Chitosan Mitigates Phytophthora Blight in Chayote (Sechium edule) by Direct Pathogen Inhibition and Systemic Resistance Induction

Abstract

Phytophthora blight, caused by Phytophthora capsici, is a destructive disease that significantly constrains the production of chayote (Sechium edule) in Mexico, leading to substantial yield and economic losses. The increasing ineffectiveness of synthetic fungicides and associated environmental concerns underscore the need for sustainable control alternatives. This study evaluated the antifungal efficacy of low molecular weight chitosan (75–85% deacetylation; Sigma-Aldrich) against P. capsici under in vitro and in vivo conditions. Chitosan solutions (0.1–3.0 g L⁻¹) were tested for their ability to inhibit pathogen growth and suppress disease symptoms. In vitro assays demonstrated a concentration-dependent inhibition of mycelial growth, with the highest dose (3.0 g L⁻¹) reducing radial expansion by 32.6%. In fruit inoculation experiments, treatment with 1.0 g L⁻¹ chitosan decreased lesion size by 50.9%, while the same concentration reduced disease severity index (DSI) by 50% in whole plants. Notably, symptom suppression was observed in tissues not directly exposed to chitosan, suggesting the activation of systemic resistance. Although the underlying molecular mechanisms were not directly assessed, the results support the dual role of chitosan as a direct antifungal agent and a potential inducer of host defense responses. These findings highlight the potential of chitosan as a biodegradable, low-toxicity alternative to synthetic fungicides and support its integration into sustainable management strategies for Phytophthora blight in chayote production systems.

1. Introduction

Sechium edule (Jacq.) Sw., commonly known as chayote, is a perennial climbing vine belonging to the Cucurbitaceae family [1]. It was domesticated in Mesoamerica over five centuries ago and is cultivated primarily for its edible fruits, shoots, and tubers [2]. In addition to its agricultural importance, chayote possesses notable nutritional and pharmacological properties, attributed to its rich composition of phenolics, flavonoids, carotenoids, and bioactive polysaccharides, which have been associated with antihypertensive, antidiabetic, and anti-inflammatory effects [3,4]. In recent years, chayote has emerged as a significant horticultural commodity in tropical and subtropical regions, including Brazil, Mexico, India, Costa Rica, and China [5,6].

Mexico is currently the world’s leading exporter of chayote, with major markets in the United States and Canada [3]. In 2023, the national industry generated over USD 70 million in production value [7], representing an important source of income and employment in rural communities [8]. However, the sustainability of chayote production is increasingly compromised by biotic constraints, including viral [9], bacterial [10], and fungal pathogens [11]. Among these, Phytophthora blight, caused by Phytophthora capsici, poses one of the most severe threats to crop yield and fruit quality [11,12,13].

P. capsici is a soilborne oomycete with a wide host range and high pathogenic variability [14]. It infects multiple plant organs—roots, crowns, stems, leaves, and fruits—and is particularly aggressive in warm, humid environments with poor drainage [15], such as those found in major chayote-producing regions like Veracruz, Oaxaca, and Chiapas [12,16]. In Veracruz, three isolates were obtained in different chayote-producing areas, showing morphological diversity, and variations in their virulence and in their resistance to metalaxyl [17]. Disease symptoms typically include foliar wilting, stem and crown rot, and white powdery lesions on fruits [11,12,13]. While quantitative estimates of yield loss in chayote are scarce, field observations indicate that P. capsici infection can result in progressive disease development and, under conducive conditions, total crop failure [18]. These losses not only diminish export potential but also threaten the livelihoods of small-holder farmers.

Current disease management strategies for Phytophthora blight in chayote are heavily dependent on the application of synthetic fungicides, primarily metalaxyl-based formulations [11,12]. However, the repeated use of such compounds has led to the emergence of fungicide-resistant P. capsici isolates, as reported in several cropping systems [14]. Moreover, increasing regulatory restrictions [19], risks of environmental contamination [20,21], and concerns regarding human health [20] and phytotoxicity [22] underscore the need for sustainable, low-risk alternatives to conventional chemical control.

