Mycologia ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/umyc20 Three new species of Gliocephalotrichum causing fruit rot on different hosts from Brazil Rildo Alexandre Fernandes da Silva , Camila Pereira de Almeida , Ailton Reis , Frederick Mendes Aguiar , Priscila Chaverri & Danilo Batista Pinho To cite this article: Rildo Alexandre Fernandes da Silva , Camila Pereira de Almeida , Ailton Reis , Frederick Mendes Aguiar , Priscila Chaverri & Danilo Batista Pinho (2020): Three new species of Gliocephalotrichum causing fruit rot on different hosts from Brazil, Mycologia, DOI: 10.1080/00275514.2020.1801017 To link to this article: https://doi.org/10.1080/00275514.2020.1801017 View supplementary material Published online: 18 Sep 2020. Submit your article to this journal View related articles View Crossmark data Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=umyc20 MYCOLOGIA https://doi.org/10.1080/00275514.2020.1801017 Three new species of Gliocephalotrichum causing fruit rot on different hosts from Brazil Rildo Alexandre Fernandes da Silva a, Camila Pereira de Almeida a, Ailton Reis Frederick Mendes Aguiar a, Priscila Chaverri c, and Danilo Batista Pinho a b , a Departamento de Fitopatologia, Universidade de Brasília, Brasília, Distrito Federal, 70910-900, Brazil; bEmpresa Brasileira de Pesquisas Agropecuárias (EMBRAPA)—Hortaliças, 70275-970, Brasília, Distrito Federal, Brazil; cEscuela de Biología and Centro de Investigaciones en Productos Naturales (CIPRONA), Universidad de Costa Rica, San Pedro, San José, Costa Rica ABSTRACT ARTICLE HISTORY The genus Gliocephalotrichum (Nectriaceae), originally described as a soil-borne fungus, has been associated with postharvest diseases, especially of tropical fruits. Taxonomic studies using both morphological and molecular phylogenetic analyses have contributed to recognition of novel species in several countries. However, in Brazil, only three isolates of Gliocephalotrichum have been collected from soil samples and roots since the late 1970s. Our study expands the sample range using many Gliocephalotrichum isolates obtained from rotting fruits of tropical plant species in different states of Brazil. Polyphasic taxonomy was assessed with phylogenetic analyses of DNA sequences from four nuclear loci, morphological comparisons, and pathogenicity tests. As a result, three known species (G. bulbilium, G. longibrachium, and G. simplex) were identified from new hosts and locations in Brazil. In addition, three new species are described—G. abrachium, G. brasiliense, and G. caryocaris. A key to all Gliocephalotrichum species worldwide is provided. Although species of Gliocephalotrichum have not been considered to be important plant pathogens, this study shows they may cause postharvest fruit rot in tropical fruits and therefore have an impact in communities that depend economically on the harvest and sale of these fruits. Received 3 October 2019 Accepted 20 July 2020 INTRODUCTION The genus Gliocephalotrichum J.J. Ellis & Hesseltine (= Leuconectria Rossman, Samuels & Lowen) was isolated for the first time from soil samples in Louisiana and proposed as a new genus by Ellis and Hesseltine (1962), with G. bulbilium as the type species. The genus was initially recognized based primarily on the presence of a penicillate conidiogenous apparatus terminating in phialides producing aseptate and ellipsoidal conidia and, at the bottom of the first branch of the conidiogenous apparatus, the development of several sterile hairs or stipe extensions. Later, Wiley and Simmons (1971) distinguished Gliocephalotrichum from Cylindrocladium Morgan (now Calonectria De Not.), transferred two Cylindrocladium taxa (Cy. simplex and Cy. simplex var. microchlamydosporum) into Gliocephalotrichum, and described one new species from soil in Thailand. The authors discussed the differences between Gliocephalotrichum and Calonectria: the latter has a stipe elongation that is dichotomously branched and with one stipe extension mostly ending in a sterile vesicle and the former with the stipe Published online 18 Sep 2020 Brazilian cerrado; Hypocreales; plant pathology; postharvest diseases; taxonomy; 3 new taxa extension developing below the first branch of the conidiogenous apparatus. The first study to integrate molecular and classical taxonomy of Gliocephalotrichum was by Decock et al. (2006). Later, Lombard et al. (2014) described five additional new species based on phylogenetic analyses of four gene regions. Other studies added species to the genus, resulting in a total of 13 accepted names in Gliocephalotrichum (Huang and Schmitt 1973; Zhuang and Luo 2008). Even though Gliocephalotrichum is referred to as a group of soil saprotrophs, several studies report that many species cause fruit rot of plants such as Nephelium lappaceum in Hawaii, Malaysia, Philippines, Puerto Rico, and Thailand (Pordesimo and Luna-Ilag 1982; Sivakumar et al. 2002; Nishijima et al. 2007; SerratoDiaz et al. 2012; Sakinah and Latiffah 2013), Psidium guajava in Hawaii (Constantelos et al. 2011), Durio graveolensi in Brunei Darassalam (Sivapalan et al. 1998), Garcinia mangostana in Thailand (Sangchote and Pongpisutta 1998), and Vaccinium macrocarpon in the United States (Constantelos et al. 2011). Until now, CONTACT Danilo Batista Pinho danilopinho@unb.br Supplemental data for this article can be accessed on the publisher’s Web site. © 2020 The Mycological Society of America KEYWORDS 2 FERNANDES DA SILVA ET AL.: NEW SPECIES OF GLIOCEPHALOTRICHUM only three Gliocephalotrichum isolates had been collected in Brazil on soil or from plant roots (Lombard et al. 2014). The diversity of Gliocephalotrichum and its pathology are poorly known in Brazil. In this study, several Gliocephalotrichum isolates were collected from various plant species in Brazil presenting fruit rot symptoms. Our aims are to (i) characterize these isolates using a polyphasic approach (multilocus phylogenetic analyses, cultural characteristics, and morphology) and (ii) determine their pathogenicity. This study will advance the knowledge of poorly known fungal groups, especially in threatened ecosystems such as the Brazilian Cerrado. MATERIALS AND METHODS Isolates.