【作者】 张建军；

【导师】 童蕴慧；

【作者基本信息】 扬州大学 ， 作物遗传育种， 2004， 硕士

【摘要】 用紫外线、亚硝基胍、和腐霉利对采自田间的对腐霉利（procymidone）敏感的5个灰葡萄孢（Botrytis cinerea）菌株（HY2-1、HY2-2、HY2-3、DF2-2、XY6-1）分生孢子进行抗药性诱导，共获得抗腐霉利菌株67个，其中高抗菌株(EC₅₀＞100μg／ml)54个，占抗性菌株总数的80.60％；中抗菌株(10μg／ml＜EC₅₀≤100μg／ml)2个，占2.98％；低抗菌株(1μg／ml＜EC₅₀≤10μg／ml)11个，占16.42％。3种诱变方法以紫外线诱变频率最高，平均为0.413×10^-6；其次为亚硝基胍，平均诱变频率为0.252×10^-6；腐霉利诱变频率为0.190×10^-6。诱导的5个抗性菌株中，以XY6-1菌株较易产生变异，变异率达0.591×10^-6，且突变菌株都为高抗菌株；HY2-3菌株较难变异，变异率仅为0.147×10^-6。 筛选出感腐霉利抗乙霉威的菌株XY6-1（Dic^S Npc^R）和抗腐霉利感乙霉威的菌株XY6-1 140（Dic^R Npc^S），将这两个菌株在PSA平板上进行对峙生长获融合菌丝，挑取融合部位菌丝在PSA平板上培养至产孢，经单孢分离和测定后得到18个Dic^R Npc^R表型的单孢菌株。测定18个单孢后代的抗性水平，其中16个菌株对腐霉利的抗性水平与抗性亲本（Dic^RNpc^S）一致，2个菌株对腐霉利的抗性水平低于抗性亲本。 将抗性菌株在不含药剂的培养基上转代培养，直至20代，测定后代的抗药水平。结果表明，有79.1％的菌株抗性水平稳定，与其亲本一致，20.9％的菌株抗性不稳定，其中部分菌株丧失了抗药性。另外，高抗菌株中有87.0％菌株的抗药性能稳定遗传，而中低抗菌株有53.8％抗药性呈不同程度降低。 从抗性稳定的高抗菌株中，挑选出6个菌株进行单孢分离，共得到455个单孢菌株。测定单孢株的抗性水平表明，有86.8％单孢株抗性水平与亲本比较没有下降，也没有出现抗药性丧失的单孢菌株。来自DF2-2 050、HY2-2 070、XY6-1 140的单孢菌株，其抗性水平都能稳定遗传，而HY2-1 200、HY2-3 050、D1-3的单孢菌株，24.3％抗性水平呈不同程度的下降。 通过菌丝生长速率、产孢能力、致病性等性状的测定，比较诱导的高抗菌株HY2-1 200、HY2-2 070、DF2-2 050、XY6-1 140与其原始菌株及野生抗性菌株D1-3与敏感菌株的生物学特性差异。发现诱导的抗性菌株的菌丝生长速率普遍显扬州大学硕士学位论文著性慢于敏感菌株;而致病性、产抱能力差异较大。Dl一3的菌丝生长率和致病性界于敏感菌株之间:产抱量显著低于敏感菌株。 用相同浓度的抗感菌株分生抱子液混合接种番茄叶片，发病后分离病斑及病斑表面的分生抱子。测定病斑和分生抱子形成的菌株对腐霉利的抗感性。试验表明，有2个抗性菌株的适合度高于其原始菌株，2个菌株低于其原始菌株。野生抗性菌株Dl一3的适合度低于敏感菌株。更多还原

【Abstract】 5 strains of Botrytis cinerea (HY2-1, HY2-2, HY2-3, DF2-2, XY6-1) sensitive to procymidone were isolated from vegetables in Jiangsu province. 67 mutants resistant to the fungicide were obtained from the sensitive strain conidia treated with ultraviolet, N-methyl-N-nitroguanidine and procymidone. Of all resistant mutants, the low-resistant (1 g/ml<EC50 10g/ml), the moderate-resistant (10 g/ml<EC50 100 g/ml )and the high-resistant(EC50>00 g/ml) ones accounted for 16.42%, 2.98% and 80.60% respectively. The mutant frequency of the strains by the treatment of ultraviolet reached 0.413 10-6 while those by the treatment of N-methyl-N-nitroguanidine and procymidone were 0.252 10-6 and 0.190 10-6 respectively. The mutant frequency of XY6-1 was 0.591 10-6, higher than that of others and all mutants from XY6-1 had high resistance. But the resistant mutation in HY2-3 was more difficult than other strains, with the low mutant frequency of 0.147 10-6.XY6-1 (DicSNpeR) with the sensitivity to procymidone and the resistance to diethofexcarb, and XY6-1 140 (DicRNpcS) with the resistance to procymidone and the sensitivity to diethofexcarb were obtained and inoculated opposite on PSA. Anastomosis region was formed between the two colonies after cultured 4 day. Anastomosis mycelia were moved and cultured for sporulation on PSA. 18 strains resistant to two fungicides (DicRNpcR) were screened out from 203 conidium progenies by single spore isolation. Of 18 resistant strains with DicRNpcR, there were 16 strains with same resistant level to procymidone as their resistant parent and two strains with the lower resistant level than the resistant parent.The resistant mutants were continuously transferred from tube to tube for 20 times or generations and each generation was grown on PSA for 3d. 79.1% of all progenies were as resistant to procymidone as the original strains and 13.4% lost resistance. Moreover, 87.0% of the high resistant mutants were stable, more than the moderate or low resistant ones (46.2%).455 single conidium progenies were obtained from 6 mutants high resistant toprocymidone. 86.8% of all progenies could keep high resistance. Moreover, the resistance of single spore progenies from DF2-2 050, HY2-2 070 and XY6-1 140 was stable while 24.3% of single spore progenies from HY2-3 050, HY2-1 200 and Dl-3 appeared some decline in resistance.The biological characters such as mycelium growth rate, sporulation capacity and pathogenicity of resistant mutants were compared those of their original sensitive strains. The results showed that the mycelia of mutants grew more slowly than their original sensitive strains. But there were no regular differences in sporulation capacity and pathogenicity between resistant mutants and their original sensitive strains.The competitive ability of resistant mutants on tomato leaves were compared by inoculating the conidial suspension mixed from resistant mutants and their original sensitive strains in proportion of 1:1. Then the conidia on lesions were isolated on PSA amended with procymidone at 100 g/ml and the natural fitness of resistant mutants was determined. The results showed that two resistant mutants was more competitive than their original strains while fitness of 2 other resistant mutants was lower than that of the original strains. Moreover, the competition of wild resistant isolate Dl-3 was lower than that of sensitive isolates.更多还原