Characterisation and pathogenicity of

Pestalotiopsis uvicola and Pestalotiopsis

clavispora causing grey leaf spot of mango
(Mangifera indica L.) in Italy
Ahmed Mahmoud Ismail, Gabriella
Cirvilleri & Giancarlo Polizzi

European Journal of Plant Pathology

Published in cooperation with the
European Foundation for Plant
Pathology
ISSN 0929-1873
Volume 135
Number 4
Eur J Plant Pathol (2013) 135:619-625
DOI 10.1007/s10658-012-0117-z

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Eur J Plant Pathol (2013) 135:619625
DOI 10.1007/s10658-012-0117-z

Characterisation and pathogenicity of Pestalotiopsis uvicola

and Pestalotiopsis clavispora causing grey leaf spot of mango
(Mangifera indica L.) in Italy
Ahmed Mahmoud Ismail & Gabriella Cirvilleri &
Giancarlo Polizzi

Accepted: 10 October 2012 / Published online: 19 October 2012

# KNPV 2012

Abstract During 2009 and 2010, twenty-one isolates of Pestalotiopsis spp. Associated with grey
patches on the leaves, twigs, and panicles of mango were collected in six orchards located in Sicily
(Italy). Morphological characteristics of colony
(colour and mycelium appearance), and conidia
(size, shape, septation, length and the number of
apical and the basal appendages) as well as phylogenetic analysis of the Internal Transcribed Spacer (ITS) region (ITS1, 5.8S gene, and ITS2) of six
representative isolates revealed the occurrence of
P. uvicola and P. clavispora. The representative
isolates of both species were pathogenic to the
artificially inoculated detached mango leaves cv.
Kensington Pride and showed significant variation
in lesion size. This is the first report worldwide of
P. uvicola and P. clavispora causing grey leaf spot
of mango.

A. M. Ismail
Agriculture Research Center,
Plant Pathology Research Institute,
12619 Giza, Egypt
G. Cirvilleri : G. Polizzi (*)
Dipartimento di Gestione dei Sistemi
Agroalimentari e Ambientali, sezione Patologia Vegetale,
University of Catania,
Via S. Sofia 100,
95123 Catania, Italy
e-mail: [email protected]

Keywords Mango diseases . Leaf spot . Pestalotiopsis

spp. . Molecular characterisation . Pathogenicity
The mango (Mangifera indica L., family Anacardiaceae) is grown throughout a wide range of frost-free
climates and is one of the worlds most important fruit
crops (Litz 1998). Pestalotiopsis mangiferae (Henn.)
Steyaert (syn. Pestalotia mangiferae Henn.) is the
casual agent of grey leaf spot and stem-end rot of
mango fruit in several countries (Mordue 1980; Lim
and Khoo 1985; Johnson 1994; Ko et al. 2007). Grey
leaf spot, although common on mango in the tropics,
is usually a minor problem (Lim 1998). P. mangiferae
is a weak pathogen that usually requires wounding in
order to infect mango. Other Pestalotiopsis species
were described on mango plants. P. mangifolia
(Guba) J. Xiang Zhang & T. Xu was founded associated to leaf spot and P. versicolor (Speg.) Steyaert was
detected on flowers (Lim 1998). In addition, P. glandicola (Castagne) Steyaert was reported as causal
agent of a post-harvest disease of mango in Bangalore
(Ullasa and Rawal 1989). Since 1990, mango cultivation was introduced to the southern Italy (Sicily) and
the main diseases present in this area are not recognised. Recently, during a survey conducted in mango
plantations in Sicily, severe symptoms of grey leaf
spot were detected in all orchards and observed on
leaves, twigs, and panicles. Grey leaf spot symptoms
were widespread with approximately 70 to 100 %
symptomatic plants per field. As a consequence, the