Chitosan, a linear polysaccharide derived from the deacetylation of chitin—commonly obtained from crustacean shell waste—has gained attention as a promising biocontrol agent [23,24]. Its favorable characteristics include biodegradability, low toxicity, environmental safety, and compatibility with integrated pest management systems [25]. These outstanding characteristics have increased the demand for chitosan and the protocols aimed at obtaining it in a sustainable and more economical way [26,27].

Chitosan exhibits broad-spectrum antimicrobial activity against bacteria, fungi, oomycetes, and nematodes [28]. In addition to its direct inhibitory effects, chitosan is known to trigger plant immune responses [29]. This immune response involves the rapid production of reactive oxygen species (ROS), activation of mitogen-activated protein kinase (MAPK) signaling cascades, and transcriptional activation of defense-related genes [30]. Furthermore, chitosan promotes salicylic acid (SA) accumulation, enhances phenylalanine ammonia-lyase (PAL) activity, and can induce systemic resistance through calcium signaling and the biosynthesis of secondary metabolites [30]. Previous studies in chili pepper and cucumber have demonstrated the efficacy of chitosan in suppressing P. capsici through both direct pathogen inhibition and the induction of host resistance [31,32,33,34].

Despite the growing interest in chitosan-based disease management, its application in chayote remains largely unexplored. In particular, the potential role of chitosan in suppressing P. capsici and activating systemic resistance in this crop has not been evaluated. Therefore, the objective of this study was to assess the antifungal activity of low molecular weight chitosan against P. capsici under in vitro conditions and to evaluate its efficacy in reducing disease severity and inducing systemic resistance in chayote fruits and plants. To the best of our knowledge, this is the first report describing the dual function of chitosan as both a direct antimicrobial compound and an elicitor of plant defense responses in chayote. The results of this study are intended to support the development of sustainable and environmentally responsible alternatives to synthetic fungicides for the management of Phytophthora blight in this economically and culturally important crop.

2. Materials and Methods

2.1. Preparation of Chitosan Solution

Low molecular weight chitosan (50–190 kDa; 75–85% deacetylation; Sigma-Aldrich 448869, Burlington, MA, USA) was used for all experiments. A 10 g L⁻¹ stock solution was prepared by dissolving chitosan in 1% (v/v) glacial acetic acid (Karal, Leon, GTO, Mexico) under continuous stirring at room temperature for 12 h. The solution was adjusted to pH 5.6 using 1 M NaOH (CTR Scientific, Monterrey, NL, Mexico) and subsequently diluted with sterile distilled water to the final concentrations required for each assay.

2.2. Pathogen Culture and Inoculum Preparation

The P. capsici isolate Pc-Tux01, previously identified and confirmed as pathogenic [35], was obtained from the strain collection of the Genetic Resources Management and Conservation Unit, Faculty of Biological and Agricultural Sciences, Universidad Veracruzana (Veracruz, Mexico). The isolate was cultured on V8 juice agar (Campbell’s, Camden, NJ, USA; Agarmex, Ensenada, BC, Mexico) and incubated at 27 °C in the dark for 7 days. Zoospore production was induced following the method described by Andrade-Luna et al. [13]. Briefly, the agar containing the Pc-Tux01 isolate was divided into four parts, each part was placed in a Petri dish and covered with sterile distilled water until it was at the same level as the agar and incubated at room temperature under constant light for 5 d. Zoospores were released by placing the Petri dishes with the mycelium at 4 °C for 30 min and then at room temperature for 1 h. Zoospore suspensions were quantified using a hemocytometer and adjusted to the desired concentration for inoculation.

2.3. In Vitro Antimicrobial Activity of Chitosan

The antifungal activity of chitosan against P. capsici was assessed using a modified version of the protocol described by Huang et al. [36]. Clarified V8 agar was amended with chitosan at final concentrations of 0.1, 0.3, 0.5, 1.0, 2.0, and 3.0 g L⁻¹. A total of four replicates (n = 4) were prepared per treatment. Control plates received 1% glacial acetic acid, matching the solvent used to dissolve chitosan. A 5 mm-diameter mycelial plug from an actively growing P. capsici culture was placed at the center of each plate. Plates were incubated in the dark at 27 °C for 10 days. Colony diameter was measured along two perpendicular axes and averaged. The inhibition rate (%) was calculated using the following formula:

Inhibition rate = [(C − T)/(C − I)] × 100

(1)

where C = mean colony diameter in the control, T = colony diameter in chitosan treatment, and I = initial diameter of the mycelial plug (5 mm).

