New Strategies for Management of Vegetable Diseases in Organic and Traditional Farms

Materials and Methods

Characteristics of P. capsici isolates in Illinois. This study was conducted to investigate morphological, pathogenic, and genetic variations among P. capsici isolates from processing pumpkin fields in Illinois. Fifty-seven single-zoospore isolates of P. capsici, collected from 22 processing pumpkin fields at six towns in central Illinois during 2000-2001, were used in this study. All 57 isolated were used to study colony characteristics, mating types, sporangial morphology, and chlamydospore formation. Twenty-four isolates were tested for their pathogenicity on pumpkin seedlings in a greenhouse by inoculating plants with P. capsici zoospores. Random amplified polymorphic DNA (RAPD) markers were employed to assess genetic variation among twenty-four isolates of P. capsici from 10 individual fields at six locations.

Red-light treatment
Greenhouse experiments. Sporangial suspensions in sterilized-distilled water (SDW) were prepared from 5- to 6-day-old culture plates of P. capsici grown on LBA at 75 F under continuous white fluorescent light. The suspension was then incubated at 68 F for 1 h to allow the sporangia to release their zoospores. Zoospores were separated from the empty sporangia by passing the suspension through a 4-layered facial tissue (Kleenex). The concentration of zoospores was adjusted to 2×105 zoospores/ml water using a haemocytometer.

Inoculum for artificial soil infestation was prepared by growing P. capsici on oat meal-V8-juice broth (V8JB) substrate in 1-L conical flasks. The substrate, consisting of 200 g oatmeal and 120 ml V8-juice broth per liter, was autoclaved for 30 min and inoculated with mycelial plugs, 7 mm in diameter, taken from the margin of a young colony of P. capsici grown on LBA. The flasks were then incubated at 75 F in darkness. After 6 weeks, the colonized oatmeal was added to steamed soil mix (soil : sand : peat; 1:1:1) and mixed thoroughly. The inoculum potential of the soil mix was 600 colony forming units (cfu) per gram of soil, which was determined by the dilution-plate-count method.

Seeds of bell pepper (Capsicum annum; Hybrid SPP 6112, Sakata Co., Morgan Hill, CA), processing pumpkin (Cucurbita moschata; cultivar Dickinson, Libby’s Pumpkin Processing Co., Morton, IL), and tomato (Lycopersicon esculentum; Hybrid XTM 6217, Sakata Co., Morgan Hill, CA) were sown in 10-cm-diameter plastic pots (one seed per pot) containing steamed soil mix and were grown on a greenhouse bench under continuous red light irradiation (600-700 nm) from fluorescent tubes (FL20S•R-F, National Co., Japan). The fluorescent tubes provided red light with an intensity of 287 µW/cm2, which was a supplement to natural daylight. Control seedlings were kept under continuous white light (WL; FL20SS•D/18, Mitsubishi, Tokyo, Japan; 380-740 nm; 473 µW/cm2) or under natural daylight (NDL) in the same greenhouse. Three- or four-week-old seedlings were transferred from red light to NDL and inoculation was accomplished by adding the suspension of motile zoospores over the surface of the soil in each pot (5 ml/seedling/pot). Control seedlings either grown in WL or NDL were also inoculated using the same procedure. An additional set of control seedlings were inoculated with SDW only. Seedlings were watered before inoculation to keep the soil wet. After inoculation, the pots were placed in plastic trays containing water that kept the soil moist for at least 12 hours. The seedlings were then placed on a greenhouse bench, on a randomized complete design, and watered twice daily. Pumpkin and tomato seedlings were also inoculated by transplanting the seedlings into artificially infested soils in pots. Each pot contained one seedling and 14 pots were used for each treatment. Beginning the 4th day after inoculation, seedlings were evaluated for damping-off symptoms every day for 15 successive days. The seedlings were also evaluated on the 20th day after inoculation. Height, and fresh and dry weight of seedlings were also measured. Dry weight was determined after drying the seedlings at 158 degrees F (70 degrees C) for 24 hrs. Data were analyzed using analysis of variance procedures (SAS Institute, Cary, NC).

