Characterization of fludioxonil-resistant and pyrimethanil-resistant phenotypes of Penicillium expansum from apple

Penicillium expansum is the primary cause of blue mold, a major postharvest disease of apple. Fludioxonil and pyrimethanil are two newly registered postharvest fungicides for pome fruit in the United States. To evaluate the potential risk of resistance development in P. expansum to the new postharvest fungicides, one isolate of each of thiabendazole-resistant (TBZ-R) and -sensitive (TBZ-S) P. expansum was exposed to UV radiation to generate fungicide-resistant mutants. Four fludioxonil highly-resistant mutants (EC(50) > 1,000 microg/ml) and four pyrimethanil-resistant mutants (EC(50) > 10 microg/ml) were tested for sensitivities to thiabendazole, fludioxonil, and pyrimethanil, and fitness parameters including mycelial growth, sporulation on potato dextrose agar (PDA), sensitivity to osmotic stress, and pathogenicity and sporulation on apple fruit. The stability of resistance of the mutants was tested on PDA and apple fruit. Efficacy of the three fungicides to control blue mold incited by the mutants was evaluated on apple fruit. Six fungicide-resistant phenotypes were identified among the parental wild-type isolates and their mutants based upon their resistance levels. All four fludioxonil highly-resistant mutants were sensitive to pyrimethanil and retained the same phenotypes of resistance to TBZ as the parental isolates. All four pyrimethanil-resistant mutants had a low level of resistance to fludioxonil with a resistance factor >15. The two pyrimethanil-resistant mutants derived from a TBZ-S isolate became resistant to TBZ at 5 microg/ml. After 20 successive generations on PDA and four generations on apple fruit, the mutants retained the same phenotypes as the original generations. All mutants were pathogenic on apple fruit at both 0 and 20 degrees C, but fludioxonil highly-resistant mutants were less virulent and produced fewer conidia on apple fruit than pyrimethanil-resistant mutants and their parental wild-type isolates. Compared with the parental isolates, all four fludioxonil highly-resistant mutants had an increased sensitivity to osmotic stress on PDA amended with NaCl, while the pyrimethanil-resistant mutants did not. Pyrimethanil was effective against blue mold caused by fludioxonil-resistant mutants at both 0 and 20 degrees C. Pyrimethanil and fludioxonil reduced blue mold incited by pyrimethanil-resistant mutants during 12-week storage at 0 degrees C but were not effective at 20 degrees C. TBZ was not effective against pyrimethanil-resistant mutants derived from TBZ-S wild-type isolates at room temperature but provided some control at 0 degrees C. The results indicate that: (i) a fitness cost was associated with fludioxonil highly resistant mutants of P. expansum in both saprophytic and pathogenic phases of the pathogen but not pyrimethanil-resistant mutants; (ii) pyrimethanil possessed a higher risk than fludioxonil in the development of resistance in P. expansum; and (iii) triple resistance to the three apple-postharvest fungicides could emerge and become a practical problem if resistance to pyrimethanil develops in P. expansum populations.     

Effect of the antagonist Candida sake on apple surface microflora during cold and ambient (shelf life) storage

This study examined the impact of the application of a biocontrol yeast, Candida sake CPA-1 (3 × 10⁶ colony forming units (cfu) ml^-1) on the resident microbial populations just prior to harvest, during 7 months cold storage and subsequent ambient shelf-life in two seasons on apples untreated or treated with a preharvest pesticide programme. The changes in populations of the antagonistic yeast ( C. sake) were also monitored. Generally, application of the antagonist had little effect on the total bacterial populations which remained very low both prior to harvest and subsequently during cold storage. White yeasts were predominant on the apples during the experimental period, with lower populations of pink yeasts. When apples were removed after 7 months to ambient conditions the yeast populations increased quickly but those apples treated with C. sake had significantly less white yeasts than untreated controls. The dominant filamentous fungi isolated were Cladosporium, Alternaria and Penicillium spp. Penicillium spp. which is responsible for major postharvest diseases was seldom isolated at preharvest but it became important during later cold storage and shelf life period. Populations of Cladosporium and Penicillium were significantly reduced by the C. sake treatment when removed from cold storage during the ambient shelf-life. In contrast, the Alternaria spp. were unaffected by the antagonist. The actual populations of C. sake applied decreased significantly immediately after application (24 h). However, they subsequently increased to a maximum after one month cold storage (10³ cfu g^-1), and populations increased again under ambient shelf-life conditions. The C. sake populations, and the resident microbial populations, were unaffected by preharvest fungicide applications. This study demonstrates that preharvest application of an antagonistic yeast such as C. sake has an impact on the resident mycoflora both during 7 months cold storage and during ambient shelf-life storage.     

