The Fate of Thiophanate-methyl Fungicide and Its Metabolites on Plant Leaves and Glass Plates

The persistence of thiophanate-methyl, dimethyl 4, 4′-o-phenylenebis (3-thioallophanate), and its metabolites on plant leaves and glass plates was examined using a radiolabeled compound. Apple and grape leaves and glass plates treated with thiophanate-methyl-phenyl-¹⁴C were kept outdoors, except on rainy days and nights when they were kept in green house.

Half lives of thiophanate-methyl on apple and grape leaves were around 15 and 12 days, respectively. On glass plates half life was around 3 days. The per cent of abundance of the parent compound and a major degraded product, methyl 2-benzirnidazolecarbamate, versus applied thiophanate-methyl at 14 days after treatment was as follows; 52.6 and 10.1% on apple leaves, 49.5 and 8.9% on grape leaves, and 5.5 and 24.1% on glass plates, respectively.     

Control of Verticillium wilt transmission in strawberry runners with systemic benzimidazole-type fungicides

Pre-planting root dips and soil drenches with benomyl, carbendazim (MBC), thiabendazole and thiophanate-methyl were used to reduce disease severity in mother plants. Transmission of the pathogen Verticillium dahliae through stolons and into young runners was prevented by two applications of benomyl or thiophanate-methyl at o-i% active ingredient applied at stolon appearance and 1 month later. Sufficient concentrations of fungistatic residues were detected in all plant tissues to suppress the internal spread of the pathogen.     

The use of fungicidal paints and gels to treat existing apple cankers

Four paints containing mercuric oxide, octhilinone, thiophanate-methyl or triad-imefon and five gel formulations (carbendazim/triadimefon in sodium alginate or xanthan gum, carbendazim/triadimefon/hypophosphorous acid and thiophanate-methyl/oxycarboxin in xanthan or PP969/alginate) were tested on natural cankers caused by Nectria galligena on apple trees. Scraping cankers decreased their size 7–9 months later due to stimulation of callus formation, even where no fungicide was applied, and decreased perithecial, but not conidial production. Only the paints were applied to scraped cankers and that containing mercuric oxide was the most effective in decreasing canker size and restricting new canker growth. All formulations except the octhilinone paint were applied to unscraped cankers. Only mercuric oxide paint and acidic carbendazim/triadimefon/xanthan gel stimulated callus formation and only the former caused a decrease in canker size. Every treatment, except triadimefon paint and PP969/alginate, reduced conidial production. Bioassay with TV. galligena showed that the carbendazim and thiophanate-methyl gels achieved some movement of fungitoxicant into plant tissue.     

In vitro studies on the effects of fungicides on beneficial fungi of peach twig mycoflora

In vitro studies were carried out to investigate a possible integrated use of chemical and biological means to control the peach twig blight pathogen,Monilinia laxa. Three fungal antagonists ofM. laxa (Penicillium purpurogenum, Penicillium frequentans andEpicoccum nigrum) and six fungicides (vinclozolin, iprodione, thiram, captan, benomyl and thiophanate-methyl) were used in the study. Sensitivity of the fungal isolates to the fungicides was determined in vitro by calculating ED50 values. Benomyl and thiophanate-methyl were the most fungitoxic compounds and captan was the least fungitoxic.M. laxa andP. purpurogenum were the most sensitive to all chemicals tested, whileE. nigrum andP. frequentans presented bigger differences in their sensitivity to chemicals compared toM. laxa. E. nigrum was consistently less sensitive to benomyl (ED50=2.26 ppm), thiophanate-methyl (ED50=9.61 ppm) and vinclozolin (ED50=3.89 ppm) than the other fungi.P. frequentans was less sensitive to captan, vinclozolin, iprodione, thiophanate-methyl and thiram thanM. laxa (8, 7, 5, 4 and 2 times respectively). These results suggest thatE. nigrum andP. frequentans could be successfully used in an integrated control programme that combines biological and chemical methods.     

