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3.5 Greening (Huanglongbing) Disease in : Present Status and Diagnostic Efforts

Das A. K. National Research Centre for Citrus, Amravati Road, Nagpur – 440010, India.

Among all diseases of citrus described to date, (CGD) (syn. Huanglongbing, HLB) is considered probably the most destructive and lethal. The disease infects citrus trees of almost all and causes substantial economic losses to . CGD is now known to occur in 40 different Asian, African, Oceanian, South and North American countries and is slowly invading new citrus growing areas (1). The causal pathogen of CGD is a fastidious, phloem-limited bacterium, belongs to the genus Candidatus Liberibacter, three species of which are currently known, Candidatus Liberibacter asiaticus, occurring in Asian countries, in Brazil and the USA (Florida), Ca. L. africanus in African countries, and Ca. L. americanus present in Brazil. The disease is graft- and vector (psyllid) -transmissible. The psyllids, Diaphorina citri and Trioza erytreae are natural vectors: the heat-sensitive African form transmitted by T. erytreae, and the heat-tolerant Asian and American form, transmitted by D. citri. CGD is attributed to one of the major causes of citrus decline in India (2). The presence of greening disease in India was first suspected in 1960s (3). Thereafter it was reported from different citrus growing regions of India and was considered to be principal cause of citrus dieback disease. A major survey and disease diagnosis project was initiated at the beginning of this decade at National Research Centre for Citrus, Nagpur. Extensive surveys were conducted during 2002- 2007 in some of the major citrus belts of the country (Vidarbha and Marathwada regions of Maharashtra, Abohar and Hosiarpur regions of , 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 (Mosambi, Sathgudi, Jaffa, Malta), mandarin (Nagpur, Kinnow, Coorg, Darjeeling), acid (Kaghzi, Vikram, Pramalini, Jayadevi) and (Assam) were surveyed. Different kinds of CGD-associated symptoms were observed viz., mottling or blotchy mottle (Fig.1, B, Fig. 2, B, C), severe chlorosis with green veins (Fig. 2, A), pale green colour in young leaves, Zinc- deficiency-like symptoms, vein yellowing and general yellowing in different cultivars of citrus. Sometimes leaves become almost chlorotic with scattered green spots (Fig. 2, D). However, ‘leaf mottling’ symptom was found to be highly diagnostic for the disease especially for sweet orange group. Typical HLB (yellow shoot)-like symptoms were often seen emerged from a part of the canopy (Fig. 1, A). Severely infected trees were found sparsely foliated affected by extensive twig dieback. Biological indexing of budwoods collected from greening- suspected plants was done on 1- year old sweet orange (Mosambi) indicator plants. Typical symptoms of disease on the indicator plants developed 4-6 months after inoculation. The disease was preliminary diagnosed through analysis of the fluorescent marker substances by thin layer chromatography (TLC). Results of survey indicated that incidence of greening was more on sweet orange and mandarin groups. Incidence of the disease ranged from 8-43% in Mosambi sweet orange, 30- 40% in Malta sweet orange, 9 – 46 % in Sathgudi sweet orange, 15-47% in Coorg mandarin, 1- 6% in Nagpur mandarin, 16-30% in Sikkim mandarin, 10-20% in Darjeeling mandarin, 10-53% in Jampui Hills mandarin, 3-15% in Kinnow mandarin, 8-38% in Assam lemon and 2-13% in acid lime.

