Sympatric species develop more efficient ectomycorrhizae in the Pinus-Laccaria symbiosis
Ibeth Rodríguez-Gutiérrez a, Daniel Ramírez-Martínez a, Roberto Garibay-Orijel a, *, Virginia Jacob-Cervantes b, Jesús Pérez-Morenoc, María del Pilar Ortega-Larrocea d, Elsa Arellano-Torres e
a Instituto de Biología, Universidad Nacional Autónoma de México, Tercer Circuito s/n, Ciudad Universitaria, Coyoacán, 04510, Ciudad de México, Mexico.
b Centro Nacional de Investigación Disciplinaria en Conservación y Mejoramiento de Ecosistemas Forestales, Instituto Nacional de Investigación Forestal, Agrícolas y Pecuarias, Av. Progreso 5, Coyoacán, 04110 Ciudad de México, Mexico.
c Colegio de Posgraduados, Campus Montecillo, carretera México-Texcoco Km. 36.5, 56230 Montecillo, Texcoco, Estado de México, Mexico.
d Instituto de Geología, Universidad Nacional Autónoma de México, Ciudad Universitaria, Coyoacán, 04510 Ciudad de México, Mexico.
e Departamento de Ecología y Recursos Naturales, Facultad de Ciencias, Universidad Nacional Autónoma de México, Circuito Exterior Ciudad Universitaria, Coyoacán, 04510 Ciudad de México, Mexico.
*Corresponding author: rgaribay@ib.unam.mx (R. Garibay-Orijel)
Abstract
The mycorrhizal symbiosis is optimal when the plant and the fungi are sympatric. However, in forest plantations the inoculum typically belongs to exotic or allopatric fungi. In this paper, the efficiency of mycorrhization was determined by evaluating the effect of 2 sympatric fungi species (Laccaria trichodermophora and L. bicolor s.l.) and 2 allopatric (L. laccata var. laccata and L. vinaceobrunnea) on the growth and nutrient contents of Pinus montezumae. We also tested the effect of the mycorrhizal helper bacteria Pseudomonas fluorescens (Pf_Ag001). After 1 year of growth, we evaluated the mycorrhization percentage, plant height, diameter at root collar, dry weight and nutrient contents (N, P, K) of aerial part and roots. The mycorrhization percentage varied from 93.5% to 98.5%. The treatments that showed higher efficiency (biomass accumulation and K contents) were those inoculated with sympatric species. All Laccaria treatments, either in the presence or absence of the bacteria, showed a better response compared to not inoculated controls. This work demonstrates the significance of using inocula of sympatric species as these are genetically predisposed to associate with their hosts, naturally adapted to the local environmental and edaphic conditions compared with those of allopatric origin.
Keywords: Ectomycorrhizal inoculum; Exotic; Laccaria laccata; Laccaria trichodermophora; Laccaria vinaceobrunnea; Laccaria bicolor; Pseudomonas fluorescens; Pinus montezumae
© 2019 Universidad Nacional Autónoma de México, Instituto de Biología. Este es un artículo Open Access bajo la licencia CC BY-NC-ND
Las especies simpátricas desarrollan ectomicorrizas más eficientes en la simbiosis Pinus-Laccaria
Resumen
La simbiosis micorrízica es óptima cuando la planta y los hongos son simpátricos. Sin embargo, en las plantaciones forestales típicamente se usa inóculo de hongos exóticos. En este trabajo, la eficacia de la micorrización se determinó mediante la evaluación del efecto de 2 especies de hongos simpátricas (Laccaria trichodermophora y L. bicolor s.l.) y 2 alopátricas (L. laccata var. laccata y L. vinaceobrunnea) en el crecimiento y contenido de nutrientes de Pinus montezumae. También evaluamos el efecto de la bacteria ayudadora de micorrizas Pseudomonas fluorescens (Pf_Ag001). Después de 1 año de crecimiento, evaluamos el porcentaje de micorrización, la altura de la planta, el diámetro en el collar de la raíz, el peso seco y el contenido de nutrientes (N, P, K). El porcentaje de micorrización varió de 93.5% a 98.5%. Los tratamientos que mostraron una mayor eficiencia fueron los inoculados con especies simpátricas. Todos los tratamientos con Laccaria, en presencia o ausencia de bacterias, mostraron una mejor respuesta en comparación con los controles no inoculados. Este trabajo demuestra la importancia de usar inóculos de especies simpátricas ya que están genéticamente predispuestas a asociarse con sus hospedadores y están naturalmente adaptadas a las condiciones ambientales y edáficas locales.
