Nan Hee Yu

Diffusible and Volatile Antifungal Compounds Produced by an Antagonistic Bacillus velezensis G341 against Various Phytopathogenic Fungi

The aim of this study was to identify volatile and agar-diffusible antifungal metabolites produced by Bacillus sp. G341 with strong antifungal activity against various phytopathogenic fungi. Strain G341 isolated from four-year-old roots of Korean ginseng with rot symptoms was identified as Bacillus velezensis based on 16S rDNA and gyrA sequences. Strain G341 inhibited mycelial growth of all phytopathogenic fungi tested. In vivo experiment results revealed that n-butanol extract of fermentation broth effectively controlled the development of rice sheath blight, tomato gray mold, tomato late blight, wheat leaf rust, barley powdery mildew, and red pepper anthracnose. Two antifungal compounds were isolated from strain G341 and identified as bacillomycin L and fengycin A by MS/MS analysis. Moreover, volatile compounds emitted from strain G341 were found to be able to inhibit mycelial growth of various phytopathogenic fungi. Based on volatile compound profiles of strain G341 obtained through headspace collection and analysis on GC-MS, dimethylsulfoxide, 1-butanol, and 3-hydroxy-2-butanone (acetoin) were identified. Taken together, these results suggest that B. valezensis G341 can be used as a biocontrol agent for various plant diseases caused by phytopathogenic fungi.

Characterization of Bacillus amyloliquefaciens DA12 Showing Potent Antifungal Activity against Mycotoxigenic Fusarium Species

In an attempt to develop a biological control agent against mycotoxigenic Fusarium species, we isolated Bacillus amyloliquefaciens strain DA12 from soil and explored its antimicrobial activities. DA12 was active against the growth of mycotoxigenic F. asiaticum, F. graminearum, F. proliferatum, and F. verticillioides both in vitro and in planta (maize). Further screening using dual culture extended the activity range of strain DA12 against other fungal pathogens including Botrytis cinerea, Colletotrichum coccodes, Endothia parasitica, Fusarium oxysporum, Raffaelea quercus-mongolicae, and Rhizoctonia solani. The butanol extract of the culture filtrate of B. amyloliquefaciens DA12 highly inhibited the germination of F. graminearum macroconidia with inhibition rate 83% at a concentration of 31.3 μg/ml and 100% at a concentration of 250 μg/ml. The antifungal metabolite from the butanol extract was identified as iturin A by thin layer chromatography-bioautography. In addition, volatile organic compounds produced by DA12 were able to inhibit mycelial growth of various phytopathogenic fungi. The volatile compounds were identified as 2-heptanone, 5-methyl heptanone and 6-methyl heptanone by gas chromatography-mass spectrometry (GC-MS) analysis. These results indicate that the antagonistic activity of Bacillus amyloliquefaciens DA12 was attributable to iturin A and volatile heptanones, and the strain could be used as a biocontrol agent to reduce the development of Fusarium diseases and mycotoxin contamination of crops.

Chemosensitization of Fusarium graminearum to Chemical Fungicides Using Cyclic Lipopeptides Produced by Bacillus amyloliquefaciens Strain JCK-12

Fusarium head blight (FHB) caused by infection with Fusarium graminearum leads to enormous losses to crop growers, and may contaminate grains with a number of Fusarium mycotoxins that pose serious risks to human and animal health. Antagonistic bacteria that are used to prevent FHB offer attractive alternatives or supplements to synthetic fungicides for controlling FHB without the negative effects of chemical management. Out of 500 bacterial strains isolated from soil, Bacillus amyloliquefaciens JCK-12 showed strong antifungal activity and was considered a potential source for control strategies to reduce FHB. B. amyloliquefaciens JCK-12 produces several cyclic lipopeptides (CLPs) including iturin A, fengycin, and surfactin. Iturin A inhibits spore germination of F. graminearum. Fengycin or surfactin alone did not display any inhibitory activity against spore germination at concentrations less than 30 μg/ml, but a mixture of iturin A, fengycin, and surfactin showed a remarkable synergistic inhibitory effect on F. graminearum spore germination. The fermentation broth and formulation of B. amyloliquefaciens JCK-12 strain reduced the disease incidence of FHB in wheat. Furthermore, co-application of B. amyloliquefaciens JCK-12 and chemical fungicides resulted in synergistic in vitro antifungal effects and significant disease control efficacy against FHB under greenhouse and field conditions, suggesting that B. amyloliquefaciens JCK-12 has a strong chemosensitizing effect. The synergistic antifungal effect of B. amyloliquefaciens JCK-12 and chemical fungicides in combination may result from the cell wall damage and altered cell membrane permeability in the phytopathogenic fungi caused by the CLP mixtures and subsequent increased sensitivity of F. graminearum to fungicides. In addition, B. amyloliquefaciens JCK-12 showed the potential to reduce trichothecenes mycotoxin production. The results of this study indicate that B. amyloliquefaciens JCK-12 could be used as an available biocontrol agent or as a chemosensitizer to chemical fungicides for controlling FHB disease and as a strategy for preventing the contamination of harvested crops with mycotoxins.

