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RESEARCH ARTICLE
Powdery mildew caused by Erysiphe
corylacearum: An emerging problem on
hazelnut in Italy
Slavica MatićID
1
*, Andrea G. Caruso
2
, Chiara D’ErricoID
1
, Camilla Sacco BottoID
1
,
Emanuela Noris
1
, Vojislav Trkulja
3
, Stefano Panno
2
, Salvatore Davino
2
, Marco Moizio
4
1Institute for Sustainable Plant Protection, National Research Council, Turin, Italy, 2Department of
Agricultural, Food and Forest Sciences, University of Palermo, Palermo, Italy, 3Agricultural Institute of
Republic of Srpska, Banja Luka, Bosnia and Herzegovina, 4SAGEA Centro di Saggio s.r.l., Castagnito
d’Alba (CN), Italy
*slavica.matic@ipsp.cnr.it
Abstract
Erysiphe corylacearum has recently been reported in northern Italy (Piedmont) and other
European countries as the causal agent of a new emerging powdery mildew on hazelnut.
This disease is much more dangerous than the common hazelnut powdery mildew caused
by Phyllactinia guttata as it significantly reduces yield and quality of hazelnuts. This study
aimed to perform morphological and molecular characterization of the fungal isolates from
powdery mildew-infected plants in the Piedmont Italian region. Additionally, genetic diversity
studies and pathogenicity tests were conducted. Thirty-six fungal isolates originating from
symptomatic hazelnut plants exhibiting specific powdery mildew symptoms on the superior
leaf side were identified morphologically as E.corylacearum. Single- and multilocus
sequence typing of five loci (ITS, rpb2,CaM,GAPDH and GS) assigned all isolates as E.
corylacearum. Multilocus and GAPDH phylogenetic studies resulted in the most efficient
characterization of E.corylacearum. Studied fungal isolates were able to cause new emerg-
ing powdery mildew disease by fulfilling Koch’s postulates. The emergence of powdery mil-
dew disease in Italy revealed the E.corylacearum subgrouping, population expansion, and
high nucleotide similarity with other recently identified E.corylacearum hazelnut isolates. To
contain this harmful disease and inhibit the fungus spread into new geographical zones, it
will be necessary to implement more rigorous monitoring in neighboring hazelnut plantations
near infected hazelnuts, use sustainable fungicides and search for new biocontrol agents.
Introduction
The powdery mildew fungi are obligate, biotrophic plant pathogens belonging to the Erysi-
phales order of the Ascomycota phylum [1]. Recently, they have been increasing in Europe
due to the spread of novel powdery mildews into new geographic areas accompanied by new
species assignment and taxonomic splitting [2,3].
Erysiphe corylacearum is one of the alien pathogens affecting hazelnut (Corylus avellana L.;
common European hazelnut) in the Middle East and East-Central and Southern Europe. This
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OPEN ACCESS
Citation: MatićS, Caruso AG, D’Errico C, Botto CS,
Noris E, Trkulja V, et al. (2024) Powdery mildew
caused by Erysiphe corylacearum: An emerging
problem on hazelnut in Italy. PLoS ONE 19(5):
e0301941. https://doi.org/10.1371/journal.
pone.0301941
Editor: Abhay K. Pandey, Tocklai Tea Research
Institute, INDIA
Received: October 16, 2023
Accepted: March 25, 2024
Published: May 28, 2024
Copyright: ©2024 Matićet al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting information
files. All DNA sequences of the studied isolates are
publicly available in the GenBank Nucleotide
database (accession numbers OQ917083-
OQ917118, OR126170- OR126205, OQ995105-
OQ995140, OR105713- OR105735, and
OR126217- OR126241). The Accession Numbers
of reference isolates used in the sequence analyses
are shown in Figs 4,5,S1 and S2.
pathogen has a negative impact on fruit production and safety (U. Braun & S. Takam.), and is
the causal agent of the new emerging hazelnut powdery mildew disease [4].
E.corylacearum has been reported for the first time on hazelnut in Turkey [4]. Before that,
it was documented only on other Corylus species in Asia, such as Asian hazel (Corylus hetero-
phylla Fisch. ex Trautv.), Japanese hazel (Corylus sieboldiana Blume) or in America on beaked
hazel (Corylus cornuta Marshall) [5–7]. After Turkey, it further spread on hazelnut from the
Middle East i.e. Iran [8] to East Europe (Ukraine [9], Romania [10]), Central Europe (Austria
[11], Hungary [12], Germany [13], Slovenia [14], Switzerland [15]), and Southern Europe
(Italy [16] and Spain [17]). Regarding Italy, it was reported for the first time in the Piedmont
region in 2020 [16,18].
