Flaccumfaciens Pv Research Articles

High Diversity of Curtobacterium flaccumfaciens in Poinsettia and Detection of Three Pathogenicity Plasmids for Identification of C. flaccumfaciens pv. poinsettiae

Outbreaks in Europe have raised concerns about poinsettia bacterial canker caused by Curtobacterium flaccumfaciens pv. poinsettiae, described in the United States. Using a semiselective medium containing aztreonam and fosfomycin and selection during isolation based on matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) spectra, 424 Curtobacterium strains were isolated from Belgian poinsettias of African and European origin. Different populations coexisted: 130 strains were identified as C. flaccumfaciens with scores ≥2.0 and 294 with scores <2.0 or as another Curtobacterium. MALDI-TOF MS libraries constructed using similar medium and extraction procedure and a pathogenicity test on poinsettia were used to screen collections from poinsettia and wheat, a possible alternative host, for C. flaccumfaciens pv. poinsettiae. The concatenated recA- gyrB partial sequences showed that 114 poinsettia or wheat strains belonged to different Curtobacterium species. Eighty-eight nonpathogenic strains and four U.S. strains from litter were intermingled with bean pathogenic C. flaccumfaciens pv. flaccumfaciens strains in the three genetic groups of C. flaccumfaciens but lacked their pathogenicity markers. Four new European recA- gyrB sequatypes of C. flaccumfaciens pv. poinsettiae, obtained from symptomatic and asymptomatic poinsettias, were distinguished from three American sequatypes. Six sequatypes were pathogenic in tests, belonged to genetic groups related to two different genomospecies, and possessed a plasmid. Sequencing of six plasmids revealed three related plasmids containing proteases and a polygalacturonase found only in strains pathogenic in pathogenicity tests and specifically identified by polygalacturonase-based PCR and loop-mediated isothermal amplification assays. Similarities between plant and litter Curtobacterium and the role of plasmids in pathogenicity and punctual transmission of pathogenicity among heterogeneous C. flaccumfaciens are suggested.

Phytopathogenic Curtobacterium flaccumfaciens Strains Circulating on Leguminous Plants, Alternative Hosts and Weeds in Russia.

Many bacterial plant pathogens have a broad host range important for their life cycle. Alternate hosts from plant families other than the main (primary) host support the survival and dissemination of the pathogen population even in absence of main host plants. Metabolic peculiarities of main and alternative host plants can affect genetic diversity within and between the pathogen populations isolated from those plants. Strains of Gram-positive bacterium Curtobacterium flaccumfaciens were identified as being causal agents of bacterial spot and wilt diseases on leguminous plants, and other crop and weed plants, collected in different regions of Russia. Their biochemical properties and susceptibility to copper compounds have been found to be relatively uniform. According to conventional PCR assays, all of the isolates studied were categorised as pathovar Curtobacterim flaccumfaciens pv. flaccumfaciens, a pathogen of legumes. However, the strains demonstrated a substantial diversity in terms of virulence on several tested host plants and different phylogenetic relationships were revealed by BOX-PCR and alanine synthase gene (alaS) sequencing.

Multiphasic investigations imply transfer of orange-/red-pigmented strains of the bean pathogen Curtobacterium flaccumfaciens pv. flaccumfaciens to a new species as C. aurantiacum sp. nov., elevation of the poinsettia pathogen C. flaccumfaciens pv. poinsettiae to the species level as C. Poinsettiae sp. nov., and synonymy of C. albidum with C. citreum

Curtobacterium flaccumfaciens (Microbacteriaceae), a plant-pathogenic coryneform species includes five pathovars with valid names and a number of proposed – but unvalidated – new members. In this study, phenotypic features and DNA similarity indexes were investigated among all C. flaccumfaciens members. Results showed that the C. flaccumfaciens pv. poinsettiae strains causing bacterial canker of Euphorbia pulcherrima in the USA as well as the orange-/red-pigmented strains of C. flaccumfaciens pv. flaccumfaciens pathogenic on dry beans in Iran are too distinct from each other and from the type strain of the species to be considered members of C. flaccumfaciens. Hence, the latter two groups were elevated at the species level as C. poinsettiae sp. nov. (ATCC 9682T = CFBP 2403T = ICMP 2566T = LMG 3715T = NCPPB 854T as type strain), and C. aurantiacum sp. nov. (50RT = CFBP 8819T = ICMP 22071T as type strain). Within the emended species C. flaccumfaciens comb. nov., yellow-pigmented strains causing bacterial wilt of dry beans and those causing bacterial canker of Euphorbia pulcherrima in Europe were retained as C. flaccumfaciens pv. flaccumfaciens and C. flaccumfaciens pv. poinsettiae, respectively; while taxonomic position of the sugar beet pathogen C. flaccumfaciens pv. beticola ATCC BAA144PT was confirmed. The newly described onion pathogen C. allii was also reclassified as C. flaccumfaciens pv. allii with the pathotype strain LMG 32517PT. Furthermore, C. flaccumfaciens pv. basellae causing bacterial leaf spot of malabar spinach (Basella rubra) was transferred to C. citreum pv. basellae with ATCC BAA143PT as pathotype.

