The Molecular Identification Of Xanthomonas Axonopodis Pv. Citri And The Establishment Of Detection Methods For The Pathogen

Citrus bacterial canker disease (CBCD), caused by Xanthomonas axonopodis pv.citri Vauterin et al. is a serious disease of most citrus species and cultivars. The pathogene have been appeared in lists of quarantine pests in many contries. Until now much attention was payed to the studies on detection and inspection, and great progresses have been made.In this study, a sample showing CBCD symptoms was collected from Hubei and the Xanthomonas axonopodis pv. citri was identificated based on its molecular information. Biological indexing, PCR based on a pair of primers corresponded to sequence of a conservation protein and immuno-fluorescence microscopy methods were employed for the detection of the pathogene. Results were showed as following.Four bacteria (X₁, X₂, X₃ and X₄) were isolated from a dry leaf of citrus from Hubei showing CBCD symptoms. A positive control, crude solution from leaf tissue with diseas lesions (X₀) and 4 isolated bacteria were analyzed by PCR. Products with a size about 413 bp as expected were amplified from positive control and X₀. A produt about 500 — 600bp was amplified from X4. No products were obtained from other samples. The amplified products were cloned and sequenced. Results showed that the fragments of isolates from Nanjin and Hubei consisted of 413 bp, and had similarities of 99% (411/413) and 100 % (413/413) with a isolated reported by Silva (2002) . Fragment amplified from isolate X4 consisted of 557 bp and had a similarity of 93% (484/515) with a Agrobacterium tumefaciens.Cultures of Xanthomonas axonopodis pv. citri with a concentrition 10³cfu/ml were inoculated to in vitro leaves of 7 citrus cultivars. These citrus leaves showed different symptoms. Among these cultivars 3 sweet orange (Citrus sinensis) and trifoliate orange (Poncitrus trifoliat) had a higher susceptibility and showed symptoms in a shorter period. Duncan grapefriut (C.paaradisi) and rough lemon (Citrus jambhiri) showed only brown necrosis.Total DNA was extracted from 3 samples by two protocols. When DNA extracted by chloroform/isoamyl alcohol was used as templates, fragments with expected size 413bp were amplified from all three isolates from Nanjin, Sichuan and Hubei. When extracts obtained by lysating with proteinas K were used as templates, the same products were also amplified, but showd weaker electrophoresis bands.Culture of Xanthomonas axonopodis pv. citri was appled to glass plates. After blocking with BSA and co-culturing with antibodies, plates were observed for...