Final Report for GNC06-067
Field and greenhouse trials were conducted to evaluate the efficacy of compost tea on Septoria lycopersici, causal agent of Septoria leaf spot on tomato, in Kansas, in 2006 and 2007. Compost tea with several additives sprayed weekly on tomato plants prior to and after disease onset in the field led to no significant difference in control of the pathogen compared to untreated controls. In the greenhouse, inclusion of specific additives did not reduce disease compared to unamended compost tea. However, inclusion of hydrolyzed fish fertilizer increased disease severity compared to other treatments.
Compost tea has been cited as an option for conventional and organic growers to suppress plant pathogens (Diver, 2002; Ingham, 2005a; Kannangara, 2006; Tsror 1999). Compost tea is an aqueous solution that results from the extraction of microorganisms, fine particulate organic matter, and soluble chemical components of compost, that is intended to maintain or increase the beneficial microorganism population of the source compost (NOSB, 2006). Beneficial microorganisms in compost tea are thought to suppress plant diseases by occupying spatial niches on the phylloplane, competing with pathogens for leaf/seed exudates, or directly antagonizing pathogens (Diver, 2002; Ingham, 2005a). Compost teas are also thought to enhance crop fertility by introducing microorganisms that might aid in soil nutrient retention and extraction, and by adding soluble nutrients, further adding to their potential value as a part of an integrated crop management plan (Diver, 2002; Ingham, 2005a; Kannangara, 2006; Merrill and McKeon, 2001).
There are several methods for producing compost tea, and variables can include aeration (injecting air into the brew tank or recirculating the contents), compost source (ex: cow manure, yard waste, bark, etc), additives (ex: molasses, fish hydrolysate, humic acid, kelp), brewing/production time, and compost-to-water ratio (Ingham, 2005b; Diver, 2002; Al-Dahmani et al, 2003; Elad and Shtienberg, 1994, Welke, 2004; Scheuerell and Mahaffee 2004).
Al-Dahmani, et al. (2003) investigated the effects of compost source (cow manure, pine bark, yard waste, and organic farm compost), compost maturity (5, 10, 16 months), aeration, compost-to-water ratio (1:1,1:3, and 1:5), and filtration on compost efficacy against the bacterial pathogen Xanthomonas vesicatoria on young tomato plants in the greenhouse. In their trials all formulations provided significant disease suppression (Al-Dahmani, et al. 2003). Elad and Shtienberg (1994) investigated the effects of various production intervals (4 hours, 1 week, and 2 weeks), the addition of a proprietary nutrient broth, pasteurization, and dilution on efficacy of a non-aerated compost tea against Botrytis cinerea on tomato leaves, pepper leaves, and grape berries in growth chamber and greenhouse studies. They found that brewing for ten to fourteen days was more effective, but addition of nutrients did not increase the suppressiveness. Pasteurization to eliminate the microflora of the compost tea had no effect on disease suppression, but dilution reduced its effectiveness (Elad and Shtienberg, 1996). Welke (2004) investigated the effects of aeration and water-to-compost ratio on the efficacy of CT against Botrytis cinerea on strawberry fruit. While both aerated and non-aerated compost tea suppressed disease, only aerated compost tea resulted in greater yields. Interestingly, 8:1 concentrations of water-to-compost resulted in significant differences in disease while 4:1 concentrations did not (Welke, 2004).
