Comparative Study of Phosphorous-Acid-Containing Products for Managing Phytophthora Blight of Bell Pepper

Abstract

Phytophthora blight of pepper caused by Phytophthora capsici is a major constraint to bell pepper (Capsicum annuum) production. The long-term effectiveness of chemicals currently in use against P. capsici is uncertain due to the development of fungicide resistance by this pathogen. Hence, the efficacy of alternative chemicals such as phosphorous-acid-containing products was evaluated in this study. In in vitro tests, ProPhyt, K-Phite, Lexx-A-Phos, Agri-Fos, and Nutri-Phite were less effective in inhibiting mycelial growth (EC₅₀ = 50.5 to 324.4 µg mL⁻¹) and sporangium formation (EC₅₀ = 6.1 to 225.7 µg mL⁻¹) of two P. capsici isolates, but more effective against zoospore germination compared with mefenoxam. Among phosphorous-acid-containing products tested, Nutri-Phite was most effective in inhibiting mycelial growth of both P. capsici isolates. In greenhouse studies, Nutri-Phite was effective against Phytophthora blight used as drench. The use of Nutri-Phite, Agri-Fos, ProPhyt, and K-Phite could induce systemic resistance against foliar blight when applied to the root and potting mix. The results indicated that some phosphorous-acid-containing products have the potential to lower disease occurrence and delay Phytophthora blight of bell pepper without phytotoxic effects. The utility of the systemic protection induced by these products is promising in Phytophthora blight management.

1. Introduction

Phytophthora blight, caused by Phytophthora capsici Leonian, is one of the most devastating diseases of bell pepper (Capsicum annuum L.) in the United States and worldwide [1,2,3]. In recent years, the incidence and severity of this disease have increased [4]. Besides bell pepper, P. capsici can attack many cucurbit and solanaceous crops, including squash, pumpkin, cucumber, tomato, and eggplant [5,6,7,8,9]. On bell pepper, P. capsici attacks the plant at any growth stage when environmental conditions are favorable to cause seedling damping off, crown rot, foliar blight, stem blight/canker, fruit rot, and root rot [10]. P. capsici can survive in the soil as oospores for several years [11]. The surviving propagules are dispersed through the soil by means of surface water, and from soil to foliage by water splash [2].

The management of Phytophthora disease relies heavily on the selective application of chemicals. Fungicides of the phenylamide group including metalaxyl (e.g., Ridomil) and mefenoxam (e.g., Ridomil Gold) were widely used in the field to control Phytophthora diseases [12]. However, the intensive application of these fungicides on bell pepper fields led to the development of phenylamide-resistant P. capsici strains. The resistance of P. capsici strains to mefenoxam was first reported in North Carolina and New Jersey [13]. Occurrences of mefenoxam-resistant strains of P. capsici were then noted in Michigan [14], Southern Italy [15], and Florida [16].

Due to the insensitivity of P. capsici strains to phenylamide fungicides, more research attention has been given to the disease suppression activity of phosphorous-acid-containing products such as phosphonate and phosphites. Currently, there are several commercial products that contain phosphorous acid phosphonate as the phosphorus carrier: Agri-Fos (Agrichem Manufacturing Industries Pty. Ltd., Loganholme, QLD, AUS), K-Phite (Plant Food Systems, Zellwood, FL, USA), Lexx-A-Phos (Foliar Nutrients, Inc., Cairo, GA, USA), Nutri-Phite (Biagro Western Sales, Inc., Visalia, CA, USA), and ProPhyt (Luxembourg-Pamol, Inc., Memphis, TN, USA). These products have been extensively used to manage plant disease and were demonstrated to be effective against various diseases caused by oomycetes [1,17,18,19]. The use of technical and commercial formulations of phosphonate (Nutri-Phite P Foliar) in a hydroponic growing system in the greenhouse significantly reduced Phytophthora root and crown rot of tomato and bell pepper caused by P. capsici [1].

The phosphorous-acid-containing products are translocated in both the xylem and the phloem [20] and are believed to have a complex mode of action by not only acting directly on the pathogen but also indirectly by acting as plant activators. As plant activators, they induce the host to produce compounds that ultimately inhibit pathogen growth [21,22]. Since they do not work solely through a direct effect on the pathogens, they may reduce the selection pressure [23] on P. capsici, which should reduce the probability that resistant strains to these products will develop [22].

