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,
R.H. Ratcliffe, R.H. Shukle, and C.E. Williams (USDA-ARS); H.W.
Ohm, 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 133,540 ha (330,000 acres) of wheat in 2002, down 14 % from 2001, continuing the downward trend during the past several years. Most of the reduction in wheat acreage in Indiana is accounted for by increased soybean acreage. According to the USDA National Agricultural Statistics Service, wheat yield in Indiana averaged 3,562 kg/ha (53 bu/acre) in 2002, only 77 % of the average yield in 2000 and 80 % of the average yield in 2001. Farmers seeded an estimated 142,000 ha (350,000 acres) of winter wheat in Indiana for the 2002 harvest season, but harvested only 133,500 ha (330,000 acres). Little acreage was abandoned because of winter kill, but some acreage was abandoned from a combination of severe BYDV infection combined with spring flooding and fungal disease, largely glume blotch, in parts of southern Indiana. Probably because of strong wheat prices during autumn 2002, seeded acreage for the 2003 harvest season is estimated by the USDA National Agricultural Statistics Service at 450,000 acres in Indiana, 29 % higher than in 2001-02. Early maturing wheat cultivars in the central and northern areas of Indiana are of interest because of the continued profitability of double cropping; producing a crop of soybeans after wheat harvest in the same season.
The new SRWW licensed cultivars, INW0101, INW0102, and INW0123, performed well and are being grown by farmers for the first time in 2002-03. INW0101 and INW0102 are early, like the cultivar Clark, and should fit into double cropping with soybeans. INW0123 is similar in maturity to the cultivar Patterson. All three cultivars have Lr37, Sr38, and Yr17 resistance to populations of B. graminis in Indiana, and they have resistance to St. nodorum, S. tritici, and SBMV.
The most widespread and damaging disease on wheat in Indiana during 2002 was yellow dwarf, caused by either of two related viruses, BYDV or cereal yellow dwarf virus. The long, warm autumn of 2001 provided ample time for aphid vectors to infest wheat, reproduce, and move about in wheat fields, resulting in widespread and uniform infection. Aphids also can transmit these viruses in the spring, but autumn infections are more damaging. In statewide wheat performance trials, yellow dwarf was particularly severe at the southwest Indiana site, and there were considerable differences among entries in the percentage of plants that showed symptoms. Some entries were very susceptible, whereas others had only a low percentage of symptomatic plants. Although yellow dwarf was especially severe in southwestern Indiana, it occurred throughout the state.
Both S. tritici and St. nodorum leaf blotch were present in many fields, but did not reach the flag leaves of susceptible cultivars until late in the season. As usually observed in cultivar trials, the range of resistance between the most and the least resistant entries was small and mainly a matter of reduced severity on the flag leaf. Leaf rust was present in only trace amounts, but its annual presence indicates that fully susceptible cultivars would be at risk. Stripe rust developed well early in the season in a variety trial in southwest Indiana and was seen in other areas. Hot, dry weather that began in June halted spread of this disease.
Fusarium head blight developed to some extent around the state, but was not severe. Epidemiological studies suggest that cold weather for several weeks before and during the flowering period reduced production of inoculum.
Wheat genes responding to Hessian fly attack (Williams, Collier, Nemacheck, Sardesai, Subramanyam, and Puthoff). The expression of 20 pathogenesis-related genes was quantified by both Northern analysis and quantitative RT-PCR. Genes included in this study have been implicated in responses of dicotyledonous plants to microbial pathogens, but little was known about the responses of a similar set of genes in monocots to either pathogens or to insects. Only a few of the genes, such as PAL and PR-1, responded as predicted during incompatible interactions. Several of the genes, although responsive in resistant dicots, appeared to respond in both resistant and susceptible wheat. The majority of the genes showed little or no response to feeding by Hessian fly larvae. These data suggest that wheat defenses against the Hessian fly may be very different from the mechanisms of resistance utilized by monocots against pathogens.
