Introduction

In a forest, most plants naturally mycorrhize, since they find propagules in the soil for their mycorrhization, such as spores, sclerotia, mycelium, mycorrhizal roots and rhizomorphs. One of the most studied propagules has been the spores, which have a considerable longevity since they can remain in the soil for several years and even decades before germinating (^{Bruns et al., 2009}; ^{Nguyen et al., 2012}). In the case of plant production in nursery, the plant is outside its natural habitat and is unable to access enough fungal propagules for its mycorrhization, so it is necessary to carry out the inoculation in the nursery.

Therefore, it is essential to know the techniques of production of inocula with ectomycorrhizal fungi and the costs involved. There are three inoculation techniques with ectomycorrhizal fungi in the nursery that are: inoculation with monte soil, inoculation based on spores and inoculation with vegetative mycelium (^{Landis et al., 1989}; ^{Rossi et al., 2007}; ^{Duponnois et al., 2011}). In Mexico, most traditional nurseries use the forest floor as a substrate for plant production and take advantage of it as the sole source of ectomycorrhizal fungal propagules. However, this method has a set of deficiencies, which include: i) the possibility of introducing pathogens; ii) the lack of a mycorrhization controlled by the heterogeneity of distribution of the ectomycorrhizal propagules; and iii) the erosion and destruction of the forest areas from which the soil is extracted.

The second method is the inoculation based on spores that consists in the application of the ectomycorrhizal inoculant in the irrigation water or it can also be mixed in the substrate. For the preparation of said inoculant, the ectomycorrhizal fungus is dehydrated or ground fresh and applied to the substrate. It is important to select an ectomycorrhizal species that is in abundance for the preparation of said inoculant, since large quantities of fresh fungi are required due to their high moisture content. The third method of inoculation is by means of fungal mycelium, this is based on the selection, isolation, purification and subsequent propagation of the ectomycorrhizal fungus in a carrier for example in a mixture of peat-vermiculite; which is applied directly to the substrate that will be used in the nursery.

The fungal mycelium can also be included in sodium alginate, in order to avoid dehydration and keep it in good condition until its application; this technique of preparation of inoculants has also given very good results (^{Pera et al., 1998}; ^{Oliveira et al., 2006}). Several factors must be taken into account for the selection of the species of ectomycorrhizal fungus to be used as an ectomycorrhizal inoculant based on spores or mycelium, such as: i) the compatibility of the fungus with the host plant; ii) the efficiency of the mycobiont to promote rapid mycorrhization, greater growth and survival of the plant; iii) the shelf life of the mycobiont; iv) quality control in the production process of inoculants; and v) the methodological and financial feasibility of production of the inoculants (^{Brundrett et al., 1996a}).

In particular, this last aspect has received little attention, mainly in the production of neotropical ectomycorrhizal inoculants. In the present study, an analysis of the costs of inoculants based on powder and liquid spores is described and carried out. The efficiency of the analyzed inoculants was evaluated in Pinus patula. Species of the genus Laccaria and Hebeloma were chosen according to previous studies which have demonstrated their abundance and biocultural importance in the center of Mexico (^{Pérez-Moreno et al., 2008}).

Materials and methods

Preparation of ectomycorrhizal inoculants based on powder spores. Species sporomes of the edible ectomycorrhizal fungi belonging to the genera Hebeloma and Laccaria (Figure 1a and 2a), were acquired in the market of Ozumba, State of Mexico located at 19° 02’ 11” north latitude and 98° 47’ 48” west longitude, during the months of august and september of 2016. Once the sporomas were acquired, they were classified by species, according to the diagnostic characteristics specified by ^{Carrasco-Hernández et al. (2010}, ²⁰¹⁵).

Figure 1 a) Lady collecting edible wild mushrooms in the market of Ozumba, State of Mexico; b) fresh mushrooms of Hebeloma spp. in the dehydrator; c) fungi of the genus Hebeloma spp. dehydrated; d) mill used for mushroom dehydration; e) Dehydrated inoculant of Hebeloma spp.; f) inoculation of Pinus greggii with Hebeloma sp. 

Figure 2 a) Wild edible mushroom collectors in the market of Ozumba, State of Mexico, also called “hongueras”; b) grinding of L. proxima for preparation of ectomycorrhizal inoculant in liquid; c) inoculant in Laccaria proxima liquid; d) application of liquid inoculant of L. proxima; e) quadrants of the Neubauer camera; f) external mycelium of L. proxima in Pinus patula. 

The stipe was cut and only the pileus was dehydrated in a dehydrator type trays with steam (brand JERSA) at a temperature of 33 ±2 °C for a period of 16 hours (Figures 1b and 1c). The already dehydrated inoculum was milled in an industrial mill (Figure 1d.), with an aperture sheet of 1 mm at the outlet, to allow homogenization of the inoculum thus produced. The inoculum obtained was placed in plastic bags of 500 g capacity and 1.5 mL Eppendorf tubes (Figures 1e and 1f). The inoculant was stored at 3 °C until use. Fresh and dry weight were recorded, and these data were used for cost analysis.

Results and discussion

One of the greatest impacts of logging is the change in the composition of the community of ectomycorrhizal fungi, since these are altered by the chemical and biological changes that occur in the soil after deforestation (^{Bradley, 2001}; ^{Jones et al., 2003}). It should be noted that the mycorrhizal association according to Dupponnois et al. (2011), 95% of native vegetation areas are present that have not been disturbed, while this is less than 1% in disturbed sites.