Optimizing red-light treatment. Experiments were conducted in the greenhouse to optimize the effectiveness of red-light treatment for control of Phytophthora blight of vegetables. The optimization involved determining irradiation duration (days), time of irradiation (number of hours per day and night time vs. day time irradiation), and intensity of irradiation. Pepper, pumpkin, and tomato plants were included in the experiments. Inoculation of plants and evaluation of plants for induced resistance are as described above.

Red-light trials in pepper field. During 2002-2004, field trials were conducted in a naturally infested field with P. capsici near Shawneetown, IL. Bell pepper cultivars California Wonder, King Arthur, Brigardier, Emerald Isle, and Paladin included in this study. ‘King Arthur,’ ‘California Wonder,’ and ‘Brigardier’ are susceptible cultivars and Emerald Isle and Paladin have been identified as resistant to Illinois isolates of P. capsici. Seedlings were either grown under continues red light for four weeks or under natural day light (control plants) in a greenhouse. Eight-week-old seedlings were transplanted in May into raised beds with drip irrigation and black plastic mulch. Disease incidence was determined as percent wilted or dead plants on a weekly schedule one week after transplanting until middle of August.

Red-light trials in pumpkin and watermelon fields. During 2002-2004, the trials were conducted in irrigated fields near Pekin, IL. Processing pumpkin cultivar Dickinson and watermelon cultivar Royal Sweet were included in this study. Seedlings were either grown under continues red light for four weeks or under natural day light (control plants) in a greenhouse. The seedlings were transplanted into the filed plots in June. Disease incidence and severity on leaves and vines were evaluated at three locations (11 sq. ft each) in each plot. Ten fruits were examined in each plot for Phytophthora infection. Disease incidence was determined as percent leaves, vines, or fruits affected. Disease severity was determined as percent area of leaf or vine affected. Numbers of uninfected and infected fruit with P. capsici were recorded at harvest in September. Weight of uninfected fruit was measured. The data were analyzed using GLM procedures of SAS.

Seed treatment.

Laboratory studies. Five isolates of P. capsici, collected from commercial processing pumpkin fields in Illinois, were used in this research. Fungicides mefenoxam (Apron XL LS, Syngenta Crop Protection, Inc., Greensboro, NC) and metalaxyl (Allegiance LS, Gustafson LLC, Plano, TX) were evaluated for their effectiveness in inhibiting mycelial growth, sporangium and zoospore germination. All of the laboratory experiments were repeated twice.

P. capsici was grown on LBA for 4 days at 75 degrees F in darkness. Mycelial plugs, 7 mm in diameter, were removed from actively growing margins of the culture and transferred onto the center of each LBA plate previously amended with mefenoxam or metalaxyl at 0, 1, 5, 10, 20, 50, 100, 150, and 200 μg a.i./ml. Plates were incubated in the dark at 75 degrees C for 6 days to evaluate mycelium growth. Colony diameter of P. capsici was measured in two directions for each individual plate and averaged.

To evaluate the effects of these fungicides on sporangium germination, the isolates were grown on LBA under continuous fluorescent light (F20T12/CW, Phillips Lighting Co., Somerset, NJ) at 75 degrees C for 7 days. Then, sporangial suspension was prepared in SDW. Aliquots of the sporangial suspension (400 μl) were immediately pipetted onto Petri plates containing LBA amended with 0, 10, 20, 50, 100, 150, or 200 μg a.i./ml of the fungicide. The plates were then incubated in the dark at 75 degrees F for 12 h. The percentage of germinated sporangia was determined by examining 100 sporangia per plate using light microscopy.

