Volume 9, Issue 12, December – 2024 International Journal of Innovative Science and Research Technology

ISSN No:-2456-2165

Metabolic Adaptation of Trichoderma Asperellum

Wild-Type to Enhance Growth Rate and Biocontrol
Efficacy against Anthracnose (Colletotrichum spp.)
In Sokra2,3,*, Chey Socheata1, Horn Meta2, Rin Chanra2, and Horn Linan 1
1
Department of Soil and Crop Production, Faculty of Agronomy, University of Kratie, Camodia
2
Department of Food Engineering, Faculty of Agro-Industry, University of Kratie, Cambodia
3
Metabolic Engineering Research Unit, School of Biotechnology, Suranaree University of Technology, 111 University Avenue,
Suranaree, Muang, Nakhon Ratchasima, 30000, Thailand

*Corresponding Author: In Sokra2*

Abstract:- Pathogenic fungi significantly damage crops losses include agricultural practices, biological factors, and
in both vegetable and fruit cultivation. Trichoderma environmental conditions (Liliane et al. 2020). Among the
asperellum is a biocontrol fungus that suppresses these most destructive factors affecting crops is fungal pathogens
pathogens and promotes plant growth. However, its that cause anthracnose, a disease that frequently occurs and
effectiveness in controlling such pathogens is limited. destroys crops annually, whether they are leafy plants or
This study aimed to enhance the biocontrol efficacy of fruit-bearing plants, despite the use of pesticides during
T. asperellum against crop-damaging fungi through planting and after harvest (Alves et al. 2015 & Peralta-Ruiz
metabolic adaptation. Serial transfer experiments were et al. 2023). In tropical regions, anthracnose causes fruit rot
conducted in potato dextrose broth (PD broth) and is commonly found on crops like pepper, tomatoes, and
containing 3% sugar, followed by plating with many other agricultural products (Zakaria 2021). To control
anthracnose to compare the inhibitory effects of the and treat this issue, farmers have been using chemical
wild-type strain and its evolved variant. The evolved pesticides, but the use of these chemicals has been found to
strain, T. asperellum TIS-11T, demonstrated have negative effects on the crops, especially by interfering
significantly improved growth and sugar utilization, with the photosynthesis process, causing a decline in the
reaching a biomass of 0.5 g/mL within 48 hours. crops’ growth and yield (Petit et al. 2012). Chemical
Additionally, the evolved strain exhibited complete pesticides also contribute to pollution, the evolution of
inhibition (100%) of anthracnose on PDA culture plates resistant pathogens, and serious health risks to humans and
within 5 days. One-way ANOVA revealed a significant other living organisms (Goswami et al. 2018). Thus,
difference (p<0.01) increase in growth inhibition by the controlling biological factors with the use of biologically
T. asperellum TIS-11T strain compared to the wild- derived pesticides has become an important requirement for
type. In conclusion, T. asperellum TIS-11T is a highly agricultural productivity (dos Santos et al. 2021). Recently,
effective biocontrol agent against anthracnose and has biological pesticides have played a key role in replacing
strong potential for industrial applications in pathogen chemical pesticides, offering effectiveness without
management. impacting human health, animals, or crops. Biological
pesticides contain beneficial organisms, including fungi and
Keywords:- Metabolic Adaptation, Trichoderma bacteria, that combat harmful fungal and bacterial diseases.
Asperellum, Growth Rate, Biocontrol, Anthracnose. Among these, Trichoderma species are popular and
effective in controlling fungal and bacterial pathogens that
I. INTRODUCTION cause diseases like rot, leaf blight, and fruit spoilage.
Moreover, Trichoderma not only protects and treats
Cambodia is an agricultural country, with about 61% diseases but also promotes plant growth. Biological
of its population living in rural areas and approximately pesticides produced from Trichoderma are safe, cost-
77% relying on agriculture, including activities such as effective, and efficient for crops (Yao et al. 2023). Despite
farming, fishing, and animal husbandry (Karamba et al. this, the effectiveness of Trichoderma remains limited for
2022). Cambodian agriculture is heavily impacted by practical use, leading farmers to continue using chemical
climate change, which brings increased temperatures, pesticides for pathogen control (Supyani et al. 2023).
irregular rainfall, and rapid weather changes (Karamba et Similarly, Ons et al. (2020) pointed out that over 78,000
al. 2022). Additionally, various crops produced in farmers worldwide require agricultural pesticides for fungal
Cambodia, including fruits and vegetables like bananas, disease treatment and still use chemical pesticides more
chilies, mangoes, and others, are exported abroad (Piseth et frequently due to their higher effectiveness. Ruangwong et
al. 2021). Despite this, all crops are vulnerable to damage al. (2021a) found that Trichoderma produces organic
during the production cycle, including planting, care, and compounds that inhibit the growth of pathogenic fungi,
harvest. Factors contributing to reduced crop yields and meaning that fungal pathogens are weakened by substances

