应用电导仪测定番茄灰霉病菌对多菌灵抗药性的初步研究

1.0 mg/L和5.0 mg/L多菌灵盐酸盐溶液处理灰霉病菌多菌灵敏感菌株和抗性菌丝体,用电导仪测定溶液电导率的动态变化,结果表明:在敏感菌株存在时,多菌灵盐酸盐溶液2 h后的电导率显著降低,而抗性菌株存在的溶液中电导率则未见下降。     

牛至精油及其主要成分香芹酚和百里酚对16种植物病原真菌的抑制活性

为评价牛至精油及其2种主要成分香芹酚和百里酚在植物真菌病害防治方面的应用前景，采用菌丝生长速率法和孢子萌发法测定3种物质对16种植物病原真菌菌丝生长和孢子萌发的抑制活性，并采用黄瓜子叶法测定其对黄瓜灰霉病的防治效果。结果表明，牛至精油在500 mg/L浓度处理下可完全抑制番茄早疫病菌Alternaria solani、黄瓜灰霉病菌Botrytis cinerea和小麦根腐病菌Bipolaris sorokiniana等12种植物病原真菌的菌丝生长，对棉花黄萎病菌Verticillium dahliae的菌丝生长抑制率也达到97.66%。牛至精油对这13种病原真菌的EC₅₀在83.09~236.58 mg/L之间，香芹酚和百里酚对这13种病原真菌的毒力均高于牛至精油，其中香芹酚对黄瓜灰霉病菌、油菜菌核病菌Sclerotinia sclerotiorum、小麦全蚀病菌Gaeumannomyces graminis、棉花黄萎病菌和小麦茎基腐病菌Fusarium pseudograminearum菌丝生长的毒力较强，EC₅₀分别为9.09、22.45、24.28、28.40和28.80 mg/L；百里酚对黄瓜灰霉病菌和小麦全蚀病菌菌丝生长的毒力较强，EC50分别为21.32 mg/L和27.08 mg/L。香芹酚和百里酚对棉花黄萎病菌、玉米大斑病菌Setosphaeria turcica、苦瓜枯萎病菌Fusarium oxysporum f.sp.momordicae、黄瓜灰霉病菌和黄瓜炭疽病菌Colletotrichum orbiculare的孢子萌发亦有较强毒力，其EC₅₀为3.78~289.07 mg/L。1 000 mg/L牛至精油对黄瓜灰霉病的预防效果与400 mg/L嘧霉胺悬浮剂的预防效果无显著差异；500 mg/L百里酚对黄瓜灰霉病的预防效果和治疗效果均与400 mg/L嘧霉胺悬浮剂的效果相当，而香芹酚的治疗效果显著优于400 mg/L嘧霉胺悬浮剂。表明牛至精油及其主要成分香芹酚和百里酚的抑菌谱广且活性强，具有作为植物源杀菌剂的开发应用潜力。     

多菌灵等4种杀菌剂对烟草灰霉病菌的室内生物活性

采用菌丝生长速率法和孢子萌发法，分别测定了烟草灰霉病菌对多菌灵、嘧霉胺、异菌脲和丙环唑的敏感性，同时通过离体叶片法评估了这4种杀菌剂对烟草灰霉病的保护和治疗作用。结果表明:4种杀菌剂对烟草灰霉病菌的菌丝生长和孢子萌发均表现出了不同程度的抑制活性，并对灰霉病同时具有保护和治疗作用。其中多菌灵对菌丝生长的抑制活性最强，EC₅₀平均值为0.06 mg/L，其次为丙环唑、嘧霉胺和异菌脲，EC₅₀平均值分别为0.36、0.53和0.60 mg/L；异菌脲和丙环唑对烟草灰霉病菌孢子萌发的抑制活性较强，EC₅₀平均值分别为2.05和2.21 mg/L，其次为嘧霉胺和多菌灵，EC₅₀平均值分别为10.56和131.23 mg/L。异菌脲和多菌灵对灰霉病的保护作用和治疗作用均最强，当药剂质量浓度为200 mg/L时，其对离体叶片的保护和治疗作用防效分别为100%、100%和98.3%、91.8%。研究结果可为烟草灰霉病的科学防治提供依据。     

