SERODIAGNOSIS OF PLUM POX VIRUS

Identification of plum pox virus (PPV) by symptom expression in woody hosts or by transmission to herbaceous hosts is either not very reliable or it is time-consuming. We therefore tried to develop a serological test, which is simple enough to be used in agricultural extension stations with a staff not trained in serological methods.

For this purpose we used the single-radial-diffusion test modified from Shepard (1972). With the single-radial-diffusion test one reactant - the antibody - is present in a layer of agar gel; the other - the antigen - diffuses radially from a well into the antibody-agar matrix. Antigen molecules precipitate when, at the proper distance from the well, proportions of antibody and antigen reach serological equivalence. Complexed antigen and antibody form a whitish ring or halo around the well. Elongated virus particles such as plum pox virus, whose length exceeds the pore diameter of the agar gel, have to be degraded to diffusible subunits.

PPV was purified according to van Oosten (1972), whom we have to thank for the PPV isolate. Antisera with titers up to 1:8000 were produced. However, for the radial diffusion tests we used antisera with titers of about 1:128 or 1:256. To avoid unspecific reactions with host plant protein or other plant substances, this antiserum was crossabsorbed with healthy plant sap according to Uyemoto et al. (1972). After this treatment the titer of the antisera was one or two twofold dilution steps lower. 2% Oxoid Ion Agar No. 2 in Tris-HC1 buffer, 0.05 M, pH 7.2, was mixed at a temperature of 45°C with the same volume of pretreated PPV antiserum diluted to a titer of 1:64. Four ml were pipetted into each clear plastic dish 4.7 to 4.5 cm in size to make a 2 mm agar layer. Holes of about 4 mm in diameter were cut in the solidified agar and filled with crude sap of PPV infected plant material.

For tests we used 0.2 to 0.7 g material from different parts of the plants. From fruits the skin with or without symptoms was ground with an equal amount (weight/volume) of 5% pyrrolidine in 0.05 M Tris-HCl buffer, pH 7.2. Pyrrolidine degrades the PPV particles into subunits. From leaves with symptoms, the spots and blotches were punched out and ground in 1.5 volumes of pyrrolidine solution. The filling of the agar wells with the resulting slimy slurry is difficult and has to be done with great care with a pasteur pipette.

The crude saps from fruit skin and leaves were neither clarified nor centrifuged.

Presence of PPV was indicated by development of whitish precipitation rings around the agar wells. When testing fruit skin, precipitation rings may start to be visible as early as about two hours; while with slimy crude sap from leaves, it takes at least six hours. Sap from known