Biological Control 60 (2012) 132–140

Contents lists available at SciVerse ScienceDirect

Biological Control
journal homepage: www.elsevier.com/locate/ybcon

Postharvest biocontrol of Monilinia laxa, Monilinia fructicola and Monilinia

fructigena on stone fruit by two Aureobasidium pullulans strains
Marta Mari ⇑, Camilla Martini, Michela Guidarelli, Fiorella Neri
CRIOF – Diproval, University of Bologna, Via Gandolfi, 19, 40057 Cadriano, Bologna, Italy

h i g h l i g h t s g r a p h i c a l a b s t r a c t

" The antagonistic effect of two yeasts

was tested in the same experiment
against M. laxa, M. fructicola and M.
fructigena.
" The two antagonists were identified
by molecular and morphological
tools as Aureobasidium pullulans.
" The washed cells of antagonists
completely inhibited M. laxa and
fructicola rots and reduced M.
fructigena infections.
" Low temperature did not influence
antagonist efficacy, M. laxa and M.
fructicola were completely inhibited
by antagonists.

a r t i c l e i n f o a b s t r a c t

Article history: The antagonistic effects of yeasts, L1 and L8, isolated from carposphere of ‘Redhaven’ peaches were tested
Received 22 April 2011 for the first time in the same experiment against three Monilinia species (Monilinia laxa, Monilinia fructi-
Accepted 22 October 2011 cola and Monilinia fructigena) in in vitro and in vivo trials. The two antagonists were selected after preli-
Available online 29 October 2011
minary assays for their ability to reduce brown rot in peaches and nectarines, and both were identified by
molecular and morphological tools as Aureobasidium pullulans. In in vivo trials, neither the autoclaved
Keywords: cells, nor the sterile culture filtrates of either antagonist showed any significant reduction of rot incidence
Peach
produced by inocula of the three Monilinia species, while the washed cells of L1 and L8 completely inhib-
Nectarine
Brown rot
ited M. laxa and M. fructicola rots and reduced M. fructigena infections by 70% and 90%, respectively. In
Yeasts other trials, nectarines treated with antagonist cells and inoculated with the pathogens were stored at
Postharvest disease 0 °C for 21 days, plus 7 days at 20 °C. The low temperature reduced brown rot development, since all fruit
were free from disease symptoms on removal from cold storage. However after 7 d at 20 °C, untreated
fruit were rotted over 45% depending on the Monilinia species but the antagonists completely inhibited
M. laxa and M. fructicola, while M. fructigena infections were reduced by 89.8% and 91.2% by L1 and L8,
respectively. For both strains, 108 CFU ml 1 was the most active concentration, although L1 showed good
activity at a concentration of 107 CFU ml 1. Isolate L8 at the concentration of 107 CFU ml 1 was ineffec-
tive against M. fructicola and M. fructigena, showing no difference between treated fruit and the control,
excepting the case of nectarines inoculated with M. laxa, where L8 at the concentration of 107 CFU ml 1
reduced the brown rot infections with respect to the control. The increase in population density of A.
pullulans strains L1 and L8 in the wounds of nectarines stored at 0° or 20 °C was low but sufficient to con-
trol brown rot. In conclusion, the present preliminary study identified two antagonistic strains of A. pullu-
lans as active ingredients for the development of biofungicides for postharvest application against three
Monilinia species that are responsible for high economic losses in stone fruit crops.
Ó 2011 Elsevier Inc. All rights reserved.

⇑ Corresponding author. Fax: +39 051765049.

E-mail address: marta.mari@unibo.it (M. Mari).

1049-9644/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.biocontrol.2011.10.013
 M. Mari et al. / Biological Control 60 (2012) 132–140 133

