Abstract:

Sugarcane is an important Brazilian commodity, being usually cultivated in soils with low natural fertility. This study aimed to isolate diazotrophic endophytes from sugarcane tissues and evaluate the morphological and physiological characteristics of their colonies as well as their plant growth-promoting (PGP) traits in select diazotrophic endophytic bacteria. Fifty-six bacterial isolates were identified in the sugarcane tissues, and these isolates presented distinct morphological and physiological traits. A total of thirty-five bacterial isolates were biochemically evaluated. Overall, Bacillus was the dominant genus. Isolates of Methylobacterium spp. and Brevibacillus agri were present only in leaves, while Herbaspirillum seropedicae occurred only in stems. Except to IPA-CF45A, all isolates were nitrogenase positive. All endophytes exhibit production of indol 3-acetic acid. Over 50% of endophytes solubilize phosphate, release N-acyl homoserine lactones, and present the activity of 1-aminocyclopropane-1-carboxylic acid deaminase, catalase, lipase and protease. The network analysis showed that isolates belonged to Burkholderia, Herbaspirillum, and Methylobacterium interact with Bacillus. Bacterial endophytes exhibited distinct morphological, physiological, and PGP traits that are useful for sustainable agriculture, highlighting the isolates IPA-CC33, IPA-CF65, IPA-CC9 and IPA-CF27. Further studies on the effects of these diazotrophic endophytes and their potential for providing microbial inoculants for improving sugarcane fields will provide valuable information to maintain the sustainability and environment quality.

Keywords:
Nitrogen-fixing bacteria; phytohormones; phosphate; indol 3-acetic acid; catalase; protease

HIGHLIGHTS

HIGHLIGHTS

INTRODUCTION

Sugarcane (Saccharum spp.) is economically important in several countries, mainly those located in Latin America. Nowadays, Brazil is the world's largest sugarcane producer, presenting an estimated area of 8.5 million hectares and an average productivity of about 75.8 Mg ha^-1 [¹1 IBGE. Levantamento sistemático da produção agrícola. Cana-de-açúcar. [cited 2018 Apr 9]. Available from: https://sidra.ibge.gov.br/home/lspa/brasil
https://sidra.ibge.gov.br/home/lspa/bras... ]. The Northeastern region of Brazil is particularly important for sugarcane production, and Pernambuco, Paraíba, and Alagoas are the most productive states, representing about 80% of all sugarcane produced in 2018/2019 [²2 CONAB. Acompanhamento da Safra Brasileira - Cana-de-açúcar - Terceiro levantamento (Safra 2019/20). [cited 2019 Mar 19]. Available from: https://www.conab.gov.br
https://www.conab.gov.br... ]. Although important, sugarcane is usually cultivated in soils with low fertility that limit its productivity and, at the same time, require the intensive use of chemical fertilizers, mainly nitrogen [³3 Bordonal RO, Carvalho JLN, Lal R, Figueiredo EB, Oliveira BG, La Scala N. Sustainability of sugarcane production in Brazil. A review. Agron Sustain Dev 2018;38(2):13.].

Nitrogen is the most required nutrient by sugarcane, and chemical fertilization is necessary to provide nitrogen to this plant species and, therefore, to increase its productivity [³3 Bordonal RO, Carvalho JLN, Lal R, Figueiredo EB, Oliveira BG, La Scala N. Sustainability of sugarcane production in Brazil. A review. Agron Sustain Dev 2018;38(2):13.]. Under Brazilian-field conditions, sugarcane absorbs 26% of the total nitrogen applied through of chemical fertilization and the remaining N is lost [⁴4 Otto R, Castro SAQ, Mariano E, Castro SGQ, Franco HCJ, Trivelin PCO. Nitrogen use efficiency for sugarcane-biofuel production: what is next? Bio Energy Res 2016;9:1272-89.]. Thus, nitrogen can be supplied to sugarcane by the biological nitrogen fixation (BNF), and this process can significantly increase sugarcane yield [⁵5 Muangthong A, Youpensuk S, Rerkasem B. Isolation and characterization of endophytic nitrogen fixing bacteria in sugarcane. Tropical Life Sci Res 2015;26(1):41-51., ⁶6 Schultz N, Pereira W, Silva PA, Baldani JI, Boddey RM, Alves BJR, et al. Yield of sugarcane varieties and their sugar quality grown in different soil types and inoculated with a diazotrophic bacteria consortium. Plant Prod Sci 2017;20(4):366-74.]. Nitrogen-fixing bacteria carry out the BNF process endophytically in sugarcane tissues and then can supply a considerable proportion of nitrogen to these plants and, therefore, promote plant growth and crop yield. According to Bordonal and coauthors [³3 Bordonal RO, Carvalho JLN, Lal R, Figueiredo EB, Oliveira BG, La Scala N. Sustainability of sugarcane production in Brazil. A review. Agron Sustain Dev 2018;38(2):13.], BNF represents an important source of N to sugarcane and contributes with more than 40 kg N ha^-1 year^-1.

The endophytic bacteria colonize different plant organs, including roots, stems, leaves, flowers, and fruits, and have been recognized as efficient plant growth-promoters [⁷7 Compant S, Samad A, Faist H, Sessitsch A. A review on the plant microbiome: Ecology, functions and emerging trends in microbial application. J Adv Res 2019;19:29-37.]. As they act endophytically, these bacteria are more efficient than those found in the rhizosphere because they are always in contact with plant tissues and, therefore, can be more beneficial [⁸8 Defez R, Andreozzi A, Bianco C. The overproduction of indole-3-acetic acid (IAA) in endophytes upregulates nitrogen fixation in both bacterial cultures and inoculated rice plants. Microb Ecol 2017;74(2):441-52.]. Schultz and coauthors [⁶6 Schultz N, Pereira W, Silva PA, Baldani JI, Boddey RM, Alves BJR, et al. Yield of sugarcane varieties and their sugar quality grown in different soil types and inoculated with a diazotrophic bacteria consortium. Plant Prod Sci 2017;20(4):366-74.] have reported that BNF, performed by diazotrophic endophytes, contributes significantly to the total of nitrogen found in sugarcane. Beyond nitrogen-fixation, these bacteria display important plant growth-promoting (PGP) traits, such as production of phytohormones (auxins, cytokinins, and gibberellins) and N-acyl homoserine lactones, as well as the protection against phytopathogens, and amelioration of abiotic stress [⁸8 Defez R, Andreozzi A, Bianco C. The overproduction of indole-3-acetic acid (IAA) in endophytes upregulates nitrogen fixation in both bacterial cultures and inoculated rice plants. Microb Ecol 2017;74(2):441-52., ⁹9 Mukherjee A, Singh B, Prakash VJ. Harnessing chickpea (Cicer arietinum L.) seed endophytes for enhancing plant growth attributes and bio-controlling against Fusarium sp. Microbiol Res 2020;237:126469.].