This fungal pathogen is much more dangerous than Phyllactinia guttata (Wallr.: Fr), the
causal agent of the so-called ’common’ hazelnut powdery mildew [19], due to its impact on
both the yield and the hazelnut plants life. Symptoms of P.guttata are visible mainly on the
lower surface of the leaf during late summer and have minimal impact on hazelnuts, requiring
little or no treatment. On the contrary, E.corylacearum is particularly harmful to hazelnut cul-
tivation, causing significant damage, that results in a strong reduction in both yield and quality
of hazelnuts, especially when associated with water stress [4]. The plants affected by the new
powdery mildew show symptoms of a white patina on the upper surface of the leaves; in the
case of an intensive attack, the symptoms also occur on young shoots, bracts, and fruits. Pow-
dery mildew lesions turn gradually from yellow to brown colour. In susceptible cultivars,
leaves dry out and fruits fall prematurely, resulting in high yield losses [4].
Currently, there are limited curative chemical interventions against E.corylacearum (e.g.
treatment with triazole fungicide Revysion). Farmers may rely on the use of preventive control
methods such as the use of sulphur-based fungicides (efficient also against P.corylicola and
gall mites) and sustainable agronomic practices (removal and destruction of the infected leaves
and other symptomatic plant material, balanced fertilization, and irrigation).
Currently, there are no available data on the impact of this emerging disease on hazelnuts
in Italy. Considering the heavy impact of E.corylacearum on hazelnut crop in countries where
it is already well established, there is a strong risk that this pathogen may profoundly compro-
mise hazelnut production in Piedmont, one of the major Italian hazelnut producers, and that
it will spread to other cultivation areas in Italy. The objective of the present study was to char-
acterize the causal agent of the new hazelnut powdery mildew disease found in the Piedmont
region. Both morphological observation and single- and multi-locus phylogenetic analyses
were performed together with pathogenicity tests to fulfill Koch’s postulates. Furthermore, the
pathogen geographical distribution in Piedmont was studied.
Materials and methods
Field survey
A field survey was performed in the Piedmont region (North-Western Italy). During the late
spring-summer period (June-August) of 2022, eight hazelnut orchards planted with the
‘Tonda Gentile delle Langhe’ (Trilobata) cultivar have been observed for E.corylacearum
symptoms in eight different locations of Torino, Cuneo and Asti provinces (Fig 1). Leaves
showed the characteristic symptoms of new powdery mildew associated with E.corylacearum
and they appeared as round, white, powdery spots visible mainly on the upper surface of the
leaves and gradually increased in size. The leaves also showed symptoms of common powdery
mildew, visible mostly on the lower part of the leaf. Symptomatic leaves were collected and
transported in paper bags to the laboratory for morphological observations and molecular
analyses.
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Funding: This research was funded by funded by
CNR project FOE-2021 (NUTRAGE).
Competing interests: The authors have declared
that no competing interests exist.
Morphological characterization
A total of 160 isolates were collected from eight locations and examined for their morphologi-
cal characteristics. Each isolate was collected from a single powdery mildew spot from the
upper or lower leaf surface of a single hazelnut plant.
To make anatomical observations, hyphae, conidiophores, conidia, and chasmothecia from
a single powdery mildew spot were stripped off from the leaf surface with a clean needle and
Fig 1. Map of the Piedmont region (North-Western Italy) showingthe presence of emerging fungus Erysiphe
corylacearum, the causal agent of powdery mildew, recently introduced in Italy. The site of samples collection
indicated by a star are those where E.corylacearum is detected during 2022 field-survey, while locations marked by a
dot correspond to the fungus-free zones.
