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References

  1. Thavong P. 2002. Effect of dirty panicle disease on rice seed vigor. Agric. Res. J. 20: 111-120.
  2. Chen D, Liu X, Li C, Tian W, Shen Q, Shen B. 2014. Isolation of Bacillus amyloliquefaciens S20 and its application in control of eggplant bacterial wilt. J. Environ. Manage 137: 120-127.
    Pubmed CrossRef
  3. Pathak KV, Keharia H. 2013. Characterization of fungal antagonistic bacilli isolated from aerial roots of banyan (Ficus benghalensis) using intact-cell MALDI-TOF mass spectrometry (ICMS). J. Appl. Microbiol. 114: 1300-1310.
    Pubmed CrossRef
  4. Alvarez F, Castro M, Príncipe A, Borioli G, Fischer S, Mori G, et al. 2011. The plant-associated Bacillus amyloliquefaciens strains MEP218 and ARP23 capable of producing the cyclic lipopeptides iturin or surfactin and fengycin are effective in biocontrol of sclerotinia stem rot disease. J. Appl. Microbiol. 112: 159-174.
    Pubmed CrossRef
  5. Zhao Y, Selvaraj JN, Xing F, Zhou L, Wang Y, Song H, et al. 2014. Antagonistic action of Bacillus subtilis strain SG6 on Fusarium graminearum. PLoS One 9: e92486.
    Pubmed PMC CrossRef
  6. Raaijmakers JM, Vlami M, de Souza JT. 2002. Antibiotic production by bacterial biocontrol agents. Antonievan Leeuwenhoek 81: 537-547.
    Pubmed CrossRef
  7. Ahmad F, Ahmad I, Khan MS. 2008. Screening of free-living rhizospheric bacteria for their multiple plant growthpromoting activities. Microbiol. Res. 163: 173-181.
    Pubmed CrossRef
  8. Ait Kaki A, KacemChaouche N, Dehimat L, Milet A, Youcef-Ali M, Ongena M, et al. 2013. Biocontrol and plant growth promotion characterization of Bacillus species isolated from Calendula officinalis rhizosphere. Indian J. Microbiol. 53: 447-452.
    Pubmed PMC CrossRef
  9. Souza Rd, Ambrosini A, Passaglia LMP. 2015. Plant growthpromoting bacteria as inoculants in agricultural soils. Genet. Mol. Biol. 38: 401-419.
    Pubmed PMC CrossRef
  10. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. 2011. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28: 2731-2739.
    Pubmed PMC CrossRef
  11. Mora I, Cabrefiga J, Montesinos E. 2015. Cyclic lipopeptide biosynthetic genes and products, and inhibitory activity of plant-associated Bacillus against phytopathogenic bacteria. PLoS One 10: e0127738.
    Pubmed PMC CrossRef
  12. Huang X, Lu Z, Bie X, Lü F, Zhao H, Yang S. 2007. Optimization of inactivation of endospores of Bacillus cereus by antimicrobial lipopeptides from Bacillus subtilis fmbj strains using a response surface method. Appl. Microbiol. Biotechnol. 74: 454-461.
    Pubmed CrossRef
  13. Loper JE, Schroth MN. 1986. Influence of bacterial sources of indole-3-acetic acid on root elongation of sugar beet. Physiol. Biochem. 76: 386-389.
    CrossRef
  14. Ruangsanka S. 2014. Identification of phosphate-solubilizing fungi from the asparagus rhizosphere as antagonists of the root and crown rot pathogen Fusarium oxysporum. ScienceAsia 40: 16-20.
    CrossRef
  15. Cappuccino JC, Sherman N. 1992, pp. 125-179. Microbiology: A Laboratory Manual, 3th Ed. Benjamin/cummings, New York.
  16. Tang J-S, Gao H, Hong K, Yu Y, Jiang M-M, Lin H-P, et al. 2007. Complete assignments of 1H and 13C NMR spectral data of nine surfactin isomers. Magn. Reson. Chem. 45: 792-796.
    Pubmed CrossRef
  17. Garbay-Jaureguiberry C, Roques BP, Delcambe L, Peypoux F, Michel G. 1978. NMR conformational study of iturin A, an antibiotic from Bacillus subtilis. FEBS. Lett. 93: 151-156.
    Pubmed CrossRef
  18. Yang H, Li X, Li X, Yu H, Shen Z. 2015. Identification of lipopeptide isomers by MALDI-TOF-MS/MS based on the simultaneous purification of iturin, fengycin, and surfactin by RP-HPLC. Anal. Bioanal. Chem. 407: 2529-2542.
    CrossRef
  19. Yu GY, Sinclair JB, Hartman GL, Bertagnolli BL. 2002. Production of iturin A by Bacillus amyloliquefaciens suppressing Rhizoctonia solani. Soil Biol. Biochem. 34: 955-963.
    CrossRef
  20. Dunlap CA, Schisler DA, Price NP, Vaughn SF. 2011. Cyclic lipopeptide profile of three Bacillus subtilis strains; antagonists of Fusarium head blight. J. Microbiol. 49: 603-609.
    Pubmed CrossRef
  21. Singh AK, Rautela R, Cameotra SS. 2014. Substrate dependent in vitro antifungal activity of Bacillus sp. strain AR2. Microb. Cell Fact. 13: 67-67.
    Pubmed PMC CrossRef
  22. Hermann A, Guenther W, Guenther J. 1984. Iturin AL: structure and derivatives of a peptidolipid with a high content of C16-iturinic acids, Liebigs Ann. Chem. 5: 854-866.
  23. Akira I, Seiji T, Shigeo M, Akinori S. 1982. Structures of β-amino acids in antibiotics iturin A. Tetrahedron Lett. 23: 3065-3068.
    CrossRef
  24. Moran S, Rai DK, Clark BR, Murphy CD. 2009. Precursordirected biosynthesis of fluorinated iturin A in Bacillus spp. Org. Biomol. Chem. 7: 644-646.
    Pubmed CrossRef
  25. Ji SH, Paul NC, Deng JX, Kim YS, Yun B-S, Yu SH. 2013. Biocontrol activity of Bacillus amyloliquefaciens CNU114001 against fungal plant diseases. Mycobiology 41: 234-242.
    Pubmed PMC CrossRef
  26. Arguelles-Arias A, Ongena M, Halimi B, Lara Y, Brans A, Joris B, et al. 2009. Bacillus amyloliquefaciens GA1 as a source of potent antibiotics and other secondary metabolites for biocontrol of plant pathogens. Microb. Cell Fact. 8: 63.
    Pubmed PMC CrossRef
  27. Torres MJ, Brandan CP, Petroselli G, Erra-Balsells R, Audisio MC. 2016. Audisio MC. 2016. Antagonistic effects of Bacillus subtilis subsp. subtilis and B. amyloliquefaciens against Macrophomina phaseolina: SEM study of fungal changes and UV-MALDI-TOF MS analysis of their bioactive compounds. Microbiol. Res. 182: 31-39.
    CrossRef
  28. Li B, Li Q, Xu Z, Zhang N, Shen Q, Zhang R. 2014. Responses of beneficial Bacillus amyloliquefaciens SQR9 to different soil borne fungal pathogens through the alteration of antifungal compounds production. Front. Microbio. 5: 1-10.
    Pubmed PMC CrossRef
  29. Liu X, Ren B, Gao H, Liu M, Dai H, Song F, et al. 2012. Optimization for the production of surfactin with a new synergistic antifungal activity. PLoS One 7: e34430.
    Pubmed PMC CrossRef
  30. Islam MR, Jeong YT, Lee YS, Song CH. 2012. Isolation and identification of antifungal compounds from Bacillus subtilis C9 inhibiting the growth of plant pathogenic fungi. Mycobiology 40: 59-66.
    Pubmed PMC CrossRef
  31. Nakano MM, Marahiel MA, Zuber P. 1988. Identification of a genetic locus required for biosynthesis of the lipopeptide antibiotic surfactin in Bacillus subtilis. J. Bacteriol. 170: 5662-5668.
    Pubmed PMC CrossRef
  32. Tapi A, Chollet-Imbert M, Scherens B, Jacques P. 2010. New approach for the detection of non-ribosomal peptide synthetase genes in Bacillus strains by polymerase chain reaction. Appl. Microbiol. Biotechnol. 85: 1521-1531.
    Pubmed CrossRef
  33. Vessey JK. 2003. Plant growth-promoting rhizobacteria as biofertilizers. Plant Soil 255: 571-586.
    CrossRef
  34. Islam S, Akanda AM, Prova A, Islam MT, Hossain MM. 2016. Isolation and identification of plant growth-promoting rhizobacteria from cucumber rhizosphere and their effect on plant growth promotion and disease suppression. Front. Microbio. 60: 1360.
    Pubmed PMC CrossRef
  35. Khalid A, Arshad M, Zahir ZA. 2004. Screening plant growth-promoting rhizobacteria for improving growth and yield of wheat. J. Appl. Microbiol. 96: 473-480.
    Pubmed CrossRef

