Carrier-Based Application of RsPod1EGY Phage to Effective Control Potato Bacterial Wilt

Abstract

Bacterial wilt, caused by Ralstonia solanacearum, poses a significant threat to Solanaceae crops, including potatoes. Traditionally, chemical bactericides have been the primary method of disease management. However, the excessive and repeated use of these chemicals has led to the emergence of resistant R. solanacearum strains and raised environmental and safety concerns. Therefore, the current study aimed to explore bacteriophages as a sustainable and eco-friendly alternative to chemical control. Specifically, we evaluated the efficacy of the bacteriophage RsPod1EGY as a biocontrol agent under greenhouse conditions to reduce disease severity and maintain potato productivity. To improve phage survival and activity, we tested six carrier types; compost, peat moss, clay soil, sandy soil, talc powder, and wheat bran. Our findings indicated that compost, talc powder, and wheat bran significantly prolonged the phage’s viability and activity, reducing disease severity by 90%, compared to 65% and 85% for peat moss and phage-alone treatments respectively. In contrast, pathogen control treatments resulted in complete plant mortality. Phage survival was enhanced in pathogen-infected soils, supporting its role in reducing R. solanacearum populations. Potato yield was preserved in compost and talc powder treatments comparable to pathogen-free controls. These results underscore the potential of integrating bacteriophage RsPod1EGY with suitable carriers for sustainable bacterial wilt management, highlighting the importance of delivery methods in agricultural applications.

1. Introduction

Potato (Solanum tuberosum L.), is one of the most important crops in the Solanaceae family and is extensively cultivated in Egypt and worldwide [1]. According to FAOSTAT, global potato production reached 383.08 million tons in 2023, with Egypt contributing 6.87 million tons during the same year [2]. Despite its economic importance, potato production faces major challenges from plant diseases, including fungal, bacterial, and viral infections. Among these, bacterial wilt, or brown rot, caused by Ralstonia solanacearum, is particularly devastating. This disease severely impacts the quality and yield of potatoes, leading to greater losses than any other bacterial disease, with annual economic impacts exceeding $950 million [3,4].

R. solanacearum is a Gram-negative phytopathogenic bacterium with a broad host range, affecting over 200 plant species [5]. Recent taxonomic revisions have categorized Ralstonia into three species, R. solanacearum, R. pseudosolanacearum, and R. syzygii. These species differ in host specificity, geographic distribution, and epidemiology [6]. Ralstonia species primarily target the root xylem tissues, entering through small wounds before spreading to the stem. Once inside the plant, Ralstonia produces exopolysaccharides that obstruct the xylem vessels, disrupting water transport and causing wilting symptoms [7]. The onset of wilting is a clear indication of bacterial colonization in the xylem, rendering curative disease control measures ineffective [7].

Despite the economic importance of bacterial wilt and its widespread occurrence in tropical and subtropical regions, effective control strategies remain elusive [8,9]. Various management approaches, including crop rotation, chemical treatments, microbial antagonists, and resistant rootstocks, have been explored to mitigate the disease [10,11,12]. However, achieving consistent disease suppression and completely eradicating the pathogen from the soil remains a significant challenge [13]. This highlights the urgent need for innovative strategies to control bacterial wilt in Solanaceae crops.

Biological control offers a promising, environmentally friendly alternative for managing plant diseases. Researchers continue to investigate biocontrol agents, their mechanisms of action, and innovative biotechnological applications [14]. Among these, bacteriophages, viruses that specifically target and lyse bacterial pathogens, have emerged as a viable strategy for bacterial wilt management [15]. These phages have demonstrated effectiveness against several bacterial pathogens, such as Pseudomonas syringae, Xanthomonas campestris, X. axonopodis, Pectobacterium carotovorum, Dickeya solani, and Erwinia amylovora [8].

