Postharvest Biology and Technology 75 (2013) 1–8

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Postharvest Biology and Technology
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A new strain of Metschnikowia fructicola for postharvest control of Penicillium expansum and patulin accumulation on four cultivars of apple
Davide Spadaro ∗ , Alessia Lorè, Angelo Garibaldi, Maria Lodovica Gullino
Centre of Competence for the Innovation in the Agro-environmental Sector (AGROINNOVA), University of Turin, Via Leonardo da Vinci 44, I-10095 Grugliasco, Turin, Italy

a r t i c l e

i n f o

a b s t r a c t
The efficacy of three antagonistic yeasts, Metschnikowia pulcherrima strain MACH1, M. pulcherrima strain GS9, and Metschnikowia fructicola strain AL27, against Penicillium expansum and patulin accumulation was evaluated on apples stored at room (22 ± 1 ◦ C for 7 days) and cold temperatures (1 ± 1 ◦ C for 56 days). To increase the potential range of application of the biocontrol agents (BCAs), their efficacy was evaluated on four cultivars of apple, i.e. ‘Golden Delicious’, ‘Granny Smith’, ‘Red Chief’ and ‘Royal Gala’. AL27 was more effective than MACH1 and GS9 in the control of blue mold rot and in the reduction of patulin accumulation. The efficacy of AL27 was in most cases similar to the chemical control used, making the antagonist as competitive as chemical fungicides. In vitro experiments showed that AL27 reduced the conidial germination and germ tube length of P. expansum more than the other strains. The three BCAs were more effective in the control of blue mold rot on ‘Golden Delicious’ apples than on the other tested cultivars. © 2012 Elsevier B.V. All rights reserved.

Article history: Received 10 July 2012 Accepted 5 August 2012 Keywords: Apple Biological control Metschnikowia fructicola Mycotoxin Penicillium expansum Yeast

1. Introduction Postharvest losses of fruit and vegetables are mainly due to attacks of pathogens during harvest, storage, transport and marketing (Snowdon, 1990). Some species of Penicillium are important plant pathogens causing decay on various fruit and vegetables, through their antioxidant proteins and hydrolytic enzymes (Bertolini and Tian, 1996; Qin et al., 2007). Penicillium expansum can particularly cause blue molds and blue rots on several plant species (Stange et al., 2002). Besides its pathogenic activity, P. expansum is able to produce patulin, a highly reactive unsaturated lacton, that may cause acute and chronic toxicity, including carcinogenic, mutagenic, and teratogenic effects (Beretta et al., 2000; Hasan, 2000; McCallum et al., 2002). The mycotoxin causes impairment of kidney functions, oxidative damage, and weakness to the immune system. It also has a negative impact on reproduction in males via interaction with hormone production (Selmanoglu and Kockaya, 2004; Fuchs et al., 2008). Patulin can be found in several typologies of fruit-derived food, including apple, pear, peach and apricot juices and nectars (Spadaro et al., 2007, 2008a). The Joint FAO/WHO Expert Committee on Food Additives (JEFCA) established a provisional maximum tolerable daily intake (PMTDI) of 0.4 g kg−1 body weight (bw) day−1 , based on a no observable effect level of 43 g kg−1 bw day−1 and a

∗ Corresponding author. Tel.: +39 011 6708942; fax: +39 011 6709307. E-mail address: davide.spadaro@unito.it (D. Spadaro). 0925-5214/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.postharvbio.2012.08.001

safety factor of 100 (World Health Organization, 1995). Based on this PMTDI, patulin is regulated in the European Union at levels of 50 mg kg−1 in fruit juices and fruit nectars, 25 mg kg−1 in solid apple products, and 10 mg kg−1 in apple-based products for infants and young children (European Commission, 2006). The use of chemical fungicides is an important strategy for controlling P. expansum in harvested commodities (Eckert and Ogawa, 1990; Janisiewicz and Korsten, 2002; Zhou et al., 2002). However, during the last decades, some fungicides have lost their efficacy due to the development of resistant strains. Several studies demonstrated resistance of P. expansum to the most common fungicides used in postharvest (Sholberg et al., 2005; Errampalli et al., 2006). Moreover, concern for public safety has resulted in the