Trichoderma Stromaticum and Its Overseas Relatives

Mycol Progress (2012) 11:215–254 DOI 10.1007/s11557-011-0743-4

ORIGINAL ARTICLE

Trichoderma stromaticum and its overseas relatives

Gary J. Samuels & Adnan Ismaiel & Jorge de Souza & Priscila Chaverri

Received: 1 October 2010 /Revised: 25 January 2011 /Accepted: 26 January 2011 /Published online: 22 February 2011 # Springer-Verlag (outside the USA) 2011

Abstract Trichoderma stromaticum, T. rossicum and new- stromaticum in tropical America. The closest relatives of T. ly discovered species form a unique lineage in Tricho- stromaticum are collected in Africa and Thailand; somewhat derma. Phylogenetic and phenotypic diversity in more distantly related are T. rossicum and T. barbatum,both Trichoderma stromaticum are examined in the light of found in north temperate regions. reported differences in ecological parameters and AFLP patterns. Multilocus phylogenetic analysis using 4 genes (tef1, Keywords Hypocreales . Theobroma cacao . Pleomorphic rbp2, cal, chi18-5) did not reveal phylogenetic basis for the fungi . Species concepts . Species complex . Hypocrea . two reported divergent AFLP patterns or for ecological Hypocreaceae . Cacao . Biological control . Biogeography. parameters; however, this analysis does indicate incomplete Systematics speciation with one supported clade derived from within T. stromaticum that corresponds to AFLP Group 2 of de Souza Taxonomic novelties Trichoderma barbatum . et al. (2006, Phytopathology 96:61–67). Trichoderma T. caesareum . T. floccosum . T. ivoriense . T. lanuginosum . stromaticum is known only from tropical America and is T. vermipilum typically found in association with Theobroma cacao infected with Moniliophthora perniciosa. It is reported here for the first time on pseudostromata of M. roreri in Peru. Strains of T. Introduction stromaticum also have been isolated as endophytes from stems of Theo. cacao. There are no recognized close relatives of T. Witches’ Broom Disease has caused considerable eco- nomic and social disruption in South America, but especially in the Brazilian state of Bahia (see reviews G. J. Samuels (*) : A. Ismaiel Systematic Mycology and Microbiology Lab, and references in de Souza et al. 2006, 2008; Loguercio United States Department of Agriculture, et al. 2009a, b). The pathogen, Moniliophthora perniciosa, Agriculture Research Service, infects cacao trees when basidiospores germinate and Rm 304, B-011A, 10300 Baltimore Ave, penetrate all meristematic tissues, including pods and Beltsville, MD 20705, USA e-mail: [email protected] flower cushions, leading to hypertrophy and hyperplasia and loss of apical dominance (witches’ broom formation) J. de Souza and loss of fruit. The pathogen ultimately produces small Centro de Ciências Agrárias Biológicas e Ambientais (CCABA), mushrooms on the infected dead brooms and infected pods Universidade Federal do Recôncavo da Bahia (UFRB), Centro, Cruz das Almas, BA 44380-000, Brazil that continue the disease cycle (Purdy and Schmidt 1996; Hebbar et al. 2002;deSouzaetal.2006, 2008; Meinhardt P. Chaverri et al. 2008). Trichoderma stromaticum colonizes dead Department of Plant Sciences and Landscape Architecture, brooms and other tissue infected with M. perniciosa and University of Maryland, 2112 Plant Sciences Building, destroys the basidiocarps of the pathogen thereby reducing College Park, MD 20742, USA the inoculum (Bastos 1996;Hjorthetal.2003). A 216 Mycol Progress (2012) 11:215–254 biological control product ‘Trichovab®,’ which is based on Morphological analyses asinglestrainofT. stromaticum, hasbeenusedonan experimental scale in Brazil since 1996 (de Souza et al. Observations of microscopic characters were made from 2006).Thestrainusedinthisproduct(‘TVC’)was cultures grown on CMD or SNA (low nutrient agar; originally isolated in the Brazilian Amazonian region Nirenberg 1976), less frequently from potato dextrose agar (Pará State) but its efficacy is subject to environmental (PDA; Difco). Cultures to be used for micromorphological conditions (Sanogo et al. 2002; Loguercio et al. 2009a, b) observations were incubated at 25°C under alternating cool and its effectiveness is greatly enhanced when it is applied white fluorescent light/darkness. in combination with farm sanitation and copper-based Material to be used for microscopic measurements was fungicides (Medeiros et al. 2010). first immersed in 3% (aq.) potassium hydroxide (KOH), de Souza et al. (2006) found that a large collection of which was replaced with water or more KOH as the strains of T. stromaticum from Brazil, Ecuador and preparation dried. Observations were made with differential Colombia could be divided between two amplified interference contrast, phase contrast or bright field micros- fragment length polymorphism (AFLP) groups that corre- copy. Helicon Focus® version 4.21.5 Pro (MP) (Helicon lated with a number of biological differences (de Souza Soft, www.heliconfocus.com) was used to produce some et al. 2006; 2008; Loguercio et al. 2009a, b)andthefact composite images. To make sections of perithecial collec- that a Hypocrea teleomorph, H. stromatica JL Bezerra tions, small pieces of substratum with one or two stromata et al. (Bezerra et al. 2003) is known for Group 2. These were rehydrated in 3% KOH. These were supported in differences suggest that T. stromaticum as it is currently Tissue-Tek® O.C.T. compound (Miles, Elkhart, IN, USA) understood is composed of at least two cryptic species. In on the stage of an IEC-CTF microtome cryostat; sections the present work, we apply multilocus phylogenetic and were made at a thickness of ca. 15 μm. Permanent classical mycological analyses to a collection of strains of preparations were made following Volkmann-Kohlmeyer T. stromaticum to clarify the taxonomy of this species. and Kohlmeyer (1996). InthecourseoftaxonomicworkwithTrichoderma/ Where possible, 30 units of each parameter were Hypocrea, we have collected or received from collabo- measured for each collection using Scion Image for rators many strains, which are routinely identified through Windows® (www.scioncorp.com). The continuous measure- DNA sequence analysis. Several of these are closely ments are reported as extremes in brackets with the range related to T. stromaticum and are included in the present calculated as mean plus and minus standard deviation. work. Computation of descriptive statistics, including 95% confidence intervals (ci), was performed using Systat 10 (Systat Software, San José, CA, USA). Materials and methods Growth rates were determined on PDA and SNA at 15, 20, 25, 30 and 35°C in darkness (with intermittent light). Cultures used Measurements were made at intervals of 24 up to 96 h. Colony characters were taken from colonies incubated on Although the primary emphasis of the present work is T. PDA and SNA at 25°C with alternating cool white stromaticum, related strains and species were included fluorescent light and darkness (12 h:12 h) after 7−10 days; when their close relationship to T. stromaticum was these conditions are referred to in descriptions as ‘under revealed during on-going taxonomic studies of Tricho- light.’ Color standards are from Kornerup and Wanscher derma. The strains used in this study are cited in Table 1. (1978, K&W). These include several cultures cited by de Souza et al. (2006) and perithecial collections. Cultures and perithecial DNA extraction, PCR and sequencing specimens of Trichoderma stromaticum and other species were collected by the authors in Brazil (Bahia), Cameroon, DNA from all strains included in this study was extracted Ecuador and Thailand. Cultures were also provided by using ArchivePure DNA cell/Tissue kit from 5 PRIME colleagues in Austria, Canada, Côte d’Ivoire, Ecuador, (Gaithersburg, MD, USA). The primers and their sequences Peru, Republic of South Africa, Russia, U.K., and USA. used in this study are given in Table 2. The primers for ITS Single ascospores were isolated from perithecial collections and α-actin (act) were described previously (Samuels and on cornmeal agar (Difco or BD BBL, Franklin Lakes, NJ, Ismaiel 2009). A portion of translation elongation factor 1- USA)+2% (w/v) dextrose (CMD) with the use of a α (tef1) was amplified using the primers Ef728M and Ef-2. micromanipulator. Representative cultures are deposited in Ef7-28 M is a modified version of EF728F (Carbone and Centraalbureau voor Schimmelcultures (CBS, Utrecht, and Kohn 1999) where nucleotide #4 is changed from C to Y= The Netherlands). C/T. This modification was necessary to make the primer yo rges(02 11:215– (2012) Progress Mycol Table 1 Trichoderma strains used in the research

Species Strain T. stromaticum strain Provenance GenBank accession numbers code and AFLP groupa cal chi18-5 rbp2 tef1 ITS

T. barbatum G.J.S. 04-308=CBS 125733 Tb USA: MI HQ342352 HQ342483 HQ342286 HQ342223 HQ342417 T. barbatum DAOM 230008 Russia: Siberia HQ342353 HQ342484 HQ342287 HQ342224 HQ342418 T. caesareum G.J.S. 01-225=CBS 124369 T Thailand: Khao Yai HQ342345 HQ342476 HQ342279 HQ342216 HQ342410 Natl Park T. floccosum G.J.S. 01-238=CBS 124372 T Thailand: Khao Yai Natl Park HQ342347 HQ342478 HQ342281 HQ342218 HQ342412 254 T. ivoriense G.J.S. 01-312=CBS 125734 T Côte d’Ivoire HQ342346 HQ342477 HQ342280 HQ342217 HQ342411 T. lanuginosum G.J.S. 01-174=CBS 126100 Cameroon: Reserve Faunal du Dja HQ342349 HQ342480 HQ342283 HQ342220 HQ342414 T. lanuginosum G.J.S. 01-176=CBS 125718 T Cameroon: Reserve Faunal du Dja HQ342350 HQ342481 HQ342284 HQ342221 HQ342415 T. medusae G.J.S. 01-171=CBS 125719 T Cameroon: Reserve Faunal du Dja HQ342343 HQ342474 HQ342277 HQ342214 HQ342408 T. medusae G.J.S. 01-166 Cameroon: Reserve Faunal du Dja HQ342344 HQ342475 HQ342278 HQ342215 HQ342409 T. rossicum G.J.S. 07-72=Berg PR26-12-6 Austria HQ342351 HQ342482 HQ342285 HQ342222 HQ342416 T. rossicum G.J.S. 98-89=DAOM 230009= Russia: Siberia HQ342356 HQ342487 HQ342289 HQ342225 HQ342420 TUB F 718 T. rossicum DAOM 230011 T Russia: Siberia HQ342354 HQ342485 HQ342288 AY937441 HQ342419 T. rossicum DAOM 230010 Russia: Siberia HQ342355 HQ342486 HQ400747 AY937424 DQ08302 T. stromaticum DISc 183A AM1, 2 Brazil: Pará HQ342292 HQ342423 HQ342228 HQ34216700 HQ342359 T. stromaticum DIS 185A AM 2, 2 Brazil: Pará HQ342308 HQ342439 HQ342244 HQ342182 HQ342375 T. stromaticum DIS 185 C Brazil: Pará HQ342296 HQ342427 HQ342232 HQ342170 HQ342363 T. stromaticum G.J.S. 00-02 Colombia: Paraíso HQ342333 HQ342464 FJ442714 FJ463361 FJ442675 T. stromaticum G.J.S. 00-102 Brazil: Pará HQ342331 HQ342462 HQ342266 HQ342203 HQ342397 T. stromaticum G.J.S. 00-107 BA 54: 1 Brazil: Bahia HQ342330 HQ342461 HQ342265 HQ342202 HQ342396 T. stromaticum G.J.S. 00-108 Brazil: Bahia HQ342328 HQ342459 EU341807 AY937436 DQ083013 T. stromaticum G.J.S. 00-110 BA 55, 1 Brazil: Bahia HQ342326 HQ342457 HQ342262 HQ342199 HQ342393 T. stromaticum G.J.S. 00-127 Brazil: Bahia HQ342298 HQ342429 HQ342234 HQ342172 HQ342365 T. stromaticum G.J.S. 00-130 Brazil: Bahia HQ342307 HQ342438 HQ342243 HQ342181 HQ342374 T. stromaticum G.J.S. 00-132 Brazil: Bahia HQ342293 HQ342424 HQ342229 HQ342168 HQ342360 T. stromaticum G.J.S. 00-141 Brazil: Bahia HQ342327 HQ342458 HQ342263 HQ342200 HQ342394 T. stromaticum G.J.S. 00-91 Brazil: Pará HQ342294 HQ342425 HQ342230 HQ342169 HQ342361 T. stromaticum G.J.S. 00-93 Brazil: Pará HQ342297 HQ342428 HQ342233 HQ342171 HQ342364 T. stromaticum G.J.S. 01-91 Brazil: Pará HQ342342 HQ342473 HQ342276 HQ342213 HQ342407 T. stromaticum G.J.S. 01-92 Brazil: Bahia HQ342311 HQ342442 HQ342247 HQ342184 HQ342378 T. stromaticum G.J.S. 01-93 Brazil: Bahia HQ342341 HQ342472 HQ342275 HQ342212 HQ342406 T. stromaticum G.J.S. 01-95 Brazil: Bahia HQ342337 HQ342468 HQ342271 HQ342208 HQ342402 T. stromaticum G.J.S. 01-97 Brazil: Bahia HQ342336 HQ342467 HQ342270 HQ342207 HQ342401 T. stromaticum G.J.S. 02-09 BA 73, 1 Brazil: Bahia HQ342334 HQ342465 HQ342268 HQ342205 HQ342399 T. stromaticum G.J.S. 03-134 Brazil: Bahia HQ342338 HQ342469 HQ342272 HQ342209 HQ342403 T. stromaticum G.J.S. 03-135 Brazil: Bahia HQ342340 HQ342471 HQ342274 HQ342211 HQ342405 T. stromaticum G.J.S. 03-47 BA 48, 1 Brazil: Bahia HQ342329 HQ342460 HQ342264 HQ342201 HQ342395 217 T. stromaticum G.J.S. 03-50 BA 59, 1 Brazil: Bahia HQ342332 HQ342463 HQ342267 HQ342204 HQ342398 218 Table 1 (continued)