Control of Blue Mold of Apples by Preharvest Application of Candida sake Grown in Media with Different Water Activity

ABSTRACT Unmodified and low water activity (a(w))-tolerant cells of Candida sake CPA-1 applied before harvest were compared for ability to control blue mold of apples ('Golden Delicious') caused by Penicillium expansum under commercial storage conditions. The population dynamics of strain CPA-1 on apples were studied in the orchard and during storage following application of 3 x 10(6) CFU/ml of each treatment 2 days prior to harvest. In the field, the population size of the unmodified treatment remained relatively unchanged, while the population size of the low-a(w)-modified CPA-1 cells increased. During cold storage, the populations in both treatments increased from 10(3) to 10(5) CFU/g of apple after 30 days, and then declined to about 2.5 x 10(4) CFU/g of apple. In laboratory studies, the low-a(w)-tolerant cells provided significantly better disease control as compared with the unmodified cells and reduced the number of infected wounds and lesion size by 75 and 90%, respectively, as compared with the non-treated controls. After 4 months in cold storage, both unmodified and low-a(w)-tolerant cells of C. sake were equally effective against P. expansum on apple (>50% reduction in size of infected wounds).     

Improved Control of Postharvest Decay of Pears by the Combination of Candida sake (CPA-1) and Ammonium Molybdate

ABSTRACT The potential enhancement of Candida sake (CPA-1) by ammonium molybdate to control blue and gray mold caused by Penicillium expansum and Botrytis cinerea, respectively, on Blanquilla pears was investigated. In laboratory trials, improved control of blue and gray molds was obtained with the application of ammonium molybdate (1, 5, 10, and 15 mM) alone or in combination with C. sake at 2 x 10(6) or 2 x 10(7) CFU ml(-1) on Blanquilla pears stored at 20 degrees C. In semicommercial trials at 1 degrees C for 5 months, the efficacy of C. sake at 2 x 10(6) CFU ml(-1) on reducing P. expansum and B. cinerea decay was enhanced more than 88% with the addition of ammonium molybdate 5 mM in the 1999-2000 season. In two seasons, the performance C. sake at 2 x 10(6) CFU ml(-1) plus ammonium molybdate was similar to or greater than that of C. sake at 2 x 10(7) CFU ml(-1). Similar control of blue mold was obtained on pears stored under low oxygen conditions. The preharvest application of ammonium molybdate did not reduce postharvest blue mold decay. The population of C. sake on pear wounds significantly decreased in the presence of ammonium molybdate 1 and 5 mM at 20 and 1 degrees C.     

Biocontrol Potential of Metchnikowia pulcherrima Strains Against Blue Mold of Apple

ABSTRACT Eight strains of Metschnikowia pulcherrima isolated over a 4-year period from an unmanaged orchard and selected for their biocontrol activity against blue mold (caused by Penicillium expansum) of apples were characterized phenotypically, genetically, and for their biocontrol potential against blue mold on apples. All strains grew well and only differed slightly in their growth in nutrient yeast dextrose broth medium at 1 degrees C after 216 h, but large differences occurred at 0 degrees C, with strain T5-A2 outgrowing other strains by more than 25% transmittance after 360 h. This strain was also one of the most resistant to diphenylamine (DPA), a postharvest antioxidant treatment. All strains required biotin for growth in minimum salt (MS) medium, although strain ST2-A10 grew slightly in MS medium containing riboflavin or folic acid, as did ST3-E1 in MS medium without vitamins. None of the strains produced killer toxins against an indicator strain of Saccharomyces cerevisiae. Analysis of Biolog data from YT plates for all eight strains using the MLCLUST program resulted in separation of the strains into one major cluster containing four strains and four scattered strains from which strain ST1-D10 was the most distant from all other strains. This was particularly apparent in 3-D and principle component analysis. Genetic differentiation of the eight strains using maximum parsimony analysis of nucleotide sequences from domain D1/D2 of nuclear large subunit (26S) ribosomal DNA resulted in detection of two clades. Strain ST1-D10 grouped with the type strain of M. pulcherrima but the remaining seven strains grouped separately, which might possibly represent a new species. All strains significantly reduced blue mold on mature Golden Delicious apples during 1 month of storage at 1 degrees C followed by 7 days at room temperature, but strains T5-A2 and T4-A2 were distinctly more effective under these conditions. Strain T5-A2 also was the most effective in tests on harvest mature apples treated with the lowest concentration of the antagonist and stored for 3 months at 0.5 degrees C. Populations of all eight strains increased in apple wounds by approximately 2 log units after 1 month at 1 degrees C followed by 5 days at 24 degrees C. Our results indicate that M. pulcherrima is an excellent candidate for biological control of postharvest diseases of pome fruit. The variation in phenotypic, genetic, and biocontrol characteristics among strains of M. pulcherrima isolated from the same orchard should make it possible to select antagonists with characteristics that are most desirable for postharvest application.     