Antifungal bioactivity of 6-bromo-4-ethoxyethylthio quinazoline

In order to create novel potent antifungal agents, the antifungal effects of 6-bromo-4-ethoxyethylthio quinazoline on plant pathogenic fungi were evaluated by mycelial growth rate method. The bioassay results showed that title compound possesses high antifungal activities on fungi with EC₅₀ values ranging from 17.47 to 70.79 μg/mL. The mechanism of action of 6-bromo-4-ethoxyethylthio quinazoline against fungi was studied in Gibberella zeae model. After treated with title compound at 100 μg/mL for 12 h, the mycelial reducing sugar, chitosan, soluble protein and pyruvate content, chitinase activity showed declining tendency.     

Synthesis of 2-amino-6-oxocyclohexenylsulfonamides and their activity against Botrytis cinerea

BACKGROUND: With the objective of exploring the fungicidal activity of 2‐oxocyclohexylsulfonamides (2), a series of novel 2‐amino‐6‐oxocyclohexenylsulfonamides (6 to 23) were synthesised, and their fungicidal activities against Botrytis cinerea Pers. were evaluated in vitro and in vivo. RESULTS: The compounds were characterised by IR, ¹H NMR and elemental analysis. Bioassay results of mycelial growth showed that compounds 6 to 23 had a moderate antifungal activity against B. cinerea. N‐(2‐methylphenyl)‐2‐(2‐methylphenylamino)‐4,4‐dimethyl‐6‐oxocyclohexenylsulfonamide (13) and N‐(2‐chlorophenyl)‐2‐(2‐chlorophenylamino)‐6‐oxocyclohexenylsulfonamide (21) showed best antifungal activities, with EC₅₀ values of 8.05 and 10.56 µg mL^?1 respectively. Commercial fungicide procymidone provided an EC₅₀ value of 0.63 µg mL^?1. The conidial germination assay showed that most of compounds 6 to 23 possessed excellent inhibition of spore germination and germ‐tube elongation of conidia of B. cinerea. For in vivo control of B. cinerea colonising cucumber leaves, the compound N‐cyclohexyl‐2‐(cyclohexylamino)‐4,4‐dimethyl‐6‐oxocyclohexenylsulfonamide (19) showed a better control effect than the commercial fungicide procymidone. CONCLUSION: The present work demonstrated that 2‐amino‐6‐oxocyclohexenylsulfonamides can be used as possible new lead compounds for further developing novel fungicides against B. cinerea. Copyright © 2011 Society of Chemical Industry     

Mechanism of action and selectivity of a novel herbicide,fenquinotrione

Fenquinotrione is a novel herbicide that can control a wide range of broadleaf and sedge weeds with excellent rice selectivity. We revealed that fenquinotrione potently inhibited the 4-hydroxyphenylpyruvate dioxygenase (HPPD) activity in Arabidopsis thaliana with an IC₅₀ of 44.7 nM. The docking study suggested that the 1,3-diketone moiety of fenquinotrione formed a bidentate interaction with Fe(II) at the active site. Furthermore, π–π stacking interactions occurred between the oxoquinoxaline ring and the conserved Phe409 and Phe452 rings, indicating that fenquinotrione competes with the substrate, similar to existing HPPD inhibitors. A more than 16-fold difference in the herbicidal activity of fenquinotrione in rice and the sedge, Schoenoplectus juncoides, was observed. However, fenquinotrione showed high inhibitory activity against rice HPPD. Comparative metabolism study suggested that the potent demethylating metabolism followed by glucose conjugation in rice was responsible for the selectivity of fenquinotrione.     

稻田土壤8种真菌对4种杀菌剂的敏感性分析

基于ITS-5.8s rDNA区序列,初步鉴定了浙江大学试验基地稻田土壤的8种常见真菌。采用菌丝生长速率法测定了井冈霉素、多菌灵、咪鲜胺和三唑酮4种常用杀菌剂对这8种土壤真菌的抑菌活性,以及多菌灵与咪鲜胺混用对禾柄锈菌、黑曲霉和长枝木霉3种真菌菌丝生长的抑制作用。结果表明:8种真菌对咪鲜胺最敏感,EC50值在0.091~3.1 mg/L之间;多菌灵的EC50值在0.15~1.8 mg/L之间;三唑酮的EC50值在0.57~8.5 mg/L之间;井冈霉素的敏感性最低,EC50在12~94 mg/L之间。由4种杀菌剂的SSDs曲线分析结果得出,不同真菌对杀菌剂敏感性存在显著差异。当多菌灵与咪鲜胺按其EC50值分别以体积比3∶2、1∶1和7∶3混用时,增效系数分别为1.615、1.039和1.042,表现出一定的协同或相加作用。本研究结果为进一步阐明杀菌剂对稻田生态系统真菌群落的生态效应及杀菌剂的科学合理使用提供了重要依据。     

