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Research article
Screening Plant Growth-Promoting Bacteria with Antimicrobial Properties for Upland Rice
1Department of Agricultural Science, Faculty of Agriculture, Natural Resources and Environment, Naresuan University, Phitsanulok 65000, Thailand
2Centre for Crop and Food Innovation, Murdoch University, 90 South St., Murdoch WA, 6150 Australia
3Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai, 50200, Thailand
4Center of Excellence in Research for Agricultural Biotechnology, Naresuan University, Phitsanulok 65000, Thailand
5Center of Excellence in Biomaterials, Naresuan University, Phitsanulok 65000, Thailand
6Department of Chemistry, Faculty of Science and Center of Excellence for Innovation in Chemistry, Naresuan University, Phitsanulok 65000, Thailand
7Department of Microbiology and Parasitology, Faculty of Medical Science, Naresuan University, Phitsanulok 65000, Thailand
8Centre of Excellence in Fungal Research, Naresuan University, Phitsanulok 65000, Thailand
J. Microbiol. Biotechnol. 2024; 34(5): 1029-1039
Published May 28, 2024 https://doi.org/10.4014/jmb.2402.02008
Copyright © The Korean Society for Microbiology and Biotechnology.
Abstract
Keywords
Graphical Abstract

Introduction
Upland rice (
The rhizosphere, comprising the endorhizosphere, rhizoplane, and ectorhizosphere, plays a critical role in shaping soil microbial communities [4]. The rhizosphere serves as a hotspot for microbial activity depending on the availability of root exudates [5]. The rhizosphere can harbor a diversity of bacteria, fungi and nematodes depending on factors such as the soil type, rice variety, rhizodeposition and environmental conditions [6, 7]. For rice rhizospheres, bacterial populations can be isolated using traditional serial dilution techniques [8]. Advances in high-throughput sequencing technology have further facilitated investigations into the structure and diversity of these bacterial communities [9]. In a comprehensive review by Vessey [10], multiple bacterial genera including
The PGPR exhibit both direct and indirect positive impacts on plants and soil through the synthesis of growth-promoting compounds, including indole-3-acetic acid and siderophores [12]. Additionally, it has been observed that bacteria secrete 1-aminocyclopropane-1-carboxylate (ACC) deaminase [13] and other organic substances in order to facilitate the dissolution of phosphate in soil [14]. The release of antagonistic agents and antibacterial chemicals can indirectly benefit crop yields by reducing damage from plant pathogens [15]. In particular, bacterial lipopeptides are pivotal in the control of some plant pathogens. For example, the surfactin, fengycin and iturin lipopeptides from
Most studies on rice-associated bacteria have been undertaken with paddy or lowland rice and there is only limited investigation into bacteria associated with rain-fed upland rice [22, 23]. As environmental conditions can differ greatly between upland regions engaged in rain-fed rice production, there is a need to target beneficial organisms to local conditions. Therefore, in this study we isolated bacteria from root tissues and rhizosphere soils from upland rice, and screened strains for their ability to solubilize insoluble calcium phosphate and produce IAA, and their antimicrobial activity against rice pathogens in vitro. In addition, crude lipopeptides produced by
Materials and Methods
Isolation of Rice Root-Associated Bacteria
Ten samples of Khao Rai Leum Pua Phetchabun rice plants at the panicle initiation stage of development were harvested from local farms in Khao Kho district, Phetchabun Province, Thailand. Root samples were soaked in phosphate-buffered saline (PBS) and run through a sonicator (CREST Ultrasonics, USA) for 30 sec to obtain rhizosphere bacteria via serial ten-fold dilutions. For obtaining endophytic bacteria, 2-3 cm pieces of roots were surface sterilized as described previously [24, 25]. The sterilized roots (ca. 1 g) were mixed with 1 ml of 0.03 M MgSO4 and ground in a mortar. Serial ten-fold dilution of ground roots was prepared in PBS to 10-3-10-7. Aliquots (100 μl) of root suspension were spread onto Luria-Bertani agar (LBA), King's B agar (KB) tryptic soya agar (TSA) and peat moss extract agar. Plates were incubated in the dark for 5 days at 28-30°C and then colonies were observed under a stereomicroscope (Optika, Italy). Bacterial strains were characterized based on colony morphology, Gram staining, and cell morphology using a scanning electron microscope (SEM) (Leo1455VP). All bacterial cultures were kept at –80°C in a 25% (v/v) glycerol solution and used for further research. In vitro assays for measuring phosphate solubilization, indole-3-acetic acid production (IAA), and antimicrobial production were determined using all 315 isolates (127 endophytes and 188 from the rhizosphere).
Screening for Phosphate Solubilization Activity
Bacterial strains were tested for phosphate solubilizing activity on Pikovskaya's medium (PVK) containing 0.5% (w/v) tricalcium phosphate (Ca3(PO4)2). Bacteria were aseptically spot inoculated on agar plates and incubated at 28°C ± 2°C for 3-7 days. Later, the diameter of the clear zone was measured (in mm) using a Vernier caliper, and the measurements used for a phosphate solubilization index (PSI) [26]. The released phosphorus was quantified using the molybdate blue/colorimetric method [27]. The ninety-one isolates that were able to solubilize insoluble calcium phosphate on PVK were investigated further. Bacterial inoculum (1 × 108 CFU/ml) was inoculated into 250 ml Erlenmeyer flasks containing 50 ml Pikovskaya's broth supplemented with 0.5% (w/v) tricalcium phosphate and incubated in a 30°C rotary shaker at 150 rpm, for 7 days. After incubation, the supernatant was collected by centrifugation (Dynamica, Velocity14 R) at 10,000 ×
Screening for Indole-3-Acetic Acid Production
The ability of all 315 isolates to produce IAA was assessed as described by Gordon & Weber [28]. Bacterial cells were cultivated in Yeast malt broth (YMB) containing 1 mg/ml L-tryptophan and shaken at 150 rpm for 4 days at room temperature (28-30°C). The supernatant was collected by centrifugation (Dynamica, Velocity14 R) at 10,000 ×
Screening for Antagonistic Bacteria Using Dual Culture Bioassay
The dual culture method was used to evaluate the antifungal activity of 43 bacterial isolates (24 from the rhizosphere and 19 from within the roots). We selected 13 isolates (KK005, KK039, KK047, KK053, KK065, KK066, KK074, KK075, KK077, KK081, KK102, KK245 and KK291) with strong ability to produce IAA, 16 isolates (KK002, KK007, KK018, KK079, KK108, KK135, KK146, KK151, KK180, KK191, KK199, KK202, KK225, KK306, KK313 and KK314) with strong ability to solubilize phosphate, and a further 14 isolates (KK024, KK067, KK073, KK145, KK149, KK157, KK184, KK231, KK232, KK243, KK269, KK275, KK281 and KK287) with different colony morphologies from the original 315 isolates. The 43 isolates were used in dual culture bioassays against two pathogenic fungi of rice:
PIRG (%) = [(R1 – R2)/R1] × 100
where R1 is the radial diameter of the control colony, and R2 is the radial diameter of the treatment colony.
