Mycologia, 107(2), 2015, pp. 284–297. DOI: 10.3852/14-206 # 2015 by The Mycological Society of America, Lawrence, KS 66044-8897 Unexpected diversity of basidiomycetous endophytes in sapwood and leaves of Hevea Rachael Martin bia, Rigidoporus, Tinctoporellus, Trametes (Polypor- Romina Gazis ales), Peniophora, Stereum (Russulales) and Coprinel- Clark University, Biology Department, 950 Main Street, lus (Agaricales), all of which have been reported as Worcester, Massachusetts 01610 endophytes from a variety of hosts, across wide Demetra Skaltsas geographic locations. Literature records on the University of Maryland, Department of Plant Science geographic distribution and host association of these and Landscape Architecture, 2112 Plant Sciences genera revealed that their distribution and substrate Building, College Park, Maryland 20742 affinity could be extended if the endophytic niche was investigated as part of fungal biodiversity surveys. Priscila Chaverri Key words: natural rubber, Polyporales, tropical University of Maryland, Department of Plant Science and Landscape Architecture, 2112 Plant Sciences fungi, white-rot fungi Building, College Park, Maryland 20742, and Universidad de Costa Rica, Escuela de Biologı´a, Apdo. 11501-2060, San Pedro, San Jose´, Costa Rica INTRODUCTION David Hibbett1 Horizontally transmitted fungal endophytes are be- Clark University, Biology Department, 950 Main Street, lieved to harbor a great portion of uncharacterized Worcester, Massachusetts 01610 fungal diversity (Fro¨hlich and Hyde 1999, Arnold et al. 2000, Arnold and Lutzoni 2007, Arnold 2008). This assemblage includes potential biological control Abstract: Research on fungal endophytes has ex- agents, sources of useful secondary metabolites panded dramatically in recent years, but little is (Higginbotham et al. 2013), plant mutualists (Arnold known about the diversity and ecological roles of et al. 2003), latent and weak pathogens (Slippers and endophytic basidiomycetes. Here we report the Wingfield 2007) and saprotrophs, including wood analysis of 310 basidiomycetous endophytes isolated decayers (Parfitt et al. 2010, Chaverri and Gazis 2011). from wild and planted populations of the rubber tree Rodriguez et al. (2009) grouped fungal endophytes genus, Hevea. Species accumulation curves were into four classes based on transmission behavior, nonasymptotic, as in the majority of endophyte colonization pattern, host range and tissue prefer- surveys, indicating that more sampling is needed to ence. The species reported in this study are class III recover the true diversity of the community. One endophytes sensu Rodriguez et al. (2009), which are hundred eighteen OTUs were delimited, represent- horizontally transmitted across a broad host range, ing nine orders of Basidiomycota (Agaricales, Athe- have a limited in planta colonization pattern and liales, Auriculariales, Cantharellales, Hymenochae- exhibit high biodiversity within the host. Communi- tales, Polyporales, Russulales, Septobasidiales, ties within this class appear to be dominated by Tremellales). The diversity of basidiomycetous endo- members of the Ascomycota, especially genera known phytes found inhabiting wild populations of Hevea to contain pathogenic species such as Colletotrichum was comparable to that present in plantations. and Pestalotiopsis or saprotrophic species like Penicil- However, when samples were segregated by tissue lium and Xylaria (Davis et al. 2003, Vega et al. 2006). type, sapwood of wild populations was found to The mycoparasitic genus Trichoderma also has been contain a higher number of species than sapwood reported as an important component of the endo- of planted trees. Seventy-five percent of isolates were phytic community in wild populations of Hevea and members of the Polyporales, the majority in the Theobroma (Samuels et al. 2006, Gazis and Chaverri phlebioid clade. Most of the species belong to clades 2010). known to cause a white-rot type of wood decay. Two Basidiomycota have been reported as endophytes, species in the insect-associated genus Septobasidium but they usually make up a small fraction of the total were isolated. The most frequently isolated genera number of endophytes isolated (Rubini et al. 2005, included Bjerkandera, Ceriporia, Phanerochaete, Phle- Arnold et al. 2007, de Errasti et al. 2010, Sakayaroj et al. 2010, Min et al. 2013). Studies that have focused Submitted 17 Aug 2014; accepted for publication 16 Dec 2014. entirely on basidiomycetous endophytes are limited 1 Corresponding author. E-mail: [email protected] (Duckett and Ligrone 2007, Rungjindamai et al. 2008, 284 MARTIN ET AL.: BASIDIOMYCETOUS ENDOPHYTES OF HEVEA 285 Pinruan et al. 2010, Tello et al. 2013). It is likely that Endophytes may switch to a pathogenic