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H. Brent Pemberton, Kevin Ong, Mark Windham, Jennifer Olson, and David H. Byrne

Rose rosette disease is incited by a negative-sense RNA virus (genus Emaravirus ), which is vectored by a wind-dispersed eriophyid mite ( P. fructiphilus ) ( Di Bello et al., 2015a ; Laney et al., 2011 ). Symptoms on roses include witches broom

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Hai-nan Liu, Jian-rong Feng, Xiao-fang Liu, Wen-hui Li, Wen-juan Lv, and Ming Luo

Biotechnology Research Center at Southwest University, Chongqing, China. Tissue culture seedlings of wild-type tobacco were used to confirm the transformation of the RNAi expression vector. Extraction of RNA and RT-PCR for SFB Pollen RNA was extracted using

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K.S. Ling, C.A. Clark, C. Kokkinos, J. R. Bohac, S.S. Hurtt, R. L. Jarret, and A. G. Gillaspie

Sweet potato virus disease (SPVD) is the most devastating virus disease on sweetpotato [Ipomoea batatas (L.) Lam] world wide, especially in East Africa. However, weather it is present in the U.S. is unknown. SPVD is caused by co-infection of sweetpotato feathery mottle virus (SPFMV) and sweetpotato chlorotic stunt virus (SPCSV). Presence of two other potyviruses, sweetpotato virus G (SPVG) and Ipomoea vein mosaic virus (IVMV) has also been confirmed in the U.S. Sweet potato leaf curl virus (SPLCV), a whitefly (Bemisia tabaci) transmitted Begomovirus, also has the potential to spread to commercial sweetpotato fields and poses a great threat to the sweetpotato industry. The U.S. collection of sweetpotato germplasm contains about 700 genotypes or breeding lines introduced from over 20 different countries. Newly introduced sweetpotato germplasm from foreign sources are routinely screened for major viruses with serology and graft-transmission onto indicator plants (Ipomoea setosa). However, a large portion of this collection including heirloom cultivars or old breeding materials has not been systemically screened for these major sweetpotato viruses. In this study, a total of 69 so-called heirloom sweetpotato PI accessions were evaluated for their virus status. We used Real-time PCR to detect five sweetpotato viruses, including four RNA viruses (SPCSV, SPFMV, SPVG, and IVMV) and one DNA virus (SPLCV). A multiplex Real-time RT-PCR system was developed to detect three RNA viruses (SPFMV, SPVG, and IVMV). Preliminary data indicated that about 15% of these heirloom sweetpotato germplasm carried at least one of these viruses tested. Details on virus infection status will be presented.

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Rebecca Grumet

One of the first major successes in the genetic engineering of useful traits into plants has been the engineering of virus resistance. The first example of genetically-engineered virus resistance was published in 1986, since then there have been more than 50 reports of genetically engineered plant virus resistance. These examples span a range of virus types, a variety of plant species, and have utilized several different types of genes. A unique feature of the genetically-engineered virus resistance is that the resistance genes came from the virus itself, rather than the host plant. Most examples have utilized coat protein genes, but more recently, replicase-derived genes have proved highly effective. Other strategies include the use of antisense or sense-defective sequences, and satellite or defective interfering RNAs. This talk will provide an overview of the different approaches, possible mechanisms, the crops and viruses to which they have been applied, and progress toward commercial applications.

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K. Heuss-La Rosa, R. Hammond, J.M. Crosslin, C. Hazel, and F. Hammerschlag

In vitro micrografting was tested as a technique for inoculating peach [Prunus persica (L.) Batsch] 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. The infected shoots served as graft bases and the source of 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 digoxigenin-labeled cRNA probed derived from PNRSV RNA 3. Rates of successful grafting were 55% and 73% in three trials and PNRSV was found in all tips analyzed. Virus concentrations approximated those found in the source shoots, suggesting that this method should be useful for screening transformed peach shoots for coat protein-mediated resistance to PNRSV.

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Kenneth C. Eastwell and Gabriel B. Kalmar

In certain cultivars of cowpea [Vigna unguiculata (L.) Walp.] that are operationally immune to cowpea mosaic virus strain SB (CPMV), coinoculation of CPMV with cowpea severe mosaic virus strain DG (CPSMV) reduces severity and delays expression of symptoms normally induced by CPSMV alone. In cultivars susceptible to both viruses, coinoculation delays development of symptoms in response to CPSMV. Using monoclonal antibodies for serological assays and virus-specific RNA probes for hybridization, it is demonstrated that the presence of CPMV in the inoculum yields a concomitant delay in the synthesis of CPSMV coat protein and replication of CPSMV RNA and restricts the transport of CPSMV out of infection centres. Only bottom component of CPMV containing RNA1 is required to offer protection against CPSMV. Destroying the integrity of CPMV RNA eliminates its protective capability. In cowpea cultivars that are operationally immune to CPMV, the presence of CPSMV in the inoculum is unable to compensate for events of CPMV replication that are inhibited. The lack of complementation suggests a high degree of specificity in the replication of these two comoviruses.

