Bioprospecting for Isoetes cangae Endophytes with Potential to Promote Plant Growth

Isoetes cangae is a native plant found only in a permanent pond in Serra dos Carajás in the Amazon region. Plant-associated microbial communities are recognized to be responsible for biological processes essential for the health, growth, and even adaptation of plants to environmental stresses. In this sense, the aims of this work were to isolate, identify, and evaluate the properties of endophytic bacteria isolated from I. cangae. The bioprospecting of potentially growth-promoting endophytes required the following steps to be taken: isolation of endophytic colonies, molecular identification by 16S rDNA sequence analysis, and evaluation of the bacterial potential for nitrogen fixation, production of indole acetic acid and siderophores, as well as phosphate solubilization and mineralization. Bacillus sp., Rhizobium sp., Priestia sp., Acinetobacter sp., Rossellomorea sp., Herbaspirillum sp., Heyndrickxia sp., and Metabacillus sp., among other bacterial species, were identified. The isolates showed to be highly promising, evidencing the physiological importance for the plant and having the potential to promote plant growth.


Introduction
Isoetes cangae is an ancient plant whose evolutionary process dates from the Devonian period [1].However, its discovery was made a few years ago by Pereira et al. [2].Te new species was named in reference to its habitat, on canga soil, in rupestrian felds.Until now, it has only been found in a permanent pond in Serra dos Carajás, Pará.Tis aquatic plant lives underwater and grows among the rocks in the pond, in an iron-rich oligotrophic environment.Morphologically, it is characterized by the presence of root, corm, and leaves.Pereira et al. [2] suggest that I. cangae be classifed as critically endangered because it was found only in a single location within a mining area, predicting a signifcant decrease in individuals.Indeed, in 2019, it was included in the IUCN red list as "Critically Endangered" [3].It is, therefore, important to understand the bacteria that live in symbiosis with these plants, promoting their health, protection, and growth.
Plant-associated microbial communities are responsible for biological processes essential for the health, growth, and even adaptation of plants to environmental stresses.Te benefts of this relationship are credited to compounds produced by bacteria, such as growth-regulating hormones [4], antibiotics and siderophores [5], as well as nitrogen fxation [6], and mineralization and solubilization of nutrients such as phosphorus [7].
Using bacteria to speed up the growth and promote the adaptation of plants to biotic and abiotic stresses is a widely used technology worldwide.Most times, this is the diference in the success of cultivation [8].Amorim and Melo [9] highlight that benefcial bacteria can help in all plant cycles, allowing increase in germination rate, reproductive organ development, and productivity.
Studies on plant growth enhancement are usually carried out using rhizobacteria, but some studies point to a greater efciency of endophytic bacteria [10].Some of the bacteria become facultative intracellular endophytes and competitively colonize plant roots.Tey ofer several benefts to the plant because they are in an environment that sufers less impact from the abiotic and biotic variations of the rhizosphere [11,12].
Tus, the aims of this study were to isolate and identify endophytic bacteria with an important role in the development of I. cangae.Te bioprospecting of endophytic bacteria with potential as growth promoters required the following steps to be taken: isolation of endophytic bacteria associated with Isoetes cangae, morphological description, characterization, and preservation of microorganisms and assessment of nitrogen fxation potential, production of indole acetic acid, and siderophores and phosphate mineralization.

Materials and Methods
Isoetes cangae is endemic of Amendoim pond, a permanent pond located in southeastern Pará State, within the limits of the Carajás National Forest-FLONA (05 °52′00″-06 °33′00″S and 049 °53′00″-050 °45′00″W).Amendoim pond has an area of 13.96 ha and a maximum depth of 7.8 m [13], with an average variation in the water column height of 2.5 m over the year.Te Chico Mendes Institute of Biodiversity of the Ministry of the Environment (ICMBio/MMA; numbers 64187 and 5972) grants the permit for all collections.

