The genus Pleurotus in Argentina: mating tests

The genus Pleurotus in Argentina: mating tests Bernardb E. Lechnerl, Jorge E. Wrighttl 1PRHIDEB-CONICET, & Edgardo Albertó2 Departamento de BBE, Facultad de Ciencias Exactas y Naturales, U~versidad de Buenos Aires (C1428EHA), Buenos Aires, Argentina and Instituto Fitotécnico de Santa Catalina, Universidad Nacional de La Plata, Facultad de q;iencias Agrarias y Forestales, Llavallol, Buenos Aires, Argentina 2 Instituto del~vestigaciones Biotecnológicas IlB-INTECH (CONICET-UNSAM), <I::. C. 164, (7130) Chascomús, Buenos Aires, Argentina Lechner, B.E., J.E. Wright & E. Albertó (2005): The genus Pleurotus in Argentina: mating tests. -Sydowia 57 (2); 233-245. In order to study the sexual compatibility, monosporic cultures from 32 strains belonging to four out of the six Pleurotus species recorded from Argentina were obtained. All tested species have a heterothallic multifactorial tetrapolar mating system. P. pulmonarius and P. ostreatus could be separated by interspecific crossings only. Our mating tests confirm the synonymy of P. smithii with P. cystidiosus. Compatibility tests confirm that in Argentina there are, so far, 5 Pleurotus species, namely P. albidus, P. cystidiosus, P. djamor, P. ostreatus and P. pulmonarius. Keywords: Argentina, Pleurotus, sexual compatibility, systematics. An exhaustive study of the genus Pleurotus for Argentina based on macro- a~d micromorphology was undertaken (Lechner et al., 2004). In order to learn about the relationships among species from the biological standpoint, we undertook mating tests among different strains, utilizing, in several cases, fruit body production to obtain a spore print and monosporic cultures. Already Bensaude (1918), Kniep (1920) and Vandredies (1923) proposed th~t mating tests studies could be useful for defining species. Boi4in (1986) pointed out that the study of sexual compatibility was a very useful tool for the study of the taxonomy, especially of wood decaying species, which are easy to culture. According to Petersen & Hughes (1999), there are three, most applicable, species concepts: the morphological, the phylogenetic and the biological. For the determination of the species concept, all data ought to be considered, but mating tests studies could be the most informative. Many author¡; have studied compatibility systems for the delimitation of species:Bqidin (1980, 1986), Boidin & Lanquetin (1965, 1977, 1984) and Hallenberg (1984) worked on several genera of Corticiaceae s.l. and other basidiomycetes; Petersen (1995a, 1995b, 1994, 1992), Nicholl & Petersen (2000) and Petersen & Hughes (1998, 1993) studied 233 mating types in agarics of the genera Omphalotus, Lentinula and Pleurotus, among others. The aim of this work is i) to contribute to a better delimitation of interesterility groups of Pleurotus in Argentina, and to ii) ascertain the accuracy of monosporic culture crossing as a tool for the identification of Pleurotus species. Materials and Methods Specimens collected were tentatively and macromorphological studies. determined based on micro- Strains used -The Argen tinean cul tures used for mating compatibility tests are listed in Tab. I, with data on their origin, collection number, substrate, date and collector . Other strains used: P. cystidiosus, MALAYSIA, Selangor, 1985, coll. K.M. Graham, MUCL 28.690. P. djamor var. roseus, BRASIL, sao Paulo, on leaf of Agavaceae in garden, coll. Edson De Paula, MUCL 35.018 (P. ostreatoroseus = P. djamor); P. ostreatus, ITALY, IX-93, commercial strain, BAFC 2067. Test strains: The following strains from the University ofTennessee (TENN) were utilized as standard strains for interspecific crossings: P. cornucopiae (PCORN) TENN 8763, Austria, 16-VI-96; P. djamor (PDJAM) TENN 6346, Malaysia, IV-95; P. ostreatus (PO) TENN 6689; Austria; P. pulmonarius (PPULM) TENN 4203, Sweden, Vastergotland, 18-IX-91, on Fraxinus sp. Fruit body production -To obtain basidiomata, traditional methods for fruiting species of Pleurotus were used (Zadrazil, 1974; Stamets, 1993). A mixture of sawdust (70%), wheat meal (10%), oatmeal (4%) and CaCO3(1%) was introduced into 40x25cm polypropylene bags and autoclaved at 120 °C for 2 hours. After cooling they were inoculated with spawn, and incubated in the dark at 25 °C.After 15 days, bags were kept at 18-20 °C with 9 h light/15 h darkness photoperiod to induce basidiome formation. Fruit bodies were obtained from strains BAFC 120, 695, 809, 1003, 2034, 2067, 2545 and 2787. Mating studies -Monosporic cultures were obtained from water dilutions of fresh spore prints on sterile aluminum paper; spores were suspended in 10 ml of sterile-distilled water containing 0.01 mL of Tween 80 to avoid agglutination. This solution was diluted to 1/10, 1/100 and 1/1000. Petri dishes containing Nobles' medium (Nobles, 1948) were inoculated with 1 ml of each of these dilutions and incubated in the dark at 25 °C. Individual monosporic cultures were isolated from germinating single spores. All isolates were examined for clamp connections under the stereomicroscope. Isolates with clamp connections were discarded, only isolates without clamp connections were used for mating studies. Self-crossings for each strain were carried out in order to obtain the mating types. Haplonts were confronted in