Integron variability in Xanthomonas arboricola pv. juglandis and Xanthomonas arboricola pv. pruni strains

The integron platform and the gene cassette arrays of 34 Xanthomonas arboricola pv. juglandis and of 47 Xanthomonas arboricola pv. pruni strains isolated from different geographical areas were screened to check their variability. Genetic variability of the strains was also tested by means of BOX-PCR. For two representative strains of the two pathovars, the integrase gene intI and part of the flanking gene ilvD were also cloned and sequenced. Whereas X. a. pv. pruni strains did not show relevant variability, six X. a. pv. juglandis strains isolated in Australia showed some differences in the gene sequences. The CLUSTALW algorithm indicated that the majority of the X. a. pv. juglandis strains are closely related to X. a. pv. pruni, whereas the X. a. pv. juglandis strains isolated in Australia were more similar to Xanthomonas hortorum pv. pelargonii. Similarly, the gene cassette array pattern of the Australian strains, as well as that of the oldest strain maintained in culture, was different from the other strains. Also, three X. a. pv. pruni strains showed a different cassette array pattern when compared with the majority of other strains but no relationships with geographical area of isolation or host plant was revealed. This study confirmed that in addition to species, integrons may generate diversity also within two X. arboricola pathovars.     

Mapping quantitative trait loci associated with resistance to bacterial spot (Xanthomonas arboricola pv. pruni) in peach

Bacterial spot, caused by Xanthomonas arboricola pv. pruni (Xap), is a serious disease that can affect peach fruit quality and production worldwide. This disease causes severe defoliation and blemishing of fruit, particularly in areas with high rainfall, strong winds, high humidity, and sandy soil. The molecular basis of its tolerance and susceptibility in peach is yet to be understood. An F₂ population of 63 genotypes derived from a cross between peaches “O’Henry” (susceptible) and “Clayton” (resistant) has been used for linkage map construction and quantitative trait loci (QTL) mapping. Phenotypic data for leaf and fruit response to Xap infection were collected over 2 years at two locations. A high-density genetic linkage map that covers a genetic distance of 421.4 cM with an average spacing between markers of 1.6 cM was developed using the International Peach Single Nucleotide Polymorphism Consortium (IPSC) 9K array v1. Fourteen QTLs with an additive effect on Xap resistance were detected, including four major QTLs on linkage groups (LG) 1, 4, 5, and 6. Major QTLs, Xap.Pp.OC-4.1 and Xap.Pp.OC-4.2, on LG4 were associated with Xap resistance in leaf; Xap.Pp.OC-5.1 on LG5 was associated with Xap resistance in both leaf and fruit, while Xap.Pp.OC-1.2 and Xap.Pp.OC-6.1 on LG1 and LG6, respectively, were associated with Xap resistance in fruit. This suggested separate regulation of leaf and fruit resistance for Xap in peach as well as participation of genes involved in general plant response to biotic stress. The potential for marker-assisted selection for Xap resistance in peach is discussed.     

Development of an efficient real-time quantitative PCR protocol for detection of Xanthomonas arboricola pv. pruni in Prunus species

Xanthomonas arboricola pv. pruni, the causal agent of bacterial spot disease of stone fruit, is considered a quarantine organism by the European Union and the European and Mediterranean Plant Protection Organization (EPPO). The bacterium can undergo an epiphytic phase and/or be latent and can be transmitted by plant material, but currently, only visual inspections are used to certify plants as being X. arboricola pv. pruni free. A novel and highly sensitive real-time TaqMan PCR detection protocol was designed based on a sequence of a gene for a putative protein related to an ABC transporter ATP-binding system in X. arboricola pv. pruni. Pathogen detection can be completed within a few hours with a sensitivity of 10(2) CFU ml(-1), thus surpassing the sensitivity of the existing conventional PCR. Specificity was assessed for X. arboricola pv. pruni strains from different origins as well as for closely related Xanthomonas species, non-Xanthomonas species, saprophytic bacteria, and healthy Prunus samples. The efficiency of the developed protocol was evaluated with field samples of 14 Prunus species and rootstocks. For symptomatic leaf samples, the protocol was very efficient even when washed tissues of the leaves were directly amplified without any previous DNA extraction. For samples of 117 asymptomatic leaves and 285 buds, the protocol was more efficient after a simple DNA extraction, and X. arboricola pv. pruni was detected in 9.4% and 9.1% of the 402 samples analyzed, respectively, demonstrating its frequent epiphytic or endophytic phase. This newly developed real-time PCR protocol can be used as a quantitative assay, offers a reliable and sensitive test for X. arboricola pv. pruni, and is suitable as a screening test for symptomatic as well as asymptomatic plant material.     

