The incidence of benomyl tolerance in Verticillium fungicola, Mycogone perniciosa and Hypomyces rosellus in mushroom crops

Of the 229 isolates of Verticillium fungicola examined 63 % were found to show some benomyl tolerance and 53% were very tolerant having EDg, values in excess of 50 ppm. There was a tendency for the tolerant isolates to have a slower growth rate than the sensitive ones on unamended potato dextrose agar (PDA) and forty-five isolates (19 %) showed growth stimulation at 5 ppm benomyl. No major differences were found in ten isolates of V. fungicola tested on benomyl, thiabendazole and thiophanate methyl agars or of twelve isolates compared on benomyl and carbendazim hydrochloride agars. Tolerant isolates of V. fungicola and higher levels of crop loss were linked in the east and north of the country but not in the south-east. Tolerance was not found in sixty-six isolates of Mycogone perniciosa or twenty-four of Hypomyces rosellus. Benomyl was used on 89 % of the farms in the survey either applied to the casing (34%) or as a spray (55 %) or both (10%).     

Strains of Colletotrichum coffeanum, the causal agent of coffee berry disease, tolerant to benzimidazole compounds in Kenya

The detection of Colletotrichum coffeanum tolerant to methyl ester of benzimidazole 2-carbamic acid (carbendazim) and a related benzimidazole compound, cypendazole, followed increases in levels of coffee berry disease observed on Coffea arabica in experimental plots sprayed for 2 yr with these compounds. Sporulation by the pathogen on naturally infected berries removed from carbendazim-, cypendazole- or benomyl-sprayed plots was not checked by a further application of 0–05 % (a.i.) of any of the compounds. Nearly all the isolates from these berries were capable of some growth on agar media containing 1000 ppm (a.i.) of either carbendazim or cypendazole. However, only a few could tolerate 1000 ppm of benomyl and the inability of this compound to reduce sporulation on berries infected with tolerant strains was presumably due to its rapid conversion to carbendazim within the host tissue. Less than 1 ppm of carbendazim, cypendazole or benomyl was needed to give 50% inhibition of conidia of the normal strain. Against the most tolerant strains, however, the LD 50 was > 100 ppm of carbendazim and about 30 ppm of benomyl. Whether isolated from unsprayed or benzimidazole-sprayed plots, all isolates of Colletotrichum acutatum, a saprophytic cohabitant of lesions initiated on berries by C. coffeanum, showed the highest degree of tolerance to benzimidazole compounds. No tolerance of either fungus to the ‘conventional’ fungicide captafol was detected.     

Systemic action of benomyl against late-wilt disease of maize.

Benomyl (methyl-I-(butyl carbamoyl)-2-benzimidazole carbamate) at 2.5 to 100 ppm completely inhibited the growth in vitro of Cephalosporium maydis Samra, Sabet & Hingorani, the causal fungus of late-wilt of maize. In pot experiments, late-wilt was controlled by applications equivalent to 10 ppm benomyl/unit wt dry soil at sowing but not by applications 30 days after sowing or by seed treatments (dusts and dips). A fungitoxic substance was detected in benomyl-treated soil, planted with maize, 120–150 days after treatment and for 150–180 days in unplanted soil. Sap expressed from maize grown in benomyl-treated soil was also fungitoxic, especially that from the roots and distal parts of the leaf blades. In the field, soil applications of benomyl to 10 kg/feddan (4200 m²) failed to control late-wilt. Tests indicated little fungitoxicity in the sap of maize plants 25 days after sowing.     

The effect of the systemic fungicide benomyl on survival and reproduction of the bird cherry aphid (Rhopalosiphum padi)

Weight gain and intrinsic rate of population increase (r_m) of Rhopalosiphum padi were measured on wheat plants given soil drenches of benomyl at various concentrations. Reduced weight gain occurred with concentrations as low as 3.125 ppm. Mortality of aphids significantly exceeded control mortality at 25 ppm, and no aphids survived concentrations above 50 ppm. Development time was extended at 12.5 ppm and above. Reductions in fecundity and r_m became statistically significant by 25 ppm. Topical application of benomyl at 100 ppm or above increased aphid mortality, which reached 97% at 400 ppm. Weight gain of aphids was also reduced by topical application at 100 ppm. A factorial experiment showed absence of interaction between the effects on aphid weight of soil drench application to plants and topical application to aphids.     

