Original Article

Isolation, characterization and activity of Azospirillum brasilense strains associated to sugarcane

Dra.C. María Laura Tortora^{1  *} 

Lic. Lucia Vera¹ 

Lic. Noel Grellet-Naval² 

Dra.C. Karina Dantur¹ 

María de los Ángeles Núñez¹ 

Micaela Alderete¹ 

Dr.C. Eduardo Raúl Romero¹ 

¹Subprograma Agronomía, Sección Caña de Azúcar, Estación Experimental Agroindustrial Obispo Colombres (EEAOC), Av. William Cross Nº3150, Las Talitas, Tucumán, Argentina

²Ledesma S.A.A.I, Libertador General San Martin, Jujuy, Argentina.Dirección postal: Av. William Cross Nº 3150, Las Talitas, Tucumán, Argentina

ABSTRACT

Sugarcane is one of the most relevant bioenergy crops in Argentina. Due to its high capacity for biomass production, the crop has a high nitrogen requirement that can be supplied using chemical fertilizers. To ensure the sustainability of bioenergy programs and reduce the environmental issues caused by synthetic fertilizers, it is necessary to replace them in a short term. In this sense, Azospirillum inoculation constitutes the most used alternatives in our region to increase sugarcane plantations yields. The objective of this work was to isolate and characterize Azospirillum bacteria from sugarcane, analyze some of their PGPB characteristics and evaluate the effect of bioproducts formulated with the obtained isolates on crop sprouting. Three endophytic isolates (Ls1, Ls2 and Ls3) and two from the rhizosphere (Tv1 and Tv2) were obtained, with potential ability to fix atmospheric nitrogen and produce total indoles. Using 16S gene restriction and sequencing, the isolates were identified as A. brasilense. Genotyping by BOX-PCR, confirmed that Ls1, Ls2 and Ls3 were different from each other and from Az39 strain, unlike observations for Tv1 and Tv2. On greenhouse bioassays, it was observed that the inoculation with Ls1 biopreparation significantly increased sugarcane germination rate, so it could be used as a biofertilizer in this crop.

Key words: bacteria; biofertilizers; nitrogen; grass

INTRODUCTION

Azospirillum is considered one of the most important plant growth promoting bacteria (PGPB) ¹. The ability of this bacterium to promote growth and increase the production of various plant species, is due to the existence of mechanisms of action such as the production of phytohormones, the biological fixation of nitrogen (FBN) and the ability to limit the growth of certain phytopathogenic organisms, such as antibiosis and the production of siderophores ². Azospirillum is able to associate with 113 plant species, 14 of which are grasses and the remaining correspond to 34 other botanical families ³. In this sense, there are numerous works that confirm the ability of Azospirillum to associate both in endophytic and rhizospheric forms to the cultivation of sugarcane ^4).

In Argentina, the production of sugarcane is one of the most important agroindustrial activities. Due to its climatic and edaphic conditions, Tucumán is the province with the highest production of sugarcane, with a harvestable area of 269,530 ha for the 2017 harvest ⁵. Sugarcane is considered one of the most potential bioenergy crops, since it allows not only sugar to be obtained as food, but also bioethanol from its fermentation and cogeneration of electricity from the use of its harvest residues. The crop has high nitrogen requirements (between 180 and 250 kg ha^-1 per year^-1), due to its large biomass production capacity associated with the prolonged duration of its cycle, part of this nutrient assimilated by the plant is provided by the mineralization of soil organic matter; however, it is not enough to satisfy the requirements of the crop, so nitrogen fertilization is a necessary agronomic practice.

Currently, urea is the most used nitrogenous fertilizer and approximately 240 kg of urea per ha per year are applied. However, only between 20 and 50 % of the applied nitrogen is used by the crop, the rest that is not assimilated is lost through leaching, denitrification and volatilization, turning the chemical fertilizer into an important source of environmental contamination. On the other hand, the synthesis of these fertilizers demands high levels of fossil energy, so in order to achieve the sustainability of bioenergy programs, it is necessary to completely replace them in the short term.

