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.

First report of black rot disease in Eruca vesicaria subsp. sativa caused by Xanthomonas campestris pv. campestris in Belgium.

Eruca vesicaria subsp. sativa (Mill.) Thell. (arugula or rocket) is a leafy vegetable originating from the Mediterranean region primarily being sold in bagged salads. From 2014 to 2017, plants (cv. Montana) exhibiting blackened leaf veins and irregular V-shaped chlorotic to necroic lesions at the leaf margins were observed in commercial greenhouses in Flanders, Belgium (Figure S1A). Symptoms started after harvest of the first cut, indicating that leaf injury favours disease development. By the last cut, infections had spread uniformly across the plots, with symptoms advanced to the point where harvesting was no longer profitable. Excised surface-sterilized necrotic leaf tissue and seeds were homogenized in phosphate buffer (PB), followed by dilution plating on Pseudomonas Agar F containing sucrose. After four days at 28°C, bright yellow round, mucoid, convex Xanthomonas-like colonies were obtained, both from leaves and seeds. For confirmation, DNA was extracted from pure cultures after which a partial fragment of gyrB was amplified and sequenced (Holtappels et al. 2022). Amplicons were trimmed to 530 nucleotides (Genbank ON815895-ON815900) according to Parkinson et al. (2007) and compared with the NCBI database. Strain GBBC 3139 shares 100% sequence identity with Xanthomonas campestris pv. campestris (Xcc) type strain LMG 568 and with RKFB 1361-1364, isolated from arugula in Serbia (Prokic et al. 2022). The other isolates from Belgian rocket - GBBC 3036, 3058, 3077, 3217 and 3236 - all have a gyrB sequence 100% identical to that of Xcc strain ICMP 4013, among others. To determine the genetic relatedness to other pathogenic Xc strains, the genomes of GBBC 3077, 3217, 3236 and 3139 were sequenced using a MinION (Nanopore) and non-clonal sequences were submitted to NCBI (BioProject PRJNA967242). Genomes were compared by calculating Average Nucleotide Identity (ANI). This revealed that the Belgian strains cluster together with Xc isolates originating from Brassica crops and separate from strains identified as Xc pv. barbareae, pv. incanae and pv. raphani (Figure S2A). Their designation as pv. campestris is supported by maximum likelihood clustering of concatenated gyrB-avrBs2 sequences (EPPO, 2021; Figure S2B,C). Finally, pathogenicity was verified on five-week-old rocket 'Pronto' plants grown in a commercial potting mix by cutting the leaves along the midrib with scissors dipped into a suspension of 108 cfu/ml of each strain or PB as control (4 plants/strain). Plants were kept in closed polypropylene boxes for 48 hr to support high humidity and facilitate infection. They were then maintained at 25 ± 2 °C. Lesions like those observed on commercial plants developed on the inoculated leaves within one week (Figure S1B). Bacterial colonies reisolated from symptomatic tissue were identified based on gyrB as the strains used for inoculation, thereby fulfilling Koch's postulates. To the best of our knowledge, this is the first report of black rot disease in arugula caused by Xcc in Belgium. Previously, Xcc on arugula has been reported in Argentina, California and Serbia as well (Romero et al. 2008; Rosenthal et al. 2017; Prokic et al. 2022). Arugula being a minor crop in Belgium, challenged by Xcc infections and strong import competition, many growers have abandoned the sector in recent years. Therefore, this study makes a strong case for early detection of disease symptoms and timely application of relevant management strategies in vulnerable crop settings.

The potential of bacteriophages to control Xanthomonas campestris pv. campestris at different stages of disease development.

Xanthomonas campestris pv. campestris (Xcc) is a vascular pathogen that invades the xylem of Brassica crops. Current chemical and antibiotics-based control measures for this bacterium are unsustainable and inefficient. After establishing a representative collection of Xcc strains, we isolated and characterized bacteriophages from two clades of phages to assess their potential in phage-based biocontrol. The most promising phages, FoX2 and FoX6, specifically recognize (lipo) polysaccharides, associated with the wxc gene cluster, on the surface of the bacterial cell wall. Next, we determined and optimized the applicability of FoX2 and FoX6 in an array of complementary bioassays, ranging from seed decontamination to irrigation- and spray-based applications. Here, an irrigation-based application showed promising results. In a final proof-of-concept, a CaCl2 -formulated phage cocktail was shown to control the outbreak of Xcc in the open field. This comprehensive approach illustrates the potential of phage biocontrol of black rot disease in Brassica and serves as a reference for the broader implementation of phage biocontrol in integrated pest management strategies.

Daring to be differential: metabarcoding analysis of soil and plant-related microbial communities using amplicon sequence variants and operational taxonomical units.

