Application of Trichoderma spp. to Control Colletotrichum sp. and Pseudopestalotiopsis spp., Causing Agents of Fruit Rot in Pomelo (Citrus maxima (Burm.) Merr.)

Abstract

Fruit rot seriously damages pomelo production. Given concerns regarding the safety of chemical agents, biological alternatives are becoming more preferable. Therefore, the experiment aimed to (i) identify the pathogens causing pomelo fruit rot disease and (ii) select Trichoderma spp. strains controlling the determined pathogens in Ben Tre, Vietnam. Three pathogenic fungal strains isolated from diseased pomelo fruits were selected. The three pathogenic fungal strains were randomly injected into 9 healthy pomelo fruits. The strain PCP-B02-A2 led to a completely rotten fruit on day 17 after infection, while strains PCP-B02-B2 and PCP-B03-A1 had infected spots whose lengths were 17.5 and 28.1 mm, became larger, and eventually led to the whole fruit rot. The pathogens were identified by the internal transcribed spacer (ITS) technique as Colletotrichum gloeosporioides PCP-B02-A2, Pseudopestalotiopsis camelliae sinensis PCP-B03-A1, and P. chinensis PCP-B02-B2. Twenty-five Trichoderma spp. strains were isolated. The ITS technique identified four strains, including Trichoderma asperellum TP-B01, T. harzianum TP-B08, T. harzianum TP-B09, and T. asperellum TP-C25. The PCP-B02-A2 strain had antagonism at 66.7–68.7%, while those of PCP-B02-B2 and PCP-B03-A1 were 64.2–71.1% and 55.7–57.4%, respectively.

1. Introduction

Pomelo (Citrus maxima (Burm.) Merr.) is native to Southeast Asia and is most widely distributed in Thailand and Malaysia [1]. In Vietnam, pomelo has high economic value, occupies a large planting area, and is popular in the consuming market [2]. In addition to its nutritional value with a high vitamin C content, pomelo also contains sugars such as sucrose, glucose, fructose, and fiber, while its by-product is pomelo peel, which is rich in ascorbic acid, essential oils, pectin, flavonoids, polyphenols, sugars, fiber, biomass, and some trace elements [3]. However, fungal diseases are now widely distributed in many citrus-growing areas where temperature and humidity conditions are suitable [4]. Postharvest fungal pathogens are one of the causes of significant reduction in citrus quality and marketable yield, and have serious economic impacts [5].

The fungus Pestalotiopsis sensu lato includes three genera, Neopestalotiopsis, Pseudopestalotiopsis, and Pestalotiopsis. The fungus is the causing agent of diseases including fruit rot, leaf blight, leaf spot, stem rot, scab, and postharvest rot [6,7,8]. Pseudopestalotiopsis spp. are a broad host species and have been reported to cause the rot of tropical fruits in Thailand such as strawberries, avocados, mangosteens, and plums [8]. In addition to causing fruit rot, the fungus also causes leaf blight of jackfruit and rambutan [8]. In China, Pseudopestalotiopsis spp. have been reported to cause leaf spot disease on Euonymus japonicus [9]. Initial symptoms appear as yellow spots ranging from 1.2 to 4.9 mm in diameter, which then enlarge into large, irregular lesions with white centers surrounded by brown halos. Under humid conditions, black dots appear in the center of the lesions [9]. Pseudopestalotiopsis spp. are the cause of leaf blight on tea (Camellia sinensis) in Malaysia and China; symptoms include the formation of small, circular to irregular brown spots that gradually enlarge, turn darker, and eventually impede plant growth [7,8,9,10].

Colletotrichum gloeosporioides causes anthracnose on citrus, affecting leaves and branches, causing fruit drop after fruit set, and fruit rot after harvest [11,12]. Colletotrichum spp. survive in the field on alternative hosts, wild plants, and plant residues on diseased fruit tissues. Transmission is mainly by rain in small areas or by wind [13]. Because the pathogen exists as spores in soil and residues during cultivation, the use of chemical pesticides is not effective in the long term and also leads to negative impacts on the environment. Integrated disease control is the most effective management method for fruit rot in crops due to the combination of basic factors for disease management, such as cultivation techniques, breeding resistant varieties, and chemical control, in which biological control is considered [14]. Using biofertilizers containing antagonistic fungal strains can control some pathogens in the soil and can be environmentally friendly [15]. On the other hand, antagonistic fungi also bring many benefits when existing in the soil by increasing crop quality and yield [15]. Trichoderma spp. can increase biomass, root length, height, branches, and number of fruits [16]. Trichoderma spp. can also stimulate plant resistance against pests and diseases, decompose organic matter, and help plants withstand abiotic stress [17].

In addition, Trichoderma spp. fungi are used to control plant diseases in sustainable disease management systems [18], because of their antibiotic, parasitic, and competitive mechanisms [19]. The ability to antagonize Trichoderma is because they can survive in various unfavorable conditions, be reproductive, and use nutrients efficiently [20]. Species of Trichoderma are widely distributed in most climates in the world and can grow and develop on a variety of substrates under different environmental conditions, which is another reason why Trichoderma spp. are widely used in the world [21,22].

Trichoderma spp. can mobilize and absorb nutrients from the soil better than other rhizosphere microorganisms. Therefore, the control of some pathogens using Trichoderma spp. involves the coordination of many mechanisms of which nutrient competition is one of the most important [23]. Siderophores secreted by Trichoderma spp. can be up to 12 to 14 types and can convert insoluble iron into soluble iron, helping plants uptake more iron, reducing the amount of iron in the environment, and thus reducing the amount of iron available for pathogenic fungi to use [24]. Besides the ability to compete for nutrients, parasitism is also one of the important antagonistic mechanisms of Trichoderma [20]. According to Kumar et al. [25], T. harzianum can parasitize via forming adhesions on the pathogenic hyphae of F. solani by gently rolling around the hyphae within 95 h. After 6 days of infection, the pathogenic fungus was completely inhibited, while T. harzianum multiplied via asexual conidiogenesis. At the same time, Trichoderma spp. grew along the host hyphae and secreted enzymes that degraded the cell wall mechanically and enzymatically during penetration [26]. Most fungal cell walls are composed of chitin and β-1,3-glucan [27]. Chitin is covalently cross-linked to the β-1,3-glucan network and contributes to the stiffness and physical strength of the fungal cell wall [28]. Trichoderma spp. secrete a variety of chitinase and β-1,3-glucanase that degrade glycosidic bonds in β-glucan and chitin polymers of fungal cell walls, thus inhibiting the growth of pathogenic fungi [28,29].

