Introduction

The ectomycorrhizal symbiosis between fungi and trees or shrubs, both Gymnosperms and Angiosperms, mainly occurs in temperate and boreal zones. This symbiosis is ecologically relevant due to the impact on the structure, composition, and functioning of plant communities (^{Pérez-Moreno & Read, 2004}; ^{Smith & Read, 2008}; ^{Umbanhowar & McCann, 2005}). Mycorrhized seedlings have advantages over non mycorrhized ones since fungi improve their nutritional status, water absorption, drought and disease resistance enhancing plant growth and fitness (^{Barroetaveña & Rajchenberg, 2003}; ^{Bonfante & Genre, 2010}; ^{Pérez-Moreno & Read, 2000}).

However, the outcome of the mycorrhizal symbiosis varies from positive to neutral (even negative) depending on the plant species, the species of fungus and their origin, as well as the soil fertility (^{Barroetaveña et al., 2016}; ^{Umbanhowar & McCann, 2005}). The origin of the participants, i.e., whether they are sympatric or allopatric, is particularly important because it determines its natural predisposition to establish the symbiosis. In previous studies with arbuscular mycorrhizal fungi, the results indicate significant effects in the local adaptation when the tests have included sympatric plants and fungi, instead of allopatric combinations (^{Hoeksema et al., 2010}; ^{Klironomos, 2003}; ^{Rúa et al., 2016}). The ectomycorrhizal symbiosis with native species has also shown better local adaptations, mostly reflected in terms of plant growth and colonization (^{Carrasco-Hernández et al., 2010}, ²⁰¹¹; ^{Carrera-Nieve & López-Ríos, 2004}; ^{Cuevas-Rangel, 1979}; ^{Martínez-Reyes et al., 2012}; ^{Méndez-Neri et al., 2011}; ^{Perea-Estrada, 2009}; ^{Quoreshi et al., 2009}; ^{Valdés et al., 1983}, ²⁰⁰⁹).

Species of the genus Laccaria (Berk & Bromme) are among the main ectomycorrhizal fungi used around the world (^{Kropp & Mueller, 1999}). Laccaria species are habitat pioneers and they have been used as model species in the study of ectomycorrhizal symbiosis (^{Khasa et al., 2009}; ^{Pera & Parladé, 2005}; ^{Quoreshi et al., 2008}; ^{Trappe, 1977}; ^{Wadud et al., 2008}, ²⁰¹⁴). Additionally, the publication of the genome sequence of the ectomycorrhizal fungus, L. bicolor (^{Martin & Selosse, 2008}), was the foundation of subsequent studies of the ectomycorrhizal interaction at genomic level (^{Larsen et al., 2011}). Species of this genus have been used to perform mycorrhization on different tree genera such as Pinus, Pseudotsuga, Betula, Quercus, among others (^{Dixon & Johnson, 1992}; ^{Gibson & Deacon, 1988}; ^{Mortier et al., 1988}; ^{Onwuchekwa et al., 2014}; ^{Parladé & Álvarez, 1993}; ^{Sudhakara & Natarajan, 1997}; ^{Zadworny et al., 2004}). Even though all species of the genus are considered good mycorrhizal candidates, the former statement is not always accurate at the species level because the symbiosis is highly specific (^{Kropp & Mueller, 1999}; ^{Molina et al., 1992}; ^{Perea-Estrada et al., 2009}; ^{Wilson et al., 2017}). Therefore, the adaptation to local conditions reflects evolutionary processes in the plant-fungal symbiosis process under specific environmental conditions within the geographic distribution of both symbionts (^{Hoeksema et al., 2010}).

