Merials and Methods
Hot-water treatment of horseradish propagative stocks (sets). A BLUE M electrical laboratory water-bath (BLUE M Electrical Company, Blue Island, Illinois, U.S.), with 30,000 cm3 capacity, was used for hot-water treatment of the horseradish sets. The water bath was filled with water to 80% of its capacity and temperature of water was set at the desired level. The bath was turned on and water temperature stabilized at desired temperature, which was monitored using a thermometer. Each time, 20 horseradish sets were placed in a perforated stainless-steel basket and immersed into the water. After re-establishing the desired temperature, the sets were kept in the water for scheduled time and taken out of bath and dried on blotter on a lab bench.
Set culturing. The first experiment of horseradish set treatment in hot water was conducted using horseradish cultivar 1590. Sets of size, 1.5-2.5 cm-diameter x 25 cm-long were heat-treated at 44, 46, 48 or 50°C, each for 10 or 30 min. In the second experiment, sets of horseradish cultivars 1573 and 1722, measuring 1.5-2.5 cm-diameter x 25 cm-long were heat-treated at temperatures 46, 47, or 48°C, each for 10, 20, or 30 min. In the third experiment, sets of cultivar Victor-7, with 0.5-1.5 cm-diameter x 25 cm-long and 1.5-3.0 cm-diameter x 25 cm-long, were heat-treated at temperatures 44°C for 30 min, and 45, 46, 47, 48 or 49°C each for 10, 20, or 30 min. The fourth trial was done using the sets of cultivars 15K, 1573 and 1722 of size 1.5-2.5 cm-diameter x 25 cm long. The sets were treated at 47°C for 20 min. Control sets that received no hot-water treatment were included in all four experiments.
In all the experiments except the third, the treated sets were cultured within one week after heat treatment. In the third experiment, the treated sets were cultured within one week and 3 months after heat treatment. Non-heat-treated sets (control) for each experiment were also cultured. The sets were cultured on acidified potato dextrose agar (A-PDA), with pH of 4.4, in Petri plates. A 5 cm-long segment from each set was cut, peeled to remove epidermis, and surface-sterilized using 6% sodium hypochlorite solution for 1 min followed by 100% ethanol for 2 min. Then, the segment was rinsed in sterilized-distilled water 3 times and blotted in sterilized paper towels. Small segments (0.5 cm-thick each) were cut from the middle section of the surface-sterilized segment and plated onto A-PDA in Petri plates (five segments in each plate). The cultured plates were incubated at 24ºC and 12 hr light/12 hr darkness. Cultured segments were examined for growing fungi 5, 10, and 15 days after plating. Fungi growing out of the cultured pieces were transferred onto PDA and later identified.
Set germination and plant vigor. Experiments were conducted in greenhouse and field to determine the effects of thermo-therapy on set germination and plant vigor. The greenhouse studies included two cultivars, 15K and 1573. Sets of size 1-2.5 cm-diameter x 20 cm-long were selected for heat-treatment. The sets were heat-treated at temperatures 46, 47, 48, 49 or 50°C, each for 10, 20 or 30 min. Control sets (without heat- treatment) were included for each cultivar. The treated sets were planted in 25 cm x 25 cm x 5 cm flats containing general purpose soil mix (1:1:1- soil:peat:perlite). Five roots were planted in each flat on 10 January 2007. The flats were placed on greenhouse benches in a split plot design arrangement with three replications (five sets per replication) for each treatment. In this design, cultivars were assigned randomly to main plots and treatments (temperature-time) to subplots. Each bench in the greenhouse room was considered as a block. The data on set germination, plant vigor and foliage weight were recorded on 15 February 2007, 35 days after planting sets. The vigor was assessed based on a fully grown plant at a given time and at a given place, using a scale of 0-4 (0= no plant growth, 1= 1-25% growth of the fully grown plant, 2= 26-50% growth of the fully grown plant, 3= 51-75% growth of the fully grown plant, and 4= 76-100% growth of the fully grown plant at a given time and at a given place). The foliage weight was the weight of freshly harvested leaves 35 days after planting sets.
