Molecular Identification Of Xanthomonas Axonopodis Pv. Citri And Selection Disease-resistant Of Resources

Citrus canker is a bacterial disease that causes serious damages on the world's citrus production.The disease is a particular infective to sweet orange.Infected citrus trees showed special symptoms on leaf and fruit,when it becomes serious,it can cause leaf and fruit drop,as well as heavy losses of the citrus production and fruit quality.Xanthomonas axonopodis pv.citri is the causal agent of citrus canker disease, and five forms,namely A,B,C,D and E have been described for the pathogen.In China the bacterium causing canker disease has been recognized as Type A,but no detail differentiation of the strains presented in various regions and different cultivars have been reported.Rep-PCR is performed using DNA amplification with primers based on short bacterial repetitive elements(ERIC,BOX ) and has used to identify the relationship between bacteria strains.To have the primary characterization of the pathogen in China,we collected 21 strains of X.axonopodis pv.citri from 7 cultivars in 10 citrus producing areas of Hunan province.Trying to characterize the strains by rep-PCR with BOX and ERIC specific primers.With the BOX primers,Xanthomonas DNA was amplified with the PCR products ranging from approximately 800 to 6,000 bp.In total,32 fragments were visualized and 28 of them were polymorphic.The ERIC primers generated PCR products that ranged from 300 to 5,000 bp.Among the 28 PCR products,20 were polymorphic.Cluster analysis with UPGMA method revealed that the citrus canker pathogen had complex differentiation in Hunan province.Taking into account 85%of similarity,6 clusters were identified.The relationship between these differentiations and the pathogenesis will be further investigated.At present,the control of citrus canker relies mainly on chemical control,but it is difficult to eradicate the disease.All the citrus genotypes up to now tested were susceptible to the disease when they were artificially inoculated.China is the most important origin center of citrus,with long history of cultivation and plenty genetic resources giving the possibility to find some genotypes resistance to the pathogen.In order to find a resistant genotype,7 types of Citrus ichangensis Swingle,which was previously reported to be resistant to canker disease,5 types of C.junos Sieb.ex Tanaka,and one citron cultivar.Some of the tested accessions were kindly supplied by Citrus Research Institute of Chinese Academy of Agricultural Sciences(Beipei, Chongqing).The highly susceptible 'Bingtang' sweet orange was included as the control in all the assay.Detached leaves from each genotype were punctured and then inoculated by Xanthomonas axonopodis pv.citri maintaining in petri dishes on wet filter papers at 28℃.The disease development was recorded every day.The in vitro assay was repeated three times.After 10 days from the inoculation,6 genotypes(3 Ichang papeda,2 C.junos and 1 citron) showed almost no typical symptoms, therefore these genotypes were selected for further in vivo assay.About 30 plants from each genotype were planted in an epidemic orchard of 'Bingtang' sweet orange. From May to August,the plants were inoculated with the pathogen for three times and the symptom recording was taken every month in all the growing season.The assay continued for 2 years.The results indicated that citron was the most resistant genotype, as no canker symptom was found on leaves in all the tests,for the 3 types of C. ichangensis,the disease incidences were 30-50%.For C.junos,Shencheng was 33.33%and Zhencheng 69.23%.Even though C.ichangensis and C.junos showed less disease incidence than sweet orange(more than 70%),they do not have the resistant gene,while citron has not shown any disease symptom implying a resistant genotype inside Citrus and offering the possibility for further breeding of resistant cultivars.