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produced by Trichoderma. Similarly, metabolic evolution B. Metabolic Adaptation

of beneficial microorganisms is a popular method to The T. asperellum wt was obtained from the
enhance microbial growth and performance (In et al. 2020). Microbiology Laboratory at the University of Kratie. Using
The culture of Trichoderma under both biotic and abiotic a serial transfer method, the T. asperellum strain was
stress conditions leads to improved strains with higher inoculated into the prepared broth and monitored daily until
capabilities, contributing positively to crop production all sucrose was depleted. Cultures were grown in 250 mL
(Cabral-Miramontes et al. 2022). flasks containing 100 mL of broth, maintained at 37°C in a
shaking incubator set to 200 rpm. The initial biomass
Thus, the purpose of this study is to enhance the concentration was 0.01 g/mL, following the protocol
growth and inhibitory activity of Trichoderma by described by In et al. (2020).
cultivating it in high-sugar (3%) potato dextrose broth
(PDA) and investigating its effectiveness in controlling C. Percentage Inhibition Measurement
anthracnose disease on crops. The T. asperellum wt and T. asperellum TIS-11T,
which were enhanced for their biocontrol capabilities, were
II. MATERIALS AND METHODS cultivated on growth media containing the anthracnose
pathogenic fungus (Colletotrichum spp.). The growth rate
A. Potato Dextrose Broth of the anthracnose pathogenic fungus was measured every
Potatoes were cleaned, sliced, and 200 grams were 24 hours until the pathogen was completely suppressed. To
boiled in water for 10 minutes. The volume was then determine the percentage of growth inhibition, the formula
adjusted to 1000 mL with water, and sucrose was added at I = (C-T)/C x 100 was used, where I represents the
concentrations of 2%, 2.5%, and 3%. The mixture was percentage of inhibition (%), C is the radial growth of
stirred thoroughly to ensure complete dissolution. The Colletotrichum spp. (in millimeters), and T is the radial
solution was transferred to a glass container and autoclaved growth of T. asperellum (in millimeters) (Rahman et al.
at 121°C and 106 kPa (1 atm) for 20 minutes. After 2009).
sterilization, the broth was cooled and made ready for
experimental use.  The Experiment was Divided into three Treatments:

 Treatment 1 (TC): Potato dextrose agar (PDA) plates

containing Colletotrichum spp.
 Treatment 2 (TB): PDA plates containing
Colletotrichum spp. and T. asperellum wt.
 Treatment 3 (TA): PDA plates containing
Colletotrichum spp. and T. asperellum TIS-11T

Fig 1: Growth Plates of the Three Factors to Illustrate the Level of Activity Inhibition by the Biocontrol Agents

D. Data Collection percentage for each factor and treatment was calculated
Primary data were collected and analyzed at each using the formula described by Rahman et al. (2009).
experimental stage to obtain numerical and quantitative
measurements. The biomass of T. asperellum wt was E. Data Analysis
measured every 24 hours until the sugar in the medium was All data were entered into Microsoft Excel
completely depleted. For each measurement, a 1 mL liquid Professional Plus 2021 for cleaning and proper
sample was taken and centrifuged using a Mini Centrifuge organization. The data were presented as mean ± standard
(Model: Mini-6KS) for 3 minutes. The supernatant was deviation (SD). The means of the treatments were analyzed
discarded, and the remaining solid residue was weighed using a One-way ANOVA with Tukey's HSD multiple
using an electronic balance (Model: HL-300LWP). The comparisons test. This analysis was performed using
inhibition level was determined by measuring the radial GraphPad Prism software (Version 10.2.0, Windows, San
growth of the anthracnose pathogenic fungus using a Diego, California, USA, www.graphpad.com), with a
millimeter-scale ruler. Measurements were taken every 24 significance level set at p < 0.01.
hours for all treatments and replicates. The inhibition

