First Report of ' Candidatus Phytoplasma citri' related strain (16SrII-C subgroup) associated with phyllody disease of cumin in India.

Cumin (Cuminum cyminum L.), is an important export-oriented seed spice crop for India. Cumin is popularly used for flavouring food, including soups, pickles and vegetables, and for herbal medicine. India is the largest producer, consumer and exporter of cumin seed with an annual production of 0.795 million tones over an area of 1.09 million hectares. During 2020-21, India exported about 0.299 million tons of cumin worth of Rs 33280 million (Anonymous, 2021). Recently, phytoplasma suspected symptoms were observed in cumin at Agricultural Research Station, Mandor, Jodhpur, Rajasthan, India from 2019. The symptoms related to phytoplasma infection were first recorded after 70-75 days of sowing in the month of January of the years 2019 to 2022. The major symptoms recorded were yellowing, phyllody, witches-broom, yellowing and deformed elongated seeds. Disease incidence was recorded as 0.25-1.0%, 0.5-1.5%, 0.5-2.5 % and 0.5-10.6% during the years 2019, 2020, 2021 and 2022, respectively using quadrate method. In 2022, among different genotypes assessed GC 4, MCU 73, MCU 105, and MCU 32 exhibited lower disease incidences ranging from 0.5% to 1.5%, while MCU 78 recorded the highest disease incidence at 10.6%. To detect the association of phytoplasma with symptomatic cumin samples, genomic DNA was extracted from four representative cumin genotypes (CuPP-MND-01 to CuPP-MND-04) and one asymptomatic cumin plant using the Qiagen DNeasy plant mini kit (Germany). The extracted DNA was amplified using nested PCR assays with universal phytoplasma detection primers for 16S rRNA gene (P1/P7 and R16F2n/R16R2) (Schneider et al., 1995; Gundersen and Lee, 1996) and secA gene specific primers (SecAfor1/SecArev3 followed by nested PCR primers SecAfor5/ SecArev2) (Hodgetts et al. 2008; Bekele et al. 2011). The amplicons of ∼1.25 kb with 16S rRNA gene and ∼600 bp with secA gene specific primers were amplified in all symptomatic cumin plant samples and positive control of brinjal little leaf. PCR amplified products from the four selected positive samples (CuPP-MND-01 to CuPP-MND-04) of 16S rRNA gene and secA gene, were sequenced from both ends. The 1,245 bp sequences were deposited in GenBank (OQ299007-10), which showed 100% sequence identity with each other and 99.4% identity with 'Candidatus Phytoplasma citri' reference strain (GenBank accession: U15442) (Rodrigues Jardim et al. 2023). The phylogenetic analysis and virtual RFLP analysis using 17 restriction enzymes of 16S rRNA gene sequences through iPhyclassifier allowed affiliating the cumin phytoplasma strains with 16SrII-C subgroup strain with a similarity coefficient of 1 to the reference pattern of 16Sr group II, subgroup C (GenBank accession: AJ293216) (Zhao et al. 2009). In addition, the phylogenetic analysis of the secA gene-based sequences (OQ305073-76) further confirmed the close association of 16SrII-C group phytoplasmas with phyllody and witches' broom disease of cumin. Earlier 16SrII-C subgroup phytoplasma has been reported from various crops and weeds in India (Rao et al. 2021). However, no phytoplasma association has been reported earlier with cumin in India and abroad. To the best of our knowledge, this is the first report on the association of 16SrII-C group phytoplasma causing phyllody, witches' broom in cumin genotypes. This report has economic and epidemiological implications and needs immediate attention to reduce export losses due to phytoplasma disease in cumin and to prevent the potential spread to other crops.