竹红菌甲素对番茄灰霉病菌的抑制作用

为研究竹红菌甲素(HA)对番茄灰霉病菌的抑制作用,分别采用菌丝生长速率法、电导率法、紫外分光光度法和电镜观察法研究了HA对番茄灰霉病菌抑制率以及其对电导率、蛋白质和核酸相对泄漏度、菌丝形态与超微结构的影响。结果表明:在黑暗条件下,HA对番茄灰霉病菌无明显抑制作用;但在12 000 lx光照条件下,抑菌作用显著,EC50值为17.16 mg/L,且随着HA浓度上升,抑菌率呈极显著增加趋势,40 mg/L下抑菌率达73.9%;番茄灰霉病菌细胞相对电导率、蛋白质与核酸相对泄漏度随HA浓度上升而明显升高;电镜观察可见,经34.32 mg/L的HA处理后,大多数菌丝出现畸形、膨大或缢缩等现象,细胞器几乎全部消失,细胞内出现大面积空腔,细胞遭到严重破坏。研究结果可为HA开发成绿色高效生物农药提供理论依据。     

1-(2-酰氧基-4,5-亚甲二氧基)苯基-2-甲基丙烯酮类化合物的合成及抑菌活性

以具有较高抑菌活性的卡枯醇衍生物1-(2-羟基-4,5-亚甲二氧基)苯基-2-甲基丙烯酮为原料,设计合成了14个未见文献报道的1-(2-酰氧基-4,5-亚甲二氧基)苯基-2-甲基丙烯酮类(2a～2n)化合物,其结构经电喷雾串联质谱(ESI-MS)和核磁共振氢谱确证。初步生物活性测试结果表明,在100 mg/L下,目标化合物对5种供试病原菌均具有较高的抑菌活性,其中, 2b和2d 对番茄灰霉病菌Botrytis cinerea和小麦赤霉病菌Fusarium graminearum的抑制率达100%。     

放线菌Lj20抗真菌物质的分离及其在病害防治中的作用

菌株Lj20是从健康辣椒植株分离得到的一株有杭真菌活性的植物内生放线菌,为了进一步明确其生物防治潜力,利用大孔吸附树脂、离心薄层层析以及柱层析等技术对Lj20代谢产物中的抗真菌活性物质进行了分离和提纯,并采用生长速率法测定了其代谢产物对黄瓜灰霉病菌的抑制作用,同时在温室中对Lj20发酵液粗提物的防病效果进行了试验.从Lj20代谢产物中分离得到一个抗真菌活性物质,该化合物时黄瓜灰霉病菌具有较强抑制作用,EC50值为174.94 mg/L;500mg/LLj20发酵液粗提物对黄瓜灰霉病的防治效果达62.5%;经鉴定其化合物结构为3,5-二叔丁基-4-羟基-卞基甲醚.结果表明,放线菌Lj20有进一步开发和利用的价值.     

Comparative antifungal activities and biochemical effects of monoterpenes on plant pathogenic fungi