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Article

Research article

J. Microbiol. Biotechnol. 2018; 28(9): 1527-1535

Published online September 28, 2018 https://doi.org/10.4014/jmb.1804.04025

Copyright © The Korean Society for Microbiology and Biotechnology.

Antagonistic Activity against Dirty Panicle Rice Fungal Pathogens and Plant Growth-Promoting Activity of Bacillus amyloliquefaciens BAS23

Sukanya Saechow 1, Anon Thammasittirong 1, 2, Prasat Kittakoop 3, 4, 5, Surasak Prachya 4 and Sutticha Na-Ranong Thammasittirong 1, 2*

1Department of Microbiology, Faculty of Liberal Arts and Science, Kasetsart University KamphaengSaen Campus, Nakhon Pathom 73140, Thailand , 2Microbial Biotechnology Unit, Faculty of Liberal Arts and Science, Kasetsart University,KamphaengSaen Campus, Nakhon Pathom 73140, Thailand, 3Chulabhorn Graduate Institute, Chemical Biology Program, Chulabhorn Royal Academy, KamphaengPhet 6 Road, Laksi, Bangkok 10210, Thailand , 4Chulabhorn Research Institute, KamphaengPhet 6 Road, Laksi, Bangkok 10210, Thailand , 5Center of Excellence on Environmental Health and Toxicology (EHT), CHE, Ministry of Education, Bangkok, Thailand

Received: April 16, 2018; Accepted: July 11, 2018

Abstract

Bacterial strain BAS23 was isolated from rice field soil and identified as Bacillus amyloliquefaciens. Based on dual culture method results, the bacterium BAS23 exhibited potent in vitro inhibitory activity on mycelial growth against a broad range of dirty panicle fungal pathogens of rice(Curvularialunata, Fusarium semitectum and Helminthosporiumoryzae). Cell-free culture of BAS23 displayed a significant effect on germ tube elongation and mycelial growth. The highest dry weight reduction (%) values of C. lunata, H. oryzaeand F. semitectum were 92.7%, 75.7% and 68.9%, respectively. Analysis of electrospray ionization-mass spectrometry (ESI-MS) and 1H nuclear magnetic resonance (NMR) spectroscopy revealed that the lipopeptides were iturin A with a C14 side chain (C14 iturinic acid) and with a C15 side chain (C15 iturinic acid), which were produced by BAS23 when it was cultured in nutrient broth (NB) for 72 h at 30°C.BAS23, the efficient antagonistic bacterium, also possessed in vitromultipletraits for plant growth promotionand improved rice seedling growth. The results indicated that B.amyloliquefaciens BAS23 represents a useful option either for biocontrol or as a plant growthpromoting agent.

Keywords: Bacillus amyloliquefaciens, Biocontrol, Lipopeptides, Plant growth promoting, Rice dirty panicle

Introduction

Rice (Oryzae sativa L.) is a major commercial crop and an important main food source for Thai people and worldwide. Dirty panicle disease is one of the most serious diseases of rice, causing yield losses and reduced seed quality such as the germination percentage [1]. Dirty panicle disease is caused by several pathogenic fungi, for example, Helminthosporium oryzae, Curvularia lunata and Fusarium semitectum. These fungal pathogens usually infect rice plants at the panicle-forming stage and after harvest.

