On-line version ISSN 1852-5962
Kurtziana vol.37 no.1 Córdoba Jan./June 2012
Molecular techniques based on ITS analysis: useful tools for the identification of wood-decay Basidiomycetes in urban trees?
Carolina A. Robles 1, Cecilia C. Carmarán 1 & Silvia E. Lopez 1
1 PROPLAME-PRHIDEB-CONICET, Departamento de Biodiversidad y Biología Experimental, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, PB II, 4to piso, (CP1428EHA) Ciudad Autónoma de Buenos Aires, Argentina. E-mail: firstname.lastname@example.org; email@example.com; firstname.lastname@example.org
The presence and identity of wood-decay Basidiomycetes affecting Platanus acerifolia were evaluated in Buenos Aires City during 2007. From 247 samples of basidiomes and wood with signs of decay, 19 strains were isolated, from which 10 rDNA sequences were obtained. The aim of this work was to analyze the scope of information obtained from ITS sequences as taxonomic tools to study local populations of wood-rotting fungi in urban trees. Phylogenetic analyses were performed under static and dynamic homologies. The techniques employed enable to: identificate Coprinellus micaceus and corroborate the identification of Bjerkandera adusta, while these same results were not achieved for Inonotus rickii, Ganoderma resinaceum, Peniophora laxitexta and Phlebiopsis gigantea due to the complexity of the genera and the lack of sequences. Lack of sequences at a regional level would be a major obstacle for the detection of wood decay agents in urban trees using ITS sequences.
Key words: Wood-decay fungi; Urban trees; Phylogenetic analyses; Bjerkandera adusta; Coprinellus micaceus; Ganoderma resinaceum; Inonotus rickii; Peniophora laxitexta; Phlebiopsis gigantea.
Técnicas moleculares basadas en análisis de ITS: ¿herramientas útiles para la identificación de Basidiomycetes degradadores de la madera en árboles urbanos?
Durante 2007 se evaluó la presencia e identidad de Basidiomycetes xilófagos que afectan a Platanus acerifolia en la Ciudad de Buenos Aires. Sobre la base de 247 muestras de basidiomas y madera con signos de pudrición se aislaron 19 cepas, a partir de las cuales se obtuvieron 10 secuencias correspondientes a rADN. El objetivo de este trabajo fue analizar la utilidad de la información brindada por las secuencias correspondientes al fragmento ITS en la identificación taxonómica de poblaciones locales de hongos xilófagos en árboles urbanos. Se realizaron análisis filogenéticos bajo homologías estáticas y dinámicas. Los resultados obtenidos fueron comparados con las identificaciones morfológicas y de cultivo previas de las cepas. Las técnicas empleadas permitieron: identificar a Coprinellus micaceus y corroborar la identificación de Bjerkandera adusta, mientras que no se lograron los mismos resultados, debido a la complejidad de los géneros y a la ausencia de secuencias, para Inonotus rickii, Ganoderma resinaceum, Peniophora laxitexta y Phlebiopsis gigantea. La falta de secuencias a nivel regional aparece como uno de los principales obstáculos para la detección de agentes de pudrición de la madera en árboles urbanos, mediante los análisis basados en secuencias ITS.
Palabras clave: Hongos de pudrición; Árboles urbanos; Análisis filogenéticos; Bjerkandera adusta; Coprinellus micaceus; Ganoderma resinaceum; Inonotus rickii; Peniophora laxitexta; Phlebiopsis gigantea.
Urban trees provide invaluable benefits for well-being of people, and their phytosanitary condition is an important issue in the urban planning. Understanding the fungi involved in tree damage can be useful to estimate the damage severity. Detection and identification of decay fungi by molecular tools were used in several recent studies (Adair et al., 2002; Jellison et al., 2003; Nicolotti et al., 2009). They are a promising alternative for specific, sensitive and rapid routine diagnoses. PCR-based methods using nuclear or mitochondrial ribosomal DNA (rDNA) loci have proven valuable for fungal detection and identification at different taxonomic levels (Guglielmo et al., 2007; 2010). One of the present trends concerning diagnosis of wood rot is the analysis of fungal DNA extracted directly from wood (Hoegger & Kües, 2007). At the same time, few studies involving DNA techniques were carried out in relation with fungi in urban trees (Guglielmo et al., 2007; 2008; 2010; Robles et al., 2011).