Assessment of Antifungal Activity Using Cell-Free Supernatant
Nine bacterial isolates, three (KK024, KK275 and KK281) with the highest fungal activity in the dual culture assay, and six (KK023, KK024, KK058, KK275, KK281 and KK312) with different colony morphology, were used for the cell-free supernatant assay. Inocula (ca. 106 CFU/ml) were cultured in 5 ml of modified Wickerhams Antibiotic Test Medium (WATM) [32] and incubated at 28°C on a shaking incubator for 5 days. The cell-free supernatants were collected by centrifugation (Dynamica, Velocity14 R) at 15,000 ×
Molecular Identification of Bacterial Strains
Three rhizosphere isolates (KK138, KK269 and KK281) and three endophytes (KK007, KK018 and KK066) were chosen for identification based on their overall ability to solubilize insoluble calcium phosphate, produce IAA, and their antimicrobial activity. The 16S rRNA gene was amplified with the 27F primer (5'-AGA GTT TGA TCM TGG CTC AG-3') and the 1492R primer (5'-TAC GGY TAC CTT GTT ACG ACT T-3') [34]. An amplification reaction was performed as previously described [35], and the purified PCR products were commercially sequenced at Macrogen, Korea. The obtained 16S rRNA gene sequences were compared to those of their phylogenetic relatives in the EzBioCloud database (http://www.ezbiocloud.net). The MEGA software package (Version 10) was used to construct and analyze phylogenetic trees using a neighbor-joining method [36]. The resultant tree topology was evaluated using bootstrap analysis with 1,000 resampled data sets. The sequences were submitted to the GenBank database to obtain accession numbers.
Extraction of Lipopeptides
Isolate KK281 was chosen to investigate antimicrobial lipopeptides due to its high antimicrobial activity in the dual culture study and its antifungal activity in the cell-free supernatant test. Crude extract containing lipopeptides was prepared using a slightly modified method from that described by Pathak
LC–ESI–MS/MS Conditions
The crude lipopeptide extract was dissolved in 70% ethanol (10 mg/ml) and standard surfactin was prepared in ethanol (5 mg/ml), sonicated and filtered using a syringe filter (0.45 μm) and the analysis parameters as described by Ma
Antifungal Activity of Lipopeptides
The antifungal activity of the extracted lipopeptides was evaluated by the disc diffusion assay against
Antibacterial Activity of Lipopeptides
The antibacterial activity of the lipopeptides was evaluated by the disc diffusion assay by preparing a 25,000 ppm stock and dissolving lyophilized fractions in 70% ethanol at three concentrations: 62.5, 125, 250, and 500 ppm. A paper disc (6 mm diameter) containing lipopeptides was placed on Nutrient agar (NA) inoculated with
Cytotoxicity Testing
Cytotoxicity testing was undertaken on the L-929 cell line [40] to establish whether the bacteria and extracts would be safe to use in future in vivo screening trials. The standard colorimetric MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay with the L-929 cell line (mouse fibroblast) was performed. The crude extracts of KK281 were prepared in a 5-80 μg/ml concentration range and were dissolved in 100 μl DMSO. A microplate spectrophotometer (Shimadzu, UV-1800) was used to measure absorbance at 570 and 650 nm. Doxorubicin was used as the positive control. Cytotoxicity was expressed as the concentration of the compound inhibiting growth by 50% (IC50).
Statistical Analysis
All experiments were carried out in triplicate except the LC-ESI- MS/MS analysis. The data were subject to analysis of variance (ANOVA) followed by Duncan’s multiple range test (
Results
Isolation of Rhizosphere and Endophytic Bacteria
A total of 315 culturable bacterial isolates were obtained, 127 from root tissues and 188 from the rhizosphere of upland rice. Ninety isolates were recovered from Luria-Bertani agar, of which 31 were endophytic bacteria and 59 were rhizospheric bacteria. Fifty of the 82 isolates from King's B agar were rhizosphere bacteria, and the remainder were endophytes. Furthermore, 81 isolates were obtained using Tryptic soya agar (38 rhizospheric, 43 endophytic) and 62 isolates (41 rhizospheric, 21 endophytic) were obtained from the peat moss extract agar. All isolates were investigated in vitro for phosphate solubilizing activity and IAA production. Selected isolates were used subsequently for antifungal, lipopeptide and other studies (Fig. 1).
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Fig. 1. Schematic diagram illustrating the steps required for isolating and screening for plant growthpromoting bacteria and assessing their potential antibacterial properties.
Indole-3-Acetic Acid Production
Out of 315 isolates, five isolates were able to produce IAA in the range of 328-362 μg/ml (Table 1), with isolate KK074 the highest producer (362.6 ± 28.0 μg/ml).
-
Table 1 . In vitro plant growth promoting properties and antifungal activity.
Bacterial ID Code IAA production (μg/ml) * Phosphate solubilization index (PSI) * pH of medium Phosphate solubilization (μg/ml) * Inhibition of pathogenic fungi (%)* 3 days 7 days 1 day 7 days C. lunata NUF001B. oryzae 2464KK007 - 3.44 ± 0.67bc 4.74 ± 0.54b 7.00 4.06 714.25 ± 12.14a 36.59 ± 1.84cd 45.06 ± 2.97bc KK024 11.59 ± 1.67d - - 7.00 4.94 139.12 ± 32.29f 58.24 ± 0.82a 45.29 ± 2.57bc KK026 16.82 ± 0.60d 3.57 ± 1.04abc 4.27 ± 0.64bc 7.00 5.48 98.17 ± 14.66g - - KK066 328.43 ± 24.10b - 2.45 ± 0.15e 7.00 5.47 151.52 ± 2.02f 30.03 ± 2.41de - KK067 - 3.48 ± 0.20bc 4.82 ± 0.34b 7.00 5.81 137.55 ± 1.34f 55.02 ± 1.75ab 52.83 ± 1.84ab KK070 - 4.09 ± 0.45ab 3.49 ± 0.16d 7.00 5.32 107.78 ± 3.46g - - KK074 362.59 ± 28.02a - - - - - - 20.49 ± 8.85d KK075 330.09 ± 15.28b - - 7.00 4.98 196.75 ± 4.65e 0.80 ± 0.15f - KK077 352.32 ± 13.72a - - - - - 27.75 ± 0.60e - KK081 348.71 ± 7.74ab - - - - - - - KK135 - - 3.85 ± 0.12cd 7.00 3.94 683.45 ± 13.51b - - KK138 - - 2.62 ± 0.03e 7.00 3.96 694.63 ± 9.12b - - KK184 14.04 ± 2.76d 3.87 ± 0.14abc 4.71 ± 0.12b 7.00 5.56 100.49 ± 2.63g 53.23 ± 1.64ab 51.47 ± 1.56ab KK225 2.82 ± 0.88d 4.51 ± 0.49a 7.85 ± 0.54a 7.00 4.40 389.55 ± 6.39c 38.60 ± 11.97c 37.59 ± 8.13c KK269 148.54 ± 1.87c 2.98 ± 0.08c 3.52 ± 0.14d 7.00 5.53 153.02 ± 3.07f 50.71 ± 2.68b 56.01 ± 3.14a KK275 6.82 ± 0.44d - - 7.00 4.91 92.11 ± 2.09g 54.14 ± 1.34ab 47.98 ± 0.47ab KK281 23.59 ± 1.02d - - 7.00 4.90 275.78 ± 2.87d 57.91 ± 0.42ab 46.62 ± 5.36b a-gThe mean values with different superscript letters in a column are statistically different from Duncan’s multiple range test (
p < 0.05).
