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The fungal isolate Isaria javanica pf185 has potential as a mycopesticide because it demonstrates insecticidal activity against the green peach aphid and antifungal activity against Colletotrichum gloeosporioides. For commercialization of this isolate, determination of the optimal and least expensive culture conditions is required; however, these data are not currently available. This study describes the conditions for optimal development of conidia and production of metabolites for the biocontrol of the fungal pathogen. The optimal culture conditions were examined using cultures on solid agar and liquid media. High growth temperature enhanced spore formation but reduced antifungal activity in both solid and liquid media. The highest spore yield was obtained in a medium containing glucose as a carbon source and yeast extract as a nitrogen source. Soybean powder and wheat bran were effective nitrogen sources that promoted spore production and antifungal activity of the isolate. These results revealed the basic, cost-effective growth media for commercial production of a biopesticide with insecticidal and antifungal properties for use in integrated pest management.

A new hybrid non-ribosomal peptide-polyketide antibiotic (serratamid) for phytoprotection was isolated from the ethyl acetate layer of tryptic soy agar culture of the soil bacterium Serratia plymuthica C1 through bioassay-guided fractionation. Its chemical structure was elucidated using instrumental analyses, such as mass and nuclear magnetic resonance spectrometry. Serratamid showed antibacterial activity against 15 phytopathogenic bacteria, with minimum inhibitory concentration (MIC) values ranging from 0.244 to 31.25 μg/mL. In vitro, it displayed strong antibacterial activity against Ralstonia solanacearum and four Xanthomonas spp., with MIC values (0.244-0.488 μg/mL) superior to those of streptomycin sulfate, oxolinic acid, and oxytetracycline. Further, serratamid and the ethyl acetate layer of S. plymuthica C1 effectively reduced bacterial wilt caused by R. solanacearum on tomato seedlings and fire blight caused by Erwinia on apple fruits in a dose-dependent manner. These results suggest that serratamid is a promising candidate as a potent bactericide for controlling bacterial diseases.

In 2019, symptoms of Botrytis mold on the peony (Paeonia lactiflora Pall.) 'Sarah Bernhardt' were observed during a survey of the commercial greenhouses of Gangjin County, South Korea. The initial symptoms, small brown spots, were observed mainly at the leaf margins. The lesions extended to the interior of leaves forming irregular spots in which abundant conidia developed. Fungal colonies were obtained from surface-sterilized tissue excised from growing edges of the lesions that were transferred to potato dextrose agar. Melanized irregular sclerotia were formed in these colonies after 40 days at 8°C. Molecular phylogeny based on sequences of genes for glyceraldehyde-3-phosphate dehydrogenase, heat-shock protein 60, and RNA polymerase subunit II were highest for the PBC-2 isolate to the type strains of Botrytis cinerea, rather than other Botrytis species associated with peony diseases. Following Koch's postulates, healthy Sarah Bernhardt plants were inoculated with a foliar application of conidial suspensions of the isolate PBC-2. Following incubation under humidity with a 12 hr photoperiod for 7 days, symptoms developed on the leaf margins that were identical to those observed in the greenhouses. This study is the first report of Botrytis blight caused by B. cinerea on peonies grown in commercial greenhouses in South Korea.

Serratia plymuthica C-1, a biocontrol agent, was isolated from soil collected from a mountain forest in Korea. Previous studies have shown that certain strains of S. plymuthica cause root rot disease in ginseng. To the best of our knowledge, this is the first report of the sequence of the circular chromosome of S. plymuthica C-1, which plays a dual role by causing root rot in ginseng and exhibiting biocontrol activity. The findings of this study will assist in analyzing the genes associated with the pathogenicity and biocontrol properties of S. plymuthica.

Pseudomonas coronafaciens X-1 was isolated from halo blight lesions on oat leaves in Korea. Currently, no genome resource on the oat halo blight pathogen is available. Here, the complete genome sequence of this strain, containing a circular chromosome and one circular megaplasmid is reported. This is the first genome sequence of the oat halo blight pathogen and will contribute to our understanding of the pathogenicity and ecological aspects of the halo blight pathogen and the interactions between P. coronafaciens and oats.

The chitinase-producing bacterium, Paenibacillus elgii HOA73, is a biocontrol agent that limits the damage caused to plants by microbial pathogens, insects, and nematodes. However, the mechanisms involved in the biocontrol of plant diseases by HOA73 have not been determined. The objective of this study was to elucidate the role of extracellular chitinase obtained from isolate HOA73 in the control of the fungal pathogen Botrytis cinerea, the causative agent of gray mold in tomato. The HOA73 strain grew efficiently in a chitin-containing broth and produced chitin oligomers through chitinase activity; protease, lipase, and Fe-chelating siderophores were also secreted by the bacterium. Cultures containing intact bacteria inhibited B. cinerea conidia germination to a greater extent than did the bacterial cells alone or the cell-free culture supernatant. The antifungal activity increased with culture age and was heat-sensitive because of chitinase-mediated production of long-chain chitin oligomers. The biocontrol efficacy of undiluted bacterial cultures against gray mold in tomato was comparable to that of a standard fungicide. This study demonstrated that P. elgii HOA73 bacterial cultures grown on chitin-based minimal medium may be an effective formulation for the integrated control of gray mold.

We present the data showing that overexpression of the Arabidopsis zinc finger homeodomain protein ZHD5 promotes shoot regeneration and confers other cytokinin-associated phenotypes. To isolate protein factors that bind to the promoter element of ABF2, which is a key regulator of ABA responsive gene expression, we carried out yeast-one-hybrid screen. Four zinc finger homeodomain (ZF-HD) protein genes were identified from the screen. Expression study showed that one of the ZF-HD proteins, ZHD5, is induced by various cytokinins (CKs). We generated and analyzed ZHD5 overexpression (OX) lines to study its function. Our results revealed that ZHD5 promoted shoot regeneration efficiency significantly, obviating CK requirement. Other CK-related phenotypes, such as enhanced cell proliferation and pathogen resistance, were also observed. At the molecular level, changes in the expression levels of a number of genes relevant to the phenotypes were observed. Collectively, our results provde strong evidence that ZHD5 is involved in shoot regeneration and other aspects of CK response.

Dual biocontrol of both insects and plant pathogens has been reported for certain fungal entomopathogens, including Beauveria bassiana and Lecanicillum spp. In this study, we demonstrate, for the first time, the dual biocontrol potential of two fungal isolates identified by morphological and phylogenetic analyses as Isaria javanica. Both these isolates caused mortality in the greater wax moth, and hence can be considered entomopathogens. Spores of the isolates were also pathogenic to nymphs of the green peach aphid (Myzus persicae), with an LC50 value of 107 spores/mL 4 days after inoculation and an LT50 of 4.2 days with a dose of 108 spores/mL. In vitro antifungal assays also demonstrated a strong inhibitory effect on the growth of two fungi that are pathogenic to peppers, Colletotrichum gloeosporioides and Phytophthora capsici. These results indicate that I. javanica isolates could be used as novel biocontrol agents for the simultaneous control of aphids and fungal diseases, such as anthracnose and Phytophthora blight, in an integrated pest management framework for red pepper.

An effective bioformulation of mixtures of chitin-degrading bacteria has been used successfully to control plant diseases and nematodes. In this study, the bioformulation approach was assessed to control damping-off disease of ginseng. In pot experiments with soils infested with dapming-off pathogens of ginseng, root-drenchings of Chrobacterium sp. C-61, Lysobacterium enzymogenes C-3, and mixture of two bacterial strains grown in chitin minimal medium were signficantly increased emergence of seeds and reduced damping-off disease incidence of seedlings. Efficacy of the bioformulated product depended on the dose and timing of application. In two-year-old ginseng field, the high control efficacies were achieved by soil drenching of two times with an undiluted product or three times with a 10-fold diluted product. In a To-jik nursery (self soil nursery), biocontrol efficacy of the undiluted product against damping-off disease were similar to that of a seed dressing with fungicide, Tolclofos-methyl WP. These results suggest that the bioformulated product containing Chromobacterium sp. C-61 and L. enzymogenes C-3 could be an effective approach to control of ginseng damping-off disease.