Species Strain T. stromaticum strain Provenance GenBank accession numbers code and AFLP groupa cal chi18-5 rbp2 tef1 ITS

T. stromaticum G.J.S. 03-76 Brazil: Bahia HQ342339 HQ342470 HQ342273 HQ342210 HQ342404 T. stromaticum G.J.S. 04-190=CBS 127462 Peru HQ342314 HQ342445 HQ342250 HQ342187 HQ342381 T. stromaticum G.J.S. 04-214 Peru HQ342319 HQ342450 HQ342255 HQ342192 HQ342386 T. stromaticum G.J.S. 04-305=CBS 126593 Ecuador: Pichilingue HQ342318 HQ342449 HQ342254 HQ342191 HQ342385 T. stromaticum G.J.S. 04-306 CBS 126773 Ecuador: Pichilingue HQ342320 HQ342451 HQ342256 HQ342193 HQ342387 T. stromaticum G.J.S. 04-327 Peru HQ342313 HQ342444 HQ342249 HQ342186 HQ342380 T. stromaticum G.J.S. 04-331 Peru HQ342310 HQ342441 HQ342246 HQ342183 HQ342377 T. stromaticum G.J.S. 05-455 AM 14, 2 Ecuador: Pichilingue HQ342316 HQ342447 HQ342252 HQ342189 HQ342383 T. stromaticum G.J.S. 06-320 Ecuador: Pichilingue HQ342315 HQ342446 HQ342251 HQ342187 HQ342382 T. stromaticum G.J.S. 06-327 Ecuador: Pichilingue HQ342317 HQ342448 HQ342253 HQ342190 HQ342384 T. stromaticum G.J.S. 07-102 BA 38, 1 Brazil:Bahia HQ342324 HQ342455 HQ342260 HQ342197 HQ342391 T. stromaticum G.J.S. 07-108 BA 5 Brazil: Bahia HQ342325 HQ342456 HQ342261 HQ342198 HQ342392 T. stromaticum G.J.S. 07-118 Brazil: Bahia HQ342312 HQ342379 HQ342248 HQ342185 HQ342379 T. stromaticum G.J.S. 07-119 Brazil: Bahia HQ342299 HQ342430 HQ342235 HQ342173 HQ342366 T. stromaticum G.J.S. 07-120 Brazil: Bahia HQ342306 HQ342437 HQ342242 HQ342180 HQ342373 T. stromaticum G.J.S. 07-76 BA 7, 1 Brazil: Bahia HQ342335 HQ342466 HQ342269 HQ342206 HQ342400 T. stromaticum G.J.S. 07-77 BA 66, 2 Brazil: Bahia HQ342305 HQ342436 HQ342241 HQ342179 HQ342372 T. stromaticum G.J.S. 07-78 AM 10, 2 Brazil: Pará HQ342304 HQ342435 HQ342240 HQ342178 HQ342371 T. stromaticum G.J.S. 07-79 Brazil: Bahia HQ342303 HQ342434 HQ342239 HQ342177 HQ342370 T. stromaticum G.J.S. 07-80 BA 21, 2 Brazil: Bahia HQ342302 HQ342433 HQ342238 HQ342176 HQ342369 T. stromaticum G.J.S. 07-88 BA 49, 1 Brazil: Bahia HQ342322 HQ342453 HQ342258 HQ342195 HQ342389 T. stromaticum G.J.S. 07-89 BA 65, 2 Brazil: Bahia HQ342300 HQ342431 HQ342236 HQ342174 HQ342367 T. stromaticum G.J.S. 07-91 BA 36, 1 Brazil: Bahia HQ342321 HQ342452 HQ342257 HQ342194 HQ342388 T. stromaticum G.J.S. 07-94 AM 13, 1 Colombia: Paraíso HQ342323 HQ342454 HQ342259 HQ342196 HQ342390 T. stromaticum G.J.S. 07-95 BA 53, 2 Brazil: Bahia HQ342301 HQ342432 HQ342237 HQ342175 HQ342368 T. stromaticum G.J.S. 97-179=ATCC 204429 Brazil: Pará HQ342295 HQ342426 HQ342231 AY937434 AY937434 T. stromaticum G.J.S. 97-180 AM 12, 2 Brazil: Rondônia HQ342290 HQ342421 HQ342226 HQ342166 HQ342357

T. stromaticum G.J.S. 97-181 AM 11, 2 Brazil: Pará HQ342291 HQ342422 HQ342227 AY937447 AY937447 11:215– (2012) Progress Mycol T. stromaticum TVC=G.J.S. 97-183=CBS AM 7, 2 Brazil: Pará HQ342309 HQ342440 HQ342245 AY937418 AY937418 101875=ATCC 204426= DAOM 231100 T T. vermipilum PPRI 3559=- CBS 127103 T Republic of South Africa HQ342348 HQ342479 HQ342282 HQ342219 HQ342413 T. virens DIS 162 Costa Rica FJ442403 HQ342489 FJ442696 FJ463367 FJ442669 T. virens G.J.S. 01-287=CBS 123790 Côte d’Ivoire FJ442404 HQ342488 EU341804 AY750894 DQ083023 a From de Souza et al (2006) b T denotes an ex-type culture c DIS cultures are stem endophytes of Theobroma species 254 Mycol Progress (2012) 11:215–254 219

Table 2 PCR and sequencing primers used in the study

Locus Primer name Primer length (b) Primer sequence cal CL1 20 5′- GAR TWC AAG GAG GCC TTC TC CAL737RM 20 5′- CAT YTT TCK KGC CAT CAT GG act TRI-ACT1 23 5′- TGG CAC CAC ACC TTC TAC AAT GA TRI-ACT2 22 5′- TCT CCT TCT GCA TAC GGT CGG A ACT-500 F 16 5′- ATT CCG TGC TCC TGA G ACT-511R 16 5′- CTC AGG AGC ACG GAA T tef1 EF-728 M 20 5′- CAT YGA GAA GTT CGA GAA GG EF-2 21 5′- GGA RGT ACC AGT SAT CAT GTT ITS ITS5 22 5′- GGA AGT AAA AGT CGT AAC AAG G ITS4 20 5′- TCC TCC GCT TAT TGA TAT GC rpb2 RPB-F2 18 5′- GAA GCG TCT GGA TYT SGC RPB-R2 18 5′- GGG GAA AGG RAT GAT ACT RPB-450R 20 5′- TCC ATR CCT CTG TTG ATC AT RPB-432 F 20 5′- ATG ATC AAC AGA GGY ATG GA chi18-5 Chit42-1a 19 5′- AGCWAGCACSGATGCCAAC Chit42-2a 19 5′- AGGTTCTGAGTYGWGTCCA

sequence match those for all Trichoderma species from all the format. The alignments were manually edited, if necessary, sections of the genus. For rpb2, we designed primers RPB2- using MacClade 4.06 (Maddison and Maddison 2003). F2 and RPB2-R2 (Table 2). For calmodulin (cal), we used the primer CL1 (O’Donnell et al. 2000) and Cal737RM. The Phylogenetic analyses latter is a modified version of cal737R (Carbone and Kohn 1999) where we changed nucleotides 4, 9, and 10 from C, T, Data for each gene were analyzed separately and concate- and G to Y(C/T), K (G/T), and K (G/T), respectively. For nated as a combined multilocus sequence (MLS). A chitinase (chi18-5), we used the primers Chi18-5-1a and reciprocal 70% bootstrap threshold (Mason-Gamer and Chi18-5-2a (Kullnig-Gradinger et al. 2002). Kellogg 1996; Reeb et al. 2004; de Queiroz 1993) was The PCR mixture (20 μL) contained 10 μL of Taq 2X used to determine whether partitions could be combined master mix (New England Biolabs, Ipswich, MA, USA), into a single phylogeny. A conflict was assumed to be

1 μL of 25 mM MgCl2, 0.5 μL of 10 mM forward and significant if two different relationships for the same taxa— reverse primers and 7.5 μL of distilled water. The reaction one being monophyletic and the other non-monophyletic, mixtures in 0.2-ml PCR tubes were placed in an MJ both with BP≥70%—were observed on each of the Research PCR PTC-200 thermo-cycler. The program used genealogies. If no conflict exists between the highly for all the genes was a touch down (Don et al. 1991) supported clades in individual gene genealogies, the genes described previously in Samuels and Ismaiel (2009) sequenced likely share similar phylogenetic history and The PCR products were prepared for sequencing using an resolution, and combining the datasets can ultimately enzymatic purification system (Exosap-IT; USB Corporation, increase clade support. Trichoderma virens was selected Cleveland, OH, USA). The purified PCR products were as outgroup because of its close relationship of the species directly sequenced using BigDye Terminator v3.1 chemistry with T. stromaticum and T. rossicum (Bissett et al. 2003). on an automated 3130xl Genetic Analyzer (Applied Bio- To infer the phylogenies of the selected taxa, four systems, Foster City, CA, USA). Both strands of each individual data sets (tef1, chi18-5, rpb2, cal) were used in amplicon were sequenced using the same primers used in Bayesian and maximum parsimony analyses. ITS and act the PCR reactions. For sequencing act and rpb2, two were not included in the analyses because of their low additional internal primers were used (Table 2). The polymorphism and thus few phylogenetically informative sequences were assembled and edited to construct a characters. Consequently, ITS and act did not provide any consensus sequence using Sequencher 4.1 (Gene Codes, resolution for the Stromaticum Clade. Madison, WI, USA). For each locus, sequences were For the individual data sets, Bayesian inference analysis preliminarily aligned using Clustal X version 1.8 (Thompson was performed as described in Samuels and Ismaiel (2009) et al. 1997) under the default settings and saved in nexus in MrBayes version 3.1.2 (Huelsenbeck and Ronquist 220 Mycol Progress (2012) 11:215–254

2001). The substitution model for each locus was determined The combined dataset was also analyzed by Bayesian using jMODELTEST (Posada 2008). The models selected for and maximum parsimony methods as described for the tef1, rbp2, cal, chi18-5 were K80, TrN+G, TrN+I+G, and individual datasets with exceptions described below. The GTR+G, respectively. Two concurrent analyses of four Bayesian analysis was performed with data partitioned into chains (one cold and three heated) were both run for 1 four, representing four loci. The substitution model for each million generations to ensure that the analyses were not locus obtained above was used with each partition. For the trapped at local optima. Random starting trees were used combined dataset, we used 5×106 generations in MrBayes. and sampled every 100 generations. Among 10,000 trees Convergence of log-likelihoods was also examined in produced, initial trees were discarded in the burn-in TRACER v1.5. phase based on the plot of log-likelihood scores of the To investigate the presence of recombination in the trees versus generation number as performed by TRACER combined data set within the isolates in the Stromaticum v.1.5 (Rambaut and Drumond 2009) to ensure that the log- clade, two tests were performed: firstly, the Partition likelihood reached stable equilibrium. The exact numbers Homogenetity Test (PHT; Farris et al. 1995) implemented discarded in burn-in phase for each locus tree are given in in PAUP 4.0b10 (Swofford 2002).Tenthousandreplicates Table 3. The remaining trees were used to construct a majority were analyzed in a heuristic search with the addition of ten rule consensus tree with posterior probability of 0.95 or random sequences and one tree was saved per replicate. greater considered significant (Leache and Reeder 2002). A P value of 0.01 (99% confidence) was used as a Maximum parsimony analysis (MP) was conducted significance threshold (Cunningham 1997); and secondly, using PAUP version 4.0b10 (Swofford 2002), using a the Phi-test as implemented in SplitsTree software (Huson heuristic search with the starting tree obtained via stepwise 1998). addition with random addition of 1,000 replicates, tree– bisection–reconnection (TBR) as the branch swapping algorithm, and MULTREES off. All characters were unordered Results and of equal weight and gaps were treated as missing data. Stability of the clades (bootstrap) was assessed with 1,000 Phylogenetic analyses replicates, using the same MP settings. Bootstrap values greater than or equal to 70% were considered significant A total of 69 strains were included in the study (Table 1). (Hillis and Bull 1993). Two strains of T. virens were used as outgroup for the