Species of Laccaria that have been used in mycorrhization processes are L. laccata s.l., L. bicolor, L. amethystina, L. proxima, and L. trichodermophora in association with Pinus as P. ayacahuite, P. banksiana, P. contorta, P. densiflora, P. douglasiana, P. greggii, P. michoacana, P. montezumae, P. oaxacana, P. patula, P. pinaster, P. pinea, P. pseudostrobus, P. radiata, P. rudis, and P. sylvestris (^{Carrasco-Hernández et al., 2010}, ²⁰¹¹; ^{Carrera-Nieve & López-Ríos, 2004}; ^{Chapela et al., 2001}; ^{Galindo-Flores et al., 2015}; ^{Hynson et al., 2013}; ^{Martínez-Reyes et al., 2012}; ^{Méndez-Neri et al., 2011}; ^{Onwuchekwa et al., 2014}; ^{Parladé & Álvarez, 1993}; ^{Pera & Parladé, 2005}; ^{Perea-Estrada et al., 2009}; ^{Perrin et al., 1997}; ^{Quoreshi et al., 2009}; ^{Sudhakara & Nataranja, 1997}; ^{Teramoto et al., 2012}; ^{Valdés et al., 2006}; ^{Zadworny et al., 2004}). However, in previous works, the efficiency of sympatric versus allopatric species with Pinus hosts has not been experimentally tested.

The ectomycorrhizal symbiosis is a tripartite partnership, where mycorrhizal helper bacteria (MHB) promote host colonization and enhance the symbiosis function (^{Aspray et al., 2013}; ^{Garbaye, 1994}; ^{Frey-Klett et al., 1999}; ^{Vik et al., 2013}). There are MHB included in the genera Enterobacter, Paenibacillus, Pseudomonas, Burkholderia, Rhodococcus and Streptomyces (^{Kumari et al., 2013}). The MHB can promote mycorrhization in the bacteria-fungus-plant interaction. However, in general the MHB favor the phase of pre-infection as they favour spore germination, mycelia growth through the soil, as well as an increase in the root susceptibility to mycorrhizal colonization. These effects have been demonstrated for Pseudomonas fluorescens in L. laccata and L. bicolor (^{Deveau et al., 2007}, ²⁰¹⁰; ^{Duponnois & Garbaye, 1991}; ^{Frey-Klett et al., 1999}).

With the aim of producing native ectomycorrhizal inocula suitable for forest plants adapted to local conditions we selected the Trans-Mexican Volcanic Belt (TMVB) as a study model. The TMVB is around 1,000 km length with irregular amplitudes ranging between 80 and 230 km. This mountain range is recognized as a center of diversification, endemism and biogeographic transition for a variety of taxa, making it one of the most heterogeneous and complex biogeographic provinces (^{Flores-Villela & Canseco-Márquez, 2007}; ^{Morrone, 2010}; ^{Morrone & Escalante, 2002}). Pinus is the most diverse ectomycorrhizal host worldwide. Mexico is an important diversification center for Pinus Lamb. with 47 species, from which 50% are distributed in the TMVB (^{Farjon, 1996}; ^{Farjon & Styles, 1997}).

To test the hypothesis that the ectomycorrhiza becomes more efficient (in terms of plant growth and nutrient content) when a sympatric relationship between fungi and plants exists, we evaluated the mycorrhization effect on P. montezumae with 2 sympatric fungi (L. trichodermophora and L. bicolor s.l.) and 2 allopatric ones (L. laccata var. laccata and L. vinaceobrunnea). We also tested the effect of P. fluorescens on the mycorrhization by the 4 fungal species.

Materials and methods

One of the most important forest trees in the TMVB is Pinus montezumae Lamb. This species is naturally distributed between 2,000 and 3,200 m asl forming large woodland areas in the National Parks. Four Laccaria species that produce sporomes in great abundance were selected (^{Garibay-Orijel et al., 2009}; ^{Montoya et al., 2005}): Laccaria trichodermophora and L. bicolor s.l. that are sympatric with P. montezumae, and L. laccata var. laccata and L. vinaceobrunnea that do not share the same habitat with this host.

Fruitbodies of L. trichodermophora and L. bicolor s.l. were collected from the Malinche National Park in the State of Tlaxcala. There, the average altitude is 3,200 m, climate is temperate sub humid with annual average temperature of 15.3 °C and an average rainfall range between 600 to 800 mm. The main vegetation are conifer forests dominated by P. montezumae, P. teocote, P. hartwegii, and Abies religiosa (^{Castillo-Guevara et al., 2012}; ^{Montoya et al., 2012}). Fruitbodies of L. laccata var. laccata and L. vinaceobrunnea, were obtained from Ixtlán de Juárez, at the Sierra Norte in the State of Oaxaca. In this area, the average elevation is 2,470 m, the predominant climate is temperate humid with annual average temperature of 15 °C and average rainfall ranging between 1,000 and 1,300 mm. The main vegetation is constituted by mixed temperate Pinus-Quercus forests dominated by P. patula, P. oaxacana, and P. douglasiana (^{UNFOSTI, 2012}, ^{Valdés et al., 2006}).

