First Report of Virulence Phenotypes of Puccinia helianthi, Causal Agent of Sunflower Rust in Illinois.

Sunflower rust caused by Puccinia helianthi (Schw.) is an autoecious and macrocyclic rust that occurs on wild perennial, wild annual, and cultivated sunflower species (Helianthus annuus L.). Severe epidemics of sunflower rust can significantly reduce yield and quality of cultivated sunflower (4). Uredinia of P. helianthi were observed in individual sunflower fields in four Illinois counties in 2008, namely; Champaign, LaSalle, McLean, and Livingston. Leaves with uredinia from each field were collected and shipped to the USDA-ARS Sunflower Pathology Laboratory in Fargo, ND. Urediniospores were harvested by rinsing leaves with Soltrol 170 isoparaffin solvent (Chevron Phillips Chemicals, Woodlands, TX). Urediniospores were increased by inoculating the spore suspension onto 21-day-old seedlings of susceptible oilseed hybrid Myc-7350 with a vacuum-pump powered atomizer. After the Soltrol 170 evaporated, plants were placed in a dew chamber for 24 h and then moved to a greenhouse at approximately 20 to 25°C with a 14-h photoperiod. When sporulating uredinia were visible, a bulk collection of urediniospores was made from samples of each location. Additionally, one single-pustule derived isolate from each location was obtained by harvesting urediniospores from a single pustule and increasing as above. To determine the virulence phenotypes, each single-pustule derived isolate and bulk collection were inoculated as above onto at least two replicated sets of the nine international differential lines (each set containing three plants per line) and evaluated after 14 days (1,2). The single-pustule isolates from LaSalle, Livingston, and McLean counties were determined to be virulence phenotype 300. The single-pustule isolate from Champaign produced no infection on any differential lines, including the susceptible, and was therefore considered not viable. The virulence phenotypes of the bulk samples were coded as 330 (Champaign), 324 (McLean), and 336 (Livingston and LaSalle). Virulence of all aforementioned virulence phenotypes was found to be common in a recent survey of 400 Midwestern P. helianthi samples collected in 2007 and 2008 (1). Although sunflower production is limited in Illinois, expansion could occur. This is particularly true in southern Illinois, where double cropping sunflower after winter wheat is a possibility. Urediniospore germination and infection is favored by free moisture and temperatures ranging from 10 to 25°C, while optimum temperature for spore development ranges from 20 to 35°C (3). These environmental conditions are often more common in Illinois than in the major sunflower-producing states in the Great Plains, where sunflower rust is a concern annually. Thus, determination of P. helianthi virulence phenotypes in Illinois may be important for future management of sunflower rust. References: (1) T. J. Gulya and S. G. Markell. National Sunflower Association. Online Publication/Gulya_RustStatus_09, 2009. (2) T. Gulya and S. Masirevic. FAO Eur. Res. Network on Sunflower. Bucarest, Romania. 31, 1995. (3) T. Gulya et al. Sunflower Diseases. Page 263 in: Sunflower Technology and Production. A. A. Schneiter, ed. American Society of Agronomy, Madison, WI, 1997. (4) S. Markell et al. Plant Dis. 93:668, 2009.

Stability of haemoglobin A1c (HbA1c) measurements from frozen whole blood samples stored for over a decade.

OBJECTIVE: Haemoglobin A1c (HbA1c), a measure of long-term glycaemic control, is at the centre of the clinical management of diabetes mellitus. However, the reproducibility of HbA1c measurements from whole blood samples which have been in long-term storage is unknown. We undertook this study to assess the reproducibility of HbA1c measurements from whole blood samples that had been in storage at -70 degrees C for over a decade. RESEARCH DESIGN AND METHODS: Three hundred and thirty-six samples of frozen whole blood from the Atherosclerosis Risk in Communities (ARIC) Study, stored at -70 degrees C for 11-14 years assayed for HbA1c using a dedicated ion-exchange HPLC assay (Tosoh A1c 2.2 Plus HPLC) were compared with measurements on these same samples conducted prior to storage (in 1990-92) using a Diamat (Bio-Rad) HPLC instrument. RESULTS: HbA1c measurements from long-term stored samples were strongly correlated with values obtained prior to long-term storage (r=0.97). The difference between HbA1c from long- and short-term stored samples had a mean of 0.35% HbA1c (sd=0.35) and a CV of 5.8%, which was approximately three times that of duplicate assays (CV 1.3 to 2.5%). CONCLUSIONS: These data demonstrate that highly correlated but more variable and slightly higher HbA1c results were obtained from frozen whole blood samples that have been in storage for more than a decade. This highly reproducible assay performance would lead to comparable ranking of individuals and unbiased estimates of relative risks and odds ratios in epidemiological studies (case-control and cohort designs), but results should be realigned when the absolute value is of interest. These results have important implications for epidemiological studies and clinical trials which have stored whole blood specimens.