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III. RESULT AND DISCUSSION

A. Metabolic Adaptation of T. Asperellum wt to T. Asperellum TIS-11T

Fig 2: Metabolic Adaptation of T. Asperellum wt in Liquid Culture Media with Sucrose Concentrations Ranging from 2% to 3%
(w/v). The Transfers were Conducted from the First to the 11th, Starting with a Biomass Level of 0.01 g/mL. The First and
Second Transfers used 2% Sucrose (Circle), the Third and Fourth Transfers used 2.5% Sucrose (Square), and Transfers 4 Through
11 used 3% Sucrose (Triangle). (a) Graph of Biomass (g/mL) and (b) Graph of Percentage sucrose Consumption during
Successive Transfers.

After culturing and isolating T. asperellum wt, 0.5 g/mL and complete sucrose utilization at the 11th
metabolic adaptation was performed through serial transfer (Fig. 2). These results align with findings from
transfers in liquid media containing sucrose concentrations Jantama et al. (2008), which highlighted that
ranging from 2% to 3% (w/v). The strain utilizing 3% microorganism grown in low-sugar environments exhibit
sucrose most efficiently and showing optimal growth was reduced metabolic activity and energy production, limiting
selected for further evaluation. T. asperellum wt was cell proliferation. The gradual increase in sucrose
initially cultured in a medium containing 2 g/mL sucrose, concentration (to 3%) in subsequent transfers supported
starting with a biomass of 0.01 g/mL. By the 11th transfer, enhanced growth and metabolic efficiency, consistent with
biomass progressively increased while sucrose was fully other studies. The final strain, T. asperellum TIS-11T,
utilized (Fig. 2). To optimize growth, a sucrose demonstrated robust growth and a high tolerance for
concentration of 2.5% was applied, enriching the substrate elevated sucrose concentrations, making it a suitable
for metabolic adaptation. By the second transfer, biomass candidate for biocontrol applications against anthracnose
levels increased as sucrose concentration rose; however, pathogens. Additionally, it exhibited superior metabolic
sucrose consumption was incomplete. By the third transfer, adaptation, enabling complete sucrose utilization and
biomass reached 0.35 g/mL, with complete utilization of sustained biomass production. High substrate
2.5% sucrose. From the fourth transfer onward, growth concentrations typically inhibit enzymatic activity in
improvements were observed, culminating in a biomass of microorganisms, as reported by Ghaly et al. (2005).

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However, this study found that T. asperellum TIS-11T In conclusion, the metabolic adaptation process
maintained high metabolic activity and growth under these resulted in the development of T. asperellum TIS-11T, a
conditions. These findings are consistent with In et al. strain with enhanced growth potential, high biomass
(2020), who showed that microorganism adaptation in production, and complete sucrose utilization. This strain
specific media can induce metabolic shifts that enhance holds significant promise for biocontrol applications and
growth. Similarly, stress conditions during culturing may further studies.
improve environmental tolerance and resilience, leading to
increased growth (Carroll & Marx 2013 & Jantama et al.
2015).

B. Percentage of Inhibition of T. Asperellum TIS-11T and T. Asperellum wt to Colletotrichum spp.

Fig 3: Cultivation of Anthracnose Pathogenic Fungus Colletotrichum spp., T. asperellum wt, and T. asperellum TIS-11T on potato
dextrose agar (PDA). TC = Colletotrichum spp. without T. asperellum, TB = Colletotrichum spp. with T. asperellum wt, TA =
Colletotrichum spp. with T. asperellum TIS-11T.

The anthracnose pathogenic fungus (Colletotrichum (2021), who showed that Colletotrichum spp. is a fungus
spp.), T. asperellum wt, and T. asperellum TIS-11T were that is difficult to control, especially without protection or
cultured on solid potato dextrose agar (PDA). The first plate, immediate treatment. Research by López-Moral et al. (2020)
inoculated with only the anthracnose pathogen, showed confirmed that Colletotrichum spp. grows well under
strong growth up to the second day with a radial growth of sufficient conditions and nutrition, leading to rapid growth
80 ± 0.01 mm, covering the entire plate (Fig. 3 and Table without protection.
1). This result is consistent with the findings of Li et al.

