyswkx200803022

    [5]

    ABD-ELGAWAD M M M. Biological control agents of plant-parasitic nematodes[J]. Egyptian Journal of Biological Pest Control, 2016, 26(2): 423-429. http://d.old.wanfangdata.com.cn/OAPaper/oai_pubmedcentral.nih.gov_3625126

    [6]

    DEGENKOLB T, VILCINSKAS A. Metabolites from nematophagous fungi and nematicidal natural products from fungi as an alternative for biological control (Part Ⅰ): metabolites from nematophagous ascomycetes[J]. Applied Microbiology and Biotechnology, 2016, 100(9): 3799-3812. doi: 10.1007/s00253-015-7233-6

    [7]

    FLOR-PEREGRÍN E, VERDEJO-LUCAS S, TALAVERA M. Combined use of plant extracts and arbuscular mycorrhizal fungi to reduce root-knot nematode damage in tomato[J]. Biological Agriculture & Horticulture, 2017, 33(2): 115-124. doi: 10.1080/01448765.2016.1261740

    [8] 孙建华. Sr18真菌代谢物抗线虫活性研究[D].天津: 天津师范大学, 2000. http://www.wanfangdata.com.cn/details/detail.do?_type=degree&id=Y348077

    SUN J H. Study on nematocidal activity of fungi Sr18 metabolites[D]. Tianjin: Tianjin Normal University, 2000. http://www.wanfangdata.com.cn/details/detail.do?_type=degree&id=Y348077

    [9] 李平, 田阳, 高丙利, 等. Sr18生防制剂的杀线虫谱研究[J].中国农学通报, 2012, 28(18): 238-245. http://d.old.wanfangdata.com.cn/Periodical/zgnxtb201218043

    LI P, TIAN Y, GAO B L, et al. The nematicidal spectrum of bio-nematicides from filamentous fungus Sr18[J]. Chinese Agricultural Science Bulletin, 2012, 28(18): 238-245. http://d.old.wanfangdata.com.cn/Periodical/zgnxtb201218043

    [10] 王博. Sr18菌发酵液小分子活性组分的制备及杀线机理的研究[D].天津: 天津师范大学, 2013.

    WANG B. Preparation and study on nematocidal mechanism of small molecule active components of fungi Sr18 metabolites[D]. Tianjin: Tianjin Normal University, 2013.

    [11] 侯金丽, 冯欣. Sr18菌代谢产物对松材线虫蛋白质和酶活力的影响[J].湖北农业科学, 2014, 53(19): 4604-4606. http://d.old.wanfangdata.com.cn/Periodical/hbnykx201419025

    HOU J L, FENG X. Effects of fungi Sr18 metabolites on the protein content and the enzyme activities in the nematode[J]. Hubei Agricultural Sciences, 2014, 53(19): 4604-4606. http://d.old.wanfangdata.com.cn/Periodical/hbnykx201419025

    [12]

    DECRAEMER W, KARANASTASI E, BROWN D, et al. Review of the ultrastructure of the nematode body cuticle and its phylogenetic interpretation[J]. Biological Reviews, 2003, 78(3): 465-510. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=10.1017/S1464793102006115

    [13] 张路平, 李纪标, 孔繁瑶.松材线虫和拟松材线虫扫描电镜结构的比较研究[J].电子显微学报, 2000, 19(5): 679-684. doi: 10.3969/j.issn.1000-6281.2000.05.003

    ZHANG L P, LI J B, KONG F Y. Comparison of structures between Bursaphelenchus xylophilus and B. mucronatus by SEM[J]. Journal of Chinese Electron Microscopy Society, 2000, 19(5): 679-684. doi: 10.3969/j.issn.1000-6281.2000.05.003

    [14] 张晓歌. Sr18菌杀线虫产物发酵工艺放大及活性组分的研究[D].天津: 天津师范大学, 2008. http://cdmd.cnki.com.cn/Article/CDMD-10065-2008072129.htm

    ZHANG X G. Study on enrichment and activity components of nematicidal metabolites of fungi Sr18[D]. Tianjin: Tianjin Normal University, 2008. http://cdmd.cnki.com.cn/Article/CDMD-10065-2008072129.htm

