【作者】 杜立新；

【导师】 曹克强； 冯书亮；

【作者基本信息】 河北农业大学 ， 植物病理学， 2004， 硕士

【摘要】 本研究采用平板对峙法测定了供试的枯草芽孢杆菌(Bacillus subtilis)BS-208、BS-209和BS-21及蜡质芽孢杆菌(Bacillus cereus)BC-01对番茄灰霉病菌(Botrytis cinerea)的抑制作用。在温室条件下测定了它们对番茄灰霉病的保护作用和治疗作用，并于2002年和2003年在田间条件下测定了BS-208和BS-209菌株制剂对番茄灰霉病的防治效果。结果表明，枯草芽孢杆菌BS-208、BS-209和BS-21及蜡质芽孢杆菌BC-01对番茄灰霉病菌的抑菌率分别为78.06％、82.49％、75.74％和73.84％；温室盆栽试验表明只有BS-208和BS-209菌株对番茄灰霉病有较好的保护作用，两菌株发酵液和菌体处理后24、48h接种病菌，防效均达75％以上，好于0h接种的防效，但无菌滤液防治效果较差；BS-208和BS-209菌株的发酵液和无菌滤液对番茄灰霉病表现出较高的治疗活性，BS-21和BC-01菌株发酵液的治疗活性很低。两年的田间试验表明枯草芽孢杆菌BS-208和BS-209制剂对番茄灰霉病均具有良好的防治效果，并且防效随浓度加大而提高，以800倍的防效最好，两菌株制剂800倍液连续三次施药后防效均达到74％以上，并且BS-208菌株的防效略高于BS-209菌株。 通过对BS-208菌株与BS-209菌株生理生化特征的测定，结果表明两菌株的生理生化特征基本上是一致的。对BS-208和BS-209菌株生长曲线测定，结果表明在PDB液体培养基中BS-208和BS-209分别在培养24h和28h达到最高的生长量，都在48h形成芽孢。通过测定化学药剂对两菌株生长影响，结果表明在烯唑醇、甲基托布津、多菌灵、粉锈宁、多抗霉素、吡虫啉、辛硫磷、阿维菌素、高效氯氰菊酯和锐劲特田间使用的最高浓度下，基本上不影响两菌株的生长和芽孢形成，而代森锰锌可以完全抑制两菌株的生长。 采用生长速率法测定BS-208和BS-209菌株对番茄灰霉病菌菌丝生长的抑制作用，结果表明，BS-208菌株的无菌滤液对番茄灰霉病菌菌丝生长的抑制率为49.58％，而BS-209菌株抑菌效果不好。用载玻片悬滴法测定两菌株的发酵液对番茄灰霉病菌分生孢子萌发的抑制作用，结果表明，在稀释10倍的菌株发酵液处理后，BS-208和BS-209的抑制率达到93.45％和92.35％，且未见有附着胞形成，经两菌株发酵液原液处理可以完全抑制孢子萌发。 在温室和田间条件下研究了BS-208和BS-209在番茄叶面和体内的定殖与传导。通过针刺接种利福平标记的BS-208和BS-209菌株，24h后就可在全株各个部位分离到目的菌。在温室和田间条件下，BS-208菌株的定殖菌量分别为11×10~8～5.6×10~8cfu/g鲜叶和15×10~8～0.02×10~8cfu/g鲜叶，BS-209菌株的定殖菌量分别为222×10~6～55×10~6cfu/g鲜叶和205×10~6～0.48×10~6cfu/g鲜叶；印象法观察发现BS-208和BS-209菌株在叶片上都能定殖，但BS-208的定殖能力强于BS-209菌株；扫描电镜观察表明，BS-208菌株在番茄叶面分布不均匀，大多定殖于伤口周围、叶面的凹陷处和绒毛的根部。 通过测定与抗病性有关的酶活性结果表明，接种BS-208菌株发酵液48h后PAL、POD活性达到最大值，随后PPO在96h达到最大值。 对BS-208菌株的无菌滤液的酸碱稳定性、热稳定性进行了测试。结果表明，BS一208菌株的无菌滤液热稳定性较强，耐酸，在强碱性条件下不稳定。 在摇瓶中对BS一208和BS一209的发酵培养基及发酵条件进行了筛选。‘结果表明，所筛选到BS一208的理论最优培养基组合为氮源A4%，淀粉1.6%，酵母粉0.5%，理论最优发酵条件组合为转速230 rpm，初始pHS，接种量0.5%，装量70 mL(500 mL三角瓶中)，发酵周期为60h;BS-209的理论最优培养基组合为氮源A3%，淀粉1.6%，酵母粉0.5%，理论最优发酵条件组合为转速230 rpm，初始pHS，接种量1%或3%，装量50 mL(500 mL三角瓶中)，发酵周期为58 ho 利用平板对峙法测定了BS一208和BS一209对19种植物病原真菌的抑菌作用。结果表明，两菌株除对苹果轮纹病菌抑菌率较低为18.34%和10.18%以外，对所测定的其余18种植物病原真菌都有较高的抑制作用。更多还原

