Differentiation of the Xanthomonas hortorum – Xanthomonas hydrangeae species complex using sensitive and selective LAMP assays

Related publications: https://doi.org/10.1099/ijsem.0.005163, https://doi.org/10.1002/ndr2.12008The seven pathovars of Xanthomonas hortorum and Xanthomonas hydrangeae, referred to as the X. hortorum – X. hydrangeae species complex, cause disease on a multitude of plants, including crops, ornamental and wild plants. Cross-pathogenicity was proven for some of the strains within this species complex. It is thus important to have highly specific and fast diagnostics methods for members of the X. hortorum – X. hydrangeae species complex. A comparative genomic analysis was conducted for representative members within the complex to identify singletons for use as genomic targets for the assays. Seven loop-mediated isothermal amplification (LAMP) diagnostics assays were developed for the detection of six clades within the X. hortorum – X. hydrangeae species complex, in addition to one assay specific for the entire species complex. Primer sets were tested on a set of 62 reference strains. The primer sets amplified their respective targets within 15 minutes. Based on the reference set, all assays had a sensitivity, specificity, and efficiency of 100%. The assays were used on a validation set of 60 strains. According to the LAMP results, out of the 60 strains, 39 strains were assigned to one of the clades within the complex, 9 were assigned to the complex but to yet undefined clades within the complex, and 12 strains were previously misclassified as X. hortorum since their genomic DNA did not yield amplification with any of the assays. The seven genome-based assays are promising for use as diagnostic tools for various members within the X. hortorum – X. hydrangeae species complex, and for assigning new and historical isolates to this complex

Xanthomonas hydrangeae sp. nov., a novel plant pathogen isolated from Hydrangea arborescens

This paper describes a novel species isolated in 2011 and 2012 from nursery-grown Hydrangea arborescens cultivars in Flanders, Belgium. After 4 days at 28 °C, the strains yielded yellow, round, convex and mucoid colonies. Pathogenicity of the strains was confirmed on its isolation host, as well as on Hydrangea quercifolia. Analysis using MALDI-TOF MS identified the Hydrangea strains as belonging to the genus Xanthomonas but excluded them from the species Xanthomonas hortorum. A phylogenetic tree based on gyrB confirmed the close relation to X. hortorum. Three fatty acids were dominant in the Hydrangea isolates: anteiso-C15 : 0, iso-C15 : 0 and summed feature 3 (C16 : 1  ω7c/C16 : 1  ω6c). Unlike X. hortorum pathovars, the Hydrangea strains were unable to grow in the presence of lithium chloride and could only weakly utilize d-fructose-6-PO4 and glucuronamide. Phylogenetic characterization based on multilocus sequence analysis and phylogenomic characterization revealed that the strains are close to, yet distinct from, X. hortorum. The genome sequences of the strains had average nucleotide identity values ranging from 94.35-95.19 % and in silico DNA-DNA hybridization values ranging from 55.70 to 59.40 % to genomes of the X. hortorum pathovars. A genomics-based loop-mediated isothermal amplification assay was developed which was specific to the Hydrangea strains for its early detection. A novel species, Xanthomonas hydrangeae sp. nov., is proposed with strain LMG 31884T (=CCOS 1956T) as the type strain

Ether bond cleavage of a phenylcoumaran beta-5 lignin model compound and polymeric lignin catalysed by a LigE-type etherase from Agrobacterium sp

A LigE-type beta-etherase enzyme from lignin-degrading Agrobacterium sp. has been identified, which assists degradation of polymeric lignins. Testing against lignin dimer model compounds revealed that it does not catalyse the previously reported reaction of Sphingobium SYK-6 LigE, but instead shows activity for a β-5 phenylcoumaran lignin dimer. The reaction products did not contain glutathione, indicating a catalytic role for reduced glutathione in this enzyme. Three reaction products were identified: the major product was a cis-stilbene arising from C−C fragmentation involving loss of formaldehyde; two minor products were an alkene arising from elimination of glutathione, and an oxidised ketone, proposed to arise from reaction of an intermediate with molecular oxygen. Testing of the recombinant enzyme against a soda lignin revealed the formation of new signals by two-dimensional NMR analysis, whose chemical shifts are consistent with the formation of a stilbene unit in polymeric lignin

Dynamics of Seed-Borne Rice Endophytes on Early Plant Growth Stages

Bacterial endophytes are ubiquitous to virtually all terrestrial plants. With the increasing appreciation of studies that unravel the mutualistic interactions between plant and microbes, we increasingly value the beneficial functions of endophytes that improve plant growth and development. However, still little is known on the source of established endophytes as well as on how plants select specific microbial communities to establish associations. Here, we used cultivation-dependent and -independent approaches to assess the endophytic bacterrial community of surface-sterilized rice seeds, encompassing two consecutive rice generations. We isolated members of nine bacterial genera. In particular, organisms affiliated with Stenotrophomonas maltophilia and Ochrobactrum spp. were isolated from both seed generations. PCR-based denaturing gradient gel electrophoresis (PCR-DGGE) of seed-extracted DNA revealed that approximately 45% of the bacterial community from the first seed generation was found in the second generation as well. In addition, we set up a greenhouse experiment to investigate abiotic and biotic factors influencing the endophytic bacterial community structure. PCR-DGGE profiles performed with DNA extracted from different plant parts showed that soil type is a major effector of the bacterial endophytes. Rice plants cultivated in neutral-pH soil favoured the growth of seed-borne Pseudomonas oryzihabitans and Rhizobium radiobacter, whereas Enterobacter-like and Dyella ginsengisoli were dominant in plants cultivated in low-pH soil. The seed-borne Stenotrophomonas maltophilia was the only conspicuous bacterial endophyte found in plants cultivated in both soils. Several members of the endophytic community originating from seeds were observed in the rhizosphere and surrounding soils. Their impact on the soil community is further discussed

A diagnostic tool for improved detection of Xanthomonas fragariae using a rapid and highly specific LAMP assay designed with comparative genomics

Molecular diagnostics of plant pathogens are crucial to prevent disease spread and to enhance food quality and security. A comparative genomics approach using genomes of different Xanthomonas species and pathovars was applied to identify highly specific targets in the genome of Xanthomonas fragariae, the causal agent of angular leaf spot of strawberry, listed under quarantine regulations in Europe. A reliable and sensitive loop-mediated isothermal amplification (LAMP) assay was designed using a unique marker providing a highly specific and rapid detection, making this technique convenient for on-site detection. Specificity of the designed assay was tested on 37 culture collection strains of X. fragariae, 82 strains of other Xanthomonas species and pathovars and 11 strains of other bacterial genera isolated from strawberry leaves. A detection limit of 10^2 fg was achieved, approximating 20 genome copies per reaction. When performing analyses with crude plant material, a consistent lower detection efficiency of 10^2 CFU ml−1 was achieved. The LAMP assay designed in this study was adapted to work on crude plant material without any prior extensive extraction steps or incubation period. Moreover, it does not require advanced analytical knowledge or a fully equipped laboratory. Results were produced within 7 to 20 min depending on the pathogen concentration, thus providing a high throughput and user-friendly method for detection and screening of plant material in support of quarantine regulations