Vol. 16(30), pp. 1619-1629, 26 July, 2017 DOI: 10.5897/AJB2017.16016 Article Number: D4EE05965435 ISSN 1684-5315 African Journal of Biotechnology Copyright © 2017 Author(s) retain the copyright of this article http://www.academicjournals.org/AJB

Full Length Research Paper

Evaluation of plant-growth promoting properties of Gluconacetobacter diazotrophicus and Gluconacetobacter sacchari isolated from sugarcane and tomato in West Central region of Colombia

Gloria M. Restrepo1, Óscar J. Sánchez2*, Sandra M. Marulanda1, Narmer F. Galeano1 and Gonzalo Taborda3

1Biological Research Group, Research Institute of and Agro-industrial Biotechnology, Universidad Católica de Manizales, Carrera 23 No. 60-63, 170002, Manizales, Colombia. 2Research Group on Food and Agro-industry, Department of Engineering, Universidad de Caldas, Calle 65 No. 26-10, 170004, Manizales, Colombia. 3Research Group on Chromatography and Related Techniques, Department of Chemistry, Universidad de Caldas, Calle 65 No. 26-10, 170004, Manizales, Colombia.

Received 1 April, 2017; Accepted 29 June, 2017

The genus Gluconacetobacter comprises different species with agricultural and industrial importance. This study aims at determining the presence of Gluconacetobacter diazotrophicus and Gluconacetobacter sacchari in sugarcane and tomato crops considering their potential biotechnological applications. were isolated from roots, stems and leaf tissues and identified using phenotypic and biochemical evaluations, and molecular and phylogenetic analyses. Isolates were characterized by the production of indolic compounds, nitrogenase activity and phosphate solubilization. For this, G. diazotrophicus ATCC 49037 was used as the reference strain. The results showed that all isolates of native G. diazotrophicus exhibited equal or better phosphate solubilization index (SI) for CaCO3 and AlPO4 than reference strain. For G. sacchari, GIBI031 isolate displayed better SI for both phosphate sources and GIBI014 isolate had better SI only for AlPO4. G. diazotrophicus GIBI029 had a greater production of indolic compounds than ATCC 49037 strain in the presence of tryptophan. All isolates except G sacchari GIBI031, showed better nitrogenase activity than the control. These results constitute the first report confirming the presence of G. diazotrophicus and G. sacchari associated with sugarcane in Colombia. In addition, this is the first report on the presence of G. sacchari in tomato under natural conditions. Finally, one G. sacchari isolate presented nitrogenase activity despite the fact that this is a differential characteristic between G. diazotrophicus and G. sacchari. These findings have ecological significance and will advance research towards the evaluation of plant-soil interactions involving these bacteria in crops other than sugarcane. The isolates found are potential candidates for the development of novel biotechnological processes for production of new alternative biofertilizers considering the significant plant-growth promotion properties determined in this work.

Key words: Sugarcane, tomato, Gluconacetobacter diazotrophicus, Gluconacetobacter sacchari, indolic compounds, phosphate solubilization, nitrogenase activity.

1620 Afr. J. Biotechnol.

INTRODUCTION cell division, and formation of the roots. They are applied during the in vitro cultivation of plant material and in The increase of world population has led to the excessive plantations (Castillo et al., 2005). use of chemical fertilizers causing soil erosion and loss of In this sense, bacteria of the genus Gluconacetobacter its physicochemical and microbiological equilibria have a huge potential. This genus was described by provoking negative environmental impacts (White and Yamada and Yukphan (2008) and includes gram Brown, 2010). For these reasons, the need to produce negative bacteria. Gluconacetobacter bacteria, belonging alternative environmentally friendly agricultural supplies to the class , order contributing to enhance the crop yield to meet the global and family , do not form endospores, food demand has arisen. An important amount of bacteria and are obligate aerobes. G. liquefaciens, G. exhibit plant-growth promoting properties and are being azotocaptans, G. diazotrophicus, G. hansenii, G. employed for production of biofertilizers alternative to johannae and G. sacchari, among others are the main chemical fertilizers. However, the production volume and species of the genus (Sievers and Swings, 2005). availability of these bio-inputs are still small so the Gluconacetobacter diazotrophicus is an endophytic plant- research in the isolation, identification, and growth promoting bacterium first reported by Cavalcante characterization of new or already discovered bacterial and Döbereiner (1988) associated with sugarcane in strains is of paramount importance to develop new Brazil. This bacterium has been isolated from sugarcane biotechnological products and processes relevant for the cultivars in different countries (Bellone et al., 1997; world agriculture. Fuentes et al., 1993; Li and Macrae, 1991). Likewise, it Plant-growth promoting rhizobacteria are soil has been recovered from other crops like coffee (Jiménez microorganisms growing in the surface of the root and in et al., 1997), pineapple (Tapia et al., 2000), carrot the space surrounding it. They are directly or indirectly (Madhaiyan et al., 2004), and rice (Muthukumarasamy et related to the growth promotion of the plants (Ahemad al., 2005). G. diazotrophicus is a subject of great interest and Kibret, 2014). The direct-action mechanisms include among nitrogen-fixing bacteria, as it fixes nitrogen under nitrogen biological fixation, phosphate solubilization, iron aerobic growth conditions (Eskin et al., 2014). This sequestering, and production of phytohormones and 1- property allows it to perform the process in non- aminocyclopropane-1-carboxylate deaminase. Main leguminous plants. Additionally, nitrates do not inhibit indirect mechanism is related to their function as nitrogenase activity; this is because G. diazotrophicus biocontrol agents, which is evidenced through the lacks the nitrate reductase enzyme (Cavalcante and competition for nutrients and space, induction of systemic Döbereiner, 1988). Another feature of interest that has resistance and production of siderophores or antifungal been attributed to this bacterium is its ability to solubilize metabolites (Glick, 2012). Biological nitrogen fixation phosphates (Stephen et al., 2015) and zinc (Crespo et associated with vascular plants is the most important al., 2011; Natheer and Muthukkaruppan, 2012). This process for nitrogen input to the natural ecosystems solubilization ability allows G. diazotrophicus to access (Monteiro et al., 2012). This process converts the insoluble nutrients and make them available for atmospheric nitrogen into ammonia. Nitrogen-fixing absorption by plants. G. diazotrophicus produces auxins bacteria have an enzyme, called nitrogenase, which such as 3-indoleacetic acid (IAA) (Eskin et al., 2014), catalyzes the breakdown of the triple covalent bond of the which highlights its potential even more. IAA has control nitrogen molecule at ambient temperature and pressure over many important developmental processes in plants, (Eskin et al., 2014). Phosphorus is the second inorganic including cell division and growth, vascular tissue nutrient necessary for all life forms. It is an essential differentiation, root initiation, apical dominance, element in molecules such as RNA, DNA, and ATP, as phototrophic and gravitropic responses, flowering, fruit well as phospholipids (Ahemad and Kibret, 2014). In ripening, leaf senescence and leaf and fruit abscission several tropical soils, phosphorus predominates in the (De Souza et al., 2015; Hernández-Rodríguez et al., form of inorganic insoluble phosphates. These insoluble 2010; Malekpoor Mansoorkhani et al., 2014). forms are not absorbed by the plants (Bashan et al., G. sacchari has been isolated from the pods of 2013). Inorganic phosphate can be divided into two sugarcane leaves and the pink sugar-cane mealy bug groups: Calcium phosphates and iron and aluminum attacking this crop. G. sacchari does not fix nitrogen as phosphates. On the other hand, phytohormones are G. diazotrophicus does (Franke et al., 1999). It is a important metabolites promoting the plant growth. The bacterium efficient for cellulose production when grown in auxins are a group of phytohormones regulating the plant glucose-based media and the yield and quality of the development and having a direct effect on the growth, cellulose obtained is comparable to those of other