The antifungal activity of twelve monoterpenes, camphene, (R)-camphor, (R)-carvone, 1,8-cineole, cuminaldehyde, (S)-fenchone, geraniol, (S)-limonene, (R)-linalool, (1R,2S,5R)-menthol, myrcene and thymol was evaluated against four plant pathogenic fungi Rhizoctonia solani, Fusarium oxysporum, Penecillium digitatum and Asperigallus niger by using mycelial growth inhibitory technique. (S)-limonene and thymol were examined for their inhibitory effects on pectin methyl esterase (PME), cellulase and polyphenol oxidase (PPO) of tested fungi. Thymol was the most potent antifungal compound against the four test fungi with EC₅₀ values of 33.50, 50.35, 20.14 and 23.80 mg/L on R. solani, F. oxysporum, P. digitatum and A. niger, respectively. The antifungal activity of thymol was comparable to a reference fungicide, carbendazim. (S)-limonene and 1,8-cineole exhibited pronounced antifungal activity against the four tested fungi. The most effective antifungal compounds thymol and (S)-limonene showed strong inhibitory effect on the activity of PME and cellulase but revealed no inhibitory effect on PPO. The results showed that PME was more sensitive than cellulase to thymol and (S)-limonene. This is the first report on the inhibitory effects of monoterpenes thymol and (S)-limonene on PME, cellulase and PPO. The results indicated that monoterpenes may cause their antifungal activity by inhibiting PME and cellulase. The strong antifungal activity of thymol, (S)-limonene and 1,8-cineole reported in this study indicated that these compounds have a potential to be used as fungicides.     

Multiple resistance of Botrytis cinerea from kiwifruit to SDHIs,QoIs and fungicides of other chemical groups

BACKGROUND: Botrytis cinerea Pers.: Fr. is a high‐risk pathogen for fungicide resistance development that has caused resistance problems on many crops throughout the world. This study investigated the fungicide sensitivity profile of isolates from kiwifruits originating from three Greek locations with different fungicide use histories. Sensitivity was measured by in vitro fungitoxicity tests on artificial nutrient media. RESULTS: Seventy‐six single‐spore isolates were tested for sensitivity to the SDHI fungicide boscalid, the QoI pyraclostrobin, the anilinopyrimidine cyprodinil, the hydroxyanilide fenhexamid, the phenylpyrrole fludioxonil, the dicarboxamide iprodione and the benzimidazole carbendazim. All isolates from Thessaloniki showed resistance to both boscalid and pyraclostrobin, while in the other two locations the fungal population was sensitive to these two fungicides. Sensitive isolates showed EC₅₀ values to boscalid and pyraclostrobin ranging from 0.9 to 5.2 and from 0.04 to 0.14 mg L^?1 respectively, while the resistant isolates showed EC₅₀ values higher than 50 mg L^?1 for boscalid and from 16 to > 50 mg L^?1 for pyraclostrobin. All QoI‐resistant isolates carried the G143A mutation in cytb. Sensitivity determinations to the remaining fungicides revealed in total eight resistance phenotypes. No isolates were resistant to the fungicides fenhexamid and fludioxonil. CONCLUSION: This is the first report of B. cinerea field isolates with resistance to both boscalid and pyraclostrobin, and it strongly suggests that there may be a major problem in controlling this important pathogen on kiwifruit. Copyright © 2010 Society of Chemical Industry     

Virtual screening identifies a novel piperazine-based insect juvenile hormone agonist

Juvenile hormone (JH) agonists constitute a subclass of insect growth regulators and play important roles in insect pest management. In this work, a multi-step virtual screening program was executed to find novel JH agonists. A database of 5 million purchasable compounds was sequentially processed with three computational filters: (i) shape and chemical similarity as compared to known JH-active compounds; (ii) molecular docking simulations against a Drosophila JH receptor, methoprene-tolerant; and (iii) free energy calculation of ligand–receptor binding using a modified MM/PBSA (molecular mechanics/Poisson–Boltzmann surface area) protocol. The 11 candidates that passed the three filters were evaluated in a luciferase reporter assay, leading to the identification of a hit compound that contains a piperazine ring system (EC₅₀=870 nM). This compound is structurally dissimilar to known JH agonists and synthetically easy to access; therefore, it is a promising starting point for further structure optimization.     