A sporangial suspension in SDW was prepared and incubated at 68 degrees F for 1 h to allow the sporangia to release their zoospores. Zoospores were separated from the empty sporangia by passing the liquid through a 4-layer facial tissue. Zoospores in SDW were induced to encyst by vortexing for 5 min. Concentration of zoospores was adjusted to 105 zoospores/ml. Aliquots (400 μl) were pipetted onto LBA plates amended with fungicides as described above. Inoculated plates were incubated in the dark at 75 degrees F for 12 h. The percentage of zoospore germination was assessed by examining 100 zoospores per plate using light microscopy.

Greenhouse studies. Effects of mefenoxam and metalaxyl on seedling damping-off of pumpkin, caused by P. capsici, were studied in a greenhouse using a naturally infested soil and artificially infested soil mix (field soil:sand; 3:1). Naturally infested soil was collected from a processing pumpkin field near Pekin, IL. The inoculum density of P. capsici in naturally infested field soil and artificially infested soil mix was determined by the soil dilution-plate method using a Phytophthora selective medium (PARPH). The inoculum density in the naturally infested field soil was 90 cfu/g soil, and the inoculum densities in artificially infested soils were 600, 1400, and 4000 colony forming unit (cfu)/g soil.

Seeds of three processing pumpkin cultivars (Dickinson, Hybrid-401, Hybrid-698) were treated with mefenoxam (0.64 fl oz Apron XL LS/100 lb seed) and metalaxyl (1.5 fl oz Allegiance LS/100 lb seed). A volume of tap water equivalent to 20% of seed weight was poured into a plastic bag and the fungicide was added to the water and mixed thoroughly. Seeds were placed in the bag and shaken for 2 min to coat the seeds with fungicide. Treated seeds were then air-dried.

Plastic pots (30-cm long × 20-cm wide × 15-cm deep) were filled with P. capsici-infested soil. Pots with non-infested soil were included as a control. Eighteen seeds were sown in each pot. The pots were arranged in a randomized complete block design, with three replications. All experiments were repeated twice. The experiments were conducted in a greenhouse maintained at 64-72 degrees F and pots were watered daily beginning the first day of seeding. Seedling emergence was assessed 10 days after sowing seeds and seedling stand was determined three weeks after seedling emergence (31 days after seeding). Diseased seedlings were examined using light microscopy and infected tissues were plated on PARPH for isolating P. capsici.

Field studies. Field experiments were conducted in irrigated pumpkin fields, naturally infested with P. capsici, near Pekin, IL, during 2001-2003. The experiments were performed in a randomized complete block design with three replications, each consisting of a 25-foot-long row. The plots were spaced 3 feet apart. Fifty seeds were planted in a single row in each plot. Seeds, either treated with mefenoxam (0.64 fl oz Apron XL LS/100 lb seed), metalaxyl (1.5 fl oz Allegiance LS/100 lb seed), or not treated (control), were sown approximately 5 cm deep. Soil samples were collected from the upper 10 cm of soil in the fields (one sample per 11 ft2 area, taken randomly), at the time of planting, using a soil auger, and mixed together. The population density of P. capsici was determined by dilution plating of soil samples on a PARPH selective medium and was ≥100 cfu per g soil. The seedlings were also sprayed with a P. capsici zoospore suspension (105 spores/ml; 150 ml/25 ft2 area) one week after seedling emergence to provide higher inoculum pressure in the plots. Application of foliar inoculum was to evaluate the efficacy of seed treatment on protecting plants against air-borne inoculum of P. capsici.

Seedling emergence was assessed 10 days after sowing seeds and seedling stand was determined 25 days after seedling emergence (35 days after seeding). Post-emergence damping-off was determined by counting plants showing girdling stem lesions with or without falling-over, wilting, and/or death of seedlings. Diseased seedlings were examined using light microscopy and infected tissues were plated on PARPH to isolate P. capsici.

