Pseudomonas Phaseolicola Research Articles

Whole-Genome Sequence Resources for Pseudomonas amygdali pv. sesami and Xanthomonas arboricola Isolated from Sesame (Sesamum indicum) in North Carolina in 2022

Sesame ( Sesamum indicum) is a specialty crop with increasing interest as an alternative, rotational crop for U.S. agricultural systems. This crop is susceptible to several pathogens, including Pseudomonas and Xanthomonas species. Two bacterial isolates were recently cultured from sesame in North Carolina symptomatic of bacterial spot and identified as Pseudomonas amygdali pv. sesami (Pas) and Xanthomonas sp. These isolates were used for hybrid whole-genome sequencing and assembly using Illumina and PacBio to develop robust genomic resources for these disease-causing strains. The results provide the most complete Pas genome available and placed the Xanthomonas sp. isolate into the X. arboricola species designation. Improved genomic resources for pathogens of sesame are needed to accurately detect, characterize, and employ timely management of the disease. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Chemical components of different solvent extracts of Asclepias curassavica L. and antibacterial effect of the extracts on tomato pathogens

The use of environmentally friendly and sustainable agricultural methods in the control of diseases and pests is of great importance. In both conventional and organic agricultural production systems, the utilization of various plant extracts as part of integrated pest management has gained significance in recent years. The chemical constituents of various solutions derived from the flowers, leaves, and roots of the Asclepias curassavica L. plant were investigated, along with the possibilities of utilizing these solutions in the control of tomato plant pathogenic bacteria. As a result of the analysis, acetic acid in 60% and 80% ethanol solutions, as well as acetic acid methy ester in 60% and 80% methanol solutions, were detected in the flowers, leaves, and roots. The effects of A. curassavica flower, leaf, and root extracts, prepared using three different solvents (water, methanol, and ethanol), were investigated on eight different pathogenic bacteria (Agrobacterium tumefaciens, Clavibacter michiganensis, Dickeya zeae, Pectobacterium caratovorum, Pseudomonas phaseolicola, Pseudomonas tomato, Pseudomonas viridiflava, and Xanthomonas euvesicatoria) that cause diseases in plants. Several main compounds such as acetic acid, acetic acid. methyl ester, Furfural, 2-Furanmethanol, 4H-Pyran-4-one. 2.3-dihydro-3.5-dihydroxy-6-methyl-, Glycerin, Benzo furan. 2.3-dihydro- and 5-Hydroxy methyl furfural were identified as analyzed by GC-MS with different concentrations of ethanol and methanol solutions used for the flower, leaves and root of A. curassavica plants. The flower extract prepared with 80% ethanol exhibited a higher inhibition zone (ranging from 1.5 mm to 5.3 mm) in all pathogens, compared to other applications. The successful suppression effect of A. curassavica flower extracts on this disease is promising, especially in organic farming areas. Additionally, since it is environmentally friendly and sustainable, it can be included in integrated control methods to prevent the loss of productivity caused by diseases.

Development of a Genome-Informed Protocol for Detection of Pseudomonas amygdali pv. morsprunorum Using LAMP and PCR.

One of the causal agents of bacterial canker is Pseudomonas amygdali pv. morsprunorum-Pam (formerly Pseudomonas syringae pv. morsprunorum). Recently detected in Chile, Pam is known to cause lesions in the aerial parts of the plant, followed by more severe symptoms such as cankers and gummosis in the later stages of the disease. This study presents the design of PCR and LAMP detection methods for the specific and sensitive identification of Pseudomonas amygdali pv. morsprunorum (Pam) from cherry trees. Twelve Pseudomonas isolates were collected, sequenced, and later characterized by Multi-locus Sequence Analysis (MLSA) and Average Nucleotide Identity by blast (ANIb). Three of them (11116B2, S1 Pam, and S2 Pam) were identified as Pseudomonas amygdali pv. morsprunorum and were used to find specific genes through RAST server, by comparing their genome with that of other Pseudomonas, including isolates from other Pam strains. The effector gene HopAU1 was selected for the design of primers to be used for both techniques, evaluating sensitivity and specificity, and the ability to detect Pam directly from plant tissues. While the PCR detection limit was 100 pg of purified bacterial DNA per reaction, the LAMP assays were able to detect up to 1 fg of purified DNA per reaction. Similar results were observed using plant tissues, LAMP being more sensitive than PCR, including when using DNA extracted from infected plant tissues. Both detection methods were tested in the presence of 30 other bacterial genera, with LAMP being more sensitive than PCR.

