INDIANA
PURDUE UNIVERSITY
Departments of Agronomy, Entomology, and Botany and Plant Pathology, and the USDA-ARS, Purdue University, West Lafayette, IN 47907, USA.
J.M. Anderson, S.E. Cambron, C.C. Collier, C. Crane, S.B. Goodwin, A. Johnson, J.A. Nemacheck, S. Scofield, B. Schemerhorn, R.H. Ratcliffe, R.H. Shukle, and C.E. Williams (USDA-ARS); H.W. Ohm, L. Kong, F.L. Patterson, H.C. Sharma, and J. Uphaus (Department of Agronomy); G. Buechley, D. Huber, G. Shaner, and J.R. Xu (Department of Botany and Plant Pathology); and J. Stuart (Department of Entomology).
Indiana farmers harvested 174,089 hectares (430,000 acres) of wheat in 2003, up 30 % from 2002. According to the USDA National Agricultural Statistics Service, wheat yield in Indiana averaged 4,439 kg/ha (66 bu/acre) in 2003, up 25 % from the average yield in 2002. Little acreage was abandoned due to winter kill. Increasing interest in early maturing wheat cultivars in central and northern areas of Indiana is because of the continued profitability of double cropping; producing a crop of soybeans after wheat harvest in the same season.
Three new SRWW licensed cultivars, INW0301, INW0304, and INW0316, were released and seed is being increased. INW0301, tested as P92226E2-5-3, has H9 and H13 for resistance to Hessian fly biotype L, Lr37-Sr38-Yr17, resistance to powdery mildew, leaf blotch, and soilborne mosaic virus, and has performed well in the midsouth U.S., likely due to its Hessian fly resistance. INW0304, tested as P97395B1-4-3-8, has moderate type-2 resistance to FHBt, resistance to glume blotch, leaf blotch, leaf rust, powdery mildew, SBWMV, and has H13. INW0316, tested as P961341A3-2-2, has resistance to yellow dwarf viruses from intermediate wheatgrass, Th. intermedium. All three cultivars are early, with heading dates similar to that of the cultivar Patterson.
Leaf and glume blotch were severe in the southern part of the state on susceptible cultivars. Septoria tritici persisted later into the season and blighted upper leaves more than in recent years, probably because of cool, wet weather during much of May. Stagonospora nodorum also was present and caused glume blotch. Stripe rust was evident as focal infections during May and early June but did not cause significant damage. Warm weather halted progress of the disease. Fusarium head blight was significant in southern Indiana this year, with up to 60 % infected spikes in some fields. In northern Indiana, up to 15 % of spikes were infected in many fields. Unusually cool weather throughout the state, especially at night during most of the grain-filling period, likely delayed development and spread of the disease after infection. Temperatures were much warmer beginning about 20 June, which was about 1 week prior to physiological maturity, and the disease began to spread significantly in infected spikes. The fungus did not develop during much of the grain-filling period because of cool temperatures, which allowed kernels to develop normally, and visibly scabby kernels were not conspicuous in harvested grain. However, fungal growth late in grain-filling produced significant toxin. Thus, levels of deoxynivalenol were higher than expected because kernels appeared fairly normal and grain quality was satisfactory.
Resistance (Wiangjun, Ayala, Thompson, Balaji, and Anderson). The incorporation of intermediate wheatgrass -derived resistance gene(s) into improved wheat germ plasm generated a wheat substitution line, P29, which is completely resistant to cereal yellow dwarf virus (CYDV). The undetectable CYDV titer in P29 and related wheat lines led many to conclude that the resistance prevented viral replication. The purpose of this study is to test the hypothesis that Th. intermedium-derived resistance allows CYDV to replicate within the initially infected cells but inhibits viral movement. To determine whether CYDV replication or movement is inhibited, we examined the inoculated leaf for replication and uninoculated leaves for systemic spread. CYDV subgenomic RNA, produced only during replication, was found within the inoculated portion of the leaves of P29 and Th. intermedium, demonstrating that viral replication was not affected. The absence from the uninoculated, newly emerging leaves of inoculated P29 and Th. intermedium plants indicated the inhibition of viral systemic infection. Resistance could be overcome, resulting in a systemic spread of CYDV, if P29 was inoculated at the 1-leaf stage or younger with 50 to 100 viruliferous aphids/plant. As these infected P29 seedlings continued to grow, the resistance phenotype was recovered. However, when 7 to 10 aphids were used, the resistance was maintained across all developmental stages tested.