Different studies have shown that sugar cane is able to obtain up to 70 % of its nitrogen requirements from the FBN ⁶, a process carried out by different bacteria, rhizospheric and endophytic, capable of associating and colonizing the sugar cane, among which are those of the genus Azospirillum. The Azospirillum-sugarcane association is the starting point for FBN programs with non-legume plants worldwide ⁶. This background, together with the growing interest in implementing sustainable agronomic systems in the management of the cane fields through the use of PGP bacteria, has led to the fact that currently, around 10,000 hectares of sugarcane plantations in Tucumán are inoculated with commercial bioproducts containing to strain A. brasilense Az39 in its composition (unpublished results). This inoculation allows to increase the sprouting and emergence of the seedlings, achieving the rapid establishment of the cane field.

Taking into account these considerations, the objective of this work was to isolate and characterize bacteria of the genus Azospirillum from sugarcane, analyze some of their growth promoting characteristics and evaluate the effect on sprouting of the crop.

MATERIALS AND METHODS

Isolation of bacteria of the genus Azospirillum

Samples were taken of rhizospheric soil and roots of sugarcane plants of the LCP variety 85-384, belonging to reed beds located in “Los Gómez” and “Las Talitas”, department Leales and Tafí Viejo, respectively. Bacteria of the genus Azospirillum were isolated following the technique described by Döbereiner et al., for the isolation of rhizospheric bacteria ⁷, three samples of rhizospheric soil 0-15 cm deep were taken from each location. Each sample evaluated, in turn, consisted of five subsamples of approximately 500 g each. From these samples, successive dilutions were made in sterile physiological solution (NaCl 0.9 % m v^-1) and aliquots of 100 μL were seeded in the semisolid culture medium free nitrogen malate (NFb) (malic acid 5 g L^-1; K2HPO4 0.5 g L^-1; MgSO₄.7H₂O 0.2 g L^-1; NaCl 0.1 g L^-1; CaCl₂.2H₂O 0.02 g L^-1; KOH 4.5 g L ^-1, 1.75 g L^-1 agar, pH 6.8 adjusted with KOH). For the isolation of endophytic bacteria, from each locality root samples were taken from five independent plants, spaced approximately 3 m apart and chosen at random. Ten roots of 5 to 15 cm were carefully taken from each plant, which were superficially disinfected with 70 % ethanol, 1 min, 2 % sodium hypochlorite (v v^-1) and five washes with sterile distilled water. The disinfected roots were cut into 1 cm fragments and transferred to the semi-solid NFb culture medium. The flasks were incubated for five days at 30 °C. The cultures that showed bacterial growth in the form of a sub-surface white film, typical of Azospirillum, were replic in solid NFb culture medium added with yeast extract (5 g L^-1) and Congo Red (15 mL L^-1 an aqueous solution 1: 400 (p v^-1), for the identification of small, dry colonies, scarlet red color ⁸.

Analysis of the promoting characteristics of growth

Biological nitrogen fixation was analyzed according to the ability of the isolates to grow in the nitrogen-free NFb culture medium and by PCR amplification of a 710 bp fragment of the nifD gene of nitrogenase, an enzyme involved in the nitrogen fixation process. As primers, nifDf and nifDr (Sigma-Aldrich, USA) were used ⁹. The ability of the isolates to produce indoles was evaluated following the colorimetric technique described by Glickmann and Dessaux ¹⁰. For this, a colony isolated in the solid NFb medium was seeded in the liquid culture medium Luria Bertani (LB) (yeast extract 5 g L^-1, triptein 10 g L^-1, NaCl 10 g L^-1). The cultures were incubated at 30 °C with shaking (45 r.p.m.). At 24, 48, 72 and 96 h, samples were taken to evaluate the bacterial growth by determining the optical density at 560 nm (DO_560nm). The concentration of total indoles produced and excreted to the culture medium was determined at 96 h of incubation, measuring the DO_530nm and extrapolating the values obtained in a calibration curve in which pure indole acetic acid (IAA) was used as standard. The Az39 strain of A. brasilense was used as reference.

