Pomegranate wilt, Soil-borne pathogens, Ceratocystis fimbriata,  Fusarium oxysporum, Fungicide efficacy. 

Pomegranate (Punica granatum L.) is a deciduous, multi-stemmed shrub or small tree reaching 5-8 m in height, cultivated for its fruits valued for both nutritional and therapeutic properties, being rich in carbohydrates and essential minerals such as calcium, iron, and sulfur (Palou and Del-Rio 2009).  Arid and semi-arid regions with 500-1000 mm of annual rainfall, hot and dry summers, and mild winters are ideal for pomegranate cultivation, as the crop requires a warm, dry climate during the fruit ripening stage. The plant thrives well in medium-deep, loamy, well-drained soils with a pH of 7.5 and prefers temperature ranging from 23 to 32 C. It is extensively grown in Asia, the Middle East and the Mediterranean region with major producers including India, Iran, China, Turkey, Afghanistan, Egypt, and Spain. In India, pomegranate cultivation is primarily concentrated in Maharashtra, Gujarat, Karnataka, and Andhra Pradesh. In contrast, Himachal Pradesh accounts for a smaller share, with cultivation spread over 2,880 hectares and an annual production of 3,271 metric tons of fruit (Anonymous, 2021a; 2021b). Its cultivation has faced increasing challenges due to a combination of biotic and abiotic stresses affecting its aerial and root systems. Foliar pathogens mainly affect photosynthesis and diminish the fruit quality and market value, whereas soil-borne pathogens pose a more serious threat by causing complete plant death. Wilt is a major soil-borne disease that causes yellowing and drying of pomegranate plant. This disease has been reported in several countries, including Armenia, North America, France (Ferrari and Pechenot 1974), Slovakia, Switzerland (Matasci and Gessler 1997), Italy (Panconesi, 1999), India (Somasekhara, 1999), China (Huang et al., 2003), and Pakistan (Alam et al., 2017). In Himachal Pradesh, it has previously been documented in the districts of Bilaspur, Kullu, Mandi, and Hamirpur affecting pomegranate orchards (Khosla et al., 2011).

This disease threatens the economic stability of farmers worldwide because of the drying of fruits, flowers, and leaves that causes complete reduction of yield from infested plants. Several pathogens have been identified to be associated with this disease, including Ceratocystis fimbriata, Fusarium oxysporum, Fusarium solani, Rhizoctonia spp., and Meloidogyne incognita (Sharma et al., 2010). However, this study specifically focuses on assessment of fungicide efficacy for managing C. fimbriata and F. oxysporum. Studies related to M. incognita have been published separately and are therefore not discussed in detail in this publication (Mukesh et al., 2024). C. fimbriata and F. oxysporum commonly enter through root injuries caused by intercultural operations. C. fimbriata invades xylem and phloem, secreting enzymes like cellulases and pectinases that degrade cell walls. F. oxysporum enters through root wounds and spreads within the xylem, where its hyphae and spores, along with host responses like tyloses and gum deposition, block water flow, leading to wilt symptoms development (Yadeta and Thomma 2013). C. fimbriata produces endoconidia, aleuroconidia, and chlamydospores, while F. oxysporum forms macroconidia, microconidia, and chlamydospores that enable both pathogens to persist in soil for long periods in the absence of a host (Nasution et al., 2019; Haware et al., 1996).

The persistence of soil-borne pathogens and their spread through irrigation water and infected plant material make their management particularly difficult. These diseases pose a serious threat to the sustainability and profitability of pomegranate orchards, especially in monoculture systems with inadequate disease control. Although biological control methods (bioagents and soil amendments) offer sustainable options when used preventively, they are generally ineffective against active infections under field conditions. In contrast, fungicides provide more reliable control of ongoing infections. Systemic fungicides are absorbed and translocated within the plant, offering internal protection, while non-systemic fungicides act externally through contact. Combination fungicides, which merge both properties, offer broader-spectrum defense. Fungicides act by inhibiting pathogen growth or interfering with microbial enzyme activity in the soil (Kenarova and Boteva 2023). However, excessive use, particularly of triazole-based compounds, may negatively impact beneficial soil microbes and enzymatic processes (Roman et al., 2021). The effectiveness of fungicides against C. fimbriata and F. oxysporum depends on their mode of action, application timing, environmental conditions, and pathogen resistance. Field management remains challenging due to variable weather, soil characteristics, and evolving pathogen resistance. This study, therefore, evaluated the performance of systemic, non-systemic, and combination fungicides under in vitro, pot, and field conditions to identify the most effective treatments for reducing fungal load in wilt-infested pomegranate tree basin soil.

The study was conducted at the department of Plant Pathology, Dr YS Parmer University of Horticulture and Forestry Nauni, Solan, Himachal Pradesh, India.

