Complete genome sequence of a novel bacteriophage vB_Pci_PCMW57 infecting phytobacteria pseudomonas cichorii.

BACKGROUND: A novel virulent bacteriophage infecting phytobacteria Pseudomonas cichorii (P. cichorii) was isolated from leafy vegetables in Brazil. P. cichorii is a Gram-negative soil phytobacterium, the causal agent of a number of economically important plant diseases worldwide. METHODS AND RESULTS: In this study, a new phage specific for P. cichorii was isolated from solid samples (lettuce, chicory and cabbage), designated vB_Pci_PCMW57. Electron microscopy revealed a small virion (~ 50-nm-diameter icosahedral capsid) with a short, non-contractile tail. The genome of vB_Pci_PCMW57 is 40,117 bp in size, with a GC content of 57.6% and encodes 49 open reading frames. The phage is genetically similar to P. syringae phages Pst_GM1 and Pst_GIL1, and the P. fluorescens phages WRT and KNP. According to electron microscopy and whole-genome sequence analysis, vB_Pci_PCMW57 should be classified as a Caudoviticetes, family Autographiviridae, subfamily Studiervirinae. CONCLUSIONS: The complete phage genome was annotated, and the sequence identity of the virus with other Pseudomonas viruses was higher than 95%. To our knowledge, this is the first report of a bacteriophage infecting Pseudomonas cichorii.

First Report of Bacterial Leaf Spot on Spinach (Spinacia oleracea) Caused by Pseudomonas sp. in South Carolina.

A large grower of Brassica leafy greens and spinach in South Carolina observed a severe outbreak of leaf spot on 150 hectares of spinach (Spinacia oleracea) in Orangeburg County, SC in 2013. The entire field was lost due to the outbreak. Symptoms appeared on 8-week old plants as tan to white necrotic spots with black centers, water-soaking and no discernable chlorotic borders. Lesions varied from 2 mm to 1 cm in diameter and often coalesced to cover >50% of the leaves. Symptomatic spinach plants cv. Vancouver were collected in 2013 from the field. Bacterial streaming was evident from the border of necrotic lesions under magnification. Lesion border regions were excised, surface-disinfested with 0.5% NaOCl, macerated in sterilized distilled water and streaked onto nutrient agar (NA) and Pseudomonas Agar F (PAF). Bacterial growth was observed on NA and PAF; several single colonies were selected and re-streaked onto PAF. Colonies fluoresced blue under UV light after 48 h at 28oC. Two of the strains were subjected to 16S rRNA sequencing (GenBank accessions OM983506 and OM983507) and Fatty Acid Methyl Ester (FAME) analysis (MIDI LABS, Newark, DE). FAME results had a best similarity index (0.788) to Pseudomonas cichorii/viridiflava. The 16S sequences were queried to Pseudomonas type-strains in GenBank resulting in best matches: P. ovata (99.23% identity with 99% coverage) and P. maditerranea (99.04% identity with 100% coverage). Additionally, sequences had 97.33% identity with 100% coverage as a P. cichorii type strain, and only 96.86% identity with 97% coverage as a P. viridiflava type strain. These two strains were tested for pathogenicity on the spinach cv. Vancouver. Bacteria were grown on PAF for 48 h, and a bacterial suspension was prepared with sterile distilled water with the addition of 0.001% Latron (Plant Health Technologies, Boise, ID) and adjusted to an optical density of 0.4 at OD600. Six-week-old plants (eight plants) were sprayed with the bacterial suspension to runoff, placed at 100% relative humidity for 72 h, and then put in a growth chamber at 25oC with a 12 h diurnal light cycle for 10 days. Eight plants of 'Vancouver' were sprayed with water and 0.001% Latron as controls. Both strains were pathogenic on 'Vancouver' and caused symptoms similar to those observed in the field. Symptoms were not observed on negative controls. The same bacterial colonies were recovered from the lesions on inoculated plants, fulfilling Koch's postulates. Comparative rep-PCR analysis using the BOXA1R primer (Versalovic et al. 1994) showed both strains had identical DNA-banding profiles. All identification methods used indicate that this is a different Pseudomonas species from the one reported on spinach in California by Koike et al (2002). The top producers of spinach in SC stopped large-scale production in 2014 as a result of this pathogen. In 2020, due to inability of processors to obtain sufficient quantities of spinach, SC growers again planted the crop. Growers experienced yield losses due to similar symptoms on the crop. BOX-PCR of isolated strains of bacteria from these plants showed a DNA banding pattern similar to the 2013 strains.

