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Journal of Plant Pathology (2015), 97 (1), 167-171


Edizioni ETS Pisa, 2015
167

Short Communication

CHARACTERISATION OF COLLETOTRICHUM SPECIES


CAUSING ANTHRACNOSE DISEASE OF MANGO IN ITALY
A.M. Ismail1, G. Cirvilleri2, T. Yaseen3, F. Epifani4, G. Perrone4 and G. Polizzi2
1 Agricultural

Research Center, Plant Pathology Research Institute, 12619 Giza, Egypt


di Agricoltura, Alimentazione e Ambiente, sezione Patologia Vegetale,
University of Catania, Via S. Sofia 100, 95123 Catania, Italy
3International Center for Advanced Mediterranean Agronomic Studies, 70100 Valenzano (Bari), Italy
4Istituto di Scienze delle Produzioni Alimentari,Via Amendola 122/O, 70126 Bari, Italy
2Dipartimento

SUMMARY

Anthracnose symptoms consisting of necrotic spots on


the leaves, twigs and branches were observed on mango
trees of cv. Kensington Pride in orchards located in the
countryside of Palermo and Milazzo (southern Italy).
Based on morphological observations and phylogenetic
analysis of the -tubulin (benA) and histone H3 (HIS3)
genes, three Colletotrichum species were identified and
recovered from diseased plants, i.e. C. karstii (nine isolates), C. kahawae subsp. ciggaro (six isolates) and C. gloeosporioides (six isolates). Following artificial inoculation,
all species induced symptoms on the leaves and fruits of
cv. Kensington Pride. To our knowledge, this is the first
report of mango anthracnose caused by C. karstii, C. kahawae subsp. ciggaro and C. gloeosporioides in Italy.
Key words: anthracnose, Colletotrichum spp., mango,
pathogenicity.

Anthracnose is a major disease of mango (Mangifera indica), especially in humid tropical and subtropical growing
areas (Arauz, 2000), where it causes considerable damage
to floral panicles, leaves, and fruits (Ploetz, 1998). Although the prevalent disease agents are Colletotrichum
gloeosporioides and C. acutatum (Prior et al., 1992; Arauz,
2000; Peres et al., 2005; Rivera-Vargas et al., 2006), other
species of this genus, i.e. C. fructicola, C. tropicale and C.
karstii and the newly described C. dianesei (Lima et al.,
2013) have also been found. Moreover, C. asianum was
reported to cause anthracnose in Sri Lanka (Krishnapillai
and Wilson Wijeratnam, 2014), Australia, Panama, Philippines, Brazil, Colombia, Japan and Thailand (Lima et
al., 2013; Weir et al., 2012) whereas Damm et al. (2012a,
Corresponding author: G. Polizzi
Fax: +39.095.7147283
E-mail: gpolizzi@unict.it

2012b) identified C. simmondsii, C. fioriniae and C. karstii, three members of the species complexes C. acutatum
and C. boninense, as the causal agents of anthracnose in
Australia.
Mango diseases in Italy were studied by Ismail et al.
(2013a, 2013b) who investigated disorders other than anthracnose. This latter disease has now been taken into consideration and, as reported in the present paper, the fungal
species associated with it were identified molecularly and
their pathogenicity determined.
Isolations were made on potato-dextrose-agar medium
(PDA) amended with streptomycin sulfate (0.1g l1). Plates
were kept at 25C in the dark and single spore cultures
were obtained. A total of 18 isolates of Colletotrichum spp.
were recovered from symptomatic samples of cv. Kensington Pride collected from orchards of the Palermo and
Milazzo areas. Six representative isolates (CO24, CO26,
CO29, CO34, CO35 and CO36) of Colletotrichum spp.
were investigated morphologically and their pathogenicity
tested. The morphological characteristics of the cultures
were recorded using the color chart of Rayner (1970) and
the conidial size determined after incubation at 25C for
12 days in the dark. Extraction of total genomic DNA was
done using the Wizard Magnetic DNA purification kit for
food (Promega, USA). The quality of genomic DNA was
determined by agarose gel electrophoresis and its amount
estimated with a ND-1000 Spectrophotometer (Thermo
Fisher Scientific, USA). The primers T1 (ODonnell and
Cigelnik, 1997) and Bt2b (Glass and Donaldson, 1995)
were used for the amplification of part of the benA, gene
and primers CYLH3F and CYLH3R (Crous et al., 2004)
for the HIS3 gene. Primer concentrations and the PCR
protocol were as described by Vitale et al. (2013). Preliminary alignment of the two sequenced loci (benA, HIS3) was
performed using the software package BioNumerics version 5.1 (Applied Maths, USA), and manual adjustment for
improvement was made wherever necessary. Phylogenetic
analysis was first conducted on the two single-locus alignments and, successively, the combined alignment of the
two loci was analyzed for deducing phylogeny. Multilocus

