Studies on the diagnosis of hop stunt viroid in fruit trees: Identification of new hosts and application of a nucleic acid extraction procedure based on non-organic solvents

A non-radioactive digoxigenin-labelled RNA probe specific for hop stunt viroid (HSVd) diagnosis has been developed. The high sensitivity and specificity of this RNA probe in dot blot hybridizations to nucleic acids from field samples, allowed the confirmation of the presence of HSVd in apricot (Prunus armeniaca L.) and its detection in two fruit tree species not previously described as hosts of this pathogen, almond (Prunus dulcis Miller) and pomegranate (Punica granatum L.). This result supports and extends the notion of the world wide distribution of HSVd, infecting cultivated fruit trees. HSVd was also found to accumulate to much higher levels in mature apricot fruits than in leaves. Additionally, a sample processing procedure which does not involve the use of organic solvents was demonstrated to render faithful results when used for viroid detection. The combined reliability and facility of use of both this extraction procedure and the non-radioactive probe will benefit agronomic investigations addressing the detection and eradication of HSVd. Other applications of the work described here, as the study of possible causal relations between specific disorders and HSVd infection, are also discussed.     

Comparison of monoclonal antibodies and polymerase chain reaction assays for the typing of isolates belonging to the d and m serotypes of plum pox potyvirus

ABSTRACT Plum pox potyvirus (PPV) isolates may be divided into four groups separated by serological, molecular, and epidemiological differences. Monoclonal antibodies specific for the two major groups of isolates, represented by the D and M serotypes of the virus, have been obtained. Polymerase chain reaction (PCR)-based assays allowing the direct detection and differentiation of PPV isolates have also been developed. We now report on a large-scale comparison of these two typing approaches. The results obtained show an overall excellent correlation between the results obtained in indirect double-antibody sandwich enzyme-linked immunosorbent assay using PPV-D- and PPV-M-specific monoclonal antibodies and those derived from either specific PCR assays or restriction fragment length polymorphism analysis of PCR fragments. Without exception, all isolates reacting positively with the PPV-M-specific monoclonal antibody were found to belong to the M serotype using the PCR-based assays, while 51 out of 53 isolates recognized by the D-specific monoclonal antibodies belonged to the D serotype according to the PCR typing results. However, failure to react with a specific monoclonal antibody did not prove as effective a predictor of the serotype of the isolate analyzed. In a few cases, the results obtained with the various techniques diverged, indicating low level variability of the epitopes recognized by the serotype-specific monoclonal antibodies. Isolates belonging to the two minor groups of PPV (El Amar and Cherry) also gave divergent results, indicating that the current typing assays are not suited for the analysis of such isolates.     

Peach red marbling and peach sooty ringspot, two new virus-like degenerative diseases of Prunus

More than 600 Prunus samples were examined by using a nonradioactive digoxigenin-labelled RNA probe specific for hop stunt viroid (HSVd). Prunus salicina and Prunus armeniaca appeared to be better hosts than Prunus persica . The weak viroid concentration in flowers and young leaves of peach trees growing in the field did not permit its detection in such samples. The diagnosis was more reliable (about 85%) with bark and leaves aged 4 months and more, from regrowths of GF 305 peach seedlings inoculated and kept in the greenhouse. Detection of HSVd in leaves and bark of apricot and Japanese plum plants aged 3 months or more also proved reliable (about 80% and 90%, respectively). HSVd could be transmitted in apricot, peach and plum nucleic acid preparations to GF 305 peach seedlings by repeated stem slashing, and to cherries ( Prunus avium and Prunus serrulata ) by approach grafting with an infected P. salicina source. The viroid was eliminated from 18% of the clones obtained after thermotherapy.
In the course of this study, 25 selected Prunus accessions suspected to be infected by unusual diseases were analysed by hybridization with a HSVd-specific probe and by indexing on GF 305 peach seedlings in the greenhouse. Fifteen of these accessions were found to be infected by HSVd, 19 induced reddish marbling, and four induced small blackish spots on the leaves aged about 4 months. Repeated assays showed that these foliar symptoms were not caused by the viroid. Peach red marbling (PRMa) has not been associated with any known virus and seems to be caused by an infectious agent not yet described. That could also be the case with the agent of peach sooty ringspot (PSRS). PRMa and PSRS symptoms were reproduced by grafting and indexing, and their causal agents eliminated by thermotherapy in a significant fraction of the treated plants. They behave like viral agents and can infect the different Prunus species studied.