【作者】 杨廷；

【导师】 周泽扬；

【作者基本信息】 西南大学 ， 微生物学， 2007， 硕士

【摘要】 烟草黑胫病（Tobacco Black Shank）是一种由烟草黑胫病菌[Phytophthora parasitica var．nicotianae（Breda de Hean）Tuker]引起的烟草病害，破坏性极强，对所有栽培烟草具有世界范围内的毁灭性危害，其防治十分困难。目前，人们主要是用化学农药来防治该病。然而，由于具有环境污染严重、残留药物对人畜危害大、容易诱导产生病虫害的耐药性等问题，传统的化学农药已经受到严重的质疑和挑战。与之相比，生物农药具有对哺乳动物毒性低、对环境压力小等优点。因此，生物农药的研制与开发具有很大价值和潜力。在研究烟草黑胫病生防菌的过程中，我们从土壤中分离得到一株产多种抗生素的假单胞菌Pseudomonas sp.,编号为20^#-5，初步研究表明其对烟草黑胫病菌有较强的抑制作用。本论文在此基础上，对目标菌株20^#-5抑制烟草黑胫病菌的平板拮抗作用、防治烟草黑胫病的温室控病试验、田间防效试验及其定殖规律进行了系统的研究。结果如下：1．平板拮抗试验分别用20^#-5菌及其发酵液内的活性物质对烟草黑胫病菌进行了平板拮抗试验。结果表明，20^#-5菌明显抑制了黑胫病菌的生长，对峙培养60h后，抑菌带宽达到0.5cm；镜检受抑制的黑胫病菌菌丝，发现菌丝生长畸形，菌丝壁消解。20^#-5菌发酵液内的活性物质（以下简称20^#-5菌发酵液）对黑胫病菌菌丝的生长也有抑制作用，活性物质浓度越高，抑制率越强。2．温室控病试验采用20^#-5菌发酵液对烟草黑胫病进行盆钵控病研究。20^#-5菌发酵液对烟草黑胫病发病前的处理和发病后的处理均有一定的防治效果，分别达到59.32％和10.17％，前者防效明显高于后者。温室盆栽试验表明，20^#-5菌发酵液对烟草黑胫病的预防效果远远好于治疗效果，这为田间防治提供了重要信息。3．田间防效试验发病初期，20^#-5菌发酵液对烟草黑胫病发病前的处理防效为64.39％，低于20％移栽灵防治效果的72.07％；高于58％甲霜灵-锰锌（防效为44.88％）；发病后的处理防效仅为39.88％。统计结果显示，各药剂处理的防效之间差异不显著。发病盛期，20^#-5菌发酵液对烟草黑胫病发病前的处理防效有所上升，达到75.68％，高于发病后的处理（防效为50.00％）、58％甲霜灵-锰锌（防效为36.58％）和20％移栽灵（防效为29.77％），但其与前二者差异不显著，与后者差异显著。两个化学药剂处理的防效均有下降趋势。发病末期，20^#-5菌发酵液对烟草黑胫病发病前的处理防效仍在上升，达79.70％，显著高于58％甲霜灵-锰锌（防效为37.84％）和20％移栽灵（防效为29.16％）；也高于发病后的处理（防效为49.02％），但差异不显著。与发病初期、盛期、末期的各对照药剂比较，20^#-5菌发酵液对烟草黑胫病发病前的处理防效和发病后的处理防效一直上升，说明其药效稳定，持效期较长。对黑胫病发病前的处理的防效明显高于发病后的处理，进一步证明20^#-5菌发酵液对烟草黑胫病主要起预防作用。4．定殖试验通过逐步提高链霉素（Streptomycin，Stre）浓度的方法，成功对20^#-5菌进行了抗链霉素标记，获得了抗200μg·ml^-1 Stre的自发突变株，记为20^#-5^*。20^#-5^*菌在牛肉膏蛋白胨培养基中连续传代10次，仍具有稳定的抗药性，且与原始菌株具有相似的平板拮抗能力和盆栽控病效果，可以作为20^#-5菌的标记株进行定殖研究。在20^#-5^*菌的定殖试验中，所用烟草品种和土壤均与盆栽控病试验一致。结果表明，菌悬液灌根处理1d后，就能在烟草根、茎、叶内部检测到20^#-5^*菌。根内部标记菌的数量在灌根后7d里逐步上升，之后有所下降；茎和叶内部标记菌的数量在灌根后12d里一直处于上升阶段，之后也有所下降。这说明20^#-5菌具有内生性。灌根后17d，仍能在烟草根、茎、叶表面检测到标记菌株，说明20^#-5菌能够长时间定殖于烟草各组织表面。从定殖数量消长动态看，20^#-5^*菌在烟草根、茎、叶内部均有一个“由升到降”的数量变化趋势。20^#-5^*菌在根内部的定殖量明显高于茎和叶，茎和叶之间相差不大，茎稍高。更多还原

