Soybean Mosaic Virus : Infection of Soybean Seed Parts and Seed Transmission

BOWERS, G. R., Jr., and R. M. GOODMAN. 1979. Soybean mosaic virus: Infection of soybean seed parts and seed transmission. Phytopathology 69: 569-572. The presence of soybean mosaic virus (SMV) (Illinois severe isolate) in cotyledons, and embryos from immature seeds of both Midwest and Merit. reproductive plant parts was compared in field-grown, manually inoculated In mature seeds, infectious virus was detected in embryos of Midwest seeds soybean (Glycine max) cultivars Merit and Midwest. Soybean mosaic virus but not in those of Merit seeds. Infectious virus was not detected in testas was detected in 100% of the flowers, immature seeds, and green pods that from mature Midwest seeds by a local lesion index, but the same testas were tested but not in any of the dry pods. The percentage of Merit seeds contained viral antigen detectable by enzyme-linked immunosorbent assay. containing detectable virus was 58% at physiological maturity and 0.8% at The relationship between the time during the growing season when the harvest maturity; the corresponding values for Midwest were 94 and 66%, plants were inoculated with SMV and the incidence of seed coat mottling, respectively. The percentage of mature seeds containing SMV was the same percent germination, and the incidence of SMV transmission through as the percentage of SMV-infected seedlings arising from those seeds. The seed was tested in field-grown Williams soybeans. Time of inoculation incidence of SMV transmission through seed was only slightly reduced after affected only virus transmission through seed, which averaged 16 or 3% if storage for 6 mo at 14 C. Infectious SMV was detected in the testas, inoculation was before or after the onset of flowering, respectively. Additional key words: embryo infection, seed transmission. Transmission of soybean mosaic virus (SMV) through soybean planting in the field on 21 May 1976. The plants were inoculated by (Glycine max [L.] Merr.) seed occurs at relatively low frequencies manually rubbing the leaves with inoculum prepared from SMV-Il(3,12). Previous investigations suggested the importance, but did S infected soybeans (cultivar Rampage) which had been inoculated not entirely resolve the roles, of several factors that may influence 14-21 days previously. The inoculum contained 4 ml of 0.05 M the incidence of SMV seed transmission. For example, time of sodium phosphate buffer, pH 7.0, per gram of tissue (fresh weight) inoculation relative to time of flowering may (14) or may not plus a small amount of 22 -mm (600-mesh) Carborundum and was (15) affect the incidence of transmission of SMV through seed. applied with a sterilized gauze pad. Similarly, 20 plants were Evidence for variation in seed transmission of SMV in different inoculated weekly for 8 wk. The first two inoculations preceded soybean germplasms has been documented (14,15,18,21), but little flowering and the others were done during various reproductive is known about its cause. stages identified according to Fehr et al (11). The third inoculation Embryo infection appears to be an important factor affecting the was at stage R 1 in which there is one flower at any node. The fourth seed transmissibility of other plant viruses (2,23). Working with a inoculation was at stage R2 when there is a flower at the node Brazilian isolate of SMV, Porto and Hagedorn (21) were able to immediately below the uppermost node with a completely unrolled detect the virus in embryos from mature Bans~i soybean seeds in leaf. The fifth inoculation was at R4 when there is a 2-cm-long pod which SMV is seed transmitted (4). at one of the four uppermost nodes with a completely unrolled leaf. We report here the results of studies on the infection by SMV of By the seventh inoculation the plants were at stage R6 (possessing a reproductive tissues of two soybean cultivars, Merit and Midwest, pod containing full-size beans at one of the four uppermost nodes previously shown to exhibit high and low incidences, respectively, with a completely unrolled leaf). of SMV transmission through mature seed (15,16). We show that All symptomless plants were removed at the end of August. this variation is not due to differences in the incidence of flower or When the plants reached maturity, each of the eight inoculation immature seed infection and that SMV infection of mature soybean sets were harvested and threshed separately. Two samples of 200 seed embryos is confined to the seed-transmitting genotype. We seeds were counted from each inoculation set, and the percentage of also report results indicating that the incidence of SMV transmottling was determined. Two hundred seeds from each set were mission through seed of Williams soybeans is lower in plants that germinated and 100 seedlings were randomly selected and indexed become infected after the onset of flowering. A preliminary report on the detached primary leaves of bean (Phaseolus vulgaris 'Top of part of this work has been published (5). Crop') as described by Quantz (22) and refined by Milbrath and Soong (20). MATERIALS AND METHODS Presence of SMV in reproductive tissue during plant development. Merit and Midwest soybeans each were planted in Effect of inoculation time. Soybean plants were inoculated at 10 hills in a field near Urbana, IL on 22 May 1976. Plants were different reproductive stages to determine the effect of time of manually inoculated with SMV-I1-S on June 17, 25 days before the inoculation upon rates of seed mottling and seed transmission. Two onset of flowering for Merit and 50 days for Midwest. Ten plants hills (10 plants per hill) of Williams soybeans were inoculated with from each cultivar were sampled during reproductive states R 1, R2, the Illinois severe isolate of SMV (SMV-I1-S) (24) 3 wk after R4, R6, R7, and R8. At stage R7 the plant is at physiological maturity with the pods and 50% of the leaves yellow. At stage R8 (harvest 00031-949X/79/000100$03.00/0 maturity) 95% of the pods are brown. Both cultivars also were ©1979 The American Phytopathological Society sampled after the seeds were germinated immediately after Vol. 69, No. 6, 1979 569 harvesting and again after 6 mo of storage at 14 C. A sample was primary leaves on Top Crop beans. The remaining half of the taken from every node of each of the 10 plants, one plant being extract from each