Table 3 Summary of sequence alignment and phylogenetic analysis

Parameters LOCUS

tef1 rpb2 chi18-5 cal MLS

Parsimony analysis data Number of strains 69 69 69 69 69 Number of parsimony informative characters 120 91 126 70 407 % Parsimony informative characters 23 8.6 16.6 14.8 14.5 % Parsimony info. characters provided to MSL 29.5 22.4 31 17.1 100 Tree length (# of steps) 209 186 179 136 735 Number of equally parsimonious trees 26 206 264 46 6 Consistency index 0.847 0.839 0.849 0.831 0.814 Homoplasy index 0.153 0.161 0.151 0.169 0.186 Retention index 0.946 0.934 0.95 0.931 0.929 MCMC parameters MCMC generations (millions) 1 1 1 1 5 Number of runs 2 2 2 2 2 Number of chains/temp 4/0.2 4/0.2 4/0.2 4/0.2 4/0.2 Number of initial trees discarded as burn-in 700 500 600 900 700 (-)LnL 1,938.29 2,863.46 2,333.934 1,600.261 8,176.807 Total tree length 0.71 8.5 12.358 12.25 0.427 Mycol Progress (2012) 11:215–254 221 phylogenetic analysis. For each strain, we sequenced six The combined tree (MLS, Fig 1e) revealed a mono- unlinked genes: five were protein-coding genes and one phyletic T. stromaticum with three highly supported non-coding (ITS). Summaries of the alignments for the internal branches (A, C, D). Clade A corresponds to six loci characterized in this study are shown in Table 2. AFLP Group 2 (including the ex-type strain ‘TVC’=G.J.S. Tef1 had the highest number of informative characters with 97-183) of de Souza et al. (2006).AFLPGroup1ofde 23% followed by chi18-5, cal, rpb2, act and ITS with Souza et al is equivalent to poorly supported Clade B. 16.6, 14.8, 8.6, 3.6 and 3.4%, respectively. Due to the Clade C, which appeared with weak support in tef1, and paucity of phylogenetically informative characters in the was supported by Bayesian analysis in the cal tree, was ITS and act datasets, these two loci were not used for highly supported in MLS; it includes only strains from further analyses. Peru and Ecuador. Clade D received strong support only in Partition homogeneity test detected significant hetero- cal and MLS; it was not seen in any other trees. geneity within the combined four-gene data set at the Clades E (Fig 1e, G.J.S. 01-171/G.J.S. 01-166) and F 99% confidence level (P=0.01). Nonetheless, we com- (G.J.S. 01-174, G.J.S. 01-176) received varying levels of bined the multi-locus sequence (MLS)dataforthe support in all single gene trees. These clades have high phylogenetic analyses because combining all genes can support by both methods in the MLS tree. Clade F, which improve the accuracy of the inferred phylogenies even in consists of two Cameroonian ascospore strains, G.J.S. thepresenceofsignificantincongruence (Cunningham 01-174 and G.J.S. 01-176, was maintained across all four 1997; Darlu and Lecointre 2002). In addition, the 70% individual gene trees with high support. Clades E and F reciprocal bootstrap indicates no topological conflicts had a sister relationship in three of four individual gene between gene genealogies and that the datasets can be trees. Only the tree based on cal did not support the sister- combined. relationship of these two species. Clade F had a sister The phylogenetic trees were obtained based on individ- relationship with single strain PPRI 3559 in the chi18-5 ual gene datasets by two methods (Bayesian inference and tree with high support. Clades E and F formed a larger maximum parsimony). The trees obtained by the two clade in three out of four gene trees plus the MLS tree. methods had essentially identical topology; therefore, for South African strain PPRI 3559 clustered with these two each individual gene sequence, the tree obtained from the clades in three of four single gene trees and in MLS. In the Bayesian inference is presented but the bootstrap support cal tree, clades E, F and PPRI 3559 were unresolved with from the maximum parsimony analysis are depicted on respect to each other and basal to T. stromaticum. This respective trees. The Bayesian trees for the individual gene large clade had a sister relationship with T. stromaticum trees are shown in Fig 1a−d. with support in three of the four individual gene trees. The monophyly of T. stromaticum (Clade A, Fig. 1a−e) Therefore these five African strains plus G.J.S. 01-225 was supported in all the individual gene trees. Of the from Thailand are considered to be the closest relatives of subclades A−C, seen in the MLS (Fig 1e), only A was T. stromaticum. supported in more than one of the individual gene trees Trichoderma rossicum and Clade G (DAOM 230008, G. (rpb2, chi18-5).Thetreebasedonrpb2 (Fig 1a)wasthe J.S. 04-308) were consistently basal to the Stromaticum only gene genealogy that recovered two lineages within clade. These are species of north temperate regions. the Stromaticum clade (subclades A and B) supported by Trichoderma rossicum was described from Siberia and has both methods. The phylogenetic tree based on chi18-5 been collected in Austria but was reported from Peru near (Fig 1b) was the closest to the rpb2 tree. Subclade A of Lake Titicaca at an elevation of ca. 3,800 m (Hoyos-Caravajal this tree was identical to that of rpb2 and was supported et al. 2009); Clade G strains were obtained from Siberia and by the two methods. However, isolates of subclade B did Michigan, respectively. Clade G was maintained in all four not form a distinct cluster. In the tef1 tree (Fig 1c), most single gene trees with high support by both methods. strains of T. stromaticum fell into a single, well-supported However, its sister-relationship with T. rossicum was clade; however, seven strains that were collected in Peru supported only in two of the single gene trees. and Ecuador (C in Fig 1c) formed a subclade that had no Between the clades of T. stomaticum and T. rossicum statistical support by either of the two methods. This three single strains, PPRI 3559, G.J.S. 01-238 and G.J.S. subclade was also present in the cal phylogeny (Fig 1d), 01-312, did not fall into any clade. These three strains fall where it was supported by only one of the methods between T. stromaticum and T. rossicum clades in a (Bayesian inference posterior probability 0.96, BS was majority of the gene trees and the MLS tree with high 63%). Like tef1,thecal tree did not support division of T. support (Fig 1a−e). stromaticum into subclades A and B, although a clade of The ITS rRNA region could distinguish most of the species four Brazilian strains found here was also found in MLS under the study except PPRI 3359, which cannot be (Fig 1d,e). distinguished from Clade E (Table 4). None of the various 222 Mycol Progress (2012) 11:215–254 subclades of T. stromaticum seen in Fig. 1 a−e were These differences are considerable given that some species diagnosed by ITS sequences. The differences in genetic of Trichoderma differ by only one base pair (Komon- distance among the strains of the Stromaticum/Rossicum Zelazowska et al. 2007; Degenkolb et al. 2008;Samuelset clade range from 0.0035 (2 bp) –0.014 (7 bp) (Table 4). al. 2009).

G.J.S. 97-180 G.J.S. 97-181 DIS 183A rpb2 G.J.S. 00-132 G.J.S. 00-91 G.J.S. 97-179 DIS 185C G.J.S. 00-93 G.J.S. 00-127 G.J.S. 07-119 G.J.S. 07-89 G.J.S. 07-95 G.J.S. 07-80 G.J.S. 07-79 G.J.S. 07-78 G.J.S. 07-77 A G.J.S. 07-120 G.J.S. 00-130 DIS 185A TVC G.J.S. 04-331 G.J.S. 01-92 G.J.S. 07-118 G.J.S. 04-327 G.J.S. 04-190 T. stromaticum G.J.S. 06-320 G.J.S. 05-455 G.J.S. 06-327 G.J.S. 04-305 G.J.S. 04-214 G.J.S. 04-306 G.J.S. 07-91 G.J.S. 07-88 G.J.S. 07-94 G.J.S. 07-102 G.J.S. 07-108 G.J.S. 00-110 G.J.S. 00-141 G.J.S. 00-108 G.J.S. 03-47 G.J.S. 00-107 G.J.S. 00-102 G.J.S. 03-50 B G.J.S. 00-02 G.J.S. 02-09 G.J.S. 07-76 G.J.S. 01-97 G.J.S. 01-95 G.J.S. 03-134 G.J.S. 03-76 G.J.S. 03-135 G.J.S. 01-93 G.J.S. 01-91 G.J.S. 01-171 G.J.S. 01-166 E G.J.S. 01-225 G.J.S. 01-174 G.J.S. 01-176 F PPRI 3559 G.J.S. 07-72 DAOM 230011 DAOM 230010 T. rossicum G.J.S. 98-89 G.J.S.04-308 DAOM 230008 G G.J.S. 01-312 G.J.S. 01-238 G.J.S. 01-287 DIS 162 1 change

Fig. 1 Majority-rule (50%) consensus tree resulting from Bayesian or BS 70%. Trichoderma virens G.J.S. 01-287 was used as outgroup. analysis of a rpb2, b chi18-5, c tef1, d cal, and e MLS dataset. Support for the branches refers to posterior probability of equal or Branches with thick black lines represent clades with posterior greater than 0.95 and maximum parsimony bootstrap of 70% or probability (PP)≥0.95 and bootstrap maximum parsimony (BS) greater (Leache and Reeder 2002; Hillis and Bull 1993). Tree statistics values≥70%. Thick gray lines represent clades with either PP≥0.95 are presented in Table 3. Text in red indicates perithecial collections Mycol Progress (2012) 11:215–254 223

G.J.S. 97-180 G.J.S. 97-181 DIS 183A chit42 G.J.S. 00-132 G.J.S. 00-91 G.J.S. 97-179 G.J.S. 00-93 G.J.S. 00-127 G.J.S. 07-119 G.J.S. 07-89 G.J.S. 07-95 G.J.S. 07-80 G.J.S. 07-79 G.J.S. 07-78 G.J.S. 07-77 G.J.S. 07-120 G.J.S. 00-130 A DIS 185A TVC G.J.S. 04-331 G.J.S. 01-92 G.J.S. 07-118 G.J.S. 04-327 G.J.S. 04-190 G.J.S. 06-320 G.J.S. 05-455 G.J.S. 06-327 G.J.S. 04-305 G.J.S. 04-306 DIS 185C G.J.S. 04-214 G.J.S. 07-91 G.J.S. 07-88 T. stromaticum G.J.S. 07-94 G.J.S. 07-102 G.J.S. 07-108 G.J.S. 00-110 G.J.S. 00-141 G.J.S. 00-108 G.J.S. 03-47 G.J.S. 00-107 G.J.S. 00-102 G.J.S. 03-50 G.J.S. 00-02 G.J.S. 02-09 G.J.S. 07-76 G.J.S. 01-97 G.J.S. 01-95 G.J.S. 03-134 G.J.S. 03-76 G.J.S. 03-135 G.J.S. 01-93 G.J.S. 01-91 G.J.S. 01-171 G.J.S. 01-166 E G.J.S. 01-225 G.J.S. 01-174 G.J.S. 01-176 F PPRI 3559 G.J.S. 01-312 G.J.S. 07-72 G.J.S. 98-89 T. rossicum DAOM 230011 DAOM 230010 G.J.S.04-308 DAOM 230008 G G.J.S. 01-238 G.J.S. 01-287 DIS 162 1 change

Fig. 1 (continued)

Species delimitation four independently clustering single strains. We interpret these lineages and single strains as taxonomic species. Multilocus phylogenetic analysis with four genes indicates Trichoderma stromaticum is clearly distinguished from that the Stromaticum/Rossicum clade is well supported and all other closely related fungi by its apparent host that it comprises six strongly supported internal lineages and specialization on Moniliophthora species that parasitize 224 Mycol Progress (2012) 11:215–254

G.J.S. 97-180 tef1 G.J.S. 97-181 DIS 183A G.J.S. 00-132 G.J.S. 00-91 G.J.S. 97-179 DIS 185C G.J.S. 00-93 G.J.S. 00-127 G.J.S. 07-119 G.J.S. 07-89 G.J.S. 07-95 G.J.S. 07-80 G.J.S. 07-79 G.J.S. 07-78 G.J.S. 07-77 G.J.S. 07-120 G.J.S. 00-130 DIS 185A TVC G.J.S. 04-331 G.J.S. 01-92 G.J.S. 07-118 G.J.S. 04-214 G.J.S. 07-91 G.J.S. 07-88 G.J.S. 07-94 G.J.S. 07-102 G.J.S. 07-108 G.J.S. 00-110 G.J.S. 00-141 G.J.S. 00-108 G.J.S. 03-47 G.J.S. 00-107 G.J.S. 00-102 G.J.S. 03-50 T. stromaticum G.J.S. 00-02 G.J.S. 02-09 G.J.S. 07-76 G.J.S. 01-97 G.J.S. 01-95 G.J.S. 03-134 G.J.S. 03-76 G.J.S. 03-135 G.J.S. 01-93 G.J.S. 01-91 G.J.S. 04-327 G.J.S. 04-190 G.J.S. 06-320 G.J.S. 05-455 C G.J.S. 06-327 G.J.S. 04-305 G.J.S. 04-306 PPRI 3559 G.J.S. 01-174 G.J.S. 01-176 F G.J.S. 01-225 G.J.S. 01-171 G.J.S. 01-166 E G.J.S. 01-312 G.J.S. 01-238 G.J.S. 07-72 DAOM 230011 T. rossicum DAOM 230010 G.J.S. 98-89 G.J.S.04-308 DAOM 230008 G G.J.S. 01-287 DIS 162 1 change Fig. 1 (continued) cacao, by the stiff, erect hairs that arise from the conidial Druzhinina (personal communication) detected it on air pustules and by the broad-celled hyphae that comprise the filters in Austria based on ITS1+2 sequences. All other pustule. Moreover, it is conclusively known only from members of the clade except T. rossicum and T. barbatum, tropical America, although Zachow et al. (2009) reported it which are northern hemisphere species (or in the case of T. from soil in the Canary Islands based on ITS1 sequence and rossicum, high elevation Andean), are paleotropical. Mycol Progress (2012) 11:215–254 225

