Wheat Fhb1 encodes a chimeric lectin with agglutinin domains and a pore-forming toxin-like domain conferring resistance to Fusarium head blight.

Fusarium head blight (FHB), caused by Fusarium graminearum, is a devastating disease of wheat and barley that leads to reduced yield and mycotoxin contamination of grain, making it unfit for human consumption. FHB is a global problem, with outbreaks in the United States, Canada, Europe, Asia and South America. In the United States alone, total direct and secondary economic losses from 1993 to 2001 owing to FHB were estimated at $7.67 billion. Fhb1 is the most consistently reported quantitative trait locus (QTL) for FHB resistance breeding. Here we report the map-based cloning of Fhb1 from a Chinese wheat cultivar Sumai 3. By mutation analysis, gene silencing and transgenic overexpression, we show that a pore-forming toxin-like (PFT) gene at Fhb1 confers FHB resistance. PFT is predicted to encode a chimeric lectin with two agglutinin domains and an ETX/MTX2 toxin domain. Our discovery identifies a new type of durable plant resistance gene conferring quantitative disease resistance to plants against Fusarium species.

Occurrence of Viruses and Associated Grain Yields of Paired Symptomatic and Nonsymptomatic Tillers in Kansas Winter Wheat Fields.

Vector-borne virus diseases of wheat are recurrent in nature and pose significant threats to crop production worldwide. In the spring of 2011 and 2012, a state-wide sampling survey of multiple commercial field sites and university-managed Kansas Agricultural Experiment Station variety performance trial locations spanning all nine crop-reporting regions of the state was conducted to determine the occurrence of Barley yellow dwarf virus-PAV (BYDV-PAV), Cereal yellow dwarf virus-RPV, Wheat streak mosaic virus (WSMV), High plains virus, Soilborne wheat mosaic virus, and Wheat spindle streak mosaic virus using enzyme-linked immunosorbent assays (ELISA). As a means of directly coupling tiller infection status with tiller grain yield, multiple pairs of symptomatic and nonsymptomatic plants were selected and individual tillers were tagged for virus species and grain yield determination at the variety performance trial locations. BYDV-PAV and WSMV were the two most prevalent species across the state, often co-occurring within location. Of those BYDV-PAV- or WSMV-positive tillers, 22% and 19%, respectively, were nonsymptomatic, a finding that underscores the importance of sampling criteria to more accurately assess virus occurrence in winter wheat fields. Symptomatic tillers that tested positive for BYDV-PAV produced significantly lower grain yields compared with ELISA-negative tillers in both seasons, as did WSMV-positive tillers in 2012. Nonsymptomatic tillers that tested positive for either of the two viruses in 2011 produced significantly lower grain yields than tillers from nonsymptomatic, ELISA-negative plants, an indication that these tillers were physiologically compromised in the absence of virus-associated symptoms. Overall, the virus survey and tagged paired-tiller sampling strategy revealed effects of virus infection on grain yield of individual tillers of plants grown under field conditions and may provide a complementary approach toward future estimates of the impact of virus incidence on crop health in Kansas.

Climate Suitability for Magnaporthe oryzae Triticum Pathotype in the United States.

Wheat blast, caused by the Triticum pathotype of Magnaporthe oryzae, is an emerging disease considered to be a limiting factor to wheat production in various countries. Given the importance of wheat blast as a high-consequence plant disease, weather-based infection models were used to estimate the probabilities of M. oryzae Triticum establishment and wheat blast outbreaks in the United States. The models identified significant disease risk in some areas. With the threshold levels used, the models predicted that the climate was adequate for maintaining M. oryzae Triticum populations in 40% of winter wheat production areas of the United States. Disease outbreak threshold levels were only reached in 25% of the country. In Louisiana, Mississippi, and Florida, the probability of years suitable for outbreaks was greater than 70%. The models generated in this study should provide the foundation for more advanced models in the future, and the results reported could be used to prioritize research efforts regarding the biology of M. oryzae Triticum and the epidemiology of the wheat blast disease.

Chromosome engineering, mapping, and transferring of resistance to Fusarium head blight disease from Elymus tsukushiensis into wheat.

