Viroid Diseases of in

P. Ramachandran, P. K. Pandey, Y. S. Ahlawat, Anupam Varma and S. P. Kapur

ABSTRACT. Exocortis disease of citrus in India has been known to occur for more than two decades but studies on its causal viroid have not been accomplished. The results of analysis of samples showing exocortis-like symptoms in R-PAGE revealed presence of two viroid species, one belonging to Indian Tomato bunchy top viroid (ITBVd) and the other to citrus B (CVd-IIa) group of viroids. This is for the first time in India that viroids have been found infecting citrus plantings.

Citrus exocortis disease was first tained in a greenhouse (max. 25-35 C described by Fawcett and Klotz (4). and rnin. 20-25 C). The leaves from symp The involvement of a viroid with this tornatic plants 6 months aRer inoculation disease was established in 1972 (10). In were used as viroid source. Young leaf India, exocortis was reported in some samples obtained from trees suspected trees in Delhi and which were to be affected with exocortis disease grafted on (5,8). These from Ludhiana were also analysed reports were based on visual symp- (Table 1). Cucumber cv. Suyo seed- toms and transmission to Rangpurlime lings (cotyledons) were used for bio- of limited samples. We therefore at- assay of the nucleic acid preparation tempted to investigate if any viroid obtained from grafted plants and sam- species is present in a disease showing ples from Ludhiana. exocortis-like symptoms in citrus plant- Six months after inoculation, con- ing~in India. The results are presented spicuous stunting was observed in in- in this paper. oculated Rangpur lime plants and epi- Budwood were collected from cit- nasty symptoms in from rus plantings in Pune (Maharashtra), the samples collected from Pune and Bangalore (Karnataka) and Delhi (Table Bangalore but not from Delhi source. 1) and graft-inoculated on rooted cut- However cucumber plants showed typ- tings of Etrog citron and Rangpur lime ical symptoms of viroid infection from seedlings. Grafted plants were main- Delhi samples.

TABLE 1 SOURCE OF PLANTING MATERIAL FOR VIROID DETECTION

Symptoms Place of Rootstock ScionVar. No. oftrees collection (RS) (sv) sampled (RS) (sv) -- -- - Ludhiana Rangpurlime Kinnow 1 BSz LY (Punjab) mandarin MaltaSwoz 1 Bs Db Etrogcitron 1 Bs St

Pune Trifoliate Mosambi Swo 1 B~P Db (Maharashtra)

Bangalore Rangpurlime (Karnataka)

Delhi Not known Trifoliate 6 No visible Rough Mosambi Swo 3 symptoms

'Swo = sweet orange, Bs = bark scaling, Bsp = bark splitting, Db die back, St = stunting.

438 Short Communications 439

Young leaves of inoculated Etrog ARer two washes in sterile distilled citron plants were used for nucleic acid water the gels were developed inafresh (NA) extraction. Total NA were pre- solution of 375 mM KOH, 2.3 mM pared by grinding 1 g of leaf tissue in sodium borate, 0.5% HCHO (37% wlv) 3 ml of extraction buffer (0.53 M and further fixed in a solution of 70 mM NH,OH, 0.013 M (EDTA) with Tris, 4 sodium carbonate. Silver stained M LiC1, and 1% purified bentonite) and bands on electrophoretograms were 4 m10.05 M Tris saturated phenol (con- evaluated based on their electrophore- taining 0.1 g of &hydroxy quinolinel tic mobility in comparison to reference 100ml). Samples and extraction buffer samples. In the present study, purified were maintained at 4-5 C throughout low molecular weight RNA from In- the extraction procedure. The samples dian tomato bunchy top viroid (ITBVd) were homogenized in liquid nitrogen and an isolate of hop stunt viroid and centrifuged (15 min, 8,000 g at 4 (HSVd) from grapevine isolated by us C, and nucleic aclds from upper aque- were used as reference samples. ous layer were precipitated (-20 C, 30 The results of R-PAGE revealed min) by adding 0.5 volume of 7.5 M differences in electrophoretic mobility ammonium acetate solution and 2.5 vol- in bands observed in different samples. ume of ethanol. The precipitate was Bands observed in citron samples from collected by centrifugation as above, Ludhiana, Mosambi sweet orange dried with a current of air, and dissol- from Pune and Chini sweet ornage ved in sterilized double glass dlstilled from Bangalore corresponded in water (100 pllg of tissue for gel elec- electrophoretic mobility to ITBVd trophoresis). while that in trifoliate and Mosambi Return Polyacrylamide Gel Elec- from Delhi to HSVd type. No bands trophoresis (R-PAGE) was used (7,9) were detected in samples of Kinnow with some modification. Aliquots of 10 mandarin and Malta sweet orange from pl were mixed with 6 p1 of a solution Ludhiana and healthy controls. containing 1% xylene cyan01 and 1% Viroids have been detected from bromophenol blue prepared in 40% suc- two type of trees - one showing typical rose. First electrophoretic separation exocortis symptoms and transmissible was at constant current 46 mA for 2.5 to its indicator hosts, whereas the hr at 20 C on 7.5% acrylamide non- other ones were without any visible denaturing slab gel in high salt buffer symptoms. Bioassay of symptomless (89 mM Tris, 89 mM boric acid , 2.5 trees on cucumber, however, showed mM EDTA, pH 8.3). Duringthe second the presence of viroid infection. R- run the buffer in both the reservoirs PAGE analysis of the test samples was replaced with a low salt buffer (118 clearly showed the presence of viroid dilution of the high salt buffer) at boil- species belonging to ITBVd (7) and ing point (95 C) and the gel left as such HSVd (2) in citrus plantings in the without passing current for 5 min. country. The band on the HSVd pat- These conditions denature the viroid. tern and its infection to cucumber cv. Later the run was resumed at constant Suyo was reported as Citrus B viroid current 46 rnA for 2 hr at 70 C with (CBVd) and was considered as a strain reversed polarity. Gels were stained of HSVd (2). CBVd is now known to using silver nitrate (6). After fixing the be identical to CVd-IIa (13). It is there- gels in a mixture of acetic acid and fore concluded that citrus plantings in ethanol for 1 to 2 hr they were kept in India carried viroid infection appar- a solution of 0.2% silver nitrate for 1hr. ently of CVd-IIa and ITBVd types.

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