Lytic bacteriophages are particularly advantageous due to their host specificity, natural abundance, eco-friendly nature, and self-replicating capability, making them an effective and sustainable option for controlling bacterial plant pathogens [15]. A previous study successfully isolated the Ralstonia phage, RsPod1EGY (GenBank accession number MG711516) from Egyptian soil [16]. This phage effectively targeted R. solanacearum race 3, biovar 2, suppressing pathogen growth and reducing exopolysaccharides synthesis in tomato plants. However, maintaining the viability of phages in field conditions remains challenging due to environmental factors such as UV radiation, fluctuations in pH, temperature variations, and desiccation.

This study aimed to evaluate the efficacy of the bacteriophage RsPod1EGY in protecting potatoes against R. solanacearum under greenhouse conditions. The research focuses on assessing disease severity, pathogen suppression in soil, and phage longevity across different soil types used as carriers. Additionally, we investigated optimal application methods to enhance phage sustainability in the soil, both in vitro and under greenhouse conditions. Potato yield was measured post-treatment to determine the practical effectiveness of phage application. By improving bacteriophage persistence in the soil, this study seeks to enhance phage activity, reduce bacterial wilt incidence, and maintain potato productivity, offering an alternative to chemical-based disease management.

2. Materials and Methods

2.1. Soil Samples

The lifespan of phage in different carrier types was evaluated using soil samples from El-Gharbia Governorate, Egypt (30.8756° N, 31.0335° E, clay soil) and El-Behira Governorate, Egypt (31.0510° N, 30.4682° E, sandy soil). Compost and peat moss were procured in 10 kg packages from local agricultural supply stores in Giza, Egypt. Before use, all soil samples were tested to confirm the absence of R. solanacearum. Talc powder was sourced from medicinal suppliers in Giza, Egypt, while wheat bran was purchased from supermarkets in Giza. The physical and chemical properties of all soil carriers were analyzed at the Soil, Water, and Environment Research Institute (SWERI) in Giza, Egypt.

2.2. Soil Chemical Analysis

The chemical properties of the soils and carriers used in this study were analyzed to determine their nutrient composition. The pH, electrical conductivity (EC), and saturation percentage (SP) were measured for different soil types. The concentrations of major anions (HCO₃⁻, Cl⁻, SO₄²⁻) and cations (Ca²⁺, Mg²⁺, Na⁺, K⁺) were determined, along with the available macro- and micro-elements, including nitrogen (N), phosphorus (P), potassium (K), copper (Cu), iron (Fe), manganese (Mn), and zinc (Zn). The results of the chemical analysis are presented in

Table 1. Chemical constituents for different soil and carriers used in this investigation.

Carrier Type	pH	EC	SP	Anions (meq/L)				Cations (meq/L)				Available Micro- and Macro- Elements in Soil Sample (mg/Kg Soil)
				CO3=	HCO3−	Cl−	SO4=	Ca++	Mg++	Na+	K+	N	K	P	Cu	Fe	Mn	Zn
Compost	-	-	-	-	-	-	-	2.7	0.81	-	-	-	-	-	76.3	1.5	521	114
Peat moss	-	-	-	-	-	-	-	1.43	0.33	-	-	-	-	-	73.0	0.25	345	114
Sand	7.32	0.9	18.5	-	0.6	7.4	0.99	1.9	2.5	4.34	0.15	29.9	83.22	31.64	0.94	6.34	0.92	1.39
Clay	7.2	1.39	40.5	-	1.5	10.5	1.59	2.5	3.65	7.32	0.24	33.1	232.4	1.57	3.52	14.1	2.3	1.28

.

2.3. Potato Tubers

A potato cultivation experiment was conducted in a greenhouse using potato tubers (Spunta cv.) obtained from the Plant Pathology Research Institute, Viral Diseases Research Department in Giza, Egypt.

