Plant growth-promoting endophytic bacteria in peach palm seedlings

– The objective of this work was to isolate endophytic bacteria from peach palm ( Bactris gasipaes var. gasipaes ) plants and to evaluate the effects of their inoculation on the plant seedlings. Bacteria were isolated from the leaves and roots of the seedlings and from the meristems of peach palm plants in vitro. The isolates were characterized phenotypically and, then, 15 of them, representing different phenotypic groups, were selected and identified by partial sequencing of the 16S rRNA gene. Afterward, these isolates and two commercial strains of Azospirillum brasilense (Ab-V5 and Ab-V6) were inoculated in the peach palm seedlings. After 76 days, the seedlings were evaluated for plant development. The following six genera were identified based on the sequencing: Pseudomonas, Enterobacter , Rhizobium , Stenotrophomonas , Klebsiella , and Erwinia . Out of the 15 inoculated isolates, 9 had a positive effect on the root dry mass of palm peach, with CNPF 77 ( Enterobacter sp.), CNPF 100 ( Rhizobium sp.), and CNP 179 and CNPF 277 ( Stenotrophomonas sp.) standing out. Peach palm seedlings harbor endophytic bacteria which are able to increase root dry matter.


Introduction
Peach palm (Bactris gasipaes Kunth var. gasipaes Henderson) is considered the most important domesticated palm species in the Neotropics because of the diversity of its products, such as edible fruit rich in starch, flour, oil, and the production of palm hearts, (Graefe et al., 2013). Since the 1990s, this species has emerged as the main crop for supplying palm hearts in the Brazilian market; and the states of São Paulo, Bahia and Paraná are its main producers and consumers Franchetti & Rozane, 2017).
The expansion of peach palm cultivation area brought new demands. The provision of sufficient quantities of seed and seedlings of genetic and sanitary quality are some of the bottlenecks in this production system of this crop (Yokomizo & Kalil Filho, 2020). For this reason, studies involving different cloning techniques have been reported, such as the rooting of basal offshoots (Isaid et al., 2018). However, few significant advances are reported for the rooting percentage and field survival of this type of propagule that could result in a technique applicable on a larger scale (Yokomizo & Kalil Filho, 2020).
Plant growth-promoting bacteria (PGPB) can act on plant growth either through direct mechanisms (biological nitrogen fixation, phytohormone productions, phosphorus solubilization, and iron sequestration by siderophore producers) or indirect mechanisms (induction of systemic resistance and competition and production of antibiotics, among others) (Olanrewaju et al., 2017;Afzal et al., 2019). Several studies for the isolation and inoculation of bacteria have shown a large number of endophytic bacteria colonizing specific niches inside plants with different responses for plant growth (Jha et al., 2013;Brusamarello-Santos et al., 2017).
Considering the PGPB benefits, their inoculation in some agricultural crops and the use of biofertilizers obtained from bacteria, when applied at early stages of plant development and in vegetative propagules, can positively influence the initiation and growth of roots and stems (Mariosa et al., 2017;Cipriano et al., 2021). For instance, promising results were obtained from bacterial isolates in the induction and formation of adventitious roots in cuttings of woody species of eucalyptus, in which Azospirillum spp. strains were inoculated (Raasch et al., 2013). These bacterial groups have the ability to produce phytohormones, such as indole acetic acid, and they can induce further development of the root system (Lana et al., 2017). Costa et al. (2019) isolated endophytic fungi and bacteria from peach palm fruit with phytopathogen inhibition properties; however, the identification of the isolates was not carried out in their study. The isolation and selection of PGPB in peach palm are of great interest to increase the survival rate of seedlings, rooting of basal offshoots, pathogen control, and productivity.
The objective of this work was to isolate endophytic bacteria from peach palm plants, and to evaluate their effects on peach palm seedlings.

