First Report of Seedling Blight Caused by Sclerotium rolfsii on Wheat in Oklahoma.

Wheat (Triticum aestivum L.) is an important crop in Oklahoma and throughout the Central Plains of the United States. The soilborne fungus, Sclerotium rolfsii, is a major pathogen on peanut (Arachis hypogaea L.) but is not known to cause major damage on wheat. During September of 1998, damping-off and rotting of young wheat seedlings were observed in breeder plots in Payne County, OK. The occurrence of symptoms was sporadic with an estimated stand reduction of 10 to 15%. Symptomatic plants were collected from the field and brought to the laboratory. Sclerotia-like bodies from the symptomatic plants were surface disinfested in aqueous 1% NaOCl for 2 min and allowed to germinate at 25 ± 2°C on sterile filter paper moistened with a 1% aqueous solution of methanol. Aerial mycelia from germinating sclerotia were transferred to potato dextrose agar amended with 100 ppm of streptomycin (SPDA) to obtain pure cultures. Pure cultures had coarse, white mycelium distinctive of S. rolfsii and produced very small (0.05 to 0.1 mm), abundant, round, brown sclerotia on the surface of the medium after 15 days of incubation. Pathogenicity was tested on three hard red winter wheat cultivars commonly grown in Oklahoma (Jagger, 2137, and 2174). Four plants of each cultivar were inoculated at the two-leaf stage (Feekes' scale stage 1) by placing a 0.5-cm agar disk removed from a 3-day-old culture onto a 1-cm diameter filter paper that was then pressed to the base of the shoot. Noninoculated plants were used as a control. After inoculation, pots were covered with polyethylene sheets to maintain 95 to 100% relative humidity and incubated at 25 ± 2°C in the greenhouse. Lesions were initially superficial, yellowish, and water soaked. Lesions expanded and resulted in damping-off of seedlings. Noninoculated plants were free of disease and remained healthy. No significant difference (P ≤ 0.05) in disease severity was observed among the cultivars. To fulfill Koch's postulates, the fungus was reisolated onto SPDA where it had the same characteristics as the initial culture. To our knowledge, this is the first report of S. rolfsii on wheat in Oklahoma. Even though S. rolfsii is not expected to pose a significant risk to wheat production, infection of wheat may enhance survival of S. rolfsii and facilitate infection and losses in a following peanut crop. This is especially important in certain areas of Oklahoma where a wheat-peanut rotation is occasionally practiced.

Determination of cellular carbohydrates in peanut fungal pathogens and baker's yeast by capillary electrophoresis and electrochromatography.

In this work, the quantitation of cellular carbohydrates, namely chitin and glucan, in peanut fungal pathogens and baker's yeast was carried out by capillary electrophoresis (CE) and capillary electrochromatography (CEC). The chitin and glucan of the fungi were hydrolyzed by the enzymes chitinase and glucanase, respectively, to their corresponding sugar monomers N-acetylglucosamine (GlcNAc) and glucose (Glc). These two monosaccharides were then tagged with 6-aminoquinoline (6-AQ) to allow their separation and detection in CE and CEC. The 6-AQ derivatives of GlcNAc and Glc formed the basis for the determination by CE and CEC of chitin and glucan in peanut fungi and baker's yeast. Several parameters affecting the separation of the 6-AQ derivatives of GlcNAc and Glc, including the separation voltage and the composition of the running electrolyte, were investigated. Under the optimized separation conditions, the contents of cellular carbohydrates including N-acetylglucosamine, chitin, glucose, and glucan in some fungi, such as Sclerotinia minor, Sclerotium rolfsii, and baker's yeast, were successfully determined. The method described here allowed the assessment of genetic differences in Sclerotium rolfsii isolates from various locations.

Effect of a Select Group of Seed Protectant Fungicides on Growth of Sclerotinia minor In Vitro and Its Recovery from Infested Peanut Seed.

