On the specificity of the in vitro and in vivo antifungal activity of benomyl

With respect to sensitivity to benomyl in vitro four categories of fungi were distinguished; taxonomically related fungi usually proved to belong to the same category. Within the form-class Deuteromycetes a correlation appeared to exist between sensitivity to this fungicide and morphogenesis of conidia.Phoma betae, in contrast to all otherPhoma spp., was shown to be highly resistant to benomyl. This, however, may be expected on account of its perfect state. The data on the in vitro antifungal activity of benomyl conformed to those on the effectiveness of benomyl against plant diseases.     

The function of an MBC-releasing deposit of benomyl and thiophanates in plant roots and soil

A comparative study has been made on the effects of short-term treatment versus long-term treatment with benomyl and three thiophanates with regard to distribution of fungitoxicant (MBC, or EBC) within plants and their protection against fungal diseases. In both treatments, plants were exposed to the fungicides for two days and then transplanted to garden soil; long-term treated plants received an additional supply of fungicide as a daily drench for one week.Results of short-term treatment of cucumber seedlings and tomato plants provided additional proof for the hypothesis, that MBC derivatives, substituted at N-1 in the benzimidazole nucleus, are retained to some extent on or in the roots, and gradually converted to MBC which then moves into the aerial parts of the plant. Correlation of infection with concentration of fungitoxicant within leaves showed 0.35 g/g fresh weight to be the limiting concentration for symptom expression of cucumber powdery mildew. Only with benomyl was MBC to be released long enough into the aerial parts of the plant to protect leaves, unfolding after termination of the treatment, for some weeks.With long-term treatment, a concentration of MBC (or EBC) sufficient for protection was found with all fungicides in all above-ground parts of the plant until the end of the experiment. Thus, a reservoir of fungicide in the planting medium guarantees a continuous supply of fungicide only then may new growth be efficaciously protected.Samenvatting Een vergelijkend onderzoek werd ingesteld naar de effecten van kortdurende behandedeling tegenover voortgezette behandeling met benomyl en drie thiophanaten wat betreft de verdeling van de eigenlijke fungitoxische verbinding (MBC, respectievelijk EBC) in planten en hun bescherming tegen schimmelziekten. Bij beide behandelingen werden de planten gedurende twee dagen aan de fungiciden blootgesteld en daarna in tuingrond geplant; in het geval van de voortgezette behandeling werd dagelijks gedurende één week een hoeveelheid fungicide in het gietwater toegevoegd.De resultaten van de kortdurende behandeling van komkommerzaailingen en tomateplanten ondersteunden de hypothese, dat MBC-derivaten, die op de N-1 plaats in de benzimidazoolring gesubstitueerd zijn, tot op zekere hoogte in de wortels worden vastgelegd, waar ze geleidelijk in MBC worden omgezet en vandaar als zodanig naar de bovengrondse delen worden getransporteerd. Correlering van infectie met concentratie van het MBC in de bladeren liet zien, dat 0,35 g/g vers gewicht de grensconcentratie is, waarboven geen symptomen van komkommermeeldauw meer optreden. Alleen in het geval van benomylbehandeling werd MBC voldoende lang naar de bovengrondse delen vervoerd om ook die bladeren die zich na beëindiging der behandeling ontplooiden, gedurende enkele weken te beschermen tegen meeldauw.Bij voortgezette behandeling werd de genoemde concentratie met alle fungiciden in alle bovengrondse plantedelen tot het einde van de onderzoeksperiode gehandhaafd. Klaarblijkelijk garandeert een zekere voorraad van het fungicide in de grond een continue toevoer van het fungicide aan de plant; alleen onder die omstandigheden kunnen nieuwgevormde plantedelen doeltreffend worden beschermd.     

Effects of benomyl,carbendazim and thiophanates on respiration and oxidative phosphorylation of Fusarium oxysporum and Saccharomyces cerevisiae

Benomyl [methyl 1-(butylcarbamoyl)-benzimidazol-2-ylcarbamate] (350 × 10^?6 M) decreased the respiration rate of Fusarium oxysporum conidia by 50% during germination. This inhibition was maintained at least 24 h after the treatment had begun. The treatment did not modify the relation between incubation time and respiration rate. Carbendazim [methyl benzimidazol-2-ylcarbamate], thiabendazole[2-(thiazol-4-yl)benzimidazole], thiophanate [1,2-di-(3-ethoxycarbonyl-2-thioureido)benzene] and thiophanate-methyl [1,2-di-(3-methoxycarbonyl-2-thioureido)benzene] were assayed using isolated mitochondria of Saccharomyces cerevisiae. These four compounds decreased mitochondrial respiration and oxidative phosphorylation rates to different extents when they were applied at a concentration of 250 × 10^?6 M . Thiophanate-methyl was the most effective since it completely suppressed the mitochondrial respiratory control at 75 × 10^?6 M .     

