Diversity of Fusarium spp. in Pomelo (Citrus maxima (Burm.) Merr.) Orchards Riskily Caused Root Rot and Yellow Leaf Disease, and the Control Approach

Abstract

Diseases caused by Fusarium spp. vary around the world. It is important to determine the causals agents and indigenous antagonists against these pathogens. Thus, this study aimed to (i) determine the pathogens of root rot and yellow leaf disease (RRYLD), (ii) select Trichoderma spp. strains to control the pathogens, and (iii) evaluate methods for preparing the antagonistic fungi. Diseased soil samples were collected from pomelo orchards in Ben Tre province, Vietnam. The experiment isolated 08 Fusarium spp. strains, with the fastest growth in PDA in FP-C16, FP-B18, FP-B16, and FP-B03 (8.33–17.3 mm) on day 4 of culture. They were identified as Fusarium fujikuroi FP-C16, F. verticillioides FP-B18, F. verticillioides FP-B16, and F. incarnatum FP-B03. On the other hand, 25 Trichoderma spp. strains were isolated from the pomelo rhizosphere. Among them, 13 Trichoderma spp. strains showed rapid growth and strong antagonistic activity against two Fusarium spp. strains under laboratory conditions. The two Trichoderma spp. strains TP-C40 and TP-G50 had antagonistic efficiencies against FP-C16 and FP-B16 at 47.7–63.5%. The two selected Trichoderma spp. strains were identified as Trichoderma asperellum TP-C40 and T. yunnanense TP-G50. The two Trichoderma spp. strains TP-C40 and TP-G50 reduced the number of leaves and roots infected by Fusarium spp.

1. Introduction

Pomelo cultivation in the Mekong Delta is facing many diseases, such as root rot and yellow leaf disease (RRYLD), anthracnose, thrips, black blight, and stem-end rot [1,2,3,4,5]. Therein, the RRYLD caused by Fusarium spp. has significantly and severely damaged citrus trees [6]. Fusarium spp. is known as a fungi that can strongly exhibit harm to many crops all over the world, damaging host plants, reducing fruit quality, and decreasing the yield and development of plants. Fusarium spp. can cause symptoms such as rot in seedlings, roots, top shoots, and stems during plant development stages [7]. Furthermore, plants start from browned roots and stems and yellow leaves only to completely wither and die. Four species of Fusarium spp. have been identified to cause diseases in citrus, which are Fusarium solani (Martius) (70.6%), F. oxysporum (17.6%), F. equiseti (8.82%), and F. brachygibbosum (2.94%) [8,9]. Likewise, Kurt et al. [10] have detected F. solani as a causal agent in citrus in Turkey. Moreover, Farhaoui et al. [11] assume that F. oxysporum is the most pathogenic species, followed by F. solani, F. equiseti, F. nygmai, F. brachygibbosum, F. proliferatum, F. culmorum, and F. falciforme. The diversity of Fusarium spp. fungi has been recorded with eight species [12]. However, the influences of these species vary. Nowadays, using microbial antagonists is well considered, and Trichoderma spp. fungi are well known for their potent biocontrol capability via antagonism, parasitism, and antibiotic production mechanisms. Moreover, Trichoderma spp. also induces plant resistance against pathogens [13]. Many studies have used Trichoderma spp. to control Fusarium spp. fungi. For instance, Trichoderma spp. reduced the disease rate of tomato wilt caused by F. oxysporum f. sp. lycopersici [14]. Furthermore, indigenous Trichoderma spp. reduced 35% of the sesame wilt caused by F. oxysporum F.28.1A [15]. In addition, T. asperellum T-SP19 and T-SP32 are found to control Lasiodiplodia theobromae S-P06 and S-P07, which cause stem-end rot in pomelo [5]. Moreover, T. asperellum IIPR Th-31 and T. afroharzianum IIPR Th-33 reduced 78–85% of the wilt rate in pigeonpea (Cajanus cajan L.) [16].

As can be seen, RRYLD has become an important constraint in pomelo production, but information on the diversity of Fusarium species associated with this disease in pomelo orchards in the Mekong Delta of Vietnam remains limited. In addition, the use of indigenous Trichoderma spp. isolated from pomelo rhizosphere soils as potential biocontrol agents against these pathogens has not been sufficiently investigated. Therefore, the novelty of this study lies in combining the identification of Fusarium spp. associated with pomelo RRYLD and the screening of native Trichoderma spp. for their antagonistic and disease-suppressive potential. Thus, the current study aimed (i) to determine the diversity of the causal agents of RRYLD in pomelo orchards, (ii) to select Trichoderma spp. strains that can antagonize Fusarium spp. pathogens under in vitro conditions, and (iii) to assess the potential of applying selected Trichoderma spp. strains to control Fusarium-induced RRYLD in pomelo seedlings under greenhouse conditions.

2. Materials and Methods

2.1. Materials

Time of the experiment: from October 2022 to April 2023.

