New Method for Screening Cowpea Germ Plasm for Resistance to Cucumber mosaic virus.

Utilizing the Cucumber mosaic virus(CMV)-resistant cowpea germ plasm line, GC-86L-98, a new method of screening for resistance in the greenhouse followed by field screening was developed. A uniform source of CMV inoculum (freeze-dried infected cowpea tissue) was diluted to provide an infection rate in GC-86L-98 similar to that observed under field conditions. Plants of test lines were mechanically inoculated with this standard inoculum rate and assessed under greenhouse conditions. Lines considered equivalent in infection percentage with GC-86L-98 were then evaluated in field tests. Test line plants were exposed to virus from susceptible cultivar Coronet when plants were at the first or second trifoliolate leaf stage, and then leaf samples were assayed by direct antigen coating-enzyme-linked immunosorbent assay. Lines with infection percentages similar to or lower than the resistant control were considered resistant. A new line (PI 441917) with CMV resistance and several lines with Blackeye cowpea mosaic virus resistance were found. Newly discovered CMV-resistant lines will help to control the serious cowpea stunt disease caused by synergism of the two viruses.

An improved genetic linkage map for cowpea (Vigna unguiculata L.) combining AFLP, RFLP, RAPD, biochemical markers, and biological resistance traits.

An improved genetic linkage map has been constructed for cowpea (Vigna unguiculata L. Walp.) based on the segregation of various molecular markers and biological resistance traits in a population of 94 recombinant inbred lines (RILs) derived from the cross between 'IT84S-2049' and '524B'. A set of 242 molecular markers, mostly amplified fragment length polymorphism (AFLP), linked to 17 biological resistance traits, resistance genes, and resistance gene analogs (RGAs) were scored for segregation within the parental and recombinant inbred lines. These data were used in conjunction with the 181 random amplified polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP), AFLP, and biochemical markers previously mapped to construct an integrated linkage map for cowpea. The new genetic map of cowpea consists of 11 linkage groups (LGs) spanning a total of 2670 cM, with an average distance of 6.43 cM between markers. Astonishingly, a large, contiguous portion of LG1 that had been undetected in previous mapping work was discovered. This region, spanning about 580 cM, is composed entirely of AFLP markers (54 in total). In addition to the construction of a new map, molecular markers associated with various biological resistance and (or) tolerance traits, resistance genes, and RGAs were also placed on the map, including markers for resistance to Striga gesnerioides races 1 and 3, CPMV, CPSMV, B1CMV, SBMV, Fusarium wilt, and root-knot nematodes. These markers will be useful for the development of tools for marker-assisted selection in cowpea breeding, as well as for subsequent map-based cloning of the various resistance genes.

Resistance to Cucumber mosaic virus in Cowpea and Implications for Control of Cowpea Stunt Disease.

Cucumber mosaic virus (CMV) and Blackeye cowpea mosaic virus (BlCMV) interact synergistically in dually infected plants of cowpea (Vigna unguiculata subsp. unguiculata) to cause cowpea stunt disease, the most damaging viral disease of this crop in the U.S. Sources of resistance to BlCMV are known and are present in cultivars of cowpea such as Pinkeye Purple Hull-BVR. However, no sources of CMV resistance have been found previously in cowpea. In 1998, PI 441918, a cowpea line growing in regeneration plots, was observed to have few viral symptoms, was not infected with BlCMV, and had a low titer of CMV when tested using direct antigen coating-enzyme-linked immunosorbent assay (DAC-ELISA). In greenhouse tests, infection of PI 441918 with CMV resulted in a lower titer of virus if the inoculated plants were from white seeds of PI 441918 than if the plants were from tan seeds of this PI, and a lower titer of virus than plants of the susceptible cultivar Coronet. In the field, with CMV- and BlCMV-infected plants of Coronet in spreader rows, plants from white seed of PI 441918 had no infection with BlCMV and a low infection rate with CMV. PI 441918 offers a high level of resistance to BlCMV and moderate resistance to CMV, which are important characteristics in a parental line to develop cultivars of cowpea resistant to cowpea stunt disease.

RT-PCR Detection of Seedborne Cowpea aphid-borne mosaic virus in Peanut.

