GrainGenes

ITEMS FROM THE UNITED STATES

 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 (USDA-ARS), W.A. Berzonsky, I.M. Dweikat, D. McFatridge, H.W. Ohm, F.L. Patterson, and H.C. Sharma (Department of Agronomy); G. Buechley, S. Goodwin (USDA-ARS), D. Huber, K. Perry, and G. Shaner (Department of Botany and Plant Pathology); R.H. Ratcliffe, C.E. Williams, S.E. Cambron, F. Maas, M. Jones, C. Collier, C. Liang, and D. Fasoula (USDA-ARS, Crop Production and Pest Control Research Unit (Department of Entomology)).

Wheat production.

Indiana farmers harvested 660,000 acres (267,000 ha) of soft red winter wheat in 1997, at an average yield of 58 bu/A (3,639 kg/ha), for a total production of 38.3 million bushels (0.972 million metric tons). Total production was 140 % of total production in 1996. Clark was the leading public cultivar, occupying 14 % of the wheat area, down from 15.9 % the previous year. The new cultivar Patterson occupied 9.4 % of the area. In total, public cultivars occupied 35.4 % of the wheat area; private cultivars and brands occupied 64.6 % of the wheat area. Some of the private brands are publicly developed lines.

Weather and disease development.

Disease development was retarded on Indiana's wheat crop because of a cool spring. Dry weather during April prevented spread of leaf blotch and other foliar diseases. Temperatures remained low during the wet weather of May, which continued to retard disease spread. When temperatures finally rose, leaf and glume blotch developed rapidly, but generally too late to do much damage. Leaf rust was reported to be abundant in states south of Indiana but very little rust developed here. Powdery mildew developed in the lower to mid canopy in some southern Indiana fields.

One disease favored by the cool weather was wheat yellow mosaic. The persistence of cool weather until after flag leaf emergence allowed mosaic and yellowing symptoms to appear on upper leaves. Although most cultivars grown in Indiana are resistant or moderately resistant to yellow mosaic, some proved to be very susceptible and most of the flag leaf area was yellow by the time wheat had finished flowering. This destruction of photosynthetic area on the upper leaves probably resulted in considerable reduction in yield.

Although rain fell during the time some wheat in Indiana was flowering, scab generally was not severe. Cool, dry weather during early spring may have prevented maturation of ascospores of Gibberella zeae, so that even though rain fell during anthesis, little inoculum was being produced. Scab could be found in many fields, but usually no more than 1 % of the heads were affected, and the incidence of infection was much lower in many fields.

Research on plant pathogens.

Scab (Buechley and Shaner). The Korean winter wheat cultivar Chokwang has shown some resistance to scab. We evaluated a recombinant inbred population of 'Chokwang x Clark' in the greenhouse. Family mean AUDPCs were approximately normally distributed and ranged from highly resistant (symptoms only on inoculated floret) to complete blighting of heads. Several foliar fungicides were applied at various growth stages from flag leaf emergence to full heading and compared for efficacy against scab. Several treatments reduced severity of powdery mildew, leaf blotch, and glume blotch, but none provided control of scab. Scab incidence was only moderate in this test (11 % incidence in the untreated check, with an average severity on scabbed heads of 68 %). Four seed treatments (Raxil-thiram, Raxil XT, LS174, and Dividend + Apron XL) applied to scabby seed (from the 1996 crop) were compared in a field trial sown in the fall of 1997. Average emergence based on live seed planted was 71 %, and no differences occurred among treatments.

Six cultivars with different degrees of resistance to scab were inoculated using six different procedures to refine greenhouse screening methods. Inoculum was introduced into a central floret with a syringe or on a small piece of cotton that had been dipped in a spore suspension. Heads subsequently were incubated by covering them with glassine or plastic bags or placing the plants in a mist chamber for 3 days. Both cultivar and inoculation method had large effects on AUDPC. The 'cultivar x treatment' interaction was also significant, but only about 1/5 the magnitude of the cultivar effect alone. Sumai 3 and Ning 7840 expressed a high degree of resistance under all treatments. Clark and Patterson had severe scab with all treatments except for the cotton-glassine bag treatment, which overall produced the least scab development. Freedom and Frontana were more variable among inoculation treatments than very susceptible or very resistant cultivars. For reliability and speed, we conclude that syringe inoculation, followed by placing plants in a moist chamber for 3 days, is best.

