Bacterial endophytes of rice (Oryza sativa L.) and their potential for plant growth promotion and antagonistic activities

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Bacterial endophytes of rice (Oryza sativa L.) and their potential for plant growth promotion and antagonistic activities Vinay Kumar*, Lata Jain, Sanjay Kumar Jain, Sameer Chaturvedi, Pankaj Kaushal ICAR - National Institute of Biotic Stress Management, Raipur 493225, Chhattisgarh, India

A R T I C L E

I N F O

Article History: Received 4 September 2019 Revised 10 February 2020 Accepted 16 February 2020 Available online xxx Edited by S Guptaitem Key words: Bacterial endophytes Rice Growth promotion Antagonistic activities Biological control

A B S T R A C T

Bacterial endophytes live inside the plant tissues and known to play a crucial role in the functioning of host plants through influencing their physiology and development. In present study, a total of 32 bacterial endophytes were isolated from four plant tissues (root, stem, leaf and grain) of six rice varieties cultivated in central-eastern and northeastern states of India. Microbial composition varied among the plant tissues studied, they were predominant in the root and leaf tissues. Biochemical and molecular characterization identified these bacterial isolates belonging to phyla Proteobacteria and Firmicutes representing 5 genera. Out of 32 isolates, (19) 59.3% Gram’s stain-positive, (17) 53.1% were able to produce indole acetic acid, (9) 28.1% siderophore development and (18) 56.2% phosphate solubilization activities indicating the plant growth-promoting (PGP) ability. The antagonistic activity of identified bacterial endophytes was determined against bacterial leaf blight disease-causing pathogen, Xanthomonas oryzae pv. oryzae (8 isolates) and soilborne fungal pathogens viz, Rhizoctonia solani (17 isolates), Fusarium verticillioides (15 isolates) and Sclerotium rolfsii (9 isolates) were exhibiting antagonistic activities against tested pathogens. Further, to confirm the probable role of lipopeptides in antagonism, PCR based detection of lipopeptide genes in the genome of bacterial endophytes showed the presence of antibacterial (surfactin) and antifungal (iturin D and bacillomycin D) genes in Bacillus subtilis (NIBSM_OsR10). This Bacillus subtilis isolate showed potential antibacterial activities against Xanthomonas oryzae pv. oryzae and strong antagonistic activities against fungal pathogens Rhizoctonia solani, Fusarium verticelloides and Sclerotium rolfsii. Potential isolates exhibiting antibacterial and antifungal activities in the present study may be used for the development of biocontrol formulations for controlling multiple biotic stresses. © 2020 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction Plant microbiomes consist of a huge diversity of micro-organisms which inhabit different plant tissues and organs such as seeds, roots, stems, leaves, flowers and fruits (Philippot et al., 2013; Berg et al., 2014). These microbial communities are present externally as epiphytes and in internal tissues and organs of a plant as endophytes, influencing plant vigor and health (Hardoim et al., 2015). Among these microbial communities, endophytes are the microbes that live inside the host plant without causing any disease symptoms or negative effect and are known to have the ability to antagonize plant pathogens (Mousa et al., 2015). Endophytes play an important role in the functioning of the host plant by influencing their physiology and developmental processes. Bacterial endophytes are known to be involved in imparting tolerance or resistance to the host plant from various biotic and abiotic stresses by releasing antimicrobial metabolites, synthesizing phytohormones, siderophores, competing with pathogens for space and nutrients and modulating the plant * Corresponding author. E-mail address: [email protected] (

resistance response (Rosenblueth and Martínez-Romero, 2006; Friesen et al., 2011; Mercado-Blanco and Lugtenberg, 2014). Studies on plant-endophytic microbe interactions are essential due to the application of beneficial microbes as plant growth-promoting agents and biocontrol agents for controlling phytopathogens (Scholthof, 2001). Endophytic microbes seem to be the possible alternatives to reduce the use of pesticides and chemical fertilizers (Bredow et al., 2015). Host-associated bacterial endophytes are useful tools for the development of biocontrol agents for phytopathogen control (Upreti and Thomas, 2015). The composition of bacterial endophytes varied among the plants, tissues, organs, genotypes, varieties, soil and location (Hardoim et al., 2015) Rice (Oryza sativa L) is the most important cereal crop for more than 50% of the world’s population (Khush, 2003). However, rice diseases limit the production of rice particularly bacterial leaf blight, blast and sheath blight are considered as major constraints in rice cultivation worldwide. To establish a sustainable agriculture system, combination of eco-friendly approaches are essentially required to control these diseases (Tian et al., 2007). Studies have been reported on the isolation of rice bacterial endophytes from wild accessions and traditional rice varieties (Elbeltagy et al., 2000) and from

https://doi.org/10.1016/j.sajb.2020.02.017 0254-6299/© 2020 SAAB. Published by Elsevier B.V. All rights reserved.

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locations (Mano and Morisaki, 2008). Endophytes have also been isolated from plant tissues, ovule and seed endosphere using culturebased and culture-independent approaches (Hardoim et al., 2012; Walitang et al., 2017) and for determination of plant growth-promoting activities (Bertani et al., 2016). Despite the numerous beneficial effects of bacterial endophytes on plant growth promotion and development, the bacterial endophytic diversity in important rice varieties grown in central-eastern and northeastern states of India had not been explored. However, keeping in view that the bacterial endophytes are host specific, their presence and performance is largely influenced by genotypes, soil properties and geographical locations. It has been well documented that the indigenous or native bacterial isolates perform better as biocontrol agent due to their wider adaptability with the host under similar climatic conditions. The Chhattisgarh state is known for a rich heritage of indigenous rice varieties that were adapted to different agro-ecosystems (Richharia, 1979) and is also known as Rice Bowl of India due to its richness in diversity for different agro-economic traits, Over 23,250 rice germplasm have been recorded in the region (Richharia, 1979; Sinha, 2016 and Sahu et al., 2017). Exploring the diversity of bacterial endophytes in rice varieties and landraces grown in this region is crucial for the identification of potential endophytes for plant growth promotion and biocontrol agents. Hence, the present study aimed to generate information on bacterial endophytic diversity in different tissues of different varieties of rice and characterize promising isolates with plant beneficial traits including antagonistic activities against bacterial and fungal pathogens. The study identified bacterial endophytes which can be effective for simultaneous controlling of more than one disease or multiple pathogens. Exploring the beneficial properties of plant endophytes will be useful in finding novel endophytes for plant growth promotion and as potential bio-control agents. 2. Materials and method 2.1. Sample collection and isolation of bacterial endophytes For isolation of bacterial endophytes, six rice varieties commonly grown in central, eastern and northeastern states of India viz, cv. Swarna, Jaighundi, Dubraj, HMT, Tulsimanjari and Vishnubhog were used. These varieties were grown in the experimental field as well as in pots at ICAR- National Institute of Biotic Stress Management, Baronda, Raipur (21.22 N and 81.49 E). Plant tissue such as root, stem, leaf and grains were collected and placed on ice from the rice field and transported to the laboratory. The samples were surface sterilized by sodium hypochlorite and inoculated on nutrient agar Petri plates for isolation of bacterial endophytes. Surface sterilization process involved washing of plant tissues in running water for 10 min and twice with distilled water for 1 min followed by dipping in 3% (v/ v) sodium hypochlorite solution for 3 min and 70% (v/v) ethanol for 3 min, finally the samples were washed thrice with sterile double distilled water for 2 min. To check complete external disinfection process 1 ml of water from last rinse was added to 10 ml of nutrient broth and incubated for 48 h. All the procedures were performed under aseptic conditions (Elbeltagy et al., 2000). Surface sterilized plant tissues were dried on sterile filter paper and cut into small pieces or segments of 0.5 to 1 cm in size, six to seven tissue segments were placed on Nutrient agar plates in triplicate and incubated at 30 °C for bacterial growth. After 2 7 days of incubation few representative colonies appearing on the Petri plates were picked-up sterile loop. Morphologically distinct colonies were selected and purified by sub-culturing. Pure cultures of these isolates were processed for further morphological, biochemical and molecular characterization. Individual bacterial colonies of each isolate were cultured on nutrient broth and stored in 20% sterile glycerol at 80 ° C for further studies.

