Next Generation Sequencing genomic analysis of bacteria from soils of the sites with naturally-occurring summer truffle (Tuber aestivum Vittad.)


  • Marta Siebyła Department of Forest Protection, Forest Research Institute, Braci Leśnej 3, Sękocin Stary, 05-090 Raszyn, Poland
  • Dorota Hilszczanska Department of Forest Ecology, Forest Research Institute, Raszyn, Poland



soil bacteria, summer truffle, NGS method, bacterial diversity


The rhizosphere is the region of soil in which the highest densities of fungi and bacteria occur. In this study, an attempt was made to assess the distribution of bacterial species in soil where the summer truffle Tuber aestivum Vittad. bears fruit in selected stands in Poland. In order to determine the bacterial metagenome, the Next Generation Sequencing (NGS) method was applied. Differences occurred in the bacterial species composition at the cluster level between soils in which summer truffle fruiting was recorded and control soils. In particular, differences in the percentage of Firmicutes bacteria were noted with an average frequency of 3.9% in truffle soil compared to 96.1% in the control soil. It was estimated that two bacterial species, namely Lysobacter antibioticus and Ensifer adhaerens had a positive effect on the occurrence of T. aestivum. Our research increased the knowledge of particular groups of bacteria accompanying truffles and their potential impact on the formation of fruiting bodies in T. aestivum.