Trichoderma spp. are used to antagonize and control anthracnose caused by C. gloeosporioides on grape leaves [30]. T. koningiopsis PSU3-2 strain effectively prevents anthracnose on postharvest chili peppers [31]. Under in vitro, in silico, and in planta experimental conditions, two fungal strains, T. harzianum MC2 and NBG can antagonize three fungal pathogens, Fusarium oxysporum f. sp. lycopersicum, C. gloeosporioides, and Rhizoctonia solani, which cause diseases such as wilt and fruit rot in solanaceous plants, causing huge yield losses in the field as well as in storage [32]. However, the antagonistic ability of Trichoderma against Pseudopestalotiopsis sp. has not been recorded to date.

Trichoderma spp. have been reported to be resistant to a wide range of citrus pathogens. T. harzianum, T. guizhouense, T. atroviride, and T. koningiopsis have been reported to be effective against Alternaria alternata, C. gloeosporioides, and Penicillium digitatum causing postharvest diseases on oranges [12]. Trichoderma has been used to antagonize the pathogen causing postharvest rot of pomelo caused by the fungus Lasiodiplodia theobromae with an efficiency of up to 84% [33]. Fusarium and Phytophthora species are also pathogens of citrus, associated with root rot and fruit rot. T. asperellum has been reported to be effective against both Fusarium and Phytophthora pathogens [34]. In Indonesia, the fungus Botryodiplodia theobromae seriously affects citrus orchards and T. asperellum Tc-Pjn-02 is proposed to replace chemical fungicides [35]. Based on the above results, a study was conducted to use Trichoderma spp. to antagonize Colletotrichum sp. and Pseudopestalotiopsis spp. causing fruit rot and to demonstrate disease control measures to improve the yield of pomelo in Ben Tre.

2. Materials and Methods

2.1. Materials

Soil samples for isolation of pathogenic fungi and Trichoderma spp. were collected from pomelo-growing soil in Ben Tre. The experiment was conducted from October 2022 to May 2023 at the Laboratory of Edible and Medicinal Mushrooms, Faculty of Crop Science, College of Agriculture, Can Tho University.

2.2. Methods

2.2.1. Isolation of Fungi Causing Pomelo Rot in Ben Tre

Sample collection and processing: A total of 12 samples with fruit showing fruit rot disease were collected in 3 districts of Giong Trom, Chau Thanh, and Binh Dai in Ben Tre province. After collection, the diseased fruit samples were brought to the laboratory and washed, the surface was disinfected with 70⁰ alcohol, then washed again with sterilized distilled water, dried on sterilized absorbent paper, placed in a cool place, and observed for diseased samples for 7 to 10 days.

Isolation and identification of fungi causing fruit rot: Isolation method according to Burgess et al. [36] and Roger and Beasley [37]. A knife was used to cut a V-shape on the diseased sample at the junction between diseased and healthy tissue. The cut sample was placed in a Petri dish containing PDA medium. Next, the sample dishes were incubated for about 3–5 days in an incubator at 25–28 °C. The fungi were purified on a PDA medium and observed for the uniformity of color, the surface of fungal colonies, and the shape of mycelia. After the fungal strains had been purified, observed, and recorded for the morphological characteristics of the colonies, the fungal morphology was determined according to Klich [38] and identified.

Inoculate the fruit (Koch’s postulates) to observe the possibility of causing disease: The fruit sample was washed with sterilized distilled water, then dried with a paper towel to dry, then sterilized with 70° alcohol for 30 s, then a sharp needle was used to puncture the pomelo peel to create a wound (6 wounds on the same fruit corresponding to 3 replications). After 10 days of culturing the fungus, sterilized distilled water was added and a glass slide was used to scrape the fungus out. The fungal suspension diluted the spore density to 10⁷ spores/mL. A micropipette was used to aspirate 200 µL of the diluted fungal suspension and inject it into the previously created wound. At the same time, sterilized distilled water was added to the control fruit. Then, the pomelo fruits were placed on a Petri dish and wrapped in a nylon bag. Notably, wet cotton must be added to provide moisture and create favorable conditions for the fungus to grow. The pomelo fruits were observed daily to determine when the disease appeared on the fruit and compare the infected fruit with the control fruit.

2.2.2. Isolation and Selection of Trichoderma spp. Fungi with the Ability to Antagonize Colletotrichum sp. and Pseudopestalotiopsis spp. Causing Fruit Rot Disease on Pomelos in Ben Tre

Sample collection and processing method: Experimental soil was collected at the surface layer of 0–20 cm from 30 households growing pomelo in 3 districts of Giong Trom, Chau Thanh, and Binh Dai, Ben Tre province. Soil samples were collected in wet areas near the roots of the trees. After collection, plant residues were removed from the soil samples and stored in cold storage at 4 °C.

Isolation of pathogenic fungi: Fungal strains were collected according to the method of Kumar et al. [39] in a specialized TSM (Trichoderma Selective Medium). Soil samples were mixed with sterilized distilled water at a ratio of 1.0 g soil to 99.0 mL sterilized distilled water, shaken well for 30 min, and left to settle for 24 h at 28–32 °C. Then, 200.0 µL of soil sample were spread evenly on the TSM medium that had been sterilized at 121 °C for 20 min. After 48 h, the samples were observed and selected for colonies. Fungal samples were separated and purified on the PDA medium. The morphological characteristics of the mycelia (color and shape of the mycelia) were observed. The fungal spores were observed under a 40× optical microscope to identify Trichoderma fungi. After determining the pure sample, the strains were stored in a refrigerator at 4 °C (In Eppendorf tubes and Petri plates containing PDA medium). Finally, the isolated fungal strains were named according to the genus Trichoderma (T) and the name of Pomelo (P) followed by the serial number of the sampling location in order from 01 to 25.

Evaluation of growth of Trichoderma spp. strains: Growth of Trichoderma strains was tested after being purified on the PDA medium. The experiment was completely randomized with 3 replications for each strain and incubated at 28 ± 2 °C. Trichoderma spp. strains were cultured on the PDA medium to observe and record colony diameter after 24, 48, and 72 h. Observations recorded the growth of mycelia and colony color, and the average yield for each strain corresponding to the growth indicators.