There is a high diversity of PGPB associated with sugarcane; however, the knowledge of this diversity and their PGP traits is limited. Commercial sugarcane plants cultivated in Thailand exhibited an endophytic bacteria community with important PGP traits, including 1-aminocyclopropane-1-decarboxylate (ACC) deaminase, indole-3-acetic (IAA) acid, nitrogen fixation, phosphate solubilization, and siderophore production [¹⁰10 Kruasuwan W, Thamchaipenet A. Diversity of culturable plant growth-promoting bacterial endophytes associated with sugarcane roots and their effect of growth by co-inoculation of diazotrophs and actinomycetes. J Plant Growth Regul. 2016;35(4):1074-87.]. In sugarcane plants grown in nitrogen-free conditions, all bacterial endophytes displayed nitrogenase activity and three of them were able to achieve high nitrogen contents in sugarcane [⁵5 Muangthong A, Youpensuk S, Rerkasem B. Isolation and characterization of endophytic nitrogen fixing bacteria in sugarcane. Tropical Life Sci Res 2015;26(1):41-51.]. Diazotrophic endophytic bacteria associated to sugarcane tissues identified by Leite and coauthors [¹¹11 Leite MCBS, Pereira APA, Souza AJ, Andrade PAM, Barbosa MV, Andreote FD, et al. Potentially diazotrophic endophytic bacteria associated to sugarcane are effective in plant growth-promotion. J Exp Agric Int 2018;21(3):1-15.] solubilized phosphate and produced IAA and exopolysaccharides. IAA-producing bacteria with antifungal properties were identified in endophytic bacteria community found in sugarcane tissues [¹²12 Rodrigues AA, Forzani MV, Soares RDS, Sibov ST, Vieira JDG. Isolation and selection of plant growth-promoting bacteria associated with sugarcane. Pesqui Agropecu Trop 2016;46(2):149-58.]. These studies reinforce that these bacterial endophytes can effectively stimulate the plant growth in different soils.

As reported above, these bacteria are easily found in plant tissues and can be isolated to enable the evaluation of their biochemical capabilities related to BNF and plant growth. Thus, these endophytic bacteria can be used to benefit sugarcane by increasing its growth and yield. This study isolated diazotrophic endophytes from leaves and stems of sugarcane and evaluated their morphophysiological and biochemical traits. The main hypothesis is that diazotrophic endophytic bacteria associated with sugarcane could produce and release useful biochemical compounds and, thus, act as plant growth-promoters. Therefore, these selected bacteria could be used to promote the growth of sugarcane and other agronomically important crops.

MATERIAL AND METHODS

Plant material and bacterial isolation

Samples of leaves and stems of sugarcane (varieties RB 1011, RB 867515, and RB 92579) from fields located in distillery Japungú (7°10'38.85"S and 34°58'12.72"W; Santa Rita, Paraiba, Brazil) and distillery Tabú (7°30'30.19"S and 34°52'33.17" W; Alhandra, Paraiba, Brazil) were used for the isolation of endophytic diazotrophic bacteria. These samples were transferred to the Laboratory of Soil Biology from the Agronomical Institute of Pernambuco (Recife, Pernambuco, Brazil). Leaves and stems (10 g each) were macerated, separately, in 5% sterile saline solution, and plated on tubes containing semisolid broth without nitrogen: LGI (Liquid Glucose Ivo) [¹³13 Baldani JI, Baldani VLD, Sampaio MJAM, Döbereiner J. A fourth Azospirillum species from cereal roots. An Acad Bras Ciênc 1984;56:265.], LGI-P (Liquid Glucose Ivo Pernambuco) [¹⁴14 Reis VM, Olivares FL, Döbereiner J. Improved methodology for isolation of Acetobacter diazotrophicus and confirmation of its habitat. World J Microb Biot 1994;10:101-4.], JMV (Johanna Mannitol Vera) [¹⁵15 Baldani VLD. Efeito da inoculação de Herbaspirillum spp. no processo de colonização e infecção de plantas de arroz e ocorrência e caracterização parcial de uma nova bactéria diazotrófica [thesis]. Seropédica (RJ): Federal Rural University of Rio de Janeiro; 1996.], JNFb (Johanna New Fabio) [¹⁶16 Baldani VLD, Baldani JI, Olivares FL, Döbereiner J. Identification and ecology of Herbaspirillum seropedicae and the closely related Pseudomonas rubrisubalbicans. Symbiosis 1992;13:65-73.] and NFb (New Fabio) [¹⁷17 Döbereiner J, Marriel IE, Nery M. Ecological distribution of Spirillum lipoferum Beijerinck. Can J Microbiol 1976;22:1464-1473.]. The full composition of each nitrogen-free semisolid culture media are specified in supplementary table 1.

The tubes containing semisolid and nitrogen-free broth were incubated at 30 °C until diazotrophic bacteria exhibit typical growth, i.e., the formation of a pellicle near the surface of the medium (microaerophilic film) [¹⁸18 Döbereiner J, Baldani VLD, Baldani JI. Meios de culturas e soluções utilizadas. In: Motta AC, Oliveira MAS, Martins FC and Quazi TSG, editors. Como isolar e identificar bactérias diazotróficas de plantas não-leguminosas. Brasília: Empresa Brasileira Pesquisa Agropecuária - Centro Nacional de Pesquisa Agrobiologia; 1995. p. 47-53.]. After re-isolation and confirmation of purity, the bacterial strains were stored in potato-P media with added sterile mineral oil. The procedure was conducted in triplicate. The potato-P media (pH 5.5) was composed by: potato filtrate (200 g L^-1); crystalized cane sugar (100 g L^-1); KOH (2.0 g L^-1); vitamin solution (1.0 mL L^-1); micronutrient solution (2.0 mL L^-1); two drops of bromothymol blue (5 g L^-1 in 0.2 N KOH); and agar (15.0 g L^-1). The potato filtrate was obtained after sliced potatoes homogenized with distilled water were boiled for 30 min. The vitamin solution was composed by biotin (10 mg L^-1) and pyridoxal-HCl (20 mg L^-1). The micronutrient solution contains: CuSO₄.5H₂O (0.04 g L^-1), ZnSO₄.7H₂O (0.12 g L^-1), H₃BO₃ (1.40 g L^-1), Na₂MoO₄.2H₂O (1.0 g L^-1) and MnSO₄.H₂O (1.175 g L^-1).

Morphological and physiological characterizations

Morphological and physiological characterizations of bacterial isolates were performed after 192 hours of bacterial growth in Petri dishes containing culture medium 79 with bromothymol blue [¹⁹19 Fred EB, Waksman SA. Laboratory Manual of General Microbiology. New York: McGraw-Hill Book; 1928.]. The following characteristics of bacterial colonies were evaluated: size/diameter (<1.0 mm or > 1.0 mm); shape (circular, lenticular or irregular); edge (whole, wavy or filamentous); surface (smooth, rough or filamentous); appearance (translucent, opaque or transparent); color (white, yellow, cream, transparent or pink); formation of acids and alkalis; formation and volume of mucus (absent or present, much or little); and growth time (fast, intermediate, or slow). The Gram stains of bacterial isolates were performed according to Yano and coauthors [²⁰20 Yano DMY, Attili DS, Gatti MSV, Eguchi SY, Oliveira UM. Técnicas de microbiologia em controle de qualidade. Campinas: Fundação Tropical de Pesquisas e Tecnologia "André Tosello"; 1991.].

