Final Report for LS06-187
Project Information
The purpose of this project was to address plant diseases in organic farming systems by targeting soil health as the fundamental principle towards achieving a healthy cucumber crop. The effect of Si [Ca2SiO4; wollastonite (Vansil W-50)] incorporation into soil at 200-600 kg Si/ ha on cucumber ‘Straight Eight’ diseases, fruit yield and quality, and Si content in root and above ground plant tissues was evaluated in multiple seasons at two organic field locations in north central Florida. Silicon application did not limit the incidence and severity of downy mildew, caused by the fungus-like pathogen Pseudoperonospora cubensis, melon worm and leaf miner, caused by Diaphania hyalinata and Lyriomyza sativae, respectively, or root knot nematode, caused by Meloidogyne spp. Yield and fruit quality were also not affected by Si soil amendment. Plant tissues tested from the field indicated no elevation in Si content in cucumber plants or cover crops (sorghum x sudangrass in spring and rye in winter) across all Si treatments and this may account for the lack of disease reduction observed. Other elements and micro-elements were also not impacted by Si amendments.
The effect of amending soil with a high rate of Si (Vansil W-50, equivalent to 600 kg Si/ha) on anthracnose (Colletotrichum orbiculare) and Si uptake was also evaluated in multiple greenhouse experiments with 2-week-old ‘Straight Eight’ cucumber plants. Soil amendment with Si significantly reduced anthracnose severity in the leaves and increased Si uptake compared with the non-amended control. The results of this research indicate that Si may play a significant role in disease control of cucumber in a greenhouse setting where high pressure from multiple pathogens may be absent and arthropod pests and environmental factors can be better controlled.
We established protocols for testing the role of plant-generated phytoalexins (low molecular weight antifungal compounds produced by plants) as a mechanism for disease resistance in cucumber. Implementation of the protocol was carried out on greenhouse-generated and plants inoculated with C. orbiculare. HPLC analyses of methanolic extracts of leaves showed that specific UV-positive peaks defined by their elution pattern induced following pathogen infection were also induced by silicon and plants exposed to a combination of silicon and the pathogen had the greatest levels of them.
1.Survey natural organic farm ecosystems for diseases infecting cucurbits amended without and with Si.
2.Determine the fate of Si as a soil amendment in the agricultural system without and with cover crops.
3.Quantitatively assess the role of Si in suppressing diseases of cucumber in greenhouse evaluations and determine whether any flavonoid phytoalexin is involved in Si-induced resistance.
4.Determine the potential economic impact of Si soil amendment on cucumber diseases through partial budgeting.
The element Si has been demonstrated to reduce many soil-borne and foliar plant diseases in a number of crops, including rice, sugarcane and certain vegetables and ornamentals. Research has also shown that the host resistance of susceptible and partially resistant cultivars can be increased by Si to the same general level as those containing complete genetic disease resistance. Structural changes in the plant’s cell wall following silicon uptake has been implicated in enhanced disease resistance through creation of a structural barrier. Si may also induce resistance by eliciting plant production of flavonoid phytoalexins (low molecular weight antifungal compounds). Previous research has ascribed such a role for silicon action in the cucumber-powdery mildew fungus interaction (Fawe et al.1998. Phytopathology 396-401).
Cooperators
Research
A. FIELD EXPERIMENTS
Soil treatments. During 2007-2009, the effect of Si on naturally-occurring cucumber diseases was tested at one commercial organic farm in Island Grove, FL and at the University of Florida’s organic research plots in Citra, FL. These sites were chosen because they were found to be very low (< 4 ppm) in Si. The soil was well drained, coarse-textured Arredondo sand. Previous research has demonstrated that soils less than 19 ppm are considered to be extremely low in this element. The cucumber cultivar Straight Eight was selected for all experiments because of its high susceptibility to a number of diseases. The experimental design consisted of three Si rates x 2 years with a two level cover crop factor as a split plot treatment. This cover crop factor occurred during the non-cash crop period when the plots were split so that one half was seeded with either ryegrass ‘Marshall’ (winter non-cash crop period) or sorghum x sudangrass (summer non-cash crop period) and the other half was left fallow. All treatments were arranged in a randomized complete block design with four replications. Si amendments were applied at three rates: 0, 200, 400 and 600 kilograms of elemental Si/ha. Wollastonite [CaSiO3; Vansil W-50 (R. T. Vanderbilt Co., Inc. Norwalk, CT)], a naturally-occurring mined mineral ore, was the silicon source used. Cucumbers were grown following the National Organic Program Standards (NOPS).
