Progress 10/01/01 to 09/30/06
Outputs
Work ceased with the retirement of the PI on September 1, 2004.
Impacts
The application of non-disease inducing viroids or TsnRNAs continues to offer the potential for reduction in vegetative growth as well as enhanced crop performance. Factors involved in the expression of the 'finger imprint' symptom on Poncirus trifoliata rootstock have been defined as well as the most probable causal agent of citrus gummy bark disease.
Publications
- No publications reported this period
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Progress 01/01/03 to 12/31/03
Outputs
A viroid etiology for citrus gummy bark (CGB) disease of sweet orange is supported by the similarity of symptom expression to cachexia disease of mandarins and tangelos caused by the hop stunt viroid (HSVd) related citrus viroid II (CVd-II) as well as the detection of CVd-II variants in CGB infected Washington navel and Dortyol sweet orange, a Turkish cultivar. A survey was made of 67 clones of CVd-II related variants recovered from severe CGB symptomatic and non-symptomatic trees of the same cultivars growing in close proximity. Only CVd-IIa, a non-cachexia inducing variant, was found in non-symptomatic Washington navel trees and no CVd-II variants were recovered from the Dortyol control. CGB infected sources contained a number of CVd-II related variants with the predominant species detected closely related to CVd-IIc, a known cachexia inducing viroid. Biological activity of representative variants from CGB sources was determined by transmission to citron (Citrus
medica) as well as by bioassay on the indexing host for cachexia, Parson's Special mandarin (Citrus reticulata). Selected citrus viroids that induce no diseases of economic impact to citrus production have been investigated for the ability of a transmissible agent to confer an economic advantage in citrus performance. Although trees containing CVd-IIIb were reduced in size by about 50%, average fruit size was increased and the distribution more homogeneous. The unusual symptom, 'finger imprint', described exclusively on Poncirus trifoliata containing CVd-IIIb has been reported in only a single field trial investigating the effects of citrus viroids on crop performance. With this, the question has persisted whether the observed growth abnormality was a 'disease symptom' induced by citrus viroid IIIb (CVd-IIIb) or a consequence of mechanical damage caused by the handling of young trees during propagation or cultural practices in the field. The reoccurrence of 'finger imprint' symptoms
on trees after five years in the field in which no abnormal growth features were previously noted supports the proposition of a viroid-induced disease. The symptom expression results from an unusual etiology of a complex relationship of the specific viroid CVd-IIIb on the specific rootstock, P. trifoliata only when supplemental water is applied by sprinkler irrigation.
Impacts
The application of non-disease inducing viroids or TsnRNAs continues to offer the potential for reduction in vegetative growth as well as enhanced crop performance. Factors involved in the expression of the 'finger imprint' symptom on Poncirus trifoliata rootstock have been defined as well as the most probable causal agent of citrus gummy bark disease.
Publications
- Semancik, J.S., Bash, J.A. and Gumpf, D.J. 2003. Induced dwarfing of citrus by transmissible small nuclear RNA (TsnRNA). Page 390- 394. Proceedings of the 15th Internationa Organization of Citrus Virologists.
- Onelge, N., Cinar, N., Szychowski, J.A., Vidalakis, G. and Semancik, J.S. 2004. Citrus Viroid II Variants Associated with 'Gummy Bark' Disease. European Journal of Plant Pathology (submitted).
- Vidalakis, G., Gumpf, D.J., Bash, J.A., and Semancik, J.S. 2004. 'Finger-Imprint' of Poncirus trifoliata: An Unusual Relationship of a Specificity of Viroid, Host and Field Management. Plant Disease (submitted).
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Progress 01/01/02 to 12/31/02
Outputs
Evidence of post-transcriptional gene silencing (PTGS) in avocado sunblotch viroid (ASBVd) infected avocado was suggested by detection of ASBVd-specific 23mer small RNAs. PTGS was observed in all infected tissues including bleached, variegated and symptomless carrier foliar sources as well as fruit with typical sunblotch disease lesions. Tissues with the different symptom expressions, characterized by the presence of different ASBVd variants, were found to induce PTGS at differential levels. Detection of the PTGS small RNA as well as intensity was directly related by symptom type and viroid titer. PTGS was also induced in Gynura aurantiaca infected with two closely-related mild and severe variants of the Pospiviroid, citrus exocortis viroid (CEVd) and could not be altered by with the induction of severe symptoms in Gynura infected with the mild variant. The detection of the PTGS small RNA fragments was not directly related to viroid titer during initiation of symptom
development in Gynura. Most unusual data was obtained from the oldest trial of TsnRNAs established in 1984 at the Lindcove Center. Valencia sweet orange on trifoliate rootstock containing TsnRNA-IIa and TsnRNA-IIIb although reduced in size by about 20 % and 50% respectively, exhibited an increase in yield of 3 to 5 fold (see table). Although most previous harvests have confirmed the performance of greater yield/vegetative growth from TsnRNA containing trees, the 2002 harvest was unique in the dramatic distinction to control trees especially considering the TsnRNA trees of half the size producing 3 fold greater yields. Also, as seen in past harvests, fruit size was enhanced by the presence of the TsnRNAs. Additional field trials both at Lindcove and UCR are being evaluated for the effect of TsnRNAs on a range of Poncirus trifoliata hybrid rootstocks for potential extension of the data already obtained. A reduction in vegetative growth has been observed on seedlings of citrumelo 1452
and citremon 1449 (see photo). In addition, a reduction in the fruiting of citrumelo 1452 was noted. How this property might affect a commercial scion will be interesting to investigate.