Potato-dextrose agar containing 100 µg of streptomycin sulfate per milliliter of medium (SPDA) was amended to establish levels of 0, 2, 4, 6, 8, and 10 µg/ml of the fungicides thiophanate-methyl, carboxin, dicloran, captan, pentachloronitrobenzene (PCNB), or thiram. Fresh mycelial plugs, dry mycelial fragments, and sclerotia of Sclerotinia minor were placed onto the amended media, and mycelial growth and sclerotial germination were determined for 7 days. Thiophanate-methyl was the most effective chemical in inhibiting growth of S. minor, followed by PCNB and dicloran. Thiophanate-methyl was the only chemical that prevented germination of sclerotia of S. minor. Peanut seed naturally infested with S. minor was treated with the fungicides alone or in various combinations; control seed was treated with talcum powder. Seed was stored in polyethylene bags for 12 weeks at 24°C. To determine the incidence of viable S. minor in treated seed, seed was removed from bags, washed for 1 min in 0.2% unscented, liquid soap solution, rinsed twice in distilled water, and then dried for 15 min prior to plating on SPDA. The incidence of S. minor in talcum and thiophanate-methyl treated seed was 3.68 and 0.0%, respectively. Incidence of S. minor in seed treated with the other compounds ranged from 0.40 to 0.56%. Subsequent tests were performed using thiophanate-methyl and some of the above chemicals, in combinations or alone. These tests confirmed that thiophanate-methyl was the only compound that consistently reduced recovery of S. minor from infested seed. Germination of thiophanate-methyl treated seed equaled that of seed treated with talcum.

High-performance liquid phase separation of glycosides. 5. Determination of individual glucosinolates in cabbage and rapeseed by laser-induced fluorescene capillary electrophoresis via the enzymatically released isothiocyanate aglycon.

A capillary electrophoresis (CE) method was developed for the profiling and determination of individual glucosinolates (GSs) via their isothiocyanate degradation products upon myrosinase digestion. The resulting isothiocyanates, the structures of which are reflective of the parent GS's, were then converted to their corresponding amines via base hydrolysis or reaction with 1, 2-benzenedithiol. Subsequently, the amines were fluorescently labeled to allow their sensitive detection by laser-induced fluorescence (LIF). The CE method involved the use of in situ charged micelles for the separation of isothiocyanates and their corresponding fluorescently labeled amines by micellar electrokinetic capillary chromatography (MECC). The term "in situ charged micelles" refers to micelles formed by complexing the polar hydroxyl groups of glycosidic surfactants with borate. The MECC method with on-column LIF detection was applied to the determination of GSs in white cabbage, rapeseed leaves, and rapeseed roots.

High-performance liquid-phase separation of glycosides. III. Determination of total glucosinolates in cabbage and rapeseed by capillary electrophoresis via the enzymatically released glucose.

A selective and sensitive method for the determination of total glucosinolates (GSs) in plant extracts by capillary electrophoresis-laser-induced fluorescence (LIF) detection was developed. It was based on the enzymatically released glucose from glucosinolates in the presence of the hydrolyzing enzyme myrosinase. The released glucose was converted to gluconic acid (GA) by the action of glucose oxidase. The resulting GA was then labeled selectively with the fluorescent tag 7-aminonaphthalene-1, 3-disulfonic acid (ANDSA). The peak area resulting from the GA-ANDSA derived from free and bound glucose was subtracted from the peak area of the GA-ANDSA resulting from the free glucose in the sample. This gave the total glucosinolates in the sample. The peak areas were normalized to the internal standard, N-acetylneuraminic acid derivatized with ANDSA. The method was validated using four different plant extracts, white cabbage leaves, rapeseed leaves, rapeseed roots, and rapeseed seeds. Furthermore, a capillary electrophoresis-UV detection method for profiling GS in plant extracts was developed. In addition to providing a fingerprint of the glucosinolates in plant extracts, the method allowed the experimenter to rapidly check the various steps involved in the extraction and sample cleanup.

Effects of Temperature and Wetness Duration on Infection of Peanut Cultivars by Cercospora arachidicola.

