Bacterial type III effector-induced plant C8 volatiles elicit antibacterial immunity in heterospecific neighbouring plants via airborne signalling.

Upon sensing attack by pathogens and insect herbivores, plants release complex mixtures of volatile compounds. Here, we show that the infection of lima bean (Phaseolus lunatus L.) plants with the non-host bacterial pathogen Pseudomonas syringae pv. tomato led to the production of microbe-induced plant volatiles (MIPVs). Surprisingly, the bacterial type III secretion system, which injects effector proteins directly into the plant cytosol to subvert host functions, was found to prime both intra- and inter-specific defense responses in neighbouring wild tobacco (Nicotiana benthamiana) plants. Screening of each of 16 effectors using the Pseudomonas fluorescens effector-to-host analyser revealed that an effector, HopP1, was responsible for immune activation in receiver tobacco plants. Further study demonstrated that 1-octen-3-ol, 3-octanone and 3-octanol are novel MIPVs emitted by the lima bean plant in a HopP1-dependent manner. Exposure to synthetic 1-octen-3-ol activated immunity in tobacco plants against a virulent pathogen Pseudomonas syringae pv. tabaci. Our results show for the first time that a bacterial type III effector can trigger the emission of C8 plant volatiles that mediate defense priming via plant-plant interactions. These results provide novel insights into the role of airborne chemicals in bacterial pathogen-induced inter-specific plant-plant interactions.

Evidence for Volatile Memory in Plants: Boosting Defence Priming through the Recurrent Application of Plant Volatiles.

Plant defence responses to various biotic stresses via systemic acquired resistance (SAR) are induced by avirulent pathogens and chemical compounds, including certain plant hormones in volatile form, such as methyl salicylate and methyl jasmonate. SAR refers to the observation that, when a local part of a plant is exposed to elicitors, the entire plant exhibits a resistance response. In the natural environment, plants are continuously exposed to avirulent pathogens that induce SAR and volatile emissions affecting neighbouring plants as well as the plant itself. However, the underlying mechanism has not been intensively studied. In this study, we evaluated whether plants "memorise" the previous activation of plant immunity when exposed repeatedly to plant defensive volatiles such as methyl salicylate and methyl jasmonate. We hypothesised that stronger SAR responses would occur in plants treated with repeated applications of the volatile plant defence compound MeSA than in those exposed to a single or no treatment. Nicotiana benthamiana seedlings subjected to repeated applications of MeSA exhibited greater protection against Pseudomonas syringae pv. tabaci and Pectobacterium carotovorum subsp. carotovorum than the control. The increase in SAR capacity in response to repeated MeSA treatment was confirmed by analysing the defence priming of the expression of N. benthamiana Pathogenesis-Related 1a (NbPR1a) and NbPR2 by quantitative reverse-transcription PCR compared with the control. We propose the concept of plant memory of plant defence volatiles and suggest that SAR is strengthened by the repeated perception of volatile compounds in plants.

Seed defense biopriming with bacterial cyclodipeptides triggers immunity in cucumber and pepper.

Seed priming is to expose seeds to specific compounds to enhance seed germination. Few studies of plant immune activation through seed priming have been conducted. Here, we introduce an emerging technology that combines seed priming with elicitation of plant immunity using biologically active compounds. This technology is named 'seed defense biopriming' (SDB). We prepared heat-stable metabolites from 1,825 root-associated Bacillus spp. isolated from the rhizosphere in South Korea. These preparations were tested for their ability to induce SDB in cucumber and pepper seeds and trigger plant immunity. SDB with heat-stable metabolites of the selected Bacillus gaemokensis strain PB69 significantly reduced subsequent bacterial diseases under in vitro and field conditions and increased fruit yield. Transcriptional analysis of induced resistance marker genes confirmed the upregulation of salicylic acid, ethylene, and jasmonic acid signaling. Mortality of the insect pest Spodoptera litura increased when larvae fed on SDB-treated cucumber tissues. Analysis of the causative bacterial metabolites identified a leucine-proline cyclodipeptide and a commercially obtained leucine-proline cyclodipeptide induced similar results as treatment with the bacterial preparation. Our results indicate that SDB treatment with the heat-stable bacterial metabolite effectively elicited immunity and controlled disease in seedlings to whole plants, thereby increasing yield even under field conditions.

Biological and chemical strategies for exploring inter- and intra-kingdom communication mediated via bacterial volatile signals.