Novel Rosaceae plant elicitor peptides as sustainable tools to control Xanthomonas arboricola pv. pruni in Prunus spp.

Fruit crops are regarded as important health promoters and constitute a major part of global agricultural production, and Rosaceae species are of high economic impact. Their culture is threatened by bacterial diseases, whose control is based on preventative treatments using compounds of limited efficacy and negative environmental impact. One of the most economically relevant examples is the pathogen Xanthomonas arboricola pv. pruni (Xap) affecting Prunus spp. The plant immune response against pathogens can be triggered and amplified by plant elicitor peptides (Peps), perceived by specific receptors (PEPRs). Although they have been described in various angiosperms, scarce information is available on Rosaceae species. Here, we identified the Pep precursor (PROPEP), Pep and PEPR orthologues of 10 Rosaceae species and confirmed the presence of the Pep/PEPR system in this family. We showed the perception and elicitor activity of Rosaceae Peps using the Prunus–Xap pathosystem as proof‐of‐concept. Treatment with nanomolar doses of Peps induced the corresponding PROPEP and a set of defence‐related genes in Prunus leaves, and enhanced resistance against Xap. Peps from the same species had the highest efficiencies. Rosaceae Peps could potentially be used to develop natural, targeted and environmentally friendly strategies to enhance the resistance of Prunus species against biotic attackers.     

Insights into the genome of the xanthan-producing phytopathogen Xanthomonas arboricola pv. pruni 109 by comparative genomic hybridization

The phytopathogenic bacterium Xanthomonas arboricola pv. pruni is the causal agent of Prunus Bacterial Spot disease that infects cultivated Prunus species and their hybrids. Furthermore, X. arboricola pv. pruni (Xap) plays a role in biotechnology since it produces xanthan gum, an important biopolymer used mainly in the food, oil, and cosmetics industry. To gain first insights into the genome composition of this pathovar, genomic DNA of X. arboricola pv. pruni strains was compared to the genomes of reference strains X. campestris pv. campestris B100 (Xcc B100) and X. campestris pv. vesicatoria 85-10 (Xcv 85-10) applying microarray-based comparative genomic hybridizations (CGH). The results implied that X. arboricola pv. pruni 109 lacks 6.67% and 5.21% of the genes present in the reference strains Xcc B100 and Xcv 85-10, respectively. Most of the missing genes were found to be organized in clusters and do not belong to the core genome of the two reference strains. Often they encode mobile genetic elements. Furthermore, the absence of gene clusters coding for the lipopolysaccharide (LPS) O-antigens of Xcc B100 and Xcv 85-10 indicates that the structure of the O-antigen of X. arboricola pv. pruni 109 differs from that of Xcc B100 and Xcv 85-10.     

A duplex-PCR method for species- and pathovar-level identification and detection of the quarantine plant pathogen Xanthomonas arboricola pv. pruni

A PCR-based method was developed for the stone fruit quarantine pathogen Xanthomonas arboricola pv. pruni (Xap), which provides rapid, sensitive and specific in planta detection and isolate identification. Primers specific for Xap were identified using random amplified polymorphic DNA (RAPD). Simplex PCR with these primers had a limit of detection per PCR reaction of approximately 10 CFU for isolate cultures and 50 CFU for plant material when used on tenfold dilutions of isolate culture or genomic DNA extracted from spiked samples, respectively. The primers were adapted as a high-throughput single-step screening based on a digoxigenin-labeled DNA probe assay with a detection limit of 4 × 10² CFU from isolate cultures. A duplex-PCR method was designed that includes the pathovar-level with species-level primers based on species-specific regions of the quinate metabolic gene qumA, increasing diagnostic confidence and offering the first molecular test for all X. arboricola pathovars.     

