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This study aims to critically assess different micromechanical analysis models applied to carbon-fiber-reinforced plastic (CFRP) composites, employing micromechanics-based homogenization to accurately predict their effective properties. The paper begins with the simplest Voigt and Reuss models and progresses to more sophisticated micromechanics-based models, including the Mori–Tanaka and Method of Cells (MOC) models. It provides a critical review of the areas in which these micromechanics-based models are effective and analyses of their limitations. The numerical analysis results were confirmed through finite element simulations of the periodic representative volume element (RVE). Furthermore, the effective properties predicted by these micromechanics-based models were validated via experiments conducted on IM7/5320-1 composite material with a fiber volume fraction of 0.62.

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 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.

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.

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.

Isaria javanica pf185 is an important entomopathogenic fungus with potential for use as an agricultural biocontrol agent. However, the effect of I. javanica pf185 on plant growth is unknown. Enhanced tobacco growth was observed when tobacco roots were exposed to spores, cultures, and fungal cell-free culture supernatants of this fungus. Tobacco seedlings were also exposed to the volatiles of I. javanica pf185 in vitro using I-plates in which the plant and fungus were growing in separate compartments connected only by air space. The length and weight of seedlings, content of leaf chlorophyll, and number of root branches were significantly increased by the fungal volatiles. Heptane, 3-hexanone, 2,4-dimethylhexane, and 2-nonanone were detected, by solid-phase micro-extraction and gas chromatography-mass spectrophotometry, as the key volatile compounds produced by I. javanica pf185. These findings illustrate that I. javanica pf185 can be used to promote plant growth, and also as a biocontrol agent of insect and plant diseases. Further studies are necessary to elucidate the mechanisms by which I. javanica pf185 promotes plant growth.

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.

Biological control is an important process for sustainable plant production, and this trait is found in many plant-associated microbes. This study reviews microbes that could be formulated into pesticides active against various microbial plant pathogens as well as damaging insects or nematodes. The focus is on the beneficial microbes that colonize the rhizosphere where, through various mechanisms, they promote healthy plant growth. Although these microbes have adapted to cohabit root tissues without causing disease, they are pathogenic to plant pathogens, including microbes, insects, and nematodes. The cocktail of metabolites released from the beneficial strains inhibits the growth of certain bacterial and fungal plant pathogens and participates in insect and nematode toxicity. There is a reinforcement of plant health through the systemic induction of defenses against pathogen attack and abiotic stress in the plant; metabolites in the beneficial microbial cocktail function in triggering the plant defenses. The review discusses a wide range of metabolites involved in plant protection through biocontrol in the rhizosphere. The focus is on the beneficial firmicutes and pseudomonads, because of the extensive studies with these isolates. The review evaluates how culture conditions can be optimized to provide formulations containing the preformed active metabolites for rapid control, with or without viable microbial cells as plant inocula, to boost plant productivity in field situations.

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.

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.

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.

Pyraclostrobin is a broad-spectrum fungicide that inhibits mitochondrial respiration. However, it may also induce systemic resistance effective against bacterial and viral diseases. In this study, we evaluated whether pyraclostrobin enhanced resistance against the bacterial spot pathogen, Xanthomonas euvesicatora on pepper (Capsicum annuum). Although pyraclostrobin alone did not suppressed the in vitro growth of X. euvesicatoria, disease severity in pepper was significantly lower by 69% after treatments with pyraclostrobin alone. A combination of pyraclostrobin with streptomycin reduced disease by over 90% that of the control plants. The preventive control of the pyraclostrobin against bacterial spot was required application 1-3 days before pathogen inoculation. Our findings suggest that the fungicide pyraclostrobin can be used with a chemical pesticide to control bacterial leaf spot diseases in pepper.

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.

This study describes the numerical simulation results of aluminum/carbon-fiber-reinforced plastic (CFRP) hybrid joint parts using the explicit finite-element solver LS-DYNA, with a focus on capturing the failure behavior of composite laminates as well as the adhesive capacity of the aluminum–composite interface. In this study, two types of adhesive modeling techniques were investigated: a tiebreak contact condition and a cohesive zone model. Adhesive modeling techniques have been adopted as a widely commercialized model of structural adhesives to simulate adhesive failure based on fracture mechanics. CFRP was studied with numerical simulations utilizing LS-DYNA MAT54 to analyze the crash capability of aluminum/CFRP. To evaluate the simulation model, the results were compared with the force–displacement curve from numerical analysis and experimental results. A parametric study was conducted to evaluate the effect of different fracture toughness values used by designers to predict crash capability and adhesive failure of aluminum/CFRP parts.