On the anti-cholinesterase activity of benomyl

The fungicide benomyl, previously reported to be an anti-cholinesterase and toxic to earthworms, has now been shown to have little or no effect on cholinesterase. However, it breaks down to form two products: butyl isocyanate. which is an irreversible inhibitor of the enzyme of comparable potency to organophosphates; and methyl benzimidazol-2-ylcarbamate (MBC, carbendazim), which does not inhibit at all. The reported inhibitory power of benomyl is therefore likely to be due to formation of small quantities of the former. It is also concluded that toxicity of benomyl and MBC to earthworms cannot be due to inactivation of cholinesterase.     

Metabolism of benomyl in carrot,strawberry and apple

The metabolism of benomyl was studied in carrots treated with high rates of benomyl, and in strawberries and apples treated at normal rates. The same metabolites were observed in both cases though, at normal rates, their concentrations were low but significant. Detected by means of gas-liquid chromatography they were: carbendazim, 2-aminobenzimidazole (2-AB), benzimidazole, 2-aminobenzonitrile, o-phenylene-diamine and the conjugates of carbendazim and 2-AB. o-Phenylenediamine has not hitherto been reported as a metabolite of benomyl. Two unidentified compounds were also observed by gas-liquid chromatography; their mass spectra were obtained by means of combined gas-liquid chromatography and mass spectrometry.     

In vitro and in vivo antifungal activity of synthetic pure isothiocyanates against Sclerotinia sclerotiorum

BACKGROUND: Isothiocyanates (ITCs) released by the enzymatic hydrolysis of glucosinolates in the Brassicaceae are potentially useful for controlling fungal pathogens. In vitro activity of pure ITCs against Sclerotinia sclerotiorum (Lib.) de Bary was studied by adding them to glass filters in petri dishes and dissolving them in the growing media. RESULTS: Methyl, allyl and benzyl ITCs were the most fungitoxic of the compounds in bioassays with S. sclerotiorum isolate Ss31. In the volatile phase, mycelial growth was completely inhibited by these three compounds. Aromatic ITCs were less toxic in the petri dishes but were more toxic than aliphatic ITCs when dissolved in the agar. Benzyl ITC exhibited the highest inhibitory effect on sclerotial germination, with an EC₅₀ value of 75.1 µmol L^?1. Butyl and benzyl ITCs reduced apothecial production of S. sclerotiorum by 92.5% at the highest concentration. In in vivo assay, only allyl and 2‐phenylethyl ITCs reduced disease incidence (by 76.7 and 70% respectively) at low concentrations. CONCLUSION: Sclerotinia sclerotiorum in the soil might be suppressed by the higher concentrations of allyl and benzyl ITCs released from decomposition of Brassica juncea, B. carinata, B. nigra and Sinapis spp. Copyright © 2011 Society of Chemical Industry     

A new extraction method for benomyl residues in soil and its application in movement and persistence studies

A simple and rapid method for extracting benomyl residues from soils was compared with previous methods. Soil was extracted by shaking for 2 h at room temperature with (1:1) acetone/M aqueous ammonium chloride followed by clean-up by solvent partition and ultraviolet absorption estimation of carbendazim. Recoveries were comparable to those obtained by refluxing with methanolic hydrochloric acid for 4 h, hitherto the most efficient method reported, and were much greater than those obtained by extraction with ethyl acetate or chloroform. The new method gave more tractable extracts than those obtained by refluxing with methanolic hydrochloric acid, which form troublesome metal hydroxide precipitates during clean-up. In field experiments with 2-[¹⁴C]-benomyl and 2-[¹⁴C]-carbendazim, no radioactivity was found more than 25 mm from the soil surface during 10 months after surface application of 1 kg/ha. Carbendazim residues in soils from three field experiments indicated that its persistence is very sensitive to soil pH. The time for 50% loss of initial dose ranged from 26 months at pH 5.5 to less than 3 months at pH 7.2. Biological effectiveness in a crop may therefore depend markedly on differences in soil pH.     