Identification of a Chlorosis-Inducing Toxin from Pyrenophora tritici-repentis and the Chromosomal Location of an Insensitivity Locus in Wheat.

ABSTRACT Culture filtrate from Pyrenophora tritici-repentis race 1 isolate 78-62 contained a genotype-specific toxin which elicited extensive chlorosis on sensitive wheat genotypes. This toxin was partially purified using gel filtration, ion exchange, and reversed-phase chromatography. The chlorosis toxin was found to be a polar, nonionic, low-molecular-weight molecule. Wheat genotypes infiltrated with crude culture filtrate and partially purified chlorosis toxin exhibited the same chlorotic symptoms seen with conidial inoculations of isolate 78-62. All tested wheat genotypes that exhibited extensive chlorosis to the toxin also exhibited extensive chlorosis to conidial inoculations, and all wheat genotypes insensitive to the toxin did not exhibit extensive chlorosis to conidial inoculations. The recombinant inbred population derived from the cross W-7984 x Opata 85 segregated for chlorosis induction from infiltration with partially purified chlorosis toxin from isolate 78-62. The locus identified by the marker XGli1, associated with resistance to conidial inoculations from race 1 isolates Pti2 and 78-62 and race 3 isolate D308, also was associated with insensitivity to infiltration of crude culture filtrate and partially purified chlorosis toxin. The marker XGli1, located on the short arm of chromosome 1A, is linked to the insensitivity locus within 5.7 cM. We propose that this chlorosis toxin be designated Ptr ToxC.

Structural and Physical Properties of a Necrosis-Inducing Toxin from Pyrenophora tritici-repentis.

ABSTRACT Cultivar-specific toxic metabolites of Pyrenophora tritici-repentis are involved in the appearance of necrotic and chlorotic foliar lesions characteristic of tan spot. A P. tritici-repentis necrosis-inducing toxin, Ptr necrosis toxin, was purified from isolate 86-124, sequenced by gas-phase amino acid microsequencing, and characterized by circular dichroism (CD) spectroscopy and isoelectric focusing. The purified protein had a similar amino acid composition and molecular weight as previously reported. Analysis of the CD spectrum from 178 to 250 nm indicated a protein consisting of 13% alpha-helix, 36% antiparallel beta-sheet, 25% turns, and 25% other structures. The Ptr necrosis toxin from isolate 86-124 has an isoelectric point near pH 10. Using overlapping proteolytic fragments obtained from the toxin, a sequence of 101 continuous amino acids was obtained, but the amino terminus was blocked and 9 to 16 amino acids could not be sequenced. Secondary structure prediction based on the amino acid sequence indicated a beta-sheet protein with little alpha-helix, which is in agreement with the structure determined by CD spectroscopy. Sequence analysis indicated the presence of a possible membrane adhesion site and several possible phosphorylation sites that may be involved in phytotoxicity.

RFLP mapping of resistance to chlorosis induction by Pyrenophora tritici-repentis in wheat.

Tan spot, caused by Pyrenophora tritici-repentis, is an economically important disease in major wheat production areas. The fungus can produce two genetically distinct symptoms on leaves of susceptible wheat genotypes: tan necrosis (nec) and extensive chlorosis (ch1). Our objectives were to determine the number of genes conditioning resistance to tan spot in a population of wheat recombinant inbred lines, and map the chromosomal location of the resistance genes using RFLPs. Conidia produced by the P. tritici-repentis isolate Pti2 (nee + chl +) were used to inoculate seedlings of 135 recombinant inbred lines derived from the cross of the synthetic hexaploid wheat W-7984 with Opata 85. A subset of the population was inoculated with conidia produced by the isolates D308 (nec - chl +) and 86-124 (nec + chl-). Inoculated seedlings were rated on a scale of 1 to 5 based on lesion type. Necrosis-inducing culture filtrate produced by the isolate 86-124 was also used to screen the entire population. A map consisting of 532 markers was employed to identify significant associations between marker loci and tan spot resistance. The entire population was insensitive to culture filtrate produced by the isolate 86-124, and the entire subset was resistant to conidial inoculation of the same isolate. The population segregated for reaction to isolates D308 and Pti2, indicating that this population segregates for resistance to extensive chlorosis only, and not to tan necrosis. RFLP analysis indicated the presence of a gene with a major effect in 1AS, a gene with a minor effect in 4AL, and an interaction between the 1AS gene and a gene in 2DL. Together, these loci explained 49.0% of the variation in this population for resistance to tan spot produced by the isolate Pti2. Two regions one in 1BL and one in 3BL, were significantly associated with resistance to extensive chlorosis, but were not significant in the multiple regression model. It should be feasible to introgress these resistance loci into adapted genetic backgrounds by using a marker-assisted selection scheme.