Phosphate Solubilization
In the primary screening, forty-six isolates were able to solubilize insoluble calcium phosphate on Pikovskaya’s plates (data not shown). Out of forty-six isolates, ten exhibited phosphate solubilization index ranged from 2.45 to 7.85, with KK225 having the highest PSI of 4.51 (3 days) and 7.85 (7 days) (Table 1). Five isolates were able to release phosphorus in the range of 275-714 μg/ml in the secondary screening with the molybdenum blue method. Isolate KK007 released the highest amount of phosphorus (714.25 ± 12.14 μg/ml).
Screening of Antagonistic Bacteria by Dual Culture Bioassay
Of the 43 isolates investigated for their antifungal activities, six isolates inhibited
Antifungal Activity of Cell-Free Supernatants
Cell-free supernatants of the nine bacterial isolates that were tested showed antifungal activity against
Traits of Bacterial Isolates Selected for Further Study
Based on indole-3-acetic acid production, phosphate solubilization and antifungal assays, isolates KK007, KK074 and KK281 were selected for further assays. Colony morphology (Fig. 2C) was characterized on TSA (Fig. 2A) and HiCrome
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Fig. 2. Isolation and characterization of bacterial isolates KK007, KK074 and KK281: (A) morphological characterization on TSA; (B) on HiCrome
Bacillus Agar; (C) colony morphology; (D) Gram staining; (E) appearance under SEM.
Identification of Bacterial Isolates Based on 16S rRNA Gene and Phylogenetic Tree
Based on screening program of plant growth promoting properties, antimicrobial activity and colony morphology observation, we identified at least six genera using molecular analysis (Table 2). Among those isolates, KK281 was selected for further assays due to its superior bioactivities. BLAST analysis revealed that the 16S rRNA gene of isolate KK281 was 99.86% identical to that of
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Table 2 . Identification of upland rice endophytic and rhizosphere bacteria based on 16S rRNA gene sequence.
TBRC ID Code Isolate KK Sequence Length (bps) NCBI Accession Closely related taxa Similarity (%) Taxonomic assignment 15994* KK007 1,466 ON406146 Pantoea stewartii subsp.Indologenes LMG 2632T99.45 Pantoea stewartii 15995* KK018 1,479 ON406147 Priestia megaterium NBRC 15308T99.93 Priestia megaterium 15999* KK066 1,400 ON406148 Enterobacter roggenkampii EN-117T99.79 Enterobacter sp.15996¥ KK138 1,464 ON406149 Acinetobacter soli CIP 110264T100.00 Acinetobacter soli 15997¥ KK269 1,468 ON406150 Pantoea allii LMG 24248T99.04 Pantoea sp.15998¥ KK281 1,473 ON406151 Bacillus siamensis KCTC 13613T99.86 Bacillus sp.Note: * = endophytic bacteria, \ = rhizosphere bacteria
Antimicrobial Activity of Lipopeptides
Antifungal Activity. Antifungal activity of the lipopeptides are presented in Fig. 3. The crude lipopeptides produced by
-
Table 3 . Inhibition zone diameter of lipopeptides extract from isolate KK281 against
Curvularia lunata NUF001 andXanthomonas oryzae pv.oryzae by the paper disc diffusion method.Sample Concentration (mg/ml) Inhibition zone diameter (mm) C. lunata NUF001 96 hXoo 72 hCrude lipopeptides 12.5 39.93 ± 2.14a 19.33 ± 1.29c 6.25 17.20 ± 0.87c 16.43 ± 0.50d 3.125 18.10 ± 2.56c 12.47 ± 1.23e 70% ethanol 0.00 ± 0.00d NA Prochloraz 500 ppm 29.65 ± 0.77b NA Streptomycin* NA 42.50 ± 0.30b Chloramphenicol* NA 53.20 ± 0.60a a-eThe mean values with different superscript letters in a column are statistically different from Duncan’s multiple range test (
p < 0.05).*Streptomycin 10 μg/disc, chloramphenicol 10 μg/disc
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Fig. 3. Inhibition zone diameter (mm) of lipopeptide extracts from isolate KK281 against fungal pathogens
Curvularia lunata NUF001. (A-E) on PDA at 28°C ± 2 for 96 h) andXanthomonas oryzae pv.oryzae (F-J) on NA plate at 28°C ± 2 for 72 h. (A,F) 12.5 mg/ml; (B,G) 6.25 mg/ml; (C,H) 3.125 mg/ml; (D) 70% ethanol; (E) Prochloraz 500 ppm; (I) streptomycin 10 μg/disc and (J) chloramphenicol 10 μg/disc.
Antibacterial Activity. Crude lipopeptides at a concentration of 12.5 mg/ml resulted in a large zone of inhibition (19.33±1.29 mm) against
Liquid Chromatography–Electrospray Ionization–Tandem Mass Spectrometry (LC–ESI–MS/MS) Analysis of Lipopeptides
The chemical profile of crude lipopeptide extract was investigated using LC-ESI-MS/MS instrument. The full scan total ion chromatogram of sample demonstrated that the main peaks began at 18 min. In the range of 18.179–18.557 min, the major peaks of surfactin were eluted at a retention time (
-
Fig. 4. LC–ESI–MS/MS spectra of surfactin. (A) Crude lipopeptides of KK281 precursors ion [M + H]+at
m/z 1036.6947 at retention time 18.296 min. (B) Standard surfactin precursors ion [M + H]+atm/z 1036.6879 at retention time 18.374 min.
