Departments of Agronomy, Entomology, and Botany and Plant Pathology, and the USDA-ARS, Purdue University, West Lafayette, IN 47907, USA.
J.M. Anderson (USDA-ARS), T. Kisha, H.W. Ohm, F.L. Patterson, and H.C. Sharma (Department of Agronomy); G. Buechley, S. Goodwin (USDA-ARS), D.M. Huber, K. Perry, and G. Shaner (Department of Botany and Plant Pathology); R.H. Ratcliffe, R. Shukle, C.E. Williams, S. Cambron, C. Collier (USDA-ARS, Crop Production and Pest Control Research Unit), and J. Stuart (Department of Entomology).
Indiana farmers harvested 510,000 acres (206,500 ha) of soft red winter wheat in 1999, at an average yield of 66 bu/acre (4,434 kg/ha), for a total production of 33.7 million bushels (0.917 million metric tons). Total production in 1999 was 94 % of production in 1998. Patterson, in its third year of production, was the leading public cultivar, occupying 16.2 % of the wheat area. Public cultivars, identified by cultivar name, occupied 30.2 % of the wheat area; private cultivars and brands, including some publicly developed lines marketed under license agreements, occupied 69.8 % of the wheat area.
Wheat was seeded in the autumn of 1998 on time, but generally dry soil conditions delayed germination. Improving soil moisture conditions coupled with unusually mild temperatures until mid to late December resulted in excellent wheat emergence and crop establishment. Night temperatures in the range of -18° to -26°C during the first 10 days of January, 1999, were preceded by snow cover over much of the state. After early January, winter temperatures were mild and there was snow cover during colder days, resulting in little or negligible winterkill. The excellent crop condition in early spring continued through the season. Take-all root rot was prevalent by early spring, but dry weather during the summer limited severe infections (obvious white heads) to early seeded fields or where wheat followed wheat or pasture in the rotation. Approximately 1-5 % of spikes were infected with F. graminearum throughout much of the state, although 25-30 % of spikes were blighted in some fields in limited areas. Powdery mildew developed early in the growing season but declined with the onset of warm, dry conditions beginning in May and continuing through harvest. Leaf and glume blotches caused by S. tritici and St. nodorum were present, but lesions did not reach upper leaves until late in the season, thus, causing little wheat yield loss. Leaf yellowing from BYDV was common, likely resulting from a large influx of aphids in early April. Leaf rust appeared soon after heading, earlier than in most seasons and developed to moderate epidemic levels, causing 2-5 % crop loss on susceptible cultivars. The warm, dry conditions continued throughout the growing season and likely limited grain fill, particularly on the later cultivars.
Biotype determination (Ratcliffe and Cambron). Hessian fly populations collected in 1999-2000 from mid-Atlantic (Delaware and Maryland); southeastern (Alabama, Georgia, and South Carolina); and northwestern states (Idaho and Washington) were evaluated for biotype composition at W. Lafayette, IN. Hessian fly populations from Delaware and Maryland and the northern third of Alabama were predominantly biotype L, which is virulent to all presently deployed resistance genes except H13. Populations from central and southern Alabama, Georgia, and South Carolina were predominantly biotype O, which is unable to survive on wheat cultivars carrying H7H8 or H13 resistance. The effectiveness of H7H8 and H13 resistance against fly populations in Georgia was demonstrated in 1999 field tests in the Uniform Hessian Fly Nursery and Georgia State Wheat trials at Griffin and Plains, GA. Hessian fly populations from northern Idaho and eastern Washington had virulence to resistance genes H3 and H6, but also consisted of 25-50% of the nonvirulent biotype GP. Thus, all presently deployed resistance genes would be moderately to highly effective to Hessian fly populations in these areas. However, virulence to H3 and H6 must be monitored carefully. Research was conducted in coöperation with personnel at the Universities of Delaware, Georgia, Idaho, and Maryland, and Auburn University, Clemson University, and Washington State University.
