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Postharvest Biology and Technology 75 (2013) 1–8

Contents lists available at SciVerse ScienceDirect

Postharvest Biology and Technology

jou rna l h omepa g e: www.elsev ier .com/ locate /postharvbio

new strain of Metschnikowia fructicola for postharvest control of Penicilliumxpansum and patulin accumulation on four cultivars of apple

avide Spadaro ∗, Alessia Lorè, Angelo Garibaldi, Maria Lodovica Gullinoentre of Competence for the Innovation in the Agro-environmental Sector (AGROINNOVA), University of Turin, Via Leonardo da Vinci 44, I-10095 Grugliasco, Turin, Italy

r t i c l e i n f o

rticle history:eceived 10 July 2012ccepted 5 August 2012

eywords:pple

a b s t r a c t

The efficacy of three antagonistic yeasts, Metschnikowia pulcherrima strain MACH1, M. pulcherrima strainGS9, and Metschnikowia fructicola strain AL27, against Penicillium expansum and patulin accumulationwas evaluated on apples stored at room (22 ± 1 ◦C for 7 days) and cold temperatures (1 ± 1 ◦C for 56days). To increase the potential range of application of the biocontrol agents (BCAs), their efficacy wasevaluated on four cultivars of apple, i.e. ‘Golden Delicious’, ‘Granny Smith’, ‘Red Chief’ and ‘Royal Gala’.

iological controletschnikowia fructicolaycotoxin

enicillium expansumeast

AL27 was more effective than MACH1 and GS9 in the control of blue mold rot and in the reduction ofpatulin accumulation. The efficacy of AL27 was in most cases similar to the chemical control used, makingthe antagonist as competitive as chemical fungicides. In vitro experiments showed that AL27 reduced theconidial germination and germ tube length of P. expansum more than the other strains. The three BCAswere more effective in the control of blue mold rot on ‘Golden Delicious’ apples than on the other testedcultivars.

. Introduction

Postharvest losses of fruit and vegetables are mainly due tottacks of pathogens during harvest, storage, transport and mar-eting (Snowdon, 1990). Some species of Penicillium are importantlant pathogens causing decay on various fruit and vegeta-les, through their antioxidant proteins and hydrolytic enzymesBertolini and Tian, 1996; Qin et al., 2007). Penicillium expansuman particularly cause blue molds and blue rots on several plantpecies (Stange et al., 2002).

Besides its pathogenic activity, P. expansum is able to pro-uce patulin, a highly reactive unsaturated lacton, that may causecute and chronic toxicity, including carcinogenic, mutagenic, anderatogenic effects (Beretta et al., 2000; Hasan, 2000; McCallumt al., 2002). The mycotoxin causes impairment of kidney functions,xidative damage, and weakness to the immune system. It also has

negative impact on reproduction in males via interaction withormone production (Selmanoglu and Kockaya, 2004; Fuchs et al.,008). Patulin can be found in several typologies of fruit-derivedood, including apple, pear, peach and apricot juices and nectarsSpadaro et al., 2007, 2008a). The Joint FAO/WHO Expert Committee

n Food Additives (JEFCA) established a provisional maximum tol-rable daily intake (PMTDI) of 0.4 �g kg−1 body weight (bw) day−1,ased 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).

925-5214/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.postharvbio.2012.08.001

© 2012 Elsevier B.V. All rights reserved.

safety factor of 100 (World Health Organization, 1995). Based onthis PMTDI, patulin is regulated in the European Union at levelsof 50 mg kg−1 in fruit juices and fruit nectars, 25 mg kg−1 in solidapple products, and 10 mg kg−1 in apple-based products for infantsand young children (European Commission, 2006).

The use of chemical fungicides is an important strategy forcontrolling P. expansum in harvested commodities (Eckert andOgawa, 1990; Janisiewicz and Korsten, 2002; Zhou et al., 2002).However, during the last decades, some fungicides have lost theirefficacy due to the development of resistant strains. Several stud-ies demonstrated resistance of P. expansum to the most commonfungicides used in postharvest (Sholberg et al., 2005; Errampalliet al., 2006). Moreover, concern for public safety has resulted inthe cancellation of some of the most effective fungicides in Europe(European Parliament, 2009) and the United States (United StatesCongress, 1996; Dayan et al., 2009). Therefore, research focused onthe development of alternative control that should be both effec-tive and economically feasible. The use of microbial antagoniststo control postharvest diseases of fruit and vegetables is one ofthe most promising alternatives to fungicides (Qin et al., 2004;Droby et al., 2009). Some components of the microbial commu-nity present on the surface of fruit and vegetables, such as bacteriaand yeasts, have been shown to have significant antagonistic activ-ity against P. expansum (Usall et al., 2001; Janisiewicz and Korsten,

2002).

