ISHS Acta Horticulturae 386: XVI International Symposium on Fruit Tree Virus diseases
NEW TRENDS IN THE DETECTION OF FRUIT TREE VIROIDS |
| Authors: | E.V. Podleckis, A. Hadidi |
| Abstract:
Viroids are small, covalently closed, circular, single-stranded RNA molecules that contain 246–371 nucleotides and possess no protein coat or mRNA activity. Apple scar skin viroid (ASSVd) (Hashimoto and Koganezawa, 1987; Hadidi et al., 1990) was the first viroid reported to infect pome fruits (Malus, Pyrus, Cydonia) and cause diseases of economic importance. Dapple apple viroid (DAVD) (Hadidi et al., 1990), hop stunt viroid (HSVd) (Shikata, 1990), pear blister canker viroid (PBCVd) (Flores et al., 1991), pear rusty skin viroid (PRSVd) (Chen et al., 1987; Hadidi and Yang, 1990; Zhu et al., 1995) and fruit crinkle viroid (Ito et al., 1993) have all also been reported to infect pome fruit trees. Peach latent mosaic viorid (PLMVd) reported to infect peach (Flores et al., 1990; Albanese et al., 1992; Minafra et al., 1993; Shamloul et al., 1995). Strains of HSVd have been detected in grapevine (Vitis vinifera), citron (Citrus medica), and cause dapple fruit disease in peach (Prunus persica) and Japanese plum (P. salicina) (Sano et al., 1989; Shikata, 1990). Citrus spp. are infected naturally by at least five distinct groups of viroids including citrus exocortis viroid (CEVd) and the hop stunt-like citrus cachexia viroid (CCaVd) (Semancik et al., 1988; Yang et al., 1992). Traditionally, viroids have been detected by inoculation of sensitive indicator plants. ASSVd and DAVd, for example, have been detected by graft-inoculation of sensitive apple indicators such as the cultivars ‘Red Delicious’ and ‘Lord Lambourne’. But, because in most apple and pear cultivars, ASSVd and DAVD infection produces only fruit blemishes and no foliar symptoms, inoculated indicator trees must produce fruit to express symptoms. These assays can require 3–5 years to complete. Howell and Mink (1992) subsequently described a bioassay for ASSVd infested apples whereby the graft-inoculated apple cultivars ‘Stark's Earliest’ and ‘Sugar Crab’ were induced to express leaf curling and epinasty symptoms in as little as 8 wk by growing the inoculated indicators under constant light. Still, bioassays are time and space consuming and are sensitive to variabilities in environmental conditions, host plant physiology and differences in virulence of the viroid strains. Because viroids lack a protein coat, the sensitive and rapid serological tests, like the enzyme-linked immunosorbent assay (ELISA), used to detect many plant viruses are not applicable. Return gel electrophoresis is used for viroid detection (Singh, 1991), but the technique involves extensive sample preparation and requires considerable technical expertise. Dot and Northern blot nucleic acid hybridization assays are the most convenient and sensitive methods commonly used to detect viroids. Originally, the probes used in the hybridization assaya were radioactively-labeled complementary DNA (cDNA). The development of plasmid transcription vectors that contain promoters for SP6, T3 and T7 bacterial RNA polymerases has resulted in the increased use of so-called "riboprobes" in viroid detection. The riboprobe system has the advantage of easily producing microgram amounts of uniformly labeled, single stranded complementary RNA (cRNA) probes that are more sensitive than similar cDNA probes (Candresse et al., 1990; Hadidi, 1988; Hadidi et al., 1990; Lakshman et al., 1986). 32P-labeled cRNA probes have been developed for the detection of ASSVd, DAVd and PRSVd from pome fruits (Hadidi et al., 1990: Zhu et al., 1995), PLMVD frompeach (Shamlouol et al., 1995; Ambrose et al., 1995), PBCVd from pear | |
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