Dual functionality of natural mixtures of bacterial volatile compounds on plant growth.

Bacteria emit volatile compounds that modulate plant growth. Previous studies reported the impacts of bacterial volatile compounds on plant growth; however, the results varied depending on bacterial nutrient availability. We investigated whether the effects of plant growth-inhibiting volatiles (PGIVs) and plant growth-promoting volatiles (PGPVs) depended on the perceived dose by evaluating the growth of Arabidopsis thaliana seedlings placed at 7, 14, and 21 cm away from Bacillus amyloliquefaciens GB03 colonies growing in rich medium. A large bacterial colony (500 µl inoculum) inhibited plant growth at 7 cm and promoted growth at 21 cm, whereas a small bacterial colony (100 µl inoculum) induced the opposite pattern of response. We identified pyrazine and 2,5-dimethylpyrazine as candidate PGIVs that significantly reduced plant growth at a distance of 7 cm. PGIV effects were validated by exposing plants to synthetic 2,5-dimethylpyrazine and bacteria emitting PGPVs, which showed that PGIVs overwhelm PGPVs to rapidly increase salicylic acid content and related gene expression. This is referred to as the defence-growth trade-off. Our results indicate that high PGIV concentrations suppress plant growth and promote immunity, whereas low PGPV concentrations promote growth. This study provides novel insights into the complex effects of bacterial volatile mixtures and fine-tuning of bacteria-plant interactions.

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.

Disruption of Firmicutes and Actinobacteria abundance in tomato rhizosphere causes the incidence of bacterial wilt disease.

Enrichment of protective microbiota in the rhizosphere facilitates disease suppression. However, how the disruption of protective rhizobacteria affects disease suppression is largely unknown. Here, we analyzed the rhizosphere microbial community of a healthy and diseased tomato plant grown <30-cm apart in a greenhouse at three different locations in South Korea. The abundance of Gram-positive Actinobacteria and Firmicutes phyla was lower in diseased rhizosphere soil (DRS) than in healthy rhizosphere soil (HRS) without changes in the causative Ralstonia solanacearum population. Artificial disruption of Gram-positive bacteria in HRS using 500-µg/mL vancomycin increased bacterial wilt occurrence in tomato. To identify HRS-specific and plant-protective Gram-positive bacteria species, Brevibacterium frigoritolerans HRS1, Bacillus niacini HRS2, Solibacillus silvestris HRS3, and Bacillus luciferensis HRS4 were selected from among 326 heat-stable culturable bacteria isolates. These four strains did not directly antagonize R. solanacearum but activated plant immunity. A synthetic community comprising these four strains displayed greater immune activation against R. solanacearum and extended plant protection by 4 more days in comparison with each individual strain. Overall, our results demonstrate for the first time that dysbiosis of the protective Gram-positive bacterial community in DRS promotes the incidence of disease.

Achieving similar root microbiota composition in neighbouring plants through airborne signalling.

The ability to recognize and respond to environmental signals is essential for plants. In response to environmental changes, the status of a plant is transmitted to other plants in the form of signals such as volatiles. Root-associated bacteria trigger the release of plant volatile organic compounds (VOCs). However, the impact of VOCs on the rhizosphere microbial community of neighbouring plants is not well understood. Here, we investigated the effect of VOCs on the rhizosphere microbial community of tomato plants inoculated with a plant growth-promoting rhizobacterium Bacillus amyloliquefaciens strain GB03 and that of their neighbouring plants. Interestingly, high similarity (up to 69%) was detected in the rhizosphere microbial communities of the inoculated and neighbouring plants. Leaves of the tomato plant treated with strain GB03-released ß-caryophyllene as a signature VOC, which elicited the release of a large amount of salicylic acid (SA) in the root exudates of a neighbouring tomato seedling. The exposure of tomato leaves to ß-caryophyllene resulted in the secretion of SA from the root. Our results demonstrate for the first time that the composition of the rhizosphere microbiota in surrounding plants is synchronized through aerial signals from plants.

