Yong Seong Lee

Biocontrol potential of Lysobacter antibioticus HS124 against the root-knot nematode, Meloidogyne incognita, causing disease in tomato

Lysobacter antibioticus HS124 is an antagonistic bacterial strain that was previously isolated from the rhizosphere soil of pepper and showed an enhanced ability to produce lytic enzymes as well as an antibiotic that was identified as 4-hydroxyphenylacetic acid (4-HPAA). In the present study, nematicidal activity of the strain and 4-HPAA against the root-knot nematode, Meloidogyne incognita, causing disease in tomato was investigated in both in vitro and in vivo conditions. For this purpose, adding different concentrations of culture filtrate, crude extract collected from extraction with ethyl acetate and 4-HPAA, in 24-well plates containing ca 500 eggs or 300 second-stage juveniles (J2), significantly decreased the rate of nematode hatch and caused higher mortality of J2 compared with the control treatments. Nematicidal activity of the bacterial strain was further confirmed by conducting pot experiments in which tomato plants were inoculated with M. incognita and the HS124 culture (BC). The control pots were treated with commercial nematicide (CN, 5% Ethoprophos), tap water (TW) or the non-inoculated bacterial culture medium (BCM). In these pot experiments, results demonstrated a strong antagonistic potential of L. antibioticus HS124 against M. incognita where the disease was significantly reduced in the pots treated with BC as compared to TW or BCM. Furthermore, the shoot fresh weight was also increased significantly, which may be attributed to the disease control ability of the strain. Hence, L. antibioticus HS124 may be further developed as a potential biocontrol of root knot nematode in the field.

Biocontrol of Late Blight (Phytophthora capsici) Disease and Growth Promotion of Pepper by Burkholderia cepacia MPC-7

A chitinolytic bacterial strain having strong antifungal activity was isolated and identified as Burkholderia cepacia MPC-7 based on 16S rRNA gene analysis. MPC-7 solubilized insoluble phosphorous in hydroxyapatite agar media. It produced gluconic acid and 2-ketogluconic acid related to the decrease in pH of broth culture. The antagonist produced benzoic acid (BA) and phenylacetic acid (PA). The authentic compounds, BA and PA, showed a broad spectrum of antimicrobial activity against yeast, several bacterial and fungal pathogens in vitro. To demonstrate the biocontrol efficiency of MPC-7 on late blight disease caused by Phytophthora capsici, pepper plants in pot trials were treated with modified medium only (M), M plus zoospore inoculation (MP), MPC-7 cultured broth (B) and B plus zoospore inoculation (BP). With the sudden increase in root mortality, plants in MP wilted as early as five days after pathogen inoculation. However, plant in BP did not show any symptom of wilting until five days. Root mortality in BP was markedly reduced for as much as 50%. Plants in B had higher dry weight, P concentration in root, and larger leaf area compared to those in M and MP. These results suggested that B. cepacia MPC-7 should be considered as a candidate for the biological fertilizer as well as antimicrobial agent for pepper plants.

Effects on Meloidogyne incognita of chitinase, glucanase and a secondary metabolite from Streptomyces cacaoi GY525

Streptomyces cacaoi GY525, isolated from liquid compost containing crab shell powder, produced a secondary metabolite 3-benzyl-1,4-diaza-2,5-dioxobicyclo[4.3.0]nonane (BDDB) and lytic enzymes including chitinase and β-1,3-glucanase. To examine the effect of the bacterial culture filtrate (CF) and BDDB on mortality of second-stage juvenile (J2) and hatch inhibition of Meloidogyne incognita, different concentrations of them were added in 24-well plates containing ca 250 eggs and 250 J2, respectively. With increasing concentrations of CF and BDDB mortality of J2 increased while hatch decreased. In pot trials, tomato plants were treated with the GY525 culture (SC), culture medium (CM), commercial nematicide (5% ethoprophos) (CN) and combination of CN and SC (CN+SC). Tap water (TW) was used as a control. During the experimental periods, growth of tomato plants treated with SC markedly increased compared with TW and CN treatments. After 7 weeks the number of egg masses in TW was over 220, while that in SC was around 40 per plant. Population of J2 in soil and the number of egg masses in plant roots in SC, CN+SC and CN were significantly lower than those in TW and CM. Our results suggest that S. cacaoi GY525 has potential as a biological control agent against root-knot nematodes.