Detection of Xanthomonas arboricola pv. pruni by PCR using primers based on DNA sequences related to the hrp genes

Efficient control of Xanthomonas arboricola pv. pruni, the causal agent of bacterial spot on stone fruit, requires a sensitive and reliable diagnostic tool. A PCR detection method that utilizes primers to target the hrp gene cluster region was developed in this study. The nucleotide sequence of the PCR product amplified with primers specific for the hrp region of the xanthomonads and genomic DNA of X. arboricola pv. pruni was determined, and the sequence was aligned with that of X. campestris pv. campestris, which was obtained from the GenBank database. On the basis of the sequence of the amplified hrp region, a PCR primer set of XapF/R specific to X. arboricola pv. pruni was designed. This primer set yielded a 243-bp product from the genomic DNA of X. aboricola pv. pruni strains, but no products from other 21 strains of Xanthomonas or from two epiphytic bacterial species. Southern blot hybridization with the probe derived from the PCR product with the primer set and X. aboricola pv. pruni DNA confirmed the PCR results. The Xap primer system was successfully applied to detect the pathogen from infected peach fruits. When it was applied in field samples, the primer set was proved as a reliable diagnostic tool for specific detection of X. aboricola pv. pruni from peach orchards.     

The ubiquitous plasmid pXap41 in the invasive phytopathogen Xanthomonas arboricola pv. pruni: complete sequence and comparative genomic analysis

The complete DNA sequence of the 41 102-bp plasmid pXap41 from the invasive plant pathogen Xanthomonas arboricola pv. pruni CFBP 5530 was determined and its 44 coding regions were annotated. Comparative analysis with 15 Xanthomonas plasmids and 19 complete genomes revealed that nearly one-fourth of this plasmid has high sequence identity to plasmid pXAC64 and an 8.8-kb chromosomal region of Xanthomonas axonopodis pv. citri strain 306 carrying genes that encode type III effectors and helper proteins. The presence of pXap41 in all X. arboricola pv. pruni genotypes was confirmed for eight strains by plasmid profiling and for 35 X. arboricola pv. pruni isolates with a new plasmid multiplex PCR assay. This plasmid was not detected in any other X. arboricola pathovars (n=12), indicating the potential for the application of the pXap41 PCR method as a pathovar-level detection and identification tool.     

QTL analysis of field resistance to Xanthomonas axonopodis pv. manihotis in cassava

We evaluated cassava bacterial blight (CBB) infection in an pair-cross population of 150 individuals derived from an intra-specific cross between two non-inbred cassava (Manihot esculenta Crantz) lines. The replicated trials were carried out in the field under high disease pressure over two consecutive crop cycles. Evaluations were conducted at 4 and 7 months after planting for the two cycles. Simple regression analysis and the nonparametric Kruskal-Wallis rank-sum test revealed that eight quantitative trait loci (QTLs) were involved in resistance. We detected changes in QTLs from crop cycle to crop cycle. The pathogen population (Xanthomonas axonopodis pv. manihotis) was also monitored over the period, using a restriction fragment length polymorphism probe and pathogenic tests. Changes in QTL detection over the 2 years could be correlated with changes in pathogen population structure. One QTL, located in linkage group D, was conserved over the two crop cycles, and in field to greenhouse evaluations. This study thus identified molecular markers useful for marker assisted-selection, a technique that can accelerate the long, multiple-season process of breeding for CBB resistance. Received: 1 January 2000 / Accepted: 25 June 2000     

Immunolocalization and Histocytopathological Effects of Xanthomonas arboricola pv. pruni on Naturally Infected Leaf and Fruit Tissues of Peach (Prunus persica L. Batsch)

Immunofluorescence and cytohistochemical studies have been performed to understand the host–parasite relationships in the pathosystem: peach–Xanthomonas arboricola pv. pruni (Xap). Using a commercial immunodetection kit, Xap cells were specifically identified in tissues from infected leaves and fruits. Sections from infected leaves showed that the pathogen penetrates the mesophyll via stomata and develops in the intercellular spaces where it degrades the cell wall components. This leads to cell collapse and consequently to the formation of necrotic lesions. The same events have been noted in sections from infected fruits. However, the contaminated zones of mesocarp parenchyma exhibited cell dedifferentiation and generated somatic embryo‐like structures. Sections from midrib samples collected at different distances from infected lamina revealed the presence of Xap cells in the sieve tubes and xylem suggesting a systemic trafficking of the pathogen. The results are discussed in terms of cytological effects and epidemiology of Xap.     