2.2. Morphological and biochemical characterization Standard identification protocols based on morphology were performed to characterize the bacterial isolates. Morphology, arrangement and appearance of the bacterial cell wall were determined by Gram’s staining (Bathlomew, 1962). Biochemical activities of bacterial endophytes viz, oxidase test, catalase test, nitrate reduction test, urease activity, citrate utilization test, methyl red, Voges Proskauer test and gas production were performed following the procedures mentioned earlier Cappuccino and Sherman (2002). 2.3. Molecular characterization The total genomic DNA from 24 h old cultures of the bacterial endophytes was isolated using Wizard Genomic DNA Purification Kit, Promega following standard protocol. The quality and quantity of isolated DNA was evaluated on 1% (w/v) agarose gel as well as using a spectrophotometer (Eppendorf AG, Hamburg, Germany). For the PCR reaction 50 ng of genomic DNA and 10 picomole each of forward and reverse primer concentration was used. 2.3.1. PCR amplification of 16S rDNA gene and sequencing Molecular characterization and identification of the bacterial endophytes was performed using amplification of 16S rDNA region using the universal forward (27 F) and reverse (1492 R) primers described by Lane (1991). PCR amplification for 16S ribosomal DNA region was performed using Thermal cycler (Mastercycler ProS, Eppendorf, USA). PCR amplified products were resolved in 1.2% (w/v)) agarose gel, visualized using UV transilluminator and documented using Gel documentation system (Protein Simple, Alfa Innotech Corporation, USA). The amplicons of about 1500 bp were selected and gel purified using MinElute Gel Extraction Kit (Qiagen, Germany) and quantified by taking absorbance at 260 nm using spectrophotometer (Eppendorf, USA). The gel purified PCR products were sequenced from both the ends by Genetic Analyzer 3130 (Applied Biosystems, CA, and USA). 2.3.2. Analysis of 16S ribosomal DNA sequences and data deposition The sequencing of 16S rDNA was performed using both forward and reverse primers. The generated sequences were aligned and merged using the BioEdit software, V. 7.2.5 (Hall, 1999) and compared with NCBI databases. The homology search was performed using Basic Local Alignment Search Tool (BLASTn) tool available at the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm. nih.gov/blast/) (Altschul et al., 1997) and nucleotide sequences showing 99% similarity were selected for the identification of respective isolates. The nucleotide sequences of 16S rDNA generated from this study have been deposited in the GenBank, NCBI database. 2.4. Screening for plant growth promoting (PGP) activities 2.4.1. Indole acetic acid (IAA) production Bacterial endophytes were screened for their capability to produce, indole acetic acid (IAA) qualitatively using the colorimetric method described by Gordon and Weber (1951). Five ml of YeastPeptone-Mannitol (YPM) broth with tryptophan (100 mg /ml) were inoculated with bacterial isolate and incubated at 30 °C with continuous shaking at 100 rpm under dark conditions after 72 h culture broth was centrifuged (Microcentrifuge, 5425R model, Eppendorf AG, Hamburg, Germany) for 10 min at 8000 rpm. 2 ml of Salkowski reagent [A mixture of 0.5 M ferric chloride (FeCl3) and 35% perchloric acid (HClO4)] was added to 2 ml of culture supernatant and incubated at room temperature for 30 min, appearance of pink color confirmed the production of IAA by bacterial endophytes. The test was performed for qualitative analysis (development of color) indicated the IAA production abilities.

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2.4.2. Phosphate solubilization test Isolated bacterial endophytes were evaluated for their ability to solubilize the insoluble phosphate. Bacterial isolates were spot inoculated at four places on Pikovskaya media containing tri-calcium phosphate in Petri dish and incubated at 28 § 2 °C for 3 4 days (Pikovskaya, 1948). The appearance and development of a clear halo zone around bacterial isolates indicated positive phosphate solubilization activity. 2.4.3. Siderophore production Endophytic bacterial isolates were screened for production of qualitative siderophore on the Chrome azurol S (CAS) agar medium following the procedure as described by Schwyn and Neilands (1987). The bacterial isolates were spot inoculated on CAS agar medium plates and incubated at 28§1 °C for 2 4 days for their growth. The appearance of orange color halo zone around the bacterial isolate indicated the siderophore production activity. 2.4.4. Hydrogen cyanide (HCN) production Exponentially grown endophytic bacterial isolates were separately streaked on glycerin agar medium following the procedure described by Bakker and Schippers (1987) with simultaneous supplementation of a filter paper soaked in picric acid (0.5%) in Na2CO3 (5%) in the upper lid of Petri plate. The plates were incubated at 28 § 2 °C for 24 to 48 h in incubator shaker (Eppendorf AG, Hamburg, Germany). Changes in the color of the filter paper from yellow to light brown color or reddish-brown indicated the HCN production activity. 2.4.5. DNase assay DNase assay was conducted for the detection of deoxyribonuclease activity of endophytes as described by (Smith et al., 1969). Bacterial isolates were spot inoculated on DNase test agar base with methyl green and incubated at 35 °C for 24 h and appearance of clear zone around the bacterial spot indicated the DNase positive activity. 2.5. Carbohydrate utilization test Carbohydrates discs of 21 different sugars namely, Sucrose, Dextrose, Lactose, Galactose, Maltose, Mannose, Raffinose, Fructose, Trehalose, Inositol, Mannitol, Salicin, Rhamnose, Inulin, Cellobiose, Adonitol, Melibiose, Arabinose, Xylose, Dulcitol and Sorbitol (HiMedia) were tested for utilization by endophytes. The phenol red agar base media plates were surface seeded with endophytic bacteria and required carbohydrate discs were aseptically placed on the surface of the plate at 3 cm away from each other and incubated at 30 °C for 18 48 h. Results were recorded twice after 18 h and 48 h. The development of yellow color around the disk indicated a positive reaction.

2.7. In vitro antagonistic activities of bacterial endophytes 2.7.1. Antagonistic activities against bacterial pathogens Isolated bacterial endophytes were qualitatively screened for their antibacterial properties against Gram- positive (Staphylococcus aureus) and Gram- negative (Xanthomonas oryzae pv. ozyzae, Escherichia coli, Klebsiella pneumoniae and Salmonella typhimurium) bacterial pathogens following the cross-streak assay method (Williston et al., 1947). Bacterial endophytes were inoculated as a single streak at the center of the nutrient agar plate and incubated at 30 °C for 5 6 days. The overnight grown cultures of the test microorganisms were thickly streaked at perpendicular to the endophyte line of growth. The plate was incubated at 30 °C in incubator shaker and observed daily for growth inhibition for 7 days. 2.7.2. Antagonistic test against soil borne fungal pathogens Bacterial endophytes were tested in vitro for their antagonistic activity toward the soil-borne plant pathogenic fungi, Rhizoctonia solani, Sclerotium rolfsii and Fusarium verticillioides using a dual culture approach. These pathogenic fungal strains were isolated and accessioned at Indian Type Culture Collection, New Delhi, India with the accession numbers viz., ITCC 8218, ITCC 8322 and ITCC 8333, respectively. A 5 mm mycelial disk of each fungal pathogen from 7-day old culture was placed on one end of a potato dextrose agar (PDA) plates and each endophytic bacterial isolate was streaked on the other end of the plate. The plates with only fungal culture at one end served a control. The PDA plates were incubated at 28 °C for 7 8 days or till fungal growth covered the plates in the control. The antagonistic activities of the isolates were indicated by the inhibition zones formed between the endophytic bacterial and fungal isolates. All dual culture assays were carried out in triplicates. 2.8. Amplification of lipopeptide genes using PCR The bacterial endophytic isolates showing strong antibacterial and/ or antifungal activities were screened further to detect the presence of lipopeptide genes namely surfactin, Itruin D, bacillomycin D and fengycin. The five pairs of gene-specific primers previously reported by Gond et al. (2015) as listed in Table 1 were selected. PCR amplification of these genes was performed with the reaction condition of initial denaturation at 95 °C for 5 min followed by denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min and extension at 72 °C for 1 min for 35 cycles and a final extension at 72 °C for 7 min.