The application of agrochemicals remains the primary method to control fungal diseases. The use of synthetic chemicals has recently raised concern due to their negative effects on the environment and human health, so that alternative and more sustainable strategies are required. Biological control is an environmentally friendly strategy for dealing with plant pathogens and shows promise in replacing or reducing the use of synthetic chemical compounds. Most of the species from the genus Bacillus strains are considered to be safe microorganisms and they possess several advantages that make them useful as biological control agents [2, 3]. These endospore-forming bacteria hold the remarkable ability of synthesizing antagonistic compounds such as iturin, fengycin and surfactin, which show antibacterial or antifungal activity against different phytopathogens [4, 5]. The production of endospores by these bacteria allows them to survive for extended periods under adverse environmental conditions and permits easy formulation for commercial purposes [6]. In the last few years, there are reports demonstrating that various bacterial species, including Bacillus, could act as biocontrol agents and plant growth-promoting agents [7, 8]. Plant growth-promoting bacteria are known to enhance plant growth by a variety of mechanisms; for example, by the production of phytohormones (indole-3-acetic acid (IAA), gibberellic acid and ethylene), nitrogen fixation and denitrification, phosphate solubilization, 1-aminocyclopropane-1-carboxylate deaminase activity and production of siderophore [9].

In this study, we performed the screening of bacteria isolated from soil as potential antagonists against the dirty panicle disease fungal pathogens of rice. The effect of the antagonistic bacterial strain and its extracellular metabolites on the pathogenic fungi were studied in vitro using various methods. The antifungal compounds produced by BAS23 were isolated using reversed-phase high performance liquid chromatography (RP-HPLC) and analyzed using electrospray ionization-mass spectrometry (ESI–MS) and nuclear magnetic resonance spectroscopy (1H NMR). In addition, plant growth-promoting activity and its effect on root and shoot lengths of rice in vitro were also reported. To our knowledge, this is the first report of this antagonistic bacterium displaying broad spectrum activities against the dirty panicle rice fungal pathogens, C. lunata, H. oryzae and F. semitectum, and also promoting rice seedling growth.

Materials and Methods

Phytopathogenic Fungi

The pathogenic fungi, C. lunata PFR12, F. semitectum PFR5, and H. oryzae PFR3, were obtained from the Plant Health Clinic, Kasetsart University Kamphaeng Saen Campus, Nakhon Pathom, Thailand. The fungi were maintained at 4°C on potato dextrose agar (PDA) medium.

Isolation and Identification of Bacteria

Soil bacteria were isolated from rice paddy fields in Nakhon Pathom province, Thailand. Soil samples (10 g each) were added to 90 ml of sterile deionized water and mixed for 5 min. Serial dilutions were made and plated on nutrient agar (NA) and incubated at 37°C for 2 days. Bacterial colonies were purified by repeated streaking on new NA plates.The bacterial strain BAS23 was identified based on its biochemical and physiological characteristics and on 16S rDNA analysis using the universal primers 27F and 1492R. Homology blast search was performed against the database and deposited in GenBank (Accession No. KY433354.1).

Phylogenetic Analysis

Multiple sequence alignments were generated with ClustalW. A phylogenetic tree was carried out on the basis of neighbor-joining method with 1,000 bootstrap replicates, using MEGA 5.0 program [10].

In Vitro Screening of Antagonistic Bacteria

The dual culture method was used to evaluate the antifungal activity of antagonist bacteria. A 5 mm plug of 5-day-old culture of each fungal pathogen was placed 2.5 cm away from the edge in a Petri dish containing PDA and incubated at 30°C for 1 day. A loopful of bacteria was streaked 4.5 cm away from the plug of the pathogenic fungus on the same Petri dish and incubated at 30°C for 10 days. The percentage of inhibition of radial growth (PIRG) was calculated using the following formula:

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. Experiments were performed in triplicate.

Effect of BAS23 on Fungal Hyphal Morphology

The hyphal morphology of mycelial growth at the edge of the fungal colony nearest to the inhibition zone after incubation for 7 days was studied using a light microscope (Olympus CX31, Japan) and a scanning electron microscope (SEM; Quanta 400, Thermo Fisher Scientific, USA) with an operating voltage of 10 kV.

Effect of Cell-Free Culture on Mycelial Growth and Spore Germination

Mycelial growth inhibition assay was performed using the dry weight determination method. The overnight culture of BAS23, growing on NA at 30°C, was seeded into 15 ml of nutrient broth (NB) and incubated at 30°C with shaking at 150 rpm for 24 h. The optical density (OD600) of BAS23 culture was adjusted to 0.5 and then 1% of the adjusted culture was inoculated into 100 ml of NB and incubated at 30°C with shaking at 150 rpm for 72 h. Cell-free culture of BAS23 was obtained by centrifugation at 29,581 ×g for 10 min at 4°C. The cell-free culture was filtered aseptically and then added to 100 ml of potato dextrose broth (PDB) containing spore suspension (1 × 106 spores/ml) of each fungal pathogen to yield final concentrations of 5%, 10%, 15%, 20%, and 30%. PDB medium without cell-free culture was used as the control. After culture at 30°C with shaking at 150 rpm for 7 days, the mycelia were filtered and dried at 55°C for 5-7 days until constant reaching a weight.

Detection of Antimicrobial Lipopeptide Genes

BAS23 was characterized using the specific primers of four lipopeptide biosynthetic gene markers, srfAA (surfactin), fenD (fengycin), bmyB (bacillomycin), and ituC (iturin A synthetase C)[11]. The PCR reaction volume was 25 μl and included 0.2 mM dNTP (Vivantis Technologies, Malaysia), 0.2 μM of each primer, 1×PCR buffer, 2.0 mM MgCl2, 2.0 U of DreamTaq DNA polymerase (Thermo Scientific, USA) and 1 μl of genomic DNA. Lipopeptide amplification was performed with an initial denaturation for 4 min at 95ºC, followed by 25 cycles of denaturation (1 min at 94ºC), annealing (1 min at 55ºC for bmyB and 58 ºC for fenD, ituC, and srfAA) and extension (1 min at 70ºC). A final extension step at 70ºC for 5 min was followed by a 4ºC soak. These sequences were compared with available lipopeptide sequence genes in the GenBank database using the BLAST tool (https://www.ncbi.nlm.gov/BLAST/).

