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Cuban Journal of Agricultural Science

Print version ISSN 0864-0408On-line version ISSN 2079-3480

Cuban J. Agric. Sci. vol.50 no.2 Mayabeque Apr.-June 2016


Cuban Journal of Agricultural Science, 50(2): 315-320, 2016, ISSN: 2079-3480




Critical levels of phosphorus in the soil for forage legumes, inoculated with arbuscular mycorrhizal fungi


Niveles críticos de fósforo en el suelo para leguminosas forrajeras inoculadas con hongos micorrízicos arbusculares



P. J. González,I J. F. Ramírez,II R. Rivera,I A. Hernández,I G. Crespo Flores,I

IInstituto Nacional de Ciencias Agrícolas, MES. Carretera de Tapaste km. 3,5. San José de Las Lajas, Mayabeque, Cuba.
IIEstación Experimental de Pastos y Forrajes de Villa Clara, MINAG, Cuba.




The effect of arbuscular mycorrhizal inoculation on critical levels of phosphorus in the soil for Neonotonia wightii and Stylosanthes guianensis was evaluated. An amount of 20 places were selected, with concentrations of assimilable phosphorus from 0 to 20 cm, which varied from 9 to 55 mg kg-1. In each place, the response of both legumes to phosphoric fertilization was evaluated with or without inoculation of the arbuscular mycorrhizal fungi Glomus cubense. Using the method of Cate and Nelson, critical levels of phosphorus in the soil were obtained for these legumes, inoculated or not. For N. wightti without inoculation, the critical level was 35 mg kg-1 (R2=0.90), but with the inoculation with G. cubense, it decreased up to 20 mg kg-1 (R2=0.87). For S. guianensis, without inoculation, the result was 20 mg kg-1 (R2=0.83). In presence of G. cubense, there was no response to phosphoric fertilization.

Key words: soils, nutrition, phosphoric fertilization, Neonotonia wightii, Stylosanthes guianensis.


Se evaluó el efecto de la inoculación micorrízica arbuscular en los niveles críticos de fósforo en el suelo para Neonotonia wightii y Stylosanthes guianensis. Se seleccionaron 20 sitios, con  concentraciones de fósforo asimilables de 0 a 20 cm de profundidad, que variaron de 9 a 55 mg kg-1. En cada sitio se evaluó la respuesta de ambas leguminosas a la fertilización fosfórica, estuvieran inoculadas o no con la especie de hongo micorrízico arbuscular Glomus cubense. Mediante el método de Cate y Nelson se obtuvieron los niveles críticos de fósforo en el suelo para estas leguminosas, inoculadas o no. Para N. wightti sin inocular, el nivel crítico fue de 35 mg kg-1 (R2=0.90), pero con la inoculación de G. cubense disminuyó a 20 mg kg-1 (R2=0.87). Para S. guianensis, sin inoculación, fue de 20 mg kg-1 (R2=0.83). En presencia de G. cubense no hubo  respuesta a la fertilización fosfórica.

Palabras clave: suelos, nutrición, fertilización fosfórica, Neonotonia wightii, Stylosanthes guianensis.




Inoculation of selected strains of arbuscular mycorrhizal fungi (AMF) is an alternative for improving yields and reducing the use of fertilizers in forage species because, this way, an effective symbiosis between microorganisms and host plants is achieved, which increase the surface of absorption of its roots and, consequently, the efficiency of utilization of nutrients (Cavagnaro et al. 2014).

These benefits of plant nutrition may influence on critical levels of soil nutrients. That is that concentrations of a determined nutrient, from those that are expected to have a high or low response of crops to the application of fertilizers (Bai et al. 2013). Taking into account that knowledge of critical levels of a nutrient is a necessary tool for a proper diagnosis on the need of fertilization, it is essential to know the effect of AMF on this indicator in inoculated crops.

This study evaluated the influence of arbuscular mycorrhizal inoculation on critical levels of phosphorus in the soil for forage legumes like glycine (Neonotonia wightii cv. Tinaroo) and stylo (Stylosanthes guianensis cv. CIAT-184).