2.4. Bacterial Strains and Growth Conditions

In our earlier research [17], the R. solanacearum Phylotype IIa, sequevar 1 (race 3, biovar 2), strains K3 and K10, were isolated from potato tubers showing brown rot symptoms. These strains were characterized based on their cultural and morphological properties, and molecular identification [17]. A standard PCR assay, using specific primers for R. solanacearum race 3, biovar 2, confirmed the identity of each strain, producing a characteristic 718 bp band as reported by Pastrik et al. (2002) [18] Additionally, R. solanacearum K10 was previously sequenced by Elhalag et al. [19], and its sequence was deposited under accession numbers OR533690–OR533691. For long-term storage, the strains were preserved in 20% glycerol at –30 °C.

2.5. The Phage RsPod1EGY Used in This Study

The phage RsPod1EGY, previously isolated from Egyptian soil and stored at 4 °C, was continuously propagated using R. solanacearum strain K10, its primary host [17]. Phage multiplication was carried out in bacterial cell cultures grown in King’s medium, followed by precipitation and suspension in SM buffer. The lytic activity of the RsPod1EGY phage against R. solanacearum was confirmed on King’s medium surface using strains K3 and K10 as a host at a concentration of 10⁸ CFU/mL, including both the spot test [20], and the plaque assay [21].

2.6. RsPod1EGY Phage Survival in Different Carrier Types

This study assessed the longevity of the RsPod1EGY phage in vitro using six carriers: compost, peat moss, clay soil, sandy soil (all autoclaved at 121 °C for 30 min), talc powder, and wheat bran. Each carrier was inoculated with 2 mL of a 100-fold diluted phage solution (10⁹ PFU/mL) and mixed thoroughly. The carriers were incubated in 200 mL containers at room temperature with six replicates per carrier type. To assess the impact of the phage host (R. solanacearum) on phage survival, two sets of clay and sandy soils were prepared, one infected with the pathogen and one not. The pathogen-infected replicates were mixed with 5 mL of a bacterial suspension (10⁸ CFU/mL) before phage inoculation. The plaque-forming units (PFU) of RsPod1EGY were counted in each carrier type at 0, 3, 5, 10, 14, 21, 30, and 60 days post-inoculation. To analyze phage survival, 1000 mg samples from each carrier were suspended in 9 mL phosphate buffer (pH 7) and incubated in a shaker at 28 °C and 125 rpm for two hours. The suspension was centrifuged at 5000 rpm for 10 min, and the supernatant was filtered using a membrane filter. For plaque assays, 100 μL aliquots of the suspension were tested on King’s B plates with 2% agar and 0.75% soft agar, as described by [21]. Plates were incubated at 28 °C for 24 to 48 h, and plaques were quantified by serial dilution.

2.7. Evaluation of Phage Activity in Various Carrier Types in a Greenhouse Potato Pot Experiment

The in vivo evaluation was conducted in a greenhouse under natural conditions at the Plant Pathology Research Institute, Giza, Egypt. Forty plastic pots, each with a diameter of 20 cm and filled with 3 kg of clay soil, were infested with a 150 mL suspension of R. solanacearum, incubated for 24 h and adjusted to 1 × 10⁸ CFU/mL, resulting in approximately 8 × 10⁷ CFU/g of dry soil. These pots were divided into 10 treatment groups, each with four replicates. The treatments included: (Rs + compost), (Rs + compost + phage), (Rs + peat moss), (Rs + peat moss + phage), (Rs + talc powder), (Rs + talc powder + phage), (Rs + wheat bran powder), (Rs + wheat bran powder + phage), phage only (control), and R. solanacearum only (control). Additionally, four non-infected pots were designated as the water control. Phage application was carried out twice: initially at planting, when 100 g of each carrier type was inoculated with 10 mL of phage lysate at the same concentration, and again 14 days later as soil drenching at the same concentration. All pots were planted with potato tuber sprouts (Spunta cv.) and irrigated regularly.

2.8. Disease Assessments

To evaluate the disease severity (DS %), the wilt symptoms on the plant foliage were rated based on the scale outlined by [22,23] as follows: 0 = No symptoms; 1 = 1–25% wilt; 2 = 26–50% wilt; 3 = 51–75% wilt; 4 = 76–100% wilt; and 5 = Dead plants.

