Rhizosphere microbial communities associated to rose replant disease: links to plant growth and root metabolites

Abstract

Growth depression of Rosa plants at sites previously used to cultivate the same or closely related species is a typical symptom of rose replant disease (RRD). Currently, limited information is available on the causes and the etiology of RRD compared to apple replant disease (ARD). Thus, this study aimed at analyzing growth characteristics, root morphology, and root metabolites, as well as microbial communities in the rhizosphere of the susceptible rootstock Rosa corymbifera ‘Laxa’ grown in RRD-affected soil from two sites (Heidgraben and Sangerhausen), either untreated or disinfected by γ-irradiation. In a greenhouse bioassay, plants developed significantly more biomass in the γ-irradiated than in the untreated soils of both sites. Several plant metabolites detected in R. corymbifera ‘Laxa’ roots were site- and treatment-dependent. Although aloesin was recorded in significantly higher concentrations in untreated than in γ- irradiated soils from Heidgraben, the concentrations of phenylalanine were significantly lower in roots from untreated soil of both sites. Rhizosphere microbial communities of 8-week-old plants were studied by sequencing of 16S rRNA, ITS, and cox gene fragments amplified from total community DNA. Supported by microscopic observations, sequences affiliated to the bacterial genus Streptomyces and the fungal genus Nectria were identified as potential causal agents of RRD in the soils investigated. The relative abundance of oomycetes belonging to the genus Pythiogeton showed a negative correlation to the growth of the plants. Overall, the RRD symptoms, the effects of soil treatments on the composition of the rhizosphere microbial community revealed striking similarities to findings related to ARD.

References

[1] Mahnkopp, F. et al. Induction and diagnosis of apple replant disease (ARD). A matter of heterogeneous soil properties? Sci. Hort. 241, 167-177 (2018).

[2] Grunewaldt-Stöcker, G., Mahnkopp, F., Popp, C., Maiss, E. & Winkelmann, T. Diagnosis of apple replant disease (ARD): microscopic evidence of early symptoms in fine roots of different apple rootstock genotypes. Sci. Hort. 243, 583-594 (2019).

[3] Mazzola, M. & Manici, L. M. Apple replant disease: role of microbial ecology in cause and control. Annu. Rev. Phytopathol. 50, 45-65 (2012).

[4] Nicola, L. et al. Fumigation with dazomet modifies soil microbiota in apple orchards affected by replant disease. Appl. Soil Ecol. 113, 71-79 (2017).

[5] Yim, B., Smalla, K. & Winkelmann, T. Evaluation of apple replant problems based on different soil disinfection treatments-links to soil microbial com- munity structure? Plant Soil 366, 617-631 (2013).

[6] Tewoldemedhin, Y. T., Mazzola, M., Labuschagne, I. & McLeod, A. A multi- phasic approach reveals that apple replant disease is caused by multiple biological agents, with some agents acting synergistically. Soil Biol. Biochem. 43, 1917-1927 (2011a).

[7] Winkelmann, T. et al. Apple replant disease: causes and mitigation strategies. Curr. Issues Mol. Biol. 30, 89-106 (2019).

[8] Spethmann, W. & Otto, G. in Encyclopedia of Rose Science (eds Roberts, A. V., Debener, T. & Gudin, S.) p. 169-180 (Elsevier, Academic Press, Oxford, 2003).

[9] Yim, B. et al. Effects of biofumigation using Brassica juncea and Raphanus sativus in comparison to disinfection using Basamid on apple plant growth and soil microbial communities at three field sites with replant disease. Plant Soil 406, 389-408 (2016).

[10] Henfrey, J. L., Baab, G. & Schmitz, M. Physiological stress responses in apple under replant conditions. Sci. Hort. 194, 111-117 (2015).

[11] Weiß, S., Liu, B., Reckwell, D., Beerhues, L. & Winkelmann, T. Impaired defence reactions in apple replant disease-affected roots of Malus domestica ‘M26’. Tree Physiol. 37, 1672-1685 (2017a).

[12] Weiß, S., Bartsch, M. & Winkelmann, T. Transcriptomic analysis of molecular responses in Malus domestica ‘M26’ roots affected by apple replant disease. Plant Mol. Biol. 94, 303-318 (2017b).

[13] Chizzali, C. & Beerhues, L. Phytoalexins of the Pyrinae: biphenyls and diben- zofurans. Beilsten J. Org. Chem. 8, 613-620 (2012).

[14] Kokubun, T. & Harborne, J. B. A survey of phytoalexin induction in leaves of the Rosaceae by copper ions. Z. f.ür. Naturforsch. C. 49, 628-634 (1994).

[15] Ayati, Z. et al. Phytochemistry, traditional uses and pharmacological profile of rose hip: a review. Curr. Pharm. Des. 24, 4101-4124 (2018).

[16] Mehboob, H. et al. A review on secondary metabolites of Rosa laevigata Michaux: an important medicinal plant. Biochem. Anal. Biochem. 6, 326 (2017).

