Persistence of Ecto- and Ectendomycorrhizal Fungi Associated with Pinus Montezumae in Experimental Microcosms

Symbiosis (2018) 74:67–78 DOI 10.1007/s13199-017-0496-1 Persistence of ecto- and ectendomycorrhizal fungi associated with Pinus montezumae in experimental microcosms Edith Garay–Serrano1,2 & Ma. del Pilar Ortega–Larrocea1 & Frédérique Reverchon3 & Iris Suárez–Quijada1 Received: 9 August 2016 /Accepted: 13 June 2017 /Published online: 30 June 2017 # Springer Science+Business Media B.V. 2017 Abstract Ectomycorrhizal (ECM) and ectendomycorrhizal Keywords ITS . Extramatrical mycelium . Morphotyping . fungal species associated with Pinus montezumae were re- Species co-existence corded in 8 year-old trees established in microcosms and com- pared with those associated with 2 year-old trees, in order to determine their persistence over the long-term. Mycorrhizal 1 Introduction root tips were morphologically and anatomically character- ized and sequenced. The extension of extramatrical mycelium Tree roots in coniferous forests are colonized by a variety of of ECM fungi with long exploration strategies was evaluated. ectomycorrhizal (ECM) and, to a lesser extent, by In total, 11 mycorrhizal species were registered. Seven mycor- ectendomycorrhizal fungi, which improve plant water uptake rhizal species were detected on both 2 and 8 year-old pines: and nutrient availability, increase the tolerance of roots to high Atheliaceae sp., Rhizopogon aff. fallax, R. aff. occidentalis, temperatures or soil acidity, and protect roots against patho- Suillus pseudobrevipes, Tuber separans, Wilcoxina mikolae gens (Horton and Van der Heijden 2008; Futai et al. 2008). and Wilcoxina rehmii.Onespecies,Thelephora terrestris, The fungal components involved in the ECM symbiotic asso- was exclusively associated with two year–old seedlings, while ciation are the intraradical mycelium (or Hartig net), the Cenococcum geophilum, Pezizaceae sp. and Pyrenomataceae extramatrical mycelium (including rhizomorphs) and resis- sp. were exclusively found on 8 year-old trees. Atheliaceae sp. tance structures as sclerotia (Futai et al. 2008). External my- was the ECM fungal species that presented the most abundant celium plays an important ecological role in seedling estab- mycelium. Finally, we report one new fungal species of lishment (Reverchon et al. 2015), as an extension of roots. It Pezizaceae occurring as a symbiont of P. montezumae. also constitutes a reservoir of carbon and an efficient way of exploring the soil, taking up and transporting water and nutri- ents as phosphorus, nitrogen and other minerals (Ekblad et al. Electronic supplementary material The online version of this article 2013; Hendricks et al. 2016). External mycelium constitutes (doi:10.1007/s13199-017-0496-1) contains supplementary material, which is available to authorized users. up to 80% of the total biomass of the ECM fungi (Wallander et al. 2001), and is thereby considered as the main organ for * Edith Garay–Serrano nutrient fluxes (Futai et al. 2008). Differences in the develop- [email protected] ment of external mycelium have been recorded between ECM fungal (ECMF) species (Agerer 2001), but the different pat- terns existing in mycelial growth have seldom been registered, 1 Instituto de Geología, Departamento de Edafología, Universidad Nacional Autónoma de México (UNAM), 04510 Ciudad de mainly due to the difficulty to study extramatrical mycelium in Mexico, Mexico field conditions (Anderson and Cairney 2007). 2 Posgrado de Ciencias de la Tierra, Universidad Nacional Autónoma The establishment of microcosms as experimental units has de México (UNAM), Ciudad de Mexico, Mexico allowed to gain insights into several ecological questions. The 3 Red de Estudios Moleculares Avanzados, Instituto de Ecología, A.C, development and persistence of extramatrical mycelium, the Carretera antigua a Coatepec, El Haya, Xalapa, strategy of substrate occupation by ECM fungi, the role of 91070 Veracruz, Mexico mycorrhizal associations in seedling growth and nutrient 68 Garay–Serrano E. et al. exchange, and the weathering activities of ECM fungi have all microcosm experimental units were maintained under green- been explored using microcosms (Donnelly et al. 2004; house conditions, at 25 °C with 45–50% relative humidity, Heinonsalo et al. 2001, 2010; Koele and Hildebrand 2011; and watered weekly with tap water; since trees were 7 years Read et al. 2004; Rosenstock 2009; Saccone et al. 2012). old, modified Melin & Norkrans solution (MMN) was applied More recently, microcosms have been used to disentangle every fortnight to the microcosms. The MMN solution was the relationship between biodiversity and ecosystem function- prepared as follows: CaCl2, 0.05 g; NaCl, 0.025 g; KH2PO4, . ing by testing the effect of intra- and interspecific identity and 0.5 g; (NH4)2HPO4, 0.125 g; MgSO4 7H2O, 0.15 g; richness of ECM fungi on the regulation of plant productivity, FeNaEDTA, 0.027 g, adjusted to 1000 ml with distilled water. soil CO2 efflux and soil nutrient retention (Hazard et al. 2017). During one year (2014), the presence of mycorrhizal Mexico is the second centre of diversity of Pinus species