Anthraquinones isolated from Cassia tora (Leguminosae) seed show an antifungal property against phytopathogenic fungi

The fungicidal activities of Cassia tora extracts and their active principles were determined against Botrytis cineria, Erysiphe graminis, Phytophthora infestans, Puccinia recondita, Pyricularia grisea, and Rhizoctonia solani using a whole plant method in vivo and were compared with synthetic fungicides and three commercially available anthraquinones. The responses varied with the plant pathogen tested. At 1 g/L, the chloroform fraction of C. tora showed a strong fungicidal activity against B. cinerea, E. graminis, P. infestans, and R. solani. Emodin, physcion, and rhein were isolated from the chloroform fraction using chromatographic techniques and showed strong and moderate fungicidal activities against B. cinerea, E. graminis, P. infestans, and R. solani. Furthermore, aloe-emodin showed strong and moderate fungicidal activities against B. cinerea and R. solani, respectively, but did not inhibit the growth of E. graminis, P. infestans, P. recondita, and Py. grisea. Little or no activity was observed for anthraquinone and anthraquinone-2-carboxylic acid when tested at 1 g/L. Chlorothalonil and dichlofluanid as synthetic fungicides were active against P. infestans and B. cinerea at 0.05 g/L, respectively. Our results demonstrate the fungicidal actions of emodin, physcion, and rhein from C. tora.     

云南省4种蕨类植物提取液的抑菌活性

为了合理开发利用蕨类植物资源,本研究利用圆纸片法分析了云南省4种蕨类植物单芽狗脊蕨（Woodwardia unigemmata）、蜈蚣蕨（Pteris vittata）、鸡足山耳蕨（Polystichum jizhushanense）和灰绿耳蕨（Polystic-hum eximium）的提取液对金黄色葡萄球菌（Staphylococus aureus）、大肠杆菌（Escherichia coli）和枯草芽孢杆菌（Bacillus subtilis）的抑菌活性。抑菌试验结果表明,当4种蕨类植物提取液浓度分别为10%、8%、6%和4%时,对照组无抑菌圈出现,单芽狗脊蕨提取液对大肠杆菌和金黄色葡萄球菌无抑菌活性,灰绿耳蕨提取液对金黄色葡萄球菌无抑菌活性,其它的蕨类植物提取液对供试微生物都表现出不同程度的抑菌活性;并且这4种蕨类植物提取液至少对一种供试细菌具有抑菌活性,抑菌活性范围为8～14mm,平均抑菌圈直径为11.2mm,4种提取液对枯草芽孢杆菌抗菌活性效果最明显。由此,我们推测本研究中的4种蕨类植物均有抗菌物质存在,并将有望成为筛选抗菌新型药物的种质资源。     

Fungicidal property of Curcuma longa L. rhizome-derived curcumin against phytopathogenic fungi in a greenhouse

Fungicidal activity of Curcuma longa rhizome-derived materials against Botrytis cineria, Erysiphe graminis, Phytophthora infestans, Puccinia recondita, Pyricularia oryzae, and Rhizoctonia solani was tested using a whole plant method in vivo. It was compared with synthetic fungicides and four commercially available compounds derived from C. longa. The response varied with the tested plant pathogen. At 1000 mg/L, the hexane extract of C. longa showed fungicidal activities against E.graminis, P. infestans, and R. solani, and the ethyl acetate extract of C. longa showed fungicidal activities against B. cineria, P. infestans, Pu. recondita, and R. solani. Curcumin was isolated from the ethyl acetate fraction using chromatographic techniques and showed fungicidal activities against P. infestans, Pu. recondita, and R. solani with 100, 100, and 63% control values at 500 mg/L and 85, 76, and 45% control values at 250 mg/L, respectively. In the test with components derived from C. longa, turmerone exhibited weak activity against E. graminis, but no activity was observed from treatments with borneol, 1,8-cineole, sabinene, and turmerone. In comparison, potent fungicidal activity with chlorothalonil against P. infestans at 50 mg/L and dichlofluanid against B. cinerea at 50 mg/L was exhibited. These results may be an indication of at least one of the fungicidal actions of curcumin derived from C. longa.     