Our data suggest that Th. intermedium-derived resistance to CYDV was primarily dosage-dependent and could be developmentally regulated if the amount of inoculum was large enough. When testing for wheatgrass-derived resistance, the seedlings must be at least at the 2-leaf stage prior to inoculation with BYDV or CYDV. Based on our data, we propose three possible models for the mechanism of Th. intermedium-derived resistance to CYDV: 1) in the phloem-restriction model, once delivered into the plants, CYDV is restricted to the initially infected companion cells where it replicates, 2) in the inhibition of long-distance transport model, CYDV can spread from the infected companion cells into adjacent sieve elements but its movement beyond this point is blocked, or 3) in the inhibition of reëntry model, CYDV can move systemically through sieve elements but cannot reënter the companion cells at distal locations for replication. The cellular localization of CYDV within the inoculated leaves of P29 is currently being investigated to test these models.
To increase the resolution of the RFLP-based map from Th. intermedium-wheat M4 recombinant lines (Crasta et al. 2000; Genome 43:698-706), we added PCR-derived markers. To validate our mapping results, several M4 recombinant lines were analyzed by GISH and repetitive FISH DNA probes. Two M4 lines also were crossed with wheat and the F2 progeny were examined for the presence of Th. intermedium segregating fragments. From this data we identified several diagnostic markers that conclusively identify field selections derived from Th. intermedium-containing material. This increased resolution also showed that, in most of the recombinant lines tested, the chromosome T7D·7E appeared as a mosaic of wheat and Th. intermedium chromatin sections. These lines are being examined in more detail to more fully understand the mosaic nature of these chromosomes.
Genetics and germ plasm (Sharma, Shen, Kong, and Ohm). We have research in progress to shorten the 7E chromosome segments in P961341 and KS24-2-11. P961341 is a Purdue line that has YDV resistance from intermediate wheatgrass and has the distal approximately three-fourths of the long arm of chromosome 7E from Th. intermedium. The YDV resistance is subtelomeric on 7EL. KS24-2-11 is a translocation line provided to us by K. Armstrong (Armstrong et al. 1993; Theor Appl Genet 85:561-567) that has FHB resistance from tall wheatgrass (Lophopyrum ponticum). The FHB resistance is proximal to the centromere on the long arm of chromosome 7El2. We are characterizing segregating populations from a cross of 'P961341/CS Ph1' deletion line and segregating populations from a cross 'KS24-2-11/CS Ph1' deletion line.
We also are characterizing and genotyping segregating populations from a cross 'K11463/K2620'. K11463 and K2620 are substitution lines (Armstrong et al. 1993; Theor Appl Genet 85:561-567). K11463 has chromosome 7El1 and is susceptible to FHB. K2620 has chromosome 7El2 and has resistance to FHB. Our objective is to achieve recombination between 7El1 and 7El2 to identify marker loci along the two chromosomes.
Mapping (Goodwin lab). Mapping of five genes for resistance to Septoria tritici blotch in wheat was completed last year. Work is now in progress to map QTL for resistance to this disease in a collaborative project with Dr. Hugh Wallwork in Australia and to backcross the five previously identified single genes into a common susceptible background to use as differential cultivars.
We also are exploring quantitative RT-PCR for identification of resistant and susceptible lines of wheat to Septoria tritici blotch. Two approaches are being pursued 1) to measure fungal biomass and 2) to quantify gene expression in the host. Analysis of resistant and susceptible wheat plants from 1-27 days after inoculation showed that fungal biomass remained low in both resistant and susceptible plants until 12-14 days after inoculation. At this time, the fungal biomass increased rapidly in susceptible plants but remained near zero in the resistant plants. This may provide an accurate method of separating resistant and susceptible plants before symptoms are expressed fully.