Isolation of Endophytic Microorganisms from I. cangae.
Te bacterial isolation was carried out using 3 plants collected from 3 diferent portions (West, North, and East) of Amendoim pound which were processed by separating root, corm, and leaves and disinfected in 70% v/v ethanol for one minute, 2.5% v/v sodium hypochlorite solution for 3 minutes, 70% v/v ethanol for 1 minute, and two rinses in sterile distilled water [14].Subsequently, the samples were aseptically macerated and cultured on Luria-Bertani (LB) agar (triptona, 1 g/L; yeast extract, 0.5 g; NaCl, 1 g; and agar, 20 g/L).Te media were sterilized by autoclaving at 121 °C for 15 min before use.Te plates were incubated at room temperature for 24-48 h.
Diferences in colony morphology of grown bacteria were observed: shape, elevation, margin (edges of colonies), color, surface appearance, density, consistency, and pigmentation [15].Afterward, the cultures were tested using Gram-staining.Conservation and maintenance procedures in the short and medium term were performed to stock the grown cultures; thus, the bacterial isolates were kept frozen at −20 °C in 20% glycerol, and a batch was kept at 4 °C on inclined test tube Luria-Bertani (LB) agar, and mineral oil was added.
For activation of cultures at the beginning of the tests, the isolates were inoculated in LB broth (tryptone, 1 g/L; yeast extract, 0.5 g; and NaCl, 1 g), modifed from Ambrosini and Passaglia [16].Te media were sterilized in an autoclave at 121 °C for 15 minutes before use.Te fasks were incubated at 28 ± 1 °C and 100 rpm for 18 h.

Nitrogen Fixation Capacity.
To identify nitrogen-fxing bacteria, the isolates were cultured in test tubes containing 10 mL of NFb culture medium (5 g of malic acid/0.5 g of K 2 HPO 4 /0.2 g of MgSO 4 .7H 2 O/0.1 g of NaCl/0.01 g of CaCl 2 .2H 2 O/4 mL of FeEDTA-1.64%/2mL of bromothymol blue-0.5%/2mL of micronutrient solution/1.75 g/L of agar; 1 L of distilled water, pH 6.8).Te fasks were incubated at 28 ± 1 °C for 7 days, and those that presented a typical aerotaxis flm close to the medium surface were considered positive for nitrogen fxation.Te test was performed in triplicate.Te presence of bacterium flm formation in NFb semi-solid medium as described by Kuss et al. [17].Te change in the medium's color from intense green to bluishgreen or blue is an indicator that biological fxation has occurred, since it indicates the reduction of atmospheric nitrogen to ammonia.
PCR was used to detect the presence of the nifH gene in the isolates selected as nitrogen fxers, using the primers Ueda19F (5′GCIWTYTAYGGIAARGGIGG 3′) [18] and R6 (5′GCCATCATYTCICCIGA 3′) [19].Each reaction was performed in a volume of 50 μl inside a tube containing 10 l 5x PCR bufer (Promega Corporation/Madison, Wisconsin, EUA), 2 mM MgCl 2 (Promega Corporation/Madison, Wisconsin, EUA), 200 μM each of the four dNTPs (Promega Corporation/Madison, Wisconsin, EUA), 0.2 mM of each primer (IDT), and 1.5 U of Taq DNA polymerase (Promega Corporation/Madison, Wisconsin, EUA).Te program used for amplifcation followed the steps: 95 °C for 5 minutes, followed by 35 cycles of 94 °C for 30 seconds, 52 °C for 45 minutes, and 72 °C for 30 seconds and a single fnal elongation step at 72 °C for 10 minutes.Te PCR products were separated by electrophoresis on a 1.2% agarose gel, stained, and visualized on a transilluminator with UV light.Fragments of approximately 455 bp were considered positive [20].