Effectiveness of H13 resistance (Ratcliffe, Ohm, and Patterson). The Purdue cultivar INW9811, which carries H13 resistance to biotype L, was tested against Hessian fly populations collected in 1999 from Alabama, Georgia, Idaho, and Washington and populations previously collected from Indiana, Illinois, Maryland, North Carolina, and Virginia (1989-96). INW9811 was highly resistant to all populations except those from eastern Maryland and Virginia. Tests with INW9811 are underway with the populations collected from Delaware, Maryland, North Carolina, and Virginia in 1999-2000 to evaluate the present range of H13 virulence in Hessian fly populations from the mid-Atlantic area.
Hessian fly/wheat interaction (Shukle, Yoshiyama, L. Zantoko, and Faghihi). The primary control of Hessian fly is through genetically resistant wheat. The widespread use of resistant wheat has resulted in the evolution of biotypes of the insect that can live on formerly resistant wheat. Continued effective control of the insect will require molecular markers for analysis of the genetic structure of field populations of the insect, an understanding of the molecular interaction between the insect and wheat, genetic manipulation of the insect's genome, and identification of transgenes for Hessian fly resistance. We have contributed toward genetic and molecular analyses that have revealed three genes controlling virulence in Hessian fly to resistance in wheat that are part of a linkage group. These data will make map-based positional cloning of these genes feasible. We are testing linkage between virulence in the insect to Marquillo resistance and the locus controlling sex ratio in the insect. Linkage between such loci can influence the evolution of virulence in the insect. A program of genetic manipulation of Hessian fly through transposon and viral vectors has been initiated. The use of mitochondrial markers to evaluate geographic populations of the fly from the Old World and North America also is being developed. Through collaborative research, we are testing genetic transformation of wheat with a proteinase inhibitor transgene for Hessian fly resistance.
Hessian fly map (SD Rider Jr. , W. Sun, Ratcliffe, and Stuart). An integrated physical and AFLP-based genetic map of the Hessian fly has been constructed. To facilitate mapping avirulence genes in the Hessian fly, a backcross mapping population from a single F1 female of the Hessian fly was used to construct a genetic map with AFLP markers. The map consists of 84 polymorphic loci on four discrete linkage groups and covers approximately 580-Kosambi cM. Given the extremely small size of the Hessian fly genome (98 Mb), this indicates that the physical distance per unit of recombination averages only 170 kb/cM in the Hessian fly. To determine if each of the four AFLP linkage groups is associated with one of the four Hessian fly chromosomes, we positioned arbitrary genomic clones on the polytene chromosomes of the Hessian fly by in situ hybridization. Two clones on each chromosomethen were sequenced partially so that we could convert them into PCR-based codominant genetic markers. These markers then were positioned on the AFLP-linkage map, demonstrating that each linkage group corresponds with a different Hessian fly chromosome. The AFLPs that are linked to the genes conditioning avirulence to Hessian fly resistance genes H6, H9, and H13 have been identified.
Molecular aspects of Hessian fly resistance (Williams, Collier, J.A. Nemacheck, and C. Liang). Genes are being cloned that respond to feeding by first instar Hessian fly larvae. One of these genes is similar in derived amino acid sequence to jacalin-like lectins. Jacalins have been shown to exhibit insecticidal properties against several types of insect pests. This gene, Whi-1, responds quickly to attempted feeding by avirulent larvae, being induced in leaves within 22 hrs of the onset of larval feeding. The mRNA levels increase fourfold over 3 days and then decrease. The induction of Whi-1 in leaves, distant from the larval feeding site at the base of the plant, suggests that a systemic signal is sent in response to attempted larval feeding. Whi-1 is not induced in response to feeding by virulent larvae.
G. Shaner and G. Buechley.
During the spring of 1999, the spore density of F. graminearum and other Fusarium species in the air was monitored by operation of a Burkard spore sampler. Spore density also was monitored by exposing wheat spikes for 24-hour periods, washing them, and plating the wash water on a selective medium. The Burkard sampler collected spores on most days from 23 May through 11 June, when sampling ceased. Although the highest number of spores collected occurred on the day with maximum rainfall, no consistent relation occurred between rainfall amount and number of spores collected. The number of spores collected as wheat was flowering was low; more spores were collected after anthesis. The wheat-head bioassay method of enumerating spores collected spores on only 2 days during anthesis. Disease development in the plot area was monitored by counting the number of diseased spikes in 20 rows 60-cm long throughout the plot area. Mean incidence of scab was 6.6 %. For those spikes that had symptoms of FHB, average severity (percentage of spikelets per spike blighted) was 30 %.
