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.     

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.     

Nutritional Enhancement of Biocontrol Activity of Candida sake (CPA-1) against Penicillium expansum on Apples and Pears

Pome fruits are poor in nitrogenous compounds and the addition of nitrogen can improve colonisation of the fruits by antagonists. Twenty-two nitrogenous compounds were evaluated for their effect on Candida sake (CPA-1) growth in vitro. Ten compounds that induced greater growth were applied with the antagonist to wounded fruits to evaluate their effect on enhancing control of Penicillium expansum. Calcium chloride and 2-deoxy-D-glucose were also tested. L-serine and L-aspartic acid enhanced biocontrol by C. sake against P. expansum on apples. On apples and pears, ammonium molybdate, calcium chloride and 2-deoxy-D-glucose improved the capacity of the antagonist to control P. expansum. The addition of ammonium molybdate at 1 mM allowed C. sake to be used on apples and pears at a lower concentration without diminishing control. Similar results were observed with the addition of calcium chloride to the antagonist. 2-deoxy-D-glucose at 6 and 18 mM enhanced biocontrol on pears by over 81%, but on apples the improvement of biocontrol was observed only at 6 mM. In cold storage, the combination of ammonium molybdate and C. sake completely eliminated the incidence of blue mould on pears, and reduced its severity and incidence by more than 80% on apples.     

Resistance of postharvest biocontrol yeasts to oxidative stress: a possible new mechanism of action

ABSTRACT We detected the generation of the reactive oxygen species (ROS) superoxide anion ( O.(-) (2)) and hydrogen peroxide (H(2)O(2)) in apple wounds 2 immediately after wounding, and assessed the relationships between (i) timely colonization of apple wounds by biocontrol yeasts, (ii) resistance of these microorganisms to oxidative stress caused by ROS, and (iii) their antagonism against postharvest wound pathogens. We analyzed a model system consisting of two yeasts with higher (Cryptococcus laurentii LS-28) or lower (Rhodotorula glutinis LS-11) antagonistic activity against the postharvest pathogens Botrytis cinerea and Penicillium expansum. LS-28 exhibited faster and greater colonization of wounds than LS-11. In contrast to LS-28, the number of LS-11 cells dropped 1 and 2 h after application, and then increased only later. In vitro, LS-28 was more resistant to ROS-generated oxidative stress. The combined application of biocontrol yeasts and ROS-deactivating enzymes in apple wounds prevented the decrease in number of LS-11 cells mentioned above, and enhanced colonization and antagonistic activity of both biocontrol yeasts against B. cinerea and P. expansum. Polar lipids of LS-11 contained the more unsaturated and oxidizable alpha-linolenic acid, which was absent in LS-28. Resistance to oxidative stress could be a key mechanism of biocontrol yeasts antagonism against postharvest wound pathogens.     

An Endophytic Actinomycete, Streptomyces sp. AOK-30, Isolated from Mountain Laurel and Its Antifungal Activity

To survey endophytic actinomycetes as potential biocontrol agents against fungal diseases of mountain laurel, young plants of mountain laurel (Kalmia latifolia L.) were used as an isolation source. From a total of 73 actinomycetes isolates obtained from leaves, stems and roots of test plants, Streptomyces sp. AOK-30 was selected, because i) it had a broad and intense antimicrobial spectrum against various yeasts and fungal pathogens of Ericaceae, ii) it grew on the multiplication and rooting media for tissue culture for this plant, and iii) the tissue-cultured seedlings that had been treated with this isolate became resistant to Pestalotia disease without any adverse growth effects such as malformation, dwarfing, discolorization and defoliation. This isolate was identified as Streptomyces sp., based on cultural, physiological and morphological characteristics. The present results indicate that isolate AOK-30 is a potential biocontrol agent against diseases of mountain laurel. Received 10 September 2002/ Accepted in revised form 22 November 2002     

Improving Control of Green and Blue Molds of Oranges by Combining Pantoea Agglomerans (CPA-2) and Sodium Bicarbonate