In a second experiment, cultivars representing a range of resistance to scab were inoculated by injecting spores into one central floret or by injecting spores into tip, middle, and basal florets. Most cultivars developed symptoms more quickly when inoculated with the 3-point method. Freedom and Ning 7840 showed a high degree of resistance regardless of inoculation method. Sumai 3 had more than twice as much scab with 3-point inoculation than with 1-point inoculation. Kernel weights were lower following 3-point inoculation compared to 1-point inoculation. Most cultivars yielded fewer grams per head following 3-point inoculation. Freedom and Ning 7840 yielded equally well with either inoculation method. Inoculation of Freedom reduced yield 39 % compared to the uninoculated control. Yield of inoculated Ning 7840 was slightly greater than the uninoculated control.

Fifty Italian wheats obtained from the USDA­NSGC were screened for scab resistance using the single floret inoculation, moist chamber method. Among 105 plants tested, AUDPC ranged from 0.08­19. Sixty-five plants with AUDPC < 11 were saved for further evaluation in the autumn of 1997. These selected lines were inoculated by the 1-point method or by spraying a spore suspension on heads at anthesis. Some lines showed resistance regardless of method of inoculation; others were resistant when inoculated by the 1-point method but moderately or fully susceptible when inoculum was sprayed onto the head. Many of the original accessions from the NSGC proved to be heterogeneous for scab reaction. Some selections that showed good resistance to both 1-point and spray inoculation were from the following accessions: Oscar V (CItr 15128), Fubav D (CItr 15163), Marhein 26 (CItr 15164), Victor V (CItr 15124), and Funo (CItr 14349). Seed of the resistant selections is being multiplied and will be deposited back in the NSGC.

Partial resistance to leaf rust (Shaner and Buechley). We completed the third year of a field study of partial resistance to wheat leaf rust in a recombinant inbred population of F8 families from a cross between cultivars CI 13227 and Suwon 92. In the greenhouse, latent period of P. recondita, a major component of partial resistance in CI 13227, is controlled by four genes with epistatic effects. The field study was conducted to determine if partial resistance would be detectable under more natural field conditions, and if degree of partial resistance was related to length of latent period. Large differences in rust development occurred among families in the field. Severity of rust in the field was correlated negatively to length of latent period as measured in the greenhouse (R = ­0.694). All families with long latent period had a low severity of rust in the field. Some families with shorter latent periods (a susceptible trait) also had a low severity of rust in the field, presumably because they possess other components of partial resistance (small pustules, fewer spores per uredinium). The fact that partial resistance was expressed clearly in the field, using single-row, 1-m-long plots, where movement of inoculum between plots would occur, indicates that partial resistance can be selected reliably in standard breeding nurseries that use the same small plot configuration.

Septoria (Goodwin). We have continued investigating the population biology of the Septoria pathogens, with the goal of determining whether gene flow occurrs among populations of the pathogens infecting the major market classes of wheat in the central United States. Isolates of M. graminicola (Septoria tritici blotch), Phaeosphaeria nodorum (Septoria nodorum blotch), and of the barley pathogen Septoria passerinii were collected in the midwestern and north central states and analyzed for RAPD variation. Most isolates were genetically distinct, except those from different lesions on the same leaf. For M. graminicola, the ascospores are probably the primary sources of inoculum, and subsequent spread on a leaf occurs by asexual pycnidiospores.