testa half was indexed for the presence of viral randomly selected from each of 10 hills during each stage for both antigen with an enzyme-linked immunosorbent assay (ELISA). cultivars. These samples are considered as 'node-plant' positions. The above procedure was repeated three times. The samples were indexed for the presence of SMV as described above. Individual plants were considered as replications. In stages RESULTS R I and R2 entire flowers were indexed, but in stage R4 pods and Time of inoculation. A significant correlation (P = 0.05) was immature green seeds were indexed together. During R6, R7, and found between the incidence of seed transmission of SMV and the R8 the pods and seeds were indexed separately. For the time of inoculation (r = -0.826), which indicated that early germination sample, two seeds from each 'node-plant' position infection resulted in more seed transmission than did later were placed on moist cellulose pads under continuous light in a infections. The incidence of seed transmission was higher when germinator operating at 25 C and 100% RH. When the seedlings infection occurred prior to the onset of flowering (Table 1). had fully-expanded primary leaves, a leaf from one plant from each However, the seed produced by plants inoculated after flowering 'node-plant' position was indexed individually. Seeds also were exhibited a low but detectable incidence of seed transmission of stored in the cold room at 14 C for 6 mo after which they were SMV. The time of inoculation had no effect upon the incidence of germinated and indexed as above. The percentage of seed coat seed coat mottling or germination (Table 1). mottling from each 'node-plant' position was determined by examEffect of stage of reproductive development. Virus was detected ining all of the seeds from the plants that produced the seeds used in in all of the young reproductive parts tested from both cultivars. the germination sample. Maturation of SMV-infected Merit and Midwest soybean seeds Presence of SMV in seed parts. Merit and Midwest soybeans was accompanied by a decrease in the proportion of seeds from were planted in five 1I-m rows in a field near Urbana, IL. On 14 June which virus was detected by indexing (Fig. 1). Statistical analysis 1977, 3 wk after planting, the plants were inoculated with SMV-I1according to Fisher's least significant difference, (FLSD) for S. Seed samples were taken from plants at three stages of plant comparison between any two means indicated significant reproductive development (11). The first sampling was at stage R6, differences (P = 0.05) between stages R6, R7, and R8 for Merit and a second was at stage R7, and the third was at stage R8. At each between stages R7 and R8 for Midwest. No significant difference (P sampling time, five plants with symptoms of each cultivar were = 0.05) existed, however, between the percentage of seeds from randomly selected from each of the five rows. Three seeds were which virus was detected before and after germination. Storage of taken from each of these 25 selected plants, one from a node near Midwest soybean seeds for 6 mo resulted in a slight, but significant, the top, a second from a node near the middle, and a third from a decrease in percentage of virus detected in seeds (P = 0.05) (Table node near the bottom of each plant. The seeds were placed 2). overnight on moist cellulose pads under continuous light in a Effect of node position. Analysis of FLSD indicated a germinator operating at 25 C and 100% RH. Each seed was then significant difference (P = 0.05) for the presence of virus in seed carefully and cleanly dissected into embryo, testa, and cotyledon. from different node positions only when Midwest soybean seeds These seed parts were separately surface-decontaminated by were germinated immediately after harvesting (Table 2). However, immersion in 10% (w/v) Na 3PO 4 for 1 min, followed by several no pattern could be established. washes with distilled water (26), and each was separately indexed Analysis by FLSD showed no significant difference (P = 0.05) on the detached primary leaves of Top Crop beans, for the percentage of mottled seed taken from different node Immediately after harvesting and again after 6 mo of storage at positions of Merit soybean plants. A significant difference (P = 14 C, two seeds from each of the three sampling positions (as described above) from 25 plants of each cultivar were germinated as described above. When the seedlings had fully expanded primary leaves, a leaf from one plant from each sampling position was I I I I I I I , , indexed individually as above. R2 R1 R2 R6 R4 Twenty-five seeds from SMV-infected Midwest soybean plants > + were harvested at stage R8 and germinated as above. The testa was 2 100 -:R6 removed from each seed and cut in half; one half was surfaceO0 Ri R4 k*•".7 decontaminated as described above and the other half was only z 90 R6 rinsed in distilled water. Each testa half was then homogenized in z 0 "", "", 0.5 ml of buffer. One half of the extract from each testa half was 0_ " z indexed for the presence of infectious SMV on the detached O 0 7 * Merit Seeds WL 60 \•' 10 [----]Me r it Pods7R TABLE 1. The effect of the time of inoculation with soybean mosaic virus 5. C-CMerit Pods R7 (Illinois severe isolate) upon seed coat mottling, germination, and seed t) 5 Midwest Seeds R7 transmission in Williams soybeans c 4030--..'Midwest PodsC Week of Developmental mottling Germination Transmission Z inoculation stagea (%)b (%)C (%)de tO20 1 69.0 90.5 18.0 U3 2 65.8 91.5 19.0 (. 10 R8 \R8 3 RI 80.5 89.5 11.0 + 4 R2 72.3 85.0 2.0 I1 5 R4 80.0 88.0 4.0 40 50 60 70 80 90 100 110 120 130 140 150 160 6 69.8 87.0 4.0 DAYS AFTER PLANTING 7 R6 77.0 85.0 3.0 8 78.5 92.0 4.0 Fig. 1. Detection of the Illinois severe isolate of soybean mosaic virus aAccording to the descriptions in reference 11. (SMV-II-S) from Midwest and Merit soybean flowers, pods, or seeds at bSample of 400 seeds. various stages of reproductive growth of artificially inoculated plants. cSample of 200 seeds. Points correspond to developmental reproductive stages RI to R8 (see dSample of 100 seeds. reference 11). Flowers were indexed in stages R 1 and R2. Seeds and pods The r value between the week of inoculation and rate of seed transmission were indexed together in stage R4. Pods and seeds were separately indexed was -0.826. in stages R6, R7, and R8.