Identification of the chemical profile and evaluation of the antimicrobial effect of Eryngium billardieri Delar essential oil component against bacterial species of agricultural and food interest.

Studies on the antibacterial activity of the essential oil of E. billardieri are limited. In this study, we identified this herb as a natural complex effective against several bacteria by employing disk diffusion and broth microdilution susceptibility methods. Primary estimation of the antimicrobial effect of this herbal compound by disk diffusion method showed that the oil could inhibit the growth of the tested bacteria by the appearance of haloes between 8.25 and 21.25 mm. In the next step, the oil was found to be active against all 24 tested Gram-negative and Gram-positive bacteria in the broth media, at minimum inhibitory concentrations ranging from 0.67 to 34.17 g L-1. Furthermore, Enterococcus faecalis and Curtobacterium flaccumfaciens pv. flaccumfaciens were the most sensitive food and plant pathogenic bacteria, respectively. Gas chromatography-mass spectrometry analysis was conducted to assign the ingredients present in the oil; 34 different components representing 95.71% of the total oil were identified, with n-hexadecanoic acid being the dominant component, followed by 2-Pentadecanone, 6,10,14-trimethyl, 1H-Indene, 1-ethylideneoctahydro-, and Cinnamyl tiglate. These findings demonstrate, for the first time, a broad spectrum of the antibacterial capacity of E. billardieri. Based on these observations, the oil could be applied as a natural preservative with the potential for designing novel products. Its bioactive agents can also be isolated for further use in the food and agricultural industries.

Priming Bean Seedlings to Boost Natural Plant Defenses Against Common Bacterial Wilt: Leaf Architecture, Leaf area, Foliage Water Content, and Plant Biomass Results (Part 3)

This greenhouse study evaluated the effects of two chemicals for priming kidney bean seedlings against bacterial wilt disease (Curtobacterium flaccumfaciens pv. Flaccumfaciens) (CFF). The premise of this study was that the oxidant properties of chlorine dioxide would mimic the signaling properties of radical oxygen species thereby initiating a cascade of molecular plant defenses. The factorial study included two levels for the foliar chlorine dioxide treatment, two levels for the bacterial wilt inoculation treatment, and two optional treatments. The biomass variables included oven dry total plant biomass, oven dry fruit biomass, and oven dry leaf biomass. Also, foliage and total plant water content data was collected, as well as total leaf area. Specific leaf area (SLA) was estimated from the leaf area and biomass data. The primers had equivalent leaf area, plant and fruit biomass as the water control for the CFF wilt inoculated plants. The EB 400 mg/l primer reduced SLA for the CFF inoculated plants. Both EB formulations increased aboveground water content in the CFF wilt inoculated plants. Multivariate tables revealed several significant correlations among leaf architecture, plant tissue water content, and biomass growth parameters for the EB primers and the water control treatment for the two CFF wilt treatments. Re-allocation of plant resources from plant growth to plant defenses due to chemical primers were estimated and discussed to determine the tradeoffs between plant yield and enhanced plant defenses. The three articles in this study show that chlorine dioxide primers can initiate a series of ROS and salicylic acid signals. This interplay of ROS signals and salicylic acid signals generated by the chlorine dioxide primers activates a long-term SAR response that protects plants against future pathogen attacks. In addition, interaction of the ROS and salicylic acid signals activates a suite of defense mechanisms that provide universal, multifaceted plant immunity that can be sustained across a crop season.

First report of Curtobacterium flaccumfaciens pv. flaccumfaciens causing bacterial tan spot of soybean in Russia.