Statistical optimization of medium components for chitinase production by Pseudomonas fluorescens strain HN1205: role of chitinase on egg hatching inhibition of root-knot nematode

In the present research, statistical Plackett–Burman design (PBD) and central composite design (CCD) were used to optimize the mediums components in order to enhance the chitinase activity of Pseudomonas fluorescens strain HN1205. Among the seven nutritional elements that were studied, crab shell powder, CaCl2·2H2O and yeast extract were proved using PBD to have significant effect on chitinase activity. An optimal medium was obtained by applying a three-factor central composite design, which consisted of crab shell powder (1.0 g/L), CaCl2 (0.5 g/L) and yeast extract (0.5 g/L), with the highest chitinase activity of 1.03 U/mL. This value was 2.87-fold higher than the activity obtained in the lowest productive medium in the design matrix. The chitinase produced by the HN1205 strain was partially purified with 80% ammonium sulphate and anion-exchange chromatography and assessed for inhibition of hatching of nematode eggs. The partially purified chitinase significantly reduced the hatch of Meloidogyne incognita eggs (8.1%), as compared to the effect of 10 mmol/L Tris-HCl buffer (pH 8.0) (49.4%) and boiled chitinase (54.7%). This study demonstrated the role of chitinolytic enzymes produced by P. fluorescens strain HN1205. The obtained optimal activity proved that the enzymes could be potential biological control agents.

Nematicidal activity of Lysobacter capsici YS1215 and the role of gelatinolytic proteins against root-knot nematodes

Lysobacter capsici YS1215 is a soil-borne strain that could inhibit the growth of phytopathogenic fungi, including Phytophthora capsici, Rhizoctonia solani and Fusarium oxysporum, as well as root-knot nematodes. The effect of different concentrations of bacterial culture filtrate (BCF) of L. capsici YS1215 on the mortality of second-stage juveniles (J2) of Meloidogyne incognita was studied using 24-well plates. The J2 mortality increased with increasing concentrations of BCF. YS1215 also produces gelatinases in the culture filtrate. To study its role in nematicidal activities, the partial purification and the characterisation of gelatinolytic proteins were done from the culture medium of the YS1215. The partially purified proteins showed three clear bands with molecular weights estimated using zymography to be 255.7, 232.1 and 146.4 kDa. The optimal pH and temperature for the proteins were 8.0 and 40°C, respectively. The activity of the proteins was inhibited by ethylenediaminetetraacetic acid, FeCl3 and 1,10-phenanthroline, whereas it was activated by MnCl2. The proteins may belong to the group of metalloproteases. Moreover, the proteins could hydrolyse skimmed milk, collagen, gelatin and bovine serum albumin (BSA) as substrates, but not casein. The proteins could induce 75% J2 mortality in five days and degrade the J2 bodies. The present study demonstrates the role of the gelatinolytic proteins in the nematicidal potential of L. capsici YS1215.

Role of Lytic Enzymes Secreted by Lysobacter capsici YS1215 in the Control of Root-Knot Nematode of Tomato Plants

Lysobacter spp. are well known as biocontrol agents acting against fungal pathogens and root-knot nematode disease, which cause serious losses in a wide range of crops. In this study, the roles of the lytic enzymes from Lysobacter capsici YS1215, isolated from soil in Korea, was investigated for the biocontrol of root-knot nematode under in vitro and in vivo conditions. Chitinase and gelatinase activities of L. capsici YS1215 were enhanced by second stage nematode eggs and juveniles of Meloidogyne incognita into medium as substrates. The activities were confirmed by SDS-PAGE with active bands in chitinase active staining and gelatin zymography. The results clearly indicated that application of bacterial cultures and crab shell powder significantly increased the chitinase and gelatinase activities, as well as the population of chitinolytic and gelatinolytic bacteria in the rhizosphere. Furthermore, L. capsici YS1215 also promoted growth of shoot due to a decrease in the severity of disease caused by M. incognita in comparison with the control (fertilizer treatment). These results demonstrated that L. capsici YS1215 and its lytic enzymes played an important role in the suppression of M. incognita infection, and are likely to provide an alternative to synthetic nematicides used for the control of root-knot nematodes infections in tomato plants.

Purification and properties of a Meloidogyne-antagonistic chitinase from Lysobacter capsici YS1215

The root-knot nematodes, Meloidogyne spp., cause serious diseases in various plants and their chemical control may lead to environmental problems. Therefore, alternative control measures against the phytopathogenic nematodes are being sought. One of the potential targets against Meloidogyne spp. may be the chitinolysis and degradation of nematode eggs. Therefore, in the present study, a chitinolytic and nematicidal strain of Lysobacter capsici YS1215 was isolated from an agricultural field in Korea. The aim of this study was to purify chitinase secreted by L. capsici YS1215 and investigate its nematicidal role against Meloidogyne incognita. The chitinase secreted by L. capsici YS1215 was purified by protein precipitation with 80% ammonium sulphate, anion-exchange chromatography with DEAE-cellulose and gel-filtration chromatography with Sephadex G-100. By chitinase-active staining of the purified enzyme, a single band was obtained with an estimated molecular mass of 43.6 kDa. The optimal pH and optimal temperature for the highest chitinase activity were 6.0 and 40°C, respectively. The purified chitinase degraded the chitin layer of the eggshells and significantly reduced hatch of second-stage juveniles. The activity of chitinase secreted by L. capsici YS1215 was not affected by CoCl2, MnCl2, MgCl2, CuSO4, CaCl2 or EDTA. The purified enzyme could also hydrolyse swollen chitin, glycol chitin, glycol chitosan and chitin powder. Thus, the role of chitinase secreted by L. capsici YS1215 against Meloidogyne spp. may be useful for further development of a biocontrol agent.

