Studies on the survival and infective ability of dicarboximide-resistant strains of Botrytis cinerea

Few dicarboximide-resistant strains of Botrytis cinerea produce macroconidia either on culture media or on natural substrates, although, the viability of such conidia was similar to those of sensitive strains. The ability of dicarboximide-resistant strains to infect strawberries was similar to that of sensitive strains and most of the resistant strains competed successfully with sensitive strains when inoculated in equal numbers onto detached strawberries and onto flowers of field-grown plants, especially after treatment with dicarboximide fungicides. All resistant strains tested survived for at least 9 months on inoculated strawberry leaf litter although their incidence was ‘diluted’ by wild type sensitive strains. Sporulation and dispersal of resistant strains from the litter was very limited resulting in a low incidence of fruit infection even after treatment with dicarboximides. Consequently, there was no significant increase of resistant strains in the plantation and control of infection was maintained with iprodione and dichlofluanid. Poor sporulation of dicarboximide-resistant strains of B. cinerea is considered to be the single most important factor in limiting their development in the field.     

Isolation of Microbial Antagonists for Biocontrol of Grey Mould Disease of Strawberries

A screening programme is described for the assessment of the potential of biocontrol agents to control grey mould of strawberries caused by Botrytis cinerea. Bacteria were isolated from strawberry fruits, leaves and flowers from a commercial field site and screened for antagonism towards B. cinerea using two in vitro and one in vivo screening techniques. From 559 microorganisms isolated, 108 inhibited pathogen growth on agar plates and 27 of these prevented spore germination on Cellophane membranes. The ability of these 27 isolates to inhibit infection of young strawberry leaves by B. cinerea on whole plants under glass was then tested. Seven isolates reduced grey mould development and were subsequently assessed in a field trial. Two isolates, one of Bacillus pumilus and one of Pseudomonas fluorescens, were as effective or more effective than standard dichlofluanid sprays and may therefore be of potential value as antagonists of B. cinerea.     

Control of tomato grey mould on unheated crops using non- systemic fungicide sprays

High volume sprays of dichlofluanid (0.1 % a.i.) reduced total fruit numbers but gave better control of Botrytis cinerea infections of tomato stems, leaves and fruit than sprays at 0.05 or 0.025% a.i. Surface residues from the sprays at 0–1, 0.05 or 0.025% a.i. were respectively 5.4, 1.6, 0.7 μg/g fresh weight of ripe fruit at harvest. Tank-mixed zineb (0.12% a.i.) and captan (0.2% a.i.) were less effective and increasing the spray intervals from 1 wk to 2 and 3 wk reduced the fungitoxicity of captan and zineb more than that of dichlofluanid. Harvest residues on ripe fruit were 7.1, 2.8 or 2.4 μg/g when dichlofluanid (0.1 % a.i.) sprays were applied at 1, 2 and 3 wk intervals respectively. Good control of B. cinerea was achieved if the whole plant was sprayed initially with dichlofluanid as soon as the second truss was flowering and subsequent sprays were restricted to the upper section of stem including the four or five youngest trusses of buds, flowers and fruit. When used as a post-infection spray there was a period of c. 8 wk before dichlofluanid markedly reduced the incidence of grey mould. Tank-mixed zineb (0.12% a.i.) controlled Botrytis fruit spotting but not leaf and stem infections. Botrytis stem lesions extended more rapidly on zineb-sprayed plants than on unsprayed plants or on plants treated with captan or dichlofluanid.     

Control of Botrytis cinerea on glasshouse tomato by high volume sprays

The new fungicides iprodione, vinclozolin and prochloraz, and also a mixture containing carbendazim and maneb, were compared with the established protectant fungicides dichlofluanid and chlorothalonil for effectiveness against grey mould ( Botrytis cinerea ) in an unheated tomato crop. Iprodione and vinclozolin gave the best control of lesions on leaves and stems but dichlofluanid was the most effective in controlling ghost-spotting of fruit. The carbendazim/maneb mixture was effective against a carbendazim-sensitive isolate but not against an insensitive isolate of B. cinerea . When applied 1 or 2 days after inoculation, all five protectant fungicides controlled the insensitive isolate better than the sensitive isolate.     