All three species (M. graminicola, P. nodorum, and S. passerinii) were distinguished easily by RAPD analysis alone. Genetic analyses of M. graminicola were performed using progeny of a cross between two Dutch isolates of the fungus provided by Dr. G. H. J. Kema (Wageningen, The Netherlands). Analyses of more than 100 putative RAPD loci on 99 progeny isolates revealed that most loci segregated according to Mendelian expectation. Several loci with probable 'codominant' alleles were identified. The putative alleles were cloned and sequenced, revealing that each in fact represented a different allele at a single genetic locus. The alleles differed by insertions or deletions of 20­60 base pairs. Specific primers now are being made to convert these into sequence-tagged sites for additional studies of the genetics and population biology of these organisms. These data are being combined with AFLP data from Dr. Kema to produce the first genetic map of this fungus. Work with the host lead to the identification of an AFLP marker that is linked to a gene for Septoria resistance in wheat at a map distance of approximately 10 cM. Work is continuing to find additional linked markers and to convert the linked AFLP marker into a sequence tagged site. Degenerate primers were used to amplify and clone 300 resistance gene analogs (RGAs) from wheat. Analyses with 4- and 6-base cutting restriction enzymes categorized the 300 clones into approximately 10 classes. Representative clones from each class were sequenced and characterized. Each had the characteristics of known resistance genes and thus was a true RGA. Work is continuing to determine whether any of these RGAs segregates with known resistance genes. For more information, see the lab web site at: http://www.btny.purdue.edu/USDA-ARS/Goodwin_lab/Goodwin_Lab.html

Barley yellow dwarf virus (Sharma, Anderson, Ohm, and Perry). Wheat germplasm P29 that carries resistance to BYDV on an introgressed group 7E chromosome from the wheatgrass Th. intermedium was released. This line was examined for the level of resistance to subgroup I and subgroup II BYDV strains. In P29 plants inoculated with two different subgroup I PAV strains, the titer of virus in leaf and stem tissue was reduced 42­52 % when compared with the BYDV susceptible cultivar Abe. P29 and Abe had the same content of PAV in roots. These results and the absence of detectable virus in inoculated Th. intermedium plants indicate that the complete wheatgrass resistance to subgroup I has not been introgressed into P29. In contrast, P29 was completely resistant throughout the plant to the subgroup II strains, NY-RPV and NY-RMV. Experiments are underway to determine if addition/substitution lines containing this 7E chromosome have enhanced resistance to subgroup I strains when in combination with resistance contained on group I or group II Th. intermedium chromosomes.

Because P29 is a substitution line containing an entire wheatgrass chromosome, it is not suitable for breeding for BYDV resistance. Consequently, this line was irradiated to induce chromosomal translocations as a means of retaining BYDV resistance, while reducing the amount of alien chromatin. Following gamma irradiation and selfing, the standard segregation analysis approach identified 17 putative translocation lines of which three were translocations as confirmed by RFLP analysis. The success rate of identifying BYDV resistant and susceptible translocation lines from irradiated seed using segregation analyses was quite low (4.0 %). However, analysis of susceptible progeny of irradiated seed with a repetitive sequence specific to the alien chromosome quickly demonstrated a high rate of deletions in the introgressed alien chromosome (58 %). A comparative RFLP analysis of susceptible lines containing alien chromatin, their resistant sister lines, and other resistant lines showed that 36 % of the progeny of gamma-irradiated seed had deletions in the alien chromosome. This approach of initially identifying BYDV-susceptible deletion lines using an alien chromosome-specific repetitive sequence followed by RFLP analysis of their resistant sister lines rapidly identified resistant translocation/deletion lines and localized the BYDV resistance to the distal end of 7EL. Several of the susceptible and resistant translocations were selected for further characterization, and the smallest resistant translocations are being incorporated into elite wheat lines.

Chromosome pairing analysis and ELISA of hybrids between the alien addition lines P114 and P25 that had been recovered from the 'wheat-intermediate wheatgrass' cross established that P114 and P25 both carry the same chromosome from the wheatgrass that imparts BYDV resistance. Additionally, P29 and P12 substitution lines were found to contain the same 7E chromosome, confirming our previous results based on RFLP analysis.

Evaluation by ELISA of 65 accessions of wild and primitive wheats and of Aegilops species did not reveal resistance to P-PAV isolate of BYDV. One accession (G 3248) of T. urartu appeared to be promising, but its resistance needs to be confirmed.

Research on Hessian fly.

