Correlation of Low Level Wheat Streak Mosaic Virus Resistance in Triumph 64 Wheat with Low Virus Titer

Seifers, D. L., and Martin, T. J. 1988. Correlation of low level wheat streak mosaic virus resistance in Triumph 64 wheat with low virus titer. Phytopathology 78:703-707. Wheat streak mosaic virus (WSMV) reduced the yield of Triumph 64 immunosorbent assay (ELISA). As incubation temperature increased from (cultivar having a low level of resistance to virus multiplication) by 18% 15 to 30 C, differences in relative titer between cultivars decreased. ELISA compared with 40% for the cultivar Centurk. Triumph 64 consistently had values decreased from the tip to the base in expanding leaves but were more a lower virus titer as determined by infectivity assay and enzyme-linked uniform from tip to base in fully expanded leaves. Wheat streak mosaic (WSM) caused by wheat streak mosaic University). ELISA was performed as described by Clark and virus (WS MV) is one of the most serious threats to wheat (Triticum Adams (3), except that 2 50-Ml volumes were used at each step. aestivum L.) production in western Kansas (7,18). High levels of Coating and conjugated antibodies (from a 1-mg/ ml stock) were resistance to WSMV have been translocated into common wheat used at 1:100 for the dilution curve and ELISA-infectivity from two Agropyron species (14,17). The Agropyron-derived correlation experiments and at 1:400 (economy of antibody usage) resistance has given complete protection from WSMV in for all other experiments. Samples were assayed in randomly mechanically inoculated and naturally infected field trials, located triplicate wells. The antibodies were conjugated with However, attempts to incorporate this high level of resistance into alkaline phosphatase (Type VIIS, Sigma Chemical Co., St. Louis, agronomically acceptable cultivars have failed because of linkages MO) at a 2:1 (enzyme:antibody) ratio (3). After a 30-min between WSMV resistance and undesirable agronomic incubation at room temperature, substrate reactions were stopped characteristics, by adding 50 M1 of 3 M NaOH per well. Absorbance was measured Low-level resistance to WSMV (sometimes referred to as at 405 nm (ELISA values) using a Titertek Multiskan microelisa tolerance) has been reported in common wheat cultivars (13,15). plate reader (Flow Laboratories, Inc., McLean, VA). Cultivars carrying this level of resistance become infected and For all experiments, the reported absorbance is the observed develop WSM symptoms, but yield is reduced less than in value minus the absorbance of the healthy control for that susceptible cultivars. This type of resistance has not been used treatment. extensively in breeding progress because of the difficulty and Infectivity assay. Inoculum was prepared by macerating inaccuracy of the screening process. Selection procedures involve diseased tissues at a 1:300 dilution in 0.02 M potassium phosphate time-consuming replicated trials comparing yield from paired buffer. The method of inoculation used throughout this plots of inoculated versus healthy plants. These trials normally investigation was reported previously (9). have to be repeated for several years to accurately identify the Inoculated plants were maintained in a greenhouse at 27 ± 3 C resistant reaction, for 2 wk, and the number of systemically infected plants was Resistance in cereal crops to plant viruses has been associated recorded. with reduced virus titer (6,16). Measurement of virus titer has been Yield loss from WSMV. A split-plot experimental design was labor intensive in the past; however, enzyme-linked immunosorbent used with cultivars as main plots and healthy versus WSMVassay (ELISA) can now be used to quantitate virus concentration inoculated plants as subplots. Each treatment was replicated four (2,4,8,11,16). times. Subplots consisted of four rows, 3.9 m long with 0.3-m row In this paper, we summarize 8 yr of data from yield trials spacing, seeded at 50 kg/ha. Planting was done in midto late mechanically inoculated with WSMV, which demonstrate the September each year. Plants were inoculated 14 days after effectiveness of resistance in Triumph 64 compared with the emergence using an air-blast inoculation technique (10). Incidence susceptible cultivar, Centurk. We have correlated the use of of WSMV infection was estimated in late April or early May, when ELISA with infectivity assays in determining relative WSMV titer symptoms could easily be observed. Subplots were hand trimmed in both cultivars and established that differences do exist between to 2.4 m just before harvest, in late June to early July. Volume these cultivars in the field and in the seedling stage in a controlled weights and yields were measured and corrected to a 12% moisture environment. basis. Data were combined over years (1973-1975, 1982-1986) for analysis of variance (ANOVA). MATERIAL AND METHODS Dilution curve. Wheat was seeded in 30X 50-cm, soil-filled flats and grown in a growth chamber (Warren/Sherer Model E138-15) Virus maintenance. Isolate KS-1 of WSMV was used in field at 22 C with a 12-hr photoperiod of fluorescent light studies before 1982; isolate H81 was used in field studies after 1981 (approximately 500 E" sec• m). Seedlings were inoculated at and in all other tests reported here. The method of virus the base of the primary leaf 6 days after planting. Third leaves of maintenance has been described previously (9). infected plants were harvested 14 days postinoculation (DPI). ELISA. Antiserum to WSMV (titer = 1:64 by the Harvested tissue was ground in a mortar and pestle at a 1:2 (w/v) microprecipitin test) was provided by J. K. Uyemoto (Kansas State dilution in PBS. Twofold serial dilutions (1:2-1:1,024) were made in PBS. Healthy tissue was treated identically in all cases. Data reported are the means of four replications, with each replication © 1988 The American Phytopathological Society done on different days. Vol. 78, No. 6,1988 703 Cultivar virus titer and ELISA-infectivity correlation. Wheat leaf/length of third leaf) varied from 0.15 to 0.85 within a single cultivars (Eagle, Centurk, and Triumph 64) were