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Dep. of Agronomy, Univ. of Kentucky, Lexington, KY 40546-0091
* Corresponding author (dtekrony{at}uky.edu).
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Abbreviations: AA, accelerated aging germination • CLA, carnation leaf agar • DAA, days after anthesis • DON, deoxynivalenol • FDS, Fusarium damaged seeds • FHB, Fusarium head blight • PDA, potato dextrose agar • PM, physiological maturity • SI, Fusarium graminearum seed infection • SG, standard germination
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Infection by F. graminearum may affect both the physical and physiological aspects of seed quality, including seed size and weight, composition, and quality. Bechtel et al. (1985) reported that even slightly infected seeds with apparently uninfected embryos exhibited reduced seed germination. A negative correlation was also reported between SI and germination (Haflon-Meiri et al., 1979; Tuite et al., 1990). A similar negative association has been shown between visual estimates of FHB and germination (Bechtel et al., 1985). Infected seed is often contaminated with DON, a mycotoxin produced by F. graminearum. Contamination of grain with DON has been significantly correlated with SI (Snijders and Krechting, 1992; Trigo-Stockli et al., 1998). Miller and Young (1985) reported that time of harvest may influence the concentration of DON in the seed.
Wheat cultivar differences in resistance to FHB were first reported in 1891. Active mechanisms of resistance to F. graminearum infection include resistance to initial infection (Type I resistance) and resistance to spread of infection within a head of the plant (Type II resistance) (Schroder and Christensen, 1963). Several studies have reported differences between susceptible and resistant cultivars both in severity of infection and modes of resistance (Bai and Shaner, 1996; Mesterhazy et al., 1999; Ribichich et al., 2000), but none of these studies assessed the effects of FHB resistance on seed quality throughout seed development in a field environment. Likewise, little information is available on when peak infection occurs during seed development and maturation and how infection levels relate to seed germination and vigor, and the production of DON.
The objective of this study was to determine the effect of infection by Fusarium graminearum during soft red wheat seed development on the production of DON and seed quality across several cultivars with variable levels of Type II resistance to FHB.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Corn seed infected with F. graminearum was distributed in the plots of each cultivar on 24 Apr. 2000 and 24 Mar. 2001. The corn seed inoculation procedure was modified from Fauzi and Paulitz (1994). Corn seed inoculated with a mixture of 13 local isolates of F. graminearum obtained from wheat fields in various production regions in Kentucky was distributed uniformly (
35.5 g m-2) among the wheat rows. Isolate mixture was obtained by planting seed on selective agar medium and transferring mycelium of cultures to carnation leaf agar (CLA) to induce sporulation. Pure culture was obtained by single-spore isolation and increased on potato dextrose agar (PDA).
Plots were six rows spaced 0.20 m apart and 36.6 m long, with seeds spaced 0.178 m apart within rows. Each plot was divided into 15 ranges (1.22 m x 1.83 m) and was established in a randomized complete block design with two replications. At anthesis, 1200 heads at the Feekes 10.2 development stage for each cultivar were tagged with an adhesive label on the pedicel from four middle rows of the 15 ranges (80 heads per range). Plots were mist irrigated at heading and irrigation continued until 26 May 2000 and 4 June 2001. Plots were irrigated during two periods daily, at 15-min intervals, for a duration of 5 min from 600 to 800 h and at 20-min intervals for 10 min from 1800 to 2000 h.
Starting 10 DAA, 75 to 80 tagged heads were randomly harvested from each cultivar by cutting the pedicel immediately below the head. Harvests continued at 4-d intervals until harvest maturity (first time the seed moisture declined to <140 g kg-1). A total of 10 or 11 harvests were made in all cultivars (concluding at 50 DAA), except in P2552 in 2000, where seven harvests were made. Harvested heads were placed in zipper bags and kept on ice during transport to the laboratory.
At each harvest, a group of 30 heads was selected at random and visually scored for FHB incidence (infected heads/number heads scored), severity (infected spikelets/number of spikelets per head), and plot severity (incidence x severity) according to Stack and McMullen (1995). (All measures of infected heads and spikelets were based on visual symptoms.) Following this evaluation, heads were dried in cloth bags at 30°C and held at 10°C before threshing and testing for seed quality.
