THERAPY AND CITRUS IMPROVEMENT Improving Therapy Methods for Citrus Germplasm Exchange

Movement of citrus germplasm between citrus-growing countries carries the risk of inadvertent spread of serious virus and viruslike pathogens. Current procedures have not been well tested for all pathogens, or are not completely efficient. The exclusion of the two pathovars of Xanthomonas campestris causing citrus canker and citrus bacterial spot, the greening pathogen, severe isolates of citrus tristeza virus (CTV) and citrus tatterleaf virus (TLV) was studied using a tissue culture quarantine methodology. Infected budsticks were cultured in vitro at 32 C. Shoot tips excised from sprouting buds were grafted in vitro to axenically grown seedlings of Troyer citrange. The citrus canker and the greening pathogens were consistently eliminated, even by grafting large shoot tips of 0.5-0.7 mm. Severe strains of CTV were consistently eliminated only by the use of small shoot tips of 0.15-0.2 mm. TLV was difficult to eliminate, but 42% of the plants propagated from shoot tips with three-leaf primordia were TLV-free. Shoot-tip grafting eliminated greening and CTV from chronically infected plants from Africa and Asia. Severe CTV isolates from Africa, Asia, South America, and North America were also eliminated easily. One isolate of naturally-spreading psorosis from Argentina was also eliminated. Movement of citrus germplasm between different citrus areas is often desirable for commercial and scientific purposes, including establishment of germplasm repositories. Uncontrolled movement of whole or propagative plant tissue carries the risk of introducing new pests and pathogens. This risk can be overcome by the introduction of new materials through quarantine stations (9). However, the classical methods of quarantine are slow and require greenhouse facilities remote from citrus production areas. Such facilities are often not available in many countries or are situated where expertise and/or financial support for citriculture are not available. An alternative citrus tissue culture system was developed for safe introduction of citrus germplasm (13). It consists of culturing imported budsticks in vitro at 32 C to induce the sprouting of lateral buds and formation of flushes from which shoot tips are excised and micrografted in vitro (shoot-tip grafting) (11). The method is being used successfully in Spain to import citrus germplasm from different growing areas (14), but it has not been experimentally tested for elimination of pathogens causing severe diseases not present in Spain. In addition, shoot-tip grafting (STG) requires a high manual dexterity to isolate the very small shoot tips needed. Grafting success increases as shoot tip size increases, but success in eliminatin pathogens declines rapidly. Discovery of two bacterial diseases of citrus in Florida (citrus canker and citrus bacterial spot diseases) has also increased concern about the hazard of introducing these pathogens via imported budwood. The efficacy of STG to retrieve disease-free plants from budwood contaminated or infected with these diseases has not been tested. Results are presented in this paper on the application of STG for elimination of the citrus canker and bacterial Mention of a trademark, warranty, proprietary product, or vendor does not constitute a guarantee by the U. S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable. Therapy and Citrus Improvement spot pathogens, greening agent, tatterleaf virus, severe citus tristeza virus isolates and naturally-spreading psorosis, and on the influence of shoot tip size, and addition of viricides and antibiotics in the budstick culture medium on elimination of bacterial and viral pathogens. MATERIALS AND METHODS Sources of infected materials Citrus canker and citrus bacterial spot. Two strains of Xanthomonas campestris were used (6). Strain XC62 of X. campestris pv. citri causes the "A" type of citrus canker. Strain F1 of X. campestris pv. citrumelo causes citrus bacterial spot disease in Florida nurseries. Tissue sources for experimentation were: a) glasshousegrown plants of Duncan grapefruit experimentally inoculated with bacterial suspensions and expressing lesions on leaves and twigs typical of the respective pathovars, and b) budsticks from glasshouse-grown healthy plants of Duncan grapefruit that were contaminated by immersion for 30 or 120 sec in bacterial suspensions of XC62 or F1 (lo8 cells/ml), dried at room temperature for 30 min, placed in plastic bags and incubated at 30 C in continuous darkness for 20 hr. Greening. Sources of greening-infected tissue were glasshouse-grown Valencia sweet orange plants infected with the greening pathogen prior to the experiment by graft-inoculation with a source of likubin from Taiwan (coded as B121). This likubin isolate was free