Host Adaptation And Pathogenicity Of Hop Stunt Viroid

Hop stunt viroid (HSVd), the only member of the Hostuviroid genus (family Pospiviroidae), hasbeen found in wide range of hosts including hop, cucumber, grapevine, citrus, plum, apricot, peach andalmond. HSVd infections cause economically important diseases such as stunting in hops, dapple fruitsymptom in plum and peach, and cachexia in citrus. Since2006, HSVd infections have beenoccasionally detected in plants like apricot, plum, and peach in China. However, there has been nosystematic effort to investigate the incidence and sequence variability of HSVd from stone fruits(apricot, peach, plum and almond), hop (Humulus lupulus) and grapevine (Vitis) and also has littleinformation on transmission, host adaptation, and pathogenicity. The purpose of this research work is toinvestigate answers for the above-mentioned problems.Initially, we developed a poly-probe for the simultaneous detection of four viroids that infectgrapevine like HSVd, Australian grapevine viroid (AGVd), Grapevine yellow speckle viroid-1and2(GYSVd-1,2), using a non-isotopic dot-blot hybridization technique. A poly-probe was constructed bycloning tandem full-length sequences of HSVd, AGVd, GYSVd-1into a single vector. The cRNApoly-probe, synthesized through in-vitro transcription of recombinant plasmid, detected all four viroidswith similar sensitivity to that obtained using individual probes. In addition, combined with a modifiedCTAB extraction protocol, a reliable and inexpensive method of nucleic acid extraction, a rapid, reliableand cost-effective approach to the simultaneous detection of four grapevine viroids was developedwhich has the potential for routine use in quarantine and certification programs for the screening ofviroid-free mother plants.Survey detailed for the incidence of HSVd infection in various plant sources like stone fruits, hopand grapevine in China was conducted using dot-blot hybridization. Out of553tested samples,127samples were positive for HSVd, indicating average infection rate of23%. Reversetranscription-Polymerase chain reaction (RT-PCR), cloning and sequencing were performed on HSVdpositive samples to analyze HSVd sequence variants.The results showed: i) the same or closely relatedHSVd variants could be isolated from different cultivars of the same host or even from different hostsfrom different regions, indicating cross transmission of HSVd between different hosts. ii) Inphylogenetic analysis, majority of the HSVd variants isolated from stone fruits, grapevine and hop wereclustered into known hop and plum groups. However, two grapevine variants, HSVd.g50andHSVd.g57,could not be clustered into any known groups, but probably formed a new phylogeneticgroup of HSVd isolates together with HSVd.apr35, which was reported to result from recombinationbetween the representative groups of known viroids. iii) HSVd.g38was the single grapevine variant thatclustered with the plum group isolates, supporting cross transmission between grapevine and stone fruitsand the heterogeneity of grapevine isolates.At last, we constructed infectious clone of HSVd-grapevine isolate, termed as KY04, andHSVd-hop isolate with the name of KFKi, which was a new variant evolved from KY04in hop.Comparison of infectivity, pathogenicity and replication between KY04and its mutant KFKi adopted tohop was performed. The results obtained by inoculation to cucumber and hop plants with the two variants indicated that: i) KFKi, the adaptive variant, was not able to stably replicate in grapevine, butreverse mutation to the original variant of KY04, indicating host-specificity of HSVd evolution. ii)KY04caused more severe stunting than KFKi, supporting the influence of evolution on HSVdpathogenicity and the involvement of the five different point mutations between HSVd-KFKi andHSVd-KY04with pathogenicity of these two variants. Moreover, competitive infection bioassaybetween the two variants showed that replication capacity of KY04is significantly higher than KFKi inboth cucumber and hop. These observations suggests that KFKi has been evolved more in its hostadoptability but not in infectivity, replication rate and its pathogenicity and implied that HSVd evolvedin principle of "Trade-off". iii) Secondary structure of KY04was more stable than KFKi during heatdenaturation. iv) The amount of HSVd-sRNA generated from KY04was more than KFKi in infectedhop plants. It indicated that KY04was under more selection pressure from RNA silencing than KFKi.The observation that selection pressure produced by RNA silencing forced KY04change its secondarystructure supported the hypothesis that RNA silencing is an important selection pressure shaping theevolution of the secondary structures of these pathogens. v) Examination of the HSVd sRNA frequencyshowed that their5' termini mapped at many positions of the genomic plus-and minus-strand RNAs,with a large fraction of the counts merging in specific regions (hot spots). Furthermore, the hot spotpatterns of the same viroid sRNAs were different between various hosts, indicating that hostdetermining these profiles.