Antibacterial activities of penicillic acid isolated from Aspergillus persii against various plant pathogenic bacteria

The emergence of pathogenic bacterial strains resistant to agrochemicals and the increasing demand for organic foods have led to the discovery of new antibacterial metabolites that can be used either directly or as a lead molecule for development of synthetic bactericides. During the screening of antibacterial fungal cultures, we found that one fungal strain, Aspergillus persii EML‐HPB1‐11, showed strong in vitro antibacterial activity against Xanthomonas arboricola pv. pruni (Xap) with a minimum inhibitory concentration (MIC) of 10% of fermentation broth filtrate. The active compound was identified as penicillic acid (PA: 3‐methoxy‐5‐methyl‐4‐oxo‐2,5‐hexadienoic acid) by mass and NMR spectroscopy. The in vitro antibacterial activity of PA was tested against 12 phytopathogenic bacteria. All of the bacterial pathogens tested were highly inhibited by PA with MIC values of 12·3–111·1 μg ml−1. It also effectively suppressed the development of bacterial spot disease in detached peach leaves, showing control values of 82·4 and 94·1% at concentrations of 111·1 and 333·3 μg ml−1 respectively. This is the first report on the production of PA by A. persii. This study suggests that PA can be used as a lead molecule for development of synthetic bactericides for control of various plant diseases.

Nematicidal activity of grammicin produced by Xylaria grammica KCTC 13121BP against Meloidogyne incognita

BACKGROUND The endolichenic fungus Xylaria grammica KCTC 13121BP showed strong nematicidal activity against Meloidogyne incognita. This study aimed to identify the nematicidal metabolites and to evaluate the efficacy of the strain as a biocontrol agent under pot and field conditions. RESULTS Bioassay-guided fractionation and instrumental analyses led to grammicin being identified as the nematicidal metabolite. Because patulin is a mycotoxic isomer of grammicin and is known to have strong antibacterial and cytotoxic activities, several biological activities of the two compounds were compared. Grammicin showed strong second-stage juvenile killing and egg-hatching inhibitory effects, with a 50% effective concentration at 72 h (EC50/72 h ) of 15.9 µg/mL and a 50% effective concentration at 14 days (EC50/14 days ) of 5.87 µg/mL, respectively, whereas patulin was virtually inactive in both respects. Patulin was strongly active toward various phytopathogenic bacteria in vitro, whereas grammicin was weakly so. Patulin at the concentration range of 0.1-10 µg/mL also showed dose-dependent cytotoxicity toward the human first-trimester trophoblast cell line SW.71, whereas grammicin was not toxic toward this cell line. In pot and field experiments, a wettable powder-type formulation and fermentation broth filtrate of X. grammica KCTC 13121BP effectively suppressed the development of root-knot nematode disease on tomato and melon plants. CONCLUSION The results suggest that X. grammica and grammicin may have potential applications for control of root-knot nematode disease of various crops.

Nematicidal activity of verrucarin A and roridin A isolated from Myrothecium verrucaria against Meloidogyne incognita

Abstract The widespread use of synthetic nematicides has caused significant problems to the environment as well as human health. To address this issue, eco-friendly control measures, such as microbial nematicides, are being developed. During the screening of Myrothecium strains with nematicidal activity against the root-knot nematode (RKN) Meloidogyne incognita , we found that the acetone extract of Myrothecium sp. KACC 40321 was highly effective against hatched juveniles of M. incognita at 7 days after exposure. The fungus was identified as Meloidogyne verrucaria . Two macrocyclic trichothecenes verrucarin A and roridin A were isolated and identified as major active metabolites by bioassay-guided fractionation and instrumental analysis. When the second-stage juveniles were treated with the chemicals, no juvenile mortality was observed. However, they effectively killed juveniles from treated eggs. The hatched juvenile mortality was used to evaluate the in vitro nematicidal activity of the compounds against M. incognita . The median effective concentrations were 1.88 μg/mL for verrucarin A and 1.50 μg/mL for roridin A. Among various liquid media, commercial malt extract broth (cMEB) was found to be the best for the production of verrucarin A and roridin A, followed by potato dextrose broth. The cMEB culture filtrate effectively reduced the formation of galls and egg masses on tomato roots in a pot experiment. In addition, the culture filtrate reduced the formation of galls on the roots of melon plants and the number of RKNs in the soils under field conditions. These results suggest that M. verrucaria KACC 40321 can be used as a biocontrol agent against RKNs in various crops. To the best of our knowledge, this is the first study to report the effectiveness of verrucarin A and rorridin A against hatched juveniles of M. incognita .