Place of the experiment: The soils were collected from the districts of Chau Thanh, Binh Dai, and Giong Trom in Ben Tre province, Vietnam. The isolation and selection of pathogens and antagonists took place in the Edible and Medicinal Mushroom Laboratory, Faculty of Crop Science, College of Agriculture, Can Tho University, Can Tho City, Vietnam.

Culture medium: Potato dextrose agar (PDA)—potato extract: 200.0 g L⁻¹, D-glucose: 20.0 g L⁻¹ (Xilong, Xinjiang, China), chloramphenicol: 25.0 g L⁻¹ (Xilong, Xinjiang, China), and agar: 20.0 g L⁻¹ (Hai Long, Ho Chi Minh City, Vietnam), pH 6.5–6.8 [17]. Trichoderma selective medium (TSM)—NH₄NO₃: 1.0 g L⁻¹ (Xilong, Xinjiang, China), K₂HPO₄: 0.90 g L⁻¹ (Xilong, Xinjiang, China), KCl: 0.15 g L⁻¹ (Xilong, Xinjiang, China), MgSO₄.7H₂O: 0.20 g L⁻¹ (Xilong, Xinjiang, China), chloramphenicol: 0.25 g L⁻¹, agar: 20.0 g L⁻¹, and glucose: 3 g L⁻¹, pH 6.5–6.8 [18].

Soil for biocontrol experiment: Alluvial soil was collected from the Experiment Sections, College of Agriculture, Can Tho University. The soil was dried, cleaned from organic residues, mixed, ground, and autoclaved twice (24 h, between one and two times). The autoclaved soil was put into a pot weighing 2.0 kg.

2.2. Methods

2.2.1. Isolation and Assessment of Pathogenicity of Fusarium spp. in Pomelo

Isolation of Fusarium spp. pathogen: A total of 10 rhizosphere and root samples of diseased pomelo trees at depths of 5–20 cm were collected from the districts of Chau Thanh, Binh Dai, and Giong Trom, Ben Tre province. The diseased samples were kept in plastic bags, labeled, brought back to the lab, and immediately isolated. The samples were isolated according to Olszak-Przybyś et al. [19]. One gram of the sample was distilled at concentrations of 1 × 10⁻¹, 1 × 10⁻², and 1 × 10⁻³ in sterilized distilled water (SDW), gently shaken for 30 min, and sedimented for one day under room temperature (28–32 °C). After autoclaving the equipment at 121 °C for 20 min, the sample was inoculated on PDA. After incubation for 2–4 days, mycelia that had the features of the pathogens were selected. The fungi were isolated according to their mycelia. The mycelia were observed for the unique features of Fusarium spp. fungi. The isolated fungi were stored in solid PDA under 4 °C

Description of pathogen morphology: The Fusarium spp. fungi were described according to Nikitin et al. [20]. Monospores were cultured in PDA under light. A PDA droplet was placed on a microscopic slide in a Petri dish (90 mm × 15 mm; Dinlab, Casablanca, Morocco). Fungal monospores were inoculated to the PDA droplet, covered by a microscopic slip, and added with some SDW droplets in the Petri dish. The sample was incubated at room temperature (25 °C) for 4 days. The mycelia surface, color, and shape and the spore size were observed by stereomicroscopy (Kruess, Hamburg, Germany) and light microscopy at 40X (Olympus, Evident Corporation, Tokyo, Japan). The fungi were arranged according to the sampling location, mycelia shape and color, and the shape of the hyphae.

Identification of the selected Fusarium spp. fungi: Strains of the Fusarium spp. had their DNA extracted from the hyphae. The spores were cultured for 7 days in PDA. Their hyphae were then put into a 2.2 mL Eppendorf and incubated at room temperature for 10 min. They were then centrifuged at 13,000 rpm for 5 min. The cell pellet was collected and rinsed with 500 µL ethanol 70% (Xilong, Xinjiang, China), centrifuged at 13,000 rpm for 5 min, and vacuum dried. The DNA was dissolved in 100 µL TE 0.1X (Merck, Darmstadt, Germany). The PCR was conducted with a primer pair of ITS1 and ITS4 [21] with the following sequences: ITS 1: 5′-TCCGTAGGTGAACCTGCGG-3′, ITS 4: 5′-TCCTCCGCTTATTGATATGC-3′. The PCR was conducted with a total volume of 50 µL via the following steps: denaturation (95 °C for 5 min, a 30-cycle reaction (denaturation at 95 °C for 90 s, annealing at 52 °C for 60 s, and elongation at 72 °C for 90 s), and termination at room temperature). The PCR amplicons were purified and sequenced by automatic sequencing. The sequences were compared with the database of GenBank in NCBI by BLASTN.

2.2.2. Isolation and Selection of Trichoderma spp.

Sampling: 25 rhizosphere samples of healthy pomelo trees were collected from districts of Chau Thanh, Binh Dai, and Giong Trom, Ben Tre province, Vietnam. The soil was collected at a depth of 5–15 cm. The samples were stored at 4 °C and transported back to the laboratory.