The Brazilian strain of Cowpea aphid-borne mosaic virus (CABMV) is a severe pathogen in peanut and a significant problem when distributing germ plasm to other countries. The virus is seedborne at approximately 0.15% in peanut, depending upon the cultivar, and its detection in seed lots would strengthen quarantine programs. Utilizing 3' sequence data (GenBank Accession #AF241233), primers were designed from the coat protein region and evaluated by reverse transcription-polymerase chain reaction (RT-PCR). Use of the forward primer 5'-CGCTCAAACCCATTGTAGAA-3' and reverse primer 5'-TATTGCTTCCCTTGCTCTTTC-3' yielded a 221-bp product. Extracts of thick seed slices and a sample size of 12 to 25 seed showed no significant advantage of RT-PCR over enzyme-linked immunosorbent assay (ELISA) in tests of large seed lots. However, RT-PCR detected more virus in seed than in the number of infected seedlings normally arising in germination tests. Also, RT-PCR was extremely sensitive and detected 1 infected leaf among 99 healthy leaves. In contrast, ELISA detected only one infected leaf among nine healthy leaves.

Sensitive Method for Testing Peanut Seed Lots for Peanut stripe and Peanut mottle viruses by Immunocapture-Reverse Transcription-Polymerase Chain Reaction.

An immunocapture-reverse transcription-polymerase chain reaction (IC-RT-PCR) method was developed for testing peanut (Arachis hypogaea) seed lots for infection by Peanut stripe virus (PStV) and Peanut mottle virus (PeMV). A small slice was removed from each seed distal to the radicle of a random 100-seed sample, the slices were extracted in buffer and centrifuged, and a portion of the supernatant was incubated in a tube that had been coated with antiserum to either PStV or PeMV. Following immunocapture of the virus, the tube was washed, the RT-PCR mix (with primers designed from conserved sequences within the capsid region of each virus) was placed in the same tubes, and the test completed. Results obtained on 15 previously untested seed lots from the collection indicated good correlation between virus detected by the IC-RT-PCR method and virus detected from the same seed lots by enzyme-linked immunosorbent assay (ELISA). The IC-RT-PCR method detected three lots infected with PeMV and none with PStV from 106 seed lots grown in Ecuador (results confirmed by ELISA). The IC-RT-PCR method is more sensitive than ELISA (currently used on samples consisting of five seeds), is useful for testing large numbers of seed lots of peanut germ plasm, and could be adapted to test other plants and detect other viruses.

RT-PCR Method for Detecting Cowpea Mottle Carmovirus in Vigna Germ Plasm.

A highly sensitive reverse transcription-polymerase chain reaction (RT-PCR) method was developed to detect cowpea mottle carmovirus (CPMoV) in newly acquired germ plasm of Vigna spp. It detected virus in tissues diluted up to 10-9. The preferred primers were designed from the RNA replicase cDNA sequence of CPMoV. These primers were able to detect CPMoV in plants infected with 10 different isolates of the virus. There were no cross-reactions with either bean mild mosaic or melon necrotic spot carmoviruses or any of the common cowpea viral pathogens tested. The RT-PCR method was up to 105 times more sensitive than direct antigen coating enzyme-linked immunosorbent assay (DAC-ELISA) in detecting CPMoV. The RT-PCR method gave no false positive reaction as is sometimes seen with ELISA.

First Report of Peanut Stunt Cucumovirus Naturally Infecting Desmodium sp.

Plant species in the genus Desmodium (Fabaceae) are used as forage and cover crops and include a number of common weeds such as beggarweed (D. tortuosum) and beggarlice (D. intortum). Accessions of the genus are part of the plant genetic resources collection maintained at Griffin, GA. Peanut stunt cucumovirus (PSV) was detected in naturally infected plants of Desmodium sp. PI 322505 (from Brazil) in a germ plasm regeneration plot by a direct antigen coating-enzyme-linked immunosorbent assay (DAC-ELISA) with an antiserum against PSV strain ER (subgroup I) originally isolated from cowpea in Georgia. The infected plants showed mild mosaic symptoms. Indicator host studies in the greenhouse revealed symptoms characteristic of PSV on Nicotiana tabacum cv. Burley 21 (ringspots and oak leaf pattern), Chenopodium album subsp. amaranticolor (chlorotic local lesions), and Vigna unguiculata (chlorotic spots followed by systemic mild mosaic). These symptomatic indicator plants tested positive for PSV by DAC-ELISA. Greenhousegrown plants of D. incanum (kaimi-clover) and D. uncinatum (Spanish tick-clover) were inoculated with the field isolate and the plants were tested for PSV by DAC-ELISA (10 infected of 10 tested and 3 infected of 9 tested, respectively). The PSV isolate infecting Desmodium spp. was found to contain satellite RNA and it generated the predicted products in reverse transcription-polymerase chain reactions (RT-PCRs) with primers based on specific PSV-ER sequences. The RT-PCR products were confirmed by restriction-enzyme digestion (1). This is the first report of PSV naturally infecting a member of the genus Desmodium. Because some members of this genus may grow as perennial weeds near peanut, cowpea, or other host crops, this genus may serve as an alternate/overwintering host for the virus. Reference: (1) R. A. Naidu et al. Phytopathology 85:502, 1995.