Systemic Migration and Establishment of Xanthomonas campestris pv. pruni in Plum Leaves and Twigs

The systemic migration of Xanthomonas campestris pv. pruni (Xcp) through vascular bundles of leaves and twigs of plum was investigated. A rifampicin-resistant strain of Xcp was inoculated into leaves located midway from the tip of new green twigs of Golden King plum trees in a glasshouse. High numbers of the pathogen were recovered 4 and 8 weeks after inoculation from sections of uninoculated and symptomless veins, petioles, and twig tissue. Symptoms of bacterial spot developedwithin 8 weeks on main and secondary veins of uninoculated leaves located as far as 13 cm from the inoculated leaf on the same twig. Weekly isolation indicated the constant presence of Xcp in apparently unaffected shields of twig tissue obtained from a naturally infected Golden King orchard. Xcp apparently enters plum twigs through veins of infected leaves and migrates systemically through twigs to leaves.     

Systemic Invasion of Plum Seed and Fruit by Xanthomonas campestris pv. pruni through Stalks

Many fruits on Golden King plum trees inoculated through the stalks with Xanthomonas campestris pv. pruni developed unusual lesions extending from the exocarp to the endocarp. A few uninoculated, diseased fruits had similar lesions. The pathogen was isolated from both inoculated and uninoculated stalks and from seeds inside fruits. Scanning electron microscopy of inoculated stalks and mature fruits with unusual lesions revealed that vascular channels of the stalk, seed coat, stony endo, carp, and mesocarp were filled with masses of X. campestris pv. pruni. Bacterial colonies also occurred in other tissues of these fruit parts but were apparently absent from the starchy endosperm or surface of the diseased exocarp. This is the first full report of systemic movement of X. campestris pv. pruni to seed and fruit through stalks.     

Identification of a pathogenicity locus in Xanthomonas campestris pv. vesicatoria

Summary Mutants of a tomato strain ofXanthomonas campestris pv.vesicatoria (XCV), causal agent of bacterial spot of tomato and pepper, were produced using the transposon Tn5 carried in the suicide plasmid pGS9. One prototrophic mutant, M461, was isolated which caused no visible reaction on tomato or pepper, but maintained the wild-type ability to induce a hypersensitive reaction (HR) on tobacco. This mutant showed similar growth characteristics to the wild-type in culture, but growth in planta was reduced. A genomic library of wild-type XCV was constructed in the broad host range cosmid vector pLAFR3. Clone p6AD4 restored pathogenicity to M461 on tomato and the ability to induce a HR on pepper. This clone contained ca. 22 kb of XCV DNA. The insertion in M461 was in a site corresponding to a 1.1 kbEcoRI fragment of p6AD4.     

The influence of thermal treatment and operational conditions on xanthan produced by X. arboricola pv pruni strain 106

The influence of thermal treatment and operational conditions (pH and stirrer speed) used in the process of xanthan production by Xanthomonas arboricola pv pruni strain 106 were evaluated through yield of xanthan, aqueous solution and fermentation broth viscosity, sodium content, pyruvate and acetyl content and molar mass. Different conditions used during the fermentation affected the xanthan characteristics. Thermal treatment decreased the final yield and pyruvate and acetyl content, and increased the xanthan aqueous solution and fermentation broth viscosities, as well as molar mass. In this study the best combination of yield and viscosity was obtained with the use of pH 7 and 400 rpm during fermentation and post-fermentation thermal treatment. Aggregation of xanthan molecules promoted by heating and detected through an increase of molar mass was apparently affected by the sodium content. As a result, a correlation between molar mass and xanthan solution viscosity could be observed.     

Genetic Diversity of Xanthomonas arboricola pv. fragariae Strains and Comparison with some other X. arboricola Pathovars using Repetitive PCR Genomic Fingerprinting

