RESUMEN
Sharka disease, caused by Plum pox virus (PPV), is probably the most important disease of stone fruits crops worldwide because of tremendous yield losses from infected trees (1). During November 2004, symptoms resembling sharka disease were observed in a plum and apricot orchard consisting of 5,000 trees in Pocito, San Juan Province, Argentina. Apricot leaves showed chlorotic spots while plum leaves showed chlorotic rings, spots, and irregular edges. Plum fruits were deformed and much smaller than those from symptomless trees. Samples collected from 70 symptomatic trees were tested using double-antibody sandwich enzyme-linked immunosorbent assays with a polyclonal antiserum anti-PPV from BIOREBA (Reinach BL1, Switzerland), and immunosorbent electron microscopy with a polyclonal antiserum from our laboratory made against a recombinant PPV capsid protein (CP). The samples were also tested using double-antibody sandwich indirect enzyme-linked immunosorbent assay using the REAL kit (Durviz, Valencia, Spain) with two different monoclonal antibodies including Mab 5b that recognizes all strains of PPV and Mab 4DG5 that is specific for PPV strain D. Samples were positive with both antibodies in 80% of the cases. Leaf extracts from symptomatic plum samples were also analyzed by immuno-capture reverse-transcription polymerase chain reaction. A 1,209-bp fragment was amplified with specific primers that anneal at the 5' end of the coat protein coding region and the viral 3' end poly A tail. The amplified fragment was cloned and the nucleotide sequence was determined for two of the resulting clones (Gen-Bank Accession Nos. DQ299537 and DQ299538). The sequences were 98% identical with the PPV-strain D from the United States (GenBank Accession No. AF360579) and Germany (GenBank Accession No. X81081). The restriction sites for AluI and RsaI, previously described (2) as typical for the PPV-D strain, were present in the expected positions. To our knowledge, this is the first report of PPV-D in Argentina. Reference: (1) M. Németh. Virus, Mycoplasma, and Rickettsia Disease of Fruit Trees. Martinus Nijhoff Publishers, Dordrecht, the Netherlands, 1986. (2) T. Wetzel et al. J. Virol. Methods 33:355, 1991.
RESUMEN
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).
