The antifungal activity of some sulphonyl derivatives of isoxazole,pyrazole, thiazole and thiophene

Fifty-three heterocyclic sulphonyl derivatives including eight sulphonamides, three sulphonyl azides, nine sulphonohydrazides and twenty sulphonohydrazones of substituted thiophenes, and a smaller range of analogous isoxazoles pryazoles and thiazoles, were tested as potential fungicides in a simple screening procedure against Mucor mucedo, Septoria nodorum, Trichoderma viride, Chaetomium globosum and Aspergillus niger. Several thiophene-2-sulphonyl based compounds exhibited a high level of antifungal activity at 100 mg litre^?1 against the five test species, especially the mono-halogen-substituted sulphonamides and sulphonohydrazines, in which a single chlorine or bromine atom was substituted in the para position of an attached phenyl ring. The most active compound, against all five species of fungus was N-(4-chlorophenyl)-N-(trichloromethylthio) thiophene-2-sulphonamide which had average MIC₅₀ and MIC₁₀₀ values of 86 and 180 μmol respectively. (MIC₅₀ and MIC₁₀₀ values are, respectively, the concentrations required to inhibit fungal growth by 50% and to inhibit it totally.) In general, the isoxazole analogues of the thiophene-2-sulphonyl compounds exhibited a much lower fungitoxic activity, whilst the pyrazole and thiazole based compounds had little or no activity. Compared with the other results, the considerable activity shown by 4-[2′-(3,4-dichlorobenzylidene)hydrazinosulphonyl]thiophene-2-carboxylic acid was unexpected.     

Synthesis and fungicidal activity of 1,4-dithiino[2,3-b]quinoxaline-2,3-dicarbonitriles

A series of eleven 1,4-dithiino[2,3-b]quinoxaline-2,3-dicarbonitriles was prepared by reaction of 2,3-dichloroquinoxalines with disodium (2)-2,3-dimercapto-2-butenedinitrile in N,N-dimethylformamide. These products were tested for in-vitro fungicidal activity by a Minimum Inhibitory Concentration (MIC) method. Several of these compounds showed broad-spectrum fungicidal activity. The activity exhibited by these compounds was greatly dependent upon the substituents of the quinoxaline ring, with the nitro-substituted derivatives showing the highest levels of antifungal activity. None of the compounds prepared, however, showed fungicidal activity comparable to that of the commercial fungicides screened.     

Quantitative structure-activity relationships of antifungal N-phenylsuccinimides and N-phenyl-1,2-dimethylcyclopropanedicarboximides

The antifungal activity of 61 N-phenylsuccinimides and 16 N-phenyl-1,2-dimethylcyclopropanedicarboximides having various benzene ring substituents was determined against Botrytis cinerea by the agar medium dilution method. The structure-activity relationships were analyzed using such physicochemical substituent parameters as hydrophobic π, electronic σ⁰, steric E₈, and HB (hydrogen bonding) values with the multiple regression technique. The π values were derived from log P (octanol-water partition coefficient) values for the N-monosubstituted-phenylsuccinimide system. The hydrophobic effect is significant only for m-substitutents. The stronger the electron withdrawal and the smaller the steric dimensions of the ring substituents, the greater is the activity. When substituents are hydrogen bond acceptors, the effect is to lower the activity. These features are almost identical between two series of compounds.     

A Rapid Microbioassay for Discovery of Novel Fungicides for Phytophthora spp

ABSTRACT A microbioassay was developed for the discovery of compounds that inhibit Phytophthora spp. This assay uses a 96-well format for high-throughput capability and a standardized method for quantitation of initial zoospore concentrations for maximum reproducibility. Zoospore suspensions were quantifiable between 0.7 and 1.5 x 10(5) zoospores per ml using percent transmittance (620 nm). Subsequent growth of mycelia was monitored by measuring optical density (620 nm) at 24-h intervals for 96 h. Full- and half-strength preparations of each of three media (V8 broth, Roswell Park Memorial Institute mycological broth [RPMI], and mineral salts medium) and four zoospore concentrations (10, 100, 1,000, and 10,000 zoospores per ml) were evaluated. Both full- and half-strength RPMI were identified as suitable synthetic media for growing P. nicotianae, and 1,000 zoospores per ml was established as the optimum initial concentration. The assay was used to determine effective concentration values for 50% growth reduction (EC(50)) for seven commercial antifungal compounds (azoxystrobin, fosetyl-aluminum, etridiazole, metalaxyl, pentachloronitrobenzene, pimaricin, and propamocarb). These EC(50) values were compared with those obtained by measuring linear growth of mycelia on fungicide-amended medium. The microbioassay proved to be a rapid, reproducible, and efficient method for testing the efficacy of compounds that inhibit spore germination in P. nicotianae and should be effective for other species of Phytophthora as well. The assay requires relatively small amounts of a test compound and is suitable for the evaluation of natural product samples.     

