Final Report for GS00-002
We investigated the potential of Brassica species as a biofumigation cover crop for control of soilborne disease in tomato production. In a laboratory jar test, Indian mustard inhibited mycelial growth of Sclerotium rolfsii, the fungal agent that causes Southern blight of tomatoes. In field trial, Brassica cover crops (B. juncea L and B. campestris) grew over winter and were tilled into the soil the following spring. In Spring 2000, marketable tomato fruit yield was significantly higher in the Brassica treated plots than in the control plots treated with rye. In Summer 2001, marketable tomato fruit yield was not significantly different between treatments. In an additional laboratory study, individual isothiocyanates (ITCs) (one of the active compounds released from the Brassica species) were assayed for activity against selected plant pathogens and potential synergistic effects were investigated. Results suggested that there were differences in pathogen sensitivity to the compounds studied. Additive and synergistic effects were observed when combinations of ITCs, suggesting that some of the effects seen with the uses of whole tissue application maybe due to a combination of chemicals instead of the dominate ITC present.
Methyl Bromide (MeBr) is necessary in tomatoes production to maintain profitable yields under intensive cultivation. This broadspectrum pesticide is extremely lethal and kills most organisms with which it comes in contact. It has no selectivity to pathogens; instead it produces a near sterile environment removing beneficial organisms as well as pathogens. In addition to the ecological imbalance it imposes on the application site, questions have been raised about its effect on surrounding ecosystems as it volatilizes into air. It is however, methyl bromide’s potential as an ozone-depleting agent that has led to the ban enforced by the EPA and parties of the Montreal Protocol (E.P.A., 1997).
Loss of MeBr may prove devastating to many agricultural communities. A study by the U.S. Department of Agriculture and state universities assessed the effects of potential restrictions of MeBr. The findings, based on agriculture in California, Florida, Georgia, Kentucky, North Carolina and Tennessee, estimated about $1 billion lost annually in growers’ net revenue and consumer cost. Regional and national effects will be visible in the lower quantity and quality of produce available for consumption (Ferguson and Padula, 1994).
The glucosinolate/myrosinase system found in Brassica spp is a natural defense mechanism (Siemens and Mitchell-Olds, 1998). Maceration of plant material causes the mixing of glucosinolate with myrosinase; myrosinase degrades glucosinolates to isothiocyanates that produce the pungent flavor associated with cole crops such as broccoli, cabbage and Brussels sprouts. In controlled laboratory experiments, the pesticidal qualities of isothiocyanates have been proven (Al-Khatib et al., 1997; Charron and Sams, 1999; Harvey et al., 2002; Mayton et al., 1996; and Mojtahedi et al., 1993).
Non-chemical alternatives to MeBr will serve to reduce the potential environmental impact of synthetic pesticides. Biofumigation with Brassica species may provide viable alternative and when incorporated within an Integrated Pest Management system may provide an effective, sustainable regime of disease control.
The overall objective of this study is to determine the feasibility of Brassica sp. green manures as a natural alternative to methyl bromide. Pursuant with this goal, many factors were investigated. This study looked at the ability of mustard-biofumigation to control individual soilborne pathogens. and the relationship of isothiocyanate production and pathogen control. The field study investigated implementation of biofumigation in field settings and looked at its integration with sustainable agricultural systems. Additionally, isolation of individual glucosinolates and determination of the lethality of each specific isothiocyanate was completed to aid in screening Brassica sp. for field use.
The laboratory component for the project consists of the determination of the glucosinolates profiles from glucosinolate producing plants and determination of lethality of individual ITCs on selected plant pathogens. This portion of the project consisted of three phases: (1) capillary electrophoretic analysis of seed extracts, (2) assay of crude seed extracts for lethality against a selected pathogen, (3) assay of individual ITCs for lethality to five plant pathogens.