Different yeasts are also able to reduce the patulin level invitro (Coelho et al., 2008; Reddy et al., 2011). Fermentative yeastsreduce patulin contamination during production of cider from

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pple juice (Harwig et al., 1973). Moss and Long (2002) showedhat Saccharomyces cerevisiae metabolizes patulin to the lessoxic E-ascladiol, whereas there are few studies on the effect ofiological control yeasts on patulin accumulation in stored pomeruit (Castoria et al., 2005; Morales et al., 2008a; Lima et al., 2011).

Several studies have revealed that fruit cultivars may differ inheir susceptibility to blue mold rots and to patulin accumula-ion (Neri et al., 2010; Konstantinou et al., 2011). Therefore, thepple cultivar should be considered a critical factor influencing theiocontrol of P. expansum and its patulin accumulation on fruit.orales et al. (2008b) found that the pH value of the apple vari-

ties was a determining factor in patulin accumulation only underold storage: ‘Golden Delicious’ apples, characterized by a lower pH,ere more prone to patulin accumulation at 1 ◦C. At room tempera-

ures, varieties of apple with higher amounts of organic acids, suchs ‘Golden Delicious’ and ‘Fuji’, accumulated more patulin. Anothertudy showed that patulin accumulation was significantly higher inGolden Delicious’ and ‘Red Delicious’ than in ‘Granny Smith’ andFuji’ apples, due to the lower acidity of the fruit (Konstantinou et al.,011).

The specific P. expansum strain may be another important factorn its pathogenicity and in its ability to synthesize patulin in theruit (Neri et al., 2010). Sommer et al. (1974) found that different P.xpansum strains produced differing patulin levels, and the levelsere not related to the virulence of the P. expansum strains (Neri

t al., 2010; Reddy et al., 2010). Beretta et al. (2000) similarly foundhat the patulin content in apples was not always related to theiameter of the rotten areas, since very high levels were sometimesetected in fruit with small rots.

The aims of the present study were to evaluate the efficacy ofhree antagonistic yeasts Metschnikowia pulcherrima strain MACH1Saravanakumar et al., 2008), M. pulcherrima strain GS9 (Spadarot al., 2008b), and Metschnikowia fructicola strain AL27, in the con-rol of P. expansum and patulin accumulation in apples stored atoom and cold temperatures. To increase the potential range ofpplication of the biocontrol agents (BCAs), their efficacy was eval-ated on four cultivars of apple, i.e. ‘Golden Delicious’, ‘Grannymith’, ‘Red Chief’ and ‘Royal Gala’.

. Materials and methods

.1. Microorganisms

M. pulcherrima strain MACH1 (Saravanakumar et al., 2008), M.ulcherrima strain GS9 (Spadaro et al., 2008b) and M. fructicolatrain AL27 were isolated from the carposphere of ‘Golden Deli-ious’ apples harvested in unsprayed orchards located in Northerntaly. The microorganism culture was stored at −20 ◦C in cell sus-ension with 65% (v/v) glycerol and 35% (v/v) of a solution of00 mM MgSO4 and 25 mM Tris (pH 8.0). The strain AL27 waseposited within the Industrial Yeasts Collection (DBVPG) on March9, 2011 with deposit designation 30P and its uses were patentedith the Italian patent application TO2011A000534, deposited on

une 20, 2011. The strains were grown in YEMS (30 g L−1 yeastxtract, 5 g L−1 d-mannitol, 5 g L−1 l-sorbose; Spadaro et al., 2010).

Inocula of the antagonists for all experiments were preparedy subculturing in 250 mL Erlenmeyer flasks containing 75 mLEMS and incubated on a rotary shaker (100 rpm) at 22 ◦C for 48 h.east cells were collected by centrifugation at 1500 rpm for 10 min,ashed and resuspended in sterilized Ringer solution (pH 6.9 + 0.1;erck, Darmstadt, Germany) and brought to a standard concentra-

ion of 108 cells mL−1 by direct counting with a hemacytometer.Four isolates of P. expansum (PEX06, PEX12, PEX25 and PEX27),

ach obtained from rotted apples harvested in Piedmont, North-rn Italy, and selected for their virulence (Reddy et al., 2010), were

and Technology 75 (2013) 1–8

used as a mixture during the experiments to ensure a high levelof disease. Each strain belongs to the AGROINNOVA collection andwere stored in tubes with potato dextrose agar (PDA; Merck) and50 mg L−1 of streptomycin (Merck) at 4 ◦C. Conidial suspensionsused for fruit inoculation were prepared by growing the pathogenson Petri dishes on PDA containing 50 mg L−1 of streptomycin. Aftera week incubation at 22 ◦C, conidia from the four strains were col-lected and resuspended in sterile Ringer’s solution. After filteringthrough eight layers of sterile cheese-cloth, conidia were countedand brought to a final concentration of 105 mL−1. The resultantsuspensions were shaken using a vortex mixer for 30 s before inoc-ulation.