Antifungal activity of 4-methyl-6-alkyl-2H-pyran-2-ones

A number of 4-methyl-6-alkyl-alpha-pyrones were synthesized and characterized on the basis of 1H NMR and mass spectroscopy. These compounds were tested in vitro against pathogenic fungi, namely, Sclerotium rolfsii Saccardo, Rhizoctonia bataticola (Taub.) Butler, Pythium aphanidermatum (Edson) Fitz., Macrophomina phaseolina (Tassi), Pythium debaryanum (Hesse), and Rhizoctonia solani Nees. Lower homologues were less effective, whereas compounds such as 4-methyl-6-butyl-alpha-pyrone, 4-methyl-6-pentyl-alpha-pyrone, 4-methyl-6-hexyl-alpha-pyrone, and 4-methyl-6-heptyl-alpha-pyrone were found effective against all of the test fungi. They inhibited mycelial growth by approximately 50% (ED50) at 15-50 microg/mL. 4-Methyl-6-hexyl-alpha-pyrone, which was found most effective, was tested against S. rolfsii in a greenhouse at 1, 5, and 10% concentrations. The 10% aqueous emulsion of 4-methyl-6-hexyl-alpha-pyrone suppressed disease development in tomato by 90-93% as compared with the untreated infested soil in the greenhouse after 35 days of treatment.     

Isolation, characterization and antifungal activity of major constituents of the Himalayan lichen Parmelia reticulata Tayl

Antifungal activity of hexane, ethyl acetate and methanol extracts of Parmelia reticulata was evaluated against soilborne pathogenic fungi, namely, Sclerotium rolfsii, Rhizoctonia solani, R. bataticola, Fusarium udum, Pythium aphanidermatum and P. debaryanum by poisoned food technique. Maximum antifungal activity was exhibited by hexane and ethyl acetate extracts against most of the test pathogens. Secondary metabolites, namely, (±)-isousnic acid, (±)-protolichesterinic acid, atranorin, evernyl, ethyl hematommate, ethyl orsellinate, methyl hematommate (3-formyl-2,4-dihydroxy-6-methylbenzoic acid methyl ester), 2-hydroxy-4-methoxy-3,6-dimethylbenzoic acid, 1-hydroxy-3,6-dimethoxy-8-methyl-xanthen-9-one, baeomycesic acid and salazinic acid, were isolated from the above extracts and identified by 1H NMR, 13C NMR and mass spectroscopic methods. When these metabolites were tested for antifungal activity against test pathogens, maximum antifungal activity was exhibited by (±)-protolichesterinic acid against R. solani (ED50=23.09 μg mL(-1)) and P. debaryanum (ED50=16.07 μg mL(-1)) and by atranorin against S. rolfsii (ED50=39.70 μg mL(-1)). The antifungal activity of protolichesterinic acid was found to be comparable to that of hexaconazole, a commercial fungicide.     

Volatile compounds emitted by sclerotia of Sclerotinia minor,Sclerotinia sclerotiorum,and Sclerotium rolfsii

Volatile compounds emitted by sclerotia of Sclerotinia minor, Sclerotinia sclerotiorum, and Sclerotium rolfsii were identified by solid phase microextraction followed by gas chromatography and mass spectometry. Both S. minor and S. sclerotiorum emitted 2-methylenebornane and 2-methylisoborneol. In addition, S. minor emitted mesityl oxide, gamma-butyrolactone, cis- and trans-linalool oxide, linalool, and trans-nerolidol. S. sclerotiorum emitted 2-methyl-2-bornene, 1-methylcamphene, and a diterpene with a molecular weight of 272. Sclerotium rolfsii did not emit any of these compounds but did emit delta-cadinene and cis-calamenene. Chemicals emitted by S. minor and S. sclerotiorum were tested to determine if they could stimulate germination of conidia of Sporidesmium sclerotivorum, a mycoparasite on sclerotia of Sclerotinia spp. Chemicals were tested at 1 part per billion to 100 parts per million, both in direct contact with conidia and near, but not in, physical contact. None of the chemicals alone nor a combination of all chemicals induced germination of conidia of S. sclerotivorum.     

