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Article

Identification, Characterization, and Growth-Promoting Effects of Bacterial Endophytes Isolated from Okra (Abelmoschus esculentus L.)

by
Ahsanul Salehin
1,
Sakiko Yamane
1,
Makoto Ueno
1 and
Shohei Hayashi
1,2,*
1
Faculty of Life and Environmental Sciences, Shimane University, 1060 Nishikawatsu, Matsue 690-8504, Japan
2
Estuary Research Center, Shimane University, 1060 Nishikawatsu, Matsue 690-8504, Japan
*
Author to whom correspondence should be addressed.
Agronomy 2023, 13(5), 1226; https://doi.org/10.3390/agronomy13051226
Submission received: 10 March 2023 / Revised: 14 April 2023 / Accepted: 23 April 2023 / Published: 26 April 2023
(This article belongs to the Special Issue How Could Microorganisms Benefit the Agriculture Environment?)
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Abstract

:
Microorganisms colonize plant roots and exhibit plant growth promotion properties, therefore functioning as biofertilizers. To effectively use plant growth-promoting rhizobacteria, understanding their colonizing behavior and ability to compete with co-existing bacteria is essential. In this study, 12 endophytic bacterial strains belonging to seven genera in four classes with 99–100% homology were isolated from the roots of okra plants (Abelmoschus esculentus L.). Four isolates (Okhm3, Okhm5-4, Okhm10, and Okhm11) were inoculated on okra seeds and their effects on plant growth and colonization with single and mixed inoculations were evaluated. Okra was cultivated using sterilized vermiculite, and the growth parameters and colonization were measured 30 d after seed inoculation. All strains exhibited plant growth promotion traits that could improve okra plant growth in pot culture experiments. Notably, Okhm5-4 and Okhm10 strains (belonging to the Ensifer and Pseudomonas genera) revealed the highest growth-promoting effects on okra plants. Both strains were detected in the endosphere and rhizosphere of okra plants. Okhm10 and Okhm5-4, with lower colonization than Okhm3, showed better growth than Okhm3. Therefore, the colonization potential does not determine the growth-promoting effects. While Okhm3 populations remained stable in both inoculation conditions, the population level of other strains decreased in the mixed inoculation. This study showed bacterial endophytes isolated from Okra can be exploited as bio-fertilizers for sustainable agriculture systems.

1. Introduction

To ensure food security, increased food production is urgently needed because of the growing global population, and modern agriculture systems are being intensified using various technologies to meet the growing global demand for food supply [1,2]. Currently, agricultural intensification and crop production largely depend on chemical fertilizers [3], and this is one of the most important ways to increase efficiency and obtain better quality products in agriculture [4]. However, in recent years, the use of chemical fertilizers has exponentially increased worldwide, causing serious environmental problems [5,6]. Therefore, it is a great challenge in agriculture to find sustainable strategies to ensure competitive crop yields, provide environmental safety, and protect the ecological balance in the agro-ecosystem to relieve the detrimental effects of intensive farming practices.
Plant growth-promoting bacteria (PGPB) are soil-borne, free-living bacteria that aggressively colonize the rhizosphere, plant roots, or both, and enhance the growth and yield of plants during cultivation [7]. The use of PGPB is becoming a widely accepted practice in intensive agriculture to enhance sustainable agricultural production in different parts of the world [8]. Bio-fertilizers are based on microorganisms that colonize the rhizosphere or the interior of plants and promote growth by enhancing the nutrient supply to the host plant when applied to seeds, plants, or the soil [9]. PGPB are gaining attention as bio-fertilizers that improve the quality and sustainability of soil and increase crop yield [10,11,12,13] as they successfully colonize the rhizosphere, rhizoplane or root interior [14]. Plant growth may be enhanced under different stress conditions through microbial inoculation, and it is assumed that these microbes enhance growth and yield in different ways, such as by regulating the nutritional and hormonal balance, producing plant growth regulators, solubilizing nutrients, and inducing resistance against plant pathogens [15,16,17]. Microorganisms play an important role as microbial resources since they can promote plant growth in several ways, including increasing the seed germination rate, root and shoot growth, yield, leaf size, chlorophyll content, nitrogen-protein content and drought tolerance, and delaying leaf aging [18,19,20,21,22].
Endophytes live inside plants, colonizing the internal tissues of host plants, and can establish a mutualistic association without harming the host plants [23,24,25]. Endophytes promote plant growth in many ways, including by phytohormone production [26,27], siderophores production [28,29], expression of 1-aminocyclopropane-1-carboxylate (ACC) deaminase [30,31], and nitrogen fixation [32,33]. In addition, some endophytes have been reported to be capable of protecting plants by producing antipathogenic substances [34,35], ameliorating disease development [36,37], and inducing resistance to biotic and abiotic stresses [23].
Okra (Abelmoschus esculentus L.) belongs to the Malvaceae family and is also known as ladies finger, okro, or gumbo [38]. It is an important vegetable crop that is native to tropical Africa. Currently, it is a widely grown vegetable crop in the tropics and sub-tropics, as well as in warmer temperate areas, due to its delicious tender fruits [39,40,41]. Okra is widely used as a vegetable crop and provides important nutrients to the human diet, including minerals, proteins, carbohydrates, and vitamins [42] and is also used commercially in medicine and industry [40,43]. Diverse endophytic bacteria have been isolated from okra plants, such as Bacillus, Pseudomonas, Azospirillum, Serratia, and Enterobacter [44,45]. Among the endophytic isolates of the okra plant, many strains have beneficial properties, such as nitrogen fixation, auxin production, antagonistic effects, phosphate solubilization, potassium solubilization, and siderophore production.
In sustainable agriculture practices, inoculation is one of the most accepted approaches to ensure the effects of plant growth promotion [46]. Inoculation with PGPB has already been studied to confirm its significant role in increasing the growth and yield of agronomically important crops, such as okra [44], maize [47], cotton [48], wheat [49], sweet potato [50,51], rice [52], canola [53] and strawberries [54].
In addition to the individual colonizing ability of PGPR, interactions with other co-existing bacteria are important for determining colonization and plant growth-promoting potential. Therefore, negative interactions with co-existing bacteria should be considered. It has been reported that co-existing bacteria inhibit colonization and reduce the inoculant population number in sugarcane, with this antagonistic behavior being observed under in vitro conditions [55]. Such interactions also decrease the plant growth-promotion effect in tomato plants [26,27,56].
To utilize PGPR efficiently and effectively, it is essential to understand its colonizing behavior and its ability to compete with co-existing bacteria. The effects of co-inoculation with multiple bacteria on plant growth have been studied, but their activity as PGPR on okra plants has not been extensively explored. The aim of this study was to isolate and identify the endophytic bacterial strains from okra roots and to evaluate their effects of PGPR inoculation on okra growth. We then aim to determine how co-inoculation of the endophytic strains affects their colonization and plant growth.