Cytotoxicity Testing
Our results showed no cytotoxic effects of the crude lipopeptide derived from
Discussion
In this study, we employed a combination of morphological observations and molecular techniques for identifying plant growth-promoting bacteria (PGPB). The 16S rRNA gene sequencing was used to identify and compare the taxonomical differences with those recorded in databases (GenBank and EzBioCloud Database). This approach allowed us to accurately categorize the isolates at the genus level, encompassing members of the genera
Phosphate solubilization is one of the most important plant growth-promoting traits due to the limited supply of available phosphate resulting from the strong adsorption of phosphate to reactive surfaces in most agricultural soils. The principal mechanism of phosphate solubilization by bacteria is the production of organic acids and enzymes which can convert tricalcium phosphate in the assay medium from insoluble to soluble forms [42, 43]. For example,
Indole acetic acid is an important phytohormone that regulates plant growth and development [48]. In our study, isolate KK074 was one of the best IAA producers, along with KK077 and KK081. Among the top three IAA-producing isolates, KK074 was uniquely effective in inhibiting the growth of
In this study, the two most potent bacterial antagonistic isolates were KK024 (
The cell-free supernatant of KK281 (
The crude extract of KK281, containing surfactin, strongly inhibited growth of
In our study, LC-ESI-MS/MS analysis revealed the presence of surfactin with
In our study, the crude lipopeptide extract was non-cytotoxic towards the L-929 cell line (mouse fibroblast). The cytotoxicity of crude lipopeptides towards cell lines can vary significantly depending on several factors, including the type of lipopeptide, the target cell line, and the concentration of the lipopeptide [62]. In the future, we plan to use purified lipopeptides to better understand the specific effects of lipopeptides on various cell lines.
Conclusion
This comprehensive study evaluated 315 bacterial isolates in in vitro assay to assess their potential to promote the growth of plants and control diseases. Overall, one isolate (
Supplemental Materials
Acknowledgments
This work was financially supported by Research and Researcher for industry (RRi), Thailand Science Research and Innovation (TSRI) and Banrai-ioon strawberry farm (Grant No. MSD62I0098). Special thanks are extended to Dr. Sakamon Devahastin for his constructive comments and to Dr. Kumrop Ratanasut for providing the phytopathogenic bacterium.
Author Contributions
Conceptualization, B. Dell, J. Jumpathong; Methodology, J. Jumpathong, N. Suphrom, K. Khamsuk; Validation, B. Dell, J. Jumpathong; Formal Analysis, K. Khamsuk, N. Suphrom; Investigation, K. Khamsuk, J. Jumpathong; Resources, K. Khamsuk, J. Jumpathong; Data Curation, K. Khamsuk, J. Jumpathong; Writing – Original Draft Preparation, K. Khamsuk, J. Jumpathong; Writing – Review & Editing, K. Khamsuk, B. Dell, W. Pathom-aree, W. Pathaichindachote, N. Suphrom, N. Nakaew and J. Jumpathong; Visualization, K. Khamsuk, J. Jumpathong; Supervision, J. Jumpathong; Project Administration, J. Jumpathong; Funding Acquisition, J. Jumpathong
Conflict of Interest
The authors have no financial conflicts of interest to declare.
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Related articles in JMB

Article
Research article
J. Microbiol. Biotechnol. 2024; 34(5): 1029-1039
Published online May 28, 2024 https://doi.org/10.4014/jmb.2402.02008
Copyright © The Korean Society for Microbiology and Biotechnology.
Screening Plant Growth-Promoting Bacteria with Antimicrobial Properties for Upland Rice
Khammool Khamsuk1, Bernard Dell2, Wasu Pathom-aree3, Wanwarang Pathaichindachote1,4, Nungruthai Suphrom5,6, Nareeluk Nakaew7,8, and Juangjun Jumpathong1,8*
1Department of Agricultural Science, Faculty of Agriculture, Natural Resources and Environment, Naresuan University, Phitsanulok 65000, Thailand
2Centre for Crop and Food Innovation, Murdoch University, 90 South St., Murdoch WA, 6150 Australia
3Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai, 50200, Thailand
4Center of Excellence in Research for Agricultural Biotechnology, Naresuan University, Phitsanulok 65000, Thailand
5Center of Excellence in Biomaterials, Naresuan University, Phitsanulok 65000, Thailand
6Department of Chemistry, Faculty of Science and Center of Excellence for Innovation in Chemistry, Naresuan University, Phitsanulok 65000, Thailand
7Department of Microbiology and Parasitology, Faculty of Medical Science, Naresuan University, Phitsanulok 65000, Thailand
8Centre of Excellence in Fungal Research, Naresuan University, Phitsanulok 65000, Thailand
Correspondence to:Juangjun Jumpathong, juangjunj@nu.ac.th
Abstract
This study explores beneficial bacteria isolated from the roots and rhizosphere soil of Khao Rai Leum Pua Phetchabun rice plants. A total of 315 bacterial isolates (KK001 to KK315) were obtained. Plant growth-promoting traits (phosphate solubilization and indole-3-acetic acid (IAA) production), and antimicrobial activity against three rice pathogens (Curvularia lunata NUF001, Bipolaris oryzae 2464, and Xanthomonas oryzae pv. oryzae) were assessed. KK074 was the most prolific in IAA production, generating 362.6 ± 28.0 μg/ml, and KK007 excelled in tricalcium phosphate solubilization, achieving 714.2 ± 12.1 μg/ml. In antimicrobial assays using the dual culture method, KK024 and KK281 exhibited strong inhibitory activity against C. lunata, and KK269 was particularly effective against B. oryzae. In the evaluation of antimicrobial metabolite production, KK281 and KK288 exhibited strong antifungal activities in cell-free supernatants. Given the superior performance of KK281, taxonomically identified as Bacillus sp. KK281, it was investigated further. Lipopeptide extracts from KK281 had significant antimicrobial activity against C. lunata and a minimum inhibitory concentration (MIC) of 3.1 mg/ml against X. oryzae pv. oryzae. LC-ESI-MS/MS analysis revealed the presence of surfactin in the lipopeptide extract. The crude extract was non-cytotoxic to the L-929 cell line at tested concentrations. In conclusion, the in vitro plant growth-promoting and disease-controlling attributes of Bacillus sp. KK281 make it a strong candidate for field evaluation to boost plant growth and manage disease in upland rice.
