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 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%).     

The effect of benomyl on the infection of tomatoes by Fusarium oxysporum f.sp. lycopersici and Botrytis cinerea

In glasshouse experiments at Auchincruive, drench applications of benomyl (100 or 142 mg per plant) to the soil surface around the stem bases of pot-grown tomato plants before inoculation with Fusarium oxysporum f.sp. lycopersici reduced the penetration of the fungus up the stems and/or decreased the development of vascular discoloration and associated severity of wilting. Similar drenches applied after establishment of the fungus in the stems either halted or considerably retarded the growth of the pathogen up the vessels. This again was reflected in reduced vascular discoloration and wilt symptoms. In experiments with benomyl over 2 years at a commercial holding in Argyll, the application of soil drenches (at the rates above) shortly after planting out and again 5 weeks later, coupled with a programme of stem and foliar sprays (at 0–05 % a.i.) during the summer, reduced the development of stem lesions caused chiefly by Botrytis cinerea and increased the general survival of plants more than did drench or spray treatments alone. There were indications that ‘ghost spotting’ of the fruit, particularly where spray applications were made, was also slightly reduced, but the magnitude of the effect was not consistent.     

Predisposing effects of Eyespot (Pseudocercosporella herpotrichoides) on Septoria nodorum infection of winter wheat

All isolates of PeniciUium simplicissimum, P. verrucosum var. cyclopium, P. brevicompactum, P. multicolor, P. oxalicum, P. paxilli, Botrytis cinerea, and of a Gliocladium sp. obtained from necrotic virus-tested narcissus twin-scales previously dtoped in benomyl were tolerant to 1000 μg/ml of this fungicide in agar. Every necrotic twin-scale examined was infected with at least one of these species. The first two species were the most frequently isolated and a similar range of species infected different narcissus clones or different cultivars. It is argued that the use of benomyl during the twin-scaling programme should be discontinued as soon as a suitable alternative fungicide is found.     

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.     

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.     

Negative cross-resistance between benomyl and MDPC in British isolates of Botrytis cinerea, Pseudocercosporella herpotrichoides and Verticillium fungicola var. fungicola

Fifteen isolates of Pseudocercosporella herpotrichoides, four isolates of Botrytis cinerea and four isolates of Verticillium fungicola var. fungicola were examined on potato dextrose agar amended with benomyl or methyl N-(3, 5-dichlorophenyl)-carbamate (MDPC). Negatively correlated cross-resistance was clearly demonstrated with the isolates of P. herpotrichoides and B. cinerea. There were indications that the same phenomenon might also operate with the isolates of V. fungicola var. fungicola.     

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.     

Tolerance in Isolates of Colletotrichum coffeanum to Prochloraz-Mn in Kenya

Coffee berry disease, caused by Colletotrichum coffeanum is a serious disease of Coffea arabica in Kenya. Control of this disease is achieved mainly through the use of fungicides which currently include chlorothalonil and prochloraz-Mn (Octave). Fungicide resistance in C. coffeanum to benzimidazoles has been well documented. Isolates of C. coffeanum highly sensitive to prochloraz-Mn have been shown to acquire tolerance to high levels of prochloraz-Mn in culture. These isolates were able to grow and sporulate in malt extract agar (MEA) amended with 250 μg ml^-1 prochloraz-Mn. The prochloraz-Mn tolerant isolates of C. coffeanum showed low level of tolerance to benomyl. The benomyl tolerant isolates of C. coffeanum equally showed low level of tolerance to prochloraz-Mn. The prochloraz-Mn and benomyl tolerant and sensitive isolates were found to be highly pathogenic and induced sporulating lesions on seedlings and berries of coffee cv. SL 28 whichis, very susceptible to C. coffeanum. Mixed inoculation tests using prochloraz-Mn and benomyl tolerant isolates and prochloraz-Mn tolerant and sensitive isolates of C. coffeanum showed that the prochloraz-Mn tolerant isolate was competitive.     