Airborne chemical signals emitted by bacteria influence the behavior of other bacteria and plants. We present an overview of in vitro methods for evaluating bacterial and plant responses to bacterial volatile compounds (BVCs). Three types of equipment have been used to physically separate the bacterial test strains from either other bacterial strains or plants (in our laboratory we use either Arabidopsis or tobacco plant seedlings): a Petri dish containing two compartments (BI Petri dish); two Petri dishes connected with tubing; and a microtiter-based assay. The optimized procedure for the BI Petri dish system is described in this protocol and can be widely used for elucidation of potential function in interactions between diverse microbes and those plant and chemical volatiles emitted by bacteria that are most likely to mediate bacterial or plant responses to BVCs. We also describe a procedure for metabolome-based BVC profiling via dynamic (i.e., continuous airflow) or static headspace sampling using solid-phase microextraction (SPME). Using both these procedures, bacteria-bacteria communications and bacteria-plant interactions mediated by BVCs can be rapidly investigated (within 1-4 weeks).

Aboveground insect infestation attenuates belowground Agrobacterium-mediated genetic transformation.

Agrobacterium tumefaciens causes crown gall disease. Although Agrobacterium can be popularly used for genetic engineering, the influence of aboveground insect infestation on Agrobacterium induced gall formation has not been investigated. Nicotiana benthamiana leaves were exposed to a sucking insect (whitefly) infestation and benzothiadiazole (BTH) for 7 d, and these exposed plants were inoculated with a tumorigenic Agrobacterium strain. We evaluated, both in planta and in vitro, how whitefly infestation affects crown gall disease. Whitefly-infested plants exhibited at least a two-fold reduction in gall formation on both stem and crown root. Silencing of isochorismate synthase 1 (ICS1), required for salicylic acid (SA) synthesis, compromised gall formation indicating an involvement of SA in whitefly-derived plant defence against Agrobacterium. Endogenous SA content was augmented in whitefly-infested plants upon Agrobacterium inoculation. In addition, SA concentration was three times higher in root exudates from whitefly-infested plants. As a consequence, Agrobacterium-mediated transformation of roots of whitefly-infested plants was clearly inhibited when compared to control plants. These results suggest that aboveground whitefly infestation elicits systemic defence responses throughout the plant. Our findings provide new insights into insect-mediated leaf-root intra-communication and a framework to understand interactions between three organisms: whitefly, N. benthamiana and Agrobacterium.

Elicitation of induced resistance against Pectobacterium carotovorum and Pseudomonas syringae by specific individual compounds derived from native Korean plant species.

Plants have developed general and specific defense mechanisms for protection against various enemies. Among the general defenses, induced resistance has distinct characteristics, such as broad-spectrum resistance and long-lasting effectiveness. This study evaluated over 500 specific chemical compounds derived from native Korean plant species to determine whether they triggered induced resistance against Pectobacterium carotovorum supsp. carotovorum (Pcc) in tobacco (Nicotiana tabacum) and Pseudomonas syringae pv. tomato (Pst) in Arabidopsis thaliana. To select target compound(s) with direct and indirect (volatile) effects, a new Petri-dish-based in vitro disease assay system with four compartments was developed. The screening assay showed that capsaicin, fisetin hydrate, jaceosidin, and farnesiferol A reduced the disease severity significantly in tobacco. Of these four compounds, capsaicin and jaceosidin induced resistance against Pcc and Pst, which depended on both salicylic acid (SA) and jasmonic acid (JA) signaling, using Arabidopsis transgenic and mutant lines, including npr1 and NahG for SA signaling and jar1 for JA signaling. The upregulation of the PR2 and PDF1.2 genes after Pst challenge with capsaicin pre-treatment indicated that SA and JA signaling were primed. These results demonstrate that capsaicin and jaceosidin can be effective triggers of strong induced resistance against both necrotrophic and biotrophic plant pathogens.

The folate precursor para-aminobenzoic acid elicits induced resistance against Cucumber mosaic virus and Xanthomonas axonopodis.