Si accumulation in cucumber was quantified at four stages of crop growth (transplant, 1st flower, 1st harvest, and last harvest). Data collection took place to determine if cover cropping with rye and sorghum x sudangrass could help maintain the levels of Si available to the subsequent cucumber crop by sequestering it in the cover crop biomass and releasing it back into the soil when the cover crop is incorporated into the soil.
Plant pest and damage surveys. Plants showing signs of damage, disease, or irregular growth were inspected in the field and if necessary samples were brought to the lab for further processing. Plant samples were viewed in the lab under stereo and compound microscopes. Diseased plant tissue was plated on a range of appropriate semi-selective media and/or placed into moist chambers to promote the development of pathogen structures that could aid in identification. Diagnostic personnel were consulted when the pests or cause of damage or disease was not readily apparent. Disease ratings were made for leaf pathogens at both sites using the 0-12 Horsfall-Barratt Scale. Disease ratings began shortly after symptoms and signs were evident in the field. The ratings were made weekly until it was time to plow under the plants (shortly after the final harvest). The primary foliar disease detected at both field locations was downy mildew (Pseudoperonospora cubensis). Root knot nematode (Meloidogyne spp.) was the predominant soil-borne pathogen in cucumber plants at all sites during this study. At the end of several seasons, the root systems of the cucumber plants were visually evaluated for gall severity. The root knot rating system used was: 1 = 0% of the root galled, 2 = 1- 25% of the root galled, 3 = 25-75% of the root galled, 4 = >75% of the root galled.
B. GREENHOUSE EXPERIMENTS
Soil treatments and disease estimation. A total of six greenhouse experiments were conducted. Two-week-old ‘Straight Eight’ cucumber seedlings were treated by amending soil (organic Fafard FOF 30, Conrad Fafard, Inc, Agawan, MA) with a high rate of Si (Vansil W50). Treatments included cucumber seeds planted into 1) Si-amended soil (equivalent to 600 kg Si/ha) + no C. orbiculare inoculation 2) Si-amended soil (equivalent to 600 kg Si/ha) with C.orbiculare inoculation, 3) non-amended soil with no C. orbiculare inoculation, and 4) non-amended soil with C. orbiculare inoculation. Each treatment included 5 replications and the experiment was repeated 4 times. Disease evaluations (Horsfall-Barrett Scale) were recorded for leaves at 3, 5-7, and 9-14 days post inoculation and Si levels in plant tissues were determined in two experiments.
Si uptake and phytoalexin analysis. Two destructive harvests were conducted: one at 7 and one at 9 days post-inoculation. Six plants were harvested at sampling time; three were destined for Si content analysis, and three for phytoalexin analysis. The harvested portions of the plants consisted of the two first true leaves sans petioles. The leaves from each plant were placed in individual labeled paper bags, in which they remained in their respective storage environments until further processing. Immediately after harvest, samples for Si tissue analysis were placed in a Fisher Scientific Isotemp Oven at 80oC until completely dry, which took a minimum of 24 hours, then stored at ambient room temperature and humidity. Samples for phytoalexin analysis using mass spectroscopy and a high performance liquid chromatography (MS-HPLC) were stored in a -80?C freezer immediately after harvest and between steps of processing. Samples for HPLC-MS analysis were ground in a blender (Hamilton Beach 51101B) with 75 ml of 80:20 methanol:water and filtered through Whatman #4 filter paper to remove large particles. 20 ml of this extract was evaporated until dry (approximately 36 h) on a forced-air evaporator (Organomation Associates Inc. N-Evap 111, Berlin MA) with the water bath for the tubes maintained at 25?C. Tubes containing extract residues were stored at -80?C until redissolution. Extract residues were redissolved with a 2:1 v/w ratio of distilled water:fresh tissue weight. Dried leaf tissues were ground individually in a Wiley mill, which was cleaned by air blast between samples for inductively coupled plasma (ICP) Si analysis sample preparation.
HPLC-Mass spectral analyses. To test whether phytoalexin compounds play a role in plant defense against the pathogen C. orbiculare following Si amendment, we examined greenhouse-grown cucumber plants that were treated four different ways: No Si amendment, no pathogen (control); Si amendment, no pathogen; no Si amendment, plus pathogen; and Si amendment, plus pathogen. Methanolic extracts of known amounts of leaves were analyzed using high performance liquid chromatography coupled to mass spectrometry. A Phenomenex (Torrace, CA) Synergi 4 µm Hydro-RP 80A (2X150 mm) column with a C18 guard column (2x4 mm) in an Agilent (Palo Alto, CA) 1100 series binary pump was used. Following the injection of the leaf extracts the column was eluted using the mobile phases 0.2% (v/v) acetic acid in water (A) and 0.2% acetic acid in methanol (B) over 105 minutes. The detector (Agilent 1100 G1314A UV/Vis) was set at 280 nm and the peak areas were analyzed quantitatively. This analysis separated 55 to 65 different peaks in these samples.