Bacterial identification

Polymerase chain reaction (PCR) amplification of repetitive bacterial DNA fingerprinting (rep-PCR) using the BOX-A1R primer was used to differentiate bacterial isolates, while ribosomal 16S rRNA gene was amplified and analyzed to identify bacterial isolates at genus or species level. Bacterial isolates were retrieved from storage by subculture on liquid DYGS (Dextrose Yeast Glucose Sucrose) culture medium [²¹21 Rodriguez Neto J, Malavolta Junior VA, Victor O. Meio Simples para isolamento e cultivo de Xanthomonas campestris pv. citri tipo B. Summa Phytopathol 1986;12(1-2):16-7.] and incubated at 30 °C (200 rpm; 48 h). Bacterial genomic DNA was extracted from colonies using Wizard® Genomic DNA Purification Kit (Promega Corporation, WI, USA) according to the manufacturer's recommendations. The extracted DNA was diluted in sterile Milli-Q water (30 ng μL^-1) and stored at-20 °C until further use.

Amplification was performed in an Applied Biosystems 2720 Thermal Cycler (Life Technologies, CA, USA) using the BOX-A1R oligonucleotide (5'-CTACGGGGCCAAGACGACGCTG-3') (Invitrogen, MA, USA). To rep-PCR, DNA template (20 ng μL^-1) was used in a 20 µL reaction mixture containing 2.0 mM MgCl₂, 0.2 mM deoxynucleotide triphosphate (dNTPs), 0.2 µM primer, 10X 10% Buffer and 0.3 U Taq polymerase. The rep-PCR program was conducted using an initial denaturation of 9 min at 94 °C, followed by 30 cycles of 94 °C for 1 min, 55 °C for 1 min and 72 °C for 5 min, with a final extension at 72 °C for 10 min. The rep-PCR products were analyzed by 1.2% agarose gel electrophoresis and visualized on LPIX HE transilluminator (Loccus Biotechnology, SP, Brazil). The resulting fingerprints were transformed into a two-dimensional binary matrix (‘1’ for the presence and ‘0’ for the absence of a band at a particular position) and analyzed by NTSYSpc 2.1 software using the unweighted pair group method with arithmetic mean (UPGMA) algorithm.

Following rep-PCR fingerprinting analysis, ribosomal 16S rRNA genes sequencing of the bacterial isolates using the FD1 (5'-AGAGTTTGATCCTGGCTCAG-3') and RD1 (5'-AAGGAGGTGATCCAGCC-3') primers was performed. PCR reaction was carried out using a 50 µL reaction mixture containing 0.2 mM dNTPs, 10X 10% Buffer, 0.2 µM of each primer, 2.0 mM MgCl₂, 0.3 U Taq DNA polymerase (5 U µL^-1) and template DNA (20 ng µL^-1). The PCR amplification was performed in an Applied Biosystems 2720 Thermal Cycler (Life Technologies, CA, USA), being initiated by incubating the reaction mixture at 94 °C for 3 min followed by 30 cycles of denaturation (94 °C for 50 s), annealing (57 °C for 50 s) and extension (72 °C for 60 s), and finished with a final extension step at 72 °C for 7 min.

The PCR products were confirmed by electrophoresis on a 1.2% agarose gel containing SybrGold® (Sigma-Aldrich, MO, USA) and viewed by exposure to UV light on transilluminator. The 1 kb Plus DNA ladder (Promega Corporation, WI, USA) was used as molecular weight marker. The PCR products were purified and sequenced by Macrogen (Seoul, Korea) and StabVida (Lisbon, Portugal). All sequences generated were compared with GenBank database for similarity search (https://blast.ncbi.nlm.nih.gov/Blast.cgi) Multiple sequence alignment was carried out with SeqMan, BioEdit, and ClustalW programs. The neighbor-joining phylogenetic analysis was performed using Molecular Evolutionary Genetics Analysis (MEGA) 5.1 program.

Plant growth-promotion and biocontrolling tests

Bacterial isolates identified as described previously were considered to biochemical characterization (plant growth-promotion and biocontrolling tests). The phosphate solubilization was tested using the National Botanical Research Institute's Phosphate (NBRIP) medium containing insoluble tricalcium phosphate as the single phosphorus source [²²22 Nautiyal C. An efficient microbiological growth medium for screening phosphate solubilizing microorganisms. FEMS Microbiol Lett 1999;170:265-70.]. After incubation at 28 °C for 72 h, all isolates forming clear halo zones were identified as phosphate-solubilizing bacteria. Based in halo zone and colony diameters, the index of phosphate solubilization was calculated. IAA production was quantified using the Salkowski-based colorimetric technique [²³23 Sarwar M, Kremer RJ. Enhanced suppression of plant growth through production of L-tryptophan-derived compounds by deleterious rhizobacteria. Plant Soil 1995;172(2):261-9.]. For this, bacteria isolates were incubated in Tryptic Soy Broth (TSB) culture media supplied with L-tryptophan under shaking condition (150 rpm; 28 °C) for 48 h. A separate TSB broth inoculated with sterile water was used as control. IAA production was quantitated spectrophotometrically at 530 nm by using Salkowski’s reagent (0.5 mM FeCl₃ and 35% HClO₄) and expressed as mg mL^-1.

The Chromeazurol-S (CAS) assay was used to detect siderophores [²⁴24 Shin SH, Lim Y, Lee SE, Yang NW, Rhee JH. CAS agar diffusion assay for the measurement of siderophores in biological fluids. J Microbiol Methods 2001;44:89-95.]. Bacterial isolates were cultured in TSB broth containing CAS for 120 h (28 °C) and isolates forming yellow halo zones around the colony were identified as siderophore-producing bacteria. N-acyl homoserine lactones (AHL) production was checked by growing bacterial isolates in TSB agar medium containing X-Gal (5-Bromo-4-chloro-3-indolyl-beta-D-galactoside) and the biosensor strain (Agrobacterium tumefaciens NT1) [²⁵25 Cha C, Gao P, Chen YC, Shaw PD, Farrand SK. Production of acyl-homoserine lactone quorum-sensing signals by gram-negative plant-associated bacteria. Mol Plant Microbe In 1998;11(11):1119-29.]. The production of AHL was confirmed by visualization of the blue halo around the well after 48 h. The ACC deaminase activity of bacterial isolates was screened based on their ability to use ACC as nitrogen source [²⁶26 Penrose DM, Glick BR. Methods for isolating and characterizing ACC deaminase-containing plant growth-promoting rhizobacteria. Physiol Plant 2003;118(1):10-5.]. All isolates were inoculated in TSB medium supplemented with 3.0 mM ACC for 72 h (28 °C). Petri plates without ACC and with 0.2% (w/v) ammonium sulfate were used as the negative and positive control, respectively. Bacterial isolates with ACC deaminase activity were identified comparing with the negative and positive controls.

Nitrogenase activity was measured using the acetylene reduction assay [²⁷27 Boddey RM, Boddey LH, Urquiaga S. A técnica de redução de acetileno na medição da fixação biológica de nitrogênio. Itaguaí: Editora Universidade Rural; 1990.]. For this purpose, aliquots of bacterial cultures (2.0 mL) were transferred into glass tubes and under a hypoxic atmosphere with 10% acetylene (C₂H₄) for 18 h. The quantification of the ethylene produced in the samples from each bottle was carried out in a gas chromatograph with flame ionization detector. Tubes without injected acetylene were used as negative control. After calculated, nitrogenase activity were expressed as ɳmol C₂H₄ hour^-1. Catalase activity was checked by placing drops of hydrogen peroxide onto the bacterial colony streaked on TSB broth and the appearance of bubbles was positive for the test [²⁰20 Yano DMY, Attili DS, Gatti MSV, Eguchi SY, Oliveira UM. Técnicas de microbiologia em controle de qualidade. Campinas: Fundação Tropical de Pesquisas e Tecnologia "André Tosello"; 1991.]. Gelatinase activity was detected in glass tubes using a TSB broth supplied with gelatin [²⁰20 Yano DMY, Attili DS, Gatti MSV, Eguchi SY, Oliveira UM. Técnicas de microbiologia em controle de qualidade. Campinas: Fundação Tropical de Pesquisas e Tecnologia "André Tosello"; 1991.]. The enzyme liquefies the growth medium by hydrolyzing gelatin and therefore gelatin liquefaction was considered a positive result.