A. Isolation and identification of C. fimbriata and F. oxysporum

Infected root samples were chopped surface sterilized, and plated on Potato Dextrose Agar (PDA). Rhizospheric soil samples (100 g each) were collected from the basins of wilt-affected pomegranate trees at a depth of 10–25 cm and pooled to form a composite sample. Fungal populations were assessed using the serial dilution plate method. One gram of composite soil was suspended in 9 ml sterile distilled water (10⁻¹ dilution), followed by serial dilutions. From each dilution, 0.1 ml was plated onto selective media. C. fimbriata was isolated on V8 juice agar (200 ml V8 juice, 3 g CaCO₃, 20 g agar per liter), and F. oxysporum on Malachite Green Agar (2.5 ppm) containing 15 g peptone, 1 g KH₂PO₄, 0.5 g MgSO₄·7H₂O, 20 g agar, and 0.0025 g malachite green oxalate per liter (Jeschke et al., 1990; Bragulat et al., 2004). Plates were incubated at 25 °C and emerging colonies were purified on PDA medium. The morphological characteristics of C. fimbriata and F. oxysporum were examined by preparing slides from mature cultures and observing spore and conidial structures under a microscope at 40× magnification (Windels, 1991). Identification was carried out using a taxonomic key and confirmed by comparing measurements with descriptions from previous studies (Hunt, 1956; Leslie and Summerell 2006).

B. Pathogenicity test

Pathogenicity test was conducted on healthy pomegranate seedlings (cv. Kandhari Kabuli) grown in plastic pots under greenhouse conditions at the Department of Plant Pathology. C. fimbriata culture was mass-multiplied on wheat bran, and F. oxysporum on maize seed medium. For culture preparation, wheat and maize seeds were boiled until softened. Excess water was drained, and the seeds were mixed with sawdust (for wheat) or sand (for maize) in a 1:3 ratio (sawdust/sand: seed) to prevent clumping. The mixtures were sterilized using the tyndallization method with three consecutive days of heating at 100°C in an autoclave for 15 minutes at 5 psi pressure (Negi and Gautam 2013). After sterilization, the media were inoculated with actively growing both fungal cultures and incubated at 25°C for 30 days to allow for proliferation. Each  pathogens inoculums was applied at a rate of 2 g/kg soil, totaling 10 g inoculum per pot, placed near the seedling roots (Chaudhary et al., 2017). To enhance infection, small cuts were made on the root surface. Pots were regularly irrigated to maintain optimal moisture. Disease development was monitored, and the number of days until first symptom appearance was recorded. Each pathogen was re-isolated from the infected roots of newly infested plants onto PDA and identified by comparing its cultural and morphological characteristics with those of the original isolates (Agrios, 2005).

C. In-vitro evaluation of various fungicides against mycelial growth of C. fimbriata and F. oxysporum

Various systemic fungicides (difenoconazole 25%EC, propiconazole 25% EC, thiophanate methyl 70% WP and fosetyl-Al 80% WP) were evaluated at 100,200 and 300ppm concentration and non systemic fungicides (chlorothalonil WP and copper oxychloride 50 WP) were tested at 250,500,1000ppm while the combi fungicide (metiram+pyraclostrobin 60 WG and carbendazim 12% +mancozeb 63% WP) were tested at 100,250, 500ppm concentration against mycelial growth of C. fimbriata and F. oxysporum using the poison food technique described by Nene and Thapliyal (1993). Each fungicide was prepared at double the desired concentration by dissolving it in 50 ml of sterile distilled water. This solution was added to 50 ml of melted double-strength PDA, thoroughly mixed, and poured into Petri plates containing a pinch of streptocycline (Hindustan Antibiotic Ltd.). For the control treatment, 50 ml of sterile distilled water was mixed with 50 ml of double-strength PDA without fungicide. A 5 mm disc of each test fungus (C. fimbriata and F. oxysporum) was placed in the center of each plate with poisoned medium at different concentrations for all treatments and incubated at 25°C. Eight fungicides with a control treatments were tested and replicated thrice for each fungus, the experiment was laid out using completely randomized design (CRD) under laboratory conditions. When fungal growth in the control treatment reached the edge (90 mm), the mycelial growth in the fungicide-treated plates was measured and per cent growth inhibition was calculated by using the formula given by Vincent (1947). 

D. Evaluation of fungicides against C. fimbriata and F. oxysporum colonies cfu/ g soil under pot conditions

This experiment was conducted on pots containing pomegranate seedlings maintained prior to the study in the greenhouse during 2019 and 2021. During year 2019, the pots containing the healthy pomegranate seedlings inoculated with a mass multiplied inoculum of C. fimbriata and F. oxysporum @ 2g/kg soil as mentioned earlier in text (Chaudhari et al., 2017). After the establishment of disease symptoms or partial wilting of pomegranate seedlings soil samples were taken from each treatment for counting of pathogens colonies/g soil under laboratory conditions. Each treatment was then drenched twice at 15 days interval with fungicide solution with 2g or 2ml dosage (1litre/pot) while the control treatment were maintained with application water without any fungicide drenching on it.  For consistency across treatments, colony counts were recorded exclusively from plates corresponding to the 10⁻⁴ dilution, as this dilution consistently provided well-isolated and countable colonies. The fungal population was expressed as colony-forming units per gram of soil (cfu/g), calculated using the following formula:

The soil samples were taken again after 30 days of 2nd drenching to count the number of colonies of each fungus cfu/g soil in each treatment. The experiment were repeated in year 2021 by following the similar method and the average data on number of colonies on each treatment before and after drenching of fungicides from both year (2019 and 2021) were pooled further analyzed using statistical analysis. A significant reduction in fungal colony counts was observed in amended treatments compared to the control. The percent reduction over control was calculated using the formula:

E. Evaluation of fungicides against C. fimbriata and F. oxysporum colonies cfu/ g soil under field conditions 

The efficacy of fungicides was evaluated in a wilt-infested pomegranate orchard at the University Model Farm during 2019 and 2021. During year 2019, partially wilted pomegranate trees were tagged for treatment, and the impact of fungicides on C. fimbriata and F. oxysporum (cfu/g soil) was assessed to determine their effectiveness in managing the disease under field conditions. The soil samples were taken from each treatment before fungicide drenching for counting of pathogens colonies in infested soil in each treatment. Fungal colonies in soil were counted through serial dilution method on specific medium as mentioned earlier. Each plant in treatment were drenched at the rate of 2g or ml/L dose where 10-15 liters of fungicide solution were drenched twice at 15 days interval on each replication according to vigour of the plant and a separate control treatment were maintained without drenching any fungicides on it. After 30 days of 2nd (second) drenching the soil samples were taken again from each treatment and the colonies of each fungal pathogen were again counted on specific medium for each. A Randomized Block Design (RBD) was used for the field experiment with three replications, while fungal colonies under in vitro conditions were counted using the serial dilution method following a Completely Randomized Design (CRD), with each treatment replicated three times. The experiment was repeated during the year 2021 by following the similar methodology. The average data on number of colonies on each treatment before and after drenching of fungicides from both year (2019 and 2021) were pooled and further analyzed using statistical tools.

F. Statistical analysis

The statistical analysis of the data collected from various experiments was performed using RStudio software (Field et al., 2013). In vitro data on mycelial growth inhibition of C. fimbriata and F. oxysporum were analyzed using two-way ANOVA, with fungicide and concentration as factors. Pot and field experiment data were log₁₀-transformed to normalize colony-forming unit (CFU) counts. Tukey's HSD was applied for multiple comparisons (Tukey, 1949). Two-way ANOVA was conducted to assess the effects of treatment, time, and their interaction. The Shapiro-Wilk test was used to evaluate the normality of residuals, while Levene’s test assessed the homogeneity of variances (Shapiro and Wilk, 1965). All analyses were carried out using R packages rstatix (for assumption tests and ANOVA), car (for model diagnostics), agricolae (for Tukey’s HSD test), and ggplot2 (for visualizations). Significant treatment effects were detected (p < 0.05) and visualizations were created using the ggplot2 package (Wickham, 2011). 

A. Disease symptoms 

The disease was typically observed in patches of 5–10 infested plants in an orchard, with symptoms becoming noticeable rainy season. Hot and dry summer followed by continuous rainfall, were found to be highly conducive to the development and spread of the disease. Both above- and below-ground symptoms were noticed. Above-ground symptoms included yellowing and drying of leaves, which eventually led to plant wilting. Initially, the yellowing was localized to a single branch or one side of the canopy and gradually spread to other parts of the plant with time (Fig. 1a) resulting in complete wilting or drying of full bearing pomegranate plant (Fig. 1b). The disease progression was observed more rapid in orchards with poor management practices and waterlogged basin soil that results in plant mortality in rapid succession, particularly under suboptimal management conditions. Infected plants also exhibited drying of fruit, flowers, and buds, significantly reducing productivity and overall plant health. Stems of infected plants showed distinct blue or black staining (Fig. 1c; 1d), while roots displayed notable changes, including discoloration of vascular tissues (Fig. 1e) and drying of xylem vessels (Fig. 1f). Pathogen isolation under laboratory conditions confirmed the presence of F. oxysporum and C. fimbriata being associated with these symptomatic roots and stems. 

Fig. 1. Symptom of pomegranate wilt disease under field conditions; a: yellowing of leaves; b: drying of  complete plant ; c;d: staining of vascular tissue of stem;  e: staining of vascular tissue of roots; f: drying of root xylem vessels.

B. Pathogen Identification 

Two fungal pathogens were consistently isolated from infected roots and stems, with C. fimbriata associated to vascular staining of roots and stem, and F. oxysporum linked to drying of xylem tissue of roots.