*Corresponding author. E-mail: [email protected].

Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Restrepo et al. 1621

cellulose-producing bacteria (Trovatti et al., 2011). samples had the same characteristics as the original culture, No studies have been published concerning the isolation in LGI-NPs medium was performed. The colonies were presence and potential use of bacteria belonging to the purified on PDA. The strain of G. diazotrophicus PAL5 (ATCC 49037) was used as a positive control. genus Gluconacetobacter under the agro-climatic conditions of Colombia. The aim of this study was to determine the plant-growth promoting properties of Biochemical characterization of isolates bacteria of the genus Gluconacetobacter obtained from tomato and sugarcane cultivars in West Central region of The biochemical test API 20NE (BioMérieux, France) was performed Colombia and identify potential isolates for the as directed by the manufacturer. Additionally, gram stains and development of biofertilizer-type inoculants for oxidase and catalase tests were completed (Cavalcante and Döbereiner, 1988; Dong et al., 1995; Gillis et al., 1989). Isolates economically important crops. were stored at -80°C in a 15% glycerol solution.

MATERIALS AND METHODS PCR identification of isolates Sampling location Preserved isolates were reactivated. One colony from each plate Samples from sugarcane (Saccharum officinarum L.) and tomato was picked and transferred to 5 mL modified DYGS broth (Solanum lycopersicum) were collected in the West Central region (Rodrigues Neto et al., 1986). Cultures were incubated at 30°C of Colombia. Sampling was performed at sites where no chemicals under stirring at 150 rpm for 72 h. Cultures were centrifuged for 10 had been recently applied. A total of 10 plants were collected at min at 2500 rpm, and pellets were washed twice with sterile 0.85% each site. A bromatological analysis of each soil sample was made. saline solution. Extraction and purification of genomic DNA was performed using The sugarcane sampling was carried out in crops at three farms ® with different planting times (270, 360 and 450 days) located at a PureLink® Genomic DNA kit (Invitrogen , USA) and the 919, 1001 and 1121 m.a.s.l., respectively; from each plant sample, recommended protocol for gram-negative bacteria. The quality of leaves, stems and roots were collected. Additionally, samples from extraction was verified by 1% agarose-ethidium bromide a greenhouse-grown tomato crop were taken at the “Tesorito” farm electrophoresis using λ/Hind III phage as a molecular weight at the University of Caldas. Sampling was performed three times at marker. different stages of the production cycle (50, 100 and 150 days). The 16S rDNA subunit was amplified via PCR from the previously Stem and root samples were collected. extracted genomic DNA. For this reaction, the universal primers RB and RM (Madhaiyan et al., 2004) were used; the expected fragment was approximately 800 bp. The final volume of the PCR reaction Culture media and growing conditions was adjusted to 25 µL (1X PCR buffer, 0.2 mM dNTPs, 1 mM MgCl2, 0.5 μM primers, 1 U of polymerase taq DNA recombinant A nitrogen-free medium that allows for the enrichment of samples polymerase by Invitrogen and 100 ng of bacterial DNA). The and the selection of diazotrophic bacteria (LGI-P semisolid medium) amplification was performed using a T100 thermocycler (Bio-Rad, was initially used (Cavalcante and Döbereiner, 1988). Isolation was USA) according to the following amplification program: initial performed using LGI-PNs agar. LGI-PNs agar has the same denaturation at 95°C for 10 min, then 35 cycles at 92°C for 1 min, composition per liter as the semisolid LGI-P medium, with the 57°C for 30 s and 72°C for 1 min. The PCR products were analyzed via 1% agarose-ethidium bromide electrophoresis. Sequencing of addition of 50 mg of yeast extract and 15 g of agar-agar. Bacteria of ® interest were purified using potato dextrose agar (PDA) (Oxoid®, the PCR products was performed by Macrogen (Republic of United Kingdom). Modified DYGS broth (Rodrigues Neto et al., Korea). 1986) was used to obtain the biomass needed for polymerase chain reaction (PCR) assays and the evaluation of indolic compound production and phosphate solubilization. Phosphate solubilization Bioinformatics analysis was evaluated using National Botanical Research Institute's Phosphate (NBRIP) medium, which was individually supplemented Quality analysis of the obtained sequences was performed using -1 ® with Ca3(PO4)2 and AlPO4 at 5 g×L (Nautiyal, 1999). the CLC Main Workbench program (Qiagen Company, USA). Sequences with a Phred value less than 30 per base and a size less than 600 bases were rejected. Isolation of Gluconacetobacter spp. The sequences were annotated using BLASTN version 2.2.27+ (Altschul et al., 1990). Sequences were compared against the nt Collected samples were washed with tap water, disinfected with database of the National Center for Biotechnology Information 0.1% Tween 80 aqueous solution and washed with sterile distilled (NCBI), with an e-value threshold of 1×10-5. These results were water (Dibut et al., 2004). The samples were macerated in 1% filtered with 80% similarity and 80% coverage of the region sucrose solution using a low-speed blender for 30 s. The amplified with RM/RB primers. Once the BLASTN descriptions were homogeneous mixture obtained was serially diluted from 10-1 to 10-3; obtained, a database of sequences belonging to the genus then, 100 μL aliquots were plated in triplicate in 6-mL vials Gluconacetobacter was built and validated using data from RDP containing 3 mL of semisolid LGI-P medium (Cavalcante and (Ribosomal Database Project). Clustal W version 2.1 (Larkin et al., Döbereiner, 1988; Madhaiyan et al., 2004). 2007) was used for alignments. Phylogenetic tree was built using Cultures were incubated at 30°C for 6 to 7 days. The following the maximum likelihood method based on the Hasegawa-Kishino- criteria were used to select isolates with typical characteristics of Yano model (Hasegawa et al., 1985). The percentage of trees in Gluconacetobacter spp. (Cavalcante and Döbereiner, 1988): Gas which the associated taxa clustered together is shown next to the production, formation of a yellow-orange film on the medium branch (bootstrap value). The analysis involved 14 nucleotide surface, and a color change. Cultures matching these criteria were sequences. All positions containing gaps and missing data were grown again in semisolid LGI-P medium. Once it was verified that eliminated. There is a total of 577 positions in the final dataset. 1622 Afr. J. Biotechnol.

