Published in Crop Sci 39:1513-1515 (1999)
© 1999 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
Crop Science 39:1513-1515 (1999)
© 1999 Crop Science Society of America
NOTES
Identification of suitable seed regeneration sites for photoperiod-sensitive cowpea germplasm
A.G. Gillaspie, Jr.a,
R.J. Goenagab,
S. Torresb and
E. Peregrinec
a Jr., USDA, ARS, Plant Genetic Resources Conservation Unit, 1109 Experiment St., Griffin, GA 30223 USA
b USDA, ARS, Tropical Crops and Germplasm Research Unit, P.O. Box 70, Mayaguez, Puerto Rico 00681
c USDA, ARS, Germplasm Introduction Research Unit, P.O. Box 3008, Kingshill, St. Croix, US Virgin Islands, 00851
s9gg{at}ars-grin.gov
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ABSTRACT
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To determine an appropriate location to regenerate photoperiod-sensitive cowpea [Vigna unguiculata (L.) Walp.], seed production and virus contamination data were measured at Isabela, PR, St. Croix, VI, and Griffin, GA for six photoperiod-sensitive cowpea accessions. The results indicate that Isabela was best suited for seed regeneration of photoperiod-sensitive cowpea. Seed production at Isabela was equal to or greater than that at either of the other two locations for these accessions. Virus infection of plants and of seeds was high at Griffin. However, little virus infection occurred at Isabela and at St. Croix. The systematic approach used in these tests can be used as a model for curators in the National Plant Germplasm System and others to determine the best environment in which to regenerate germplasm.
Abbreviations: BlCMV, blackeye cowpea mosaic potyvirus • CCMV, cowpea chlorotic mottle bromovirus • CMV, cucumber mosaic cucumovirus • CSMV, cowpea severe mosaic comovirus • DAC-ELISA, direct antigen coated-enzyme-linked immunosorbent assay • NPGS, National Plant Germplasm System • NSSL, National Seed Storage Laboratory • SBMV, southern bean mosaic sobemovirus
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INTRODUCTION
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THE NATIONAL PLANT GERMPLASM SYSTEM
acquires, maintains, evaluates, and distributes crop germplasm for U.S. agriculture. The Vigna collection is maintained at Griffin, Georgia (Jarret et al., 1990) with a secondary collection at the NSSL at Ft. Collins, Colorado. Two problems emerge in the curation of cowpea germplasm in Georgia and other areas of similar latitude: (i) regeneration of seed of photoperiod-sensitive lines, and (ii) contamination of seed by seedborne viruses during regeneration. Some cowpea germplasm accessions do not flower or flower too late in the season for the production of mature seeds in Georgia, and a similar phenomenon has been reported in California (Ehlers and Hall, 1996; Patel and Hall, 1990). This problem is important because the NPGS has a limited number of locations available for regeneration of such photoperiod-sensitive crops. Research at the University of California, Riverside, showed that fall planting of photoperiod-sensitive cowpea lines at Coachella Valley in California is effective for obtaining seed production (A.E. Hall and J.D. Ehlers, 1996, personal communication). The germplasm accessions that will not produce seeds in the summer in the field in Georgia will do so in the late fall and winter in the greenhouse. However, production of seed in the greenhouse is expensive and space is often limited.
Seedborne viruses in cowpea are a serious problem worldwide. At least 15 viruses have been reported to be seedborne in this crop (Hampton, 1983; Mali and Thottappilly, 1986). Methods have been devised to eliminate seedborne viruses from cowpea germplasm (Gillaspie et al., 1995), but germplasm may become re-contaminated during seed regeneration. The desired seed regeneration involves the production of numerous virus-free seeds to allow adequate seed storage at the NSSL and for public distribution without risk of virus dissemination.
In the experiment reported here, seed production and the contamination by seedborne viruses of six cowpea germplasm accessions grown in Georgia was compared with those grown in Isabela, PR and St. Croix, VI, the most southern locations operated by USDA-ARS. These locations were chosen because their southern latitudes potentially allow the appropriate photoperiods for flowering and seed production. Also, it was possible that their isolated locations would reduce recontamination with insect-transmitted viruses. The six accessions had previously been observed to produce few seeds in Georgia.
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Materials and Methods
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Field Experiments
Field experiments were established with PIs 527259, 582744, 582800, 582803, 582830, and 582846 at Isabela, PR (18° 30' N lat. and 67° W long.), St Croix, VI (17° 45' N lat. and 64° 45' W long.), and Griffin, GA (33° 30' N lat. and 84° 30' W long.) in 1997 (Table 1)
. The experiments in all three locations were planted in a randomized complete block design with four replications. Plots contained two rows, each 3 m long by 3 m wide.
