【作者】 李敏；

【导师】 杨谦；

【作者基本信息】 哈尔滨工业大学 ， 环境工程， 2008， 博士

【摘要】 由于化学农药造成了严重的环境污染,生物农药越来越受到人们的关注。但是,生物农药防治效果较慢,使农业生产承担了很大的风险。因此,化学农药与生物农药复合使用已成为目前植物病害防治的一条新途径。哈茨木霉(Trichoderma harzianum)是一种优良的植物病害防治真菌,由于对苯并咪唑类杀菌剂的高度敏感性,限制了其与杀菌剂的复合使用。因此获得具有杀菌剂抗性的优良菌株,研究抗性菌株与杀菌剂协同作用防治植物病害,对新型菌药混配剂的开发和应用具有重要的理论与实践意义。使用简并PCR和反向PCR方法,从哈茨木霉(T. harzianum)基因组DNA中扩增出了α-、β1-、β2-和γ-微管蛋白基因序列。BlastP比对结果显示,4条基因推测的氨基酸序列与其它真菌的相应氨基酸序列均具有较高的同源性,与粗糙脉孢菌(Neurospora crassa)的同源性分别达93%、92%、92%和90%。通过序列分析确定了各微管蛋白氨基酸的保守结构域和典型基序的位置,对蛋白的二级结构和三级结构进行了预测。采用PEG-CaCl2介导的原生质体法,成功地将BenR基因转化到哈茨木霉(T. harzianum)β2-微管蛋白基因位点,获得了具有多菌灵抗性的菌株。Southern blot分析结果表明, BenR基因已经整合到哈茨木霉( T. harzianum)基因组DNA中,并稳定遗传;抗性水平检测结果显示,抗性菌株可以在1 500μg/mL的多菌灵浓度下生长,多菌灵抑制菌丝生长的有效中浓度达471.26μg/mL,抑制孢子萌发的有效中浓度达307.21μg/mL,比原菌株提高了1 200倍以上。对哈茨木霉(T. harzianum)抗性菌株的主要生物学特性进行了研究。结果表明,各抗性菌株的最适生长温度、最适产孢温度及分生孢子萌发的适宜温度均为25℃~30℃;各菌株对pH的适应范围较广,但偏酸性条件有利于菌丝生长、分生孢子产生及孢子萌发;光照对产孢具有明显的促进作用;在测定的多菌灵浓度下各菌株可以生长、产孢并孢子萌发,但随多菌灵浓度的升高逐渐降低;菌株TS1分生孢子产生的最佳固体发酵条件为:麸皮与秸秆比例为2∶1、培养基含水量为55%、接种量为25%,培养温度为26℃,在此条件下发酵产生的分生孢子可达15.42×108个/g;抗性菌株对噻菌灵、苯菌灵和甲基托布津表现正交互抗药性。通过室内拮抗实验及室内防效测定,得出以下结论:哈茨木霉(T.harzianum)各抗性菌株抑菌谱广,对供试的8种植物病原菌均具有一定的抑制作用,其中抗性菌株TS1对水稻立枯病病原尖孢镰刀菌(Fusarium oxysporum)和立枯丝核菌(Rhizoctonia solani)菌丝生长抑制率分别达81.34%和86.19%;抗性菌株TS1与多菌灵复合处理对水稻苗期立枯病的室内防效最高,为82.25%,比单独处理分别提高了22.82%和11.24%。生防真菌对水稻幼苗根部与抗病相关的主要防御酶的诱导研究表明,抗性菌株TS1和多菌灵复合处理的POD、PAL、PPO及SOD活性均高于对照,但除SOD外,各酶活性与用菌株TS1单独处理时相比无显著差异,这表明复合处理可以激发水稻的防卫反应,诱导系统获得抗性,但对酶活的诱导无普遍增效作用;与原菌株相比,哈茨木霉(T. harzianum)抗性菌株TS1对POD、PAL及PPO活性的诱导持续期延长。研究了抗性菌株TS1与多菌灵复合处理对水稻苗期立枯病的田间防效及菌株TS1在水稻根际的定殖能力。结果表明,3种配比的复合处理对病害防治均具有增效作用,但配比不同增效程度不同,其中6∶4配比增效作用最显著,对尖孢镰刀菌(F. oxysporum)和立枯丝核菌(R. solani)的相对防效分别为87.80%和84.08%;根际定殖能力测定结果表明,菌株TS1可以在水稻根际和根表定殖;多菌灵对菌株的根际定殖能力在灭菌土壤中未表现出明显的促进作用。更多还原