A plethora of compounds stimulate protective mechanisms in plants against microbial pathogens and abiotic stresses. Some defense activators are synthetic compounds and trigger responses only in certain protective pathways, such as activation of defenses under regulation by the plant regulator, salicylic acid (SA). This review discusses the potential of naturally occurring plant metabolites as primers for defense responses in the plant. The production of the metabolites, hexanoic acid and melatonin, in plants means they are consumed when plants are eaten as foods. Both metabolites prime stronger and more rapid activation of plant defense upon subsequent stress. Because these metabolites trigger protective measures in the plant they can be considered as “vaccines” to promote plant vigor. Hexanoic acid and melatonin instigate systemic changes in plant metabolism associated with both of the major defense pathways, those regulated by SA- and jasmonic acid (JA). These two pathways are well studied because of their induction by different microbial triggers: necrosis-causing microbial pathogens induce the SA pathway whereas colonization by beneficial microbes stimulates the JA pathway. The plant’s responses to the two metabolites, however, are not identical with a major difference being a characterized growth response with melatonin but not hexanoic acid. As primers for plant defense, hexanoic acid and melatonin have the potential to be successfully integrated into vaccination-like strategies to protect plants against diseases and abiotic stresses that do not involve man-made chemicals.

The integration of a complex of pathways involving growth promotion and stress tolerance in response to bacterial volatile organic compounds (VOCs) is remarkable in enhancing plant health. VOCs are emitted from microbes associated with the plant. In this chapter, we focus on the mechanisms involving the array of structurally diverse VOCs that are active in maintaining a balance for the plant under abiotic and biotic stress. Certain VOCs boost plant growth, act as direct deterrents of pests, or are activators of systemic plant defense mechanisms. The responses to the VOCs involved in plant health engage many different trophic levels and advantageously can occur at distance from the original signal. Thus, VOC sensing has a holistic aspect for plant welfare.

Pseudomonas chlororaphis O6 induces systemic tolerance in plants against drought stress. A volatile, 2R, 3R-butanediol, produced by the bacterium causes partial stomatal closure, thus, limiting water loss from the plant. In this study, we report that applications of extracellular polymeric substances (EPS) from P. chlororaphis O6 to epidermal peels of leaves of Arabidopsis thaliana also reduce the size of stomatal openings. Growth of A. thaliana seedlings with applications of the EPS from P. chlororaphis O6 reduced the extent of wilting when water was withheld from the plants. Fluorescence measurements showed photosystem II was protected in the A. thaliana leaves in the water stressed EPS-exposed plants. These findings indicate that P. chlororaphis O6 has redundancy in traits associated with induction of mechanisms to limit water stress in plants.

Many root-colonizing microbes are multifaceted in traits that improve plant health. Although isolates designated as biological control agents directly reduce pathogen growth, many exert additional beneficial features that parallel changes induced in animal and other hosts by health-promoting microbes termed probiotics. Both animal and plant probiotics cause direct antagonism of pathogens and induce systemic immunity in the host to pathogens and other stresses. They also alter host development and improve host nutrition. The probiotic root-colonizing pseudomonads are generalists in terms of plant hosts, soil habitats and the array of stress responses that are ameliorated in the plant. This article illustrates how the probiotic pseudomonads, nurtured by the carbon (C) and nitrogen (N) sources released by the plant in root exudates, form protective biofilms on the root surface and produce the metabolites or enzymes to boost plant health. The findings reveal the multifunctional nature of many of the microbial metabolites in the plant-probiotic interplay. The beneficial effects of probiotics on plant function can contribute to sustainable yield and quality in agricultural production.

The GacS/GacA system in the root colonizer Pseudomonas chlororaphis O6 is a key regulatory system of many traits relevant to the plant probiotic nature of this bacterium. The work in this paper elucidates proteins using proteomics approach in P. chlororaphis O6 under the control of the cytoplasmic regulatory protein, GacA. A gacA mutant of P. chlororaphis O6 showed loss in production of phenazines, acyl homoserine lactones, hydrogen cyanide, and protease, changes that were associated with reduced in vitro antifungal activity against plant fungal pathogens. Production of iron-chelating siderophore was significantly enhanced in the gacA mutant, also paralleling changes in a gacS mutant. However, proteomic analysis revealed proteins (13 down-regulated and 7 upregulated proteins in the mutant compared to parental strain) under GacA control that were not apparent by a proteomic study of a gacS mutant. The putative identity of the downregulated proteins suggested that a gacA mutant would have altered transport potentials. Notable would be a predicted loss of type-VI secretion and PEP-dependent transport. Study of mutants of these GacA-regulated proteins will indicate further the features required for probiotic potential in this rhizobacterium.

Commercial biocontrol of microbial plant diseases and plant pests, such as nematodes, requires field-effective formulations. The isolate Pseudomonas chlororaphis O6 is a Gram-negative bacterium that controls microbial plant pathogens both directly and indirectly. This bacterium also has nematocidal activity. In this study, we report on the efficacy of a wettable powder-type formulation of P. chlororaphis O6. Culturable bacteria in the formulated product were retained at above 1 × 108 colony forming units/g after storage of the powder at 25 °C for six months. Foliar application of the diluted formulated product controlled leaf blight and gray mold in tomato. The product also displayed preventative and curative controls for root-knot nematode (Meloidogyne spp.) in tomato. Under laboratory conditions and for commercially grown melon, the control was at levels comparable to that of a standard commercial chemical nematicide. The results indicated that the wettable powder formulation product of P. chlororaphis O6 can be used for control of plant microbial pathogens and root-knot nematodes.

Root-knot nematodes (Meloidogyne spp.) are parasites that attack many field crops and orchard trees, and affect both the quantity and quality of the products. A root-colonizing bacterium, Pseudomonas chlororaphis O6, possesses beneficial traits including strong nematicidal activity. To determine the molecular mechanisms involved in the nematicidal activity of P. chlororaphis O6, we constructed two mutants; one lacking hydrogen cyanide production, and a second lacking an insecticidal toxin, FitD. Root drenching with wild-type P. chlororaphis O6 cells caused juvenile mortality in vitro and in planta. Efficacy was not altered in the fitD mutant compared to the wild-type but was reduced in both bioassays for the mutant lacking hydrogen cyanide production. The reduced number of galls on tomato plants caused by the wild-type strain was comparable to that of a standard chemical nematicide. These findings suggest that hydrogen cyanide-producing root colonizers, such as P. chlororaphis O6, could be formulated as “green” nematicides that are compatible with many crops and offer agricultural sustainability.

Pseudomonas chlororaphis O6, isolated from the roots of dryland, field-grown commercial wheat in the USA, enhances plant health and therefore it is used in agriculture as a biofertilizer and biocontrol agent. The me tabolites produced by this pseudomonad stimulate plant growth through direct antagonism of pathogens and by inducing systemic resistance in the plant. Studies upon P. chlororaphis O6 identify the pathways through which defined bacterial metabolites generate protection against pathogenic microbes, insects, and nematodes. P. chlororaphis O6 also triggers plant resistance to drought and salinity stresses. The beneficial de terminants are produced from bacterial cells as they form biofilms during root colonization. Molecular control these processes in P. chlororaphis O6 involves the global regulatory Gac/Rsm signaling cascade with cross-talk between other global regulatory pathways. The Gac/Rsm regulon allows for coordinate phasing of expression of the genes that encode these beneficial traits among a community of cells. This review provides insights on the Gac/Rsm regulon in expression of beneficial traits of the P. chlororaphis O6 which can contribute to help yield enhancement and quality in agricultural production.

