Phenotypic and Genotypic Characterization of Xanthomonas campestris pv. zinniae Strains

During 1997 and 1998, serious outbreaks of bacterial leaf spot disease were observed on zinnia plants grown in home and commercial gardens in Ohio, USA. Twenty-two strains of Xanthomonas campestris pv. zinniae, isolated from diseased zinnia plants and contaminated seeds, were identified based on morphological, physiological and biochemical tests, fatty acid methyl ester analyses and pathogenicity tests on zinnia cv. Scarlet. Host range studies indicated that all of the X. campestris pv. zinniae strains were pathogenic on zinnia and tomato, but not on cabbage, lettuce, pepper and radish. The phenotypic and genotypic relationships among the strains determined based on serological reaction pattern, fatty acid profiles, repetitive extragenic palindromic-polymerase chain reaction (rep-PCR) fingerprints and sequence analysis of the 16S–23S rDNA spacer region suggested that X. campestris pv. zinniae strains were closely related to each other, but clearly distinct from other Xanthomonas species including X. campestris pv. campestris, X. axonopodis pv. vesicatoria, X. vesicatoria and X. hortorum pv. vitians tested in this study. The results also demonstrated that rep-PCR fingerprinting is rapid, reliable and the most practical method for routine detection and identification of X. campestris pv. zinniae strains.     

Induction of Systemic Acquired Resistance in Pepper Plants by Acibenzolar-S-methyl against Bacterial Spot Disease

The ability of acibenzolar-S-methyl to induce resistance in pepper plants against Xanthomonas campestris pv. vesicatoria was investigated in both growth chamber and open field conditions. Growth chamber experiments showed that acibenzolar-S-methyl (300M) treatment protects pepper plants systemically and locally against X. campestris pv. vesicatoria. Evidence for this was a reduction in the number and diameter of bacterial spots and bacterial growth in planta. Systemic protection was also exerted by the acibenzolar-S-methyl acid derivative, CGA 210007, which may be produced by hydrolysis in the plant. The efficacy of acibenzolar-S-methyl was also found in open field conditions, where both leaves and fruit were protected from the disease. The highest efficacy (about 67%) was obtained by spraying the plants 6–7 times every 8–12 days with a mixture of acibenzolar-S-methyl and copper hydroxide (2.5 + 40ghl^–1 active ingredient). Persistence and translocation data obtained from the growth chamber experiments revealed a persistence of acibenzolar-S-methyl lasting five days after treatment with rapid translocation and negligible levels of acid derivative formation. Since the protection exerted by acibenzolar-S-methyl against bacterial spot disease was observed when the inducer was completely degraded, it would appear to be due to SAR activation.     

Detection and identification of the crucifer pathogen, <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv. <Emphasis Type="Italic">campestris</Emphasis>, by PCR amplification of the conserved Hrp/type III secretion system gene <Emphasis Type="Italic">hrcC</Emphasis>

The development of a rapid detection method for Xanthomonas campestris pv. campestris (Xcc) in crucifer seeds and plants is essential for high-throughput certification purposes. Here we describe a diagnostic protocol for the identification/detection of Xcc by PCR amplification of fragments from the pathogenicity-associated gene hrcC. Under stringent conditions of amplification, a PCR product of 519 bp from hrcC was obtained from a collection of 46 isolates of Xcc, with the exception of two isolates from radish. No amplicons were obtained from 39 pure cultures of the phytopathogenic bacteria Xanthomonas campestris pv. cerealicola, X. campestris pv. juglandis, X. campestris pv. pelargonii, X. campestris pv. vitians, X. arboricola pv. pruni, X. axonopodis pv. phaseoli, X. axonopodis pv. vesicatoria, X. vesicatoria, Pseudomonas syringae pv. phaseolicola, P. syringae pv. syringae, P. syringae pv. tomato, P. fluorescens, P. marginalis, Pectobacterium atrosepticum, P. carotovorum subsp. carotovorum. In addition, PCR reactions were negative for fifty unidentified environmental isolates purified from the surface of crucifers. The PCR fragment was obtained from four strains previously classified as X. campestris pv. aberrans, X. campestris pv. armorociae, X. campestris pv. barbarae and X. campestris pv. incanae using pathogenicity assays. Our PCR protocol specifically detected Xcc in inoculated leaves, seeds and naturally infected leaves of crucifers.     

