INTRODUCTION

The ectomycorrhiza is a mutualistic symbiosis of enormous functional importance in boreal, temperate and some subtropical and tropical ecosystems (Pérez-Moreno and Read, 2001a, b; Read and Pérez-Moreno, 2003). Today, the use of ectomycorrhizal fungi, particularly of edible mushroom species, has gained immense importance in forest tree production (Smith and Read, 2010). Species of edible ectomycorrhizal mushrooms in Hebeloma and Laccaria are found worldwide (Cairney and Chambers, 1999). The worldwide numbers of recognized species for these edible ectomycorrhizal genera are 75 species for Laccaria (Mueller, 1985), and 150 and 124 species for Hebeloma, according to Kirk et al. (2008) and Index Fungorum (2017), respectively.

The generic name of Hebeloma comes from the ancient Greek word Hebe, meaning ‘youth’ or ‘puberty,’ and the suffix - loma, meaning ‘a fringe’ or ‘youthful veil,’ in reference to the fungal veil that is only seen in immature stages of basidiomata of this genus. Both genera have great potential for use in nursery-based plant inoculation programs because they include pioneer species that thrive in low fertility conditions and that are associated with a wide range of host trees (Obase et al., 2009; Trocha et al., 2007), including species of great forest importance in the genera Eucalyptus, Larix, Picea, Pinus and Pseudotsuga (Danielson, 1984; Baar et al., 1994; Wong et al., 1989).

Several studies have reported on the basidiomata production of edible ectomycorrhizal mushrooms, such as L. laccata (Scop) Cooke, H. cylindrosporum Romagn. and H. sarcophyllum (Peck) Sacc. (Debaud and Gay, 1987; Wang and Hall, 2004). Additionally, some other species of edible ectomycorrhizal mushrooms have sporadically produced basidiomata under controlled conditions; for example, Cantharellus cibarius Fr., Lactarius deliciosus (L.) Gray, Tricholoma portentosum (Fr) Quél and Rhizopogon roseolus (Corda) Th. Fr. (Danell and Camacho, 1997; Danell, 2002; Guerin-Laguette et al., 2000; Yamada et al., 2001).

It has previously been reported that the factors influencing basidiomata formation in edible ectomycorrhizal mushrooms associated with their plant hosts are mainly temperature, fertilization regime (Debaud and Gay, 1987; Ellis et al., 1999), relative humidity (Yamada et al., 2007), the contribution of host carbohydrates and even electrical impulses (Ohga et al., 2001).

It has also been reported that light regulates the fungal development and behavior of some molds and yeasts that belong to the Ascomycota. For example, a blue light-activated complex has been identified in Neurospora crassa as the product of the WC-1 and WC-2 genes (Linden et al., 1997); and the hyphal cell growth of Schizosaccharomyces japonicus is a response to the light which is dependent on the WC-1 and WC-2 orthologs. Additionally, previous studies showed the importance of some wavelengths in the cultivation of saprobic Basidiomycetes including Pleurotus tuber-regium, P. ostreatus, Hypsizygus marmoreus, and Lentinula edodes (Jang et al., 2013; Kuforiji and Fasidi, 2009; Nakazawa et al., 2008). However, to our knowledge, the influence of light wavelength on basidiomata formation in edible ectomycorrhizal mushrooms has not been reported yet.

The aim of this study was to evaluate the effect of two light filters, yellow and red, on basidiomata formation in two edible ectomycorrhizal mushrooms, L. bicolor (Maire) P. D. Orton and H. leucosarx P. D. Orton, associated with two neo-tropical pines of great economic importance: P. greggii Engelm. ex Parl. and P. montezumae Lamb. In addition, the full developmental process of these basidiomata, undescribed so far, from mycelial aggregates of a primordium to hymenophore maturation of a matured basidioma is reported and illustrated in detail.

