Molecular detection and characterization of Citrus tristeza virus and ‘Candidatus Liberibacter asiaticus’ related with citrus decline in India

Citrus (Citrus spp.) is the third most important fruit crop after banana and mango in India which is grown in about 9.35 lakh hectors with an annual production of 115.1 lakh metric tons (NHB, 2015-16). The average yield of citrus fruits in India is about 12.3t/ ha, which is far lower than the citrus yield in developed countries like USA, Brazil, China etc. where the yield goes up to 30 t/ha. Diseases caused by virus and virus-like pathogens is the primary factor responsible for such gloomy scenario. Citrus tristeza virus (CTV) and ‘Candidatus Liberibacter asiaticus’ (Ca. L. asiaticus) bacterium are two most devastating systemic pathogens of Indian citrus industry. Tristeza disease is caused by CTV, a phloem limited, monopartite, and singlestranded RNA virus whereas citrus greening disease (HLB) caused by three different species of a Gram negative, non-cultured, alpha proteobacteria ‘Candidatus Liberibacter'. Among the three species ‘Ca. L. asiaticus’ is the most serious and widely distributed pathogen in Indian subcontinent. In the present study, samples were collected from the four major citrus growing states of the India viz. Maharashtra, Assam, Sikkim and West Bengal. The leaves and twig samples were collected from citrus plants showing the characteristic and probable symptoms of tristeza and citrus greening disease. Based on PCR-based molecular detection the incidences of CTV in all four states were found in the range of 1.5% 40% whereas HLB incidence was found in the range of 3% -35%. Molecular diagnosis by PCR using pathogen specific primers showed that around 12%-15% of citrus trees in the grove were mixed infected with both pathogens. During survey, presence of Brown citrus aphids (vector of CTV) and citrus psylla (vector of HLB) were observed in the citrus orchards. The highest infection of both pathogens was found in mosambi whereas least infection was found in Acid lime and Pomelo. Standardized RT-PCR, LAMP, PCR, duplex PCR techniques were successfully used for the detection of CTV and ‘Ca. L. asiaticus’. Sequence of coat protein, RNA binding protein and p18 protein gene of CTV isolates showed 97% to 100% sequence identity with CTV sequences available in the GenBank. Phylogenetic analysis carried out using coat protein gene of all twenty eight sequenced CTV isolates grouped them into eleven clades indicating the presence of at least eleven different CTV genotypes in citrus trees of Maharashtra, Assam, Sikkim and West Bengal. Similarly, nucleotide sequence of 16S rRNA and A2J5 gene of ‘Ca. L. asiaticus’ showed 96% to 100% identity with sequences of other reported isolates. Phylogenetic analysis of the 16S rRNA gene and A2J5 indicated that HLB isolates associated with citrus decline in the present study belong to ‘Ca. L. asiaticus’ species only. The tandem repeats numbers (TRN) analysis revealed that class II was most prevalent, followed by class I and class III. PCR/RT-PCR technique developed for the rapid and accurate detection of both pathogens can be used by different laboratories for survey, epidemiological studies, plant quarantine, resistance breading and finally for citrus budwood certification program. Moreover the rapid, low cost, robust and highly sensitive CTVRT-LAMP technique developed in the present study may be used at remote location and mobile pathology centers having minimum facility for detection of CTV and will facilitate the implementation of citrus bud-wood certification program in these remote places.