Pileus from fruitbodies were dried at 35 °C and manually grinded to obtain the inoculum. To know the concentration of spores in the inoculum of each species, we performed triplicate counts in a Neubauer chamber. We also conducted spore viability tests following the protocol of ^{Moreno-Martínez (1984}) and ^{Santiago-Martínez et al. (2003}) using 1.0% of tetrazolium buffer. We prepared 1 L of 1.0% 2, 3, 5 trifeniltetrazolium chloride in a buffer solution. We mixed 400 mL of KH₂PO₄ and 600 mL of NaH₂PO₄-H₂O solution; we added 10 g of tetrazolium salt, adjusted to pH 6 with KOH. In 1.5 mL Eppendorf tubes we placed a sample of each inoculum, we re-suspended them for 1 min with a vortex and incubated them for 30 min at room temperature. We then counted the total number of metabolically active spores in a Neubauer Chamber.

We acquired the MHB (strain P. fluorescens Pf_ Ag001) from BIOqualitum that sells it under the tradename BactoCROP guaranteeing minimum concentration as 100 millions of bacteria per gram.

Seeds of P. montezumae were collected from the surroundings of the Iztaccíhuatl Volcano in the State of Mexico located in the TMVB. They were surface-sterilized with hydrogen peroxide (H₂O₂) 30% and 20 mL of Tween-20 in 500 mL distiled water, and subsequently washed with running water and placed 24 h in water for pre-germination. We used a substrate composed of a 1:1 mixture of peat and agrolite and 134 mL containers. Peat was sterilized with 50 kiloGrays of Gamma radiation at the Institute of Nuclear Sciences, UNAM, as it has been shown it contains ectomycorrhizal fungi spores resistant to pasteurization (^{Ángeles-Argáiz et al., 2016}). At the beginning of the experiment, each plant was inoculated with 10⁷ spores placed in water solution added to the substrate. Bacteria treatments included 0.1 g of BactoCROP per container, diluted with the fungal inoculum. All plants remained 365 days in the greenhouse without any fertilization and watered with tap water every third day up to the saturation point; all treatments were randomly rotated every week.

The experimental design included 2 factors: (1) the ectomycorrhizal fungal species, including 4 levels (L. trichodermophora, L. bicolor s.l., L. laccata var. laccata and L. vinaceobrunnea); (2) the bacterial inoculum, including 2 levels (presence or absence of P. fluorescens). We also included a treatment only with bacterial inoculum and a negative control without inoculum. In total, the experiment had 10 treatments, with 13 seedlings each, comprising a total of 130 experimental units, each one constituted by one plant.

After a year of growth, we measured each plant height from the root collar and the root collar diameter (RCD); subsequently plants were dehydrated at 80 °C in an oven during 48 h to evaluate the dried shoots and roots weight. We determined total P, N and K content of the aerial and root parts for 5 randomly selected pines by treatment. Phosphorus was determined by colorimetry, N by wet digestion (^{Bremner, 1975}), and K by flame photometry ammonium acetate extraction (^{Chapman & Parker, 1986}).

The mycorrhization percentage in the root system for each seedling was randomly calculated. We divided the root system in 3 equal fractions (upper, middle and lower) and randomly selected 4 secondary roots per fraction, thus twelve secondary roots per plant were analyzed (^{Carrasco-Hernández et al., 2011}), counting the number mycorrhizal and non-mycorrhizal root tips in each under a stereoscopic microscope.

Evaluation of the mycorrhization effect and promotion by MHB on each response variable was conducted using two-factor variance analysis. When significant differences were obtained, we looked for the homogeneous groups by Tukey tests with the software Statistica 8 (StatSoft ver.2008).