【Abstract】 Inhibition rates of Bacillus subtilis, strains BS-208, BS-209 and BS-21, Bacillus cereus, strain BC-01 against Botrytis cinerea were tested by dual culture on potato dextrose agar. Results showed that inhibition effects of 4 strains were 78.06%, 82.49%, 75.74% and 73.84%, respectively. The protective and curative effects of Bacillus subtilis BS-208, BS-209, BS-21 and Bacillus cerus BC-01 against B. cinerea were assayed in vivo under greenhouse conditions. Control efficacies of preparations of strains BS-208 and BS-209 were also assayed against B. cinerea in the field in 2002 and 2003. The greenhouse experiment result showed that the fermentation liquid and spore of strains BS-208 and BS-209 had control efficacy above 75% with inoculating after 24 and 48 hours, but the filtrate had poor control efficacy. Result of two years field experiment in the field indicated that preparations of strains BS-208 and BS-209 could effectively control tomato gray mould. The control efficacy became better with the increasing of concentration and 800-fold treatment showed the best control efficacy. After three times treatment with the 800-fold preparation continuously, the control efficacy reached above 74% and effect of strain BS-208 was better than that of BS-209.The result of biochemical characters of strains BS-208 and BS-209 showed that the characters of two strains were basically consistent. The growth curve of the two strains was assayed, results showed that the time of strain BS-208 and BS-209 reached the highest amount in PDB liquid media were 24 hours and 28 hours after cultivation respectively and both of the two strains could form spore 48 hours after cultivation. Two strains could be cultured together with diniconazole, thiophanate-mthyl,carbendazim, triadimefon, polyoxin, imidacloprid, phoxim, abamectin, beta-cypermethrin and fipronil with the maximal concentrations used in field. Mancozeb could inhibit entirely the growth of strains BS-208 and BS-209.Inhibition effects of strains BS-208 and BS-209 to B. cinerea were tested by means of mycelium growth inhibition method. The filtrate of strain BS-208 could inhibit mycelium growth of B. cinerea with inhibition rate of 49.58%. The inhibition efficacy of strain BS-209 was poor. Two strains were tested for their effects on conidia germination of B. cinerea. After diluting 10-fold fermentation liquid of strains BS-208 and BS-209 the suppressive rate of spore germination were 93.45% and 92.35%. No formation of appressorium was found.Colonization and conduction of antagonist B. subtilis strains BS-208 and BS-209 on phylloplane and inside of tomato body were studied by using mutant strains of BS-208 and BS-209 resistant to rifampicin under greenhouse and field conditions. Within 24 hours after injection on the stem, target bacteria were isolated from all parts of tomato body. Results showed that population of strain BS-208 in greenhouse and field were 11 x 108~5.6 x 108cfu/g and 15 x 108~0.02 x 108cfu/g fresh weight respectively, whereas that of strain BS-209 in greenhouse and field were 222 x 106~55 x 106cfu/g and 205 x 106~0.48 x 106 cfu/g fresh weight, respectively. Impression observation method result showed that strain BS-208 and BS-209 could colonize on the leaf surface and the colonization ability of BS-208 was better than that of BS-209. Scanning electron microscopy observation exposed that strain BS-208 distributed asymmetrically on phylloplane of tomato and most of them colonized near the wound, in the sunken place and the base of floss.Induction of systemic resistance and related enzymes activities of superoxide dismutase (SOD), peroxidase (POD), polyphenoase (PPO) and phenylalanine ammonia lynse (PAL) by inoculation of BS-208 fermentation and B. cinerea were studied. Results indicated that activities of PAL and POD reached the maximum 48 hours after inoculation, while that of PPO reached the maximum 96 hours after inoculation.The stability of filtrate of BS-208 was measured after being treated at different pH values and temperatures. Results showed that it更多还原