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Chad Finn and Robert Martin

Cuttings from Rubus ursinus Cham. & Schlecht, the trailing blackberry, were collected in Oregon, Washington, and British Columbia from 21 sites. The cuttings were rooted and placed in pots in the greenhouse. After the plants began to grow, leaves were harvested for ELISA testing using standard procedures. Each sample represented three clones from a site. Plants from 18 sites were represented by five samples and two sites were represented by three samples. None of the samples tested positive for the presence of raspberry bushy dwarf virus or tomato ringspot virus. Forty-four percent of the samples tested positive for tobacco streak virus. Only 33% of the sites on the Pacific coast tested positive for tobacco streak, whereas, 100% of the Cascade Mountain sites and 88% of the sites in the coastal range type environment tested positive. The only site in the Willamette Valley had no positive tests. With one exception, all of the sites that tested negative for the virus were also low elevation sites 0-90 m.

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R. Manshardt, S. Lius, D. Gonsalves, M. Fitch, J. Slightom, and J. Sanford

Transgenic papaya lines carrying the coat protein gene (CP) of papaya ringspot virus (PRV) strain HA 5-1 display PRV reactions ranging from complete susceptibility (39-3 & 39-4), to slight delay in onset of symptoms (39-1) and attenuation of symptoms (60-3), to high-level resistance (55-1, 63-1). Normal Mendelian segregation of transgene expression was lost in R1 of 39-3 and 39-4, and inbred R1 60-3 gave an aberrant 1:1 ratio. R0 55-1 plants were resistant in the field (Hawaii) for 2 years following manual and/or aphid inoculation, and the high-level resistance remained stable in the R1 after repeated manual inoculations in the greenhouse and graft inoculation for up to 1 year (Cornell). However, inoculation with PRV HA-Oahu strain produced symptoms in some plants at Cornell (9% after 6 weeks) and in Hawaii (50% after 1 year). Two 55-1 and one 60-3 plant subsequently underwent remission of symptoms and became ELISA-negative (Hawaii). Transmission of PRV isolates from symptomatic 55-1 plants to other CP+ 55-1 bioassay plants was unsuccessful.

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Wendy Wagoner, J. Stamp, H. Matthews, J. Kellogg, and R. Bestwick

Ethylene is a known causal factor in the decay and senescence of fruits and vegetables. The aim of the present study was to incorporate a gene for control of ethylene biosynthesis in order to prevent or delay the senescence of the cauliflower curds. We first developed a reproducible transformation system using marker genes for beta glucuronidase (GUS) and antibiotic resistance. Brassica oleraceae L. var. botrytis was transformed by inoculating hypocotyl explants with the Agrobacterium tumefaciens strains C58 or EHA101 containing plasmids pAG5110, pAG5420, or pAG5520. The plasmid pAG5110 contains the genes for neomycin phosphotransferase II (NPTII) and GUS. The plasmids pAG5420 and pAG5520 contain a functional gene for S-adenosylmethionine hydrolase (SAMase) under an ethylene or wound inducible promoter, respectively. Hypocotyl explants were screened on regeneration medium with kanamycin for selection of transformants. Shoot regeneration occured within 4-6 weeks and morphologically normal plants developed within 3-4 months. The transgenic nature of the plants was confirmed by histochemical GUS assay, an ELISA based NPTII assay and Southern blot analysis. Transgenic plants outplanted in the greenhouse are being evaluated and selfed to study expression and inheritance pattern of the introduced trait.

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E. Bruton Strange, Todd C. Wehner, and Zvezdana Pesic-Van Esbroeck

Watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai] is a major crop in the southern U.S., where the most important virus diseases are papaya ringspot virus (PRSV), watermelon mosaic virus-2, and zucchini yellow mosaic. The most economical control of virus diseases of watermelon is probably through genetic resistance. Watermelon has not been screened extensively for resistance to PRSV. The objective of this research was to develop a suitable method for screening watermelons for resistance to PRSV and then to screen the USDA germplasm collection. To date, we have developed an effective method and have nearly completed the screening. Several of the 1283 accessions have shown resistance to the virus. Methods tests involved 10 isolates of PRSV, several watermelon accessions and multiple inoculation procedures. Seedlings were screened in greenhouse flats with six replications per test. Tests were rated visually on a 0 to 9 scale (0 = no damage, 9 = plant dead), as well as with ELISA to detect the presence of virus. The watermelon germplasm collection was screened in four separate runs of 1283 accessions with `Charleston Gray' as the susceptible check. This research will be useful for those interested in effective screening methods, and sources of resistance for development of improved watermelon cultivars.