Indoleacetic Acid (IAA) Production.
Te evaluation of IAA production by endophytic species of I. cangae was performed according to Bent et al. [21] with modifcations.Te colonies were grown in LB medium and shaken at 100 rpm and at 30 °C, for 18 h.A 100 μL aliquot of the LB medium with colonies was inoculated into 3 mL of King's B medium (20 g/L protease peptone; 1.5 g/L K 2 HPO 4 ; 1.5 g/L MgSO 4 .7H 2 O; 20 g/L agar, pH 7.2), supplemented with Ltryptophan (0.25 g/L) as a precursor for IAA synthesis [22].Afterward, the vial was incubated shaking (100 rpm) at 28 °C in the dark, for 72 hours.Ten, a 1.5 mL aliquot of cell suspension was transferred to 2 mL microtubes and Te development of pink color indicates the production of qualitative IAA, being the intensity of the staining directly proportional to the concentration of IAA present in the medium [17].Te absorbance was read in a spectrophotometer at 530 nm.King's B medium without inoculum was used as the negative and white control.Te IAA produced was determined by building up a standard curve (adjustment R 2 � 0.988) with 0, 1, 3, 5, 7, 10, and 25 μg/mL IAA concentrations.
2.4.Siderophore Production.Production of siderophores was analyzed according to Schwyn and Neilands [23].Te colonies were grown in LB broth, at 30 °C and 100 rpm, for 48 hours.A 5 μL aliquot of the isolates grown on LB broth was spot inoculated on CAS medium (King's B medium, supplemented with chromoazurol S + FeCl 3 + hexadecyltrim ethylammonium) and incubated at 28 °C for 4 to 7 days.Te results of the test for the identifcation of isolates producing siderophores were considered positive when clear halos were observed around the colonies.Te assay was performed in triplicate.

Inorganic Phosphorus Solubilization Capacity.
Te cultures were reactivated in LB solid medium.Aliquots of 0.5 mL of bacterial suspension (10 8 cells/mL) were individually transferred to a 250 mL Erlenmeyer fask containing NBRIP liquid culture medium [24] added with 2 g/L of FePO 4 .Te culture medium and culture medium added with inoculum-free iron phosphate were established as controls.Te treatments were incubated at 28 °C and shaken for 10 days.After this incubation period, the cultures were centrifuged at 5,000 ×g for 10 min, and the supernatant was fltered on Whatman no.42 flter paper.To quantify the soluble P, the colorimetric method of Murphy and Riley [25] was used, subtracting the soluble P contained in the control sample (culture medium with iron phosphate without inoculation).Te assay was performed in triplicate.Te pH of the fltered supernatant of all samples, including the controls, was also determined.
2.6.Organic Phosphorus Mineralization.Te organic phosphorus mineralization test was performed according to Dash et al. [26], with modifcations.Cultures grown in LB medium (10 g/L) were spot inoculated on Petri dishes containing selective agar sodium phytate medium (10 g/L Dglucose, 4 g/L sodium phytate, 2 g/L CaCl.2H 2 O, 5 g/L NH 4 NO 3 , 0.5 g/L KCl, 0.5 g/L MgSO 4 , 0.01 g/L MnSO 4 , 15 g/L agar, pH 7.00 at 25 °C) for identifcation of the potential for organic phosphorus mineralization.Te plates were incubated at 28 °C for 15 days.Te formation of a clear halo zone around the colonies during the 15 days shows phosphate mineralization.Te assay was performed in triplicate.

Genomic DNA Isolation, PCR, and 16S rDNA Sequencing.
Te sequencing of the 16S rRNA gene, a preserved region used for phylogenetic studies, was carried out for the molecular identifcation of selected bacterial isolates from diferent parts of the plant.
Te total genomic DNA of the isolates was extracted with the Wizard ® Genomic DNA Purifcation Kit (Promega Corporation/Madison, Wisconsin, EUA).Te purifed DNA was quantifed in NanoDrop and used as a template to amplify a segment of the 16S rRNA gene by PCR using the universal prokaryotic primers 27f and 1492r [27,28].Each reaction was performed in a volume of 50 μl in a tube containing 10 μl of 5X PCR bufer (Promega Corporation/ Madison, Wisconsin, EUA), 2 mM MgCl 2 (Promega Corporation/Madison, Wisconsin, EUA), 200 μM of each of the four dNTPs (Promega Corporation/Madison, Wisconsin, EUA), 0.2 μM of each primer (IDT), and 1.5 U of Taq DNA polymerase (Promega Corporation/Madison, Wisconsin, EUA).Te program used for amplifcation was as follows: 95 °C for 5 min, followed by 25 cycles of 95 °C for 1 min, 50 °C for 1 min, and 72 °C for 1 min and 30 s, and one single fnal elongation step at 72 °C for 7 min.
Te fragments obtained from the PCR reactions were separated by electrophoresis on 1% agarose gel, stained, and visualized in a transilluminator with ultraviolet light.At the end, the amplifed fragments were stored in a freezer at −20 °C.