cancellation of some of the most effective fungicides in Europe (European Parliament, 2009) and the United States (United States Congress, 1996; Dayan et al., 2009). Therefore, research focused on the development of alternative control that should be both effective and economically feasible. The use of microbial antagonists to control postharvest diseases of fruit and vegetables is one of the most promising alternatives to fungicides (Qin et al., 2004; Droby et al., 2009). Some components of the microbial community present on the surface of fruit and vegetables, such as bacteria and yeasts, have been shown to have significant antagonistic activity against P. expansum (Usall et al., 2001; Janisiewicz and Korsten, 2002). Different yeasts are also able to reduce the patulin level in vitro (Coelho et al., 2008; Reddy et al., 2011). Fermentative yeasts reduce patulin contamination during production of cider from

Merck) and 50 mg L−1 of streptomycin (Merck) at 4 ◦ C. 2005. Altschul et al. 2010).2 D.. used as a mixture during the experiments to ensure a high level of disease. As control. 0. 2 L of 10× buffer (Taq DNA Polymerase. After a week incubation at 22 ◦ C. each obtained from rotted apples harvested in Piedmont.0% agarose gel in TBE buffer. ‘Granny Smith’. 72 ◦ C. (1974) found that different P. expansum (PEX06. i. and the levels were not related to the virulence of the P. ‘Red Chief’ and ‘Royal Gala’. Materials and methods 2. Oensingen. 72 ◦ C. 5. 100 L of the conidial suspension (5 × 106 conidia mL−1 ) of the pathogen in Ringer’s solution was added to 5 mL of PDB. 2008b) and M. Darmstadt. conidia from the four strains were collected and resuspended in sterile Ringer’s solution. 2011 with deposit designation 30P and its uses were patented with the Italian patent application TO2011A000534. 5 g L−1 d-mannitol. Sommer et al.. Molecular and morphological identification The yeast antagonist Metschnikowia fructicola strain AL27 was identified by sequencing the internal transcribed spacer 1 (ITS1). 5 × 108 . accumulated more patulin. pulcherrima strain GS9 (Spadaro et al. expansum strains produced differing patulin levels. characterized by a lower pH. Konstantinou et al. 1990) for similarity. the apple cultivar should be considered a critical factor influencing the biocontrol of P. M. The microscope observation of the cell and colony morphology was complementary to the molecular analysis. (1990) and the D1/D2 domain at the 5 end of the LSU rRNA gene according to Kurtzman and Robnett (1998). 30 cycles: 94 ◦ C. or 5 × 109 cells mL−1 ). pulcherrima strain GS9 were previously identified (Saravanakumar et al.. Moss and Long (2002) showed that Saccharomyces cerevisiae metabolizes patulin to the less toxic E-ascladiol. and then stained with SYBR SAFE (Invitrogen. were added to the test tube. expansum was assessed in 5 mL of potato dextrose broth (PDB. 2011). and Metschnikowia fructicola strain AL27. Sharnbrook. 2. United Kingdom). Yeast cells were collected by centrifugation at 1500 rpm for 10 min. 2011..8S ribosomal RNA gene. 55 ◦ C 30 s. Switzerland) and Kingfisher magnetic particle processor (Thermo Labsystems.2. 2010). M. Lima et al. After filtering through eight layers of sterile cheese-cloth. Merck. The ITS regions were amplified using genomic DNA as a template and universal primers ITS1 (5 -TCCGTAGGTGAACCTGCGG-3 ) and ITS4 (5 TCCTCCGCTTATTGATATGC-3 ). Germany) and brought to a standard concentration of 108 cells mL−1 by direct counting with a hemacytometer.e. United Kingdom) following the manufacturers’ protocols.. M. on conidial germination and on germ tube length of P. At room temperatures. pulcherrima strain GS9 (Spadaro et al. PEX12. Spadaro et al. PCRs were performed using a TGradient thermal cycler (Biometra. 2010). Another study showed that patulin accumulation was significantly higher in ‘Golden Delicious’ and ‘Red Delicious’ than in ‘Granny Smith’ and ‘Fuji’ apples. and internal transcribed spacer 2 (ITS2) according to White et al. 1973). and selected for their virulence (Reddy et al. expansum strain may be another important factor in its pathogenicity and in its ability to synthesize patulin in the fruit (Neri et al. Morales et al. 5 g L−1 l-sorbose. Italy) using an ABI PRISM 3730XL DNA Sequencer (AME Bioscience. Each strain belongs to the AGROINNOVA collection and were stored in tubes with potato dextrose agar (PDA. varieties of apple with higher amounts of organic acids. 