Characteristics of a PCR-Based Assay for In Planta Detection of Xanthomonas campestris pv. pelargonii

Polymerase chain reaction (PCR) amplification was carried out with a primer pair targeting a sequence in the genome of Xanthomonas campestris pv. pelargonii , the causative agent of bacterial blight in geraniums. PCR amplification with the primer pair XcpMl/XcpM2 using total nucleic acid preparations from 22 geographicallydiverse isolates of X. campestris pv. pelargonii generated a major 197 bp DNA product. In contrast, no major amplification products were consistently generated from 12 other pathovars of X. campestris or from 19 isolates representing 10 different plant pathogenic bacteria, including two other bacterial pathogens of geraniums, Corynebacterium fascians and Pseudomonas cichorii . After PCR using this primer pair, between 1380 and 13800 copies of the X, campestris pv. pelargonii bacterial DNA target as template were detected by ethidium bromide staining of agarose gels, and between 13.8 and 138 copies by blot hybridization to a pathovar-specific biotinylated probe. Similarly, between 630 and 6300 colonyforming units (CFU) of X. campestris pv. pelargonii could be detected after ethidium bromide staining of agarose gels, and between 63 and 630 CFU after blot hybridization. The PCR-based assay was used to identify X. campestris pv. pelargonii in diseased geraniums; whereas discrete amplification products were not obtained with healthy plants.     

Sensitive and specific detection of Xanthomonas campestris pv. pelargonii with DNA primers and probes identified by random amplified polymorphic DNA analysis.

The random amplified polymorphic DNA method was used to distinguish strains of Xanthomonas campestris pv. pelargonii from 21 other Xanthomonas species and/or pathovars. Among the 42 arbitrarily chosen primers evaluated, 3 were found to reveal diagnostic polymorphisms when purified DNAs from compared strains were amplified by the PCR. The three primers revealed DNA amplification patterns which were conserved among all 53 strains tested of X. campestris pv. pelargonii isolated from various locations worldwide. The distinctive X. compestris pv. pelargonii patterns were clearly different from those obtained with any of 46 other Xanthomonas strains tested. An amplified 1.2-kb DNA fragment, apparently unique to X. campestris pv. pelargonii by these random amplified polymorphic DNA tests, was cloned and evaluated as a diagnostic DNA probe. It hybridized with total DNA from all 53 X. campestris pv. pelargonii strains tested and not with any of the 46 other Xanthomonas strains tested. The DNA sequence of the terminal ends of this 1.2-kb fragment was obtained and used to design a pair of 18-mer oligonucleotide primers specific for X. campestris pv. pelargonii. The custom-synthesized primers amplified the same 1.2-kb DNA fragment from all 53 X. campestris pv. pelargonii strains tested and failed to amplify DNA from any of the 46 other Xanthomonas strains tested. DNA isolated from saprophytes associated with the geranium plant also did not produce amplified DNA with these primers. The sensitivity of the PCR assay using the custom-synthesized primers was between 10 and 50 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differentiation of Xanthomonas campestris pvs. pelargonii and hederae by Polymerase Chain Reaction

Geranium isolates of Xanthomonas campestris pv. pelargonii ( Xcp ) and English ivy isolates of X. campestris pv. hederae ( Xch ) were tested by polymerase chain reaction (PCR) to determine whether the two pathovars could be discriminated using amplification conditions developed to identify and detect Xcp in infected geraniums. Using PCR, other workers reported that the genomes of Xcp and Xch were indistinguishable. The objective of this study was to determine whether the two pathovars have sufficient sequence diversity to allow them to be distinguished by molecular means. Three primer pairs were used for PCR amplification. Two of the primer pairs (REP and XcpM1/XcpM2) were able to distinguish between Xch and Xcp , whereas amplification with the third primer pair (ERIC) did not allow discrimination between the two pathovars. Based on PCR amplification, Xcp and Xch are distinctly different pathovars. Additionally, all three primer pairs showed discrimination between Xcp and Acidovorax , a bacterial pathogen that induces leaf spot on geranium.     

