Folia Microbiol DOI 10.1007/s12223-013-0290-2
Antifungal properties of the human Metschnikowia strain IHEM 25107 Maurizio Sisti & Vincenzo Savini
Received: 15 July 2013 / Accepted: 30 October 2013 # Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2013
Abstract Metschnikowiaceae may show natural, straindependent antifungal properties, so they are employed in agriculture as natural, safe alternatives to pesticides. With this paper, we documented the ability of the recently described Metschnikowia IHEM 25107 to inhibit growth of several molds, including diverse Aspergillus species and dermatophytes. This biocontrol activity enables pulcherriminproducing strains to naturally antagonize competing microorganisms. Keywords Metschnikowia . Antifungal . Biocontrol
Introduction The genus Metschnikowia belongs to the family Metschnikowiaceae that includes water and terrestrial ascomycetous fungi inhabiting plants, pests, and animals (Giménez-Jurado et al. 1995; Hazen 1995; Molnár and Prillinger 2005; Sipiczki 2006). Metschnikowia pulcherrima (anamorphic state: Candida pulcherrima) is the most studied species (Giménez-Jurado et al. 1995; Hazen 1995) and may behave as a biocontrol agent against food-borne microorganisms and fruit and vegetable pathogens responsible for postharvest decay (Krutzman and Droby 2001; Spadaro et al. 2002; Leverentz et al. 2006; Prodorutti et al. 2006). The antifungal property relies on the strain-dependent production of pulcherrimin, a reddish pigment that forms a M. Sisti (*) Department of Biomolecular Science, Section of Hygiene, University of Urbino Carlo Bo, Via S. Chiara, 27, 61029 Urbino, PU, Italy e-mail: [email protected] V. Savini Clinical Microbiology and Virology, Spirito Santo Hospital, Pescara, PE, Italy
chelate complex with iron ions (Kluyver et al. 1953; Türkel and Ener 2009), thus immobilizing the metal in the growth medium; mutants that do not produce pulcherrimin lack in fact the antimicrobial activity (Sipiczki 2006; Türkel and Ener 2009). M. pulcherrima was the first terrestrial member of the genus to be described (Pitt and Miller 1968); also, it was until recently the only Metschnikowia species cultivated from man (Hazen 1995). In 2012, however, we described a human, pigment-producing Metschnikowia non-pulcherrima strain (Savini et al. 2012), potential biocontrol property of which is investigated in the present work. Particularly, we studied the strain’s activity against several molds, including dermatophytes and Aspergillus species and, based on results we obtained, we suggest this novel isolate might be employed in agriculture to antagonize postharvest pathogens (Krutzman and Droby 2001; Prodorutti et al. 2006). Moreover, we would like to emphasize that pigment production could enable producing strains, like IHEM 25107, to survive in the environment as well as in the human host. The analyses performed in Savini et al. (2012) placed strain IHEM 25107 into an uncertain taxonomic position. Furthermore, classification of Metschnikowiaceae is still under definition (Sipiczki et al. 2013) and, although genomebased results indicated that the studied yeast belonged to the phylogenetic group of M. pulcherrima , it did not match, genetically, with any previously described species; conversely, physiological and metabolic properties were those of Metschnikowia sinensis that, however, has never been reported to produce pigment (Savini et al. 2012; Xue et al. 2006). We tested strain IHEM 25107 antagonism against six dermatophyte isolates (Epidermophyton floccosum, Tricophyton mentagrophytes , Tricophyton rubrum , Tricophyton shoenleinii , Tricophyton tonsurans , and Tricophyton violaceum ) collected in our laboratories from human onychomycosis cases and skin infections and eight clinical
Folia Microbiol
and environmental molds belonging to the genus Aspergillus, including Aspergillus flavipes, Aspergillus flavus, Aspergillus fumigatus , Aspergillus nidulans , Aspergillus niger , Aspergillus terreus , Aspergillus ustus , and Aspergillus versicolor.
Materials and methods Isolation of molds and of strain IHEM 25107 Dermatophytes were isolated from human onychomycosis cases and skin infections while Aspergillus strains were collected from clinical and environmental samples. IHEM 25107 was isolated as a putative commensal from the sputum of a leukemia patient, as described in Savini et al. (2012). Mold identification was performed according to standard methods (Murray et al. 1999). Filamentous fungi and strain IHEM 25107 were grown on potato dextrose agar (PDA) and Sabouraud dextrose agar, respectively, at 35 °C and subcultured twice to ensure purity and viability.
strain IHEM 25107 were confirmed by inoculating serial suspension dilutions on PDA plates.