Insect surveys (Cambron and Ratcliffe). Twenty-six Hessian fly-resistant wheat cultivars or germplasm lines were evaluated in Uniform Hessian Fly Nursery Trials in Alabama (1), Arkansas (2), Georgia (2), Indiana (3), and South Carolina (4). Wheats carrying Hessian fly resistance genes H9, H12, H13, H16, H21, or H26 were the most effective, although H9 was less effective in the trial at Griffin, Georgia. Twenty-six wheat lines or cultivars from the Uniform Hessian Fly Nursery and 33 wheat lines each from the Uniform Southern and Uniform Eastern Soft Red Winter Wheat Nurseries were evaluated for response to Hessian fly biotypes GP, B, C, D, E, and L in laboratory tests. The response of wheat lines to the Hessian fly biotypes will be published in 1998 in the USDA­ARS, Special Report 'Hessian Fly Status Report for Crop Year 1996­97', available from S. Cambron. Hessian fly populations from Alabama, Florida, and Mississippi were tested for biotype composition. Biotype L was identified from populations in northern Alabama and southern Mississippi but not in southern Alabama or northern Florida. Several thousand wheat germplasm lines and plant introduction (PI) accessions from federal, state, and commercial programs were evaluated for resistance to various Hessian fly biotypes as part of an ongoing effort to identify new sources of resistance in common wheat germplasm, and to assess progress in incorporating Hessian fly resistance in soft winter wheat breeding lines and cultivars. Results of tests with PI accessions will be available from the Germplasm Resources Information Network (GRIN).

Tolerance in wheat to Hessian fly (Ratcliffe and Gumaelius). Progeny of four selections from Pioneer 2555 soft red winter wheat that demonstrated tolerance to Hessian fly biotype E at 18°C were tested for antibiosis and tolerance to fly biotypes E and L at 18° and 26°C. Plants demonstrating antibiosis, tolerance, or both antibiosis and tolerance were selected from each group of progeny. Tolerant plants were not stunted and had normal green color even though infested with normally developing fly larvae. Plants expressing antibiosis always contained dead first instar larvae, and, in some cases, both dead and living larvae. Resistance was considered to be associated only with tolerance when living, but not dead, larvae were found on phenotyically resistant plants. The resistance gene(s) associated with antibiosis was not identified in selections of Pioneer 2555, but loss of antibiosis resistance in some plants when tested at 26°C indicated the presence of a temperature-sensitive gene(s). The percentages of plants from progeny of the Pioneer 2555 selections that demonstrated tolerance to Hessian fly biotypes E and L were 17.4 % and 4.7 %, respectively. Fifty-eight percent of the 'tolerant' plants were susceptible to biotype GP, indicating the absence of antibiosis genes (H3, H5, H6, and H7H8) in these selections.

Seneca wheat, which carries the Hessian fly resistance gene combination H7H8 and is susceptible to biotype L, demonstrated a form of tolerance when tested against biotype L at 27°C. Phenotypically, Seneca was susceptible to biotype L at 18°, 24°, and 27°C, but at 27°C, 75 % of the infested plants appeared to 'grow out' of the stunting caused by larval feeding, even though larvae survived. Monon (H3), Magnum (H5), and Caldwell (H6), which also are susceptible to Hessian fly biotype L, were stunted severely at all temperatures.

Hessian fly genetics (Ratcliffe in cooperation with J.J. Stuart, Department of Entomology, Purdue University). Pupae of male Hessian flies were exposed to four different doses of gamma radiation (0, 15, 30, 40, and 60 Gy) and mated to untreated females. The fertility of females and their F1, F2, and F3 offspring were evaluated. Incomplete fertility (semisterility) in several lines segregated as a dominant genetic marker, indicating that semisterility could be used as a dominant phenotypic marker to follow the inheritance of chromosome rearrangements in these lines. Polytene chromosomes of 26 progenies produced by semisterile females derived from 40 Gy-exposed males were examined for the presence of chromosome rearrangements. Three independent rearrangements were identified among the larvae in these progenies. Therefore, radiation-induced semisterility can be used as a dominant genetic marker for chromosome rearrangements in the Hessian fly.

Molecular markers linked to Hessian fly-resistance genes in wheat (Williams, Jones, Liang, Fasoula, Collier in cooperation with H. Ohm and F. Patterson). AFLP markers were identified for a new Hessian fly resistance gene that was introgressed from durum into soft red winter wheat. Five new wheat populations were established to map the location of this and other new resistance genes. Hessian fly tests demonstrated that resistance of each of these five sources is conferred by a single unique locus. Crosses have been, made and RFLP polymorphisms have been identified between parents of a mapping population containing resistance gene H20.

