Citrus Virologists Conference Proceedings ( 1957-2010 ) Title Detection of a Necrotic Strain of Citrus Ringspot in Star Ruby Grapefruit in Spain Permalink

A 6-yr-old planting of Star Ruby grapefruit on sour orange rootstock propagated from illegally introduced budwood was inspected for ringspot symptoms. Yellow blotches, chlorotic mottling and ring-like patterns were observed in mature leaves and fruits of some plants. In the spring flush, some young shoots had shock symptoms and young leaves showed different chlorotic patterns. None of the trees showed bark scaling, but a few of them had blisters on some young twigs. Graft-inoculated seedlings of sweet orange, sour orange, Mexican lime, Duncan grapefruit, Dweet tangor and Etrog citron developed a severe shock reaction with leaf abscission and dieback in the first flush. Necrotic pin points or etching appeared in some leaves. Inoculated grapefruit and citron seedlings grew poorly in subsequent flushes and some plants showed dieback, and finally died. This ringspot isolate shows similarities with necrotic strains described in Texas and Florida. The new isolate was readily transmitted to C. quinoa inducing chlorotic to necrotic local lesions. Transmission was not obtained using Aphis gossypii or A. eitricola as vectors. Partially purified extracts from infected plants had infectivity associated to two fractions in a sucrose density gradient. A 48-kd protein was present in the two fractions, but not in equivalent extracts from healthy plants. A 38-kd protein was sometimes found associated with infectious fractions, usually when purifying from young shoots at the shock stage. Virus particles were not observed in infectious fractions. Index words. Purification, protein, double stranded RNA, aphid transmission, immunoelectron microscopy, herbaceous hosts. A number of graft-transmissible diseases of citrus that induce different chlorotic patterns in young leaves have been currently grouped and named as psorosis diseases (19, 23). The members of this group, which includes psorosis A, psorosis B, ringspot (CRSV), concave gum-blind pocket, impietratura and cristacortis, cause different field symptoms, but they are difficult to identify by indexing on indicator plants. Mechanical transmissibility to Chenopodium quinoa and other herbaceous hosts (20) has been associated to most ringspot isolates, psorosis B and, in general, to the presence of severe bark scaling symptoms in the field trees (8, 18). Citrus plants mechanically inoculated with different local lesion isolates of CRSV reproduced the characteristic leaf and shoot symptoms (8) and, at least with one isolate, bark scaling was also obtained (9). The etiology of the psorosis disease has not been established as yet but an unusual virus has been associated with infectious fractions of partially purified extracts from CRSV infected citrus plants (5) . These infectious fractions contained a 48-kd protein, presumed to be the viral coat protein. Psorosis diseases are primarily transmitted through budwood, but natural spread of psorosis-ringspot isolates has been observed in Argentina (15, 17) and to a lesser extent in Texas (16, 18). In recent years, an illegal budwood introduction of Star Ruby grapefruit took place in Spain and some plantings were propagated from this material before release of virus-free budwood by the Citrus Quarantine Station. Some lines of this cultivar in Florida and Texas were known to carry a necrotic strain of CRSV (10,16). The possibility that trees of Star Ruby grapefruit, propagated from budwood of uncontrolled origin, could be infected with a CRSV strain of this type, prompted us to survey one of these plantings and to test for presence of this pathogen. MATERIALS AND METHODS Graft-inoculation tests. Indicator plants were grownin asteam-sterilized artificial potting mix (50% sand and 50% peat moss) and fertilized according Eleventh ZOCV Conference to a standard procedure (1). Inoculated plants were incubated in a temperature-controlled greenhouse kept at 1826 C, with the only exception of agroup of Etrog citron plants (Arizona 861-SI), used to index for viroids of the exocortis group, that was incubated in a warm greenhouse (26-32 C). A minimum of five plants of each of the following species and cultivars were inoculated to observe symptoms: Madame Vinous sweet orange, Dweet tangor, Mexican lime, Duncan grapefruit, Etrog citron and sour orange. ELISA. ELISA tests for tristeza were done using the monoclonal antibody 3DF1 in the conditions previously described (21). Mechanical transmission. Young leaves showing symptoms were selected to use as inoculum source to transmit the pathogen from citrus to herbaceous hosts. Carborundumdusted leaves were rubbed with leaf extracts prepared in cold TACM buffer (0.05 M Tris-C1, 0.1% ascorbic acid, 0.1% L-cysteine and 0.5% 2-mercaptoethanol, pH 8.0) (5) immediately before inoculation (8). Different fractions during the purification process were also assayed on C. quinoa. Aphid transmission. Aphis gossypii and A. citricola were tried as potential vectors of the disease following the procedure described by Hermoso de Mendoza et al. (11). Madame Vinous sweet orange seedlings were used as donors and as receptor plants. The donor plants were decapitated and the aphids fed in new succulent shoots showing symptoms. The aphid-inoculated shoots on receptor plants were allowed to grow for 6 months and then pruned every three months to force new growth. Analysis of double-stranded RNA (dsRNA). DsRNAs were purified from several types of tissues and analyzed following the general procedure described by Dodds et al. (6). The gels were stained with ethidium bromide and with silver nitrate (12). Partial purification and characterization. Partial purification of the CRSV isolate used in this study was done following the general procedure described by Derrick et al. (5) using young symptomatic leaves of Duncan grapefruit, Mexican lime, Madame Vinous sweet orange or sour orange. Clarified and concentrated extracts were layered on a linear 10-40% suerose gradient and centrifuged for 3.5 hr at 27,000 rpm in a Sorvall AH-627 rotor using 12 ml tubes. The gradients were fractionated from the top into 0.6 ml fractions using an ISCO gradient fractionator. A group of fractions, selected for having infectivity on C. quinoa, were pooled and precipitated by centrifugation at 300,000 g for l hr. The pellet was resuspended and electrophoresed in 0.5% agarose at 50 V for 4 hr as described by Derrick et al. (5). The agarose block comprised between 0.2 and 1.2 cm from the top edge of the agarose gel, was sliced, added with 0.4 ml of protein denaturing mixture (0.25 M Tris-C1 pH 8.0, 5.7% SDS, 14.3% (vIv) mercaptoethanol and 28.6% (vlv) glycerol) and heated for 4 min in boiling water. Proteins in this fraction were separated by polyacrylamide slab gel electrophoresis (SDS-PAGE) (3). The gels were stained with Coomassie blue and then with silver nitrate. Immunoelectron microscopy (IEM). IEM was performed by the methods described by Derrick et al. (4, 5). Grids were coated for 30 min with 0.02 mglml of protein A and then for 1 hr with an antiserum prepared to the CRSV-4 ringspot isolate from Florida (5) (kindly provided by K. S. Derrick, Univ. Florida, CREC, Lake Alfred). After incubation for 3 hr with the antigen solution, the grids were positively stained with 2% uranyl acetate for 10 min and blotted with filter paper.