G.J.S. 04-327 cal G.J.S. 04-190 G.J.S. 06-320 G.J.S. 05-455 C G.J.S. 06-327 G.J.S. 04-305 G.J.S. 04-306 G.J.S. 04-214 G.J.S. 07-91 G.J.S. 07-88 G.J.S. 07-102 G.J.S. 07-108 G.J.S. 00-110 G.J.S. 00-141 G.J.S. 00-108 G.J.S. 03-47 G.J.S. 00-107 G.J.S. 03-50 G.J.S. 02-09 G.J.S. 07-76 G.J.S. 01-97 G.J.S. 01-95 G.J.S. 03-76 G.J.S. 03-135 G.J.S. 01-93 G.J.S. 01-91 G.J.S. 97-180 G.J.S. 97-181 DIS 183A G.J.S. 00-132 G.J.S. 00-91 G.J.S. 97-179 DIS 185C G.J.S. 00-93 G.J.S. 00-127 G.J.S. 07-119 G.J.S. 07-89 T. stromaticum G.J.S. 07-95 G.J.S. 07-80 G.J.S. 07-79 G.J.S. 07-78 G.J.S. 07-77 G.J.S. 07-120 G.J.S. 00-130 DIS 185A TVC G.J.S. 04-331 G.J.S. 01-92 G.J.S. 07-118 G.J.S. 07-94 G.J.S. 00-102 G.J.S. 00-02 D G.J.S. 03-134 G.J.S. 01-174 G.J.S. 01-176 F PPRI 3559 G.J.S. 01-171 G.J.S. 01-166 E G.J.S. 01-225 G.J.S.04-308 DAOM 230008 G G.J.S. 01-238 G.J.S. 01-312 G.J.S. 07-72 G.J.S. 98-89 DAOM 230011 T. rossicum DAOM 230010 G.J.S. 01-287 DIS 162 1 change Fig. 1 (continued) 226 Mycol Progress (2012) 11:215–254

G.J.S. 97-180 MLS G.J.S. 97-181 DIS 183A G.J.S. 00-132 G.J.S. 00-91 G.J.S. 97-179 G.J.S. 00-93 G.J.S. 00-127 G.J.S. 07-119 G.J.S. 07-89 G.J.S. 07-95 G.J.S. 07-80 G.J.S. 07-79 G.J.S. 07-78 G.J.S. 07-77 G.J.S. 07-120 G.J.S. 00-130 DIS 185A TVC A G.J.S. 04-331 G.J.S. 01-92 G.J.S. 07-118 G.J.S. 04-327 G.J.S. 04-190 G.J.S. 06-320 G.J.S. 05-455 G.J.S. 06-327 C G.J.S. 04-305 G.J.S. 04-306 DIS 185C T. stromaticum G.J.S. 04-214 G.J.S. 07-94 G.J.S. 07-102 G.J.S. 00-02 D G.J.S. 03-134 G.J.S. 07-91 G.J.S. 07-88 G.J.S. 07-102 G.J.S. 07-108 G.J.S. 00-110 G.J.S. 00-141 G.J.S. 00-108 G.J.S. 03-47 B G.J.S. 00-107 G.J.S. 03-50 G.J.S. 02-09 G.J.S. 07-76 G.J.S. 01-97 G.J.S. 01-95 G.J.S. 03-76 G.J.S. 03-135 G.J.S. 01-93 G.J.S. 01-91 G.J.S. 01-171 G.J.S. 01-166 T. medusae E G.J.S. 01-225 T. caesarium G.J.S. 01-174 G.J.S. 01-176 T. lanuginosum F PPRI 3559 T. vermipilum G.J.S. 01-312 T. ivoriense G.J.S. 01-238 T. floccosum G.J.S. 07-72 G.J.S. 98-89 DAOM 230011 T. rossicum DAOM 230010 G.J.S. 04-308 DAOM 230008 T. barbatum G G.J.S. 01-287 DIS 162 5 changes Fig. 1 (continued)

De Souza et al (2006) and Loguercio et al. (2009b) Moniliophthora perniciosa) than members of Group 1. distinguished two groups within T. stromaticum based on Loguercio et al. (2009b) observed that generally members AFLP patterns; Group 1 corresponds to Clade B (Fig 1) of Group 1 were able to sporulate better in the tree canopy here, and Group 2 corresponds to Clade A. They attributed than members of Group 2, and this increased sporulation biological and ecological differences to the respective was correlated with lower incidence of M. perniciosa. groups. We have included in this study several of the When representatives of the two groups were experimen- strains studied by de Souza et al (Table 1). Members of tally inoculated into cacao trees in the field, members of Group 2 sporulated more profusely on rice and pieces of both groups were able to develop an endophytic relation- ‘dry broom’ (dead, proliferated branches of cacao that ship in sapwood of the trees, but Group 2 members were formed following infection of meristematic tissue by recovered up to 120 days after inoculation whereas Group 1 Mycol Progress (2012) 11:215–254 227

members were not reisolated (de Souza et al. 2008). Loguercio et al. (2009a) found that in the field Group 1 DAOM 230008 members more effectively reduced survival of M. perniciosa in hanging brooms than did members of Group 2. They concluded that, allowing for strain differences, overall members of Group 1 were more effective biological control G.J.S. 04-308 agents than Group 2 members. We observed the following differences between the phylogenetic groups corresponding to AFLP Groups 1 and 2: conidial pustules of Group 1 are

DAOM 230011 slightly larger (95% ci 0.5−0.6 vs 0.3−0.4mm)andhairs arising from pustules of Group 1 are slightly longer (105−113 vs 80−85 μm). De Souza et al. (2006) reported longer conidia inGroup2thaninGroup1butaftermeasuring700and600

G.J.S. 01-238 conidia from the respective collections, we did not observe any difference in conidial length. To add to these differences, we have observed perithecia only for Clade A of T. stromaticum and a subset of subclade A, subclade C (Fig 1), 01-312 only includes strains from Peru and Ecuador. Ascospores of subclade C are longer and narrower than in subclade A and members of subclade C were isolated from the pseudostroma

strains from different clades of M. roreri, cause of Frosty Pod Rot of cacao (Evans et al.

PPRI 3559 G.J.S. 2003a), in addition to M. perniciosa. Can the two AFLP groups be recognized taxonomically? Although in de Souza et al. (2006) and Loguercio et al. Trichoderma (2009b), AFLP groups 1 (clade B here) and 2 (clade A here) are biologically distinct, and perithecia have not been G.J.S. 01-176 found for one of those groups (Group 1, Clade A) multilocus phylogenetic data (Fig. 1e) support this clustering only weakly (Clade A is highly supported, but not clade B), and De Respinis et al. (2010) did not observe any

G.J.S. 01-174 differences between the groups in MALDI-TOF MS analysis of the proteome. Various studies have discussed comparisons and reliability of AFLP data versus phylogenetic (Meudt and Clarke 2007; Althoff et al. 2007). Many phylogeneticists are skeptical about the reliability of AFLPs G.J.S. 01-225 for phylogenetic inferences because of homology issues (Althoff et al. 2007; Bussell et al. 2005). Because AFLP fragments are anonymous and sorted by size, there is a

G.J.S. 01-166 potential for homoplasy of fragments, producing different results from those of phylogenetic analyses. In contrast, other authors conclude that AFLP data may contain dependable phylogenetic signal and is best for examining G.J.S. 01-171 within species relationships such as phylogeographic Species patterns or genotyping, or to study recently diverged species (Meudt and Clarke 2007; Althoff et al. 2007). - distance) expressed as number of nucleotide differences per site between p Trichoderma stromaticum Clades A and B are possibly in

Trichoderma stromaticumG.J.S. medusae05-455 medusae caesariatum lanuginosum lanuginosum vermipilum ivoriense floccosum rossicum barbatum barbatum the process of speciation. In support of this, we examined whether there was genetic exchange among the various clades of the species. When only the combined four-gene dataset for T. stromaticum was used, partition homogeneity ITS uncorrected ( test provided no evidence for heterogeneity of the data at the level of P=0.1, and the PHI test, which is used for G.J.S. 01-171G.J.S. 01-166 0.0071 G.J.S. 01-225 0.0071G.J.S. 01-174 0.0035G.J.S. 01-176 0.0053PPRI 3559 0 0.0053G.J.S. 01-312 0.0035G.J.S. 01-238 0.0018 0.0070 0.0088DAOM 0.0035 230011 0.00178 0.011G.J.S. 0.0018 04-308 0.0141 0.0018DAOM 230008 0 0.0070 0.0018 0.0141 0.014 0.0018 0.0124 0.012 0.0070 0 0.012 0.012 0.0070 0.012 0 0.012 0.0089 0.012 0.012 0.012 0.0088 0.0035 0.012 0.0106 0.012 0.0141 0.0088 0.0018 0.0141 0.0107 0.0141 0.0141 0.0070 0.0018 0.0141 0.01241 0.0141 0.012 0.0071 0.012 0.012 0.0035 0.0070 0.0070 0.0070 0.0106 0.012 0.0035 0.0035 0 Strain no. G.J.S. 05-455 Table 4 detection of recombination in the data, was also insignif- 228 Mycol Progress (2012) 11:215–254 icant. These results suggest the absence of recombination Fig. 2 Colonies of Trichoderma species grown on PDA (above) and b and hence incipient isolation. Various authors hypothesize SNA (below) under light for 1 week at 25°C unless noted otherwise. a T. barbatum (G.J.S. 04-208, 10 days). b T. floccosum (G.J.S. 01-238). that even if isolation and speciation is effective immediate- c T. ivoriense (G.J.S. 01-312). d T. lanuginosum (G.J.S. 01-176). e T. ly, the time required for evolutionary changes to appear in medusae (G.J.S. 01-171). fT. rossicum (DAOM 230011, 96 h). g T. two distinct lineages might not be enough to allow for their vermipilum (PPRI 3559, 10 days). h−j T. stromaticum showing recognition (Knowles and Carstens 2007; O'Meara 2010). variation in groups. h Clade A (Fig 1e, G.J.S. 07-76). i Clade B (Fig 1e, G.J.S. 07-77). j Clade C (Fig 1e, G.J.S. 05-455) As is the case of several species of Trichoderma (e.g., T. harzianum, T. hamatum, T. viridescens, T. asperelloides, etc.), species radiation seems to be an active and rapid clades differ in characters of the anamorphs and growth rates. process (Chaverri et al. 2003; Druzhinina et al. 2010), and Conidial pustules of the two members of Clade F (Fig 9a−c; T. stromaticum is just another example. Using the two see Trichoderma lanuginosum, below) are conspicuously criteria of Dettman et al. (2003), the phylogenetic data do not wooly whereas pustules in Clade E produce more discrete support division of T. stromaticum into two phylogenetically sterile hairs (Fig 11b, c; see Trichoderma medusae,below). distinct lineages. According to the genealogical concordance Conidia of Clade E are somewhat longer and narrower (95% criterion, the subclades must exist in the majority of single ci 4.5−4.7×2.7−2.8 μm, L/W=1.7−1.8) than conidia of gene trees if they are to be considered distinct. Here, we Clade F (95% ci 3.8−4.0×2.5−2.7, L/W=1.5−1.6). A observed the division of T. stromaticum into two clades in verticillium-like synanamorph formed in SNA cultures of only one single gene tree (rpb2) and thus the phylogeny of Clade F (Fig 9g−j; see Trichoderma lanuginosum,below) the individual gene trees does not fulfill this criterion. while Clade E lacks a synanamorph. Ascospores of Clade E According to the genealogical non-disconcordance criterion, are larger than those of Clade F. Finally, cultures of Clade E the clades must be supported in at least one individual gene grow much faster on PDA and SNA at 30°C than cultures of tree and not be contradicted in any other single locus tree at Clade F. We recognize Clade E below as the new species T. the same level of support. The two clades of the rpb2 tree medusae and Clade F as T. lanuginosum. (A, B) did not exist with support in any other trees. Members of Clade G cannot be separated from T. Moreover this result was contradicted in the cal tree, in rossicum on the basis of their microscopic morphology, which there are two additional supported subclades that do and actually strain DAOM 230008 was identified as T. not match the clades of the rpb2 tree; therefore, the rossicum in Bissett et al. (2003). However, the two nondisconcordance criterion was not met either. available cultures of Clade G grow considerably faster than While biological and AFLP data suggest the existence of any of the six cultures of T. rossicum that we have studied, more than one taxon in T. stromaticum, the lack of differences the difference especially noticeable on PDA and SNA at in the sequences of peptides in theproteome(DeRespinisetal. 30°C. Despite the similarity in their phenotypes, T. 2010) and the multilocus phylogenetic analysis lead us to rossicum and T. barbatum are phylogenetically distinct conclude that T. stromaticum is undergoing speciation, but we and Clade G is described as a new species, T. barbatum. cannot recognize taxonomic partition of the species at this time. These two species are sympatric in central Europe and are All of the members of the Stromaticum/Rossicum clade known from temperate regions. produce hairs from their conidial pustules and there are Four single strain lineages (PPRI 3359, G.J.S. 01-225, G.J. essentially two distinct morphologies among these hairs. In T. S. 01-238, G.J.S. 01-312) cluster independently in the stromaticum, the hairs are more or less awl-shaped, relatively Stromaticum/Rossicum Clade and are recognized here as short and often produce phialides from their tips. Trichoderma distinct taxonomic species. The closest relationship of G.J.S. stromaticum is further distinguished by the anatomy of its 01-225 is with T. medusae (Fig. 1e) but it differs from that pustules, which are formed of broad, short hyphae. Most of speciesinitsmuchfastergrowthrateonPDAandSNA;itis the remaining members of the clade produce more or less described as T. caesareum. PPRI 3559 is closely related to T. wooly pustules and the hairs are relatively long; they tend to lanuginosum but differs from that species in its larger conidia be variously coiled or sinuous and most often are sterile, the and faster growth rate; PPRI 3559 is described as T. exception being strains of Clade F (Fig 1). Each of the clades vermipilum. Two single strain lineages, G.J.S. 01-312 and representedinFig1e, including the single strains G.J.S. G.J.S. 01-238 do not have any close relationships within the 01-225, G.J.S. 01-312, G.J.S. 01-238 and PPRI 3359 represents Stromaticum/Rossicum Clade but they are morphologically a distinct species. These species are distinguished as follows. consistent with the other members of the clade in anamorph The two strains comprising each of the Clades E and F were and teleomorph (G.J.S. 01-238) characters. Strain G.J.S. derived from Hypocrea specimens collected on decorticated 01-312, isolated from soil in Côte d’Ivoire, is distinguished wood in close proximity to each other in a single more or by its small conidia, the smallest conidia in the group, and is less undisturbed Cameroonian forest. The Hypocrea tele- described as T. ivoriense. Despite the relative phylogenetic omorphs of these collections were indistinguishable but the isolation of strain G.J.S. 01-238, an ascospore isolate from the Mycol Progress (2012) 11:215–254 229 230 Mycol Progress (2012) 11:215–254