2.2. Molecular and morphological identification

The yeast antagonist Metschnikowia fructicola strain AL27 wasidentified by sequencing the internal transcribed spacer 1 (ITS1),5.8S ribosomal RNA gene, and internal transcribed spacer 2 (ITS2)according to White et al. (1990) and the D1/D2 domain at the 5′

end of the LSU rRNA gene according to Kurtzman and Robnett(1998). The DNA, coming from antagonist cell suspensions grownin YPD for 48 h, was extracted using NucleoMag 96 Plant Kit(Macherey Nagel, Oensingen, Switzerland) and Kingfisher mag-netic particle processor (Thermo Labsystems, Basingstoke, UnitedKingdom) following the manufacturers’ protocols. The ITS regionswere amplified using genomic DNA as a template and universalprimers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′). The D1/D2 domains were amplifiedusing the primers NL-1 (5′-GCATATCAATAAGCGGAGGAAAAG-3′)and NL-4 (5′-GGTCCGTGTTTCAAGACGG-3′) on the genomic DNA.PCRs were performed using a TGradient thermal cycler (Biome-tra, Göttingen, Germany). Each 20 �L PCR contained 1 �L of DNAtemplate (50 ng), 200 mM of each deoxynucleotide triphosphate,2 �L of 10× buffer (Taq DNA Polymerase, Qiagen, Chatsworth, CA,USA), 0.7 mM each primer, and 1.0 U Taq DNA Polymerase (Qia-gen). PCR program for ITS regions was: 95 ◦C, 3 min; 34 cycles:94 ◦C, 15 s; 55 ◦C, 45 s; 72 ◦C, 55 s; 72 ◦C, 7 min; 4 ◦C. PCR pro-gram for D1/D2 domain was: 95 ◦C, 10 min; 30 cycles: 94 ◦C, 30 s;55 ◦C 30 s; 72 ◦C, 45 s; 72 ◦C, 7 min; 4 ◦C. A 10 �L aliquot of PCRproducts from each reaction was electrophoresed in 2.0% agarosegel in TBE buffer, and then stained with SYBR SAFE (Invitrogen,Eugene, OR, USA). Gel images were acquired with a Gel Doc 1000System (Bio-Rad Laboratories, Hercules, CA, USA). PCR amplifica-tion products were cloned into the PCR4 TOPO vector (Invitrogen)using the TOPO TA cloning kit following the manufacturer pro-tocol and sequenced by BMR Genomics (Padova, Italy) using anABI PRISM 3730XL DNA Sequencer (AME Bioscience, Sharnbrook,United Kingdom). The sequences were analyzed by using the soft-ware BLASTn (Basic Local Alignment Search Tool; Altschul et al.,1990) for similarity. The microscope observation of the cell andcolony morphology was complementary to the molecular analysis.M. pulcherrima strain MACH1 and M. pulcherrima strain GS9 werepreviously identified (Saravanakumar et al., 2008; Spadaro et al.,2008b).

2.3. Antagonism in vitro

The effect of the isolates of Metschnikowia spp. on conidial ger-mination and on germ tube length of P. expansum was assessedin 5 mL of potato dextrose broth (PDB, Merck). A conidial suspen-sion (100 �L; 5 × 106 conidia mL−1) of P. expansum strain PEX06was added to a 10 mL test tube. Living cells of each antago-

nistic yeast (100 �L of a suspension containing 5 × 107, 5 × 108,or 5 × 109 cells mL−1), were added to the test tube. As control,100 �L of the conidial suspension (5 × 106 conidia mL−1) of thepathogen in Ringer’s solution was added to 5 mL of PDB. After

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2 h incubation of the 45◦ sloping tubes at 22 ± 1 ◦C on a rotaryhaker (100 rpm), 100 conidia per replicate were observed micro-copically and their germination was evaluated. The treatmentsere replicated three times. The experiment was carried out

wice.

.4. Efficacy on four cultivars of apples

Apples (Malus × domestica), ‘Golden Delicious’, ‘Granny Smith’,Red Chief’ and ‘Royal Gala’, harvested in an Italian orchard grownccording to integrated pest management practices, were disin-ected in sodium hypochlorite (NaClO, 1.0% as chlorine) and rinsednder tap water, dried at room temperature and punctured with

sterile needle at the equatorial region (3 mm depth; 3–4 mmide; 3 wounds per fruit). Fruit were exposed to treatments with

0 �L of the cell suspension (108 mL−1) of M. pulcherrima strainACH1, strain GS9 or M. fructicola strain AL27 per wound. Chem-

cal treatment consisted in the application into each inoculatedound (10 �L) of a suspension (1.25 mL L−1 water) of imazalil andyrimethanil (Philabuster 400SC®, Decco Italia srl, Belpasso, Italy;

mazalil 17.2% a.i.; pyrimethanil 17.2% a.i.). An inoculated con-rol was also performed: after 3 h at room temperature, 10 �L ofhe conidial suspension mixture of P. expansum (105 mL−1) wereipetted into the apple wounds. Apples were randomly packed inommercial plastic trays and stored either at 22 ± 1 ◦C for 7 days ort 1 ± 1 ◦C for 56 days.