BACKGROUND: Microorganisms are not only indispensable to ecosystem functioning, they are also keystones for emerging technologies. In the last 15 years, the number of studies on environmental microbial communities has increased exponentially due to advances in sequencing technologies, but the large amount of data generated remains difficult to analyze and interpret. Recently, metabarcoding analysis has shifted from clustering reads using Operational Taxonomical Units (OTUs) to Amplicon Sequence Variants (ASVs). Differences between these methods can seriously affect the biological interpretation of metabarcoding data, especially in ecosystems with high microbial diversity, as the methods are benchmarked based on low diversity datasets. RESULTS: In this work we have thoroughly examined the differences in community diversity, structure, and complexity between the OTU and ASV methods. We have examined culture-based mock and simulated datasets as well as soil- and plant-associated bacterial and fungal environmental communities. Four key findings were revealed. First, analysis of microbial datasets at family level guaranteed both consistency and adequate coverage when using either method. Second, the performance of both methods used are related to community diversity and sample sequencing depth. Third, differences in the method used affected sample diversity and number of detected differentially abundant families upon treatment; this may lead researchers to draw different biological conclusions. Fourth, the observed differences can mostly be attributed to low abundant (relative abundance < 0.1%) families, thus extra care is recommended when studying rare species using metabarcoding. The ASV method used outperformed the adopted OTU method concerning community diversity, especially for fungus-related sequences, but only when the sequencing depth was sufficient to capture the community complexity. CONCLUSIONS: Investigation of metabarcoding data should be done with care. Correct biological interpretation depends on several factors, including in-depth sequencing of the samples, choice of the most appropriate filtering strategy for the specific research goal, and use of family level for data clustering.

Effect of in feed administration of different butyrate formulations on Salmonella Enteritidis colonization and cecal microbiota in broilers.

Butyrate has been used extensively as a feed additive to improve gut health and to decrease Salmonella colonization in poultry. Salmonella mainly colonizes the ceca so butyrate concentrations should be increased in this gut segment. Discrepancies on the effects of butyrate on Salmonella colonization, described in the scientific literature, could thus be due to butyrate release location effects. In this study, newly developed butyrate formulations were evaluated for their effect on cecal butyrate concentrations and on colonization by Salmonella Enteritidis. In a first trial, broilers were randomly allocated to 7 dietary treatment groups with formulations based on different approaches to modify the butyrate release profile: release from wax matrices based on diffusion/erosion; micropellets supposedly release butyrate around pH 7 in the colon; tributyrin is based on the hydrolysis of esters in the small intestine. Fat-protected butyrate was included as a reference, because of its known effect on reduction of Salmonella colonization. Four days after infection, the number of cfu Salmonella per g cecal content and spleen were determined. Butyrate formulations in a wax matrix significantly reduced the Salmonella colonization in cecal content. In a second trial, wax and fat-protected butyrate treatments were replicated and results from the first trial were confirmed. Compared to the control group a higher proportion of butyrate concentration was observed in ceca for those groups with reduced Salmonella colonization. This was associated with a beneficial shift in the cecal microbiota. In conclusion, formulations that increase cecal butyrate concentrations are superior in protecting against Salmonella Enteritidis colonization.

Differential efficiency of wild type rhizogenic strains for rol gene transformation of plants.

Rhizogenic agrobacteria induce extensive root proliferation, in several economically valuable, dicotyledonous plant species, a phenomenon referred to as "hairy roots." Besides their pathogenic nature, agrobacteria have proven to be a valuable asset in biotechnology and molecular plant breeding. To assess the potential of frequently used rhizogenic strains, growth in yeast extract glucose broth and antibiotic resistance was analyzed. Growth curves were established for Arqua1, NCPPB2659, LMG150, LMG152, and ATCC15834; and regression analysis of the exponential growth phase resulted in a reliable and standardized method for preparation of a bacterial suspension for inoculation. Cell density did not correlate with the timing of hairy root emergence. The highest number of hairy roots was obtained with an inoculum of 1 × 108 CFU ml-1 for Arqua1, NCPPB2659, and LMG152. Cell density of ATCC15834 did not affect the number of hairy roots formed. The identity of the rhizogenic strains for plant transformation was verified in phylogenetic analysis using average nucleotide identity (ANI), which also provided insight in their genetic diversity within the Rhizobium taxon.

Reduced-Particle-Size Wheat Bran Is Efficiently Colonized by a Lactic Acid-Producing Community and Reduces Levels of Enterobacteriaceae in the Cecal Microbiota of Broilers.

