Characterization of Novel Strains of Citrus Canker Bacterium from Citrus in Taiwan

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(1)Characterization of Citrus Canker Bacteria in Taiwan 台灣農業研究 (J. Taiwan Agric. Res.) 57(4):265–278 (2008). 265. Characterization of Novel Strains of Citrus Canker Bacterium from Citrus in Taiwan1 Hsin-Cheng Lin2,5, Hsiang Chang3, and Kuo-Ching Tzeng4 ABSTRACT Lin, H. C., H. Chang, and K. C. Tzeng. 2008. Characterization of novel strains of citrus canker bacteria from citrus in Taiwan. J. Taiwan Agric. Res. 57:265–278.. Two strains of Xanthomonas axonopodis pv. citri isolated from citrus plants in Taiwan were examined for pathogenicity on the leaves of Mexican lime (Citrus aurantifolia), grapefruit (C. paradisi), Liucheng (C. sinensis), and lemon (C. limon).. The results showed. that two strains induced flated necrotic lesions with watersoaked margin on the leaves of four Citrus species. Based on physiological, biochemical, genetic and proteinic characterizations including NaCl tolerance, hydrolysis of gelatin, oxidation of carbon sources, polymerase chain reactions with primers specific to X. axonopodis pv. citri (Xac), rep-PCR, and SDS-PAGE analysis, these two strains were identified as Xac and grouped into Xac-Ap type. Both strains could be differentiated from X. axonopodis pv. citrumelo (pathotype E) by oxidation of carbon sources, pectolytic activity, and amplified DNA profiles of PCR, while they could be only differentiated from type Xac-Af strain by a distinct pathogenicity on the leaves of Mexican lime. Taiwan.. Our data indicated they appeared to be novel strains of Xac in. We designated these two atypical symptoms-inducing strains as Xac-Ap type which. are different from strains of types Xac-A , Xac-Af, Xac-Ar, Xac-A* and Xac-Aw described previously. Key words: Citrus canker, pthA gene, Rep-PCR, SDS-PAGE, Xanthomonas axonopodis pv. Citri.. 1. Contribution NO. 2335 from Agricultural Research Institute, Council of Agriculture. Accepted: November 5, 2008. 2. Assistant professor, Department of Biotechnology, Chung Chou Institute of Technology, Changhua, Taiwan, ROC. 3. Senior Research Fellow, Biotechnology Division, ARI, Wufeng, Taichung, Taiwan. ROC. 4. Professor, Department of Plant Pathology, National Chung Hsing University, Taichung, Taiwan, ROC. 5. Corresponding author, e-mail: lin1368@dragon.ccut.edu.tw; Fax: (04)8394070..

(2) 266. 台灣農業研究. 第 57 卷. 第4期. INTRODUCTION There are distinct types of citrus canker disease caused by various pathovars and variants of the bacterium Xanthomonas axonopdis. Because symptoms are generally similar, the separation of these forms of the bacterial pathogen is based on host range and other phenotypic and genotypic characteristics of the strains. The Asiatic type of citrus canker (pathotype A), caused by the X. axonopodis pv. citri (Xac), is by far the most widespread and severe form of the disease. This is the strain that causes the disease most often referred to as ‘citrus canker’(Stall & Civerolo 1991). Cancrosis B (pathotype B) caused by the X. axonopodis pv. aurantifolii is a minor canker disease of diminishing importance on lemons in Argentina, Paraguay, and Uruguay. Mexican lime, sour orange and pummelo are also susceptible (Civerolo 1984). Cancrosis C (pathotype C) also caused by the X. axonopodis pv. Aurantifolii that can only infect infected Mexican lime in Brazil (Schubert et al. 2001). Strains in pathotypes A, B and C induce raised canker lesions on citrus plants. However, Cancrosis E (pathtype E) was originally described in Florida, induce flat necrotic lesions with watersoaked margins on citrus species (Graham & Gottwald 1991; Stall & Civerolo 1991). The disease was renamed citrus bacterial