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mounted in water. The remaining portion of the sample (isolate) was conserved and used for
molecular study if necessary. Slides were observed under a Leica DM750 biological microscope
(Leica Microsystems GmbH, Wetzlar, Germany) equipped with a Leica EC4 camera (Leica
Microsystems GmbH, Wetzlar, Germany). Anatomical features were measured using Scope-
Image 9.0 with 40×objective. Microscopic observations of all isolates were carried out, deter-
mining the shape and size of conidia, asci, ascospores, chasmothecia and appendages. The
obtained results were compared with the species descriptions reported by Braun and Cook [7]
and Bradshaw et al. [20]. Based on morphological observations, 36 random fungal isolates
were selected and used in subsequent molecular analyses.
DNA extraction, PCR and sequencing
Mycelia containing both conidia and chasmothecia of a single powdery mildew spot were
scraped from the leaf surface and used for DNA extraction with 5% Chelex (Bio-Rad, Hercules,
California, USA), as described previously by Hirata and Takamatsu [21]. DNA extracts were
used directly for PCR amplification of the following DNA regions: internal transcribed spacer
(ITS), RNA polymerase second largest subunit (rpb2), calmodulin (CaM), glyceraldehyde-
3-phosphate dehydrogenase (GAPDH), and glutamine synthetase (GS). The ITS region was
amplified using either primers ITS1 and ITS4 [22] or ITS1 and PM6 [23]. The other four genes
were amplified as described by Bradshaw et al. [24], using the following primers: PmRpb2_4
and PMRpb2_6R (rpb2), PMCAM1 and PMCAM4R (CaM), PMGAPDH1 and PMGAPDH3R
(GAPDH), and GSPM2 and GSPM3R (GS). PCR products were purified using a QIAquick
PCR purification kit (Qiagen, Hilden, Germany), in accordance with manufacturer’s instruc-
tions and sequenced in both directions at the BMR Genomics Centre (Padua, Italy). The
obtained sequences were deposited in the NCBI GenBank database under the following acces-
sion numbers: OQ917083- OQ917118 for ITS, OR126170- OR126205 for rpb2, OQ995105-
OQ995140 for CaM, OR105713- OR105735 for GAPDH, and OR126217- OR126241 for GS
(Table 1).
Sequence analyses
The ITS sequences of all 36 isolates obtained were aligned with the reference Erysiphe
sequences available in the GenBank database, using the BLAST software package (www.ncbi.
nlm.nih.gov). Subsequently, Maximum Likelihood (ML) phylogenetic analyses were per-
formed on both single and concatenated sequences of five loci (ITS, rpb2,CaM,GAPDH and
GS). Concatenated phylogenetic analyses were also performed on the basis of Bayesian infer-
ence (BI) using Geneious Prime v. 2020.1.2 (Biomatters Ltd., New Zealand). Heating parame-
ter in BI phylogenetic analyses was adjusted to 0.2 by sampling the trees at every 1000
generations. BI analyses were terminated when the mean standard deviation of the split fre-
quencies was less than 0.01. The ITS phylogenetic analyses were carried out including 26 refer-
ence sequences of E.corylacearum and 11 reference sequences of close Erysiphe phylogenetic
species infecting Corylus spp., such as E.pseudocorylacearum,E.cornutae,E.coryli-ameri-
canae,E.corylicola, and E.syringae [20]. Other single and multilocus-sequence phylogenetic
analyses included only the 36 isolates from this study, as gene sequences of reference E.coryla-
cearum isolates were not available. In all phylogenetic analyses, E.necator (FH00941202)
sequences were used as the outgroup. Concatenated dataset of five loci included a total of
2,397 bp. The best-fit nucleotide model of each dataset for a ML analysis was determined using
Findmodel (http://www.hiv.lanl.gov/content/sequence/findmodel/findmodel.html): JC: Jukes-
Cantor for ITS, TrN: Tamura-Nei plus Gamma for CaM and GAPDH and concatenated tree,
and HKY: Hasegawa-Kishino-Yano for rpb2 and GS. The ML analyses were carried out with
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MEGA 11 software [25]. The most appropriate nucleotide model of each partition was identi-
fied by means of MrModeltest v.2.3 [26] and used for BI analyses.
DNA polymorphism parameters (haplotype diversity, nucleotide diversity, number of poly-
morphic sites, mutations and nucleotide differences) were determined by means of DNA
Sequence Polymorphism v. 6 software [27].
Tajima’s D, Fu’s and Li’s D, and Fu’s and Li’s F tests determined departures from the null
hypothesis of neutral evolution. Significant values of these tests can indicate the presence of
population changes such as change in size, population expansion, population contraction, and
population subdivision [28,29].
