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The potential of bacillus subtilis BAS114 for in vitro biocontrol of Fusarium oxysporum.

ABSTRACT

Among 140 soil bacterial isolates, Bacillus subtilis BAS114 showed the highest inhibitory activity against Fusarium oxysporum using the dual culture method. Cell-free culture of B. subtilis BAS114 also efficiently inhibited the growth and spore germination of F. oxysporum. In culture media containing 20-30% cell-free culture, the inhibition of F. oxysporum was up to 73.08-76.92% in terms of dry weight and 67.38-68.15% inhibition of diameter growth. B. subtilis BAS114 can inhibit a broad range of rice pathogenic fungi, including Fusarium semitectum, Helminthosporium oryzae and Rhizoctonia solani and also the asparagus pathogenic fungus, Phomopsis asparagi. In addition, B. subtilis BAS114 can inhibit the rice bacterial blight, Xanthomonas oryzae. These findings suggested that B. subtilis BAS114 can be a powerful biological control agent.

KEYWORDS: Bacillus subtilis Fusarium oxysporum Biological control

INTRODUCTION

A soil-borne plant fungal pathogen, Fusarium oxysporum, is the causal agent of root rot or wilt diseases in rice and several plant species, such as tomato, potato, eggplant, chili, sugarcane, maize, banana and tobacco [1-3]. Colonization of plants by F. oxysporum leads to the necrosis of the infected tissues, a subsequent collapse of vascular vessels and decay of the plant [4]. The disease causes the death of adult plants, with consequent economic losses [5]. Fungal diseases are usually controlled using an integrated strategy, incorporating the use of fungicides and host plant resistance. However, the increasing use of chemical fungicides has generated negative effects on the environment and on human health. Biological control has been described as a non-hazardous strategy to reduce crop damage caused by plant pathogens [6-8]. The mechanisms of biocontrol may involve and be divided into antibiosis, competition, parasitism, cell wall degrading enzymes and induced resistance [9]. The bacterial antagonists, Bacillus, Pseudomonas, and Burkholderia, have been reported as efficient biological control agents [10, 11]. Several strains of Bacillus have been found to produce wide-spectrum antibiotics. These antagonistic bacteria show advantages over other antagonists, since they are resistant to adverse environmental conditions because they produce endospores [12]. The current study aimed to screen a powerful bacterial antagonist against F. oxysporum. The effect of the antagonistic bacterial strain and its extracellular metabolites on this pathogenic fungus was studied in vitro using various methods. In addition, the selected antagonist, B. subtilis BAS114, was studied for its ability to antagonize the growth of pathogenic fungi and a bacterium causing the major rice diseases, dirty panicle disease, sheath blight disease and leaf blight disease, and Phomopsis blight in asparagus.

MATERIALS AND METHODS

Pathogenic microorganisms:

The pathogenic fungus, Fusarium oxysporum, was isolated from diseased rice collected from paddy fields in Nakhon Pathom province, Thailand. Morphological characterization and nucleotide sequence analysis of the D1/D2 region of the LSU rRNA gene were performed for fungal identification. Helminthosporium oryzae, F. semitectum, Rhizoctonia solani, Phomopsis asparagi and Xanthomonas oryzae were obtained from the Plant Health Clinic, Kasetsart University Kamphaeng Saen campus, Nakhon Pathom, Thailand. The pathogenic fungi and bacterium were maintained at 4[degrees]C on potato dextrose agar (PDA) medium and nutrient agar (NA) medium, respectively.

Soil bacteria:

The 140 bacterial isolates used in this work were all isolated from soil in various agricultural fields in Nakhon Pathom, Thailand [13], and they were maintained at 4[degrees]C on NA medium.

Dual culture antifungal activity of B. subtilis BAS114:

The antifungal activity of bacterial antagonists was evaluated using the dual culture method on PDA medium. A mycelial plug (5 mm diameter) of F. oxysporum was placed 2.5 cm away from the edge in a Petri dish and incubated at 30[degrees]C for one day. A loopful of bacteria was then streaked 4.5 cm away from the plug of F. oxysporum on the same Petri dish. Plates were incubated at 30[degrees]C for 10 days. Inhibition of mycelial growth was determined by measuring the colony radial growth after 7 days of inoculation. The percentage of inhibition of radial growth (PIRG) was calculated using the following formula [14, 15].