Materials and Methods
Ten grams of root and leaf tissues obtained from four peach palm seedlings approximately 8 months of age were used for the evaluation of the density and isolation of bacteria. These seedlings were produced from seed in a commercial nursery (MM Mudas, Eldorado, SP, Brazil) in plastic bags containing substrate. The roots and leaves were collected, and they were first washed in running tap water and superficially disinfected with alcohol 70% for 30 s, followed by sodium hypochlorite 6% (active chlorine) for 2 min and 30 s, and six washes in sterile deionized water. To ensure the isolation of only endophytes, 100 μL water from the last wash was plated to verify the absence of external bacteria. The plant material was placed in 90 mL of sterile saline solution (NaCl) at 0.85%, and ground in a blender at full power for 1 min, representing a 10 -1 dilution. An aliquot of 500 μL was taken from each sample, which was then added to a test tube containing 4.5 mL of sterile saline solution, representing a dilution of 10 -2 , followed by the same procedure until a 10 -7 dilution was attained. In Petri dishes containing solid culture medium (DYGS) with dextrose, yeast, and glutamate (Rodrigues Neto et al., 1986), 100 μL of each dilution was inoculated with three replicates each. The plates were incubated at 28°C for 10 days. Different colonies (approximately 10 colonies per plate) diluted from 10 -4 to 10 -7 were then selected and subcultured until purification. In addition to the isolates obtained from roots and leaves, 11 isolates obtained from meristems of peach palm grown in vitro, maintained at the laboratory of tissue culture and transformation, at Embrapa Florestas, were also included. All isolates were morphologically characterized in the DYGS medium.
For that, the morphology of at least three colonies isolated from each isolate was evaluated for the growth time (very fast, fast, intermediate, slow or very slow), form (punctiform, circular, or irregular), elevation (flat, convex, raised, pulvinate, or umbonate), margin (smooth, undulate, lobate, erose, or filamentous), surface (smooth, or rough), mucus production (sparse, little, moderate, or abundant), mucus transparency (opaque, transparent, or translucent), homogeneity, and color (Hungria & Silva, 2011). The similarity of the bacterial isolates was calculated using the Jaccard coefficient with NTSYS-pc software, version 2.1t (Rohlf, 2000). A dendrogram grouping the bacteria from roots, leaves, and meristems was generated using the UPGMA method. Bacterial isolates representative of the different phenotypic groups, with at least 75% dissimilarity, were selected with three isolates obtained from meristems (Table 1); then, they were subjected to partial sequencing of the 16S rRNA gene and to inoculation in seedlings. All isolates were stored in glycerol at -20°C and in mineral oil, at room temperature, in the laboratory of soil microbiology of Embrapa Florestas.
The isolated bacteria were cultured in solid DYGS medium for 48 hours, and the DNA from isolated colonies was extracted using the PureLink Genomic DNA mini kit (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA), following the manufacturer's instructions. The amplification of the 16S rRNA gene was performed using the primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') (Lane, 1991), and the partial sequencing was performed using the primer 27F. Sequencing was performed by Macrogen Inc., located in Seoul, South Korea, on an Applied Biosystems 3730xl Genetic Analyzer (Thermo Fisher Scientific, Waltham, MA, USA). The ends showing low quality were manually edited using the MEGA version X software (Kumar et al., 2018). Then, the sequences were subjected to the basic local alignment search tool (BLAST) in NCBI (2022a), using only alignment with type strains of the databank. The 16S rRNA gene sequences were deposited in the GenBank under the accession number OM912645-OM912659.