The genetic relationship within 26 Xanthomonas arboricola pv. fragariae strains and between this pathovar and 20 strains of X. arboricola pv. corylina, 22 strains of X. arboricola pv. juglandis and 16 strains of X. arboricola pv. pruni has been assessed by means of repetitive polymerase chain reaction (rep‐PCR) using Enterobacterial Repetitive Intergenic Consensus), BOX (BOXA subunit of the BOX element of Streptococcus pneumoniae) and repetitive extragenic palindromic primer sets. Cluster analysis was performed by means of unweighted paired group method using arithmetic average (UPGMA). Upon rep‐PCR and UPGMA cluster analysis, a relevant genetic diversity was found within the strains. The overall similarity, however, was high (i.e. 80%). The four X. arboricola pathovars showed similar but clearly different genomic patterns and clustered into four different groups, with X. arboricola pv. corylina and X. arboricola pv. juglandis more closely related to X. arboricola pv. fragariae. Representative strains of X. arboricola pv. fragariae and the putative xanthomonads isolated from strawberry leaves showing leaf blight symptoms underwent pathogenicity tests. After artificial inoculation, X. arboricola pv. fragariae induced necrotic spots accompanied, sometimes, by a chlorotic halo. The blackening of the leaf veins and peduncle was, sometimes, also observed. The four putative xanthomonads isolated from diseased strawberry leaves and not inducing symptoms after artificial inoculation, clustered apart from X. arboricola pathovars.     

A "defeated" rice resistance gene acts as a QTL against a virulent strain of Xanthomonas oryzae pv. oryzae

The genetic components responsible for qualitative and quantitative resistance of rice plants to three strains (CR4, CXO8, and CR6) of Xanthomonas oryzae pv. oryzae (Xoo) were investigated using a set of 315 recombinant inbred lines (RILs) from the cross Lemont (japonica) × Teqing (indica) and a complete linkage map with 182 well distributed RFLP markers. We mapped a major gene (Xa4) and ten quantitative trait loci (QTLs) which were largely responsible for segregation of the resistance phenotype in the RILs. The Teqing allele at the Xa4 locus, Xa4 ^T , acted as a dominant resistance gene against CR4 and CXO8. The breakdown of Xa4 ^T -associated resistance mediated by the mutant allele at the avrXa4 locus in the virulent strain CR6 results from significant changes in both gene action (lose of dominance) and the magnitude of gene effect (≈50% reduction). Nevertheless, Xa4 ^T still acted as a recessive QTL with a significant residual effect against CR6. The mutant alleles at the avrXa4 locus in CXO8 and CR6 that lead to a reduction in effect, or “breakdown”, of Xa4 ^T were apparently accompanied by corresponding penalties for their fitness. The quantitative component of resistance to Xoo in the RILs was largely due to a number of resistance QTLs. Most resistance QTLs mapped to genomic locations where major resistance genes and/or QTLs for resistance to Xoo, blast and sheath blight were identified in the same cross. Most QTLs showed consistent levels of resistance against all three Xoo strains. Our results suggest that a high level of durable resistance to Xoo may be achieved by the cumulative effects of multiple QTLs, including the residual effects of “defeated” major resistance genes. Received: 28 April 1998 / Accepted: 9 October 1998     

A “defeated” rice resistance gene acts as a QTL against a virulent strain of Xanthomonas oryzae pv. oryzae

The genetic components responsible for qualitative and quantitative resistance of rice plants to three strains (CR4, CXO8, and CR6) of Xanthomonas oryzae pv. oryzae (Xoo) were investigated using a set of 315 recombinant inbred lines (RILs) from the cross Lemont (japonica) × Teqing (indica) and a complete linkage map with 182 well distributed RFLP markers. We mapped a major gene (Xa4) and ten quantitative trait loci (QTLs) which were largely responsible for segregation of the resistance phenotype in the RILs. The Teqing allele at the Xa4 locus, Xa4 ^T , acted as a dominant resistance gene against CR4 and CXO8. The breakdown of Xa4 ^T -associated resistance mediated by the mutant allele at the avrXa4 locus in the virulent strain CR6 results from significant changes in both gene action (lose of dominance) and the magnitude of gene effect (≈50% reduction). Nevertheless, Xa4 ^T still acted as a recessive QTL with a significant residual effect against CR6. The mutant alleles at the avrXa4 locus in CXO8 and CR6 that lead to a reduction in effect, or “breakdown”, of Xa4 ^T were apparently accompanied by corresponding penalties for their fitness. The quantitative component of resistance to Xoo in the RILs was largely due to a number of resistance QTLs. Most resistance QTLs mapped to genomic locations where major resistance genes and/or QTLs for resistance to Xoo, blast and sheath blight were identified in the same cross. Most QTLs showed consistent levels of resistance against all three Xoo strains. Our results suggest that a high level of durable resistance to Xoo may be achieved by the cumulative effects of multiple QTLs, including the residual effects of “defeated” major resistance genes.