Over 100 Hessian fly-responsive genes from wheat were identified by GeneCalling (Curagen Corp.). Approximately 20 % of the sequences had no match in the databases, ~ 17 % matched wheat genes of known function, and 11 % matched genes of known function from other plants. The remaining 52 % matched plant genes of unknown function. When compared to GeneCalling results from wheat/microbe interactions, the few genes that responded to Hessian fly also responded to the pathogens, indicating that undefined modes of resistance may protect wheat from the Hessian fly.
Hessian fly biotypes and evaluation of wheat germ plasm and barley lines for resistance (Cambron, Ratcliffe, and Shukle). We screened 4,675 wheat germ plasm lines and 262 barley lines for Hessian fly resistance against various laboratory-maintained biotypes. Germ plasm evaluated was received from four university breeding programs, three private companies, and three uniform nurseries that are distributed in eastern U.S. Of those, 1,226 wheat lines were screened against biotype O, which has recently been collected from the field and purified. Biotype O is avirulent to H7H8, which is still effective in the southern areas of Georgia and Alabama.
The Uniform Hessian fly Nursery was sent to 14 coöperators in the eastern U.S. with 13 nurseries returned for evaluation of Hessian fly infestation. These data are available in the 200102 Uniform Hessian fly Report.
Two Hessian fly populations, one virulent to H9 and one virulent to H13, have been isolated and increased for use in genetics studies and germ plasm evaluation. A recent acquisition of a Hessian fly biotype from Israel is being increased for use in germ plasm evaluation. This biotype has virulence to all resistance genes that have been deployed in the U.S.
Hessian fly genetics (Stuart, Behura, and Valicente). An AFLP-based genetic map has been amended and improved to correspond with the Hessian fly polytene chromosomes. The DNA sequences of 52 AFLP markers were determined and used as probes to screen a Hessian fly BAC library. Selected BAC clones corresponding to each marker were then used as probes in FISH experiments to determine the corresponding position of each AFLP on the polytene chromosomes of the Hessian fly. These data have been used to establish the correspondence between chromosomes and linkage groups and to identify chromosome regions that show high rates of genetic recombination. The region with the greatest rate of recombination is associated with the tip of the small arm of chromosome X2 in proximity to vH13, the gene conditioning virulence and avirulence to Hessian fly resistance gene H13 in wheat. A region of recombination suppression was discovered that includes and extends beyond the pericentromeric region of autosome 2, a region associated with vH3 and vH5, the genes that condition avirulence to Hessian fly resistance genes H3 and H5, respectively. Chromosome maintenance (Cm), a locus that controls paternal X chromosome elimination and sex determination in Hessian fly embryos, was positioned within 1 cM of markers on the genetic map and determined to reside near the tip of the long arm of Hessian fly autosome 1.
Nuclear and mitochondrial DNA sequence divergence in Hessian fly (Shukle, Zantoko, Yoshiyama, and Johnson). Analysis of sequence divergence in the Hessian fly mitochondrial 12S rRNA gene in populations from the United States and the Old World (southwest Asia, the Middle East, North Africa, and southern Spain) has been completed. The analyses have revealed patterns in the geographic distribution of mtDNA haplotypes in North America and in the Old World. Analysis of Wolbachia DNA in Hessian fly populations has shown that the biogeographic patterns observed for mtDNA haplotypes are not due to Wolbachia infection influencing inheritance of mtDNA variants. Several hypotheses for the biogeographic distribution of mtDNA haplotypes can be proposed for testing in future studies. The distribution of mtDNA haplotypes observed in the United States and Canada may reflect genealogical relationships among introduced populations and ancestral populations in Europe and Asia. Results can provide a basis for evaluation of evolutionary history and genetic variation in host-adapted alleles (virulence alleles) within and among populations in the United States.
The ribosomal DNA ITS2 region from Hessian fly has been cloned and intraspecific sequence variation documented. Sequence variation in introns from several nuclear genes also has been identified. Data from nuclear and mitochondrial genes can be used to develop an intraspecific phylogeny and combined with markers, such as microsatellites, to assess genetic structure and gene flow within and among Hessian fly populations in future studies.