Table 1. List of Erysiphe corylacearum isolates used for the molecular identification and of their corresponding accession numbers.
Isolate Site of collection ITS rpb2 CaM GAPDH GS
1_PdIT Serravalle Langhe (Cuneo province, Piedmont region) OQ917083 OR126170 OQ995105 OR105700 OR126206
2_PdIT OQ917084 OR126171 OQ995106 OR105701 OR126207
3_PdIT OQ917085 OR126172 OQ995107 OR105702 OR126208
4_PdIT OQ917086 OR126173 OQ995108 OR105703 OR126209
5_PdIT OQ917087 OR126174 OQ995109 OR105704 OR126210
6_PdIT OQ917088 OR126175 OQ995110 OR105705 OR126211
7_PdIT OQ917089 OR126176 OQ995111 OR105706 OR126212
8_PdIT OQ917090 OR126177 OQ995112 OR105707 OR126213
9_PdIT OQ917091 OR126178 OQ995113 OR105708 OR126214
10_PdIT OQ917092 OR126179 OQ995114 OR105709 OR126215
11_PdIT OQ917093 OR126180 OQ995115 OR105710 OR126216
38_PdIT OQ917108 OR126195 OQ995130 OR105725 OR126231
39_PdIT OQ917109 OR126196 OQ995131 OR105726 OR126232
40_PdIT OQ917110 OR126197 OQ995132 OR105727 OR126233
41_PdIT OQ917111 OR126198 OQ995133 OR105728 OR126234
42_PdIT OQ917112 OR126199 OQ995134 OR105729 OR126235
43_PdIT OQ917113 OR126200 OQ995135 OR105730 OR126236
44_PdIT OQ917114 OR126201 OQ995136 OR105731 OR126237
45_PdIT OQ917115 OR126202 OQ995137 OR105732 OR126238
46_PdIT OQ917116 OR126203 OQ995138 OR105733 OR126239
47_PdIT OQ917117 OR126204 OQ995139 OR105734 OR126240
48_PdIT OQ917118 OR126205 OQ995140 OR105735 OR126241
23_PdIT Benevello (Cuneo province, Piedmont region) OQ917094 OR126183 OQ995116 OR105713 OR126217
24_PdIT OQ917095 OR126185 OQ995117 OR105715 OR126218
25_PdIT OQ917096 OR126184 OQ995118 OR105714 OR126219
26_PdIT OQ917097 OR126186 OQ995119 OR105716 OR126220
27_PdIT OQ917098 OR126181 OQ995120 OR105711 OR126221
28_PdIT OQ917099 OR126187 OQ995121 OR105717 OR126222
29_PdIT OQ917100 OR126188 OQ995122 OR105718 OR126223
30_PdIT OQ917101 OR126189 OQ995123 OR105719 OR126224
31_PdIT OQ917102 OR126190 OQ995124 OR105720 OR126225
32_PdIT OQ917103 OR126191 OQ995125 OR105721 OR126226
33_PdIT OQ917104 OR126182 OQ995126 OR105712 OR126227
34_PdIT OQ917105 OR126192 OQ995127 OR105722 OR126228
35_PdIT OQ917106 OR126193 OQ995128 OR105723 OR126229
36_PdIT OQ917107 OR126194 OQ995129 OR105724 OR126230
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Pathogenicity assays
Two-years old healthy hazelnut plants of ‘Tonda Gentile delle Langhe’ cv. were used for the
pathogenicity assays. Plants were maintained in 20 L plastic pots containing a sterilized mix-
ture of 80% peat and 20% perlite in a greenhouse.
Three plants were inoculated with powdery mildew on three randomly selected branches,
using 10 μL of a conidial suspension (1 ×10
5
spores mL
−1
) obtained by washing conidial
spores from the field-collected heavily infected leaves with powdery mildew symptoms on the
upper leaf side.
Plants were then maintained under growth chamber conditions: 25 ˚C (day), 19˚C (night),
12-h photoperiod and once daily watering. Three non-inoculated plants were kept away from
inoculated ones under the same conditions in a greenhouse and served as control plants.
Re-isolations were performed from leaves artificially inoculated with powdery mildew fun-
gus and from control plants. Macro- and micro-morphological characteristics of the hyphae,
conidiophores and conidia stripped off from leaves of the inoculated plants were observed.
