Xanthomonas campestris pv. campestris (cause of black rot of crucifers) in the genomic era is still a worldwide threat to brassica crops

Background Xanthomonas campestris pv. campestris (Xcc) (Pammel) Dowson is a Gram-negative bacterium that causes black rot, the most important disease of vegetable brassica crops worldwide. Intensive molecular investigation of Xcc is gaining momentum and several whole genome sequences are available. Taxonomy Bacteria; Phylum Proteobacteria; Class Gammaproteobacteria; Order Xanthomonadales; Family Xanthomonadacea; Genus Xanthomonas; Species X. campestris. Host range and symptoms Xcc can cause disease in a large number of species of Brassicaceae (ex-Cruciferae), including economically important vegetable Brassica crops and a number of other cruciferous crops, ornamentals and weeds, including the model plant Arabidopsis thaliana. Black rot is a systemic vascular disease. Typical disease symptoms include V-shaped yellow lesions starting from the leaf margins and blackening of the veins. Race structure, pathogenesis and epidemiology Collections of Xcc isolates have been differentiated into physiological races based on the response of several brassica species lines. Black rot is a seed-borne disease. The disease is favoured by warm, humid conditions and can spread rapidly from rain dispersal and irrigation water. Disease control The control of black rot is difficult and relies on the use of pathogen-free planting material and the elimination of other potential inoculum sources (infected crop debris and cruciferous weeds). Major gene resistance is very rare in B. oleracea (brassica C genome). Resistance is more readily available in other species, including potentially useful sources of broad-spectrum resistance in B. rapa and B. carinata (A and BC genomes, respectively) and in the wild relative A. thaliana. Genome The reference genomes of three isolates have been released. The genome consists of a single chromosome of approximately 5 100 000 bp, with a GC content of approximately 65% and an average predicted number of coding DNA sequences (CDS) of 4308. Important genes identified Three different secretion systems have been identified and studied in Xcc. The gene clusters xps and xcs encode a type II secretion system and xps genes have been linked to pathogenicity. The role of the type IV secretion system in pathogenicity is still uncertain. The hrp gene cluster encodes a type III secretion system that is associated with pathogenicity. An inventory of candidate effector genes has been assembled based on homology with known effectors. A range of other genes have been associated with virulence and pathogenicity, including the rpf, gum and wxc genes involved in the regulation of the synthesis of extracellular degrading enzymes, xanthan gum and lipopolysaccharides

Xanthomonas campestris pv. campestris race 1 is the main causal agent of black rot of Brassicas in Southern Mozambique

Severe outbreaks of bacterial black rot caused by Xanthomonas campestris pv. campestris (Xcc) were observed in Brassica production fields of Southern Mozambique. The causal agent of the disease in the Mahotas and Chòkwé districts was identified and characterised. In total, 83 Xanthomonas-like strains were isolated from seed samples and leaves of cabbage and tronchuda cole with typical symptoms of the disease. Forty-six out of the 83 strains were found to be putative Xcc in at least one of the tests used: Classical biochemical assays, enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies, Biolog identification system, polymerase chain reaction (PCR) with specific primers and pathogenicity tests. The ELISA tests were positive for 43 strains. Biolog identified 43 strains as Xanthomonas, but only 32 as Xcc. PCR tests with primers targeting a fragment of the hrpF gene were positive for all 46 strains tested. Three strains were not pathogenic or weakly pathogenic and all other strains caused typical black rot symptoms in brassicas. Race type differentiation tests revealed the Xcc strains from Mozambique as members of race 1. The prevalence of this pathogenic race of the Xcc pathogen in Mozambique should be considered when black rot resistant cultivars are evaluated or introduced into the production regions of this country

Production of embryos from microspore cultures of Portuguese tronchuda cabbage landraces

Twelve accessions of tronchuda cabbage landraces were tested for their ability to produce embryos through microspore culture in NLN-13 medium. A sample from all the isolations was stained with DAPI for determination of microspore developmental stage. The relationship between the microspore developmental stage and the production of microspore-derived embryos was evaluated. Embryos were obtained from all the accessions. Considerable variation was observed between isolations with different developmental stage of microspores, accessions and plants within the same accession. The best embryogenic responses occurred in microspore populations with approximately 10 to 89% of binucleate pollen. The highest embryo yield was obtained with one of the ‘Couve de Valhascos’ accessions (0.41 embryos / 1 000 microspores) and the lowest yields with the accessions of ‘Couve Portuguesa’ (less than 0.07 embryos / 1 000 microspores)