DS % was calculated using the formula:

$$\mathrm{DS} (\%)={\displaystyle \frac{\sum R.T}{5\times N}}\times 100$$

T = Total number of plants in each severity category; R = Disease rating; N = Total number of plants tested. Additionally, to calculate the percentage reduction in disease severity, the following formula was used:

$$\mathrm{Percent} \mathrm{Reduction} \left(\mathrm{PR}\right)={\displaystyle \frac{C-T}{C}}\times 100$$

C = Control T = treatment.

Disease severity (DS) and the populations of R. solanacearum and the phage were assessed at 0, 20, and 40 days post-soil inoculation, until the complete death of the pathogen in the control group. R. solanacearum counts were determined using SMSA media, and phage counts were obtained through a King’s plaque assay, employing a serial dilution technique. Additionally, the Area Under the Disease Progress Curve (AUDPC) was calculated as described by [24].

2.9. Potato Yield Calculation

At the end of the experiment, 80 days following the pathogen inoculation, the average fresh weighted (FW) potato yield was calculated for each pot and expressed as grams per pot.

2.10. Statistical Analysis

The Survival of RsPod1EGY phage in different carrier types was assessed using a split-plot design. In this design carrier types (treatments) were allocated to the main plots, while time points were assigned to the sub-plots. Analysis of variance (ANOVA) was performed to identify significant differences between the main effects of the carrier types (p_Treatments), time points (p_time), and the interaction between them (p_{Treatment × time}). To further investigate, Tukey’s honestly significant difference (HSD) test was applied to the interaction’s p-value (p_{Treatment × time} ≤ 0.05). Subsequent experiments were conducted using a completely randomized design (CRD). Data analysis was performed using ANOVA, and the HSD test (p < 0.05) was applied for post hoc analysis and pairwise comparisons, all conducted using JMP Data Analysis Software, version 14.

3. Results

3.1. Soil Chemical Properties

Table 1 presents the chemical composition of various soils and carriers, highlighting differences in pH, electrical conductivity (EC), ions, and nutrient content. Clay soil exhibits higher electrical conductivity (1.39 ms/cm) and better nutrient retention compared to sand. It contains greater levels of calcium, magnesium, sodium, and potassium. Additionally, clay has more nitrogen (33.1 mg/kg), potassium (232.4 mg/kg), and iron (14.1 mg/kg) than sand. While compost and peat moss do not provide complete ion data, they are rich in essential micronutrients such as copper, iron, manganese, and zinc, which can enhance soil fertility. These findings suggest that clay retains nutrients more effectively than sand and that compost and peat moss can improve phage survival in soil applications.

3.2. Lytic Activity of RsPod1EGY Phage Against R. solanacearum

The activity of RsPod1EGY was confirmed against both strains of R. solanacearum K3, and K10, using plaque and spot assays. Since both strains showed similar results, a representative image of strain K10 is presented. Clear zones indicating lytic activity were observed in both assays, appearing as plaques (Figure 1A) and halo spots (Figure 1B).

3.3. Survival of RsPod1EGY Phage in Different Carrier Types

Table 2. Effect of carrier types on the survival of RsPod1EGY phage (CFU/mL).