Genetic rearrangements in Pseudomonas amygdali pathovar aesculi shape coronatine plasmids.

Plant pathogenic Pseudomonas species use multiple classes of toxins and virulence factors during host infection. The genes encoding these pathogenicity factors are often located on plasmids and other mobile genetic elements, suggesting that they are acquired through horizontal gene transfer to confer an evolutionary advantage for successful adaptation to host infection. However, the genetic rearrangements that have led to mobilization of the pathogenicity genes are not fully understood. In this study, we have sequenced and analyzed the complete genome sequences of four Pseudomonas amygdali pv. aesculi (Pae), which infect European horse chestnut trees (Aesculus hippocastanum) and belong to phylogroup 3 of the P. syringae species complex. The four investigated genomes contain six groups of plasmids that all encode pathogenicity factors. Effector genes were found to be mostly associated with insertion sequence elements, suggesting that virulence genes are generally mobilized and potentially undergo horizontal gene transfer after transfer to a conjugative plasmid. We show that the biosynthetic gene cluster encoding the phytotoxin coronatine was recently transferred from a chromosomal location to a mobilizable plasmid that subsequently formed a co-integrate with a conjugative plasmid.

First report of wildfire disease caused by Pseudomonas amygdali pv. tabaci on perilla in Korea

Perilla (Perilla frutescens) is an important crop which produces edible leaves and oil from its seeds which is used as a seasoning in Korean food (Kim et al., 2009). Perilla is cultivated throughout the year in greenhouses in southern Korea. In November 2020, irregular brown spots with halos that led to necrosis on most upper leaves were observed in Miryang (35°32´04.6˝N; 128°46´01.9˝E) (Figure 1-3). The symptoms were very similar to wildfire disease in soybeans. The disease spread to up to 20–30% of the perilla plants cultivated in two greenhouses. Five diseased plants were collected and the leaves of each plant were removed. To isolate the causal pathogen, supernatants of surface-sterilised and homogenised leaves were plated on King's B medium (Sigma-Aldrich, USA) and incubated at 28°C for two days. Fluorescent colonies occupied approximately 60% of the area covered by bacterial colonies, indicating that a fluorescent bacterium was the predominant isolate from the diseased perilla. One isolate (designated strain MY1) was collected for analyses of 16S rRNA gene sequences and multilocus sequence analysis (MLSA) using sequences of the gltA, gyrB, and rpoD genes (Hwang et al., 2005). A BLAST search showed that the 16S rRNA gene sequence of strain MY1 (GenBank Accession No. ON810536) corresponded to Pseudomonas amygdali strain DEB with 99% identity. Moreover, strain MY1 was closest to P. amygdali pv. tabaci strain ATCC 11528 in a phylogenetic tree constructed using MLSA (Yun & Kim, 2021) (Figure 4). The physiological and biochemical characteristics of strain MY1 were determined based on levan production, oxidase activity, potato soft rot, arginine dihydrolase activity and tobacco hypersensitive response (LOPAT) tests. In the tests, strain MY1 was positive for levan production and tobacco hypersensitivity, but negative for oxidase activity, potato soft rot and arginine dihydrolase activity. Based on the LOPAT and MLSA analyses, strain MY1 belongs to P. amygdali pv. tabaci 1a (Sun et al., 2021). To substantiate the pathogenicity of strain MY1, a bacterial suspension of the strain (adjusted to 108 CFU/ml) was inoculated onto ten four-week-old perilla and incubated at 28°C. After seven days, the inoculated perilla leaves showed similar symptoms to those observed originally in the greenhouse. Completing Koch's postulates, bacteria with fluorescent colonies were re-isolated from symptomatic tissue and the 16S rRNA sequence of the isolates was identical to P. amygdali. To the best of our knowledge, this is the first report of wildfire disease caused by P. amygdali pv. tabaci on perilla in Korea and globally. Perilla wildfire disease reduces the quality of leaves, so early detection and prevention are essential to reduce the spread of disease. S.Y. Choi and J.I. Kim contributed equally to this work. This work was supported by “Cooperative Research Program for Agriculture Science & Technology Development project (PJ01425901)” Rural Development Administration, Korea.

