Introduction

The phosphorus (P) is one of the limiting nutrients in the soil due to its low availability and low mobility among macro nutrients (^{Souchie et al., 2006}). In the plant, phosphorus is required in the formation of nucleic acids, phosphates of sugars and membranes, protein synthesis and formation of the new protoplasm, so an adequate supply of this element in the soil is necessary (^{Condron and Tiessen, 2005}). The absorption of P in plants is carried out through diacid or monoacid ions of the substrate, due to the low solubility of the phosphate compounds, there is a tendency of displacement of the equilibrium towards the solid phase, so that the concentration of P in solution at a specific time can become very low (0.02 to 0.1 mg L^-1) (^{Chesworth, 2008}).

These characteristics have led the plants to develop different physiological mechanisms (accumulation of carbohydrates in the root) and biochemical (root exudates) to increase the acquisition of nutrients, especially P. One such mechanism is the formation of the symbiotic association with arbuscular mycorrhizal fungi (HMA; ^{Smith and Smith, 2011}), because it is known that plants can acquire P by (1) direct absorption that occurs through transporters in the epidermis of the root and (2) mycorrhizal absorption translocating P from the extra radical hyphae of the fungi to the interior of the root (^{Smith et al., 2004}). In soils with limited phosphorus content, plants can completely depend on the mycorrhizal association to acquire this nutrient (^{Smith et al., 2003}).

Arbuscular mycorrhizae are associations of the mutualistic type between plants and a great variety of fungi (^{Smith and Read, 2008}); the HMA are soil organisms that form symbiosis with 80% of terrestrial plants (^{Smith et al., 2004}). This association benefits the plant by increasing the availability of nutrients with low availability or low soil mobility, has biocontrol effects against some pathogenic microorganisms, increasing nutritional status, tolerance to water stress, and other benefits (^{Smith et al., 2011}). At the same time, this association plays an important role on soil physical characteristics, increasing particle aggregation and soil stability (^{Khan, 2006}).

Numerous studies have demonstrated the advantages of inoculation with arbuscular mycorrhizal fungi in low fertility soils (^{Herrmann et al., 2015}). The most important effect of HMA on crops is an increase in the uptake and translocation of soil nutrients such as N, P, K, Ca, and Mg (^{Säle et al., 2015}), which translates into higher growth and development of plants, as well as a higher percentage of transplant survival (^{Rodríguez et al., 2017}).

Under conventional production schemes optimum crop development demands high application of mineral fertilizers and pesticides (^{Weih et al., 2011}). Phosphate fertilizer application is sometimes superior to the needs of such crops, resulting in excessive accumulation of readily available phosphorus in the soil (^{Withers et al., 2001}). Therefore, it is necessary to know the optimum phosphorus dose so that the plant and the mycorrhizal association can be efficiently developed, so that the benefits that this symbiosis can be obtained (^{Taffouo et al., 2014}).

Mexico is the center of origin of avocado (Persea americana Mill; ^{Gutiérrez et al., 2010}) with a great diversity of species, up to 20 different and three races have been reported: Mexican, West Indian and Guatemalan (^{Chen et al., 2009}). It has been documented that avocado is a myotrophic culture that responds favorably to mycorrhizal inoculation (^{Barcenas et al., 2007}; ^{Bañuelos et al., 2013}). However, it is known that there is a great genetic and functional diversity of HMA species (^{Helgason and Fitter, 2009}), and that the genera of HMA associated to the same host species present differences in relation to the level and capacity of sporulation (^{Bever, 2002}), ability to colonize roots (^{Klironomos and Hart, 2002}) and to acquire phosphorus (^{Smith et al., 2000}).

In addition, varieties of the same plant may also differ in their response to mycorrhization. In wheat, colonization by Glomus intraradices varied between 16 and 37% between cultivars (^{Zhu et al., 2001}) and in barley, two varieties inoculated with G. intraradices differed in more than 100% in acquisition of inorganic phosphorus (Pi) (^{Zhu et al., 2003}).

In avocado cultivation the results may be compelling, for example, in a study to evaluate the functional differences between arbuscular mycorrhizal fungi Glomus intraradices, Scutellospora heterogama and their effect on avocado plants (Persea americana), it was found that the joint inoculation with G. intraradices and S. heterogama reduced plant growth rates and the absorption of P, zinc (Zn) and iron (Fe) in relation to plants inoculated with G. intraradices, evidencing in this case the absence of functional complementarity (^{Violi et al., 2008}).

In contrast to these results, inoculation with consortia of HMA species (Glomus fasciculatum, Glomus constrictum, Glomus tortuosum, Glomus geosporum, Acaulospora scrobiculata Glomus mosseae and Glomus cubense) favored a greater development in Mexican creole avocado plants (Persea americana var. drymifolia) with respect to control plants, increasing plant height up to 54%, stem diameter (up to 36%), number and leaf length (48% and 40% respectively), as well as fresh root weight up to 85%) (^{Castro et al., 2013}).

It is known that avocado cultivation is susceptible to attack caused by root pathogens, and that in the nursery stage the soil is sterilized prior to planting, thereby eliminating all types of microorganisms, so that reintroduction of beneficial microorganisms such as arbuscular mycorrhizal fungi may play an important role in the production of this plant. Due to the above, the present work has as objective: to evaluate the effect of different concentrations of phosphorus fertilization, in the development of avocado plants with and without mycorrhizal inoculant, expressed in growth variables.