Twenty-five additional heads were threshed and 100 fresh seeds were selected randomly and evaluated visually for Fusarium damage and infection. Visual ratings of Fusarium damaged seeds (FDS) were conducted by classifying seeds into normal (plump, normal color, no visual infection), moderately damaged (discolored or slightly shriveled with normal color) and severely damaged (shriveled, pinkish-white—referred to as tombstone). From the same 25 heads in each cultivar, 100 fresh seeds were plated for evaluation of SI. Following surface sterilization with NaOCl (1% available chlorine) for four minutes (Haflon-Meiri et al., 1979), seeds were plated on modified PDA (39.5 g PDA + 1 L water + 1.5 g pentachloronitrobenzene [nitropentachlorobenzene] and 0.5 g chloramphenicol {2,2-Dichloro-N-[2-hydroxy-1-(hydroxymethyl)-2-(4-nitrophenyl)ethyl]acetamide}). Plates were cultured at 25°C under incandescent light and examined at 7 and 14 d for SI using gross colony morphology as described by Tuite et al. (1990). Several representative colonies from infected seeds in 2001 were speciated on CLA according to Nelson et al. (1983) to assist in F. graminearum colony identification.
Twenty-five spikes from each harvest were also analyzed for DON content. Seed and chaff were separated from spikes by hand threshing and dried at 30°C to 12% moisture content before grinding the seeds. Five-gram samples were submitted to the laboratory of L. P. Hart, Michigan State University in 2000 for analysis of DON as described previously (Hart and Braselton, 1983). In 2001, 5-g seed samples were ground in a coffee mill for 12 to 15 s, stored in sealed plastic bags at 10°C before analyzing for DON using direct competitive enzyme-linked immunosorbent assay with an EZ-Quant Vomitoxin (DON) plate kit (Beacon Analytical Systems Inc., Portland, ME). Twenty-five milliliters of sterile distilled water was added to the 5-g ground sample (a 5:1 ratio of water to seed sample was used if 5 g of seed was not available). Samples were shaken for 10 s in 50-mL plastic centrifuge tubes, allowed to settle for 2 to 3 min, and filtered through no. 4 Whatman filter paper into glass vials. Although the amount of filtered aliquot varied among samples, a minimum of 100 µL was required for analysis of DON content.
Standard germination was determined on four 50-seed samples (from a 30-head sample) in rolled towels at 20°C for 7d following a prechilling treatment of imbibed seeds at 10°C for 5 d as described in Association of Official Seed Analysts (AOSA, 2000). A first count was made 4 d after placement into 20°C by recording those normal seedlings with a coleoptile > 2 cm in length. A final count was made at 7 d for normal and abnormal seedlings and dead seed as described by AOSA (2000). In 2001, the fungicide tebuconazole {alpha-[2-(4-chlorophenyl)ethyl]-alpha-(1,1-dimethylethyl)-} was applied to subsamples at a rate of 1 mL 1000 g-1 seeds before SG testing.
Accelerated aging germination, a stress vigor test, was conducted by placing 20 g of seed over 50 mL deionized water and aging at 43°C for 72 h (Hampton and TeKrony, 1995) before testing for germination as described previously. For immature seeds from the early harvests, 200 seeds were counted, weighed, and the dry seed (up to 5 g) were placed in fine mesh screens inside a larger screen. The remainder of the larger screen was filled with a control wheat seed lot to bring total seed sample for aging to 20 g. Following aging, planted seeds were prechilled at 10°C for 5 d, then tested for germination. The conductivity vigor test was used to determine membrane integrity by placing four replications of 50 seeds in 75 mL of deionized water, covered with aluminum foil and holding at 25°C for 24 h before measuring electrical conductivity as described by Hampton and TeKrony (1995).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Physiological maturity (maximum dry seed weight, PM) occurred at 28 (Coker 9474), 29 (Roane), 31 (P25R18), and 32 DAA (P2552) (data not shown). Seed moisture at PM ranged from 430 g kg-1 for P2552 to 470 g kg-1 for P25R18 and Coker 9474 on a fresh weight basis. Similar rates of dry weight accumulation occurred across cultivars during development, ranging from 1.2 (Coker 9474) to 1.1 (P25R18) mg per seed per day (data not shown).