168

Colletotrichum on mango in Italy

Journal of Plant Pathology (2015), 97 (1), 167-171

Fig. 1. A. Anthracnose symptoms on the leaves of a naturally infected mango. Dark brown to black lesions coalesce forming large
patches that lead to apical and marginal scorching. B. Symptoms on a detached artificially inoculated mango leaf. C,D. Acervuli
and conidia of Colletotrichum kahawae subsp. ciggaro. E,F. An acervulus and conidia of C. karstii. G,H,I. Acervuli, setae and conidia of C. gloeosporioides. Scale bars = 20m.

alignment was performed using the Clustal W algorithm


in MEGA version 5 (Tamura et al., 2011). Phylogenetic
and molecular evolutionary analyses were inferred using
the Maximum Likelihood method based on the TamuraNei model (1993). A discrete Gamma distribution was
used to model evolutionary rate differences among sites
(5 categories (+G, parameter=0.4299). The rate variation
model allowed for some sites to be evolutionarily invariable
([+I], 45.7103% sites). All positions with less than 95% site
coverage were eliminated. All positions containing gaps
and missing data were eliminated. The analysis involved
69 nucleotides with a total of 815 positions in the final
dataset. The sequence of 51 different Colletotrichum species representing the three main Colletotrichum complexes
(C. gloeosporioides, C. boninense and C. acutatum) were retrieved from GenBank and included in the analysis.
Pathogenicity tests were conducted using the six representative isolates on detached mango leaves of cv. Kensington Pride as described by Ismail et al. (2013a). In addition,
mycelial plugs taken from the margin of actively growing
colonies of the six isolates were placed on abraded areas
of the leaf blades of 1-year-old seedlings and of detached
fruits of the same cultivar. Twenty-four inoculation points
were used for each isolate. Sterile PDA discs were used to
inoculate controls. Inoculated seedlings and fruits were
placed in plastic bags to maintain the humidity high, and
incubated at room temperature (25C) in the dark. The
bags were removed after 48h and seedlings and fruits were
kept at the same temperature.
Fungal isolates identified as C. karstii produced colonies with moderately dense, cottony, lobate mycelium,
initially white in the centre then turning olivaceous buff
on the upper surface and greenish olivaceous on the reverse side of the plate. Conidia were hyaline, smooth,
straight, cylindrical, had a round apex and a base with

a prominent hilum (Fig.1F), and measured 14-17.94.76.1m (average=15.85.3m).