Keywords: Lipopeptides, surfactin, TBRC 15998, root-associated bacteria
Introduction
Upland rice (
The rhizosphere, comprising the endorhizosphere, rhizoplane, and ectorhizosphere, plays a critical role in shaping soil microbial communities [4]. The rhizosphere serves as a hotspot for microbial activity depending on the availability of root exudates [5]. The rhizosphere can harbor a diversity of bacteria, fungi and nematodes depending on factors such as the soil type, rice variety, rhizodeposition and environmental conditions [6, 7]. For rice rhizospheres, bacterial populations can be isolated using traditional serial dilution techniques [8]. Advances in high-throughput sequencing technology have further facilitated investigations into the structure and diversity of these bacterial communities [9]. In a comprehensive review by Vessey [10], multiple bacterial genera including
The PGPR exhibit both direct and indirect positive impacts on plants and soil through the synthesis of growth-promoting compounds, including indole-3-acetic acid and siderophores [12]. Additionally, it has been observed that bacteria secrete 1-aminocyclopropane-1-carboxylate (ACC) deaminase [13] and other organic substances in order to facilitate the dissolution of phosphate in soil [14]. The release of antagonistic agents and antibacterial chemicals can indirectly benefit crop yields by reducing damage from plant pathogens [15]. In particular, bacterial lipopeptides are pivotal in the control of some plant pathogens. For example, the surfactin, fengycin and iturin lipopeptides from
Most studies on rice-associated bacteria have been undertaken with paddy or lowland rice and there is only limited investigation into bacteria associated with rain-fed upland rice [22, 23]. As environmental conditions can differ greatly between upland regions engaged in rain-fed rice production, there is a need to target beneficial organisms to local conditions. Therefore, in this study we isolated bacteria from root tissues and rhizosphere soils from upland rice, and screened strains for their ability to solubilize insoluble calcium phosphate and produce IAA, and their antimicrobial activity against rice pathogens in vitro. In addition, crude lipopeptides produced by
Materials and Methods
Isolation of Rice Root-Associated Bacteria
Ten samples of Khao Rai Leum Pua Phetchabun rice plants at the panicle initiation stage of development were harvested from local farms in Khao Kho district, Phetchabun Province, Thailand. Root samples were soaked in phosphate-buffered saline (PBS) and run through a sonicator (CREST Ultrasonics, USA) for 30 sec to obtain rhizosphere bacteria via serial ten-fold dilutions. For obtaining endophytic bacteria, 2-3 cm pieces of roots were surface sterilized as described previously [24, 25]. The sterilized roots (ca. 1 g) were mixed with 1 ml of 0.03 M MgSO4 and ground in a mortar. Serial ten-fold dilution of ground roots was prepared in PBS to 10-3-10-7. Aliquots (100 μl) of root suspension were spread onto Luria-Bertani agar (LBA), King's B agar (KB) tryptic soya agar (TSA) and peat moss extract agar. Plates were incubated in the dark for 5 days at 28-30°C and then colonies were observed under a stereomicroscope (Optika, Italy). Bacterial strains were characterized based on colony morphology, Gram staining, and cell morphology using a scanning electron microscope (SEM) (Leo1455VP). All bacterial cultures were kept at –80°C in a 25% (v/v) glycerol solution and used for further research. In vitro assays for measuring phosphate solubilization, indole-3-acetic acid production (IAA), and antimicrobial production were determined using all 315 isolates (127 endophytes and 188 from the rhizosphere).
Screening for Phosphate Solubilization Activity
Bacterial strains were tested for phosphate solubilizing activity on Pikovskaya's medium (PVK) containing 0.5% (w/v) tricalcium phosphate (Ca3(PO4)2). Bacteria were aseptically spot inoculated on agar plates and incubated at 28°C ± 2°C for 3-7 days. Later, the diameter of the clear zone was measured (in mm) using a Vernier caliper, and the measurements used for a phosphate solubilization index (PSI) [26]. The released phosphorus was quantified using the molybdate blue/colorimetric method [27]. The ninety-one isolates that were able to solubilize insoluble calcium phosphate on PVK were investigated further. Bacterial inoculum (1 × 108 CFU/ml) was inoculated into 250 ml Erlenmeyer flasks containing 50 ml Pikovskaya's broth supplemented with 0.5% (w/v) tricalcium phosphate and incubated in a 30°C rotary shaker at 150 rpm, for 7 days. After incubation, the supernatant was collected by centrifugation (Dynamica, Velocity14 R) at 10,000 ×
Screening for Indole-3-Acetic Acid Production
The ability of all 315 isolates to produce IAA was assessed as described by Gordon & Weber [28]. Bacterial cells were cultivated in Yeast malt broth (YMB) containing 1 mg/ml L-tryptophan and shaken at 150 rpm for 4 days at room temperature (28-30°C). The supernatant was collected by centrifugation (Dynamica, Velocity14 R) at 10,000 ×
Screening for Antagonistic Bacteria Using Dual Culture Bioassay
The dual culture method was used to evaluate the antifungal activity of 43 bacterial isolates (24 from the rhizosphere and 19 from within the roots). We selected 13 isolates (KK005, KK039, KK047, KK053, KK065, KK066, KK074, KK075, KK077, KK081, KK102, KK245 and KK291) with strong ability to produce IAA, 16 isolates (KK002, KK007, KK018, KK079, KK108, KK135, KK146, KK151, KK180, KK191, KK199, KK202, KK225, KK306, KK313 and KK314) with strong ability to solubilize phosphate, and a further 14 isolates (KK024, KK067, KK073, KK145, KK149, KK157, KK184, KK231, KK232, KK243, KK269, KK275, KK281 and KK287) with different colony morphologies from the original 315 isolates. The 43 isolates were used in dual culture bioassays against two pathogenic fungi of rice:
PIRG (%) = [(R1 – R2)/R1] × 100
where R1 is the radial diameter of the control colony, and R2 is the radial diameter of the treatment colony.
Assessment of Antifungal Activity Using Cell-Free Supernatant
Nine bacterial isolates, three (KK024, KK275 and KK281) with the highest fungal activity in the dual culture assay, and six (KK023, KK024, KK058, KK275, KK281 and KK312) with different colony morphology, were used for the cell-free supernatant assay. Inocula (ca. 106 CFU/ml) were cultured in 5 ml of modified Wickerhams Antibiotic Test Medium (WATM) [32] and incubated at 28°C on a shaking incubator for 5 days. The cell-free supernatants were collected by centrifugation (Dynamica, Velocity14 R) at 15,000 ×
Molecular Identification of Bacterial Strains
Three rhizosphere isolates (KK138, KK269 and KK281) and three endophytes (KK007, KK018 and KK066) were chosen for identification based on their overall ability to solubilize insoluble calcium phosphate, produce IAA, and their antimicrobial activity. The 16S rRNA gene was amplified with the 27F primer (5'-AGA GTT TGA TCM TGG CTC AG-3') and the 1492R primer (5'-TAC GGY TAC CTT GTT ACG ACT T-3') [34]. An amplification reaction was performed as previously described [35], and the purified PCR products were commercially sequenced at Macrogen, Korea. The obtained 16S rRNA gene sequences were compared to those of their phylogenetic relatives in the EzBioCloud database (http://www.ezbiocloud.net). The MEGA software package (Version 10) was used to construct and analyze phylogenetic trees using a neighbor-joining method [36]. The resultant tree topology was evaluated using bootstrap analysis with 1,000 resampled data sets. The sequences were submitted to the GenBank database to obtain accession numbers.