Impacts
Evidence for gene silencing in viroid infection indicates that the non-translated viroid genome interacts with the host similar to a translated viral genome. The application of non-disease inducing viroids remains an effective means of reduction in vegetative growth and increased performance of citrus.
Publications
- Hadidi, A, Flores, R, Randles, J.W. and Semancik, J.S. (eds). 2003. VIROIDS. CSIRO Publishing, Australia, 370pp.
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Progress 01/01/01 to 12/31/01
Outputs
A viroid etiology has been suggested for citrus gummy bark (CGB) disease of sweet orange based on the similarity of symptoms with cachexia. The consistent detection of hop stunt viroid related Group II citrus viroids found in CGB infected Washington navel and the Turkish cultivar, Dortyol, while not in asymptomatic controls further supports this view. A total of 47 clones constructed from RT-PCR products employing CVd-IIa specific primers were analyzed by sequence homology. The 6 clones derived from the asymptomatic Washington navel were closely related in size (302 nucleotides [nts.]) and sequence to the non-cachexia variant CVd-IIa. The 22 clones produced from CGB infected Washington Navel were almost equally divided between the CVd-IIa class of 301-304 nts. (9 clones) and the cachexia CVd-IIc class of 296 nts. (11 clones) with only single clones of 295 and 299 nts. related to the CVd-IIb class. A more complex collection occurred in the 19 clones derived from CGB
infected Dortyol. Three clones of the CVd-IIa class were found among the predominant cachexia related CVd-IIb class of 11 clones (298-300 nts.) and CVd-IIc class of 5 clones (296 nts.). Transmission to citron by transcripts of representative clones from all clusters described by homology has been successful with CGB bioassay and indexing tests currently in progress. Citrus, as a single plant group, harbors a diverse collection of five viroid populations. Although variants from each of the five viroids express symptoms in the indexing and bioamplification host, citron (Citrus medica), only two diseases of economic impact to citrus production, exocortis and cachexia, have been attributed to viroids. This relationship facilitated the proposal for the division of viroid-like molecules into two distinct classes. A class that induce diseases impeding agricultural performance, the viroids, and a class that share physical and biological properties in the absence of pathogenic responses
harmful to the crop productivity, the transmissible small nuclear RNA (TsnRNA). Although the process of viroid pathogenesis is still to be clearly defined, it has been conjectured that TsnRNA molecules may affect host metabolism to effect responses of economic advantage. Accordingly, the association for the reduced vegetative growth of scions on Poncirus trifoliata and related rootstocks with the presence of CVd-III variants has progressively gained significance in many citrus growing regions. Performance trials sweet orange on trifoliate rootstock over a period of about 10 years has indicated a consistent increase in fruit yield/canopy from trees reduced in size by 20% with TsnRNA-IIa or 50% with TsnRNA-IIIb.
Impacts
The characterization of citrus gummy bark disease as a possible cachexia-related disorder has been tested by assay of selected CVd-II variants. The application of non-disease inducing viroids has been effective for the modification of citrus vegetative growth.
Publications
- N. Onelge, A. Cinar, J.A. Szychowski and J.S. Semancik. 2002. Cachexia-Related Viroids Associated with Citrus Gummy Bark. Proc. 15th IOCV, Riverside, (in press).
- J.S. Semancik, J.A. Bash and D.J. Gumpf. 2002. Induced dwarfing of citrus by transmissible small RNA (TsnRNA). Proc. 15th IOCV, Riverside, (in press).
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Progress 01/02/00 to 12/31/00
Outputs
Natural selection of two new variants of citrus exocortis viroid (CEVd) was detected by observing tissues displaying both severe and mild symptoms from a single GYNURA AURANTIACA. The variants, CEVd-S (severe) and CEVd-M (mild) differing by only 5 nucleotides confined to the pathogenic (P) domain, remained stable when propagated by rooted cuttings or from successive plants inoculated with tissue extracts or transcripts from cDNA clones. CEVd-S induces a very severe reaction in Gynura that is consistent throughout a range of environmental conditions. However, symptoms resulting from CEVd-M infection can vary from a non-symptomatic condition to a severe reaction when grown at 40 degree centigrade. This differential response was confined to a single host, GYNURA AURANTIACA, and expressed under standard growing conditions. The distinct host responses induced by these variants could not be correlated with any changes in sequence or conformation of the dominant viroid
variant as predicted by molecular modeling. Therefore, the variable symptom expression appears to be associated with a specific temperature sensitive response of GYNURA AURANTIACA.
Impacts
The distinctly different symptom expression of variants of CEVd differing by only five nucleotides in a specific host was demonstrated to be governed by a temperature-sensitive host response and not the conformation of the viroid molecule.
Publications
- Skoric, D., M. Conerly, J.A. Szychowski and J.S. Semancik. 2001. CEVd induced symptom modification as a response to a host-specific temperature-sensitive reaction. Virology 280:115-123.
- Semancik, J.S. 2001. Avocado sunblotch viroid. Global Crop Protection Compendium. CAB International.
