[en] Understanding the role of root microbiota is crucial in sustainable forest management but remains challenging, especially for tropical trees. We developed an efficient and low-toxicity method to extract and amplify the fungal DNA associated with Aucoumea klaineana Pierre fine roots. To improve DNA quality, we optimized a commercial extraction kit by incorporating activated charcoal and modifying incubation periods. This enhanced protocol, combined with bovine serum albumin during PCR, effectively mitigated inhibitors present in A. klaineana tree root samples. This approach opens new perspectives for studying the microbiota of tropical trees.
Guidosse, Quentin ; Université de Liège - ULiège > TERRA Research Centre
Roy, Mélanie; University Paul-Sabatier Toulouse III, Toulouse, France, IRL IFAECI Instituto Franco-Argentino para el Estudio del Clima y sus Impactos, CNRS, CONICET, UBA, IRD, Ciudad Autónoma de Buenos Aires, Argentina
Becerra, J.X., On the factors that promote the diversity of herbivorous insects and plants in tropical forests. Proc. Natl. Acad. Sci. USA 112:19 (2015), 6098–6103, 10.1073/pnas.1418643112.
Born, C., Vignes, H., Muloko, N., Wickings, E.J., Hossaert-Mckey, M., Chevallier, M.H., Isolation and characterization of polymorphic microsatellite loci from Aucoumea klaineana Pierre (Burseraceae), a tropical rainforest tree of Central Africa. Mol. Ecol. Notes 6:4 (2006), 1054–1056, 10.1111/j.1471-8286.2006.01431.x.
De Nicolai, J., Rodrigues, T.M., Cell wall thickenings and tylosoid: developmental morphology reveals novelties for secretory canals in Protium ovatum (Burseraceae). J. Plant Res. 135:2 (2022), 247–257, 10.1007/s10265-021-01365-6.
Demeke, T., Jenkins, G.R., Influence of DNA extraction methods, PCR inhibitors and quantification methods on real-time PCR assay of biotechnology-derived traits. Anal. Bioanal. Chem. 396:6 (2010), 1977–1990, 10.1007/s00216-009-3150-9.
Díaz-Vallejo, E.J., Seeley, M., Peyton, Smith A., Marín-Spiotta, E., A meta-analysis of tropical land-use change effects on the soil microbiome: emerging patterns and knowledge gaps. Biotropica 53 (2021), 738–752.
Doyle, J.J., Doyle, J.L., A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem. Bull. 19 (1987), 11–15.
Gardes, M., Bruns, T.D., ITS primers with enhanced specificity for basidiomycetes - application to the identification of mycorrhizae and rusts. Mol. Ecol. 2:2 (1993), 113–118, 10.1111/j.1365-294X.1993.tb00005.x.
Guidosse, Q., du Jardin, P., White, L.J.T., Lassois, L., Doucet, J.-L., Gabon's green gold: a bibliographical review of thirty years of research on okoumé (Aucoumea klaineana Pierre). Biotechnol. Agron. Soc. Environ. 26:1 (2022), 30–42, 10.25518/1780-4507.19458.
Haas, B.J., Gevers, D., Earl, A.M., Feldgarden, M., Ward, D.V., Giannoukos, G., Ciulla, D., Tabbaa, D., Highlander, S.K., Sodergren, E., Methé, B., De Santis, T.Z., The Human Microbiome Consortium, Petrosino, J.F., Knight, R., Birren, B.W., Chimeric 16S rRNA sequence formation and detection in sanger and 454-pyrosequenced PCR amplicons. Genome Res. 21:3 (2011), 494–504, 10.1101/gr.112730.110.
Hansen, M.C., Potapov, P.V., Moore, R., Hancher, M., Turubanova, S.A., Tyukavina, A., Thau, D., Stehman, S.V., Goetz, S.J., Loveland, T.R., Kommareddy, A., Egorov, A., Chini, L., Justice, C.O., Townshend, J.R.G., High-resolution global maps of 21st-century forest cover change. Science 342:6160 (2013), 850–853, 10.1126/science.1244693.
Hu, T., Chitnis, N., Monos, D., Dinh, A., Next-generation sequencing technologies: an overview. Hum. Immunol. 82:11 (2021), 801–811, 10.1016/j.humimm.2021.02.012.
Kruskal, W.H., Wallis, W.A., Use of ranks in one-criterion variance analysis. J. Am. Stat. Assoc. 47 (1952), 583–621.
Morgan, B.S.T., Egerton-Warburton, L.M., Barcoded NS31/AML2 primers for sequencing of arbuscular mycorrhizal communities in environmental samples. Appl. Plant Sci., 5(8), 2017, 1700017, 10.3732/apps.1700017.
Onguene, N.A., Tsimi, J.P.M., Balla, M.J.E., Statut mycorhizien de l'okoumé (Aucoumea klaineana Pierre) en régénération artificielle au sud Cameroun. Tropicultura 20:3 (2002), 104–108.
Peršoh, D., Plant-associated fungal communities in the light of meta'omics. Fungal Divers. 75:1 (2015), 1–25, 10.1007/s13225-015-0334-9.
Porter, T.M., Golding, G.B., Are similarity- or phylogeny-based methods moreappropriate for classifying internal transcribed spacer(ITS) metagenomic amplicons?. New Phytol. 192 (2011), 775–782.
Richards, L.A., Dyer, L.A., Forister, M.L., Smilanich, A.M., Dodson, C.D., Leonard, M.D., Jeffrey, C.S., Phytochemical diversity drives plant–insect community diversity. Proc. Natl. Acad. Sci. USA 112:35 (2015), 10973–10978, 10.1073/pnas.1504977112.
Simmons, T., Caddell, D.F., Deng, S., Coleman-Derr, D., Exploring the root microbiome: extracting bacterial community data from the soil, rhizosphere, and root Endosphere. JoVE, 135, 2018, 57561, 10.3791/57561.
Vancov, T., Keen, B., Amplification of soil fungal community DNA using the ITS86F and ITS4 primers. FEMS Microbiol. Lett. 296:1 (2009), 91–96, 10.1111/j.1574-6968.2009.01621.x.
Vroh, Bi I., Harvengt, L., Chandelier, A., Mergeai, G., du Jardin, P., Improved RAPD amplification of recalcitrant plant DNA by the use of activated charcoal during DNA extraction. Plant Breed. 115 (1996), 205–206.
Waud, M., Busschaert, P., Ruyters, S., Jacquemyn, H., Lievens, B., Impact of primer choice on characterization of orchid mycorrhizal communities using 454 pyrosequencing. Mol. Ecol. Resour. 14:4 (2014), 679–699, 10.1111/1755-0998.12229.
White, T.J., Bruns, T.D., Tayloer, J.W., Amplification and direct sequencing of fungal ribosomal RNA Genes for phylogenetics. PCR - Protocols and Applications - A Laboratory Manual, 1990, Academic Press, 315–322.