Inoculants Application on I. cangae Growth: Preliminary
Studies.Aiming to promote growth on I. cangae and evaluate the potential of some isolated bacterial inoculants, initial studies were carried out in a greenhouse.For aquatic plant cultivation was applied 1 : 10 proportion (substrate : water).In addition to the control, the bacterial colonies used in the tests were selected from diferent parts of the plant, totaling 6 isolates of diferent endophytes originating from the leaf (Acinetobacter soli EL5), corm (Acinetobacter soli WC10 and Bacillus sp.EC3), and root (Bacillus sp.NR2, Priestia sp.WR9, and Bacillus sp.ER4).Such microorganisms were selected, taking into account both nitrogen International Journal of Microbiology  International Journal of Microbiology fxation potential, siderophore production, and easy growth in LB medium.Te endophytic bacteria used as inoculants were activated in LB broth at a rate of 10% v/v and incubated at 28 °C at 100 rpm for 18−24 h.Subsequently, the selected microorganisms grew in 79 broth for 24 h at 30 °C [16].Subsequently, the PGPR bacteria were inoculated into the plant substrate at 1% v/v.1).

Selection of Representative Profles of Isolated I. cangae
Colonies.Te disinfection of the plants to remove the epiphytic population was efective, since no bacterial growth was observed in plates with noncrushed plants.A total of 109 colonies were isolated from crushed plants in the LB agar and tested for morphological characteristics, classifying them according to the portion of the Amendoim pond where plants were collected from (West, North, or East) and the part of the plant (leaf, corm, or root).Te isolates were coded according to their origem east (E), west (W), and north (N) from Amendoim pond and from diferent I. cangae parts: corm (C), leave (L), and root (R).Among the isolated colonies and after the reactivation of the frozen cultures of the selected microorganisms only 49 isolated colonies remained viable to growth in LB medium for the following tests to evaluate the potential promotion of vegetal growth.

Nitrogen Fixation of Endophytic Microorganisms Isolated from I. cangae.
Most of the isolates showed to be capable of fxing N 2 .36 isolates (75% of the total tested) formed an aerotactic band near the surface of NFb medium, selective for nitrogen-fxing strains.Only the Chromobacterium sp.(EL4) strain showed no growth in NFb medium and was not evaluated (Table 1).

Phosphate Mineralization by Endophytic Microorganisms
Isolated from I. cangae.Most of the isolates tested (78.7%) were capable of organic phosphate solubilization forming clear halo zones around the colonies in the sodium phytate agar medium (Figure 2).Isolates Acinetobacter soli (EL5) and Acinetobacter soli (WC10) showed the largest zone of solubilization.Chromobacterium sp.(EL4) and Herbaspirillum sp.(WC9) isolates lost viability and could not be evaluated.

Multifunctional Potential of Isolates.
In total evaluated isolates, 89% were positive for the IAA production, 75% of the total tested was positive for nitrogen fxation, 78.7% were able to mineralize phosphate, and only 14.3% produced siderophores, as shown in Figure 3.

Inoculants Application on I. cangae Growth: Preliminary
Studies.Te inoculant application in Isoetes cangae enabled improvements in plant growth, such as an increase in the number of leaves, as well as a more robust/elongated leaf area and root.Te treatments that used the colonies Acinetobacter soli WC10 followed by Bacillus sp.NR2 were more promising in the plant cultivation (Figure 4) promoting positive changes in the plant.Te plant cultivation response variables were improved in the presence of inoculants, promoting an increase of up to 5 times the leaves number, as well as average root length in up to 4 and 4.5 times the length leaves, difering signifcantly (p < 0.05) using the test ANOVA performed with the 3 treatments.

