Uso de la secuenciación de segunda generación (NGS) para descubrir la diversidad de hongos degradadores de la madera en los bosques Andino Patagónicos: Hongos degradadores de la madera en los bosques Andino Patagónicos

Lucia Molina; María Belén Pildain

doi:10.30550/j.lil/2022.59.S/2022.09.22

Authors

Lucia Molina Centro de Investigación y Extensión Andino Patagónico (CIEFAP), Esquel, Chubut, Argentina https://orcid.org/0000-0003-1073-8810
María Belén Pildain CONICET, CIEFAP. Esquel, Chubut, Argentina https://orcid.org/0000-0002-0777-8232

DOI:

https://doi.org/10.30550/j.lil/2022.59.S/2022.09.22

Keywords:

Decay, Hymenochaetales, Illumina, Nothofagus, Polyporales

Abstract

Fungi are the main degraders of wood in forest ecosystems, contributing significantly to the global carbon cycle. Metagenomic approaches based on a specific amplicon (metabarcoding) constitute a powerful tool for their prospecting and study. The main objective of this study was to characterize, through second-generation sequencing NGS, communities of degrading fungi in the sapwood of two species of Nothofagus from the forests of northern Patagonia, to evaluate diversity patterns in sites, seasons, hosts, plant compartments and health conditions, as a contribution to the autoecology of the species of this genus. Our study comprised three main methodological steps: (i) sampling of wood from healthy and diseased living trees of the forest species N. dombeyii and N. pumilio; (ii) DNA extraction, amplification and sequencing of the ITS1 region on the MiSeq Illumina platform, (iii) read processing and data extraction of the orders Polyporales and Hymenochaetales and (iv) data analysis and interpretation. A total of 35 molecular operational taxonomic units amplicon sequence variant -ASV- were obtained, to 23 putative fungal genera in 15 families. Postia pelliculosa was the most frequently detected species in the study. The host was the strongest factor among the variables studied in terms of its effect on the structure and composition of the fungal community analyzed. For N. dombeyi, which is distributed in a wide range of climatic conditions, the site was the strongest shaper of its communities, while for N. pumilio a greater susceptibility to changes in temperature and seasonality was discovered, which are, in fact, relevant factors for the conservation of forests in the current scenario of climate change. This is the first study to use NGS as a rapid, large-scale strategy to elucidate the diversity of wood-degrading fungi in Patagonian temperate forests.

Downloads

Download data is not yet available.

References

Altschul, S., Madden, T. L., Schäffer, A. A., Zhang, J., Zhang, Z., Miller, W. y Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 25 (17): 3389-3402. https://doi.org/10.1093/nar/25.17.3389

Anderson, M. J. (2017). Permutational Multivariate Analysis of Variance (PERMANOVA ). En Wiley StatsRef: Statistics Reference Online (pp. 1–15). Wiley. https://doi.org/10.1002/9781118445112.stat07841

Anslan, S., Nilsson, R. H., Wurzbacher, C., Baldrian, P., Tedersoo, L. y Bahram, M. (2018). Great differences in performance and outcome of high-throughput sequencing data analysis platforms for fungal metabarcoding. MycoKeys 39: 29-40. https://doi.org/10.3897/mycokeys.39.28109

Arantes, V, y Goodell, B. (2014). Current understanding of brownrot fungal biodegradation mechanisms: a review. In: Deterioration and protection of sustainable biomaterials. ACS Publications; p. 3–21.

Bahram, M., Küngas, K., Pent, M., Põlme, S., Gohar, D. y Põldmaa, K. (2022). Vertical stratification of microbial communities in woody plants. Phytobiomes Journal 6 (2): 161-168 . https://apsjournals.apsnet.org/doi/10.1094/PBIOMES-06-21-0038-R

Baldrian, P. (2016). Forest microbiome: diversity, complexity and dynamics. FEMS Microbiology Reviews 41 (2): 109-130. https://doi.org/10.1093/femsre/fuw040

Barroetaveña, C., Salomón, M. E. S. y Bassani, V. (2019). Rescuing the ectomycorrhizal biodiversity associated with South American Nothofagaceae forest, from the 19th century naturalists up to molecular biogeography. Forestry: An International Journal of Forest Research 92 (5): 500-511. https://doi.org/10.1093/forestry/cpz047

Baselga, A., Orme, D., Villéger, S., De Bortoli, J., Leprieur, F. y Logez, M. (2022). Partitioning beta diversity into turnover and nestedness components. En R package version 1.5.6. https://cran.r-project.org/package=betapart

