© 2013 Plant Management Network.
Evaluation of Biorational Products for Management of Phytophthora Blight of Bell Pepper Transplants
Camilla Yandoc Ables, National Academy of Sciences, Washington, DC 20418; Jason C. Hong, Nancy Kokalis-Burelle, and Joseph P. Albano, USDA-ARS, United States Horticultural Research Laboratory, Fort Pierce, FL 34945; Elizabeth M. Lamb, New York State Integrated Pest Management Program, Cornell University, Geneva, NY 14456; and Erin N. Rosskopf, USDA-ARS, United States Horticultural Research Laboratory, Fort Pierce, FL 34945
Yandoc Ables, C., Hong, J. C., Kokalis-Burelle, N., Albano, J. P., Lamb, E. M., and Rosskopf, E. N. 2013. Evaluation of biorational products for management of Phytophthora blight of bell pepper transplants. Online. Plant Health Progress doi:10.1094/PHP-2013-0226-01-RS.
There is a critical need for pest control products that are compatible with sustainable agricultural practices, such as those based on natural antagonists or plant defense activators. Four separate, repeated experiments were conducted in which commercially available biopesticides and phosphonate-containing products were evaluated in the greenhouse for the management of Phytophthora blight of bell pepper caused by Phytophthora capsici. The phosphonate-containing product, FNX-100, applied as a soil drench, was found to be the most effective treatment for decreasing the incidence of stem and crown rot. A single soil drench of FNX-100 applied at the lowest concentration provided disease control that was as effective as multiple applications of the highest rate. Foliar applications of FNX-100 at any concentration or frequency were ineffective for disease control, and caused pronounced phytotoxicity when compared to the FNX-100 soil drench. The phosphorous acid K-Phite was found to be less effective than FNX-100, but disease symptoms were minimal in plants treated with a soil drench of that product. While the efficacy of three other phosphonate-containing products, Prophyt, Phostrol, and FNX-2500, were inconsistent, it was observed that these products were also more effective in managing disease when applied as a soil drench rather than as a foliar spray.
Bell pepper (Capsicum annuum) is an important horticultural crop grown worldwide. In the US alone, bell pepper was reportedly cultivated on approximately 21,300 ha in 2010 (22). In the United States, Florida ranks first in cash receipts for bell pepper with a 2010 production area of 7,163 ha and a farm gate value of more than $295 million (22). A major pathogen of peppers is the soilborne oomycete, Phytophthora capsici, which can infect every part of the plant and can cause root rot, foliar blight, and pod rot (25). The pathogen was first described on bell pepper in New Mexico in 1922 (17). Symptoms of root rot are stem girdling at the soil line followed by sudden wilt and death. Infected plants often have brown to black discolored roots and/or crowns (15). Symptoms are most severe when weather conditions are warm, 25 to 30°C, and humid. Significant yield reductions due to this disease have been reported in many bell pepper-growing areas, including New Jersey and Florida, where losses reach 20-25% and 30-40%, respectively (19).
Historically, pepper production has been highly dependent upon soil fumigation with methyl bromide or combinations of other fumigants (26). The 2013 Methyl Bromide Critical Use Nomination (CUN) for this crop in the southeastern United States has been reduced to 5,673 kg for preplant soil application in pepper production (10), compared to the nearly 2 million kg used annually in the 1990s. While considerable resources are being invested in the search for chemical fumigant alternatives to methyl bromide for pepper production, few feasible options are currently available. Many registered methyl bromide alternatives are of limited use due to health or environmental impacts, regulatory issues, and lack of efficacy for certain pests. The most promising alternative for bell pepper production, particularly since the rescinding of the United States registration of methyl iodide, is a carefully timed combination of 1,3-dichloropropene, chloropicrin, and metam sodium or metam potassium, and is applied to preformed raised beds prior to transplanting (18). While this three-chemical combination is currently the most promising approach, there remains a significant need for other integrated approaches, which may include combinations of fungicides, although limitations exist for the use of these materials as well (1). Reports of resistance to fungicides commonly used for control of P. capsici (i.e., mefenoxam and metalaxyl) are on the rise (13). Even with effective fumigant application, pathogen-contaminated irrigation water that is applied can cause devastating infestations (14). Development of a range of alternative approaches, including non-chemical control, will provide more options for growers.
