Citrus canker is one of the serious diseases in citrus and some citrus relatives. This disease has been caused by Xanthomonas axonopodis pv. citri (Xac) bacteria or Xac pathotype A (Xac-A) which is the most wide spread pathogen in Asia and other citrus growing areas (Verniere et al., 1998). Most commercial citrus including Mexican lime, grapefruits and commercial lime in Thailand (Pan lime) are susceptible host for Xac. This pathogen can infect leaves, twigs and fruits through stomata and wounds (Graham et. al., 2004). Moreover, this disease directly reduce fruit quality and quantity of yield by defoliation, blemished fruit, premature fruit drop, die-back of twigs and general debilitation of the tree (Das, 2003). Normally, the pathogenic bacteria can survive from one crop season to the next as latent infection in propagation organs or epiphytic populations on plant surfaces. The equipments for harvesting are also the cause of disease spread (Graham et al., 2000). Therefore, the method for their early detection is necessary. Currently, many methods have been developed and are available in laboratories. Polymerase chain reaction (PCR) method is a high sensitive detection method (Cubero et al., 2001) but, it requires laboratory equipments and specialized training. In contrast, immunodiagnostic test kit is an easy method to use at the site where disease is suspected. It does not require neither special equipment nor training to perform the detection. The immunodiagnostic test is based on the ability of an antibody to recognize and bind to a specific antigen, a substance associated with the plant pathogen. This method is known as enzyme-linked immunosorbent assay (ELISA). In this study the specificity and sensitivity of serological and molecular tools for the detection of citrus canker pathogen (X. axonopodis pv. citri) were investigated and compared.

1. Results and discussions
1.1 Bacteria isolation and pathogenicity test
Xanthomonas axonopodis pv. citri (Xac) was isolated from lesion lime leaves. Total of nineteen bacterial isolates were isolated from three different regions in Thailand; Banpaew, Samut Sakhon, Nakhon Ratchasima and Phichit.  All bacterial isolates were initially selected on KCD semi-selective medium. The Gram stain indicated that, all isolates were Gram-negative rod bacteria (data not shown). The bacterial isolates were also confirmed the strain by Xac specific primers (Coletla-Filho, 2006).  The PCR results indicated that, 12 (included 10 isolated from Samut Sakhon (BP102, BP104, BP105, BP107, BP109, BP201, BP202, BP203, BP205 and BP210) and 2 isolates from Phichit (PJ01 and PJ03) contained the 581 bp Xac regulation of pathogenicity factors (rpf) gene fragments which encoded pathogenicity effecter protein (Coletla-Filho, 2006). But this gene was not detected in samples from Nakhon Ratchasima (K01, K02, SUT02 and SUT06) and unrelated bacteria such as E. coli, Sinorhizobium and Agrobacterium that were used as negative control (Figure 1). These primers were also not able to produce specific PCR product from X. axonopodis pv. vesicatoria,  X. campestris pv. campestris, X. axonopodis pv. phaseoli, X. axonopodis pv. glycine, X. oryzae pv. oryzae template (Figure 2).  Non-target DNA bands were detected in isolated from Nakhon Ratchasima and some from Phichit (Figure 1). 
 

Figure 1 Specific amplification of Xac target (581 bp fragment) using XAC01 and XAC02 specific primers

Figure 2 Specific amplification of Xac target by PCR

The bacterial isolates were inoculated to both susceptible (Pan) and resistance limes (Nam Hom and M33) for pathogenicity test. Ten bacterial isolates from Samut Sakhon and 2 isolates from Phichit could infect both susceptible and resistance lime tested. These 12 pathogenic bacteria should be Xanthomonas species since the Xac specific genes product were observed and they can infect limes. The lesion caused by isolated BP104 and BP210 on both susceptible (Pan) and resistance limes (Nam Hom and M33) slowly turned brown after 3 week post-infection. The BP104 and BP210 isolates seem to be virulence pathogenic bacteria. These two virulence pathogenic bacteria were identified by 16S rRNA gene sequencing. The accession number HQ875739 and HQ875740 were obtained. The 16S rRNA gene sequences confirmed that both strains were X. axonopodis pv. citri (Xac). In this experiment, Xac BP210 isolate was used as bacterial antigen for further antibody production.