Curtobacterium flaccumfaciens pv. flaccumfaciens (H.) Collins & Jones is known as a pathogen of different legume crops, including soybean (Glycine max (L.) Merr.) (Hedges 1922; Dunleavy 1983). OEPP/EPPO (2011) considers C. flaccumfaciens pv. flaccumfaciens as present in Russia based on reports of the disease on common beans in two regions of Russia (North Caucasus and Far East) made without proper pathogen identification. During the summer of 2020 and the spring of 2021, soybean plants with tan spot disease (10-40% of plants) were reported during routine assays of several fields in Stavropol Krai (44.72°N, 43.29°E). After harvest in 2021, we inspected 48 soybean seed lots collected in different regions of Russia for the presence of C. flaccumfaciens pv. flaccumfaciens. Seed testing was performed using the OEPP/EPPO (2011) protocol. For bacteria isolation, seed extracts were spread on MSCFF agar plates (Maringoni et al. 2006). After 5 days of incubation at 28°C potential, C. flaccumfaciens pv. flaccumfaciens colonies were used for further tests on NSA and SSM agar (Tegli et al. 2017, Maringoni et al. 2016). Six seed lots produced in Stavropol, Ryazan (53.95°N, 40.62°E), Orel (52.39°N, 37.69°E) and Amur (51.31°N, 128.22°E) regions were suspect. Ten isolates (SB1 to SB4 from Stavropol, F-125-1 to F-125-3 from Ryazan, and F-30-1 to F-30-3 from Amur) were selected, and further identified by morphological, physiological, and biochemical properties, MALDI TOF MS, 16S rRNA sequences, and specific primers CffFOR2 and CffREV4 (Tegli et al. 2017). Isolates consistently formed yellow, circular, smooth colonies on agar, and were identical to C. flaccumfaciens pv. flaccumfaciens type strain DSM 20129T in diagnostic physiological properties (Tegli et al. 2017). DNA was isolated from the bacteria by the CytoSorb Kit (Sintol, Moscow). All tested strains were positive in the PCR assay (Fig. 1). 16S rRNA fragments were amplified using primers 27F/1492R (Marchesi et al. 1998) and PCR products were sequenced (Evrogen, Moscow, Russia). The obtained 16S rRNA sequences (1473 bp, Accession No. OL539808.1-OL539817.1) were 100% identical to DSM 20129T (AM410688.1) according to a BLAST NCBI search. A pathogenicity test was done by leaf-cutting with scissors wetted with inoculum (for soybeans) or by injecting 5 microliters of the bacterial suspension (108 CFU/ml) into the stem (for common beans). All ten isolates for the inoculum were grown on nutrient agar for 72 h at 28°C. Soybean cv. Kasatka plants (stage V1) were used for inoculation, and common bean (cv. Purpurnaya) plants were inoculated as well to confirm multi-host virulence. Sterile water served as a control. Ten plantlets were used as replicates for each treatment. The plants were incubated at 24°C, 80% RH, and a 14 hour light/10 hour dark cycle. Tan spots (soybean) and wilt (beans) have developed 7-21 d.p.i (Fig. 2.1-2.6). Control plants remained asymptomatic. Seed inoculation by soaking them in the same bacterial suspension repeatedly produced twisted primary root (Fig. 2.7-2.8), but typical disease symptoms on leaves developed in 4-5 weeks only. The pathogen was successfully reisolated from all infected plants and not from the controls, thus fulfilling Koch's postulates. The identity of the reisolated strains was confirmed using morphological and physiological characteristics and the DNA sequence data for the 16S rRNA. These results indicated that a causal agent of the tan spot is present on soybean in three important agricultural areas of Russia (South, Central, and the Far East). To the best of our knowledge, this is the first report of C. flaccumfaciens pv. flaccumfaciens causing a bacterial tan spot of soybean in Russia.

