邻烯丙基苯酚对番茄灰霉病菌全细胞呼吸的影响

 利用Oxygraphy液相氧电极研究了植物源杀菌剂邻烯丙基苯酚对番茄灰霉病菌(Botrytis cinerea)全细胞呼吸的影响。结果显示,邻烯丙基苯酚处理浓度为5、10 mg/L,菌丝细胞的呼吸速率较对照分别增加了19.58%和24.56%;处理浓度为150 mg/L,可显著抑制菌丝细胞的呼吸,抑制率达到77.49%,表明邻烯丙基苯酚具有低浓度刺激而高浓度抑制灰霉病菌细胞呼吸的双重影响。用药剂处理萌芽期、非萌芽期的分生孢子,测定呼吸的结果表明,药剂对孢子的呼吸作用效果与对菌丝的相似,但以萌芽期分生孢子最为敏感。以0.01 mol/L琥珀酸钠作为病菌细胞呼吸底物时,10 mg/L邻烯丙基苯酚对病菌细胞的呼吸速率具有抑制作用,而添加0.1 mol/L葡萄糖和0.01 mol/L丙酮酸钠两种底物时却表现出刺激病菌细胞呼吸的作用,表明邻烯丙基苯酚对细胞呼吸的影响和氧化底物密切相关。反应液中单独添加呼吸电子传递链特异性位点抑制剂鱼藤酮20μmol/L、嘧菌酯10 mg/L和叠氮化钠0.01 mol/L后,病菌菌丝细胞呼吸均受到显著抑制,抑制率分别为48.94%、33.08%和39.18%。随后再添加10 mg/L邻烯丙基苯酚,不改变这种抑制效果。单独添加旁路氧化酶抑制剂水杨肟酸2 mM不影响病菌菌丝的呼吸,随后再添加10 mg/L邻烯丙基苯酚后,两者协同作用则可显著抑制菌丝的呼吸。     

Characterisation of QoI‐resistant field isolates of Botrytis cinerea from citrus and strawberry

BACKGROUND: In 2004, field isolates of Botrytis cinerea Pers. ex Fr., resistant to strobilurin fungicides (QoIs), were first found in commercial citrus orchards in Wakayama Prefecture, Japan. Subsequently, QoI‐resistant isolates of this fungus were also detected in plastic strawberry greenhouses in Saga, Ibaraki and Chiba prefectures, Japan. Biological and molecular characterisation of resistant isolates was conducted in this study. RESULTS: QoI‐resistant isolates of B. cinerea grew well on PDA plates containing kresoxim‐methyl or azoxystrobin at 1 mg L^?1, supplemented with 1 mM of n‐propyl gallate, an inhibitor of alternative oxidase, whereas the growth of sensitive isolates was strongly suppressed. Results from this in vitro test were in good agreement with those of fungus inoculation tests in vivo. In resistant isolates, the mutation at amino acid position 143 of the cytochrome b gene, known to be the cause of high QoI resistance in various fungal pathogens, was found, but only occasionally. The heteroplasmy of cytochrome b gene was confirmed, and the wild‐type sequence often present in the majority of resistant isolates, indicating that the proportion of mutated cytochrome b gene was very low. CONCLUSION: The conventional RFLP and sequence analyses of PCR‐amplified cytochrome b gene are insufficient for molecular identification of QoI resistance in B. cinerea. Copyright © 2009 Society of Chemical Industry     

Inhibitory effect of bionic fungicide 2‐allylphenol on Botrytis cinerea (Pers. ex Fr.) in vitro

BACKGROUND: 2‐Allylphenol is a registered fungicide in China to control fungal diseases on tomato, strawberry and apple. It is synthetic and structurally resembles the active ingredient ginkgol isolated from Ginkgo biloba L. bark. 2‐Allylphenol has been used in China for 10 years. However, its biochemical mode of action remains unclear. An in vitro study was conducted on the biochemical mechanism of 2‐allyphenol inhibiting Botrytis cinerea (Pers. ex Fr.). RESULTS: The inhibition was approximately 3 times stronger when the fungus was grown on non‐fermentable source, glycerol, than that on a fermentable carbon source, glucose. Inhibition of B. cinerea and Magnaporthe oryzae (Hebert) Barr mycelial growth was markedly potentiated in the presence of salicylhydroxamic acid (SHAM), an inhibitor of mitochondrial alternative oxidase. Furthermore, at 3 h after treatment with 2‐allylphenol, oxygen consumption had recovered, but respiration was resistant to potassium cyanide and sensitive to SHAM, indicating that 2‐allylphenol had the ability to induce cyanide‐resistant respiration. The mycelium inhibited in the presence of 2‐allylphenol grew vigorously after being transferred to a fungicide‐free medium, indicating that 2‐allylphenol is a fungistatic compound. Adenine nucleotide assay showed that 2‐allylphenol depleted ATP content and decreased the energy charge values, which confirmed that 2‐allylphenol is involved in the impairment of the ATP energy generation system. CONCLUSION: These results suggested that 2‐allylphenol induces cyanide‐resistant respiration and causes ATP decrease, and inhibits respiration by an unidentified mechanism. Copyright © 2009 Society of Chemical Industry     