pairs in Petri dishes using 7 mm diameter blocks as inoculum. Plates were incubated in the dark at 25 °C,and after one 234 week. Mycelia were screened for clamp connections within the contact zone and on the obverse sides of donor inoculum blocks under the microscope (Burnett, 1968). Presence of clamp connections is considered as a positive mating result, which means that both strains are compatible (+); absence of clamp connections indicates incompatibility (-). For interspecific crossings, mating types of monosporic cultures obtained from morphologically determined collections were confronted with test strains The results of these crossings allowed us to confirm or reject such identifications. To confirm identification of Pleurotus collections with similar micro- and macromorphology, two or three monosporic isolates were confronted. When monosporic cultures of doubtful species were lacking, di-mon crossings (the Buller phenomenon) were undertaken (Boidin, 1980)~ Table I: List of Pleurotus isolatesfrom Argentina usedin the study. Species BAFC N° P. albidus 2787 Locality Substrate Date Collector 809 t315 101 1221 695 136 235 Species BAFC N° P. djamor P. djamor A. Martinez 10.06.1989 1100 Capital Federal, University City Córdoba, La Punilla Misiones, Pto. Libertad On fallen trunk 25. 08. 1999 E. Lechner 73 On Populus sp. 25.02.2001 On dead tree of Araucaria angustifolia N. Manero & B.J. Lechner 10.04. 1985 J. Deschamps Buenos Aires, On Platanus sp. March 1994 H. Spinedi La Plata Capital On dead zone 18. 04. 2000 S. Frachia Federal, Barrio of Platanus sp. La Paternal 821 Misiones, El Palmital del cruce On Ficus sp. fallen trunks 28. 05. 2001 E. Albertó 815 Misiones, On fallen trunk 28. 05. 2001 E. Albertó Unknown 18. 04. 1994 Pablo Pica On trunk of Araucaria araucana March djamor var. Collector OnCordylinesp. 188 var. Date Capital Federal 215 cy$tidiosus Substrate 2545 190 P. Locality Palmital roseus El del cruce P. ostreatus 2034 Capital Federal 120 Neuquen, Moquehue Populmonarius 1003 Buenos Aires, Ezeiza 76 Misiones, San Pedro 263 San Pedro, S 26° 32', W 54° 04' 1993 J. del Vas On living 25.07 .1987 J. Deschamps declining tree of Populus sp. On branches 27. 05. 2001 E. Albertó & of Araucaria O. Popoff angustifolia On branches of 27. 05. 2001 D. Krueger A. angustifolia Results Mating types were assigned after se1f-crossingof the 126 obtained monosporic cultures (Tab. II). Among the strains tested, seven strains had four mating types, while three others (BAFC 1003, 815 and 821) had three mating types. Self-crossings from eight monosporic cultures from BAFC 809 resulted all negative; the same occurred with ten monosporic cultures from BAFC 76. However, crossings between 236 monosporic cultures of these strains and monosporic cultures of P. albidus and P. pulmonarius, respectively, resulted positive. The charts of intra- and interspecific confrontations between different cultures are illustrated in Figs. 1-5. AlI four species studied, Pleurotus albidus, P. djamor, P. ostreatus and P. pulmonarius, exhibited a heterothallic tetrapolar sexual compatibility system. Table II. Mating types assigned to monosporic cultures (P. aJbjdus: BAFC 1221, 1350 and 1354, 1399; P. djamor: BAFC 815 and 821; P. ostreatus: BAFC 120,2067; P. pu1monarjus: BAFC 263 and 1003). Monosporic cultures are denoted as numbers of each strain. Mating type of monosporic cultures Species BAFC Monosporic cultures used A,B, A,B2 A2Bl A2B2 Pleurotus albjdus (Berk.) Pegler Pairing among mating types obtained from self-crossing (BAFC 1221, 1350, 1354 and 1399), and monosporic cultures (BAFC 695, 809, 2545, and 2787) with test strains of P. ostreatus, P. djamor, P. pulmonarius and P. cornucopiae (Figs. 1-4) showed no compatible matings. Pairing among Pleurotus albidus strains BAFC 1221 with 1350,1354, 1399,695,809 and 2545 resulted positive (Figs. 2-3). Di-mon crossings between monosporic and polysporic cultures were undertaken (Fig. 5) to confirm taxonomic delimitation of several strains that had tentatively been identified as P. albidus based on morphological characteristics such as pileus of fragile nature, white to cream color and remarkable laciniate-crenate margin (Lechner et al, 2004). Positive results were obtained for BAFC 136, 215, 575, 607, 695, 809, 317, 1221, 1315, 1319, 1330, 1350, 2545 and 2787. These strains constitute clearly an interesterility group within Pleurotus. Several specimens showed great morphological variability, observed 237 not only in collections from nature, but also in fruit bodies obtained in culture. Due to their anastomosed lamellae on the stipe (BAFC 809) some resemble P. cornucopiae, whereas others (e.g. BAFC 136) form more plane and shorter basidiomes, without such characteristics. These variations might suggest the existence of two different species. However, mating tests allowed us to conclude that these morphological variations are intraspecific, and may be possibly caused by environmental constraints. Pleurotus cystidiosus o. K. Miller It was not possible to obtain monosporic cultures ofP. cystidiosus from Argentina since fruit bodies obtained in culture did not produce viable spores. Positive resu1ts of pairings were observed when BAFC 73, determined as P. smithii by Spinedi (1995), and BAFC 188 were BAFC 2067 ~1~¡~O-Ij1-! o (\I ... :)1 + + (J I!. o( al + PO TEN 6689 413611516 o ('I ~ 01 + + u 10. ct al + + BAFC 1354 3 I 9 I 8 I 7 1'.0 o N U LL ct aJ 10 11 Fig. 1: Crossings of monosporic cultures of Pleurotus ostreatus with P. pulmonarius and P. albidus. First row: P. ostreatus (BAFC 120) crossed with P. ostreatus (BAFC 2067) and P. albidus (BAFC 1354 and BAFC 1350); Second row: P. ostreatus (BAFC 120) crossed with P. ostreatus (TEN 6689), P. pulmonarius (TEN 4203) and with P. ostreatus (BAFC 2034); Third row: P. ostreatus (BAFC 2067) crossed with P. albidus (BAFC 1354 and BAFC 1350) and P. pulmonarius (TEN 4203). 