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Craig S. Charron and Carl E. Sams

The U.S. Clean Air Act bans the use of methyl bromide after 2005. Consequently, the development of alternative methods for control of soilborne pathogens is imperative. One alternative is to exploit the pesticidal properties of Brassica L. species. Macerated leaves (10 g) from `Premium Crop' broccoli [B. oleracea L. (Botrytis Group)], `Charmant' cabbage [B. oleracea L. (Capitata Group)], `Michihili Jade Pagoda' Chinese cabbage [B. rapa L. (Pekinensis Group)], `Blue Scotch Curled' kale [B. oleracea L. (Acephala Group)], Indian mustard [B. juncea (L.) Czerniak, unknown cultivar] or `Florida Broadleaf' mustard [B. juncea (L.) Czerniak] were placed in 500-mL glass jars. Petri dishes with either Pythium ultimum Trow or Rhizoctonia solani Kühn plugs on potato-dextrose agar were placed over the jar mouths. Radial growth of both fungi was suppressed most by Indian mustard. Volatiles were collected by solid-phase microextraction (SPME) and analyzed by gas chromatography-mass spectrometry. Allyl isothiocyanate (AITC) comprised >90% of the volatiles measured from `Florida Broadleaf' mustard and Indian mustard whereas (Z)-3-hexenyl acetate was the predominant compound emitted by the other species. Isothiocyanates were not detected by SPME from `Premium Crop' broccoli and `Blue Scotch Curled' kale although glucosinolates were found in freeze-dried leaves of all species. When exposed to AITC standard, P. ultimum growth was partially suppressed by 1.1 μmol·L-1 (μmol AITC/headspace volume) and completely suppressed by 2.2 μmol·L-1 R. solani was partially suppressed by 1.1, 2.2, and 3.3 μmol·L-1 AITC. Use of Brassica species for control of fungal pathogens is promising; the presence of AITC in both lines of B. juncea suppressed P. ultimum and R. solani but some Brassicas were inhibitory even when isothiocyanates were not detected.

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Craig S. Charron and Carl E. Sams

There has been significant interest in the glucosinolate-myrosinase system in plants of the Brassicaceae due to accumulating evidence that some glucosinolate degradation products are anticarcinogenic and/or suppressive to plant pathogens. Because glucosinolate hydrolysis is catalyzed by endogenous myrosinase, characterization of myrosinase activity is important for elucidating the potential bioactivity of crop glucosinolates. We measured the specific activity in citrate-phosphate buffer extracts across the pH range 4.5–6.5 of two cultivars each of five Brassica groups grown during two fall and two spring seasons. Specific activity in two kale cultivars was highly variable, but tended to have highest activity from pH 5.0–6.0. In both cauliflower cultivars from Fall 2000, Fall 2001, and Spring 2002, optimal pH was around pH 6.0. In Spring 2000, however, specific activity was highest at pH 5.0. Maximum specific activity in both cabbage cultivars occurred in the pH range 5.5–6.0 in Fall 2000, Fall 2001, and Spring 2002. In Spring 2000, specific activity in `Red Acre' cabbage was uniform across the range pH 4.5–5.5 and maximum specific activity was at pH 5.0 for `Early Round Dutch' cabbage. Both brussels sprouts cultivars had pH maxima around pH 5.5–6.0 and significantly lower activity at pH 4.5. Specific activity in broccoli was much like that of cauliflower in that highest activity occurred around pH 5.5–6.0 in Fall 2000, Fall 2001, and Spring 2002, but in Spring 2000, maximum activity was at pH 5.0. These results indicate that in most cases, pH optima were in the range 5.5–6.0, but varied somewhat with season and genotype.

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Craig S. Charron and Carl E. Sams

Crops of the Brassicaceae contain glucosinolates(GSs), which when hydrolyzed by the enzyme myrosinase, generate products involved in cancer chemoprotection, plant defense, and plant-insect interactions. A rapid-cycling base population of B. oleracea L. was grown in a hydroponic system in a controlled environment to determine the roles of temperature, photosynthetic photon flux (PPF), and photoperiod in GS concentration and myrosinase activity. The concentration of total GSs in leaves was 44% and 114% higher at 12 and 32 °C respectively than at 22 °C under constant light of 300 μmol·m-2·s-1. The concentration of glucoraphanin, the precursor to sulforaphane, a compound with chemoprotective properties, was 5-fold higher at 32 than at 22 °C. Total GSs were ≈50% lower in roots at 12 °C and 32 than at 22 °C. Total GSs in leaves decreased 20% when PPF was increased from 200 to 400 μmol·m-2·s-1. Myrosinase activity on a fresh weight basis (activity-FW) was ≈30% higher in leaves and stems at 12 and 32 °C than at 22 °C, and ≈30% higher in leaves grown at 200 and 400 μmol·m-2·s-1 than at 300 μmol·m-2·s-1. Consideration of climatic factors that influence the glucosinolate-myrosinase system may be necessary to optimize the planting and cultivation of Brassica crops for maximum health benefits.