MATERIALS AND METHODS

Vegetative and fungal material and plant inoculation

Basidiomata of Laccaria and Hebeloma were obtained in a market in Ozumba, State of México, México, and identified according to macro and microscopic features, following conventional methods (Largent, 1973; Mueller, 1992). Species obtained included L. laccata, L. bicolor, L. proxima (Bound) Pat., H. mesophaeum (Pers.) Quél., H. alpinum (J. Favre) Bruchet and H. leucosarx P.D. Orton. Of these species, L. bicolor and H. leucosarx were selected for this study because they have a great economic and cultural importance in central México, and also have a great potential in the production of inoculum for trees of two neo-tropical species native to México, P. greggii (Domínguez et al., 2001) and P. montezumae (Calderón et al., 2006). Both pines are of great forest importance and used in reforestation in degraded sites due to their high survival percentage and economic importance as timbers in México and Central America.

Seeds of both pines came from natural forests in Xochicoatlán, Hidalgo and Cofre de Perote, Veracruz. Before planting, the seeds were sterilized with hydrogen peroxide (H₂O₂) at 30 % for 20 min and rinsed three times with sterile distilled water; the 140 cm³ black plastic cylindrical containers (Merck, Baltimore, EUA) were also cleaned and disinfected with 70 % ethanol prior to being used. The substrate was added to 90 % of the total volume of the containers, and then spore inoculum was applied in a homogeneous layer.

Then, the seeds of the species were placed according to the treatments, just after the spore addition, and they were covered with a surface layer of the same substrate, and finally, a layer of “tezontle” (ground volcanic rock) was added. The substrate used consisted of a mixture of sand, pine bark and forest soil at a 2:2:1 ratio; which was sterilized in an autoclave (Aesa CV-250, Puebla, México) at 103 kPa and a temperature of 125 ºC for 5 h. One hundred and twenty mL of this substrate were added to each container.

The pilei of L. bicolor and H. leucosarx were cut from their stipes, since pilei contain spores in their hymenophores. These pilei were dehydrated in an oven at 35 ºC for 48 h, then ground in a mill (Thomas Wiley, Minnesota, USA) and finally sieved in a 1.19 mm opening mesh to homogenize the size of the particles obtained. The inoculum was kept in 1.5 mL Eppendorf plastic vials at a temperature of 5 °C until use. Each plant was inoculated with 10⁷ to 10⁸ spores, which were counted in a Neubauer chamber (Marienfeld, Lauda-Konigshofen, Germany).

After a year, the plants were transferred to 1000 cm³ pots containing the same substrate and remained there in the greenhouse for the next four years, being watered every other day with sterile distilled water. P. greggii and P. montezumae trees with a mycorrhizal colonization percentage of 90 % or more, were selected for transplantation into pots with a capacity of 7200 cm³ (Venecia, Chicoloapan, México), containing the same substrate. To evaluate the ectomycorrhizal colonization, a modification of the method proposed by Grand and Harvey (1982) was used. In this case, three 98 cm³-cylindrical (20 x 1.25 cm, height and radium, respectively) soil cores were extracted from the pots where the trees were grown for four years, by using PVC tubes; and the percentage of colonization was evaluated in them.

Experimental design and statistical analysis

The experimental design used was completely randomized with eight treatments. Two pine species, P. greggii and P. montezumae, were inoculated with each of L. bicolor or H. leucosarx, separately. The soil surface of the 7200 cm³ pots used for each of the plant fungus combinations was covered with a yellow paper filter (Manila paper, Lumen, Toluca, México) with a wavelength of ~ 590 nm, or a red one with a wavelength of ~ 660 nm, leaving the above-ground part uncovered. For each of the 8 treatments thus generated, there were four replicates, giving a total of 32 pots, each one constituted by a tree. Treatments were arranged in a randomized experimental design. Data analyses for the number of basidiomata produced per pot consisted in the estimation of 95 % confidence limits (Snedecor and Cochran, 1989) for each treatment using Statgraphic Plus 5.1.

