Baldoni DB1*, Coelho G2, Jacques RJS3, Silveira RMB4 and Antoniolli ZI5
1*Programa de Pós-graduação em Ciências do Solo. Universidade Federal de Santa Maria. Av. Roraima nº 1000 -
Campus - Bairro Camobi - CEP: 97105-900, Santa Maria – Rio Grande do Sul, Brasil. E-mail: [email protected]
2Departamento de Fundamentos da Educação, Universidade Federal de Santa Maria 4Departamento de Botânica, Universidade Federal do Rio Grande do Sul
3,5Departamento de Solos, Universidade Federal de Santa Maria
Baldoni DB, Coelho G, Jacques RJS, Silveira RMB and Antoniolli ZI – Sarcoporia
polyspora: a rare wood-degrading Basidiomycota is newly recorded to South America
Mycosphere 1, 1–8
Abstract
Brown-rotting fungi and coniferous forests have a possible common and interesting co- evolutionary history particularly in the North Hemisphere. These fungi degrade almost exclusively wood cellulosic materials participating heavily in the carbon cycle and soil formation, although being less diverse than white-rotters, in their ecosystems and exotic forests known as important sources of energy and wood production. Studying wood- inhabiting fungi in cultivated pine forests in Southern Brazil, we have collected and analyzed morphologically and molecularly (ITS region) a rare fungus named Sarcoporia polyspora, which has shown to be an aggressive decompositor previously unreported from South America.
Key words – biodegradation – macrofungus – Pinus sp. wood – soil humus
Article Information
Received *** Accepted ***
Published online ****
*Corresponding author: Daiana Bortoluzzi Baldoni – e-mail – [email protected]
Introduction
Fungi are able to modify their substrates secreting enzymes that act extracellularly and other compounds; they find their niche in forests either in the leaf litter, in soil organic matter (humus) or in wood, as in the case of most Basidiomycota, which attack its primordial compounds – cellulose, hemicelluloses and lignin. Brown rotters degrade almost exclusively cellulosic materials, are not predominant in the group, but present a possible interesting co- evolutionary history particularly within coniferous forests, where they participate heavily in the carbon cycle and soil formation.
Species associated with brown rots were firstly assigned as representing about 7% of American wood-rotting Basidiomycota (120 from 1700 sp), 85% occurring on coniferous substrates, and estimated as being less than 10 % in Europe. The total number of brown- rotting polypores was reported as being 22% (77 from 322) in Europe and 18% (79 from
about 439) in North America, whereas a lower percentage of 14% (98 from 704) was found in China, for instance. In all cases, the majority of brown-rotting polypores were found on gymnosperm wood in boreal forests; except to a higher percentage of 38,7% (25 from 62) reported to Patagonian Andes (Southern Argentina) mostly on Nothofagus species, Fagaceae, (Gilbertson 1980, Gilbertson and Ryvarden 1986, Ryvarden & Gilbertson 1993, Rajchenberg 2006, Dai 2011).
Sarcoporia polyspora P. Karsten is an aggressive brown-rotting polypore characterized
by producing dextrinoid spores. Currently included in the family Fomitopsidaceae (Kirk et al. 2008), it was originally described in 1894, but after redescribed or recombined under different names, as morphologically discussed by Vlasak and Kout (2010). The species has been most referred under the name Parmastomyces transmutans (Overh.) Ryvarden & Gilb. (1984, 1994), especially after Ryvarden (1991), who stated that Sarcoporia would be morphologically more similar to Hapalopilus, suggesting its synonymy with a polypore genus characterized by lacking dextrinoid reaction in spores and producing white rot.
The species has been considered as common to North America, with several records to Asia, but rare in Europe and North Africa. The aim of this study is to contribute to the knowledge about this rare and potentially useful species providing morphological and molecular insights, besides extending its geographic distributional data to cultivated pine forests in Southern Brazil.
Methods
Fungal material collections and morphological characterization
Basidiomes were collected in cultivated areas with Pinus spp. at FEPAGRO/Florestas (Fundação Estadual de Pesquisa Agropecuária) in Santa Maria, Rio Grande do Sul State, Southern Brazil. Specimens were observed, photographed and collected in plastic pots or absorbent paper for transporting to Prof. Marcos Rubens Fries Soil Biology and Environmental Microbiology Laboratory, CCR/UFSM; after they were analyzed and preserved. Specimens are extant at Instituto de Ciência Naturais - UFRGS, Brasil, ICN Herbarium (acronyms are according to: http://sweetgum.nybg.org/ih/). Munsell Soil Color Charts (1994) were used for naming colors. Microscopical analyses were performed through handmade sections of fresh basidiomes using a razor blade and mounting on glass slides with drops of KOH 5% plus aqueous phloxine or drops of Melzer’s reagent. Statistical analysis of measurements was performed with EXCEL® (MS Office 2003), its abbreviations are presented conform Coelho (2005). Authors of fungal names follow Kirk & Ansell (2008). Coniferous substrates are abbreviated as: Pt. for P. taeda L. and P.sp for unidentifyed Pinus species.
