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%).     

Effect of Four Fungicides on Barley Net Blotch Caused by Drechslera teres

The effect of four fungicides (benomyl, carbendazim, triadimefon and sulp hur), used in intensive barley cropping on the epidemiology of Drechslera teres f. maculata and f. teres, was investigated under a controlled environment. Two fungicides (benomyl and carbendazim) increased disease development, especially at the recommended field rate, sulphur had the same effect at lower concentrations and triadimefon promoted net blotch caused by D. teres f. teres. At the recommended field rate, carbendazim enhanced sporulation in the two pathogen forms, whereas benomyl and sulphur enhanced 14 sporulation only on the maculata form. The results showed that, under controlled conditions, most of the fungicides promotednet blotch development and that sporulation increased either as a result of the pathogen form or of the concentration of the active ingredient.     

The control of apple canker (Nectria galligena) in a young orchard with established infections

In a factorial experiment on canker control the efficiency of phenylmercurie nitrate (PMN) applied at leaf fall, before bud burst or both was compared with the application of dodine, dithianon, triforine, thiabendazole, benomyl or carbendazim in May and June. Of the total number of cankers which developed in unsprayed trees 76–78% resulted from infections in April to August of each year of the experiment. Infection was reduced significantly by all of the fungicides applied in summer. Of these carbendazim was outstanding, controlling both summer and autumn infections. Sporulation throughout the summer was suppressed by carbendazim and to a lesser extent benomyl, but whereas the suppressant effect of carbendazim persisted until long after leaf fall that of benomyl was evident only until August. Dodine, dithianon, triforine and thiabendazole had no significant effect on spore production. Dithianon and dodine showed highest toxicity to the germination of Nectria galligena spores of all fungicides used in summer. All of the fungicides controlled apple scab (Venturia inaequalis) although thiabendazole was the least effective. Autumn applications of PMN reduced canker incidence but their value alone was not as great as carbendazim, dithianon, benomyl or dodine applied in summer. PMN applied in spring reduced the number of cankers in trees receiving no other fungicides but tended to increase the incidence of infections, particularly in autumn, when used in conjunction with fungicides applied in May and June. PMN applied before bud burst reduced sporulation of N. galligena for a few weeks, after which production resumed and in late summer and autumn actually exceeded the controls. None of the fungicides had any direct effect on leaf fall. Infection of the crotch and basal leaf scars was more common in summer than in autumn whereas infection of leaf scars above the basal region was more common in autumn than in summer. The rootstocks of many of the trees became infected via callus tissue associated with adventitious root development and this was controlled by the carbendazim treatment.     

In vitro and In vivo Effects of Some Fungicides against the Chickpea Blight Pathogen, Ascochyta rabiei

The effects of various fungicides on mycelial growth and spore germination of Ascochyta rabiei were determined by incorporating them into potato dextrose agar and measuring colony diameter and observing colony growth and spore germination at 20 ± 2°C. Eight fungicides prevented spore germination of the pathogen at concentrations of 0.125–2 μg/ml, three hindered mycelial growth at 2–4 μg/ml and seven failed to inhibit mycelial growth even at 128 μg/ml. The reference fungicide for the pathogen, chlorothalonil, stopped conidial germination at low rates but did not prevent mycelial growth at 128 μg/ml. Thirteen fungicides were tested against seed infections of the pathogen, and benomyl + thiram, carbendazim and carbendazim + chlorothalonil seed treatments gave more than 85% inhibition on both vacuum‐infiltrated and naturally infected seeds. Coating the seeds with polymers did not increase the effectiveness of fungicides. Three fungicides; (azoxystrobin, chlorothalonil and mancozeb), gave the highest protection in the field but protection decreased with increased inoculum pressure. Addition of humic acid to fungicide suspensions did not affect their performance.     

