Diversity Among Sub-Isolatesof Cross-Protecting Citrus Tristeza VirusIsolates in South Africa

Citrus tristeza virus (CTV) disease is endemic in Southern Africa and its principle natural vector is the aphid, Toxoptera citricida. Aphid transmission occurs in a semi-persistent manner. The effect of the disease is minimized by deliberate infection of virus-free citrus cultivars with approved mild CTV isolates before release to the industry. Presently, three CTV isolates have been approved as pre-immunizing agents on several hundred cultivars and selections. These isolates were derived from field trees, and it is suspected that each of them is constituted of different strains. Each strain may have different effects depending on host and environment. This creates problems in South Africa due to the large variety of cultivars as well as the variation in climatic conditions of the citrus producing areas of the country. Single aphid transmissions of CTV were performed from two cross-protecting isolates, GFMS 12, without the seedling yellows (SY) component, and LMS 6, with the SY component, in attempts to identify different strains in each isolate. The percentage positive transmissions were 8% for GFMS 12 and 16% for LMS 6. Evaluation of the sub-isolates on two CTV sensitive hosts (Mexican lime and Marsh grapefruit) revealed that some of them were significantly milder or more severe than the original isolate regarding growth and stem pitting development. Significant differences occurred between sub-isolates as well. The SY component was present in 38% of the LMS 6 sub-isolates. Sub-isolates were also differentiated by their restriction fragment length polymorphism (RFLP) patterns and single strand conformation polymorphism (SSCP) of the coat protein gene. It is possible that some sub-isolates contain more than one CTV strain. The results confirm the presence of different CTV strains in the pre-immunizing isolates. Citrus tristeza virus (CTV) causes stem pitting and decline diseases of citrus and is one of the most important pathogens world-wide (2). The disease is endemic in Southern Africa due to the occurrence of the most efficient insect vector, the brown citrus aphid, Toxoptera citricida . In South Africa many strains of CTV exist, and they usually occur as mixtures in a host (11). The use of decline tolerant rootstock/scion combinations has limited economical losses. However, citrus cultivars such as grapefruit and lime do not produce satisfactorily in the presence of CTV even if propagated on resistant rootstocks. To reduce the effects of the virus on these sensitive cultivars, cross protection by mild CTV isolates is used (14, 16). The development of severe stem pitting on grapefruit trees propagated from pre-immunized budwood indicated a breakdown of protection or a segregation of strains that may be present in the pre-immunizing isolates (8). When severe strains occur within an isolate, serious repercussions may be encountered in different climatic conditions, since the severe strains within the pre-immunizing isolate may become dominant (5). In this study, we attempted to discriminate between strains that may occur within two pre-immunizing isolates in South Africa by subculturing using single aphid transmissions. The sub-isolates were evaluated for symptom expression on sensitive biological hosts and by molecular biology techniques. MATERIALS AND METHODS Virus isolates. Two CTV isolates, GFMS 12 and LMS 6, which are approved as pre-immunizing isolates in the Southern Africa Citrus Improvement Program were sub-cultured by single aphid transmissions. GFMS 12 was derived from grapefruit and contains only 104 Fourteenth IOCV Conference, 2000—Citrus Tristeza Virus the stem pitting component of CTV while LMS 6 which was derived from lime, contains stem pitting as well as a mild form of SY. The isolates were maintained on Mexican lime plants in an insect-free screenhouse at 24 ° to 28 ° C. The plants were pruned lightly to force new growth which is favored by the aphids prior to conducting vector tests. The aphid-transmitted isolates are referred to as sub-isolates. Aphid transmissions. Apterous brown citrus aphids were collected in a Eureka lemon orchard and transferred to virus-free actively growing Mexican lime plants in an insect cage in the laboratory at 20 ° to 24 ° C. The aphids were transferred three times, with 24 h intervals, to new virus-free plants in order to obtain non-viruliferous insects. In the transmission studies, groups of 50 to 80 aphids were transferred to each of the two Mexican lime plants containing the CTV isolates for a virus acquisition period of 24 h. Aphids were then placed singly on virus-free Mexican lime plants. A total of 111 aphids were transferred from GFMS 12 and 50 from LMS 6. After an inoculation access period of 24 h, the aphids were killed by spraying with a suitable insecticide. Plants were then kept in a screenhouse at 20 ° to 24oC. Transmission efficiency was determined as the ratio between the number of infected plants and the total number of inoculated plants. Enzyme-linked immunosorbent assay (ELISA). CTV infection from aphid transmissions was determined after 3 mo by double antibody sandwich ELISA using polyclonal antiserum (1). Reactions were read on a Titertek ELISA plate reader at 405 nm. A positive reaction was defined as an optical density reading of more than three times that of the negative control. Biological indexing. Mexican lime which is sensitive to stem pitting and Marsh grapefruit which is sensitive to SY were used to assess the severity of the sub-isolates. Three seedlings of each of the two indicators were inoculated by budding and then maintained in a screenhouse at 24 ° to 28oC. Plants were trained to develop a single shoot. After 6 mo the grapefruit plants were inspected for seedling yellows symptoms, the growth of both indicators was measured and the bark peeled to assess stem pitting. Stem pits were counted under a stereo microscope at 6x magnification and calculated as number of pits per cm 2 . Molecular characterization. Four grams of bark and leaf midrib tissue of Mexican lime plants was frozen in liquid nitrogen and pulverized to powder with a mortar and pestle. Double-stranded (ds) RNA was isolated using the CF-11 cellulose chromatography procedure (10). The coat protein (CP) gene was amplified in a one-tube reverse transcription polymerase chain reaction (RT-PCR) in a thermal cycler (6). The resulting amplified products were analyzed on 4% agarose gels after digestion with the restriction enzyme Hin f 1 to obtain restriction fragment length polymorphism (RFLP) patterns (12). Single-strand conformation polymorphism (SSCP) analysis was performed directly on the denatured PCR products of the CP gene of the different CTV subisolates (13).