DNA extraction and ITS region gene sequencing: DNA of fruit rot fungi PCP-B02-A2, PCP-B02-B2, PCP-B03-A1, and Trichoderma spp. culture after 7 days on PDA medium was extracted according to Doyle and Doyle [40]. Fungal spores on PDA medium were put into 2.2 mL Eppendorf, shaken well, incubated at room temperature for 10 min, centrifuged at 13,000 rpm for 5 min to collect the supernatant, transferred to a new Eppendorf, washed with 500.0 µL of 70% ethanol, centrifuged at 13,000 rpm for 5 min, and vacuum dried. Next, the DNA was dissolved in 100 uL of TE 0.1X. Then, the PCR reaction was carried out with primer pair ITS 1: 5′-TCCGTAGGTGAACCTGCGG-3′; ITS 4: 5′-TCCTCCGCTTATTGATATGC-3′ [41]. The PCR reaction was carried out in a total volume of 50 µL through the following reaction stages: Initial denaturation (95 °C for 5 min), a 30-cycle procedure of denaturation (95 °C for 90 s), annealing (52 °C for 60 s), and extension (72 °C for 90 s), final extension (72 °C for 5 s), and termination at room temperature.

Sequence analysis and identification of Trichoderma spp. PCR products were purified and sequenced using an automated sequencing system. Based on the sequences, they were compared with the database in the Genbank using BLAST version 2.15.0 at NCBI. The fungal sequences were used to construct a phylogenetic tree using MEGA 6.0 software.

2.2.3. Antagonistic Ability of Trichoderma spp. Against Pathogenic Fungi

According to Bell et al. [42] to evaluate the antagonistic activity of Trichoderma strains against Colletotrichum sp. and Pseudopestalotiopsis spp., the double culture method should be used. The experiment was arranged in a completely randomized design, with 3 replications. Trichoderma spp. and Colletotrichum spp. strains or Pseudopestalotiopsis spp. were cultured in the same Petri dish, with a center distance of 3 cm between two colonies at opposite ends of the PDA Petri dish (90 mm in diameter) for 7 days. The Petri dishes were placed under uniform light conditions, with a temperature of 28 ± 2 °C. The antagonistic ability of Trichoderma spp. strains was monitored through the colony radius of Colletotrichum spp. and Pseudopestalotiopsis spp. and the antagonistic efficiency was determined at 24, 48, 72, and 96 h after incubation according to the formula of Kakraliya et al. [43].

$$AE\left(\%\right)={\displaystyle \frac{(C-T)}{C}}\times 100$$

in which: C is the radius of the pathogenic fungal colony in the control; T is the radius of the pathogenic fungal colony corresponding to the treatment with Trichoderma spp.

2.3. Data Processing

Data were processed using SPSS 13.0 software. Analysis of variance (ANOVA) was used to determine the difference between treatments using Duncan’s test at a significance level of 5%.

3. Results

3.1. Isolation of Fungi Causing Pomelo Fruit Rot Disease in Ben Tre

3.1.1. Morphological Characteristics of Fungal Strains Causing Pomelo Fruit Rot

Figure S1 shows the results of isolation and purification of 19 fungal strains causing fruit rot on PDA medium from 12 fruit samples showing signs of fruit rot collected from Chau Thanh, Binh Dai, and Giong Trom districts, Ben Tre province. The morphology of the fungal strains and the percentage of infected fruit samples at different locations were very diverse. That is, the mycelium, color, ability to develop fungal colonies, and ability to cause disease of the selected strains were very distinct (

Table 1. Colony characteristics of pathogenic fungal strains at 8 days of culture in PDA medium.

Strain	Shape	Surface Shape	Surface Color	Diameter (cm)
PCP-B02-A2	Evenly radiated	The center of the fungus was pinkish white, raised, and winding in a wavy shape	White-gray, initially pinkish	4.35
PCP-B02-B1	Almost round	The black and gray center then wander out evenly	Blackish gray	8.80
PCP-B02-B2	Almost round to round	Hyphae grew thick and raised	Creamy white	4.57
PCP-B02-C1	Evenly rounded	In the form of cotton foam, hyphae protrude from the medium	Gray	8.90
PCP-B02-C2	Radiance	The center of the fungus was initially black, then spread evenly	Darkish gray	90
PCP-B02-D1	Almost round	The hyphae grew thick with blackish hyphae	Blackish gray	8.70
PCP-B03-A1	Spread evenly to almost round	The white hyphae were as porous as cotton, rising and then radiating	Milky white, light yellow in the center	6.72
PCP-B03-A2	Evenly rounded	White hyphae grew thickly, gradually turned gray	Light gray	90
PCP-B15-A2	Almost round	Mycelia thickened and raised to form circles	Creamy white	6.25
PCP-B17-A1	Radiance	Thick hyphae grew close to the medium	Gray	8.95
PCP-B17-A2	Almost round	Mycelia grew thickly and were sticking to the medium	Blackish brown	8.54
PCP-B17-B2	Radiance	The hyphae grew thickly, protruding from the medium	Grayish white	8.90
PCP-B17-C1	Evenly rounded	White hyphae spreads evenly	Gray	8.95
PCP-B17-C2	Evenly rounded	Thick and silky mycelia	Black interspersed with gray	90
PCP-B17-D1	Radiance	Hyphae grew tightly together	Light gray	90
PCP-B17-D2	Almost round	The center of the fungus was initially black, then spread outward.	Darkish gray	8.75
PCP-C02-A1	Evenly rounded	Mycelia grew in a circle, beyond the edge of the colony protruding	Black interspersed with gray	8.90
PCP-C02-A2	Evenly radiated	Blackish brown to black from the center to the edge of the plate, fungal foci appeared	Blackish gray	8.85
PCP-C08-A1	Almost round	White alternating with irregular blackish brown	Blackish brown	8.70

Notes: P: Pomelo; C: Colletotrichum; P: Pseudopestalotiopsis; C: Chau Thanh; B: Binh Dai; A, B, C, and D: Fruit code.

).

3.1.2. Growth of Fungal Strains Causing Pomelo Fruit Rot on PDA Medium

Table 2. Growth rate of fungal strains causing fruit rot on pomelo on PDA medium 3 days after transplantation.