Impact of bacterial spot outbreaks on the phytosanitary quality of tomato and pepper seeds

The impact of disease outbreaks on the phytosanitary quality of seeds was investigated for two pathosystems: tomato–Xanthomonas vesicatoria and pepper–Xanthomonas euvesicatoria. This study, which was performed in Italy and Serbia, aimed to evaluate the season‐to‐season transmission of phytopathogenic regulated bacteria associated with phytosanitary risks posed by seeds produced in areas where bacterial infections are possible. For each pathosystem, field plots were experimentally inoculated to simulate an initial infection rate of 1%, 5% and 15%. The area under the disease progress curve (AUDPC) was calculated for each field plot, the seeds produced were analysed to determine the contamination level and rate, and the plant‐to‐seed transmission was evaluated by a seedling grow‐out (SGO) assay. To investigate transmission under field conditions, a second‐year experiment was performed, wherein seeds collected from the first year were used to establish new field plots. During the first growing season, AUDPC values were positively correlated with the percentages of initial infection for each pathosystem. Seed contamination levels in pepper ranged from 34 to 100 CFU g^?1, and the contamination rate ranged from 1.50% up to 3.17% for X. euvesicatoria, whereas processing and fresh market tomato seeds produced both in Italy and Serbia were not infected by X. vesicatoria. During SGO assays and the second cropping year, no symptoms were observed in either tomato or pepper plants. Therefore, the calculated pepper seed contamination rate for X. euvesicatoria appeared to be less than the threshold necessary to initiate a disease outbreak. Finally, all seeds obtained during the second cropping year were uninfected.     

Complementary bacterial enrichment techniques for the detection ofPseudomonas syringae pv.tomato andXanthomonas campestris pv.vesicatoria in infested tomato and pepper seeds

A scheme for routine seed testing forXanthomonas campestris pv.vesicatoria andPseudomonas syringae pv.tomato in pepper and tomato seeds was developed. The scheme is based on different bacterial enrichment techniques. As few as 1000 and 10–100 colony forming units per gram of seeds were detected using a liquid enrichment technique or leaf enrichment technique, respectively. Relatively large amounts of saprophytes on the seed surfaces did not interfere with the detection of the pathogens.     

Analysis of the toxicity of purothionins and hordothionins for plant pathogenic bacteria

Purothionins (PTHs) and hordothionins (HTHs) were purified by cation-exchange chromatography from petroleum-ether extracts of wheat and barley flour respectively. The HTHs could be separated into two fractions, HTH-1 and HTH-2. Radial diffusion assays and micro-plate broth dilution assays with a number of plant pathogenic bacteria showed that these proteins were toxic forClavibacter michiganensis subsp.michiganensis, the causal agent of bacterial canker on tomato,C. m. subsp.sepedonicus, the causal agent of ring rot on potato, andXanthomonas campestris pv.vesicatoria, the causal agent of a spot disease on tomato and pepper. Only minor differences in toxicity between PTHs and HTHs, and between HTH-1 and HTH-2, were detected. Minor differences in toxicity of these thionins were also detected for different strains of these bacteria. The use of these plant proteins for engineering bacterial disease resistance into solanaceous crops will be discussed.     

Genetic relationships among strains of <Emphasis Type="Italic">Xanthomonas campestris</Emphasis>pv. <Emphasis Type="Italic">campestris</Emphasis>revealed by novel rep-PCR primers