Biological control for suppressing Phytophthora diseases has received considerable interest in recent years. Biological agents that have shown potential for controlling P. capsici include: Trichoderma harzianum, Streptomyces rochei (11,29), Muscodor albus (20), Gliocladium virens, and Burkholderia cepacia (19). Availability of many biologically-based products is limited by market constraints and issues with regard to registration of B. cepacia with the US Environmental Protection Agency. A current listing of microbial products can be found at the American Phytopathological Biological Control Committee webpage (www.oardc.ohio-state.edu/apsbcc).
Another new and promising method for controlling Phytophthora species is the use of phosphonates. These organic compounds containing C-PO(OH)2 or C-PO(OR)2 groups have direct and indirect effects on oomycetes, including plant defense activation (4,12). Phosphonates can suppress diseases caused by P. infestans (late blight of potato) (5), P. citricola (avocado stem canker) (9), P. cinamomi (avocado root rot) (7,24), P. palmivora, P. megakarya (black pod of cacao) (23), P. nicotianae (Phytophthora blight of periwinkle) (31), and P. capsici on cucurbit crops (pumpkin) (30).
In this study, several commercially available biopesticides and phosphonate-containing products were tested for their efficacy in controlling P. capsici on greenhouse-grown bell pepper transplants at several inoculum levels and soil fertilization rates. The effects of concentration, application method, and application frequency on the efficacy of phosphonates against Phytophthora root rot of bell pepper were also evaluated.
Test plant propagation. In all experiments, seeds of the Phytopthora-susceptible pepper cultivar Enterprise (Seminis Vegetable Seeds, Oxnard, CA) were sown in plastic flats (35 × 67 cm) containing a greenhouse potting medium composed of sphagnum peat, processed pine bark, vermiculite, and perlite (Fafard 4P Mix, Fafard Inc., Anderson, SC). Seedlings were transplanted at the four-leaf stage into 10-cm diameter plastic pots with potting medium. All plants were watered and fertilized daily; unless otherwise indicated, fertilization consisted of half-strength Peters 20-20-20 plus micronutrients fertilizer (Peters Professional Water Soluble Fertilizer, Scotts-Sierra Horticultural Products Co., Marysville, OH). Plants were maintained in a greenhouse with an average temperature of 24°C and relative humidity of 96%.
Inoculum production. Production of zoospores was based on the Dah-Wu and Zentmyer method (6). Cultures of a virulent isolate of P. capsici, strain Cp-32, cultures were grown on corn meal agar amended with pimaricin, ampicillin, rifampicin, pentachloronitrobenzene, and hymexasol (PARP-H) and then transferred to V8 juice agar (250 ml clarified V8 juice, 3 g CaCO2, 750 ml water). Fifteen 5-mm mycelial disks were obtained from 5-day-old cultures and placed in Petri dishes containing 20 ml quarter-strength V8 juice. Petri dishes were incubated at 27°C (in the dark) for 48 h, after which the disks were rinsed with sterile water four times to remove the V8 juice. After rinsing, 20 ml of 10-4 M 2[N-morpholinolethanesulfonic acid] (MES, Sigma-Aldrich Co., St. Louis, MO) solution was added to each plate. The plates were then incubated for 48 h in a 25°C incubator with continuous light. Following incubation, the zoospores were suspended in sterile deionized water and filtered through two layers of cheesecloth. The final zoospore concentration was estimated using a hemacytometer after chilling the suspension to slow spore movement.