Analysis of gene function in Hessian fly (Shukle and Yoshiyama). Microinjection has been used for delivery of DNA and dsRNA into Hessian fly embryos prior to formation of the blastoderm. A piggyBAC-based transformation vector has been used for transformation of Hessian fly with a GFP marker gene. Expression of GFP has been documented in G1 individuals but validation of transformation through genetic analysis or cytological detection of the GFP gene on polytene chromosomes in situ has not been done. Use of RNAi appears to hold promise for suppressing gene expression and generating loss-of-function phenotypes to reveal putative function. We have used RNAi to evaluate the role of a Hessian fly glutathione S-transferase (GST) gene during development of larvae on the host plant. Injection of dsRNA for the GST into embryos led to a failure of larvae developing from the embryos to survive the first instar on the host plant. Larvae developing from buffer-injected control embryos or embryos injected with a dsRNA control (dsRNA for the transposase of an endogenous Hessian fly mariner element) completed the first instar on the host plant in a normal manner. No effect from the GST dsRNA was observed on embryonic development or on the ability of hatchling larvae to infest the host plant. The deleterious effect of the GST dsRNA on larval development occurred after larvae infested the host plant. We propose that these results indicate a role for GSTs in the ability of Hessian fly larvae to deal with general defense responses of the host plant.
Expression of Hessian fly genes during interactions with wheat (Shukle, Johnson, and Yoshiyama). A program to identify Hessian fly genes differentially expressed during compatible interactions with wheat has been initiated. During the next year a pool of sequences differentially expressed in first-instar larvae during the compatible interaction will be developed. Homology searches (tBLASTx) will be used to identify known and unknown/novel sequences.
An ABC transporter gene, a glutathion S-transferase gene, and a vermilion gene from Hessian fly have been cloned and characterized. Expression of these genes during development has been documented by RT-PCR. Double-stranded RNA interference has been used to assess the role of GST expression in the interaction of Hessian flies with wheat. Results support a role for GSTs in the compatible interaction with wheat.
Markers for resistance genes (Goodwin). A bulked-segregant analysis with AFLP and microsatellite markers has lead to the mapping of five genes for resistance to S. tritici leaf blotch in wheat. The genes and their approximate chromosomal locations are Stb1 (5BL), Stb2 (3BS), Stb3 (6DS), Stb4 (7D) near the centromere, and Stb8 (7BL). Each gene has at least one linked microsatellite marker and Stb4 and Stb8 also have linked AFLPs. Stb8 is a new gene; the others were known previously but not mapped with microsatellites. The mapping populations were RIL or DH lines developed by collaborators including Stb1 (RIL population developed by Greg Shaner and George Buechley at Purdue University). Stb2 and Stb3 (DH populations supplied by Hugh Wallwork, South Australian Research and Development Institute, Adelaide, South Australia), Stb4 (RIL population provided by Jorge Dubcovsky, University of California-Davis), and Stb8 (the standard ITMI RIL mapping population).
We currently are developing quantitative RT-PCR for identification of resistant and susceptible lines in wheat. Preliminary results look promising, and we anticipate that this will provide a method to discriminate resistant from susceptible lines within 7-10 days after inoculation, well before the usual expression of Septoria tritici blotch symptoms at 18-21 days after inoculation. Our tests could then be performed on seedlings and greatly increase the amount of material tested per year.
In a collaborative project with Dr. Joe Anderson, we have used quantitative RT-PCR to determine that the protein disulfide isomerase (PDI) may be associated with the resistance response in wheat. This is the first time that PDI has been implicated in resistance in plants, although it appears to be involved in signal-transduction events in animal systems. Several other pathogenesis-related genes also were tested and were shown to be more strongly induced in resistant plants following inoculation compared to uninoculated controls.
For more information see the Goodwin lab web site at: http://www.btny.purdue.edu/USDA-ARS/Goodwin_lab/Goodwin_Lab.html, and the USDAARS/Purdue University wheat genomics web site: http://www.btny.purdue.edu/usda-ars/wheatgen/.