Scheuerell and Mahaffee (2004) investigated the effects of aeration, additives, and compost source on compost tea efficacy against cucumber seedling damping off caused by Pythium ultimum. Their investigations reported that ACT and NCT significantly reduced the occurance of damping off, but only ACT reduced its occurance consistently. Their results indicated that NCT without additives resulted in no significant reduction of damping-off while NCT produced with either fungal (seaweed powder, humic acids, and rock dust) or bacteria (a proprietary bacterial nutrient solution) promoting additives significantly, but inconsistently, reduced damping-off. Aerated CT produced without additives or with the putative bacteria promoting additive resulted in inconsistent reduction of damping-off, while aerated CT produced with the putative fungal additives consistently gave significant control of the pathogen (Scheuerell and Mahaffee, 2004). Septoria lycopersici Speg., the causal agent of Septoria leaf spot on tomato (Lycopersicon esculentum Mill.), is an important fungal disease of tomato (Parker, et al. 1997). Symptoms are circular lesions up to 1/8 inch in diameter that begin as yellow areas that then turn brown, sometimes with a light or dark border (Sherf and Macnab, 1986/ compendium). After several days, lesions may begin to produce black pycnidia, which distinguish this disease from others (Sherf and Macnab, 1986). Septoria leaf spot occurs in most U.S. states where tomato is grown and can cause severe defoliation and yield loss (Sherf and Macnab, 1986). All released tomato cultivars are susceptible to this disease (Parker, et al. 1995; Sugha and Kumar, 1998). Control of this pathogen has been achieved through cultural controls and fungicide applications (Blum, 2000; Elmer and Fernandino, 1995; Parker, et al. 1995; Tu, et al. 1998). In the Midwest/Great Plains, a number of fungicides are labeled for preventative use for foliar diseases of tomato (Egel, et al. 2007). For organic growers, fungicide options are limited. Copper fungicides (Bordeaux mixes, copper hydroxide, copper oxide, copper oxychloride, and copper sulfate) are currently labeled for control of S. lycopersici in organic production, but their use is controversial because they are toxic to many microorganisms at recommended rates (Diver, et al. 1999). Because these fungicides have provided adequate control, historically there has been little work done to develop resistant cultivars (Tu and Poysa, 1990), though breeding programs have recently begun to target this disease (Tu, et al. 1998). In the meantime, CT may provide a means of controlling Septoria leaf spot in tomato production.
Blum (2000) demonstrated that it is possible to reduce the incidence of this disease through the introduction of bacteria and yeast isolates onto the phylloplane. Kashyap (1978) inhibited leaf necrosis due to Septoria lycopersici through the introduction of antagonists Trichoderma viride strain 3, Acremonium charticola, and Cladosporium sphaerospermum strain 3. Silva, et al (2004) reported that when used alone, Bacillus cereus moderately lowered area under the disease progress curve (AUDPC) values of Septoria leaf spot as well as early and late blight on tomato. Gangaiah (2005) studied the efficacy of CT against Septoria leaf spot and early blight on tomato. He reported greater control and marketable yields with CT and mancozeb than untreated plots, though the treated plots had high (>80%) disease severity.
The objectives of this research were to re-evaluate the efficacy of the general compost tea formulation investigated by Gangaiah (2005) in the field, and to investigate the role of different additives in the greenhouse.
Trials were conducted in the summers of 2006 and 2007 at the Kansas State University Horticulture Research and Extension Center near Olathe, KS, and at Thowe Farms (a commercial farm) in Manhattan, KS, in 2007. The field trials investigated the efficacy of a compost tea with several additives (Gangiah et al).
In both years an open field area at Olathe was tilled to remove weeds. The soil type was Kennebec silt loam, previously cropped to pumpkins in 2005. In 2006, no pre-plant amendments were added prior to bed formation. In 2007, Early Bird Chicken Manure Compost (3-4-2) (CMPP, Inc., High Point, MO, USA) was applied at a rate of 70 pounds of nitrogen per acre and was thoroughly incorporated into the soil prior to bed formation. Twenty-inch-wide raised beds were formed using a Nolt’s Compact plastic mulch layer (Nolt’s Produce Supplies, Leola, PA, USA). In 2006, transplants of tomato cultivar ‘Celebrity’ were purchased from a commercial garden center on June 1 and planted in the field on June 3 and supported using the stake-and-weave method. In 2007, seeds of tomato cultivar ‘Rutgers’ were sown on April 21 in a 200 cell plug tray filled with Metro-Mix 200 (Sun Gro Horticulture, Bellevue, WA, USA) and placed under intermittent mist in the greenhouse. Seedlings were transplanted into 10.16cm (4 inch) round pots on May 3, and watered as needed with 125 ppm 20-10-20 Peters Professional Peat-Lite Special water-soluble fertilizer (Scotts Company, Marysville, OH, USA) until planted in wire cages in the field on May 17.
