Co-inoculation with Azospirillum brasilense promotes growth in forage legumes 1

Studies have shown the synergism of co-inoculation with symbiotic and non-symbiotic N2-fixing bacteria, efficiently contribute to plant development. However, for crops such as forage legumes, data on the use of co-inoculation are still incipient, requiring studies to identify the contribution of this technique for application in pasture areas, thus ensuring greater sustainability. The objective of this study was to evaluate the effects of co-inoculation of Azospirillum brasilense (Ab-V5) and native rhizobia isolated from two forage legumes, Crotalaria spectabilis and Lupinus albus . Two experi - ments were installed in pots with 8dm³. Both were conducted in a randomized block design (DBC) with four replications and an 8 x 2 factorial scheme with six bacterial strains and two control treatments without inoculation (with presence or absence of mineral nitrogen), co-inoculated or not with the Ab-V5 strain. Plants were cultivated until the time of flowering when shoot (SDM) and root dry matter (RDM), number (NN) and nodule dry matter (NDM), and relative symbiotic efficiency (RSE) were evaluated. Isolates 05-21 and 06-03, for C. spectabilis and L. albus , respectively, indicated higher potential to promote plant growth when co-inoculated with Ab-V5, showing potential to act as substitutes for nitrogenous chemical fertilizers, reducing costs and increasing the sustainability of production


INTRODUCTION
_______________________________________________ Submmited on August 13 th , 2021 and accepted on May 30 th , 2023. 1 This work was extracted from the first author's Master's Dissertation.This study was financed by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior -Brasil (CAPES). 2 Universidade Prof. Edson Antônio Velano (UNIFENAS), Programa de Pós-graduação em Ciência Animal, Alfenas, MG, Brazil.anabeatriz.terra@hotmail.com;adauton.rezende@unifenas.br;ligiane.florentino@unifenas.br *Corresponding author: anabeatriz.terra@hotmail.com The increase in the world population, associated with a higher demand for food, has raised concerns about the sustainability of the production chains.In Brazil, pastures are characterized by extensive grazing, and it is commonly responsible for soil degradation and productivity losses over the years.The use of chemical fertilizers is a direct technique to recover these areas (Carvalho et al., 2017).However, it is considered unsustainable from an economic and environmental point of view.
With increasingly demanding consumers, there is the necessity of new alternatives to reduce and optimize the use of nitrogen fertilizers (Aguirre et al., 2020).These alternatives can promote improvements in soil characteristics and increases production.In this context, the use of N2-fixing bacteria stands out, which can significantly contribute with nitrogen (N) to the plant metabolic demand and the enrichment of this element to the soil (Moreira & Siqueira, 2006).Recently, studies have shown the feasibility of using co-inoculation of N 2 -fixing bacterial strains (Hungria et al., 2015;Galindo et al., 2018;Wang et al., 2019).
Ana Beatriz Carvalho Terra et al.According to Hungria et al. (2015), co-inoculation can improve crop performance, respecting sustainability demands in economic, social, and environmental terms.
Inoculation with Azospirillum may represent a key component for degraded pasture recovery programs, providing a higher accumulation of nitrogen in the biomass, in addition to helping with carbon sequestration (Hungria et al., 2016).Aguirre et al. (2020) highlight the use of the associative bacterium Azospirillum brasilense, which, in addition to its ability to fix nitrogen, also produces phytohormones responsible for root growth, increasing the density, length, and volume of roots (Moreira et al., 2010).Considering that, in Brazil, there are about 175 million hectares of pasture (Lapig, 2017), the impact of any increase in the efficiency of N is considered significant, both in terms of production and recovery of these areas (Aguirre et al., 2020).
Co-inoculation with Azospirillum, by promoting root growth, consequently, increases the infection sites for rhizobia strains (Cassán et al., 2009), providing better productivity in legumes such as soybean (Glycine max) and common beans (Phaseolus vulgaris L.) (Hungria et al., 2013).Galindo et al. (2018) also found a positive effect of co-inoculation with A. brasilense in two soybean cultivars, positively influencing not only yield but also crop profitability.Most studies concerning co-inoculation are related to soybean crops, especially because they are of economic interested to agribusiness.
However, the plant growth promoting bacteria (PGPR) which increases the efficiency in one legume may noy necessarily show the same response with different species (Korir et al., 2017).Therefore, these unstable responses to co-inoculation emphasize the need of identifying appropriate combinations of rhizobia strain and PGPR.This research aimed to evaluate the effects of co-inoculation of Azospirillum brasilense (Ab-V5) and native rhizobia isolated from two forage legumes, Crotalaria spectabilis and Lupinus albus.

