Final Report for LS03-147
Greenhouse production is an excellent means of augmenting income for farms, as it can provide spring and fall cash crops that are not weather dependent. A careful system of exclusion, sanitation, and environment management has allowed us to eliminate the use of pesticides in our modified hydroponics greenhouses. Many diseases can be controlled through cultural practices; however, species of water-borne pathogens, specifically Pythium and Phytophthora, remain potential threats in a hydroponic greenhouse production system. The objective of the research funded by this planning grant was to investigate the impact of three types of alternative biorationals on production of tomatoes in a modified hydroponic system. Botanical alternatives (bioactive herbage), and two types of microbial alternatives (endophytic Beauveria and PGPR) were applied alone and in combination to tomatoes grown in hydroponic culture. Tomatoes were grown under standard conditions for modified substrate culture. Herbage and PGPR treatments were applied directly to the substrate at transplant; Beauveria was applied as a seed treatments. Tomatoes were harvested April 27, 2004 through June 15, 2004. At harvest, tomatoes were weighed and graded. Protocols for exclusion, sanitation and environment management were sufficient to eliminate the need for commercial pesticide applications. For fresh market tomatoes, tomato yield was significantly impacted by interaction of Monarda herbage and herbage rate. There was a significant interaction between Pythium and herbage at the high rate for yield of fresh market tomatoes. There were no significant differences in Processing Tomato or Total Marketable Tomato yields due to Pythium and herbage. Treatment with Roman chamomile herbage resulted in reduced weight of Fresh Market Tomatoes. Treatment with Roman chamomile did not affect weight of Processing Tomatoes or Total Marketable Tomatoes. There was no effect of the interaction among Beauveria, Pythium and PGPR on Fresh Market Tomatoes or Total Marketable Tomatoes when all cultivars were considered but there was a significant decrease in the weight of Processing Tomatoes in treatments with Beauveria in some cultivars. Within all data sets, effects of Pythium treatments were rarely significant, but when they were, plants treated with Pythium produced more fruit. Based on these data, we have identified biorational combinations that not only have the potential to control Pythium disease in hydroponic tomatoes but also to increase yield. A proposal based on the data collected in this research was submitted to Southern SARE in response to the 2005 Call for Proposals for Research and Education grants.
Originally, the objective of this planning grant was to investigate the use of Monarda bioactive herbage on disease control in tomato transplant production in float beds. The market research study that was conducted in the initial stages of this project found that, due to grower belief that float bed production systems produce tomato transplants of markedly inferior quality, there is unlikely to be a market for this product. At that point, research goals were redirected in order to provide data that would most benefit growers, consumers, and other stakeholders.
The objective of this research was to investigate the impact of three types of alternative biorationals on production of tomatoes in a modified hydroponic system. Botanical alternatives (bioactive herbage), and two types of microbial alternatives (endophytic Beauveria and PGPR) were applied alone and in combination to tomatoes grown in hydroponic culture.
Greenhouse production is an excellent means of augmenting income for farms, as it can provide spring and fall cash crops that are not weather dependent. Greenhouse tomato production in modified hydroponic substrate culture is standard throughout the southeastern United States (Ray et al., 2004, Snyder, 2001). Greenhouse crops can be a boon to the organic food supply because they can generate the copious amounts of produce desired by consumers and processors. For example, field tomatoes produce between 18,000-24,000 lbs of tomato fruit per acre (Diver et al., 1999), but in our greenhouses (in which no pesticides are used) 10,500 lbs in 0.066 acres or a staggering 160,000 lbs/acre is not uncommon (Ray et al., 2004).
Greenhouse environments are perceived to be sterile, but the same conditions that are optimal for propagating plants are usually optimal for their pathogens. Pathogen exclusion is the preferred method for control of diseases and insects in greenhouse crops, but in many systems weekly pesticide applications are used (Snyder, 2001) either as preventatives or because exclusion methods fail. When proper techniques are applied, greenhouse growers can produce tomatoes without pesticides, thus providing the consumer with pesticide residue-free produce. Producers receive the two-fold benefit of value-added eco-labeling and reduced production costs. Hydroponically-grown plants are often isolated from many pathogens that cause tremendous losses in the field [e.g., Fusarium wilt of basil (Garibaldi et al., 1997)]. In contrast, pathogens that only sporadically cause disease in the field may cause tremendous losses in hydroponic culture.