[17] Yim, B., Winkelmann, T., Ding, G. C. & Smalla, K. Different bacterial communities in heat and gamma irradiation treated replant disease soils revealed by 16S rRNA gene analysis-contribution to improved aboveground apple plant growth? Front. Microbiol. https://doi.org/10.3389/fmicb.2015.01224 (2015).

[18] Yim, B., et al. Effects of soil pre-treatment with Basamid^® Granules, Brassica juncea, Raphanus sativus and Tagetes patula on bacterial and fungal com- munities at two apple replant disease sites . Front. Microbiol. 8, 1604, https://doi. org/ 10.3389/fmicb.2017.01604 (2017).

[19] Franke-Whittle, I. H., Manici, L. M., Insam, H. & Stres, B. Rhizosphere bacteria and fungi associated with plant growth in soils of three replanted apple orchards. Plant Soil 395, 317-333 (2015).

[20] Tilston, E. L. et al. Candidate causal organisms for apple replant disease in the United Kingdom. Phytobiomes 2, 261-274 (2018).

[21] Radl, V. et al. Reduced microbial potential for the degradation of phenolic compounds in the rhizosphere of apple plantlets grown in soils affected by replant disease. Environ. Microbiol. 14, 8 (2019).

[22] Otto, G., Winkler, H. & Szabo, K. Investigations about the course of infestation of rootlets of apple seedlings by root pathogenic actinomycetes in soils with specific apple replant disease. Zentralblatt für Mikrobiol. 148, 467-476 (1993).

[23] Emmett, B., Nelson, E. B., Kessler, A. & Bauerle, T. L. Fine-root system develop- ment and susceptibility to pathogen colonization. Planta 239, 325-340 (2014).

[24] Kelderer, M., Manici, L. M., Caputo, F. & Thalheimer, M. Planting in the ‘inter-row’ to overcome replant disease in apple orchards: a study on the effectiveness of the practice based on microbial indicators. Plant Soil 357, 381-393 (2012).

[25] Mazzola, M. Elucidation of the microbial complex having a causal role in the development of apple replant disease in Washington. Phytopathol 88, 930-938 (1998).

[26] Tewoldemedhin, Y. T., Mazzola, M., Mostert, L. & McLeod, A. Cylindrocarpon species associated with apple tree roots in South Africa and their quantifi- cation using real-time PCR. Eur. J. Plant Pathol. 129, 637-651 (2011b).

[27] Mai, W. F., Werwin, I. A. & Abawi, G. S. Diagnosis, etiology and management of replant disorders in New York cherry and apple orchards. Acta Hort. 363, 33-41 (1994).

[28] Popp, C., Grunewaldt-Stöcker, G. & Maiss, E. A soil-free method for assessing pathogenicity of fungal isolates from apple roots. J. Plant Dis. Prot. 126, 329-341 (2019).

[29] Otto, G., Winkler, H. & Szabo, K. Proof of actinomycetes in rootlets of species of Rosaceae from a SARD soil-a contribution to the specificity of replant dis- eases. Acta Hort. 363, 43-48 (1994).

[30] Szabo, K. Investigations into the specificity of specific replant diseases and possibilities to overcome it: I. Are the replant diseases of rose and apple caused by the same pathogen?. Zeitschrift für Pflanzenkrankheiten und Pflan- zenschutz 106, 237-243 (1999).

[31] Grunewaldt-Stöcker, G., et al. Observations on early fungal infections with relevance for replant disease in fine roots of the rose rootstock Rosa cor- ymbifera “Laxa”. Sci. Rep. (submitted, 2020).

[32] Popp, C., Wamhoff, D., Winkelmann, T., Maiss, E. & Grunewaldt-Stöcker, G . Molecular identification of Nectriaceae in infections of apple replant disease affected roots collected by Harris Uni-Core punching or laser microdissection. J Plant Dis. Protect. 127, 571-582 (2020)

[33] Braun, P. G. Effects of Cylindrocarpon and Pythium species on apple seedlings and potential role in apple replant disease. Can. J. Plant Pathol. https://doi.org/ 10.1080/07060669509500672 (1995).

[34] Reim, S. et al. Genes involved in stress response and especially in phytoalexin biosynthesis are upregulated in four Malus genotypes in response to apple replant disease. Front. Plant Sci. 10, 1724 (2020).

[35] Hoffmann, T. et al. Histochemical screening, metabolite profiling and expression analysis reveal Rosaceae roots as the site of flavan-3-ol biosynthesis. Plant Biol. 14, 33-40 (2012).

[36] Gosch, C., Halbwirth, H. & Stich, K. Phloridzin: biosynthesis, distribution and physiological relevance in plants. Phytochemistry 71, 838-843 (2010).

[37] Barreca, D., Bellocco, E., Laganà, G., Ginestra, G. & Bisignano, C. Biochemical and antimicrobial activity of phloretin and its glycosilated derivatives present in apple and kumquat. Food Chem. 160, 292-297 (2014).

[38] Lucini, L., Pellizzoni, M., Pellegrino, R., Molinari, G. P. & Colla, G. Phytochemical constituents and in vitro radical scavenging activity of different Aloe species. Food Chem. 170, 501-507 (2015).

