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Cuban Journal of Agricultural Science

versión impresa ISSN 0864-0408versión On-line ISSN 2079-3480

Cuban J. Agric. Sci. vol.53 no.3 Mayabeque jul.-set. 2019  Epub 01-Sep-2019

 

PASTURE SCIENCE

Yield of Mulato II grass inoculated with Bradyrhizobium sp. and Glomus cubense under agricultural drought conditions

C. J. Bécquer Granados1  * 

R. Reyes Rosseaux2 

D. Fernández Milanés2 

P. J. González Cañizares3 

F. Medinilla Nápoles4 

1Estación Experimental de Pastos y Forrajes de Sancti Spíritus, Apdo. 2255, ZP. 1, C. P. 62200, Sancti Spíritus, Cuba

2Instituto de Investigaciones de Pastos y Forrajes, Sede. Fontanar, Rancho Boyeros, La Habana, Cuba.

3Instituto Nacional de Ciencias Agrícolas, Carretera a Tapaste, km 3 ½, San José de las Lajas, Mayabeque, Cuba

4Centro Meteorológico Provincial de Sancti Spíritus, Sancti Spíritus, Cuba

ABSTRACT

A field experiment was carried out, under conditions of agricultural drought, to evaluate the effect of an isolate of Bradyrhizobium sp. and a strain of Glomus cubense in Brachiaria hibrido cv. CIAT 36087 (Mulato II), in order to select the best treatment for its subsequent evaluation under different edaphoclimatic conditions and cultivars of this species. In 1 ha, cultivated with Mulato II for three years, five divisions of 0.20 ha each were carried out. After deep grazing, treatments were applied. The inoculation was carried out according to established methodologies. The experimental design was of control plots, with 5 treatments and 5 replications. ANOVA analysis was performed. Differences among means were determined by Duncan test. Treatments consisted of simple applications of inoculants, the combination of these, absolute control and fertilized control. In the 1st cut, yield of aerial biomass with Ho5 + INCAM4 (6.14 t ha-1) was superior to the rest of inoculated treatments and to control, although lower than the fertilized control. In the 2nd cut, this combination was superior to the rest of treatments (6.56 t ha-1), except for the fertilized control, which was repeated in the 3rd cut, with a yield of 6.35 t ha-1. CP content was higher in Ho5 + INCAM4 (9.01%), with respect to the inoculated treatments and to the control, although inferior to the fertilized control. In nutrient extraction, with this combination, as well as with the simple application of INCAM4, statistical superiority was observed. In general, it can be concluded that the combination of Bradyrhizobium sp. and G. cubense, in the three cuts, showed superior results in the aerial biomass yield and in the CP content of plants, although the inoculation only with G. cubense, and in combination with Bradyrhizobium sp., led to a greater P extraction.

Key words: inoculants; Brachiaria hibrido; aerial biomass; extraction

INTRODUCTION

Feeding of cattle in Cuba is mainly based on the use of grasses. Forage species of the Poaceae family (grasses) are the most important group of plants for humans (Aguado et al. 2004), due to availability as food, which is a factor that influences significantly on production systems (Rubio et al. 2013). However, low fertility of the soils dedicated to livestock and the impossibility of having sufficient quantities of fertilizers to guarantee an adequate nutrition of these crops, due to their high prices in the international market, limit the yields and quality of the biomass that cattle consumes and, consequently, reduce their productivity. As a palliative to this situation, new drought-resistant species with greater productive potential and better quality have been introduced. Brachiaria genus, in recent times, has been enriched with hybrid species of high forage importance, such as Brachiaria hibrido cv. CIAT 36087 (Mulato II), due to its high adaptability and persistence in acid soils, of medium and low fertility, for its efficient growth and durability, even under drought conditions, its high production of good quality biomass and the high degree of acceptance by animals (Argel et al. 2007).

