Mycorrhiza https://doi.org/10.1007/s00572-017-0808-6

ORIGINAL PAPER

Microscopic characterization of orchid mycorrhizal fungi: as a putative novel orchid mycorrhizal of Vanilla in different crop systems

Ma. del Carmen A. González-Chávez1 & Terry J Torres-Cruz2 & Samantha Albarrán Sánchez 1 & Rogelio Carrillo-González1 & Luis Manuel Carrillo-López3 & Andrea Porras-Alfaro2

Received: 1 July 2017 /Accepted: 9 November 2017 # Springer-Verlag GmbH Germany, part of Springer Nature 2017

Abstract Vanilla is an orchid of economic importance widely been reported as a mycorrhizal symbiont in orchids. Direct cultivated in tropical regions and native to Mexico. We sam- amplification of pelotons also yielded common plant patho- pled three species of Vanilla (V. planifolia, V. pompona,and gens, including Fusarium and Pyrenophora seminiperda,es- V. insignis) in different crop systems. We studied the effect of pecially in those sites with low colonization rates, and where crop system on the abundance, type of fungi, and quality of large numbers of degraded pelotons were observed. This re- pelotons found in the roots using light and electron microsco- search reports for the first time the potential colonization of py and direct sequencing of mycorrhizal structures. Fungi Vanilla by Scleroderma, as a putative orchid mycorrhizal sym- were identified directly from pelotons obtained from terrestrial biont in four sites in Mexico and the influence of crop system roots of vanilla plants in the flowering stage. Root samples on mycorrhizal colonization on this orchid. were collected from plants in crop systems located in the Totonacapan area in Mexico (states of Puebla and Veracruz). Keywords Mycorrhizal colonization . Pelotons . DNA was extracted directly from 40 pelotons and amplified Scleroderma sp. . Vanilla insignis . V.planifolia . V.pompona using ITS rRNA sequencing. Peloton-like structures were ob- served, presenting a combination of active pelotons character- ized by abundant hyphal coils and pelotons in various stages of degradation. The most active pelotons were observed in Introduction crop systems throughout living tutors (host tree) in compari- son with roots collected from dead or artificial tutors. Fungi Vanilla, a terrestrial and epiphytic orchid, is of vital economic identified directly from pelotons included Scleroderma importance for the production of natural vanillin, a fragrant areolatum, a common ectomycorrhizal fungus that has not product used in the food and cosmetic industry. In global trade, there is a growing demand for natural vanilla essence flavor. This plant represents a gastronomic legacy to the world Electronic supplementary material The online version of this article and is a unique product of Mexico’s cultural heritage. Vanilla (https://doi.org/10.1007/s00572-017-0808-6) contains supplementary has several uses in food, pharmaceutical, and other industries material, which is available to authorized users. (Havkin-Frenkel and Belanger 2011). Mexico, as the center of origin of Vanilla (IMPI 2016), * Andrea Porras-Alfaro [email protected] represents a major diversity hotspot that provides resources that could improve the production of vanilla. But most of the 1 Programa de Edafología, Colegio de Postgraduados, Campus information available for this plant has been collected from Montecillo. Carr. México-Texcoco, 56230 Montecillo, Mexico State, sites where Vanilla was introduced, including Costa Rica, Mexico Colombia, Belize, Madagascar, and Puerto Rico (Porras- 2 Department of Biological Sciences, Western Illinois University, Alfaro and Bayman 2007; Gretzinger and Dean 2011; Macomb, IL 61455, USA Lubinsky et al. 2011). Although Vanilla planifolia Andrews 3 Facultad de Zootecnia y Ecología, CONACYT-Universidad is preferred for its flavor (Ehlers and Pfister 1997;Sreedhar Autónoma de Chihuahua, 31453 Chihuahua State, Mexico et al. 2007), other cultivated species such as V. tahitensis and Mycorrhiza