spot (CBS) and the bacterium was reclassified as X. axonopodis pv. citrumelo (Vauterin et al. 1995). The typical canker lesion caused by X. axonopodis pv. citri is erumpent, with watersoaked, or oily tan or brown colored margin, often surrounded by a chlorotic halo. However, several distinct phenotypes of Xac have been reported (Verniere et al. 1998; Mohammadi et al. 2001; Sun et al. 2004; Lin et al. 2005). Strains from southwest Asia (Xac-A*) and Florida (Xac-Aw) elicit typical canker lesions on Mexican lime and induce flat watersoaked lesions on grapefruit (Verniere et al. 1998; Sun et al. 2004). These new strains have a limited host range in Mexican lime that differs from Xac strains which have a wide host range. Recently, atypical symptoms-inducing strains of type Xac-Af and type Xac-Ar were identified in Taiwan (Lin et al. 2005). A strain of Xac-Af induces flat necrotic lesion with watersoaked margin and light chlorotic halo on leaves of grapefruit, lemon and Liucheng, while induces typical canker lesions on Mexican lime. Strains of Xac-Ar induce restricted and raised corky lesions with no watersoaked margin and light chlorotic halo on leaves of citrus species. Several approaches have been employed in studying the taxonomy and discriminating between strains of X. axonopodis belonging to various pathotypes including physiological and biochemical assays (Verniere et al. 1991; Verniere et al. 1993), serological approaches (Civerolo & Fan 1982; Alvarez et al. 1991), phage typing (Verniere et al. 1998), protein profiles (Vauterin et al. 1991), fatty acid analysis (Graham et al. 1990; Vauterin et al. 1991), DNA-DNA hybridization (Vauterin et al. 1991), restriction-fragment length polymorphism (Hartung & Civerolo 1989; Graham et al. 1990), plasmid DNA fingerprints (Pruvost et al. 1992), and specific primer pairs (Hartung et al. 1993; Cubero & Graham 2002; Sun et al. 2004). In addition, Southern hybridization with a pthA probe reveals distinct profiles among strains in pathotype A, pathotype B, pathotype C, and pathotype E (Swarup et al. 1992). And rep-PCR with BOX and ERIC primer pairs has been used to separate pathotypes of Xac or to differentiate strains in.

(3) Characterization of Citrus Canker Bacteria in Taiwan. 267. the same pathotype. The rep-PCR technique also allows to evaluate the diversity of Xac in certain geographic areas of the world (Cubero & Graham 2002). In this study, we examined the pathogenicity of Xac strains from Taiwan on the leaves of Mexican lime, grapefruit, Liucheng, and lemon. We found both of the strains could induce atypical symptoms and further characterized these strains based on their physiological, biochemical, genetic, and proteinic characterizations. The results revealed that these atypical symptoms-inducing strains are novel strains of Xac.. MATERIALS AND METHODS BACTERIA STRAINS AND CULTURE CONDITION A total of six X. axonopodis strains were used in this study, including two typical Asiatic type strains XW19 and 2863 (Xac-A), one atypical symptoms-inducing strains XW47 (Xac-Af type), one citrus bacterial spot (CBS) strain X. axonopodis pv. citrumelo F2 (pathotype E) (Lin et al. 2005), and newly isolated strains X. axonopodis pv. citri XL16 and XL38 from Taiwan. All strains were stored in YPD broth (yeast extract 7 g, bactopeptone 7 g, dextrose 7g, distilled water 1000 mL, pH 7.2) (Verniere et al. 1991) cotaining 20% glycerol at -80℃. When required, each bacterial strain was streaked out from the glycerol stock at -80℃ and cultured on YPD agar plates at 30℃ for 3 days.. PATHOGENICITY TEST Citrus plants (C. paradisi, grapefruit; C. aurantifolia, Mexican lime; C. sinensis, Liucheng; C. limon, lemon) grown in pots under greenhouse conditions