Seeds produced in 2000 showed few visible signs of F. graminearum infection until late in development. Likewise, SI remained very low (<16%) before PM in all cultivars except P2552 and increased to maximum levels after PM (Fig. 1). Infection was highest in P2552, the most susceptible cultivar, exceeding 60% from 48 DAA onward and lowest for Coker 9474 (<25% at all harvests). Deoxynivalenol concentration during development remained low in all cultivars, ranging from <0.5 mg kg-1 for Coker 9474 to 4.2 mg kg-1 for P2552 (Fig. 1).
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2001
Seed infection was severe in 2001, which provided for visual assessment of disease severity and the measurement of SI and seed quality under epidemic FHB conditions. Physiological maturity occurred from 30 to 32 DAA for all cultivars. Seed moisture at PM ranged from 420 g kg-1 for Roane to 470 g kg-1 for P25R18 and declined steadily during development (data not shown). Dry seed weight means in all cultivars ranged from 22 to 28 mg per seed at PM, which was significantly lower than dry seed weight of control samples grown in noninoculated plots.
Favorable temperatures and irrigation during sporulation produced an abundance of primary inoculum in 2001. Fusarium head blight infection symptoms were visible at 5 to 7 DAA, including brown, water-soaked lesions and whitening of infected spikelets. Incidence of FHB increased rapidly in all cultivars and exceeded 80% by 27 DAA (data not shown). Average FHB severity in harvested heads also increased rapidly during the early stages of seed development (Harvests 1 to 5, when symptoms were visible) and ranged from 7% for Coker 9474 at 11 DAA to 37% for P2555 at 27 DAA (Fig. 2A). Visual assessment of seeds for Harvests 3 to 10 showed a consistent increase in the severely FDS at each harvest for all cultivars (Fig. 2B). Seed damage was most evident in P2552 reaching a maximum of 63% at 45 DAA, whereas Coker 9474 and Pioneer 25R18 had the fewest damaged seeds, which did not exceed 30% at any harvest date.
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20% at 10 DAA to maximum levels of >95% at 37 to 40 DAA, which were maintained until the final harvest (50 DAA) (Fig. 3). Seed infection increased rapidly between 18 and 36 DAA, exceeding 65% in all cultivars at 30 DAA (PM). Seed infection for Coker 9474 was slightly lower than the other cultivars until 30 DAA, when all cultivars exhibited similar infection levels. There were significant correlations between SI and visual ratings of FHB severity of head infection (r = 0.67, P < 0.01) and Fusarium-damaged seeds (r = 0.64, P < 0.01) across all cultivars (Table 1).
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Standard germination of untreated seed of the four cultivars from early harvests was highly variable (Fig. 4A), ranging from <40% for Coker 9474 at 10 DAA, to >85% for P25R18 at 15 DAA. Germination of all cultivars declined to unacceptable commercial quality (<80%) by 25 DAA and continued to decline to 30% at the last harvest. Dead seeds comprised >50% of untreated seed samples in Roane and Coker 9474 from 40 to 50 DAA and >60% in P2552 (data not shown). There was a significant negative relationship between SG of untreated seed and SI (Fig. 5).
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31 DAA) the SG of treated seed was lowest in P2552 and highest in P25R18 (52 and 74%, respectively), but below acceptable commercial quality for all cultivars. In contrast to untreated seed, there was little decline in the SG of treated seed after PM in all cultivars. Accelerated aging germination was variable among cultivars across the early harvest dates ranging from 39% for P2552 to 78% for Coker 9474 (Fig. 6). The AA did not decline after PM (>31 DAA), which was similar to the trends for SG of treated seed (Fig. 5B) and the two tests were highly correlated (r = 0.92) across all sampling dates. However, there was little relationship between AA and SI, DON, or SG of untreated seeds across all cultivars and harvest dates (Table 1).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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High levels of F. graminearum present during seed development and maturation in 2001 resulted in unacceptable seed germination and vigor. Standard germination declined to below acceptable commercial quality (<80%) early in development (22 DAA) when SI was <20% (Fig. 3) and continued to decline to near 30% SG for all cultivars at the final harvest. This resulted in a significant negative relationship between SG of untreated seed and SI (Fig. 5). Fungicide seed treatment significantly improved the germination of all cultivars as SI was increasing from PM to harvest (Fig. 4). Unfortunately, germination of treated seed was still below acceptable commercial quality for planting seed purposes.
