April - June 2007 - Kasetsart University

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Kasetsart J. (Nat. Sci.) 41 : 262 - 273 (2007) Novel PCR Primers for Specific Detection of Xanthomonas citri subsp. citri the Causal Agent of Bacterial Citrus Canker Udomsak Lertsuchatavanich 1 , Ampaiwan Paradornuwat 1 , Junlapark Chunwongse 2 , Norman W. Schaad 3 and Niphone Thaveechai 1 * ABSTRACT The new primers were developed for specific detection of Xanthomonas citri subsp. citri (Hasse) (Xcc) [syn. X. axonopodis pv. citri (Xac)], the causal agent of Asiatic citrus canker disease. Twenty three strains of Xcc and 34 strains of other xanthomonads including X. fuscans subsp. aurantifolii, X. alfalfae subsp. citrumelonis, X. campestris pv. campestris, X. campestris pv. glycines, X. citri subsp. malvacearum and X. fuscans subsp. fuscans were tested for specificity of new primers by classical PCR. The results showed that these 354 F/R primers specifically amplified all of Xcc strains but not other xanthomonad strains. The 354-bp PCR fragment was sequenced and its nucleotide sequences were compared for similarity with Genbank database. The 354-bp nucleotide sequences were 99.7% similar to gene XAC2443 of Xac strain 306 (Accession AE011881). The sensitivity of these specific primers for detection of viable cells and total DNA of Xcc were 70 CFU/µl and 50 pg/µl, respectively. Therefore, these novel primers can be used as an alternative application for rapid and specific detection of Xcc. Key words: Xanthomonas, bacterial citrus canker, detection, polymerase chain reaction INTRODUCTION Bacterial canker of citrus is a serious disease of most citrus species and cultivars in many citrus-producing areas worldwide. Five forms of the disease have been described, cankers A, B, C, D, and E. Canker A or A-strain (Asiatic canker) is the most common and most damaging of the citrus canker strains (Schubert et al., 2001). It was originally found in Asia and is by far the most widespread. Recently, information based upon DNA sequences comparison or alignment of 16S- 23S internal transcribed spacers (ITS) regions with amplified fragment length polymorphism (AFLP) analysis of the five recognized forms of citrus canker was demonstrated by Schaad et al. (2005, 2006). Citrus pathogens were reclassified into three pathovars of Xanthomonas campestris (or X. axonopodis): pathovars citri for strain “A”, aurantifolii for strains “B/C/D” and citrumelo for strain “E”, which were revealed as taxon I including all “A” strains; taxon II containing all “B”, “C”, and “D” strains; and taxon III containing all “E” strains. The taxa I, II and III citrus strains were reinstated with new names, respectively as Xanthomonas citri subsp. citri (Hasse, 1915), 1 Department of Plant Pathology, Faculty of Agriculture, Kasetsart University, Bangkok 10900, Thailand. 2 Department of Horticulture, Faculty of Agriculture, Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom 73140, Thailand. 3 USDA-ARS FDWSRU, 1301 Ditto Avenue, Fort Detrick, MD 21702-5023, USA. * Corresponding author, e-mail: agrnpt@ku.ac.th Received date : 31/05/06 Accepted date : 24/10/06
Xanthomonas fuscans subsp. aurantifolii (Gabriel et al., 1989), and Xanthomonas alfalfae subsp. citrumelonis (Riker et al., 1935). A new strain of X. citri subsp. citri, designated A*, was identified in southwest Asia and has a restricted natural host range to Mexican lime (Verniere et al., 1998). Another Aw-strain, which behaves similarly, has recently been discovered in Florida. This strain has a restricted host range that includes Mexican lime and alemow (Citrus macrophylla) (Sun et al., 2004). The A-strain is the target of international quarantine efforts, in which the development of rapid and reliable procedures for the diagnosis of this pathogen has been a priority. The polymerase chain reaction (PCR) is a principle for plant disease diagnosis (Henson and French, 1993). However, routine application of PCR for detection of plant pathogens can result in false-positive diagnosis when PCR primers are non-specific to the pathogen. Several sets of primers have been developed for diagnosis of Xcc. Non-specific amplification of Hartung’s primers (Hartung et al., 1993) for detection of Xcc were reported by Zaccardelli and Mazzucchi (1997). Miyoshi et al. (1998) studied similarity of the intergenic spacer region between 16S-23S rRNA genes among X. citri subsp. citri, X. campestris pv. glycines, X. alfalfae subsp. alfalfae (X. c. pv. alfalfae), X. c. pv. physalidicola, X. c. pv. pisi, X. c. pv. pruni, X. c. pv. cucurbitae and X. c. pv. vesicatoria, and designed primers XCF and XCR based on the data from this study. Primers XCF- XCR were not only for detection of Xcc but also for X. c. pv. glycines. Kingsley et al. (2000) developed fluorogenic PCR assay for specific detection of Xcc A and A*-strain using the forward/reverse primers and probes designed from unique RAPD fragment to target 126 bp amplicon. Mavrodieva et al. (2004) designed primers, VM3 and VM4, for real-time PCR by selecting to amplify the pthA gene family and run experiments to compare with Kingsley’s primers, KF and KR. The results showed that Xcc (A, A* and Aw) and Kasetsart J. (Nat. Sci.) 41(2) 263 X. fuscans subsp. aurantifolii (B and C) reacted and gave expected product sizes with VM3-VM4 primers. On the other hand, Kingsley’s primers gave prominent band with Xcc A and A*-strain but the reaction with A w -strain and X. fuscans subsp. aurantifolii (B and C) were inconsistent and also gave more primer-dimer products when compared with VM3-VM4 primers. The purpose of this study was to design specific PCR primers of Xcc from genomic DNA especially gene XAC2443 (Accession AE011881) in order to apply them for detection of this international quarantine bacterial pathogen of citrus. MATERIALS AND METHODS Bacterial strains To obtain original local strains to be used in this study, canker lesions on lime (Citrus aurantifolia), mandarin (C. reticulata), sweet orange (C. sinensis) and leach lime (C. hystrix) were collected from leaves, twigs, and fruit from each major citrus growing area in Thailand. The corky-like raised surface lesions, surrounded by a yellow halo were washed in running water for 1-2 minutes, sprayed with 70% ethyl alcohol, and airdried. Each lesion was removed from the leaf and cut into 4-5 pieces then soaked in 0.85% NaCl for 20 minutes. A loop of the suspension was streaked onto Fieldhouse and Sasser (FS) agar (Schaad et al., 2001) and incubated at 30°C. After 3-4 days, plates were examined for small green-colored starch hydrolyzing colonies typical of Xcc. Promising colonies of Xcc were transferred onto nutrient agar (NA) (Schaad et al., 2001) twice and stored either in sterile distilled water at room temperature or on NA slants at 4°C, and in 50% glycerol at -80°C. Several bacterial strains from Japan and the United States of America were included in this experiment (Table 1). PCR primers The new primer pair namely 354F-354R