Table 1 List of PCR primers used for amplification of lipopeptide genes. S. No

Antibiotics

Target gene

Sequences (50 30 )

Amplicon size (bp)

1

Surfactin

Sfp gene

675

2

Fengycin

Fen D

3

Surfactin

Srf C

4

Iturin A

ItuD

5

Bacillomycin D

Bam C

F-ATGAAGATTTAC GGAATTTA R-TTATAAAAGCT CTTCGTACG F-TTTGGCAGCAGG AGAAGTTT R-GCTGTCCGTTC TGCTTTTTC F-ACAGTATGG AGGCATGGTC R-TTCCGCCACTT TTTCAGTTT F-GATGCGATCTC CTTGGATGT R-ATCGTCATGT GCTGCTTGAG F-GAAGGACACG GAGAGAGTC R-CGCTGATGAC TGTTCATGCT

2.6. Antibiotic sensitivity profiling An antibiotic sensitivity test was conducted using antibiotics discs (6 mm diameter) with their concentrations mentioned in parenthesis namely Ampicillin (10 microgram [mcg]), Methicillin (5 mcg), Cefixime (5 mcg), Ceftriaxone (30 mcg), Vancomycin (30 mcg), Polymyxin B (300 unit), Tetracycline (30 mcg), Streptomycin (10 mcg), Gentamicin (10 mcg), Azithromycin (15 mcg), Gatifloxacin (5 mcg), Ciprofloxacin (5 mcg), Nalidixic acid (30 mcg) and Trimethoprim (5 mcg) were used. Overnight grown bacterial cultures in nutrient broth were surface seeded with on Muller Hinton agar plates and discs were placed 3 4 mm apart as mentioned in Kirby Bauer disk-diffusion assay (Bauer et al., 1996). The diameter of the inhibition zone was recorded and organisms were categorized as resistant and sensitive following the manufacturer’s manual.

3

964

441

647

875

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The PCR products were resolved on 1.2% (w/v) agarose gel for the presence of specific amplicon. 3. Results 3.1. Isolation of bacterial endophytes More than 60 bacterial endophytes were isolated from the plant tissues namely root, leaf, stem and grain (seed) from the six rice varieties of which, 32 isolates were finally selected on the basis of morphological and functional traits for further characterization. The highest number of bacterial endophytes were isolated from the root (16) followed by the stem (7), leaf and grain yielded (5) and (4) isolates, respectively. The morphological, biochemical and molecular characterization of 32 bacterial isolates represented 2 phyla (Proteobacteria and Firmicutes) and five genera viz. Bacillus (19), Enterobacter (10), Klebsiella (1), Leclercia (1) and Xanthomonas (1). The analyzed tissues differed with respect to the richness of microbial diversity, the number genera present in the different plant tissues namely roots (4) leaves (3) grain (2) and stem (1) were recovered. Bacterial endophyte, Bacillus cereus was found in all the plant tissues (root, stem, leaf and grain), while B. subtilis was present in root, leaf and grain. Enterobacter cloacae was shared between the root and leaf and E. tabaci, B. stratosphericus were found in grain and root tissues while B. pumilus was found in leaf and stem tissues only. The tissue-specific endophytes were (4) in the root namely Leclercia adecarboxylata, Klebsiella pneumoniae, Enterobacter asburiae, Bacillus thermophilus, (2) in leaf tissues Bacillus xiamenensis, Xanthomonas sacchari and B. velezensis (1) in stem tissues while none of the isolates were found specific to the grain (Fig. 1). 3.2. Morphological and molecular characterization Morphological characterization based on bacterial colonies showed different colony colors viz., white, cream and yellow. The Gram staining showed 19 isolates (59.3%) endophytes found to be Gram-positive whereas 13 isolates (40.6%) were Gram-negative. The Gram-positive isolates belong to the Bacillus species, whereas the Gram-negative was represented by members of Enterobacter, Klebsiella, Leclercia and Xanthomonas. The tissue-wise distribution of Grampositive isolates was from root (6 in number), stem (7), leaf (3) and grain (3) whereas Gram-negative isolates were found in the root (10), leaf (3) and grain (1). None of the isolates from the stem was Gram-negative. Out of 32 bacterial endophytes 59.3% and 40.6% isolates belonged to the phyla Firmicutes and Proteobacteria, respectively (Table 2). PCR amplification of 16S rDNA region using forward (27 F) and reverse (1492 R), primers produced ~1500 bp amplicon size. The amplified PCR products were sequenced bi-directionally using forward (27 F) and reverse (1492 R) primers. The Nucleotide sequences

generated were aligned to get about 1400 1500 bp fragment. The aligned nucleotide sequences of 16S rDNA fragments were deposited in the GenBank database under the accession numbers, KY911276, KY927393-KY927399, KY927846-KY927850, KY930332-KY930334 and KY930702-KY930716. Based on the BLASTn analysis of nucleotide sequences of 16S rRNA gene sequence, endophytic bacterial isolates were identified and the detail of the isolates and their nearest relative based on the sequence are given in Table 3. The bacterial endophytes were finally identified as Bacillus cereus, B. pumilus, B. subtilis, B. velezensis, B. thermophilus, B. xiamenensis B. stratosphericus belonging to the phylum Firmicutes. Isolates belonging to the phylum Protobacteria included Klebsiella pneumoniae, Enterobacter cloacae, E. tabaci, E. asburiae, Xanthomonas sacchari and Leclercia adecarboxylata. 3.3. Biochemical characterization of the endophytic bacterial isolates A total of 28 isolates (87.5%) were found to be catalase-positive. All the Bacillus isolates were catalase-positive except B. velezensis (NIBSM_OsS6) and B. cereus (NIBSM_OsL3) isolated from stem and leaf tissues, respectively. However, almost all the Gram-negative isolates except, E. cloacae (NIBSM_OsR16) and L. adecarboxylata (NIBSM_OsR11) were catalase positive. Nineteen isolates (59.3%) were found to be oxidase positive including all the Bacillus isolates. While Klebsiella pneumoniae (NIBSM_OsR6), L. adecarboxylata (NIBSM_OsR11) and all the Enterobacter isolates were oxidase-negative except E. cloacae (NIBSM_OsR16) isolate (Table 2). Majority of the isolates showed the ability to convert glucose into acidic end products like lactate, acetate and formate as revealed by Methyl Red (MR) positive and Voges Proskauer (VP) negative tests results. A total of 27 isolates (84.3%) showed MR positive test, whereas only 5 isolates were MR negative. In the VP test, only 5 isolates namely, E. cloacae (NIBSM_OsR3), B. cereus (NIBSM_OsR5), K. pneumoniae (NIBSM_OsR6), E. cloacae (NIBSM_OsR9) and B. cereus (NIBSM_OsL3) were found to be positive and MR negative (Table 2). In the Nitrate reductase (NR) test, 25 isolates (78.1%) were positive having the ability to perform nitrification. Among them, all the Bacillus isolates showed positive reaction except B. stratosphericus (NIBSM_OsR1). Other isolates namely, L. adecarboxylata (NIBSM_OsR11), K. pneumoniae (NIBSM_OsR6) and all the Enterobacter sp. isolated from root were NR positive except E. cloacae (NIBSM_OsR3, NIBSM_OsR12 and NIBSM_OsR16). However, none of Enterobacter isolates from stem, leaf and grain showed NR positive activity. Nine out of 32 isolates were able to produce gas, of which 4 isolates belong to the Bacillus group viz. B. subtilis (NIBSM_OsS2), B. velezensis (NIBSM_OsS6), B. cereus (NIBSM_OsS7) and B. cereus (NIBSM_OsL3) showed gas-producing ability. In addition to these, 5 isolates namely, L. adecarboxylata (NIBSM_OsR11), E. tabaci (NIBSM_OsR7), E. cloacae (NIBSM_OsR13), E. asburiae (NIBSM_OsR14) and E. cloacae (NIBSM_OsR16) isolated from root showed gas production ability. However, none of the Bacillus isolates from root and grain were able to produce gas. A total of 23 isolates (71.8%) showed citrate utilization activity (Table 2). Among them all the Enterobacter isolate showed citrate utilization ability except for E. cloacae (NIBSM_OsR3) and E. tabaci (NIBSM_OsG1) isolated from root and grain, respectively. Out of eight B. cereus isolates, none of the isolates from root and grain showed citrate positive activity, however, B. cereus isolated from stem and leaf showed citrate utilization activity. 3.4. Screening for plant growth promotion traits

Fig. 1. Venn diagram depicting the bacterial isolates shared among different tissues.

3.4.1. Indole acetic acid production A total of 17 isolates (53.1%) were found to have IAA production ability, belonging to the Bacillus group (10 isolates), Enterobacter group (5 isolates) and of L. adecarboxylata (1) and K. pneumoniae (1) isolate (Table 2).