First occurrence of Xanthomonas campestris pv. raphanion wallflower (Erysimum cheiri) in the United Kingdom

In a study of diseases caused by Xanthomonas campestris on UK nursery-grown wallflowers (Erysimum cheiri), one isolate (P764) obtained in 1990 from Sussex differed from the others. The affected plants (cv. Bedder Mixed), showed distorted growth with chlorotic and necrotic spots, but no sectored V-shaped wilting, the most common symptom associated with xanthomonads in wallflowers. Other X. campestris isolates (including P763), were obtained from wallflowers with typical wilting. Infected plant material was comminuted in sterile 0.1% peptone solution and loopfuls streaked onto yeast dextrose chalk agar and nutrient dextrose agar (Lelliott & Stead, 1987). Plates were incubated for up to 72 hours at 28°C. Xanthomonas-like colonies were purified by re-streaking and isolates were maintained at -80°C (Protect System, UK)

Occurrence and diversity of Xanthomonas campestris pv. campestris in vegetable brassica fields in Nepal

Black rot caused by Xanthomonas campestris pv. campestris was found in 28 sampled cabbage fields in five major cabbage-growing districts in Nepal in 2001 and in four cauliflower fields in two districts and a leaf mustard seed bed in 2003. Pathogenic X. campestris pv. campestris strains were obtained from 39 cabbage plants, 4 cauliflower plants, and 1 leaf mustard plant with typical lesions. Repetitive DNA polymerase chain reaction-based fingerprinting (rep-PCR) using repetitive extragenic palindromic, enterobacterial repetitive intergenic consensus, and BOX primers was used to assess the genetic diversity. Strains were also race typed using a differential series of Brassica spp. Cabbage strains belonged to five races (races 1, 4, 5, 6, and 7), with races 4, 1, and 6 the most common. All cauliflower strains were race 4 and the leaf mustard strain was race 6. A dendrogram derived from the combined rep-PCR profiles showed that the Nepalese X. campestris pv. campestris strains clustered separately from other Xanthomonas spp. and pathovars. Race 1 strains clustered together and strains of races 4, 5, and 6 were each split into at least two clusters. The presence of different races and the genetic variability of the pathogen should be considered when resistant cultivars are bred and introduced into regions in Nepal to control black rot of brassicas

Chitosan coating functionalized with flaxseed oil and green tea extract as a bio-based solution for beef preservation

Ecological and safe packaging solutions arise as pivotal points in the development of an integrated system for sustainable meat production. The aim of this study was to assess the effect of a combined chitosan (Ch) + green tea extract (GTE) + essential oil (thyme oil, TO; flaxseed oil, FO; or oregano oil, OO) coating on the safety and quality of vacuum-packaged beef during storage at 4 °C. An optimized bio-based coating formulation was selected (2% Ch + 2% GTE + 0.1% FO) to be applied to three fresh beef cuts (shoulder, Sh; knuckle, Kn; Striploin, St) based on its pH (5.8 ± 0.1), contact angle (22.3 ± 0.4°) and rheological parameters (viscosity=0.05 Pa.s at shear rate > 20 s−1). Shelf-life analysis showed that the Ch–GTE–FO coating delayed lipid oxidation and reduced total viable counts (TVC) and Enterobacteriaceae growth compared with uncoated beef samples over five days. In addition, Ch–GTE–FO coating decreased total color changes of beef samples (e.g., ∆E*=9.84 and 3.94, for non-coated and coated Kn samples, respectively) for up to five days. The original textural parameters (hardness, adhesiveness and springiness) of beef cuts were maintained during storage when Ch–GTE–FO coating was applied. Based on the physicochemical and microbial characterization results, the combination of the Ch–GTE–FO coating developed was effective in preserving the quality of fresh beef cuts during refrigerated storage along with vacuum packaging.This research was funded by ETHICAL MEAT—Integrated System for Sustainable Meat Production project (POCI-01-0247-FEDER-072254), co-financed by the European Regional Development Fund (ERDF) through Compete 2020–Competitiveness and Internationalisation Operational Programme (POCI). This work was also financially supported by FCT under the scope of the strategic funding of UIDB/04469/2020 unit, and by LABBELS—Associate Laboratory in Biotechnology, Bioengineering and Microelectromechanical Systems, LA/P/0029/2020.info:eu-repo/semantics/publishedVersio