酵母挥发性物质环辛四烯抑制灰霉菌的研究

为了探明生防酵母菌菌株C410产生的挥发性物质对灰霉菌的抑制作用机理,本文以挥发性物质的主要成分之一环辛四烯为研究对象,测定其对灰霉菌的糖、蛋白、DNA、RNA、细胞的形态及细胞膜通透性的影响。结果表明,在16.5μL/L环辛四烯的作用下,灰霉菌生长速率显著降低,总糖含量基本不变;还原糖含量显著降低;蛋白含量变化不显著;RNA的完整性显著降低。在49.5μL/L环辛四烯的作用下,灰霉菌的生长几乎完全停止,但细胞通透性不受影响,不导致细胞死亡。环辛四烯孵育下灰霉菌DNA分子没有被打断。由此推测还原糖和RNA代谢过程是环辛四烯作用于灰霉菌的重要靶标,抑制其生长及侵染。     

Mode of action of the anilino-pyrimidine fungicide pyrimethanil. 2. Effects on enzyme secretion in Botrytis cinerea

The effect of pyrimethanil on the levels of cell wall degrading enzymes secreted by Botrytis cinerea Pers. was investigated in diseased plant tissues and in liquid B. cinerea cultures. Total proteinase activity isolated from infected carrot slices which were treated with 5.0 μM pyrimethanil was decreased by 76%, 3 d after inoculation. Polygalacturonase, cellulase, proteinase and laccase activities were all decreased in the medium of three day-old cultures grown in the presence of pyrimethanil. The pyrimethanil concentrations resulting in 50% reduction in total enzyme activities (IC₅₀) were approximately 0.25 μM for polygalacturonase, cellulase and proteinase, and approximately 1.0 μM for laccase. No significant growth inhibition was observed at these pyrimethanil concentrations. Pyrimethanil did not inhibit the enzymes directly, nor did it inhibit the synthesis of cytosolic proteins. Therefore, it was proposed that the fungicide inhibits protein secretion at a post-translational stage in the secretory pathway. Large differences were found in the effects of pyrimethanil on the growth of B. cinerea in liquid cultures and on agar plates, depending on the composition of the medium. In liquid media containing cellulose and protein as carbon and nitrogen sources, growth inhibition occurred at 5.0 μM pyrimethanil, whilst no growth inhibition was observed with 50 μM pyrimethanil in malt extract. Similarly, growth occurred on potato/dextrose agar (PDA) at 0.5 μM pyrimethanil, but no growth was seen at this concentration on agars containing cellulose and protein. Thus it appears that pyrimethanil is most active in media where the fungus has to utilise extracellular enzymes to mobilise the nutrients it requires for growth.     

Modulators of membrane drug transporters potentiate the activity of the DMI fungicide oxpoconazole against Botrytis cinerea

Modulators known to reduce multidrug resistance in tumour cells were tested for their potency to synergize the fungitoxic activity of the fungicide oxpoconazole, a sterol demethylation inhibitor (DMI), against Botrytis cinerea Pers. Chlorpromazine, a phenothiazine compound known as a calmodulin antagonist, appeared the most potent compound. Tacrolimus, a macrolide compound with immunosuppressive activity, was also active. The synergism of chlorpromazine negatively correlated with the sensitivity of the parent strain and mutants of B. cinerea. The synergism was highest in a mutant that overexpressed the ATP-binding cassette transporter BcatrD, known to transport DMI fungicides such as oxpoconazole. The synergism of chlorpromazine positively correlated with its potency to enhance the accumulation of oxpoconazole in BcatrD mutants. These results indicate that chlorpromazine is a modulator of BcatrD activity in B. cinerea and suggest that mixtures of DMI fungicides with modulators may represent a perspective for the development of new resistance management strategies.     

