Viral satellite RNA associated with cucumber mosaic virus (CMV) is know to modulate CMV symptomology. Virulent CMV associated RNA 5 (CARNA 5) satellites may intensify crop disease. Naturally occurring variants of these satellites, however, attenuate CMV symptoms. Satellite transgenic tomato plants expressing the S-CARNA 5 or 1-CARNA 5 ameliorating forms of the satellite were evaluated under simulated CMV epidemic conditions in USDA–APHIS approved field trials. Trials conducted at Beltsville, Md., in 1994 and 1995 demonstrated that CMV can be effectively controlled under field conditions in satellite transgenic plants. Yields of transgenic lines infected with CMV were 50%–65% greater than that of non-transgenic infected controls. Yields of noninfected transgenic lines ranged from 5% greater than, to 33% less than, noninfected nontransgenic controls. Expression of CARNA 5 in inoculated transgenic plants greatly reduced CMV foliar symptoms and virus titers when compared to inoculated control plants. Levels of CARNA 5 were detected at varying levels in infected transgenic plants throughout the growing season. Virus or satellite was not detected in samples collected from tomato border plants and weeds growing inside and outside a nonhost crop border surrounding the test plot. Field tests conducted in 1996 will evaluate transgenic tomato plants with a double construct coding for the CMV coat protein gene and 1-CARNA 5 satellite.
John R. Stommel, Marie E. Tousignant, Thanda Wai, and Jacobus M. Kaper
A.Q. Villordon, C.A. Clark, R.A. Valverde, R.L. Jarret, and D.R. LaBonte
Previous work by our group has detected the presence of a heterogeneous population of Ty1-copia-like reverse transcriptase retrotransposon sequences in the sweetpotato genome. Recently, we detected the presence of putatively active Ty1-copia-like reverse transcriptase sequences from a virus-infected `Beauregard' sweetpotato clone. In the current study, we report the differential detection of putatively stress-activated sequences in clones from seedling 91-189. The clones were infected with different combinations of virus isolates followed by extraction of leaf RNA samples at three sampling dates (weeks 2, 4, and 6) after inoculation. After repeated DNAse treatments to eliminate contaminating DNA, the RNA samples were subjected to first strand cDNA synthesis using random decamer primers followed by PCR analysis utilizing Ty1-copia reverse transcriptase-specific primers. Through this approach, we detected amplified fragments within the expected size range (280-300 bp) from clones infected with isolates of sweetpotato leaf curl (SPLC) and feathery mottle viruses (FMV) (week 2 and 6) and FMV (week 4). We were unable to detect PCR products from the noninfected clones or the other infected samples. The data suggests that specific viruses may be involved in the expression of these Ty1-copia-related reverse transcriptase sequences. It also appears that sampling at various dates is necessary to detect putative activity over time. This preliminary information is essential before proceeding to the construction and screening of cDNA libraries to isolate and fully characterize the putatively active sweetpotato Ty1-copia-like retrotransposon sequences. Through the partial or complete characterization of sweetpotato Ty1-copia elements, sequences that correspond to cis-regulatory element(s) can be identified and further studied for their roles in responding to specific stress factors.
Binoy Babu, Gary Knox, Mathews L. Paret, and Francisco M. Ochoa-Corona
single-stranded negative-sense RNA virus ( Laney et al., 2011 ). The virus is transmitted by the eriophyid mite species Phyllocoptes fructiphilus ( Amrine et al., 1988 ; Laney et al., 2011 ) and by grafting ( Amrine et al., 1988 ). The mites do not fly
Sara Spiegel, Dan Thompson, Aniko Varga, and Delano James
An apple chlorotic leaf spot virus (ACLSV) isolate was detected by TAS-ELISA and RT-PCR in an ornamental dwarf flowering almond (Prunus glandulosa Thunb.). This plant, maintained at the Centre for Plant Health, Sidney, B.C., Canada, has been showing transient leaf symptoms during the spring seasons. A 390-bp fragment and a 1,350-bp product, in the RNA polymerase and the coat protein viral coding regions, respectively, were amplified by RT-PCR from the infected plant. A sequence comparison of the 390-bp fragment of this ACLSV isolate (designated as AL1292) with other published isolates, revealed a similarity of 81% to 84% at the nucleotide level and 88% to 100% at the amino acid level. In contrast to other ACLSV isolates, AL1292 has an exceptionally narrow range of experimental herbaceous and woody hosts, as determined by mechanical and graft inoculation assays. These standard bioassays may not be effective for the detection of the AL1292 isolate because of its limited host range. The results we report in this paper confirm P. glandulosa as a natural host of this virus. Currently it is not known how ACLSV is spread, other than by bud-grafting and possibly by root grafts. The use of virus-tested source plants for the preparation of planting material will minimize its spread.
