Abbreviations: Cb, carbenicillin; CMV, cucumber mosaic virus; GUS, (β -glucuronidase; Km, kanamycin; MS, Murashige and Skoog NPT II, neomycin phosphotransferase II; NOS, nopaline synthase. We thank Krystal A. Fober for plant care in the greenhouse
Paula P. Chee and Jerry L. Slightom
Cucumber mosaic virus (CMV) is an aphid-transmitted virus that infects snap bean growing regions in New York State and Wisconsin. The core collection of common bean accessions (Phaseolus vulgaris), the complete collection of scarlet runner bean accessions (Phaseolus coccineus) and snap/dry bean cultivars were screened for resistance to CMV. Although variation in foliar symptom expression was observed, no resistance was observed in 93 snap bean and16 dry bean cultivars tested, and only one of the 406 accessions from the core collection (PI 309881) was symptomless. PI 309881 did not have common bean characteristics, and was later identified as a tepary bean (Phaseolus acutifolius) accession based on morphology and PCR-RFLP of chloroplast DNA. Screening of 260 P. coccineus accessions was inaccurate when a visual rating of foliar symptoms was used. It was necessary to determine infection using ELISA and test plant screening with grey zuccini. Using this approach it was determined that 80 P. coccineus accessions were susceptible to CMV; however, the remaining accessions provided possible sources for transfer of CMV resistance to snap bean. Crosses of P. coccineus accessions were made to breeding line 5-593 and backcrossed to 5-593 and snap bean cultivar `Hystyle'. PI 309881 was crossed with ICA Pijao in order to develop interspecific hybrids. Populations were developed from the interspecific crosses/backcrosses and evaluated for CMV resistance using ELISA and visual ratings of foliar symptoms.
Yiping Zhang, Vince Lackney, and Molly Kyle
We report the detection of molecular markers linked to polygenic recessive resistance to cucumber mosaic virus (CMV) in pepper using distributional extreme analysis. A total of 132 mapped tomato genomic, cDNA, and pepper genomic clones from different linkage groups in two existing pepper maps, especially from the ones showing tentative CMVR QTLs, were selected as hybridization probes for Southern blots, in which DNA from the 12 most resistant and 15 most susceptible individuals from a large segregating F2 were probed. Several clones appeared to cosegregate with CMV resistance phenotype. Further analysis is being done to place these markers on existing molecular linkage maps. The precise locations of resistance genes can be defined by examining additional markers within the region. The availability of closely linked DNA markers may facilitate marker-aided selection in pepper CMV resistance breeding programs.
P.B. McGarvey, M.S. Montasser, and J.M. Kaper
Transgenic tomato plants (Lycopersicon esculentum Mill.) expressing cucumber mosaic virus (CMV) satellite RNA fused to a gene for β-glucuronidase were produced using Agrobacterium-mediated transformation. The R1 progeny of self-crossed R0 plants were challenge-inoculated with virion or RNA preparations of CMV or tomato aspermy virus (TAV). The transgenic plants challenged with CMV-1 showed mild disease symptoms in the first 2 weeks postchallenge followed by a decrease in symptoms, resulting in little difference between the transgenic and uninfected control group by the fourth week. Enzyme-linked immunosorbent assay results showed about a 10-fold decrease in virus accumulation in the transgenic plants compared to controls. Tolerance was evident only in plants that contained the recombinant insert and produced mature unit-length satellite RNA after CMV infection. Plants challenged with TAV showed no significant tolerance to virus-induced symptoms.
R.C. Grube, Y. Zhang, B. Huang, and M.M. Kyle
Resistance to cucumber mosaic virus (CMV) in Capsicum from two sources is being transferred into three commercial types (bell, jalapeno, and Anaheim) using a backcross breeding scheme. We have optimized our CMV seedling screening protocol, which involves multiple inoculations beginning at the cotyledon stage with a severe CMV serogroup I isolate. Both sources of resistance, C. annuum `French Perennial' and a C. frutescens accession (BG2814-6), exhibit oligogenic recessive inheritance and share some but not all resistance alleles. Selection for type in the BCF1 generation had no effect on the frequency of resistant individuals in the BCF2 generation. We have determined that it is necessary to self-pollinate every other backcross generation to screen for resistance. Occasionally disease symptoms appear in adult plants that were initially resistant to multiple inoculations at the seedling stage, and we are investigating the correlation between seedling resistance and adult plant resistance. We are also exploring the extent to which the different sources of resistance behave differently as a function of genetic background. Additionally, we are mapping quantitative trait loci (QTLs) for CMV resistance in pepper with the goal of converting RFLP and/or RAPD markers into PCR-based markers to facilitate molecular marker-assisted selection for CMV resistance.