【Abstract】 As chemical pesticides have resulted in serious pollution, biopesticides have attracted more and more attentions. But, control effects of biopesticides were slow, which bring great risk to agriculture. Consequently, employment of biopesticides with chemical pesticides has become a new pathway in plant disease control. Trichoderma harzianum is an excellent biocontrol agent against a range of fungal plant pathogens. Because of its sensitive to benzimidazole fungicides, it’s difficult to work with the chemicals in plant disease integrated control program. Therefore, to obtain excellent strains resistant to these fungicides and research their cooperations in disease control would appear significant for the exploration and application of novel biocontrol agent and chemical mixtures.α,β1,β2 andγtubulin gene were cloned from T. harzianum by degenerate PCR and inverse PCR. The results of BLASTP alignments showed that the four predicted proteins show high degree of homology with other fungal tubulins. T. harzianumα,β1,β2 andγtubulin amino acid sequences are found with similarities of 93%, 92%, 92% and 90% with the corresponding sequences of Neurospora crassa. The positions of the conserved domains and motifs were identified. Simultaneity, the secondary structures and tertiary structures were predicted.In order to construct biocontrol strain with carbendazim resistance, BenR gene from N. crassa was specifically integrated intoβ2 tubulin site of T. harzianum using PEG-CaCl2 method. Southern blot analysis showed that the BenR gene was integrated into the genome of T. harzianum and the transformants were all genetically stable. Sensitivity tests showed that transformants can grow at 1500μg/mL carbendazim. The effective concentrations were up to 471.26μg/mL for mycelial growth and 307.21μg/mL for conidial germination, which appear 1200-fold higher than those of sensitive strain.Main biological characteristics of T. harzianum resistant strains were studied. The results showed that optimal temperatures for mycelial growth, conidial production and conidial germination are 25 to 30℃. pH values are not crucial, but slight acidity is beneficial to mycelial growth, conidial production and conidial germination. Light can stimulate conidial production significantly. Each resistant strains can growth, sporulation and germination at tested carbendazim concentrations, though biomass, sporulation and germination rate decreased. The optimal sporulation of resistant strain TS1 can be obtained on culture media of bran and straw at ratio of 2 to 1 with 55% water content, 25% inoculum sizes and temperature of 26℃. Under the optimal conditions, the production of conidia reached a concentration of 15.42×108 spores/g. Resistant strains appear positive cross-resistance to triabendazole, benomyl, and thiophanate-methyl.Antagonism and indoor control effect were studied. The results showed that T. harzianum resistant strains were proposed with broad-spectrum antifungal activities, which displayed strong vitro antagonistic abilities against eight plant pathogens tested. The inhibitory rate of resistant strain TS1 against plant pathogen Fusarium oxysporum and Rhizoctonia solani were 81.34% and 86.19% respectively. Control efficiency of resistant strain TS1 and carbendazim, in combination, was 82.22% against rice seedling blight, and 20.3% and 48.9% higher than treated groups single with TS1 and carbendazim respectively.Systemic induction of biocontrol fungi on main defense enzymes in rice seeding roots was studied. The results showed that the activities of POD, PAL, PPO and SOD of roots treated by strain TS1 and carbendazim in combination were higher than that in control. Except for SOD, the activities of defense enzymes treated by combination were not different significantly with that treated by strain TS1 only. Results indicated that combination can stimulate rice defense reaction, induce systemic resistance, but there is no synergistic effects were observed. Compared with T. harzianum T88, inducing duration of POD, PAL and PPO in rice seedling roots treated by strain TS1 were prolonged.Field control efficacies of resistant strain TS1 and carbendazim in combination against rice seedling blight were studied, and colonization ability of strain TS1 was conducted. The results showed that combination at three ratios all have synergistic effects on disease control, but the degree of synergism was distinct at different ratios. Significant synergistic effect could be found took place at ratio of 6 to 4, whose control efficiency against plant pathogen F. oxysporum and R. solani were 87.80% and 84.08% respectively. Strain TS1 was found can colonize on rice rhizosphere and rhizoplane, but carbendazim have no significant effect on colonization ability in sterilized soil.更多还原