Abstract
The steroidal saponin from cayenne pepper, CAY-1, was tested as a potential fungicide in detached-leaf assays and field trials. Efficacy of CAY-1 against strawberry anthracnose was compared to the commercial fungicide azoxystrobin. Both fungicides prevented anthracnose leaf lesions when applied to the host 24 hours prior to inoculation with Colletotrichum fragariae, but neither prevented lesion development when applied 24 hours after inoculation. CAY-1 reduced the growth of several fungal pathogens in laboratory assays and prevented anthracnose development in detached-leaf assays, but it did not control foliar or fruit-rot diseases of strawberry in field trials.
INTRODUCTION
Anthracnose fruit rot and crown rot (Colletotrichum spp.) (CitationSmith, 1998a Citation–Citationc), Botrytis gray mold (Botrytis cinerea), leather rot (Phytophthora cactorum), and stem- end rot (Gnomonia comari) (CitationEllis and Madden, 1998; CitationMaas, 1998; CitationSutton, 1998) are severe diseases that cause significant yield losses to the strawberry industry in the United States. The strawberry industry in the United States represents 32 billion dollars annually and accounts for over a quarter of the total production of strawberries in world. Control of these diseases has been achieved by applications of site-specific fungicides as well as multisite inhibitors (CitationWedge et al., 2007). In the past two decades fungicides including anilinopyrimidines (pyrimethanil and cyprodinil), phenylpyrroles (fludioxonil), hydroxyanilides (fenhexamide), and quinine-outside inhibitors (azoxystrobin) with novel modes of action have been introduced to treat these diseases worldwide (CitationGold et al., 1996; CitationRosslenbroich and Stuebler, 2000). Controlling strawberry diseases has become challenging as the efficacy of some fungicides has diminished due to development of resistant strains of fungi (CitationDekker, 1995; CitationPeres et al., 2002, Citation2004), and other fungicides have lost their registration for use on strawberries in order to comply with the mitigation measures determined by the Environmental Protection Agency to lessen human health risks and ecological effects (CitationLegard et al., 2001). However, indiscriminate use of some of these site-specific inhibitors has led to pathogen strains resistant to benzimidazoles, dicarboximides, and anilinopyrimidines (CitationLaMondia, 1995; CitationMoorman and Lease, 1992; CitationMyresiotis et al., 2007; CitationSmith and Black, 1991, Citation1993). The introduction of new fungicides without cross-resistance with current fungicides represents an alternative to overcome limitations in disease control (CitationWedge et al., 2007). Furthermore, CitationWedge et al. (2007) suggest an individualized spray program according to the needs of the grower.
Natural product-based fungicides are being developed as alternatives to traditional fungicides for use on strawberries (CitationWedge et al., 2007). CAY-1, a steroidal saponin with a molecular weight of 1243.35 Da, may represent an option against Colletotrichum fragariae, C. acutatum, C. gloeosporioides, Botrytis cinerea, Phytophthora cactorum, and Gnomonia comari. The biochemical mode of action of CAY-1 has been associated with the detergent properties of the saponins. It interacts with membrane sterols leading to changes in the membrane composition causing leakage of cell components and ultimately to cell death (CitationPolacheck et al., 1991). CAY-1 exhibits a broad spectrum of antifungal in vitro activity against a number of plant pathogenic species, including C. acutatum, C. fragariae, C. gloeosporoides, Fusarium oxysporum (CitationAbril et al., 2008), Aspergillus flavus, A. fumigatus, A. parasiticus, A. niger, Pneumocystis carinii, and Candida albicans (CitationDe Lucca et al., 2002). The purpose of this study was to evaluate the efficacy of the natural product-based fungicide, CAY-1, in greenhouse and field trials.
MATERIALS AND METHODS
Fungicides
CAY-1 is a fungicidal steroidal saponin isolated from the ground fruit of cayenne pepper (Capsicum frutescens). The commercial fungicides azoxystrobin, benomyl, captan, and iprodione () were used as industry standards for efficacy comparison against CAY-1 in laboratory (CitationAbril et al., 2009), greenhouse, and field trials.