Oat, a gluten-free source of vitamins, minerals, fiber, and antioxidants required for human health (Rasane et al., 2015), is increasingly being cultivated in South Korea (Ju et al. 2011). In May 2020, ~30 % Avena sativa (cv. Samhan) leaves of a commercial oat field in Jeongeup, Korea (35.3859°N, 126.5607°E), displayed a leaf spot disease at the tillering growth stage. Small, brown spots appeared initially on the lower leaves, but progressed to dark-brown oval or irregular, necrotic spots with a light-yellowish border. To isolate the pathogen, tissue excised from growing edges of about 50 lesions was surface sterilized (immersion in 70 % ethanol then 1 % sodium hypochlorite), rinsed and transferred to potato dextrose agar. Five isolates were purified using the single-spore culture method on water agar plates (Choi et al. 1999). Conidia from the PDA cultures (n = 50) were elongated, ellipsoidal, and obclavate, with one to five septa (primarily transverse), 18 to 35 μm × 7 to 12 μm. The morphological characteristics of the fungus from each culture were consistent with those observed for Alternaria alternata (Chen et al. 2020; Simmons 2007). To confirm the identity of the fungal pathogen, the internal transcribed spacer (ITS) region was amplified with universal primers ITS1/ITS4 and sequenced (White et al. 1990). BLAST analysis of the resulting nucleotide sequence of one representative isolate, Oaa-2 (GenBank MT786424), showed 100 % identity with A. alternata-type strains, such as CBS916.96. Further, nine nuclear protein-coding genes from the Oaa-2 isolate were amplified using previously described primers (Lawrence et al. 2013; Wouderengerg et al. 2015). These sequences were deposited under the following GenBank accession numbers: MT795660 (rpb2), MT795656 (His), MT795657 (CML), MT795658 (ACT), MT787343 (CHSD), MT795655 (GAPDH), MT795661 (endoPG), and MT795662 (TEF1). The nine sequences of the Oaa-2 isolate showed 99%-100 % identity to those of A. alternata-type strain CBS916.96 in NCBI. Fig. S2 shows the phylogenetic trees for the ITS and GAPDH sequences indicating high homology. Pathogenicity of this isolate was confirmed by spraying leaves of surface-sterilized, glasshouse-grown, "Samhan" oats with suspensions (5 × 106 spores/ml) of Oaa-2 conidia. Experimental seedlings were grown for 2 weeks in a chamber at 20 ± 3 °C in 60 % relative humidity with a 10-h light/14-h dark cycle (200 µmol/m2/s). Conidial suspensions were prepared for inoculation by flooding with sterile water, followed by filtering the suspension through three layers of sterile cheesecloth to remove debris. Three pots each containing twenty oat seedlings were sprayed with the Oaa-2 isolate conidial suspension (5 × 106 conidia/ml) and incubated for 10 days at 20 ± 3 °C in 100 % humidity with a 10-h photoperiod. Three healthy seedlings were sprayed with sterile water as a control. After 10 days, dark brown spots developed in the inoculated plants; however, no spots were observed in the controls. Fungi with the same conidial characteristics as Oaa-2 were isolated from the infected plants, and the ITS sequence and the nine nuclear protein-encoding genes of these plant isolates was identical to those of Oaa-2. Pathogenicity tests also were conducted with isolate Oaa-2 with two oat cultivars, Sunyang and Joyang, with the same result. To our knowledge, A. alternata has not been previously reported to cause disease on oats in South Korea, although it does cause economic losses in China (Chen et al. 2020).

Biosurfactants have been used for crop protection in integrated pest management programs, as they are generally biodegradable and environmentally friendly. While screening for insecticidal bacteria against green peach aphid, Myzus persicae, we found that a Bacillus isolate, Y9, exhibited strong surface tension activity and produced insecticidal metabolites. The insecticidal metabolites with surface tension activity were extracted from the cell-free supernatant of Y9 and subjected to repetitive column chromatography and preparative high performance liquid chromatography (HPLC). Based on data from instrumental analyses, the metabolites were identified as surfactin isomers consisting of C14[Leu7], C14[Val7] and C15[Leu7]. The isomers with leucine showed higher insecticidal activity than the isomers with valine. Y9 was capable of producing biosurfactants as insecticidal metabolites against M. persicae, and showed potential for use as a biocontrol agent in an integrated pest management program.

Most biocontrol agents for plant diseases have been isolated from sources such as soils and plants. As an alternative source, we examined the feces of tertiary larvae of the herbivorous rhino beetle, Allomyrina dichotoma for presence of biocontrol-active microbes. The initial screen was performed to detect antifungal activity against two common fungal plant pathogens. The strain with strongest antifungal activity was identified as Bacillus amyloliquefaciens KB3. The inhibitory activity of this strain correlated with lipopeptide productions, including iturin A and surfactin. Production of these surfactants in the KB3 isolate varied with the culture phase and growth medium used. In planta biocontrol activities of cell-free culture filtrates of KB3 were similar to those of the commercial biocontrol agent, B. subtilis QST-713. These results support the presence of microbes with the potential to inhibit fungal growth, such as plant pathogens, in diverse ecological niches.

Chromobacterium sp. strain C61 has strong biocontrol activity; however, the genetic and biochemical determinants of its plant disease suppression activity are not well understood. Here, we report the identification and characterization of two new determinants of its biocontrol activity. Transposon mutagenesis was used to identify mutants that were deficient in fungal suppression. One of these mutants had an insertion in a homologue of depD, a structural gene in the dep operon, that encodes a protein involved in non-ribosomal peptide synthesis. In the second mutant, the insertion was in a homologue of the luxI gene, which encodes a homoserine lactone synthase. The luxI- and depD- mutants had no antifungal activity in vitro and a dramatically reduced capacity to suppress various plant diseases in planta. Antifungal production and biocontrol were restored by complementation of the luxI- mutant. Other phenotypes associated with effective biological control, including motility and lytic enzyme secretion, were also affected by the luxI mutation. Biochemical analysis of ethyl acetate extracts of culture filtrates of the mutant and wild-type strains showed that a key antifungal compound, chromobactomycin, was produced by wild-type C61 and the complemented luxI- mutant, but not by the luxI- or depD- mutant. These data suggest that multiple biocontrol-related phenotypes are regulated by homoserine lactones in C61. Thus, quorum sensing plays an essential role in the biological control potential of diverse bacterial lineages.