Synthesis and in-vitro antifungal activity of 6-trifluoromethyipyrazolo[3,4-6d]pyrimidin-4(5H)-thiones

Twelve 6-trifluoromethylpyrazolo[3,4-d]pyrimidin-4(5H)-thiones were prepared by the reaction between 4-thiocarbamoyl-5-aminopyrazoles and trifluoroacetic anhydride. They were tested in vitro for antifungal activity against a series of phytopathogenic fungi of different taxonomic classes. The EC50 and MIC values of four compounds were comparable or inferior to those of reference commercial fungicides in controlling Sclerotinia minor, Corticium solani and Phytium ultimum.     

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.     

苹果树腐烂病菌拮抗放线菌JPD-1的筛选及鉴定

为筛选对苹果树腐烂病菌Valsa mali具有拮抗作用的放线菌,从苹果树根际土壤中分离获得放线菌,利用平板对峙法和生长速率法筛选拮抗活性较高的菌株,通过菌株培养特征、形态特征、生理生化特性及16S rDNA序列分析确定其分类地位,并采用离体枝条烫伤接种法测定所筛选获得的拮抗菌株对苹果树腐烂病的防效。结果表明,从苹果树根际土壤中共分离获得28株放线菌,其中菌株JPD-1对苹果树腐烂病菌的拮抗作用最强,抑制率为72.50%,该菌在高氏合成1号培养基上生长最好,培养7 d时菌落生长旺盛,气生菌丝白色,基生菌丝深粉红色,无可溶性色素产生;经形态特征观察、生理生化试验及16S rDNA序列分析,将菌株JPD-1鉴定为公牛链霉菌Streptomyces tau‐ricus。菌株JPD-1发酵滤液对苹果树腐烂病菌的抑制率及对离体枝条上苹果树腐烂病的防效均随稀释倍数的增大而减弱,发酵滤液原液使苹果树腐烂病菌菌丝膨大、末端畸形,抑制率为78.14%,对离体枝条上苹果树腐烂病的防效为76.37%。表明分离到的放线菌菌株JPD-1可以用作苹果树腐烂病的生防材料,具有很好的开发及应用前景。     

吖啶橙诱变提高枯草芽孢杆菌G3抗真菌活性

 枯草芽孢杆菌G3菌株正在登记为防治番茄叶霉病的生物杀菌剂。为改进该菌的抗真菌活性,用诱变剂吖啶橙诱变得到20个突变株,其中5个突变株Ga1、Ga8、Ga12、Ga1和Ga19在肉汤和PDA平板上形成粘液状菌落,完全不同于出发菌株。PDA平板抑菌圈试验,突变株对番茄叶霉菌和黄瓜灰霉菌产生比野生株G3更大的透明抑菌圈。其中Ga1菌株对番茄叶霉菌和黄瓜灰霉菌的抑菌带宽分别是G3的1.71和1.69倍。用番茄叶霉菌为指示菌的生物测定结果表明,突变株固体培养物或摇瓶培养物以最小抑菌浓度(M IC)和有效抑菌中浓度(EC₅₀)表示的抗真菌活性皆高于G3菌株。抗真菌活性以Ga1 > Ga8 > Ga19 > Ga12 > Ga13 > G3为序。参试菌株培养物的抗真菌活性与菌量(CFU)不相关,而与甲醇抽提代谢物的干重,尤其是伊枯草菌素A含量呈一定的正相关。突变株Ga1抗真菌活性最强,固体培养物或摇瓶培养物均是G3的3倍左右。摇瓶培养时,伊枯草菌素A动态分泌曲线表明,Ga1菌株产生比G3更多的伊枯草菌素A。Ga1菌株增强了合成伊枯草菌素A的能力。     

Involvement of antifungal compounds from rockmelon fruit rind (<Emphasis Type="Italic">Cucumis melo</Emphasis> L.) in resistance against the fruit rot pathogen <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">melonis</Emphasis>.

