Location

This study was carried out at the Morelia campus of the Universidad Autónoma Chapingo (UACH), located at 19º 68’ 69”-101º 23’ 80”. The climate is temperate subhumid with rains in summer and average humidity, C (w1). Average annual temperature of 17.5 °C and precipitation of 773.5 mm per year. The dominant winds come from the southwest and northeast, varying in July and August with intensities of 2 to 14.5 km h^-1 (^{SMN, 2015}).

Vegetal material

Four-month-old seedlings of the Biloxi variety were used that were micropropagated in vitro at the School of Agrobiology of the Michoacan University of San Nicolás de Hidalgo (UMSNH) their sanitary quality was confirmed in order to be used in the trials.

Inoculum sources

To investigate the colonization by mycorrhizae of cranberry cv. Biloxi pots with native mycorrhizae inoculated substrate of a wild Vaccinium confertum cranberry species were used, a species of the Gaultheria sp. Genus and the Endospor^® commercial product. The details of inoculum sources are listed below.

1. Endospor^® endomicorritic inoculant contains endomicorrizas forming fungi, such as Gigasporas margarita, Glomus mosseae, G. clarum, G. deserticolae, G. etunicatum, G. brasilianum, G. intraradices, Trichoderma and Gliocladium. It is recommended in vegetables, fruit trees, flowers, grasses and shrubs. The inoculants were prepared and applied the same day. The application was carried out according to the dosage indicated on the label and dissolving it in 250 ml of water (^{Bactiva, 2015}).

2. The native mycorrhizae source were the rhizospheres (soil + roots) of the 3 year old wild cranberry (V. confertum) and a Gaultheria species collected in a forest adjacent to Morelia city of colonized with endomicorrizas and following the methodology proposed by ^{Carrillo (2015)}. Applying 1 kg of soil in 3.6 L of water so that the same day of inoculation it was extracted from this population.

In the trial the pots contained three liters of peat moss plus perlite mixture (v/v 1:1) inoculated with both commercial and native mycorrhiza. The peat moss has the ability to retain moisture, good aeration and high content of organic matter, and the perlite retains water so it is longer available for the plant in addition of being inert. This mixture guarantees adequate moisture seeking the effectiveness of inoculation with native beneficial microorganisms (^{Scagel et al., 2005}; ^{Bizabani and Dames, 2015}). They were stablished in a completely randomized experimental design with 12 replicates.

Presence of mycorrhizae

In order to distinguish mycorrhizal fungi, a root staining protocol proposed by ^{Phillips and Hayman (1970)} was followed from a root sample. The analyzes were carried out in the agroecology laboratory of the ecosystems research center in UNAM (CIECO), Campus Morelia, Michoacán.

Evaluation of the cranberry plants growth

For the evaluation of cv. Biloxi blueberry growth (Vaccinium spp.) under the three sources of inoculum plus the control, plants were measured. Measurements were taken 161 days after the transplant (March 13, 2014).

Three measurements of seedling growth were made.

Plant height: measured from the neck of the main stem to the apex.

Number of stems: the number of stems or shoots at the base of the seedling were counted; secondary sprouts produced during the growth period.

Number of leaves: the number of leaves of the main stem was counted, from the base to the apex.

Length and width of the main stem leaf: the length and width of the main leaf of the main stem were measured with measuring tape (Figure 1).

Figure 1 Growth measurements of blueberry cv. Biloxi once inoculated with mycorrhizae.  

For each variable, analyzes of variance (ANOVA) and tests of means comparison (Tukey α <0.05) were performed inn order to analyze its effect.

Achievements

Since the seedlings used were multiplied in vitro their sanity was confirmed by tissue tests as follows: small fractions of branches, stems and roots were cut into petri dishes with potato-dextrose agar (PDA) (8 g potato starch, 20 g dextrose, 20 g agar) and following the morphological keys of Barnet and Hunter (1972) to identify pathogens. All seedlings were free of bacteria and fungi.