Table 1: Measurement of Anthracnose Pathogenic Fungus Growth Levels for all Three Treatments.
Day TC ± SD TB ± SD TA ± SD PI.TB (%) PI.TA (%)
(mm) (mm) (mm)
1 60.30 ± 1.53 54.70 ± 0.58 55.33 ± 0.58 10.38 9.29
2 80.00 ± 0.00 51.30 ± 1.15 29.00 ± 1.00 35.83 63.75
3 80.00 ± 0.00 33.70 ± 3.21 15.33 ± 0.58 57.92 80.83
4 80.00 ± 0.00 16.30 ± 1.53 3.67 ± 1.53 79.58 95.42
5 80.00 ± 0.00 8.00 ± 2.65 0.00 90.00 100.00

TC = Colletotrichum spp. without T. asperellum On the second day, the plate with T. asperellum wt
TB = Colletotrichum spp. with T. asperellum wt showed inhibition of the anthracnose pathogen, with a
TA = Colletotrichum spp. with T. asperellum TIS-11T growth of 51.3 ± 1.15 mm, indicating an inhibition
PI.TB = Percentage of inhibition of TB percentage of 35.23%. By the final day, T. asperellum wt
PI.TA = Percentage of inhibition of TA reduced the radial growth of Colletotrichum spp. to 8 ± 2.65
SD = Standard deviation mm, corresponding to an inhibition percentage of 90%. In
the plate with T. asperellum TIS-11T, the growth of the
anthracnose pathogen was inhibited to 29 ± 1 mm on the
second day. On subsequent days, the growth of

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Colletotrichum spp. was completely inhibited by T. anthracnose pathogen, with complete mycelial destruction in
asperellum TIS-11T by the fifth day, with 100% inhibition just 5 days following its enhanced metabolic capacity. The
(Table 1). increased sucrose concentration of 3% enhanced the growth
and metabolism of T. asperellum TIS-11T. This finding is in
The growth of the anthracnose pathogen across the line with the study by In et al. (2020), which showed an
three treatments differed significantly (p<0.01). The growth increase in biomass following metabolic adaptation. The
of T. asperellum wt was optimal at a sucrose concentration bioactive compounds produced by T. asperellum TIS-11T
of 20 g/L, which led to effective inhibition of anthracnose showed an increase in antifungal activity, contributing to a
disease in this experiment (Ajam et al. 2023). A study by stronger inhibition of anthracnose. The study by Shang et al.
Intana et al. (2021) found that T. asperellum produces (2020) also demonstrated that T. asperellum is highly
bioactive compounds that are effective against both fungal effective in protecting and treating anthracnose disease. The
and bacterial pathogens. This is why the presence of T. results of this experiment indicate that T. asperellum TIS-
asperellum slowed down the growth of the anthracnose 11T has improved efficacy in inhibiting anthracnose
pathogen and gradually destroyed its mycelial structure. T. compared to T. asperellum wt, which had not yet shown
asperellum TIS-11T exhibited high efficacy in inhibiting the enhanced capabilities.

C. Comparison of T. asprellum TIS-11T to Recent Studies

Table 2: Comparison of T. asperellum TIS-11T with Other Microorganisms in Inhibiting Colletotrichum spp. Activity.
Strain Media Time (Day) Percentage (%) References
T. asperellum wt PDAa 5 90 This study
T. asperellum TIS-11T PDA 5 100
T. kingingiopsis PDA 5 79.57 Ruangwong et al. (2021b)
T. harzianum PDA 7 64.2 Sutarman et al. (2021)
T. asperellum PDA 7 80 Kim et al. (2023)
T. viride PDA 10 62.61 Agnihotri et al. (2023)
T. asperellum PDA 7 76.47 Manzar et al. (2021)
T. harzianum PDA 5 83 Intana et al. (2007)
Paenibacillus polymyxa AT4 NAb 7 76.44 Tram et al. (2023)
Bacillus velezensis AK-0 NA 7 80.70 Kim et al. (2021)
Paenibacillus polymyxa C1 NA 7 100 Suprapta (2022)
a
Media for fungi (potato dextrose agar)
b
Media for bacteria (nutrient agar)

