Characterization and Antagonistic Control Measures of Pichia kudriavzevii Yeast like Fungus in Brown Rot Disease of Citrus sinensis (L.) Osbeck

Abstract

Citrus sinensis is one of the most important Vitamin C containing fruit all over the world which is badly infected by brown rot disease (BRD). The present research work was designed to isolate and characterize the pathogenic fungus responsible for BRD of C. sinensis as well as evaluation of its antagonistic control measures. The causal agent of BRD was isolated and characterized using physiological, morphological and molecular methods. The pathogenic fungus was isolated from the skin of sweet orange. The isolated fungus was grown in different nutrients media, temperature, pH, sugar concentrations and different carbohydrates. The highest growth of the fungi was observed in 5% fructose containing PD broth media at 37C, pH 7.5 and 0.05gm/L NaCl concentration. PCR amplification of the isolated fungal DNA by internal transcribed spacer (ITS) region products showed approximately 650bp size of clear cut band. sequencing analysis showed that the amplified its region of the fungus (GenBank Submission# SUB5672073) has 99.80% similarities with the sequences of Pichia kudriavzevii isolate 11-577 its-1 partial sequence. In pathogenicity assay, artificially infected P. kudriavzevii fungus in lemon, malta and apple fruits showed a typical brown rot disease symptoms and the isolates showed similar size of 650bp clear band for PCR products. In antifungal activity assay, the methanol extracts of Allium sativum displayed highest 17.18±0.66 mm diameter zone of inhibition against the isolated fungus. Rhizobium leguminosarum and Alternaria spp. showed significant antagonistic effect in coculture with the isolated fungus. The present research work would be helpful to control the devastating brown rot disease of Citrus sinensis.