Some quality parameters were assessed on healthy fruit of everyultivar. Firmness was measured on each fruit at two oppositeites along the equatorial region with a FT327 – Fruit Pressureester with an 11 mm probe (EFFEGI, Alfonsine, Italy). The probeescended towards the sample at 1.0 mm/s and the maximumorce (N) was defined as firmness. Total soluble solids (TSS) wereetermined by measuring the refractive index of pressed juiceLarrigaudière et al., 2002) with a digital refractometer (DBR95,ingapore) and the results were expressed as percentages (g/100 gruit weight). Acidity was measured by titration with 0.1 N NaOHo pH 8.0: 5 mL of pressed juice diluted with 5 mL of distilled waterere evaluated. Titratable acidity was calculated as percent malic

cid (Wright and Kader, 1997).Each treatment was replicated three times. Twenty fruit per

eplication were used (60 inoculation sites). The severity of theiseases was determined by measuring the mean lesion diame-er on the rotted apples and the percentage of rot (fresh weightf rot/fresh weight of fruit). The experiments were carried outwice.

.5. Patulin analysis

Patulin was extracted from rot caused by P. expansum on applesreated and stored at 22 ◦C and at 1 ◦C. The extraction proceduresed was modified by AOAC Official Method 2000.02 Patulin inlear and Cloudy Apple Juices and Apple Puree. Twenty gramsf sample were placed in a centrifuge tube to which 20 dropsf pectinase enzyme solution (Sigma Chemical Co., St Louis, MO,SA; 5 U/g of juice) and 10 mL of water were added. The mix-

ure was left at 40 ◦C for 2 h and then centrifuged at 4500 rpmor 5 min. Ten milliliters of clear juice was placed into 100 mLeparating funnel; patulin was extracted with 30 mL of ethylcetate shaking for 1 min. The organic layer was separated fromhe water layer. The procedure was repeated three times. Therganic phase was dehydrated with 25 g of sodium sulphatenhydrous and then evaporated to dryness (Rotavapor Laborota

000, Heidolph®, Schwaback, Germany). The residual was resumedith 2 mL of acidic water (pH 4.0) and transferred into a HPLC

ial. The HPLC apparatus was an Agilent 1100 series equippedith G1379 degasser, G1313A autosampler, G1316A column

and Technology 75 (2013) 1–8 3

thermostat set at 30 ◦C, G1315B UV diode array detector setat 276 nm, G1311 quaternary pump and Agilent ChemstationG2170AA Windows XP operating system (Agilent®, Waldbronn,Germany). A stainless steel analytical column (250 × 4.6 mm i.d.,4 �m, Synergy Hydro-RP C18; Phenomenex®, Torrance, CA, USA)preceded by a guard column (4 × 3 mm i.d.) with the samestationary phase was used. The mobile phase, eluting at aflow rate of 0.800 mL/min, consisted of an isocratic mixture ofwater–acetonitrile–perchloric acid (95:4:1) for 20 min, followedby a washing step with an isocratic mixture of water–acetonitrile(35:65). One hundred microliters of sample was injected onto theHPLC column and the retention time of patulin was about 15 min.The amount of patulin in the final solution was determined by usinga calibration graph of concentration versus peak area and expressedas ng/mL, achieved by injection onto the HPLC column of 100 �L ofstandard solutions of patulin (Sigma Chemical Co., St Louis, MO,USA). The standard solutions had concentrations of 500 ng mL−1,400 ng mL−1, 250 ng mL−1, 100 ng mL−1 and 50 ng mL−1 of patulin.The recovery was determined on a blank apple puree spiked at threeconcentrations of patulin (10, 50 and 100 ng g−1). Each test was per-formed three times and the mean recovery values were respectively90.9%, 91.9% and 100.9%. The repeatability ranged from 1.0% to 6.2%for duplicate analyses. The limit of detection (LOD) and the limitof quantification (LOQ), based on the IUPAC definition (Thompsonet al., 2002), were respectively 1.04 and 1.57 ng g−1. The high valueof the regression coefficient (R2 ≥ 0.99) obtained indicated a goodlinearity of the analytical response.

2.6. Statistical analysis

For the efficacy experiments, data from at least two experimen-tal trials were pooled. For the mycotoxin experiments, the analyseswere carried out in triplicate and the values represented the meanvalues. The statistical analysis was performed by one-way analysisof variance (ANOVA), using SPSS-WIN software (17.0), and Dun-can’s multiple range test was employed; p < 0.05 was consideredsignificant.