On the other hand, the use of microbial biofertilizers based on mycorrhizae and growth-promoting bacteria allow the pastures to persist, adapt and increase productivity through the beneficial properties of microorganisms. This facilitates greater and more efficient nutrient uptake, such as the greater acquisition of phosphorus and solubilization of mineral elements or mineralization of organic compounds (Druille et al. 2015 and Ramos et al. 2015). There are reports on the superior effect of biofertilizers in the production of aerial biomass and increase of the foliar chemical-bromatological composition in Urochloa ruziziensis plants (Silva et al. 2015). Also Bécquer et al. (2017) reported that the combination of Bradyrhizobium sp. and Trichoderma harzianum caused the increase in aerial biomass and dry weight of the spike of Cenchrus ciliaris, in the presence of stress due to drought.

The objective of the experiment was to evaluate the effect of a native isolate of Bradyrhizobium sp. and a commercial strain of Glomus cubense in Mulato II grass, under agricultural drought conditions, in order to select the best treatment for its subsequent evaluation in different edaphoclimatic conditions and cultivars of this species.

MATERIALS AND METHODS

Experimental period and location. The experiment was carried out in the period from April to September 2018, in areas of the Niña Bonita genetic livestock enterprise, located at 23.00° N and 88.55° W.

Basic characteristics of experimental soil (table 1). The soil was identified as lixiviated red ferralitic (Hernández et al. 2015), which presented a slightly acidic pH, low contents of assimilable phosphorus (P2O5) and medium contents of interchangeable potassium (K +) and organic matter. Ca2+, Mg2+ and base exchange capacity (BEC) showed characteristic values of this type of soil (Paneque and Calaña 2001). Prior to the assembly of the experiment, 10 soil samples were taken in the area by the zigzag method at a depth of 0-20 cm for their chemical characterization.

Soil analyzes were carried out using methods established in the soil, organic fertilizers and vegetable tissue laboratory of the department of biofertilizers and plant nutrition of the National Institute of Agricultural Sciences (INCA, initials in Spanish).

Table 1 Chemical characteristics of soil (0-20 cm deep) 

Type of soil

  • pH H2O

OM (%) P2O5 (mg 100 g-1) Ca2+ Mg2+ Na+ K+ BEC
(cmol kg-1)
Lixiviated red Ferralitic 6.3 3.25 2.8 9.7 2.2 0.15 0.34 12.40
SD ± 0.2 0.11 0.3 0.8 0.3 0.02 0.07 0.42

Climate data during the experimental period.Figure 1 shows that, in the period from January to April (month of the beginning of the experiment), rainfall was scarce, with monthly accumulations lower than the historical one (73.6% in April), so the application of inoculants was carried out in a stressful environment for plants and microorganisms.

However, precipitations started to increased in May, with a monthly accumulation superior to the historical one (183.9%), which fell to 37% in July, with 115.2% in August and dropped to 38.6% in September, month of the third cut.

Figure 1 Monthly and historical precipitations, and % regarding historical precipitations January-September/2018 

Regarding the intensity of the agricultural drought during the time the experiment lasted (table 2), it is considered that it varied from very severe (April, 1st and 2nd tens of May, 2nd and 3rd tens of July, 1st and 2nd tens of August), until light (3rd tens of June, and 1st tens of September) and very light (2nd and 3rd tens of September). This data indicates that the experiment, in general, took place under agricultural drought conditions, which was accentuated in the months of April, May and August (CMP 2018).

Table 2 Category of the agricultural drought in the EPG Niña Bonita, April- September/2018 (CMP 2018

Month/tens Agricultural drought intensity category Key
April 01 5 Very severe drought
April 02 5 Very severe drought
April 03 5 Very severe drought
May 01 5 Very severe drought
May 02 5 Very severe drought
May 03 4 Severe drought
June 01 4 Severe drought
June 02 4 Severe drought
June 03 2 Light drought
July 01 4 Severe drought
July 02 4 Severe drought
July 03 5 Very severe drought
August 01 5 Very severe drought
August 02 5 Very severe drought
August 03 3 Moderate drought
September 01 2 Light drought
September 02 1 Very light drought
September 03 1 Very light drought

Plant material. Brachiaria hibrido cv. CIAT 36087 (Mulato II), as established grass

Microorganisms, inocula preparation and grass inoculation. HMA strain. EcoMic® product was used, with a formulation based on the HMA Glomus cubense strain, which was selected for its high efficiency index, according to previous tests conducted under similar conditions to those of this experiment (González et al. 2007).