2. Materials and Methods

2.1. Sample Collection and Isolation of Endophytic Bacteria

The collected okra plant (Abelmoschus esculentus) was cultivated from June to October 2015 in an agricultural field in Shimane prefecture, Higashi Mochida-cho, Japan. After collection, the plant sample was aseptically transported to the laboratory. The sample was washed with running tap water for 10 min, and the water was wiped off with a paper towel. Then, the thick part of the root was cut with a sterilized knife, and the surface was sterilized with 70% ethanol for 1 min followed by 3% sodium hypochlorite for 3–4 min. For the isolation of endophytic bacteria, the root parts were macerated, serially diluted, plated on yeast mannitol agar (YMA) medium [57], and incubated at 26 °C for 4–5 days (Table S1). The efficiency of the washing procedure was evaluated by stamping the surface of the sterilized root onto YMA agar media. After incubation, 12 bacterial colonies were picked, and repeated culturing was performed for isolation.

2.2. Identification of Endophytic Bacteria

The HM medium in Minamizawa et al. [58] was used in this study (Table S2). The 16S rRNA genes of the isolated strains were PCR-amplified using KOD plus polymerase (Toyobo Co., Ltd., Osaka, Japan) with the primers fD1 (5′-AGAGTTTGATCCTGGCTCAG-3′) and rP2 (5′-ACGGCTACCTTGTTACGACTT-3′) [59]. The PCR amplification mixture and PCR running conditions are summarized in Tables S3 and S4, respectively. A part of the colony of an isolated strain was added to the mixture as a template. After the confirmation of amplification of the 16S rRNA gene (about 1500 bp), the dideoxy sequencing was performed at Eurofins Genomics K.K. (Tokyo, Japan). The primer fD1-rP2 was used to determine the partial nucleotide sequence of all 12 strains. The closest sequence for each isolate was assigned using the database (https://www.ddbj.nig.ac.jp/ (accessed on 5 April 2023)) by a BLAST [60] search. A multiple sequence alignment was constructed using ClustalW 2.1 [61]. The alignments were manually edited, and the neighbor-joining methods based on partial sequences of the 16S rRNA gene (1354 bp) with 1000 bootstrap replicates were used to construct phylogenetic trees using MEGA 11 [62]. The substitution model was maximum composite likelihood. All sequences data were deposited with the DDBJ Nucleotide Submission System under the accession numbers LC745574 to LC745585.