Preparation and Analysis of Lipopeptides

Crude extract containing lipopeptides was prepared using a slightly modified method from that described by Chen et al. [2] and Huang et al. [12]. BAS23 was grown in NB medium at 30°C for 72 h; the culture broth was centrifuged at 2,103 ×g for 20 min. Lipopeptides were precipitated by adding 6.0 M HCl to a final pH of 2 and stored overnight at 4°C. The precipitate was collected using centrifugation at 2,103 ×g for 20 min, washed twice with deionized water (adjusted to pH 2.0 with 6.0 M HCl) and extracted three times using methanol. The combined extracts were evaporated at 30°C under vacuum to dryness in a rotary evaporator to yield a pale-yellow extract of lipopeptides. Lipopeptides were isolated using RP-HPLC and analyzed using ESI-MS and 1H NMR spectra. Crude extract (157.0 mg) of BAS23 was separated using RP-HPLC with a C8 reversed-phase column (Sepax GP-C8, 21.2 mm × 250 mm, particle size 5 μm). The RP-HPLC product was eluted with 65% MeOH:H2O, using a flow rate of 12 ml/min (UV detector set at 225 nm). Two lipopeptides, iturin A with the C14 and C15 side chains, were eluted at the retention times of 38.1 min and 54.2 min, respectively.

Indole-3-Acetic Acid Production

Indole-3-acetic acid (IAA) production was performed following the method of Loper and Schroth [13]. A 2 ml supernatant of the BAS23 culture, which had been grown in NB containing 500 μg/ml of L-tryptophan at 30°C and 150 rpm for 48 h, was mixed with two drops of orthophosphoric acid and 4 ml of Salkowski reagent (50 ml, 35% of perchloric acid, 1 ml of 0.5 M FeCl3 solution). The mixtures were incubated at room temperature for 30 min. The absorbance of the developing pink color was measured at 530 nm using a UV-Vis spectrophotometer (GENESYS 10S UV-Vis, Thermo Scientific, USA). Pure IAA (Sigma-Aldrich Co, USA) was used as the standard.

Phosphate Solubilization

The quantitative bioassay was carried out in Erlenmeyer flasks (125 ml) containing 20 ml of National Botanical Research Institute’s phosphate growth medium (NBRIP) supplemented with Ca3(PO4)2 and inoculated with 1 ml of BAS23 culture. Bacterial cultures were grown for 7, 14, and 21 days at 30°C on a shaker at 150 rpm. The cultures were harvested using centrifugation at 29,581 ×g for 10min at 4°C. The supernatant was then filtered through a 0.45 μm membrane and the amount of soluble phosphate was measured following the ascorbic acid method. A reagent (0.48 ml) containing 5 M H2SO4, antimony potassium tartrate, ammonium molybdate and 0.1 M ascorbic acid was added to the supernatant filtrate (3 ml). The samples were incubated at room temperature for 20 min and their absorbance measured at 880 nm using the UV-Vis spectrophotometer. The amount of soluble phosphate was determined using a potassium dihydrogen phosphate standard [14].

Ammonia Production

BAS23 was cultured in 10 ml of peptone water and incubated at 30°C for 48-72 h. The ammonia production was detected by adding 0.5 ml/tube of Nessler’s reagent. The appearance of a brown-to-yellow color was a positive test for ammonia production [15].

Catalase Test

BAS23 was cultivated overnight and a small amount of bacterial colony was then smeared on a clean glass slide and treated with one drop of 3% H2O2. The formation of gas bubbles within a few seconds indicated catalase activity.

Rice Seed Treatment and Germination

Rice seeds (cultivar Suphanburi 1, a high-yielding, good-quality grain cultivar, widely cultivated in central Thailand) were soaked in distilled water for 15 h, sterilized in 10% clorox for 30 min and washed five times with sterilized distilled water. The surface sterilized seeds were soaked for 1 h in cell-suspension. The seeds soaked in sterilized distilled water were used as the control. A sample of incubated seeds (20 seeds) was blotted dry and then placed on wet blotters and incubated in a growth chamber. The percentage of seed germination and shoot and root lengths were measured every 12 h for 5 days. All experiments were performed in triplicate with five replications.

Results

In Vitro Screening of Antagonistic Bacteria and Identification of Bacterial Antagonist

Sixty bacterial isolates obtained from the rice field soil samples were investigated using the dual culture method for their antifungal activities to inhibit the mycelial growth of dirty panicle fungal pathogens of rice, C. lunata, F. semitectum, and H. oryzae. In total, 13 isolates showed antagonistic activity against a broad range of rice fungal pathogens. Notably, BAS23 showed the highest inhibitory activity against these pathogenic fungi (Table S1 and Fig. S1) and was selected for further studies. BAS23 is Gram-positive, catalase-positive, endospore-forming, rod-shaped and motile. The results of morphological, biochemical, 16S rDNA sequence analysis and phylogenetic analysis supported that BAS23 (GenBank Accession No. KY433354.1) was B. amyloliquefaciens (Fig. 1).

Figure 1. Phylogenetic tree of B. amyloliquefaciens BAS23 based on 16S rRNA sequence analysis.

Effect of BAS23 on Hyphal Morphology of Pathogenic Fungi

The effect of BAS23 on hyphal morphology was examined using microscopy as shown in Fig. 2. Microscopic observations of C. lunata and H. oryzae hyphae from the margins of the inhibition zones showed swelling and bulb formation (Figs. 2A and 2B). Morphological changes in F. semitectum hyphae treated with BAS23 were not observed under light microscopy; however, the SEM micrograph showed hyphal swelling (Fig. 2C). Otherwise, the hyphae of the three fungal pathogens from the untreated control were intact and elongated with smooth surfaces (Figs. 2D-2F).

