Kyaw Wai Naing

Characterization of antifungal activity of Paenibacillus ehimensis KWN38 against soilborne phytopathogenic fungi belonging to various taxonomic groups

Soilborne fungal phytopathogens cause significant losses in many economically important crops and vegetables. The only way to control these devastating pathogens is by using higher doses of fungicides which not only increase the cost of production but also cause significant damage to the environment. Therefore alternate control measures are always looked for. In the present study, an antagonistic strain was isolated from the soil of the pepper fields around the seashore of Jellanamdo, South Korea and identified as Paenibacillus ehimensis KWN38 based on 16S rRNA sequencing. The strain showed high antifungal activity against six tested fungal pathogens belonging to various taxonomic groups on dual culture plates. Furthermore, the strain produced volatile antimicrobial compounds which had strong fungal growth inhibitory effect. The strain also showed high chitinase, cellulase, glucanase and protease activities. The hyphal morphologies of Rhizoctonia solani AG-1 (IA), Fusarium oxysporum f.sp. lycopersici and Phytophthora capsici were significantly destroyed by the crude enzymes and butanol extract from the culture supernatant and the affected hyphae showed abnormal bending, tip curling, and irregular branching. Hence, Paenibacillus ehimensis KWN38 is considered as a potential biocontrol agent of the soil-borne fungi causing plant diseases which is an important perspective of the present study.

Biocontrol Potential of Streptomyces griseus H7602 Against Root Rot Disease (Phytophthora capsici) in Pepper

Corporation of Nature and People, Gunsan 573-952, Korea(Received on March 29, 2012; Revised on May 7, 2012; Accepted on May 24, 2012)The root rot of pepper ( Capsicum annuum L.) caused byPhytophthora capsici is one of the most important di-seases affecting this crop worldwide. This work presentsthe evaluation of the capacity of Streptomyces griseusH7602 to protect pepper plants against Phytophthoracapsici and establishes its role as a biocontrol agent. Inthis study, we isolated an actinomycete strain H7602from rhizosphere soil, identified it as Streptomyces griseusby 16S rRNA analysis and demonstrated its antifungalactivity against various plant pathogens including P.capsici. H7602 produced lytic emzymes such as chitinase,β-1,3-glucanase, lipase and protease. In addition, crudeextract from H7602 also exhibited destructive activitytoward P. capsici hyphae. In the pot trial, results showedthe protective effect of H7602 against pepper from P.capsici. Application of H7602 culture suspension reduced47.35% of root mortality and enhanced growth ofpepper plants for 56.37% in fresh root and 17.56% g infresh shoot as compared to control, resulting in greaterprotection to pepper plants against P. capsici infestation.Additionally, the enzymatic activities, chitinase and β-1,3-glucanase, were higher in rhizosphere soil and rootsof pepper plants treated with H7602 than other treatedplants. Therefore, our results indicated a clear potentialof S. griseus H7602 to be used for biocontrol of root rotdisease caused by P. capsici in pepper. Keywords : antifungal activity, biocontrol, Phytophthoracapsici, Streptomyces griseusBased on increasing public concern about residues offungicides in food products, as well as about their soil-degrading effects, availability of a sustainable and environ-mentally friendly method for disease control in pepper ishighly desirable. Technical, economical and environmentalfactors are forcing to adopt new sustainable methods, suchas use of microbial antagonists for the control of soilbornepathogens. Recently, the interest in biological control bybeneficial microorganisms has increased consistently as analternative disease control to substitute for various chemicalcontrols against airborne or soilborne plant pathogens (Anet al., 2010; Mukherjee and Sen, 2006; Sang et al., 2011),and antagonistic microorganisms have been shown toinhibit the growth and proliferation of various phytopatho-gens with little or no side effects (Arthurs et al., 2009).Phytophthora root rot (PRR), caused by Phytophthoracapsici, is one of the most devastating soilborne diseases inthe world. Due to a lack of resistant cultivars, control ofsoilborne pathogens of pepper is mainly aimed at P. capsiciand involves numerous applications of fungicides bothbefore and after transplanting in the field (Hwan and Kim,1995). To combat wilt caused by P. capsici in pepper,cultural practices based on the biology and ecology of thepathogen have been suggested (Ristaino and Johnston,1999). One of the strategies is use of biocontrol agents inorder to reduce the number of applications of fungicidesand the pathogen population resistant to the fungicides(Sang et al., 2008).There have been considerable efforts to find biologicalcontrol agents against PRR of pepper and several potentialcandidates have been reported including: Penicilliumstriatisporum (Ma et al., 2008), Pseudomonas fluorescens(Paul and Sarma, 2006), and Streptomyces rochei (Ezziyyaniet al., 2007). The mechanisms of disease suppression byantagonistic bacteria have been extensively studied andthey involved the production of antibiotics, extracellularenzymes, biosurfactant or cyanide production and/or the

Antagonism of antifungal metabolites from Streptomyces griseus H7602 against Phytophthora capsici

