Formulation and Evaluation of an Eco-Friendly Allamanda Microemulsion Biofungicide for the Control of Anthracnose in Papaya

Abstract

An eco-friendly microemulsion biofungicide derived from Allamanda cathartica was developed for the control of papaya anthracnose caused by Colletotrichum gloeosporioides. The formulation was prepared by blending surfactants, carrier oil, and water and optimized using ternary phase diagrams to identify stable microemulsion systems. All selected formulations exhibited surface tension values ranging from 29 to 31 mN/m, while particle sizes ranged from 51.79 to 1801.05 nm. The optimized formulation, coded as AM8, consisted of 35% Allamanda concentrated liquid crude extract (ACLCE), 26% water, 13% alkyl polyglucoside surfactant, and 26% dimethylamide oil. Papaya fruits coated with the formulations showed significant reductions (p < 0.05) in anthracnose incidence caused by C. gloeosporioides. Control fruits treated with water showed 75% disease incidence, whereas fruits treated with benomyl showed 42% disease incidence. Disease incidence, severity, and disease index decreased with increasing formulation concentration, and fruits treated with the eight formulations at 10% concentration exhibited significantly lower disease incidence (0–17%) and disease index (0–17%), with disease severity consistently scored as zero. The Allamanda formulation demonstrated strong antifungal activity with EC₅₀ and EC₉₅ values of 1.839 and 7.067 mg/mL (w/v), respectively, at the 95% confidence level. The optimized formulation AM8 remained stable for up to one year and showed superior disease control performance compared with the conventional fungicide benomyl. In addition, the formulation maintained fruit quality by preserving firmness, peel color, and soluble solids concentration, thereby extending papaya shelf life up to 30 days without adversely affecting the natural ripening process. These findings demonstrate the potential of Allamanda-based microemulsion formulations as sustainable biofungicides for postharvest control of papaya anthracnose and provide a promising alternative to conventional synthetic fungicides.

1. Introduction

Papaya (Carica papaya L.) is one of the most important tropical fruits cultivated worldwide, with significant economic value in both domestic consumption and international trade. Globally, papaya production exceeds 13 million tonnes annually, with major producing countries including India, Brazil, Indonesia, Mexico, and Nigeria [1]. In Malaysia, papaya is among the major fruit crops cultivated for both local consumption and export markets. Although production levels fluctuate due to environmental conditions and disease pressures, recent reports indicate that papaya production reached approximately 38,883 metric tons in 2023, highlighting its continued importance within the Malaysian horticultural sector [2]. Maintaining the competitiveness of the papaya industry therefore requires continuous improvement in fruit quality, postharvest handling, and disease management strategies.

Papaya is a climacteric fruit characterized by a rapid increase in respiration rate and ethylene production during ripening. These physiological changes accelerate softening and senescence of the fruit, thereby increasing susceptibility to postharvest fungal diseases. Among these diseases, anthracnose caused by Colletotrichum gloeosporioides is one of the most destructive diseases affecting papaya production, resulting in severe yield losses and reduced marketability due to the development of dark, sunken necrotic lesions on fruit surfaces [3,4,5].

Traditionally, papaya anthracnose has been managed using thermal and chemical treatments. Although these methods can reduce disease incidence, they are often associated with several limitations, including potential heat injury to fruits, the development of fungicide resistance in pathogen populations, and concerns regarding chemical residues that may pose risks to human health and the environment [6]. Consequently, increasing attention has been directed towards the development of safer and environmentally friendly alternatives for postharvest disease control [7].

Plant-derived extracts have gained considerable interest as potential natural antifungal agents because they contain diverse bioactive compounds with antimicrobial properties [5,6]. Among these plants, species of the genus Allamanda (Apocynaceae) have attracted attention due to their reported biological activities and traditional medicinal uses. Allamanda species are widely grown as ornamental plants throughout tropical regions, including Malaysia, although they are native to South America. Previous studies have reported that extracts from Allamanda leaves contain diverse bioactive compounds and exhibit a range of biological activities, including antifungal, antimicrobial, and antimitotic properties [8,9,10,11,12,13]. Notably, these extracts have also demonstrated strong antifungal activity against C. gloeosporioides [14]. Concentrations above 5% completely inhibited fungal growth, and microscopic observations revealed severe damage to fungal structures. In vivo studies further showed that papaya fruits treated with chloroform extracts of A. cathartica var. Jamaican Sunset at concentrations of 5 and 7 mg/mL significantly reduced anthracnose incidence. Phytochemical analysis using GC–MS revealed the presence of several bioactive compounds, including campesterol, β-sitosterol, stigmasterol, plumericin, squalene, and α-tocopherol. Bioautography-guided isolation subsequently identified plumericin (C₁₅H₁₄O₆; MW 290), a sesquiterpene lactone, as the major active antifungal compound responsible for the inhibitory activity against C. gloeosporioides [15].