Evolutionary analysis were conducted in MEGA7 (Kumar et al., Statistical analysis 2016). Analysis of variance was performed using the GLM procedure of SAS (SAS Institute, Cary, USA) to determine the occurrence of Assessment of growth-promoting properties significant differences among treatments for all evaluated variables. The comparison of means was made using the Duncan test (p- value <0.05). The evaluation of the growth-promoting properties of isolates was carried out using inocula obtained by transferring colonies to 5 mL tubes containing DYGS medium (pH 6.5). These inoculates were incubated at 30°C with stirring at 150 rpm for 4 days. The cells were RESULTS washed three times with 0.85% isotonic saline solution, and the 8 -1 pellet was resuspended to a cell concentration of 10 cells×mL Isolation, biochemistry, characterization and (OD = 0.9 - 1). 600 molecular identification of Gluconacetobacter spp.

Nitrogenase activity Soils used for evaluated crops were rich in organic matter (between 13.0 and 20.4%), with pH values between 4.4 Bacterial nitrogenase activity was determined using the nitrogen- and 5.9 and nitrogen levels between 0.40 and 0.68%, free semisolid LGI-P medium and the acetylene-reduction assay according to analyses performed by the Laboratory of (ARA) as described by Muthukumarasamy et al. (1999), Videira et Chemistry and Soil Fertility at the Universidad de Caldas. al. (2007) and Boddey et al. (2007) with some modifications. A 8 -1 Morphotypes matching the selection criteria, that is, the suspension of 20 μL at cell concentration of 10 cells×mL was colonies obtained from a yellow-orange surface film that used to inoculate 5 mL of LGI-P medium. Cultures were incubated at 30°C for 48 h. Subsequently, 10% v/v of the culture vial caused clearance on LGI-PN culture medium and formed atmosphere was replaced with acetylene gas. After incubation at brown colonies in PDA medium (Figure 1), were 30°C for 24 h, 500 µL gas samples were analyzed on a GCMS- considered as presumptive Gluconacetobacter spp. A QP2010 Plus (Shimadzu®) gas mass chromatography flame total of 16 of the 21 isolated colonies from sugarcane ionization detector using a GS-Alumina column (Agilent presented the characteristics described previously. Only Technologies ®). Each sample was evaluated six times. The results four of the 33 isolates from tomato presented this were expressed as nmol reduced acetylene×mL-1×h-1 and nmol reduced acetylene×108 cells×h-1. pigmentation in LGI-P, LGI-PN and PDA culture media. PCR with the RB/RM universal primers resulted in the amplification of products of the expected size (800 bp) for Assessment of indolic compounds 54 presumptive isolates. Through a BLASTN analysis of the 54 PCR products amplifying the 16S rRNA gene, two Total indolic compounds were determined by spectrophotometry isolates were identified as G. diazotrophicus and three using Salkowski reagent (Glickmann and Dessaux, 1995; Gordon isolates as G. sacchari. The two G. diazotrophicus and Weber, 1951; Sarwar and Kremer, 1995). A recovered colony isolates from sugarcane were obtained from root (coded grown in PDA medium was transferred to the DYGS medium and incubated at 30°C under stirring at 150 rpm for 24 to 48 h. Two as GIBI025) and stem (coded as GIBI029) samples with washes were performed with sterile distilled water, and the a BLAST identity (BI) of 99.37% in both cases and global inoculum was prepared at a final concentration of approximately 108 alignment identity (GI) of 98.42 and 98.24%, respectively. cells×mL-1. Two of the three G. sacchari isolates were recovered Indolic compounds production was induced by adding L- ® from sugarcane: GIBI014 (BI 100% and GI 98.78%) and tryptophan (1.01%) (Panreac , USA) to the DYGS medium. GIBI031 (BI 100% and GI 98.95%); the third isolate was Cultures were incubated at 30°C and 150 rpm for 60 h. The determination of indolic compounds was made in the supernatant recovered from tomato and coded as GIBI141 (BI 99.83% using Salkowski reagent and by reading absorbance at a and GI 99.65%). For greater identification reliability, wavelength of 540 nm. Each sample was analyzed in triplicate. The phylogenetic analyses supported by the scientific levels of indolic compounds were estimated from a standard curve literature were performed with reported reference of 150 ppm of indolic acetic acid (Merck®, USA) and were -1 sequences. An alignment was performed over 543 bp expressed in µg×mL . that included the sequences obtained and 8

representative sequences from different species from the genus Gluconacetobacter. The tree was rooted using the Phosphate solubilization 16S rRNA sequence of Burkholderia cepacia NR 114491

Phosphate solubilization was assessed using NBRIP medium with as shown in Figure 2. In this figure, two groups among bromocresol green (22 mg×L-1) (Nautiyal, 1999), which was Gluconacetobacter isolates can be observed. One group -1 supplemented individually with 5 g×L of Ca3(PO4)2 or AlPO4. To includes G. johanae, G. azotocaptans, and G. each plate, 20 µL volume of the inoculum was added in triplicate. diazotrophicus. The other cluster is formed by G. Readings at 24, 48 and 72 h were performed. The diameter of the liquefaciens and G. sacchari. These findings are colony and the size of solubilization halo were determined in order consistent with the classification of the Gluconacetobacter to calculate the solubilization index SI (Kumar and Narula, 1999) and the percentage of solubilization efficiency SE (Nguyen et al., genus described by Yamada and Yukphan (2008). 1992). Additionally, the production of organic acids was evaluated Isolates were positive for catalase and indolic by noting the changes in the pH of the culture medium. compounds and negative for oxidase and the liquefaction Restrepo et al. 1623

Figure 1. (a) Orange-yellow surface film and clearance on the LGI-PN culture medium. (b) Orange colonies in the LGI-PN medium incubated during 8 days. (c) Brown colonies from potato dextrose medium incubated during 8 day.