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 metabolites 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 determinants 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.

Bacillus amyloliquefaciens LM11 was isolated from the feces of larvae of the rhino beetle and showed strong antifungal activities against various phytopathogenic fungi by producing biosurfactants. In this study, our overall goal was to determine relationship between biosurfactants produced from the LM11 strain and its role in growth inhibition of phytopathogenic fungi. Production and expression levels of B. amyloliquefaciens LM11 biosurfactants were significantly differed depending on growth phases. Transcriptional and biochemical analysis indicated that the biosurfactants of the LM11 strain were greatly enhanced in late log-phase to stationary phase. Inhibitions of phytopathogenic mycelial growth and spore germination were directly correlated (P

Chitinase-producing Paenibacillus elgii strain HOA73 has been used to control plant diseases. However, the antimicrobial activity of its extracellular chitinase has not been fully elucidated. The major extracellular chitinase gene (PeChi68) from strain HOA73 was cloned and expressed in Escherichia coli in this study. This gene had an open reading frame of 2,028 bp, encoding a protein of 675 amino acid residues containing a secretion signal peptide, a chitin-binding domain, two fibronectin type III domains, and a catalytic hydrolase domain. The chitinase (PeChi68) purified from recombinant E. coli exhibited a molecular mass of approximately 68 kDa on SDS-PAGE. Biochemical analysis indicated that optimum temperature for the actitvity of purified chitinase was 50ºC. However, it was inactivated with time when it was incubated at 40ºC and 50ºC. Its optimum activity was found at pH 7, although its activity was stable when incubated between pH 3 and pH 11. Heavy metals inhibited this chitinase. This purified chitinase completely inhibited spore germination of two Cladosporium isolates and partially inhibited germination of Botrytis cinerea spores. However, it had no effect on the spores of a Colletotricum isolate. These results indicate that the extracellular chitinase produced by P. elgii HOA73 might have function in limiting spore germination of certain fungal pathogens.

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.

The radio frequency (RF) coil is one of the key components of the magnetic resonance imaging (MRI) system. It has a significant impact on the performance of the nuclear magnetic resonance (NMR) detection. Among numerous practical designs of RF coils for NMR imaging, the birdcage RF coil is the most popular choice from low field to ultra-high field MRI systems. In the transmission mode, it can establish a strong and homogeneous transverse magnetic field B1 for any element at its Larmor frequency. Similarly, in the reception mode, it exhibits extremely high sensitivity for the detection of even faint NMR signals from the volume of interest. Despite the sophisticated 3D structure of the birdcage coil, the developments in the design, analysis, and implementation technologies during the past decade have rendered the development of the birdcage coils quite reasonable. This article provides a detailed review of the recent progress in the birdcage RF coil technology for the MRI system.

A novel analytical solution for the designing of the birdcage RF coil has been demonstrated in this paper. A new concept of dominant resonance path has been introduced in this paper which is used to identify the specific closed current loop in the birdcage RF coil which is responsible for the dominant resonance frequency mode. This concept is used to determine the precise numerical values of the lumped capacitance deployed in the legs and/or end-rings of the birdcage RF coil for its proper operation at the desired resonance frequency. The analytical solution presented in this paper has been established by performing the two-port network based equivalent circuit modeling of the birdcage RF coil. The proposed analytical solution uses T-matrix theory and develops a relationship between the input impedance of the birdcage coil and the impedances of its leg and end-ring segments. The proposed analytical solution provides the information about the resonance frequency spectrum of the birdcage RF coil and solves the issue of its interfacing with external circuits without affecting its resonance characteristics. Based upon the proposed analysis and designing strategy presented in this paper, the low pass, high pass and band pass configurations of the birdcage RF coil were successfully implemented with FPCB (Flexible Printed Circuit board) technique for small volume NMR imaging applications at 1.5 T and 3.0 T MRI system. The results obtained for the implemented birdcage coils using the proposed analysis and designing technique are in closed agreement with already established methods.

The chitinase-producing bacterial strain C-1 is one of the key chitinase-producing biocontrol agents used for effective bioformulations for biological control. These bioformulations are mixed cultures of various chitinolytic bacteria. However, the precise identification, biocontrol activity, and the underlying mechanisms of the strain C-1 have not been investigated so far. Therefore, we evaluated in planta biocontrol efficacies of C-1 and determined the draft genome sequence of the strain in this study. The bacterial C-1 strain was identified as a novel Serratia sp. by a phylogenic analysis of its 16S rRNA sequence. The Serratia sp. C-1 bacterial cultures showed strong in planta biocontrol efficacies against some major phytopathogenic fungal diseases. The draft genome sequence of Serratia sp. C-1 indicated that the C-1 strain is a novel strain harboring a subset of genes that may be involved in its biocontrol activities.