London plane (Platanus acerifolia) is one of the most frequent urban tree species in Buenos Aires City (Filippini et al., 2000) and it is one of the most affected species by fungal pathogens (Sede & Lopez, 1999).
The aim of this work was to analyze the scope of information obtained from ITS sequences as taxonomic tools to study local populations of wood-rotting fungi in urban trees.
Material and methods
A survey was carried out in Buenos Aires City during 2007. Wood samples, basidiomes and conidiomata were removed from a total of 247 London plane trees showing cankers, cavities or debarking areas in their trunks or lower branches (Robles et al., 2011). All the samples obtained were processed and plated onto Malt Extract Agar (MEA) 1.2%. The cultures were examined every 2-3 days in order to detect Basidiomycetes isolations. The criteria applied for the selection of potential Basidiomycetes strains were the lack of sporulation, the presence of clamp connections and the appearance of the colony. A total of 19 axenic strains were obtained and identified by means of keys based on mycelial characters (Deschamps & Wright, 1975; Nobles, 1965; Stalpers, 1978) and morphological characters of basidiomes. These strains were incorporated to the culture collection of the Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires (BAFCcult).
A total of sixteen strains were selected for DNA extractions and the ITS1 and ITS2 regions were amplified. The nucleotide sequences determined in the present study were deposited in the GenBank DNA sequence database. Their accession numbers are listed in Tables 1-6.
Bjerkandera sp. strains, GenBank accession number and origin of the sequences. N.d.: no data available*: unpublished paper.
Coprinopsis sp., Coprinellus sp. and Psathyrella sp. strains, GenBank accession number and origin of the sequences. N.d.: no data available.*: unpublished paper. +: published only in database.": direct submission.
Ganoderma sp. strains, GenBank accession number and origin of the sequences. N.d.: no data available.*: unpublished paper.
Inonotus sp. and Phellinus sp. strains, GenBank accession number and origin of the sequences. N.d.: no data available*: unpublished paper.": direct submission.
Peniophora sp. strains, GenBank accession number and origin of the sequences. N.d.: no data available.*: unpublished paper.": direct submission.
Phlebiopsis sp. and Phlebia sp. strains, GenBank accession number and origin of the sequences. *: unpublished paper.
For genotypic analyses, the cultures used for the molecular studies were grown on MEA 1.2%. The cultures were incubated in the dark at 25°C for 21 days. DNA was extracted from the cells by using the UltraCleanTM Microbial DNA Isolation Kit (MO BIO laboratories inc., Solana Beach, USA), according to the manufacturer's instructions. The ITS region of the strains was amplified by using the universal primers ITS1 and ITS4 (White et al., 1990) under the following PCR conditions: (1) 94 °C for 5:00 min; (2) 94 °C for 1:00 min; (3) annealing at 58 °C for 1:00 min; (4) extension at 72 °C for 1:30 min; (5) 35 times to 2; and (6) a final extension of 72 °C for 7:00 min. PCR reaction mix was prepared as follows: dNTPs (1.85 µl) at 1 mM, primers ITS1 and ITS4 (0.5 µl ) at 10 mM each, Taq polymerase (0.2 µl) at 5 U/µl, MgCl2 (1.25 µl) at 50 mM and the reaction buffer supplied by the manufacturer (2.5 µl). In some cases, best amplification results were achieved by adding 1 µl of 2 % bovine serum albumin (BSA, Promega Corp.) to the PCR reaction mix. PCR products were purified using a QIAGEN Gel Extraction kit (QIAGEN Inc.). Both strands of each fragment were sequenced by Macrogen DNA sequencing service. Sequences obtained were compared with sequences from GenBank (Tables 1-6).
Following the guidelines expressed by Hibbet et al. (2011) and Nilsson et al. (2011), environmental samples of NCBI BLAST were tested in order to identify sequences with high similarity, without significant results.
DNA sequences were edited with the software program BioEdit sequence alignment editor, version 188.8.131.52 (Hall, 1999). A sequence of Amanita muscaria (EU346871) was chosen as an outgroup in all the analyses. The phylogenetic analysis of the sequence data under static homologies was performed using the parsimony method and NONA version 2.0 (Goloboff, 1997) with all the characters equally weighted and gaps scored as missing data. To determine the support for each clade, bootstrap analyses were performed with 1000 replications. Phylogenetic analysis under dynamic homologies was performed using the software program POY version 4.0.2467 Beta (Wheeler, 2006). All the characters were equally weighted and auto_sequence_partition, build (10) and swap (100) were used as parameters. Bremer support values were calculated for each clade.
