【作者】 张艳军；

【导师】 周明国；

【作者基本信息】 南京农业大学 ， 植物病理学， 2009， 博士

【摘要】 Fusarium graminearum(有性态为Gibberella zeae)可以引起小麦赤霉病,产生DON毒素污染谷物,对人类和牲畜健康造成极大威胁。为此,我国政府制定标准规定：在食用小麦面粉中DON的毒素含量不得超过1mg kg-1。为了防治小麦赤霉病并减轻其造成的DON毒素污染,人们从综合防治角度开发了许多防治策略,而化学防治在整个防控体系中起着非常重要的作用。在中国,苯并咪唑类杀菌剂多菌灵已被用于防治小麦赤霉病达40年之久。但是,由于多菌灵的单位点专化作用机理,F. graminearum已对该杀菌剂产生严重抗药性,并导致了化学防治效果的下降甚至丧失。因此,寻找和开发多菌灵的替代药剂是我们现在所面临的严峻挑战。而且有关多菌灵抗性产生后对F.graminearum DON毒素产生的影响的研究未见报道,而这些信息对于准确评估F.graminearum对多菌灵产生抗性的风险是必需的。此外,人们对杀菌剂影响小麦衰老过程的关注也较少。基于以上情况,本文的主要研究结果如下所述。利用实时定量PCR(RQ-PCR)和配备电子捕获检测器的气相色谱(GC-ECD)评估新型杀菌剂氰烯菌酯、戊唑醇、戊·福混剂(25%)、嘧菌酯、多菌灵和福美双在大田接种条件下对小麦赤霉病和DON毒素污染的控制效果。测定指标有：病花率(%)、禾谷镰孢菌侵染菌量(Tri5 DNA)和总DON毒素含量(包括DON.3-ADON和15-ADON).结果显示总DON毒素含量与病花率或禾谷镰孢菌侵染菌量成正相关关系。杀菌剂氰烯菌酯、戊唑醇和戊·福混剂(25%)能够明显降低病花率、禾谷镰孢菌侵染菌量和总DON毒素污染水平。但是与对照相比,嘧菌酯、多菌灵和福美双并不能有效降低病花率、禾谷镰孢菌侵染菌量和总DON毒素污染水平；嘧菌酯和多菌灵处理甚至明显加剧了总DON毒素污染程度,原因可能在于嘧菌酯和多菌灵提高了单位禾谷镰孢菌侵染菌量总DON毒素产生的能力。总之,杀菌剂氰烯菌酯、戊唑醇和戊·福混剂(25％)既能够有效防治小麦赤霉病的发生又能降低总DON毒素污染水平。测定了67株F. graminearum菌株在摇培液体培养基或大田麦穗上的产DON能力,菌株包括30株对多菌灵抗性的田间菌株、30株对多菌灵敏感的田间菌株、3株对多菌灵抗性的点突变体(β2-微管167位密码子突变)、3株对多菌灵敏感的点突变体(β2-微管240位密码子突变)和突变体的出发菌株ZF21。此外,试验还测定了这67个菌株在小麦上的病花率和谷粒中F. graminearum DNA的侵染量(AFgDNA).无论是液体摇培还是田间麦穗接种试验,结果表明多菌灵抗性基因会增加F. graminearum菌株的产DON能力。尽管多菌灵抗性基因不会改变F. graminearum菌株在小麦上的病花率,但却可以增加谷粒中F. graminearum DNA的侵染量。在离体摇培条件下,多菌灵抗性F.graminearum菌株中毒素合成基因Tri5的表达得到增强；F. graminearum菌株产DON能力与Tri5的表达水平呈指数关系。此外,小麦赤霉病病花率和谷粒中总DON含量与F.graminearum DNA侵染量比值之间却没有明显关系。这些结果表明,谷粒中总DON含量是由谷粒中F. graminearum的菌丝量决定的,而不受F. graminearum菌株产DON能力强弱的影响。大田条件下,在小麦生长期[ZGS]57(小麦出穗率3／4),按照推荐剂量叶面喷施4种杀菌剂氰烯菌酯(2-氰基-3-氨基-3-苯基丙烯酸乙酯)(新型杀菌剂)、嘧菌酯(甲氧基丙烯酸酯类)、戊唑醇(三唑类)和多菌灵(苯并咪唑类)处理小麦植株(品种,南农9918),评估杀菌剂对冬小麦衰老过程生理生化和谷物产量的影响。杀菌剂处理显著增加旗叶叶绿素(CHL)和可溶性蛋白(SP)含量,减少丙二醛含量(MDA)和电解质渗透。此外,杀菌剂处理也明显增加超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和过氧化物酶(POD)的活性,与之相应的是提高了小麦旗叶中过氧化氢(H2O2)的含量和降低了超氧阴离子(O2-)的含量。所有结果表明,杀菌剂处理能够延缓小麦衰老,原因在于杀菌剂处理可以提高抗氧化酶的活性以保护小麦免受活性氧(AOS)的危害。与未处理对照相比,杀菌剂处理可增加小麦谷物产量。在所有测试杀菌剂中,氰烯菌酯、嘧菌酯和戊唑醇表现出类似的延缓小麦衰老和增加小麦产量的作用,但是氰烯菌酯的效果是最好的更多还原