Control of Botrytis cinerea and Botrytis squamosa in overwintered salad onions by fungicide sprays

In 1974/75, 13 sprays of 0·2% a.i. thiram applied at 14 day intervals to overwintered salad onions reduced the incidence of Botrytis cinerea and significantly increased onion yields. In 1977/78 both B. cinerea and B. squamosa occurred, and 12 iprodione sprays at 0·1% a.i. applied at 14-day intervals or 6 sprays at 0·2% a.i. applied at 28-day intervals gave good control of B. cinerea and B. squamosa and significantly increased onion yields. Benomyl (0·1% a.i. at 14-day intervals, or 0·2% a.i. at 28-day intervals) failed to control either pathogen because of the development of carbendazim-insensitive strains of the fungi. Effective control of both pathogens and increased yields were obtained with an application of 0·4% a.i. thiram in October and November followed by an application of 0·2% a.i. iprodione in December and January.     

Comparative Resistance Patterns of Fludioxonil and Vinclozolin in Botryotinia fuckeliana

Fludioxonil is a new non-systemic phenylpyrrole fungicide. It is a derivative of the antibiotic pyrrolnitrin and is highly active against Ascomycetes. Basidiomycetes and Deuteromycetes. In the present study we determined the baseline sensitivity of B. fuckeliana for fludioxonil in comparison to the dicarboximide vinclozolin. Subsequently these baselines were compared with the sensitivity of dicarboximide-resistant field isolates and with resistant laboratory strains selected on media amendedwith vinclozotin or fludioxonil, respectively. Additionally, sensitivities of genetically homogeneous ascospore progenies of sexual crosses of sensitive isolates with dicarboximide-resistant field isolates or resistant laboratory strains were compared to, the baselines. Baseline ECst, values for the inhibition of germination ranged from 0.07 to 0.20 μg ml^-1 of vinclozolin and for mycelial growth from 0.003 to 0.016 μg ml’ of fludioxonil and from 1.7 to 2.0 μg ml^-1 of vinclozolin and for mycelial growth from 0.003 to 0.016 μg ml^-1 of fludioxonil and from 0.13 to 0.27 μg ml^-1 of vinclozolin. Sensitivity to fludioxonil of all dicarboximide-resistant field isolates did not differ from their baseline. However, resistant laboratory strains that were selected either on media amended with fludioxonil or vinclozolin showed decreased sensitivities to both active ingredients. All sexual progenies of the crosses described above were of the parental phenotypes. We found no independent segregation of the resistance markers.     

The effect of fungicides, irrigation and plant density on the development of Peronospora sparsa, the cause of downy mildew in rose and blackberry

Downy mildew of rose (Rosa spp.) and blackberry (Rubus fructicosus), caused by Peronospora sparsa, can cause significant losses in production. In a series of experiments, fungicides with different modes of action to the commonly used phenylamide‐based products were examined for efficacy on both crops. Cymoxanil + mancozeb + oxadixyl and fluazinam gave good downy mildew control on both rose and blackberry. On outdoor, container‐grown rose, high volume sprays of fosetyl‐aluminium were also effective, but on young micropropagated blackberry plants, application as a drench treatment was better than as a spray. Good control was also achieved on blackberry with chlorothalonil and with metalaxyl in formulation with either thiram or mancozeb. There was no evidence of loss of control with phenylamide fungicides due to resistance. Irrigation regimes were also examined on blackberry and sub‐irrigation of plants in propagation on a sand bed led to significantly lower disease levels than those found where plants had been irrigated by overhead sprinklers. Reducing the density of container‐grown rose plants had a measurable effect on the progress of downy mildew, though this was small and temporary when compared with the effect of fungicide treatment. Fungicide programmes which gave season‐long control of rose downy mildew resulted in significantly improved extension growth.     