Results and Discussion
Ten DNA sequences were obtained, which under phylogenetic analyses produced the following results described and discussed below. Some notes about the species recognized are given.
Bjerkandera adusta (Willd.) Karst. Meddn Soc. Fauna Flora fenn. 5: 38, 1879
Culture descriptions: See Nobles (1965) and Stalpers (1978).
ARGENTINA. Buenos Aires City, C. A. Robles, VII-2007. On Platanus acerifolia. BAFC cult3301.
The strain BAFCcult 3301 (GenBank nº FJ850965) was identified as Bjerkandera adusta by cultural characters. The phylogenetic analyses included sequences from Belice, Canada, China, Japan, Latvia, Lithuania, Poland and Sweden.
Analysis under static homologies resulted in three most parsimonious trees, tree length = 416, CI = 0.94, RI = 0.97. Analysis under dynamic homologies resulted in one retained tree, Cost = 749 (Fig. 1).The results suggest a division of the strains in two main clades without geographical or host correlation. The Argentinean strain appears in the same clade that a Sweden one from Picea abies (Vasiliauskas et al., 2005) with low support.
Fig. 1. The strict consensus trees obtained from Bjerkandera sequences based on ITS sequence data and maximum parsimony analysis under static homologies. Boostrap support values for 1000 replications are shown above branches. Bremer support values for default values are shown below branches. * indicates collapsed clades when Boostrap support was performed. □ indicates clades recovered by both analyses under static and dynamic homologies. In bold type is presented the sequence obtained in this work. The tree was rooted to Amanita muscaria (EU346871).
This is first sequence reported from Argentinean collection of B. adusta.
The position of the strain assigned to Bjerkandera fumosa (GenBank nº FJ903396) suggests a misidentification.
Coprinellus micaceus (Bull.) Vilgalys, Hopple & Jacq. Johnson, Col. Fig. Engl. Fung. Vol. 3, pl. 261, 1800
Culture description: see Robles et al. (2011)
ARGENTINA. Buenos Aires City, C. A. Robles, I, V, VIII-2007. On Platanus acerifolia. BAFC cult 3218 (GenBank nº FJ850970), BAFC cult 3311 (GenBank nº FJ850971), BAFC cult 3312 (GenBank nº FJ850969), BAFC cult 3313 (GenBank nº FJ850968).
Phylogenetic analyses enable identification since it was not possible to obtain basidiomes and there were not previous culture descriptions of this species. We included sequences from Australia, China, Czech Republic, Hawaii, Spain, Sweden and USA. Analysis under static homologies resulted in twenty-eight most parsimonious trees, tree length = 930, CI= 0.61, RI = 0.79. Analysis under dynamic homologies resulted in two retained trees, Cost = 1725. The results suggest division of clades in relation with the species involved without a geographical correlation (Fig. 2). Analyses under both static and dynamic homologies show that all Argentinean strains are located in the same clade, together with members of C. micaceus, from Australia, Hawaii, Spain and unknown origins.
Fig. 2. The strict consensus trees obtained from Coprinellus, Coprinopsis and Psathyrella sequences based on ITS sequence data and maximum parsimony analysis under static homologies. Boostrap support values for 1000 replications are shown above branches. Bremer support values for default values are shown below branches. * indicates collapsed clades when Boostrap support was performed. □ indicates clades recovered by both analyses under static and dynamic homologies. In bold type are presented the sequences obtained in this work. The tree was rooted to Amanita muscaria (EU346871).
Ganoderma resinaceum Boud., in Patouillard, Bull. Soc. mycol. Fr. 5: 72, 1890 
Culture descriptions: See Bazzalo & Wright (1982) and Stalpers (1978).
ARGENTINA. Buenos Aires City, C. A. Robles V, XII-2007. On Platanus acerifolia. BAFC cult 3296, BAFC cult3297.