Fungal isolation: The Trichoderma spp. fungi were isolated according to Alwadai et al. [22] in TSM. The soil samples were diluted with SDW with the ratio of 1 g soil and 99 mL SDW, shaken for 30 min, and sedimented for a day at room temperature (28–32 °C). The soil solution was spread on TSM [23]. After 48 h, the samples were observed to select and purify fungal strains. The characteristics of hyphae (color and shape) and spores were observed by light microscopy at 40× to detect Trichoderma spp. strains. The pure samples were stored at 4 °C in Eppendorf and Petri dishes containing PDA.

Morphology of Trichoderma spp.: The morphological characteristics of the Trichoderma spp. strains were observed on PDA according to Matas-Baca et al. [24]. Hyphae (6 mm) of Trichoderma spp. isolated in TSM were inoculated to PDA and incubated at 28 ± 2 °C for 7 days. The diameters of mycelia were measured at hours 24, 48, 72, and 96 after inoculation. Other characteristics, such as pigments, green spores, odor, and mycelia, were also recorded according to Kumar et al. [25].

Biocontrol capacity of Trichoderma spp. against Fusarium spp. causing RRYLD under in vitro conditions: The antagonistic activity of Trichoderma spp. against Fusarium spp. was evaluated using the dual-culture technique on PDA. Strains of both Trichoderma spp. and Fusarium spp. were cultured in the same PDA Petri dish (φ = 9.5 mm), with the distance between the two centers of the two mycelia (φ = 5 mm) of 3 cm (Figure 1). The diameters of each mycelium were measured every day to determine the percent inhibition of mycelial growth (PIMG) of Trichoderma spp. strains. The dish with only Fusarium spp. was used as the control. Each treatment was arranged with three replicates each of which was a Petri dish, and the experiment was repeated independently. Colony radius was recorded at 24, 48, 72, and 96 h after inoculation.

The PIMG was calculated according to Krutmuang et al. [26], as follows:

• PIMG = {(R1 − R2)/R1} × 100%

Therein, the following holds:

• R1: The radii of Fusarium spp. in the negative control;
• R2: The radii of Fusarium spp. co-inoculated with Trichoderma spp.

The inhibition zone is where the Trichoderma spp. mycelia is overlapping the pathogenic mycelia.

Biocontrol capacity of Trichoderma spp. against pomelo RRYLD by Fusarium spp. under greenhouse conditions: The experiment was completely randomized, with 3 replications, each of which contained four trees corresponding to 4 pots. The treatments included the following:

• Treatment 1: no pathogens;
• Treatment 2: four pathogenic strains;
• Treatment 3: four pathogenic strains + spray two Trichoderma spp. strains at the same time as the infection;
• Treatment 4: four pathogenic strains + spray two Trichoderma spp. strains 3 days after the infection;
• Treatment 5: four pathogenic strains + spray two Trichoderma spp. strains 6 days after the infection.

The suspension (2 × 10⁶ spores/seedlings) of the selected Fusarium spp. strains was poured in Pomelo seedlings. The pathogens were inoculated on days 7, 14, 21, and 28 after infection.

The parameters of evaluation included the RRYLD’s time of appearance and the number of infected leaves and roots. The level of disease was considered on day 40 after planting.

The identification of the selected Trichoderma spp. strains: Trichoderma spp. was identified in the same manner as Fusarium spp.

2.3. Statistical Analysis

Data were subjected to analysis of variance (ANOVA) using SPSS version 13.0. Mean comparisons were performed using Duncan’s multiple range test at the 5% probability level. Differences were considered statistically significant at p < 0.05. The results are presented as mean values, and treatments sharing the same letter within a column are not significantly different.

3. Results

3.1. Morphology and Growth of Fusarium spp. Strains Causing RRYLD in Pomelo

3.1.1. Morphology of Fusarium spp.

Eight Fusarium spp. strains causing RRYLD were isolated in PDA from soil samples collected from the three districts of Chau Thanh, Binh Dai, and Giong Trom. All of the fungi covered the whole Petri dish on days 9–10 of culture. In general, the fungi were white (FP-B06 and FP-B10), darkish brown (FP-B03), from yellow to white (FP-C110 and FP-C16), from purple to light yellow (FP-B16 and FP-B18), and yellowish purple (FP-C05). The surface structure was different. The mycelia grew into concentric circles. The border was white and undulate. The lower side of the mycelia was flat, white, and light yellow, white, and dark in the middle (Figure 1 and Figure S1). The mycelia of the Fusarium spp. strains were yellow in the middle and then turned into white, purple, or light yellow, which were the features of each fungal strain (

Table 1. Mycelia characteristics of Fusarium spp. causing root rot and yellow leaf disease on day 10 after inoculation on PDA.