Please cite this article as: <.t.i.O.p.g.a.d.-h.-n.y. Kumar et al., Bacterial endophytes of rice (Oryza sativa L.) and their potential for plant growth promotion and antagonistic activities, South African Journal of Botany (2020), https://doi.org/10.1016/j.sajb.2020.02.017

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Detail of bacterial endophytes

Biochemical attributes

Strain/isolates

Isolation source

Grams Stain

Oxidase

Catalase

Methyl Red (MR)

1

Bacillus stratosphericus Bacillus cereus Enterobacter cloacae Bacillus cereus Bacillus cereus Klebsiella pneumoniae Enterobacter tabaci Enterobacter cloacae Enterobacter cloacae Bacillus subtilis Leclercia adecarboxylata Enterobacter cloacae Enterobacter cloacae Enterobacter asburiae Bacillus thermophilus Enterobacter cloacae Bacillus subtilis Bacillus subtilis Bacillus cereus Bacillus cereus Bacillus pumilus Bacillus velezensis Bacillus cereus Bacillus pumilus Enterobacter cloacae Bacillus cereus Xanthomonas sacchari Bacillus xiamenensis Enterobacter tabaci Bacillus cereus Bacillus subtilis Bacillus stratosphericus Total Positive (Nos) % Positive

NIBSM_OsR1

Root

+

+

+

+

NIBSM_OsR2 NIBSM_OsR3 NIBSM_OsR4 NIBSM_ OsR5 NIBSM_OsR6

Root Root Root Root Root

+

+

+

+ +

+ +

+ + + + +

NIBSM_OsR7 NIBSM_OsR8 NIBSM_OsR9 NIBSM_OsR10 NIBSM_OsR11

Root Root Root Root Root

+ + + +

+ +

NIBSM_OsR12 NIBSM_OsR13 NIBSM_OsR14 NIBSM_OsR15 NIBSM_OsR16 NIBSM_OsS1 NIBSM_OsS2 NIBSM_ OsS3 NIBSM_ OsS4 NIBSM_OsS5 NIBSM_OsS6 NIBSM_OsS7 NIBSM_OsL1 NIBSM_OsL2 NIBSM_OsL3 NIBSM_OsL4

Root Root Root Root Root Stem Stem Stem Stem Stem Stem Stem Leaf Leaf Leaf Leaf

NIBSM_OsL5 NIBSM_OsG1 NIBSM_OsG2 NIBSM _OsG3 NIBSM_OsG4

Leaf Grain Grain Grain Grain

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

+

+

+

+

+ + + + + + + +

+ + + + + + + +

+

+

+

+

+ + + 19 59.3

+ + + + + + + + + + + +

Voges Proskauer (VP)

Gas production

Citrate utilization

Nitrate reductase

Urease

Indole Acetic Acid production

Phosphate solubilization

+ +

Siderophore production

DNase activity

+

+

+

+

+

+ +

HCN production

+ + + + + + + +

+

+ + + + + + + + + + + + + +

+ + + +

+ +

+ +

+

+ + +

+ + + + +

+ + + + +

19 59.3

28 87.5

27 84.3

5 15.6

+

9 28.1

+

+ + +

+ + + + +

+ + + + +

+ + + + + + +

+ + + + + +

+ + + + + + + + + + +

+ + + + + + + + + + + +

+ +

+

+ + + + +

+ + +

+ + + + +

+

+ +

+

+

+

+

+

+ +

+ + +

+ +

+

23 71.8

25 78.1

18 56.2

17 53.1

+ +

+

+

+ +

+ + +

+ + +

+ + +

+

+ + + + + +

+ +

9 28.1

+ +

18 56.2

+ + + + + +

+ +

+

12 37.5

4 12.5

5

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Name of the bacterial endophytes

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Plant growth promoting (PGP) traits

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promotion and antagonistic activities, South African Journal of Botany (2020), https://doi.org/10.1016/j.sajb.2020.02.017

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Table 2 Biochemical and functional characterization (plant growth promoting activities) of bacterial endophytes.

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Table 3 Molecular characterization of bacterial endophytes and their identities. S. No. Name of the bacterial endophytes

Strain/isolates

NCBI accession numbers

Name of the isolates matching with NCBI database and their accession numbers

% Identity

Rice varieties/cultivars used for isolation

1 2 3

Bacillus stratosphericus Bacillus cereus Enterobacter cloacae

NIBSM_OsR1 NIBSM_OsR2 NIBSM_OsR3

KY911276 KY930702 KY930703

99 99 95

Tulsimanjari Jaigundi Vishnunhog

4 5 6 7 8

Bacillus cereus Bacillus cereus Klebsiella pneumoniae Enterobacter tabaci Enterobacter cloacae

NIBSM_OsR4 NIBSM _OsR5 NIBSM_OsR6 NIBSM_OsR7 NIBSM_OsR8

KY930704 KY930705 KY930706 KY930707 KY930708

99 99 99 99 99

Jaigundi Swarna Swarna Swarna Tulsimanjari

9

Enterobacter cloacae

NIBSM_OsR9

KY930709

99

Jaigundi

10

Bacillus subtilis

NIBSM_OsR10

KY930710

95

Vishnubhog

11 12

Leclercia adecarboxylata Enterobacter cloacae

NIBSM_OsR11 NIBSM_OsR12

KY930711 KY930712

99 99

Dubraj HMT

13

Enterobacter cloacae

NIBSM_OsR13

KY930713

99

Swarna

14 15 16 17 18 19 20 21 22 23 24 25

Enterobacter asburiae Bacillus thermophilus Enterobacter cloacae Bacillus subtilis Bacillus subtilis Bacillus cereus Bacillus cereus Bacillus pumilus Bacillus velezensis Bacillus cereus Bacillus pumilus Enterobacter cloacae

NIBSM_OsR14 NIBSM_OsR15 NIBSM_OsR16 NIBSM_OsS1 NIBSM_OsS2 NIBSM _OsS3 NIBSM _OsS4 NIBSM_OsS5 NIBSM_OsS6 NIBSM_OsS7 NIBSM_OsL1 NIBSM_OsL2

KY930714 KY930715 KY930716 KY927393 KY927394 KY927395 KY927396 KY927397 KY927398 KY927399 KY927846 KY927847

98 95 99 99 99 99 99 99 99 99 99 99

Dubraj Vishnubhog Tulsimanjari Swarna Jaigundi Jaigundi Jaigundi Jaigundi Jaigundi Jaigundi Swarna Swarna

26 27 28 29 30 31 32

Bacillus cereus Xanthomonas sacchari Bacillus xiamenensis Enterobacter tabaci Bacillus cereus Bacillus subtilis Bacillus stratosphericus

NIBSM_OsL3 NIBSM_OsL4 NIBSM_OsL5 NIBSM_OsG1 NIBSM_OsG2 NIBSM OsG3 NIBSM_OsG4

KY927848 KY927849 KY927850 KY930332 KY930333 KY930334 KY962816

Bacillus stratosphericus [NR_042336.1] Bacillus cereus strain [NR_115714.1] Enterobacter cloacae subsp. Dissolvens [NR_118011.1] Bacillus cereus [NR_115714.1] Bacillus cereus [NR_115714.1] Klebsiella pneumoniae [NR_117686.1] Enterobacter tabaci [NR_146667.2] Enterobacter cloacae subsp. Dissolvens [NR_044978.1] Enterobacter cloacae subsp. dissolvens [NR_118011.1] Bacillus subtilis subsp. inaquosorum strain [NR_104873.1] Leclercia adecarboxylata [NR_104933.1] Enterobacter cloacae subsp. dissolvens strain [NR_118011] Enterobacter cloacae subsp. dissolvens [NR_118011.1] Enterobacter asburiae [NR_042349.1] Bacillus thermophilus [NR_109677.1] Enterobacter ludwigii [NR_042349.1] Bacillus subtilis[NR_027552.1] Bacillus subtilis [NR_027552.1] Bacillus cereus [NR_115714.1] Bacillus cereus [NR_074540.1] Bacillus xiamenensis [NR_148244.1] Bacillus velezensis [NR_075005.2] Bacillus cereus [NR_115714.1] Bacillus stratosphericus [NR_042336.1] Enterobacter cloacae subsp. dissolvens NR_118011.1] Bacillus cereus [NR_074540.1] Xanthomonas sacchari [NR_026392.1] Bacillus xiamenensis [NR_148244.1] Enterobacter tabaci [NR_146667.2] Bacillus cereus [NR_115714.1] Bacillus subtilis [NR_027552.1] Bacillus stratosphericus [NR_118441.1]

99 99 99 99 99 99 98

Swarna Swarna Jaigundi Swarna Jaigundi Jaigundi Jaigundi

3.4.2. Phosphate solubilization assay Nine isolates (28.1%) produced a clear zone around the streaked area on Pikovskaya media confirming the potential phosphate solubilization activity (Table 2). Among them 6 isolates belonging to the Bacillus group were isolated from the root (2), stem (3) and leaf (1) tissues. Other isolates include 2 Enterobacter isolates and one K. pneumoniae (NIBSM_OsR6) were isolated from the root (2) and leaf (1) tissues. However, none of the isolates from stem and grain tissues showed phosphate solubilization activity.