TABLE 1 Chemical and Trade Names, Chemical Class, Mode of Action, Fungicide Resistance Action Committee (FRAC) Group, and Commercial Rates of Fungicides
Preparation of Pure CAY-1 for Greenhouse Studies
CAY-1 was prepared and purified as previously reported (CitationDe Lucca et al., 2002, Citation2006). Crude, dry, ground cayenne pepper was aqueously extracted (4:1, water:pepper) overnight (4°C). The slurry was centrifuged (5,000 RPM, 10 minutes, 4°C) and the supernatant passed through six layers of cheesecloth. The pelleted pepper was resuspended in water, allowed to equilibrate for 10 minutes, and centrifuged as before. The resulting supernatant was passed through cheesecloth, and the filtered supernatants combined. This extract was then centrifuged at high speed (10,000 RPM, 10 minutes, 4°C), and the supernatant then passed through a Supor AcroPak 200 filter (0.2 µM, Pall Corp, Ann Arbor, MI, USA). The filtered liquid was then freeze dried. The crude, freeze-dried material was dissolved in water (1:4, extract:water) and eluted through a 400 gram C18 (125 Å, 55–105 µM, Waters Corp, Milford, MA, USA) gravity column using a step gradient of methanol (MeOH):water (0, 25, 50, 75, 100% MeOH). The semipure CAY-1 was eluted in the 75% MeOH eluate. The methanol was removed under vacuum, and the remaining liquid was freeze-dried.
CAY-1 was purified using preparative-scale high-pressure liquid chromatography (HPLC) and purity was confirmed by mass spectrometry. HPLC analyses were performed using a Waters UV-VIS 996 detector and a Waters Radial Compression Bondapack C18 (25 mM x 100 mM; 10 µM particle size) reverse-phase column. Two column segments were combined using a PrepLC 25 mM extension kit (Waters). Elution was carried out at a flow rate of 8 mL/min−1 with the following solvent system: 65 minutes, 5% to 100% B; followed by holding for 15 minutes at 100% B; wherein solvent A is 0.1% trifluoroacetic acid in water (treated with a Millipore system) and solvent B is acetonitrile (Aldrich Chemical Company, Milwaukee, WI, USA). Separation of the CAY-1 (molecular mass: 1,243) from contaminating compound was achieved with the use of mass spectrometry. The mass spectrometer utilized was a Finnigan MAT LCQ ion trap (Finnigan, San Jose, CA, USA) equipped with an electrospray ionization interface. HPLC effluent was split with a 0.5 mL/min−1 introduced directly into the electrospray interface. Positive ion mode was used with a spray voltage of 3.5 kV and a capillary temperature of 200°C. Full scan spectra from m/z 100 to 2,000 were measured using 300 ms for collection time and three microscans were summed. CAY-1 was identified by an intense (M+H)+ ion at m/z 1,244 at 13.86 minutes.
Semipure CAY-1 Used for Field Studies
It was not possible to produce pure CAY-1 in sufficient quantities for field studies, so semipure CAY-1 was prepared and used in place of the pure compound. This material was prepared as described above and remained in a dried condition until sprayed on the field crop. The concentration of the CAY-1 in the semipurified material was calculated from HPLC/MS data, and the concentration of CAY-1 in the tank mix solution applied to the field crop was also calculated from this data.
Greenhouse Studies
Conidial suspension preparation
Colletotrichum fragariae isolate CF-75 (CitationSmith and Black, 1990) was grown on Potato Dextrose Agar. Conidia were harvested from 7- to10-day-old cultures and adjusted to a final concentration of 106 conidia/mL with a hemacytometer.
Source of plants
‘Chandler’ strawberry plants were purchased from a commercial nursery and planted in 10 ¥ 10 cm plastic pots containing a 1:1 (v/v) mixture of Jiffy-Mix (JPA, West Chicago, IL, USA) and pasteurized sand. Strawberry plants were grown for 6 weeks in a greenhouse maintained at 30°C day/18°C night ± 6° with a 16 hour photoperiod. The second or third youngest leaf without visible signs of injury or symptoms of disease was removed from each plant no more than 4 hours before fungicide treatment and/or inoculation and placed in a sealed container at 100% RH and 12°C. Leaves were washed in distilled water and transferred into sterile distilled water in 10 x 150 mM tissue-culture tubes. All three leaflets of a leaf were inoculated by misting the adaxial surface with a hand pump to the point of runoff with a conidial suspension of C. fragariae isolate CF-75.
