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Differential Susceptibility of Bean Pods, Bell Pepper Fruits, and

Soybean Leaves Inoculated With Colletotrichum Capsici Enzymes

Jolina A. Enardecido*

1,2

, Maricel J. Didal

1,3

, Ismael T. Cabalinan

1,3

, and Merlina H. Juruena

University of Southeastern Philippines, Tagum-Mabini Campus, Tagum City, 8100, Philippines

Monkayo College of Arts, Sciences, and Technology, Monkayo, Davao de Oro, 8805, Philippines

Department of Agriculture, Regional Crop Protection Center, Trento, Agusan del Sur, 8505, Philippines

*Corresponding Author

DOI: https://doi.org/10.51244/IJRSI.2025.120800352

Received: 05 Sep 2025; Accepted: 12 Sep 2025; Published: 14 October 2025

ABSTRACT

Colletotrichum spp. causes anthracnose diseases in various crops by producing cell wall-degrading enzymes

(CWDE’s). Although these enzymes are known to play a role in pathogenesis, their direct effects on host

tissues and contribution to disease development remain poorly understood. In this study, Colletotrichum

capsici was isolated from bell pepper, and its enzymatic activities were tested on bean pods, bell pepper fruits,

and soybean leaves to determine the role of enzymes in disease development. The study focused on

characterizing the effects of enzymatic tissue degradation based on symptom appearance, disease incidence

percentage, and the infection severity level. Pathogen discs (10 mm) were cultured in Potato Dextrose Broth

(PDB), and crude filtrates (CF) were obtained through sequential filtration. Plant tissues were inoculated with

CF concentrations of 25%, 50%, 75%, and 100%, arranged in a Completely Randomized Design (CRD), and

analyzed using analysis of variance (ANOVA). Results showed that symptom expression occurred as early as

12 hours post-inoculation. At 100% CF, symptoms appeared at 1.75, 2.75, and 0.50 days in bean pods, bell

pepper fruits, and soybean leaves, respectively. Disease incidence reached 93.75%, 58.33%, and 100%, while

severity reached 26.25%, 21.67%, and 68.33% in bean pods, bell pepper fruits, and soybean leaves,

respectively. The study demonstrated that higher CF concentrations consistently accelerated symptom onset

and increased both disease incidence and severity. These results confirm that the pathogenicity of C. capsici is

closely associated with its enzymatic activity, underscoring the critical role of fungal enzymes in host tissue

degradation. This knowledge provides valuable insights for resistance breeding and the development of

enzyme-targeted disease management strategies.

Keywords: Colletotrichum, anthracnose disease, enzymes, crude filtrate, tissue degradation

INTRODUCTION

Colletotrichum spp. are among the major fungal plant pathogens responsible for anthracnose diseases, which

affects a wide range of hosts (Freeman et al., 1998), including peppers (Capsicum spp.). These pathogens can

infect multiple plant organs, including roots, stems, leaves, and fruits. Most crops grown worldwide are

susceptible to one or more Colletotrichum species (Dowling et al., 2020), making anthracnose a significant

global economic constraint to crop production. The disease symptoms are typically characterized by sunken

lesion of varying colors on leaves, stems, fruits, or flowers. These lesions often enlarge, usually leads to

wilting, withering, and eventual death of infected plant tissues (Mehbub et al., 2006). The pathogen is

cosmopolitan in distribution, with primary inoculum disseminated by wind or rain (Ayaa, 2021). During host

invasion, Colletotrichum spp. employ diverse strategies, ranging from intracellular hemibiotrophy to

subcuticular intramural necrotrophy (Jeyaraj et al., 2023). Moreover, they develop specialized infection

structures, including germ tubes, appressoria, intracellular hyphae, and secondary necrotrophic hyphae.

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Enzymes play a critical role in the pathogenicity of Colletotrichum spp. The pathogen produces an arrays of

cell wall-degrading enzymes (CWDEs), such as cellulases, hemicelluloses, pectinases, and proteases, which

breakdown the plant cell wall and facilitate fungal penetration into host tissues (Thaker, 2022). Similarly,

Zhang et. (2022), stated that the pathogen produces cutinases and lipases to breach the cuticle of the host plant,

degrading the waxy cuticle, and thereby allowing the fungus to access the plant tissue. Moreover,

Colletotrichum spp. secretes enzymes involved in the degradation of complex plant polymers, such as

ligninases and xylanases, to degrade complex polymers of the host tissues (Kachroo, 2022). During

colonization of host tissues, Colletotrichum spp. can modify the extracellular matrix of the host plant by

secreting enzymes such as glucanases and chitinase, which facilitates hyphal growth and dissemination within

the host (Riseh et al., 2024).