Antagonistic activity test IHEM 25107 antifungal activity was tested through a disk diffusion method (Jorgensen et al. 1999). For this purpose, the PDA Petri dish (60 mm of diameter) was previously seeded with 100 μL (approximately 105 CFU per plate) of conidial inoculum suspension. Sterile filter paper disks (6.3 mm in diameter) were positioned at the plate center; they were previously dried, then impregnated with 10 μL (approximately 104 CFU per disk) of strain IHEM 25107 inoculum suspension. Plates were incubated at 30 °C for 72 h and antifungal activities evaluated manually by measuring inhibition zone (IZ) diameters; IZ sizes (expressed in millimeters) were calculated from strain IHEM 25107 colony edge to that of fungal lawn. Experiments were repeated independently three times for each strain.
Pigment production
Results
Strain IHEM 25107-related pigment was confirmed to be pulcherrimin by growing a lawn of the yeast on yeast nitrogen base (Bergman 2001) agar with 1 % glucose with a steep concentration gradient of ferric citrate. Growth was inhibited at the highest concentration and the pigment was produced abundantly in a zone corresponding to nontoxic iron levels. The remaining growth was free of pigmentation.
Figure 1 shows strain IHEM25107 antagonistic activity versus T. mentagrophytes, while Figs. 2 and 3 report IZ diameters produced by the test inoculum. Notably, the highest value was found to be 5 mm and was obtained with A. nidulans, A. ustus, T. mentagrophytes, T. shoenleinii , and T. violaceum ; a 4 mm diameter was instead achieved with A. flavus , A. versicolor, and T. tonsurans. Again, a 2 mm IZ was observed with A. fumigatus, A. rubrum, and A. terreus while, for A. flavipes , growth inhibition was revealed by a 1 mm-IZ. Finally, only A. niger and E. floccosum displayed a complete resistance, as indicated by the absence of any IZs.
Preparation of conidial and yeast inoculums suspension Conidial inoculum suspensions were prepared by flooding each culture slant with 1 mL sterile 0.85 % NaCl solution containing 0.05 % Tween 80 and gently probed with a pipette tip. The resulting mixture was withdrawn and the heavy particles allowed to settle for 3–5 min. The upper homogeneous suspensions containing the mixture of conidia and hyphal fragments were vortexed for 15 s. The transmittances of mixture suspensions were adjusted according to the CLSI M38-A protocol (NCCLS 2002) using a spectrophotometer set at a wavelength of 530 nm to provide a final test inoculum of about 106 CFU/mL. Strain IHEM 25107 inoculum suspensions were prepared by selecting five colonies of 1 mm diameter (at least), from a 24-h culture and suspending them in 5 mL of sterile 0.85 % NaCl solution. Cell suspension turbidity was adjusted according to the CLSI M27-A protocol (NCCLS 1997) using a spectrophotometer set at a wavelength of 530 nm to provide a final test inoculum of 106 CFU/mL. The inoculum titres of dermatophytes, Aspergillus spp. and
Fig. 1 Strain IHEM25107 antagonism versus T. mentagrophytes; the reddish pigment is visible at the IZ edge
Folia Microbiol Fig. 2 Strain IHEM25107 antagonism versus dermatophytes; results are means of three independent experiments that agree within 10 %
Discussion Metschnikowiaceae may display a natural, strain-dependent antimicrobial activity related to pulcherrimin production; particularly, the pigment binds iron ions in the growth medium, thus inhibiting bacterial and fungal survival as well as spore germination (Kluyver et al. 1953; Sipiczki 2006). Biocontrol properties exerted by Metschnikowia non-pulcherrima yeasts are poorly reported in the literature, and only include those presented in this work along with previously published observations on M. fructicola (Prodorutti et al. 2006; Karabulut et al. 2003). In this context, our results show that strain IHEM 25107 antagonizes the growth of almost all the studied molds; A. nidulans, A. ustus, T. mentagrophytes, T. shoenleinii, and T. violaceum appeared to be the most susceptible fungi among those tested; conversely, growth of E. floccosum and A. niger has not been inhibited. Lack of A. niger susceptibility is in agreement with Sipiczki’s findings (although these were obtained with M. pulcherrima; Sipiczki 2006), but not with those by Türkel (Türkel and Ener 2009); given the absence of standardized methodologies for testing, however, it is hard to compare results.