1.2 Serological studies
1.2.1 Determination of antiserum titer
Antisera were raised from dead cells of Xac BP210 in two New Zealand white rabbits. The titer of Xac BP210 antisera were determined by indirect ELISA. Serial dilutions of the antisera up to 1:16,000 were reacted with dead and live cells of Xac BP210 at 10⁴~10⁶ CFU/mL.  The suspension of E. coli at 10⁵ CFU/mL and pre-immune sera were used as negative control. The results showed that, 1:4,000 or lower dilutions of both antisera were able to detected dead cells of Xac at 10⁵ CFU/mL or more. Whereas, 1:2,000 diluted antiserum were reactive against live cells of 10⁶ CFU/mL and dead cells at 10⁵ CFU/mL or more. The highly concentrate antiserum (1:1,000 dilution) strongly reacted against both live and dead cells (10⁵ CFU/mL) of Xac. However, antiserum 1 showed stronger reaction than antiserum 2 (Table 1). These results indicated that, at least 1:2,000 dilution of these antisera were able to detected dead cell of Xac at 10⁵ CFU/mL and live cells at 10⁶ CFU/mL. Nevertheless, dead cells of bacteria showed stronger reaction with antisera than live cells. Since these antisera were produced from injection with heat killed cells which might have broken some cells. 
 

Table 1 ELISA for antiserum 1 and antiserum 2 titer test

1.2.2 Sensitivity of X. axonopodis pv. citri detection
Antiserum 1 and antiserum 2 at the dilution of 1:2,000 were tested for the sensitivity with live and dead cells of BP210 at different concentrations (10³~10⁸ CFU/mL). The positive result is the number which is at least double the reaction intensity of negative control (10⁶ CFU/mL of E. coli and pre-immune serum reaction). The bacterial suspension of live cells at 10⁶ CFU/mL and dead cells at 10⁵ CFU/mL showed positive reaction in ELISA (Table 2). Stronger reaction can be observed with the increase of bacterial densities in both antisera tested.  Both antisera have the sensitivity of detection at 10⁶ CFU/mL for live cell and 10⁵ CFU/mL for dead cells. The sensitivity of ELISA for X. axonopodis pv. citri (Jin et al., 2001), Clavibacter michiganensis subsp. michiganensis (de Leon et al., 2008), C. michiganensis subsp. sepedonicus and Erwinia amylovora (Kokoskova and Mraz, 2008) have also been reported to be at the level of 10⁵~10⁶ CFU/mL. The detection efficiency of ELISA is limited by the level of the pathogen population and dependent upon the immunological properties of the antiserum used. The antisera produced in this study have the sensitivity as high as those produced in other reports.
 

Table 2 Sensitivity of ELISA in detecting live and dead cells of Xac BP210

1.2.3 Cross-reaction of antiserum to other Xanthomonas sp.
Antisera dilutions (1:1,000 and 1:2,000) were tested for the cross-reactivity against live cells of other five Xanthomonas sp. and E. coli at 10⁶ CFU/mL. The results in Table 3 showed that, dilution of 1:1,000 antisera strongly cross-react against X. axonopodis pv. vesicatoria and weakly reacted against X. axonopodis pv. phaseoli and X. campestris pv. campestris. While, more diluted antisera (1:2,000) cross-reacted with only X. axonopodis pv. vesicatoria but not with others Xanthomonas (Table 3). These results suggested that, antisera diluted to 1:2,000 are the suitable condition for detection of lived target bacteria of at least 10⁶ CFU/ml or 10⁵ cell/well. Although, these antisera showed cross-reactivity with X. axonopodis pv. vesicatoria but this bacterium does not infect citrus plant.They only infect tomato and Capsicum pathogen (Mirik and Aysan, 2009). Therefore, the cross-reaction detection should not be of alarm. The antigenic molecules from the normal cell surface components are often including in polyclonal antibodies (or antiserum). This mixing of antibodies has multiple specificities and cross-reactivities with unrelated bacteria species. This study attempt to diluted multiple specific antibodies and retain dominant antibodies which showed acceptable specificity for a given pathogen. 
 