3. Results

3.1. Molecular and morphological identification

The strain AL27 was identified by sequencing the ribosomalregions ITS1–5.8S–ITS2 with universal primers ITS-1 and ITS-4 andsequencing the D1/D2 domain with the primers NL-1 and NL-4.The sequences of the amplified regions were deposited in Gen-Bank. The BLAST analysis of the ITS sequence (accession numberHQ682194.2; amplimer size: 251 bp) showed that the ampliconof AL27 showed 99% (249/251) identity with the sequences ofMetschnikowia fructicola. The analysis of the D1/D2 domain (acces-sion number HQ682195; amplimer size: 448 bp) confirmed that thePCR product of AL27 had 99% (447/448) identity with the sequencesof Metschnikowia fructicola, while the identity with strains of M.pulcherrima was lower (98%; 437/444). The observation of themorphological (colony morphology) and microscopic (cell shapeand size) characteristics of AL27 confirmed the rDNA sequencingresults. Colonies are milky white, cells are ovoid and they measure1.66 × 3.30 to 2.54 × 7.21 �m.

3.2. Antagonism in vitro

The effect of M. fructicola strain AL27, M. pulcherrima strain GS9and M. pulcherrima strain MACH1 was evaluated on conidial ger-mination and germ tube length of P. expansum (Table 1). In thecontrol, 98.0% of the conidia germinated and the average germ tube

4 D. Spadaro et al. / Postharvest Biology

Table 1Conidial germination (%) and germ tube length (�m) of Penicillium expansum co-cultivated with a cell suspension of antagonistic yeast in PDB at 22 ± 1 ◦C for 12 h.

Treatment Penicillium expansum

Conidialgermination (%)a

Germ tubelength (�m)a

AL27 1 × 106 cfu mL−1 16.7 b 32.5 fAL27 1 × 107 cfu mL−1 8.7 a 23.7 eAL27 1 × 108 cfu mL−1 5.0 a 2.3 aGS9 1 × 106 cfu mL−1 78.0 g 31.0 fGS9 1 × 107 cfu mL−1 69.3 f 25.0 eGS9 1 × 108 cfu mL−1 56.7 e 18.7 cdMACH1 1 × 106 cfu mL−1 58.7 e 20.9 dMACH1 1 × 107 cfu mL−1 40.7 d 16.9 cMACH1 1 × 108 cfu mL−1 29.3 c 11.2 bControl 98.0 h 96.1 g

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ength was 96.1 �m. The three microorganisms were able to sig-ificantly reduce the germination rate and the germ tube lengthf P. expansum at each concentration tested. Each microorganismhowed a higher inhibition capability when co-cultivated at theighest concentration (108 cells mL−1), than when applied at 107

r 106 cells mL−1. AL27 was more effective than the other twoicroorganisms in reducing the conidial germination at each of

he three concentrations tested. In particular, when co-cultivatedith AL27 at 108 and 107 cells mL−1, the germination rates were

nly 5.0% and 8.7% respectively. The highest germinations, respec-ively 78.0% and 69.3%, were observed when co-cultivating withS9 at 106 and 107 cells mL−1. The smallest germ tube length

2.3 �m) was observed in presence of 108 cells mL−1 of AL27, fol-

owed by 108 cells mL−1 of MACH1 (11.2 �m). The germ tubes wereonger when reducing the concentration of the yeast cells. Longererm tubes where observed in presence of 106 cells mL−1 of AL2732.5 �m) and GS9 (31.0 �m).

ig. 1. Blue mold rot diameter (mm) caused by Penicillium expansum on apples ‘Golden Dulcherrima strain MACH1, M. pulcherrima strain GS9 and M. fructicola strain AL27 and stohe same cultivar and the same storage trial, followed by the same letter, are not statisticalln the application into each inoculated wound (10 �L) of a suspension (1.25 mL L−1 watermazalil 17.2% a.i.; pyrimethanil 17.2% a.i.

and Technology 75 (2013) 1–8

3.3. Efficacy on four cultivars of apples

The efficacy of the antagonist yeasts was evaluated on ‘GoldenDelicious’, ‘Granny Smith’, ‘Red Chief’, and ‘Royal Gala’ apples,stored at room (22 ± 1 ◦C for 7 days) and low temperature (1 ± 1 ◦Cfor 56 days). Blue mold rot was evaluated as rot diameter (Fig. 1)and as percentage of rot weight (Fig. 2). The mixture of imazalil andpyrimethanil was chosen as a chemical control because it is regis-tered in several European countries for use against postharvest rotson apple.