MATERIAL AND METHODS
The rhizobia strains used in the experiment were isolated by Terra (2018) from the legumes Crotalaria spectabilis and Lupinus albus, classified as efficient, intermediate, and inefficient in the previous authentication and cross inoculation experiments.Table 1 shows the origin of the strains, classification in terms of efficiency, and morphological characteristics in medium 79 (Fred & Waksman, 1928).
The rhizobia strains were cultivated in liquid medium 79 for three days until the log phase of growth, with approximately 10 8 cells mL -1 .The A. brasilense strain, used in the co-inoculation, was cultivated in FAM liquid medium (Magalhães & Döbereiner, 1984) for three days, enough time to reach the log growth phase.
Two different experiments were installed in a greenhouse, the first for C. spectabilis isolates and the second for L. albus (Table 1).The experiments were in a randomized block design (DBC), with four replications, in a factorial scheme (8x2), with six treatments with the rhizobia mentioned in table 1, and two control treatments, one with mineral nitrogen (70 mg.L -1 N-NH 4 NO 3 ) and another without nitrogen and inoculation, co-inoculated or not with the Ab-V5 strain.
Liming was done with dolomitic limestone according to the method of raising the base saturation to 60%.The soil was moistened and incubated in plastic bags to increase the limestone reaction speed.After 30 days, macronutrients (phosphorus and potassium) were added following the recommendation by Novais et al. (1991).
Ten days after fertilization, four seeds were sown per pot, and after germination, only two plants were left per pot.The inoculated treatments applied one mL of bacterial suspension per seed, and the co-inoculated one, there was also one mL of A. brasilense bacterial suspension.The treatment with mineral nitrogen had two applications, 10 and 25 days after plant emergence, of 35 mg.L -1 N-NH 4 NO 3 , totaling 70 mg.L -1 N-NH 4 NO 3 during cultivation.
After the flowering period, the following parameters were evaluated: shoot dry matter (SDM) and root dry matter (RDM), number (NN) and nodule dry matter (NDM), and relative symbiotic efficiency (RSE).
An adaptation of the formula by Bergersen et al. (1971) was used to calculate the relative symbiotic efficiency (RSE%).In this research, the SDM value of the treatment containing mineral N and inoculated with A. brasilense (Ab-V5) was adopted, divided by the others, and multiplied by 100, as indicated below.