Comparison of fungicides for the control of Rhizoctonia solani causing datnping-off of mung bean (Phaseohis aureus)

Of 41 fungicides tested in the laboratory, copper carbonate, copper sulphate, mercuric chloride, Agrosan GN, quintozene, kasugamycin, carboxin, pyracar-bolid, carbendazim, chloroneb, benomyl, Ohric, RH 893 (2-n-octyl-4-isothiazole-3-one) and Terrazole were most inhibitory to the mycelial growth of Rhizoctonia solani on Czapek's agar plates and had EC₅₀ values of less than 1 μg a.i./ml, while copper oxychloride, Udonkor, zineb, ziram, F 319 (3-hydroxy-5-methyl isoxazole) and anilazine were much less toxic, ziram being least inhibitory with an EC₅₀ of 214 μg a.i./ml. Of 17 fungicides tested in the greenhouse as seed treatments, thiabendazole, carbendazim, benomyl, thiophanate-methyl, dichlozoline and Ohric gave 80–90% control of damping-off of mung bean seedlings. A single soil drench with thiophanate-methyl and two drenches with benomyl gave about 90% disease control, More seedlings with R. solani infection survived when thiophanate-methyl was used as a post-inoculation soil drench than when benomyl or chloroneb were used.     

The modes of action of two benzimidazoles and thiophanate-methyl used for the control of Verticillium wilt of strawberry

Benomyl, thiabendazole and thiophanate-methyl were found to be fungistatic against Verticillium dahliae in vitro. All materials suppressed wilt symptoms on artificially inoculated strawberry plants when applied as a root dip, soil drench or a foliar application. Quantitative bioassays of the amount of toxic residues and fungal colonization in tissues of treated plants after root treatment revealed that these materials or their toxic breakdown products were readily translocated to crown and leaves: they suppressed the internal spread of the pathogen throughout the plant. After foliar application there was no marked basipetal movement of material within the plant: fungal colonization was greatly reduced in treated leaves, but the roots and crowns were colonized to a similar extent to those of untreated plants.     

Screening of epoxide hydrolase producing microorganisms for biotransformation of deoxynivalenol

Deoxynivalenol (DON) transformation products from selected time course experiments were analyzed by thin-layer chromatography. With the strainAlternaria alternata f. sp.lycopersici AS27-3, one major metabolite of DON in ethyl acetate was observed. This unidentified metabolite was more polar than DON and has a R_f value of 0.71. Derivatization indicated that this metabolite was probably an unidentified trichothecene. Screening of 29 other microbial isolates (bacteria, yeast, filamentous fungi) for DON transformation did not result in any active organism. Presented at the 26^th Mykotoxin-Workshop in Herrching, Germany, May 17–19, 2004     

Effects of chemical combinations on the induction of aneuploidy in Saccharomyces cerevisiae

Nocodazole, ethyl acetate, acetone and methyl ethyl ketone all are known to induce aneuploidy. Treatment of yeast strain D61.M with mixtures containing ineffective low levels of nocodazole and ineffective low levels of these solvents was highly effective in inducing aneuploidy. Ineffective low levels of nocodazole mixed with ineffective low levels of methyl 2-benzimidazolecarbamate also gave elevated frequencies of aneuploidy. Dimethyl formamide, a solvent that does not induce aneuploidy, mixed with low levels of nocodazole gave no increase in aneuploidy frequency above those levels seen in controls.     

Control of apple canker using fungicidal paints and gels

Paints containing mercuric oxide or thiophanate-methyl were applied to either scraped or unscraped cankers caused by Nectria galligena on apple trees at two sites. Three gel formulations (carbendazim/triadimefon in alginate or xanthan and carbendazim/imazalil in xanthan) and a solvent-based PP969 formulation were applied to unscraped cankers only. Assessments were made 9 and 21 months after treatment. Mercuric oxide was more effective than thiophanate-methyl after 21 months on both scraped and unscraped cankers. All gel formulations reduced spore production and fungitoxicant could still be detected in bark and wood after 21 months. The solvent-based PP969 formulation did not perform as well as the gels.     

Microbial Transformation of Methyl 5(6)-Butyl-2-Benzimidazolecarbamate

The anthelmintic agent methyl 5(6)-butyl-2-benzimidazolecarbamate (Parbendazole) was transformed to two of its animal metabolites, methyl 5(6)-(4-hydroxybutyl)-2-benzimidazolecarbamate and methyl 5(6)-3-(carboxypropyl)-2-benzimidazolecarbamate, by the filamentous fungus Cunninghamella bainieri ATCC 9244. The transformation pathway was shown to be through the 4-hydroxybutyl product to the 3-carboxypropyl product. The reaction favored accumulation of the latter product.     

