Natural occurrence of 'Candidatus Phytoplasma ziziphi' isolates in two species of jujube trees (Ziziphus spp.) in India

Ziziphus jujuba known as Indian jujube or Ber is an important plant cultivated for its fleshy fruits rich in sugars and vitamins. In India, the ripe fruits are mostly consumed raw, but are sometimes stewed. The dried ripe fruit is a mild laxative; seeds are sedative and are taken to halt nausea, vomiting, and abdominal pains in pregnancy. Ziziphus nummularia known as Jharber commonly grows along roadsides and uncultivated land in India. The tender parts of both the species are favoured fodder of sheep, goats, and cattle. During October 2007, phytoplasma-like symptoms were observed on Z. jujuba and Z. nummularia plants growing at Payagpur, Bahraich district of Uttar Pradesh, India. Z. nummularia showed rosetting, proliferation of axillary shoots, witches' broom-like appearance and little leaves malformed and golden yellow (Fig.1a). Z. jujuba exhibited severe rosetting, but little leaves remained green (Fig.1b).

The total DNA from samples of infected plants was isolated and subjected to nested PCR using universal primers specific to the 16S rRNA gene: P1/P6 (Deng & Hiruki, 1991) and R16F2n/R16R2n (Gundersen & Lee, 1996). This resulted in positive amplicons of ~1.5 and ~1.2 kbp, respectively, in infected samples (2/2) of Z. jujuba and Z. nummularia, but not from the healthy ones. The ~1.2 kbp amplicons (two) from each sample were sequenced and data deposited in GenBank, Accession Nos. EU375487 (Z. nummularia) and EU366162 (Z. jujuba).

BLAST comparisons of both 16S rDNA sequences of EU375487 and EU366162 shared 99% identity with each other, and 98% with those of members of the 16SrV elm yellows group, including accessions AB052875, AB052877 and AB052879. These accessions describe the new Candidatus Phytoplasma species, 'Candidatus Phytoplasma ziziphi', associated with jujube witches' broom in China, Japan and Korea (Jung et al, 2003). Therefore, phytoplasmas associated with witches' broom disease in Z. jujuba and Z. nummularia in Bahraich district, in India, are considered isolates of 'Ca. Phytoplasma ziziphi'. To our knowledge, this is the first record of jujube witches'-broom phytoplasma in Z. jujuba and Z. nummularia in India.