2.4. Evaluation of Chitosan Against Phytophthora Blight in Chayote Fruits

Fruits of S. edule var. virens levis at horticultural maturity (18 ± 2 days post anthesis) were selected, surface-sterilized with 70% ethanol for 30 s, and rinsed with sterile distilled water. Fruits were immersed for 30 s in chitosan solutions at 0.5, or 1.0 g L⁻¹ or 1% glacial acetic acid (control). After air drying for approximately 5 h, each fruit (n = 10 per treatment) was inoculated by placing a 5 mm P. capsici mycelial plug on the equatorial region. Inoculated fruits were incubated at 27 °C in the dark. Lesion diameters were measured at 7 days post inoculation (dpi) using a digital vernier.

2.5. Evaluation of Chitosan Against Phytophthora Blight in Chayote Plants

Seeds were aseptically extracted from mature fruits and immersed for 30 s in chitosan solutions (0.1, 0.25, 0.5, and 1.0 g L⁻¹) or 1% acetic acid (control). Treated seeds were sown in pots (15 cm diameter) filled with a sterile 1:1 (v/v) peat–perlite mixture. Plants were grown in a greenhouse under controlled conditions (27 ± 2 °C, natural photoperiod) without fertilization and irrigated as needed. At 7 and 14 days after sowing, the plants were foliar sprayed with 1 mL of the corresponding chitosan concentrations until runoff. Once dried (1 h), the base of each plant stem (n = 10 per treatment) was inoculated with 1 mL of a zoospore suspension of P. capsici (1 × 10⁵ zoospores mL⁻¹). Disease severity was assessed at 12 dpi using a 0–4 scale: 0 = healthy; 1 = stem lesions; 2 = partial leaf wilting; 3 = complete foliar wilting; 4 = plant death. A DSI was calculated according to González-Peña et al. [37].

DSI (%) = [Σ(class frequency × score of rating class)/(total number of plants × maximal disease index)] × 100

(2)

2.6. Statistical Analysis

All experiments were conducted in triplicate. Data from in vitro and fruit inoculation assays were analyzed using one-way analysis of variance (ANOVA), followed by Tukey’s honestly significant difference (HSD) test at p ≤ 0.05. Non-parametric data from the plant assay were analyzed using the Kruskal–Wallis test, followed by pairwise comparisons using the Mann–Whitney U test with Bonferroni correction. Statistical analyses were performed using R software (version 4.3.1; R Core Team, Vienna, Austria) with the packages agricolae, rstatix, and ggplot2.

3. Results

3.1. Chitosan Inhibited Mycelial Growth of P. capsici

Chitosan significantly inhibited the mycelial growth of P. capsici in vitro in a dose-dependent manner (Figure 1A–C). After 6 dpi, the pathogen fully colonized the control plates, whereas chitosan-amended media exhibited a marked reduction in colony diameter. At the highest concentration tested (3 g L⁻¹), chitosan reduced mycelial growth by 32.6% compared to the control. Lower concentrations of 0.3, 0.5, 1, and 2 g L⁻¹ also inhibited growth, reducing colony diameter by 5.2, 10.3, 15.2, and 24.1%, respectively. All chitosan concentrations tested significantly differed from the control (p < 0.05). These findings confirm that low molecular weight chitosan exerts a measurable antifungal effect on P. capsici in vitro, supporting its potential as a biocontrol agent.

3.2. Chitosan Reduced Phytophthora Blight Symptoms in Chayote Fruits

The application of chitosan significantly reduced symptom severity in chayote fruits inoculated with P. capsici (Figure 2A–C). At 7 dpi, control fruits developed extensive white lesions characteristic of Phytophthora blight [15], averaging 10.2 cm in diameter. In contrast, fruits treated with 1.0 g L⁻¹ chitosan exhibited lesions of 5.0 cm, corresponding to a 50.9% reduction in lesion size. Treatment with 0.5 g L⁻¹ chitosan also significantly reduced lesion diameter to 8.6 cm, representing a 15.7% decrease relative to the control (p < 0.05). These results demonstrate that chitosan effectively restricts lesion expansion and symptom development in infected chayote fruits.

