Final Report for GNC07-074
This study was conducted to develop an effective method for management of internal discoloration of horseradish roots. Internal discoloration of horseradish is a disease complex caused by at least three fungi, Verticillium dahliae, V. longisporum, and Fusarium solani. These fungi are carried in the propagating roots (set-borne inoculum) and also survive in the soil (soil-borne inoculum). The first step for management of the disease was to develop a reliable method to eradicate set-borne inoculum of the pathogen. This was achieved by treating the sets in hot water. The most effective treatment for eradication of set-borne inoculum, without adversely effecting set germination or plant vigor, was determined to be hot-water treatment of horseradish sets at 47ºC for 20-30 min. The Second step for management of the disease was protecting plants against soil-borne inoculum in the fields. This goal was fulfilled by set treatment with the fungicide fludioxonil (Maxim 4FS or Maxim Potato WP)or biocontrol agent Trichoderma virens (SoilGard 12G or G-41) prior to planting sets. By combining the hot-water treatment of the sets and application of the fungicide or biocontrol agent onto the sets the internal discoloration of horseradish roots was effectively managed.
Horseradish (Armoracia rusticana Gaertn. Mey. & Scherb.), a root crop in the Brassicaceae family, is cultivated for its white, pungent, fleshy root. Approximately half of the total commercial horseradish supply of the United States (US) is from the Mississippi River Valley, near East St. Louis, Illinois. Eau Claire, Wisconsin, and Tule Lake, California, are the other two major production areas of horseradish with each producing about 20% of the total horseradish supply in the US. Horseradish is a high-value crop with the value of processed-products exceeding $ 6,000 per acre.
Internal discoloration of roots, a complex disease of horseradish, is a major limiting factor in horseradish production. Verticillium wilt of horseradish and the associated root deterioration have been known since the middle of the 19th century in Europe. Verticillium wilt was first reported in the US in 1931 in Michigan where it was reported to have reduced yields by 20%. Verticillium wilt and root deterioration resulted in a substantial yield loss in Wisconsin in 1973.
Over the past 20 years, the growers in Illinois and other parts of North America have experienced substantial reductions in marketable yield of horseradish due to the internal discoloration of roots. Yield losses of up to 100%, caused by the internal root discoloration, have frequently occurred in Illinois. There is no effective strategy to control this disease complex.
Potschke (1923) was the first to determine that Verticillium spp. causing internal discoloration of horseradish roots. Eastburn and Chang (1994) reported Verticillium dahliae as the primary causal agent of internal discoloration of horseradish roots in Illinois. Percich and Johnson (1990) and Babadoost et al. (2004) reported that internal discoloration of horseradish roots is a complex disease. At least three fungal species, V. dahliae, V. longisporum and Fusarium solani were identified as the causal agents of the internal root discoloration (Babadoost et al., 2004). It is also likely that additional bacterial and/or fungal pathogens are involved in causing internal discoloration horseradish roots.
Gerber et al. (1983) related the internal discoloration problem to soil and suggested that soil sterilization would help alleviate the problem. There has been, however, little success in controlling the disease through application of fungicides or soil fumigation. Attempts to control the disease using plant resistance also met with practically no success, as no resistant cultivars are commercially available and are not expected to be available in the near future. The practice of crop rotation alone is not effective due to set-borne nature of the disease and pathogen’s wide host range and long-term survival in the soil.
The growers save their horseradish sets from previous harvest to plant in the next season. Most of the sets the growers save are apparently clean (asymptomatic), but infected. Set-borne inoculum of Verticillium and Fusarium species is considered important because infected sets usually give rise to severely infected root, which is unmarketable. Thus, starting horseradish production from pathogen-free sets is essential.
Pathogen-free sets of horseradish can be generated by tissue culturing of horseradish leaves. However, there have been major constraints to large scale deployment of tissue culture. Tissue culture is frequently more expensive than other forms of propagation, it is more labor intensive, and requires more specialized environmental control throughout the numerous stages of development. Pathogen-free sets may also be produced by treating the sets in hot water. However, an effective temperature and treatment time have to be established in order to achieve eradication of set-borne pathogens without reducing germination of sets or vigor of resultant plants.