Fungicides
CAY-1 and azoxystrobin (Abound, Syngenta Crop Protection, Inc, Greensboro, NC, USA) were prepared at concentrations of 625, 1250, and 2,500 ppm in 50% ethanol and applied with a vacuum/pressure pump (Welch Pump, Model # 25228-01, Skokie, IL, USA) at 5 psi and a 10 mL TLC sprayer head (Kontes Glass Company, Vineland, NJ, USA). A noninoculated, treated control and non-treated, inoculated control were included in each trial. The experiment was repeated twice.
Fungicide treatments
Pre- and postinoculation treatments were conducted. In the pre-inoculation treatment, leaflets were sprayed with fungicides and placed in racks in a dew chamber (Percival Scientific, Model I-60DL, Boone, IA, USA) in complete darkness for 24 hours at 30°C. Then leaflets were allowed to air dry, inoculated with the conidial suspension of C. fragariae, and placed in the dew chamber for an additional 48 hours. In the postinoculation treatment, leaflets were inoculated with a conidial suspension and placed in racks in a dew chamber for 24 hours at 30°C. Then leaflets were allowed to air dry, sprayed with the fungicides, and returned to the dew chamber for an additional 24 hours. Leaflets were incubated in the dew chamber for a total of 48 hours after inoculation, then they were transferred to 18-gallon sealed plastic containers at 100% RH, 28°C, and continuous fluorescent light for an additional 3 days before assessing disease symptoms. Photographs of each leaflet for posterior macroscopic disease assessment (quantitative) were taken with a DXC-151A color video camera (Hitachi Instruments, Inc., Houston, TX, USA) attached to a BH-2 Olympus light microscope (Olympus Corporation, Marietta, GA, USA) and captured with Bioquant 98™ image-analysis software package (R&M Biometrics, Inc., Nashville, TN, USA).
Disease severity on the leaflets was assessed as percentage area infected by the fungi expressed in an arbitrary scale of 1 to 3. A score of 0 indicated no symptoms of anthracnose and a score of 3 indicated the most severe anthracnose lesions (totally covering the leaf surface). Phytotoxicity was assessed as percentage of necrotic area on an arbitrary scale of 1 to 5. A score of 0 indicated no phytotoxicity and a score of 5 indicated the most severe phytotoxicity symptoms (totally covering the leaf surface).
Field Studies
Field trials were conducted at the Hammond Research Station, Hammond, LA, for fruit-rot disease control on strawberries. Two concentrations of CAY-1 were compared with an untreated control and azoxystrobin as part of a larger study including 12 other commercial fungicides (CitationWedge et al., 2007). Plots were established on a fine, sandy loam soil on raised beds mulched with black plastic. Plot size was 1.2 M × 6.4 M with four replications per fungicide treatment. Drip irrigation was applied as needed through drip tape laid under the plastic mulch. ‘Camarosa’ strawberry plants purchased from a commercial nursery were transplanted on October 22, 2002, on 30.5 cm spacing with two rows of plants per bed and 40 plants per plot.
CAY-1 was applied to strawberries at rates of 0.37 and 0.74 kg/ha, and the commercial fungicide azoxystrobin was applied at 0.13 liter a.i./ha. Fungicide treatments were applied every 7 to 10 days (or as soon after rainfall as possible) starting at or near full-bloom stage and continuing until one week before harvesting ceased. Berries were harvested twice a week throughout the entire season. Total fruit count and weights were obtained for both marketable and cull fruits, along with average weight per berry and percentages of marketable and diseased cull fruit per acre. Fruit were separated into marketable fruit, physical (deformed or small fruit) culls, and diseased culls. The total number of berries from each plot with symptoms of gray mold (Botrytis cinerea), anthracnose (C. acutatum), and stem-end rot (Gnomonia comari) was noted on four occasions: February 27, March 20, April 3, and April 24, 2003. Foliar disease was evaluated on April 24, 2003, and expressed on an arbitrary scale of 1 to 5. A score of 0 indicated no symptoms of foliar disease, and a score of 5 indicated the most severe foliar lesions.