1. Introduction lans. M. fructicola was inhibited by strains of A. pullulans on nectar-

ines Prunus persica (L.) Batsch. var. laevis (Janisiewicz et al., 2010),
Brown rot is one of the most important fungal diseases in com- while the diameter of decay on peaches P. persica (L.) Batsch in-
mercial Prunus species worldwide (Batra, 1991). The disease can be fected by M. laxa was reduced by half by a treatment based on A.
caused by three species: Monilinia laxa (Aderhold and Ruhland) pullulans strain PL5 (Zhang et al., 2010). Additional efforts must
Honey, Monilinia fructicola (Winter) Honey and Monilinia fructigena therefore be made to screen and develop new BCAs and to make
(Aderhold and Ruhland) (Ogawa et al., 1995). M. fructigena is pre- effective biofungicide products against brown rot available on
valent on pomefruits. M. fructicola is the most common pathogen the market.
on stone fruits in North America, Australasia, Japan and Brazil, The aim of the present study was the isolation of effective BCAs
while it has been identified only recently in European countries against brown rot on peaches and nectarines. Antagonists are gen-
(Lichou et al., 2002; De Cal et al., 2009; Pellegrino et al., 2009), erally selected for their activity against one pathogen species,
moving from A1 to A2 in the list of quarantine organisms (EPPO, while in our experiments two effective strains of A. pullulans (L1
2003). M. laxa is the most common species in European and South and L8) were tested in vitro and in vivo conditions against three
African stone fruit orchards. All three pathogens can cause severe species of Monilinia (M. laxa, M. fructicola and M. fructigena), all de-
losses in stone fruits, more often after harvest during storage and rived from Monilinia populations present in the Emilia–Romagna
transport than in the field. In Europe, postharvest losses caused Region of Italy. The effect of BCA concentrations and fruit storage
by M. laxa sometimes reach high values (59%) (Larena et al., temperatures on biocontrol efficacy was also tested. Finally, the
2005), while M. fructicola in the USA, when under favorable condi- population dynamics of the antagonists on wounded tissues of
tions for disease development, produces high postharvest losses, in fruit kept at room temperature and under refrigeration was also
some cases reaching values of 80–90% (Hong et al., 1997). Few assessed.
losses produced by M. fructigena on stone fruit have been reported,
but recently it has been detected frequently in Italy on cherries and
2. Materials and methods
peaches (Mari, personal communication).
Rainfall near harvest, high humidity and warm temperatures
2.1. Pathogens and their molecular identification by PCR reaction
are favorable to brown rot development, and an Italian standard
schedule to control the pathogen recommends two fungicides
One strain of M. laxa, one of M. fructicola, and one of M. fructige-
applications in the field, the first during bloom and the second just
na from our collection were used. The isolates were grown on
before harvest. In Europe, no chemical treatments are allowed on
Potato Dextrose Agar (PDA) (Sigma St. Louis, MO, USA) at 25 °C for
stone fruit after harvest (Casals et al., 2010), thus frequently
10 days and identified by morphological analysis and sequencing
favouring brown rot development during storage, transport, retail
of ribosomal DNA ITS regions. PCR was performed using universal
and at consumer sites. Monilinia control depends on an integrated
primers ITS1 (GCCGTAGGTGAACCTGCGG) and ITS4 (GCCTCCGCTT
strategy based on orchard fungicide spray programs and cultural
ATTGATATGC) directly from mycelia in pure culture, using the pro-
practices and maintenance of proper storage conditions in packing-
tocols described by Iotti and Zambonelli (2006). PCR reactions
houses and during distribution. However, to overcome the issues
were conducted in 50 ll volume reactions containing 10 mM
related to the use of fungicides and their residues in fruit, other
Tris/HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 150 lM for each
pathogen control strategies have been investigated. These include
dNTP, 300 lM for each primer, and 1.5 U of TaKaRa Taq DNA poly-
treatments based on biocontrol agents (BCAs) (Karabulut and Bay-
merase (Takara, Otsu, Japan). Twenty microgram of Bovine Serum
kal, 2003; Larena et al., 2005; Zhang et al., 2010), chemical prod-
Albumin (BSA) (Fermentas, Vilnius, Lithuania) were added to the
ucts with low toxicity (Gregori et al., 2008), hot water (Karabulut
reaction tubes containing the template fragment of the fungal
et al., 2004, 2010) and natural substances (Neri et al., 2007; Mari
mycelia before adding the other PCR reagents. The amplification
et al., 2008). Within BCAs, yeasts naturally occurring on the surface
reactions were carried out in a T gradient thermal cycler (Biometra,
of fruits or vegetables have usually been preferred for the control
Gottingen, Germany) with an initial denaturation at 95 °C for 6 min
of postharvest diseases such as brown rot, and Metschnikowia pul-
followed by 30 cycles of 94 °C for 30 s, 55 °C for 30 s, 72 °C for
cherrima (Pitt & M.W. Mill.) (Grebenisan et al., 2008), Pseudozyma
1 min and a final step of 72 °C for 10 min. PCR products were run
fusiformata (Buhagiar) (Zhang et al., 2010), Cryptococcus laurentii
on 1.5% agarose gels, stained with ethidium bromide and visual-
(Kuff.) C.E. Skinner (Yao and Tian, 2005) are examples of the yeasts
ized under UV light. The amplified DNA was purified by Nucleospin
tested against M. laxa or M. fructicola on stone fruits. Yeasts and
Extracts II Kit (Macherey–Nagel, Germany) and finally sequenced
yeast-like microorganisms deserve particular attention as BCAs,
by BMR Genomics (Padova, Italy; http://www.bmrgenomics.it).
since their activity is not related to antibiotic production, have
The ITS sequences were compared to those of the GenBank data-
no negative environmental or toxicological impact, are also easily
base (http://www.ncbi.nlm.nih.gov/BLAST/) using the BLASTN
cultivated for large scale production in cheap and simple nutrient
search. For the infection trials, pathogens were grown on V-8 agar
media. Aureobasidium pullulans (De Bary) Arnaud, a yeast-like mi-
(V8A: 250 ml of pure V8 juice and 40 g of agar in 1 l of distilled
crobe, resides in different environments such as the surface of fruit
water). Petri dishes were incubated at 25 °C with 12-h dark: 12-h
from the early stages of their development to maturity (Janisiewicz
light cycles for 10 days. Conidial suspensions were prepared by
et al., 2010) or in woody tissues and leaves (Gonzalez and Tello,
washing the colonies with sterile distilled water containing 0.05%
2011). Its preventive activity against fungal diseases in storage
(v/v) Tween 80, quantified with a hemacytometer and diluted to
and in the field has been widely reported: Botrytis cinerea Pers.
the concentrations required for each experiment.
and Penicillium expansum Link. on apples (Malus domestica Borkh)
(Castoria et al., 2001; Weiss et al., 2006; Zhang et al., 2010), Rhizo-
pus stolonifer (Ehrenb.) Vuill. and Aspergillus niger (Tiegh.) on table 2.2. Fruit
grapes (Vitis vinifera L.) and P. italicum and P. digitatum on citrus
fruits (Lima et al., 1999) were significantly controlled by applica- Nectarines, and peaches were obtained from local packinghous-