BACKGROUND AND AIMS: The use of vitamins including vitamin B1, B2 and K3 for the induction of systemic acquired resistance (SAR) to protect crops against plant pathogens has been evaluated previously. The use of vitamins is beneficial because it is cost effective and safe for the environment. The use of folate precursors, including ortho-aminobenzoic acid, to induce SAR against a soft-rot pathogen in tobacco has been reported previously. METHODS: In the present study, para-aminobenzoic acid (PABA, also referred to as vitamin Bx) was selected owing to its effect on the induction of SAR against Xanthomonas axonopodis pv. vesicatoria in pepper plants through greenhouse screening. KEY RESULTS: Dipping of pepper seedlings in a 1 mm PABA solution in field trials induced SAR against artificially infiltrated X. axonopodis pv. vesicatoria and naturally occurring cucumber mosaic virus. Expression of the Capsicum annuum pathogenesis-related 4 gene was primed in response to pathogen infection as assessed by quantitative real-time PCR. The accumulation of cucumber mosaic virus RNA was reduced in PABA-treated pepper plants at 40 and 105 d post-treatment. Unexpectedly, fruit yield was increased in PABA-treated plants, indicating that PABA-mediated SAR successfully protected pepper plants from infection by bacterial and viral pathogens without significant fitness allocation costs. CONCLUSIONS: The present study is the first to demonstrate the effective elicitation of SAR by a folate precursor under field conditions.

Chitinophaga eiseniae sp. nov., isolated from vermicompost.

A Gram-negative, rod-shaped bacterial strain, YC6729(T), was isolated from vermicompost collected at Masan, Korea, and its taxonomic position was investigated by a polyphasic taxonomic approach. Strain YC6729(T) grew optimally at 30 °C and at pH 6.5-8.5. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain YC6729(T) belongs to the genus Chitinophaga in the family Chitinophagaceae. It was related most closely to Chitinophaga terrae KP01(T) (96.4 % 16S rRNA gene sequence similarity), Chitinophaga ginsengisegetis Gsoil 040(T) (96.1 %), Chitinophaga arvensicola IAM 12650(T) (96.1 %) and Chitinophaga pinensis DSM 2588(T) (93.3 %). Strain YC6729(T) contained MK-7 as the major menaquinone and homospermidine as the major polyamine. The fatty acids of strain YC6729(T) were iso-C(15 : 0), C(16 : 1)ω5c, iso-C(17 : 0) 3-OH, C(16 : 0), anteiso-C(18 : 0) and/or C(18 : 2)ω6,9c, iso-C(15 : 0) 2-OH and/or C(16 : 1)ω7c, C(14 : 0), iso-C(15 : 0) 3-OH, iso-C(15 : 1) G, C(18 : 1)ω5c, iso-C(15 : 1) I and/or C(13 : 0) 3-OH, C(13 : 0) 2-OH, C(16 : 0) 3-OH and unknown fatty acid ECL 13.565. The polar lipid profile contained phosphatidylethanolamine, unknown aminolipids and unknown lipids. The total DNA G+C content of strain YC6729(T) was 48.9 mol%. The phenotypic, chemotaxonomic and phylogenetic data showed that strain YC6729(T) represents a novel species of the genus Chitinophaga, for which the name Chitinophaga eiseniae sp. nov. is proposed. The type strain is YC6729(T) ( = KACC 13774(T)  = DSM 22224(T)).

Nocardioides caricicola sp. nov., an endophytic bacterium isolated from a halophyte, Carex scabrifolia Steud.

A Gram-staining-positive, coccoid to rod-shaped bacterium, designated strain YC6903(T), was isolated from a halophytic plant (Carex scabrifolia Steud.) collected from sand dunes at Namhae Island, Korea, and its taxonomic position was investigated by using a polyphasic approach. Strain YC6903(T) grew optimally at 30 °C and at pH 8.0. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain YC6903(T) belongs to the genus Nocardioides in the family Nocardioidaceae. Strain YC6903(T) was related most closely to Nocardioides pyridinolyticus OS4(T) (97.0 % 16S rRNA gene sequence similarity), Nocardioides dokdonensis FR1436(T) (96.6 %), Nocardioides aquiterrae GW-9(T) (96.6 %) and Nocardioides hankookensis DS-30(T) (96.6 %). The cell-wall peptidoglycan contained LL-diaminopimelic acid and MK-8(H(4)) was the major respiratory quinone. The mean (±SD) level of DNA-DNA relatedness between strain YC6903(T) and N. pyridinolyticus OS4(T) was 53.5±5.5 %. The predominant cellular fatty acid of strain YC6903(T) was iso-C(16 : 0) (28.9 %). The DNA G+C content was 71.7 mol%. Phenotypic, phylogenetic and chemotaxonomic data indicated that strain YC6903(T) represents a novel species of the genus Nocardioides, for which the name Nocardioides caricicola sp. nov. is proposed. The type strain is YC6903(T) (=KACC 13778(T) =DSM 22177(T)).