Inhibition of egg development by phosphine in the cosmopolitan pest of stored products Liposcelis bostrychophila (Psocoptera: Liposcelididae)

Phosphine-induced delay in development of eggs was investigated as a mechanism of resistance to this fumigant in Liposcelis bostrychophila Badonnel. One-day-old eggs of a susceptible and a strongly resistant strain of L bostrychophila were exposed to a range of phosphine concentrations for 6days at 30 (+/- 1) degrees C and 70 (+/- 2)% RH. Delay in mean hatching period occurred in both susceptible and resistant eggs, although it was more pronounced in the latter. A maximum delay of 2.65 days was recorded for eggs of the susceptible strain at 0.01 mg litre(-1) (the highest concentration at which eggs survived) and 13.39 days for the resistant strain at 1 mg litre(-1) (the highest concentration tested). Delay in egg development time was positively correlated with increasing phosphine concentration. Our results reveal that the most successful strategy to control resistant L bostrychophila is to apply relatively low concentrations of phosphine for extended exposure times (eg 0.05 mg litre(-1) for 16 days) that allow all eggs to hatch to the much less tolerant nymph stage.     

Control of post‐harvest decay of apples by pre‐harvest and post‐harvest application of ammonium molybdate

Ammonium molybdate was tested as a potential fungicide for use in apples (cv Golden Delicious) against blue and grey mould, important post‐harvest diseases of pome fruits. In tests in vivo at 20 °C, ammonium molybdate (15 mM ) reduced lesion diameters of Penicillium expansum, Botrytis cinerea and Rhizopus stolonifer by 84%, 88% and 100% respectively. When apples treated with ammonium molybdate were stored at 1 °C for three months, a significant reduction in severity and incidence of P expansum and B cinerea was observed in both years of study (1998 and 1999). In the second year of the experiment the reduction in disease severity was greater than 88% for both pathogens, and the level of control was similar to, or greater than, that observed with the fungicide imazalil. When ammonium molybdate was applied as a pre‐harvest treatment, a significant reduction in blue mould decay was observed after three months in cold storage. In vitro, ammonium molybdate greatly inhibited spore germination of P expansum and B cinerea, although better inhibition was obtained against grey mould. Ammonium dimolybdate, sodium molybdate and potassium molybdate were also tested in vitro in comparison with ammonium molybdate as inhibitors of spore germination, but only ammonium molybdate inhibited spore germination by more than 50%. © 2001 Society of Chemical Industry     

Influence of phosphine on hatching of Cryptolestes ferrugineus (Coleoptera: Cucujidae), Lasioderma serricorne (Coleoptera: Anobiidae) and Oryzaephilus surinamensis (Coleoptera: Silvanidae)

The hatching and mortality response of 0- to 48-h-old eggs of field strains of the stored-product insects Cryptolestes ferrugineus (Stephens), Lasioderma serricorne (F) and Oryzaephilus surinamensis (L) following phosphine fumigation for 24, 48 or 120 h at 27 (+/- 2) degrees C was investigated. Hatching was delayed and reduced in the first few days in a phosphine-resistant strain of C ferrugineus that was treated with 2.0-7.0 mg litre(-1) doses for 48 h (5-80% mortality) and with 1.0-2.0 mg litre(-1) for 120 h (44-84% mortality). In both the exposures there were significant increases in hatching on later days when compared with the corresponding controls. Developmental delay was, however, not evident in susceptible strains of C ferrugineus, L serricorne and O surinamensis that were exposed to phosphine for 24 h.     