Blanchette, R. A. (1991). Delignification by Wood-Decay Fungi. Annual Review of Phytopathology 29 (1): 381-403. https://doi.org/10.1146/annurev.py.29.090191.002121

Brown, S. P., Veach, A. M., Rigdon-Huss, A. R., Grond, K., Lickteig, S. K., Lothamer, K., Oliver, A. K. y Jumpponen, A. (2015). Scraping the bottom of the barrel: are rare high throughput sequences artifacts? Fungal Ecology 13: 21-225. https://doi.org/10.1016/j.funeco.2014.08.006

Buchanan, P. K. y Hood, I. A. (1992). New species and new records of Aphyllophorales (Basidiomycetes) from New Zealand. New Zealand Journal of Botany 30 (1): 95-112. https://doi.org/10.1080/0028825X.1992.10412888

Callahan, B. J., McMurdie, P. J., Rosen, M. J., Han, A. W., Johnson, A. J. A. y Holmes, S. P. (2016). DADA2: High-resolution sample inference from Illumina amplicon data. Nature Methods 13 (7): 581-583. https://doi.org/10.1038/nmeth.3869

Goodell, B., Winandy, J. E. y Morrell, J. J. (2020). Fungal Degradation of Wood: Emerging Data, New Insights and Changing Perceptions Coatings 10 (12): 1210. https://doi.org/10.3390/coatings10121210

Cunningham, G. H. (1965). Polyporaceae of New Zealand. En D. of S. y I. Research (Ed.), Transactions. https://doi.org/10.2307/3756787

Dai, Y.-C. (2005). Illustrations of Pathogenic Wood-Decaying Fungi in China. Science Press.

Daniels, L. D. y Veblen, T. T. (2004). Spatiotemporal influences of climate on altitudinal treeline in northern Patagonia. Ecology 85 (5): 1284-1296. https://doi.org/10.1890/03-0092

de Errasti, A., de Beer, Z. W., Coetzee, M. P. A., Roux, J., Rajchenberg, M. y Wingfield, M. J. (2016). Three new species of Ophiostomatales from Nothofagus in Patagonia. Mycological Progress 15 (2): 1-17. https://doi.org/10.1007/s11557-016-1158-z

de Errasti, A., de Beer, Z. W., Rajchenberg, M., Coetzee, M. P. A., Wingfield, M. J. y Roux, J. (2015). Huntiella decorticans sp. nov. (Ceratocystidaceae) associated with dying Nothofagus in Patagonia. Mycologia 107 (3): 512-521. https://doi.org/10.3852/14-175

Donoso Zegers, C. (1993). Estructura, variación y dinámica de bosques templados de Chile y Argentina. Ecología Forestal. Editorial Universitaria, Santiago, Chile.

Douanla-Meli, C. (2007). Fungi of Cameroon. Ecological diversity with emphasis on the taxonomy of Non-gilled Hymenomycetes from the Mbalmayo forest reserve. En Bibliotheca Mycologica (Vol. 202).

Doyle, J. J. y Doyle, J. L. (1990). Isolation of plant DNA from fresh tissue. Focus 12: 13-15.

Dumolin, S., Demesure, B. y Petit, R. J. (1995). Inheritance of chloroplast and mitochondrial genomes in pedunculate oak investigated with an efficient PCR method. Theoretical and Applied Genetics 91 (8): 1253-1256. https://doi.org/10.1007/BF00220937

Edgar, R. C. (2010). Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26 (19): 2460-2461. https://doi.org/10.1093/bioinformatics/btq461

Edgar, R. C. (2016). SINTAX: a simple non-Bayesian taxonomy classifier for 16S and ITS sequences. bioRxiv 074161. https://doi.org/10.1101/074161

Edgar, R. C. y Flyvbjerg, H. (2015). Error filtering, pair assembly and error correction for next-generation sequencing reads. Bioinformatics 31 (21): 3476-3482. https://doi.org/10.1093/bioinformatics/btv401

Frøslev, T. G., Kjøller, R., Bruun, H. H., Ejrnæs, R., Brunbjerg, A. K., Pietroni, C. y Hansen, A. J. (2017). Algorithm for post-clustering curation of DNA amplicon data yields reliable biodiversity estimates. Nature Communications 8 (1): 1188. https://doi.org/10.1038/s41467-017-01312-x