Fig. 3 Trichoderma barbatum. a−c Pustules on CMD. d−f Hairs on conidial pustules. g−i Conidiophores arising at the base of sterile hairs. j Phialides. k Conidia. All from G.J.S. 04-308. Scale bars (a) 1 mm, (b, c) 0.5 mm, (d) 100 μm, (e−h)20μm, (i−k)10μm Mycol Progress (2012) 11:215–254 231 same Thai forest as T. caesareum, no single character of its found in the eastern Brazilian states of Bahia and Pará. The anamorph or teleomorph distinguishes it from other species in unresolved Clade A (Fig. 1), which is equivalent to AFLP the Stromaticum/Rossicum Clade. However, its rate of growth Group 1 of de Souza et al. (2006), includes strains almost on SNA at 30°C is most similar to Andean collections of T. exclusively from the Bahia, exceptions being one strain stromaticum, T. barbatum, and T. ivoriense. It can be from the state of Pará (G.J.S. 00-102) and one strain from distinguished from T. stromaticum on the basis of its Colombia (AM13 of de Souza et al. 2006=G.J.S. 00-02). morphology; geography distinguishes it from the north Clade B, Group 2 of de Souza et al. (2006), includes a temperate T. barbatum, and it has larger conidia than T. mixture of strains from Bahia and Pará. Clade C, which is ivoriense. The strain G.J.S. 01-238 is named below as T. resolved in cal, tef1 and MLS (Fig 1c,d, and e, respectively) floccosum. is known only from Amazonian Peru and Ecuador. This clade includes the only known strains that occur on both Biogeography cacao pathogens Moniliophthora perniciosa and M. roreri. All others are found on M. perniciosa; T. stromaticum is Trichoderma stromaticum is known conclusively only from isolated rarely as an endophyte from sapwood of cacao. The Latin America. The greatest diversity of the species is closest relatives of T. stromaticum are strains collected in,

Fig. 4 Trichoderma caesareum. a, b Pustules from SNA. The wooly Fertile branch with phialides. g Conidia. All from G.J.S. 01-225. Scale nature of the pustule is evident in (b). c, d Hairs arising from the bars (a) 1 mm, (b) 0.5 mm, (c) 100 μm, (d, e)20μm, (f, g)10μm pustule. e Fertile branches arising from the base of a sterile hair. f 232 Mycol Progress (2012) 11:215–254 Mycol Progress (2012) 11:215–254 233

R Fig. 5 Trichoderma caesareum, Hypocrea teleomorph. a, b Stromata. protruding white hairs. Hairs extending beyond the c Surface of the stroma. d Section through a stroma showing four perithecia in median longitudinal section. e Stroma surface showing surface of the pustule, sinuous tending to spiraled, perithecial ostiolar canal. f Stroma surface in section. g Cells of the septate, smooth, infrequently branched, base ca. 5 μm interior of a stroma below perithecia. h Asci. i Ascospores. All from diam, tip subacute to acute, sterile. Fertile branches G.J.S. 01-225. Scale bars (a) 1 mm, (b) 0.5 mm, (c, e−h)20μm, (d) arising from near the base of the hairs, paired or 200 μm, (i)10μm solitary, typically a few cells long, but longer with distance from the tip, ca. 5 μm wide, producing phialides directly or producing unicellular 2º branches respectively, West (Côte d’Ivoire), Central (Cameroon), and bearing phialides. Phialides flask-shaped, (3.7−)5.0−7.5 South (Republic of South Africa) Africa, and Thailand. (−10.0) μm long, (2.7−)3.2−4.2(−5.0) μmatthewidest Somewhat more distantly related are T. rossicum and T. point, (1.7−)2.2−3.5(−4.2) μm at the base, L/W=(1.1−) barbatum, both found in north temperate regions (Siberia, 1.3−2.1(−3.3), arising from a cell (2.7−)3.5−4.5(−5.0) μm Austria, Andean Peru, USA: Michigan). Trichoderma diam, arising from 1º and 2º branches, solitary, paired or in lanuginosum and T. medusae were collected as Hypocrea dense botryose heads of several. Conidia (n=152) oblong to specimens in close proximity in a more or less undisturbed narrowly ellipsoidal, (4.0−)4.2−5.5(−9.5)×(2.2−)2.5−3.5 Cameroonian rainforest (Reserve Faunal du Dja), and T. (−6.5) μm, L/W=(1.4−)1.5−1.9(−2.0) (95% ci=4.8−5.0× caesareum and T. floccosum were collected as Hypocrea 2.9−3.1 μm, L/W=1.6−1.7), smooth. Chlamydospores not specimens in an undisturbed forest in southern Thailand observed on CMD or SNA within 10 days. (Khao Yai National Park). Trichoderma rossicum and T. barbatum are sympatric in soil in Austria and are only Etymology ‘Barbatum ’ from Latin ‘barbatus’ meaning known from soil collected in North temperate regions of bearded, with reference to the long hairs arising from the Asia (Siberia), Europe (Austria) and the United States pustules. (Michigan, T. barbatum). Trichoderma rossicum has previously been known only from Siberia (Bissett et al. 2003) Habitat Soil, roots of strawberry (Fragaria). and Andean Peru (Hoyos-Carvajal et al. 2009); strain DAOM 230008 was originally identified as T. rossicum Known distribution USA (Michigan), Russia (Siberia). (Bissett et al. 2003). Holotype USA, Michigan, Michigan State University, Horticulture Farm, isolated from roots of strawberry, date Descriptions of the species unknown, R. Olatinwo and A. Schilder T-10 (BPI 881029 G.J.S. 04-308; holotype designated herewith). Live Trichoderma barbatum Samuels, sp. nov. ex-type culture CBS 125733. Figs. 2a and 3 Additional material examined Russia, Siberia, Krasnoyarsk Trichodermati rossici simile sed in agaris dictis PDA vel region, isolated from soil under apple, Oct 1997, G. SNA temperatura 30°C magis celeriter crescens. Pustulae Szakacs, TUB F698 (Culture DAOM 230008). conidiales lanosae; conidia anguste ellipsoidea, (4.0−)4.2−5.0 (−5.7)×(2.2−)2.5−3.5(−6.5) μm, LW=(1.4−)1.5−1.9(−2.0). Comments The culture DAOM 230008 (98-90) was con- Holotype BPI 881029, designated here sidered to be T. rossicum by Bissett et al. (2003). Mycobank 519539 The ex-type strain (T-10) of T. barbatum was originally identified as T. stromaticum by Olatinwo et al. Telemorph None known (2004) because of a similarity between the two species in their ITS sequences. These authors isolated the strain Characteristics in culture Optimum temperature for from healthy strawberry roots. It was found to reduce growth on PDA and SNA at 25−30°C. Colony radius after severity of root lesions caused by Rhizoctonia fragariae 96 h on PDA at 25−30°C 60−68 mm, on SNA 44−55 mm as compared to untreated control. When applied as a root (n=2 cultures). Not growing at 35°C. After 10 days at 25°C dip to strawberry transplants at two sites in Michigan, the under light on PDA forming a continuous lawn of conidia, total fruit weight and number of berries the following K&W 28D−F8 (Deep Green, Dark Green), no distinctive season was significantly greater in treated plants than odor or diffusing pigment; on SNA pustules forming in untreated plants or plants treated with PlantShield (T. abundance around the periphery of the colony. Pustules harzianum) at one of the sites. Olatinwo et al. (2004) pulvinate to hemispherical, 0.5−1 mm diam, easily suggested that pre-planting treatment with T-10 might be removed from the agar, gray green, with abundant beneficial to strawberries. 234 Mycol Progress (2012) 11:215–254

Trichoderma caesareum Samuels, sp. nov. Figs. 4 and 5 walled, lacking long hyphal elements, (4−)7−14(−18)×(4−) 5−8(−10) μm. Asci cylindrical, (77−)80−92(−103)×(4.0−) Pustulae conidiales lanosae; conidia (4.0−)4.5−5.2 4.7−6.2(−7.5) μm(n=30), apex with a conspicuous dis- (−6.0)×(2.0−)2.5−3.0, L/W=1.5−2.1(−2.4). Trichodermati charge ring, ascospores uniseriate. Part ascospores hyaline, medusae Samuels simile sed in agaris dictis PDA vel SNA conspicuously warted, dimorphic; distal part subglobose or magis celeriter crescens. conical, 4.0−5.0(−5.2)×(3.5−)3.7−4.2(−4.5) μm; proximal Holotype BPI 863896, designated here part oblong to wedge-shaped or ellipsoidal, (3.7−)4.2(−5.0 Mycobank 519540 (−5.5)×(2.7−)3.0−3.5(−4.0) μm.

Telemorph Hypocrea sp. Etymology ‘caesareum’ from Latin, in reference to the long hairs arising from the conidial pustule. Characteristics in culture Optimum temperature for growth on PDA and SNA 25°C, colony radius after 96 h on PDA Habitat Trichoderma caesareum is known only from 65−70 mm, on SNA 35−50 mm. Typically not growing at cultures derived from ascospores of one Hypocrea speci- 35°C. On SNA after 1 week at 25°C pustules forming in men collected in primary forest; the Hypocrea develops on abundance throughout the colony. Pustules conspicuous, bark. hemispherical, 1−2 mm diam, grayish green (K&W 28D5), with abundant protruding white hairs largely obscuring the Known distribution Thailand, known only from the original mass of conidia. Hairs extending beyond the surface of the collection. pustule, sinuous to spiraled or straight, septate, smooth or warted, infrequently branched, base ca. 5 μm wide, tip Holotype Thailand, Prachinburi Province, Khao Yai National subacute. Fertile branches arising from near the base of Park, along trail betweern Khlong E-Thao (14º28′N, 101º20′ the hairs, typically 1 or few cells in length, longer with E, elev. 750 m) and 14º28′N, 101º12′E, elev. 800 m, on bark, distance from the tip and producing unicellular 2º 18 Aug 201, G.J.S. 9074, R. Nasit (BPI 863896, a dry culture branches, ca. 5 μm wide. Phialides doliform or flask- ex-ascospore isolation). Live ex-type culture G.J.S. 01-225= shaped, (3.7−)4.7−6.2(−7.2) μm long, (3.0−)3.5−4.0 CBS 124369). (−4.2) μm at the widest point, L/W=(1.2−)1.3−1.8 (−2.1), (1.5−)2.0−3.2) μm at the base, arising directly Trichoderma floccosum Samuels, sp. nov. from a cell (3.0−)3.2−4.5(−5.0) μm wide and terminating Figs. 2b, 6 and 7 1º and 2º fertile branches; all branches terminating in 1 to several densely clustered phialides. Conidia (n=30) oblong, Trichodermati medusae Samuels simile sed in agaris (4.0−)4.5−5.2( −6.0)×(2.0−)2.5−3.0 μm, L/W=1.5−2.1(−2.4) dictis PDA vel SNA temperatura 30°C magis celeriter (95% ci=4.5−5.0×2.6−2.8 μm,L/W=1.7−1.9), smooth, gray crescens. Pustulae conidiales lanosae. Conidia ellipsoidea green. Chlamydospores not observed. (3.5−)4.0−5.0(−5.5)×(2.5−)2.7−3.5(−3.7) μ m, L/W=(1.3−) 1.5−1.7(−1.9). Characteristics of the teleomorph Stromata scattered, gre- Holotype BPI 871616, designated here garious, discoidal, ca. 1 mm diam, broadly attached, hyphae Mycobank 519541 not visible, surface plane to convex, perithecial elevations appearing as low tubercles, perithecial openings appearing as Telemorph Hypocrea sp. darker dots against the surrounding tissue, yellowish brown to brown, not reacting to 3% KOH. Cells of the stroma surface in Characteristics in culture Optimum temperature for growth face view pseudoparenchymatous, elliptical in outline, 8−12 on PDA 25°C, on SNA 25−30°C. Colony radius after 96 h (−15) μm diam, thin-walled. Perithecia circular in section, on PDA at 25°C ca. 60 mm, on SNA at 25/30°C 40−45 (n=10) (225−)230−250(−265) μm high, 150−185(−200) μm (−60) mm. Typically not growing at 35ºC. After 10 days at wide, ostiolar canal 75−90 μm long. Perithecial papilla 25°C under light on PDA pustules forming in abundance formed of small cells, clavate elements lacking. Surface around the margin of the colony; on SNA few large region distinguished from the internal tissue of the stromata pustules forming at the margin of the colony. On SNA by pigmentation in the outermost 2−3 layers of cells; cells of pustules conspicuous, pulvinate to nearly hemispherical, the stroma surface in section pseudoparenchymatous, 1−2 mm diam, gray green with abundant protruding white (5−)7−10(−12) μm diam, thin-walled. Tissue below the hairs largely obscuring the mass of conidia. Hairs extending stroma surface, between perithecia pseudoparenchymatous, beyond the surface of the pustule, sinuous; septate; smooth, thin-walled, lacking hyphal elements. Tissue of the stroma infrequently branched, base ca. 5 μm diam, tip subacute, below perithecia, perithecia textura epidermoidea, thin- sterile. Fertile branches arising from near the base of the Mycol Progress (2012) 11:215–254 235