The extraction and separation of intact GS using capillary electrophoresis (CE) require three steps: (1) crude extraction, (2) clean-up using solid phase extractions (SPE), and (3) separation using CE. The extraction procedure for intact GS is a modification of the method outlined by Declercq and Daun (1989). Seed material was selected based on GS content as reported in the literature (Daxenbichler et al., 1991; Fahey et al., 2001). Seeds analyzed included: Alyssum “Saxatile” (Lobularia maritime (L.) Desv.), Arugula (Eruca vesicaria ssp. Sativa (L.) Cav.), Broccoli “Decicco” (Brassica oleracea [Botrytis Group] L.), Brussels sprouts “Long Island” (B. oleracea [Gemmifer Group] L.), Cabbage “Late Flat Dutch” (B. oleracea [Capitata Group] L.), Caper Bush (Capparis spinosa ssp. Inermis L.), Collards “Vates” (B. oleracea [Acephala Group] L), Dames Rocket (Hesperis matronalis L.), English Wallflower (Cheiranthus cheiri L.), Fall Raab “Salade” (B. rapa [Ruvo Group] L.), Kohlrabi “Early Vienna Purple” (B. oleracea [Gongylodes Group] L.), Mustard “Florida Broadleaf” (B. juncea (L.) Czern.), Mustard “Southern Giant Curled” (B. juncea (L.) Czern.), Peppergrass (Lepidium sativum L.), Pokeweed (Phytolacca americana L.), Radish “Scarlet Globe” (Raphanus sativus [Radicula Group] L.), Radish “White Icicle,” Rutabaga (B. napus [Neobrassica Group] L.), Turnip “Seven Top” (B. rapa [Rapifera Group]L.), Upland Cress (Barbarea verna (Miller) Asch.), Watercress (Nasturtium officinalis L.), Weld (Reseda luteola L.), Woad (Isatis tinctoria L.). All seeds were obtained from commercial sources. Seeds were stored at 5 degrees C until used.
Crude extracts were prepared by grinding 200 mg of seed, adding 4 mL boiling water, heating at 95 degrees C, centrifuging and collecting the supernatant. This was repeated twice per 200 mg of seed. Supernatants from the two extraction steps were combined and the volume adjusted to 10 mL. Purification of crude extracts was carried out using ion exchange chromatography on a prepared DEAE-Sephadex A-25 column
Separation of intact GS was preformed by modifing the MECC (micellar electorkinetic capillary chromatography) method described by Michaelsen et al. (1992). Separation was performed utilizing a Hewlett-Packard (Palo Alto, CA) 3D CE in MECC mode. GS elutioned from the DEAE column were injected by pressure (50 mbars) for 10 seconds. Separation was preformed at 30ºC and 20 kV, with negative polarity at the injection end. Ultraviolet (UV) detection at 235 nm using a deuterium lamp was utilized. The capillary column was 720 mm in total length and the length from injection end to detector was 500 mm. Quantitation of the response from each GS detected was calculated based on the internal standards. Tentative identification was based on comparison of relative retention times.
Seed profiles were verified using the desulfo-GS method for high performance liquid chromatograph (HPLC) published by Spinks et al. (1984). Analysis was preformed on Hewlett Packard 1050 HPLC (Palo Alto, CA) on a Hypersil C18 column 250 x 4.6 mm (Phenomenex, Torrance, CA).
The crude seed extracts used for CE analysis were applied in a growth inhibition assay on S. rolfsii. Mycelial plugs (4.5-cm dia) of S. rolfsii were placed on potato dextrose agar (PDA) in microplates (35 x 15 mm). Filter sterilized seed extracts (100 uL) were applied to the PDA, 1 cm from the plug along with 30 ug myrosinase (aqueous) (Sigma-Aldrich; St. Louis, MO). After 48 h, results were recorded as net radial growth.