Despite the promising antifungal activity of plant extracts, their practical application in agriculture is often limited by issues related to poor stability, low solubility of hydrophobic compounds, and inconsistent performance under field or storage conditions. Therefore, the development of suitable formulation systems is essential to improve the stability, delivery, and efficacy of plant-derived bioactive compounds [16].

Microemulsion systems have received considerable attention in agrochemical formulations due to their ability to enhance the solubility, stability, and bioavailability of hydrophobic active ingredients [16]. Microemulsions are thermodynamically stable isotropic systems composed of oil, water, and surfactants, typically forming droplets in the nanometer size range (10–100 nm) [17]. These systems provide several advantages, including improved dispersion, enhanced surface wetting, and efficient delivery of active ingredients to target surfaces [18].

However, despite the reported antifungal properties of Allamanda extracts, studies on microemulsion-based formulations of Allamanda-derived biofungicides for postharvest disease control remain limited. Therefore, the study aimed to develop and evaluate an eco-friendly microemulsion biofungicide derived from Allamanda for the control of anthracnose caused by C. gloeosporioides in papaya. The study further investigated the physicochemical characteristics of the formulations, their antifungal efficacy, and their effects on fruit quality and storage life.

2. Materials and Methods

2.1. Preparation of Formulations Using a Ternary Phase Diagram

The formulations were prepared using the aqueous titration method and a three-component phase diagram system with varying ratios of oil to surfactant (100:0, 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, and 0:100), as shown in

Table 1. Surfactant combinations for phase diagram construction.

Formulation	Surfactant Phase
AM1	Agnique MBL 510H
AM2	Agnique MBL 530H
AM3	10% Agnique MBL 530H:90% Agnique MBL 510H
AM4	10% Agnique MBL 530H:90% Agnique MBL 510H
AM5	20% Agnique MBL 530H:80% Agnique MBL 510H
AM6	30% Agnique MBL 530H:70% Agnique MBL 510H
AM7	40% Agnique MBL 530H:60% Agnique MBL 510H
AM8	50% Agnique MBL 530H:50% Agnique MBL 510H

. The oil and surfactants used in all AM formulations (AM1–AM8), namely Agnique AMD 810, Agnique MBL 510H, and Agnique MBL 530H, were obtained from BASF SE, Ludwigshafen, Germany. Each mixture containing precise quantities of oil and surfactant (0.5 g) was placed into a 10 mL screw-cap glass tube, after which water was incrementally added to determine the stable phases. The systems were visually assessed for clarity and stability at room temperature. These samples were then centrifuged at 3500 rpm for 15 min to evaluate stability, where stable formulations maintained a transparent one-phase appearance. The results were plotted on ternary phase diagrams to identify the isotropic regions, indicating balanced microemulsion systems. From each phase diagram constructed, different formulations were selected to allow incorporation of the active ingredient (Allamanda concentrated liquid crude extract, ACLCE). AM3 and AM4 were obtained from the 90% Agnique MBL 510H:10% Agnique MBL 530H surfactant mixture within the Agnique AMD 810/water microemulsion phase diagram, but with different composition points selected within the isotropic region. The selected formulations were subsequently evaluated for stability, surface tension, and particle size.

2.2. Characterization of Formulations

2.2.1. Stability Test

Selected formulations were centrifuged at 3500 rpm for 30 min and kept at room temperature for four weeks. The formulations were then evaluated for the ability to retain a transparent one-phase appearance after four weeks.

2.2.2. Surface Tension Analysis

Surface tension was measured using a KRÜSS^® Tensiometer K6 (KRÜSS GmbH, Hamburg Germany) based on the Du Noüy ring method. The instrument was calibrated using deionized water (72 mN m⁻¹) prior to measurement. For analysis, 20 mL of formulation samples at concentrations of 1%, 3%, 5%, and 7% (v/v) were placed in the test vessel, and the platinum ring was immersed approximately 5 mm below the liquid surface. The surface tension was recorded at the point of lamella detachment. The ring was cleaned with acetone and flame-sterilized between measurements to prevent contamination. All measurements were performed in triplicate.