The effect of benomyl on growth and ultrastructure of two isolates of Phytophthora infestans from Egypt

The effect of benomyl as a fungicide on the growth rate and ultrastructure of two isolates (P1319 and P623) of Phytophthora infestans is compared. Benomyl caused a concentration-dependent inhibition of the mycelial growth of both isolates. The isolate P1319 was found to be more sensitive to benomyl than the isolate P623. Ultarstructural studies confirmed these observations. The hyphae of isolate P1319 subjected to 100 and 500 ppm benomyl showed more severe changes in the cytoplasm than those of isolate P623. An increase in lipid bodies and vacuoles in the hyphal cytoplasm was the characteristic phenomenon after treatment with benomyl, particularly at a concentration of 500 ppm.     

Tradeoffs between reproduction and mycelium production in the unit-restricted decomposer Coprinus cinereus

A laboratory experiment was performed which examined tradeoffs between production of mycelium and reproduction (using stipe dry weight as an estimator of spore production) in the coprophilous mushroom species Coprinus cinereus. Isolates of the fungus taken from a single dikaryotic mycelium were grown in Petri plates containing yeast extract agar. Plates varied in diameter and resource density, but the total volume of agar was kept constant. Isolates grown in 100 mm and 150 mm diameter plates produced significantly less mycelium compared to isolates grown in 60 mm diameter plates. Within 60 mm plates there was no correlation between the efficiency of mycelium production and fruit body production, but in larger plates there was a significant negative correlation between the two. These results indicate that isolates grown on larger plates were less efficient at using resources than isolates grown on small plates, and that mycelium production is curtailed on larger plates to maintain spore production.     

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.     

Growth of helminthosporia on media containing the fungicide benomyl and the assay of benomyl using Botrytis cinerea

A bioassay using Botrytis cinerea detected 0–05 ppm benomyl in potato dextrose agar. Nine species of Cochliobolus, fourteen of Drechslera, four of Helminthosporium and five of Pyrenophora tolerated the presence of benomyl to the same extent, and grew on agar plates containing 40 ppm of the compound.     

Benomyl tolerance in isolates of Botrytis cinerea from tomato plants

Three hundred and forty-nine isolates of Botrytis cinerea were collected from tomato crops on forty-one nurseries and 173 (40/6 %) were found to be tolerant to benomyl. There was no obvious association between disease incidence and the occurrence of tolerance. In a fungicide comparison experiment on tomatoes in 1973, twenty of the sixty-four (31 %) isolates examined were benomyl tolerant, the majority of these were from benomyl sprayed plants. In 1974 in a similar experiment, 384 of the 394 (97-5 %) isolates examined were tolerant. Tolerance was monitored in two tomato experiments in relation to a spray programme in which benomyl and dichlofluanid were used in various combinations. There was no marked effect of the spray programmes on the incidence of tolerance on either site. In the experiments B. cinerea was controlled and significant increases in yield were obtained with benomyl in 1973 but not in 1974. This difference is attributed to the change in the pathogen population with a large increase in the incidence of tolerance on the experimental site in 1974.     

Occurrence of strains of Colletotrichum coffeanutn resistant to methyl benzimidazol-2-ylcarbamate (carbendazim) and chemically-similar compounds

Certain strains of Colletotrichum coffeanum (the causal fungus of coffee berry disease) which developed in coffee plots sprayed in 1973 and 1974 with carbendazim formulations Bavistin and Derosal and with Folicidin (cypendazol) were resistant to these fungicides and also to Benlate (benomyl). Strains resistant to cypendazol developed in plots sprayed with benomyl but these were not resistant to benomyl itself. There were also indications of strains resistant to carbendazim amongst the population of saprophytic species (C. acutatum and C. gloeosporoides) colonizing coffee bark. Resistant isolates of C. coffeanum in culture produced colour variants by sectoring more frequently than normal isolates. These sectors were equally pathogenic to coffee berries and their growth was slightly enhanced on media containing carbendazim.     