The potential of using Pantoea agglomerans (strain CPA-2) alone, or in combination with sodium bicarbonate or sodium carbonate solutions, for control of Penicillium digitatum (green mold) and Penicillium italicum (blue mold) on oranges was investigated under ambient (20 °C) and cold storage (3 °C) conditions. P. agglomerans controlled both pathogens on oranges at 2 × 10⁸ cfu ml^-1. The biocontrol agent was found to be completely tolerant to 2% sodium bicarbonate at room temperature, although its culturability was reduced by > 1000-fold after 30 min in 2% sodium carbonate. The efficacy of P. agglomerans for control of green mold was improved when combined with sodium bicarbonate, resulting in complete and 97.6% reduction of decay incidence at 3 °C and 20 °C, when compared to untreated controls. Satisfactory results were also obtained with the combined treatment for control of blue mold. P. agglomerans grew well inside wounds on oranges at both 20 °C and 3 °C. In contrast, it showed a reduced growth on the surface of intact fruit. Sodium bicarbonate at 2% concentration did not noticeably affect antagonist population development. Thus, use of bicarbonate treatment at 2% followed by the antagonist P. agglomerans CPA-2 could be an alternative to chemicals for control of postharvest diseases on oranges.     

Enhancement of biocontrol efficacy of antagonistic yeasts by salicylic acid in sweet cherry fruit

Salicylic acid (SA) and two yeast antagonists, Rhodotorula glutinis and Cryptococcus laurentii, were investigated separately and together for controlling Penicillium expansum and Alternaria alternata in sweet cherry fruit. Applied separately, both SA (0·5 mm, pH 3·6) and antagonists inhibited decay caused by P. expansum and A. alternata. Biocontrol activity of R. glutinis was enhanced in combination with SA, whereas the efficacy of C. laurentii was not affected. Application of SA did not affect the growth of R. glutinis and C. laurentii in cherry wounds. Results of in vitro studies showed that SA at low concentrations had little effect on the growth of the yeasts or the pathogens. SA treatment induced a significant increase in polyphenoloxidase, phenylalanine ammonia-lyase, and β-1,3-glucanase activity in cherry fruit, but did not alter the levels of peroxidase. The mechanism by which SA enhanced the biocontrol efficacy of the antagonistic yeast may be related to its ability to induce biochemical defense responses in sweet cherry fruit rather than its fungitoxicity effects on the pathogens.     

Studies on the biological control of plant pathogens in Finland1

Studies on biological control of plant pathogens in Finland were started by Pohjakallio in the 1950s, concentrating especially on the microbes parasitizing fungal sclerotia. At the turn of the 70s, Kallio studied the protective effect of Finnish isolates of Phlebia gigantea against the most important forest pathogen Heterobasidion annosum with positive results. The preparation was put on the market. Later in the 1970s, the observation by Tahvonen that Finnish light-coloured peat had an inhibitory effect on the infection of plants by some seed and soil-borne fungal pathogens led to further investigations. Of the microbes isolated from peat, Streptomyces spp. proved the most effective antagonists in both in vitro and in vivo tests. Studies are in progress with S. griseoviridis and several other fungal and bacterial antagonists as potential biocontrol agents against some seed and soil-borne pathogens and storage diseases.     

Long shelf life ofTalaromyces flavus in coating material of pelleted seed

Spores of the biocontrol agentTalaromyces flavus were recovered from coating material of chinese aster and tomato seeds in which they were incorporated 17 years before. The seeds had been stored at room temperature. About 20% of the ascospores had retained their heat resistance and survived treatment in aqueous suspension at 60°C for 30 min. None of the chinese aster seeds and 90% of the tomato seeds germinated after the storage period. Presence ofT. flavus during storage had not affected germinability of the seeds.     

Ozone for control of post-harvest decay of table grapes caused byRhizopus stolonifer