Table 3 Cross-reaction of ELISA to other Xanthomonas species and unrelated microorganism

Many studies reported that, the ELISA sensitivity 10⁵~10⁶ CFU/mL is sufficient for identification of bacteria pathogens from symptomatic plants and colonies on selective media (Jin et al., 2001; Alvarez, 2004; de Leon et al., 2008; Kokoskova and Mraz, 2008). However, boiling the bacteria samples could improve the sensitivity of detection.  Several previous reports have mention that using extraction buffer containing EDTA and lysozyme to removed cell surface component could improve the sensitivity of ELISA detection (Jones et al., 1997, Alvarez, 2004). However, EDTA was not used in this study but the sensitivity of detection was still high.  

1.2.4 Detach leaf assay
Different concentrations of canker bacteria (10⁷, 10⁶ and 10⁵ CFU/mL) were inoculated to Pan lime leaves using detach leaf assay. The pathogen on the infected leaves was detected by ELISA method every 2 days post inoculation. The results showed that, infected leaves on the first day of inoculation (day zero) showed negative reactions in ELISA test whether the bacterial cells were killed by heat treatment or not (Table 4). The inoculation of this experiment were 20 µL of bacterial suspensions dropped on each area. Thus, bacteria populations on the leaves were 10⁵, 10⁴ and 10³ cells on the inoculated leaves area. The sensitivity of the ELISA (10⁵ CFU/mL or 10⁴ cells/well for dead cells) should be sufficient for detection of dead cells. However, protein or other polysaccharide molecules from the leaf may interfered with the bacteria binding to the plate therefore, low detection of target bacteria were observed. 
 

Table 4 ELISA detection of bacteria on infected leaf using detached leaf assay

Infected leaves on day two showed positive reaction when the leaves were inoculated with 10⁵ cells. Consequently, infected leaves 4 days post inoculation showed positive reaction with all bacteria concentration (10⁵, 10⁴ and 10³ cells) whether the cells were boiled or not.  The boiled bacteria (dead cell) from infected leaves showed higher reaction in ELISA test than lived cells. These results were in accordance with the results of antibody titer and sensitivity test (Table 1 and Table 2). After day 4 post inoculation, symptoms on the infected areas were observed as slightly raised blister-like lesions.  Thus, these results indicated that, ELISA assay is a well-established method for identification of bacterial pathogens from symptomatic plants. However, enrichment techniques can enhance ability of pathogen detection (Jin et al., 2001) on natural samples but more time is needed for the culturing period. 

1.2.5 Molecular detection of Xac from canker lesions
Canker pathogen on symptom and non-symptom plant materials (leaf and twig) in infected areas were detected by PCR amplification using Xac specific primers. The results showed that, the Xac specific PCR products were detect in all samples of symptom plant materials and some non-symptom plant materials (Table 5).
 

Table 5 Xac specific amplification of canker pathogen from symptom and non-symptom plant materials

These specific primers were also used to detect canker pathogen on infected leaves from the detach leaf experiment. The results showed that, canker pathogen were detected by these specific primers on the first day of inoculation while ELISA was unable (Table 4). The amplification assay using specific primers have been reported to detect at low pathogen population (10²~10³ CFU/mL) (Cubero et al., 2001, Cubero and Graham, 2002, Coletla-Filho, 2006, Park et al., 2006, Golmohammadi et al., 2007, de Leon et al., 2008). The sensitivity of PCR amplification of this study showed very high sensitivity at 10³ CFU/mL or 1 cell in the PCR reaction (Figure 3). Moreover, this methodology showed no cross-reactivity with other Xanthonomas species (Figure 2). However, PCR method required laboratory equipment, special reagents and skill to perform.
 