from CTV based on ELISA and by indexing to citrus indicators. At the time of budwood selection, the source plants had moderate greening symptoms and infection was verified by graft inoculation to Cleopatra mandarin seedlings. Limited experiments were done with glasshouse-grown plants of several hosts chronically infected with greening isolates from Reunion (Bl), South Africa (B49), Taiwan (B121), Philippines (B140 and B1421, and China (B144). These plants had very severe symptoms of greening. All sources, except B121, were coinfected with CTV. Citrus tristeza virus (CTV). Sources of CTV-infected budwood were glasshouse-grown plants of Valencia and Washington navel sweet orange plants graft-inoculated several months prior to the experiments with a seedling yellows (SY) source of CTV (B28). Limited experiments were done with glasshouse-grown plants of several hosts chronically infected with CTV isolates from Brazil (B77), California (B6), China (B81, B82, B144), Colombia (B128, B131), Japan (B31), Peru (B133), Philippines (B140, B142), Reunion (Bl), and South Africa (B49). These all cause stem pitting in grapefruit andlor sweet orange. The source plants for B1, B140, B142, B144 and B149 were coinfected with greening. All chronically infected plants were showing CTV andlor greening symptoms. Tatterleaf virus (TLV). The sources of TLV-infected tissue were glasshouse-grown plants of Valencia and Washington navel sweet oranges graft-inoculated with an isolate of TLV which had been passaged through Nicotiana clevelandii by mechanical inoculation (4). The TLV-infected plants were coinfected by graft-inoculation with the CTV isolate B28 (see above). Naturally-spreading psorosis. The source was Duncan grapefruit plants graft-inoculated with isolate B84 (free from CTV) from Concordia, Argentina. Tissue culture techniques Budstick culture in vitro. Pencilsized budsticks of triangular to circular cross section which contained four to eight buds were collected from glasshouse-grown source plants. They were washed with detergent and tap water and then disinfested by immersion for 10 min in a 1.5% solution of sodium hypochlorite which contained 0.1% Tween 20 wetting agent. The sticks were rinsed three times with sterile, Eleventh ZOCV Conference deionized water, and cultured individually in 38 x 200-mm test tubes containing 50 ml of the plant cell culture salt solution of Murashige and Skoog (10) solidified with 1.2% Bacto agar. To study the influence of antibiotics on greening in newly formed side shoots, 0.5 g11 penicillin and 1.25 gA tetracycline were added to the culture medium. In one test, CTVand TLV-infected budwood was cultured in medium amended with 50 mgA Virazole [I-A-Dribofuranosyl-1H-l,2,4-triazole-3-carboxamide (ribavirin)] (5) to study the influence of this viricide on CTV and TLV elimination. Cultures were placed in an incubator at a constant 32 C and exposed to 45 p,E ~ ' m ~ light for 16 hr a day. Shoot-tip grafting (STG) in vitro. The standard technique of STG (10,14) was used in all experiments. Grafting was done on Troyer citrange seedlings 4 to 6 cm tall and 1.6 to 1.8 mm in diameter at the point of decapitation. This size was reached 9-10 days after seed germination at 30 C in continuous darkness. Young flushes from sprouting buds on the budsticks cultured i n vitro were surface sterilized by immersion for 5 min in a 0.25% sodium hypochlorite solution which contained 0.1% Tween 20 wetting agent. They were rinsed three times with sterile, deionized water and used as the source of shoot tips. Shoot tips composed of the apical meristem plus three-leaf primordia (0.15-0.2 mm) were used inmost experiments. Shoot tips composed of the apical meristem plus six-leaf primordia, (0.5-0.7 mm) were also used in the experiments on the influence of shoot tip size on pathogen elimination. Shoot tips with three-leaf primordia were grafted inside an inverted-T incision made at the point of decapitation of the rootstock, whereas the shoot tips with six-leaf primordia were grafted inside a "window" made by excising a 1-mm2 piece of bark of the rootstock 3-4 mm below the decapitation point. Shoottip-grafted cultures were placed in a culture room at 25 C and exposed 16 hr daily to 40 p,E ~ l m ~ light. Transplanting to soil. Successfully grafted plants were transferred from the test tubes to pots containing a commercial potting mix (Metro Mix 500, W. R. Grace Co.). The pots were covered with a transparent plastic cup to maintain humidity and incubated under a bank of artificial lights at approximately 25 C. Ventilation in the transparent cover was gradually increased over a several-week acclimatization period, and the cover was removed when the plants did not show stress when exposed to normal room humidity conditions. The plants were transferred to a glasshouse and evaluated for growth.