Cowpea cultivars Early Ramshorn and Dixie Queen, reported to be resistant to cucumber mosaic virus (CMV) by Sinclair and Walker in 1955, were reexamined for the existence of individual CMV-resistant genotypes within seedling populations. CMV-inoculated populations of these two cultivars became CMV-infected at rates of 60% and 80%, respectively, as determined by DAS-ELISA. CMV-free plants were grown to maturity, seeds from these sources were planted, and second-generation seedlings were inoculated again with CMV. Rates at which these seedlings became CMV-infected were 10% and 24%, respectively. The complementary 90% and 76% of these populations remained completely free of ELISA-detectable CMV and were saved to maturity. Evaluations of third-generation populations are being performed. Twenty additional cultivars and breeding lines also are being evaluated. Seedlings of 14 of the 22 cultivars/lines became 95% to 100% infected when inoculated with CMV. Results from eight of the 22 suggested that seedling populations contain CMV-susceptible and -resistant plants. Near isogeneic sublines of susceptible and resistant plants per cultivar/line are being conveyed to cowpea breeders for genetic analyses and breeding purposes.
Yiping Zhang, Vince Lackney, and Molly Kyle
Genetic resistance to cucumber mosaic virus (CMV) in pepper (Capsicum spp.) is recessive, polygenic and, therefore, has been difficult to transfer in breeding programs. Although a few varieties have been released with some resistance, in our tests, these develop severe symptoms that are eventually indistinguishable from the susceptible reactions. Furthermore, accurate and consistent screens for the disease can be relatively difficult; therefore, we report on the detection molecular markers linked to two CMV resistance genes using distributional extreme analysis to identify the relevant quantitative trait loci (QTL). The 12 most resistant and 15 most susceptible individuals were selected from a segregating F2 population of 316 individuals that were derived from the interspecific cross (C. annuum `Jupiter' × C. frutescens BG2814-6). A total of 132 tomato genomic, cDNA, and pepper genomic clones were hybridized to filters with DNA extracted from the distributional extremes. These clones included framework markers representing all pepper linkage groups and also selected clones from regions of the genome identified in a preliminary analysis as possibly involved with CMV resistance. Several clones from the two regions of the genome previously identified appear to be nonrandomly cosegregating with the CMV resistance phenotype in this larger population. Further analysis will be done by adding more markers in the regions and refining the positions of the resistance QTL.
John F. Murphy, Edward J. Sikora, Bernard Sammons, and Wojciech K. Kaniewski
Three processing tomato (Lycopersicon esculentum Mill.) lines engineered to express the cucumber mosaic virus (CMV) capsid protein (CP) gene were evaluated in the summers of 1995 and 1996 under high levels of naturally occurring CMV disease pressure. One tomato line expressed the capsid protein gene from a subgroup II isolate of CMV (line 11527), whereas two lines (12261 and 12295) expressed the capsid protein genes from a CMV subgroup I and a subgroup II isolate. Evaluation of CMV incidence based on symptomatic plants revealed that only 9% and 8% of the plants in line 11527 were infected in 1995 and 1996, respectively, 5 weeks after being transplanted. None of the plants in line 12261 developed symptoms in 1995, whereas 26% were symptomatic in 1996. There were no symptomatic plants in line 12295 in either the 1995 or the 1996 trial. In contrast to the CMV transgenic lines, 96% and 95% of the susceptible control plants were symptomatic by the 5-week rating period. CMV incidence in the CMV transgenic lines was much higher when infection was based on detection of virus by enzyme-linked immunosorbent assay (ELISA). This was particularly true in the 1996 trial where no less than 97% of the plants within a treatment were determined to be infected. Though a relatively high percentage of the transgenic plants were infected, the amount of CMV that accumulated in these plants was significantly less than in the susceptible controls, which may explain the occurrence of the attenuated symptoms. Despite CMV infection of the transgenic lines in the Alabama field trials, the performance of these lines could be of practical value to growers.
Michael Mazourek, George Moriarty, Michael Glos, Maryann Fink, Mary Kreitinger, Elizabeth Henderson, Greg Palmer, Ammie Chickering, Danya L. Rumore, Deborah Kean, James R. Myers, John F. Murphy, Chad Kramer, and Molly Jahn
‘Peacework’ is a new open-pollinated, early red bell pepper cultivar with Cucumber mosaic virus (CMV) resistance developed for and within organic systems. Development of this cultivar was conducted at Cornell University's Department of Plant
Shin Je Kim, Kyung-Hee Paek, and Byung-Dong Kim
A cDNA clone of cucumber mosaic virus (CMV) 117 N-satellite RNA driven by the cauliflower mosaic virus (CaMV) 35S transcript promoter, was stably integrated into the genome of Petunia hybrida `Bluepicoti' tissues by Agrobacterium tumefaciens Ti plasmid-mediated transformation. Transgenic plants producing CMV satellite RNA showed delayed disease development when inoculated with CMV-Y, a helper virus for the I17N-satellite RNA. Furthermore, transgenic petunia plants showed delayed disease development against tobacco mosaic virus (TMV), a tobamovirus not related to CMV. Northern blot analysis revealed that large amounts of unit length satellite RNA (335 bp) were produced in CMV-infected transgenic petunia plants; whereas, mainly transcripts driven by the CaMV 35S promoter (approximately 1 kb) were produced in TMV-infected transgenic plants. SDS-PAGE and Western blotting showed that symptom reduction was correlated with a reduction in the amount of viral coat protein in transgenic plants.