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.     

Control of blue mold (Penicillium expansum) by fludioxonil in apples (cv Empire) under controlled atmosphere and cold storage conditions

A reduced risk fungicide, fludioxonil, was tested for its efficacy against blue mold caused by thiabendazole-resistant and -sensitive Penicillium expansum (Link) Thom in apples under three storage conditions. In a co-treatment, fludioxonil and inoculum were applied together to test the protective activity of the fungicide on wounds that had been aged for 1 or 2 days. The fungicide was also tested for its curative activity in post-inoculation treatment on apples that had been inoculated for 1 or 2 days. Fludioxonil was very effective as co-treatment and as post-inoculation treatment. At a concentration of 300 mg litre(-1), fludioxonil gave complete control of post-harvest blue mold caused by the thiabendazole-resistant and -sensitive P expansum for 105 days in controlled atmosphere (CA) storage at 2 (+/-1) degrees C, for 42 days in common cold storage at 4 (+/-1) degrees C and also in a shelf-life study for 6 days at 20 (+/-1) degrees C. Comparison on the effect of fludioxonil in CA storage and common cold storage showed that higher concentrations of fungicide were needed in cold storage than in CA storage. Fludioxonil at a concentration of 450 mg litre(-1), gave 98 and 92% control of blue mold of apples in the simulated shelf-life studies after CA and common cold storages, respectively. Fludioxonil has a potential to be incorporated in the fungicide resistance management strategies for control of blue mold in apples stored for 105 days.     

Prestorage heat treatment reduces pathogenicity of Penicillium expansum in apple fruit

Penicillium expansum is one of the main postharvest pathogens of apples in Israel. Heating apple fruit inoculated with P. expansum for 96 h at 38°C completely inhibited decay development. Fruit held for 24 h at 42°C or 12 h at 46°C had significantly reduced decay after an additional 14 days incubation at 20°C, compared with unheated inoculated control fruit. Mycelial growth and percentage spore germination in vitro were inversely proportional to length of time of exposure to various temperatures. The ET₅₀ for spore germination was 42, 34 and 20 h at 38, 42 and 46°C, respectively, while the ET₅₀ for mycelial growth was 48, 44 and 36 h at those temperatures. When Penicillium spores were incubated on crude extract prepared from the peel of apple fruits held 4 days at 38°C, germ tube elongation was significantly reduced, while the walls of the tubes were thicker, compared with germ tubes from spores incubated on crude extract prepared from peel of non-heated fruit. The evidence presented here supports the hypothesis that the effect of heating on the decay of apples caused by P. expansum is not only the result of direct inhibition of fungal germination and growth by high temperature, but is also partly due to the formation of an inhibitory substance in the heated peel.     

Effect of the Biocontrol Yeast Rhodotorula glutinis Strain LS11 on Patulin Accumulation in Stored Apples

ABSTRACT Contamination of apples (Malus domestica) and derived juices with fungicide residues and patulin produced by Penicillium expansum are major issues of food safety. Biocontrol agents represent an alternative or supplement to chemicals for disease control. Our data show that these microbes could also contribute to actively decreasing patulin accumulation in apples. Three biocontrol agents, Rhodotorula glutinis LS11, Cryptococcus laurentii LS28, and Aureobasidium pullulans LS30, were examined for their in vitro growth in the presence of patulin and for their capability to decrease mycotoxin recovery from the medium. Strain LS11 yielded the highest growth rates and the greatest decrease of toxin recoveries. Further, it caused the appearance of two major spots on thin-layer chromatography (TLC) plates, suggesting possible metabolization of the mycotoxin. In vivo, i.e., in the low percentage of LS11-pretreated apples infected by P. expansum, patulin accumulation was significantly lower than in nontreated infected fruits. Yeast cells survived and increased in infected apples and, in a model system emulating decaying apple, resulted in accelerated breakdown of patulin and the production of the same TLC spots as those detected in vitro. These data suggest that biocontrol yeast cells surviving in decaying apples could metabolize patulin and/or negatively affect its accumulation or synthesis. To our knowledge, this is the first report describing the effect of a biocontrol agent on patulin accumulation in vivo.     

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.     

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.     

Effects of Ca2+ and Mg2+ on Botrytis cinerea and Penicillium expansum in vitro and on the biocontrol activity of Candida oleophila