Among the Colletotrichum species, C. capsici is one of the most frequently reported fungal plant pathogens in

the Philippines (Balendres, 2023). It is a filamentous phytopathogenic fungus, and it secretes several enzymes

during the infection process, primarily targeting the host plant cell wall as part of its initial infection activity,

leading to anthracnose disease (Gu et al., 2024). In this study, Colletotrichum capsici was isolated to determine

the effects of the different concentration level of crude filtrate (CF) containing enzymes on disease

development. Inoculated tissues of bean pods, bell pepper fruits, and soybean leaves were evaluated for the

symptom appearance, disease incidence, and severity of infection. Understanding fungal enzymatic activity in

disease development is essential for advancing knowledge of plant-fungal enzymes interactions and

developing effective disease management.

MATERIALS AND METHODS

Preparation of Culture Medium

The commercial Potato Dextrose Agar (PDA) medium was prepared following the procedure in the label using

39g PDA powder per liter of distilled water. The PDA was sterilized at 15 psi for 20 minutes. This was

conducted at the PCR Laboratory of the University of Southeastern Philippines, Tagum- Mabini Campus,

Apokon, Tagum City, Philippines.

Isolation of the Pathogen

The fungus was isolated from infected bell pepper fruits showing symptoms of anthracnose disease from the

nearby public market. The fruits were washed with tap water, and small sections (3-6 mm long) of the tissue

with lesions were cut off and dipped into previously sterilized petri plates containing 1% sodium hypochlorite

(NaOCl) for one minute, then rinsed thrice with sterile distilled water (SDW) following the method of Peraza-

Sanchez et al., (2005). The disinfected tissue was isolated using the PDA medium and incubated at room

temperature. A microscopic examination was done to confirm the identity of the fungus based on morphology.

Pure culture of C. capsici was characterized by a slow to medium growth, and have olive brown to pinking

colony. The aerial mycelium was soft, sticky, and becoming flat on the upper surface. Conidiophores were

formed as short single phiallides on hyphae. Spores were whitish and elongated.

Preparation of Broth Medium

Potato Dextrose Broth (PDB) was prepared following the procedure of Yokota et al., (2010) using 24g of

commercial PDB added with 1 liter of distilled water and boiled. The broth was filtered using gauze cloth and

supplemented with 10g of dextrose powder. The mixture was transferred to a 250ml Erlenmeyer flask and

sterilized at 15 psi for 20 minutes.

Preparation of Fungal Enzyme

Seven-day old Colletotrichum capsici pure culture was utilized for fungal enzyme production. Agar bits of C.

capsici culture were inoculated and allowed to grow in the sterilized PDB medium. After 7 days, the fungal

mycelia were removed from the medium by filtering, using 3 layers of gauze cloth above and 3 layers of filter

paper below, this will allow the filtrate to drain and then it was centrifuged to separate the supernatant from the

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pellet. The filtrate (supernatant) was collected using a syringe microfilter (0.2um) and used in the tissue

maceration experiment to retain the broth with enzymes. First filtration used triple layered cheesecloth, second

filtration used 3 layered filter paper, and third filtration used a syringe filter. Then, the presence of spores was

microscopically examined to ensure there were no single spores in the filtrate. This was conducted at the

Regional Crop Protection Center (RCPC), DA-Caraga Region.