Badura L., 2004. Mikroorganizmy glebowe i ich znaczenie w ekosystemach degradowanych przez człowieka. Inży- nieria ekologiczna 12: 14-15.Barbieri E., Bertini L., Rossi I., Ceccaroli P., Saltarel- li R., Guidi C., Zambonelli A., Stocchi V., 2005. New evidence for bacterial diversity in the ascoma of the ectomycorrhizal fungus Tuber borchii. FEMS Micro- biology Letters 247: 23–35. femsle.2005.04.027Beals E.W., 1984. Bray-Curtis ordination: an effective strategy for analysis of multivariate ecological data. Ad- vances in Ecological Research 14.Bolyen E., Rideout J.R., Dillon M.R., Bokulich N.A., Ab- net C.C., Al-Ghalith G.A., ... & Caporaso J.G., 2019. Reproducible, interactive, scalable and extensible mi- crobiome data science using QIIME 2. Nature Biotech- nology, 37(8), 852-857. 019-0209-9Carrig C., Rice O., Kavanagh S., Collins G., O’Flaherty V., 2007. DNA extraction method affects microbial com- munity profiles from soils and sediment. Applied Mi- crobiology and Biotechnology 77:955–964. https://doi. org/10.1007/s00253-007-1219-yCerigini E., Palma F., Barbieri E., Buffalini M., Stoc- chi V., 2008. The Tuber borchii fruiting body-specific protein TBF-1, a novel lectin which interacts with as- sociated Rhizobium species. FEMS Microbiology Let- ters 284(2): 197-203. 6968.2008.01197.xChao A., 1984. Nonparametric estimation of the number of classes in a population. Scandinavian Journal of sta- tistics: 265-270.Courtois S., Cappellano C.M., Ball M., Francou F.X., Nor- mand P., Helynck G., Martinez A., Kolvek S.J., Hopke J., Osburne M.S., August P.R., Nalin R., Guérineau M., Jeannin P., Simonet P., Pernodet J.L., 2003. Recombi- nant environmental libraries provide access to micro- bial diversity for drug discovery from natural products. Applied and Environmental Microbiology 69: 49-55. T.O., Robe P., Cecillon S., Clark I.M., Constan- cias F., Simonet P., Hirsch P.R., Vogel T.M., 2011. Ac- cessing the soil metagenome for studies of microbial di- versity. Applied and Environmental Microbiology 77(4): 1315-1324.èche S., Philippot L., David M.M., Navarro E., Vo- gel T.M., Simonet P., 2009. Characterization of denitri- fication gene clusters of soil bacteria via a metagenomic approach. Applied and Environmental Microbiology 75(2): 534-537. A., Antony-Babu S., Le Tacon F., Robin C., Frey- Klett P., Uroz S., 2016. Temporal changes of bacterial communities in the Tuber melanosporum ectomycorrhi- zosphere during ascocarp development. Mycorrhiza 26(5): 389-399. D.P., 1992. Conservation evaluation and phylogenet- ic diversity. Biological Conservation 61(1): 1-10. Forests Database Bank ( tal/mapy).Frey-Klett P., Garbaye J.A., Tarkka M., 2007. The mycorrhiza helper bacteria revisited. New Phytol- ogist 176(1): 22-36. 8137.2007.02191.xGans J., Wolinsky M., Dunbar J., 2005. Computational improvements reveal great bacterial diversity and high metal toxicity in soil. Science 309(5739): 1387-1390. J., Churin J.L., Duponnois R., 1992. Effects of substrate sterilization, fungicide treatment, nd mycorrhi- zation helper bacteria on ectomycorrhizal formation of pedunculate oak (Quercus robur) inoculated with Lac- caria laccata in two peat bare-root nurseries. Biology Fertility Soils 13 (1): 55-57.Gryndler M., Hršelová H., 2012. Isolation of bacteria from ectomycorrhizae of Tuber aestivum Vittad. Acta Mycologica 47 (2): 155-160. am.2012.018Gryndler M., Soukupová L., Hršelová H., Gryndlerová H., Borovička J., Streiblová E., Jansa J., 2013. A quest for indigenous truffle helper prokaryotes. Environmen- tal Microbiology Reports 5 (3): 346-352. https://doi. org/10.1111/1758-2229.12014Heip C., 1974. A new index measuring evenness. Journal of the Marine Biological Association of the UK 54(3): 555-557. Hilszczańska D., Rosa-Gruszecka A., Sikora K., SzmidlaH. 2013. First report of Tuber macrosporum occurrence in Poland. Scientific Research and Essays 8 (23): 1096- 1099. de Aza C., Armenteros S., McDermott J., Mauceri S., Olaizola J., Hernández-Rodríguez M., & Mediavil- la O., 2022. Fungal and bacterial communities in Tuber melanosporum plantations from Northern Spain. Fore- sts, 13(3): 385.ńska D., 2016. Polskie trufle skarb odzyskany.CILP, Warszawa (54).Hilszczańska D., Szmidla H., Sikora K., Rosa-Gruszecka A. 2019a. Soil properties conducive to Tuber aestivum Vitt. fruiting bodies formation in the Nida Basin stands. Polish Journal of Environmental Studies 28(3): 1713- 1718.ńska D., Rosa-Gruszecka A., Gawryś R., Horak J., 2019b. Eff of soil properties and vegetation characteris- tics in determining the frequency of Burgundy truffl fruit- ing bodies in Southern Poland. Écoscience 26(2): 113-122. H., Navarro R., Takeshita K., Tago K., Hayatsu M., Hori T., Kikuchi Y., 2014. Bacterial population succes- sion and adaptation affected by insecticide application and soil spraying history. Frontiers in Microbiology 5:457. Jaccard J., Turrisi R., 2003. Interaction effects in multiple regression. Thousand OKS, Sage 72, London (104).Ko H.S., Jin R.D., Krishnan H.B., Lee S.B., Kim K.Y., 2009. Biocontrol ability of Lysobacter antibioticus HS124 against Phytophthora blight is mediated by the production of 4-hydroxyphenylacetic acid and several lytic enzymes. Current Microbiology 59(6): 608-615. J.R., Viger M., Arnold E.C., Harris Z.M., Ventura M., Miglietta F., Girardin C., Edwards R.J., Rumpel C., Fornasier F., Zavalloni C., Tonon G., Alberti G., Taylor G., 2017. Biochar alters the soil microbiome and soil function: results of next‐generation amplicon sequenc- ing across Europe. Global Change Biology Bioenergy 9(3): 591-612. D.Y., Crouch J.A., 2009. Bacterial/fungal inter- actions: from pathogens to mutualistic endosymbionts. Annual Review of Phytopathology 47: 63-82. https://Ławrynowicz M., Krzyszczyk T., Fałdziński M., 2008. Oc- currence of black truffles in Poland. Acta Mycol. 