search for




 

Indigenous Trichoderma spp. fungi that can antagonize Fusarium spp. fungi causing pineapple stem rot§
Korean J. Microbiol. 2024;60(3):138-151
Published online September 30, 2024
© 2024 The Microbiological Society of Korea.

Ly Ngoc Thanh Xuan1, Tran Tri Thong1, Phan Tan Nhut2, Nguyen Duc Trong2, Vo Minh Thuan2, Tran Chi Nhan1, Le Thi My Thu2, Do Thi Xuan3, Le Thanh Quang2, and Nguyen Quoc Khuong2*

1Experimental and Practice Section, An Giang University, Vietnam National University Ho Chi Minh City, An Giang 90116, Vietnam
2Faculty of Crop Science, College of Agriculture, Can Tho University, Can Tho 94115, Vietnam
3Institute of Food and Biotechnology, Can Tho University, Can Tho 94115, Vietnam
Correspondence to: E-mail: nqkhuong@ctu.edu.vn;
Tel.: +84-942-679-867

§Supplemental material for this article may be found at http://www.kjom.org/main.html
Received January 3, 2024; Revised May 12, 2024; Accepted June 18, 2024.
Abstract
In this study, we selected indigenous Trichoderma spp. fungi that antagonized best against Fusarium spp. fungi, which caused the pineapple stem rot disease (PSRD). The experiments consisted of the following: (i) selecting fungi causing the PSRD from rhizosphere and stem of infected pineapple; (ii) evaluating the control ability of Trichoderma spp. fungi selected from soils of healthy pineapple plants to the Fusarium spp. fungi. The selection resulted in 10 out of 20 Fusarium spp. fungi that caused the disease by over 20% severity after six weeks of infection. From 50 Trichoderma spp. fungi, the three Trichoderma strains, T. asperellum T-LM-12, T. asperellum T-LM-22, and T. hamatum T-LM-42, isolated from pineapple farms produced chitinase contents at 208.1, 243.9, and 147.2 U/ml, respectively, which were higher than those of other fungi. Furthermore, the average antagonizing indices of the T-LM-12, T-LM-22, and T-LM-42 strains against the 10 selected Fusarium spp. fungi (Fusarium equiseti strains, Fs-LM-02, Fs-LM-04, Fs-LM-07, Fs-LM-10, Fs-LM-13, and Fs-LM-14; F. incarnatum strains, Fs-LM-09; F. oxysporum strain, Fs-LM-16, and Fs- LM-18; and F. solani strain, Fs-LM-19) were 52.1%, 52.4%, and 46.2%, respectively, 144 h after of inoculation.
Keywords : Fusarium spp., Trichoderma spp., biological control, pineapple stem rot disease
Body

Climate change, environmental pollution, and the use of chemicals in agriculture over recommendation have resulted in difficulties in recent pineapple cultivation (Haridas and Prathap, 2023). Moreover, facing biotic and abiotic stresses could reduce pineapples’ productivity and fruit quality (Arshad et al., 2023). The biotic factor is usually related to fungi, bacteria, viruses, and nematodes. In particular, more than 10,000 pathogenic fungal species, such as Fusarium, Phytophthora, Pythium, and others (Iqbal et al., 2022; Jeevalatha et al., 2022), were found responsible for severe diseases in crops (Sood et al., 2020). The stem rot disease (or bud rot) has been determined to be caused 79% by Fusarium spp. and 21% by Talaromyces stollii, meaning that Fusarium spp. are the common pathogens for the pineapple stem rot disease (PSRD) (Barral et al., 2020) and pathogens mainly present in the soil. Therefore, many plant protection products are used to reduce pathogens (Edlinger et al., 2022). However, long-term overuse of chemicals in agriculture brings about low efficiency and environmental contamination (Kaur et al., 2019). Another approach to environmentally friendly control of pathogens is the application of biological control (Yassin et al., 2022). Many microbial species can suppress pathogens, popularly known as Bacillus (Mulk et al., 2022), Pseudomonas (Lal et al., 2022), and Trichoderma (Go et al., 2023). Trichoderma spp. has been applied to protect crops from pathogenic fungi and bacteria (Caracciolo et al., 2023; Khuong et al., 2023). Indigenous Trichoderma spp. fungi were found to be able to present many pathogenic fungi, such as the Moniliophthora roreri fungus, causing fruit rot disease on cocoa (Leiva et al., 2020); Phytophthora spp. fungi causing defoliation on rubber trees (Sirikamonsathien et al., 2023); and Fusarium spp. that is one of the pathogens causing serious diseases in pineapple (Bezerra et al., 2019; Ibrahim et al., 2020). Recent studies pointed out that well-known plant diseases, such as root rot, waterlogging, wilt, fruit rot, and other crop diseases, can be controlled by Trichoderma spp. (Olowe et al., 2022; Singh et al., 2022). Thus, this study aimed to (i) determine the strongest Fusarium spp. fungi that caused the PSRD; (ii) and select indigenous Trichoderma spp. fungal strains that control the selected Fusarium spp. fungi with the most substantial growth.