2.2.3. Particle Size Measurement

Particle size of the transparent formulations was measured using a Nanophox Sympatec photon cross-correlation sensor (Sympatec GmbH, Clausthal-Zellerfeld, Germany) equipped with Windox 5 software based on the 3D cross-correlation technique. Samples were illuminated using a vertically polarized helium–neon laser (λ = 632.8 nm) at 25 °C, and measurements were recorded at a scattering angle of 90°. Approximately 1–2 mL of sample was placed in the measurement vial and analyzed within 20 min of preparation. Each formulation was measured in five replicates.

2.3. Evaluation of the Formulations Regarding Anthracnose Development

Papaya (Carica papaya cv. ‘Sekaki’) fruits at color stage two (green with yellow traces) were surface sterilized with 70% ethanol and rinsed with distilled water before treatment application. The experimental design was completely randomized with 26 treatments resulting from the combination of three dilution levels (5, 7, and 10% (v/v)) and eight formulations (AM1-AM8, Table 1) plus two additional treatments, a positive and a negative control. The number of replicates was three, with six fruits per replicate. In the negative control, the fruits were immersed in sterilized distilled water, and in the negative one, they were treated with the commercial fungicide benomyl (50% WP, 151 0.33 g L⁻¹). Each fruit was wounded (3 mm deep and 5 mm diameter) at the mid region. Each of the wounds was then inoculated with 30 µL conidial suspension of C. gloeosporioides (1 × 10⁶ spores mL⁻¹) and held at room temperature for an hour. Treated fruits were placed in cardboard boxes and incubated (22 °C, 53% RH) for 10 days. At the end of the 8-day storage period, disease incidence and disease severity were evaluated.

Disease Assessment

Disease incidence was calculated using the following formula: Disease incidence (%) = [(Number of infected fruits/Total number of fruits per treatment) × 100]. During storage, disease severity was evaluated using a 0–4 scale, where 0 = apparently healthy fruit without lesions; 1 = 1–25% of the fruit surface covered with water-soaked lesions; 2 = 26–50% of the fruit surface covered with water-soaked lesions and soft rot symptoms; 3 = 51–75% of the fruit surface covered with water-soaked lesions and necrosis around the lesions; and 4 = 76–100% of the fruit surface covered with water-soaked lesions, with a distinct halo zone and concentric rings of dark acervuli. The disease index (DI) was calculated using the formula DI (%) = [Σ(i × Ni)/(N × S)] × 100, where i represents the disease severity rating (0–4), Ni is the number of fruits with a rating of i, N is the total number of fruits assessed, and S is the highest disease severity scale (4). The effective concentrations required to inhibit 50% (EC₅₀) and 95% (EC₉₅) of C. gloeosporioides were determined using dose–response analysis. The antifungal activity of the formulation at different concentrations was expressed as percentage inhibition relative to the untreated control. The data were analyzed using Probit analysis with POLO Plus, and EC₅₀ and EC₉₅ values were estimated from the fitted model.

2.4. Influence of Allamanda Microemulsion on the Physicochemical Characteristics of Papaya Fruits

The physicochemical characteristics of papaya fruits coated with selected Allamanda microemulsion formulations (AM) were assessed. The experiment was conducted in a completely randomized design with six replicates per treatment. Treatments consisted of AM1, AM2, and AM8 at 7% (v/v), with sterile distilled water and commercial fungicide benomyl serving as the negative and positive controls, respectively. Papaya fruits at color stage two were immersed in the respective treatments and allowed to air dry for 30 min. The treated fruits were packed in cardboard boxes and stored at 10 °C and 80% RH for 30 days. Data were recorded at 0, 5, 10, 15, 20, 25, and 30 days of storage. The number of replicates varied depending on the physicochemical parameter evaluated and is stated in the corresponding subsections below.

2.4.1. Weight Loss

Fruit weight was measured every five days using a top pan balance. Each treatment consisted of six replicates, with ten fruits in each replicate, and the same fruits were monitored throughout the experimental period. The percentage of weight loss was calculated using the following formula: Weight loss (%) = [(W1 − W2)/W1] × 100, where W1 is the initial weight of the fruit at day 0 and W2 is the weight of the fruit at the respective storage period.

2.4.2. Firmness

Firmness of the fruits in all six treatments was determined using an Instron Universal Testing Machine (Model 5543, Instron Corp, Norwood, MA, USA), supported by Instron Merlin Software version M12-13664-EN, using compression mode. Three replicates were used for each treatment. The diameter of the cylindrical probe used was 6 mm, which was programmed to penetrate in a normal direction at a crosshead speed of 20 mm min⁻¹. Measurements were recorded on slices with 2 cm thick peel and pulp cut horizontally. The mean compression force was measured at the maximum peak of recorded force expressed as Newton (N).