For its application, a certified solid inoculant containing 25 spores g-1 of substrate was used, produced in the department of biofertilizers and plant nutrition of INCA, and applied a broadcast sowing, at a rate of 50 kg ha-1.

Rhizobium isolate. Ho5 isolate was used, which belong to the genus Bradyrhizobium sp. (Bécquer et al. 2016), microsimbion of Centrosema virginianum, legume from an arid livestock ecosystem of Holguín, Cuba. It was selected based on previous results obtained in different crops, inoculated with that isolate (Bécquer et al. 2018).

The isolate grew in a yeast-mannitol solid medium (Vincent 1970) and was resuspended in a yeast-mannitol liquid medium until a viable cell concentration of 107-108 CFU / mL was achieved. The inoculation was done with a sprayer backpack with the spout directed to the furrow. To do this, first, the base inoculum was mixed with common water, at a ratio of 1:10 up to reaching 12 L of the final suspension (each backpack), and it was applied at a rate of 30 L/ha. This activity was carried out in the cool hours of the morning.

The microbial inoculants were only applied at the beginning of the experiment. Three cuts were made, at intervals of approximately 60 days each, the first cut was made on June 1, the second, on August 1, and the third, on September 30, 2018. Broadcasting N fertilizer was also applied, at the beginning of the experiment and after the first cut (50 kgN ha-1 cut-1).

Agrotechnical procedures. One hectare dedicated to grazing was used, which had been cultivated for three years with Mulato II and it was divided into five plots of 0.20 ha (100m x 20m) each. In April 2018, coinciding with the beginning of the first spring rains, an intensive grazing was carried out and then the treatments were applied.

Experimental design and statistical analysis. The experimental design consisted on control plots (Lerch 1976), of 2000m2 each, with 5 treatments and 10 replications, which consisted of 10 samples taken at random in each plot, with frames of 1 m2. An ANOVA analysis was performed. The differences among means were determined by Duncan (1955) comparison test. The statistical program StatGraphics (Statistical Graphics Corporation, version 2.0.0.0.) was used.

Treatments

  1. Absolute control

  2. Fertilized control (50 kgN ha-1 cut-1)

  3. INCAM4 strain (Glomus cubense)

  4. Isolated Ho5 (Bradyrhizobium sp.)

  5. INCAM4 + Ho5 (moment of the cut)

Evaluated variables. Yield of aerial biomass (DM, t ha-1) was evaluated, as well as crude protein content (CP, %) and concentration of P (g kg-1) and K (g kg-1) in the biomass aerial (averages of three cuts). Yield of dry mass (DM) of the aerial biomass (kg ha-1) was estimated from the percentage of DM and the yield of green mass (GM) of each plot. It was cut at a height of 10 cm from the soil and the GM of the aerial part of the plants, that were located in the calculation area of ​​each plot, was weighed with a balance of 0.25 kg of precision and a sample of 200 g was taken, which was taken to an air circulation oven at 70ºC, until reaching a constant mass to determine DM percentage, according to the formula:

DM (%) = [DM of the sample (g)/GM of the sample (g)] x 100

DM yield was estimated from the formula:

DM (kg plot1) = [GM (kg plot1) x DM (%) / 100] (This data was subsequently extrapolated to t ha1)

The extraction of N, P or K (g kg-1), was calculated as follows:

[Dry mass (DM) aerial part (g kg1) x% of the element in the DM aerial part] / 100

Crude protein content (%) was calculated by the formula (Kalra 1998):

% Protein =% Nitrogen x 6.25

The analyses of soil and of nutrient concentrations in the biomass were carried out according to the analytical techniques established in the soil and plant tissue laboratory of Biofertilizers and Plant Nutrition Department of the INCA (Paneque et al. 2011).