Fusarium rot caused by Fusarium oxysporum f. sp. melonis, causes significant postharvest losses in rockmelon crops. Although latent infection is often present in the field, symptoms of the disease may not appear until fruit maturity. The susceptibility of different-aged rockmelon fruit cv. “Colorado” was determined by inoculating fruit at different stages of development with a spore suspension of F. oxysporum f. sp. melonis. Disease symptoms appeared first and were more severe in older fruit compared to younger fruit. Disease symptoms on fruit 35 DAA (Days After Anthesis) and 42 DAA appeared within 3 days of inoculation and rapidly covered the fruit within 5 days. In contrast, disease symptoms on fruit 7 DAA appeared 6 days after inoculation and grew slowly. Extraction of antifungal compounds without involving acid hydrolysis from 7 DAA fruit rind did not show antifungal activity on TLC plates. However, hydrolysis of the ethyl acetate fraction resulted in a strong fungal inhibitory zone on agar plates against colonies of F. oxysporum f. sp. melonis. Separation of the hydrolysed crude extracts on TLC plates indicated the presence of two distinct antifungal zones with R_f 0.36 and 0.13 in young fruit 7, 14 and 21 DAA. The area of fungal inhibition of compound R_f 0.36 was greater than that of R_f 0.13 on the TLC plate. Extracts from mature fruit of 35 and 42 DAA did not have detectable levels of antifungal compounds. The decrease in the susceptibility of rockmelon fruit during maturity may be correlated to a decrease in the antifungal compounds in the fruit with maturity.     

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.     

Photo-induced fungicidal activity elicited by naturally occurring thiophene derivatives

Photo-induced fungitoxicity was observed in Alternaria alternata, Aspergillus niger, Cladosporium variabile, Colletotrichum sp., Rhizopus nigricans, Pythium aphanidermatum, and Saprolegnia sp., elicited by 5-(but-3-en-l-ynyl)-2,2′-bithienyl, 2,2′: 5′,2″-terthienyl, and 2-chloro-4-[5-(penta-1,3-diynyl)-2-thienyl]but-3-ynyl acetate in the presence of near ultraviolet (u.v.) radiation (320–380 nm). Conidiogenesis in A. niger, and sporangiosporogenesis in R. nigricans were depressed on media treated with 2,2′: 5′,2″-terthienyl and irradiated with near u.v. light. Radial growth of mycelia of all fungi tested was dramatically reduced by all three compounds in the presence of near u.v. light. The viability of conidia of A. niger, and R. nigricans was unaffected by 2,2′: 5′,2″-terthienyl, both in the dark and in near u.v. light. Newly emergent germ-tubes were most sensitive to toxicity mediated by u.v. light. The oomycetous fungi tested were the most sensitive to the photo-activated toxicity generated by the three compounds used. The results indicate that ED₅₀ values are dependent upon the species and the presence or absence of near u.v. light. Dramatically lowered ED₅₀ values were always observed in all systems treated with near u.v. light. The dose of near u.v. light used had no effect on the radial growth of fungi in the absence of the three compounds.     

Insect growth inhibition,antifeedant and antifungal activity of compounds isolated/derived from Zingiber officinale Roscoe (ginger) rhizomes

Fresh rhizomes of Zingiber officinale (ginger), when subjected to steam distillation, yielded ginger oil in which curcumene was found to be the major constituent. The thermally labile zingiberene‐rich fraction was obtained from its diethyl ether extract. Column chromatography of ginger oleoresin furnished a fraction from which [6]‐gingerol was obtained by preparative TLC. Naturally occurring [6]‐dehydroshogaol was synthesised following condensation of dehydrozingerone with hexanal, whereas zingerone and 3‐hydroxy‐1‐(4‐hydroxy‐3‐methoxyphenyl)butane were obtained by hydrogenation of dehydrozingerone with 10% Pd/C. The structures of the compounds were established by ¹H NMR, ¹³C NMR and mass (EI‐MS and ES‐MS) spectral analysis. The test compounds exhibited moderate insect growth regulatory (IGR) and antifeedant activity against Spilosoma obliqua, and significant antifungal activity against Rhizoctonia solani. Among the various compounds, [6]‐dehydroshogaol exhibited maximum IGR activity (EC₅₀ 3.55 mg ml ^?1) while dehydrozingerone imparted maximum antifungal activity (EC₅₀ 86.49 mg litre^?1). © 2001 Society of Chemical Industry     

