First report of a ‘Candidatus Phytoplasma australasia’‐related strain (16SrII‐D) associated with crown choking of areca palm in India

Abstract

Areca palm (Areca catechu) is an economically important crop that is grown primarily in South Asia and South India, mainly in the states of Karnataka, Kerala and Maharashtra. With 0.73 million hectares cultivated and 1.2 million tonnes produced, India ranks first in world areca nut production. The state of Karnataka alone accounts 80% of annual production in India. During January 2021, crown choking symptoms (locally named “Hidimundige”), which resemble phytoplasma symptoms were observed on areca palm plantations. The highest disease incidence (50%) was recorded in Ajjarani, Sirsi Taluk and Uttara Kannda, followed by 30% in Haralakatta, Channagiri Taluk, Davanagere, Kelaginuru and Honnavara Taluk, in the district of Uttara Kannada (Karnataka state). The lowest disease incidence was 1% for the Dakashina Kannada and Tumkur districts in Karnataka.The most prominent symptom in infected palms was short, dark green leaves with a wavy leaf lamina. Leaflets failed to unfold properly leading to a bunchy top or witches’ broom appearance, and became crinkled and brittle. Reduced internodal length, drying of inflorescences and tapering of stem at the apex were also recorded. The roots became black, crinkled and brittle, and a pinkish or brownish discoloration was observed in the phloem. Affected plants failed to produce new leaves and existing leaves fell leading to a bare stem and death of the plant in quick succession (Figs. 1-3). About 80–100% of the plants died in affected areas. Seven leaf samples were collected from diseased palms growing in the Zonal Agricultural & Horticultural Research Station, Keladi Shivappa Nayaka University of Agricultural and Horticultural Sciences in Shivamogga, Karnataka (Latitude: 13° 55' 47.748“. Longitude: 75° 34' 5.1636”) during March 2022. Typical phytoplasma pleomorphic bodies lacking cell walls were seen in the sieve tube cells of leaf midrib sections from diseased areca palms, when examined by transmission electronic microscopy (Spurr, 1969) at the Sri Sai Histology Center, Hyderbad India (Fig. 4). The phytoplasma cell sizes ranged from 500 - 2000 nm. Seven leaf and three inflorescence samples from diseased palms and two leaf samples from asymptomatic palms were collected from the same location. Total DNA was extracted by the CTAB method and subjected to direct PCR using primers P1 (Deng & Hiruki, 1991) and P7 (Schneider et al., 1995) and nested PCR with R16F2n/R16R2 primers (Gundersen & Lee, 1996) that amplify the phytoplasma 16S rRNA gene. The expected direct (1.8 Kb) and nested (1.25 Kb) PCR products were obtained from all the diseased plant samples and positive reference controls but not from the symptomless plants. The R16F2n/R2 PCR products were cloned and sequenced directly at Biokart Pvt. Ltd., Karnataka, India, and the sequence deposited in GenBank (Accession No. ON606488). The R16F2n/R2 sequence was subjected to BLAST analysis and showed the highest nucleotide identity (99.92%) with 16SrII phytoplasma isolates, ‘Candidatus Phytoplasma aurantifolia’ (MT555412, MT555411) identified in Croton bonplandianus in Maharashtra, India. Virtual RFLP analysis of the R16F2n/R2 sequence with iPhyClassifier yielded an RFLP pattern identical to that of the 16Sr group II, subgroup D (Y10097) with a similarity coefficient of 1.00. Phylogenetic analysis (MEGA 7.0.18) using the neighbour-joining algorithm with selected phytoplasma reference strains confirmed that the areca palm phytoplasma clustered within the clade of phytoplasma strains of the 16SrII group (Fig. 5). In India, the cultivation of areca nut is mostly limited to the states of Assam, Karnataka and Kerala, and palms in these areas are affected by a yellowing disease named yellow leaf disease (YLD). YLD is one of the most important diseases of this palm species (Nambiar, 1949) and it is associated with the 16SrXI phytoplasma group, ‘Ca. P. oryzae’. (Chaithra et al., 2014). ‘Ca. P. aurantifolia’ is known to infect several plant species in India (Rao et al., 2017). However, this is the first report of a ‘Ca. P. aurantifolia’- related strain causing crown choking of Areca catechu in India. The impact of crown choking on areca nut yield, the geographic distribution of the disease and its epidemiological implication in India are currently under investigation.