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Carl E. Sams, Dilip R. Panthee, Craig S. Charron, Dean A. Kopsell and Joshua S. Yuan

Glucosinolates (GSs) and carotenoids are important plant secondary metabolites present in several plant species, including arabidopsis (Arabidopsis thaliana). Although genotypic and environmental regulation of GSs and carotenoid compounds has been reported, few studies present data on their regulation at the molecular level. Therefore, the objective of this study was to explore differential expression of genes associated with GSs and carotenoids in arabidopsis in response to selenium fertilization, shown previously to impact accumulations of both classes of metabolites in Brassica species. Arabidopsis was grown under 0.0 or 10.0 μM Na2SeO4 in hydroponic culture. Shoot and root tissue samples were collected before anthesis to measure GSs and carotenoid compounds and conduct gene expression analysis. Gene expression was determined using arabidopsis oligonucleotide chips containing more than 31,000 genes. There were 1274 differentially expressed genes in response to selenium (Se), of which 516 genes were upregulated. Ontology analysis partitioned differentially expressed genes into 20 classes. Biosynthesis pathway analysis using AraCyc revealed that four GSs, one carotenoid, and one chlorophyll biosynthesis pathways were invoked by the differentially expressed genes. Involvement of the same gene in more than one biosynthesis pathway indicated that the same enzyme may be involved in multiple GS biosynthesis pathways. The decrease in carotenoid biosynthesis under Se treatment occurred through the downregulation of phytoene synthase at the beginning of the carotenoid biosynthesis pathway. These findings may be useful to modify the GS and carotenoid levels in arabidopsis and may lead to modification in agriculturally important plant species.

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Christina L. Pierson, Carl E. Sams, Dennis E. Deyton and Craig S. Charron

Biofumigation is an alternative to traditional methods of soil sterilization such as methyl bromide. Biofumigation utilizes volatile, pesticidal compounds in soil incorporated plant material from various Brassica species. Three experiments were conducted to study the degradation of allyl isothiocyanate (AITC) generated from the breakdown of glucosinolates present in Oriental mustard (Brassica juncea L. Czerniak). Mustard seed meal was incorporated into a sandy clay loam soil in all experiments. In the first experiment, samples were hydrated and then held in an incubator at 20 ± 0.2 °C. Samples were taken periodically for 7 days or until AITC was not detectable. For the second experiment, hydrated samples were removed from the incubator after 4 hours and 5 mL of ethyl acetate was added. The samples were then placed in a refrigerator at 4 ± 0.2 °C and samples were taken periodically over 77 days. For the third experiment, samples were taken from a strawberry plot experiment grown in a randomized complete block design. Samples were taken and 5 mL of ethyl acetate was added. Then samples were placed into a cooler until returning to the laboratory. The incubator experiment was repeated and showed that the highest concentration of AITC occurred between 2 and 8 hours after hydration. The storage experiment showed a stable relationship between time and AITC degradation. AITC was still present after 77 days. The strawberry plot experiment showed rapid AITC degradation similar to the incubator experiment. Future research will be done to confirm the effects of temperature and glucosinolate content on the amount of allyl isothiocyanate present.

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Heather D. Toler, Craig S. Charron, Carl E. Sams and William R. Randle

Glucosinolates are sulfur-containing secondary plant metabolites commonly found in the family Brassicaceae. The presence of selenium in soils increases the uptake of sulfur and inhibits the production of glucosinolates in brassicaceous plants. This study was undertaken to determine the extent of selenium's impact on sulfur uptake and glucosinolate production in Brassica oleracea L. Rapid-cycling B. oleracea plants were grown hydroponically in half-strength Hoagland's nutrient solution with selenium treatments delivered as sodium selenate concentrations of 0.0, 0.5, 0.75, 1.0, and 1.5 mg·L−1. Elevated sulfur treatments of 37 mg·L−1 sulfate and 37 mg·L−1 sulfate/0.75 mg·L−1 selenate were incorporated to compare with selenium treatments. Plants were harvested and freeze-dried 1 day before anthesis. Selenium and sulfur content of plant tissue was determined by flame atomic absorption spectrophotometry and a carbon–nitrogen–sulfur analyzer. Glucosinolate content of leaf tissue was determined by high-performance liquid chromatography. Selenium and sulfur uptake in plants positively correlated with selenium concentration in the nutrient solution. The sulfur concentration of plants exposed to selenium equaled or exceeded the sulfur concentration of plants exposed to elevated sulfur. Despite higher sulfur concentrations, there occurred a statistically significant decrease in production of five of the seven glucosinolates analyzed in selenium-enriched plants. Plants that underwent elevated sulfur treatments had higher glucosinolate production than selenium-treated plants. These results suggest that selenium either upregulates or prevents the downregulation of sulfur uptake in B. oleracea. In addition, the presence of selenium within the plant appears to have a negative impact on the production of certain glucosinolates despite adequate availability of sulfur.