(i) C. fimbriata. The culture of C. fimbriata exhibited a grayish-white coloration with a distinct growth pattern on PDA medium and displayed a dense, single growth structure with a light brown center surrounded by grayish-white growth along the rim, characterized by pinhead-like structures and an undulating border (Fig. 2). The colony margins ranged from regular to irregular, and complete mycelial growth (90 mm) was achieved after 18 days of incubation at 25°C. The growth pattern was observed dense and mat-like, contributing to the colony’s grayish-white appearance. Microscopic examination revealed that the mycelium of C. fimbriata consisted of septate, branched hyphae that were hyaline when young but darkened with age. Morphologically, the fungus produced oval, olive-brown endoconidia while the aleuroconidia were smaller, truncate at the base, and golden-brown (Fig. 2). The presence of black to dark-brown perithecia was observed (Fig. 4), and the ascospores were hyaline and hat-shaped.

(ii) F. oxysporum. The culture of F. oxysporum on PDA medium initially exhibited whitish mycelial growth, which later developed pale purple to reddish-pink pigments, accompanied by fluffy aerial hyphae and discrete orange sporodochia (Fig. 2). The colony margins were observed from regular to irregular, reaching a diameter of approximately 90 mm after 12 days of incubation at 25°C. Microscopic examination revealed septate, hyaline (non-pigmented), and branched mycelium. The fungus produced a large number of macroconidia and microconidia. Macroconidia, predominantly formed in sporodochia, varied in size and shape, ranging from sickle-shaped to slightly or strongly curved, with 3–5 septa (Fig. 2) while the microconidia were observed oval, elliptical, reniform, or allantoid in shape. Chlamydospores were observed as thick-walled, round to oval in shape, found singly, in pairs, or occasionally in short chains, and formed either intercalary within the hyphae or terminally at their ends. 

Fig. 2. Cultural and spore characteristics of C. fimbriata (aleuroconidia, endoconidia) and F. oxysporum (macroconidia, microconidia) on PDA.

C. Pathogenicity test

The pathogenicity test confirmed the virulence of both pathogens isolated from wilt-affected pomegranate plants. Under pot conditions, C. fimbriata showed a shorter incubation period, with leaf yellowing observed around 17 days after inoculation (DAI) and complete plant mortality by 36 DAI. In comparison, F. oxysporum caused symptoms more slowly, with yellowing at an average of 22 DAI and complete wilting by 38 DAI. When both pathogens were co-inoculated, disease progression accelerated, with complete wilting occurring by 35 DAI, suggesting a possible synergistic interaction that intensified disease severity. Distinct pathogenic mechanisms were observed: C. fimbriata caused vascular discoloration with blue-black streaks in roots, while F. oxysporum led to xylem drying and disruption of water transport. Re-isolation and morphological confirmation of both fungi from symptomatic tissues validated their role as the causal agents. The faster progression of symptoms in plants inoculated with both pathogens highlights the potential for enhanced disease development under co-infection conditions poses a significant challenge to disease management and necessitate a deeper understanding of their synergistic effects.

D. In vitro evaluation of fungicides against mycelial growth of C. fimbriata 

The in vitro evaluation of fungicides against the mycelial growth of C. fimbriata revealed significant differences in efficacy, influenced by the mode of action and concentration. Among the systemic fungicides, Propiconazole 25% EC demonstrated the highest average mycelial inhibition at all concentrations (100, 200, and 300 ppm), with an inhibition rate of 94.44% at 300 ppm (Fig. 3) formed a distinct Tukey group (a), indicating its superior efficacy closely followed by the combi-fungicide Carbendazim 12% + Mancozeb 63% WP, and Difenoconazole 25% EC, with average inhibition rates of 89.07% and 88.33% at 500ppm, respectively placed in group under Tukey group (ab), showing significant control over the pathogen (Table 1). 

Fig. 3. In vitro inhibition of mycelial growth of C. fimbriata  by Difenoconazole 25% EC, Propiconazole 25% EC, Carbendazim + Mancozeb WP and Copper Oxychloride 50 WP at different concentrations.

Thiophanate methyl 70% WP exhibited moderate efficacy with an average inhibition of 75.37%, also belonging to Tukey group (ab). Non-systemic fungicides, including Chlorothalonil WP, and Copper oxychloride 50% WP, showed lower inhibition rates, ranging from 70.62% to 71.66% at 1000ppm, and formed Tukey group (c), indicating their comparatively moderate performance. Fosetyl-Al 80% WP and Metiram + Pyraclostrobin 60 WG exhibited a similar inhibition rate to the non-systemic fungicides. The untreated control showed no inhibition, forming a distinct Tukey group (d). Fig. 5 depicted mycelial growth inhibition (%) by fungicides against C. Fimbriata. The ANOVA revealed significant effects of both fungicide type and concentration on the mycelial growth of C. fimbriata under in vitro conditions (Table S1, Appendix-I, supplementary data). The effect of fungicides was highly significant (F = 102.39, p < 0.001), indicating substantial differences among the treatments in suppressing fungal growth. Likewise, concentration also had a significant effect suggesting that the efficacy of fungicides varied depending on their dosage. The low residual mean square further supports the consistency of the treatment effects.

