INTRODUCTION

Avocado (Persea americana Mill.) is a fruit crop of high economic importance in Ecuador, with 6536 ha under cultivation (INEC-ESPAC, 2017). The main areas of avocado production in Ecuador are the provinces of Carchi, Imbabura, Pichincha, Tungurahua, Azuay and Loja (Viera et al., 2016a). Fuerte (green avocado) and Hass (black avocado) are the main commercial varieties grown (Viera et al., 2016b), but both varieties are grafted onto rootstocks from the cultivar Criollo (local germplasm) (Viera et al., 2017a). Therefore, the production of seedlings in the nursery to be used as rootstocks is fundamental and will be of vital importance to the success or failure of an avocado plantation (Andrade et al., 2012). To use local material for propagation of rootstocks is the most recommended (Rodríguez et al., 2016), but genetic variation of cultivar Criollo seed causes variability in both the rate of germination and vegetative growth in the nursery (Viera et al., 2017a). To minimize the impact of this variation and to promote vegetative growth, we suggest that agronomic management (fertilization and irrigation) plus the use of beneficial microorganisms can be used to promote better development of the seedlings, through improving their nutrition.

Nutrient absorption by the plant will depend on its metabolic need and is determined by complex interactions at the plant-soil interface (Reddy et al., 2014). Mineral nutrients taken from the soil are incorporated into a variety of important compounds with structural and physiological roles in the plant (Ohkama &Wasaki, 2010).

Increased plant nutrient absorption after application of microorganisms has been reported for a number of plant species (Gyaneshwar et al., 2002;Berg, 2009;Richardson et al., 2009) and the efficient assimilation in the plant of these mineral elements will be reflected in better plant development (Forde et al., 2004) and increased plant biomass (Jobbagy & Jackson, 2004). Native mycorrhiza have been shown to increase biomass and the uptake of phosphorus in avocado plants (Viera et al, 2017b.); on the other hand, the fungus Trichoderma has been shown to promote plant growth (Zhang et al., 2016) as well as increase the uptake of mineral elements by the plant (Mehetre & Mukherjee, 2015; Rui et al., 2018).

The application of bio-inputs to reduce the use of agrochemicals is currently being promoted. For this reason, plant response to the inoculation of microorganisms at the nutritional level has been documented by several studies (Adesemoye & Klopper, 2009;Richardson et al., 2009;Jacoby et al., 2017). This study examined the effect of a mycorrhizal fungus (Glomus iranicum var tenuihypharum) and a growth promoter fungus (Trichoderma harzianun) on the absorption of nutrients by the avocado seedling.

MATERIALS AND METHODS

Location of the experiment.

The study was carried out at the Tumbaco Experimental Farm of the National Institute of Agricultural Research (INIAP), located in the province of Pichincha, at an altitude of 2348 meters, latitude 0°12’00”S and longitude 78°24'00"W. The experiment was conducted inside a plastic covered greenhouse, using mesh shade (100%) to cover the ground and prevent the plant from having direct contact with the soil. There was an average temperature of 23°C, a relative humidity of 41% and equipped with a drip irrigation system.

Vegetal material.

Seeds of avocado cultivar Criollo were sown in plastic bags of 2 L capacity that contained disinfected substrate made up of black soil and pumice rock (ratio 2:1). Irrigations of 500 ml/plant were carried out twice weekly. For plant nutrition, 50 ml plant^-1 of a solution composed of the following elements and quantities (ppm): 160 Ca²⁺, 48 Mg²⁺, 234 K⁺, 14 NH₄⁺, 196 NO₃^-, 64 SO₄^2-, 0.6 Fe²⁺, 0.5 Mn⁴⁺, 0.02 Cu²⁺, 0.05 Zn²⁺, 0.5 B- and 0.01 Mo⁴⁺ was applied every 10 days (Hoagland and Arnon 1938). The solution was free of phosphorus.

Inoculation of the mycorrhizal fungus.

Two inoculations were made. The first at 20 days after sowing and the second 75 days later. In each inoculation, 5 g of mycorrhiza (Glomus iranicum var . tenuihypharum) plant^-1 was applied at a concentration of 120 propagules per gram of commercial product, diluted in 200 ml of water and applied in drench in four holes made to a depth of 15 cm around the emerging plant (50 ml per hole). Micorrhizal colonization was confirmed by estimating the colonization rate (Trouvelot et al., 1986).