Diagnosis and management of Sclerotinia stem rot (white mould) of lentils in Greece

The paper describes the field-level symptoms, the identification and management of Sclerotinia stem rot of lentil caused by the soilborne plant pathogen Sclerotinia sclerotiorum in Greece. Regarding symptoms at a field level, initially plants before flowering turn yellow with roots and the base of the plants become brown; then rotten plants exhibit a dry stem and die. On the diseased tissue, at the base of the stem, the typical white mycelium and the resting bodies (sclerotia) were observed. According to our pathogenicity studies in vitro, on the infected plant tissues the fungus first develop a characteristic fluffy white mycelium which will give rise to large black sclerotia, the most obvious evidence of plants infected with S. sclerotiorum. Finally, concerning evaluation of fungicides, isolates of S. sclerotiorum were sensitive to thiophanate-methyl and to triazole fungicides. Thiophanate-methyl and triazole fungicides proved to be most effective in controlling the disease emerged from mycelium or sclerotia.     

Characterization of the fungicidal activity of Calothrix elenkinii using chemical methods and microscopy

An investigation was directed towards biochemical characterization of cyanobacterium Calothrix elenkinii and analysis of the chemical nature and mode of action of its fungicidal metabolite(s) against oomycete Pythium debaryanum. Biochemical characterization of the culture in terms of carbohydrate utilization revealed the facultative nature of C. elenkinii. Unique antibiotic markers were also found for this strain. 16S rDNA sequencing of the strain revealed 98% similarity with Calothrix sp. PCC7101. The fungicidal activity was tested by disc diffusion assay of different fractions of the culture filtrate. A minimum inhibitory concentration of 10 μl was recorded for ethyl acetate fraction of the 7-weeks old culture filtrates. HPLC, followed by NMR spectral analysis demonstrated the presence of a substituted benzoic acid in the ethyl acetate fraction. Microscopic examination revealed distinct granulation, followed by disintegration of the hyphae of Pythium sp., indicating the presence of an active metabolite in the culture filtrates of Calothrix sp. The fungicidal activity of C. elenkinii can be attributed to the presence of 3-acetyl-2-hydroxy-6-methoxy-4-methyl benzoic acid. This is the first report of a benzoic acid derivative having fungicidal activity in cyanobacteria.     

Genetic analysis of resistant mutants to antimitotic benzimidazole compounds in Schizosaccharomyces pombe

Summary Mutants resistant to the antimitotic compounds thiabendazole and methyl-2-benzimidazolecarbamate were isolated and analyzed genetically in the fission yeast, Schizosaccharomyces pombe. They comprised three groups in terms of genetic linkage. Mutants in one linkage group (ben1) differed phenotypically from those in the other two (ben2 and ben3). The former were resistant to the compounds at any physiological temperature tested, whereas the latter exhibited temperature dependent resistance. Through tetrad analysis, ben1 was mapped at the rightmost part of chromosome II, and ben2 was mapped near the centromere of the same chromosome. Haploidization experiments revealed the location of ben3 on chromosome II. By analogy with Aspergillus nidulans, it is suggested that one of these ben genes may code for tubulin.     

Plant growth-promoting and non-promoting rhizobacteria from avocado trees differentially emit volatiles that influence growth of Arabidopsis thaliana

Microbial volatile organic compounds (mVOCs) play important roles in inter- and intra-kingdom interactions, and they are also important as signal molecules in physiological processes acting either as plant growth-promoting or negatively modulating plant development. We investigated the effects of mVOCs emitted by PGPR vs non-PGPR from avocado trees (Persea americana) on growth of Arabidopsis thaliana seedlings. Chemical diversity of mVOCs was determined by SPME–GC–MS; selected compounds were screened in dose–response experiments in A. thaliana transgenic lines. We found that plant growth parameters were affected depending on inoculum concentration. Twenty-six compounds were identified in PGPR and non-PGPR with eight of them not previously reported. The VOCs signatures were differential between those groups. 4-methyl-2-pentanone, 1-nonanol, 2-phenyl-2-propanol and ethyl isovalerate modified primary root architecture influencing the expression of auxin- and JA-responsive genes, and cell division. Lateral root formation was regulated by 4-methyl-2-pentanone, 3-methyl-1-butanol, 1-nonanol and ethyl isovalerate suggesting a participation via JA signalling. Our study revealed the differential emission of volatiles by PGPR vs non-PGPR from avocado trees and provides a general view about the mechanisms by which those volatiles influence plant growth and development. Rhizobacteria strains and mVOCs here reported are promising for improvement the growth and productivity of avocado crop.