Materials and Methods

Soil collection

A total of 20 soil samples from 20 different healthy pineapple farms were collected to isolate Trichoderma spp. fungi at the depth of 10–15 cm (200 g/sample) surrounding a pineapple stem. Plant and soil samples from 10 farms with plants infected by the PSRD in each location were collected to isolate Fusarium spp. The samples ranged in amount from two to three, and they had to be new and exhibit typical disease symptoms. Those samples were stored in plastic bags and labeled. The sampling locations were Vinh Vien town and Vinh Vien A commune, Long My district, Hau Giang province, Vietnam.

Media preparation

The isolation medium for the Trichoderma fungi was the TSM (Trichoderma Specific Medium), containing MgSO4・7H2O 0.2 g, KH2PO4 1.18 g, KC1 0.15 g, NH4NO3 1.0 g, glucose 0.5 g, agar 20 g in a liter of distilled water with a pH adjusted to 6.5–6.8 (Merck). The Potato Dextrose Agar (PDA) was used to isolate the pathogen of the PSRD and contained the following: potato infusion 200 g, dextrose 20 g, agar 20 g in a liter of distilled water with a pH adjusted to 6.5–6.8 (Merck).

Isolation of fungi causing the pineapple stem rot disease

Infected pineapple stems were rinsed and chopped into small square fragments with a side of 4–5 mm for the plant sample. The infected fragments were surface-sterilized using ethanol 70% for 30 sec. Subsequently, the samples were rinsed thrice with sterile distilled water and dried using sterilized tissue paper. Cut infected samples were placed in Petri dishes of solid PDA media and incubated at 25°C in three days. They were observed until the advent of hyphae, which were then inoculated into another Petri dish of PDA media until the fungi were pure.

The rhizosphere sample of infected plants

The samples were diluted with sterilized distilled water (SDW) with a ratio of 1 g soil and 99 ml of SDW. The diluted samples were shaken well for 30 min and left to sediment for one day at 28–32°C. Soil extracts were well spread on sterilized PDA media (autoclaved at 121°C in 20 min). After three days, the samples were checked and colonies were selected. Net isolation and purification of the samples depended on the morphology of hyphae. The morphological observation of the hyphae (colors and shapes), spores, and sclerotia was conducted under a 40X light microscope to detect Fusarium spp. Subsequently, pure samples were stored at 4°C in vial tubes, and Petri dishes of PDA media were utilized for the following experiments. Moreover, fungal strains were named after the Fusarium genus (F), the sampling location (LM), and the sampling position.

Growth of the Fusarium spp. fungi

The experimental design was a completely randomized design (CRD) with three replications, each of which was a Petri dish. Strains of Fusarium spp. had been cultured seven days before the experiment. The strains from the Eppendorf tubes were inoculated to PDA media in Petri dishes and incubated at 25°C. Radii of colonies in the Petri dishes (ф = 90 mm) were measured at 24, 48, 72, and 96 h of inoculation.

Evaluating the ability of the Fusarium spp. fungi to cause the pineapple stem rot disease

This experiment was also conducted in a CRD with four replications, each of which was a pineapple pot. Pineapple plants for a tissue culture of 20 days were artificially infected with the disease by watering with a suspension of Fusarium spp. spores with a density of 106 spores/ml. Each pineapple pot contained 6 kg of sterilized soil and was watered with 5.0 ml of the fungal suspension under the greenhouse condition. The rate of infected pineapple leaves was determined at 1, 2, 3, 4, 5, and 6 weeks after infection (WAI). Infection rate (%) = (number of infected leaves)/(total number of leaves) × 100%

Isolation of Trichoderma spp. fungi from acid sulfate soil for pineapple

Trichoderma spp. fungi were isolated according to Kumar et al.’s method (2012). First, 1 g of soil was mixed with 99 ml of SDW, shaken for 30 min, and left to sediment for a day. Then, 0.1 ml of the solution was dropped on each Petri dish of TSM. The dishes were incubated at 28 ± 2°C in 96 h. When hyphae were observed, fungi were inoculated to PDA media until pure. The strains were named after the Trichoderma genus (T), the sampling location (LM), and the sampling order.