KEY MESSAGE: This manuscript describes the transfer and molecular cytogenetic characterization of a novel source of Fusarium head blight resistance in wheat. Fusarium head blight (FHB) caused by the fungus Fusarium graminearum Schwabe [telomorph = Gibberella zeae (Schwein. Fr.) Petch] is an important disease of bread wheat, Triticum aestivum L. (2n = 6x = 42, AABBDD) worldwide. Wheat has limited resistance to FHB controlled by many loci and new sources of resistance are urgently needed. The perennial grass Elymus tsukushiensis thrives in the warm and humid regions of China and Japan and is immune to FHB. Here, we report the transfer and mapping of a major gene Fhb6 from E. tsukushiensis to wheat. Fhb6 was mapped to the subterminal region in the short arm of chromosome 1E(ts)#1S of E. tsukushiensis. Chromosome engineering was used to replace corresponding homoeologous region of chromosome 1AS of wheat with the Fhb6 associated chromatin derived from 1E(ts)#1S of E. tsukushiensis. Fhb6 appears to be new locus for wheat as previous studies have not detected any FHB resistance QTL in this chromosome region. Plant progenies homozygous for Fhb6 had a disease severity rating of 7 % compared to 35 % for the null progenies. Fhb6 has been tagged with molecular markers for marker-assisted breeding and pyramiding of resistance loci for effective control of FHB.

Lipid profiles in wheat cultivars resistant and susceptible to tan spot and the effect of disease on the profiles.

Lipid profiles in wheat leaves and the effects of tan spot on the profiles were quantified by mass spectrometry. Inoculation with Pyrenophora tritici-repentis significantly reduced the amount of leaf lipids, including the major plastidic lipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), which together accounted for 89% of the mass spectral signal of detected lipids in wheat leaves. Levels of these lipids in susceptible cultivars dropped much more quickly during infection than those in resistant cultivars. Furthermore, cultivars resistant or susceptible to tan spot displayed different lipid profiles; leaves of resistant cultivars had more MGDG and DGDG than susceptible ones, even in noninoculated plants. Lipid compositional data from leaves of 20 noninoculated winter wheat cultivars were regressed against an index of disease susceptibility and fitted with a linear model. This analysis demonstrated a significant relationship between resistance and levels of plastidic galactolipids and indicated that cultivars with high resistance to tan spot uniformly had more MGDG and DGDG than cultivars with high susceptibility. These findings suggest that lipid composition of wheat leaves may be a determining factor in the resistance response of cultivars to tan spot.

Preliminary Assessment of Resistance Among U.S. Wheat Cultivars to the Triticum Pathotype of Magnaporthe oryzae.

Magnaporthe oryzae is the causal agent of blast disease on several graminaceous plants. The M. oryzae population causing wheat blast has not been officially reported outside South America. Wheat production in the United States is at risk to this pathogen if it is introduced and established. Proactive testing of U.S. wheat cultivars for their reaction to blast and identification of resistance resources is crucial due to the national and global importance of the U.S. wheat industry. In this preliminary study, the phenotypic reaction of 85 U.S. wheat cultivars to M. oryzae (Triticum pathotype) was determined. Although there was a significant correlation in the reaction to blast at the seedling and adult plant stages, only 57% of the head reaction was explained by the seedling reaction. Because of the importance of disease development at the head stage in the field, assessment of all 85 cultivars occurred at the head stage. Among cultivars tested, a continuum in severity to head blast was observed; cultivars Everest and Karl 92 were highly susceptible with more than 90% disease severity, while cultivars Postrock, JackPot, Overley, Jagalene, Jagger, and Santa Fe showed less than 3% infection. No evidence of the presence of physiological races among isolates T-7, T-12, T-22, and T-25 was found.

Association study of resistance to Soilborne wheat mosaic virus in U.S. winter wheat.