Calcium application

Calcium on pumpkin and watermelon. Field trials were conducted during 2004-2004 to evaluate the effectiveness of calcium application, calcium application plus seed treatment, red-light treatment plus calcium application plus applications of a cooper fungicide (Cuprofix DF), and calcium application plus seed treatment plus application of fungicide Cuprofix DF for control of Phytophthora blight of pumpkin and watermelon. Processing pumpkin cultivar Dickinson and watermelon cultivar Royal Sweet were used in this study. The experiments were conducted in irrigated pumpkin fields near Pekin, IL. The fields, naturally infested with P. capsici, were planted in May and harvested in September. Seeds were treated with Apron XL LS (0.64 fl oz per 100 lb seed) prior to planting. For red light treatment, seedlings were grown under continues red light for four weeks in the greenhouse and transplanted into the plots.

Gypsum at the rate of 800 lb calcium per acre was applied at planting seed. Calcium chloride at the rate of 40 lb calcium per acre (one half at first female flower set and another half at fruit sizing) was applied. Calcium content of leaves in the plots with calcium application and in untreated control plots was determined. To determined calcium content of leaves, fully expanded leaves, closest to the vine growing tips, were collected at the growth stages of fruit set and one week before harvest and tested. Samples were digested in boiling HNO3, with sequential addition of 30% H2O2. Calcium content in each sample was determined by atomic absorption spectroscopy (AAS). To determine calcium content in soil, samples of soil were collected prior to application of Ca into soil. Soil samples were collected from top 0-8 inches. Samples were extracted with 1 N ammonium acetate (NH3OAc) with pH of 7.0 and determined by atomic absorption spectroscopy (AAS).

Disease incidence and severity on leaves, vines, and fruit were assessed at biweekly intervals beginning one week after seeding until harvest.

Calcium on pepper. Field trials were conducted during 2003-2004 to evaluate the effectiveness of calcium application, red-light treatment plus calcium application, and red-light treatment plus calcium application plus application of fungicide Cuprofix for control of Phytophthora blight of pepper. The experiments were conducted in an irrigated field near Pekin, IL. The field was naturally infested with P. capsici. Bell pepper cultivar California Wonder, a highly susceptible cultivar to P. capsici, was used in this study. Seedlings were grown in a greenhouse and eight-week-old seedlings were transplanted into the field.

Gypsum at the rate of 800 lb calcium per acre was applied at planting seedlings. Calcium chloride at the rate of 40 lb calcium per acre (one half prior to fruit set and another half three weeks after fruit set) was applied onto plants. Calcium content of leaves and soil was determined as described above. Disease incidence was determined as percent wilted or dead plants on a weekly schedule one week after transplanting until the end of the season.

Screening pumpkin and pepper cultivars/lines for resistance to P. capsici

Screening pumpkins. Six processing pumpkin cultivar and 22 jack-o-lantern pumpkin cultivars were screened for resistance to P. capsici. Plants were grown in the greenhouse and four-week-old seedlings were inoculated by adding 5 ml of a zoospore suspension (2 x 105 spores/ml of water) onto the soil surface around the stem of each plant in the pot. Disease development on the plants was assessed daily until 21 days after inoculation.

Screening peppers. Sixty-eight cultivars/lines of bell pepper were evaluated for resistance against P. capsici in the greenhouse and field. Seedlings were grown in a greenhouse at 64 to 77 degrees F. Eight-week-old seedlings were inoculated with P. capsici by adding 5 ml of zoospore suspension in SDW (2 x 105 spores/ml) at the base of each seedling. Control seedlings were treated with only SDW.

The pots were watered before inoculation to keep the soil wet. After inoculation, the pots were watered three times per day. Beginning the 4th day after inoculation, seedlings were evaluated for development of Phytophthora lesions on the lower section of stems, loosing leaves, wilting, and death of the seedlings. The evaluation of disease development on the seedlings was continued until 24 days after inoculation, when no more infection was observed.

