Joana G. Vicente

Identification and Origin of Xanthomonas campestris pv. campestris Races and Related Pathovars.

ABSTRACT One hundred sixty-four isolates of Xanthomonas campestris pv. campestris and other X. campestris pathovars known to infect cruciferous hosts (X. campestris pvs. aberrans, raphani, armoraciae, and incanae) were inoculated onto a differential series of Brassica spp. to determine both pathogenicity to brassicas and race. Of these, 144 isolates were identified as X. campestris pv. campestris and grouped into six races, with races 1 (62%) and 4 (32%) being predominant. Other races were rare. The remaining 20 isolates from brassicas and other cruciferous hosts were either nonpathogenic or very weakly pathogenic on the differential series and could not be race-typed. Five of these isolates, from the ornamental crucifers wallflower (Cheiranthus cheiri), stock (Matthiola incana) and candytuft (Iberis sp.), showed clear evidence of pathovar-like specificity to the hosts of origin. A gene-for-gene model based on the interaction of four avirulence genes in X. campestris pv. campestris races and four matching resistance genes in the differential hosts is proposed. Knowledge of the race structure and worldwide distribution of races is fundamental to the search for sources of resistance and for the establishment of successful resistance breeding programs.

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. USEFUL WEBSITE http://www.xanthomonas.org/

Sources and Origin of Resistance to Xanthomonas campestris pv. campestris in Brassica Genomes

ABSTRACT Two hundred and seventy-six accessions of mainly Brassica spp. were screened for resistance to Xanthomonas campestris pv. campestris races. In Brassica oleracea (C genome), the majority of accessions were susceptible to all races, but 43% showed resistance to one or more of the rare races (2, 3, 5, and 6) and a single accession showed partial resistance to races 1, 3, 5, and 6. Further searches for resistance to races 1 and 4, currently the most important races worldwide, and race 6, the race with the widest host range, were made in accessions representing the A and B genomes. Strong resistance to race 4 was frequent in B. rapa (A genome) and B. napus (AC genome), indicating an A genome origin. Resistance to races 1 and 4 was present in a high proportion of B. nigra (B genome) and B. carinata (BC genome) accessions, indicating a B genome origin. B. juncea (AB genome) was the most resistant species, showing either strong resistance to races 1 and 4 or quantitative resistance to all races. Potentially race-nonspecific resistance was also found, but at a lower frequency, in B. rapa, B. nigra, and B. carinata. The combination of race-specific and race-nonspecific resistance could provide durable control of black rot of crucifers.

Discrimination of Pseudomonas syringae isolates from sweet and wild cherry using rep-PCR

Bacterial canker is one of the most important diseases of cherry (Prunus avium). This disease can be caused by two pathovars of Pseudomonas syringae: pv. morsprunorum and pv. syringae. Repetitive DNA polymerase chain reaction-based fingerprinting (rep-PCR) was investigated as a method to distinguish pathovars, races and isolates of P. syringae from sweet and wild cherry. After amplification of total genomic DNA from 87 isolates using the REP (repetitive extragenic palindromic), ERIC (enterobacterial repetitive intergenic consensus) and BOX primers, followed by agarose gel electrophoresis, groups of isolates showed specific patterns of PCR products. Pseudomonas syringae pv. syringae isolates were highly variable. The differences amongst the fingerprints of P. syringae pv. morsprunorum race 1 isolates were small. The patterns of P. syringae pv. morsprunorum race 2 isolates were also very uniform, with one exception, and distinct from the race 1 isolates. rep-PCR is a rapid and simple method to identify isolates of the two races of P. syringae pv. morsprunorum; this method can also assist in the identification of P. syringae pv. syringae isolates, although it cannot replace inoculation on susceptible hosts such as cherry and lilac.

Inheritance of race-specific resistance to Xanthomonas campestris pv. campestris in Brassica genomes

ABSTRACT The inheritance of resistance to three Xanthomonas campestris pv. campestris races was studied in crosses between resistant and susceptible lines of Brassica oleracea (C genome), B. carinata (BC genome), and B. napus (AC genome). Resistance to race 3 in the B. oleracea doubled haploid line BOH 85c and in PI 436606 was controlled by a single dominant locus (Xca3). Resistance to races 1 and 3 in the B. oleracea line Badger Inbred-16 was quantitative and recessive. Strong resistance to races 1 and 4 was controlled by a single dominant locus (Xca1) in the B. carinata line PI 199947. This resistance probably originates from the B genome. Resistance to race 4 in three B. napus lines, cv. Cobra, the rapid cycling line CrGC5, and the doubled haploid line N-o-1, was controlled by a single dominant locus (Xca4). A set of doubled haploid lines, selected from a population used previously to develop a restriction fragment length polymorphism map, was used to map this locus. Xca4 was positioned on linkage group N5 of the B. napus A genome, indicating that this resistance originated from B. rapa. Xca4 is the first major locus to be mapped that controls race-specific resistance to X. campestris pv. campestris in Brassica spp.