Specific symptoms of powdery mildew and disease severity were evaluated at three time-points
starting 10 days post-inoculation (dpi) (10, 15 and 20 dpi).
Disease severity (DS) as the percentage of visually infected leaf area was evaluated by aver-
aging three inoculated plants. Statistical analyses for the pathogenicity assay were performed
using PAST v. 4.03 software [30]. Differences in DS between different time-points were ana-
lyzed by a one-way ANOVA, and in case of significant results (P <0.05), the Turkey HSD was
used for mean separation.
Results
Field survey
Out of eight hazelnut orchards inspected, typical E.corylacearum symptoms were observed in
two orchards located in the Benevello and Serravalle Langhe areas of the Cuneo province.
Symptoms associated with E.corylacearum were not found in the remaining sites in the prov-
inces Torino and Asti (Fig 1). In these two provinces, only symptoms specific to common
powdery mildew caused by P.guttata were observed.
Symptoms caused by E.corylacearum have been observed from late spring through the
summer. These symptoms consisted of small circular, powdery white spots on the adaxial leaf
surface, measuring a few millimeters in size, with pale to chlorotic leaf areas. Lesions of yellow
to brown color corresponding to the upper white spots were observed on the lower leaf surface.
Powdery spots progressively increased to 1–1.5 cm in size (Fig 2A), and also occurred on
young branches and fruit bracts at later inspections (Fig 2B). Infected leaves tended to a pur-
ple-bronze color (Fig 2C). Finally, heavily infected leaves wilted prematurely, and affected
fruits dried and occasionally fell.
Morphological characterization
Morphological observations of 160 samples from eight locations confirmed the presence of E.
corylacearum only in samples from powdery mildew spots from the upper leaf side in two
orchards; Benevello and Serravalle Langhe. All other samples collected from the remaining six
locations where common powdery mildew was observed did not show the presence of E.cory-
lacearum. In these samples, the presence of P.guttata was only observed (data not shown).
These morphological results confirmed the results of the field survey and indicated that E.cor-
ylacearum is predominantly found on the ‘upper’ powdery spots of the hazelnut leaves, and P.
guttata is found on the ‘lower’ ones.
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Microscopic observation of 36 selected E.corylacearum samples mainly revealed a myce-
lium on the upper side of leaves, with the following characteristics: 2–5 μm wide, white,
branched, hyaline, septate, and thin-walled. The size of conidiophores was around
69 ±4×7.1 ±1.2 μm. Size and shape of conidia were similar among all isolates, showing an
ellipsoid to ovoid shape, hyaline, with size of 32.3 ±0.8 ×20.4 ±0.7 μm, and they were pro-
duced individually on the conidiophores (Fig 3).
Chasmothecia were scattered to gregarious on the lower side of leaves, with a diameter of
88 ±10 μm; 6 to 14 hyaline appendages for each chasmothecium. Appendages were long about
the chasmothecial diameter, equatorial, with a thick base and thinner upper part, and with reg-
ularly dichotomously branched apices and curved tips. Each chasmothecium contained 3–5
asci of obovoid to ellipsoid shape with a size of 44 ±1×33.2 ±0.8 μm, containing 5–7 hyaline
ascospores of ellipsoid to ovoid shape with a size of 20.0 ±0.7 ×12.4 ±0.3 μm (Fig 3). Follow-
ing the key for the identification to the species of Erysiphe on Corylus hosts [20], all studied iso-
lates were identified as E.corylacearum.
Based on morphological observations, 36 E.corylacearum isolates were selected and used in
subsequent molecular analyses. Among them, 22 isolates originated from Serravalle Langhe
location and 14 isolates from Benevello location (Table 1).
Molecular identification and phylogenetic analyses
The alignment of 36 ITS sequences from this study with the reference Erysiphe spp. sequences
available in the GenBank database showed the highest identity (99.6–100%) with E.coryla-
cearum, while a lower identity (98.9–99.2%) was observed with E.pseudocorylacearum, the
closest phylogenetic relative of E.corylacearum [20].
Phylogenetic analyses based on ITS sequences of the studied isolates and of 11 reference iso-
lates of six Erysiphe species grouped all 36 isolates with E.corylacearum (Fig 4).
