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a Dep. of Plant Pathology, North Dakota State Univ., Fargo, ND 58105
b Dep. of Plant Sciences, North Dakota State Univ., Fargo, ND 58105
c Agriculture and Agri-Foods Canada, Winnipeg, MB, R3T 2M9 Canada
d USDA-ARS, Northern Crops Research Laboratory, Fargo, ND 58105
* Corresponding author (rstack{at}ndsuext.nodak.edu)
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Abbreviations: FDK, Fusarium damaged kernels • FHB, Fusarium head blight • LDN(DIC), Langdon durum- T. dicoccoides substitution lines • QTL, quantitative trait loci • TDIC, Triticum turgidum var. dicoccoides
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Outbreaks of FHB on wheat are unpredictable and highly dependent on weather conditions during and just following anthesis, when plants are at the most susceptible stage (Sutton, 1982). On spring wheat (Triticum aestivum L.) and durum (T. turgidum L. var. durum) in the northern Great Plains region of North America, the epidemics of 1993, 1994, and 1997 were very severe, with more localized losses occurring in other years between 1995 and 2000. Previous outbreaks, on a more limited scale, had occurred sporadically in the region since the early 1980s (McMullen et al., 1997; Wilcoxson et al., 1988; Windels, 2000).
Management practices such as crop rotation have been ineffective in limiting the disease. Most researchers agree that incorporation of resistance is the best long-term answer to FHB (Meidaner, 1997). Sources of resistance to FHB are available in hexaploid wheat (Mesterhazy, 1995); however, transfer of that resistance from hexaploid wheat to durum has not been reported. Durum wheat cultivars differ in their response to FHB from moderately to highly susceptible (McMullen et al., 1994; Stack, 1988; Stack and Elias, 1995–2000, unpublished).
The greatest progress to date in identification of resistance to FHB has been with resistance expressed as limitation of the spread of infection within the spike (Wang and Miller, 1988). This phenotypic expression has been termed "Type II" resistance in the literature (Meidaner, 1997; Mesterhazy, 1995). Molecular markers for quantitative trait loci (QTL) associated with Type II resistance have been identified in hexaploid wheat (Anderson et al., 2001; Waldron et al., 1999).
Triticum turgidum L. var. dicoccoides (TDIC) possesses many interesting traits, including resistance to stem rust (Puccinia graminis Pers.) (Miller et al., 1998), resistance to stripe rust (P. striiformis Pers.) (Reinhold et al., 1983), resistance to FHB (Miller et al., 1998), and grain quality factors (Joppa et al., 1991). A set of disomic chromosome substitution lines from TDIC in the background of Langdon durum had been developed as described by Joppa and Williams (1988) using the TDIC line FA-15-3 ("Israel A"). A gene for high grain protein concentration in this population has been studied (Joppa and Cantrell, 1990; Steiger et al., 1996) and mapped (Joppa et al., 1997). These lines have also been evaluated for agronomic traits and grain quality (Cantrell and Joppa, 1991; Elias et al., 1996; Joppa et al., 1991).
In a search for potentially useful sources of resistance to FHB in durum, we tested FHB reactions of several hundred durum lines and accessions (Stack and Elias, 1994, unpublished). In one such screening test in 1994, we included several of the disomic chromosome substitution lines developed for high grain protein concentration (Joppa and Williams, 1988).
On the basis of results from those preliminary tests, we decided to examine the entire set of the LDN(DIC) disomic chromosome substitution lines for reaction to FHB. The purpose of this study was to determine if any of the LDN(DIC) lines showed useful levels of resistance to FHB. This was done by inoculation with F. graminearum by a method and under conditions which minimized the effect of environmental and plant development factors. A preliminary report of a portion of the work presented in this paper has appeared (Stack et al., 1999).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The TDIC line FA-15-3 ("Israel A"), the source of the substituted chromosomes, was not included in the tests of the LDN(DIC) lines, but was tested for FHB reaction separately in replicated experiments evaluating TDIC accessions. Those experiments were done under conditions similar to the tests of the LDN(DIC) lines (Miller et al., 1998).