Viridenepoxydiol, a new pentasubstituted oxiranyldecene produced by Trichoderma viride

A new pentasubstituted oxiranyldecene, named viridenepoxydiol, has been isolated (0.9 mg/L) from the culture filtrate of a strain of Trichoderma viride showing in vitro and in vivo antagonistic activity against Sclerotium rolfsii, which is the causal agent of crown and stem rot of artichoke. Viridenepoxydiol was characterized as 3,5,9-trimethyl-2-oxiranyl-dec-8-ene-2,5-diol (3) using spectroscopic methods. It showed inhibitor effect on mycelial growth of S. rolfsii and its minimum inhibitory concentration (over 90% inhibition) was found to be 396 mug/mL. This is the first time that viridenepoxydiol was reported.     

An efficient method for the purification and characterization of nematicidal azadirachtins A,B, and H,using MPLC and ESIMS

Azadirachtin A enriched concentrate containing 60% active ingredient (a.i.) was prepared from the methanolic extract of the de-fatted neem (Azadirachta indica A. Juss) seed kernels. Azadirachtins A, B, and H, the three major bioactive constituents of neem seed kernel, were purified from this methanolic concentrate by employing reverse phase medium-pressure liquid chromatography (MPLC), using methanol-water solvent system as an eluant. The three pure azadirachtin congeners thus obtained were characterized by their unique mass spectral fragmentation, using electrospray probe in positive ion mode (ESI). All three azadirachtins exhibited nematicidal and antifungal activities. Azadirachtin B was the most effective against the reniform nematode Rotylenchulus reniformis (EC(50) 96.6 ppm), followed by Azadirachtin A (119.1 ppm) and H (141.2 ppm). At 200-ppm concentration, the test compounds caused 50-65% mortality of Caenorhabditis elegans nematode. Azadirachtin H showed the highest activity against the phytophagous fungi Rhizoctonia solani (EC(50) 63.7 ppm) and Sclerotium rolfsii (EC(50) 43.9 ppm), followed by B and A. The isolation of pure azadirachtins A, B, and H directly by MPLC purification from its concentrate and their characterization by ESIMS are unique and less time-consuming.     

Determination of cellular carbohydrates in peanut fungal pathogens and baker's yeast by capillary electrophoresis and electrochromatography.

In this work, the quantitation of cellular carbohydrates, namely chitin and glucan, in peanut fungal pathogens and baker's yeast was carried out by capillary electrophoresis (CE) and capillary electrochromatography (CEC). The chitin and glucan of the fungi were hydrolyzed by the enzymes chitinase and glucanase, respectively, to their corresponding sugar monomers N-acetylglucosamine (GlcNAc) and glucose (Glc). These two monosaccharides were then tagged with 6-aminoquinoline (6-AQ) to allow their separation and detection in CE and CEC. The 6-AQ derivatives of GlcNAc and Glc formed the basis for the determination by CE and CEC of chitin and glucan in peanut fungi and baker's yeast. Several parameters affecting the separation of the 6-AQ derivatives of GlcNAc and Glc, including the separation voltage and the composition of the running electrolyte, were investigated. Under the optimized separation conditions, the contents of cellular carbohydrates including N-acetylglucosamine, chitin, glucose, and glucan in some fungi, such as Sclerotinia minor, Sclerotium rolfsii, and baker's yeast, were successfully determined. The method described here allowed the assessment of genetic differences in Sclerotium rolfsii isolates from various locations.     

