J Phytopathol
SHORT COMMUNICATION
Arthrinium phaeospermum, Phoma cladoniicola and Ulocladium
consortiale, New Olive Pathogens in Italy
Sandra Lo Piccolo, Vincenzo Mondello, Selene Giambra, Gaetano Conigliaro, Livio Torta and
Santella Burruano
Dipartimento di Scienze Agrarie e Forestali, University of Palermo, Viale delle Scienze 4, 90128 Palermo, Italy
Keywords
decline syndrome, Olea europaea, Sicily,
weak pathogens
Correspondence
S. Burruano, Dipartimento di Scienze Agrarie e
Forestali, University of Palermo, Italy.
E-mail: santella.burruano@unipa.it
Received: May 7, 2013; accepted: August 13,
2013.
doi: 10.1111/jph.12179
Abstract
In recent years, leaf necrosis and twig dieback in the olive crop have been
detected in Sicily (Italy). In this article, we identify the predominant
fungal species associated with symptomatic leaves and twigs, using
morphological features and DNA sequencing of the internal transcribed
spacer (ITS) region, as Alternaria alternata, Arthrinium phaeospermum,
Phoma cladoniicola and Ulocladium consortiale. The pathogenicity of these
four species was tested on olive plants cv. Biancolilla. All species were
pathogenic on leaves, but only U. consortiale produced cortical lesions on
twigs, thus suggesting its main role in the Olea europaea twig dieback. To
our knowledge, this is the first report of A. phaeospermum, P. cladoniicola
and U. consortiale as olive pathogens.
Introduction
Materials and Methods
Olive (Olea europaea L. var. sativa) is historically grown
in most of the Mediterranean basin countries, where
it represents an economically important tree crop.
Recent investigations have highlighted the incidence
of common O. europea diseases worldwide, such as
cercosporiosis, anthracnose, peacock spot, sooty
mould, fruit rot, verticillium wilt and phoma dieback
(Tosi and Zazzerini 2005; Moral et al. 2008; Trapero
et al. 2009). Additionally, a new decline syndrome in
olive trees has been recently observed in Sicily (Italy).
The syndrome consists of foliar chlorosis, associated
with necrotic irregular marginal or apical spots, which
then flowed resulting in withering of leaf tips. Symptomatic twigs show apical defoliations, cortical necrosis and withering (Fig. 1). A preliminary aetiological
study conducted in two Sicilian olive orchards
allowed us to ascertain the constant association
between fungal microorganisms and symptomatic
organs of declining olive trees (Ferraro et al. 2010).
Thus, the aim of this study was to identify the most
prevalent fungal taxa associated with symptoms and
to test their pathogenicity.
Samplings were conducted during spring, summer
and autumn of 2007 and 2008 in two Sicilian olive
orchards, located on a hill (Racalmuto, Agrigento,
south-western Sicily) and on plane (San Cipirello,
Palermo, north-western Sicily). For each survey, samples of symptomatic leaves and twigs were collected
and subjected to isolation tests. The plant material
was surface-sterilized by sequential washing in 5%
NaOCl for 5 min, 95% EtOH for 1 min and 5% H2O2
for 3 min and then rinsed three times in distilled sterile water. Fragments at the margin between the
healthy and affected tissue were aseptically excised
and placed on 2% malt extract agar (MEA; Oxoid,
Milano, Italy). Petri dishes were incubated at
25 2°C in the dark and daily observed for fungal
development during two weeks. Hyphal tips or spores
of representative fungal isolates were transferred to
2% MEA for a preliminary morphological identification at genus level on the basis of their macroscopic
and microscopic features (Domsch et al. 1980; Barnett
and Hunter 1998; Pitt and Hocking 1999).
Microscopic characteristics were examined using a
Ó 2013 Blackwell Verlag GmbH
1
New fungal pathogens on olive
(a)
(b)
Fig. 1 Symptomatic olive trees in Sicily: withering of twigs (a); chlorosis
and necrosis on leaves (b).