In the second experiment, individual isothiocyanates of interest were purchased from LKT Laboratories, Inc (St. Paul, MN) and Aldrich (St. Louis, MO). The ITCs selected included: allyl ITC, alyssin, benzyl ITC, berteroin, cheirolin, erucin, 3-indomethanol, methyl ITC, phenyl ITC, phenethyl ITC, and 3-phenylpropyl. Brassinin, a non-isothiocyanate phytoalexin from cabbage, also was included in this study. The plant pathogens selected for the assay include the fungal pests Botrytis cinerea (strain ATCC 90870), Sclerotium rolfsii, Penicillium expansum, and Pythium myriotylum. In addition, Agrobacterium tumefaciens (strain C58), the bacteria responsible from grown gall, was included as an assay subject.
Test compounds were dissolved in dimethyl sulfoxide (DMSO) and multiple dilutions were made to achieve a range of concentrations (approximately 0.1 uMoles/mL to 2 mMoles/mL). Small petri plates containing 4.5 mL of potato dextrose agar (PDA) or clarified V-8 agar were used in this assay. Hyphal plugs (4.5-cm dia) were cut from the growing margins of pathogen cultures. Sclerotium rolfsii, P. expansum and B. cinerea plugs were placed on PDA, while P. myriotylum plugs were placed on clarified V-8 agar. After placing a hyphal plug in the center of the plate, 10 µL of the diluted compound was pipetted onto the agar 1 cm from the plug. The process was repeated for each dilution and pathogen. Due to the size of this experiment, it was broken down into a series of assays.
The first assay (assay 1) was conducted with only allyl ITC (AITC) against P. expansum and S. rolfsii to determine any potential problems. After placing the hyphal plugs, 10 uL of each dilution was pipetted onto the agar 1 cm from the plug. This affected a series of treatments of AITC at 0.11-, 0.22-, 0.44-, 0.64- and 0.87 mMoles/mL . Two control sets were established; one receiving no treatment compounds and another receiving the solvent DMSO. Mycelial growth was measured after 24 h for S. rolfsii and after 48 h for P. expansum.
The second assay was conducted in a similar manner. Benzyl, 3-phenylproply and phenyl ITC were diluted (w/v) to produce dosages of 1115.6-, 111.6-, 11.2-, 0.1 uMoles /mL agar. As in assay 1, the resulting dilutions were tested against both S. rolfsii and P. expansum.
The third assay consisted of the pathogens P. myriotylum and B. cinerea. The compounds assayed for inhibitory activity included: methyl ITC, allyl ITC, 3-indomethanol, erucin, cheirolin, berteroin, brassinin and alyssin. The 10 uL application of each dilution resulted in treatments of 0.002-, 0.02-, 0.22-, and 2.22 uMoles/mL , respectively. For B. cinerea, allyl ITC dosages did not include a 0.002 uMoles /mL concentration and 3-indomethanol dilutions consisted of only 2.2- and 0.22 uMoles /mL concentration. The same dosages were used for the P. myriotylum plates except that the allyl ITC and methyl ITC included only the 0.22 uMoles /mL concentration. Growth was determined after 24 h for P. myriotylum and after 48 h for B. cinerea.
Based on the results of assay 2 and assay 3, compounds and concentrations were selected for a fourth assay including B. cinerea, P. myriotylum, P. expansum and S. rolfsii. Allyl-, methyl-, and phenethyl ITC were diluted such that the 10 uL application resulted in dosages of 2.2-, 22.2-, 111.1-, 222.2-, and 2222.2 uMoles/mL . Due to limited availability, cheirolin treatment dosages were limited to 2.2-, 22.2-, 111.1-, and 177.6 uMoles/mL . Brassinin dosages were limited to 2.2-, 22.2-, and 111.1 uMoles/mL , while erucin was tested at the limited dosages of 2.2-, 22.2-, and 35.6 uMoles/mL . Concentrations responsible for 50 and 90 % inhibition (IC50 and IC90, respectively) were calculated for each compound and pathogen using regression statistics (SigmaPlot, 2000).