The nursery experiment was installed in February 2020 using peach palm seedlings provided by the company MM Mudas (Eldorado, SP, Brazil) of about 60 cm height and approximately 8 months of age. The seedlings were transplanted into 3.8 L pots containing a mixture of nonsterile soil and expanded vermiculite of medium texture at 1:1 (v/v). The soil chemical characteristics were the following: pH 6.29 in CaCl 2 ; 0.0 cmol dm -3 Al +3 ; 3.13 cmol dm -3 H+Al +3 ; 8.26 cmol c dm -3 Ca +2 ; 5.85 cmol c dm -3 Mg +2 ; 0.21 cmol c dm -3 K + ; 2.1 mg dm -3 P; and 2.85% total orgnic carbon. The seedlings were subjected to the inoculation of 15 bacterial isolates obtained from peach palm tissues and two strains of Azospirillum brasilense (Ab-V5 and Ab-V6). Bacteria were previously cultured in liquid DYGS medium for 48 hours, in an incubator at 28°C, under 150 rpm agitation. For that, 5 mL of each isolate was inoculated at the base of the seedlings (treatment), and also in the control treatment (5 mL of water). The strains of A. brasilense were inoculated at a proportion of 1:1 (v/v) both together and individually.
The statistical design was completely randomized with 5 replicates, and each one represented by a pot with one seedling. Fertilization was applied fortnightly following the recommendation of Morsbach et al. (1998). Intermittent nebulization was activated every 3 hours for 3 min. At 76 days after transplanting, the plant height (PH) and the number of leaves (NL) were evaluated. Aerial parts and roots were separated and dried at 60°C until they reached a constant weight. Shoot dry matter (SDM), root dry matter (RDM), and the root dry matter/shoot dry matter ratio (RDM/SDM) were evaluated. For all variables, the Shapiro-Wilk's normality test was performed, at 5% probability. The NL data did not show a normal distribution and were transformed to (x+1) 0.5 before the analysis of variance. The means were compared using the Scott-Knott's test (1974), at 5% probability. Statistical analyses were performed by the R software version 3.2.2 (R Core Team, 2015), using the statistical package ExpDes.pt version 1.1.2 (Ferreira et al., 2013).

Results and Discussion
Colony-forming units (CFU) from roots and leaves of the palm peach seedlings were estimated between 30 and 300 colonies. The bacterial densities were 3.40 x 106 CFU g -1 and 1.14 x 108 CFU g -1 for leaves and roots, respectively. After the purification, 207 bacterial isolates were obtained, including 58 from leaves and 138 from roots. There was no bacterial growth after roots and leaves surface washing, which suggests Pesq. agropec. bras., Brasília, v.57, e02962, 2022 DOI: 10.1590/S1678-3921.pab2022.v57.02962 that the isolated bacteria are endophytic. A group of 5 isolates from roots and 7 isolates from leaves were selected based on their phenotypic similarities, and these isolates together with 3 isolates from meristems were grouped into 15 phenotypic groups (Table 1).
There were no significant differences between the inoculation treatments and the control without inoculation for SDM in seedlings (Table 2). Because the experiment was carried out using seedlings, there was genetic variability between them, which may have resulted in the absence of a difference between inoculation and noninoculation treatments. Statistical differences among the treatments were observed only for RDM. Higher RDM values were observed for CNPF 77 (Enterobacter sp.), CNPF 100 (Rhizobium sp.), CNPF 179 (Stenotrophomonas sp.) and CNPF 277 (Stenotrophomonas sp.). The RDMs were also higher for five isolates -CNPF 90 (Klebsiella sp.), CNPF 94 (Rhizobium sp.), CNPF 105 (Klebsiella sp.), CNPF 118 Among the genera isolated from meristems, Erwinia is a genus among plant pathogens (Hauben et al., 1998) that cause a great damage to important crops; however, a recently identified strain as E. gerundensis showed evidence of plant growth promotion, especially regarding the acquisition of nutrients (Saldierna Guzmán et al., 2021). In our work, the isolate CNPF 110 (Erwinia sp.) did not cause any damage to seedlings. Increases of the RDM were observed when isolates belonging to the genus Klebsiella sp.