were used for examining the pathogenicity of X. axonopodis pv. citri strains. To prepare inoculum, bacterial cells grown overnight in YPD broth were harvested by centrifugation and resuspended in sterile distilled water to a concentration of approximately 108 colony-forming units (CFU)/mL. Prior to inoculation, six wounds per 1-cm2 on young fully expanded citrus leaves were made with a standard 26-gauge needle. An aliquot (20 μL) of bacterial suspension was dropped onto each of six wounds on leaves, and the inoculum drops were wiped off with sterile cotton just after inoculation. Citrus plants inoculated with various X. axonopodis strains were moved into a growth chamber with 65–90% relative humidity and 12-hr light at 30℃ and 12-hr dark at 25℃. Visual symptom was recorded and examined with dissection microscopy.. PHYSIOLOGICAL AND BIOCHEMICAL CHARACTERIZATION Physiological and biochemical characteristics used to differentiate pathovars of X. axonopodis associated with citrus plants were performed to characterize the X. axonopodis pv. citri strains isolated in Taiwan. Salt tolerance was tested by growing bacteria on YPDA plates containing 3% NaCl (Verniere et al. 1998). Hydrolysis of gelatin was performed as described in Lelliott et al. (Lelliott et al. 1966). Oxidation of carbon sources was carried out on Biolog® GN microplates (Biolog Inc., Hayward, CA) as described by Verniere et al. (Verniere et al. 1993). Pectolytic activity was tested on Hildebrand’s medium with pH values at 5.0, 7.0, and 8.5 (Hildebrand 1971)..

(4) 268. 台灣農業研究. 第 57 卷. 第4期. TOTAL DNA EXTRACTION Total DNAs of xanthomonad strains were extracted using the method described by Sambrook et al. (1989). PCR was performed with a thermocycler (GeneAmp PCR System 2400, Perkin-Elmer, CT).. IDENTIFICATION OF Xanthomonas STRAINS WITH SPECIFIC PRIMER PAIRS Primer pairs 2/3 and 4/7 designed from sequence of plasmid of the X. axonopodis pv. citri (Hartung et al. 1993), and primer pairs J-pth1/J-pth2, J-RXg/J-RXc2 designed based on sequences of nuclear localization signal in pthA (Cubero & Graham 2002) and rDNA (Cubero & Graham 2002) of X. axonopodis pv. citri were used to identify or differentiate the Xac strains tested in this study.. REP- AND ERIC-PCR ANALYSES The genetic diversity among the Xac strains was analyzed by REP- and ERIC-PCR. The REP-PCR was carried out as described by Versalovic et al. (Versalovic et al. 1991) with some modifications. A 25 µL reaction mixture contained 150 ng template DNA, 1× Taq buffer, 3 mM MgCl2, 1.25 µM each of primers REP1-1 and REP2-1, 250 µM each of deoxynucleoside triphosphates and 0.8 U Taq polymerase (DyNAzymeTM II DNA Polymerase, Finnzymes Oy Inc. Finland). The amplification condition consisted of 94℃ for 1 min, 44℃ for 1 min, and 65℃ for 8 mins for 30 cycles plus an initial step of 95℃ for 7 mins and a final step of 65℃ for 15 mins. The ERIC-PCR was carried out with primer pair ERIC1R/ERIC2 in a 25 µL reaction mixtures in which the other ingredients were the same as that for REP-PCR; the amplification condition consisted of 94℃ for 1 min, 52℃ for 1 min, and 65℃ for 8 mins for 30 cycles plus an initial step of 95℃ for 7 mins and a final step of 65℃ for 15 mins. PCR products of the REP- and ERIC-PCR were analyzed by 2% agarose gel electrophoresis in 0.5× TAE buffer at 90 V for 4 hrs and were stained with ethidium bromide. REP- and ERIC-PCR fingerprint profiles were converted to binary form (0 = absence; 1 = presence), and Dice coefficient was calculated to determine the similarity among the bacterial strains tested (Dice 1945).. PATHOGENICITY (pthA) GENE DETECTION The primers pthAP7/AR2 described Lin et al. (2005) were used to amplify the region corresponding to nucleotide of