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ThesisItem Restricted Characterization of citrus greening pathogen(s) and its integrated management(Punjab Agricultural University, 2023) Katoch, Sonali; Arora, AnitaSurveys conducted during 2019-21 to assess the distribution and intensity of greening disease in citrus orchards of Punjab revealed that its prevalence was high in the district Ludhiana (33.33%) followed by Hoshiarpur (32.60%), Fazilka (27.59%) and Faridkot (19.31%). The maximum mean disease incidence (16.60%) was observed in Hoshiarpur and the maximum mean disease index (11.98%) was observed in Ludhiana. Leaf mottling, yellowing, green islands and bi-coloured fruit symptoms of disease were observed on different cultivars of citrus. Multilocus sequence typing using 16S rDNA, CLIBASIA gene locus and tufB-secE-nusG-rplKAJL-rpoBC gene cluster was followed to identify and characterize the pathogen. Three primers (OA1/OI2c, CGB F/R and Las606/LSS) corresponding to bacterial 16S rDNA sequence were used on a total of 153 samples, and positive reactions were observed with all the three primer pairs. However, CGB F/R and Las606/LSS had higher detection rates (69.9%) than the OA1/OI2c primer (37.8%), indicating more robustness of the former. Similar results were observed with nursery samples of Punjab. The root samples from greening positive plants collected in the month of February were also found positive with five primer pairs (OA1/OI2c, CGB F/R, rplJ/K F/R, Las606/LSS and LapGP F/R) whereas, the root samples collected in May and September months showed no amplification. In addition, the CLIBASIA gene locus based primer pairs corresponding to sequences coding for hypothetical, heamolysin and type-I secretary system protein and one primer based on tandem repeats were used on 15 isolates of greening bacterium. All the samples showed positive reaction with five primer pairs. The disease was characterized by sequencing of 16S rDNA and CLIBASIA gene locus from five isolates with CGB F/R, three with Las606/LSS, two clones with OA1/OI2c and four with CLIBASIA gene locus which showed 99-100% similarity with reported isolates of Candidatus Liberibacter asiaticus (Ca. Las) from Asia. The phylogenetic analysis of these isolates showed 99-100% similarity with Ca. Las. The isolation of Ca. Las from leaves of PCR positive Kinnow plants was attempted on Liber A media, but it could not be cultured. The cultivars viz., Kinnow, Daisy, Musambi and Kagzi Lime were observed to be the best indicator plants for indexing. For integrated management of citrus greening, the treatment consisting of zinc sulphate + manganese sulphate + boric acid, tetracycline hydrochloride and 2,4-dichlorophenoxyacetic acid (2,4-D sodium salt) was proved significantly superior to other treatments in reducing the percent disease index (13.99) and providing 59.29 percent disease control with significant increase in yield (54.87 kg/tree) as compared to control (38.32 kg/tree).