Figure 2. Phylogenetic tree of Gluconacetobacter spp. of 16S rRNA gene. The tree was rooted using the sequence of Burkholderia cepacia (NR 114491). G diaz KX96238 (GIBI029); G diaz KX96237 (GIBI025); G sacc KX965664 (GIBI014); G sacc KX965665 (GIBI031); G sacc KX965666 (GIBI141). Accession numbers correspond to GenBank nomenclature (Appendix 1). G. azotocaptans (G azot); G. johannae (G joha); G. diazotrophicus (G diaz); G. liquefaciens (G liqu); G. sacchari (G sacc); B. cepacia (B cepa). 1624 Afr. J. Biotechnol.

Table 1. Nitrogenase activity of Gluconacetobacter spp.

Nitrogenase activity Isolate nmol reduced acetylene×mL-1×h-1 nmol reduced acetylene×108 cells-1×h-1 Mean Std dev Mean Std dev GIBI025 0.007b 0.0005 0.037c 0.0005 GIBI029 0.010a 0.0015 0.053b 0.0070 GIBI014 0.006c 0.0010 0.028d 0.0050 GIBI031 0.001d 0.0010 0.004e 0.0040 GIBI141 0.012a 0.0000 0.060a 0.0015 ATCC 49037 0.005c 0.0005 0.029d 0.0020

Values obtained are averages of three replicates. Identical letters indicate the absence of statistically significant differences. Std dev: Standard deviation.

Table 2. Indolic compound production after 60 h of incubation and phosphate solubilization after 72 h of incubation.

Indolic compounds Ca3(PO4)2 AlPO4 Isolate (µg×mL-1) SIa SEa APIa Mean Std dev Mean Std dev Mean Std dev Mean Std dev GIBI025 17.75d 1.75 3.333ab 0.297 233.3ab 29.737 13.33b 1.154 GIBI029 78.55a 0.00 3.619a 0.357 261.9a 35.952 14.93ab 1.616 GIBI014 48.28c 4.80 2.587c 0.075 158.3c 7.216 13.33b 1.154 GIBI031 46.61c 4.27 3.547a 0.144 254.2a 14.433 15.87a 1.616 GIBI141 7.75d 0.27 1.613d 0.105 61.3d 10.764 5.47c 0.161 ATCC 49037 64.28b 13.53 3.097b 0.218 209.5a 21.823 14.00ab 0.000

Values obtained are averages of three replicates. Identical letters indicate the absence of statistically significant differences. API: Acid production index; SI: Solubilization index; SE: Solubilization efficiency; Std dev: Standard deviation.

of gelatin. Additionally, isolates grew at pH 5.5 and 30°C. compounds (78.547 µg×mL-1) including the reference The biochemical characteristics of G. diazotrophicus strain (64.28 µg×mL-1). The GIBI141 and GIBI025 isolates isolates are similar to the reference strain ATCC 49037 presented the lowest concentrations of indolic compounds (Sievers and Swings, 2005). (7.75 and 17.74 µg×mL-1, respectively). Statistically significant differences between GIBI029 and GIBI025 isolates were observed (p-value < 0.05) (Table 2). Growth-promoting properties

Nitrogenase activity Phosphate solubilization capacity

Table 1 shows the test results for nitrogenase activity for Phosphate solubilization halos were not present in plates the Gluconacetobacter spp. isolates compared to the with aluminum phosphate; only color change in halos was characteristics of the reference strain. The GIBI141 strain present, which indicates acid production (Table 2). In this presented higher nitrogenase activity than the other test, the rate of acid production was calculated by isolates and the reference strain while the GIBI031 comparing the halo diameter for the color change to the isolate exhibited values lower than the reference strain. diameter of the bacterial colony. To determine the These isolates showed statistically significant differences efficiency of the tricalcium phosphate solubilization, the at 5% significance level. percentage of solubilization efficiency was evaluated after 72 h of incubation. Only one G. sacchari isolate (GIBI031) showed better solubilization efficiency than the Production of indolic compounds reference strain with a value statistically equal to the best G. diazotrophicus isolate (GIBI029). In the case of According to the measurements, isolate GIBI029 aluminum phosphate, GIBI031 and GIBI029 isolates produced the highest concentration of total indolic exhibited a performance statistically equal to that of the Restrepo et al. 1625