3.4.3. Siderophore production CAS agar assay was used to determine the potential of endophytes to produce siderophores. A total of (18) 56.2% isolates showed siderophore production ability (Table 2). Among them 10 isolates belong to the genus Bacillus isolated from the root (4), stem (4), leaf (1) and grain (1) tissues. Four Enterobacter isolates and L. adecarboxylata isolated from roots, one each isolate of Enterobacter from leaf and grain also produced siderophore.

3.4.4. Urease test The urease test was conducted to identify the bacterial endophytes for having the potential to breakdown urea to other forms of nitrogen that can readily be absorbed by the plants. A total of 18 isolates (56.2%) showed urease activity, of which 10 belonged to Bacillus group. The remaining eight isolates were represented by L. adecarboxylata, X. sacchari and members of Enterobacter group (Table 2).

The number of bacterial isolates showed urease activities were recovered from the root (9) followed by stem (4), leaf (3) and grain (2). 3.4.5. DNase assay Eighteen (37.5%) isolates showed potential DNase activity (Table 2), among them 11 belonged to the Bacillus group representing B. stratosphericus (NIBSM_OsR1) and B. thermophilus (NIBSM_OsR15) isolated from root and all Bacillus isolated from stem tissue except B. cereus (NIBSM_OsS4) and two isolates of B. cereus (NIBSM_OsG2), B. subtilis (NIBSM OsG3) from grain tissues while none of the isolates from leaf tissue showed DNase activity. 3.4.6. Hydrogen cyanide (HCN) production Only four isolates showed HCN production ability, which included three isolates of Bacillus viz., B. subtilis (NIBSM_OsS1), B. cereus (NIBSM_OsL3) and B. subtilis (NIBSM OsG3) isolated from stem, leaf and grain tissues respectively (Table 2). One isolate E. cloacae (NIBSM_OsL2) from leaf tissues showed HCN production. None of the root isolates produced HCN. 3.5. Carbon source/ carbohydrate utilization pattern The bacterial endophytes isolated from rice were screened for their carbohydrate utilization patterns using 21 different sugars. Among them the highest utilized sugars were maltose 65.5% (21 isolates) followed by fructose and cellobiose 59.3% (19 isolates), sucrose and dextrose 53.1% (17 isolates) trehalose and salicin 46.8% (15

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Fig. 2. Carbohydrate utilization pattern of bacterial endophytes isolated from rice.

isolates) (Fig. 2). However, the least utilized sugars were inulin, dulictol and adonitol used by only one isolate and galactose by two isolates. The bacterial isolates which fermented the highest number of (fourteen) sugars included B. stratosphericus (NIBSM_OsG4) and E. cloacae (NIBSM_OsL2) followed by 10 sugars by B. cereus (NIBSM_OsS7), B. cereus (NIBSM_OsG2) and 9 by B. pumilus (NIBSM_OsL1). It was found that the bacterial endophytes isolated from leaf and grains were able to utilize higher number of sugars. The endophytes isolated from leaf were able to use dextrose, fructose and trehalose. Sugar utilization pattern of bacterial endophytes recovered from different tissues, root, stem, leaf and grain were able to use sugars maltose (10), fructose (6), (dextrose, fructose and trehalose) and cellobiose (4, all isolates), respectively. While none of the root isolates were able to use dulcitol, inulin and leaf isolates (galactose, inositol, inulin, dulcitol, adonitol and arabinose), Stem isolates (galactose, raffinose, inositol, inulin and adonitol) and grain isolates (adonitol, melibiose and dulcitol). Inulin was only fermented by B. subtilis (NIBSM_OsG3) and adonitol by B. cereus (NIBSM_OsR5).

3.6. Antibiotic sensitivity test Antibiotic sensitivity pattern against fourteen broad-spectrum antibiotics was used for screening. These antibiotics belong to four major groups namely, Beta-lactam antibiotics, cell wall synthesis inhibition (AMP, MET, CFM, CTR, VA, PB); protein synthesis inhibition (TE, S, GEN, AZM) and nucleic acid synthesis inhibition (GAT, CIP, NA) folate synthesis inhibition TR. Antibiotic screening showed that most of the isolates were resistant to methicillin (28), cefixime (27) and ampicillin (20) while susceptible to gentamycin (31), gatifloxacin (28), ciprofloxacin (25), streptomycin (24) and tetracycline (24) (Fig. 3). The bacterial isolates B. cereus (NIBSM_OsR4), E. cloaceae (NIBSM_OsR8), E. cloaceae (NIBSM_OsR9) showed higher resistance to 12 tested antibiotics followed by B. cereus (NIBSM_OsR5) and B. cereus (NIBSM_OsG2).The bacterial endophytes showing the least resistance for 1 antibiotic were B. subtilis (NIBSM_OsS2), B. velezensis (NIBSM_OsS6) and for 2 antibiotics B. subtilis (NIBSM_OsS1), B. xiamensis (NIBSM_OsL5) and for 3 antibiotics E. cloaceae (NIBSM_OsR16) and B. subtilis (NIBSM_OsG3).

Fig. 3. Antibiotics sensitivity pattern of rice endophytes.

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The antibiotic sensitivity pattern revealed that all the bacterial isolates from root tissues showed susceptibility to gentamicin and resistance to methicillin except for E. cloaceae (NIBSM_OsR8). All isolates from stem tissues showed resistance to cefixime except B. subtilis (NIBSM_OsS2) and were susceptible to streptomycin, gentamycin and gatifloxacin. All leaf isolates (5) were resistant to methicillin and susceptible to gentamycin, azithromycin and gatifloxacin. The obtained antibiogram showed that the Firmicutes (Bacillus group) were found to be resistant to beta-lactam antibiotics except for vancomycin as compared to other groups. Protein synthesis inhibiting antibiotics were found to be most susceptible and maximum sensitivity was found for aminoglycosides followed by fluoroquinolones. Among Proteobacteria (Enterobacteriaceae group) maximum isolates were resistant to beta-lactam group except for CTR and PB. Maximum sensitivity was found for aminoglycosides followed by fluoroquinolones. 3.7. In vitro antagonistic activities of bacterial endophytes 3.7.1. Antagonistic activities against bacterial pathogens All the bacterial endophytes were assessed for their antibacterial properties against five bacterial pathogens namely, Xanthomonas oryzae pv. oryzae (Xoo), Escherichia coli, Salmonella typhimurium, Staphylococcus aureus and Klebsiella pneumoniae and (Table 4). A total of 25% (8), 15.6% (5), 12.5% (4) and 3.1% (1) bacterial endophytes showed antagonistic activities against the test organisms Xanthomonas oryzae pv. oryzae, S. aureus, E. coli and K. pneumoniae, respectively. None of the bacterial isolates showed antagonistic effect against S. typhimurium. The Bacillus isolates showing antagonistic effect against Xanthomonas oryzae pv. oryzae were B. subtilis (NIBSM_OsR10), B. thermophiles (NIBSM_OsR15) isolated from root and B. subtilis (NIBSM_OsS1, NIBSM_OsS2), B. velezensis (NIBSM_OsS6) isolated from stem tissues. The Bacillus isolates from the root tissues showed the highest antagonistic activities followed by the stem isolates. None of the leaf and grain isolates showed any antagonistic activities. Enterobacter asburiae (NIBSM_OsR14), E. cloacae (NIBSM_OsR16; NIBSM_OsL2) also showed antagonistic activities against Xanthomonas oryzae pv. oryzae. A total of 5 bacterial isolates showed antagonistic activities against S. aureus, of which four belong to Bacillus group namely B. stratosphericus (NIBSM_OsR1), B. subtilis (NIBSM_OsS1), B. subtilis (NIBSM_OsS2) and B. pumilus (NIBSM_OsL2). The root isolate E. cloacae (NIBSM_OsR8) also exhibited antagonistic activities whereas none of the Enterobacter isolates from grain tissues showed antagonistic activities. Four bacterial isolates showed antagonistic activities against E. coli of which two were Bacillus subtilis from the stem (NIBSM_OsS2) and grain (NIBSM_OsG3). None of the Bacillus isolates from root and leaf showed antagonistic activities. E. cloacae isolated from root (NIBSM_OsR4) and leaf (NIBSM_OsL2) exhibited antagonistic activities against E. coli Only one isolate E. cloacae (NIBSM_OsL2) showed antagonistic activity against K. pneumoniae. This isolate also showed antagonistic activities against Xanthomonas oryzae pv. oryzae E. coli, Fusarium verticillioides and have the ability for siderophore and HCN production. 3.7.2. Antagonistic activities against fungal pathogens Antagonistic effect of bacterial endophytic isolates was tested against fungal phytopathogen namely, Rhizoctonia solani, Sclerotium rolfsii and Fusarium verticillioides. A total of 53.1% (17), 28.1% (9) and 46.8% (15) bacterial isolates showed antagonism against R. solani, S. rolfsii and F. verticillioides, respectively, as observed by dual culture assay. The presence of inhibition zones (halos) and inhibition of mycelial growth were evaluated on PDA plates and most of the tested pathogens showed mycelial growth inhibition and halo formation

