High Species Diversity in Colletotrichum Associated with Citrus Diseases in Europe

Persoonia 39, 2017: 32–50 ISSN (Online) 1878-9080 www.ingentaconnect.com/content/nhn/pimj RESEARCH ARTICLE https://doi.org/10.3767/persoonia.2017.39.02 High species diversity in Colletotrichum associated with citrus diseases in Europe V. Guarnaccia1, J.Z. Groenewald1, G. Polizzi 2, P.W. Crous1,3,4 Key words Abstract Species of Colletotrichum are considered important plant pathogens, saprobes, and endophytes on a wide range of plant hosts. Several species are well-known on citrus, either as agents of pre- or post-harvest infections, Anthracnose such as anthracnose, postbloom fruit drop, tear stain and stem-end rot on fruit, or as wither-tip of twigs. In this study Citrus we explored the occurrence, diversity and pathogenicity of Colletotrichum spp. associated with Citrus and allied multi-locus sequence typing genera in European orchards, nurseries and gardens. Surveys were carried out during 2015 and 2016 in Greece, pathogenicity Italy, Malta, Portugal and Spain. A total of 174 Colletotrichum strains were isolated from symptomatic leaves, fruits, petals and twigs. A multi-locus phylogeny was established based on seven genomic loci (ITS, GAPDH, ACT, CAL, CHS1, HIS3 and TUB2), and the morphological characters of the isolates determined. Preliminary pathogenicity tests were performed on orange fruits with representative isolates. Colletotrichum strains were identified as members of three major species complexes. Colletotrichum gloeosporioides s.str. and two novel species (C. helleniense and C. hystricis) were identified in the C. gloeosporioides species complex. Colletotrichum karstii, C. novaezelandiae and two novel species (C. catinaense and C. limonicola) in the C. boninense species complex, and C. acutatum s.str. was also isolated as member of C. acutatum species complex. Colletotrichum gloeosporioides and C. karstii were the predominant species of Colletotrichum isolated. This study represents the first report of C. acutatum on citrus in Europe, and the first detection of C. novaezelandiae from outside New Zealand. Pathogenicity tests revealed C. gloeosporioides s.str. to be the most virulent species on fruits. The present study improves our understanding of species associated with several disease symptoms on citrus fruits and plants, and provides useful information for effective disease management. Article info Received: 3 March 2017; Accepted: 7 April 2017; Published: 7 June 2017. INTRODUCTION stress conditions, or placed in postharvest storage (Crous et al. 2016a). Appressoria that develop from germinating spores, Colletotrichum is one of the most important genera of plant start plant infection by penetration of the cuticle (Deising et al. pathogenic fungi, responsible for several diseases in many 2000) and occasionally also of the epidermal cells via fungal crops worldwide (Sutton 1992, Cannon et al. 2000, 2012, Cai hyphae (Bailey & Jeger 1992). et al. 2009, Udayanga et al. 2013). Colletotrichum spp. were The taxonomy of Colletotrichum species has recently been recently included in the list of the 10 most important plant reviewed in several impactful studies (Cannon et al. 2008, Cai pathogenic fungi in the world, based on perceived scientific and et al. 2009, Damm et al. 2009, 2012a, b, 2013, 2014, Weir et economic importance (Dean et al. 2012). Agricultural produc- al. 2012, Liu et al. 2014, 2015, 2016). Before the molecular era, tion losses caused by Colletotrichum spp. involve important morphological characters such as size and shape of conidia staple food crops grown in developing countries throughout and appressoria, presence or absence of setae, aspect, colour the tropics and subtropics (Dean et al. 2012). Colletotrichum and growth rate of the colonies, formed the basis to study and species can infect more than 30 plant genera (Perfect et al. compare the taxonomy of Colletotrichum species (Von Arx 1957, 1999, Farr et al. 2006, Damm et al. 2012a, b, Farr & Rossman Sutton 1980, 1992). Modern studies demonstrated that these 2017), causing anthracnose disease and postharvest decay characters are not reliable for species level identification due to on a wide range of tropical, subtropical and temperate fruits, their variability under changing environmental conditions (Cai grasses, vegetable crops and ornamental plants (Bailey & Jeger et al. 2009, Liu et al. 2016). 