Inoculation of Fungal Enzyme in Filtrate

After a week of fungal cultivation in PDB, each plant tissues were inoculated with different concentrations of

culture filtrate (CF) of C. capsici by drenching the suspension. Test plant tissues were monitored for a week

until symptoms appeared. The number of days from inoculation of the fungal enzyme to the first appearance of

symptoms was counted and recorded. The incidence was evaluated and recorded regularly until the termination

of the study with the formula %Disease Incidence = No. infected tissue/Total no, of tissue assessed x 100. The

severity of the disease was assessed using the arbitrary rating scale of Miller-Butler et al., (2019) with slight

modifications, where 0-no disease infection, 1- very slight (1-20%) infection symptoms, 2- slight (21-40%)

infection symptoms, 3- moderate (41-60%) infection symptoms, 4- moderately heavy (61-80%) infection

symptoms, 5-heavy (81-100%) infection symptoms. And based on the standard rating scale of Mohamed et al.,

(2000), degree of infection (% Disease Index) was computed as % Disease Index = 0n

+1n

+2n

+3n

+4n

+5n

/ (maximum grade) total population x 100, where: 0n

+1n

…. +5n

refers to the number of samples showing

the rating scale of 0,1,2,3,4, and 5.

Statistical Analysis

This experiment was laid out in a Completely Randomized Design (CRD) with five treatments replicated four

times in varying concentration level of crude filtrate (CF): T

-Untreated, T

-25% CF, T

-50% CF, T

-75% CF,

-100% CF. Data collected was analyzed using the analysis of variance, and further test were done using

Tukey’s Honest Significant Difference (THSD).

Ethical Considerations

The study was conducted in a controlled laboratory setting, and all experiments were designed to minimize

waste and prevent environmental harm. The researchers ensured that all chemicals and materials used in the

study were handled and disposed in accordance with local regulations and guidelines. By acknowledging and

addressing these ethical considerations, the researchers aimed to ensure the integrity of the research process

and contribute to the development of responsible and sustainable agricultural practices.

RESULTS AND DISCUSSION

Days of Symptom Appearance

Bean pods, bell pepper fruits, soybean leaves exhibited symptoms within 120 hours of observation. In bean

pods, the earliest symptoms were observed at 100% crude filtrate (CF), with an average of 1.75 days after

inoculation, while the latest symptoms appeared at 25% CF, averaging 3.88 days after inoculation. These

results suggest that higher concentrations of fungal enzymes accelerated symptom development. In bell pepper

fruits, symptoms appeared at 2.75 days with 100% and 75% CF, whereas 50% CF resulted in symptom

appearance after 4 days, and 25% CF produced no symptoms. Although statistical analysis revealed no

significant differences among treatments, the trend indicated that higher CF concentrations reduced the time to

symptom appearance. A similar pattern was observed in soybean leaves, where 100% CF induced symptoms at

0.50 days, while 25% CF delayed symptom expression to 2.01 days. Again, no significant differences were

detected among treatments (Table 1). Overall, fungal enzymes in the CF influenced the rate of symptom

development differently depending on host tissue and enzyme concentration. In general, higher CF

concentrations tended to accelerate symptom appearance across all hosts, with soybean leaves displaying the

highest sensitivity, showing symptoms earlier and at lower concentrations compared to bean pods and bell

pepper fruits.

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Table 1. Mean number of days of symptom appearance after inoculation of fungal enzymes in the crude filtrate

on PDB filtrate to bean pods, bell pepper fruits, and soybean leaves

TRT

Bean pods

Bell pepper fruits

Soybean leaves

Control

none

25 % CF

3.88

none

2.01

50 % CF

3.06

4.00

2.00

75 % CF

3.01

2.75

1.58

100 % CF

1.75

2.75

0.50

Values are the means of each treatment with four replications; means of the same superscript are not

significantly different;

means not significant; and none means no symptoms.

The number of days to cause symptoms inoculated with enzymes of C. capsici differs depending on several

factors such what enzyme is present, the genetic make-up of the host plant, and the environmental conditions

(Wijesundera et al. 1989). Enzymatic tissue degradation activity of C. capsici relative to symptom appearance

totally occurs within 5-7 days of post-inoculation. As for bean pods and soybean, similar trend was observed

from the study of Prajapati et al., (2014) in which symptom appeared around 4-6 days of post-inoculation of

pathogen filtrate. Another study from Silva et al., (2021), the infection symptoms of tomato inoculated with the

Colletotrichum coccodes enzymes, appeared before 7 days after inoculation, mainly in fruits inoculated with

artificial wound. The morphology of the different plant organs differs, thus soybean leaves with larger, and

have higher number of stomates where the mean stomatal frequency on the adaxial surface was 130; on the

abaxial surface, it was 316 stomata/mm

(Ciha & Brun, 1975), was infected early as compared to bean pods,

and bell pepper fruits. This phenomenon implied that enzymatic tissue degradation activities in the

pathogenicity of C. capsici is crucial to understand for the disease management establishment.