Gardes, M. y Bruns, T. D. (1993). ITS primers with enhanced specificity for basidiomycetes - application to the identification of mycorrhizae and rusts. Molecular Ecology 2 (2): 113-118. https://doi.org/10.1111/j.1365-294X.1993.tb00005.x

Grossart, H.-P., Wurzbacher, C., James, T. Y. y Kagami, M. (2016). Discovery of dark matter fungi in aquatic ecosystems demands a reappraisal of the phylogeny and ecology of zoosporic fungi. Fungal Ecology 19: 28-38. https://doi.org/10.1016/j.funeco.2015.06.004

Hatakka, A. (2005). Biodegradation of lignin . En S. Alexander (ed.), Biopolymers Online. Wiley-VCH Verlag GmbH & Co. https://doi.org/10.1002/3527600035.bpol1005

Harrison, J. G. y Griffin, E. A. (2020). The diversity and distribution of endophytes across biomes, plant phylogeny and host tissues: how far have we come and where do we go from here? Environmental Microbiology 22 (6): 2107-2123. https://doi.org/10.1111/1462-2920.14968

Hibbett, D., Abarenkov, K., Kõljalg, U., Öpik, M., Chai, B., Cole, J., Wang, Q., Crous, P., Robert, V., Helgason, T., Herr, J. R., Kirk, P., Lueschow, S., O’Donnell, K., Nilsson, R. H., Oono, R., Schoch, C., Smyth, C., Walker, D. M., … Geiser, D. M. (2016). Sequence-based classification and identification of Fungi. Mycologia 108 (6): 1049-1068. https://doi.org/https://doi.org/10.3852/16-130

Hood, I. A., McDougal, R. L., Somchit, C., Kimberley, M. O., Lewis, A. S. R. y Hood, J. O. L. (2019). Fungi decaying the wood of fallen beech ( Nothofagus ) trees in the South Island of New Zealand. Canadian Journal of Forest Research 49 (1): 1-17. https://doi.org/10.1139/cjfr-2018-0179

Johnston, P. R., Johansen, R. B., Williams, A. F. R., Paula Wikie, J. y Park, D. (2012). Patterns of fungal diversity in New Zealand Nothofagus forests. Fungal Biology 116 (3): 401-412. https://doi.org/10.1016/j.funbio.2011.12.010

Johnston, P. R., Park, D. y Smissen, R. D. (2017). Comparing diversity of fungi from living leaves using culturing and high-throughput environmental sequencing. Mycologia 109 (4): 1-12. https://doi.org/10.1080/00275514.2017.1384712

Küngas, K., Bahram, M. y Põldmaa, K. (2020). Host tree organ is the primary driver of endophytic fungal community structure in a hemiboreal forest. FEMS Microbiology Ecology 96 (2): 1-10. https://doi.org/10.1093/femsec/fiz199

Kunkel, V., Wells, T. y Hancock, G. R. (2016). Soil temperature dynamics at the catchment scale. Geoderma 273: 32-44. https://doi.org/10.1016/j.geoderma.2016.03.011

McMurdie, P. J. y Holmes, S. (2013). phyloseq: An R Package for Reproducible Interactive Analysis and Graphics of Microbiome Census Data. PLoS ONE 8 (4): e61217. https://doi.org/10.1371/journal.pone.0061217

McMurdie, P. J. y Paulson, J. N. (2016). biomformat: An interface package for the BIOM file format. En R/Bioconductor Package.

Molina, L. (2022). Sanidad de los bosques de Nothofagus. Caracterización de sitio y organismos asociados a la muerte agrupada. Universidad de Buenos Aires.

Molina, L., Rajchenberg, M., de Errasti, A., Aime, M. C. y Pildain, M. B. (2020). Sapwood-inhabiting mycobiota and Nothofagus tree mortality in Patagonia: Diversity patterns according to tree species, plant compartment and health condition. Forest Ecology and Management 462: 117997. https://doi.org/10.1016/j.foreco.2020.117997

Nilsson, R. H., Anslan, S., Bahram, M., Wurzbacher, C., Baldrian, P. y Tedersoo, L. (2019). Mycobiome diversity: high-throughput sequencing and identification of fungi. Nature Reviews Microbiology 17 (2): 95-109. https://doi.org/10.1038/s41579-018-0116-y

Oksanen, J., Blanchet, F. G., Friendly, M., Kindt, R., Legendre, P., McGlinn, D., Minchin, P. R., O’Hara, R. B., Simpson, G. L., Solymos, P., Stevens, M. H. H., Szoecs, E. y Wagner, H. (2020). vegan: Community Ecology Package (2.5.7). https://cran.r-project.org/package=vegan