45 s. CA.. Hercules. Chatsworth. Northern Italy. Basingstoke.. 4 ◦ C. Beretta et al. their efficacy was evaluated on four cultivars of apple. USA).. expansum and patulin accumulation in apples stored at room and cold temperatures. were .. 2010). 2011).3. conidia were counted and brought to a final concentration of 105 mL−1 . Conidial suspensions used for fruit inoculation were prepared by growing the pathogens on Petri dishes on PDA containing 50 mg L−1 of streptomycin.. PCR amplification products were cloned into the PCR4 TOPO vector (Invitrogen) using the TOPO TA cloning kit following the manufacturer protocol and sequenced by BMR Genomics (Padova. since very high levels were sometimes detected in fruit with small rots. were more prone to patulin accumulation at 1 ◦ C. 2008b). coming from antagonist cell suspensions grown in YPD for 48 h. (2000) similarly found that the patulin content in apples was not always related to the diameter of the rotten areas. The microorganism culture was stored at −20 ◦ C in cell suspension with 65% (v/v) glycerol and 35% (v/v) of a solution of 100 mM MgSO4 and 25 mM Tris (pH 8.0 U Taq DNA Polymerase (Qiagen). fructicola strain AL27 were isolated from the carposphere of ‘Golden Delicious’ apples harvested in unsprayed orchards located in Northern Italy. 2011). Living cells of each antagonistic yeast (100 L of a suspension containing 5 × 107 . / Postharvest Biology and Technology 75 (2013) 1–8 apple juice (Harwig et al. Antagonism in vitro The effect of the isolates of Metschnikowia spp. ‘Golden Delicious’. A 10 L aliquot of PCR products from each reaction was electrophoresed in 2. The resultant suspensions were shaken using a vortex mixer for 30 s before inoculation. 2008). 2. expansum strains (Neri et al. 2008). 34 cycles: 94 ◦ C. 2008. 5 × 106 conidia mL−1 ) of P. such as ‘Golden Delicious’ and ‘Fuji’. 4 ◦ C. CA. Eugene.. 7 min. 2008b). Inocula of the antagonists for all experiments were prepared by subculturing in 250 mL Erlenmeyer flasks containing 75 mL YEMS and incubated on a rotary shaker (100 rpm) at 22 ◦ C for 48 h. Therefore.. was extracted using NucleoMag 96 Plant Kit (Macherey Nagel. 45 s. 55 s.. Microorganisms M.. PCR program for D1/D2 domain was: 95 ◦ C. pulcherrima strain MACH1 and M. After 2. 3 min. 55 ◦ C. 72 ◦ C. in the control of P. deposited on June 20. Morales et al. The sequences were analyzed by using the software BLASTn (Basic Local Alignment Search Tool. PEX25 and PEX27).1. Göttingen. Spadaro et al. Spadaro et al. To increase the potential range of application of the biocontrol agents (BCAs). expansum strain PEX06 was added to a 10 mL test tube.. Several studies have revealed that fruit cultivars may differ in their susceptibility to blue mold rots and to patulin accumulation (Neri et al. A conidial suspension (100 L. Qiagen.. pulcherrima strain MACH1 (Saravanakumar et al. 2010. PCR program for ITS regions was: 95 ◦ C..0).7 mM each primer. The DNA. The specific P. Reddy et al. and 1. whereas there are few studies on the effect of biological control yeasts on patulin accumulation in stored pome fruit (Castoria et al. Germany). The strains were grown in YEMS (30 g L−1 yeast extract. 72 ◦ C.9 + 0. 200 mM of each deoxynucleotide triphosphate. washed and resuspended in sterilized Ringer solution (pH 6. Gel images were acquired with a Gel Doc 1000 System (Bio-Rad Laboratories. expansum and its patulin accumulation on fruit. The D1/D2 domains were amplified using the primers NL-1 (5 -GCATATCAATAAGCGGAGGAAAAG-3 ) and NL-4 (5 -GGTCCGTGTTTCAAGACGG-3 ) on the genomic DNA. 10 min. 2010. Four isolates of P. 2008a. due to the lower acidity of the fruit (Konstantinou et al. USA). USA). OR. 30 s.. The aims of the present study were to evaluate the efficacy of three antagonistic yeasts Metschnikowia pulcherrima strain MACH1 (Saravanakumar et al. (2008b) found that the pH value of the apple varieties was a determining factor in patulin accumulation only under cold storage: ‘Golden Delicious’ apples. Each 20 L PCR contained 1 L of DNA template (50 ng). 15 s. Merck).. 7 min. The strain AL27 was deposited within the Industrial Yeasts Collection (DBVPG) on March 29.1.