Effects of solarization of soil on nematode and fungal pathogens at two sites in victoria

The effect of covering soil with transparent polyethylene sheets, known as soil solarization, on the viability of plant pathogens was determined. The treatment was tested in mid-summer on sandy loams in N.W. and S. Victoria. Columns of moist soil were inoculated with one of a variety of pathogens, viz. Fusarium oxysporum, Pythium irregulare, Plasmodiophora brassicae, Sclerotium cepivorum, S. rolfsii, Sclerotinia minor, Verticillium dahliae and the nematodes Macroposthania xenoplax, Meloidogyne javanica, Pratylenchus penetrans and Tylenchulus semipenetrans. Columns were placed vertically in soil, and then treated either for 4 weeks in N.W. Victoria, or 6 weeks in S. Victoria.Preliminary laboratory tests showed that pathogens were killed by temperatures within the range 38–55°C. The relative sensitivities of pathogens to fluctuating soil temperatures were similar at both sites. The most sensitive were the nematodes, and the fungi V. dahliae, S. cepivorum and S. minor, while F. oxysporum, P. irregulare and P. brassicae were the least sensitive. In N.W. Victoria treatment effects were apparent to 26 cm and most pathogens were not recovered from 0 to 11 cm. In S. Victoria treatment effects were apparent to a depth of 16cm and most pathogens were not recovered from 0 to 6cm.     

Viridepyronone, a new antifungal 6-substituted 2H-pyran-2-one produced by Trichoderma viride

A new antifungal 6-substituted 2H-pyran-2-one, named viridepyronone, has been isolated from a cultural filtrate of a strain of Trichoderma viride showing antagonistic activity in vitro toward Sclerotium rolfsii, which is the causal agent of crown and stem rot of artichoke. Viridepyronone was characterized as 6-(4-oxopentyl)-2H-pyran-2-one 2 with spectroscopic methods. Bioassays showed that viridepyronone had a good antifungal activity against S. rolfsii, and its minimum inhibitory concentration (over 90% inhibition) was found to be 196 microg/mL. This is the first report of viridepyronone produced by any species of fungi.     

Characterization of cyclodextrin glycosyltransferase of the same gene expressed from Bacillus macerans, Bacillus subtilis, and Escherichia coli

The plasmid pHG contains a cyclodextrin glycosyltransferase (CGTase) gene (cgt) derived from Bacillus macerans. Two transformants, Bacillus subtilis (pHG) and Escherichia coli (pHG), were found to produce CGTases with the same primary structure as the enzyme from B. macerans. However, the beta-cyclodextrin coupling activity of the CGTase from E. coli (pHG) was 14-fold higher than that of the enzymes from the other strains. By contrast, no differences in alpha-cyclodextrin coupling activities were observed among these CGTases. CGTase from E. coli (pHG) was found to be less thermostable than the other CGTases. When the CGTase produced by B. subtilis was treated with increasing urea concentrations (10-1000 mM) to promote increasing degrees of protein unfolding, a bell-shaped beta-cyclodextrin coupling activity profile was obtained. Subtle differences in the conformation of the CGTase produced by E. coli are therefore proposed to be responsible for the markedly increased beta-cyclodextrin coupling activity of this enzyme.     

Echinacea species and alkamides inhibit prostaglandin E(2) production in RAW264.7 mouse macrophage cells

