The variability of hop latent viroid as induced upon heat treatment

We have previously shown that heat treatment of hop plants infected by hop latent viroid (HLVd) reduces viroid levels. Here we investigate whether suc h heat treatment leads to the accumulation of sequence variability in HLVd. We observed a negligible level of mutated variants in HLVd under standard cultivation conditions. In contrast, the heat treatment of hop led to HLVd degradation and, simultaneously, to a significant increase in sequence vari ations, as judged from temperature gradient-gel electrophoresis analysis an d cDNA library screening by DNA heteroduplex analysis. Thirty-one cDNA clon es (9.8%) were identified as deviating forms. Sequencing showed mostly the presence of quadruple and triple mutants, suggesting an accumulation of mut ations in HLVd during successive replication cycles. Sixty-nine percent of base changes were localised in the left half and 31% in the right half of t he secondary structure proposed for this viroid. No mutations were found in the central part of the upper conserved region. A 'hot spot" region was id entified in a domain known as a 'pathogenicity domain" in the group represe ntative, potato spindle tuber viroid. Most mutations are predicted to desta bilise HLVd secondary structure. All mutated cDNAs, however, were infectiou s and evolved into complex progeny populations containing molecular variant s maintained at low levels. (C) 2001 Academic Press.