Chitinophaga vermicomposti sp. nov., with antifungal activity, isolated from vermicompost.

A Gram-negative, rod-shaped bacterial strain, YC6729T, was isolated from the vermicompost (VC) collected at Masan, Korea and its taxonomic position was investigated by a polyphasic taxonomic approach. Strain YC6729T grew optimally at 30 degrees C and at pH 6.5-8.5. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain YC6729T belongs to the genus Chitinophaga in the family Chitinophagaceae. Most closely related species are Chitinophaga terra KP01T (96.4 %), Chitinophaga ginsengisegetis Gsoil 040T (96.1 %) and Chitinophaga arvensicola IAM 12650T (96.1 %). Strain YC6729T contained MK-7 as the major menaquinone and homospermidine as the major polyamine. The major fatty acids of strain YC6729T C15:0 iso, C16:1omega5c and C17:0 iso 3-OH. The total DNA G+C content was 48.9 mol%. The phenotypic, chemotaxonomic and phylogenetic data showed that strain YC6729T represents a novel species of the genus Chitinophaga, for which the name Chitinophaga vermicomposti sp. nov. is proposed. The type strain is YC6729T (= KACC 13774T = DSM 22224T).

Sphingosinicella vermicomposti sp. nov., isolated from vermicompost, and emended description of the genus Sphingosinicella.

A Gram-stain-negative, rod-shaped bacterium, designated strain YC7378(T) was isolated from vermicompost (VC) collected at Masan, Korea, and its taxonomic position was investigated by using a polyphasic approach. Strain YC7378(T) grew optimally at 30 degrees C and at pH 6.5-8.5. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain YC7378(T) belongs to the genus Sphingosinicella in the family Sphingomonadaceae. The most closely related strains are Sphingosinicella soli KSL-125(T) (95.7 %), Sphingosinicella xenopeptidilytica 3-2W4(T) (95.6 %) and Sphingosinicella microcystinivorans Y2(T) (95.5 %). Strain YC7378(T) contained ubiquinone Q-10 as the major respiratory quinone system and sym-homospermidine as the major polyamine. The major fatty acids of strain YC7378(T) were C(18 : 1)omega7c, C(16 : 1)omega7c and/or iso-C(15 : 0) 2-OH, C(14 : 0) 2-OH and C(16 : 0). The major polar lipids were sphingoglycolipid, diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The total DNA G+C content was 59.4 mol%. The phenotypic, phylogenetic and chemotaxonomic data showed that strain YC7378(T) represents a novel species of the genus Sphingosinicella, for which the name Sphingosinicella vermicomposti sp. nov. is proposed. The type strain is YC7378(T) (=KCTC 22446(T) =DSM 21593(T)).

Eiseniicola composti gen. nov., sp. nov., with antifungal activity against plant pathogenic fungi.

A Gram-negative, short rod-shaped bacterial strain, YC06271T, was isolated from the vermicompost (VC) collected at Masan, Korea and its taxonomic position was investigated by a polyphasic taxonomic approach. Strain YC06271T grew optimally at 28-30 degrees C and at pH 7.0-9.0. The 16S rRNA gene sequence of strain YC06271T was most closely related to members of the genera Bordetella (96.4-95.8 %), Achromobacter (96.0-95.7 %), Alcaligenes (96.0-94.2 %), Pusillimonas noertemannii (95.9 %), Pigmentiphaga (95.8-95.5 %) and less than 95.5 % similarity with the members of the other genera of the family Alcaligenaceae. Strain YC06271T contained ubiquinone-8 (Q-8) as the major respiratory quinone system and putrescine as the major polyamine. The major fatty acids of strain YC06271T were C16:1omega7c and/or C15:0 iso 2-OH and C16:0. The major polar lipids were phosphatidylglycerol and phosphatidylethanolamine. The G+C content of the genomic DNA was 55.4 mol%. Phylogenetic analysis, biochemical, chemotaxonomic and phenotypic characteristics strongly supported the differentiation of strain YC06271T from the validly published genera of the family Alcaligenaceae. Therefore, it is proposed that strain YC06271T represents a novel species within a novel genus, with the name Eiseniicola composti gen. nov., sp. nov. The type strain is YC06271T (= KCTC 22250T = DSM 21045T).