RESULTS AND DISCUSSION
For both legume species the co-inoculation had positive effect (Tables 2 and 3).For C. spectabilis, the 05-21 strain (Table 2) stands out, which when co-inoculated, presented superior results in all parameters, including when compared to the control treatment with mineral N. Regarding SDM and RDM there was an increase of 16% and 36%, respectively, when the strain 05-21 was co-inoculated with A. brasilense in comparison with the N control treatment.
This strain, associated with A. brasilense, showed a symbiotic efficiency (RSE) of 116,41%.Without co-inoculation the same strain had a RSE of 75,71%, indicating a positive response to co-inoculation and showing potential for replacement of mineral N, attending the nutritional needs of the plant, and promoting its growth.
L. albus also had a positive interaction, with higher values in the presence of co-inoculation (Table 3).The strain 06-03 had better results, being inferior only to the treatment with mineral N, in the parameters of RDM and SDM.Regarding SDM and RDM the strain when co-inoculated with A. brasilense had results that represent 91,24% and 78,41% of the values found in N mineral treatment.
The strain still obtained an RSE of 92,39% when compared to the control treatment with mineral N, highlighting its potential to supply nitrogen to the plants as well as their development.Azospirillum brasilense is mainly due to the bacteria's capacity to produce growth hormone (Bárbaro et al., 2008).
According to Ferlini (2006), the co-inoculation of Bradyrhizobium japonicum and Azospirillum brasilense in soybean increases production.The same was observed in this study for L. albus and C. spectabilis.There were some treatments where co-inoculation resulted in higher values of MSPA and MSR, highlighting their potential not only in fixing nitrogen but also in stimulating plant growth and production.Reaction of pH in culture medium after the growth of the strain, evaluated by the color change of the indicator (N -Neutral; Ac -Acid; Al -Alkali).
Corroborating these results, Molla et al. (2001), in a laboratory experiment to evaluate the potential to improve root growth and nodulation in soybean co-inoculated with Azospirillum and Bradyrhizobium, observed that Azospirillum has the potential to stimulate root growth significantly.
These results are relevant, because root growth is an important parameter as it contributes to water and nutrient absorption by plants and enhances soil structure reducing compaction problems commonly found in degraded pasture areas.Also, the higher production of shoot dry matter contributes to biomass production and consequently improves nutrients availability in these regions.For NN and NDM (Table 2 and 3), in both legumes the co-inoculated treatments were superior to those in which the rhizobia acted in isolation.Cassán et al. (2009) support these results, reporting that the number of nodules and the percentage of nodulated plants was higher in soybean plants co-inoculated with B. japonicum and A. brasilense, attributing these results to the excretion of metabolic products by A. brasilense.This was also observed by Molla et al. (2001), where Azospirillum improved nodule initiation and development in soybean plants by co-inoculation with Bradyrhizobium.Thus, there is an increase in nodulation and a higher root growth in response to the positive interaction between bacteria (Ferlini, 2006).

CONCLUSION
For the co-inoculation analysis, treatments 06-03 and 05-21, for L. albus and C. spectabilis, respectively, presented higher values of SDM, RDM, and RSE.Thus, indicating a better potential for nitrogen supply to the plant, as well as promoting plant growth when co-inoculated with Azospirillum brasilense.
Plant and root growth, in association with improvements in the physicochemical and biological characteristics of the soil, as a consequence of co-inoculation with plant growth-promoting bacteria, contribute to the recovery of degraded areas and constitute an alternative to more sustainable agricultural practices.
with Azospirillum brasilense promotes growth in forage legumes Data on SDM, RDM, NN, NDM, and RSE were submitted to analysis of variance using the statistical analysis program Sisvar, version 5.3.Treatment means were compared by the Scott-Knott test at 5% probability.

Cassán
et al. (2009) verified that A. brasilense produces root growth regulator compounds, such as indoleacetic acid (IAA), justifying better responses in co-inoculated treatments, both for L. albus and C. spectabilis.The results of SDM and RDM with co-inoculation also show that the beneficial effect of the association of isolated rhizobia with

Table 1 :
Identification, origin, efficiency data, and morphological characteristics in medium 79 of Crotalaria spectabilis and Lupinus albus isolates used in the co-inoculation experiment with Azospirillum brasilense

Table 2 :
Shoot dry matter (SDM) and root dry matter (RDM), number (NN) and nodule dry matter (NDM), and relative efficiency (RSE) values of Crotalaria spectabilis cultivated with different treatments co-inoculated or not with Azospirillum brasilense, and in the presence or absence of N mineral Means followed by the same letter, uppercase in the column and lowercase in the row, do not differ statistically by the Scott-Knott test (5%).

Table 3 :
Table3: Shoot dry matter (SDM) and root dry matter (RDM), number (NN) and nodule dry matter (NDM), and relative efficiency (RSE) values of Lupinus albus cultivated with different treatments co-inoculated or not with Azospirillum brasilense, and in the presence or absence of N mineral Means followed by the same letter, uppercase in the column and lowercase in the row, do not differ statistically by the Scott-Knott test (5%).In general, the results found in this study corroborate several other studies that have been carried out in Brazil, especially with soybeans.The practice of co-inoculation can be considered a key component of degraded pasture recovery programs, representing a new technique to improve crop productivity, and contributing to sustainable practices in agricultural systems.