In vitro and In vivo Activity of Cyprodinil and Pyrimethanil on Botrytis cinerea Isolates Resistant to other Botryticides and Selection for Resistance to Pyrimethanil in a Greenhouse Population in Greece

No cross-resistance was observed between pyrimethanil or cyprodinil and the fungicides benomyl, iprodione or carbendazim + diethofencarb. In vitro, both anilinopyrimidine fungicides were effective against strains of Botrytis cinerea resistant to benzimidazoles and/or dicarboximides and against a wild type strain insensitive to diethofencarb (EC₅₀ values ranged between 0.03–0.19 and 0.006–0.054gml^–1 for pyrimethanil and cyprodinil, respectively). Preventive applications of anilinopyrimidines completely protected young cucumber plants and fruits that were inoculated with all strains of B. cinerea. The effectiveness of pyrimethanil against grey mould was studied in greenhouse grown tomatoes in relation to (a) the type of infection and the progress of the disease on different plant parts and (b) the response of the naturally occurring B. cinerea population to the selection pressure caused by eight successive applications of this fungicide. Pyrimethanil effectively controlled grey mould on leaves, fruits and stems but did not significantly reduce the number of dead plants and fruits with 'ghost spot' symptoms. The selection pressure caused by the consecutive applications of pyrimethanil resulted in reduction of its effectiveness on leaves that became apparent after the sixth application. This was correlated with a shift of the B. cinerea population (not previously exposed to anilinopyrimidines) towards reduced sensitivity, probably due to the development of a low level of resistance (R _L = 7.7). Pyrimethanil delayed the onset of the disease but it did not reduce the infection rate.     

甲氧基丙烯酸酯类新化合物ZJ1621的生物活性

对由国家南方农药创制中心浙江基地合成的甲氧基丙烯酸酯类新化合物ZJ1621(2- -3-甲氧基丙烯酸甲酯)进行了温室杀菌活性及田间防效的研究。温室及田间药效试验结果表明:在100 mg/L 剂量下,该化合物能有效防治小麦白粉病、黄瓜白粉病、黄瓜霜霉病等多种植物病害,防效在80％以上。该化合物在植株叶片之间不具有内吸传导性,但持效期可达14 d 以上,在25~200 mg/L剂量范围内对试验作物安全。     

Inhibition of plant pathogens in vitro and in vivo with essential oil and organic extracts of Cestrum nocturnum L.

The efficacy of the essential oil and various organic extracts from flowers of Cestrum nocturnum L. was evaluated for controlling the growth of some important phytopathogenic fungi. The oil (1000 ppm) and the organic extracts (1500 μg/disc) revealed antifungal effects against Botrytis cinerea, Colletotrichum capsici, Fusarium oxysporum, Fusarium solani, Phytophthora capsici, Rhizoctonia solani and Sclerotinia sclerotiorum in the growth inhibition range of 59.2-80.6% and 46.6-78.9%, respectively, and their MIC values were ranged from 62.5 to 500 and 125 to 1000 μg/mL. The essential oil had a remarkable effect on spore germination of all the plant pathogens with concentration and time-dependent kinetic inhibition of P. capsici. Further, the oil displayed remarkable in vivo antifungal effect up to 82.4-100% disease suppression efficacy on greenhouse-grown pepper plants. The results obtained from this study may contribute to the development of new antifungal agents to protect the crops from fungal diseases.     

冬青卫矛内生放线菌YDG17菌株发酵液抑菌活性研究

从冬青卫矛植株中筛选得到一株内生链霉菌YDG17,其发酵液对多种植物病原真菌有较强的抑制作用。室内生物测定结果表明,YDG17菌株发酵液对供试的11种植物病原真菌菌丝生长均有一定的抑制作用,其中对小麦赤霉病菌Fusarium graminearum、番茄灰霉病菌Fulvia fulvum和番茄早疫病菌Alternaria solani的抑制作用最强,EC50值分别为259.98、336.13和100.72 mg/L;对供试的3种植物病原真菌孢子萌发也均有一定的抑制作用,其中对番茄灰霉病菌孢子萌发的抑制作用最强,EC50值为87.84 mg/L。离体子叶法测定结果表明,发酵液原液对番茄灰霉病的保护效果为97.62%,治疗效果为79.63%。盆栽试验结果表明,发酵液原液对番茄灰霉病的保护效果为71.34%,治疗效果为64.23%。捷克八溶剂系统纸色谱测定结果表明,该发酵液的主要活性物质为碱性水溶性抗生素。选用弱酸性离子交换树脂吸附法对YDG17发酵液活性成分进行了初步分离,并对其活性馏分进行ESI-MS/MS分析,表明其活性物质主要为链丝菌素类化合物,该类化合物为首次从植物内生菌中分离得到。     