Effect of temperature and humidity on dynamics and transmission of Pseudomonas amygdali pv. lachrymans aerosols.

Cucumber angular leaf spot (ALS) disease, caused by Pseudomonas amygdali pv. lachrymans (Pal), is an emerging disease with a high incidence that causes severe damage to cucumber worldwide. Bacterial aerosols play a crucial role in the epidemiology of greenhouse ALS disease. However, little is known about the influence of temperature and relative humidity (RH) on the dynamics of Pal in aerosols. A study was conducted to investigate the relationships between the concentration of Pal aerosols and their dependence on temperature and RH in aerosol chambers and greenhouses. The results demonstrated that temperature and RH are both significant factors influencing the release amount, survival time and infectivity of Pal in aerosols, while RH has a greater influence on particle size than temperature across the range of conditions tested. The release amount and survival time of Pal in aerosols under high RH (95%) and low temperature (≤ 25°C) conditions were significantly higher than those under low RH (35%) and high temperature (35°C) conditions. The highest release amount of Pal aerosol (96 CFU/m3) and highest survival rate (98.41%) were found at 18°C and 95% RH, while the highest disease index (DI = 60.9) caused by Pal aerosol was found at 25°C and 95% RH. In addition, Pal aerosols presented a larger diameter (4.7->7.0 μm) under high RH (95% RH) than under dry conditions (≤ 65% RH). These findings will play a crucial role in elucidating the influence of environmental parameters on the dynamics and transmission of Pal in aerosols. Based on our findings, preliminary recommendations for controlling airborne Pal spread involve controlling air temperature and RH, which will contribute to the effective alleviation and control of cucumber ALS disease.

Humidity Reduces Rapid and Distant Airborne Dispersal of Viable Viral Particles in Classroom Settings.

The transmission of airborne pathogens is considered to be the main route through which a number of known and emerging respiratory diseases infect their hosts. While physical distancing and mask wearing may help mitigate short-range transmission, the extent of long-range transmission in closed spaces where a pathogen remains suspended in the air remains unknown. We have developed a method to detect viable virus particles by using an aerosolized bacteriophage Phi6 in combination with its host Pseudomonas phaseolicola, which when seeded on agar plates acts as a virus detector that can be placed at a range of distances away from an aerosol-generating source. By applying this method, we consistently detected viable phage particles at distances of up to 18 feet away from the source within 15 min of exposure in a classroom equipped with a state of the art HVAC system and determined that increasing the relative humidity beyond 40% significantly reduces dispersal. Our method, which can be further modified for use with other virus/host combinations, quantifies airborne transmission in the built environment and can thus be used to set safety standards for room capacity and to ascertain the efficacy of interventions in closed spaces of specified sizes and intended uses.

Effector-triggered inhibition of nodulation: A rhizobial effector protease targets soybean kinase GmPBS1-1.

Type III protein secretion systems of nitrogen-fixing rhizobia deliver effector proteins into leguminous host cells to promote or inhibit the nodule symbiosis. However, mechanisms underlying effector-triggered inhibition of nodulation remain largely unknown. Nodulation outer protein T (NopT) of Sinorhizobium sp. NGR234 is an effector protease related to the Pseudomonas effector Avirulence protein Pseudomonas phaseolicola B (AvrPphB). Here, we constructed NGR234 mutants producing different NopT variants and found that protease activity of NopT negatively affects nodulation of smooth crotalaria (Crotalaria pallida). NopT variants lacking residues required for autocleavage and subsequent lipidation showed reduced symbiotic effects and were not targeted to the plasma membrane. We further noticed that Sinorhizobium fredii strains possess a mutated nopT gene. Sinorhizobium fredii USDA257 expressing nopT of NGR234 induced considerably fewer nodules in soybean (Glycine max) cv. Nenfeng 15 but not in other cultivars. Effector perception was further examined in NopT-expressing leaves of Arabidopsis (Arabidopsis thaliana) and found to be dependent on the protein kinase Arabidopsis AvrPphB Susceptible 1 (AtPBS1) and the associated resistance protein Arabidopsis Resistance to Pseudomonas syringae 5 (AtRPS5). Experiments with Nicotiana benthamiana plants indicated that the soybean homolog GmPBS1-1 associated with AtRPS5 can perceive NopT. Further analysis showed that NopT cleaves AtPBS1 and GmPBS1-1 and thus can activate these target proteins. Insertion of a DKM motif at the cleavage site of GmPBS1-1 resulted in increased proteolysis. Nodulation tests with soybeans expressing an autoactive GmPBS1-1 variant indicated that activation of a GmPBS1-1-mediated resistance pathway impairs nodule formation in cv. Nenfeng 15. Our findings suggest that legumes face an evolutionary dilemma of either developing effector-triggered immunity against pathogenic bacteria or establishing symbiosis with suboptimally adapted rhizobia producing pathogen-like effectors.