Synthesis and in vitro antifungal activity of 1-amino-3,4-dialkylnaphthalene-2-carbonitriles and their analogues

Twenty-four 3- and/or 4-alkyl-substituted 1-aminonaphthalene-2-carbonitriles and their analogues were prepared and evaluated for growth-inhibiting activity against four phytopathogenic fungi: Fusarium culmorum, Alternaria brassicicola, Botrytis cinerea and Penicillium expansum. The results obtained were compared with the activity of a commercial fungicide. The highest fungistatic activity was revealed by amino nitriles having hydrogen atoms or only one short alkyl group (CH3, C2H5) at the 3- or 4-position of the naphthalene system. The minimum values of calculated EC50 and EC95 indexes were 1.1 and 5.1 mg litre-1, respectively. These values were considerably lower than those for the reference fungicide.     

Influence of artificial rainfall and washing on the benomyl and thiophanate-methyl residue content in lettuce

Experiments have been carried out to investigate the influence of artificial rainfall, applied during the growing season of lettuce in a glasshouse, and washing of the sample prior to analysis on the residue content of the crop treated with the systemic fungicides benomyl and thiophanate-methyl. The results show that if artificial rainfall is applied, treatment with a threefold dose of thiophanate-methyl, compared to benomyl, results in about the same residue content at harvest time. Applying artificial rainfall during the growing season leads to a residue level on the average 54.7% lower for benomyl and 60.2% for thiophanate-methyl compared to no sprinkling. If the sample is thoroughly rinsed with water prior to analysis the figures obtained are 34.6% with and 39.3% without artificial rainfall for benomyl and 56.9 and 71.3% respectively for thiophanate-methyl. The combined effect of both treatments is a residue content of 70.4 % less for benomyl and 88.8 % for thiophanate-methyl.     

Evaluation of benomyl and thiophanate-methyl for the control of Verticillium wilt of strawberry in the Netherlands

In pot experiments the fungicides benomyl and thiophanate-methyl controlledVerticillium wilt of strawberry when applied as a soil drench after planting. Both compounds were ineffective as foliar sprays and as root dips prior to planting. Soil drenches applied to commercially grown runner plants in the waiting field (August) and to the same plants in the greenhouse (December or January) increased the yield. On infested ground, a soil drench with thiophanate-methyl promoted the occurrence of crown rot caused byPhytophthora cactorum.     

The effect of benomyl and thiophanate-methyl on earthworm populations in apple orchards

The use of the fungicides benomyl and thiophanate-methyl on experimental orchard plots for 3 years and of benomyl on a commercial orchard for 1 year resulted in a drastic reduction of earthworm populations. The species of earthworms affected were Lumbricus terrestris, L. castaneus, L. festivus, L. rubellus, Allolobophora caliginosa, A. chlorotica, A., longa, A. rosea, A. tuberculata and Octolasium cyaneum. L. terrestris and A. chlorotica were more affected than the other species, both being virtually eliminated after 2 years' spraying. The depleted populations of species other than L. terrestris and A. chlorotica recovered to normal levels after a 2-year rest period following a single year's treatment with benomyl (1.40 kg/ha). The role of the surface-feeding L. terrestris is discussed in relation to the spray fall-out on the sward and to grass management.     

Resistance to benomyl and some chemically related compounds in strains of Penicillium species

TwoPenicillium species, vizP. brevicompactum andP. corymbiferum, were isolated from senescent petioles of cyclamen and from bulbs of lilies, respectively, both samples treated previously with benomyl. The isolates turned out to be very resistant to this fungicide when grown on malt agar, supplied with the fungicide; at a concentration of 2000 g/ml they were less inhibited than randomly chosen isolates of the same species on agar with 1 g/ml.The strains retained their resistance at the same level for at least 3 months after repeated subculturing on fungicide-free agar.Resistance to benomyl coincided with resistance to methyl-thiophanate and, to a lesser extent, also to thiabendazole and furidazol.Samenvatting Uit afstervende bladstelen van cyclamen en uit schubben van leliebollen, welke eerder met benomyl waren behandeld, konden respectievelijkPenicillium brevicompactum enPenicillium corymbiferum worden geïsoleerd. De isolaten blekenin vitro zeer resistent tegen het fungicide. De myceliumgroei van deze isolaten werd op moutagar met 2000 g/ml benomyl minder geremd dan die van willekeurige isolaten van dezelfde soorten op agar met 1 g/ml (Fig. 1).De isolaten bleven gedurende tenminste 3 maanden resistent na regelmatig overenten op voedingsbodems zonder het fungicide.De resistente stammen van de beidePenicillium-soorten bleken eveneens resistent tegen methyl-thiophanaat en in mindere mate ook tegen thiabendazol en furidazol (Tabel 1). De volgorde van de groeiremmende werking van deze fungiciden was voor de willekeurig gekozen (gevoelige) isolaten: benomyl>thiabendazol>methyl-thiophanaat >furidazol. Voor de resistente stammen was deze: thiabendazol en furidazol >benomyl>methyl-thiophanaat. In het feit dat een dergelijke verandering in volgorde van remmend effect ook voorBotrytis cinerea geldt, ligt een aanwijzing, dat de wijze waarop de resistentie werkt, voor deze schimmels gelijk is.     