Amylase activity was detected in TSB broth containing 1% soluble starch [²⁸28 Vedder EB. Starch agar: A new culture medium for the gonococcus. J Infect Dis 1915;16: 385.]. The Petri dishes were incubated in a growth chamber (23°C; 5 days) under dark conditions. Amylase was identified by a translucent halo around the colony in contrast with a purple background. To chitinase activity [²⁹29 Renwick A, Campbell R, Coe S. Assessment of in vivo screening systems for potential biocontrol agents of Gaeumannomyces graminis. Plant Pathol 1991;40:524-32.], the isolates were grown in TSB broth supplied with 0.5% colloidal chitin and positive cultures were identified by observing transparent halo surrounding the colony. Lipase activity was observed in a TSB culture media supplemented with 1.0% Tween 20 and incubated at 23 °C in the dark for five days [²⁹29 Renwick A, Campbell R, Coe S. Assessment of in vivo screening systems for potential biocontrol agents of Gaeumannomyces graminis. Plant Pathol 1991;40:524-32.]. Lipase activity was detected by the formation of a clear halo around the bacterial colony. The protease activity was measured using TSB culture medium supplied with casein and casein hydrolysis was visualized by the formation of a transparent halo [³⁰30 Dees C, Ringelberg STC, Phelps TJ. Characterization of the cellulose-degrading bacterium NCIMB 10462. Appl Biochem Biotechnol 1995;51-52:263-74.]. The isolates were grown in TSB culture media supplied with urea incubated for 48 h (20 °C) to measure urease activity [³¹31 Roberge MR, Knowles R. Ureolysis, immobilization and nitrification in black spruce (Picea mariana Mill.)-humus. Soil Sci Soc Am Proc 1966;30:201-4.]. After incubation, the presence of pink halo zones was positive for the test.

Statistical analysis

The results were subjected to analysis of variance (ANOVA) preceded by the F test at the 1% and 5% probability levels. The data were analyzed using the Shapiro-Wilk test to evaluate normality. Scott-Knott test (p < 0.05) was used to analyze the quantitative data (phosphate solubilization, IAA production, and nitrogenase activity), and statistical analyses were performed using the SAS software version 8.2. The network was built by using Pearson correlation coefficients with the Euclidian dissimilarity indices using the free statistics Past software version 4.0 (https://folk.uio.no/ohammer/past/)

RESULTS

Isolation of bacterial endophytes

Fifty-six bacterial isolates were obtained from leaves (21 isolates) and stems (35 isolates) of sugarcane (Supplementary tables 2 and 3). The isolates only grew in the NFb, JNFb, and LGI-P, while no isolates were found in the LGI or JMV broths. A total of 30 and 18 bacterial isolates were, respectively, grown in NFb and JNFb broths, while eight bacterial isolates were grown in LGI-P broth (IPA-CC49, IPA-CC51, IPA-CC52, IPA-CC55, IPA-CC56, IPA-CF61, IPA-CF62, and IPA-CF65).

Morphological and physiological traits

The morphological and physiological traits of bacterial isolates are summarized in Figure 1. Thirty-nine colonies of bacterial isolates were smaller than 1.0 mm, including 13 isolates from leaves and 26 isolates from stems, while 17 colonies were larger than 1.0 mm (Figure 1A). The majority of colonies isolated in leaves (78%) and stems (90%) were circular (Figure 1B), with a whole border (more than 70% of all isolates) and a smooth surface (85% and 92% of isolates from leaves and stems, respectively) (Figure 1B). Filamentous borders were found only in three bacterial colonies isolated from stems (isolates IPA-CC11, IPA-CC30B, and IPA-CC30C), and only one bacterial colony (IPA-CC11 from stems) displayed a filamentous surface. In general, bacterial colonies from leaves were translucent (60%), while bacterial colonies from stems were proportionally opaque (37%), translucent (29%), or transparent (34%). As showed in Figure 1C, yellow and cream were most prevalent colors observed in the bacterial colonies. The majority of bacterial isolates were able to produce acids (Figure 1D) and unable to produce mucus (Figure 1E). Furthermore, bacterial colonies displayed fast growth (88%) and were Gram-positive (63%) (Figure 1E and 1F).

Biochemical traits

Thirty-five bacterial isolates, 15 endophytes from leaves and 20 isolates from stems, were biochemically evaluated and presented characteristics of diazotrophic and plant growth-promoting bacteria (PGPB). In leaves, three isolates were recognized as diazotrophs and 12 bacterial isolates were recognized as PGPB. In contrast, 12 and 14 isolates from stems were identified as diazotrophs and PGPB, respectively. Isolates of Brevibacillus agri and Methylobacterium organophilum were present only in leaves, while isolates of Herbaspirillum seropedicae were found only in stems (Figure 2D). All bacterial isolates from the leaves and stems of the sugarcane plants synthesized IAA (Figure 3A). Except in IPA-CF45A, all bacterial isolates showed a positive nitrogenase activity (Figure 3B).

Bacterial isolates from leaves (80%) and stems (55%) solubilized phosphate (Figure 3C). Six isolates were identified as efficient siderophore-producing bacteria (Figure 4A and 4B). Fifty-four percent of diazotrophic endophytes (60% and 50% from leaves and stems, respectively) synthesized N-acyl homoserine lactones (AHL) (Figure 4C). Overall, 71% of diazotrophic bacterial endophytes were positive for ACC deaminase (67% and 75% of endophytes from leaves and stems, respectively). Over 50% of endophytes displayed activity of catalase, lipase and protease. Additionally, 17%, 23%, 14%, and 26% of bacterial isolates were positive for amylase, gelatinase, chitinase, and urease, respectively. The network analysis showed correlations between diazotrophic endophytes and PGPB (Figure 5).

