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JOURNAL OF ENVIRONMENTAL SCIENCES ISSN 1001-0742 CN 11-2629/X
Journal of Environmental Sciences 2013, 25(Suppl.) S122–S126
www.jesc.ac.cn
Screening of endophytic bacteria against fungal plant pathogens Tatsuya Ohike1, ∗, Kohei Makuni2 , Masahiro Okanami1,2 , Takashi Ano1,2 1. Graduate School of Biology-Oriented Science and Technology, Kinki University, 930 Nishimitani, Kinokawa, Wakayama 649-6493, Japan 2. Department of Biotechnological Science, Faculty of Biology-Oriented Science and Technology, Kinki University, 930 Nishimitani, Kinokawa, Wakayama 649-6493, Japan
Abstract Bacterial endophytes were found from 6 plant leaves among 35 plant leaves screened. Two of the isolated bacteria showed antagonistic activity against fungal plant pathogens. An isolate named KL1 showed the clear inihibition against plant pathogens, Fusarium oxysporum and Rhizoctonia solani, on PDA as well as TSA plate. Supernatant of the bacterial culture also showed the clear inhibition against the fungal growth on the plate and the antibiotic substance was identified as iturin A by HPLC analysis. KL1 was identified as Bacillus sp. from the 16S rRNA gene analysis. Very thin hyphae of R. solani was miccroscopically observed when the fungus was co-cultivated with KL1. Key words: biocontrol; endophyte; plant pathogens
Introduction Agriculture is a main industry to produce food, and the bacteria in soil assumed to be involved in the role of efficient food production. On the other hand other microbes such as Fusarium and Rhizoctonia species are widely distributed soil-borne pathogens and cause diseases on a wide range of economically important plant species (Garcia et al., 2006). Rhizoctonia solani is known to reduce the production of crops such as rice (Mew and Rosales, 1986), cucumber (Trillas et al., 2006) and lettuce (Grosch et al., 2011). Strategies to control Rhizoctonia diseases are limited and the disease is spreading all over the world. Fusarium oxysporum also invades roots and causes wilt diseases through colonization in xylem tissue of host plants, and reduces the production of crops. Synthetic chemical fungicides have long been used as active agents in reducing the plant diseases. However, they are costly, can cause environmental pollution, and may induce drug-resistant pathogens. Considering the limitations of chemical fungicides, it seems appropriate to search for a supplemental control strategy. Biological control, or the use of microorganisms to prevent plant diseases, offers an attractive alternative or supplement to chemical pesticides (San-Lang et al., 2002). Therefore, developing new biological control agents using antagonistic bacteria seems to be an ideal solution for this problem. We reported that endophyte have the * Corresponding author. E-mail: [email protected]
potential of used as new isolation source of biocontrol agent.
1 Materials and methods 1.1 Plant leaf samples Endophytic bacteria were isolated from 35 different plant leaf samples in Wakayama prefecture in Japan. 1.2 Screening of endophytes Surface of the leave samples was disinfected in 70% ethanol for 60 sec, followed by 1.0% NaOCl for 5 min, and 70% ethanol for 30 sec, thoroughly washed with sterile distilled water. After sterilization, leaf samples were cut into a 5-mm square and put on the potato dextrose broth agar medium (PDA) and trypto-soya broth agar medium (TSA), and incubated at 24°C or 30°C, respectively. Isolated endophytic bacteria were assayed for antifungal activity against F. oxysporum or R. solani on PDA and TSA plates. 1.3 Antifungal activity of endophytes The dual culture technique was employed to test antagonistic effect of KL1 on the growth of R. solani and F. oxysporum (Zhou et al., 2011). An agar plug of 0.7cm diameter from actively growing fungal mycelium was placed on the center of the PDA plate, and then KL1 were inoculated on the plate at 4 equidistance sites, 3 cm apart from the colony of R. solani or F. oxysporum in the center. Another plates were inoculated with the same
Suppl.