All studied isolates clustered in the same cluster with E.corylacearum from Italy, Spain,
Austria, Switzerland, Germany, Slovakia, Slovenia, Hungary, Romania, Azerbaijan, Turkey,
Georgia, and Iran. Within this major group, only E.corylacearum Chinese isolates HMNWA-
FU-CF2012020 and Dai13042 from Asian hazel, and Hungarian KK_3_219 isolate from hazel-
nut formed a separate subcluster.
Fig 2. Symptoms of Erysiphe corylacearum on hazelnut leaves. (A) 0.5–1 cm circular, powdery white spots and pale to yellow leaf areas on upper leaf
surface; (B) White to grayish mycelium growth on fruit bracts; (C) Progressive growth of the powdery spots and turning the leaf color to purple-bronze
in late infection stage.
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Phylogenetic analyses carried out for the single-locus sequences (rpb2,CaM and GAPDH)
permitted a further distinction of the studied isolates in distinct E.corylacearum subclusters,
while the GS analysis grouped all studied isolates together as the ITS region (S1 Fig).
Finally, concatenated ML phylogenetic analyses based on all five loci (ITS, rpb2,CaM,
GAPDH and GS) were performed and the obtained results allowed a more robust differentia-
tion of the studied isolates discerning 4 different subclusters when compared to single loci (Fig
5). The first subcluster included predominantly isolates from the Serravalle Langhe orchard
with two isolates from the Benevello orchard, while the other three subclusters contained iso-
lates exclusively from one orchard, either Serravalle Langhe or Benevello. Concatenated BI
Fig 3. Morphological characteristics of Erysiphe corylacearum isolates from hazelnut in this study. (A) Conidia; (B) Chasmothecium realising the
asci with ascospores; (C) Ascospores; (D) Chasmothecium and its appendages. Scale bar = 20 μm (A-C), 50 μm (D).
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Fig 4. Phylogenetic analysis of Erysiphe corylacearum isolates from hazelnut obtained on the basis of ITS inferred
from Maximum Likelihood analysis. The values at the nodes indicate bootstrap support values based on 1000
replicates. For each isolate, the isolate name, the Accession Number, the host affiliation, and the geographic origin are
shown. The isolates of E.corylacearum from this study are shown in bold and their collection site is shown in
parentheses; (BE) = Benevello, (SL) = Serravalle Langhe. The isolate FH00941202 of Erysiphe necator was used as
outgroup.
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phylogenetic analyzes confirmed the results of grouping and subgrouping of the studied iso-
lates obtained by ML analyses (S2 Fig).
Overall, the ITS region revealed 4 haplotypes within Italian and reference isolates with a
low degree of haplotype diversity (Hd) (0.125), as well as a low degree of nucleotide diversity
per site (π) (0.00120) (S1 Table). Tajima’s D, Fu’s and Li’s D, and Fu’s and Li’s F tests were sig-
nificantly negative which may indicate the population expansion or selective processes. Two
independent studies, including this one and one performed by Mezzalama et al. [18] found
higher DNA polymorphism among reference isolates originating from eleven countries com-
pared to the Italian isolates originating from Piedmont. The haplotypes identified in this study
were mainly associated with singleton variable sites. On the other hand, no InDel haplotypes
were found in the examined region by the DNASP software, and they were not associated with
insertion/deletion mutations.
Pathogenicity assays
The first symptoms became visible on inoculated hazelnut branches at 10 dpi, consisting of
tiny white powdery spots on the upper leaf surface. White spots gradually extended along with
the onset of chlorotic leaf areas. On the lower leaf side, yellow to brown areas corresponding to
the upper powdery spots were discerned. Inoculated hazelnut plants showed typical powdery
mildew symptoms at 15–20 dpi, while control plants were asymptomatic (Fig 6).
During the development of the powdery mildew disease, E.colyracearum specific symptoms
were observed during all three observation periods (10,15 and 20 dpi), and DS increased grad-
ually during the time course of the disease. Symptoms between all three-time course periods
were statistically significant and the highest incidence of the disease was observed at 20 dpi
when DS reached about 40%. (S3 Fig).
Fig 5. Phylogram based on ITS, rpb2,CaM,GAPDH and GS sequences of the studied Erysiphe corylacearum isolates. The site of their
collection is shown in parentheses; (BE) = Benevello, (SL) = Serravalle Langhe. The concatenated phylogenetic tree was obtained by
Maximum Likelihood analysis using the Tamura-Nei plus Gamma model. The isolate FH00941202 of Erysiphe necator was used as
outgroup.