Effects of oximecarbamate, organophosphate and benzimidazole nematicides on life cycle stages of root-knot nematodes, Meloidogyne spp.

A study was made of the effects of concentrations of 2 to 32 ppm of oximecarbamate, organophosphate and benzimidazole nematicides on the hatch, larval viability and migration of Meloidogyne javanica, M. incognita and M. hapla and on development of M. javanica in roots. Aldicarb at less than 8 ppm had little effect on hatch and methomyl markedly affected only the hatch of M. hapla. As little as 2 ppm of fenamiphos or thionazin markedly reduced hatch of all three species but less than 8 ppm ethoprophos significantly reduced only the hatch of M. incognita and phorate had little effect on hatch. Benomyl and thiabendazole had no significant effects on hatch. When egg masses of M. incognita were transferred from nematicides which suppressed hatch to water, hatching occurred, but aldicarb, fenamiphos, ethoprophos and thionazin significantly reduced total hatch. None of the nematicides killed larvae of the three species immersed in 16 and 32 ppm solutions of them for 3 days. Aldicarb at 2 ppm reduced migrations of all three species; the effects of methomyl, fenamiphos or thionazin on migration varied according to species, while phorate, ethoprophos, benomyl or thiabendazole had little or no effect on migration. Aldicarb or thionazin at 2 ppm stopped development of M. javanica in roots of tomato seedlings while methomyl, ethoprophos or fenamiphos at 4 ppm reduced development by 60% and at 8 ppm of ethoprophos or fenamiphos or 16 ppm of methomyl, development was stopped. Phorate had little effect on development and benomyl or thiabendazole had no effect. Nematicide concentrations which reduced development prevented the normal orientation of larvae in the roots and reduced or prevented giant cell formation.     

The production of toxic metabolites by Chaetomium spp. isolated from solis of permanent pasture.

One hundred and two isolates of Chaetomium spp. have been identified from 2563 soil samples collected from permanent pasture at Nappan, Nova Scotia. Chaetomium umbonatum was the Chaetomium species most commonly isolated. Fifty-six of the Chaetomium isolates were grown in the laboratory and the cultures examined for the production of toxic metabolites. The culture filtrates of 12, and extracts of mycelium of 18, of these isolates inhibited bacterial growth. Chetomin was detected in nine mycelium extracts and isolated from four of the mycelium extracts. Chaetoglobosins were isolated from three mycelium extracts.     

Effects of soil treatments and cultivars on 'early dying' disease of potatoes caused by Globodera rostochiensis and Verticilliurn dahliae

Plots in a field infested with Globodera rostochiensis and Verticillium dahliae were treated in 1971 with methyl bromide, dazomet, aldicarb, benomyl or aldicarb + benomyl and potatoes (cv. Pentland Dell) were grown in four consecutive years. In 1971, all treatments delayed the development of haulm symptoms, decreased soil populations of G. rostochiensis and increased yields. In the second year yields were increased by all treatments except dazomet whereas in the third and fourth years only benomyl or aldicarb + benomyl increased yields. Soil populations of V. dahliae were decreased by methyl bromide in 1971 but not by other treatments or in later years. In 1976, Pentland Dell and Pentland Crown were grown on an infested field in plots fumigated with methyl bromide. Fumigation slightly delayed shoot emergence but increased plant height, ground cover and the size and persistence of the leaves; development of haulm symptoms was delayed and yield increased. Fumigating with methyl bromide at five times the normal rate was more beneficial to Pentland Dell than to Pentland Crown. In a comparison of 10 cultivars in 1975, symptoms developed sooner and yields were smaller on a field infested with G. rostochiensis and V. dahliae than on a non-infested field and on both sites Maris Peer plants died soonest and yielded least. Pentland Crown yielded most on the non-infested field and Maris Piper on the infested field.     

