Treatments and crop establishment

A completely randomized design was used with eight treatments and three replicates to evaluate the arbuscular mycorrhizal fungus (AMF) strains and Spiruvin leaf bioproducts. Bean seeds (n = 10) inoculated with rhizobia (Azofert⁽, Mayabeque, Cuba) and AMF per pot were sown for a total of 720 plants in 72 pots. The AMF strains (Rhizophagus irregularis, Glomus cubense and Funneliformis mosseae) and Spiruvins were studied with a dose of 2 L ha^-1 composed of seven amino acids, 11 types of vitamins and oligopeptides besides a control group with the application of Azofert as indicated by the technical norms of bean cultivation in Cuba.

The treatments were (T1) Azofert without AMF (control group); (T2) Azofert without AMF + 2 L ha^-1 of Spiruvins; (T3) Azofert + Rhizophagus irregularis; (T4) Azofert + Glomus cubense; (T5) Azofert + Funneliformis mosseae; (T6) Azofert + Rhizophagus irregularis + 2 L ha^-1 of Spiruvins; (T7) Azofert + Glomus cubense + 2 L ha^-1 of Spiruvins; (T8) Azofert + Funneliformis mosseae + 2 L ha^-1 of Spiruvins.

The AMF strains were obtained from the Arbuscular Mycorrhizal Fungus collection of the Plant Biofertilizer and Nutrition Department of the Instituto Nacional de Ciencias Agrícolas (INCA), based on the certified EcoMic⁽, (Mayabeque, CU) product, with 40 spores/g of inoculant and 50% of non-toxic and pathogen-free radicle colonization. Additionally, the inoculant Azofert (Mayabeque, CU) was used with a concentration of 1 × 10⁷ - 1 × 10⁸ UFC mL^‑1, containing native rhizobium species that induce high concentrations of nodulation factors in bacteria, favoring BNF. Azofert (Mayabeque, CU) belongs to the Collection of Bioproducts of the Plant Biochemistry and Physiology Department at INCA.

The biofertilizer inoculation (rhizobia and AMF) and the combined use of the AMF strains with the leaf biostimulant (Spiruvin) are described as follows. The biofertilizers were applied previous to sowing following the cover crop seeding method (^{Fernández et al., 2000}). A dose of 0.045 kg ha^-1 of EcoMic⁽, (Mayabeque, CU), equivalent to 10% of seed weight was used. Firstly, the seeds were soaked with the bacterial (Azofert, Mayabeque, CU) inoculant for three min; after that, the seeds were covered completely with the AMF strains, dried at the shade for five min, and subsequently proceeded to sowing. As mentioned previously, the Spiruvin biostimulant originates from the humid biomass of Spirulins and vinasse, which are natural products; through their bioactive compounds, they stimulate the assimilation of the macro-elements provided to the soil (by the radicle), as well as to the plant natural capacity to produce its own hormones, enzymes, and other products based on amino acids. Then, at 15 and 30 days after sowing (DAS) and the flowering stage, a dose of 2 L ha^-1 was applied to the leaves with a spray model Batlle 730061 UNID (Barcelona, ES) of 400 mL, which guaranteed application evenness on all the plants.

Variables assessed

A total of 15 plants per treatment were selected, and plant growth variables were measured as follows: plant height and stem diameter (cm) at 15, 30 and 45 DAS. The yield components were number of pods and number of grains per pod, weight of 100 grains (g) and yield (Mg ha^-1), which were determined at the moment of harvesting. Additionally, the mycorrhizal function variables, colonization and visual density expressed in %, were assessed. From the roots sampled, 200 mg were weighed and stained following the method of ^{Phillips and Hayman (1970)}. The mycorrhizal colonization percentage was quantified with the intercept method (^{Giovannetti and Mosse, 1980}). Visual density was determined following the methodology described by ^{Trouvelot et al. (1986)}.

Statistical analysis

The data were subjected to an analysis of variance (ANOVA). When significant differences occurred among treatments, Tukey’s (P ≤ 0.05) range test was performed with Statgraphics Centurión XVI statistical program.

Plant height

Plant height increased with the inoculation of the AMF strains, which were applied by covering the seeds; likewise, height increased with the combined AMF and Spiruvin treatments at 30 and 45 das (P ≤ 0.05), highlighting the combination of the Rhizophagus irregularis (Table 2). This response could have been attributed to the effect of the bioproducts, which increased the metabolic functions related with plant growth and development. This positive effect could have been related to AMF and the biostimulants with bioactive compounds that promote mechanisms of nutrient absorption and translocation in plants (^{Abdel-Fattah et al., 2016}; ^{Martínez-Sánchez et al., 2017}).