Fig. 3 IHEM25107 antagonism versus Aspergillus spp; results are means of three independent experiments that agree within 10 %
In nature, antimicrobial properties of Metschnikowiaceae are thought to permit yeasts to bear hostile environments through the inhibition of competing microorganisms while, in agriculture, pigment-producing strains are accurately selected to be used as natural alternatives to chemical pesticides (Savini et al. 2012). Likewise, although further study is required to more deeply understand IHEM 25107 properties, the studied strain might represent a further Metschnikowia nonpulcherima isolate showing biocontrol potential. From a biotechnological point of view, it might be a future challenge to define whether antifungal Metschnikowia strains may be used as natural treatments of mycoses in mammals; also, it is likely that pulcherrimin incorporation into selective laboratory media could represent another field of investigation in the next years. It is therefore indispensable that methods to test isolates’ antimicrobial activity be standardized. From a biological perspective, finally, we could suppose that strain IHEM 25107 intrinsical activity might have permitted the yeast, by antagonizing resident microorganisms, to survive on the mucosa from which it was cultivated. Future investigations will shed more light on life cycle of Metschnikowiaceae, along with the ecologic importance of
Folia Microbiol
pulcherrimin, both in the natural environment and in the human host. Acknowledgments Authors are grateful to Prof. Marc-André Lachance for characterizing the studied pigment as pulcherrimin.
References Bergman L W (2001) Growth and maintenance of yeast. In: P N. MacDonald (eds). Methods in molecular biology. Two-hybrid systems: methods and protocols, vol. 177. Humana Press Inc.: Totowa, NJ. Giménez-Jurado G, Valderrama MJ, Sá-Nogueira I, Spencer-Martins I (1995) Assessment of phenotypic and genetic diversity in the yeast genus Metschnikowia. Antonie Van Leeuwenhoek 68:101–110 Hazen KC (1995) New and emerging yeast pathogens. Clin Microbiol Rev 8:462–478 Jorgensen JH, White T, Pfaller MA (1999) Antifungal agents and susceptibility tests. In: Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yolken RH (eds) Manual of clinical microbiology. ASM Press, Washington DC, pp 1526–1543 Karabulut OA, Smilanick JL, Gabler FM, Mansour M, Droby S (2003) Near-harvest applications of Metschnikowia fructicola, ethanol, and sodium bicarbonate to control postharvest diseases of grape in central California. Plant Dis 87:1384–1389 Kluyver AJ, van der Walt JP, van Triet AJ (1953) Pulcherrimin, the pigment of Candida pulcherrima. Proc Natl Acad Sci U S A 39:583–593 Krutzman CP, Droby S (2001) Metschnikowia fructicola, a new ascosporic yeast with potential for biocontrol of postharvest fruit rots. Syst Appl Microbiol 24:395–399 Leverentz B, Conway WS, Janisiewicz W, Abadias M, Kurtzman CP, Camp MJ (2006) Biocontrol of the food-borne pathogens Listeria monocytogenes and Salmonella enterica serovar Poona on fresh-cut apples with naturally occurring bacterial and yeast antagonists. Appl Environ Microbiol 72:1135–1140 Molnár O, Prillinger H (2005) Analysis of yeast isolates related to Metschnikowia pulcherrima using the partial sequences of the large subunit rDNA and the actin gene; description of Metschnikowia andauensis sp. nov. Syst Appl Microbiol 28:717–726
Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yolken RH (1999) Manual of clinical microbiology, 7th edition. American Society for Microbiology: Washington, D.C. National Committee for Clinical Laboratory Standards (NCCLS) (1997) Reference method for broth dilution antifungal susceptibility testing of filamentous fungi. Approved standard M38-A. National Committee for Clinical Laboratory Standards: Wayne, PA. National Committee for Clinical Laboratory Standards (NCCLS) (2002) Reference method for broth dilution antifungal susceptibility testing of yeast. Approved standard M27-A. National Committee for Clinical Laboratory Standards: Wayne, PA. Pitt JI, Miller MW (1968) Sporulation in Candida pulcherrima, Candida reukaufii