The set of 13 LDN-DIC lines were tested for FHB response in experiments across five environments. The five experiments were conducted from 1996 through 1998 in a controlled-environment greenhouse. Each experiment was arranged in a randomized complete block design with experimental units consisting of a single row of plants of a particular genotype. The LDN(DIC) lines and durum checks were randomly assigned to the rows of the replicate blocks.
Three experimental durum lines from the NDSU durum breeding program were used as checks. D91103 was included in all five tests as a moderately resistant entry. The NDSU breeding lines D87450 and D88541 were included as susceptible checks. D91103 has consistently shown one of the lowest FHB severity scores among numerous durum lines in many screening trials since 1995 (R.W. Stack, 1999, unpublished). D91103 was not released as it lacks acceptable grain quality (E.M. Elias, 1997, unpublished). Similarly, D87450 and D88541 have been used in numerous trials as susceptible check lines. All three lines also grow well in the greenhouse environment during the off-season (R.W. Stack, 1999, unpublished).
Plant Culture
In each of the five repeated experiments (Table 1), plants were grown in a temperature controlled greenhouse at 18 ± 2°C through the early stages of plant growth. From anthesis onward [after Feekes 10.0 (Simmons et al., 1985)], the greenhouse controls were set to maintain 20 to 25°C where possible. Actual temperatures during the incubation period for each experiment are given in Table 1.
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Plants were grown in a soil mix or a commercial soil-less medium as specified in Table 1. During preparation, both substrates received additions of ground dolomitic limestone (692 g m-3), magnesium-ammonium-phosphate fertilizer (166 g m-3 7-17-6 +12 Mg) ("MagAmp-K" W.R. Grace & Co., Columbia, MD). The pH was adjusted to 6.5 with Ca(OH)2 on the basis of a soil test.
Throughout each experiment, plants were watered as needed, usually twice weekly, and fertilized weekly with 3 g L-1 of a complete soluble fertilizer (15-30-15, "Miracle Gro", Scotts Miracle Gro Products, Port Washington, NY) applied to runoff. Supplemental lighting to increase daylength to 16 h was provided by lighting fixtures spaced 2.5 m apart each way in a grid located 3 m above the soil surface and fitted with 400-W wide spectrum high pressure sodium bulbs ("Lucalox LU400", General Electric Co., Cleveland, OH). Plants were grown to anthesis and inoculated. After inoculation, plants were maintained under the same greenhouse conditions through the incubation period and to maturity.
Inoculum Preparation
Preparation of inoculum and inoculation of plants generally followed published methods (Stack, 1989). Briefly, cultures of F. graminearum were grown in the laboratory on petri dishes of carnation leaf agar or half strength potato dextrose agar for 15 to 20 d at room temperature (22–24°C) under fluorescent lights. To prepare conidiospore inoculum, petri dish cultures were flooded with sterile distilled water and gently agitated; the resultant spore suspension was poured off and strained through several layers of sterile cheesecloth to remove hyphal fragments. Concentration was adjusted to 50 000 conidiospores mL-1. Freshly prepared spore suspension was held on crushed ice and used within 4 h of preparation.
Three strains of F. graminearum were used in all experiments. Each strain was isolated originally from symptomatic plants and had been tested repeatedly for pathogenicity under a range of environments. Each strain was maintained, cultured, and prepared separately, then spore suspensions of equal concentration were mixed together immediately before use. All three cultures have maintained strong disease-causing ability by these methods (Mitchell Fetch et al., 1998; Stack et al., 1997; Waldron et al., 1999).
Inoculation
Inoculations were done by the single spikelet method, in which inoculum is placed into a single spikelet near the middle of each spike at anthesis (Stack, 1989). This method of inoculation selectively targets the kind of FHB physiological resistance expressed as a limitation of spread of infection within the spike, also called "Type II" resistance (Mesterhazy, 1995; Wang and Miller, 1988). Spikelet inoculation also excludes differences due to morphological and developmental factors—either genotypic or phenotypic—which can produce differences in FHB expression.