A careful system of exclusion, sanitation, and environment management has allowed us to eliminate the use of pesticides in our greenhouses. Many diseases can be controlled through cultural practices; however, species of water-borne pathogens, specifically Pythium and Phytophthora, remain potential threats in a hydroponic greenhouse production system. Pythium, which cannot be excluded from greenhouse systems and for which there are no registered pesticides available to greenhouse growers, contaminates water sources (Paulitz and Berlanger, 2001; Koohakan et al., Ingram, 2004). This organism can survive water treatment procedures and viable propagules have been found in municipal water systems. The spread of both Pythium and Phytophthora through water systems is a likely scenario in the Southern Appalachians. Pythium has been isolated from farm well systems. Phytophthora parasitica was transmitted from a single plant on the drain end of an ebb-and-flow system. (Strong et al., 1997). Pythium disease, in particular, has the potential to cause total loss in tomato production greenhouses.
Alternative biorationals are defined in this report as chemicals derived from plants, fungi, bacteria, or other non-man-made synthesis and which can be used for pest control and certain microorganisms that are effective in controlling target pests. These agents usually do not have toxic effects on animals and people and do not leave toxic or persistent chemical residues in the environment (www.epa.gov/pesticides). Botanicals are plant-derived materials that are generally short-lived in the environment because they rapidly decay in the presence of light, water and air. Microbial pesticides are microorganisms or their by-products.
Species of Monarda are sources of essential oils of great chemical diversity (Marshall and Scora, 1972; Mazza and Marshall, 1992; Mazza et al., 1993). Essential oils are highly volatile substances isolated from an odiferous plant; the term essential was used because these oils were thought to contain the essence of odor and flavor (Linskens and Jackson, 1991). The genus Monarda (Labiatae) contains at least 16 annual or perennial herbs valued for their essential oil content (Marshall and Scora, 1972). High content of two essential oils used in the perfume industry, geraniol and citral, makes these plants potentially high-value crops. There are myriad ethnobotanical uses for Monarda species (Vogel, 1970; Duke, 1992; Duke 2004) - many of which are related to the bioactive properties (antibacterial, antifungal, and antioxidant) of the components of the essential oils. At least 56 phytochemicals with antifungal or herbicidal activity have been isolated from M. didyma, at least 36 have been isolated from M. fistulosa, and at least 26 isolated from M. punctata. Plant essential oils are well known for their antifungal properties and have been proposed as natural, safe pesticides (Bauske et al., 1994b; Deans, 1991; Tsao and Zhou, 2000; Thompson, 1989). Several key essential oils have been shown to inhibit the growth of significant soilborne fungal pathogens. Fusarium and Sclerotinia (Dube et al., 1989; Meleo et al., 2002; authors, unpublished), Pythium (Bauske et al., 1994a), and Rhizoctonia and Verticillium (Pitzarokili et al., 1999) have all exhibited growth inhibitions when exposed to various plant essential oils, many of which are present in Monarda spp.
In our laboratory and under controlled greenhouse conditions, essential oils of plants in the genus Monarda (Labiatae) inhibit the growth of economically important plant pathogenic soilborne fungi. When added to planting medium in the form of bioactive herbage (dried and ground leaves and flowers), the growth of Fusarium and Rhizoctonia is inhibited, and sclerotia of Sclerotinia are killed (Gwinn et al. 2003, unpublished). Three Monarda cultivars of differing chemistry were selected for a study on the impact of herbage on Rhizoctonia seedling disease: ‘Elsie’s Lavender’, ‘Marshall’s Delight’, and ‘Sioux’. Differences between treatment combinations were due to the main effects of Monarda or R. solani. In Rhizoctonia-infested media, percent germination and plant height were increased significantly with the addition of ‘Elsie’s Lavender’ and ‘Marshall’s Delight’ herbage. Disease index (values 1-4) (Seth, 2001) was evaluated for two cultivars. A high disease index indicates a greater loss due to disease. In Rhizoctonia-infested medium, disease index was decreased significantly with the addition of ‘Elsie’s Lavender’ but not with ‘Sioux’ (Gwinn et al. 2003).