All the tests corresponding to the determination of the growth promoting characteristics were carried out in triplicate for each evaluated strain. The data were analyzed statistically by analysis of variance (ANOVA) and the Fisher LSD test, with the InfoStat program (Statistical Software, 2010) for Windows.

Molecular characterization of the isolated

Amplification by PCR, sequencing and ARDRA of the 16S rDNA gene

In order to confirm that the isolates obtained belong to the genus Azospirillum, their molecular characterization was carried out by means of PCR amplification of a 1450 bp fragment of the 16S rDNA gene, using the primers 27f and 1492r (Biodynamics, Argentina). The amplified products, on the one hand, were sent to the Reference Center for Lactobacillus (CERELA) for sequencing, and analyzed using the BLAST program (Basic Local Alignment Search Tool). On the other hand, the digestion of the amplified products was carried out following the ARDRA (Amplified Ribosomal DNA Restriction Analysis) technique with the restriction enzyme Alul (Thermo Scientific, USA). The obtained fragments were placed on a 1.5 % agarose gel (p v^-1) in order to compare the restriction profiles.

Genotyping by BOX-PCR

Finally, to identify the different isolates at the strain level, the BOX-PCR technique was used using the boxa1r primer (Sigma Aldrich, USA) ¹¹. This technique is based on the PCR amplification of intergenic DNA sequences, using a primer that hybridizes specifically with repetitive sequences (rep sequences) of the bacterial genome ¹². The profiles of bands generated are unique among species, even between strains. The conditions to carry out the amplification were: denaturation at 95 °C 2 min, followed by 35 cycles of 94 °C 3 s, 92 °C 30 s, 50 °C 1 min, 65 °C 2 min, with an extension final at 65 °C for 8 min. The amplified products were separated by polyacrylamide gel electrophoresis, with coupling to Li-Cor (Biosciences, USA) for detection. To perform the molecular characterization of the new isolates, the Az39 strain of A. brasilense was used as reference.

Selection of isolates and formulation of biofertilizers

The isolates of Azospirillum that showed differences in their genetic fingerprint after carrying out the BOX-PCR, were selected and sent to a biofertilizer producing company for the formulation of different bioproducts.

Bioassay on sugar cane

The effect of four bioproducts (Bio) formulated from the isolates Ls1, 2 and 3 and from the reference strain Az39 (BioLs1, BioLs2, BioLs3 and BioAz39, respectively) was evaluated on the sprouting speed of the sugar cane culture. For this, uninodal stakes extracted from the middle third of stalks of seed cane of the LCP 85-384 variety were inoculated by irrigation with 5 mL of each biofertilizer. Considering that the optimum concentration for inoculation with biofertilizers based on Azospirillum corresponds to 1 x 106 cfu mL^{-1 (13}, and that the bioproducts had an average concentration of 1 x 109 cfu mL^-1 the corresponding dilutions were made (1 : 1000 v v^-1) of each bioproduct before its application. For each evaluated treatment, 12 uninodal stakes were planted in plastic trays using sterile fine sand as substrate. After the inoculation, the trays were kept in a hothouse at 25 °C. The trays were placed in an experimental design of completely randomized blocks. We worked with three repetitions per treatment, and as a positive control we used the biofertilizer based on the Az39 strain of A. brasilense (BioAz39). The results were expressed as sprouting velocity index (IVB) (14), calculated from the following formula:

$IVB= \left(B1/N1 + B2/N2 +\dots + Bn/Nn\right)$

Where: B is the number of shoots, and N the days after planting.

