Mexico is the leading exporter of fresh papaya to the United States and Canada (OEC, 2022). The state of Oaxaca ranks first in national production, with an average yield of 100 t ha⁻¹. Within the state, the Costa region stands out for its significant contribution, accounting for about 80% of total production. The main producing municipalities-Villa de Tututepec, Santa María Huazolotitlán, Santiago Pinotepa Nacional, and Santiago Jamiltepec-together comprise a harvested area of 2 787 ha (SIAP, 2024). Papaya cultivation in this region represents an important source of economic activity, largely due to its high demand for labor. Each hectare is estimated to require around 500 workdays per production cycle, underscoring its regional economic impact (Alarcón-Pulido et al., 2022). Papaya production faces major phytosanitary challenges, particularly anthracnose, caused by Colletotrichum species. This disease is characterized by dark lesions on fruits accompanied by acervuli arranged in concentric rings (Tapia-Tussell et al., 2008). It can affect up to 40% of total yield (Maharaj and Rampersad, 2012), leading to the intensive use of highly toxic fungicides, mainly to control C. gloeosporioides (Landero-Valenzuela et al., 2016). However, recent evidence suggests that the disease may involve a complex of Colletotrichum species, highlighting the need for further research to confirm this (Zakaria,

2021).

Anthracnose management in tropical crops such as papaya, mango (Mangifera indica), avocado (Persea americana), and banana (Musa × paradisiaca) has relied primarily on synthetic fungicides. This dependence raises concerns about toxic residues in fruits and underscores the lack of comprehensive management strategies. Although biocontrol agents offer a more sustainable alternative, they still face challenges related to consistency and effectiveness (Crouch et al., 2014). In this context, the present study aimed to identify the causal agent of anthracnose in papaya and assess the efficacy of biological and chemical products in reducing disease severity.

Collection of infected fruits. Fruits of papaya showing typical anthracnose symptoms were collected from 12 sites located in the municipalities of Villa de Tututepec de Melchor Ocampo and Santa María Huazolotitlán, in the Costa region of Oaxaca.

Fungal isolates. Fruits showing visible symptoms were washed several times with sterile distilled water and liquid soap to remove dust and impurities, then disinfected with 96% ethyl alcohol for three minutes. Brown to black lesions with orange-black acervuli were selected. From each lesion, a 1-mm fragment of symptomatic tissue was excised and placed on Petri dishes containing Potato Dextrose Agar (PDA, BIDICO®) medium. The plates were labeled and incubated for seven days to promote fungal growth. All plates were incubated at 25 °C in complete darkness. After four days of growth, actively growing hyphal tips from disinfected tissues were subcultured onto fresh PDA plates to obtain axenic cultures.

Taxonomic identification. Identification of the isolates was carried out at the Soil Laboratory of CIIDIR-IPN, Oaxaca. For cultural and morphological characterization, the microculture technique described by Aquiahuatl-Ramos et al. (2012) was used. This method involves culturing small portions of mycelium on PDA agar blocks placed on microscope slides inside a humid chamber, allowing microscopic observation of hyphae, conidiophores, and reproductive structures with minimal disturbance and under conditions resembling natural growth. Taxonomic identification was performed using the keys of Ainsworth et al. (1973), Sutton (1992), and Barnett and Hunter (1998).

In vitro sensitivity and effectiveness analysis. To evaluate in vitro sensitivity, the agar disk diffusion method was used following the procedure described by Davis and Stout (1971), as adapted by Adaskaveg and Hartin (1997). Three commonly used field fungicides and three biological control agents were tested, each applied at three concentrations (high, medium, and low). The specific doses for each treatment are detailed in Table 1.

Table 1

Product / Microorganism	High concentration	Medium concentration	Low concentration
	g L-1	L-1	g L-1
Prochloraz	3	2	1
Benomyl	0.6	0.4	0.3
Azoxystrobin	0.3	0.2	0.1
Trichoderma sp.	1.5	1.2	1
	9.0 × 106 conidia mL-1	7.2 × 106 conidia mL-1	6.0 × 106 conidia mL-1
Bacillus subtilis	4.0 × 109 UFC mL-1	3.0 × 109 UFC mL-1	2.0 × 109 UFC mL-1
Bacillus cereus	1.5 × 108 UFC	1.2 × 108 UFC	1.0 × 108 UFC

Two previously identified fungal strains (C. gloeosporioides and C. truncatum) were used. PDA plates were inoculated with 200 µL of a spore suspension adjusted to 1 × 10⁶ spores mL⁻¹, prepared by counting in a Neubauer chamber and adjusted to the required concentration using the dilution equation:

V 2 = C 1 *   V 1 C 2

Where C₁ is the initial concentration, V₁ the initial volume, C₂ the desired final concentration, and V₂ the required final volume.