2.4.3. Peel Color

Peel color was measured using a Chroma Meter (Model CR-300, Minolta Corp., Tokyo, Japan), calibrated against a standard white tile (L* = 97.30, C* = 1.88, h° = 85.8). Three replicates were used for each treatment. Measurements were taken at three positions on each fruit: the stem end, equatorial region, and blossom end. Color parameters were recorded as lightness (L*), chroma (C*), and hue angle (h°), representing brightness, color intensity, and hue of the fruit surface, respectively.

2.4.4. Soluble Solids Concentration (SSC)

Concentration of soluble solids was determined every five days until the 30th day of storage. Fruit juice was prepared by blending the fruit tissue. The SSC was measured using a palette digital refractometer (Atago Co., Ltd., Tokyo, Japan). Filtrated juice drops were placed on the glass prism of the refractometer to obtain the SSC reading (%). The readings were recorded at room temperature after standardization using distilled water. Three replicates per treatment were used and results were expressed as °Brix.

2.4.5. Statistical Analysis

Data were subjected to analysis of variance (ANOVA) using SAS software version 9.4. When significant differences were detected at the 0.05 significance level, treatment means were separated using the least significant difference (LSD) test. The number of replicates used for each physicochemical parameter was as described in the corresponding subsections.

3. Results

3.1. Ternary Phase Diagrams and Point Selection of Allamanda Formulations

Ternary phase diagrams were constructed to determine the isotropic microemulsion regions formed by the surfactant, oil, and water components. Seven phase diagrams consisting of surfactants (Agnique MBL 510H and/or Agnique MBL 530H), oil (Agnique AMD 810), and water were obtained (Figure 1). The constructed phase diagrams showed isotropic regions ranging from 33% to 66%. The largest one-phase region (66%) was observed in the system containing 90% Agnique MBL 510H and 10% Agnique MBL 530H with Agnique AMD 810 and water, whereas the smallest one-phase region (33%) was obtained in the system containing Agnique MBL 530H, Agnique AMD 810, and water. Larger isotropic regions were generally observed in systems containing mixed surfactants (Agnique MBL 510H and Agnique MBL 530H) compared with systems containing a single surfactant. Based on the isotropic regions obtained from the seven phase diagrams, eight formulation points were selected. The selected formulations were coded as AM1, AM2, AM3, AM4, AM5, AM6, AM7, and AM8.

3.2. Stability, Surface Tension, and Particle Size Measurements of Formulations

From the seven phase diagrams constructed, eight formulations (AM1, AM2, AM3, AM4, AM5, AM6, AM7, and AM8) were obtained, and all formulations remained stable after four weeks. At room temperature, the surface tension of all selected formulations was significantly lower than that of the control (water), which had a surface tension of 72 mN/m (

Table 2. Composition and characterization of formulations.

Formulation	Composition (%w/w)						Surface Tension (mN/m)	Particle Size (nm)	Stability (>4 Weeks)
	Ag. MBL 510H	Ag. MBL 530H	Ag. AMD 810	Water	* ACLCE	Total
AM1	19.5	-	19.5	26	35	100	30.5	88.83	√
AM2	-	13	19.5	32.5	35	100	29.2	59.67	√
AM3	11.7	1.3	32.5	19.5	35	100	30	1801.05	√
AM4	5.85	0.65	45.5	13	35	100	29.1	124.9	√
AM5	10.4	2.6	26	26	35	100	29.2	1151.71	√
AM6	13.65	5.85	13	32.5	35	100	30	59.77	√
AM7	7.8	5.2	26	26	35	100	29.5	152.94	√
AM8	6.5	6.5	26	26	35	100	29.5	51.79	√

* ACLCE = Allamanda concentrated liquid crude extract; √ = stable under room temperature.

). AM1 showed the highest surface tension (31.2 mN/m) among the formulations. The lowest surface tension value (28.5 mN/m) was observed in AM2, AM3, and AM7, followed by AM4 and AM6 (29.0 mN/m), AM8 (29.2 mN/m), and AM5 (29.3 mN/m). Particle sizes of the selected formulations ranged from 51.79 to 1800 nm (Table 2). The smallest particle size was detected in AM8 (51.79 nm). AM2 and AM6 showed similar particle sizes of 59.67 and 59.77 nm, respectively. Larger particle sizes ranging from approximately 100 to 1800 nm were observed in AM4, AM7, AM5, and AM3. Among the formulations tested, AM8 exhibited the smallest particle size while maintaining relatively low surface tension.