Novel primers for rep-PCR were developed with the original software and based on `ancient diverged periodical sequences'. Rep-PCR with these primers was applied to study genetic relationships among 51 Xanthomonas campestris strains. The strains were collected from different countries including Russia, Japan, UK, Germany and Hungary. Reference strains of three X. campestrispathovars and five other Xanthomonas species were included. Based on qualitative differences in amplification profiles, the strains were divided into four major groups. Two subgroups recognised within X. campestrispopulation were similar to RFLP haplotypes. The third subgroup included strains of two other pathovariants and Japanese isolates of X. campestris pv. campestriswhile the fourth group comprised the other species of Xanthomonas. The analysis of the diversity within X. campestris resulted in the conclusion that isolates belong to distinct clonal populations (subgroups). The differences between the subgroups of X. campestris were only slightly smaller than between species of Xanthomonas. A PCR fragment about 600 bp amplified by primer KRPN2 was found in nearly all tested strains of X. campestris.SCAR primers designed for this marker produced a single specific band for strains of X. campestris, but not for other Xanthomonas, Pseudomonas and Erwiniastrains tested. Application of the new primer set for rep-PCR offers a rapid, simple and reproducible method for identification of bacterial strains. The X. campestris-specific SCAR primers may be used in diagnostics of this important plant pathogen.     

Specificity of polycllonal and monoclonal antibodies for the identification of Xanthomonas campestris pv. campestris

Polyclonal and monoclonal antibodies (PCAs and MCAs), produced to whole cells and flagellar extracts ofXanthomonas campestris pv.campestris (Xcc), respectively, were tested for specificity. In immunofluorescence microscopy (IF) the three PCAs tested, reacted at low dilutions with all Xcc strains, some other xanthomonads and non-xanthomonads. At higher dilutions most cross-reactivity with non-xanthomonad strains disappeared. However, the cross-reactivity with strains ofX. c. pv.vesicatoria (Xcv),X. c. pv.amoraciae (Xca) andX. c. pv.phaseoli var. fuscans (Xcpf) remained.Six MCA-producing cell clones viz. 20H6, 2F4, 18G12, 10C5, 17C12 and 16B5 were selected for specificity tests with an enzyme immunoassay (EIA), IF and a dot-blot immunoassay (DBI). None of the MCAs reacted with all Xcc strains in IF and EIA. In DBI, only MCAs 17C12 and 16B5 reacted with all Xcc strains. All six MCAs tested, cross-reacted in one of either tests with other pathovars ofX. campestris, such as Xcv or Xca. The MCAs were also tested in immunoblotting experiments using total bacterial extracts, cell envelope and flagellar extracts. MCAs 20H6, 2F4, 18G12 and 10C5 reacted with the lipopolysaccharide (LPS) of Xcc. MCAs 16B5 and 17C12 reacted with a 39 kilodalton and a 29 kilodalton protein, respectively.It is concluded that the PCAs and MCAs discussed in this study may be used for routine identification and differentiation of (a group of) Xcc strains. The significance of the cross-reactions with other pathovars ofX. campestris needs to be determined by testing seed lots.     

Multilocus sequence analysis reveals a novel phylogroup of Xanthomonas euvesicatoria pv. perforans causing bacterial spot of tomato in Iran

A multilocus sequence analysis (MLSA) was performed on five housekeeping genes (fusA, gapA, gltA, lacF and lepA) of 22 Xanthomonas euvesicatoria strains recently isolated from alfalfa, pepper and tomato plants in Iran. In addition, 161 strains isolated worldwide from pepper, poinsettia, rose and tomato plants were included in the analysis. All X. euvesicatoria pv. perforans isolates from tomato plants in Iran clustered in a monophyletic group, although five MLSA haplotypes were detected among them. The Iranian tomato strains presented 10 nucleotide differences in the lepA gene sequences compared to the known worldwide population of X. euvesicatoria pv. perforans. Statistical analyses revealed a recombination event that had occurred in the lepA gene of the strains isolated from tomato in Iran. BOX‐PCR analysis confirmed the inclusion of Iranian tomato strains within X. euvesicatoria pv. perforans. Furthermore, X. euvesicatoria pv. euvesicatoria strains isolated from pepper in Iran differed in one nucleotide in the lepA gene sequence from the known worldwide population of the pathovar, and clustered in a group containing strains isolated in Nigeria. The strains isolated from alfalfa in Iran clustered with the type strain of X. euvesicatoria pv. alfalfae. Altogether, the results reveal the existence of a phylogenetically novel population of X. euvesicatoria pv. perforans in Iran which needs further in‐depth analysis to pinpoint the epidemiological impact of these strains.     