Priming Bean Seedlings to Boost Natural Plant Defenses Against Common Bacterial Wilt: Gas Exchange, and Fluorescence Results (Part 2)

This greenhouse study evaluated the effects of two chemical primers for kidney bean seedlings against a bacterial wilt (Curtobacterium flaccumfaciens pv. Flaccumfaciens) (CFF). The premise of this study was that the oxidant primers would mimic the signaling properties of radical oxygen species and initiate a cascade of molecular defenses. The factorial study included two levels for the foliar chlorine dioxide treatment, and two levels for the bacterial wilt inoculation treatment, plus two supplemental chemical treatments. The foliage response variables were gas exchange and fluorescence. There was a 36, 154, and 70% reduction in Pn, gs, and E, respectively, at 39 DAT when comparing the inoculated control to the non-inoculated control. The chlorine dioxide primers lowered leaf temperatures and leaf vapor pressure deficit in the CFF wilt inoculated plants. The chlorine dioxide primers improved gas exchange at 39 DAT when compared to the water treatments. Part 1 and 2 of this series conclude that the chlorine dioxide primers can activate a long-term, systemic acquired resistance (SAR) response in kidney bean plants infected with the CFF wilt. The Part 2 article also concludes that the EB treatments caused several inexplicable correlations among the gas exchange responses. A structured water premise was proposed as an explanation for the gas exchange anomalies due to the EB treatments. Intuitively, this study suggests that chlorine dioxide primers can initiate a series of ROS and salicylic acid signals that activate a suite of mechanisms that provide universal, multifaceted plant immunity that is sustained across a crop season.

First Report of Curtobacterium flaccumfaciens pv. flaccumfaciens Causing Bacterial Wilt and Blight on Sunflower in Russia.

In the summer of 2018, wilt and leaf spots were observed on sunflower (Helianthus annuus L.) plants in fields near Kursk (51.74°N, 36.02°E) in Russia. In the following years, incidence of this disease was 5 to 20% in the inspected fields. Marginal chlorosis on seedling leaves developed into wilt and necrosis about one week later (Fig. 1). Mature plants had leaves with blight and reduced height compared to symptomless plants. Pathogen isolation from seeds was done by the method of Tegli et al. (2002) with modifications. Bacteria from diseased plants were isolated by streaking inoculum from symptomatic tissues on nutrient dextrose agar (NDA) (Schaad et al. 1988). The plates were incubated at 30°C for 7 to 10 days. Isolates consistently formed slow-growing, yellow, circular, smooth colonies without soluble pigment. The isolated bacteria were aerobic, gram-positive, and rod-shaped. Eight strains, CF-20 to CF-26 from plants, and Curt1 and Curt3 from seeds, were identified by MALDI TOF MS analysis as Curtobacterium flaccumfaciens pv. flaccumfaciens or C. flaccumfaciens pv. poinsettiae. All strains had GENIII MicroPlate (BIOLOG) test results identical to C. flaccumfaciens pv. flaccumfaciens strain DSM20129T. Further analysis was done by specific PCR (Tegli et al. 2002) and 16S rDNA, gyrB, and atpD gene sequencing. For PCR amplification, DNA was extracted by the CitoSorb Kit (Syntol Co., Moscow). Primers 27F/1492R (16S rRNA) (Marchesi et al. 1998), 2F/6R (gyrB) (Richert et al. 2005), and aptD2F/aptD2R (Jacques et al. 2012) were used to amplify the target gene sequences. The PCR products were sequenced by Evrogen (Moscow). The 16S rRNA sequences (OL584192.1 to OL584199.1) were identical to that of C. flaccumfaciens pv. flaccumfaciens strain DSM20129T (AM410688.1; 1,477/1,477 bp). The phylogenetic tree of concatenated gyrB (560 bp) and atpD (716 bp) sequences (OL548915.1 to OL548922.1 and OL548923.1 to OL548930.1, respectively) clustered the strains from sunflower among C. flaccumfaciens pv. flaccumfaciens, C. flaccumfaciens pv. betae, and C. flaccumfaciens pv. oortii (Fig. 2) with high genetic similarity to other C. flaccumfaciens strains: 96.3 to 100% for atpD and 95 to 100% for gyrB. A pathogenicity test for each of the strains was performed by injecting 5 μl of a bacterial suspension (108 CFU/ml) grown for 72 h on NDA into the stems of five plantlets (four true leaf stage) of the sunflower cv. Tunka (Limagrain, France) and soybean cv. Kasatka (VIM, Russia). Strain DSM20129T was a positive control, while sterile water was a negative control. The plants were incubated at 24°C, 80% relative humidity, and 14-h light/day. Wilting and blight on sunflower (Fig. 3) and tan spots on soybean were observed in 15 to 20 days after inoculation for all sunflower strains and strain DSM20129T. The negative control plants were asymptomatic. The bacteria re-isolated from the inoculated plants exhibited the same morphological characteristics and 16S rDNA sequence as the original culture, thus fulfilling Koch's postulates. The presence of C. flaccumfaciens pv. flaccumfaciens in sunflower seeds indicated that the bacterium was transmitted via seed. Sunflower has been previously reported as a host for the pathogen (Harveson et al. 2015). The presence of C. flaccumfaciens pv. flaccumfaciens on beans in Russia was suggested from the disease symptoms (Nikitina and Korsakov 1978), but, to our knowledge, this is the first report of the pathogen affecting sunflower in Russia. Phytosanitary categorization placed C. flaccumfaciens pv. flaccumfaciens in the EPPO A2 list (EPPO 2011). Thus, sunflower seeds should be tested to protect pathogen-free areas from introduction of this pathogen.