Inheritance of seed and rhizosphere microbial communities through plant-soil feedback and soil memory.

Since the discovery of the role of microbes in the phytobiome, microbial communities (microbiota) have been identified and characterized based on host species, development, distribution, and condition. The microbiota in the plant rhizosphere is believed to have been established prior to seed germination and innate immune development. However, the microbiota in seeds has received little attention. Although our knowledge of the distribution of microbiota in plant seeds and rhizosphere is currently limited, the impact of these microbiota is likely to be greater than expected. This minireview suggests a new function of microbial inheritance from the seed to root and from the first generation of plants to the next. Surprisingly, recruitment and accumulation of microbiota by biotic and abiotic stresses affect plant immunity in the next generation through plant-soil feedback and soil memory. To illustrate this process, we propose a new term called 'microbiota-induced soil inheritance (MISI).' A comprehensive understanding of MISI will provide novel insights into plant-microbe interactions and plant immunity inheritance.

Beyond the two compartments Petri-dish: optimising growth promotion and induced resistance in cucumber exposed to gaseous bacterial volatiles in a miniature greenhouse system.

BACKGROUND: Bacterial volatiles promote plant growth and elicit immunity responses in plants grown in two-compartment Petri dishes. Due to the limitations of bacterial volatile compound (BVC) treatments such as their high evaporation rates, it is convenient to apply BVCs in closed systems such as greenhouses. However, the concentrations of BVCs must be optimised. We therefore attempted to optimise BVC emissions from bacteria grown on solid medium and synthetic BVC treatment in order to maximise plant growth and induced resistance in a miniature greenhouse system. RESULTS: We cultivated the model BVC emitter Bacillus subtilis GB03 on complex medium for continuous treatment, which we placed near 1-week-old cucumber seedlings in a miniature greenhouse. Aboveground and belowground plant growth parameters were significantly increased at 1 and 2 weeks after treatment with BVCs released by B. subtilis GB03. Moreover, this treatment protected cucumber seedlings against the angular leaf spot pathogen Pseudomonas syringae pv. lachrymans. In addition, cucumber shoot growth was promoted in response to the slow release of BVCs from filter paper that had absorbed 1000 and 10 µM synthetic 2,3-butanediol, a key BVC from B. subtilis strain GB03. However, induced resistance was only elicited when 10 plates containing 10 µM 2,3-butanediol were utilised in the miniature greenhouse. The mechanism of induced resistance appears to involve the activation of the jasmonic acid signalling pathway. CONCLUSIONS: To overcome the difficulties associated with treatment using a single application of BVC in the greenhouse, we optimised conditions for BVC application via consistent exposure in a slow-release system.

Plant growth-promoting archaea trigger induced systemic resistance in Arabidopsis thaliana against Pectobacterium carotovorum and Pseudomonas syringae.

Archaea have inhabited the earth for a long period of time and are ubiquitously distributed in diverse environments. However, few studies have focused on the interactions of archaea with other organisms, including eukaryotes such as plants, since it is difficult to cultivate sufficient numbers of archaeal cells for analysis. In this study, we investigated the interaction between soil archaea and Arabidopsis thaliana. We demonstrate for the first time that soil archaea promote plant growth and trigger induced systemic resistance (ISR) against the necrotrophic bacterium Pectobacterium carotovorum subsp. carotovorum SCC1 and biotrophic bacterium Pseudomonas syringae pv. tomato DC3000. Ammonia-oxidizing archaeon Nitrosocosmicus oleophilus MY3 cells clearly colonized the root surface of Arabidopsis plants, and increased resistance against both pathogenic species via the salicylic acid-independent signalling pathway. This mechanism of bacterial resistance resembles that underlying soil bacteria- and fungi-mediated ISR signalling. Additionally, volatile emissions from N. oleophilus MY3 were identified as major archaeal determinants that elicit ISR. Our results lay a foundation for archaea-plant interactions as a new field of research.

