摘要
植物内生菌(endophyte)是指生长在健康植物组织、器官内部,不造成明显症状的正常微生物,并可以从经过表面消毒的植物内部分离得到。植物内生菌是一个十分丰富的类群,而抗菌内生菌作为其中的一类,能够产生很多新的抗菌活性物质,不但可以用于医疗上新药的研发,还可以用于农业植物病害的防治,但现阶段对植物抗菌内生菌的研究才刚刚开始,对其特性的认识还不够深入。传统的药用植物作为一类新的内生菌来源材料,正在成为内生菌研究者争相研究的对象。本文选择了药用植物华重楼(Paris polyphylla var. chinensis Franch)作为内生菌的分离材料,分离、筛选抗菌微生物,并对抑菌效果显著的内生菌进行了传统鉴定和分子鉴定,初步研究了抗菌物质的粗分条件及其相关性质。
从药用植物华重楼(Paris polyphylla var. chinensis Franch)地下块茎中分离得到79株内生菌,初步分为7株真菌,3株放线菌和69株细菌。通过对细菌病原菌的初步抑菌筛选,得到了21株内生菌对一种或两种病原菌具有抑菌活性,进一步对植物病原菌进行了拮抗筛选,有13株内生细菌对一种或几种植物病原菌具有拮抗活性,其中5株内生细菌对试验的5种植物病原菌均有较好的抑菌活性,并选择了抑菌效果最好的内生细菌Iun35进行了以后的一系列研究工作。
对菌株Iun35进行了形态学和生理生化特征的鉴定,确定该菌为枯草芽孢杆菌(Bacillus subtitls),该菌对水稻稻瘟病菌( Pyricularia oryzae Cav. )、松赤枯病菌( Pestalotia funereal)、玉米纹枯病菌(Rhizoctonia solani Kuha)、玉米弯胞病菌[ Curvularia lunata (Walk) Boed ]、小麦赤霉病菌[Gibberelle zeae ( Schw. ) Petch ]菌丝生长的抑制率分别达到80.5%、71.9%、70.5%、83.3%、57.1%。
菌株Iun35产生的抑菌物质可经(NH_4)_2SO_4沉淀,其溶解液再经丙酮低温沉淀后,丙酮上清液经减压旋转蒸发后得到粗提液。粗提液的温度、pH稳定性及蛋白酶敏感性试验表明抑菌成分是一种相当稳定的物质。粗提液经透析袋(22mm ,14 000D)透析后,抑菌物质仍存在于透析袋内,说明其分子量不小于10kD。粗提液具有广泛的抑菌谱,对受试的玉米纹枯菌(Rhizoctonia cerealis)、油菜菌核病(Sclerotinia sclerotiorum)等13株病原菌都有明显的抑制效果,说明该菌在生物农药和抗生素生产中具有很好的应用前景。
Plant endophyte, a natural microorganism, inhabiting in the tissues and apparatus of plant and having no obvious harm to plant itself, can be separated from the inner part of disinfected plant. Plant endophyte is a big group with various kinds. As a kind of endophyte, antimicrobial endophyte is a potential source of novel chemistry and biology to be applied in not only new drug research and development of the medical, but also the biological control of plant diseases in agriculture. Now we are just at the beginning of the research on endophytes, and many of their characters have not been understood deeply. As a novel materials inhabited by endophytes, the traditional medicinal plants are becoming the objectives of the endophytic researchers. In this scientific dissertation, the medicinal plants Paris polyphylla var. chinensis Franch were chosen as the materials for separation of endophytes. The antimicrobial endophytes were screened and the strain with remarkable antimicrobial effect was identified by the traditional characterization and the molecular characterization. It was studied preliminarily about the crude separated conditions of antibiotic substance and its related properties.
79 strains of endophytes have been separated from the underground tuber tissues of Paris polyphylla var. chinensis Franch, a traditional medicinal plant which is collected at Pengzhou county, Sichuan province in southwest China, included 7 fungi, 3 actinomycetes and 69 bacteria. By preliminary antimicrobial screening against pathogenic bacteria, 21 endophytes which had antimicrobial activity against one or two kinds of pathogenic bacteria were obtained. Through further screening against plant pathogenic fungi, there were 13 endophytic bacteria with antimicrobial activity against one or several kinds of plant pathogenic fungi, in which 5 endophytic bacteria had better antimicrobial activity against all plant pathogenic fungi in test. The strain Iun35 with best antimicrobial activity was selected to the next series of studies.
Based on the characteristics of morphology, physiology and biochemistry as well as the 16S rDNA analysis, the strain Iun35 was classified into Bacillus subtilis. The strain was tested for antagonism toward 5 plant pathogenic fungi by measuring its growth rate. The results showed that its inhibitory rates against Pyricularia oryzae Cav, Pestalotia funereal, Rhizoctonia solani Kuha , Curvularia lunata (Walk) Boed. and Gibberelle zeae ( Schw. ) Petch were 80.5%、71.9%、70.5%、83.3%、57.1% respectively.
The antibiotic substance produced by the strain Iun35 can be fractionally precipitated by ammonium sulfate with series of concentration, then the solution of precipitation was precipitated again by acetone under low temperature. The crude extracts containing antibiotic substance was obtained from the supernatant treated with acetone by vacuum rotary evaporation. Through the stability test of pH and temperature and the proteinase sensitivity test, it was obvious that the antibiotic substance is a quite stable material. After dialysed by dialysis-bag (22mm, 14 000D), the antibiotic substance was still presented in the dialysis-bag which showed that the molecular weight of the antibiotic substance was no lower than 10kD. The crude extracts have a widely antibiotic spectrum, and possessed inhibitory effect against 13 plant pathogenic fungi, included Rhizoctonia cerealis, Sclerotinia sclerotiorum and Physalospora piricala etc. The results demonstrate that the strain Iun35 has a wonderfully potential application in biological pesticide and antibiotic production.
引文
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