Interactions Among Citrus Viroids and Phytophthora citrophthora

Reports from the laboratory of V. Rossetti in the early 1980’s indicated that exocortis infected sweet orange and lemon displayed an “. induced greater resistance . ” to lesion development by Phytophthora citrophthora . Severe strains of exocortis were described by Rossetti as having “. a disastrous effect on trees . ” but little different from mild strains in the resistance response to P. citrophthora . Citrus materials containing selected variants of the more widely occurring citrus viroids CVd-Ia, CVd-IIa, and CVd-IIIb, known to induce only mild to moderate responses on Poncirus trifoliata rootstocks and citron were tested for Phytophthora lesion development on Washington navel sweet orange grafted on Carrizo citrange and viroid infected for 2 yr, rooted cuttings of Lisbon lemon from infected field trees infected with viroids for 10 yr and field trees of Valencia sweet orange on P. trifoliata infected with viroids for 20 yr. Data from greenhouse and field inoculations with P. citrophthora verified a significant reduction in lesion development in some viroid infected sweet orange but not in lemon. However, a specific and consistent relationship with a single citrus viroid could not be identified. Among the most serious soilborne diseases of citrus are those caused by Phytophthora spp. Although most rootstocks are moderately tolerant, most scion tissues are susceptible to infection with sweet orange and lemon cultivars being especially affected (13). A now classical observation by Rossetti et al. (8) in 1980 reported that Hamlin sweet orange scions on Rangpur lime rootstocks infected with exocortis displayed resistance to Phytophthora infection. Although infection by severe strains of exocortis seriously inhibited the growth and vigor of trees, some “mild” strains that “. did not induce scaly bark symptoms . ” also conferred a similar level of resistance to Phytophthora . Further studies reporting that exocortis infection in Sicilian lemons reduced P. citrophthora lesions extended the initial observations and, in addition, correlated the reduced development of Phytophthora lesions with “. stunted trees . ” (9). “Tolerance” to P. citrophthora was suggested (12, and unpublished data) as occurring more specifically in Citrus exocortis viroid (CEVd) infected citrus and was conjectured to be possibly related to the anti-fungal properties of the pathogenesis related (PR) protein P23. Investigations with transgenic citrus linked the in vivo activity of P23 against P. citrophthora and the possible development of systemic acquired resistance (SAR) (5). When the initial observation of a relationship between exocortis disease and resistance or tolerance to Phythophthora infection was made (8), a comprehensive understanding of the complexity of the viroid population in citrus was not yet appreciated. At times, any moderation in exocortis-like symptoms both in susceptible rootstocks and the indexing host, Etrog citron, were interpreted as induced by “mild” or “moderate” strains of the exocortis disease agent. With the recognition of five distinct viroids as potential components of any viroid population in citrus, this relationship has been clarified (3). Investigation of the development of P. citrophthora lesions in the presence of single citrus viroids as influenced by diverse experimental 448 Sixteenth IOCV Conference, 2005—Short Communications conditions and plant materials is the subject of this report. Earlier studies focused on citrus viroids with the potential for the improvement of citrus performance (1, 2, 6, 7, 10, 11) in the absence of viroid induced diseases of economic importance. Representative viroids known to induce only mild to moderate effects on two prime indicator hosts, Poncirus trifoliata and Etrog citron, include the well characterized citrus viroids Citrus viroid Ia (CVd-Ia), Citrus viroid IIa (CVd-IIa) and Citrus viroid IIIb (CVd-IIIb), which occur with wide frequency in citrus growing regions (4). Consistent with the observations of Rossetti et al. (8), the reduction of vegetative growth on scion cultivars grafted on P. trifoliata rootstock caused by these specific variants has also been documented (10, 11). Host species highly susceptible to the citrus viroids and P. citrophthora were selected . These differed in age and were maintained under either greenhouse or field conditions (Table 1). Testing was accomplished over a 1-yr period. Experiments 1 and 2 were conducted during January through April under standard greenhouse conditions at UC-Riverside while Experiment 3 entailed a field study from March to June at the Lindcove Research and Experimental Center in central California. All scion cultivars were derived from disease-tested sources from the Citrus Clonal Protection Program (CCPP) at UC-Riverside. However, preparation of test plants required varied treatment and time for propagation. Materials for Experiments 1 and 3 were prepared as budded seedlings and graft inoculated. Rooted cuttings were made for Experiment 2 from field grown Lisbon lemon that had been infected with the respective individual viroids for 10 yr, requiring two additional years of greenhouse culture to reach a size suitable for Phytophthora inoculation. Even though both sweet orange and lemon have been characterized as excellent reservoirs for systemic viroid infection, all sources were verified for the presence of the respective viroids. For greenhouse tests, 914 replicates were employed while the field experiment included 8-12 replicate inoculations. P. citrophthora (R. E. Sm. & E. H. Sm.) Leonian (Isolate M189 from a citrus nursery in San Diego, California) was used for the inoculations after growing on clear V8 juice agar for 4 days. Two to six hours prior to inoculation, 4 mm and 5 mm discs of agar containing hyphae (for greenhouse and field inoculations respectively) were transferred to distilled water to stimulate sporangial formation. Bark flaps of a size equal to the agar discs were cut and folded back for both viroid free (controls) and viroid treated plants. An agar disc was inserted between the bark and the wood and the bark was replaced. Moistened cheesecloth covering the inoculation site was sealed with Parafilm and held in place with duct tape. The cheesecloth was kept moist for approximately 48 h to ensure the production and release of the infectious zoospores from sporangia. Approximately 1.5-3 mo post inoculation, the bark was stripped to