Disease Incidence (%)

Percentage of disease incidence was evaluated to assess the extent and severity of disease within the

population of the specific crop. Table 2 shows the means of the disease incidence percentage on bean pods

after 12, 24, 48, 72, 96 and 120 hours of inoculation of fungal enzyme in the crude filtrate. Results revealed

that 50%, 75%, and 100% CF exhibited disease incidence on the 48

hour after inoculation with 12.50%,

18.75% and 75%, respectively. Tissues inoculated with 25% exhibited disease incidence on the 72

hour after

inoculation with 6.25% while other treatments progressed rapidly. Disease incidence progressed until 120

hour after inoculation with 100% CF bearing the highest disease incidence of 93.75%. It was followed by 75%

CF, 50% CF and 25% CF with 81.25%, 68.75% and 62.50%, respectively. Statistically, results of disease

incidence on bean pods bear significant difference among treatments. More so, it was observed that, higher

concentrations of fungal enzymes result in higher disease incidence percentages

Table 2. Means of the disease incidence percentage after 120 hours of inoculation of fungal enzyme in crude

filtrate on bean pods

Treatments

Hours of Incubation

12h

24h

48h

72h

96h

120h

T1-control

0.00

T2-25%

0.00

6.25

43.75

62.50

T3-50%

0.00

12.50

56.25

68.75

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T4-75%

0.00

18.75

50.00

75.00

81.25

T5-100%

0.00

75.00

93.75

Pr (> F)

0.0046

0.0000

0.0014

0.0002

Values are means of each treatment with four replications; means of the same superscript are not significantly

different; and

– not significant

Similar trend was also observed in bell pepper fruit as presented in Table 3 which revealed that crude filtrate

with fungal enzymes inoculated on bell pepper fruits, resulted to no disease incidence in all treatment during

the first 48 hours after inoculation. 75% and 100% CF exhibited disease incidence on the 72

hour after

inoculation with 41.67% and 58.33%, respectively, with no progress on the next succeeding period. 50% CF

exhibited disease incidence on the 96

hour with 8.33%, and 25% CF did not exhibit disease incidence at all.

Statistically, results revealed significant difference among treatments only after 120 hours of observation.

Results further revealed that, higher concentrations of unboiled fungal enzymes result in higher disease

incidence percentages in bell pepper. Meanwhile, in soybean leaves, Table 4 showed that 50%, 75% and 100%

CF exhibited 8.33%, 75% and 100% disease incidence after 12 hours of inoculation, respectively. 25% CF

exhibited disease incidence after 48 hours with initial rate of 58.33%. After 120

hours, 50%, 75% and 100%

CF exhibited 100% disease incidence while 25% CF remained at 66.67%. Statistically, inoculation with lower

concentration (25%) CF has significant difference among concentration treatments.

Table 3. Means of the disease incidence percentage after 120 hours of inoculation of fungal enzymes in crude

filtrate inoculated on bell pepper

Treatments

Hours of Incubation

12h

24h

48h

72h

96h

120h

T1-control

0.00

T2-25% CF

0.00

T3-50% CF

0.00

8.33

T4-75% CF

0.00

41.67

T5-100% CF

0.00

58.33

Pr (> F)

0.0056

0.0119

Values are means of each treatment with four replications; means of the same superscript are not significantly

different; and

means not significant

Study of Prajapati et al., (2014), discusses how the extracellular glucoamylase of Colletotrichum sp. KCP1

produced by solid state fermentation, and purified by ammonium sulphate and gel permeation chromatography,

resulted to the stability of the enzymes over wide pH range and at 40-50

C temperature at 120 minutes. The

varied cellular temperature and pH of the different plant tissues influenced the enzymatic activity of the

pathogen relative to tissue degradation where enzymes exhibited maximum activity at 50

C with pH 5.0

(Prajapati et al., 2014). Additionally, fungal plant pathogens release various extracellular enzymes to degrade

cell wall polymers from plants to obtain nutrients and ensure infection during invasion process of plant tissues

(Kubicek et al., 2014).