Extraction of Lipopeptides
Isolate KK281 was chosen to investigate antimicrobial lipopeptides due to its high antimicrobial activity in the dual culture study and its antifungal activity in the cell-free supernatant test. Crude extract containing lipopeptides was prepared using a slightly modified method from that described by Pathak
LC–ESI–MS/MS Conditions
The crude lipopeptide extract was dissolved in 70% ethanol (10 mg/ml) and standard surfactin was prepared in ethanol (5 mg/ml), sonicated and filtered using a syringe filter (0.45 μm) and the analysis parameters as described by Ma
Antifungal Activity of Lipopeptides
The antifungal activity of the extracted lipopeptides was evaluated by the disc diffusion assay against
Antibacterial Activity of Lipopeptides
The antibacterial activity of the lipopeptides was evaluated by the disc diffusion assay by preparing a 25,000 ppm stock and dissolving lyophilized fractions in 70% ethanol at three concentrations: 62.5, 125, 250, and 500 ppm. A paper disc (6 mm diameter) containing lipopeptides was placed on Nutrient agar (NA) inoculated with
Cytotoxicity Testing
Cytotoxicity testing was undertaken on the L-929 cell line [40] to establish whether the bacteria and extracts would be safe to use in future in vivo screening trials. The standard colorimetric MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay with the L-929 cell line (mouse fibroblast) was performed. The crude extracts of KK281 were prepared in a 5-80 μg/ml concentration range and were dissolved in 100 μl DMSO. A microplate spectrophotometer (Shimadzu, UV-1800) was used to measure absorbance at 570 and 650 nm. Doxorubicin was used as the positive control. Cytotoxicity was expressed as the concentration of the compound inhibiting growth by 50% (IC50).
Statistical Analysis
All experiments were carried out in triplicate except the LC-ESI- MS/MS analysis. The data were subject to analysis of variance (ANOVA) followed by Duncan’s multiple range test (
Results
Isolation of Rhizosphere and Endophytic Bacteria
A total of 315 culturable bacterial isolates were obtained, 127 from root tissues and 188 from the rhizosphere of upland rice. Ninety isolates were recovered from Luria-Bertani agar, of which 31 were endophytic bacteria and 59 were rhizospheric bacteria. Fifty of the 82 isolates from King's B agar were rhizosphere bacteria, and the remainder were endophytes. Furthermore, 81 isolates were obtained using Tryptic soya agar (38 rhizospheric, 43 endophytic) and 62 isolates (41 rhizospheric, 21 endophytic) were obtained from the peat moss extract agar. All isolates were investigated in vitro for phosphate solubilizing activity and IAA production. Selected isolates were used subsequently for antifungal, lipopeptide and other studies (Fig. 1).
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Figure 1. Schematic diagram illustrating the steps required for isolating and screening for plant growthpromoting bacteria and assessing their potential antibacterial properties.
Indole-3-Acetic Acid Production
Out of 315 isolates, five isolates were able to produce IAA in the range of 328-362 μg/ml (Table 1), with isolate KK074 the highest producer (362.6 ± 28.0 μg/ml).
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Table 1 . In vitro plant growth promoting properties and antifungal activity..
Bacterial ID Code IAA production (μg/ml) * Phosphate solubilization index (PSI) * pH of medium Phosphate solubilization (μg/ml) * Inhibition of pathogenic fungi (%)* 3 days 7 days 1 day 7 days C. lunata NUF001B. oryzae 2464KK007 - 3.44 ± 0.67bc 4.74 ± 0.54b 7.00 4.06 714.25 ± 12.14a 36.59 ± 1.84cd 45.06 ± 2.97bc KK024 11.59 ± 1.67d - - 7.00 4.94 139.12 ± 32.29f 58.24 ± 0.82a 45.29 ± 2.57bc KK026 16.82 ± 0.60d 3.57 ± 1.04abc 4.27 ± 0.64bc 7.00 5.48 98.17 ± 14.66g - - KK066 328.43 ± 24.10b - 2.45 ± 0.15e 7.00 5.47 151.52 ± 2.02f 30.03 ± 2.41de - KK067 - 3.48 ± 0.20bc 4.82 ± 0.34b 7.00 5.81 137.55 ± 1.34f 55.02 ± 1.75ab 52.83 ± 1.84ab KK070 - 4.09 ± 0.45ab 3.49 ± 0.16d 7.00 5.32 107.78 ± 3.46g - - KK074 362.59 ± 28.02a - - - - - - 20.49 ± 8.85d KK075 330.09 ± 15.28b - - 7.00 4.98 196.75 ± 4.65e 0.80 ± 0.15f - KK077 352.32 ± 13.72a - - - - - 27.75 ± 0.60e - KK081 348.71 ± 7.74ab - - - - - - - KK135 - - 3.85 ± 0.12cd 7.00 3.94 683.45 ± 13.51b - - KK138 - - 2.62 ± 0.03e 7.00 3.96 694.63 ± 9.12b - - KK184 14.04 ± 2.76d 3.87 ± 0.14abc 4.71 ± 0.12b 7.00 5.56 100.49 ± 2.63g 53.23 ± 1.64ab 51.47 ± 1.56ab KK225 2.82 ± 0.88d 4.51 ± 0.49a 7.85 ± 0.54a 7.00 4.40 389.55 ± 6.39c 38.60 ± 11.97c 37.59 ± 8.13c KK269 148.54 ± 1.87c 2.98 ± 0.08c 3.52 ± 0.14d 7.00 5.53 153.02 ± 3.07f 50.71 ± 2.68b 56.01 ± 3.14a KK275 6.82 ± 0.44d - - 7.00 4.91 92.11 ± 2.09g 54.14 ± 1.34ab 47.98 ± 0.47ab KK281 23.59 ± 1.02d - - 7.00 4.90 275.78 ± 2.87d 57.91 ± 0.42ab 46.62 ± 5.36b a-gThe mean values with different superscript letters in a column are statistically different from Duncan’s multiple range test (
p < 0.05)..