Beauveria bassiana is a soilborne fungus with entomopathogenic properties. Isolates of this fungus occur worldwide and have an extensive host range of insects at all stages of development. Efficacy against insect pests, such as citrus root weevil, Artipus floridanus (Eyal et al., 1994), Colorado potato beetle, Leptinotarsa decemlineata (Jaros-Su et al., 1999), European corn borer, Ostrinia nubilalis (Bing and Lewis, 1991; Feng et al., 1988), greenhouse whitefly, Trialeurodes vaporariorum (Poprawski et al., 2000), lesser stalk borer, Elasmopalpus lignosellus (McDowell et al., 1990), and sweet potato whitefly, Bemisia tabaci (Eyal et al., 1994), has been demonstrated.
Commercial preparations of B. bassiana are produced as conidial suspensions and applied as foliar sprays for insect control by direct contact of conidia with the insect, followed by parasitism. However, B. bassiana has been shown to colonize plants endophytically, and control insects systemically with no harmful effects to the plant host. Following foliar applications of B. bassiana, endophytic colonization has been demonstrated in corn (Bing and Lewis, 1991; Bing and Lewis, 1992a, 1992b; Lewis et al., 1996; Jones, 1994), potato and cotton (Jones, 1994). In corn, colonization by B. bassiana was correlated with a reduction in tunneling by European corn borer (Bing and Lewis, 1991). With light and electron microscopy, Wagner and Lewis (2000) confirmed that B. bassiana, applied as a conidial suspension to foliage, was an endophyte of corn. Conidia germinated and hyphae entered through natural openings such as stomates, or by direct penetration via enzymatic activity and mechanical pressure. Hyphae of B. bassiana were observed in epidermal regions, between palisade parenchyma cells, and within xylem elements. Wagner and Lewis (2000) suggested that movement within vascular bundles may enable the fungus to travel within the plant, and ultimately provide overall insecticidal protection. Other endophytic fungi, such as Neotyphodium coenophialum, which infects tall fescue, are known to adversely affect both plant pathogens and insect pests of their plant hosts (Bernard et al., 1997; Gwinn et al., 1992).
In addition to activity against insects (Leckie, 2002; authors, unpublished) we have shown that B. bassiana can control Rhizoctonia damping-off in tomato seedlings when applied as a seed treatment. In greenhouse studies, B. bassiana 11-98 provided significant protection against damping-off caused by R. solani (Ownley et al., 2000; Ownley et al., unpublished data). In two trials, using soil infested with R. solani, seed treated with B. bassiana had significantly greater plant stands (68 to 75%) than untreated seed (13 to 25% plant stand). In the second trial, the percent plant stand from seeds treated with B. bassiana and planted in R. solani-infested soil was 75%, which was not significantly different from untreated seed planted in pathogen-free soil (82%). In pathogen-free soil, from both trials, the percent plant stand from seeds coated with B. bassiana was 89 to 92%. This was similar to the percent plant stand from untreated seeds in pathogen-free soil (82 to 85%).
In a separate greenhouse experiment, seeds of ‘Mountain Spring’ and ‘Mountain Pride’ tomatoes, were treated with B. bassiana 11-98 conidia in 2.5% MC solution and planted in potting soil infested with R. solani (Ownley, 2004). Controls in infested soil were: untreated seed or seed treated with 2.5% MC. Additional controls were untreated seed, and seed treated with 2.5% MC (trial 2 only; Table 1) sown in pathogen-free soil. In both trials, for both cultivars in infested soil, seed treated with B. bassiana resulted in significantly higher plant stands than untreated seed. In our studies, we have determined that B. bassiana 11-98 is endophytic in tomato (Leckie, 2002). In current studies we are determining the importance of this endophytic behavior in insect and disease control in tomato and other crops, evaluating different formulations and application methods of B. bassiana for efficacy against Rhizoctonia damping-off and greenhouse pests, and determining its spectrum of activity against other soilborne pathogens.
Plant Growth Promoting Rhizobacteria.