On each plate, four filter paper disks (6 mm) were placed and impregnated with 10 µL of each treatment, leaving one disk with distilled water as a control. The plates were incubated at 25 ± 1 °C for seven days, after which the inhibition halos were measured. Effectiveness (%) was calculated using Abbott’s formula (1925), comparing the growth of each treatment with that of the control.

normalE normalf normalf normale normalc normalt normali normalv normale normaln normale normals normals   ( % ) = ( X c - X t X c ) * 100

Where X _c = value observed in the control treatment, and X _t = value observed in the tested treatment.

In vivo assays in papaya for the control of Colletotrichum spp. under laboratory conditions. Five fruits per treatment were selected at ripeness stage 5 according to Salazar (2002). After washing and disinfection, the fruits were allowed to dry for 10 minutes, and the treatments described in Table 1 were applied by spraying; the control consisted of fruits treated only with sterile distilled water. The fruits were incubated for 10 days at 25 ± 1 °C and 75% relative humidity. The experimental design was factorial, considering six antifungal treatments and four evaluation times. Product effectiveness was determined after 10 days based on the number of positive infections per fruit, using Abbott’s formula (1925). Disease severity was assessed by measuring the diameter of lesions caused by C. gloeosporioides and C. truncatum and classifying them according to the five-grade scale of Castellanos et al. (2014), based on the affected area (mm²) (Table 2).

Table 2

Grade	Injury area (mm)	Damage description
1	0	No visible damage
2	1 – 15	Very small injuries
3	16 – 30	Moderate injuries
4	31 – 45	Extensive injuriess
5	> 45	Very severe injuries

Treatment effectiveness was evaluated 10 days after inoculation using Abbott’s formula (1925). Disease severity was quantified by measuring lesion diameter and classifying fruits according to the five-grade scale of Castellanos et al. (2014). In addition, the severity index was calculated using the formula of Anculle and Álvarez (1999), considering both the frequency and degree of damage in the evaluated fruits:

I S ( % ) = ( false∑ n F x g ) N * G * 100

Where IS = severity index; Nf = number of fruits in each grade; G = damage grade; N = total number of fruits evaluated; and Gₘₐₓ = highest grade on the scale.

Data analysis. Data on in vitro and fruit inhibition were transformed (√(x+1) and cosine, respectively) and analyzed under a completely randomized design. For the fruit assay, a factorial arrangement (6 × 4) was applied, followed by ANOVA and mean comparison (Tukey or Duncan, p ≤ 0.05) using SAS® 9.4. The severity index was used solely as a descriptive indicator.

Identification of isolates. C. gloeosporioides was identified in 66.7% of the sites and C. truncatum in the remaining 33.3%, based on morphological characteristics, as associated with anthracnose in papaya fruits. Colletotrichum gloeosporioides produced cylindrical- fusiform conidia (14.5-17.02 µm) and white-to-black colonies (29.55-56.38 mm after seven days). In contrast, C. truncatum exhibited curved, hyaline conidia (22.8-23.8 µm) and white-to-black colonies with radial growth (22.3-41.1 mm after seven days) (Figure 1). Molecular identification of the isolates is necessary for confirmation.

Figure 1

In vitro sensitivity and effectiveness of fungicides and biological agents. Table 3 shows the results of the in vitro assays, which revealed clear differences in the sensitivity of C. gloeosporioides and C. truncatum to the evaluated treatments. Prochloraz was the most effective, producing inhibition halos of 14 mm (48.66%) in C. gloeosporioides and 25-27 mm (86.42%) in C. truncatum.