reference strain (p-value <0.05). by Cavalcante and Döbereiner (1988) and Baldani et al. (2014). The recovered isolates presumably formed exopolysaccharides (EPS), which were evidenced by the DISCUSSION mucoid and shiny appearance of the colonies. Cultures in liquid media also increased in viscosity between 60 and The strains evaluated in this study were isolated from the 72 h of incubation as described by Meneses et al. (2011). roots (GIBI025), leaves (GIBI014) and stems (GIBI029, Exopolysaccharide biosynthesis is required for the biofilm GIBI031) of a 270-day-old sugarcane crop; GIBI141 formation during plant colonization by the endophytic isolate was recovered from tomato stems. This is bacterium G. diazotrophicus (De Souza et al., 2015). consistent with reported studies in which G. For the isolates obtained, macroscopic and microscopic diazotrophicus bacteria were recovered from sugarcane features and the results of the biochemical tests were in Brazil (Cavalcante and Döbereiner, 1988), Australia (Li consistent with the most parsimonious tree built using the and Macrae, 1991), Mexico (Fuentes et al., 1993) and 16S rRNA gene. Clustering of the genus Argentina (Bellone et al., 1997). The source of the G. Gluconacetobacter (bootstrap> 50) was evidenced when sacchari isolates (GIBI014 and GIBI031) corresponds to compared to sequences from different genera (data not that reported by Franke et al. (1999). In addition, this shown). The tree containing Gluconacetobacter spp. species was also recovered from tomato stems (GIBI141) (Figure 2) showed clustering between G. of a 150-day-old crop, for which there is no any work diazotrophicus and taxonomically related bacteria such reporting this antecedent in the available scientific as G. azotocaptans and G. johannae. These findings literature. It is worthy to highlight that the isolation of G. were supported by a bootstrap value greater than diazotrophicus and G. sacchari has not been previously 50 (Matsutani et al., 2010) and also were consistent with reported in Colombia. G. diazotrophicus and G. sacchari the similarities found among the groups with some GI are endophytic bacteria whose occurrence has been values below 98.65%, the threshold suggested by Kim et proven in the sugarcane tissues by Beneduzi et al. (2013) al. (2014) to be considered within the same species. In and Franke-Whittle et al. (2005), respectively. the cluster of G. sacchari and G. liquefaciens isolates, the The high content of assimilable sugars favors the grouping and bootstrap values are in agreement with the presence of G. diazotrophicus. In natural environments, identity percentage, which was above 98.78% for the these bacteria use the organic acids in sugarcane juice three isolates. This provides a greater certainty on the and other sugars present (like glucose) in the taxonomic allocation of G. sacchari isolates. Despite the aboveground part of the plant as energy sources. This is results of the tree were not conclusive for the genomic evidenced in vitro by the growth of this species in culture region used, the BLAST results (identities greater than media with 10% sucrose at pH 5.5. These conditions are 98.65% in the 16S rRNA gene) let us infer that the similar to those of sugarcane juice (Baldani et al., 2014). isolates can be associated to the G. diazotrophicus Additionally, the isolates of the genus Gluconacetobacter species with a high degree of certainty. They were from sugarcane were obtained from a 270-day-old crop. associated with reference sequences of the species G. Samples from such plants have lower percentage of azotocaptans and G. diazotrophicus. Although the 16S lignified roots and stems compared to samples taken ribosomal gene provides evolutionary information, it from older sugarcane crops. In coffee crops, Jiménez et should be noted that only a comparison of complete al. (1997) observed greater recovery of endophytic genomes could precisely define evolutionary relationships bacteria from young plant tissues. This was because between closely related microorganisms (Mende et al., processing those samples facilitates tissue homo- 2013). The use of the 16S rRNA gene facilitates the genization and the release of associated bacteria. In the identification of microorganisms from a broader present study, this aspect could have influenced the perspective. failure to obtain isolates from 360- and 450-day-old The nitrogenase activity of the isolates was assessed sugarcane crops. by the reduction of acetylene to ethylene. Reference The isolation of G. diazotrophicus and G. sacchari from strain ATCC 49037 and strains GIBI014 and GIBI025 tomato crops has not been previously reported in the showed lower enzyme activity than the strains GIBI029 literature. The only work relating G. diazotrophicus to and GIBI141. For this assay, cultures were started for 8 -1 tomato crops corresponded to the evaluation of each isolate from a density of 10 cells×mL . The colonization and yield promotion of tomato by the nitrogenase activity of G. diazotrophicus is influenced inoculation of G. diazotrophicus (Luna et al., 2012). This under in vitro and in vivo conditions by various factors, represents a great potential for production of biofertilizers such as the presence of inorganic nitrogen sources for crops other than sugarcane for which commercial (Fuentes et al., 1993), the aeration of the culture medium inoculants are being developed in Brazil and India. used for biomass production (Fuentes et al., 1993; Orange colonies obtained from LGI-PN medium and Muthukumarasamy et al., 2002), and the plant genotype. brown colonies formed in PDA (sugar 10%) are typical of It has been estimated that G. diazotrophicus can fix up to the bacteria under study. Such colonies were described 150 kg N×ha-1×year-1 in sugarcane (Boddey et al., 1991). 1626 Afr. J. Biotechnol.