i. 50 and 100 ng g−1 ).66 × 3. The sequences of the amplified regions were deposited in GenBank. Colonies are milky white. Molecular and morphological identification The strain AL27 was identified by sequencing the ribosomal regions ITS1–5. Belpasso. The probe descended towards the sample at 1. MO.9%. The analysis of the D1/D2 domain (accession number HQ682195. Each test was performed three times and the mean recovery values were respectively 90. Phenomenex® . Patulin analysis Patulin was extracted from rot caused by P. 5 U/g of juice) and 10 mL of water were added. For the mycotoxin experiments. USA. were disinfected in sodium hypochlorite (NaClO. The organic phase was dehydrated with 25 g of sodium sulphate anhydrous and then evaporated to dryness (Rotavapor Laborota 4000. harvested in an Italian orchard grown according to integrated pest management practices.. Twenty fruit per replication were used (60 inoculation sites). The procedure was repeated three times. using SPSS-WIN software (17.05 was considered significant. Total soluble solids (TSS) were determined by measuring the refractive index of pressed juice (Larrigaudière et al. The high value of the regression coefficient (R2 ≥ 0.0% to 6.6. The mobile phase. pulcherrima strain MACH1.2. cells are ovoid and they measure 1. patulin was extracted with 30 mL of ethyl acetate shaking for 1 min. 2002) with a digital refractometer (DBR95. G1316A column thermostat set at 30 ◦ C. Synergy Hydro-RP C18. The experiments were carried out twice. Italy).4. imazalil 17. Waldbronn. The mixture was left at 40 ◦ C for 2 h and then centrifuged at 4500 rpm for 5 min. Results 3. G1315B UV diode array detector set at 276 nm.d.. Twenty grams of sample were placed in a centrifuge tube to which 20 drops of pectinase enzyme solution (Sigma Chemical Co.0) and transferred into a HPLC vial. the analyses were carried out in triplicate and the values represented the mean values.i. The treatments were replicated three times. 2. The statistical analysis was performed by one-way analysis of variance (ANOVA).8S–ITS2 with universal primers ITS-1 and ITS-4 and sequencing the D1/D2 domain with the primers NL-1 and NL-4. fructicola strain AL27.04 and 1.0% as chlorine) and rinsed under tap water. data from at least two experimental trials were pooled. Antagonism in vitro The effect of M. Torrance. Decco Italia srl. Germany). 98.6 mm i. G1313A autosampler. 3. The amount of patulin in the final solution was determined by using a calibration graph of concentration versus peak area and expressed as ng/mL. Statistical analysis For the efficacy experiments. dried at room temperature and punctured with a sterile needle at the equatorial region (3 mm depth.2.0). Italy.54 × 7. The severity of the diseases was determined by measuring the mean lesion diameter on the rotted apples and the percentage of rot (fresh weight of rot/fresh weight of fruit).800 mL/min. 437/444).). pulcherrima strain GS9 and M. The standard solutions had concentrations of 500 ng mL−1 . Alfonsine. pulcherrima strain MACH1 was evaluated on conidial germination and germ tube length of P. Acidity was measured by titration with 0. 1. G1311 quaternary pump and Agilent Chemstation G2170AA Windows XP operating system (Agilent® . CA.9%.D. Schwaback. St Louis. The recovery was determined on a blank apple puree spiked at three concentrations of patulin (10. pyrimethanil 17.2% for duplicate analyses.. The HPLC apparatus was an Agilent 1100 series equipped with G1379 degasser.) with the same stationary phase was used.5.2% a. M. The BLAST analysis of the ITS sequence (accession number HQ682194. eluting at a flow rate of 0.1. fructicola strain AL27 per wound. achieved by injection onto the HPLC column of 100 L of standard solutions of patulin (Sigma Chemical Co. 4 m. Germany). Spadaro et al. p < 0.99) obtained indicated a good linearity of the analytical response.2% a. A stainless steel analytical column (250 × 4. USA). The observation of the morphological (colony morphology) and microscopic (cell shape and size) characteristics of AL27 confirmed the rDNA sequencing results. expansum (105 mL−1 ) were pipetted into the apple wounds. were respectively 1. The limit of detection (LOD) and the limit of quantification (LOQ)..0% of the conidia germinated and the average germ tube . and Duncan’s multiple range test was employed. Singapore) and the results were expressed as percentages (g/100 g fruit weight).02 Patulin in Clear and Cloudy Apple Juices and Apple Puree. based on the IUPAC definition (Thompson et al.. Some quality parameters were assessed on healthy fruit of every cultivar. Each treatment was replicated three times. 100 conidia per replicate were observed microscopically and their germination was evaluated. 3 wounds per fruit). amplimer size: 251 bp) showed that the amplicon of AL27 showed 99% (249/251) identity with the sequences of Metschnikowia fructicola. ‘Granny Smith’. The repeatability ranged from 1. St Louis. while the identity with strains of M. pulcherrima was lower (98%. consisted of an isocratic mixture of water–acetonitrile–perchloric acid (95:4:1) for 20 min. Titratable acidity was calculated as percent malic acid (Wright and Kader. Apples were randomly packed in commercial plastic trays and stored either at 22 ± 1 ◦ C for 7 days or at 1 ± 1 ◦ C for 56 days.. ‘Red Chief’ and ‘Royal Gala’. 