Comparative Analyses of Four Complete Genomes in Pseudomonas amygdali Revealed Differential Adaptation to Hostile Environments and Secretion Systems.

Pseudomonas amygdali is a hemibiotrophic phytopathogen that causes disease in woody and herbaceous plants. Complete genomes of four P. amygdali pathovars were comparatively analyzed to decipher the impact of genomic diversity on host colonization. The pan-genome indicated that 3,928 core genes are conserved among pathovars, while 504–1,009 are unique to specific pathovars. The unique genome contained many mobile elements and exhibited a functional distribution different from the core genome. Genes involved in O-antigen biosynthesis and antimicrobial peptide resistance were significantly enriched for adaptation to hostile environments. While the type III secretion system was distributed in the core genome, unique genomes revealed a different organization of secretion systems as follows: type I in pv. tabaci, type II in pv. japonicus, type IV in pv. morsprunorum, and type VI in pv. lachrymans. These findings provide genetic insight into the dynamic interactions of the bacteria with plant hosts.

HopAZ1, a type III effector of Pseudomonas amygdali pv. tabaci, induces a hypersensitive response in tobacco wildfire-resistant Nicotiana tabacum 'N509'.

Pseudomonas amygdali pv. tabaci (formerly Pseudomonas syringae pv. tabaci; Pta) is a gram‐negative bacterium that causes bacterial wildfire disease in Nicotiana tabacum. The pathogen establishes infections by using a type III secretion system to inject type III effector proteins (T3Es) into cells, thereby interfering with the host__s immune system. To counteract the effectors, plants have evolved disease‐resistance genes and mechanisms to induce strong resistance on effector recognition. By screening a series of Pta T3E‐deficient mutants, we have identified HopAZ1 as the T3E that induces disease resistance in N. tabacum ‘N509’. Inoculation with the Pta ∆hopAZ1 mutant did not induce resistance to Pta in N509. We also found that the Pta ∆hopAZ1 mutant did not induce a hypersensitive response and promoted severe disease symptoms in N509. Furthermore, a C‐terminal truncated HopAZ1 abolished HopAZ1‐dependent cell death in N509. These results indicate that HopAZ1 is the avirulence factor that induces resistance to Pta by N509.

Complete genome sequence of the kiwifruit bacterial canker pathogen Pseudomonas savastanoi strain MHT1

BackgroundPseudomonas savastanoi is an important plant pathogen that infects and causes symptoms in a variety of economically important crops, causing considerable loss of yield and quality. Because there has been no research reported to date on bacterial canker of kiwifruit (Actinidia chinensis) plants caused by P. savastanoi and, in particular, no in-depth studies of the complete genome sequence or pathogenic mechanism, long-lasting and environmentally friendly control measures against this pathogen in kiwifruit are lacking. This study therefore has both theoretical value and practical significance.ResultsWe report the complete genome sequence of P. savastanoi strain MHT1, which was first reported as the pathogen causing bacterial canker in kiwifruit plants. The genome consists of a 6.00-Mb chromosome with 58.5% GC content and 5008 predicted genes. Comparative genome analysis of four sequenced genomes of representative P. savastanoi strains revealed that 230 genes are unique to the MHT1 strain and that these genes are enriched in antibiotic metabolic processes and metabolic pathways, which may be associated with the drug resistance and host range observed in this strain. MHT1 showed high syntenic relationships with different P. savastanoi strains. Furthermore, MHT1 has eight conserved effectors that are highly homologous to effectors from P. syringae, Pseudomonas amygdali, and Ralstonia solanacearum strains. The MHT1 genome contains six genomic islands and two prophage sequences. In addition, 380 genes were annotated as antibiotic resistance genes and another 734 as encoding carbohydrate-active enzymes.ConclusionThe whole-genome sequence of this kiwifruit bacterial canker pathogen extends our knowledge of the P. savastanoi genome, sets the stage for further studies of the interaction between kiwifruit and P. savastanoi, and provides an important theoretical foundation for the prevention and control of bacterial canker.