Palabras clave: Inóculo ectomicorrízico; Exótico; Laccaria laccata; Laccaria trichodermophora; Laccaria vinaceobrunnea; Laccaria bicolor; Pseudomonas fluorescens; Pinus montezumae
© 2019 Universidad Nacional Autónoma de México, Instituto de Biología. This is an open access article under the CC BY-NC-ND license
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.
Table 1
Growth, biomass accumulation and mycorrhization of Pinus montezumae inoculated with different Laccaria species and P. fluorescens.
Origin |
Treatment |
Height (cm) |
Root collar diam (cm) |
Root dry weight (mg) |
Shoot dry weight (mg) |
Total dry weight (mg) |
M% |
Sympatric |
L.b |
16.5abc |
2.9a |
8.7a |
20.0a |
28.7a |
93.5a |
L.b/P.f |
16.3c |
2.8a |
8.0a |
20.0a |
28.0a |
98.4a |
|
L.t |
15.5c |
2.5ab |
5.8bc |
13.5b |
19.3b |
98.5a |
|
L.t/P.f |
16.7abc |
2.7ab |
6.2b |
12.0b |
18.2b |
98.5a |
|
Allopatric |
L.v/P.f |
16.8abc |
2.7ab |
4.0de |
8.0c |
12.0c |
94.5a |
L.l |
16.7abc |
2.5ab |
4.5cd |
7.3c |
11.8c |
97.6a |
|
L.v |
17.1ab |
2.4ab |
3.7de |
7.0c |
10.7c |
95.7a |
|
L.l/P.f |
17.9a |
2.2ab |
4.1de |
6.5c |
10.6c |
97.1a |
|
Controls |
C- |
16.4abc |
2.0ab |
4.2de |
7.3c |
11.5c |
0b |
P.f |
16.6abc |
1.7b |
2.6e |
3.9d |
6.5d |
0b |
L.t/P.f: Laccaria trichodermophora + Pseudomonas fluorescens, L.t: L. trichodermophora, L.v/P.f: L. vinaceobrunnea + P. fluorescens, L.v: L. vinaceobrunnea, L.l./P.f: L. laccata var. laccata + P. fluorescens, L.l: L. laccata var. laccata, L.b/P.f: L. bicolor + P. fluorescens, L.b: L. bicolor, C-: no-inoculated negative control, P.f: P. fluorescens, root collar diam: root collar diameter, M%: mycorrhization percentage. Different letters show statistically significant differences on post hoc Tukey test (p < 0.05). n = 13.
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).
Table 2
Nutrient contents (mg) in Pinus montezumae inoculated with different Laccaria species and P. fluorescens.