Screening of endophytic bacteria against fungal plant pathogens
size agar plug of fungal colony in the absence of KL1 as control. These plates were incubated at 24°C. And then the antagonistic effect of the KL1 on R. solani and F. oxysporum was observed. 1.4 Antifungal activity of KL1 supernatant and its thermal stability examination Antifungal activity and thermal stability of cell free supernatant from KL1 were tested by measuring the ability to inhibit fungal growth of R. solani on PDA agar plates. KL1 was grown in 300 mL conical flasks containing 60 mL of No. 3S medium (Ohno et al., 1995) at 30°C and for 120 hr. Cell free supernatant was obtained by centrifugation at 16,000 ×g at 24°C for 10 min followed by filtration through 0.20-µm cellulose acetate filter. An agar piece (0.7-cm diameter) of R. solani was placed at the center of Petri dishes containing PDA. Each of the 200 µL, 150 µL, 100 µL, 50 µL of cell free supernatant was put into a penicillin-cup placed on the PDA, respectively. Thermal stability of the cell free supernatant was examined as follows: 300 µL of cell free supernatant was heat treated in a dry heat block (Dry Thermo Bath MG-2200 Eyela, Tokyo, Japan) for 15 min at 50°C, 55°C, 60°C, 65°C, 70°C or 90°C. After the heat treatment, each supernatant was tested for the remaining antifungal activity. 1.5 Antifungal activity by PDA medium containing 5%–15% KL1 supernatant KL1 supernatant was sterilized by filtration through 0.20µm membrane filter (DISMIC-25cs Cellulose Acetate 0.20-µm, Advantec, Tokyo, Japan) and added to sterilized PDA (5%–15%, V/V) of final concentration) (Coda et al., 2011). After mixing of the samples, aliquots of 10 mL were poured into petri plates (50-mm diameter). Control plates contained PDA alone. The assay was carried out by placing 0.7-cm-diameter plugs of growing R. solani onto the center of petri dishes containing the culture medium. Plates were incubated aerobically at 24°C. Mycelia growth inhibition was calculated as: I=
C−T × 100 C
(1)
where, I (%) is the mycelia growth inhibition, C (mm) is the mycelia diameter in control, and T (mm) is the mycelia diameter in PDA containing KL1 supernatant (Forchetti et al., 2007). 1.6 Extraction and HPLC analysis of iturin A and surfactin For extraction of iturin A and surfactin, culture fluid was centrifuged at 16,000 ×g for 10 min. Then 500 µL of the supernatant was mixed with 500 µL of extraction buffer (CH3 CN:10 mmol/L ammonium acetate solution of 35:65 (V/V), and the mixture was vortexed for 10 min. The mixture was filtered through a 0.20-µm pore size polytetrafluoroethylene (PTFE) membrane (DISMIC-
S123
13JP, Advantec, Tokyo, Japan), and then injected into a high performance liquid chromatography (HPLC). HPLC analysis was performed using a JASCO LC-2000 series HPLC system (JASCO, Tokyo, Japan) equipped with a Chromolith Performance RP18e column at a flow rate of 2.0 mL/min. For separation, 10 mmol/L ammonium acetate aqueous solution and acetonitrile were used as mobile phase. The elution profile comprised an initial isocratic phase of 35% (iturin) or 45% (surfactin) acetonitrile for 10 min. Lipopeptide were detected by absorbance at 205 nm. Purified iturin A purchased from Sigma-Aldrich Co., USA and purified surfactin were used as standard samples for the HPLC. 1.7 Identification of antagonistic endophyte Bacterial 16S rRNA genes were PCRamplified with primers 357FWD-Bam (5′ -GGGGATCCTCCTACGGGAGGCAGCAG-3′ ) and 1100RV-Bam (5′ -CCGGATCCGGGTTGCGCTCGTTG3′ ). The sequences of the amplified product were compared by BLAST search and identification was done based on similarity up to species level.
2 Results and discussion To make sure the bacteria were isolated from the inside of the leaves, the surface sterilization was confirmed to be perfect by touching the surface of the leaves on TSA and PDA plates. One example is shown in Fig. 1. No colonies from the surface of the plant leaves were detected on the touching plates (Fig. 1a, c), but the colonies were emerged from inside of the leaves (Fig. 1b, d). Six endophytic bacteria were isolated from 35 different plant leaves, and two were further screened and an isolate named KL1 having been isolated from the chestnut tree (Castanea crenata) leaves showed a strong antifungal activity as shown in Fig. 2. Supernatant of the culture of KL1 also showed the strong inhibitory effect against the fungi, and the antifungal activity of supernatant was stable even after heating at 90°C for 15 min (Fig. 3). In addition, growth of R. solani was inhibited on PDA medium containing supernatant of KL1 (Fig. 4). The inhibition effect was kept even after the 14 days and stability of the suppressive effect is a suitable characteristic as a biocontrol agent. 16S rRNA gene of KL1 was amplified using FWD357 and RV1100 primers. Based on partial 16S rRNA gene sequence, the isolated strain KL1 showed high sequence identity (99%) to Bacillus sp. by BLAST analysis. From these features, it is suggested that the compound extracted from KL1 supernatant might be cyclic lipopeptide iturin A. Therefore, qualitative analysis of iturin A and surfactin by HPLC were performed for the extract of the supernatant of KL1. As a result, clear iturin A homolog peaks, which
Journal of Environmental Sciences 2013, 25(Suppl.) S122–S126 / Tatsuya Ohike et al.