Immunomagnetic isolation of Xanthomonas campestris pv. pelargonii

Immunomagnetic fishing was developed as an improved procedure for increasing the bacterial target to non-target recovery ratio in suspensions containing mixtures of target and non-target organisms. A cell suspension containing the target Xanthomonas campestris pv. pelargonii and non-target organisms, is treated with rabbit polyclonal antiserum against X.c. pv. pelargonii and incubated for 1 h. The suspension is then mixed with paramagnetic iron oxide particles coated with goat anti-rabbit antibodies (immunomagnetic particles). After incubation, the polished surface of a 14 mm diameter neodymium supermagnet is placed at the air-water interace and the magnetic particles are attracted to the magnet. After all visible magnetic particles have attached to the bottom of the magnet, the magnet is dipped in sterile buffer to remove non-target organisms. The magnet with attached magnetic particles is rubbed evenly over an agar surface to dislodge the particles and attached bacteria. Conventional immunomagnetic isolation (immunomagnetic attraction) and immunomagnetic fishing were compared, for the recovery of the target organism in geranium leaf washings spiked with X.c. pv. pelargonii. With immunomagnetic attraction and immunomagnetic fishing, bacterial non-target organisms were reduced to 11.4 and 1.5% of the initial population, respectively, whereas the target was only reduced to 63.7 and 53.8%.     

Simultaneous PCR Detection of the Two Major Bacterial Pathogens of Geranium

Xanthomonas campestris pv. pelargonii ( Xcp ) and Ralstonia solanacearum ( Rs ) are the two most important bacterial pathogens of commercially cultivated geraniums ( Pelargonium spp.), both causing bacterial wilt and leaf spot. Asymptomatic infections are important reservoirs of infections in commercial growing facilities. Our objective was to design a multiplex PCR (Polymerase Chain Reaction) assay to detect infection by either or both of these pathogens. We used a previously characterized PCR primer pair for Xcp that amplifies a region of 200 bp. In addition, we designed a new primer pair specific for Rs that amplifies a region of 822 bp. With these two primer pairs, we could detect either or both pathogens. As geranium tissue extracts frequently contain inhibitors of the PCR process, a negative PCR could result from either an accurate indication that the plant was pathogen-free or from a false negative assay. We therefore designed `amplification competence' primers, targeting a portion of the geranium 18 s rRNA gene, and generating a 494-bp amplification product that confirms amplification competence and validates a negative assay result. Thus, the triple primer pair multiplex PCR screens for the two most important bacterial pathogens of geraniums simultaneously confirms amplification competence for each geranium sample.     

Detection of Puccinia pelargonii‐zonalis‐infected Geranium Tissues and Urediniospores

The occurrence of geranium rust (caused by Puccinia pelargonii‐zonalis) in commercial greenhouses can result in unmarketable plants and significant economic losses. Currently, detection of geranium rust relies solely on scouting for symptoms and signs of the disease. The purpose of this research was to develop a rapid detection assay for P. pelargonii‐zonalis‐infected tissues or urediniospores on greenhouse‐grown geraniums. Two oligonucleotide primers were designed based on internal transcribed spacer sequence data from three isolates of P. pelargonii‐zonalis. The primers amplified a 131‐bp product from genomic DNA from each isolate of P. pelargonii‐zonalis but did not amplify a product from genomic DNA from twelve other rust fungi or four other plant pathogenic fungi. A PCR product was amplified consistently from solutions that contained 1 ng or 100 pg/ml of purified P. pelargonii‐zonalis DNA in conventional PCR and at 1 pg/ml using real‐time PCR. The detection threshold was 10² urediniospores/ml for real‐time PCR and 10⁴ urediniospores/ml for conventional PCR using urediniospores collected by vacuum from sporulating lesions. Puccinia pelargonii‐zonalis DNA was amplified by real‐time PCR from urediniospores washed from a single inoculated leaf, but recovered urediniospores were below detection threshold from one inoculated leaf with 5, 10, 25 and 50 non‐inoculated leaves. Conventional and real‐time PCR did not detect P. pelargonii‐zonalis in infected leaf tissues, presumably due to PCR inhibitors in the geranium leaf tissue. The inhibition of both conventional and real‐time PCR by geranium tissues suggests that a detection assay focusing on urediniospore recovery and microscopic examination with subsequent species verification by PCR may be the most efficient method for assessing the presence of geranium rust in greenhouses.     