The effect of ozone on post-harvest decay of table grapes was studied both with regard to its effectiveness and its possible mode of action. Ozone concentrations fell rapidly upon contact with organic matter and the amount which reacted with grape berries and the microflora on their surface was about 0.1mgg^-1, when supplied at a rate of 8mgmin^-1for 20min. The dose applied could be increased by longer periods of exposure, but symptoms of toxicity were observed on certain cultivars. The number of colony forming units (cfu) of fungi, yeasts and bacteria naturally present on the berry surface was considerably reduced by a 20min exposure to ozone. Ozone treatments significantly reduced the extent of berry decay caused by fungi following cold storage and increased shelf-life. A significant decrease in decay was observed in berries that were treated with ozone either before or after being inoculated withRhizopus stolonifer.This finding indicates that, in addition to its sterilizing effect, ozone also induced resistance to post-harvest decay development. The phytoalexins resveratrol and pterostilbene were elicited by ozone treatments, at levels similar to those produced by uv-c irradiation. Resveratrol accumulated in greater quantities than pterostilbene. Inoculation withR. stoloniferin addition to ozone treatment, raised the levels of both stilbenes even more. Exposing berries to ozone was almost as effective as SO₂fumigation for the control of storage decay caused byR. stoloniferand no deleterious effects were observed on the appearance of the grape cluster. Ozone treatments can therefore be considered as a possible substitute for SO₂fumigation for the control of post-harvest fungal decay.     

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     

Studies on Endophytic Actinomycetes ( I ) <Emphasis Type="Italic">Streptomyces </Emphasis>sp. Isolated from Rhododendron and Its Antifungal Activity

To survey endophytic actinomycetes as potential biocontrol agents against fungal diseases of rhododendron, young plants of rhododendron were surface-sterilized for use as an isolation source. Nine, six and two isolates, with distinguishing characteristics based on the macroscopic appearance of colonies, were obtained from roots, stems and leaves, respectively, suggesting that various species of actinomycetes grow in the respective organs of this plant as symbionts or parasites. On an agar medium, only isolate R-5 commonly formed a clear growth-inhibition zone against two major fungal pathogens of rhododendron, Phytophthora cinnamomi and Pestalotiopsis sydowiana, indicating that this isolate can produce antifungal material(s). Acetone extracts of a liquid culture of R-5 had a broad antimicrobial spectrum against Gram-positive bacteria, yeast and filamentous fungi. Isolate R-5 was identified as a Streptomyces sp. based on morphological, physiological and chemotaxonomical characteristics. The present results indicate that isolate R-5 is a suitable candidate for the biocontrol of diseases of rhododendron. Received 25 March 2000/ Accepted in revised form 18 May 2000     

Molecular Approaches to Assist the Screening and Monitoring of Postharvest Biocontrol Yeasts

Nineteen yeast isolates obtained from the surface of several fruits and vegetables grown in Southern Italy and Israel were compared by molecular analysis using arbitrarily primed polymerase chain reaction (AP-PCR) and random amplified polymorphic DNA technique (RAPD-PCR). Genetic analysis made it possible to distinguish between closely-related genitically different strains which had the same morphological characteristic, and to discard isolates which were genetically identical. Following PCR characterisation, 6 isolates were selected and tested for their biocontrol activity against major postharvest pathogens (Penicillium digitatum on grapefruit, Botrytis cinerea, Rhizopus stolonifer and Aspergillus niger on table grape and B. cinerea and R. stolonifer on cherry tomato). All the isolates showed a good biocontrol efficacy on both wounded and non-wounded fruits. Furthermore, the preharvest application of the most effective antagonist (LS15) on table grape resulted in a significant reduction in grey mold ranging from 28.3% to 38.2% compared to the untreated control. The RAPD-PCR technique was also useful for identifying and monitoring the survival of the antagonist after field application.     

RETRACTED ARTICLE: Effect of calcium chloride on the biocontrol efficacy of two antagonistic yeasts against <Emphasis Type="Italic">Penicillium expansum</Emphasis> on apple fruit

Two antagonistic yeasts, Candida membranaefaciens and Pichia guilliermondii, were evaluated for the control of the blue mold of apple caused by Penicillium expansum. Dual culture, cell-free metabolite and volatile tests were used for in vitro assay. Yeast strains of two genera inhibited growth of P. expansum; inhibition varied from 30.27% to 44.19% in dual culture, from 79.40% to 90.57% in the volatile metabolite test, and from 72.99% to 88.77% in the cell-free metabolite test. Calcium chloride (2% w/v) significantly inhibited the growth of the pathogen P. expansum, but did not affect the colony-forming units (CFU) of the yeasts C. membranaefaciens and P. guilliermondii in potato dextrose broth. The concentration of yeast suspension influenced spore germination and germ tube growth of P. expansum in vitro, as well as disease incidence and lesion development in fruits. There were significant negative relationships between the suspension concentrations of the yeasts and the growth as well as infectivity of the pathogen. The addition of calcium resulted in lower spore germination rates and slower growth of germ tubes in vitro, as well as in lower disease incidences and smaller lesion diameters compared with treatments with yeast antagonists alone. When yeast cell suspensions reached a concentration of 10⁷ CFU ml^-1, growth of the pathogen was completely limited in vitro, and no infection was found in apple fruits treated with or without calcium. This article has been retracted because part of the data shown has already been published before, by different authors. An erratum to this article can be found at     