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Progress 01/01/99 to 12/31/99
Outputs
Citrus viroid (CVd) Group II is comprised of hop stunt viroid (HSVd) related variants of 295-302 nts. Included in this group are the cachexia disease-inducing agents, citrus cachexia viroid (CCaVd) or CVd-IIb, CVd-IIc, Ca-903, and Ca-909 as well as the non-cachexia inducing variant, CVd-IIa. The cachexia indexing hosts Parson's Special mandarin and Orlando tangelo as well as Citrus macrophylla responded with symptoms of gumming, discoloration and stem pitting when infected by CVd-IIb, CVd-IIc or Ca-903. However, Palestine sweet lime (PSL), the indicator host used to describe the xyloporosis disease, displayed a distinctly different fine pitting reaction in the absence of any discolorations or gumming with infection by the same viroids. Cachexia-inducing variants contain a number of nucleotide changes more similar to hope-type HSVd sequences than the citrus-type as typified by CVd-IIa. The nucleotide sequence of CVd-IIc was identical to CVd-II isolates common to trees
expressing xyloporosis. Experimental evidence indicates that either CVd-IIb or CVd-IIc can cause citrus diseases known as cachexia and xyloporosis and that the two disease designations reflect the distinct responses of different indexing hosts to the same viroids. A cachexia disease specific oligonucleotide has been developed which may be employed as a probe in molecular hybridization as well as primer in reverse transcriptase-polymerase chain reaction (RT-PCR) procedures. The cachexia-specific probe demonstrates the ability to discriminate between the severe cachexia variants, CVd-IIb and CVd-IIc, and moderate to mild variants, Ca-903 and Ca-909. These tools can segregate cachexia-inducing citrus viroids (CVd) from the non-cachexia variant, CVd-IIa, known to introduce commercially desirable effects on tree growth and performance.
Impacts
These studies more clearly describe the relationship between viroids and citrus diseases as well as the definition of cachexia and xyloporosis as specific diseases of citrus. Laboratory protocols have been developed for the indexing of cachexia disease that contributes to a more rapid and efficient detection to replace bioassay requiring long incubation periods.
Publications
- Reanwarakorn, K. and Semancik, J.S. 1998. Correlation of hop stunt viroid variants to cachexia and xyloporosis diseases of citrus. Phytopathology 89:568-574.
- Semancik, J.S. and Duran-Vila, N. 1999. Viroids in plants: Shadows and footprints of a primitive RNA. IN: Origin and Evolution of Viruses, Domingo, Webster and Holland (eds.), Academic Press, London. Pp. 37-64.
- Reanwarakorn, K. and Semancik, J.S. 1999. Discrimination of cachexia disease agents among citrus variants of hop stunt viroid. Annals of Applied Biology 135:481-487.
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Progress 01/01/98 to 12/01/98
Outputs
Two hop stunt viroid-related Group II citrus viroids are characterized as either a cachexia disease non-pathogenic variant (CVd-IIa) or a pathogenic variant (CVd-IIb). Viroid cDNA clones were transmissible to citron (CITRUS MEDICA L.) and LUFFA AEGYPTIACA Mill. To describe cachexia pathogenicity and symptom expression in LUFFA, chimeric viroid cDNA clones were constructed. Symptoms induced on two bioassay hosts reflected the differential response with CVd-IIa and -IIb. Constructs with the C-V-T2 domain from clone-IIa induced severe symptoms on LUFFA typical of CVd-IIa, but were non-symptomatic on mandarin, a bioassay host for the cachexia disease. Constructs with the same region from clone-IIb genome induced mild symptoms on LUFFA, but a severe reaction on mandarin. Specific site-directed mutations in the V-domain of the CVd-IIa clone constructed conversions to the CVd-IIb sequence. With the introduction of six site-specific changes into the V-domain of the clone-IIa,
cachexia pathogenicity was acquired. Selections of VITIS VINIFERA, Zinfandel (-1A) and Mission, were found to harbor different grapevine yellow speckle viroid-1 (GYSVd-1) variants. The variants were shown to contain homogenous populations of 'type 1' and 'type 2' GYSVd-1 variants. Symptoms of yellow speckle disease were induced only in vines containing 'type 2'. Pagadebit selections from Italy revealed the non-symptomatic variant was essentially identical to Zinfandel-1A while symptomatic selections were unique. Changes (43) were spread throughout the T1, C, V, and T2 domains from symptomatic selections.
Impacts
(N/A)
Publications
- SZYCHOWSKI, J.A., CREDI, R., REANWARAKORN, K., and SEMANCIK, J.S. 1998. Population diversity in grapevine yellow speckle viroid-1 and the relationship to disease expression. Virology 248: 432-444.
- REANWARAKORN, K. and SEMANCIK, J.S. 1998. Regulation of pathogenicity in hop stunt viroid-related group II citrus viroids. J. of Gen.
