Molecular Identification and Characterization of Citrus Greening Bacterium, Candidatus Liberibacter Asiaticus Associated With Decline of Citrus

Abstract

Citrus is an important fruit which is grown worldwide. Indian ranks 4th in cultivation and production of oranges in the world. Citrus production is suffering heavy economic loss in recent years due to citrus greening diseases or Huanglongbing (HLB), which has become one of the greatest challenges for citrus growers across the world. Characteristic symptoms include yellowing of leaf veins and adjacent tissues, followed by premature defoliation and dieback of twigs. In this present study, HLB symptomatic and asymptomatic citrus tree leaves (n=60) were collected from Sardar Vallabhbhai Patel University of Agriculture & Technology, ICARIndian Institute of Farming Systems Research (IIFSR) and Parikshitgarh, Meerut. Genomic DNA was isolated using CTAB method and PCR amplification of 16S rDNA was carried out with primer set Las606/LSS and OI1/OI2c which resulted in amplification of ~500bp (n=45) and ~1160bp (n=2) respectively. The amplicons were cloned in pTZ57R/T and sequenced. The sequence obtained from 16S rDNA was submitted to GenBank, NCBI with accession no. MH473377 to MH473403, MH481540 to MH481543 and MH481746. BLAST analysis of sequence obtained with primer Las606/LSS, MH473377 to MH481403 showed 99% homology with Ca. L. asiaticus, china (MH36877), Italy (KY990822) and Brazil (JF819898). BLAST analysis of sequence obtained with primer OI1/OI2c, the Ca. L. asiaticus, MH481746 showed 99% homology with the Ca. L. asiaticus Mexico (JQ867437), Japan (AB555706) and China (DQ157273). Phylogenetic analysis revealed that the sequences from the present study Ca. Liberibacter (MH473377 to MH473403, MH481540 to MH481543) clustered in the same clade with the other Ca. L. asiaticus reported from other places worldwide. Similarly, Ca. L. americanus and Ca. L. africanus were clustered in different clade with their respective strains. The sequence identity matrix with nucleotide sequences from Las606/LSS showed an identity of 70 to 100% and OI1/OI2c primers showed 51.6 to 100% sequence identity with other Candidatus Liberibacter asiaticus strains. Analysis of 16S rDNA clearly indicated that isolates from the study fits in to Candidatus Liberibacter asiaticus group. It was concluded that in amplification of HLB using primer Las606/LSS gave better results as compared to OI1/OI2c. as Huanglongbing affects all citrus cultivars, regardless of root stock and there is no resistance within the major commercial citrus cultivars. The present can be used for the diagnostics and production of disease free citrus planting material.