In May 2019, halo blight symptoms were observed on leaves of oats (Avena sativa L. ‘Joyang’) at the tillering stage. Plants were cultivated in experimental fields in Wanju County (latitude 35.842006°, longitude 127.049588°), located in the southern region of South Korea. Symptoms consisted of water-soaked spots, which progressed into blotches or streaks with brown oval to spindle-shaped centers surrounded by yellow halos. These symptoms were similar to those reported for oats infected by Pseudomonas coronafaciens in other parts of the world (Haber and Harder 1992; Harder and Harris 1973; Wilkie 1972). To isolate the causal agent of the symptoms, 1- to 3-cm-long pieces of symptomatic leaves from 10 different oat plants were surface sterilized with 70% ethanol for 30 s and macerated in sterile distilled water. The macerates were streaked onto nutrient agar plates and grown at 28°C for 48 h. In total, four pure colonies were selected and were cream-colored, wet, and shiny, with convex surfaces. The isolated colonies did not produce fluorescent siderophores when cultured on King’s medium B. We selected two representative colonies (SP-1 and X-1) for further characterization. The 16S rRNA, gyrB, and rpoD loci were amplified using the primers 27F/1492R (Lane 1991), gyrB+271ps/gyrB-1022ps, and rpoD+147p/rpoD-1222ps, respectively (Sarkar and Guttman 2004). The sequences were deposited in GenBank with the following accession numbers: MN134544 (SP-1) and MN134571 (X-1) for the 16S rRNA gene, MN599033 (SP-1) and MN599034 (X-1) for the gyrB gene, and MN599035 (SP-1) and MN599036 (X-1) for the rpoD gene. Analyses of these sequences indicated that the two isolates belong to the same clade of pathotypes as P. coronafaciens. These pathotypes formed a clade distinct from that of other P. syringae pathovars, as described recently by Dutta et al. (2018). For confirmation, PCR analysis with two different specific primer sets was performed. The 498-bp products from SP-1 and X-1 were amplified using a specific primer pair for P. coronafaciens, Pc-12-F and Pc-12-R (An et al. 2015), and 600-bp products with primers, P1 and P2, to detect the sequence within the plasmid pCOR1 harboring the genes for toxin production (Dutta et al. 2018). Consequently, the bacteria isolated from the lesions on the oat plants were P. coronafaciens strains. To fulfill Koch’s postulates, pathogenicity tests were conducted in which isolates SP-1 and X-1 were inoculated onto surface-sterilized leaves of 2-week-old, disease-free, greenhouse-grown Joyang oat plants. Leaves were inoculated using a 1-ml needle syringe with 400 µl of a bacterial suspension (10⁸ CFU/ml) or 400 µl of sterile water (for the control group). The plants were incubated for 1 week at 23°C in 100% relative humidity under a 10-h photoperiod. After 5 days, the inoculated leaves began to exhibit typical brown necrotic spots surrounded by yellow halos. Bacteria were isolated from these diseased leaves, and the 16S rRNA gene sequences of the isolates were the same as those from the blighted field-grown leaves. The pathogenicity test was repeated twice. These findings indicated that P. coronafaciens strains accounted for the symptoms observed in field-grown oats. To the best of our knowledge, this is the first report of oat halo blight disease caused by P. coronafaciens in South Korea. The pathogen has the potential to impair plant health and result in economic losses, as reported for other global locations (Elliott 1920; Harder and Harris 1973; Wilkie 1972).

Commercial biocontrol of microbial plant diseases and plant pests, such as nematodes, requires field-effective formulations. The isolate Pseudomonas chlororaphis O6 is a Gram-negative bacterium that controls microbial plant pathogens both directly and indirectly. This bacterium also has nematocidal activity. In this study, we report on the efficacy of a wettable powder-type formulation of P. chlororaphis O6. Culturable bacteria in the formulated product were retained at above 1 × 108 colony forming units/g after storage of the powder at 25 °C for six months. Foliar application of the diluted formulated product controlled leaf blight and gray mold in tomato. The product also displayed preventative and curative controls for root-knot nematode (Meloidogyne spp.) in tomato. Under laboratory conditions and for commercially grown melon, the control was at levels comparable to that of a standard commercial chemical nematicide. The results indicated that the wettable powder formulation product of P. chlororaphis O6 can be used for control of plant microbial pathogens and root-knot nematodes.

Because bacterial isolates from only a few genera have been developed commercially as biopesticides, discovery and characterization of novel bacterial strains will be a key to market expansion. Our previous screen using plant bioassays identified 24 novel biocontrol isolates representing 12 different genera. In this study, we characterized the 3 isolates showing the best biocontrol activities. The isolates were Pantoea dispersa WCU35, Proteus myxofaciens WCU244, and Exiguobacterium acetylicum WCU292 based on 16S rRNA sequence analysis. The isolates showed differential production of extracellular enzymes, antimicrobial activity against various fungal or bacterial plant pathogens, and induced systemic resistance activity against tomato gray mold disease caused by Botrytis cinerea. E. acetylicum WCU292 lacked strong in vitro antimicrobial activity against plant pathogens, but induced systemic resistance against tomato gray mold disease. These results confirm that the trait of biological control is found in a wide variety of bacterial genera.

Because bacterial isolates from only a few genera have been developed commercially as biopesticides, discovery and characterization of novel bacterial strains will be a key to market expansion. Our previous screen using plant bioassays identified 24 novel biocontrol isolates representing 12 different genera. In this study, we characterized the 3 isolates showing the best biocontrol activities. The isolates were Pantoea dispersa WCU35, Proteus myxofaciens WCU244, and Exiguobacterium acetylicum WCU292 based on 16S rRNA sequence analysis. The isolates showed differential production of extracellular enzymes, antimicrobial activity against various fungal or bacterial plant pathogens, and induced systemic resistance activity against tomato gray mold disease caused by Botrytis cinerea. E. acetylicum WCU292 lacked strong in vitro antimicrobial activity against plant pathogens, but induced systemic resistance against tomato gray mold disease. These results confirm that the trait of biological control is found in a wide variety of bacterial genera

Plant has possessed diverse stress signals from outside and maintained its fitness. Out of such plant responses, it is well known that mitogen-activated protein kinase (MAPK) cascade plays important role in wounding and pathogen attack in most dicot plants. However, little is understood about its role in wounding response for the economically important monocot rice plant. In this study, therefore, the involvement of MAPK was investigated to understand the wounding signaling pathway in rice. The OsMPK1 was rapidly activated by wounding within 10 min, and OsMPK1 was also activated by challenge of rice blast fungus. Further analysis revealed that OsMKK4, the upstream kinase of OsMPK1, phosphorylated OsMPK1 by wounding in vivo. Furthermore, OsMPK1 directly interacted with a rice defense-related transcription factor OsWRKY53. To understand a functional link between MAPK and its target transcription factor, we showed that OsMPK1 activated by the constitutively active mutant OsMKK4(DD) phosphorylated OsWRKY53 in vitro. Taken together, components involving in the wounding signaling pathway, OsMKK4-OsMPK1-OsWRKY53, can be important players in regulating crosstalk between abiotic stress and biotic stress.

Hot pepper is one of the economically important crops in Asia. A large number of gene sequences, including expressed sequence tag (EST) and genomic sequences are publicly available. However, it is still a daunting task to determine gene function due to difficulties in genetic modification of a pepper plants. Here, we show the application of the virus-induced gene silencing (VIGS) repression for the study of 459 pepper ESTs selected as non-host pathogen-induced cell death responsive genes from pepper microarray experiments in Nicotiana benthamiana. Developmental abnormalities in N. benthamiana plants are observed in the 32 (7%) pepper ESTs-silenced plants. Aberrant morphological phenotypes largely comprised of three groups: stunted, abnormal leaf, and dead. In addition, by employing the combination of VIGS and Agrobacterium-mediated transient assays, we identified novel pepper ESTs that involved in Bax or INF1-mediated cell death responses. Silencing of seven pepper ESTs homologs suppressed Bax or INF1-induced cell death, five of which suppressed both cell death responses in N. benthamiana. The genes represented by these five ESTs encode putative proteins with functions in endoplasmic reticulum (ER) stress and lipid signaling. The genes represented by the other two pepper ESTs showing only Bax-mediated cell death inhibition encode a CCCH-type zinc finger protein containing an ankyrin-repeat domain and a probable calcium-binding protein, CML30-like. Taken together, we effectively isolated novel pepper clones that are involved in hypersensitive response (HR)-like cell death using VIGS, and identified silenced clones that have different responses to Bax and INF1 exposure, indicating separate signaling pathways for Bax- and INF1-mediated cell death.