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Progress 01/01/91 to 07/01/97
Outputs
A catalogue for the 10 known citrus viroids (CV) utilizing size and sequence homology has been developed. Interference between the mild isolate, CVd-IIa, and the severe cachexia agent, CVd-IIb, occurs in citrus. This offers a potential practical approach for the control of cachexia by "viroid interference." Citrus viroids, CVd-Ia, Cvd-IIa, and CVd-IIIb, reduced tree size of Valencia orange on PONCIRUS TRIFOLIATA rootstock by 20-50%. The yield per canopy volume was significantly enhanced in all trees when compared with viroid-free trees. Although pathogenic agents in some hosts, selected viroids may be viewed as TRANSMISSIBLE, SMALL, NUCLEAR RIBONUCLEIC ACIDS (Tsn-RNAs) in specific hosts which do not express a disease syndrome. Synthesis of cDNA probes by random-priming of a viroid template display specificity to all members included within a single viroid group. Mapping the loci for probe transcription suggests that a similar stem-loop structured conformation may be
responsible for group specificity. An unusual variant of citrus exocortis (CEV) was detected in hybrid tomato (LYCOPERSICON ESCULENTUM MILL. X L. PERUVIANUM). The 92 nucleotide variant, CEV D-92, contained two repeated sequences spanning the V and T2 domains. Moderation of symptoms in Gynura accompanied the incorporation of the repeats. Four major groups of grapevine viroids were defined: (i) CEV-g, an isolate of citrus exocortis viroid, (ii) GVd-c, recovered from cucumber, and AGVd, Australian grapevine viroid, (iii) GYSVd-1 and GYSVd-2, inducing yellow speckle disease and (iv) HSVd-g, hop stunt viroid.
Impacts
(N/A)
Publications
- SEMANCIK, J. S., RAKOWSKI, A. G., BASH, J. A., and GUMPF, D. J. 1997. Applications of selected viroids for dwarfing and enhancement of production of 'Valencia' orange. Journal of Horticultural Science 72,
- WOLPERT, J. A., SZYCHOWSKI, J. A., and SEMANCIK, J. S. 1996. Effect of viroids on growth, yield, and maturity indices of Cabernet Sauvignon grapevines. American Journal of Enology and Viticulture 47,
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Progress 01/01/96 to 12/30/96
Outputs
Citrus viroids, CVd-Ia CVd-IIa, and CVd-IIIb, resulted in a statistically significant reduction of 20-50% in tree size of Valencia orange, CITRUS SINENSIS (L.) Osb., on PONCIRUS TRIFOLIATA (L.) Raf. rootstock. Reduction in canopy volume was accompanied by an increase in yield per tree in the presence of CVd-IIa, whereas net decreases in yield resulted with either CVd-Ia or CVd-IIIb. The yield per canopy volume was significantly enhanced in all viroid-containing trees when compared with the viroid-free trees. Although pathogenic agents in some hosts, selected viroids may also be viewed as TRANSMISSIBLE, SMALL, NUCLEAR RIBONUCLEIC ACIDS (TSN-RNAS) in those specific hosts which do not express a disease syndrome. The effect of viroids was tested by comparing shoot tip cultured (STC), viroid-free Cabernet Sauvignon to STC vines reinoculated with two grapevine yellow speckle viroids (GYSVd-1 and GYSVd-2) and hop stunt viroid (HSVd-g). Growth and yield components were
measured over a three year-period (1991-1993) on mature vines in the Napa Valley. On average, vegetative growth was greater on STC vines as a result of heavier shoots. Viroids did not affect yield components but fruit of STC vines was higher in titratable acidity and lower in pH than STC + viroids. In these own-rooted vines, viroids do not cause recognizable "disease".
Impacts
(N/A)
Publications
- SEMANCIK, J. S., RAKOWSKI, A. G., BASH, J. A., and GUMPF, D. J. 1997. Applications of selected viroids for dwarfing and enhancement of production of 'Valencia' orange. Journal of Horticultural Science 72,
- WOLPERT, J. A., SZYCHOWSKI, J. A., and SEMANCIK, J. S. 1996. Effect of viroids on growth, yield, and maturity indices of Cabernet Sauvignon grapevines. American Journal of Enology and Viticulture 47,
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Progress 01/01/95 to 12/30/95
Outputs
Synthesis of cDNA probes by random-priming of a viroid template displays the unusual property of specificity to all members included within a single citrus viroid Group. Mapping the loci for probe transcription from the CEVD, CVd-IIb, and CVd-IV genomes suggests that a similar structured conformation may be responsible for group specificity. A stem-loop configuration in the viroid template and hybridization target sites can be proposed to be responsible for the availability of the group-specific sequences. A viroid present in very low titres was isolated from symptomless field broad bean plants was identified as a variant of citrus exocortis viroid. Serial passage through tomato and back to the host of origin, broad bean, resulted in major changes in replication efficiency, host range and pathogenicity. The effects of these sequence variations on viroid secondary structure result in non-pathogenic viroid variants which can remain unnoticed in certain plant species but
may act as reservoirs of viroid disease.
Impacts
(N/A)
Publications
- FRANCIS, M. I., SZYCHOWSKI, J. A., and J. S. SEMANCIK. 1995. Structural sites specific to citrus viroid groups. J. Gen. Virol. 76: 1081-1089.
- FAGOAGA, C., SEMANCIK, J. S., and N. DURAN-VILA. 1995. A citrus exocortis viroidvariant from broad bean (VICIA FABA L.): infectivity and pathogenesis. J. Gen. Virol. 76: 2271-2277.