Colletotrichum spp. is known as an enzyme producing pathogen during infection processes especially in

tropical and subtropical fruits such as the high valued crops as mango, strawberry, avocado, citrus, papaya,

cashew, and passion fruits (Lakshmi et al., 2011), thus the symptom development with the application of the

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crude filtrate of Colletotrichum spp. infected the crops in this study, and the symptoms was prevalent.

Moreover, the fungal pathogen is reported to produce cutinases, allowing the pathogen to penetrate through the

cuticle of the host plant. In addition, Colletotrichum spp. also produces pectate lyase, proteinases, lipases,

cellulases, amylases, and esterases (Martinez and Maicas, 2021).

Table 4. Means of the disease incidence percentage after 120 hours of inoculation of fungal enzyme in the

crude filtrate on soybean leaves

Treatments

Hours of Incubation

12h

24h

48h

72h

96h

120h

T1-control

0.00

T2-25% CF

0.00

58.33

66.67

T3-50% CF

8.33

58.33

91.67

100.00

T4-75% CF

75.00

83.33

91.67

100.00

T5-100% CF

100.00

Pr (> F)

0.0000

0.0009

0.0000

Values are means of each treatment with four replications; means of the same superscript are not significantly

different; and

means not significant

Bell pepper fruits resulted to lower disease incidence as compared to beans and soybean due to the

CaChiIII7 chitinase gene that has been identified and isolated from pepper plants by Ali et al., (2020), where

the transient expression of CaChiIII7 gene in pepper increases the basal resistance to C. acutatum by

significantly expressing several defense response genes and the hypersensitive response (HR), accompanied by

an induction of H

biosynthesis. As for the bean pods, lower disease incidence percentage can be influenced

by the proteinaceous inhibitors of the enzymes released by the C. capsici as discussed by Wijesundera et al.,

(1989), where they observed that Polygalacturonase produced by Colletotrichum lindemuthianum race γ have

no activity detected after inoculation. Whereas, the 100% incidence in soybean leaves were entirely due to

increase photosynthetic activity contributing to increase energy usage inhibiting the establishment of defense

mechanism, and the tissue morphology where there are larger and hinger number of stomates (Ciha & Brun,

1975), thereby contributing to the rapid infection, as the pathogen developed and produces a wide array of

enzymes and toxins contributing to successful pathogenesis (Pring et al., 1995).

Disease Severity (%)

On bean pods (Figure 1), it was observed that on 12 and 24 hours after inoculation, results revealed that 50%,

75%, and 100% CF exhibited infection with 2.50%, 2.50%, and 15% severity, respectively (Table 5). 25% CF

exhibited disease severity only after 72 hours while other CF concentration progressed rapidly. Slower

progress was noted on 50% CF and 100% CF after 96 hours with the severity rate of 13.75 and 21.25% from

previous 11.25 and 20% respectively, while rapid progress was observed on 25% and 75% CF with 8.75 and

16.25% from previous 1.25 and 8.75%, respectively. After 120 hours of CF inoculation, slow progress was

observed on 25%, 75%, and 100% CF with 12.50%, 18.75%, and 26.25%, respectively, while samples applied

with 50% CF remained at 13.75%. Statistical analysis revealed that 100% CF has no significant difference to

75% CF but differed significantly to the rest of the treatments, while 25% CF and 50% CF and Treatment

showed no significant difference with each other.

Bean plants has cuticle as a protective outer layer to pathogen attack, containing proteins such as pectin,

cellulose, and hemicelluloses that can be a substrate for the enzymes produced by Colletotrichum capsici

(Huang, 2013). These enzymes degrade the cell wall components of bean, thus allowing the fungus to

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penetrate inside its host’s tissues (Pring et al., 1995). During the infection process of C. capsici, its spores may

land on the bean pod surface, then germ tube and appressoria may develop facilitate penetration into the cuticle

and cell wall. Enzymes such as the pectinases and cellulases breaks down those cell wall components.

Symptoms of the C. capsici infecting bean plants is usually water-soaked lesions which advance and turn dark

brown or black. These small lesions may coalesce which eventually leads to severe disease damage (Azad et

al., 2020).