【Abstract】 Tobacco black shank caused by Phytophthora parasitica var. nicotianae is a serious plant disease which distributes widely in the world. It has drastic destruction to all cultural tobacco and is very difficult to be controlled. For a long time, people have been trying to control this disease with chemical pesticides, mainly including metalaxyl, yizailing. However, because of disadvantages such as contamination to environment, harm to human body, inducing resistance of the pest, chemical pesticides had been challenged. While biological pesticides have the merits of low toxicity to mammal and little pressure to environment. Therefore, the development of biological pesticides is valuable and potential.Pseudomonas sp. 20^#-5 strain is an antibiotics-producing bacterium. Preliminary study indicated that its metabolites could inhibit the growth of Phytophthora parasitica var. nicotianae. In this paper, we reported the petri dish antagonism test, greenhouse disease-control experiment, field control effect of this strain to tobacco black shank and its colonization in tobacco plant body.1. Antagonism test in petri dish20^#-5 strain and its ferment products were added to the medium respectively to test their antagonism to Phytophthora parasitica var. nicotianae. Results showed that both the strain and active compound of its ferment products （we call it ferment products of 20^#-5 strain for short below） obviously inhibited the growth of P. parasitica. The inhibition zone was 0.5cm after cultivation for 60h. While tested under microscope, the mycelia of the P. parasitica were found to grow abnormally and their walls had been destructed.2. Disease-control experiment in greenhouseArtificial inoculation test in greenhouse demonstrated that ferment products of 20^#-5 strain could control tobacco black shank efficiently. The control efficiency of the treatment with ferment products before disease was 59.32％, while that after disease was only 10.17％. This result indicated that the efficiency of ferment products of 20^#-5 strain to tobacco black shank was "prevention" but not "therapy".3. Field control effect testControl efficiency of each treatment was high at the beginning of disease because of the low attack rate. The efficiency of the treatment with ferment products before disease was 64.39％. It was a little lower than the efficiency of yizailing （72.07％）, but higher than metalaxyl. The efficiency of the treatment with ferment products after disease was only 39.88％. Statistical analysis indicated that they were not significant one another. The efficiency of the treatment with ferment products before disease upgraded to 75.68％, higher than that of ferment products after disease （50.00％）, metalaxyl （36.58％） and yizailing （29.77％） at intermediate stage of disease. The statistical analysis showed significant difference when compared with yizailing. Notably, the efficiency of two chemical pesticides had decreased.The efficiency of the treatment with ferment products before disease increased to 79.70％at the end of disease. It was significantly high when compared with those of metalaxyl and yizailing, which was 37.84％and 29.16％, respectively; but it wasn’t distinct when compared with that of treatment with ferment products after disease.Compared with other treatments, the efficiency of the treatment with ferment products of 20^#-5 strain before and after tobacco black shank upgraded consecutively, which indicated that it had a steady efficiency and long persistence. The efficiency of the treatment before disease was higher than that of after disease proved that the effect of ferment products of 20^#-5 strain was "prevention".4. Colonization testSpontaneous mutant strain 20^#-5* resisting 200μg-ml^-1 streptomycin was obtained by increasing antibiotic concentration in the culture step by step. After being transferred 10 generations on the plate medium, it still maintained the resistance to Stre and the antagonism activity to P. parasitica in petri dish and greenhouse control effects. This mutant strain was used for the colonization analysis.In the colonization test, the tobacco variety and soil were the same with those in the greenhouse disease-control experiment. Results indicated that strain 20^#-5* could be detected in the inner part of tobacco roots, stems and leaves one day after watering roots with the suspension. The amounts had been upgrading for seven days in the inner roots while twelve days in the inner stems and leaves, then decreased respectively. This suggested that 20^#-5 strain had the endogenousness. Besides, 20^#-5* strain could colonize on the surface of tobacco roots, stems and leaves seventeen days after watering roots. It suggested that they could retain for a long time on the surface of tobacco tissues.The amounts of 20^#-5 colonized in tobacco roots, stems and leaves had the tendency of first increasing and then decreasing. The population of 20^#-5* in the roots was more than those in other tissues. The amounts in stems were slightly higher than those in leaves.更多还原