Resistance to F. graminearum in wheat cultivar Chokwang (Shaner and Buechley). We evaluated a recombinant-inbred population from the cross 'Chokwang/Clark' for resistance to F. graminearum. F2-derived F8 families were inoculated in the greenhouse when plants reached mid anthesis (GS 63) by applying a droplet containing conidia to a floret at the middle of the spike. The AUDPC for the progress of severity over time was the statistic used to characterize resistance. The distribution of family mean AUDPCs was bimodal. Neither mode corresponded to the parental mean, although the higher mode was only slightly below the mean for Clark. The lower mode was considerably higher than the mean for Chokwang. The pattern of inheritance is quite distinct from that observed for an inbred recombinant population derived from the cross 'Ning 7840/Clark', which leads us to suspect that the genes for resistance in Chokwang are different from those in Ning 7840.
Control of FHB and other diseases of wheat with fungicides (Shaner and Buechley). Fungicide treatments, in common with trials in several states, were applied to wheat plots at two locations in Indiana when wheat was in the early flowering stage. Some additional treatments were applied at flag leaf emergence. Incidence and severity of FHB were recorded for each treatment, as well as severity of foliar diseases. Plots were harvested for yield and grain quality assessment. Although powdery mildew was light at both locations, several fungicide treatments reduced severity compared to the untreated control, and some totally prevented development of the disease. Leaf blotch was only moderately severe. An application of BAS 500 at GS 61 or two applications of Folicur + Induce at GS 37 and GS 61 held leaf blotch to the lower canopy. Glume blotch developed at the southwest Indiana site. Most treatments that included an application at GS 61 reduced severity on spikes substantially compared to the untreated control . Treatments applied only at GS 37 were ineffective against glume blotch. Fusarium head blight occurred at low incidence at both sites. At the southwest Indiana site, where scab was somewhat more severe, no treatment had significantly less incidence than the control. Yields were high at both sites, compared to historical yields at these locations. At the west-central Indiana site, eight treatments had a significantly higher yield than the untreated control, which had the lowest yield in the trial. Four treatments at the southwest Indiana site yielded more than the control. Test weights were only fair at both sites.
T. Kisha, S. Goodwin, and H.W. Ohm.
A detached-leaf assay (Benedikz et al. 1981; Ecker et al. 1989) for St. nodorum blotch consistently separated resistant wheat lines from susceptible ones. Lesion size measured 10 days after inoculation was the most effective indicator. Latent period, the time from inoculation to the appearance of pycnidia, was almost twice as long (~ 2 weeks) in the resistant wheat lines including Coker 84-27, Cotipora, and Roazon compared to susceptible cultivar Clark. The Benzimidazole agar media used for the detached-leaf assay (Ecker et al. 1989) contained 20 g/l agar, 50 ppm benzimidazol (0.05 g/l), and 0.25 g/l chloramphenicol (add below 50°C). The agar was poured into '100 x 15 mm' petri dishes to a depth of 10 mm. Just prior to use, a '30 x 50 mm' rectangle was cut from the agar. Leaves were sampled at the three-leaf stage. The basal portion of the second leaf was cut to approximately 40 mm in length and inserted across the gap to form a bridge. Leaves were inoculated with a 2 µl drop (20,000 spores) of a suspension of 107 pycnidia spores/ml from an Indiana isolate of St. nodorum isolated from the cultivar Clark. Petri dishes were incubated at 20°C with a day/night cycle of 12h/12h. The lesion area and area of chlorosis were measured at 3, 5, 7, 10, and 14 days. Leaves were monitored daily for the formation of pycnidia.
Cultivars and lines historically observed as resistant in the field were identified consistently as resistant by the detached leaf test, whereas those known to be susceptible in the field also stood out as susceptible by the detached leaf assay . One exception was the cultivar Patterson. In the field, Patterson shows severe symptoms of St. nodorum infection but was moderately resistant in the detached-leaf assay. The leaves of Patterson are moderately resistant, but it exhibits severe symptoms of glume blotch. Resistance in glumes has been reported as genetically distinct from that in leaves (Fried and Meister 1987; Bostwick et al. 1993). A test similar to the detached-leaf assay but using detached spikelets is being evaluated. Latent period, the time from inoculation to the first appearance of pycnidia, was about 6 days in the susceptible wheats, but ranged from 11-15 days in those showing some resistance. Early appearance of secondary inoculum in susceptible wheat lines may contribute to the increased severity of symptoms observed in the field.