Table 5. Means of the disease severity percentage after 120 hours of inoculation of crude filtrate with fungal

enzyme on bean pods

TREATMENTS

Hours of Incubation

12h

24h

48h

72h

96h

120h

T1-control

0.00

T2-25% CF

0.00

1.25

8.75

12.50

T3-50% CF

0.00

2.50

11.25

13.75

T4-75% CF

0.00

2.50

8.75

16.25

18.75

T5-100% CF

0.00

15.00

20.00

21.25

26.25

Pr (> F)

0.0046

0.0000

0.0025

0.0005

Values are the means of each treatment with four replications; means of the same superscript are not

significantly different; and

means not significant

Study of Armesto et al., (2020), revealed that fungal plant pathogens are capable of secreting enzymes,

enabling them to infect its host tissue. In their study, Colletotrichum gloeosporioides related to anthracnose

disease were evaluated in producing hydrolytic enzymes. Their study resulted that all the enzymes mentioned

were detected from the pathogen, and also obtained highest disease severity indexes, which suggested a

relationship between enzymes and its aggressiveness of the isolates. Therefore, the present study further

proved the activity of enzymes in disease development.

Figure 1. Bean pods (T1-T5) inoculated with different concentration of Colletotrichum capsici crude filtrate

after 120 hours of inoculation

Meanwhile, for bell peppers (Figure 2), it revealed no infection on the first 48 hours (Table 6). On 72 hours,

75% CF, and 100% CF exhibited 6.67% and 11.67%, respectively. 50% CF exhibited 1.67% disease severity

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while 75% CF and 100% CF remained. On 120 hours, 75% CF and 100% CF progressed with 13.33% and

21.67%, respectively while 50% CF remained at 1.67%. 25% CF remained no infection at all, and that the

statistical analysis revealed that 100% CF differed significantly among all treatments except for 75% CF.

Table 6. Means of the disease severity percentage after 120 hours of inoculation of crude filtrate with fungal

enzyme on bell pepper

TREATMENTS

Hours of Incubation

12h

24h

48h

72h

96h

120h

T1-control

0.00

T2-25% CF

0.00

T3-50% CF

0.00

1.67

T4-75% CF

0.00

6.67

13.33

T5-100% CF

0.00

11.67

21.67

Pr (> F)

0.0153

0.0282

0.0413

Values are means of each treatment with four replications; means of the same superscript are not significantly

different; and

means not significant

Bell peppers has cuticle as barrier against fungal pathogen’s infection in which the fruit tissues are rich in

pectin and other polysaccharides, however, it can be degraded by the pathogen’s enzymes. C. capsici infects

bell peppers through penetration in the fruit’s surface which is usually the stomata as the natural opening or

through wounds as an artificial portal of entry (Krasnow and Ziv, 2022). Enzymes produced by the fungal

pathogen particularly the pectinases and proteases break down the cell walls of its host, thereby disrupting the

cellular integrity aiding to further invasion and colonization of the pathogen within the bell pepper fruit. Bell

pepper infected with C. capsici exhibit sunken, and white to dark lesion which may be covered with fungal

spores contributing to disease spread (Sangeetha et al., 2021).

Figure 2. Bell pepper fruits (lengthwise cut) from T1-T5 inoculated with different concentration of

Colletotrichum capsici crude filtrate after 120 hours of inoculation

On the other hand, similar trend was observed in soybean (Figure 3) where 50%, 75%, and 100% CF exhibited

disease severity at 12 hours and progressed until 120 hours (Table 7). 25% CF exhibited disease severity on 48

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hours and progressed until 120 hours. After 120 hours, 100% CF showed the highest disease severity with

68.33%, followed by 50% CF with 51.57%, while 25% CF showed the lowest with 26.67%. Statistically, the

results revealed that all treatments are comparable to each other with 100% CF as the highest implying that

higher crude filtrate concentration with fungal enzymes aid in successful infection. Thus, understanding the

interaction of the host plant and the enzymes secreted by the pathogen would contribute to disease

management.

