Characteristic of Monilinia spp. fungi causing brown rot of pome and stone fruits in Poland

Abstract

Brown rot, caused by fungi belonging to the genus Monilinia, is one of the most important diseases of stone and pome trees in the world. During the summers of 2010 and 2011, a total of 670 Monilinia spp. isolates were obtained from infected fruits. They were collected from different commercial stone and pome fruit orchards, located in northern, southern and central Poland. All isolates were identified using multiplex PCR. Twenty isolates obtained from plum, peach and apple fruits were identified as M. polystroma and 5 isolates from plums as M. fructicola. The remaining isolates were identified as M. fructigena or M. laxa. The identification of the isolates was also confirmed on the basis of growth characteristics in culture according to the EPPO standard PM 7/18. A comparison of morphological features of four Monilinia spp. growing on two selective growth media, APDA-F500 and CHA, indicated significant differences between these species. In artificial inoculation of fruits, all the examined Monilinia spp. isolates were pathogenic. The species affiliation of M. polystroma and M. fructicola isolates collected from orchards in Poland was confirmed on the base of phylogenetic and sequence analysis of the internal transcribed spacer (ITS1/5.8S rDNA/ITS2) region of ribosomal DNA.

Full text

Characteristic of Monilinia spp. fungi causing brown rot
of pome and stone fruits in Poland
Anna Poniatowska &Monika Michalecka &
Anna Bielenin
Accepted: 15 November 2012 /Published online: 29 November 2012
#KNPV 2012
Abstract Brownrot,causedbyfungibelongingto
the genus Monilinia, is one of the most important
diseases of stone and pome trees in the world.
During the summers of 2010 and 2011, a total of
670 Monilinia spp. isolates were obtained from
infected fruits. They were collected from different
commercial stone and pome fruit orchards, located
in northern, southern and central Poland. All iso-
lates were identified using multiplex PCR. Twenty
isolates obtained from plum, peach and apple fruits
were identified as M.polystroma and 5 isolates
from plums as M.fructicola. The remaining iso-
lates were identified as M.fructigena or M.laxa.
The identification of the isolates was also con-
firmed on the basis of growth characteristics in
culture according to the EPPO standard PM 7/18.
A comparison of morphological features of four
Monilinia spp.growingontwoselectivegrowth
media, APDA-F500 and CHA, indicated signifi-
cant differences between these species. In artificial
inoculation of fruits, all the examined Monilinia
spp. isolates were pathogenic. The species affilia-
tion of M.polystroma and M.fructicola isolates
collected from orchards in Poland was confirmed
on the base of phylogenetic and sequence analysis
of the internal transcribed spacer (ITS1/5.8S
rDNA/ITS2) region of ribosomal DNA.
Keywords Stone and pome fruits .PCR .ITS
sequencing .Pathogenicity test .Morphological
characterization
Introduction
Brown rot of fruit trees occurs in all major fruit grow-
ing areas of the world. The disease causes significant
losses in pome and stone fruit crops each year. Typical
disease symptoms, caused by fungi belonging to the
genus Monilinia Honey (anamorph Monilia), are sim-
ilar on all stone and pome trees, and include blossom
and twig blight, cankers and fruit rot. Among the
over 30 species within this genus, three are con-
sidered to be of great economic importance in fruit
production, and include M.laxa,M.fructigena and
M.fructicola.M.laxa and M.fructigena are com-
mon pathogens in Europe, while M.fructicola is
known mainly in some countries of Asia, North,
Central and South America, as well as in Australia
and New Zealand. Based on the sequence analysis
of ribosomal DNA internal transcribed spacer
(ITS) region of M.fructigena ribosomal DNA
(Fulton et al. 1999) and morphological differences
between different M.fructigena isolates originating
Eur J Plant Pathol (2013) 135:855–865
DOI 10.1007/s10658-012-0130-2
A. Poniatowska :M. Michalecka (*):A. Bielenin
Pomology Division, Research Institute of Horticulture,
Pomologiczna 18 Str,
96-100 Skierniewice, Poland
e-mail: monika.michalecka@inhort.pl
A. Poniatowska
e-mail: anna.poniatowska@inhort.pl

from Japan and Europe, van Leeuwen et al. (2002)
identified the new species M.polystroma.Further-
more, other differences between these species were
identified based on polymorphisms observed in a
DNA region with unknown function (Petróczy and
Palkovics 2009). In 2010 M.polystroma was also
reported in China (Zhu and Guo 2010). As a conse-
quence of increased international trade of fruit and nurs-
ery material, the risk of spreading M.fructicola and
M.polystroma in Europe has increased. M.fructicola
is a quarantine pest within the EU (EPPO A2 list).