3.3. Effect of Allamanda Formulations on Anthracnose Disease in Papaya

Papaya fruits coated with Allamanda biofungicide formulations significantly reduced (p < 0.05) anthracnose incidence caused by C. gloeosporioides compared with untreated fruits and fruits treated with the commercial fungicide benomyl. Control fruits treated with water showed 75% disease incidence, whereas fruits treated with benomyl showed 42% disease incidence. Disease incidence, severity, and disease index decreased with increasing concentrations of the Allamanda formulations. All fruits treated with the eight formulations at 10% concentration showed significantly lower disease incidence (0–17%) and disease index (0–17%), while the disease severity score remained at 0 (Figure 2;

Table 3. Effect of Allamanda emulsion formulations on the severity score of anthracnose disease in papaya after 8 days of storage at ambient temperature.

Concentrations (%)	Allamanda Emulsion Formulations
	* C−	* C+	AM1	AM2	AM3	AM4	AM5	AM6	AM7	AM8
5	2	1	1	1	0	0	0	0	0	0
7	2	1	1	1	0	0	0	1	0	0
10	2	1	0	0	0	0	0	0	0	0

* C⁻ = negative control (water); C+ = positive control (Benomyl).

). No significant differences were observed among the formulation concentrations tested (5, 7, and 10% v/v). Among the formulations, AM4 showed complete suppression of anthracnose development with 0% disease incidence. However, fruits treated with AM4 showed undesirable quality characteristics, including shrivelling and slower peel color development. AM8 also showed strong antifungal activity compared with the control treatments (water and benomyl). The visual appearance of untreated and AM8-treated papaya fruits is shown in Figure 3.

Disease severity scores (Table 3) were consistently low across treatments, with most values recorded as 0. Due to the limited variability and presence of zero variance in several treatments, statistical comparison was constrained and results are presented descriptively. The disease index (DI) further confirmed the antifungal efficacy of the Allamanda microemulsion formulations (

Table 4. Disease index (%) of anthracnose in papaya fruits treated with Allamanda formulations.

Treatment	Disease Index (%) *
Negative Control (water)	75
Positive Control (Benomyl)	42
Allamanda formulations	0–17

* Disease index values were derived from severity scoring and expressed as percentage disease intensity. Values are presented as ranges due to minimal variability among replicates.

). Untreated control fruits exhibited a high disease index of 75%, indicating severe infection by C. gloeosporioides. In contrast, fruits treated with the synthetic fungicide benomyl showed a reduced disease index of 42%. Notably, papaya fruits treated with Allamanda formulations across all tested concentrations exhibited markedly lower disease index values ranging from 0 to 17%, indicating substantial suppression of disease development. These results are consistent with the observed reductions in disease incidence and severity, confirming the effectiveness of the formulation in controlling anthracnose.

The antifungal efficacy of the Allamanda microemulsion formulation was further quantified through dose–response analysis. The formulation exhibited a clear concentration-dependent inhibitory effect against C. gloeosporioides, with increasing concentrations resulting in progressively greater reductions in disease incidence and severity. Based on Probit analysis, the EC₅₀ and EC₉₅ values were estimated at 1.839 and 7.067 mg/mL (w/v), respectively, indicating strong antifungal activity of the formulation (

Table 5. Effective concentration (EC) values of Allamanda microemulsion formulation against Colletotrichum gloeosporioides.

Effective Concentration (EC) *	Value (mg/mL, w/v)
EC50	1.839
EC95	7.067

* Values were determined from probit analysis using POLO Plus.

). The relatively low EC₅₀ value demonstrates high potency, as effective inhibition was achieved at low concentrations. These findings are consistent with the observed reduction in disease parameters and confirm the effectiveness of the optimized formulation (AM8) as a promising biofungicide for controlling papaya anthracnose.