238 crossed with monosporic cultures of strain P. cystidiosus MUCL 28690, showing that both belong to P. cystidiosus interesterility group (Fig.5). Macro- and micromorphological studies are consistent with this result. The collections of P. smithii showed the presence of pileocystidia (Lechner et al., 2004), absent in the original description of this speciesgiven by Guzman (1975). PJeurotus djamor(Fr.) Boedijn Monosporic cultures of two collections (BAFC 815, BAFC 821) were confronted with test strains of P. djamor. Positive results confirmed our morphological identification (Fig. 4). Di-mon crossing between the monosporous culture of P. djamor from TENN and MUCL 35018, identified as P. ostreatoroseus Singer, resulted positive tOO. BAFC 2034 8 I 10 I 4 I 5 I-"' o N U 11. ~ ~j al + 11l PPULM TEN l10 ¡ 1 114 I 9 r-ID ~ C.) ~ a3 ~ 7 10 11 BAFC 1221 5 "" II) M .- U IL < ID I 6 I 9 112 BAFC 5 /13 1350 11~1-IJ PO TEN 6689 413611516 + + I + + Fig. 2: Crossings of monosporic cultures of Pleurotus strains. First row: Pleurotus ostreatus crossed with P. ostreatus (BAFC 2034), P. albidus (BAFC 695 and BAFC 2545) and P. pulmonarius (BAFC 1003); Second row: P. ostreatus crossed with P. pulmonarius (TEN 4203), P. albidus (BAFC 1399, BAFC 809, BAFC 2787); Third row: P. albidus crossed with P. albidus (BAFC 1221, BAFC 1350), and P. ostreatus (TEN 6689) 239 PJeurotus ostreatus (Jacq.: Fr.) Kummer Results of intraspecific crossing showed that the three collections (BAFC 120, 2034 and 2067) belong to the same species (Figs.1-2). BAFC 1003, which initially was identified as P. ostreatus based exclusively on morphological studies, resulted negative in crossing with BAFC 2067 (Fig. 2). Pleurotus pulmonarjus (Fr.) Quélet Pairing among test strains of P. pulmonarius with BAFC 76, 263 and 1003 resulted positive (Fig.4). Pairing between BAFC 263 and 2067 of P. ostreatus resulted not compatible (Fig.4). We selected interspecific crossing between strains of P. ostreatus and P. pulmonarius, because both species are very similar. It is very difficult, if ~ 1399 AFC 5 8 7 Fig. 3: Crossings of monosporic cultures of Pleurotus strains. First row: P. albidus (BAFC 1354), crossed with P. djamor (TEN 6346), P. pulmonarius (TEN 4203) and with P. cornucopiae (TEN 8763); Second row: P. albidus crossed with P. albidus (BAFC 1350), P. cornucopiae (TEN 8763) and P. pulmonarius (TEN 4203); Third row: P. albidus (BAFC 1221) crossed with P. albidus (BAFC 1399, BAFC 695, BAFC 809, BAFC 2545). 240 not impossible to separate P. ostreatus from P. pulmonarius based exclusively on micro- and macroscopic features. Discussion Our mating compatibility studies allow to confirm five interesterility groups in Argentina: Pleurotus albidus, P. cystidiosus, P. djamor, P. ostreatus and P. pulmonarius. The P. albidus interesterility group is attributed to a larger sample of isolates that were available from a wide variety of locations and substrates throughout Argentina. In previous studies on mating relationships in Pleurotus, extensive intracollection mon-mon pairings (e.g. Anderson et al., 1973; Hilber, 1982; Bresinsky et al., 1987, Petersen, 1995b) and dimon pairings (e.g. Vilgalys et al., 1993) were performed to determine BAFC 76 BAFC 1350 15 11011317 "' <0 N U IL <C ID 12 13 11 Fig. 4: Crossings of monosporic cultures of Pleurotus strains. First row: P. pulmonarius (TEN 4203) crossed with P. pulmonarius (BAFC 76, BAFC 1003, BAFC 263); Second row: P. pulmonarius (BAFC 263) crossed with P. albidus (BAFC 1350), P. pulmonarius (BAFC 263) and P. ostreatus (BAFC 2067); Third row: P. djamor (TEN 6346) crossed with P. djamor (BAFC 821, BAFC 815) and P. albidus (BAFC 1221). 241 mating types from each individual prior to performing intercollection matings. In this study, we attempted to maximize the number of intercollection matings by using mon-mon and di-mon pairing with testers from known interesterility groups in Pleurotus. The extensive collection data available for most isolates from this study support our view that the interesterility groups represent good species that differ in a number of significant morphological characteristics (Lechner et al., 2004). P. cystidiosus is a widespread species covering large regions of the northern hemisphere (North America, Europe and Asia) as well as the southern part of Africa (Zervakis, 1998). For Central and South America there is a record of P. smithii in Mexico (Guzmán, 1975), MONOSPORIC BAFC I BAFC 809 6 I BAFC 136- I BAFC 215 + I BAFC 695 18 + 14 15 1221 16 BAFC 2067 I BAFC 1350 11215113 + CUL TURE (n) l4T7l1O + + I + BAFC 575 BAFC 607 BAFC 809 c + .s w ~ ~ ~ ~ u 1Z o ~ ct u c BAFC 317 BAFC 1315 BAFC 1319 1 BAFC 1330 BAFC 254!; BAFC 278~r BAFC 203'~ BAFC 2067 BAFC 188 BAFC 73 + + + + BAFC 100:3 MUCL 35018 Fig.5: Di-mon crossing between monosporic cultures of P. albidus (BAFC 809, BAFC 695, BAFC 1221, BAFC 1350), P. ostreatus (BAFC 2067), P. pulmonarius (TEN 4203), P. djamor (TEN 6346) and P. cystidiosus (MUCL 28690) with polysporic cultures of P. albidus (BAFC 136, BAFC 215, BAFC 575, BAFC 607, BAFC 809, BAFC 317, BAFC 1315, BAFC 1319, BAFC 1330, BAFC 2545, BAFC 2787), P. ostreatus (BAFC 2034, BAFC 2067), P. cystidiosus (BAFC 188, BAFC 73), P. pulmonarius (BAFC 1003) and P. djamor (MUCL 35018). 