and Chlamydozyma species: their relationships with Metschnikowia. Mycologia 60:663–685 Prodorutti D, Ferrari A, Pertot I (2006) Efficacy of Shemer (Metschnikovia fructicola) against post-harvest rot of small fruits [Trentino]. Italian Plant Protection Association. Biennial meeting, Riccione, Rimini (Italy), 27–29 Mar 2006. ISMEA 2007601926, pt. 2, p. 423–427. Savini V, Hendrickx M, Sisti M, Masciarelli G, Favaro M, Fontana C, Pitzurra L, Arzeni D, Astolfi D, Catavitello C, Polilli E, Farina C, Fazii P, D’Antonio D, Stubbe D (2012) An atypical, pigmentproducing Metschnikowia strain from a leukaemia patient. Med Mycol 51:438–443 Sipiczki M (2006) Metschnikowia strains isolated from botrytized grapes antagonize fungal and bacterial growth by iron depletion. Appl Environ Microbiol 72:6716–6724 Sipiczki M, Pfliegler WP, Holb IJ, 2013. Metschnikowia species share a pool of diverse rRNA genes differing in regions that determine hairpin-loop structures and evolve by reticulation. Plos One, e67384. Spadaro D, Vola R, Piano S, Gullino ML (2002) Mechanisms of action, efficacy and possibility of integration with chemicals of four isolates of the yeast Metschnikowia pulcherrima active against postharvest pathogens on apples. Postharvest Biol Tech 24:123–134 Türkel S, Ener B (2009) Isolation and characterization of new Metschnikowia pulcherrima strains as producers of the antimicrobial pigment pulcherrimin. Z Naturforsch 64:405–410 Xue ML, Zhang LQ, Wang QM, Zhang JS, Bai FY (2006) Metschnikowia sinensis sp. nov., Metschnikowia zizyphicola sp. nov. and Metschnikowia shanxiensis sp. nov., novel yeast species from jujube fruit. Int J Syst Evol Microbiol 56:2245–2250
Antifungal properties of the human Metschnikowia strain IHEM 25107 Maurizio Sisti & Vincenzo Savini
Received: 15 July 2013 / Accepted: 30 October 2013 # Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2013
Abstract Metschnikowiaceae may show natural, straindependent antifungal properties, so they are employed in agriculture as natural, safe alternatives to pesticides. With this paper, we documented the ability of the recently described Metschnikowia IHEM 25107 to inhibit growth of several molds, including diverse Aspergillus species and dermatophytes. This biocontrol activity enables pulcherriminproducing strains to naturally antagonize competing microorganisms. Keywords Metschnikowia . Antifungal . Biocontrol
Introduction The genus Metschnikowia belongs to the family Metschnikowiaceae that includes water and terrestrial ascomycetous fungi inhabiting plants, pests, and animals (Giménez-Jurado et al. 1995; Hazen 1995; Molnár and Prillinger 2005; Sipiczki 2006). Metschnikowia pulcherrima (anamorphic state: Candida pulcherrima) is the most studied species (Giménez-Jurado et al. 1995; Hazen 1995) and may behave as a biocontrol agent against food-borne microorganisms and fruit and vegetable pathogens responsible for postharvest decay (Krutzman and Droby 2001; Spadaro et al. 2002; Leverentz et al. 2006; Prodorutti et al. 2006). The antifungal property relies on the strain-dependent production of pulcherrimin, a reddish pigment that forms a M. Sisti (*) Department of Biomolecular Science, Section of Hygiene, University of Urbino Carlo Bo, Via S. Chiara, 27, 61029 Urbino, PU, Italy e-mail: [email protected] V. Savini Clinical Microbiology and Virology, Spirito Santo Hospital, Pescara, PE, Italy
chelate complex with iron ions (Kluyver et al. 1953; Türkel and Ener 2009), thus immobilizing the metal in the growth medium; mutants that do not produce pulcherrimin lack in fact the antimicrobial activity (Sipiczki 2006; Türkel and Ener 2009). M. pulcherrima was the first terrestrial member of the genus to be described (Pitt and Miller 1968); also, it was until recently the only Metschnikowia species cultivated from man (Hazen 1995). In 2012, however, we described a human, pigment-producing Metschnikowia non-pulcherrima strain (Savini et al. 2012), potential biocontrol property of which is investigated in the present work. Particularly, we studied the strain’s activity against several molds, including dermatophytes and Aspergillus