Soilborne wheat mosaic virus (SBWMV) is one of the most important winter wheat pathogens worldwide. To identify genes for resistance to the virus in U.S. winter wheat, association study was conducted using a selected panel of 205 elite experimental lines and cultivars from U.S. hard and soft winter wheat breeding programs. Virus symptoms were evaluated twice in virus-infected fields for the panel at Manhattan, KS in spring 2010 and 2011 and for a subpanel of 137 hard winter wheat accessions at Stillwater, OK in spring 2008. At the two locations, 69.8 and 79.5% of cultivars were resistant or moderately resistant to the disease, respectively. After 282 simple-sequence repeat markers covering all wheat chromosome arms were scanned for association in the panel, marker Xgwm469 on the long arm of chromosome 5D (5DL) showed a significant association with the disease rating. Three alleles (Xgwm469-165bp, -167bp, and -169bp) were associated with resistance and the null allele was associated with susceptibility. Correlations between the marker and the disease rating were highly significant (0.80 in Manhattan at P < 0.0001 and 0.63 in Stillwater at P < 0.0001). The alleles Xgwm469-165bp and Xgwm469-169bp were present mainly in the hard winter wheat group, whereas allele Xgwm469-167bp was predominant in the soft winter wheat. The 169 bp allele can be traced back to 'Newton', and the 165 bp allele to Aegilops tauschii. In addition, a novel locus on the short arm of chromosome 4D (4DS) was also identified to associate with the disease rating. Marker Xgwm469-5DL is closely linked to SBWMV resistance and highly polymorphic across the winter wheat accessions sampled in the study and, thus, should be useful in marker-assisted selection in U.S. winter wheat.

Genetic differentiation at microsatellite loci among populations of Mycosphaerella graminicola from California, Indiana, Kansas, and North Dakota.

Mycosphaerella graminicola causes Septoria tritici blotch (STB) in wheat (Triticum aestivum) and is considered one of the most devastating pathogens of that crop in the United States. Although the genetic structures of M. graminicola populations from different countries have been analyzed using various molecular markers, relatively little is known about M. graminicola populations from geographically distinct areas of the United States and, in particular, of those from spring versus winter wheat. These are exposed to great differences in environmental conditions, length and season of host-free periods, and resistance sources used in geographically separated wheat breeding programs. Thus, there is more likely to be genetic differentiation between populations from spring versus winter wheat than there is among those within each region. To test this hypothesis, 330 single-spore isolates of M. graminicola representing 11 populations (1 from facultative winter wheat in California, 2 from spring wheat in North Dakota, and 8 from winter wheat in Indiana and Kansas) were analyzed for mating type frequency and for genetic variation at 17 microsatellite or simple-sequence repeat (SSR) loci. Analysis of clone-corrected data revealed an equal distribution of both mating types in the populations from Kansas, Indiana, and North Dakota, but a deviation from a 1:1 ratio in the California population. In total, 306 haplotypes were detected, almost all of which were unique in all 11 populations. High levels of gene diversity (H = 0.31 to 0.56) were observed within the 11 populations. Significant (P ≤ 0.05) gametic disequilibrium, as measured by the index of association (rBarD), was observed in California, one Indiana population (IN1), and three populations (KS1, KS2, and KS3) in Kansas that could not be explained by linkage. Corrected standardized fixation index (G″(ST)) values were 0.000 to 0.621 between the 11 populations and the majority of pairwise comparisons were statistically significant (P ≤ 0.001), suggesting some differentiation between populations. Analysis of molecular variance showed that there was a small but statistically significant level of genetic differentiation between populations from spring versus winter wheat. However, most of the total genetic variation (>98%) occurred within spring and winter wheat regions while <2% was due to genetic differentiation between these regions. Taken together, these results provide evidence that sexual recombination occurs frequently in the M. graminicola populations sampled and that most populations are genetically differentiated over the major spring- and winter-wheat-growing regions of the United States.

Effects of Integrating Cultivar Resistance and Fungicide Application on Fusarium Head Blight and Deoxynivalenol in Winter Wheat.