242 Peru (Guzmán et al., 1980), Cuba (Rodríguez-Hernández & CaminoVilaró 1990), Brazil (Capelari, 1999) and Argentina (Spinedi, 1995). Capelari & Fungaro (2003) studied the growth rate in cu1ture, dikaryon-monokaryon matings, and genetic variability. They concluded that the criteria used to separate P. cystidiosus from P. smithii are unsatisfactory and shou1d be considered synonym. The macroand micromorphological study of Spinedi 's collections made in a previous work (Lechner, et al. 2004) and positive results of pairings between cultures of P. smithii and P. cystidiosus here obtained, permit us confirm the supposed synonymy of these two taxa, and to reaffirm that P. cystidiosus is the only synnematoid (producing an anamorph with synnemata) Pleurotus species in South America. Some Pleurotus species can be characterized by a unique range of distribution in Argentina. For example, the P. cystidiosus group is distributed a1ongeastern Argentina, in a temperate zone; the P. ostreatus group extends from southern to centra1Argentina, the cold to temperate area; the P. djamor group occurs in the subtropical northeast Argentina; P. pulmonarius is distributed over two broad geographic regions in both northern (subtropical) and central (temperate) Argentina. P. albidus was collected from central to north of Argentina, in great diversity of substrates from diverse species of living trees to stumps and fallen trunks. According to Albertó et al. (2002) P. albidus fruits from Central America to central Argentina. Pleurotus cystidiosus, P. djamor, P. ostreatus and P. pulmonarius intersterility groups show strong preferences for hosts tree species (Tab. I): P. albidus (also known in Argentina as P. laciniatocrenatus Speg.) was collected in great diversity of substrates from diverse species of living trees to stumps and fallen trunks. In summary, interspecific crossings permit to separate morphologically very similar species like P. pulmonarius and P. ostreatus (Lechner et al., 2002). Moreover, mating tests confirmed the synonymy of P. smithii with P. cystidiosus, and helped to unambiguously identify strains with differing micromorphology within P. albidus. Sexual compatibility studies confirmed the heterothallic tetrapolar mating type of all Argentinean Pleurotus species,with the presence of mu1tiple alleles as previously reported by Hilber (1982) and Petersen (1995a). Acknowledgements We wish to thank the curators of BAFC and MUCL culture collections for making available strains. Mario Rajchenberg (Esquel, Argentina), who provided the collection and strain of P. ostreatus from Patagonia, Argentina. Dr. R.H. Petersen (Knoxville, TN) for supplying monosporic cultures of several species of Pleurotus. The Argentina National Park is thanked for permission to collect in protected areas. This research was supported by PRHIDEB-CONICET/Argentina and PIP 2500 (CONICET). This is paper n° 161 of the PRHIDEB-CONICET series. 243 References Albertó, E., R.H. Petersen, K. W. Hughes & B.E. Lechner (2002). "Miscellaneous notes on Pleurotus". -Persoonia 18 (1): 55-69. Anderson, N.A., S.S. Wang, & J. W. Schwandt (1973). The Pleurotus ostreatussapidus species complex. -Mycologia 65: 28-35. Bensaude, M. (1918) Recherche sur le cycle évolutif et la sexualité chez les Basidiomycetes. -These, Paris, 156 p. Boidin, J. (1980). La notion d'espece III: le critere d'interfertilité ou intercompatibilité: resultats et problemes. -Bulletin de la Société Mycologique de France 96 (1): 43-57. Boidin, J. (1986). Intercompatibility and the species concepts in the saprobic Basidiomycotina. -Mycotaxon 26: 319-336. Boidin, J. & P. Lanquetin (1965). Hétérobasidiomycetes saprophytes et Homobasidiomycetes résupinés X. Nouvelles données sur la polarité dite sexuelle. -Revue Mycologique 30: 3-16. Boidin, J. & P. Lanquetin (1977). Peniophora (subgen. Duportella) malenconii, nov. sp. (Basidiomycetes, Corticiaceae), espece méditerranéenne partiellement interstérile avec son vicariant californien. -Revue Mycologique 41: 119-128. Boidin, J. & P. Lanquetin (1984). Repertoire des données utiles pour effectuer les tests d'intercompatibilité chez les Basidiomycetes III. Aphyllophorales non porées- Cryptogamie Mycologie 5: 193-245. Burnett, J.H. (1968). Fundamentals of mycology. -Edward Arnold. Ltd. London. 