CHARACTERISATION OF COLLETOTRICHUM SPECIES CAUSING ANTHRACNOSE DISEASE OF MANGO IN ITALY

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. 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, 12619 Giza, Egypt 2 Dipartimento di Agricoltura, Alimentazione e Ambiente, sezione Patologia Vegetale, University of Catania, Via S. Sofia, 95123 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, 70126 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: +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 (0.1 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

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 = 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 = 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 (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 14-17.9 4.7-6.1 µm (average = 15.8 5.3 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 15.6-19.7 3.2-4.6 µm (average = 17.2 4 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 11.2-13.8 4-5.7 µm (average = 12.6 4.9 µm). 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 %, whereas the remaining six isolates clustered in the C. kahawae subsp. ciggaro CBS 115194 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

Journal of Plant Pathology (2015), 97 (1), 167-171 Ismail et al. 169 79 CO29 CO34 CO28 CO24 99 CO22 CO19 CO18 CO13 CO1 93 Colletotrichum karstii CBS 128500 99 Colletotrichum phyllanthi CBS 175.67 Colletotrichum annellatum CBS 129826 Colletotrichum petchii CBS 378.94 Colletotrichum boninense complex Colletotrichum novae-zelandiae CBS 128505 Colletotrichum brasiliense CBS 128501 Colletotrichum hippeastri CBS 125376 82 Colletotrichum parsonsiae CBS 128525 Colletotrichum beeveri CBS 128527 85 Colletotrichum colombiense CBS 129818 Colletotrichum brassicicola CBS 101059 94 Colletotrichum boninense CBS 123755 Colletotrichum torulosum CBS 128544 99 Colletotrichum cymbidiicola CBS 123757 Colletotrichum oncidii CBS 129828 Colletotrichum constrictum CBS 128504 Colletotrichum dacrycarpi CBS 130241 CO26 CO27 CO20 Colletotrichum gloeosporioides CBS 112999 Colletotrichum kahawae subsp. ciggaro 115194 CO12 Colletotrichum gloeosporioides complex CO14 CO25 CO33 CO35 CO36 Colletotrichum anthrisci CBS 125334 Colletotrichum pseudoacutatum CBS 436.77 Colletotrichum orchidophilum CBS 632.80 Colletotrichum kinghornii CBS 198.35 Colletotrichum phormii CBS 118194 70 Colletotrichum australe CBS 116478 Colletotrichum acerbum CBS 128530 Colletotrichum rhombiforme CBS 129953 Colletotrichum salicis CBS 607.94 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 88 Colletotrichum tamarilloi CBS 129814 97 Colletotrichum lupini CBS 109225 Colletotrichum cuscutae IMI 304802 Colletotrichum acutatum complex 83 Colletotrichum limetticola CBS 114.14 Colletotrichum melonis CBS 159.84 89 Colletotrichum paxtonii IMI 165753 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 13.5 mm. The other isolates produced lesions of similar size (6.5-10.2 mm). Seven days post inoculation, all isolates caused small lesions (3.2-4.1 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 (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 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 HG972854 HG972860 Colletotrichum kahawae subsp. ciggaro CO35 HG972855 HG972861 CO36 HG972856 HG972862 Colletotrichum karstii CO24 HG972851 HG972857 CO29 HG972852 HG972858 CO34 HG972853 HG972859

170 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. REFERENCES Aiello D., Carrieri R., Guarnaccia V., Vitale A., Lahoz E., Polizzi G., 2015. Characterization and pathogenicity of Colletotrichum gloeosporioides and C. karstii causing preharvest disease on Citrus sinensis in Italy. Journal of Phytopathology 163: 168-177. Arauz L.F., 2000. Mango anthracnose: Economic impact and current options for integrated management. Plant Disease 6: 600-611. Crous P.W., Groenewald J.Z., Risède J.M., Simoneau P., Hywel- Jones N., 2004. Calonectria species and their Cylindrocladium anamorphs: species with sphaeropedunculate vesicles. Studies in Mycology 50: 415-430.