V. pompona are also commercially important for the produc- the trees in the lower strata of vegetation. There is no external tion of natural vanilla flavor in some places (Havkin-Frenkel input of fertilizers or organic matter, but the natural recycling and Dorn 1997). of leaves is common. Vanilla is a pantropical genus that is both terrestrial and The live tutors no shade house crop system (Puntilla epiphytic. Most orchids have been reported to associate with Aldama) uses live trees but vegetation is removed and more mycorrhizal fungi in the form-genus Rhizoctonia managed than the ‘acahual’ system. Two field sites were sam- (), though many orchids are known to associate pled. One of the two Puntilla Aldama crop systems is a small with other groups of Basidiomycota and Ascomycota fungi plot production (site 1). Pachira macrocarpa Aubl. is used as (Kristiansen et al. 2001; Rasmussen 2002; Rasmussen and Vanilla support tree for V. insignis and V. planifolia.For Rasmussen 2014). Unlike temperate orchids, mycorrhizal re- V. planifolia, rows of support trees are separated 2.5 m and lationships of tropical terrestrial orchids are largely unex- four plants are planted in each support tree. Abundant organic plored (Pereira et al. 2005). In vanilla, there is little informa- matter is added that comes from decaying leaf litter. The other tion about natural mycorrhizal fungal communities in associ- Puntilla Aldama crop system (site 2) has V. pompona using ation with roots (Porras-Alfaro and Bayman 2003; Porras- C. sinensis as the support tree. Trees are planted in rows 3.5 m Alfaro and Bayman 2007;Baymanetal.2011). apart, one plant per tree. Ceratobasidium (anamorph Rhizoctonia), Thanathephorus The shade house-dead tutors crop system (20 Soles field (anamorph Rhizoctonia), and Tullasnella (anamorph site) produces vanilla using a 50% luminosity shade house Epulorhiza)havebeenreportedascommonmycorrhizalfungi established at 3 m height, with all sides covered. Vanilla’s of Vanilla roots (Porras-Alfaro and Bayman 2007), but these support trees were planted in rows 1.5 m apart. Support trees studies have been conducted in sites where Vanilla is non- come from dead branches of trees of Gliricidia sepium (Jacq.) native. Steud. from a plantation near the site. Plant density varied Mycorrhizal roots in orchids, including Vanilla,frequently from one to two plants per tree. Organic matter, such as leaves contain a combination of non-colonized sections, active from trees, is recycled to provide nutrients. pelotons, and clumps of degenerate hyphae (Mejstrik 1970; The shade house-concrete tutors crop system (50% lumi- Barmicheva 1989; Masuhara and Katsuya 1994;Porras- nosity; Pantepec field site) is stretched above the plot (3.5 m Alfaro and Bayman 2003, 2007;Zettler1997; Zettler et al. high), covering all sides. In this crop system, vanilla tutors are 2001; Batty et al. 2002; Peterson and Massicotte 2004). The made of concrete posts for plant support, one plant per pole. degraded or collapsed pelotons are normally confined to the Additional nutrients are added through compost from crop center of the cell with a patchy distribution in the cortex cells, residues, cow manure, and sawdust from Cedrela odorata L. and the hyphae forming these structures are difficult to distin- We hypothesize that levels of colonization and pelo- guish (Huynh et al. 2004;Làtretal.2008). This patchy distri- ton condition will be influenced by crop system. More bution of pelotons is likely a result of multiple events of col- intense farming and stressful conditions (i.e., dead tu- onization. Pereira et al. (2005) and Valadares et al. (2012) tors) will limit mycorrhizal colonization in comparison suggested that pelotons found in external cortical cells might with Vanilla plants grown in a more natural environ- serve as an inoculum source for recolonization of inner cortex ment (i.e., forested area with higher levels of organic cells, seeds, and other roots. matter and potential mycorrhizal symbionts). We also The overall goal of this study was to analyze mycor- expect to find little differences in peloton morphology rhizal fungal colonization in crop systems in Vanilla. for the different Vanilla species. A previous report We evaluated mycorrhizal structures in Vanilla under showed overlap on Rhizoctonia-like mycorrhizal symbi- different crop systems in Mexico, ranging from natural onts and morphology of pelotons isolated from different conditions to the use of shade houses to more intense sites and different Vanilla species (Porras-Alfaro and farming practices using concrete tutors. The peloton- Bayman 2003, 2007). forming fungi were identified using direct sequencing. The ‘acahual’ crop system (Primero de Mayo field site) follows an agricultural management where a more natural Materials and methods setting is maintained. Tree species are used as support trees (tutors) for vanilla plants. Some examples of these trees are as Root sampling follows: Citrus sinensis (L.) Osbeck., Erythrina americana Mill., Gliricidia sepium (Jacq.) Kunth ex Walp., Terrestrial roots were collected from four vanilla crop Tabenaemontana sp., Litsea glaucescens Kunth., Eugenia systems (‘acahual’, live tutors, shade house-dead tutors, capuli (Schldl. & Cham.) Hook. & Arn., and Hamelia erecta shade house-concrete tutors) located at five field sites in Jacq. Shade is managed by cutting branches or trees to allow Totonacapan area (Puebla and Veracruz), Mexico sunshine to reach the Vanilla plants, which are planted next to (Table 1;Fig.S1). We collected V. planifolia G. Jack, Mycorrhiza