     

Plumieride from Allamanda cathartica as an inhibitory compound to plant pathogenic fungi

Allamanda leaf extract (Allamanda cathertica) was made in water at room temperature (25?± 2?°C) as well as in a number of less polar to highly polar solvents like methylene chloride, benzene, chloroform and ethyl acetate at their boiling point, that means, at refluxing temperature (40?± 2?°C). Methylene chloride, benzene, chloroform, ethyl acetate and water extracts were applied to determine their growth inhibition against Phomopsis vexans, Phytophthora capsici, Fusarium oxysporum, Rhizoctonia solani and Sclerotium rolfsii. Results of these extracts showed that refluxing methanol, ethanol and ethyl acetate extracts of Allamanda were statistically similar for inhibition of mycelial growth of all fungi tested. But effect of 50% ethanol extract is different; it inhibited 100% mycelial growth of P. vexans, P. capsici and F. oxysporum; 83.33% of R. solani and 88.63% of S. rolfsii. Effort was also made to find out the compound in Allamanda to be responsible for such antifungal activity. Thin layer chromatography (TLC) of Allamanda extracts showed the presence of a number of compounds having polarity very high to low. The R_f values of compounds in 37–42 fractions were calculated and from these six fractions, crystals were separated. These crystals were more or less white. Melting point of these crystals was determined by ordinary and digital melting point apparatus that ranged from 145.5–162 C. Structural determination of the compound was done by Infra-red (IR) spectral study. The finger print region was 700–1400?cm^?1. The strong band at 1612.4, 1633.6, 1693.4, 1655 and 2850.6?cm^?1 indicated the presence of conjugated double bond (–C=C–C=C–), non-conjugated double-bond (–C=C–C–C–C=C–), carbonyl group attached to carbon–carbon double (–CO–C=C), ester (–COOR) and C–H stretching, respectively. Mass spectra of separated compounds gave molecular weight 470. All these characters are typical to pumieride as described previously. Again, In vitro screening of plumieride against P. vexans, P. capsici, F. oxysporum, R. solani and S. rolfsii were found effective in inhibiting radial mycelial growth of these fungi at 1:2 w/v concentration.     

华石斛化学成分研究(Ⅱ)

海南特有植物华石斛(Dendrobium sinense)的化学成分和药理活性研究报道较少。为了深入研究华石斛的化学成分,该研究采用MCI小孔树脂柱色谱、硅胶柱色谱和Sephadex LH-20柱色谱等多种现代分离纯化技术,从其全草乙醇提取物的乙酸乙酯部位中分离纯化了10个化合物。结果表明:对分离纯化得到的10个化合物鉴定为罗汉松脂素(1)、4,5-二羟基-2,3-二甲氧基-9,10-二氢菲(2)、华石斛素C(3)、对甲氧基苯乙醇(4)、顺式对羟基肉桂酸乙酯(5)、对羟基苯丙酸乙酯(6)、丁香醛(7)、3-羟基苯甲醛(8)、3,9-dihydroxy-megastigma-5-ene(9)和(9Z,12Z)-9,12-二烯十八碳酸甲酯(10)。其中,首次从华石斛中分离得到的有化合物1、4-6、8-10。通过体外抑制乙酰胆碱酯酶活性实验发现,化合物2、4和化合物9能够使乙酰胆碱酯酶活力下降。     

Some effects of differently-acting nematicides on the cereal cyst-nematode (Heterodera avenae) and on the appearance of ''scorch'' in spring wheat on light loamy sand

In fungitoxicity tests against Phytophthora cinnamomi on Chamaecyparis lawsoniana cv. Ellwoodii, a drench of furalaxyl (1000 mg a.i./l) applied to the compost in which 1-yr-old plants were growing, 1 wk before they were inoculated with 650 000 zoospores, controlled disease for at least 12 months. With an inoculum dose of 650 zoospores/plant, furalaxyl at 500 mg a.i./l controlled disease even when inoculation was 12 wk after fungicide treatment. Aluminium tris (ethyl phosphonate) (2000 mg a.i./l) applied as a drench 1 wk before inoculation with 650 000 zoospores/plant did not prevent root infection but delayed foliar symptoms for 9 months: the same treatment, using etridiazole (500 mg a.i./l) only slightly reduced disease incidence. When applied as a single drench 2 days before inoculation, prothiocarb (2000 mg a.i./l) and cuprammonium compounds (200 mg a.i./l) were much less effective than furalaxyl (1200 mg a.i./l), sodium ethyl phosphonate (1500 mg a.i./l), aluminium tris (ethyl phosphonate) (1500 mg a.i./l) or etridiazole (500 mg a.i./l). However, a drench of furalaxyl at 1000 mg a.i./l, aluminium tris (ethyl phosphonate) at 2000 mg a.i./l or etridiazole at 500 mg'a.i./l did not eradicate P. cinnamomi from compost containing infected root debris. Pre-planting drenching of the compost was ineffective. All fungicide treatments were non-phototoxic to 1-yr-old C. lawsoniana cv. Ellwoodii. These results are of special relevance to the control of P. cinnamomi on container-grown woody ornamentals.     