PIRG (%) = [(R1 - R2)/R1] x 100

where, R1 is the radial diameter of F. oxysporum in the control plate and R2 is the radial diameter of F. oxysporum with the antagonistic bacteria. Experiments were performed in triplicate.

The selected bacterium, B. subtilis BAS114, was also tested for its ability to inhibit the growth of the rice pathogenic fungi, F. semitectum, H. oryzae and R. solani, and the asparagus pathogenic fungus, P. asparagi, using the dual culture method as described above. In addition, the agar-well diffusion method was used to evaluate the antibacterial activity of BAS114 against X. oryzae. The pathogenic bacterium inoculum was spread using a sterile cotton swab on nutrient glucose agar (NGA). Then cell suspension of B. subtilis BAS114 was added into the well. The culture plates were inoculated at 37[degrees]C for 48 h. The inhibition zone was measured in millimeters.

Effect of B. subtilis BAS114 cell-free culture on mycelial growth of F. oxysporum:

The antifungal activity of B. subtilis BAS114 cell-free culture was evaluated using the poison agar method and the dry-weight determination method. B. subtilis BAS114 was grown in 100 ml of nutrient broth (NB) medium for 3 days at 30[degrees]C with shaking at 150 rpm. Cell-free culture was obtained using centrifugation at 15,000 rpm at 4[degrees]C for 10 min. Streptomycin (1 mg/ml) and ampicillin (1 mg/ml) were then added to the supernatant. A 5 mm agar plug of F. oxysporum was placed on the center of PDA media that contained different concentrations of cell-free culture (5%, 10%, 15%, 20% and 30%). PDA medium without cell-free culture was used as a control. Mycelial growth was determined by measuring the colony diameter growth after 7 days of inoculation at 30[degrees]C. The percentage of inhibition of diameter growth (PIDG) was calculated using the following formula [16, 17]. PIDG (%) = [(C-E)/C] x 100%

where, C is the diameter of the control colony and E is the diameter of the treated colony. Experiments were performed in triplicate.

Dry-weight determination of mycelial growth was performed according to Saechow et al. (2016) [13]. A spore suspension (1x1[0.sup.7] spores/ml) of F. oxysporum was inoculated into potato dextrose broth (PDB) medium that contained cell-free culture of BAS114 (5%, 10%, 15%, 20% and 30%) and incubated at 30[degrees]C with 150 rpm shaking for 7 days. PDB medium without cell-free culture was used as a control. The filtrated mycelia were dried at 55[degrees]C until constant weight.

Effect of B. subtilis BAS114 cell-free culture on spore germination:

Cell-free culture of BAS114 (5%, 10%, 15%, 20% and 30%) was supplemented into PDB medium for culturing F. oxysporum as described above. The spore germination of F. oxysporum was determined by counting 100 spores in some random fields under a light microscope (400x) after 7-48 h of incubation and reported as percentage of spore germination (PSG).

Effect of B. subtilis BAS114 on hyphal morphology

A mycelial plug (5 mm diameter) of F. oxysporum was placed on the center of a PDA plate and BAS114 was streaked in a square pattern around the agar plug at 2.5 cm distance. Mycelial growth from the edge of the fungal colony nearest to the inhibition zone after incubation for 7 days at 30[degrees]C was examined under a light microscope (1,000x).

Results:

In vitro antagonistic ability:

Of the 140 isolated bacteria, 17 isolates were inhibitory to F. oxysporum using the dual culture method. BAS114 showed the highest value of PIRG (53.33%; Table 1). This antagonistic bacterium was identified as B. subtilis [13]. The effect of B. subtilis BAS114 on hyphal morphology was observed under a light microscope. The micromorphology of F. oxysporum hyphae showed swelling, thickening and bulb formation. Conversely, in the untreated control, pathogen hyphae were intact and elongated with a smooth surface (Fig. 1).