Table 7. Means of the disease severity percentage after 120 hours of inoculation of crude filtrate with fungal

enzyme on soybean leaves

TREATMENTS

Hours of Incubation

12h

24h

48h

72h

96h

120h

T1-control

0.00

T2-25% CF

0.00

11.67

25.00

26.67

T3-50% CF

1.67

11.67

25.00

40.00

43.33

51.67

T4-75% CF

15.00

16.67

33.33

40.00

46.67

48.33

T5-100% CF

20.00

30.00

40.00

56.67

61.67

68.33

Pr (> F)

0.0000

0.0001

0.0000

Values are means of each treatment with four replications; means of the same superscript are not significantly

different; and

means not significant

Soybeans have a robust cuticle and its cell wall is composed with pectin, cellulose, and hemicelluloses which

can be degrade during the infection process of C. capsici through producing enzymes, however as defense

mechanism of the plant, soybean produces phytoalexins and other chemical compounds that could inhibit the

growth of the pathogen as part of their defense to survive. Symptoms of anthracnose caused by C. capsici in

soybean can be characterized as dark, and sunken lesions in which if it became severe, yield loss will be

inevitable (Saxena et al., 2016). Study of Naveen et al., (2021), demonstrated that the fungal enzymes of

Colletotrichum spp. such as the cellulase, pectin methylesterase, and ascorbate peroxidase was involved to the

virulence of the pathogen. Their study resulted that all the mentioned enzymes were correlated with their

pathogenicity which causes severe losses to the treated crops under favorable conditions, concluding the

specific use of those enzymes as an indication of virulence of the pathogen.

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Figure 3. Soybean leaves (trifoliate) from T1-T5 inoculated with different concentration of Colletotrichum

capsici crude filtrate after 72 hours of inoculation

The symptom characteristic developed on the crops used in this study can be further explained by the

production of macerating enzymes of the pathogen which enabling them to successfully infect the host plants.

For example, according to Anand et al., (2008), Colletotrichum spp. produces enzyme cellulases that catalyzes

the host cell wall degradation. More so, according to Joshi, (2018), the fungal pathogen can produce

pectinolytic enzyme, endo-polygalacturonases, protein kinases, glucanases, and chitinase. Furthermore,

according to Villafana and Rampersad, (2020), the ability of Colletotrichum spp. to produce and secrete

cutinase required to dismantle the cuticle of the host plant during penetration which is very crucial to the

necrotrophic stage of their infection strategy. Cell wall-degrading enzymes (CWDEs) are critical for the initial

infection processes as they degrade the complex carbohydrates components in the host plants’ cell wall (Liao

et al., 2012). And as the infection process is advancing, continuous degradation of pectin by the pectinases

allows the pathogen to move, thereby spreading the infection such as the study of Prusky et al., (2007) which

showed that the pectinases production of the microorganism is correlated with the extent of tissue colonization

leading to symptom development of infected plants.

CONCLUSION

This study demonstrates that crude filtrates (CF) of Colletotrichum capsici accelerate symptom development,

increase disease incidence, and intensify severity in bean pods, bell peppers, and soybean leaves in a

concentration-dependent manner. Higher CF levels consistently reduced the time to symptom appearance and

led to greater infection levels, with soybean leaves showing the highest susceptibility compared to bean pods

and bell peppers. These findings underscore the pivotal role of fungal enzymes in driving host tissue

degradation and pathogenicity. The differential responses among hosts highlight the importance of host traits,

such as stomatal density and defense gene expression, in influencing disease outcomes. Overall, the results

provide new insights into enzyme-mediated infection processes of C. capsici and point to potential

applications in resistance breeding and enzyme-targeted disease management strategies.

RECOMMENDATION

The findings of this study can inform Integrated Pest Management (IPM) strategies by reducing reliance on

synthetic fungicides and mitigating their environmental impact. Practical applications include modifying

irrigation practices to lower humidity that favors fungal growth and adopting crop rotation to disrupt the life

cycle of C. capsici. Given the global relevance of Colletotrichum spp. as pathogens of economically important

crops, understanding their host interactions under changing climate conditions is essential. Future research

should examine how temperature influences enzymatic activity and disease dynamics, with implications for

food security, particularly in developing countries.

ACKNOWLEDGMENT

The authors gratefully acknowledge the University of Southeastern Philippines (USeP), Tagum-Mabini

Campus, and the DA-Caraga Region RCPC for providing the resources and facilities necessary to conduct this

research. Sincere appreciation is also extended to the authors’ families and friends for their constant

encouragement, understanding, and support throughout the duration of this work.

Conflict of Interest

The authors declare that they have no conflict of interest in the publication of this research paper. No financial

or personal relationships with other people or organizations have influenced the conduct of this research or the

preparation of this manuscript.

Data Availability

The data supporting the findings of this study are available from the corresponding author upon reasonable

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request, as the dataset is not publicly archived.  
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