Experimental setup and evaluation of variables

The five-year old inoculated P. greggii and P. montezumae trees, covered with the two filter colors, remained for an additional period of a year in the greenhouse in the 7200 cm³ containers and were irrigated every other day. Assessments were made of the basidiomata formed during four months (from October 2013 to January 2014), because their formation was only detected at this time of the year. The age of the trees at the end of the experiment was thus 6 years old. A total number of basidiomata formed in each treatment was counted twice a week, and a detailed description was made of the basidioma development of L. bicolor and H. leucosarx, from the mycelial aggregates of a juvenile primordium to the fertile mature basidiomata (Table 1). Photographs of the developmental stages of the basidiomata were taken with an Olympus SZ61, SZ2-LGB model stereoscopic microscope (USA). Glass slides were placed under those basidiomata to collect basidiospores, to record the color of spore print and to evaluate the spore micromorphology and size. The morphology and size of the spores were evaluated with a LEICA MD1000 model microscope (Germany).

RESULTS

Frequency of basidiomata formation

A total of 102 mature basidiomata (each with a fully developed hymenophore) of L. bicolor and H. leucosarx was recorded. Ninety percent of them corresponded to H. leucosarx and 10 % to L. bicolor. Fifty-five basidiomata were associated with P. montezumae and 47 were associated with P. greggii. Basidiomata formation was recorded from 280 to 330 d after transplantation into the 7200 cm³ pots. The first basidioma formation in H. leucosarx was observed associated with P. montezumae.

Effect of light on basidiomata formation

There was a conspicuous effect of two light filters on the basidiomata formation of Laccaria and Hebeloma. A greater number of H. leucosarx than L. bicolor basidiomata, was observed regardless of the host plant. The number of Hebeloma basidiomata was 2.6 and 2.1 times higher when pot soil was exposed to the yellow filter as opposed to the red filter, in P. montezumae and P. greggii, respectively. The number of L. bicolor basidiomata recorded was relatively small: eight and one basidiomata in association with P. greggii exposed to red filter and yellow filter, respectively. No basidiomata of L. bicolor were observed in association with P. montezumae (Figure 1). The longevity of L. bicolor basidiomata, from a small visible primordium until senescent basidiomata, averaged 18 d. By contrast, the time from the mycelial aggregates to the senescence of H. leucosarx basidiomata averaged 37 d. In the case of L. bicolor, abundant whitish spore print was observed on glass slides 14 d after the appearance of visible primordia.

Developmental pattern of H. leucosarx and L. bicolor basidiomata

H. leucosarx. At 280 d after transplantation into the 7200 cm³ pots, white mycelial aggregates were observed on the soil surface with P. montezumae (Figures 2c, 2e). After this initial phase, the small, white to whitish primordia (Figure 2f), began to widen at their stipe base while the pileus began to form immature basidiomata (Figures 2f and 2g). The stipe secondarily elongated (Figure 2h). The cream-colored gills of the young basidiomata turned light brown as the pileus matured and expanded symmetrically (Figures 2h and 2i).

The shape of the pileus ranged from subglobose to hemispherical, later convex and flat in fully mature basidiomata (Figures 2i and 2j). Spores (Figure 2k) were almond-shaped [9.36-12.05 (Lm = 10.95 µm) x 5.27-7.39 (Wm = 6.225 µm) µm and Q = 1.63-1.77 (Qm = 1.76)] where Q means the ratio of length/width for all spores measured. This is the first description of the developmental pattern of basidiomata of H. leucosarx. In general, there is little information on H. leucosarx, as most Hebeloma articles focus on H. crustuliniforme and H. cylindrosporum (Cairney and Chambers, 1999).

L. bicolor. At 280 d after transplantation, white mycelial aggregates were observed at the stem base of P. greggii trees (Figure 3a). After this initial phase, the small primordia (Figures 3b and 3d), began to widen at their stipe base and developed violet-colored young basidiomata (Figure 3e). Matured basidiomata showed subglobose pileus (Figure 3f), later convex and finally flat (Figure 3g), finely fibrous or scaly, with pink gills (Figure 3f). The stipe’s base was deep violet but its top displayed a lighter color. Spore print was white to whitish, spores were globose, and spiny ornamentation was observed (Figure 3h). The above-described features of L. bicolor match the description given for field basidiomata by Mueller (1992). The basidiomata formation was recorded along with the ectomycorrhizal structures, the host roots, including mantle, Hartig net and external hyphae (Figure 3i).