Antimicrobial activities of an oxygenated cyclohexanone derivative isolated from Amphirosellinia nigrospora JS-1675 against various plant pathogenic bacteria and fungi

To evaluate the antimicrobial activities of an active compound isolated from the culture broth of Amphirosellinia nigrospora JS‐1675 against various plant pathogenic bacteria and fungi.

Development of a Biofungicide Using a Mycoparasitic Fungus Simplicillium lamellicola BCP and Its Control Efficacy against Gray Mold Diseases of Tomato and Ginseng

To develop a commercial product using the mycoparasitic fungus Simplicillium lamellicola BCP, the scaleup of conidia production from a 5-l jar to a 5,000-l pilot bioreactor, optimization of the freeze-drying of the fermentation broth, and preparation of a wettable powder-type formulation were performed. Then, its disease control efficacy was evaluated against gray mold diseases of tomato and ginseng plants in field conditions. The final conidial yields of S. lamellicola BCP were 3.3 × 109 conidia/ml for a 5-l jar, 3.5 × 109 conidia/ml for a 500-l pilot vessel, and 3.1 × 109 conidia/ml for a 5,000-l pilot bioreactor. The conidial yield in the 5,000-l pilot bioreactor was comparable to that in the 5-l jar and 500-l pilot vessel. On the other hand, the highest conidial viability of 86% was obtained by the freeze-drying method using an additive combination of lactose, trehalose, soybean meal, and glycerin. Using the freeze-dried sample, a wettable powder-type formulation (active ingredient 10%; BCP-WP10) was prepared. A conidial viability of more than 50% was maintained in BCP-WP10 until 22 weeks for storage at 40°C. BCP-WP10 effectively suppressed the development of gray mold disease on tomato with control efficacies of 64.7% and 82.6% at 500- and 250-fold dilutions, respectively. It also reduced the incidence of gray mold on ginseng by 65.6% and 81.3% at 500- and 250-fold dilutions, respectively. The results indicated that the new microbial fungicide BCP-WP10 can be used widely to control gray mold diseases of various crops including tomato and ginseng.

Antibacterial Activity of Pharbitin, Isolated from the Seeds of Pharbitis nil, against Various Plant Pathogenic Bacteria

This study aimed to isolate and characterize antibacterial metabolites from Pharbitis nil seeds and investigate their antibacterial activity against various plant pathogenic bacteria. The methanol extract of P. nil seeds showed the strongest activity against Xanthomonas arboricola pv. pruni (Xap) with a minimum inhibition concentration (MIC) value of 250 μg/ml. Among the three solvent layers obtained from the methanol extract of P. nil seeds, only the butanol layer displayed the activity with an MIC value of 125 μg/ml against Xap. An antibacterial fraction was obtained from P. nil seeds by repeated column chromatography and identified as pharbitin, a crude resin glycoside, by instrumental analysis. The antibacterial activity of pharbitin was tested in vitro against 14 phytopathogenic bacteria, and it was found to inhibit Ralstonia solanacearum and four Xanthomonas species. The minimum inhibitory concentration values against the five bacteria were 125-500 μg/ml for the n-butanol layer and 31.25-125 μg/ml for pharbitin. In a detached peach leaf assay, it effectively suppressed the development of bacterial leaf spot, with a control value of 87.5% at 500 μg/ml. In addition, pharbitin strongly reduced the development of bacterial wilt on tomato seedlings by 97.4% at 250 μg/ml, 7 days after inoculation. These findings suggest that the crude extract of P. nil seeds can be used as an alternative biopesticide for the control of plant diseases caused by R. solanacearum and Xanthomonas spp. This is the first report on the antibacterial activity of pharbitin against phytopathogenic bacteria.