tion of A. pullulans. Despite these encouraging results, only a few es; different cultivars were used according to availability. Fruits
studies have reported on the control of Monilinia spp. by A. pullu- free of visible wounds and rot and homogeneous in maturity and
size were stored at 0 °C and used for experiments within 5 days
134 M. Mari et al. / Biological Control 60 (2012) 132–140
of collection. Fruits were wounded by a sterile nail (3  3  3 mm)
on the equator (one wound per fruit).
2.3. Yeast isolation, identification and preliminary selection for activity
against M. laxa
The strains L1 and L8 used in this study were originally isolated
from the surface of ‘Redhaven’ peaches collected from an organic
orchard of the Agricultural Faculty (University of Bologna, Italy).
A fruit sample was washed in 1 l-beaker containing 200 ml of
phosphate buffer (0.05 M, at pH 6.8) by shaking in a rotary shaker
for 15 min at 140 rpm. The washing was centrifuged for 20 min at
4800 rpm, the supernatant discarded, except for a small amount
(2 ml) of washing that was diluted in serial with phosphate buffer.
One-hundred microliter of the diluted suspensions were trans-
ferred to Petri dishes containing PDA amended with streptomycin
sulfate and neomycin (0.1%) to avoid bacterial isolates. The plates
were incubated at 25 °C for up to 3 days, the developing colonies
were observed under light microscope and yeasts with different
characteristics were selected and purified by triple re-streaking
of single colonies on Nutrient Yeast Dextrose Agar (NYDA: 8 g of
nutrient broth, 5 g of yeast extract, 10 g of dextrose and 15 g of
agar in 1 l of distilled water). A preliminary selection of antagonists
based on their activity against M. laxa was made directly on fruit by
testing the isolates on wounded ‘Springcrest’ peaches and ‘Rita
Stark’ nectarines according to availability, as described by Bonater-
ra et al. (2003). Aliquots of 20 ll of antagonist suspension
(107 CFU ml 1) were introduced into each wound site. After 2 h
at room temperature, the wounds were inoculated with 20 ll of
M. laxa of suspensions containing of 103 conidia ml 1. Fruits trea-
ted with 20 ll of distilled water and inoculated with a pathogen
represented the control. When dried, fruits were incubated at
20 °C for 5 d and the percentages of infected wounds were re-
corded. The sample unit was represented by 10 fruits for each iso-
late of candidate antagonist. The experiment was conducted two
times. Selected antagonists (effectiveness ratings greater than
50%) were used for additional trials in five rounds of trial screening.
After these preliminary trials, two antagonists (L1 and L8) were se-
lected and subsequently identified through microscopic observa-
tion of cell and colony morphology and by sequencing of domain
D1/D2 of 26S ribosomal DNA and the Internal Transcribed Spacers
(ITS 1 and 2 region) according to White et al. (1990) as described
above. Purified colonies were maintained on NYDA slants at 4 °C
until use.
2.4. Antifungal activity of L1 and L8 against M. laxa, M. fructicola and
M. fructigena on fruit stored at 20 °C and 0 °C
The effects of the selected antagonists, L1 and L8, were tested
against M. laxa, M. fructicola and M. fructigena on ‘Stark Red Gold’
nectarines. Each antagonist was grown in 250 ml Erlenmeyer flasks
containing 75 ml NYDB (NYDA without agar) incubated on a rotary
shaker (140 rpm) at 25 °C for 48 h. Yeast cells were harvested by
centrifugation at 4800g for 10 min and re-suspended in sterile dis-
tilled water (SDW). One sample (5 ml) of cells from each isolate
was autoclaved (120 °C per 20 min) and represented the auto-
claved cell suspension (ACS); another sample (5 ml) was centri-
fuged again and re-suspended twice in SDW to remove culture
media and represented the washed cell suspension (WCS)
(108 CFU ml 1). The concentrations of cells in all suspensions were
adjusted to108 CFU ml 1, with a haemocytometer, unless other-
wise stated. The supernatant of the first centrifugation was steril-
ized by filtration with a sterile microfilter (0.45 lm) and
represented the culture filtrate (CF). Fruits were wounded as de-
scribed above and treated with 20 ll of ACS or WCS or CF or sterile
distilled water as control (CK). After air drying (2 h), 20 ll of
103 conidia ml 1 suspensions of each of the three pathogens were
pipetted into the wounds. Fruits were kept at 20 °C after treatment
and inoculation. The disease incidences (percentage of infected
wounds) were recorded 5 d after inoculation. Each treatment was
represented by five fruits and each treatment was replicated five
times. The experiment was conducted three times.
In addition, to determine the effect of storage temperature
(0 °C) on efficacy of L1 and L8, artificially wounded fruits, as de-
scribed above, were treated with 20 ll of a cell suspension of each
antagonist (108 CFU ml 1) and after 2 h at room temperature were
inoculated with the same quantity of a conidial suspension of the
three pathogens (103 conidia ml 1). The control samples were
inoculated only with conidial suspensions of the pathogen. Treated
nectarines were incubated at 0 °C for 21 days and then incubated
at 20 °C for 7 days at which time the percentages of infections were
recorded. The sample unit was represented by four replicates of 20
fruits each. The experiment was conducted two times.
2.5. In vitro antagonistic activity assays
The L1 and L8 selected strains were evaluated for their interac-
tions with M. laxa, M. fructicola and M. fructigena in culture. For this
purpose, disks (6-mm diameter) were cut from PDA dishes and
wells filled with 105-ll of 108 CFU ml 1 of WCS or ACS, or CF of
L1 or L8 strains obtained as described above, or sterile distilled
water as control (Zhang et al., 2008). One hour later, an aliquot
of 35-ll of 105 spores ml 1 of single pathogen was inoculated onto
each well. The dishes were then sealed with parafilm to retain
humidity and to avoid cross-contamination and incubated at
25 °C for 6 days at which time the diameters of the colonies were
recorded. The sample unit was represented by five dishes for each
pathogen and antagonist interactions. The experiment was con-
ducted three times.
2.6. Effect of antagonist concentration on control of M. laxa, M.
fructicola and M. fructigena
Cell suspensions of L1 and L8 yeast were prepared from colonies
grown on NYDA after 48 h at 25 °C. The final concentrations of 106,
107 and 108 CFU ml 1were prepared by dilution with SDW. ‘Guerri-
era’ nectarines were wounded as described above and treated with
20 ll of L1 or L8 treatments. After 2 h air drying, fruits were inoc-
ulated by introducing 20 ll of suspension of each pathogen
(103 conidia ml 1) into the wounds. Fruits were kept at 20 °C for
4 d after inoculation, at which time the disease incidence (percent-
age of infected wounds) was recorded. Each treatment was repre-
sented by five fruits replicated five times. The experiment was
conducted three times.
2.7. Population dynamics of yeasts L1 and L8
In order to study the population dynamics of antagonists, cv
‘Guerriera’, nectarines were wounded as described above and
20 ll of cell suspension (108 CFU ml 1) of L1 or L8 were introduced
into each wound. Treated fruits were kept at 20 °C and collected
after 0, 6, 24, 48, 72, 96, 120 h from treatment or kept at 0 °C
and collected after 0, 7, 14, 21 d plus 7 d at 20 °C (shelf-life). The
number of yeast cells in wounds was determined as described by
