Occurrence of Witches'-Broom, a New Phytoplasma Disease of Acid Lime (Citrus aurantifolia) in India

In India, acid lime (Citrus aurantifolia (L.) Swingle) is one of the most important citrus fruits grown. It constitutes nearly 20% of the total citrus production. During 1995, an unusual type of disease was observed on a 6-year-old acid lime plant in an orchard in the Nagpur District in eastern Maharashtra. It was named witches'-broom disease (WBD) to reflect the most conspicuous symptom. Other symptoms included small chlorotic leaves, highly proliferated shoots, and shortened internodes. Leaves dropped prematurely and infected twigs were distorted. In advanced stages, infected branches had dieback symptoms. WBD of lime has been reported from Oman and UAE (1) and the causal phytoplasma was designated “Candidatus Phytoplasma aurantifolia” (2). Subsequent surveys in 1995-1998 revealed disease incidences as high as 5% in Maharashtra and in other major acid-lime-growing states—Andhra Pradesh, Tamilnadu, and Karnataka. After the grafting of infected acid lime shoots, disease symptoms developed on Troyer citrange, rough lemon, and Rangpur lime, but not on sweet orange (mosambi), mandarin (Nagpur), or trifoliate orange. The WBD agent was transmitted from infected acid lime to periwinkle (Catharanthus roseus) plants and vice versa by dodder (Cuscuta reflexa). Ultrathin sections of leaf midrib of infected acid lime plants were fixed on copper grids, stained with uranyl acetate and lead acetate, and examined in a JEM 100S transmission electron microscope. Numerous bodies having the characteristic morphology of phytoplasmas were observed in phloem sieve tubes of acid lime in diseased but not in healthy leaves. The phytoplasmal bodies ranged from 100 to 800 nm in diameter and were bounded by a poorly defined membrance. Freehand transverse sections of young internode regions of a WBD-infected periwinkle plant were stained in DAPI (4′, 6 diamidino-2-phenylindole; 1.0 μg/ml) and were observed with a fluorescent microscope (Leica). An intense bluish-white fluorescence in the phloem elements of diseased periwinkle and its absence in healthy samples were consistent with the presence of phytoplasmas. This is the first report of phytoplasma-induced witches'-broom disease of acid lime in India. References: (1) M. Garnier et al. Plant Dis. 75:546, 1991. (2) L. Zreik et al. Int. J. Syst. Bacteriol. 45:449,1995.