Fungus Strain	Growth Rate of Fungal Strain (cm)
	24 h	48 h	72 h
PCP-B02-A2	1.55 g	2.75 j	3.65 g
PCP-B02-B1	4.60 b	7.40 d–f	8.50 e
PCP-B02-B2	1.13 h	2.73 k	3.55 g
PCP-B02-C1	4.05 cd	7.45 c–f	8.80 ab
PCP-B02-C2	4.05 cd	7.66 bc	8.55 de
PCP-B02-D1	3.67 e	7.65 bc	8.65 b–e
PCP-B03-A1	1.15 h	2.65 l	3.25 i
PCP-B03-A2	4.55 b	7.50 b–e	8.80 ab
PCP-B15-A2	0.64 i	2.70 l	5.35 f
PCP-B17-A1	5.20 a	7.60 b–d	8.75 a–c
PCP-B17-A2	3.57 e	7.70 ab	8.85 a
PCP-B17-B2	5.25 a	7.25 fg	8.50 e
PCP-B17-C1	2.67 f	7.90 a	8.55 de
PCP-B17-C2	2.46 f	7.05 gh	8.65 b–e
PCP-B17-D1	3.80 de	7.50 b–e	8.60 c–e
PCP-B17-D2	4.15 c	7.30 ef	8.50 e
PCP-C02-A1	3.65 e	7.50 b–e	8.70 a–d
PCP-C02-A2	4.70 b	6.80 i	8.30 f
PCP-C08-A1	4.50 b	6.95 hi	8.65 b–e
F	*	*	*
CV (%)	8.20	5.10	3.00

Note: The mean numbers in the same column followed by one or more identical letters do not differ statistically at 5% on the Duncan test. (*) a 5% difference with statistical significance.

shows that the colony growth diameter significantly differed by 5% at 24, 48, and 72 h after inoculation on the PDA medium. At 72 h after inoculation, the PCP-B17-B2 strain had the fastest growth (5.25 cm), followed by the PCP-B17-C1 strain (5.23 cm). In addition, the PCP-B15-A2 strain (0.64 cm) was assessed to have slower growth than the other strains examined.

3.1.3. Morphological Classification of Fungal Strains Causing Pomelo Fruit Rot

Based on the morphological and color characteristics of the fungal strains after isolation, it was found that nearly 85% of the total 19 isolated fungal strains had morphological characteristics as well as color and growth rates that matched the Lasiodiplodia spp. strains causing post-harvest fruit stem rot on pomelos. In addition, only three fungal strains, PCP-B02-A1, PCP-B02-B2, and PCP-B03-A1, after isolation and observation of morphology and growth rate, were found to be different from the remaining strains. These strains were used to perform the following experiments (Table 1 and Table 2).

3.1.4. Evaluation of the Pathogenicity of Selected Fungal Strains

The results in

Table 3. Length of lesions (mm) on pomelo caused by Colletotrichum gloeosporioides PCP-B02-A2, Pseudopestalotiopsis camelliae-sinensis PCP-B02-B2, and Pseudopestalotiopsis chinensis PCP-B03-A1.

Fungus Strain	Length of Lesions (mm) on the Fruit in Days After Infection
	Day 7	Day 9	Day 12	Day 14	Day 17
PCP-B02-A2	12.3	14.2 b	18.2 b	21.8 b	nd
PCP-B02-B2	12.8	14.1 b	15.5 c	16.6 c	17.5 b
PCP-B03-A1	11.8	16.7 a	19.9 a	23.9 a	28.1 a
F	ns	*	*	*	*
CV (%)	10.4	10.2	7.13	9.27	3.83

Note: The mean numbers in the same column followed by one or more identical letters do not differ statistically at 5% on the Duncan test. nd: not determined, * difference by significance of 5%, ns: no significance.

show that on day 7 after inoculation, the PCP-B02-A2, PCP-B02-B2, and PCP-B03-A1 strains were all toxic with lesion diameters ≥ 10 mm, of which the largest diameter was the PCP-B02-B2 strain reaching 12.8 mm. On day 9 after inoculation, the diameter of the rapidly developing lesion was PCP-B03-A1 (16.7 mm). On day 14 after inoculation, the pomelo inoculated with C. gloeosporioides completely rotted. When cutting the pomelo, it was seen that the fungus had penetrated deep into the fruit, causing the fruit to rot (Figure S2a). At this time, the pomelo inoculated with Ps. camelliae-sinensis had lesions that were deep into the fruit peel, and yellow spots appeared on the surface of the fruit. For the pomelo inoculated with Ps. chinensis around the lesion, a pale-yellow hernia appeared surrounding the lesion.

On day 17 after inoculation, the fruit inoculated with Ps. camelliae-sinensis completely rotted, white fungal filaments appeared on the pomelo peel, and the fruit flesh was severely rotten and began to ooze water (Figure S2b). The fruit inoculated with Ps. chinensis had no significant changes around the lesion with a surrounding yellowish border (Figure S2c).

3.1.5. Identification of Fungi Causing Pomelo Fruit Rot

Sequencing of the ITS region gene showed that the PCP-B02-A2 strain was identified as Colletotrichum gloeosporioides with 100% identity (GenBank database accession number OQ824978). Meanwhile, the PCP-B03-A1 strain was identified as Pseudopestalotiopsis camelliae-sinensis and the PCP-B02-B2 strain was Pseudopestalotiopsis chinensis with 100% identity, with accession numbers OQ824977 and OQ824976, respectively (Figure 1).

3.2. Isolation of Trichoderma spp. from Pomelo Farms in Ben Tre

3.2.1. Isolation of Trichoderma spp. from Pomelo Cultivation Soil

Twenty-five strains of Trichoderma spp. were isolated from 30 samples of soil from the rhizosphere of pomelo trees collected in 30 pomelo farms in 3 districts of Giong Trom, Chau Thanh, Binh Dai, in Ben Tre province. Results in

Table 4. General characteristics of Trichoderma spp. on PDA after 8 days of culture.