The experiment was set up as randomized complete block design with four replications. The three treatments were 1) weekly applications of compost tea 2) weekly applications of chlorothalonil Bravo Weatherstik, Syngenta Corp., Greensboro, NC, USA and 3) an untreated control.
Each block consisted of one 33.53 m (110 ft) row and blocks were spaced 12.2 m (40 ft) apart. Five-plant plots of each treatment were spaced 12.2 m (40 ft) apart in each block. Within each plot, plants were spaced 0.6 m (2 ft) apart. Sorghum sudangrass (Sorghum x drummondii) seed was drill-planted between blocks (approximately 1.52 m (5 ft) and 5.49 m (18 ft) from rows in 2006 and 2007, respectively) at a rate of 112 kg/hectare (100 lbs/acre) within two weeks after tomato transplant to minimize plot-to-plot interference.
The Manhattan site was cleared of weeds and a pre-plant amendment of 18-46-0 fertilizer (acquired from the local farmer cooperative) was incorporated into the soil. Raised beds were covered with black plastic mulch and plastic drip tape was laid under the plastic mulch. Three-week-old seedlings of tomato cultivar ‘Mountain Spring’ were planted on May 12 and were supported using the stake and weave system. The experiment was set up as a RCBD with three replications. One row of tomato plants was segmented into three blocks, each with two five-plant plots. The two treatments were 1) weekly applications of compost tea and 2)an untreated control. Six untreated plants separated each five-plant plot, and the ends of the row were bordered with five untreated plants. All plants were spaced at 0.6 m (2 ft). S. lycopersici was not introduced into the field in an effort to avoid unnecessary economic injury to the farmer. However, this pathogen was naturally present due to several consecutive years of tomato grown in the same location.
In 2006 the Olathe plots were subject to natural infection. In 2007, the Olathe plots were inoculated on July 5 using a spore suspension. Inoculum was produced as follows. Lesions of S. lycopersici were excised from infected tomato leaves (Manhattan, KS, community garden), surface sterilized in a 10% bleach solution for 30 seconds and incubated on moist filter paper in glass Petri dishes for 48 hours at room temperature to stimulate production of cirrhi from pycnidia. Conidia were stored on 1/4-strength potato dextrose agar slants at 4.5 degrees Celsius (40º F) (Sundin et al., 1999). Two weeks before field inoculation, slants of S. lycopersici were removed from storage and spores were streaked onto 9-cm plates of modified V8 agar (150 ml V8 (Campbell Soup Company, Camden, NJ, USA), 3.0 g CaCO3, 15 g agar, 850 ml distilled water) and incubated in the dark at room temperature. After 5 days plates were flooded with 25 ml of sterile distilled water and a flamed loop was used to agitate cirrhal masses. The concentration of the spore suspension was determined with a hemacytometer and was adjusted to 1x105 spores ml-1and 1 ml of the suspension was spread on each of two new V8 plates. Inoculated plates were stored unsealed, in a dark drawer at room temperature until pycnidia production (approximately 5 days).
On the day of inoculation plates were flooded and spore concentration adjusted to 1x105 in a final volume of 500 mL. 2.06 g of Knox gelatin (NBTY, Inc., Bohemia, NY) was dissolved under heat in 25 ml of sterile distilled water, cooled, and added to the spore suspension to replace the natural adhesive characteristics of the pycnidia that are lost through the addition of water to the spores. A trigger bottle was used to apply the spore suspension to the bottom 1/3 of plants at a rate of approximately 40 ml per plot.
Compost Tea Production
Forty gallons of CT were brewed weekly using an Alaska Giant tea brewer (Alaska Bounty, Palmer, AK, USA) beginning on June 28 and June 7 in 2006 and 2007, respectively. The components included: 151 L (40 gal) water, 1.8 kg (4 lbs) vermicompost (Rising Mist Organic Farm, Belvue, KS, USA), 95 ml (0.4 C) unsulfured molasses, 95 ml (0.4 C) hydrolyzed fish fertilizer (2-4-1) (Neptune’s Harvest Fertilizer, Gloucester, MA, USA), 0.4 kg (0.8 lbs) alfalfa-based fertilizer (3-1-5) (Bradfield Industries, Springfield, MO, USA), and 16 ml (0.07 C) (Humisolve TM7 (BioAg Corp., Carson City, NV, USA).