Fig. 6 Trichoderma floccosum. a, b Pustules on SNA. The wooly seen in (c) and (f). f Sterile hair. g Fertile branch with phialides. nature of the pustule is evident in (b). c−f Sterile hairs arising from the h conidia. All from G.J.S. 01-238. Scale bars (a) 1 mm, (b) 0.5 mm, pustules. Fertile branches arising from the base of the hairs can be (c−f)20μm, (g, h)10μm 236 Mycol Progress (2012) 11:215–254 Mycol Progress (2012) 11:215–254 237

R Fig. 7 Trichoderma floccosum, Hypocrea teleomorph. a, b Stromata. Habitat Trichoderma floccosum is known only from c Surface of the stroma showing ostiolar openings. d Section through a stroma showing several perithecia in median, longitudinal section. e cultures derived from ascospores of an unnamed Hypocrea Cells of the stroma surface in section. f Stroma surface region showing species collected in primary forest; the Hypocrea develops the ostiolar opening of a perithecium. g Cells of the interior of a on decorticated wood. stroma below perithecia. i Asci. j Ascospores. All from G.J.S. 01-238. Scale bars (a, b) 1 mm, (c, e−g)20μm, (d) 200 μm, (i, j)10 μm Known distribution Thailand, known only from the original collection.

hairs, typically 1 or few cells in length, longer with Holotype Thailand, Nakhorn Nayok Province, Khao Yai distance from the tip, ca. 5 μmwide,producing2º National Park, Mo Sing To trail from shops at park branches. Phialides flask-shaped, (3.7−)4.7−6.2(−7.2) μm headquarters, on decorticated wood, 4 Sep 2001, G.J.S. long, (3.2−)3.5−4.5(−4.7) μm at the widest point, L/W= 9202, R. Nasit (BPI 871616, a dry culture ex-ascospore (1.3−)1.5−1.7(−1.9), base (1.2−)2.2−3.5(−4.0), arising isolation). Live ex-type culture G.J.S. 01-238=CBS 124372. from a cell (3.2−)3.7−4.7(−5.0) μm wide; forming directly from and terminating 1º and 2º fertile branches; Trichoderma ivoriense Samuels, sp. nov. Figs. 2c and 8 all branches terminating in 1 to several densely clustered phialides. Conidia (n=30) ellipsoidal, (3.5−)4.0−5.0 Pustulae conidiogenae lanosae, griseo-virides, pilei ex (−5.5)×(2.5−)2.7−3.5(−3.7) μm, L/W=(1.3−)1.5−1.7 pustulis conidiogenis exorientes, steriles, sinuosi; conidia (−1.9) (95% ci=4.4−4.7×2.9−3.1 μm, L/W 1.5−1.6), ellipsoidea, (3.0−)3.2−3.7(−4.0)×(2.0−)2.2−2.7 μm, L/W= smooth, gray green. Chlamydospores abundant on CMD and (1.1−)1.2−1.6(−1.7). SNA reverse, terminal and intercalary, subglobose, (6−)7−17 Holotype BPI 881030, designated here (−30) μmdiam. Mycobank 519542

Characteristics of the teleomorph Stromata scattered, Characteristics in culture Optimum temperature for growth gregarious, discoidal, 1−1.5 mm diam, broadly attached, on PDA and SNA 25°C, on PDA colony radius ca. 60 mm hyphae not visible, surface plane to convex, perithecial after 96 h at 25°C, on SNA colony radius ca. 50 mm. Not elevations appearing as low tubercles, perithecial open- growing at 35°C. On PDA and CMD after 10 days at 25°C ings appearing as darker dots against the surrounding under light colonies producing a continuous layer of white tissue, yellowish brown to brown, not reacting to 3% mycelium, no pigment diffusing through the agar, no KOH. Cells of the stroma surface in face view distinctive odor; conidia forming throughout the colony on pseudoparenchymatous, elliptical in outline, (10−)13−21 macronematous, mononematous conidiophores, conidia yel- (−25)×(8−)11−17(−19) μm, thin walled. Perithecia cir- lowish green (K&W 29D7). On SNA conidia forming in cular to elliptic in section, (n=12) (228−)240−285(−300) pustules dispersed in a 2−3 cm broad band around the margin μm high, (143−)165−205(−212) μm high, ostiolar canal of a 9-cm-diam Petri plate; mononematous conidiophores not 70−100(−124) μm long. Perithecial papilla formed of observed. Pustules 0.5−1 mm diam, yellowish green (K&W small cells, clavate elements lacking. Surface region 28D−E7), hemispherical, remaining discrete; long, sterile, distinguished from the internal tissue of the stromata by sinuous to loosely coiled, white hairs arising in abundance pigmentation in the outermost 2−3 layers of cells; cells of from each pustule. Conidiophores in pustules comprising a the stroma surface in section pseudoparenchymatous, (6−) long, sterile hair with short fertile branches arising from its 8−14(−16)×(5−)7−11(−12) μm, thin-walled. Tissue of the base. Sterile hairs tapering from 5−7 μm at the base to an acute stroma below perithecia, perithecia textura epidermoidea, tip, and producing shorter sterile branches, often roughened, lacking long hyphal elements, thin-walled, (4−)9−17 septate, tending to coil near the tip. Fertile branches arising at (−21)×(3−)5−9(−12) μm. Asci cylindrical, (n=30), (89−) right angles from the base of the hairs, typically comprising a 94−112(−124)×(4.5−)5.5−6.7(−7.5) μm, ascospores uni- single basal cell with phialides clustered at its tip or producing seriate, apex with a conspicuous discharge ring. Part 2º cells, each with terminal phialides; branches more distant ascospores spinose, hyaline, dimorphic; distal part sub- from the tip 2 or 3 cells long. Phialides doliform, (3.7−) globose or conical, (4.0−)4.5−5.2( −6.0)×(3.5−)3.7−4.5 4.7−6.0(−7.5) μm long, (3.0−)3.2−4.0(−4.5) μm at the widest (−4.7) μm; proximal part oblong to wedge-shaped or point, (1.7−)2.0−3.0(−5.0) μm at the base, L/W (1.1−)1.3−1.7 ellipsoidal, (3.5−)4.0−5.2( −6.2)×(2.7−)3.0−3.7(−4.0) μm. (−2.1) (n=30); arising from a cell (3.2−)3.5−4.5(−4.7) μm wide. Conidia (n=30) ellipsoidal, (3.0−)3.2−3.5(−4.0)× Etymology ‘floccosum’ from Latin, a lock or flock of wool (2.0−)2.2−2.5(−2.7) μm, L/W (1.1−)1.2−1.6(−1.7) (95% ci on something such as clothing or fruit; in this case, on the 3.3−3.5×2.5−2.7 μm, L/W 1.5−1.6). On CMD a gliocladium/ pustule. verticillium-like synanamorph forming from erect hyphae, 238 Mycol Progress (2012) 11:215–254

Fig. 8 Trichoderma ivoriense. a, b Pustules on CMD (a) and SNA and phialides. g−i Synanamorph (CMD). j Conidia. k Chlamydospores. (b). c−e Sterile hairs. Fertile branches arising from the base of the All from G.J.S. 01-312. Scale bars (a)1mm,(b, g) 0.5 mm, (c−e, h, I, hairs seen in (d) and (e). Fertile branch arising from the base of a hair k)20μm, (f, j)10μm Mycol Progress (2012) 11:215–254 239

Fig. 9 Trichoderma lanuginosum. a−c Pustules formed on SNA. d h, j, k from G.J.S. 01-174; c, g, i, k from G.J.S. 01-176. Scale bars Sterile hairs arising from a pustule. e, f Hairs with fertile branches (a−c)0.5mm,(d−f, h, i)20μm, (g) 150 μm, (k)10μm arising from the base. g−j Synanamorph on SNA. k Conidia. a, b, d−f, 240 Mycol Progress (2012) 11:215–254

40−170 μmlong,4−6 μm wide at base, phialides 5−15 μm Fig. 10 Trichoderma lanuginosum, Hypocrea teleomorph. a−c b − − μ Stromata. d Cells at the stroma surface; a perithecial opening visible long; conidia ellipsoidal, 2.5 4.0×2.0 3.2 m, held in in the upper left corner. e Section through a stroma showing a glistening green heads. Chlamydospores abundant on SNA perithecium in median longitudinal section. f Surface of a stroma reverse, terminal, obovoidal to fusiform, (4.2−)5.2−8.0 showing a perithecial opening. g Cell of the stroma interior below a (−10.0)×(4.0−)4.7−5.2(−9.2) μm. perithecium. h−j Asci. A ring can be seen in the ascal apex in (i) and (j). k Discharged part ascospores. Images a, c−f, i−k from G.J.S. 01- 174; g, h, k from G.J.S. 01-176. Scale bars (a) 1 mm; (b, c) 0.5 mm, Etymology Named for the country of origin of the type, (d, f−h)20μm, (e) 200 μm, (i−k)10μm Côte d’Ivoire.

Habitat Soil. soidal, (n=60) (3.7−)4.0−6.5(−8.2)×(2.5−)2.7−4.2(−5.2) μm, with a protuberant, flat basal abscission scar. Pustules Known distribution Côte d’Ivoire, known only from the type. hemispherical, 0.25−0.5 mm diam, with abundant protruding white hairs, hairs largely obscuring the mass Holotype Côte d’Ivoire, isolated from soil, Oct 2001, I. of conidia. Hairs extending beyond the surface of the Kibbe 164 (BPI 881030). Live ex-type Culture: G.J.S. 01- pustule, sinuous to straight, only a slight tendency to 312=CBS 125734. coiled or spiraled, sterile, septate, smooth, infrequently branched, base 3.5−4.5 μm wide, tip blunt. Fertile Comments On the basis of morphology it would be very branches arising from near the base of the hairs, difficult to distinguish T. ivoriense from the only distantly typically 1 or few cells in length, longer with distance related T. spirale Bissett. The only known strain of T. from the tip and producing unicellular 2º branches, ivoriense is slower growing than T. spirale and produces far 3.5−4.5 μm wide. Phialides doliform to lageniform, fewer chlamydospores. Cultures of T spirale may produce (4.2−)5.2−7.2(−9.7) μm long, (3.0−)3.5−4.2(−4.5) μm only mononematous conidiophores similar to those found at the widest point, L/W (1.0−)1,4−2.0(−2.7), arising in PDA and CMD cultures of T. ivoriense. from a cell (3.2−)3.5−4.5−5.0) μm wide; arising directly from, and terminating 1º and 2º fertile branches; all branches Trichoderma lanuginosum Samuels,sp.nov.Figs.2d, 9 and 10 terminating in 1 to several densely clustered phialides. Conidia (n=60) ellipsoidal, (3.0−)3.5−4.2(−4.7)×(2.0−)2.2−3.0(−3.5) Trichodermati medusae Samuels simile sed conidia (3.0−) μm, L/W (1.2−)1.3−1.7(−2.0) (95% ci 3.8−4.0×2.5−2.7 μm, 3.5−4.2(−4.7)×(2.0−)2.2−3.0(−3.5) μm, L/W=(1.2−)1.37−1. L/W 1.5−1.6), smooth, yellow green. Chlamydospores not (−2.0) et in agaro dicto SNA synanamorphosis verticillio observed. simile formans. Holotype BPI 863853, designated here Characteristics of the teleomorph Stromata scattered, gre- Mycobank 519543 garious, discoidal, ca. 1 mm diam, broadly attached, hyphae not visible, surface plane to convex, perithecial elevations Telemorph Hypocrea sp. appearing as low tubercles, perithecial openings appearing as darker dots against the surrounding tissue or concolo- Characteristics in culture Optimum temperature for growth rous, yellowish brown to brown, not reacting to 3% KOH. on PDA 20−25°C, colony radius after 96 h 28−42 mm; on Cells of the stroma surface in face view pseudoparenchy- SNA 25°C, colony radius after 96 h 30−35 mm. Typically matous, elliptical in outline, (5−)8−15(−18) μm diam, thin- not growing at 35°C. On PDA after 1 week at 25°C under walled. Perithecia circular to elliptical in section, (n=15) light a continuous lawn of conidia forming in the aerial (188−)200−225(−240) μm high, (100−)115−160(−180) μm mycelium over the entire colony, margin sterile; pustules wide, ostiolar canal 50−75 μm long. Perithecial papilla forming after two weeks; conidia grayish yellow green (K&W formed of small cells, clavate elements lacking. Surface 28d6−7). Colony on SNA after 10 days under the same region distinguished from the internal tissue of the conditions producing mononematous conidiophores abun- stromata by pigmentation in the outermost 2−3layers dantly throughout the colony in the aerial mycelium; pustules of cells; cells of the stroma surface in section pseudo- just beginning to form. Mononematous conidiophores, (n= parenchymatous, (4−)5−9(−11)×4−8(−11) μm diam, thin- 38) 41−120(−187) μm long, (2.5−)3−5(−6.7) μmwideat walled. Tissue of the stroma below perithecia, perithecia base, verticillium-like, with 1−3 verticils of phialides and 2 textura epidermoidea to t. angularis, lacking long hyphal branches. Phialides awl-shaped, (n=60) (8−)12−21(−34) μm elements, cells thin-walled, (6−)10−20(−30)×(6−)8−15(−20) long, (2.0−)2.2−3.7(−4.5) μm wide at the base, arising μm. Asci cylindrical, (65−)70−85(−97)×(2.7−)3.7−5.2(−6.5) singly from the condiophore or in verticils of 2−4. μm, ascospores uniseriate, apex with a conspicuous discharge Conidia arising from mononematous conidiophores ellip- ring. Part ascospores dimorphic, spinose, hyaline; distal part Mycol Progress (2012) 11:215–254 241 242 Mycol Progress (2012) 11:215–254