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 OsMKK4DD 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.

The GacS/GacA two component system regulates various traits related to the biocontrol potential of plant-associated pseudomonads. The role of the sensor kinase, GacS, differs between strains in regulation of motility. In this study, we determined how a gacS mutation changed cell morphology and motility in Pseudomonas chlororaphis O6. The gacS mutant cells were elongated in stationary-phase compared to the wild type and the complemented gacS mutant, but cells did not differ in length in logarithmic phase. The gacS mutant had a two-fold increase in the number of flagella compared with the wild type strain; flagella number was restored to that of the wild type in the complemented gacS mutant. The more highly flagellated gacS mutant cells had greater swimming motilities than that of the wild type strain. Enhanced flagella formation in the gacS mutant correlated with increased expression of three genes, fleQ, fliQ and flhF, involved in flagellar formation. Expression of these genes in the complemented gacS mutant was similar to that of the wild type. These findings show that this root-colonizing pseudomonad adjusts flagella formation and cell morphology in stationary-phase using GacS as a major regulator.

A liquid bio-formulation containing chitinolytic bacteria, chitin and their products was assessed for its potential biological control against root-knot nematodes on cucumber. The bio-formulation was prepared by cultures of three chitinolytic bacteria, Chromobacterium sp. strain C-61, Lysobacter engymogenes and Serratia plymuthica in minimal medium supplemented with chitin. Under pot conditions, the bio-formulation showed better growth of cucumber plants, and less root galls and population density of Meloidogyne spp. than control media without the bio-formulation. In a greenhouse, 75-fold diluted bio-formulations were treated instead of water around cucumber plants through hoses for drip irrigation six times at 5-day intervals from the transplanting date. After 30 and 60 days, the treatment provided about 7% and 10% enhancement in the plant height and about 78% and 69% reduction in population density of Meloidogyne spp. in the rhizosphere, respectively. In addition, the experiments showed that the control effects occurred only in the soils contacted with the bio-formulation. Undiluted bio-formulations were drenched three times at 10-day intervals around cucumber plants severely infested with Meloidogyne spp. The treatment showed about 37% plant enhancement without dead plants compared with 37% death in the untreated control, and about 82% nematode reduction. These results suggest that the bio-formulation can be practically used to control the root-knot nematode on cucumber.

The GacS/GacA system in the root colonizer Pseudomonas chlororaphis O6 is a key regulator of many traits relevant to the biocontrol function of this bacterium. Proteomic analysis revealed 12 proteins were down-regulated in a gacS mutant of P. chlororaphis O6. These GacS-regulated proteins functioned in combating oxidative stress, cell signaling, biosynthesis of secondary metabolism, and secretion. The extent of regulation was shown by real-time RT-PCR to vary between the genes. Mutants of P. chlororaphis O6 were generated in two GacS-regulated genes, trpE, encoding a protein involved in tryptophan synthesis, and prnA, required for conversion of tryptophan to the antimicrobial compound, pyrrolitrin. Failure of the trpE mutant to induce systemic resistance in tobacco against a foliar pathogen causing soft rot, Pectobacterium carotovorum SCCI, correlated with reduced colonization of root surfaces implying an inadequate supply of tryptophan to support growth. Although colonization was not affected by mutation in the prnA gene, induction of systemic resistance was reduced, suggesting that pyrrolnitrin was an activator of plant resistance as well as an antifungal agent. Study of mutants in the other GacS-regulated proteins will indicate further the features required for biocontrol-activity in this rhizobacterium.

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.

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.

The stationary-phase sigma factor, RpoS, influences the expression of factors important in survival of Pseudomonas chlororaphis O6 in the rhizosphere. A partial proteomic profile of a rpoS mutant in P. chlororaphis O6 was conducted to identify proteins under RpoS regulation. Five of 14 differentially regulated proteins had unknown roles. Changes in levels of proteins in P. chlororaphis O6 rpoS mutant were associated with iron metabolism, and protection against oxidative stress. The P. chlororaphis O6 rpoS mutant showed increased production of a pyoverdine-like siderophore, indole acetic acid, and altered isozyme patterns for peroxidase, catalase and superoxide dismutase. Consequently, sensitivity to hydrogen peroxide exposure increased in the P. chlororaphis O6 rpoS mutant, compared with the wild type. Taken together, RpoS exerted regulatory control over factors important for the habitat of P. chlororaphis O6 in soil and on root surfaces. The properties of several of the proteins in the RpoS regulon are currently unknown.