During the last few years this species was detected
in 11 countries in Central and Western Europe.
M.polystroma was recently detected in a single
commercial orchard in Hungary (Petróczy and
Palkovics 2009). M.fructicola infects a wide range
of rosaceous stone fruit trees and to a lesser extent
apple (Malus spp.) and pear trees (Prunus spp.).
Moreover, the fungus has also been found on
flowering quinces (Chaenomeles spp.), hawthorns
(Crataegus spp.), quinces (Cydonia spp.), loquat
(Eriobotrya japonica), blackberries (Rubus fruticosus)
(CABI/EPPO 2010; OEPP/EPPO 2010) and grapes
(Vitis vinifera) (OEPP/EPPO 1997). M.polystroma
was previously isolated only from apples (van Leeuwen
et al. 2002; Petróczy and Palkovics 2009) and plums
(Zhu and Guo 2010).
Molecular detection of Monilinia spp. is mainly
based on the analysis of the ribosomal ITS region
(Fulton et al. 1999; Snyder and Jones 1999; Hughes
et al. 2000; Ioos and Frey 2000; Förster and Adaska-
veg 2000; Gell et al. 2007). Côté et al. (2004) devel-
oped an identification method using multiplex PCR,
which enables the differentiation of four Monilinia
species (M.laxa,M.fructigena,M.polystroma and
M.fructicola) on the basis of different lengths of PCR
products derived from a genomic region of unknown
function. More recently, a real-time PCR method has
been developed that allows for the differentiation of
these four brown rot-causing Monilinia spp. (van
Brouwershaven et al. 2010). Morphological identifi-
cation is primarily based on the examination of cultur-
al characters on the PDA medium, according to the
key described by Lane (2002) or to the EPPO standard
PM 7/18. Amiri et al. (2009) have developed a new
selective medium for isolation and enumeration of
M.fructicola,M.laxa and M.fructigena, however,
no research has been conducted to determine whether
morphological differences among the four Monilinia
spp. can be detected on this selective medium. Van
Leeuwen et al. (2002) showed that the differences in
spore size and the intensity of stroma formation on
CHA medium is a good discriminating morphological
character between M.polystroma and M.fructigena
species.
The objective of this study was to determine the
primary causal agents of brown rot disease in Poland
through the analysis of molecular and morphological
characteristics. This study provides the first report of
M.polystroma and M.fructicola in Poland. The ability
to properly identify and characterize Monilinia species
associated with development of brown rot will be
helpful for the determination of the pathogen preva-
lence and potential for economic damage in this re-
gion, which is necessary for establishing effective
disease control measures.
Material and methods
Sample collection and fungus isolation
The Monilinia spp. fungi were isolated from infected
apple, peach, cherry, sour cherry and plum fruits
showing typical brown rot symptoms. A total of 670
isolates was collected from various geographical
regions in Poland during the summers of 2010 and
2011. From each fruit sample, a small fragment from
the border of healthy and diseased parenchymal tissue
was aseptically excised and placed on 4% potato dex-
trose agar medium (PDA, Difco™, Becton Dickinson,
Sparks, MD, USA) or on acidified PDA medium
(pH=3.6) amended with fosetyl-aluminum (APDA-
F500 medium, Amiri et al. 2009). The samples
were incubated at 22°C until fungal mycelium
growth was observed.
DNA extraction and amplification in multiplex PCR
Total fungal DNAwas extracted from the Monilinia spp.