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Fig. 1 Isolation endophytic bacteria from a leaf of Castanea crenata on TSA (a, b) and PDA (c, d) plates. Surface sterilized leaves were put on the plate directly (a, c) to confirm the sterilization, and after that the leaves were cut into a 5-mm square and put on TSA (b) and PDA (d).
a
b
Fig. 2 Inihibition of the growth of Rhizoctonia solani (a) and Fusarium oxysporum (b) on PDA plate, by a newly isolated endophytic bacterium named KL1.
a
b
c
d
e
f
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h
Fig. 3 Thermal stability of crude fungicides produced by an endophyte KL1. (a) inhibition of the growth of R. solani by KL1 cell free supernatant on PDA. Each stainless cup contains the supernatant, and the volume is 50, 100, 150, and 200 µL respectively clockwise from top-left. Inhibition of R. solani by the heat treated supernatant. Each 200 µL of the supernatant was heat treated for 15 min at 25°C (b), 50°C (c), 55°C (d), 60°C (e), 65°C (f), 70°C (g), 90°C (h), respectively, followed by the antifungal assay on PDA against R. solani.
5% 10% 15%
Inhibition ratio (%)
100 90 80 70 60 50 40 30 20 10 0
3
4
5
6
7 8 9 10 11 12 13 14 Incubation time (day) Fig. 4 Supernatant fluid of endophyte KL1 inhibited growth of R. solani. Inhibition of the growth of R. solani by the cell-free supernatant of the culture broth of KL1.
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Screening of endophytic bacteria against fungal plant pathogens
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Time (min) Fig. 5 HPLC analysis of the lipopeptides produced by KL1. Homologs of iturin A were detected in the culture broth of KL1 (b), but, no surfactin was detected (d), in comparison with standard iturin A (200 mg/L) and surfactin (500 mg/L). Several peaks of iturin A (a) and surfactin (c) correspond to the lipopeptide with a fatty acid of different length of carbon chain.
a
b
Fig. 6
Microscopic analysis of the control R. solani (a) and R. solani in the boundary region co-cultivated with KL1 (b).
correspond to the lipopeptide with a C14 -C17 β-amino fatty acid, were detected (Fig. 5a, b), however, another lipopeptide surfactin, which is found in other Bacillus species, was not detected in the extract of KL1 (Fig. 5c, d). Microscopic analysis of the boundary region of cocultivation of KL1 with R. solani showed thinner and smooth hyphae of the fungus (Fig. 6a, b). As KL1 was able to suppress R. solani and F. oxysporum, which cause serious damages on many economically important agricultural crops, and is an endophyte, having more affinity for plants than other bacterial biocontrol agents, it was suggested that KL1 can be a good candidate as a biocontrol agent.
3 Conclusions We have screened endophytes in plant leaves. The isolated bacteria showed antagonistic activity against fungal plant pathogens. An isolate named KL1, showed stronger inihibition than other 34 isolates against plant pathogens (data not shown), Fusarium oxysporum and Rhizoctonia solani, on PDA as well as TSA plate. KL1 was identified as Bacillus sp. from the 16S rRNA gene analysis. These results suggest that KL1 can be a good candidate of a biocontrol agent, and as it was isolated from a plant leave it may have good affinity with plants, which is a suitable characteristic in the agriculture.
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Journal of Environmental Sciences 2013, 25(Suppl.) S122–S126 / Tatsuya Ohike et al.