3.4. Effect of Allamanda Formulations on Fruit Quality and Storage Life of Papaya

A significant (p < 0.05) reduction in weight loss was observed in AM1 (4.2%), followed by AM8 (5.4%), AM2 (5.4%), and benomyl (5.5%), compared with the control (6.0%) at the end of the storage period (Table 4). AM8 maintained the highest fruit firmness (3.6 N) after storage. Fruits treated with AM8 also showed lower L* and C* values and a higher h° value, indicating slower peel color development during ripening. Coating with Allamanda formulations delayed the ripening process during storage (10 °C, 80% RH) under commercial packaging conditions. Treated fruits showed reduced weight loss, slower changes in peel color, and higher firmness compared with the control (

Table 6. Physicochemical characteristics of papaya after being treated with Allamanda microemulsion biofungicide (AM1, AM2, and AM8) after 30 days of storage in commercial packaging at 10 °C, 80% RH.

Treatments	Weight Loss (%)	Firmness (N)	L*	C*	h°	SSC (°Brix)
Control	6.0 a	1.55 a	44.86 a	23.27 a	109.93 b	3.33 a
Benomyl	5.5 b	2.43 c	43.43 b	20.35 b	108.52 b	2.21 a
AM1	4.2 c	1.77 ab	43.22 b	20.31 b	107.82 b	2.07 a
AM2	5.4 b	2.69 d	41.16 c	18.21 c	107.81 b	2.11 a
AM8	5.4 b	3.61 e	40.53 c	16.51 c	117.69 a	2.17 a

L* = lightness; C* = chroma; h° = hue angle. z Within each respective treatment, means followed by the same letters are not significantly different (LSD test at p < 0.05).

). These treatments helped maintain papaya quality for up to 30 days of storage without affecting soluble solids concentration (SSC).

4. Discussion

4.1. Formulation Development Based on Ternary Phase Diagrams

Formulation of antifungal substances is an important aspect in the development of biofungicides because the successful delivery of active compounds, shelf life, stability, and effectiveness under commercial conditions depend largely on the quality of the formulation. A suitable formulation should be economical to produce, contain sufficient active ingredient to be effective, and be easy to handle and apply to fruits. In commercial applications, the success of biological control agents depends greatly on formulation quality, which determines product stability, delivery efficiency, and field performance [16].

Ternary phase diagrams were constructed using alkyl polyglucoside (APG) surfactants (Agnique MBL 510H and/or Agnique MBL 530H), dimethylamide carrier oil (Agnique AMD 810), and water to identify isotropic microemulsion regions suitable for formulation development. The wider homogeneous isotropic regions obtained with mixtures of Agnique MBL 510H and Agnique MBL 530H indicated greater efficiency of the mixed surfactant system compared with single-surfactant systems. In contrast, Agnique MBL 510H or Agnique MBL 530H used alone showed poorer miscibility with water and narrower one-phase regions. This suggests that interactions between the mixed surfactants improved interfacial packing and reduced interfacial tension, leading to broader isotropic microemulsion regions [17,19].

Among the tested systems, the mixed surfactant system containing Agnique MBL 510H and Agnique MBL 530H produced the widest isotropic region, whereas the single-surfactant system produced the smallest region. The wider isotropic regions obtained with mixed surfactants may be associated with improved surfactant packing and increased hydrophilicity of the mixtures. Since microemulsion points were selected from these isotropic regions, the broadening of the one-phase area was advantageous for identifying stable and practical formulations. In addition, the use of a relatively high oil content may reduce the amount of surfactant required, thereby contributing to more cost-effective formulations. The oil phase also plays an important role in enhancing formulation performance because suitable carrier oils can improve the solubilization and dispersion of hydrophobic active ingredients within microemulsion systems [20].

4.2. Stability, Surface Tension, and Particle Size of Allamanda Formulations

All eight formulations selected from the isotropic regions remained stable after four weeks, indicating acceptable short-term physical stability. In addition, all microemulsion and nano-sized formulations containing the active ingredient (ACLCE) remained stable at room temperature for up to one year, demonstrating good physicochemical stability during storage. Surface tension is an important characteristic of microemulsion formulations because it influences wetting, spreading, and penetration on the fruit surface. In this study, all formulations exhibited much lower surface tension values (29–31 mN/m) than water (72 mN/m), indicating improved interfacial activity. Lower surface tension facilitates improved wetting and spreading of droplets across the fruit surface, thereby enhancing surface coverage and improving contact between the formulation and the target surface during application [21]. Although the formulations showed relatively similar surface tension values, the substantial reduction compared with water suggests that all selected systems possessed improved application potential.