Treatment	Time from Soil Amendment (Days)								Mean ± SD
	0	3	5	10	14	21	30	60
Compost	7.91 ± 0.02 a	7.86 ± 0.01 a	7.83 ± 0.02 a	7.77 ± 0.01 a	7.76 ± 0.01 a	6.69 ± 0.02 b	5.61 ± 0.01 de	4.12 ± 0.04 gh	6.95 ± 1.39 a
Talc powder	7.92 ± 0.01 a	7.86 ± 0.02 a	7.81 ± 0.01 a	7.77 ± 0.01 a	7.75 ± 0.02 a	6.71 ± 0.02 b	5.62 ± 0.01 de	4.12 ± 0.06 gh	6.95 ± 1.41 a
Peat moss	7.91 ± 0.01 a	7.85 ± 0.01 a	7.54 ± 0.05 a	6.32 ± 0.05 bc	6.10 ± 0.09 bcd	4.00 ± 0.07 gh	1.53 ± 0.56 k	0.00 ± 0.00 l	5.15 ± 3.00 c
Clay soil	7.92 ± 0.01 a	7.85 ± 0.003 a	6.07 ± 0.05 bcd	4.87 ± 0.11 f	3.27 ± 0.03 i	2.58 ± 0.02 j	0.00 ± 0.00 l	0.00 ± 0.00 l	4.07 ± 3.16 d
Wheat bran	7.87 ± 0.03 a	7.82 ± 0.03 a	7.76 ± 0.002 a	7.72 ± 0.006 a	7.44 ± 0.04 a	5.66 ± 0.02 cde	4.55 ± 0.02 fg	0.57 ± 0.08 l	6.17 ± 2.57 b
Sandy soil	7.87 ± 0.01 a	6.34 ± 0.01 bc	5.03 ± 0.08 ef	3.68 ± 0.07 hi	1.64 ± 0.77 k	0.43 ± 0.39 l	0.00 ± 0.00 l	0.00 ± 0.00 l	3.12 ± 3.07 e
Time point (Mean ± SD)	7.90 ± 0.02 a	7.60 ± 0.61 b	7.01 ± 1.19 c	6.36 ± 1.75 d	5.66 ± 2.56 e	4.34 ± 2.55 f	2.88 ± 2.68 g	1.45 ± 2.07 h
PTreatment	<0.0001
Ptime	<0.0001
PTreatment x time	<0.0001

Different letters indicate significant differences between treatments according to Tukey’s HSD test at p < 0.05.

and Figure 2 offer complementary insights into the survival dynamics of RsPod1EGY under various conditions. As shown in Table 2, compost and talc maintained the highest phage viability measuring 6.95 log CFU/mL, followed by wheat bran at 6.17 log CFU/mL. In contrast, peat moss, clay soil, and sandy soil showed a significant decline, with phage counts in clay and sandy soil dropping to undetectable levels by day 60. The highly significant p values (<0.0001) confirm that both carrier type and time significantly influence phage survival. Figure 2 further illustrates the rapid decline of RsPod1EGY in clay and sandy soil, with a slight survival advantage in the presence of R. solanacearum. Statistical annotations indicate significant differences between time points, reinforcing the time-dependent nature of phage viability. These findings underscore the importance of selecting appropriate carrier materials for phage delivery.

3.4. Assessment of the Phage Activity Using Different Carrier Types Under Greenhouse Conditions

Phage treatments using different carriers were assessed in greenhouse conditions to control bacterial wilt disease in potato plants. Compost and talc powder significantly improved phage survivability and reduced disease severity by 90%. In comparison, wheat bran reduced severity by 85%, while peat moss and phages alone resulted in a 65% reduction (

Table 3. Impact of RsPod1EGY phage using different carrier on disease severity and disease progress of potato bacterial wilt under greenhouse conditions.

Treatment	Disease Severity%	DS Reduction (%)	AUDPC
C + RS C + RS + Ph	100 ± 0.02 a 10 ± 0.22 d	-- 90	1357.36 ± 40.04 a 55.00 ± 12.90 d
PM + RS PM + RS + Ph	100± 0.16 a 35 ± 0.50 b	-- 65	1328.87 ± 65.530 a 154.25 ± 17.80 bc
WB + RS WB + RS + Ph	100± 0.24 a 15 ± 0.28 c	-- 85	1374.88 ± 69.70 a 58.00 ± 9.120 cd
TP + RS TP + RS + Ph	100 ± 0.03 a 10 ± 0.02 d	-- 90	1335.27 ± 31.13 a 69.75 ± 10.34 cd
Ph	35± 0.04 b	65	177.0 ± 27.72 b
NC	0.0 ± 0.0 e	--	0.00 ± 0.00 d
PC	100 ± 0.06 a	--	1392.00 ± 39.00 a
	p < 0.0001	--	p < 0.0001

All values represent means of three replicates ± SD. C: Compost; PM: Peat moss; WB: Wheat bran; TP: Talc powder; Ph: Phage; NC: Negative control (No bacteria); PC: Positive control (bacteria); RS: R. solanacearum; (AUDPC): Area under disease progress curve. Different letters indicate significant differences between treatments according to Tukey’s HSD test at p < 0.05.