In vitro, in planta, and comparative genomic analyses of Pseudomonas syringae pv. syringae strains of pepper (Capsicum annuum var. annuum).

Pseudomonas syringae pv. syringae (Pss) is an emerging phytopathogen that causes Pseudomonas leaf spot (PLS) disease in pepper plants. Pss can cause serious economic damage to pepper production, yet very little is known about the virulence factors carried by Pss that cause disease in pepper seedlings. In this study, Pss strains isolated from pepper plants showing PLS symptoms in Ohio between 2013 and 2021 (n = 16) showed varying degrees of virulence (Pss populations and disease symptoms on leaves) on 6-week-old pepper seedlings. In vitro studies assessing growth in nutrient-limited conditions, biofilm production, and motility also showed varying degrees of virulence, but in vitro and in planta variation in virulence between Pss strains did not correlate. Comparative whole-genome sequencing studies identified notable virulence genes including 30 biofilm genes, 87 motility genes, and 106 secretion system genes. Additionally, a total of 27 antimicrobial resistance genes were found. A multivariate correlation analysis and Scoary analysis based on variation in gene content (n = 812 variable genes) and single nucleotide polymorphisms within virulence genes identified no significant correlations with disease severity, likely due to our limited sample size. In summary, our study explored the virulence and antimicrobial gene content of Pss in pepper seedlings as a first step toward understanding the virulence and pathogenicity of Pss in pepper seedlings. Further studies with additional pepper Pss strains will facilitate defining genes in Pss that correlate with its virulence in pepper seedlings, which can facilitate the development of effective measures to control Pss in pepper and other related P. syringae pathovars. IMPORTANCE: Pseudomonas leaf spot (PLS) caused by Pseudomonas syringae pv. syringae (Pss) causes significant losses to the pepper industry. Highly virulent Pss strains under optimal environmental conditions (cool-moderate temperatures, high moisture) can cause severe necrotic lesions on pepper leaves that consequently can decrease pepper yield if the disease persists. Hence, it is important to understand the virulence mechanisms of Pss to be able to effectively control PLS in peppers. In our study, in vitro, in planta, and whole-genome sequence analyses were conducted to better understand the virulence and pathogenicity characteristics of Pss strains in peppers. Our findings fill a knowledge gap regarding potential virulence and pathogenicity characteristics of Pss in peppers, including virulence and antimicrobial gene content. Our study helps pave a path to further identify the role of specific virulence genes in causing disease in peppers, which can have implications in developing strategies to effectively control PLS in peppers.

Synergistic effect of Cordia curassavica Jacq. essential oils association against the phytopathogen Xanthomonas campestris pv. campestris.

The increased use of pesticides applied to treat diseases caused by bacteria has caused serious environmental problems. There are few fungicides/bactericides for the treatment of plant diseases caused by Xanthomonas campestris pv. campestris (Xcc), and only two natural products with general bactericidal/fungicidal use are available on the market. Thus, this study evaluated the antimicrobial activity of essential oils (EOs), and their combinations, from five distinct genotypes of Cordia curassavica (Jacq.) Roem. & Schult (Syn. Varronia curassavica Jacq.) (CCUR) against Xcc. GC/MS chemical analysis revealed α-pinene, sabinene, (E)-caryophyllene, ar-curcumene, ß-sesquiphellandrene, 7-cyclodecen-1-one, and ar-Turmerone as the major compounds of the five EOs of CCUR. All EOs showed growth inhibition of Xcc with minimum inhibitory concentration between 500 and 1000 µg mL-1. The associations between two EOs from different CCUR genotypes showed that 70% of the total combinations had an additive effect. However, the combinations between CCUR-002 × (-302, -202) and CCUR-302 × (-601) showed a synergistic effect, with mean fractional inhibitory concentration FIC50 values of 0.28, 0.42, and 0.40, respectively. This study demonstrates that combinations of C. curassavica EOs have antimicrobial activity and a potential to be used in the control of black rot. Graphical abstract.

Phylogenetic Analyses of Xanthomonads Causing Bacterial Leaf Spot of Tomato and Pepper: Xanthomonas euvesicatoria Revealed Homologous Populations Despite Distant Geographical Distribution.