An amount of 20 places were selected, with different antecedents of P fertilizartion, belonging to the Empresa Pecuaria Genética “Niña Bonita”, in Artemisa, and to the Cooperativa de Créditos y Servicios Fortalecida “Orlando López”, in Havana. These facilities are located in lixiviated red ferrallitic soils, lixiviated yellow ferrallitic soil and red brown fersialitic soil (Hernández et al. 2015), with contents of assimilable P that had a growing variation, from 9 to 56 mg kg-1. In each place, the response of glycine (Neonotonia wightii cv. Tinaroo) and stylo (Stylosanthes guianensis cv. CIAT-184) to phosphoric fertilization was determined, with or without arbuscular mycorrhizal inoculation.

A field with 1,000 m2 of surface was defined in each place. An amount of 10 subsamples were taken from the soil, from 0 to 20 cm deep, according to the zigzag method, in order to form a compound sample, as a representation of this area. The content of P was determined in the samples regarding the method of Bray and Kurtz I (van Reeuwijk 2002).

Later, plots of glycine and stylo, with a surface of 500 m2 each, were established in the selected places. Through a completely randomized design with ten reptitions, three treatments were distributed in those plots. These treatments were composed by a control without phosphorus and without mycorrhizal inoculation, another without phosphorus and with inoculation with Glomus cubense, and the last treatment with only the application of 50 kg ha-1 of P2O5. This dose is traditionally used in both units for phosphoric fertilization of protein banks. G. cubense was used because of its high efficiency in improving yield of crops under the conditions of the soil used for this study (Rivera et al. 2015).

Legumes were sown in June, 2013, in furrows separate at 70 cm and light stream, with doses of 8 kg of total seeds ha-1 (2 kg of pure germinable seed ha-1). Inoculation was performed at the moment of sowing using the method of seed covering. For that purpose, these seeds were submerged into a fluid paste, product of a mixture of an amount of solid inoculum equivalent to   10 % of their weight (800 g) and 300 mL of water. Once the seeds were covered and the inoculum was solid, the sowing started.

The inoculum was obtained from a clayey substratum, sterilized in an autoclave at 120 °C for an hour for three days, with Brachiaria decumbens cv. Basilisk as host plant. It contained 35 spores g-1 of substratum, abundant fragments of hyphae from INCAM-4 strain, belonging to Glomus cubense species (Y. Rodr. and Dalpé) (Rodríguez et al. 2012) and rootlets from the host plant. The strain belonged to the collection of the Instituto Nacional de Ciencias Agrícolas (INCA) from the Republic of Cuba.

Three cuts were performed, the first at 120 d after sowing, the second and the third at intervals of 60 and 90 d between cuts, depending on biomass availability. In each cut, yield of dry mass (DM) of the aerial part was evaluated through the distribution at random of 10 frame of 1 m2 per each treatment, and the green mass (GM) was cut at 10 cm high the over soil surface. Samples of 200 g were taken from each frame, and were put into a circulated air oven at 70 ºC until reaching a constant mass, in order to determine the percentage of dry mass (DM) and estimate DM yield.

The experiment was conducted under dry conditions. Due to the lack of efficient isolates of ryzobium and to not limit the response to phosphoric fertilization, 35 kg of N were applied at the moment of sowing and after each cut. Urea and triple superphosphate were used as N and P sources, respectively. There was no application of potassium fertilization because the contents of this element in the soil were considered as enough for these legumes.

In each place, relative yield (RY) of both species, in presence and absence of arbuscular mycorrhizal inoculation, was calculated using the following formulas:

RY (%) in absence of inoculation = [yield of DM (kg ha-1) of the treatment without P2O5 / yield of DM (kg ha-1) with the application of 50 kg P2O5 ha-1] x 100.

RY (%) in presence of inoculation = [yield of DM (kg ha-1) of the treatment without P2O5 + G. cubense / yield of DM (kg ha-1) with the application of 50 kg P2O5 ha-1] x 100.