Detecting the heavy metal tolerance level in ectomycorrhizal fungi <Emphasis Type="Italic">in vitro</Emphasis>

Summary Eight isolates of ectomycorrhizal fungi namely, Laccaria fraterna (EM-1083),Laccaria laccata (EM-1191), Pisolithus tinctorius (EM-1081),Pisolithus tinctorius (EM-1293), Scleroderma cepa (EM-1233),Scleroderma flavidum (EM-1235),Scleroderma verucosum, (EM-1283) and Hysterangium incarceratum (EM-1185) were grown on specially designed cocktail media prepared by adding various concentrations of different heavy metals namely Al, As, Cd, Cr, Ni and Pb. The heavy metals were selected keeping in view their relative abundance in coal ash and potential toxicity. The fungal isolates were grown on such designed cocktail media. The colony diameter was used for the measurement of the fungal growth. Total heavy metal accumulated in the mycelia was assayed by atomic absorption spectrophotometry.In relation to metal tolerance ability in general,Hysterangium incarceratum (EM-1185) showed maximum tolerance with respect to growth, Laccaria fraterna (EM-1083) and Pisolithus tinctorius (EM-1293) also showed considerable tolerance to the heavy metals tested. In relation to metal uptake in particular, Pisolithus tinctorius (EM-1293), has reported maximum uptake of Al (34642.58 ppm), Cd (302.12 ppm) and Pb (3501.96 ppm). In Laccaria fraterna (EM-1083), As (130.57 ppm) and Cr (402.38 ppm) uptake was recorded maximum; and Hysterangium incarceratum (EM-1185) has recorded maximum Ni (2648.59 ppm) uptake among the three suitable isolates documented here.     

Ultrastructural changes in the spore and mycelia of Ascosphaera apis after treatment with benomyl (Benlate 50 W)

In Ascosphaera apis, after 8 days growth in darkness at 28° C, numerous sporocysts were observed, within which mature spores were seen aggregated into a spore ball. The mature spore of A. apis had a thick spore wall with an electron-opaque outer layer, a spore membrane with many depressions, and sporoplasm containing numerous ribosomes and mitochondria. In the cytoplasm of the mycelium, mitochondria with well-defined cristae and numerous ribosomes were observed. At a concentration of 1 g/ml of culture medium, benomyl appeared to inhibit colony growth of A. apis, but some sporocysts containing deformed spores were found. Deformed spores possessed a thick spore wall with a grainy matrix, and depressions were no longer detected in the spore membrane. Ribosomes were lacking in the sporoplasm and mitochondria appeared degenerate. The mycelium from the treated culture contained mitochondria with an electron-lucid matrix and no well defined cristae, while ribosomes were completely depleted. The significance of these observations in relation to the use of benomyl to control chalkbrood disease in the honey bee is discussed.     

The effect of carbendazim-generating fungicides with and without a mineral oil additive on tomato stem lesions caused by carbendazim-sensitive and tolerant strains of Botrytis cinerea

Isolates of Botrytis cinerea were obtained from tomatoes in several localities in the West Scotland. Some isolates grew on agar containing 100 mg/1 benomyl (carbendazim-tolerant), while others did not (carbendazim-sensitive). Pot-grown tomato plants treated with benomyl and other carbendazim-generating fungicides, applied either as sprays or soil drenches, were inoculated on the leaf scars with some of these isolates. On treated plants the carbendazim-tolerant isolates formed lesions which were about as large as those on untreated plants. Sensitive isolates formed much smaller lesions on treated plants. There was evidence that the increase in lesion size during the period 7–14 days after inoculation with a carbendazim-sensitive isolate was less on plants sprayed with benomyl or carbendazim with added mineral oil (2% Actipron) than on plants to which the fungicides alone had been applied. No such effect was recorded with thio-phanate-methyl. There was also an indication that the addition of Actipron to a benomyl spray improved the effect of the fungicide against two tolerant isolates, though there was no effect on the relative increase in lesion size during the second week after inoculation. In two tests the addition of 2% and 4% Actipron to benomyl soil drenches did not improve the level of leaf scar lesion control.     