To describe the ectomycorrhizae morphology, we conducted the characterization of structures as proposed by ^{Agerer (1987-2002}). Colors were recorded according to ^{Kornerup and Wanscher (1978}). Photographs were taken with the aid of a multifocal automatic microscope (Leica Z16 APOA) with an 8 mega-pixel camera (Leica DFC490); 3D images were assembled in Leica application systems V4.3.0. Scanning electron photographs were taken with a scanning electron microscope (JEOL JSM-5310LV); anatomic characteristics were photographed with an Olympus BX51 microscope.

Results

The percentage of mycorrhization varied from 93.5% to 98.5%, we did not find significant differences between sympatric and allopatric species. However, we found significant differences (F = 55.95, p < 0.0001) between all the mycorrhizal treatments and the 2 control treatments (C and C/PF), which did not develop any mycorrhizae (Table 1).

The height of plants was similar among treatments. There were significant differences (F = 3.17, p < 0.002) only between the treatments of Laccaria laccata var. laccata with P. fluorescens (x- = 17.9 cm) compared to those of L. trichodermophora and L. bicolor s.l. with P. fluorescens (x- = 15.5 and 16.3, respectively). The remaining treatments produced heights ranging from 16.3 to 16.8 cm. The root collar diameter (RCD) showed significant differences (F = 2.49, p < 0.012) between L. bicolor s.l. (x- = 2.9 cm), and L. bicolor s.l. with P. fluorescens (x- = 2.8 cm) treatments compared to the control with only P. fluorescens (x- = 1.7 cm). Although the negative control plants were thinner (x- = 2.0 cm) than treatments inoculated with fungi, differences were not significant (Table 1).

The 3 variables used to evaluate biomass (i.e., root dry weight, shoot dry weight and total dry weight) showed the same trend (Table 1). The best treatments were those inoculated with L. bicolor with or without P. fluorescens (28.7 and 28.0 mg, respectively), having a significantly greater biomass (F = 127.33, p = 0.0001) than the rest of the treatments. Laccaria trichodermophora treatments (with or without bacteria) had the second best total dry weight (19.3, 18.2 mg respectively) being significantly higher than the allopatric species that did not presented significant differences than the no-inoculated control. The control inoculated only with P. fluorescens showed the lowest total dry biomass ( x- = 6.5 mg) (Table 1).

The P roots contents did not show significant differences among the treatments (F = 1.27, p > 0.284). However, both the P content in the shoots (F = 1.99, p < 0.067) and in the whole plant (F = 2.18, p < 0.044) showed significant differences and followed a similar trend, with L. trichodermophora with P. fluorescens always with higher values. Significant differences were observed in the total P content between plants mycorrhized with L. trichodermophora with P. fluorescens ( x- = 180.3 mg) compared to both P. fluorescens control ( x- = 108.4 mg), and the no-inoculated control ( x- = 106.5 mg) treatments. The N content in shoots (F = 2.48, p > 0.024), roots (F = 0.67, p > 0.728) and total (F = 0.93, p > 0.513) parts of the plant did not show any significant differences between treatments and compared to the negative control. This was also true for the K content in the roots (F = 0.93, p > 0.509). Mycorrhizal plants inoculated with L. bicolor showed the highest concentration of K in the shoots (x- = 34.0 mg), followed by L. bicolor with P. fluorescens (x- = 26.6 mg) and L. trichodermophora (x- = 26.1 mg) treatments, showing significant differences (F = 7.00, p = 0.0001) relative to no-inoculated control (x = 14.4 mg). Total K of mycorrhizal plants with L. bicolor (x- = 60.7 mg), L. bicolor and P. fluorescens (x- = 51.4 mg), L. trichodermophora (x- = 50.4 mg), L. trichodermophora with P. fluorescens (x- = 44.7 mg), and L. laccata with P. fluorescens (x- = 43.0 mg) showed higher significant concentrations (F = 4.76, p = 0.0001) than the negative control (x- = 32.1 mg) (Table 2).