In the present study, we investigated whether reducing the particle size of wheat bran affects the colonizing microbial community using batch fermentations with cecal inocula from seven different chickens. We also investigated the effect of in-feed administration of regular wheat bran (WB; 1,690 µm) and wheat bran with reduced particle size (WB280; 280 µm) on the cecal microbial community composition of broilers. During batch fermentation, WB280 was colonized by a lactic acid-producing community (Bifidobacteriaceae and Lactobacillaceae) and by Lachnospiraceae that contain lactic acid-consuming butyric acid-producing species. The relative abundances of the Enterobacteriaceae decreased in the particle-associated communities for both WB and WB280 compared to that of the control. In addition, the community attached to wheat bran was enriched in xylan-degrading bacteria. When administered as a feed additive to broilers, WB280 significantly increased the richness of the cecal microbiota and the abundance of bacteria containing the butyryl-coenzyme A (CoA):acetate CoA-transferase gene, a key gene involved in bacterial butyrate production, while decreasing the abundances of Enterobacteriaceae family members in the ceca. Particle size reduction of wheat bran thus resulted in the colonization of the bran particles by a very specific lactic acid- and butyric acid-producing community and can be used to steer toward beneficial microbial shifts. This can potentially increase the resilience against pathogens and increase animal performance when the reduced-particle-size wheat bran is administered as a feed additive to broilers.IMPORTANCE Prebiotic dietary fibers are known to improve the gastrointestinal health of both humans and animals in many different ways. They can increase the bulking capacity, improve transit times, and, depending on the fiber, even stimulate the growth and activity of resident beneficial bacteria. Wheat bran is a readily available by-product of flour processing and is a highly concentrated source of (in)soluble dietary fiber. The intake of fiber-rich diets has been associated with increased Firmicutes and decreased Proteobacteria numbers. Here, we show that applying only 1% of a relatively simple substrate which was technically modified using relatively simple techniques reduces the concentration of Enterobacteriaceae This could imply that in future intervention studies, one should take the particle size of dietary fibers into account.

High-Quality Draft Genome Sequences of Five Xanthomonas arboricola pv. fragariae Isolates.

Xanthomonas arboricola pv. fragariae was described in 2001 as the causal agent of strawberry bacterial leaf blight. We report here the first draft whole-genome sequences of five X. arboricola pv. fragariae isolates from Italy and France.

Comparative genome analysis of pathogenic and non-pathogenic Clavibacter strains reveals adaptations to their lifestyle.

BACKGROUND: The genus Clavibacter harbors economically important plant pathogens infecting agricultural crops such as potato and tomato. Although the vast majority of Clavibacter strains are pathogenic, there is an increasing number of non-pathogenic isolates reported. Non-pathogenic Clavibacter strains isolated from tomato seeds are particularly problematic because they affect the current detection and identification tests for Clavibacter michiganensis subsp. michiganensis (Cmm), which is regulated with a zero tolerance in tomato seed. Their misidentification as pathogenic Cmm hampers a clear judgment on the seed quality and health. RESULTS: To get more insight in the genetic features linked to the lifestyle of these bacteria, a whole-genome sequence of the tomato seed-borne non-pathogenic Clavibacter LMG 26808 was determined. To gain a better understanding of the molecular determinants of pathogenicity, the genome sequence of LMG 26808 was compared with that of the pathogenic Cmm strain (NCPPB 382). The comparative analysis revealed that LMG 26808 does not contain plasmids pCM1 and pCM2 and also lacks the majority of important virulence factors described so far for pathogenic Cmm. This explains its apparent non-pathogenic nature in tomato plants. Moreover, the genome analysis of LMG 26808 detected sequences from a plasmid originating from a member of Enterobacteriaceae/Klebsiella relative. Genes received that way and coding for antibiotic resistance may provide a competitive advantage for survival of LMG 26808 in its ecological niche. Genetically, LMG 26808 was the most similar to the pathogenic Cmm NCPPB 382 but contained more mobile genetic elements. The genome of this non-pathogenic Clavibacter strain contained also a high number of transporters and regulatory genes. CONCLUSIONS: The genome sequence of the non-pathogenic Clavibacter strain LMG 26808 and the comparative analyses with other pathogenic Clavibacter strains provided a better understanding of the genetic bases of virulence and adaptation mechanisms present in the genus Clavibacter.

Draft genome sequence of Xanthomonas fragariae reveals reductive evolution and distinct virulence-related gene content.

BACKGROUND: Xanthomonas fragariae (Xf) is a bacterial strawberry pathogen and an A2 quarantine organism on strawberry planting stock in the EU. It is taxonomically and metabolically distinct within the genus Xanthomonas, and known for its host specificity. As part of a broader pathogenicity study, the genome of a Belgian, virulent Xf strain (LMG 25863) was assembled to draft status and examined for its pathogenicity related gene content. RESULTS: The Xf draft genome (4.2 Mb) was considerably smaller than most known Xanthomonas genomes (~5 Mb). Only half of the genes coding for TonB-dependent transporters and cell-wall degrading enzymes that are typically present in other Xanthomonas genomes, were found in Xf. Other missing genes/regions with a possible impact on its plant-host interaction were: i) the three loci for xylan degradation and metabolism, ii) a locus coding for a ß-ketoadipate phenolics catabolism pathway, iii) xcs, one of two Type II Secretion System coding regions in Xanthomonas, and iv) the genes coding for the glyoxylate shunt pathway. Conversely, the Xf genome revealed a high content of externally derived DNA and several uncommon, possibly virulence-related features: a Type VI Secretion System, a second Type IV Secretion System and a distinct Type III Secretion System effector repertoire comprised of multiple rare effectors and several putative new ones. CONCLUSIONS: The draft genome sequence of LMG 25863 confirms the distinct phylogenetic position of Xf within the genus Xanthomonas and reveals a patchwork of both lost and newly acquired genomic features. These features may help explain the specific, mostly endophytic association of Xf with the strawberry plant.