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).

Sweet scents from good bacteria: Case studies on bacterial volatile compounds for plant growth and immunity.

Beneficial bacteria produce diverse chemical compounds that affect the behavior of other organisms including plants. Bacterial volatile compounds (BVCs) contribute to triggering plant immunity and promoting plant growth. Previous studies investigated changes in plant physiology caused by in vitro application of the identified volatile compounds or the BVC-emitting bacteria. This review collates new information on BVC-mediated plant-bacteria airborne interactions, addresses unresolved questions about the biological relevance of BVCs, and summarizes data on recently identified BVCs that improve plant growth or protection. Recent explorations of bacterial metabolic engineering to alter BVC production using heterologous or endogenous genes are introduced. Molecular genetic approaches can expand the BVC repertoire of beneficial bacteria to target additional beneficial effects, or simply boost the production level of naturally occurring BVCs. The effects of direct BVC application in soil are reviewed and evaluated for potential large-scale field and agricultural applications. Our review of recent BVC data indicates that BVCs have great potential to serve as effective biostimulants and bioprotectants even under open-field conditions.

Bacterial RNAs activate innate immunity in Arabidopsis.

The common molecular patterns of microbes play a critical role in the regulation of plant innate immunity. However, little is known about the role of nucleic acids in this process in plants. We pre-infiltrated Arabidopsis leaves with total RNAs from Pseudomonas syringae pv. tomato DC3000 (Pto DC3000) and subsequently inoculated these plants with the same bacterial cells. Total Pto DC3000 RNAs pre-infiltrated into Arabidopsis leaves elicited plant immune responses against Pto DC3000. However, sheared RNAs and RNase A application failed to induce immunity, suggesting that intact bacterial RNAs function in plant innate immunity. This notion was supported by the positive regulation of superoxide anion levels, callose deposition, two mitogen-activated protein kinases and defense-related genes observed in bacterial RNA-pre-treated leaves. Intriguingly, the Pto DC3000 population was not compromised in known pattern recognition receptor mutants for chitin, flagellin and elongation factor-Tu (EF-Tu). Plant defense-related mutant analyses further revealed that bacterial RNA-elicited innate immunity was normally required for salicylic and jasmonic acid signaling. Notably, among total RNAs, the abundant bacterial RNA species 16S and 23S ribosomal RNAs were the major determinants of this response. Our findings provide evidence that bacterial RNA serves as a microbe-associated molecular pattern in plants.

Root Exudation by Aphid Leaf Infestation Recruits Root-Associated Paenibacillus spp. to Lead Plant Insect Susceptibility.

Aphids are a large group of hemipteran pests that affect the physiology, growth, and development of plants by using piercing mouthparts to consume fluids from the host. Based an recent data, aphids modulate the microbiomes of plants and thereby affect the overall outcome of the biological interaction. However, in a few reports, aboveground aphids manipulate the metabolism of the host and facilitate infestations by rhizosphere bacteria (rhizobacteria). In this study, we evaluated whether aphids alter the plant resistance that is mediated by the bacterial community of the root system. The rhizobacteria were affected by aphid infestation of pepper, and a large population of gram-positive bacteria was detected. Notably, Paenibacillus spp. were the unique gram-positive bacteria to respond to changes induced by the aphids. Paenibacillus polymyxa E681 was used as a rhizobacterium model to assess the recruitment of bacteria to the rhizosphere by the phloem-sucking of aphids and to test the effect of P. polymyxa on the susceptibility of plants to aphids. The root exudates secreted from peppers infested with aphids increased the growth rate of P. polymyxa E681. The application of P. polymyxa E681 to pepper roots promoted the colonization of aphids within 2 days of inoculation. Collectively, our results suggest that aphid infestation modulated the root exudation, which led to the recruitment of rhizobacteria that manipulated the resistance of peppers to aphids. In this study, new information is provided on how the infestation of insects is facilitated through insect-derived modulation of plant resistance with the attraction of gram-positive rhizobacteria.

