CHARACTERISATION OF COLLETOTRICHUM SPECIES CAUSING ANTHRACNOSE DISEASE OF MANGO IN ITALY

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1 Journal of Plant Pathology (2015), 97 (1), Edizioni ETS Pisa, Short Communication CHARACTERISATION OF COLLETOTRICHUM SPECIES CAUSING ANTHRACNOSE DISEASE OF MANGO IN ITALY A.M. Ismail 1, G. Cirvilleri 2, T. Yaseen 3, F. Epifani 4, G. Perrone 4 and G. Polizzi 2 1 Agricultural Research Center, Plant Pathology Research Institute, Giza, Egypt 2 Dipartimento di Agricoltura, Alimentazione e Ambiente, sezione Patologia Vegetale, University of Catania, Via S. Sofia, Catania, Italy 3 International Center for Advanced Mediterranean Agronomic Studies, 70 Valenzano (Bari), Italy 4 Istituto di Scienze delle Produzioni Alimentari,Via Amendola 122/O, Bari, Italy 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 β-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: 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 (0.1 g l 1 ). Plates were kept at 25 C 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 25 C 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-0 Spectrophotometer (Thermo Fisher Scientific, USA). The primers T1 (O Donnell 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

2 168 Colletotrichum on mango in Italy Journal of Plant Pathology (2015), 97 (1), 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 = 20 μm. 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 Tamura- Nei model (1993). A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = ). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], % 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 (25 C) in the dark. The bags were removed after 48 h 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 µm (average = µm). 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 µm (average = µm). 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 µm (average = µm). The phylogenetic tree with the highest likelihood value ( ) 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 , 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 ), were highly supported with a bootstrap value of %, whereas the remaining six isolates clustered in the C. kahawae subsp. ciggaro CBS clade with a bootstrap value of %. 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 22.5 mm, followed by CO34 with

3 Journal of Plant Pathology (2015), 97 (1), Ismail et al CO29 CO34 CO28 CO24 99 CO22 CO19 CO18 CO13 CO1 93 Colletotrichum karstii CBS Colletotrichum phyllanthi CBS Colletotrichum annellatum CBS Colletotrichum petchii CBS Colletotrichum boninense complex Colletotrichum novae-zelandiae CBS Colletotrichum brasiliense CBS Colletotrichum hippeastri CBS Colletotrichum parsonsiae CBS Colletotrichum beeveri CBS Colletotrichum colombiense CBS Colletotrichum brassicicola CBS Colletotrichum boninense CBS Colletotrichum torulosum CBS Colletotrichum cymbidiicola CBS Colletotrichum oncidii CBS Colletotrichum constrictum CBS Colletotrichum dacrycarpi CBS CO26 CO27 CO20 Colletotrichum gloeosporioides CBS Colletotrichum kahawae subsp. ciggaro CO12 Colletotrichum gloeosporioides complex CO14 CO25 CO33 CO35 CO36 Colletotrichum anthrisci CBS Colletotrichum pseudoacutatum CBS Colletotrichum orchidophilum CBS Colletotrichum kinghornii CBS Colletotrichum phormii CBS Colletotrichum australe CBS Colletotrichum acerbum CBS Colletotrichum rhombiforme CBS Colletotrichum salicis CBS Colletotrichum pyricola CBS Colletotrichum godetiae CBS Colletotrichum johnstonii CBS Colletotrichum acutatum CBS Colletotrichum fioriniae CBS Colletotrichum costaricense CBS Colletotrichum tamarilloi CBS Colletotrichum lupini CBS Colletotrichum cuscutae IMI Colletotrichum acutatum complex 83 Colletotrichum limetticola CBS Colletotrichum melonis CBS Colletotrichum paxtonii IMI Colletotrichum simmondsii CBS Colletotrichum sloanei IMI Colletotrichum chrysanthemi CBS Colletotrichum cosmi CBS Colletotrichum walleri CBS Colletotrichum indonesiense CBS Colletotrichum guajavae IMI Colletotrichum scovillei CBS Colletotrichum laticiphilum CBS Colletotrichum brisbanense CBS Colletotrichum nymphaeae CBS ,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 13.5 mm. The other isolates produced lesions of similar size ( mm). Seven days post inoculation, all isolates caused small lesions ( mm) 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 9.9 mm followed by CO35 with a mean lesion diameter of 8.7 mm. The other isolates produced lesions of about the same size ( ). 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 β-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 HG HG Colletotrichum kahawae subsp. ciggaro CO35 HG HG CO36 HG HG Colletotrichum karstii CO24 HG HG CO29 HG HG CO34 HG HG972859

4 170 Colletotrichum on mango in Italy Journal of Plant Pathology (2015), 97 (1), 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. REFERENCES Aiello D., Carrieri R., Guarnaccia V., Vitale A., Lahoz E., Polizzi G., Characterization and pathogenicity of Colletotrichum gloeosporioides and C. karstii causing preharvest disease on Citrus sinensis in Italy. Journal of Phytopathology 163: Arauz L.F., Mango anthracnose: Economic impact and current options for integrated management. Plant Disease 6: Crous P.W., Groenewald J.Z., Risède J.M., Simoneau P., Hywel- Jones N., Calonectria species and their Cylindrocladium anamorphs: species with sphaeropedunculate vesicles. Studies in Mycology 50:

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