The objectives of this study were to (1) compare the efficacy of different phosphorous-acid-containing products in inhibiting the growth of P. capsici in vitro and in vivo and (2) determine whether or not these products have the ability to induce systemic resistance in bell pepper against P. capsici.

2. Materials and Methods

2.1. P. capsici Isolates and Chemicals

Two P. capsici isolates used in this study were PPC1 (A1 mating type) and PPC6 (A2 mating type). These isolates were isolated from bell pepper in Tifton, GA, USA [24].

The five phosphorous acid compounds evaluated in the laboratory and in the greenhouse are presented in

Table 1. Chemicals evaluated in this study.

Commercial Name	Active Ingredients
Agri-Fos	Mono- and di-potassium salt of phosphorous acids (45.8%) (Agrichem Manufacturing Industries Pty. Ltd., Loganholme, QLD, AUS)
K-Phite	Mono- and di-potassium salts of phosphoric acid (53%) (Plant Food Systems, Zellwood, FL, USA)
Lexx-A-Phos	Di-potassium phosphate (22.7%), di-potassium phosphonate (22.4%) (Foliar Nutrients, Inc., Cairo, GA, USA)
Nutri-Phite	Total phosphoric acid (60%), soluble potash (K2O) (5%) (Biagro Western Sales, Inc., Visalia, CA, USA)
ProPhyt	Potassium phosphate (54.5%) (Luxembourg-Pamol, Inc., Memphis, TN, USA)
Ridomil Gold 480 EC 1	Mefenoxam (phenylamide) (480 g L−1) (Syngenta Crop Protection, Inc., Greensboro, NC, USA)
Actigard 2	Acibenzolar-S-methyl (50%) (Syngenta Crop Protection, Inc., Greensboro, NC, USA)

¹ Reference chemical against Phytophthora spp. ² Positive control for plant activators or resistance inductors.

. Ridomil Gold 480 EC was used as the standard fungicide control against P. capsici. A plant activator Actigard (Acibenzolar-S-methyl) was used as the positive control for the induction of host resistance.

2.2. In vitro Tests

2.2.1. Mycelial Growth

P. capsici isolates PPC1 and PPC6 were grown on 10% V8 agar (V8A; 100 mL of V8 juice, 1 g CaCO₃, 17 g agar, and 900 mL of distilled water) at 25 °C for 4 days. One 7 mm diameter plug with mycelia was transferred to potato dextrose agar (PDA) amended with phosphorous-acid-containing products at 0, 3.5, 17.5, 35, 85, 175, 350, 850, 1750, and 2600 µg mL⁻¹. Three replicate plates per treatment were tested, and the experiment was repeated twice. After 7 days of incubation at 25 °C in the dark, two perpendicular colony diameters were measured per plate. For each fungicide, the effective concentration at which mycelial growth is reduced by 50% (EC₅₀) was calculated by regressing mycelial relative growth values against the log of fungicide concentrations [25].

2.2.2. Zoospore Germination

Ten agar plugs (7 mm in diameter) from 4-day-old cultures of isolates PPC1 and PPC6 as described above were transferred to Petri dishes containing 10 mL of sterile 10% V8 juice and incubated for 24 h at 25 °C in the dark. The V8 juice was then pipetted out and rinsed once with sterile distilled water. Ten milliliters of non-sterile soil extract were then added to the Petri dish and incubated for 48 h at 25 °C under continuous light. Zoospore formation was induced by chilling at 4 °C for 20 min and, then, rewarming at room temperature for another 20 min. A 100 µL of zoospore suspension was plated onto PDA amended with phosphorous acid compounds at the above concentrations. Three plates were prepared for each concentration of each treatment, and the experiment was performed three times. The plates were kept at room temperature for 2 h before 100 encysted zoospores were randomly examined for germination at a magnification of 100× with an SZX16 Olympus stereo microscope. Germination for each treatment was determined by dividing the number of germinating zoospores by the total number of zoospores examined before multiplying by 100. EC₅₀ values representing the concentration of fungicides that inhibits the germination of P. capsici zoospores by 50% were obtained by linear regression of inhibitions against logarithmic fungicide concentrations [25].