Effect of a biocontrol agent (<Emphasis Type="Italic">Bacillus subtilis</Emphasis>) and modified atmosphere packaging on postharvest decay control and quality retention of litchi during storage

The efficacy of biological control and two types of modified atmosphere packaging (MAP) alone and in combinations was evaluated under cold storage as well as simulated market-shelf conditions to control decay and pericarp browning on litchi cv. ‘McLean’s Red’. Fruits were dipped for 2 min at 15°C inBacillus subtilis or prochloraz separately, packed in MAP [low density polyethylene (LDPE) or polypropylene (PP)], heat sealed and stored at 2°C and 90% r.h. for 18 days followed by 2 days at 14°C and 75% r.h. to simulate market-shelf conditions. A commercially adopted sulfur dioxide treatment was included as a comparative control. Fruits treated withB. subtilis + PP or prochloraz + PP and stand-alone PP treatment did not show decay or browning at 2°C. Decay and browning were controlled significantly after 2 days at 14°C inB. subtilis + PP or prochloraz + PP treatments. However, the prochloraz + PP affected the natural pinkish-red color of the pericarp and gave higher h° (hue angle) values. The stand-alone PP treatment (∼14% O₂, ∼5% CO₂) showed 11.3% decay due mainly toAlternaria alternata andCladosporium spp. at 14°C. The effectiveness of the MAP was improved at 14°C whenB. subtilis was combined with PP, controlling decay and pericarp browning and retaining the fruit color and quality.B. subtilis survived in PP at 2° and 14°C, but not in LDPE. Stand-alone LDPE (∼3% O₂, ∼10% CO₂) and combination treatmentsB. subtilis + LDPE or prochloraz + LDPE failed to control decay and pericarp browning. Higher yeast populations were observed in LDPE orB. subtilis + LDPE at both 2° and 14°C.Candida, Cryptococcus andZygosaccharomyces spp. were the predominant yeasts in all LDPE treatments. Reprints ofS. Afr. Litchi Growers’ Assoc. Yearb. references can be obtained from D. Sivakumar.     

Can phyllosphere yeasts explain the effect of scab fungicides on russeting of Elstar apples?

In 1999 and 2000, the effects of scab fungicides on yeast composition and russeting of Elstar apples were assessed. Yeast composition of fungicide-treated and untreated young apple fruit with or without russet symptoms was investigated and enzyme activity of the yeasts was studied. Cryptococcus albidus, C.laurentii,Rhodotorula glutinis, Sporobolomyces roseus andMetschnikowia pulcherrima dominated the apple fruit surface. Russeted apple fruitlets had a higher red yeast density than non-russeted fruitlets. Invitro fungicide susceptibility of the dominant yeast species varied. Dithianon and dodine were active against all tested species, captan and tolylfluanid showed specificity for certain species, whereas pyrimethanil and bupirimate were largely ineffective. Fungicide treatment in the field had a clear effect on the yeast composition. Metschnikowia etschnikowia pulcherrima was eliminated from the phyllosphere and cryptococco$ï species diminished by both captan and dithianon. The red yeast population was not significantly changed by either fungicide. Captan, dithianon, tolylfluanid and pyrimethanil reduced russet in the field in both years, dodine and kresoxim-methyl only in 2000. All yeast species had cutinolytic activity, all but M.pulcherrimaand Debaryomyces hansenii were lipolytic, and some of the isolates showed proteolytic activity.     