S. Lo Piccolo et al.
The 40-ll reaction volume contained 50–100 ng of
DNA template, 2 mM MgCl2, 0.2 mM dNTP, 0.3 lM of
each primer, 0.5 U of Taq DNA polymerase (Dream
Taq, Fermentas, Italy) and 1 9 Dream Taq buffer
(Fermentas). The amplification reaction was carried
out as follows: initial denaturation at 95°C for 2 min;
followed by 35 cycles of denaturation at 95°C for 30 s,
annealing at 55°C for 20 s and extension at 72°C for
1 min; and a final extension at 72°C for 5 min. PCR
products were sequenced in both directions using the
same primers that were used for PCR. The nucleotide
sequences were compared to those found in GenBank
using the BLAST program (Altschul et al. 1997). The
same isolates used for DNA extraction were tested for
pathogenicity on five 3-year-old healthy olive plants
(cv. Biancolilla). For each plant, the inoculations
were performed on three twigs (in three different points) and on five leaves for each twig. In detail,
a 6-mm-diameter mycelium plug derived from a
7-day-old colony grown on malt extract agar (MEA,
Oxoid, UK) was placed both on leaves wounded in
three points with sterile needles and on properly
peeled twigs. Three control plants were inoculated
with non-colonized plugs of MEA. Plants were then
kept in greenhouse under controlled conditions
(T = 22 1°C; R.H. = 70–80%), watered fortnightly
and checked for symptoms. The length of leaf lesions
was measured 2 weeks after inoculation, while the
length of cortical lesions, after 20 weeks. The data
were subjected to ANOVA and the Tukey’s test. To fulfil
the Koch’s postulates, re-isolation assays from
symptomatic tissues were carried out on MEA.
Results and Discussion
light microscope (Axioskop; Zeiss, Jena, Germany)
coupled to an AxioCam MRc5 (Zeiss) digital camera,
and images were captured using the software AxioVision 4.6 (Zeiss). For each sampling, the isolation frequency (IF) of each fungal genus in both olive groves
was calculated. The species-level identification of the
most prevalent fungal genera was performed on the
basis of criteria given by Simmons (1998, 2007), Samson et al. (2000) and Diederich et al. (2007). One representative isolate for each fungal species identified
morphologically was used for genomic DNA extraction using the standard cetyltrimethylammonium
bromide (CTAB)-based protocol (O’Donnell et al.
1998). The internal transcribed spacer (ITS) regions
(ITS1, 5.8S gene, ITS2) of the ribosomal DNA operon
were amplified by polymerase chain reaction (PCR)
using the primers ITS1F (fungal specific; Gardes and
Bruns 1993) and ITS4 (universal; White et al. 1990).
2
The most prevalent fungal genera associated with
leaf necrosis and twig dieback in both olive orchards
were Alternaria, Arthrinium, Phoma and Ulocladium
(Table 1). Other genera isolated in only one of the
two olive groves were Aureobasidium, Camarosporium
and Septoria. Moreover, ubiquitous genera such as
Cladosporium and Penicillium were isolated with low IF
in both locations. The specific identification of the isolates referable to the four predominant genera
showed the presence of only one species for each
genus. The isolates of Alternaria genus produced colonies that grew rapidly (5–7 cm diameter at 25°C for
7 days on MEA) and appeared floccose, whitish to
grey brown, with the reverse brown to black. Conidiophores were septate and pale brown, simple or