醚菌酯对番茄灰霉病菌的生物活性及其防病方式研究

离体试验表明,醚菌酯对番茄灰霉病菌菌丝生长、孢子萌发、孢子产量和菌核产量均有一定的抑制作用,其EC50分别为935.670、0.667、0.479和6.975mg/L。其中对菌丝生长的抑制作用较低。但是,用10mg/L和20mg/L的醚菌酯叶片喷雾处理前24h,接种灰霉病菌菌碟,醚菌酯对灰霉病的保护效果分别达到92.3%和100%;处理后24h接种,治疗效果分别达83.3%和100%,说明醚菌酯对番茄灰霉病具有很好的保护和治疗作用。醚菌酯具有优异的内吸活性,能在植物体内向顶传导。     

Relationship between chemical structure and biological activity of triazole fungicides against Botrytis cinerea

The inhibitory activity of commercial and experimental triazole fungicides on the target enzyme, sterol 14α-demethylase (P450_14DM), was studied in a cell-free sterol synthesis assay of Botrytis cinerea Pers. ex Fr. In order to assess structure-activity relationships, the inhibitory activities of the compounds on radial growth of the fungus were tested as well. The EC₅₀ values (concentrations of fungicide inhibiting radial growth of B. cinerea on PDA by 50%) of all triazoles tested ranged between 10^?8 and 10^?5 m. IC₅₀ values (concentrations of fungicide inhibiting incorporation of [2-¹⁴C]mevalonate into C4-desmethyl sterols by 50%) generally ranged between 10^?9 and 10^?7 M and correlated with inhibition of radial mycelial growth. However, differences in IC₅₀ values did not reflect quantitatively the observed differences in EC₅₀ values, since the ratio between EC₅₀ and IC₅₀ increased with decreasing fungitoxicity. For a limited number of compounds the correlation between intrinsic inhibitory activity and fungitoxicity was low. Both in-vitro tests were used to investigate structure-activity relationships for stereoisomers of cyproconazole, SSF-109 and tebucona-zole. Fungitoxicity and the potency to inhibit cell-free C4-desmethyl sterol synthesis correlated for all stereoisomers tested. Mixtures of isomers of tebucona-zole or cyproconazole were slightly less active than the most potent isomer. The high activity of several commercial triazoles in both experiments implies that poor field performance of triazole fungicides against B. cinerea is due neither to insensitivity of the P450_14DM nor to low in-vitro sensitivity of the fungus.     

Strong resistance to the fungicide fenhexamid entails a fitness cost in Botrytis cinerea, as shown by comparisons of isogenic strains

BACKGROUND: Fenhexamid, a sterol biosynthesis inhibitor effective against Botrytis, inhibits the 3‐ketoreductase (Erg27) involved in C‐4 demethylation. Several fenhexamid‐resistant phenotypes have been detected in Botrytis cinerea populations from French vineyards. The field isolates with the highest resistance levels display amino acid changes in Erg27 (F412S, F412I or F412V). RESULTS: Fenhexamid‐resistant mutants were generated by site‐directed mutagenesis of the erg27 gene in a sensitive recipient strain to overcome the impact of different genetic backgrounds. The wild‐type erg27 allele was replaced by the three mutated alleles (erg27^F412S/I/V) by homologous recombination. These isogenic strains were shown to be fenhexamid‐resistant and were used to quantify the impact of F412 mutations on fungal fitness. Several parameters, including radial growth, the production of sclerotia and conidia, freezing resistance and aggressiveness, were quantified in laboratory conditions. Analysis of variance demonstrated significant differences between the mutant and parental strains for some characters. In particular, the mutants grew more slowly than the wild‐type strain and displayed variations in the production of sclerotia and conidia with temperature and susceptibility to freezing. CONCLUSIONS: The results highlight a moderate but significant impact of F412 mutations on the survival capacity of B. cinerea strains displaying high levels of resistance to fenhexamid in laboratory conditions, potentially limiting their dispersal and persistence, particularly in terms of overwintering, in field conditions. Copyright © 2011 Society of Chemical Industry     