E.  In vitro evaluation of fungicides against mycelial growth of F. oxysporum 

The in vitro evaluation of fungicides against the mycelial growth of F. oxysporum revealed significant variations in efficacy depending on the fungicide type and concentration (Table 1).  Among the treatments, Carbendazim 12% + Mancozeb 63% WP demonstrated the highest average inhibition (81.85%) at all three fungicide concentration tested and was classified under Tukey group (a), indicating its superior performance in controlling the pathogen (Fig. 4). Propiconazole 25% EC also exhibited strong inhibition with an average value of 72.78% and was grouped under Tukey group (ab). Other systemic fungicides, including Difenoconazole 25% EC and Thiophanate methyl 70% WP, showed moderate efficacy, with average inhibition rates of 61.36% and 57.22%, respectively, forming Tukey groups (bc and bcd). Non-systemic fungicides including Chlorothalonil WP and Copper oxychloride 50 WP, exhibited moderate to low inhibition, with averages of 48.46% and 39.38%, respectively, and were categorized under Tukey groups (cde and de). Metiram + 60 WG exhibited the lowest inhibition among the fungicides tested (36.05%, e) and was comparable to the non-systemic treatments.  Figure 5 depicted nhibition of mycelial growth inhibition (%) by fungicides against F. Oxysporum. The untreated control treatment revealed  no inhibition (f), emphasizing the necessity of fungicide application in reducing the mycelial growth of F. oxysporum. The ANOVA demonstrated both fungicide and concentration effects were also highly significant (Table S1, Appendix-I, supplementary data). The fungicide factor showed a strong influence on fungal inhibition (F = 44.45, p < 0.001), demonstrating notable variation in efficacy among the tested fungicides. Similarly, the effect of concentration was significant, indicating dose-dependent suppression of mycelial growth. The residual variance was slightly higher than that observed for C. fimbriata, suggesting relatively more variability in response.

Fig. 4. In vitro inhibition of mycelial growth of F. oxysporum by Difenoconazole 25% EC, Propiconazole 25% EC, Carbendazim + Mancozeb WP and Copper Oxychloride 50 WP, at different concentrations.

Table 1: In vitro evaluation of fungicides against mycelial growth of C. fimbriata and F. oxysporum.

*C1, C2, C3 for Systemic fungicides: 100, 200 and 300 ppm, Non systemic fungicides: 250, 500 and 1000ppm; Combi-fungicides; 100, 250 and 500 ppm respectively.

Fig. 5. Inhibition of mycelial growth (%) by fungicides against C. fimbriata and F. oxysporum.

F. Evaluation of fungicides against C. fimbriata colonies/g soil under pot conditions  

Application of different fungicides significantly affected the population of C.  fimbriata in pot soil, as reflected by changes in log₁₀ cfu/g values before and after treatment revealed all fungicide treatments led to reductions (Table 2). While the control treatment showed an increase in fungal population (from 6.68 to 6.99 log₁₀ cfu/g). The most effective treatment was Propiconazole 25% EC, which resulted in the highest reduction (Δlog₁₀ = –1.03) and a 95.9% decrease in population compared to the control. This was followed by Carbendazim + Mancozeb  WP (–0.90 Δlog₁₀; 94.0%), Difenoconazole 25% EC (–0.84; 93.5%), and Metiram + Pyraclostrobin 60 WG (–0.75; 91.3%). Thiophanate methyl 70% WP, Fosetyl-Al 80% WP, Chlorothalonil WP, and Copper oxychloride 50 WP also showed considerable reductions. Tukey’s HSD grouping indicated significant differences among treatments, with Propiconazole, Carbendazim + Mancozeb, and Difenoconazole forming distinct groups from the less effective treatments and the control. Two-way ANOVA (Table S2, Appendix-I, supplementary data) revealed highly significant effects of treatment (F = 45.32, p < 2e–16), time (F = 1034.06, p < 2e–16), and their interaction (F = 41.38, p = 7.01e–16) on fungal population, confirming that the reduction was both time- and treatment-dependent. Normality testing using the Shapiro–Wilk test (Table S3, Appendix-I, supplementary data) confirmed that most treatments had normally distributed residuals (p > 0.05), except for Fosetyl-Al and Metiram + Pyraclostrobin, which slightly deviated from normality (p = 0.000). Levene’s test for homogeneity of variance (Table S4, Appendix-I, supplementary data) indicated that variances across treatments were homogeneous (F = 0.6293, p = 0.7432), validating the assumptions of ANOVA. 

Table 2: Effect of fungicides on C. fimbriata population under pot condition.

