A real-time qPCR assay to quantify Fusarium graminearum biomass in wheat kernels.

AIMS: To develop a real-time PCR assay to quantify Fusarium graminearum biomass in blighted wheat kernels. METHODS AND RESULTS: Primers designed to amplify a gene in the trichothecene biosynthetic cluster (TRI6) were evaluated for sensitivity and specificity. Primer pair Tri6_10F/Tri6_4R specifically and consistently amplified a 245-bp DNA fragment from F. graminearum. A workflow was developed and validated to extract DNA from infested grain. The assay detected as little as 10 µg of F. graminearum mycelia in 1 g of ground wheat grain with a high correlation between fungal biomass and cycle threshold values (R(2) = 0·9912; = 0·004). In field-inoculated grain, qPCR measurements of biomass correlated closely with deoxynivalenol levels (R = 0·82, P < 0·0001) and two visual techniques to assess grain quality (R = 0·88, P < 0·0001 and R = 0·81, P < 0·0001). CONCLUSIONS: The qPCR assay provided accurate and precise assessments of the amount of F. graminearum biomass in blighted wheat kernels. This method represents a technical advance over other approaches to quantify kernel colonization and real-time PCR detection methodologies for F. graminearum that do not correlate quantification of fungal genomic DNA to biomass. SIGNIFICANCE AND IMPACT OF THE STUDY: Quantifying F. graminearum biomass, especially low levels of growth associated with kernels that are visually asymptomatic, represents a new approach to screen for resistance to kernel infection, an understudied yet potentially important avenue to reduce the impact of head blight.

Emergence of a novel population of Puccinia striiformis f. sp. tritici in Eastern United States.

The geographic range of stripe rust of wheat, caused by Puccinia striiformis f. sp. tritici, has increased dramatically since 2000 in the United States. Yield losses to the disease have been most severe in the eastern United States, where measurable yield loss had been rare prior to 2000. The objective of this study was to examine the phenotypic and genotypic variation among isolates of P. striiformis f. sp. tritici collected from populations in the eastern United States before and since 2000. Virulence phenotype and amplified fragment length polymorphism (AFLP) markers were used to examine 42 isolates collected between 1960 and 2004. In addition, the genetic structure of 59 isolates collected in 2005 using a hierarchical sampling strategy was examined. The data indicated that the contemporary isolates (collected since 2000) were very distinct from older isolates (collected before 2000) based on virulence and AFLP markers, and that the old population prevalent before 2000 may have been replaced by the contemporary population. The old and new populations appear to be genetically distinct and may represent an exotic introduction rather than a mutation in isolates of the old population.

Aggressiveness of Puccinia striiformis f. sp. tritici Isolates in the South-Central United States.

Although stripe rust, caused by Puccinia striiformis f. sp. tritici, has been an occasional problem on wheat in the south-central United States from 1941 until 1999, the disease has been consistently severe in the region since 2000. Furthermore, since 2000, the geographic range of stripe rust in the eastern United States has expanded, and the old population of races has been replaced by a new population. The objective of this study was to determine whether new isolates of the pathogen were more aggressive and better adapted to warmer temperatures than old isolates. In all, 6 old isolates (collected before 2000) and 14 new isolates (collected since 2000) were evaluated at 12 and 18°C for latent period on wheat seedlings and urediniospore germination on Noble agar. At 12°C, old and new isolates had similar latent periods and spore germination percentages. However, at 18°C, new isolates averaged 2 days less for latent period and double the spore germination compared with old isolates. Therefore, the new isolates are better adapted and, thus, more aggressive at warmer temperatures than the old isolates. These differences may have contributed to the severity of recent epidemics in the region and to the expanded geographic range for stripe rust.

Evaluation of Components of Fusarium Head Blight Resistance in Soft Red Winter Wheat Germ Plasm Using a Detached Leaf Assay.