湖南省草莓灰霉病菌对4种杀菌剂的抗药性检测

为明确湖南省草莓灰霉病菌菌株的抗药性状况,2013-2015年从湖南省不同地区草莓灰霉病病果或病叶上经单孢分离获得草莓灰霉病菌454株,采用最小抑制浓度法(MIC)检测不同地区草莓灰霉病菌株对多菌灵、腐霉利、异菌脲、嘧霉胺的抗药性。结果表明:草莓灰霉病菌对多菌灵、腐霉利、异菌脲、嘧霉胺的抗性频率分别为55.73%,77.31%、3.52%和77.31%;所测菌株对4种杀菌剂的敏感性类型有Cbz~RPcm~RIpd~RPmt~R、CbzSPcm~RIpd~RPmt~R、Cbz~RPcm~RIpdSPmt~R、Cbz~SPcm~RIpdSPmt~S、Cbz~RPcm~SIpd~SPmt~R、Cbz~SPcm~RIpd~SPmt ~R等6种,其所占比例分别为33.26%、5.07%、41.41%、4.63%、3.96%、11.67%,未发现对4种杀菌剂均敏感的类型,表明湖南省草莓灰霉病菌已对多菌灵、腐霉利和嘧霉胺产生抗药性,对异菌脲的抗药性较低。     

来源于植物的短肽LD-1对番茄灰霉病的抑制作用及其抗病性研究

短肽LD-1是从牛心朴子草根中新分离的具有抗菌活性的多肽。本文以番茄灰霉病为供试菌株,研究了短肽LD-1对番茄灰霉病的抑制作用及其抗病性。结果表明:短肽LD-1对番茄灰霉病菌菌丝生长及孢子萌发具有较好的抑制作用。离体试验中,短肽LD-1对番茄灰霉病菌菌丝生长抑制作用的EC50在24、48、72h分别为28.3051、30.2134、32.7427mg/L,对孢子萌发的EC50为16.6919mg/L;活体试验中,短肽LD-1对番茄灰霉病具有较好的防治效果,90mg/L短肽LD-1对番茄灰霉病防治效果达到74.24%。短肽LD-1能导致病菌芽管发育畸形,不能正常发育。短肽LD-1对番茄种子的萌发和根系生长具有明显的促进效果,并且经短肽LD-1处理后的番茄须根发达,须根数目明显增多,促进了番茄的生长发育,提高了抗病性。     

β-Methoxyacrylates; synthesis of new types of strobilurin fungicides with extended side chains

A series of novel oxime ether β-methoxyacrylates have shown good fungicidal activity against a number of fungal pathogens. These were designed by introducing a one- or two-atom spacer group adjacent to the oxime ether group. Best fungicidal activity was obtained when an amino spacer group was used. ©1999 Society of Chemical Industry     

生防菌株SW11在番茄植株上的定殖能力及其对番茄灰霉病的防控效果

通过抗生素抗性标记法结合室内拮抗和小区防效试验,研究生防菌株SW11在番茄植株及其根围土壤中的定殖能力,及其对番茄灰霉病的防控效果.结果显示菌株SW11在番茄根、茎、叶和根围土壤中均能够较长时间定殖寄生,施药后5d在番茄植株内定殖量达到最高值,在根围土壤和植株表面则呈先多后少、逐渐下降的动态变化趋势,施药30 d后,菌株SW11抗Rif标记菌株在根面的存在量为1.31×104 cfu/g,根围土壤中为4.88×102 cfu/g,叶面为1.84×102 cfu/g,茎外为0.87×102 cfu/g,根、茎和叶内为20～65 cfu/g.当菌液浓度为1.57×108 cfu/mL时,施药7d后对番茄灰霉病的预防效果可达88.15％,治疗效果可达70.50％.研究结果证明生防菌株SW11可以成为番茄植株微生态环境中的有益微生物之一,能够有效控制番茄灰霉病的发生和为害,为其开发应用和番茄灰霉病的生物防治提供了理论依据和有效途径.     

河南省番茄灰霉病菌对3种杀菌剂的抗药性检测

为了明确河南省番茄灰霉病菌对苯并咪唑类杀菌剂多菌灵、二甲酰亚胺类杀菌剂腐霉利和氨基甲酸酯类杀菌剂乙霉威的抗药性状况,2013年从河南省不同保护地中采集番茄灰霉病病果或病叶,经单孢分离共获得番茄灰霉病菌菌株139株。采用最低抑制浓度法（MIC）测定了其对多菌灵、腐霉利和乙霉威的抗药性。结果显示：番茄灰霉病菌对多菌灵、腐霉利和乙霉威产生抗性菌株的频率分别为81. 29%、80.58%和93.53%; 所测菌株对3类杀菌剂的抗性类型有BenRDicSNPCS、BenRDicRNPCS、BenRDicSNPCR、BenSDicRNPCR、BenSDicSNPCR和BenRDicRNPCR 6种,所占比例分别为2. 88%、3.60%、3.60%、5.04%、13.67%和71. 22%。表明河南省番茄灰霉病菌已对多菌灵、腐霉利和乙霉威产生抗药性,迫切需要筛选新的杀菌剂防治番茄灰霉病。     