A fungistatic chromene from<Emphasis Type="Italic">Ageratum conyzoides</Emphasis>

Ageratum conyzoides L. is an annual herb in the tropics and subtropics whose extracts are known to possess pharmacological and biocidal activity. We report on the bioactivity of a secondary metabolite (a chromene) isolated from the shoots ofA. conyzoides against some plant pathogenic fungi. Organic solvent extracts from the shoots were tested for antifungal activity against the plant pathogenic fungiRhizoctonia solani, Sclerotium rolfsii, Botryodiplodia theobromae, Phomopsis theae andFusarium species growingin vitro on potato dextrose agar medium. The cruden-hexane extract completely inhibited the growth ofR. solani andS. rolfsii. Then-hexane extract was chromatographed over a column of silica gel followed by activity-guided fractionation to give an antifungal principle. Structure elucidation by detailed analysis of¹H,¹³C NMR and mass spectroscopy identified the active compound as precocene II. The growth ofR. solani andS. rolfsii was completely inhibited by precocene II at a concentration of 80–100 ppm. The sclerotia ofR. solani andS. rolfsii were also completely suppressed by 150 ppm of precocene II. Sub-culture of these inhibited fungi onto precocene II-free medium restored growth of the fungus, indicating that precocene II is fungistatic. Crude or refined extracts fromA. conyzoides offer the possibility of biocontrol of plant pathogenic fungi. http://www.phytoparasitica.org posting Feb. 11, 2004.     

Synthesis and antifungal activity of new nitro-alcohol derivatives

Thirty-six new nitro-alcohol derivatives were synthesised. Tested in vitro against Helminthosporium sativum, the compounds had high antifungal activities; the EC₅₀ values varied between 10^?2 and 10^?6 M , and for the majority of the compounds between 10^?4 and 10^?6 M . The EC₅₀ values were calculated by probit analysis, except for the compounds with low activity, for which the values were estimated. Interesting relationships between the structure and antifungal action of the compounds were established.     

Microbiological and Chemical Analysis of Neem (Azadirachta indica) Extracts: New Data on Antimicrobial Activity

The antimicrobial effects of extracts of neem seed (Azadirachta indica A. Juss.) were investigated using microbial growth inhibition assays. A laboratory-prepared neem seed extract along with a commercially available formulated product, were characterized using HPLC, and shown to be effective against a range of bacteria in an agar diffusion assay. The active ingredient,i.e., the unformulated seed extract of the commercial product, also showed activity and this was further investigated in a biochromatogram, using the sensitive bacteriumBacillus mycoides. Results showed antibacterial activity as three discrete inhibition zones that did not correspond to the R_f of the major neem metabolites, azadirachtin, nimbin and salannin. This suggests that these compounds were not antibacterial. The colony radial growth rates of the fungal pathogens that cause ‘take-all’ and ‘snow mould’ disease were both significantly affected when the commercial, unformulated, neem seed extract was incorporated into the growth medium. Experiments in liquid culture suggested that the effect was fungistatic. Conidial germination of the commercially important obligate pathogenSphaerotheca fuliginea (powdery mildew) was reduced to 11%. The results show that neem seed extracts possess antimicrobial activity with notable effects on some fungal phytopathogens. This Work demonstrates that neem seed extracts have potential for controlling both microbial and insect pests. http://www.phytoparasitica.org posting Sept. 16,2001.     

On a difference in the antifungal activity of tridemorph and its formulated product Calixin

The antifungal effects of tridemorph and its formulated product Calixin were compared in vitro on Ustilago maydis, Saccharomyces cerevisiae, Torulopsis candida, Botrytis allii, and Cladosporium cucumerinum. MIC values for both products were about the same. In liquid media the products were somewhat more effective than on solid media. T. candida proved sensitive only at pH 5, but the other organisms were as sensitive at pH 7 as at pH 5. Whereas tridemorph even at high concentrations did not affect oxygen consumption of these organisms, Calixin at concentrations slightly above the MIC values appeared to inhibit respiration. This effect of Calixin could be explained by the presence of Nekanil LN in the formulation. This compound inhibited both growth and respiration of the organisms at high concentrations; however, in the simultaneous presence of tridemorph a synergistic effect on oxygen consumption was observed.     