Figure 2. Effect of BAS23 on the hyphal morphology of C. lunata (A), H. oryzae (B), and F. semitectum (C) co-inoculated with BAS23 and normal hyphae of C. lunata (D) H. oryzae (E) and F. semitectum (F). Arrows indicate swollen and bulb structures.

Effect of BAS23 Cell-Free Culture on Fungal Growth

The inhibitory activity on fungal growth by cell-free culture (5%, 10%, 15%, 20%, and 30%) of BAS23 was determined using the dry weight method. As shown in Table 1, the extracellular metabolites in the cell-free culture of BAS23 inhibited the growth of the three fungal pathogens. Based on the dry weight reduction (%), cell-free culture of BAS23 showed the best inhibitory effect on C. lunata growth, followed by H. oryzae and F. semitectum, respectively. The degree of fungal growth inhibition corresponded to the increase in the concentration of the cell-free culture. BAS23 cell-free culture also affected the germ tube elongation of the fungal pathogens. The germ tube of C. lunata showed abnormal swelling and could not extend forward, especially in the presence of 30% cell-free culture (Fig. 3A). In the presence of 5-30% cell-free culture, germ tubes of H. oryzae and F. semitectum could elongate but the hyphae showed abnormal swelling and bulb formation, especially at 30%(Figs. 3B and 3C). It was noted that spores of untreated pathogenic fungi germinated normally and the germ tubes developed into long hyphal strands (Figs. 3D-3F). These results indicated that cell-free culture of BAS23 displayed antifungal activity against dirty panicle disease pathogenic fungi of rice.

Table 1 . Effect of BAS23 cell-free culture on dry weight of C. lunata, F. semitectum, and H. oryzae..

Concentration of cell-free culture (%)Dry weight reduction (%)

C. lunataF. semitectumH. oryzae
581.2±0.7c53.8±0.6c54.9±0.3e
1087.6±1.2b59.1±0.5b63.9±0.3d
1590.7±0.9a66.1±1.7a69.8±0.5c
2091.7±0.1a67.5±1.3a72.3±1.6b
3092.7±0.2a68.9±0.8a75.7±0.1a

Data represent the mean ± standard deviation. Means in each column with the same lowercase superscript letter are not significantly different (p < 0.05 )..



Figure 3. Effect of BAS23 cell-free culture (30%) on spore germination and germ tube elongation of pathogenic fungi C. lunata (A), H. oryzae (B) and F. semitectum (C) and untreated spores of C. lunata (D), H. oryzae (E) and F. semitectum (F).

Analysis of Lipopeptides

The presence of lipopeptide genes in BAS23 was determined using PCR with specific primers. A sequence analysis indicated that the fragment of lipopeptide gene markers was 99% similar to ituC (iturin A synthetase C), fenD (fengycin), and srfAA (surfactin), and 100% similar to bmyB (bacillomycin) (Table S2). These findings suggested that BAS23 was positive for the presence of lipopeptide genes. To further characterize the lipopeptide profiles of BAS23, antifungal substances were extracted from the supernatant, isolated using RP-HPLC and analyzed using ESI-MS and 1H NMR spectroscopy. Two lipopeptides were obtained after RP-HPLC separation, and the 1H NMR spectra (Figs. 4A and 4C) suggested that they were iturin A with C14 and C15 side chains [16, 17]. The 1H NMR spectrum of iturin A with the C14 side chain (Fig. 4A) showed characteristic signals of α-protons of amino acids in a peptide; these protons had 1H NMR resonances at a chemical shift (δ) of 3.70-4.70 ppm. Fig. 4A also showed signals of a methyl group (at δ 0.85 ppm) and a number of methylene protons (at δ 1.30 ppm) of the C14 fatty acid side chain in iturin A. Moreover, the 1H NMR spectrum in Fig. 4A showed signals of aromatic protons of tyrosine amino acid at δ 6.72 (doublet) ppm and 7.07 (doublet) ppm. The 1H NMR spectrum of iturin A with the C15 side chain (Fig. 4C) displayed characteristic signals for α-protons of amino acids in a peptide at δ 3.73-4.75 ppm, a side chain of fatty acid at δ 0.85 ppm (methyl group) and at 0.86 ppm (methyl protons), and aromatic protons of tyrosine at δ 6.71 ppm and 7.05 ppm (both doublet).

Figure 4. Identification of iturin A with C14 side chain using 1H NMR (in CD3OD) spectroscopy (A) and ESI-MS (B) and identification of iturin A with C15 side chain by 1H NMR (in CD3OD) spectroscopy (C) and ESI-MS (D).

Table 2 shows the observed m/z values in the ESI-MS mass spectra for the antifungal lipopeptides, iturin A with the C14 and C15 side chains, obtained from the literature. The positive ions typically observed in these experiments are the parent molecule plus a cation, such as, [M+H]+ or [M+Na]+. Intense signals in the m/z range 1,000-1,100 were obtained in the ESI-MS mass spectra of the crude lipopeptides. The mass peaks at m/z 1,065.5 and 1,079.5 were in accordance with the calculated mass values of sodium adducts of the C14 and C15 homologues of iturin A, respectively, while m/z 1,043.5 and 1,057.5 were the calculated mass values of the proton adduct of the C14 and C15 iturin A, respectively (Figs. 4B and 4D and Table 2). No peaks corresponding to the calculated mass of the various surfactin homologues, fengycin homologues or to those of bacillomycin homologues could be detected under these conditions. It is worth mentioning that C14 and C15 side chains of iturin A are also known as C14 iturinic acid and C15 iturinic acid, respectively [22-24]. However, there have been a number of articles that have published incorrect structures, particularly regarding the aliphatic side chain. The correct structures of iturin A with the C14 and C15 side chains are shown in Fig. 5.

Table 2 . Assignments of major m/z peaks observed in mass spectra of lipopeptides of iturin A with the C14 and C15 side chains, reported in the literature..

Typem/zAssignmentReferences
Iturin A1043.5iturin A C14 [M+H]+[2, 18-19]
1057.5iturin A C15 [M+H]+[3, 18]
1065.5iturin A C14 [M+Na]+[2, 18, 20-21]
1079.5iturin A C15 [M+Na]+[18, 20-21]


Figure 5. Structure of iturin A with C14 side chain (A) and C15 side chain (B).

