Abstract
Phytoplasma infected acid lime plants in India develop characteristic symptoms like small chlorotic leaves, multiple sprouting and shortened internodes. Leaves drop prematurely and infected branches have distorted twigs resembling witches’ broom appearance which eventually show die-back symptoms. During its first report in 1999, witches’ broom disease identification was made on the basis of symptomatology and electron microscopy. However, molecular techniques have proved to be more accurate and reliable for phytoplasma detection than the conventional methods. During survey in the year 2010 six samples were collected from infected acid lime plants showing typical field symptoms from Vidarbha region of Maharastra. Initially, phytoplasma bodies were observed in phloem tissues of all six symptomatic samples under JEM 100S transmission electron microscope and all these six samples were subsequently screened using different set of phytoplasma specific universal primers by nested PCR, a widely recommended molecular technique for phytoplasma detection. In the present study P1/P7 “universal” phytoplasma-primer set was used for first round of PCR and amplified products were processed separately for nested PCR with three different nested primer pairs viz. R16F2n/R16R2, R16mF2/R16mR1 and fU5/rU3. The presence of phytoplasma was confirmed in all six suspected samples and one representative ~1.2 kb size amplicon was sequenced and deposited in GenBank as Candidatus Phytoplasma species AL-M (JQ808143). This is the first report of PCR based molecular detection of phytoplasma-induced witches’ broom disease of acid lime (WBDL) in India. Further molecular evaluation to determine the identity to the species level is in progress.
Abbreviations
- WBDL:
-
Witches’ Broom Disease of Acid Lime
- UAE:
-
United Arab Emirates
- RFLP:
-
Restriction fragment length polymorphism
- EM:
-
Electron microscopy
- PVPP:
-
Polyvinylpolypyrrolidone
References
Ahrens U, Seemuller E (1992) Detection of DNA of plant pathogeneic mycoplasma like organisms by a polymerase chain reaction which amplifies a sequence of the 16S rRNA gene. Phytopathology 82:828–832
Alhudaib K, Arocha Y, Wilson M, Jones P (2009) Molecular identification, potential vectors and alternative host of the phytoplasma associated with a lime decline disease in Saudi Arabia. Crop Prot 28:13–18
Bertaccini A, Duduk B (2009) Phytoplasma and phytoplasma diseases: a review of recent research. Phytopathology 48:355–378
Bove JM (1986) Witches’ broom disease of lime. FAO Plant Prot Bull 34:217–218
Bove JM, Danet JL, Bananej K, Hassan ZN, Taghizadeh M, Salehi M, Garnier M (2000) Witches’ broom disease of lime in Iran. In: Proceedings of the 14th conferences of IOCV, Riverside, USA, pp. 207–212
Chen J, Pu X, Deng X, Liu S, Li H, Civerolo E (2009) A Phytoplasma related to ‘Candidatus Phytoplasma asteris’ detected in citrus showing Huanglonbing (Yellow Shoot Disease) symptoms in Guangdong, P. R. China. Phytopathology 99:236–242
Davis RE, Lee IM (1993) Cluster-specific polymerase chain reaction amplification of 16S rDNA sequences for detection and identification of mycoplasma-like organisms. Phytopathology 83:1008–1011
Firrao G, Garcia-Chapa M, Marzachi C (2007) Phytoplasma genetics, diagnosis and relationships with the plant and insect host. Front Biosci 12:1352–1375
Garnier M, Zreik L, Bove JM (1991) Witches’ broom a lethal mycoplasmal disease of lime in the sultanate of Oman and the United Arab Emirates. Plant Dis 75:546–551
Ghosh DK, Das AK, Singh S, Singh SJ, Ahlawat YS (1999a) Occurrence of witches’ broom – a new Phytoplasmal disease of Acid lime (Citrus aurantifolia) in India. Plant Dis 83:302
Ghosh DK, Das AK, Singh S, Singh SJ, Ahlawat YS (1999b) Association of phytoplasma with witches’ broom, a new disease of acid limes. Curr Sci 77:174–177
Kumar B, Mistry NC, Singh B, Gandhi CP (2011) Indian Horticulture Database 2011. National Horticulture Board, Gurgaon. Aristo Printing Press, New Delhi, pp 44–67
Lee IM, Hammond RW, Davis RE, Gundersen DE (1993) Universal amplification and analysis of pathogen 16S rDNA for classification and identification of mycoplasma-like organism. Phytopathology 83:834–842
Lee IM, Gundersen-Rindal DE, Devis RE, Bartoszyk IM (1998) Revised classification scheme of phytoplasmas based on RFLP analyses of 16S rRNA and ribosomal protein gene sequences. Int J Syst Bacteriol 48:1153–1169
Lee IM, Devis RE, Gundersen-Rindal DE (2000) Phytoplasma: phytopathogenic mollicutes. Ann Rev Microbiol 56:1593–1597
Manimekalai R, Soumya VP, Kumar RS, Selvarajan R, Reddy MK, Baranwal VK (2010) Molecular detection of 16srXI group phytoplasma associated with root wilt disease of coconut (Cocos nucifera) in India. Plant Dis 94:636
Manimekalai R, Nair S, Soumya VP, Roshna OM, Thomas GV (2011) Real-time PCR technique-based detection of coconut root (wilt) phytoplasma. Curr Sci 101:1209–1213
Mannan S, Ibrahim M, Chohan SN, Holford P (2010) Detection of phytoplasma in citrus orchards of Pakistan. J Phytol 2:49–54
Santos-Cervantes ME, Chavez-Mediana JA, Mendez-Lozano J, Leyva-Lopez NE (2008) Detection and molecular characterization of two little leaf phytoplasma strains associated with Pepper and Tomato diseases in Guanajuato and Sinaloa, Mexico. Plant Dis 92:1007–1011
Seemueller E, Marcone C, Lauer U, Ragozzino A, Goeschl M (1998) Current status of molecular classification of the phytoplasmas. Plant Pathol 80:3–26
Zreik L, Carle P, Bove JM, Garnier M (1995) Characterization of the mycoplasma-like organism associated with witches’ broom disease of lime and proposition of a Candidatus taxon for the organism, ‘Candidatus Phytoplasma aurantifolia. Int J Syst Bacteriol 45:449–453
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Ghosh, D., Bhose, S., Manimekalai, R. et al. Molecular detection of Candidatus Phytoplasma spp. causing witches’ broom disease of acid lime (Citrus aurantifolia) in India. J. Plant Biochem. Biotechnol. 22, 343–347 (2013). https://doi.org/10.1007/s13562-012-0160-z
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DOI: https://doi.org/10.1007/s13562-012-0160-z