Discussion
Te assays showed that the endophytic species of I. cangae are diversifed and promising as potential bacteria to International Journal of Microbiology promote plant growth through inoculation.According to Pramanic et al. [30], the endophytic microbiome increases the potential of plant-based systems, providing improvements in the ecological efciency.Te study of endophytes and their inherent functions at specifc spatial and ecological locations will also aid in understanding the growth of specifc plants in that location and ways of controlling their growth through endophytic microbiome modelling.
Makino et al. [31] isolated aquatic plant growthpromoting bacteria from Lemna mino, a water lentils, verifying that the Pelomonas sp.strain promoted IAA production, which is also observed in known land plants, while other traits, such as siderophore production and phosphate solubilization.Te authors indicated that additional candidate PGPB for duckweeds could be obtained by screening bacterial isolates from diverse aquatic plants.Pramanic et al. [30] detailed the studies on microbiomes and diversity in microbial communities inhabiting the three common free-foating aquatic plants of tropical regions viz., duckweed, water hyacinth, and water lettuce, widely implicated for their bioremediation potential.Studies conducted till date reveal the prevalence and dominance of diferent Bacillus, Rhodanobacter, Pseudomonas, Rhizobium, Achromobacter, Serratia, Actinobacteria, Proteobacteria, Klebsiella, and Acidobacteria, have also been prominently reported.According to O'Brien et al. [32], duckweedassociated microorganisms were proposed to aid in a number of host-related functions, including plant defense, increased nutrient availability, phytohormones production, phytoremediation, and prevention of abiotic stress.Shehzadi et al. [33] reported that the genus Microbacterium, Bacillus, and Halomonas were found to be associated with wetland plants, Typha domingensis, Pistia stratiotes, and Eichhornia crassipes.
Castro et al. [34] isolated endophytic microorganisms from two mangrove species, Rhizophora mangle and