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C.F. Lunde, M.S. Mehlenbacher, and D.C. Smith

A survey of hazelnut (Corylus avellana L.) genotypes for response to the eastern filbert blight pathogen [Anisogramma anomala (Peck) E. Müller] was performed. Seven varieties were discovered that did not display disease signs or symptoms when subjected to severe inoculation with A. anomala in the greenhouse and assayed for infection. These cultivars are `Closca Molla', `Ratoli', `Yoder #5', `Potomac', `Medium Long', `Grand Traverse' and `Zimmerman'. `Ratoli' and `Closca Molla', both minor varieties from Spain, are superior agronomic types to the resistant cultivar Gasaway, which has been the main resistance source used in the breeding program. Only `Zimmerman' carries the RAPD marker linked to resistance in populations segregating for the `Gasaway' gene. Three populations were created using, `Zimmerman', as the pollen parent in controlled crosses. These populations were inoculated with spores of the pathogen and assayed by indirect ELISA and by observation of canker incidence. Resistant phenotypes make up 84% of the populations, indicating that `Zimmerman' possesses resistance either distinct from or additional to that found in, `Gasaway'. A RAPD marker linked to the resistance gene in crosses with `Gasaway' cosegregates with the resistant phenotype in all three populations (0 cM, 3 cM, 4 cM). Mechanisms to explain the distortion in these populations are discussed. Further studies are required to characterize the mechanism and inheritance resistance in these other clones.

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Wenhao Dai, Bingcheng Sheng, and Zhen Zhang

`Xiao Fang Shi' is a rare, dwarf cultivar of persimmon (Diospyros kaki Linn cv.) recently found north of Shanghai, China. The tree starts to bear fruit at 2 years of age, while standard trees start fruiting at 5 or 6 years of age. Dwarf and standard cultivars have about equal spring shoot growth, but the dwarf cultivar has little fall growth. To determine the mechanisms of dwarfness and early fruiting, enzyme-linked immunosorbant assay (ELISA) was used to analyze the endogenous indoleacetic acid (IAA), gibberellic acid (GA1+3) and abscicic acid (ABA) contents in leaves and shoot tips of dwarf (`Xiao Fang Shi') and standard (`Da Fang Shi' and `Zhu Sha Hong') persimmon. The measurement was done during the entire growing season. The results showed that IAA, GA1+3, and ABA contents were influenced by cultivars, ages of trees, and phenophases. The dwarf cultivar `Xiao Fang Shi' has lower IAA and GA1+3 but higher ABA contents than the two standard cultivars during the growing season. These correlations are especially evident when the fruit is ripening. The correlation coefficiency between contents of IAA and GA1+3 and tree height was 0.9704, while that between ABA content and tree height is –0.9697. The low IAA and GA, and high ABA contents may be responsible for little shoot growth of the dwarf cultivar in the fall.

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Eric W. Mercure, Carol A. Auer, and Mark H. Brand

Tissue proliferation (TP) is characterized primarily by the formation of galls or tumors at the crown of container-grown rhododendrons propagated in vitro. However, TP of Rhododendron `Montego' is observed initially in in vitro shoot cultures and it is characterized by the formation of multiple shoots with small leaves and nodal tumors. The formation of shoots in `Montego' TP (TP+) shoot cultures occurs without the presence of exogenous cytokinin in the medium, unlike normal `Montego' (TP–) shoot cultures, which require cytokinin for shoot growth. Structural studies have shown that tumors are composed of many adventitious buds and parenchyma cells, suggesting that TP is a result of abnormal cytokinin regulation that is controlling tumor and shoot formation. Two approaches are being used to determine if differences in cytokinin concentration and/or metabolism exist between TP+ and TP– shoot cultures. In the first approach, shoot cultures are grown in vitro for 1 week in the presence of tritiated isopentenyladenine (iP). Cytokinin uptake and metabolism are analyzed using HPLC and other analytical methods. Experiments suggest that extensive degradation and N-glucoside conjugation occur in TP+ and TP– shoots, resulting in the removal of most of the exogenous iP. In the second approach, the levels of endogenous cytokinins such as iP, isopentenyladenosine, zeatin, and zeatin riboside, are being measured in TP+ tumors and shoots and in TP– shoots by an ELISA method.