Resistance in wheat to Fusarium graminearum (Shaner and Buechley). We tested 49 wheat lines for resistance to FHB with both single-floret (point) and whole-spike (spray) inoculation. Disease was usually more severe with spray than with point inoculation, but a few lines showed the opposite pattern. Four lines were highly resistant after point inoculation but fully susceptible after spray inoculation, suggesting that they have a high degree of resistance to spread of the pathogen but no resistance to primary infection. We evaluated F3 progeny from tested F2 plants with point inoculation to evaluate the reliability of single-plant selection in early segregating generations. The correlation between the mean rating of the progeny and the rating of the parent plant was poor (r = 0.115, n = 243). Of the more than 2,200 heads inoculated, 48 % developed no blight symptoms. Many of these were progeny of apparently resistant plants, but some of these plants may have been escapes. Partially recessive resistance also could explain the poor correlation. We identified AFLP and microsatellite markers that are closely linked and flank a major QTL for FHB resistance on chromosome 3BS in an RIL population derived from cross 'Ning 7840/Clark'. Ning 7840 derives most of its resistance from Sumai 3. We identified additional QTL for FHB resistance on chromosomes 2BL and 2AS.
Epidemiology of Fusarium head blight. A brief warm spell in mid April was followed by 5 weeks of unusually cool weather. Although moisture was adequate for production of inoculum just before flowering, it was too cold. As temperatures rose, rainfall was insufficient for infection. We used a weather-based model developed from previous years' data to compare predicted with observed incidence of FHB. Model I, which uses weather for 7 days before anthesis, predicted less than a 25 % probability of an epidemic (defined as >10 % incidence) for all cultivar-planting date combinations. Model II, which includes both pre- and postanthesis weather, predicted less than a 20 % probability for a severe epidemic.
Fungicide research. In a fungicide trial, four treatments applied at early anthesis reduced incidence of FHB compared to the untreated control but did not eliminate the disease.
Mapping of Fusarium head blight resistance: Ning 894037 and Fundulea 201R (Shen and Ohm). QTL mapping was conducted in two wheat RIL populations derived from the crosses of the Chinese wheat line 'Ning 894037/Alondra' and 'Patterson/F201R'. Response to F. graminearum was evaluated for disease spread in greenhouse and field experiments after inoculation of a single floret in spikes. Using SSR markers and BSA, a major resistance QTL was identified in Ning 894037 on chromosome arm 3BS, in the region of markers Xgwm493, Xbarc133, and Xgwm533. A QTL with moderate effect also was identified in the moderately susceptible parental line Alondra on chromosome 2DS. These two QTL explained 42.5 % (3BS) and 12.1 % (2BS) of the phenotypic variation in the 'Ning 894037/Alondra' population. An Additional QTL with small effect was also suggested on 6B of Ning 894037. In the 'Patterson/F201R' population, four QTL were identified on chromosomes 1B, 3A, 5A, and 3D; three of which are from F201R. The QTL on 1B near Xbarc8 and 3A near Xgwm674 and Xbarc67-3, derived from F201R, account for 18.7 % and 13.0 % of the variation of resistance for FHB, respectively.
Huapei 57-2 and Bizel (Bourdoncle and Ohm). A population of RILs was developed by SSD from a cross 'Patterson/Chinese line Huapei 57-2'. RILs were evaluated for resistance to spread of the disease after inoculation of a single floret in spikes in one field experiment and two greenhouse tests. A major QTL was identified on chromosome 3BS that is flanked by markers Xgwm493 and Xgwm533 and cosegregates closely with Xbarc133 and Xbarc147. Additional QTL of smaller effects also were identified on chromosomes 3A, 3BL, and 5B. The QTL on 3BS is likely the same as the one already identified using lines derived from Sumai 3. Given its stability across populations and environments, we concluded that MAS for this QTL will be efficient.
The wheat cultivar Bizel is resistant to FHB. Given its pedigree, Blé Bohémien/rye//Oro/3/variant of Hauters, it was characterized using telomeric and dispersed rye-specific repetitive DNA sequences. We have shown conclusively that Bizel does not contain rye chromatin. Therefore, FHB resistance in Bizel is not derived from its rye progenitor. In addition, SSR markers designed for wheat and mapped across the entire genome can be used for gene tagging of FHB resistance QTL in Bizel.