A Cucumber Disease Caused by Alternaria alternata and its Control

A severe leaf spot disease of cucumber caused by a pathotype of Alternaria alternata (Fr.) Keissler was recorded recently in plastic houses in Crete. Lesions ranged in size of a pin point to over 5 cm in diameter, with necrotic tissue on most of their area and a surrounding yellow zone. The pathogen grew satisfactorily on PDA at temperatures between 5 °C–40 °C and spore germination occurred in the range less than 10 °C to over 37 °C. Optimum temperature in both cases was near 26 °C. Of,13 fungicides tested in vitro, sodium omadine, etem, dichlofluanid, captan and folpet were the most inhibitory on spore germination, and iprodione, sodium omadine and dichlofluanid on mycelial growth. Of 25 fungicides applied on two leaf cucumber plants 24 h before inoculation, maneb, etem, dichlofluanid and chlorothalonil were the most effective. When the last fungicides, plus mancozeb, were applied 24 h after inoculation only maneb was effective. In greenhouse experiments, iprodione, prochloraz-manganese-complex, chlorothalonil, dichlofluanid, guazatine, maneb and etem were the most effective for disease control, while mancozeb was less effective. The local cucumber cv. Knossos and the Dutch F₁ hybrids Evadan, Frella, Herta, Malfa, Mazourka, Pepinex 69 and Renova were all susceptible to infection.     

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.     

Laboratory studies on the effects of fungicides, acaricides and insecticides on the entomopathogenic fungus, Verticillium lecanii

Tests on agar suggested that several fungicides, insecticides and acaricides could be recommended for use with the fungus, Verticillium lecanii (Zimm) Viégas; some chemicals, benodanil, oxycarboxin, iprodione, vinclozolin, dinocap, carbaryl, dicofol, dienochlor, pirimicarb, permethrin and white oil are in theory sufficiently innocuous to V. lecanii for spraying simultaneously with is spores while separate aphlications would be recommended for diflubenzuron which is slightly more toxic. Results of tests on aphids with some of the compounds of greater but intermediate toxicity on agar, suggested that separate applications are advisable with bupirimate, pyrazaphos, triforine, cyhexatin, dioxathion and tetradifon but any use of zineb is best avoided. The most toxic compounds on agar in this study were thiram, captan, maneb, mancozeb, chlorothalonil, dichlofluanid, fenarimol, imazalil, BTS 40542, quinomethionate, fluotrimazole and diazinon. Of these compounds tested with V. lecanii on aphids, captan, chlorothalonil, dichlofluanid, quinomethionate and thiram proved to be incompatible with V. lecanii but fenarimol proved relatively harmless. On the basis of the results of this study, careful selection of pesticides and fungicides would permit the combined use of V. lecanii and chemicals in integrated control programmes.

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Résumé Des tests sur agar ont suggéré que plusieurs fongicides, insecticides et acaracides pouvaient être recommandés pour une utilisation simultanée avec le champignon Verticillium lecanii (Zimm) Viégas; quelques substances chimiques — benodanil, oxycarboxine, iprodione, vinelozoline, dinocap, carbaryl, dicofol, dienochlor, pirimicarb, permethrine et huile blanche — sont en théorie suffisamment innoffensives contre V. lecanii pour être pulvérisées simultanément avec ses spores, tandis que des applications séparées devraient être préconisées pour le diflubenzuron qui est légèrement plus toxique. Les résultats d'essais sur agar, contre des aphides, avec quelques produits de toxicité plus élevée mais cependant moyenne, ont conduit à suggérer des applications séparées pour: bupirimate, pyrazaphos, triforine, cyhexatine, dioxathion et tetradifon, mais d'éviter toute utilisation de zineb. Les produits les plus toxiques sur agar ont été: thiram, captan, maneb, mancozeb, chlorothalonil, dichlofluanide, fenarimol, imazalil, BTS 40542, quinomethionate, fluotrimazole et diazinon. Après essais contre aphides, avec V. lecanii, captan, chlorothalonil, dichlofluanide, quinomethionate et thiram ont montré leur incompatibilité avec V. lecanii, tandis que fenarimol se montrait relativement sans nocivité. Sur la base des résultats de cette étude, une sélection soigneuse des pesticides et des fongicides devrait permettre l'utilisation combinée de V. lecanii et de produits chimiques dans des programmes de lutte intégrée.