2.2.3. Sporangium Formation

Isolates PPC1 and PPC6 were grown on V8A at 25 °C for 4 days, and three agar plugs (1 cm in diameter) were transferred to 6 cm Petri dishes containing 10 mL of sterile 10% V8 juice amended with phosphorous-acid-containing products at the above concentrations. Three plates were prepared for each concentration of each treatment and the experiment was repeated twice. The plates were incubated for 48 h at 25 °C under continuous light. The sporangia were counted at 100× with an SZX16 Olympus stereo microscope. Linear regression analysis of the relative sporangium values against logarithmic fungicide concentrations was used to estimate the concentration of fungicides that inhibits the formation of P. capsici sporangia by 50% (EC₅₀) [25].

2.3. Greenhouse Tests

2.3.1. Control of Phytophthora Blight of Bell Pepper by Phosphorous-Acid-Containing Products

The ability of phosphorous-acid-containing products to suppress Phytophthora blight of bell pepper in an artificially infested growing medium was examined. Seeds of bell pepper cv. Camelot (Seminis Vegetable Seeds, Inc., Oxnard, CA, USA) were planted in 128-cell flats filled with moisture control growing medium (Scotts Miracle-Gro Company, Marysville, OH, USA). Plants were maintained in the greenhouse at 25–28 °C for 2 months. Mycelial inoculum of P. capsici was prepared for growing mix infestation according to the method described by Sujkowski et al. [26] with some modifications. Fifteen agar plugs (5 mm in diameter) taken from the edge of 5-day-old V8A cultures of P. capsici isolate PPC1 were inoculated into each flask containing 500 mL of vermiculite and 250 mL of 10% V8 broth (with CaCO₃ 1 g L⁻¹). The cultures were incubated at 25 °C for 2 weeks. Plastic bags with 2058 mL of growing medium were prepared, and 42 mL of the mycelial inoculum and 315 mL of sterile distilled water were added accordingly. The growing medium and inoculum were thoroughly mixed to ensure uniform distribution of the inoculum. The infested growing media were treated with phosphorous-acid-containing products at concentrations of 0.25% and 0.10% (wt/wt). A healthy control was treated with the same volume of sterile V8 broth-vermiculite and distilled water. A non-treated control, which was inoculated with mycelium of P. capsici and treated with sterile distilled water, was included. An amount of 150 mL of treated growing medium was transferred onto each plastic tube (4 cm in diameter, 20.5 cm long). After that, a 2-month-old bell pepper seedling was then transplanted into each tube. Tubes were arranged in a randomized block design with four replications. A replicate for each treatment consisted of three tubes. Disease incidence representing the percentage of plants showing Phytophthora blight symptoms was recorded every five days until one month after transplanting. Plant height and shoot and root weights were measured after disease evaluation to evaluate the phytotoxic effect of fungicide on bell pepper seedlings.

2.3.2. Induction of Systemic Resistance in Bell Pepper by Phosphorous-Acid-Containing Products

The ability of phosphorous-acid-containing products to induce systemic resistance against Phytophthora leaf blight of bell pepper was studied. One-month-old bell pepper seedlings were transplanted to 10 cm diameter plastic pots and maintained in the greenhouse. Phosphorous acid products were applied at two concentrations (0.10% and 0.25%) to the growing medium as a drench (25 mL per pot) 12 and 5 days prior to inoculation. A zoospore suspension of P. capsici PPC1 was prepared as described previously. The concentration of zoospores was adjusted to 4 × 10⁴ mL⁻¹. Fifty microliters of zoospore suspension were used to inoculate the first fully expanded leaf of each tested seedling. Growing media treated with distilled water alone were used as healthy control, whereas growing media inoculated with zoospores of P. capsici PPC1 and treated with distilled water were used as non-treated control. All plants were then kept in a plastic chamber with 80–90% moisture for 12 h in a randomized block design. Each treatment was replicated four times, and each replicate contained three plants. The severity of Phytophthora blight was determined three days after inoculation based on a 0–5 disease severity rating scale described by Candole et al. [27] in which: 0 = symptomless, 1 = small circular or irregular spots on upper leaves, 2 = leaf enlarged symptoms with brownish lesions beginning to appear on stems and <25% of the plant wilted, 3 = leaves defoliated with lesions on leaves covering half of a leaf and 25–50% of the plant wilted moderately, 4 = leaves defoliated or dried, with rapidly expanding stem lesions and 50–70% of the plant wilted severely, and 5 = dead plant. Percent disease index was calculated at the end of the tests.