Fusarium head blight (FHB) or scab, incited by Fusarium graminearum, can cause significant economic losses in small grain production. Five field experiments were conducted from 2007 to 2009 to determine the effects on FHB and the associated mycotoxin deoxynivalenol (DON) of integrating winter wheat cultivar resistance and fungicide application. Other variables measured were yield and the percentage of Fusarium-damaged kernels (FDK). The fungicides prothioconazole + tebuconazole (formulated as Prosaro 421 SC) were applied at the rate of 0.475 liters/ha, or not applied, to three cultivars (experiments 1 to 3) or six cultivars (experiments 4 and 5) differing in their levels of resistance to FHB and DON accumulation. The effect of cultivar on FHB index was highly significant (P < 0.0001) in all five experiments. Under the highest FHB intensity and no fungicide application, the moderately resistant cultivars Harry, Heyne, Roane, and Truman had less severe FHB than the susceptible cultivars 2137, Jagalene, Overley, and Tomahawk (indices of 30 to 46% and 78 to 99%, respectively). Percent fungicide efficacy in reducing index and DON was greater in moderately resistant than in susceptible cultivars. Yield was negatively correlated with index, with FDK, and with DON, whereas index was positively correlated with FDK and with DON, and FDK and DON were positively correlated. Correlation between index and DON, index and FDK, and FDK and DON was stronger in susceptible than in moderately resistant cultivars, whereas the negative correlation between yield and FDK and yield and DON was stronger in moderately resistant than in susceptible cultivars. Overall, the strongest correlation was between index and DON (0.74 ≤ R ≤ 0.88, P ≤ 0.05). The results from this study indicate that fungicide efficacy in reducing FHB and DON was greater in moderately resistant cultivars than in susceptible ones. This shows that integrating cultivar resistance with fungicide application can be an effective strategy for management of FHB and DON in winter wheat.

A Novel Source of Resistance in Wheat to Pyrenophora tritici-repentis Race 1.

Tan spot, caused by the fungus Pyrenophora tritici-repentis, causes serious yield losses in wheat (Triticum aestivum) and many other grasses. Race 1 of the fungus, which produces the necrosis toxin Ptr ToxA and the chlorosis toxin Ptr ToxC, is the most prevalent race in the Great Plains of the United States. Wheat genotypes with useful levels of resistance to race 1 have been deployed, but this resistance reduces damage by only 50 to 75%. Therefore, new sources of resistance to P. tritici-repentis are needed. Recombinant inbred lines developed from a cross between the Indian spring wheat cvs. WH542 (resistant) and HD29 (moderately susceptible) were evaluated for reaction to race 1 of the fungus. Composite interval mapping revealed quantitative trait loci (QTL) on the short arm of chromosome 3A explaining 23% of the phenotypic variation, and the long arm of chromosome 5B explaining 27% of the variation. Both resistance alleles were contributed by the WH542 parent. The QTL on 5BL is probably tsn1, which was described previously. The 3AS QTL (QTs.ksu-3AS) on 3AS is a novel QTL for resistance to P. tritici-repentis race 1. The QTL region is located in the most distal bin of chromosome 3AS in a 2.2-centimorgan marker interval. Flanking markers Xbarc45 and Xbarc86 are suitable for marker-assisted selection for tan spot resistance.

Number of Experiments Needed to Determine Wheat Disease Phenotypes for Four Wheat Diseases.

Disease phenotypes for winter wheat cultivars were determined in numerous inoculated greenhouse and field experiments over many years. For four diseases, Fusarium head blight, tan spot, Septoria leaf blotch, and Stagonospora leaf blotch, at least 20 cultivars each had been evaluated in a minimum of five experiments. Reference cultivars of known disease reaction were included in each experiment, which allowed transformation of the percent disease severity data to a 1-to-9 scale for comparisons between experiments. Variations in scale values obtained for individual cultivars among the different experiments were used to calculate standard deviations for disease phenotype data. Standard deviations were used to calculate the number of experiment repetitions needed within each disease to achieve different levels of accuracy (margins of error). A margin of error of ±1.5 for the 1-to-9 scale was chosen as the best level of accuracy. Rounding values within this range would put the estimated disease phenotype within ±1 unit of the actual phenotype. To achieve a margin of error of ±1.5 for Fusarium head blight, tan spot, Septoria leaf blotch, and Stagonospora leaf blotch would require a mean that was calculated from a minimum of five, five, seven, and eight experiments, respectively. Personnel who report disease phenotype data to wheat producers or breeders should be aware of the number of experiments upon which they are basing their reports and adjust any disclaimers accordingly. Similarly, wheat breeders should be aware of the inherent variability in phenotyping these four wheat diseases and make appropriate adjustments to their selection protocols. With a minimum of five experimental repetitions, disease phenotype values obtained from inoculated greenhouse and field experiments had very high correlations (r = 0.81 to 0.92, P < 0.0001) with published Kansas State University Research and Extension ratings obtained from commercial fields.