(Table 4 and Fig. 4). The strongest inhibition was observed for S. rolfsii and R. solani that presented clear inhibition zones with the tested isolates. Out of seventeen bacterial isolates showing antagonistic activities against R. solani, 13 belonged to Bacillus group. These included 6 root isolates viz. B. stratosphericus (NIBSM_OsR1), B. cereus (NIBSM_OsR2, NIBSM_OsR4, NIBSM_OsR5), B. subtilis (NIBSM_OsR10), B. thermophillus (NIBSM_OsR15), 5 stem isolates B. subtilis (NIBSM_OsS1, NIBSM_OsS2), B. cereus (NIBSM_OsS4, NIBSM_OsS7), B. pumilus (NIBSM_OsS5) and one each of leaf (B. pumilus NIBSM_OsL2) and grain (B. stratosphericus NIBSM_OsG4) isolate. In case of Enterobacter group, the only root isolates showed antagonistic activity were E. cloacae (NIBSM_OsR3, NIBSM_OsR13, NIBSM_OsR16) and K. pneumoniae (NIBSM_OsR6). Nine bacterial isolates showed antagonistic effect against S. rolfsii, of which 7 belonged to Bacillus group including 3 root isolates representing B. cereus (NIBSM_OsR2), B. subtilis (NIBSM_OsR10), B. thermophillus (NIBSM_OsR15), 2 isolates each from stem B. subtilis (NIBSM_OsS1), B. velezensis (NIBSM_OsS6) and grain B. subtilis (NIBSM_OsG1), B. cereus (NIBSM_OsG2). Bacillus isolates from leaf did not show any response. In addition to the Bacillus group, only 2 root isolates viz., E. cloacae (NIBSM_OsR3) and K. pneumoniae (NIBSM_OsR6) also showed antagonistic activities against S. rolfsii. The bacterial isolates showing antagonistic activities against Fusarium verticillioides were 15 in number, of which 10 belonged to Bacillus group. Five isolates from stem tissues viz., B. subtilis (NIBSM_OsS1, NIBSM_OsS2), B. cereus (NIBSM_OsS4), B. pumilus (NIBSM_OsS5) and B. velezensis (NIBSM_OsS6), 2 isolates each from leaf B. pumilus (NIBSM_OsL1), B. cereus (NIBSM_OsL2) and root B. subtilis (NIBSM_OsR10), B. stratosphericus (NIBSM_R15), only one grain B. subtilis (NIBSM_OsG3) showed antagonistic activities. In case of Enterobacter group E. tabaci (NIBSM_OsR7), E. cloacae (NIBSM_OsR8, NIBSM_OsR16) from root tissues showed antagonistic activity against F. verticillioides. 3.8. Detection of lipopeptide genes in the bacterial endophytes The PCR amplification of lipopeptide genes using gene-specific primer showed the presence of surfactin, Bacillomycin D and antifungal iturin genes in the genome of bacterial endophytes (Fig. 5). The surfactin (Srf) gene-specific primer produced the desired amplicon of 675 bp in the bacterial isolates Bacillus subtilis isolated from stem tissues only. Similarly the Iturin and Bacillomycin D specific primers produced desired amplicons of 647 bp and 875 bp, respectively only in B. subtilis (NIBSM_PR10) isolated from root tissues while none of the isolates produced desired amplicon for surfactin C and fengycin genes. This suggested that Bacillus endophytes from rice might secrete lipopeptides in the host plant either on their surfaces or inside the plant tissues which provides protection from various pathogenic microorganisms. The Bacillus endophytes isolated from root tissues showed the strongest antagonistic potential against tested fungal and bacterial pathogens. 4. Discussion In this study, we reported the diversity of bacterial endophytes living inside the different tissues of rice, their plant growth promotion traits and potential antagonistic activities. There were relatively diverse groups of bacterial endophytes inhabiting rice root, leaf, stem, and grains (Fig. 1, Tables 2 and 3). Among the 32 bacterial endophytes isolated in this study, the tissue specific presence of bacterial endophytes showed that the abundance of Gram-positive isolates in the root while Gram negative in stem and leaf tissues. At the genus level, Bacillus (19) isolates were the predominant ones found in all the tissues followed by Enterobacter found in all tissues except stem. Sengupta et al. (2017) also reported that the Bacillus species was

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S. No

Name of the bacterial endophytes

Strain

Bacterial pathogens X. oryzae pv. oryzae (Xoo)

Bacillus stratosphericus Bacillus cereus Enterobacter cloacae Bacillus cereus Bacillus cereus Klebsiella pneumoniae Enterobacter tabaci Enterobacter cloacae Enterobacter cloacae Bacillus subtilis Leclercia adecarboxylata Enterobacter cloacae Enterobacter cloacae Enterobacter asburiae Bacillus thermophilus Enterobacter cloacae Bacillus subtilis Bacillus subtilis Bacillus cereus Bacillus cereus Bacillus pumilus Bacillus velezensis Bacillus cereus Bacillus pumilus Enterobacter cloacae Bacillus cereus Xanthomonas sacchari Bacillus xiamenensis Enterobacter tabaci Bacillus cereus Bacillus subtilis Bacillus stratosphericus Total Positive (Nos) % Positive

NIBSM_OsR1 NIBSM_OsR2 NIBSM_OsR3 NIBSM _OsR4 NIBSM _OsR5 NIBSM_OsR6 NIBSM_OsR7 NIBSM_OsR8 NIBSM_OsR9 NIBSM_OsR10 NIBSM_OsR11 NIBSM_OsR12 NIBSM_OsR13 NIBSM_OsR14 NIBSM_OsR15 NIBSM_OsR16 NIBSM_OsS1 NIBSM_OsS2 NIBSM _OsS3 NIBSM _OsS4 NIBSM_OsS5 NIBSM_OsS6 NIBSM_OsS7 NIBSM_OsL1 NIBSM_OsL2 NIBSM_OsL3 NIBSM_OsL4 NIBSM_OsL5 NIBSM_OsG1 NIBSM_OsG2 NIBSM _OsG3 NIBSM_OsG4

E .coli (Ec)

S. typhimurium (St)

Fungal pathogens K. pneumoniae (Kp)

+

R. solani (Rs) +++ ++ +++ +++ ++ ++

S. rolfsii (Sr)

Fusarium verticillioides (Fv)

+++

++ + +

+ + ++

+++

++

+++

+++ +++ +++

+++ + + ++

+ + +++ + + +

+ +

++ +++ +++ +++

+

+++ ++ ++

+++ ++ +++

+ +

+

+ + ++ + + +

+

+ + 8 25

5 15.6

4 12.5

0 0

1 0.03

+ 17 53.1

++ +++

++

9 28.1

15 46.8

Abbreviations: X. oryzae: Xanthomonas oryzae pv. oryzae (Xoo), S. aureus: Stephyllococus aureus (Sa), E .coli: Escherichia coli, S. typhimurium: Salmonella typhimurium (St), K. pneumoniae: Klebsiella pneumoniae (Kp), S. rolfsii: Sclerotium rolfsii (SR), F. verticillioides: Fusarium verticillioides (Fv), R. solani: Rhizoctonia solani (Rs) *Inhibition zone : No activity, +: activity present, ++: moderate activity, +++: strong/high activity.

