Collection and preparation of the fungal material

The Russula delica sporomes were obtained in a pine-oak forest in the municipality of Pueblo Nuevo, Durango, at the coordinates 23°44.89' N and 105°32.27' W and at an altitude of 2 836 m. The material was transported in ice boxes to the forest nursery of the Valle del Guadiana Experimental Station of INIFAP in Durango, Dgo. (23°44.39' N - 104°37.42' W, 1 879 m). The material was cleaned, dissected in pieces of homogenous size, and placed in a solar dehydrator at a temperature of 35 °C until it reached a crisp consistency. Susbsequently, it was ground in a Thomas Model 4 Wiley^® Forage Mill and passed through a 1 mm sieve in order to homogenize the grain size.

The presence of spores in a suspension prepared with 0.1 g of ground sporomes in Polysorbate-80 at 0.1 % was verified; this suspension was stirred with a Bortex mixer for one minute and observed through an American Optical^® Microstar One-Ten microscope (40x). The spores were counted using a Neubauer chamber. The average number was 162 x 10⁶ spores per gram.

Plant production

The plants were grown in the forest nursery of the Valle del Guadiana Experimental Station (INIFAP). The substrate mixture utilized consisted of 50 % peat moss, 40 % composted pine bark, and 10 % expanded perlite; controled-release fertilizer was added (8 month release, 18-6-12 N-P-K + microelements), in a proportion of 4 kg m^-3 of substrate, a dose generally used for growing Pinus engelmannii in the forest nurseries of the state of Durango. The containers were polysterene trays with 77 cavities (162 mL per cavity), disinfected with a 10 % chlorine solution and impregnated with copper sulfate (4 kg in 100 L water).

The seeds, previously soaked in water for 24 hours and disinfected with a 10 % chlorine solution during 15 minutes, were planted in October 2013. Once the germination was established, the seedlings were watered every other day. Furthermore, hydrosoluble fertilizers were applied twice a week in a proportion of 1 g L^-1 water, according to the conventional practices for pine plant production. During the first three months, the NPK 7-40-19 fertilizer was administered; the NPK 2010-20 formula was added in the course of the subsequent months, and the NPK 4-25-35 formula, during the last three months.

Treatments and experimental design

Two application methods (in substrate and in irrigation water) and four doses of inoculum (0, 0.75, 1.50 and 2.25 g per tray) were evaluated (Table 1). The low dose was calculated based on the inoculation of 100 000 plants with 1 kg inoculum; medium and high doses were determined by duplicating and triplicating the low dose, respectively. The experimental design was random, with a 2 x 4 factorial treatment arrangement (two application methods and four doses). For each treatment four repetitions were carried out, each in a tray with 77 plants.

Table 1 Induced inoculation treatments with Russula delica Fr. evaluated in Pinus engelmannii Carr. plants. 

*Application during the preparation of the substrate. **Divided into three applications (42, 135 and 198 days after planting).

Response variables and statistical analysis

Two months (358 days) after planting, destructive sampling was carried out, with the extraction of four plants selected at random in each repetition, in order to evaluate the morphological variables: root neck diameter (mm), dry root biomass (g), and Dickson quality index (QI) (^{Dickson et al., 1960}). In addition, the root mycorrhiza percent infection was estimated using Grand and Harvey method (^{Carrera and López, 2004}), which consists in determining the ratio of the number of roots with mychorrhiza infection to the total number of roots multiplied by 100.

The data were subjected to a variance analysis, previously verifying the normalcy assumption and variance homogeneity. The following statistical model was utilized:

Where:

In the case of the variables that had significant differences with a 95 % probability, a multiple mean comparison was carried out with Tukey's test. The statistical analysis was performed using the GLM procedure of the SAS 9.3 software (2009).

The variance analysis evidenced significant differences (p<0.05) between application methods and between doses of inoculum for all the assessed morphological variables. The percentage of mycorrhization displayed highly significant differences (p<0.0001) between application methods, the doses and their interactions (Table 2).

Table 2 P values of the variance analysis for the effect of inoculation on the quality of the Pinus engelmannii Carr. plants grown in nursery. 

DRB = Dry root biomass; QI = Dickson's Quality Index; MP = Mycorrhiza percent infection.

The morphological variables of the plant quality, root neck diameter, dry root biomass and Dickson quality index (QI) registered higher averages with the application in irrigation water; however, their response differed according to the dose of the inoculum. The root neck diameter was larger in the control, with significant differences compared to the high dose. Conversely, for the anhydrous biomass of the root and QI; the treatment with the best results was the low dose, with significant differences in relation to the medium and high doses, as well as to the control (Table 3).

Table 3 P values of the variance analyses for the effect of inoculation with Russula delica Fr. on the quality variables of the Pinus engelmannii Carr. 

DRB = Dry root biomass; QI = Dickson's Quality Index. Different letters in the same column indicate significant differences between levels of each factor using Tukey's test (p<0.05).