In this study, evidences for antagonism were established by production of antifungal metabolites from Streptomyces griseus H7602, which were active to inhibit mycelial growth of Phytophthora capsici in the in vitro assays. Mycelial growth and zoosporangia formation of P. capsici was strongly inhibited in the medium containing the cell free culture filtrate of S. griseus H7602. Antifungal metabolites from the cell free culture filtrate of S. griseus H7602 showed substantial antagonistic effects on P. capsici. In addition, a purified antifungal compound was separated from the antifungal metabolites of S. griseus H7602 and identified to be 1H‐pyrrole‐2‐carboxylic acid (PCA) by spectra analyses. PCA showed strong antifungal activity and was evaluated for the first time for its antagonism against P. capsici under in vitro conditions. Minimum inhibitory concentration (MIC) value of PCA was low (4 µg ml−1), and the mycelial growth of P. capsici was almost inhibited at concentration of 64 µg ml−1. This study suggests that the PCA may be useful as biofungicides against P. capsici, and the prominent antagonism of antifungal metabolites from S. griseus H7602 highlights it as a candidate for biocontrol of P. capsici.

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.

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.

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.

Antagonistic potential of Paenibacillus elgii HOA73 against root-knot nematode (Meloidogyne incognita)

This study was conducted to evaluate the nematicidal potential of Paenibacillus elgii strain HOA73 against the root-knot nematode, Meloidogyne incognita, under both in vitro and in vivo conditions. The novel strain HOA73 (GenBank accession number JQ12069) produced hydrolytic enzymes including gelatinase and chitinase. Exposure of M. incognita to various concentrations (10-30%) of the bacterial culture filtrate (BCF) or 0.05-0.4 mg ml−1 of the crude enzymes produced by P. elgii HOA73 significantly reduced hatch of the second-stage juvenile (J2) and caused substantial mortality under in vitro conditions. The hatch inhibition and J2 mortality rate was enhanced with the increase in the concentration of BCF and the crude enzymes. Similarly, the antagonistic effects also increased significantly over the time for which the treatments were given. Moreover, the crude enzymes partially destroyed cuticle layers of eggs and juveniles at 0.4 mg ml−1 concentration. Furthermore, the pot experiment indicated that addition of HOA73 culture into potted soil significantly reduced the root galling, number of egg masses and nematode population by 62.1%, 69.8% and 53.0%, respectively, as compared to the control at 7 weeks after M. incognita infestation. HOA73 inoculation also promoted tomato plant fresh shoot weight by 17.9% as compared to control. Hence, the present results demonstrated the ability of P. elgii HOA73 as a potential biocontrol candidate against the root-knot nematode as well as a plant growth promoter for tomato.

Isolation and characteristics of protocatechuic acid from Paenibacillus elgii HOA73 against Botrytis cinerea on strawberry fruits

This study was undertaken to describe purification, identification, and characteristics of protocatechuic acid (PCA) isolated for the first time from Paenibacillus elgii HOA73 against Botrytis cinerea (the cause of gray mold disease on strawberry fruit). PCA was purified by different chromatographic techniques and identified as PCA (3,4‐dihydroxybenzoic acid) by nuclear magnetic resonance and liquid chromatography–mass spectrometry analyses. PCA displayed potent antifungal activity against B. cinerea and Rhizoctonia solani. However, the antifungal activities were not sufficient to inhibit mycelial growth of Phytophthora capsici and Fusarium oxysporum. The minimum inhibitory concentration of PCA to inhibit any visible mycelial growth of both B. cinerea and R. solani was 64 µg ml−1. Most B. cinerea conidia displayed altered shape and absence of germination, or were degraded after treatment with 50 and 100 µg ml−1 PCA, respectively. Moreover, gray mold formation on strawberry fruit was almost or completely inhibited by these PCA concentrations 7 days following infection with B. cinerea conidia, respectively. PCA may be a promising alternative to chemical fungicides as a potential biofungicide to prevent growth of B. cinerea in strawberry fruit disease management.

Biological Control of Root-knot Nematode by Streptomyces sampsonii KK1024

Streptomyces sampsonii KK1024 having strong chitinolytic activity was isolated from crab-shell rich soil at Muan, Jeolanamdo. The KK1024 produced chitinase, protease, gelatinase and lipase. When 50% of KK1024 culture broth was treated to juveniles and eggs of root-knot nematode, juvenile mortality at 3 days was 81.67% and egg hatch rate at 5 days was 2.00%. When of crude enzyme produced by KK1024 was treated, juvenile mortality at 3 days was 96.00% and egg hatch rate at 5 days was 5.33%. At 1% of butanol extract from KK1024, juvenile mortality was highest with 90.00% and egg hatch rate was lowest with 0%. The comparison of the effect of KK1024 culture broth with only medium, synthetic fertilizer, and commercial nematicide on tomato growth and nematode infection was examined in pot trials. KK1024 culture broth showed lower number of egg mass and gall in plant, and population of juveniles in soil compared with only medium and synthetic fertilizer treatment, but not in commercial nematicide. However, the highest shoot weight and length was discovered in KK1024 culture broth. These results suggest that Streptomyces sampsonii KK1024 producing lytic enzymes and nematicidal compounds can be one of candidates for biocontrol agents against root-knot nematodes.

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.