MOLECULAR AND BIOCHEMICAL CHARACTERIZATION OF XANTHOMONAS Citri Subsp. citri.
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Date
2019
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College of Agriculture Nagpur, Dr. Panjabrao Deshmukh Krishi Vidyapeeth, Akola
Abstract
Citrus is one of the important tropical and subtropical fruits in the world as well as in India which belongs to the family Rutaceae and sub-family Aurantioedae. Among all factors responsible for decline in citrus production, citrus canker is one of the most serious problem which is caused by Xanthomonas citri subsp. citri. The bacterium infects the twigs, petioles, fruit stalks and fruits, resulting in both qualitative and quantitative loss in acid lime. The disease is characterized by raised corky necrotic lesions, surrounded by yellowish halo on the leaves. Xanthomonas citri subsp. citri was successfully isolated on the nutrient agar medium, from the fresh lesions on the leaves of naturally canker infected acid lime leaves collected from different agroclimatic zones of Maharashtra. The fourteen isolates were designated from Xcc-1 to Xcc-14 representing the agroclimatic zone. Pathogenic ability of
all different isolates of Xcc were confirmed and found that isolate Xcc-6, Xcc-7 Xcc-10, Xcc11 and Xcc-12 showed highly pathogenic to initiate minute canker lesion and fully developed symptoms after 17-22 days. While Xcc-1, Xcc-2, Xcc-3, Xcc-4, Xcc-5, Xcc-8, Xcc-9 and Xcc-13 were found to produce very poor in virulence symptoms. The studies on physiological characteristics of Xanthomonas citri subsp. citri. showed the maximum growth of pathogen observe at temperature 300C and at pH level 7.
A several bio-chemical test were conducted to differentiate the isolate. All isolates of bacteria were found negative test for gram’s staining while positive for starch hydrolysis, potassium hydroxide test, catalase test, hydrogen sulphide production, gelatin liquefaction, indole production, nitrate reduction acid and gas production tests to performed to characterized the Xanthomonas citri subsp. citri.
Molecular detection by polymerase chain reaction with specific primers J-pth1/Jpth-2, J Rxg/Rxc2,Xac-01/Xac-02, M+/M- and Ms+/Ms- was performed on fourteen isolates. The patterns showed by PCR analysis were identical for all the isolates tested confirming their identity as Xanthomonas citri subsp. citri. The genetic variability was studied by using five inter simple sequence repeat and randomly amplified polymorphic DNA primers. A total of 16 amplicons were obtained with the 5 UBC series primers. Out of 16 bands 14 were found to be polymorphic and 2 were monomorphic and the level of average polymorphism was 87.50 per cent. Isolate Xcc-11 (Nagpur) had higher value of similarity coefficient (0.85) with Xcc-10 (Wardha), whereas Xcc-12 (Gadchiroli), had lower value of similarity coefficient (0.42) with Xcc-1 (Sindhudurg) with high degree of polymorphism while two primers OPF-4 and OPF-2 failed to produce scorable bands. A total of 28 amplicons were obtained with the 3 primers. All the bands produced were polymorphic with 100 per cent polymorphism. Isolate Xcc-11 (Nagpur) had higher value of similarity coefficient (0.81) with Xcc-10 (Wardha), whereas Xcc-2 (Dapoli), had lower value of similarity
coefficient (0.41) with Xcc-13 (Gondia).
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ISOKAR, SHUBHAM SUBHASH. (2019). Molecular and biochemical characterization of Xanthomonas citri subsp. citri. Department of plant pathology, Dr. Panjabrao Deshmukh Krishi Vidyapeeth, Akola. M. Sc. 2019. Print. xiii, 91p. (unpublished).