Seed germination and development of orchid seedlings (Cyrtopodium saintlegerianum) with fungi

Fungi of Rhizoctonia complex are mycorrhizal of orchids and may to germinate yours seeds and development the seedlings. In this context, our objective was to select a fungal isolate to promote seed germination and seedling development of Cyrtopodium saintlegerianum. Pelotons were found in the roots and three mycorrhizal fungi were isolated. We tested mycorrhizal isolates obtained from C. saintlegerianum roots and six mycorrhizal fungi from other orchids as well three pathogenic isolates (of rice and bean) to germinate the seeds in oatmeal-agar medium. Seeds not inoculated were used as control. The isolates En07 (Waitea circinata), Cs10 (Tulasnella sp.) and Ro88 (Rhizoctonia oryzae) were efficient to promote seed germination, but only En07 differing statistically of the control. The non-specific isolate En07 promoted germination in 81% of seeds and the specific isolate (Cs10) promoted 60%, evidencing the non-specificity mycorrhizal association in this orchid during germination. Axenic seedlings were inoculated with four mycorrhizal fungi (non-inoculated seedlings control). After six months, the isolates En07 and Cs10 were efficient in the interaction with the seedlings, but did not differ to the control. Therefore, our results suggested that fungi of the Rhizoctonia complex can be used in the germination and seedling development of C. saintlegerianum.


Introduction
Mycorrhizal associations are essential to the life cycle of orchids in natural habitat. The interaction begins during seed germination when the mycorrhizal fungi infect basal cells in embryo. The hyphal coils formed into these cells are digested by the orchid to obtain carbon and nutrients necessary for its developmental initial phase. The seed produces a heterotrophic structure called the protocorm, which then forms the seedling. The seedling produces its first root and fungi can colonize its cortical cells. From this phase, symbiosis with mycorrhizal fungi facilitates the acquisition of nutrients from the substrate (Peterson et al. 2004;Rasmussen & Rasmussen 2009;Dearnaley et al. 2012).
Orchids Mycorrhizal Fungi (OMF) verified in Brazilian orchids belong to the Rhizoctonia complex and were identified as Ceratobasidium D.P. Rogers, Thanathephorus Donk and Tulasnella Schroet teleomorphic genera (Nogueira et al. 2005;Pereira et al. 2011Pereira et al. , 2015Silva et al. 2016). And studies have shown that mycorrhizal isolates promote in vitro seed germination and seedling development better than axenic commercial media for orchid propagation (Pereira et al. 2011;Guimarães et al. 2013;Jiang et al. 2015). In this way, the mycorrhizal fungi inoculation has been highlighted as a promising strategy to improve orchid seedling production (Cribb et al. 2003).
Due to differences in specificity observed during mycorrhizal orchid interactions, fungal isolation has been required to select the suitable isolate to promote seed germination and seedling development (Dearnaley et al. 2012). Some orchids have a narrow specificity with some fungal genera. During seed germination experiments, Coppensia doniana (Batem. ex W. Baxter) Campacci and Oncidium flexuosum Sims demonstrated preference for the mycorrhizal fungi Ceratorhiza sp., anamorphic form of Ceratobasidium genera . In contrast, Epidendrum secundum Jacq. and Cyrtopodium glutiniferum Raddi preferred fungi Epulorhiza sp., mycorrhizal anamorphic form of Tulasnella genera (Pereira et al. 2009(Pereira et al. , 2015. Understanding such specificities is valuable for selection of the symbiont that assure propagation and commercialization of healthy orchid seedlings. Some mycorrhizal fungi also associate with non-host orchids. For example, the seeds of Tolumnia variegata (Sw.) Braem associated with the mycorrhizal isolate of Ionopsis utricularioides (Sw.) Lindl. (Otero et al. 2004); seeds of Epidendrum nocturnum Jacq. interacted with Spiranthes brevialabris Lind. mycorrhizal fungi (Zettler et al. 2007); and seeds of Spathoglottis plicata Blume had better germination with two mycorrhizal isolates of Dendrobium anosmum Lind. and Paphiopedilum sukhakulii Schoser & Senghas (Aewsakul et al. 2013). The performance of non-specific mycorrhizal fungi may be evaluated to select the suitable fungi for propagating of some orchids (Zettler et al. 2007).