Identification of Rhodococcuserythropolis isolates capable of degrading the fungicide carbendazim

Continuous enrichment cultures were used to identify bacterial isolates capable of degrading the fungicide carbendazim. The bacteria originated from sites that had been repeatedly treated with the structurally related parent fungicide, benomyl, over a period of several years. Six isolates were identified as carbendazim degraders on the basis of their ability to produce diffusion-clearing zones on a minimal salts solid medium spray-coated with a 0.1% solution of carbendazim, their ability to grow in a minimal salts broth supplemented with carbendazim as the sole carbon source, and their ability to reduce carbendazim levels in liquid cultures. All six isolates were identified as Rhodococcus erythropolis or a closely related species by analyses of nutritional utilization and whole-cell fatty acid methyl ester profiles. A chemically induced mutant of R. erythropolis isolate B2E was identified that was no longer capable of degrading carbendazim, as determined by negative results in all three assays. Further characterization of these strains provides an opportunity for their development in bioremediation of the compound. Received: 22 July 1996 / Received revision: 16 December 1996 / Accepted: 10 January 1997     

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 effect of seed treatment with benzimidazole-based fungicides on infection of the foliage of overwintered salad onions by Botrytis cinerea

Seed treatments of carbendazim (Bavistin 50% W.p.) and thiophanate-methyl (Mildothane 50% W.p.) applied to overwintered salad onions at 250 g a.i./kg seed protected the foliage of plants from infection by Botrytis cinerea during the seasons 1973–1976. Crop establishment and yield were also improved. Seed treatment with calomel was not effective. Chemical analysis of treated non-viable seeds, retrieved from the soil, indicated that 73% carbendazim and 46% thiophanate-methyl remained attached to the seeds after 9 months in the soil. Analyses of onion leaves revealed that each fungicide was represented by similar quantities of carbendazim, 5 μg/g fresh weight in October 1975 reducing to 1 μg/g fresh weight in May 1976. Bioassay tests showed that the fungicide was acropetally distributed and was present in all leaves early in the season (October) but was absent from some new leaves formed in the following spring. Carbendazim-insensitive isolates of B. cinerea occurred after three seasons' use of this chemical. Sensitive isolates failed to grow on agar containing 1 μg/ml benomyl but all insensitive isolates (31 total) grew normally at this concentration and some were capable of growth on agar containing 1000 μg/ml benomyl. The emergence of foliar isolates of the fungus insensitive to the benzimidazole-based compounds used in the treatment of seeds indicated that these fungicides did not provide a permanent solution to the disease problem.     

The control of wet bubble disease of mushrooms caused by Mycogone perniciosa

In experiments on the control of Mycogone perniciosa there was a good relationship between yield, disease incidence and the rate of benomyl applied to the casing. Casing treatment controlled the disease irrespective of time of inoculation. Benomyl controlled the disease in all experiments and good results were also achieved with thiophanate methyl and to a lesser extent with thiabendazole. Bioassay of sporophores showed the presence of an inhibitory substance from benomyl and thiophanate methyl treatments even after 6 wk of cropping.     

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.     

Efficacy of benzimidazole and related fungicides against Rhizoctonia solani and R. bataticola

Five formulations of four benzimidazole derived fungicides, carbendazim, benomyl, thiophanate methyl and methyl 4-[2-(2-dimethylamino acetamide) phenyl]-3-thioallophanate were compared for their toxicity towards two pathogenic isolates of Rhizoctonia solani and three of R. bataticola. The isolates of two fungi showed significant differences in mycelial growth inhibition by the five fungicides. Benomyl and carbendazim were most inhibitory to all isolates of both fungi while the sesame isolate of R. bataticola was least sensitive to all fungicides. Disease control (90%) was obtained with low concentrations of benomyl against root rot of cowpea caused by R. solani, and with thiophanate methyl against root rot of sesame and sunflower, and leaf blight of mung bean caused by R. bataticola. The spread of stalk-end rot of sunflower heads was best checked with a spray of thiophanate methyl. The results suggest that benzimidazole fungicides having similar toxophores act differently for disease control in different host-parasite combinations.     

Double resistance by citrus green mould Penicillium digitatum to the fungicides guazatine and benomyl

In vitro dosage response data with different isolates of Penicillium digitatum and the fungicide guazatine indicated an approximate 10-fold shift in tolerance when compared with wild type strains. ED₅₀ values for resistant strains were approximately 0.5 μg/ml compared to approximately 0.05, μg/ml for the wild type strains. Colony growth of guazatine resistant isolates on selective media containing carbendazim showed that they were also resistant to the benzimidazole group of fungicides. In vivo tests in inoculated oranges with strains previously characterised by in vitro tests confirmed resistance to guazatine and benomyl. A combined treatment of these fungicides at 400 /μ/ml and 500 μg/ml respectively, which normally gives protection against decay, also failed to provide adequate mould control. Growth and pathogenicity of the resistant strains in these tests in oranges were indistinguishable from that of wild type strains.     