Figure 3 Xac specific PCR amplification from a tenfold dilution series of X. axonopodis pv. citri cultured cells

2 Conclusion
The sensitivity and specificity of serological and molecular tools have been investigated. The molecular tool is more effective than serological tool due to the high sensitivity (at least 1 cell) and specificity. Therefore, molecular detection able to detect the present of pathogen in infected leave even before the appearance of symptom. However, serological test from this study was not inferior to other reports and sufficient for pathogen detection from symptomatic tissue.

3 Materials and Methods
3.1 Isolation of Xanthomonas axonopodis pv. citri (Xac)
Infected lime leaves from three different regions in Thailand; Samut Sakhon, Nakhon Ratchasima and Phichit which are about 300~500 km apart were used in this study. Different Xac isolates were isolated from infected lime leaves tissue by commonly used methods (OEPP/EPPO, 2005). Briefly, the infected leaves were washed with sterile water and surface sterilize by soaked in 1% sodium hypochlorite for 3 min. Then, the lesions were rinsed in sterile water several times and excised with scalpel.  Xac isolates were initially selected by streaked the water-soak tissue from the lesion margins on sterile semi-selective media (KCD medium, nutrient agar (NA) supplemented with Kasugamycin (16 µg/mL), Cephalexin (16 µg/mL) and Daconil (Chlorothalonil) (12 µg/mL) prior to enrichment on NA media without antibiotics. The bacteria were grown at 28℃ for 24~48 h. The bacterial colonies were collected for Gram staining and further used for pathogenicity tests. 

Other Xanthomonas strains include X. axonopodis pv. vesicatoria, X. campestris pv. campestris, X. axonopodis pv. phaseoli, X. axonopodis pv. glycine, X. oryzae pv. Oryzae obtained from Chulabhorn Research Institute (CRI, Thailand) were used as negative control in strain identification and cross-reaction tests.

3.2 Specific primers identification and 16S rRNA gene sequencing
Single colony of Xac isolates and other Xanthomonas strains were resuspened in 1 mL DI-water and boiled for 10 min. Then, the suspensions were used as template for PCR reaction, with specific XAC01 and XAC02 primers for Xac identification (Coletta-Filho, 2006). The PCR reactions were performed with 50 µL reaction mixture, containing 1 µL of boiled cell suspension, 1× reaction buffer (Promega GoTaq), 1.5 mM MgCl₂, 0.2 mM dNTPs, 0.2 µM primers (XAC01: 5’-CGC CAT CCC CAC CAC CAC CAC GAC-'3; XAC02: 5’-AAC CGC TCA ATG CCA TCC ACT TCA-'3), 1.25 U Taq DNA polymerase (Promega). Unrelated bacterial DNA included Escherichia coli, Sinorrhizobium and Agrobacterium were also subjected to PCR reaction as negative control. PCR conditions were as followed; an initial cycle of 94°C for 5 min followed by 35 cycles of 94°C for 45 s, 60°C for 45 s, and 72°C for 45 s, with a final step of 72°C for 5 min. The PCR products were observed under UV light after electrophoresis through 1.0 % (w/v) agarose gels and stained with ethidium bromide.

The full length 16S rRNA gene of Xac isolates BP104 and BP210 were sequenced by Macrogen (Korea) (http://dna.macrogen.com/eng/). The sequence results were applied to BLAST program for gene comparison with data in the GenBank (NCBI). 

3.3 Pathogenicity test
3.3.1 Plant samples
M33 hybrid limes (highly resistant to canker) were from Phichit horticulture research center, Samut Sakorn and Prachinburi (Thailand), Nam Hom limes (highly resistant to canker) were from Phichit horticulture research center and Pan limes (susceptible to canker) were from Samut Sakorn. These lime plants were planted in the test field at Suranaree University of Technology, Nakhon Ratchasima, Thailand.   