Inhibition of prostaglandin E(2) (PGE(2)) production in lipopolysaccharide-stimulated RAW264.7 mouse macrophage cells was assessed with an enzyme immunoassay following treatments with Echinacea extracts or synthesized alkamides. Results indicated that ethanol extracts diluted in media to a concentration of 15 microg/mL from E. angustifolia, E. pallida, E. simulata, and E. sanguinea significantly inhibited PGE2 production. In further studies, PGE2 production was significantly reduced by all synthesized alkamides assayed at 50 microM, by Bauer alkamides 8, 12A analogue, and 14, Chen alkamide 2, and Chen alkamide 2 analogue at 25 microM and by Bauer alkamide 14 at 10 microM. Cytotoxicity did not play a role in the noted reduction of PGE2 production in either the Echinacea extracts or synthesized alkamides. High-performance liquid chromatography analysis identified individual alkamides present at concentrations below 2.8 microM in the extracts from the six Echinacea species (15 microg/mL crude extract). Because active extracts contained <2.8 microM of specific alkamide and the results showed that synthetic alkamides must have a minimum concentration of 10 microM to inhibit PGE2, it is likely that alkamides may contribute toward the anti-inflammatory activity of Echinacea in a synergistic or additive manner.     

Retention of alkamides in dried Echinacea purpurea

Different drying methods, such as freeze-drying (FD), vacuum microwave drying (VMD), and air-drying (AD), were applied to fresh roots and leaves of Canadian-grown Echinacea purpurea to determine the optimal method for preserving alkamide levels. Using HPLC, six alkamide fractions (alkamides 1, 2, 3, 6a/6, 7, 8/9) were quantitated in dried roots, whereas four alkamide fractions (alkamides 1, 2, 3, 8/9) were measured in dried leaves. Different elution conditions used in HPLC for alkamide analysis did not affect the eluted fractions nor the quantitation of different alkamides. Individual alkamide concentrations in roots and leaves were affected by the drying methods used. To preserve higher levels of total alkamides, FD was found to be the best method, VMD was a superior method for drying roots than AD at 70 degrees C, while AD at 50 degrees C was the preferred method for drying leaves of E. purpurea.     

甘薯茎线虫(Ditylenchus destructor)β-1,4内切葡聚糖酶基因(Dd-eng-1b)cDNA全长的克隆与序列分析

β-1,4内切葡聚糖酶是植物寄生线虫口针分泌的一类细胞壁降解酶,在植物线虫的侵染过程中具有重要的作用。本文以甘薯茎线虫为材料,用RT-PCR和RACE方法,获得了β-1,4-内切葡聚糖酶基因的cDNA全长,并将该基因命名为Dd-eng-1b (GenBank登录号为FJ430142)。此cDNA全长序列为1640bp,包括一个1443bp的完整ORF,编码一含481个氨基酸的蛋白,其理论分子量与等电点分别为50.89KD,pI为6.94。序列比对分析表明含有糖基水解酶的保守结构域,属于纤维素酶第五家族成员,N端具有19个氨基酸残基组成的信号肽,C端含有细菌式样的纤维素结合域(CBDⅡ)。cDNA与基因组DNA重叠分析表明,此基因包含4个内含子,长度分别为36bp、76bp、187bp和344bp,切割点符合5‘-GT...AG-3’的规律。系统进化树分析表明,该基因与细菌 Bacillus.subtilis和Erwina carotovora 分泌的纤维素酶属于同一支。     

Fungichromin: a substance from Streptomyces padanus with inhibitory effects on Rhizoctonia solani

Streptomyces padanus strain PMS-702 is an antagonist of Rhizoctonia solani AG-4, the causal agent of damping-off of cabbage. Treatment of cabbage seeds with the culture filtrate of S. padanus strain PMS-702 was effective in reducing the incidence of damping-off of cabbage. The major active ingredient from the culture filtrate of S. padanus strain PMS-702 was purified by silica gel column chromatography and identified as the polyene macrolide, fungichromin, by NMR and mass spectral data. Bioassay studies showed that fungichromin had a strong antifungal activity against R. solani AG-4, and its minimum inhibitory concentration (over 90% inhibition) was found to be 72 microg/mL. This is the first report of fungichromin from S. padanus as an active ingredient for the control of Rhizoctonia damping-off of cabbage.     