Mycorrhizae morphological description. L. trichodermophora + P. fluorescens + P. montezumae (Fig. 1A): dichotomous mycorrhizae with lateral branches of the same length, with straight edges and branches. The mantle presented reflective white patches over an orange base, it also showed emerging hyphae varying in quantity at the base and the apex. The base was yellowish brown (5D8 (^{Kornerup & Wanscher (1978})), the tips and apices were bright orange (5A6). Mantle plectenchymatous with palmate Hartig net widely distributed and with individual hyphae. L. trichodermophora + P. montezumae (Fig. 1B, C): same as before with 2 main differences: the superficial mantle showed a cottony texture and the mycorrhiza showed a strong orange color (5A8).

Figure 1 Morphology of mycorrhizae formed between: Pinus montezumae, Laccaria spp. and Pseudomonas fluorescens. A: L. trichodermophora + P. fluorescens + P. montezumae; B-C: L. trichodermophora + P. montezumae; D: L. laccata var. laccata + P. fluorescens + P. montezumae; E-F: L. laccata var. laccata + P. montezumae; G: L. vinaceobrunnea + P. fluorescens + P. montezumae; H-I: L. vinaceobrunnea + P. montezumae; J: L. bicolor s.l. + P. fluorescens + P. montezumae; K-L: L. bicolor s.l + P. montezumae; M: P. montezumae + P. fluorescens; N-O: P. montezumae non mycorrhizal roots. 

L. laccata var. laccata + P. fluorescens + P. montezumae (Fig. 1D): dichotomous mycorrhizae with lateral branches of the same length golden yellow (5B7), with straight edges and branches. Cotton-like superficial mantle, with emerging hyphae in some parts, surrounding the apices. Mantle plectenchymatous with anastomosed hyphae in the middle parts. L. laccata var. laccata + P. montezumae (Fig. 1E, F): same as before with 2 main differences: it showed abundant emerging hyphae from all the mycorrhiza, with a fan-like shape and orange (5B8) mantle.

L. vinaceobrunnea + P. fluorescens + P. montezumae (Fig. 1G): dichotomous mycorrhizae with lateral branches of the same length, with straight edges and branches. Apex orange (5A6), the rest of the mycorrhiza was brownish yellow (5E8). Cottony superficial mantle with emerging hyphae in certain parts; apex is mantle-free. Mantle pseudoparenchymatous, with palmate Hartig net widely distributed and showing individual hyphae. L. vinaceobrunnea + P. montezumae (Fig. 1H, I): same as before with 2 main differences: it presented constrictions at the base of the branch, in the middle and before reaching the apices and the whole mycorrhiza was orange brown (6C8).

L. bicolor s.l. + P. fluorescens + P. montezumae (Fig. 1J): dichotomous mycorrhizae with side branches of the same length; straight edges and branches, reddish brown (7E8). Cotton-like superficial mantle with rarely emanating hyphae. Mantle pseudoparenchymatous with palmate Hartig net widely distributed. L. bicolor s.l + P. montezumae (Fig. 1K-L): same as before with one main difference: the mycorrhiza was orange (5B8).

Roots of P. montezumae + P. fluorescens (Fig. 1M) and roots of P. montezumae (Fig. 1N-O): roots lack mantle and showed root hairs without superficial or intraradical hyphae.

Discussion

All the inoculated plants developed mycorrhizas and mycorrhization percentages in all treatments were high, greater than 93.5%. This is explained by the fact that the 4 Laccaria species used are ectomycorrhizal pine symbionts and all are native from Mexican forests as also is P. montezumae. As we will discuss later, the main differences found between sympatric and allopatric species are not evident in their ability to colonize the roots, but in their effect to improve the symbiosis efficiency.

The mycorrhiza helper bacteria P. fluorescens did not improve the mycorrhization percentage of any of the Laccaria species. This contrasts with previous reported positive effects of P. fluorescens in mycorrhization percentage (^{Frey-Klett et al., 1999}) and increase in root biomass of L. laccata (^{Duponnois & Garbaye, 1991}).

Pseudomonas fluorescens comprises a complex of genetic species with around 50% of genomic divergence between strains. In consequence, it might be expected that different strains exhibit a diverse spectrum of genetic traits involved in multi-trophic interactions with plants and other microbes (^{Loper et al., 2012}). As is has been recently shown by ^{Barragán-Soriano et al. (2018}), MHB do increase the growth and physiological quality of P. montezumae, so further research is needed to find compatible strains of MHB-sympatric Laccaria- and P. montezumae.