The strains corresponding to BAFCcult 3296 (GenBank nº FJ850967) and 3297 (GenBank nº FJ850966) were identified as Ganoderma resinaceum by cultural and morphological characters. The phylogenetic analyses included sequences from China, England, Finland, Germany, Netherlands and USA. Analysis under static homologies resulted in five most parsimonious trees, tree length = 366, CI = 0.7, RI = 0.81. Analysis under dynamic homologies resulted in four retained trees, Cost = 1048. The results suggest a division of clades without a clear relation with the species involved. Analyses show a clade formed with strains of G. resinaceum and Ganoderma pfeifferi; a second clade containing strains of Ganoderma lucidum and Ganoderma tsugae and a third clade formed with strains of Ganoderma tsugae, Ganoderma australe and Ganoderma adspersum. Argentinean strains locate separately from European and Chinese G. resinaceum's strains, in a separate clade from all other species. There are partial ITS sequences of Ganoderma strains from Argentina. However, these were not detected by BLAST and due to their small size they were not considered in the analyses. Taking into consideration the macro and micro-morphological characters and given the complexity of the relations inside the genus, already noted by Gottlieb et al. (2000), a conservative position was adopted and the strains of this study were located inside the species Ganoderma resinaceum. The above-mentioned complexity might be solved by testing the level of resolution of alternative genes (Fig. 3).
Fig. 3. The strict consensus trees obtained from Ganoderma sequences based on ITS sequence data and maximum parsimony analysis under static homologies. Boostrap support values for 1000 replications are shown above branches. Bremer support values for default values are shown below branches. * indicates collapsed clades when Boostrap support was performed. □ indicates clades recovered by both analyses under static and dynamic homologies. In bold type are presented the sequences obtained in this work. The tree was rooted to Amanita muscaria (EU346871).
Inonotus rickii (Pat.) D.A. Reid, Kew Bull. 12: 141, 1957.
Culture description: See Wright & Iaconis (1955).
ARGENTINA. Buenos Aires City, C. A. Robles, V, IX-XII-2007. On Platanus acerifolia. BAFC cult 3319.
This Inonotus strain (BAFCcult 3319; GenBank nº GU016326), showed cultural and morphological characters similar to those of I. rickii (Robles et al., 2011). The phylogenetic analyses included sequences from Argentina, Canada, China, Germany, Spain and USA. Analysis under static homologies resulted in five most parsimonious trees, tree length = 1248, CI = 0.57, RI = 0.65. Analysis under dynamic homologies resulted in one retained tree, Cost = 2135. Under static homologies, the strain of this study is located in the same clade as the rest of the strains of I. rickii, these from China and Argentina, together with two strains of Inonotus patouillardii, one of them from Argentina (Fig. 4). These results support those obtained by Gottlieb et al. (2002) and indicate that this technique is not able to solve the conflict between I. rickii and I. patouillardii's identification.
Fig. 4. The strict consensus trees obtained from Inonotus and Phellinus sequences based on ITS sequence data and maximum parsimony analysis under static homologies. Boostrap support values for 1000 replications are shown above branches. Bremer support values for default values are shown below branches. * indicates collapsed clades when Boostrap support was performed. □ indicates clades recovered by both analyses under static and dynamic homologies. In bold type is presented the sequence obtained in this work. The tree was rooted to Amanita muscaria (EU346871).
Peniophora laxitexta C.E. Gómez, Darwiniana 20(1-2): 195, 1976.
Culture description: See Gómez & Loewenbaum (1976).
ARGENTINA. Buenos Aires City, C. A. Robles, IX-2007. On Platanus acerifolia. BAFC cult 3309.
The strain corresponding to BAFCcult 3309 (GenBank nº FJ882040) was identified as Peniophora laxitexta by cultural characters. The phylogenetic analyses included sequences of several species of the genus from the Northern Hemisphere, Lithuania and Sweden. Analysis under static homologies resulted in one most parsimonious tree, tree length = 531, CI = 0.86, RI = 0.87. Analysis under dynamic homologies resulted in one retained tree, Cost = 992.
The topology of the tree sustains the species recognized for the genus. The position of the Argentinean strain is consistent with the identification of BAFCcult 3309 as a different species inside the Peniophora genus. To our knowledge, this is the first report of a DNA sequence of P. laxitexta (Fig. 5).
Fig. 5. The strict consensus trees obtained from Peniophora sequences based on ITS sequence data and maximum parsimony analysis under static homologies. Boostrap support values for 1000 replications are shown above branches. Bremer support values for default values are shown below branches. * indicates collapsed clades when Boostrap support was performed. □ indicates clades recovered by both analyses under static and dynamic homologies. In bold type is presented the sequence obtained in this work. The tree was rooted to Amanita muscaria (EU346871).