Two graphs were built for each legume. These graphs contained the relative yield obtained in each place, in absence of mycorrhizal inoculation and in presence of G. cubense, respectively, with their own content of soil P. Two perpendicular lines were located using the graphic method of Cate and Nelson (1965). The horizontal line cut the Y axis and divided relative yields that showed a high response to P from those that had a low or null response to the application of this element. The vertical line cut the X axis and indicated the critical level of P in the soil. It was statistically calculaed from the following division of the population in two groups, and was located in the point where the highest correlation coefficient (R2) was reached between both , according to Cate and Nelson (1971).

Figures 1 and 2 show critical levels of soil P for glycine and stylo, respectively, inoculated or not with G. cubense. For glycine, with or without inoculation, and for stylo without inoculation, the location of most of the points in the left inferior and right superior quadrants, which form the perpendicular lines of graphics, indicated that soil P contents and relative yield of legumes were related (Cate and Nelson 1965). Consequently, the used method was valid for determining the critical levels of this element, with or without arbuscular mycorrhizal inoculation.

According to Cate and Nelson (1971), crop yield, under field conditions, is affected by a group of edaphic and climatic conditions that are inherent to each locality. However, the fact that even though a division between two populations may be established, one with high and another with low or null response to the application of an specific nutrient, indicates that the content of this nutrient in the soil is more important than those variables that cannot be controlled, in order to determine the response of these crops to its application.

In absence of mycorrhizal inoculation, the critical level of soil P for glycine was 35 mg kg-1. However, after inoculating with G. cubense, this level decreased to 20 mg kg-1. For stylo, there was a critical level of 20 mg kg-1 of P in absence of G. cubense, and there was no response of this crop to phosphoric fertilization with the presence of this AMF species.

According to these results, in absence of arbuscular mychorrizal inoculation, a response of glycine to phosphoric fertilization may be expected when the tenors of soil P are lower than 35 mg kg-1. However, after inoculating it with G. cubense, inferior values indicate that plants are sufficiently provided of P. In this case, a response to the application of this element should be expected when its content within the soil is inferior to 20 mg kg-1.

In stylo without inoculation, a response to phosphoric fertilization may be obtained when the contents of soil P are inferior to 20 mg kg-1, but, with the presence of G. cubense, its application is unnecessary, apart from the indicators of this element in the soil. This way, it may be inferred that, with arbuscular mycorrhizal inoculation, this species may obtain all the P necessary for fulfilling its requirements.

The effect of G. cubense on the modification of critical levels of P for both forage legumes, may be related to the increase of absorption surface of roots of inoculated plants, which could explore a superior volume of soil and access to less available forms of this element. It has been demonstrated that the inoculation of AMF species, previously selected due to their ability of improving productivity of crops, may produce an effective mycorrhizal functioning that improves absorption efficiency of nutrients, increases yields and decreases the requirements of fertilizers  (Taffouo et al. 2014).

Phosphorus is a macronutrient within the soil in low concentrations, and it is characterized by its low mobility. Under these conditions, the introduction of efficient AMF species is a determinant element for phosphoric nutrition of plants, which roots increase the places of absorption with higher affinity for this element, and may access to less mobile forms of it (Cozzolino et al. 2013). 

The difference between the critical levels of P in both forage legumes may be caused by the difference in their requirements of this nutrient. Glycine needs soils of high fertility, provided of nutrients, mainly P, for a good development. Stylo is better developed in soils of low fertility and high acidity. In fact, it is stated that this species may be cultivated under low P availability conditions, reaching the maximum growth with relatively low doses of this element (Lopes et al. 2011).  

It can be concluded the arbuscular mycorrhizal inoculation may modify the critical levels of soil P for forage legumes. Under the conditions of this study, for glycine without inoculation, the critical level was 35 mg kg-1, but with the presence of G. cubense, it was reduced to 20 mg kg-1. For stylo, the critical level of P was 20 mg kg-1without inoculation. In presence of G. cubense, there was no response to phosphoric fertilization.



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Received: 21/7/2015
Accepted: 1/6/2016



P. J. González, Instituto Nacional de Ciencias Agrícolas, MES. Carretera de Tapaste km. 3,5. San José de Las Lajas, Mayabeque, Cuba. Email:

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