Calcium salts have been reported to play an important role in the inhibition of postharvest decay of apples and in enhancing the efficacy of postharvest biocontrol agents. Therefore, the present study was conducted in order to examine and compare the effects of calcium and magnesium salts on the germination and metabolism of the postharvest pathogens Botrytis cinerea and Penicillium expansum , and to determine the effects of these salts on the biocontrol activity of two isolates (182 and 247) of the yeast Candida oleophila. Increasing concentrations of CaCl₂ (25–175 mM) resulted in decreased spore germination and germ-tube growth of both pathogens. The greatest effect was observed in the case of B. cinerea. The inhibitory effect could be overcome by the addition of glucose to the germination medium. MgCl₂ (25–175 mM) had no effect on germination or germ-tube growth of either pathogen, indicating that the calcium cation rather than the chloride anion was responsible for the inhibition. The pectinolytic activity of crude enzyme obtained from the culture medium of both pathogens was also inhibited by 25–175 mM CaCl₂, with the greatest effect on the crude enzyme from P. expansum. Biocontrol activity of isolate 182 was enhanced by the addition of 90 or 180 MM CaCl₂, whereas there was no effect on the biocontrol activity of isolate 247. This was apparently due to the inability of isolate 247 to proliferate in apple wounds. It is postulated that enhanced biocontrol activity of isolate 182 of the yeast C. oleophila in the presence of Ca²⁺ ions is directly due to the inhibitory effects of calcium ions on pathogen spore germination and metabolism, and indirectly due to the ability of isolate 182 to maintain normal metabolism in the presence of"toxic" levels of calcium.     

Studies on thiabendazole resistance of Penicillium expansum of pears: pathogenic fitness and genetic characterization

Isolates of Penicillium expansum recovered from stored pears were scored for resistance to the fungicide thiabendazole (TBZ) and for pathogenic fitness. Out of 50 isolates, nine were sensitive (S) and 41 resistant (R or RR). Seven of these resistant isolates (RR) germinated with a higher percentage on TBZ-amended medium than on unamended medium. Six S isolates and six RR isolates were chosen at random for further analysis. S and RR isolates had similar in vitro growth fitness, although RR isolates were characterized by higher infection severity on fruits. Laboratory-induced resistant isolates were generated by UV-irradiating S strains, and a similar correlation between the induced TBZ resistance and pathogenic fitness was observed. The β-tubulin gene of RR and S isolates was amplified and sequenced; mutations correlating with TBZ resistance were identified at residues Phe 167 and Glu 198. Analogous mutations were detected in the laboratory-induced resistant isolates.     

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     

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.     

A simple method for long-term cryopreservation of <Emphasis Type="Italic">Calonectria ilicicola</Emphasis> on barley grains

We developed a simple method for long-term preservation of the soybean red crown rot fungus, Calonectria ilicicola, using barley grains. Autoclaved barley grains were inoculated with the fungus, then incubated at 25°C for 1 month. After incubation, grains were dried to approximately 3% moisture content, and stored at 4, −20, or −80°C for 3 years. C. ilicicola preserved on barley grains at −80°C remained viable without any change in mycelial growth and virulence. These results showed that C. ilicicola can be successfully cryopreserved for extended periods on barley grains at −80°C. We also confirmed that cultures preserved on barley grains are suitable for direct use without further manipulation as inocula in pathogenicity tests.     

黄瓜霜霉病菌保存方法

 Pseudoperonospora cubensis(Berk. & M. A. Curtis) Rostovzev, the causal agent of cucumber downy mildew, is an obligate parasitic fungus. Up to now it can not be preserved on culture medium. In this study, the sporangia of P. cubensis were preserved in protective substances of 10% dimethyl sulfoxide plus 5% skim milk, 10% dimethyl sulfoxide plus 10% skim milk, sterilized water and leaves in vitro, and stored at -20℃, -70℃, and preliminary freezing at -20℃ for 24 h prior to -70℃ respectively. The results showed that the sporangia preserved in 10% dimethyl sulfoxide plus 5% skim milk, preliminary freezing at -20℃ for 24 h prior to -70℃ were still highly pathogenic after 12 months preservation. The percentage of germination of sporangia, incidence and disease index were 46%, 50.0% and 40.0 respectively 9 d after inoculation. This method solved the problem of P. cubensis preservation.     

Alternative disease control agents induce resistance to blue mold in harvested 'red delicious' apple fruit

ABSTRACT Alternative control agents, including UV-type C (254 nm) irradiation, yeasts antagonistic to fungal growth, chitosan and harpin, were evaluated for their ability to induce resistance in cv. Red Delicious apple fruit against postharvest blue mold caused by Penicillium expansum. Freshly harvested and controlled atmosphere (CA)-stored fruit were treated with these agents at different doses and concentrations or with paired combinations of the agents. Treated fruit were inoculated with P. expansum 24, 48, or 96 h following treatment, and stored at 24 degrees C in the dark. The fruit were evaluated for development of disease every 2 days for 14 days by measuring the diameter of lesions that formed. The area under the disease progress curve (AUDPC) was calculated and analyzed statistically. All treatments were effective in reducing the AUDPC; UV-C was most effective, followed by harpin, chitosan, and the yeasts, respectively. Regardless of treatment, fresh fruit were more responsive to treatments than CA-stored fruit. There was a clear time-dependent response of the fruit to the treatments, in which treatments applied 96 h before inoculation provided the best results. In a few situations, the combinations of agents did provide an additive effect, but no synergistic effects were detected. Moreover, disease severity in fruit treated by any combination was markedly better than that in the controls. Although the combinations of treatments was overall less effective than the single treatments, they did provide significant reductions of the progress of disease in comparison with the controls. Because the fungus did not come into contact with any of the control agents, this study showed conclusively that the agents studied were able to induce resistance in the fruit rather than merely inhibit the pathogen directly. It also showed, for the first time, that harpin is able to induce resistance in harvested apple fruit. The use of these control agents may minimize the costs of control strategies and reduce the risks associated with the excessive use of fungicides in harvested apple fruit.     