Based on assay 4, three combinations were investigated for potential synergistic activity for the pathogens B. cinerea, and S. rolfsii. The two compounds with greatest inhibition were added to the plates at 5 uL each. The same was done for the two compounds with the least inhibitory activity. The third combination consisted of one high and one low inhibition compound. To look for synergistic activity, the pathogens also were treated with the individual compounds for comparison. The combinations were selected specifically for each pathogen and concentrations were chosen to produce less than optimal inhibition and within the linear range of the compounds, such that synergistic activity could be more readily assessed. Treatment combinations selected for assay against B. cinerea included: (A) methyl ITC (55.6 uMoles/mL) and allyl ITC (55.6 uMoles/mL), (B) cheirolin (88.8 uMoles/mL) and erucin (17.6 uMoles/mL), and (C) methyl ITC (111.1 uMoles/mL) and cheirolin (88.8 uMoles/mL). Treatment combinations selected for assay against S. rolfsii included: (A) phenethyl ITC (11.2 uMoles/mL) and erucin (17.6 uMoles/mL), (B) methyl ITC (55.6 uMoles/mL) and brassinin (55.6 uMoles/mL), and (C) phenethyl ITC (11.2 uMoles/mL) and brassinin (55.6 uMoles/mL.
With the same concentration used in assay 4, a fifth assay was developed to investigate the toxicity of these compounds on A. tumefaciens. This was accomplished using liquid culture and measuring the absorbance. Bacterial growth was measured using a Shimadzu UV-260 spectrophotometer (Columbia, MD.) as a function of absorbance at a wavelength of 500 nm.
Capillary electrophoretic separation of intact GS produced results similar to the desulfo-GS method on HPLC. The extract from collard seed produced the highest concentration of GS at 241.5 uMole/g seed weight with the majority being epiprogoitrin and sinigrin (allyl GS). Watercress seed also yielded a high GS concentration of 224.5 uMole/g. Gluconasturtiin comprised 88% of the watercress GS profile. Some seed extracts, such as radish and Brussels sprouts, had diverse profiles consisting of over six identified GS. In contrast, profiles of extracts from peppergrass and caper bush seeds showed little diversity with only one GS detected.
The extracts from seed of GS containing plants inhibited mycelial growth of S. rolfsii. Peppergrass was the most effective (P0.05).
Assay 1 was used primarily to determine if DMSO exhibited any inhibitory activity on growth of the pathogens. Mycelial growth of P. expansum among control plugs and DMSO treated plugs were not significantly different (P< 0.001). Sclerotium rolfsii plugs had 10% more growth when treated with DMSO (P<0.01). The AITC treatments significantly impacted mycelial growth of both pathogens across dosage (P<0.001). Dosages in excess of 652 uMoles/mL caused growth inhibition of 95% for S. rolfsii. Penicillium expansum experienced only 63 % inhibition with 652 uMole/mL.
The four compounds selected for use in assay 2 have similar chemical structures, differing only in a methyl addition. However, significant differences among the treatments were observed. Benzyl ITC provided the best control with 100% inhibition of mycelial growth from Sclerotium rolfsii plugs across all dosages. Phenethyl and 3-phenylpropyl ITC had inhibition levels significantly different from benzyl and phenyl ITC, inhibiting growth by 78.2 ± 28.3 and 71.4 ± 36.7%, respectively, across dosages (P< 0.05). Phenyl ITC exhibited the least amount of activity, inhibiting growth by 41.7 ± 37.5% across dosage levels. A dosage response was observed for these ITC with the exception of Benzyl ITC (due to the 100% inhibition).
Similar results were obtained when these compounds were tested on P. expansum. Penicillium expansum appeared more resistant to phenethyl, phenyl, and 3-phenylproply ITC than did S. rolfsii. Benzyl ITC provided 100% control of mycelial growth across all dosages and was significantly different from the other compounds (P< 0.05). Although phenyl ITC exhibited the least amount of activity, inhibiting growth by only 33.8 ± 38.4% across dosages, its level of inhibition was not significantly different from phenethyl and 3-phenylpropyl. These ITC had inhibition across dosages at 64.2 ± 39.5 and 59.2 ± 42.87%, respectively. A dosage response within treatment was observed except with benzyl ITC.