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Progress 01/01/94 to 12/30/94
Outputs
Variants of avocado sunblotch viroid (ASBVd) of between 247 and 250 nucleotides in length have been recovered from diseased avocado tissues. The sunblotch syndrome covers a complex pattern of disease symptoms which are associated with infection by variants of ASBVd. The viroid species are designated ASBVd-B, ASBVd-V, and ASBVd-Sc from their association with bleached, variegated, or symptomless carrier tissues, respectively. Host-viroid interactions and structural relationships among the variants suggest a transition in sunblotch disease from a severe acute to a persistent mild form of infection. The nucleotide sequence and secondary structure of two representative variants from the Group III citrus viroids, CVd-IIIa (297 bases) and CVd-IIIb (294 bases) were determined. The variants are related to the apple scar skin viroid (ASSVd) family. Although smaller in size than any of the ASSVd-related viroids, the central conserved region as well as most of the terminal
conserved region of ASSVd is retained. The rod-like structural configuration (characteristic of ASSVd) of the variants is predicted by minimum free energy analysis.
Impacts
(N/A)
Publications
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Progress 01/01/93 to 12/30/93
Outputs
Isolates of citrus exocortis viroid (CEV) from sweet orange have been selected by sequential passage through the alternate hosts citron, GYNURA AURANTIACA, a hybrid tomato of LYCOPERSICON ESCULENTUM X L. PERUVIANUM and disorganized callus culture of the hybrid tomato. Distinctions in symptom expression, titer, and electrophoretic mobility among the CEV isolates, termed, CEVc (citron), CEVg (Gynura), CEVt (tomato), and CEVcls (callus) are supported by different nucleotide sequences. The nucleotide sequence of full length cDNA clones of CEVc purified from citron displays exchanges not reported for any previously described CEV variant. An unusual number of exchanges have been localized in the terminal domains. A common pattern of nucleotide exchanges, described as a "tomato signature", can be detected in all of the isolates derived from hybrid tomato tissues. An unusual variant of citrus exocortis viroid (CEV) was detected when an inoculum source from GYNURA AURANTIACA
D.C. was used to infect a hybrid tomato (LYCOPERSICON ESCULENTUM MILL. X L. PERUVIANUM). The 92 nucleotide enlarged variant, CEV D-92, contained two repeated sequences spanning the V and T2 domains. A dramatic moderation of symptom expression in Gynura accompanied the incorporation of these repeated sequences.
Impacts
(N/A)
Publications
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Progress 01/01/92 to 12/30/92
Outputs
Systemic interactions of long duration among the Group II Citrus Viroids, CVd-IIa and CVd-IIb were investigated by grafting buds from established citron (CITRUS MEDICA L.) sources containing single viroids to a common healthy seedling. In healthy tissues, the two viroids became established in approximately equal titres as a mixed infection. By contrast, tissues that grew from the CVd-IIa or CVd-IIb source buds contained only trace amounts of the heterologous invading viroids. The interference between CVd-IIa and CVd-IIb indicates that the mild and relatively innocuous isolate, CVd-IIa, can interfere with the replication and/or accumulation of the severe cachexia agent, CVd-IIb, in citrus. This offers a potential practical approach for the control of cachexia in commercial plantings by "viroid interference". Valencia/trifoliate planting inoculated with pure isolates of citrus viroids (CV) showed specific reactions of: "Trifoliate deep pit" a symptom caused by citrus
viroid Ia which induces deep pits in the wood of the trifoliate trunk. "Trifoliate mild bark cracking" a symptom caused by citrus viroid IIa which induces mild bark cracking in the trifoliate trunk. "Trifoliate finger imprint" a symptom caused by citrus viroid IIIb which induces grooving in the trifoliate trunk. These three new and distinct symptoms in PONCIRUS TRIFOLIATA and their association with representative members of the citrus viroid groups I, II and III are described with effects of these viroids on tree dimensions.
Impacts
(N/A)
Publications
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Progress 01/01/90 to 12/30/90
Outputs
Variation in tree size occurred in grapefruit trees budded in trifoliate orange rootstock and inoculated with a graft-transmissible dwarfing agent (GTD) from grapefruit. Extracts from `Etrog' citron displayed a profile of five viroid bands. These results indicate that a segregation of the viroid complex in grapefruit budwood was a major factor in the variation seen among trees inoculated from the GTD source 225-T. Cross protection assays using CEV-129 as a "protecting" strain against the severe type strain of CEV demonstrated that a mild strain of CEV could provide apparent `protection' against challenge inoculations with the severe strain. The protection effect, however, displayed a variability which ranged from only a brief delay to almost total impairment of symptom expression. Analyses of California and European grapevine sources indicated a ubiquitous occurrence of viroids in these materials. Hybridization results indicated homology to both GV-1 and GV-3 for
viroids of varieties grown in California as well as from European sources. Wine and rootstock varieties contained a greater proportion of the more common GV-1 plus GV-3 viroid profile, whereas the table varieties contained a larger proportion of the relatively unusual viroid profile of GV-1, -2, and -3. An unexpected divergence of four viroid profiles emerged in the rootstock species. These profiles were 1) Gv-1, -2, and -3, 2) GV-1 plus GV-3, 3) GV-3, and 4) viroid-free.
