FEMS Microbiology Ecology, 96, 2020, fiaa178 doi: 10.1093/femsec/fiaa178 Advance Access Publication Date: 26 August 2020 Research Article RESEARCH ARTICLE Downloaded from https://academic.oup.com/femsec/article/96/11/fiaa178/5897353 by [email protected] on 28 October 2020 Soil P reduces mycorrhizal colonization while favors fungal pathogens: observational and experimental evidence in Bipinnula (Orchidaceae) Mar´ıa Isabel Mujica1,2,*,†,Mar´ıa Fernanda Perez´ 1,2, Marcin Jakalski3,‡, Florent Martos3 and Marc Andre´ Selosse3,4 1Departamento de Ecolog´ıa, Pontificia Universidad Catolica´ de Chile, Alameda 340, Santiago, Chile, 2Instituto de Ecolog´ıa and Biodiversidad (IEB), Alameda 340, Santiago, Chile, 3Department of Plant Taxonomy and Nature Conservation, University of Gdansk, Wita Stwosza 59, 80–308, Gdansk, Poland and 4Institut de Systematique,´ Evolution,´ Biodiversite´ (UMR 7205–MNHN, CNRS, Sorbonne Universite,´ EPHE, Universite´ des Antilles), 45 rue Buffon, 75005 Paris, France ∗Corresponding author: Alameda 340, Santiago, 8331150 Chile. Tel: +56 987671304; E-mail: [email protected] One sentence summary: In situ P addition reduces mycorrhizal colonization in Bipinnula fimbriata in the short term, and this is coupled with an increase in pathogen richness. Editor: Petr Baldrian †Mar´ıa Isabel Mujica, http://orcid.org/0000-0003-0826-6455 ‡Marcin Jakalski, http://orcid.org/0000-0002-5481-9148 ABSTRACT Little is known about the soil factors influencing root-associated fungal communities in Orchidaceae. Limited evidence suggests that soil nutrients may modulate the association with orchid mycorrhizal fungi (OMF), but their influence on non-mycorrhizal fungi remains unexplored. To study how nutrient availability affects mycorrhizal and non-mycorrhizal fungi associated with the orchid Bipinnula fimbriata, we conducted a metagenomic investigation within a large population with variable soil conditions. Additionally, we tested the effect of phosphorus (P) addition on fungal communities and mycorrhizal colonization. Soil P negatively correlated with the abundance of OMF, but not with the abundance of non-mycorrhizal fungi. After fertilization, increments in soil P negatively affected mycorrhizal colonization; however, they had no effect on OMF richness or composition. The abundance and richness of pathotrophs were negatively related to mycorrhizal colonization and then, after fertilization, the decrease in mycorrhizal colonization correlated with an increase in pathogen richness. Our results suggest that OMF are affected by soil conditions differently from non-mycorrhizal fungi. Bipinnula fimbriata responds to fertilization by altering mycorrhizal colonization rather than by switching OMF partners in the short term, and the influence of nutrients on OMF is coupled with indirect effects on the whole fungal community and potentially on plant’s health. Keywords: orchid mycorrhiza; fungal root community; fungal guilds; orchid fertilization; soil nutrients; Bipinnula INTRODUCTION communities that colonize plant roots can be highly diverse (Vandenkoornhuyse et al. 2002). Great focus has been placed on Almost all plant species have fungal root associates in all their mycorrhizal fungi, which increase plant nutrient uptake (van tissues (Rodriguez et al. 2009; Hardoim et al. 2015) and fungal Received: 20 April 2020; Accepted: 24 August 2020 C The Author(s) 2020. Published by Oxford University Press on behalf of FEMS. All rights reserved. For permissions, please e-mail: [email protected] 1 2 FEMS Microbiology Ecology, 2020, Vol. 96, No. 11 der Heijden et al. 2015). They have been crucial for the evolution with mycorrhizal colonization and OMF species richness, while of plants (Selosse and Le Tacon 1998; Strullu-Derrien et al. 2018) soil nitrate negatively correlated with OMF phylogenetic diver- and greatly influence plant fitness (Brundrett 2007; Smith and sity. However, given that it was a correlational approach, where Read 2008), plant interactions and long-term plant dominance in addition only OMF were analyzed, experimental studies com- (Van Der Heijden 2002; van der Heijden et al. 2015), as well as bined with assessment of the total fungal diversity are needed to tolerance to fluctuating environmental conditions (McCormick draw conclusions regarding the impact of soil nutrients on OMF et al. 2006). Yet, endophytes, i.e. fungi colonizing roots in a loose communities and to elucidate their effect on non-mycorrhizal pattern, without visible damage nor true mycorrhizal morpho- endophytic and pathogenic associates. This study is a closer genesis (Wilson 1995), also can confer fitness benefits on the experimental investigation of a large population of Bipinnula fim- host plants, including tolerance to heat, disease, and drought