Fifty terrestrial and epiphytic species of Cyrtopodium Rchb. f. have been reported from South America, thirty of which occur in area the Cerrado (Batista & Bianchetti 2005). Some species of the genus Cyrtopodium have been widely explored as sources for raw material for small medical industries and ornamental gardens (Barreto & Parente 2006;Dutra et al. 2009;Vogel & Macedo 2011;Pereira et al. 2015). Cyrtopodium saintlegerianum Rchb. f. occurs as an epiphyte on species of palm in the Brazilian Cerrado (Batista & Bianchetti 2006;Romero-Gonzáles et al. 2008;BFG 2018), and has been propagated in media axenic containing phytoregulators (Rodrigues et al. 2015;Silva et al. 2017). This species has been used in the ornamentation of gardens as well as to prepare dermatological plasters.
Little information has been reported about mycorrhizal association in these genera orchids. Recently, seeds and seedlings of C. glutiniferum exhibited satisfactory development when inoculated with mycorrhizal fungi of the genus Tulasnella (Guimarães et al. 2013;Pereira et al. 2015). However, more studies are required to determine the presence of mycorrhizal fungi in situ, their isolation and to evaluate fungi potential during in vitro symbiotic seed germination. In view of this aspect, the objective in present study was to select a suitable fungal to promote seed germination and seedling development of C. saintlegerianum. To achieve this aim, we first confirm root mycorrhizal colonization of C. saintlegerianum throw root anatomical analysis. Its mycorrhizal fungi were isolated and identified morphologically. The seed germination test was performed co-inoculating C. saintlegerianum seeds with different isolates: its own mycorrhizal fungi, others mycorrhizal isolates and some pathogenic Rhizoctonia-like fungi. Isolates that promoted the development of the embryo as well as other specific isolates were inoculated in axenic seedlings of C. saintlegerianum to test their potential to support the ex vitro development.

Capsule and root collection
Capsules and roots of C. saintlegerianum (Fig. 1a) were collected during March 2010 to August 2011 from three different plants growing on three palms in pasture areas in the Brazilian Cerrado (16 o 07'66.6''S and 50 o 10'04.4''W). The biological material was transported to the Laboratório de Genética de Microrganismos (LGM) at the Universidade Federal de Goiás (UFG) Brazil. Some root fragments were fixed in FAA70 (Formaldeyde -Acetic acid -Alcohol 70%) (Johansen 1940) for two days and stored in ethanol solution (70%) until anatomical characterization of mycorrhizae. Others fragments were reserved to mycorrhizal fungi isolation. The capsules were stored into flask containing silica gel and kept at 4 °C until germination experiments.

Mycorrhizae microscopy characterization, fungal isolation and identification
Root fragments of C. saintlegerianum were sectioned by freehand for optical microscopy (OM) observation. The sections were cleared and subjected to 1% aqueous safranin and 0.3% astra blue (Krauss & Arduin 1997) for cell roots and fungal structures coloring. Root sections were prepared to Scanning Eletronic Microscope (SEM) observation according to Silva et al. (2016) in Laboratório Multiusuário de Microscopia de Alta Resolução -LabMic, Physics Institute, UFG.
The mycorrhizal fungi were isolated according to Gonçalves et al. (2014) using the PDA medium (Potato Dextrose Agar, composed of 200 g of potato, 20 g of dextrose, 20 g of agar and 1 L of water - Otero et al. 2004). Fungal isolates were cultivated on PDA plates under continuous fluorescent light for five days at room temperature (26±2 °C). Isolates with morphological characteristics of OMF (Currah & Zelmer 1992) were maintained in a growth chamber at 26±2 °C with a 16 h photoperiod.
The identification of OMF were performed using features described by Currah & Zelmer (1992), Nogueira et al. (2005), Pereira et al. (2005) and Silva et al. (2016). The cultural characteristics evaluated were colony diameter, growth tax, number of nuclei per cell, hyphal diameter, width and length of monilioid cells and polyphenol oxidase (PPO) production. The cultural characteristics (color, aerial mycelium, mycelium shape and size, and colony diameter) were evaluated after 72 and 336 h on PDA or CMA medium (Corn Meal Agar, composed of 15 g agar and 1 L broth obtained from cooking 30 g of corn meal).
The number of nuclei per cell and hyphal diameter were evaluated according to Meinhardt et al. (2001). The images of hyphae were captured using a Leica DMI6000 optical microscope (OM) with an epifluorescence accessory and processed in Leica IM50 editor. The width and length of monilioid cells of each isolate were assessed in fungal colonies cultured in CMA for two months and were measured from images taken under BelPhotonics microscope cells using the BelAnalyzer MicroImage software.