isolates cultivated on PDA, following the protocol pro-
posed by Aljanabi and Martinez (1997). The quality and
purity of DNA were evaluated on the basis of UV spec-
trum using Nano-Drop Spectrophotometer ND-1000
with v.3.1.2 software. Subsequently, DNA was amplified
in multiplex PCR using the reverse primer MO368-5 (5′-
GCAAGGTGTCAAAACTTCCA-3′), which is specific
for Monilinia spp., and three species-specific forward
856 Eur J Plant Pathol (2013) 135:855–865

primers: MO368-8R (5′-AGATCAAACATCGTC-
CATCT-3′,forM.fructigena and M.polystroma),
MO368-10R (5′-AAGATTGTCACCATGGTTGA-3′,
for M.fructicola)andLaxa-R2(5′-TGCACATCA-
TATCCCTCGAC-3′,forM.laxa) (Côté et al. 2004), in
order to amplify the non–coding region of Monilinia
spp. with unknown function. PCR mixtures were pre-
pared in a reaction volume of 15 μl, containing 15 ng of
genomic DNA, 0.2 μM of each primer, 200 μMofeach
dNTP (Fermentas, Vilnius, Lithuania), 2.5 mM MgCl
2
and 0.4U of DyNAzyme™II DNA Polymerase (Finn-
zymes, Espoo, Finland) in 1x Optimized DyNAzyme™
Buffer. Amplification reactions were carried in a
Biometra T3000 thermocycler (Biometra, Germany),
using amplification conditions consisting of denatur-
ation at 94°C for 2 min, followed by 35 cycles of
denaturation step at 94°C for 30 s, an annealing step at
60°C for 20 s, and an extension step at 72°C for 1 min,
with a final extension step at 72°C for 3 min. In order to
verify the applicability of the multiplex PCR assay in
identification of unknown samples, four reference iso-
lates of Monilinia spp. were used in amplification reac-
tions: M.fructicola (CBS 127259), M.laxa (CBS
488.50), M.fructigena (CBS 101500) and M.poly-
stroma (CBS 122306). The PCR products specific for
M.laxa,M.fructigena,M.fructicola and M.polystroma
were separated by electrophoresis in 2% agarose gels
run in 1x Tris-Borate-EDTA (TBE) buffer. The gels
were stained with ethidium bromide and the products
were visualized and photographed under UV light.
PCR amplification of rDNA-ITS region
from Monilinia spp. isolates
The species affiliation of all M.fructicola and M.poly-
stroma isolates was also confirmed by a second PCR
assay. The DNA of M.fructicola was amplified in reac-
tions using the primer ITS4Mfcl (5′-TGGGTTTTGGCA-
GAAGCACACT-3′), while DNA of M.polystroma was
amplified with the ITS4Mfgn primer (5′-
GGTGTTTTGCCAGAAGCACACT-3′, Ioos and Frey
2000). Both primers were complementary to the
corresponding ITS1-5.8S-ITS2 region of M.fructicola
and M.polystroma, respectively, and were used in PCR
with the common ITSMonilia primer (5′-GGTA-
GACCTCCCACCCTTGTGTA-3′; Petróczy and Pal-
kovics 2009; Petróczy et al. 2012). PCR mixtures were
prepared in reaction volume of 50 μl, containing 15 ng of
genomic DNA, 0.2 μM of each primer, 200 μM of each
dNTP, 2.5 mM MgCl
2
, and 0.4 U of DyNAzyme™
Polymerase (Finnzymes, Espoo, Finland) in 1x Optimized
DyNAzyme™Buffer. The amplification parameters were:
an initial denaturation at 94°C for 2 min, followed by 35
cycles with denaturation at 94°C for 30 s, annealing at
55°C for 1 min, extension at 72°C for 1 min 30 s and with
a final extension at 72°C for 10 min. All the obtained PCR
products were separated in 1.5% agarose gel, stained with
ethidium bromide and photographed on UV transillumi-
nator. Clear, distinct bands were excised from the agarose
gel and purified with Nucleid Acid Purification Kit (Axy-
gen, Union City, CA, USA).
Sequence analysis of rDNA-ITS region
The purified PCR products were subjected to DNA se-
quencing (Genomed, Warsaw, Poland). For each ana-
lyzed species, the consensus sequences, covering the
rDNA-ITS region were constructed, using DNASTAR’s
Lasergene software (DNASTAR Inc., Madison, USA).
The resulting sequences were used to screen similar
fungal sequences, available in the GenBank database,
using BLAST N program (blast.ncbi.nlm.nih.gov). Sev-
eral representative ITS sequences of isolates of M.fructi-
cola,M.laxa,M.fructigena,M.polystroma and M.mali
were selected and downloaded from the GenBank data-
base. On the basis of these selected ITS sequences and
consensus sequences obtained in this study the phyloge-
netic tree was constructed (MEGA v4.1 software, Center
for Evolutionary Functional Genomics, The Biodesign
Institute, Tempe, USA), performing neighbour-joining
phylogenetic analysis. The tree was evaluated with
1,000 bootstrap replications to test the clade stability.
Additionally, the DNA ITS sequences of four standard
isolates were also included to the tree. The ITS DNA
sequences of M.polystroma and M.fructigena isolates
obtained in this study and several isolates selected from
GenBank database and the corresponding ITS sequence
of the M.polystroma standard isolate were analyzed
using Multiple Sequence Alignment tool in ClustalW2
software (www.ebi.ac.uk/Tools/msa/clustalw2/), in order
to obtain detailed information about nucleotide substitu-
tions in this region within and between these two species.