2.3. Characterization of Endophytic Bacteria

Twelve representative isolates of nine genera in four classes were selected based on their phylogenies (Table 1 and Figure 1), and four isolates were used to characterize their plant growth-promoting and endophytic abilities.
The indole-3-acetic acid (IAA) production ability of the four selected isolates was determined using the Salkowski assay [63] on HM medium (Table S2). The isolates were cultivated in HM liquid medium supplemented with 200 μg/mL L-tryptophan at 26 °C with continuous shaking at 150 rpm. Uninoculated samples were used as controls. After four days of incubation, an aliquot of the supernatant was collected from the centrifuged culture at 10,000× g for 10 min at 4 °C. Then, by adding double the volume of Salkowski reagent, the absorbance was measured at 530 nm using a UV-VIS spectrophotometer (UV-1700, Shimadzu, Kyoto, Japan) after 30 min in the dark.
The Phosphate solubilization assays were performed by streaking the bacterial isolates on Pikovskaya’s medium containing Ca3(PO4)2 (5 g/L) as the phosphate source, NH4NO3 (0.50 g/L), MgSO4 (0.10 g/L), NaCl (0.20 g/L), dextrose (10 g/L), yeast extract (0.50 g/L), and agar (15 g/L) [64]. After incubation for three days at 26 °C, a clear halo zone around the bacterial colonies confirmed the solubilization of mineral phosphate.
For the cellulase assay, isolates were spotted on carboxymethyl cellulose (CMC) agar medium [65]. After incubation for six days at 26 °C, a clear zone around the point of inoculation was examined by staining the remaining CMC with Congo red [66]. For the pectinase assay, the bacterial strains were spotted on nutrient agar (DIFCO Laboratories, Detroit, MI, USA) supplemented with 0.5% pectin. After incubation at 28 °C for three days, the remaining pectin was stained with cetyltrimethylammonium bromide (CTAB) to visualize the clear zone around the inoculated bacteria [67].
To determine the dinitrogen-fixing potential of the isolates, PCR amplification of the nifH gene, which encodes nitrogenase reductase, was performed using a small amount of culture sample as a template. The primers PolF (5′-TGCGAYCCSAARGCBGACTC-3′) and PolR (5′-ATSGCCATCATYTC RCCGGA-3′), which were designed to match a broad range of bacterial nifH genes [68], were used for PCR detection (Table S5).
The ACC deaminase assay was performed by streaking the bacterial isolates on DF salt minimal medium [69], and the presence of growth in the media after incubation at 26 °C for three days was considered positive. The ingredients of DF salt minimal medium are listed in Table S6.

2.4. Effect of Inoculation on Plant Growth Promotion

Four selected bacterial strains (Table 2 and Figure S1) were used in the inoculation experiment. To prepare the bacterial inoculum, each bacterial strain was cultivated separately in HM liquid medium with continuous shaking at 150 rpm at 26 °C for four days. The culture was harvested by centrifugation (10,000× g for 10 min at 4 °C), washed twice with sterilized distilled water, and the cell pellet was resuspended in sterilized distilled water at 109 colony-forming units CFU/mL to prepare an inoculum based on OD–CFU/mL correlated linear equations prepared for each strain. Co-inoculation was performed with all four strains. The inoculum for co-inoculation was prepared by culturing the four strains under the same conditions described above, and cell suspensions of each strain were mixed to obtain the same density. In order to avoid antagonistic interactions between the strains, their strain compatibility for growing together was assessed by HM liquid medium and further plating. The four strains on HM agar medium were separately counted based on naked-eye observation.
In this study, okra (Abelmoschus esculentus) was used as the test plant. Okra seeds of cv. Early five (AOK001, Takii & Co., Ltd., Kyoto, Japan) were surface-sterilized by dipping them in 70% ethanol for 1 min, followed by 1% sodium hypochlorite (NaOCl) with 3–4 drops of Tween-20 for 13 min. The seeds were then washed 7–8 times with sterilized distilled water and inoculated by dipping in each bacterial inoculum solution at ~109 CFU/mL for 8 h. The same number of seeds was soaked in sterilized distilled water as a control. The inoculated seeds were sown in a Leonard jar [70] whose upper pot was filled with water-soaked sterile vermiculite, and the lower pot was supplied with a sterilized Hoagland solution [71]. A cotton wick was connected to supply liquid nutrient solution to the top pot. The Leonard jar was placed in a ventilated (<0.2 mm pore size) transparent plastic bag (Sun bag, Sigma-Aldrich, Tokyo, Japan), and the inoculated plant was aseptically grown in a phytotron (Model-LH 220S, Nippon Medical and Chemical Instruments Co., Ltd., Osaka, Japan) at 28/25 °C (14 h/10 h, day/night) with 6000 to 7000 lux light intensity under white fluorescent light conditions for 30 days. The experiment was conducted twice, using six plants for each treatment. After 30 days of cultivation, the okra plants were harvested, and initial plant growth parameters (fresh weight and length of the root and shoot, chlorophyll, and leaf number) were measured.