Phlebiopsis gigantea (Fr.) Jülich, Persoonia 10 (1): 137. 1978.
Descriptions: See Deschamps & Wright (1975) and Nakasone (1990).
ARGENTINA. Buenos Aires City, C. A. Robles, VIII-2007. On Platanus acerifolia. BAFC cult 3318.
Cultural studies suggested that the strain corresponding to BAFCcult 3318 (GenBank nº FJ850972) belongs to Phlebiopsis gigantea. The phylogenetic analyses included sequences of specimens from gymnosperms and from forest soil without any additional information about the host. The reports are from Canada, Finland, Lithuania, Poland, Sweden and USA. There are some reports for this species in the Southern Hemisphere (Deschamps & Wright, 1975; Vainio, 2008). Analysis under static homologies resulted in three most parsimonious trees, tree length = 315, CI = 0.75, RI = 0.59. Analysis under dynamic homologies resulted in two retained trees, Cost = 821. In both phylogenetic analyses, the Argentinean strain is associated, with a high support value, with the clade that contains most of the P. gigantea strains incorporated in the analyses. This result suggests that the Argentinean strain belongs to P. gigantea (Fig. 6). However, the strain BAFCcult 3318 remains in a separated clade from the rest of strains analyzed. P. gigantea is quite common in the gymnosperm-dominated forests of the northern hemisphere, so new additions of regional strains to these analyses are necessary to determinate if this topology indicates that Argentinean strains, or maybe those from the Southern Hemisphere, represents a cluster, and analyze the taxonomic implications of it.
Fig. 6. The strict consensus trees obtained from Phlebiopsis and Phlebia sequences based on ITS sequence data and maximum parsimony analysis under static homologies. Boostrap support values for 1000 replications are shown above branches. Bremer support values for default values are shown below branches. * indicates collapsed clades when Boostrap support was performed. □ indicates clades recovered by both analyses under static and dynamic homologies. In bold type is presented the sequence obtained in this work. The tree was rooted to Amanita muscaria (EU346871).
Regarding the use of molecular data as a tool in fungal identification, it should be noted that Vialle et al (2009) evaluated several mitochondrial genes as DNA barcode but pointed out that no single mitochondrial gene gave a better taxonomic resolution than the ITS, the region already widely used in fungal taxonomy. The internal transcribed spacer (ITS) of nuclear DNA (nrDNA) has been proposed (Bellemain et al., 2010) as the official primary barcoding marker for fungi (Deliberation of 37 mycologists from 12 countries at the Smithsonian's Conservation and Research Centre, Front Royal, Virginia, May 2007).
Our work shows some of the scopes of ITS region as taxonomic tool. The results exhibit dissimilar situations. In the case of Coprinellus micaceus, phylogenetic analyses lead to the identification without basidiome or previous culture descriptions. For example in Peniophora laxitexta, molecular tools were not useful because there were not previous sequences of this species. In this case the study is limited by the lack of sequences in the databases. In other cases these limitations lies at regional level, as in the case of Inonotus rickii, Ganoderma resinaceum and Phlebiopsis gigantea. We consider that the latter is probably the commonest obstacle in the development of studies in the frame of detection of wood decay in urban trees. However, the type of analysis tested here, combined with the knowledge of the host species, can be useful for a good approach at genus level of wood-rotting fungi.
In the future, direct analyses of DNA (environmental samples) on wood would be useful only if they could be compared with sequences from correctly identified specimens, with metadata about host, information about georefence and the access to the final results in the published paper. In order to come to this point it is required to increase the size of databases with molecular data from local populations. However, it is still necessary to conduct studies involving morphological and culture aspects to complement diagnosis of wood-rot fungi to ensure the taxonomic positions of obtained sequences.
This work was supported by CONICET PIP 1482, ANPCyT PICT 2007/01346 and University of Buenos Aires, Argentina UBACYT X122. The authors gratefully thank Dr. Gerald Walter for his assistance with the sequences of Coprinellus micaceus. Publication number 188 PRHIDEB CONICET.
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Original recibido el 30 de Junio de 2011;
primera decisión: 19 de Agosto de 2011;
aceptado el 17 de Octubre de 2011.
Editor responsable: Gerardo Robledo.