Mari et al. (1996) with some modifications. Three samples of fruit
tissue were taken from around the wound with a sterile 10 mm
diameter cork-borer (about 10 mm deep) and transferred into ster-
ile stomacher bags containing 10 ml of SDW and Tween 80 (0.05%)
and homogenized by a stomacher for 10 min (Bag Mixer 400;
Interscience, St Nom, France). The resulting slurry with prior dilu-
tion in SDW was surface-plated on NYDA for enumeration of
antagonist cells. Petri dishes were incubated at 25 °C for 2 d. Each
 M. Mari et al. / Biological Control 60 (2012) 132–140
treatment, containing three fruits, was replicated three times. The
experiment was conducted two times.
2.8. Data analysis
All data regarding infected fruit were subjected to a one-way
analysis of variance (ANOVA) using the statistical package Statisti-
ca for Windows (Statsoft Inc.). Separation of means was performed
using the least significant difference (LSD) test, at P < 0.05. Before
analysis of data, homogeneity of variance was tested by the Krus-
kal–Wallis test. In order to improve the homogeneity of variances,
data of population dynamics (CFU wound 1) were transformed to
logarithms. All experiments were carried out in a completely ran-
domized block design.
3. Results
3.1. Yeast isolation and preliminary selection for activity against M.
laxa
Eighty-six isolates of yeasts and yeast-like organisms, different
in color and shape, were isolated from the surface of ‘Red Haven’
peaches. A selection process was carried out directly on the fruit
by treating wounds of peaches and nectarines with a cell suspen-
sion of the isolates and subsequently inoculating them with
M. laxa. Approximately, 5.8% of these possible antagonists showed
a reduction of brown rot incidence over 50% with respect to un-
treated fruit (data not shown). At the end of five rounds of trials,
two antagonists (L1 and L8) were selected for additional trials be-
cause of their better antifungal activity at the concentration of
107 CFU ml 1. TheL1 strain showed the highest levels of activity
in the control of M. laxa in peaches and nectarines (P93%) while
the L8isolate was also effective, butits activity was lower than that
of L1 (P60%) (Table 1).
3.2. Pathogen and antagonist molecular identification
The sequence analysis of the ITS rDNA regions identified the
three pathogen strains used in this study as M. laxa, M. fructicola
and M. fructigena. Moreover, both the sequencing of D1/D2 domain
and ITS rDNA regions recognized the two antagonist yeast strains
(L1 and L8) as A. pullulans, with a sequence homology percentage
of 100% with A. pullulans CBS 584.75T (accession number
FJ150906) (Fig. 1).
The results of microscopic observations of both fungal conidia
and yeast colony morphology were compared with the identifica-
tion keys of Batra (1991) and Barnett et al. (2000) respectively
and were complementary to the molecular analysis. Our yeast
identifications were also confirmed by Industrial Yeasts Collection,
Department of Applied Biology, Perugia University, Perugia, Italy
(unpublished information).
Table 1
Biocontrol activity of two selected antagonistsa in reducing the incidence of brown rot
caused by Monilinia laxa on peaches and nectarines. Fruits were stored at 20 °C for
5 days.
Treatment ‘Springcrest’ peaches ‘Rita Star’ nectarines
Infected fruits (%) b
EI (%) Infected fruits (%) EI (%)
Control 80ac – 52a –
L1 4c 95 3.8c 93
L8 32b 60 16.7b 67.9
a inoculated with M. laxa significantly reduced the brown rot infec-
Antagonists were applied at 107 CFU ml 1.
b
EI = Effectiveness Index (Control Treated)/Control  100.
c
Data on peaches and nectarines are the mean of three and two trials respec-
tively. Values followed by the same letter are not significantly different by LSD test
(P < 0.05).
135
3.3. In vitro antagonistic activity assays
The effects of both A. pullulans strains applied as WCS, ACS and
CF on growth of all three Monilinia species were investigated in
tests on PDA plates. After 6 days incubation at 25 °C, the diameter
of colonies without antagonist was 38, 68 and 57 mm for M. laxa,
M. fructicola, M. fructigena, respectively (Table 2). Both antagonists
applied as WCS completely inhibited the growth of M. laxa and
M. fructigena, however L1 and L8 WCS reduced M. fructicola colony
diameter by 63.2% and 36.8%, respectively. In addition the CF of
both antagonists significantly inhibited the growth of M. fructigena
with respect to control, reducing growth by 59.6% for L1 and 57.9%
for L8.
3.4. Antifungal activity of L1 and L8 against M. laxa, M. fructicola and
M. fructigena
The activities of L1 and L8 were tested on ‘Star Red Gold’ nectar-
ines at 20 °C. Artificial inoculation of fruits with M. laxa, M. fructi-
cola and M. fructigena (103 conidia ml 1) resulted in 85%, 70%,
and 100% incidence of infection, respectively, after 5 days of incu-
bation (Fig. 2). The washed cells of the antagonists were effective
against all three Monilinia species, completely inhibiting M. laxa
and M. fructicola and reducing M. fructigena by 70% (L1) and 90%
(L8). Autoclaved cell suspensions and culture filtrates of both
antagonists were ineffective against brown rot, since treated fruit
showed an incidence of disease not significantly different from
the control. In fruit treated with L8 ACS and CF and inoculated with
M. fructicola, the incidence of infected fruit was significantly higher
than the control.
In a second trial, ‘Star Red Gold’ nectarines were stored at 0 °C
for 21 days followed by incubation for 7 days at 20 °C. The low
temperature influenced brown rot, since all pathogens remained
quiescent and no disease symptoms were observed on fruit on re-
moval after 3 weeks of storage. When nectarines were moved to
20 °C, M. laxa, M. fructicola and M. fructigena resumed growth and
rotted control fruit to the extent of 65%, 53%, and 49%, respectively,
after 7 d at this temperature (Table 3). Both strains completely
inhibited M. laxa and M. fructicola, while M. fructigena infections
were reduced by 89.8% and 91.2% by L1 and L8, respectively.
3.5. Effect of antagonist concentration on control of M. laxa, M.
fructicola and M. fructigena
Three antagonist cell concentrations (108, 107 and 106 CFU ml 1)
were tested against the three Monilinia species. Both antagonists
provided the highest control of pathogens (0% of infected fruits
after 5 days at 20 °C) at the highest concentration (108 CFU ml 1)
(Fig. 3). When L1 concentrations were reduced by one log unit
(107 CFU ml 1), its efficacy was reduced against M. laxa and
M. fructigena from 100% to 84.6% and 64.3% respectively.
Nevertheless, statistical differences were observed with respect to
reduction of disease obtained at the highest concentration
(108 CFU ml 1). While, fruit treated with L1 at 107 CFU ml 1and
inoculated with M. fructicola were significantly more infected than
fruit treated with L1 108 CFU ml 1and inoculated with the same
pathogen. The middle concentration of L8 (107 CFU ml 1) was inef-
fective for the control of M. fructicola and M. fructigena, showing no
significant differences between treated fruit and the inoculated
controls. Treatment of nectarines with the L8 strain (107 CFU ml 1)
tions with respect to the inoculated control (36% against 76%
respectively). The lowest concentrations of both antagonists
(106 CFU ml 1) were not effective against all the three Monilinia
species.
136 M. Mari et al. / Biological Control 60 (2012) 132–140