A Simple DNA Extraction Method for PCR-based Detection of Xanthomonas campestris pv. pelargonii in Geraniums

A simple method for PCR-based plant clinical diagnosis of bacterial blight of geraniums caused by Xanthomonas campestris pv. pelargonii is described. The method entails maceration of infected tissues in water or 10mM Tris- HCI, pH 8.0 buffer, followed by treatment of the macerate with a commercially-available extraction matrix (GeneReleaser^TM) in which nucleic acid is released by brief microwave heating. Nucleic acid prepared in this manner served directly as template for PCR amplification with primers targeting a sequence in the genome of the bacterium. Using this protocol, it was possible to quickly identify X. campestris pv. pelargonii in infected geraniums, whereas amplification products were not obtained with nucleic acid preparations from noninfected plants, or from plants infected with the bacterial pathogens, Corynebacterium fascians or Pseudomonas cichorii .     

PCR-based detection of Xanthomonas campestris pv. phaseoli var. fuscans in plant material and its differentiation from X. c. pv. phaseoli

A PCR-based method was developed for the specific detection of Xanthomonas campestris pv. phaseoli var. fuscans from plant material. Primers Xf1 and Xf2, based on a sequence conserved amplified region (SCAR) derived from RAPD PCR analysis of X. c. pv. phaseoli var. fuscans , amplified a DNA fragment of 450 bp from all such isolates. In contrast, no amplification product was obtained from any X. c. pv. phaseoli isolates, or from any other DNAs tested. As few as 10 cells of X. c . pv. phaseoli var. fuscans (equivalent to about 100 fg DNA) could be detected in vitro . In planta , following an initial inoculation of as little as one cell, an amplification product was generated after only 2 d of incubation, allowing highly sensitive detection 10 d before disease symptoms were observed. Moreover, the failure to amplify DNA from X. c . pv. phaseoli isolates shows that these primers provide a rapid, improved method to differentiate these two varieties using PCR.     

Accumulation of Salicylic Acid and 4-Hydroxybenzoic Acid in Phloem Fluids of Cucumber during Systemic Acquired Resistance Is Preceded by a Transient Increase in Phenylalanine Ammonia-Lyase Activity in Petioles and Stems

Cucumber (Cucumis sativa) leaves infiltrated with Pseudomonas syringae pv. syringae cells produced a mobile signal for systemic acquired resistance between 3 and 6 h after inoculation. The production of a mobile signal by inoculated leaves was followed by a transient increase in phenylalanine ammonia-lyase (PAL) activity in the petioles of inoculated leaves and in stems above inoculated leaves; with peaks in activity at 9 and 12 h, respectively, after inoculation. In contrast, PAL activity in inoculated leaves continued to rise slowly for at least 18 h. No increases in PAL activity were detected in healthy leaves of inoculated plants. Two benzoic acid derivatives, salicylic acid (SA) and 4-hydroxybenzoic acid (4HBA), began to accumulate in phloem fluids at about the time PAL activity began to increase, reaching maximum concentrations 15 h after inoculation. The accumulation of SA and 4HBA in phloem fluids was unaffected by the removal of all leaves 6 h after inoculation, and seedlings excised from roots prior to inoculation still accumulated high levels of SA and 4HBA. These results suggest that SA and 4HBA are synthesized de novo in stems and petioles in response to a mobile signal from the inoculated leaf.     