六种杀菌剂对烟草赤星病菌菌丝生长和分生孢子萌发的抑制作用

采用菌丝生长速率法和孢子萌发法测定了烟草赤星病菌Alternaria alternata对6种杀菌剂(氟硅唑、异菌脲、菌核净、嘧菌酯、醚菌酯及多菌灵)的敏感性。结果表明:6种杀菌剂对烟草赤星病菌菌丝生长和分生孢子萌发均表现出了不同程度的抑制活性。其中,对菌丝生长抑制活性最强的是氟硅唑,EC50平均值为(0.34±0.11)mg/L,其次为菌核净[(1.13±0.68)mg/L]和异菌脲[(2.08±0.24)mg/L];对分生孢子萌发抑制活性最强的是嘧菌酯协同100 mg/L水杨肟酸,EC50平均值为(0.28±0.09)mg/L,其次分别为单独处理的嘧菌酯[(5.79±2.76)mg/L]、醚菌酯[(11.93±3.93)mg/L]、菌核净[(19.95±15.94)mg/L]和异菌脲[(23.33±11.33)mg/L]。多菌灵对烟草赤星病菌菌丝生长和分生孢子萌发的抑制活性均最差,不宜用于烟草赤星病的防治,而异菌脲、氟硅唑、嘧菌酯和醚菌酯均有必要进一步针对烟草赤星病进行田间防效试验。     

Effects of plant essential oils and components from Oriental sweetgum (Liquidambar orientalis) on growth and morphogenesis of three phytopathogenic fungi

Commercial plant essential oils obtained from 40 plant species were tested for their antifungal activity against Phytophthora cactorum, Cryphonectria parasitica, and Fusarium circinatum. Strong antifungal activity against Phytophthora cactorum was achieved with the essential oil derived from Oriental sweetgum, Liquidambar orientalis at 28 × 10⁻³ mg/mL air concentration. In a test with C. parasitica, inhibition rate of patchouli was 51.0%, whereas the other essential oils showed weak activity. Essential oils of manuka (Leptospermum scoparium) and patchouli (Pagostemon patchouli) showed moderate activity against F. circinatum. Analysis by gas chromatography-mass spectrometry led to identification of 11 compounds in the oil of L. orientalis. The antifungal activity of identified compounds was tested singularly by using standard or synthesized compounds. Inhibition rates of cinnamyl aldehyde and benzaldehyde were 100% against P. cactorum at 28 × 10⁻³ mg/mL air concentration. There was a significant morphological alternation in three phytopathogenic fungi after oil or compound treatment.     

Isolation and partial characterization of an antifungal protein from the endophytic Bacillus subtilis strain EDR4

An antifungal protein E2, from the culture filtrate of the endophytic Bacillus subtilis strain EDR4 of wheat with a high activity against numerous fungal species in vitro and take-all in wheat caused by Gaeumannomyces graminis var. tritici in vivo, was purified by (NH₄)₂SO₄ precipitation, hydrophobic-interaction chromatography, anion-exchange chromatography and polyacrylamide gel electrophoresis (PAGE). The molecular mass of the protein was about 377.0 kDa determined by gel permeation chromatography (GPC) using a Superdex 200 10/300 GL pre-packed column and the pI value of the protein detected by isoelectric focusing PAGE was 6.59. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) the antifungal protein showed a band with a molecular mass of 39.1 kDa, which suggest that the native protein consists of multi-subunits. The amino acid sequences of three peptides from the antifungal protein were obtained by using a nano-ESI-MS/MS (Q-TOF2) System. The protein isolated may be regarded as a new protein according to amino acid sequences of three peptides. The purified protein exhibited inhibitory activity on mycelium growth of e.g. Fusarium graminearum, Macrophoma kuwatsukai, Rhizoctonia cerealis, Fusarium oxysporum f.sp. vasinfectum, Botrytis cinerea and G. graminis var. tritici (Ggt). Scanning electron microscopy showed that hyphae of Ggt treated with the antifungal protein were severely deformed. The antifungal protein E2 exhibited ribonuclease and hemagglutinating activities as well as a trifle protease activity. However, no β-1,3-glucanase, β-1,4-glucanase, chitinase or protease inhibitory activities were detected.