2.4. Statistical Analysis

Each experiment was repeated, and since data from all repetitions were homogeneous according to Levene’s test, they were pooled for analysis. Because all data sets met the conditions of normality and homogeneity of variances according to the Kolmogorov–Smirnov test, analysis of variance was performed using the general linear models procedure, and means were separated using Fisher’s least significant difference test (p = 0.05). All statistical analyses were performed in SAS 9.4 (SAS Institute, Cary, NC, USA).

3. Results

3.1. Effect of Phosphorous-Acid-Containing Products on Mycelium Growth, Zoospore Germination, and Sporangium Formation of P. capsici

Phosphorous-acid-containing products were less effective than Ridomil Gold 480 EC in inhibiting mycelial growth and sporangium formation of P. capsici (

Table 2. EC₅₀ values of different phosphorous-acid-containing products for Phytophthora capsici mycelial growth, sporangium formation, and zoospore cyst germination *.

Fungicides	EC50 (µg mL−1)
	PPC1	PPC6
Mycelial growth
Agri-Fos	139.4 ± 3.1	324.4 ± 14.6
Nutri-Phite	50.5 ± 1.9	94.5 ± 2.8
Lexx-A-Phos	246.4 ± 10.1	195.5 ± 5.9
K-Phite	112.4 ± 8.2	181.5 ± 12.6
ProPhyt	120.3 ± 6.1	164.2 ± 5.4
Ridomil Gold 480 EC	7.6 ± 0.6	13.8 ± 2.5
Zoospore germination
Agri-Fos	39.5 ± 2.4	13.9 ± 1.3
Nutri-Phite	49.1 ± 2.2	12.5 ± 4.5
Lexx-A-Phos	280.5 ± 8.6	58.9 ± 2.7
K-Phite	52.0 ± 2.9	27.9 ± 1.1
ProPhyt	61.1 ± 3.2	5.2 ± 0.6
Ridomil Gold 480 EC	>100	>240
Sporangium formation
Agri-Fos	149.4 ± 11.5	34.0 ± 1.2
Nutri-Phite	58.1 ± 4.5	12.1 ± 2.4
Lexx-A-Phos	127.2 ± 6.3	42.4 ± 1.5
K-Phite	217.9 ± 5.9	45.9 ± 1.6
ProPhyt	6.1 ± 0.7	225.7 ± 5.9
Ridomil Gold 480 EC	9.3 ± 1.2	2.3 ± 0.2

* Each treatment consisted of three replicates. All experiments were repeated twice and data from three repeats were pooled before analysis. Data are expressed as the mean of nine replicate plates ± standard deviation.

). The estimated EC₅₀ values for inhibition of mycelial growth of P. capsici were higher for the phosphorous-acid-containing products (ranged from 50.5 ± 1.9 to 246.4 ± 10.1 for PPC1 and from 94.5 ± 2.8 to 324.4 ± 14.6 for PPC6) compared to the EC₅₀ values of Ridomil Gold 480 EC (EC₅₀ = 7.6 ± 0.6 for PPC1 and EC₅₀ = 13.8 ± 2.5 µg mL⁻¹ for PPC6). Among the phosphorous acid products tested, Nutri-Phite exhibited the best inhibition of mycelial growth of PPC1 and PPC6, with EC₅₀ values of 50.5 ± 1.9 and 94.5 ± 2.8 µg mL⁻¹, respectively. Ridomil Gold 480 EC also had the lowest EC₅₀ value for inhibiting sporangium formation of P. capsici PPC6, whereas ProPhyt appeared to be the most effective product to inhibit the formation of PPC1 sporangia. Data in this table also show that Ridomil Gold 480 EC barely affected zoospore cyst germination of P. capsici. On the other hand, the intermediate inhibition effect of Agri-Fos, Nutri-Phite, K-Phite, and ProPhyt on the germination of P. capsici was recorded.

