Immunocapture reverse transcription-polymerase chain reaction combined with nested PCR greatly increases the detection of Prunus necrotic ring spot virus in the peach.

A detection system based on nested PCR after IC-RT-PCR (IC-RT-PCR-Nested PCR) was developed to improve indexing of Prunus necrotic ringspot virus in peach trees. Inhibitory effects and inconsistencies of the standard IC-RT-PCR were overcome by this approach. IC-RT-PCR-Nested PCR improved detection by three orders of magnitude compared with DAS-ELISA for the detection of PNRSV in leaves. Several different tissues were evaluated and equally consistent results were observed. The main advantages of the method are its consistency, high sensitivity and easy application in quarantine programs.

Fluctuations of Prunus Necrotic Ringspot Virus (PNRSV) at Various Phenological Stages in Peach Cultivars.

Fluctuations in Prunus necrotic ringspot virus (PNRSV) concentration were researched in single plants of six peach (Prunus persicae) cultivars-Kurakata, Red Haven, Nectar Red, Start Delicious, Meadowlark, and Loadel-by double antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA) of dormant buds (May, June), flowers (September), new sprouts (November), and mature leaves (January) (Southern Hemisphere). The optimum extract dilution (sample weight per buffer volume) to detect the virus was also quantified. The average absorbance patterns of the six cultivars show a steady increase in virus concentration, ranging from A 405nm 0.61 in May to A 405nm 0.86 in July for dormant buds, to A 405nm 1.22 in September in flowers, to 1.53 in November in new sprouts, where the highest concentration was found. Virus concentrations in mature leaves drop to values similar to those of noninfected plants in January ( A405nm 0.12). The yearly average (six noninfected peach trees) ranged from A405nm 0.04 to A405nm 0.08. This drop coincides with an increase in summer temperature and attenuates foliation symptoms caused by PNRSV. Analysis of dormants buds, flowers, or new sprouts with 5-cm-long leaves was reliable to differentiate infected from noninfected plants. Cluster analysis of absorbance profiles for single plants of cvs. Loadel and Meadowlark, however, showed a comparatively low profile, with a drop at flowering time (A405nm 0.20 in September) close to the average of healthy controls. The difference between infected and healthy plants did not become apparent in all cultivars from the analysis of plants at a given phenological stage, for example by the analysis of flower only, the material most preferred to diagnose the virus. Therefore, plants should be analyzed during flowering and sprouting or flowering and dormancy (dormant buds).