Effect of benomyl on the potato cyst nematode,Heterodera rostochiensis

Benomyl is a strong fungistat against certain groups of fungi and it does not have much effect on most plant-parasitic nematodes.Heterodera spp., however, are affected, and the literature indicated a strong effect onHeterodera rostochiensis in the field. Laboratory tests demonstrated that hatching of larvae fromH. rostochiensis cysts is strongly inhibited by benomyl in vitro. In a pot trial, benomyl effectively suppressed formation of new cysts on the roots of potatoes grown in sand, clay soil or lightsandy loam. There was no such effect in soil rich in organic matter probably because of strong adsorption of benomyl. Control ofH. rostochiensis by benomyl in soil could be due to suppressed hatch or to other mechanisms, such as inhibition of larval penetration of roots. In view of its long persistence in the soil and of its various side-effects, benomyl does not seem very promising for practical control ofHeterodera rostochiensis.Samenvatting Benomyl is bekend als een middel met sterk fungistatische eigenschappen ten aanzien van bepaalde groepen schimmels. Het heeft in het algemeen weinig effect op planteparasitaire aaltjes. Cystevormende aaltjes echter, en in het bijzonderHeterodera rostochiensis, zijn, volgens aanwijzingen uit de literatuur gevoeliger voor benomyl.Onder laboratoriumomstandigheden bleek het uitkomen van de larven vanH. rostochiensis uit de cysten sterk door benomyl te worden geremd (Tabel 1 en 2). In een potproef werd door een grondbehandeling met benomyl de vorming van nieuwe cysten op aardappelwortels onderdrukt, afhankelik van de grondsoort (Tabel 3). Naarmate de grond meer organische stof of klei bevat, is het effect minder groot. Bijgebruik van rivierzand trad reeds bij een dosering van ongeveer 1 ppm benomyl een volledige on derdrukking van cystevorming op. Maar bij potgrond (59% organische stof) was er, ook bij een tien maal hogere dosering, geen effect. Verder onderzoek zal moeten uitwijzen of het effect in grond, berust op de remming van het uitkomen van larven uit de cysten, of (ook) op de beïnvloeding van andere aspecten van het parasitair gedrag van dit aaltje, zoals de penetratie in de wortels.Gezien de grote persistentie van benomyl in de grond, en de nevenwerkingen, lijkt het middel minder geschikt voor toepassing in de praktijk.Guest worker of TNO, Netherlands Organization of Applied Scientific Research.     

Resistance of Venturia nashicola to thiophanate-methyl and benomyl: build-up and decline of resistance in the field

Changes in the proportion of benzimidazole-resistant strains in populations of Venturia nashicola were monitored in pear orchards. Successive applications of thiophanate-methyl increased the level of resistance in the populations. When the application of benzimidazoles was stopped and other fungicides were applied, the proportion of highly resistant strains gradually decreased and the proportion of intermediately resistant, weakly resistant and sensitive strains increased. It is suggested that this phenomenon is an example of genetic homeostasis within microbial populations.     

Uptake and translocation of benomyl and carbendazim (methyl benzimidazol-2-yl carbamate) in the symplast

Cytoplasmic uptake of carbendazim (methyl benzimidazol-2-yl carbamate; MBC) from an aqueous solution was demonstrated with isolated mesophyll cells. About 2.5% of the labelled MBC (ring-2-[¹⁴C]) in the treatment solution (1.85 μg/ml) was taken up in 44 h. When cotyledons of cucumber seedling were treated with either 347 or 36 μg [¹⁴C]-MBC/plant 1.11 and 0.13% were extracted, respectively, from the remainder of the plant, 5 days after treatment. Greatest amounts were detected in shoot apices. Likewise, when MBC and benomyl were applied at the dose of 2 μmol, 0.34 and 0.57% were detected in the untreated part of the plant with a bioassay procedure. Foliar application with 347 or 36 μg[¹⁴C]-MBC/leaf resulted in the translocation of 1.68 and 0.11% out of the treated area. By scalding the living cells of the petiole translocation was prevented suggesting that long distance movement occurred in the symplast. During a period of 14 days 1.56% of [¹⁴C]-MBC applied to cucumber leaves was metabolised and respired as CO₂. This degradation was assumed to occur enzymically within the symplast.     