3.2. Application of Phosphorous-Acid-Containing Products as Drench in Artificially Infested Growing Medium to Suppress Phytophthora Blight

Data from both experiments that evaluated the effects of phosphorous-acid-containing products on the development of Phytophthora blight are shown in Figure 1. Among the phosphorous materials, the application of Nutri-Phite as drench at 0.25% was the most effective in terms of disease incidence reduction and was not significantly different from the healthy control and Ridomil Gold 480 EC control. Agri-Fos, K-Phite, and Lexx-A-Phos reduced the disease incidence, but the efficacy was not significantly different from the non-treated control.

3.3. Induction of Systemic Resistance in Bell Pepper against Phytophthora Blight by Phosphorous-Acid-Containing Products

The disease incidence of bell pepper treated with ProPhyt, K-Phite, and Nutri-Phite at both concentrations and Agri-Fos at a high concentration were significantly lower than the non-treated control and were not significantly different with the chemical activator Actigard. As shown in Figure 2, the application of these products considerably reduced the disease incidence from 83.3% in non-treated control to 38.9% or lower. Treatment of Lexx-A-Phos was not effective as a plant activator against Phytophthora blight.

3.4. Effect of Phosphorous-Acid-Containing Products on the Growth of Bell Pepper Seedlings

Plant height, fresh roots, and dry shoots and roots were not affected by the application of phosphorous-acid-containing products, indicating that they were not phytotoxic to pepper plants. On the other hand, a low concentration (0.10%) of ProPhyt and a high concentration (0.25%) of Agri-Fos and K-Phite reduced the fresh shoot weight compared with the healthy control (

Table 3. Effect of phosphorous-acid-containing products on plant height, fresh and dry shoots, and roots of bell pepper seedlings *.

Treatments	Concentration (%)	Height (cm)	Fresh Weight (g)		Dry Weight (g)
			Shoots	Roots	Shoots	Roots
Healthy control	0	21.7 ± 1.2 a	12.0 ± 0.4 a	6.3 ± 1.5 ab	1.7 ± 0.5 a	0.7 ± 0.1 bcd
Non-treated control	0	17.6 ± 1.3 a	9.6 ± 0.1 ab	5.6 ± 1.1 b	1.2 ± 0.2 ab	0.5 ± 0.1 d
Ridomil Gold 480 EC	0.10	16.8 ± 2.2 a	7.2 ± 0.5 b	4.5 ± 1.3 b	1.0 ± 0.3 ab	0.4 ± 0.1 d
ProPhyt	0.25	18.9 ± 1.9 a	8.5 ± 0.6 ab	6.7 ± 0.6 ab	1.5 ± 0.4 ab	0.7 ± 0.1 bcd
	0.10	17.1 ± 2.8 a	8.1 ± 0.2 b	5.8 ± 0.4 b	1.1 ± 0.3 ab	0.6 ± 0.1 abcd
Agri-Fos	0.25	21.6 ± 1.1 a	8.2 ± 0.3 b	8.2 ± 2.2 a	1.4 ± 0.2 ab	0.9 ± 0.2 a
	0.10	18.4 ± 0.8 a	9.0 ± 0.5 ab	5.9 ± 0.3 b	1.3 ± 0.1 ab	0.6 ± 0.1 abcd
K-Phite	0.25	20.3 ± 1.4 a	8.3 ± 0.1 b	7.0 ± 0.9 ab	1.4 ± 0.3 ab	0.7 ± 0.2 abc
	0.10	18.4 ± 2.5 a	8.7 ± 0.4 ab	5.8 ± 0.5 b	1.3 ± 0.1 ab	0.6 ± 0.1 bcd
Lexx-A-Phos	0.25	21.9 ± 1.5 a	9.5 ± 0.3 ab	6.4 ± 2.1 ab	1.6 ± 0.4 ab	0.8 ± 0.1 ab
	0.10	16.5 ± 0.8 a	8.7 ± 0.9 ab	4.9 ± 1.7 b	1.1 ± 0.3 ab	0.5 ± 0.1 d
Nutri-Phite	0.25	20.9 ± 2.1 a	10.7 ± 1.1 ab	5.4 ± 0.8 b	1.6 ± 0.5 ab	0.5 ± 0.1 d
	0.10	20.7 ± 0.8 a	10.3 ± 0.7 ab	6.1 ± 1.6 ab	1.6 ± 0.3 ab	0.7 ± 0.2 abcd

