A method is described for the improved detection of bean yellow mosaic virus (BYMV) in gladioli leaves. Specific sequences of BYMV RNA, present in total RNA extracts of infected plants were detected following amplification by the polymerase chain reaction (PCR). The viral RNA was initially reverse-transcribed into cDNA, then specific sequences were amplified by PCR using specific oligonucleotides as primers. Detectable amounts of virus RNA in BYMV-infected plant tissue by PCR were approximately three to four orders of magnitude lower as compared with those detectable by ELISA and molecular hybridisation. Combining PCR with molecular hybridisation (using a 32P-labelled transcript of viral sequences as a probe), further increased the sensitivity of this method to a gain of four to five orders of magnitude as compared with direct molecular hybridisation, and enabled the detection of up to single picogram quantities of the virus.
Peach (Prunus persica (L.) Batsch) shoot-cultures infected with prunus necrotic ringspot virus (PNRSV) were selected for evaluating the responses of in uitro grown shoots of cvs Hermosa and Summerset to thermotherapy. The survival of shootcultures during thermotherapy was improved by selection of the optimum concentration of 6-benzylamino purine in the medium and optimum age of shoots for treatment. Alternating high and low temperature thermotherapy regimes were more effective in decreasing virus titre than constant high temperatures. Of the regimes tested, the most effective inhibition of PNRSV combined with a high survival of shoots was obtained by applying 38 "C for 16 h in light alternating with 28 "C for 8 h in darkness for 18 days for Hermosa and 22 days for Summerset. Following this treatment 90% of Hermosa and 40% of Summerset shoot-cultures were virus-free as determined by enzyme-linked immunosorbent assay. Relatively large (about 10 mm) apices excised from these shots regenerated into virus-free plants. The advantage of the in oitro system for thermotherapy is discussed.
SUMMARY
Bean yellow mosaic virus (BYMV) could not be detected in corms of infected gladioli unless they were cut 6–60 days before testing. Detection after cutting was time‐ and temperature‐dependent, was restricted to the cut area, and varied among cultivars. Virus could be recovered from uncut corms after storage for over 2 yr at 6 oC. BYMV in corms could be detected by enzyme‐linked immunosorbent assay and by immunosorbent electron microscopy with antisera against a gladiolus isolate purified from gladiolus leaves or corms. It could not be detected in corms with antiserum against a lupin isolate which readily detected BYMV in gladiolus leaves. Protein subunits of corm‐BYMV banded in SDS‐PAGE as a single 31 000 dalton polypeptide, while leaf‐BYMV produced a major 34 000 and several smaller polypeptides. Both major polypeptides retained the different serological properties of their source virions but their peptide maps indicated a common origin. It is suggested that the smaller polypeptide from corm‐BYMV is a stable cleavage product of the intact leaf‐BYMV coat subunits. Corm‐BYMV, although lacking some of the antigenic properties of leaf‐BYMV, was still infective.
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