Pathotypic diversity of Hyaloperonospora brassicae collected from Brassica oleracea

Downy mildew caused by Hyaloperonospora brassicae is an economically destructive disease of brassica crops in many growing regions throughout the world. Specialised pathogenicity of downy mildews from different Brassica species and closely related ornamental or wild relatives has been described from host range studies. Pathotypic variation amongst Hyaloperonospora brassicae isolates from Brassica oleracea has also been described; however, a standard set of B. oleracea lines that could enable reproducible classification of H. brassicae pathotypes was poorly developed. For this purpose, we examined the use of eight genetically refined host lines derived from our previous collaborative work on downy mildew resistance as a differential set to characterise pathotypes in the European population of H. brassicae. Interaction phenotypes for each combination of isolate and host line were assessed following drop inoculation of cotyledons and a spectrum of seven phenotypes was observed based on the level of sporulation on cotyledons and visible host responses. Two host lines were resistant or moderately resistant to the entire collection of isolates, and another was universally susceptible. Five lines showed differential responses to the H. brassicae isolates. A minimum of six pathotypes and five major effect resistance genes are proposed to explain all of the observed interaction phenotypes. The B. oleracea lines from this study can be useful for monitoring pathotype frequencies in H. brassicae populations in the same or other vegetable growing regions, and to assess the potential durability of disease control from different combinations of the predicted downy mildew resistance genes

Edible films based on black chia (Salvia hispanica l.) seed mucilage containing Rhus microphylla fruit phenolic extract

Functional films based on black chia (Salvia hispanica L.) seed mucilage (BCm) containing Rhus microphylla (Rm) fruit phenolic extract were built and characterized. A hydro-alcohol extract (HAE) of Rm was incorporated as the bioactive agent due to its noteworthy phenolic profile, and good antioxidant and antifungal activities. The effects of the BCm concentration (0.2% and 0.4%, w/v), HAE incorporation, and their interaction with glycerol (1.0%, w/v) and calcium chloride (0.05%, w/v) on the films physicochemical properties were evaluated. The filmogenic solutions successfully fitted to the HerschelBulkley model (R2 0.05) changed by the HAE addition, but their surface tension was enhanced (p < 0.05), which could favor their coating ability. The polyanionic nature of the systems (zeta potential-Zp values from 26.9 to 33.3 mV) allowed them to interact with Ca2+ cations, thus forming stable and resistant films. All the films showed low water solubility (15.0% to 22.3%) and high permeability (3.7 to 4.0 × 1010 g m1 s1 Pa1), as well as high biodegradability (moisture content from 66.0% to 80.9%); although the moisture content was reduced (p < 0.05) with HAE addition. The combination of higher BCm ratio and HAE addition (BCm0.4+Rm) led to a more resistant, thick, opaque, and dark film compared with the others obtained. This study reveals the BCm-based films potential, highlighting those with HAE, representing a novel alternative to improve the quality of food products.Financial support from Universidad Autónoma Agraria Antonio Narro (UAAAN) is gratefully acknowledged by the authors. Zlatina Genisheva thanks to Foundation for Science and Technology (FCT) for the financial support (ref. SFRH/BPD/108868/2015) and to the project COMPETE 2020 (POCI-01-0145-FEDER-029145). This study was supported by FCT under the scope of the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. The authors would also like to thank to Pablo Virgen of Biocampo S.A. de C.V. and MSc Fidel Peña-Ramos from UAAAN, for their assistance during this study.info:eu-repo/semantics/publishedVersio