In all the treatments, average diameters were estimated to be above 6.0 mm; these values are a good indicator of the quality of the plant, as, according to the recommendations of ^{Prieto et al. (2009)}, plants with a diameter larger than 5 mm are more resistant to bending and tolerate the damages due to pests and noxious fauna better. Furthermore, the diameter determines the robustness of the stem and is directly related to the vigor and survival rate of the plants in the field.

The highest mycorrhiza percent infection was observed after inoculation of the substrate, with highly significant differences with regard to application in irrigation water. This variable increased with the dose of inoculum, with high differences in relation to the control. Likewise, a significant effect of the interaction between the application method and the dose of inoculum was evident, an indication that the response induced by the dose of inoculum will depend on how this is administered. Figure 1 shows that the dose of inoculum does not show significant differences when applied in irrigation water, with mycorrhization values ranging from 6.1 to 7.9 %; however, when applied in the substrate, the mycorrhiza percent infections varied significantly (30.1 to 40.8 %) according to the dose utilized.

Different letters indicate significant differences between treatments, determined using Tukey's test (p<0.05).

Figure 1 Mycorrhiza percent infection in Pinus engelmannii Carr. plants in response to induced inoculation with Russula delica Fr. 

The results showed a tendency to reduce the plant quality - particularly the root neck diameter- as the doses of inoculum increased. Conversely, ^{Montes et al. (2001)} cite, in a similar study, larger diameters in P. engelmannii plants inoculated with Glomus intraradices N. C. Schenck & G. S. Sm., a fungus forming arbuscular mycorrhiza, compared to those that were not inoculated. However, according to ^{Chilvers et al. (1987)}, ectomycorrhizae have a better capacity than arbuscular mycorrhiza to effect secondary inoculations because of their propagation across the roots through hyphae.

On the other hand, the root biomass increased when the plant was inoculated with a low dose, compared to the control; this has been attributed to the fact that the mycorrhiza modifies the root and increases the dry weight and is therefore considered to be a good indicator, as plants with more biomass and plants with a larger root biomass have a higher survival rate in field (^{Davis and Jacobs, 2005}). Conversely, when medium and high doses were used, the survival rate was lower and even showed no significant differences in relation to the control. These results agree with the findings of ^{Chávez et al. (2009)}, who inoculated Pinus radiata D. Don with Rhizopogon luteolus Fr. & Nordholm, Suillus bellinii (Inzenga) Watling infection and Suillus luteus L. (Fries) Gray in doses of up to 1 x 10⁷ spores per plant, and registered insignificant differences for the root biomass. The same effects occurred for the QI, and a better plant quality was obtained with low doses of inoculums. This is related to the fact that the variables of dry matter are the most closely correlated with the QI (^{Bionotto et al., 2010}), which indicates that inoculation with high doses not always results in a good development of the plant and may limit its growth. In this respect, low values in mycorrhizal plants may be ascribed to the high demand of the fungi for carbohydrates (^{Dosskey et al., 1991}). The same behavior has been documented in various species of conifers (^{Rincón et al., 2005}; ^{Menkis et al., 2010}), in which a reduction of the growth rates has been observed in plants with abundant mycorrhizae. ^{García et al. (2010)} inoculated Eucalyptus urophylla S. T. Blake with Glomus intraradices y Pisolithus tinctorius (Pers.) Coker & Couch and found higher QIs in plants without mycorrhizae.

In this study, there is nevertheless a noteworthy difference between the use of inoculum and its omission. A low dose is proven to be advisable to stimulate the formation of mychorrizae and to improve the condition of the plant for its establishment in field. Various authors have shown that mychorrhizal plants have a higher probability in the plantation sites because mycorrhizae increase the availability of nutrients (Menkis et al., 201) and improve the hydric status of the plants (^{Ortega et al., 2004}); however, the efficiency of the ectomycorrhiza also depends on the physical and chemical characteristics of the soil (^{Teste et al., 2004}).

Finally, a large difference was observed in the formation of mychorrhizae between application methods; although inoculation in irrigation water generated higher plant quality indices, inoculation in the substrate is an appropriate option to render the inoculation more effective, even with lower doses, resulting in a reduced use of inoculum. Based on this, the new research must focus on determining the best cultural practices, such as the use of substrates, containers, fertilization routines and irrigation to propitiate effective colonization by ectomycorrhizal fungi, as well as on evaluating the performance of mycorrhizal plants in the plantation sites.

The application method and the dose of Russula delica inoculum were determined to have a significant effect on the quality of the plant in the nursery. The addition of a low dose (0.75 g per tray) in the irrigation water promotes a better morphological quality of the plant. The highest mycorrhiza percent infection was obtained with inoculation in the substrate; this suggests the possibility of trying lower doses to ensure colonization by ectomycorrhizal fungi.

Conflict of interest

The authors declare no conflict of interest.

Contributions by author

Laura Elena Martínez Nevárez: establishment and management of the assay in the nursery, data collection and analysis, and drafting of the manuscript; Homero Sarmiento López: definition of the experimental design and treatments; José Ángel Sigala Rodríguez: data analysis, development of charts and tables and bibliographical documentation; Sergio Rosales Mata: fungi recollection and utilization, and establishment of the assay in the nursery; José Bernardo Montoya Ayón: review of the manuscript.