摘要
对灰葡萄孢等病菌拮抗能力强、防病效果好的3株细菌，经形态、培养性状观察和生理生化测定，鉴定为2属2种，即W10为地衣芽孢杆菌[Bacillus licheniformis(weigmann)Chester]；W3和Y2-11-1为多粘类芽孢杆菌[Paenibacillus polymyxa(Prazmowski)Ash et al]。这两种对蔬菜灰霉病的高效拮抗细菌为国内首次报道和应用。
3株拮抗细菌的生长条件研究表明，通常菌量达到最大时的培养时间为24～72h；生长温度为27～30℃；培养液pH值为7～8。
根据试验结果及经济因素，拮抗细菌的最佳营养发酵配方是：W10菌株为1％麦芽糖，4％黄豆饼粉，0.2％硝酸钠，0.02％磷酸氢二钾和0.05％酵母膏：W3菌株为2％蔗糖，4％黄豆饼粉，0.4％酵母膏；Y2-11-1菌株为1％麦芽糖，2％黄豆饼粉，0.2％硝酸钠，0.02％磷酸氢二钾以及0.2％酵母膏。
3株拮抗细菌通过抗药性诱导，获得抗利福平350μg／ml的标记菌株，并用于检测在番茄植株体表的定殖能力。拮抗细菌在番茄植物体表的定殖与定殖部位、引进浓度、接种灰霉病菌时间和环境因子有关。在番茄叶表拮抗细菌处理后10～15d仍可检测到一定的菌量。拮抗细菌在番茄果表的定殖能力与最初引进的细菌浓度呈正相关，即引进的浓度越高，定殖时间越长，定殖的菌量也越大。除W10菌株外，在高浓度(10~9cfu／ml)下，拮抗细菌处理后10～15d也能检测到少量细菌。
与对照相比，接种灰霉病菌对拮抗细菌的定殖有一定的影响。但是，先接种拮抗细菌后接种灰霉病菌(间隔1d)要比拮抗细菌与灰霉病菌同时接种、先接种灰霉病菌后接种拮抗细菌影响小。这表明灰霉病菌与拮抗细菌间存在营养、空间的竞争，也显示拮抗细菌在灰霉病害发生前使用要比发生后使用效果好。
温度、湿度、光照等环境因子对拮抗细菌的定殖均有影响。拮抗细菌定殖的最佳条件一般为：高湿(RH 95～100％)、光照12h和温度25～35℃。在一定的温度
扬州大学硕士学位论文
范围内(15一35℃)，温度愈高，越有利于拮抗细菌的生长和定殖。光照对拮抗细
菌的定殖也影响很大，黑暗或持续光照24h条件下，拮抗细菌的定殖要比光照12h
差。一般来说，拮抗细菌的生长和定殖需要高湿(RH 95一10俄)条件，湿度越高，
定殖越好。
Three strains of bacteria antagonistic highly against Botrytis cinerea Per. were isolated from tomato leaves and soil. According to the morphological. physiological and biochemical features, the isolate W10 was identified as Bacillus licheniformis (Weigmann) Chester and the isolates W3 and Y2-11-1 belonged to Paenibacillus polymyxa (Prazmowski) Ash et al. It is first reported that these bacterial species had antagonism to B. cinerea on vegetables in China. The growth conditions of these antagonistic bacteria had been studied. The optimum culture time for antibiotic bacteria ranged from 24 to 72 hours. The optimum growth temperature was from 27 to 30癈. The optimum growth pH values in the media was from 7 to 8.
According to the experimental results and economical factors, the best commercial production medium of strain W10 was made up of maltose 1%. soybean cake powder 4%. NaNO3 0.2%, K2HPO4 0.02% and yeast extract 0.05%; that of W3 consisted of sucrose 2%, soybean cake powder 4% and yeast extract 0.4%; that of Y2-11-1 was composed of maltose 1%, soybean cake powder 2%, NaNO3 0.2%, K2HPO40.02% and yeast extract 0.2%.
Three antagonistic bacteria were induced to be resistant to rifampicin and used to study the colonization on the surface of tomato plants. The colonizing ability of antagonistic bacteria was related to plant parts, introduced bacterial concentration and environmental factors. About 10-15 days after introducing antagonistic bacteria, they could be detected from tomato phylloplane. The colonizing capacity of antagonistic bacteria on tomato fruit surface had positive Riationship to the introduced bacterial concentration. The higher bacterial concentration introduced, the bigger population and
the longer time of bacterial colonization. Except strain W10, other antagonistic bacteria could be detected from 10 to 15 days after introduction at high concentration (109cfu/ml).
Contrasted with inoculating antagonistic bacteria only, inoculation of Botrytis cinerea had some influence on the colonization of antagonistic bacteria. But the effect of inoculating antagonistic bacteria before B.cinerea was smaller than that of inoculating B.cinerea and bacteria together and inoculating antagonistic bacteria after B.cinerea. This showed that there were competitions in nutrition and space between antagonistic bacteria and B.cinerea. It also indicated that the prevention of antagonistic bacteria was better than their cure for grey mould.
Temperature, relative humidity and illumination had significant effects on the colonization of antagonistic bacteria. The optimum conditions of three strains were consistent as a whole. The optimum relative humidity was from 95% to 100%. The optimum light time was 12 hours for each day. The optimum temperature ranged from 25℃ to 35℃. In lower temperature the bacteria could not grow and colonize well on tomato plant. In the condition of continuous darkness or illumination, the colonization of bacteria was weaker than that of light for 12 hours a day. In general, the growth and colonization of antagonistic bacteria required high humidity. The higher relative humidity, the better colonization of antagonistic bacteria.
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