Tap water was added to the brew tank approximately 24 hours prior to use to allow for volatilization of chlorine. All other ingredients were added to a 19 L (5 gal) bucket fitted with multiple perforations, a diaphragm for generation of fine bubbles, and a hook on the base for ensuring submersion. Air was pumped vigorously into the diffusion bucket with a Whitewater LT19 linear air pump (Alaska Bounty, Palmer, AK, USA) for the entire 24-hour brew period. Prior to application, CT was filtered through two layers of nylon stocking to remove particles that could obstruct the spraying apparatus.
In Olathe, CT was applied weekly with a 4-gallon, piston pump backpack sprayer (SP Systems, LLC, Santa Monica, CA, USA) at rates ranging from 0.5-1.0 gallons (undiluted) per plot to achieve complete coverage of plants, with rates increasing with increasing plant size. Chlorothalonil was applied with a 4-gallon, piston pump backpack sprayer (Solo Company, Newport News, VA, USA) at a rate of 2 pounds active ingredient per acre. Control plots received no treatment. Initial applications occurred on June 29 and June 8 for a total of 10 and 11 applications in 2006 and 2007, respectively.
Each week after compost tea was prepared and applied at Olathe the tea was transported to Manhattan and applied within six hours of Olathe plots, with a 4-gallon, piston pump backpack sprayer (Solo Company, Newport News, VA, USA). The initial application occurred on June 9 for a total of 9 applications.
Assessments and analysis:
Disease severity was assessed weekly after the onset of symptoms in the field (approximately 9 weeks after transplanting). One leaf approximately 38 cm (15 in) above ground was randomly chosen on each plant and was marked for repeated measurement. In 2006, visual measurements were taken to reflect the percent lesion coverage per leaf. In 2007, visual measurements were taken to reflect the number of lesions per leaf and the percent lesion coverage per leaf. The area under disease progress curve (AUDPC) was determined by the trapezoidal method (Madden, et al., 2007). The general linearized model (GLM) procedure of SAS version 9.1 (SAS Institute Inc., Cary, NC, USA) was used to complete analysis of variance of overall disease severity and weekly and total yield. Individual rating dates were analyzed with the GLM procedure of SAS and mean separation was by Tukey’s studentized range (HSD) tests (alpha=0.05).
In 2007 yield data in Olathe were collected weekly by counting and weighing U.S. #1, U.S. #2, and cull fruits per plant. Yield data could not be taken in Manhattan due to ongoing harvest by the grower.
Greenhouse studies were conducted in 2007 to examine the effects of individual additives. Plants were treated twice at 1-week intervals with compost tea or chlorothalonil, then inoculated 4-5 days later following the methods described below.
Seeds of tomato cultivar ‘Rutgers’ were produced as described above and treatments (described below) were initiated when plants were 2-3 weeks old (4-6 leaves). The experiment was set up as a completely randomized design, with plants 30.5 cm apart.
The treatments included six compost teas, three concentrations of chlorothalonil, and untreated controls.
Due to logistical constraints the six compost tea formulations could not all be tested simultaneously. Thus, in each repetition of the experiment we tested three compost tea formulations, either 1, 2, and 3 (repeated four times) or 4, 5, and 6 (repeated twice). Within each replication, two independent copies of each tea formulation were produced to help assess lot-to-lot variability. The untreated control and the chlorothalonil treatment were included in each repetition. (The different chlorothalonil concentrations were used due to some observed phytotoxicity in preliminary trials.) In each repetition of the experiment, each treatment was applied to four plants.
Tea production and application:
Compost tea for greenhouse experiments was produced by filling six 1 L, wide-mouth glass bottles with 900 ml distilled water, adding the additives outlined above, and aerating continuously through a dual output Whisper 2000 air pump (Carolina Biological Supply Company, Burlington, NC, USA) with six-way gang valves. The compost teas were brewed for 24 hours, filtered through two layers of cheesecloth, and sprayed to run-off in the greenhouse onto the abaxial and adaxial sides of all leaves on each plant.