Data Analysis
Data were evaluated by analysis of variance (ANOVA) using the general linear model procedure (GLM) of Statistical Analysis Systems (SAS) software (Version 9.1). Significant factors were separated and tested using Fisher's protected least significant difference (LSD) tests (P < 0.05).
RESULTS AND DISCUSSION
Greenhouse Studies
CAY-1 and azoxystrobin had slight or no effect on the incidence of anthracnose disease when applied 24 hours after inoculation (). In contrast, CAY-1 (at all three concentrations) and azoxystrobin (at 1250 ppm) provided protection against anthracnose when applied 24 hours before inoculation. Direct microscopic observation of germination and appressorial formation confirmed that the pre-inoculation treatment provided protection. Appressorial formation was visible with CAY-1 and azoxystrobin at the lowest concentration tested, 625 ppm (data not shown). CAY-1 prevented fungal germination at the medium and highest concentrations, 1,250 and 2,500 ppm. Azoxystrobin prevented fungal germination particularly at its medium concentration, 1,250 ppm. Sporadic phytotoxicity was observed with CAY-1 and azoxystrobin on both the pre-inoculation and post-inoculation treatments at all concentrations (). These results demonstrated that the natural product-based fungicide, CAY-1, and the commercial fungicide, azoxystrobin, both provided protection against anthracnose when applied 24 hours before the leaves were inoculated with the fungal pathogen. However both fungicides had little or no effect on the incidence of the disease when applied 24 hours after inoculation. This supports the findings of CitationWong and Wilcox (2001) who reported a protectant activity of azoxystrobin when applied before fungal inoculation, but little effect when applied after fungal inoculation. These greenhouse screening trials and our microscopic observations of the germinated conidia suggest a protectant mode of action for CAY-1 against C. fragariae similar to that documented for azoxystrobin (i.e., inhibition of conidial germination; CitationBertelsen et al., 2001; CitationWong and Wilcox, 2001). These promising greenhouse studies suggested that field studies of CAY-1 were warranted.
TABLE 2 Effects of Azoxystrobin and CAY-1 Applied to Detached ‘Chandler’ Strawberry Leaves Before and After Inoculation with Colletotrichum fragariae Isolate CF-75 on Anthracnose Lesion Development and Phytotoxicity Rating
Field Studies
Significant differences were observed in marketable yield, diseased yield, and size of strawberries as a result of fungicide treatments (). Plots treated with azoxystrobin had higher marketable yields than untreated plots or the two CAY-1 treated plots. There were no significant differences in berry size due to fungicide treatments. The most prevalent diseases were Botrytis gray mold, anthracnose fruit rot, and stem-end rot; and total disease incidence increased as the season progressed (data not shown). Botrytis gray mold was more prevalent on March 20 than on the other dates while anthracnose was more prevalent later in the season. The lowest incidence of Botrytis gray mold was on plots treated with azoxystrobin and CAY-1 at half rate (). Plots treated with CAY-1 at full rate had Botrytis gray-mold levels as high as the untreated plots. The number of berries with anthracnose symptoms was lowest on plots treated with azoxystrobin, followed by plots treated with CAY-1 at half rate. Plots treated with azoxystrobin had the lowest incidence of stem-end rot and foliar diseases ().
TABLE 3 Effect of Fungicide Treatment on Yield and Size of ‘Camarosa’ Strawberry Fruit in Field Trials Conducted in Hammond, Louisiana
TABLE 4 Average Number of Strawberry Fruit with Fruit Rot Symptoms from Four Replications and Rating Dates, and Average Foliar Disease Score Obtained from Field Trials Conducted in Hammond, Louisiana
Results of these field studies were disappointing because plots treated with the two rates of CAY-1 exhibited as much disease as the untreated control plots. The higher levels of disease on the CAY-1 treatments were probably due to injury of fruit and foliage. This damage by CAY-1 (a saponin) could have been due to (a) its detergent-like properties affecting the receptacle in the case of the fruit and the cuticle in the case of the leaves of the strawberry or (b) other compounds present in the semipurified CAY-1 used in the field trials that were not present in the purified CAY-1 used in the in vitro and detached-leaf trials. Improvements in the formulation of CAY-1 might prevent this injury and result in better disease control.
Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the U.S. Dept. of Agriculture and does not imply its approval to the exclusion of other products or vendors that also may be suitable.
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