Impacts
(N/A)
Publications
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Progress 01/01/89 to 12/30/89
Outputs
Nucleic acid extracts from citrons (Citrus medica cv. Etrog) displaying mild andmoderate symptoms associated with the exocortis disease were analyzed by sequential PAGE which revealed the presence of several viroids. Citrus viroids were characterized by the physical parameters of electrophoretic mobility, chromatography on CF-11 cellulose and hybridization to cDNA probes. These characteristic properties combined with biological distinctions in the host range and symptoms expression suggested a scheme for the organization of the citrus viroids into five major groups. Highly purified citrus cachexia viroid (CCaV) was obtained by CF-11 cellulose chromatography. When tested against a broad range of other citrus viroids, the CCaV cDNA hybridized to only one, CV-IIa, which has been identified as the casual agent of a mild form of the citrus exocortis disease. These two viroids have been classified by bioassay reactions as the casual agents of two distinct citrus diseases,
an "exocortis-like" syndrome and cachexia. An organelle-free fraction which synthesized citrus exocortis viroid (CEV) RNA was prepared from nuclei-rich smples taken from CEV-infected Gynura aurantiaca D.C. leaf tissue. Sucrose gradient sedimentation analysis of the sarkosyl supernatant (SSN) components before and after the RNA synthesis showed that the bulk of the progeny remained associated with the viroid complex which sedimented faster than viroid RNA.
Impacts
(N/A)
Publications
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Progress 01/01/88 to 12/30/88
Outputs
Recent evidence indicates an almost ubiquitous occurrence of grapevine viroids in California. Of fundamental importance is an understanding of how this general distribution of grapevine viroids has been accomplished. GV(subscript 1), GV(subscript 2) and GV(subscript 3) were demonstrated to be transmissible into Emperor seedlings and viroid-free, shoot tip cultured Cabernet Sauvignon. A survey of samples taken from the rootstock collection at UCD detected at least one grapevine viroid in all varieties tested. Citron tissue infected with severe isolates of cachexia, known to be free of all other citrus diseases and to induce a strong reaction in Parson's special mandarin, contained a new species of viroid RNA. A viroid RNA of about 300 nucleotides, designated as the putative citrus cachexia viroid (CCaV), was detected. No homology to either citrus exocortis viroid (CEV) or CV-I was observed with hybridization of CCaV to cDNA made to CEV and CV-I. The circular form of
CCaV was isolated from denaturing gels and transmitted to healthy citron. Secondary inoculations to Parson's Special mandarin from these citron sources confirmed the new viroid as the cachexia disease agent. Citrons inoculated with different field sources of the exocortis disease, displayed a variety of symptoms. Nucleic acid preparations were analyzed by sequential polyacrylamide gel electrophoresis.
Impacts
(N/A)
Publications
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Progress 01/01/87 to 12/30/87
Outputs
Rooted cuttings of Etrog citron (Citrus medica) were inoculated with the citrus exocortis viroid (CEV). Floral morphology was abnormal from that of healthy plants in that the corolla failed to open. Fruits that developed from these flowers were characterized by a ringed constriction resembling an asymmetrical "hourglass" that contained a high percentage of seeds that aborted and lacked viability. A survey of sample grapevines of both commercial importance and historical interest has been made for the presence of viroid-like RNAs. Three viroid-like RNAs, designated grapevine viroid 1, 2, and 3 (GV(1), GV(2), GV(3)), ranging in size between 300 and 370 nucleotides, and displaying both circular and linear forms were detected by polyacrylamide gel electrophoresis. All of the 24 vines tested contained at least one of the viroid-like RNAs. The largest, GV(1), and the smallest, GV(3), were the most widely distributed. GV(2) was identified in only four of the vines. No
definitive correlation could be made between the viroid-like RNAs and any of the well-known grape diseases. A series of viroids and viroid-like RNAs extracted from citron (Citrus medica), grapevine Vitis vinifera), and avocado (Persea americanum) displayed differential binding capacities to cellulose in the presence of ethanol. This nonionic interaction was influenced by the presence of magnesium ions suggesting variations in conformation among the viroid RNAs and corresponding differences in reactivity with cellulose.
Impacts
(N/A)
Publications
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Progress 01/01/86 to 12/30/86
Outputs
The isolated walls from citrus exocortis viroid (CEV) infected cells have a higher proportion of arabinosyl and galactosyl residues and a lower proportion of xylosyl, mannosyl and glucosyl residues than do corresponding walls from healthy cells. Three kinds of arabinosyl residues and hydroxyproline, indicators of wall extension, were consistently higher in the walls of CEV infected tomato cells. A greater content of 3-linked glucosyl residues were detected in the walls of CEV infected cells suggesting that the accumulation of B-1,3-glucans may contribute to the inhibition in expansive cell growth. Three additional viroid RNA species smaller than CEV have been identified in citron which display mild symptoms of the exocortis disease. Hybridization studies with CEV-cDNA indicates no significant homology between CEV and the three viroids which also failed to replicate in the preferred herbaceous host for CEV, Gynura aurantiaca. A discontinuous-pH polyacrylamide gel
electrophoresis system has been described which enhances the separation of low molecular weight circular and linear RNA molecules by increasing the pH differential between the gel and the running buffer. The conditions of electrophoresis in low salt and 8 M urea also permit the effective transfer of RNA molecules directly to nylon-based membranes without additional denaturation treatment.
