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Edizioni ETS Pisa, 2015
167
Short Communication
SUMMARY
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
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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.
Ismail et al.
169
CO29
CO34
CO28
CO24
99 CO22
CO19
93
CO18
CO13
CO1
99
100
82
85
94
100
99
100
100
CO20
Colletotrichum gloeosporioides CBS 112999
79
70
100
100
100
88
97
83
87
96
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
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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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
Ismail et al.
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