Fungal Strain	Location	Mycelia and Spores
		Color	Hyphae	Spore
FP-B03	Binh Dai	From darkish brown to white	Tilt, crossed	Transparent, oval
FP-B06	Binh Dai	White	Tilt, crossed	Transparent, oval
FP-B10	Binh Dai	White	Tilt, crossed	Transparent, oval
FP-B16	Binh Dai	From purple to light yellow	Tilt, crossed	Transparent, oval
FP-B18	Binh Dai	From yellow to white	Tilt, crossed	Transparent, oval
FP-C05	Chau Thanh	From yellow to yellowish pink	Tilt, crossed	Transparent, oval
FP-C10	Chau Thanh	From yellow to white	Tilt, crossed	Transparent, oval
FP-C16	Chau Thanh	From yellow to white	Tilt, crossed	Transparent, oval

F: Fusarium spp., P: Citrus maxima (pomelo), C: Chau Thanh, B: Binh Dai.

).

3.1.2. Growth of Fusarium spp. on PDA

At hour 24 after inoculation on PDA, two strains, FP-B16 and FP-B18, had the fastest growing diameter with 17.3 mm and 16.7 mm compared with 5.33–10.7 mm of the other strains (

Table 2. Growth of Fusarium spp. causing root rot and yellow leaf disease on PDA.

Fungal Strain	Mycelia Growth (mm)
	24 h	48 h	72 h	96 h
FP-B03	10.7 b	17.7 b	31.7 b	39.7 c
FP-B06	5.33 d	12.3 e	20.3 e	29.3 e
FP-B10	8.67 c	14.6 d	22.7 d	30.1 e
FP-B16	17.3 a	28.3 a	45.7 a	60.3 a
FP-B18	16.7 a	27.3 a	44.3 a	57.3 b
FP-C05	9.01 c	14.3 d	19.3 e	25.1 f
FP-C10	6.67 d	16.1 c	25.6 c	34.7 d
FP-C16	8.33 c	12.3 e	22.1 d	32.3 e
F	*	*	*	*
CV (%)	8.03	5.50	5.22	4.15

^a–f In the same column, numbers with the same following letters are not significantly different according to the Duncan test. * Different at a 5% significance level.

). At hour 48 after inoculation, strains FP-B16 (28.3 mm) and FP-B18 (27.3 mm) had greater growth diameters than the other strains (12.3–17.7 mm). At hour 72 after inoculation, the strains FP-B16 and FP-B18 showed significant growth differences from the other strains, with 45.7 mm and 44.3 mm compared with 19.3–31.7 mm (Table 2). At hour 96 after inoculation, the growth diameter of FB-B16 was 60.3 mm and greater than those of the other strains (25.1–57.3 mm). Ultimately, the strains FP-B03, FP-B16, FP-B18, and FP-C16 were chosen for identification and artificial infection.

3.1.3. Identification of Fusarium spp. Causing RRYLD in Pomelo

The strains FP-B03, FP-B16, FP-B18, and FP-C16 were identified as Fusarium fujikuroi, F. verticillioides, F. verticillioides, and F. incarnatum, respectively, with the corresponding accession numbers of OQ928087, OQ928088, OQ928089, and OQ928090, at 99% similarity (Figure 2).

3.2. Morphology and Growth of Trichoderma spp. Strains

3.2.1. Morphology of Trichoderma spp.

Twenty-five Trichoderma spp. strains were collected from 25 rhizosphere samples from 25 pomelo orchards in Chau Thanh, Binh Dai, and Giong Trom districts, Ben Tre province (Figure 3 and Figure S2). The mycelia covered the dish surface on day 5 after inoculation and turned light or dark green (

Table 3. Mycelia characteristics of Trichoderma spp. on day 7 of culture on PDA.

Fungal Strain	Shape	Surface	Color
TP-B26	Round	Roughly green forming concentric circle	Green, white
TP-B27	Round	Roughly green	Green, white
TP-B28	Nearly round	From white to light yellow, in the middle of the mycelia, a green circle appeared	Green, light yellow
TP-B29	Even round	Green, round white hyphae forming concentric circle	Green, white
TP-B30	Round	Roughly green, white hyphae in the concentric circle	Green, white
TP-B31	Nearly round	Smoothly green, white concentric circle	Dark green, white
TP-B32	Even round	Roughly green, thick white hyphae, altered green hyphae	Green, white
TP-C33	Even round	Roughly green, light yellow	Green, light yellow
TP-C34	Round	Roughly green, green center with altered white hyphae	Dark green, white
TP-C35	Uneven round	Fibrous white, green center	Green, white
TP-C36	Uneven round	Roughly green center, from green to light yellow border	Green, light yellow
TP-C37	Round to nearly round	Roughly green, white center, green and white surrounding	Dark green, white
TP-C38	Nearly round	Roughly green, white hyphae on green mycelia	Dark green, white
TP-C39	Round to nearly round	Roughly white and roughly green, green center	Dark green, roughly white
TP-C40	Even round	Roughly green, thick white hyphae on green mycelia	Dark green, white
TP-C41	Round to nearly round	Smoothly green center, white hyphae on green mycelia	Green, white
TP-C42	Irregular	Roughly green	Green
TP-C43	Nearly round	Fibrous white on green mycelia in the center, green surrounding	Dark green, white
TP-C44	Round to nearly round	Roughly white on green mycelia	Green, white
TP-G45	Nearly round	Roughly green with altered white hyphae	Dark green, white
TP-G46	Even round	Smoothly green, raised white center	Green, white
TP-G47	Even round	Roughly green, white center	Dark green, white
TP-G48	Even round	Fibrous light-yellow border, roughly green center, raised roughly white surrounding	Green, from white to light yellow
TP-G49	Irregular	Roughly green, white hyphae on green mycelia in the center, light yellow border	Dark green, from white to light yellow border
TP-G50	Even round	Raised fibrous white, roughly green border	Dark green, white

T: Trichoderma spp., P: Citrus maxima (pomelo), B: Binh Dai, C: Chau Thanh, G: Giong Trom.

