Serological Detection of Cherry Leafroll Virus in English Walnut Trees

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Abstract
Direct (D) and indirect (I) enzyme-linked immunosorbent assays (ELISA) were compared for relative reliability in detecting the walnut strain of cherry leaf-roll virus (CLRV-W), the causal agent of blackline disease in English walnuts. The lowest concentrations of purified CLRV-W8 detected by I-ELISA and D-ELISA with peroxidase-conjugated .gamma.-globulin were 4 ng/ml and 48 ng/ml, respectively, of the virus in phosphate buffer, pH 6.5. Likewise, based on the highest dilution of CLRV-W-infected walnut inner bark tissue and pollen in PBS-Tween buffer at which the virus was detected, I-ELISA was 8 and 16 times more sensitive than D-ELISA, respectively. I-ELISA with peroxidase and alkaline phosphatase was more sensitive than I-ELISA with glucose oxidase for detection of the known concentrations of purified CLRV-W8 or of the virus in infected walnut tissues. I-ELISA with .gamma.-globulin-peroxidsae conjugate is efficient and reliable for indexing English walnut seedlings in nurseries for CLRV-W. It also was reliable in detecting CLRV-W in naturally infected orchard trees when a relatively large amount of pollen from each tree (average 80 g/tree) was collected from which 0.1 g was used for I-ELISA tests. The detection of CLRV-W by I-ELISA in orchard trees was not reliable when a relatively small amount of pollen from each tree (average 5 g/tree) was collected for the assay. The relatively large amount of pollen from each tree was necessary for reliable I-ELISA tests to compensate for the uneven and erratic distribution of CLRV-W in naturally infected walnut orchard trees.