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林业科学 ›› 2017, Vol. 53 ›› Issue (5): 82-87.doi: 10.11707/j.1001-7488.20170510

• 论文与研究报告 • 上一篇    下一篇

无菌和带菌松材线虫对赤松的致病性

林丽, 周蕾, 潘珺, 康李鹏, 叶建仁, 朱丽华   

  1. 南方现代林业协同创新中心 南京林业大学林学院 南京 210037
  • 收稿日期:2016-09-23 修回日期:2016-12-11 出版日期:2017-05-25 发布日期:2017-06-22
  • 通讯作者: 朱丽华
  • 基金资助:
    江苏省高校自然科学研究重大项目(15KJA220003);江苏高校优势学科建设工程资助项目(PAPD)。

Pathogenicity of Aseptic and Germ-Carrying Bursaphelenchus xylophilus on Pinus densiflora

Lin Li, Zhou Lei, Pan Jun, Kang Lipeng, Ye Jianren, Zhu Lihua   

  1. Co-Innovation Center for Sustainable Forestry in Southern China College of Forestry, Nanjing Forestry University Nanjing 210037
  • Received:2016-09-23 Revised:2016-12-11 Online:2017-05-25 Published:2017-06-22

摘要: [目的] 探讨无菌和带菌松材线虫对赤松的致病性,明确松材线虫在松树萎蔫病中的地位,为致病机制研究及病害防控提供依据。[方法] 以赤松胚性愈伤组织为材料,每块组织接种无菌松材线虫250条,培养12天后,利用2,3,5-三苯基氯化四氮唑(TTC)染色法对愈伤组织进行染色,分析无菌松材线虫对赤松愈伤组织细胞活性的影响。以赤松组培苗和4年生盆栽实生苗为对象,每株接种无菌松材线虫和带菌松材线虫(组培苗250条,4年生苗3 000条),分别于18天和35天后统计2种类型线虫接种所致松苗萎蔫率,并对植株体内线虫进行再分离,分析2种类型松材线虫对赤松幼苗的致病能力。[结果] 无菌松材线虫接种导致赤松胚性愈伤组织严重水渍化,TTC染色为米黄色或淡粉色,证明愈伤组织已失去活性。无菌松材线虫和带菌松材线虫接种均能导致赤松组培苗萎蔫,接种18天后,萎蔫率分别为70%和60%;萎蔫植株中均可再分离到松材线虫,其每株平均分别为599±567条和365+240条,二者差异显著(P<0.01)。剩余外观健康的赤松组培苗中也分离到线虫,但数量较少(仅10~20条)。2种类型松材线虫接种引起赤松盆栽苗同等程度的萎蔫,接种35天后的萎蔫率均达80%,无菌和带菌松材线虫致萎蔫植株中均可再分离到松材线虫,其每株平均分别为34 733±34 162条和25 057±21 410条,但差异不显著(P=0.508)。其余外观尚健康的松苗也进行线虫分离,2株接种无菌松材线虫的植株体内线虫分别为486条和22条;接种带菌松材线虫的2株松苗中,1株分离到646条线虫,另1株没有分离到线虫。[结论] 无菌和带菌松材线虫均对赤松具有致病性,松材线虫是赤松萎蔫的直接原因,伴生细菌并非赤松萎蔫的必须因子。

关键词: 松材线虫病, 松材线虫, 无菌, 致病性, 赤松

Abstract: [Objective]The aim of this study is to explore the pathogenicity of aseptic Bursaphelenchus xylophilus (pine wood nematode, PWN) and non-aseptic PWN on Pinus densiflora, to better understand the role of PWN in pine wilt disease development, and to provide useful information on pathogenicity mechanism and disease control. [Method] The embryogenic calli of P. densiflora were inoculated with aseptic PWNs under aseptic conditions and cultured for 5 days. The effect of aseptic PWNs on the activity of embryogenic callus cells were evaluated by staining the embryogenic calli with the triphenyltetrazoliumchloride (TTC) solution. Tissue-cultured microshoots and 4-year-old seedlings cultured in green house were inoculated with aseptic PWNs and non-aseptic PWNs, respectively. In 18 and 35 days after inoculation, the pine wilt ratio was recorded and the PWNs were isolated from inoculated seedlings. The pathogenicity of aseptic PWN and non-aseptic PWN on P. densiflora was analyzed. [Result]The inoculation by aseptic PWNs caused severe water stain in embryogenic calli of P. densiflora, and the TTC assay showed beige/light pink of embryogenic calli, revealing that the embryogenic cells lost viability. The embryogenic calli of control treatment remained healthy and showed bright red in TTC assay. Both the aseptic PWN and non-aseptic PWN wilted the tissue-cultured microshoots of P. densiflora. The wilting rates of the microshoots were 70% and 60% in 18 days after inoculation with aseptic PWNs and non-aseptic PWNs, respectively. PWNs were recovered from all wilted microshoots with average number of PWNs 599+567/microshoot for aseptic PWNs inoculation and 365+240/microshoot for non-aseptic PWNs. The number of nematodes recovered from wilted microshoots showed significant difference (P < 0.01) between aseptic PWN and non-aseptic PWN treatments. Fewer PWNs (10-20/microshoot) were also recovered from the remaining healthy-looking microshoots. The aseptic PWN could induce wilting of potted-seedlings of P. densiflora, as the same as non-aseptic PWN, with average of 80% wilting ratio in 35 days after inoculations. PWNs were recovered from wilted seedlings in both treatments. The number of recovered PWNs per seedling was 34 733±34 162 and 25 057±21 410 for aseptic PWNs and non-aseptic PWNs inoculations, respectively. No significant difference was found in number of recovered PWNs between the two treatments (P = 0.508). The number of recovered PWNs in two healthy-looking seedlings of aseptic PWNs treatment was 486 and 22, while only one healthy-looking seedlings of non-aseptic PWNs treatment contained 646 PWNs. [Conclusion] The aseptic and non-aseptic PWN could cause wilting of P. densiflora. The PWN is the main factor causing wilt of Japanese red pine, while the bacteria carried by the PWN are not necessary for the development of PWD.

Key words: pine wilt disease, Bursaphelenchus xylophilus, asepsis, pathogenicity, Pinus densiflora

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