Genomic variability and plasticity of Pseudomonas causing coffee leaf spots in Minas Gerais state, Brazil

AbstractFoliar spots caused by Pseudomonas coronafaciens pv. garcae (Pcg), Pseudomonas amygdali pv. tabaci (Pat) and Pseudomonas cichorii (Pch) are major bacterial diseases that can reduce coffee production. However, little is known about the genetic diversity and molecular mechanisms underlying the pathogenicity to coffee plants of these bacteria. In this study, genome sequences of Pcg, Pat and Pch strains isolated from coffee plants in Minas Gerais state, Brazil, were used to assess their variability and plasticity, and compare their type III secretion system (T3SS) and apoplastic effector repertoires as well as tabtoxin biosynthetic/detoxification genes. Genomic diversity was found for all three phytopathogens, among which Pch possesses the highest number of exclusive proteins. The Pcg genome is the most stable whereas that of Pch is the most plastic, which is related to their host ranges. When compared with those of Pseudomonas syringae pv. tomato DC3000, hrp/hrc gene sequences are more conserved in Pcg and Pat than in Pch, which also possesses the smallest T3SS and the largest apoplastic effector repertoires. The only T3SS effector family common to all three pathogens is AvrE, suggesting that, as for other plant–Pseudomonas interactions, it may play a crucial role for pathogenicity towards coffee plants. Apoplastic proteins associated with maintaining the redox balance and degrading proteins/peptides not previously described as important in plant–bacteria interactions were found. Gain/loss of the tabtoxin biosynthetic cluster with retention of the antitoxin gene was observed, indicating that tabtoxin production is not a limiting factor for the occurrence of mixed infections.

Guttation Fluids Containing Pseudomonas amygdali pv. lachrymans Are a Potential Source of Secondary Infection in Cucumber.

Guttation is a common feature of cucumber leaves under high relative humidity conditions; however, little is known about the role of guttation in the transmission of Pseudomonas amygdali pv. lachrymans, which is the pathogen of cucumber angular leaf spot disease. In this study, experimental evidence for the transmission of P. amygdali pv. lachrymans inside cucumber plants and through guttation was provided, and the results proved that P. amygdali pv. lachrymans can be transmitted from the bottom leaf to the upper leaves inside the plant and excreted from the upper leaves through guttation. After that, the third leaf of cucumber was inoculated with P. amygdali pv. lachrymans bacterial suspension, P. amygdali pv. lachrymans was detected on the fifth leaf, the petiole, and the stem and in guttation drops. Healthy cucumber seedlings were infected by P. amygdali pv. lachrymans in the guttation droplets, indicating that guttation fluids containing P. amygdali pv. lachrymans could become a potential source of secondary infection. The results from this study verified the hypothesis that guttation is a potential route for P. amygdali pv. lachrymans excretion from cucumber plants and may be a source of secondary transmission under high relative humidity.

An Ancestry Perspective of the Evolution of PBS1 Proteins in Plants.