Fig. 1. Nucleotide sequences comparison of the internal transcribed spacer ITS 1 and 2 region of Aureobasidium pullulans yeast (strains L1 and L8) with the sequence of A.
pullulans CBS 584.75T, accession number FJ150906, identification in BLASTN search. The alignment was performed using ClustalX program.
 M. Mari et al. / Biological Control 60 (2012) 132–140 137

Table 2
Effect of L1 and L8 Aureobasidium pullulans strains on the growth of Monilinia spp. on PDA plates. Colony diameter was measured after 6 days of incubation at 25 °C. Each values is
the means of five plates (replicates) ± standard errors. In the same column data followed by asterisk are significantly different from the control by LSD test (P < 0.05).

Treatments Monilinia laxa Monilinia fructicola Monilinia fructigena

L1 WCS (108 CFU ml 1
) 0 ± 0 27 ±7.13 0 ± 0
L1 ACS 36 ± 1.8 66 ± 0.75 56 ± 0.25
L1 CF 36 ± 1.05 68 ± 0.95 23 ± 0.63
L8 WCS (108 CFU ml 1
) 0 ± 0 43 ± 3.55 0 ± 0
L8 ACS 37 ± 0.34 65 ± 0.63 54 ± 0.87
L8 CF 36 ± 2.29 68 ± 0.41 24 ± 0.75
Control 38 ± 1.60 68 ± 0.75 57 ± 1.03

Table 3
Effect of cold storage on Aureobasidium pullulans L1 and L8 strainsa activity against
Monilinia rot on ‘Star Red Gold’ nectarines. Infected fruits (%) after 21 d at 0 °Cb + 7 d
at 20°C.

Pathogen Control L1 L8
Monilinia laxa 65 ± 5ax 0b 0b
Monilinia fructicola 53 ± 19.2a 0b 0b
Monilinia fructigena 49 ± 12.9a 5 ± 5b 4.3 ± 6.2b
a
Antagonists were applied at 108 CFU ml 1.
b
On removal from cold storage no fruit showed brown rot symptoms.
x
Data are the mean of four replicates of 20 fruit each. Within the same row,
values followed by the same letter are not significantly different by LSD test
(P 60.05).

However, no significant differences were observed between the

two antagonist populations. On fruits stored at 0 °C, the popula-
tions of L1 and L8 remained the same during the first 7 days after
treatment, then increased until the 21st day of storage in refriger-
ated temperature, reaching 1.7  106 CFU ml 1 and
2  106 CFU ml 1 respectively. When fruits were moved to 20 °C,
the populations declined slowly to 1.5  106 CFU ml 1 for L1 and
1.3  106 CFU ml 1 for L8. There was no significant difference be-
tween the L1 and L8 A. pullulans strains in this experiment.