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.

Field evaluation of the bacterial volatile derivative 3-pentanol in priming for induced resistance in pepper.

Plants are defended from attack by emission of volatile organic compounds (VOCs) that can act directly against pathogens and herbivores or indirectly by recruiting natural enemies of herbivores. However, microbial VOC have been less investigated as potential triggers of plant systemic defense responses against pathogens in the field. Bacillus amyloliquefaciens strain IN937a, a plant growth-promoting rhizobacterium that colonizes plant tissues, stimulates induced systemic resistance (ISR) via its emission of VOCs. We investigated the ISR capacity of VOCs and derivatives collected from strain IN937a against bacterial spot disease caused by Xanthomonas axonopodis pv. vesicatoria in pepper. Of 15 bacterial VOCs and their derivatives, 3-pentanol, which is a C8 amyl alcohol reported to be a component of sex pheromones in insects, was selected for further investigation. Pathogens were infiltrated into pepper leaves 10, 20, 30, and 40 days after treatment and transplantation to the field. Disease severity was assessed 7 days after transplantation. Treatment with 3-pentanol significantly reduced disease severity caused by X. axonopodis and naturally occurring Cucumber mosaic virus in field trials over 2 years. We used quantitative real-time polymerase chain analysis to examine Pathogenesis-Related genes associated with salicylic acid (SA), jasmonic acid (JA), and ethylene defense signaling. The expression of Capsicum annuum Pathogenesis-Related protein 1 (CaPR1), CaPR2, and Ca protease inhibitor2 (CaPIN2) increased in field-grown pepper plants treated with 3-pentanol. Taken together, our results show that 3-pentanol triggers induced resistance by priming SA and JA signaling in pepper under field conditions.

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.

Two volatile organic compounds trigger plant self-defense against a bacterial pathogen and a sucking insect in cucumber under open field conditions.

Systemic acquired resistance (SAR) is a plant self-defense mechanism against a broad-range of pathogens and insect pests. Among chemical SAR triggers, plant and bacterial volatiles are promising candidates for use in pest management, as these volatiles are highly effective, inexpensive, and can be employed at relatively low concentrations compared with agrochemicals. However, such volatiles have some drawbacks, including the high evaporation rate of these compounds after application in the open field, their negative effects on plant growth, and their inconsistent levels of effectiveness. Here, we demonstrate the effectiveness of volatile organic compound (VOC)-mediated induced resistance against both the bacterial angular leaf spot pathogen, Pseudononas syringae pv. lachrymans, and the sucking insect aphid, Myzus persicae, in the open field. Using the VOCs 3-pentanol and 2-butanone where fruit yields increased gave unexpectedly, a significant increase in the number of ladybird beetles, Coccinella septempunctata, a natural enemy of aphids. The defense-related gene CsLOX was induced by VOC treatment, indicating that triggering the oxylipin pathway in response to the emission of green leaf volatiles can recruit the natural enemy of aphids. These results demonstrate that VOCs may help prevent plant disease and insect damage by eliciting induced resistance, even in open fields.

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.

Draft genome sequence of the plant growth-promoting bacterium Bacillus siamensis KCTC 13613T.

Bacillus siamensis KCTC 13613(T), a novel halophilic Bacillus species isolated from a salted Thai food, produced antimicrobial compounds against plant pathogens and promoted plant growth by volatile emission. We determined the 3.8-Mb genome sequence of B. siamensis KCTC 13613(T) to reveal the plant-beneficial effect at the genomic level.

Genome sequence of the Shiga toxin-producing Escherichia coli strain NCCP15657.

Shiga toxin-producing Escherichia coli causes bloody diarrhea and hemolytic-uremic syndrome and serious outbreaks worldwide. Here, we report the draft genome sequence of E. coli NCCP15657 isolated from a patient. The genome has virulence genes, many in the locus of enterocyte effacement (LEE) island, encoding a metalloprotease, the Shiga toxin, and constituents of type III secretion.