Induced resistance is an enhanced defense capacity developed by a plant when stimulated by pathogen, plant-growth promoting rhizobacteria (PGPR), or other appropriate entity. Inducing a plant’s own defense systems is an area of growing interest for plant disease control industries. These methods use organisms or chemicals that are environmentally benign to stimulate disease resistance. Plants acquire a state of general resistance in response to an initial stimulus; this phenomenon is termed systemic acquired resistance (SAR) (Metraux et al., 2001). Salicylic acid, a simple phenolic compound, is necessary for SAR regulation, but it is not the mobile signal as was once believed (Metraux et al., 2001). The SAR response requires a necrotizing response of the plant. Plant growth-promoting rhizobacteria (PGPR) are naturally occurring root-colonizing bacteria that can induce increased plant growth (Cleyet-Marcel et al., 2001; Kloepper, 1992; Glick, 1995), often with concomitant reductions in plant diseases. The PGPR induce resistance in distant portions of the plant; this is termed induced systemic response or ISR (Raupach and Kloepper, 2000; Jetiyanon and Kloepper, 2002). In ISR, the response is independent of salicylic acid, but requires responsiveness to plant growth regulators, jasmonic acid and ethylene. The beneficial effects of PGPR for disease control have been reported for many crops and pathogens (Cleyet-Marcel et al., 2001; Kloepper, 1992; Raupach et al., 1996; Raupach and Kloepper, 1998; Reddy et al., 1999). Since disease control by any single strain of PGPR is typically less than that of fungicides, much research has been devoted to mixtures of PGPR to optimize disease control. BioYield and Subtilex are commercial preparations of PGPR. The PGPR products are incorporated into the planting mix used to grow transplants and contain species of spore-forming PGPR Bacillus strains. Treated transplants show increased shoot and root growth leading to more rapid development than untreated transplants. An ISR response is frequently observed. Insect herbivory is altered by PGPR colonization of some plants; colonization may lead to shifts in host metabolism and alteration of defense compounds (Zehnder et al., 2001). Also, PGPR treatment can result in enhanced plant growth. Simultaneous activation of ISR and SAR results in synergistic additive protection (Pieterse et al., 2001). Some PGPR produce salicylic acid and effectively induce resistance (Audenaert et al., 2002).
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Twelve alternative biorationals (alone and in various combinations) were evaluated for control of Pythium disease. Eight cultivars of Monarda spp., were tested – ‘Cerise’, ‘Croftway Pink’, ‘Elsie’s Lavender’, ‘Marshall’s Delight’,’ Puerto Purification’, ‘Sioux’, ‘Stone’s Throw Pink’, and ‘Violet Queen’. Herbage was also collected from Roman chamomile. A list of all treatments is on file with SARE. Foliage and flowers (herbage) were collected from the Monarda Evaluation Garden located in the UT Gardens, Knoxville, TN. This garden contains four replicated blocks of 52 Monarda species or cultivars. These plants have been sampled monthly for three growing seasons to monitor essential oil content and composition. Two isolates of Beauveria bassiana were used - Botaniguard (BioAgriculture Corporation, Butte, MT) and 11-98 (Bonnie Ownley, University of Tennessee). Beauveria bassiane conidia (106 colony forming units/mL) were suspended in an aqueous methyl cellulose solution (0.02g/ mL) and used to coat tomato seeds. Seeds were allowed to dry and planted within one week. Commercial preparations of PGPR, “Bioyield” (Gustafson LLC, Plano, Texas) and “Subtilex” (Becker Underwood, Ames, IA), were used.
Due to the complex nature of the treatments used in these experiments, several abbreviations were used. All Monarda treatments were two letter abbreviations. The following Monarda cultivars were used as treatments: NH = No herbage control; CE = ‘Cerise’; CP = ‘Croftway Pink’; EL = ‘Elsie’s Lavender’; MD =’ Marshall’s Delight’; PP = ‘Puerto Purification’; SI =‘Sioux’; SP = ‘Stone’s Throw Pink’ and VQ = ‘Violet Queen’. Roman chamomile (RC) was also used as a herbage treatment. When the cultivar abbreviation is followed by – L, then the low rate of herbage applies; otherwise the high rate was used. If no two letter abbreviations are used, then herbage was not a factor in the experiment. The following pathogen treatments were used: +P = Pythium zoospores added at transplanting and –P = no zoospores added at transplanting. Three letter abbreviations were used for microbial treatments. The following microbial alternatives were tested: BTG = Botaniguard, a commercially-available isolate of B. bassiana; UTN = B. bassiana 11-98 (Bonnie Ownley); BYD = Bioyield,; and SBL = Subtilex. A plus sign (+) before the abbreviation indicates that the treatment was included and a minus sign (–) indicates the control.