Palmer, J. M., Jusino, M. A., Banik, M. T. y Lindner, D. L. (2018). Non-biological synthetic spike-in controls and the AMPtk software pipeline improve mycobiome data. PeerJ 6 (5): e4925. https://doi.org/10.7717/peerj.4925

Pearson, K. (1900). X. On the criterion that a given system of deviations from the probable in the case of a correlated system of variables is such that it can be reasonably supposed to have arisen from random sampling. The London, Edinburgh, and Dublin Philosophical Magazine and Journal of Science 50 (302): 157-175. https://doi.org/10.1080/14786440009463897

Pildain, M. B., Coetzee, M. P. A., Rajchenberg, M., Petersen, R. H., Wingfield, M. J. y Wingfield, B. D. (2009). Molecular phylogeny of Armillaria from the Patagonian Andes. Mycological Progress 8 (3): 181-194. https://doi.org/10.1007/s11557-009-0590-8

Rajchenberg, M. (2006). Los políporos (Basidiomycetes) de los bosques Andino Patagónicos de Argentina. Schweizerbart’sche Verlagsbuchhandlung.

Raup, D. M. y Crick, R. E. (1979). Measurement of faunal similarity in paleontology. Journal of Paleontology 53 (5): 1213-1227. https://doi.org/https://www.jstor.org/stable/1304099

Riley, R., Salamov, A.A., Brown, D.W., Nagy, L.G., Floudas, D., Held, B.W. y Grigoriev, I.V. (2014). Extensive sampling of basidiomycete genomes demonstrates inadequacy of the white-rot/brown-rot paradigm for wood decay fungi. Proceedings of the National Academy of Sciences 111 (27). https://doi.org/10.1073/pnas.1400592111

Rim, S. O., Roy, M., Jeon, J., Montecillo, J. A. V., Park, S.-C. y Bae, H. (2021). Diversity and Communities of Fungal Endophytes from Four Pinus Species in Korea. Forests 12 (3): 302. https://doi.org/10.3390/f12030302

Robinson, R. M., Morrison, D. J. y Jensen, G. D. (2004). Necrophylactic periderm formation in the roots of western larch and Douglas-fir trees infected with Armillaria ostoyae. II. The response to the pathogen. Forest Pathology 34 (2): 119-129. https://doi.org/10.1111/j.1439-0329.2004.00354.x

Ryvarden, L. y Johansen, I. (1980). A preliminary polypore flora of East Africa. En A preliminary Polypore flora of East Africa. Fungiflora.

Tuor, U., Winterhalter, K. y Fiechter, A. (1995). Enzymes of white-rot fungi involved in lignin degradation and ecological determinants for wood decay. Journal of Biotechnology 41 (1): 1-17. https://doi.org/10.1016/0168-1656(95)00042-O

U’Ren, J. M., Lutzoni, F., Miadlikowska, J., Zimmerman, N. B., Carbone, I., May, G. y Arnold, A. E. (2019). Host availability drives distributions of fungal endophytes in the imperilled boreal realm. Nature Ecology & Evolution 3 (10): 1430-1437. https://doi.org/10.1038/s41559-019-0975-2

Vo?íšková, J., Brabcová, V., Cajthaml, T. y Baldrian, P. (2014). Seasonal dynamics of fungal communities in a temperate oak forest soil. New Phytologist 201 (1): 269-278. https://doi.org/10.1111/nph.12481

Wang, H. H., Chu, H. L., Dou, Q., Feng, H., Tang, M., Zhang, S. X. y Wang, C. Y. (2021). Seasonal Changes in Pinus tabuliformis Root-Associated Fungal Microbiota Drive N and P Cycling in Terrestrial Ecosystem. Frontiers in Microbiology 11: 526898. https://doi.org/10.3389/fmicb.2020.526898

White, T. J., Bruns, T., Lee, S. y Taylor, J. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. En T. J. White, T. Bruns, S. J. W. T. Lee, J. Taylor, M. A. Innis, D. H. Gelfand, y J. J. Sninsky (Eds.), PCR protocols: a guide to methods and applications (pp. 315–322). Academic Press.

Wickham, H. (2016). ggplot2: Elegant Graphics for Data Analysis (3.3.3; Vol. 2, Número 1, pp. 1–189). Springer-Verlag. https://ggplot2.tidyverse.org