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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 indicating the presence of the causal bacterium and the size of PCR products obtained were found similar to that amplified from Candidatus Liberibacter asiaticus (Fig. 4, A). Further, digestion of the 16S rDNA PCR product (amplified fragment using primers OI 1/ OI 2c) yielded two fragments of 640 and 520 bp as reported only for Ca. L. asiaticus (4) (Fig. 4, B). To ascertain the nature of the amplicon, the amplified DNA fragments were purified from the agarose gel using QIAquick gel extraction kit (Qiagen, Gmbh, Germany). The purified PCR products were cloned into the pGEM-TTM easy vector (Promega Biosciences, Calif.) and sequenced at the commercially available automated DNA sequencing facility (Genei, Bangalore, India). Search for homologies in GenBank databases (http://www.ncbi.nlm.nih.gov/blast) were carried out using the BLAST programme. The sequencing and subsequent phylogenetic analyses (using the Neighbour-Joining method, conducted in MEGA software version 4.0) (5) confirmed amplification of ‘Ca. L. asiaticus’ DNA from the GenBank database (Fig. 5). The DNA nucleotide sequences of Ca. Liberibacter asiaticus Nagpur mandarin isolate and sweet orange isolate have been deposited in the GenBank database under accsession numbers EU 939452 and FJ 177536 respectively. The psyllid vector of greening, Diaphorina citri was found present in most of the areas surveyed. Ca. L. asiaticus could also be detected by PCR from psyllids (Fig. 3,A, B) collected in HLB-affected orchards using OI1/ OI2c primer pairs. Efforts are however continuing to improve every steps of the diagnostic protocol for developing more robust and highly sensitive diagnostic tools.

Fig 1. A, Emergence of CGD symptoms from the upper canopy of a Mosambi sweet orange (C. sinensis) tree. B, Classic blotchy mottle symptoms in Mosambi sweet orange leaves.

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Fig 2. CGD symptoms in different varieties of citrus in India. A, Nagpur mandarin (C. reticulata) B, Kaghzi lime (C. aurantifolia) C, Assam lemon (C. limon) and D, Pummelo (C. grandis).

Fig 3. A, Psyllid (Diaphorina citri) nymphs on the new flushes of Nagpur mandarin tree. B, Adult psyllids feeding on a CGD-infected Nagpur mandarin leaf.

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640 bp

1160 bp

520 bp

Fig. 4. PCR detection of CGD bacterium, Ca. L. asiaticus in India. A, Agarose gel electropheresis of DNA extracted from citrus leaf midribs and bark tissues amplified with the OI1/OI2c primers and B, Digestion of the amplified product with the restriction enzyme Xba 1. M : 1 Kb ladder (MBI Fermentas, Hanover, MD, USA), lane1, extracts from healthy citrus, lane 2-5, extracts from CGD-infected citrus plants.

LasNagpur I EU 939452 I LasJapan|AB008366.1|  LasFLDade|EU130552.1|

LasChina|DQ157274.1| 100 LasMalayasia|EU224394.1| LasChina|DQ303210.1|

LasFLNassau|EU130553.1| LasFLPalmBeach|EU130554.1| LasSPBrazil|AY919311.1|

LasMalayasia|EU224393.1| LafNelspruit SAfrica|L22533.1| 100 Lafcapensis SAfrica|AF137368.1|

LamSP Brazil|AY742824.1|

0.015 0.010 0.005 0.000 Fig. 5. Phylogenetic tree constructed from alignment of 16S rRNA gene sequences from “Ca. Liberibacter”. Genbank accession nos. are given in parentheses. Bootstrap values (based on 1000 replications) are indicated at the nodes. The phylogenetic tree was linearized assuming equal evolutionary rates in all lineages. Las= Ca. Liberibacter asiaticus, Laf = Ca. L. africanus, Lam = Ca. L. americanus,

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Literature cited

Bové JM. 2006. Huanglongbing : a destructive, newly emerging, century-old disease of citrus. J. Plant Pathol. 88: 7-37. Ahlawat YS. 1997. Viruses, greening bacterium and viroids associated with citrus (Citrus spp.) decline in India. Indian J. Agril. Sci. 67: 51-57. Fraser LR, Singh D. 1966. Greening virus, a threat to citrus industry. Indian Hort. 10 : 21-22. Jagoueix S, Bové JM, Garnier M. 1996. PCR detection of the two liberobacter species associated with greening disease of citrus. Mol. Cell. Probes 10: 43 - 50. Tamura K, Dudley J, Nei M, Kumar S. 2007. MEGA 4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol. Bio. Evol. 24: 1596-1599.

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