pthA. Polymerase chain reaction for pthA gene was performed in a 50 μL mixtures containing 150 ng template DNA, 1× Taq buffer, 1 μM primer pthAP7, 1 μM primer pthAR2, each deoxynucleoside triphosphate at a concentration of 300 μM, 1U of Taq plus DNA polymerase, and 5 μL DMSO. The amplification condition consisted of 94℃ for 1 min, 63℃ for 1 min, and 72℃ for 5 mins for 35 cycles plus an initial step of 94℃ for 10 mins and a final step of 72℃ for 10 mins.. SDS-PAGE ANALYSIS Electrophoresis of cellular proteins was carried out in a discontinuous system under the denaturing condition following the method of Laemmli (Laemmli 1970). A bacterial suspension with an optical density of 1.0 at 630 nm was prepared in 1 mL distilled water in an Eppendorf tube from each strain cultured on nutrient agar medium cantaining 1% glucose. Each sample was spun down at 1000 rpm for 5 mins at 4℃. The bacterial pellet was washed in sterile distilled water and then resuspended in sample.

(5) Characterization of Citrus Canker Bacteria in Taiwan. 269. buffer at 0.2× volume, boiled for 4 mins and centrifuged at 10000 rpm for 10 mins. Fifty microliters of soluble proteins from each sample was loaded into slots of stacking gel (5% w/v). Proteins were fractioned in 12% resolving gel. Electrophoresis was carried out in a vertical gel slab unit (Sigma Chemical Co., London, UK) at a constant voltage of 140 V untile the tracking dye reached near the bottom. Gel was stained in Coomassie brilliant blue R250 and destained in acetic acid: methanol solution without the dye.. RESULTS PATHOGENICITY TEST Based on the symptoms induced on leaves of the four Citrus species, the strains XL16 and XL38 tested were grouped into Xac-Ap type (Table 1). In the Xac-Ap type strains induced flat necrotic lesions with watersoaked margins and light chlorotic haloes on the leaves of Mexican lime (Fig. 1C), grapefruit (Fig. 1F), Liucheng and lemon. The size of lesions induced by strains XL16 and XL38 on leaves of four citrus species was not significantly different from that induced by XW19. The lesion size was in a range from 1.9 to 2.6 mm in diameter 24 days after inoculation.. PHYSIOLOGICAL AND BIOCHEMICAL CHARACTERIZACTION Type Xac-Ap strains XL16 and XL38 were able to grow in the presence of 3% NaCl, possessed hydrolysis ability of gelatin and were positive in utilization of L-fucose, D-galactose and alaninamide in the Biolog® GN plate. They also were positive for pectolytic activity on Hildebrand’s medium at pH 7.0 and pH 8.5 the same as reference strains of types Xac-A and Xac-Af (Table 2), whereas X. axonopodis pv. citrumelo (pathotype E) strain F2 did not utilize L-fucose and with no pectolytic activity on the Hildebrand’s medium at various pH values (Table 2).. IDENTIFICATION OF Xanthomonas STRAINS WITH SPECIFIC PRIMER PAIRS Xac-specific primers 2/3, 4/7, J-pth1/J-pth2 and J-RXg/J-RXc2 were used to identify strains XL16 and XL 38. Specific PCR products were detected from these two strains as well as reference strains of Xac-A 19 and 2863. No specific amplified product was detected from DNA of X. axonopodis pv. citrumelo F2 with these primer pairs (Table 2).. REP- AND ERIC-PCR ANALYSES The similarity coefficient of both atypical symptoms-inducing strains XL16 and XL38 was 0.9–1.0 to Xac reference strains XW19 or 2863 based on REP- and ERIC-PCR analyses (Fig. 2). The size of amplification products ranged from 800 to 2500 base pairs (bp) for ERIC and from 600 to 2000 bp for REP.. PATHOGENICITY (pthA) GENE DETECTION A 3.8 kb DNA product containing a full length of pthA gene of Xac strain was amplified with primer pair pthAP7/pthAR2 from each of Xac strains XL16, XL38 and XW19 but not from X. axonopodis pv. citrumelo strain F2. (Fig. 3)..