seeded, replication of a single genotype because of normal asynchronous inoculated, and maintained under the same conditions as the plant growth. Third leaves from plants that had leaf ratios of 0.25. seedlings used in the dilution curve experiments. Control plants, 0.50, and 0.75 were harvested from each of four replications for inoculated with buffer and abrasive only, were placed in the same each cultivar (six leaves/ratio/cultivar/replication). The tissue growth chambers. The third leaf of infected plants was harvested was ground in a mortar and pestle and diluted to 1:600 for ELISA. from each cultivar when they were fully expanded (14 DPI) in the The procedure was repeated twice and data were subjected to first two trials. The fourth leaf (half the length of leaf 3) was used in ANOVA to determine if differences in virus titer existed between trial 3. The tissue was ground in PBS (1:15, w/v) and applied to leaf ratio classes within cultivars. microelisa plates. The remaining extract was further diluted to a Within-leaf virus distribution. Third leaves of infected Centurk final dilution of 1:900 and applied to the same plates. The test was and Triumph 64 seedlings were sampled at 7 DPI (third leaf repeated three times with four replications of each cultivar in each one-half the length of second leaf) and 14 DPI (third leaf fully test. Data from each test were analyzed separately. expanded and twice the length of fourth leaf). Six leaves, per Equivalent infected leaves from other test plants in the same treatment per replication, were cut into thirds based on leaf length planting were harvested for infectivity assay. Infectivity data from (base, middle, and tip). Tissue was ground in a mortar and pestle all three trials were combined for analysis of variance. Simple and diluted with PBS to 1:600 for ELISA. Data presented are correlation coefficients were generated from infectivity data and means of four experiments and are compared using an LSD (P= ELISA values of each test. 0.05). Determination of virus titer in field-grown plants. During the spring of 1984, WSMV-inoculated field plots of Centurk and RESULTS Triumph 64 were sampled to determine relative virus titer with the ELISA. Six flag leaves were harvested from each replication and Yield loss due to WSMV. WSMV reduced average (8 yr) yield maintained on ice or in a refrigerator at 5 C until they were ground and volume weight of Centurk by 40.7 and 10.6%, respectively, and in a mortar and pestle in PBS (1:20, w/v). Two hours or less of Triumph 64 by 18.3 and 2.8%, respectively (Table 1). Loss elapsed between sample harvest and ELISA processing. A final difference between cultivars was highly significant as indicated by a sample dilution of 1:600 (w/v) was applied to ELISA plates. Four significant cultivar X inoculation interaction (P,< 0.01). There was replications of each cultivar were sampled on two dates (8 and 22 no significant year X inoculation X cultivar interaction, indicating May). Flag leaves were about 50% emerged on the first date and consistency in cultivar response to WSMV over the 8 yr. Both fully developed on the second date (5 days postanthesis). cultivars averaged 90% infection in inoculated plots over 8 yr. Temperature study. Wheat was seeded into metal flats. Healthy plots had from 1 to 5% WSMV-infected plants (average Seedlings of Centurk and Triumph 64 were inoculated and kept in 2%). 15, 20, 25, and 30 C chambers having the same lighting condition as Dilution curve. The dilution curve was the most linear from the described previously. Leaf length measurements on 25 plants from 1:64 to the 1:1,024 dilutions (Fig. 1). Within this range of dilutions, each treatment were made every 3 days and on days when samples the ELISA value doubled in response to every doubling of virus for ELISA were taken. Six leaves from each treatment were concentration. This corresponds to a relative virus concentration harvested 7, 14, 21, and 28 DPI. Harvested tissue was ground in a of 1.6-0.1, assuming that the initial concentration in the leaves was mortar and pestle and diluted to 1:600 for ELISA. The 1:600 100. An ELISA value was considered positive if it was four times dilution used for this study against the 1:400 dilution antibody greater than the corresponding healthy extract response. phases was previously determined to be in the linear response range Cultivar virus titer and ELISA-infectivity correlation. Triumph of the ELISA dilution curve for the antiserum used. The 64 had lower mean ELISA values compared with Eagle and experiment was repeated three times. ELISA values of individual Centurk at the 1:15 sample dilution (Table 2). However, Triumph leaves of Centurk and Triumph 64, on a given sampling day and 64 was significantly lower than Eagle only in trials I and 3 and was temperature, were subjected to ANOVA to determine the never significantly lower than Centurk at the 1:15 sample dilution. statistical reliability of the cultivar differences. A weighted mean When samples were diluted to 1:900 (within the linear range of the (average ELISA value of all leaves present, corrected for fresh dilution curve), Triumph 64 was significantly lower than Eagle and weight of each leaf) was calculated for each plant at each sampling Centurk in all trials (Table 2). time and temperature. This mean was used in ANOVA to The infectivity assay confirmed the ELISA results with the 1:900 determine if significant interactions occurred between cultivar dilution. The average percents of infection for the three trials were response and incubation temperature. 75, 66, and 45% for Eagle, Centurk, and Triumph 64, respectively. Asynchronous plant growth effects. The lengths of the third and Triumph 64 was significantly lower than Eagle and Centurk, fourth leaves of systemically infected plants of Centurk and whereas Eagle and Centurk were not different at P < 0.05. Rank Triumph 64 (14 DPI) were measured. Leaf ratios (length of fourth correlations between the 1:900 dilution ELISA and the infectivity TABLE 1. Grain yield and volume weight of WSMV-infected and healthy Centurk and Triumph 64 wheat cultivars over 8 yr at Hays, KS