Antibacterial Activity of Origanum vulgare and Rosmarinus officinalis standardized extracts Against Curtobacterium and Xanthomonas

Diseases caused by Curtobacterium and Xantomonnas species represent an agricultural problem in crops and can generate economic impacts on the commercialization of seeds and food. Origanum vulgare L. (oregano) and Rosmarinus officinalis L. (rosemary) have rosmarinic acid and others phenolics that can lead to the control of phytopathogenic bacteria in common bean (Phaseolus vulgaris L.). This study aimed to evaluate the in vitro and in vivo antibacterial activity of O. vulgaris and R. officinalis extracts, standardized in rosmarinic acid, against Curtobacterium flaccumfaciens pv. flaccumfaciens, Xanthomonas axonopodis pv. phaseoli, Xanthomonas fuscans subsp. fuscans and Xanthomonas sp. The antibacterial effect of the extracts in bean seed was also investigated. The content of rosmarinic acid was 8.55 % for O. vulgare and 16.30 % for R. officinalis extract. It was verified the complete in vitro inhibition of the bacteria studied by both extracts at 0.8% (w/w) with exception of Xanthomonas axonopodis pv. phaseoli BRM 025302 that was completely inhibited at 1.2% (w/w) of oregano. In addition, no symptom of phytotoxicity were noted in detached bean leaves treated with them. Under greenhouse conditions, some reduction on severity of Curtobacterium wilt by both extracts at 1% (w/w) was noted to bean cultivars BRS Sublime and BRS Estilo. Under the experimental conditions these extracts were not efficient to control the common bacterial blight caused by X. axonopodis pv. phaseoli. Both extracts are promising in the treatment of seeds, specially in related to contamination by Fusarium spp., whose percentage decreased on average an average from 94% to 10%. In addition, these bean seeds maintained the germination percentage adequate to that required by legislation. Further studies must be conducted to better investigate the potential of these standardized extracts as a bioproduct for agriculture.

Antimicrobial Diterpenes from Rough Goldenrod (Solidago rugosa Mill.).

Solidago rugosa is one of the goldenrod species native to North America but has sporadically naturalized as an alien plant in Europe. The investigation of the root and leaf ethanol extracts of the plant using a bioassay-guided process with an anti-Bacillus assay resulted in the isolation of two antimicrobial components. Structure elucidation was performed based on high-resolution tandem mass spectrometric and one- and two-dimensional NMR spectroscopic analyses that revealed (-)-hardwickiic acid (Compound 1) and (-)-abietic acid (Compound 2). The isolates were evaluated for their antimicrobial properties against several plant pathogenic bacterial and fungal strains. Both compounds demonstrated an antibacterial effect, especially against Gram-positive bacterial strains (Bacillus spizizenii, Clavibacter michiganensis subsp. michiganensis, and Curtobacterium flaccumfaciens pv. flaccumfaciens) with half maximal inhibitory concentration (IC50) between 1 and 5.1 µg/mL (5-20 times higher than that of the positive control gentamicin). In the used concentrations, minimal bactericidal concentration (MBC) was reached only against the non-pathogen B. spizizenii. Besides their activity against Fusarium avenaceum, the highest antifungal activity was observed for Compound 1 against Bipolaris sorokiniana with an IC50 of 3.8 µg/mL.