    [15] 徐飞, 李桂水, 楼文君.膜分离技术在发酵液提取浓缩中的应用[J].过滤与分离, 2006, 16(2): 26-29. doi: 10.3969/j.issn.1005-8265.2006.02.008

    XU F, LI G S, LOU W J. Applications of membrane separation technology in extraction and concentration of fermentation liquor[J]. Journal of Filtration & Separation, 2006, 16(2): 26-29. doi: 10.3969/j.issn.1005-8265.2006.02.008

    [16]

    MAMIYA Y. The life history of the pine wood nematode, Bursaphelenchus lignicolus[J]. Japanese Journal of Nematology, 1975, 5: 16-25.

    [17] 刘维志.植物病原线虫学[M].北京:中国农业出版社, 2000.

    LIU W Z. Plant pathogenic nematodes[M]. Beijing: China Agriculture Press, 2000.

    [18]

    KAWAZU K, SUN J H, KANZAKI H. Screening of fungal cultures for nematicidal activity and preliminary fractionation of a nematicidal fungal culture[J]. Scientific Reports of the Faculty of Agriculture Okayama University, 1996, 85: 1-6.

    [19] 郭素枝, 章淑玲, 陈玉芬, 等.甘薯茎线虫的扫描电镜制样方法[J].福建农林大学学报(自然科学版), 2005, 34(1): 43-45. http://d.old.wanfangdata.com.cn/Periodical/fjnydxxb200501009

    GUO S Z, ZHANG S L, CHEN Y F, et al. Sampling method of Ditylenchus destructor specimens for scanning electron microscopy[J]. Journal of Fujian Agriculture and Forestry University (Natural Science Edition), 2005, 34(1): 43-45. http://d.old.wanfangdata.com.cn/Periodical/fjnydxxb200501009

    [20] 张灿, 张路平.松材线虫和拟松材线虫食道腺及其分泌物超微结构观察[J].北京林业大学学报, 2006, 28(3): 119-122. doi: 10.3321/j.issn:1000-1522.2006.03.021

    ZHANG C, ZHANG L P. Ultrastructural observation on esophageal glands and their secretory granules in Bursaphelenchus xylophilus and Bursaphelenchus mucronatus by TEM[J]. Journal of Beijing Forestry University, 2006, 28(3): 119-122. doi: 10.3321/j.issn:1000-1522.2006.03.021

    [21] 宇克莉, 孟庆敏, 邹金华.镉对玉米幼苗生长、叶绿素含量及细胞超微结构的影响[J].华北农学报, 2010, 25(3): 118-123. http://d.old.wanfangdata.com.cn/Periodical/hbnxb201003026

    YU K L, MENG Q M, ZOU J H. Effects of Cd2+ on seedling growth, chlorophyll contents and ultrastructures in maize[J]. Acta Agriculturae Boreali-Sinica, 2010, 25(3): 118-123. http://d.old.wanfangdata.com.cn/Periodical/hbnxb201003026

    [22] 曹峰, 杨玲, 龚姝榕, 等.松材线虫内生细菌与病原细菌的互作关系分析[J].北京林业大学学报, 2016, 38(9): 25-33. doi: 10.13332/j.1000-1522.20160082

    CAO F, YANG L, GONG S R, et al. Relationships between pathogenic bacterium and the endophytic bacteria isolated from Bursaphelenchus xylophilus[J]. Journal of Beijing Forestry University, 2016, 38(9): 25-33. doi: 10.13332/j.1000-1522.20160082

    [23] 赵迪, 刘彬, 李玲玉, 等.白僵菌及其伴生菌发酵液对线虫的毒力研究[J].农药学学报, 2013, 15(2): 178-182. doi: 10.3969/j.issn.1008-7303.2013.02.09

    ZHAO D, LIU B, LI L Y, et al. Nematicidal activity of Beauveria bassiana and the accompanying fungi using fermentation filtrate[J]. Chinese Journal of Pesticide Science, 2013, 15(2): 178-182. doi: 10.3969/j.issn.1008-7303.2013.02.09

    [24]

    COOPER D, ELEFTHERIANOS I. Parasitic nematode immunomodulatory strategies: recent advances and perspectives[J]. Pathogens, 2016, 5(3): 58. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5039438/