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S. aureus (Sa)

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Table 4 Antagonistic activities of bacterial endophytes against bacterial and fungal pathogens.

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Fig. 4. Antagonistic potential of rice bacterial endophytes against fungal pathogens. A: Fusarium verticilloides control. B: NIBSM_OsR15. C: NIBSM_R10. D: Rhizoctonia solani control. E: NIBSM_OsR15. F: NIBSM_OsS4. E: Sclerotium rolfsii control. F: NIBSM_OsR15. G: NIBSM_OsG2.

most abundant in the root samples in rice. Bacterial endophytic diversity in 12 wild species and cultivated rice identified 28 strains belonging to 13 genera (Elbeltagy et al., 2000). Actinobacterial

endophyte diversity in stem and root tissue of rice was reported and found that few actinobacterial populations of stem were correlated with those present in the root (Tian et al., 2007).

Fig. 5. Agarose gel showing amplification of lipopeptide genes. Gene-specific primers used for amplification: sfp: surfactin (675 bp), ituD- Iturin (647 bp), Bam C- Bacillomycin D gene (875 bp), M: 100 bp ladder; 1. Bacillus stratosphericus (NIBSM_OsG4), 2. B. subtilis (NIBSM_OsS1), 3. B. subtilis (NIBSM_OsS2), 4. B. subtilis (NIBSM OsG3), 5. B. pumilus (NIBSM_OsS5), 6. B. subtilis (NIBSM_OsR10).

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Diversity of endophytes in different crops has been reported and it was also recorded that within the same host plant bacterial endophytes are not limited to single species only but have several different genera and species living inside (Rosenblueth and MartinezRomero, 2006). Bacterial endophytes of the genera namely Bacillus species, Enterobacter, Pseudomonas, and Agrobacterium were most commonly isolated from the plants (Hallmann et al., 1997). A total of 28 isolates (87.5%) were catalase-positive having ability to convert hydrogen peroxide, the end product of various metabolites and toxin, thus assist in the providing protection to cell from oxidative damage caused by the reactive oxygen species. Majority of the isolates showed the ability to convert glucose into acidic end products like lactate, acetate and formate as revealed by Methyl Red (MR) positive and Voges Proskauer (VP) negative tests results. 4.1. Plant growth promoting (PGP) traits Seventeen isolates showed indole acetic acid (IAA) production ability in this study and the majority of them were isolated from root tissues. Bacterial endophytes have been reported to promote plant growth by various mechanisms viz. by the synthesis of phytohormones including Indole acetic acid (Mendes et al., 2007) by increasing the distribution and size of the roots thereby enhancing uptake of nutrient from the soil (Li et al., 2008). Klebsiella pneumoniae (NIBSM_OsR6) isolated from root tissues showed the production of IAA and phosphate solubilization activity. The results corroborated with findings of Fouts et al. (2008) that showed the involvement of Klebsiella endophyte in nitrogen fixation in the maize and wheat crops. In the present study, Leclercia adecarboxylata (NIBSM_OsR11) isolated from root tissue was found to produce phytohormone, IAA, siderophore and activities like nitrate reductase, urease. Phosphorous is a crucial macronutrient required for the growth and development of the plants, majority of (95 99%) phosphorous found in soil as insoluble organic and inorganic phosphates which are released into the soil solution very slowly (Mengel and Kirkby, 1978) that is not directly available to the plant. Phosphate solubilization has been considered an important trait by which bacteria can promote plant growth by enhancing availability and uptake of phosphate. PGPR utilize these insoluble forms of phosphates and convert them into soluble and available forms for plants (Shaikh et al., 2016). In this study, bacterial endophytes of genera, Bacillus and Enterobacter showed potential to solubilize phosphorous. The bacteria have the machinery to solubilize phosphate mineral (insoluble from) through reducing pH by the excretion of protons and organic acids (Gyaneshwar et al., 1999; Mullen, 2005). In the present study, 18 rice bacterial endophytes (56.2%) were urease positive and showed capability of converting urea to simpler forms which can easily be utilized by the host plant. Urease activity was also reported from rice endophytes (Tan et al., 2001). The results are in agreement with previous reports where Klebsiella, Enterobacter and Leclercia genera have been reported to have the capability for nitrogen fixation, phosphate solubilization and synthesis of phytohormones, production of siderophores and antimicrobial compounds (Melo et al., 2016). A bacterial strain, Enterobacter cloacae was reported to produce IAA in higher amount (Coulson and Patten, 2015). Bacillus group of microbes possess extra advantages over the other microbes due to endospore formation which can tolerate adverse conditions. Bacillus subtilis, Bacillus licheniformis and Bacillus pumilis are being utilized as bio-fertilizer and bio-control agents for controlling phytopathogens (Raaijmakers and Mazzola, 2012). Bacterial endophytes known to contain genes for the carbohydrate metabolism (central carbon metabolism), including glycolysis pathway, pyruvate oxidation pathways, pentose phosphate pathway, glyoxylate cycle and tricarboxylic acid cycle. The bacteria attached to the root have been reported to enhance growth and productivity in several crops though modulation in the concentration of soluble

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sugar (Paul and Lade, 2014). Due to the presence of the genes for metabolic pathways in bacterial endophytes have ability to metabolize carbohydrates and other carbon sources present in root exudates of the plant. The utilization of sucrose as carbon source by the bacteria in relationship with the plant has also been reported (Sprenger and Lengeler, 1988; Bogs and Geider, 2000). Sugar signaling have been reported for cellular adjustment to shifting environments and found to be essential in responses to diverse stimuli, more specifically to the status of carbohydrate of the plants (OHara et al., 2013). 4.2. Antibiotic sensitivity patterns Plant-associated soils (rhizosphere) harbor several microbes including bacteria and fungi which produce antibiotic with broadspectrum activities of specific to coexisting microbes. To verify the strength of isolates as a source of new antibiotics, bacterial endophytic isolates were screened for their antibiotics sensitivity profile using 14 broad-spectrum antibiotics showed that the Firmicutes were found to be resistant to beta-lactam antibiotics except vancomycin while maximum sensitivity was found for aminoglycosides followed by fluoroquinolones. Among proteobacteria, maximum were resistant to beta lactam group except ceftriaxone and polymyxin B maximum sensitivity was found for aminoglycosides followed by fluoroquinolones. The antibiotic sensitivity pattern of bacterial endophytes isolated from different tissues revealed that all the bacterial isolates from root tissues showed resistance to methicillin, whereas except for E. cloaceae (NIBSM_OsR8), all root isolates showed susceptibility to gentamicin. The characteristics of antibiotics resistance in bacterial endophytes reported from Andrographis paniculata (Arunachalam and Gayathri, 2010), Paederia foetida (Pal et al., 2012), Curcuma longa (Kumar et al., 2016) and actinomycetes (Passari et al., 2015). The biocontrol bacteria identified till date represented diverse genera, including Bacillus, Agrobacterium, Pantoea, Burkholderia and Serratia known to produce numerous antibiotics with overlapping or diverse spectrum of activities against pathogenic fungi and many of them exhibiting broad-spectrum activity (Raaijmakers and Mazzola, 2012). In addition, the functional and ecological importance of the presence of antibiotics implicit to provide a survival benefit to the bacteria known to produce antibiotics in the highly competitive but resource-limited rhizospheric conditions (Raaijmakers and Mazzola, 2012). The presence of ampicillin resistance in Enterobacter provides an added advantage to compete with coexisting microorganisms in the rhizosphere and soil environments. 4.3. Antagonistic activities Bacterial endophytes have the ability to prevent or reduce or the harmful effects of certain pathogenic microorganisms. The bacterial endophytes providing beneficial effects to their host plant by involving diverse mechanisms including antibiosis, growth-promotion, induced resistance, competition, quorum sensing and parasitism (Amer and Utkhede, 2000; Collins and Jacobsen, 2003; Ryan et al., 2008; Jorjani et al., 2011; Mansoori et al., 2013). Bacterial endophytic microbes exhibit a combination of numerous bio-control properties/ mechanisms (Ongena et al., 2007). Our findings showed that some of the endophytic bacterial isolates possess both antibacterial and antifungal activities against multiple pathogens. In this study, 15 Bacillus isolates were found to exhibit antagonism against bacterial and/or fungal pathogens screened in dual culture assay. Among them, B. subtilis (NIBSM_OsS2) showed antagonistic activities against multiple bacterial pathogens such as Xanthomonas oryzae pv. oryzae (Xoo), S. aureus, E. coli and fungal pathogens namely, R. solani and F. verticillioides. Similarly, B. subtilis (NIBSM_OsR10), B. thermophilus (NIBSM_OsR15) isolates showed antagonism against bacterial pathogen, X. oryzae pv. oryzae (Xoo), and R. solani, S rolfsii and F. verticillioides whereas B. velezensis