To identify resistant plants even sooner, we are looking at the expression of 30 host genes during a time course and intensively at 12 hours after inoculation. Preliminary results indicate large differences between resistant and susceptible plants for the levels of gene expression, with a much higher expression in resistant plants compared to susceptible controls depending on the specific gene analyzed. Our goal is to identify resistant and susceptible plants at 12 hours after inoculation, without having to wait for 18 or more days for symptoms to be expressed. A rapid PCR test could greatly increase the throughout and efficiency of our resistance screening efforts compared to the usual greenhouse tests. However, it may be necessary to tailor the technique for each specific resistance gene analyzed.
Chemical control (Shaner and Buechley). Fungicides were evaluated for efficacy against FHB. Corn residue was the source of inoculum; mist irrigation promoted inoculum production and infection by G. zeae. Fungicides were applied at Feekes GS 10, 10.5, 10.51, or 10.52. All fungicide treatments, even those applied at GS 10, reduced incidence of head blight. Only the JAU6476 and V-10116 treatments applied during flowering reduced blight severity (the percentage of the head blighted on those heads that showed any blight). Most treatments reduced the number of Fusarium-damaged kernels. Several treatments applied at flowering reduced the DON level in grain. Three treatments increased yield significantly.
Epidemiology (Shaner and Buechley). We compared the effect of three densities of corn residue on the soil surface on abundance of G. zeae inoculum and FHB development. Early in the season, more spores of G. zeae were captured with Burkard volumetric samplers in plots with corn residue than in the plot with no residue. Later, no differences in spore capture among residue treatments were observed. Residue density had no effect on incidence or severity of FHB across three cultivars of different maturity that had each been planted at two dates. The general favorable weather for head blight largely obscured differences in local inoculum density.
Ratings in state wheat variety trials at four locations revealed differences among entries in incidence and severity of FHB. Correlations between Fusarium-damaged kernels and FHB index (an overall rating of head blight intensity, based on both incidence and severity) were low. Likewise, correlations between DON and FHB index or Fusarium-damaged kernels were low. None of these correlations had predictive value, meaning that grain quality, in terms of visible damage or DON, could not be predicted from head blight intensity, nor could DON content be predicted from amount of Fusarium-damaged kernels.
Germ plasm (Shaner and Buechley). We found a moderate but significant correlation between type-II and type-I resistance in repeated experiments (r = 0.75, P = 0.0000) among lines originally selected for type-II resistance. Between the two experiments, expression of type-II resistance was reasonably consistent (r = 0.63, P = 0.0002), but the expression of type I resistance was inconsistent (r = -0.04, P = 0.83). The poor correlation between expression of type-I resistance between the two experiments was largely the result of several lines that had a low severity in the first experiment but a high severity in the second experiment, which may have been related to use of different kinds of plastic bags used to cover heads for 48 h after inoculation.
For evaluation of type-I resistance, we sprayed heads with a suspension of conidia of F. graminearum. We compared two inoculum levels (2 x 10^4^ versus 4 x 10^4^ conidia/milliliter), two growth stages (GS 10.52 versus 10.54), and two patterns of inoculation (spray one side of head versus spray both sides). Cultivars with a range of resistance were used. Main effects of each factor were highly significant. A few interactions between cultivar and the other variables also were significant.
We evaluated type-II resistance in a population
of RILs derived from a cross between wheat cultivars Chokwang
(moderately resistant) and Clark (susceptible). The correlation
between experiments for number of blighted spikelets 22 days after
inoculation was 0.51. Transgressive segregation for greater resistance
in Clark was greater than that shown by Chokwang.
Wheat Hessian fly interactions (Collier, Nemacheck, Puthoff, Sardesai, Subramanyam, Giovanini, and Williams). The expression of two closely related Hessian fly-responsive wheat genes, responding in incompatible interactions, were compared, using quantitative RT-PCR. One gene, Wci-1, was found to be a general stress-response gene that is up-regulated by aphids, virus infection, wounding, and various chemical elicitors of plant defense responses, in addition to the Hessian fly. Hfr-1, however, responded in a more specific way to Hessian fly and SA only. The up-regulation of Hfr-1 was most dramatic in leaf sheath regions adjacent to larval feeding sites, although some systemic response was evident in leaf blades. These findings were confirmed in plant lines containing four different single Hessian fly-resistance genes infested with three different fly genotypes.