2.25 mL L−1 water) of imazalil and pyrimethanil (Philabuster 400SC® . 250 ng mL−1 . 2. The experiment was carried out twice. One hundred microliters of sample was injected onto the HPLC column and the retention time of patulin was about 15 min. MO.9% and 100.0: 5 mL of pressed juice diluted with 5 mL of distilled water were evaluated. Fruit were exposed to treatments with 10 L of the cell suspension (108 mL−1 ) of M. expansum (Table 1). / Postharvest Biology and Technology 75 (2013) 1–8 3 12 h incubation of the 45◦ sloping tubes at 22 ± 1 ◦ C on a rotary shaker (100 rpm). 400 ng mL−1 . The residual was resumed with 2 mL of acidic water (pH 4. Ten milliliters of clear juice was placed into 100 mL separating funnel. 100 ng mL−1 and 50 ng mL−1 of patulin.d. expansum on apples treated and stored at 22 ◦ C and at 1 ◦ C.21 m. Heidolph® . 91.1 N NaOH to pH 8. amplimer size: 448 bp) confirmed that the PCR product of AL27 had 99% (447/448) identity with the sequences of Metschnikowia fructicola. 2002). An inoculated control was also performed: after 3 h at room temperature. 3. USA) preceded by a guard column (4 × 3 mm i. Efficacy on four cultivars of apples Apples (Malus × domestica). Firmness was measured on each fruit at two opposite sites along the equatorial region with a FT327 – Fruit Pressure Tester with an 11 mm probe (EFFEGI. ‘Golden Delicious’. Chemical treatment consisted in the application into each inoculated wound (10 L) of a suspension (1. 1997).30 to 2. 10 L of the conidial suspension mixture of P. The extraction procedure used was modified by AOAC Official Method 2000.0 mm/s and the maximum force (N) was defined as firmness. strain GS9 or M.57 ng g−1 . 3–4 mm wide. followed by a washing step with an isocratic mixture of water–acetonitrile (35:65). In the control. The organic layer was separated from the water layer.

and ‘Royal Gala’ apples. respectively 78. The average values of some quality parameters have been measured on the apples before storage (Table 2). 2). than when applied at 107 or 106 cells mL−1 . Chemical treatment consisted in the application into each inoculated wound (10 L) of a suspension (1. MACH1 and GS9 showed a higher control of the rot lesion diameter on ‘Golden Delicious’.0% on ‘Golden Delicious’. Italy. ‘Granny Smith’. the three biocontrol agents (BCAs) were able to significantly reduce the blue mold rot diameter and weight compared to the control. Treatment Penicillium expansum Conidial germination (%)a AL27 1 × 106 cfu mL−1 AL27 1 × 107 cfu mL−1 AL27 1 × 108 cfu mL−1 GS9 1 × 106 cfu mL−1 GS9 1 × 107 cfu mL−1 GS9 1 × 108 cfu mL−1 MACH1 1 × 106 cfu mL−1 MACH1 1 × 107 cfu mL−1 MACH1 1 × 108 cfu mL−1 Control 16. pulcherrima strain GS9 and M.3 c 98. followed by 108 cells mL−1 of MACH1 (11.05). The smallest germ tube length (2.8% on ‘Granny Smith’.7 d 29.i. AL27 was more effective than the other two microorganisms in reducing the conidial germination at each of the three concentrations tested. The rot weight was only 0.3 m) was observed in presence of 108 cells mL−1 of AL27. Spadaro et al.9 c 11.7 cd 20. The highest germinations.5 m) and GS9 (31. ‘Red Chief’. The values of firmness did not differ among the cultivars. In particular. GS9 and MACH1.3. when co-cultivated with AL27 at 108 and 107 cells mL−1 . 1). AL27 was the most effective antagonist and provided an efficacy in reducing the rot diameter statistically similar to imazalil + pyrimethanil on ‘Golden Delicious’.7% respectively. ‘Royal Gala’ and ‘Red Chief’ (Fig.3% on ‘Red Chief’ and 3. and ‘Royal Gala’. the three BCAs significantly reduced the blue mold lesion diameter.0 f 25. expansum at each concentration tested. AL27.7 a 5. AL27 reduced more than the other two BCAs the rot weight and its effect was similar to the application of imazalil + pyrimethanyl.7 b 8. M. . On the other hand.3 f 56. the germination rates were only 5. Values of the same cultivar and the same storage trial.2% a.i. The three microorganisms were able to significantly reduce the germination rate and the germ tube length of P.0% and 69. Again. 1.2% a. fructicola strain AL27 and stored at 22 ± 1 ◦ C for 7 days (dark grey) or at 1 ± 1 ◦ C for 56 days (light grey). In the trials carried out at 22 ± 1 ◦ C for 7 days. expansum. were observed when co-cultivating with GS9 at 106 and 107 cells mL−1 . Efficacy on four cultivars of apples The efficacy of the antagonist yeasts was evaluated on ‘Golden Delicious’.0 g 69.1 g 3. Fig.3 a 31. Decco Italia srl.7 e 58.2 m). Blue mold rot was evaluated as rot diameter (Fig. length was 96. stored at room (22 ± 1 ◦ C for 7 days) and low temperature (1 ± 1 ◦ C for 56 days).0% and 8.4 D.2 b 96.7 e 40.0 e 18. When apples were stored at 1 ± 1 ◦ C for 56 days.9% on ‘Royal Gala’ apples. Among the cultivars tested. imazalil 17. total soluble solids and titratable acidity were significantly different among the a Values in the same column followed by the same letter are not statistically different by Duncan’s Multiple Range Test (p < 0. but AL27 was the most effective in reducing the rot diameter on all the apple cultivars (Fig. Philabuster 400SC® . ‘Granny Smith’. On the ‘Granny Smith’ apples. The germ tubes were longer when reducing the concentration of the yeast cells.25 mL L−1 water) of imazalil and pyrimethanil.5 f 23. 