2.5. Colonization of Endophytes in Okra Plant

The populations of the inoculated strains in the root, shoot, and rhizosphere were determined in all six replicates for each treatment. For the rhizosphere sample, the root part was dipped in 150 mL of sterilized distilled water and gently shaken to suspend the inoculants in the rhizosphere, using samples from the six replicates. Then, the root and stem parts were separated, washed 6–7 times in sterilized distilled water to remove most of the surface-attached bacteria, macerated with sterilized distilled water using a sterilized motor and pestle, and then subjected to dilution plating (incubation at 26 °C for three days on HM medium) for the determination of CFU/g. The identification of the bacterial strains in the colonization assay was based on naked-eye observation. The morphologies of the colonies of the co-inoculated strains were clearly different when they were counted separately. At the same time, an aliquot of the final washing solution was plated directly, and no colonies were detected.

2.6. Statistical Analysis

Statistical analysis of the data regarding plant growth and population of the inoculant was performed using the IBM SPSS software package version 28 (IBM Co., Armonk, NY, USA). The data were subjected to Tukey’s test after one-way ANOVA.

3. Results

3.1. Isolation and Identification of Endophytic Bacteria

Different morphologies were observed among the bacterial colonies on the YMA medium, and 12 endophytic bacterial strains were isolated and identified from the roots of okra plants. 16S rRNA sequences of 1289 to 1382 bp were determined in this study. Based on partial 16S rRNA gene sequencing analysis, 12 endophytic bacterial isolates were assigned to their close relatives through BLAST analysis using the DDBJ database. These isolates belonged to seven bacterial genera in four classes, which showed 99–100% homology, as presented in Table 1 and Table S7; their phylogenetic tree is presented in Figure 1. The α-proteobacteria class was predominantly identified among the four classes. According to the DDBJ database sequences, Okhm3 and Okhm11 are associated with Rhizobium; Okhm4, Okhm5-2, Okhm7, and Okhm8 are Agrobacterium; Okhm5-1 and Okhm12 are Bacillus; Okhm5-3 and Okhm5-4 are Ensifer; Okhm9 are Variovorax; and Okhm10 are Pseudomonas expressing 99–100% similarity.

3.2. Selection and Characterization of Isolated Endophytic Bacteria

Four isolates (Okhm3, 5-4, 10, and 11) which tended to promote okra growth in a preliminary inoculation experiment were selected among the 12 isolates for the experiment to evaluate the plant growth-promoting properties on okra plants. The most relative closets for the selected four isolates, Okhm3, Okhm5-4, Okhm10, and Okhm11, were identified as Rhizobium paranaense, Ensifer adhaerens, Pseudomonas gessardii, and Rhizobium herbae, respectively, by 16S rRNA gene analysis, and their plant growth-promoting activities are presented in Table 2. Each of the four isolates exhibited ACC deaminase activity, but none showed either nifH amplification or cellulose activity. IAA was only produced by Okhm10, and pectinase activity was only observed in Okhm3. Okhm3, Okhm10, and Okhm11 demonstrated a phosphorus solubilization zone around the endophyte colonies. The selected four bacterial isolates were cultivated together and none of them showed antagonistic activity against each other.

3.3. Effect of Plant Growth Promotion of Isolated Strains in Okra Plant

The effects of single inoculation and co-inoculation with the isolated strains on the growth of okra plants are presented in Figure 2. With a single inoculation, all the isolated strains showed plant growth-promoting ability. Okhm5-4 and Okhm10 showed the highest plant growth-promoting abilities among the inoculated and control strains. All growth parameters of Okhm5-4 and Okhm10 in okra plants were significantly higher than those in the control. Okhm3 and Okhm11 also enhanced okra plant growth compared with the control. The root and shoot weights after inoculation were significantly increased for Okhm3 and Okhm11, respectively, compared with the control. In okra plants inoculated with Okhm3, Okhm5-4, Okhm10, and Okhm11, root weight, shoot weight, root length, and shoot length increased by 0.7–2.0, 0.7–1.8, 5.3–16.7, and 1.1–5.6-fold, respectively, compared with the control.
Co-inoculation of the isolated strains also enhanced okra plant growth. The root and shoot weights were significantly increased by co-inoculation compared with the control, whereas the growth was lower than that of the single inoculation with Okhm5-4 and Okhm10. In okra plants co-inoculated with Okhm3, Okhm5-4, Okhm10, and Okhm11, root weight, shoot weight, root length, and shoot length increased by 1.1, 1.1, 7.4, and 3.5- fold, respectively, compared with the control.
More lateral root development was observed in inoculated okra plants than in control plants. No differences were observed in the leaf number or chlorophyll content between the inoculated and control plants (Figure S2). The experiment was conducted twice, and the same results were obtained.