The dilutions selected for the compounds in the third assay produced less inhibition of P. myriotylum and B. cinerea than expected. Of the eight compounds assayed against P. myriotylum, only cheirolin and erucin caused inhibition (16.3 ± 13.7 and 17.2 ± 8.0 %, respectively), across dosages, significantly different from the control (P<0.05). Against B. cinerea, only methyl and allyl ITC elicited levels of inhibition significantly different from the control (16.6 ± 14.4 and 22.5 ± 11.8 %, respectively) when examined across all dosages (P0.05). Berteroin had a similar response; 0.02 uMoles/mL inhibited growth by 26.5 ± 5.5 %, while 2.22 uMoles/mL only induced 4.3 ± 7.0 % inhibition. These erratic results may be the result of the near-threshold levels used in this assay. Difference in response may represent genetic and environmental difference in the specific plugs used in the assay. The extremely low dosages may have magnified these differences.
The fourth assay conducted utilized compounds that had demonstrated activity in one of the previous assays. Selected compounds were applied to all four fungal pathogens. Botrytis cinerea was sensitive to the allyl, methyl, and phenethyl treatments. Treatments suppressed radial growth significantly across dosage levels (P<0.05). Plugs treated with 222.2- and 2222.2 uMoles/mL of allyl ITC exhibited 80.2 ± 24.5 and 100.0 ±0.0 %, respectively. Brassinin at111.1 uMoles/mL significantly affected mycelial growth by reducing it 37.7 ± 9.4 %. Both methyl and phenethyl ITC treatments at dosages of 111.1-, 222.2-, and 2222.2 uMoles/mL significantly reduced growth of B. cinerea relatived to the control (P<0.05).
All compounds, at all dosages reduced P. expansum growth significantly. At the lowest application of 2.2 uMoles/mL, these compounds suppressed mycelial growth by 71-78 %. Complete suppression (100%) was achieved at 111.1 uMoles/mL of phenethyl ITC and at 222.2 uMoles/mL of allyl and methyl ITC.
Pythium myriotylulm mycelial growth was suppressed by all of the compounds across dosage levels (P<0.05). Only allyl ITC, brassinin and methyl ITC at the 2.2 uMoles/mL dose did not significantly reduce pathogen growth. Treatments applied in excess of 222.2 uMoles/mL induced 100% inhibition. Cheirolin, erucin and phenethyl ITC provided the greatest level of suppression at the low dosage of 22.2 uMoles/mL with inhibition of 84.8 ± 26.3, 93.4 ± 3.7 and 100.0 ± 0.0 %, respectively.
Mycelial growth of S. rolfsii was suppressed by all of the compounds across dosage levels (P<0.05). A dosage effect however, was observed. Erucin produced the greatest level of suppression (81.8 ± 4.7 %) at the lower dosage of22.2 uMoles/mL , while phenethyl ITC affected 75.7 ± 20.1% inhibition. The other compounds demonstrated less than 20 % inhibition at the same dosage.
Agrobacterium tumefaciens was utilized also in this series of assays. Allyl, methyl and phenethyl ITC, as well as cheirolin treated cultures, exhibited suppressed growth, and differed significantly from the control cultures across dosage (P<0.05). Phenethyl ITC exhibited the most control of bacterial growth with 48.0 ± 4.4 % inhibition at the 111.1 uMoles/mL dosage and 92.5 ± 0.3 % at 222.2 uMoles/mL dosage. Allyl ITC also demonstrated control of bacterial growth at the 222.2- and 2222.2 uMoles/mL dosages, inhibiting bacterial growth 41.0 ± 3.1 and 93.6 ± 0.5 %, respectively.