Apoplastic extracts from a transgenic wheat line exhibiting lesion-mimic phenotype have multiple pathogenesis-related proteins that are antifungal.

A transgenic wheat line constitutively expressing genes encoding a class IV acidic chitinase and an acidic beta-1,3-glucanase, showed significant delay in spread of Fusarium head blight (scab) disease under greenhouse conditions. In an earlier work, we observed a lesion-mimic phenotype in this transgenic line when homozygous for transgene loci. Apoplastic fluid (AF) extracted from the lesion-mimic plants had pathogenesis-related (PR) proteins belonging to families of beta-1,3-glucanases, chitinases, and thaumatin-like proteins (TLPs). AF had growth inhibitory activity against certain fungal pathogens, including Fusarium graminearum and Gaeumannomyces graminis var. tritici. Through a two-step ion-exchange chromatography protocol, we recovered many PR proteins and a few uncharacterized proteins. Three individual protein bands corresponding to a TLP (molecular mass, 16 kDa) and two beta-1,3-glucanases (molecular mass, 32 kDa each) were purified and identified by tandem mass spectrometry. We measured the in vitro antifungal activity of the three purified enzymes and a barley class II chitinase (purified earlier in our laboratory) in microtiter plate assays with macroconidia or conidiophores of F. graminearum and Pyrenophora tritici-repentis. Mixtures of proteins revealed synergistic or additive inhibitory activity against F. graminearum and P. tritici-repentis hyphae. The concentrations of PR proteins at which these effects were observed are likely to be those reached in AF of cells exhibiting a hypersensitive response. Our results suggest that apoplastic PR proteins are antifungal and their antimicrobial potency is dependent on concentrations and combinations that are effectively reached in plants following microbial attack.

Inheritance of Resistance to Stagonospora nodorum Leaf Blotch in Kansas Winter Wheat Cultivars.

Stagonospora nodorum blotch can cause serious yield and quality losses of wheat (Triticum aestivum) in many countries worldwide. Although there are other control methods, host resistance is the most desirable. Three recent Kansas winter wheat cultivars (Betty, Heyne, and 2163) have been developed with moderate levels of resistance to the leaf phase of Stagonospora nodorum blotch. To determine inheritance of resistance and allelism, these cultivars were crossed with one of three susceptible lines (Larned, KS96WGRC39, or Newton) and intercrossed in all possible combinations, including reciprocals. The parents, F1, F2, and F3 generations were tested for resistance to S. nodorum in the greenhouse as 4-week-old seedlings. Cytoplasmic effects were not detected in any cross. The mean levels of infection in the F1s of the two crosses Betty × Larned and Heyne × KS96WGRC39 indicated resistance was dominant. The observed phenotypic ratios of F2 plants for both crosses were not significantly different from the expected ratio for a single dominant gene. The ratio observed for F3 lines in the Betty × Larned cross fit that expected for a single dominant gene. However, the observed ratio of the F3 lines from the cross Heyne × KS96WGRC39 did not fit the ratio expected for a single dominant gene. The allelism test for Betty and Heyne indicated that they have different resistance genes. The F1 mean rating of the cross 2163 × Newton was intermediate between the two parents, indicating the absence of dominance for resistance in 2163. The phenotypic ratio observed in the F2 plants from this cross did not fit the ratio expected for a single dominant gene. The simple genetic control of resistance in cv. Betty makes it a useful source of resistance for wheat breeding programs.

