National Research Centre for Citrus, Amravati Road, Nagpur-440010, India.
Abstracts of the papers presented at the International Conference of Indian Virological Society on “Emerging and Re-emerging viral Diseases of the Tropics and Subtropics” at Indian Agricultural Research Institute, New Delhi, India, December 11–14, 2007.
Of all diseases of citrus described to date, citrus Huanglongbing (HLB) or greening disease is considered probably the most destructive and lethal. HLB is now known to occur in 40 different Asian, African, Oceanian, South and North American countries and is slowly invading new citrus growing areas. The causal pathogen of HLB is a fastidious, phloem-limited bacterium, a member of the subdivision of the phylum Proteobacteria. The HLB bacterium belongs to the genus Candidatus Liberibacter, three species of which are currently known, Candidatus Liberibacter asiaticus, occurring in Asian countries and, to a lesser extent, in Brazil and the USA (Florida), Candidatus Liberibacter africanus with its subspecies “capensis”, recorded from African countries, and Candidatus Liberibacter americanus present in Brazil. The disease is graft- and vector (psyllid) -transmissible. It has been difficult to consistently detect the Liberibacters using traditional biological assays, detection of fluorescent compounds, light or electron microscopy, or ELISA. This is presumably because of the low concentration and the uneven distribution of the pathogens in host plants and vector insects. In addition, the non-specific nature of foliar symptoms makes the disease difficult to distinguish from nutrient deficiencies or other diseases. In recent times, different PCR-based molecular approaches (one-step, nested, multiplex and real time) are developed to detect and differentiate Ca. Liberibacter species using species-specific primers based on 16S rRNA gene or rplKAJL-rpoBC operon sequences.
HLB is attributed to one of the major causes of citrus decline in India. A major survey and disease diagnosis project was initiated at the beginning of this decade at NRCC, Nagpur. Extensive surveys were conducted during 2002- 2006 in some of the major citrus belts of the country (Vidarbha and Marathwada regions of Maharashtra, Abohar and Hosiarpur regions of Punjab, Chettalli, Gudur and Periyakulum regions of Southern India and different parts of North-East India) to record the incidence and distribution of this disease. Commercially important citrus cultivars like sweet orange (Mosambi, Sathgudi, Jaffa), mandarin (Nagpur, Kinnow, Coorg, Khasi, Darjeeling) and acid lime (Kaghzi, Vikram, Pramalini, Jayadevi)) and lemon (Assam) were surveyed. Results of survey indicated that incidence of greening was more on sweet orange and Coorg mandarin compared to other cultivars. Different kinds of symptoms were observed viz., mottling or blotchy mottle, severe chlorosis with green veins, pale green colour in young leaves, Zinc-deficiency-like symptoms, vein yellowing and general yellowing. In severe cases leaves become almost chlorotic with scattered green spots. However, ‘leaf mottling’ symptom was found to be highly diagnostic for the disease. The disease was preliminarily diagnosed through biological indexing and analysis of the fluorescent marker substances by thin layer chromatography. To confirm the presence of this bacterium through polymerase chain reaction (PCR), DNA was extracted from leaf midrib and bark tissues by CTAB procedure/Qiagen DNeasy TM Plant mini kit. PCR was performed with different sets of greening-specific primers for amplification of 16S rDNA (OI1/OI2c), ribosomal protein genes (A2/J5) and 16S/23S intergenic regions (OI2/23S1). All the infected samples yielded specific amplification products, sizes of which were found similar to that amplified from Ca. L. asiaticus. PCR amplicons were sequenced and the sequencing also confirmed amplification of Ca. L. asiaticus DNA from the genebank database. Digestion of the 16S rDNA PCR product yielded two fragments of 640 and 520 bp as reported only for Ca.
L. asiaticus. The psyllid vector of greening, Diaphorina citri Kuwayama was found present in most of the areas surveyed. Ca. L. asiaticus could also be detected by PCR from psyllids (subdivided in batches of 10 insects) collected in HLB-affected sweet orange orchards. The PCR-based diagnostic assay is being routinely used for screening mother plants against HLB disease in centre's on-going programme of commercial production of disease-free nursery plants.