2).3%. Belpasso. 2).0 h Germ tube length ( m)a 32. Blue mold rot diameter (mm) caused by Penicillium expansum on apples ‘Golden Delicious’.0 m). treated with Metschnikowia pulcherrima strain MACH1.9 d 16. AL27 was as effective as the chemical in reducing the rot weight (Fig. are not statistically different by Duncan’s Multiple Range Test (p < 0. 1). The mixture of imazalil and pyrimethanil was chosen as a chemical control because it is registered in several European countries for use against postharvest rots on apple.0 a 78. 1. / Postharvest Biology and Technology 75 (2013) 1–8 Table 1 Conidial germination (%) and germ tube length ( m) of Penicillium expansum cocultivated with a cell suspension of antagonistic yeast in PDB at 22 ± 1 ◦ C for 12 h. pyrimethanil 17.05). 1) and as percentage of rot weight (Fig. By considering the reduction of the rot weight (Fig. followed by the same letter. Each microorganism showed a higher inhibition capability when co-cultivated at the highest concentration (108 cells mL−1 ). 2.1 m. all the BCAs were effective against P. Its efficacy was statistically similar to the chemical control and higher than the other two antagonists. Longer germ tubes where observed in presence of 106 cells mL−1 of AL27 (32. ‘Red Chief’.7 e 2..

0 ng g−1 at 22 ◦ C and 67.. ‘Granny Smith’.2 ng g−1 on ‘Granny Smith’.5 ng g−1 on ‘Royal Gala’). The patulin level observed in the apples treated with AL27 was similar to the level of the chemical control on ‘Golden Delicious’. / Postharvest Biology and Technology 75 (2013) 1–8 5 Fig.7 b Firmness (N)* 70. stored either at 1 ± 1 ◦ C for 56 days or at 22 ± 1 ◦ C for 7 days. several isolates belonging to the yeast genus Metschnikowia were isolated from different sources and selected for their efficacy against Table 2 Average values of quality parameters on apples ‘Golden Delicious’. because they rapidly colonize and survive on fruit surfaces for long periods of time under different conditions. and ‘Royal Gala’ used during the trials of storage at 22 ± 1 ◦ C for 7 days or at 1 ± 1 ◦ C for 56 days. AL27 was the most effective BCA on all the apple cultivars. fructicola strain AL27 and stored at 22 ± 1 ◦ C for 7 days (dark grey) or at 1 ± 1 ◦ C for 56 days (light grey).5 a 8. Patulin reduction The patulin produced was significantly lower in the trials performed at low temperature compared to the experiments carried out at 22 ± 1 ◦ C for 7 days.0 ng g−1 on ‘Golden Delicious’.4 ng g−1 at 22 ◦ C and 16. 4.6 ng g−1 at 1 ◦ C).3 a Titratable acidity (g malic acid/100 mL* ) 0.235 0. “±” stands for standard error of the means. 29. 24. 4. Values followed by the same letter are not statistically different by Duncan’s Multiple Range Test (p < 0.5 68.5 c 0. 2.348 ± ± ± ± 0. The higher total soluble solids in ‘Golden Delicious’ apples could be related to a higher efficacy of the BCAs. See Fig. 2009).05). than on apples treated with imazalil + pyrimethanil (0. Discussion Biocontrol agents can be applied as an alternative to fungicides to prevent and control postharvest diseases. while the highest titratable acidity was observed in ‘Granny Smith’ apples.027 b 0. was the patulin level in the fruit treated with AL27 (78. of apples. Yeasts are suitable biocontrol agents against postharvest diseases. expansum.3 12.8 11. The highest concentrations of patulin were observed in the fruit treated with GS9. when the fruit where kept at 1 ± 1 ◦ C for 56 days. 1. 3. and ‘Royal Gala’. ‘Granny Smith’. In particular. ‘Granny Smith’ and ‘Royal Gala’. Apple cultivar Golden Delicious Granny Smith Red Chief Royal Gala Total soluble solids (%)* 14. ‘Golden Delicious’ apples had higher total soluble solids contents (14.0 ng g−1 on ‘Royal Gala’).1 ng g−1 at 1 ◦ C) higher than the level in the chemical control (56. . and in particular P.2 ng g−1 on ‘Granny Smith’. In particular. Blue mold rot percentage (fresh weight) caused by Penicillium expansum on apples ‘Golden Delicious’. M.811 0. Only on ‘Red Chief’.4 a 9. except for the cv Red Chief. 3). use available nutrients to proliferate rapidly.6 74. where patulin content was significantly higher on the apples stored at 1 ± 1 ◦ C for 56 days (Fig.026 c * The results are the means of two independent experiments.6 75. In general. limit nutrient availability to the pathogen and generally are unaffected by fungicides used commercially (Droby et al.2 ± ± ± ± 0. treated with Metschnikowia pulcherrima strain MACH1.5%).7 ng g−1 on ‘Golden Delicious’.1 a 7.013 d 0.5 ± ± ± ± 8.4. pulcherrima strain GS9 and M. ‘Red Chief’. Previously.6 c 0. the patulin level was lower on apples treated with AL27 (0.034 a 0. Spadaro et al.509 0. cultivars. the three antagonists were able to significantly reduce the patulin content compared to the control.5 10.8 a 0. ‘Red Chief’.D. 1. which was also the least effective antagonist.

