中国农业科学 ›› 2015, Vol. 48 ›› Issue (5): 889-899.doi: 10.3864/j.issn.0578-1752.2015.05.07

• 植物保护 • 上一篇    下一篇

病原真菌降解两种蚧虫体壁过程中胞外酶作用

董晶1,谢映平1,刘卫敏2,牛秀萍1,薛皎亮1   

  1. 1山西大学生命科学学院,太原 030006
    2山西大学应用生物学研究所,太原 030006
  • 收稿日期:2014-09-27 出版日期:2015-03-01 发布日期:2015-03-01
  • 通讯作者: 谢映平,Tel/Fax:0351-7018092;E-mail:xieyingping@eyou.com
  • 作者简介:董晶,E-mail:dongjing003@163.com
  • 基金资助:
    国家自然科学基金(31070584)、高等学校博士学科点专项科研基金(20101401110008)

Role of Extracelluar Enzymes of the Entomopathogenic Fungi During Degrading the Integument of Two Species of Scale Insects

DONG Jing1, XIE Ying-ping1, LIU Wei-min2, NIU Xiu-ping1, XUE Jiao-liang1   

  1. 1College of Life Science, Shanxi University, Taiyuan 030006
    2Research Institute of Applied Biology, Shanxi University, Taiyuan 030006
  • Received:2014-09-27 Online:2015-03-01 Published:2015-03-01

摘要: 【目的】探究病原真菌降解蚧虫体壁过程中胞外酶的作用及其毒力效应,为应用病原菌对蚧虫进行生物防治提供依据。【方法】选用4株昆虫病原真菌—蜡蚧霉(Lecanicillium lecanii)菌株V3.4505、V3.4504、噬菌蚧霉(L. fungicola)菌株HEB02和镰刀菌Fusarium incarnatum-equiseti菌株HEB01,以日本龟蜡蚧(Ceroplastes japonicus Green)和沙里院褐球蚧(Rhodococcus sariuoni Borchsenius)为靶标害虫,以这两种蚧虫的表皮作为病原菌培养基的唯一碳源,测定培养过程中各菌株4种胞外酶的活性变化,包括脂肪酶、类枯草杆菌蛋白酶(Pr1酶)、几丁质酶和N-乙酰-氨基葡萄糖苷酶(NAG酶),并据此分析真菌入侵蚧虫体壁过程中胞外酶的作用。同时,测定两种蚧虫被4株病原真菌感染8 d后的死亡率,用于评价菌株和胞外酶的毒力效应。【结果】4株病原菌在降解两种蚧虫的体壁过程中,4种胞外酶的活性都发生了明显的变化,呈现先升高后降低趋势。脂肪酶的活性高峰出现得最早,在培养2 d后就达到最大值,且在日本龟蜡蚧上各菌株脂肪酶的活性明显高于沙里院褐球蚧。菌株的Pr1酶活性高峰出现在3—4 d,而几丁质酶和NAG酶的活性高峰分别出现在6 d和4—5 d。4个菌株相比较,V3.4505菌株对日本龟蜡蚧和沙里院褐球蚧的致死率均为最高,分别是73%和81%,且与HEB02菌株和HEB01菌株有差异显著。4个株菌的Pr1酶活性平均值与它们对日本龟蜡蚧和沙里院褐球蚧感染8 d后累计死亡率的线性相关性显著,线性方程分别为:y=0.082x+5.822(R2=0.823)和y=0.119x+14.75(R2=0.764);4个株菌的几丁质酶活性平均值与两种蚧虫累计死亡率的线性相关也较显著,线性方程分别为:y=-0.148x+15.89(R2=0.645)和y =0.095x+10.46(R2=0.762)。【结论】在对两种蚧虫的感染过程中,病原真菌的4种胞外酶均参与了对蚧虫蜡质和体壁的降解。脂肪酶降解虫体表面的蜡质,酶活性高峰出现最早,在蜡壳厚的日本龟蜡蚧上其酶活性表现得更高。在其余3种酶中,先由Pr1酶作用,降解蚧虫原表皮中的蛋白质,再由几丁质酶和NAG酶作用,降解几丁质。酶作用高峰时间与酶活性大小与其底物在蚧虫体壁中的排列层次与含量相吻合。菌株的酶活性和蚧虫致死率相关性分析说明,Pr1酶和几丁质酶可作为菌株的毒力指示因子。4个菌株相比较,蜡蚧霉V3.4505菌株表现出较好的致病特性,说明其对蚧虫的毒力较高,可考虑作为蚧虫生物防治应用的病原菌种。