G. Evaluation of fungicides against F. oxysporum colonies/g Soil under pot conditions 

Application of different fungicidal treatments had a significant effect on the soil population of F. oxysporum, as observed from the log₁₀-transformed cfu/g values before and after treatment (Table 3).  The untreated control showed an increase in the fungal population, with log₁₀ cfu rising from 6.66 to 7.01, indicating active multiplication of the pathogen in the absence of any intervention. In contrast, all fungicide treatments led to a decrease in cfu levels post-application, with varying degrees of suppression among treatments. Among the tested fungicides, treatment Propiconazole 25% EC proved most effective, causing the highest reduction in population (Δlog₁₀ = –0.81) and achieving a 94.3% decrease relative to the control (Figure 6) This was closely followed by Carbendazim + Mancozeb WP (–0.74 Δlog₁₀; 93.2%) and Difenoconazole 25% EC (–0.62 Δlog₁₀; 92.1%). Copper oxychloride 50 WP was comparatively less effective, showing the lowest reduction among fungicides (Δlog₁₀ = –0.42; 85.6%). Tukey's HSD test distinguished these treatments into statistically significant groupings, where Propiconazole and Carbendazim + Mancozeb were placed in distinct groups from less effective treatments and the control. The two-way ANOVA (Table S5, Appendix-I, supplementary data) revealed that all main effects and interactions were highly significant (p < 0.001). Specifically, the treatment effect (F = 70.07), time effect (F = 952.03), and treatment × time interaction (F = 50.65) significantly influenced the log₁₀-transformed cfu values of F. oxysporum, indicating that both the type of fungicide and the timing of application strongly determined the pathogen suppression outcome. Assumptions of ANOVA were examined through normality and homogeneity tests. The Shapiro–Wilk normality test (Table S6, Appendix-I, supplementary data) indicated that most treatments met the normality assumption (p > 0.05), except for Fosetyl-Al and Propiconazole, which showed deviations (p = 0.000), suggesting slight distributional skewness in those treatments. However, Levene’s test for homogeneity of variance (Table S7, Appendix-I, supplementary data) indicated a violation of the equal variance assumption across treatment groups (F = 10.156, p = 5.82 × 10⁻⁸), suggesting heteroscedasticity in the data. Despite this, the ANOVA results remain valid due to the robustness of the test to moderate deviations. The findings clearly demonstrate that specific fungicides—particularly Propiconazole 25% EC and Carbendazim + Mancozeb WP are highly effective in reducing F. oxysporum populations in soil among all treatments. The results support the potential use of these fungicides in integrated wilt management strategies targeting soil-borne Fusarium infections. The Q-Q plot comparing residuals from the two-way ANOVA models for F. oxysporum and C. fimbriata demonstrated that both datasets largely follow the normality line, indicating that residuals are approximately normally distributed (Figure 7). While minor deviations are observed at the tails, slightly more for Fusarium and the central distribution aligns well with the theoretical quantiles. Residuals for Ceratocystis show slightly better adherence to normality. Overall, the log₁₀ transformation and balanced design help meet the ANOVA normality assumption, supporting the validity of the statistical analysis.

Table 3: Effect of fungicides on F. oxysporum population under pot condition.

Fig. 6. Reduction in Log₁₀ CFU of C. fimbriata (X) and F. oxysporum (Y) after fungicide treatment under pot conditions, Positive Δlog₁₀ CFU in the control reflects an increase in pathogen population.

Fig. 7. Q-Q Plot of residuals from Two-Way ANOVA under pot conditions for C. fimbriata and F. oxysporum treated with fungicides.

In the pot experiment, all fungicide treatments reduced the severity of wilt symptoms in pomegranate plants. Treated plants exhibited delayed disease progression and healthier foliage compared to untreated controls treatments which demonstrated rapid disease development, severe wilting, and complete drying at later stages, leading to total plant collapse.

H. Evaluation of fungicides against C. fimbriata colonies/g soil under field condition 

Application of different fungicidal treatments significantly influenced the soil population of C. fimbriata under field conditions, as measured by changes in log₁₀-transformed colony-forming units per gram (CFU/g) of soil (Table 4).  All fungicides tested resulted in a notable reduction in pathogen population compared to the untreated control, which exhibited an increase in cfu levels post-treatment (from 6.58 to 7.03 log₁₀ CFU/g, i.e., a +0.455 log unit change). Among the treatments, Propiconazole 25% EC exhibited the highest suppressive effect with a Δlog₁₀ CFU of -0.778 and a corresponding 95.1% reduction over the control treatment, followed closely by Carbendazim + Mancozeb WP and Difenoconazole 25% EC, demonstrating 93.6% and 93.2% reductions, respectively (Figure 8). The two-way ANOVA (Table S8, Appendix-I, supplementary data) confirmed a highly significant effect of treatment (F = 80.86, p < 2e-16), time (before vs. after application; F = 1936.39, p < 2e-16), and their interaction (F = 75.61, p < 2e-16) on C. fimbriata population, indicating that not only individual fungicide efficacy but also the change over time and specific treatment-time combinations were critical in influencing fungal dynamics. Levene’s test for homogeneity of variances yielded a non-significant result (F = 0.9298, p = 0.5487), validating the assumption of equal variance across groups (Table S10, Appendix-I, supplementary data) However, the Shapiro-Wilk normality test indicated that post-treatment log₁₀ cfu values deviated significantly from normal distribution (Table S11, Appendix-I, supplementary data). Tukey’s HSD test grouped the treatments based on their log₁₀ cfu means, with the control being statistically distinct (group 'a') and highly suppressive treatments like Propiconazole and Carbendazim + Mancozeb forming separate lower groupings (group 'd' and 'cd'), reflecting their superior efficacy in suppressing C. fimbriata population in soil. 

