New Disease Reports
New Disease Reports (2008) 18, 41.
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First report of a 'Candidatus Phytoplasma asteris'-related strain associated with a yellows disease of black pepper (Piper nigrum) in India 

C.R. Adkar-Purushothama, P. Casati, F. Quaglino, G. Durante and P.A. Bianco*

*ero.bianco@unimi.it

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Accepted: 23 Dec 2008

Black pepper (Piper nigrum) yellows is a newly recognized disease in Coorg (Kodagu) district of Karnataka State in southern India, where pepper is grown in 10,690 ha with a production of 2360 metric tonnes. Symptoms include yellowing and curling of the leaves. In the advanced stage, vines become yellow and slender with generalized decline and reduction in the yield noted (Fig.1).

Based on observation of symptoms, an association of a phytoplasma with the disease was suspected. Leaf samples from symptomatic and asymptomatic vines were collected from black pepper plants during December 2007 and January 2008. Total DNA was extracted as previously described (Adkar-Purushothama et al., 2007), and assayed for phytoplasma infection through nested polymerase chain reaction (PCR) employing primer pairs P1/P7 (Deng & Hiruki, 1991) and R16F2n/R16R2 (F2n/R2) (Gundersen & Lee, 1996), for the amplification of phytoplasma 16S rDNA. DNAs extracted from ‘Candidatus Phytoplasma asteris’-related strain AAY (group 16SrI) and ‘Ca. Phytoplasma ulmi’-related strain EY1 (group 16SrV), maintained in periwinkle (Catharanthus roseus), were used as positive controls. Reaction mixtures containing DNA extracted from healthy periwinkle and reaction mixture without template DNA were used as negative controls. All samples from symptomatic pepper plants along with positive controls showed 1250bp PCR products (F2n/R2 fragment), which were absent in asymptomatic samples (Fig.2).

A representative phytoplasmal F2n/R2 fragment was cloned, sequenced and deposited in GenBank (FJ462798). The 16S rDNA nucleotide sequence of the phytoplasma identified in yellows-diseased black peppers [Coorg Black Pepper Yellows (CBPY) phytoplasma] shared >99% sequence identity with that of the Sugarcane yellows phytoplasma type I (EU423900) from group 16SrI, and more than 98% with those of other related strains including AAY (X68373) and OY-M (AP006628). Moreover, CBPY phytoplasma showed 98% of identity with the BPP phytoplasma (AY823413), previously associated with phyllody of black pepper in India (Bhat et al., 2006). To our knowledge, this is the first molecular evidence for the association of a phytoplasma with a yellows disease of black pepper, and the identification of a ‘Ca. Phytoplasma asteris’-related strain associated with such disease in India.

Figure1+
Figure 1: (a) Healthy black pepper plant and (b) yellows diseased black pepper plant
Figure 1: (a) Healthy black pepper plant and (b) yellows diseased black pepper plant
Figure2+
Figure 2: Phytoplasmal 16S rDNA F2n/R2 fragments (1.25Kbp) visualized through electrophoresis on 1% agarose gel. L: 1Kbp plus DNA ladder (Invitrogen, Carlsbad, CA, USA); 1-5: yellows diseased black pepper plants; 6-8: asymptomatic black pepper plants; AAY: ‘Ca. Phytoplasma asteris’-related strain AAY; EY1: ‘Ca. Phytoplasma ulmi’-related strain EY1; HP: healthy periwinkle; W1: direct PCR without template DNA; W2: nested PCR without template DNA.
Figure 2: Phytoplasmal 16S rDNA F2n/R2 fragments (1.25Kbp) visualized through electrophoresis on 1% agarose gel. L: 1Kbp plus DNA ladder (Invitrogen, Carlsbad, CA, USA); 1-5: yellows diseased black pepper plants; 6-8: asymptomatic black pepper plants; AAY: ‘Ca. Phytoplasma asteris’-related strain AAY; EY1: ‘Ca. Phytoplasma ulmi’-related strain EY1; HP: healthy periwinkle; W1: direct PCR without template DNA; W2: nested PCR without template DNA.

References

  1. Adkar-Purushothama CR, Gottravalli-Ramanayaka J, Sano T, Casati P, Bianco PA, 2007. Are phytoplasmas the etiological agent of yellow leaf disease of Areca catechu in India? Bulletin of Insectology 60, 161-162.
  2. Bhat AI, Madhubala R, Hareesh PS, Anandaraj M, 2006. Detection and characterization of the phytoplasma associated with a phyllody disease of black pepper (Piper nigrum L.) in India. Scientia Horticulturae 107, 200-204.
  3. Deng S, Hiruki C, 1991. Amplification of 16S rRNA genes from culturable and non-culturable mollicutes. Journal of Microbiological Methods 25, 53-61.
  4. Gundersen DE, Lee IM, 1996. Ultrasensitive detection of phytoplasma by nested-PCR assays using two universal primer pairs. Phytopathologia Mediterranea 35, 144-151.
This report was formally published in Plant Pathology

©2008 The Authors