Projects to analyze the genetics and population biology of the S. tritici blotch pathogen (Mycosphaerella graminicola) are continuing. Phylogenetic analyses of the internal-transcribed spacer (ITS) region of the ribosomal DNA of M. graminicola and other fungi identified a large monophyletic group within the genus Mycosphaerella. This group contained fungi in the asexual genera Septoria, Cladosporium, and Cercospora, among others. The analysis revealed that the closest relative of M. graminicola is S. passerinii from barley. Six isolates of S. passerinii from cultivated barley were identical for ITS sequence, but a seventh, from the wild barley H. jubatum, was different and probably represents a new, undescribed species of Septoria that is separated from S. passerinii by host specificity. This analysis also confirmed that the gray leaf-spot disease of maize is caused by two different species of Cercospora. Both of these species were in a monophyletic Cercospora group within Mycosphaerella. Therefore, these species either have, or evolved from a species that did have, a Mycosphaerella sexual stage.
Other work with M. graminicola identified a transposable element that apparently was acquired recently. This element appears to be active during both sexual and asexual reproduction. Movement of transposable elements could be an important source of genetic variability within M. graminicola. Work is underway to obtain the complete DNA sequence of the transposable element and to test whether it can be used to analyze gene function in this organism.
Genetic analyses of M. graminicola-resistance genes in wheat are continuing. A possible new gene for resistance was identified in the 'Opata 85/synthetic' mapping population. Additional inoculation experiments identified materials segregating for the Stb4 resistance gene from Tadinia. Work is continuing to make isogenic lines for the Stb1-Stb4 resistance genes in a susceptible, spring wheat background.
Differential display analysis identified a number of genes that may be expressed during the resistance response of the cultivar Tadinia to M. graminicola. One of these clones was similar to the Xa21 gene for resistance to bacterial sheath blight in rice, another was similar to the pathogenesis-related PR10 gene from common bean, and a third was a protein disulfide isomerase that was not implicated previously in disease resistance. Northern analysis to confirm differential expression of these and other genes is continuing.
For more information, see the lab web site at: http://www.btny.purdue.edu/USDAARS/Goodwin_lab/Goodwin_Lab.html.
D. Huber, I. Thompson (graduate student in Botany & Plant Pathology), D. Schulze (Agronomy); and coöperators A. Sutton and S. Bajt, (Brookhaven and Argonne National Laboratories), M. Halsey (Monsanto Company), and S. Xiulan, P. Yufa, S. Ruiqing, and Z. Rong (China P.R.).
This research has focused on understanding the effect of cultural conditions affecting take-all, microbial interactions involved in biological control, the role of manganese in resistance of plants to take-all, and Mn oxidation as a virulence factor for Ggt.
All of the cultural conditions affecting the incidence or severity of take-all have been shown to affect the availability of Mn. Conditions such as specific precrops (oats or rice) that reduce disease severity in a subsequent wheat crop, increase Mn availability in the soil, whereas those conditions that reduce Mn availability (liming and nitrification), increase take-all severity. Manganese availability increases following oats (gray-speck resistant varieties) through inhibition of Mn-oxidizing microorganisms in the soil and a persistent effect for a subsequent wheat crop. The mechanism is quite different following paddy rice, where the anaerobic soil conditions favor Mn reduction and its increased availability for plant uptake similar to the effect of a firm seedbed.
All organisms evaluated for potential biological control of take-all increase Mn availability for the plant. Seed treatment with strong Mn-oxidizing organisms such as Agrobacterium radiobacter (may comprise from 14-20 % of the rhizosphere microbial population under severe disease conditions) increase the severity of take-all. In contrast, seed treatment with Bacillus cereus or other Mn-reducing organisms that increase Mn availability in the rhizosphere for plant uptake may reduce take-all severity. Resistance of rye to take-all is a function of its efficiency for uptake of manganese and other micronutrients.