Biocontrol of Postharvest Grey Mould on Tomato by Yeasts

The fungal pathogen Botrytis cinerea causes severe rots on tomato fruit during storage and shelf life. Biological control of postharvest diseases of fruit may be an effective alternative to chemical control. Yeasts are particularly suitable for postharvest use, proving to be highly effective in reducing the incidence of fungal pathogens. Yeast fungi isolated from the surface of solanaceous plants were evaluated for their activity in reducing the postharvest decay of tomato caused by B. cinerea. Of 300 isolates, 14 strains of Rhodotorula rubra and Candida pelliculosa were found to be strongly antagonistic to the pathogen in vitro and were selected for further storage experiment. The antagonists were evaluated for their effect on the biological control of postharvest grey mould. Artificially wounded fruits were treated by means of a novel technique: small sterile discs of filter paper imbibed separately in suspensions of each yeast and the pathogen were superposed onto each wound. After 1‐week, 11 isolates were significantly effective in reducing the diameter of lesions by more than 60% compared to the control treated with B. cinerea alone. Total protection was obtained with the strain 231 of R. rubra on fruits challenged with pathogen spores. To our knowledge, R. rubra and C. pelliculosa have not been described as biocontrol agents against grey mould caused by B. cinerea. Our data demonstrate that the application of antagonistic yeasts represents a promising and environmentally friendly alternative to fungicide treatments to control postharvest grey mould of tomato.     

Effects of treating seed potatoes from commercial and stem cutting stocks with benomyl, thiabendazole and 2-aminobutane on yield and disease

Potato seed tubers of six cultivars from commercial stocks and from stocks derived from stem cuttings (healthier seed) were fumigated with 2-aminobutane 2 wk after lifting or treated with benomyl or thiabendazole in January. 2-aminobutane prevented skin spot and gangrene developing on treated tubers. Experiments were planted at Rothamsted (clay with flints soil) and at Woburn (sandy loam soil) in 1973–75. Healthier seed produced more stems/plant than commercial stocks and yielded on average 8% more at Rothamsted in 1973 and 1974 and respectively 5 and 10% more at Woburn in 1973 and 1975. Seed treatments did not consistently affect stem numbers or increase yield although all treatments tended to decrease tuber size. Infection of stem bases and tubers by Polyscytalum pustulans and Rhizoctonia solani was usually less from healthier than from commercial seed and was decreased by benomyl and thiabendazole in 1973 and 1974. Infection by Helminthosporium solani of the skin around tuber eyes was greater from healthier than from commercial seed but was decreased by benomyl and thiabendazole. 2-aminobutane sometimes decreased infection of tubers by P. pustulans and R. solani but neither of stem bases nor of tubers by H. solani. Gangrene on tubers uniformly wounded at lifting was not consistently affected by seed source or seed treatment. Treating seed with benomyl or thiabendazole in 1975 decreased skin spot and black scurf in tubers stored until March 1976. These treatments also decreased silver scurf on the produce of commercial seed at Rothamsted but gangrene was not consistently affected by seed treatments.     

Occurrence and management of fungicide resistance in Botrytis cinerea on tomato from greenhouses in Hebei,China

Grey mould, caused by the fungal pathogen Botrytis cinerea, is one of the most devastating tomato diseases, and the control of this disease is mainly by the application of chemicals. In this study, 512 isolates of B. cinerea were collected from tomato grown in greenhouses at 10 locations in 10 cities of Hebei Province from 2011 to 2016 and tested for their sensitivities to carbendazim (Car), diethofencarb (Die), iprodione (Ipr) and pyrimethanil (Pyr). Of these tested isolates, 95.7%, 95.2%, 31.6% and 89.4% were resistant to Car, Die, Ipr and Pyr, respectively. There were nine fungicide‐resistant phenotypes in the tested isolates. Car^RPyr^RDie^RIPR^S and Car^RPyr^RDie^RIPR^R were the most common phenotypes, accounting for 59.6%, and 31.1% of the tested isolates, respectively. The field trials showed that the control efficacies (CE) of carbendazim + diethofencarb (WP, 25% + 25%), pyrimethanil (EC, 40%) and iprodione (WP, 50%) at the recommended doses were 22.75%–29.23%, 58.44%–64.19% and 61.02%–65.17%, respectively, significantly lower than those of boscalid (WG, 50%) and pyrisoxazole (EC, 25%). The resistance management trial conducted from 2015 to 2017 indicated that the CE of tomato grey mould in the experimental fields was higher than 90% and the sensitivity to carbendazim, diethofencarb and pyrimethanil of B. cinerea isolates from the experimental fields increased on a yearly basis. These results showed that the frequency of resistance to Car, Die, Ipr and Pyr was high, and these four fungicides could not effectively control tomato grey mould. Tomato grey mould could be controlled by using biopesticides and newly synthesized fungicides with different modes of action. Our findings would be useful in designing and implementing fungicide resistance management spray programmes for the control of tomato grey mould.     