References Coda R, Cassone A, Rizzello C G, Nionelli L, Cardinali G, Gobbetti M, 2011. Antifungal activity of Wickerhamomyces anomalus and Lactobacillus plantarum during sourdough fermentation: Identification of novel compounds and longterm effect during storage of wheat bread. Applied and Environmental Microbiology, 77: 3484–3492. Forchetti G, Masciarelli O, Alemano S, Alvarez D, Abdala G, 2007. Endophytic bacteria in sunflower (Helianthus annuus L.): isolation, characterization, and production of jasmonates and abscisic acid in culture medium. Applied Microbiology and Biotechnology, 76: 1145–1152. Garcia V G, Onco M A P, Susan V R, 2006. Review. Biology and systematics of the form genus Rhizoctonia. Spanish Journal of Agricultural Research, 4: 55–79. Grosch R, Schneider J H M, Kofoet A, Feller C, 2011. Impact of continuous cropping of Lettuce on the disease dynamics of bottom rot and genotypic diversity of Rhizoctonia solani AG 1-IB. Journal of Phytopathology, 159: 35–44.
Vol. 25
Ohno A, Ano T, Shoda M, 1995. Production of a lipopeptide antibiotic, surfactin, by recombinant Bacillus subtilis in solid state fermentation. Biotechnology and Bioengineering, 47: 209–214. Mew T W, Rosales A M, 1986. Bacterization of rice plants for control of sheath blight caused by Rhizoctonia solani. Phytopathology, 76: 1260–1264. San-Lang W, Shih I L, Wang C H, Tseng K C, Chang W T, Twu Y K et al., 2002. Production of antifungal compounds from chitin by Bacillus subtilis. Enzyme and Microbial Technology, 31: 321–328. Trillas M I, Casanova E, Cotxarrera L, Ordovas J, Borrero C, Aviles M, 2006. Composts from agricultural waste and the Trichoderma asperellum strain T-34 suppress Rhizoctonia solani in cucumber seedlings. Biological Control, 39: 32– 38. Zhou X, Lu Z, Lv F, Zhao H, Wang Y, Bie X, 2011. Antagonistic action of Bacillus subtilis strain fmbj on the postharvest pathogen Rhizopus stolonifer. Journal of Food Science, 76: M254–M259.
JOURNAL OF ENVIRONMENTAL SCIENCES ISSN 1001-0742 CN 11-2629/X
Journal of Environmental Sciences 2013, 25(Suppl.) S122–S126
www.jesc.ac.cn
Screening of endophytic bacteria against fungal plant pathogens Tatsuya Ohike1, ∗, Kohei Makuni2 , Masahiro Okanami1,2 , Takashi Ano1,2 1. Graduate School of Biology-Oriented Science and Technology, Kinki University, 930 Nishimitani, Kinokawa, Wakayama 649-6493, Japan 2. Department of Biotechnological Science, Faculty of Biology-Oriented Science and Technology, Kinki University, 930 Nishimitani, Kinokawa, Wakayama 649-6493, Japan
Abstract Bacterial endophytes were found from 6 plant leaves among 35 plant leaves screened. Two of the isolated bacteria showed antagonistic activity against fungal plant pathogens. An isolate named KL1 showed the clear inihibition against plant pathogens, Fusarium oxysporum and Rhizoctonia solani, on PDA as well as TSA plate. Supernatant of the bacterial culture also showed the clear inhibition against the fungal growth on the plate and the antibiotic substance was identified as iturin A by HPLC analysis. KL1 was identified as Bacillus sp. from the 16S rRNA gene analysis. Very thin hyphae of R. solani was miccroscopically observed when the fungus was co-cultivated with KL1. Key words: biocontrol; endophyte; plant pathogens
Introduction Agriculture is a main industry to produce food, and the bacteria in soil assumed to be involved in the role of efficient food production. On the other hand other microbes such as Fusarium and Rhizoctonia species are widely distributed soil-borne pathogens and cause diseases on a wide range of economically important plant species (Garcia et al., 2006). Rhizoctonia solani is known to reduce the production of crops such as rice (Mew and Rosales, 1986), cucumber (Trillas et al., 2006) and lettuce (Grosch et al., 2011). Strategies to control Rhizoctonia diseases are limited and the disease is spreading all over the world. Fusarium oxysporum also invades roots and causes wilt diseases through colonization in xylem tissue of host plants, and reduces the production of crops. Synthetic chemical fungicides have long been used as active agents in reducing the plant diseases. However, they are costly, can cause environmental pollution, and may induce drug-resistant pathogens. Considering the limitations of chemical fungicides, it seems appropriate to search for a supplemental control strategy. Biological control, or the use of microorganisms to prevent plant diseases, offers an attractive alternative or supplement to chemical pesticides (San-Lang et al., 2002). Therefore, developing new biological control agents using antagonistic bacteria seems to be an ideal solution for this problem. We reported that endophyte have the * Corresponding author. E-mail: [email protected]
potential of used as new isolation source of biocontrol agent.