In the trials carried out at 22 ± 1 ◦C for 7 days, the three bio-control agents (BCAs) were able to significantly reduce the bluemold rot diameter and weight compared to the control. AL27 wasthe most effective antagonist and provided an efficacy in reduc-ing the rot diameter statistically similar to imazalil + pyrimethanilon ‘Golden Delicious’, ‘Royal Gala’ and ‘Red Chief’ (Fig. 1). On the‘Granny Smith’ apples, AL27 was as effective as the chemical inreducing the rot weight (Fig. 2).

When apples were stored at 1 ± 1 ◦C for 56 days, the three BCAssignificantly reduced the blue mold lesion diameter, but AL27 wasthe most effective in reducing the rot diameter on all the applecultivars (Fig. 1). Its efficacy was statistically similar to the chemicalcontrol and higher than the other two antagonists, GS9 and MACH1.By considering the reduction of the rot weight (Fig. 2), all the BCAswere effective against P. expansum. Again, AL27 reduced more thanthe other two BCAs the rot weight and its effect was similar to theapplication of imazalil + pyrimethanyl. The rot weight was only 0.8%on ‘Granny Smith’, 1.0% on ‘Golden Delicious’, 2.3% on ‘Red Chief’and 3.9% on ‘Royal Gala’ apples.

Among the cultivars tested, AL27, MACH1 and GS9 showed ahigher control of the rot lesion diameter on ‘Golden Delicious’. The

average values of some quality parameters have been measuredon the apples before storage (Table 2). The values of firmness didnot differ among the cultivars. On the other hand, total solublesolids and titratable acidity were significantly different among the

elicious’, ‘Granny Smith’, ‘Red Chief’, and ‘Royal Gala’, treated with Metschnikowiared at 22 ± 1 ◦C for 7 days (dark grey) or at 1 ± 1 ◦C for 56 days (light grey). Values ofy different by Duncan’s Multiple Range Test (p < 0.05). Chemical treatment consisted) of imazalil and pyrimethanil. Philabuster 400SC® , Decco Italia srl, Belpasso, Italy;

D. Spadaro et al. / Postharvest Biology and Technology 75 (2013) 1–8 5

Fig. 2. Blue mold rot percentage (fresh weight) caused by Penicillium expansum on apples ‘Golden Delicious’, ‘Granny Smith’, ‘Red Chief’, and ‘Royal Gala’, treated withM la strag

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etschnikowia pulcherrima strain MACH1, M. pulcherrima strain GS9 and M. fructicorey). See Fig. 1.

ultivars. In particular, ‘Golden Delicious’ apples had higher totaloluble solids contents (14.5%), while the highest titratable acidityas observed in ‘Granny Smith’ apples. The higher total soluble

olids in ‘Golden Delicious’ apples could be related to a higherfficacy of the BCAs.

.4. Patulin reduction

The patulin produced was significantly lower in the trials per-ormed at low temperature compared to the experiments carriedut at 22 ± 1 ◦C for 7 days, except for the cv Red Chief, where pat-lin content was significantly higher on the apples stored at 1 ± 1 ◦Cor 56 days (Fig. 3). In general, the three antagonists were able toignificantly reduce the patulin content compared to the control.L27 was the most effective BCA on all the apple cultivars, storedither at 1 ± 1 ◦C for 56 days or at 22 ± 1 ◦C for 7 days. The pat-lin level observed in the apples treated with AL27 was similar

o the level of the chemical control on ‘Golden Delicious’, ‘Grannymith’ and ‘Royal Gala’. In particular, when the fruit where kept at

± 1 ◦C for 56 days, the patulin level was lower on apples treatedith AL27 (0.0 ng g−1 on ‘Golden Delicious’, 1.2 ng g−1 on ‘Granny

able 2verage values of quality parameters on apples ‘Golden Delicious’, ‘Granny Smith’, ‘Red C

± 1 ◦C for 56 days.

Apple cultivar Total soluble solids (%)*

Golden Delicious 14.5 ± 0.8 a

Granny Smith 10.8 ± 0.6 c

Red Chief 11.3 ± 0.5 c

Royal Gala 12.2 ± 0.7 b

* The results are the means of two independent experiments. “±” stands for standard ey Duncan’s Multiple Range Test (p < 0.05).

in AL27 and stored at 22 ± 1 ◦C for 7 days (dark grey) or at 1 ± 1 ◦C for 56 days (light

Smith’, 24.0 ng g−1 on ‘Royal Gala’), than on apples treated withimazalil + pyrimethanil (0.7 ng g−1 on ‘Golden Delicious’, 4.2 ng g−1

on ‘Granny Smith’, 29.5 ng g−1 on ‘Royal Gala’). Only on ‘Red Chief’,was the patulin level in the fruit treated with AL27 (78.0 ng g−1 at22 ◦C and 67.1 ng g−1 at 1 ◦C) higher than the level in the chemicalcontrol (56.4 ng g−1 at 22 ◦C and 16.6 ng g−1 at 1 ◦C). The highestconcentrations of patulin were observed in the fruit treated withGS9, which was also the least effective antagonist.

