~ 173 ~ Journal of Pharmacognosy and Phytochemistry 2020; 9(5): 173-176 E-ISSN: 2278-4136 P-ISSN: 2349-8234 www.phytojournal.com JPP 2020; 9(5): 173-176 Received: 24-07-2020 Accepted: 25-08-2020 Pradeep Manyam Department of Plant Pathology, University of Agricultural Sciences, Dharwad, Karnataka, India Nargund VB Department of Plant Pathology, University of Agricultural Sciences, Dharwad, Karnataka, India Corresponding Author: Pradeep Manyam Department of Plant Pathology, University of Agricultural Sciences, Dharwad, Karnataka, India Diagnosis of Xanthomonas axonopodis pv citri isolates, the causal agent of citrus canker by pathogenicity and PCR based methods Pradeep Manyam and Nargund VB Abstract Citrus canker caused by Xanthomonas axonopodis pv citri (Xac) is one of most destructive diseases of citrus in terms of damage to trees and fruit quality and remained as one of the important quarantine disease. Under field conditions, though Asiatic citrus canker disease is relatively easy to diagnose, the accurate detection of pathogen for various pathological studies is necessary. In present study, a total of 13 plant samples collected from various parts of Karnataka state were diagnosed by an integrated approach using pathogenecity, isolation and molecular techniques. Five isolates were able grow on nutrient glucose agar medium characteristic of Xanthomonas axonopodis pv. citri.. The pathogenicity of all the above five isolates (XAC 1, 2, 3, 4 and 5) was proved with typical canker lesions on leaves of citrus after five to eight days of incubation period. All five isolates of Xac were confirmed at molecular level by amplifying highly conserved nucleotide sequence est A region (777 bp) using Xc-lip primer pair that distinguishes Xanthomonads from bacteria. The study revealed that integrated approach as reliable technique for detection of Xac for laboratory analysis. Keywords: Citrus canker, pathogenicity, isolation, PCR, xanthomonads, Xanthomonas axonopodis pv. citri. 1. Introduction In India, citrus species occupies third position among fruits after mango and banana in term of production and Asiatic citrus canker is one of the major constraints of its cultivation. Among five pathotypes of cirus canker pathogen that are geographically distributed in different continents except Europe, cankerous form A which is also known as true or Asiatic canker disease is the most devastating form and can infect most commercial citrus varieties and citrus relatives (Vauterin et al., 1995) [12] . The diseased plants are characterized by the occurrence of conspicuous raised necrotic lesions that develop on leaves, twigs and fruits. Severe infection results in defoliation, die-back, deformation of fruit and premature fruit drop (Stall and Seymour, 1983). Canker causes fruit losses ranging from premature fruit drop due to abscission to non-marketable quality due to lesions. The bacterium Xacis rod-shaped measuring 1.5-2.0 x 0.5-0.75 mm, Gram-negative and has a single polar flagellum. Colonies on culture media were usually yellow as a result of Xanthomonadin pigment production (Chand and Pal, 1982 and Goto, 1992) [2, 4] . Xac survives primarily in naturally occurring lesions. Cankerous leaves, twigs and branches constitute the main source of inoculum (Nirvan, 1963) [9] . Bacterial cells ooze from existing lesions during wet weather to provide inoculum for further disease development. Infection by Xacoccurs, like many other bacterial diseases, primarily through stomata and wounds produced during strong winds and by insects. Methods of detecting Xac from natural habitats include leaf-infiltration, bacteriophage, fluorescent antibody and ELISA (Goto, 1992) [4] . The polymerase chain reaction and dot blot immunobinding assay (DIA) were developed for rapid, sensitive, and specific detection of the pathogen. (Wang and Liu, 2004; Mavrodieva et al., 2004) [7] . Golmohammadiet al. (2007) [3] and Yin et al. (2007) [14] found real-time PCR to be more effective at detecting Xac, and up to 100-1000 times as sensitive. Since the nucleotide sequence of estA is highly conserved in Xanthomonads, the sequence was used to design a specific PCR primer set, Xc-lip-F2-Xc-lip- R2. 2. Materials Andmethods 2.1 Isolationandidentification of the pathogenic bacteria A total of 13 samples of citrus diseased leaves were collected for the study from eleven places