The AVRPPHB SUSCEPTIBLE1 (PBS1) and RESISTANCE TO PSEUDOMONAS SYRINGAE 5 (RPS5) proteins are involved in signal transduction to evoke innate plant immune response. In Arabidopsis, PBS1 is cleaved by the AvrPphB (Pseudomonas phaseolicola Avirulence protein B) protease, activating RPS5 and turning in a hypersensitive response (HR). We searched for PBS1 orthologs to trace their origin and evolution. PBS1 orthologs were found in embryophytes and in other plant taxa but with lower similarity. PBS1 phylogenetic analysis indicates high divergence, suggesting that the decoy function described for Arabidopsis PBS1 might be associated with a small fraction of orthologs. Ancestral reconstruction analysis suggests an elevated diversity in the amino acid sequence within the described motifs. All the orthologs contain the conserved PBS1 kinase subdomains, whereas the cleavage motif is present in several embryophyte orthologs but absent in most other taxa. The putative resistance recognition motifs in PBS1 orthologs are highly diverse. PBS1 cleavage site motif is exposed in some 3D structure predictions, whereas it is not in others, suggesting different modes of regulation and functions in PBS1 orthologs. Our findings suggest that PBS1 originated in the lineage that gave rise to embryophytes, with the angiosperm sequences forming a separate clade from pteridophyte proteins.

Biochemical pathways and associated microbial process of di-2-ethyl hexyl phthalate (DEHP) enhanced degradation by the immobilization technique in sequencing batch reactor

ABSTRACT A bacterial strain ASLT-13 was successfully isolated from activated sludge and identified as Pseudomonas amygdali. Gas chromatograph-mass spectrometer (GC-MS) analysis indicated that strain ASLT-13 could completely mineralize di 2-ethyl hexyl phthalate (DEHP). DEHP was first metabolized from the longer side chain of the benzene ring into shorter branches (Phatlalic mono-esters) like Dibutyl phthalate (DBP) under the action of degrading genes. DBP was then converted into di-methyl phthalate (DMP), and then hydrolysed to phthalic acid (PA). PA was eventually converted to CO2 and H2O through the TCA cycle. The optimal conditions for immobilization were the sodium alginate (SA) concentration of 6%, CaCl2 concentration of 5%, ratio of bacteria and SA of 1:1, crosslinking time of 6 h. Bacterial quantity and community structure in sequencing batch reactors (SBRs) was investigated by q-PCR and high-throughput sequencing. The results indicated that DEHP removal efficiency was significantly enhanced by immobilization. Arthrobacter, Acinetobacter, Bacillus and Rhodococcus were the predominant genera for DEHP degradation. This study suggested that the cell immobilization technology had a potential application in DEHP wastewater treatment.

Infection of cucumber seedling roots by Pseudomonas amygdali pv. lachrymans following artificial substrate inoculation

There are few studies of cucumber roots at the seedling stage infected by Pseudomonas amygdali pv. lachrymans (Pal). We used growth chamber assays to determine the infection of cucumber seedling roots by Pal following artificial substrate inoculation. Symptoms on cucumber roots, leaves, and stems were observed over time. We used real-time fluorescent quantitative PCR (qPCR) to obtain accurate numbers of Pal copies in infected roots, and then statistically assessed the dynamics of Pal copies in roots, stems, and leaves. Results indicated that Pal not only caused root decay, but also damaged the whole plant. Following root infection, the bacteria were transmitted through stems to the leaves. Cucumber seedlings in a moist planting substrate showed lodging, leaf chlorosis and wilt, and rotted roots. This study provides evidence that in moist substrate, Pal can infect cucumber roots and be transmitted throughout the host plant. In addition, the results also provide a foundation for further research on the transmission and proliferation of Pal in cucumber plants under field conditions.

Ethiopian coffee germplasm is a valuable resistance gene pool to brazilian Pseudomonas syringae PVS garcae and tabaci

ABSTRACT Seven wild accessions of Coffea arabica from Ethiopia prospected by FAO Coffee Mission 1964-1965 were investigated concerning the resistance to 18 Brazilian strains and two Kenyan strains of Pseudomonas syringae pv. garcae and four P. syringae pv. tabaci strains, causal agents of bacterial halo blight and bacterial leaf spot, respectively. The cultivars of C. arabica IPR 102, resistant to the diseases, and Mundo Novo IAC 376-4, susceptible, were used as experimental controls. Our results indicated that the Ethiopian accessions presented high levels of resistance to all Brazilian strains of P. syringae pv. garcae but were susceptible to infection caused by Kenyan strains, which causes different levels of severity in wild accessions and experimental controls. Ethiopian accessions were also considered resistant to the four P. syringae pv. tabaci strains, with low susceptibility observed, one point on the severity scale, in access E-268 in response to a strain of the bacterium.