     

Fungicide programmes for the control of spur blight, powdery mildew and grey mould on raspberry

Five sprays of dichlofluanid (0–1% a. i.) reduced spur blight (Didymella applanata) on canes and powdery mildew (Sphaerotheca macularis) on fruit more than five sprays of captan (0–1% a. i.). Omission of the first three applications resulted in little increase in disease but when the fourth (full bloom) application also was omitted the incidence of disease was comparable with that on untreated plants. The final (fruitlet stage) application did not further reduce disease incidence. Five sprays of dichlofluanid reduced grey mould (Botrytis cinerea) on stored fruit more than five sprays of captan. There was an increase in grey mould with progressive reductions in the number of sprays. Incidence on canes was slight and was unaffected by treatments. A programme of five dichlofluanid sprays applied at 14-day intervals until c. 2 wk before harvest and supplemented by a single early full bloom spray of either dinocap (0–025% a. i.), binapacryl (0–05% a. i.), triforine (0–025% a. i.), benomyl (0–025% a. i.) or replaced by benomyl (0–025% a. i.) all halved both the number of fruits infected by powdery mildew and the number of spur blight lesions on canes, compared with unsprayed controls. Six applications of any of the following fungicides at 7-day intervals from 23 May until 29 June 1977 reduced powdery mildew incidence on fruit, the last fungicide being particularly effective: dinocap (0–0125%), dichlofluanid (0–075%), binapacryl (0–025%), triforine (0–0125%), bupirimate (0–0075%), ditalimfos (0–01875%) and fenarimol (0–0036%). Only triforine and bupirimate reduced the incidence of mildew on shoots. Dichlofluanid, binapacryl, triforine and fenarimol all reduced the amount of spur blight on canes. The largest fruit occurred on plants sprayed with bupirimate, ditalimfos and fenarimol, and late ripening was associated with triforine treatments.     

Effects of Selected Fungicides and the Timing of Fungicide Application on Beauveria bassiana-Induced Mortality of the Colorado Potato Beetle (Coleoptera: Chrysomelidae)

Four fungicides used for controlling foliar diseases of potato (Solanum tuberosum) were evaluated under field and laboratory conditions for their effects on the infectivity and sporulation of Beauveria bassiana when used as a control for the Colorado potato beetle, Leptinotarsa decemlineata (CPB). We investigated the direct effects of fungicides on B. bassiana-induced CPB mortality and the effect of time between fungicide and B. bassiana application. Effects of fungicide on conidial survival in the soil and on foliage were examined in the field. Significantly more larval mortality was observed when larvae were sprayed with B. bassiana than with the water control. Fungicide had no significant effect on larval mortality in the field. In the laboratory, survival of larvae was significantly lower among larvae fed fungicide-treated foliage. B. bassiana-induced mortality in the laboratory was observed only when larvae were fed foliage treated with copper hydroxide or water. Larvae fed mancozeb- or chlorothalonil-treated foliage experienced high mortality regardless of B. bassiana treatment. While there was no significant effect of fungicide on B. bassiana sporulation on cadavers in the field, a pattern emerged that indicated higher proportions of cadavers producing conidia in plots sprayed with water or copper hydroxide than in plots sprayed with chlorothalonil or mancozeb. Survival of B. bassiana conidia in the soil and on foliage was significantly greater in plots treated with copper hydroxide or water than in plots treated with mancozeb or chlorothalonil. Fungicides such as copper hydroxide may be less deleterious to the fungus than mancozeb and chlorothalonil.     

Effect of fungicides on the mycelial growth of soft fruit spoilage fungi

The in vitro activity of a range of fungicides was tested on strains of Botrytis cinerea, Mucor mucedo, Rhizopus stolonifer, and R. sexualis. Di-chlofluanid and dichloran were more active than the other fungicides against all of the strains of B. cinerea, while dichloran, dichlorophen and the dithiocarbamate fungicides were the most active against the Rhizopus species. Only dichlorophen and thiram markedly inhibited the growth of M. mucedo.     