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Discussion
A recent taxonomic study on powdery mildew disease agents caused by Erysiphe species on
Corylus hosts in the world includes six species; E.cornutae,E.corylacearum,E.coryli-ameri-
canae,E.corylicola,E.pseudocorylacearum, and E.syringae [20]. Beside them, P.guttata is a
globally widespread powdery mildew agent on hazelnut, of no particular economic concern
for hazelnut growers [4,7,19,31].
In this study, 36 new strains, originating from one geographic zone (Cuneo province in the
Piedmont region) isolated from hazelnut plants showing powdery mildew disease symptoms
on the upper leaf side, were identified as E.corylacearum. We demonstrated that these strains
were the causal agent of the new powdery mildew disease. On the other hand, surveyed hazel-
nut orchards in Turin and Asti provinces with typical symptoms of common powdery mildew
did not show the presence of E.corylacearum, and only the presence of P.guttata was found in
them.
The results of our study are of particular importance because E.corylacearum is increas-
ingly damaging hazelnut cultivation not only in the Piedmont region, but also throughout the
European Union, where it is considered as an emerging pathogen [32]. Originally found only
in Asia and America on other hazelnut species (Asian, Japanese and beaked hazelnut), it was
Fig 6. Pathogenicity test with Erysiphe corylacearum: (a) an inoculated and (b) a non-inoculated hazelnut plant. Powdery mildew symptoms
observed 20 days post-inoculation.
https://doi.org/10.1371/journal.pone.0301941.g006
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able to spread in less than a decade to hazelnut trees from Turkey and Iran to Eastern, Central
and Southern Europe, including Italy [7]. Moreover, it will probably expand further into new
geographic regions.
All the isolates examined in this study were identified morphologically as E.corylacearum
following the identification key for the Erysiphe species on Corylus hosts [20]. However, this
key follows the strict host specialization and, due to a possible host range expansion of Erysiphe
spp., confirmatory molecular analyses were required for the correct identification of the
isolates.
Molecular characterization confirmed the morphological identification in this study. Given
that ITS-based identification is not always sufficient for the precise identification of powdery
mildew species [33] and considering that the majority of previous studies on E.corylacearum
were based exclusively on the ITS molecular characterization [8,10–13,15,18], we undertook
identification using also other loci [24]. The GS phylogenetic analyses grouped all 36 isolates
with E.corylacearum, in a way similar to the ITS marker. However, when the rpb2,CaM and
GAPDH loci were used in phylogenetic analyses, it was possible to better differentiate studied
isolates separating them into two distinct E.corylacearum subclusters. This confirmed the use-
fulness of the markers rpb2,CaM and GAPDH reported by results of Bradshaw and coworkers
[24] allowing an improved broad and fine scale phylogenetic analyses of powdery mildew.
Finally, concatenated ML and BI phylogenetic analyses based on all five loci (ITS, rpb2,
CaM,GAPDH and GS) permitted the most robust phylogenetic scaling of E.corylacearum, as
already reported in similar studies of fungal identification [24,34–36]. This analysis enabled
not only a differentiation into two separate clusters, but also made possible to separate the
major cluster into four different subclusters. The most similar results with the combined phy-
logenetic analysis were achieved with the GAPDH marker, which indicates its potential and
usefulness in future phylogenetic analyses of E.corylacearum. High-resolution genetic typing
approach of MLST in this study reflected the results of previous MLST works of hazelnut pow-
dery mildews [24], but for more comprehensive concatenated phylogenetic studies, it will be
necessary the multi-locus sequencing of E.corylacearum from other countries.
The recent emergence of E.corylacearum in Italy and throughout many European countries
may be associated with the accidental import of infected common hazelnut propagating mate-
rial from Asia, which is susceptible to this powdery mildew. Another possibility is that it was
present in latent form on Japanese hazel that was imported in Europe at the beginning of the
twentieth century [37] or on some other exotic hazelnuts. Since host jumps and/or host range
expansions may happen in powdery mildews [38], it might be that E.corylacearum started to
spread from exotic on other well-established Corylus spp. in Europe such as common and
Turkish hazelnut (Corylus colurna). One additional hypothesis is that E.corylacearum gradu-
ally spread from its initial infection zone (Turkey and Iran) towards to Europe via wind. Obli-
gate biotrophs such as powdery mildews are known for their long-distance dispersal habit.