Inoculation of Trichoderma.

The first inoculation was made 20 days after sowing with subsequent monthly applications up to 150 days. The inoculation used a Trichoderma harzianum suspension with a dose of 0.18 g product plant^-1 (concentration of 1.53 × 10⁹ conidia/g), together with the adjuvant Silwet L-77 (0.15 ml l^-1). Drench (50 ml) of the suspension was placed in each hole as above. The establishment of T. harzianum in the substrate was confirmed by re-isolation of the fungus from the potting soil.

Application of the phosphorus source.

Monopotassium phosphate (NPK 0-52-34, ULTRASOL® MKP, manufactured by SQM) was applied at a dose of 200 g L^-1. Six ml of the solution was applied per plant, with subsequent applications every 15 days until 150 days after sowing.

Macro and micronutrient analysis.

The avocado seedling was separated into its aerial part (stem and leaves) and the roots. An analysis of the macro and micronutrient content was carried out using the official methods of the Association of Official Analytical Chemist (AOAC, 2016). The Dumas method was used for the determination of nitrogen (N); the atomic absorption method for potassium (K), calcium (Ca), magnesium (Mg), iron (Fe), manganese (Mn), copper (Cu) and zinc (Zn); and the colorimetric method for phosphorus (P), boron (B) and sulfur (S).

Experimental design.

The experiment was performed using a randomized complete design (DBCA) block with 3 replications (three plants of the same treatment were considered as one replication). The factors evaluated were: Trichoderma (with and without inoculation), mycorrhiza (with and without inoculation), and source of phosphorus (with and without phosphorus application). Two hundred days after the seeds were sown, a chemical analysis was undertaken to determine the content of macro and micronutrient in the aerial parts and in the roots of the avocado seedling. An analysis of variance was performed to determine differences between treatments and the Tukey test at 5% was used to determine differences among means. In addition, a correlation analysis (Pearson) was carried out on the mineral elements to determine relationships between them. The data were analyzed using the statistical software R version 3.5.1.

RESULTS AND DISCUSSION

Berg (2009) showed that microorganisms increased nutrient absorption by the plant when they had established in the soil or developed a symbiosis with the plant host. Re-isolation of T. harzianum (Fig.1), showed the fungus had achieved an average population of 1.3 × 10^-5 CFU per gram of substrate. In addition, symbiosis of G. iranicum var tenuihypharum with the avocado root (Fig. 2) was confirmed microscopically with an average colonization of 21.55%. These results indicate that the microorganisms had established in the area of influence of the rhizosphere of the avocado seedling.

1. Root of avocado seedling

The microorganisms under evaluation influenced the absorption of macronutrients, principally N⁵⁺, Ca²⁺ and Mg²⁺, in the root of the avocado seedling (Table 1).

N has a greater influence on plant growth than any other essential nutrient in the plant and it is a structural component of cell walls (Fageria, 2009). Rudresh et al (2005) reported the nitrogen absorption in roots increases with the inoculation of Trichoderma spp. On the other hand, magnesium is a mineral element that also influences plant growth because it is part of the structure of the chlorophyll molecule and is associated with the activation of enzymatic molecules (Chesworth, 2008). In this research, plants treated with T. harzianum showed higher amounts of N⁵⁺ (1.29%) and Mg²⁺ (0.17%) compared to the control (1.19% and 0.15%, respectively).

G. iranicum var tenuihypharum influenced the absorption of Ca²⁺, an element that has a beneficial effect on the vigor of the plants. It is one of the structural components, promoting the absorption of ions and the formation of mitochondria in the roots (Fageria, 2009). It was observed that the treated plants contained a greater amount (0.20%) of this mineral compared to the control (0.18%). It has been reported that mycorrhiza improves the uptake of calcium and its translocation in the plant (Talaat and Shawky, 2011). In general, there was a positive increase in the amount of macronutrients in the plants treated with the mycorrhiza (Table 1). Improvement in the uptake of macronutrients with the application of mycorrhizal fungi has previously been reported by Sokolovski et al. (2002) and Briccoli et al. (2015).