3.4. Effect of Colonization of Isolated Strains in Okra Plant

The populations of the inoculated endophytic strains in the rhizosphere, roots, and shoots of the okra plants are presented in Figure 3. All the isolated strains were colonized and detected in the rhizosphere, root, and shoot of okra plants 30 days after seed inoculation in both the single- and co-inoculated treatments. No colonies were observed on the control plates.
In the single inoculation treatments, the rhizospheric populations of Okhm3 were significantly higher than those of the other inoculated strains (Figure 3a). The populations of Okhm3 were 3.2, 3.5, and 2.7-fold higher in the rhizosphere than those of Okhm5-4, Okhm10, and Okhm11, respectively, after single inoculation. In the case of co-inoculation, all the strains colonized the okra plants, resulting in a large population, in which the rhizospheric population of Okhm3 was also significantly larger. Its population was 2.9, 3.3, and 2.8-fold higher than Okhm5-4, Okhm10, and Okhm11, respectively. The population size of the inoculated strains in the rhizosphere tended to decrease by 0.4–0.8-fold compared with single inoculation.
In the case of a single inoculation, the root populations of Okhm3 were also significantly larger than those of the other inoculated strains (Figure 3b). After single inoculation, the populations of Okhm3 were 2.4, 1.8, and 1.9-fold higher in the roots than those of Okhm5-4, Okhm10, and Okhm11, respectively. In the co-inoculation treatments, the root population of Okhm3 was also significantly larger, and was 4.1, 4.2, and 3.3-fold larger than Okhm5-4, Okhm10, and Okhm11, respectively. In the co-inoculation treatment, Okhm3 maintained its population; however, the populations of Okhm10 and Okhm11 significantly decreased. Therefore, the populations of Okhm3 in the root were similar to those with the single inoculation, while, the population decreased by 1.7–2.5-fold compared with the single inoculation of Okhm5-4, Okhm10, and Okhm11, respectively.
In the single inoculation treatment, the shoot populations of Okhm3 were also significantly higher, being 2.9, 2.4, and 2.3-fold higher than those of Okhm5-4, Okhm10, and Okhm11, respectively (Figure 3c). With co-inoculation, the populations of Okhm3 in the root were also significantly larger, 4.3, 4.0, and 3.7-fold higher than Okhm5-4, Okhm10, and Okhm11, respectively. The populations of Okhm3 were maintained levels similar to those for the individual inoculation, whereas the populations decreased by 1.4–1.7-fold for Okhm5-4, Okhm10, and Okhm11.
Furthermore, the populations of inoculated strains (Okhm3, Okhm5-4, Okhm10, and Okhm11) in the rhizosphere were 3.0–4.4, 2.3–4.2, 1.4–3.4, and 2.2–4.1-fold higher with single inoculation and 2.5–3.9, 3.6–5.2, 3.4–4.6, and 3.0–4.7-fold higher with co-inoculation than in the roots and shoots, respectively.