Morphological examination of selected Monilinia
spp. isolates
On the basis of results obtained in PCR reactions, five
representative isolates of each species were selected
Eur J Plant Pathol (2013) 135:855–865 857

for morphological characterization. Fungal cultures
were incubated on PDA at 22°C using a 12 h UV
light/12 h dark regime for 7–10 days, in order to
induce spore production. Then a conidial water sus-
pension was prepared and distributed evenly onto the
surface of PDA medium, and the plates were incubat-
ed under the same conditions as described above, until
the fungal growth was observed. From the edge of
several monoconidial fungal colonies a 4-mm-
diameter plug of mycelium was cut and placed on
new PDA medium. Then the cultures were incubated
for 10 days under the same conditions, in order to
assess the capacity of isolates to form black stromatal
plates. The morphological characterization of Monili-
nia spp. isolates was performed according to EPPO
standard PM 7/18 on the 7
th
day of incubation. Addi-
tionally, the size of 40 spores of each isolate was
measured under a Nikon H550S optical microscope
(Nikon Corporation, Kawasaki, Japan) at 100x mag-
nification. The morphological characterization of iso-
lates was also evaluated on the basis of fungal growth
on APDA-F500 medium (Amiri et al. 2009) and on
cherry decoction agar (CHA medium), prepared
according to the method of Gams et al. (1998), but
with the cherry juice at half of the recommended
concentration. The monoconidial isolates were incu-
bated on APDA-F500 medium and on CHA medium
at 22°C in the dark for 10 days. The color of the
colonies, the presence of conidia, the growth pattern
and growth rate per 24 h of incubation were deter-
mined. Each culture experiment was performed two
times with two replicates for each isolate. The growth
rate in mm per 24 h was calculated for each replicate
within experiment, then averaged for all replicates
across all five isolates of an individual species exam-
ined in the experiment. Mean growth rate obtained for
each Monilinia species was compared between spe-
cies. All data were analyzed using analysis of variance
and the Tukey honest significant difference test.
Pathogenicity test and reisolation of the causal agents
of brown rot
Pathogenicity of all four Monilinia spp. was tested on
mature plums. The fruit were surface sterilized by
submersion in 70% ethanol for 1 min. Subsequently,
fruit were rinsed two times with sterile water and
dried. A plug of mycelium from the edge of mono-
conidial cultures growing on PDA was inserted into a
small hole punched in the fruit peel. Five representa-
tive isolates of each examined Monilinia spp. were
used for inoculation of plum fruit. Each isolate was
tested on five plums in one experiment. 20 fruit
grafted with plugs from sterile PDA agar were used
as a negative control. Inoculated fruit were incubated
for 7 days in a sterilized glass container at room
temperature. The experiment was repeated twice. All
four causal agents of brown rot were re-isolated from
inoculated fruit on PDA medium and incubated at
22°C under 12 h UV light/12 h dark for 7 days. Their
identification, based on examination of morphological
features, was performed according to EPPO standard
PM 7/18.
Results
Multiplex PCR amplification
The molecular analysis performed according to the
protocol described by Côté et al. (2004) on 670 Moni-
linia spp. isolates resulted in the identification of 433
isolates of M.fructigena, 212 isolates of M.laxa,20
isolates of M.polystroma and five isolates of
M.fructicola, originating from various locations in
Poland (Table 1). The obtained PCR product sizes
were: 535 bp for M.fructicola, 402 bp for M.fructi-
gena,421bpforM.polystroma and 352 bp for
M.laxa (Fig 1.). The resulting PCR product sizes of
examined and reference isolates were consistent with
product sizes proposed by Côté et al. (2004), making
accurate classification possible within the Monilinia
species.
PCR amplification of rDNA-ITS region
Amplification of M.fructicola DNA using the primers
ITS4Mfcl and ITSMonilia and M.polystroma DNA
with the primers ITS4Mfgn and ITSMonilia yielded a
374 bp size amplicon. In both PCRs products of spe-
cific size were obtained (Petróczy and Palkovics 2009;
Petróczy et al. 2012).
Sequencing and analysis of rDNA-ITS region
Multiple sequence alignment was performed for the
374 bp rDNA-ITS region of M.polystroma and in-
cluded the following isolates: 20 examined in this
858 Eur J Plant Pathol (2013) 135:855–865

study, UFT isolate from Hungary (GenBank acc. No.