Effect of culture filtrates ofXanthomonas campestris pv.pelargonii and extracts of geranium stems inoculated withX. c. pv.pelargonii on geranium callus and seedlings

Experiments were designed to determine whetherXanthomonas campestris pv.pelargonii produces a toxin which induces symptoms of bacterial blight in geranium, and is active at the cellular level. Culture filtrates ofX. c. pv.pelargonii were prepared by ethyl acetate extraction and ultrafiltration of the aqueous fraction. Culture filtrates adjusted to several pH values induced maximum disease ratings on geranium seedlings in the pH range 7–10. Geranium callus growth was significantly reduced by the filtrate in the same pH range. An active fraction could also be isolated from diseased tissue. A thin-layer chromatography-callus bioassay system detected toxin activity in the culture filtrate and in extracts of geranium stems inoculated withX. c. pv.pelargonii. Callus growth inhibition was located at R_f = 0.2–0.3 for both sources of toxin. These results suggest thatX. c. pv.pelargonii produces a toxin which causes disease symptoms, is present in diseased tissues, and inhibits callus growth. This opens the possibility of developing resistance to this pathogen by selecting cells insensitive to the toxin and regenerating plants from these cells.     

A nucleic acid sequence-based amplification system for detection of Listeria monocytogenes hlyA sequences.

A nucleic acid sequence-based amplification system primarily targeting mRNA from the Listeria monocytogenes hlyA gene was developed. This system enabled the detection of low numbers (< 10 CFU/g) of L. monocytogenes cells inoculated into a variety of dairy and egg products after 48 h of enrichment in modified listeria enrichment broth.     

Specific detection of Xanthomonas axonopodis pv. dieffenbachiae in anthurium (Anthurium andreanum) tissues by nested PCR

Efficient control of Xanthomonas axonopodis pv. dieffenbachiae, the causal agent of anthurium bacterial blight, requires a sensitive and reliable diagnostic tool. A nested PCR test was developed from a sequence-characterized amplified region marker identified by randomly amplified polymorphic DNA PCR for the detection of X. axonopodis pv. dieffenbachiae. Serological and pathogenicity tests were performed concurrently with the nested PCR test with a large collection of X. axonopodis pv. dieffenbachiae strains that were isolated worldwide and are pathogenic to anthurium and/or other aroids. The internal primer pair directed amplification of the expected product (785 bp) for all 70 X. axonopodis pv. dieffenbachiae strains pathogenic to anthurium tested and for isolates originating from syngonium and not pathogenic to anthurium. This finding is consistent with previous studies which indicated that there is a high level of relatedness between strains from anthurium and strains from syngonium. Strains originating from the two host genera can be distinguished by restriction analysis of the amplification product. No amplification product was obtained with 98 strains of unrelated phytopathogenic bacteria or saprophytic bacteria from the anthurium phyllosphere, except for a weak signal obtained for one X. axonopodis pv. allii strain. Nevertheless, restriction enzyme analysis permitted the two pathovars to be distinguished. The detection threshold obtained with pure cultures or plant extracts (10(3) CFU ml(-1)) allowed detection of the pathogen from symptomless contaminated plants. This test could be a useful diagnostic tool for screening propagation stock plant material and for monitoring international movement of X. axonopodis pv. dieffenbachiae.     

Detection of Xanthomonas oryzae pv. oryzae in artificially inoculated and naturally infected rice seeds and plants by molecular techniques

A polymerase chain reaction (PCR) technique was developed for detecting the presence of Xanthomonas oryzae pv. oryzae, the bacterial leaf blight (BLB) pathogen in rice seed and for studying the transmission of this bacterium from seed to plant. Primers TXT and TXT4R from an insertion sequence (IS1113) of the pathogen were used to amplify a 964-bp DNA fragment. A combined biological and enzymatic amplification (BIO-PCR) technique was used to detect the pathogen in naturally infected seed. The level of detection of TXT and TXT4R primers was 55 fg DNA of X. o. pv. oryzae, which is roughly the equivalent of seven cells (and four cells in pure culture suspension) of X. o. pv. oryzae. Hybridization of IS1113 with the amplified DNA fragment in Southern blot analysis confirmed that the 964-bp DNA fragment was amplified from X. o. pv. oryzae. The presence of the IS1113 element in strains of X. o. pv. oryzae from 16 rice-growing countries was confirmed by DNA dot blot analysis. X. o. pv. oryzae was detected from the seed washes and DNA extracted from the seed washes of naturally infected seeds of cvs Jaya and TN1. When stored at 4 degrees C, the pathogen was recovered up to 4 months and 9 months from naturally infected seeds of cvs Jaya and TN1, respectively. The BLB bacterium was also detected in seedlings, mature plants and seeds collected from plants raised from naturally infected seeds.     