The data were analyzed statistically by the analysis of variance (ANOVA) and the Fisher LSD test, with the program InfoStat (Statistical Software, 2010) for Windows.

RESULTS AND DISCUSSION

Isolation and Characterization

Three endophytic isolates with typical characteristics of the genus Azospirillum were obtained from sugarcane roots of the Leales department, which were denominated Ls1, Ls2 and Ls3, and two rhizospheres from the Tafí Viejo department, which were denominated Tv1 and Tv2. The association of Azospirillum with the roots of sugarcane can only be successful if the bacterium is able to survive in the soil and reach significant populations in the root system, so these results confirm the ability of this bacterial genus to colonize naturally the cultivation of sugar cane ¹⁵.

Although bacteria of the genus Azospirillum are generally considered rhizosphere bacteria, some strains develop specific differences in the way they colonize the roots. Predominantly they colonize the root surface and only a few strains are able to colonize the inside of the roots. These endophytic strains, being in an environment protected from environmental conditions, have specific mechanisms to communicate and interact with the plant much more efficiently than the rhizospheric strains.

Biological fixation of nitrogen (FBN)

All the isolates were able to grow in NFb medium, forming a white subsurface film ⁷. In addition, the presence of the nifD gene of nitrogenase provides evidence of the potential capacity of these isolates to fix atmospheric nitrogen (Figure 1), in agreement with that reported by other authors ⁹.

The amplification corresponds to the rhizospheric strains of Tafí del Valle Tv1 and Tv2 (lines 1 and 2), strain Az39 used as reference (line 3), molecular weight marker 1Kb (Promega, USA) (lines 4 and 8), endophytic strains of Loyalists Ls1, Ls2 and Ls3 (lines 5, 6 and 7)

Figure 1 PCR amplification of the nifD gene (710 bp) of the isolates obtained 

Currently, the contribution of Azospirillum to nitrogen requirements of sugarcane through the FBN is a controversial issue. Some research reports claim that the crop is able to obtain up to 70 % of its nitrogen requirements from the FBN and that this is the main mechanism for promoting the growth of bacteria of this genus ¹⁶, others assume that Azospirillum it uses nitrogen from the FBN for its own use and the contribution of nitrogen to the crop is related to the assimilation of nitrates ¹⁷.

Producción de indoles totales

Figure 2 shows the production of total indoles expressed in μg mL^-1 as a function of bacterial growth, evaluated by measuring the optical density at 560 nm.

Equal letters indicate no statistically significant differences (LSD, p ≤ 0.05)

Figure 2 Growth (DO₅₆₀) (circles) and production of total indoles (μg mL^-1) (bars) of the different isolates at 96 hs incubation at 30 ºC 

As can be seen in Figure 2, all the isolates synthesized and excreted total indoles, in amounts similar to the Az39 strain of A. brasilense. The production of phytohormones is one of the most important PGPB characteristics of the genus Azospirillum¹⁸. These bacteria produce and release to the culture medium different phytohormones, during the stationary phase of growth, among which auxins, gibberellins, abscisic acid, ethylene and salicylic acid have been found. Although these phytohormones significantly affect the growth of the root, resulting in a better absorption of moisture and nutrients, they have also been shown to participate in increasing the growth of the cultures when Azospirillum is applied in a foliar manner ². For this reason, the production of phytohormones is important not only for the production of inoculants at an industrial level, but also for the action of these bacteria in the phyllosphere, in the rhizosphere and even within plants. It has been observed that inoculation with Azospirillum and the phytohormones produced by these rhizobacteria induce a greater growth promotion response in seeds and seedlings ². The results obtained with the new isolates, producers of indoles, are important for the selection and subsequent formulation of possible biofertilizers ¹⁹.

Molecular characterization

Amplification by PCR, sequencing and ARDRA of the 16S rDNA gene

Figure 3 shows the PCR amplification of the 16S gene of the rDNA of the different isolates obtained and its subsequent digestion with the restriction enzyme AluI. The obtained profiles were compared with those of the Az39 strain of A. brasilense used as reference.