Table 1 Percentage of mycorrhizal colonization in Site System Variety % root Vanilla growing under different colonization a crop systems range (average)

Primero de Mayo, Papantla, traditional (acahual) V. planifolia 8–70 (33, a) Veracruz Pantepec, Puebla shade house/concrete V. planifolia 0–48 (25, a) tutors 20 Soles, Papantla, Veracruz shade house/dead tutors V. planifolia 13–23 (18, a) Puntilla Aldama, Veracruz, site1 shade house/alive tutors V. planifolia 58–70 (65, a) Puntilla Aldama, Veracruz, site 1 shade house/alive tutors V. insignis (n =1) 23 Puntilla Aldama, Veracruz, site 1 alive tutors V. planifolia ‘rayada’ 70 (n =1) Puntilla Aldama, Veracruz, site 2 alive tutors Vanilla pompona (n =2) 23–68 (45, a)

n = 5 plants per vanilla crop system and 10 segments per each plant; except with V. insignis and V. planifolia ‘rayada’ (only one plant was available) and two plants for V. pompona a No significant differences were observed among sites or plant species represented by same letter after the average

V. planifolia ‘rayada’, V. pompona Schiede, and V.insignis Percentage of fungal colonization in Vanilla roots Ames. These species bloom between March and April, except for V. insignis, which was collected in June. Samples were Five vanilla plants (30 roots per plant) in each crop system were collected from five different sites: Primero de Mayo, analyzed for fungal colonization. Ten transversal root segments Papantla de Olarte Ocampo, Veracruz (20° 17′ 43″ N97°15′ (1–3 mm) of each plant were analyzed for a total of 50 root 54.4″ W, 196 m.a.s.l, 4-year-old crop), Pantepec, Puebla (20° segments per crop system. Each plant was used as a replicate. 30′ 0.8″ N97°53′ 13.8″ W, 205 m.a.s.l, 3-year old crop), 20 Roots were stained with 1% toluidine in 50% glycerol and put in Soles, Papantla de Olarte, Veracruz (20° 25′ 39″ N97°18′ 47″ a concave glass slide, covered with a cover slide and observed at W, 193 m.a.s.l, 4-year old crop), and two crop systems at 400× magnification under a light microscope according to Puntilla Aldama, San Rafael, Veracruz (20° 14′ 0.5″ N96° Rasmussen and Whigham (2002). 54′ 14″ W, 17 m.a.s.l and 20° 11′51″ N 96° 54′.24″ W; The rate of fungal colonization was estimated as the percent- 15 m.a.s.l., respectively, 4-year old crops) (Figs. S1 and S2). age of the volume of cortical root space occupied by pelotons per Wild native Vanilla was not collected due to the lack of acces- segment of vanilla root, according to the following adapted for- sible locations near the study sites. mula (Boddington and Dodd 1998):