briata using next-generation sequencing to assess whether (i) (Rodriguez et al. 2009). The functions of fungal root communities nutrient availability affects the mycorrhizal fungi of B. fimbri- Downloaded from https://academic.oup.com/femsec/article/96/11/fiaa178/5897353 by [email protected] on 28 October 2020 are highly influenced by the diversity level and fungal species ata differently, (ii) nutrient addition influences OMF communi- involved (Alzarhani et al. 2019), so that a main issue is to under- ties and mycorrhizal colonization as expected from our previous stand how environmental factors determine the diversity and regional correlation study and (iii) nutrient addition influences composition of root fungal communities, including mycorrhizal the non-mycorrhizal fungi associated with B. fimbriata.Toinves- and endophytic fungi. tigate this, we (i) evaluated the relationships between soil nutri- Root fungal communities are greatly influenced by abiotic ents and the diversity of the whole fungal community associated and biotic factors (David, Seabloom and May 2016), including with the Bipinnula fimbriata population before any treatment, host plants (Roy et al. 2013), climate (Tedersoo et al. 2012)and and (ii) conducted a P fertilization to evaluate how experimental soil conditions (Blaalid et al. 2012;Hugginset al. 2014; Van Geel nutrient addition affects the fungal root community associated et al. 2016; Boeraeve, Honnay and Jacquemyn 2018). Particularly, with Bipinnula fimbriata. soil nutrients play a major role in regulating diversity and tax- onomic composition of fungal root associates (Peay, Garbelotto and Bruns 2009;Polme˜ et al. 2013;Yaoet al. 2013). For example, MATERIALS AND METHODS fungal diversity declined significantly under increasing P addi- Orchid species and study site tion (Liu et al. 2012) and short-term P fertilization produced shifts in root-associated fungal communities (Fabianska´ et al. 2019). In Bipinnula Comm. ex Juss. (subtribe Chloraeinae, Orchidoideae) is addition, nutrient addition can also affect mycorrhizal coloniza- a genus of terrestrial, photosynthetic orchids endemic to south- tion, which tends to decrease under nitrogen (N) and phospho- ern South America, comprising a separate group of five species rus (P) fertilization (Treseder 2004; Bechem and Alexander 2012; endemic to Chile (Cisternas et al. ;Novoaet al. 2015). Bipinnula fim- Balzergue et al. 2013). briata (Poepp.) Johnst. is the most frequent of these five species; Little is known about the effect of soil nutrients on fungal it is distributed in lowland (< 500 m) coastal areas from 29 to ◦ root associates in Orchidaceae. Orchids commonly form mycor- 35 S(Novoaet al. 2015), preferably on sandy stabilized soils, in rhizal associations with fungi belonging to at least three families open sites exposed to sunlight and marine breezes (Elortegui´ of basidiomycetes (Tulasnellaceae, Serendipitaceae, and Cerato- and Novoa 2009). It has fasciculate fleshy roots and it flowers basidiaceae; Dearnaley, Martos and Selosse 2012); and in addi- from July to November. According to previous studies, this orchid tion to these orchid mycorrhizal fungi (OMF), a large diversity forms mycorrhizal associations with members of Ceratobasidi- of endophytic fungi associate with orchid roots, some of which aceae and Tulasnellaceae (Steinfort et al. 2010; Mujica et al. 2016; maybepathogenic(e.g. Julou et al. 2005; Bayman and Otero Herrera et al. 2019). 2006;Shahet al. 2019). Limited evidence suggests that soil nutri- The study was conducted in a large B. fimbriata population ents play a key role in orchid mycorrhizas. Bunch et al. (2013), located at the ‘La Cruz’ hill, near the town of Zapallar, on the ◦ ◦ for example, showed that the composition of mycorrhizal fungi coast of central Chile (32 33 S, 71 28W;30mabovesealevel; varies among populations of the orchid Cypripedium acaule in Fig. 1). The climate is Mediterranean with a mean annual tem- ◦ association with soil pH and percentage of organic matter, C and perature of 14.2 and mean annual precipitation of 384 mm (Lue- N, whereas Mujica et al. (2016) found a significant correlation bert and Pliscoff ) and the soil type is alfisol, whereas the geolog- between soil phosphorus (P) and nitrogen (N) and the composi- ical substrate is marine terraces (Casanova et al. 2013). The vege- tion and diversity of mycorrhizal fungi in two Bipinnula species. tation of the hill is coastal Mediterranean and consists mainly of However, the effect of soil nutrients on the diversity and com- sclerophyllous shrubs such as Baccharis macraei and Puya chilen- position of orchid non-mycorrhizal fungal associates remains sis, perennial herbs as Bahia ambrosioides and Happlopapus foliosus unknown. It has been suggested