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Progress 01/01/85 to 12/30/85
Outputs
Citrus exocortis (CEV) replication is sustained in tomato cell suspension cultures. Incorporation of P by pulse-labelling or continuous feeding of CEV-containing cells demonstrated the viroid to be a persistent and well-regulated component of the host nucleic acid profile. As the suspension cells approached stationary phase and senescence in the growth cycle, the relative proportion of CEV molecules to host RNA increased. Cells were grown in the presence of specific inhibitors to further examine the coordination between host nucleic acid and CEV synthesis. Actinomycin D in low concentrations (1 mu g/ml) inhibited about 80% of host rRNA synthesis, but did not affect CEV replication. Analysis by polyacrylamide gel electrophoresis of nucleic acid preparations, obtained from several varieties of grapevine by a procedure designed to isolate and purify viroids, revealed the presence of RNA species with characteristic physical properties of viroids. Under non-denaturing
conditions, a band with a mobility faster than that of citrus exocortis viroid (CEV) was detected, and under denaturing conditions two bands were observed, one co-migrating with the circular forms of CEV and a second migrating faster than the linear forms of CEV. Some grapevine preparations were infective for Gynura aurantiaca, inducing symptoms similar to those caused by CEV, and the appearance of an RNA which had the same mobility as CEV in electrophoretic systems and hybridized with cDNA to CEV.
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Progress 01/01/84 to 12/30/84
Outputs
A pathogenic RNA with a molecular weight of about 105,000 daltons has been detected in extracts from citron (Citrus medica) by polyacrylamide gel electrophoresis. Under denaturing conditions two bands can be observed which migrate faster respectively than the circular and linear molecules of the citrus exocortis viroid (CEV). Leaf curling symptoms in citron similar to those produced by CEV are favored by high temperature and high light intensity . No evidence for cross-protection in citron was observed when CEV and a number of mild exocortis isolates were tested. The new pathogenic RNA was readily transmitted to citron by mechanical inoculation but not to known herbaceous hosts of CEV. Therefore, these properties suggest that the isolate may be a new viroid which we have named, citron variable viroid (CVaV) because of the irregular appearance of leaf curling symptoms in citron. This discovery raises a question concerning the practical implication for the efficacy
of citron and Gynura Aurantiaca as indicator plants for CEV and viroid disease of citrus in general.
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Progress 01/01/83 to 12/30/83
Outputs
Treatment of nucleic acid preparations from citrus exocortis viroid (CEV) infected tissues with CayRG, CoyRG, MnyRG, and ZnyRG results in rapid precipitation of all large RNA molecules and selective precipitation of low-molecular-weight RNA species. Analysis of the viroid-RNA by polyacrylamide gel electrophoresis is greatly enhanced in the absence of any additinal treatment with 2M LiCl. The ratio of circular to linear molecules as well as the relative infectivity of MnyRG treated viroid preparations remain unaffected. Cell-free synthesis of CEV in nuclei-rich preparations from infected Gynura aurantiaca was found to be optimum at 18-24c. Incubation of reaction mixtures at higher temperatures (30-36C) resulted in an increase of CEV linear molecules and the recovery of incomplete or nicked newly synthesized RNA species. Although the MgyRG ion optimum for CEV synthesis paralleled that of total [DTyP] CMP incorporation, the response to Mny+ with a pronounced reduction (80%)
in 5mM MyRG, CEV synthesis was maintained at a near optimum level in 1-15 mM MnyRG. Inhibition of alpha-amanitin-sensitive CEV synthesis in 200 mM (NH(4)(2)SO(4) resembles the reaction to similar conditions of RNA polymerase II on a free nucleic acid template. However, detection of trace levels of alpha-amantin-resistant CEV synthesis activity inhibited by low (NH(4))(2)SO(4) concentrations (25mM) suggests also the possible involvement of RNA polymerase I-like activity.
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Progress 01/01/82 to 12/30/82
Outputs
Cell and protoplast-derived callus and suspension cultures from healthy and citrus exocortis viroid (CEV)-infected tomato tissue have been established. The growth rates of CEV-containing cells is not affected by the presence of the viroid. In addition, the viroid containing cells demonstrate a higher temperature tolerance for growth. The cell wall structure of infected tissue apparently differs from healthy cells as suggested by the differential release of protoplasts and the increased yields of protoplasts from infected cells in the presence of Beta-1,3-glucanase. This biochemical distinction also persists in cell suspension cultures. The relationship between the cell wall anomaly and the increased frequency of plasmalemmasomes in CEV-containing callus cells is discussed. Partially purified nuclei from citrus exocortis viroid (CEV)-infected Gynura aurantiaca are able to synthesize linear and circular viroid molecules. Pretreatment of the nuclei with actinomycin D
or deoxyribonuclease did not affect viroid synthesis, whereas the synthesis of other cellular RNAs was severely reduced. These observations support the essential role of CEV complementary RNA sequences in viroid replication. However, when Alpha-amanitin was included in this In Vitro synthesis system, CEV replication was markedly reduced by concentrations of 10 nM or greater. Taken together, these data support the proposition that viroid synthesis is catalyzed by a DNA-dependent RNA polymerase acting on a RNA template.
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Progress 01/01/81 to 12/30/81
Outputs
The process of pathogenesis accompanying viroid infection offers a unique systemfor investigating the interactions of a host cell with a minimal infectious molecule. Since symptom expression mimics a hormonal imbalance response, leaf discs, and stem segments as well as callus cultures from healthy and citrus exocortis viroid (CEV) infected plants of a hybrid of Lycopersicon esculentum and Lycopersicon peruvianum were cultured in vito under different hormone regimes. The differences in response observed when the media was supplemented with auxins, alone or in combinations with cytokinins, suggest that the inability of CEV-infected cells to respond to auxins might be involved in the development of the pathogenic syndrome caused by the viroid. Of the five small RNA's unique to extracts purified from tissue of avocado infected with the avocado sunblotch viroid (ASV), the 70,000 dalton, ASV(5), was strongly implicated as the infectious agent of the disease. ASV-RNAs
were monitored in symptomatic and asymptomatic less carrier host. Only symptomatic tissues contained detectable concentrations of ASV(1)-ASV(5) RNAs, whereas, ASV(5) was present in relatively high concentrations in all tissues of only the symptomless carrier. The complete ASV(1)-ASV(5) complement was not seen in asymptomatic hosts tissue. It remains unclear whether these small RNAs associated with the sunblotch diseases are viroid-specified or simply host-induced species.