4. Discussion

In the present study, 12 endophytic bacterial strains were isolated from the roots of okra plants and α-Proteobacteria was the dominant class among the isolated Proteobacterial strains, followed by β- and γ-Proteobacteria (Table 1). In previous studies on okra endophytes, Proteobacteria, including α-, β-, and γ-Proteobacteria, and Bacilli were also predominant among the isolated strains [44,45]. These results suggest that the endophytic communities of okra plants consist of bacteria belonging to common phyla. The genera Rhizobium and Pseudomonas in Proteobacteria and Bacillus in Bacilli have been reported as endophytes [44,45] in okra plants. The most dominant genera, Agrobacterium as well as Ensifer, and Variovorax identified in this study, have not been reported as okra endophytes. On the other hand, the common genera in other studies, Enterobacter sp., Azospirillum sp., and Serratia sp. [44,72] were not isolated in our study. These results suggest the presence of diverse bacterial endophytes of okra with some common genera, as well as the effects of okra cultivar and environmental conditions at the okra growing site. Gaiero et al. [73] reported that soil type and environmental factors, such as the microbial community, influenced the endophytic community structure, which may explain the differences between our study and previous studies.
In this study, four endophytic bacterial strains isolated from the roots of okra were examined for their growth-promoting activities in inoculated plants. All the isolated strains exhibited significant plant growth promotion ability, with Okhm5-4 and Okhm10 showing the highest plant growth promotion ability among the inoculated strains (Figure 2). The four selected strains were identified as Rhizobium, Ensifer, and Pseudomonas (Table 2), indicating that their growth-promoting ability is not exclusive to a particular genus. The isolated strains used in our study showed some plant growth-promoting properties, and the same PGPR properties in okra plant growth have been previously reported [44,45,74,75]. Hence, multiple pathways exist to increase the growth of okra, and endophytic bacteria have different combinatorial growth-promoting properties. Ji et al. [76] reported that inoculation with PGPR has a growth-promoting effect on plants, and inoculation with beneficial microbes could be used in agriculture to enhance growth and protect crops against disease. Furthermore, in our study, we observed a positive effect of inoculating isolated strains of okra on growth without displaying any signs of disease. In this study, no significant difference was observed in plant chlorophyll content as compared with the uninoculated treatment (Figure S2). Eleiwa et al. [77] reported that inoculation of wheat grains with the biofertilizers Bacillus polymyxa or Azospirillum brasilinseas significantly increased the chlorophyll a, chlorophyll b, and carotenoids compared with control.
The present study showed that the inoculants stimulated the lateral root growth of okra plants (Figure 2), which led to higher root weights. The inoculation with Okhm10, which had IAA-producing activity, improved lateral root growth and resulted in a significant increase in the fresh root weight of okra, suggesting that the production of IAA by Okhm10 is one of the factors that promote plant growth. Similar changes in root morphology were observed after inoculation with IAA-producing Klebsiella sp. in tomato plants [26], whereas IAA-non-producing Bacillus sp. did not affect the lateral root growth of tomato plants [27]. IAA has similar effects on okra plants, where inoculation with IAA-producing Pseudomonas spp. significantly improved okra plant growth parameters [44,45]. Furthermore, IAA production in the rhizosphere has been reported as an important plant growth-promoting factor that stimulates lateral root growth and absorption of nutrients [78], and inoculation with other IAA-producing plant-associated bacteria has shown similar effects on inoculated strawberries [79] and mung bean [80]. Therefore, it is concluded that IAA production by endophytic bacteria is one of the pathways that promote growth, mainly the lateral root growth of okra.
Three inoculated strains, which did not have IAA-producing activity (Table 2), also showed okra growth-promoting ability (Figure 2), indicating that there are other ways to induce okra growth. In addition to the IAA production, other mechanisms of plant growth promotion have been reported as follows: ACC deaminase production [75,81], phosphate-solubilizing abilities [44,74,82], cellulase-pectinase activity [26,83], siderophores production [44,84], nitrogen fixation [81], organic acid production, and production of plant hormones such as IAA [44,74,75], gibberellic acid (GA3) [85], and cytokinins [74,86]. In the present study, each of the four isolates exhibited ACC deaminase activity, with Okhm3, Okhm10, and Okhm11 demonstrating phosphorus solubility (Table 2), suggesting that there are multiple pathways to promote okra plant growth. It was also found that inoculation with Bacillus sp., Pseudomonas sp., Azospirillum sp., and Serratia sp., which had multiple plant growth-promoting traits, enhanced the growth of okra plants, as observed in our study [44,74].
In this study, all the inoculation strains colonized the rhizosphere and endosphere of the okra plants (Figure 3), resulting in colonization potential. Although similar results of colonization by PGPR inoculation have also been reported in several studies on sweet potato and tomato plants [26,27], this study is the first to evaluate the colonization of inoculated bacteria in okra. Among the inoculated bacteria, Okhm3 population densities were higher by 2.7–3.5, 1.7–2.4, and 2.3–2.9-fold in the roots, rhizosphere, and shoots of the plants, respectively (Figure 3), indicating the high colonization ability of Okhm3. The pectinase-producing ability of Okhm3 may facilitate the entry of the inoculant into okra plant tissues and subsequent colonization. It has been reported that Rhizobium and Azospirillum strains produce hydrolytic enzymes that enable them to enter white clover and colonize [83]. Despite the high colonization of Okhm3, it did not show higher growth promotion than Okhm5-4 and Okhm10, suggesting that high colonization does not always result in high plant growth. In addition, these results indicate that the potential of a single cell of Okhm5-4 and Okhm10 is higher than that of Okhm3.
Plant growth-promoting effects were also observed with the co-inoculation of the isolated strains; however, the growth promotion was lower compared with the single inoculation of Okhm5-4 and Okhm10 (Figure 2). Inoculation with multiple beneficial bacteria has been reported to have a higher potential than inoculation with a single bacterial inoculant [47,55], while cancelation of the positive effects [87,88,89], and negative effects of co-inoculation have also been reported [26,27,56]. The effects of co-inoculation seem to depend on the combination of the strains. In our study, plant growth was observed to be lower in the co-inoculation condition than in the single inoculations of Okhm5-4 and Okhm10. The reason might be the neutralization of the phytostimulation ability of the other inoculated strains. It has been reported that plant growth promotion is inhibited by Bacillus sp. F-33 when it was co-inoculated with other endophytes [27], which is similar to our results. These results provide useful information for evaluating the effects of growth promotion in practical use because the actual cropping area contains numerous microorganisms.
All the inoculated strains colonized the rhizosphere and endosphere of the okra plants in the co-inoculation treatment (Figure 3). The population of Okhm3 was maintained; however, the populations of the other inoculated strains were 0.4–2.5-fold decreased compared with those in the single inoculation. The high colonizing potential of Okhm3 seemed unaffected by other inoculants under co-inoculation conditions. These results suggest that the competitive interactions among the inoculated strains for colonization might be the reason for the decrease in the population, except in the case of Okhm3. It is hypothesized that several functions, such as pectinase-producing ability and hydrolytic enzymes, may be involved in plant colonization ability and that PGPR have different interactions with plants when establishing their populations in the rhizosphere and/or endosphere. However, the majority of studies that examined the effects of co-inoculation with PGPR did not examine changes in PGPR populations. Using limited examples, Dhungana et al. [26] found that co-inoculation of Klebsiella sp. and Herbaspirillum sp. decreased plant growth promotion in radish plants while maintaining their PGPR population. Salehin et al. [27] reported that tomato plant growth was reduced by Klebsiella sp. inoculation following Bacillus sp., whereas PGPR populations of Bacillus sp. decreased after inoculation.
The four selected isolates were characterized and used to inoculate okra plants, and thus, their plant growth-promoting activity and colonization abilities were observed in this study. The four isolates belonged to three different genera with different characteristics. The current results provide useful information that could be helpful for sustainable agriculture.
Although the value of plant growth cannot be assessed until the yield is measured, principal plant growth is considered a very important stage for plant development. This study investigated the effects on the initial growth of okra and significant okra growth has been observed.
While Okhm5-1 was closely related to Bacillus mobilis, this strain was also related to Bacillus cereus. Bacillus cereus is a ubiquitous soil bacterium responsible for food-associated gastrointestinal diseases [90] and is estimated to be responsible for 1.4–12% of all food poisoning outbreaks worldwide [91]. Therefore, when inoculating it, it is necessary to investigate its virulence and toxicity.