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Eur J Plant Pathol (2013) 135:619625

vegetative growth and the fruit production of young

mango plants were reduced (Fig. 1). Thus, the objectives of this work was to determine the etiology of the
grey leaf spot of mango in southern Italy.
Surveys were performed during 2009 and 2010 in
six mango orchards cv. Kensington Pride located in
Catania (2 orchards), Messina (2 orchards), Palermo (1
orchard), and Ragusa (1 orchard). Twenty five samples of symptomatic leaves, twigs, and panicles were
collected. Plant materials were surface disinfected by
sequential washing in 70 % EtOH for 30 s and 10 %
bleach solution (0.5 % NaOCl) (for 1 min), and then
rinsed in distilled sterile water (DSW). Small pieces
between the healthy tissues and infected ones were cut
into 24 mm2 and placed in 9-cm-diameters Petri
dishes containing potato dextrose agar medium
(PDA) (Difco Laboratories, Detroit, MI)) amended
Fig. 1 Common observed
symptoms of grey leaf spot
during a survey in mango
plantation. a, large grey necrotic lesions along the
branch (arrow) b lesions
coalesce to form grey
patches and abundant black
acervuli were formed on the
necrotic tissues. Colony and
conidia of P. uvicola (c, d)
and P. clavispora (e, f).
Scale bars: d-f020 m

with streptomycin sulphate (0.1 gl1) to inhibit any

bacterial growth. Plates were incubated at 252 C
in the dark for 5 days until fungal growth appeared.
The most prevalent fungal species associated with
grey leaf spot were Pestalotiopsis spp. Twenty-one
pure cultures of Pestalotiopsis spp. were grown on
PDA at 25 C in the dark for 710 days (Table 1)
and were identified on the basis of morphological
criteria such as conidia size, septation, number of
appendages as well as colony texture and colour
(Keith et al. 2006; Espinosa et al. 2009; Sutton
1980). The isolates were initially separated into two
groups according to their appearance on culture
(Fig. 1). In all Pestalotiopsis isolates five-celled conidia were observed, of which apical and basal cells
were thin-walled hyaline to pale olivaceous and the
three median cells were thick-walled light to dark

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Eur J Plant Pathol (2013) 135:619625
Table 1 Pestalotiopsis isolates
obtained in this study and their
origin

621

Isolates (GeSA No)a

Plant organ

Origin (Province)

2, 3, 4, 5b, 6

twigs

Catania-orchard 1

7b, 9

leaves

Catania-orchard 1

P. uvicola

15, 17

twigs

Catania-orchard 2

16

leaves

Catania-orchard 2

25b

leaves

Ragusa-orchard 3

26

twigs

Ragusa-orchard 3

P. clavispora
a

GeSA: Dipartimento di Gestione dei Sistemi Agroalimentari

e Ambientali, Catania, Italy

Isolates used for molecular

analysis and pathogenicity test

10b, 11, 12, 13, 14

twigs

Catania-orchard 2

18b

twigs

Palermo-orchard 1

20, 21

panicles

Messina-orchard 1

22b

leaves

Messina-orchard 2

brown. In the first group of the isolates resembling to

P. uvicola conidia were smooth, fusiform, straight,
measuring 24.61.6 to 25.11.7 m mean length
and 7.2 to 7.90.6 m mean width. Two to three
hyaline apical appendages and one basal appendage
were usually observed. The apical appendages lengths
ranged from 14.1 to 19.5 m mean length, while the
basal appendages measured 6.4 to 7.2 m mean
length. The colony was initially white to greenish
(front side of plates) and pale saffron colour in the

centre of colony diffused beyond colony margins (reverse side). The acervuli were black and appeared
moderately concentrated in the centre of the plate. In
the second group of the isolates resembling to P.
clavispora, conidia were smooth, fusiform, and wider
at the middle than apex and base measuring, 22.31.4
to 24.71.9 m mean length and 7.60.5 to 9.6
0 m mean width. Two to four (three being the most
frequently observed) apical hyaline straight and slightly swollen at apex appendages measuring 21.8 to

Table 2 Pestalotiopsis isolates retrieved from the GenBank used for phylogenetic analysis
Species

Culture No.

Location

Host

ITS GenBank Accession No.