546p. Bresinsky A., M. Fisher, B. Meixner & W. Paulus (1987). Speciation in Pleurotus. Mycologia 79: 234-245. Capelari, M. (1999). First record of Antromycopsis macrocarpa for Brazil. Mycotaxon 72: 101-105. Capelari, M. & M. H. P. Fungaro (2003). Determination of biological species and analysis of genetic variability by RAPD of isolates of Pleurotus subgenus Coremiopleurotus. -Mycological Research 107: 1050-1054. Guzmán, G. (1975). New and interesting species of Agaricales of México. -Beihefte zur Nova Hedwigia 51: 99 -118. Guzmán, G., R. Valenzuela & A. Canale (1980). Primer registro de Pleurotus smithii de América del Sur y obtención de la fase asexual de la cepa mexicana. Boletín de la Sociedad Mexicana de Micología 14: 17- 26. Hallenberg, N. (1984). Compatibility between species of Corticiaceae s.l. (Basidiomycetes) from Europe and North America. -Mycotaxon 21: 335-388. Hilber, O. (1982) Die Gattung Pleurotus. -Bibliotheca Mycologica 87: 1-448. Kniep, H. (1920) Uber morphologische und physiolgische Gesch1echtedifferenzierung (Untersuchungen an Basidiomyceten). -Verh. Physik. Mediz. Ges. Würzburg 46: 1-28. Lechner, B. E., J. E. Wright & E. Albertó (2004). The genus Pleurotus in Argentina. Mycologia 96: 845-858. Lechner, B.E., R. Petersen, M. Rajchenberg & E. Albertó (2002). Presence of Pleurotus ostreatus in Patagonia, Argentina. -Rev. Iberoam. Micol. 19: 111114. Nicholl, D.B.G. & R.H. Petersen (2000). Phenetic plasticity in Pleurotus djamor Mycotaxon 76: 17 -37. Nobles, M. K. (1948). Studies in forest pathology VI. Identification of cultures of wood-rotting fungi. -Canadian Journal Research 26: 281-431. Petersen, R. H. (1992). Mating systems in three New Zealand agarics. -New Zealand Journal of Botany 30: 189-197. 244 Petersen, R.H. (1994). Notes on mating systems of Auriscalpium vulgare and A. villipes. -Mycological Research 98: 1427-1430. Petersen, R.H. (1995a). There is more to a mushroom than meets the eye. Mating studies in the Agaricales. -Mycologia 87: 1-17. Petersen, R. H. (1995 b ). Contributions of mating studies to mushroom systematics. Canadian Journal of Botany 73 (Suppl. 1): S 831-S 842. Petersen, R.H. & K. W. Hughes (1993). Intercontinental interbreeding collections of Pleurotus pulmonarius with notes of P. ostreatus and other species. Sydowia Annales Mycologici 45: 139-152. Petersen, R.H. & K. W. Hughes (1998). 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(Manuscript accepted lO January 2005) 245 

The genus Pleurotus in Argentina: mating tests Bernardb E. Lechnerl, Jorge E. Wrighttl 1PRHIDEB-CONICET, & Edgardo Albertó2 Departamento de BBE, Facultad de Ciencias Exactas y Naturales, U~versidad de Buenos Aires (C1428EHA), Buenos Aires, Argentina and Instituto Fitotécnico de Santa Catalina, Universidad Nacional de La Plata, Facultad de q;iencias Agrarias y Forestales, Llavallol, Buenos Aires, Argentina 2 Instituto del~vestigaciones Biotecnológicas IlB-INTECH (CONICET-UNSAM), <I::. C. 164, (7130) Chascomús, Buenos Aires, Argentina Lechner, B.E., J.E. Wright & E. Albertó (2005): The genus Pleurotus in Argentina: mating tests. -Sydowia 57 (2); 233-245. In order to study the sexual compatibility, monosporic cultures from 32 strains belonging to four out of the six Pleurotus species recorded from Argentina were obtained. All tested species have a heterothallic multifactorial tetrapolar mating system. P. pulmonarius and P. ostreatus could be separated by interspecific crossings only. Our mating tests confirm the synonymy of P. smithii with P. cystidiosus. Compatibility tests confirm that in Argentina there are, so far, 5 Pleurotus species, namely P. albidus, P. cystidiosus, P. djamor, P. ostreatus and P. pulmonarius. Keywords: Argentina, Pleurotus, sexual compatibility, systematics. An exhaustive study of the genus Pleurotus for Argentina based on macro- a~d micromorphology was undertaken (Lechner et al., 2004). In order to learn about the relationships among species from the biological standpoint, we undertook mating tests among different strains, utilizing, in several cases, fruit body production to obtain a spore print and monosporic cultures. Already Bensaude (1918), Kniep (1920) and Vandredies (1923) proposed th~t mating tests studies could be useful for defining species. Boi4in (1986) pointed out that the study of sexual compatibility was a very useful tool for the study of the taxonomy, especially of wood decaying species, which are easy to culture. According to Petersen & Hughes (1999), there are three, most applicable, species concepts: the morphological, the phylogenetic and the biological. For the determination of the species concept, all data ought to be considered, but mating tests studies could be the most informative. Many author¡; have studied compatibility systems for the delimitation of species:Bqidin (1980, 1986), Boidin & Lanquetin (1965, 1977, 1984) and Hallenberg (1984) worked on several genera of Corticiaceae s.l. and other basidiomycetes; Petersen (1995a, 1995b, 1994, 1992), Nicholl & Petersen (2000) and Petersen & Hughes (1998, 1993) studied 233 mating types in agarics of the genera Omphalotus, Lentinula and Pleurotus, among others. The aim of this work is i) to contribute to a better delimitation of interesterility groups of Pleurotus in Argentina, and to ii) ascertain the accuracy of monosporic culture crossing as a tool for the identification of Pleurotus species. Materials and Methods Specimens collected were tentatively and macromorphological studies. determined based on micro- Strains used -The Argen tinean cul tures used for mating compatibility tests are listed in Tab. I, with data on their origin, collection number, substrate, date and collector . Other strains used: P. cystidiosus, MALAYSIA, Selangor, 1985, coll. K.M. Graham, MUCL 28.690. P. djamor var. roseus, BRASIL, sao Paulo, on leaf