Strain Name	Shape	Surface Shape	Surface Color	Diameter (cm)
TP-B01	Radiance	Light green granules, forming concentric circles	Green, white	8.90
TP-B02	Almost round	Green mixed with yellow granules	Green, light yellow	9.0
TP-B03	Almost round to round	In the form of green granules, the center of the fungus forms a silky white circle	Green, white	9.0
TP-B04	Evenly rounded	In the form of green granules, radiating evenly to form concentric circles	Dark green	8.70
TP-B05	Radiance	White, thickly grown, alternating green hyphae	White, green	9.0
TP-B06	Uneven roundness	Fine green granules, the center of the mycelia was white	Dark green, white	8.85
TP-B07	Evenly rounded	Green granules, alternating white	Green, White	8.90
TP-B08	Evenly radiated	Granules were green, white and light yellow	Light yellow, white	9.0
TP-B09	Round to near-round	In the form of granules, the center of the colony was green, alternating in the middle of the hyphae with white	Green, White	9.0
TP-B10	Evenly radiated	Silky, thickly grown mycelia with a light yellowish color mixed with green	Green, white, light yellow	8.80
TP-B11	Uneven roundness	Green granular form	Dark green, white	9.0
TP-B12	Uneven roundness	In the form of green hyphae, the hyphae radiated unevenly	Green	8.90
TP-G13	Radiance	White hyphae with green granules in the middle	Green, White	8.95
TP-G14	Almost round	White granules mixed with green granules	White, green center	8.86
TP-G15	Evenly radiated	Light yellow hyphae mixed with white hyphae, scattered fungus grew thickly adhering to the medium	Brownish yellow, white	9.0
TP-G16	Uneven roundness	Fine granular form with a dark green color	Dark green	9.0
TP-G17	Radiance	In the form of green granules, around the center of the fungus there were protruding white hyphae	Green alters white, green in the center	8.85
TP-G18	Almost round to round	White mixed with yellowish green hyphae	Light green, yellow-brown, white	8.90
TP-G19	Uneven roundness	White hyphae thickly grew around the border with green granules	White, green, and green center	9.0
TP-C20	Almost round	The hyphae were green mixed with white	Green, white	8.70
TP-C21	Almost round to round	Green granules, with raised colony margins	Green, white	8.90
TP-C22	Round to round	In the form of green granules, the center of the fungus had white granules	Dark green mixed with white	9.0
TP-C23	Evenly rounded	Light yellow hyphae, the center of the fungi had green granules	Yellow-brown, green center	9.0
TP-C24	Evenly radiated	White hyphae densely grew around the border with green granules	White, green	8.90
TP-C25	Radiance	Dense white hyphae gradually turned green	White mixed with green	9.0

after isolating and purifying the Trichoderma spp. strains showed that the initial colonies were white and filamentous, then gradually turned green or brown. After 48 h of inoculation, the isolated strains had fast-growing, slightly porous mycelia, spreading in concentric circles on the surface of the plate. The strains formed long sporangiophores and had short and thick lateral branches. The top of the branches had a short bottle-shaped fruiting body. The appearance of conidia was egg-shaped (oval) in small numbers (Figure S3).

3.2.2. Growth of Trichoderma spp. on PDA Medium

The growth of Trichoderma spp. was significantly different by 5% after 24, 48, and 72 h of inoculation. At hour 48 of inoculation, TP-B09, TP-G18, and TP-C22 strains had diameters ranging from 4.20 to 4.46 (cm) which were significantly different by 5% compared to the other strains. In addition, the strains with slow growth rates were TP-C20 and TP-B04 with diameters of only 2.70 (cm). At hour 48 after inoculation, TP-G14, TP-G19, and TP-C24 strains had large fungal colony diameters ranging from 7.70 to 7.83 (cm). In addition, strains with small fungal colony diameters included strains TP-B04 and TP-C20 with values of 5.20 and 5.36 (cm). At hour 72 after inoculation, the observed Trichoderma strains had covered the surface of the plate, except for strains with slow growth rates such as TP-B04, TP-G16, and TP-C20 with fungal colony diameters of 7.90–7.60–7.30 (cm), respectively, significantly different by 5% compared to strains TP-B01, TP-B03, TP-B09, TP-B10, and TP-C25 (

Table 5. Average growth rate of Trichoderma spp. on PDA medium.

Fungal Strain	Growth Rate of Fungal Strain (cm)
	24 h	48 h	72 h
TP-B01	3.56 a–c	7.76 a	8.70 ab
TP-B02	3.06 e–g	7.36 d–f	8.63 ab
TP-B03	2.80 hi	6.90 hi	8.76 ab
TP-B04	2.36 j	5.20 n	7.90 e
TP-B05	3.63 a–c	7.63 a–c	8.80 ab
TP-B06	3.50 bc	7.60 a–d	8.36 c
TP-B07	2.43 j	5.56 m	8.20 cd
TP-B08	3.20 ef	7.10 gh	8.66 ab
TP-B09	2.66 i	7.30 e–g	8.80 ab
TP-B10	2.90 gh	7.50 b–e	8.66 ab
TP-B11	3.13 ef	6.56 k	8.30 cd
TP-B12	3.76 a	7.80 a	8.70 ab
TP-G13	3.70 ab	7.60 a–d	8.76 ab
TP-G14	3.50 bc	7.70 ab	8.70 ab
TP-G15	3.20 ef	7.20 fg	8.33 cd
TP-G16	2.16 k	5.86 l	7.60 f
TP-G17	3.03 fg	6.80 ij	8.26 cd
TP-G18	3.26 de	7.40 c–f	8.60 b
TP-G19	3.56 a–c	7.83 a	8.60 b
TP-C20	2.80 hi	5.36 mn	7.30 g
TP-C21	3.10 ef	6.63 jk	8.36 c
TP-C22	2.66 i	6.86 i	8.16 d
TP-C23	3.43 cd	7.20 fg	8.36 c
TP-C24	3.66 ab	7.76 a	8.66 ab
TP-C25	3.73 a	7.66 ab	8.83 a
F	*	*	*
CV (%)		5.05	3.61

Note: The mean numbers in the same column followed by one or more identical letters do not differ statistically at 5% on the Duncan test. * difference by significance of 5%.

).

3.3. Antagonistic Ability of Trichoderma spp. Against Fruit Rot Fungi

The results of

Table 6. Antagonistic performance of Trichoderma spp. with Colletotrichum gloeosporioides PCP-B02-A2 causing pomelo fruit rot.