Treatment |
Ps |
Pr |
Pt |
Ns |
Nr |
Nt |
Ks |
Kr |
Kt |
L.t/P.f |
132.9a |
47.4a |
180.3a |
0.9a |
1.0a |
1.9a |
21.4bc |
23.3a |
44.7ab |
L.t |
80.7ab |
39.1a |
119.8ab |
1.2a |
0.8a |
2.0a |
26.1ab |
24.3a |
50.4ab |
L.v/P.f |
92.9ab |
49.8a |
142.7ab |
1.1a |
1.0a |
2.1a |
16.6bc |
22.0a |
38.6bc |
L.v |
95.5ab |
40.0a |
135.5ab |
1.2a |
0.9a |
2.1a |
18.0bc |
22.6a |
40.6bc |
L.b/P.f |
96.3ab |
35.4a |
131.7ab |
1.1a |
1.0a |
2.1a |
26.6ab |
24.8a |
51.4ab |
L.b |
95.4ab |
44.6a |
140.0ab |
1.1a |
0.8a |
1.9a |
34.0a |
26.7a |
60.7a |
L.l/P.f |
81.3ab |
37.3a |
118.6ab |
0.8a |
0.8a |
1.6a |
17.3bc |
25.7a |
43.0ab |
L.l |
106.7ab |
33.4a |
140.1ab |
1.1a |
0.8a |
1.9a |
19.2bc |
20.5a |
39.7bc |
P.f |
62.4b |
46.0a |
108.4b |
1.1a |
0.9a |
2.0a |
19.5bc |
24.3a |
43.8ab |
C- |
70.0ab |
36.5a |
106.5b |
0.9a |
0.7a |
1.6a |
14.4c |
17.7a |
32.1c |
L.t/P.f: Laccaria trichodermophora + Pseudomonas fluorescens, L.t: L. trichodermophora, L.v/P.f: L. vinaceobrunnea + P. fluorescens, L.v: L. vinaceobrunnea, L.l./P.f: L. laccata var. laccata + P. fluorescens, L.l: L. laccata var. laccata, L.b/P.f: L. bicolor + P. fluorescens, L.b: L. bicolor, C-: negative control, P.f: P. fluorescens, Ps: phosphorous in shoot, Pr: phosphorous in roots, Pt: total phosphorous, Ns: nitrogen in shoot, Nr: nitrogen in roots, Nt: total nitrogen, Ks: potassium in shoot, Kr: potassium in roots, Kt: total potassium. Different letters show statistically significant differences on post hoc Tukey test (p < 0.05). n = 5.
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).
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.
Acknowledgements
Authors appreciate the thoughtful comments and reviews by Joaquín Cifuentes-Blanco and Roberto Lindig-Cisneros. We thank the support provided by Iris Suárez-Quijada. We thank the Laboratory of Microscopía y Fotografía de la Biodiversidad, in particular to the areas of Electron Microscopy to Berenit Mendoza Garfias, and Multifocal Photography to Susana Guzman, both at the Instituto de Biología, UNAM. Finally, we thank Mephi Boseth Álvarez-Sánchez for graphic design and photographic assistance. IR-G thanks to the Posgrado en Ciencias Biológicas of the Universidad Nacional Autónoma de México (UNAM) for financial support during the development of this work, as well as the National Council of Science and Technology (Conacyt) for the scholarship granted during her doctoral studies. This project was partially funded by UNAM PAPIIT IN210217 to RGO.
References
Agerer, R. (Ur. 1987-2002). Color Atlas of Ectomycorrhiza. Germany Einhorn-Verlag, Schwäbisch Gmünd.
Ángeles-Argáiz, R. E., Flores-García, A., Ulloa, M., & Garibay-Orijel, R. (2016). Commercial Sphagnum peat moss is a vector for exotic ectomycorrhizal mushrooms. Biological Invasions, 18, 89–101. https://doi.org/10.1007/S10530-015-0992-2
Aspray, T. J., Jones, E. E., Davies, M. W., Shipman, M., & Bending, G. D. (2013). Increased hyphal branching and growth of ectomycorrhizal fungus Lactarius rufus bay the helper bacterium Paenibacillus sp. Mycorrhiza, 23, 403–410. https://doi.org/10.1007/s00572-013-0483-1
Barragán-Soriano, J. L., Pérez-Moreno, J., Almaraz-Suárez, J. J., Carcaño-Montiel, M. G., & Medrano-Ortiz, K. I. (2018). Inoculation with an edible ectomycorrhizal fungus and bacteria increases growth and improves the physiological quality of Pinus montezumae Lamb. Revista Chapingo Serie Ciencias Forestales y del Ambiente, 24, 3–16. https://doi.org/10.5154/r.rchscfa.2017.01.010
Barroetaveña, C., & Rajchenberg, M. (2003). Las micorrizas y la producción de plántulas de Pinus ponderosa Dougl. ex Laws. en la Patagonia, Argentina. Bosque (Valdivia), 24, 17–33. https://doi.org/10.4067/S0717-92002003000100002
Barroetaveña, C., Bassani, V. N., Monges, J. I., & Rajchenberg, M. (2016). Field performance of Pinus ponderosa seedlings inoculated with ectomycorrhizal fungi planted in steppe-grasslands of Andean Patagonia, Argentina. Bosque (Valdivia), 37, 307–316. https://doi.org/10.4067/S0717-92002016000200009
Bois, G., & Coughlan, A. P. (2009). Ectomycorrhizal inoculation for boreal forest ecosystem restoration following oil sand extraction: the need for an initial three-step screening process. In D. Khasa, Y. Piché, & A. P. Coughlan (Eds.), Advances in mycorrhizal science and technology (pp. 129–137). Ottawa: NRC Research Press.