Effect of native mycorrhizae and commercial Endospor ^R product on plant growth and mycorrhizal colonization

The effect of inoculation on seedlings was measured both in the growth it produced in the plant and in the colonization in roots. Statistical analysis of both types of variables was analyzed using an Anova. Table 1 presents the results. In general, we can point out that among the four treatments, the one that was inoculated with Gaultheria sp, produced a seedling height, number of leaves and frequency and intensity of mycorrhizal colonization greater than the rest of treatments. The average seedling growth of this treatment after 161 days was 22.818 cm in height and 17.636 leaves per plant and of frequency and intensity of colonization of 8.6% and 15%, respectively. There were no significant differences between treatments in number of stems, leaf length and width (Table 1).

Table 1 Results of seedling growth measurements and colonization by mycorrhizae according to the inoculum sources tested in cranberry cv Biloxi. 

^z= tratamientos con la misma letra estadísticamente son iguales.

Table 1 shows that for plant height the treatment with Vaccinium confertum inoculum and the uninoculated control had an average height of 16.545 and 16.5 cm respectively, which were similar to those obtained with the inoculation with Gaultheria sp. while the seedlings inoculated with the commercial product Endospor registered the lowest value. For number of leaves per plant, the treatment inoculated with Gaultheria sp. and the control, presented similar values and very different to those obtained with the V. confertum inoculum and the product Endospor. For the rest of the variables the treatments did not show notable differences; the seedlings inoculated or not show similar values in number of stems, length and width of leaf.

Figure 2 shows the root with mycorrhizae of the Gaultheria sp. species and Figure 3 shows the data plotted in Table 1. The results show that for the nursery stage, the blueberry seedlings cv Biloxi inoculated with the tested treatments can reach a low colonization and a relatively low growth. This coincides with Callejas-Ruíz et al. (2009), which found no effect when inoculating with mycorrhizae of the Glomus genus in gourd. Nor does Scagel et al. (2008) show an important effect of mycorrhizal inoculation of blueberry seedlings in the early stages of the plant. This author suggests that colonization may depend on environmental, management or substrate factors and not just on the presence or absence of the correct mycorrhizae.

Figure 2 Presence of mycorrhizae in the root of blueberry cv Biloxi. 

Figure 3 Variables of seedling growth and colonization by mycorrhizae in cranberry cv Biloxi, in response to different inoculum sources. The actual values of the variables were centered for graphing purposes  

An opposite result was reported by ^{Ávila et al. (2009)}, who found a positive effect of the mycorrhizae of the commercial product Micorrizafer^® (Agrosafer Company, Medellín, Colombia), which contains mycorrhizal spores of Glomus, Entrophospora, Scutellospora and Acaulospora on micropropagated stems. In the Andean blueberry species V. meridionale, colonization causes the plants to develop short roots unless they are added growing promoters such as indole-butyric acid or indole-acetic acid in the culture medium. The use of the promoters is also contradictory because ^{Rodríguez (2006)} also found positive effects of mycorrhizae by multiplying stem cuttings and inoculating them with the commercial product Mycosym Tri-Ton (Hymenoscyfus ericae) from the Chilean company Biotritón S. A. This author points out that interaction with the effect of growth hormones may be negative, and does not recommend their use when inoculating cranberry with mycorrhizae.

In terms of inoculum sources, this paper coincides with that of ^{Carrillo et al. (2015)} who obtained colonization of mycorrhizae from propagules of Gaultheria pumila, a Chilean species. After 6 months the roots of cv Brigitta of V. corymbosum (high blueberries of the north) were colonized with these mycorrhiza although not so widely. It may be due because for these authors as in our case, the effect of the Peat Moss substrate or peat should be investigated; authors such as ^{Scagel et al. (2005)}; ^{Farías et al. (2014)}; Bezabani and Dames (2015) point out that the relationship between the nature of the inoculum, the nutrition, the substrate and the observation period of the measurements may be factors that vary the results, although all agree that the mycorrhizae reduce the application of nutrients and make viable their use in organic farming systems.

Conclusions

The test allowed to distinguish the positive effect of the inoculum of Gaultheria sp. as a source of mycorrhizae for cv Biloxi cranberries. Although low colonization (frequency of 8.3% and intensity of 15%) can be considered, it produced a growth of 22.8 cm in length on stem and 17.6 leaves per plant after 161 days of transplantation. The Endospor^® product; as well as the rhizosphere of V. confertum did not produce a different effect to the uninoculated control.