Euphorbia helioscopia a Putative Plant Reservoir of Pathogenic Curtobacterium flaccumfaciens

The endophyte EHF3 strain was isolated in Algeria from an Euphorbia helioscopia plant growing in a fallow field. The strain was characterized by biochemical and physiological tests and assayed for the production of secondary metabolites involved in biocontrol, for plant growth promotion ability and for pathogenicity. The strain was identified by BIOLOG test as Curtobacterium flaccumfaciens. Biochemical and physiological characterization revealed that the strain was able to secrete protease, caseinase and amylase enzymes, to grow up to 37°C and at pH values 5 to 9. C. flaccumfaciens EHF3 strain was incapable of solubilizing phosphorus and to produce IAA, HCN siderophores and phenazine compounds. The strain showed a moderate swimming and swarming motility and produced biofilm. EHF3 strain was positive at the hypersensitivity test on tobacco plants and induced symptoms on three varieties of bean resembling to those of the bacterial wilt disease induced by C. flaccumfaciens pv. flaccumfaciens. The preliminary data reported in this study, regarding the detection of a pathogenic C. flaccumfaciens strain, as endophyte of a E. helioscopia plant, highlight the role of non-host plants as reservoir of this bacterial pathogen.

Protective Properties of Copper-Loaded Chitosan Nanoparticles against Soybean Pathogens Pseudomonas savastanoi pv. glycinea and Curtobacterium flaccumfaciens pv. flaccumfaciens.

Soybeans are a valuable food product, containing 40% protein and a large percentage of unsaturated fatty acids ranging from 17 to 23%. Pseudomonas savastanoi pv. glycinea (Psg) and Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff) are harmful bacterial pathogens of soybean. The bacterial resistance of soybean pathogens to existing pesticides and environmental concerns requires new approaches to control bacterial diseases. Chitosan is a biodegradable, biocompatible and low-toxicity biopolymer with antimicrobial activity that is promising for use in agriculture. In this work, a chitosan hydrolysate and its nanoparticles with copper were obtained and characterized. The antimicrobial activity of the samples against Psg and Cff was studied using the agar diffusion method, and the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined. The samples of chitosan and copper-loaded chitosan nanoparticles (Cu2+ChiNPs) significantly inhibited bacterial growth and were not phytotoxic at the concentrations of the MIC and MBC values. The protective properties of chitosan hydrolysate and copper-loaded chitosan nanoparticles against soybean bacterial diseases were tested on plants in an artificial infection. It was demonstrated that the Cu2+ChiNPs were the most effective against Psg and Cff. Treatment of pre-infected leaves and seeds demonstrated that the biological efficiencies of (Cu2+ChiNPs) were 71% and 51% for Psg and Cff, respectively. Copper-loaded chitosan nanoparticles are promising as an alternative treatment for bacterial blight and bacterial tan spot and wilt in soybean.

EVIDENCE OF POST-FORMED STRUCTURES DURING SYSTEMIC Curtobacterium flaccumfaciens pv. flaccumfaciens Infection In Resistant Phaseolus vulgaris GENOTYPES

Bacterial wilt of the common bean caused by Curtobacterium flaccumfaciens pv. flaccumfaciens results in economic losses. The aim of this study was to analyze the colonization of C. flaccumfaciens pv. flaccumfaciens in resistant, moderately resistant, and susceptible genotypes of common bean plants. The genotypes Ouro Branco and IPA 9 (resistant), Diacol Calima (moderately resistant), and CNFRS 11997 and CNFP 10429 (susceptible) were inoculated in the epicotyl, with 100 µL of bacterial suspension of the BRM 14933(Cff25). Disease severity was evaluated 21 days after inoculation (DAI), on a scale from 1 to 9. Plant samples were prepared for scanning electron microscopy analyses. Ouro Branco and IPA 9 (resistant) plants exhibited low colonization, the formation of filaments surrounding bacterial cells and vestures more developed in the pit the xylem vessels. Diacol Calima (moderately resistant) plants presented lower levels of colonization and filament formation than that of resistant cultivars. CNFC 10429 and CNFRS 11997 (susceptible) showed high levels of colonization in the xylem and vessel obstruction, preventing water and nutrient flow, which explains the symptoms of wilt and plant death. Thus, resistance to C. flaccumfaciens pv. flaccumfaciens can be explained by plant’s capacity to limit pathogen propagation as a post-formed defense mechanism in this pathosystem.

Development of a multiplex real-time PCR method for the detection of Pseudomonas savastanoi pv. glycinea and Curtobacterium flaccumfaciens pv. flaccumfaciens in soybean seeds.