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 ...

Pseudomonas chlororaphis O6 possesses many beneficial traits involved in biocontrol of plant diseases. In this paper, we examined the effect of a mutation in rpoS encoding a stress-related alternative sigma factor to better understand the regulation of these traits. Biochemical studies indicated that production of acyl homoserine lactones was altered and phenazine was increased in the P. chlororaphis O6 rpoS mutant. The rpoS mutation reduced hydrogen cyanide levels, but the rpoS mutant still displayed a level of in vitro antifungal activity against Fusarium graminearum and Alternaria alternata. Tomato root colonization by the rpoS mutant was lower than that by the wild type at 5, 7, and 13 days after inoculation. The rpoS mutant was less effective than the wild type in induction of systemic resistance to two foliar pathogens after root inoculation of the tomato plants. Our findings demonstrate that the stationary-phase sigma factor RpoS regulates production of several key factors involved in the biocontrol potential of P. chlororaphis O6, some independently of the global regulator GacS.

Root colonization by Pseudomonas chlororaphis O6 induces systemic drought tolerance in Arabidopsis thaliana. Microarray analysis was performed using the 22,800-gene Affymetrix GeneChips to identify differentially-expressed genes from plants colonized with or without P. chlororaphis O6 under drought stressed conditions or normal growth conditions. Root colonization in plants grown under regular irrigation condition increased transcript accumulation from genes associated with defense, response to reactive oxygen species, and auxin- and jasmonic acid-responsive genes, but decreased transcription factors associated with ethylene and abscisic acid signaling. The cluster of genes involved in plant disease resistance were up-regulated, but the set of drought signaling response genes were down-regulated in the P. chlororaphis O6-colonized under drought stress plants compared to those of the drought stressed plants without bacterial treatment. Transcripts of the jasmonic acid-marker genes, VSP1 and pdf-1.2, the salicylic acid regulated gene, PR-1, and the ethylene-response gene, HEL, also were up-regulated in plants colonized by P. chlororaphis O6, but differed in their responsiveness to drought stress. These data show how gene expression in plants lacking adequate water can be remarkably influenced by microbial colonization leading to plant protection, and the activation of the plant defense signal pathway induced by root colonization of P. chlororaphis O6 might be a key element for induced systemic tolerance by microbes.

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.

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.

Recent unusual weather due to global warming causes shortage of daily sunlight and constitutes one of the primary reasons for agricultural damages. LED light sources are frequently utilized to compensate for the shortage of sunlight in greenhouse agriculture. The present study is aimed at evaluating formations of phytochemicals as well as growth characteristics of mature strawberry fruits (‘Daewang’ cultivar) during cultivation in a closed growth chamber equipped with artificial LED light as a sole light source. Each LED light of blue (448 nm), red (634 and 661 nm) or mixed blue plus red (blue:red = 3:7) was separately supplied and the intensity of each light was adjusted to 200 ± 1 μmol·m -2 ·s -1 at plant level with a photoperiod consisted of 16 hours light and 8 hours darkness. Strawberries grown under mixed LED light of blue and red wavelengths showed a higher production of fruits than those grown under other LED treatments. Fructose, one of the free sugars, increased in mixed LED light-grown fruits. Anthocyanin contents were elevated remarkably in the mixed LED light-grown fruits compared with those in other LED treatments. Contrastingly, contents of total phenolics and flavonoids were not of much different from one another among the fruits treated with various LED lights. On the other hand, ripening of strawberry fruits was found to be faster when grown under blue LED light compared with other LED treatments. Moreover, antioxidant activities of blue or red LED light-grown fruits, respectively, were significantly higher than those of mixed LED light-grown fruits. We suggest that when daylight is in shortage during cultivation in a greenhouse, supplementation of sunlight with LED light, which is composed of blue and red wavelengths, could be useful for the enhancement of productivity as well as of free sugar content in strawberry fruits. In addition, for the strawberry culture in the plant factory, selective adoption of LED light wavelength would be required to accomplish the purpose of controlling fruit maturation time as well as of enhancing contents of sugars and antioxidants of fruits.

2R,3R-Butanediol, a volatile compound produced by certain rhizobacteria, is involved in induced drought tolerance in Arabidopsis thaliana through mechanisms involving stomatal closure. In this study, we examined the involvement of nitric oxide and hydrogen peroxide in induced drought tolerance, because these are signaling agents in drought stress responses mediated by abscisic acid (ABA). Fluorescence-based assays showed that systemic nitric oxide and hydrogen peroxide production was induced by 2R,3R-butanediol and correlated with expression of genes encoding nitrate reductase and nitric oxide synthase. Co-treatment of 2R,3R-butanediol with an inhibitor of nitrate reductase or an inhibitor of nitric oxide synthase lowered nitric oxide production and lessened induced drought tolerance. Increases in hydrogen peroxide were negated by co-treatment of 2R,3R-butanediol with inhibitors of NADPH oxidase, or peroxidase. These findings support the volatile 2R,3R-butanediol synthesized by certain rhizobacteria is an active player in induction of drought tolerance through mechanisms involving nitric oxide and hydrogen peroxide production.

Phytohormone balance is increasingly recognized as central to the outcome of plant–pathogen interactions. Differential screening for genes induced by a non-host pathogen in pepper plants (Capsicum annuum) identified a putative gibberellin 2-oxidase gene, CaGA2ox1. Analysis of the deduced amino acid sequence of CaGA2ox1 showed 53 and 50 % amino acid identity to Pisum sativum PsGA2ox2 and Arabidopsis AtGA2ox6, respectively. Expression in pepper plants of CaGA2ox1 was preferentially increased in response to non-host pathogen inoculation and during the host resistance response. CaGA2ox1 expression increased following treatment with salicylic acid and ethephon (albeit with different induction patterns), but remained unchanged following treatment with methyl jasmonate and abscisic acid. The gene product of CaGA2ox1 is predicted to catalyze the metabolism of GA4, and does so in recombinant E. coli extracts. Further PEG-mediated transient expression studies showed that CaGA2ox1 fused with soluble modified green fluorescent protein localized to the cytosol in chili pepper protoplasts. Interestingly, the transcript level of CaGA2ox1 was not affected by treatments of either pepper with bioactive GA4+7 or paclobutrazol, an inhibitor of GA biosynthesis. Taken together, these results provide the first evidence that a GA 2-oxidase, which is important in GA metabolism, may also play a role in plant defense signaling and plant–microbe interactions.