—Isolates were obtained from rotting fruits of different plant hosts originating in the states of Minas Gerais, Distrito Federal, Piauí, Tocantins, and Goiás, Brazil. We used direct and indirect isolation methods described by Alfenas et al. (2016). All isolates are listed in TABLE 1, including location and host. Isolates were preserved in mineral oil at the Culture Collection at the University of Brasilia (CCUB). Dry cultures were deposited at the Herbarium of the University of Brasilia (UB). DNA extraction, PCR, and sequencing.—Total genomic DNA was extracted from cultures grown on malt extract agar (MEA) after 7 d using the Wizard Genomic DNA Purification Kit (Promega, Madison, Wisconsin), with modifications described by Pinho et al. (2012). Portions of four loci were amplified and sequenced: (i) translation elongation factor 1-α (TEF1) as the primary barcode using primers EF-1F and EF-2R (Jacobs et al. 2004); (ii) β-tubulin (BTUB) with primers T1 (O’Donnell et al. 2007) and Bt2b (Glass and Donaldson 1995); (iii) histone H3 (HIS3) using primers CYLH3F and CYLH3R (Crous et al. 2004); and (iv) nuclear 18S-5.8S-28S partial rDNA including internal transcribed spacers ITS1 and ITS2 with primers LR5 (Vilgalys and Hester 1990) and V9G (Hoog and Ende 1998). Amplification was performed with an initial denaturation temperature of 95 C for 5 min, followed by 35 cycles of denaturation at 95 C for 30 s, annealing at 56 C (for TEF1 and BTUB), 52 C (ITS), or 53 C (HIS3) for 45 s, and extension at 72 C for 45 s, and final extension at 72 C for 5 min. Polymerase chain reaction (PCR) products were sent to Macrogen (Seoul, Korea) for purification and sequencing. Phylogenetic analyses.—First, a phylogenetic analysis of TEF1 was carried out, and then selected isolates were used in the multigene analyses. A separate phylogenetic analysis was made for each partial gene obtained in this study to test for possible topological conflicts. Newly generated sequences were assembled and annotated using the forward and reverse strands with MEGA7 (Kumar et al. 2016). The BLAST algorithm was used to first identify isolates using TEF1 sequences. Then, representative sequences of all species of Gliocephalotrichum were downloaded together with Calonectria candelabra (CPC 1675), which was used as an outgroup. All sequences were deposited in GenBank (TABLE 1). Alignments were carried out in MAFFT 7 (Katoh and Standley 2013) using the algorithm MUSCLE and the default parameters. Phylogenetic analyses were performed using Bayesian inference (BI) and maximum likelihood (ML) criteria. For BI analyses, the best substitution models for each partial gene were determined with MrModeltest (Nylander 2004). The Web portal CIPRES (Miller et al. 2010) was used to run MrBayes 3.2.2 (Ronquist et al. 2012). The Markov chain Monte Carlo (MCMC) analysis was run with a total of 10 million generations and one tree sampled every 1000 generations. The convergence of the log likelihoods was confirmed using TRACER 1.7.1 (Rambaut and Drummond 2018). The first 25% of saved trees were discarded as the burn-in, with posterior probabilities (PPs) calculated for the remaining trees. The ML analysis was performed using RAxML 8.2.9 (Stamatakis 2014) through the CIPRES portal, starting with a randomized stepwise-addition parsimony tree under a GTR+GAMMA model for each gene region and the four-gene combined data set and 1000 bootstrap (BS) replicates under the same model. All trees were edited in FigTree 1.4 (Rambaut 2018). The nucleotide matrices and phylogenetic trees from all five data sets (individual and four-gene combined) are available at TreeBASE (25086). Morphological characterization.—Isolates were characterized morphologically based on examination of monoconidial cultures after 7 d of growth on MEA and synthetic nutrient agar (SNA; Nirenberg 2011) with sterile carnation leaves maintained at room Temperature (ca. 26 C). Microscopical characteristics were examined after 7 d by mounting fungal structures in clear lactoglycerol and polyvinylactoglycerol (PVLG). At least 30 measurements for each morphological trait were made using a Leica DM2500 microscope (Leica Microsystem, Nassloch, Germany). Color determinations followed Rayner (1970). Colony characteristics were from 7-d-old MEA cultures grown at 26 C. Measurements were summarized as 95% confidence intervals and average, minimum, and maximum values. Table 1. Gliocephalotrichum species and isolates included in this study. GenBank accession numbersb Species G. abrachium G. bacillisporum G. bulbilium G. brasiliense G. caryocaris G. cylindrosporum G. grande G. humicola Isolates CCUB10 CCUB1018 CCUB1022 CCUB1041 CCUB301 CCUB9 CCUB1042 CBS 250.91 CBS 126572 CBS 132042 CBS 118.68 CBS 562.75 CBS 451.92 CBS 104.95 CBS 113467 CPC 13577 CPC 21866 CPC 21867 CPC 23321 CPC 23322 CPC 23323 CPC 23324 CPC 23325 CPC 23334 CPC 23335 CPC 23336 CPC 23337 CPC 23339 CCUB13 CCUB12 CCUB1253 CCUB232 CCUB355 CCUB381 CCUB229 CCUB231 CBS 902.70 CBS 903.70 CBS 904.70 HMAS CBS 135945 CBS 135946 CPC 23340 CPC 23344 CPC 23345 CPC 23347 CBS 126571 CBS 132043 CCUB17 BTUB MN508721 MN508733 MN508734 MN508735 MN508728 MN508720 MN508736 KF513182 DQ374413 DQ374414 KF513183 KF513184 KF513185 KF513186 KF513187 KF513188 DQ377830 DQ377829 KF513189 KF513190 KF513191 KF513192 KF513193 KF513194 KF513195 KF513196 KF513197 KF513198 MN508723 MN508722 — MN508730 MN508731 MN508732 