Table 3

Source of variation	DF	Day
		3	4	5	6	7	8	9	10
Product	5	1.141**	3.225**	4.588**	7.880**	9.209**	13.157**	17.345**	17.985**
Injury	3	1.393**	5.048**	8.410**	9.234**	8.945**	13.873**	17.345**	24.967**
Product*
Injury	15	0.318ns	0.321ns	0.365ns	0.720ns	0.703ns	1.229ns	17.345ns	1.773ns
Mistake	96	0.306	0.43	0.991	1.204	1.504	1.716	17.345	2.213
Total	119
CV (%)		46.723	47.071	55.33	58.024	59.319	57.459	50.705	48.269

Where: (P < 0.05) according to Tukey’s multiple range test; * = significant at 0.05, ** = highly significant at 0.001, ns = not significant.

In the experiment with C. gloeosporioides, high-dose azoxystrobin produced similar inhibition halos (14 mm; 29.81%), while B. subtilis and B. cereus achieved 11-13 mm (73.47% and 50.87%, respectively). Trichoderma sp. showed intermediate inhibition against C. gloeosporioides (9-11 mm; 27.65%), whereas benomyl and low-dose azoxystrobin were the least effective (<7 mm; <20%). For C. truncatum, B. subtilis (25-27 mm; 28.97%) and Trichoderma sp. (20-23 mm; 71.34%) were the most notable, with prochloraz being the most effective treatment against both species. Overall, B. subtilis and Trichoderma sp. exhibited greater control potential for C. truncatum (Figure 2A and B).

Figure 2

Effectiveness and severity of fungicides in Carica papaya var. Maradol fruits. Analysis of variance revealed highly significant differences (P < 0.001) among treatments throughout the evaluation period (3-10 days after inoculation), indicating a marked effect on the inhibition and progression of lesions caused by C. truncatum. Disease severity also showed a continuous and significant increase (Table 3).

For C. gloeosporioides, analysis of variance showed significant differences among treatments from day 5 post-inoculation (P < 0.05; P < 0.001), indicating their effect on lesion progression. The lesion factor was highly significant, with no treatment × lesion interaction (P > 0.05), reflecting uniform behavior across sites. The coefficients of variation (44.9-54.3%) correspond to the natural variability of fresh tissue (Table 4).

Table 4

Source of variation	DF	Day
		3	4	5	6	7	8	9	10
Product	5	0.563ns	1.505*	3.969**	4.229*	7.361**	6.744*	5.882*	8.296*
Injury	3	3.378**	8.904**	17.604**	22.457**	30.895**	32.674**	40.082**	43.353**
Product*Injury	15	0.206ns	0.4655ns	0.420ns	0.880ns	0.990ns	1.206ns	1.555ns	1.170ns
Mistake	96	0.442	0.669	0.958	1.351	1.705	2.207	2.153	2.606
Total	119
CV (%)		49.726	52.811	52.284	54.337	54.134	53.807	46.713	44.897

Where: (P < 0.05) according to Tukey’s multiple range test; * = significant at 0.05, ** = highly significant at 0.001, ns = not significant.

In fruits inoculated with C. truncatum, significant differences were observed among treatments. B. cereus showed the largest lesions (2.74-20.02 mm), whereas prochloraz exhibited the least progression (<1.0 mm initially; 1.54-5.58 mm on day 10), followed by benomyl. Trichoderma sp. and B. subtilis presented intermediate values, with site 1 being the most affected (17.73 mm) compared to site 4 (6.28 mm) (Table 5).