Differentiation of Xanthomonas species Pathogenic to Sugarcane by PCR-RFLP Analysis

The PCR-RFLP of the 16S-23S rDNA spacer region was used to differentiate Xanthomonas species pathogenic to sugarcane. Strains of X. albilineans, X. campestris pv. vasculorum Types A and B, X. sacchari and Xanthomonas sp. from Trinidad, South Africa and India were examined. The amplification products were digested with Alu I, Hae III, Hpa II and Mbo I and the results showed that the different groups of bacterial strains exhibited distinct RFLP patterns for each tested endonuclease, except X. albilineans and X. sacchari which could only be differentiated from each other by the digestion with Hpa II. The results also allowed the separation of X.c. pv. vasculorum Type A from X.c. pv. vasculorum Type B and strongly suggested that the analyzed Xanthomonas sp. strains belong to X. sacchari. Nine X. campestris (pv. not determined) strains included in this study showed identical profiles to X.c. pv. vasculorum Type A group and DNA–DNA hybridization experiments confirmed these results. PCR-RFLP of the 16S-23S rDNA spacer region could be applied as a reliable method for differentiating the xanthomonads pathogenic to sugarcane.     

Structural conservation of the <Emphasis Type="Italic">hrp</Emphasis> gene cluster in <Emphasis Type="Italic">Xanthomons oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis>

The clustered hrp genes encoding the type III secretion system in the Japanese strains MAFF301237 and MAFF311018 of Xanthomonas oryzae pv. oryzae were sequenced and compared. The strains differ in their pathogenicity, location, and year of isolation. A 30-kbp sequence comprising 29 open reading frames (ORFs) was identical in its structural arrangement in both strains but differed from X. campestris pv. campestris, X. axonopodis pv. citri, and X. axonopodis pv. glycines in certain genes located between the hpaB-hrpF interspace region. The DNA sequence and the putative amino acid sequence in each ORF was also identical in both X. oryzae pv. oryzae strains as were the PIP boxes and the relative sequences. These facts clearly showed that the structure of the hrp gene cluster in X. oryzae pv. oryzae is unique.     

Differentiation of Xanthomonas campestris pv. Phaseoli from Xanthomonas campestris pv. Phaseoli var. Fuscans by PFGE and RFLP

Xanthomonas campestris pv. phaseoli and X. campestris pv. phaseoli var. fuscans, the causal agents of the common and fuscous bacterial blight of beans, appear to be phenotypically identical except that the latter can produce a melanin-like pigment in culture. Ten isolates of X. campestris pv. phaseoli and 12 isolates of X. campestris pv. phaseoli var. fuscans were examined using pulsed-field gel electrophoresis (PFGE) and restriction fragment length polymorphism (RFLP). The average genome sizes for X. campestris pv. phaseoli and X. campestris pv. phaseoli var. fuscans were 3850.6±48.9 and 3584.3±68.1kb respectively. The genetic relatedness of the isolates was determined from macrorestriction patterns generated using XbaI. Cluster analysis indicated that the non-fuscous and fuscous strains are distinct. RFLP results, based on the highly conserved hrp genes and a pectate lyase gene from Xanthomonas, also indicated that the two bacteria are genetically different. The results obtained in this study suggest that this pathovar can be segregated into two subgroups under a recently proposed reclassification of the Xanthomonas genus.     

Investigation of Genomic Variability of Xanthomonas Arboricola Pv. juglandis by AFLP Analysis

Xanthomonas arboricola pv. juglandis is the causal agent of walnut blight, one of the most important and widespread diseases of Persian (English) walnut (Juglans regiaL.), causing severe damage to leaves, twigs and nuts. To investigate the genomic variability of X. arboricolapv. juglandis, 66 isolates obtained from different countries (England, France, Italy, The Netherlands, Romania, Spain, USA, and New Zealand) were analysed using the Amplified Fragment Length Polymorphism (AFLP) technique. EcoRI and MseI were used as restriction endonucleases. Primers with a core sequence including endonuclease recognition sites and a 3^prime-terminal cytosine selective base for MseI primer, or no selective base for EcoRI primer, were used. Data were analysed by means of a multiple correspondence analysis. A total of 76 amplified polymorphic DNA fragments were used to compute relationships among isolates. The AFLP profiles of X. arboricola pv. juglandis isolates appeared to be reliably distinguishable from X. arboricola pv. pruni and X. arboricola pv. corylina, and from other Xanthomonas species, i.e. X. campestris pv. campestris, X. fragariae, X. hortorum, X. axonopodis pv. vesicatoria. Though this pathogen is associated with one single host genus, a high level of genomic diversity was found. This diversity might be partly explained by the geographic origin. Nevertheless, isolates with different patterns were collected within one country, and similar molecular patterns were found in isolates collected at different sites. However, genetic diversity might be influenced by exchanging vegetative material from different countries. Mixing of X. arboricola pv. juglandis isolates might have partly concealed the influence of the geographic location from which the bacteria were isolated.     