Inoculation. Bell pepper plants were inoculated with P. capsici
zoospores 10 days after treatments were applied for all experiments described
below. Plants were watered in excess 24 h before inoculation. Inoculation was
performed by pipetting 10 ml of the zoospore suspension (which ranged from
5000-6,000 spores/ml depending on the experiment) onto potting soil adjacent to the
plant crown. Control plants received 10 ml of sterile deionized water applied in
a similar manner. To enhance disease development, high soil moisture was
maintained by placing all pots in plastic saucers filled with water for three
days post inoculation. Plants were maintained in the greenhouse for the duration
of experiments, where temperature and humidity were maintained as described
Disease and phytotoxicity severity assessment and
data analysis. Unless
otherwise indicated, disease severity (DS) was assessed 21 days after
inoculation on an ordinal 0 to 4 scale: 0 = no disease; 1 = presence of stem
lesion or girdling; 2 = stem lesion or girdling plus wilting of leaves adjacent
to the lesion; 3 = stem lesion or girdling plus wilting of the lower and upper
leaves; 4 = dead plant. Phytotoxicity was assessed based on an ordinal scale:
0 = no damage; 1 = small chlorotic lesions or tip/edge burn; 2 = few leaves w/large
burn areas; 3 = more leaves with large burn areas; 4 = all leaves burned and plant
wilting; 5 = plant dead. Nonparametric data analysis was done with SAS Proc Mixed
(SAS Institute Inc., Cary, NC), as detailed previously (27). Relative treatment
effects and their confidence interval limits were calculated by the SAS LD_CI
Effect of zoospore concentration on the efficacy of selected biopesticides and dipotassium phosphonates against Phytophthora blight of bell peppers. This experiment was conducted as a completely randomized design (CRD) with four replications per treatment, which consisted of combinations of biopesticides and zoospore concentrations. The biopesticides tested were FNX-100 (Foliar Nutrients Inc., Cairo, GA), DieHard (Horticultural Alliance Inc., Sarasota, FL), MBI600 (Microbio Ltd, a subsidiary of Becker Underwood, Ames, IA), and Primastop (AgBio Development Inc. Westminster, CO). Additional information on the biopesticides and the application rates are provided in Table 1. Each replication consisted of one pot containing a single bell pepper plant. The efficacy of the four biopesticides was tested against increasing concentrations (50, 500, and 5,000 zoospores/plant) of P. capsici (Cp-32). Controls consisted of inoculated non-treated plants, which did not receive biopesticide treatment, and non-inoculated treated plants. Biopesticides were applied during transplanting and plants were inoculated with P. capsici zoospores10 days after transplant/biopesticide application. The experiment was performed twice.
Table 1. Biorational materials tested against Phytophthora capsici.
Due to significant interactions with trial, data from the two trials were analyzed separately. For trial 1, biopesticides had a significant effect on stem rot severity, but no significant effect on disease severity was observed for zoospore concentration, and no interaction between treatment and zoospore concentration occurred (Table 3a). For trial 2, treatment and zoospore concentration effects were both significant, but there was no interaction between these factors (Table 3b). In both trials only plants treated with FNX-100 had a reduction of root rot (Fig. 1). Disease severity levels were similar in control plants and plants treated with Diehard, MBI, and Primastop. In trial 2, median disease severity was significantly different in plants that received 50, 500, or 5,000 zoospores (Table 3b, Fig. 1). Plants inoculated with 50 zoospores had a median disease severity of zero while plants inoculated with 500 or 5,000 zoospores had a median DS of 4. Control plants showed no symptoms of infection.
Table 3a. Test statistics for the effects of treatment and zoospore concentration on the severity of stem rot of pepper caused by Phytophthora capsici (Trial 1).
* Significant at P = 0.05.
dfN = numerator degrees of freedom; dfD = denominator degrees of freedom.
Table 3b. Test statistics for the effects of treatment and zoospore concentration on the severity of stem rot of pepper caused by Phytophthora capsici (Trial 2).
* Significant at P = 0.05.