关键词: 病原真菌, 胞外酶, 日本龟蜡蚧, 沙里院褐球蚧, 体壁, 毒力

Abstract: 【Objective】The objective of this study is to explore the effect of extracellular enzymes and their virulence during entomopathogenic fungi degrading the integument of scale insects and to provide evidence for biological control by applying entomopathogenic fungi. 【Method】 The strains of the entomopathogenic fungi, Lecanicillium lecanii V3.4505, V3.4504, L. fungicola HEB02, and Fusarium incarnatum-equiseti HEB01 were used and the two species of scale insects, Ceroplastes japonicus Green and Rhodococcus sariuoni Borchsenius, as the target were studied. The cuticle of the two scale insects was used as the sole carbon source in the medium for fungal culturing. The activities of four extracellular enzymes, including lipase, protease (Pr1), chitinase, and N-acetyl-D-glucosaminidase (NAGase), produced by the four strains were determined, and based on this, the function of the extracelluar enzymes during the strains penetrating the integument of scale insects was analyzed. Meanwhile, the cumulative mortalities of the two scale insects infected with the four strains for eight days were assayed to evaluate the virulence of the strains and the extracellular enzymes. 【Result】 The activities of the four enzymes all changed significantly during the scale insect integument-degrading process, their changing trend all exhibited rising in the first few days and falling in the later. The activity peak of lipase appeared earliest that was at two days after inoculation, and the lipase activity of the strains cultured on the cuticular medium of C. japonicus was obviously higher than that on R. sariuoni. The activity peak of Pr1 came after 3-4 d while the activity peak of chitinase and NAGase appeared at 6 d and 4-5 d, respectively. Among the four strains, V3.4505 strain caused the highest mortality rate of 73% for C. japonicus and 81% for R. sariuoni, respectively, and that showed significant difference with other two strains, HEB02 and HEB01. The average values of Pr1 activity of four strains were significantly related to the cumulative mortality of the two scale insects, and the linear equations were y=0.082x+5.822 (R2=0.823) for C. japonicas and y=0.119x+14.75 (R2=0.764) for R. sariuoni. Similarly, the average chitinase activity of four strains were also significantly related to the cumulative mortality of the two scale insects, the linear equations were y=-0.148x+15.89 (R2=0.645) for C. japonicas and y=0.095x+10.46 (R2=0.762) for R. sariuoni. 【Conclusion】 The four extracellular enzymes worked together to degrade the wax and the integument of the scale insects in the infecting process. The lipase degraded the wax on the scale insects and its peak came earliest, and its activity of the four strains cultured on the cuticular medium of C. japonicus was obviously higher than that on the R. sariuoni because C. japonicus has a thicker wax layer. Among other three enzymes, Pr1 worked first to degrade the protein in the procuticle of scale insects then chitinase and NAGase worked together to degrade the chitin in the integument. The activity peak and level of each enzyme were corresponding to the distribution and content of its substrate in the integument of scale insects. The correlation analysis on the enzyme activities of the strains and mortality of the scale insects indicated that Pr1 and chitinase should be used as a virulence indication for the strains. Among the four strains, L. lecanii V3.4505 showed better pathogenic characteristics, which means this strain possesses higher virulence against scale insects and it can be considered as a better pathogenic fungus for biological control.

Key words: entomopathogenic fungi, extracellular enzymes, Ceroplastes japonicus Green, Rhodococcus sariuoni Borchsenius, integument, virulence