The standards correspond to the strain Az39 used as reference (line 1), rhizospheric strains of TafÍ del Valle Tv1 and Tv2 (lines 2 and 3), endophytic strains of Loyal Ls1, Ls2 and Ls3 (lines 4, 5 and 6), marker of molecular weight of 100 bp (Promega, USA) (line 7)

Figure 3 Electrophoresis profile of the fragments obtained after restriction of the 16S rDNA with the AluI restriction enzyme 

It was observed that the isolates presented the same restriction profile as the Az39 reference strain of A. brasilense, so it is inferred that they could correspond to this species of the genus Azospirillum. The sequencing of the 16S gene of the rDNA and its subsequent analysis using the BLAST program allowed confirming it (Table 1).

Table 1 Sequence analysis of the isolates obtained 

Isolation	Description	Max score	Total score	Query cover	e-value	Ident	Accesion
Tv 1	A. brasilense strain Az39 plasmid AbAZ39	183	362	14 %	2.00E-42	99 %	CP007797.1
Tv 2	A. brasilense strain Az39 plasmid AbAZ40	547	1032	56 %	5.00E-152	93 %	CP007797.1
Ls 1	A. brasilense partial 16S rRNA strain Gr54	368	368	35 %	4.00E-98	96 %	FR667893.1
Ls 2	A. brasilense partial 16S rRNA strain Gr54	723	723	70 %	0	96 %	FR667893.1
Ls 3	A. brasilense 16S rRNA cole M7	363	363	35 %	2.00E-96	95 %	HE646772.1

Genotyping by BOX-PCR

The polymorphism patterns generated by BOX-PCR have been used for the differentiation of bacterial strains of the genus Azospirillum²⁰. Figure 4 shows the different genetic fingerprints of the isolates, obtained by BOX-PCR.

The standards correspond to the rhizospheric strains of Tafí del Valle Tv1 and Tv2 (lines 1 and 2), strain Az39 used as reference (line 3), endophytic strains of Loyal Ls1, Ls2 and Ls3 (lines 4, 5 and 6, respectively) and line 7, molecular weight marker of 1Kb (Promega, USA)

Figure 4 Genetic fingerprint of the different isolates obtained by BOX-PCR 

The BOX-PCR technique showed that Tv1 and Tv2 isolates have the same band profile as Az39 from A. brasilense, so we can confirm that it is the same strain. On the contrary, the profiles of Ls1, Ls2 and Ls3 are different from each other and with the profile of the Az39 strain, so it can be confirmed that they are different strains.

Bioassay on sugar cane

The isolates Ls1, Ls2 and Ls3 were selected for being endophytes and for presenting differences in their genetic fingerprint with respect to strain Az39, for the formulation of bioproducts that were named BioLs1, BioLs2 and BioLs3. In addition, BioAz39 was used as a positive control for growth induction. The sprouting velocity index (IVB according its acronyms in Spanish) of the sugar cane stakes inoculated with the different biofertilizers is shown in Figure 5.

The IVB values correspond to the average of three determinations and the error bars indicate standard deviation (SD). Different letters indicate significant differences (LSD, p≤0.05)

Figure 5 Sprouting velocity index (IVB) of the uninodal sugarcane cuttings inoculated with the biofertilizers formulated from the selected isolates 

