Citrus Virologists Conference Proceedings ( 1957-2010 ) Title The Grouping of Citrus Viroids : Additional Physical and Biological Determinants and Relationships with Diseases of Citrus Permalink

A consensus catalogue for the grouping of citrus viroids (CV) utilizing the physical parameters of size and nucleotide sequence homology as tested with group-specific cDNA probes has been furthered developed. Analysis of cachexia disease sources by sequential polyacrylamide gel electrophoresis (sPAGE) has revealed a third member of the CV-I1 Group, designated as CV-IIc. A viroid characterized by a similar size and sequence homology is common to all "xyloporosis" disease sources analyzed. Chrysanthemum, which has been identified as a selective host for CEV and the Group I1 viroids, produces a leaf spotting reaction when inoculated with CV-IIa, the viroid inducing the most mild reaction in citron. Mixed infections of viroids from different groups has indicated a range of responses on citron including: 1) both enhanced and delayed severe dwarfing symptoms of CEV, 2) synergism resulting in severe dwarfing and epinasty mimicking CEV symptoms, and 3) a variation in leaf symptoms ranging from no effect to severe epinasty. Field testing of pure citrus viroids and mixed viroid inoculations of commercial scion on exocortis sensitive rootstock combinations has been initiated. Analysis of the viroid profiles of citrons inoculated with field sources of some diseases of suspected viroid etiology such as, "xyloporosis" of Palestine sweet lime, "gummy bark" of navel orange, and "Kassala" of grapefruit, indicated the presence of a complex pattern of several viroids. Index words. Citrus viroids, Etrog citron, cDNA viroid probes, molecular hybridization, electrophoresis, sequential PAGE, exocortis, cachexia. With the revelation of the large collection of citrus viroids (6), an attempt has been made to rationalize these viroids into specific groupings on the basis of physical and biological properties. The existence of viroidlike pathogenic agents in citrus was originally detected by a symptom reaction on Etrog citron, the bioassay host for exocortis (4, 19). These were, therefore, assumed to be isolates of the citrus exocortis viroid (CEV). The citrus viroids have now been demonstrated to be distinctly different from the 371 nucleotide CEV and to comprise a diverse collection of independently-transmissible RNA molecules in a size range of 275-340 nucleotides. In addition to the type isolate of classical CEV, four discrete groups of the citrus viroids were proposed on the basis of molecular size, conformation, and sequence homology as well as host range and symptom reactions on Etrog citron (7, 8). In the studies reported here, a complete series of cDNA probes representative of each citrus viroid group has been tested against a standard range of citrus viroids. The homology reactions verify the specificity of the distinct groups. Chrysanthemum is introduced as an additional selective host which may be useful in detection of the CV-I1 group noted for the most mild symptom reaction and lowest titer in citron. With the collection of over 12 citrus viroids and the identification of exocortis and cachexia as the only two citrus diseases induced by viroids, the significance of the citrus viroids as agents of specific diseases or possibly even more subtle physiological conditions affecting citrus production remains unclear. Viroids and Viroid Induced Diseases DEFINITION OF CITRUS VIROID GROUPS BY PHYSICAL PROPERTIES Tissue selection and sequential PAGE. Essential to the description of the citrus viroids was the utilization of Etrog citron as the common host for detection and analysis of viroids from various citrus sources. The standard procedure adopted in these studies involved the primary inoculation into citron of a bud or tissue piece of source materials derived from commercial plantings or disease collections. After a period of 3 to 6 months, the new flush of tissue was collected, the nucleic acids extracted (8) and analyzed by sequential polyacrylamide gel electrophoresis (sPAGE) (17). If it was desirable to either transport or store samples, freshly collected tissue was frozen in liquid nitrogen, pulverized to small fragments to assure random sampling, and lyophilized. With this treatment, excellent recovery of biologically-active viroid preparations could be assured from tissues maintained under dry conditions without any special temperature requirements. A representative of each citrus viroid group is presented in Fig. 1 as a discrete silver stained band in a denaturing gel, the final stage in the detection of viroids by sPAGE. With the exception of CEV and CV-IV which comprise single viroid groupings, the CV-I, -11, and -111 Groups each contain 2-4 viroids. Furthermore, it is not unexpected that additions to these families will be made as viroid surveys employing the standard sPAGE procedure are extended to the different citrus growing regions. An additional Group-I1 viroid, designated as CV-IIc, was found when analyzing the cachexia disease source collection maintained at UCRiverside. CV-IIc is a pure single viroid isolate contained in the cachexia source Ca 905 which originated from Prior Lisbon lemon. Although CV-IIc induces a more moderate (6.5110) reaction in Parson Special mandarin Fig. 1. Polyacrylamide (5%) gel containing 8M urea and silver stained after processing by the sequential PAGE procedure of CEV (A), CV-I (B), CV-I1 (C), and CV-I11 + CV-IV mixture (D). than the severe reaction (8110) produced by the type citrus cachexia viroid (CCaV) or CV-IIb (26, 27), both CV-IIb and CV-IIc must be considered as cachexia disease agents even though they represent physically separable molecular entities. Group specific sequence homology by molecular hybridization. Using viroid templates, cDNA probes were synthesized by the random priming procedure in the presence of 32P. TO test the group specificity of the probes, a standard range of citrus viroids were electrotransferred from denaturing gels to nytran membranes, hybridized to the radioactive cDNA probes, and autoradiographed. In this way, the relative intensity of the homology reaction of the specific citrus viroids, as defined by the characteristic electrophoretic mobility, could be monitored. The marked specificity of the citrus viroid groups was essentially sustained throughout the test (Table 1). The sole intergroup reaction observed was a very weak signal CV-IV produced when probed with the CEVcDNA. This suggests that a closer relationship may exist between the genome of CEV, the largest (371 nucleotides), and CV-IV, the smallest (275 nucleotides) of the citrus viroids than among the almost continuous array of viroids contained in Groups I, 11, and 111. From this, it can be speculated that a more significant 180 Eleventh IOCV Conference