山东蓬莱葡萄灰霉菌对7种杀菌剂的抗药性检测

为了明确葡萄灰霉菌对啶酰菌胺、多菌灵、咯菌腈、异菌脲、腐霉利、嘧霉胺的抗药性和对抑霉唑的敏感性,本试验采用菌丝生长速率法和孢子萌发法检测了采自山东蓬莱地区的69株葡萄灰霉菌对上述前6种杀菌剂的抗药性、对抑霉唑的敏感性及抑霉唑与其他6种杀菌剂的交互抗性关系。结果表明,抑霉唑对这69株葡萄灰霉菌的EC₅₀分布在0.403～28.76μg/mL之间,平均值为(9.34±10.34)μg/mL;葡萄灰霉菌菌株中抗啶酰菌胺(BosR)、多菌灵(CarR)、咯菌腈(FluR)、异菌脲(IprR)、嘧霉胺(PyrR)、腐霉利(ProR)的比例分别为100%、100%、9.47%、97.18%、100%、89.20%,测试菌株的抗药性均为多抗类型,没有单抗菌株,其中对3种杀菌剂(啶酰菌胺、多菌灵、嘧霉胺)、对4种杀菌剂(啶酰菌胺、多菌灵、异菌脲、嘧霉胺)、对5种杀菌剂(啶酰菌胺、多菌灵、异菌脲、嘧霉胺、腐霉利或啶酰菌胺、多菌灵、咯菌腈、异菌脲、嘧霉胺)和对6种杀菌剂(啶酰菌胺、多菌灵、咯菌腈、异菌脲、嘧霉胺、腐霉利)的抗性频率分别为2.33%、9.30%、79.07%、2.33%、6.97%,表明啶酰菌胺、多菌灵、嘧霉胺对测试葡萄菌株完全丧失防效,建议在该葡萄产区停止使用这些药剂,测试菌株对腐霉利、异菌脲的抗性频率高,建议采取限制使用、禁止单独使用等措施,测试菌株对咯菌腈的抗性频率较低,可以继续使用但需按照科学使用规则进行。抑霉唑与其他6种杀菌剂间不存在交互抗性关系,说明其可以和其他药剂同时使用但建议减少使用。     

Genetic population structure and fungicide resistance of Botrytis cinerea in pear orchards in the Western Cape of South Africa

Botrytis cinerea isolates from pear blossoms (Pyrus communis) in South Africa were collected from four orchards in two production areas in the Western Cape. The cryptic species status based on vegetative‐incompatibility alleles of the Bc‐hch gene indicated that all the isolates belonged to B. cinerea. A microsatellite analysis of B. cinerea populations was performed to assess the genetic population structure. Total gene diversity (H) was high, with a mean of 0.69 across all populations. Some genotype flow was evident between orchards as indicated by the spread of microsatellite multilocus genotypes, in agreement with the moderate, but significant population differentiation among orchards (mean φ_PT = 0.118, P = 0.001). Index of association analyses (I_A and r?_d) suggest that the populations reproduce mostly asexually, even though mating type distribution did not differ significantly from a 1:1 ratio, suggesting frequency‐dependent selection. Isolates resistant to benomyl were evident in one orchard only. This orchard was also significantly differentiated from all other populations, suggesting infrequent localized selection for benomyl resistance. Overall, the findings of this study highlight the dangers of a mixed reproduction system, and stress the importance of regularly monitoring fungicide resistance levels towards developing more efficient management practices.     

多霸2诱导番茄挥发性物质及对番茄灰霉病和晚疫病的防治效果

研究了多功能制剂多霸2对保护地番茄灰霉病和晚疫病的防治效果,测定了其对番茄挥发性物质的诱导效应。结果表明:多霸2对番茄灰霉病的相对防效较木霉菌药剂高43.83%,使番茄晚疫病发病率较霜脲氰低53.67%。番茄经多霸2诱导后,挥发性物质的总含量较对照组的增加75%;氧合酯类、芳香类化合物的含量分别较对照组提高95%和21%;含量最高组分(E)-2-己烯醛诱导后较对照提高95%;其他一些组分含量也显著提高,表明多功能制剂多霸2防治病害与提高番茄自身产生挥发抗菌物质有关。