Therefore, for future research, the effect of adding the that K416 and 16G6 mutants were able to solubilize isolates found on the interaction with sugarcane plantlets some of the insoluble phosphate sources tested although under regional conditions should be assessed. with less ability, compared to PAL5. 16D10 mutant was G. sacchari is not considered a nitrogen-fixing completely unable to solubilize any insoluble phosphate bacterium since it does not exhibit any nitrogenase source tested. G. diazotrophicus and G. sacchari isolates activity measured by ARA (Franke-Whittle et al., 2005). used in this study, ATCC 49037 (PAL5), GIBI029, However, in this work, G. sacchari GIBI141 isolate GIBI025, GIBI014, GIBI031, and GIBI 141, exhibited SI presented the highest acetylene-reduction activity with tricalcium phosphate lower than those reported for compared to the other isolates of the same species with PAL5 in the above-cited work (3.10, 3.62, 3.33, 2.59, an activity statistically equal to that of the G. 3.55 and 1.61, respectively); NBRIP medium and shorter diazotrophicus GIBI029 isolate. In the phylogenetic tree, incubation period of 72 h was used. Aluminum phosphate GIBI141 isolate is found in a group separate of the other solubilization was not observed. However, the production two G. sacchari isolates (Figure 2). This could explain its of acids was evidenced by the change in the medium differential behavior. color. It is important to perform laboratory tests in liquid The recovered isolates had the ability to produce media with other sources of phosphorus before indolic compounds. Works performed by Chaves et al. performing field assessments. It is also important to (2015) and Ríos et al. (2016) also demonstrated the complete the characterization of the acids involved in ability of G. diazotrophicus to produce auxin compounds. solubilization (Bashan et al., 2013; Restrepo et al., 2015). Isolate GIBI029 produced an amount of indolic In the case of G. sacchari, reports on the evaluation of compounds similar to that produced by the reference phosphate solubilization were not found. Nevertheless, strain ATCC 49037. It is recommended that the specific G. sacchari GIBI031 isolate presented the highest values determination of indolic acetic acid be performed via of solubilization among the phosphorus sources (SI: Ca3 chromatographic techniques such as HPLC since (PO4)2, 3.55 and AlPO4, 15.87) evaluated in this work spectrophotometric determination only allows for the within this species. This performance should be quantification of total indolic compounds. G. sacchari confirmed through phosphate solubilization in liquid isolates produced indolic compounds in an amount equal medium as mentioned previously. or higher than that of G. diazotrophicus GIBI025. It is necessary to assess this behavior in field to determine the potential of these isolates for promoting plant growth. Conclusion In this way, the application spectrum of this bacterium can be broadened considering that most reports on G. In this work, two native strains of G. diazotrophicus and sacchari are aimed at synthesis of bacterial cellulose three strains of G. sacchari were isolated from the roots (Trovatti et al., 2011). and stems of sugarcane. G. sacchari strains were Phosphate solubilization was evaluated using two recovered from tomato crop. This is the first report on the sources of phosphorus, tricalcium phosphate and isolation of G. diazotrophicus and G. sacchari in aluminum phosphate. The NBRIP medium was Colombia. G. diazotrophicus promotes plant growth and supplemented with bromocresol green to improve the has a great potential as an active principle of visualization of the test. After 72 h, the solubilization of biofertilizers. The isolates from sugarcane recovered in tricalcium phosphate was observed in both of the native this study (GIBI029 and GIBI031) showed growth- isolates of G. diazotrophicus and in G. sacchari. This promoting properties, such as indolic compounds finding demonstrated the potential of the recovered production and phosphate solubilization, with values isolates with the exception of the GIBI141 isolate, which above or close to those of the reference strain ATCC had the lowest values of tricalcium phosphate 49037. For G. sacchari, the highest acetylene-reduction solubilization and acid production in the presence of activity was observed in GIBI141 isolate that is an aluminum phosphate. Phosphorus solubilization has important finding considering that this activity has not been observed in several G. diazotrophicus isolates been reported before; the activity value achieved was recovered from sugarcane. Delaporte-Quintana et al. statistically equal to that of the G. diazotrophicus GIBI029 (2016) reported the solubilization of insoluble phosphate isolate. The evaluation of phosphate solubilization in (dicalcium phosphate, tricalcium phosphate, Gluconacetobacter isolates reported in this study showed hydroxyapatite, basis slag, and ferric phosphate) by four statistically significant differences being G. diazotrophicus different strains of G. diazotrophicus (PAL5 and its Tn5- GIBI029 and G. sacchari GIBI031 the isolates with the derivative mutants K416, 16D10 and 16G6 defective in best phosphate solubilization. All these isolates are organic acid production) in NBRIP solid minimal medium. therefore promising for the development of commercial The PAL5 strain solubilized all the sources of insoluble inoculants. phosphate with SI of 9.25 (dicalcium phosphate), 6.84 The specific determination of indolic acetic acid by (tricalcium phosphate), 2.82 (hydroxyapatite), 1.11 (basis chromatographic techniques such as HPLC is recom- slag), and 1.20 (ferric phosphate). These authors indicate mended, because spectrophotometric determination only Restrepo et al. 1627