Regulation of defense-related enzymes associated with bacterial spot resistance in Tomato

Twenty tomato (Solanum lycopersicon) cultivars were screened for resistance against bacterial spot disease incited byXanthomonas axonopodis pv.vesicatoria under field conditions with and without pathogen infection. Screening was done by artificially inoculating aX. axonopodis pv.vesicatoria suspension to 4-week-old tomato seedlings and observing them for typical symptoms of the disease. Seedlings were categorized into highly resistant, resistant, susceptible and highly susceptible cultivars on the basis of disease incidence. Tomato cultivars were screened for defense-related enzymes, total phenols and lignin contents. The temporal patterns of all these enzymes were estimated with a moderately susceptible tomato cultivar. Native PAGE analysis of both peroxidase (POX) and polyphenol oxidase (PPO) was carried out for the time course of enzyme activities and also by selecting three different tomato cultivars, following infection with the pathogen. Based on the inducible amounts of these enzymes upon pathogen infection, the tomato cultivars were correlated with the disease incidence under field conditions. A significant (P≤0.05) correlation was observed between the degree of host resistance and the enzyme levels. In highly resistant tomato cultivars the enzyme levels, total phenols and lignin contents were increased in comparison with highly susceptible tomato cultivars. Isoform analysis of POX and PPO enzymes indicated a clear difference between resistant and susceptible tomato cultivars in the number of isoforms and also in the intensity of each isoform in the presence of pathogen infection. The possible regulation of defense-related enzymes in imparting host resistance is discussed. http://www.phytoparasitica.org posting March 11, 2008.     

Metabolic Diversity of Xanthomonas axonopodis pv. vignicola, Causal Agent of Cowpea Bacterial Blight and Pustule

Fifty-five strains of Xanthomonas axonopodis pv. vignicola, isolated from blight and pustule symptoms of cowpea leaves, originating from 11 countries, were characterized for their carbon-source metabolization pattern using the Biolog GN microplate system. Great variation was found between strains according to origin. Dextrin, glycogen and succinamic acid were not used by strains from Benin, Uganda or Thailand, but by all the other strains (excluding two strains from Mozambique), whereas N-acetyl-D-glucosamine and malonic acid were used by the strains from Benin, Uganda and Thailand, but generally not by the other strains. The strains from Benin, Uganda and Thailand, as well as strains from Venezuela, Brazil and Mozambique, clustered separately from the others in multivariate analysis. Nineteen substrates were used by all the strains, 47 not by any strain and 29 only by some strains. No considerable differences were found between strains isolated from blight symptoms and from pustules. Virulence of strains was not related to the metabolic pattern. The Biolog database was not representative of the diversity of X. axonopodis pv. vignicola, since all strains were identified as Xanthomonas campestris, although belonging to eight pathovars, while only eight of nine strains from Benin and both strains from Thailand were identified as X. campestris pv. vignicola. The Biolog system appeared to be useful for characterizing the diversity of X. axonopodis pv. vignicola strains. A set of representative strains based on metabolic and molecular diversity, virulence and geographic origin is suggested for screening for resistant cowpea cultivars.     