Table 5

Product	Day
	3	4	5	6
B. cereus	2.739±1.096a	5.006±1.263a	7.441±1.475a	9.26±1.885a
B. subtilis	0.920±0.646ab	0.702±0.533b	3.329±1.256ab	4.414±1.530ab
Benomyl	0.686±0.523ab	1.0775±0.614b	1.795±0.735b	1.548±0.854b
Azoxytrobin	0.284±0.284b	1.3975±0.652b	4.241±1.205ab	5.301±1.497ab
Trichoderma	0±0b	0.979±0.56b	3.07±0.985b	4.094±1.172ab
Prochloraz	0±0b	0.341±0.341b	0.977±0.551b	0.521±0.52b
Injury:
1	1.9243±0.633ab	3.987±0.962a	6.464±0.881a	7.844±1.141a
2	0.842±0.639ab	1.724±0.565b	3.69±0.932ab	3.392±0.877b
3	0.842±0.319ab	0.329±0.2349b	2.073±0.915b	3.264±1.377b
4	0±0b	0.2943±0.294b	1.674±0.825b	2.257±1.0359b
Product	Day
	7	8	9	10
B. cereus	10.488±1.831a	13.469±2.395a	17.736±2.355a	20.024±2.826a
B. subtilis	6.812±1.897ab	7.145±2.103a	11.137±2.342ab	13.954±2.477ab
Benomyl	1.724±0.986b	2.123±1.161ab	4.043±1.501bc	5.579±1.921b
Azoxytrobin	5.62±1.532ab	6.793±1.783b	8.102±1.948bc	11.048±2.398ab
Trichoderma	4.851±1.440ab	8.346±1.761ab	8.892±2.267bc	15.273±2.444a
Prochloraz	1.541±1.064b	1.844±1.271b	3.014±1.773c	5.311±2.286b
Injury:
1	8.105±1.397a	10.564±1.486a	14.717±1.897a	17.726±1.743a
2	6.386±1.23ab	7.878±1.466ab	10.922±1.920a	14.584±2.091ab
3	3.506±1.237b	4.605±1.543b	5.167±1.439b	8.868±2.065bc
4	2.694±1.235b	3.433±1.675b	4.475±1.442b	6.281±2.124c

Means within columns followed by the same letter are not significantly different (P < 0.05) according to Tukey’s multiple ange test. where: * = significant at 0.05; ** = highly significant at 0.001; ns = not significant.

In fruits inoculated with C. gloeosporioides, significant differences were observed among treatments (Table 6). Prochloraz produced the largest lesion diameters throughout the evaluation period (2.38-24.58 mm), whereas benomyl and Trichoderma sp. recorded the smallest values (1.10-8.23 mm). Site 1 exhibited the highest severity (23.99 mm) and site 4 the lowest (6.36 mm). In terms of effectiveness, prochloraz was the most efficient (50-75%), followed by benomyl (45-65%), while Bacillus spp. and azoxystrobin showed limited control (<40%) and higher severity (>70%).

Table 6

Product	Day
	3	4	5	6
Prochloraz	2.38±0.69a	4.19±1.04a	7.61±1.19a	9.07±1.76a
B. subtilis	1.06±0.50a	2.89±0.95a	3.37±1.27ab	5.82±1.73ab
B. cereus	0.94±0.55a	2.26±0.94a	4.25±1.46ab	6.77±1.67ab
Trichoderma	1.57±0.84a	1.72±0.95a	2.70±1.35	2.63±1.51b
Azoxytrobin	0.61±0.42a	1.36±0.74a	3.63±1.21ab	5.26±1.58ab
Benomyl	1.1±0.61a	1.25±0.69a	2.04±0.82b	3.47±1.00b
Injury:
1	3.10±0.654a	5.42±0.90a	8.50±1.22a	10.87±1.40a
2	1.12±0.497b	2.51±0.72b	5.07±0.96b	7.09±1.27ab
3	0.55±0.388b	0.82±0.47b	1.62±0.71c	3.10±1.07bc
4	0.34±0.241b	0.36±0.36b	0.54±0.40c	0.95±0.56c
Product	Day
	7	8	9	10
Prochloraz	13.22±2.24a	15.56±2.56a	18.72±2.80a	24.58±3.90a
B. subtilis	6.550±1.95ab	9.55±2.32ab	12.96±2.48ab	16.88±2.85ab
B. cereus	8.07±1.99ab	6.97±2.26b	11.93±2.53ab	15.71±2.77ab
Trichoderma	4.63±2.01b	6.61±2.45b	9.05±2.63b	13.67±2.78b
Azoxytrobin	8.54±2.16ab	10.46±2.49ab	11.61±2.52ab	14.47±2.58bc
Benomyl	3.34±1.06b	5.66±1.26b	7.93±1.61b	8.23±1.78b
Injury:
1	13.6±1.62a	15.59±1.76a	19.66±1.51a	23.99±1.53a
2	9.85±1.58a	12.13±2.06ab	15.33±2.16a	19.07±2.50ab
3	4.52±1.45b	6.65±1.72bc	8.73±1.88b	12.94±2.46bc
4	1.56±1.02b	2.17±1.05c	4.40±1.49b	6.36±1.95c

Means within columns followed by the same letter are not significantly different (P < 0.05) according to Tukey’s multiple range test. where: * = significant at 0.05; ** = highly significant at 0.001; ns = not significant.