Materials and methods

Results and discussion

The mycorrhizal inoculants had significant effects, as well as the different levels of phosphorus in measured variables and only one effect was found by interaction of both factors in height and growth rate (Table 1).

ns= no significativo; ^*= p≤ 0.05; ^**= p≤ 0.01; ^***= p≤ 0.001; PF-A= peso fresco parte aérea; PF-R= peso fresco raíz; PS-A= peso seco parte aérea; PS-R= peso seco raíz; n= 4.

Table 1. Multivariate ANOVA for each growth variable. 

When phosphorus was not added, Rizofermic-UV inoculant showed the highest number of leaves and leaf area. Plants inoculated with P. scintillans had an increase in diameter, height, and leaf area at 20 ppm, compared to the other treatments. Both inoculants increased leaf number, fresh shoot weight, and growth rate at 20 ppm, compared to uninoculated treatment, although this effect was already observed at higher doses of phosphorus. In general, when comparing the effect on the increase of the measured variables between inoculants at different doses of phosphorus, the effect of P. scintillans was more noticeable at 20 ppm, and the effect of Rizofermic-UV occurred at 40 ppm (Table 2).

±= indica desviación estándar; PF-A= peso fresco parte aérea; PF-R= peso fresco raíz; PS-A= peso seco parte aérea, PS-R= peso seco raíz; n= 4. Letras distintas muestran diferencias estadísticas entre inóculos por nivel de fertilizante (Fischer, p≤ 0.05); †, ^***; ‡, **; +, *= muestra diferencias entre los niveles de fósforo por inoculante.

Table 2 Comparative analysis of the effect of the interaction of two mycorrhizal inoculants and the application of four doses of phosphorus in the development of avocado plants. 

It was found that mycorrhizal colonization decreased with the addition of P doses for both inoculants. Statistical differences (p< 0.05) were observed among the inoculate for this variable. Both the Rizofermic-UV and P. scintillans consortium had a significant decrease in mycorrhizal colonization from 80 ppm, with Rizofermic-UV the inoculant having the highest percentages of root colonization at 20 ppm (78.34%) (Figure 1).

Figure 1 Colonization percentages of various inoculants avocado roots (Persea americana). Different letters indicate comparative differences between all treatments at different concentrations of phosphorus and HMA. n= 4 indicates ± standard deviation  

Discussion

According to the results observed in this experiment, the high doses of P (160 ppm) caused the plants to die. It has been reported that high P availability may lead to plant growth depression (^{Jiménez and Fernández, 2016}). In this sense, ^{Marschner (2008)} states that high levels of P produce toxicity in the plant by altering the absorption of Zn and Fe. On the other hand, ^{Li et al. (2004)} indicate that Zn or Fe deficiencies are more frequent when there are high concentrations of P in the soil, so it is likely that studies that have described micronutrient deficiencies are in fact a reflection of toxicity symptoms due to to high levels of P (^{Shane et al., 2003}).

It was found that the addition of P and mycorrhization promoted plant growth. Within the different doses of P tested, the dose of 20 ppm of P had an effect on the increase of biomass, coinciding with that found by ^{Montoya (2007)}. Of the two HMA inoculums tested, the plants inoculated with the Rizofermic-UV consortium showed a tendency in the increase of the growth variables, essentially in leaf area (p< 0.05), unlike Pacispora scintillans, which did not have a growth compared to the control.

The differences in effects between HMA strains tested on avocado seedlings, especially in the case of leaf area could be due to functional complementarity between the different species within the Rizofermic-UV consortium and avocado. It is known that HMA species differ widely in their strategies for acquiring P and promoting host plant development (^{Thonar et al., 2011}). In this sense Jaizme and Azcón (1995) found differences of complementarity among HMA species associated with avocado plants.

Some studies report that increases in P levels reduce the development of mycorrhizal symbiosis; However, the levels at which such a reduction occurs are variable (^{Almaca and Ortas, 2010}) and are based on the identity of the HMA species and the plant genotype (^{Lacerda et al., 2011}). In this case, the interactions between mycorrhizal inoculation and the addition of phosphate fertilizer showed different effects. For example, P. sintillans treatment plus 20 ppm of P increased growth variables more than treatments individually, similar results have been reported by (^{Dutt et al., 2013}3). In this sense, the addition of P. scintillans could make fertilizer use more efficient and represent a step forward in reducing the current fertilizer doses used for avocado cultivation.

It is known that mycorrhizal colonization begins to occur at three days after inoculation and strengthened from 21 days (^{Mustafa et al., 2010}); however, both studies conclude that mycorrhizal inoculation needs time to show its beneficial effect. Since avocado plants have a rich nutrient reserve, it is possible that the benefits of the mycorrhizal association do not manifest themselves at an early stage of inoculation (^{Osorio et al., 2012}), so the inclusion of these symbionts must occur at a key low lignification stage (^{Leskovar et al., 1991}) and high susceptibility (^{Afek et al., 1990}) of the plant.

Conclusions

In the present work and under the experimental conditions presented here, it is suggested the fertilization with a minimum dose of phosphorus in interaction with HMA, since in the results obtained here was the concentration where a significant increase in the variables was observed and where there was a increase in height and relatively greater leaf area.

The present study showed that inoculation with HMA, essentially with the strain Pascispora scintillans, promotes greater growth in avocado seedlings when they are fertilized with 20 ppm of phosphorus.

For the fresh and dry weight the interaction of the HMA with the P were those that had more relevance. P. scintillans promoted higher growth in plants compared to plants inoculated with the Rizofermic-UV consortium and control plants.