Two Homologues of the Global Regulator Csr/Rsm Redundantly Control Phaseolotoxin Biosynthesis and Virulence in the Plant Pathogen Pseudomonas amygdali pv. phaseolicola 1448A.

The widely conserved Csr/Rsm (carbon storage regulator/repressor of stationary-phase metabolites) post-transcriptional regulatory system controls diverse phenotypes involved in bacterial pathogenicity and virulence. Here we show that Pseudomonas amygdali pv. phaseolicola 1448A contains seven rsm genes, four of which are chromosomal. In RNAseq analyses, only rsmE was thermoregulated, with increased expression at 18 °C, whereas the antagonistic sRNAs rsmX1, rsmX4, rsmX5 and rsmZ showed increased levels at 28 °C. Only double rsmA-rsmE mutants showed significantly altered phenotypes in functional analyses, being impaired for symptom elicitation in bean, including in planta growth, and for induction of the hypersensitive response in tobacco. Double mutants were also non-motile and were compromised for the utilization of different carbon sources. These phenotypes were accompanied by reduced mRNA levels of the type III secretion system regulatory genes hrpL and hrpA, and the flagellin gene, fliC. Biosynthesis of the phytotoxin phaseolotoxin by mutants in rsmA and rsmE was delayed, occurring only in older cultures, indicating that these rsm homologues act as inductors of toxin synthesis. Therefore, genes rsmA and rsmE act redundantly, although with a degree of specialization, to positively regulate diverse phenotypes involved in niche colonization. Additionally, our results suggest the existence of a regulatory molecule different from the Rsm proteins and dependent on the GacS/GacA (global activator of antibiotic and cyanide production) system, which causes the repression of phaseolotoxin biosynthesis at high temperatures.

Quantitative real-time PCR assay for rapid detection of Pseudomonas amygdali pv. lachrymans in cucumber leaf rinse

Pseudomonas amygdali pv. lachrymans (Pal) is a bacterium that causes angular leaf spot disease of cucumbers. There is no direct protection against this pathogen, only prevention. Previously published detection systems are labour-intensive and time-consuming. The aim of this study was to create a rapid and reliable diagnostic assay for the detection of Pal in cucumber plants without the need to destroy or damage the plant. The created method is based on washing the bacteria off the leaf surface with saline solution and using the sample directly in a quantitative real-time PCR assay. This detection system is based on the specific amplification of a small coding region of the bacteriophage N4 adsorption protein B gene. The assay was tested on 120 plant samples inoculated with seven different Pal strains. The detection limit of the assay was 2.60 × 102 CFU ml−1, and the concentration of Pal in the leaf rinse ranged from 2.71 × 102 to 6.94 × 107 CFU ml−1 depending on the particular strain. This assay is a reliable and rapid diagnostic method for the detection of Pal from cucumber leaves and is valuable for contributing to angular leaf spot management.

Control of Pseudomonas amygdali pv. loropetali on Metal, Wood, and Loropetalum chinense Stem Surfaces

Clorox Germicidal Bleach, Cuproxat, Green-Shield II, KleenGrow, Virkon S, and ZeroTol 2.0 were evaluated for their ability to inactivate Pseudomonas amygdali pv. loropetali on stainless steel (SS), pressure-treated wood (PtW), and loropetalum stem sections. Clorox at 11% product and Virkon S at 1.0% product eliminated P. amygdali pv. loropetali on SS surfaces and nearly eliminated it on PtW surfaces. Green-Shield II at 0.5% product and KleenGrow at 0.8% product nearly eliminated the bacterium on SS, while causing a significant reduction without elimination on PtW. Cuproxat and ZeroTol 2.0 were not effective against this bacterium in these applications. Clorox and KleenGrow were evaluated further for their ability to kill P. amygdali pv. loropetali on loropetalum stems, which was used as a preliminary surrogate for vegetative stem cuttings. Only Clorox eliminated bacteria from stem surfaces, but the bacterial inoculum level appeared to affect efficacy. Several disinfestants are commercially available that kill P. amygdali pv. loropetali on production surfaces.