The fungal PPO production was evaluated in Petri plates containing YEA medium (Yeast Extract Agar, composed of 15 g Yeast Extract 15 g, 15 g Agar 15 g in water 1 L) with addition of tannic acid (5 g), according to Zelmer & Currah (1995). Petri plates without tannic acid were used as a control. The production of PPO was detected by the presence of an amber-colored halo around the colonies after five days.

In vitro symbiotic germination and ex vitro development
Seeds collected from a 12-month mature capsule were sterilized as described for Silva et al. (2016). After capsule opening, 50% of the seeds were used to test the viability of the embryo with triphenyltetrazolium chloride (TTC) and 50% were used for symbiotic cultivation. A TTC solution was used to evaluate seed viability adopting modifications to orchid seeds (Vujanovic et al. 2000). Ten sample (with approximately 0.001 g of seed) were transferred into microtubes containing 2 mL of 1% TTC solution and kept in a water bath at 40 °C in continuous darkness to prevent TTC precipitation. The embryos were observed for the capture of images under light microscopy (BelView software).
We tested mycorrhizal isolates obtained from C. saintlegerianum roots and six mycorrhizal fungi from other orchids (fungal isolates belonging to the LGM library). Three pathogenic isolates, taxonomically related to some mycorrhizal fungi (two of Rhizoctonia oryzae Ryker & Gooch and one of Rhizoctonia solani Kühn, pathogenics of rice and bean) were supplied by the Collection of Functional Microorganisms of Embrapa Arroz e Feijão (CNPAF) -Brazil (Tab. 1). All isolates selected were grown in plates with oatmeal-agar medium (OMA -Zettler et al. 2007;Steinfort et al. 2010). A 9-mm mycelial disc was taken from the edge of each isolate colony and placed in plates with OMA and containing approximately 150 seeds. Plates containing seeds without fungi were maintained as controls. The experimental design was completely randomized with ten treatments and eight replicates (Tab. 1). The plates were incubated in a germination chamber at 26±2 °C and with a 16 h photoperiod. The data obtained from the seed viability test were normalized by square root transformation, whereas the germination data were transformed using arcsin.
To compare each treatment we performed an analysis of variance and a posteriori Tukey test (at 5% probability) using the software R v 2.11.0 (Díaz & Álvarez 2009;Steinfort et al. 2010). Germination was assessed every two weeks, under a light microscope coupled to a digital camera, using the parameters: 0 -no germination, 1 -swelled embryo and rupture of testa (germination), 2 -continued embryo enlargement and production of rhizoid (Fig. 2) adapted of Stewart & Zettler (2002). The final evaluation occurred nine months after sowing. Seeds were observed under an optical microscope to assess the presence of pelotons, which were stained according to Chutima et al. (2010). Seeds at different stages of germination were collected for SEM observation. Seed preparation was performed as suggested by Chou & Chang (2004) and visualization was done in the same mode as the root fragments.
For ex vitro mycorrhization assessment, asymbiotic seedlings with thirteen months were obtained from in vitro seed germination using MS medium (Silva et al. 2017). The mycorrhizal fungi were Tulasnella sp. isolates (Cs02, Cs10 and Cs21) obtained from C. saintlegerianum and Waitea circinata Warcup & Talbot (En07) from E. nocturnum (Tab. 1). Seedlings of C. saintlegerianum were inoculated with 0.2 g of mycelium of each isolate. The seedlings were grown in axenic substrate (Sphagnum sp.) in a complete randomized experimental design with five treatments and five replicates. Number of shoots, stem diameter (0.5 cm above the base), stem vigor in region of pseudobulb formation (1 cm above the base) and survival of the seedlings were evaluated after six months. To compare each treatment (isolate and control) we performed an analysis of variance and a posteriori Tukey test (at 5% probability) using the software R v 2.11.0 .