Table 4: Effect of fungicides on C. fimbriata population under field condition.

I. Evaluation of fungicides against F. oxysporum colonies/g soil under field condition conditions  

Application of various fungicidal treatments significantly reduced the soil population of F. oxysporum compared to the untreated control (Table5). The control plots showed an increase in pathogen load, with cfu values rising from 6.58 to 7.03 log₁₀ cfu/g, reflecting a +0.444 log unit change. In contrast, all fungicidal treatments led to a notable decline in fungal population. Propiconazole 25% EC exhibited the strongest suppressive effect, reducing the fungal population by -0.741 log units and achieving a 93.9% reduction relative to the control, followed closely by Difenoconazole 25% EC (-0.670 log, 93.1%) and Carbendazim + Mancozeb WP (-0.619 log, 92.0%). These results confirm the broad-spectrum efficacy of systemic and contact fungicides in managing soil-borne F. oxysporum under field conditions. The statistical analysis supported these observations. A two-way ANOVA (Table S9, Appendix-I, supplementary data) revealed that treatment (F = 127.20, p < 2e-16), time (before vs. after drenching; F = 1757.80, p < 2e-16), and their interaction (F = 110.30, p < 2e-16) were all highly significant, indicating that both the fungicidal interventions and the time of application had substantial and interactive effects on fungal suppression. Levene’s test (Table S10, Appendix-I, supplementary data) confirmed the homogeneity the homogeneity of variances among groups (F = 0.6335, p = 0.8422), thereby validating the assumptions required for ANOVA. However, the Shapiro -Wilk test for normality of post-treatment log₁₀ cfu values (Table S11, Appendix-I, supplementary data) indicated a significant deviation from normality (W = 0.6179, p = 3.37e-07), suggesting that the data distribution was skewed after treatment, possibly due to the sharp population reductions in several treatments. Tukey’s HSD test further classified the treatments based on their post-drenching log₁₀ cfu means. The untreated control formed a distinct group (‘a’), while the most effective treatments such as Propiconazole and Difenoconazole fell into the lower groupings (‘d’ and ‘cd’), indicating statistically significant reductions in fungal load compared to other fungicides and the control. The Q–Q plots of residuals from the ANOVA models for C. fimbriata and F. oxysporum under field conditions indicated that the assumption of normality was reasonably met (Figure 9). For C. fimbriata, the residuals closely followed the theoretical quantiles with only slight deviations at the extremes, suggesting that the residuals are approximately normally distributed. In the case of F. oxysporum, the residuals also generally aligned with the theoretical line, though a mild deviation was observed in the upper tail, indicating a slight right-skew. However, these deviations are minimal and fall within acceptable limits for field experiment data. 

Table 5: Effect of fungicides on F. oxysporum population under pot condition.

Fig. 8. Reduction in Log₁₀ CFU of C. fimbriata (x) and F. oxysporum (y) after fungicide treatment under field conditions.

Fig. 9. Q-Q Plot of residuals from Two-Way ANOVA under field conditions for C. fimbriata and F. oxysporum treated with fungicides.

Under field conditions, each fungicide treatment led to a considerable reduction in disease severity in wilt-infected pomegranate plants. Treated plots showed improved plant vigor, delayed symptom onset, and better survival rates compared to untreated controls. Control plants, in contrast, displayed extensive wilting, progressive canopy yellowing, and ultimately dried completely during the later stages of disease development. The results demonstrate that the fungicide applications were effective in mitigating wilt under natural conditions, offering a practical approach for managing the disease in pomegranate orchard.

DISCUSSION

Pomegranate wilt, primarily caused by C. fimbriata and F. oxysporum, poses a severe threat to orchard productivity due to rapid disease progression and complete plant mortality. Initial symptoms such as yellowing, stunting, and wilting indicate systemic vascular disruption, often resulting from fungal colonization that blocks water and nutrient flow (Huang et al., 2003). C. fimbriata infection is distinguished by blue or black vascular streaks, while F. oxysporum cause browning and drying of xylem vessels, both reflecting distinct pathogenic mechanisms (Sharma et al., 2010; Yadeta and Thomma 2013). The post-monsoon environment and waterlogged soils likely enhance disease spread, as previously reported (Park, 1959). Morphological and cultural analyses of C. fimbriata matched previous descriptions, including hat-shaped ascospores and dark perithecia (Hunt, 1956; Raja et al., 2017), suggesting survival adaptations. The pigmentation and sporulation traits of F. oxysporum aligned with taxonomic descriptions by Leslie and Summerell (2006), and prior findings by Patel et al. (2020); Burgess et al. (1989) confirming its diagnostic features and adaptive capacity. Pathogenicity tests revealed that C. fimbriata  induced faster disease onset compared to F. oxysporum, and co-inoculation led to even earlier wilting, indicating a synergistic interaction that amplified disease severity. These results align with earlier studies (Xu et al., 2011; Alam et al., 2016; Mohmad et al., 2020), reinforcing the importance of understanding dual infections for disease management. 