3.3. Chitosan Mitigated Phytophthora Blight in Chayote Plants and Suggests Induction of Systemic Resistance

Chitosan applications also conferred protection against Phytophthora blight in chayote plants under greenhouse conditions (

Table 1. Effect of different chitosan concentrations on DSI in chayote plants inoculated with P. capsici.

Chitosan Treatments (g L−1)	Mean of Rank	Disease Severity Index (%)	Disease Inhibition Rate (%)
Control	35.3	96	0
0.1	27.6	84	12.5
0.25	30.9	86	10.4
0.5	18.4	64	33.3
1	15.3 *	48	50

* Significant difference in the chitosan treatment compared to the control (p < 0.05, Kruskal–Wallis test followed by pairwise Mann–Whitney U tests with Bonferroni correction). There were no significant differences between the control and all other chitosan treatments.

, Figure 3). Plants treated with 1.0 g L⁻¹ chitosan exhibited a significantly lower disease severity index (DSI = 48) compared to the untreated control (DSI = 96), representing a 50% reduction (p < 0.05). Although treatments with 0.1, 0.25, and 0.5 g L⁻¹ resulted in DSIs of 84, 86, and 64, respectively, these reductions were not statistically significant. Notably, we observed disease suppression in tissues not directly treated with chitosan, indicating a systemic effect. This suggests that chitosan may trigger systemic defense responses in chayote, consistent with its role as an elicitor of induced resistance [29]. These results collectively support the dual function of chitosan as both an antifungal compound and a modulator of host immunity.

4. Discussion

This study evaluated the antifungal efficacy of low molecular weight chitosan against P. capsici, the causal agent of Phytophthora blight in chayote, a disease of growing concern in major production areas of Mexico [11,12,16]. Chitosan was applied at concentrations ranging from 0.1 to 3.0 g L⁻¹ under controlled conditions. The key findings indicate that chitosan suppressed mycelial growth in vitro in a dose-dependent manner and significantly reduced disease severity in fruits and whole plants in vivo.

The in vitro assays demonstrated that chitosan at 1.0 g L⁻¹ inhibited mycelial growth by 15.2%, with a maximum inhibition of 32.6% observed at 3.0 g L⁻¹ (Figure 1A–C). These results align with previous reports indicating the antifungal activity of chitosan against Phytophthora spp., including P. infestans [30,36,38], P. nicotianae [37], and P. capsici [31,34,39,40]. The dose-dependent inhibition is consistent with prior studies that emphasize the importance of concentration and physicochemical characteristics such as molecular weight and degree of deacetylation in determining antifungal efficacy [41]. In this study, a commercially available chitosan with a molecular weight 50–190 kDa and 75–85% deacetylation was used. Despite its effectiveness, the inhibition levels were lower than those reported in studies using higher molecular weight chitosan or oligochitosan. For example, Xu et al. [40] reported 74% inhibition of P. capsici at 1.0 g L⁻¹ using 300–500 kDa chitosan, and up to 92% inhibition with oligochitosan, suggesting that polymer size significantly influences antifungal activity. Furthermore, Mohammadi et al. [42] observed 100% inhibition of Phytophthora drechsleri with the same chitosan concentration used in our study, highlighting the influence of pathogen species and assay conditions on efficacy outcomes.

In vivo assays supported the protective role of chitosan, with 1.0 g L⁻¹ reducing lesion diameter in chayote fruits by 50.9% (Figure 2A–C) and decreasing the disease severity index in whole plants by 50% (Table 1, Figure 3). These results are consistent with similar studies in other crops: Zohara et al. [32] reported 85% inhibition in cucumber seedlings treated with 0.5 g L⁻¹ chitosan, and Esyanti et al. [33] observed approximately 84% disease reduction in pepper using comparable concentrations. However, the lower efficacy observed in our study relative to these findings may be attributed to host-specific factors, differences in experimental design, or variability in chitosan formulation. For example, Torres et al. [34] reported complete inhibition in Capsicum annuum fruits using 160 kDa chitosan applied via pre-inoculation immersion, suggesting that both timing and method of application can modulate performance. Similarly, Atia et al. [38] achieved >98% inhibition in tomato leaves using 15 kDa chitosan, reinforcing the influence of molecular weight on bioactivity.