3.3.2 Xac Inoculation on resistance and susceptible limes
Half of the M33, Nam Hom and Pan limes leaves were wounded with needles and celite, the other halves were not wounded (healthy). One ml of the bacterial isolate suspension (10⁸ CFU/mL) was sprayed on the leaves. The plants were then covered with plastic bags overnight. In the morning, the bags were removed and the plants were left in natural condition for 2~3 weeks. The virulent canker disease apparent on each leaf was evaluated.

3.4 Serological studies
3.4.1 Preparation of antigen
Antigen was prepared from the cells of Xac isolate BP210. The bacterial cells were enriched on NB (nutrient broth) at 28℃ for 48 h. The bacterial cells were collected by low-speed centrifugation (4,200 ×g) and washed once in sterile 0.85% NaCl. The bacterial suspension was adjusted spectrophotometrically to A₆₀₀ 0.2 (about 10⁸ CFU/mL) and heated to 85ËšC for 15 min. The killed cell suspension was used as antigen for antiserum production.

3.4.2 Antiserum production
Antiserum against Xac (BP210) was prepared using two New Zealand white female rabbits. 1 mL of antigen mixed with Freund’s complete adjuvant (1:1 v/v) was immunized to each rabbit by subcutaneous injection on the first day. Then, 1 mL of antigen (mixed with Freund’s incomplete adjuvant (1:1 v/v)) was injected subcutaneously 7 day later. After that, 1 mL of bacterial antigen without adjuvant was injected intravenous on day 14 and day 21. One week after the last injection, the blood was drawn from the central ear artery then kept in refrigerator overnight. The antiserum was clarified by centrifugation at 3,000×g for 15 min at 4℃ and stored at -80℃ for future use. This antiserum was used for indirect ELISA assay for the antibody titer, the sensitivity and the cross reaction of the antiserum. The pre-immune serum was bleed before immunization to use as negative control in the ELISA assay. Absorbance values of at least double of the control were considered positive (OEPP/EPPO, 2005).

3.4.3 Indirect ELISA assay
The indirect enzyme-linked immunosorbent assay (ELISA) was carried out as described by Alvarez et al (1991). The bacterial concentrations were adjusted from 10³~10⁸ CFU/mL in 0.05 M carbonate coating buffer (0.015 M Na₂CO₃, 0.035 M NaHCO₃, 0.003 M NaN₃; pH 9.6).  Half volume of each diluted bacterial suspension were treated at 85℃ for 15 min to kill the cells and used as dead cells antigen.  100 μL of each diluted bacterial suspensions (both live and dead cells antigen) were coated in microtiter plate well at 4℃ overnight. The plate was washed three times with phosphate buffer saline+0.05%Tween20 (PBST) and 200 μL of 2% skim milk were added and incubated for 1 h at 37℃. Then, the plates were washed as mentioned earlier and 100 µL of diluted antiserum were added to the wells. The ELISA plates were incubated at 37℃ for 1 h then, three times washing were performed.  Goat anti-rabbit gamma globulin conjugated to alkaline phosphatase (whole molecule, enzymatic activity 1 mg/mL) obtained from Sigma Chem. Co St Louis, Mo (Production#F0382) was diluted to 1:10,000 and 100 µL was added in each well, followed by one hour incubation at 37℃. The wells were repeatedly washed and 100 μL of enzyme substrate (1 mg/mL p-Nitrophenyl-phosphate (PNPP) in 10 mM Diethanolamine; Thermo Scientific#34047) were added in each well. The reaction were stopped by adding equal volume of 0.75 M NaOH and the OD at 405 nm were measured after 15~30 min incubation in the dark at room temperature. The live and dead cells of E. coli (10⁶ CFU/mL) and pre-immune serum were used as negative control. 