43 (2): 143-151. C., Knight R., 2005. UniFrac: a new phyloge- netic method for comparing microbial communities. Applied and Environmental Microbiology 71(12): 8228-8235. M., Olivier J.M., 1992. Effect of soil Pseudomo- nads on colonization of hazel roots by the ecto-mycor- rhizal species Tuber melanosporum and its competitors. Plant Soil 139: 265-273.Medinger R., Nolte V., Pandey R.V., Jost S., Ottenwael- der B., Schloetterer C., Boenigk J., 2010. Diversity in a hidden world: potential and limitation of next‐gener- ation sequencing for surveys of molecular diversity of eukaryotic microorganisms. Molecular Ecology 19: 32-40. A., Ding G.C., Piceno Y. M., Tom L.M., DeSantis T.Z., Andersen G.L., Smalla K., Bonfante P., 2013. Truf- fle brûlés have an impact on the diversity of soil bacte- rial communities. PLoS One 8(4): e61945. https://doi. org/10.1371/journal.pone.0061945Monaco P., Toumi M., Sferra G., Tóth E., Naclerio G.,& Bucci A., 2020. The bacterial communities of Tuber aestivum: preliminary investigations in Molise region, Southern Italy. Annals of Microbiology, 70(1): 37. S.E., Cosart T.F., Johnson J.V., Holben W.E., 2008. Extensive phylogenetic analysis of a soil bacte- rial community illustrates extreme taxon evenness and the effects of amplicon length, degree of coverage, and DNA fractionation on classification and ecological pa- rameters. Applied and Environmental Microbiology 75: 668-675. C., Mello A., Bonfante P., 2008. Dissecting the rhizosphere complexity: the truffle-ground study case. Rendiconti Lincei. Scienze Fisiche Naturali 19: 241-259.łociniczak M., Biniecka P., Bondarczuk K., Piotrowska-Seget Z., 2020. Metagenomic functional profiling reveals differences in bacterial composition and function during bioaugmentation of aged petro- leum-contaminated soil. Frontiers in Microbiology 11:2106. J., Gunasekaran P., 2008. Strategies for access- ing soil metagenome for desired applications. Biotech- nology Advances 26: 576-590. biotechadv.2008.08.002Roesch L., Fulthorpe R.R., Riva A., Casella G., Hadwin A.K.M., Kent A.D., Daroub S.H., Camargo F.A.O., Far- merie W.G., Triplett E.W., 2007. Pyrosequencing enumer- ates and contrasts soil microbial diversity. ISME Journal 1: 283-290. A., Hilszczańska D., Szmidla H. 2014. Warunki środowiskowe sprzyjające występowaniu trufli (Tuber spp.) na historycznych stanowiskach w Polsce. Leśne Prace Badawcze 75 (1): 5-11.Sandhya V.S.K.Z., Ali S.Z., Grover M., Reddy G., Ven- kateswarlu B., 2010. Effect of plant growth promoting Pseudomonas spp. on compatible solutes, antioxidant status and plant growth of maize under drought stress. Journal of Plant Growth Regulation 62(1): 21-30. C., Agnolucci M., Bedini S., Lepera A., Toffanin A., Giovannetti M., Nuti M.P., 2002. Diversity of cul- turable bacterial populations associated to Tuber borchii ectomycorrhizas and their activity on T. borchii mycelial growth. FEMS Microbiology Letters 211 (2): 195-202. P.D., Handelsman J., 2003. Biotechnological pros- pects from metagenomics. Current Opinion in Biotech- nology 14(3): 303-310. C.E., 1948. A mathematical theory of communication. Bell Labs Technical Journal 27(3): 379-423.Siebyła M., Szyp-Borowska I., 2021. Comparison of bac- terial communities in roots of selected trees with and without summer truffle (Tuber aestivum) ectomycorrhi- za. Folia Forestalia Polonica, 63(2): 97-111. https://doi. org/10.2478/ffp-2021-0011Simpson E.H., 1949. Measurement of diversity. Nature 163: 688.Staley C., Unno T., Gould T.J., Jarvis B., Phillips J., Cot- ner J.B., Sadowsky M.J., 2013. Application of Illumina next‐generation sequencing to characterize the bacterial community of the Upper Mississippi River. Journal of Applied Microbiology 115(5): 1147-1158. https://doi. org/10.1111/jam.12323Team R.C., 2013. A language and environment for statisti- cal computing. 1-1706.Torsvik V., Ovreas L., 2002. Microbial diversity and func- tion in soil: from genes to ecosystems. Current Opinion in Microbiology 5 (3): 240-245. S1369-5274(02)00324-7Vahdatzadeh M., Deveau A., Splivallo R., 2015. The role of the microbiome of truffles in aroma formation: a meta-analysis approach. Applied and Environmental Microbiology 81: 6946-6952. AEM.01098-15Yutani M., Taniguchi H., Borjihan H., Ogita A., Fujita K.I., Tanaka T., 2010. Alliinase from Ensifer adhaerens and its use for generation of fungicidal activity. AMB Ex- press 1(1): 1-8. L., Vaughan-Martini A., Angelini P., 2003. Yeast distribution in a truffle-field ecosystem. Annals of Mi- crobiology 53: 275-282.Zhou G.C., Wang Y., Zhai S., Ge F., Liu Z.H., Dai Y.J., Yuan S., Hou Y.J., 2013. Biodegradation of the neonic- otinoid insecticide thiamethoxam by the nitrogen-fixing and plant-growth-promoting rhizobacterium Ensifer ad- haerens strain TMX-23. Applied Microbiology and Bio- technology 97(9): 4065-4074. s00253-012-4638-3






Research article