Acidified peel of litchi fruits selects for postharvestPenicillium decay

Litchi fruits are fumigated after harvest with sulfur dioxide (SO₂) to prevent their rapid browning. SO₂ blocks enzymatic activity but bleaches the fruits and, if this process is followed by dipping the fruit in dilute hydrochloric acid, the appealing red color is regained. Hot water brushing (HWB) is among the alternative methods that were developed to replace the use of SO₂. HWB reduced fungal population size on the surface of the fruit peel after treatment but did not eliminate fruit infection after storage. Whereas untreated fruits were infected with a variety of fungal species,Penicillium sp. was the only fungus that developed on the pericarp after storage in fruits that had been dipped in 1.5M HCl. Fruit treated by HWB followed by handling and storage under sterile conditions suffered greater decay than fruit stored under non-sterile conditions but with more ventilation. APenicillium sp. isolated from litchi grew well in liquid medium acidified to the pH range reported for SO₂ and HCl-treated litchi fruits. Morphological analysis identified fungal isolates asP. aurantiogriseum. Internal transcribed spacer sequence analysis of five isolates suggested a sequence similarity toP. commune. Our data support the hypothesis that dipping litchi fruit in hydrochloric acid eliminates infection by common opportunistic fungi and selects forPenicillium species that tolerate low pH. http://www.phytoparasitica.org posting April 30, 2004.     

Biological Control of Sclerotinia pseudotuberosa and Other Fungi During Moist Storage of Quercus robur Seeds

The fungal pathogen Sclerotinia pseudotuberosa is a major cause of deterioration during storage of Quercus robur seeds (acorns) and along with other, mainly saprotrophic fungi, contributes to the decline of viability and vigour in the acorn population. Hot-water thermotherapy (HWT; 41 °C for 2.5 h) killed the fungal pathogen S. pseudotuberosa and prolonged the storage life of acorns. The addition of the systemic fungicide benomyl to the HWT and/or the broad-spectrum fungicide thiram as a seed dressing further enhanced the storage life of acorns. Three fungal antagonists, Coniothyrium minitans, Trichoderma sp. (KW3) and Trichoderma virens (G20), were also applied as a film-coating to acorns using a polyvinylacetate sticker achieving ca. 10⁷–10⁸ viable conidia per acorn. The biological treatments provided protection against infection and the spread of infection of S. pseudotuberosa and other fungi on the acorns during storage over several months. All treated and stored acorns grew normally following sowing in seedbeds. The Trichoderma species were more effective than C. minitans, with T. virens being the most effective. T. virens reduced pathogen spread from acorns infected with S. pseudotuberosa to `clean' acorns when T. virens coated infected and 'clean' acorns were mixed together. However, T. virens was less effective than HWT at preventing the proliferation of this pathogen within individual acorns that were infected before coating. A combination of HWT and subsequent coating with T. virens provided a more effective control against both S. pseudotuberosa and saprotrophic fungi than when either treatment was applied alone.     

Biological control of potato soft rot caused by Dickeya solani and the survival of bacterial antagonists under cold storage conditions

Dickeya and Pectobacterium are responsible for causing blackleg of plants and soft rot of tubers in storage and in the field, giving rise to losses in seed potato production. In an attempt to improve potato health, biocontrol activity of known and putative antagonists was screened using in vitro and in planta assays, followed by analysis of their persistence at various storage temperatures. Most antagonists had low survival on potato tuber surfaces at 4 °C. The population dynamics of the best low-temperature tolerant strain and also the most efficient antagonist, Serratia plymuthica A30, along with Dickeya solani as target pathogen, was studied with TaqMan real-time PCR throughout the storage period. Tubers of three potato cultivars were treated in the autumn with the antagonist and then inoculated with D. solani. Although the cell densities of both strains decreased during the storage period in inoculated tubers, the pathogen population was always lower in the presence of the antagonist. The treated tubers were planted in the field the following growing season to evaluate the efficiency of the bacterial antagonist for controlling disease incidence. The potato endophyte S. plymuthica A30 protected potato plants by reducing blackleg development on average by 58.5% and transmission to tuber progeny as latent infection by 47–75%. These results suggest that treatment of potato tubers with biocontrol agents after harvest can reduce the severity of soft rot disease during storage and affect the transmission of soft rot bacteria from mother tubers to progeny tubers during field cultivation.     

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.