½Š#ðÞþ0% #ðÞþ25% #ðÞþ50% #ðÞþ75% #ðÞ100% %Colonizationby pelotons ¼ Total number of segments

where B#^ represents the number of segments colonized by Scanning electron microscopy analysis pelotons in roots, the percentage of root surface occupied by the fungi is shown in parenthesis. Only one Vanilla plant from Three root segments of each one of the five plants from each V. planifolia ‘rayada’ and V. insignis was under cultivation at vanilla species from the four crop systems, except for varieties the Puntilla Aldama site. Hence, only ten segments from each V. planifolia ‘rayada’ and V. insignis, were fixed (see below) of these two plants were analyzed. and mounted on a scanning electron microscope (SEM) sam- We compared root colonization among the five sites ple holder. Three root samples from rayada variety were where Vanilla was collected using ANOVA and Tukey mounted since only one plant was growing in the crop system test (α = 0.05). Comparison in colonization between at Puntilla Aldama, Veracruz. No SEM analysis was per- V. planifolia and V. pompona was also made. Samples formed for V. insignis due to the reduced amount of root seg- on alive tutors were compared against the samples col- ments available for this plant species. lected from dead or artificial tutors. V. planifolia Specimen preparation was performed according to Bozzola ‘rayada’ and V. insignis were excluded from the analysis and Russell (1999). Vanilla root samples were washed with since only one plant was available. deionized water and fixed in 2.5% glutaraldehyde in a Mycorrhiza

Sorensen phosphate buffer (pH 7.2). Subsequently, the roots the Basic Local Alignment Search Tool (BLAST) (Altschul were washed with Sorensen phosphate buffer (pH 7.2) to et al. 1997) and UNITE (Kõljalg et al. 2013). Sequences were eliminate glutaraldehyde residues. Dehydration was per- deposited to GenBank under accession numbers KX953680– formed in an ethanol series (30, 40, 50, 60, 70, 80, 90, 2x- KX953706. 100%) after incubating for 40 min. Root samples were dried at critical point using transitional fluid carbon dioxide in a Phylogenetic analysis Samdri-780A Tousimis dryer (Pathan et al. 2010). An accel- erating voltage of 15 kV was used. The analysis was per- ITS sequences of Scleroderma generated during this study formed using the JEOL JSM 6390 SEM in the Electron were combined into a dataset with additional GenBank se- Microscopy Unit at Colegio de Postgraduados, Montecillo, quences and a previous phylogenetic study of Scleroderma Mexico. (Nouhra et al.2012). The outgroup taxa Pisolithus sp. (AJ629887) and Suillus sp. (GQ267488) were used for anal- Identification of fungi from pelotons ysis. Alignments were performed using the multiple sequence alignment software MUSCLE (ebi.ac.uk/tools/msa/muscle). Vanilla roots were abundantly washed with tap water in order The phylogenetic reconstruction was carried out in MEGA6 to eliminate soil particles. The velamen was removed and (Filipski et al. 2013). A maximum likelihood analysis was areas colonized by fungi were identified as brown zones along performed using Tamura-Nei substitution model as deter- the length of the roots according to Porras-Alfaro and Bayman mined using model selection in MEGA. One thousand boot- (2003). The presence of pelotons inside the brown zones in the strap replicates were performed to assess the level of support cortex of vanilla roots was verified with an optical microscope for each node. Nodes that showed a bootstrap support < 70% (Olympus, CX31 model) at 400× magnification by checking were not considered significantly supported. Alignment was 2–3-mm transversal root cuts. Superficial root disinfection submitted to TreeBASE (S19988). was carried out in 1-cm root segments with 70% ethanol for 1 min, 20% sodium hypochloride and two drops of Tween-20 for 3 min and at least five sterile distilled water rinses (mod- Results ified from Otero and Bayman 2009; Porras-Alfaro and Bayman 2003). Disinfected root segments (3–5 mm) were Fungal colonization put in microtubes containing some drops of sterilized distilled water, and pelotons were forced to separate from root cells Vanilla roots presented characteristic peloton colonization in using water suction with a sterile Pasteur pipet. Pelotons were the root cortex. In general, all plants show some level of col- transferred to new microtubes containing 1 mL of a onization with the exception of one plant from Pantepec, Streptomycin and Tetracycline solution (50 μgmL−1)forat Puebla, in which pelotons were not observed in any of the least 1 h. roots. Colonization percentages vary from 0 to 70% After surface sterilization, individual pelotons were collect- (Table 1). The highest level of colonization (average 33– ed for DNA extraction and direct sequencing. DNA from sep- 65%) was observed in plants where alive tutors were used arated pelotons was extracted using the Wizard genomic DNA (e.g., Puntilla Aldama and Primero de Mayo) (Fig. S2a,c) in purification kit (Promega, Madison, WI) following the manu- comparison with sites with dead tutors (18%, e.g., 20 Soles facturer’s instructions. PCR was conducted using the ITS1- and Pantepec) (Table 1;Fig.S3a,d) as expected based on our 5.8S–ITS2 nrRNA (ITS barcode) with primers ITS1F (Gardes hypothesis. However, due to the high variability among roots, and Bruns 1993) and ITS4 (White et al. 1990). Each 25 μLof differences were not significant between dead vs. alive tutors. PCR mixture contained 12.5 μL of PCR master mix Characteristic colonization by pelotons was observed in (Promega, Madison, WI), 3 μL of 1% bovine serum albumin Vanilla roots from all the different crop systems including a (BSA), 1 μLofeachprimer(5μM), 6.5 μLofnuclease-free variety of pelotons on different stages of degradation (Fig. 1 water, and 1 μLofDNAforeachsample.ThePCRwasrun and 2, Fig. S3). No differences in colonization were observed under the following conditions: 95 °C for 5 min, 35 cycles of between Vanilla species. Microscopy showed characteristic 94 °C for 30 s, annealing at 53 °C for 30 s, and extension at Bcoil^ structures in orchids with active hyphae crossing cortex 72 °C for 45 s, followed by a final extension at 72 °C for cells (Fig. 2b). In some cases, hyphae were easily distin- 7 min. PCR products were checked using gel electrophoresis guished; however, in other cases, hyphae were strongly (1.2% agarose in Tris-acetate-EDTA buffer). All samples were compacted inside cells (Fig. 1 and 2). Crystals were common- sequenced using the forward primer at Beckman Coulter ly observed in vanilla roots (Fig. 1c). Genomics (Danvers, MA), and sequences were trimmed and Overall colonization of transversal sections varies for each edited in Sequencher (Gene Codes, Ann Arbor, MI, USA). section. In some cases, the majority of the cells were colonized Closest relatives of fungal sequences were determined using by pelotons (Fig. 1a, b and Fig. 2f), while other transversal Mycorrhiza