Among the evaluated bioproducts, BioLs1 was the one that showed the greatest capacity to increase the budding of uninodal stakes of sugarcane, after its application (Figure 5). BioLs1 presented statistically significant differences in comparison with BioLs3, BioAz39 and with the control without inoculation. On the other hand, BioLs1 and BioLs2 bioproducts significantly increased the IVB compared to the uninoculated control. The IVB was 2.30 for the control, while for BioLs1 and BioLs2 it was 5.02 and 4.01 respectively. It is important to point out that the BioLs1 bioproduct, formulated from the resident isolate A. brasilense Ls1, showed greater capacity to increase the IVB of sugarcane than BioAz39, which contains A. brasilense Az39, the most used strain in our region for the formulation of inoculants for sugar cane. The importance of the FBN during the initial growth of sugarcane, is due to the fact that the N absorption rate of the crop is highest in the first months after sprouting. In this period the crop absorbs more N than it uses for its growth and development, storing the excess as organic substances in its tissues (pods and leaf blades). Then, that N is remobilized to zones of active growth to satisfy, along with the N contributed from the ground, the high requirements of the phase of great growth. This behavior represents a strategy of biological administration of N, which guarantees that it does not compromise growth ²¹. For this reason, the use of bioproducts based on PGP bacteria capable of carrying out the FBN and of producing and excreting phytohormones allowing the rapid establishment of the plant in its initial stages of growth and development, is of fundamental importance considering that poor and prolonged emergencies affect the effective fulfillment of the following phenological phases of the crop, significantly reducing the production and yield of the sugarcane ²¹

CONCLUSIONS

• It is possible to isolate and characterize different strains of A. brasilense associated with the cultivation of sugar cane, with potential capacity to fix atmospheric nitrogen and synthesize indoles. The profiles of bands obtained by BOX-PCR confirm that the strains Ls1, Ls2 and Ls3 are strains different from each other and to the strain Az39. The BioLs1 and BioLs2 bioproducts show a positive effect on the IVB of the sugarcane, in comparison with the control without inoculating, in bioassays carried out under controlled conditions. The BioLs1 bioproduct also causes an increase in the sprouting rate of the inoculated seedlings, in comparison with BioAz39.

• In this sense, the isolates A. brasilense Ls1 and Ls2 with potential capacity to fix nitrogen and produce indoles, are promising strains associated with sugar cane that could be used for the formulation of biofertilizers, in order to increase yields and production of the crop.

BIBLIOGRAFÍA

1. Cassán F, Diaz-Zorita M. Azospirillum sp. in current agriculture: from the laboratory to the field. Soil Biology and Biochemistry. 2016;103:117-30. doi:10.1016/j.soilbio.2016.08.020

2. Fukami J, Cerezini P, Hungria M. Azospirillum: benefits that go far beyond biological nitrogen fixation. AMB Express. 2018;8(1):73. doi:10.1186/s13568-018-0608-1

3. Pereg L, de-Bashan LE, Bashan Y. Assessment of affinity and specificity of Azospirillum for plants. Plant and Soil. 2016;399(1-2):389-414. doi:10.1007/s11104-015-2778-9

4. Vera L, Grellet Naval N, Nuñ M de los A, Alonso L, Leggio Nem F, Fernández González P, et al. Estudio de la cepa Azospirillum brasilense Az39 como biofertilizante en caña de azúcar. Revista Industrial y Agrícola de Tucumán. 2017;94(2):1-11.

5. Fandos C, Scandaliaris J, Scandaliaris P, Carreras JI, Soria F. Relevamiento satelital de cultivos en la provincia de Tucumán. Reporte Agroindustrial. 2017;138:1-10.

6. Urquiaga S, Cruz KHS, Boddey RM. Contribution of nitrogen fixation to sugar cane: nitrogen-15 and nitrogen-balance estimates. Soil Science Society of America Journal. 1992;56(1):105-14. doi:10.2136/sssaj1992.03615995005600010017x

7. Döbereiner J, Baldani VLD, Baldani JI. Microorganismos diazotróficos aerobios. In: Como isolar e identificar bactérias diazotróficas de plantas não-leguminosas [Internet]. 1ra ed. Brasilia, Brasil: Embrapa SPI; 1995 [cited 2019 Feb 7]. p. 11-25. Available from: https://www.scirp.org/(S(351jmbntvnsjt1aadkposzje))/reference/ReferencesPapers.aspx?ReferenceID=1508971

8. Cáceres EAR. Improved medium for isolation of Azospirillum spp. Applied and Environmental Microbiology. 1982;44(4):990-1.