MN508727 MN508729 DQ377841 KF513208 DQ377842 EU984072 KF513209 KF513210 KF513211 KF513212 KF513213 KF513214 DQ377835 DQ377836 MN508726 HIS3 MN508738 MN508750 MN508751 MN508752 MN508745 MN508737 MN508753 KF513323 KF513324 KF513325 KF513327 KF513328 KF513329 KF513330 KF513331 KF513332 KF513333 KF513334 KF513335 KF513336 KF513337 KF513338 KF513339 KF513340 KF513341 KF513342 KF513343 KF513344 MN508740 MN508739 MN508754 MN508747 MN508748 MN508749 MN508744 MN508746 KF513353 KF513354 KF513355 — KF513356 KF513357 KF513358 KF513359 KF513360 KF513361 KF513367 KF513368 MN508743 ITS MN450200 MN450212 MN450213 MN450214 MN450207 MN450199 MN450215 KF513251 DQ374408 DQ374409 KF513252 KF513253 KF513254 KF513255 KF513256 KF513257 DQ374406 DQ374407 KF513258 KF513259 KF513260 KF513261 KF513262 KF513263 KF513264 KF513265 KF513266 KF513267 MN450202 MN450201 MN450216 MN450209 MN450210 MN450211 MN450206 MN450208 DQ366705 KF513277 DQ366706 EF121859 KF513278 KF513279 KF513280 KF513281 KF513282 KF513283 DQ278422 DQ278422 MN450205 TEF1 MN508678 MN508706 — MN508713 MN508691 MN508677 MN508714 KF513405 KF513406 KF513407 KF513409 KF513410 KF513411 KF513412 KF513413 KF513414 KF513415 KF513416 KF513417 KF513418 KF513419 KF513420 KF513421 KF513422 KF513423 KF513424 KF513425 KF513426 MN508681 MN508680 MN508719 MN508698 MN508701 MN508705 MN508689 MN508692 KF513435 KF513436 KF513437 HM054075 KF513438 KF513439 KF513440 KF513441 KF513442 KF513443 KF513449 KF513450 MN508685 Substrate/host Caryocar brasiliense Garcinia mangostana Spondias purpurea S. purpurea C. brasiliense C. brasiliense C. brasiliense Plant root Leaf litter Leaf litter Air Flacourtia sp. Clusia sp. Soil Soil Nyssa sylvatica Leaf litter Leaf litter Nephelium lappaceum N. lappaceum N. lappaceum N. lappaceum N. lappaceum N. lappaceum N. lappaceum N. lappaceum N. lappaceum N. lappaceum Syzygium cumini Cyrtostachys renda S. purpurea D. madagascariensis S. purpurea Spondias mombin C. brasiliense C. brasiliense Soil Soil Soil Leaf litter Soil Soil Soil Soil Soil Soil Leaf litter Leaf litter C. brasiliense Place Piauí, Brazil Distrito Federal, Brazil Distrito Federal, Brazil Goiás, Brazil Minas Gerais, Brazil Distrito Federal, Brazil Tocantins, Brazil Brazil French Guiana French Guiana Central African Republic Indonesia Puerto Rico Brazil Thailand USA French Guiana French Guiana Puerto Rico Puerto Rico Puerto Rico Puerto Rico Puerto Rico Mexico Mexico Mexico Mexico Mexico Distrito Federal, Brazil Distrito Federal, Brazil Distrito Federal, Brazil Distrito Federal, Brazil Distrito Federal, Brazil Distrito Federal, Brazil Minas Gerais, Brazil Minas Gerais, Brazil Thailand Thailand Thailand China Taiwan Taiwan Taiwan Taiwan Taiwan Taiwan French Guiana French Guiana Distrito Federal, Brazil Collector A. Reis A. Reis A. Reis A. Reis A. Reis A. Reis A. Reis L. Pfenning C. Decock & V. Robert C. Decock & V. Robert J. Nicot I. Gandjar W.R. Buck L. Pfenning M. Reblova T. Sutton C. Decock & V. Robert C. Decock & V. Robert L.M. Serrato-Diaz L.M. Serrato-Diaz L.M. Serrato-Diaz L.M. Serrato-Diaz L.M. Serrato-Diaz L.M. Serrato-Diaz L.M. Serrato-Diaz L.M. Serrato-Diaz L.M. Serrato-Diaz L.M. Serrato-Diaz A. Reis A. Reis A. Reis A. Reis A. Reis A. Reis A. Reis A. Reis C. Klinsukont S. Chomchalow S. Chomchalow W.Y. Zhuang & Y. Nong P.W. Crous P.W. Crous P.W. Crous P.W. Crous P.W. Crous P.W. Crous C. Decock & V. Robert C. Decock & V. Robert A. Reis Reference This study This study This study This study This study This study This study Lombard et al. 2014 Lombard et al. 2014 Lombard et al. 2014 Lombard et al. 2014 Lombard et al. 2014 Lombard et al. 2014 Lombard et al. 2014 Lombard et al. 2014 Lombard et al. 2014 Lombard et al. 2014 Lombard et al. 2014 Lombard et al. 2014 Lombard et al. 2014 Lombard et al. 2014 Lombard et al. 2014 Lombard et al. 2014 Lombard et al. 2014 Lombard et al. 2014 Lombard et al. 2014 Lombard et al. 2014 Lombard et al. 2014 This study This study This study This study This study This study This study This study Lombard et al. 2014 Lombard et al. 2014 Lombard et al. 2014 Lombard et al. 2014 Lombard et al. 2014 Lombard et al. 2014 Lombard et al. 2014 Lombard et al. 2014 Lombard et al. 2014 Lombard et al. 2014 Lombard et al. 2014 Lombard et al. 2014 This study 3 (Continued) MYCOLOGIA G. longibrachium a 4 GenBank accession numbersb Species G. mexicanum G. microchlamydosporum G. nephelii G. ohiense G. queenslandicum G. simmonsii G. simplex Calonectria candelabrum Gliocladiopsis tenuis a Isolatesa CBS 135947 CBS 135948 CBS 345.64 CPC 21862 CPC 21863 CBS 135949 CBS 135950 CBS 567.73 CBS 112956 CBS 114868 CBS 135951 CBS 135952 CBS 135953 CBS 267.65 CBS 983.69 CBS 511.81 CCUB15 CCUB16 CPC 1675 CBS 114147 BTUB KF513220 KF513221 DQ374410 DQ374411 DQ374412 KF513222 KF513223 DQ374415 KF513224 KF513225 KF513226 KF513227 KF513228 DQ377838 KF513229 KF513230 MN508724 MN508725 FJ972426 JQ666148 HIS3 KF513369 KF513370 KF513371 — — KF513372 KF513373 — KF513374 KF513375 KF513376 KF513377 KF513378 KF513379 KF513380 KF513381 MN508741 MN508742 FJ972476 JQ666038 ITS KF513289 KF513290 DQ366699 DQ366700 DQ366701 KF513291 KF513292 DQ366707 KF513293 KF513294 KF513295 KF513296 KF513297 DQ366702 KF513298 KF513299 MN450203 MN450204 GQ280557 JQ666069 TEF1 KF513451 KF513452 KF513453 KF513454 KF513455 KF513456 KF513457 KF513458 KF513459 KF513460 KF513461 KF513462 KF513463 KF513464 KF513465 KF513466 MN508683 MN508684 FJ972525 JQ666114 Substrate/host N. lappaceum N. lappaceum Soil — — N. lappaceum N. lappaceum Soil Eleaeocarpus angustifolius E. angustifolius N. lappaceum N. lappaceum N. lappaceum Soil Soil Musa sp. D. madagascariensis D. madagascariensis Eucalyptus sp. Soil Place Mexico Mexico Zaïre Zaïre South Africa Guatemala Guatemala USA Australia Australia Guatemala Guatemala Guatemala South Africa Brazil New Zealand Distrito