Molecular analysis
DNA was extracted from parts of the basidiomes using a DNeasy Plant Mini Kit® (Qiagen, São Paulo, Brazil). The complete ITS region in nrDNA (ITS1-5.8S-ITS2) was amplified with ITS1 and ITS4 primers (White et al. 1990). Amplification reaction was performed conform Lupatini et al. (2008). After amplification, electrophoresis was performed to check the amplification in 1.5% agarose gel and 1X TBE buffer (90 mM Tris-borate, 2 mM EDTA, pH 8.0). DNA was stained with BlueGreen Loading Dye I® (LGC Biotecnologia, Cotia, Brazil) and observed in ultraviolet light. PCR products were purified with the Gen Elute PCR clean-up Kit® (Sigma, Saint Louis, USA) following manufacturer's instructions and sequencing was carried out in Mega BACE sequencer 500 (Amersham Biosciences).
Sequenced fragments were analyzed using the program Staden Package 2.0.0b (Staden et al. 2003). A BLASTn search of the National Center for Biotechnology Information
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databases verified that the sequence obtained from Sarcoporia polyspora was affiliated to the Polyporaceae. Fungal sequences (ITS1-5.8s-ITS2) obtained in our laboratory were aligned with other sequences from GenBank using ClustalX (Larkin et al. 200 ) and in BioEdit (Hall ) to obtain a final alignment (Table ). The sequence was deposited at enBank (Altschul et al. ) under the accession number 26 52 .
The phylogeny was reconstructed by ITS1-5.8S-ITS2 analyses. The General Time Reversible model nucleotide substitution model was estimated using FindModel run in May, 11, 2012. The Maximum Likelyhood analysis was performed in MEGA 5.0 (Tamura et al. 2011) where 1000 bootstrap replicates were used in all reconstructions. Pseudomerulius
curtisii GU187536 and GU187533 sequences were used as outgroups.
Table 1 Specimens of fungi included in this study. In bold the accession Genbank
number referred to the sequences obtained from Sarcoporia polyspora in Santa Maria, Southern Brazil.
Species Strain Locality
GenBamk acession number Antrodia carbonica (Overh.) Ryvarden & Gilb. FP 105585-R Taiwan EU232211
Antrodia sinuosa (Fr.) P. Karst. RLG1182R Taiwan AY966450
Antrodia sinuosa (Fr.) P. Karst. 155753 Sweden JQ518273
Antrodia sp. P. Karst. AFTOL-ID 1504 USA DQ484059
Antrodia xantha (Fr.) Ryvarden FCUG 100 Taiwan EU232209
Antrodia xantha (Fr.) Ryvarden P209 (G185) Turkey AJ415569
Oligoporus placentus (Fr.) Gilb. & Ryvarden P100 Germany AJ416069
Oligoporus placentus (Fr.) Gilb. & Ryvarden P120 (FPRL 280) Germany AJ249267
Parmastomyces mollissimus (Maire) Pouzar Dai10193 China FJ627250
Sarcoporia polyspora P. Karst. DBB20 Brazil JX268521
Postia placenta (Fr.) M.J. Larsen & Lombard JV0509/174 USA JN592502
Postia placenta (Fr.) M.J. Larsen & Lombard FPRL 280 Germany EF524035
Pseudomerulius curtisii (Berk.) Redhead & Ginns DJL-DR-4
Dominican
Republic GU187536
Pseudomerulius curtisii (Berk.) Redhead & Ginns REH8912 Australia GU187533
Results
Sarcoporia polyspora P. Karsten
= Polyporus subcartilagineus Overh. Overholts L.O. Mycologia 33(1), 1941. = Polyporus transmutans Overh. Mycologia 44: 226, 1952. = Tyromyces kravtzevianus Bondartsev & Parm. In Parm. Mycotheca Estonica I. No. 25. 1957. = Tyromyces mollissimus Maire. In Maire R. Bull. Soc. Hist. Nat. Afrique N. 36. 1945(Figs. 1-2).