of Agavaceae in garden, coll. Edson De Paula, MUCL 35.018 (P. ostreatoroseus = P. djamor); P. ostreatus, ITALY, IX-93, commercial strain, BAFC 2067. Test strains: The following strains from the University ofTennessee (TENN) were utilized as standard strains for interspecific crossings: P. cornucopiae (PCORN) TENN 8763, Austria, 16-VI-96; P. djamor (PDJAM) TENN 6346, Malaysia, IV-95; P. ostreatus (PO) TENN 6689; Austria; P. pulmonarius (PPULM) TENN 4203, Sweden, Vastergotland, 18-IX-91, on Fraxinus sp. Fruit body production -To obtain basidiomata, traditional methods for fruiting species of Pleurotus were used (Zadrazil, 1974; Stamets, 1993). A mixture of sawdust (70%), wheat meal (10%), oatmeal (4%) and CaCO3(1%) was introduced into 40x25cm polypropylene bags and autoclaved at 120 °C for 2 hours. After cooling they were inoculated with spawn, and incubated in the dark at 25 °C.After 15 days, bags were kept at 18-20 °C with 9 h light/15 h darkness photoperiod to induce basidiome formation. Fruit bodies were obtained from strains BAFC 120, 695, 809, 1003, 2034, 2067, 2545 and 2787. Mating studies -Monosporic cultures were obtained from water dilutions of fresh spore prints on sterile aluminum paper; spores were suspended in 10 ml of sterile-distilled water containing 0.01 mL of Tween 80 to avoid agglutination. This solution was diluted to 1/10, 1/100 and 1/1000. Petri dishes containing Nobles' medium (Nobles, 1948) were inoculated with 1 ml of each of these dilutions and incubated in the dark at 25 °C. Individual monosporic cultures were isolated from germinating single spores. All isolates were examined for clamp connections under the stereomicroscope. Isolates with clamp connections were discarded, only isolates without clamp connections were used for mating studies. Self-crossings for each strain were carried out in order to obtain the mating types. Haplonts were confronted in pairs in Petri dishes using 7 mm diameter blocks as inoculum. Plates were incubated in the dark at 25 °C,and after one 234 week. Mycelia were screened for clamp connections within the contact zone and on the obverse sides of donor inoculum blocks under the microscope (Burnett, 1968). Presence of clamp connections is considered as a positive mating result, which means that both strains are compatible (+); absence of clamp connections indicates incompatibility (-). For interspecific crossings, mating types of monosporic cultures obtained from morphologically determined collections were confronted with test strains The results of these crossings allowed us to confirm or reject such identifications. To confirm identification of Pleurotus collections with similar micro- and macromorphology, two or three monosporic isolates were confronted. When monosporic cultures of doubtful species were lacking, di-mon crossings (the Buller phenomenon) were undertaken (Boidin, 1980)~ Table I: List of Pleurotus isolatesfrom Argentina usedin the study. Species BAFC N° P. albidus 2787 Locality Substrate Date Collector 809 t315 101 1221 695 136 235 Species BAFC N° P. djamor P. djamor A. Martinez 10.06.1989 1100 Capital Federal, University City Córdoba, La Punilla Misiones, Pto. Libertad On fallen trunk 25. 08. 1999 E. Lechner 73 On Populus sp. 25.02.2001 On dead tree of Araucaria angustifolia N. Manero & B.J. Lechner 10.04. 1985 J. Deschamps Buenos Aires, On Platanus sp. March 1994 H. Spinedi La Plata Capital On dead zone 18. 04. 2000 S. Frachia Federal, Barrio of Platanus sp. La Paternal 821 Misiones, El Palmital del cruce On Ficus sp. fallen trunks 28. 05. 2001 E. Albertó 815 Misiones, On fallen trunk 28. 05. 2001 E. Albertó Unknown 18. 04. 1994 Pablo Pica On trunk of Araucaria araucana March djamor var. Collector OnCordylinesp. 188 var. Date Capital Federal 215 cy$tidiosus Substrate 2545 190 P. Locality Palmital roseus El del cruce P. ostreatus 2034 Capital Federal 120 Neuquen, Moquehue Populmonarius 1003 Buenos Aires, Ezeiza 76 Misiones, San Pedro 263 San Pedro, S 26° 32', W 54° 04' 1993 J. del Vas On living 25.07 .1987 J. Deschamps declining tree of Populus sp. On branches 27. 05. 2001 E. Albertó & of Araucaria O. Popoff angustifolia On branches of 27. 05. 2001 D. Krueger A. angustifolia Results Mating types were assigned after se1f-crossingof the 126 obtained monosporic cultures (Tab. II). Among the strains tested, seven strains had four mating types, while three others (BAFC 1003, 815 and 821) had three mating types. Self-crossings from eight monosporic cultures from BAFC 809 resulted all negative; the same occurred with ten monosporic cultures from BAFC 76. However, crossings between 236 monosporic cultures of these strains and monosporic cultures of P. albidus and P. pulmonarius, respectively, resulted positive. The charts of intra- and interspecific confrontations between different cultures are illustrated in Figs. 1-5. AlI four species studied, Pleurotus albidus, P. djamor, P. ostreatus and P. pulmonarius, exhibited a heterothallic tetrapolar sexual compatibility system. Table II. Mating types assigned to monosporic cultures (P. aJbjdus: BAFC 1221, 1350 and 1354, 1399; P. djamor: BAFC 815 and 821; P. ostreatus: BAFC 120,2067; P. pu1monarjus: BAFC 263 and 1003). Monosporic cultures are denoted as numbers of each strain. Mating type of monosporic cultures Species BAFC Monosporic