because one of the main mechanisms of action exploited by yeast strains is .. due to their genetic background (Spadaro et al. which is a mixture of two active ingredients commercially available in several European markets. In the current research a new yeast strain. fructicola strain AL27 and stored at 22 ± 1 ◦ C for 7 days (dark grey) or at 1 ± 1 ◦ C for 56 days (light grey). but its biocontrol capability was lower against blue mold rot (Saravanakumar et al. resulting in a higher aggressiveness (Morales et al. was isolated from the surface of ‘Golden Delicious’ apples. particularly chitinases (Saravanakumar et al.. See Fig. named MACH1. The efficacy of AL27 was in most cases similar to the chemical control used. showing lower efficacy compared to MACH1. as previously demonstrated for other antagonistic microorganisms (Hofstein et al. A.. 2002). AL27 was more effective than MACH1 and GS9 in the control of blue mold rot. in recent years. was selected for its efficacy against P. expansum (Spadaro et al. Janisiewicz et al. the capacity to reduce the patulin accumulation on apple was considered as an important feature for the antagonist selection.. indicating that the mycotoxin was not absorbed but completely biodegraded (Reddy et al. Spadaro et al... patulin was not detected in the growth medium nor in the yeast cell wall. pulcherrima and M. 2008b).6 D. performed at 22 ± 1 ◦ C for 7 days and at storage temperature. named GS9. this is the first report describing the efficacy of M. grapes. named AL27. M. pulcherrima strain GS9 and M. was previously isolated from a ‘Golden Delicious’ apple and evaluated for its biocontrol against B. and a higher inhibition was obtained in presence of higher concentrations of antagonist cells. alternata and P. expansum. fructicola effectively reduced the development of postharvest rots of grapes and strawberries (Kurtzman and Droby.. and selected for its efficacy against B. Moreover. 2008a). 2009).. where different strains of the same yeast species showed different biocontrol capabilities. 2010).Generally. Another strain of M. During shelf life. expansum more than the other strains.The in vitro experiments showed that AL27 reduced the conidial germination and germ tube length of P. The yeast cell concentration was an important factor in determining the inhibition. The yeast strain was identified as M.. Spadaro et al. One strain of M. P.. The higher total soluble solids in ‘Golden Delicious’ could be related to a higher efficacy of the BCAs. 2004).. Patulin concentration (ng g−1 ) produced by Penicillium expansum on apples ‘Golden Delicious’. / Postharvest Biology and Technology 75 (2013) 1–8 Fig. pulcherrima. showed high efficacy as a BCA against postharvest decays of apples. expansum on four apple cultivars. Karabulut et al. ‘Red Chief’. either yeast or bacteria (Morales et al. The strain showed a good efficacy against grey mold and alternaria rot. 2000. The same strain was evaluated for its capability to biodegrade patulin when grown in vitro: after 48 h growth of the yeast. These results are in agreement with previous studies.. To the best of our knowledge. and ‘Royal Gala’. cinerea. the efficacy of the three biocontrol agents was higher when the fruit were stored at 1 ◦ C than at 22 ◦ C. 2011). Also M. 2010). M. ‘Golden Delicious’ apples had higher total soluble solids contents. expansum may take advantage of the optimal conditions of growth and increase the growth rate. 3. Previous studies showed that low storage temperatures resulted in a higher efficacy of the antagonists. postharvest diseases (Zhang et al. pulcherrima.. pulcherrima MACH1 was significantly higher on apples stored at 1 ± 1 ◦ C for 56 days.. 2001. In particular. pulcherrima. 1994). 2001. either when the lesion diameter or the rot weight was considered as parameters. The results obtained in vitro were confirmed by the results of the trials on fruit. the efficacy of M. 2003. cinerea and P.The three BCAs were more effective in the control of blue mold rot on ‘Golden Delicious’ apples than on the other cultivars. harvested in organic orchards located in Piedmont. By considering the quality parameters of the fruit. Its mechanism of action was mainly based on competition for nutrients and release of hydrolases. treated with Metschnikowia pulcherrima strain MACH1. fructicola in reducing the accumulation of patulin on apples. 2008). grapefruit and tomatoes (Schena et al. fructicola through its morphological characteristics and through sequencing of the ITS region and the D1/D2 domain. 2011). 1. ‘Granny Smith’. 2008b) and its capacity to completely biodegrade patulin in vitro within 72 h (Reddy et al. isolated from the surface of ‘Golden Delicious’ apples.