Development of a sensitive ELISA using three monoclonal antibodies against lipopolysaccharides to detect <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv. <Emphasis Type="Italic">vesicatoria</Emphasis>, the causal agent of bacterial spot of tomato and pepper

Bacterial spot, caused by Xanthomonas campestris pv. vesicatoria (Doidge 1920) Dye 1978 (Xcv), is responsible for severe production losses in tomato and pepper. Three specific monoclonal antibodies (MABs), 7AH10, 5HB3, and 4AD2, which are reactive to lipopolysaccharides of the bacteria, were tested in three kinds of enzyme-linked immunosorbent assays (ELISAs). The most sensitive ELISA was a competitive one able to detect populations of Xcv as low as 10³–10⁴cfu/ml. Bacterial growth in artificially inoculated tomato and pepper plants could be successfully monitored, and assays on field-collected samples reconfirmed the diagnostic utility of the MABs. Studies on the accessibility of the MABs to their lipopolysaccharide epitopes confirmed that sample preparation can be improved to further increase the sensitivity of the assay. Immunoblotting of differential extracts of the bacterial cells and immunoelectron microscopy demonstrated that the epitopes were present throughout the cell wall and involved in complexes that can be loosened through the action of proteinase K or lysozyme.     

Cytokinin induces bacterial pathogen resistance in tomato

Phytohormones are involved in the regulation of plant responses to biotic stress. How a limited number of hormones differentially regulate defence responses and influence the outcome of plant–biotic interactions is not fully understood. In recent years, cytokinin (CK) was shown to induce plant resistance against several pathogens. In the present study, we investigated the effect of CK in inducing tomato resistance against the hemibiotrophic pathogenic bacteria Xanthomonas campestris pv. vesicatoria (Xcv) and Pseudomonas syringae pv. tomato (Pst). We demonstrate that CK enhances tomato resistance to Xcv and Pst through a process that relies on salicylic acid and ethylene signalling. CK did not directly affect the growth or biofilm formation ability of these pathogens in vitro. Overall, our work provides insight into the underlying mechanisms of CK-induced immune responses against bacterial pathogens in tomato.     

Protein electrophoresis and DNA:DNA hybridizations of xanthomonads from grasses and cereals1

More than 120 Xanthomonas campestris strains pathogenic for grasses and cereals were compared by polyacrylamide gel electrophoresis (SDS-PAGE) of their whole-cell proteins. Genotypic relationships between representative strains of the electrophoretic groups were determined by DNA:DNA hybridizations. Two major groups of bacteria were delineated. The first included X. campestris pv. graminis, pv. arrhenatheri and some isolates from Bromus, which could be differentiated from each other by their protein fingerprints, and also the following pathovars which it was impossible to differentiate by SDS-PAGE: cerealis, hordei, poae, secalis, translucens and undulosa. DNA:DNA hybridizations indicated that significant degrees of DNA-binding (>60% D) exist between all these pathovars. In the second group, strains of X. campestris pv. holcicola, pv. vasculorum and pv. oryzae were related at 40–45% DNA-binding, while strains of pv. oryzae and pv. oryzicola were genotypically highly related (85% D). All the pathovars of this second group could be differentiated from each other by their protein electrophoretic fingerprints.     

Development of a specific molecular tool for detecting Xanthomonas campestris pv. musacearum

A specific and rapid diagnostic tool has been developed to detect Xanthomonas campestris pv. musacearum, the causal agent of bacterial wilt of banana. PCR primers were developed from intergenic regions of X. campestris pv. musacearum following its partial sequence. A total of 48 primers were tested for specificity to X. campestris pv. musacearum strains collected from various regions in Uganda. These were also tested for specificity against related Xanthomonas species from the vasicola group, Xanthomonas species pathogenic to other crops, and against those existing saprophytically on banana plants. Seven primer sets (Xcm12, Xcm35, Xcm36, Xcm38, Xcm44, Xcm47 and Xcm48) were found to be very specific to X. campestris pv. musacearum. These primer sets directed the amplification of the expected product for all 52 strains of X. campestris pv. musacearum collected from different locations in Uganda. No amplification products were obtained with unrelated phytopathogenic bacteria or endophytic/epiphytic bacteria from banana. A detection limit of 10³ CFU mL^?1 corresponding to about four cells per PCR reaction was observed when X. campestris pv. musacearum cells were used for all the seven primer sets. The DNA samples from symptomless plant tissues also tested positive with primer set Xcm38. The specific PCR method described here is a valuable diagnostic tool which can be used to detect the pathogen at early stages of infection and monitor disease.