Phosphate Solubilization
In the primary screening, forty-six isolates were able to solubilize insoluble calcium phosphate on Pikovskaya’s plates (data not shown). Out of forty-six isolates, ten exhibited phosphate solubilization index ranged from 2.45 to 7.85, with KK225 having the highest PSI of 4.51 (3 days) and 7.85 (7 days) (Table 1). Five isolates were able to release phosphorus in the range of 275-714 μg/ml in the secondary screening with the molybdenum blue method. Isolate KK007 released the highest amount of phosphorus (714.25 ± 12.14 μg/ml).
Screening of Antagonistic Bacteria by Dual Culture Bioassay
Of the 43 isolates investigated for their antifungal activities, six isolates inhibited
Antifungal Activity of Cell-Free Supernatants
Cell-free supernatants of the nine bacterial isolates that were tested showed antifungal activity against
Traits of Bacterial Isolates Selected for Further Study
Based on indole-3-acetic acid production, phosphate solubilization and antifungal assays, isolates KK007, KK074 and KK281 were selected for further assays. Colony morphology (Fig. 2C) was characterized on TSA (Fig. 2A) and HiCrome
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Figure 2. Isolation and characterization of bacterial isolates KK007, KK074 and KK281: (A) morphological characterization on TSA; (B) on HiCrome
Bacillus Agar; (C) colony morphology; (D) Gram staining; (E) appearance under SEM.
Identification of Bacterial Isolates Based on 16S rRNA Gene and Phylogenetic Tree
Based on screening program of plant growth promoting properties, antimicrobial activity and colony morphology observation, we identified at least six genera using molecular analysis (Table 2). Among those isolates, KK281 was selected for further assays due to its superior bioactivities. BLAST analysis revealed that the 16S rRNA gene of isolate KK281 was 99.86% identical to that of
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Table 2 . Identification of upland rice endophytic and rhizosphere bacteria based on 16S rRNA gene sequence..
TBRC ID Code Isolate KK Sequence Length (bps) NCBI Accession Closely related taxa Similarity (%) Taxonomic assignment 15994* KK007 1,466 ON406146 Pantoea stewartii subsp.Indologenes LMG 2632T99.45 Pantoea stewartii 15995* KK018 1,479 ON406147 Priestia megaterium NBRC 15308T99.93 Priestia megaterium 15999* KK066 1,400 ON406148 Enterobacter roggenkampii EN-117T99.79 Enterobacter sp.15996¥ KK138 1,464 ON406149 Acinetobacter soli CIP 110264T100.00 Acinetobacter soli 15997¥ KK269 1,468 ON406150 Pantoea allii LMG 24248T99.04 Pantoea sp.15998¥ KK281 1,473 ON406151 Bacillus siamensis KCTC 13613T99.86 Bacillus sp.Note: * = endophytic bacteria, \ = rhizosphere bacteria.
Antimicrobial Activity of Lipopeptides
Antifungal Activity. Antifungal activity of the lipopeptides are presented in Fig. 3. The crude lipopeptides produced by
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Table 3 . Inhibition zone diameter of lipopeptides extract from isolate KK281 against
Curvularia lunata NUF001 andXanthomonas oryzae pv.oryzae by the paper disc diffusion method..Sample Concentration (mg/ml) Inhibition zone diameter (mm) C. lunata NUF001 96 hXoo 72 hCrude lipopeptides 12.5 39.93 ± 2.14a 19.33 ± 1.29c 6.25 17.20 ± 0.87c 16.43 ± 0.50d 3.125 18.10 ± 2.56c 12.47 ± 1.23e 70% ethanol 0.00 ± 0.00d NA Prochloraz 500 ppm 29.65 ± 0.77b NA Streptomycin* NA 42.50 ± 0.30b Chloramphenicol* NA 53.20 ± 0.60a a-eThe mean values with different superscript letters in a column are statistically different from Duncan’s multiple range test (
p < 0.05)..*Streptomycin 10 μg/disc, chloramphenicol 10 μg/disc.
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Figure 3. Inhibition zone diameter (mm) of lipopeptide extracts from isolate KK281 against fungal pathogens
Curvularia lunata NUF001. (A-E) on PDA at 28°C ± 2 for 96 h) andXanthomonas oryzae pv.oryzae (F-J) on NA plate at 28°C ± 2 for 72 h. (A,F) 12.5 mg/ml; (B,G) 6.25 mg/ml; (C,H) 3.125 mg/ml; (D) 70% ethanol; (E) Prochloraz 500 ppm; (I) streptomycin 10 μg/disc and (J) chloramphenicol 10 μg/disc.
Antibacterial Activity. Crude lipopeptides at a concentration of 12.5 mg/ml resulted in a large zone of inhibition (19.33±1.29 mm) against
Liquid Chromatography–Electrospray Ionization–Tandem Mass Spectrometry (LC–ESI–MS/MS) Analysis of Lipopeptides
The chemical profile of crude lipopeptide extract was investigated using LC-ESI-MS/MS instrument. The full scan total ion chromatogram of sample demonstrated that the main peaks began at 18 min. In the range of 18.179–18.557 min, the major peaks of surfactin were eluted at a retention time (
-
Figure 4. LC–ESI–MS/MS spectra of surfactin. (A) Crude lipopeptides of KK281 precursors ion [M + H]+at
m/z 1036.6947 at retention time 18.296 min. (B) Standard surfactin precursors ion [M + H]+atm/z 1036.6879 at retention time 18.374 min.
Cytotoxicity Testing
Our results showed no cytotoxic effects of the crude lipopeptide derived from
Discussion
In this study, we employed a combination of morphological observations and molecular techniques for identifying plant growth-promoting bacteria (PGPB). The 16S rRNA gene sequencing was used to identify and compare the taxonomical differences with those recorded in databases (GenBank and EzBioCloud Database). This approach allowed us to accurately categorize the isolates at the genus level, encompassing members of the genera
Phosphate solubilization is one of the most important plant growth-promoting traits due to the limited supply of available phosphate resulting from the strong adsorption of phosphate to reactive surfaces in most agricultural soils. The principal mechanism of phosphate solubilization by bacteria is the production of organic acids and enzymes which can convert tricalcium phosphate in the assay medium from insoluble to soluble forms [42, 43]. For example,
Indole acetic acid is an important phytohormone that regulates plant growth and development [48]. In our study, isolate KK074 was one of the best IAA producers, along with KK077 and KK081. Among the top three IAA-producing isolates, KK074 was uniquely effective in inhibiting the growth of
In this study, the two most potent bacterial antagonistic isolates were KK024 (
The cell-free supernatant of KK281 (
The crude extract of KK281, containing surfactin, strongly inhibited growth of
In our study, LC-ESI-MS/MS analysis revealed the presence of surfactin with
In our study, the crude lipopeptide extract was non-cytotoxic towards the L-929 cell line (mouse fibroblast). The cytotoxicity of crude lipopeptides towards cell lines can vary significantly depending on several factors, including the type of lipopeptide, the target cell line, and the concentration of the lipopeptide [62]. In the future, we plan to use purified lipopeptides to better understand the specific effects of lipopeptides on various cell lines.