Federal, Brazil Distrito Federal, Brazil Amazonas, Brazil Vietnam Collector L.M. Serrato-Diaz L.M. Serrato-Diaz J.A. Meyer — — L.M. Serrato-Diaz L.M. Serrato-Diaz L.H. Huang I. Steer & B. Paulus I. Steer & B. Paulus L.M. Serrato-Diaz L.M. Serrato-Diaz L.M. Serrato-Diaz H.J. Swart C. Ram H.J. Boesewinkel A. Reis A. Reis A.C. Alfenas P.W. Crous Reference Lombard et al. 2014 Lombard et al. 2014 Lombard et al. 2014 Lombard et al. 2014 Lombard et al. 2014 Lombard et al. 2014 Lombard et al. 2014 Lombard et al. 2014 Lombard et al. 2014 Lombard et al. 2014 Lombard et al. 2014 Lombard et al. 2014 Lombard et al. 2014 Lombard et al. 2014 Lombard et al. 2014 Lombard et al. 2014 This study This study Alfenas et al. 2015 Lombard and Crous 2012 CBS = CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands; CPC = Pedro W. Crous working collection house date; CCUB = Coleção de Culturas da Universidade de Brasília, Universidade de Brasília, Brasília, Brazil. Ex-type strains indicated in bold. FERNANDES DA SILVA ET AL.: NEW SPECIES OF GLIOCEPHALOTRICHUM Table 1. (Continued). MYCOLOGIA Pathogenicity tests.—Pathogenicity assays were performed on mature fruits of Caryocar brasiliense, Spondias purpurea, Syzygium cumini, and Syzygium jambos. Six Glicephalotrichum isolates, one of each species from different locations and hosts, were evaluated (CCUB12, CCUB13, CCUB231, CCUB232, CCUB301, and CCUB353). To obtain inoculum, each isolate was grown in Petri dishes containing 2% MEA and 20–30 sterile toothpicks for 7–10 d at room temperature. Then, the colonized toothpicks were drilled into the fruit’s surface until reaching the mesocarp. A control was toothpick-drilled without the fungus. Inoculated fruits were maintained in moisture chambers with 90% relative humidity at room temperature. At 7 d post inoculation, the presence or absence of symptoms was evaluated. If symptoms were present, reisolation was carried out to fulfill Koch’s postulates. RESULTS Isolates.—Fifty-one Gliocephalotrichum isolates were obtained from various fruits collected in five states of Brazil, i.e., Distrito Federal, Minas Gerais, Goiás, Tocantins, and Piauí. Isolates were found causing fruit rot in Caryocar brasiliense (Caricaceae), Cyrtostachys renda, Dypsis madagascariensis, Syagrus romanzoffiana (Arecaceae), Garcinia mangostana (Clusiaceae), Spondias mombin, S. purpurea (Anacardiaceae), Syzygium cumini, and Sy. jambos (Myrtaceae). The symptoms and signs observed in the field included intense white to light purple or brown sporulation on the fruit, otherwise appearing cottony when the mass of spores was absent. In the same fruits, darkening and wilting was observed, mainly in fruits of Spondias purpurea and S. mombin. Phylogenetic analyses.—TEF1 amplicons were obtained for 51 isolates, generating sequences of approximately 700 bp. The TEF1 matrix included 101 taxa (51 isolates from this study and 50 taxa from GenBank), composed of 647 sites (347 conserved) and 268 parsimony-informative characters. In the TEF1 tree, our isolates segregated into five distinct clades. Based on this analysis, 18 representative isolates of different clades, hosts, and locations were chosen for the multilocus analysis. No topological conflicts were found between the four partial genes based on a reciprocal 70% bootstrapping threshold. Therefore, the data sets were concatenated. For the multilocus analysis, 69 taxa were used in the ingroup and two isolates of Calonectria candelabra (CPC 1675 and CPC 1679) as outgroups. The 5 concatenated matrix corresponded to four partial gene regions: 358 sites for BTUB, 528 for HIS3, 569 for ITS, and 638 for TEF1, for a total of 2093 sites after alignment. Of the concatenated matrix, 1426 characters were conserved, 652 variable, and 607 (29%) phylogenetically informative (SUPPLEMENTARY TABLE 1). For BI analysis, a GTR+G+I model was selected for TEF1 and HIS3, HKY+I for BTUB, and SYM+I for ITS. The BI and ML tree topologies were not in conflict. The multilocus phylogenetic tree confirmed that Gliocephalotrichum was well supported phylogenetically in a clade distinct from Calonectria. Our Gliocephalotrichum isolates grouped into six species-level clades (FIG. 1). Three of these represented known species, but the others represented undescribed taxa. One of the three known species is G. simplex, which is represented by three isolates collected on fruits of Cyrtostachys renda, Syzygium cumini, and S. purpurea in Distrito Federal. A second known species is G. bulbilium, represented by isolates collected on Cyrostachys renda, Spondias purpurea, Syzygium cumini, and Sy. jambos. One isolate collected on Caryocar brasiliense clustered with G. longibrachium (with poor support) and is referred hereafter as G. cf. longibrachium. Gliocephalotrichum abrachium, sp. nov., is represented by several isolates collected on Caryocar brasiliense fruits in different states of Brazil and on Garcinia mangostana and S. purpurea in Distrito Federal and Goiás. Isolates of G. caryocaris, sp. nov., were collected also on C. brasiliense in Distrito Federal and Minas Gerais. Gliocephalotrichum brasiliense, sp. nov., was most diverse in terms of host and geographic origin. Pathogenicity tests.