In our evaluation of more than 60 isolates of Ggt from every major wheat production area of the world in coöperation with Claudia Heppner in Belgium, all virulent isolates were strong Mn oxidizers and could render Mn physiologically unavailable to crop plants. Some isolates of Ggt are temperature sensitive for both virulence and Mn oxidation, and a loss of virulence is associated with a loss of Mn-oxidizing ability. Using high energy, X-ray fluorescent analysis (XRF and XANES techniques), we have been able to document Mn oxidation along hyphae in bulk soil and along runner hyphae of Ggt. A Mn deficiency at the infection site can be induced by Ggt whereby resistance of the plant is compromised (Mn is required for function of the shikimic acid pathway). The loss of virulence either at specific temperatures or in general and interaction of various biological control organisms are independent of growth of the pathogen, but determined by Mn interactions in the soil and plant. We currently are evaluating various approaches to stabilize or enhance Mn availability for plant uptake.
The new seed treatment fungicide, MON65500, was highly effective in reducing take-all and increasing wheat yields in 4 years of coöperative research in China with Sheng Xiulan and researchers at the Gansu Institute of Plant Protection, Sheng Ruiqing and researchers at the Ningxia Institute of Plant Protection, Peng Yufa at the Chinese Academy of Agricultural Science, and Mark Halsey of Monsanto Company.
K. Perry and H.W. Ohm.
Ten wheat cultivars and breeding lines were evaluated to determine if they could be infected by WSBMV and WSSMV under field conditions in Indiana, Illinois (F. Kolb), and Kansas (R. Sears). Of six SRWWs tested, Clark shows high resistance to infection by both viruses. Abe and Patterson show intermediate levels of infection, and Caldwell, Cardinal, and Pioneer 2548 are relatively susceptible to infection. Two pairs of HRWW differentials were tested, and their respective resistances were effective in different cropping environments. Sierra and KS93U140 were highly resistant to infection by WSBMV, but susceptible to WSSMV. Century and KS92WGR22 were highly resistant to infection by WSSMV, but susceptible to WSBMV.
H.C. Sharma, J. Anderson, and H.W. Ohm.
Two BYDV-resistant addition lines, developed at the Beijing Academy of Agricultural Science, with either a Th. intermedium group-1 or group-2 chromosome pair were kindly provided to Purdue University by Professor Zhiyong Xin. These addition lines were crossed to cultivar Patterson and the seed irradiated with gamma rays. Approximately 213 M2 families were tested for resistance/susceptibility to BYDV subgroup 1 P-PAV. From these results, progeny of M2 families were selected and the M3 generation was tested. Currently, M3 plants are being examined molecularly to identify and characterize group-1 and group-2 translocations. In conjunction with these analyses, over 1,000 M4 plants from selected M3 families currently are being tested for BYDV resistance. The goal is to pyramid translocations of these two wheatgrass chromosomes with P29-derived BYDV-resistant translocations to achieve a high level of resistance in wheat to all BYDV strains.
In collaboration with Richard Wang and J. Zhang, genomic in situ hybridizations conclusively demonstrated that the group-7 wheatgrass chromosome in P29 is a 7E-genome chromosome and not an St-genome chromosome.
Desirable translocations with BYDV resistance from group-7 chromosome of Th. intermedium developed from germ plasm line P29, and its sister addition and substitution lines are being incorporated for germ plasm and cultivar development. Translocation lines derived from the BYDV-resistant P29 line have been identified that contain small translocations and are BYDV resistant. Field trials are underway to determine the effectiveness of this resistance under field conditions.
In our attempt to identify additional sources of BYDV-resistance genes in the close relatives of wheat, susceptible accessions and a potentially resistant accession of T. urartu are being retested for confirmation of reaction to BYDV. The original bulk seed of the accession that had shown resistance turned out to have both resistant and susceptible plants in a large-scale test. Thus, single plant progenies are being tested.
Ismail Dweikat accepted a position of sorghum breeding at the University of Nebraska. Victoria Zismann, technician in the lab of S. Goodwin, left to accept a position at The Institute for Genomic Research (TIGR).