Conclusion
This comprehensive study evaluated 315 bacterial isolates in in vitro assay to assess their potential to promote the growth of plants and control diseases. Overall, one isolate (
Supplemental Materials
Acknowledgments
This work was financially supported by Research and Researcher for industry (RRi), Thailand Science Research and Innovation (TSRI) and Banrai-ioon strawberry farm (Grant No. MSD62I0098). Special thanks are extended to Dr. Sakamon Devahastin for his constructive comments and to Dr. Kumrop Ratanasut for providing the phytopathogenic bacterium.
Author Contributions
Conceptualization, B. Dell, J. Jumpathong; Methodology, J. Jumpathong, N. Suphrom, K. Khamsuk; Validation, B. Dell, J. Jumpathong; Formal Analysis, K. Khamsuk, N. Suphrom; Investigation, K. Khamsuk, J. Jumpathong; Resources, K. Khamsuk, J. Jumpathong; Data Curation, K. Khamsuk, J. Jumpathong; Writing – Original Draft Preparation, K. Khamsuk, J. Jumpathong; Writing – Review & Editing, K. Khamsuk, B. Dell, W. Pathom-aree, W. Pathaichindachote, N. Suphrom, N. Nakaew and J. Jumpathong; Visualization, K. Khamsuk, J. Jumpathong; Supervision, J. Jumpathong; Project Administration, J. Jumpathong; Funding Acquisition, J. Jumpathong
Conflict of Interest
The authors have no financial conflicts of interest to declare.
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Table 1 . In vitro plant growth promoting properties and antifungal activity..
Bacterial ID Code IAA production (μg/ml) * Phosphate solubilization index (PSI) * pH of medium Phosphate solubilization (μg/ml) * Inhibition of pathogenic fungi (%)* 3 days 7 days 1 day 7 days C. lunata NUF001B. oryzae 2464KK007 - 3.44 ± 0.67bc 4.74 ± 0.54b 7.00 4.06 714.25 ± 12.14a 36.59 ± 1.84cd 45.06 ± 2.97bc KK024 11.59 ± 1.67d - - 7.00 4.94 139.12 ± 32.29f 58.24 ± 0.82a 45.29 ± 2.57bc KK026 16.82 ± 0.60d 3.57 ± 1.04abc 4.27 ± 0.64bc 7.00 5.48 98.17 ± 14.66g - - KK066 328.43 ± 24.10b - 2.45 ± 0.15e 7.00 5.47 151.52 ± 2.02f 30.03 ± 2.41de - KK067 - 3.48 ± 0.20bc 4.82 ± 0.34b 7.00 5.81 137.55 ± 1.34f 55.02 ± 1.75ab 52.83 ± 1.84ab KK070 - 4.09 ± 0.45ab 3.49 ± 0.16d 7.00 5.32 107.78 ± 3.46g - - KK074 362.59 ± 28.02a - - - - - - 20.49 ± 8.85d KK075 330.09 ± 15.28b - - 7.00 4.98 196.75 ± 4.65e 0.80 ± 0.15f - KK077 352.32 ± 13.72a - - - - - 27.75 ± 0.60e - KK081 348.71 ± 7.74ab - - - - - - - KK135 - - 3.85 ± 0.12cd 7.00 3.94 683.45 ± 13.51b - - KK138 - - 2.62 ± 0.03e 7.00 3.96 694.63 ± 9.12b - - KK184 14.04 ± 2.76d 3.87 ± 0.14abc 4.71 ± 0.12b 7.00 5.56 100.49 ± 2.63g 53.23 ± 1.64ab 51.47 ± 1.56ab KK225 2.82 ± 0.88d 4.51 ± 0.49a 7.85 ± 0.54a 7.00 4.40 389.55 ± 6.39c 38.60 ± 11.97c 37.59 ± 8.13c KK269 148.54 ± 1.87c 2.98 ± 0.08c 3.52 ± 0.14d 7.00 5.53 153.02 ± 3.07f 50.71 ± 2.68b 56.01 ± 3.14a KK275 6.82 ± 0.44d - - 7.00 4.91 92.11 ± 2.09g 54.14 ± 1.34ab 47.98 ± 0.47ab KK281 23.59 ± 1.02d - - 7.00 4.90 275.78 ± 2.87d 57.91 ± 0.42ab 46.62 ± 5.36b a-gThe mean values with different superscript letters in a column are statistically different from Duncan’s multiple range test (
p < 0.05)..
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Table 2 . Identification of upland rice endophytic and rhizosphere bacteria based on 16S rRNA gene sequence..
TBRC ID Code Isolate KK Sequence Length (bps) NCBI Accession Closely related taxa Similarity (%) Taxonomic assignment 15994* KK007 1,466 ON406146 Pantoea stewartii subsp.Indologenes LMG 2632T99.45 Pantoea stewartii 15995* KK018 1,479 ON406147 Priestia megaterium NBRC 15308T99.93 Priestia megaterium 15999* KK066 1,400 ON406148 Enterobacter roggenkampii EN-117T99.79 Enterobacter sp.15996¥ KK138 1,464 ON406149 Acinetobacter soli CIP 110264T100.00 Acinetobacter soli 15997¥ KK269 1,468 ON406150 Pantoea allii LMG 24248T99.04 Pantoea sp.15998¥ KK281 1,473 ON406151 Bacillus siamensis KCTC 13613T99.86 Bacillus sp.Note: * = endophytic bacteria, \ = rhizosphere bacteria.
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Table 3 . Inhibition zone diameter of lipopeptides extract from isolate KK281 against
Curvularia lunata NUF001 andXanthomonas oryzae pv.oryzae by the paper disc diffusion method..Sample Concentration (mg/ml) Inhibition zone diameter (mm) C. lunata NUF001 96 hXoo 72 hCrude lipopeptides 12.5 39.93 ± 2.14a 19.33 ± 1.29c 6.25 17.20 ± 0.87c 16.43 ± 0.50d 3.125 18.10 ± 2.56c 12.47 ± 1.23e 70% ethanol 0.00 ± 0.00d NA Prochloraz 500 ppm 29.65 ± 0.77b NA Streptomycin* NA 42.50 ± 0.30b Chloramphenicol* NA 53.20 ± 0.60a a-eThe mean values with different superscript letters in a column are statistically different from Duncan’s multiple range test (
p < 0.05)..*Streptomycin 10 μg/disc, chloramphenicol 10 μg/disc.
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