The use of sodium carbonate to improve curing treatments against green and blue moulds on citrus fruits

The effectiveness of curing oranges and lemons at 33 degrees C for 65h followed by storage under ambient and cold-storage conditions was investigated. This treatment effectively reduced the incidence of Penicillium digitatum (Pers) Sacc and P italicum Wehmer decay on inoculated and naturally infected oranges and lemons stored at 20 degrees C for 7 days. However, it failed to control green and blue mould infections on fruits placed in long-term cold storage, except green mould on oranges, which was effectively controlled. Dipping fruits in a sodium carbonate solution (20 g litre(-1)) for 2.5 min following a curing treatment at 33 degrees C for 65 h satisfactorily reduced green and blue mould incidence during subsequent long-term storage at 4 degrees C on oranges and at 10 degrees C on lemons. The efficacy was greater on injured fruits inoculated after the combination of treatments was applied, achieving a 60-80% reduction in decay in comparison with the curing treatment alone in all cases. A significant reduction of blue mould was also observed on fruits inoculated both before the treatments and on those re-inoculated after the treatments, demonstrating both protectant and eradicant activity. Thus, combining curing at 33 degrees C for 65 h with sodium carbonate treatment effectively controlled these post-harvest diseases on artificially inoculated citrus fruits and protected against re-infection. With naturally inoculated lemons, curing followed by sodium carbonate significantly reduced both green and blue mould incidence, but was not superior to curing alone. With naturally infected oranges, curing significantly reduced blue mould, but decay was not reduced further when followed by sodium carbonate treatment.     

Fungicide seed treatment efficacy against Microdochium nivale and M. majus in vitro and in vivo

BACKGROUND: Seed-borne Microdochium majus (Wollenweber) and M. nivale Fries are the primary pathogens responsible for Fusarium seedling blight in the UK. The two species show differences in pathogenicity, host preference and sensitivities to temperature, but their relative sensitivities to fungicide seed treatments are unknown. The aim was firstly to determine the efficacy of fungicide seed treatments towards single-spore isolates of M. majus and M. nivale using in vitro experiments, and subsequently to determine efficacy in vivo over a range of temperatures. RESULTS: Differences in EC(50) values between all seed treatments were evident from the in vitro experiments and ranged from 0.028 mg L(-1) for fludioxonil to 22.8 mg L(-1) for carboxin + thiram. The two seed treatments that showed best performance in vitro were used to examine efficacy towards seed-borne infection in vivo at 4, 8, 12 and 16 degrees C. Generally, seedling emergence improved and the severity of stem-base disease symptoms on emerged seedlings was reduced for both species through the use of the fungicides. The combination of fludioxonil + difenconazole showed improved performance compared with fludioxonil alone. Significantly less severe symptoms were observed through the use of fludioxonil and fludioxonil + difenconazole compared with bitertanol + fuberidazole at 12 degrees C and for all except one M. nivale infected seed lot at 8 degrees C. CONCLUSIONS: Differences in fungicide sensitivity between the two species in vitro were not evident in vivo. This is the first report of the effect of fungicide seed treatments on the control of seedling blight caused by M. majus and M. nivale.     

Antifungal vapour-phase activity of allyl-isothiocyanate against Penicillium expansum on pears

The fungitoxicity of allyl-isothiocyanate (AITC) vapour against Penicillium expansum , the causal agent of blue mould on pears (cvs Conference and Kaiser), was evaluated. The best control of blue mould was obtained by exposing fruits for 24 h in a 5 mg L⁻¹ AITC-enriched atmosphere, the extent of control depending on the inoculum density. Lesion diameter was inversely related to AITC concentration. In treated fruits the percentage of infected wounds increased with conidial concentration, with fewer than 20% affected at 1 × 10³ conidia mL⁻¹ to almost 80% at 1 × 10⁶ conidia mL⁻¹. In comparison, >98% of wounds were infected in untreated fruits irrespective of conidial concentration. AITC treatments were effective up to 24 h after inoculation for Conference and 48 h for Kaiser. AITC treatments also controlled a thiabendazole-resistant strain of P. expansum , reducing the incidence of blue mould by 90% in both cultivars. The use of AITC produced from pure sinigrin or from Brassica juncea defatted meal may be an economically viable alternative to synthetic fungicides against P. expansum .     