Transplants were grown in float beds; seeds were transplanted in December 2003 and seedlings were transplanted to bags of perlite in February 2004. All alternative biorational treatments except Beauveria, which was applied as a seed treatment, were added at the time of transplant. For treatments except those designated as low rate, Monarda herbage (6.75 g) was packaged in commercial tea bags (GMBH&CO. Hamburg, Germany); approximately one half the amount of herbage (3.33 g) was used in low rate treatments. An opening was created in the perlite by hand, then the tea bag was inserted into this opening, and the tea bag was covered by the displaced perlite. A suspension of P. myriotylum zoospores (15 mL) was added directly adjacent to the stem. BioYield or Subtilex (10mL) treatments were applied directly adjacent to the side of the stem opposite to where Pythium was applied. Each row served as a replicate, and each treatment was replicated eight times. Fertigation protocols developed in earlier research (Ray, 2004) were used. Protocols for exclusion, sanitation, and environment management were also followed.
At harvest, tomatoes were weighed and graded. Grade 1 tomatoes (Jumbo) were at least 8.2 cm in diameter. Grade 2 tomatoes (Extra Large) ranged from 6.5 to 8.2 cm. Grade 3 (Large) tomatoes ranged from 5.8-6.5 cm; Grade 4 (Medium) ranged from 5.4-5.8 cm. Tomatoes were designated as Grade 5 (Small) if they ranged from 4.6 to 5.4 cm. Tomatoes that were smaller than Grade 5, damaged or off-color were classified as culls. Data for Grades 1 and 2 were combined and the new data set was termed Fresh Market Tomatoes. Data for Grades 3-5 were combined and the new data set was called Processing Tomatoes because they could be sold to processing operations and home canners. Tomatoes were harvested April 27, 2004 through June 15, 2004 and data were combined into 8 harvest dates – Harvest 1 (April 23 and 30); Harvest 2 (May 5); Harvest 3 (May 11); Harvest 4 (May 18); Harvest 5 (May 18 and 25); Harvest 6 (May 28 and June 2); Harvest 7 (June 4 and 11) and Harvest 8 (June 15).
Protocols for exclusion, sanitation and environment management were sufficient to eliminate the need for commercial pesticide applications. There were no incidences of insect damage. There were occasional infections by the gray mold fungus (Botrytis cinerea), but these were contained by sanitation. Consistent with previous experiments in the greenhouse, there was a row effect. Yield in the outer rows was significantly higher than in the internal rows so data from these rows were eliminated from the data set.
In order to have as many treatments as possible, the experiments were not designed in a complete factorial. The large data set was divided into subsets for analysis. For fresh market tomatoes, tomato yield was significantly impacted by interaction of herbage and herbage rate (P= 0.0526). PP had the greatest yield (5.37 lb/plant) and it was significantly different from all treatments except EL and CE-L. The MD treatment was significantly lower than PP, EL, and CE-L. There was no impact on yield of Total Marketable Tomatoes. There was a significant interaction between Pythium and herbage at the high rate (P= 0.0038) for yield of fresh market tomatoes. Tomato weight was greater for PP+P (3.98 lb/plant) than all other treatments except CP-P (3.98 lb/plant). Conversely, yield of plants with PP-P (2.43 lb/plant) was significantly lower than PP+P, CP+ P, EL-P (4.07 lb/plant), and NH – P (3.89 lb/plant). MD-P had lower yields (2.62 lb/plant) than PP+ P, CP-P, and El-P. There were no significant differences in Processing Tomato or Total Marketable Tomato yields due to Pythium and herbage. In future experiments, we plan to focus on four herbage treatments NH, CP, EL, and PP.
Treatment with Roman chamomile resulted in reduced weight of Fresh Market (P=0.0355); NH –P (4.27 lb./plant) was less than RC-P (2.74 lb/plant), but neither was different from treatments to which Pythium was added – RC+P (3.4 lb/plant) and NH +P (2.94 lb/plant). Treatment with RC did not affect weight of Processing Tomatoes or Total Marketable Tomatoes. Because of the high reduction of yield in the presence of RC, we will not include this treatment in future experiments.