feeding much of the basidiomycetous endophyte diversity strategy when suitable conditions occur, such as remains undetected due to sampling biases (Arnold decreased water content or increased nutrient or et al. 2007, Hyde and Soytong 2008). There are oxygen availability or when host defenses are low multiple challenges that limit the detection of (Hendry et al. 2002, Sieber 2007, Alvarez-Loayza et al. basidiomycetous endophytes with traditional culture- 2011). Endophytes also may have the potential to based methods, such as their slow growth in compar- become saprotrophic upon host senescence or ison to most ascomycetes (Crozier et al. 2006, Arnold abscission of leaves or branch fall (Parfitt et al. et al. 2007) and their lack of informative morpholog- 2010). Some species isolated as endophytes have been ical characters (Evans et al. 2003, Wang et al. 2005), shown to produce wood decay enzymes, including which may explain why culture-based studies have xylanases, b-mannanases, cellulases, polygalacturonas- reported a low number of basidiomycetous endo- es and laccases (Urairuj et al. 2003, Oses et al. 2006, phytes or grouped them under unidentified or sterile Promputtha et al. 2010). Such observations lend mycelia. support to the ‘‘latent pathogen or saprotroph The majority of endophyte research has focused on hypothesis’’, which suggests that many endophytic species living within leaf tissue (Arnold and Lutzoni species may invade living trees as a strategy for early 2007, Unterseher et al. 2013). Fewer studies have substrate colonization (Promputtha et al. 2007, Parfitt examined endophytes inhabiting stem or trunk et al. 2010, Porras-Alfaro and Bayman 2011). Many tissues of plants (Evans et al. 2003, Rubini et al. genera have been isolated from both living and dead 2005, Oses et al. 2006, Gazis and Chaverri 2010, Parfitt substrates (Unterseher et al. 2013), but only in a few et al. 2010, Gazis et al. 2012, Jin et al. 2013). Even cases has the connection at the species level been though the number of studies focusing on sapwood confirmed through molecular data. Examples include endophytes has been lower than those targeting leaf Guignardia mangiferae (Promputtha et al. 2007) and endophytes, the diversity found in this habitat may be Perisporiopsis lateritia (Chaverri and Gazis 2011). of comparable magnitude (Gazis 2012). Fungal The aims of this study were to identify 310 endophytes may colonize leaves in the same way as endophytic basidiomycetous strains isolated from pathogens, entering through direct penetration or leaves and sapwood of Hevea spp. populations through stomata or wounds (Bayman et al. 1998, distributed in wild and managed habitats, compare Arnold and Herre 2003, Santamarı´a and Bayman the diversity and composition of these communities 2005, Saunders et al. 2010). Less is known about the and explore other endophytic basidiomycetous com- mode of entry of sapwood endophytes, which are munities inhabiting a diverse array of hosts and thought to colonize the stem of plants mainly habitats. We generated sequences of the internal through the root system or by migrating from the transcribed spacer (ITS) region of nuclear ribosomal crown through the vascular system (Boddy 1994, Oses RNA (rRNA) genes from Hevea endophytes and et al. 2006, De Errasti et al. 2010). constructed phylogenies that included (i) reference Most studies of sapwood endophytes have been sequences from published multigene phylogenies, conducted in temperate regions (Petrini and Fisher (ii) well identified sequences of taxa that are closely 1988, 1990; Fisher and Petrini 1990; Oses et al. 2006; related to Hevea endophytes and (iii) sequences of Giordano et al. 2009). Tropical hosts that have been diverse basidiomycete endophytes, most of which sampled for sapwood endophytes are limited to Hevea were identified only partially. We segregated Hevea (Gazis and Chaverri 2010, Gazis 2012), Theobroma endophyte samples according to management regime (Evans et al. 2003, Rubini et al. 2005, Crozier et al. (wild vs. managed) and tissue of origin (leaf vs. 2006, Thomas et al. 2008), Coffea (Vega et al. 2008) sapwood), and performed comparisons of community and Garcinia (Sim et al. 2010). Many of the diversity and composition. Finally we assessed the host basidiomycetous endophytes detected in sapwood and geographic ranges of Hevea endophytes with belong to taxonomic groups that are considered to reference to basidiomycete endophytes sampled in be wood decayers (Oses et al. 2006, Parfitt et al. 2010). other studies by mining public sequence repositories Nevertheless these taxonomic groups also have been and their associated metadata. isolated from leaves, including members of the Xylariales, such as Daldinia, Hypoxylon, and Xylaria (Rubini et al. 2005,