5. Conclusions

In the present study, all the strains isolated from okra roots exhibited growth-enhancing features, such as IAA, phosphate solubilization, ACC deaminase production, and pectinase activity, which could contribute to the improvement of okra plant growth parameters in pot culture experiments when applied individually or in combination. In particular, Okhm5-4 and Okhm10 showed the greatest growth-promoting effects on okra. All the inoculated strains colonized and detected the endosphere and rhizosphere of okra plants in both treatments.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy13051226/s1, Table S1: Ingredients of yeast mannitol agar (YMA) medium, Table S2: Ingredients of HM agar medium, Table S3: PCR ingredients for amplification of 16S rRNA, Table S4: PCR running conditions, Table S5: PCR ingredients for amplification of nifH gene, Table S6: Ingredients of DF salt minimal medium, Table S7: Relevant data about BLAST results of isolated endophytic bacterial strains from okra plants, Figure S1: Selected four endophytic bacterial strains used for inoculation experiment (a); okra plants in phytotron (b), Figure S2: The effects of inoculation with the isolated strains on the chlorophyll (a) and leaf number (b) of okra plants. The okra plants were cultivated using sterilized vermiculite, and the parameters were measured 30 days after seed inoculation. CTL represents the control samples. The bars represent the standard deviation (n = 6), and different letters indicate significant differences at p < 0.05 by Tukey’s test.

Author Contributions

Conceptualization, A.S., M.U. and S.H.; methodology, A.S. and S.H.; software, A.S. and S.H.; validation, A.S. and S.H.; formal analysis, A.S. and S.H.; investigation, A.S., S.H. and S.Y.; resources, A.S., S.Y. and M.U.; data curation, A.S. and S.H.; writing—original draft preparation, A.S. and S.H.; writing—review and editing, A.S. and S.H.; visualization, A.S. and S.H.; supervision, S.H.; project administration, S.H.; funding acquisition, S.H. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

The data presented in this study are available in the article.

Acknowledgments

The authors thank the faculty of Life and Environmental Sciences at Shimane University for their financial support for the publication of this report.

Conflicts of Interest

The authors declare no conflict of interest.