Fig. 1 Scanning electron micrographs of V. planifolia at a, b Primero de Mayo, c, d 20 soles, and e, f Pantepec. a, b Active hyphae, pelotons colonizing complete cells. e, f More degraded and compacted pelotons, with no active hyphae

sections show lower levels of colonization with more degrad- observed (Fig. 2e, g). Mycorrhizal hyphae were observed ed pelotons (Fig. 1f and Fig. 2a, d). V. planifolia collected crossing plant cell walls in the cortex (Fig. 2b). In the majority from Primero de Mayo alive tutors showed high levels of of cases, coils seem to be composed of a single hyphal type, intracellular colonization in cortex cells with very large and but in a few cases, additional hyphae of potentially different active hyphal masses, forming pelotons that almost fill the fungi in the cells were observed (Fig. 2c), which is expected, cells completely (Fig. 1a, b). In contrast, V. planifolia from since other fungal endophytes and plant pathogens can colo- 20 Soles and Pantepec (dead or concrete tutors) showed more nize orchid roots, but not necessarily form pelotons. compacted and degraded pelotons (Fig. 1c, e, f). V. planifolia, V. pompona,andV. planifolia ‘rayada’ all are Direct sequencing of pelotons colonized by characteristic orchid mycorrhizal structures with little differences in peloton structure among Vanilla species Direct sequencing from pelotons was done as an attempt to (Fig. 2). These samples were collected from Puntilla identify potential fungi responsible of the formation of Aldama, in which alive tutors were used. Pelotons were more pelotons. Forty pelotons were sequenced with a success rate compacted than the ones observed in Primero de Mayo in of 65% (a clean sequence was obtained) (Table 2). Several of V. planifolia, but they still appeared more active than pelotons the fungal taxa recovered from pelotons represent plant path- observed in sites with dead tutors. Hyphal segments ogens or saprophytic fungi, including Pyrenophora connecting the peloton to the plant cell wall were easily seminiperda (46%), Fusarium spp. (12%), Aureobasidium Mycorrhiza