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Table 1 Temperature and moisture conditions at Griffin, Isabela, and St. Croix during the months of the regeneration experiment
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The soil at Isabela, PR, is a well-drained Oxisol (clayey, kaolinitic, isohyperthermic, Tropeptic Eutrustox) with a pH of 5.7. At St Croix, VI, the soil is a well-drained Fine-loam (carbonatic isohyperthermic Typic Calciustolls) with a pH of 8.3. At Griffin, GA, the soil is a moderately- well-drained Cecil sandy loam (clayey, kaolinitic, thermic Typic Kanhapludults) with a pH of 6.1.
All seeds were obtained after one cycle of virus elimination (Gillaspie et al., 1995). Treflan1
(Dow AgroSciences, Indianapolis, IN; trifluralin; a,a,a-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine) herbicide was applied at, or just before hand-planting in mid to late May. Seedlings were thinned to an in-row spacing of 15 cm. Plots were irrigated as necessary. Leaf samples were taken from five random plants per row (two samples per plot) each week beginning when the plants were at the second trifoliolate-leaf stage and continued for 5 to 6 wk. The leaf samples collected in Isabela and St. Croix were sent by overnight mail to Griffin for testing by direct antigen coated-enzyme-linked immunosorbent assay (DAC-ELISA). The five leaves from each row were combined for testing. A weekly application of insecticide (carbaryl [1-naphthyl N-methylcarbamate] and malathion [0,0-dimethyl phosphorodithoate of diethyl mercaptosuccinate] at the recommended rates) was made from the time of flowering through pod maturity to reduce infestation by cowpea curculio borers (Chalcodermus aeneus Boheman). Pods were harvested by hand as they matured, dried at 21°C, 25% RH for 1 wk, and shelled. Seeds were counted and weighed. Random 100-seed samples from each replication of each accession were planted in flats in the greenhouse. These seedlings were sampled at the third-trifoliolate-leaf stage and tested by DAC-ELISA.
Seed production data were analyzed by a two-way analysis of variance with accessions and locations as the two factors. Accessions were compared within each location.
Dac-elisa
A disk (18-mm diam.) of leaf tissue was excised from each leaflet with a cork borer. For viral assays by DAC-ELISA, infected or control leaf tissue was triturated at 1:9 dilution (w/v) in extraction buffer, pH 7.1 (Gillaspie et al., 1995) and further dilution was done in this buffer, if necessary. Polyclonal rabbit antisera were diluted at the indicated rates (CSMV-PVAS-470 [1:10000], American Type Culture Collection, Rockville, MD; CCMV [1:5000], CMV [1:5000], and SBMV [1:20000], donated by O.W. Barnett, Clemson University; and BlCMV [1:20000], contributed by J.W. Demski, University of Georgia, Griffin, GA) in healthy plant extract (1:50 w/v healthy cowpea tissue and conjugate buffer), and the tests were conducted as previously published (Gillaspie et al., 1995).
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Results and Discussion
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Mean seed production for these accessions was nine times greater at Isabela than at Griffin and 40 times greater than at St. Croix (Table 2)
. At St. Croix, there were no differences among accessions, but at Isabela, seed yield of PI 527259 was significantly higher than all accessions except PI 582800. At Griffin, yield of PI 582846 was significantly higher than the other five accessions, whereas PI 527259 and PI 582803 produced no or very few seed.
St. Croix is the most southerly location and should have produced greater seed yield, but yield at St. Croix was low for several reasons other than daylength. The soil pH at St. Croix is 8.3, which must limit growth and survival of some genotypes as noted for PI 582744. In addition, the soil is mainly coral mixed with a little true soil, allowing water and nutrients to leach through rapidly. At Griffin, the seed yield of several of these cowpea accessions were higher than observed in past years (unpublished data). PI 527259 flowered late and the pods did not mature before frost. Average minimum and maximum daily temperatures ranged from 9.5°C to 31.7°C at Griffin during the growing period, but at the other locations temperatures were more moderate (Table 1). The combination of soil fertility and texture, temperature, and daylength at Isabela was the best of the three locations.