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Agronomy 13 01226 g001 550
Figure 1. Phylogenetic tree based on the partial sequence of 16S rRNA gene of isolates and related strains. The phylogenetic trees were constructed using the neighbor joining method with 1000 bootstrap replicates in the software program MEGA11. Bootstrap values above 60% are shown at the nodes. The sequences of the inoculated strains are boldfaced. The accession numbers are indicated to the right of the strain name. Methanobacterium thermoautotrophicum (AB020530.1) constituted an outgroup.
Figure 1. Phylogenetic tree based on the partial sequence of 16S rRNA gene of isolates and related strains. The phylogenetic trees were constructed using the neighbor joining method with 1000 bootstrap replicates in the software program MEGA11. Bootstrap values above 60% are shown at the nodes. The sequences of the inoculated strains are boldfaced. The accession numbers are indicated to the right of the strain name. Methanobacterium thermoautotrophicum (AB020530.1) constituted an outgroup.
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Figure 2. The effects of inoculation with isolated strains on the growth of okra plant root weight (a), shoot weight (b), root length (c), and shoot length (d). The okra plant was cultivated using sterilized vermiculite, and the parameters were measured 30 days after seed inoculation. CTL represents the control samples. The bars represent the standard deviation (n = 6), and different letters indicate significant differences at p < 0.05 by Tukey’s test.
Figure 2. The effects of inoculation with isolated strains on the growth of okra plant root weight (a), shoot weight (b), root length (c), and shoot length (d). The okra plant was cultivated using sterilized vermiculite, and the parameters were measured 30 days after seed inoculation. CTL represents the control samples. The bars represent the standard deviation (n = 6), and different letters indicate significant differences at p < 0.05 by Tukey’s test.
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Figure 3. Colonization of inoculated strains in the rhizosphere (a), root (b), and shoot (c) of the okra plants, and the effects of single inoculation and co-inoculation of seeds with isolated strains Okhm3, Okhm5-4, Okhm10, and Okhm11 on colonization. The okra plant was cultivated using sterilized vermiculite, and colonization was examined 30 days after seed inoculation. No colony appeared in the control samples. The bars represent the standard deviation (n = 6), and different letters indicate significant differences at p < 0.05 by Tukey’s test.
Figure 3. Colonization of inoculated strains in the rhizosphere (a), root (b), and shoot (c) of the okra plants, and the effects of single inoculation and co-inoculation of seeds with isolated strains Okhm3, Okhm5-4, Okhm10, and Okhm11 on colonization. The okra plant was cultivated using sterilized vermiculite, and colonization was examined 30 days after seed inoculation. No colony appeared in the control samples. The bars represent the standard deviation (n = 6), and different letters indicate significant differences at p < 0.05 by Tukey’s test.
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Table
Table 1. The closest relatives of isolated endophytic bacterial strains from okra plants.
Table 1. The closest relatives of isolated endophytic bacterial strains from okra plants.
StrainsCloset Relative aAcc. NoId. (%)Class
Okhm3Rhizobium paranaense PRF 35NR_134152.199α-proteobacteria
Okhm4Agrobacterium tumefaciens UQM 1685NR_116306.199α-proteobacteria
Okhm5-1Bacillus mobilis MCCC 1A05942NR_157731.1100Bacilli
Okhm5-2Agrobacterium tumefaciens UQM 1685NR_116306.199α-proteobacteria
Okhm5-3Ensifer adhaerens LMG 20216NR_042482.199α-proteobacteria
Okhm5-4Ensifer adhaerens NBRC 100388NR_113893.199α-proteobacteria
Okhm7Agrobacterium tumefaciens UQM 1685NR_116306.199α-proteobacteria
Okhm8Agrobacterium tumefaciens UQM 1685NR_116306.199α-proteobacteria
Okhm9Variovorax paradoxus NBRC 15149NR_113736.199β-proteobacteria
OKhm10Pseudomonas gessardii CIP 105469NR_024928.199γ-proteobacteria
Okhm11Rhizobium herbae CCBAU 83011NR_117530.199α-proteobacteria
Okhm12Peribacillus frigoritolerans DSM 8801NR_117474.199Bacilli
a Based on the 16S rRNA gene sequence in the database.
Table
Table 2. Plant growth-promoting activities of bacterial endophytes isolated from Okra plants.
Table 2. Plant growth-promoting activities of bacterial endophytes isolated from Okra plants.
IsolatesCloset RelativePlant Growth-Promoting Properties
IAA aPhosphate SolubilizationnifHCellulasePectinaseACC Deaminase b
Okhm3Rhizobium paranaense-+--++
Okhm5-4Ensifer adhaerens-----+
Okhm10Pseudomonas gessardii++---+
Okhm11Rhizobium herbae-+---+
a Indole-3-acetic acid; b 1-aminocyclopropane-1-carboxylate deaminase; (+) denotes positive results and (-) denotes negative results.
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Salehin, A.; Yamane, S.; Ueno, M.; Hayashi, S. Identification, Characterization, and Growth-Promoting Effects of Bacterial Endophytes Isolated from Okra (Abelmoschus esculentus L.). Agronomy 2023, 13, 1226. https://doi.org/10.3390/agronomy13051226

AMA Style

Salehin A, Yamane S, Ueno M, Hayashi S. Identification, Characterization, and Growth-Promoting Effects of Bacterial Endophytes Isolated from Okra (Abelmoschus esculentus L.). Agronomy. 2023; 13(5):1226. https://doi.org/10.3390/agronomy13051226

Chicago/Turabian Style

Salehin, Ahsanul, Sakiko Yamane, Makoto Ueno, and Shohei Hayashi. 2023. "Identification, Characterization, and Growth-Promoting Effects of Bacterial Endophytes Isolated from Okra (Abelmoschus esculentus L.)" Agronomy 13, no. 5: 1226. https://doi.org/10.3390/agronomy13051226

APA Style

Salehin, A., Yamane, S., Ueno, M., & Hayashi, S. (2023). Identification, Characterization, and Growth-Promoting Effects of Bacterial Endophytes Isolated from Okra (Abelmoschus esculentus L.). Agronomy, 13(5), 1226. https://doi.org/10.3390/agronomy13051226

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