April - June 2007 - Kasetsart University

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270 Southern blot hybridization The amplified PCR products from all strains of Xcc (Table 2) were hybridized with 354bp probe but not with other xanthomonads (Figure 1B). Sequencing of target PCR product The 354 bp, expected PCR fragment from Xcc was amplified by 354 F/R primers. The sequences obtained from 354 F/R cloned were blast(N) in Genbank database at National Center for Biotechnology Information (http://www.ncbi. nlm.nih.gov/BLAST/). Searching results showed that sequences of 354 bp of expected product were 99.7% similar to sequence of Xac strain 306 gene XAC 2443 (Accession AE011881) (Figure 2). DISCUSSION Xanthomonas citri subsp.citri (Xcc) is the causal agent of citrus bacterial canker disease, an important pathogen of Citrus species, and it is important to international phytosanitary quarantine in many citrus producing countries worldwide (OEPP/EPPO, 2005). Other bacterial citrus pathogens, X. fuscans subsp. aurantifolii and X. alfalfae subsp. citrumelonis are closely related to Xcc and X. alfalfae subsp. citrumelo and have been <strong>Kasetsart</strong> J. (Nat. Sci.) 41(2) easily misidentified as Xcc (Schoulties et al., 1987). Effective control and eradication of citrus canker needs a rapid, specific, and sensitive detection techniques. The polymerase chain Figure 3 PCR amplification products of 354 F/ R primers on 1% agarose gel 0.5x TBE buffer. Lane 1) marker DNA 1 kb (Fermentas ? ), 2-7) chromosomal DNA of X. citri subsp. citri at concentration from 50 ng to 50 fg per microliter by ten-fold dilution. Table 3 Sensitivity of classical PCR for detecting viable cells and purified DNA of Xanthomonas citri subsp. citri strain T7. Dilution a Cell/µl PCR results c DNA concentration b PCR results c 0.1 OD 600 nm 7.0×10 4 + 50 ng/µl + 10 -1 7.0×10 3 + 5 ng/µl + 10 -2 7.0×10 2 + 500 pg/µl + 10 -3 7.0×10 + 50 pg/µl + 10 -4 7.0 - 5 pg/µl - 10 -5 0 - 500 fg/µl - 50 fg/µl - a Cell suspension of Xcc was adjusted to turbidity 0.1 O.D. of wavelength 600nm which gave 7.0×104 cell/µl and ten-fold serially diluted to 10-5 . The number of cell per microliter was counted by haemacytometer. b DNA of Xcc was adjusted by ten-fold dilution from 50 ng/µl to 50 fg/µl. c PCR specific amplification with 354 F/R primers were performed following the reaction mix and amplification program in methods. Presence (+) or absence (-) of unique predicted PCR product size after agarose gel electrophoresis.
eaction technique has been used for rapid and reliable detection for many plant pathogens (Henson and French, 1993). Thus, this PCR technique is suitable for routine assay in international quarantine which requires a rapid and sensitive method for routine assay. Plasmid (pthA gene family) and chromosomal DNA of Xcc have been used to design specific PCR primers (Hartung et al., 1993, Kingsley et al., 2000, Mavrodieva et al., 2004) for detection of Xcc. In this experiment, new specific primers, 354 F/R primers, were designed from a fragment in chromosomal DNA of Xcc by substractive hybridization that translated to conserved hypothetical protein (gene XAC2443). The results were that 354 primers showed specific DNA amplification of all strains of Xcc. These Xcc strains were isolated from different hosts and geographical areas in Thailand including strains from Japan and Saudi Arabia which gave the expected 354 bp PCR fragment but not from other xanthomonads (Table 2). This is the first report of using sequences from a conserved hypothetical protein in chromosomal DNA to design specific PCR primers for detection of Xcc. The PCR primers from conserved hypothetical protein region showed more specificity than primers from plasmid DNA (VM3-VM4 and 2-3 primers, Table 2). The primers designed from chromosomal DNA, KF-KR, had specific amplification with all strains of Xcc in this experiment. The primers also produced prominent band with Xcc A and A*strain but the reactions with A w -strain and X. fuscans subsp. aurantifolii (B and C-strain) were inconsistent and also gave more primer-dimer products (Mavrodieva et al., 2004). At present, PCR product fragment of KF-KR primers still cannot be identified when searching with Genbank database by using BLAST program provided by National Center for Biotechnology Information (NCBI). The primers designed from plasmid <strong>Kasetsart</strong> J. (Nat. Sci.) 41(2) 271 DNA, 2-3 primers and pthA gene family, VM3- VM4 primers, amplified not only all strains of Xcc but also other xanthomonad strains. Primers 2-3 cross-reacted with X. citri subsp. malvacearum and primers VM3-VM4 cross-reacted with X. fuscans subsp. aurantifolii, X. citri subsp. malvacearum and X. campestris pv. glycines (Table 2) because these primers were designed for detection of avirulence or pathogenicity genes which are commonly found in the genus Xanthomonas (Gabriel, 1997). The plasmid DNA has been reported as being easily cured, frequently mutants within the internal sequence and not present in all pathogens (Miyoshi, 1998). The propose of VM3-VM4 primers is to develop universal detection of Xsc and X. fuscans subsp. aurantifolii. However, results in this experiment showed that the primers did not completely detect all target strains of xanthomonad. They detected one third from X. fuscans subsp. aurantifolii (B-strain), all 4 strains of X. fuscans subsp. aurantifolii (C-strain) but did not detect any of X. fuscans subsp. aurantifolii (Dstrain). Nine strains of X. citri subsp. malvacearum and X. campestris pv. glycines also reacted with VM3-VM4 primers. The pthA, pthB and pthC, members of pthA gene family, belong to a family of avirulence or pathogenicity genes found in the genus Xanthomonas (the avrBs3/pthA gene family; Leach and White, 1996; Gabriel, 1997) and these may be transferred horizontally on plasmids between Xcc and X. fuscans subsp. aurantifolii (Brunings and Gabriel, 2003). The results of this experiment also confirmed that the avrBs3/pthA gene family is distributed in X. citri subsp. malvacearum and X. campestris pv. glycines. The assay of 354 F/R primers with classical PCR had the ability to detect a lower limit of about 70 CFU/µl of viable cells and the lower limit of detection of 50 pg/µl of purified Xcc total DNA. Sensitivity of other primers to detect viable cells of Xcc and purified Xcc total DNA were 10 CFU/µl and 25 pg/µl for 2-3 and 10 CFU/µl and 1
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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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pineapple fruit allowed the accumul
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Kasetsart J. (Nat. Sci.) 41(2) 215
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Cluster analysis Cluster analysis u
Page 19 and 20: Kasetsart J. (Nat. Sci.) 41(2) 219
Page 21 and 22: Table 3 (Continued) 1 2 3 4 5 6 7 8
Page 25 and 26: CONCLUSION AFLP markers classified
Page 27 and 28: Kasetsart J. (Nat. Sci.) 41 : 227 -
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 and 62: Kuhlmann, V. and B. Foroughi-Wehr.
Page 63 and 64: Xanthomonas fuscans subsp. aurantif
Page 67 and 68: was collected and washed with 70% e
Page 69: expected size was amplified with 35
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
Page 113 and 114: GC. Analytical methods Total carboh
Page 115 and 116: ash. The crude hot water polysaccha
Page 117 and 118: 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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