Particle size further differentiated the formulations. Considerable variation in droplet size was observed among the systems, with some formulations producing nano-sized droplets while others formed much larger dispersed structures. Formulations such as AM8, AM2, and AM6 were characterized by relatively small droplet sizes, whereas AM3 and AM5 exhibited substantially larger droplets. The formation of larger droplets may be attributed to instability of the interfacial film, aggregation, and coalescence during dilution, which can lead to the development of larger dispersed structures [15,21]. Several formulations exhibited droplet sizes below 100 nm and could therefore be considered nano-sized systems. In this study, the term microemulsion refers to the formulation system based on phase behavior, whereas nano-sized formulations refer to those with droplet sizes below 100 nm. Smaller droplet sizes are desirable because they generally improve solubilization of hydrophobic active compounds and may enhance their delivery and dispersion efficiency. Among the tested formulations, AM8 combined the smallest particle size with acceptable surface tension and stability, supporting its selection as the best formulation.

4.3. Effect of Allamanda Formulations on Anthracnose Control in Papaya

The success of a biofungicide depends not only on the stability and delivery efficiency of its formulation but also on its ability to effectively suppress the target pathogen under practical storage and postharvest handling conditions [7]. All Allamanda-based formulations effectively reduced anthracnose incidence and severity compared with untreated and benomyl-treated fruits. These findings demonstrate the strong antifungal potential of the Allamanda formulations in controlling C. gloeosporioides during storage. The observed reduction in disease development may be attributed to the formation of a protective coating on the fruit surface, which can act as a physical barrier that limits pathogen attachment, germination, and penetration. Such protective effects have been widely reported for fruit coatings and bio-based formulations used in postharvest disease management [9]. In addition to acting as a barrier, the coating may also influence fruit physiology by slowing senescence and reducing tissue susceptibility to infection [8].

Although AM4 showed the greatest disease suppression, it was not considered the most suitable formulation because coated fruits exhibited undesirable quality changes such as shrivelling and delayed peel color development. Similar effects have been reported where excessive coating concentrations may negatively influence fruit appearance and ripening behavior [22]. This finding highlights that the most biologically active formulation is not always the most commercially practical. In contrast, AM8 provided strong disease control while maintaining acceptable fruit quality, making it the most promising formulation. This is further supported by the low disease index values (0–17%) observed in Allamanda-treated fruits, indicating effective suppression of disease progression. The predominance of zero severity scores across treatments limited statistical differentiation but clearly reflects the strong antifungal efficacy of the formulation. The antifungal efficacy of AM8 was further supported by toxicity analysis, where relatively low concentrations were sufficient to achieve substantial inhibition of anthracnose development. Moreover, lesion development in AM8-treated fruits progressed more slowly than in both benomyl-treated and untreated fruits, indicating its potential as an effective alternative biofungicide for postharvest anthracnose management. Such coating-based strategies are widely reported to reduce disease development and delay ripening by forming protective barriers and modifying physiological processes during storage [23,24].

The dose–response analysis demonstrated that the Allamanda microemulsion formulation exhibited strong antifungal activity against C. gloeosporioides, as reflected by the relatively low EC₅₀ value (1.839 mg/mL). This indicates that effective inhibition of the pathogen can be achieved at comparatively low concentrations, highlighting the high potency of the formulation. The EC₉₅ value (7.067 mg/mL) further confirms that near-complete suppression of fungal growth is attainable within a practical concentration range. Such concentration-dependent antifungal responses are commonly reported in plant-derived bioactive compounds [25]. The observed antifungal efficacy is likely attributed to the presence of bioactive compounds in A. cathartica, such as iridoids, flavonoids, and phenolic constituents, which have been widely reported to exhibit anti-microbial properties [26]. In addition, the microemulsion system may enhance the bioavailability and penetration of these active compounds, thereby improving their interaction with fungal cells and increasing overall efficacy [27]. These findings are consistent with the concentration-dependent reductions in disease incidence and severity observed in treated papaya fruits, supporting the reliability of the EC values obtained through Probit analysis. Overall, the results demonstrate that the optimized formulation (AM8) has strong potential as an effective and sustainable biofungicide for the postharvest control of papaya anthracnose.

4.4. Effect of Allamanda Formulations on Fruit Quality and Shelf Life

In addition to effective disease control, preservation of fruit quality is a critical requirement for any postharvest treatment intended for commercial application. The Allamanda-based formulations improved the storage performance of papaya fruits by maintaining key quality attributes during storage.

Weight loss is a major factor influencing postharvest quality because harvested fruits continuously lose water through transpiration, leading to shrinkage and deterioration of visual appearance during storage [28]. The reduced weight loss observed in coated fruits suggests that the microemulsion formulations formed a semi-permeable barrier on the fruit surface, thereby limiting water vapor diffusion and reducing moisture loss. Edible coatings and bio-based films are widely reported to function as protective barriers against moisture and gas exchange, thereby reducing dehydration and extending the storage life of fresh produce [24,29].