On the other hand, we demonstrate the efficiency of peat sterilization with Gamma rays, since both the negative control and the treatment with only P. fluorescens showed no mycorrhizae. The former means that we managed to eliminate the viability of resistant ectomycorrhizal fungi spores present in the peat (^{Ángeles-Argáiz et al., 2016}).

Overall, we did not find significant differences in growth parameters, although pines mycorrhized with allopatric species were little higher and smaller root collar diameter than those plants mycorrhized with sympatric species. The most important differences occurred in total biomass accumulation, as both sympatric species (L. bicolor followed by L. trichodermophora) promoted the enhancement of plant biomass (Table 1). Also, the increase in biomass accumulation in these treatments was independent of the presence of P. fluorescens. By comparison, allopatric species did not show any increase in biomass accumulation compared to the negative control.

Regarding nutrient contents, we did not find a significant relationship between the plants N content, in any part of the plant, and the mycorrhization treatments. We also did not find differences in P and K content in the roots between treatments. However, plants mycorrhized with the sympatric species L. trichodermophora with P. fluorescens were the unique treatment with significant higher P concentration in the total plant than the no-inoculated controls. Similarly, treatments with sympatric species, especially L. bicolor, showed a higher K concentration of shoot and total plant.

Our experimental data confirm the hypothesis that the sympatric mycorrhizal species are more efficient to accumulate biomass and nutrients (K) in the host plant. This was shown in an artificial substrate were nutrients came form organic matter (peat). However, an enhanced effect of the ability of sympatric fungi should be expected if this symbiosis was grown in the natural soils where it develops. The capacity of mycorrhizas to exploit and transfer soil nutrients to plants is related with ecological adaptations as particular soil ecotypes (^{Hoeksema et al., 2010}; ^{Klironomos, 2003}; ^{Rúa et al., 2016}).

The mycorrhizae morphology in the presence of P. fluorescens showed differences, the mantle shape and general coloring, in contrast with the mycorrhizae synthetized without bacteria. In the case of L. trichodermophora, the mantle shape was cottony with strong orange color; whereas, in the presence of P. fluorescens, the mycorrhiza had few hyphae with pale orange color. L. vinaceobrunnea presented a reduced mantle of yellow-orange coloring; whereas in the presence of bacteria the mycorrhiza had a cottony mantle pale orange in color at the base. While branching type of this symbiotic partners is similar to previous descriptions of L. bicolor s.l. with P. pseudostrobus in the absence of P. fluorescens; they are differences in the color from the brown color previously reported (^{Carrasco-Hernández et al., 2010}; ^{Santiago-Martínez et al., 2003}). The mycorrhiza of L. trichodermophora matches the type of branching but not in the coloring (strong orange), which is different from the pale yellow mycorrhizas reported previously (^{Galindo-Flores et al., 2015}). Consistency in the branching pattern is a main feature that characterizes the Pinus-Laccaria association (^{Agerer, 1987, 2002}), while differences in color may be due to particular metabolic pathways related with the specific species association and also the maturing of the symbiosis.

The widespread use of Laccaria species in mycorrhization is explained because they are pioneers with the ability to colonize a variety of important forest trees (^{Bois & Coughlan, 2009}; ^{Fortin & Lamhamedi, 2009}; ^{Parent & Moutoglis, 2009}; ^{Quoreshi et al., 2009}; ^{Sudhakara & Natarajan, 1997}). However, in this study, we have shown that the plant compatibility with its ectomycorrhizal fungus is differential, even though with phylogenetically related (within the same genus) fungal species. The fungus ability to colonize and remain in the host roots is evidenced through physiological and morphological responses of the plant (^{Karst et al., 2014}; ^{Onwuchekwa et al., 2014}; ^{Perrin et al., 1997}; ^{Quoreshi et al., 2008}). However, the ectomycorrhizal efficiency depends on the origin of the participants in the symbiosis. The local adaptation potential of sympatric species plays a fundamental role in the successful development in the field (^{Rúa et al., 2016}). Therefore, when selecting ectomycorrhizal fungi inoculum to grow forest plants, we should prioritize sympatric species to increase survival and growth success of plants and to ensure minimal disruption and disturbance of the natural communities.