The Molecular Basis of Host Specialization in Bean Pathovars of Pseudomonas syringae

Biotrophic phytopathogens are typically limited to their adapted host range. In recent decades, investigations have teased apart the general molecular basis of intraspecific variation for innate immunity of plants, typically involving receptor proteins that enable perception of pathogen-associated molecular patterns or avirulence elicitors from the pathogen as triggers for defense induction. However, general consensus concerning evolutionary and molecular factors that alter host range across closely related phytopathogen isolates has been more elusive. Here, through genome comparisons and genetic manipulations, we investigate the underlying mechanisms that structure host range across closely related strains of Pseudomonas syringae isolated from different legume hosts. Although type III secretion-independent virulence factors are conserved across these three strains, we find that the presence of two genes encoding type III effectors (hopC1 and hopM1) and the absence of another (avrB2) potentially contribute to host range differences between pathovars glycinea and phaseolicola. These findings reinforce the idea that a complex genetic basis underlies host range evolution in plant pathogens. This complexity is present even in host-microbe interactions featuring relatively little divergence among both hosts and their adapted pathogens.

Identification and Discrimination of Pseudomonas syringae Isolates from Wild Cherry in England

A survey of wild cherry (Prunus avium) woodland plantations and nurseries was carried out in 2000/01. Trees with symptoms of bacterial canker were found in 20 of the 24 plantations visited and in three of seven nurseries. Fifty-four Pseudomonas syringae isolates from wild cherry together with 22 representative isolates from sweet cherry and 13 isolates from other Prunus spp., pear and lilac were characterised by physiological, biochemical, serological and pathogenicity tests. Isolates from wild cherry were predominantly P. syringae pv. syringae (Pss), but P. syringae pv. morsprunorum (Psm) races 1 and 2 were also found. Physiological and biochemical tests discriminated Psm races 1 and 2 from other P. syringae isolates. Agglutination and indirect-enzyme-linked immunosorbent assay tests with three different antisera showed that Psm race 1 and race 2 were very uniform and indicated high variability amongst other P. syringae isolates. However, pathogenic Pss isolates could not be distinguished from non-pathogenic isolates of P. syringae on the basis of physiological, biochemical or serological tests. Pathogenicity tests on rooted lilac plants and on micropropagated plantlets of lilac and two wild cherry clones differentiated Pss and Psm isolates and demonstrated a range of aggressiveness amongst Pss isolates. Serological tests could be used as an alternative to the classical physiological and biochemical tests to increase the speed of detection and discrimination of isolates, but pathogenicity tests are still necessary to discriminate the pathogenic Pss isolates.

Identification of Isolates that Cause a Leaf Spot Disease of Brassicas as Xanthomonas campestris pv. raphani and Pathogenic and Genetic Comparison with Related Pathovars

ABSTRACT Twenty-five Xanthomonas isolates, including some isolates received as either X. campestris pv. armoraciae or pv. raphani, caused discrete leaf spot symptoms when spray-inoculated onto at least one Brassica oleracea cultivar. Twelve of these isolates and four other Xanthomonas isolates were spray- and pin-inoculated onto 21 different plant species/cultivars including horseradish (Armoracia rusticana), radish (Raphanus sativus), and tomato (Lycopersicon esculentum). The remaining 13 leaf spot isolates were spray-inoculated onto a subset of 10 plant species/cultivars. The leaf spot isolates were very aggressive on several Brassica spp., radish, and tomato causing leaf spots and dark sunken lesions on the middle vein, petiole, and stem. Based on the differential reactions of several Brassica spp. and radish cultivars, the leaf spot isolates were divided into three races, with races 1 and 3 predominating. A differential series was established to determine the race-type of isolates and a gene-for-gene model based on the interaction of two avirulence genes in the pathogen races and two matching resistance genes in the differential hosts is proposed. Repetitive-DNA polymerase chain reaction-based fingerprinting was used to assess the genetic diversity of the leaf spot isolates and isolates of closely related Xanthomonas pathovars. Although there was variability within each race, the leaf spot isolates were clustered separately from the X. campestris pv. campestris isolates. We propose that X. campestris isolates that cause a nonvascular leaf spot disease on Brassica spp. should be identified as pv. raphani and not pv. armoraciae. Race-type strains and a neopathotype strain for X. campestris pv. raphani are proposed.

Characterisation of disease resistance gene-like sequences in Brassica oleracea L.

Abstract Several cloned disease resistance genes from a wide range of plant species are known to share conserved regions with similar structural motifs. Degenerate primers based on conserved sequences of the nucleotide binding site of the genes RPS2, N and L6 were used for polymerase chain reaction (PCR) amplification from genomic DNA of two doubled haploid lines of Brassica oleracea. Sequences of amplified products were highly variable, but most of them showed similarity to known disease resistance genes, including RPS5, RPS2 and N, and to disease resistance gene-like sequences (RGLs) from different species. Primers based on B. oleracea sequences amplified five groups of RGLs. Products were mapped through cleaved amplified polymorphic sequence assays onto four different linkage groups of B. oleracea. PCR amplification from cDNA and allele analysis indicated that four locus-specific RGL fragments are expressed in cauliflower. Screening of a B. oleracea bacterial artificial chromosome library (BAC) with four B. oleracea RGL probes identified a small number of clones, suggesting that the four RGLs may not be highly copied. Screening of a BAC library of A. thaliana with the same probes identified clones that mapped onto four different chromosomes. These map positions correspond to known disease resistance loci of A. thaliana.

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.