Draft Genome Sequences of Four Dickeya dianthicola and Four Dickeya solani Strains.

Dickeya dianthicola and "Dickeya solani" are currently the dominant bacterial pathogens of potatoes in Europe. Here, we present the draft genome sequences of four strains of each pathogen.

Sequence diversity in the Dickeya fliC gene: phylogeny of the Dickeya genus and TaqMan® PCR for 'D. solani', new biovar 3 variant on potato in Europe.

Worldwide, Dickeya (formerly Erwinia chrysanthemi) is causing soft rot diseases on a large diversity of crops and ornamental plants. Strains affecting potato are mainly found in D. dadantii, D. dianthicola and D. zeae, which appear to have a marked geographical distribution. Furthermore, a few Dickeya isolates from potato are attributed to D. chrysanthemi and D. dieffenbachiae. In Europe, isolates of Erwinia chrysanthemi biovar 1 and biovar 7 from potato are now classified in D. dianthicola. However, in the past few years, a new Dickeya biovar 3 variant, tentatively named 'Dickeya solani', has emerged as a common major threat, in particular in seed potatoes. Sequences of a fliC gene fragment were used to generate a phylogeny of Dickeya reference strains from culture collections and with this reference backbone, to classify pectinolytic isolates, i.e. Dickeya spp. from potato and ornamental plants. The reference strains of the currently recognized Dickeya species and 'D. solani' were unambiguously delineated in the fliC phylogram. D. dadantii, D. dianthicola and 'D. solani' displayed unbranched clades, while D. chrysanthemi, D. zeae and D. dieffenbachiae branched into subclades and lineages. Moreover, Dickeya isolates from diagnostic samples, in particular biovar 3 isolates from greenhouse ornamentals, formed several new lineages. Most of these isolates were positioned between the clade of 'D. solani' and D. dadantii as transition variants. New lineages also appeared in D. dieffenbachiae and in D. zeae. The strains and isolates of D. dianthicola and 'D. solani' were differentiated by a fliC sequence useful for barcode identification. A fliC TaqMan®real-time PCR was developed for 'D. solani' and the assay was provisionally evaluated in direct analysis of diagnostic potato samples. This molecular tool can support the efforts to control this particular phytopathogen in seed potato certification.

Molecular detection of Puccinia horiana in Chrysanthemum x morifolium through conventional and real-time PCR.

Puccinia horiana Henn. is a quarantine organism and one of the most important fungal pathogens of Chrysanthemum x morifolium cultivars grown for cut flower or potted plant production (florist's chrysanthemum) in several regions of the world. Highly specific primer pairs were identified for conventional, nested, and real-time PCR detection of P. horiana based on the specific and sensitive PCR amplification of selected regions in the internal transcribed spacers (ITS1 and ITS2) of the nuclear ribosomal DNA (rDNA). Using these different PCR versions, 10 pg, 10 fg, and 5 fg genomic DNA could be detected, respectively. When using cloned target DNA as template, the detection limits were 5000, 50, and 5 target copies, respectively. These detection limits were not affected by a background of chrysanthemum plant DNA. The DNA extraction method was optimized to maximize the recoverability of the pathogen from infected plant tissue. A CTAB extraction protocol or a selection of commercial DNA extraction methods allowed the use of 10 ng total (plant+pathogen) DNA without interference of PCR inhibitors. Due to the specificity of the primers, SYBR Green I technology enabled reliable real time PCR signal detection. However, an efficient TaqMan probe is available. The lowest proportion of infected plant material that could still be detected when mixed with healthy plant material was 0.001%. The real-time PCR assay could detect as few as eight pure P. horiana basidiospores, demonstrating the potential of the technique for aerial detection of the pathogen. The amount of P. horiana DNA in plant tissue was determined at various time points after basidiospore inoculation. Using the real-time PCR protocol, it was possible to detect the pathogen immediately after the inoculation period, even though the accumulation of pathogen DNA was most pronounced near the end of the latent period. The detection system proved to be accurate and sensitive and could help not only in pathogen diagnosis but also in pathogen monitoring and disease forecasting systems.