Plant Growth-Promoting Traits of BAS23

BAS23 was evaluated for plant growth-promoting metabolite production. The results revealed that BAS23 was positive for multiple plant growth-promotion traits including IAA, ammonia and catalase productions (Table 3). For phosphate-solubilization, BAS23 was able to solubilize phosphate and showed maximum phosphate solubilization of 0.33 mg/l at 30°C for 21 days in NBRIP liquid medium, therefore a longer period of growth increased the phosphate-solubilizing activity.

Table 3 . In vitro production of plant growth-promoting metabolites..

IAA* at 500 µg/ml of tryptophan (µg/ml)Phosphate solubilization (mg/l)NH3Catalase

7 days14 days21 days
16.36±0.910.01±0.000.08±0.010.33±0.01++

*IAA: Indole-3-acetic acid.



Effect of BAS23 on Rice Seedling Growth

The effect of BAS23 on rice seedling growth was investigated. Compared with the control, rice seed treatment with cell-suspension of BAS23 resulted in enhanced root and shoot lengths by 40.64% and 39.06%, respectively, evaluated on 5-day-old rice seedlings (Fig. 6). There were no significant differences in seed germination between the control and treated seed with a cell-suspension of BAS23 (data not shown).

Figure 6. Efficacy of BAS23 on root and shoot lengths (A) and growth of rice seedlings (5 days after incubation) from treated and untreated seed with BAS23 (B).

Discussion

Members of Bacillus are often considered as important biological control agents [2, 3, 5, 25]. In this study, using the dual culture method, the most potent bacterial antagonistic isolate identified as Bacillus amyloliquefaciens BAS23 showed a strong antifungal activity against the dirty panicle rice fungal pathogens, C. lunata, F. semitectum, and H. oryzae. BAS23 induced morphological abnormalities, such as hyphal swelling and bulb formation, in the mycelia of all rice fungal pathogens.

Cell-free culture of BAS23 displayed an inhibitory effect on the mycelial growth of these dirty panicle rice fungal pathogens. The degree of fungal growth inhibition corresponded to an increased concentration of the cell-free culture. The highest dry weight reduction values of C. lunata, H. oryzae, and F. semitectum using 30% cell-free culture were 92.7%, 75.7%, and 68.9%, respectively. In addition, inhibitory germ tube elongation of C. lunata spore was observed with cell-free culture of BAS23, while the germ tubes of H. oryzae and F. semitectum could elongate but the hyphae showed abnormal swelling and bulb formation. These results revealed that certain extracellular metabolites generated by BAS23 affected mycelial growth and germ tube elongation. Some Bacillus species are known to produce numerous antimicrobial compounds which have been well characterized genetically and biochemically in vitro [5, 26]. Among these antagonistic compounds, the predominant lipopeptides of the surfactin, iturin and fengycin families have been well reported for their potential against a wide range of plant pathogens [3, 4, 27]. The presence of lipopeptide genes was related to antimicrobial activity in several Bacillus spp. [4, 5, 11]. In the current study, genes involved in three families of lipopeptides, surfactin family, iturin family and fengycin family, were detected in BAS23 based on PCR experimentation. Regarding the ESI-MS and 1H NMR spectroscopic analysis, iturin A with the C14 and C15 side chains was only detected in crude lipopeptides extracted from BAS23 when it was cultured in NB for 72 h at 30°C. Similar results were obtained by Alvarez et al. [4] showing that the peptide synthetase genes for bacillomycin in B. amyloliquefaciens ARP23, and fengycin and surfactin in B. amyloliquefaciens MEP218, were detectable using PCR, but these lipopeptides were not detected in the culture supernatants from these strains. According to the literature, mutation of an essential gene, in appropriate culture conditions, the aggregation state of the culture medium where the bacterial cells developed or interaction between the bacterial antagonist and fungal pathogen have been reported as influencing factors for lipopeptide production [27-32]. Iturin is known for its strong antifungal property and hemolytic activity [4, 11]. Therefore, iturin production would be important for the efficiency of rice fungal pathogen control using BAS23.

In addition to the potential antagonistic effects against rice fungal pathogens, BAS23 also displayed multiple plant growth-promoting traits, such as IAA, ammonia and catalase productions and phosphate solubilization. In the current study, a seedling growth experiment was performed in vitro to avoid any interference from environmental factors. Rice seed bacterization with BAS23 exhibited significant increases in root and shoot lengths compared with the untreated control. IAA is a well-known growth hormone that enhances plant growth by stimulating plant cell elongation or cell division [33]. Seed treatment with IAA-producing bacteria significantly enhanced root and shoot growth in vitro [34] and in vivo [34, 35]. This may imply that the IAA produced by BAS23 caused improved rice seedling growth. In conclusion, B. amyloliquefaciens BAS23 showed strong antifungal activity against dirty panicle rice fungal pathogens as well as plant growth promotion activities in vitro. Consequently, the ability of B. amyloliquefaciens BAS23 to inhibit dirty panicle disease and improve rice seedling growth should be studied in the greenhouse and field trials.

Supplemental Materials

Acknowledgments

This work was financially supported by a Graduate Program Scholarship from the Graduate School (Grant year 2014), Kasetsart University, Bangkok, Thailand and partially supported by the Department of Microbiology (Grant year 2017), Faculty of Liberal Arts and Science, Kasetsart University. P. Kittakoop was supported by the Center of Excellence on Environmental Health and Toxicology, Science & Technology Postgraduate Education and Research Development Office (PERDO), Ministry of Education, Thailand.

Conflict of Interest


The authors have no financial conflicts of interest to declare.

Fig 1.

Figure 1.Phylogenetic tree of B. amyloliquefaciens BAS23 based on 16S rRNA sequence analysis.
Journal of Microbiology and Biotechnology 2018; 28: 1527-1535https://doi.org/10.4014/jmb.1804.04025

Fig 2.

