Detection Of Xanthomonas Axonopodis Pv Research Articles

Development of simple and quick DNA release protocol for conventional PCR based detection of Xanthomonas axonopodis pv. punicae causing bacterial blight in pomegranate

An economical, simple, rapid, and culture independent method was developed for routine analyses and detection of Xanthomonas axonopodis pv. punicae (Xap) that causes bacterial blight in pomegranate. Five DNA release buffers (B1-B5) were optimized for extracting bacterial genomic DNA (gDNA) directly from (a)symptomatic pomegranate leaves followed by conventional polymerase chain reaction (PCR)-based detection of Xap. B1, B3, and B4 were found suitable to release gDNA, which was subjected to PCR using universal primers for 16S rRNA and rpsL genes, and pathogen specific xopQ primers. DNA released from B1 and B4 successfully produced amplicons of expected sizes. Additional analyses found that DNA released using B4 buffer was stable up to 45 days at −20 °C/−80 °C and 35 days at 4 °C and 8-800 pg DNA could be detected by the PCR-based assay. B4 was further validated for versatility by extracting DNA of Xanthomonas spp. causing citrus canker and sorghum shoot stripe disease.

Correction to: Loop-mediated isothermal amplification assay for pre-symptomatic stage detection of Xanthomonas axonopodis pv. punicae infection in pomegranate

Correction to: Loop-mediated isothermal amplification assay for pre-symptomatic stage detection of Xanthomonas axonopodis pv. punicae infection in pomegranate

Loop-mediated isothermal amplification assay for pre-symptomatic stage detection of Xanthomonas axonopodis pv. punicae infection in pomegranate

Bacterial blight in pomegranate caused by Xanthomonas axonopodis pv. punicae (Xap) is an increasing threat to pomegranate cultivation in India. To prevent economic losses, it is pivotal to detect the infection in latent stages rather than in symptomatic stages. We have developed an enhanced loop-mediated isothermal amplification (LAMP) technique for the detection of latent Xap infection in pomegranate using six sets of pathovar-specific primers. The DNA-intercalating dye ethidium bromide was standardized for visualizing positive amplification in the LAMP assay. The optimum reaction time was 30 min or more at 65 °C, and assay sensitivity was enhanced down to femtogram (fg) amounts of template DNA (0.153 fg) in the LAMP assay. In artificially inoculated plants, the enhanced LAMP assay detected Xap on the 7th day post inoculation (dpi), while PCR detected Xap only after the 11th dpi. Finally, the specificity of the LAMP assay for Xap was validated against 31 bacterial strains belonging to 22 species. The LAMP assay developed in this study is highly specific, sensitive and robust and can be used as an improved alternative for PCR-based methods for the early detection of Xap infection in pomegranate. Early detection of Xap in pomegranate ensures quality fruits with increased yield by adopting precautionary measures that ensure improved monetary benefit to farmers.

Detection of Xanthomonas axonopodis pv. vignicola, causal agent of bacterial blight of cowpea in seeds by non-serological methods

Among the diseases infecting cowpea, bacterial blight caused by Xanthomonas axonopodis pv. vignicola (Burkholder,1944). Vauterin et al . (1995) is a major production constraint. First necrotic lesions are formed on leaves and later the stem is attacked and the pathogen reaches vascular bundles and the disease becomes systemic. In the present study, attempted have been made to develop the suitable methods for the detection of the pathogen in the seeds and to find out the nature of transmission using selective and semi-selective media and compare them for their efficacy. Results indicated NSCAA medium to be more efficient in recovering the colonies of seed borne bacterium Xanthomonas axonopodis pv. vignicola with 13 2 ×10 5 cfu/ml as compared to 75×10 5 cfu/ml of colonies on Nutrient agar. The next best medium was SIBU agar (126×10 5 cfu/ml) followed by XTS medium (118×10 5 cfu/ml). All the media for isolation of plant pathogenic bacterium from seeds revealed that the cowpea bacterium is seed borne in nature. Another method employed to detect pathogen in seeds is Van Vuurde et al.(1983) method and results revealed that unsterilized seeds of susceptible cultivar C-152 yielded more number of colonies 35×10 2 cfu/ml by direct plating the seed extract on NSCAA whereas the resistant germplasm, DCS 47-1 yielded very less number of colonies 2×10 2 from diseased unsterilized seeds confirming its seed borne nature.