Likewise, the relative efficiency index (IEI, %) was calculated based on DM yield of aerial biomass, CP content and P concentration, according to the formula (Santillana et al. 2012):

IEI: [Inoculated TreatmentAbsolute Control / Absolute Control] x100

In addition, the relative agronomic efficiency (EAR, %) was calculated, based on the DM yield of aerial biomass, CP content and P concentration, according to the formula (Matheus et al. 2007):

EAR: [Inoculated TreatmentAbsolute Control / Fertilized controlAbsolute control] x100

RESULTS AND DISCUSSION

Aerial biomass yield, inoculation efficiency index (%) and relative agronomic efficiency (%), 1st, 2nd and 3rd cut. Table 3 shows that aerial biomass yield, in the inoculated treatments, in the 1st cut, was higher in the Ho5 + INCAM4 treatment (6.14 t ha-1) compared to the rest of inoculated treatments and absolute control, although inferior to the fertilized control (6.82 t ha-1). On the other hand, in the 2nd cut, the results were similar, and it is observed that aerial biomass yield in the inoculated treatments was higher with the combination of both microorganisms (6.56 t ha-1), which exceeded also to absolute control (4.60 t ha-1), although lower than the fertilized control (7.39 t ha-1). The efficiency index of the inoculation confirmed the previous results of the statistical analysis, where the indexes reached by the treatment Ho5 + INCAM4 in the 1st cut (53.12%), as well as in the 2nd cut (48.61%), were uperior to the rest. On the other hand, the highest relative agronomic efficiency was shown by this treatment, with 75.80% (1st cut) and 70.25% (2nd cut). Although the cuts were made 60 days apart, there is a similarity in efficiency values in the inoculated treatments, when comparing the results of one cut, in relation to the other, which supports the possibility of a stability of the microbial population in the soil, until the 2nd cut, without the reinoculation of treatments.

Table 3 Aerial biomass yield (t ha-1 DM) of grass, index of inoculation efficiency (%) and relative agronomic efficiency (%), 1st, 2nd and 3rd cut 

Treatments t ha-1 DM IEI, % EAR, % t ha-1 DM IEI, % EAR, % t ha-1 DM IEI, % EAR (%)
1st cut 2nd cut 3rd cut
Absolute control 4.01 e 4.60 e 4.35 e
Fertilized control 6.82 a 7.39 a 7.22 a
INCAM4 5.33 c 32.92 46.98 5.88 c 27.83 45.88 5.63 c 29.43 44.60
Ho5 4.65 d 15.96 22.78 5.19 d 12.83 21.15 4.78 d 9.89 14.98
Ho5 + INCAM4 6.14 b 53.12 75.80 6.56 b 42.61 70.25 6.35 b 45.98 69.69
SE ± 0.21** 0.23** 0.22**

abcdeDifferent letters indicate significant differences p<0.001

The obtained values of dry matter yield were superior in all the treatments to those reported by Guiot (2005) for this cultivar, in different soils, with nitrogen and phosphoric chemical fertilization.

It was also observed that the single application of each of these microorganisms, although with inferior results to those of their combination, was superior to absolute control yield, where the treatment inoculated with the INCAM4 strain was highlighted, with 5.33 t -1 (1st cut) and 5.88 t ha-1 (2nd cut). This type of result agrees with reports of González et al. (2011), when inoculating Mulato II grass with Glomus hoi-like in a lixiviated red ferralitic soil. Likewise, it coincides with Bécquer et al. (2017), who made a simple inoculation with Ho5 isolate, in the Cenchrus ciliaris L. (Buffel cv. Formidable), under drought stress, which is an environmental condition that coincides with those of the present experiment.

However, there are interrelationships among microorganisms in ecosystems, such as synergistic, antagonistic, biochemical and physical competition. Multifunctionality of these microorganisms in agricultural systems is expressed in accordance with biotic factors, as well as edaphoclimatic factors (Salinas and Soriano 2014). Everything seems to indicate that the combination of the two microorganisms used in the present experiment led to a synergy by positive interaction, which should have influenced on its superiority in the studied variable. Bécquer et al. (2017) obtained higher values ​​of the aerial biomass of Cenchrus ciliaris L., in comparison with the absolute control, when inoculating the Ho5 isolate (Bradyrhizobium sp.), in combination with Funneliformis mosseae, under water stress conditions. Also, in similar stressful environmental conditions, Bécquer et al. (2018), obtained superior results in Cynodon dactylon, when inoculated with the Ho5 isolate, combined with a strain of Trichoderma harzianum.