Fungal Strain	Antagonistic Efficiency (%)			Diameter of Colletotrichum gloeosporioides PCP-B02-A2 (cm)
	48 h	72 h	96 h	48 h	72 h	96 h
TP-B01	35.7 bc	49.1 d–f	67.7 a	1.73 ef ± 0.06	1.85 b–d ± 0.05	1.55 d ± 0.05
TP-B02	33.3 cd	53.2 b–d	61.4 bc	1.80 de ± 0.10	1.70 d–f ± 0.10	1.85 b ± 0.05
TP-B03	19.7 ef	39.2 g	61.1 bc	2.16 bc ± 0.06	2.21 a ± 0.08	1.85 bc ± 0.05
TP-B05	12.9 g	36.8 g	55.2 d	2.35 a ± 0.05	2.30 a ± 0.10	2.15 a ± 0.05
TP-B08	44.4 a	60.6 a	66.7 a	1.50 g ± 0.10	1.43 g ± 0.06	1.60 d ± 0.10
TP-B09	44.4 a	52.6 b–d	68.0 a	1.50 g ± 0.10	1.72 d–f ± 0.07	1.53 d ± 0.12
TP-B10	20.9 ef	46.4 ef	62.5 b	2.13 bc ± 0.06	1.95 bc ± 0.15	1.80 c ± 0.10
TP-B12	29.6 d	51.1 cd	62.5 b	1.90 d ± 0.10	1.77 de ± 0.11	1.80 c ± 0.10
TP-G13	22.2 e	56.3 ab	59.3 c	2.10 c ± 0.10	1.59 f ± 0.08	1.95 b ± 0.05
TP-G14	38.8 b	50.5 c–e	63.5 b	1.65 f ± 0.05	1.80 c–e ± 0.10	1.75 c ± 0.05
TP-G18	32.0 cd	50.5 c–e	62.5 b	1.83 de ± 0.12	1.80 c–e ± 0.10	1.80 c ± 0.10
TP-G19	16.6 fg	45.0 f	63.9 b	2.25 ab ± 0.05	2.00 b ± 0.10	1.73 c ± 0.06
TP-C24	32.0 cd	56.0 b	61.8 bc	1.83 de ± 0.06	1.60 f ± 0.10	1.83 bc ± 0.06
TP-C25	33.3 cd	55.1 bc	68.7 a	1.80 de ± 0.10	1.63 ef ± 0.06	1.50 d ± 0.10
F	*	*	*	*	*	*
CV (%)	55.7	36.2	20.4	6.08	7.05	5.83

Note: The mean numbers in the same column followed by one or more identical letters do not differ statistically at 5% on the Duncan test. * difference by significance of 5%.

show that at hour 48 after inoculation, the antagonistic efficiency of Trichoderma spp. with the fungal strains C. gloeosporioides PCP-B02-A2, Ps. camelliae-sinensis PCP-B03-A1, and Ps. chinensis PCP-B02-B2 were not statistically different. At hour 72 after inoculation, dead mycelia appeared, the antagonistic efficiency was at an average level, and the difference was statistically significant at 5%. At hour 96 after inoculation, the pathogenic fungi were completely inhibited, and the antagonistic efficiency of Trichoderma spp. reached a high level of resistance with 3 pathogenic fungal strains.

3.3.1. Antagonistic Ability of Trichoderma spp. Against Colletotrichum gloeosporioides PCP-B02-A2 on PDA Medium

Table 6 shows the antagonistic performance at hour 48 after inoculation with the equivalent antagonistic ability of 44% including TP-B08 and TP-B09 strains. At hour 72 after inoculation, the strains with the highest antagonistic performance were TP-B08 (60.6%) with a statistically significant difference of 5% compared to the remaining strains. At hour 96 after inoculation, the highest antagonistic performance was TP-C25 (68%). Next, the TP-B01, TP-B08, and T-B09 strains achieved an efficiency of 66.7–68%. In addition, low antagonistic performance was recorded in TP-B05 (55.2%) and TP-G13 (59.3%). The best antagonistic strains selected were TP-C25, TP-B09, TP-B01, and TP-B08 (Figure S4).

3.3.2. Antagonistic Ability of Trichoderma spp. Against Pseudopestalotiopsis chinensis PCP-B02-B2 on PDA Medium

All Trichoderma spp. strains had antagonistic efficiency against P. chinensis. Therein, TP-B08 (49.2%) and TP-G14 (63.2%) strains had a statistically significant difference of 5% compared to the remaining strains at hours 48 and 72 after inoculation. At hour 96 after inoculation, Trichoderma spp. strains had high antagonistic efficiency against pathogenic fungi, reaching 50%, especially TP-B08 with an antagonistic efficiency of up to 71.1% (

Table 7. Antagonistic performance of Trichoderma spp. with the fungal strain Pseudopestalotiopsis chinensis PCP-B02-B2 causing pomelo fruit rot.

Fungal Strain	Antagonistic Efficiency (%)			Diameter of Pseudopestalotiopsis chinensis PCP-B02-B2 (cm)
	48 h	72 h	96 h	48 h	72 h	96 h
TP-B01	19.9 f	38.6 g	64.2 b	2.05 a ± 0.05	2.0 b ± 0.10	1.30 f ± 0.10
TP-B02	42.7 b–d	52.4 b–d	54.6 d	1.46 cef ± 0.06	1.55 e–g ± 0.05	1.65 d ± 0.05
TP-B03	37.5 de	54.0 b	53.2 d	1.60 bc ± 0.10	1.50 g ± 0.10	1.70 cd ± 0.10
TP-B05	18.0 f	24.8 h	40.9 f	2.10 a ± 0.10	2.45 a ± 0.05	2.15 a ± 0.05
TP-B08	49.2 a	55.0 b	71.1 a	1.30 f ± 0.10	1.46 g ± 0.06	1.05 g ± 0.05
TP-B09	33.6 e	47.8 d–f	66.1 b	1.70 b ± 0.10	1.70 c–e ± 0.10	1.23 f ± 0.06
TP-B10	45.3 ab	48.9 c–f	64.2 b	1.40 ef ± 0.10	1.66 c–f ± 0.06	1.30 f ± 0.10
TP-B12	43.3 bc	46.3 ef	58.7 c	1.45 de ± 0.05	1.75 cd ± 0.05	1.50 e ± 0.10
TP-G13	16.0 f	35.6 g	42.3 f	2.15 a ± 0.05	2.10 b ± 0.10	2.10 a ± 0.10
TP-G14	43.3 bc	63.2 a	67.0 b	1.45 de ± 0.05	1.20 h ± 0.10	1.20 f ± 0.10
TP-G18	38.3 c–e	44.8 f	49.1 e	1.57 b–d ± 0.04	1.80 c ± 0.10	1.85 b ± 0.05
TP-G19	41.4 b–d	50.2 b–e	60.1 c	1.50 cef ± 0.10	1.62 d–g ± 0.07	1.45 e ± 0.05
TP-C24	46.6 ab	50.9 b–e	49.6 e	1.36 ef ± 0.06	1.60 d–g ± 0.10	1.83 bc ± 0.06
TP-C25	35.5 e	52.9 bc	64.2 b	1.65 b ± 0.05	1.53 fg ± 0.12	1.30 f ± 0.10
F	*	*	*	*	*	*
CV (%)	49.0	38.1	28.8	6.08	6.40	6.23

Note: The mean numbers in the same column followed by one or more identical letters do not differ statistically at 5% on the Duncan test. * difference by significance of 5%.