Institute of Environmentally-Friendly Agriculture, Department of Plant Biotechnology, Chonnam National University, Gwangju500-757, Korea (Received on November 13, 2011; Revised on January 5, 2012; Accepted on January 11, 2012)Root colonization by the rhizobacterium Pseudomonaschlororaphis O6 in Arabidopsis thaliana Col-0 plantsresulted in induced tolerance to drought and salinitycaused by halide salt-generated ionic stress but not byosmotic stress caused by sorbitol. Stomatal aperturesdecreased following root colonization by P. chlororaphisO6 in both wild-type and ABA-insensitive Arabidopsismutant plants. These results suggest that an ABA-independent stomatal closure mechanism in the guardcells of P. chlororaphis O6-colonized plants could be akey phenotype for induced systemic tolerance todrought and salt stress. Keywords : abiotic stress, induced tolerance to environ-mental stress, priming, stomatal closurePlants possess various survival systems against abioticstresses, such as cold, drought, and salinity (Zhu, 2001).Under environmental stresses, the level of the planthormone, abscisic acid (ABA) increases triggering adaptiveresponses essential for survival (Zhu, 2001). Duringdrought and salt stresses, ABA induces stomatal closure tominimize water loss through transpiration (Leung andGiraudat, 1998). Consequently, ABA-biosynthesis mutants(aba mutants) and some of the ABA-response plantmutants (i.e. the ABA-insensitive abi mutants) aresusceptible to drought stress, due to problem of stomatalaperture regulation (Leung and Giraudat, 1998; Schroederet al., 2001). However, another plant growth regulator,jasmonate (Evans, 2003; Suhita et al., 2004), also stimu-lates stomatal closure under drought conditions (Creelmanand Mullet, 1997).Root colonization of certain plant-associated microbeselicits physiological and biochemical change to enhancesystemic resistance against various biotic and abioticstresses in plants (Kim et al., 2011; Yang et al., 2010),termed to as “induced systemic resistance (ISR)” or“induced systemic tolerance (IST)”. IST against drought orsalt stresses in plants can be induced with systemicapplication of certain rhizobacteria including Gram-positive Bacillus strains (Ryu et al., 2004; Timmusk andWagner 1999; Zhang et al., 2010), an endophytic fungalisolate, Trichoderma harziarum (Bae et al., 2010), andcertain Gram-negative bacterial isolates, such as ACC-deaminase producing bacteria (Mayak et al., 2004). Rootcolonization by Pseudomonas chlororaphis O6 inducedsystemic resistance against a broad spectrum of plantdiseases caused by viral, bacterial, and fungal pathogens invarious plants by jasmonic acid-ethylene related pathways(Cho et al., 2008; Kim et al., 2004; Ryu et al., 2007;Spencer et al., 2003). Additionally, root colonization by P.chlororaphis O6 induced systemic tol erance against drought,a process accompanied by stomatal closure. Applying2R,3R-butanediol, a volatile produced by P. chlororaphisO6, resulted in induced tolerance through a salicylic acid(SA), jasmonic acid (JA) and ethylene-dependent mech-anism (Cho et al., 2008). However, mechanisms involvedin microbe-mediated IST against drought have not beencharacterized or elucidated.In this study, we tested the hypothesis that P. chloro-raphis O6 induces tolerance to other abiotic stresses, suchas salinity, osmotic pressure, cold and heat. We used addi-tional Arabidopsis mutants altered in ABA signalingpathways to identify the role of ABA in induced abioticstress tolerance. Parental A. thaliana ecotypes Columbia(Col-0) or Landsberg erecta (Ler), and mutant and trans-genic lines were obtained from the Ohio State University

Jeonnam Agricultural Extension Service Center, Naju 520-715, Korea(Received on November 5, 2011; Revised on December 27, 2011; Accepted on January 1, 2012)Trifloxystrobin is a strobilurin fungicide, which possessesbroad spectrum control against fungal plant diseases.We demonstrated that pre-treating red pepper plantswith trifloxystrobin resulted in increased plant growthand leaf chlorophyll content compared with those incontrol plants. Relative water content of the leaves andthe survival rate of intact plants indicated that plantsacquired systemic tolerance to drought stress followingtrifloxystrobin pre-treatment. The recovery rate by re-hydration in the drought treated plant was better inthose pre-treated with trifloxystrobin than that in watertreated plants. Induced drought tolerance activity bytrifloxystrobin was sustained for 25 days after initialapplication. The trifloxystrobin treated red pepper plantsalso had induced systemic tolerance to other abioticstresses, such as frost, cold, and high temperature stresses.These findings suggest that applying the chemical fungi-cide trifloxystrobin induced systemic tolerance to cer-tain abiotic stresses in red pepper plants. Keywords : cold and freezing stresses, drought stress, hightemperature stress, induced systemic tolerance, plant growthpromotion Plants rely on many adaptive strategies against differentabiotic and biotic stresses because of their immobility.Applications of certain chemicals and plant associatedmicrobes are known to induce resistance pathways toabiotic and biotic stresses (Sticher et al., 1997; Schreiberand Desveaux, 2008; Yang et al., 2010). In systemicacquired resistance (SAR), plants develop a broad spectrumof systemic resistance against plant diseases or abioticstresses upon pathogen attack or treatment of chemicals(Ryals et al., 1996). Root colonization by biocontrolmicrobes also elicits systemic resistance, known as inducedsystemic resistance (ISR), against abiotic stresses and abroad spectrum of diseases caused by bacterial, fungal, andviral pathogens (Kim et al., 2011). Recent studies suggest that certain chemical applicationscan induce systemic resistance against abiotic or bioticstresses (Schreiber and Desveaux, 2008). Plants treatedwith one chemical inducer, β-aminobutyric acid (BABA),show dual induction for resistance to abiotic and bioticstresses (Ton et al., 2005). Application of strobilurin totobacco induces resistance against Tobacco Mosaic Virusand wildfire diseases (Herms et al., 2002). Soil applicationof a chemical pesticide, imidacloprid, reduces the incidenceof citrus canker disease (Graham and Myers, 2011). Induc-tion of drought stress tolerance in plants occurs with thefungicides, ketoconazole and propiconazole, and with theinsecticide, imidacloprid (Abdul Jaleel et al., 2007b; Han etal., 2010; Manivannan et al., 2007). Soil applications ofpropiconazole also induce tolerance to drought and inducetolerance to salinity (Adul Jallel et al., 2008), and imidaclo-prid-treated plants are tolerant to high temperature (Goniaset al., 2008). Induced tolerance to flooding and salinityoccurs with treatments with the plant regulator paclobutra-zol (Abdul Jaleel et al., 2007c; Lin et al., 2005). Trifloxystrobin, a strobilurin fungicide, is used to controla wide range of plant diseases because of its broad spectrumantifungal activity (Reuveni et al., 2000). Trifloxystrobinblocks electron transfer and inhibits mitochondrial re-spiration in the target fungus (Bartlett et al., 2002). In thisstudy, we examined whether trifloxystrobin could inducesystemic tolerance against the abiotic stresses of drought,low temperature, freezing, and high temperature in redpepper plants. We also examined the growth promotioneffects of trifloxystrobin and induced systemic resistance toplant diseases.Effect of trifloxystrobin pretreatment on plant growthand chlorophyll content. Surface-sterilized red pepperseeds were planted in pots to determine the effect of asuspension concentrate type formulation of trifloxystrobin(SC) on plant growth and photosynthesis. TrifloxystrobinSC was obtained from Bayer Cropscience Korea and was

Biocontrol microbes have mainly been screened among large collections of microorganisms via. nutrient-rich in vitro assays to identify novel and effective isolates. However, thus far, isolates from only a few genera, mainly spore-forming bacilli, have been commercially developed. In order to isolate field-effective biocontrol microbes, we screened for more than 200 oligotrophic bacterial strains, isolated from rhizospheres of various soil samples in Korea, which induced systemic resistance against the soft-rot disease caused by Pectobacterium carotovorum SCC1; we subsequently conducted in planta bioassay screening. Two oligotrophic bacterial strains were selected for induced systemic disease resistance against the Tobacco Mosaic Virus and the gray mold disease caused by Botrytis cinerea. The oligotrophic bacterial strains were identified as Pseudomonas manteilii B001 and Bacillus cereus C003 by biochemical analysis and the phylogenetic analysis of the 16S rRNA sequence. These bacterial strains did not exhibit any antifungal activities against plant pathogenic fungi but evidenced several other beneficial biocontrol traits, including phosphate solubilization and gelatin utilization. Collectively, our results indicate that the isolated oligotrophic bacterial strains possessing induced systemic disease resistance could provide useful tools as effective biopesticides and might be successfully used as cost-effective and preventive biocontrol agents in the field.