Biological Control of Postharvest Diseases of Peaches and Nectarines by Yeasts

Abstract Biocontrol activities of a total of 103 yeast isolates were tested against postharvest diseases of peaches. Seven isolates, with the best efficacy in reducing the number of infected wounds or lesion sizes of Penicillium expansum and Botrytis cinerea, were selected for further large‐scale experiments. In large‐scale experiments, all selected isolates were significantly effective (P ≤ 0.05) in reducing the number of infected wounds and the diameter of lesions caused by P. expansum, B. cinerea, and Monilinia fructicola. DR52 was significantly superior to all the other yeasts in effectiveness against all three pathogens. The efficacy of the other yeast antagonists against B. cinerea and P. expansum was almost equal, while the control of M. fructicola was inferior. DR52 was selected for further storage experiment because its efficacy was higher against the three pathogens and it also showed a different random amplified polymorphic DNA‐polymerase chain reaction pattern compared with other isolates. DR52 was identified by Centraalbureau voor Schimmeelcultures (Baarn, The Netherlands) as Kloeckera apiculata. K. apiculata completely controlled both pathogens after 30 days of storage. Its efficacy declined to an 83.4% reduction in B. cinerea incidence and an 87.5% reduction in P. expansum incidence after 45 days of storage.     

Biological control of Botrytis cinerea on tomato using naturally occurring fungal antagonists

Abstract

In order to evaluate the potential of naturally occurring filamentous fungi having potential as biocontrol agents effective against grey mould and post-harvest fruit rot caused by Botrytis cinerea on tomato, fungal saprophytes were isolated. They were obtained from leaves, fruits and flowers belonging to different species of cultivated and spontaneous Solanaceous plants collected at the horticultural area of La Plata, Argentina. Of 300 isolates screened for inhibition of B. cinerea using the dual culture technique on agar plate, 12 strains inhibited strongly mycelial growth of the pathogen. Among the antagonists one isolate of Epicoccun nigrum (126), four of Trichoderma harzianum (110, 118, 248 and 252) and four isolates of Fusarium spp. decreased the spore germination of B. cinerea between 30 and 70%. These isolates were probed on tomato fruits to evaluate their biocontrol activity against post-harvest grey mould. In growth chamber tests, E. nigrum (27), F. equiseti (22, 105) and T. harzianum (118, 252) reduced the diameter of fruit lesions by 50 – 90% and were selected for further biocontrol assays of tomato plants in the greenhouse. Although there were not significant differences between the treatments and the control, F. equiseti (105), E. nigrum (27) and T. harzianum (118) reduced by 20, 22 and 22 respectively the disease on whole plants. The targeted application of isolates of E. nigrum, T. harzianum and F. equiseti provides a promising alternative to the use of fungicide spray to control B. cinerea on tomatoes.     

Screening tomato-associated bacteria for biological control of grey mold on tomato

Aiming at discovering effective biocontrol agents (BCAs) against grey mold on tomato caused by Botrytis cinerea Pers., we selected 819 bacterial isolates from the surface as well as the interior of the roots, stems, and leaves of tomato plants grown in B. cinerea-infested fields. In a dual-culture assay, 116 isolates (14.16%) showed antagonism against B. cinerea and fewer ones against five additional tomato-associated fungal pathogens – Pythium ultimum, Phytophthora capsici, Fusarium oxysporum f. sp. lycopersici, Sclerotinia sclerotiorum and Ralstonia solanacearum. Thirty-one isolates with antagonism to B. cinerea and at least one of the five additional pathogens were assessed for their efficacy in controlling grey mold on tomato in a greenhouse test. Thirteen of them attained the efficacy over 50% and were subjected to the second greenhouse test, in which 12 isolates consistently accomplished the biocontrol efficacy over 50%, with isolates ABc28 and ABc22 achieving the efficacy of 66.71% and 64.90%, respectively. Under greenhouse conditions, the above two as well as isolates ABc2, ABc11 and ABc17 increased tomato biomass by more than 20% in comparison with the control. The 12 antagonistic isolates accomplishing the biocontrol efficacy over 50% in both greenhouse tests were considered potential BCAs against grey mold, which were identified as Pseudomonas spp., Pantoea spp., Bacillus spp. and Chryseobacterium spp. Ten of them were found to produce at least one of the three hydrolytic enzymes (protease, cellulase and chitinase) and/or siderophore, which might be involved in their mechanisms of suppressing the disease. Based on the origin of these 12 strains, the leaf tissue, especially the leaf interior, of tomato plants grown in a B. cinerea-infested field appears to be a good source of potential BCAs against grey mold.     