DISCUSSION

Isolation of bacterial endophytes

In this study, 56 bacterial isolates were obtained from sugarcane tissues, being 21 and 35 isolates found in leaves and stems, respectively. Interestingly, we found a higher number of endophytic bacterial isolates in sugarcane tissues than those found in previous studies with different crops, such as sorghum [³²32 Silva JF, Silva TR, Escobar IEC, Fraiz ACR, Santos JWM, Nascimento TR, et al. Screening of plant-growth promotion ability among bacteria isolated from field-grown sorghum under different managements in Brazilian drylands. World J Microbiol Biotech 2018;34:186.] and sugarcane [³³33 Alam IT, Nesa SR, Alam KM, Begum A, Akhter H. Phenotypic and molecular characterization of diazotrophic bacteria associated with sugarcane in Bangladesh. Org Agr 2019;9(3):331-43.]. For instance, Patel and Archana [³⁴34 Patel JK, Archana G. Diverse culturable diazotrophic endophytic bacteria from Poaceae plants show cross-colonization and plant growth promotion in wheat. Plant Soil 2017;417:99-116.] isolated, through NFb broth, 31 endophytic diazotrophic bacteria in leaves, stems, and roots from Poaceae plants. By comparison, Leite and coauthors [¹¹11 Leite MCBS, Pereira APA, Souza AJ, Andrade PAM, Barbosa MV, Andreote FD, et al. Potentially diazotrophic endophytic bacteria associated to sugarcane are effective in plant growth-promotion. J Exp Agric Int 2018;21(3):1-15.] found 24 endophytic diazotrophic bacteria in stems and roots of sugarcane by using LGI-P broth, while Alam and coauthors [³³33 Alam IT, Nesa SR, Alam KM, Begum A, Akhter H. Phenotypic and molecular characterization of diazotrophic bacteria associated with sugarcane in Bangladesh. Org Agr 2019;9(3):331-43.] found 21 bacterial isolates in roots and stems of sugarcane by using LGI broth. A total of 30, 18 and 8 bacterial isolates grown, respectively, in NFb, JNFb and LGI-P broths. These results showed that the numbers of bacterial isolates varied considerably according to the different types of culture media. Previously, Magnani and coauthors [³⁵35 Magnani GS, Didonet CM, Cruz LM, Picheth CF, Pedrosa FO, Souza EM. Diversity of endophytic bacteria in Brazilian sugarcane. Genet Mol Res 2010;9:250-8.] reported variations in the number of bacterial isolates obtained with different types of culture media and stated that NFb and JNFb were more effective than LGI.

Morphological and physiological traits

According to the morphological and physiological analyses, the majority of bacterial colonies were smaller than 1.0 mm. In sugarcane, endophytic bacterial isolates (C2HL2, C34MR1, and UT3R1) with a size between 2.0 to 3.0 mm were previously described by Muangthong and coauthors [⁵5 Muangthong A, Youpensuk S, Rerkasem B. Isolation and characterization of endophytic nitrogen fixing bacteria in sugarcane. Tropical Life Sci Res 2015;26(1):41-51.]. As shown in Figure 1B, the majority of colonies isolated in leaves (78%) and stems (90%) were circular. This result agrees with Patel and coauthors [³⁴34 Patel JK, Archana G. Diverse culturable diazotrophic endophytic bacteria from Poaceae plants show cross-colonization and plant growth promotion in wheat. Plant Soil 2017;417:99-116.], who found 58% of endophytic bacterial colonies from roots of Brassica rapa being circular. Additionally, more than 70% of all bacterial isolates were translucent and displayed whole border and smooth surface (Figure 1B). Interestingly, only endophytes isolated from stems exhibited bacterial colonies with filamentous border. This variation in colony appearance is common and a previous study showed significant variation in endophytic isolates [⁹9 Mukherjee A, Singh B, Prakash VJ. Harnessing chickpea (Cicer arietinum L.) seed endophytes for enhancing plant growth attributes and bio-controlling against Fusarium sp. Microbiol Res 2020;237:126469.].

The production of pigments by various organisms is a common phenomenon and can represent an evolutionary advantage for many species. In this study, bacterial colonies exhibited different colors, being yellow and cream the most prevalent (Figure 1C). Kumar and coauthors [³⁶36 Kumar V, Jain L, Jain SK, Chaturvedi S, Kaushal P. Bacterial endophytes of rice (Oryza sativa L.) and their potential for plant growth promotion and antagonistic activities. S Afr J Bot. 2020;https://doi.org/10.1016/j.sajb.2020.02.017
https://doi.org/10.1016/j.sajb.2020.02.0... ] found only three colors (white, cream, and yellow) in bacterial colonies isolated from different rice plant tissues. Three bacterial isolates from stems had colorless colonies (transparent) (isolates IPA-CC30B, IPA-CC30C, and IPA-CC56). Interestingly, only IPA-CF19 produced pink colonies (Figure 1C). According to Tuli and coauthors [³⁷37 Tuli HS, Chaudhary P, Beniwal V, Sharma AK. Microbial pigments as natural color sources: current trends and future perspectives. J Food Sci Technol 2015;52(8):4669-78.], canthaxanthin is a pigment found in pink bacterial colonies that displays important biological attributes, such as photo-protectant, antioxidant, and antimicrobial activities. Thus, this isolate could have these characteristics and could be a potential plant growth-promoter.

Bromothymol blue was used to identify the bacterial ability to produce acids and alkalis in the culture broth, and the majority of bacterial isolates were able to produce acids (Figure 1D). The ability to produce acids contributes to pH stability and is particularly important to bacterial endophytes living in plant apoplast. The sugarcane apoplast maintains substantial levels of carbohydrates, organic acids, and amino acids, and these compounds can provide energy and nutrients to endophytic bacteria [⁸8 Defez R, Andreozzi A, Bianco C. The overproduction of indole-3-acetic acid (IAA) in endophytes upregulates nitrogen fixation in both bacterial cultures and inoculated rice plants. Microb Ecol 2017;74(2):441-52., ³⁸38 Silva PRA, Vidal MS, Soares CP, Polese V, Tadra-Sfeir MZ, Souza EM, et al. Sugarcane apoplast fluid modulates the global transcriptional profile of the diazotrophic bacteria Paraburkholderia tropica strain Ppe8. Plos One 2018;13(12):e0207863]. According to Geilfus [³⁹39 Geilfus CM. The pH of the apoplast: dynamic factor with functional impact under stress. Mol Plant 2017;10(11):1371-86.], the apoplast is slightly acidic (pH 5.5) and can be alkalinized in response to biotic and abiotic stresses. Therefore, the bacterial endophytes, capable of producing acids, can symbiotically live in sugarcane apoplast without causing a pathogenic effect. Indeed, Silva and coauthors [³⁸38 Silva PRA, Vidal MS, Soares CP, Polese V, Tadra-Sfeir MZ, Souza EM, et al. Sugarcane apoplast fluid modulates the global transcriptional profile of the diazotrophic bacteria Paraburkholderia tropica strain Ppe8. Plos One 2018;13(12):e0207863] reported that Paraburkholderia tropica, an endophytic diazotrophic bacteria with optimum growth in pH 5.0 to 5.8, altered its gene expression pattern in sugarcane apoplast, which allowed it to use the apoplast energy sources while avoiding host plant defense mechanisms.

Except to six endophytes from stems (IPA-CC4, IPA-CC22A, IPA-CC22B, IPA-CC31, IPA-CC32, and IPA-CC34), bacterial isolates were unable to produce mucus (Figure 1E). Mucus or extracellular polymeric substances (EPS), such as polyamides, polyesters, inorganic polyanhydrides, and polysaccharides, are polymers with different and important biological functions that are produced and excreted by many bacterial strains [³⁹39 Geilfus CM. The pH of the apoplast: dynamic factor with functional impact under stress. Mol Plant 2017;10(11):1371-86.]. Although EPS can protect the bacterial strains against abiotic stress, the exact physiological function of these biomolecules for endophytic bacteria is poorly understood. Tian and coauthors [⁴⁰40 Tian B, Zhang C, Ye Y, Wen J, Wu Y, Wang H, et al. Beneficial traits of bacterial endophytes belonging to the core communities of the tomato root microbiome. Agr Ecosyst Environ 2017;247:149-6.] report that EPS can create a favorable microenvironment and, therefore, contributes to the survival of endophytes inside the host plant. The EPS from endophytic bacteria helps to mitigate water loss by the host plant, which is critical in protecting the integrity of the xylem vessels in stems [⁷7 Compant S, Samad A, Faist H, Sessitsch A. A review on the plant microbiome: Ecology, functions and emerging trends in microbial application. J Adv Res 2019;19:29-37.].