morphotypes was registered monthly on the roots of pines with 49 of the 120 species reported worldwide, of which 22 growing in the 19 experimental units. Mycorrhizal tips were are endemic (Gernandt and Pérez-de la Rosa 2014). As pines collected by removing one of the plates of the microcosms. As are obligate ECM symbionts (Pérez-Moreno and Read 2004), mycorrhizas may differ in color depending on their age, the it is important to gain knowledge about the composition of identity of ECM morphotypes was confirmed through molec- their fungal symbionts and about the development of ular identification. associated extramatrical mycelium under different conditions. Reverchon et al. (2010, 2012) studied the ECMF 2.2 Description of mycorrhizal morphotypes communities established in neotropical forests dominated by Pinus montezumae, in Corredor Biológico Chichinautzin The sampled ECM root tips were described using criteria such (CBC), central México. The authors also established bioassays as branching morphology, color, presence of cystidia, emanat- with P. montezumae growing in microcosms filled with soil ing hyphae, rhizomorphs, and characteristics of mantle sur- from CBC, in order to identify the ECMF propagules able to face, as described by Agerer (1987–2002). Descriptions were colonize the roots of this pine species (Reverchon et al. 2015). recorded in microphotographic images taken under a stereo- Those pines have been maintained in microcosms for 8 years scopic microscope. Longitudinal sections of ectotrophic my- and this study aimed at assessing the persistence and coexis- corrhizas were made by hand cutting and mounted with poly- tence of ECMF species associated with P. montezumae grow- vinyl alcohol – lactic acid – glycerol (PVLG), either stained ing in microcosms, and at evaluating the extramatrical myce- with 0.01% acid fuchsin in lactic acid or stain free. lium development of those ECMF species presenting long- Microscopic characteristics (mantle type with presence or ab- distance exploration type of external mycelia. Furthermore, sence of gelatinous matrix, latex and anatomical features of we aimed at describing in detail the morphological and ana- external elements and Hartig net type) were recorded and tomical characteristics of ECMF morphotypes associated with photographed under optical Leica DME and/or petrographic P. montezumae, since such morphology features may differ Olympus BX-51 microscopes. depending on the host and soil type (Ma et al. 2010). 2.3 Molecular identification of ECM morphotypes and sequence analysis 2 Materials and methods Total DNAwas extracted from 143 collected root tips with the 2.1 Pinus maintenance in microcosms and sampling RedExtract–N–Amp Plant PCR kit (Sigma-Aldrich, Mexico) of ECM root tips as described in Reverchon et al. (2015). Internal transcribed spacer 1 (ITS1) and 2 (ITS2), and 5.8S rDNA were amplified Two year-old trees of P. montezumae were established in mi- by PCR. The 25–μl PCR mixture contained 3 μl of DNA crocosms in 2008, with non-sterilized soil as a substrate, as (diluted in distilled water 1/10), 2.5 μlof10Xbuffer,2.5μl reported by Reverchon et al. (2015). The soil used to set up the of 25 mM MgCl2,2.5μl of 2 mM dNTPs, 0.25 μlof50μM microcosms was sampled in the CBC, under mature neotrop- ITS1F primer; 0.25 μlof50μM ITS4 primer, 0.35 μlof5U ical forests dominated by P. montezumae, at an altitude of Taq polymerase (Axygen, California) and 13.65 μlofsterile 3100 m. The characteristics of this volcanic soil are described deionized ultrapure water. PCR cycling parameters were as in Reverchon et al. (2015). Briefly, soil pH ranged from 4.8 to follows: 1 cycle at 95 °C for 2 min, followed by 35 cycles 5.7, soil total carbon content from 7.7 to 17.8 kg m−2,soiltotal with a denaturation step at 95 °C for 1 min, an annealing step nitrogen content from 0.42 to 1.04 kg m−2, and soil available at 55 °C for 1 min, and an extension step at 72 °C for 1 min, phosphorus from 1.38 to 2.39 g m−2. Microcosms were then finally 1 cycle at 72 °C for 8 min for final extension. scaled up and trees were transferred to bigger units consisting Amplified PCR products were sent to High Throughput of acrylic plates of 50 × 50 cm, filled with additional sterilized Genomics Center in Washington University for purification soil from the CBC, with one tree per microcosm. Nineteen and sequencing reactions. Persistence of ecto- and ectendomycorrhizal fungi 69 The obtained fungal ITS sequences were manually edited ecto- and ectendomycorrhizal fungi were identified from the and assembled using the Sequencher software (V4.1, Gene obtained sequences (Supplementary Table T1). Five ECMF Codes, Ann Arbor, MI, USA). The consensus sequences were species were identified to the species level: Cenococcum contrasted with sequences of the ITS region in NCBI and geophilum, Rhizopogon aff. fallax, R. aff. occidentalis, UNITE gene databases and species names were assigned Suillus pseudobrevipes and Tuber separans;furthermore, when the 97% sequence similarity criterion, recommended two of the identified fungal species were previously reported by Tedersoo et al.

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