(6) 270. 台灣農業研究. 第 57 卷. 第4期. SDS-PAGE ANALYSIS OF TOTAL SOLUBLE PROTEINS The reproducibility of the electrophoresis technique used in this study was checked by preparing protein extracts in duplicate and running all of the extracts in at least two gels. In all cases, a level of reproducibility of more than 0.9 was obtained. A high homogeneity in SDS protein profiles was observed among Xac strains XL16, XL38, XW47, and XW19 and there were no detectable differences among these strains tested (Fig. 4). Table 1. Symptoms induced by Xanthomonas axonopodis pv. citri strains on four Citrus species C. aurantifolii. C. paradisi. C. sinensis. C. limon. Type. Strains. (Mexican lime). (Grapefruit). (Liucheng). (Lemon). A. Canker. Canker. Canker. FW. FW. FW. FW. FW. FW. XW19. Canker z. f. XW47. Canker. p. XL16,XL38. A. A z. FW. Canker: Typical erumpent canker lesion with watersoaked margin and light chlorotic halo; FW: Flat necrotic lesion with watersoaked margin and light chlorotic halo.. Table 2. Physiological, biochemical and genetic characteristics of Xanthomonas axonopodis pv. citri type Ap strains XL16 and XL38 Item. XL16. Growth on NaCl (3%). +. Hydrolysis of gelatin. +. v. XL38. XW47. XW19 w. 2863. +. +. +. +. +. +. +. +. +. +. F2. Oxidation of carbon sources z D-Galactose. +. +. +. +. +. +. Alaninamide. +. +. +. +. +. +. +. +. +. +. +. －. Hildebrand’s medium (pH5.0). －. －. －. －. －. －. Hildebrand’s medium (pH7.0). +. +. +. +. +. －. Hildebrand’s medium (pH8.5). +. +. +. +. +. －. 2/3. +. +. +. +. +. －. 4/7. +. +. +. +. +. －. J-pth1/J-pth2. +. +. +. +. +. －. J-RXg/J-RXc2. +. +. +. +. +. －. L-Fucose Pectolytic activity on. y. PCR with primers x. z. Oxidation of carbon sources was done in the Biolog GN plates as described by Verniere et al. (1993). Pectolytic activity was tested on Hildebrand’s medium (Hildebrand 1971). x The primer pairs 2/3 and 4/7 are specific for DNA fragments in the X. axonopodis pv. citri plasmid (Hartung et al. 1993); The primer pair J-pth1/J-pth2 is specific for the nuclear localization signal in pthA of X. axonopodis pv. citri (Cubero & Graham 2002); The primer pair J-RXg/J-RXc2 is specific for rDNA of X. axonopodis pv. citri (Cubero & Graham 2002). w Strains XW19 and 2863 were reference strains of type Xac-A for X. axonopodis pv. citri; XW47 was a reference strain of type Xac-Af for X. axonopodis pv. citri ; F2 was a reference strain for X. axonopodis pv. citrumelo (pathotype E). v ＋: positive reaction; －: negative reaction. y.