The wheat gene Hfr-2 is up-regulated during compatible interactions, primarily near larval feeding sites. The expression of this gene increases dramatically in proportion to the level of larval infestation. The sequence of this gene appears to encode two domains, one similar to seed-storage proteins and the other similar to pore-forming proteins. This gene is not expressed in developing seeds, however. This gene may be partially responsible for the disruption of cell membrane integrity leading to leakage of water and nutrients for larval consumption.
Defense-response genes involved in hypersensitive response and oxidative burst appear to contribute little to wheat defense against the Hessian fly. Genes encoding several key enzymes such as NADPH oxidase, catalase, and superoxide dismutase do not respond to Hessian fly feeding. No response was visible when infested tissues were stained for the accumulation of peroxide with DAB (diaminobenzidine), an indication that the hypersensitive response is not active. Also, DPI (diphenylene iodonium), an inhibitor of the production of reactive oxygen species, did not allow the survival of avirulent larvae on treated resistant plants.
Avirulence genes (Stuart). Close linkage (3±2 cM) was discovered between Hessian fly avirulence genes vH3 and vH5 on Hessian fly autosome 2 (A2). Bulked-segregant analysis discovered two DNA markers (28-178 and 23-201) within 10 cM of these loci and only 3±2 cM apart. However, 28-178 was located in the middle of the short arm of Hessian fly chromosome A2, whereas 23-201 was located in the middle of the long arm of chromosome A2, suggesting the presence of severe recombination suppression over the proximal region of A2. To further test that possibility, an AFLP-based genetic map of the Hessian fly genome was constructed. Fluorescence in situ hybridization of 20 markers on the genetic map to the polytene chromosomes of the Hessian fly indicated there was good correspondence between the linkage groups and the four Hessian fly chromosomes. The physically anchored genetic map is the first of any gall midge species. The proximal region of mitotic chromosome A2 makes up 30 % of its length but corresponded to less than 3 % of the chromosome A2 genetic map.
Hessian fly populations (Alber, Johnson, Lu, Mittapalli, Schemerhorn, and Shukle). The historic record states there was a single introduction of Hessian fly into the United States. However, to date this hypothesis has not been tested. We are using mitochondrial genes and transposable element display (TE display) to reveal relationships within and between fly populations in the United and between these populations and populations in the Old World. Results are testing hypotheses concerning a single introduction into the U.S. and the presumed center of origin for the pest in the southern Caucasus of southwest Asia.
The salivary glands and midgut are of importance in the feeding of Hessian fly larvae on wheat. The role of salivary glands is being addressed by the ARS team at Manhattan, KS. We are cloning and characterizing midgut genes encoding enzymes involved in digestion. We hope to reveal basic information concerning digestive physiology in the pest and apply this knowledge to develop novel strategies toward achieving more durable resistance in wheat. We are using RNAi in experiments directed toward silencing/suppressing expression of midgut proteases in Hessian fly larvae and in testing compensation by the midgut proteases. Additionally, we hope to utilize WSMV to express the Bowman-Birk protease inhibitor in wheat and assess its effect on the development of Hessian fly larvae. If successful, this will establish WSMV as a transient expression vector to bioassay potential transgenes for Hessian fly resistance in wheat. This coupled with knowledge emerging from the C. E. Williams laboratory identifying promoters for wheat genes up-regulated in the compatible interaction of Hessian fly larvae with wheat may allow site specific expression of transgenes for resistance in the plant, greatly facilitating the specificity and efficacy of transgenic resistance.
We have brought into culture two Hessian fly populations from Israel that display virulence to a wide range of resistance genes in wheat. From these populations we are selecting Hessian fly lines pure breeding for virulence to new resistance genes (e.g., H31 and H25). These lines will be used by the Stuart laboratory at Purdue to develop mapping populations for fly genes controlling virulence to the resistance genes and by the Williams laboratory to reveal gene expression in wheat containing the resistance genes during compatible and incompatible interactions.
William Bourdoncle, Ph.D. student with Herb Ohm, completed degree requirements and has accepted a corn breeding position with the Monsanto company in France. David Drake, Ph.D. student with Herb Ohm, completed degree requirements and is Extension Agronomist, Utah State University, Richfield, UT. Ousmane Boukar, Ph.D. student with Herb Ohm, completed degree requirements and is Scientific Director, IRAD Agriculture Research Station, Maroua, Cameroon.