Detection of Xanthomonas axonopodis pv. phaseoli in bean seeds by flow cytometry, immunostaining and direct viable counting

Flow cytometric analysis of immuno-stained cells (immuno-FCM) was compared to immunofluorescence microscopy (IF) and dilution plating on a semi-selective medium, for quantitative detection of Xanthomonas axonopodis pv. phaseoli (Xap) in bean seed extracts. Cell concentrations of Xap between 103-107 CFU/mL were added to healthy bean seed extracts. A flow cytometry sorting procedure was developed to separate immuno-stained Xap cells from crude seed extracts and confirming by PCR. FCM was evaluated for direct viable counting (DVC) of Xap using combinations of propidium iodide (PI) and carboxy fluorescein diacetate (cFDA) or PI and SYTO 9 and also the combination of immuno-FCM and PI. Dilution plating and IF allowed detection of Xap in bean seed extracts in a range of 103-106 CFU/mL and immuno-FCM from 104-106 CFU/mL. Sorted cells could be detected in crude seed extracts by PCR without further extraction. FCM also allowed quantification of viable cells of Xap after DVC procedures; the red fluorescent dye propidium iodide was used to identify dead cells in combination with the green fluorescent dyes cFDA or SYTO 9, these identifying live cells. The combination of immuno-FCM and PI could be more promising and reliable to detect this pathogen in seeds.

콩 종자에서 Xanthomonas axonopodis pv. glycines의 검출을 위한 Direct PCR 방법 개발

콩 불마름병을 일으키는 Xanthomonas axonopodis pv. glycines를 종자에서 DNA 추출없이 바로 검출하는 방법에 대하여 연구하였다. 콩 종자에서 X. axonopodis pv. glycines를 특이적으로 검출하기 위해 특이적인 유전자로 알려져 있는 glycinecin A로부터 증폭 size가 401 bp인 primer Xag F1 & Xag R1을 고안하였다. Xag Fl과 Xag R1 primer는 콩 종자와 잎에서 분리한 균주와 KACC에서 분양받은 X. axonopoids pv. glycines 균주를 증폭시켰으나 근연종인 X. axonopodis pv. citri, X. axonopodis pv. vesicatoria 등은 증폭되지 않았다. 콩 종자에 존재하는 것으로 알려진 다른 세균들 역시 증폭되지 않았다. 고안된 Xag F1 & Xag R1 primer를 이용한 X. axonopodis pv. glycines의 검출 한계 농도 측정은 genomic DNA와 세포 현탁액을 이용하였다. X. axonopodis pv. glycines의 genomic DNA는 200 fg까지 증폭이 되었으며, 세포현탁액은 <TEX>$OD_{600nm}$</TEX> 0.1로 농도를 조정한 뒤, <TEX>$10^{-8}$</TEX>까지 희석하여 측정한 결과 <TEX>$10^{-6}$</TEX>인 <TEX>$1.8{\times}10^3$</TEX> cfu/ml까지 증폭이 확인되었다. 자연 감염된 종자에서 병원균을 검출하기 위해 종자를 육안상 건전종자와 불건전한 종자(변색립, 피해립)로 구분하여 direct PCR 방법으로 실험 한 결과 육안상 건전종자에서는 병원균이 검출되지 않았으나 불건전한 종자에서는 병원균의 검출이 확인되었다. 또한 진탕 배양 시간에 따른 병원균의 검출여부를 조사한 결과 진탕 배양 2시간부터 병원균의 검출이 확인되었으며 시간이 지날수록 증폭 밴드가 더욱 선명해 지는 것을 확인할 수 있었다. 그러므로 고안된 Xag Fl & Xag R1 primer를 이용한 direct PCR 방법은 다른 많은 미생물들로 오염되어진 콩종자에 있는 X. axonopodis pv. glycines를 신속하고 민감하게 검정할 수 있는 효과적인 방법으로 활용될 수 있을 것이다. Direct Polymerase Chain Reaction (PCR) method that combines biological and enzymatic amplification of PCR targets was developed for the detection of Xanthomonas axonopodis pv. glycines on soybeen seeds without DNA isolation. Primers Xag F1 and Xag R1 were designed to specifically amplify a 401 bp fragment of the glycinecin A gene of X axonopodis pv. glycines. Xag F1 and Xag R1 were used to carry out the PCR analysis with genomic DNA from 45 different bacterial strains including phylogenetically related bacteria with X axonopodis pv. glycines, and other bacterial strains of different genus and species. The PCR assay using this set of primers were able to detect X axonopodis pv. glycines with DNA concentration as low as 200 fg and <TEX>$1.8{\times}10^3$</TEX> cfu/ml. The Xag was detected from the seed samples incubated for 2 hrs with shaking and the intensity of the band was increase with the incubation time of seeds. The Direct PCR assay method without DNA isolation makes detection of X. axonopodis pv. glycines on soybean seeds easier and more sensitive than other conventional methods. The developed seed assay using direct PCR method will be useful for the specific detection of X. axonopodis pv. glycines in soybean seed samples.