A. FIELD EXPERIMENTS
The plant diseases, arthropod pests and damage in cucumber plants observed at two organic production sites in north central Florida are listed in Table 1. Based on the extremely high density of root knot nematodes (Meloidogyne spp.) at the Island Grove location, research at this site was discontinued after the initial season in Spring 2008. Si application did not limit the incidence and severity of downy mildew, melon worm and leaf miner or root knot nematode and did not increase plant tolerance to abiotic stresses. Yield and fruit quality were also not affected by Si soil amendment. Elemental tissue analyses indicated that Si soil amendments did not significantly elevate Si in cucumber or cover crop tissues at Citra. The non-Si treated tissues had equivalent Si levels as the treatments with 200, 400, and 600 kg Si/kg amendment (Figure 1), and this might help to explain why no differences were detected between treatments. Two graphs are included to illustrate the similarity in data for all treatments including 0 and 600 kg Si/ha; tissue analyses at 200, 400 and 600 kg Si/hectare were very similar. For all Si treatments, there appeared to be a dip in tissue Si presence at the final cucumber fruit harvest. Results from other element analyses indicated no significant differences or trends for sulfur, magnesium, nitrogen, phosphorus, potassium, or calcium. Tissue testing also provided data on additional elements of boron, zinc, manganese, iron, and copper. For all treatments, trends indicated a similar spike in elemental ppm for cover crops (sorghum and rye) with reduction in each of the minor elements during the cucumber crop. Si amendments at the second research site in Island Grove resulted in similar tissue results with respect to Si and other elements (data/figures not shown).
B. GREENHOUSE EXPERIMENTS
Si was consistently detected at higher levels in leaf tissue of cucumber plants grown in Si-amended soil compared with plants from non-amended soil (data not shown). Significant differences in anthracnose severity were observed at 5-7 days after inoculation in each experiment and at 8-14 days after inoculation in five of six experiments. Si treatment reduced disease severity in leaves by 20-60% when compared to the non-treated control (Figure 2). The area under the disease progress curve (AUDPC) for anthracnose was also reduced by treatment with Si.
Phytoalexin analyses generated peaks that indicated the following general pattern (Figures 3A-C):
1. The first 5 minutes of elution contained salts and sugars and the next 10 minutes had amino acids (especially phenylalanine and tryptophan) (Figure 4A).
2. The largest UV peaks mostly due to flavonoids of molecular weight range 700 to 1000 range were eluted between 30 and 42 minutes (Figure 4B).
3. Flavonoids with one less sugar, and of molecular weight range 700 were eluted between 43 and 55.5 minutes (Figure 4C).
4. Between 66.5 and 80 minutes, UV positive compounds of molecular weight range of 290 to 350 were eluted (Figure 4C).
There were both several qualitative and quantitative differences between the samples of the four treatments. In particular, the pathogen treatment produced five UV positive peaks eluting between 66 and 75 minutes of our elution protocol (Figure 4C). These five peaks were absent in the controls where Si and the pathogen were not added (Figure 4C). Si treatment alone induced these peaks to some degree but the peak areas for these five peaks significantly increased upon both Si and the pathogen together compared to Si alone or pathogen alone (Figure 4C). Qualitative and quantitative differences between these peaks suggest that one or many of these could be phytoalexin compounds. Currently, a quantitative analysis of multiple HPLC traces and identification of specific compounds via the analysis of mass spectral data are in progress. Figure 3A-C are presented below in that order:
- Table 1. Survey of disease, arthropod pest and damage in field-grown cucumber ‘Straight Eight’
- Figure 3. HPLC phytoalexin analysis in cucumbers following Si application
- Figure 1. Elemental analysis of field-grown cucumbers treated with Si
- Figure 2. Effect of Si on anthracnose in greenhouse-grown cucumbers
Educational & Outreach Activities
Participation Summary:
Datnoff, L.E. 2008. Silicon soil amendments for enhancing disease resistance while improving overall crop health for cucurbits in organic farming systems. Florida Organic Growers’ Newsletter, Summer 2008. http://www.foginfo.org/docs/Foghorn_2008.pdf.