ABSTRACT The effects of temperature and duration of wetness (relative humidity >/=95%) on infection of three peanut cultivars by Cercospora arachidicola were determined under controlled conditions. Plants of the Spanish cv. Spanco and the runner cvs. Florunner and Okrun were exposed to constant temperatures of 18 to 30 degrees C during 12-h periods of wetness each day that totaled 12 to 84 h following inoculation of leaves with conidia. Severity of disease, measured by either lesion density (number per leaf) or lesion size (diameter), was greatest for 'Spanco', intermediate for 'Florunner', and lowest for 'Okrun' in each of two experiments. Lesion density was evaluated further because it was an indicator of both the occurrence and degree of infection. Nonlinear regression analysis was employed to evaluate the combined effects of temperature (T) and wetness duration (W) on lesion density (Y). In the regression model, the Weibull function characterized the monotonic increase of Y with respect to W, while a hyperbolic function characterized the unimodal response of Y with respect to T. Parameters for the intrinsic rate of change with respect to W (b), the intrinsic rate of change with respect to T (f), the optimal value of T (g), and the upper limit (e) when T is optimum (T = g) were estimated for each cultivar and experiment. The effect of cultivar was characterized primarily by differences in the upper limit parameter e. In each experiment, e was greatest for 'Spanco', intermediate for 'Florunner', and least for 'Okrun'. The effect of cultivar on b followed a pattern similar to that for e in experiment 1, but not in experiment 2. Differences among cultivars for estimates of f and g were small and inconsistent. Estimates for g were precise for each cultivar and experiment and fell within the range of 22.3 to 23.2 degrees C. Cultivar responses to T and W were further evaluated using data pooled over the two experiments. Parameter e was estimated for each cultivar, but common values of b, f, and g were estimated. At e = 22.8 degrees C, lesion density approached an upper limit of 96, 17, and 6 lesions per leaf for the cvs. Spanco, Florunner, and Okrun, respectively. These fitted values approximated the observed values of 86, 25, and 9 lesions per leaf for the respective cultivars. Cultivars varied in their response to W at a given T. At 22.8 degrees C, one lesion per leaf was expected following 26, 30, and 36 h of wetness for 'Spanco', 'Florunner', and 'Okrun', respectively. If temperature was increased to 28 degrees C, one lesion per leaf was expected following 36, 44, and 54 h of wetness for the respective cultivars.

Ethylene Production and Leaflet Abscission of Three Peanut Genotypes Infected with Cercospora arachidicola Hori.

Ethylene can induce abscission of leaves and other plant organs. Increased ethylene production by plant tissues can occur after invasion by microorganisms. The fungus Cercospora arachidicola Hori, attacks peanut leaflets and causes defoliation. Our objective was to determine if ethylene was involved in this defoliation. Leaves of three peanut, Arachis sp., genotypes were inoculated with C. arachidicola. Two genotypes, ;Tamnut 74' and PI 109839, produced ethylene and were defoliated. The third genotype, PI 276233, a wild species, did not produce ethylene above control levels and was not defoliated. Increase in ethylene production by Tamnut 74 and PI 109839 coincided with appearance of disease symptoms. Tamnut 74 produced the most ethylene, but PI 109839 was equally defoliated. Thus, less overall ethylene production did not necessarily indicate a more resistant genotype in this system unless ethylene production remained at control levels, as it did for PI 276233. Ethylene sufficient to initiate abscission could have been produced by the seventh day after inoculation when it was similar for both Tamnut 74 and PI 109839, but 3 to 4 times control amounts. This occurred before the rapid increase in ethylene production and before disease symptoms were visible. Silver ion, a potent inhibitor of ethylene action, was sprayed at three concentrations on intact Tamnut 74 plants. All rates reduced abscission and 150 mg/liter Ag(I) decreased abscission to below 10%. The data indicate that ethylene produced by peanut leaves in response to C. arachidicola infection initiates abscission and that ethylene action can be blocked by Ag(I) in such a host-pathogen interaction.