6
International Journal of Microbiology Avicennia nitida, found in streams of two mangrove systems in Bertioga and Cananéia, SP, Brazil.Te authors reported that Bacillus sp. was the most frequently isolated genus, comprising 42% of the species isolated from Cananéia and 28% of the species from Bertioga.Here, we emphasize the importance of the endophytic genus Bacillus in I. cangae (approximately 40%) in comparison with other bacterial genera.Ando et al. [35] also isolated a large number of Bacillus sp. from mangrove sediments in Japan and reported the possible ability of these isolates to degrade polluting organic compounds by fermentation.Among the isolates, the authors identifed two endophytes, B. thuringiensis (MB4) and B. pumilus (MB8), which were able to control many bacterial and fungal pathogens.Chen et al. [36] reported that endophytic bacteria were isolated from 4 species of aquatic plants: Phragmites communis, Potamogeton crispus, Nymphaea tetragona, and Najas marina.Te isolated bacteria were classifed into 12 genera in the Gammaproteobacteria, Bacilli, Alphaproteobacteria, Flavobacteria, and Actinobacteria.In addition, diferent strains were isolated from diferent parts of the 4 plants, suggesting the diferent parts of the 4 plants harbored different endophytic bacteria, similar to this work that isolated bacteria from leaf, root, and corm of I. cangae.Chen et al. [36] found that endophytic bacteria Pseudomonas sp., Enterobacter sp., Aeromonas sp., Flavobacterium sp., Klebsiella sp., Pantoea sp., and Paenibacillus sp. have a Psolubilization capacity and could be used as inoculants and promoters to increase P-uptake by plants, as suggested in this work by Enterobacter genus.Pontes et al. [37] isolated bacteria from Hordeum vulgare L. with potential to be used as inoculant in the same species, producing indolic compounds and siderophores, as well as phosphate solubilization.
Te results of the nitrogen fxation capacity assays indicate that endophytic species have an important role in nitrogen fxation as well, covering 75% of the isolated species, which favors the growth and physiology of the plant.Moreira and Siqueira [38] highlighted the great relevance of nitrogen-fxing microorganisms not only in the plant rhizosphere but also in the soil and philosphere.In the aquatic environment, where I. cangae lives, nitrogen fxation supports primary productivity [39,40] and plays a critical role in maintaining the balance of the nitrogen pool in combination with denitrifcation [41,42].
In this study, the nifH gene was found in 20.4% of the colonies.Other genes are also important for the molecular indication of this feature.For example, in K. pneumoniae, 20 unidentifed genes are grouped together in a very compact organization in a chromosomal region encompassing 24,206 base pairs organized in 8 operons: nifJ, nifHDKTY, nifENX, nifUSVWZ, nifM, nifF, nifLA, and nifBQ [43].In addition, Kuklinsky-Sobral et al. [44] and Zehr et al. [45] reported that the nifH gene could not be amplifed from some symbiotic bacteria, which may be due to variability of the nifH gene sequence.
Te IAA tests were very satisfactory regarding the potential of isolated colonies in relation to plant health, since this substance afects the root morphology, increasing the length and root hair number (Figure 3).Among auxins, IAA is the most studied and produced by bacteria [46], with practical application in plant growth promotion [47].Tere is also increasing evidence that IAA is a signaling molecule in microorganisms that can act as a reciprocal signaling molecule in microbe plant interactions, as it afects gene expression in some microorganisms [48].Leite et al. [49] also evaluated that 78% of the isolates from cassava produced IAA and 31% of the isolates were able to solubilize inorganic phosphate.Te identifcation of 19 isolates allowed the grouping into six bacterial genera, namely: Achromobacter, Bacillus, Burkholderia, Enterobacter, Pantoea, and Pseudomonas.It should be noted that the genera Bacillus sp. and Enterobacter sp. were reported and sequenced in the present work, and 78.7% of the isolates were able to mineralize organic phosphate as indicated above.
Te endophytic bacterial strains were important for siderophore production as well, improving the Fe availability to the associated plant.Pontes et al. [37] highlighted that endophytic bacterial strains that produce siderophores are important for the synthesis of the bacterial consortium, since they can reduce phytopathogens proliferation by iron chelation.In addition, these compounds can reduce the availability of metals in the rhizosphere, reducing their toxicity to plants and their own microorganisms [50].Tis fnding is important to understand the ecology of the plant that lives in an iron-rich region such as Canga, in Carajás.In this sense, the isolated bacteria showed great ecological importance and adaptation to impacted environments, in addition to the possibility of being used in bioremediation techniques.Dimkpa et al. [51] suggest that siderophoreproducing and auxin-producing bacteria simultaneously can be potential candidates for microbe-assisted phytoremediation of metal contamination; they found representative Streptomyces sp.species producer of three hydroxamate siderophores that promote auxin synthesis in the presence of some ions by chelating these metals and making them unavailable for uptake by the plant and inhibiting the synthesis of auxins.
Te assays for organic phosphate mineralization by the isolated colonies were promising, confrming that about 50% of bacterial isolates were capable of sodium phytate mineralization.A reduced availability of phosphorus limits plant growth, being essential for its metabolism [52][53][54].Tus, it is important for the health of the plant to contain endophytic microorganisms that have this ability.Mineralization is measured by phosphatase enzymes produced by plant and microbe in the soil, catalyzing organic phosphate hydrolysis [55] and releasing inorganic phosphorus for plant uptake [56].Te results were similar to those reported by Junior and Oliveira [57], who observed that 33.2% of rhizobium isolates collected in Amazonian soil produced a hydrolysis halo zone, while Pedrinho et al. [58] obtained 46.55% of solubilizing bacterial isolates.Fernández et al. [59] highlighted that the diversity of microorganisms capable of solubilizing inorganic phosphate varies across each type of soil and environment.Te same authors verifed in their study that only 0.06% of the total bacteria had the potential to solubilize inorganic phosphate.
When bioprospecting for endophytic bacteria in I. cangae, the genus Bacillus was the most observed, being a complex group for species diferentiation.Besides being cosmopolitans, they are related to biogeochemical cycles, such as carbon and nitrogen.Several studies confrmed the potential of Bacilli as biocontrol agents and encourage their use in agriculture [60].Tey are capable of synthesizing antimicrobial substances such as ethylparaben, lipopeptides [61], and enzymes that attack the phytopathogenic fungi [62].