Department of Plant Pathology, The Ohio State University, OARDC, 1680 Madison Avenue, Wooster, Ohio 44691, USA(Received on May 11, 2011; Revised on July 21, 2011; Accepted on July 21, 2011)Root colonization of Arabidopsis thaliana with Pseudo-monas chlororaphis O6 induces systemic tolerance againstdiverse pathogens, as well as drought and salt stresses. Inthis study, we demonstrated that 11 genes in the leaveswere up-regulated, and 5 genes were down-regulated asthe result of three- to five-days root colonization by P.chlororaphis O6. The identified priming genes wereinvolved in cell signaling, transcription, protein synthesis,and degradation. In addition, expression of selectedpriming genes were induced in P. chlororaphis O6-colonized plants subjected to water withholding. Genesencoding defense proteins in signaling pathways re-gulated by jasmonic acid and ethylene, such as VSP1and PDF1.2, were additional genes with enhancedexpression in the P. chlororaphis O6-colonized plants.This study indicated that the expression of priminggenes, as well as genes involved in jasmonic acid- andethylene-regulated genes may play an important role inthe systemic induction of both abiotic and biotic stressdue to root colonization by P. chlororaphis O6. Keywords :disease defense, drought stress, induced systemicresistance, priming Induced resistance or tolerance against abiotic and bioticstresses is stimulated in plants by two systems. In systemicacquired resistance (SAR), plants develop a broad spectrumof systemic resistance against plant diseases upon pathogenattack. SAR involves elevated salicylic acid (SA) levels andboth local and systemic accumulation of pathogenesis-related (PR) proteins (Van Loon et al., 2006). Expression ofPR genes, PR-1 in particular, is used as a marker for SARinduction (Durrant and Dong, 2004) and a key regulatoryprotein is NPR1 (Pierterse and Van Loon, 2004). Aciben-zolar-S-methyl is a chemical that activates the SARpathway (Bostock, 2005).Colonization of roots by microbes also elicits systemicresistance against abiotic stresses and a broad spectrum ofdiseases caused by bacterial, fungal, and viral pathogens(Kim et al., 2011). Induced systemic resistance (ISR) causesthe plant to induce different array of genes than SAR. Onerhizobacterium, Pseudomonas chlororaphis O6, inducesISR for many plant diseases and enhanced drought tolerance(Han et al., 2006; Kim et al., 2004a; Ryu et al., 2007;Spencer et al., 2003). Three days of root colonization by P.chlororaphis O6 reduces opening of leaf stomata (Cho etal., 2008), thereby demonstrating a rapidly induced systemiceffect.Enhanced plant gene activation was observed in oilseedrape treated with the ISR-inducers P. thivervalensis MLG45and Bacillus amyloliquefaciens (Carieaux et al., 2003;Sarosh et al., 2009), in response to abiotic stress and afterchallenge by insects (Conrath et al., 2002). Differentialexpressed cucumber genes were induced in P. chlororaphisO6-colonized cucumber after challenge with the fungalpathogen Corynespora cassicola (Kim et al., 2004a). Oneinduced plant gene encoding galactinol synthase (CsGolS1),when over expressed in tobacco, increases galactinol levels,enhances disease resistance against Botrytis cinerea andPectobacterium carotovorum, and provides drought andsalinity tolerance (Kim et al., 2008). Another study ofplants inoculated with the ISR-inducer P. fluorescens WCS417r, showed 81 genes to increase in expression afterchallenge inoculation with the bacterial speck pathogen P.syringae pv. tomato (Verhagen et al., 2004). Few changes ingene expression were noted in plants colonized by P.fluorescens WCS 417r without pathogen challenge (VanWees et al., 1999; Verhagen et al., 2004). This enhancedgene activation in the colonized plants by ISR-inducingrhizobacteria is referred to as priming. However, the plant

A bacterial artificial chromosome library of Pectobacterium carotovorum 35 was constructed to characterize the genome and to sequence its hrp region. The hrp cluster of P. carotovorum 35 consisted of 26 open reading frames in five operons. A promoter-based green fluorescent protein technology was used to identify the genes regulated by the alternative sigma factor, HrpL, in P. carotovorum 35. The majority of the selected clones contained the hrpJ operon promoter sequence, which harbors a hrp box, but no putative hrp boxes were detected within the promoter sequences of two other hrpL-regulated genes encoding for pectate lyase and large repetitive protein. Although the promoters of five other hrp operons also contained hrp boxes, their expression was not HrpL-dependent in the promoter-based selection in E. coli. However, transcriptional analysis showed that expression from all operons harboring hrp boxes, except for the hrpN operon, was reduced significantly in the hrpL mutant. The severity of soft-rot symptoms when the hrpL mutant was applied to the surface of tobacco leaves, mimicking natural infection, was greatly attenuated. These results indicate that the hrpL gene of P. carotovorum 35 may be involved in the development of soft-rot symptoms.

On plants, microbial populations interact with each other and their host through the actions of secreted metabolites. However, the combined action of diverse organisms and their different metabolites on plant health has yet to be fully appreciated. Here, the multifactorial nature of these interactions, at the organismal and molecular level, leading to the biological control of plant diseases is reviewed. To do so, we describe in detail the ecological significance of three different classes of secondary metabolites and discuss how they might contribute to biological control. Specifically, the roles of auxin, acetoin, and phenazines are considered, because they represent very different but important types of secondary metabolites. We also describe how studies of the global regulation of bacterial secondary metabolism have led to the discovery of new genes and phenotypes related to plant health promotion. In conclusion, we describe three avenues for future research that will help to integrate these complex and diverse observations into a more coherent synthesis of bacterially mediated biocontrol of plant diseases.

Lysobacter antibiocus HS124 is a chitinase-producing rhizobacterium with proven capacities to suppress plant diseases. Bacterial cultures of L. antibioticus HS124 showed strong biocontrol efficacies against various plant diseases compared to those of bacterial cultures of Bacillus subtilis QST713 which is an active ingredient of a commercial biopesticide, Serenade. Here, we report the draft genome sequence and automated annotation of strain HS124. This draft genome sequence indicates the novelty of L. antibiocus HS124 and a subset of gene functions that may be related to its biocontrol activities

A linear cascade experimental apparatus often consists of only a few cascade blades. Advantages to this experimental arrangement are increased by the use of larger cascade blades, a lower mass flow rate, a corresponding decrease in required power, and easier optical access within the cascade passage. However, fewer cascade blades in the cascade row make it difficult to establish periodic flow conditions between blades compared to infinite cascade model experiments. Generally, removing fluid from the cascade walls or adjusting tailboards located downstream of the cascade are common methods to establish periodic flow conditions through the cascade blades. In this study, a passage for cascade experiments is designed to satisfy infinite cascade flow conditions without any flow control or tailboards. A one-pitch at cascade row is adopted as its width and only a single cascade blade is installed within the passage. The surface isentropic Mach number distribution on the blade is chosen for the existence of infinite cascade flow conditions, and 14 geometric design variables related to the passage shape are applied to the design of a one-pitch passage by using a genetic algorithm. Flow structures within a passage designed using a genetic algorithm match with those obtained with the infinite cascade flow condition. Computed results obtained with a single cascade blade show that infinite cascade flow conditions can be obtained by modifying only the passage walls of the cascade experimental apparatus.

Imidacloprid is a systemic insecticide which has been used widely in various crops to control insects. In the present study, we demonstrated that pre-treatment of imidacloprid significantly induced tolerance to drought in plant. Relative water content, chlorophyll levels, and recovery rate upon rehydration after drought stress in tobacco plants pre-treated with imidacloprid were higher levels than the control plants. Induced drought tolerance by imidacloprid treatments in red pepper was also demonstrated by measurement of recovery rate and fresh weight upon drought stress. Taken together, our results suggest that imidacloprid, in addition to exerting direct insecticidal activity, may also protect plants by induced tolerance to drought in plant.