Table 2 also shows that the yield of aerial biomass, in the inoculated treatments, in the 3rd cut, as in the previous cuts, was higher in the Ho5 + INCAM4 treatment (6.35 t ha-1) compared with the rest of the inoculated treatments and absolute control (4.35 t ha-1), although inferior to the fertilized control (7.22 t ha-1). On the other hand, the index of efficiency of inoculation highlights the indexes reached by Ho5 + INCAM4 treatment (45.98%), superior to the rest. The highest relative agronomic efficiency was shown for this treatment, with 69.69%.

The beneficial effect of the mycorrhizal inoculation on the increase of aerial biomass yield could be related to the influence of the strain introduced in the improvement of the nutritional status of plants. It is known that the addition of efficient strains of HMA can increase the effectiveness of the absorption of soil nutrients, and this is translated into an increase in grass biomass production (González 2014). On the other hand, the synthesis of auxins by rhizobia, especially indoleacetic acid, promotes the radical development and improves water and soil nutrient absorption and, therefore, plant development (Caballero-Mellado 2006).

It is important to observe in figures 2 and 3, that the values obtained in the 3rd cut, in all treatments, are lower than in the 1st cut (inoculation efficiency index), and even lower than those of the 2nd cut (relative agronomic efficiency). However, the treatment that showed the greatest decrease was the simple application of Ho5. This result could be taken into consideration to recommend a second inoculation, from the 3rd cut, although future studies, in different environments and cultivars of Brachiaria hibrido, are needed to make a better explained recommendation.

Figure 2 Inoculation efficiency index of inoculated treatments in the three cuts 

Figure 3 Relative agronomic efficiency of inoculated treatments in the three cuts 

CP content in the aerial biomass (average of three cuts), inoculation efficiency index (IEI, %) and relative agronomic efficiency (EAR, %). In table 4, CP content demonstrated the superiority of the Ho5 + INCAM4 treatment (9.01%), with respect to the rest of inoculated treatments, and to the control (7.29%), although it was lower than the fertilized control (9.93%). The inoculation efficiency index and the relative agronomic efficiency showed higher values in this combination (23.59% and 65.15%, respectively). As in the previous variable, the application of the two microorganisms, separately, resulted in lower values than those of the fertilized control and those of the combined treatment, but, in turn, they showed higher values than those of absolute control.

Table 4 CP content in the aerial biomass (average of three cuts), inoculation efficiency index (IEI, %) and relative agronomic efficiency (EAR, %) 

Treatments CP (%) IEI (%) EAR (%)
Absolute control 7.29 e
Fertilized control 9.93 a
INCAM4 8.35 c 14,54 40,15
Ho5 7.82 d 7,27 20,08
Ho5 + INCAM4 9.01 b 23,59 65,15
SE ± 0.17**

abcdeDifferente letters indicate significant differences p<0.001

These results suggest that the plants presented a higher nitrogen supply, due to the increase in the use of soil nutrients, and humidity, from the increase of the mycorrhizal structures of the grass, and the effect of the rhizobium isolate in its root development. The HMAs improve the extraction of nutrients from the soil and their efficiency (Bitterlich and Franken 2016), which brings higher exports. On the other hand, growth promotion of non-legume plants, through rhizobia inoculation, may be related to the increase of root system, proliferation of root hairs and absorption of water and nutrients from the soil and, therefore, with the development of infected plants and a more efficient use of nitrogen and other nutrients (Biswas et al. 2000).