Bacterial leaf spot of tomato and pepper (BLS), an economically important bacterial disease caused by four species of Xanthomonas (X. euvesicatoria (Xe), X. vesicatoria (Xv), X. gardneri (Xg), and X. perforans (Xp)), is a global problem and can cause over 50% crop loss under unfavorable conditions. Among the four species, Xe and Xv are prevalent worldwide. Characterization of the pathogens is crucial for disease management and regulatory purposes. In this study, we performed a multilocus sequence analysis (MLSA) with six genes (hrcN, dnaA gyrB, gapA, pdg, and hmbs) on BLS strains. Other Xanthomonas species were included to determine phylogenetic relationships within and among the tested strains. Four BLS species comprising 76 strains from different serological groups and diverse geographical locations were resolved into three major clades. BLS xanthomonads formed distinct clusters in the phylogenetic analyses. Three other xanthomonads, including X. albilineans, X. sacchari, and X. translucens pv. undolusa revealed less than 85%, 88%, and 89% average nucleotide identity (ANI), respectively, with the other species of Xanthomonas included in this study. Both antibody and MLSA data showed that Xv was clearly separated from Xe and that the latter strains were remarkably clonal, even though they originated from distant geographical locations. The Xe strains formed two separate phylogenetic groups; Xe group A1 consisted only of tomato strains, whereas Xe group A2 included strains from pepper and tomato. In contrast, the Xv group showed greater heterogeneity. Some Xv strains from South America were closely related to strains from California, while others grouped closer to a strain from Indiana and more distantly to a strain from Hawaii. Using this information molecular tests can now be devised to track distribution of clonal populations that may be introduced into new geographic areas through seeds and other infected plant materials.

Diversity of pectobacterium strains by biochemical, physiological, and molecular characterization / Diversidade de cepas de Pectobacterium por caracterização bioquímica, fisiológica e molecular

Pectobacterium is a complex taxon of strains with diverse characteristics. It comprises several genera, including Erwinia, Brenneria, Pectobacterium, Dickeya, and Pantoea. Pectobacterium and Dickeya cause diseases in a wide range of plants, including potatoes, where they are causative agents of soft rot in tubers and blackleg in field-grown plants.Characterizing Pectobacterium species allows for the analysis of the diversity of pectinolytic bacteria, which may support control strategies for plant bacterial diseases. The aim of this study was to perform biochemical, physiological, and molecular characterizations of Pectobacteriumspp. from different sites and host plants. The isolated strains were characterized by the glucose fermentation test, Gram staining, catalase activity, oxidase activity, growth at 37 ºC, reducing substances from sucrose, phosphatase activity, indole production, acid production from different sources (sorbitol, melibiose, citrate, and lactose), pathogenicity in potato, and hypersensitivity reactions. Molecular characterization was performed with species-specific primers ECA1f/ECA2r and EXPCCF/EXPCCR, which identify P.atrosepticum and P.carotovorum subsp. carotovorum (Pcc), respectively, and with primers 1491f/L1RA/L1RG and Br1f/L1RA/L1RG that differentiate Pcc from Dickeya chrysanthemi and from P. carotovorum subsp. brasiliensis. The strains were identified as belonging to the genus Pectobacterium, though they did not fit the biochemical nor the molecular classification standards for subspecies differentiation, indicating significant diversity among the strains.

Pectobacterium é um táxon complexo de isolados bacterianos com características diversas. Compreende vários gêneros como Erwinia, Brenneria, Pectobacterium, Dickeya e Pantoea. Pectobacterium e Dickeya causam doenças em ampla variedade de plantas, incluindo a batateira, na qual são os agentes etiológicos da podridão mole dos tubérculos e da canela-preta de plantas cultivadas em campo.A caracterização de espécies de Pectobacterium permite a análise da diversidade de bactérias pectolíticas, podendo auxiliar estratégias de controle de doenças bacterianas em plantas. O objetivo deste trabalho foi caracterizar bioquímica, fisiológica e molecularmente isolados de Pectobacterium sp. provenientes de diferentes locais e hospedeiros. Os isolados foram caracterizados pelos testes de fermentação de glicose, Gram, catalase, oxidase, crescimento à 37 ºC, redução de substâncias a partir de sacarose, atividade da fosfatase, produção de indol, produção de ácido a partir de sorbitol, melibiose, citrato e lactose, patogenicidade em batata e reação de hipersensibilidade. Para a caracterização molecular, foram utilizados os pares de primersECA1f/ECA2r e EXPCCF/EXPCCR [específicos para P. atrosepticum e P.carotovorum subsp. carotovorum(Pcc), respectivamente] e as tríades de primers 1491f/L1RA/L1RG e Br1f/L1RA/L1RG, para diferenciar Pcc de Dickeya chrysanthemi e de P. carotovorum subsp. brasiliensis. Os isolados foram identificados como pertencentes ao gênero Pectobacterium, no entanto, não se enquadraram na classificação bioquímica e tampouco molecular para diferenciação das subespécies, demonstrando a grande diversidade dos mesmos.