Resistance gene analogs (Dweikat and Ohm). Several research groups have reported cloning of resistance genes to bacterial, fungal, or viral pathogens in various plant hosts. What has linked several of these studies is the identification of genes that appear to encode members of a signal transduction pathway that share the unusually similar features of leucine-rich repeats as well as nucleotide binding domains. Because the majority of plant resistance genes that have been cloned contain similar sequence motifs, screening the genome of wheat for the presence of genes containing such motifs will allow us to test for cosegregation of these resistance gene analogs with already identified Hessian fly resistance genes. To test this, bulked genomic DNAs prepared from 11 near isolines, each containing a different Hessian fly resistance gene, were used for degenerate primer PCR amplification designed to target regions containing sequence motifs similar to those identified in several resistance genes previously cloned by others.

We have produced a collection of PCR products migrating at approximately 500 bp in size. The 500 bp amplification products were extracted from an agarose gel and ligated to vector, producing approximately 1,300 transformants. Clones have been derived from the PCR amplification products, and several have been sequenced and tested by DNA gel blot hybridizations and specific PCR amplifications using cultivar Newton, the susceptible parent, and near-isolines. Mapping of identified PCR-based polymorphisms has detected complete linkage of three polymorphisms to resistance genes H6, H11, and H16 in F2 populations of 50 progeny each.

Personnel.

William Berzonsky has accepted an Assistant Professor position, responsible for hard white spring wheat breeding and genetics, at North Dakota State University, Fargo. Oswald Crasta, postdoctoral scientist with J. Anderson, has accepted a Research Scientist position with Curagen, a gene discovery company in Conneticut. David Drake completed the M.S. degree and is continuing graduate studies with H. Ohm. Ann Greene, postdoctoral scientist with J. Anderson, has accepted a postdoctoral position at North Carolina State University. Luke Gumaelius began studies toward the M.S. degree under the direction of R. Ratcliffe. Michael Francki, postdoctoral scientist with J. Anderson, accepted a Senior Research Scientist position in the Department of Botany at University of Western Australia at Nedlands. Xueyi Hu, postdoctoral scientist with S. Goodwin, accepted a Research Scientist position with The Quaker Oats Company, Inc. and will be located at Hohhot, China. Maria Elisa Manetti completed the M.S. degree with H. Ohm and has returned to Argentina.

Publications.

Anderson JM, Bucholtz DL, Greene AE, Francki MG, Gray SM, Sharma HC, Ohm HW, and Perry KL. 1998. Characterization of wheatgrass-derived barley yellow dwarf virus resistance in a wheat alien chromosome substitution line. Phytopathology 88:(Accepted December 4, 1997).

Anderson JM, Bucholtz DL, Crasta O, Greene AE, Francki MG, Sharma HC, and Ohm HW. 1998. Effectiveness of wheatgrass-derived barley yellow dwarf virus resistance and identification of resistant translocation lines. 7th Inter Cong Plant Path, Edinburgh, Scotland, UK.

Anderson JM, Crasta O, Francki MG, Bucholtz DL, Sharma HC, and Ohm HW. 1998. Molecular and cytogenetic analysis of barley yellow dwarf virus resistant translocation lines containing Thinopyrum intermedium chromosomal segments. PAGV, San Diego, CA. Abstract P151.

Berzonsky WA, Francki MG, Anderson JM, and Ohm HW. 1997. A comparison of wheat/rye centromere regions. Agron Abstr 89:151.

Boukar O and Ohm HW. 1997. Relation between floret opening and incidence of scab in wheat. Agron Abstr 89:86.

Collier CC, Ohm HW, Jones MJ, and Williams CE. 1998. Molecular markers to track the pyramiding of several new Hessian fly resistance genes into a single line of common wheat. PAGV, San Diego, CA. Abstract P159.

Cook VM and Ohm HW. 1997. DNA markers associated with resistance to barley yellow dwarf virus in oats. Agron Abstr 89:83.

Dweikat IM and Ohm