Pestalotiopsis matildae

CBS118155

South Africa

Thamnochortus spicigerus

DQ278916

Pestalotiopsis matildae

CBS118143

South Africa

T. spicigerus

DQ278917

Pestalotiopsis microspora

TC-13

India

Terminalia chebula

AY924287

Pestalotiopsis microspora

AZ-71

India

Azadirachta indica

AY924291

Pestalotiopsis disseminata

CY236

USA-Texas

Cyphomyrmex wheeleri

HQ608049

Pestalotiopsis disseminata

CY152

USA-Texas

Cyphomyrmex wheeleri

HQ607992

Pestalotiopsis heterocornis

Endo358

China

Podocarpus macrophyllus

AY681489

Pestalotiopsis heterocornis

Endo391

China

Podocarpus macrophyllus

AY681491

Pestalotiopsis theae

Endo80

China

Lucuma nervosa

DQ813433

Pestalotiopsis theae

Endo46

China

Dracontomelon duperreanum

DQ813432

Pestalotiopsis clavispora

P6

Canada

Argania spinosa

HQ414541

Pestalotiopsis clavispora

PALUC-12

Chile

Persea americana

HQ659767

Pestalotiopsis uvicola

UCD2568AR

USA

Vitis vinifera

HQ288239

Pestalotiopsis uvicola

Isolate 173189

China

Taxus chinensis

JN198506

Guignardia mangiferae

CBS 115051

Brazil

Spondias mombin

FJ538325

Guignardia citricarpa

CBS 102374

Brazil

Citrus aurantium

FJ538313

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622
Fig. 2 One of the 17 consensus trees obtained by
PUAP maximum parsimony
analysis from partial ITS region (ITS1, 5.8S gene and
ITS2) of rDNA data sequence (TL0252, CI0
0.921, RI00.957, RC0
0.881, HI00.079). Parsimony bootstrap values from
1,000 replicates are indicated on the branches nodes.
The tree was rooted to outgroup taxa Guignardia
mangiferae CBS115051 and
Guignardia citricarpa
CBS102374. Isolates in bold
are obtained from mango

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Eur J Plant Pathol (2013) 135:619625

623

Fig. 3 Lesions induced by

inoculation of the six representative isolates of P. uvicola and P. clavispora on
detached mango leaves cv.
Kensington Pride. Data
within columns are the mean
of six lesions developed on
two detached mango leaves.
Means values within columns followed by same letter are not significantly
different according to Least
Significant Different test
(LSD) at P<0.05

33.9 m mean length and one basal appendage

measuring 5.5 to 8.1 m mean length. The colony
was white to creamy with dense mycelium, circular growth appearance (front side) and pale luteos
to saffron colour diffused in the medium (reverse
side). The acervuli were black and appeared
throughout the entire plate.
Six representative isolates (GeSA-5, GenBank No
JX875592; GeSA-7, GenBank No JX875593; GeSA10, GenBank No JX875594; GeSA-18, GenBank No
JX875595; GeSA-22, GenBank No JX875596 and
GeSA-25, GenBank No JX875597), were selected
and total DNA was extracted using Puregene Genomic DNA Purification Kit following the manufacturers instructions (Gentra Systems, Minnesota.
U.S.A). Part of the internal transcribed spacer (ITS)
region of the ribosomal DNA operon was amplified
with the universal primers ITS1 and ITS4 (White et al.
1990). PCR reaction was carried out in 25 l containing 1 PCR buffer (Invitrogen-Life Technologies),
1 mg/ml BSA, 2 mM MgCL 2 , 1 mM dNTPs,
0.5 M of each primer, 1U Taq polymerase and 1 l
of DNA template. After an initial hot start (94 C for
3 min), 35 PCR cycles were performed on a Gene
Amp. PCR System 9700 thermocycler (Foster City,
USA) using the following conditions: a denaturation
step of 94 C for 30 s followed by annealing at 50 C
for 50 s and extension at 72 C for 1 min followed by
final extension at 72 C or 10 min. PCR products of
the ITS region (ITS1, 5.8S gene, and ITS2) were
sequenced using the same primers (ITS1 and ITS4)
of the initial amplification. DNA purification and sequencing was performed at BMR Genomics (Padova,