species and, based on results we obtained, we suggest this novel isolate might be employed in agriculture to antagonize postharvest pathogens (Krutzman and Droby 2001; Prodorutti et al. 2006). Moreover, we would like to emphasize that pigment production could enable producing strains, like IHEM 25107, to survive in the environment as well as in the human host. The analyses performed in Savini et al. (2012) placed strain IHEM 25107 into an uncertain taxonomic position. Furthermore, classification of Metschnikowiaceae is still under definition (Sipiczki et al. 2013) and, although genomebased results indicated that the studied yeast belonged to the phylogenetic group of M. pulcherrima , it did not match, genetically, with any previously described species; conversely, physiological and metabolic properties were those of Metschnikowia sinensis that, however, has never been reported to produce pigment (Savini et al. 2012; Xue et al. 2006). We tested strain IHEM 25107 antagonism against six dermatophyte isolates (Epidermophyton floccosum, Tricophyton mentagrophytes , Tricophyton rubrum , Tricophyton shoenleinii , Tricophyton tonsurans , and Tricophyton violaceum ) collected in our laboratories from human onychomycosis cases and skin infections and eight clinical
Folia Microbiol
and environmental molds belonging to the genus Aspergillus, including Aspergillus flavipes, Aspergillus flavus, Aspergillus fumigatus , Aspergillus nidulans , Aspergillus niger , Aspergillus terreus , Aspergillus ustus , and Aspergillus versicolor.
Materials and methods Isolation of molds and of strain IHEM 25107 Dermatophytes were isolated from human onychomycosis cases and skin infections while Aspergillus strains were collected from clinical and environmental samples. IHEM 25107 was isolated as a putative commensal from the sputum of a leukemia patient, as described in Savini et al. (2012). Mold identification was performed according to standard methods (Murray et al. 1999). Filamentous fungi and strain IHEM 25107 were grown on potato dextrose agar (PDA) and Sabouraud dextrose agar, respectively, at 35 °C and subcultured twice to ensure purity and viability.
strain IHEM 25107 were confirmed by inoculating serial suspension dilutions on PDA plates.
Antagonistic activity test IHEM 25107 antifungal activity was tested through a disk diffusion method (Jorgensen et al. 1999). For this purpose, the PDA Petri dish (60 mm of diameter) was previously seeded with 100 μL (approximately 105 CFU per plate) of conidial inoculum suspension. Sterile filter paper disks (6.3 mm in diameter) were positioned at the plate center; they were previously dried, then impregnated with 10 μL (approximately 104 CFU per disk) of strain IHEM 25107 inoculum suspension. Plates were incubated at 30 °C for 72 h and antifungal activities evaluated manually by measuring inhibition zone (IZ) diameters; IZ sizes (expressed in millimeters) were calculated from strain IHEM 25107 colony edge to that of fungal lawn. Experiments were repeated independently three times for each strain.
Pigment production
Results
Strain IHEM 25107-related pigment was confirmed to be pulcherrimin by growing a lawn of the yeast on yeast nitrogen base (Bergman 2001) agar with 1 % glucose with a steep concentration gradient of ferric citrate. Growth was inhibited at the highest concentration and the pigment was produced abundantly in a zone corresponding to nontoxic iron levels. The remaining growth was free of pigmentation.
Figure 1 shows strain IHEM25107 antagonistic activity versus T. mentagrophytes, while Figs. 2 and 3 report IZ diameters produced by the test inoculum. Notably, the highest value was found to be 5 mm and was obtained with A. nidulans, A. ustus, T. mentagrophytes, T. shoenleinii , and T. violaceum ; a 4 mm diameter was instead achieved with A. flavus , A. versicolor, and T. tonsurans. Again, a 2 mm IZ was observed with A. fumigatus, A. rubrum, and A. terreus while, for A. flavipes , growth inhibition was revealed by a 1 mm-IZ. Finally, only A. niger and E. floccosum displayed a complete resistance, as indicated by the absence of any IZs.