4. Discussion

Biocontrol agents can be applied as an alternative to fungicidesto prevent and control postharvest diseases, and in particular P.expansum, of apples. Yeasts are suitable biocontrol agents againstpostharvest diseases, because they rapidly colonize and surviveon fruit surfaces for long periods of time under different condi-tions, use available nutrients to proliferate rapidly, limit nutrient

availability to the pathogen and generally are unaffected by fungi-cides used commercially (Droby et al., 2009). Previously, severalisolates belonging to the yeast genus Metschnikowia were iso-lated from different sources and selected for their efficacy against

hief’, and ‘Royal Gala’ used during the trials of storage at 22 ± 1 ◦C for 7 days or at

Firmness (N)* Titratable acidity (g malic acid/100 mL*)

70.6 ± 8.4 a 0.509 ± 0.027 b74.5 ± 9.1 a 0.811 ± 0.034 a68.6 ± 7.5 a 0.235 ± 0.013 d75.5 ± 8.3 a 0.348 ± 0.026 c

rror of the means. Values followed by the same letter are not statistically different

6 D. Spadaro et al. / Postharvest Biology and Technology 75 (2013) 1–8

Fig. 3. Patulin concentration (ng g−1) produced by Penicillium expansum on apples ‘Golden Delicious’, ‘Granny Smith’, ‘Red Chief’, and ‘Royal Gala’, treated with Metschnikowiap d stor

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ulcherrima strain MACH1, M. pulcherrima strain GS9 and M. fructicola strain AL27 an

ostharvest diseases (Zhang et al., 2010). M. pulcherrima, in recentears, showed high efficacy as a BCA against postharvest decaysf apples, grapes, grapefruit and tomatoes (Schena et al., 2000;anisiewicz et al., 2001; Spadaro et al., 2002). Also M. fructicola effec-ively reduced the development of postharvest rots of grapes andtrawberries (Kurtzman and Droby, 2001; Karabulut et al., 2003,004).

One strain of M. pulcherrima, named MACH1, was isolatedrom the surface of ‘Golden Delicious’ apples, harvested in organicrchards located in Piedmont, and selected for its efficacy against. cinerea, A. alternata and P. expansum. The strain showed a goodfficacy against grey mold and alternaria rot, but its biocontrolapability was lower against blue mold rot (Saravanakumar et al.,008). Its mechanism of action was mainly based on competi-ion for nutrients and release of hydrolases, particularly chitinasesSaravanakumar et al., 2009). The same strain was evaluated forts capability to biodegrade patulin when grown in vitro: after 48 hrowth of the yeast, patulin was not detected in the growth mediumor in the yeast cell wall, indicating that the mycotoxin was notbsorbed but completely biodegraded (Reddy et al., 2011). Anothertrain of M. pulcherrima, named GS9, was previously isolated from

‘Golden Delicious’ apple and evaluated for its biocontrol against. cinerea and P. expansum (Spadaro et al., 2008b) and its capacityo completely biodegrade patulin in vitro within 72 h (Reddy et al.,011), showing lower efficacy compared to MACH1.

In the current research a new yeast strain, named AL27, iso-ated from the surface of ‘Golden Delicious’ apples, was selectedor its efficacy against P. expansum on four apple cultivars. More-ver, the capacity to reduce the patulin accumulation on apple

as considered as an important feature for the antagonist selec-

ion. The yeast strain was identified as M. fructicola through itsorphological characteristics and through sequencing of the ITS

egion and the D1/D2 domain. To the best of our knowledge, this

ed at 22 ± 1 ◦C for 7 days (dark grey) or at 1 ± 1 ◦C for 56 days (light grey). See Fig. 1.

is the first report describing the efficacy of M. pulcherrima and M.fructicola in reducing the accumulation of patulin on apples.Thein vitro experiments showed that AL27 reduced the conidial ger-mination and germ tube length of P. expansum more than theother strains. The yeast cell concentration was an important fac-tor in determining the inhibition, and a higher inhibition wasobtained in presence of higher concentrations of antagonist cells,as previously demonstrated for other antagonistic microorganisms(Hofstein et al., 1994). The results obtained in vitro were confirmedby the results of the trials on fruit, performed at 22 ± 1 ◦C for 7 daysand at storage temperature. AL27 was more effective than MACH1and GS9 in the control of blue mold rot, either when the lesion diam-eter or the rot weight was considered as parameters. These resultsare in agreement with previous studies, where different strains ofthe same yeast species showed different biocontrol capabilities, dueto their genetic background (Spadaro et al., 2008b). The efficacy ofAL27 was in most cases similar to the chemical control used, whichis a mixture of two active ingredients commercially available inseveral European markets.Generally, the efficacy of the three bio-control agents was higher when the fruit were stored at 1 ◦C than at22 ◦C. In particular, the efficacy of M. pulcherrima MACH1 was sig-nificantly higher on apples stored at 1 ± 1 ◦C for 56 days. Previousstudies showed that low storage temperatures resulted in a higherefficacy of the antagonists, either yeast or bacteria (Morales et al.,2008a). During shelf life, P. expansum may take advantage of theoptimal conditions of growth and increase the growth rate, result-ing in a higher aggressiveness (Morales et al., 2010).The three BCAswere more effective in the control of blue mold rot on ‘Golden Deli-cious’ apples than on the other cultivars. By considering the quality