All three pathogens (V. dahliae, V. longisporum and Fusarium solani) causing the internal discoloration of horseradish roots survive in soil for several years. Crop rotations have not had significant effect on controlling the disease, as the pathogens have wide host range. Attempts for controlling the discoloration problem by soil fumigation and fungicide treatment of plant stock have resulted in failure. Our preliminary trials had showed that fungicide fludioxonil (Maxim 4FS or Maxim Potato WP) and biocontrol agent Trichoderma virens (SoilGard 12G or G-41) were effective to protect plants in the field against soil-borne inoculum of the pathogens, when they were applied onto pathogen-free sets.
The goal of this study was to develop effective strategy for management of the internal discoloration of horseradish roots.
This was a two-year project to develop an effective strategy for management of the internal discoloration of horseradish roots. The specific objectives of this research project were:
(i) to evaluate and demonstrate the effectiveness of thermo-therapy for control of set-borne inoculum of the internal root discoloration;
(ii) to demonstrate the effectiveness of the biofungicides for control of the internal root discoloration;
(iii) to demonstrate effectiveness of an IPM approach to solve the complex internal discoloration disease of horseradish root; and
(iv) to establish a sustainable horseradish production system.
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).
The percentage of the sets with fungal colonies decreased as the hot-water treatment temperature was raised and duration of treatment increased. The percentage of untreated sets (control) with Fusarium was significantly higher than percentage of heat-treated sets with Fusarium. There was a negative linear response of percentage of sets with Fusarium to temperature.
In the hot-water treatment experiment with cultivar 1590, Fusarium was detected only when the sets were heat-treated at 44°C for 10, or 30 min; 46°C for 10; min and in untreated sets. No Fusarium or Verticillium was detected when the sets were heat-treated at 46°C for 30 min; 48°C for 10 or 30 min; and 50°C for 10 or 30 min. Verticillium was detected when the sets were heat-treated at 44°C for 10 or 30 min; and also in untreated sets.
In cultivar 1573, no Fusarium or Verticillium was detected when sets were heat-treated at ≥47°C. In cultivar 1722, no Fusarium was detected when sets were heat-treated at temperatures above 46°C for 20 min. In cultivars 1573 and 1722, percentage of sets with Verticillium was significantly higher for control treatment as compared to other treatments but not 46°C for 10 min.
In cultivar Victor-7, when the sets were cultures 5 days after treatment, the percentage of sets with Fusarium was significantly higher for untreated control as compared to treatments at 46°C for 10 min. When the sets of same size were cultured 3 months after heat treatment, percentage of sets with Fusarium was significantly higher for untreated control when compared to other treatments. No Fusarium was detected in any of the sets treated at temperatures above 46°C for 30 min.
The percentage of sets with Fusarium was significantly higher in the sets of cultivars 15K and BTW that received no heat treatment as compared to those that were treated at 47°C for 20 min. For cultivars 1573 and BTW, no Fusarium was detected when the sets were treated at 47°C for 20 min. No Verticillium was detected in any of the sets treated at 47°C for 20 min.
Set Germination and Plant Vigor
The greenhouse study showed that germination of sets of both 15K and 1573 was not significantly affected when they were treated at temperatures of 46, 47, 48, or 49°C for 10, 20, or 30 min and at 50°C for 10, and 20 min. The treatment at 50°C for 30 min, however, significantly reduced the percentage of germinated sets. The vigor and foliage weight of plants grown from untreated sets were lower than those of heat-treated sets.
In 2007, set treatment at 48°C for 20 or 30 min significantly reduced percentage of germination in cultivars 15K and BTW compared to untreated sets and the sets treated at 46°C and 47°C. Similarly, set treatment at 48°C for 20 or 30 min significantly reduced percentage of germination in cultivars 15K and BTW compared to untreated sets and the sets treated at 46°C, 47°C, and 48°C for 10 min. For cultivar 1573, however, only set treatment at 48°C for 30 min reduced percentage of germination significantly when compared to all the other treatments and control. For cultivar 1722, set treatment at 48°C for 30 min affected germination significantly compared to other treatments but not untreated control.