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Kathleen Heuss-LaRosa, Rosemarie Hammond, James M. Crosslin, Christine Hazel', and Freddi A. Hammerschlag

In vitro micrografting was tested as a technique for inoculating peach [Prunus persica (L.) Batsch] shoot cultures with Prunus necrotic ringspot virus (PNRSV). Cultured `Suncrest' shoots derived from a naturally infected tree (as indicated by ELISA testing) maintained virus in vitro, with virus concentrations in growing tips and folded leaves being several times those of fully expanded leaves. Infected shoots served as graft bases and source of the virus. Grafted tips were derived from `Suncrest' trees that had tested negative for the virus. Leaf samples were collected from the tips following grafting and analyzed for the presence of virus by slot-blot hybridization with a (DIG)-labeled cRNA probe derived from PNRSV RNA 3. Rates of successful grafting ranged from 55% to 73% in three trials and PNRSV was found in all tips analyzed. Virus concentrations approximated those found in source shoots, suggesting that in vitro micrografting should be useful for screening transformed peach shoots for coat protein-mediated resistance to PNRSV. Chemical name used: digoxigenin (DIG).

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Richard Hampton

Vectors with specific vector-virus relationships (e.g., aphid, beetle, thrip, nematode) commonly cause short-range dissemination of cowpea viruses. However, viruses that are seed-borne in cowpea can be disseminated around the world in a single year through seed shipments. Likewise, increased world emphasis on germplasm collection and exchange, for development of improved crop cultivars, increases the risk of disseminating seed-borne viruses in germplasm. Seed-borne cowpea viruses that are not reported in the U.S.A., but are apt to occur in Vigna unguiculata from world centers of cowpea origin include COWPEA APHID-BORNE MOSAIC, COWPEA MILD MOTTLE, COWPEA MOSAIC, and COWPEA MOTTLE VIRUSES. All of these viruses were detected by ELISA serology in V. unguiculata seedlots processed as potential germplasm introductions, in collaboration with the government of Denmark. Germplasm-borne viruses, once introduced into breeding programs, may be seed-transmitted directly into breeding progenies, along with genes derived from the germplasm source. Such viruses also may be spread by insect vectors to other breeding lines, and could cause disease outbreaks to nearby commercial cowpea crops.

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M.J. Díez, S. Roselló, F. Nuez, J. Costa, M.S. Catalá, and A. Lacasa

Seedlings of three tomato (Lycopersicon esculentum Mill.) cultivars [`RDD', carrier of the Sw5 gene, which confers resistance to tomato spotted wilt virus (TSWV); `Pitihué', tolerant to the virus; and the susceptible cultivar Rutgers] were placed at the four- to five-leaf stage in cages containing a population of viruliferous thrips (Frankliniella occidentalis Perg.), and remained there for 0, 7, or 15 days. Plants were subsequently transplanted either into the open field or in tunnels protected with a mesh of 14 × 10 threads/cm. Systemic symptoms and number of dead plants were recorded and enzymelinked immunosorbent assays (ELISA) were performed. `Rutgers' exhibited severe systemic symptoms regardless of treatment and a high number of plants died. The level of infected plants remained low when protective measures were applied to seedlings of `Pitihué' and acceptable yields were obtained. In open air cultivation, where seedling infection was severe, <20% of `RDD' plants became infected and high yields were obtained; protected cultivation did not reduce yield. Although the percentage of infected plants was higher when cultivated under mesh, the yield of all three cultivars was greater than in the open field. The environment created under mesh stimulated growth, neutralizing the effect of the infection.