Fig. 2 Scanning electron micrographs of Vanilla on alive tutors at Puntilla Aldama. a–c V. planifolia. d, e V. pompona. f, g V. planifolia ‘rayada’

sp. (4%), Didymellaceae sp. (4%), and Trichocomaceae sp. sites that have dead or artificial tutors. The different Vanilla (4%). species were colonized by the characteristic peloton structures Scleroderma areolatum (Basidiomycota) was detected as a of orchid mycorrhizal fungi. This study describes for the first common fungus in the four study sites from direct sequencing time Scleroderma areolatum (an ectomycorrhizal fungus) as a of pelotons from V. planifolia and V. planifolia ‘rayada’, putative mycorrhizal fungus in orchids, showing the potential representing 33% of the sequences. The phylogenetic analysis influence of nearby vegetation as a source of mycorrhizal for the ITS nrRNA region confirmed that sequences of inocula. Scleroderma in Vanilla cluster in the same clade of Pelotons have a short lifespan in colonized roots, and dur- S. areolatum obtained from different ectomycorrhizal diversi- ing lysis, the hyphal content becomes disorganized and the ty and phylogenetic studies associated with Picea, Pinus, walls take on flattened or angular profiles. In the final stages, Quercus,andCastanea dentata (Fig. 3). To our knowledge, the wall material clumps together, forming an irregular mass this is the first study reporting S. areolatum as a putative (Fig. 1c, f) (reviewed by Smith and Read 1997). The rapid orchid mycorrhizal fungus in Vanilla. degradation of pelotons in roots seems to be a significant mechanism to gain nutrients (C and N) from the fungus to the host cell (Kuga et al. 2014). Orchids maintain a strict Discussion control of their fungal symbionts, likely limiting regions of colonization to areas where the fungus can provide specific This study confirms that the levels of colonization and density ecological services (i.e., acquire nutrients and water) (Leake of hyphal coils inside Vanilla root cells vary greatly by crop and Cameron 2012). For example, in Vanilla, fungi are found system type. Vanilla planifolia from the sites with more natu- in higher abundance in terrestrial roots with respect to roots ral conditions (alive tutors) tend to have the highest levels of attached to the tree cortex or those that are not in contact with colonization and most active pelotons in comparison with the substrate (Porras-Alfaro and Bayman 2003, 2007). Fungal Mycorrhiza

Table 2 Sequences directly obtained from pelotons Isolate Accession number Organism Collection site Host colonizing vanilla species in different crop systems SAPA5 KX953703 Aureobasidium sp. Pantepec V. planifolia SAPA2 KX953686 Didymellaceae Pantepec V. planifolia SAPA3 KX953694 Fusarium sp. Pantepec V. planifolia SAPA9 KX953706 Fusarium sp. Pantepec V. planifolia SAPA12 KX953683 Fusarium sp. Pantepec V. planifolia SAPA8 KX953705 Pyrenophora seminiperda Pantepec V. planifolia SAPA11 KX953682 Pyrenophora seminiperda Pantepec V. planifolia SAPA21 KX953687 Pyrenophora seminiperda Primero de Mayo V. planifolia SAPA22 KX953688 Pyrenophora seminiperda Primero de Mayo V. planifolia SAPA27 KX953692 Pyrenophora seminiperda Puntilla Aldama V. planifolia SAPA32 KX953696 Pyrenophora seminiperda Puntilla Aldama V. planifolia SAPA39 KX953700 Pyrenophora seminiperda Puntilla Aldama V. pompona SAPA40 KX953701 Pyrenophora seminiperda Puntilla Aldama V. pompona SAPA41 KX953702 Pyrenophora seminiperda Puntilla Aldama V. planifolia SAPA6 KX953704 Pyrenophora seminiperda Pantepec V. planifolia SAPA29 KX953693 Pyrenophora seminiperda Puntilla Aldama V. planifolia SAPA31 KX953695 Pyrenophora seminiperda Puntilla Aldama V. planifolia SAPA10 KX953681 Scleroderma areolatum Pantepec V. planifolia SAPA13 KX953684 Scleroderma areolatum 20 Soles V. planifolia SAPA14 KX953685 Scleroderma areolatum 20 Soles V. planifolia SAPA23 KX953689 Scleroderma areolatum Primero de Mayo V. planifolia SAPA25 KX953690 Scleroderma areolatum Primero de Mayo V. planifolia SAPA26 KX953691 Scleroderma areolatum Primero de Mayo V. planifolia SAPA37 KX953698 Scleroderma areolatum Puntilla Aldama V. planifolia ‘rayada’ SAPA38 KX953699 Scleroderma areolatum Puntilla Aldama V. planifolia ‘rayada’ SAPA36 KX953697 Trichocomaceae Puntilla Aldama V. planifolia ‘rayada’