Fruit firmness was also better maintained in coated fruits compared with untreated fruits. Maintenance of firmness is an important quality attribute because it reflects delayed softening and improved resistance to mechanical damage during handling and transportation. Fruit softening during storage is generally associated with enzymatic degradation of cell wall components and metabolic changes occurring during ripening [30]. Edible coatings have been shown to slow these processes by inhibiting the activity of softening enzymes and reducing oxidative stress, thereby helping preserve the structural integrity of the fruit tissue [31]. The improved firmness observed in fruits treated with the Allamanda formulations therefore suggests that the microemulsion coating not only suppressed pathogen development but also moderated physiological processes associated with ripening.

Peel color parameters further indicated that the coatings influenced ripening behavior during storage. The lower L* and C* values and higher h° values observed in coated fruits suggest a slower transition from green to yellow compared with untreated fruits. Such delayed color development is commonly associated with reduced respiration rates and modified internal gas exchange resulting from the semi-permeable barrier formed by the coating [23]. By moderating oxygen diffusion and metabolic activity, edible coatings can slow chlorophyll degradation and pigment synthesis, thereby delaying visible ripening [23,31].

In contrast, soluble solids content was not significantly affected by the treatments, indicating that the sweetness and eating quality of the fruits were preserved despite delayed ripening [23,24]. Maintenance of soluble solids during storage is a desirable commercial attribute because it suggests that fruit flavor and consumer acceptability are not compromised by the treatment [28,31]. Overall, the results indicate that the Allamanda-based formulations, particularly AM8, were able to extend the postharvest storage life of papaya for up to 30 days while maintaining key quality attributes. This highlights their potential as dual-function treatments providing both disease control and quality preservation.

4.5. Toxicity and Formulation Stability

Based on formulation characteristics, antifungal efficacy, and fruit quality performance, AM8 was identified as the most suitable formulation. Although some formulations, particularly AM4, showed slightly stronger disease suppression, AM8 provided the best overall balance between effective anthracnose control and maintenance of marketable fruit quality. This balance is particularly important for commercial postharvest treatments, where both disease management and preservation of fruit appearance are essential [6,22].

The selected formulation also demonstrated favorable stability characteristics. Physicochemical stability of the microemulsion system was maintained for up to one year at room temperature, while effective disease-control performance was retained for at least six months of storage. Such stability is a key requirement for practical application of biofungicides, as successful postharvest biological treatments must remain stable during storage, transportation, and commercial handling while maintaining biological activity [32].

The strong performance of AM8 may also be associated with its nano-sized droplet characteristics. Formulations containing droplets in the nanometer range provide a large interfacial area and improved dispersion of hydrophobic active compounds, which can enhance the solubilization, distribution, and retention of bioactive ingredients on treated surfaces [33]. Improved surface coverage may therefore facilitate more effective interaction between the antifungal compounds and the pathogen [34], contributing to the observed inhibition of C. gloeosporioides.

These findings further support earlier reports demonstrating the antifungal activity of A. cathartica extracts against C. gloeosporioides [14,15] and highlight the advantage of microemulsion formulation in improving the delivery and practical application of plant-derived antifungal compounds. Overall, the results demonstrate that the optimized Allamanda-derived formulation represents a promising eco-friendly microemulsion biofungicide for the postharvest control of papaya anthracnose. In addition to effective disease suppression, the formulation maintained key fruit quality attributes and extended storage life, indicating its suitability for sustainable postharvest disease management. The successful integration of Allamanda-derived antifungal compounds into a stable microemulsion system highlights the potential of formulation technology to enhance the practical application of botanical biofungicides in postharvest disease management.

5. Conclusions

A novel botanical biofungicide based on Allamanda cathartica was successfully developed using a microemulsion formulation system. The optimized formulation demonstrated strong antifungal activity against Colletotrichum gloeosporioides, the causal agent of papaya anthracnose, while maintaining fruit quality during storage. Application of the Allamanda nanoemulsion reduced disease development without adversely affecting fruit firmness or soluble solids content, thereby contributing to extended postharvest storage life. These findings highlight the potential of Allamanda-based microemulsion formulations as eco-friendly and sustainable alternatives to conventional chemical fungicides for postharvest disease management in papaya.

6. Patents

The optimized formulation AM8 developed in this study is protected under a Malaysian patent (Patent No. PI2011004439).