—Four days post inoculation, all six Gliocephalotrichum isolates inoculated started to produce symptoms in fruits of C. brasiliense, Sy. cumini, Sy. jambos, and S. purpurea. At 7–10 d post inoculation, the fungi had colonized the surface of the fruits in which the species was previously found and also the other fruits in cross-inoculation (FIG. 2). The symptoms of Gliocephalotrichum on Sy. jambos fruits were circular to oblong lesions, with brown coloring in its center of the lesion, and the edges with a soaked appearance of color pale brown; the inner part of the lesion presented conidiophores and conidia. In a cut section of the fruit, it was observed that the exocarp, mesocarp, and endocarp appeared soaked and with shades of brown to light brown from the inside to the edges. It was common to detect the presence of mycelium inside the fruit that reached the seed (FIG. 2C–K). In S. purpurea, all isolates of Gliocephalotrichum grew very fast, overlapping the symptoms on the surface of the fruit. In infected areas, 6 FERNANDES DA SILVA ET AL.: NEW SPECIES OF GLIOCEPHALOTRICHUM Figure 1. Bayesian inference consensus tree of the concatenated four-gene sequences. Accessions in bold represent isolates from this study. Posterior probability (PP)/maximum likelihood (ML) bootstrap values are indicated at nodes. Thicker nodes represent PP/ML values above 0.99/99. The scale bar represents the number of expected changes per site. it was possible to observe the intense darkening of tissues followed by abundant sporulation that gave a mucoid aspect to the surface of the infected fruit (FIG. 2A–I). In C. brasiliense and Sy. cumini fruits, due to their natural coloration (i.e., dark green and purplish, respectively), the initial symptoms were almost imperceptible MYCOLOGIA 7 Figure 2. Pathogenicity assays on fruits of Spondias purpurea (A, E, I), Caryocar brasiliense (B, F, J), Syzygium jambos (C, G, K), and Syzygium cumini (D, H, L) with the species Gliocephalotrichum abrachium CCUB301 (A–D), G. brasiliense CCUB232 (E–H), and G. caryocaris CCUB231 (I–L) 10 d post inoculations. All species of Gliocephalotrichum managed to infect the fruits; in all fruits (except Sy. jambos), the mycelial growth and sporulation were fast and intense, respectively, overlapping the symptoms. In Sy. jambos, the growth was slower, showing symptom of waterlogging and darkness of the skin. and appeared as only a slight darkening of the affected area and abundant mycelium (FIG. 2B–J, D–L). In all inoculated fruits, it was observed that the production of stipe extensions depended on the isolate or the species of Gliocephalotrichum. Koch’s postulates were confirmed by pathogenicity assays, which presented the same symptoms in the laboratory as in the field, and by the reisolation of the fungus from the inoculated and affected areas. TAXONOMY Following the genealogical concordance phylogenetic species recognition criterion (GCPSR) (Laurence et al. 2014), all novel species proposed constituted monophyletic groups (FIG. 2). Morphological characteristics of those species clearly distinguished them from other known species (see dichotomous key). Therefore, these new species were also identified using morphology. 8 FERNANDES DA SILVA ET AL.: NEW SPECIES OF GLIOCEPHALOTRICHUM Gliocephalotrichum abrachium R.A. Fernandes, C.P. FIG. 3 Almeida & Pinho, sp. nov. MycoBank MB832548 Typification: BRAZIL. MINAS GERAIS: Brasília de Minas, on rotting fruit of Caryocar brasiliense, 2 Dec 2016, F.M. Aguiar (holotype UB23871). Ex-type living culture CCUB301. GenBank: ITS = MN450207; HIS3 = MN508745; TEF1 = MN508691; TUB = MN508728. Etymology: abrachium (Latin), referring to the absence of a stipe extension. Description: Sexual stage not observed. Colonies fast-growing (70 mm in 4 d on MEA 2%), pale luteous to ochre, with a diffusible pale luteous to black ochre pigment in the agar, aerial mycelium absent, abundant conidiophores present on the entire surface of the colony, sporulation intense; chlamydospores visible to naked eye as dark points. Conidiophores abundant, solitary, erect, hyaline, arising from submerged hyphae, stipe extension absent; stipe septate, hyaline, pale luteous to light luteous, smooth, 282–420 × 11–18 µm (avg. 318 × 15.5 µm), followed by a penicillate conidiogenous apparatus. Conidiogenous apparatus densely penicillate, consisting of a whorl of fertile branches, 30–100 µm long (avg. 62 µm), 32–104 µm wide (avg. 63 µm); first branch aseptate, 39–64 × 5.5–9.5 µm (avg. 40 × 9.5 µm); secondary branch aseptate, 12–28 × 4.5–7.5 µm (avg. 19 × 6 µm); additional branches (–3), aseptate, 8–24.5 × 2.5–6 µm (avg. 17.5 × 4.5 µm), each terminating in a whorl of 4–6 phialides. Phialides aseptate, cylindrical, slightly swollen, hyaline, 10.5–22 × 3–5.5 µm (avg. 16 × 4.5 µm), apex with minute periclinal thickening and inconspicuous collarette. Conidia hyaline, smooth, ellipsoid to oblong, (5–)6–10(–12) × (2–)3–4.5 µm (avg. 8 × 3 µm), forming a mucoid drop at the apex of conidiophore, turning luteous to orange with age. Chlamydospores forming bulbiloid aggregates immersed and superficial in the medium, umber to dark umber, 198–400 × 222–382 µm (avg. 300 × 302 µm), formed by globose to ellipsoid cells, 19–28 × Figure 3. Gliocephalotrichum abrachium (ex-type CCUB301). A. Conidiophores on SNA. B–C. Conidiophores. D–E. Conidiogenous apparatus with conidiophore branches and phialides. F. Ellipsoid to oblong conidia. G. Chlamydospore aggregates with globose to ellipsoid cells. Bars: A = 0.1 mm; B, C = 20 μm; D–G = 10 μm. MYCOLOGIA 14–22 µm (avg. 24 × 19 µm); chlamydospores absent. Ecology and known distribution: On rotting fruits of Caryocar brasiliense, Garcinia mangostana, and Syagrus oleracea, Brazil (Distrito Federal, Goiás, Minas Gerais, Piauí, and Tocantins). Additional specimens examined: BRAZIL. DISTRITO FEDERAL: Brasília and Riacho Fundo, on fruits of Caryocar brasiliense, 15 Dec 2016, A. Reis (cultures CCUB2, CCUB5, CCUB6, CCUB7, CCUB23). GOIÁS: Cabeceiras and Padre Bernardo, on fruits of Caryocar brasiliense, 24 Nov 2016, A. Reis (cultures CCUB1, CCUB8, CCUB11, CCUB21, CCUB1041). MINAS GERAIS: Jequitibá, on fruits of Caryocar brasiliense, 15 Dec 2016, F.M. Aguiar (culture CCUB18); Montes Claros, on fruits of Caryocar brasiliense, 2 Sep 2016, F. M. Aguiar (cultures CCUB19, CCUB302); Brasília de Minas, on fruits Caryocar brasiliense, 16 Dec 2016, F. M. Aguiar (cultures CCUB382, CCUB383); Unaí, on fruits of Caryocar brasiliense, 23 Nov 2016, A. Reis (cultures CCUB3, CCUB4). TOCANTINS: Palmas, on fruits of Caryocar brasiliense, 12 Apr 2017, A. Reis (cultures CCUB1020, CCUB1024). PIAUÍ: Monte Alegre on fruits of Caryocar brasiliense, 16 Dec 2016, A. Reis (culture CCUB19). Commentary: Gliocephalotrichum abrachium is a sister species to G. longibrachium but can be distinguished by the absence of a stipe extension, the number of fertile branches, smaller variation in the number of phialides, and conidial shape and size. Gliocephalotrichum brasiliense R.A. Fernandes, C.P. Almeida & Pinho, sp. nov. FIG. 4 MycoBank MB832549 Typification: BRAZIL. DISTRITO FEDERAL: Águas Claras, sample of Dypsis madagascariensis fruit, 17 Dec 2016, A. Reis (holotype UB23870). Ex-type culture CCUB232. GenBank: ITS = MN450209; HIS3 = MN508747; TEF1 = MN508698; TUB = MN508730. Etymology: brasiliense (Latin), in reference to the geographic origin in Brazil. Description: Sexual stage not observed. Colonies fastgrowing (70 mm in 3 d on MEA 2%), aerial mycelium absent, conidiophores and sporulation abundant, diffusible pale luteous to light luteous pigment in the agar. Conidiophores solitary, erect, hyaline, emerging directly from hyphae, consisting of stipe and stipe extensions emerging immediately below fertile branches, followed by a penicillate conidiogenous apparatus; stipe septate, hyaline, pale luteous to luteous, with small verrucose ornamentations at the base, 137–190 × 7–10 µm (avg. 157.5 × 8.5 µm), stipe branching 2–4 times, directly subtending penicillate at right angles, progressively 9 bending upward, hyaline, septate, 78–191 µm long (avg. 126 µm), 2.5–10 µm wide (avg. 6.5 µm) at the base, ending in a vesicle clavate to somewhat clavate. Conidiogenous apparatus densely penicillate, consisting of a whorl of fertile branches, 21–48 µm long (avg. 37.5 µm), 22–78.5 µm wide (average 58.0 µm); first branch aseptate, 12–30 × 4–8.5 µm (avg. 23.0 × 6.0 µm); secondary branch aseptate, 6–13 × 2–4.5 µm (avg. 9 × 3.5 µm); tertiary branch aseptate, 6.5–7.5 × 2–3.5 µm (avg. 7 × 3.0 µm), each terminating in a whorl of 4–8 phialides. Phialides aseptate, cylindrical, slightly ventricose, 6.5–10 × 1.5–3 µm (avg. 8 × 2.5 µm), apex with minute periclinal thickening and inconspicuous collarette. Conidia hyaline, smooth, sigmoid to ellipsoid, beveled at one or both ends, (5.5–)6.5–8(–8.5) × (1.5–)2–3 µm (avg. 7 × 2.5 µm), forming a mucoid drop at the apex of conidiophore, turning luteous to orange with age. Chlamydospores formed singly, terminal, immersed in the agar, abundant, pale luteous to light luteous, 24–32 µm diam (avg. 28 µm), globose to subglobose, wall 1.5–3.5 µm thick (avg. 2.5 µm). Ecology and known distribution: Rotting fruits of Caryocar brasiliense, Dypsis madagascariensis, Spondias purpurea, Spondias mombin, and Syagrus romanzoffiana, Brazil (Distrito Federal, Goiás, and Tocatins). Additional specimens examined: BRAZIL. DISTRITO FEDERAL: Brasília, on fruits of Syagrus romanzoffiana, 15 Dec 2016, A. Reis (culture CCUB304); Gama, on fruits of Spondias purpurea, 17 Dec 2016, A. Reis (cultures CCUB1021, CCUB1022, CCUB1043). GOIÁS: Nova Veneza, on fruits of Spondias mombin, 23 Nov 2016, A. Reis (cultures CCUB1023, CCUB1025, CCUB1044, CCUB1045, CCUB1050). TOCANTINS: Palmas, on fruits of Caryocar brasiliense, 15 Dec 2016, A. Reis (cultures CCUB1019, CCUB1042). Commentary: Gliocephalotrichum brasiliense represents a sister group to G. bulbilium. Morphologically, both are similar, but the former has more variation in the length of the stipe and presents no deviation in the number of branches, has conidia smaller and wider than those of G. bulbilium, and forms solitary and terminal chlamydospores. Gliocephalotrichum caryocaris R.A. Fernandes, C.P. FIG. 5 Almeida & Pinho, sp. nov. MycoBank MB832550 Typification: BRAZIL. MINAS GERAIS: Montes Claros, sample of C. brasiliense fruit, 28 Jan 2017, A. Reis (holotype UB23976). Ex-type culture CCUB231. GenBank: ITS = MN450208; HIS3 = MN508746; TEF1 = MN508692; TUB = MN508729. Etymology: caryocaris (Latin), referring to the genus Caryocar, the most common host of this species. 10 FERNANDES DA SILVA ET AL.: NEW SPECIES OF GLIOCEPHALOTRICHUM Figure 4. Gliocephalotrichum brasiliense (ex-type CCUB232). A. Conidiophores on SNA. B–C. Conidiophores. D–E. Conidiogenous apparatus with conidiophore branches and phialides. F–G. Stipe extensions. H. Terminal and intercalary chlamydospores. I. Conidia. Bars: A = 0.1 mm; B–E = 20 μm; F–I = 10 μm. Description: Sexual morph not observed. Colonies fastgrowing (70 mm in 3 d in MEA 2%), aerial mycelium absent, conidiophores and sporulation abundant, turning dark brown after 10 d, diffusible pale dark brown pigment in the agar. Conidiophores solitary, erect, hyaline, emerging directly from hyphae, consisting of stipe and stipe extensions emerging immediately below fertile branches, followed by a penicillate conidiogenous apparatus; stipe septate, hyaline, slightly luteous, 103–180.5 × 8.5–12.5 µm (avg. 156.5 × 10.5 µm), stipe branching 2–5 times, directly subtending penicillate at right angles, progressively bending upward, hyaline, septate, 127.5–191 µm long (avg. 160.5 µm), 3.5–5.5 µm wide (avg. 5 µm) at the base, ending in a vesicle slightly clavate. Conidiogenous apparatus densely penicillate, consisting of a whorl of fertile branches, 21.5–42.5 µm long (avg. 30 µm), 30.5–64.5 µm wide (avg. 50.5 µm); first branch aseptate, 8–18 × 3–5.5 µm (avg. 13 × 4.5 µm); secondary branch aseptate, 5.5–9.5 × 2–4.5 µm (avg. 8 × 3.5 µm); tertiary branch aseptate, 6–9 × 2–4.5 µm (avg. 7.5 × 3 µm), each ending in a whorl of 5–8 phialides. Phialides aseptate, cylindrical, slightly swollen, hyaline, 7–9.5 × 1.5–2.5 µm (avg. 8.5 × 3.5 µm), apex with minute