AM937114), HML-3 isolate from China (GenBank
acc. No. GU067539) and CBS 122306 isolate from
Japan. No differences were detected in the sequences
obtained for this region among these isolates. A com-
parison of the ITS DNA sequences for M.polystroma
strains evaluated in this study and M.fructigena
sequences available in GenBank (acc. Nos. Z73780,
EF207428, HQ166417, AM937113; CBS 101500ITS)
allowed for the identification of five nucleotides
distinguishing these two species. Moreover, the
ITS region of six M.fructicola isolates examined
in this study was in 100% similar to the sequences
of selected M.fructicola isolates (GenBank acc.
Nos. FJ411110, GU967379; CBS 127259ITS). The
ITS sequences obtained for AP1 M.polystroma
Table 1 Number of M.fructigena,M.polystroma,M.laxa and M.fructicola isolates obtained from pome and stone fruits and different
locations in Poland
Species Fruits Number
of isolates
(total 670)
Location
Skierniewice Dąbrowice Nowy
Dwór
Biała
Rawska
Komorowo Julków Zawada Miłobądz
M.fructigena apple 56 x x x x x x x x
pear 12 x x x x
quince 20 x x x
apricot 17 x x x x x
peach 15 x x x x
plum 275 x x x x
sour cherry 17 x x x
sweet cherry 21 x x x
M.polystroma apple 4 x x
peach 1 x
plum 15 x
M.laxa apricot 14 x x x x x
peach 12 x x x x
plum 148 x x x x
sour cherry 25 x x
sweet cherry 13 x x x
M.fructicola plum 5 x
Fig. 1 Specific products obtained in multiplex PCR amplifica-
tion with MO368-5, MO368-8R, MO368-10R and Laxa-R2
primers and with DNA isolated from M.fructicola,M.laxa,
M.fructigena and M.polystroma pure cultures. Lines 1, 6, 11
and 16 correspond to standard Monilinia isolates. The remaining
lines represent the products obtained for selected examined
isolates. NC - is a negative control (NC), containing the reaction
mixture with double deionized water instead of DNA. M –
molecular DNA ladder (O’Gene Ruler™100 bp DNA Ladder
Plus, Fermentas, Vilnius, Lithuania)
Eur J Plant Pathol (2013) 135:855–865 859

isolate and M1PL M.fructicola isolate were depos-
ited in GenBank (Accession Nos. JF820317,
JX312665).
The constructed phylogenetic tree (Fig. 2), includ-
ing 25 ITS sequences of Monilinia isolates from
Poland, 15 ITS sequences of Monilinia spp. selected
strains (GenBank) and ITS fragments from four stan-
dard isolates, grouped all the sequences into two clus-
ters. Within each cluster, two well supported groups
were delimited. One of the groups in the first cluster
contained the M.polystroma isolates from Poland
(isolates AP1-AP3, SP1-SP15, TP1, and PP1) as well
as three selected isolates of M.polystroma originating
from Hungary (GenBank acc. No. AM937114), China
(GenBank acc. No. GU067539) and Japan (CBS
122306ITS). The second group included only Gen-
Bank sequences of M.fructigena isolates (GenBank
acc. Nos. EF207424, AM937113, HQ166417) and the
sequence of M.fructigena isolate from CBS (CBS
101500ITS). In the second cluster, one group con-
sisted of GenBank sequences of M.laxa (GenBank
acc. Nos. AF150676, EF153016, EU042149) and
the sequence of M.laxa isolate form CBS (CBS
488.50ITS). The second group in this cluster
comprised the sequences of M.fructicola isolates
M1PL-M5PL from Poland together with GenBank
sequences of M.fructicola (GenBank acc. Nos.
FJ411110, GU967379) and the sequence of the
reference M.fructicola isolate from CBS (CBS
127259ITS).
Fig. 2 Dendrogram depict-
ing relationships among M.
fructicola,M.laxa.M.fruc-
tigena,M.polystroma and
M.mali isolates based on
nuclear rDNA ITS1/5.8S
rDNA/ITS2 region. Frag-
ments M1PL-M5PL, AP1-
AP3, SP1-SP15, TP1, PP1
were sequenced in this
study, the others were re-
trieved from nucleotide se-
quence databases
(www.ncbi.nlm.nih.gov).