The disappearance of benomyl from mushroom casing

The failure of Benlate (50% benomyl) to control Mycogone perniciosa on mushroom farms where isolates of the organism were benomyl sensitive was investigated. A comparison was made of carbendazim levels, the major fungitoxic breakdown product of benomyl, in unsterilised casing, autoclaved casing and autoclaved casing mixed with a small proportion (1.7%) of casing previously treated with benomyl but from which all detectable levels of carbendazim had disappeared. Added benomyl was effective in controlling M. perniciosa in autoclaved casing, less effective in unamended casing and ineffective in the amended casing mixture. The recovery of carbendazim from these treatments was directly related to disease occurrence. Studies on farms where benomyl or other carbendazim generating fungicides had been used indicated that a lack of control of M. perniciosa was associated with the disappearance of fungicide from the casing before cropping began. This was not so on a farm where carbendazim generating fungicides had not been used before these investigations. Thiabendazole, a fungicide closely related to the carbendazim generators showed only a slight decline in concentration in casing capable of degrading benomyl. Bacteria isolated from the casing were examined in vitro for their ability to degrade benomyl. Five groups of bacteria were compared and three bacteria, Pseudomonas putida, Pseudomonas aeruginosa and an unidentified member of the Entero-bacteriaceae were most able to degrade benomyl.     

Mycotoxin production by Fusarium species isolated from New Zealand maize fields

Forty Fusarium isolates obtained from maize fields were screened for moniliformin production on maize kernels. Twelve isolates, including seven of F. subglutinans, were found to produce moniliformin at levels ranging from 0.4 to 64 ppm. Twenty six isolates were also screened for production of deoxynivalenol, diacetoxyscirpenol, T-2 toxin and zearalenone. Of these, 22, including all 11 isolates of F. graminearum, produced zearalenone at levels ranging from 0.1 to 96.0 ppm, while 13 produced T-2 toxin at low levels, (<1.1 ppm). Deoxynivalenol and diacetoxyscirpenol were each produced by six isolates, also at low levels (<1.0 ppm). Three isolates of F. graminearum and one of F. sambucinum produced four toxins simultaneously.     

Isolation and Properties of Phenol Utilizing Microorganisms with Special Reference to Catechol 1,2-Oxygenase

Phenol utilizing yeasts were isolated from soil. The relationship were examined between distribution of phenol uptake rate using intact cells and distribution of the activities of catechol 1,2-oxygenase which is one of the key enzymes in phenol metabolism. Two of the isolates showed catechol 1,2-oxygenase activity even when grown in glucose medium, though the enzyme activity was about 1% of the full activity induced by phenol. Partially constitutive mutants for catechol 1,2-oxygenase were obtained by mutagenesis of an inducible strain. The level of mutant enzyme activity was close to that of the isolated constitutive strain. One isolate, Trichosporon cutaneum, preferentially utilized phenol to glucose in medium containing both phenol (200 ppm) and glucose (0.1%), until the concentration of phenol decreased to 10–20 ppm.     

In vitro sensitivity of Verticillium fungicola to selected fungicides

Twenty isolates of Verticillium fungicola var. fungicola collected from diseased fruit-bodies of Agaricus bisporus from prochloraz-treated crops, were exposed to a range of concentrations of six chemicals (benomyl, chlorothalonil, formaldehyde, iprodione, prochloraz-Mn-complex and prochloraz + carbendazim) in vitro. EC₅₀ values were determined for each fungus-fungicide combination. All isolates were more sensitive to prochloraz-Mn-complex (EC₅₀ values less than 5 mg 1^–1) than to the remainder fungicides, and only seven isolates were moderately sensitive (EC₅₀ values between 5 and 50 mg 1^–1) to prochloraz + carbendazim. All isolates were moderately sensitive to formaldehyde, whereas the majority of isolates were very resistant to the other three fungicides (benomyl, chlorothalonil and iprodione).