Multiplex real-time PCR with TaqMan® probes has been developed for the simultaneous detection of soybean pathogens Pseudomonas savastanoi pv. glycinea and Curtobacterium flaccumfaciens pv. flaccumfaciens. The method specificity has been confirmed using 25 strains of target bacteria and 18 strains of other bacteria common to soybean seeds as endophytes. The multiplex real-time PCR developed has been shown to have high sensitivity - a positive result was achieved at 0.01 ng/µl of DNA for both target organisms, and at 100 CFU/ml of bacteria in soybean seed homogenate. The robustness of the multiplex real-time PCR developed has been verified by the detection of the pathogens in 25 commercial seed stocks, in comparison with previously published PCR protocols. In all tests, three seed stocks were positive and 22 were negative. The multiplex real-time PCR can be applied in diagnostic practice for the simultaneous detection of two important pathogens of leguminous plants.

Using of Essential Oils and Plant Extracts against Pseudomonas savastanoi pv. glycinea and Curtobacterium flaccumfaciens pv. flaccumfaciens on Soybean.

The bacteria Pseudomonas savastanoi pv. glycinea (Coerper, 1919; Gardan et al., 1992) (Psg) and Curtobacterium flaccumfaciens pv. flaccumfaciens (Hedges 1922) (Cff) are harmful pathogens of soybean (Glycine max). Presently, there are several strategies to control these bacteria, and the usage of environmentally friendly approaches is encouraged. In this work, purified essential oils (EOs) from 19 plant species and total aqueous and ethanolic plant extracts (PEs) from 19 plant species were tested in vitro to observe their antimicrobial activity against Psg and Cff (by agar diffusion and broth microdilution method). Tested EOs and PEs produced significant bacterial growth inhibition with technologically acceptable MIC and MBC values. Non-phytotoxic concentrations for Chinese cinnamon and Oregano essential oils and leather bergenia ethanolic extract, which previously showed the lowest MBC values, were determined. Testing of these substances with artificial infection of soybean plants has shown that the essential oils of Chinese cinnamon and oregano have the maximum efficiency against Psg and Cff. Treatment of leaves and seeds previously infected with phytopathogens with these essential oils showed that the biological effectiveness of leaf treatments was 80.6-77.5% and 86.9-54.6%, respectively, for Psg and Cff. GC-MS and GC-FID analyzes showed that the major compounds were 5-Methyl-3-methylenedihydro-2(3H)-furanone (20.32%) in leather bergenia ethanolic extract, cinnamaldehyde (84.25%) in Chinese cinnamon essential oil and carvacrol (62.32%) in oregano essential oil.

Bactericidal Efficacy of Oxidized Silver against Biofilms Formed by Curtobacterium flaccumfaciens pv. flaccumfaciens.

Bacterial wilt is a re-emerging disease on dry bean and can affect many other crop species within the Fabaceae. The causal agent, Curtobacterium flaccumfaciens pv. flaccumfaciens (CFF), is a small, Gram-positive, rod-shaped bacterium that is seed-transmitted. Infections in the host become systemic, leading to wilting and economic loss. Clean seed programs and bactericidal seed treatments are two critical management tools. This study characterizes the efficacies of five bactericidal chemicals against CFF. It was hypothesized that this bacterium was capable of forming biofilms, and that the cells within biofilms would be more tolerant to bactericidal treatments. The minimum biocide eradication concentration assay protocol was used to grow CFF biofilms, expose the biofilms to bactericides, and enumerate survivors compared to a non-treated control (water). Streptomycin and oxysilver bisulfate had EC95 values at the lowest concentrations and are likely the best candidates for seed treatment products for controlling seed-borne bacterial wilt of bean. The results showed that CFF formed biofilms during at least two phases of the bacterial wilt disease cycle, and the biofilms were much more difficult to eradicate than their planktonic counterparts. Overall, biofilm formation by CFF is an important part of the bacterial wilt disease cycle in dry edible bean and antibiofilm bactericides such as streptomycin and oxysilver bisulfate may be best suited for use in disease management.