With respect to the previous information, ^{Colás-Sánchez et al. (2018)} reported a similar effect to that in this study since the bean plants with arbuscular mycorrhizal fungus and rhizobium inoculation had a height increase over 50% at 21 das compared with the control group. Likewise, ^{Pérez-Peralta et al. (2019)} indicated that the previous rhizobium inoculation in bean seeds increased the plant aerial biomass. On the other hand, ^{Rivera et al. (2015)} obtained a significant increase in bean plant growth with AMF + Azofert inoculation.

Stem diameter

In stem diameter (Table 3), differences (P ≤ 0.05) were observed in the combined treatments (AMF with Spiruvins). A greater efficiency was observed with the Rhizophagus irregularis strain at 15, 30 and 45 DAS. The positive results in the combined treatments could be inferred to the effect of the biodproducts on the plant physiological processes and to a greater nutrient absorption by plant roots (^{Tamayo-Aguilar et al., 2019}).

The results of this study agree with those of ^{Aguirre-Medina et al. (2019)}, who reported significant differences in stem diameter of Tabebuia donnell-smithii by combining AMF strains and cattle manure in greenhouse conditions. Nevertheless, they did not find statistical differences by the effect of mycorrhizal fungi without cattle manure in the first plant stages. On the other hand, ^{Mujica et al. (2017)} reported an increase in stem diameter in the crop of Arachis hypogaea with the combined use of biofertilizers compared with the control group when they assessed the effect of AMF and plant growth promoter bacteria.

Yield components

Differences (P ≤ 0.05) were observed in the yield components, number of pods and number of grains per pod and weight of 1000 grains with the combination of the bioproducts (Table 4). These results agree with those reported by ^{Calero et al. (2019)} who also found differences in the yield components of bean crop with the combined application of efficient microorganisms and Rhizobium.

In addition to the previous information, ^{Tamayo-Aguilar et al. (2019)} reported that the combined used of bioproducts in Vigna unguiculata (L.) cultivation increased with respect to the treatments applied individually. ^{Colás-Sánchez et al. (2018)} found that the yield components increased compared with the control group when they evaluated the effect of biofertilization with the previous inoculation of arbuscular mycorrhizal fungus and rhizobium strains.

Bean yield

Differences (P ≤ 0.05) were observed in yield (Table 5). The combinations of the bioproducts AMF + Spiruvins increased bean yield. The greatest yield was 2.11 Mg ha^-1, obtained with R. irregularis combined with Spiruvin leaf bioproduct. This response may have been attributed to the specificity of this strain with the type of soil used, which favored the efficiency of the mycorrhizal inoculation. It is worth to highlight that the yield previously mentioned, obtained with the combination Azofert + R. irregularis + Spiruvinas, represented 37.9%, which was more than the national yield. These results showed that R. irregularis was the ideal AMF strain to increase growth and yield in the conditions of this study.

With the results obtained, a certain functional compatibility may be inferred among the plant, substrate, biostimulant, and AMF, which favored growth and yield. In this sense, ^{Tamayo and Bernal (2018)} reported bean yield increase with the combined inoculations of mycorrhizal fungi and rhizobia; likewise, they concluded that the combined actions of the bioproducts had a synergic effect in plant growth. The results of this study agree with that indicated by ^{Llanes et al. (2019)} with respect to the use of bioproducts and their benefits in Cuban agriculture since the majority of the producers use biostimulants and biofertilizers jointly with cattle manure for crop nutrition.

Fungal function

The mycorrhizal function in bean cultivation was observed in mycorrhizal colonization and visual density (Table 6). The mycorrhizal activity increased in the combined treatments with AMF and Spiruvin with differences (P ≤ 0.05) among them. The R. irregularis strain was again more efficient than the other commercial strains. These results might have been due to the plant response to assimilate efficiently the combined bioproducts related to the mycorrhizal symbiotic process (^{Tamayo-Aguilar et al., 2019}). It is worth to point out that the presence of visual density and colonization found in the treatments without AMF could be the expression of some AMF residents in the soil used to fill the pots; however, its mycorrhizal effect was less than with the commercial strains studied.

According to ^{Williams et al. (2017)} the establishment of mycorrhizal symbiosis depended on the interaction of biotic and abiotic factors, which should be considered in the agronomic cultivation that imply the combined use of biofertilizers and biostimulants with the purpose of strengthening the benefits of their interaction with plants. On the other hand, ^{Vital-Vilchis et al. (2018)} found that in mycorrhizal symbiosis in sunflower cultivation in pot conditions at 20 DAS, mycorrhizal colonization was not established and no plant growth effect was observed; however, starting from 30 DAS, plant growth was favored because of the symbiotic interaction effect between the fungi and plant.

In comparison with the plant treatment inoculated only with the AMF consortium in this study, ^{Chiquito-Contreras et al. (2018)} found significant differences (P ≤ 0.05) in the increase of the mycorrhizal colonization percentage and number of spores per gram of soil (35 and 66%, respectively) when they evaluated AMF consortium co-inoculated with the marine bacterium Stenotrophomonas rhizophila in basil plants.