4. Discussion

Two strains, L1 and L8, of A. pullulans isolated from the surface

Fig. 2. Effect of two strains of Aureobasidium pullulans on control of (1) Monilinia
laxa, (2) Monilinia fructicola, (3) Monilinia fructigena in wounded ‘Star Red Gold’
nectarines. Data were recorded after 5 days of incubation at 20 °C. Autoclaved cell
suspensions contained 1  108 CFU ml 1 (ACS); washed cell suspensions contained
1  108 CFU ml 1 (WCS) and culture filtrates (CF) were used. The controls were
treated with sterile distilled water. The data are the means of four replicates of five
fruits each, 20 fruits per treatment. For each pathogen, values followed by different
letters (capital letter – L1; lower-case – L8) were significantly different according to
LSD test, P 6 0.05.
3.6. Population dynamics of yeasts L1 and L8
The populations of A. pullulans strains L1 and L8 in the wounds
of nectarines stored at 20 °C doubled during the first 3 days (Fig. 4),
reaching 3.7  106 CFU ml 1 and 2.7  106 CFU ml 1 for L1 and L8,
respectively. The concentration of L8 in wounds peaked
(4.4  106 CFU ml 1) at the end of trial, 7 days after application,
while theL1 population remained constant (3.7  106 CFU ml 1).
of ‘Redhaven’ peaches, were identified in this study by molecular
tools and assayed for their activity against three Monilinia spp.
The sequencing of the D1/D2 domain of the large subunit (26S)
ribosomal DNA was previously used to identify Aureobasidium
(Sasahara and Izumori, 2005) and the results were confirmed by
sequencing of the ribosomal regions ITS1 and ITS2.
Various isolates and strains of Aureobasidium sp. have been re-
ported to be effective against a number of postharvest diseases
on fruits such as sweet cherries (Schena et al., 2003), strawberries
(Lima et al., 1997) and apples (Lebinger et al., 1997), but few stud-
ies have reported the activity of Aureobasidium sp. against M. laxa
on peaches (Zhang et al., 2010) and M. fructicola on nectarines
(Janisiewicz et al., 2010). To our knowledge, this is the first
study demonstrating the biocontrol of M. laxa, M. fructicola and
M. fructigena in the same experiments by the same antagonists
and there are no reports on the control of M. fructigena on peaches
and nectarines by microbial antagonists.
The most important pathogen responsible for brown rot of
stone fruits in Europe is M. laxa, followed by M. fructigena. Larena
et al. (2005) showed that in stone fruit affected by brown rot,
10–15% of infection was caused by M. fructigena, and in our moni-
toring work on Italian populations of Monilinia spp., 7% of decay
was caused by M. fructigena out of a sample of 125 brown rotted
fruits (data not reported). Since M. fructicola is now present in
138 M. Mari et al. / Biological Control 60 (2012) 132–140
Fig. 3. Effect of different concentrations of two strains (t L1 and L8) of
Aureobasidium pullulans on control of (1) Monilinia laxa, (2) Monilinia fructicola,
(3) Monilinia fructigena in wounded ‘Star Red Gold’ nectarines. Data were recorded
after five days of incubation at 20 °C. (A) 108; (B) 107; (C) 106 CFU ml 1 cell
suspension. The controls were treated with sterile distilled water. The data are the
means of five replicates of five fruits each, 25 fruits per treatment. For each
pathogen, values followed by different letters (capital letter – L1; lower-case – L8)
were significantly different according to LSD test, P 6 0.05.
European stone fruit orchards together with M. laxa and M. fructi-
gena, testing the antagonist activity against all three pathogens
could be a necessity to develop new biofungicides. BCAs screening
can be carried out by in vitro and in vivo trials (Janisiewicz and
Korsten, 2002), although in vivo trials are preferable as they are
generally more reliable and transferable to field and postharvest
conditions. In addition, the antagonist effectiveness in vitro and
in vivo cannot always be compared if the predominant BCAs mech-
anism of action is the competition for nutrient and space, as in vitro
tests do not reflect the nutritional environment at the host wound
site. In our experiments, yeast-like microorganisms isolated from
the carposphere of peaches were first assayed directly on peaches
and nectarines and only a small percentage of antagonists proved
to be effective against brown rot (data not shown). The in vitro tri-
als were made to evaluate the direct interaction between antago-
nists and pathogens without hosts (peach). The test does not
take into account the fruit and its nutritional environment,
although the test is fast and simple, it has to be considered as pre-
liminary and useful only to confirm the antagonist activity of BCA
observed in vivo. The in vitro results were slightly comparable with
those obtained in vivo; in fact, washed cells showed a significant
Fig. 4. Population dynamics of two strains (L1 and L8) of Aureobasidium pullulans in
wounds of ‘Star Red Gold’ nectarines incubated at 20 °C for 7 days (A) and for
21 days at 2 °C + 7 days at 20 °C (B). Each point represents the mean of the number
of colony forming units (CFUs) from three replicated fruits + error bars, each plated
in triplicate at each sampling time.
control of all Monilinia spp. with respect to the control, although
the concentration of pathogens was higher (105 conidia ml 1) than
that used for the in vivo trials (103 conidia ml 1), furthermore auto-
claved cells showed no inhibitory effects on pathogens. Culture fil-
trates of both antagonists reduced significantly only the colony
diameter of M. fructigena. These results appear quite different from
those obtained in vivo trials, since washed cells of L1 and L8 Aure-
obasidium strains completely controlled M. laxa and M. fructicola on
peaches after 5 days at 20 °C and no reduction of brown rot was
observed in fruit treated with both culture filtrates of antagonists.
Our data suggest that the two antagonists, grown on PDA, might
produce metabolites toxic to M. fructigena different from that pro-
duced in vivo; similar results were also obtained by Spadaro et al.
(2002) with four isolates of the yeast M. pulcherrima against post-
harvest pathogens on apples. Additional investigations are re-
quired to understand this different behavior, although our data
confirmed the discrepancy between in vitro and in vivo tests as re-
ported by other authors (Janisiewicz, 1987; Dal Bello et al., 2008)
suggesting that the in vitro trials are generally carried out to a pre-
liminary evaluation of BCAs mode of action. Loss of activity by cul-
ture filtrate is in agreement with results obtained with
Aureobasidium by Zhang et al. (2010), prompting the exclusion of
toxic metabolite production as a mode of action of the antagonist.
However, an antifungal cyclic depsipeptide (aureobasidin A) pro-
duced by A. pullulans was found to inhibit spore germination, germ
tube elongation and hyphal growth of M. fructicola in a in vitro
study (Xiaoping et al., 2007), although the authors tested pure
product and the experimental conditions applied were not compa-
rable with those in the present work. Previous studies on the
modes of action showed that Aureobasidium spp. act against fungal
pathogens through competition for nutrients (Bencheqroun et al.,
 M. Mari et al. / Biological Control 60 (2012) 132–140
2007) and resistance induction (Ippolito et al., 2000), while no di-
rect physical interaction (parasitism) was observed between
antagonist cells and pathogen hyphae (Castoria et al., 2001). Our
preliminary data on the antifungal activity of A. pullulans strains
L1 and L8 exclude antibiotic production in the in vivo tested condi-
tions, although their mode of action has not yet been investigated