). Plants treated with phages exhibited minimal wilting and remained healthier, whereas untreated controls experienced severe yellowing, wilting, and ultimately death due to R. solanacearum infection (Figure 3).

3.5. Effect of Soil Amendments and Phage Application on Pathogen Population, and Phage Survival

Table 4. Impact of RsPod1EGY phage using carrier types on the Ralstonia solanacearum count under greenhouse conditions.

Treatment	LOG10 CFU RS in Soil (T0)	LOG10 CFU RS in Rhiz (T = 20)	LOG10 CFU RS in Rhiz (T = 40)	PCR for RS in Root (T = 80)
C + RS	7.81 ± 0.48 a	6.79 ± 0.33 b	6.61 ± 0.13 a	+
C + RS + Ph	7.85 ± 0.13 a	4.49 ± 0.30 d	1.93 ± 0.31 c	-
PM + RS	7.84 ± 0.15 a	6.71 ± 0.28 b	6.56 ± 0.29 a	+
PM + RS + Ph	7.87 ± 0.24 a	4.48 ± 0.42 d	3.04 ± 0.23 b	+
WB + RS	7.83 ± 0.02 a	7.75 ± 0.27 a	6.81 ± 0.48 a	+
WB + RS + Ph	7.81 ± 0.26 a	4.52 ± 0.12 d	2.86 ± 0.11 b	-
TP + RS	7.83 ± 0.02 a	7.84 ± 0.12 a	6.77 ± 0.16 a	+
TP + RS + Ph	7.82 ± 0.08 a	4.42 ± 0.09 d	1.13 ± 0.42 d	-
Ph	7.86 ± 0.62 a	5.30 ± 0.05 c	3.45 ± 0.04 b	+
NC	0.00 ± 0.00 b	0.00 ± 0.00 e	0.00 ± 0.00 e	-
PC	7.81 ± 0.01 a	7.87 ± 0.01 a	6.85 ± 0.14 a	+
	p < 0.0001	p < 0.0001	p < 0.0001

All values represent means of three replicates ± SD. C: Compost; PM: Peat moss; WB: Wheat bran; TP: Talc powder; Ph: Phage; NC: Negative control (No bacteria); PC: Positive control (bacteria); RS: R. solanacearum; (AUDPC): Area under disease progress curve. Different letters indicate significant differences between treatments according to Tukey’s HSD test at p < 0.05.

presents the effect of the RsPod1EGY phage, applied with different carrier types, on R. solanacearum counts under greenhouse conditions. The initial bacterial load in the soil (T = 0) remained consistent across all treatments. By T = 20 and T = 40, treatments incorporating bacteriophages significantly reduced the CFU levels of R. solanacearum compared to their respective non-phage controls. Among the tested carriers, talc powder mixed with phage exhibited the greatest bacterial suppression, followed closely by compost with phage. Since the bacterial count became too low for direct quantification in some treatments, PCR analysis was used to detect the presence of R. solanacearum in root samples at T = 80. The results indicate that the pathogen was still detectable in treatments without phage application while no amplification was observed in treatments where RsPod1EGY was applied. Furthermore, plaque-forming unit (PFU) analysis over time demonstrated improved phage survival in the soil when combined with compost, talc powder, and wheat bran (

Table 5. Effect of different carriers on the longevity and activity of the RsPod1EGY phage to control potato bacterial wilt under greenhouse conditions.