Figure 2.Effect of BAS23 on the hyphal morphology of C. lunata (A), H. oryzae (B), and F. semitectum (C) co-inoculated with BAS23 and normal hyphae of C. lunata (D) H. oryzae (E) and F. semitectum (F). Arrows indicate swollen and bulb structures.
Journal of Microbiology and Biotechnology 2018; 28: 1527-1535https://doi.org/10.4014/jmb.1804.04025

Fig 3.

Figure 3.Effect of BAS23 cell-free culture (30%) on spore germination and germ tube elongation of pathogenic fungi C. lunata (A), H. oryzae (B) and F. semitectum (C) and untreated spores of C. lunata (D), H. oryzae (E) and F. semitectum (F).
Journal of Microbiology and Biotechnology 2018; 28: 1527-1535https://doi.org/10.4014/jmb.1804.04025

Fig 4.

Figure 4.Identification of iturin A with C14 side chain using 1H NMR (in CD3OD) spectroscopy (A) and ESI-MS (B) and identification of iturin A with C15 side chain by 1H NMR (in CD3OD) spectroscopy (C) and ESI-MS (D).
Journal of Microbiology and Biotechnology 2018; 28: 1527-1535https://doi.org/10.4014/jmb.1804.04025

Fig 5.

Figure 5.Structure of iturin A with C14 side chain (A) and C15 side chain (B).
Journal of Microbiology and Biotechnology 2018; 28: 1527-1535https://doi.org/10.4014/jmb.1804.04025

Fig 6.

Figure 6.Efficacy of BAS23 on root and shoot lengths (A) and growth of rice seedlings (5 days after incubation) from treated and untreated seed with BAS23 (B).
Journal of Microbiology and Biotechnology 2018; 28: 1527-1535https://doi.org/10.4014/jmb.1804.04025

Table 1 . Effect of BAS23 cell-free culture on dry weight of C. lunata, F. semitectum, and H. oryzae..

Concentration of cell-free culture (%)Dry weight reduction (%)

C. lunataF. semitectumH. oryzae
581.2±0.7c53.8±0.6c54.9±0.3e
1087.6±1.2b59.1±0.5b63.9±0.3d
1590.7±0.9a66.1±1.7a69.8±0.5c
2091.7±0.1a67.5±1.3a72.3±1.6b
3092.7±0.2a68.9±0.8a75.7±0.1a

Data represent the mean ± standard deviation. Means in each column with the same lowercase superscript letter are not significantly different (p < 0.05 )..


Table 2 . Assignments of major m/z peaks observed in mass spectra of lipopeptides of iturin A with the C14 and C15 side chains, reported in the literature..

Typem/zAssignmentReferences
Iturin A1043.5iturin A C14 [M+H]+[2, 18-19]
1057.5iturin A C15 [M+H]+[3, 18]
1065.5iturin A C14 [M+Na]+[2, 18, 20-21]
1079.5iturin A C15 [M+Na]+[18, 20-21]

Table 3 . In vitro production of plant growth-promoting metabolites..

IAA* at 500 µg/ml of tryptophan (µg/ml)Phosphate solubilization (mg/l)NH3Catalase

7 days14 days21 days
16.36±0.910.01±0.000.08±0.010.33±0.01++

*IAA: Indole-3-acetic acid.