9. Potrich DP, Passaglia LM, Schrank IS. Partial characterization of nif genes from the bacterium Azospirillum amazonense. Brazilian Journal of Medical and Biological Research. 2001;34(9):1105-13.

10. Glickmann E, Dessaux Y. A critical examination of the specificity of the salkowski reagent for indolic compounds produced by phytopathogenic bacteria. Applied and Environmental Microbiology. 1995;61(2):793-6.

11. Versalovic J, Schneider M, Bruijn FJD, Lupski JR. Genomic fingerprinting of bacteria using repetitive sequence-based polymerase chain reaction. Methods in Molecular and Cellular Biology. 1994;5(1):25-40.

12. Sabat AJ, Budimir A, Nashev D, Sá-Leão R, van Dijl J m, Laurent F, et al. Overview of molecular typing methods for outbreak detection and epidemiological surveillance. Euro Surveillance. 2013;18(4):20380. doi:10.2807/ese.18.04.20380-en

13. Bashan Y. Enhancement of wheat root colonization and plant development by Azospirillum brasilense Cd. following temporary depression of rhizosphere microflora. Applied and Environmental Microbiology. 1986;51(5):1067-71.

14. Souza RHV de, Villela FA, Aumonde TZ. Methodologies based on seedling performance for vigor assessment of pumpkin seeds. Journal of Seed Science. 2013;35(3):374-80. doi:10.1590/S2317-15372013000300015

15. Vejan P, Abdullah R, Khadiran T, Ismail S, Nasrulhaq Boyce A. Role of plant growth promoting rhizobacteria in agricultural sustainability-A review. Molecules. 2016;21(5):573-90. doi:10.3390/molecules21050573

16. Volfson V, Fibach-Paldi S, Paulucci NS, Dardanelli MS, Matan O, Burdman S, et al. Phenotypic variation in Azospirillum brasilense Sp7 does not influence plant growth promotion effects. Soil Biology and Biochemistry. 2013;67:255-62. doi:10.1016/j.soilbio.2013.09.008

17. Bremer E, Janzen HH, Gilbertson C. Evidence against associative N2 fixation as a significant N source in long-term wheat plots. Plant and Soil. 1995;175(1):13-9. doi:10.1007/BF02413006

18. Castillo P, Molina R, Andrade A, Vigliocco A, Alemano S, Cassán FD. Phytohormones and Other Plant Growth Regulators Produced by PGPR: The Genus Azospirillum. In: Cassán F, Okon Y, Creus CM, editors. Handbook for Azospirillum [Internet]. Suiza: Springer, Cham; 2015 [cited 2019 Feb 11]. p. 115-38. doi:10.1007/978-3-319-06542-7_7

19. Gouda S, Kerry RG, Das G, Paramithiotis S, Shin H-S, Patra JK. Revitalization of plant growth promoting rhizobacteria for sustainable development in agriculture. Microbiological Research. 2018;206:131-40. doi:10.1016/j.micres.2017.08.016

20. Reem G, Burdman S, Okon Y. Methods for studying phenotypic variation in Azospirillum. In: Cassán F, Okon Y, Creus CM, editors. Handbook for Azospirillum [Internet]. Suiza: Springer, Cham; 2015 [cited 2019 Feb 11]. p. 231-9. doi:10.1007/978-3-319-06542-7_12

21. Bassi D, Menossi M, Mattiello L. Nitrogen supply influences photosynthesis establishment along the sugarcane leaf. Scientific Reports. 2018;8(1):23-7. doi:10.1038/s41598-018-20653-1

Received: September 17, 2018; Accepted: January 24, 2019

^*Autor para correspondencia. [e-mail]