Preparation of conidial and yeast inoculums suspension Conidial inoculum suspensions were prepared by flooding each culture slant with 1 mL sterile 0.85 % NaCl solution containing 0.05 % Tween 80 and gently probed with a pipette tip. The resulting mixture was withdrawn and the heavy particles allowed to settle for 3–5 min. The upper homogeneous suspensions containing the mixture of conidia and hyphal fragments were vortexed for 15 s. The transmittances of mixture suspensions were adjusted according to the CLSI M38-A protocol (NCCLS 2002) using a spectrophotometer set at a wavelength of 530 nm to provide a final test inoculum of about 106 CFU/mL. Strain IHEM 25107 inoculum suspensions were prepared by selecting five colonies of 1 mm diameter (at least), from a 24-h culture and suspending them in 5 mL of sterile 0.85 % NaCl solution. Cell suspension turbidity was adjusted according to the CLSI M27-A protocol (NCCLS 1997) using a spectrophotometer set at a wavelength of 530 nm to provide a final test inoculum of 106 CFU/mL. The inoculum titres of dermatophytes, Aspergillus spp. and
Fig. 1 Strain IHEM25107 antagonism versus T. mentagrophytes; the reddish pigment is visible at the IZ edge
Folia Microbiol Fig. 2 Strain IHEM25107 antagonism versus dermatophytes; results are means of three independent experiments that agree within 10 %
Discussion Metschnikowiaceae may display a natural, strain-dependent antimicrobial activity related to pulcherrimin production; particularly, the pigment binds iron ions in the growth medium, thus inhibiting bacterial and fungal survival as well as spore germination (Kluyver et al. 1953; Sipiczki 2006). Biocontrol properties exerted by Metschnikowia non-pulcherrima yeasts are poorly reported in the literature, and only include those presented in this work along with previously published observations on M. fructicola (Prodorutti et al. 2006; Karabulut et al. 2003). In this context, our results show that strain IHEM 25107 antagonizes the growth of almost all the studied molds; A. nidulans, A. ustus, T. mentagrophytes, T. shoenleinii, and T. violaceum appeared to be the most susceptible fungi among those tested; conversely, growth of E. floccosum and A. niger has not been inhibited. Lack of A. niger susceptibility is in agreement with Sipiczki’s findings (although these were obtained with M. pulcherrima; Sipiczki 2006), but not with those by Türkel (Türkel and Ener 2009); given the absence of standardized methodologies for testing, however, it is hard to compare results.
Fig. 3 IHEM25107 antagonism versus Aspergillus spp; results are means of three independent experiments that agree within 10 %
In nature, antimicrobial properties of Metschnikowiaceae are thought to permit yeasts to bear hostile environments through the inhibition of competing microorganisms while, in agriculture, pigment-producing strains are accurately selected to be used as natural alternatives to chemical pesticides (Savini et al. 2012). Likewise, although further study is required to more deeply understand IHEM 25107 properties, the studied strain might represent a further Metschnikowia nonpulcherima isolate showing biocontrol potential. From a biotechnological point of view, it might be a future challenge to define whether antifungal Metschnikowia strains may be used as natural treatments of mycoses in mammals; also, it is likely that pulcherrimin incorporation into selective laboratory media could represent another field of investigation in the next years. It is therefore indispensable that methods to test isolates’ antimicrobial activity be standardized. From a biological perspective, finally, we could suppose that strain IHEM 25107 intrinsical activity might have permitted the yeast, by antagonizing resident microorganisms, to survive on the mucosa from which it was cultivated. Future investigations will shed more light on life cycle of Metschnikowiaceae, along with the ecologic importance of
Folia Microbiol
pulcherrimin, both in the natural environment and in the human host. Acknowledgments Authors are grateful to Prof. Marc-André Lachance for characterizing the studied pigment as pulcherrimin.