allows for the quantification of total indolic compounds. avaliação da fixação biológica de N2 associada a leguminosas e não- The continued evaluation of phosphate solubilization by leguminosas utilizando a técnica da redução do acetileno: histórica, teoria e prática (The evaluation of N2 biological fixation associated to G. diazotrophic and G. sacchari using liquid culture media leguminous and non-leguminous plants using the acetylene reduction with other sources of phosphate is also recommended. technique: History, theory, and practice, in Portuguese). This will enable a more efficient evaluation of Documentos, Vol. 245. Embrapa, Seropedica, Brazil. solubilization properties in tomato crops at the seedling Boddey RM, Urquiaga S, Reis VM, Döbereiner J (1991). Biological nitrogen fixation associated with sugar cane. Plant Soil 137:111-117. level (seeder) and in greenhouse-grown tomatoes. Castillo G, Altuna B, Michelena G, Sánchez-Bravo J, Acosta M (2005). The development of biotechnology-based inoculants Cuantificación del contenido de ácido indolacético (AIA) en un caldo from these bacteria would allow for a long-term reduction de fermentación microbiana (Quantification of the indole acetic acid in chemicals used in agriculture and promote the content (IAA) in a microbial fermentation broth, in Spanish). Anal. Biol. 27:137-142. development of environmentally friendly agronomic Cavalcante VA, Döbereiner J (1988). A new acid-tolerant nitrogen-fixing strategies. However, the success of these bacterial bacterium associated with sugar cane. Plant Soil 108:23-31. inoculants depends on the selection of native strains Chaves VA, Santos SGd, Schultz N, Pereira W, Sousa JS, Monteiro efficient in this soil type. Such strains must have the RC, Reis VM (2015). Desenvolvimento inicial de duas variedades de cana-de-açúcar inoculadas com bactérias diazotróficas (Initial ability to colonize the rhizosphere and maintain biological development of two sugarcane varieties inoculated with diazotrophic activity. In this sense, the isolates obtained have great bacteria, in Portuguese). Rev. Bras. Ciênc. Solo 39(6):1595-1602. potential for the subsequent commercial development of Crespo JM, Boiardi JL, Luna MF (2011). Mineral phosphate an innovative biotechnological inoculant for crops of solubilization activity of Gluconacetobacter diazotrophicus under P- limitation and plant root environment. Agric. Sci. 2(1):16-22. tomato and even other vegetables. De Souza R, Ambrosini A, Passaglia LMP (2015). Plant growth- promoting bacteria as inoculants in agricultural soils. Genet. Mol. Biol. 38(4):401-419. CONFLICT OF INTERESTS Delaporte-Quintana P, Grillo-Puertas M, Lovaisa NC, Rapisarda VA, Teixeira KR, Pedraza RO (2016). Solubilization of different sources of insoluble phosphate of Gluconacetobacter diazotrophicus. Rev. The authors have not declared any conflict of interests. Agron. Noreste Argent. 36(1):33-35. Dibut B, Martínez R, Ortega M, Rios Y, Fey L (2004). Presencia y uso de microorganismos endófitos en plantas como perspectiva para el mejoramiento de la producción vegetal (Presence and use of ACKNOWLEDGEMENTS endophytic microorganisms in plants as a perspective for the improvement of plant production, in Spanish). Cultiv. Tropic. 25, 13- The authors of this work are grateful for funding from the 17. Colombian Administrative Department of Science, Dong Z, Heydrich M, Bernarad K, McCully M (1995). Further evidence that the N2-fixing endophytic bacterium from the intercellular spaces Technology and Innovation (Colciencias), the Office of of sugarcane stems is diazotrophicus. Appl. Environ. Research and Graduate Studies of the Universidad Microbiol. 61(5):1843-1846. Católica of Manizales and the Vice-rectorate of Research Eskin N, Vessey K, Tian L (2014). Research progress and perspectives and Graduate Studies of the Universidad de Caldas for of nitrogen fixing bacterium, Gluconacetobacter diazotrophicus, in monocot plants. Int. J. Agron. 2014(Article ID 208383):13 pages. the project entitled “Preparation and evaluation of a Franke I, Fegan M, Hayward C, Leonard G, Stackenbrandt E, Sly L promoting-growth inoculum of tomato and carrot crops (1999). Description of Gluconacetobacter sacchari sp. nov., a new based on G. diazotrophicus” (grant 112752128333). species of acetic acid bacterium isolated from the leaf sheath of sugar cane and from the pink sugar cane mealy bug. Int. J. Syst. Bacteriol. 49:1681-1693. Franke-Whittle IH, O'Shea MG, Leonard GJ, Webb R, Sly LI (2005). REFERENCES Investigation into the ability of Gluconacetobacter sacchari to live as an endophyte in sugarcane. Plant Soil 271:285-295. Ahemad M, Kibret M (2014). Mechanisms and applications of plant Fuentes LE, Jiménez T, Abarca IR, Caballero J (1993). Acetobacter growth promoting rhizobacteria: Current perspective. J. King Saud diazotrophicus, an indoleacetic acid producing bacterium isolated Univ. Sci. 26(1):1-20. from sugarcane cultivars of México. Plant Soil 154:145-150. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990). Basic Gillis M, Kersters K, Hoste B, Janssens D, Kroppenstedt RM, Stephan local alignment search tool. J. Mol. Biol. 215:403-410. MP, Teixeira KRS, Döbereiner J, De Ley J (1989). Acetobacter Baldani JI, Reis VM, Videira SS, Boddey LH, Baldani VLD (2014). The diazotrophicus sp. nov., a nitrogen-fixing acetic acid bacterium art of isolating nitrogen-fixing bacteria from non-leguminous plants associated with sugarcane. Int. J. Syst. Evol. Microbiol. 39:361-364. using N-free semi-solid media: a practical guide for microbiologists. Glick BR (2012). Plant growth-promoting bacteria: Mechanisms and Plant Soil 384(1):413-431. applications. Scientifica 2012(Article ID 963401):15 pages. Bashan Y, Kamnev AA, de-Bashan LE (2013). Tricalcium phosphate is Glickmann E, Dessaux Y (1995). A critical examination of the specificity inappropriate as a universal selection factor for isolating and testing of the Salkowski reagent for indolic compounds produced by phosphate-solubilizing bacteria that enhance plant growth: a proposal phytopathogenic bacteria. Appl. Environ. Microbiol. 61(2):793-796. for an alternative procedure. Biol. Fertil. Soils 49:465-479. Gordon SA, Weber RP (1951). Colorimetric estimation of indoleacetic Bellone CH, De Bellone SDVC, Pedraza RO, Monzon MA (1997). Cell acid. Plant Physiol. 26(1):192-195. colonization and infection thread formation in sugar cane roots by Hasegawa M, Kishino H, Yano TA (1985). Dating of the human-ape Acetobacter diazotrophicus. Soil Biol. Biochem. 29(5-6):965-967. splitting by a molecular clock of mitochondrial DNA. J. Mol. Evol. Beneduzi A, Moreira F, Costa PB, Vargas LK, Lisboa BB, Favreto R, 22(2):160-174. Baldani JI, Passaglia LMP (2013). Diversity and plant growth Hernández-Rodríguez A, Heydrich-Pérez M, Diallo B, El Jaziri M, promoting evaluation abilities of bacteria isolated from sugarcane Vandaputte OM (2010). Cell-free culture medium of Burkholderia cultivated in the South of Brazil. Appl. Soil Ecol. 63:94-104. cepacia improves seed germination and seedling growth in maize Boddey LH, Boddey RM, Rodríguez BJ, Urquiaga S (2007). A (Zea mays) and rice (Oryza sativa). Plant Growth Regul. 60:191-197. 1628 Afr. J. Biotechnol.