A comparison of fungicides for the control of raspberry spur blight

Effective control of raspberry spur blight (Didymella applanata) and increased crop yields in the following season were obtained in a plantation of cv. Mailing Jewel with a programme of four or five sprays commencing when the new canes first appeared. Treatments were applied at 14-day intervals and continued until c. 2 wk before harvest. Thiram (0.16% a.i), benomyl (0.025% a.i.), dichlofluanid (0.10% a.i.) and captan (0.10% a.i.) gave good control of the disease but as benomyl and dichlofluanid were also effective in controlling powdery mildew (Sphaero-theca macularis) and Botrytis cinerea on the berries these fungicides appear to be the most suitable.     

Benzoic acid induces tolerance to biotic stress caused by Phytophthora cinnamomi in Banksia attenuata

Banksia attenuata plants were treated with soil drenches or foliar sprays of benzoic acid (BZA) to determine induced resistance to Phytophthora cinnamomi. Stems of B. attenuata were inoculated with the pathogen 1 week after treatment with BZA. Resistance was estimated by measuring P. cinnamomi lesions on stems. Treatment with 0.10 mM, 0.25 mM or 0.50 mM BZA caused a reduction in lesion size with 0.50 mM BZA applied as a soil drench being the most effective treatment at suppressing the development of lesions. This is the first report of BZA induced host resistance in any plant species to any pathogen.     

Glasshouse experiments on apple scab

Dilutions of various fungicides were compared on potted MM 109 clonal rootstocks in the glasshouse for protective (pre-infection) and curative (post-infection) activity against apple scab. The materials, as commercial preparations, included captan, copper (as ‘dry’ Bordeaux mixture), dichlofluanid, didecyldimethylammonium bromide (DDAB), dispersible sulphur, dithianon, dodine acetate, lime-sulphur (LS), phenylmercury chloride (PMC), and thiram; they were assessed on the basis of percentage active ingredient in the dilutions. When acting at normal rates of use, many of the fungicides were much more effective protectively than curatively, but this contrast was less applicable to DDAB, dodine acetate, LS, and dichlofluanid. PMC, however, was an especially notable exception in proving curatively superior at unusually low rates. In addition, it was particularly active in vapour form, and was effectively translocated not only from one leaf surface to the other, but, especially at 0.005%, from sprayed to younger unsprayed leaves on the same shoots. Other standard fungicides showed little or no translocated activity, but isobutyl-o-coumarate was effective at the growing shoot-tips in spring from a spray applied to the lower leaves. PMC reduced infection when applied once at any time during the scab-incubation period, and especially 24 hr. after infection, when the control was almost complete. Longer intervals before spraying reduced the curative action, but not in proportion to the intervals; the infection pattern showed multi-peak fungicidal activity and, after the later sprays, many lesions were practically infertile. The curative properties of PMC were matched by several other formulated phenylmercury salts compared at equivalent percentage mercury content, but the dimethyldithiocarbamate proved rather less effective. A preliminary attempt to supplement the curative properties of PMC with the protective properties of LS, by mixing these fungicides at low rates, showed evidence of fungicidal antagonism, and this needs further investigation. Several fungicides, notably the inorganic sulphurs, thiram, the phenylmercury compounds, dichlofluanid, and DDAB showed incidental activity against natural infection by powdery mildew.     

Pathogenicity of fungi associated with wheat and barley seedling emergence and fungicide efficacy of seed treatment