Rapid and sensitive detection of Campylobacter spp. in chicken products by using the polymerase chain reaction.

The polymerase chain reaction (PCR) after a short enrichment culture was used to detect Campylobacter spp. in chicken products. After the 16S rRNA gene sequence of Campylobacter jejuni was determined and compared with known sequences from other enterobacteria, a primer and probe combination was selected from the region before V3 and the variable regions V3 and V5. With this primer set and probe, 426-bp fragments from C. jejuni, Campylobacter coli, and Campylobacter lari could be amplified. The detection limit of the PCR was 12.5 CFU. Chicken samples inoculated with 25 CFU of Campylobacter spp. per g were PCR positive after an 18-h enrichment, which resulted in 500 CFU/ml of culture broth. This PCR-culture assay was compared with the conventional method on naturally infected chicken products. Both methods detected the same number of positive and negative samples; however, the results of the PCR-culture assay were available within 48 h.     

Rapid and sensitive detection of Campylobacter spp. in chicken products by using the polymerase chain reaction.

The polymerase chain reaction (PCR) after a short enrichment culture was used to detect Campylobacter spp. in chicken products. After the 16S rRNA gene sequence of Campylobacter jejuni was determined and compared with known sequences from other enterobacteria, a primer and probe combination was selected from the region before V3 and the variable regions V3 and V5. With this primer set and probe, 426-bp fragments from C. jejuni, Campylobacter coli, and Campylobacter lari could be amplified. The detection limit of the PCR was 12.5 CFU. Chicken samples inoculated with 25 CFU of Campylobacter spp. per g were PCR positive after an 18-h enrichment, which resulted in 500 CFU/ml of culture broth. This PCR-culture assay was compared with the conventional method on naturally infected chicken products. Both methods detected the same number of positive and negative samples; however, the results of the PCR-culture assay were available within 48 h.     

Characterization of Xanthomonas campestris pv. pruni strains from different hosts by pathogenicity tests and analysis of whole-cell fatty acids and whole-cell proteins

Strains of Xanthomonas campestris pv. pruni obtained from Prunus armeniaca. P. domestica, P. persica and P. salicina in different geographical areas were compared for pathogenicity, fatty acid and wholecell protein analysis. Four strains, one per each host plant, were inoculated at the same time, on the foliage of P. armeniaca, P. avium, P. persica and P. salicina cultivars . Mean content of fatty acids of X.c. pv. pruni strains were also compared with those of many strains of X.c. pv. campestris , pv. graminis , pv. hyacinthii , pv. pelargonii and pv. vasculorum. Strains showed a remarkable homogeneity in fatty acids content and whole-cell protein profiles and principal component and cluster analysis did not reveal any grouping according to original host or geographical origin. However, X.c . pv. pruni strains can be grouped apart from the other X. campestris pathovars. There appears to be no pathogenic specialization among the strains tested, however, they varied in aggressiveness to host plants and host plant in susceptibility. The most of the strains were able to cross-infect species other that from where they were originally isolated, although, P. avium did not show any symptom of disease. P. persica cv. Sentry and P. salicina cv. Globe Sun, recently licensed as resistant to X.c. pv. pruni. were infected, although to a lesser extent, by some strains.     