Preliminary study on the effect of benomyl on the fungal flora in a greenhouse soil

Samenvatting Monsters van een humusarme zandgrond uit een kas in Naaldwijk werden gemengd met benomyl (10 ppm). Dit gebeurde 6 maanden, 3 maanden, 6 en 3 maanden en éen week voor de schimmelanalyse. Saprofytische schimmels werden geïsoleerd van gewassen gronddeeltjes,Pythium-soorten van gezeefde grondkruimels. De isolatiepercentages van de op organische deeltjes meest voorkomende schimmels en vanPythium op grondkruimed zijn in Tabel 2 weergegeven. Een verschuiving in de schimmelpopulatie ten gunste van de benomyl-ongevoelige soorten kon niet worden aangetoond. Dit kan verklaard worden door de lage stofwisselingsaktiviteit van de bodemschimmelflora.     

Persistence of benomyl and thiophanate compounds in soil and various plants following soil application

Control ofSclerotinia sclerotiorum in muskmelons was obtained by a single soil drench with benomyl. The material was found in the apical parts of the plant and in the soil until the end of the growing season. Benomyl [methyl l-(butylcarbamoyl)-2-benzi-midazolecarbamate] and thiophanate methyl-NF 44 [1.2- bis (3 methoxycarbonyl-2-thioureido) benzene], applied to seedbeds, were taken up by and then persisted in tomato, pepper and eggplant for approximately 8 weeks after application. Thiophanate-NF 35 [1.2-bis (ethoxycarbonyl-2-thioureido) benzene] could be detected only in the bottom leaves of pepper and eggplant, for up to 4 weeks after application.     

Sensitivity of populations ofBotrytis cinerea to triazoles,benomyl and vinclozolin

Sensitivity of field isolates (121) ofBotrytis cinerea from France (1992), Germany (1979–1992), Israel (1990) and the Netherlands (1970–1989) to the triazoles tebuconazole and triadimenol, the benzimidazole benomyl and the dicarboximide vinclozolin were tested in radial growth experiments. Resistance to benomyl (in 21 to 100% of isolates tested) and vinclozolin (in 25 to 71% of isolates tested) was common in most countries. EC₅₀s (concentrations of fungicides inhibiting radial mycelial growth ofB. cinerea on B5-agar by 50%) for tebuconazole and triadimenol ranged between 0.01–1.64 and 0.4–32.6g ml^–1, respectively, and were log-normally distributed. The variation factor (ratio between EC₅₀s of the least and most sensitive isolate tested) amounts 164 and 82 for tebuconazole and triadimenol, respectively. These values are comparable to those for azole fungicides applied in control of other pathogens. Hence, variation in sensitivity to triazoles can probably not explain limited field performance of triazoles towardsB. cinerea. Isolates from south west Germany (1992) were significantly less sensitive to tebuconazole than isolates collected earlier in Germany, Israel and the Netherlands. Such less sensitive populations may contribute to the limited field performance of DMI fungicides towardsB. cinerea. The sensitivity of isolates from south west Germany to tebuconazole was similar to that of DMI-resistant mutants generated in the laboratory. These mutants displayed stable resistance with Q-values (ratio between EC₅₀ of resistant mutant and wild type isolate) between 5 and 20. Sensitivity of field isolates and laboratory mutants to tebuconazole and triadimenol was correlated.     

Lack of cross resistance to benomyl and thiabendazole in some strains of Aspergillus nidulans

Samenvatting Verworven resistentie inAspergillus nidulans tegen benomyl en thiabendazol bleek te berusten op één gen, gelocaliseerd op chromosoom VIII, op een afstand van 34 eenheden van ts-D 15 en 5 eenheden van orn-B 7. Hoewel kruisresistentie de regel is, blijkt dat sommige, mutanten tegen slechts één van beide fungiciden resistent zijn, en dat resistantie tegen thiabendazol zelfs gepaard kan gaan met extra gevoeligheid voor benomyl (Fig. 1 en 2).