Replication and Properties of Cowpea Chlorotic Mottle Virus in Resistant Cowpeas

WYATT, S. D., and C. W. KUHN. 1979. Replication and properties of cowpea chlorotic mottle virus in resistant cowpeas. Phytopathology 69:125129. Although cowpea chlorotic mottle virus (CCMV) caused no symptoms pool of RNA formed soon after inoculation. Several properties of CCMV on cowpea P1 186465, virus could be isolated from the inoculated primary produced in the resistant plants were similar to the inoculum (type strain): leaves but not from trifoliolate ones. Virus accumulation was continuous host range, serology, specific infectivity, and electrophoretic mobility. and linear in the primary leaves through a 52-day infection period. The However, based on electrophoresis and buoyant density experiments, the average rate of accumulation (4.4 Mg/g/24 hr) was 12 to 20 times less than RNA species 3 was reduced about 90% in virus purified from plants of PI the rate measured during the rapid phase of replication in susceptible 186465 in comparison to virus purified from California Blackeye. This California Blackeye cowpeas. Incorporation studies with 32 P0 4 suggested alteration was not found in the RNA of progeny resulting from back that CCMV RNA encapsidated late in the infection period came from a inoculation to California Blackeye. Additional key words: virus movement. Most studies of virus replication in plants are conducted with cycles of ultracentrifugation and analysis with the spectrophotosusceptible plants or tissues thereof (15,18), in which the virus meter (E2-0 : 5.8 mg/ml). This method was inconsistent and infects readily and moves rapidly to adjacent cells or to noninoculated portions of the plant. We theorized that different inaccurate for measurement of the low virus concentrations found and new types of information could be acquired if virus replication in the resistant host. Therefore, the clarified preparation was and its products were compared in susceptible plants with a high centrifuged on sucrose density gradients (10-40% w/v) for 4 hr at virus-replicating capacity and in resistant plants with reduced 27,000 rpm in a Spinco SW 27 rotor. Gradient profiles were replicating capacity. For a model system, we used cowpea chlorotic integrated with a planimeter to determine virus quantity. mottle virus (CCMV) and cowpeas, Vigna unguiculata (L.) Walp. Serology. Antisera were prepared by injecting rabbits subsp. unguiculata. Cowpea plant introductions resistant to intravenously and intramuscularly at weekly intervals. The animals CCVwere reported earlier (17). Preliminary tudies (20,21) we e bled during the fo rth week wh n the maximum titer was CCMV ate repor ted had Prenar studies attained. Gel double diffusion tests were run in 0.8% purified Bacto indicated that the model system had potential for elucidating agar prepared in 0.2 M acetate buffer (pH 5.0), 0.85% NaCl, and mechanisms that control virus replication and movement. 0.01% sodium azide. MATERIALS AND METHODS Isotope methods. To label virus with P0 4, stems of 10 plants per treatment, with primary leaves only, were cut above the soil line Virus manipulation. The type strain (T) of CCMV was and placed in tubes containing the isotope (0.04 mCi/ml) diluted maintained in cowpea cultivar California Blackeye. Inoculum for with neutral potassium phosphate (0.25 mM). After plants took up these experiments was either expressed sap, diluted 1:10, or the isotope for 3 hr, they were transferred to distilled water and purified virus obtained from cowpea plants which had been incubated at 27 C at 10,000 lux for an additional 21 hr. Virus was inoculated with single local lesions from soybeans, Glycine max extracted and clarified as described above. To minimize loss of (L.) Merr. 'Bragg'. The latter host also was used for infectivity small quantities of CCMV, southern bean mosaic virus (SBMV) (2assays. 4 mg) was added before ultracentrifugation. Following the Virus replication studies were conducted with the susceptible concentration step, the preparation was centrifuged on sucrose cultivar California Blackeye, and the resistant plant introduction gradients (10-40%) in which separation of CCMV and SBMV was (P1) 186465 (17). Plants were grown in 10-cm-diameter plastic pots accomplished easily (11). The CCMV zone was collected, and the containing a mixture of soil-sand-vermiculite (2:1:1, v/ v) amended po 4 activity was counted either by Cherenkov radiation in a with a complete fertilizer. The primary leaves of plants were scintillation counter or with a Nuclear Chicago thin-window gas inoculated 7 to 9 days after seeding; they were. maintained in the flow planchet counter. Activities were corrected for half life and greenhouse (21-30 C) or at 27 C with approximately 10,000 lux quenching. illumination for 16 hr per day. To retard abscission of the inoculate Physical methods. The RNA was isolated from CCMV primary leaves, the new trifoliolate leaves routinely were removed preparations by phenol-sodium dodecyl sulfate (SDS) extraction from all plants. of pronase-digested virus (19). After electrophoresis in 2.7% The virus was extracted from leaves in 0.2 M acetate buffer (pH acrylamide gels (14), the distribution of ultraviolet-absorbing 4.5) containing