The antimicrobial activity of B. subtilis BAS114 against a broad range of plant pathogenic fungi and a bacterium was studied, and the results are shown in Fig. 2 and Table 2. BAS114 showed inhibitory activity against the rice pathogenic fungi and the asparagus pathogenic fungus using the dual culture method. In addition, the results of the agar-well method indicated that Bacillus subtilis BAS114 can inhibit the bacterial blight, X. oryzae.

Effect of B. subtilis BAS114 cell-free culture on fungal growth:

Cell-free culture (5%, 10%, 15%, 20% and 30%) of B. subtilis BAS114 was used to study the inhibitory activity of fungal growth using the poison agar method and the dry-weight determination method. Cell-free culture (20-30%) showed the highest PIDG values (67.38-68.15%) compared to the control (Fig. 3). In addition, there was 73.08-76.92% reduction in the dry weight of F. oxysporum grown with 20-30% of cell-free culture of BAS114 compared to the control (Table 3).

Effect of B. subtilis BAS114 cell-free culture on spore germination:

Spores of F. oxysporum were treated with different concentrations (5%, 10%, 15%, 20% and 30%) of cell-free culture of B. subtilis BAS114. Spore germination was significantly reduced as a response to the concentration of cell-free culture (Fig. 4). The results revealed that extracellular metabolites were inhibitory to F. oxysporum growth.

Discussion:

The use of beneficial microorganisms is considered one of the most promising strategies for controlling plant diseases. Among 140 bacterial isolates, B. subtilis BAS114 showed the highest antifungal activity against F. oxysporum using the dual culture method. Light microscopy revealed that F. oxysporum hyphae in the margin of the inhibition zones showed swellings and bulb formations, which demonstrated that B. subtilis BAS114 induced morphological abnormalities in the hyphae of F. oxysporum. The inhibitory activity of mycelial growth in response to cell-free culture of BAS114 was studied using the poison agar method and the dry-weight determination method. The results revealed that with an increase in the concentration of the BAS114 cell-free culture, the PIDG gradually increased and dry weight of F. oxysporum reduced. Spore germination was also reduced as a response to the concentration of cell-free culture. These results suggested that the active substances generated by B. subtilis BAS114 affected mycelial growth and spore germination.

B. subtilis BAS114 also showed its ability to antagonize various plant pathogens in vitro-it could strongly inhibit the growth of dirty panicle fungal pathogens of rice, F. semitectum and H. oryzae. Its antifungal activity against Curvularia lunata (dirty panicle disease) was also noted in our previous report [13]. Dirty panicle disease fungi severely reduce the production and quality of rice in Thailand and worldwide [18]. In addition, B. subtilis BAS114 can inhibit R. saloni and X. oryzae, an important fungus and bacterium, respectively, that cause sheath blight disease and leaf blight disease of rice, respectively. P. asparagi, which causes Phomopsis blight in asparagus, was also inhibited by B. subtilis BAS114. Similar antagonistic activity has been reported for the B. subtilis strain JA, which can inhibit a broad range of fungal pathogens [19].

Bacillus species are often considered to be important biocontrol agents. B. subtilis is one of the important antagonists used in the biocontrol of plant diseases. It has been reported that B. subtilis produces antimicrobial compounds that inhibit pathogenic growth [19, 20]. Generally, cyclic lipopeptides (cLPs) have efficacy as biocontrol compounds. According to their amino acid sequences, CLPs can be classified into three families: iturins, fengycins and surfactins [20]. In our previous report, genes involved in the synthesis of cLPs, such as ituC (iturin A synthetase C), bmyB (bacillomycin), fenD (fengycin) and srfAA (surfactin) were detected in B. subtilis BAS114 in a polymerase chain reaction (PCR) experiment [13]. These results suggested that the inhibitory activity of B. subtilis BAS114 cell-free culture against F. oxysporum may be due to the toxic effect of the cLP components.

Conclusion:

In this study, the antagonistic effects of B. subtilis BAS114 were evaluated against F. oxysporum. In vitro assay with the antagonist and its cell-free culture showed that the BAS114 strain effectively inhibited the mycelial growth of F. oxysporum. Additionally, the results demonstrated that B. subtilis BAS114 had a broad spectrum of antimicrobial activity against five plant pathogenic fungi and a plant pathogenic bacterium. These results suggest that the B. subtilis BAS114 can be an effective biological control agent. However, the antimicrobial substances of B. subtilis BAS114 against plant pathogenic microorganisms should be further studied.