Basidiome annual, resupinate, effuse-reflexed to pileate, rarely imbricate, fragile, soft,
fleshy, watery, jelly and putrescent upon aging, firm upon drying, 250 × 80 × 20 mm. Pileus when present broadly-attached, usually limited a small reflexed part, fleshy; pilear surface white (8/1 5YR), (Fig. 1C,D), with shades of yellowish red (5/6–4/6 5YR) to dark reddish brown (3/4 5YR) especially when bruised; pileus surface cottony to fibrous, not very regular, having wrinkles and depressions, sometimes forming discrete concentric zones.
Hymenophore poroid, concolorous to the pileus surface; (Fig. 1A, B), pores round to
polygonal, (1–)2(–3)/mm, Pm= 2.00, n= 97/3; dissepiments thin, velutinous, slightly dentate;
margin of resupinate basidiomes more conspicuous, sterile, broad, up to 20 mm, becoming jelly on aging. Tube layer concolorous to the hymenophore, up to 20 mm thick, dense, forming a palisade, representing the major part of the basidiome. Context concolorous to the hymenophore, thin, up to 1 mm thick, homogeneous, fleshy to gelatinous, easily to macerate, sometimes difficult to distinguish from tube layer.
Hyphal system monomitic. Tramal generative hyphae clamped, (Fig. 2d), thin-
walled to slightly thick-walled, sparsely branched, hyaline, (2–)2.4–4(–4.4) m diam., Dm =
2.9, n = 60/1. Gloeopleurous hyphae wider than tramal hyphae , easily found when intensely stained in phloxine, usually observed as conspicuously hyaline wider hyphae, thin-walled to thick-walled, usually tortuous in outline, often solid, clamped, (5.6–)8.0–20 m diam., Dm =
11.3, n = 60/1; Dm = 2.6, n = 60/1.
Fig. 1. Basidiomes of Sarcoporia polyspora P. Karsten on decayed fallen trunk of Pinus spp. A and B Details from white pored hymenophores. C and D Effused-reflexed basidiomes with
white to reddish brown pilear surface. This picture is copyright of Gilberto Coelho.
Contextual generative hyphae clamped, thin-walled to slightly thick-walled, hyaline
to whitish opaque, sparsely branched, (2.8–)3.2–5.6(–6) m diam., Dm = 4.2, n = 60/1.
Gloeoplerous hyphae wider than tramal hyphae, (Fig. 2e), usually with a denser contents
than contextual hyphae and intensely staining in phloxine, usually observed as conspicuously hyaline wider hyphae, thin-walled to thick-walled, usually tortuous in outline, often solid, clamped, usually difficult to observe, (2.8–)4.4–12(–17.6) m diam., Dm = 7.7, n = 60/1.
Hymenium with basidia clavate (Fig. 2a), hyaline, four-sterigmate, (13.6–)17.6–
22.4(–25.6 × (5.2–)5.6–6.4(–7.6) m, Lm × Wm= 20.01 ± 2.33 × 5.90 ± 0.36; Qr= 2.27–4.62;
Qm= 3.42 ± 0.44, n= 60/1. Basidiospores ellipsoid, (Fig. 2c), narrowly ellipsoid to
subcylindrical, abundant, moderately thick-walled, strongly dextrinoid, often guttulate, (4– )4.4–5.6(–6.0) × (2.0–)2.4–2.8(–3.2) m, Lm × Wm = 4.8 ± 0.49 × 2.49 ± 0.26, Qr= 1.57– 2.40,
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Qm = 1.95 ± 0.21, n = 63/1. Hyphidia inconspicuous, long, hyaline, sometimes ventricose,
narrowing towards the apex, with simple to branched apex, 11–24 x 3.5–6.5 m, n = 8.
Associated wood rot: brown.
Substrate: on fallen coniferous wood on the forest ground, usually in advanced stages
of decomposition, on Pinus taeda L. in the study area.
Sexuality and cultural characters: heterothallic and tetrapolar; see Nobles (1965 as
P. subcartilagineus, see synonymy below species name); Stalpers (1978, as P. kravtzevianus);
nuclear behavior heterocytic (Hibbett & Donoghue 2001, Rajchenberg 2011).
Specimens examined: Brazil, Santa Maria, Boca do Monte, FEPAGRO, leg. G.
Coelho, 03.II.2011, NºGC 338-9 (ICN 139765), on Pinus taeda L.