Morales et al.. 2003. A. Commission Regulation 1107/2009 of 21 October 2009 concerning the placing of plant protection products on the market.. Postharvest Biology and Technology 7. A.). Boca Raton. F. Hoffmann. expansum. 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs. Pinedo-Rivilla. W.. sodium carbonated and sodium bicarbonate dips. Droby. Moreover.. proteomic and metabolomics studies” granted by the Italian Ministry of Education. Hirooka. S. F. Konstantinou. F. G. This study considered the efficacy of a yeast biocontrol agent both against P. essential steps to be undertaken to develop a biofungicide with commercial application.. Pagnocca. 65–108.. C.V. Annual Review of Phytopathology 40. 2010). Acknowledgments This research was funded by the project “SafeFoodControl – Development of innovative systems and technologies for the production. Coelho.. Patulin in apple-based foods: occurrence and safety evaluation. Plant Disease 95. There are recent studies on the effect of antagonists on patulin accumulation on fruit (Castoria et al. V. A. Biological Control of Postharvest Diseases – Theory and Practice. M... Pagnocca. 331–371. 2011. 1398–1407... two abundant mycotoxins. Biocontrol Science and Technology 14. 2011) or ‘Annurca’ (Castoria et al. Duke.Y. Lima et al. G. / Postharvest Biology and Technology 75 (2013) 1–8 7 competition for nutrients.. 1271–1278. Canadian Institute of Food Science and Technology Journal 6. could also enhance patulin accumulation in the fruit (Morales et al.. since very high patulin levels could be associated with small rots. A.. Bhatnagar. C. Wilson. Lima et al. but rarely was the effect on patulin accumulation tested. S. H... Hasan. In our study. Brubacher. D. Wosiacki. 1384–1389.).. 2008. Food Additives and Contaminants 17.R. 513–521. 325–331. 5–24. Near-harvest applications of Metschnikowia fructicola. by lactic acid bacteria. semi-commercial and commercial trials will be performed to evaluate the efficacy of M. Finally. Gish. Sataque Ono. R... Karabulut. Galli.. 2000. 2007. Torres. (Eds. an environment where the antagonists were already able to grow.W. L.R. 399–406. Sabino... L. J. European Parliament. M. L. Pons. C.. Eckert. E. (2011) were performed at room temperature. E. Wiess. cultivar susceptibility. L.M. Chen. 403–410. 1098–1108. Future prospects involve the study of the mechanisms of actions used by the antagonistic yeast AL27 to control the development of P. G.C. Basic local alignment search tool.W. F. Miller. F.. Further studies will involve the optimization of the fermentation and stabilization processes.Y. S. N.A. Anna Poli for the molecular identification of the yeast species.. Daus..O. The studies of Castoria et al.. Official Journal of European Union L365. 725–733.. Journal of Molecular Biology 5. Acta Horticulturae 269. Biological control of postharvest diseases of fruits.The concentration of the mycotoxin in apples stored at cold temperature was generally lower than in apples stored at room temperature. Dayan.J.. 2001. Effect of the biocontrol yeast Rhodotorula glutinis strain LS11 on patulin accumulation in stored apples. Duran-Patron. Fuchs. Harwig. Hoffmann. 2002. W.. R. Errampalli. Calvo. D. 2010). Chalutz. 2006.P. BCAs or fungicides. MACH1 was effective against postharvest pathogens but inefficient in reduction of patulin accumulation. Kurtzman. Mycotoxins in Agriculture and Food Safety. A.. Droby. Droby.R.. R. pp.. Hirooka. D. C. Phytopathology 91. B. S. Toxigenic Alternaria species of economic importance. Korsten... The patulin level was more markedly reduced by the three antagonistic yeasts in ‘Golden Delicious’ apples than on the other cultivars...L. Bardas. Wilson. Restani.C.. 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