They may be transported as structures able to cause an infection up to �1000 km from initial
sites, threating the plant health and yield [39] if they are supported by the production of huge
numbers of wind-dispersed spores from one plant to another, essential for the survival of these
obligately biotrophic fungi [40,41]. Moreover, powdery mildews asci show ‘explosive dis-
charge’, known to move in nature with speed exceeding 30 m/s (or 100 km/h) [42].
Regarding the neutrality tests in this study and taking in consideration that all E.coryla-
cearum (studied and reference) isolates showed negative values for Fu and Li’s D and Fu and
Li’s F statistics, the expansion of population size or population selection may be suggested sim-
ilar to reports for other emergent fungi [43–45]. However, negative but not significant values
of neutrality tests among the known Italian E.corylacearum isolates may indicate that the fun-
gus recently arrived, but that further expansion throughout the country has not yet initiated.
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It will be interesting to have more E.corylacearum gene sequences available from other
countries and the whole fungal genome in the GenBank database. This would allow to search
for adaptive founder events which frequently occur in powdery mildew genomes, such as sin-
gle nucleotide variations, transposition events, and genome rearrangements. These events
might be also one of the possible drivers of adaptation of powdery mildews, including E.cory-
lacearum, to new plant hosts and environmental conditions [46,47].
Conclusion
Conclusively, this study has shown that the emerging hazelnut powdery mildew in Northern
Italy is caused by E.corylacearum. Wind-spread, infected planting material, susceptibility of
‘Tonda Gentile delle Langhe’, the major cultivar of hazelnut grown in Piedmont, to E.coryla-
cearum are potential key aspects that should be considered in efficient management of new
emerging plant diseases. Moreover, more surveys in neighboring hazelnut plantations near
infected plantations, the use of sustainable fungicides and the search for new biocontrol agents
will be necessary for an efficient containment of this disease. These initiatives, together with
the concomitant search of resistant cultivars and the use of fungicides with different modes of
action, may contribute to a successful management of this new emerging and dangerous pow-
dery mildew disease.
Supporting information
S1 Fig. Phylogenetic analyses of 36 Erysiphe corylacearum isolates based on single sequences
from rpb2 (a), CaM (b), GAPDH (c), and GS (d). Each phylogenetic tree was obtained by Maxi-
mum Likelihood analysis. Reference strains included in the phylogenetic analyses of 5 loci are
indicated in bold. Bootstrap values of less than 50% are not presented. The tree was rooted to
Erysiphe necator (FH00941202).
(TIF)
S2 Fig. Consensus phylogram based on ITS, rpb2,CaM,GAPDH and GS sequences of the
studied Erysiphe corylacearum isolates. The concatenated phylogenetic tree was obtained by
Bayesian analysis using the GTR substitution model. The isolate FH00941202 of Erysiphe neca-
tor was used as outgroup.
(TIF)
S3 Fig. Powdery mildew disease severity on artificially infected hazelnut plants at 10, 15
and 20 days post-inoculation (dpi). The means and standard deviations of 9 replicates were
shown.
(TIF)
S1 Table. Molecular parameters of the isolates of Erysiphe corylacearum obtained from
partial ITS sequences.
(DOCX)
Acknowledgments
Authors thank C. Boccaccio (University of Tuscia, Viterbo, Italy) for helping in DNA extrac-
tion from plant samples.
Author Contributions
Conceptualization: Slavica Matić, Vojislav Trkulja, Marco Moizio.
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Emerging hazelnut disease caused by Erysiphe corylacearum
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Data curation: Slavica Matić, Andrea G. Caruso, Camilla Sacco Botto, Stefano Panno, Marco
Moizio.
Funding acquisition: Emanuela Noris.
Investigation: Slavica Matić.
Methodology: Slavica Matić, Camilla Sacco Botto, Stefano Panno, Marco Moizio.
Software: Chiara D’Errico.
Supervision: Slavica Matić.
Visualization: Marco Moizio.
Writing – original draft: Slavica Matić, Andrea G. Caruso, Chiara D’Errico, Emanuela Noris,
Vojislav Trkulja, Salvatore Davino, Marco Moizio.
Writing – review & editing: Slavica Matić, Marco Moizio.
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