periclinal thickening and inconspicuous collarette. Conidia hyaline, smooth, cylindrical to bacilliform, 7.5–8.5 (–9) × 1.5–2 (–2.5) µm (avg. 8 × 2 µm), forming a mucoid drop hyaline at the apex of MYCOLOGIA 11 Figure 5. Gliocephalotrichum caryocaris (ex-type CCUB231). A–B. Conidiophores on SNA. C–D. Conidiophores. E–F. Conidiogenous apparatus with conidiophore branches and phialides. G. Cylindrical to bacilliform conidia. H. Chlamydospore aggregates with globose to ellipsoid cells. I–J. Stipe extension. Bars: A, B = 0.1 mm; C–F = 20 μm; G–J = 10 μm. conidiophore. Chlamydospores abundant, brown to dark brown, superficial and immersed in the agar, forming bulbiloid aggregates, 207.5–388 × 124.5–236 µm (avg. 298 × 170.5 µm), consisting of globose to ellipsoid cells; solitary chlamydospores absent. Ecology and known distribution: Rotting fruits of Caryocar brasiliense, Brazil (Distrito Federal and Minas Gerais). Additional specimen examined: BRAZIL. DISTRITO FEDERAL: Brasília, Asa Norte, on fruits of Caryocar brasiliense, 17 Dec 2016, A. Reis (culture CCUB22). Commentary: Isolates representing G. caryocaris are morphologically similar to G. longibrachium but can be distinguished by a small variation in stipe ramification, size of the conidiogenous apparatus, smaller phialides, and chlamydospores present composed of a greater number of individual cells. Gliocephalotrichum caryocaris is also different from G. abrachium by the presence of stipe ramifications. KEY TO SPECIES OF GLIOCEPHALOTRICHUM WORLDWIDE 1. Chlamydospores forming bulbiloid aggregates on surface of culture medium; multicellular and dark brown ......................................................................... 2 1′. Chlamydospores immersed in the culture medium; solitary and hyaline ...................................................... 8 12 FERNANDES DA SILVA ET AL.: NEW SPECIES OF GLIOCEPHALOTRICHUM 2. Stipe extensions present on conidiophores .............. 3 2′. Stipe extensions absent on conidiophores.................... ......................................................................... G. abrachium 3. Conidiophores with stipe extensions between 80 and 200 µm ........................................................................... 4 3′. Conidiophores with stipe extensions between 120 and 400 µm ................................................................... 5 4. Conidiophores stipe measuring >120 and <330 µm long and with ever more than 10 µm wide.................. ..................................................................... G. simmonsii 4′. Conidiophore stipes shorter than 168 µm long and less than 16 µm wide ...................................... G. mexicanum 5. Conidia oblong to slightly ellipsoid...... G. bulbilium 5′. Conidia cylindrical to bacilliform.............................. 6 6. Stipe extension longer than 200 µm............................. ............................................................ G. longibrachium 6′. Stipe extension shorter than 200 µm........................ 7 7. Conidiogenous apparatus 22–42 × 30–64 µm, with 3 fertile branches.......................................... G. caryocaris 7′. Conidiogenous apparatus 33–73 × 37–88 µm, with 4 fertile branches ............................ G. queenslandicum 8. Conidia cylindrical ........................................................ 9 8′. Conidia ellipsoid to bacilliform................................ 11 9. Conidia cylindrical, 9–13 µm long G. cylindrosporum 9′. Conidia cylindrical, <9 µm long............................... 10 10. Conidiophores with 1–3 stipe extensions emerging 12–32 µm below the conidiogenous apparatus.......... ........................................................................ G. simplex 10′. Conidiophores with 2–6 stipe extensions and directly subtending the conidiogenous apparatus..... ....................................................................... G. nephelii 11. Conidiophores with stipes 100–200 µm long and 7-15 µm wide ......................................................... 12 11′. Conidiophore with stipe 60–136 µm long and 9–16 µm wide ..................................................................... 13 12. Conidiophores with 5–7 stipe extensions.................... ............................................................... G. bacilisporum 12′. Conidiophores with 2–4 stipe extensions.................. ................................................................... G. brasiliense 13. Phialides 6.5–8 µm long and conidia >10 µm long... .................................................. G. microcladydospoum 13′. Phialides 7–12 µm long and conidia <9 µm long 14 14. Quaternary fertile branches on luteous conidiophores ......................................................................... G. humicola 14′. Quaternary fertile branches on brown conidiophores ........................................................................ G. ohiense DISCUSSION Until now only three isolates of Gliocephalotrichum were known from soil or plant roots in Brazil (Lombard et al. 2014). The present study expands the number of Gliocephalotrichum isolates and describes three new species, their occurrences, and host ranges in Brazil. In addition, the presence of six species, three known and three undescribed, was revealed. Recent phylogenetic studies of Gliocephalotrichum have substantially influenced the taxonomy of the genus and contributed to the description of nine additional new species. Lombard et al. (2014) conducted a study with 30 isolates of Gliocephalotrichum that were isolated from Nephelium lappaceum (rambutan) fruits from Central America and found seven species displaying symptoms of fruit rot. Until now, N. lappaceum was considered the main host of Gliocephalotrichum. In the present study, fruit rot