).

3.2.2. Growth of Trichoderma spp. on PDA

At hour 24 after inoculation, the strain TP-G47 had a growth of 36.0 mm, which was equivalent to those of TP-B30, TP-C36, and TP-G48 (35.0–35.7 mm). The mycelia diameters at hour 48 after inoculation were equivalent among strains TP-B30, TP-G47, and TP-G48 (67.0 mm) (

Table 4. Growth of Trichoderma spp. strains on PDA.

Fungal Strain	Growth (mm)
	24 h	48 h	72 h
TP-B26	33.3 de	63.3 de	88.3 ab
TP-B27	32.0 efg	61.3 f	86.3 c
TP-B28	28.0 klm	57.0 hi	83.5 d
TP-B29	33.8 cd	66.0 ab	89.3 ab
TP-B30	35.5 ab	67.0 a	89.5 ab
TP-B31	30.0 hij	57.8 ghi	83.2 d
TP-B32	29.5 ijk	58.5 gh	83.6 d
TP-C33	33.5 cde	63.0 de	88.7 ab
TP-C34	33.3 de	63.3 de	88.0 b
TP-C35	27.7 lmn	56.7 ij	82.7 de
TP-C36	35.0 abc	66.0 ab	89.0 ab
TP-C37	26.3 nop	55.3 jk	80.7 f
TP-C38	26.0 op	55.0 k	80.3 f
TP-C39	28.7 j–m	57.7 ghi	82.7 de
TP-C40	28.3 klm	57.0 hi	82.5 de
TP-C41	29.0 i–l	58.5 gh	84.0 d
TP-C42	31.5 fgh	62.5 def	83.0 ab
TP-C43	30.5 ghi	62.0 ef	88.0 b
TP-C44	29.3 ijk	59.0 g	83.0 de
TP-G45	33.0 def	64.0 cd	89.3 ab
TP-G46	34.0 bcd	65.0 bc	89.4 ab
TP-G47	36.0 a	67.0 a	90.0 a
TP-G48	35.7 ab	67.0 a	90.0 a
TP-G49	25.3 p	54.7 k	78.7 g
TP-G50	27.3 mno	57.7 ghi	81.3 ef
F	*	*	*
CV (%)	0.49	0.04	0.03

^a–p In the same column, numbers with the same following letters are not significantly different according to the Duncan test. * Different at the 5% significance level.

). At hour 72 after inoculation, the strains covered the dish surface, but some strains grew more slowly, including TP-B28, TP-B32, TP-B32, TP-C35, TP-C37, TP-C38, TP-C39, TP-C40, TP-C41, TP-C42, TP-C44, TP-G49, and TP-G50 (24.0–25.5 mm) (Table 4). Therefore, the other 13 Trichoderma spp. strains, including TP-B26, TP-B28, TP-B30, TP-B32, TP-C34, TP-C36, TP-C38, TP-C40, TP-C42, TP-C44, TP-C46, TP-C48, and TP-G50, were selected for antagonism against pomelo RRYLD.

3.3. Biocontrol Capacity of Trichoderma spp. Against Fusarium spp. Under In Vitro Conditions

3.3.1. Antagonism of Trichoderma spp. Against Fusarium incarnatum FP-C16 Under In Vitro Conditions

The antagonism of the 13 Trichoderma spp. strains against Fusarium incarnatum FP-C16 are illustrated in Figure 4. In general, all of the Trichoderma spp. suppressed the growth of Fusarium incarnatum FP-C16 at different levels.

At hour 48 after inoculation, moderate PIMG of the strains accounted for 42.0%. The PIMG of the TP-B26 (59.5%) and TP-C38 (56.5%) strains were equivalent to the TP-C36 strains (50.2%) but greater than the others’ (22.1–47.0%). Weak PIMG was roughly 22.1–37.7% and included TP-B30 (28.3%), TP-C34 (22.1%), TP-C42 (22.1%), and TP-C44 (37.7%) strains (

Table 5. Percent inhibition of mycelial growth of Trichoderma spp. against Fusarium incarnatum FP-C16 causing root rot yellow leaf in pomelo.