Phosphate-solubilizing microorganisms are ubiquitous in soils and play an important role in the supply of phosphorus to plants in an environmentally sustainable manner, although their effect in the field is highly variable (Gyaneshwar, 2002). In this study, no statistical difference in the content of phosphorus was observed in the plants treated with G. iranicum var tenuihypharum. However, a trend toward an increase in this mineral was observed in plants treated with the mycorrhiza (4.2%); but this trend was not observed in plants treated with T. harzianum. On the other hand, the application of a source of phosphorous in the substrate significantly increased the amount of this element (8.1%) (Table 1).

Several studies have indicated that mycorrhiza have a positive influence on phosphorus absorption in avocado (Castro et al., 2013;Bañuelos et al., 2017) and Viera et al. (2017b) reported that native mycorrhizal fungi influenced phosphorous absorption in avocado seedlings. In this study, a commercial mycorrhiza (without specificity) was used and, despite root colonization, there was no significant difference in phosphate uptake by the plants. It may be that a longer period of evaluation is needed to detect higher differences in phosphorous absorption in the plant or a specific mycorrhiza is needed. Nevertheless, a positive tendency toward an increase in the content of this element after the 200 days-evaluation was observed, as mentioned above.

In terms of S, it was observed that there was stability in the absorption of this mineral both in the root and in the aerial part of the plant (Table 1 and 3); thus it is inferred that the microorganisms did not affect in any way the assimilation of this element. S concentrations vary based on the N/S ratio (Prosser et al., 2001). This ratio in the root was 6.8 while in the aerial part it was 8.9. Consequently the lack of variation in these values indicates the stability of S content in the whole plant.

Trichoderma inoculation showed a response in root nutrient absorption for some minerals in comparison to the mycorrhiza inoculation and the phosphorus application. The latter did not produce any statistical differences in uptake of any of the elements.

With regard to micronutrient, significant differences were observed only for Fe³⁺ absorption in the plants treated with mycorrhiza (1008.08 mg kg^-1) compared to the untreated ones (837.33 mg kg^-1) (Table 2), a result that agrees with that reported by Liu et al. (2000). Because iron is highly insoluble in the forms found in the soil (Fageria, 2009), the activity of the mycorrhiza as a bridge for the absorption of this element into the plant is a great benefit.

None of the interactions produced any statistically significant differences except for Trichoderma × Mycorrhiza × Phosphorus which presented a high value (1.36%) for the absorption of N. However, Ortiz et al. (2018) reported that a degradation of the mycorrhiza outer spore layer is observed when interacting with T. harzianum; for this reason, the application of both microorganisms at the same time is not recommended.

Aerial part of the avocado seedling

Application of the microorganisms increased foliar absorption of the macronutrient N⁵⁺, Ca²⁺ and Mg²⁺ (Table 3), the same as had occurred in the root of the avocado seedling.

Mehetre and Mukherjee (2015) reported that Trichoderma spp. improves uptake of minerals. In this study, plants treated with T. harzianum showed higher content of N⁵⁺ (1.79%), Ca²⁺ (1.39%) and Mg²⁺ (0.43%) than untreated plants (1.68%, 1.26% and 0.39% respectively). Concentration of N in the leaf is a significant physiological parameter for detecting health of crop plants (Zhao et al., 2005). In addition, it is important that the plant has good Ca content because this element is involved in the metabolism of N (Fageria, 2009); it has been reported that N concentrations of all plant organs decreased with calcium deficiency (Banath et al., 1966).

Plants treated with G. iranicum var tenuihypharum showed a trend towards an increase in the content of N⁵⁺ (1.74%) compared to those not treated (1.68%). Högberg (1997) reported the effect of mycorrhizal fungi helping plants in nitrogen uptake. Nitrogen concentration was higher in leaves than in the root, confirming that nitrogen is easily transported from the roots upwards, contrary to Fe which was found in greater amounts in the root cells (Kabata-Pendias, 2001). In addition, this trend was also observed for Ca²⁺ absorption; 1.35% in treated plants and 1.26% in untreated plants. The increase in concentration of this element after inoculation with mycorrhiza has also been reported by Talaat and Shawky (2011).