cultures used A,B, A,B2 A2Bl A2B2 Pleurotus albjdus (Berk.) Pegler Pairing among mating types obtained from self-crossing (BAFC 1221, 1350, 1354 and 1399), and monosporic cultures (BAFC 695, 809, 2545, and 2787) with test strains of P. ostreatus, P. djamor, P. pulmonarius and P. cornucopiae (Figs. 1-4) showed no compatible matings. Pairing among Pleurotus albidus strains BAFC 1221 with 1350,1354, 1399,695,809 and 2545 resulted positive (Figs. 2-3). Di-mon crossings between monosporic and polysporic cultures were undertaken (Fig. 5) to confirm taxonomic delimitation of several strains that had tentatively been identified as P. albidus based on morphological characteristics such as pileus of fragile nature, white to cream color and remarkable laciniate-crenate margin (Lechner et al, 2004). Positive results were obtained for BAFC 136, 215, 575, 607, 695, 809, 317, 1221, 1315, 1319, 1330, 1350, 2545 and 2787. These strains constitute clearly an interesterility group within Pleurotus. Several specimens showed great morphological variability, observed 237 not only in collections from nature, but also in fruit bodies obtained in culture. Due to their anastomosed lamellae on the stipe (BAFC 809) some resemble P. cornucopiae, whereas others (e.g. BAFC 136) form more plane and shorter basidiomes, without such characteristics. These variations might suggest the existence of two different species. However, mating tests allowed us to conclude that these morphological variations are intraspecific, and may be possibly caused by environmental constraints. Pleurotus cystidiosus o. K. Miller It was not possible to obtain monosporic cultures ofP. cystidiosus from Argentina since fruit bodies obtained in culture did not produce viable spores. Positive resu1ts of pairings were observed when BAFC 73, determined as P. smithii by Spinedi (1995), and BAFC 188 were BAFC 2067 ~1~¡~O-Ij1-! o (\I ... :)1 + + (J I!. o( al + PO TEN 6689 413611516 o ('I ~ 01 + + u 10. ct al + + BAFC 1354 3 I 9 I 8 I 7 1'.0 o N U LL ct aJ 10 11 Fig. 1: Crossings of monosporic cultures of Pleurotus ostreatus with P. pulmonarius and P. albidus. First row: P. ostreatus (BAFC 120) crossed with P. ostreatus (BAFC 2067) and P. albidus (BAFC 1354 and BAFC 1350); Second row: P. ostreatus (BAFC 120) crossed with P. ostreatus (TEN 6689), P. pulmonarius (TEN 4203) and with P. ostreatus (BAFC 2034); Third row: P. ostreatus (BAFC 2067) crossed with P. albidus (BAFC 1354 and BAFC 1350) and P. pulmonarius (TEN 4203). 238 crossed with monosporic cultures of strain P. cystidiosus MUCL 28690, showing that both belong to P. cystidiosus interesterility group (Fig.5). Macro- and micromorphological studies are consistent with this result. The collections of P. smithii showed the presence of pileocystidia (Lechner et al., 2004), absent in the original description of this speciesgiven by Guzman (1975). PJeurotus djamor(Fr.) Boedijn Monosporic cultures of two collections (BAFC 815, BAFC 821) were confronted with test strains of P. djamor. Positive results confirmed our morphological identification (Fig. 4). Di-mon crossing between the monosporous culture of P. djamor from TENN and MUCL 35018, identified as P. ostreatoroseus Singer, resulted positive tOO. BAFC 2034 8 I 10 I 4 I 5 I-"' o N U 11. ~ ~j al + 11l PPULM TEN l10 ¡ 1 114 I 9 r-ID ~ C.) ~ a3 ~ 7 10 11 BAFC 1221 5 "" II) M .- U IL < ID I 6 I 9 112 BAFC 5 /13 1350 11~1-IJ PO TEN 6689 413611516 + + I + + Fig. 2: Crossings of monosporic cultures of Pleurotus strains. First row: Pleurotus ostreatus crossed with P. ostreatus (BAFC 2034), P. albidus (BAFC 695 and BAFC 2545) and P. pulmonarius (BAFC 1003); Second row: P. ostreatus crossed with P. pulmonarius (TEN 4203), P. albidus (BAFC 1399, BAFC 809, BAFC 2787); Third row: P. albidus crossed with P. albidus (BAFC 1221, BAFC 1350), and P. ostreatus (TEN 6689) 239 PJeurotus ostreatus (Jacq.: Fr.) Kummer Results of intraspecific crossing showed that the three collections (BAFC 120, 2034 and 2067) belong to the same species (Figs.1-2). BAFC 1003, which initially was identified as P. ostreatus based exclusively on morphological studies, resulted negative in crossing with BAFC 2067 (Fig. 2). Pleurotus pulmonarjus (Fr.) Quélet Pairing among test strains of P. pulmonarius with BAFC 76, 263 and 1003 resulted positive (Fig.4). Pairing between BAFC 263 and 2067 of P. ostreatus resulted not compatible (Fig.4). We selected interspecific crossing between strains of P. ostreatus and P. pulmonarius, because both species are very similar. It is very difficult, if ~ 1399 AFC 5 8 7 Fig. 3: Crossings of monosporic cultures of Pleurotus strains. First row: P. albidus (BAFC 1354), crossed with P. djamor (TEN 6346), P. pulmonarius (TEN 4203) and with P. cornucopiae (TEN 8763); Second row: P. albidus crossed with P. albidus (BAFC 1350), P. cornucopiae (TEN 8763) and P. pulmonarius (TEN 4203); Third row: P. albidus (BAFC 1221) crossed with P. albidus (BAFC 1399, BAFC 695, BAFC 809, BAFC 2545). 