Endophytic bacteria from sugarcane leaves and stems showed fast growth and were mainly Gram-positive (Figure 1F and 1G). In contrast, Magnani and coauthors [³⁵35 Magnani GS, Didonet CM, Cruz LM, Picheth CF, Pedrosa FO, Souza EM. Diversity of endophytic bacteria in Brazilian sugarcane. Genet Mol Res 2010;9:250-8.] and Alam and coauthors [³³33 Alam IT, Nesa SR, Alam KM, Begum A, Akhter H. Phenotypic and molecular characterization of diazotrophic bacteria associated with sugarcane in Bangladesh. Org Agr 2019;9(3):331-43.] reported that most of the endophytic bacteria from sugarcane are Gram-negative. On the other hand, the majority of bacterial endophytes from different plant tissues of rice were Gram-positive [³⁷37 Tuli HS, Chaudhary P, Beniwal V, Sharma AK. Microbial pigments as natural color sources: current trends and future perspectives. J Food Sci Technol 2015;52(8):4669-78.]. These results showed that Gram-positive bacteria are better able to adapt to stressful environments and can form spores when surrounding conditions are unfavorable, for example, in soil with low fertility. Thus, Gram-positive bacteria can survive and predominate in acidic environments, such as the apoplast.

Biochemical traits

After the morphophysiological characterization, 15 bacterial endophytes from leaves and 20 isolates from stems were biochemically evaluated. Previously, these isolates were molecularly characterized as belonging to phyla Proteobacteria and Firmicutes (Figure 2). According to Compant and coauthors [⁷7 Compant S, Samad A, Faist H, Sessitsch A. A review on the plant microbiome: Ecology, functions and emerging trends in microbial application. J Adv Res 2019;19:29-37.], Proteobacteria and Firmicutes are common endophytes of various plant tissues. This result agrees with Kumar and coauthors [³⁶36 Kumar V, Jain L, Jain SK, Chaturvedi S, Kaushal P. Bacterial endophytes of rice (Oryza sativa L.) and their potential for plant growth promotion and antagonistic activities. S Afr J Bot. 2020;https://doi.org/10.1016/j.sajb.2020.02.017
https://doi.org/10.1016/j.sajb.2020.02.0... ] and Tian and coauthors [⁴⁰40 Tian B, Zhang C, Ye Y, Wen J, Wu Y, Wang H, et al. Beneficial traits of bacterial endophytes belonging to the core communities of the tomato root microbiome. Agr Ecosyst Environ 2017;247:149-6.], who found Proteobacteria and Firmicutes as predominant phyla in rice and tomato, respectively. In other studies, endophytic bacterial communities associated with maize, rice, sorghum, and wheat were more diverse, and members of Proteobacteria, Firmicutes, Actinobacteria, and others were identified [⁸8 Defez R, Andreozzi A, Bianco C. The overproduction of indole-3-acetic acid (IAA) in endophytes upregulates nitrogen fixation in both bacterial cultures and inoculated rice plants. Microb Ecol 2017;74(2):441-52., ³⁴34 Patel JK, Archana G. Diverse culturable diazotrophic endophytic bacteria from Poaceae plants show cross-colonization and plant growth promotion in wheat. Plant Soil 2017;417:99-116.].

Overall, it is possible observe characteristics of diazotrophic and PGPB in bacterial endophytes isolated from sugarcane tissues. In leaves, three isolates were recognized as diazotrophs and 12 bacterial isolates as PGPB. In contrast, 12 and 14 isolates from stems were identified as diazotrophs and PGPB, respectively. Bacillus was the predominant genus of endophyte in sugarcane tissues (Figure 2). The existence of tissue-specific endophytes was observed when the endophytic bacterial communities from leaves and stems of sugarcane were compared. It is noteworthy that isolates of Brevibacillus agri and Methylobacterium organophilum were present only in leaves, while isolates of Herbaspirillum seropedicae were found only in stems (Figure 2D). Methylobacterium and Herbaspirillum are genera of diazotrophs belonging to the phylum Proteobacteria. This phylum includes bacterial endophytes that perform nitrogen-fixation in different tissues of monocots, including rice, maize, sorghum, and sugarcane [⁸8 Defez R, Andreozzi A, Bianco C. The overproduction of indole-3-acetic acid (IAA) in endophytes upregulates nitrogen fixation in both bacterial cultures and inoculated rice plants. Microb Ecol 2017;74(2):441-52., ³³33 Alam IT, Nesa SR, Alam KM, Begum A, Akhter H. Phenotypic and molecular characterization of diazotrophic bacteria associated with sugarcane in Bangladesh. Org Agr 2019;9(3):331-43., ³⁴34 Patel JK, Archana G. Diverse culturable diazotrophic endophytic bacteria from Poaceae plants show cross-colonization and plant growth promotion in wheat. Plant Soil 2017;417:99-116.].

Biochemical traits are important to plant growth, and recent studies have reported these characteristics in bacterial endophytes [⁹9 Mukherjee A, Singh B, Prakash VJ. Harnessing chickpea (Cicer arietinum L.) seed endophytes for enhancing plant growth attributes and bio-controlling against Fusarium sp. Microbiol Res 2020;237:126469., ³⁷37 Tuli HS, Chaudhary P, Beniwal V, Sharma AK. Microbial pigments as natural color sources: current trends and future perspectives. J Food Sci Technol 2015;52(8):4669-78.]. For instance, the ability to synthesize IAA, solubilize minerals (such as P and K), exudate siderophores, and produce AHL are important biochemical traits [³⁷37 Tuli HS, Chaudhary P, Beniwal V, Sharma AK. Microbial pigments as natural color sources: current trends and future perspectives. J Food Sci Technol 2015;52(8):4669-78.]. In this study, all bacterial isolates from sugarcane were able to synthesize IAA (Figure 3A). As a primary auxin in higher plants, IAA has marked effects on plant development. Previous studies on several plant species, such as chickpea, maize, rice, sorghum, sugarcane, tomato, and wheat, have reported bacteria releasing IAA and promoting plant growth [⁹9 Mukherjee A, Singh B, Prakash VJ. Harnessing chickpea (Cicer arietinum L.) seed endophytes for enhancing plant growth attributes and bio-controlling against Fusarium sp. Microbiol Res 2020;237:126469., ³³33 Alam IT, Nesa SR, Alam KM, Begum A, Akhter H. Phenotypic and molecular characterization of diazotrophic bacteria associated with sugarcane in Bangladesh. Org Agr 2019;9(3):331-43., ³⁴34 Patel JK, Archana G. Diverse culturable diazotrophic endophytic bacteria from Poaceae plants show cross-colonization and plant growth promotion in wheat. Plant Soil 2017;417:99-116., ⁴⁰40 Tian B, Zhang C, Ye Y, Wen J, Wu Y, Wang H, et al. Beneficial traits of bacterial endophytes belonging to the core communities of the tomato root microbiome. Agr Ecosyst Environ 2017;247:149-6.].