Multiple phylogenetic studies and comparative genomic analyses have been conducted to clarify the taxonomy of the Bacillus genus, according to Gupta et al. [63].Tus, recent research indicates that Heyndrickxia sp., Priestia sp., and Rossellomorea sp. are products of the genus Bacillus sp., corresponding to 4%, 20%, and 4% of the genera described in this study (Table 1), respectively.
Nutaratat et al. [64] identifed the genus Enterobacter sp.DMKU-RP206 in a semiaquatic plant, a rice phyllosphere bacterium that possesses plant growth-promoting traits.Te bacterium was assessed on plant growth-promoting traits including indole-3-acetic acid (IAA) production.Phosphate solubilization, ammonia production, and antagonism to fungal plant pathogens, as well as siderophore production, were shown by this bacterium.In this work, the genus Enterobacter was also identifed and was efective in the IAA production.
Acinetobacter soli was characterized in this work in two bacterial isolates that presented expressive results in the assays.Tis species has huge metabolic and nutritional versatility and a high degree of hostile and diverse environmental adaptation ability.It uses diferent carbon sources and can grow in diverse temperature and pH conditions, it is disinfectant-resistant, tolerates low humidity rates, can adhere to and form bioflms in soil grains, which contribute to its persistence in diferent environments [65][66][67][68].Corroborating our observations, studies carried out by Kleingesinds [69] demonstrate that Acinetobacter soli can potentially be used as an inoculant in Sacrum sp., as well as its ability for nitrogen fxation and IAA production.
Silva et al. [70] analyzed a high number of colonies isolated from Aloe Vera belonging to the phyla Proteobacteria and Firmicutes, followed by Actinobacteria and Bacteroides.Teir results were similar to those found in this study, in which the genera Acinetobacter sp. and Bacillus sp. were identifed as members of the phyla Proteobacteria and Firmicutes, respectively.Tese genera have previously been reported as growth promoters, with the potential to be used in phytoremediation [71,72], biostimulation, biocontrol, and biofertilization [73,74].
According to Anudechakul et al. [75], Acinetobacter sp., isolated from the roots of Pontederia, was found to facilitate and enhance the removal of chlorpyrifos by the aquatic plant.Shiomi et al. [74] performed bioprospecting to discover endophytic bacteria for biological control of cofee leaf rust and identifed the genus Acinetobacter sp., as well as species of the genus Bacillus sp., among other endophytic species.Te authors highlighted the fact that the endophytic bacteria isolates showed activity when applied before the pathogen, suggesting that these isolates may act by antibiosis, lysis of pathogen, competition, or induction of systemic resistance in the host.Ishizawa et al. [76] reported that Acinetobacter calcoaceticus P23 and Pseudomonas fulva Ps6 increased growth and biomass production when inoculated 8 International Journal of Microbiology in hydroculture systems.Yamaga et al. [77,78] reported that the PGPB for duckweeds, Acinetobacter calcoaceticus P23, which increases the number of duckweed fronds (leaf-like structures), was isolated from Lemna sp.Te genus Acinetobacter sp. was also evidenced in this work, indicating its promise for promoting plant growth due to its properties to establish, produce IAA, and mineralize phosphate.Herbaspirillum sp., also identifed in this study, can usually fx atmospheric N 2 in microaerobic conditions and grow in the presence of N 2 as only nitrogen source.It is mainly associated with grasses, endophytically colonizing roots, stems, and leaves.However, according to Dobritsa et al. [79], Herbaspirillum aquaticum (IEH 4430, ATCC BAA-1628, DSM 21191) cannot fx N 2 and neither grows in JNFb medium, which is mainly used to isolate N 2 -fxing bacteria, such as Azospirillum sp. and Herbaspirillum sp.[80].Te study corroborates our result, not confrming N fxation by this species, as highlighted in Table 1.According to Stoltzfus et al. [81], the nifH and nifD genes were not identifed by the PCR technique with the primers in preconized conditions and neither the nitrate reductase enzyme was observed.
Te genus Rhizobium, which is traditionally considered as legume endosymbionts and has generally been isolated from nodules [82], was also identifed in this work.However, large populations of rhizobia are found in the soil and rhizospheres and are defned as endophytic species.Rhizobia have great environmental importance, since they induce root nodules and fx atmospheric N 2 in most legume species in exchange for carbon [83].As in the present study, Lin et al. [82] identifed a Rhizobium straminoryzae strain, investigating the bacterial diversity from rice straw in Taiwan.Te researchers analyzed the presence of nifH, but the gene was not detected with the primers FGPH19/PolR and AQER/PolF.Te results of nitrogen fxation for this species were not positive, as seen in Table 1.However, the performance of this species regarding the mineralization of phosphate, siderophore production, as well as IAA production was relevant.
According to Pramanic et al. [30], there are many studies on the taxonomical and functional aspects of microorganisms associated with terrestrial plants.However, the microbiome of aquatic plants is not much explored.In future trials the endophytic microorganisms can be used to control diseases caused by bacteria, fungi, and nematodes, since they compete for colonization of the similar niches occupied by these pathogens and stimulate the activation of plant defense response, leading to an increase in resistance [84].Te endophytic bacteria can act in synergism, favoring the growth and development of I. cangae and improving the health and performance of plants.
Te preliminary study using the bacterial inoculants was very promising.Te selected bacteria that have the ability to make the phosphorus present in the soil soluble and make it available to the plant, among other properties already described in this report, promoted up to 5-fold improvements in I. cangae growth.Future steps consist of optimizing the inoculant consortium through diferent implementation techniques in the culture substrate, such as cell immobilization or centrifugation of the cultivated bacterial species.When inoculants are applied in consortium, the antibiosis exerted by inoculant bacteria on pathogens acts through diferent mechanisms, such as synthesis of antimicrobial substances, competition for space and nutrients, secretion of lytic enzymes, change in pH, and/or synthesis of volatile compounds, representing losses in the cultivation of such species [5,33,55,61,62,85,86].
Tus, in the present study, techniques were applied to identify bacterial isolates with an important role in the construction of an inoculant consortium to be implemented in the I. cangae preservation, according to Chai et al. [87] and Olanrewaju and Babalola [88].In addition, the activities presented by the bacterial isolates deserve to be investigated from the perspective of biotechnology resources.