References

  1. Thavong P. 2002. Effect of dirty panicle disease on rice seed vigor. Agric. Res. J. 20: 111-120.
  2. Chen D, Liu X, Li C, Tian W, Shen Q, Shen B. 2014. Isolation of Bacillus amyloliquefaciens S20 and its application in control of eggplant bacterial wilt. J. Environ. Manage 137: 120-127.
    Pubmed CrossRef
  3. Pathak KV, Keharia H. 2013. Characterization of fungal antagonistic bacilli isolated from aerial roots of banyan (Ficus benghalensis) using intact-cell MALDI-TOF mass spectrometry (ICMS). J. Appl. Microbiol. 114: 1300-1310.
    Pubmed CrossRef
  4. Alvarez F, Castro M, Príncipe A, Borioli G, Fischer S, Mori G, et al. 2011. The plant-associated Bacillus amyloliquefaciens strains MEP218 and ARP23 capable of producing the cyclic lipopeptides iturin or surfactin and fengycin are effective in biocontrol of sclerotinia stem rot disease. J. Appl. Microbiol. 112: 159-174.
    Pubmed CrossRef
  5. Zhao Y, Selvaraj JN, Xing F, Zhou L, Wang Y, Song H, et al. 2014. Antagonistic action of Bacillus subtilis strain SG6 on Fusarium graminearum. PLoS One 9: e92486.
    Pubmed KoreaMed CrossRef
  6. Raaijmakers JM, Vlami M, de Souza JT. 2002. Antibiotic production by bacterial biocontrol agents. Antonievan Leeuwenhoek 81: 537-547.
    Pubmed CrossRef
  7. Ahmad F, Ahmad I, Khan MS. 2008. Screening of free-living rhizospheric bacteria for their multiple plant growthpromoting activities. Microbiol. Res. 163: 173-181.
    Pubmed CrossRef
  8. Ait Kaki A, KacemChaouche N, Dehimat L, Milet A, Youcef-Ali M, Ongena M, et al. 2013. Biocontrol and plant growth promotion characterization of Bacillus species isolated from Calendula officinalis rhizosphere. Indian J. Microbiol. 53: 447-452.
    Pubmed KoreaMed CrossRef
  9. Souza Rd, Ambrosini A, Passaglia LMP. 2015. Plant growthpromoting bacteria as inoculants in agricultural soils. Genet. Mol. Biol. 38: 401-419.
    Pubmed KoreaMed CrossRef
  10. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. 2011. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28: 2731-2739.
    Pubmed KoreaMed CrossRef
  11. Mora I, Cabrefiga J, Montesinos E. 2015. Cyclic lipopeptide biosynthetic genes and products, and inhibitory activity of plant-associated Bacillus against phytopathogenic bacteria. PLoS One 10: e0127738.
    Pubmed KoreaMed CrossRef
  12. Huang X, Lu Z, Bie X, Lü F, Zhao H, Yang S. 2007. Optimization of inactivation of endospores of Bacillus cereus by antimicrobial lipopeptides from Bacillus subtilis fmbj strains using a response surface method. Appl. Microbiol. Biotechnol. 74: 454-461.
    Pubmed CrossRef
  13. Loper JE, Schroth MN. 1986. Influence of bacterial sources of indole-3-acetic acid on root elongation of sugar beet. Physiol. Biochem. 76: 386-389.
    CrossRef
  14. Ruangsanka S. 2014. Identification of phosphate-solubilizing fungi from the asparagus rhizosphere as antagonists of the root and crown rot pathogen Fusarium oxysporum. ScienceAsia 40: 16-20.
    CrossRef
  15. Cappuccino JC, Sherman N. 1992, pp. 125-179. Microbiology: A Laboratory Manual, 3th Ed. Benjamin/cummings, New York.
  16. Tang J-S, Gao H, Hong K, Yu Y, Jiang M-M, Lin H-P, et al. 2007. Complete assignments of 1H and 13C NMR spectral data of nine surfactin isomers. Magn. Reson. Chem. 45: 792-796.
    Pubmed CrossRef
  17. Garbay-Jaureguiberry C, Roques BP, Delcambe L, Peypoux F, Michel G. 1978. NMR conformational study of iturin A, an antibiotic from Bacillus subtilis. FEBS. Lett. 93: 151-156.
    Pubmed CrossRef
  18. Yang H, Li X, Li X, Yu H, Shen Z. 2015. Identification of lipopeptide isomers by MALDI-TOF-MS/MS based on the simultaneous purification of iturin, fengycin, and surfactin by RP-HPLC. Anal. Bioanal. Chem. 407: 2529-2542.
    CrossRef
  19. Yu GY, Sinclair JB, Hartman GL, Bertagnolli BL. 2002. Production of iturin A by Bacillus amyloliquefaciens suppressing Rhizoctonia solani. Soil Biol. Biochem. 34: 955-963.
    CrossRef
  20. Dunlap CA, Schisler DA, Price NP, Vaughn SF. 2011. Cyclic lipopeptide profile of three Bacillus subtilis strains; antagonists of Fusarium head blight. J. Microbiol. 49: 603-609.
    Pubmed CrossRef
  21. Singh AK, Rautela R, Cameotra SS. 2014. Substrate dependent in vitro antifungal activity of Bacillus sp. strain AR2. Microb. Cell Fact. 13: 67-67.
    Pubmed KoreaMed CrossRef
  22. Hermann A, Guenther W, Guenther J. 1984. Iturin AL: structure and derivatives of a peptidolipid with a high content of C16-iturinic acids, Liebigs Ann. Chem. 5: 854-866.
  23. Akira I, Seiji T, Shigeo M, Akinori S. 1982. Structures of β-amino acids in antibiotics iturin A. Tetrahedron Lett. 23: 3065-3068.
    CrossRef
  24. Moran S, Rai DK, Clark BR, Murphy CD. 2009. Precursordirected biosynthesis of fluorinated iturin A in Bacillus spp. Org. Biomol. Chem. 7: 644-646.
    Pubmed CrossRef
  25. Ji SH, Paul NC, Deng JX, Kim YS, Yun B-S, Yu SH. 2013. Biocontrol activity of Bacillus amyloliquefaciens CNU114001 against fungal plant diseases. Mycobiology 41: 234-242.
    Pubmed KoreaMed CrossRef
  26. Arguelles-Arias A, Ongena M, Halimi B, Lara Y, Brans A, Joris B, et al. 2009. Bacillus amyloliquefaciens GA1 as a source of potent antibiotics and other secondary metabolites for biocontrol of plant pathogens. Microb. Cell Fact. 8: 63.
    Pubmed KoreaMed CrossRef
  27. Torres MJ, Brandan CP, Petroselli G, Erra-Balsells R, Audisio MC. 2016. Audisio MC. 2016. Antagonistic effects of Bacillus subtilis subsp. subtilis and B. amyloliquefaciens against Macrophomina phaseolina: SEM study of fungal changes and UV-MALDI-TOF MS analysis of their bioactive compounds. Microbiol. Res. 182: 31-39.
    CrossRef
  28. Li B, Li Q, Xu Z, Zhang N, Shen Q, Zhang R. 2014. Responses of beneficial Bacillus amyloliquefaciens SQR9 to different soil borne fungal pathogens through the alteration of antifungal compounds production. Front. Microbio. 5: 1-10.
    Pubmed KoreaMed CrossRef
  29. Liu X, Ren B, Gao H, Liu M, Dai H, Song F, et al. 2012. Optimization for the production of surfactin with a new synergistic antifungal activity. PLoS One 7: e34430.
    Pubmed KoreaMed CrossRef
  30. Islam MR, Jeong YT, Lee YS, Song CH. 2012. Isolation and identification of antifungal compounds from Bacillus subtilis C9 inhibiting the growth of plant pathogenic fungi. Mycobiology 40: 59-66.
    Pubmed KoreaMed CrossRef
  31. Nakano MM, Marahiel MA, Zuber P. 1988. Identification of a genetic locus required for biosynthesis of the lipopeptide antibiotic surfactin in Bacillus subtilis. J. Bacteriol. 170: 5662-5668.
    Pubmed KoreaMed CrossRef
  32. Tapi A, Chollet-Imbert M, Scherens B, Jacques P. 2010. New approach for the detection of non-ribosomal peptide synthetase genes in Bacillus strains by polymerase chain reaction. Appl. Microbiol. Biotechnol. 85: 1521-1531.
    Pubmed CrossRef
  33. Vessey JK. 2003. Plant growth-promoting rhizobacteria as biofertilizers. Plant Soil 255: 571-586.
    CrossRef
  34. Islam S, Akanda AM, Prova A, Islam MT, Hossain MM. 2016. Isolation and identification of plant growth-promoting rhizobacteria from cucumber rhizosphere and their effect on plant growth promotion and disease suppression. Front. Microbio. 60: 1360.
    Pubmed KoreaMed CrossRef
  35. Khalid A, Arshad M, Zahir ZA. 2004. Screening plant growth-promoting rhizobacteria for improving growth and yield of wheat. J. Appl. Microbiol. 96: 473-480.
    Pubmed CrossRef