The isolates IPA-CC36 (30.8 µg mL^-1) and IPA-CC35 (0.01 µg mL^-1) exhibited highest and lowest IAA production levels, respectively (Figure 3A). These results are similar to previous studies that found values of IAA varying from 0.3 to 20.2 µg mL^-1 with diazotrophic endophytic bacteria from sugarcane roots [¹²12 Rodrigues AA, Forzani MV, Soares RDS, Sibov ST, Vieira JDG. Isolation and selection of plant growth-promoting bacteria associated with sugarcane. Pesqui Agropecu Trop 2016;46(2):149-58.]. Silva and coauthors [³²32 Silva JF, Silva TR, Escobar IEC, Fraiz ACR, Santos JWM, Nascimento TR, et al. Screening of plant-growth promotion ability among bacteria isolated from field-grown sorghum under different managements in Brazilian drylands. World J Microbiol Biotech 2018;34:186.] reported bacterial endophytes from sorghum, producing about 71.2 to 192.8 µg mL^-1 IAA. In chickpea, Mukherjee and coauthors [⁹9 Mukherjee A, Singh B, Prakash VJ. Harnessing chickpea (Cicer arietinum L.) seed endophytes for enhancing plant growth attributes and bio-controlling against Fusarium sp. Microbiol Res 2020;237:126469.] found endophytes in seeds releasing a significant amount of IAA, with the highest value being 58.9 µg mL^-1. This is an important finding since the synthesis and liberation of IAA improve cell elongation and stimulate the growth of roots and shoots [⁸8 Defez R, Andreozzi A, Bianco C. The overproduction of indole-3-acetic acid (IAA) in endophytes upregulates nitrogen fixation in both bacterial cultures and inoculated rice plants. Microb Ecol 2017;74(2):441-52., ¹⁰10 Kruasuwan W, Thamchaipenet A. Diversity of culturable plant growth-promoting bacterial endophytes associated with sugarcane roots and their effect of growth by co-inoculation of diazotrophs and actinomycetes. J Plant Growth Regul. 2016;35(4):1074-87.]. In legumes, inoculation with rhizobia and endophytes, such as Azospirillum, Bacillus, and Paenibacillus, contributed to the production and secretion of IAA and improved nodulation [⁹9 Mukherjee A, Singh B, Prakash VJ. Harnessing chickpea (Cicer arietinum L.) seed endophytes for enhancing plant growth attributes and bio-controlling against Fusarium sp. Microbiol Res 2020;237:126469.].

IAA produced by endophytes benefits the host plant and can also stimulate nitrogenase activity, i.e., high levels of IAA positively affect the N-fixing capability of the bacterial endophytes [⁸8 Defez R, Andreozzi A, Bianco C. The overproduction of indole-3-acetic acid (IAA) in endophytes upregulates nitrogen fixation in both bacterial cultures and inoculated rice plants. Microb Ecol 2017;74(2):441-52.]. In this study, nitrogenase activity, by acetylene reduction assay, was measured in all bacterial isolates. Except in IPA-CF45A, all bacterial isolates showed a positive nitrogenase activity, and IPA-CC33 (Pseudomonas sp.), IPA-CF65 (Bacillus megaterium), and IPA-CC9 (Herbaspirillum seropedicae) were the most promising (Figure 3B). Additionally, this study found a positive correlation between IAA levels and nitrogenase activity (r = 0.99). This result confirms that IAA can stimulate nitrogenase activity in these endophytes, as reported by Defez and coauthors [⁸8 Defez R, Andreozzi A, Bianco C. The overproduction of indole-3-acetic acid (IAA) in endophytes upregulates nitrogen fixation in both bacterial cultures and inoculated rice plants. Microb Ecol 2017;74(2):441-52.]. Muangthong and coauthors [⁵5 Muangthong A, Youpensuk S, Rerkasem B. Isolation and characterization of endophytic nitrogen fixing bacteria in sugarcane. Tropical Life Sci Res 2015;26(1):41-51.] detected nitrogenase activity in all endophytic bacteria from sugarcane and observed that the two isolates with the highest activity (C2HL2 and C7HL1) were also able to achieve high nitrogen contents in sugarcane leaves growing in N-free substrate.

In this study, the majority of diazotrophic bacterial endophytes were able to solubilize phosphate, being IPA-CC1B (Bacillus sp.) the most efficient (Figure 3C). Previous studies have found bacterial endophytic communities able to solubilize phosphate in leaves, stems, and roots of rice [³⁶36 Kumar V, Jain L, Jain SK, Chaturvedi S, Kaushal P. Bacterial endophytes of rice (Oryza sativa L.) and their potential for plant growth promotion and antagonistic activities. S Afr J Bot. 2020;https://doi.org/10.1016/j.sajb.2020.02.017
https://doi.org/10.1016/j.sajb.2020.02.0... ], in roots and stems of sugarcane [³³33 Alam IT, Nesa SR, Alam KM, Begum A, Akhter H. Phenotypic and molecular characterization of diazotrophic bacteria associated with sugarcane in Bangladesh. Org Agr 2019;9(3):331-43.], in seeds of chickpea [⁹9 Mukherjee A, Singh B, Prakash VJ. Harnessing chickpea (Cicer arietinum L.) seed endophytes for enhancing plant growth attributes and bio-controlling against Fusarium sp. Microbiol Res 2020;237:126469.], and in roots of sorghum [³²32 Silva JF, Silva TR, Escobar IEC, Fraiz ACR, Santos JWM, Nascimento TR, et al. Screening of plant-growth promotion ability among bacteria isolated from field-grown sorghum under different managements in Brazilian drylands. World J Microbiol Biotech 2018;34:186.]. The Bacillus genera encompass various phosphate-solubilizing microorganisms, and their ability to increase the soluble phosphate in soil and then promote adequate and vigorous plant growth have been reported [⁸8 Defez R, Andreozzi A, Bianco C. The overproduction of indole-3-acetic acid (IAA) in endophytes upregulates nitrogen fixation in both bacterial cultures and inoculated rice plants. Microb Ecol 2017;74(2):441-52.]. The application of efficient phosphate-solubilizers as microbial inoculants can increase the phosphorus-uptake in numerous crops [⁷7 Compant S, Samad A, Faist H, Sessitsch A. A review on the plant microbiome: Ecology, functions and emerging trends in microbial application. J Adv Res 2019;19:29-37.].