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.

Diverse bacteria are known to colonize plants. However, only a small fraction of that diversity has been evaluated for their biopesticide potential. To date, the criteria for sampling and selection in such bioprospecting endeavors have not been systematically evaluated in terms of the relative amount of diversity they provide for analysis. The present study aimed to enhance the success of bio-prospecting efforts by increasing the diversity while removing the genotypic redundancy often present in large collections of bacteria. We developed a multivariate sampling and marker-based selection strategy that significantly increase the diversity of bacteria recovered from plants. In doing so, we quantified the effects of varying sampling intensity, media composition, incubation conditions, plant species, and soil source on the diversity of recovered isolates. Subsequent sequencing and high-throughput phenotypic analyses of a small fraction of the collected isolates revealed that this approach led to the recovery of over a dozen rare and, to date, poorly characterized genera of plant-associated bacteria with significant biopesticide activities. Overall, the sampling and selection approach described led to an approximately 5-fold improvement in efficiency and the recovery of several novel strains of bacteria with significant biopesticide potential.

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.

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.

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

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.

Root colonization by a plant-beneficial rhizobacterium, Pseudomonas chlororaphis O6, induces disease resistance in tobacco against leaf pathogens Erwinia carotovora subsp. carotovora SCC1, causing soft-rot, and Pseudomonas syringae pv. tabaci, causing wildfire. In order to identify the bacterial determinants involved in induced systemic resistance against plant diseases, extracellular components produced by the bacterium were fractionated and purified. Factors in the culture filtrate inducing systemic resistance were retained in the aqueous fraction rather than being partitioned into ethyl acetate. Fractionation on high-performance liquid chromatography followed by nuclear magnetic resonance mass spectrometry analysis identified the active compound as 2R, 3R-butanediol. 2R, 3R butanediol induced systemic resistance in tobacco to E. carotovora subsp. carotovora SCC1, but not to P. syringae pv. tabaci. Treatment of tobacco with the volatile 2R, 3R-butanediol enhanced aerial growth, a phenomenon also seen in plants colonized by P. chlororaphis O6. The isomeric form of the butanediol was important because 2S, 3S-butandiol did not affect the plant. The global sensor kinase, GacS, of P. chlororaphis O6 was a key regulator for induced systemic resistance against E. carotovora through regulation of 2R, 3R-butanediol production. This is the first report of the production of these assumed fermentation products by a pseudomonad and the role of the sensor kinase GacS in production of 2R, 3R-butanediol.

We provide here a comparative genome analysis of ten strains within the Pseudomonas fluorescens group including seven new genomic sequences. These strains exhibit a diverse spectrum of traits involved in biological control and other multitrophic interactions with plants, microbes, and insects. Multilocus sequence analysis placed the strains in three sub-clades, which was reinforced by high levels of synteny, size of core genomes, and relatedness of orthologous genes between strains within a sub-clade. The heterogeneity of the P. fluorescens group was reflected in the large size of its pan-genome, which makes up approximately 54% of the pan-genome of the genus as a whole, and a core genome representing only 45-52% of the genome of any individual strain. We discovered genes for traits that were not known previously in the strains, including genes for the biosynthesis of the siderophores achromobactin and pseudomonine and the antibiotic 2-hexyl-5-propyl-alkylresorcinol; novel bacteriocins; type II, III, and VI secretion systems; and insect toxins. Certain gene clusters, such as those for two type III secretion systems, are present only in specific sub-clades, suggesting vertical inheritance. Almost all of the genes associated with multitrophic interactions map to genomic regions present in only a subset of the strains or unique to a specific strain. To explore the evolutionary origin of these genes, we mapped their distributions relative to the locations of mobile genetic elements and repetitive extragenic palindromic (REP) elements in each genome. The mobile genetic elements and many strain-specific genes fall into regions devoid of REP elements (i.e., REP deserts) and regions displaying atypical tri-nucleotide composition, possibly indicating relatively recent acquisition of these loci. Collectively, the results of this study highlight the enormous heterogeneity of the P. fluorescens group and the importance of the variable genome in tailoring individual strains to their specific lifestyles and functional repertoire.