Inoculation: Plants were inoculated four or five days after the second and final treatment application. Inoculum was produced as described for the field inoculations. All plants were inoculated on the abaxial and adaxial sides of all leaves with a trigger-bottle sprayer, allowed to air dry, placed in a mist chamber in the greenhouse in a randomized order, then sprayed with tap water to create free water on the leaf surface. The chamber provided 60 seconds of mist every ten minutes. Plants were maintained in the chamber for 48 hours and then returned to the greenhouse bench.
Assessment and analysis: After 10-15 days, lesion development was evaluated by counting the total number of lesions per plant. Since the effects of time were not significant, the repetitions were combined for analysis. The GLM procedure of SAS version 9.1 was used to evaluate differences in number of lesions per plant, and means were separated using Tukey’s studentized range (HSD) test (alpha=0.05).
Field Trial Results:
In Olathe in 2006, only one week showed a significant difference among treatments. During the tenth week after planting, the fungicide treated plots had significantly lower disease severity than the control plots and compost tea treated plots. There were no significant differences between the compost tea treated plots and the control plots at any point in 2006. By the end of the season all treatments had greater than 80% disease severity. The fungicide treated plots tended to be less affected by the disease but this difference was not always significant. There were no significant differences in AUDPC values among treatments.
In Olathe in 2007 disease development was not significantly different among treatments early in the epidemic but at 12 and 13 weeks after planting, fungicide treated plots exhibited significantly lower disease severity. There were no significant differences between the compost tea treated plots and the control plots at any point in 2007 (Fig 2). By the end of the experiment, infection of selected leaves in both the CT treated plots and the control plots was close to 100%. The fungicide treated plots resulted in a significantly lower AUDPC value than the compost tea and the control plots. Data collected as number of lesions per leaf gave comparable results, but are not presented herein.
Few significant differences were found for yield data. Total fruit counts, regardless of grade, were significantly higher on untreated control plants but total fruit weights were not significantly different, indicating a smaller average fruit size on control plants. Among grades (U.S. No.1, U.S. No.2, and cull), no significant differences were found for number of fruit or weight of fruit among the treatments. The limited data collected for yield analysis in 2007 is not indicative of expected, whole-season yields because of the short period of time that fruits were harvested.
In Manhattan there were no significant differences between compost tea treated plots and control plots at any point throughout the investigation. There were no significant differences in the AUDPC.
Greenhouse Results: Most compost tea treatments led to disease severity levels that were not significantly different from chlorothalonil-treated or control plants. However, the fish fertilizer compost tea (CT-E) increased disease severity compared to the controls, with more than double the number of lesions observed in most other treatments. The plants treated with the complete compost tea (CT-F, all additives) had disease levels that were not significantly different from the fish fertilizer tea or the controls. Chlorothalonil-treated plants exhibited the numerically lowest levels of disease severity, but levels were not significantly lower than the control plants.
The compost tea treatments did not increase or decrease disease levels in any of the field studies. These results were in contrast to those of Gangaiah (2005), who used a similar compost tea and observed consistent inhibition of S. lycopersici on tomato plants, with control comparable to that obtained by the application of mancozeb. Dillard, et al. (1997) found that chlorothalonil controlled Septoria leaf spot better than mancozeb.
Though the recipe used in our trials was quite similar to those investigated by Gangaiah (2005), some differences in formulations may have contributed to the differences in results. For instance, we obtained vermicompost from a different source, and used an alfalfa-based fertilizer as opposed to alfalfa pellets. Because microbial populations are considered to be the most significant factor in CT suppressiveness (Scheuerell and Mahaffee, 2002), constituents of a CT recipe that alter the availability of microbial populations (e.g. compost) no doubt have an impact on efficacy. Our investigation into the field performance of CT against S. lycopersici on tomato further adds to the body of evidence reporting variability in disease suppression by CT that has become a hallmark of research in this area (Scheuerell and Mahaffee, 2002).
