A New Viroid is the Causal Agent of the Citrus Cachexia Disease

Citron tissue infected with severe isolates of cachexia (xyloporosis), known to be free of all other citrus diseases and to induce a strong reaction in Parson's special mandarin, contained a new species of viroid RNA. Symptomless tip tissue of plants infected with cachexia (Ca) 902 or 908 (UCR virus collection) was phenol extracted and processed by LiCl partitioning and Cf-11 cellulose chromatography. These nucleic acid extracts, when analyzed by sequential polyacrylamide gel electrophoresis in native and denaturing conditions, displayed an RNA of about 300 nucleotides not observed in healthy extracts. Both circular and linear forms of a viroid RNA, designated as the putative citrus cachexia viroid (CCaV), were detected. No homology to either citrus exocortis viroid (CEV) or citron variable viroid (CVaV) was observed with hybridization of CCaV to cDNA made to CEV and CVaV. When other cachexia sources (Ca 903,904,907), known to comprise mixtures of the cachexia agent and mild forms of the "exocortis disease" were similarly analyzed, both a common CCaV component and additional viroid RNAs were detected. The circular form of CCaV was isolated from denaturing gels by electroelution and transmitted to healthy citron. Secondary inoculations to Parson's Special mandarin from these citron sources have been made and the induced host reactions have confirmed the identity of CCaV as the cachexia disease agent. Nucleic acid extracts from cachexia sources were also inoculated to other hosts for viroids, such as Gynura aumntiaca, Lycopersicon esculentum cv. Rutgers, and Cucumis sativus cv. Suyo. Although all inoculated plants remained symptomless, the CCaV could be recovered only from cucumber after 30-45 days incubation. These properties suggest that the cachexia disease source material contains a previously undescribed viroid which is the causal agent of the disease. "Cachexia" is a term introduced by Childs (2) to describe a condition of Orlando tangelo characterized by gumming and browning of phloem tissues, wood pitting, and bark cankers. The disease was shown to be graft transmitted (3) and to also affect mandarins, mandarin hybrids, tangelos, kumquats and Citrus macrophylla. Although the cachexia disease can be found in most citrus growing regions of the world, many citrus varieties, including grapefruit, sweet orange and lemon are symptomless carriers of the agent. Mild, moderate and severe isolates of the disease exist. Severe isolates inducing severe leaf chlorosis, stunting, and dieback can be lethal to citrus on susceptible rootstocks. Comparison of symptoms and wood specimens (3) suggested a possible relationship between the cachexia disease and the xyloporosis disease affecting Palestine sweet lime in Palestine reported earlier by Reichert and Perlberger (9). However, a definitive comparative study of both diseases has never been reported (11). The widespread occurrence of cachexia coupled with the lengthy and laborious bioassay, originally requiring incubation periods of 2-7 yr in Orlando tangelo and improved to 6 months to 1 yr in Parson's Special mandarin (12), has stimulated a search for the causal agent in order to improve detection and indexing procedures. The viroid hypothesis was introduced by Roistacher et al. (13) because of the similarity in transmission properties between the cachexia agent and the citrus exocortis viroid (CEV). Additional data supporting this include: 1) the absence of any evidence of vector transmission; 2) the ineffectiveness of thermotherapy (1); and 3) the ease of elimination of the agent by shoot tip grafting (14). This report presents evidence for the isolation and purification of a small, transmissible, viroid-like RNA Tenth ZOCV Conference from cachexia disease sources which reproduced the disease symptoms on inoculated index plants. We propose the name Citrus Cachexia Viroid (CCaV) for the causal agent of the cachexia disease. MATERIALS AND METHODS Source, transmission and bioassay hosts for the cachexia disease. The principal cachexia disease isolate used in these studies was derived from an old line navel orange tree in 1963 from the University of California-Riverside (UCR) collection and designated as Ca 902. This isolate has been maintained in sweet orange and consistently bioassayed in Parson's Special mandarin as a severe cachexia isolate with a rating of 8 on a scale where 10 is most severe. The Valencia orange host indexed negative for all other known citrus diseases including severe and mild forms of the exocortis disease. The cachexia agent was transmitted by bud inoculation to the symptomless host, citron, which on the basis of transmission studies, appears to maintain a high titre of the agent. Nucleic acid extractions and viroid purification were made from citron. Extracted preparations suspected of containing the cachexia agent were inoculated by razor slashing the stems of citron seedlings. After 6-12 weeks incubation, the plants were monitored for positive transmission by both extraction and detection of the putative cachexia viroid and by bioassay on Parson's Special mandarin. Secondary inoculations to herbaceous hosts were made by stem slashing Gynura aurantiaca or needle puncture through an inoculum drop on the hypocotyl of young cucumber (Cucumis sativus cv. 'Suyo') plants as the first true leaves were emerging. After 3 weeks, cucumber plants were trimmed to 2-3 nodes and all foliage removed. The regrowth was collected and extracted after an additional 3 weeks. Ultimately, all nucleic acid fractions or purified viroid preparations to be tested for the cachexia agent were bioassayed on Parson's Special mandarin. This procedure involved mechanical transmission to healthy citron seedlings and subsequent bud inoculation from these sources to Parson's Special mandarin forced on rough lemon rootstock. Plants were first observed for the browning reaction at the budunion after 6-7 months and subsequently a t irregular intervals up to one year after inoculation. Nucleic acid extraction and viroid detection. Actively-growing tip tissue of symptomless carrier citron was collected and either extracted immediately or frozen and powdered in liquid nitrogen and stored at -20 C for use later. Tissue extraction and nucleic acid isolation were as previously reported (4, 19). This included homogenization in the presence of phenol, Tris-HC1 buffer, SDS, EDTA, and mercaptoethanol followed by concentration by ethanol precipitation and 2M LiCl salt partitioning. The 2M LiCl soluble nucleic acid fraction was subjected to CF-11 cellulose chromatography and selective elution to enhance the concentration of specific viroid RNA (16). Viroid detection was accomplished by sequential polyacrylamide gel electrophoresis (PAGE) as described by Semancik and Harper (18) and modified by the application of the low pH denaturing gel system (10). These procedures followed by silver staining (2) provided for detection of circular viroid molecules to a sensitivity of about 100 pg. Infectious viroid molecules were recovered from denaturing gels by electroelution in an IBI Model UEA unidirectional electroelutor after gels were stained with ethidium bromide, visualized on a UV transilluminator and the gel piece containing the viroid RNA excised. Electrotransfer hybridization. Viroid molecules were transferred directly from denaturing polyacrylamide gels containing 8M urea onto Diseases Induced by Viroids and Viroidlike Pathogens 127 nylon-based membranes (Nytran) using an LKB Transphor apparatus. Membranes were hybridized with complementary DNA (cDNA) probes made to the major citrus viroids, citrus exocortis viroid (CEV), the CV-Ib component of the "citron variable viroid" (CVaV) isolate, and the citrus cachexia viroid (CCaV). Randomprimed cDNA probes were made essentially by the procedure as reported by Maniatis et al. (8) using the cloned Moloney Murine Leukemia virus reverse transcriptase enzyme (Bethesda Research Laboratories). Conditions of hybridization were as presented by Garger et al. (6).