The tree was constructed
using the neighbour-joining
algorithm, with the M.mali
ITS sequence as an out-
group. Bootstrap values are
shown above or below
branches. The numbers near
each branch represent per-
centages out of 1,000 boot-
strap replications
860 Eur J Plant Pathol (2013) 135:855–865

Morphological characterization
Table 2summarizes all morphological features which
were examined on selected Monilinia spp. isolates
growing on three media: PDA, APDA-F500 and
CHA. Culturing of Monilinia spp. isolates on PDA
and APDA-F500 media showed significant differen-
ces in culture growth morphology (Table 2and Fig. 3).
On PDA medium M.fructicola isolates demonstrated
the highest growth rate (7.27 mm/day ±0.032 SEM
and 7.28 mm/day ±0.035 SEM, P < 0.001), resulting
in the largest colony diameter range (80–85 mm) after
7 days of incubation. Sporulation of this species was
profuse, with characteristic, grayish concentric rings,
while sporulation of M.fructigena,M.polystroma and
M.laxa isolates was sparse. Conidia of M.fructigena
were the largest among the examined species (24 μm×
13 μm). After 10 days of incubation, all M.polystroma
isolates and only some M.fructigena isolates started to
produce black stromatal plates, which spread random-
ly on the medium surface, while the other two exam-
ined species did not show any significant changes in
their morphology. The examined morphological fea-
tures made it possible to classify all the tested isolates
growing on PDA medium to Monilinia spp. according
to EPPO standard PM 7/18. The APDA-F500 medium
suppressed bacterial growth and inhibited or signifi-
cantly limited the growth of other fungi commonly
encountered on fruits more effectively than PDA me-
dium. The fungicidal activity of fosetyl-AL at stable
pH (3.6) was higher in the cases of M.fructicola and
M.laxa than for the other two species, thus the mean
growth rate of these species was almost two times
lower than that of M.fructigena and M.polystroma
cultures. The combination of colour of the cultures and
character of their growth and margin was unique and
characteristic for every species. During the first days
of incubation, M.fructicola and M.laxa cultures were
almost white and rosetted. After 6 days, M.laxa cul-
tures began to change colour. After 10 days, they were
olivaceous to hazel, while M.fructicola isolates
remained white.
On CHA medium all fungi grew very fast. Sporu-
lation of M.fructicola was profuse, while M.fructi-
gena and M.polystroma sporulated sparsely or not at
all. M.laxa isolates did not produce conidia on CHA
medium. After 10 days of incubation, only M.poly-
stroma isolates formed black stromatal plates in con-
trast to the other three species.
Pathogenicity test and fungus reisolation
After 7 days of incubation, all inoculated plum fruit
showed symptoms of brown rot, in contrast to the fruit
injected with sterile agar plugs, which remained
healthy. M.fructigena,M.polystroma and M.laxa
caused the rot covering the entire fruit surface in
comparison to M.fructicola isolates, with lower
growth rate, which induced a lower level of fruit rot.
Each of the examined species of Monilinia produced
characteristic reproductive and vegetative structures
on inoculated plums. M.fructigena sporodochia were
numerous, large, compact and yellow, while M.poly-
stroma structures were similar, but almost white. The
fruit infected with M.fructicola and M.laxa,had
minor and grey sporodochia on the surface, but in case
of M.fructicola they were sparsely distributed. After
14 days of incubation, sporodochia of M.polystroma
isolates created a white –yellow hyphal mantle of
stroma on the fruit surface, characteristic for this spe-
cies. All isolates of Monilinia spp. used in these inoc-
ulation experiments were successfully re-isolated and
characterized on PDA medium, fulfilling Koch
postulates.
Discussion
Among Monilinia isolates recovered from rotted fruits
collected in the orchards in Poland, during the sum-
mers of 2010 and 2011, M.fructigena was the most
frequently isolated species –in 64% of samples. This
frequency of detection might be explained by the
preference of this fungus to infect ripening fruit, caus-
ing rot and decay. In contrast, M.laxa comprised 32%
of the isolates obtained from the rotted fruits, perhaps
as a result of the fact that this species mainly causes
blossom and twig blight and decay. This preliminary
screening conducted in several orchards located in
northern, southern and central Poland confirmed the
presence of M.fructigena and M.laxa (Lenartowicz
1979) in this country. However, this study also
revealed the presence of two species not reported in
Poland so far: M.fructicola and M.polystroma.