The assay to investigate potential synergistic interactions was completed using the treatments listed in the materials and methods section. When 55.6uMoles/mL each of methyl and allyl ITC were added to a B. cinerea plate, inhibition increased 86.5 % compared to either compound separately at 111.1 uMoles/mL . The combination of 88.8 uMoles/mL cheirolin and 17.6 uMoles/mL erucin increased inhibition by 6.6 % over the individual compounds at 177.6 and 35.2 uMoles/mL , respectively. Only a 2 % increase was observed with the 55.6 uMoles/mL methyl ITC –and 88.80 uMoles/mL cheirolin combination. Two of the combinations tested against S. rolfsii caused a marked increase in inhibition. The combination of 55.6 uMoles/mL brassinin and 11.2 uMoles/mL phenethyl caused a 12 % increase in inhibition. The combination of 55.6 uMoles/mL methyl ITC and 55.6 uMoles/mL brassinin demonstrated a 144 % increase in inhibition over the individual compounds. Increased inhibition was also caused by the combinations tested against A. tumefaciens. While the combination of 55.6 uMoles/mL phenethyl ITC and 55.6 uMoles/mL brassinin and the combination 111.1 uMoles/mL allyl and 55.6 uMoles/mL phenethyl ITC produced a modest 8 % increase in inhibition, the combination of 55.6 uMoles/mL brassinin and 55.6 uMoles/mL cheirolin affected a 90% increase in inhibition.
Regression statistics were used to calculate the IC50 and IC90 for each compound against each pathogen from assay 4. As expected, the pathogens had different sensitivities to the different compounds. Methyl ITC was the most effective inhibitor of B. cinerea mycelial growth with an IC90 of 153 uMoles/mL . In contrast, it would require 253 uMoles/mL allyl ITC and 483 uMoles/mL of phenethyl ITC to obtain similar levels of suppression. Botrytis cinerea was however the exception to the rule; the other pathogens are highly sensitive to phenethyl. Less than 30 uMoles/mL of phenethyl ITC were need to produce 90% inhibition of the fungi, P. myriotylum, P. expansum and S .rolfsii. They in turn were more tolerant of methyl ITC. For both P. expansum and S. rolfsii, the IC90 for methyl ITC was in excess of 250 uMoles/mL . Agrobacterium tumefaciens was also more sensitive to phenethyl ITC (IC90 = 213 uMoles/mL ) than to methyl ITC (IC90 = 2095 uMoles/mL ).
Educational & Outreach Activities
CONFERENCE PRESENTATIONS & SEMINARS:
Specific Inhibition of Individual Isothiocyanates on Selected Plant Pathogens and Potential Synergistic Effects. Southern Region ASHS (Oral). 02/2003
Chemoprotective Properties of Glucosinolates Containing Plants Evaluated Utilizing an Agrobacterium tumefaciens-Potato assay. International Horticultural Congress, 26th (Poster) 08/2002
Glucosinolates, Isothiocyanates, and Biofumigation: a potential alternative of controlling soilborne pests. University of Tennessee, Knoxville (Exit Seminar - Oral) 07/2002
Capillary Electrophoretic Isolation and Quantitation of Individual Glucosinolates from Seeds of Sixteen Plant Species and the Relative Toxicity of their Isothiocyanate Derivatives to Sclerotium rolfsii. Southern Region ASHS (Oral) 02/2002
Biofumigation Utilizing Brassica sp. Increases Marketable Tomato Yield. ASHS 98th Annual Conf. & Expo. (Oral) 07/2001
Allyl isothiocyanates released from Brassica juncea suppresses Sclerotium rolfsii. Proc. Methyl bromide Alternatives Conference. (Poster) 01/2001
Effects of allyl isothiocyanate on mycelial growth from germinating sclerotia of Sclerotium rolfsii. ASHS 97th Annual Conference & Exposition. (Poster) 07/2000
AITC released from Indian mustard suppresses mycelia growth of S. rolfsii. Sigma Xi Paper Competition, UT Knoxville (Oral) 03/2000
GROWER AND FIELD DAY PRESENTATIONS:
Allyl isothiocyanate released from Brassica juncea suppresses Sclerotium rolfsii. Tennessee Green Industry Field Day. Tennessee State Univ. (Poster) 08/2001
Allyl isothiocyanate released from Brassica juncea suppresses Sclerotium rolfsii. Tennessee Agricultural Production Association (TAPPA). (Poster) 02/2001
Noble, R. R. P., Harvey, S. G., and Sams, C. E. 2002. Toxicity of Indian mustard and allyl isothiocyanate to masked chafer beetle larvae. Online. Plant Health Progress doi:10.1094/PHP-2002-0610-01-RS.