Greenhouse and field testing of transgenic wheat plants stably expressing genes for thaumatin-like protein, chitinase and glucanase against Fusarium graminearum.

Genes encoding pathogenesis-related (PR-) proteins isolated from a cDNA library of Fusarium graminearum-infected wheat spikes of scab-resistant cultivar 'Sumai-3' were transformed into susceptible spring wheat, 'Bobwhite' using a biolistic transformation protocol, with the goal of enhancing levels of resistance against scab. Twenty-four putative transgenic lines expressing either a single PR-protein gene or combinations thereof were regenerated. Transgene expression in a majority of these lines (20) was completely silenced in the T(1) or T(2) generations. Four transgenic wheat lines showed stable inheritance and expression of either a single transgene or transgene combinations up to four generations. One line co-expressing a chitinase and beta-1,3-glucanase gene combination, when bioassayed against scab showed a delay in the spread of the infection (type II resistance) under greenhouse conditions. This line and a second transgenic line expressing a rice thaumatin-like protein gene (tlp) which had moderate resistance to scab in previous greenhouse trials, along with susceptible and resistance checks were evaluated for resistance to scab under field conditions. None of the transgenic lines had resistance to scab in the field under conditions of strong pathogen, suggesting these plants lacked effective resistance to initial infection (type I resistance) under these conditions. As far as is known, this is the first report of field evaluation of transgenic wheat expressing genes for PR-proteins against disease resistance.

Temperature-Sensitive Reaction of Winter Wheat Cultivar AGSECO 7853 to Stagonospora nodorum.

Stagonospora blotch is an important foliar and head disease of wheat (Triticum aestivum) in many regions of the world. To determine the reaction of winter wheat cultivars to Stagonospora blotch at different temperatures, seedlings of the hard winter wheat cvs. Newton, AGSECO 7853, and Heyne were inoculated with three isolates of Stagonospora nodorum and exposed to three temperature regimes (high, 29 and 21°C [day and night]; medium, 25 and 17°C; and low, 18 and 10°C). Heyne was resistant at all temperatures in all three experiments. The reaction of AGSECO 7853, when averaged over all isolates, was intermediate between Heyne and Newton at high temperature, as susceptible as Newton at medium temperature, but more susceptible than Newton at low temperature. Therefore, the reaction of AGSECO 7853 relative to Newton was temperature sensitive. This finding is important in the evaluation of wheat lines for resistance to Stagonospora blotch because the relative ranking of one cultivar may differ from another depending upon the temperature.

Effect of Placement of Inoculum of Gaeumannomyces graminis var. tritici on Severity of Take-all in Winter Wheat.

Take-all, caused by Gaeumannomyces graminis var. tritici, is one of the most important root diseases of wheat worldwide. Because of the lack of highly effective chemical control, cultural practices, such as crop rotation, play a major role in managing disease severity. In Kansas, many producers do not use these measures and continue to suffer losses from take-all. Greenhouse and field experiments were established to assess the effect of horizontal versus vertical distribution of G. graminis var. tritici inoculum on disease severity. Oat kernel inoculum was placed at 0 (seed level), 5, 10, or 15 cm below the wheat seed or 5, 10, or 15 cm to the side of the wheat seed at a depth of 5 cm. Inoculum spatial location and distance greatly influenced take-all. Experiments showed more severe losses due to take-all when inoculum was placed below the seed than to the side of the seed. Regression analyses were used to develop take-all risk models relating inoculum distance from the seed to yield loss. Quadratic models were a better fit for data from experiments where inoculum was placed to the side of the seed, whereas linear models significantly fit data from experiments where inoculum was positioned below the seed. Within the same direction, take-all decreased as the inoculum was placed at greater distances from the seed, often to insignificant levels at 10 to 15 cm. According to the regression models, significant reduction (≥50%) in take-all might be achieved by plowing under the infested residues (crowns) to depths greater than 15 cm, or placing seed >6.0 cm to the side of inoculum. Therefore, under no-till conditions, sowing parallel to and exactly between the previous years' stubble rows (inoculum) might help manage take-all. These possibilities need to be investigated under field conditions.