Palenchar, J.; Taber, S.; Datnoff. L.E.; and Gevens, A.J. 2009. The impact of silicon soil amendments on cucumber anthracnose in the greenhouse. Joint Meeting of the Florida Phytopathological Society and the Caribbean Division American Phytopathological Society Holiday Inn Hotel & Suites Universal Studios, Orlando, FL. May 17, 60 attendees. (Palenchar: 3rd place graduate student award)
Palenchar, J.; Treadwell, D.; Datnoff, L.E.; and Gevens, A.J. 2009. Cucumber Anthracnose in Florida. Institute of Food and Agricultural Services. University of Florida. EDIS Publication PP266. Online (http://edis.ifas.ufl.edu/pdffiles/PP/PP26600.pdf).
Taber, S., Garrick, T., Rathinasabapathi, B. and McGovern, R. J. 2010. Plant Nutrition and Disease: Highlights and Resources for Growers. University of Florida-IFAS Small Farms Conference. Osceola Heritage Park, Kissimmee, FL, July 31-Aug. 1, 775 attendees. The poster presented information on the impact of silicon on plant diseases including anthracnose in cucumber.
Project Outcomes
Phytoalexin analyses generated peaks that indicated the following general pattern (Figures 3A-C):
1. The first 5 minutes of elution contained salts and sugars and the next 10 minutes had amino acids (especially phenylalanine and tryptophan) (Figure 4A).
2. The largest UV peaks mostly due to flavonoids of molecular weight range 700 to 1000 range were eluted between 30 and 42 minutes (Figure 4B).
3. Flavonoids with one less sugar, and of molecular weight range 700 were eluted between 43 and 55.5 minutes (Figure 4C).
4. Between 66.5 and 80 minutes, UV positive compounds of molecular weight range of 290 to 350 were eluted (Figure 4C).
There were both several qualitative and quantitative differences between the samples of the four treatments. In particular, the pathogen treatment produced five UV positive peaks eluting between 66 and 75 minutes of our elution protocol (Figure 4C). These five peaks were absent in the controls where Si and the pathogen were not added (Figure 4C). Si treatment alone induced these peaks to some degree but the peak areas for these five peaks significantly increased upon both Si and the pathogen together compared to Si alone or pathogen alone (Figure 4C). Qualitative and quantitative differences between these peaks suggest that one or many of these could be phytoalexin compounds. Currently, a quantitative analysis of multiple HPLC traces and identification of specific compounds via the analysis of mass spectral data are in progress. Figure 3A-C are presented below in that order:
Economic Analysis
PARTIAL BUDGET ANALYSIS
Because soil amendment with Si did not affect cucumber disease and other plant Stressors, partial budget analysis for this practice in the field was not possible. However,we did develop a cost comparison between foliar fungicide application and soil-incorporation/application of two different forms of Si, calcium silicate and potassium silicate, for management of anthracnose in a greenhouse setting. Calcium silicate, wollastonite (Vansil W-50) the form of Si used in this research, is a natural product that is potentially approvable for use in organic vegetable production. Potassium silicate is currently permitted for use in organic vegetable production in the U.S.A. Our budget estimates are based on a single application of the materials using the operation costs of a greenhouse facility in north central Florida that grows an average of 26,000 cucumber plants/ha using 92 m3 of artificial media. The data presented in Table 2 indicate that both methods of Si application are potentially less costly than a single foliar application of a copper fungicide for anthracnose management. Brecht et. al, (2004) found that soil incorporation of calcium silicate eliminated the need for multiple fungicide applications in management of gray leaf spot in St. Augustinegrass caused by the fungus Magnaporthe grisea (Plant Dis. 88:338-344).
Farmer Adoption
Additional research and grower education is needed to facilitate use of the disease management strategy developed through this project for routine organic vegetable production.
Areas needing additional study
Additional research is needed to expand evaluation of the efficacy of Si amendments for reduction of foliar and fruit diseases of greenhouse- and organically-grown vegetables. Possible pathosystems for future research include anthracnose caused by Colletotrichum spp. in tomato and pepper and leaf spot/blight and fruit root in vegetables caused by Alternaria, Botrytis, Cercospora spp. and the causal agents of downy and powdery mildew. It would also be useful to assess integration of Si amendments with cultivar resistance/tolerance and/or fungicides permitted for use in organic vegetable production such as sulfur, copper, Neem extracts, etc. Data is also needed on the horticultural and yield impacts of Si application on vegetable crops.