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In recent years, the inclusion of beneficial microorganisms in the fertilization strategies of agricultural crops has been of great interest, as an economically and ecologically viable alternative to improve the biological properties of soils, the efficiency of the use of nutrients and, consequently, reduce the use of mineral fertilizers (Sahgal and Srivastava 2020).
Among these microorganisms is Gluconacetobacter diazotrophicus, an endophytic bacterium, with the ability to fix atmospheric nitrogen, stimulate plant growth and induce response mechanisms to biotic and abiotic stresses (El-Shouny et al. 2020). They have also showed their potential as biofertilizers and as protectors against pathogenic agents, the arbuscular mycorrhizal fungi (AMF), whose structures facilitate the absorption of nutrients and water, in addition to other benefits such as the solubilization of elements that are in the soil poorly available to plants (Chandrasekaran 2020).
However to the previous, few studies deal with the effect of co-inoculation with both microorganisms, in terms of efficiency in the use of nutrients and crop yield, as well as in the reduction of mineral fertilizers. Studies on biofertilization with G. diazotrophicus, alone or combined with AMF, are also limited.
Based on these premises, this study makes a preliminary evaluation of the effect of simple and combined application of G. diazotrophicus bacterium and the AMF species Funneliformis mosseae on biomass production, the frequency of mycorrhizal colonization and the concentrations of N in the biomass of the aerial part of Guinea grass (Megathyrsus maximus cv. Likoni).
The study was carried out under semi-controlled conditions, at the Experimental Station of Pastures and Forages of Cascajal, located at 22° 39' North latitude and 80° 24' West longitude, in the Santo Domingo municipality, Villa Clara province, Cuba. Five treatments were studied, made up of simple and combined inoculation with G. diazotrophicus and F. mosseae, the application of nitrogenous fertilizer, at a rate of 25 mg N kg-1 of soil, and a control without biofertilizers or nitrogenous fertilization, in a completely random design with five repetitions.
The treatments were placed in plastic pots with a capacity of 3.5 L, previously perforated at the bottom to facilitate drainage. They were filled with 3 kg of soil, from the Station, classified as petroferric ferruginous nodular gley (Hernández et al. 2015). To fill the pots, the soil was taken to a depth of 0-20 cm and sieved with a 5-mm mesh. The soil pH in H2O was strongly acidic (4.8), with low organic matter content (2.50 %), and very low assimilable P (5 mg kg-1) and exchangeable K (0.13 cmolc kg-1).
For the application of G. diazotophicus, the commercial inoculant ICIBIOP-GLU, produced by the Instituto Cubano de Investigaciones de los Derivados de la Caña de Azúcar (ICIDCA) was used, with a concentration of 109 CFU mL-1. A solution composed of a mixture of the biofertilizer and water, in a 1:1 vv ratio, was prepared, where the seeds were immersed for 15 min. Subsequently, they were dried in the shade and sowing was carried out.
For the application of the mycorrhizal biofertilizer, the INCAM-2 strain of Funneliformis mosseae species (C. Walker & A. Schüßler), from the INCA collection, was used. The inoculum contained 30 spores per gram of substrate, as well as abundant fragments of rootlets from the host plant used to produce the inoculum (Urochloa decumbens), and a rate of 5 g per pot was applied.
For sowing, 15 guinea grass seeds (Megathyrsus maximus cv. Likoni) with 50 % germination were placed in holes 0.5 cm deep that were opened on the soil surface of each pot, close to the center, and later they were covered with the soil. In the treatments where F. mosseae was applied, the inoculant was deposited under the seeds at the time of sowing.
Eight days after sowing, a thinning was carried out and five plants per pot were left. Fertilization was immediately applied in all treatments, at a rate of 37 and 25 mg of P2O5 and K2O kg-1 of soil, equivalent to 25 and 50 kg ha-1 of one and another nutrient, respectively. Triple superphosphate and potassium chloride were used as carriers. In the treatment corresponding to nitrogen fertilization, 25 mg of N kg-1 of soil was applied, equivalent to 50 kg of N ha-1, using urea as a carrier. Fertilizers were administered in a 2 cm deep circle around the set of plants, and covered with soil. The pots were watered every three days to maintain the soil at 80 % of field capacity.
