Genetic Diversity Of Coleus Viroids And The Relationship With Their Hosts

Viroids, the smallest autonomous infectious nucleic acids known today, do not encode any proteinand depend completely on host enzymes for replication. Coleus blumei (coleus), which was foundoriginally in Indonesia, is an ornamental plant grown worldwide, and both its stem and leaf are knownto be succulent. Coleus is susceptible to infection by several Coleus blumei viroids (CbVds) species inthe genus Coleviroid, family Pospiviroidae. Up to now, six main viroids infecting coleus have beenreported: Coleus blumei viroid-1～Coleus blumei viroid-6(CbVd-1～CbVd-6). The CbVd infectioncan be either asymptomatic or symptomatic including dwarfing or slight chlorosis, depending on thecultivar. As members of the family Pospiviroidae, all of the six known coleus viroids share a commoncentral conserved region (CCR) sequence and recombination among the coleus viroids are common.Although CbVd-1has been reported in many countries in the world, the information of the other fivecoleus viroids are limited and no specific bioassays have been established for CbVd-5and CbVd-6.The present study intends to develop rapid detection methods for coleus viroids, investigate theoccurrence and distribution of CbVds in China, analyze the genetic diversity of CbVds and study therelationship between CbVd sequences and host cultivars. The sequence information of coleus viroids inthis study would be enriched in GenBank, and our studies would be of great significance to theclarification of recombination and pathogenicity mechanism for coleus viroids.Here, two rapid detection methods, Sap-direct RT-PCR method and rapid hybridization methodwere developed for coleus viroids. In the Sap-direct RT-PCR method, the sap directly obtained fromcoleus plants using a pipettor were used as the template of RT-PCR and a pair of universal primers forCbVds was designed to detect all the coleus viroids. In the hybridization method, a universal probe(8CCR-probe), which was performed using an octamer of32-nucleotide sequence derived from theCCR region of coleus viroids, can be used to detect at least four coleus viroids by Dot-blot and northernhybridization simultaneously. Both of the two rapid detection methods for coleus viroids are reliable,rapid and low-cost, and they are effective ways to survey the occurrence of coleus viroids and providereference for the development of rapid detection method for other viroids, and even viruses.888coleus samples were collected from China, India and Indonesia. Detection resultsdemonstrated that CbVd-1and CbVd-5have been widely spread in the three countries, and CbVd-2,CbVd-3and CbVd-6have only been detected in China. All the samples were negative to CbVd-4. Theresults in this study are the first reports of CbVd-1and CbVd-5in Indonesia, the first reports of CbVd-2and CbVd-3in China, and the first report of CbVd-5in India.In this study, the biological properties of CbVd-1, CbVd-3, CbVd-5and CbVd-6were confirmedusing infectious clones as inoculum sources. The first detection time for the four CbVds was distinctlydifferent, ranging from45days to300days. Molecular characterization of their progenies revealed thatthe genetic diversity of CbVds populations is not only dependent on the infected host species, but alsoon the viroid species. In addition, we also confirmed that CbVd-5and CbVd-6can be transmitted viaseeds.