Isolation and characterization of an antimicrobial lipopeptide produced by Paenibacillus ehimensis MA2012.

In this study, a novel lipopeptide antibiotic was isolated from the culture supernatant of Paenibacillus ehimensis strain MA2012. After analyses by mass spectrometry (MS), nuclear magnetic resonance (NMR), and high resolution mass spectrometry (HR‐MS/MS) the compound was identified to be polypeptin C consisting of 3‐hydroxy‐4‐methyl‐hexanoic acid moiety and nine amino acids as peptide body. It has the same molecular mass (1115 Da) with that of polypeptin A and B but the amino acid positions differ. A relatively low concentration (125 ppm) of polypeptin C lowered the surface tension of water from 72.2 to 36.4 mN/m. It showed antimicrobial activity against several plant pathogenic bacteria and fungi. When the polypeptin C was applied to the ripe pepper fruits previously inoculated with conidia of Colletotrichum gloeosporioides, the hyphal growth on the fruit was significantly suppressed. Moreover, the hyphal morphology of C. gloeosporioides was greatly affected by the purified compound. All these data suggest the great potential of P. ehimensis MA2012 to control plant fungal and bacterial diseases.

Isolation and Biocontrol Potential of Bacillus amyloliquefaciens Y1 against Fungal Plant Pathogens

This study was performed to investigate thermophilic bacteria from soil having broad antifungal spectrum against Rhizoctonia solani, Colletotrichum gloeosporioides, Phytophthora capsici, Fusarium oxysporum f.sp. lycopersici, and Botrytis cinerea. One isolate selected could resist heat shock of 60°C for one hour, and had broad antifungal activity in dual culture assay against all tested fungal pathogens and was identified as Bacillus amyloliquefaciens Y1 using 16S rRNA gene sequence. Further investigation for antifungal activity of bacterial culture filtrate (BCF) and butanol crude extract (BCE) of various concentrations showed broad spectrum antifungal activity and fungal growth inhibition significantly increased with increasing concentration with highest growth inhibition of 100% against R. solani with 50% BCF and 11 mm of zone of inhibition against R. solani with 4 mg BCE concentration. Treatment of butanol crude extract resulted in deformation, lysis or degradation of C. gloeosporioides and P. capsici hyphae. Furthermore, B. amyloliquefaciens Y1 produced volatile compounds inhibiting growth of R. solani (70%), C. gloeosporioides (65%) and P. capsici (65-70%) when tested in volatile assay. The results from the study suggest that B. amyloliquefaciens Y1 could be a biocontrol candidate to control fungal diseases in crops.

Promotion of Growth and Biocontrol of Brown Patch Disease by Inoculation of Paenibacillus ehimensis KWN38 in Bentgrass

Brown patch disease, caused by Rhizoctonia solani, is one of the most serious diseases of turf grasses including bentgrass (Agrostis palustris). This disease is usually controlled by different formulations of synthetic fungicides. In this study, two pot trials were carried out to investigate the biocontrol potential of the antagonistic strain Paenibacillus ehimensis KWN38 against R. solani, and to examine the growth response of bentgrass to the inoculated strain. Bentgrass mats with root system in plastic boxes were treated with winter grass medium (W), W plus P. ehimensis KWN38 inoculation (WP), summer grass medium (S), S plus P. ehimensis KWN38 inoculation (SP), fertilizer (F), and F plus fungicide (FF) once every 10 days for three months. After the first month, all pots were infected with R. solani. Sixty days after infection, the leaves of bentgrass in all control treatments (W, S, and F) showed high levels of infection, with significantly higher values than those of WP, SP and FF. The leaf fresh and dry weights of bentgrass innoculated with P. ehimensis KWN38 were all greater than the control treatments W, S and F. Similarly, both fresh and dry weights of the roots and the chlorophyll contents of bentgrass in WP and SP were significantly higher than those of W, S, F and FF. Moreover, bentgrass innoculated with P. ehimensis KWN38 grew faster and showed greater growth performance as compared to the other treatments in the second pot trial with infected bentgrass. A butanol extract of the P. ehinmensis KWN38 culture supernatant significantly reduced the number of fungal colonies initiating from sclerotial fragments on potato dextrose agar. The growth of monilioid cells of R. solani was delayed by treatment with P. ehimensis KWN38 culture broth, while they elongated earlier into thinner structures in the control treatment. In conclusion, the antagonist P. ehimensis KWN38 can be considered as an effective biocontrol agent against brown patch disease, as well as a promising plant growth promoting bacteria of bentgrass.