Bonfante, P., & Genre, A. (2010). Mechanisms underlying beneficial plant-fungus interactions in mycorrhizal symbiosis. Nature Communications, 1, 1–11. https://doi.org/10.1038/ncomms1046
Bremner, J. M. (1975). Total nitrogen. In C. A. Black (Ed.), Methods of soil analysis (pp. 1149–1178). Madison: American Society of Agronomy Madison.
Carrasco-Hernández, V., Pérez-Moreno, J., Espinosa-Hernández, V., Almaraz-Suárez, J. J., Quintero-Lizoala, R., & Torres-Aquino, M. (2010). Caracterización de micorrizas establecidas entre dos hongos comestibles silvestres y pinos nativos de México. Revista Mexicana de Ciencias Agrícolas, 4, 657–577.
Carrasco-Hernández, V., Pérez-Moreno, J., Espinosa-Hernández, V., Almaraz-Suárez, J., Quintero-Lizaola, R., & Torres-Aquino, M. (2011). Contenido de nutrientes e inoculación con hongos ectomicorrízicos comestibles en dos pinos neotropicales. Revista Chilena de Historia Natural, 83, 83–96.
Carrera-Nieve, A., & López-Ríos, G. F. (2004). Manejo y evaluación de ectomicorrizas en especies forestales. Revista Chapingo Serie Ciencias Forestales y del Ambiente, 10, 93–98.
Castillo-Guevara, C., Lara, C., & Pérez, G. (2012). Mycophagy by rodents in a temperate forest of Central Mexico. Revista Mexicana de Biodiversidad, 83, 772–777. https://doi.org/rmb.27445
Chapela, I. H., Osher, L. J., Horton, T. R., & Henn, M. R. (2001). Ectomycorrhizal fungi introduced with exotic pine plantations induce soil carbon depletion. Soil Biology & Biochemistry, 33, 1733–1740. https://doi.org/10.1016/S0038-0717(01)00098-0
Chapman, H. D., & Parker, F. P. (1986). Métodos de análisis para suelos, plantas y agua. México D.F.: Trillas.
Cuevas-Rangel, R. A. (1979). Prueba de inoculación con el hongo micorrízico Pisolithus tinctorius (Pers.) Coker and Couch en plantas de Pinus montezumae Lamb. en suelos de vivero. Ciencia Forestal, 4, 46–62.
Deveau, A., Brulé, C., Palin, B., Champmartin, D., Rubini, O., Garbaye, J. et al. (2010). Role of fungal trehalose and bacterial thiamine in the improved survival and growth of the ectomycorrhizal fungus Laccaria bicolor S238N and the helper bacterium Pseudomonas fluorescens BBc6r8. Environmental Microbiology Reports, 2, 560–568. https://doi.org/10.1111/j.1758-2229.2010.00145.x
Deveau, A., Palin, B., Delaruelle, C., Peter, M., Kohler, A., Pierrat, J. C. et al. (2007). The mycorrhiza helper Pseudomonas fluorescens BBc6r8 has a specific priming effect on the growth, morphology and gene expression of the ectomycorrhizal fungus Laccaria bicolor S238N. New Phytologist, 175, 743–755. https://doi.org/10.1111/j.1469-8137.2007.02148.x
Dixon, R. K., & Johnson, P. S. (1992). Synthesis of ectomycorrhizae on Northern red oak seedlings in a Michigan nursery. Journal of Arboriculture, 18, 266–272.