A DNA-barcode biodiversity standard analysis method (DNA-BSAM) reveals a large variance in the effect of a range of biological, chemical and physical soil management interventions at different sites, but location is one of the most important aspects determining the nature of agricultural soil microbiology

There are significant knowledge gaps in our understanding of how to sustainably manage agricultural soils to preserve soil biodiversity. Here we evaluate and quantify the effects of agricultural management and location on soil microbiology using nine field trials that have consistently applied different soil management practices in the United Kingdom using DNA barcode sequence data. We tested the basic hypothesis that various agricultural management interventions have a significant and greater effect on soil bacterial and fungal diversity than geographic location. The analyses of soil microbial DNA sequence data to date has lacked standardisation which prevents meaningful comparisons across sites and studies. Therefore, to analyse these data and crucially compare and quantify the size of any effects on soil bacterial and fungal biodiversity between sites, we developed and employed a post-sequencing DNA-barcode biodiversity standard analysis method (DNA-BSAM). The DNA-BSAM comprises a series of standardised bioinformatic steps for processing sequences but more importantly defines a standardised set of ecological indices and statistical tests. Use of the DNA-BSAM reveals the hypothesis was not strongly supported, and this was primarily because: 1) there was a large variance in the effects of various management interventions at different sites, and 2) that location had an equivalent or greater effect size than most management interventions for most metrics. Some dispersed sites imposed the same organic amendments interventions but showed different responses, and this combined with observations of strong differences in soil microbiomes by location tentatively suggests that any effect of management may be contingent on location. This means it could be unreliable to extrapolate the findings of individual trials to others. The widespread use of a standard approach will allow meaningful cross-comparisons between soil microbiome studies and thus a substantial evidence-base of the effects of land-use on soil microbiology to accumulate and inform soil management decisions.Agriculture and Horticulture Development Board (AHDB); British Beet Research Organisation (BBRO

Rabbit derived VL single-domains as promising scaffolds to generate antibody–drug conjugates

© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.Antibody-drug conjugates (ADCs) are among the fastest-growing classes of therapeutics in oncology. Although ADCs are in the spotlight, they still present significant engineering challenges. Therefore, there is an urgent need to develop more stable and effective ADCs. Most rabbit light chains have an extra disulfide bridge, that links the variable and constant domains, between Cys80 and Cys171, which is not found in the human or mouse. Thus, to develop a new generation of ADCs, we explored the potential of rabbit-derived VL-single-domain antibody scaffolds (sdAbs) to selectively conjugate a payload to Cys80. Hence, a rabbit sdAb library directed towards canine non-Hodgkin lymphoma (cNHL) was subjected to in vitro and in vivo phage display. This allowed the identification of several highly specific VL-sdAbs, including C5, which specifically target cNHL cells in vitro and present promising in vivo tumor uptake. C5 was selected for SN-38 site-selective payload conjugation through its exposed free Cys80 to generate a stable and homogenous C5-DAB-SN-38. C5-DAB-SN-38 exhibited potent cytotoxicity activity against cNHL cells while inhibiting DNA-TopoI activity. Overall, our strategy validates a platform to develop a novel class of ADCs that combines the benefits of rabbit VL-sdAb scaffolds and the canine lymphoma model as a powerful framework for clinically translation of novel therapeutics for cancer.This work was supported by the Portuguese Funding Agency, Fundação para a Ciência e Tecnologia, FCT IP (SAICT/2017/32085, PTDC/QUI-OUT/3989/2021 and Ph.D. fellowship SFRH/BD/131468/2017 to ASA and SFRH/BD/90514/2012 to JD). CIISA has provided support through Project UIDB/00276/2020, funded by FCT and LA/P/0059/2020-AL4AnimalS. Research Institute for Medicines (iMed.ULisboa) acknowledges the financial support of Fundação para a Ciência e Tecnologia (Projects: PTDC/QUI-OUT/3989/2021; UIDB/04138/2020 and UIDP/04138/2020). The NMR spectrometers are part of the National NMR Network (PTNMR) and are partially supported by Infrastructure Project Nº 022161 (co-financed by FEDER through COMPETE 2020, POCI and PORL and FCT through PIDDAC).info:eu-repo/semantics/publishedVersio