M.fructicola causes both blossom blight and fruit
rot (Anonymous 2009), while M.polystroma is a
pathogen specialized in fruit infections (van Leeuwen
et al. 2002). Villarino et al. (2010) observed in Spain
that M.fructicola exhibited a higher level of virulence
Eur J Plant Pathol (2013) 135:855–865 861

Table 2 Comparison of morphological features of Monilinia spp. cultures on standard PDA medium and two selective media APDA-F500 and CHA. All the data were collected from
two similar experiments
Medium Potato dextrose agar PDA Acidified PDA medium amended with
fosetyl-aluminum APDA-F500
Cherry decoction agar medium CHA
The examined
features
After 7 days of incubation at 22°C for 12h near UV
light/12h dark
After 10 days of incubation at 22°C in the dark After 10 days of incubation at 22°C in the dark
M.fructigena M.polystroma M.laxa M.fructicola M.fructigena M.polystroma M.laxa M.fructicola M.fructigena M.polystroma M.laxa M.fructicola
Color of the culture yellow/cream yellow/hazel grey grey cream grey hazel/olivaceous white/grey cream hazel yellow white/grey
Range of cultures
diameters
75–80 mm 75–80 mm 65–70 mm 80–85 mm 80–85 mm 80–85 mm 55–60 mm 55–60 mm 85 mm 85 mm 85 mm 85 mm
Average growth
rate/24 h
5.32 mm±
0.034
SEM
5.57 mm±
0.016
SEM
5.25 mm±
0.040
SEM
7.27 mm±
0.032
SEM
5.04 mm±
0.016
SEM
6.22 mm±
0,069
SEM
3.20 mm±
0.120
SEM
3.30 mm±
0.130
SEM
7.13 mm±
0.041
SEM
6.10 mm±
0.058
SEM
7.31 mm±
0.099
SEM
5.18 mm±
0.030
SEM
5.32 mm±
0.037
SEM
5.58 mm± 0.018
SEM
5.24 mm±
0.043
SEM
7.28 mm±
0.035
SEM
5.05 mm±
0.030
SEM
6.22 mm±
0.069
SEM
3.14 mm±
0.140
SEM
3.32 mm±
0.140
SEM
7.14 mm±
0.036
SEM
6.10 mm±
0.062
SEM
7.31 mm±
0.076
SEM
5.18 mm±
0.033
SEM
Character of the
growth of
culture’s margin
entire lobed lobed
rosetted
entire entire entire lobed
rosetted
lobed
rosetted
entire entire entire entire
Sporulation sparse sparse sparse abundant no no no no sparse sparse no abundant
Concentric ring
of sporulation
no no no yes no no no no no no no no
The average size
of 40 conidia
24× 13 μm16×10μm17×11μm16×10μm── ─ ─24× 13 μm16×10μm─16 ×10 μm
Formation of black
stromatal arcs
after 10 days
of incubation
rare yes no no no no no no no abundant no no
862 Eur J Plant Pathol (2013) 135:855–865

in peach orchards in comparison to M.laxa and
M.fructigena. That could result from the fact, that
M.fructicola has a greater parasitic ability, which
correlates with the production of larger quantities of
germinated conidia and longer germ tubes than
M.laxa and M.fructigena. Therefore, Villarino et al.
(2010) suggested that M.fructicola may be able to
displace M.fructigena –with a lower virulence –in
Fig. 3 Morphology of four Monilinia spp. isolates on three artificial media: PDA, APDA-F500 and CHA
Eur J Plant Pathol (2013) 135:855–865 863

some habitats and will compete only with M.laxa in
the brown rot pathosystem. However, Villarino and
others (2010) did not take into account the risk of
introducing M.polystroma as the fourth brown rot
causal agent in Europe. The pathogenicity test con-
ducted on plums in this study showed the ability of
M.polystroma isolates to produce the hyphal mantle
of stroma on the host’s cuticle, in contrast to other
examined species. Also van Leeuwen et al. (2002)
observed the ability of this fungus to form the ectos-
troma mantle on the fruit’s surface and proposed its
role in protection of the colonized fruit against degra-
dation and decomposition by abiotic and biotic fac-
tors. Inhibition of rapid decomposition of infected fruit
may enhance the survival of this pathogen and poten-
tially increase the amount of primary inoculum for the
next season (Byrde and Willetts 1977). Therefore,
potential competition in host colonization between
these new and previously reported species for Poland
and their spread among other commercial orchards
and natural habitats should be monitored. It is likely,
that the contribution of isolates with potentially lower
or higher virulence or survival potential will change
within monitored areas. The potential differences in
pathogenicity and parasitic fitness among M.fructi-
gena,M.laxa,M.fructicola and M.polystroma needs
to be evaluated under in vitro and in vivo conditions.
