Mode of access: World Wide Web. / Thesis (Ph. D.)--University of Hawaii at Manoa, 2005. / Includes bibliographical references. / Electronic reproduction. / Also available by subscription via World Wide Web / xv, 222 leaves, bound col. ill 29 cm

Odontoglossum ringspot virus (ORSV) is one of the most prevalent orchid viruses that infects greenhouse-grown orchids worldwide. In order to prevent the spread of viruses in greenhouses and to cultivate clones from virus-free mother plants, it is necessary to develop a more sensitive technique for the detection of viruses in orchids. SYBR green real-time RT-PCR is a highly sensitive technique that can specifically detect ORSV in orchid tissue. By harvesting tissue at the inoculation site and at specific distances from the inoculation site at different times past inoculation, this technique can also be used to study the rate of spread of ORSV in orchids. Orchid clones were inoculated with ORSV and other clones were mock-inoculated with molecular grade water. Leaf tissue was harvested from the ORSV-inoculated and mock-inoculated clones at the site of inoculation and at specific distances from this site at 16 h, 24 h, and 72 h past inoculation. Total RNA was extracted from the harvested tissue. Competitive RTPCR was going to be used for the quantification and detection of ORSV in the samples, but attempts at cloning an ORSV fragment into a vector in order to form a competitive standard were unsuccessful. Instead, a highly sensitive qualitative approach called SYBR green real-time RT-PCR was used for the detection of ORSV. ORSV was detected in all virus-inoculated orchids, except for one. Therefore, all of the ORSV inoculated plants except for one were infected with the virus. Unexpectedly, ORSV was also detected in all of the mock-inoculated orchids. Most likely the orchids were previously infected with ORSV, but the viral titer was too low to be detected by commercial techniques. However, there is a small possibility that the orchids were contaminated during experimentation, despite careful technique. The rate of spread of the virus could not be studied because the mock-inoculated samples also contained the virus. Although viral amplification was demonstrated in the mock-inoculated plants, SYBR green real-time RT-PCR is still a sensitive and consistent method for ORSV detection in orchids. With additional controls, this method could prove to be the ideal method for reliable detection of ORSV in commercially-grown orchids. / Department of Biology

Regelmäßige Bonituren haben gezeigt, dass virusverdächtige Symptome an Stieleichen, die zu etwa 90 % als chlorotische Ringflecken auftreten, im nord- und mitteldeutschen Raum weit verbreitet sind. In der vorliegenden Arbeit sollte der Erreger dieser Symptome isoliert und näher charakterisiert werden. Aus zwei Blattproben mit chlorotischen Ringflecken konnten stäbchenförmige Viruspartikeln mit einer Länge von ca. 450 nm isoliert und auf krautige Indikatoren übertragen werden. In einer RT-PCR mit Hüllprotein bzw. Transportprotein-sequenzspezifischen Primern wurden diese als Tobacco mosaic virus (TMV)- bzw. Tomato mosaic virus (ToMV)- Isolate identifiziert. Eine Infektion der Stieleichen mit weiteren bekannten Viren von Gehölzen, wie dem Cherry leaf roll virus (CLRV) oder dem Erreger der Ebereschenringfleckigkeit konnte mittels ELISA und RT-PCR ausgeschlossen werden. DsRNAs der Größen 1.5 und 1.6 kb sowie 1.8 und 2.0 kb konnten symptomunabhängig aus Rindengewebe, Knospen und Blättern von Stieleichen isoliert werden. Mit Hilfe der RT-DOP-PCR und der cDNA-Klonierung gelang es, Teile des 1.5/1.6 kb dsRNA-Moleküls zu charakterisieren. Die Sequenz von 479 Aminosäuren (1437 Nukleotiden) wies eine Identität von 56 % zur RNA-abhängigen RNA-Polymerase (RdRp) des Beet cryptic virus 3 (BCV 3) auf. Der spezifische Nachweis dieser Sequenz gelang mittels RT-PCR sowohl in dsRNA-Proben, als auch in angereicherten Nukleokapsiden symptomloser und symptomatischer Stieleichen. In Nested-PCR-Analysen konnte das Fragment jedoch nicht nur in Gesamt-RNA von Stieleichen, sondern auch in Gesamt-RNA und DNA verschiedenster gesunder Pflanzen amplifiziert werden. Phylogenetische Vergleiche mit ausgewählten RdRps viralen und pflanzlichen Ursprungs zeigten die engste Verwandtschaft der Stieleichen-dsRNA-Sequenz zu den Partitiviren, zu denen sich neben BCV 3 auch die endogene dsRNA aus Pyrus und aus Chloroplasten von Bryopsis gruppiert. Diese Erkenntnisse lassen in der charakteristischen Doppelbande von 1.5/1.6 kb das Vorliegen einer endogenen dsRNA vermuten. Hiermit ist in dieser Arbeit das Auftreten verschiedener Viren in Eichen nachgewiesen worden, von denen die meisten höchstwahrscheinlich nicht im direkten ursächlichen Zusammenhang mit der chlorotischen Ringfleckigkeit der Eiche stehen. / Ratings of oak populations revealed that around 90 % of all oak trees affected by viruslike symptoms showed chlorotic ringspots and that these symptoms are widely spread in oaks in north and central Germany. In this study the putative agent of these symptoms should be isolated and specified. Rod-shaped particles with a length of 450 nm were recovered from two different samples of leaves displaying chlorotic ringspots by mechanical inoculation of herbaceous indicator plants. These particles were identified to be Tobacco mosaic virus (TMV)- and Tomato mosaic virus (ToMV)- isolates by RT-PCR analyses of the coat- and movement protein genes. Infections with other well known viruses of forest trees, like Cherry leaf roll virus (CLRV) and the agent causing ringspots in European mountain ash, were excluded by ELISA and RT-PCR. DsRNA fragments of 1.5 and 1.6 kb as well as 1.8 and 2.0 kb were extracted from leaves, inner bark and bulbs of all symptomatic and asymptomatic samples of common oak. The nucleotide sequence of the 1.5 and 1.6 kb dsRNA fragment was partially characterised by reverse transcription degenerated oligonucleotide primed (DOP)-PCR and cDNA cloning. The obtained nucleotide sequence of 1437 nt encoding a putative protein of 479 amino acids revealed an identity of 56 % with the RNA-dependent RNA polymerase (RdRp) of Beet cryptic virus 3 (BCV 3). PCR amplification of the RdRp coding nucleotide sequence was possible using a number of different dsRNA samples as well as concentrated nucleocapside preparations. The same sequence was also amplified successfully by Nested-PCR not only in total RNA extracted from symptomatic and asymptomatic oak samples but also from total RNA and DNA of diverse plants. Phylogenetic analysis revealed further similarities to RdRp´s of endogenous dsRNA of Pyrus and chloroplasts of Bryopsis, both members of the Partitiviridae as well as BCV 3. These results strongly indicate that the 1.5/1.6 kb dsRNA of oak is endogenous dsRNA. In summary, it has been shown that oaks in Germany are commonly infected by a variety of different viruses most of them possibly unrelated to the wide-spread ringspot symptoms of oaks.