Isolates identified as C. kahawae subsp. ciggaro had
colonies similar to those of C. gloeosporioides, which produced orange masses of conidia released from semi-immersed acervuli. Conidia were hyaline, fusiform, pointed
end from one side (Fig. 1D) and measured 15.6-19.73.24.6m (average=17.24m).
The isolate identified as C. gloeosporioides produced
colonies with dense, raised, cottony mycelium, initially
white, then turning pale purplish-grey on the upper surface, and purplish grey on the reverse side of the plate.
Conidia were hyaline, cylindrical to ellipsoid with rounded
or obtuse ends on both sides (Fig. 1I) and measured 11.213.84-5.7m (average=12.64.9m).
The phylogenetic tree with the highest likelihood value
(5872.0382) is shown in Fig. 2. Initial tree(s) for the heuristic search were obtained applying the Neighbor-Joining
method to a matrix of pairwise distances estimated using
the Maximum Composite Likelihood (MCL) approach.
Nine out of the 18 isolates investigated in this study belonged to the C. boninense complex and clustered together
in a clade containing C. karstii CBS 128500, supported by
a bootstrap value of 99%, whereas the other nine isolates
belonged to the C. gloeosporioides complex. Three isolates
grouping with C. gloeosporioides species (CBS 112999),
were highly supported with a bootstrap value of 100%,
whereas the remaining six isolates clustered in the C. kahawae subsp. ciggaro CBS 115194 clade with a bootstrap
value of 100%. GenBank accession Nos. of six representative isolates are shown in Table 1.
The six representative isolates induced symptoms identical to those observed in the field (Fig. 1b, Fig. 3b). On detached leaves the most aggressive isolate was CO36 with a
mean lesion diameter of 22.5mm, followed by CO34 with

Journal of Plant Pathology (2015), 97 (1), 167-171

Ismail et al.

169

CO29
CO34
CO28
CO24
99 CO22
CO19

93

CO18
CO13
CO1

Colletotrichum karstii CBS 128500


Colletotrichum phyllanthi CBS 175.67
Colletotrichum annellatum CBS 129826
Colletotrichum petchii CBS 378.94

99

100

82

85
94

100

Colletotrichum colombiense CBS 129818


Colletotrichum brassicicola CBS 101059
Colletotrichum boninense CBS 123755
Colletotrichum torulosum CBS 128544

99

100

Colletotrichum boninense complex

Colletotrichum novae-zelandiae CBS 128505


Colletotrichum brasiliense CBS 128501
Colletotrichum hippeastri CBS 125376
Colletotrichum parsonsiae CBS 128525
Colletotrichum beeveri CBS 128527

Colletotrichum cymbidiicola CBS 123757

Colletotrichum oncidii CBS 129828


Colletotrichum constrictum CBS 128504
Colletotrichum dacrycarpi CBS 130241
CO26
100 CO27

100

CO20
Colletotrichum gloeosporioides CBS 112999

Colletotrichum kahawae subsp. ciggaro 115194


CO12
CO14
CO25
100
CO33
CO35
CO36

Colletotrichum gloeosporioides complex

Colletotrichum anthrisci CBS 125334

79

70

100

Colletotrichum pseudoacutatum CBS 436.77


Colletotrichum orchidophilum CBS 632.80
Colletotrichum kinghornii CBS 198.35
Colletotrichum phormii CBS 118194
Colletotrichum australe CBS 116478
Colletotrichum acerbum CBS 128530

Colletotrichum rhombiforme CBS 129953


Colletotrichum salicis CBS 607.94

100

Colletotrichum pyricola CBS 128531


Colletotrichum godetiae CBS 133.44
Colletotrichum johnstonii CBS 128532
Colletotrichum acutatum CBS 112996
Colletotrichum fioriniae CBS 125396
Colletotrichum costaricense CBS 330.75

100

88

97

83

Colletotrichum tamarilloi CBS 129814


Colletotrichum lupini CBS 109225
Colletotrichum cuscutae IMI 304802

Colletotrichum acutatum complex

Colletotrichum limetticola CBS 114.14


Colletotrichum melonis CBS 159.84
Colletotrichum paxtonii IMI 165753
89
96
Colletotrichum simmondsii CBS 12212

Colletotrichum sloanei IMI 364297


Colletotrichum chrysanthemi CBS 126518

87

Colletotrichum cosmi CBS 853.73

Colletotrichum walleri CBS 125472


Colletotrichum indonesiense CBS 127551

96

Colletotrichum guajavae IMI 350839

Colletotrichum scovillei CBS 126529


Colletotrichum laticiphilum CBS 112989
Colletotrichum brisbanense CBS 292.67
Colletotrichum nymphaeae CBS 515.78