(7) Characterization of Citrus Canker Bacteria in Taiwan. 271. Fig. 1. Symptoms induced on leaves of Mexican lime (A, B, C) and grapefruit (D, E, F) by strains of X. axonopodis pv. citri 24 days after inoculation. (A and D): Typical erumpent canker lesions with watersoaked margin and light chlorotic halo induced by type Xac-A strain XW19; (B): Typical erumpent canker lesions with watersoaked margin and light chlorotic halo induced by type Xac-Af strain XW47; (E): Flat necrosis lesions with watersoaked margin and light chlorotic halo induced by type Xac-Af strain XW47; (C and F): Flat necrosis lesions with watersoaked margin and light chlorotic halo induced by type Xac-Ap strain XL38. (Bars = 1 mm)..

(8) 272. 台灣農業研究. 第 57 卷. 第4期. DISCUSSION According to symptoms induced by Xac on leaves of four Citrus species, we found type Xac-Ap strains XL16 and XL38 failed to induce any erumpent canker lesions typical of Xac-A on the leaves of Mexican lime, grapefruit, Liucheng, and lemon but they induced flat necrotic with watersoaked lesions on the leaves of four Citrus species. Symptom induced by type Xac-Ap strains on the leaves of Mexican lime were different from those induced by strains of types Xac-A, Xac-Af, Xac-Ar, Xac-A*, and Xac-Aw (Verniere et al. 1998; Sun et al. 2004; Lin et al. 2005). To our knowledge, there is no any other Xac strain which is pathologically similar to strains of type Xac-Ap.. Fig. 2. Rep-PCR patterns of Xanthomonas axonopodis pv. citri strains using ERIC- (A) and REP- primer pair (B). Lanes 1-5: DNA templates from stains XL16, XL38, XW47, XW19 and 2863, respectively; lane M: Bio 100 DNA ladder..

(9) Characterization of Citrus Canker Bacteria in Taiwan. 273. Fig. 3. Amplification profiles of DNAs from Xanthomonas axonopodis strains by polymerase chain reaction with primer pair pthAP7/pthAR2 (A). Lanes 1-3: X. axonopodis pv. citri strains XL16, XL38 and XW19, respectively; lane 4: X. axonopodis pv. citrumelo F2; lane M: Gen-KB DNA ladder. The predicted 3.8 kb DNA fragment containinng an entire length of pthA gene is marked with an arrow in the margin.. Fig. 4. SDS-PAGE analysis of total soluble proteins from different strains of Xanthomonas axonopodis pv. citri Lanes 1-4: X. axonopodis pv. citri strains XL16, XL38, XW47 and XW19, respectively; lane M: the protein molecular weight markers..

(10) 274. 台灣農業研究. 第 57 卷. 第4期. In physiological and biochemical tests, the atypical symptoms-inducing strains of type Xac-Ap could be grown on 3% NaCl, hydrolyze gelatin and utilize L-fucose, D-galactose and alaninamide. These strains shared typical Xac group profiles of assimilation of these three carbon sources as reported by Verniere et al. (1993). This study also showed that the reference strain X. axonopodis pv. citrumelo F2, a CBS strain, could utilize D-galactose and alaninamide but not L-fucose. Therefor, type Xac-Ap could be differentiated from X. axonopodis pv. citrumelo by the carbon source utilization (Table 2). Similarity coefficients obtained from REP- and ERIC-PCR analyses grouped Xac-Ap strains XL16 and XL38 with the reference strains of types Xac-A and Xac-Af into the same group. REP- and ERIC-PCR analyses did not display any major difference among the Xac strains used in this investigation (Fig. 2). Specific primers have been designed to identify or differentiate of Xanthomonas spp. associated with citrus plant (Hartung et al. 1993; Cubero & Graham 2002). In this study, strains in Xac-Ap could be identified as Xac with primer pairs 2/3, 4/7, J-pth1/J-pth2, J-RXg/J-RXc2, and pthAP7/pthAR2. However, strain F2 can not be detected with these primer pairs. Thus, these primer pairs could be used to differentiate Xac-Ap strains from X. axonopodis pv. citrumelo strain. Strains of Xac-Aw can not be detected with primer pairs 2/3 (Cubero & Graham 2002) and 4/7 (Sun et al. 2004), and a distinct DNA profiles could be amplified from Xac-A* strains with primer pair pthAP7/pthAR2 (Lin et al. 2005). Thus, the primer pairs 2/3, 4/7 and pthAP7/pthAR2 could be used to differentiate type Xac-Ap strains from types Xac-Aw or Xac-A* strains. Sequences of lrp gene have been used to characterize the relationship among strains in different pathovars of X. axonopodis and other Xanthomonas species (Cubero & Graham 2004). Based on the lrp gene sequences, atypical symptoms-inducing strains XL16 and XL38 in type Xac-Ap were grouped into the same cluster with typical symptoms-inducing strains XW19 and 2863 in type Xac-A (unpublished data). A pathogenicity gene, pthA, is required, and present in all X. axonopodis strains tested causing citrus canker disease, and absent in non-canker causing xanthomonds isolated from citrus. The result suggested that the presence or absence of a pthA allele is therefore diagnostic for strains having capacity to cause erumpent canker lesions on citrus. Althought Xac-Ap type strains XL16 and XL38 induced flated necrotic lesions with watersoaked margin on the leaves of four Citrus species, a 3.8 kb DNA fragment containing entire pthA gene was amplified from total DNAs of type Xac-Ap strains XL16 and XL38 with primer pair pthAP7/pthAR2. It has been shown that for avrBs3 family of avirulence gene, which includes pthA from Xac (Swarup et al. 1991; Swarup et al. 1992; AI-Saadi et al. 2007) that the central region of such avr genes is composed of a number of 102 bp direct repeats are key factors determining the interaction with plant resistance genes (Bonas et al. 1993). Novel host specificities have been reported based on pthA constructs engineered with altered numbers of 102 bp repeats (Yang & Gabriel 1995). Thus, such a rearrangement, occurring spontaneously in a variant clonal subgroup of Xac, could account for the origin of Xac-Ap group of strains. The studies on the mechanism of atypical symptoms-inducing between Xac-Ap strains with citrus plants have been undertaken..

(11) Characterization of Citrus Canker Bacteria in Taiwan. 275. In the pathogenicity tests, we found new strains in type Xac-Ap which induced atypical symptoms on leaves of Citrus species tested. Based on physiological, biochemical, genetic and proteinic analyses, strains of type Xac-Ap were characterized as Xac. Type Xac-Ap strains could be differentiated from strains of type Xac-Af, Xac-A* and Xac-Aw by a distinct pathogenicity on the leaves of Mexican lime. Moreover, Xac-Ap can also be differentiated from a pathologically similar strain X. axonopodis pv. citrumelo by oxidation of carbon sources, pectolytic activity and amplified DNA profiles of PCR with specific primer pair for Xac.. 引用文獻 (Literature cited) AI-Saadi, A., J. D. Reddy, Y. P. Duan, A. M. Brunings, Q. Yuan, and D. W. Gaberiel. 2007. All five host-range variants of Xanthomonas citri carry one pthA homolog with 17.5 repeats that determines pathogenicity on citrus, but none determine host-range variation. Mol. Plant-Microbe Interact. 20: 934-943. Alvarez, A. M., A. A. Benedict, C. Y. Mizumoto, L. W. Pollard, and E. L. Civerolo. 1991. Analysis of Xanthomonas campestris pv. citri and X. c. citrumelo with monoclonal antibodies. Phytopathology 81:857-865. Bonas, U., J. Conrads-Strauch, and I. Balbo. 1993. Resistance in tomato to Xanthomonas campestris pv. vesicatoria is determined by alleles of the pepper-specific avirulence gene avrBs3. Mol. Gen. Genet. 238:261-269. Civerolo, E. L. 1984. Bacterial canker disease of citrus. J. Rio Grande Valley Hortic. Soc. 37:127-145. Civerolo, E. L. and F. Fan. 1982. Xanthomonas campestris pv. citri detection and identification by enzyme-linked immunoabsorbent assay. Plant Dis. 66:231-236. Cubero, J. and J. H. Graham. 2002. Genetic relationship among world-wide strains of Xanthomonas causing canker in citrus species and design of new primers for their identification by PCR. Appl. Environ. Microbiol. 68:1257-1264. Cubero, J. and J. H. Graham. 2004. The leucine-responsive regulatory protein (lrp) gene for characterization of the relationship among Xanthomonas species. Int. J. Syst. Evol. Microbiol. 54:429-437. Dice, L. R. 1945. Measures of the amount of ecologic association between species. Ecol. 26:297-302. Graham, J. H. and T. R. Gottwald. 1991. Research perspectives on eradication of citrus bacterial canker diseases in Florida. Plant Dis. 75:1193-1200. Graham, J. H., J. S. Hartung, R. E. Stall, and A. R. Chase. 1990. Pathological, restriction-fragment length polymorphism, and fatty acid profile relationships between Xanthomonas campestris from citrus and noncitrus hosts. Phytopathology 80:829-836..