parameters of the fruit, ‘Golden Delicious’ apples had higher totalsoluble solids contents. The higher total soluble solids in ‘GoldenDelicious’ could be related to a higher efficacy of the BCAs, becauseone of the main mechanisms of action exploited by yeast strains is

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ompetition for nutrients, and in particular for carbon sources, suchs sugars (Spadaro et al., 2010). A higher efficacy on ‘Golden Deli-ious’ apples could be also related to the source of isolation of thehree BCAs, which was the apple surface, an environment wherehe antagonists were already able to grow. Several BCAs showedood efficacy against P. expansum, but rarely was the effect on pat-lin accumulation tested. There are recent studies on the effect ofntagonists on patulin accumulation on fruit (Castoria et al., 2005;orales et al., 2008a; Lima et al., 2011). The studies of Castoria et al.

2005) and Lima et al. (2011) were performed at room temperature,nd not in cold storage conditions. In our study, MACH1 was effec-ive against postharvest pathogens but inefficient in reduction ofatulin accumulation. In contrast, AL27 was effective both in theiocontrol of the pathogen and in the reduction of patulin accumu-

ation. Yeast can be effective in reducing patulin accumulation inpples through their indirect effect on the reduction of P. expansumrowth and their direct effect in the patulin biodegradation (Coelhot al., 2007; Reddy et al., 2011). The metabolism of patulin to E-scladiol or Z-ascladiol by Saccharomyces cerevisiae (Moss and Long,002), or to desoxypatulinic acid by Rhodosporidium kratochvilovaeCastoria et al., 2011) has been previously reported.

This study considered the efficacy of a yeast biocontrol agentoth against P. expansum and patulin accumulation on more thanne cultivar of apple. Previous studies were just performed on oneultivar of apple, such as ‘Golden Delicious’ (Morales et al., 2008a;ima et al., 2011) or ‘Annurca’ (Castoria et al., 2005).

The analysis of the patulin content showed that its accumu-ation in apples was not always correlated with the severity ofhe blue mold rots, since very high patulin levels could be asso-iated with small rots. BCAs or fungicides, though able to limitathogen growth, could also enhance patulin accumulation in theruit (Morales et al., 2007). Patulin, as with other mycotoxins, isroduced in response to stress, and biocontrol or chemical appli-ations can be considered stress factors for the fungal pathogenBottalico and Logrieco, 1998; Calvo et al., 2002).The concentrationf the mycotoxin in apples stored at cold temperature was generallyower than in apples stored at room temperature, confirming thathe temperature may affect the activity of P. expansum, includinghe mycotoxin production (Santos et al., 2002).

The patulin level was more markedly reduced by the threentagonistic yeasts in ‘Golden Delicious’ apples than on the otherultivars. In ‘Granny Smith’, the patulin accumulated was lowerhan in the other apple cultivars, probably due to their high acid-ty (Konstantinou et al., 2011). Finally, in ‘Red Chief’ apples, a highatulin content in the control could be related to the average low

evel of titratable acidity of the fruit (Morales et al., 2010).Future prospects involve the study of the mechanisms of actions

sed by the antagonistic yeast AL27 to control the developmentf P. expansum and to reduce the patulin accumulation on apple.oreover, semi-commercial and commercial trials will be per-

ormed to evaluate the efficacy of M. fructicola AL27 on largecale applications. Further studies will involve the optimization ofhe fermentation and stabilization processes, essential steps to bendertaken to develop a biofungicide with commercial application.

cknowledgments

This research was funded by the project “SafeFoodControl –evelopment of innovative systems and technologies for the pro-uction, storage, processing and valorization of Piedmontese fruitnd vegetables” granted by the Piedmont Region and by the projectInduction of resistance as a strategy to control fungal pathogens of

pple in postharvest: biochemical, transcriptomic, proteomic andetabolomics studies” granted by the Italian Ministry of Educa-

ion, University and Research. The authors thank Dr. Anna Poli forhe molecular identification of the yeast species.

and Technology 75 (2013) 1–8 7

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