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April - June 2007 Volume 41 Number
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KASETSART JOURNAL NATURAL SCIENCE T
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Kasetsart J. (Nat. Sci.) 41 : 205 -
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harvesting time which started from
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y Chen and Paull (2000). Figure 1 a
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Page 17 and 18: Cluster analysis Cluster analysis u
Page 21 and 22: Table 3 (Continued) 1 2 3 4 5 6 7 8
Page 25 and 26: CONCLUSION AFLP markers classified
Page 29 and 30: difference of soil texture used in
Page 31 and 32: under wet soil condition planting.
Page 33 and 34: tropics. Following the expansion, w
Page 35 and 36: sealed in a plastic box with 1 cm w
Page 37 and 38: moisture content increment after so
Page 39 and 40: Frequency Frequency 30 20 10 0 20.0
Page 41 and 42: School, Kasetsart University. The a
Page 43 and 44: for combining ability of lines (Lam
Page 45 and 46: selective mass sibbing within indiv
Page 47 and 48: Although most of S 2-interfamily hy
Page 49 and 50: composite sets as used in this stud
Page 53 and 54: days. The numbers of calli producin
Page 55 and 56: the report of Ching (1982) showing
Page 57 and 58: clearly amplified bands generated b
Page 59 and 60: caused by either the recombination
Page 61: Kuhlmann, V. and B. Foroughi-Wehr.
Page 67 and 68: was collected and washed with 70% e
Page 69 and 70: expected size was amplified with 35
Page 71 and 72: eaction technique has been used for
Page 73 and 74: Schaad, N.W., D. Opgenorth and P. G
Page 75 and 76: MATERIALS AND METHODS Model descrip
Page 79 and 80: calculated TF value calculated TF v
Page 81 and 82: sincere gratitude and deep apprecia
Page 83 and 84: 1989). In monogastrics, the inclusi
Page 85 and 86: of all LLS samples in this study we
Page 87 and 88: Melbourne, Parkville, Victoria. Goe
Page 89 and 90: considered responsible for low prod
Page 93 and 94: mineral supplementation suggested b
Page 95 and 96: Body weight chane (kg/head) 32 30 2
Page 97 and 98: CONCLUSION AND RECOMMENDATION With
Page 99 and 100: SAS. (Statistical Analysis System).
Page 101 and 102: genes covering a variety of desirab
Page 103 and 104: mg/l NAA and 0.5 mg/l zeatin) incub
Page 105 and 106: 5. Purification by various sucrose
Page 107 and 108: as the culture period increased. So
Page 109 and 110: culture, pp. 1-20. In L.C. Fowke an
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GC. Analytical methods Total carboh
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ash. The crude hot water polysaccha
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spirulan (H-SP), and a desulfated c
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cells often displayed an increased
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LITERATURE CITED Bell, T.A. and D.V
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for alternative sources of supply.
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BR2.1.2 formed the same clade with
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supplement with glutamate (Iida et
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optimal for S. limacinum BR2.1.2 fo
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fluorescent lamp at 1.5 kLux develo
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Wisconsin, USA). Total extracted RN
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RESULTS Cloning and sequencing of m
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Expression of rmIL-2 and purificati
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other strains of mice have differen
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dose interleukin-2 therapy promotes
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The chitosano-oligosaccharides are
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enzyme and cells were maximized by
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of crude chitosanases. Fortunately,
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Relative activity (%) 100 80 60 40
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Uchida, K. Tateishi, O. Shida and K
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MATERIALS AND METHODS 1. Materials
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without coating and coated with pec
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in Table 5-7. It was found that the
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the cultures at 10,200 × g for 15
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incubation and reached the final pH
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y lysine- and tyrosine-decarboxylas
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during the stages of ripening as su
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As the 25 companies were divided in
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All the data comes up with informat
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as piece “A” or straight as pie
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et al. (1998); Zeng (2000); and Tal
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p 1, i * Kasetsart J. (Nat. Sci.) 4
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c ≤ c2 jq2 j n q p k p * 2, j , ,
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algorithm would consider RM 5, prio
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Kasetsart J. (Nat. Sci.) 41(2) 389
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the maximum deviation of about 10%.
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Stock Quantities Using Heuristic Op
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which dynamically and automatically
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2. Framework architecture and compo
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Risk (VaR) calculation which was im
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Throughput (job/sec) 7 6 5 4 3 2 1
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Speed up 40 35 30 25 20 15 10 5 0 F
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and discovery, resource selection a
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April - June 2007 - Kasetsart University

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