The results demonstrated variability in the efficacy of the evaluated treatments for managing C. gloeosporioides and C. truncatum in papaya fruits. Overall, chemical fungicides showed better performance than biological agents. Prochloraz stood out as the most effective treatment, achieving 75% effectiveness against C. truncatum and 50% against C. gloeosporioides, with relatively low severity levels (60-75%), confirming its ability to reduce disease progression and limit visible fruit damage. Benomyl showed intermediate performance, with 65% effectiveness against C. truncatum and 45% against

C. gloeosporioides, while also maintaining low severity values (60-73%), suggesting that it remains a viable alternative under postharvest conditions, although less effective than prochloraz.

In contrast, the biological treatments showed limited effectiveness. Trichoderma sp. and Bacillus spp. exhibited control percentages below 35% for both pathogens, reflecting a reduced ability to halt infection progression at this phenological stage of the fruit. Moreover, the associated severity values were high, particularly for B. subtilis and Trichoderma sp., which reached 93 and 73% in C. gloeosporioides, and 86 and 93% in C. truncatum, respectively. These results indicate that, under postharvest evaluation conditions, their efficacy was insufficient to contain lesion development-possibly due to the rapid colonization of fruit tissue by Colletotrichum spp., whose aggressiveness poses a challenge for biological agents when applied in a curative manner.

The values recorded for azoxystrobin and B. cereus place these treatments at an intermediate response level, with 30-40% effectiveness and severity ranging from 75- 86%, indicating some suppressive activity, although insufficient for effective postharvest control (Table 7).

Table 7

Treatments	Effectiveness in fruit (%)		Severity (%)
	C. gloeosporioides	C. truncatum	C. gloeosporioides	C. truncatum
Benomyl	45	65	73	60
Trichoderma sp.	35	15	73	93
Procloraz	50	75	75	60
Azoxytrobin	30	40	86	80
B. cereus	30	10	86	75
B. subtilis	25	30	93	86

Overall, the results suggest that the preventive application of chemical fungicides such as prochloraz and benomyl remains the most effective strategy for managing anthracnose in papaya, while biocontrol agents could serve as complementary tools within an integrated management program, particularly at preharvest stages or under repeated application schemes to enhance their establishment and antagonistic activity.

Anthracnose severity in papaya fruits was significantly influenced by treatment and time after inoculation (P < 0.001), with progressive lesion development observed up to day 10. Prochloraz was the most effective treatment, achieving 75% control against C. truncatum and 50% against C. gloeosporioides, followed by benomyl with intermediate efficacy. In contrast, azoxystrobin and the biological agents (Bacillus spp. and Trichoderma sp.) exhibited effectiveness below 50% and severity above 70%, indicating limited postharvest control. These results confirm that systemic fungicides remain the most effective strategy for curative postharvest management, whereas biocontrol agents may be better suited for preventive applications within integrated disease management programs.

Anthracnose in papaya fruits from the coastal region of Oaxaca was caused by C. gloeosporioides in 66.7% of the sampled sites and by C. truncatum in 33.3% of the sites, with both species differing in their response to fungicides and biological antagonists. In the in vitro assays, prochloraz was the most effective treatment, showing 14 mm inhibition in

C. gloeosporioides (48.66%) and 25-27 mm in C. truncatum (86.42%). High-dose azoxystrobin produced 14 mm inhibition in C. gloeosporioides (29.81%). The biological agents exhibited intermediate to low control: in C. gloeosporioides, B. subtilis (11-13 mm; 73.47%) and B. cereus (11-13 mm; 50.87%), while Trichoderma sp. reached 9-11 mm (27.65%). In C. truncatum, Trichoderma sp. performed best with 20-23 mm inhibition (71.34%), surpassing B. subtilis (25-27 mm; 28.97%).

In fruit assays, prochloraz was the most effective treatment, achieving 75% control against C. truncatum and 50% against C. gloeosporioides, followed by benomyl, which showed moderate control. The biological agents exhibited low postharvest effectiveness (<35%) and high disease severity (>70%), particularly B. subtilis and B. cereus. Overall, the results confirm that prochloraz is the most reliable alternative for curative postharvest management, whereas biocontrol agents require preventive applications within an integrated management program to improve their effectiveness.