For cultivar 15K, there was significant reduction in plant vigor when sets were treated at 48°C for 20 or 30 min. For cultivar 1722, heat treatment at 48°C for 30 min reduced plant vigor significantly. For cultivar 15K, plant vigor was significantly reduced when sets were treated at 48°C when compared to other treatments, but not untreated sets. For cultivars 1573 and BTW, heat treatment of sets at 48°C for 20 and 30 min reduced plant vigor as compared to other treatments and untreated sets. For cultivar 1722, 48°C for 30 min reduced plant vigor in comparison with other treatments, but not untreated sets.
Incidence of the internal root discoloration. The results of the field studies on using fungicides and biocontrol agents showed that hot-water treatment of sets reduced the incidence and severity of internal discoloration of horseradish roots.
Both fungicides (Maxim 4FS and Maxim Potato) and biocontrol agents SoilGard and G-41 protected plants against the pathogens causing internal discoloration of roots in fields without any adverse effects on set germination or plant vigor. Serenade MAX reduced germination of sets when applied onto hot-water treated sets. Disease incidence (percentage of roots affected) and disease severity (percentage of root surface area discolored at the cross section) were 68.75 and 7.51%, respectively, in plants grown from non-heat treated and non-fungicide/biocontrol-agent treated sets compared to 13.75 and 0.87% of the plants grown from hot-water treated and G-41 treated sets, and 20.0 and 1.06% of the plants grown from hot-water treated and Maxim Potato treated sets in Wisconsin trials. In Illinois, disease incidence and severity of plants grown from non-heat treated and non-fungicide/biocontrol-agent treated sets were 57.5 and 3.17%, respectively, compared to 4.17 and 0.21% of plants grown from sets treated with hot-water treated and G-41, and 7.50 and 0.46% of the plants grown from sets grown with hot-water and Maxim Potato.
Starting horseradish production from pathogen-free sets is essential for management of internal discoloration of horseradish roots. Pathogen-free horseradish sets can be prepared by hot-water treatment. Preparation of pathogen-free sets by hot-water treatment is feasible. This technique is a simple, safe, reliable, and cost/effective commercial method. It is safe because no chemical is used during set treatment. The method is simple because it can be achieved by using simple equipments/tools that are readily available. It is reliable because it eradicates pathogenic fungi carried in the sets and has no adverse effect on set germination or plant vigor at the effective temperature-time for eradication of the pathogens. Implementation of the hot-water treatment is commercially feasible because it costs approximately $50 per acre and the growers can use it without any license or complexity.
The results of this study showed that temperatures below 46ºC do not effectively control set-borne fungal pathogens. Also, temperatures above 48ºC reduce set germination and/or plant vigor. Treating the sets at 47ºC for 20-30 min effectively controls set-borne inoculum of the pathogenic fungi and has no adverse effect on either set germination or plant vigor. Even in some cases the percentages of set germination of treated sets were higher than those of control sets. This could be due to the elimination of negative effects of some organisms carried in or on the sets during the heat-treatments.
Hot-water treatments of seeds and plant materials are classical thermo-physical methods of plant protection. Crouse et al. (2001) studied the effect of hot-water treatment on fungi in apparently healthy grapevine cuttings and found that no living fungal pathogens existed inside hot-water treated grapevine tissues. Many grapevine nurseries are presently employing hot-water treatment for propagation material as a prophylactic measure, which is effective in eliminating the most well-known fungal pathogens and endophytes from grapevine tissues (Crouse et al., 2001). Nega et al. (2003) found that seed-borne pathogens could be reduced without significant losses of germination by hot-water treatments. At higher temperature, however, treatment time must be lowered to avoid reduced germination of sensitive crops. This study also indicates that hot-water treatment of horseradish propagative stocks (sets) at optimum temperature-time eradicates set-borne pathogens without having adverse effect on set germination or plant vigor.
If hot-water treated horseradish sets are planted in the fields without Verticillium or Fusarium infestation, additional practices may not be needed to control the internal discoloration of roots. But, if the heat-treated sets are considered to be planted in a field with a history of the internal root discoloration of horseradish or a history of Verticillium and/or Fusarium diseases in other crops, additional practices will be needed to protect the plants against the soil-borne inoculum of the pathogens.