Due to the above, it is essential that in the nursery the appropriate ectomycorrhizals are introduced that adapt to the plant species and also to the new conditions of the area that is to be regenerated, especially in highly degraded areas. In this sense, it is very important to know the technologies for preparation of ectomycorrhizal inoculants and the costs that would imply for their application in a large-scale nursery.

In the species studied, the average humidity percentage varied from 89 to 91% for the Laccaria case and from 91.6 to 94.4% for the Hebeloma species. The remaining percentage corresponds to dry matter. In the case of Laccaria, an average of 4.8 to 5.5% was obtained for the pileus and 2.6 to 5.3% for the stipe of dry matter. Regarding the Hebeloma species, the dry matter pile percentage was 4.3 to 5% and from 1.2 to 2.7% for the stipe. In both cases the highest percentage of dry matter was recorded in the pileus.

It should be noted that only the pileus is used for preparation of ectoomycorrhizal inoculant of Laccaria spp. and Hebeloma spp. because in the sheets is where the spores are, which are the reproductive structures from which the mycelium with ectomycorrhizal colonization capacity is produced. The data in Table 1 and 2 show the amount of fresh fungi collected for Laccaria spp. and Hebeloma spp. respectively, as well as the quantities of dry pileus (inoculum) used for the cost analysis. The final cost of producing the ectomycorrhizal powder inoculant for the species evaluated was 2 pesos per gram of ectomycorrhizal inoculant.

In previous works this type of inoculant (3g seedling^-1), in P. greggii, P. patula and P. pseudostrobus has been applied at a spore concentration of 10⁶ to 10⁸ per gram and high efficiency results have been obtained with percentages high mycorrhization that have varied from 70 to 90% (^{Carrasco-Hernández, 2011}; ^{Mendez-Neri et al., 2011}; ^{Martínez-Reyes, 2012}). If 3 g of inoculant powder-based inoculant are applied per plant, the cost of inoculation per plant is $6. The cost of producing one kilogram of inoculant ectomycorrhizal powder is $2 000, if you consider applying 3 g by plant this would reach to inoculate 333 plants. However, if the dose is reduced by half 1.5 g per seedling, the cost of the inoculant would be $3, which has also been shown to be effective (^{Rendón et al., 2014}). The cost of production of P. greggii and P. pseudostrobus is 2.72 and 2.98 pesos respectively in the nursery of the Autonomous University of Chapingo.

The plant that is offered at this price is a 10-month plant for P. greggii and 12 months for P. pseudostrobus and are produced in plastic tubes (black virgin polypropylene containers with a capacity of 140 mL) in a peat substrate-vermiculite-perlite in a proportion of 50, 30 and 20 respectively. To these plants were applied eight fertilizations and four fumigations per month. The fertilizers used and the doses of N varied according to the stage of the pine and were the following: as initiator: 9-45-15 to 50 ppm of N, in its growth stage: calcium nitrate combined with 20-20-20 to 200 ppm of N and as finalizer: 4-25-35 to 50 ppm of N.

From the above it is deduced that the cost of a mycorrhized plant with the powder inoculum, would have a price of $5.72 for P. greggii and of $5.98 for P. pseudostrobus at a dose of 1.5 g of inoculum per plant. It is important to mention that studies are needed related to the minimum doses of ectomycorrhizal inoculant powder, which can reach levels of mycorrhization abundant (higher than 70%) and reduce costs. It should be noted that ^{CONAFOR (2010)} takes into account mycorrhiza as an important quality index and that it must cover at least 40% of the root ball so that it is considered an optimum quality plant to be taken to the field.

An alternative form of preparation of ectomycorrhizal inoculant is to take the fresh pileus and grind it for its application as a liquid inoculant. The cost per ml of liquid ectomycorrhizal inoculant was $0.05 per mL and $50 per liter. The concentrations of spores varied according to the species (Table 3). The ectomycorrhizal inoculant based on spores in liquid prepared in the present study was inoculated in Pinus patula to test its effectiveness. The fungi inoculated were: L. proxima, H. mesophaeum and S. pseudobrevipes and the majority of pines presented high percentages of mycorrhization (75% to 100%), which demonstrates their effectiveness (Table 4).

According to evaluations developed in the present work with 10 mL of liquid inoculum per plant, optimal mycorrhization values are obtained and the price would be $0.50 per plant, which substantially reduces the cost compared with the ectomycorrhizal powder inoculant.

The cost of the powder ectomycorrhizal inoculant evaluated in the present work was of $2 000 kg^-1, which is lower compared to the Ecto-Rhyza product of the PHC company, although if the amount of inoculum needed per plant is considered, the product is more expensive than those handled by this company (Table 5). It should be noted that the inoculum evaluated in the present work has not been tested in smaller quantities, which would substantially reduce the cost per plant. In addition to this, the PHC company does not show greenhouse bioassays on the label of its products where it can be confirmed that the concentrations they manage can reach high mycorrhization percentages (greater than 70%).

The cost of production of the inoculum in liquid evaluated in the present work is $0.05 per mL and $50 pesos per liter, which would reach 100 plants in a dose of 10 mL per plant. This price was similar to the product Mycogrow soluble and PHC Ectorhyza and slightly lower in cost to the product Ectoplant irrigation (Table 5).