Kisung Ko, John L. Norelli, Jean-Paul Reynoird, Herb S. Aldwinckle, and Susan K. Brown
Genes encoding lysozyme (T4L) from T4 bacteriophage and attacin E (attE) from Hyalophora cecropia were used, either singly or in combination, to construct plant binary vectors, pLDB15, p35SAMVT4, and pPin2Att35SAMVT4, respectively, for Agrobacterium-mediated transformation of `Galaxy' apple, to enhance resistance to Erwinia amylovora. In these plasmids, the T4L gene was controlled by the cauliflower mosaic virus 35S promoter with duplicated upstream domain and the untranslated leader sequence of alfalfa mosaic virus RNA 4, and the attE gene was controlled by the potato proteinase inhibitor II (Pin2) promoter. All transgenic lines were screened by polymerase chain reaction (PCR) for T4L and attE genes, and a double-antibody sandwich enzyme-linked immunosorbent assay for neomycin phosphotransferase II. Amplification of T4L and attE genes was observed in reverse transcriptase-PCR, indicating that these genes were transcribed in all tested transgenic lines containing each gene. The attacin protein was detected in all attE transgenic lines. The expression of attE under the Pin2 promoter was constitutive but higher levels of expression were observed after mechanical wounding. Some T4L or attE transgenic lines had significant disease reduction compared to nontransgenic `Galaxy'. However, transgenic lines containing both attE and T4L genes were not significantly more resistant than nontransgenic `Galaxy', indicating that there was no in planta synergy between attE and T4L with respect to resistance to E. amylovora.
Richard Manshardt and Dennis Gonsalves
Transgenic papaya line 55-1 with resistance to papaya ringspot virus (PRSV) originated in 1989 by particle bombardment of cultivar Sunset with the coat protein gene (cp) of mild mutant Hawaii PRSV strain HA 5-1. Hemizygous (+/cp) R0 clones of 55-1 displayed resistance to the virulent Hawaii HA strain in greenhouse tests in New York in 1991 and to local strains in a field trial in Hawaii from 1992 to 1994. In the R1 generation produced by crossing the pistillate R0 55-1 with `Sunset', up to 50% of the hemizygous transgenic segregants were susceptible to a local Oahu PRSV strain when inoculated as seedlings but not as mature plants. Similar inoculation experiments in New York showed that hemizygous R1 transgenics were susceptible in differing degrees to PRSV strains from regions other than Hawaii. Homozygous (cp/cp) R2, R3, and R4 populations planted in various locations in Hawaii since 1994 have consistently demonstrated high-level resistance to local strains at all stages of development. When inoculated in New York with eight non-Hawaii PRSV strains, homozygous R3 seedlings were resistant to all but a Thai strain. Transgenic resistance is the result of a complex interaction involving the stage of plant development, transgene dosage, the degree of homology between transgene and challenge virus, and environmental variables. Papaya plants transformed with nontranslatable versions of various cp genes are also highly resistant to PRSV, indicating that the resistance mechanism operates at the RNA level. No loss of resistance due to the appearance of resistance-breaking virus strains or to transgene inactivation has been noted thus far.
Misaki Ishibashi, Takeshi Nabe, Yoko Nitta, and Yuichi Uno
.L. 2011 Evaluation of different RNA-extraction kits for sensitive detection of Hepatitis A virus in strawberry samples Food Microbiol. 28 38 42 Boom, R. Sol, C.J.A. Salimans, M.M.M. Jansen, C.L. Wertheim van Dillen, P.M.E. van der Noordaa, J. 1990 Rapid
Beatrice Nesi, Debora Trinchello, Sara Lazzereschi, Antonio Grassotti, and Barbara Ruffoni
Lilies have become the most important floral crop in Italy in the last 20 years and this has created a need to produce high-quality, healthy bulbs free from virus. More than one hundred million lily bulbs are imported from abroad every year, so the
Cecilia E. McGregor, Douglas W. Miano, Don R. LaBonte, Mary Hoy, Chris A. Clark, and Guilherme J.M. Rosa
and quantitative real-time polymerase chain reaction (Q-RT-PCR) for virus titer determination. Leaf material was ground to a fine powder in liquid nitrogen with a mortar and pestle and ≈50 mg was used to extract total RNA using the RNeasy Mini Kit
Sahar Eid, Keri L. Druffel, Dayle E. Saar, and Hanu R. Pappu
Register of Dahlia Names, 1969 et seq. ). There are more than a dozen viruses that infect dahlia ( Albouy, 1995 ). All of these viruses are RNA viruses except one DNA virus, Dahlia mosaic caulimovirus (DMV). DMV is widely prevalent in cultivated dahlias