Control of postharvest pathogens and colonization of the apple surface by antagonistic microorganisms in the field

ABSTRACT Selected isolates of Aureobasidium pullulans, Rhodotorula glutinis, and Bacillus subtilis reduced the size and number of lesions on wounded apples caused by the postharvest pathogens Penicillium expansum, Botrytis cinerea, and Pezicula malicorticis. Combinations of the antagonistic microorganisms were applied to apple trees in the field late in the growing season of two consecutive years. The population dynamics of the introduced microorganisms and the incidence of fruit decay were determined. Population sizes of introduced antagonists on apple surfaces increased in the field following application of treatments until harvest. After transfer of the fruit from the field into cold storage, the populations of the introduced antagonists remained higher than in the control treatments. Identification of the applied isolates of A. pullulans and R. glutinis during the experiments was achieved by isolate-specific DNA probes generated from random amplified polymorphic DNA. A combination of two strains of A. pullulans and one strain of R. glutinis suppressed rotting of apple to the same extent as the commonly used fungicide Euparen. Our data demonstrate that the application of antagonistic microorganisms in the field represents a promising alternative to fungicide treatments to control post-harvest diseases of apple.     

Effect of imidacloprid on the reproduction of acaricide-resistant and susceptible strains of Tetranychus urticae Koch (Acari: Tetranychidae)

Occasional reports linking neonicotinoid insecticide applications to field population outbreaks of the two-spotted spider mite, Tetranychus urticae Koch, have been a topic of concern for integrated pest management (IPM) programmes, particularly in apples. In order to shed light on the factors which may contribute to the occasional field population increase of T. urticae following the application of neonicotinoid insecticides, greenhouse experiments have been set up. Four different T. urticae strains, namely GSS (acaricide-susceptible), WI (organophosphate-selected), USA (a largely uncharacterised strain) and Akita (METI (mitochondrial electron transport inhibitor) acaricide-resistant and cross-resistant to dicofol), were compared for their fecundity without insecticide treatment and for their ovipositional response to foliar and drench applications of the field-relevant dose of imidacloprid (100 mg litre(-1)). Without insecticide treatment, strain GSS laid significantly more eggs (162.50 (+/- 5.43)) than the multiple resistant strain Akita (139.90 (+/- 5.54)) during a 16 day oviposition period. With imidacloprid treatment the highest effect was observed with GSS, with a significantly reduced number of eggs in drench (143.40 (+/- 4.22)) and foliar (144.60 (+/- 5.85)) applications. For strains Akita and USA, no significant differences were observed in oviposition between imidacloprid treatments and controls. The proportion of F1 female offspring decreased significantly with drench application for GSS and WI, while no differences were observed among strains in the survival of F1 immature stages, except for strain USA. The viability of eggs was relatively high (from 82.9 (+/- 4.5)% for USA to 95.2 (+/- 1.2)% for GSS) and not affected by imidacloprid treatments.     

韭菜灰霉病防治烟剂的筛选与评价

为筛选适合制备安全、高效防治韭菜上灰霉病烟剂的杀菌剂品种,从原药抑菌活性、烟雾毒力作用、成烟率、安全性和田间防效5个方面,对9种常用杀菌剂制备成韭菜灰霉病防治烟剂的可行性进行了评价.室内毒力测定结果表明:氟啶胺、咯菌腈和啶菌噁唑抑制韭菜灰霉病菌菌丝生长的EC50值分别为0.13、0.05和0.12 mg/L;吡唑萘菌胺...     

Effect of Metschnikowia pulcherrima and methyl jasmonate on apple blue mold disease and the possible mechanisms involved

Phytoparasitica - Metschnikowia pulcherrima alone and in combination with methyl jasmonate [MeJA] was applied for control of blue mold of apple caused by Penicillium expansum. In vitro, MeJA at...     

腐霉利和咯菌腈混用对黄瓜灰霉病菌的联合毒力及药剂残留动态

为探究腐霉利和咯菌腈混用对黄瓜灰霉病病原菌灰葡萄孢的联合毒力,进而通过减少用药量及施药后残留动态分析,提升黄瓜中这两种农药的风险防控水平。采用菌丝生长速率法,测得腐霉利和咯菌腈对灰霉病菌菌丝生长的有效抑制中浓度(EC₅₀)分别为0.069 mg/L和0.103 mg/L;而将两种农药以质量比1∶1混用时,EC₅₀值为0.016 mg/L,增效系数(synergistic ratio,SR)达到5.0,表现为强增效作用。腐霉利和咯菌腈单独处理3 d后的灰霉病菌菌丝经电镜扫描观察,分别表现为菌丝干瘪和胞内物质溢出;而经两种农药以质量比1∶1混合处理后,灰霉病菌同时出现了菌丝干瘪和胞内物质溢出现象,混用对菌丝的损伤符合叠加增强特征。为进一步分析腐霉利和咯菌腈混配施用后的动态残留情况,对可同时检测这两种农药残留的高效液相色谱-串联质谱(HPLC-MS/MS)方法进行了优化。所用方法下,腐霉利和咯菌腈的添加回收率分别为100%～102%和94%～96%,相对标准偏差(RSD)为1.7%～5.3%和2.8%～3.4%,定量限(LOQ)为0.01 mg/k...     