Medicago truncatula-derived calcium oxalate crystals have a negative impact on chewing insect performance via their physical properties

Plant structural traits often act as defenses against herbivorous insects, causing them to avoid feeding on a given plant or tissue. Mineral crystals of calcium oxalate in Medicago truncatula Gaertn. (Fabaceae) leaves have previously been shown to be effective deterrents of lepidopteran insect feeding. They are also inhibitors of conversion of plant material into insect body mass during or after consumption. Growth of beet armyworm, Spodoptera exigua Hübner (Lepidoptera: Noctuidae), larvae was correspondingly greater on calcium oxalate‐defective (cod) mutants of M. truncatula with lower levels of crystal accumulation. Data presented here show that insects feeding on M. truncatula leaves with calcium oxalate crystals experience greater negative effects on growth and mandible wear than those feeding on artificial diet amended with smaller amorphous crystals from commercial preparations. Commercial calcium oxalate can be added to insect artificial diet at levels up to 7.5‐fold higher than levels found in wild‐type M. truncatula leaves with minimal effect on insect growth or lepidopteran mandibles. These data suggest that negative impacts of calcium oxalate in the diet of larvae are due to physical factors, and not toxicity of the compound, as high levels of the commercial crystals are readily tolerated. In contrast to the dramatic physical effects that M. truncatula‐derived crystals have on insect mandibles, we could detect no damage to insect peritrophic gut membranes due to consumption of these crystals. Taken together, the data indicate that the size and shape of prismatic M. truncatula oxalate crystals are important factors in determining effects on insect growth. If manipulation of calcium oxalate is to be used in developing improved insect resistance in plants, then our findings suggest that controlling not only the overall amount, but also the size and shape of crystals, could be valuable traits in selecting desirable plant lines.     

Identification ofS-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-3-mercaptopyruvic acid with a metabolic intermediate betweenS-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-l-cysteine andS-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-3-mercaptolactic acid

Summary S-[2-Carboxy-1-(1H-imidazol-4-yl)ethyl]-3-mercaptopyruvic acid (I) was chemically synthesized in 15% yield by incubating a reaction mixture oftrans-urocanic acid and 3-fold excess of 3-mercaptopyruvic acid at 45°C for 6 days. The synthesized compound was characterized by fast-atom-bombardment mass spectrometry and high-voltage paper electrophoresis. CompoundI was identified with a product of an enzymatic reaction ofS-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-l-cysteine (II) with rat liver homogenate in a phosphate buffer, pH 7.4. CompoundI was degraded toS-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-3-mercaptolactic acid (III), a compound previously found in human urine [Kinuta et al. (1994) Biochem J 297: 475–478], by incubation with rat liver homogenate. From these results, we suggest that compoundI is a metabolic intermediate for the formation of compoundIII from compoundII. The present pathway follows a formation of compoundII fromS-[2-carboxy-1-(1H-imidazol-4-yl)ethyl] gluthathione [Kinuta et al. (1993) Biochim Biophys Acta 1157: 192–198], a proposed metabolite ofl-histidine.     

Specific induction of secondary product formation in transgenic plant cell cultures using an inducible promoter

This paper describes the artificial induction of secondary metabolite production in transgenic plant cell cultures using a recombinant, inducible plant promoter. The bacterial gene ubiC from Escherichia coli encodes the enzyme chorismate pyruvate lyase (CPL) which catalyses the conversion of chorismate to 4-hydroxybenzoate (4HB). This gene was fused to the tetracycline-inducible plant promoter Triple-Op. After transformation into Nicotiana tabacum W38 TET, transgenic cell cultures were established. Addition of chlorotetracycline to the medium led to specific induction of CPL activity. The optimal chlorotetracycline concentration was approximately 2 mg/l medium. Three to 5 h after induction, the ubiC mRNA concentration reached a maximum, while highest specific CPL activity was detected after 8 days. The artificial secondary metabolite 4HB was converted to glucosides, and their accumulation reached maximum levels after 5 weeks of subculture. The induction was reversible. Received: 31 May 1997 / Revision received: 22 August 1997 / Accepted: 30 September 1997