Fungal Strain	PIMG (%)		Inhibition Diameter of Fusarium incarnatum FP-C16 (cm)
	48 h	72 h	48 h	72 h
TP-B26	56.5 a	58.6 abc	0.93 ef ± 0.20	1.13 cde ± 0.12
TP-B28	47.0 bcd	58.6 abc	1.13 cde ± 0.23	1.13 cde ± 0.23
TP-B30	28.3 ef	22.1 e	1.53 ab ± 0.23	2.13 a ± 0.23
TP-B32	47.0 bcd	61.1 ab	1.13 cde ± 0.40	1.07 de ± 0.12
TP-C34	22.1 f	22.1 e	1.67 a ± 0.23	2.13 a ± 0.31
TP-C36	50.2 abc	61.1 ab	1.07 def ± 0.23	1.07 de ± 0.20
TP-C38	59.5 a	51.3 c	0.86 f ± 0.31	1.33 c ± 0.23
TP-C40	47.1 bcd	63.5 a	1.13 cde ± 0.23	1.00 e ± 0.12
TP-C42	22.1 f	36.7 d	1.33 bc ± 0.35	1.73 b ± 0.12
TP-C44	37.7 de	24.6 e	1.67 a ± 0.12	2.06 a ± 0.23
TP-C46	40.8 cd	53.8 bc	1.27 cd ± 0.12	1.27 cd ± 0.20
TP-C48	47.1 bcd	61.1 ab	1.13 cde ± 0.23	1.07 de ± 0.12
TP-G50	40.8 cd	58.6 abc	1.27 cd ± 0.12	1.13 cde ± 0.23
F	*	*	*	*
CV (%)	92.3	65.0	11.5	10.4

^a–f In the same column, numbers with the same following letters are not significantly different according to the Duncan test. * Different at the 5% significance level. PIMG: percent inhibition of mycelial growth.

).

At hour 72 after inoculation, high PIMG consisted of T-SA10 with a PIMG up to 63.5%, which was significantly greater than those of strains of TP-B26 (58.6%), TP-B28 (58.6%), TP-B32 (61.1%), TP-C36 (61.1%), TP-G48 (61.1%), and TP-G50 (58.6%). Therein, the strains with moderate PIMG were TP-C38 (51.3%) and TP-G46 (53.8%). On the other hand, the strains with weak PIMG consisted of TP-B30 (22.1%), TP-C34 (22.1%), TP-C42 (36.6%), and TP-C44 (24.4%) (Table 5).

The pathogenic hyphae were eliminated on day 9 of antagonism on PDA. The mycelia of Trichoderma spp. covered all the mycelia of the pathogens (Figure 4).

3.3.2. Antagonism of Trichoderma spp. Against Fusarium verticillioides FP-B16 Under In Vitro Conditions

The Trichoderma spp. fungi antagonized F. verticillioides FP-B16 on PDA (Figure 5). At hour 48 of inoculation, the Trichoderma spp. TP-G50 had the greatest PIMG (44.1%). Strains with moderate PIMG were TP-B26 (36.1%), TP-B26 (36.1%), TP-B30 (30.1%), TP-B32 (36.2%), TP-C38 (36.1%), TP-C40 (30.1%), TP-C42 (30.1%), TP-G46 (32.1%), and TP-G48 (38.1%). Strains of weak PIMG included TP-C34 (18.2%), TP-C36 (26.1%), and TP-C44 (20.2%). At hour 72 after inoculation, the PIMG of TP-G50 (62.6%) was significantly greater than those of TP-B26 (55.2%), TP-B28 (53.7%), TP-B32 (56.6%), TP-C36 (44.7%), TP-C38 (47.7%), TP-C40 (47.7%), TP-C42 (43.2%), TP-G46 (49.2%), and TP-G48 (53.7%). In addition, the weak PIMG happened in strains of TP-B30 (38.7%), TP-C34 (35.7%), and TP-C44 (34.2%) (

Table 6. Percent inhibition of mycelial growth of Trichoderma spp. against F. verticillioides FP-B16 causing root rot yellow leaf in pomelo.

Fungal Strain	PIMG (%)		Inhibition Diameter of Fusarium verticillioides FP-B16 (cm)
	48 h	72 h	48 h	72 h
TP-B26	36.1 ab	55.2 b	2.13 cd ± 0.20	2.00 f ± 0.12
TP-B28	36.1 ab	53.7 bc	2.13 cd ± 012	2.07 def ± 0.23
TP-B30	30.1 bc	38.7 ef	2.33 bc ± 0.20	2.73 ab ± 0.12
TP-B32	36.2 ab	56.6 b	2.13 cd ± 0.12	1.93 f ± 0.23
TP-C34	18.2 d	35.7 f	2.73 a ± 0.12	2.93 a ± 0.20
TP-C36	26.1 d	44.7 d	2.47 ab ± 0.12	2.47 c ± 0.20
TP-C38	36.1 ab	47.7 cd	2.13 cd ± 0.12	2.33 cd ± 0.23
TP-C40	30.1 cb	47.7 cd	2.33 bc ± 0.23	2.33 cd ± 0.12
TP-C42	30.1 bc	43.2 de	2.33 bc ± 0.12	2.53 bc ± 0.31
TP-C44	20.2 d	34.2 f	2.67 a ± 0.20	2.86 a ± 0.12
TP-G46	32.1 bc	49.2 cd	2.27 bc ± 0.12	2.27 cde ± 0.12
TP-G48	38.1 ab	53.7 bc	2.07 cd ± 0.23	2.07 def ± 0.20
TP-G50	44.1 a	62.6 a	1.86 d ± 0.12	1.67 g ± 0.20
F	*	*	*	*
CV (%)	8.01	7.66	10.63	9.68

^a–g In the same column, numbers with the same following letters are not significantly different according to the Duncan test. * Different at the 5% significance level. PIMG: percent inhibition of mycelial growth.