Elaboration of methods for detection of Xanthomonas axonopodis pv. phaseoli on bean seeds

Xanthomonas axonopodis pv. phaseoli detection on artificially inoculated bean seeds was investigated. The method of the International Seed Federation - ISF (2006) was used. It included bacteria extraction from seeds and isolation on semiselective media with pathogenicity test of the investigated isolates. ELISA and PCR were used for verification of results. The results showed that the semiselective media MT (Milk Tween Agar) and XCP1 (Xanthomonas campestris pv. phaseoli Agar) were very suitable for isolation of X. a. pv. phaseoli. Pathogenicity was confirmed on young bean plants. ELISA test and PCR confirmed that all investigated isolates and reisolates belong to the bacterium X. a. pv. phaseoli.

Deteksi Cepat Penyakit Pustul Bakteri pada Kedelai Menggunakan Teknik PCR dengan Primer Spesifik

A rapid polymerase chain reaction (PCR)-based procedure was developed for detection of Xanthomonas axonopodis pv. glycines, the causal agent of bacterial pustule disease on soybean. A set of primers was designed from partial sequence of the pathogenicity gene of X. axonopodis pv. glycines strain YR32. Specific PCR product of 490 base pairs was produced from strains of X. axonopodis pv. glycines originally from Indonesia as well as from Taiwan. No other pathovars and bacterial species among those tested showed amplification product under optimized PCR conditions. Shaking infected soybean leaves in phosphate buffer saline during six hours was proved to be an essential in order to increase cell number of the bacterial. The procedure was applicable and reliable for detecting of pathogens in infected plant materials. The procedure was proved to be more effective than that of conventional detection and could be of great help for monitoring of pustule bacterial disease in the soybean fields.

Detection of Xanthomonas axonopodis pv. citri on Satsuma Mandarin Orange Fruits Using Phage Technique in Korea

A phage technique for detection of Xanthomonas axonopodis pv. citri, a causal bacterium of canker on Sastuma mandarin fruits was developed. Phage and ELISA techniques were compared for their sensitivity for detection of Xanthomonas axonopodis pv. citri on orange fruits. Both of techniques revealed a similar efficiency for the bacterial detection; the pathogenic bacteria were observed in pellet from the fruits with over one canker spot with below 2 mm in diameter. In field assays, the increase of phage population(120%) on surface of the fruits related to the disease development one month later indicated that the bacterial pathogens inhabit on the surface. The procedure will be effectively used for detection of only living bacterial pathogen on fruit surfaces of Satsuma mandarin and for the disease forecasting.

Primers based on the rpf gene region provide improved detection of Xanthomonas axonopodis pv. citri in naturally and artificially infected citrus plants

To have a PCR-based detection method for Xanthomonas axonopodis pv. citri (Xac) using primers designed in a specific region of its genome. A Xac-specific region was identified inside the rpf gene cluster of strain IAPAR 306 in an analysis of its complete genomic sequence. Two primers were designed, Xac01 and Xac02, which, when used in a standard PCR assay, direct the amplification of a 581 bp fragment from DNA of strains belonging to Xac from different regions around the world including unusual American and Asian strains. This product was not observed when DNA from strains of the closely related X. a. aurantifolli and X. a. citrumelo were used as templates. Extracts prepared from 28 xanthomonads of other species, and epiphytic bacteria isolated from citrus also failed to produce products with these primers. Amplification was obtained from cells grown in vitro, from extracts of both fresh and dried citrus canker lesions and from washes of inoculated but asymptomatic leaf surfaces. In sensitivity tests, this PCR technique detected as few as 100 cells. Primers Xac01 and Xac02 provide specific and sensitive detection of Xac in all citrus tissues where the pathogen is found. This PCR-based diagnostic test is suitable for monitoring asymptomatic plants in areas where the bacteria is endemic, in plant quarantine and regulatory situations, and also for obtaining an accurate diagnosis in a very short time. These are important characteristics for any assay to be used for the management of citrus canker disease.