The concentration of P⁵⁺ increased to 6.9% in the plants after application of a source of phosphorous (Table 3), a trend that also occurred in the root although there it was not statistically significant. This is attributed to the fact that fertilization with this element results in an increase in its uptake into the plant (Xu et al., 2016).

Copper is essential for photosynthesis and mitochondrial respiration, for carbon and nitrogen metabolism, for oxidative stress protection and is required for cell wall synthesis; while manganese is fundamental for plant metabolism and development (Hansch and Mendel, 2009). T. harzianum had a significant effect on absorption of these micronutrient (Cu²⁺ and Mn²⁺) (Table 4). In general, there was a trend towards an increase in the amount of all micronutrient except B in the plants treated with this fungus, results that would confirm that Trichoderma improves the efficiency of nutrient uptake in crop plants as reported by Mehetre and Mukherjee (2015). On the other hand, mycorrhiza had no effect on the absorption of micronutrient.

As was observed in the root, there was a tendency towards an increase in the amounts of all the micronutrient in the plants with the application of a source of P⁵⁺, with the exception of Fe³⁺ (non-statistical difference). These results support the conclusions of Power et al. (1961) who reported that fertilization with phosphorous promotes plant nutrient uptake. However, statistically significant differences in uptake were recorded in the results obtained with the Trichoderma and mycorrhizal inoculation but not for the phosphorous application.

The interactions that showed statistical differences were: Trichoderma × Without Mycorrhiza × Without Phosphorus (238.90 mg kg^-1) and Trichoderma × Without Mycorrhiza × Phosphorus (245.37 mg kg^-1) in the absorption of Mn²⁺. In addition, the interaction Without Trichoderma × Without Mycorrhiza × Phosphorus obtained a high value (35.83 mg kg^-1) in terms of B³⁺ absorption. These results indicate that the application of phosphorous encourages the uptake of these minerals.

Correlation analysis

Renata (2012) suggested that there are significant relationships between concentrations of the various nutrients in the plant. This indicates that determination of interactions among minerals in the plant could help identify key elements involved in plant development. Pearson Correlation Analysis showed that mainly concentrations of the macronutrients such as N⁵⁺ and P⁵⁺ correlated significantly with micronutrient in the root of the plant (Table 5). Moderately high correlation values (> 0.6) indicated that a greater amount of the first element will also increase the second. In this study, the correlation value between N⁵⁺ and Fe³⁺ was 0.62, a result that supports the results of Pich et al. (2001) who reported that nitrogen positively affected an increase in absorption of iron into the plant. Additionally, the correlation between P⁵⁺ and Zn²⁺ was 0.67, a result that corroborates the results of Wasaki et al. (2003) who considered that zinc has a role in regulating the absorption of phosphorous. In the aerial part of the plant, further significant correlations were observed between the elements (Table 5). A high positive correlation (0.74) was observed between Ca²⁺ and Mg²⁺, which is expected as a negative correlation would have suggested that an increase in calcium would have caused the inhibition of magnesium absorption (Fageria, 2009).

적 요

아보카도는 에콰도르에서 경제적으로 중요한 과수이다. 건 강한 종묘 생산은 아보카도 식물의 생산에서 중요한 단계이며 유익한 미생물의 처리는 식물체의 영양을 향상 시키는데 사용 될 수 있다. 아보카도 묘목에 처리한 미생물의 효과를 검정한 결과, 묘목의 뿌리부분과 지상부의 양분 흡수를 증가시켰다. T. harzianum은 뿌리에서 N과 Mg의 흡수를 유의적으로 증가 시켰으며, 지상부에서는 N, Ca, Mg, Mn 및 Cu의 흡수를 증 가시켰다. 반면에, G. iranicum var tenuihypharum은 뿌리에서 Ca와 Fe의 흡수를 유의적으로 증가시켰으며, 지상부에서는 N 흡수를 증가시켰다. 또한, mycorrhiza의 접종으로 아보카도 묘 목의 뿌리 부분과 지상부에서 P 양을 증가시키는 경향이 긍 정적으로 나타났다. 두 미생물 모두 S의 흡수에 영향을 미치 지 않았는데, 이는 식물 전체에서 균일한 비율을 나타냈다. 또 한 인산질 비료(일인산 칼륨)의 시용은 미량 영양소의 흡수에 긍정적인 영향을 미쳤다.