Jiménez T, Fuentes LE, Tapia A, Macarua M, Martínez E, Caballero J Natheer ES, Muthukkaruppan S (2012). Assessing the in vitro zinc (1997). Coffea arabica L., a new host plant for Acetobacter solubilization potential and improving sugarcane growth by diazotrophicus, and isolation of other nitrogen-fixing acetobacteria. inoculating Gluconacetobacter diazotrophicus. Ann. Microbiol. Appl. Environ. Microbiol. 63(9):3676-3683. 62(1):435-441. Kim M, Oh H-S, Park S-C, Chun J (2014). Towards a taxonomic Nautiyal CS (1999). An efficient microbiological growth medium for coherence between average nucleotide identity and 16S rRNA gene screening phosphate solubilizing microorganisms. FEMS Microbiol. sequence similarity for species demarcation of prokaryotes. Int. J. Lett. 170:265-270. Syst. Evol. Microbiol. 64(2):346-351. Nguyen C, Yan W, Le Tacon F, Lapeyrie F (1992). Genetic variability of Kumar S, Stecher G, Tamura K (2016). MEGA 7: Molecular phosphate solubilizing activity by monocaryotic and dicaryotic Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol. mycelia of the ectomycorrhizal fungus Laccaria bicolor (Maire) P.D. Biol. Evol. 33(7):1870-1874. Orton. Plant Soil 143:193-199. Kumar V, Narula N (1999). Solubilization of inorganic phosphates and Restrepo GM, Marulanda S, de la Fe Y, Díaz A, Baldani VL, growth emergence of wheat as affected by Azotobacter chroococcum Hernández-Rodríguez A (2015). solubilizadoras de fosfato mutants. Biol. Fertil. Soils 28:301-305. y sus potencialidades de uso en la promoción del crecimiento de Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, cultivos de importancia económica (Phosphate-solubilizing bacteria McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson and their potential for use in promoting the growth of economically JD, Gibson TJ, Higgins DG (2007). ClustalW and ClustalX version important crops, in Spanish). Rev. CENIC, Cienc. Biol. 46:63- 76. 2.0. Bioinformatics 23(21):2947-2948. Ríos Y, Rojas M, Ortega M, Dibut B, Rodríguez J (2016). Aislamiento y Li RP, Macrae IC (1991). Specific association of diazotrophic caracterización de cepas de Gluconacetobacter diazotrophicus acetobacters with sugarcane. Soil Biol. Biochem. 23(10):999-1002. (Isolation and characterization of Gluconacetobacter diazotrophicus Luna MF, Aprea J, Crespo JM, Boiardi JL (2012). Colonization and yield strains, in Spanish). Cultiv. Tropic. 37:34-39. promotion of tomato by Gluconacetobacter diazotrophicus. Appl. Soil Rodrigues Neto J, Malavolta Jr. VA, Victor O (1986). Meio simples para Ecol. 61:225-229. isolamento e cultivo de Xanthomonas campestris pv. citri tipo B Madhaiyan M, Saravanan VS, Bhakiya D, Hyoungseok Lee, Thenmozhi (Simple media for isolation and cultivation of Xanthomonas R, Hari K, Sa T (2004). Occurrence of Gluconacetobacter campestris pv. citri type B, in Portuguese). Summa Phytopathol. diazotrophicus in tropical and subtropical plants of Western Ghats, 12(1-2):2-16. India. Microbiol. Res. 159:233-243. Sarwar M, Kremer RJ (1995). Determination of bacterially derived Malekpoor Mansoorkhani F, Seymour GB, Swarup R, Moeiniyan auxins using a microplate method. Lett. Appl. Microbiol. 20:282-285. Bagheri H, Ramsey RJ, Thompson AJ (2014). Environmental, Sievers M, Swings J (2005). Genus VIII. Gluconacetobacter Yamada, developmental, and genetic factors controlling root system Hoshino, and Ishikawa 1998 b, 32VP. In. Brenner D, Krieg N, Garrity architecture. Biotechnol. Genet. Eng. Rev. 30(1-2):95-112. G, Staley J (eds) Bergey’s Manual® of Systematic Bacteriology. Matsutani M, Hirakawa H, Yakushi T, Matsushita K (2010). Genome- Volume Two. The . Part C. 2nd edn. Springer, New wide phylogenetic analysis of Gluconobacter, Acetobacter, and York. Pp. 72-77. Gluconacetobacter. FEMS Microbiol. Lett. 315(2):122-128. Stephen J, Shabanamol S, Rishad KS, Jisha MS (2015). Growth Mende DR, Sunagawa S, Zeller G, Bork P (2013). Accurate and enhancement of rice (Oryza sativa) by phosphate solubilizing universal delineation of prokaryotic species. Nat. Methods 10:881- Gluconacetobacter sp. (MTCC 8368) and Burkholderia sp. (MTCC 884. 8369) under greenhouse conditions. 3 Biotech 5(5):831-837. Meneses CHSG, Rouws LFM, Simões-Araújo JL, Vidal MS, Baldani JI Tapia A, Bustillos MR, Jiménez T, Caballero J, Fuentes LE (2000). (2011). Exopolysaccharide Production Is Required for Biofilm Natural endophytic occurrence of Acetobacter diazotrophicus in Formation and Plant Colonization by the Nitrogen-Fixing Endophyte pineapple plants. Microb. Ecol. 39:49-55. Gluconacetobacter diazotrophicus. Mol. Plant Microbe Interact. Trovatti E, Serafim LS, Freire CSR, Silvestre AJD, Neto CP (2011). 24(12):1448-1458. Gluconacetobacter sacchari: An efficient bacterial cellulose cell- Monteiro RA, Balsanelli E, Wassem R, Marin AM, Brusamarello-Santos factory. Carbohydr. Polym. 86(3):1417-1420. LCC, Schmidt MA, Tadra-Sfeir MZ, Pankievicz VCS, Cruz LM, Videira S, Simões JL, Baldani V (2007). Metodologia para isolamento e Chubatsu LS, Pedrosa FO, Souza EM (2012). Herbaspirillum–plant posicionamento taxonômico de bactérias diazotróficas oriundas de interactions: microscopical, histological and molecular aspects. Plant plantas não-leguminosas (Methodology for isolation and taxonomic Soil 356:175-196. positioning of native diazotrophic bacteria from non-leguminous Muthukumarasamy R, Cleenwerck I, Revathi G, Vadivelu M, Janssens plants, in Portuguese). Documentos, vol 234. Embrapa, Seropedica, D, Hoste B, Ui Gum K, Park K-D, Young Son C, Sa T, Caballero- Brazil. Mellado J (2005). Natural association of Gluconacetobacter White PJ, Brown PH (2010). Plant nutrition for sustainable development diazotrophicus and diazotrophic Acetobacter peroxydans with and global health. Ann. Bot. 105:1073-1080. wetland rice. Syst. Appl. Microbiol. 28(3):277-286. Yamada Y, Yukphan P (2008). Genera and species in acetic acid Muthukumarasamy R, Revathi G, Lakshminarasimhan C (1999). bacteria. Int. J. Food Microbiol. 125:15-24. Influence of N fertilisation on the isolation of Acetobacter diazotrophicus and Herbaspirillum spp. from Indian sugarcane varieties. Biol. Fertil. Soils 29:157-164. Muthukumarasamy R, Revathi G, Loganathan P (2002). Effect of inorganic N on the population, in vitro colonization and morphology of Acetobacter diazotrophicus (syn. Gluconacetobacter diazotrophicus). Plant Soil 243:91-102.

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Appendix 1. Accession numbers corresponding to GenBank nomenclature.

Microorganisms Abrev. Accession Size (bp) Date of arrival G. azotocaptans G azot AY958232 1706 22-AUG-2006 G. johannae G joha JF793992 1358 29-MAR-2013 G. diazotrophicus G diaz KP969073 1463 11-AUG-2015 G. diazotrophicus G diaz AY230812 1395 02-APR-2003 G. diazotrophicus G diaz JX987291 1354 16-DEC-2012 G. diazotrophicus G diaz AY230808 1379 02-APR-2003 G. diazotrophicus G diaz KX096237 651 01-JUN-2016* G. diazotrophicus G diaz KX096238 673 01-JUN-2016* G. liquefaciens G liqu KP715459 1353 14-JUL-2015 G. sacchari G sacc LC108744 1483 07-JAN-2016 G. sacchari G sacc KX965666 620 11-DEC-2016* G. sacchari G sacc KX965665 590 11-DEC-2016* G. sacchari G sacc KX965664 587 11-DEC-2016* B. cepacia B cepa NR_114491 1462 03-FEB-2015

*Isolates obtained in this work.