References Bergman L W (2001) Growth and maintenance of yeast. In: P N. MacDonald (eds). Methods in molecular biology. Two-hybrid systems: methods and protocols, vol. 177. Humana Press Inc.: Totowa, NJ. Giménez-Jurado G, Valderrama MJ, Sá-Nogueira I, Spencer-Martins I (1995) Assessment of phenotypic and genetic diversity in the yeast genus Metschnikowia. Antonie Van Leeuwenhoek 68:101–110 Hazen KC (1995) New and emerging yeast pathogens. Clin Microbiol Rev 8:462–478 Jorgensen JH, White T, Pfaller MA (1999) Antifungal agents and susceptibility tests. In: Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yolken RH (eds) Manual of clinical microbiology. ASM Press, Washington DC, pp 1526–1543 Karabulut OA, Smilanick JL, Gabler FM, Mansour M, Droby S (2003) Near-harvest applications of Metschnikowia fructicola, ethanol, and sodium bicarbonate to control postharvest diseases of grape in central California. Plant Dis 87:1384–1389 Kluyver AJ, van der Walt JP, van Triet AJ (1953) Pulcherrimin, the pigment of Candida pulcherrima. Proc Natl Acad Sci U S A 39:583–593 Krutzman CP, Droby S (2001) Metschnikowia fructicola, a new ascosporic yeast with potential for biocontrol of postharvest fruit rots. Syst Appl Microbiol 24:395–399 Leverentz B, Conway WS, Janisiewicz W, Abadias M, Kurtzman CP, Camp MJ (2006) Biocontrol of the food-borne pathogens Listeria monocytogenes and Salmonella enterica serovar Poona on fresh-cut apples with naturally occurring bacterial and yeast antagonists. Appl Environ Microbiol 72:1135–1140 Molnár O, Prillinger H (2005) Analysis of yeast isolates related to Metschnikowia pulcherrima using the partial sequences of the large subunit rDNA and the actin gene; description of Metschnikowia andauensis sp. nov. Syst Appl Microbiol 28:717–726
Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yolken RH (1999) Manual of clinical microbiology, 7th edition. American Society for Microbiology: Washington, D.C. National Committee for Clinical Laboratory Standards (NCCLS) (1997) Reference method for broth dilution antifungal susceptibility testing of filamentous fungi. Approved standard M38-A. National Committee for Clinical Laboratory Standards: Wayne, PA. National Committee for Clinical Laboratory Standards (NCCLS) (2002) Reference method for broth dilution antifungal susceptibility testing of yeast. Approved standard M27-A. National Committee for Clinical Laboratory Standards: Wayne, PA. Pitt JI, Miller MW (1968) Sporulation in Candida pulcherrima, Candida reukaufii and Chlamydozyma species: their relationships with Metschnikowia. Mycologia 60:663–685 Prodorutti D, Ferrari A, Pertot I (2006) Efficacy of Shemer (Metschnikovia fructicola) against post-harvest rot of small fruits [Trentino]. Italian Plant Protection Association. Biennial meeting, Riccione, Rimini (Italy), 27–29 Mar 2006. ISMEA 2007601926, pt. 2, p. 423–427. Savini V, Hendrickx M, Sisti M, Masciarelli G, Favaro M, Fontana C, Pitzurra L, Arzeni D, Astolfi D, Catavitello C, Polilli E, Farina C, Fazii P, D’Antonio D, Stubbe D (2012) An atypical, pigmentproducing Metschnikowia strain from a leukaemia patient. Med Mycol 51:438–443 Sipiczki M (2006) Metschnikowia strains isolated from botrytized grapes antagonize fungal and bacterial growth by iron depletion. Appl Environ Microbiol 72:6716–6724 Sipiczki M, Pfliegler WP, Holb IJ, 2013. Metschnikowia species share a pool of diverse rRNA genes differing in regions that determine hairpin-loop structures and evolve by reticulation. Plos One, e67384. Spadaro D, Vola R, Piano S, Gullino ML (2002) Mechanisms of action, efficacy and possibility of integration with chemicals of four isolates of the yeast Metschnikowia pulcherrima active against postharvest pathogens on apples. Postharvest Biol Tech 24:123–134 Türkel S, Ener B (2009) Isolation and characterization of new Metschnikowia pulcherrima strains as producers of the antimicrobial pigment pulcherrimin. Z Naturforsch 64:405–410 Xue ML, Zhang LQ, Wang QM, Zhang JS, Bai FY (2006) Metschnikowia sinensis sp. nov., Metschnikowia zizyphicola sp. nov. and Metschnikowia shanxiensis sp. nov., novel yeast species from jujube fruit. Int J Syst Evol Microbiol 56:2245–2250