Journal of Plant Pathology (2015), 97 (1), 167-171 Ismail et al. 171 Damm U., Cannon P.F., Woudenberg J.H.C., Crous P.W., 2012a. The Colletotrichum acutatum species complex. Studies in Mycology 73: 37-113. Damm U., Cannon P.F., Woudenberg J.H.C., Johnston P.R., Weir B.S., Tan Y.P., Shivas R.G., Crous P.W., 2012b. The Colletotrichum boninense species complex. Studies in Mycology 73: 1-36. Glass N.L., Donaldson G.C., 1995. Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Applied Journal of Environmental Microbiology 61: 1323-1330. Ismail A.M., Cirvilleri G., Polizzi G., 2013a. Characterisation and pathogenicity of Pestalotiopsis uvicola and Pestalotiopsis clavispora causing grey leaf spot of mango (Mangifera indica L.) in Italy. European Journal of Plant Pathology 135: 619-625. Ismail A.M., Cirvilleri G., Polizzi G., Crous P.W., Groenewald J.Z., Lombard L., 2013b. Characterisation of Neofusicoccum species causing mango dieback in Italy. Journal of Plant Pathology 95: 549-557. Krishnapillai N., Wilson Wijeratnam R.S., 2014. First Report of Colletotrichum asianum causing anthracnose on Willard mangoes in Sri Lanka. New Disease Report. Internet Resource: http://dx.doi.org/10.5197/j.2044 0588.2014.029.001. Lima N.B., de A. Batista M.V., De Morais Jr M.A., Barbosa M.A.G., Michereff S.J., Hyde K.D., Câmara M.P.S., 2013. Five Colletotrichum species are responsible for mango anthracnose in northeastern Brazil. Fungal Diversity 61: 75-88. Liu F., Damm U., Cai L., Crous P.W., 2013. Species of the Colletotrichum gloeosporioides complex associated with anthracnose diseases of Proteacea. Fungal Diversity 61: 89-105. Nelson S.C., 2008. Mango anthracnose (Colletotrichum gloeosporiodes). Department of Plant and Environmental Protection Sciences. University of Hawai at Manoa. http://www. ctahr.hawaii.edu/oc/freepubs/pdf/pd-48.pdf. O Donnell K., Cigelnik E., 1997. Two divergent intragenomic rdna ITS2 types within a monophyletic lineage of the fungus Fusarium are nonorthologous. Molecular Phylogenetic and Evolution 7: 103-116. Onyeani C.A., Osunlaja S., Oworu O.O., Sosanya O., 2012. First Report of Fruit Anthracnose in Mango caused by Colletotrichum gloeosporioides in Southwestern Nigeria. International Journal of Scientific and Technology Research 1: 30-34. Peres N.A., Timmer L.W., Adaskaveg J.E., Correll J.C., 2005. Lifestyles of Colletotrichum acutatum. Plant Disease 89: 784-796. Phoulivong S., Cai L., Chen H., Mckenzie E.H.C., Abd-Elsalam K., Chukeatirote E., Hyde K.D., 2010. Colletotrichum gloeosporioides is not a common pathogen on tropical fruits. Fungal Diversity 44: 33-43. Ploetz R.C., 1998. Anthracnose. In: Ploetz R.C., Zentmyer G.A., Nishijima W.T., Rohrbach K.G. Ohr H.D. (eds) Compendium of Tropical Fruit Diseases. APS Press, St. Paul, Minnesota, USA. Prior C., Elango F., Whitewell A., 1992. Chemical control of Colletotrichum infection in mangoes. In: Bailey J.A. Jeger M.J. (eds) Colletotrichum: Biology, Pathology and Control, pp. 326-336. CABI, Wallingford, UK. Rayner R.W., 1970. A Mycological Colour Chart. CMI and British Mycological Society, Kew, UK. Rivera-Vargas L.I., Lugo-Noel Y., McGovern R.J., Seijo T., Davis M.J., 2006. Occurrence and distribution of Colletotrichum spp. on Mango (Mangifera indica L.) in Puerto Rico and Florida, USA. Plant Pathology Journal 5: 191-198. Sangeetha C.G., Rawal R.D., 2009. Temperature requirement of different isolates of Colletotrichum gloeosporioides isolated from Mango. American-Eurasian Journal of Scientific Research 4: 20-25. Scarito G., Pane A., Raudino F., Frisullo S., Cacciola S.O., 2003. Colletotrichum gloeosporioides causal agent of olive rot in Sicily. Journal of Plant Pathology 84: 310. Tamura K., Nei M., 1993. Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Molecular Biology and Evolution 10: 512-526. Tamura K., Peterson D., Peterson N., Stecher G., Nei M., Kumar S., 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution 28: 2731-2739. Vitale A., Castello I., D Emilio A., Mazzarella R., Perrone G., Epifani F., Polizzi G., 2013. Short-term effects of soil solarization in suppressing Calonectria microsclerotia. Plant and Soil 368: 603-617. Weir B.S., Johnston P.R., Damm U., 2012. The Colletotrichum gloeosporioides species complex. Studies in Mycology 73: 115-180. Received June 25, 2014 Accepted October 21, 2014