DISCUSSION

This study found an abundant formation of mature fertile basidiomata of L. bicolor and H. leucosarx associated with around 6-year-old neo-tropical P. greggii and P. montezumae seedlings, using ground pilei containing spores as inoculum source. There have been previous reports of basidiomata formation in several edible ectomycorrhizal fungal species using mycelium as inoculum source: basidiomata of H. cylindrosporum have formed with P. pinaster (Debaud and Gay, 1987), L. laccata with P. patula, L. bicolor with P. strobus (Lamhamedi et al., 1994) and Lactarius deliciosus with P. sylvestris (Guerin-Laguette et al., 2000). Massicotte et al. (2005) reported the early stages of the ontogeny of L. bicolor basidiomata associated with P. sylvestris in solid medium; however, they did not report whether these basidiomata were fertile or not, but the features described are similar with regard to the shape of the mycelial aggregates and primordia found in the developmental pattern of L. bicolor basidiomata in the present study.

Basidiomata formation in ectomycorrhizal fungi is a complex phenomenon because a set of specific physical, nutritional and biotic factors is required, as well as the obligate dependency towards the host plant. In general, these conditions have been poorly studied in the case of edible ectomycorrhizal mushrooms (Kües and Liu, 2000; Wessels, 1993; Yamada et al., 2007).

There is a previous report of primordium formation for L. laccata, associated with P. sylvestris and P. resinous (Massicotte et al., 2005). In the genus Hebeloma, various studies have reported the fruiting phenomenon: Debaud et al. (1981) reported mycorrhizal synthesis between an arctic-alpine evergreen shrub, Dryas octopetala L., and H. alpinum and H. marginatulum (J. Favre) Bruchet, which fruited after eight months in culture. Debaud and Gay (1987) reported primordium formation in H. cylindrosporum associated with P. pinaster, a year and a half after inoculation in sterile conditions using peat moss and vermiculite as substrate. However, this fungus did not complete its life cycle in the absence of the host plant in the same culture conditions and only produced primordia.

Ohta (1998) succeeded in producing basidiomata, with spore formation, of H. radicosum and Hebeloma sp. [later on described as H. radicosoides Sagara, Hongo et Y. Murak. by Sagara et al. (2000)] in culture medium, and reported the nutrients that promote mycelial growth and basidiomata production for these two species. Here, the production of primordial and fertile basidiomata of H. leucosarx on inoculated trees is reported for the first time. The basidiomata formation of Laccaria and Hebeloma in the present study has a great biotechnological potential for the use of these species as inoculants of neo-tropical trees of forest importance. Indeed, these fungi are pioneer species that produce beneficial effects in terms of growth and nutrient content (N, P, K, Ca and Mg) of their associated hosts (Carrasco-Hernández et al., 2011; Martínez-Reyes et al., 2012) and are widely marketed for consumption in the markets of central and southeastern México (Pérez-Moreno et al., 2008).

Several studies have shown that environmental conditions are crucial to basidioma formation in ectomycorrhizal fungi, including temperature, carbon dioxide concentrations in the atmosphere, moisture, salinity (Kües and Liu, 2000; Wessels, 1993), fertilization regime (Debaud and Gay, 1987; Godbout and Fortin, 1990), relative humidity (Yamada et al., 2007), the contribution of host carbohydrates (Lamhamedi et al., 1994), and the abundance of ectomycorrhizal colonization of the fungal species of interest (Guerin-Laguette et al., 2013; 2014; Pilz and Molina, 2001).