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M. Zeidan, Noga Sikron, J. Cohen, and A. Gera

Petunia vein clearing virus (PVCV), a possible member of the caulimovirus group, was detected in several cultivars of vegetatively propagated petunias (Petunia ×hybrida Hort. Volm.-Andr.) grown in commercial nurseries. Leaf dip preparations and ultrathin sections of leaf tissue were analyzed by transmission electron microscopy (TEM). Spherical virus particles, 45-50 nm in diameter, were observed in samples taken from symptomatic petunia plants. The virus was purified and a polyclonal antiserum was prepared. In immuno-specific electron microscopy (ISEM), the PVCV antiserum-treated samples reacted with a distinct decoration on the virus suspect particles. A polymerase chain reaction (PCR)-based assay was used to detect PVCV in total nucleic acid extracts derived from infected petunia plants. Two primer pairs were designed to flank a 736-base-pair sequence located in the RNA-dependent RNA polymerase gene of the PVCV genome. A DNA fragment of predicted size was visualized in agarose gels. The authenticity of the amplified DNA fragment was confirmed by restriction analysis and by hybridization with the virus-specific PVCV DNA probe. The virus could be detected efficiently in high dilutions of sap extracted from infected petunia plants.

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Kathleen Heuss, Qingzhong Liu, Rosemarie Hammond, and Freddi Hammerschlag

As part of our program to develop transgenic peach cultivars with improved disease resistance, we showed that grafting of in vitro cultured `Suncrest' peach [Prunus persica (L.) Batsch] tips `onto decapitated stems of Prunus necrotic ringspot virus (PNRSV) infected `Suncrest' shoot cultures, resulted in consistent transfer of virus across grafts as demonstrated by RNA hybridization analysis, suggesting that such a system could be useful for measuring resistance to PNRSV in peach shoot cultures. We have extended these studies to include grafts of `Springcrest' and `Nemaguard' test tips onto `Suncrest' stocks. RNA hybridization analysis showed that PNRSV persists in shoot cultures for 18 months after initiation from PNRSV-infected `Suncrest' trees and after 16 weeks of treatment of 4°C in the dark, suggesting that a supply of infected shoot cultures could be maintained for repeated use. Graft success rates for grafts of `Springcrest' onto `Suncrest' and `Nemaguard' onto `Suncrest', equaled or exceeded success rates for `Suncrest' onto `Suncrest'. Virus was transmitted from infected stocks into `Suncrest', `Springcrest', and `Nemaguard' test tips by 2 weeks in most successful micrografts. There was no significant difference in the virus concentrations among the three scions at 2, 4, and 6 weeks after grafting, suggesting that there is equal efficacy of virus transfer through grafts from `Suncrest' to the three cultivars, and that no differences in resistance to PNRSV exist among these cultivars.

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K. Heuss, Q. Liu, F.A. Hammerschlag, and R.W. Hammond

As part of a program to develop transgenic peach (Prunus persica L. Batsch) cultivars with resistance to Prunus necrotic ringspot virus (PNRSV), we are testing a system for measuring virus in peach shoot cultures. Micrografting in vitro is used for inoculation and slot-blot hybridization, with a digoxigenin (DIG)-labeled cRNA probe complementary to the 5′ open reading frame (ORF) of PNRSV RNA 3, for detection. In this study, we investigated whether infected shoots maintain virus infection over long periods of culture at 4 °C and if PNRSV-infected `Suncrest' shoot cultures can serve as graft bases to transmit virus equally well into cultivars Nemaguard, Springcrest, and Suncrest. The results of RNA hybridization analysis showed that virus was present in extracts of leaf samples from 2-year-old PNRSV-infected `Suncrest' shoots that had been subjected to varying lengths of incubation at 4 °C in the dark, suggesting that infected shoots can be maintained for repeated use. Rates of graft success were higher in heterografts between `Suncrest' bases and tips of `Springcrest' or `Nemaguard' than in autografts between `Suncrest' and `Suncrest', and there was equal efficacy of graft inoculation from `Suncrest' into these three cultivars.

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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).