Genome-Wide Association Study and Genomic Prediction for Bacterial Wilt Resistance in Common Bean (Phaseolus vulgaris) Core Collection

Common bean (Phaseolus vulgaris) is one of the major legume crops cultivated worldwide. Bacterial wilt (BW) of common bean (Curtobacterium flaccumfaciens pv. flaccumfaciens), being a seed-borne disease, has been a challenge in common bean producing regions. A genome-wide association study (GWAS) was conducted to identify SNP markers associated with BW resistance in the USDA common bean core collection. A total of 168 accessions were evaluated for resistance against three different isolates of BW. Our study identified a total of 14 single nucleotide polymorphism (SNP) markers associated with the resistance to BW isolates 528, 557, and 597 using mixed linear models (MLMs) in BLINK, FarmCPU, GAPIT, and TASSEL 5. These SNPs were located on chromosomes Phaseolus vulgaris [Pv]02, Pv04, Pv08, and Pv09 for isolate 528; Pv07, Pv10, and Pv11 for isolate 557; and Pv04, Pv08, and Pv10 for isolate 597. The genomic prediction accuracy was assessed by utilizing seven GP models with 1) all the 4,568 SNPs and 2) the 14 SNP markers. The overall prediction accuracy (PA) ranged from 0.30 to 0.56 for resistance against the three BW isolates. A total of 14 candidate genes were discovered for BW resistance located on chromosomes Pv02, Pv04, Pv07, Pv08, and Pv09. This study revealed vital information for developing genetic resistance against the BW pathogen in common bean. Accordingly, the identified SNP markers and candidate genes can be utilized in common bean molecular breeding programs to develop novel resistant cultivars.

Whole Genome Resources of 17 Curtobacterium flaccumfaciens Strains Including Pathotypes of C.flaccumfaciens pv. betae, C. flaccumfaciens pv. oortii, and C. flaccumfaciens pv. poinsettiae.

Curtobacterium flaccumfaciens complex species in the family Microbacteriaceae encompasses a group of plant pathogenic actinobacterial strains affecting annual crops and ornamental plants. The species includes five pathovars namely C. flaccumfaciens pv. betae, C. flaccumfaciens pv. flaccumfaciens, C. flaccumfaciens pv. ilicis, C. flaccumfaciens pv. oortii, and C. flaccumfaciens pv. poinsettiae. Despite the economic importance of C. flaccumfaciens, its members have rarely been investigated for their phylogenetic relationships, molecular characteristics and virulence repertories due in part to the lack of whole genome resources. Here we present the whole genome sequence of 17 C. flaccumfaciens strains representing members of four pathovars isolated from different plant species in a diverse geographical and temporal span. The genomic data presented in this study will pave the way of research on the comparative genomics, phylogenomics and taxonomy of C. flaccumfaciens, and extend our understanding of the virulence features of the species.

ОЦЕНКА УСТОЙЧИВОСТИ СОРТОВ СОИ К БАКТЕРИАЛЬНЫМ БОЛЕЗНЯМ НА ИСКУССТВЕННОМ ИНФЕКЦИОННОМ ФОНЕ

Bacterial blight (P. savastanoi pv. glycinea (Psg)) is one of the main bacterial diseases reducing the profitability of soybean cultivation. Another soybean disease recently discovered in Russia is rustybrown bacterial spot (and wilting) (C. flaccumfaciens pv. flaccumfaciens). The harm caused by these diseases, the emergence of antibiotic-resistant strains of bacterial phytopathogens and the imperfection of available protection necessitate the search for alternative strategies, one of which is the use of resistant or low-strucking cultivars. In this work, the optimal method of artificial inoculation of plants was determined and 47 domestic soybean cultivars were screened for susceptibility to different strains of the two pathogens. It is shown that the optimal method of inoculation is cutting with scissors soaked in a suspension of bacteria with a concentration of 108 CFU/mL, which produces typical disease symptoms with a maximum damage area in 10 days after inoculation. The least affected cultivar was Soer 4, and the most susceptible cultivars were Nordica and Osmon. An atypical hypersensitivity reaction was noted in the form of abortion of a triple leaf with a petiole during Psg inoculation in cultuvars Nordica and Opus and cultivar Maxus after Psg and Cff inoculation

Innovative Detection of the Quarantine Plant Pathogen Curtobacterium flaccumfaciens pv. flaccumfaciens, Causal Agent of Bacterial Wilt of Leguminous Plants.

Specific, sensitive, and rapid detection of quarantine and regulated plant pathogens is pivotal for the control of the diseases they cause. Here, we describe the PCR-based methods which have been developed for Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff), quarantine plant pathogenic bacterium for EU and causal agent of bacterial wilt of Leguminous plants. These methods include an end-point and a real-time PCR test, and a LAMP assay. Their threshold analytical limits range from 100 to 10fg of DNA template per reaction and are currently used worldwide for routine testing for Cff from laboratory to field scale.