colonization was observed in terrestrial roots of almost all the among trees (Dell et al. 1994; Yamada and Katsuya Vanilla plants sampled in this study, showing that mycorrhizal 1995;Chenetal.2006). Although it is well known that fungi are common in different crop systems in Mexico. Vanilla form mycorrhizal associations with members of Nevertheless, fungal colonization varied widely among root Rhizoctonia-like fungi (Porras-Alfaro and Bayman 2003, sections, collection sites, and vanilla species (Table 1). Porras- 2007), we did not detect any of these taxa from direct Alfaro and Bayman (2003) reported similar findings with var- amplifications of pelotons. Rhizoctonia-like fungi are iation in colonization rates among different vanilla species and difficult to isolate and to amplify in Vanilla (Porras- collection sites in Costa Rica, Puerto Rico, and Cuba, but little Alfaro and Bayman 2007). According to Zhu et al. differences between plant species. This study demonstrates (2008) and Kohout et al. (2013), the selection of live that tutor types that are dead or artificial have lower coloniza- pelotons can help yield true mycorrhizal fungi from or- tion rates (22%) than alive ones (46%), and dead tutors likely chid roots, but this is sometimes challenging due to the limit the benefits of these fungal-plant partnerships due to the mixture of active and degraded pelotons in the same conditions of this substrate (i.e., lack of access to nutrients, root segment, as observed in this study. desiccation of roots and hyphae, increase in the number of ITS primers have shown bias against Tullasnella pathogens due to stress, etc.). (Kristiansen et al. 2001; Porras-Alfaro and Bayman Our results show for the first time that S. areolatum, 2007), and it is possible that the high degradation stages an ectomycorrhizal Basidiomycota fungus, can be direct- of the pelotons, in addition to potential primer bias, ly amplified from individual Vanilla pelotons. make amplifications difficult, underestimating fungal di- Scleroderma associates mainly with eucalypt forests, versity in orchids. Fungal species from the genera Pinus and Quercus trees and shows low specificity Tulasnella, Ceratobasidium,andSebacina are among Mycorrhiza

Fig. 3 Maximum likelihood tree of Scleroderma for the ITS region. Tree support. Scale bar indicates the horizontal branch length, expressed as was generated using Pisolithus sp. and Suillus sp. as outgroups (Nouhra the hypothesized number of nucleotide substitutions per site. The isolates et al. 2012). Values associated with branches indicate the bootstrap obtained in this study are bolded the most common orchid mycorrhizal taxa detected in pelotons and roots (Shefferson et al. 2007;Taylorand photosynthetic orchids (Otero et al. 2002; Moncalvo McCormick 2008; Roy et al. 2009), and by the study of et al. 2006;Kohoutetal.2013). Studies in Vanilla have orchids in their native environments. beenmainlyfocusedonculturesofRhizoctonia fungi in In addition to Scleroderma, as a putative novel mycorrhizal places where Vanilla is non-native. This study represents fungus in Vanilla, a number of fungi were also detected in the first one to use direct sequencing on Vanilla roots direct amplifications from pelotons, including multiple puta- from Mexico, the center of origin of this crop. Many tive pathogens and saprophytic taxa not known to form my- orchids are colonized by fungi from other taxonomic corrhizal associations with plants. It is likely that these fungi groups (Rasmussen 1995;Baymanetal.2011), and were detected as opportunistic or pathogenic colonizers in the range of mycorrhizal fungi associated to adult or- areas with degraded pelotons. Pyrenophora seminiperda was chids is gradually increasing, due to the use of modern the most abundant putative pathogen (Table 2) and occurred in molecular methods, including direct sequencing from four of the five Vanilla crop systems sampled (Tables 1 and 2). Mycorrhiza