水稻白叶枯病抗性基因Xa23鉴别菌株突变体库的构建及无毒基因突变体的筛选

利用in vivo转座技术构建了白叶枯病抗性基因Xa23鉴别菌株的突变体库,特异性引物PCR扩增和转座子插入位点旁侧序列分析结果表明转座子插入到白叶枯病菌的基因组中。经人工接种鉴定,筛选到4个毒力发生变化的突变体。为进一步克隆Xa23无毒基因提供了条件。     

Specific Detection of Xanthomonas axonopodis pv. dieffenbachiae in Anthurium (Anthurium andreanum) Tissues by Nested PCR

Efficient control of Xanthomonas axonopodis pv. dieffenbachiae, the causal agent of anthurium bacterial blight, requires a sensitive and reliable diagnostic tool. A nested PCR test was developed from a sequence-characterized amplified region marker identified by randomly amplified polymorphic DNA PCR for the detection of X. axonopodis pv. dieffenbachiae. Serological and pathogenicity tests were performed concurrently with the nested PCR test with a large collection of X. axonopodis pv. dieffenbachiae strains that were isolated worldwide and are pathogenic to anthurium and/or other aroids. The internal primer pair directed amplification of the expected product (785 bp) for all 70 X. axonopodis pv. dieffenbachiae strains pathogenic to anthurium tested and for isolates originating from syngonium and not pathogenic to anthurium. This finding is consistent with previous studies which indicated that there is a high level of relatedness between strains from anthurium and strains from syngonium. Strains originating from the two host genera can be distinguished by restriction analysis of the amplification product. No amplification product was obtained with 98 strains of unrelated phytopathogenic bacteria or saprophytic bacteria from the anthurium phyllosphere, except for a weak signal obtained for one X. axonopodis pv. allii strain. Nevertheless, restriction enzyme analysis permitted the two pathovars to be distinguished. The detection threshold obtained with pure cultures or plant extracts (10³ CFU ml⁻¹) allowed detection of the pathogen from symptomless contaminated plants. This test could be a useful diagnostic tool for screening propagation stock plant material and for monitoring international movement of X. axonopodis pv. dieffenbachiae.     

EVALUATION OF 5'NUCLEASE BASED DETECTION ASSAYS TO DETECT ESCHERICHIA COLI O157:H7 FROM FOOD PRODUCTS

Two 5'nuclease-based PCR methods (PCR-LS-50B and PCR-7200) were evaluated to determine their sensitivity for detecting Escherichia coli O157:H7 from pure cultures and in food samples enriched in different media and after different incubation periods. The PCR-7200 method was able to detect E. coli O157:H7 at ± 10² CFU/mL in pure culture in both mECB and EEB. In spiked meat samples, the PCR-7200 procedure was capable of detecting the eaeA gene at lower concentrations than the PCR-LS-50B procedure, regardless of the meat type or enrichment medium. Escherichia coli O157:H7 spiked at 0.3 CFU/mL was detectable after 9 h in EEB, but it was not detected in mECB within 24 h. An enrichment time of 4 h in mECB was needed to detect E. coli O157:H7 when spiked at higher levels (41 CFU/mL). The detection levels reported in this study are similar with other reported PCR-based detection techniques for E. coli O157:H7, however, the 5'nuclease-based assays are less labor intensive and capable of higher sample throughput because of their automated detection and analysis steps.     

Simultaneous detection of Salmonella strains and Escherichia coli O157:H7 with fluorogenic PCR and single-enrichment-broth culture

A multiplex fluorogenic PCR assay for simultaneous detection of pathogenic Salmonella strains and Escherichia coli O157:H7 was developed and evaluated for use in detecting very low levels of these pathogens in meat and feces. Two sets of primers were used to amplify a junctional segment of virulence genes sipB and sipC of Salmonella and an intragenic segment of gene eae of E. coli O157:H7. Fluorogenic reporter probes were included in the PCR assay for automated and specific detection of amplified products. The assay could detect <10 CFU of Salmonella enterica serovar Typhimurium or E. coli O157:H7 per g of meat or feces artificially inoculated with these pathogens and cultured for 6 to 18 h in a single enrichment broth. Detection of amplification products could be completed in 相似文献