).

3.3.3. Identification of Trichoderma spp. Antagonizing Fusarium spp.

Two fungal strains TP-C40 and TP-G50 greatly antagonized the two pathogenic Fusarium spp. FP-C16 and FP-B16 under in vitro conditions were identified as Trichoderma asperellum FP-C16 and T. yunnanense FP-B16 with 99% similarity (Figure 6).

3.4. Effectiveness of Trichoderma spp. in Controlling Pomelo RRYLD Caused by Fusarium spp. Under Greenhouse Conditions

The disease symptoms by the four pathogens appeared via leaves (Figure 7). On day 7 after infection, the symptoms did not appear. On day 14 after infection, symptoms appeared in leaves. On day 28 after infection, among Fusarium spp., the rates of disease were more obvious. Therein, the rates of diseased leaves peaked in the treatment without Trichoderma spp., and then in the treatment sprayed with Trichoderma spp. 6 days after infection. The lowest disease rate was in the treatment sprayed with Trichoderma spp. right after the infection and in the treatment sprayed with Trichoderma spp. 3 days after infection. In particular, the number of diseased leaves was 36 leaves/plant in treatment 2, 4 leaves/plant in treatment 3, 10 leaves/plant in treatment 4, and 20 leaves/plant in treatment 5 (

Table 7. Number of yellow leaves in Pomelo caused by F. fujikuroi FP-B03, F. verticillioides FP-B16, F. verticillioides FP-B18, and F. incarnatum FP-C16.

Treatment	Number of Yellow Leaves
	7 Days	14 Days	21 Days	28 Days
Treatment 1	0	0	0	0
Treatment 2	0	8	18	36
Treatment 3	0	3	4	4
Treatment 4	0	4	6	10
Treatment 5	0	6	15	20

). Likewise, the number of diseased roots was reduced when the Trichoderma spp. were sprayed at the same time with the pathogens (

Table 8. Role of Trichoderma spp. in controlling root rot and yellow leaf disease in pomelo caused by F. fujikuroi FPB03, F. verticillioides FPB16, F. verticillioides FPB18, and F. incarnatum FPC16.

Treatment	Number of Rotten Roots
	Number of Primary Roots	Number of Diseased Primary Roots	Number of Secondary Roots	Number of Diseased Secondary Roots
Treatment 1	35	0	385	0
Treatment 2	42	26	630	52
Treatment 3	38	7	532	0
Treatment 4	30	13	330	13
Treatment 5	48	25	432	36

). This shows that the pathogenicity of the four Fusarium spp. strains was inhibited by the Trichoderma spp. (Figure 7).

4. Discussion

At hour 72 after inoculation, the growth of FP-B16 and FP-B18 reached 45.7 mm and 44.3 mm, which were significantly different from those of the other strains ranging from 19.3 to 31.7 mm (Table 2). Thus, the strains FP-B16 and FP-B18 were chosen for strong growth isolated from two different locations (Table 2). According to Uddin et al. [27], the colony diameter of the mycelia of F. oxysporum on PDA was roughly 3.0 cm at hour 72 after inoculation. Furthermore, for Fusarium spp. isolated from rotten tangerine fruits, white mycelia appeared on PDA and spores appeared on the third day [28]. Because of the fast growth of pathogens, biocontrol approaches should be applied during the plantation of pomelo to receive proper prevention. Moreover, in citrus, previous studies have frequently highlighted F. solani and F. oxysporum as major causes of dry root rot and decline [8,9,10]. However, the present study identified F. fujikuroi, F. verticillioides, and F. incarnatum among the isolates associated with pomelo RRYLD, suggesting that the disease etiology in pomelo orchards in Ben Tre province may be broader than previously recognized. This finding expands the current understanding of Fusarium diversity associated with pomelo decline in Vietnam.

Four strains causing RRYLD with the strongest rate consisted of F. fujikuroi FP-B03, F. verticillioides FP-B16, F. verticillioides FP-B18, and F. incarnatum FP-C16 (Table 2). In maize, Harish et al. [29] noted 10 Fusarium spp. strains that caused stem rot, belonging to F. acutatum, F. verticillioides, and F. andiyazi. On the other hand, in sunflowers, strains of Fusarium spp., including F. oxysporum, F. culmorum, F. graminearum, F. avenaceum, and F. solani, can cause root rot, flower rot, bud rot, and leaf blight. Therein, F. solani caused the most severe damage [30]. In addition, according to Piasai et al. [31], the causing factors for durian fruit rot are L. theobromae, Phomopsis sp., and F. solani. Ultimately, Fusarium spp. cause damages to many crops, in which F. solani is the causing agent for RRYLD in citrus. This indicates the severity of the disease in pomelo because the identified pathogen is the most pathogenic fungi. In the current study, some species of F. fujikuroi FP-B03, F. verticillioides FP-B16, F. verticillioides FP-B18, and F. incarnatum FP-C16 are risks causing RRYLD in pomelo.