on nine new plant hosts were identified. All plant host species are native to tropical regions, and four of these are native to the Brazilian Cerrado. The increase in the geographic range of Gliocephalotrichum suggests that more species may be discovered as more plant hosts and areas are sampled. Most isolates causing fruit rot in Caryocar brasiliense were identified as G. abrachium, G. brasilensis, and G. longibrachium. This host is endemic to the Brazilian Cerrado, and due to its nutritional properties, it is used in the cosmetic and food industries. Currently, there are no commercial plantations, and the fruit harvest relies on the extraction from natural habitats by local communities (Ferreira et al. 2019). Other fungi reported to cause diseases on C. brasiliense include Calonectria clavatum, Capillaureum caryovora, Ceratocystis fimbriata, Cerotelium giacomettii, Colletotrichum spp., Diaporte spp., Milesia caryocae, Oidium sp., and Periconiella smilacis (Ferreira et al. 2019). These pathogens can affect the productivity, viability of fruits, and even plant death. In addition, the wide host range of some species of Gliocephalotrichum suggest a generalist habit. Of the previously known species of Gliocephalotrichum in Brazil (G. bacilisporum, G. bulbilium, and G. simplex), only G. bacillisporum was not found in the present study. Gliocephalotrichum bulbilium and G. simplex are known pathogens of Nephelim leppaceum in Guatemala, Hawaii, Mexico, and Puerto Rico (Nishijima et al. 2007; SerratoDiaz et al. 2012; Lombard et al. 2014). However, in Brazil, neither species was associated with a host; they were found in soil samples (Lombard et al. 2014). Gliocephalotrichum bulbilium and G. simplex have also been reported in other six countries on various plant MYCOLOGIA hosts, such as Clusia spp., Flacourtia spp., Musa spp., and Nissa sylvatica, and from soil, but never in species such as C. renda, D. madagascariensis, S. cumini, S. purpurea, or Sy. jambos. Gliocephalotrichum longibrachium was initially described from French Guiana on leaf litter by Decock et al. (2006), and to date only two isolates are known. The present study expanded its occurrence to Brazil and on a new host. The first phylogenetic study with Gliocephalotrichum was carried out by Decock et al. (2006), whose analyses supported two informal groups based on their phylogenetic relationships and the position of the stipe extensions on the conidiogenous apparatus. Wiley and Simmons (1971) also proposed these two informal groups, but only based on morphology. However, Lombard et al. (2014) mentioned that multilocus phylogenetic analyses were not able to resolve the informal groups, and this is confirmed by the present work. Our study is concordant with the phylogeny of Lombard et al. (2014), which correlates with certain morphological characters, mainly, conidial shape. One group that includes G. bulbilium and G. brasiliense is characterized by having conidia ellipsoid to oblong. In addition, the former species has chlamydospores forming bulbiloid aggregates, whereas the latter has solitary or intercalary chlamydospores. Another clade is composed by G. bacillisporum, G. humicola, G. microchlamydosporum, and G. ohiensis, all of which have ellipsoidal conidia and solitary chlamydospores. A third group includes G. abrachium, G. caryocaris, and G. longibrachium, which have bacilliform conidia and bulbiloid aggregates of chlamydospores much larger than the first group. The last and largest group consists of G. cylindrosporum, G. grande, G. mexicanum, G. nephelii, G. queenlandicum, G. simmonsii, and G. simplex, with cylindrical to slightly ellipsoid conidia and intercalary to aggregated chlamydospores. Results of the pathogenicity tests determined that Gliocephalotrichum cause lesions on fruits of all plant species inoculated, fulfilling Koch’s postulates, and confirm that G. abrachium, G. brasiliense, G. bulbilium, G. caryocaris, G. longibrachium, and G. simplex are causal agents of fruit rot in Brazil. The pathogenicity tests also showed that Gliocephalotrichum usually produces stipe extensions on the host, with the exception of G. abrachium, which could be used for rapid identification to the genus level and without microscopy techniques or molecular analysis. Because Gliocephalotrichum was not associated with plant disease in Brazilian territories until the present study, information on these pathogens is very limited. Inter- and intraspecific diversity, epidemiology, ecology, geographic distribution, and methods of control are areas that need further study. 13 ACKNOWLEDGMENTS The authors wish to thank an anonymous reviewer and Brandon Matheny for their reviews and suggestions that improved earlier versions of the manuscript. FUNDING This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brasil (CAPES), Finance Code 001. We also acknowledge the financial support of the Conselho Nacional de Pesquisa (CNPq) and Fundação de Apoio a Pesquisa do Distrito Federal (FAP-DF) through grant 0193.000825/2015. P. Chaverri undertook part of this work as a visiting research scientist at the Universidade de Brasília, funded by FAP-DF (grant no. 193.000.512/201857). D. B. Pinho and A. Reis acknowledge Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the research productivity fellowships. ORCID Rildo Alexandre Fernandes da Silva http://orcid.org/00000001-8183-125X Camila Pereira de Almeida http://orcid.org/0000-00023666-0874 Ailton Reis http://orcid.org/0000-0002-5705-3002 Frederick Mendes Aguiar http://orcid.org/0000-00026644-4830 Priscila Chaverri http://orcid.org/0000-0002-8486-6033 Danilo Batista Pinho http://orcid.org/0000-0003-2624302X LITERATURE CITED Alfenas RF, Lombard L, Pereira OL, Alfenas AC, Crous PW. 2015. 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