Fig. 11 Trichoderma medusae. a−c Pustules from CMD. e Sterile h Conidia. Images a, c, e, h from G.J.S. 01-166; b, c, f, g from G.J. hairs arising from a pustule. d−f Hairs with fertile branches arising S. 01-171; b provided by D Farr. Scale bars (a, b)1mm,(c−e) from the base. g A fertile branch arising from the base of a hair. 20 μm, (f−h)10μm Mycol Progress (2012) 11:215–254 243

Fig. 12 Trichoderma medusae, Hypocrea teleomorph. a−c Stromata. apical ring can be seen in the apex of asci in i, j. Mature part d Cells of the stroma surface showing a perithecial opening. e Section ascospores. Images a, b, h−j from G.J.S. 01-166; c−g from G.J.S. 01- through a stroma showing median longitudinal sections through two 171. Scale bars (a, b) 1 mm, c 0.5 mm, (d, f, h)20μm, (e) 100 μm; perithecia. f Stroma surface region showing a perithecial opening. g (g, i, j)10μm Cells of the interior of a stroma below a perithecium. h, i Asci. An 244 Mycol Progress (2012) 11:215–254 Mycol Progress (2012) 11:215–254 245

R Fig. 13 Trichoderma rossicum. a, b Pustules from SNA. c, d Sterile hemispherical, 0.5−1 mm diam, with protruding hairs. hairs arising from pustules. e−g Sterile hairs with fertile branches Hairs white, protruding beyond the surface of the arising from near the base. h A fertile branch with phialides. g Conidia. All images from DAOM 230011. Scale bars (a) 1 mm, (b) pustule, sinuous to somewhat spiraled, especially 0.5 mm, (c−g)20μm, (h, i)10μm toward the tip, smooth, septate, tapering slightly from 3.5−4.5 μm at the base, tip blunt, unbranched or infrequently branched, sterile (rarely producing a single phialide). Fertile branches arising near the base of the hairs, subglobose or conical, (n=60) (3.2−)3.7−4.2(−4.7)×(2.7−) 1 to a few cells in length, longer with distance from the tip; 3.2−3.7(−4.0) μm; proximal part oblong to wedge-shaped or phialides arising directly from the main branch or unicellular ellipsoidal, (3.0−)3.7−4.7(−5.0)×(2.2−)2.5−3.2(−3.5) μm. 2º branches arising from the 1º fertile branches. Phialides subglobose to lageniform, (3.7−)4.7−6.7(−8.2) μm, (2.5−) Etymology ‘lanuginosum’ from Latin, in reference to the 3.5−4.5(−5.0)μm at the widest point, L/W (0.8−)1.1−1.7 hairs that appear as wooly down on the surface of the pustule. (−2.4), (1.7−)2.2−3.5(−4.5) μm wide at base, arising from a cell (2.2−)3.2−4.5(−6.0) μm wide; from arising directly from, Habitat Trichoderma lanuginosum is known only from and terminating 1º and 2º fertile branches; all branches cultures derived from ascospores of Hypocrea specimens; terminating in 1 to several densely clustered phialides. the Hypocrea teleomorph on rotting wood with bark. Conidia (n=60) ellipsoidal to nearly oblong, (3.7−)4.2−5.0 (−5.7)×(2.2−)2.5−3.0(−3.5) μm, L/W 1.5−1.9(−2.5) (95% ci Known distribution Cameroon, known only from the type 4.5−4.7×2.8−3.0 μm, L/W 1.−1.8), smooth, gray green. locality. Chlamydospores not observed.

Holotype Cameroon, Provinces du Sud et de l’Est, Dept. Characteristics of the teleomorph Stromata scattered to Haut-Nyong, Reserve Faunal du Dja, vic Somalomo, ca. gregarious, discoidal, ca. 1 mm diam, broadly attached, 30 km E of Somalomo, Mintoum, 1.5 h walk S in forest, in hyphae not visible, surface plane to slightly convex, primary forest, 03º18’N, 12º58’E, elev. 620 m, on bark, 13 perithecial elevations not evident or appearing as low Jul 2001, G.J.S. 9021, D. Begoude (BPI 863853, culture G. tubercules, perithecial openings appearing as darker dots J.S. 01-176=CBS 125718). against the surrounding tissue, yellowish brown to brown, not reacting to 3% KOH. Cells of the stroma surface in face Additional specimen examined Data as the holotype except view pseudoparenchymatous, (6−)8−12(−16) μmdiam, collected on bark of rotten wood, G.J.S. 9019, live culture thin-walled. Perithecia circular to elliptical in section, (n= 01-174=CBS 126100. 6) (185−)210−275 μm high, (137−)140−180(−185) μm wide, ostiolar canal 75−90 μm long. Perithecial papilla Trichoderma medusae Samuels sp. nov. Figs. 2e, 11 and 12 formed of small cells, clavate elements lacking. Surface region distinguished from the internal tissue of the stromata Trichodermati lanuginosi Samuels simile sed synana- by pigmentation in the outermost 2−3 layers of cells; cells morphosis verticillio simile abest et conidia (3.7−)4.2−5.0 of the stroma surface in section pseudoparenchymatous, (−5.7)×(2.2−)2.5−3.0(−3.5) μm , L/W 1.5−1.9(−2.5). (4−)5−9(−11) μm diam, thin-walled. Tissue of the stroma Holotype BPI 863841, designated here. below the surface pseudoparenchymatous, lacking hyphal Mycobank 519544 elements. Tissue of the stroma below perithecia textura epidermoidea, lacking long hyphal elements, thin-walled, Telemorph Hypocrea sp. (6−)10−18(−24)×(6−)8−15(−21) μm. Asci cylindrical, (69−)80−104(−118)×(4.0−)5.7−6.5(−8.0) μm(n=60), Characteristics in culture Optimum temperature for ascospores uniseriate, apex with a conspicuous discharge growth on PDA 20−30º, on SNA 25−30°C; on PDA ring. Part ascospores dimorphic, spinose, hyaline; distal and SNA colony radius 25−35 mm after 96 h at 25°C. part subglobose or conical, (n=60) (3.7−)4.2−5.2(−6.2)× Typically not growing at 35°C. On PDA after 1 week (3.5−)4.0−4.5(−5.0); proximal part oblong to wedge- at 25°C under 12 h cool white fluorescent light/12 h shaped or ellipsoidal, (4.0−)5.0−6.0(−8.7)×(2.5−)3.2−4.0 darkness colony tan, margin scalloped or lobbed, (−4.7) μm. lacking diffusing pigment or distinctive odor; conidia forming in one or two nearly continuous rings around Etymology ‘medusae’ from Latin, in reference to the the original inoculum, grayish green (K&W 26c−d4); conidial pustules with ‘snake like’ hairs suggestive of on SNA conidial pustules abundantly forming in broad Medusa, a gorgon of mythology that has snake-like hairs concentric rings around the original inoculum. Pustules and turns beholders to stone. 246 Mycol Progress (2012) 11:215–254 Mycol Progress (2012) 11:215–254 247

R Fig. 14 Trichoderma stromaticum. a Pustules in nature on a dead Hairs sinuous, thin-walled, septate, smooth, infrequently cacao tissue infected with Moniliophthora perniciosa (‘dry broom’). − μ b−e Pustules on SNA (b, d), PDA (c)andCMD(d). f Tissue of a branched, base 5 7 m diam, tip subacute. Fertile pustule showing broad hyphae terminating in phialides (on the left). branches arising from near the base of the hairs, paired g−1 Awl-like hairs arising from a pustule with fertile branches or solitary, typically 1 or few cells in length, longer arising from near the base. The hair in (h)terminatesintwo with distance from the tip, 4−5 μm wide, producing phialides. j Phialides clustered on a fertile branch. k, l Synanamorph from SNA. Verticillium-like branching of the synanamorph seen in phialides directly, sometimes producing 2º branches (l). m Conidia. n, o Chlamydospores. Images a from G.J.S. 97-181; typically comprising a single cell. Phialides flask- b, G.J.S. 07-76; c, G.J.S. 01-92; d, G.J.S. 05-455; e, G.J.S. 04-327; shaped to subglobose with a short contricted neck, f, G.J.S. 03-140; g, i, G.J.S. 00-132; h, G.J.S. 04-301; j, l, G.J.S. 04- (4.5−)5.0−7.0(−7.7) μm long, (2.7−)3.5−4.5(−4.7) μm 190; k, G.J.S. 97-181; m, G.J.S. 03-134; n, o, G.J.S. 00-02. Scale − − − μ bars (a, b) 1 mm, (c)0.5mm,(d, e) 0.5 mm, (f−i, l, n, o)20μm at the widest point, (1.7 )2.2 3.2( 4.2) matthebase, L/W=(1.2−)1.3−1.9(−2.2), arising from a cell (3.5−)4.0−5.2 (−6.0) μm diam; arising directly from and terminating 1º and 2º fertile branches; all branches terminating in 1 to several densely clustered phialides. Conidia (n=120) oblong, (4.0−) Habitat Trichoderma medusae known only from cultures 4.2−5.0(−5.7)×(2.2−)2.7−3.0(−3.2) μm, LW (1.4−)1.5−1.7 derived from ascospores of Hypocrea specimens collected (−2.0)(95% ci 4.5−4.7×2.8−3.0 μm, LW 1.6−1.7), smooth. in primary forest; the Hypocrea teleomorph develops on Chlamydospores not observed (few chlamydospores ob- wood with bark. served in the protologue).

Known distribution Cameroon, known only from the type Habitat Soil locality. Known distribution Russia (Siberia), Austria, Peru (Puño Holotype Cameroon, Provinces du Sud et de l’Est, Dept. State). Haut-Nyong, Reserve Faunal du Dja (a World Heritage site), 6 km E of Dja River, in forest a 2-h quick walk south Holotype Russia, Siberia, Krasnoyarsk region, soil in apple from route to Bourneville, 03º17′N, 12º47′E, elev. 600 m, orchard, October 1997, G. Szakacs (DAOM 230011). Not on small branches, 14 Jul 2001, G.J.S. 9006, D. Begoude, A examined, ex-type culture DAOM 230011 studied. Guinwith, Pascal Togo (BPI 863841, a dry culture ex- ascospore isolation). Live ex-type culture G.J.S. 01-171= Additional material examined See Table 1. CBS 125719. Comments Bissett et al (2003) compared this species to the Additional collection Same collecting data as the holotype, unrelated species T. longipile Bissett (Bissett 1991), which except on termite infested wood, G.J.S. 9001 (BPI 863836; has somewhat broader conidia. It would be difficult to live culture G.J.S. 01-166). distinguish these species on the basis of their morphology alone. Trichoderma rossicum Bissett, Kubicek & Szakacs, Can J Culture Berg PR26-12-6 (=G.J.S. 07-72) is cited in Bot 81:578. 2003. Figs. 2f and 13 Grosch et al (2006,asT. viride) as being antagonistic to Rhizoctonia solani. Teleomorph not known. Trichoderma stromaticum Samuels & Pardo-Schultheiss, Characteristics in culture Optimum temperature for Mycol Res 104: 762. 2000. Figs. 2 h−j, 14 and 15 growth on PDA and SNA 25°C. On PDA colony radius after96hat25ºC56−65 mm, on SNA 36−40 mm; considerably slower at 30°C (in 96 h on PDA 12−25 Teleomorph Hypocrea stromatica Bezerra et al., Fitopatol mm, on SNA 12−18 mm, n=6 strains studied); not Brasiliera 28:409. 2003. growing at 35°C. After 10 days at 25°C under light on PDA a continuous lawn of conidiophores covers the Characteristics in culture Optimum temperature for growth Petri plate, conidia K&W 30 D−E 8 (Deep Green, on PDA 25°C. Colony radius after 96 h at 25°C on PDA (54−) Parrot Green), no distinctive odor or diffusing pigment 55−65(−68) mm, at 30°C 37−53(−61) mm; at 15°C 3−7(−16) noted; on SNA pustules forming in concentric rings, no mm, at 20°C 15−20(−31) mm (n=9 strains). On SNA synanamorph noted. Pustules 0.25−0.5 mm diam, optimum temperature for growth 25−30°C. After 96 h at hemispherical, discrete to confluent, easily removed 25 and 30°C on SNA (25−)30−42(−52) mm, slightly slower from the agar, with hairs arising abundantly from each. at 30°C than at 25°C; 20°C 15−20(−30) mm, at 15°C 5−7 248 Mycol Progress (2012) 11:215–254