Table 2 presents a comparison of the effectiveness of include Paenibacillus polymyxa AT4, Bacillus velezensis
the inhibition of anthracnose disease activity between T. AK-0, and Paenibacillus polymyxa C1. Paenibacillus
asperellum TIS-11T and other recent studies. The level of polymyxa AT4 showed a 76.44% inhibition, Bacillus
inhibition was measured based on the percentage of activity velezensis AK-0 had an 80.70% inhibition, and
inhibition and the duration of the process. Among the Paenibacillus polymyxa C1 showed 100% inhibition in 7
biocontrol fungi Trichoderma spp. and other bacteria, T. days (Kim et al. 2021; Suprapta et al. 2022; Tram et al.
asperellum TIS-11T showed the highest effectiveness, with 2023).
a 100% inhibition level achieved in 5 days. This result
aligns with previous studies, which showed that T. When comparing biocontrol agents, both bacteria and
asperellum wt is a biocontrol agent capable of inhibiting Trichoderma species, T. asperellum TIS-11T in this study
the growth of anthracnose-causing fungi, with a 90% demonstrated the highest efficacy and the shortest time for
inhibition level in 5 days. inhibiting anthracnose disease activity.

Other Trichoderma species such as T. kingingiopsis, IV. CONCLUSION

T. harzianum, and T. viride showed moderate levels of
inhibition. T. kingingiopsis exhibited a 79.57% inhibition The study on the enhancement of growth capacity
level in 5 days (Ruangwong et al. 2021), T. harzianum through metabolic adaptation of T. asperellum TIS-11T was
showed 83% inhibition in 5 days (Intana et al. 2007), and T. conducted to increase the effectiveness of inhibiting
viride had a 62.61% inhibition level in 10 days (Agnihotri anthracnose disease activity. The results showed that T.
et al. 2022). asperellum wt growth increased from 0.28 g/mL to 0.50
g/mL, and sugar consumption increased from 2% to 3%.
In addition to biocontrol fungi, certain bacteria have Regarding the effectiveness in controlling anthracnose
also shown high efficacy in inhibiting anthracnose disease disease, a significant increase was observed, with T.
activity. However, the level of inhibition and the time asperellum TIS-11T being able to completely eliminate the
required were lower compared to the biocontrol agent T. disease within 5 days. T. asperellum TIS-11T demonstrated
asperellum TIS-11T. Notable bacteria with high efficacy higher effectiveness compared to recent studies on

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anthracnose disease inhibition. We can conclude that T. [5]. Carroll SM, & Marx CJ (2013). Evolution after
asperellum TIS-11T is a highly potential biocontrol agent introduction of a novel metabolic pathway
for managing anthracnose fungal diseases and promoting consistently leads to restoration of wild-type
sustainable agricultural practices. physiology. PLoS Genetic 9(4): 1–12.
https://doi.org/10.1371/journal.pgen.1003427
This study demonstrates the potential use of T. [6]. dos Santos GAC, da Silva RR, Moreira SI, Vicentini
asperellum TIS-11T in laboratory experiments. However, a SNC, & Ceresini PC (2021). Biofungicides: An Eco-
limitation of this study is that it has not yet been applied Friendly approach for plant disease management. In
directly to crops. In general crop cultivation, it is difficult Ó. Zaragoza & A. Casadevall (Eds.), Encyclopedia
to control all environmental conditions. Additionally, other of mycology (pp. 641–649). Elsevier.
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limitation of this research. Therefore, future research modeling of continuous submerged fermentation of
should address these limitations by studying the direct cheese whey for single-cell protein production.
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ACKNOWLEDGMENT nternational Journal of Agriculture, Environment
and Biotechnology 11: 1011–1019.
This study received funding from the competition [9]. In S, Khunnonkwao P, Wong N, Phosiran C,
project of the Samdech Techo Hun Sen Award on Jantama SS, & Jantama K (2020). Combining
Sustainable Agriculture. The authors would like to express metabolic engineering and evolutionary adaptation
their sincere gratitude to the editors and expert reviewers of in Klebsiella oxytoca KMS004 to significantly
the International Journal of Innovative Science and improve optically pure D-(-)-lactic acid yield and
Research Technology for their valuable feedback and specific productivity in low nutrient medium.
suggestions to improve this manuscript. All data and Applied Microbiology and Biotechnology 104 (22):
opinions presented in this article are solely the 9565–9579. https://doi.org/10.1007/s00253-020-
responsibility of the authors and do not necessarily reflect 10933-0
the views or policies of any affiliated organization. [10]. Intana W, Kheawleng S, & Sunpapao A (2021).
Trichoderma asperellum T76-14 released volativle
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