* Values followed by the same letter in a column are not significantly different according to Fisher’s least significant difference test (p = 0.05).

).

4. Discussion

Phosphorous acid had high efficacy against Phytophthora, with a reported EC₅₀ value for in vitro inhibition of mycelial growth at 2.5 to 5.4 µg mL⁻¹ against P. capsici [28]. However, the results of our in vitro tests demonstrated that phosphorous-acid-containing products were less effective against mycelial growth and sporangium formation. All of them reduced zoospore germination of P. capsica, whereas Ridomil Gold 480 EC was not effective against zoospores. The two most effective fungicides to inhibit mycelial growth and sporangium formation were Ridomil Gold 480 EC and Nutri-Phite. The difference in efficacy of phosphorous-acid-containing products and Ridomil Gold 480 EC on mycelial growth, sporangium formation, and zoospore germination suggests the possibility of combining these products to enhance their control effects towards P. capsici.

P. capsici is a heterothallic pathogen with two mating types, designated A1 and A2. Isolates belonging to different mating types are genetically distinct [29]. This might explain the differences in sensitivity of P. capsici isolates PPC1 (A1 mating type) and PPC6 (A2 mating type) to phosphorous acid fungicides. Whereas sporangium formation and zoospore germination of isolate PPC1 were less sensitive to these products, its mycelial growth was more impacted, as compared to those of isolate PPC6. These observations also highlight the need of identifying the mating types present in a field for optimal effects of these phosphorous-acid-containing products in the management of Phytophthora blight of bell pepper.

Nutri-Phite significantly reduced Phytophthora blight when it was used as a drench to artificially infested growing medium compared with non-treated control. This is in agreement with previous findings, which indicated that Nutri-Phite had additional benefits in reducing Phytophthora root rot [1]. The application of Nutri-Phite, Agri-Fos, ProPhyt, and K-Phite also provided significant systemic protection of bell pepper plants against P. capsici when the products were drenched to the root and growing medium prior to foliar inoculation. Whereas a high concentration of Agri-Fos was required to induce systemic resistance, dose-dependent responses were not demonstrated in treatments with Nutri-Phite, ProPhyt, and K-Phite. Guest and Bompeix [30] reported that Phosphite (salts of phosphonic acid) treatment induces a strong and rapid defense response in the challenged host plants. Phosphite exhibits a complex mode of action, acting directly on the pathogen and indirectly in stimulating host defense responses to stop the pathogen’s spread in the host and ultimately inhibit pathogen growth [22].

One of the first considerations of applying phosphorous-acid-containing products to a plant species is its phytotoxicity. Phosphite has been reported to cause foliar phytotoxicity in many crops [31,32,33]. Hence, the concentration of phosphorous-acid-containing products applied is important in agricultural production. In our study, there was no phytotoxicity observed on plants at the 0.10% application level. However, there was a significant reduction in fresh weight of shoots at 0.25% Agri-Fos and K-Phite.

Although this study was performed in the laboratory and greenhouse with one pepper cultivar, our results demonstrated the direct and indirect effects of the phosphorous-acid-containing chemicals against P. capsici. Results of this study need to be verified under field conditions. The results provide further evidence that these phosphorous-acid-containing chemicals are activators of the bell pepper plant’s defense systems against P. capsici, which can be taken into consideration in formulating large-scale management strategy studies in the field. The results also indicate that an application concentration of 0.25% Nutri-Phite, Agri-Fos, ProPhyt, and K-Phite could be used as a soil drench to evaluate the efficacy of these products against Phytophthora blight under field conditions.