Anthesis is recognized as the time of peak susceptibility to FHB infection. To reduce the effect of differences in maturity, individual rows were inoculated as they flowered. Within each row of plants, 10 spikes at mid- to late anthesis (Feekes 10.52) were selected. Spikes to be inoculated were marked with colored paper tags so each could be identified later for disease scoring at the proper time. A 10-µL droplet of F. graminearum conidial suspension was placed into one flowering spikelet near the middle of each selected spike with a repeating syringe dispenser (Nichiryo model #8100, Nichiryo, Ltd., Tokyo, Japan). At the conidiospore concentration used (50 000 mL-1), each droplet contained approximately 500 conidiospores. This concentration was chosen to give maximum incidence while not obscuring expression of Type II resistance. Spikes in each replicate row of each line were inoculated only once, as that row flowered.
Following inoculation, plants were lightly misted with a hand fogging nozzle ("Fogg-it" Dramm Corp., Manitowoc, WI) attached to a watering hose. To maintain high humidity after misting, plants were covered with a plastic tent. Misting and covering were done on three successive nights after inoculation. The plastic tent was opened during the day to prevent overheating. Following the third night, no further misting or covering was done. In all experiments, numerous noninoculated spikes provided a check on both inoculation technique and for unintended secondary spread of infection.
FHB Evaluation
At 3 to 3.5 wk post-inoculation, plants were evaluated for FHB. Each inoculated spike was individually examined and the extent of blight scored on a 0 to 100% scale (Stack and McMullen, 1995). Disease incidence was recorded as the percentage of inoculated spikes in the row showing any symptoms of FHB. A representative selection of the noninoculated spikes also was examined for symptoms in the same manner. Following scoring, plants were grown to maturity. In three of the five experiments, inoculated spikes were harvested at maturity and threshed. The proportion of Fusarium damaged kernels (FDK) in the harvested grain was visually determined (Clear and Patrick, 2001) as a second measure of disease severity (Ittu et al., 2000; Meidaner, 1997)
Data Analysis
For each experiment, analysis of variance was performed on the FHB scores and mean separation was determined by use of Fisher's protected least significant difference (LSD) at 
= 0.05. A combined analysis of variance was also done and the experiment x genotype interaction variance was used to test for the presence of significant experiment to experiment variation. In addition, Bartlett's test for homogeneity of error variance was done. The correlation between the proportion of Fusarium damaged kernels in each line and the FHB severity scores of those lines was determined for each experiment where both measures were taken.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Langdon durum (FHB severity = 51%) showed an intermediate reaction relative to the durum check lines. The mean FHB severity score of all the LDN(DIC) lines for each experiment did not differ from Langdon (Table 3). In three separate experiments, TDIC FA-15-3 was tested for FHB along with many TDIC accessions. TDIC FA-15-3 showed a highly susceptible reaction in these tests. The FHB severity of FA-15-3 was 100% in a range of 10 to 100%, 82.4% in a range of 11 to 83%, and 88.4% in a range of 19 to 99% (R.W. Stack and J.D. Miller, 1997, 2000, unpublished).
Comparisons were made between the LDN(DIC) substitution lines and the Langdon durum parent. The LDN(DIC-3A) line showed the lowest FHB severity score in all experiments, significantly lower than Langdon in those experiments where both were present (Table 3). The FHB scores of three other lines, LDN(DIC-6B), LDN(DIC-4B), and LDN(DIC-1A) were greater than LDN(DIC-3A) but significantly less than Langdon in some individual experiments (Table 3).
LDN(DIC-2A) was the most susceptible to FHB in all experiments (Table 3). LDN(DIC-2A) also had a significantly greater FHB score than the most susceptible durum check line D88541. Three other lines, LDN(DIC-7A), LDN(DIC-1B), and LDN(DIC-6A), were significantly more susceptible than Langdon in some experiments (Table 3).
