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.     

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.     

Triterpenoid saponins from Schefflera arboricola

Nine triterpenoid saponins were isolated from the leaves and stems of Schefflera arboricola. The saponins were characterised, on the basis of chemical and spectral evidence, as 3-O-[alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucuronopyranosyl] oleanolic acid, 3-O-[alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucuronopyranosyl] echinocystic acid, 3-O-[beta-D-apiofuranosyl-(1-->4)-beta-D-glucuronopyranosyl] oleanolic acid 28-O-beta-D-glucopyranosyl ester, 3-O-alpha-L-ramnopyranosyl-(1-->4)-[alpha-L-arabinopyranosyl-(1-->2)-] beta-D-glucuronopyranosyl oleanolic acid, 3-O-alpha-L-rhamnopyranosyl-(1-->4)-[alpha-L-arabinopyranosyl-(1-->2)-] beta-D-glucuronopyranosyl oleanolic acid 28-O-beta-D-glucopyranosyl ester, 3-O-alpha-L-rhamnopyranosyl-(1-->4)-[beta-D-galactopyranosyl-(1-->2)-] beta-D-glucuronopyranosyl oleanolic acid, 3-O-alpha-L-rhamnopyranosyl-(1-->4)-[beta-D-galactopyranosyl-(1-->2)-] beta-D-glucuronopyranosyl oleanolic acid 28-O-beta-D-glucopyranosyl ester, 3-O-beta-D-apiofuranosyl-(1-->4)-[alpha-L-arabinopyranosyl-(1-->2)-] beta-D-glucuronopyranosyl oleanolic acid and 3-O-beta-D-apiofuranosyl-(1-->4)-[alpha-L-arabinopyranosyl-(1-->2)-] beta-D-glucuronopyranosyl oleanolic acid 28-O-beta-D-glucopyranosyl ester.     

Identification of a major QTL for Xanthomonas arboricola pv. pruni resistance in apricot

Xanthomonas arboricola pv. pruni causes bacterial spot of stone fruit resulting in severe yield losses in apricot production systems. Present on all continents, the pathogen is regulated in Europe as a quarantine organism. Host resistance is an important component of integrated pest management; however, little work has been done describing resistance against X. arboricola pv. pruni. In this study, an apricot population derived from the cross “Harostar” × “Rouge de Mauves” was used to construct two parental genetic maps and to perform a quantitative trait locus analysis of resistance to X. arboricola pv. pruni. A population of 101 F1 individuals was inoculated twice for two consecutive years in a quarantine greenhouse with a mixture of bacterial strains, and disease incidence and resistance index data were collected. A major QTL for disease incidence and resistance index accounting respectively for 53 % (LOD score of 15.43) and 46 % (LOD score of 12.26) of the phenotypic variation was identified at the same position on linkage group 5 of “Rouge de Mauves.” Microsatellite marker UDAp-452 co-segregated with the resistance, and two flanking microsatellites, namely BPPCT037 and BPPCT038A, were identified. When dividing the population according to the alleles of UDAp-452, the subgroup with unfavorable allele had a disease incidence of 32.6 % whereas the group with favorable allele had a disease incidence of 21 %, leading to a reduction of 35.6 % in disease incidence. This study is a first step towards the marker-assisted breeding of new apricot varieties with an increased tolerance to X. arboricola pv. pruni.     

A new species of the Italian tick fauna. II. Ixodes arboricola Schulze and Schlottke, 1929]

The bird-tick Ixodes (Pholeoxiodes) arboricola Schulze and Schlottke, 1929, is reported for the first time in Italy with larvae and nymphs parasitizing Passer domesticus italiae (Vieillot) and Phoenicurus ph. phoenicurus L. Variation, natural bird hosts and presumed distribution also in Italian Alps and Apennines are discussed.     

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.     

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.     

Saccharomyces eubayanus and Saccharomyces arboricola reside in North Island native New Zealand forests

Saccharomyces is one of the best‐studied microbial genera, but our understanding of the global distributions and evolutionary histories of its members is relatively poor. Recent studies have altered our view of Saccharomyces’ origin, but a lack of sampling from the vast majority of the world precludes a holistic perspective. We evaluate alternate Gondwanan and Far East Asian hypotheses concerning the origin of these yeasts. Being part of Gondwana, and only colonized by humans in the last ～1000 years, New Zealand represents a unique environment for testing these ideas. Genotyping and ribosomal sequencing of samples from North Island native forest parks identified a widespread population of Saccharomyces. Whole genome sequencing identified the presence of S. arboricola and S. eubayanus in New Zealand, which is the first report of S. arboricola outside Far East Asia, and also expands S. eubayanus’ known distribution to include the Oceanic region. Phylogenomic approaches place the S. arboricola population as significantly diverged from the only other sequenced Chinese isolate but indicate that S. eubayanus might be a recent migrant from South America. These data tend to support the Far East Asian origin of the Saccharomyces, but the history of this group is still far from clear.     

The polyacetylenic falcarinol as the major allergen in schefflera arboricola

Schefflera arboricola has been reported to cause allergic contact dermatitis. The major allergen is now isolated and identified as falcarinol, heptadeca-1,9(Z)dien-4,6-diyn-3-ol. The isolation of (E)-β-farnesene, phytol and 24β-ethylcholesta-5,22(E)-diene-3β-ol (poriferasterol) is also reported.     

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.     

Detection of Xanthomonas fragariae and presumptive detection of Xanthomonas arboricola pv. fragariae, from strawberry leaves, by real-time PCR

Real-time (TaqMan) PCR assays were developed to detect the strawberry angular leaf spot pathogen Xanthomonas fragariae (Xf) and the strawberry bacterial blight pathogen Xanthomonas arboricola pv. fragariae (Xaf). The Xf PCR (Xf gyrB) was designed within regions of the gyraseB gene, unique to Xf, after generating gyraseB DNA sequence data from Xf and other closely related strains. The Xaf PCR (Xaf pep) was designed within regions of the pep prolyl endopeptidase gene that were unique to Xaf, after generating pep DNA sequence data from Xf and Xaf strains. The Xf gyrB PCR detected only Xf strains amongst a panel of 20 Xanthomonas-related spp. and pathovars. The Xaf pep PCR assay detected all Xaf strains tested plus two other (of three tested) X. arboricola pathovars. An existing genomic DNA extraction protocol was modified to facilitate detection of both pathogens to 10(3) cells per strawberry leaf disc.     

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.     

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.     

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.     

Influences of plant spacing on root tensile strength of Schefflera arboricola and soil shear strength

Mechanical root reinforcement depends not only on root biomechanical properties but also on root biomass. Although it is known that plant spacing can affect root growth, it is not clear how it affects root tensile strength. We interpreted a set of field data to study the effects of spacing of Schefflera arboricola on root area ratio (RAR), root tensile strength and their combined effects on soil shear strength (also termed root cohesion). S. arboricola was transplanted into compacted silty sand at a spacing of 0.5 m, 0.8 m and 1.1 m. After 20 months of growth in the field, the root systems were excavated for root tensile testing and post-test trait measurements. Plant spacing affected the growth and tensile strength of roots. More closely spaced plants had higher RAR but lower root tensile strength, especially for roots 0.5–2 mm in diameter. According to the existing root breakage and fibre bundle models used in this study, which calculate root cohesion as the product of RAR and root tensile strength, the effects of plant spacing on root cohesion were minimal for most soil depths apart from 0.4- to 0.5-m depth.

     

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.     

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.     

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.