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(14) 278. 台灣農業研究. 第 57 卷. 第4期. 台灣柑橘潰瘍病菌新菌株之特性分析. 1. 林信成2,5 張翔3 曾國欽4 摘. 要. 林信成、張翔、曾國欽。2008。台灣柑橘潰瘍病菌新菌株之特性分析。台灣農業研究 57:265–278。將台灣柑橘植株所分離到之柑橘潰瘍病菌 (Xanthomonas axonopodis pv. citri) 菌株，在墨西哥萊姆 (Citrus aurantifolia)、葡萄柚 (C. paradisi)、柳橙 (C. sinensis) 及檸檬 (C. limon) 等四種不同品種之柑橘植株葉片上分別測試其病原性。測試結果顯示這些菌株中有兩株菌株可在測試之四種柑橘植株葉片上，引起具水浸狀邊緣之扁平壞疽病斑。依據耐鹽性、明膠水解作用、碳素源利用、應用柑橘潰瘍病菌專一性引子對之聚合酵素連鎖反應、重複性序列聚合酵素連鎖反應及十二烷基硫酸鈉聚丙烯醯胺膠體電泳 (SDS-PAGE) 分析等生理、生化、基因及蛋白質特性分析，顯示這兩株菌株係屬於 X. axonopodis pv. citri (Xac)，並將歸類為 Xac-Ap 類型菌株。利用碳素源利用、明膠水解作用及 pthAP7/pthAR2、2/3、4/7、J-pth1/J-pth2、J-RXg/J-RXc2 等引子對之聚合酵素連鎖反應的 DNA 圖譜，可將這兩株菌株與 X. axonopodis pv. citrumelo (E 病原型) 菌株區分開；然而僅能利用在墨西哥萊姆上之病原性差異將這些菌株與 Xac-Af 類型菌株區分開。數據顯示出由台灣分離到可引起非典型病徵之菌株為 X. axonopodis pv. citri 之新特殊菌株，並將其命名為 Xac-Ap 以有別於 Xac-A， Xac-Af，Xac-Ar，Xac-A*及 Xac-Aw 等類型菌株。關鍵詞︰柑橘潰瘍病、pthA 基因、重複性序列聚合酵素連鎖反應、聚丙烯醯胺膠體電泳、柑橘潰瘍病菌。. 1. 2. 3. 4. 5.. 行政院農業委員會農業試驗所研究報告第 2335 號。接受日期 97 年 11 月 5 日。私立中州技術學院生物技術系助理教授。台灣彰化縣員林鎮。本所生技組聘用高級研究員。台灣台中縣霧峰鄉。國立中興大學植物病理學系教授。台灣台中市。通訊作者，電子郵件：lin1368@dragon.ccut.edu.tw；傳真：(04)8394070。.

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