P and K concentration in the aerial biomass (average of three cuts), inoculation efficiency index (IEI, %) and relative agronomic efficiency (EAR, %). Table 5 shows, that, P concentration, in the treatment inoculated with the INCAM4 strain (2.68 g kg-1) and in the combined treatment Ho5 + INCAM4 (2.59 g kg-1), had superior values regarding the rest of the treatments, which include absolute control (2.11 g kg-1) and the fertilized control (2.24 g kg-1). Also, these results are supported by the inoculation efficiency index, with values of 27.01 % in INCAM4, and 22.75 %, in the combination of the two microorganisms, and with similar results in the relative agronomic efficiency, which showed very high values for the treatment inoculated with INCAM4 (448.46 %), and for the combined treatment (369.23 %). On the other hand, the treatment inoculated with the Ho5 isolate, showed no differences in the concentration of this element (2.25 g kg-1), with respect to absolute control and fertilized control, which indicates a null or poor activity phosphate solubilizer of the isolate.

Table 5 P and K concentration in the aerial biomass (average of three cuts), inoculation efficiency index (IEI, %) and relative agronomic efficiency (EAR, %). 

Treatments P (g kg-1) IEI (%) EAR (%) K (g kg-1) IEI (%) EAR (%)
Absolute control 2.11 b 18.3
Fertilized control 2.24 b 17.8
INCAM4 2.68 a 27,01 448,46 18.5 1,1 -
Ho5 2.25 b 6,64 107,69 17.2 - -
Ho5 + INCAM4 2.59 a 22,75 369,23 18.6 1,64 -
SE 0.1** 0.3

abcdeDifferente letters indicate significant differences p<0.01

In this regard, Rivera and Fernández (2003), in experiments carried out on red ferralitic and brown with carbonates soils, found that mycorrhizal symbiosis directly increased the absorption of the three primary macroelements, through the increase in the concentration of these nutrients. The fact of conducting the experiment on an area dedicated to cattle grazing, should have influenced on the availability of organic matter, from animal depositions, which is rich in macronutrients that are not always easily assimilated by plants. In addition, it is known that HMA not only improve soil nutrient extraction, but also reduce their losses due to washing (Bender et al. 2015). Likewise, Cavagnaro et al. (2015) consider that the exploration of a greater volume of soil, the extensive networks of external mycelia that are formed, as well as the effective uptake of nutrients and immobilization of various ions in plants and fungal tissues, are some of the key mechanisms to reduce the washing of P and N through the HMA.

However, in the extraction of K, there were no differences among treatments, with insignificant results, or null in both indexes, which is in contradiction with some of the previously mentioned benefits. It is possible that, for K being the most soluble of the minerals, which is transferred into the soil through reflux and diffusion (Pradhan et al., 2017), its presence in the plant was due to greater availability at a given moment, when soil humidity allowed its easy absorption by the root system, regardless of the treatment in question. On the other hand, by not increasing the K content in the plant biomass with the application of biofertilizers, this could suggest a low participation of the applied microorganisms in the absorption of this macroelement, because its content in the soil, apparently, was enough to satisfy the requirements of the grass.

It should be taken into account that the application of biofertilizers to the crop was carried out in the month of lowest cumulative rainfall in the experimental period (figure 1), as well as one of the periods that showed a very severe agricultural drought intensity (table 2), without negatively affecting their beneficial effect on the plants, both on the yield of aerial biomass and on their bromatological composition, which may mean a tolerance of the microorganisms that composed this bioproduct, towards humidity scarcity in the soil, which is a characteristic that makes them promising for cattle rearing in Cuba.

It is concluded that, although the single application of Glomus cubense had a superior effect on the yield of the aerial biomass than that of Bradyrhizobium sp., the combination of these two microorganisms, in the three cuts, showed superior results in the yield of the aerial biomass and in the CP content of plants. Decrease in inoculation efficiency indexes was observed from the 1st cut, especially for the treatment inoculated only with Ho5. On the other hand, the inoculation with G. cubense, alone, and in combination with Bradyrhizobium sp. led to a greater extraction of P by the plants, which did not happen in the extraction of K.

It is recommended to evaluate the effect of the combination of Ho5 isolate (Bradyrhizobium sp.) with INCAM4 strain (Glomus cubense), under different edaphoclimatic conditions and cultivars of Brachiaria hibrido.

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Received: December 03, 2018; Accepted: July 01, 2019

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