240 not impossible to separate P. ostreatus from P. pulmonarius based exclusively on micro- and macroscopic features. Discussion Our mating compatibility studies allow to confirm five interesterility groups in Argentina: Pleurotus albidus, P. cystidiosus, P. djamor, P. ostreatus and P. pulmonarius. The P. albidus interesterility group is attributed to a larger sample of isolates that were available from a wide variety of locations and substrates throughout Argentina. In previous studies on mating relationships in Pleurotus, extensive intracollection mon-mon pairings (e.g. Anderson et al., 1973; Hilber, 1982; Bresinsky et al., 1987, Petersen, 1995b) and dimon pairings (e.g. Vilgalys et al., 1993) were performed to determine BAFC 76 BAFC 1350 15 11011317 "' <0 N U IL <C ID 12 13 11 Fig. 4: Crossings of monosporic cultures of Pleurotus strains. First row: P. pulmonarius (TEN 4203) crossed with P. pulmonarius (BAFC 76, BAFC 1003, BAFC 263); Second row: P. pulmonarius (BAFC 263) crossed with P. albidus (BAFC 1350), P. pulmonarius (BAFC 263) and P. ostreatus (BAFC 2067); Third row: P. djamor (TEN 6346) crossed with P. djamor (BAFC 821, BAFC 815) and P. albidus (BAFC 1221). 241 mating types from each individual prior to performing intercollection matings. In this study, we attempted to maximize the number of intercollection matings by using mon-mon and di-mon pairing with testers from known interesterility groups in Pleurotus. The extensive collection data available for most isolates from this study support our view that the interesterility groups represent good species that differ in a number of significant morphological characteristics (Lechner et al., 2004). P. cystidiosus is a widespread species covering large regions of the northern hemisphere (North America, Europe and Asia) as well as the southern part of Africa (Zervakis, 1998). For Central and South America there is a record of P. smithii in Mexico (Guzmán, 1975), MONOSPORIC BAFC I BAFC 809 6 I BAFC 136- I BAFC 215 + I BAFC 695 18 + 14 15 1221 16 BAFC 2067 I BAFC 1350 11215113 + CUL TURE (n) l4T7l1O + + I + BAFC 575 BAFC 607 BAFC 809 c + .s w ~ ~ ~ ~ u 1Z o ~ ct u c BAFC 317 BAFC 1315 BAFC 1319 1 BAFC 1330 BAFC 254!; BAFC 278~r BAFC 203'~ BAFC 2067 BAFC 188 BAFC 73 + + + + BAFC 100:3 MUCL 35018 Fig.5: Di-mon crossing between monosporic cultures of P. albidus (BAFC 809, BAFC 695, BAFC 1221, BAFC 1350), P. ostreatus (BAFC 2067), P. pulmonarius (TEN 4203), P. djamor (TEN 6346) and P. cystidiosus (MUCL 28690) with polysporic cultures of P. albidus (BAFC 136, BAFC 215, BAFC 575, BAFC 607, BAFC 809, BAFC 317, BAFC 1315, BAFC 1319, BAFC 1330, BAFC 2545, BAFC 2787), P. ostreatus (BAFC 2034, BAFC 2067), P. cystidiosus (BAFC 188, BAFC 73), P. pulmonarius (BAFC 1003) and P. djamor (MUCL 35018). 242 Peru (Guzmán et al., 1980), Cuba (Rodríguez-Hernández & CaminoVilaró 1990), Brazil (Capelari, 1999) and Argentina (Spinedi, 1995). Capelari & Fungaro (2003) studied the growth rate in cu1ture, dikaryon-monokaryon matings, and genetic variability. They concluded that the criteria used to separate P. cystidiosus from P. smithii are unsatisfactory and shou1d be considered synonym. The macroand micromorphological study of Spinedi 's collections made in a previous work (Lechner, et al. 2004) and positive results of pairings between cultures of P. smithii and P. cystidiosus here obtained, permit us confirm the supposed synonymy of these two taxa, and to reaffirm that P. cystidiosus is the only synnematoid (producing an anamorph with synnemata) Pleurotus species in South America. Some Pleurotus species can be characterized by a unique range of distribution in Argentina. For example, the P. cystidiosus group is distributed a1ongeastern Argentina, in a temperate zone; the P. ostreatus group extends from southern to centra1Argentina, the cold to temperate area; the P. djamor group occurs in the subtropical northeast Argentina; P. pulmonarius is distributed over two broad geographic regions in both northern (subtropical) and central (temperate) Argentina. P. albidus was collected from central to north of Argentina, in great diversity of substrates from diverse species of living trees to stumps and fallen trunks. According to Albertó et al. (2002) P. albidus fruits from Central America to central Argentina. Pleurotus cystidiosus, P. djamor, P. ostreatus and P. pulmonarius intersterility groups show strong preferences for hosts tree species (Tab. I): P. albidus (also known in Argentina as P. laciniatocrenatus Speg.) was collected in great diversity of substrates from diverse species of living trees to stumps and fallen trunks. In summary, interspecific crossings permit to separate morphologically very similar species like P. pulmonarius and P. ostreatus (Lechner et al., 2002). Moreover, mating tests confirmed the synonymy of P. smithii with P. cystidiosus, and helped to unambiguously identify strains with differing micromorphology within P. albidus. Sexual compatibility studies confirmed the heterothallic tetrapolar mating type of all Argentinean Pleurotus species,with the presence of mu1tiple alleles as previously reported by Hilber (1982) and Petersen (1995a). Acknowledgements We wish to thank the curators of BAFC and MUCL culture collections for making available strains. Mario Rajchenberg (Esquel, Argentina), who provided the collection and strain of P. ostreatus from Patagonia, Argentina. Dr. R.H. Petersen (Knoxville, TN) for supplying monosporic cultures of several species of Pleurotus. The Argentina National Park is thanked for permission to collect in protected areas. 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(Manuscript accepted lO January 2005) 245