Duponnois, R., & Garbaye, J. (1991). Effect of dual inoculation of Douglas fir with the ectomycorrhizal fungus Laccaria laccata and mycorrhization helper bacteria (MHB) in two bare-root forest nurseries. Plant and Soil, 138, 169–176.
Farjon, A. (1996). Biodiversity of Pinus (Pinaceae) in Mexico: speciation and paleoendemism. Botanical Journal of the Linnean Society, 121, 365–384.
Farjon, A., & Styles, B. T. (1997). Pinus (Pinaceae). Flora Neotropical Monograph V75. New York: The New York Botanical Garden.
Flores-Villela, O., & Canseco-Márquez, L. (2007). Riqueza de la herpetofauna. In I. Luna-Vega, J. J. Morrone, & D. Espinosa (Eds.), Biodiversidad de la Faja Volcánica Transmexicana (pp. 407–420). Ciudad de México: Comisión Nacional para el Conocimiento y Uso de la Biodiversidad/ Universidad Nacional Autónoma de México.
Fortin, J. A., & Lamhamedi, M. S. (2009). Ecophysiology of sporocarp development of ectomycorrhizal basidiomycetes associated with boreal forest gymnosperms. In D. Khasa, Y. Piché, & A. P. Coughlan (Eds.), Advances in mycorrhizal science and technology (pp. 161–173). Ottawa: NRC Research Press.
Frey-Klett, P., Churin, J. L., Pierrat, J. C., & Garbaye, J. (1999). Dose effect in the dual inoculation of an ectomycorrhizal fungus and a mycorrhiza helper bacterium in two forest nurseries. Soil Biology and Biochemistry, 31, 1555–1562. https://doi.org/10.1016/S0038-0717(99)00079-6
Galindo-Flores, G., Castillo-Guevara, C., Campos-López, A., & Lara, C. (2015). Caracterización de las ectomicorrizas formadas por Laccaria trichodermophora y Suillus tomentosus en Pinus montezumae. Botanical Sciences, 93, 1–9. https://doi.org/10.17129/botsci.200
Garbaye, J. (1994). Helper bacteria: a new dimension to the mycorrhizal symbiosis. New Phytologist, 128, 197–210. https://doi.org/10.1111/j.1469-8137.1994.tb04003.x
Garibay-Orijel, R., Martínez-Ramos, M., & Cifuentes, J. (2009). Disponibilidad de hongos comestibles en los bosques de pino-encino de Ixtlán de Juárez, Oaxaca. Revista Mexicana de Biodiversidad, 80, 521–534. http://dx.doi.org/10.22201/ib.20078706e.2009.002.615
Gibson, F., & Deacon, J. W. (1988). Experimental study of the establishment of ectomycorrhizas in different regions of birch root systems. Transactions of the British Mycological Society, 91, 239–251. https://doi.org/10.1016/S0007-1536(88)80211-0
Hoeksema, J. D., Chaudhary, V. B., Gehrin, C. A., Johnson, N. C., Kars, J., Koide, R. T. et al. (2010). A meta-analysis of context-dependency in plant response to inoculation with mycorrhizal fungi. Ecology Letters, 13, 394–407. https://doi.org/10.1111/j.1461-0248.2009.01430.x
Hynson, N. A., Merckx, V. S. F. T., Perry, B. A., & Treseder, K. K. (2013). Identities and distributions of the co-invading ectomycorrhizal fungal symbionts of exotic pines in the Hawaiian Islands. Biological Invasions, 15, 2373–2385. https://doi.org/10.1007/s10530-013-0458-3
Karst, J., Randall, M. J., & Gehring, C. A. (2014). Consequences for ectomycorrhizal fungi of the selective loss or gain of pine across landscapes. Botany, 92, 855–865. https://doi.org/10.1139/cjb-2014-0063
Khasa, D., Piché, Y., & Coughlan, A. P. (2009). Advances in mycorrhizal science and technology. Ottawa: NRC-Research Press.