The other LDN(DIC) lines showed intermediate responses. Within that intermediate group, rankings varied from experiment to experiment but none was significantly different from Langdon. Since each LDN(DIC) line differs by a chromosome pair, the results suggest that the substitution lines with intermediate FHB scores do not carry genes for FHB reaction that differ from those in Langdon.
Another measure of plant damage from FHB infection is proportion of Fusarium damaged kernels (FDK) observed in the grain harvested from infected spikes. In three of the trials, harvested grain was visually examined for FDK. In our tests, the proportion of such kernels in the grain closely reflected the FHB disease severity scores for all the lines. The correlations between percent FDK and FHB severity for the genotype means within the three experiments were: r = 0.86 (P < 0.001, n = 15) for Exp. 1; r = 0.87 (P < 0.001, n = 16) for Exp 2; and r = 0.95 (P < 0.001, n = 16) for Exp 5.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The presence of progeny with a high level of resistance in a cross between a moderately susceptible (Langdon) and a highly susceptible (FA-15-3) line may be due to transgressive segregation. In hexaploid wheat, the widely used FHB resistance source ‘Sumai-3’ is itself derived from such a cross (Liu and Wang, 1990), and other examples have been reported in this pathosystem (Ittu et al., 2000).
The low phenotypic FHB score of LDN(DIC-3A) may result from a gene or genes conditioning resistance located on chromosome 3A, in which case it was derived from the TDIC parent. Alternatively, the gene or genes conditioning resistance reside on one or more other chromosomes in Langdon, and the chromosome 3A of Langdon has some factor that is epistatic or inhibitory to the expression of that resistance. Substituting the TDIC 3A would then remove that factor, allowing the resistance to be expressed. Two lines of evidence support the former hypothesis. First, using another set of disomic substitution lines in which corresponding D genome chromosomes from a hexaploid ‘Chinese Spring’ wheat were substituted for Langdon A or B chromosomes (Joppa and Williams, 1988), we found that the LDN(3D-3A) line did not differ in FHB severity from Langdon (R.W. Stack, 2000, unpublished). Second, the marker studies of Otto et al. (2002), examining the segregation of resistance in a population derived from Langdon/LDN(DIC-3A), also support the hypothesis that a gene conditioning resistance to FHB is present on chromosome 3A of TDIC FA-15-3 (Otto et al., 2002).
TDIC FA-15-3 shows a highly susceptible phenotypic reaction despite the apparent presence of a strong gene for resistance to FHB. The substitution of TDIC 2A for Langdon 2A produces a line (LDN(DIC-2A)) in which the FHB score is significantly higher than Langdon itself. This chromosome may contain a factor that is epistatic to other factors that control or limit the expression of the FHB resistance on 3A, resulting in the susceptible phenotypic response observed in TDIC FA-15-3. Gilbert et al. (2000) found evidence of gene interaction for FHB response in hexaploid wheat. Alternatively, Langdon 2A may be the site of gene(s) conditioning the intermediate resistance of Langdon to FHB. Testing of the LDN(2D-2A) substitution line (Joppa and Williams, 1988) might help to clarify this.
Recently, chromosomal locations of QTL responsible for FHB resistance in hexaploid wheat have been proposed (Anderson et al., 2001; Bai et al., 1999; Waldron et al., 1999). Chromosome 3A was not among those identified by those authors, but was reported by Buerstmayr et al. (1997) in one of two genotypes. The resistance found in LDN(DIC-3A) may or may not be at the same locus. If not, it may have value for additional gene pyramiding for FHB resistance in hexaploid wheat as well as durum. That possibility is further strengthened since the resistance is expressed in the same manner (Type II) as the major resistance on other chromosomes in hexaploid wheat.
Further study is needed to confirm if any of the other three lines [LDN(DIC-6B), LDN(DIC-4B), and LDN(DIC-1A)], which tend toward lower FHB severity also possess heritable resistance. The factor on chromosome 2A that appears to confer increased susceptibility should also be studied. If it were found to be widespread in durum lines, it might help to explain why so many durums are susceptible to FHB.
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ACKNOWLEDGMENTS |
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Received for publication March 23, 2001.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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