ACKNOWLEDGEMENTS

This work was financially supported by a Graduate Program Scholarship from the Graduate School, Kasetsart University, Bangkok, Thailand and partially supported by the Department of Microbiology (grant year 2016), Faculty of Liberal Arts and Science, Kasetsart University, Thailand.

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(1) Sukanya Saechow, (1,2) Anon Thammasittirong (1,2) Sutticha Na-Ranong Thammasittirong

(1) Department of Microbiology, Faculty of Liberal Arts and Science, Kasetsart University Kamphaeng Saen campus, Nakhon Pathom 73140, Thailand

(2) Microbial Biotechnology Unit, Faculty of Liberal Arts and Science, Kasetsart University Kamphaeng Saen campus, Nakhon Pathom 73140, Thailand

Address For Correspondence:

Sutticha Na-Ranong Thammasittirong, Department of Microbiology, Faculty of Liberal Arts and Science, Kasetsart University Kamphaeng Saen campus, Nakhon Pathom 73140, Thailand.

This work is licensed under the Creative Commons Attribution International License (CC BY).

http://creativecommons.org/licenses/by/4.0/

Received 18 September 2016; Accepted 14 January 2017; Available online 26 January 2017

Table 1: PIRG of B. subtilis BAS114 against F. oxysporum using dual
culture method

Isolate        PIRG (%)                 Isolate  PIRG (%)

BAS4    47.22[+ or -]2.5[5.sup.cd]  BAS114   53.33[+ or -]1.6[7.sup.a]
BAS12   46.33[+ or -]0.3[4.sup.d]   BAS115   48.89[+ or -]1.9[2.sup.bcd]
BAS25   47.00[+ or -]0.0[0.sup.cd]  BAS129   51.85[+ or -]2.5[7.sup.ab]
BAS29   40.00[+ or -]0.8[9.sup.e]   BAS131   51.67[+ or -]1.6[7.sup.ab]
BAS54   30.00[+ or -]3.3[3.sup.g]   BAS132   50.55[+ or -]2.5[5.sup.abc]
BAS95   48.89[+ or -]1.9[2.sup.bcd] BAS135   51.11[+ or -]3.4[7.sup.ab]
BAS96   36.11[+ or -]0.9[6.sup.f]   BAS137   49.44[+ or -]0.9[6.sup.bcd]
BAS100  32.78[+ or -]0.9[6.sup.fg]  BAS138   50.56[+ or -]0.9[6.sup.abc]
BAS110  32.78[+ or -]0.9[6.sup.fg]

Different lowercase, superscript letters indicate significant
differences between bacterial isolates (p < 0.05).
Data represent the mean [+ or -] standard deviation from three
independent experiments.

Table 2: Antimicrobial activity spectrum of B. subtilis BAS114

Plant pathogenic microorganisms  PIRG (%)           Inhibition zone (mm)

H. oryzae                        61.03[+ or -]0.89      -
F. semitectum                    60.81[+ or -]2.30      -
R. solani                        47.95[+ or -]0.89      -
P. asparagi                      52.07[+ or -]1.53      -
X. oryzae                         -                    26.0[+ or -]0.27

Different lowercase, superscript letters indicate significant
differences between bacterial isolates (p < 0.05).
Data represent the mean [+ or -] standard deviation from three
independent experiments.

Table 3: Effect of B. subtilis BAS114 cell-free culture on mycelia
growth of F. oxysporum using dry weight determination method

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

 0                                      0.52[+ or -]0.0[1.sup.e]
 5                                      0.41[+ or -]0.0[2.sup.d]
10                                      0.28[+ or -]0.0[2.sup.c]
15                                      0.17[+ or -]0.0[1.sup.b]
20                                      0.14[+ or -]0.0[2.sup.a]
30                                      0.12[+ or -]0.0[1.sup.a]
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Author:Saechow, Sukanya; Thammasittirong, Anon; Thammasittirong, Sutticha Na-Ranong
Publication:Advances in Environmental Biology
Article Type:Report
Geographic Code:9THAI
Date:Jan 1, 2017
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