Mycorrhizal colonization, fungal morphological characterization and identification
The pelotons were stained dark blue due to the presence of chitin in the hyphae (Fig. 1bc). Pelotons were mostly intact with the hyphae occupying the cortical cells of the roots (Fig.  1b). Connective hyphae between pelotons within neighboring cells were found (Fig. 1c). Intact (Fig.  1d) and degraded (Fig. 1e) pelotons within cortical cells roots were observed. From C. saintlegerianum roots were obtained three isolates (Cs02, Cs10 and Cs21 - Fig. 1f-h) with Rhizoctonia characteristics (Currah & Zelmer 1992). The isolate Cs02 presented faster mycelial growth than Cs10 and Cs21 in BDA medium. The mycelium of isolates Cs02 and Cs21 was white while Cs10 is brown (Fig. 1f-h). Only in CMA medium, these isolates produced round monilioid cells with chains containing up to five cells (Fig. 1ij). The isolates presented two nuclei per cell and we registered two nuclei in one new monilioid cell of isolate Cs02 (Fig. 1k). After five days of growth on PDA microculture, the isolate Cs10 formed pelotons or hyphae bundles (Fig. 1l). None of the isolates (Cs02, Cs10 and Cs21) formed an amber-colored halo, indicating absence of PPO production. Based on morphological characterization, these isolates were identified as Tulasnella sp. (Epulorhiza sp., anamorphic phase).

In vitro germination and ex vitro development
Embryos of C. saintlegerianum seeds were stained at 3 h and the viability test demonstrated that 77% of the seeds were viable as indicated by their dark red color (Fig. 3a). The isolate non-specific En07 highlights from the other fungi providing seed tegument rupture and rhizoid formation (Stage 2) after two months (Fig. 3b). In the control (without fungus), there was no rhizoid formation (Fig. 3c). After nine months, the treatment with the En07 isolate provided 81% of germinated seeds (Stages 1 and 2). However, it was not statistically different from the isolates Cs10 (C. saintlegerianum specific) and Ro88 (pathogenic to rice), which presented 60 and 52% germinated seeds, respectively. The treatment with En07, CS10 and Ro88 showed a larger number of germinated seeds and differed statistically from the control (Tab. 2).
In SEM we observed cracks in the coat of seeds inoculated with isolate En07, indicating seed coat rupture in consequence of embryo swelling ( Fig. 3g-h). The seeds from the control showed intumescence, but did not germinate because there was no differentiation in the embryo or tegument rupture (Fig. 3i).
In the ex vitro symbiotic development, the isolates En07 and Cs10 (Fig. 3j-k) promoted thicker and vigorous stems in axenic seedlings of C. saintlegerianum after six months, but did not differ statistically from the control (Fig. 3l; Tab. 3). All isolates promoted shoot formation, but only En07 treatment presented a shoot number significantly higher than the control. Survival percentage of seedlings in association with fungi isolates was not differed from the control (Tab. 3).

Discussion
This is the first report of symbiotic germination and seedling development of C. saintlegerianum. Our study showed that embryos without fungi just exhibited intumescence, without rhizoids formation.  Figure 3 -Cyrtopodium saintlegerianum seeds and seedlings -a. viable (v) and non-viable (nv) embryos of seeds submitted to tetrazolium test (TTC); b. seed inoculated with isolate En07 (Waitea circinata of Epidendrum nocturnum) with rhizoids after two months; c. seed without fungi (control) were swollen after nine months of growth in oatmealagar media; d. seed germinated after nine months with En07 (showed many rhizoid -Stage 2); e. seeds showed rhizoids with isolate Cs10 (Tulasnella sp.); f. rhizoid emerging from the embryo and peloton inside (seed with En07); g. swollen seed showing a crack in the seed coat, suggesting that isolate En07 colonized the embryonic cells (Stage 2); h. non-germinated seeds (0) and swollen seeds (1) derived from the association with isolate En07; i. seeds with swollen embryos, but non-germinated (control -Stage 0); j. seedling six months after of ex vitro mycorrhization with isolate En07 (non-specific); k. seedling with isolate Cs10 (specific); l. seedling without fungal (control). Bars = 1 cm (j-l), 200 μm (b-d,h), 100 μm (g,i), 50 μm (a,e), 10 μm (f). Mycorrhizal fungi Tulasnella sp. and W. circinata promoted embryo development up to produce protocorm with rhizoids and ruptured coat, which confirm dependence of C. saintlegerianum during seed germination. Additionally, W. circinata isolate support better seedling development. Indeed, W. circinata is a potential isolate to be applied during symbiotic cultivation of C. saintlegerianum. We observed a large number of intact and degraded pelotons in parenchyma cells of the root cortex in adult plants of C. saintlegerianum (Fig.  1b-e). These observations confirm the maintenance of the mycorrhizal association at adult phase. Thus, the presence of pelotons indicates the orchid needs fungal interaction to acquire nutrients. In addition, the pelotons degradation may be associated with the flowering period when roots were collected, in which the plant has a higher nutritional demand.