【Abstract】 Fusarium graminearum (teleomorph, Gibberella zeae) causes head blight of cereals and contaminates grains with deoxynivalenol (DON) toxin that are harmful to humans and domesticated animals. To safeguard human health, the People’s Republic of China Ministry of Health advises a limit of less than 1 mg kg-1 for finished flour products. Various strategies have been developed to control Fusarium head blight (FHB) and to reduce DON contamination of cereals, with chemical control having an important role in an integrated FHB control program. Benzimidazole fungicides, particularly carbendazim (MBC), have been used to control FHB in China for over 40 years. However, F. graminearum has seriously developed resistance to MBC with a site-specific mode of action, leading to inefficacy of this fungicide. Therefore, there is a challenge now to find alternative fungicides for MBC in China. Besides, there are no data on the effect of MBC-resistance on DON production by F. graminearum strains. These data are needed to assess the risk of resistance of F. graminearum to MBC. Moreover less attention has been addressed towards the effect of fungicide on the senescence process of wheat treated with the fungicides. So the major results of the present study are shown as below.We used real-time quantitative PCR (RQ-PCR) and gas chromatography with electron capture detector (GC-ECD) to evaluate the efficacies of JS399-19, tebuconazole, a mixture of tebuconazole and thiram, azoxystrobin, MBC, and thiram on the development of FHB and DON contamination of winter wheat after artificial inoculation under field conditions with Fusarium graminearum. The incidence of spikelets infected (ⅡS), amount of F. graminearum Tri5 DNA (Tri5 DNA), and total DON [containing DON,3-acetyl-(3-ADON) and 15-acetyl-deoxynivalenol (15-ADON)] concentration were quantified in 2006 and 2007. A strong positive correlation was found betweenⅡS or Log10Tri5 DNA and total DON concentration in the harvested grain. The JS399-19, tebuconazole, and the mixture of tebuconazole and thiram significantly reducedⅡS of FHB, amount of Tri5 DNA, and total DON within the grain. Azoxystrobin, MBC, and thiram had no effect on the occurrence of F. graminearum compared with those of the untreated controls. Surprisingly, azoxystrobin and MBC significantly increased the total DON content in the harvested grain because they might have stimulated the amount of total DON production per Tri5 DNA. The fungicides JS399-19, tebuconazole, and the mixture of tebuconazole and thiram were the most effective in controlling FHB and reducing DON contamination of the wheat.Sixty-seven strains were evaluated for DON production in shake culture or in the field. The strains included 60 field strains (30 MBC-resistant and 30 MBC-sensitive), three MBC-resistant site-directed mutants at codon 167 inβ2-tubulin, three MBC-sensitive site-directed mutants at codon 240 inβ2-tubulin, and their MBC-sensitive progenitor strain ZF21. The incidence of infected spikelets and the amount of F. graminearum DNA in field grain (AFgDNA) also were evaluated for all strains. MBC resistance increased DON production ability in shake culture or in the field. Although MBC resistance did not change the incidence of infected spikelets, it did increase AFgDNA. Tri5 gene expression increased in MBC-resistant strains grown in shake culture. We found a significant exponential relationship between DON production ability and Tri5 gene expression in shake culture. However, no association existed between the the incidence of infected spikelets and the ratio of total DON content to the amout of F. graminearum DNA in field grain. These indicated that total DON content was determined by the amount of F. graminearum mycelia in field grain and not influenced by the DON production ability of F. graminearum.The impact of four fungicides JS399-19 (2-cyano-3-amino-3-phenylancryic acetate) (a novel fungicide), azoxystrobin (a strobilurin), tebuconazole (a triazole) and MBC (a benzimidazole), applied as foliar spray at the recommended field rate, on the physiology and biochemistry of the senescence process and grain yield was studied in winter wheat (Triticum aestivum L. cv.’Nannong No.9918’) under natural environmental conditions. Fungicide treatments to wheat plants at growth stage [ZGS] 57 (3/4 of head emerged) significantly increased the CHL and SP content and decreased the MDA content and electrolyte leakage. Additionally, activities of the antioxidative enzymes SOD, CAT and POD in flag leaves of the fungicide-treated plants were also higher than that in untreated plants. These coincided with elevated levels of H2O2 and reduced level of O2- in the fungicide-treated plants. The results suggested that the fungicide-induced delay of senescence was due to an enhanced antioxidant enzyme activity protecting the plants from harmful active oxygen species (AOS). Because all fungicides can induce the delay of wheat senescence, fungicide-treated wheat shown higher grain yield than untreated wheat. Of all tested fungicides, JS399-19, azoxystrobin and tebuconazole showed similar effects on delaying senescence of wheat and enhancing the grain yield of wheat, but JS399-19 was more efficient in general.更多还原