Klironomos, J. N. (2003). Variation in plant response to native and exotic arbuscular mycorrhizal fungi. Ecology, 84, 2292–2301. https://doi.org/10.1890/02-0413
Kornerup, A., & Wanscher, J. H. (1978). Methuen handbook of colour. London: E. Methuen.
Kumari, D., Sudhakara, R., & Ramseh, C. U. (2013). Diversity of cultivable bacteria associated with fruiting bodies of wild Himalayan Cantharellus spp. Annals of Mirobiology, 63, 845–853. https://doi.org/10.1007/s13213-012-0535-3
Kropp, B. R., & Mueller, G. M. (1999). Laccaria. In J. W. G. Cairney , S. M. Chambers (Eds.), Ectomycorrhizal Fungi: key genera in profile (pp. 65–88). Berlin: Springer-Verlag.
Larsen, P. E., Sreedasyam, A., Trivedi, G., Podila, G. K., Cseke, L. J., & Collart, F. R. (2011). Using next generation transcriptome sequencing to predict an ectomycorrhizal metabolome. BMC Systems Biology, 5, 70. https://doi.org/10.1186/1752-0509-5-70
Loper, J. E., Hassan, K. A., Mavrodi, D. V., Davis, I. I. E. W., Lim, C. K., Shaffer, B. T., et al. (2012). Comparative genomics of plant-associated Pseudomonas spp.: insights into diversity and inheritance of traits involved in multitrophic interactions. Plos Genetics, 8, e1002784. https://doi.org/10.1371/journal.pgen.1002784
Martin, F., & Selosse, M. A. (2008). The Laccaria genome: a symbiont blueprint decoded. New Phytologist, 180, 296–310. https://doi.org/10.1111/j.1469-8137.2008.02613.x
Martínez-Reyes, M., Pérez-Moreno, J., Villarreal-Ruiz, L., Ferrera-Cerrato, R., Xoconostle-Cázarez, B., Vargas-Hernández, J. J. et al. (2012). Crecimiento y contenido nutrimental de Pinus greggii Engelm. inoculado con el hongo comestible ectomicorrízico Hebeloma mesophaeum (Pers.) Quél. Revista Chapingo Serie Forestales y del Ambiente, 18, 183–192. https://doi.org/10.5154/r.rchscfa.2010.11.112
Méndez-Neri, M., Pérez-Moreno, J., Quintero-Lizaola, R., Hernández-Acosta, E., & Lara-Herrera, A. (2011). Crecimiento y contenido nutrimental de Pinus greggii inoculado con tres hongos comestibles ectomicorrízicos. Terra Latinoamericana, 29, 73–81.
Molina, R., Massicotte, H., & Trappe, J. M. (1992). Specificity phenomena in mycorrhizal symbiosis: community-ecological consequences and practical implications. In M. R. Allen (Ed.), Mycorrhizal functioning: an integrative plant-fungal process (pp. 357–423). New York: Chapman & Hall.
Montoya, A., Kong, A., Estrada-Torres, A., Cifuentes, J., & Caballero, J. (2005). Useful wild fungi of La Malinche National Park, Mexico. Fungal Diversity, 17, 115–14.
Montoya, A., Torres-García, E., Kong, A., Estrada-Torres, A., & Caballero, J. (2012). Gender differences and regionalization of the cultural significance of wild mushrooms around La Malinche volcano, Tlaxcala, Mexico. Mycologia, 104, 826–834. https://doi.org/10.3852/11-347
Moreno-Martínez, E. (1984). Análisis físico y biológico de semillas agrícolas. Mexico D.F.: Universidad Nacional Autónoma de México.