1 Materials and methods 1.1 Plant leaf samples Endophytic bacteria were isolated from 35 different plant leaf samples in Wakayama prefecture in Japan. 1.2 Screening of endophytes Surface of the leave samples was disinfected in 70% ethanol for 60 sec, followed by 1.0% NaOCl for 5 min, and 70% ethanol for 30 sec, thoroughly washed with sterile distilled water. After sterilization, leaf samples were cut into a 5-mm square and put on the potato dextrose broth agar medium (PDA) and trypto-soya broth agar medium (TSA), and incubated at 24°C or 30°C, respectively. Isolated endophytic bacteria were assayed for antifungal activity against F. oxysporum or R. solani on PDA and TSA plates. 1.3 Antifungal activity of endophytes The dual culture technique was employed to test antagonistic effect of KL1 on the growth of R. solani and F. oxysporum (Zhou et al., 2011). An agar plug of 0.7cm diameter from actively growing fungal mycelium was placed on the center of the PDA plate, and then KL1 were inoculated on the plate at 4 equidistance sites, 3 cm apart from the colony of R. solani or F. oxysporum in the center. Another plates were inoculated with the same
Suppl.
Screening of endophytic bacteria against fungal plant pathogens
size agar plug of fungal colony in the absence of KL1 as control. These plates were incubated at 24°C. And then the antagonistic effect of the KL1 on R. solani and F. oxysporum was observed. 1.4 Antifungal activity of KL1 supernatant and its thermal stability examination Antifungal activity and thermal stability of cell free supernatant from KL1 were tested by measuring the ability to inhibit fungal growth of R. solani on PDA agar plates. KL1 was grown in 300 mL conical flasks containing 60 mL of No. 3S medium (Ohno et al., 1995) at 30°C and for 120 hr. Cell free supernatant was obtained by centrifugation at 16,000 ×g at 24°C for 10 min followed by filtration through 0.20-µm cellulose acetate filter. An agar piece (0.7-cm diameter) of R. solani was placed at the center of Petri dishes containing PDA. Each of the 200 µL, 150 µL, 100 µL, 50 µL of cell free supernatant was put into a penicillin-cup placed on the PDA, respectively. Thermal stability of the cell free supernatant was examined as follows: 300 µL of cell free supernatant was heat treated in a dry heat block (Dry Thermo Bath MG-2200 Eyela, Tokyo, Japan) for 15 min at 50°C, 55°C, 60°C, 65°C, 70°C or 90°C. After the heat treatment, each supernatant was tested for the remaining antifungal activity. 1.5 Antifungal activity by PDA medium containing 5%–15% KL1 supernatant KL1 supernatant was sterilized by filtration through 0.20µm membrane filter (DISMIC-25cs Cellulose Acetate 0.20-µm, Advantec, Tokyo, Japan) and added to sterilized PDA (5%–15%, V/V) of final concentration) (Coda et al., 2011). After mixing of the samples, aliquots of 10 mL were poured into petri plates (50-mm diameter). Control plates contained PDA alone. The assay was carried out by placing 0.7-cm-diameter plugs of growing R. solani onto the center of petri dishes containing the culture medium. Plates were incubated aerobically at 24°C. Mycelia growth inhibition was calculated as: I=
C−T × 100 C
(1)
where, I (%) is the mycelia growth inhibition, C (mm) is the mycelia diameter in control, and T (mm) is the mycelia diameter in PDA containing KL1 supernatant (Forchetti et al., 2007). 1.6 Extraction and HPLC analysis of iturin A and surfactin For extraction of iturin A and surfactin, culture fluid was centrifuged at 16,000 ×g for 10 min. Then 500 µL of the supernatant was mixed with 500 µL of extraction buffer (CH3 CN:10 mmol/L ammonium acetate solution of 35:65 (V/V), and the mixture was vortexed for 10 min. The mixture was filtered through a 0.20-µm pore size polytetrafluoroethylene (PTFE) membrane (DISMIC-
S123