A notable observation in the present study was the suppression of disease symptoms in tissues not directly exposed to chitosan, suggesting the activation of systemic resistance mechanisms. Although this study did not measure molecular markers of induced resistance, the systemic effect is consistent with the known role of chitosan as a microbe-associated molecular pattern (MAMP) [29]. Chitosan can activate pattern-triggered immunity (PTI) via recognition by pattern recognition receptors (PRRs) such as chitin elicitor receptor kinase 1 (CERK1) and flagellin sensing 2 (FLS2), leading to ROS production, MAPK cascade signaling, and transcriptional activation of defense-related genes, including pathogenesis-related protein 1 (PR1), Pto interacting protein 5 (Pti5), and WRKY26 [30]. Chitosan has also been shown to induce accumulation of SA and enhance PAL activity, both key components in systemic acquired resistance (SAR) [30]. Based on these findings, it is plausible that SA-dependent signaling pathways contribute to the observed systemic protection in chayote, although this remains to be verified experimentally.

From an applied perspective, the use of chitosan at 1.0 g L⁻¹ appears to be both technically feasible and agronomically relevant. Chitosan is biodegradable, non-toxic, and approved for agricultural use, making it suitable for integration into sustainable production systems, particularly for smallholder and organic growers [25]. Its ability to reduce disease severity without reliance on synthetic fungicides represents an important step toward ecologically sound plant protection strategies; furthermore, the use of chitosan intercalated or in combination with other antimicrobial agents such as nanomaterials that can alter P. capsici membrane or serve as a vehicle for slow delivery of agrochemicals [43,44] will allow to greater control the damage caused by this oomycete.

Nonetheless, several limitations of the current study must be acknowledged. First, all experiments were conducted under controlled environmental conditions using a single chayote cultivar, which limits extrapolation to field conditions. Second, only one chitosan formulation was evaluated, and batch-to-batch variability in commercial products may affect reproducibility. Third, the proposed induction of systemic resistance was inferred based on symptom reduction but was not supported by molecular or biochemical data. Finally, the study did not assess the potential for synergistic interactions between chitosan and other biocontrol agents. Previous studies have shown that combining chitosan with beneficial microbes such as Bacillus subtilis or Trichoderma spp. can enhance efficacy and broaden the spectrum of protection [45,46].

Future research should address these limitations by: (i) validating chitosan efficacy under open-field conditions, on a longer timeline, and across multiple chayote genotypes; (ii) characterizing transcriptomic and metabolomic responses to chitosan to confirm the involvement of defense pathways, including SA and jasmonic acid (JA) signaling; (iii) comparing formulations differing in molecular weight, degree of polymerization, and delivery systems (e.g., nanoparticles, oligochitosan); and (iv) evaluating synergistic effects of chitosan in combination with microbial or botanical biocontrol agents. These efforts will contribute to the development of robust, biologically based disease management strategies suitable for use in integrated crop protection programs.

5. Conclusions

This study provides novel evidence that low molecular weight chitosan (50–190 kDa; 75–85% deacetylation) can reduce the severity of Phytophthora blight in chayote caused by P. capsici under controlled conditions. Chitosan applied at 1.0 g L⁻¹ reduced mycelial growth by 15.2% in vitro, decreased lesion diameter in fruits by 50.9%, and halved the disease severity index in whole plants. These findings suggest that chitosan exhibits both direct antifungal activity and may contribute to systemic protection in treated plants.

The concentration tested is practical and environmentally safe, and its performance under greenhouse conditions supports its integration into sustainable disease management strategies; chitosan applications can be during plant growth and also after fruit harvest to prolong its shelf life. However, variations in efficacy compared to other crops and formulations highlight the need to optimize key factors, including chitosan molecular weight, application method, and treatment timing.

Further studies should validate chitosan’s effectiveness under field conditions, confirm activation of plant immune responses through molecular assays, and assess formulation enhancements, including combinations with microbial or botanical control agents. Taken together, these findings support the potential of chitosan as a valuable tool for reducing reliance on synthetic fungicides and improving the sustainability of chayote production.