First report of a Jujube witches’‐broom phytoplasma (16SrV) strain associated with witches’‐broom and little leaf disease of Solanum melongena in India

Abstract

For molecular detection of phytoplasma, total DNA was isolated from diseased and symptomless leaf samples (100 mg). The phytoplasma 16S rRNA gene was amplified by direct PCR using P1/P6 primers (Deng & Hiruki, 1991) and nested PCR with universal primers R16F2n/R16R2 (Gundersen & Lee, 1996), producing amplicons of the expected size (c. 1.2 kb) for the diseased (4/4) samples but not from the symptomless plant tested. The consensus partial 16S rRNA gene sequences were identical and the sequence data was submitted to GenBank, Accession Nos.

Solanum melongena (aubergine or brinjal; family Solanaceae) is a popular vegetable grown extensively in India, Bangladesh, Pakistan, China, Japan, Philippines, Egypt, France, Italy, and the United States (Chadha et al., 1993). Brinjal's cultivation is hampered by the Brinjal little leaf (BLL) phytoplasma. A phytoplasma survey was conducted in various brinjal production areas of Hoshangabad in Central India (Figure 1) during May 2019. Phytoplasma-like symptoms such as proliferation of axillary shoots, witches’- broom, excessive branching, little leaf and the absence of flowers and fruits when compared to symptomless brinjal plants were observed (Figure 2).

FIGURE 1. Field view of a brinjal plantation showing withches’-broom and little leaf disease growing at Hoshangabad, Madhya Pradesh.

FIGURE 2. Brinjal plants showing symptoms of witches’-broom and little leaf (b) compared to a symptomless brinjal plant (a).

For molecular detection of phytoplasma, total DNA was isolated from diseased and symptomless leaf samples (100 mg). The phytoplasma 16S rRNA gene was amplified by direct PCR using P1/P6 primers (Deng & Hiruki, 1991) and nested PCR with universal primers R16F2n/R16R2 (Gundersen & Lee, 1996), producing amplicons of the expected size (c. 1.2 kb) for the diseased (4/4) samples but not from the symptomless plant tested. Two nested PCR amplicons were purified (Wizard SV gel extraction kit; Promega, USA) and sequenced bidirectionally (Bioinovations Pvt. Ltd., India). The consensus partial 16S rRNA gene sequences were identical and the sequence data was submitted to GenBank, Accession Nos. MW025971 (BR01) and MW025257 (BR02).

BLASTn analysis showed that the sequence shared 99% sequence identity with the 16S rRNA gene sequence of Jujube witches'-broom phytoplasma (MH972556, MH972553, MH972548) and ‘Candidatus Phytoplama balanitae’ (HG937644, LT558785, MH819290), Periwinkle yellows phytoplasma (EU375835) and the Elm yellows group (16SrV). Phylogenetic analysis (MEGA v 7.1) showed the closest relationships with strains of Jujube witches'-broom phytoplasma (MH972556, H744152 and MH972548) and ‘Ca. P. balanitae’ (LT558785), a member the group 16SrV, ‘Ca. P. ulmi’ (Figure 3).

FIGURE 3. Phylogenetic analysis based on 16S rRNA gene sequences MW025971 (BR01) and MW025257 (BR02) with 16SrV phytoplasma strains, ‘Ca. P. balanitae’ and other phytoplasma groups. Phylogenetic tree was generated with MEGA software version 7.1 using the neighbour-joining method and the NJ plot programme in a bootstrap test (1000 replicates).

Six phytoplasma ribosomal groups (16SrI, 16SrII, 16SrIII, 16SrVI, 16SrIX, 16SrXII) have been associated with S. melongena globally. BLL phytoplasmas were first reported in India in 1995 (Schneider et al., 1995). In India and Japan, a BLL phytoplasma was identified as a member of the group 16SrI, ‘Ca. P. asteris’ (Kumar et al., 2012). The group 16SrVI, ‘Ca. Phytoplasma trifolii’ was also associated with BLL symptoms in India (Yadav et al., 2015). However, this is the first report of a ‘Ca. P. ulmi’ strain (group 16SrV), associated with a witches'-broom disease of brinjal in India.

This finding has a significant impact for future epidemiological studies since brinjal could be an alternative host for 16SrV phytoplasma strain which affect several other important vegetable crops cultivated in Hoshangabad, Central India.

ACKNOWLEDGEMENTS

The authors would like to thank to the Head of the Department of Microbiology and Vice Chancellor, Barkatullah University, Bhopal for support and encouragement.