A Semi-Selective agar medium to detect the presence of Xanthomonas axonopodis pv. malvacearum in naturally infected cotton seed

A semi-selective agar medium was developed for detection of Xanthomonas axonopodis pv. malvacearum (Xam) in cotton (Gossypium hirsutum) seed. The basic medium was peptone-sucrose-agar (PSA). Criteria for the semi-selective medium were the typical colony characters of Xam and its pathogenicity on cotton. Several systemic fungicides and antibiotics in different concentrations were tested alone or in combination with others. The final composition of the semi-selective agar medium was established after several attempts in order to inhibit most of the fungal and bacterial saprophytes and favour the development of Xam. It contained PSA + cyclohexamide, cephalexin, pencycuron, triadimenol and tolylfluanid. The bacteria were recovered from naturally infected seeds by the direct plating of 2,000 surface disinfected seeds on the semi-selective medium. The recovery of the pathogen from naturally infected leaf tissues and in dilution plating, on semi-selective medium and on nutrient agar, were comparable. Among the three detection methods tested, the semi-selective medium was found to be the most reliable and quantifiable. Degree of severity of angular leaf spot in the field was not always correlated with the level of infection in the seed. This is the first report of a semi-selective agar medium to detect the presence of Xam in naturally infected cotton seed.

Sensitive and specific detection of Xanthomonas axonopodis pv. citri by PCR using pathovar specific primers based on hrpW gene sequences

A sensitive and specific assay was developed to detect citrus bacterial canker caused by Xanthomonas axonopodis pv. citri, in leaves and fruits of citrus. Primers XACF and XACR from hrpW homologous to pectate lyase, modifying the structure of pectin in plants, were used to amplify a 561 bp DNA fragment. PCR technique was applied to detect the pathogen in naturally or artificially infected leaves of citrus. The PCR product was only produced from X. axonopodis pv. citri among 26 isolates of Xanthomonas strains, Escherichia coli (O157:H7), Pectobacterium carotovorum subsp. carotovorum, and other reference microbes.

Sensitive detection of Xanthomonas axonopodis pv. dieffenbachiae on Anthurium andreanum by immunocapture-PCR (IC-PCR) using primers designed from sequence characterized amplified regions (SCAR) of the blight pathogen

One of the most devastating Xanthomonas diseases affecting the Anthurium cut flower industry worldwide is the bacterial blight caused by Xanthomonas axonopodis pv. dieffenbachiae (Xad). The disease can be spread through latently infected tissue-cultured plants that are used for the propagation of Anthurium worldwide. Current disease diagnostic techniques involve the use of semi-selective media and serological tests. This study describes the development of a PCR tool combined with a genus-specific monoclonal antibody for the sensitive detection of the pathogen directly from plants. It was demonstrated that the immunocapture PCR (IC-PCR) was more sensitive than the conventional PCR and even more sensitive than indirect ELISA for the detection of the pathogen. Latently infected plants could be positively screened for the presence of the pathogen. Three sets of primers were designed from DNA probes that were reported to show some specificity to the pathovar dieffenbachiae. The use of all three sets of primers in a single reaction successfully amplified the three individual loci when bacterial DNA was used as a template. The multiplex PCR generated PCR profiles that could differentiate between the reference strains of X. axonopodis pv. dieffenbachiae from other control bacteria. The new primers could therefore be used both for the diagnosis of Anthurium blight in single PCR reactions and also for the profiling of Xanthomonas. pv. dieffenbachiae strains using the multiplex PCR technique.

The Citrus Canker Epidemic in Florida: The Scientific Basis of Regulatory Eradication Policy for an Invasive Species

The Citrus Canker Epidemic in Florida: The Scientific Basis of Regulatory Eradication Policy for an Invasive Species

Specific Detection of Xanthomonas axonopodis pv. manihotis with a DNA Hybridization Probe

AbstractA dot‐blot assay was developed for the detection of Xanthomonas axonopodis pv. manihotis (Xam), the causal agent of cassava bacterial blight. A 898 bp DNA fragment unique to Xam strains was cloned and evaluated as a diagnostic DNA probe. The DNA probe (p898) hybridized with the total DNA of 362 Xam strains tested but not with any of the 40 other Xanthomonas strains tested or with saprophytes associated with the cassava plant. A quick colony hybridization procedure was developed to identify Xam strains. The probe also detected Xam strains in crude extracts of leaf and stem lesions, and in cassava fruits and sexual seeds that were naturally infected. The overall sensitivity of the dot blot method for detecting Xam in stem and leaf samples was about 103 colony‐forming units per reaction. Because the dot‐blot hybridization technique is sensitive and rapid, it can be easily used for culture indexing.