Viroids in Australian citrus

Viroids are the smallest infectious agents known, being unencapsidated RNAs of 240-380 bases. Citrus exocortis viroid (CEV) causes poor tree performance, especially when infected scions are rafted to trifoliate orange or citrange rootstocks. To eliminate infected budwood trees, various methods were used to detect CEV, including field inspections, bud inoculation of Etrog citron indicators (in which CEV causes severe epinasty) and hybridisation with CEV cDNA. A large number of trees with exocortislike symptoms such as dwarfing and/or bud union abnormalities produced only mild epinasty when grafted on Etrog citron and did not hybridise to the CEV probe. Upon purification and electrophoretic analysis, the presence of viroids other than CEV was demonstrated.
Screening of over 1800 trees resulted in the detection of four groups of viroids as determined by RNA homology and length in nucleotides. CEV was the only member of the first group and was the largest viroid detected, with 371 bases. CEV almost invariably occurred with other viroids of lower molecular weight. The second group, CV I, contained two viroids, of 325 and 332 bases, which were not homologous to CEV or hop stunt viroid (HSVd). Group CV I1 contained three viroids, of 297, 299 and 302 bases, all of which were homologous to HSVd. The CV I11 group contained two viroids, of 290 and 295 bases, which were not homologous to CEV or HSVd. The CV I1 and CV I11 groups were strongly associated with various field symptoms, including the disease cachexia (CV IIb), and dwarfing of trees on trifoliate orange rootstock. To confirm any causal relationship between individual viroids and field symptoms, viroids representative of each group were purified and used to inoculate Etrog citron. Tissue from these citrons was used to inoculate young field trees with viroids, singly and in combination. Parson's Special mandarin inoculated with CV IIb showed symptoms of cachexia after 2 years, confirming CV IIb as a causal agent of this disease. It will probably be 5 years before the effect of other inoculations can be assessed.