cysteine-HCl (0.01 M), sodium diethyldithiocarbamaterial was measured by scanning unstained gels at 254 nm with a mate (0.01 M), and MgCl 2 (0.01 M). Following chloroformPhotovolt densitometer. SDS (0.5%w/v) was included routinely in butanol clarification, two procedures were used for virus both the sample and electrolyte. concentration and quantitation. The routine method was two Equilibrium density gradient centrifugation of CCMV was conducted in RbCl (1.360 g/ml) in a Spinco Model E analytical ultracentrifuge (44,000 rpm for 20 hr at 25 C). The virus RbCl 00031-949X/79/000021$03.00/0 preparation was'in 0.02 M acetate buffer (pH 5.0) and 0.01 M © 1979 The American Phytopathological Society MgC12. Virus components were analyzed with ultraviolet optics. Vol. 69, No. 2, 1979 125 RESULTS accumulated rapidly in the susceptible host until 7 days after inoculation; accumulation then ceased and virus concentration Resistant reaction. The type strain of CCMV caused necrotic declined during the next 25 days (Fig. 1-A). In contrast, virus was etching on inoculated primary leaves and bright chlorotic mottle on detected first in the resistant host 4 days after inoculation (Fig. 1trifoliolate leaves of the susceptible cowpea cultivar California A), a time at which virus in the susceptible host had already reached Blackeye. The same inoculum caused no symptoms on either 75% of its maximum. The accumulation of virus in PI 186465 was primary or trifoliolate leaves of resistant PI 186465. Attempts to slow but constant throughout the infection period. A similar linear detect starch lesions were negative. However, based on sap increase was observed in eight individual time course studies inoculation tests to Bragg soybeans and California Blackeye conducted over a period of 1 yr. In these studies, which varied from cowpeas at 7, 14, and 21 days after inoculation, it was clear that the 32 to 52 days, the maximum amount of virus in the resistant host primary leaves of P1 186465 contained infectious CCMV and the was less than 30% of that in California Blackeye at its maximum at trifoliolate leaves did not. 7 days. Factors affecting infection. In initial studies, not all the PI The rate of virus accumulation was calculated for a 5-day period 186465 plants became detectably infected when inoculated with (2-7 days after inoculation) for the susceptible host and from 4 days CCMV-T. Therefore, various methods were used in an attempt to after inoculation until the end of the experiment for the resistant improve the incidence of infection. Although the use of young host. This allowed for latent periods of replication in each host. In plants (7-9 days old) and preinoculation shading were beneficial, eight experiments, the average accumulation rate in PI 186465 was the most important factor affecting infection was inoculum 4.4,ug/g/24 hrand the range was 1.4to 6.8pg/g/24 hr. The average concentration. The maximum number of infections (100%) and accumulation rate in California Blackeye cowpeas was 75 A g/g/24 highest accumulation of virus (isolated by ultracentrifugation) were obtained when inoculum was 0.1 mg/ml or higher (Table 1). When inoculum was 0.001 mg/ml or less, not all plants became infected, virus accumulation was reduced significantly, and the first I I I I I appearance of virus was delayed. The low levels of virus A.5accumulation associated with low inoculum concentration could be partially overcome by extending the length of infection. In one 0 study, for example, the ratio of virus accumulation for 0.1 and N .4 0.001 mg/ ml inoculum concentrations changed from about 40:1 tov0 3:1 (0.1:0.001 mg/ml) at 7 and 14 days after inoculation, D respectively. t2_ .3 Inoculum concentration affected the resistant PI 186465 and the local lesion host Bragg soybean similarly. No infection occurred in .2 either host with 0.0001 mg/ml (Table 1) or less. Susceptible (0 California Blackeye cowpeas, however, were more sensitive to ._ CCMV infection than were PI 186465 plants; to initiate infection 10 times less virus inoculum was needed (Table 1), to cause maximum virus accumulation 10 to 100 times less inoculum was needed (Table 1), and to cause all plants to become infected about 1,000 times less inoculum was needed. B Time-course studies showed that the rate of virus accumulation I 6 in PI 186465 cowpeas was approximately the same at 0 postinoculation temperatures of 21, 27, and 33 C. r5 Serology. Serologically, the virus from CCMV-T-inoculated LL X leaves of P1 186465 cowpeas appeared to be identical to CCMV-T ' 4 from California Blackeye plants. No spurs developed in double 0 4 diffusion tests, and the antiserum titer in ring precipitin tests was 2 the same for virus preparations from both hosts. Furthermore, no (9 3 serological reaction was detectable after the antiserum was cross.N. absorbed with either virus preparation. (0) 0f Virus accumulation. To insure infection of the resistant PI D 2 plants, the primary leaves were rubbed with inoculum sufficiently (if infectious to cause several hundred local lesions on Bragg soybean. > 1 The amount of virus in CCMV-T-inoculated primary leaves of resistant (PI 186465) and susceptible (California Blackeye) cowpeas was measured with the sucrose gradient technique. Virus