Effect of fertilization on the efficacy of biopesticides against P. capsici in bell pepper. The experiment was a CRD consisting of a two-way factorial with four replications per treatment. Treatments consisted of six biopesticides and an untreated control with four fertilizer concentrations. Each replicate consisted of one pepper plant. The biopesticides tested were FNX-100, FNX-2500, Actigard (Syngenta Crop Protection, Greensboro, NC), Mycostop (Verdera Oy, Espoo, Finland), Primastop, DieHard, Soilgard (Certis USA L.L.C., Columbia, MD), MBI600, and DiTera WDG (Valent BioSciences Corp., Libertyville, IL). Information on the biopesticides and the application rates and application schedule are provided in Table 1. The fertilizer concentrations were 0 (no fertilizer), 0.5, 1.0, and 2.0× fertilizer solution, which was formulated based on a modified Hoagland solution (15). The 1.0× treatment contained the following essential plant elements in mg/liter: 200 N (79% NO3-N and 21% NH4-N), 62 P, 168 K, 120 Ca, 49 Mg, 64 S, 1 Fe; and the following essential plant elements in μg/liter: 500 Mn, 500 B, 50 Zn, 50 Mo, and 20 Cu, derived from KNO3, KH2PO4, MgSO4, Ca(NO3)2, NH4NO3, NH4H2PO4, H3BO3, H2MoO4, FeEDTA, MnEDTA, ZnEDTA, and CuEDTA. A 10.0× stock solution was prepared and dilutions of the concentrated stock were made with elements proportionally diluted to yield solutions of 100 mg/liter (0.5×), 200 mg/liter (1.0×), and 400 mg/liter (2.0×) N. Water treated by a reverse osmosis system was used to prepare the fertilizer solutions and for the 0.0× treatment. The pH of the fertilizer solution dilutions was adjusted to 5.8-6.0 with NaOH or HCl. Plants were fertilized with 100 ml Hoagland solution per week for 5 weeks, starting at 5 days after transplanting until the end of the experiment. One set of biopesticide-treated test plants was inoculated with 25,000 zoospores at 10 days after transplant; the other set was not inoculated and was designated as the treated non-inoculated control, with four replications for each treatment. The experiment was performed twice.
For both trials, only the biopesticide treatment had a significant influence on the severity of disease (Table 4a and 4b). Among the seven biopesticides tested, only FNX-100 consistently suppressed disease symptoms (Fig. 2), and median DS rating was zero in both trials. Fertilizer level had no significant effect on the DS.
Table 4a. Test statistics for the effects of treatment and fertilizer level on the severity of root/stem rot of pepper caused by Phytophthora capsici. (Trial 1)
* Significant at P = 0.05
Table 4b. Test statistics for the effects of treatment and fertilizer level on the severity of root/stem rot of pepper caused by Phytophthora capsici. (Trial 2)
* Significant at P = 0.05
Effect of the concentration, application method, and application frequency of phosphonate on the severity of Phytophthora blight of bell pepper. A completely randomized experimental design (CRD) with four (trial 1), or five (trial 2), replications per treatment was conducted to test three concentrations of FNX-100, applied as a soil drench (0.5, 1.0, and 1.5%), or as a foliar spray (1.0%, 2.0%, and 3%), either once or three times during the course of the experiment. The drench and foliar concentrations used were based on results of preliminary evaluations of phytotoxicity caused by applications of higher drench or spray concentrations. Plants that received one-time drenches or sprays were treated at 14 days before pathogen inoculation. Plants that received three applications were treated at 14 and 3 days before inoculation, and at 6 days after inoculation. Inoculation was performed at 10 days after transplant. Each test plant received 60,000 zoospores. The experiment was performed twice.
In this experiment, only FNX-100 application method and concentration combination had a significant effect on phytotoxicity (Table 5a) and disease severity (Table 5b). Frequency of application did not have a significant effect on phytotoxicity or disease severity, and there was no treatment and frequency interaction for phytotoxicity and disease severity (Tables 5a and 5b). Plants sprayed with 2.0 and 3.0% FNX-100 exhibited greater phytotoxicity (burned leaves, wilting, or defoliation) compared to plants that were sprayed with 1.0% FNX-100 or drenched with 0.5, 1.0, or 1.5% FNX-100. Drench application of 1.0% and 1.5% FNX-100 significantly reduced disease in pepper, without causing any phytotoxic damage (Figures 2 and 3).
Table 5a. Test statistics for effects of treatment and application frequency on phytotoxicity on pepper.
* Significant at P = 0.05.
Table 5b. Test statistics for the effects of treatment and application frequency on the severity of stem rot of pepper caused by Phytophthora capsici.
* Significant at P = 0.05.
Efficacy of selected phosphonate-containing products applied as soil drench or foliar spray against Phytophthora blight of bell pepper. Five commercially produced phosphonate-containing products (K-Phite, Prophyt, Phostrol, FNX-100, and FNX-2500) were applied as a soil drench and as foliar sprays. All products were applied according to label recommendations. The product information and application rates are provided in Table 2. Treatments were applied one day after peppers were transplanted. One set of treated plants was inoculated with 10,000 zoospores per pot at 10 days after products were applied; the second set was not inoculated and was used to check for any phytotoxic effects caused by the products. Water applied as a soil drench or foliar spray served as the control. This experiment was performed in a completely randomized design (CRD) with five replications per treatment with one pepper plant per replication. The experiment was performed twice.