Phosphate solubilization and siderophore production by endophytes can increase the accessibility and absorption of different types of nutrients [⁸8 Defez R, Andreozzi A, Bianco C. The overproduction of indole-3-acetic acid (IAA) in endophytes upregulates nitrogen fixation in both bacterial cultures and inoculated rice plants. Microb Ecol 2017;74(2):441-52.]. Siderophores are metal chelating agents produced and secreted by bacteria that sequester and solubilize iron and other metals [⁷7 Compant S, Samad A, Faist H, Sessitsch A. A review on the plant microbiome: Ecology, functions and emerging trends in microbial application. J Adv Res 2019;19:29-37.]. In this study, IPA-CF44 and IPA-CC49 (Burkholderia sp.), IPA-CC8 and IPA-CC9 (Herbaspirillum seropedicae), and IPA-CC22A (Paenibacillus sp.) were identified as efficient siderophore-producing bacteria (Figure 4A and 4B). The results support previous studies in bacterial diazotrophic endophytes from maize, wheat, sorghum, and rice [³²32 Silva JF, Silva TR, Escobar IEC, Fraiz ACR, Santos JWM, Nascimento TR, et al. Screening of plant-growth promotion ability among bacteria isolated from field-grown sorghum under different managements in Brazilian drylands. World J Microbiol Biotech 2018;34:186., ³⁴34 Patel JK, Archana G. Diverse culturable diazotrophic endophytic bacteria from Poaceae plants show cross-colonization and plant growth promotion in wheat. Plant Soil 2017;417:99-116.]. The ability to produce siderophores by Proteobacteria has been reported [³⁴34 Patel JK, Archana G. Diverse culturable diazotrophic endophytic bacteria from Poaceae plants show cross-colonization and plant growth promotion in wheat. Plant Soil 2017;417:99-116.], and this is an important trait in beneficial microbes, whether they are endophytes or not [⁸8 Defez R, Andreozzi A, Bianco C. The overproduction of indole-3-acetic acid (IAA) in endophytes upregulates nitrogen fixation in both bacterial cultures and inoculated rice plants. Microb Ecol 2017;74(2):441-52.]. Microbial siderophores can supply the iron demand of plants by increasing its bioavailability, and also inhibit attacks from phytopathogens [⁷7 Compant S, Samad A, Faist H, Sessitsch A. A review on the plant microbiome: Ecology, functions and emerging trends in microbial application. J Adv Res 2019;19:29-37.]. Thus, the siderophores produced by endophytes can be the basis of a sustainable strategy to help plants tolerate abiotic stresses.

Beneficial bacteria able to synthesize AHL and release ACC deaminase enzymes are potentially useful for promoting better plant development in adverse environmental conditions [⁸8 Defez R, Andreozzi A, Bianco C. The overproduction of indole-3-acetic acid (IAA) in endophytes upregulates nitrogen fixation in both bacterial cultures and inoculated rice plants. Microb Ecol 2017;74(2):441-52.]. In this study, over 50% of diazotrophic bacterial endophytes were able to synthesize AHL (Figure 4C). Additionally, the majority of diazotrophic bacterial endophytes were positive for ACC deaminase, with this trait being registered in over 65% of bacterial endophytes from leaves and stems (Figure 4C). While AHL induces systemic resistance responses and contributes to disease suppression, ACC deaminase improves abiotic stress tolerance by diminishing ethylene levels while simultaneously increasing ammonia contents [¹⁰10 Kruasuwan W, Thamchaipenet A. Diversity of culturable plant growth-promoting bacterial endophytes associated with sugarcane roots and their effect of growth by co-inoculation of diazotrophs and actinomycetes. J Plant Growth Regul. 2016;35(4):1074-87.]. Thus, the endophytes from the sugarcane microbiome can benefit plant health and growth and are a promising tool for mitigating the negative effects of environmental stress.

The diazotrophic bacterial isolates were also tested for catalase, an enzyme involved in the antioxidant system, and considered an important indicator of stress tolerance in plants. The majority of the isolates were catalase positive and, therefore, showed the ability to convert hydrogen peroxide into water and molecular oxygen. In general, 47% of endophytes from leaves and 55% of isolates from stems were found to be catalase positive, and Bacillus isolates were predominant in this group (Figure 4). Previous studies have found that most bacterial endophytes in sugarcane [¹²12 Rodrigues AA, Forzani MV, Soares RDS, Sibov ST, Vieira JDG. Isolation and selection of plant growth-promoting bacteria associated with sugarcane. Pesqui Agropecu Trop 2016;46(2):149-58.], rice [³⁶36 Kumar V, Jain L, Jain SK, Chaturvedi S, Kaushal P. Bacterial endophytes of rice (Oryza sativa L.) and their potential for plant growth promotion and antagonistic activities. S Afr J Bot. 2020;https://doi.org/10.1016/j.sajb.2020.02.017
https://doi.org/10.1016/j.sajb.2020.02.0... ] and chickpea [⁹9 Mukherjee A, Singh B, Prakash VJ. Harnessing chickpea (Cicer arietinum L.) seed endophytes for enhancing plant growth attributes and bio-controlling against Fusarium sp. Microbiol Res 2020;237:126469.] are catalase positive. Plant exposure to a variety of biotic and abiotic stresses results in the production of reactive oxygen species (ROS), including hydrogen peroxide. Catalase prevents cell toxicity and can be useful protection against oxidative damage by ROS.

Commonly, inhibition of the growth of plant pathogens is related to the ability of endophytic bacteria to release various extracellular enzymes [⁸8 Defez R, Andreozzi A, Bianco C. The overproduction of indole-3-acetic acid (IAA) in endophytes upregulates nitrogen fixation in both bacterial cultures and inoculated rice plants. Microb Ecol 2017;74(2):441-52.]. In this study, it was observed that bacterial isolates were positive for amylase, gelatinase, chitinase, urease, lipase and protease (Figure 4). Additionally, the isolates of Bacillus, Paenibacillus and Pseudomonas were able to secrete three or more of these enzymes (Figure 4A and 4B). The penetration, infection, and colonization of plant tissues by endophytes are reportedly assisted by the extracellular enzymes they produce [³⁹39 Geilfus CM. The pH of the apoplast: dynamic factor with functional impact under stress. Mol Plant 2017;10(11):1371-86.]. These extracellular enzymes can also improve the uptake and turnover of nutrients [⁸8 Defez R, Andreozzi A, Bianco C. The overproduction of indole-3-acetic acid (IAA) in endophytes upregulates nitrogen fixation in both bacterial cultures and inoculated rice plants. Microb Ecol 2017;74(2):441-52.]. More bacterial isolates positive for chitinase, lipase, and protease were observed in the stem than in the leaves (Figure 4). Stems are responsible for transporting water and nutrients between roots and leaves and, therefore, the extracellular enzymes from endophytes can be useful in translocating nutrients between different parts of the plant and provide better nutrition and carbon gain to the host. Interestingly, the network analysis showed the isolates of Burkholderia, Herbaspirillum, and Methylobacterium to have biochemical correlations with Bacillus, mainly in stems (Figure 5).

Overall, we demonstrated that bacterial endophytes isolated in sugarcane leaves and stems presented distinct morphological, physiological and biochemical traits. Interestingly, except to isolate IPA-CF45A, all bacterial endophytes were positive for nitrogenase activity, which is an important trait related to biological nitrogen fixation. All bacterial endophytes exhibited important plant growth-promoting traits, such as phosphate solubilization, production of IAA, AHL, and siderophores, as well as ACC deaminase, catalase and extracellular enzyme activity. Further studies should explore the potential of these endophytes for sustainable agriculture, providing nutrients, mainly phosphorus and nitrogen, protecting against phytopathogens, and promoting plant growth, particularly in sugarcane fields.

Acknowledgments:

The authors are grateful to the National Council for Scientific and Technological Development (CNPq) for financial support (CNPq-Universal grant 426655/2018-4). Jadson E. L. Antunes thanks the Coordination for the Improvement of Higher Education (CAPES) for providing a scholarship. The authors sincerely thank to Agronomic Institute of Pernambuco (IPA, Recife/PE) to support this study. Ana D. S. Freitas, Carolina E. R. S. Santos, Ademir S. F. Araujo and Marcia V. B. Figueiredo are research fellows of CNPq.

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