3.4.4 Antibody titer and sensitivity tests
The antiserum was diluted to different dilution (1:1,000, 1:2,000, 1:4,000, 1:8,000 and 1:1,600 v/v) in conjugate buffer (2% polyvinyl pyrrolidone (PVP)+2% ovul albumin in PBST) and tested with both live and dead cells of bacterial antigen (10⁴~10⁸ CFU/mL) by indirect ELISA assay to test the titer of the antiserum. For sensitivity testing, the 1:2,000 (v/v) diluted antiserum was used to test both live and dead cells of bacterial antigen (10³~10⁸ CFU/ml Xac BP210) by indirect ELISA assay as described above. All experiments were done in duplicated and repeated at least 3 different times.

3.4.5 Cross-reaction test
The antiserum was diluted to 1:2,000 (v/v) and used to test the cross reactivity with other Xanthomonas bacteria at the concentration of 10⁶ CFU/mL by indirect ELISA assay as described above. The percent of cross-reaction was calculated by; {[A₄₀₅ of each Xanthomonas species - A₄₀₅ of negative control] / [A₄₀₅ of positive control - A₄₀₅ negative control]} x100.

3.5 Detached leaf assay
3.5.1 Xac inoculation
Full expanded young leaves of lime were washed in running tap water and surface sterilized in 1% sodium hypochlorite for 1~4 min. The leaves were aseptically rinsed thoroughly with sterile distilled water.  Each leaf was divided into 4 parts for treatment separation, included 10⁵, 10⁶ and 10⁷ CFU/mL infected parts and one uninfected part. Each part of the leaf was wounded by puncturing with a small needle, through the lower surface (5 needle punctured wounds per part). Each wounded leaf was placed on 1% water agar in a Petri dish, with the back of the leaf up. 20 µL of the different diluted bacterial suspension (10⁵~10⁷ CFU/mL Xac BP210) were dropped on each part of the wounded leaf. The part of uninfected leaf, 20 µL of sterile 0.85% NaCl was dropped to use as negative control. The infected leaves were maintained in an incubator at 28℃. 

3.5.2 Serological detection of Xac in inoculated lime tissues
The canker pathogen on each part of the infected leaf was detected by ELISA method. The leaf was aseptically cut and each part of the infected leaf was ground in 150 µL coating buffer. 100 µL of leaf extract was coated in one microtiter plate well and incubated at 4℃ overnight before detection with 1:2000 (v/v) diluted antiserum as mention above. 

3.6 Molecular studies using specific primers
The sensitivity of PCR amplification was performed with Xac isolate BP210 in tenfold dilution series (10⁸~10¹ CFU/mL in 0.85% NaCl). 1 µL of each dilution was used as template in the PCR reaction. The detection of Xac in inoculated lime tissue was also performed in Xac specific PCR amplification as described above. 

3.7 Canker detection from field samples
Symptom and non-symptom plant materials (leaf and twig) from Pan, Nam Hom and M33 limes were taken from the field and washed in 25 mL 0.85% NaCl and shaked for 30 min. Then, the plant material was removed, and the washing solution was boiled for 10 min. 1 µL of the boiled washing solution were used as template for specific primers (XAC01 and XAC02) amplification. The PCR condition was performed as described above. The Xac BP210 isolate was used as positive control. The detection results of inoculated tissue were demonstrated as percentage (% detection), calculated by; [number of sample that can be detect by Xac primers/total samples tested] x100.

Authors' contributions
PS carried out all experiments as her M.Sc. Thesis work. She also drafted the manuscript. MKC was the PI of the lab. She conceived the study, participated in the experimental design, coordinated, drafted and edited the manuscript. Both authors have read and proved the final manuscript.

Acknowledgements
S. Loprasert (CRI) is thanks for the Xanthomonas reference strains. PS was supported by SUT scholarship. This research was funded by SUT Research and Development Fund.

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