branched, geniculate and bored conidia in chains at
the apex. Conidia (7–10 9 23–34 lm), up to nine in
a chain, were brown, ovoid to ellipsoidal, often with a
Ó 2013 Blackwell Verlag GmbH
S. Lo Piccolo et al.
Ó 2013 Blackwell Verlag GmbH
Table 1 Isolation frequency (%) of fungal taxa isolated from symptomatic leaves and twigs of declining olive trees from two Sicilian olive orchards during 2007 and 2008
Racalmuto
San Cipirello
Leaves
Twigs
2007
2008
Leaves
2007
2008
Twigs
2007
2008
2007
2008
Fungal taxa
Sp
Su
Au
Sp
Su
Au
Sp
Su
Au
Sp
Su
Au
Sp
Su
Au
Sp
Su
Au
Sp
Su
Au
Sp
Su
Au
Alternaria alternata
Arthrinium phaeospermum
Aureobasidium
Camarosporium
Cladosporium
Penicillium
Phoma cladoniicola
Septoria
Ulocladium consortiale
TOT
56.8
―
―
5.6
8.2
0.8
―
―
―
71.4
2.1
―
―
―
―
―
6.2
―
―
8.3
16.6
2.8
0.9
4.6
6.4
1.4
8.3
―
2.3
43.3
1.1
0.5
2.1
1.6
0.5
1.1
11.6
―
1.1
19.6
0.6
0.6
―
―
―
―
―
―
0.6
1.8
6.3
24.2
2.0
0.8
2.0
0.4
1.2
―
30.6
67.5
4.5
0.5
―
2.9
6.0
1.5
―
―
0.5
15.9
―
―
―
―
―
―
―
―
―
―
2.9
5.7
3.7
2.9
0.9
0.9
0.9
―
5.7
23.6
―
―
1.9
―
―
―
―
―
―
1.9
0.8
―
0.7
―
―
―
―
―
12.1
13.6
―
―
―
―
―
1.7
―
―
6.9
8.6
54.1
―
―
―
3.5
―
―
―
―
57.6
38.8
5.2
―
―
4.5
―
1.1
0.2
0.2
50.0
51.4
1.2
―
―
7.5
―
9.2
6.1
―
75.4
26.7
3.6
―
―
4.1
0.3
2.4
3.8
―
40.9
12.5
0.8
―
―
0.6
―
5.3
1.4
0.3
20.9
23.1
13.4
―
―
8.0
4.8
―
1.1
18.2
68.6
―
―
―
―
6.1
―
―
―
―
6.1
2.4
―
―
―
3.3
0.8
―
5.3
8.6
20.4
―
―
―
―
―
―
―
―
3.8
3.8
5.7
―
―
―
0.9
―
―
―
3.4
10.0
―
―
―
―
2.6
―
―
0.5
―
3.1
―
0.5
―
―
―
―
0.9
―
0.5
1.9
(c)
(a)
(f)
(d)
(b)
New fungal pathogens on olive
(e)
(h)
short beak, muriform with up to six transverse and
2–3 longitudinal or oblique septa, smooth or roughened. These isolates were identified as Alternaria alternata (Fr.) Keissl (Fig. 2a,b). Arthrinium isolates were
characterized by rapidly growing colonies (5–7 cm
diameter at 25°C for 7 days on MEA), with floccose
whitish aerial mycelium and sporulating dark spots
on the surface. Conidiophores were hyaline or pale
brown, produced clusters of dark brown, lenticular,
smooth conidia (5–8 9 9–11 lm), and provided with
(g)
Fig. 2 Morphology of a 7-day-old colony on MEA and conidia of Alternaria alternata (a, b), Arthrinium phaeospermum (c, d), Phoma cladoniicola (e, f) and Ulocladium consortiale (g, h).
3
New fungal pathogens on olive
S. Lo Piccolo et al.
an equatorial germ slit. These isolates were identified
as Arthrinium phaeospermum (Corda) M.B. Ellis
(Fig. 2c,d). Phoma isolates, identified as Phoma cladoniicola Diederich, Kocourk., & Etayo (Fig. 2e,f),
showed fast-growing colonies (6–8 cm diameter at
25°C for 7 days on MEA) that appeared flat, powdery
to velvety, initially white and later olive grey, dark
brown to black in reverse. Black pycnidia with a subspherical to pyriform shape (50–100 lm) and ostiolate
were observed. Conidiogenous cells lining in the
inner wall of the pycnidial cavity were short, ampulliform, hyaline and smooth. Conidia (1.5–3 9 3–6 lm)
(a)
(b)
(c)
were ellipsoid, hyaline, smooth and biguttulate. The
isolates of Ulocladium produced colonies growing
mildly fast (4–6 cm diameter at 25°C for 7 days on
MEA) that appeared effuse, powdery, olive brown to
nearly black and brown in reverse. Conidiophores
were simple or branched, pale brown, smooth and
strongly geniculate. Conidia (20–35 9 15–20 lm),
single or in short chains, were obovoid or ellipsoidal,
golden brown, smooth or slightly rough and muriform with 1–5 transverse and 1–2 longitudinal or oblique septa. These isolates were identified as Ulocladium
consortiale (Th€
um.) E.G. Simmons (Fig. 2g,h). The ITS
(d)
(e)
(f)
(g)
4
Fig. 3 Pathogenicity on 3-year-old olive plants
(cv. Biancolilla) of Alternaria alternata, Arthrinium phaeospermum, Phoma cladoniicola and