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Andrew J. Price, Craig S. Charron, Arnold M. Saxton and Carl E. Sams

A study was conducted to quantify volatiles generated from Indian mustard (Brassica juncea L. Czerniak) tissue incorporated into soils under controlled conditions. Mustard residues were incorporated into noncovered and covered soils that varied by texture, temperature, moisture, pH, or sterility (autoclaved or nonautoclaved). Sandy loam soil had 38% more allyl isothiocyanate (AITC) than clay loam soil. AITC concentration in 45 °C soil was 81% higher than in soil at 15 °C, and 56% higher in covered compared to noncovered treatments. The microbial catabolism of AITC was suggested by the result that AITC concentration in autoclaved soils was over three times that measured in nonautoclaved soils. The highest AITC level detected (1.71 μmol·L–1) occurred in the autoclaved covered soil. Several factors also influenced CO2 evolution. At 30 or 45 °C, CO2 concentration was at least 64% higher than at 15°C. The covered soil had over twice the CO2 found in the noncovered soil, and the nonautoclaved soil treatment yielded twice the CO2 measured in the autoclaved soil. There were no main effect differences among soil moisture, soil pH, and soil texture treatments for CO2 concentrations. This information could be helpful in defining ideal soil conditions for field scale experiments. Additionally, this study demonstrates a sampling technique for testing fumigation potential of biofumigation and solarization systems that may have the potential to replace methyl bromide.

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Barbara Kocourkova, Craig S. Charron, Charles Graves, Norman K. Lownds and Carl E. Sams

High glucosinolate content in brassica meal is a limiting factor in consumption of rapeseed. In recent years canola cultivars of rapeseed with decreased glucosinolate content have been developed. However, environmental and nutritional factors are also believed to influence glucosinolate content. This study was conducted to determine the relationships among water stress, B nutrition, and glucosinolate content in canola. Two canola cultivars (`Cyclone' and `American A112') were grown in a continuously recirculating hydroponic system with modified Hoagland solution (0.6 ppm B). Water stress was induced gradually (2% per day using polyethylene glycol 8000) starting when plants were 4 weeks old. Osmotic potential was maintained at –0.1 MPa (high stress level), –0.085 MPa (medium stress), or 0.05 MPa (control). Treatments were arranged in a randomized incomplete-block design, with three blocks, four replications, two cultivars, and three treatments. Upper leaves (no. 15 and higher) were collected and analyzed by inductively coupled plasma emission spectrometry for B content. Total and indole glucosinolate content of seeds were measured colorimetrically and by HPLC. The leaf B content of stressed plants decreased by 55% in `Cyclone' and 29% in `American A112'. Total glucosinolate content increased 28% and 12%, respectively, in stressed plants of `Cyclone' and `American A112'. Indole glucosinolate content was 44% and 13% higher in the same plants. The interaction between cultivar and water stress was not significant for glucosinolate content but was significant for B content of the leaves.

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Craig S. Charron, Daniel J. Cantliffe, Raymond M. Wheeler, Ara Manukian and Robert R. Heath

A system and methodology were developed for the nondestructive qualitative and quantitative analysis of volatile emissions from hydroponically grown `Waldmann's Green' leaf lettuce (Lactuca sativa L.). Photosynthetic photon flux (PPF), photoperiod, and temperature were automatically controlled and monitored in a growth chamber modified for the collection of plant volatiles. The lipoxygenase pathway products (Z)-3-hexenal, (Z)-3-hexenol, and (Z)-3-hexenyl acetate were emitted by lettuce plants after the transition from the light period to the dark period. The volatile collection system developed in this study enabled measurements of volatiles emitted by intact plants, from planting to harvest, under controlled environmental conditions.

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Craig S. Charron, Daniel J. Cantliffe, Raymond M. Wheeler, Ara Manukian and Robert R. Heath

To investigate the effects of environment on plant volatile emissions, `Waldmann's Green' leaf lettuce was cultivated under different levels of photosynthetic photon flux (PPF), photoperiod, and temperature. A modified growth chamber was used to sample plant volatile emissions nondestructively, over time, and under controlled conditions. Total volatile emission rates were significantly higher from lettuce cultivated under PPF of 360 or 200 μmol·m-2·s-1 compared to 105 μmol·m-2·s-1, and significantly higher under a 16-h photoperiod than an 8-h photoperiod. No differences were detected among emission rates from different temperature treatments. In controlled environments, emissions could be regulated by adjusting environmental conditions accordingly.