). In summary, the strains with good antagonistic efficiency against pathogenic fungi were TP-B08, TP-B09, TP-B01, and TP-C25 (Figure S5).

3.3.3. Antagonistic Ability of Trichoderma spp. Against Pseudopestalotiopsis camelliae-sinensis PCP-B03-A1 on PDA Medium

In the result in

Table 8. Antagonistic performance of Trichoderma spp. with Psedopestalotiopsis camelliae-sinensis PCP-B03-A1 causing pomelo fruit rot.

Fungal Strain	Antagonistic Efficiency (%)			Diameter of Pseudopestalotiopsis camelliae-sinensis PCP-B03-A1 (cm)
	48 h	72 h	96 h	48 h	72 h	96 h
TP-B01	28.0 bc	50.6 a	57.4 a	1.90 de ± 0.10	1.50 f ± 0.10	1.63 e ± 0.06
TP-B02	19.2 de	24.3 f	47.9 b	2.13 bc ± 0.06	2.30 a ± 0.10	2.00 d ± 0.10
TP-B03	18.6 de	38.2 bc	42.7 c	2.15 bc ± 0.05	1.87 de ± 0.07	2.20 c ± 0.10
TP-B05	24.2 cd	32.5 c–e	34.9 e	2.00 cd ± 0.10	2.05 b–d ± 0.05	2.50 a ± 0.10
TP-B08	17.9 e	41.9 b	56.5 a	2.16 b ± 0.15	1.76 e ± 0.06	1.66 e ± 0.06
TP-B09	26.1 bc	43.0 b	55.7 a	1.95 de ± 0.05	1.76 e ± 0.15	170 e ± 0.10
TP-B10	31.8 ab	27.6 ef	40.1 cd	1.80 ef ± 0.10	2.20 ab ± 0.10	2.30 bc ± 0.10
TP-B12	11.0 f	24.3 f	37.5 de	2.35 a ± 0.05	2.30 a ± 0.10	2.40 ab ± 0.10
TP-G13	30.5 ab	29.2 d–f	34.9 e	1.83 ef ± 0.06	2.15 a–c ± 0.05	2.50 a ± 0.10
TP-G14	26.1 bc	27.6 ef	39.2 cd	1.95 de ± 0.05	2.20 ab ± 0.10	2.33 bc ± 0.06
TP-G18	31.8 ab	34.2 cd	49.9 b	1.80 ef ± 0.10	2.0 cd ± 0.10	1.92 d ± 0.07
TP-G19	16.7 e	33.1 c–e	40.1 cd	2.20 b ± 0.10	2.03 b–d ± 0.12	2.30 bc ± 0.10
TP-C24	35.6 a	30.9 de	40.1 cd	1.70 f ± 0.10	2.10 bc ± 0.10	2.30 bc ± 0.10
TP-C25	35.6 a	40.8 b	56.5 a	1.70 f ± 0.10	1.80 e ± 0.10	1.66 e ± 0.06
F	*	*	*	*	*	*
CV (%)	66.9	54.2	34.1	6.37	6.71	6.17

Note: The mean numbers in the same column followed by one or more identical letters do not differ statistically at 5% on the Duncan test. * difference by significance of 5%.

at hour 96 after inoculation, strains TP-B01, TP-B08, TP-B09, and TP-C25 had the highest antagonism level ranging from 55.7% to 57.4% compared to strains TP-B05, TP-G13, and TP-G14 (34.9–39.2%). Thus, for the antagonism between Trichoderma spp. and Ps. camelliae-sinensis, strains with high antagonism efficiency against pathogenic fungi were selected, such as TP-B01, TP-B08, TP-B09, and TP-C25 (Figure S6).

3.4. Identification of Trichoderma spp. Fungi that can Antagonize Fungi Causing Pomelo Fruit Rot

The TP-B01 and TP-C25 strains were identified as Trichoderma asperellum with accession numbers in the Genbank database of OR225702 and OR225705, respectively. Meanwhile, the TP-B08 and TP-B09 strains were identified as Trichoderma harzianum with accession numbers of OR225703 and OR225704, respectively, with 100% similarity (Figure 2).

3.5. Ability to Produce Enzymes

TP-B01, TP-B08, TP-B09, and TP-C25 strains produced chitinase (0.217–0.301 UI mL⁻¹), endo-β-1,3-glucanase (0.019–0.064 UI mL⁻¹), and exo-β-1,3 glucanase (0.007–0.020 UI mL⁻¹). These enzymes are considered as a mechanism to prevent pathogen growth (

Table 9. Ability to produce enzymes of selected Trichoderma spp.

Strains	Chitinase	Endo-β-1,3-Glucanase	Exo-β-1,3 Glucanase
	(UI mL−1)
TP-B01	0.301 a	0.064 a	0.020 a
TP-B08	0.217 c	0.032 c	0.007 c
TP-B09	0.251 b	0.049 b	0.017 b
TP-B25	0.223 c	0.019 d	0.007 c
F	*	*	*
CV (%)	4.10	13.3	8.60

Note: The mean numbers in the same column followed by one or more identical letters do not differ statistically at 5% on the Duncan test. * difference by significance of 5%.

).