Italy) according to the manufacturers protocol. Sequence data matrix was aligned using ClustalW option
in MEGA version 5 (Tamura et al. 2011) and corrected
the alignment where necessary. The phylogenetic relationship was determined using PAUP (Phylogenetic
Analysis Using Parsimony) v. 4.0b10 (Swofford
2002). Maximum parsimony was performed using
the heuristic search option with 1,000 random taxa
additions and tree bisection and reconnection (TBR)
as the branch-swapping algorithm. MAXTREE set to
1,000, branches of zero length were collapsed and
equally parsimonious trees were saved. Bootstrap support values were evaluated using 1,000 bootstrap replicates to determine tree length (TL), consistency
index (CI), rescaled consistency index (RC), retention
index (RI), and the homoplasy index (HI) (Hillis and
Bull 1993). PCR amplicons of all isolates were ~510
to 600 bp. The manually aligned and adjusted ITS data
set contained 22 sequences of which six sequences
were obtained from mango in this study and 18 were
retrieved from GenBank (Table 2). From total 533
characters including gaps, 337 were constant and 35
variable characters were parsimony-uninformative
while, 161 were parsimony-informative characters.
After heuristic search, maximum parsimony analysis of the 161 informative characters resulted in
17 trees (TL 0252, CI 00.921, RI 00.957, RC 0
0.881, HI 00.079). Results of the phylogenetic
analysis indicated that Pestalotiopsis species
obtained from M. indica can be assigned in two
well-separated clades of which, P. clavispora isolates were clustered with strains (P6 and PALUC
12) obtained from Argania spinosa and Persea

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americana respectively, in a clade strongly supported with bootstrap value 100 %; whereas, P.
uvicola isolates from mango were sub-grouped in
a clade poorly supported with bootstrap value 51 %
within isolates (Isolate 173189 and UCD2568AR)
obtained from Taxus chinensis and Vitis vinifera
respectively (Fig. 2).
The representative six isolates of Pestalotiopsis spp.
were tested for pathogenicity on detached mango leaves
cv. Kensington Pride disinfected by 10 % bleach solution (0.5 % NaOCl) for 2 min. Three leaves were used
for each isolates. A 6-mm of mycelium plugs from a 7days-old culture was placed on three wounded points.
Leaves inoculated with only PDA plugs served as controls. Inoculated leaves were maintained in high relative
humidity, and incubated at 25 C. Mean lesion sizes
were registered after 7 days of incubation. The trial
was repeated once. Symptoms consisted of light to dark
brown lesions surrounding the inoculation sites with
irregular dark brown margins, later, become greyish in
colour and numerous acervuli were developed on the
necrotic tissues. To fulfill Kochs postulates, diseased
tissues were placed on PDA. Pestalotiopsis spp. were
re-isolated with 100 % frequency and the colonies were
morphologically identical to those from original isolates. All the tested isolates of Pestalotiopsis were proven to be pathogenic to mango leaves. P. clavispora
isolates produced significantly larger lesion sizes than
P. uvicola isolates (Fig. 3).
Since the introduction of mango in Italy, little is
known concerning the presence of fungal diseases and
their etiology. In the present study we reported the
occurrence on mango orchards of P. uvicola and P.
clavispora identified on the basis of morphological
features as well as molecular analysis and we demonstrated the pathogenicity of both species on mango
leaves. Pestalotiopsis spp. generally are not hostspecific and can infect a wide range of plants (Steyaert
1953; Suto and Kobayashi 1993; Taguchi et al. 2001).
P. uvicola was previously reported in Sicily as the
causal agent of leaf spot and stem blight of bay laurel
(Vitale and Polizzi 2005). Moreover, P. clavispora was
reported in China and in Chile as responsible for
leaf spot, canker and twig dieback of blueberry (Luan
et al. 2008; Espinoza et al. 2009) and more recently
causing post-harvest stem end rot of avocado in Chile
(Valencia et al. 2011). To our knowledge, this is the
first report worldwide of P. uvicola and P. clavispora
causing grey leaf spot on mango orchards.

Eur J Plant Pathol (2013) 135:619625

Acknowledgments The authors thank T. Yasseen for helpful
contribution to collection of plant samples and G. Scuderi and R.
Faedda for techical assistance in molecular analysis of the strains.

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