Morrone, J. J. (2010). Fundamental biogeographic patterns across the Mexican Transition Zone: an evolutionary approach. Ecography, 33, 355–361. https://doi.org/10.1111/j.1600-0587.2010.06266.x
Morrone, J. J., & Escalante, T. (2002). Parsimony Analysis of Endemicity (PAE) of Mexican terrestrial mammals at different area units: when size matters. Journal of Biogeography, 29, 1095–1104. https://doi.org/10.1046/j365-2699.2002.00753.x
Mortier, F., Le Tacon, F., & Garbaye, J. (1988). Effect of inoculum type and inoculation dose on ectomycorrhizal development, root necrosis, and growth of Douglas fir seedlings inoculated with Laccaria laccata in a nursery. Annals of Forest Science, 45, 301–310. https://doi.org/10.1051/forest:19880401
Onwuchekwa, N. E., Zwuiazek, J. J., Quoreshi, A., & Khasa, D. P. (2014). Growth of mycorrhizal Jack pine (Pinus banksiana) and White spruce (Picea glauca) seedling planted in oil sands. Mycorrhiza, 24, 431–441. https://doi.org/10.1007/s00572
Parent, S., & Moutoglis, P. (2009). Industrial perspective of applied mycorrhizal research in Canada. In D. Khasa, Y. Piché & A. P. Coughlan (Eds.), Advances in mycorrhizal science and technology (pp. 105–113). Ottawa: NRC Research Press.
Parladé, J., & Álvarez, I. F. (1993). Coinoculation of aseptically grown Douglas fir with pairs of ectomycorrhizal fungi. Mycorrhiza, 3, 93–96.
Pera, J., & Parladé, J. (2005). Inoculación controlada con hongos ectomicorrízicos en la producción de plantas destinadas a repoblaciones forestales: estado actual en España. Investigación Agraria, Sistemas y Recursos Forestales, 14, 419–433.
Perea-Estrada, V. M., Pérez-Moreno, J., Villarreal-Ruíz, L., Trinidad-Santos, A., De la I de Bauer, M. L., Cetina-Alcalá, V. M. et al. (2009). Humedad edáfica, nitrógeno y hongos ectomicorrízicos comestibles en el crecimiento de pino. Revista Fitotecnia Mexicana, 32, 93–102.
Pérez-Moreno, J., & Read, D. J. (2000). Mobilization and transfer of nutrients from litter to tree seedlings via the vegetative mycelium of ectomycorrhizal plants. New Phytologist, 145, 301–309. https://doi.org/10.1046/j.1469-8137.2000.00569.x
Pérez-Moreno, J., & Read, D. J. (2004). Los hongos ectomicorrízicos, lazos vivientes que conectan y nutren a los árboles en la naturaleza. Interciencia, 29, 239–247.
Perrin, E., Parladé, X., & Pera, J. (1997). Receptiveness of forest soils to ectomycorrhizal association: concept and method as applied to the symbiosis between Laccaria bicolor (Maire) Orton and Pinus pinaster Art or Pseudotsuga menziesii (Mirb.) Franco. Mycorrhiza, 6, 469–476.
Quoreshi, A. M., Kernaghan, G., & Hynt, G. A. (2009). Mycorrhizal fungi in Canadian forest nurseries and field performance of inoculated seedlings. In D. Khasa, Y. Piché & A. P. Coughlan (Eds.), Advances in mycorrhizal science and technology (pp. 115–127). Ottawa: NRC Research Press.
Quoreshi, A. M., Piché, Y., & Khasa, D. (2008). Field performance of conifer and hardwood species 5 years after nursery inoculation in the Canadian Prairie provinces. New Forests, 35, 235–253. https://doi.org/10.1007/s11056-007-9074-3
Rúa, M. G., Antoninka, A., Antunes, P. M., Chaudhary, V. B., Gehring, C., Lamit, L. J. et al. (2016). Home-field advantage? Evidence of local adaptation among plants, soil, and arbuscular mycorrhizal fungi through meta-analysis. BMC Evolutionary Biology, 16, 122. https://doi.org/10.1186/s12862-016-0698-9
Santiago-Martínez, G., Estrada-Torres, A., Varela, L., & Herrera, T. (2003). Crecimiento de siete medios nutritivos y síntesis in vitro de una cepa de Laccaria bicolor. Agrociencias, 37, 575–584.