13JP, Advantec, Tokyo, Japan), and then injected into a high performance liquid chromatography (HPLC). HPLC analysis was performed using a JASCO LC-2000 series HPLC system (JASCO, Tokyo, Japan) equipped with a Chromolith Performance RP18e column at a flow rate of 2.0 mL/min. For separation, 10 mmol/L ammonium acetate aqueous solution and acetonitrile were used as mobile phase. The elution profile comprised an initial isocratic phase of 35% (iturin) or 45% (surfactin) acetonitrile for 10 min. Lipopeptide were detected by absorbance at 205 nm. Purified iturin A purchased from Sigma-Aldrich Co., USA and purified surfactin were used as standard samples for the HPLC. 1.7 Identification of antagonistic endophyte Bacterial 16S rRNA genes were PCRamplified with primers 357FWD-Bam (5′ -GGGGATCCTCCTACGGGAGGCAGCAG-3′ ) and 1100RV-Bam (5′ -CCGGATCCGGGTTGCGCTCGTTG3′ ). The sequences of the amplified product were compared by BLAST search and identification was done based on similarity up to species level.
2 Results and discussion To make sure the bacteria were isolated from the inside of the leaves, the surface sterilization was confirmed to be perfect by touching the surface of the leaves on TSA and PDA plates. One example is shown in Fig. 1. No colonies from the surface of the plant leaves were detected on the touching plates (Fig. 1a, c), but the colonies were emerged from inside of the leaves (Fig. 1b, d). Six endophytic bacteria were isolated from 35 different plant leaves, and two were further screened and an isolate named KL1 having been isolated from the chestnut tree (Castanea crenata) leaves showed a strong antifungal activity as shown in Fig. 2. Supernatant of the culture of KL1 also showed the strong inhibitory effect against the fungi, and the antifungal activity of supernatant was stable even after heating at 90°C for 15 min (Fig. 3). In addition, growth of R. solani was inhibited on PDA medium containing supernatant of KL1 (Fig. 4). The inhibition effect was kept even after the 14 days and stability of the suppressive effect is a suitable characteristic as a biocontrol agent. 16S rRNA gene of KL1 was amplified using FWD357 and RV1100 primers. Based on partial 16S rRNA gene sequence, the isolated strain KL1 showed high sequence identity (99%) to Bacillus sp. by BLAST analysis. From these features, it is suggested that the compound extracted from KL1 supernatant might be cyclic lipopeptide iturin A. Therefore, qualitative analysis of iturin A and surfactin by HPLC were performed for the extract of the supernatant of KL1. As a result, clear iturin A homolog peaks, which
Journal of Environmental Sciences 2013, 25(Suppl.) S122–S126 / Tatsuya Ohike et al.
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a
b
c
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Fig. 1 Isolation endophytic bacteria from a leaf of Castanea crenata on TSA (a, b) and PDA (c, d) plates. Surface sterilized leaves were put on the plate directly (a, c) to confirm the sterilization, and after that the leaves were cut into a 5-mm square and put on TSA (b) and PDA (d).
a
b
Fig. 2 Inihibition of the growth of Rhizoctonia solani (a) and Fusarium oxysporum (b) on PDA plate, by a newly isolated endophytic bacterium named KL1.
a
b
c
d
e
f
g
h
Fig. 3 Thermal stability of crude fungicides produced by an endophyte KL1. (a) inhibition of the growth of R. solani by KL1 cell free supernatant on PDA. Each stainless cup contains the supernatant, and the volume is 50, 100, 150, and 200 µL respectively clockwise from top-left. Inhibition of R. solani by the heat treated supernatant. Each 200 µL of the supernatant was heat treated for 15 min at 25°C (b), 50°C (c), 55°C (d), 60°C (e), 65°C (f), 70°C (g), 90°C (h), respectively, followed by the antifungal assay on PDA against R. solani.
5% 10% 15%
Inhibition ratio (%)
100 90 80 70 60 50 40 30 20 10 0
3
4
5
6
7 8 9 10 11 12 13 14 Incubation time (day) Fig. 4 Supernatant fluid of endophyte KL1 inhibited growth of R. solani. Inhibition of the growth of R. solani by the cell-free supernatant of the culture broth of KL1.