Ulocladium consortiale. Irregular necrotic
lesions on leaves caused by A. alternata (a),
A. phaeospermum (b), P. cladoniicola (c) and
U. consortiale (d) 2 weeks after inoculation.
Cortical lesion produced by U. consortiale (e)
20 weeks after inoculation: detail of lesion on
twig (f) and brown internal necrosis visible in
longitudinal section (g); white arrows indicate
the point of inoculation.
Ó 2013 Blackwell Verlag GmbH
S. Lo Piccolo et al.
New fungal pathogens on olive
Table 2 Mean lesion length on olive leaves cv. Biancolilla, 2 weeks after
inoculation with four fungal species
Species
Mean lesion length (cm) SEa
Alternaria alternata
Arthrinium phaeospermum
Phoma cladoniicola
Ulocladium consortiale
Control
2.2
3.5
2.6
3.4
0.0
0.3 bB
0.4 cC
0.4 bBC
0.2 cC
0.0 aA
1971) and decay of caraway seedlings (Mazur and
Nawrocki 2004). To our knowledge, this is the
first report of A. phaeospermum, P. cladoniicola and
U. consortiale as O. europaea pathogens.
Acknowledgements
We thank Giancarlo Polizzi for his valuable suggestions and for critically reading the manuscript.
a
Mean and standard error based on 75 replicates per treatment. Values
followed by equal letters are not significantly different. Lowercase and
uppercase letters refer to P ≤ 0.05 and P ≤ 0.01, respectively.
sequences of the four selected isolates, compared with
those available in GenBank, confirmed the morphological identifications. Particularly, the isolates
belonging to genera Alternaria (GenBank accession
number KC577267), Arthrinium (KC577268), Phoma
(KC577269) and Ulocladium (KC577270) showed
99% homology with A. alternata (JN673372), 100%
with A. phaeospermum (KC253945), 99% with
P. cladoniicola (JQ238629) and 99% with U. consortiale
(HM036619), respectively.
Pathogenicity tests showed that all the assayed
fungi caused irregular necrotic leaf spots, which
spread from the inoculum point to the leaf apex
(Fig. 3a–d). Ulocladium consortiale was the only fungus
able to produce cortical lesions (3–3.5 cm long) on
twigs (Fig. 3e–g); no symptoms were observed in controls. The length of leaf lesions produced by four fungi
differed significantly from the control (P ≤ 0.05 and
P ≤ 0.01; Table 2). Moreover, A. phaeospermum and
U. consortiale produced significantly larger lesions than
A. alternata and P. cladoniicola. Alternaria alternata,
A. phaeospermum, P. cladoniicola and U. consortiale
were re-isolated from all the symptomatic organs, and
the colonies were morphologically identical to those
of the original isolates.
In this study, we report the pathogenicity of A. alternata, A. phaeospermum, P. cladoniicola and U. consortiale on olive trees. The results indicate the four
tested fungi as pathogens on leaves and the main role
of U. consortiale in the twig dieback. On olive, only
A. alternata has already been reported as pathogen of
leafy cuttings of shoots (Bourbos et al. 1999). Arthrinium phaeospermum has been described as an endophyte in blueberry (Munitz et al. 2013) and maize
seeds (Kido et al. 2012) and is associated with flower
malformation in Aloe zebrina (Niaz and Dawar 2009),
while P. cladoniicola has been described as a lichenicolous fungus (Urbanavichus and Urbanavichene
2011). Moreover, U. consortiale has been reported as a
causal agent of black lesions on tomato fruit (Bartz
Ó 2013 Blackwell Verlag GmbH
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