The suitability of PDA and APDA-F500 media for
the isolation of Monilinia spp. from rotted fruits was
examined. Amiri et al. (2009) showed that APDA-
F500 was a useful medium for the isolation of fungi
causing brown rot of stone and pome fruits. Similarly,
in this study we found the medium to be useful for
Monilinia spp. isolation from fresh and frozen rotted
fruits. Moreover, we noticed that cultivation of these
fungi on APDA-F500 medium can be helpful in dis-
tinguishing the four examined Monilinia species based
on their characteristic growth patterns. Cultivation of
all Monilinia spp. isolates on CHA media for at least
10 days in the dark revealed that only M.polystroma
isolates were able to form numerous black arcs. This
finding is in concordance with the observations of van
Leeuwen et al. (2002).
Fulton et al. (1999) found five nucleotides in the ITS
region, what allowed for distinguishing M.polystroma
from M.fructigena. Based on this difference, we classi-
fied 20 isolates to the M.polystroma species. Moreover,
the phylogenetic analysis of ITS region of isolates se-
quenced in this study confirmed the close relationship
between M.fructicola and M.laxa species and between
M.fructigena and M.polystroma species (Fig. 2).
Molecular analysis of a genomic region with un-
known function carried out by Petróczy and Palkovics
(2009) showed the occurrence of repetitive sequence
motifs: CAT, CCT, TAGTCCA and TAGTCCC within
a 421 bp DNA fragment of M.polystroma isolate UFT
from Hungary. Sequencing of the corresponding region
in M.polystroma isolate AP1 from Poland and multiple
sequence alignment analysis performed for this region
with M.polystroma isolate UFT showed no differences
between these sequences (data not shown). This com-
parison is not sufficient for the determination of the
genetic relationship between these isolates, thus the
analysis of different genome regions of a larger number
of available isolates should be conducted. The phyloge-
netic analysis of M.polystroma isolates derived from
Poland, Hungary and Asian countries should help in
determining their origin.
On the basis of multiplex PCR (Côté et al. 2004),
phylogenetic analysis of the ITS region (Ioos and Frey
2000; Petróczy and Palkovics 2009; Petróczy et al.
2012) and morphological characterization (EPPO
standard PM 7/18), four species, M.fructigena,M.laxa,
M.fructicola and M.polystroma,wereidentifiedasthe
causal agents of brown rot of pome and stone fruits in
Poland. During the last 10 years, M.fructicola was
introduced and has spread widely in western and central
Europe. Recent reports of the occurrence of this
pathogen have come from Italy (Pellegrino et al.
2009),Spain(DeCaletal.2009), Switzerland
(Hilber-Bodmer et al. 2010), Slovenia (Munda
and Viršček Marn 2010) and Slovakia (Ondejková
et al. 2010), where the fungus was found in com-
mercial stone fruit orchards. In Poland this first
detection of M.fructicola was made in a commer-
cial plum orchard located in the central part of
the country (Dąbrowice, Table 1). M.fructicola,
M.fructigena and M.laxa are well-known, world-
wide pathogens (Byrde and Willetts 1977), in contrast
to M.polystroma, which until recently was only
reportedinJapan(vanLeeuwenetal.2002). In
2006 the pathogen was also detected in an apple
orchard in Hungary (Petróczy and Palkovics 2009)
and in 2008 in a sweet plum orchard in China (Zhu
and Guo 2010). The first isolate of M.polystroma in
Poland was obtained from rotted apples, collected
from a commercial orchard located in the south of
Poland (Zawada, Table 1).
864 Eur J Plant Pathol (2013) 135:855–865

On the basis of the number of M.fructicola and
M.polystroma isolates obtained in this study, it is
difficult to estimate the impact of these new species
on fruit production in Poland. However, the risk of
spreading these new pathogens in Poland is high and
mainly associated with fungal adaptation, the wide-
spread presence of host plants and human activity.
Acknowledgments Anna Poniatowska wishes to thank Profes-
sor László Palkovics and Dr. Marietta Petróczy for the opportunity
to perform the molecular analysis of some fungal isolates used in
this study during the Short Scientific Training (Cost Action 864) at
Corvinus University, Budapest, Hungary, in November 2010. The
authors highly appreciate the help and useful suggestions from Dr.
Grażyna Szkuta and are also grateful for the possibility of
performing some parts of this study in Central Laboratory of Main
Inspectorate of Plant Health and Seed Inspection in Toruń,Poland.
The authors wishes to thank to Dr. Mark Mazzola (USDA-ARS,
Tree Fruit Research Lab Wenatchee, WA, USA) for the English
correction and valuable remarks.
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