In ginseng fields, Alternaria blight, caused by Alternaria panax, and anthracnose, caused by Colletotrichum gloeosporoides, have become serious problems in Korea, and control of these diseases relies mainly on intensive applications of fungicides. In an effort to develop an effective environmentally-friendly control system, we utilized preventative control approaches including rain shelter shading plates and the removal of dead plants, as well as a curative control approach, such as the application of microbial agents. In the presence of rain shelter shading plates, the occurrence of Alternaria blight and anthracnose decreased significantly compared to that seen with polyethylene shading nets. In addition, the eradication of dead ginseng plants, which harboured abundant spores of the pathogens, significantly reduced the incidence of both diseases. In fields with rain shelter shading plates and in which dead plants were eradicated, four applications of a bioformulated product containing chitinolytic bacterial strains in a simple medium containing chitin provided control similar in effect to that observed with the application of the fungicide under low disease pressure. The efficacy of the bioformulated product was decreased slightly under severe disease pressure. These findings indicate that integration of the three disease management measures might constitute a new effective and environmentally-friendly system for the control of Alternaria blight and anthracnose in Korean ginseng fields.

Root colonization by Pseudomonas chlororaphis O6 in cucumber elicited an induced systemic resistance (ISR) against Corynespora cassiicola. In order to gain insight into O6-mediated ISR, a suppressive subtractive hybridization technique was applied and resulted in the isolation of a cucumber galactinol synthase (CsGolS1) gene. The transcriptional level of CsGolS1 and the resultant galactinol content showed an increase several hours earlier under O6 treatment than in the water control plants following C. cassiicola challenge, whereas no difference was detected in the plants without a pathogen challenge. The CsGolS1-overexpressing transgenic tobacco plants demonstrated constitutive resistance against the pathogens Botrytis cinerea and Erwinia carotovora, and they also showed an increased accumulation in galactinol content. Pharmaceutical application of galactinol enhanced the resistance against pathogen infection and stimulated the accumulation of defense-related gene transcripts such as PR1a, PR1b, and NtACS1 in wild-type tobacco plants. Both the CsGolS1-overexpressing transgenic plants and the galactinol-treated wild-type tobacco plants also demonstrated an increased tolerance to drought and high salinity stresses.

Root colonization of plants with certain rhizobacteria, such as Pseudomonas chlororaphis O6, induces tolerance to biotic and abiotic stresses. Tolerance to drought was correlated with reduced water loss in P. chlororaphis O6-colonized plants and with stomatal closure, indicated by size of stomatal aperture and percentage of closed stomata. Stomatal closure and drought resistance were mediated by production of 2R,3R-butanediol, a volatile metabolite of P. chlororaphis O6. Root colonization with bacteria deficient in 2R,3R-butanediol production showed no induction of drought tolerance. Studies with Arabidopsis mutant lines indicated that induced drought tolerance required the salicylic acid (SA)-, ethylene-, and jasmonic acid-signaling pathways. Both induced drought tolerance and stomatal closure were dependent on Aba-1 and OST-1 kinase. Increases in free SA after drought stress of P. chlororaphis O6-colonized plants and after 2R,3R-butanediol treatment suggested a primary role for SA signaling in induced drought tolerance. We conclude that the bacterial volatile 2R,3R-butanediol was a major determinant in inducing resistance to drought in Arabidopsis through an SA-dependent mechanism.

Skin sooty dapple disease, a fungal disease that lowers Asian pear fruit quality, has emerged recently in Korea but has not yet been thoroughly characterized. This disease affects the surface of fruit, leaves, and young shoots of the Asian pear, typically appearing as a dark or pale black dapple on the fruit surface. The disease initiates on the fruit with small circular lesions that become bigger, eventually spreading to form large circular or indefinite lesions. Sparse dark or flourishing white-greyish aerial mycelia and appearance of a dark or pale black dapple on the fruit surface are typical signs of this disease. The disease was severe during cold storage of the Niitaka and Chuhwangbae varieties, but more limited on the Gamcheonbae and Hwangkeumbae varieties. To identify causal pathogens, 123 fungal isolates were obtained from lesions. The fungi that caused typical skin sooty dapple disease symptoms in our bioassay were identified. Based on their morphological characteristics, 74% of the isolates were Cladosporium sp. and 5-7 % of the isolates were Leptosphaerulina sp., Tripospermum sp., or Tilletiopsis sp. None of the isolates caused severe soft rot by injection to a wound plug, but some of the Cladosporium sp. isolates caused mild maceration. Therefore this microbiol complex cannot account for the soft rot also observed in stored fruits. The high frequency of isolation of Cladosporium sp. from disease tissues and bioassay on pear fruit surface suggest that Cladosporium sp. could be a major pathogen in the microbial complex associated with skin sooty dapple disease of the Asian pear in Korea.

Plants guard themselves against pathogen attack using multi-layered defense mechanism. Calcium represents an important secondary messenger during such defense responses. Upon examination of a pepper cDNA library, we observed that the gene CaSRC2-1 (Capsicum annum SRC2-1) was upregulated significantly in response to infection with the type II non-host pathogen Xanthomonas axonopodis pv. glycines 8 ra, which elicits a hypersensitive response. CaSRC2-1 encodes a protein that contains a C2 domain and it exhibits a high degree of homology to the protein Soybean genes regulated by cold 2 (SRC2). However, little is known about how SRC2 expression is elicited by biotic stresses such as pathogen challenge. Further sequence analysis indicated that the CaSRC2-1 C2 domain is unique and contain certain amino acids that are conserved within the C2 domains of other plants and animals. CaSRC2-1 transcription was up-regulated under both biotic and abiotic stress conditions, including bacterial and viral pathogen infection, CaCl(2) and cold treatment, but unaffected by treatment with plant defense-related chemicals such as salicylic acid, methyl jasmonic acid, ethephone, and abscisic acid. Intriguingly, under steady state conditions, CaSRC2-1 was expressed only in the root system. A CaSRC2-1-GFP fusion protein was used to determine localization to the plasma membrane. A fusion protein lacking the C2 domain failed to target the membrane but remained in the cytoplasm, indicating that the C2 domain plays a critical role in localization. Thus, CaSRC2-1 encodes a novel C2 domain-containing protein that targets the plasma membrane and plays a critical role in the abiotic stress and defense responses of pepper plants.

SUMMARY Using cDNA microarray analysis, we isolated a cDNA clone, CaATL1 (Capsicum annuum L. Bukang AT-hook-Like gene 1), from a chili pepper plant incompatibly interacting with bacterial pathogens. The deduced amino acid sequence has a potential nuclear localization sequence and an AT-hook DNA binding motif which can bind AT-rich sequence elements. Expression of CaATL1 was specifically induced in host- and non-host-resistant responses against bacterial and viral pathogens in pepper plants. In addition, CaATL1 transcripts also increased following salicylic acid and ethephone treatment but were only mildly induced by methyl-jasmonate treatment. CaATL1::smGFP (soluble-modified green fluorescent protein) fusion protein localized to nuclei in tobacco BY2 protoplasts. The C-terminal region of the CaATL1 protein fused to the LexA DNA binding domain was able to activate reporter gene expression in yeast. To analyse further the role of the CaATL1 in pathogen defence response, we generated CaATL1-over-expressing transgenic tomato plants. These transgenic plants showed enhanced disease resistance against bacterial and oomycete pathogens. Taken together, these results provide the first evidence of a role for a plant AT-hook motif-containing transcription factor in pathogen defence response.