Cultivars/lines, that showed resistant to P. capsici in the greenhouse trials, were tested in field trials. California Wonder, Arthur King, and Maxi Bell, susceptible bell pepper cultivars to P. capsici, were included in the field trials as control checks.

Host range of P. capsici. This study was conducted to determine the host range of P. capsici isolates from pumpkin. The pathogenicity of P. capsici isolates from pumpkin was evaluated on 45 species of herbaceous plants, including 36 species of crops grown in rotation sequences with pumpkin and nine species of weeds that commonly grow in pumpkin fields in Illinois. Plants were grown in the greenhouse and four-week-old seedlings were inoculated by adding 5 ml of a zoospore suspension (2 x 105 spores/ml of water) onto the soil surface around the stem of each plant in the pot. Disease development on the plants was assessed daily until 22 days after inoculation.

Fungicide application. More than 35 fungicides, with potential in controlling Phytophthora diseases, were tested in the laboratory and field for controlling Phytophthora blight and fruit rot of pumpkin, watermelon, and pepper, caused by P. capsici. Fungicides were tested in the laboratory at concentrations of 5 to 100 ppm active ingredient (a.i.) and in the field at recommended rates by the manufacturers.

For pumpkin and watermelon, field experiments were conducted in both irrigated and non-irrigated fields near Pekin, IL, naturally infested with P. capsici. Seeds of the pumpkin cultivar Dickinson and watermelon cultivar Royal Sweet were used in the studies. Soil-drenching fungicides were applied at planting. Foliar-applications of fungicides were made at weekly intervals starting from 2nd, 3rd, 4th, and 5th week after planting at weekly intervals. The fungicides were applied with a backpack sprayer using 50 gallons of water per acre. Plants were examined on biweekly intervals for symptoms of damping-off, Phytophthora foliar blight, and fruit rot beginning second week after planting seeds until harvest. Disease incidence and severity on leaves and vines were evaluated at three locations (11 ft 2 each) in each plot. Ten fruits were examined in each plot for Phytophthora infection. Disease incidence was determined as percent leaves, vines, or fruits affected. Disease severity was determined as percent area of leaf or vine affected. Also, numbers of uninfected and infected fruit with P. capsici were recorded at the harvest. Weight of uninfected fruit was measured. The data were analyzed using GLM procedures of SAS.

For pepper, field experiments were conducted in commercial fields, infested with P. capsici. near Shawneetown (southern Illinois) and near Pekin (central Illinois) to evaluate the effectiveness of selected fungicides for control of Phytophthora blight. Seedlings of bell pepper cultivar California Wonder, highly susceptible to P. capsici, were grown in a greenhouse. Eight-week-old seedlings were transplanted into raised beds with drip irrigation and black plastic mulch. Application of fungicides began second week after transplanting. Fungicides were applied with a backpack sprayer, using 50 gallons of water per acre. Disease incidence was determined as percent wilted or dead plants on a weekly schedule.

Commercial names of tested fungicides on pumpkin, watermelon, and pepper were: A1662 480SC, Acrobat 50WP, Acrobat MZ, Actigard 50WG, AgriFos 400FL, Aliette 80WDG, Apron XL LS, ATOFAP, Bravo Ultrex 82.5WG, Cuprofix 20DF, Cuprofix 40DF, Curzate 60DF, DPX-GFJ52 46.1WG, Forum 4.16SC, Gavel 75DF, Kocide-2000, Manex 37F, NOA-446510 250SC, Nova 40W, Omega 500F, Pencozeb75DF, Phostrol 53.5F, Previcur Flex 66.5F, Pristine 38WG, ProPhyt 4.2F, Quadris 2.08SC, Ranman 400SC, Reason 500SC, Ridomil Gold Bravo, Ridomil Gold Copper, Ridomil Gold EC, Ridomil Gold MZ, Silwet L-77, Tanos 50WG, TD-2389-02, and USF-2001 520SC.