The morphological characteristics and no production of PPO confirm identification of C. saintlegerianum mycorrhizal isolates as Tulasnella sp. (Currah & Zelmer 1992;Zelmer & Currah 1995;Athipunyakon et al. 2004). We observed morphological differences among our Tulasnella (Fig. 1f-l), which propel us to select the three Tulasnella isolates to in vitro seed germination experiment. These isolates presented different results during seed germination and ex vitro seedling cultivation, corroborating with Pereira et al. (2009;, who observed that morphologically different Tulasnella can present divergent results in seed germination experiments. Some species interact with mycorrhizal fungi just during seed germination, although other species maintain the interaction during the adult phase (Peterson et al. 2004;Zettler et al. 2007;Rasmussen & Rasmussen 2009). Zettler et al. (2007) reported symbiosis maintenance between Tulasnella fungi and orchid S. brevilabris from embryo stage until adult phase. In the same way, Látalová & Baláz (2010) and Gonçalves et al. (2014) demonstrated interaction between orchid and mycorrhizal fungus from protocorm until adult stage. Silva et al. (2016) observed that W. circinata increases the vigor of Epidendrum nocturnum seedlings in vitro and a potential interaction was observed with seeds and seedlings (in vitro and ex vitro, respectively) of C. saintlegerianum too. Thus, our isolates can support the mycotrophism of this plant during in vitro and ex vitro propagation, even in natural habitat.
Earlier investigations have shown that Tulasnella sp. is associated with many tropical orchids as C. vernun, C. glutiniferum, E. secundum, In light of this, screening non-natural and natural mycorrhizal fungi is an important strategy to select a suitable isolate to orchid symbiotic cultivation. Some orchids present better development in association with their own OMFs. It is probably in consequence of narrow mycorrhizal specificity (Valadares et al. 2010;Silva et al. 2016). Other orchids respond better to OMFs of other plants, suggesting broad mycorrhizal specificity (Otero et al. 2004), regarding with present study. The ability of R. oryzae and R. solani isolates (Tab. 2), pathogenic fungi of rice and bean plants, to promote in vitro seed germination of C. saintlegerianum have highlighted. Masuhara et al. (1993) had no success in seed germination of Spiranthes sinensis var. amoena with pathogenic isolates of R. solani and R. oryzae. However, Masuhara & Katsuya (1994) suggested that Rhizoctonia spp. pathogens of rice (Oryza sativa) would also germinate orchid seeds. It is an evidence that orchids suppress pathogenic potential of these fungi and use them as nutrient source during embryo and seedling development.
The En07 and CS10 improved seedlings vigor, shoot number and stem diameter of C. saintlegerianum seedling (Tab. 3; Fig. 2j-l), but through the evaluations carried out it was not possible to verify statistical difference. These improvements are indispensable to plant longevity and establishment during acclimatization. Hence, in future approaches on the increases from seedlings association with fungi, more refined methods of measurement will be needed. Benefits of mycorrhization during orchid seedling establishment have been reported to C. glutiniferum (Guimarães et al. 2013), Phalaenopsis sp. (Moreno et al. 2000;Wu et al. 2011) e Spathoglottis plicata Blume (Aewsakul et al. 2013). Additionally, mycorrhizal associations can also suppress biotic agents, such as plant pathogens (Peterson et al. 2004;Rasmussen & Rasmussen 2009). OMFs induced resistance in rice plants to pathogenic soil fungal (Mosquera-Espinosa et al. 2013) and our isolate En07 (W. circinata) demonstrated potential as biocontrol agent against Magnaphorte oryzae, a rice blast pathogen (Carvalho et al. 2015).
Our results support the use of mycorrhizal fungi in germination and development of C. saintlegerianum. This orchid has little mycorrhizal specificity, facilitating its interaction with fungi from other plants. Some Rhizoctonia isolates may supporte seed germination, plant vigor, greater longevity and resistance to environmental factors. Thereby, we advocate the use of fungi during C. saintlegerianum propagation and suggest testing the inoculation of these in other orchid seeds and seedlings. Future investigations are necessary in order to better understanding the orchid-fungal interactions as well as the evaluation of the increment of the application of the fungi in axenic seedlings. * Stem vigor in region of pseudobulb formation (1 cm above the base) and stem diameter (0.5 cm above the base); ** Means followed by the same lowercase letters in the same row did not differ from each other according to the Tukey's test (P < 0.05).