It has also been shown that electric impulses promote basidiomata formation in L. laccata, because they cause ruptures in the hyphae or enzyme activation through the application of high voltage, which acts as a stimulator for basidiomata formation (Ohga and Ida, 2001; Ohga et al., 2004; Takaki et al., 2009a). Takaki et al. (2009a; 2009b; 2010), reported a similar trend in the production of the edible saprophytic mushrooms Pholiota nameko, Lentinula edodes, known as shiitake, and Lyophyllun decastes using electrical impulses.

The present study found that different light wavelengths (590 nm versus 660 nm) differentially influenced the number of L. bicolor and H. leucosarx basidiomata produced. Previously, Lamhamedi et al. (1994) showed that the higher the photosynthetic flux densities the bigger the size of basidiomata of L. bicolor associated with P. strobus L. Additionally, they showed that greater net photosynthesis produced higher basidiomata biomass; and that the removal of basidiomata resulted in a quick decrease of net photosynthesis and stomatal conductance of the host trees. It is generally known in saprotrophic Coprinopsis and Lentinus that different light intensities produce excellent basidiomata production (Ellis et al., 1999; Kitamoto et al., 1972; Kües, 2000).

An interesting observation in the present study was the difference in the size of L. bicolor and H. leucosarx basidiomata in relation to the size of the containers. When the plants were in the 1000 cm³ containers, the formation of L. bicolor basidiomata with a maximum height of 4 cm was observed, while the basidiomata formed in the 6 kg pots were up to 7 cm in height. Similarly, in the case of H. leucosarx, the basidiomata formed had a maximum height of 6 cm in the 1000 cm³ containers, while in the 7200 cm³ containers the basidiomata formed were up to 18 cm in height, which is their natural size when collected in the field.

One reason that could explain the smaller size of the basidiomata in the smaller containers could be a reduced supply of nutrients and water, as noted by Kikuchi et al. (2009). Another interesting observation was that the L. bicolor and H. leucosarx primordia obtained in our experiments sometimes developed at the base of the root system, near the drainage holes of the container. A similar phenomenon was reported for C. cibarius inoculated to P. sylvestrisDanell and Camacho (1997), and for L. bicolor primordia associated with white pine seedlings (Godbout and Fortin, 1990). In our case, basidiomata did not show positive gravitropism when they grew near the drainage holes as their stipes remained upright with their pilei at the bottom. Guerin-Laguette et al. (2000) also observed the lack of positive gravitropism with L. deliciosus growing upside down underneath containerized, mycorrhized, P. sylvestris seedlings.

CONCLUSIONS

To the best of our knowledge, this study demonstrates for the first time that light wavelength is a factor which influences basidiomata formation in ectomycorrhizal fungi, light wavelength differentially influencing the basidiomata formation depending on the fungal species. For H. leucosarx, a statistically higher number of basidiomata was observed when the pots were covered with a yellow filter, compared to a red filter, regardless of the associated tree species. An opposite trend was observed for L. bicolor, where a statistically higher number of basidiomata was recorded in P. greggii covered with a red filter.

Additionally, the host tree species influenced basidiomata formation differentially. The number of basidiomata of H. leucosarx associated with P. montezumae was higher than the number of basidiomata recorded in association with P. greggii. In the case of L. bicolor, basidiomata were only recorded when it was associated with P. greggii. Finally, the developmental process of H. leucosarx basidiomata is described for the first time. The developmental process of both L. bicolor and H. leucosarx basidiomata was recorded in detail.

ACKNOWLEDGMENTS

The first author is grateful to both CONACyT and COMECyT for a scholarship. We thank Dra. Magdalena Martínez-Reyes for her valuable suggestions and technical advice, and to Greta Hanako Rosas Saito for her assistance in the scanning electron microscopy. Financial support from the Project CONACyT 246674 “Biotecnologías de los hongos comestibles ectomicorrízicos y su impacto en la mitigación del cambio climático y desarrollo forestal sustentable” is acknowledged. We also acknowledge the valuable suggestions made by two anonymous referees and the editor.

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Author notes

jperezm@colpos.mx