Presented study focused on the influence of Cochliobolus sativus isolates origin on pathogenicity towards wheat and barley seedlings in comparison with pathogenicity of certain Fusarium species and Microdochium nivale. The efficacy of fungicide seed treatment against C. sativus was estimated. The C. sativus isolates were collected from different locations and were isolated from wheat, barley and sunflower seeds. The pathogenicity of C. sativus, Fusarium species and M. nivale towards germinating seedlings were expressed as germination (GA) retardation and coleoptile growth rate retardation (CGR). Of wheat only, the CGR was significantly influenced by the isolate origin. The C. sativus isolates obtained from sunflower seeds were the most aggressive. Of the barley seeds, the barley isolates were the most aggressive. Barley was significantly more susceptible to damage by C. sativus isolates than wheat. The pathogenicity of tested fungal species declined in the order: F. culmorum, F. graminearum, C. sativus, F. avenaceum, M. nivale, F. poae for both barley and wheat. The results highlighted high pathogenicity potential of C. sativus equal to that of F. avenaceum and M. nivale. The symptoms of C. sativus on coleoptile and roots were very similar or the same as the symptoms caused by Fusarium species and M. nivale, except of white, pink or red colours. Of wheat sprouts, the fungicide efficacy (FE) against C. sativus declined in the order: tebuconazole + thiram, carboxin + thiram, quazatine, difenoconazole, iprodione + triticonazole (in term of GA) and carboxin + thiram, iprodione + triticonazole, tebuconazole + thiram, difenoconazole, quazatine (in term of CGR). In barley, the FE declined in the order: carboxin + thiram, iprodione + triticonazole, tebuconazole + thiram, difenoconazole, quazatine (in term of GA) and carboxin + thiram, tebuconazole + thiram, difenoconazole, iprodione + triticonazole, quazatine (in term of CGR).     

The ABC transporter BcatrB from Botrytis cinerea exports camalexin and is a virulence factor on Arabidopsis thaliana

Arabidopsis thaliana is known to produce the phytoalexin camalexin in response to abiotic and biotic stress. Here we studied the mechanisms of tolerance to camalexin in the fungus Botrytis cinerea , a necrotrophic pathogen of A. thaliana . Exposure of B. cinerea to camalexin induces expression of BcatrB , an ABC transporter that functions in the efflux of fungitoxic compounds. B. cinerea inoculated on wild-type A. thaliana plants yields smaller lesions than on camalexin-deficient A. thaliana mutants. A B. cinerea strain lacking functional BcatrB is more sensitive to camalexin in vitro and less virulent on wild-type plants, but is still fully virulent on camalexin-deficient mutants. Pre-treatment of A. thaliana with UV-C leads to increased camalexin accumulation and substantial resistance to B. cinerea. UV-C-induced resistance was not seen in the camalexin-deficient mutants cyp79B2/B3 , cyp71A13 , pad3 or pad2 , and was strongly reduced in ups1 . Here we demonstrate that an ABC transporter is a virulence factor that increases tolerance of the pathogen towards a phytoalexin, and the complete restoration of virulence on host plants lacking this phytoalexin.     

Activity of Fluazinam against Strains of Botrytis cinerea Resistant to Benzimidazoles and/or Dicarboximides and to a Benzimidazole-phenylcarbamate Mixture

Fluazinam is a new active ingredient for the control of grey mould, belonging to the novel broad spectum phenylpyridinamine fungicides. The effect of fluazinam was studied on one wild type and four strains of Botrytis cinerea , which had been isolated from vegetable crops in Greece, and were resistant to benzimidazoles and/or dicarboximides and to the mixture of benzimidazoles (carbendazim) + phenylcarbamates (diethofencarb). In vitro fluazinam was found to be highly active against strains of B. cinerea which were sensitive or resistant to benzimidazoles or exhibited multiple resistance to benzimidazoles, dicarboximides and to the mixture carbendazim + diethofencarb [EC₅₀ and EC₉₅ values (concentration of active ingredient that suppresses mycelial growth to 50 and 95%, respectively, of that of the fungus on fungicide-free agar medium) calculated with probit analysis, ranged from 0.044 to 0.069 and 0.58 to 1.6 μg/ml, respectively]. No cross-resistance was observed between fluazinam and the market products benomyl, iprodione or carbendazim + diethofencarb. Preventive applications of fluazinam in vivo completely inhibited infections of cucumber seedlings by all the above-mentioned resistant strains of B. cinerea . Benomyl and iprodione did not effectively control the benzimidazole- and dicarboximide-resistant strains. The mixture of carbendazim + diethofencarb insufficiently controlled the strain of B. cinerea with moderate resistance to benzimidazoles. The results of this investigation indicate that it should be possible to use fluazinam as an alternative in resistance management programmes against grey mould.