0,02

Fig. 2. Phylogenetic tree constructed with the combined sequences of BenA and HIS-3 genes showing the phylogentic relationship
of the Colletotrichum isolates from Italian mangoes with species belonging to C. boninense, C. gloeosporioides and C. acutatum
complexes.

a mean lesion diameter of 13.5mm. The other isolates produced lesions of similar size (6.5-10.2mm). Seven days post
inoculation, all isolates caused small lesions (3.2-4.1mm)
on undetached leaves without significant differences
among them. All the isolates induced typical anthracnose
lesions also on detached fruits (Fig. 3C, D), the most aggressive being CO24 with a mean lesion diameter 9.9mm
followed by CO35 with a mean lesion diameter of 8.7mm.
The other isolates produced lesions of about the same size
(4.1-6.4). Isolations from diseased tissues yielded colonies
whose identity with those used for inoculum was confirmed by morphological and molecular analyses.
Phylogenetic analysis of the combined data of -tubulin
(benA) and histone H3 (HIS3) genes revealed the occurrence of three species of Colletotrichum in diseased mangoes. The most prevalent was C. karstii, a member of the
C. boninense complex recently reported as responsible
of citrus anthracnose in Italy (Aiello et al., 2015), which

constitutes a new record for mango in this country. Notwithstanding its wide geographical distribution, C. karstii
has been found in mango only in Australia (Damm et al.,
2012 b) and Brazil (Lima et al., 2013). The finding of this
species in Italy may be indicative of the expansion of its
Table 1. GenBank accession numbers of the six representative isolates of Colletotrichum spp. causing mango anthracnose in Italy.
Species

Culture No. GenBank accession No.


BenA

His3

Colletotrichum gloeosporioides

CO26

HG972854

HG972860

Colletotrichum kahawae
subsp. ciggaro

CO35
CO36

HG972855
HG972856

HG972861
HG972862

Colletotrichum karstii

CO24
CO29
CO34

HG972851
HG972852
HG972853

HG972857
HG972858
HG972859

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Colletotrichum on mango in Italy

Journal of Plant Pathology (2015), 97 (1), 167-171

Fig. 3. Pathogenicity tests on cv. Kensington Pride leaves and fruits: A. Inoculation procedure used for leaves. B. Incipient anthracnose lesions on leaves inoculated with Colletotrichum karstii isolate CO24. C. Lesions on mango fruits inoculated with C.
kahawae subsp. ciggaro isolate CO35. D. Lesions caused by C. karstii isolate CO24. E. Control.

geographical distribution, which may threat mango production in other growing areas.
C. kahawae subsp. ciggaro was first proposed as a novel
subspecies genetically distinct from C. kahawae subsp.
kahawae (Weir et al., 2012). It has been reported from numerous hosts in Australia, Europe, South Africa, and USA
(Weir et al., 2012; Liu et al., 2013), but this is its first record
of this species on mango in Italy and worldwide.
Earlier studies (Arauz, 2000; Rivera-Vargas et al., 2006;
Nelson, 2008; Sangeetha and Rawal, 2009) and the recent
one by Onyeani et al. (2012), have shown that C. gloeosporioides is a common agent of mango and other tropical
fruit trees diseases, contrary to Phoulivong et al. (2010)
claim that this species is not. In our study, three C. gloeosporioides isolates were recovered from mango and the
one whose pathogenicity was tested proved to be little aggressive on detached leaves and fruits, suggesting that this
species is not the major responsible for mango anthracnose
in Italy. This likelihood finds support in a paper by Lima
et al. (2013) who reported that C. gloeosporioides is not a
mango pathogen in Brazil. Furthermore, this species has
been also reported to be less dominant and virulent on

olive fruits in Sicily (insular Italy) (Scarito et al., 2003). To


our knowledge, this is the first record of Colletotrichum
species causing anthracnose of mango in Italy. Additional
data on a larger set of isolates are needed for a more precise assessment of the prevalence of the species involved
in this disease.
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Accepted October 21, 2014

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