(−15) mm. Colonies grown on PDA 1 week at 25 C under Fig. 15 Trichoderma stromaticum, Hypocrea stromatica teleomorph. b − a, b Stromata and conidial pustules on a dead cacao tissue infected light producing pustules abundantly in 2 4 concentric rings, with Moniliophthora perniciosa (‘dry broom’). c Cells of the stroma pustules in the center of the colony often with gold pigment. surface showing a perithecial opening. d Section through a stroma On SNA pustules forming abundantly in a broad marginal showing median longitudinal sections through several perithecia. e band, less typically pustules uniformly distributed through- Section through a stroma showing details of the stroma surface and a perithecium in median longitudinal section. f Section through the out the colony. Individual pustules on PDA and SNA surface region of a stroma showing the ostiolar canal of a perithecium. typically fully fertile within 2 weeks, easily dislodged g Cells of the interior of a stroma below a perithecium. h−j Asci and from the agar, on SNA 0.2−0.5(−1.0) mm diam; conidia part ascospores. A ring can be seen in the apex of asci in (i). Warted dark green but often yellow; hairs typically not visible ascospores are seen in (j). Images a, c from G.J.S. 04-300, b from G.J. S. 04-305; c−j from G.J.S. 04-300 on immature pustules, at maturity hairs arising from pustules, (45−)62−100(−135) μm long, (3.7−)4.7−6.7 (−10.0) μm wide at the base, stiff, erect, septate, thin- walled, sterile or producing a single terminal phialide. with M. perniciosa (‘dry brooms) or fruit bodies of M. Fertile branches arising at the base of hairs, typically perniciosa. Collections from Peru were found on the one or a few cells in length, often densely clustered, pseudostroma of M. roreri. Isolated rarely as an endophyte producing unicellular lateral branches; phialides termi- from trunk of Theobroma cacao. nating all branches, densely clustered. Phialides ampulli- form, (3.5−)5.3−7.7(−10.2) μm long, (1.0−)3.2−4.5 Known distribution Brazil (Bahia, Pará), Colombia, Ecuador, (−5.5) μm at the widest point, (1.7−)2.5−3.7(−4.2) μm Peru. at the base, arising from a cell (3.0−)3.7−5.0(−7.0 μm wide. Conidia (n=1,522) ellipsoidal, (3.0−)3.7−4.5 Holotype Brazil, Para, Belem, from dead cocoa broom, C. (−7.2)×(1.7−)2.5−3.0(−3.7) μm, LW (1.0−)1.4−1.8(−) 2.3 N. Bastos, TVC, G.J.S. 97-183 (BPI 746496; live cultures: (95% ci 4.3−4.4×2.80−2.82 μm, L/W 1.5−1.6), smooth. ATCC 204426, CBS 101875). Chlamydospores (4.2−)6.5−9.7(−13.5) μmdiam. Comments This species was fully described and illustrated Characteristics of the teleomorph Stromata dispersed in Samuels et al (2000). among conidiomata, scattered or gregarious, discoidal, Bezerra et al (2003) described Hypocrea stromatica 1.0−1.5 mm diam, broadly attached, hyphae not visible, Bezerra et al as the teleomorph of T. stromaticum. We have surface plain, perithecial elevations appearing as low observed perithecia in Clade B and C (Fig 1e) but not in tubercules, perithecial openings not visible, not reacting unresolved Clade A, which includes the ex-type culture to 3% KOH. Cells of the stromasurfaceinfaceview (TVC=G.J.S. 97-183). We have not studied the ex-type pseudoparenchymatous, elliptical in outline, (4.5−)9−17 culture of H. stromatica, which was collected in Pará State, (−20) μm diam, thin-walled. Perithecia circular in Brazil, but following our phylogenetic analysis it is likely section, (185−)210−260(−300) μm high, (100−)130−190 that it too will cluster in clade B. (−210) μm wide, ostiolar canal 60−90 μm long. Perithecial papilla formed of small cells, clavate elements Trichoderma vermipilum Samuels, sp. nov. Figs. 2g and 16 lacking. Surface region distinguished from the internal tissue of the stromata by pigmentation in the outermost Trichodermati lanuginoso simile sed ob conidia (3.5−)4.0−5.2 2−3 layers of cells; cells of the stroma surface in section (−6.0)×(2.5−)2.7−3.0(−3.5) μmmensa,L/W(1.3−) pseudoparenchymatous, (5−)8−13(−19) μm diam, thin- 1.5−1.9−2.0) differt, et in agaris dictis PDA vel SNA magis walled. Tissue of the stroma below perithecia pseudo- celeriter crescens. parenchymatous, thin-walled, lacking long hyphal ele- Holotype BPI 881031, designated here. ments, (13−)15−27(−34)×(8−)10−16(−20) μm. Asci Mycobank 519545 cylindrical, (64−)75−90(−100)×(3.2−)4.5−6.5(−9.0) μm, apex with a conspicuous discharge ring, ascospores Telemorph Not known uniseriate. Part ascospores hyaline, dimorphic; conspicuously warted, distal part conical to subglobose, (2.7−)3.7−5.2 Characteristics in culture Optimum temperature for growth (−6.5)×(2.0−)3.5−4.5(−5.5) μm; proximal part wedge- on PDA 25°C, on SNA 30ºC; colony radius after 96 h on shaped to ellipsoidal, (2.2−)3.7−5.0(−6.0)×(2.0−)3.0−4.02 PDA at 25ºC ca. 55 mm, on SNA at 30°C ca. 60 mm. On (−4.7) μm. PDA lacking diffusing pigment or distinctive odor. Typi- cally not growing at 35°C. On PDA after 10 days at 25°C Habitat Constantly associated with Moniliophthora perni- under 12 h cool white fluorescent light/12 h darkness ciosa on Theobroma cacao, on dead cacao tissue infected conidia forming in a continuous mat of densely disposed, Mycol Progress (2012) 11:215–254 249 250 Mycol Progress (2012) 11:215–254

Fig. 16 Trichoderma vermipilum. a, b Pustules from SNA. Note the near the base of a hair. j Conidia. k chlamydospores. All from PPRI wooly nature of the pustule in (b). c Hairs arising from a pustule. d−h 3559. Scale bars (a) = 1 mm, (b) 0.5 mm, (c−h, k)20μm, (i, j) Hairs with fertile branches arising from near the base. Phialides are 10 μm produced at the tip of hairs in (d, e). i Fertile branches arising from Mycol Progress (2012) 11:215–254 251 pulvinate pustules around the periphery of the Petri plate; of de Souza et al. (2006, 2008), Loguercio et al. (2009a, b), conidia grayish green (K&W 30D5−8); on SNA producing Medeiros et al. (2010) and Sanogo et al. (2002) suggests numerous pustules scattered around the periphery of the that strains of AFLP Group 1 (Clade B here) are better Petri plate. Pustules pulvinate, 0.25−1.0 mm diam, with suited for biological control than are strains of Group 2 numerous protruding hairs. Hairs white, variable in length (Clade A here). Although T. stromaticum is very easily (50−175 μm), sinuous to somewhat spiraled, mostly recognized through its morphology, the differences in producing a single terminal phialide, often producing a phenotype between the two AFLP are not diagnostic. The terminal verticil of phialides and sometimes producing a only practical ways to determine to which group a newly few solitary, lageniform phialides laterally, or sterile, collected strain of T. stromaticum belongs is through the tapering uniformly from 4−5 μm at the base. Fertile AFLP procedure or through sequencing tef1, using the branches arising near the base of the hairs, each one or a primers described here, followed by comparison with few cells in length, longer fertile branches rebranching to sequences that we have deposited in GenBank. In the produce unicellular secondary branches; phialides arising in present work, we have found strains of T. stromaticum dense clusters at the tips of all fertile branches and giving a isolated from the pseudostromata of the destructive cacao botryose aspect. Phialides nearly subglobose to flask- pathogen Moniliophthora roreri. This is the first report of T. shaped with a broad base, (4.7−)5.5−7.5(−9.5) μm long, stromaticum occurring on this pathogen, and these strains (3.7−)4.0−5.0(−5.5) μm at the widest, L/W (1.0−)1.2−1/6 may provide a new source of control of the Frosty Pod Rot (−2.1), (2.2−)2.7−3.5(−4.2) μm at the base, arising from a cell pathogen (Clade C, Fig 1e). (2.5−)2.7−4.5(−5.5) μmwide.Conidia(n=30) nearly oblong, The literature abounds in reports of Trichoderma species (3.5−)4.0−5.2(−6.0)×(2.5−)2.7−3.2(−3.5) μm(95%ci that can be mycoparasites, including strains of species that 4.5−4.9×2.8−3.0 μm, L/W 1.6−1.7) smooth, gray green. have been isolated as endophytes, but of all these we only Chlamydospores abundant on SNAwithin 1 week, intercalary know two species that are host specific: T. aggressivum in hyphal cells and conforming to the shape of the cell. Samuels et al (Samuels et al. 2002), cause of green mold disease of Agaricus mushrooms and T. stromaticum Etymology ‘vermipilum’ from Latin, reference to the worm- Samuels et al. (2000), a parasite of Moniliophthora like hairs arising in abundance from conidial pustules. perniciosa, the cause of witches’ broom of cacao. There are more examples of more or less host specificity for Habitat Known only from the original collection, man- perithecial formation, including Hypocrea latizonata Peck, grove soil. which is only found on basidiomata of Cyathus species (Sundberg and Kost 1989), and members of Trichoderma Known distribution Republic of South Africa sect. Hypocreanum Bissett, which occur only on members of the basidiomycete order Aphyllophorales (e.g. Hypocrea Holotype Republic of South Africa, KwaZulu-Natal, Dur- americanum Overton on Fomitopsis pinicola and Piptopo- ban, isolated from soil under beachwood mangrove, 1986, rus betulinus (Overton et al. 2006b), H. sulphurea (Schw.) R.Y. Auerlich s.n.(BPI 881031; ex-type culture PPRI 3359= Sacc. on basidiomata or mycelium of Exidia nucleata CBS 127103). (Overton et al. 2006a), or H. pulvinata Fuckel on various polypores (Overton et al. 2006a)). Trichoderma stromati- Comments Using the sequence similarity search tool cum is a parasite of Moniliophthora species and is utilized implemented in TrichoBLAST (www.ISTH.info) strain G. in a biological control program for Witches’ Broome J.S. 01-312 (T. ivoriense, described here) could be Disease of cacao (‘Trichovab’; Samuels et al. 2000; identified as T. vermipilum. Strain PRI 3559 was reported Medeiros et al. 2010). It is host specific. It is always as a Trichoderma sp. from South Africa by Kindermann et associated with cacao and, as was noted above, is almost al. (1998) and Kullnig et al. (2000). always found growing on cacao tissue infected with the witches’ broom pathogen M. perniciosa; it is rarely isolated from sapwood of cacao or found on cacao pods infected Discussion with the Frosty Pod Rot pathogen of cacao, M. roreri.To our knowledge it has never been isolated from soil, which Despite AFLP data, and phenotypic and biological evi- is the typical habitat of Trichoderma (Klein and Eveleigh dence suggesting the existence of cryptic species in T. 1998). Hoyos-Carvajal et al. (2009) did not list it among the stromaticum, neither multilocus phylogenetic analysis nor many species of Trichoderma that they found in soil in MALDI-TOF MS peptide analysis (De Respinis et al. Colombia and adjacent countries, and we did not find it 2010) could support more than one taxon in this species. among approximately 800 cultures of Trichoderma isolated However, despite a lack of taxonomic resolution, the work from under wild cacao trees in Peru (Samuels, unpublished). 252 Mycol Progress (2012) 11:215–254

Has T. stromaticum coevolved with its host, Monilioph- forest (Mata Atlantica) of Brazil may be a reservoir of T. thora perniciosa on cacao? The center of origin, and the stromaticum and that either there was a host switch to highest diversity of cacao is thought to be in the upper Moniliophthora species or that T. stromaticum infecting Amazon region of South America (Bartley 2005; Moto- species in genera other than Moniliophthora has been mayor et al. 2008) and the highest diversity of M. overlooked by agaricologists. The range of possible hosts perniciosa, at least in Brazil, is in the eastern, lower for T. stromaticum and its relatives is physically and Amazon regions of the states of Amazonas and Pará taxonomically wide but unexplored. (Rincones et al. 2006). Moniliophthora perniciosa is presumed to have coevolved with cacao in Amazonian Acknowledgments The following individuals contributed cultures America (Purdy and Schmidt 1996; Evans et al. 2003b). used in this research: Adriaana Jacobs and Michael Wingfield (Republic of South Africa); Ismaiel Kibbe (Côte d’Ivoire), John However in extensive exploration for wild cacao and its Bissett (Canada), Christian Kubicek and Irina Druzhinina (Austria), pathogens in several river systems in Amazonian Peru Carmen Suarez (Ecuador), Whillys Soberanis and Enrique Arevalo- (Arevalo-Giardini, Meinhardt and Samuels, unpublished) T. Giardini (Peru), Alan Pomella (Brazil); Harry Evans, Keith Holmes stromaticum was not encountered despite the presence of and Sarah Thomas (UK); Rabio Olatinwo, Annemieke Schilder and Prakash Hebbar (USA). Didier Begoude (Cameroon) enabled collect- M. perniciosa. Moniliophthora perniciosa is quite variable ing in the Reserve Faunal du Dja. Nigel Hywell-Jones and Rungtip (Purdy and Schmidt 1996; de Arruda et al. 2005). In Nasit facilitated collecting in the UNESCO World Heritage Site Khao addition to pathogenic variations in cacao (‘C’ biotype) Yai National Park (Thailand). Orlando Petrini corrected the Latin there are populations adapted to other plant species descriptions. Mention of trade names or commercial products in this ‘ ’ publication is solely for the purpose of providing specific information including the S biotype, which is adapted to solanaceous and does not imply recommendation or endorsement by the U.S. hosts, and the ‘L’ biotype that is adapted to the liana Department of Agriculture. USDA is an equal opportunity provider Arrabidaea verrucosa (Bignoniaceae). The ‘H’ biotype was and employer. recently removed from M. perniciosa as the new species Crinipellis brasiliensis MCC de Arruda et al. 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