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Progress 01/01/80 to 12/30/80
Outputs
The definition of the functional significance of citrus exocortis viroid (CEV) complementary RNA sequences as intermediary molecules of viroid synthesis and/or pathogenesis is of fundamenal importance to understanding viroid disease. The absence of inhibition of viroid synthesis by Actinomycin D concentration which inhibited 5S RNA synthesis by 80% supports the primary role of CEV complementary RNA as a template for viroid replication. Partial characterization of viroid complementary sequences indicates molecular heterogeneity which might suggest that these viroid-related species may be involved in pathogenesis. Efforts on the purification and characterization of the scrapie disease agent in hamsters with the posibility of extending the biological significance of viroids have demonstrated the importance of a protein moiety for infectivity. The scrapie agent therefore now appears to be comprised of DNA-protein complex.
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Progress 01/01/79 to 12/30/79
Outputs
Molecular hybridization with in vitro labelled citrus exocortis viroid (CEV) RNAhas been used to survey nucleic acid preparations from infected plants for viroid complementary molecules. The characterization of complementary sequences indicated the presence of viroid complementary-RNA which may function as a template for viroid synthesis or pathogenesis. In an attempt to extend the viroid model to other biological systems, the causal agent of a transmissible encephalopathy (scrapie) has been investigated. On the basis of deductions made from infectivity data, the electrophoretic mobility of the minimal scrapie infectious unit is characteristic of a low molecular weight DNA which suggests that a viroid-like molecule may be critical to infectivity of this animal disease agent.
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Progress 01/01/78 to 12/30/78
Outputs
Molecular hybridization with 1 2 5I-labeled citrus exocortis viroid (CEV) RNA demonstrated the presence of complementary RNA sequences in infected Gynura aurantiaca and tomato. The complementary RNA which forms RNA-RNA duplex molecules with CEV is detected in nuclear and soluble subcellular fractions. The presence of CEV in tumors induced by Agrobacterium tumefaciens in CEV-infected tomato suggest a similarity of neoplastic cells with apex tissue. The CEV-P, protein has been partially purified and characterized. The biological function of this protein which accumulates as a result of viroid infection remains unclear.
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Progress 01/01/77 to 12/30/77
Outputs
Current research is continuing in three areas. The mechanism and agent of scrapie infection in rodents is an important area of concern. Recent work has shown that activity may be selectively precipitated with ammonium sulfate. Further fractionation of this sample is now being undertaken. The molecular mechanism of CEV replication in infected cells of tomato and Gynura is being investigated by attempting to culture CEV in transformed cells of tomato and selective hybridization PG-21I-CEV to specific cell fractions.
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Progress 01/01/76 to 12/30/76
Outputs
A novel class of pathogenic RNA's, the viroids, isolated from diseased plant tissues has been characterized as low molecular weight (difference 101 daltons) highly-structured molecules that exhibit unusual thermal stability and high specific activity. A nuclear site of synthesis is suggested by the recovery of viroid RNA as well as the detection of DNA complementary to the citrus exocortisviroid. Aberrations of the plasma membrane which are correlated with the presence of the viroid RNA constitute the only subcellular pathology. Since no evidence exists for the translation of the viroid, pathogenesis may result from the action of the viroid as a direct regulator of genetic expression of the host. Extension of viroid diseases to animal systems have not yet been reported, however, remain a distinct possibility.
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Progress 01/01/75 to 12/30/75
Outputs
The physical properties of the pathogenic RNA (Viroid) from exocortis disease ofcitrus suggest a G-C rich T-RNA-like structure on the basis of compontional, hyperchromicity & NMR analysis. The viroid is associated with a nuclei-rich preparation as well as a plasmamembrane-like component of of the endomenbrane system suggesting a possible phase of synthesis at the nucleus level & a site ofaccumulation (or pathological lesion). This dichotomy is supported by the evidence of a DNA complementary to viroid RAN localized in the nucleus as well as the prescence of cytopathic structures at the plasma membrane.
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Progress 01/01/74 to 12/30/74
Outputs
The physical properties of the pathogenic RNA (VIROID) from exocortis diseases indicated a nucleotide composition of CMP, 29.4%; AMP, 21.5% GMP 28.8% and UMP, 19.9%. The per cent hyperchromicity (22%) resembled a tRNA-like molecule and not dsRNA, while the Tm (52) in 0.1 SSC was greater than the pure tRNA species tested. We have also introduced low field NMR spectroscopy as a new technique in the study of pathogenic RNA's of unknown sequences. The ability of this RNA to serve as a messenger molecule, or as an amino acid acceptor was compared withthat of other small RNA molecules from plant viruses. While the viral RNA's showed both of these activities, the citrus exocortis viroid had neither, contrasting the nature of this pathogen with that of plant viruses.
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