1992, Bernstein et al. 1995, Freeman & Shabi 1996, Crouch et al. 2009, Lima et al. 2011, Damm et al. 2012a, b, Anderson et Following adoption of the use of multi-gene phylogenetic ana- al. 2013, Crous et al. 2016b, Guarnaccia et al. 2016, De Silva lysis, the polyphasic protocols for studying the genus Colleto et al. 2017). Moreover, many Colletotrichum species are latent trichum significantly changed the classification and species plant pathogens, endophytes, epiphytes or saprobes, switch- concepts in Colletotrichum (Cannon et al. 2012, Damm et al. ing to a pathogenic lifestyle when host plants are subjected to 2012a, b, 2013, 2014, Weir et al. 2012). Several systematic studies of nearly all acknowledged species have led to the 1 Westerdijk Fungal Biodiversity Institute, Uppsalalaan 8, 3584 CT Utrecht, identification of 11 Colletotrichum species complexes, and The Netherlands; more than 20 singleton species (Cannon et al. 2012, Liu et corresponding author e-mail: [email protected]. al. 2014, 2016, Marin-Felix et al. 2017). In plant pathology the 2 Dipartimento di Agricoltura, Alimentazione e Ambiente, sezione Patologia most important species are members of the C. gloeosporioides Vegetale, University of Catania, Via S. Sofia 100, 95123 Catania, Italy. (Cannon et al. 2008, Phoulivong et al. 2010, Weir et al. 2012), 3 Department of Microbiology & Plant Pathology, Forestry & Agricultural Biotechnology Institute, University of Pretoria, Pretoria, South Africa. C. acutatum (Marcelino et al. 2008, Shivas & Tan 2009, Damm 4 Microbiology, Department of Biology, Utrecht University, Padualaan 8, 3584 et al. 2012a, Baroncelli et al. 2015), C. boninense (Moriwaki et CH Utrecht, The Netherlands. al. 2003, Yang et al. 2009, Damm et al. 2012b) and C. truncatum © 2017 Naturalis Biodiversity Center & Westerdijk Fungal Biodiversity Institute You are free to share - to copy, distribute and transmit the work, under the following conditions: Attribution: You must attribute the work in the manner specified by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). Non-commercial: You may not use this work for commercial purposes. No derivative works: You may not alter, transform, or build upon this work. For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at http://creativecommons.org/licenses/by-nc-nd/3.0/legalcode. Any of the above conditions can be waived if you get permission from the copyright holder. Nothing in this license impairs or restricts the author’s moral rights. V. Guarnaccia et al.: Colletotrichum on citrus in Europe 33 (Damm et al. 2009, Cannon et al. 2012) complexes. The use et al. 2016). In these studies, Colletotrichum species belonging of multi-locus phylogenetic analyses revealed many cases, in to the C. acutatum species complex were never found associ- which certain Colletotrichum spp. that were historically con- ated with citrus. However, C. acutatum s.lat. was reported in sidered to be causal agents of economically important plant Mediterranean countries causing diseases on several hosts disease, were then revealed to be different species, such as such as Fragaria × ananassa (Garrido et al. 2008), Arbutus C. alienum which seems to be the most important species in unedo (Polizzi et al. 2011) and Olea europaea (Talhinhas et al. Proteaceae cultivation (Liu et al. 2013), and not C. gloeospori 2011). Because of the commercial yield losses in citrus orchards oides s.str. as previously assumed (Lubbe et al. 2004). caused by Colletotrichum infections, the recent findings and the The citrus industry is one of the most important fruit industries changes in the species concepts, new surveys are required to worldwide. The Mediterranean countries are second only to study the Colletotrichum species diversity related to citrus and China for fruit production, and are the largest fruit exporter after their occurrence and association with foliar and fruit diseases. South Africa (FAO 2016). Therefore, the study and knowledge of The current study aimed to investigate the major citrus produc- all the pathogens affecting this crop is imperative. The use of a tion areas in Europe by large-scale sampling, and to identify polyphasic approach in the past revealed many cryptic and new isolates via morphology and multi-locus phylogeny based on Colletotrichum species associated with citrus, belonging to four modern taxonomic concepts. In 2015 and 2016 several surveys species complexes, namely: the C. boninense species com- were conducted in commercial nurseries, citrus orchards, gar- plex (C. boninense, C. citricola, C. constrictum, C. karstii and dens, backyards and plant collections to determine the occur- C. novaezelandiae) (Damm et al. 2012b, Huang et al. 2013); rence of Colletotrichum spp. associated with Citrus and allied the C. acutatum species complex (C. abscissum, C. acutatum, genera (Atlantia, Fortunella, Microcitrus, Murraya, Poncirus). C. citri, C. godetiae, C. johnstonii, C. limetticola and C. sim In particular the objectives of the present study were: mondsii) (Damm et al. 2012a, Huang et al. 2013, Crous et i) to conduct extensive surveys for

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