in depth. The production of toxic metabolites by antagonists could
have a negative environmental impact and for this reason yeasts or
yeast-like microorganisms are preferred to bacteria such as Bacillus
sp. and Pseudomonas sp. (Sharma et al., 2009). The biocontrol activ-
ity of the tested A. pullulans strains against Monilinia spp. was
dependent on cell concentrations. In all cases, the highest concen-
tration of the antagonist (108 CFU ml 1) provided the best control
of all tested pathogens, although no significant difference was
found in disease control in fruit treated with L1 at a lower concen-
tration (107 CFU ml 1) and inoculated with M. laxa and M. fructige-
na. Our results are in agreement with other studies that considered
107–108 cells ml 1 the antagonist concentrations most appropriate
to obtain a high decay reduction (McLaughlin et al., 1990; El-
Ghaouth et al., 2004) and higher concentrations are rarely
required. M. fructigena was controlled by L1 and L8 less than the
other two Monilinia species; its inhibition rate was, in fact, 70%
and 90% for L1 and L8 respectively. An explanation of the lower
antagonist activity against M. fructigena could be related to the
conidia size of this pathogen, larger (average 21  13 lm) than
conidia of M. laxa and M. fructicola (average 15  9 lm) (Mordue,
1979). For this reason, M. fructigena conidia could compete more
effectively for the space towards L1 and L8 A. pullulans strains.
However the large size of M. fructigena conidia suggests also a
higher content of nutrient reserves that may play an important role
in the competition for nutrients. However both of antagonists are
effective against Monilinia spp., L1 seems more active than L8,
when applied at the concentrations lower than 108 CFU ml 1
(Fig. 3). In the future, the use of the antagonist mixtures could be
an attractive way to increase the range of the pathogens controlled,
exploiting an eventual additive effect. As previously reported by
Weiss et al. (2006), the active ingredients of Blossom Protect FBTM
were two strains of A. pullulans (CF10 and CF40) isolated from
apple surface in southwest Germany. In addition, as L1 and L8
belong at the same species, they will need only one registration,
saving time and money.
Few data are currently available regarding the antagonist–path-
ogen interaction in terms of competition for limiting nutrients
essential for pathogenesis (Sharma et al., 2009). However, it ap-
pears that a rapid colonization of fruit wounds by the antagonist
is critical for decay control. Previous studies indicated that yeast
populations such as Rhodotorula glutinis (Fresen.) F.C. on orange in-
creased by approximately 10,000-fold during the first 3 days at
20 °C and 40,000-fold after 7 days 4 °C (Zheng et al., 2005). A
marked increase of M. pulcherrima and Hanseniaspora uvarum (Nie-
haus) populations was also observed on apples (Piano et al., 1997)
and grapes (Liu et al., 2010) respectively. In contrast, in the present
work, A. pullulans population increased weakly in wound cavities of
fruit both at 20 °C and 0 °C, and L1 and L8 growth rate was compa-
rable to that of A. pullulans L47 isolate, observed on apple wounds
by Ippolito et al. (2000). Nevertheless, in both studies the results
obtained showed good control (more than 70%) of fruit decay, sug-
gesting that A. pullulans can adapt well to the wound environment
and inhibit intrusion from pathogens.
Aureobasidium sp. is part of the microbial flora commonly resid-
ing on stonefruit and pomefruit growing in temperate regions (De
Hoog and Yurlova, 1994). Its use as a biocontrol agent of posthar-
vest diseases of fruit can be promising only if it can be combined
with routine postharvest practices. In this study, the suitability of
L1 and L8 strains to critical postharvest conditions such as low
temperature was demonstrated, since the antagonists survived in
139
the fruit wounds for long periods of time at 0 °C without showing
a reduction in population; indeed a moderate increase of viable
cells was observed after 21 days at 0 °C. The efficacy of L1 and L8
strains in decay control was also tested in a trial simulating fruit
commercial conditions (cold storage followed by simulation of re-
tail at ambient temperature) in ‘Star Red Gold’ nectarines stored at
0 °C for 21 days plus 9 days of shelf-life at 20 °C. In general, low
temperature storage delays fungal growth, while pathogens re-
sume growth and develop rapidly into decay lesions when fruits
are moved from the refrigerated room to the handling and market-
able areas. ‘Star Red Gold’ is a cultivar suited to long storage peri-
ods and can be stored for 3 weeks or more at 0 °C without
remarkable detrimental effects on quality parameters. No decay
was observed on control fruits previously inoculated with M. laxa,
M. fructicola and M. fructigena at the end of cold storage (21 days at
0 °C), while a high percentage of infections (65%, 53%, and 49%
respectively) developed in these fruit safer 7 days at 20 °C. Both
of our strains were very effective in brown rot control, since their
application completely inhibited M. laxa and M. fructicola infections
after both cold storage and shelf-life and markedly reduced M. fruc-
tigena rots (>90%). These findings make biocontrol of brown rot
feasible under commercial conditions after an appropriate formu-
lation of the assayed antagonists.
In summary, the present study identified for the first time two
antagonists (L1 and L8 A. pullulans strains) as active ingredients for
the development of biofungicides for postharvest application
against three Monilinia species, all responsible for high economic
losses in stone fruit.
References
Barnett, J.A., Payne, R.W., Yarrow, D., 2000. Yeasts: Characteristics and
Identification. Cambridge University Press, UK, 1139 pp.
Batra, L.R., 1991. World Species of Monilinia (fungi) Their Ecology, Biosystematics
and Control. Mycologia Memoir No. 16. J. Cramer, Berlin.
Bencheqroun, S.K., Bajji, M., Massart, S., Labhilili, M., El Jaafari, S., Jijakli, M.H., 2007.
In vitro and in situ study of postharvest apple blue mold biocontrol by
Aureobasidium pullulans: evidence for the involvement of competition for
nutrients. Postharvest Biology and Technology 46, 128–135.
Bonaterra, A., Mari, M., Casalini, L., Montesinos, E., 2003. Biological control of
Monilinia laxa and Rhizopus stolonifer in postharvest of stone fruit by Pantoea
agglomerans EPS125 and putative mechanisms of antagonism. International
Journal of Food Microbiology 84, 93–104.
Casals, C., Vinas, I., Landl, A., Picouet, P., Torres, R., Usall, J., 2010. Applications of
radio frequency heating to control brown rot on peaches and nectarines.
Postharvest Biology and Technology 58, 218–224.
Castoria, R., De Curtis, F., Lima, G., Caputo, L., Pacifico, S., De Cicco, V., 2001.
Aureobasidium pullulans (LS-30) an antagonist of postharvest pathogens of
fruits: study on its modes of action. Postharvest Biology and Technology 22, 7–
17.
Dal Bello, G., Monaco, S., Rollan, M.C., Lampugnani, G., Arteta, N., Abramoff, C.,
Ronco, L., Stocco, M., 2008. Biocontrol of postharvest grey mould on tomato by
yeasts. Journal of Phytopathology 156, 257–263.
De Cal, A., Gell, I., Usall, J., Viñas, I., Melgarejo, P., 2009. First report of brown rot
caused by Monilinia fructicola in peach orchards in Ebro Valley, Spain. Plant
Disease 93, 763.
De Hoog, G.S., Yurlova, N.A., 1994. Conidiogenesis, nutritional physiology and
taxonomy of Aureobasidium and Hormonema. Antonievan Leeuwenhoek 65, 41–
54.
El-Ghaouth, A., Wilson, C.L., Wisniewski, M.E., 2004. Biologically based alternatives
to synthetic fungicides for the postharvest diseases of fruit and vegetables. In:
Naqvi, S.A.M.H. (Ed.), Diseases of Fruit and Vegetables, vol. 2. Kluwer Academic
Publishers, The Netherlands, pp. 511–535.
EPPO Bulletin, 2003. Moniliniafructicola, vol. 33, pp. 281–288.
Gonzalez, V., Tello, M.L., 2011. The endophytic mycota associated with Vitis vinifera
in central Spain. Fungal Diversity 47, 29–42.
Grebenisan, I., Cornea, P., Meteescu, R., Cimpeanu, C., Olteanu, V., Campenu, Gh.,
Stefan, L.A., Oancea, F., Lupu, C., 2008. Metschnicowia pulcherrima, a new yeast
with potential for biocontrol of postharvest fruit rots. Acta Horticulturae 767,
355–360.
Gregori, R., Borsetti, F., Neri, F., Mari, M., Bertolini, P., 2008. Effects of potassium
sorbate on postharvest brown rot of stone fruit. Journal of Food Protection 71,
1626–1631.
Hong, C., Holtz, B.A., Morgan, D.P., Michailides, T.J., 1997. Significance of thinned
fruit as a source of the secondary inoculum of Monilinia fructicolain California
nectarine orchards. Plant Disease 81, 519–524.
140 M. Mari et al. / Biological Control 60 (2012) 132–140
Iotti, M., Zambonelli, A., 2006. A quick and precise technique for identifying
ectomycorrhizas by PCR. Mycological Research 110, 60–65.
Ippolito, A., Ghaouth, A.E., Wilson, C.L., Wisniewski, M., 2000. Control of postharvest
decay of apple fruit by Aureobasidium pullulans and induction of defense
response. Postharvest Biology and Technology 19, 265–272.
Janisiewicz, W.J., 1987. Postharvest biological control of blue mold on apples.
Phytopathology 77, 481–485.
Janisiewicz, W.J., Korsten, L., 2002. Biological control of postharvest diseases of
fruits. Annual Review of Phytopathology 40, 411–441.
Janisiewicz, W.J., Kurtzman, C.P., Buyer, J.S., 2010. Yeasts associated with nectarines
and their potential for biological control of brown rot. Yeast 27, 389–398.
Karabulut, O.A., Baykal, N., 2003. Biological control of postharvest diseases of
peaches and nectarines by yeasts. Journal of Phytopathology 151, 130–134.
Karabulut, O.A., Gabler, F.M., Mansour, M., Smilanick, J.L., 2004. Postharvest ethanol
and hot water treatments of table grapes to control gray mold. Postharvest
Biology and Technology 34, 169–177.
Karabulut, O.A., Smilanick, J.L., Crisosto, C.H., Palou, L., 2010. Control of brown rot of
stone fruits by brief heated water immersion treatments. Crop Protection 29,
903–906.
Larena, I., Torres, R., De Cal, A., Linan, M., Melgarejo, P., Domenichini, P., Bellini, A.,
Mandrin, J.F., Lichou, J., Ochoa de Eribe, X., Usall, J., 2005. Biological control of
postharvest brown rot (Monilinia spp.) of peaches by field applications of
Epicoccum nigrum. Biological Control 32, 305–310.
Lebinger, W., Breuker, B., Hahn, M., Mendgen, K., 1997. Control of postharvest
pathogens and colonization of the apple surface by antagonistic
microorganisms in the field. Phytopathology 87, 1103–1110.
Lichou, J., Mandrin, J.F., Breniaux, D., Mercier, V., Giauque, P., Desbrus, D., Blanc, P.,
Belluau, E., 2002. Une nouvelle moniliose. Phytoma 547, 22–25.
Lima, G., Ippolito, A., Nigro, F., Salerno, M., 1997. Effectiveness of Aureobasidium
pullulans and Candida oleophila against postharvest strawberry rots. Postharvest
Biology and Technology 10, 169–178.
Lima, G., Arru, S., De Curtis, F., Arras, G., 1999. Influence of antagonist, host fruit and
pathogen on the biological control of postharvest fungal diseases by yeasts.
Journal of Industrial Microbiology and Biotechnology 23, 223–229.
Liu, H.M., Guo, J.H., Luo, L., Liu, P., Wang, B.Q., Cheng, Y.J., Deng, B.X., Long, C.A., 2010.
Improvement of Hanseniaspora uvarum biocontrol activity against grey mold by
the addition of ammonium molybdate and the possible mechanisms involved.
Crop Protection 29, 277–282.
Mari, M., Guizzardi, M., Pratella, G.C., 1996. Biological control of gray mold in pears
by antagonistic bacteria. Biological Control 7, 30–37.
Mari, M., Leoni, O., Bernardi, R., Neri, F., Palmieri, S., 2008. Control of brown rot on
stonefruit by synthetic and glucosinolate-derived isothiocyanates. Postharvest
Biology and Technology 47, 61–67.
McLaughlin, R.J., Wilson, C.L., Chalutz, E., Kurtzman, W.F., Osman, S.F., 1990.
Characterization and reclassification of yeasts used for biological control of
postharvest diseases of fruit and vegetables. Applied and Environmental
Microbiology 56, 3583–3586.
Mordue, J.E.M., 1979. CMI Descriptions of Pathogenic Fungi and Bacteria.
Commonwealth Mycological Institute, Ferry Lane, Kew, Surrey, England,
616,617,619.
Neri, F., Mari, M., Brigati, S., Bertolini, P., 2007. Fungicidal activity of plant volatile
compounds for controlling Monilinia laxa in stone fruit. Plant Disease 91,
30–35.
Ogawa, J.M., Zehr, E.I., Biggs, A.R., 1995. Diseases caused by fungi. In: Ogawa, J.M.,
Zehr, E.I., Bird, G.W., Ritchie, D.E., Uriu, K., Uyemoto, J.K. (Eds.), Compendium of
Stone Fruit Diseases. American Phytopathological Society, St. Paul, Minn, pp. 7–
10.
Pellegrino, C., Gullino, M.L., Garibaldi, A., Spadaro, D., 2009. First report of brown rot
of stone fruit caused by Monilinia fructicola in Italy. Plant Disease 93, 668.
Piano, S., Neyrotti, V., Migheli, Q., Gullino, M.L., 1997. Biocontrol capability of
Metschnikowia pulcherrima against Botrytis cinerea postharvest rot of apple.
Postharvest Biology and Technology 11, 131–140.
Sasahara, H., Izumori, K., 2005. Production of L-sorbitol from L-fructose by
Aureobasidium pullulans LP23 isolated from soy sauce mash. Journal
Bioscience Bioengineering 100, 223–226.
Schena, L., Nigro, F., Pentimone, I., Ligorio, A., Ippolito, A., 2003. Control of
postharvest rots of sweet cherries and table grapes with endophytic isolates
of Aureobasidium pullulans. Postharvest Biology and Technology 30, 209–
220.
Sharma, R.R., Singh, D., Singh, R., 2009. Biological control of postharvest diseases of
fruits and vegetables by microbial antagonists: a review. Biological Control 50,
205–221.
Spadaro, D., Vola, R., Piano, S., Gullino, M.L., 2002. Meccanisms of action and efficacy
of four isolates of the yeast Metschnikowia pulcherrima active against
postharvest pathogens on apples. Postharvest Biology and Technology 24,
123–124.
Weiss, A., Gudrun, M., Kunz, S., 2006. Development of ‘Boni-Protect’ a Yeast
Preparation for use in the Control of Postharvest Diseases of Apple. <http://
orgprints.org/8838/>.
White, T.J., Bruns, T., Lee, S., Taylor, J., 1990. Amplification and direct sequencing of
fungal ribosomal RNA genes for phylogenetics. In: Innis, M.A., Gelfans, D.H.,
Sninsky, J.J., White, T.s. (Eds.), PCR Protocols. A Guide to Methods and
Applications. Academic Press, San Diego, CA, USA, pp. 315–322.
Xiaoping, L., Jiye, W., Ping, G., Cungui, M., Zhu, Z.R., Hongye, L., 2007. In vitro
inhibition of postharvest pathogens of fruit and control of gray mold of
strawberry and green mold of citrus by aureobasidin A. International Journal of
Food Microbiology 119, 223–229.
Yao, H.J., Tian, S.P., 2005. Effect of a biocontrol agent and methyl jasmonate on
postharvest diseases of peach fruit and the possible mechanisms involved.
Journal of Applied Microbiology 98, 941–950.
Zhang, H., Wang, L., Dong, Y., Jiang, S., Zhang, H., Zheng, X., 2008. Control of
postharvest pear diseases using Rhodotorula glutinis and its effects on
postharvest quality parameters. International Journal of Food Microbiology
126, 167–171.
Zhang, D., Spadaro, D., Garibaldi, A., Gullino, M.L., 2010. Selection and evaluation of
new antagonists for their efficacy against postharvest brown rot of peaches.
Postharvest Biology and Technology 55, 174–181.
Zheng, X.D., Shang, H.Y., Sun, P., 2005. Biological control of postharvest green mold
decay of oranges by Rhodotorula glutinis. European Food Research Technology
220, 353–357.