Evaluation of formulations of Bacillus licheniformis for the biological control of tomato gray mold caused by Botrytis cinerea

Bacillus licheniformis N1, which has previously exhibited potential as a biological control agent, was investigated to develop a biofungicide to control the gray mold of tomato caused by Botrytis cinerea. Various formulations of B. licheniformis N1 were developed using fermentation cultures of the bacteria in Biji medium, and their ability to control gray mold on tomato plants was evaluated. The results of pot experiments led to the selection of the wettable powder formulation N1E, based on corn starch and olive oil, for evaluation of the disease control activity of this bacterium after both artificial infection of the pathogen and natural disease occurrence under production conditions. In plastic-house artificial infection experiments, a 100-fold diluted N1E treatment was found to be the optimum biofungicide spray formulation. This treatment resulted in the significant reduction of symptom development when N1E was applied before Bo. cinerea infection, but not after the infection. Both artificial infection experiments in a plastic house and natural infection experiments under production conditions revealed that the N1E significantly reduced disease severity on tomato plants and flowers. The disease control value of N1E on tomato plants was 90.5% under production conditions, as compared to the 77% conferred by a chemical fungicide, the mixture of carbendazim and diethofencarb (1:1). The prevention of flower infection by N1E resulted in increased numbers of tomato fruits on each plant. N1E treatment also had growth promotion activity, which showed the increased number of tomato fruits compared to fungicide treatment and non-treated control and the increased fruit size compared the non-treated control under production conditions. This study suggests that the corn starch-based formulation of B. licheniformis developed using liquid fermentation will be an effective tool in the biological control of tomato gray mold.     

Somatic hybridization experiments in solanaceous species

Summary Protoplasts were isolated from leaves of shoot cultures ofNicotiana tabacum var. Xanthi andPetunia hybrida, nuclei fromPetunia protoplasts and subprotoplasts from the liquid part of tomato pericarp. They were submitted in several combinations to agglutination by polyethyleneglycol (Kao and Michayluk, 1974) and to fusogenic treatment at pH 9 (Keller and Melchers, 1973) in calcium nitrate solution (Binding, 1974a; Schieder, 1974a). The fate of heterospecific symplasms was investigated during subsequent culture in medium NT-A (Nagata-Takebe medium, modification A: Binding, 1975) and in medium V47 (Binding, 1974b). Cell divisions occurred in tobacco +Petunia andPetunia + tomato symplasms which contained only a few tomato chromoplasts. Incompatibility is supposed to be responsible for the failure of divisions in tobacco + tomato symplasms and inPetunia + tomato symplasms containing a large tomato subprotoplast. The advantage of subprotoplasts for hybridization experiments is discussed in comparison to protoplast fusion and organell transplantation. The experiments have been carried out in the Max-Planck-Institut für Züchtungsforschung (Erwin-Bauer-Institut), Abteilung Straub, in K?ln-Vogelsang.     

MBC tolerance in aggressive and non-aggressive isolates of Ceratocystis ulmi

In selection experiments, tolerance to 0–5 ppm methyl benzimidazole-2-ylcarbamate (MBC) occurred at a frequency of c. 1 in 1–3 ×10⁸ conidia in both aggressive and non-aggressive isolates of Ceratocystis ulmi. The tolerant strains were inhibited by 5 ppm MBC, however, and attempts to select strains tolerant to 10 ppm were unsuccessful. In each of three isolates examined, tolerance remained stable after fifteen successive transfers on fungicide-free medium. Genetic control was nuclear and probably conditioned by a single gene. It is thought unlikely that the appearance of tolerant strains in nature will jeopardize the use of MBC for the control of Dutch elm disease.     

Mildew reinfection in adjacent and separated plots of sprayed barley

In latin square experiments in 1973 and 1974, reinfection of sprayed barley, by mildew (Erysiphe graminis f.sp. hordei) was decreased by widely spacing the plots. In 1974, when yields differed between plots which were either contiguous or separated, the results suggested different times to spray for best yield increase.     

Attachment microbes antagonistic against Botrytis cinerea—biological control and scanning electron microscope studies in vivo

Bacteria and yeasts, selected by an attachment assay for their ability to adhere to Botrytis cinerea hyphae or conidia, were evaluated for biocontrol potential against B. cinerea on excised tomato stems. Eight of the 12 bacteria and seven of the eight yeast isolates conferred 90% to 100% biocontrol activity when antagonist populations were applied at three to 80 times the pathogen inoculum density. Biocontrol was maintained at similar levels when biocontrol agent (BCA) application was delayed up to 48 h after pathogen challenge. Scanning electron microscopy showed extensive colonisation of B. cinerea mycelium or conidia by many of these isolates and also evidence of pathogen degradation. The biocontrol efficacy and potential for the assay are discussed with respect to cell-to-cell adhesion as a vehicle to deliver antagonistic mechanisms to highly specific pathogen sites.