4. Discussion

Postharvest diseases are one of the problems affecting both the fresh citrus and citrus juice industries, mainly caused by fungal pathogens [11]. Fungi can infect fruit before, during, or after harvest, but the disease develops after the fruit is harvested, causing serious economic losses to the citrus industry [44]. In this study, three highly virulent fungal strains—Colletotrichum gloeosporioides PCP-B02-A2, Pseudopestalotiopsis chinensis PCP-B02-B2, and P. camelliae-sinensis PCP-B03-A1—were identified as causal agents of fruit rot in pomelo. Importantly, all three pathogens showed rapid lesion development, indicating high aggressiveness and their potential to cause significant postharvest losses. Postharvest anthracnose of citrus fruits is caused by different Colletotrichum species, especially C. gloeosporioides [11]. Anthracnose of citrus can also be a field disease, affecting leaves and branches and causing fruit drop after flowering. However, C. gloeosporioides is a weak pathogen of citrus fruits. Asexual spores are spread by rain or overhead irrigation to developing fruits [11]. In contrast, conidia are less numerous but are airborne, participating in long-distance dispersal [11]. Lasiodiplodia sp. is a species that has been reported to cause fruit rot on many citrus trees such as lemon, pomelo, and orange [33,45,46]. The primary cell wall of the fruit is composed of approximately 10% protein and 90% polysaccharides, which can be divided into three groups: cellulose, hemicellulose, and pectin [47]. Many cell wall-degrading enzymes can be secreted by invading pathogens and use the plant cell wall as a source of nutrients, reducing postharvest shelf life and ultimately leading to undesirable quality loss, softness, and decay. Some of the notable polysaccharide-degrading enzymes include xylanase, polygalacturonase, cellulase, and α-amylase [48]. Cell wall polysaccharides in fruits play an important role in resistance to invasion by L. theobromae and some other pathogens [49]. Spore-forming fungi invade plant tissue by secreting enzymes that degrade cell wall polysaccharides (cellulases, β-galactosidases, polygalacturonases, and pectinesterases). Enzymatic cleavage of cell wall polysaccharides is the essential pathway for L. theobromae to infect harvested fruit and thus lead to disease onset and fruit softening [49]. In addition to Colletotrichum sp. and Lasiodiplodia sp., other agents responsible for fruit rot have been reported, including Phomopsis, Phytophthora, Alternaria, and Penicillium [12,50,51].

The results in Table 6 and Figures S4–S6 show that the antagonistic efficiency of Trichoderma spp. reached a high level of resistance against 3 pathogenic fungi. Table 6 shows that Trichoderma spp. strains had good antagonistic rates and inhibitory efficiency against C. gloeosporioides PCP-B02-A2 (68%), P. chinensis PCP-B02-B2 (71.1%), and Ps. camelliae-sinensis PCP-B03-A1 (55.7–57.4%) at hour 96 after inoculation. These strains were selected and identified as T. asperellum TP-B01, T. harzianum TP-B08, T. harzianum TP-B09, and T. asperellum TP-C25 (Figure 2). This result is consistent with the study by Nasreen Musheer and Shabbir Ashraf [52] where the antagonistic efficiency of the Trichoderma viride strain against pathogenic fungus C. gleosporiodes under laboratory conditions was 69.79%. Furthermore, T. koningiopsis PSU3-2 was found to inhibit the growth of C. gloeosporioides isolated from chili pepper up to 79.57% [31]. To control Pseudopestalotiopsis spp., T. asperellum also showed remarkable results [53]. On mango, T. asperellum KUFA 0042 was used to control both L. theobromae and C. gloeosporioides [54].

In addition, Trichoderma strains have a mechanism directly related to the antagonistic efficiency against pathogenic fungi with fast growth rate, good nutritional competition, living space with pathogenic fungi [55,56] and secrete chitinase and glucanase that destroy the cell wall of pathogenic fungi [57]. The study by Kucuk and Sharma [58] applied biological control measures on the antagonistic ability of T. harzianum against Aspergillus ustus (71.90%). According to Rajendiran et al. [59] the antagonism rates against A. niger, A. flavus, and A. fumigatus were 55%, 51%, and 52%, respectively. In addition, Padder and Sharma [60] showed that the antagonism rate against C. lindemuthianum causing anthracnose on beans was 59.48%. Apart from microbial antagonists, plant extracts are also candidates to control Pseudopestalotiopsis spp. [8]. Bacteria, such as Burkholderia theae [61] and B. subtilis [54], also exhibit antagonism against Colletotrichum spp. However, compared to other biological control agents, Trichoderma spp. are used the most because of their diverse traits as antagonists [62]. Notably, in the study by Widyaningsih and Triasih [63], a combined application of both T. asperellum and endophytic bacteria increased the resistance of strawberry against a disease.

Compared to chemical fungicides, which can lead to resistance and environmental hazards, the use of Trichoderma offers a sustainable, eco-friendly alternative for disease management. This supports the growing need for efficient, low-cost, and eco-friendly biocontrol options in agriculture [62], aligning with integrated pest management (IPM) principles. Field application of such strains—either as seed treatments, soil amendments, or postharvest dips—could significantly reduce the incidence of fruit rot without harmful residues [64]. Future research should evaluate the efficacy of these promising Trichoderma strains under field and storage conditions to optimize delivery methods and formulations. Integration into IPM programs could also include their combination with other biocontrol agents, such as Bacillus spp. or plant extracts, for synergistic effects which would produce promising results [65]. In summary, this study contributes novel insights into the biological control of Colletotrichum and Pseudopestalotiopsis spp. using Trichoderma spp., expanding the range of effective microbial agents and supporting the transition to sustainable disease management in citrus production systems.

5. Conclusions

The 3 selected strains of fungi causing fruit rot of pomelos included Colletotrichum gloeosporioides PCP-B02-A2, Pseudopestalotiopsis camelliae-sinensis PCP-B03-A1, and Pseudopestalotiopsis chinensis PCP-B02-A2. Twenty-five strains of Trichoderma spp. were isolated from 30 soil samples collected in 3 districts of Chau Thanh, Giong Trom, and Binh Dai, in Ben Tre province. Four strains of antagonists were selected, including Trichoderma asperellum TP-B01, Trichoderma harzianum TP-B08, Trichoderma harzianum TP-B09, and Trichoderma asperellum TP-C25 to antagonize 3 strains of pathogenic fungi C. gloeosporioides PCP-B02-A2, Ps. camelliae-sinensis PCP-B03-A1, and Ps. chinensis PCP-B02-B2. The achieved antagonistic efficiencies were 66.7–68.7%, 55.7–57.4%, and 64.2–71.1%, respectively.