Conclusions
Te cultivation and isolation of microorganisms present in the leaf, root, and corm of the I. cangae species, from different collection points, allowed the identifcation of several endophytic bacterial isolates that were evaluated in relation to their potential application as plant growth-promoting microorganisms.In the present study, 49 bacterial cultures were isolated and identifed.Among them, several showed potential for biological nitrogen fxation (35), detection of the nifH gene (10), siderophore producers (7), phosphate mineralizers (37), and auxin producers (44).Tis study represents the frst efort to investigate a tropical endemic and ancestry plant associated with rare and oligotrophic environments, attesting to the well-known relevance of endophytic bacteria biodiversity.Tese endophytic bacteria represent, as in other groups, a very important technology to increase plant production, potentiating the conservation eforts of I. cangae.

Table 1 :
Molecular identifcation with similarity value in BLASTN programs in the period 09/2022, GenBank database with the isolates accession numbers, and microbiological quantifcation tests for N fxation-capacity, P mineralization, siderophores, IAA production, and nifH presence of endophytes isolated from I. cangae in Brazil in the period 07/2018.
NG: no growth in culture medium; NV: no viability; I: indeterminate.Te isolates were coded according to their origem from the Amendoim pond as E (east), W (west), and N (north) and from diferent I. cangae parts: corm (C), leaf (L), and root (R).
Isolated from I. cangae.Te identifcation of endophytes isolated from I. cangae in Brazil in the period 07/2018 was based on the sequencing of the 16S rDNA gene.Because of the fragments sizes and similarity values in BLASTN Programs in the period 09/ 2022, the classifcation of most sequences was built to the taxonomic genus level.Te prevailing genus (until now, because NC1, EC2, and WC7 are missing) was Bacillus sp.