Fusarium was also detected. This is not a surprise, since some of these plants grow under very limiting light conditions Fusarium and other pathogenic fungi are commonly isolated on the forest soil or attach to tree trunks with large shaded from commercial orchids, including Vanilla (Lee 2002; areas. This work reveals the need to continue the study of Porras-Alfaro and Bayman 2007; Adame-Garcia et al. 2015). orchid-tree overlapping mycorrhizal partners that could have Ectomycorrhizal fungi are commonly detected on non- the potential to improve the production and conservation of photosynthetic orchids, including fungi in the families Vanilla and other orchids. Thelephoraceae, Russulaceae, Inocybaceae, Hymenogastraceae, Polyporaceae, and Hymenochaetaceae (Kristiansen et al. 2001; Acknowledgements Authors thank SAGARPA-CONACYT-SNITT Taylor et al. 2002; Roy et al. 2009; Roche et al. 2010; Lee et al. fund for their support through the Subproject 03 and Megaproject 2012- 04-190442; and Vanilla producers for the opportunity to sample in their 2015), but fewer reports are available for non-Rhizoctonia my- commercial plots. SAS thanks for her scholarship from this project. corrhizal fungi in photosynthetic terrestrial orchids. This is likely due to the extensive focus on Rhizoctonia in cultured-based stud- ies, the short persistence of active pelotons in orchid roots, and References the difficulties of isolating ectomycorrhizal fungi in pure culture. Shefferson et al. (2008) reported that the terrestrial photosynthetic Adame-García J, Rodríguez-Guerra R, Iglesias-Andreu LG, Ramos- orchid Epipactis atrorubens, colonizing Estonian mine tailing Prado JM, Luna-Rodríguez M (2015) Molecular identification and hills, was associated to the ectomycorrhizal ascomycetes pathogenic variation of Fusarium species isolated from Vanilla – Trichopaea woolhopeia and Geopora cooperi. Pellegrino and planifolia in Papantla Mexico. Bot Sci 93:669 678. 10.17129/ botsci.142 Bellusci (2009) sequenced an Ascomycete-like and Rusulaceae Altschul SF, Madden T, Schäffer AA, Zhang J, Zhang Z, Miller W, uncultured fungus colonizing roots of Dactylorhiza sambucina. Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new gener- Species of orchids from the Epipactis genus were colonized by ation of protein database search programs. Nucleic Acids Res Tuber spp. and Wilcoxina spp., as well as other ectomycorrhizal 25(17):3389–3402. https://doi.org/10.1093/nar/25.17.3389 ascomycetes and basidiomycetes (Bidartondo et al. 2004; Barmicheva KM (1989) Ultrastucture of Neottia nidus-avis mycorrhizas. Agric Ecosyst Environ 29:23–27. https://doi.org/10.1016/0167- Selosse et al. 2004). 8809(90)90248-C Sporocarps of Scleroderma have been found in the states of Batty AL, Dixon KW, Brundrett MC, Sivasithamparam K (2002) Orchid Puebla and Veracruz, Mexico (Córtez-Pérez 2011;Guzmán conservation and mycorrhizal associations. In: Sivasithamparam K, et al. 2013) where this study was conducted. Besides, many Dixon KW, Barrett RL (eds) Microorganisms in plant conservation of the tree tutors are known to host arbuscular mycorrhizal and biodiversity. Springer, Science+Business Media, Dordrecht, pp 195–226 fungi, all the crop production systems in this study were Bayman P, Mosquera-Espinosa AT, Porras-Alfaro A (2011) Mycorrhizal surrounded by or are located near secondary vegetation from relationship of Vanilla and prospects for biocontrol of root rots. In: disturbed tropical forest. It is likely that ectomycorrhizal fungi Havking-Frenkel D, Belanger FC (eds) Handbook of Vanilla science – are present in some of the nearby tree species but the mycor- and technology, 1st edn. Blackwell Publishing Ldt, Oxford, pp 266 280 rhizal communities in these rural regions in Mexico are poorly Bidartondo MI, Burghardt B, Gebauer G, Bruns TD, Read DJ (2004) documented. Scleroderma was consistently identified in four Changing partners in the dark: isotopic and molecular evidence of of the five Vanilla sites in this study, showing that this putative ectomycorrhizal liaisons between forest orchids and trees. 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