冬青卫矛内生放线菌Streptomyces flavofuscus G1 发酵液中抑菌活性成分研究

从冬青卫矛内生放线菌Streptomyces flavofuscus G1的发酵液中分离得到化合物G13和G19。经核磁共振波谱及质谱分析,并结合相关文献,两个化合物分别被鉴定为phencomycin(G13)和4'-deacetyl-(-)-griseusin A(G19)。抑菌活性测定结果表明:G19对水稻白叶枯病菌Xanthomonas oryzae pv.oryzae、铜绿假单孢菌Pseudomonas aeruginosa(1.203 1)、蜡状芽孢杆菌Bacillus cereus(1.184 6)及金黄色葡萄球菌Staphyloccocus aureus(1.008 9)等供试病原细菌具有强烈的抑制作用,其最低抑菌浓度(MIC)值均为1.56μg/m L;对苹果树腐烂病菌Valsa mali和小麦赤霉病菌Fusarium graminearum菌丝生长亦有明显的抑制作用,其IC50值分别为14.70和24.35μg/m L。而G13对植物病原真菌油菜菌核病菌Sclerotinia sclerotiorum和苹果树腐烂病菌V.mali菌丝生长有强烈的抑制作用,其IC50值分别为5.21和4.82μg/m L。     

Aspergillus niger and a Penicillium sp. are not directly involved in the degradation of chlorsulfuron

When Aspergillus niger or a Penicillium sp. were grown in potato dextrose broth supplemented with chlorsulfuron, the herbicide concentration decreased by 97–99% within 78–96 h and the pH of the medium fell from 7.2 to between 2.4 and 3.1. If, during growth, the pH was maintained at or near neutrality, no decrease in herbicide concentration occurred. When sterile medium containing the herbicide was titrated with acid to mimic acid formation during fungal growth, the herbicide concentration again declined. Precipitation was not responsible for the observed decrease. The data indicate that, in laboratory media, these fungi do not directly metabolise the herbicide as previously thought. Chlorsulfuron degradation in perfused soil cores was not enhanced by inoculation with A. niger. Our results show that A. niger and a Penicillium sp. do not degrade chlorsulfuron.     

Antifungal capacity of major phenolic compounds of Olea europaea L. against Phytophthora megasperma Drechsler and Cylindrocarpon destructans (Zinssm.) Scholten

The phenolic composition of olive roots and stems was studied by high performance liquid chromatography–mass spectrometry. The in vivo levels of the principal phenolic compounds found in olive plants infected by Phytophthora megasperma Drechsler and Cylindrocarpon destructans (Zinssm.) Scholten differed from the levels observed in non-infected plants. When the antifungal activity of these compounds against both fungi was studied in vitro, the most active were quercetin and luteolin aglycons, followed by rutin, oleuropein, p-coumaric acid, luteolin-7-glucoside, tyrosol and catechin. Microscopic study showed that these phenolic compounds affected the growth, morphology and ultrastructure of the fungi. Taken together, these findings suggest that the phenolic compounds present in olive plants play an active role in the protection against pathogen attack.     

棉花黄萎病拮抗细菌H14的筛选鉴定及其拮抗机理分析

为筛选对大丽轮枝菌Verticillium dahliae Kleb.具有拮抗活性的根际细菌,以大丽轮枝菌为靶菌,分离获得棉花根际细菌,利用平板对峙法和琼脂扩散法筛选具有较高拮抗活性的菌株,采用室内盆栽法测定筛选所得菌株对棉花黄萎病的防效,并通过形态特征、生理生化特性和16S r DNA基因序列分析确立其分类地位,采用底物降解法和抗菌肽基因克隆法检测其产生水解酶和抗菌肽的能力。结果显示,试验共分离获得182株棉花根际细菌,筛选得到3株对大丽轮枝菌抑菌率大于50.00%且抑菌圈直径大于15 mm的菌株,其中菌株H14的抑菌率和抑菌圈直径最大,分别为54.25%和18.10 mm。该菌能在0～9%NaCl和pH 4.5～9.0的NB培养基上生长;经形态特征观察、生理生化试验及16S rDNA基因序列分析,最终将其鉴定为莫哈韦芽胞杆菌Bacillus mojavensis;菌株H14对大丽轮枝菌孢子萌发的抑制率和对棉花黄萎病的盆栽防效分别为89.55%和74.57%;菌株H14能合成蛋白酶,含有srfAA、fenD、bacA脂肽类抗生素合成基因。表明莫哈韦芽胞杆菌菌株H14能够合成蛋白酶和脂肽类拮抗物质,具有良好的抑菌和防病能力。