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.     

Studies on the fungicidal control of stem lesions caused by Botrytis cinerea on glasshouse tomatoes

In glasshouse pot experiments, uptake of benomyl, thiophanate-methyl and carbendazim from equivalent soil applications (in the range 0–003– 0–035 %^a-i- ^atarateof ²&4 ml/plant) gave equal levels of control of Botrytis lesions developing from inoculations of freshly exposed leaf scars on tomato stems. Spray applications of benomyl to exposed leaf scars controlled infection at concentrations down to 0025 % a.i. The effect of lower concentrations of the stem spray could be markedly enhanced either by the addition of a mineral oil (2 % Actipron) or by a prior soil application of benomyl at a low rate which on its own had little effect on lesion development. Protectant spray applications of glycophene and vinclozolin gave levels of control quite comparable to that of benomyl at equivalent concentrations. Evidence was obtained that the lesions formed at the artificially-inoculated leaf scars at the top of the stems of young pot-grown tomato plants were larger than those lower down. In spite of this, the level of disease control with soil applications of fungicides containing or generating carbendazim (MBC) was greater at the top than at the bottom, probably because of the normal migration of the fungicides and their accumulation at the extremities of the plant. In an observation trial in a commercial crop of tomatoes, benomyl applied either as five soil drenches at approximately monthly intervals, or as two drenches followed by five sprays at three-weekly intervals, or as five sprays alone gave marked reductions in plant loss and number of Botrytis stem lesions in both cvs Eurocross BB and Cudlow Cross. Those stem lesions which did develop, however, were generally as large as those on untreated control plants. Five sprays of dichlofiuanid gave similar levels of disease control. All the treatments gave apparently higher yields (statistically untested) in Eurocross BB, but less consistent responses were recorded in Cudlow Cross.     

Influence of fungicides and insecticides on the entomogenous fungus Metarhizium anisopliae a pathogen of the vine weevil,Otiorhynchus sulcatus

In the laboratory, the fungicides chlorothalonil and zineb prevented germination of Metarhizium anisopliae conidia when incorporated into Sabouraud dextrose agar (SDA) at the commercial concentration (based on the manufacturers’ recommended rates for horticultural crops). Twelve other fungicides and six insecticides had no effect on spore germination when applied at the same rate. Mycelial growth of M. anisopliae on SDA plates containing the recommended rate of all the pesticides (except propamocarb) was reduced compared with SDA alone. Two fungicides, benomyl and carbendazim, totally inhibited growth at 0.1 times the recommended rate. Growth was also completely prevented by the fungicides etridiazole, triforine and zineb, and the insecticides dichlorvos and hostathion, at 10 times the recommended rate. In a glasshouse experiment, a prophylactic drench of M. anisopliae conidia reduced vine weevil (Otiorhynchus sulcatus) populations on Impatiens plants by 88%. This level of control was not significantly reduced by subsequent application (7 days after egg infestation) of any of the pesticides at the recommended concentration. Larval control in pots treated with M. anisopliae plus any one of the 12 fungicides and four insecticides examined, ranged from 82% to 98%. The insecticide diazinon applied alone reduced larval numbers by 100%. Two other insecticides, dichlorvos and cypermethrin, and the fungicide pyrazaphos, also reduced weevil populations by over 50%. These experiments demonstrate the limitations of laboratory based in vitro screening programmes for assessing the chemical compatibility of M. anisopliae.     

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).     

Gel formulations for the treatment of pruning wounds.

Gels incorporating carbendazim and triadimefon were prepared from sodium alginate, a xanthan gum or an esterified alginic acid and evaluated as wound treatments on apple and plum trees. Fungicide movement and persistence were similar with sodium alginate and xanthan gels but movement was reduced and persistence increased with the esterified material. Callus tissue formation was not inhibited by any formulation. Adding acid to fungicides to form salts had little effect on fungicide movement or persistence and caused unacceptable phytotoxicity. Thiophanate-methyl or thiabendazole were not superior to carbendazim, nor imazalil to triadimefon, for protection against Nectria galligena and Chondroster-eum purpureum respectively. Sealants containing octhilinone, carbendazim plus captan, or mercuric oxide quickly established sufficient fungicide in the wood to eradicate N. galligena but possibly insufficient to protect adequately against C. purpureum. Gel formulations are valid alternatives to sealants as fresh wound pathogen treatments but fungicide persistence is insufficient for them to be recommended for treatment against mature wound pathogens.     

