First report in South Korea of a bacterial leaf blight of oats caused by Pseudomonas salomonii.

In March 2020, approximately 20% of leaves in a commercial oat field growing Avena sativa (cv. Samhan) in Jeongeup, Korea (35.3859°, 126.5607°), displayed leaf blight in the seedling stage of development. The lesions observed in 2020 were of a yellow discoloration that spread from the leaf tip downward with minimal brown delineation (Fig. S1A). These symptoms differed from the haloes delineated at the edges by extensive brown necrosis caused by Pseudomonas coronofaciens (Kim 2020a; Fig. S1C). To isolate the causal agent, 3-cm-long pieces of symptomatic leaves from ten different oat plants were disinfected by submersion in 70% ethanol for 5 min followed by immersion in 1% sodium hypochlorite for 5 min and rinsing extensively with sterile distilled water. The air-dried segments were transferred intact to nutrient agar, and only one colony type (yellow-colored, wet, with a shiny convex surface) was observed. After single colony isolation, three isolates from different 2020 field-grown diseased leaves, termed 2007, 2009, and 2011, were selected at random. The isolates produced fluorescent siderophores on King's medium B and triggered a hypersensitive response (HR) when infiltrated into tobacco (cv. Xanthi) leaves (Fig. S1D). Multilocus sequence typing analysis with four housekeeping genes was used for taxonomic identification of these isolates (Maiden et al. 1998). The 16S rRNA sequences were amplified with the 27F/1492R universal primers (Weisburg et al. 1991). The primers for three housekeeping genes were designed using genome sequence of P. coronaficiens X-1 causing halo blight disease in Korea (NZ_CP050260.1, Kim, 2020b). The products obtained had sizes of 640 bp for gltA (using primers F: 5'-CCT GGT AGC CAA GAT GCC GAC-3'; R: 5'-CAA AGA TCA CGG TGA ACA TGC TGG-3'), 710 bp for gyrB (with primers F: 5'-TCG GCA GCC GAG GTC ATC ATG AC-3'; R: 5'-TTG TCT TTG GTC TGC GAG CTG AA-3'), and 870 bp for rpoD ( with primers F: 5'-GTG AAG GCG AAA TCG AAA TCG-3'; R: 5'-CCG ATG TTG CCT TCC TGG ATC AG-3'). The concatenated sequences (2,353 bp/2,376 bp) had 99% identity with the gene sequences from P. salomonii type strain (AY091528.1, NZ_FNOX01000003, LC486864.1, LC486849.1), but lesser identity (90%) with P. coronafaciens (Fig. S2). Pathogenicity of the Korean isolates was confirmed by fulfilling Koch's postulates using leaves of 2 week-old greenhouse-grown 'Samhan' seedlings. Plants (n=50) were sprayed with 108 cfu/ml bacterial suspensions in water or with sterile water as controls. The plants were incubated for a week at 23 °C in 100% relative humidity under a 10 h light/14 h dark photoperiod. Five days after bacterial inoculation, yellow discoloration appeared at the leaf tips which progressed downward with time (Fig. S1B). Three bacterial isolates extracted from yellowed, inoculated leaves had 16S rRNA gene sequences identical to that of P. salomonii Korean isolates, 2007, 2009 and 2011, and they caused the anticipated symptoms when inoculated into oat leaves. These findings indicate that P. salomonii should be added to the potential pathogens of oats grown in Korea. Understanding whether spring weather conditions (warmth and humidity) boost this oat disease will help devise disease alert systems for farmers (Anderson 2004; Chakraborty 2005).

Complete Genome Resource of Serratia plymuthica C-1 that Causes Root Rot Disease in Korean Ginseng.

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.

Complete Genome Resource of Pseudomonas coronafaciens X-1 Causing Halo Blight Disease in Oats.

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.

First Report of Leaf Spots on Oat Caused by Alternaria alternata in South Korea.

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).

Hydrogen cyanide produced by Pseudomonas chlororaphis O6 is a key aphicidal metabolite.

A biocontrol bacterium, Pseudomonas chlororaphis O6 promotes plant health through multifaceted mechanisms. In this study, we used P. chlororaphis O6 mutants to examine metabolites with aphicidal activity. Direct application of intact P. chlororaphis cells to the surface of second-instar nymphs of the green peach aphid resulted in no mortality. However, nymphs died when exposed only to the volatiles produced by the P. chlororaphis O6 wild-type strain grown on rich media. Mutants lacking the production of two antibiotics, phenazine and pyrrolnitrin, or the insect toxin FitD retained the aphicidal potential of the wild-type strain. However, the volatiles produced by mutants deficient in the production of hydrogen cyanide (HCN) or defective in the synthesis of the global regulator GacS, which regulates HCN synthesis, showed no aphicidal activity. Direct application of potassium cyanide caused mortality of green peach aphid nymphs. These results indicate that HCN production by a plant probiotic is involved in preventing insect growth.

An Electroactive, Tunable, and Frequency Selective Surface Utilizing Highly Stretchable Dielectric Elastomer Actuators Based on Functionally Antagonistic Aperture Control.

An active, frequency selective surface utilizing a silver-nanowire-coated dielectric elastomer with a butterfly-shaped aperture pattern is realized by properly exploiting the electroactive control of two antagonistic functions (stretching vs compression) on a patterned dielectric elastomer actuator.

Ag nanoparticles generated using bio-reduction and -coating cause microbial killing without cell lysis.