A large environmental influence on phenotypic estimates of disease resistance and the complex polygenic nature of Fusarium head blight (FHB) resistance in wheat (Triticum aestivum) are impediments to developing resistant cultivars. The objective of this research was to investigate the utility of a detached leaf assay, inoculated using inoculum from isolates of Microdochium nivale var. majus, to identify components of FHB resistance among 30 entries of U.S. soft red winter wheat in the 2002 Uniform Southern FHB Nursery (USFHBN). Whole plant FHB resistance of the USFHBN entries was evaluated in replicated, mist-irrigated field trials at 10 locations in eight states during the 2001-2002 season. Incubation period (days from inoculation to the first appearance of a dull gray-green water-soaked lesion) was the only detached leaf variable significantly correlated across all FHB resistance parameters accounting for 45% of the variation in FHB incidence, 27% of FHB severity, 30% of Fusarium damaged kernels, and 26% of the variation in grain deoxynivalenol (DON) concentration. The results for incubation period contrasted with previous studies of moderately resistant European cultivars, in that longer incubation period was correlated with greater FHB susceptibility, but agreed with previous findings for the Chinese cultivar Sumai 3 and CIMMYT germ plasm containing diverse sources of FHB resistance. The results support the view that the detached leaf assay method has potential for use to distinguish between specific sources of FHB resistance when combined with data on FHB reaction and pedigree information. For example, entry 28, a di-haploid line from the cross between the moderately resistant U.S. cultivar Roane and the resistant Chinese line W14, exhibited detached leaf parameters that suggested a combination of both sources of FHB resistance. The USFHBN represents the combination of adapted and exotic germ plasm, but four moderately resistant U.S. commercial cultivars (Roane, McCormick, NC-Neuse, and Pat) had long incubation and latent periods and short lesion lengths in the detached leaf assay as observed in moderately FHB resistant European cultivars. The dichotomy in the relationship between incubation period and FHB resistance indicates that this may need to be considered to effectively combine exotic and existing/adapted sources of FHB resistance.

Effect of Previous Crop, Seedborne Inoculum, and Fungicides on Development of Stagonospora Blotch.

Stagonospora blotch of wheat has been difficult to control in the eastern United States. The objectives of this research were to evaluate the effects of field inoculum, seedborne inoculum, and seed treatment and foliar fungicides on Stagonospora blotch development and to develop more effective management strategies. In 1995 and 1996, similar experiments were established in "infested" and "clean" fields using two seed lots of Coker 9543 ("low" and "high" levels of seed infection) and six seed or foliar fungicide treatments. Planting in clean fields, planting seed with a low level of seedborne inoculum, treating seed with difenoconazole or triadimenol + thiram, and applying propiconazole or tebuconazole to the foliage all contributed toward reducing leaf infections by Stagonospora nodorum, severity of leaf and glume blotch, and incidence of S. nodorum in the harvested seed. Propiconazole alone was the least effective treatment. Planting in an infested field tended to negate the beneficial effects of low level of seed infection and fungicide seed treatments. Crop rotations and tillage that allow wheat debris to decompose before the next wheat crop along with difenoconazole or triadimenol seed treatment to reduce seedborne inoculum should be sufficient to avoid serious losses. In fields where wheat is grown every year, tillage and seed treatment would still be helpful, but a foliar fungicide at GS 8 may be necessary for adequate control. Applying an effective fungicide to seed appears to be a more efficient means of reducing seedborne inoculum than does producing seed with low levels of inoculum.

Efficacy of Bacterial Seed Treatments for Controlling Pythium Root Rot of Winter Wheat.

Pythium root rot, caused by various Pythium spp., is a widespread disease of wheat. The objective of this study was to identify bacterial strains from wheat roots in Arkansas that suppressed Pythium root rot and to compare their efficacy with that of bacterial strains from other areas. Bacterial strains (applied as seed treatments) that suppressed Pythium root rot in growth chamber assays were evaluated further for in vitro antibiosis against three Pythium spp. and for efficacy under field conditions. Pseudomonas fluorescens strain 2-79R, Burkholderia cepacia strain 1-23, and Pseudomonas sp. strain 1-30 were the most effective for suppressing Pythium root rot under field conditions and significantly (P = 0.10) increased yield in one experiment. Strains that were effective in the field also expressed in vitro antibiosis to at least two of three Pythium spp.; however, strains expressing the highest levels of antibiosis were not effective in the field. In the field, root rot suppression and yield enhancement were inconsistent across experiments and generally small in magnitude. Therefore, these strains have little potential for commercial use under the conditions in which they were tested.