苦参与百部提取物对几种植对几种植物病原菌的抑制作用 研究

试验研究苦参与百部农药活性提取物及其复配物对几种植物病原菌的抑制作用结果表明,各农药活性提取物及其复配物对稻胡麻叶斑病菌、立枯丝核菌、稻恶苗病菌、白菜黑斑病菌、十字花科蔬菜黑腐病病菌与十字花科蔬菜软腐病病菌、茄科蔬菜青枯病菌均有不同抑制作用;各提取物抑制效果随提取物浓度的提高而相应增强,最低抑制浓度多<0 .5mg/ mL ,并对白菜黑斑病菌孢子萌发也有明显抑制作用。与单一提取物相比,复配物对稻胡麻叶斑病菌和十字花科蔬菜软腐病病菌的抑制效果有所增强,但对十字花科蔬菜黑腐病病菌的作用较小,对稻恶苗病菌和茄科蔬菜青枯病菌的作用反而减弱     

Volatile metabolites from Salvia fruticosa as antifungal agents in soilborne pathogens

The volatile metabolites of Salvia fruticosa plants, growing wild in 15 localities scattered across Greece, were analyzed by means of GC and GC-MS. The essential oil content ranged from 0.69 to 4.68%, and the results of the analyses showed a noticeable variation in the amounts of the five main components [1,8-cineole, alpha-thujone, beta-thujone, camphor, and (E)-caryophyllene]. The antifungal activities of the essential oils from two localities, belonging in two different groups of cluster and principal component analysis, and their main components (1,8-cineole and camphor) were evaluated in vitro against five phytopathogenic fungi. Both oils were slightly effective against Fusarium oxysporum f. sp. dianthi and Fusarium proliferatum, whereas against Rhizoctonia solani, Sclerotinia sclerotiorum, and Fusarium solani f. sp. cucurbitae the oils exhibited high antifungal activities.     

Green-leaf-derived C6-aroma compounds with potent antibacterial action that act on both Gram-negative and Gram-positive bacteria

All eight C6-aliphatic alcohol and aldehyde compounds in naturally occurring green leaves showed bacteriostatic effects against Staphylococcus aureus IFO 12732, methicillin-resistant S. aureus, Escherichia coli IFO 3301, E. coli O157:H7, and Salmonella enteritidis, with bacteriostatic activities of less than 12.5 microg mL(-1). In this study, the susceptibility of Gram-positive bacteria tested was observed to be greater than that of Gram-negative bacteria. The bactericidal action of the aldehyde compounds was found to be much stronger than that of the alcohol compounds under both liquid and gaseous conditions. The most effective compound was (3E)-hexenal at concentrations of 0.1 and 1 microg mL(-1), which killed 2.1 x 10(5) cfu mL(-1) of S. aureus IFO 12732 and 1.4 x 10(5) cfu mL(-1) of E. coli IFO 3301, respectively, by direct contact with the compound. Lethality of (3E)-hexenal against S. aureus IFO 12732 and E. coli IFO 3301 was also observed as a result of gaseous contact at concentrations of 3 and 30 microg mL(-1), respectively. The bactericidal effects of 30 microg mL(-1) (3E)-hexenal were thoroughly maintained throughout periods of 2 days and 1 day against S. aureus IFO 12732 and E. coli IFO 3301, respectively, by a complex formation with alpha-cyclodextrin.     

Allium white rot and its control

Abstract. White rot ( Sclerotium cepivorum ) causes serious losses in Allium crops throughout the world. The pathogen produces sclerotia which survive for long periods and are the main source of inoculum. Sclerotial germination is stimulated by the host and new sclerotia are produced on the host near the soil surface. Allium crops are cultivated in various systems and environments and no one method of control is effective. There is increasing interest in control strategies based on combinations of treatments which decrease the populations of sclerotia in the soil, thereby improving the effectiveness of present methods of control. Materials and methods being tested for inclusion in programmes of integrated control include germination stimulants, soil fumigants, solar heating, roguing, aerobic composting, microbial control and combined chemical/microbial control with fungicide-resistant micro-organisms.