[39] Liu, F. et al. Soil indigenous microbiome and plant genotypes cooperatively modify soybean rhizosphere microbiome assembly. BMC Microbiol. 19, 201 (2019).

[40] Berg, G. & Smalla, K. Plant species and soil type cooperatively shape the structure and function of microbial communities in the rhizosphere. FEMS Microbiol. Ecol. 68, 1-13 (2009).

[41] Bailén, M., Bressa, C., Larrosa, M. & González-Soltero, R. Bioinformatic strategies to address limitations of 16rRNA short-read amplicons from different sequencing platforms. J. Microbiol. Methods 169, 105811 (2020).

[42] Lerat, S., Simao-Beaunoir, A.-M. & Beaulieu, C. Genetic and physiological determinants of Streptomyces scabies pathogenicity. Mol. Plant Pathol. 10, 579-585 (2009).

[43] Vurukonda, S., Giovanardi, D. & Stefani, E. Plant growth promoting and bio- control activity of Streptomyces sp. as endophytes. International. J. Mol. Sci. 19, 952 (2018).

[44] Mazzola, M. & Zhao, X. Brassica juncea seed meal particle size influences chemistry but not soil biology-based suppression of individual agents inciting apple replant disease. Plant Soil 337, 313-324 (2010).

[45] Bruggen, A. H. C., Francis, I. M. & Jochimsen, K. N. Non-pathogenic rhizosphere bacteria belonging to the genera Rhizorhapis and Sphingobium provide specific control of lettuce corky root disease caused by species of the same bacteria genera. Plant Pathol. 63, 1384-1394 (2014).

[46] Sánchez-González, M. et al. Analysis of phenol-adapted microbial community: degradation capacity, taxonomy and metabolic description. J. Appl. Microbiol. 126, 771-779 (2018).

[47] Thines, M. & Choi, Y.-J. Evolution, diversity, and taxonomy of the Per- onosporaceae, with focus on the genus Peronospora. Phytopathology 106, 6-18 (2016).

[48] Uzuhashi, S., Tojo, M. & Kakishima, M. Phylogeny of the genus Pythium and description of new genera. Mycoscience 51, 337-365 (2010).

[49] Doan, H. K. & Davis, R. M. Pythiogeton manoomin, a new species causing root and basal stalk rot of wild rice in the United States. Mycology 10, 243-249 (2019).

[50] Schreiter, S. et al. Effect of the soil type on the microbiome in the rhizosphere of field-grown lettuce. Front. Microbiol. 5, 1-13 (2014).

[51] Sundberg, C. et al. 454 pyrosequencing analyses of bacterial and archaeal richness in 21 full-scale biogas digesters. FEMS Microbiol. Ecol. 85, 612-626 (2013).

[52] Ihrmark, K. et al. New primers to amplify the fungal ITS2 region-evaluation by 454-sequencing of artificial and natural communities. FEMS Microbiol. Ecol. 82, 666-677 (2012).

[53] Sapp, M. et al. Site-specific distribution of oak rhizosphere-associated oomy- cetes revealed by cytochrome c oxidase subunit II metabarcoding. Ecol. Evol. https://doi.org/10.1002/ece3.5577 (2019).

[54] Sapp, M., Ploch, S., Fiore-Donno, A. M., Bonkowski, M. & Rose, L. E. Protists are an integral part of the Arabidopsis thaliana microbiome. Environ. Microbiol. 20, 30-43 (2018).

[55] Haas, B. J., Chin, M., Nusbaum, C., Birren, B. W. & Livny, J. How deep is deep enough for RNA-Seq profiling of bacterial transcriptomes? BMC Genomics 13, 734 (2012).

[56] Wang, Q., Garrity, G. M., Tiedje, J. M. & Cole, J. R. Naïve Bayesian Classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl. Environ. Microbiol. 73, 5261-5267 (2007).

[57] Edgar, R. C. Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26, 2460-2461 (2010).

[58] Cole, J. R. et al. Ribosomal Database Project: data and tools for high throughput rRNA analysis. Nucl. Acids Res. 42, D633-D642 (2014). (Database issue).

[59] Gweon, H. S. et al. PIPITS: an automated pipeline for analyses of fungal internal transcribed spacer sequences from the Illumina sequencing platform. Meth. Ecol. Evol. 6, 973-980 (2015).

[60] Mistry, J., Finn, R. D., Eddy, S. R., Bateman, A. & Punta, M. Challenges in homology search: HMMER3 and convergent evolution of coiled-coil regions. Nucleic Acids Res. 41, e121 (2013).

[61] Shannon, C., Petigara, N. & Seshasai, S. A mathematical theory of commu- nication. Bell Syst. Tech. J. 27, 379-423 (1948).

[62] Chao, A. Nonparametric estimation of the number of classes in a population. Scand. J. Stat. 11, 265-270 (1984).

[63] Hammer, Ø., Harper, D. A. T. & Ryan, P. D. PAST: Paleontological statistics software package (2001).