The ELISA results for samples collected in June at Isabela and St. Croix indicated the presence of CCMV in one sample from Isabela and of CSMV in two samples from St. Croix (Table 3)
. No virus was detected in samples collected in these plots in July. Since no plants were detected with CCMV after the first sampling, we concluded that different plants were sampled and no beetles were present other than during the early portion of the test. Thus, no further movement of CCMV occurred in the plots. Apparently some weed hosts of the virus were present near the plot area for the initial infection source. No seed transmission has been reported for CCMV (Bancroft, 1971) so this virus did not represent a problem. At Griffin, samples were infected with BlCMV, CCMV(3), and CMV by July, and by August several samples were infected with BlCMV and CMV (Table 3). Beginning in August, symptoms of cowpea stunt, a synergistic interaction between CMV and BlCMV (Pio-Ribeirol et al., 1978), were detected. Plants at Griffin grew more slowly than those in the other two locations and no plants were large enough to sample in June. By the end of the growing season most plants of many accessions at Griffin had visible symptoms of BlCMV and CMV, but the plants at both Isabela and St. Croix were still free of visible symptoms. Plants in St. Croix were chlorotic because of nutrient deficiencies associated with high soil pH; thus, symptoms could have been masked during the latter part of the season. Plants at Isabela and Griffin produced significant vegetative growth and appeared normal, so virus symptoms could easily be seen on these plants. The insecticide applications from the time of flowering until pod maturity was not of any importance in control of virus transmission. Most of the virus transmission occurs when the plants are young and succulent. Also, CMV and BlCMV are the most frequently occurring viruses and they are nonpersistent, stylet-borne viruses that are transmitted rapidly during aphid probing, before the insects can be killed by the chemicals.
Tests for seedborne virus contamination of seed produced from all plots were in agreement with the leaf sample results (Table 3). Only one seedling from an Isabela seed lot had a marginal SBMV positive (i.e., an absorbance reading near to the cut-off for judging positives in ELISA tests). Seedlings from two seed lots from the St. Croix plots had CSMV infection. These were seeds from the same replications from which this virus was detected in young plants. Seedlings from 10 seed lots from the Griffin plots were infected with BlCMV and/or CMV. However, none of the seedlings tested had both viruses nor did they have symptoms of cowpea stunt, even though it is known that both viruses can be transmitted in the same seed (Pio-Ribeirol et al., 1978). The Griffin results were lower than one might have expected from the final numbers of infected plants. The later of the infection of these plants may have reduced the amount of seeds containing virus, but there are no tests to support this hypothesis.
St. Croix is an island with little or no agriculture so that there is little source of virus inoculum to infect plants in the regeneration plots. The area around Isabela does have agriculture and historically the site has been used for winter nursery growouts of many legumes. This site was used during the summer months to avoid the numerous possible sources of inoculum provided by these nurseries, and this strategy appears to have been successful. The Griffin area has had a history of viral-infection of plants in regeneration plots, probably from virus that has overwintered in perennial weeds or from viraliferous airborne vectors from other fields. The virus elimination program has minimized the number of contaminated seeds by starting with virus-free seeds.
In conclusion, regeneration of photoperiod-sensitive cowpea germplasm can be accomplished best in Isabela, PR where seed yields are high and virus infection during seed regeneration is minimal. The approach detailed herein is adequate for use by curators who want to evaluate regeneration sites before these sites are developed for their use. We feel that based on results from previous years of regeneration with other accessions at Griffin and St. Croix, and based on the large differences in yield at Isabela, additional years of testing would not alter the general conclusion that Isabela is the best location for regeneration of photoperiod-sensitive accessions.
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ACKNOWLEDGMENTS
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We acknowledge the knowledgeable input and technical support of J.H. Chalkley at Griffin and the input of Victor Chew, USDA, ARS, Gainesville, FL, concerning the statistical analysis of these data.
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NOTES
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1 Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be available. 
Received for publication September 4, 1998.
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REFERENCES
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- Bancroft, J.B. 1971. Cowpea chlorotic mottle virus. C.M.I./A.A.B. Descriptions of Plant Viruses. No. 49. Association of Applied Biologists, Wellesbourne, England.
- Gillaspie A.G., Jr., Hopkins M.S., Pinnow D.L., Hampton R.O. Seedborne viruses preintroduction cowpea seed lots and establishment of virus-free accessions. Plant Dis. 1995;79:388-391.
- Ehlers J.D., Hall A.E. Genotypic classification of cowpea based on responses to heat and photoperiod. Crop Sci. 1996;36:673-679.[Abstract/Free Full Text]
- Hampton R.O. Seedborne viruses in crop germplasm resources: Disease dissemination risks and germplasm reclamation technology. Seed Sci. Technol. 1983;11:535-546.
- Jarret R.L., Spinks M., Lovell G., Gillaspie A.G. The S-9 plant germplasm collection at Griffin, Georgia. Diversity 1990;6(2):23-25.
- Mali V.R., Thottappilly G. Virus diseases of cowpea in the tropics. Rev. Trop. Plant Pathol. 1986;34:421-522.
- Patel P.N., Hall A.E. Genotypic variation and classification of cowpea for reproductive responses to high temperature under long photoperiods. Crop Sci. 1990;30:614-621.[Abstract/Free Full Text]
- Pio-Ribeirol G., Wyatt S.D., Kuhn C.W. Cowpea stunt: A disease caused by a synergistic interaction of two viruses. Phytopathology 1978;68:1260-1265.
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ABSTRACT
INTRODUCTION