Seed vigor as measured by AA remained low (50–70%), but levels stabilized after PM in 2001, as did the SG of treated seed. Despite SI levels in excess of 95%, AA did not decline following PM, implying that physiological seed quality of the remaining viable seed was relatively robust (Fig. 6). Thus, when the pathogen was eliminated either by seed treatment or high temperature stress during AA, there were few abnormal seedlings and germination improved compared with germination of untreated seeds. A high percentage of the nongerminated seed at the conclusion of both the AA and SG (treated seed) tests were dead, indicating that the embryos were infected and nonviable.
Conductivity readings from infected seeds in 2001 were high early in seed development, but declined during seed maturation, as reported by Rasyad et al. (1990) for disease-free seed. High levels of SI that occurred late in seed development (Fig. 3) had little effect on membrane permeability since conductivity levels remained consistently low after PM. This suggests that the mature pericarp remains intact and membrane integrity remains high even in severely infected seeds. It also indicates that conductivity is not an effective test for measuring seed vigor of wheat seed infected with FHB, which had 30% dead seed in the sample.
Moderate to high levels of Type II resistance to FHB in Coker 9474, Roane, and P25R18 did not reduce SI in 2001 and resulted in only marginal improvement in germination and seed vigor (AA) compared with the highly susceptible cultivar P2552. The level of SI was similar in both susceptible and resistant cultivars (Fig. 3), indicating Type II resistance did not slow the rate of SI in blighted heads. When FHB plant infection occurred in experimental plots in 2002 (David VanSanford, 2002, personal communication) under natural field conditions, similar levels of SI (50 to 60%) occurred across the same four cultivars. Thus, Type II resistance would seem to be of limited value in preventing SI in the field or improving seed germination and vigor.
Type II resistance was more closely associated with DON contamination in 2001 than in 2000, with the most susceptible cultivar P2552 having the highest levels of DON at all harvests compared with the lowest levels observed in the resistant P25R18 (Fig. 3). These results support the findings of Mirocha et al. (1994) that resistant cultivars had lower DON levels than susceptible cultivars. Snijders and Krechting (1992) also speculated that DON was more likely to be excluded from the seeds of resistant genotypes. However, DON levels in all cultivars during seed development and maturation in 2001 were well in excess of the acceptable limits for finished grain products (1–2 mg kg-1).
Contamination by DON was not closely associated with SI and seed quality during seed development and maturation across all cultivars in 2001. Although DON was present at significant levels as early as 10 DAA, it remained within a narrow range throughout seed development in Coker 9474, Roane, and P25R18. These results conflict with those reported by Trigo-Stockli et al. (1998) and Snijders and Krechting (1992), where DON was significantly correlated with SI. Likewise, Tuite et al. (1990) showed a significant negative relationship between DON and SG. However, most of the previous relationships between DON, SG, and SI were reported for fully developed, mature seed, not for seed harvested at all stages of seed development and maturation. Our data indicate rapid movement of DON into the assimilate stream and translocation throughout the wheat spike in the field environment. This result agrees with Savard et al. (2000), who also reported rapid movement of DON in the wheat spike following point inoculation.
Severe disease pressure and subsequent SI resulted in unacceptable seed quality in 2001. Although type II resistance reduced DON and visual seed damage in resistant cultivars, the advantages were not apparent in reducing SI. Therefore, Type II resistance may function to increase grain quality and mitigate yield loss, but did not reduce SI, and had little effect on improving subsequent seed germination and vigor during a severe FHB epidemic. Thus, a seed producer must use the same management practices (reduce inoculum, timely harvest, and fungicide seed treatment) for susceptible and resistant cultivars in an attempt to reduce SI and improve seed quality.
Received for publication August 23, 2002.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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