Treatment	LOG10 PFU Phage in Soil (T = 0)	LOG10 PFU Phage in Rhizo (T = 20)	LOG10 PFU Phage in Rhizo (T = 40)
C + RS	0.00 ± 0.00 b	0.00 ± 0.00 b	0.00 ± 0.00 f
C + RS + Ph	7.81 ± 0.17 a	6.83 ± 0.49 a	4.33 ± 0.29 b
PM + RS	0.00 ± 0.00 b	0.00 ± 0.00 b	0.00 ± 0.00 f
PM + RS + Ph	7.83 ± 0.22 a	6.73 ± 0.31 a	1.76 ± 0.38 d
WB + RS	0.00 ± 0.00 b	0.00 ± 0.00 b	0.00 ± 0.00 f
WB + RS + Ph	7.80 ± 0.29 a	6.81 ± 0.65 a	3.35 ± 0.13 c
TP + RS	0.00 ± 0.00 b	0.00 ± 0.00 b	0.00 ± 0.00 f
TP + RS + Ph	7.79 ± 0.37 a	6.77 ± 0.82 a	5.68 ± 0.07 a
Ph	7.81 ± 0.21 a	5.78 ± 0.81 a	0.78 ± 0.26 e
NC	0.00 ± 0.00 b	0.00 ± 0.00 b	0.00 ± 0.00 f
PC	0.00 ± 0.00 b	0.00 ± 0.00 b	0.00 ± 0.00 f
	p < 0.0001	p < 0.0001	p < 0.0001

All values represent means of three replicates ±SD. C: Compost; PM: Peat moss; WB: Wheat bran; TP: Talc powder; Ph: Phage; NC: Negative control (No bacteria); PC: Positive control (bacteria); RS: R. solanacearum; (AUDPC): Area under disease progress curve. Different letters indicate significant differences between treatments according to Tukey’s HSD test at p < 0.05.

).

3.6. Impact of Phage and Soil Amendments on Potato Yield

Figure 4 shows the effect of RsPod1EGY phage with different carriers on potato yield under greenhouse conditions. R. solanacearum infection significantly reduced yield in untreated groups, while phage treatments improved yield across all carriers. Talc powder with phage and compost with phage showed the highest yields, comparable to the negative control. Wheat bran with phage also enhanced yield, while peat moss with phage had a moderate effect. The significant p-value (<0.0001) confirms the phage’s effectiveness in reducing yield losses, with carrier type influencing its impact.

4. Discussion

The potato production sector in Egypt faces significant challenges due to bacterial wilt caused by R. solanacearum, which threatens crop yields and export markets [23]. Bacteriophages offer a promising, eco-friendly alternative to chemical bactericides and have the potential for sustainable disease management [25]. However, the field application of phages is limited by environmental factors such as desiccation, UV radiation, pH, and temperature fluctuations, which affect their survival [26].

This study evaluated the activity and longevity of bacteriophage RsPod1EGY in six different carriers: compost, peat moss, clay soil, sandy soil, talc powder, and wheat bran. The survival of the phage was monitored over 60 days following soil amendment. RsPod1EGY remained active in all the carriers, but its longevity varied. Compost, talc, and wheat bran were the most effective at supporting phage survival for the full 60 days, although a decline in viability began after 21 days. In contrast, the phage’s viability dropped significantly within 3, 5, and 10 days in peat moss, clay soil, and sandy soil, respectively. Our findings are consistent with previous studies, indicating that phages persisted in the rhizosphere and plant roots, even though there was a 10- to 100-fold reduction over 14 days [27,28,29].

The strong protein-binding properties of humic materials in compost may enhance phage stability. Additionally, clay soil promotes phage longevity compared to sandy soil, highlighting the role of soil composition in facilitating phage-bacteria interactions [29]. Previous studies have highlighted the effectiveness of phages in controlling diseases. Kalpage and De Costa (2015) demonstrated that phages can significantly reduce the impact of pathogens [30]. Additionally, Wang et al. (2019) reported an 80% decrease in the incidence of R. solanacearum disease in tomato plants after phage treatment [31]. The survival of phages relies on the presence of host bacteria, with survival rates observed between 0.2 × 10³ to 3.5 × 10⁴ PFU/g of soil 15 days after application [31]. These findings highlight the potential of phage-based biocontrol methods in agriculture, particularly in favorable soil conditions.