The harvest was carried out 45 d after sowing. The green mass of the aerial and root parts of the plants was weighed, and both, after washing the roots to remove the remains of soil, were taken to an air circulation oven at 70 ºC for 72 h, to determine the yields of dry mass of the aerial part, roots and total dry mass (g pot-1).The N concentrations in aerial biomass were calculated according to the manual of analytical techniques of Instituto Nacional de Ciencias Agrícolas (INCA) (Paneque et al. 2010). Rootlets were sampled at harvest time to determine the frequency of mycorrhizal colonization (Giovanetti and Mosse 1980). For statistical processing, the normality of the data was checked using the Kolmogorov-Smirnov test and the homogeneity of variances using the Levene test. When fulfilling both requirements, the analysis of variance was carried out on the original data. The mean values of the treatments were compared according to Tukey's test (P < 0.05). In all cases, the statistical program SPSS 25 (SPSS 2017) was used.
G. diazotrophicus as well as F. mosseae increased the dry mass yields of the aerial, radical and total part of the grass. Among these treatments, the highest values corresponded to G. diazotrophicus. However, with the co-inoculation with both biofertilizers, values significantly higher than those reached for each one separately, and similar to those obtained with nitrogen fertilization, were recorded (table 1).
Table 1 Effect of treatments on guinea Likoni yields
| Treatments | Dry mass aerial part | Radical dry mass | Total dry mass |
|---|---|---|---|
| (g/pot) | |||
| Control | 4.18 d | 4.02 d | 8.21 d |
| 30 mg kg-1 N | 7.32 a | 7.13 a | 14.45 a |
| 6.52 b | 6.40 b | 12.92 b | |
| 5.22 c | 5.10 c | 10.33 c | |
|
|
7.45 a | 7.38 a | 14.83 a |
| ES | 0.16 | 0.20 | 0.34 |
| P | 0.003 | 0.001 | 0.002 |
Means with different letters in the same column significantly differ, according to Tukey's test (P < 0.05)
The frequency of mycorrhizal colonization significantly increased in the treatments inoculated with F. mosseae. However, the highest values were reached with the joint application of both biofertilizers (figure 1 a). The rest of treatments showed similar values to the control, without nitrogen or biofertilizers, which reflected the root occupation level of the AMF resident in the soil used in the experiment.
Regarding the concentrations of N in the biomass of the aerial part (figure 1 b), there was a similar effect to the biomass yields. That is: G. diazotrophicus as well as F. mosseae increased the values of this indicator. When comparing both treatments, the highest values corresponded to G. diazotrophicus. However, the joint application of biofertilizers produced significantly higher N concentrations than those obtained with the inoculation of one or the other separately, and similar to the treatment with nitrogenous fertilizer.
a)- Colonization frequency- Control b)- N(g/kgDM) -ControlTreatments with different letters significantly differ, according to Tukey's test (P < 0.05). ** P < 0.01.Figure 1 Effect of the treatments on the frequency of mycorrhizal colonization (1 a) and the concentrations of N in the biomass of the aerial part of Guinea Likoni (1 b).
The positive effect of the inoculation with G. diazotrophicus on the yields of different agricultural crops has been reported by several authors, and they attribute it to its ability to fix atmospheric nitrogen and produce growth-stimulating phytohormones (Padwar et al. 2020). The increase in the production of aerial and radical biomass, as well as the concentrations of N in the biomass, obtained in the biofertilization with this microorganism, seem to confirm such affirmations in the case of Guinea grass.
It has been shown that inoculation with effective strains of AMF increases mycorrhizal colonization levels, macronutrient concentrations and biomass yields in grasses, as a function of better use of soil nutrients and fertilizers (Silva et al. 2021). But the most outstanding has been that with the joint application of both biofertilizers, guinea grass reached yields and N concentrations in the biomass of the aerial part, similar to those obtained with the application of nitrogenous fertilizer, especially in a soil with very low fertility and low organic matter content, indicative of a low availability of this nutrient.
This suggests an important contribution of G. diazotrophicus to the biological fixation of N, enhanced by co-inoculation with F. mosseae, from the synergistic effect of both microorganisms on plants. In this sense, Khan et al. (2020) argued that this synergy is reflected in an increase in root growth and, in fact, in mycorrhizal colonization of the inoculated strain, whose activity in plants is stimulated by the biological fixation of N and the production of phytohormones by the bacterium; in addition, in a greater effectiveness of the bacteria from the photosynthates produced by the fungus.
It is concluded that joint inoculation with G. diazotrophicus and F. mosseae is effective in improving nitrogenous nutrition and aerial and root biomass yields of Megathyrsus maximus cv. Likoni. However, it is suggested to continue the studies on the joint application of both biofertilizers and their contribution to the reduction of the use of nitrogenous fertilizers in grasses.