Resistance to yellow dwarf viruses (Ohm, Sharma, Ayala, Balaji, and Anderson). The severe natural epidemic of yellow dwarf virus in Indiana in 2002, the most severe since 1976, enabled us to identify several advanced soft winter wheat lines that have excellent resistance to yellow dwarf transferred from wheatgrass (Th. intermedium). In field plots, the resistant lines were scored 0.5 and wheat lines without the wheatgrass-derived resistance were typically scored 3-6 (0 = no symptoms to 9 = severe plant stunting and leaf discoloration) in nurseries at Lafayette, IN. Yellow dwarf symptoms were most severe in our head-row nurseries, which were seeded 10 days earlier than the yield trials the previous autumn, our typical practice to maximize disease establishment, including yellow dwarf viruses, in head-row nurseries. In our head-row nurseries at Lafayette, lines with wheatgrass-derived resistance were scored 0.5-0.7 and wheat lines without wheatgrass resistance scored 4-8. One of the advanced wheat lines with wheatgrass resistance was in performance trials in 2001, a season in which yellow dwarf viruses infection was negligible, and its grain yield averaged 6,586 kg/ha compared to that of Patterson at 6,250 kg/ha, (LSD0.05 = 530 kg/ha). In 2002, the yield of the yellow dwarf viruses-resistant line averaged 6,498 kg/ha compared to 5,772 kg/ha for Patterson (LSD.05 = 376). Yellow dwarf viruses symptom scores for the resistant line and Patterson in 2002 averaged 0.5 and 5, respectively. We initiated seed increase of yellow dwarf viruses-resistant lines in 2002-03 for possible cultivar release.
A quantitative, reverse-transcriptase RT-PCR technique was used to detect the coat protein genes of BYDV-PAV and CYDV-RPV and examine the level of virus accumulation following infection in a yellow dwarf viruses-resistant wheatgrass, a yellow dwarf viruses-resistant wheat line, a susceptible wheat line, and a susceptible oat line. BYDV-PAV and CYDV-RPV was detected as early as 2 and 6 hrs, respectively, in susceptible oat compared to detection by ELISA at 4 and 10 days post infestation. BYDV-PAV RNA accumulated more rapidly and to a higher level than CYDV-RPV in both oat and wheat, which may account for PAV being a more prevalent, and more severe viral disease then CYDV. This technique is reproducible, sensitive, and has the potential to be used for examining susceptibility and resistance and as a rapid diagnostic tool for yellow dwarf viruses.
Several types of markers have been used to characterize Th. intermedium translocations in wheat backgrounds. Morphological markers, when available, allow the selection of individuals with foreign heterochromatin (Banks et al 1995, Genome 38:395-405) but do not delineate their genetic constitution. SSRs, although greatly facilitating the study of traits in wheat, are genome specific. When using wheat-derived SSRs, the absence of a particular wheat band has been interpreted as presence of Th. intermedium DNA. However, this means that we cannot identify heterozygous individuals. Most wheat maps have been developed using RFLPs because of their consistency. These maps have become the framework of choice for incorporating new markers. However, RFLPs typically identify just one or two polymorphisms, are laborious and time consuming. Consequently they are not the best choice when testing large numbers of individuals. In contrast, AFLPs are reproducible, reveal a number of polymorphisms in a single gel but do not readily identify the map position of such polymorphisms. To take advantage of both marker systems, we are testing a technique to develop specific markers based on AFLP using primers designed from previously mapped RFLP probes. This technique is allowing us to direct our search for polymorphisms to previously mapped sites known to contain polymorphic zones and to design specific PCR primers to use in large-scale screening.
Dr. Steve Scofield joined the small grains
group in the USDA-ARS position of biochemical geneticist and is
adjunct associate professor in the Department of Agronomy. His
research involves a genomics approach to host-pathogen interactions.
Dr. Charles Crane joined the small grains group in the USDA-ARS
position of bioinformatics specialist. Dr. Roger Ratcliffe retired
and continues to reside in the Lafayette, IN, area (we certainly
hope he continues to stay involved). Dr. Brandon Schemerhorn,
a recent graduate of the University of Notre Dame, plans to join
the Hessian fly research group as a research entomologist, USDA-ARS.
She plans to utilize molecular techniques to investigate topics
including population genetics and the evolution of virulence in
the Hessian fly. William Smith is working with D. Huber and D.
Schulze on the effect of crop sequence on Mn-transition states
in various soil types and disease incidence.