Suppl.
Screening of endophytic bacteria against fungal plant pathogens
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Crude extract by KL1 Relative intensity
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Time (min) Fig. 5 HPLC analysis of the lipopeptides produced by KL1. Homologs of iturin A were detected in the culture broth of KL1 (b), but, no surfactin was detected (d), in comparison with standard iturin A (200 mg/L) and surfactin (500 mg/L). Several peaks of iturin A (a) and surfactin (c) correspond to the lipopeptide with a fatty acid of different length of carbon chain.
a
b
Fig. 6
Microscopic analysis of the control R. solani (a) and R. solani in the boundary region co-cultivated with KL1 (b).
correspond to the lipopeptide with a C14 -C17 β-amino fatty acid, were detected (Fig. 5a, b), however, another lipopeptide surfactin, which is found in other Bacillus species, was not detected in the extract of KL1 (Fig. 5c, d). Microscopic analysis of the boundary region of cocultivation of KL1 with R. solani showed thinner and smooth hyphae of the fungus (Fig. 6a, b). As KL1 was able to suppress R. solani and F. oxysporum, which cause serious damages on many economically important agricultural crops, and is an endophyte, having more affinity for plants than other bacterial biocontrol agents, it was suggested that KL1 can be a good candidate as a biocontrol agent.
3 Conclusions We have screened endophytes in plant leaves. The isolated bacteria showed antagonistic activity against fungal plant pathogens. An isolate named KL1, showed stronger inihibition than other 34 isolates against plant pathogens (data not shown), Fusarium oxysporum and Rhizoctonia solani, on PDA as well as TSA plate. KL1 was identified as Bacillus sp. from the 16S rRNA gene analysis. These results suggest that KL1 can be a good candidate of a biocontrol agent, and as it was isolated from a plant leave it may have good affinity with plants, which is a suitable characteristic in the agriculture.
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Journal of Environmental Sciences 2013, 25(Suppl.) S122–S126 / Tatsuya Ohike et al.
References Coda R, Cassone A, Rizzello C G, Nionelli L, Cardinali G, Gobbetti M, 2011. Antifungal activity of Wickerhamomyces anomalus and Lactobacillus plantarum during sourdough fermentation: Identification of novel compounds and longterm effect during storage of wheat bread. Applied and Environmental Microbiology, 77: 3484–3492. Forchetti G, Masciarelli O, Alemano S, Alvarez D, Abdala G, 2007. Endophytic bacteria in sunflower (Helianthus annuus L.): isolation, characterization, and production of jasmonates and abscisic acid in culture medium. Applied Microbiology and Biotechnology, 76: 1145–1152. Garcia V G, Onco M A P, Susan V R, 2006. Review. Biology and systematics of the form genus Rhizoctonia. Spanish Journal of Agricultural Research, 4: 55–79. Grosch R, Schneider J H M, Kofoet A, Feller C, 2011. Impact of continuous cropping of Lettuce on the disease dynamics of bottom rot and genotypic diversity of Rhizoctonia solani AG 1-IB. Journal of Phytopathology, 159: 35–44.
Vol. 25
Ohno A, Ano T, Shoda M, 1995. Production of a lipopeptide antibiotic, surfactin, by recombinant Bacillus subtilis in solid state fermentation. Biotechnology and Bioengineering, 47: 209–214. Mew T W, Rosales A M, 1986. Bacterization of rice plants for control of sheath blight caused by Rhizoctonia solani. Phytopathology, 76: 1260–1264. San-Lang W, Shih I L, Wang C H, Tseng K C, Chang W T, Twu Y K et al., 2002. Production of antifungal compounds from chitin by Bacillus subtilis. Enzyme and Microbial Technology, 31: 321–328. Trillas M I, Casanova E, Cotxarrera L, Ordovas J, Borrero C, Aviles M, 2006. Composts from agricultural waste and the Trichoderma asperellum strain T-34 suppress Rhizoctonia solani in cucumber seedlings. Biological Control, 39: 32– 38. Zhou X, Lu Z, Lv F, Zhao H, Wang Y, Bie X, 2011. Antagonistic action of Bacillus subtilis strain fmbj on the postharvest pathogen Rhizopus stolonifer. Journal of Food Science, 76: M254–M259.