Cost-effective "green" methods of producing Ag nanoparticles (NPs) are being examined because of the potential of these NPs as antimicrobials. Ag NPs were generated from Ag ions using extracellular metabolites from a soil-borne Pythium species. The NPs were variable in size, but had one dimension less than 50 nm and were biocoated; aggregation and coating changed with acetone precipitation. They had dose-dependent lethal effects on a soil pseudomonad, Pseudomonas chlororaphis O6, and were about 30-fold more effective than Ag(+) ions. A role of reactive oxygen species in cell death was demonstrated by use of fluorescent dyes responsive to superoxide anion and peroxide accumulation. Also mutants of the pseudomonad, defective in enzymes that protect against oxidative stress, were more sensitive than the wild type strain; mutant sensitivity differed between exposure to Ag NPs and Ag(+) ions demonstrating a nano-effect. Imaging of bacterial cells treated with the biocoated Ag NPs revealed no cell lysis, but there were changes in surface properties and cell height. These findings support that biocoating the NPs results in limited Ag release and yet they retained potent antimicrobial activity.

Comparative genomics of plant-associated Pseudomonas spp.: insights into diversity and inheritance of traits involved in multitrophic interactions.

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.

The global regulator GacS regulates biofilm formation in Pseudomonas chlororaphis O6 differently with carbon source.

An aggressive root colonizer, Pseudomonas chlororaphis O6 produces various secondary metabolites that impact plant health. The sensor kinase GacS is a key regulator of the expression of biocontrol-related traits. Biofilm formation is one such trait because of its role in root surface colonization. This paper focuses on the effects of carbon source on biofilm formation. In comparison with the wild type, a gacS mutant formed biofilms at a reduced level with sucrose as the major carbon source but at much higher level with mannitol in the defined medium. Biofilm formation by the gacS mutant occurred without phenazine production and in the absence of normal levels of acyl homoserine lactones, which promote biofilms with other pseudomonads. Colonization of tomato roots was similar for the wild type and gacS mutant, showing that any differences in biofilm formation in the rhizosphere were not of consequence under the tested conditions. The reduced ability of the gacS mutant to induce systemic resistance against tomato leaf mold and tomato gray mold was consistent with a lack of production of effectors, such as phenazines. These results demonstrated plasticity in biofilm formation and root colonization in the rhizosphere by a beneficial pseudomonad.

A Comparative Study of Micromechanical Analysis Models for Determining the Effective Properties of Out-of-Autoclave Carbon Fiber-Epoxy Composites.

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.

The GacS-regulated sigma factor RpoS governs production of several factors involved in biocontrol activity of the rhizobacterium Pseudomonas chlororaphis O6.

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.

Identification of novel pepper genes involved in Bax- or INF1-mediated cell death responses by high-throughput virus-induced gene silencing.

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.

Draft genome sequence of Pantoea ananatis B1-9, a nonpathogenic plant growth-promoting bacterium.

Pantoea ananatis B1-9 is an endophytic Gram-negative rhizobacterium that was isolated for its ability to promote plant growth and improve crop yield in the field. Here we report the draft genome sequence of P. ananatis B1-9. Comparison of this sequence to the sequenced genome of a plant-pathogenic P. ananatis strain, LMG20103, indicated that the pathogenesis-related genes were absent, but a subset of gene functions that may be related to its plant growth promotion were present.

Root-Associated Bacteria Are Biocontrol Agents for Multiple Plant Pests.

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.

Draft genome sequence of the biocontrol bacterium Pseudomonas putida B001, an oligotrophic bacterium that induces systemic resistance to plant diseases.

Pseudomonas putida B001 is a rhizobacterium that was isolated on the basis of its abilities to grow under low-nutrient conditions and induce systemic resistance against bacterial, fungal, and viral diseases of plants. Here we report the draft genome sequence and automatic annotation of strain B001. Comparison of this sequence to the sequenced genome of P. putida KT2440 points to a subset of gene functions that may be related to the defense-inducing functions of B001.

Draft genome sequence of the biocontrol bacterium Chromobacterium sp. strain C-61.

Chromobacterium sp. strain C-61 is a plant-associated bacterium with proven capacities to suppress plant diseases. Here, we report the draft genome sequence and automatic annotation of strain C-61. A comparison of this sequence to the sequenced genome of Chromobacterium violaceum ATCC 12472 indicates the novelty of C-61 and a subset of gene functions that may be related to its biocontrol activities.

The multifactorial basis for plant health promotion by plant-associated bacteria.

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.

Nematicidal activity of a nonpathogenic biocontrol bacterium, Pseudomonas chlororaphis O6.

Bacterial culture filtrates of an aggressive rhizobacterium, Pseudomonas chlororaphis O6, displayed strong nematicidal activity. The nematicidal activity of P. chlororaphis O6 was markedly reduced in the gacS mutant of P. chlororaphis O6 grown in the presence of glycine, but no reduction of nematicidal activity in the gacS mutant was noted in the absence of glycine. The results of bioassay with P. chlororaphis O6 mutants showed that phenazine and pyrrolnitrin production was not a major factor, but the effects of glycine in the culture medium suggest that formation of hydrogen cyanide might be important. Assessments in greenhouse studies with tomatoes growing in nematode-infested soils confirmed that the application of P. chlororaphis O6 resulted in the control of the root-knot nematode. Our results demonstrated that P. chlororaphis O6 could be employed as a biocontrol agent for the control of the root-knot nematode, and the global regulator, GacS, functions as a positive regulator of the expression of nematicidal compounds and enzymes in P. chlororaphis O6.

The Plant-Stress Metabolites, Hexanoic Aacid and Melatonin, Are Potential "Vaccines" for Plant Health Promotion.

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.

Crash Analysis of Aluminum/CFRP Hybrid Adhesive Joint Parts Using Adhesive Modeling Technique Based on the Fracture Mechanics.

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.