    [25] 金娜, 刘倩, 简恒.植物寄生线虫生物防治研究新进展[J].中国生物防治学报, 2015, 31(5): 789-800. http://d.old.wanfangdata.com.cn/Periodical/zgswfz201505019

    JIN N, LIU Q, JIAN H. Advances on biological control of plant-parasitic nematodes[J]. Chinese Journal of Biological Control, 2015, 31(5): 789-800. http://d.old.wanfangdata.com.cn/Periodical/zgswfz201505019

    [26]

    VOS C, CLAERHOUT S, MKANDAWIRE R, et al. Arbuscular mycorrhizal fungi reduce root-knot nematode penetration through altered root exudation of their host[J]. Plant and Soil, 2012, 354(1): 335-345. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=e1d1e41d62fec870446def1d26bb5882

    [27]

    STADLER M, ANKE H, STERNER O. Metabolites with nematicidal and antimicrobial activities from the ascomycete Lachnum papyraceum(Karst.) Karst V. production, isolation and biological activities of bromine-contaning mycorrhizin and lachnumon derivatives and four additional new bioactive metabolites[J]. Journal Antibiotics, 1995, 48(2): 149-153. doi: 10.7164/antibiotics.48.149

    [28]

    SCHOUTEN A. Mechanisms involved in nematode control by endophytic fungi[J]. Annual Review of Phytopathology, 2016, 54(1): 121-142. doi: 10.1146/annurev-phyto-080615-100114

    [29] 刘杏忠, 张克勤, 李天飞.植物寄生线虫生物防治[M].北京:中国科学技术出版社, 2004.

    LIU X Z, ZHANG K Q, LI T F. Biological control of plant-parasitic nematode[M]. Beijing: Science and Technology of China Press, 2004.

    [30]

    SULOIMAN M A. Acetylcholinesterase in selected plant-parasitic nematodes: inhibition, kinetic and comparative studies[J]. Pesticide Biochemistry and Physiology, 2008, 90(1): 19-25.

    [31]

    SERGIO M. Antioxidant enzymes in (a)virulent populations of root-knot nematodes[J]. Nematology, 2009, 11(5): 689-697. doi: 10.1163/156854108X399317

    [32] 丁中, 彭德良, 高必达.线虫乙酰胆碱酯酶研究进展[J].植物保护, 2008, 34(3): 18-21. doi: 10.3969/j.issn.0529-1542.2008.03.004

    DING Z, PENG D L, GAO B D. Advances in the studies on nematode acetylcholinesterases[J]. Plant Protection, 2008, 34(3): 18-21. doi: 10.3969/j.issn.0529-1542.2008.03.004

    [33] 徐小明, 于芹, 徐坤, 等.南方根结线虫侵染对茄子砧木幼苗根系活性氧代谢及相关酶活性的影响[J].园艺学报, 2008, 35(12): 1767-1772. doi: 10.3321/j.issn:0513-353X.2008.12.007

    XU X M, YU Q, XU K, et al. Effects of infection with Meloidogyne incognitaon on reactive oxygen metabolism and correlated enzyme activities in roots of eggplant rootstock seedlings[J]. Acta Horticulturae Sinica, 2008, 35(12): 1767-1772. doi: 10.3321/j.issn:0513-353X.2008.12.007

    [34]

    KONSTANTINOS A, SUHA J. Metabolomics, a robust bioanalytical approach for the discovery of the modes-of-action of pesticides: a review[J]. Pesticide Biochemistry and Physiology, 2011, 100(2): 105-117. https://www.sciencedirect.com/science/article/abs/pii/S0048357511000472

  • 期刊类型引用(1)

    1. 李恩杰,李娜,王青华,张永安,王玉珠,曲良建. 伊氏杀线真菌与苏云金芽孢杆菌对松材线虫的联合毒力研究. 林业科学研究. 2019(01): 106-111 . 百度学术

    其他类型引用(3)

图(5)
计量
  • 文章访问数:  4169
  • HTML全文浏览量:  454
  • PDF下载量:  46
  • 被引次数: 4
出版历程
  • 收稿日期:  2017-02-21
  • 修回日期:  2017-04-19
  • 发布日期:  2017-06-30

目录

/

返回文章
返回