The results of this study confirmed the results of previous years that set-borne inoculum of the pathogens causing internal discoloration of horseradish roots is important. The sets may appear symptomless by naked-eye observation, they could carry the pathogens. Planting such sets could result in development the internal root discoloration. No fungicide or biofungicide is available to control the set-borne inoculum of the pathogens. But, the set-borne inoculum can effectively be eradicated from sets. Protection of the plants against soil-borne inoculum of Verticillium and Fusarium species could be achieved by application of fungicide fludioxonil (Maxim 4FS or Maxim Potato WP) or biocontrol agent Trichoderma virens (SoilGard 12G or G-41) prior to planting. Thus, hot-water treatment of horseradish sets followed by treatment with fungicide fludioxonil or biocontrol agent Trichoderma virens effectively control internal discoloration of horseradish roots.
The findings of this study offer an effective strategy for management of the internal discoloration of horseradish root. Implementing this strategy will help to have sustainable horseradish production in Illinois and other horseradish growing areas in the US.
Educational & Outreach Activities
1. Babadoost, M., Eranthodi, A., Jurgens, A., Hippard, K., and Wahle, E. 2007. Thermo-therapy and use of biofungicides and fungicides for management of internal discoloration of horseradish roots, 2006. Pp. 25-31. In 2007 Horseradish Res. Rev. & Proc. Horseradish Growers School.
2. Eranthodi, A., and Babadoost, M. 2007. Thermo-therapy for eradication of fungi pathogens in propagative root-stocks (sets) of horseradish. Phytopathology (supplement) 97 (7): S33.
3. Eranthodi, A., and Babadoost, M., Trierweiler, B., Wahle, E., and Skirvin, R. M. 2008. Thermo-therapy for eradication of fungal pathogens in propagative root-stock (sets) of horseradish. Pages 1-10. In Horseradish Res. Rev. & Proc. Horseradish Growers School.
4. Babadoost, M., Jurgens, A., and Wahle, E. 2009. 2008 Illinois field trial: hot-water treatment and use of fungicides and biofungicides for management of internal discoloration of horseradish roots. Pages 11-18. In Horseradish Res. Rev. & Proc. Horseradish Growers School.
5. Babadoost, M., Eranthodi, A., and Skirvin, R. M. 2009. Evaluating efficacy of fungicides and biofungicides for control of internal discoloration of roots of horseradish plants grown from tissue culture-generated sets, Illinos-2008. Pages 25-28. In Horseradish Res. Rev. & Proc. Horseradish Growers School.
The findings of our studies were presented in the Winter Schools of North American Horseradish Growers in 2007, 2008, and 2009, which were held at Collinsville, Illinois in January of each year. The Collinsville School is the only horseradish growers’ meeting in North America. Also, field trials were showcased to the growers during growing seasons. In addition, the results of our studies were presented to plant pathologists in the Annual Meeting of the American Phytopathological Society in San Diego in 2008.
The research is expected to help to have a sustainable horseradish production in Illinois, as well as other horseradish growing areas in the US. The strategy developed in this research will be widely implemented because:
(i) there is no effective method for control of internal discoloration of horseradish roots is available,
(ii) the proposed strategy can easily be implemented by the growers,
(iii) the method is very cost/effective (about 2% of farm-gate value of the crop),
(iv) the management approach is environmentally safe and can be used in organic horseradish production too, and
(v) all materials used in implementing the strategy are commercially available.
Horseradish is a high-value crop. Farm-gate value of horseradish ranges from $3,000 to more than $6,000 per acre (~ $7,400 – $15,000 per ha). Gross value of horseradish products ranges from $6,000 to more than $10,000 (~ $15,000 – $25,000 per ha). Horseradish has nutritional and medical values. The growers are eager to increase yield and quality of the crop. The findings of this study are expected to have very positive impact on grower’s income, economy of the horseradish grower’s communities, and agricultural economy of Illinois and other states. The costs of implementing this strategy is approximately $50 per acre ($124 per ha), which is very cost/effective.
Farmers started adopting the developed strategy. However, effective implementation of the suggested methods needs developing appropriate technology for rapid hot-water treatment and fungicide/biocontrol agent application.
Areas needing additional study
Developing appropriate technology for speedy treatment of high quantity of horseradish sets with hot water is needed.