Sudhakara, R. M., & Natarajan, K. (1997). Coinoculation efficacy of ectomycorrhizal fungi on Pinus patula seedlings in a nursery. Mycorrhiza, 7, 133–138.
Smith, S. E., & Read, D. J. (2008). Mycorrhizal symbiosis. New York: Academic Press
Teramoto, M., Wu, B., & Hogetsu, T. (2012). Transfer of 14C-photosynthate to the sporocarp of an ectomycorrhizal fungus Laccaria amethystina. Mycorrhiza, 22, 219–225. https://doi.org/10.1007/s00572-011-0395-x
Trappe, J. M. (1977). Selection of fungi for ectomycorrhizal inoculation in nurseries. Annual Review of Phytopathology, 15, 203–222. https://doi.org/10.1146/annurev.py.15.090177.001223
Umbanhowar, J., & McCann, K. (2005). Simple rules for the coexistence and competitive dominance of plants mediated by mycorrhizal fungi. Ecology Letters, 8, 247–252. https://doi.org/10.1111/j.1461-0248.2004.00714.x
UNFOSTI (2012). Programa de manejo forestal para el aprovechamiento persistente de los recursos forestales maderables. Comunidad de Ixtlán de Juárez, Oaxaca. Oaxaca: México.
Valdés, M., Asbjornsen, H., Gómez-Cárdenas, M., Juárez, M., & Vogt, K. A. (2006). Drought effects on fine-root and ectomycorrhizal-root biomass in managed Pinus oaxacana Mirov stand in Oaxaca, Mexico. Mycorrhiza, 2, 117–124. https://doi.org/10.1007/s00572-005-0022-9
Valdés, M., Pereda, V., Ramírez, P., Valenzuela, R., & Pineda, R. M. (2009). The ectomycorrhizal community in a Pinus oaxacana forest under different silvicultural treatments. Journal of Tropical Forest Science, 21, 88–97.
Valdés, M., Piña, F., & Grada, R. (1983). Inoculación micorrízica y crecimiento de plántulas de pino en suelo erosionado. Boletín de la Sociedad Mexicana de Micología, 18, 56–70.
Vik, U., Logares, R., Blaalid, R., Halvorsen, R., Carlsen, T., Bekke, I. et al. (2013). Different bacterial communities in ectomycorrhizal and surrounding soil. Scientific Reports, 3, 1–8. https://doi.org/10.1038/srep03471
Wadud, M. A., Lian, C. L., Nara, K., Reza, M. S., & Hogetsu, T. (2008). Below ground genet differences of an ectomycorrhizal fungus Laccaria laccata infecting Salix stands in primary successional stage. Journal of Agroforestry and Environment, 2, 1–6.
Wadud, M. A., Nara, K., Lian, C. L., Ishida, A. T., & Hogetsu, T. (2014). Genet dynamics and ecological functions of the pioneer ectomycorrhizal fungi Laccaria amethystina and Laccaria laccata in volcanic desert on Mount Fuji. Mycorrhiza, 24, 551–563. https://doi.org/10.1007/s00572-014-0571.x
Wilson, A. W., Hosaka, K., & Mueller, G. M. (2017). Evolution of ectomycorrhizas as a driver of diversification and biogeographic patterns in the model mycorrhizal mushroom genus Laccaria. New Phytologist, 213, 1862–1873. https://doi.org/10.1111/nph.14270
Zadworny, M., Werner, A., & Idzikowska, K. (2004). Behavior of the hyphae of Laccaria laccata in the presence of Trichoderma harzianum in vitro. Mycorrhiza, 14, 401–409. https://doi.org/10.1007/s00572-004-0323-4