Greenhouse experiments evaluating phage efficacy in controlling bacterial wilt in potatoes revealed that compost and talc powder enhanced phage survival and effectiveness compared to peat moss and phage-only treatments. Compost amendments have been shown to suppress soil-borne pathogens while promoting viral communities beneficial for disease control [32,33]. Additionally, combining phages with the organic compound Acibenzolar-S-methyl (ASM) has improved bacterial disease management in tomato plants [34]. Moreover, the disease severity reduction varied among treatments, with wheat bran, peat moss, and phage alone reducing severity by 85%, 65%, and 65%, respectively. Compost and talc powder were the most effective, reducing severity by 90%, consistent with prior studies demonstrating phage-mediated disease suppression in tomatoes and tobacco [30,35]. Similar findings were reported in field trials, where phage treatments reduced bacterial spot incidence by 10–20% [36]. However, challenges remain in optimizing phage delivery, as soil adsorption and UV inactivation can hinder effectiveness [37].

The integration of RsPod1EGY with compost, talc powder, and wheat bran significantly reduced R. solanacearum populations in soil, supporting previous research on phage-carrier formulations for plant disease control [38]. Wheat bran, in particular, offers advantages in ease of handling, shelf life, and compatibility with agrochemicals, making it an ideal carrier under greenhouse conditions. In contrast, pathogen populations remained high in untreated controls, demonstrating the necessity of phage application for effective disease suppression [39,40].

Talc powder significantly enhanced RsPod1EGY stability, likely due to its mineral composition, which has been previously validated as an effective carrier for bacterial inoculants [41,42]. Talc-based formulations have successfully controlled plant pathogens, including Alternaria solani in tomatoes and Rhizoctonia solani in rice [43,44].

Additionally, soil-applied phage cocktails have effectively managed soft rot pathogens such as Pectobacterium atrosepticum and P. carotovorum [45]. While RsPod1EGY has shown effectiveness in controlling bacterial wilt, further research is needed to examine the advantages of using phage mixtures to enhance disease management. Previous studies have indicated that phage mixtures can improve effectiveness against Ralstonia solanacearum. For instance, a mixture of two Podoviridae J2 and ФRSB2 bacteriophages effectively lysed R. solanacearum cells in contaminated soil, while treatment with J2 alone prevented disease in tomato plants [46].

Phage combination treatments effectively reduce the incidence of Ralstonia wilt disease in tomato plants by 80% in both greenhouse and field conditions [47]. These biocontrol strategies utilize either highly stable single-phage treatments with strong lytic activity or phage cocktails designed to enhance their persistence and manage bacterial resistance. Furthermore, mixtures of different bacteriophages reduced the incidence of bacterial spots caused by Xanthomonas campestris pv. vesicatoria by 9% to 20%, while also helping to mitigate bacterial resistance to phages [36].

Despite these promising results, optimizing phage delivery and stability in field conditions remains challenging. Soil type, UV exposure, and limited adsorption to clay can impact efficacy. However, using compost and talc as carriers enhanced phage performance, improving wilt suppression and potato yields. These findings highlight the potential of phage-based biocontrol as a sustainable strategy against R. solanacearum. Future research should focus on field validation, combination treatments, and mitigating phage resistance.

5. Conclusions

The present study highlights the promising potential of the bacteriophage RsPod1EGY as an effective biocontrol agent against bacterial wilt caused by R. solanacearum under greenhouse conditions. Using compost and talc powder as carriers significantly improved phage stability, activity, and disease suppression. When applied with these carriers, phage treatments reduced bacterial wilt severity by up to 90% while preserving potato yield. These results underscore the potential of phage-based strategies as a sustainable alternative for managing bacterial diseases in agriculture. However, additional field trials and direct comparisons with conventional control methods are essential to fully evaluate the practicality and long-term effectiveness of phage-based biocontrol solutions.