-CNPF 90 and CNPF 105 -were inoculated in the seedlings. Bacteria belonging to the genus Klebsiella are commonly associated with plants and show growth-promoting characteristics and pathogen control (Dey et al., 2019), siderophore (Mowafy et al., 2021), phosphorus solubilization, and indolic acid compound production (Chalita et al., 2019) properties, among others. Bacteria of the Enterobacter genus, such as E. cloacae, have been reported to increase the germination rate, seed vigor, and shoot and root dry mass in rice (Oryza sativa), peanut (Arachis hypogaea), black bean (Vigna mungo) and canola (Yaish et al., 2015). Among the four bacterial isolates identified as Enterobacter, only CNPF 77 increased the root system in seedlings of peach palm. Isolates of the genus Pseudomonas were also found to be associated with peach palm in this work. Although bacteria of the Pseudomonas genus are plant growth promoters (Sah et al., 2021), among the three bacteria used in this study, only one promoted the increase of the root system, in comparison with the control, although at lower rates than those of the other isolates. Plants subjected to the inoculation of Stenotrophomonas, represented by the CNPF 179 and 277 isolates, also showed higher RDMs. The genus Stenotrophomonas comprises species which have been also reported to have growth-promoting abilities. When inoculated in tomato plants, bacteria of this   genus have also enhanced the tolerance to biotic stress, besides increasing the growth of the root system, (Alijani et al., 2020). Mucuna utilis seedlings subjected to the inoculation of Stenotrophomonas maltophilia also showed an increased tolerance to the biotic stress caused by Fusarium, besides a 30% increase of plant growth (Aeron et al., 2020). Stenotrophomonas isolates also incremented the Populus plants tolerance to abiotic stresses, besides increasing the growth of aerial parts and roots of in vitro cultivated plants and promoting the adventitious rooting of cuttings (Ulrich et al., 2021). The beneficial effects observed by the inoculation of Stenotrophomonas sp. in plants are attributed to rather direct effects, such as the production of AIA and phosphate solubilization, or indirect effects, such as the production of the enzyme ACC deaminase and siderophores (Aeron et al., 2020). These results are corroborated by the findings obtained in the present work, in which the inoculation of peach palm seedlings with two Stenotrophomonas isolates resulted in the increase of the root system, in comparison with the control treatment. Regarding the RDM/SDM ratio (Table 2), there were significant differences among treatments. However, for all treatments, the values were balanced and most were close to 1, indicating that the seedlings were healthy, although imbalanced values of the RDM/SDM ratio can occur under stress (Agathokleous et al., 2019). In peach palm, this is the first report on the occurrence of Rhizobium as an endophytic microorganism, and it was observed both in roots and leaves. However, the function of the Rhizobium genus in this culture still needs to be investigated, and further studies are required to understand the mechanisms involved in the plant growth promotion. Despite this lack of information, a positive effect of Rhizobium inoculation was observed on the production of root dry matter in peach palm. This fact becomes quite interesting, since this genus has long been characterized as being capable of establishing symbiosis with legumes, with which it is involved in the biological nitrogen fixation. Until recently, it was believed that the Rhizobium life cycle involved growth in the legume nodule as a symbiont, and in the soil as a saprophyte (Yanni et al., 1997). However, the genus Rhizobium has also been associated with various nonleguminous plants (Díez-Méndez et al., 2021). In our work, it increased the RDM of peach palm. Some isolates that showed the ability to increase the RDM were identified within genera with species that can cause damage to human and animal health (Enterobacter, Stenotrophomonas, Klebsiella, and Pseudomonas) (Martin & Bachman, 2018;Keswani et al., 2019;Ambreetha et al., 2022) and which should be carefully evaluated. These isolates need a complete characterization, at species and strain levels, by using polyphasic approaches for microbial classification prior to bioinoculant development and environmental release (Keswani et al., 2019). However, two isolates, identified as Rhizobium, are more viable candidates for short-term development because this genus shows low risk and is widely used in agriculture in Brazil.

Conclusion
Peach palm (Bactris gasipaes) seedlings harbor endophytic bacteria that are able to increase root dry matter, with four isolates belonging to the genera Enterobacter, Rhizobium, and Stenotrophomonas standing out.