The level of dissolved oxygen during production might affect efficacy. Ingham (2005b) stated that CT production systems that allow DO levels to drop below 6 ppm can result in the loss of filamentous fungi, protozoa and beneficial nematodes causing the CT to become less suppressive to plant pathogens. We measured dissolved oxygen content every 10 minutes for 24 hours on one day using a dissolved oxygen probe (Sensorex Corporation, GardenGrove, CA, USA), and the concentration fell below 6 ppm for several hours. But, the role of dissolved oxygen is not clear. Scheuerell and Mahaffee (2002) stated that it is not clear that there should be a minimum oxygen level requirement for compost tea because non-aerated compost teas can be suppressive.
In the greenhouse studies we found that none of our additives led to reduced disease development compared to unamended compost tea or controls. The only effect of an additive was that the addition of hydrolyzed fish fertilizer increased disease. The fish fertilizer was included in the compost tea used in the field.
We expected that CTF-treated plants would develop S. lycopersici lesions in a manner similar to that of control plants, because that is what was seen in field experiments. However, we know that in vitro studies are often poor predictors of field performance of biological control agents. In order to assess the utility of a screening method, there should be data that correlates screening results to disease suppression in the field (Scheuerell and Mahaffee, 2002). The results obtained in this screening study do not necessarily correlate to field performance, but perhaps through manipulation of some of the methods of this protocol, better correlation could be observed. For instance, the relatively long period of time over which this study was conducted could have had an impact on the variability that was observed. Had these experiments been conducted in growth chambers, under more controlled conditions, seasonal changes would not have affected the rate of growth and development of tomato plants and S. lycopersici as it did in the greenhouse.
This illustrates a need to identify microorganisms that compete, antagonize, induce resistance, or inhibit specific pathogens. Identification of effective biological control agents and research leading to CT production that allows for higher concentrations of these agents may lead to greater CT efficacy. Because investigations into the efficacy of CT in suppressing plant disease through field studies can deplete resources and may not provide usable results, steps should be taken to identify these biological control agents through a more feasible means.
Results of this study are similar to those found by Olanya and Larkin (2006), who found that CT generally had no effect on inhibiting foliar disease severity caused by Phytophthora infestans on potato. They offered several potential explanations for the inefficacy of their CT, including a lack of microbial persistence/retention on the leaf surface and low establishment of potential biological control agents (Olanya and Larkin, 2006). These reasons for the inefficacy of CT are in line with the results of work done by Sturz, et al. (2006), who found that establishment of microbial communities in CT might not provide characteristics necessary for microbial occupancy on the phylloplane of potato. To further identify potential sources of their CT inefficacy, Olanya and Larkin (2006) stated that the microbial constituents of their CT might not have been effective suppressors of P. infestans. Elad and Shtienberg (1994) found that individual strains of bacteria found in CT, when applied independently, controlled Botrytis cinerea on tomato as well as CT did, indicating that the suppressive activity of CT may be due to relatively few organisms present in the CT.
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Educational & Outreach Activities
A manuscript of this research is being prepared for submission to Plant Health Progress, a peer-reviewed journal of applied plant health.
At the conclusion of this experiment, the researchers had the opportunity to evaluate potentially better ways of investigating various compost tea recipes. We were uncertain as to whether or not we would be able to infect very young tomato plants with S. lycopersici, but because we could, we were able to develop a time-saving protocol that could prove to be quite valuable in the identification of effective compost tea recipes/ingredients.
As mentioned in the discussion, field trials were labor intensive and costly. In our trials, the compost tea that was studied in the field over two seasons seemed to have no effect on disease suppression. The investment put forth to evaluate this recipe in the field could have been better placed in a controlled, rapid screening of candidate recipes in the greenhouse or growth chamber. Future research on the development of effective compost teas should focus heavily on the identification of ingredients that promote the growth of the most suppressive beneficial microorganisms through the developed screening assay.
Because a compost tea was not identified as effective in controlling the target pathogen, no economic analysis can be done.
Areas needing additional study
The value of the current research will only be realized with the continuation of similar projects. For researchers looking for valuable topics of investigation, we suggest looking into the potential of compost tea as a plant health management tool.
There is still much more research that needs to be done to identify appropriate antagonists for most plant pathogens. As more research is done to address the most devastating diseases that growers face, especially when focused on sustainable control techniques, we can increase the profitability of these growers.