Fig. 2 - Microscopical characters of Sarcoporia polyspora P. Karsten. a Basidia. b
Cystidiolum. c Basidiospores. d Tramal and contextual generative hyphae. e Gloeopleurous- hyphae from context. This picture is copyright of Gilberto Coelho.
Distribution: To a more complete distributional picture see Vlasak and Kout (2010);
in summary, it was considered as rare in Europe, common in North America (usually as
Parmastomyces transmutans), and reported several times to Asia (in Russia, as P. kravtzevianus, and China, as P. molissimus), (Fig. 3).
Fig. 3 – Geographical distribution of Sarcoporia polyspora. The stars mark the occurrence of
the species on continents, according to a literature review.
Discussion
Sarcoporia polyspora is characterized by presenting basidiomes white to very pale
brown, fleshy to almost gelatinous, resupinate to effuse-reflexed, not rhizomorphic, and large pores (1–3/mm). It also presents a monomitic hyphal system dominated by clamped generative hyphae throughout the basidiomes and gloeopleurous hyphae mainly in the context; a remarkable character is the abundant, ellipsoid to subcylindrical, and dextrinoid basidiospores. The latter feature differentiates it from other brown-rotting species of
Antrodia, Fibroporia, Amyloporia, and Postia (Oligoporus) with white to brownish or
yellowish white resupinate basidiomes.
Hibbett & Binder (2002) formerly found Sarcoporia polyspora (referred there as P.
transmutans) grouped in a polyporoid clade in their study primarily based on 18S rDNA, but
using a small species dataset. In contrast, other multi-locus study performed by Binder et al. (2005) using a larger dataset (656 species, mostly represented only by nrLSU sequences), shown S. polyspora (P. transmutans) into the Antrodia clade; result revisited by Lindner & Banik (2008).
The ML analysis of the same sequence dataset yielded similar phylogenetic trees (Fig. 4). The analyzed sequence from Brazil fell well-supported in an isolated position closely related to that of its synonym P. molissimus (FJ627250), which was elected for inclusion in this study by its recognized morphological similarity.
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Antrodia alpina (EU232207) Antrodia xantha (EU232209) Antrodia sp. (DQ484059)
Antrodia xantha (AJ415569) Antrodia carbonica (EU232211) Antrodia sinuosa (AY966450)
Antrodia sinuosa (JQ518273) Postia placenta (JN592502) Oligoporus placentus (AJ249267) Oligoporus placentus (AJ416069) Postia placenta (EF524035)
Sarcoporia polyspora DBB20 Parmastomyces mollissimus (FJ627250)
Pseudomerulius curtisii (GU187533) Pseudomerulius curtisii (GU187536)
1 0 0 9 9 9 9 9 1 9 9 1 0 0 9 8 7 0 0 .0 5
Fig. 4 – Phylogenetic reconstruction of the Sarcoporia polyspora obtained from ITS1-5.8S-
ITS2 sequences. Bootstrap values (in %) are from maximum likelihood (ML) analyses (1000 bootstraps). Only topologies with bootstrap values of at least 50% are shown.
It was obtained in this study an evident separation between Brazilian sequence, DBB20, and that of P. mollissimus supported by a high boostrap value. It must be noted that it was found an evident distinction between these sequences manifested by a low similarity score of 84% in ITS1-5.8S-ITS2 region; but also between our specimen and the others from the dataset chosen among the most similar sequences retrieved with BLAST program. Furthermore, sequences of ITS1-5.8S-ITS2 region of Sarcoporia polyspora are almost inexistent at GenBank database, becoming difficult to get a more accurate molecular identification and phylogenetic assessment of the specimen.
This study represents a preliminary attempt to access the phylogenetic relationships of
Sarcoporia starting from Brazilian specimens. More studies, however, using ITS and other
genomic regions need to be carried out in order to elucidate phylogenetic relationships and possible internal speciation of a genus supposed to be monotypic (Vlasak & Kout 2010). Conspecificity between our S. polyspora and P. mollissimus (GenBank) sequences was not sufficiently elucidated in this study based on ITS1-5.8S-ITS2 region due the low similarity involved, so there is a need for generating more sequences in the genus and introducing larger datasets in order to obtain a better phylogenetic understand. Additional morphological, biochemical, and molecular studies are still necessary for resolving the status of specimens so widely distributed in the world. These records of Sarcoporia polyspora are the first one from South America.
Acknowledgements
The authors wish to acknowledge the National Council for Scientific and Technological Development (CNPq - Brazil) and the Coordination for the Improvement of Higher Education Personnel (CAPES - Brazil) for their financial support.
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