In vitro assays demonstrated that fungicide efficacy was significantly influenced by both active ingredient and concentration. Propiconazole 25% EC was most effective against C. fimbriata, while Carbendazim + Mancozeb WP showed highest inhibition of F. oxysporum. These results were statistically supported by ANOVA (p < 0.001), and corroborated by previous studies (Ismail et al., 2018; Khan et al., 2017; Khosla and Bhardwaj 2011; Sharma and Khosla 2020; Subhani et al., 2011; Sahar et al., 2013; Maitlo et al., 2014; Kubde et al., 2022). Pot experiments further validated the effectiveness of these fungicides in reducing soil populations. Propiconazole 25% EC demonstrated the highest suppression, followed by Carbendazim + Mancozeb WP and Difenoconazole 25% EC while the untreated control exhibited increased pathogen loads, highlighting the fungi’s aggressive nature in conducive conditions. ANOVA confirmed significant effects of treatment and time, with slight deviations from normality warranting cautious interpretation. These findings align with earlier pot trials (Ahmad et al., 2021; Bhimani et al., 2018; Raja et al., 2023). Field evaluations mirrored pot results, with systemic fungicides, particularly Propiconazole, showing strong suppression of both pathogens. The untreated control again showed pathogen proliferation, emphasizing the importance of timely interventions. Significant treatment effects and consistent group separation in Tukey’s test reaffirmed these outcomes. Although some post-treatment data deviated from normality (Shapiro–Wilk), Q–Q plots and Levene’s test supported the robustness of the analysis. These results validate the field applicability of systemic fungicides and support their integration into disease management strategies, in line with findings from Sonyal et al. (2016); Khosla (2013); Raja et al. (2023).  In both pot and field trials, fungicide treatments significantly reduced the severity of pomegranate wilt compared to untreated controls. Treated plants exhibited delayed symptom onset, reduced vascular browning, and improved overall health and survival. In contrast, control plants showed rapid disease progression, severe wilting, and complete drying at later stages. The consistent performance of fungicides under controlled and natural conditions highlights their potential as an effective disease management strategy. These results suggest that timely application of suitable fungicides can play a crucial role in minimizing losses due to wilt, particularly in disease-prone orchards and during early stages of plant establishment. While fungicides demonstrated significant efficacy in suppressing C. fimbriata and F. oxysporum populations under both, pot and field conditions, their use for managing soil-borne pathogens poses inherent limitations. Repeated application can lead to the development of fungicide resistance, reduced soil microbial diversity, and potential environmental contamination. Additionally, systemic fungicides may have limited efficacy in reaching deeply established inoculum in the rhizosphere or in poorly drained soils. These challenges underscore the need to adopt an integrated disease management (IDM) approach that combines chemical control with cultural practices, organic amendments, resistant cultivars, and biological agents. Such a tiered strategy not only enhances disease suppression but also ensures long-term sustainability, reduces chemical dependency, and preserves soil health.

LIMITATIONS

The study was the disrupted by the COVID-19 pandemic in 2020, which led to the complete lockdown and closure of laboratories. As a result, the planned pot and field trials were delayed, with the first trial conducted in 2019 and the second-year trials postponed to 2021. This interruption affected the continuity of data collection and experimentation, potentially impacting the consistency and scope of the study. Additionally, the inability to conduct field evaluations during the critical period of disease progression in orchards limited the integration of real-time environmental factors into the findings, highlighting the challenges of conducting agricultural research during such unprecedented global disruptions.

Given the limitations of sole reliance on fungicides for managing soil-borne pathogens like C. fimbriata and F. oxysporum, future research should focus on developing integrated and eco-friendly disease management strategies. Emphasis should be placed on exploring and validating the synergistic use of biological control agents (e.g., Trichoderma spp., PGPR), organic amendments, resistant pomegranate varieties, and soil health-improving practices. Long-term field studies are needed to evaluate the cumulative effects of these strategies on pathogen suppression, soil microbiome resilience, and crop productivity. The development of decision-support systems and tailored management packages for different agro-climatic zones will further enhance the effective and sustainable control of pomegranate wilt disease.

Mukesh, Kishore Khosla, Satish K. Sharma, Anju Sharma, Saijal Khosla  and Abhishek Sharma (2025). Assessment of Fungicide Efficacy in Managing Pomegranate wilt Disease caused by Ceratocystis fimbriata and Fusarium oxysporum. Biological Forum, 17(6): 38-52.