Control of Postharvest Decay of Apple Fruit with Candida saitoana and Induction of Defense Responses

ABSTRACT The ability of Candida saitoana to induce systemic resistance in apple fruit against Botrytis cinerea was investigated. To separate the antagonistic activity of C. saitoana from its ability to induce resistance, the antagonist and the pathogen were applied in spatially separated wounds. In fresh apples, C. saitoana applied 0 or 24 h before inoculation with B. cinerea showed no effect on lesion development caused by B. cinerea. When applied 48 to 72 h preinoculation with B. cinerea, however, C. saitoana reduced lesion diameter by more than 50 and 70%, respectively, compared with wounding. C. saitoana had no effect on lesion development on stored apples, regardless of the lag period between yeast treatment and inoculation with B. cinerea. In addition to inducing systemic resistance, C. saitoana increased chitinase and beta-1,3-glucanase activities with a higher accumulation in fresh than in stored apples. In fresh apples, the onset of systemic resistance to B. cinerea coincided with the increase in chitinase and beta-1,3-glucanase activity in systemically protected tissue. These studies show that C. saitoana is capable of inducing systemic resistance in apple fruit and indirectly suggest that antifungal hydrolases are involved in the observed systemic protection.     

Integrated control of apple postharvest pathogens and survival of biocontrol yeasts in semi-commercial conditions

The biocontrol yeast isolates Rhodotorula glutinis LS11, Cryptococcus laurentii LS28 and Aureobasidium pullulans LS30 were tested against Botrytis cinerea and Penicillium expansum on apples artificially inoculated and stored at 3 and 20 °C. Isolates LS28 and LS30 were most effective, consistently resulting in high reductions of fungal decay, while isolate LS11 was effective only on apples stored at 3 °C. The yeasts showed good in vitro resistance to dicarboximides and copper fungicides, while they were inhibited by triazoles. Isolate LS11, in contrast to LS28 and LS30, was also inhibited by benzimidazoles. The yeasts were tested on naturally-infected apples in semi-commercial conditions for 2 years. They were applied twice: soon after harvesting and 20 days later, at the beginning of the cold storage. The antagonists significantly reduced fungal decay when combined with a low dosage of benomyl showing an activity comparable to that exerted by the fungicide alone at full dosage. Periodical monitoring of the epiphytic biocontrol yeast populations in both the field and cold room showed a good rate of survival of the antagonists on the skin of treated apples. Specific fingerprints relying on amplified restriction length polymorphism (AFLP) were used to integrate the morphology-based monitoring of the yeasts.     

Erwinia amylovora can pass through the abscission layer of fruit-bearing twigs and invade apple fruit during fruit maturation

Invasion of apple fruit by Erwinia amylovora from fruit-bearing twigs through the abscission layer at fruit maturation was examined. Erwinia amylovora (ca. 10⁵ cfu) tagged with bioluminescence genes from Vibrio fischeri was deposited in artificial wounds on fruit-bearing twigs of apple trees grown in a containment greenhouse on September 22, 27, or October 5, 2004. On October 22, 176 apples were harvested and cut horizontally in half. The upper halves were stamped on plates of selective medium, and the lower halves were flooded with iodine solution to assess maturity. All fruit were symptomless and fully mature. The pathogen was recovered from 19 (10.8%) apples. The result showed that if at least ca. 10⁵ cfu of E. amylovora are present in fruit-bearing twigs at the time of fruit maturation, the bacteria can pass through the abscission layer into the fruit, even though the mature fruit lack symptoms.     

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.     

Ultrastructural and Cytochemical Aspects of the Biological Control of Botrytis cinerea by Candida saitoana in Apple Fruit

ABSTRACT Biocontrol activity of Candida saitoana and its interaction with Botrytis cinerea in apple wounds were investigated. When cultured together, yeast attached to Botrytis sp. hyphal walls. In wounded apple tissue, C. saitoana restricted the proliferation of B. cinerea, multiplied, and suppressed disease caused by either B. cinerea or Penicillium expansum. In inoculated apple tissue without the yeast, fungal colonization caused an extensive degradation of host walls and altered cellulose labeling patterns. Hyphae in close proximity to the antagonistic yeast exhibited severe cytological injury, such as cell wall swelling and protoplasm degeneration. Colonization of the wound site by C. saitoana did not cause degradation of host cell walls. Host cell walls in close contact with C. saitoana cells and B. cinerea hyphae were well preserved and displayed an intense and regular cellulose labeling pattern. In addition to restricting fungal colonization, C. saitoana induced the formation of structural defense responses in apple tissue. The ability of C. saitoana to prevent the necrotrophic growth of the pathogen and stimulate structural defense responses may be the basis of its biocontrol activity.