Great PIMG was observed in the TP-G50 strain (62.6%) at hour 72 after inoculation (Table 6). The antagonistic capacity of T. asperellum against F. oxysporum reduces 50% of the Disease Severity Index in Asparagus at 25 °C [32]. Their antagonistic performance is consistent with previous reports showing that Trichoderma spp. effectively suppress Fusarium spp. through multiple mechanisms. For instance, according to Ferreira and Musumeci [33], Trichoderma spp. control and directly affect the pathogens by parasitism, competition, and production of antibiotics and degrading enzymes. As per Boat et al. [34], strains of Trichoderma spp. produce volatile and non-volatile metabolites, such as hydrolysis enzymes (chitinase, cellulase, protease, and lipase) to degrade pathogens’ cell walls. Some other cell wall degrading enzymes produced by Trichoderma spp. can hydrolyze small polymers, such as proteins, β-1,6-glucans, and α-1,3-glucans, which can eliminate hyphae and spores of pathogenic fungi [35]. In the study by Modrzewska et al. [36], Trichoderma spp. strains can suppress the production of toxins such as deoxynivalenol 73–98%, nivalenol 87–100%, and zearalenone 12–100% produced by Fusarium spp. depending on the types of pathogens and antagonists. Ultimately, the high inhibition observed in the present study indicates that indigenous Trichoderma strains from pomelo rhizosphere soils may be particularly well adapted to local environmental conditions, which is an advantage for future practical application. In addition, the use of native antagonists may improve establishment and persistence in orchard soils compared with non-indigenous strains.

The treatments were sprayed with Trichoderma spp. inhibited fungal pathogens in Pomelo seedlings under greenhouse conditions (Table 7). This agrees with previous studies showing that Trichoderma spp. can reduce Fusarium wilt. As per Awad-Allah et al. [37], treating Trichoderma spp. on soils contaminated with Fusarium spp. causing wilt disease can remarkably reduce the disease rate of tomatoes. In particular, T. viride and T. harzianum reduced the disease rate by 57.1% and 42.3%. Moreover, according to Innocenti et al. [38], the antagonistic efficiency of T. harzianum T22 against F. oxysporum f. sp. lactucae 365.07 was 57% and 78% under dry and wet seasons, respectively. Moreover, using T. harzianum T-78 from the fermentation of citrus waste reduced the infection rate of F. oxysporum in cucumber [39]. On chili pepper, using Trichoderma sp. MHT1134 reached the wilt disease rate caused by Fusarium spp. of 50.0% compared with the negative control [40]. According to Awad-Allah et al. [37], Trichoderma sp. can antagonize F. solani under greenhouse conditions by single or mixed applications. Therefore, using Trichoderma spp. is efficient in controlling crop pathogens. Notably, the best suppression was obtained when Trichoderma spp. were applied at the time of pathogen inoculation, indicating that early establishment of the antagonist is important for effective disease prevention. The protective effect observed in the present study may be attributed not only to direct antagonism against Fusarium spp. but also to early rhizosphere colonization and possible induction of host resistance. From a practical perspective, these results suggest that selected Trichoderma strains could be developed as preventive biocontrol agents for nursery seedlings or early orchard application rather than being used only after severe disease symptoms have already developed.

Overall, the study demonstrates a clear relationship between pathogen identification and biological control selection: diverse Fusarium spp. were associated with pomelo RRYLD, and indigenous Trichoderma spp. strains showed promising antagonistic activity against representative pathogenic isolates under both in vitro and greenhouse conditions. Nevertheless, further field-scale studies are needed to confirm the consistency of disease suppression under orchard conditions, where soil properties, microbial competition, and environmental fluctuations may influence biocontrol efficacy.

5. Conclusions

Four strains that caused the most severe RRYLD were Fusarium fujikuroi FP-B03, F. verticillioides FP-B16, F. verticillioides FP-B18, and F. incarnatum FP-C16. Two strains Trichoderma asperellum TP-C40 and T. yunnanense TP-G50 were selected from 13 Trichoderma spp. strains for growing well and effectively antagonizing F. verticillioides FP-B16 and F. incarnatum FP-C16 with corresponding PIMG of 59.6–63.9% and 51.4–62.4%. Fundamentally, these findings improve current knowledge of the diversity of Fusarium spp. associated with pomelo decline and highlight the value of indigenous rhizosphere-associated Trichoderma spp. as promising biological antagonists. Practically, the results suggest that the selected Trichoderma strains have potential for development as preventive biocontrol agents for pomelo seedling production and orchard disease management. Further studies should evaluate formulation methods, shelf life, application timing, and field performance before large-scale recommendation.