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.     

In vitro susceptibility to fungicides by invertebrate-pathogenic and saprobic fungi

The effect of five fungicides, benomyl (1 mg/l), dodine (50 mg/l), manzate (100 mg/l), cupric sulphate (200 mg/l) and thiabendazole (4 mg/l) was tested under in␣vitro conditions on development of 15 isolates of fungi pathogenic for insects and␣other invertebrates (Beauveria brongniartii, Culicinomyces clavisporus, Duddingtonia flagrans, Hirsutella thompsonii, two Metarhizium anisopliae, Nomuraea rileyi, two Isaria/Paecilomyces spp., and Sporothrix insectorum) and 13 isolates of contaminant fungi (five Aspergillus spp., Cladosporium cladosporioides, Cunninghamella echinulata, Fusarium roseum, Gliocladium sp., Mortierella isabellina, Mucor plumbeus, Rhizopus arrhizus and Trichothecium roseum) originating mostly from tree-hole breeding sites of mosquitoes. Most pathogenic and contaminant fungi had clear patterns of susceptibility or resistance to tested concentration of the fungicide. Development of both pathogenic and contaminant fungi on fungicide-supplemented medium varied among fungi and fungicides tested. Minimal inhibition of pathogenic fungi was found for cupric sulphate, benomyl, dodine, thiabendazole < manzate. The highest inhibition of contaminants was obtained with thiabendazole > benomyl and dodine > manzate and cupric sulphate. Thiabendazole was the most appropriate fungicide to isolate fungi pathogenic to invertebrates from substrates with high water contents and rich in organic material. The results underline the importance of adapting both a fungicide and its concentration for a selective medium for isolating specific target fungi and while selecting against possible contaminants.     

Fungicidal control of Rhizoctonia solani in relation to soil texture, organic matter and clay minerals

In pot tests, MEMC, quintozene, captafol, carboxin, thiabendazole, carbendazim, benomyl and thiophanate-methyl used as seed treatments gave much better control of cowpea seedling rot in light-textured sandy and loamy sand soils than in heavy-textured loam and silt loam soils inoculated with Rhizoctonia solani. Disease control by chloroneb was not altered by soil texture. Amendment of sandy soil with montmorillonite reduced disease control with all fungicides, except chloroneb and carboxin; similar amendments with kaolinite decreased efficacy of MEMC and captafol. Green manuring with cluster bean reduced disease control by MEMC, captafol, benomyl and thiophanate-methyl; sunnhemp reduced efficacy of MEMC. Most fungicides gave poor disease control when farm yard manure or biogas sludge was added to soil, the sludge having the more marked effect. All the fungicides tested, except carboxin, were inactivated to different extents by humic acid extracted from farm yard manure.     

Bioremediation of Fungicides by Spent Mushroom Substrate and Its Associated Microflora

Experiments were conducted both under in vitro and in situ conditions to determine the biodegradation potential of button mushroom spent substrate (SMS) and its dominating microbes (fungi and bacteria) for carbendazim and mancozeb, the commonly used agricultural fungicides. During 6 days of incubation at 30 ± 2°C under broth culture conditions, highest degradation of carbendazim (17.45%) was recorded with B-1 bacterial isolate, while highest degradation of mancozeb (18.05%) was recorded with Trichoderma sp. In fungicide pre-mixed sterilized SMS, highest degradation of carbendazim (100.00–66.50 μg g⁻¹) was recorded with mixed inoculum of Trichoderma sp. and Aspergillus sp., whereas highest degradation of mancozeb (100.00–50.50 μg g⁻¹) was with mixed inoculum of Trichoderma sp., Aspergillus sp. and B–I bacterial isolate in 15 days of incubation at 30 ± 2°C. All these microbes both individually as well as in different combinations grew well and produced extracellular lignolytic enzymes on SMS, which helped in fungicides degradation. Under in situ conditions, among three different proportions of SMS (10, 20 and 30%, w/w) mixed with fungicide pre-mixed soil (100 μg g⁻¹ of soil), the degradation of carbendazim was highest in 30% SMS treatment, while for mancozeb it was in 20% SMS treatment. The residue levels of both fungicides decreased to half of their initial concentration after 1 month of SMS mixing.