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(NIBSM_OsS6) showed antagonism against X. oryzae pv. oryzae (Xoo), S. rolfsii, and F. verticillioides and B. stratosphericus (NIBSM_OsR1) found antagonistic to S. aureus and R. solani. It was found that the bacterial endophytes isolated from root and stem tissues showed a broad spectrum antimicrobial activities against several bacterial and fungal pathogens. Bacterial leaf blight disease is devastating and of high economic importance disease of rice worldwide. In the present study, eight endophytic isolates were identified which showed antagonistic activity against Xanthomonas, Bacillus subtilis (NIBSM_OsR10) showed broad-spectrum antagonistic activities for Xanthomonas and all three tested fungal pathogens. The presence of surfactin is a strong antibacterial and antifungal iturin and bacillomycin D genes were confirmed in the genome of Bacillus subtilis (NIBSM_PR10). In most of the previous reports, a combination of one or two lipopeptides has been reported from the Bacillus isolates having antagonistic activities Gond et al. (2015) reported that the presence of iturin D and fengycin D genes were responsible for antifungal activity in Bacillus. Our study showed that the isolate, NIBSM_PR10 have antibacterial and antifungal activities for multiple pathogens suggesting its possible defensive role in rice plant and this can further be deployed for development of bio-formulations. Bacillus species dominates among the bio-control agents due to their ability to form spore that allows them to grow and survive under various ecological stress conditions including protection against plant pathogens. Bacillus group of microbes also offers additional benefits to combat soil-borne fungal pathogens or under extreme adverse conditions. Bacillus is commonly found as endophyte in plants and these species possess genes for biosynthesis of auxin and production of siderophores which may play crucial role in providing antagonistic activities phytopathogens, synthesis of auxin and gibberellin phytoharmones and stimulation of plant growth (Forchetti et al., 2007; Chaudhry et al., 2017).The members of Bacillus genera known to exhibit antifungal property which may be due the production of lipopeptides (Ongena and Jacques 2008). Other bacterial endophytes of Enterobacteraceae family were also found to have antifungal and antibacterial properties (Table 4). The bacterial endophytes of Enterobacteriaceae family namely Enterobacter, Leclercia and Klebsiella have been reported in several crop species and they have shown as nitrogen-fixers, phosphate-solubilizers, and production of various antimicrobial compounds, siderophores and phytohormones (Melo et al., 2016). Previous report by Tian et al. (2004) found that 50% of the most frequently isolated streptomycetes were antagonistic to rice pathogens (Xanthomonas oryzae pv. oryzae, Magnaporthe grisea, Rhizoctonia solani and F. moniliforme). Siderophores known to play an important role in biological control of pathogens by removing or limiting the availability of iron from the media and enhancing or ensuring its availability to the plant and modulation plant immunity by triggering induced systemic resistance (ISR) by plant growth-promoting bacteria (Aznar and Dellagi, 2015). Siderophore producing isolates were identified in this study and these isolates may play a crucial role in promoting plant development in iron limiting conditions and controlling the phytopathogens. These microbes hold potential to be explored for bio-control properties for combating multiple bacterial and fungal pathogens. However, the isolates showing strong antifungal activities against the specific pathogens can be explored for the bio-control of respective pathogens as an alternative approach to the chemical pesticides. In this study, bacterial endophytic isolates exhibited strong antagonistic activities against the important fungal pathogens S. rolfsii, R. solani and F. verticillioides. It is assumed that the antifungal activity of the isolates may be due to secretions like production of lytic enzymes, chitinase, lipopeptides and production of certain antibiotics. Bacillus spp. has been reported to produce various lytic enzymes including chitinases which degrades chitin, a key component of the fungal cell wall (Kumar et al., 2012; Castillo et al., 2013).

The Antimicrobial peptides (AMPs) or lipopeptides are an important and novel class of potent versatile metabolites to manage or control various types of plant pathogenic microbes (Ongena and Jacques, 2008; Fira et al., 2018). To confirm the presence of lipopeptide genes in the bacterial endophytes having antagonistic activities PCR based detection was conducted. It was found that the two B. subtilis isolates from stem tissues showed the presence of surfactin gene. While B. subtilis isolated from root tissues showed the presence of Iturin D and Bacillomycin D gene. The endophytic bacterial isolates showed the presence of Iturin and Bacillomycin gene have been found to produce strong antifungal and antibacterial properties against tested pathogens (Ongena et al., 2007; Ongena and Jacques, 2008). The lipopeptides appear to be promising biopesticides to and reduce the use of chemical pesticide resulting overcoming the increasing chemical resistance in agriculture Bacillus species including B. subtilis, B. pumilis, B. licheniformis and B. amyloliquefaciens are being exploited for biological control of plant diseases and plant growth promotion activities (Raaijmakers and Mazzola, 2012). Endophytes have been reported to have the ability to accelerate the emergence of seedling, inhibition of growth of plant pathogens, plant growth promotion activities thus, they are considered as more appropriate as biological control agents (Ryan et al., 2008; Santoyo et al., 2016; Eljounaidi et al., 2016). Functional and plant growth- promoting traits such as production of indole acetic acid, siderophores, HCN, ammonia, nitrogen fixation and phosphate solubilization are the major activities of interest for the application of bacterial endophytes in crops namely maize, rice and tomato (Szilagyi-Zecchin et al., 2014; Hameed et al., 2015; Upreti and Thomas, 2015).

5. Conclusion In the present study, bacterial endophytes were found in all the plant tissues/ parts of rice with a considerably higher density in the roots and leaf. The present investigation demonstrated that the bacterial isolates have the potential to be employed as plant growth-promoters as well as can be used in biological control against pathogens. Bacterial endophytes have been identified which have broad range of antagonistic activities against bacterial and fungal pathogens. Additionally, it emerges as an attractive opportunity for further molecular studies on host plant endophyte pathogen interactions, identification and isolation of antimicrobial genes and novel antimicrobial compounds. The study reported antifungal and antibacterial activity of bacterial endophytes against different phytopathogens by direct dual culture assay. This indicates that one or more antifungal compounds are produced by these bacterial isolates that require further analysis to characterize them at an individual level. These endophytes can be further explored for their potential role in enhanced crop nutrition and management of different biotic stresses, leading to higher crop productivity through low cost input.

Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgment The authors are thankful to the Director, ICAR- National Institute of Biotic Stress Management, Raipur for providing necessary facilities for the conducting this research work. This manuscript is contribution ICAR-NIBSM/RP-23/2019-5/35 from ICAR-NIBSM, Raipur.

Please cite this article as: <.t.i.O.p.g.a.d.-h.-n.y. Kumar et al., Bacterial endophytes of rice (Oryza sativa L.) and their potential for plant growth promotion and antagonistic activities, South African Journal of Botany (2020), https://doi.org/10.1016/j.sajb.2020.02.017

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Supplementary materials Supplementary material associated with this article can be found in the online version at doi:10.1016/j.sajb.2020.02.017.

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Please cite this article as: <.t.i.O.p.g.a.d.-h.-n.y. Kumar et al., Bacterial endophytes of rice (Oryza sativa L.) and their potential for plant growth promotion and antagonistic activities, South African Journal of Botany (2020), https://doi.org/10.1016/j.sajb.2020.02.017