Sorption, mineralization and mobility of N-(phosphonomethyl)glycine (glyphosate) in five different types of gravel

Sorption, mineralization and mobility of glyphosate were studied in six substrates: the five types of gravel most frequently used as surfacing in Denmark and a sandy agricultural soil from Simmelkaer that served as a reference soil. Cumulative mineralization of [methyl-14C]glyphosate in batch studies was highest in coarse gravel, amounting to 14% after 4 days at 30 degrees C and 32% after 31 days. Mineralization was slowest in the sandy reference soil, amounting to only 2% after 31 days. The adsorption coefficient (Kd) of glyphosate in gravel ranged from 62 to 164 litre kg(-1), while that in the sandy reference soil was 410 litre kg(-1). The results indicate that the relatively low Kd in gravel allows a relatively high rate of glyphosate mineralization by the biomass. When Kd is high, in contrast, mineralization is slow. Lowering the temperature to 10 degrees C decreased mineralization by 50% in one of two gravels. The leaching of glyphosate was screened in simple columns of gravel or soil in which precipitation events (20 mm over a 2-h period) were simulated on three occasions, starting either immediately after or 2 days after application of glyphosate. [14C]Glyphosate was applied as a tracer mixed with the commercial product Roundup Garden at the recommended rate of 2.4 kg glyphosate ha(-1), equivalent to 1 microg g(-1) soil. The highest concentration of [14C] compounds (expressed in terms of glyphosate concentration) in leachate from the columns exceeded 1300 microg litre(-1), and was detected in rounded gravel after the first rain event. No glyphosate was detected in leachate from the sandy reference soil.     

Factors affecting the efficacy of post-harvest fungicide applications for the control of blue mould (Penicillium expansum) in stored apples

The effects of apple fruit maturity, temperature of fruit or dip solution, period of time in fungicidal dip, storage conditions, and spore inoculum concentration, on the efficacy of fungicides for control of blue mould ( Penicillium expansum ) were examined in various experiments.
Iprodione and imazalil were only effective when inoculum concentration was low, whilst prochloraz was highly effective in controlling rot on fruit inoculated with 3 × 10⁶ spores/ml. There was no consistent effect of dip temperature or fruit maturity on the efficacy of the fungicides. Iprodione was more effective on warm fruit (19°C) than cold fruit (6°C) whilst the reverse was true of imazalil. Extended periods of immersion in the fungicides slightly reduced the incidence of rotting but not to any useful degree.
The incidence of rotting in fruit treated with prochloraz and etaconazole was less in fruit stored under controlled-atmosphere cold storage conditions than in fruit stored in air cold-storage. Both fungicides were also effective for short-term storage at 20 C in air. Captan, benomyl, captan plus benomyl or vinclozolin were either ineffective or of poor efficacy under all storage conditions.     

Liquid Formulation of the Postharvest Biocontrol Agent Candida sake CPA-1 in Isotonic Solutions

ABSTRACT Viability of the postharvest biocontrol agent Candida sake CPA-1 stored as liquid formulation was evaluated by studying the effect of growth, preservation medium, and temperature. C. sake was grown in molasses medium with unmodified water activity (a(w)) and in the same with a(w) modified to 0.98 with the addition of several solutes. Cells were preserved with isotonic solutions of different substances. Efficacy of liquid formulations stored for different periods was tested against infection by Penicillium expansum on apples. The best growth media were the (unmodified one and those modified to 0.98 a(w) with the addition of glycerol or sorbitol. For all growth media, the best preservation medium was the isotonic solution prepared with trehalose. When the effect of trehalose concentration in the preservation medium was studied, generally, at trehalose concentrations below the isotonic one, C. sake viabilities increased with increased trehalose. However, the best results were obtained when cells were preserved with the trehalose solution which was isotonic with cells. After 7 months of storage at 4 degrees C, cells that were grown in the sorbitol-modified medium and preserved with the isotonic solution of trehalose (0.96 M) maintained their viability and efficacy against P. expansum infection of apples.