Harvey, Stephanie G., Heather N. Hannahan and Carl E. Sams. 2002. Indian mustard and allyl isothiocyanate inhibit Sclerotium rolfsii J. Amer. J. Hort. Sci. 127:27-31.
Harvey, Stephanie G. and Carl E. Sams. 2001. Brassica biofumigation increases marketable tomato yield. Proc. Methyl Bromide Alternatives Conference.
Harvey, Stephanie G. and Carl E. Sams. 2000. Allyl isothiocyanates released from Brassica juncea suppresses Sclerotium rolfsii. Proc. Methyl Bromide Alternatives Conference.
The objectives of this project were to examine to potental of biofumigation as an alternative method of controlling soilborne pathogens. The volatiles released from 2.0 g Indian mustard into a headspace volume of 1 L (equaling AITC at 1.45 umol/L) proved effective for lethal inhibition of S. rolfsii mycelial growth. It was projected that, based on average soil porosity and Indian mustard production of leaf biomass at 12 t/ha, the biomass needed for fumigation (approximately 1.68 t/ha) was achievable in field plantings. Inhibition of S. rolfsii sclerotial germination is more difficult to achieve with AITC than the inhibition of actively growing mycelia. At concentrations approaching 200 times those required to kill mycelia, sclerotia were only suppressed.
Biofumigation with Brassica spp. appears beneficial in tomato production under disease pressure and may be a viable alternative for controlling soilborne pathogens in organic or “green” systems. When integrated with an IPM system, biofumigation could increase control of soilborne pathogens in tomato production systems and may provide an alternative for use in organic systems.
Glucosinolates show extensive genetic variation and therefore results in breakdown products including isothiocyanates exhibit an equally extensive diversity. The differences in inhibitory activity among the ITC tested against these five pathogens suggest that there might be benefits to using mixtures of plant material. A diverse selection of ITC may provide better control soilborne pathogens and other pests than a single species biofumigation cover. In field settings, with multiple plant pathogens and pest present, a mixture of ITC would increase the spectrum of effectiveness. In addition, the potential additive and synergistic effects of mixtures may provide further protection.
An economic analysis was not performed. In general however, the cost associated with biofumigation would include the purchase of the covercrop seed, fuel and time for planting and cultivation, and plastics covers if not used is standard cultivation. Additionally, there is the delay in planting spring crops. This could result in later harvests and the potential for missing market windows.
Areas needing additional study
The effort to find alternative methods of pest control has increased the interest in non-synthetic alternatives. Biofumigation is one potential alternative. Identifying naturally occurring compounds with biocidal activity can help improve the viability of this system. Additional work identifying the individual compounds is needed. The impact of environmental conditions on the effectiveness of these compounds also needs extensive research. With more knowledge on the toxicity, specificity, and synergistic reactions of these compounds, breeding to modify GS content can be directed to the formulation of a "supercover" to increase efficacy of biofumigation. The vast array of naturally occurring GS, their corresponding ITC, and the many isoenzymes of myrosinase, are a resource that is just beginning to be investigated.