There was no effect of the interaction among Beauveria, Pythium and PGPR on Fresh Market Tomatoes or Total Marketable Tomatoes when all cultivars were considered but there was a significant decrease in the weight of Processing Tomatoes in treatments with Beauveria (P= 0.0030) in some cultivars (MD, EL, SI). In this data set, yields of control (7.2 lb/plant) were greater than both +BTG (5.8 lb/plant) and +UTN (5.2 lb/plant). Weights of Fresh Market and Total Marketable Tomato were not significantly different. Lack of disease pressure, however, may bias these results so we plan additional experiments with BTG and UTN. In experiments where there were differences, BYD typically outperformed SBL so we plan to use only BYD in future tests.
Within all data sets, effects of Pythium treatments were rarely significant, but when they were, plants treated with Pythium produced more fruit. Although not intuitively obvious this result is not without precedent in the literature. Greenhouse tomato roots generally fill the bag and they do not have a high concentration of root hairs. Pythium in small quantities may serve as a ‘pruning’ agent for these plants allowing the increased production of root hairs and an associated increased uptake.
Educational & Outreach Activities
Greene, S.E. 2005. Bioactive Natural Products from Monarda for Control of Tomato Disease. M.S. Thesis. University of Tennessee. Knoxville, TN.
Gwinn, K.D., Ownley, B.H., Wills, J.B., Davis, J.M., Collins-Shepard, M.H., and Baird, J. 2004. Greenhouse Production of Organic Crops: Nitrogen Source and Biorational Pest Management Systems. Proposal to Southern SARE.
We have identified biorational combinations that not only have the potential to control Pythium disease in hydroponic tomatoes but also to increase yield. Based on $1.00/lb price for fresh market tomatoes (Grades 1 and 2), several alternative biorationals will increase grower profit an estimated $1,642 /greenhouse. Weight of processing tomatoes (Grades 3-5) was not significantly different for any treatment; the difference between the highest treatment and the control was less than $100.
Based on the number of commercial greenhouses in Tennessee, these products have the potential to increase farm receipts over $1 million/crop or $2million/year in this state. This number does no include the value added by eco-labeling the produce as pesticide-free. This could increase revenues by an additional 10—20%.
A proposal based on the data collected in this research was submitted to Southern SARE in response to the Call for Proposals for Research and Education grants.
Areas needing additional study
This is the final report of a planning grant. More research is needed to ensure that we can fully develop this technology and provide the benefits to tomato producers in the Southeast. The research data that were accumulated as a result of the successful funding of this grant were used to develop proposals that, when funded, will allow us to further investigate the use of these biorational alternatives in tomato crops.
We also plan to add fertigation systems that can be certified as organic in order to fully utilize this technology and provide the additional value-added premiums that are possible through organic production. The alternative biorational treatments used in this study are consistent with organic standards. Although large-scale corporate hydroponic farms generate vegetable and flower crops for export and domestic use, there is controversy within the organic farming community over whether hydroponics are compatible with organic production. Furthermore, current National Organic Standards do not address hydroponic systems, specifically. Hydroponic operations are certified by many, but not all, certifying agents. If these crops are grown organically, they can bring a price with a 10 to 30% premium in the marketplace. However, at this time, we lack the basic scientific evidence to assist in certification of most greenhouse crops. Greenhouse growers also have few options that are consistent with the aim of on-farm sustainability. For growers who want to limit or eliminate pesticide use, with the long-term aim of higher value organic greenhouse production, greenhouse crop management options are inadequate. Scientific data that would provide them with management tools to overcome the two major hurdles to organic transition, i.e., organic fertilization regimes and effective organic control strategies for pests and pathogens, are lacking. Since pest management systems that optimize effective disease and pest control are essential to maintaining productive, profitable operations, the lack of information prevents the transitioning of greenhouse growers to organic production.
In preliminary experiments, we found no significant losses due to Pythium myriotylum; therefore, we need to determine best methods for inducing disease losses due to this pathogen so that we may effectively test control methods. Even though we had no losses due to disease, this pathogen periodically causes devastating losses in commercial hypdroponic greenhouse production systems.
Based on the research in tomatoes, we believe that we can extend the use of the bioactive alternatives described in this report to other crops. We have partnered with Dr. Jeanine Davis at the North Carolina State University Mountain Crops Research Station to bring this technology to hydroponic herb production.