Table 2. Phosphonate-containing products tested.
The interaction of treatment, phosphonate-containing product, and application method (drench or foliar) on the median disease severity rating and the level of phytotoxic damage (Figure 4A and B) on pepper plants was significant (Table 6). Only FNX-100 applied as a soil drench consistently suppressed Phytophthora stem rot in two trials, without any severe phytotoxic damage (see Table 7). None of the other products applied as foliar sprays or soil drenches provided consistent disease control (Figure 4A and B), although K-phite treatment also resulted in disease severity that was significantly lower than the untreated inoculated control.
Table 6. Test statistics for the effect treatment and method of application on the level of disease severity on pepper.
x *Significant at P = 0.05
Table 7. Damage caused by phosphonate-containing products on peppers. Observations taken from non-inoculated, treated plants at 21 days after drench or foliar application.
In the absence of methyl bromide, there is a critical need for the development of more environmentally safe products that are compatible with sustainable agriculture. Products that are based on natural antagonists or on plant defense activators are of particular interest. Several commercially available biopesticides and phosphonate-containing products were evaluated for their efficacy against root rot and stem blight on bell pepper caused by P. capsici. Regardless of the concentration of zoospores or fertilization method, the biological control products tested were unable to suppress disease symptoms. However, phosphonate-containing products were more effective in managing the pathogen, especially FNX-100. Applications of FNX-100 applied as a soil drench resulted in significant disease suppression consistently throughout all of the experiments regardless of concentration or frequency of application. However, phytotoxicity was proportional to the concentration of the product applied to foliage.
Neither Phytophthora zoospore nor fertilizer concentration significantly affected disease suppression with the biopesticides. Comparison of the two Phytophthora zoospore concentration trials revealed that FNX-100 consistently suppressed disease symptoms, while results between the two trials varied for Primastop, MBI, and Diehard. Even at the concentration of 60,000 zoospores per pepper plant, those treated with 0.5% FNX-100 by soil drench did not show disease symptoms. Previous studies have shown that the severity of infection is proportional to the concentration of inoculum (21); however, even at low concentration, P. capsici caused a high percentage of pepper plant mortality when plants were inoculated by suspending zoospores in water coupled with flooding above the soil surface (2). The same researchers also reported that 75 and 90% of plants inoculated with 10 and 25 zoospores, respectfully, died. Previous reports vary on the effectiveness of increased fertilization on the control of Phytophora diseases. In a few studies, it has been observed that the incidence of Phytophora root rot on soybean was proportional to fertilizer application rates (8,30). However, Sharma et al. (28) observed a significant decrease in disease incidence caused by P. infestans between tomato plants treated with organic fertilizer and those not treated. However, in our studies, fertilization did not have an impact on disease development.
Incidence of Phytophora blight was affected by the application of phosphonate-containing products, and the most effective treatment was FNX-100 applied as a soil drench. Increasing the concentration or number of applications of FNX-100 applied as a soil drench did not significantly improve disease control. Since the material can be applied to plants after transplant and may act via plant defense activation, it would be a useful tool for growers who have the potential for inoculum to come into the field after the initial soil disinfestations treatment, be it fumigant or non-fumigant. Foliar applications of FNX-100 at any concentration or frequency were also ineffective for controlling disease symptoms, and phytotoxicity was significantly increased when plants were treated with foliar applications compared to the soil drench. Although K-Phite was less effective than FNX-100 in one of two trials, disease symptoms were minimal for plants treated with a soil drench of that product. Although inconsistent, the results for Prophyt, Phostrol, and FNX-2500 indicate that these products were also more effective in managing disease when applied as a soil drench compared to a foliar spray.
Acknowledgments and Disclaimer
This research was funded in part by the IR-4 Biopesticide Program. The authors would like to thank Lauren Walsh, Janny Peña, Larry Markle, Kate Rotindo, Bernardette Stange, Jackie Markle, Jeff Smit,h and the late John Taylor for their assistance with this project. We also thank Denis Shah for teaching statistics courses at the annual meeting of the American Phytopathological Society and continuing to provide guidance for years after.
Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the United States Department of Agriculture.
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