Characterization of a Severe Strain of Cucumber Mosaic Cucumovirus Seedborne in Cowpea.

A new seedborne strain of cucumber mosaic cucumovirus (CMV) that induces severe symptoms on many cowpea genotypes was detected in Georgia in 1994. This strain, designated CMV-Csb, is asymptomatic on tobacco, but it produces more severe cowpea stunt symptoms when present in combination with blackeye cowpea mosaic potyvirus than do the more prevalent CMV isolates. The new strain is seedborne in cowpea (1.5 to 37%), has no associated satellite RNA, and is classified as a member of subgroup I of CMV strains based on nucleic acid hybridization assays.

Characteristics of a Latent Potyvirus Seedborne in Guar and of Guar Green-Sterile Virus.

A symptomless, seedborne potyvirus was isolated from guar (Cyamopsis tetragonoloba) germ plasm in Griffin, Georgia. The host range and serology were similar to those reported for guar green-sterile virus (GGSV) and guar symptomless virus. Biological, serological, and molecular comparisons of the Georgia isolate and the South African GGSV indicate they are similar and are closely related to bean common mosaic potyvirus (BCMV). The Georgia isolate is seed-transmitted at a rate of up to 94% in guar line PI 340385. Sequence analysis of the capsid protein (CP) gene and the 3'-untranslated region (3'-UTR) showed that both isolates are 96% homologous. GenBank searches indicate that both are related to various strains of BCMV. The highest CP nucleotide sequence and 3'-UTR identities of 91 and 93%, respectively, were with those of BCMV-NL4. On this basis, both isolates from guar should be considered as strains of BCMV.

Characteristics of a Potyvirus of the Bean Yellow Mosaic Virus Subgroup in Sesbania speciosa Germ Plasm.

A plant of Sesbania speciosa with leaf mosaic and distortion symptoms was identified in a germ plasm regeneration plot at Griffin, Georgia. The Sesbania virus produced mild or moderate mosaic symptoms on Glycine max cvs. Bragg and Tracy M, Lupinus albus, Nicotiana benthamiana, Pisum sativum cv. Perfected Wales, Phaseolus vulgaris cvs. Black Turtle, Bountiful, and Pinto, and did not infect N. tabacum. Bean yellow mosaic potyvirus (BYMV) and pea mosaic potyvirus (PMV) do not infect Perfected Wales pea and they produce mosaic, distortion, and necrosis on white lupine. The PMV strain tested produced much more severe symptoms on the three green beans, with top necrosis on Pinto. BYMV produced local latent infection of N. tabacum and BYMV and PMV produced mosaic with distortion on N. benthamiana. The Sesbania virus was seed-transmitted at a low rate in S. speciosa. Indirect-enzyme-linked immunosorbent assay tests with a general potyvirus monoclonal antibody and BYMV and white lupine mosaic virus (WLMV) polyclonal antisera were strongly positive. Tests of the Sesbania virus against a monoclonal antibody panel suggests that it is not BYMV or any of the previously described subgroup members, but is a member of the BYMV subgroup. This is the first report of a seedborne BYMV-like virus of Sesbania spp.

Ratoon stunting disease of sugarcane: isolation of the causal bacterium.

A small coryneform bacterium was consistently isolated from sugarcane with ratoon stunting disease and shown to be the causal agent. A similar bacterium was isolated from Bermuda grass. Both strains multiplied in sugarcane and Bermuda grass, but the Bermuda grass strain did not incite the symptoms of ratoon stunting disease in sugarcane. Shoot growth in Bermuda grass was retarded by both strains.