In 2007, four cultivars, 15K, 1573, 1722, and Big Top Western (BTW) were included in the field trials. The sets (1-2.5 cm-diameter x 25 cm-long)) were heat-treated at temperatures 46, 47 or 48°C, each for 10, 20, or 30 min. The experiments were conducted near Collinsville, Illinois, and Eau Clair, Wisconsin. The designs of the experiments were split plot with randomized complete block with 4 replications (10 sets per replication). The sets were planted on 18 May in a commercial field near Collinsville and 30 May in a commercial field near Eau Claire. The space between adjacent sets within the row was 60 cm and the space between the adjacent rows was 90 cm. The data on set germination and plant vigor were recorded in June, July, and August in the plots near Collinsville and in August and September in the plots near Eau Claire.
Field trials. During 2007-2008, field trials were conducted in commercial fields in Illinois (near Collinsville) and Wisconsin (near Eau Claire) to evaluate effectiveness of fungicides and biofungicides for control of the internal discoloration of horseradish roots. Three horseradish cultivars, 15K, 1573 and BTW, were used in the field trials. The sets (1-2.5 cm-diameter x 25 cm-long) cut, washed with tap water, and heat treated at 47°C for 20 min. Then, the sets were treated with the following materials: 1) fungicide Maxim 4FS (fludioxonil); 2) fungicide Maxim Potato WP (fludioxonil); 3) biocontrol agent SoilGard (Trichoderma virens12G); 4) biocontrol agent G-41 (Trichoderma virens G-41); and biocontrol agent Serenade MAX Bacillus subtilis QST 713). The sets were treated with fungicide or biocontrol agent few days prior to planting in the fields.
Set-treatment with fungicide Maxim 4FS. Two hundred milliliters of tap water was poured into a 2-gallon zip-lock plastic bag and 0.2 ml of the fungicide Maxim 4FS (fludioxonil) was added to water in the bag and mixed. Twenty sets were placed in the bag and shaken for 2 min. Treated sets were drained and dried in an exhaust hood.
Set-treatment with fungicide Maxim Potato (WP). Two hundred milliliter of tap water was poured into a 2-gallon zip-lock plastic bag and 10 g of the fungicide Maxim Potato WP (fludioxonil) was added to water in the bag and mixed thoroughly. Twenty sets were placed in the bag and shaken for 2 min. Treated sets were drained and dried in an exhaust hood.
Set-treatment with biocontrol agents. The sets were dipped in tap water, and then placed in 2-gallon zip-lock plastic bag containing the biofungicide. The bag was gently shaken for 30 seconds. Twenty sets were thoroughly covered with the biofungicide. Treated sets were dried in an exhaust hood.
Field trial. The sets were late May or early Jun in the commercial field near Collinsville (Illinois) and Eau Claire (Wisconsin). Each trial included hear-treated and non-heat-treated sets. Fungicides and biocontrol agents were applied onto heat-treated and non-heat-treated sets. Six treatments (Maxim 4FS, Maxim Potato WP, SoilGard 12G, G-41, Serenade MAX, control), two harvesting dates (July and October), and four replications (10 sets each) for each treatment combination were considered. Sets were planted 24-inch apart within the rows spaced 36-inch apart. Each plot consisted of one 20-foot-long row with 10 sets. The plots were arranged in a split-plot design, cultivar being as the main plots, heat-treatment as sub-plot, treatment (fungicides and biocontrol agents) as sub-sub-plots, and harvesting dates as sub-sub-sub-plots. Four replications of main-plots, sub-plots, sub-sub-plots, and sub-sub-sub plots were arranged in a complete block design.
During the season, weeds were controlled by cultivation and hand weeding. The fields were not irrigated. Number of plants in each plot was recorded and vigor of the plants in each plot was assessed monthly (Illinois) and bi-monthly (Wisconsin). Plants were harvested in early August (mid-season harvest) and in October (late-season harvest). Harvested roots were assessed for incidence and severity of the internal discoloration. Each root was sectioned at 1/3 (upper section) and 2/3 (lower section) of the length from the top and severity of discoloration was assessed at the cross sections. Also, two lateral roots were section and severity of the discoloration was assessed at the sections.
Data analysis. The data collected on fungal detection in sets, set germination, plant vigor, and disease incidence and severity of the roots were analyzed using ANOVA of SAS (Version 9.1, SAS Institute, Carry, North Carolina).