The ability of the Trichoderma spp. fungi to produce chitinase: this experiment was completely designed in a randomized design with four replications, each of which was a tube. Trichoderma fungi cultured on PDA media for five days were utilized to propagate in 50.0 ml tubes containing 10.0 ml of liquid TSM (added with 0.5% chitin), which had a density of 107 spores/ml, and shaken at 120 rpm at room temperature. Moreover, 10 days after incubation, 2 ml of a culture was centrifuged at 5000 rpm in 20 min. Biomass was removed, and the extract was utilized as a source of crude exopolymeric enzymes of the strains. The enzymatic activity of chitinase was determined according to Nelson et al.’s method (1944). Furthermore, 1 ml of the enzyme was reacted with 1.0 ml of chitin 1% in acetate (pH 6.0) buffer at 40°C in 60 min, the amount of reduced sugar via a metabolism with the Nelson–Somogyi reagent, which turned brick red, and the arseno-molybdate reagent, which turned sky blue, was measured at the 520 nm wavelength using a spectrophotometer (UV1800, Shimadzu). Unit of enzymatic activity (U/ml) was the amount of reduced sugar produced in 1 min per ml of the enzyme at 40°C and pH = 6.0.

Evaluating the antagonism of the Trichoderma spp. fungi against the Fusarium spp. fungi causing the pineapple stem rot disease

Experiment design: the experiment was a CRD with four replications—each of which had two Petri dishes (an antagonistic dish and a control one). Slices of gel with equal size (5 × 5 mm) contained the pathogen, Fusarium spp. fungi, and the antagonist, Trichoderma spp. fungi. Subsequently, the two slices were placed 3.0 cm apart on a Petri dish of PDA media and incubated at 25°C. Fungal radii on Petri dishes of the control treatment and the antagonistic one were measured at 48, 72, 96, 120, and 144 h after inoculation (HAI) to determine the inhibition percentage of mycelial growth (IPMG) of the Trichoderma spp. against the Fusarium spp.

According to Imtiaj and Lee (2008) and Sallam et al. (2009) IPMG of fungi was calculated as the following formula:

IPMG = (R1-R2)/R1 × 100%,

where R1 was the radius of the Fusarium spp. colony in the control treatment; R2 was the radius of the Fusarium spp. colony in the antagonistic treatment.

Identification of the Trichoderma spp. and Fusarium spp. fungi

The selected Trichoderma spp. strains were identified at the ITS regions: Trichoderma spp. strains’ DNAs were extracted from fungal colonies. In particular, spores from colonies after a 7-day incubation on PDA of the fungi were transferred into a 2.2 ml Eppendorf tube, well shaken, and incubated at room temperature for 10 min. Then, the tube was centrifuged at 13,000 rpm for 5 min to collect an extract, which was later moved to another Eppendorf. The new tube with the extract was rinsed with 500 µl of ethanol 70%, centrifuged at 13,000 rpm for 5 min, and vacuum-dried. DNAs were dissolved in 100 µl of TE 0.1X. Subsequently, a polymerase chain reaction (PCR) was performed with the primers pair of ITS 1: 5’-TCCGTAGGTGAACCTGCGG-3’; and ITS 4: 5’-TCCTCCGCTTATTGATATGC-3’ (White et al., 1990). The PCR volume was 50 µl in total, and the thermal cycle was as follows: denaturation (95°C for 5 min); 30 cycles of denaturation at 95°C for 90 sec, annealing at 52°C for 60 sec, and elongation at 72°C for 90 sec; and termination at room temperature. The PCR amplicons were purified and sequenced by an automatic sequencing system. The sequences were compared to the GenBank database in the National Center for Biotechnology Information (NCBI) by the Basic Local Alignment Search Tool (BLAST). The Fusarium spp. strains were similarly identified. All of the sequences were aligned by the CLUSTALW program. The neighbor-joining phylogenetic tree was constructed by MEGA 6.06 with 1,000-bootstrap resampling method. The evolutionary distance matrix was measured according to the Jukes–Cantor model.

Statistical analysis

Data were subjected to one-way variance analysis (ANOVA) to detect differences between treatments with the Duncan test at a 5% significance level by the SPSS 13.0 software.

Results

Isolation, selection, and identification of Fusarium spp. causing the stem rot disease

Isolation of the Fusarium spp. causing the pineapple stem rot disease: a total of 20 Fusarium spp. strains were derived from PDA media of 20 samples of stems and soils for pineapple plants infected with the PSRD at 20 farmers’ farms and labeled from Fs-LM-01 to Fs-LM-20. The morphology of the hyphae was recorded as follows: white hyphae turned into light yellow (15%), light purple (30%), and white (55%) at 96 HAI on PDA media (Table 1). On the lower side of the dish, colors varied from dark yellow to dark purple (Fig. 1A). Mycelia were white, silky, crisscrossed with two concentric circles, and spores were oval, slightly strange, colorless, and monocellular and contained 3–5 walls at four days after inoculation (DAI) (Fig. 1B).

Colors of the Fusarium spp. strains causing the pineapple stem rot disease isolated in Long My, Hau Giang

No. Strain Color No. Strain Color
1 Fs-LM-01 White 11 Fs-LM-11 White-Light purple
2 Fs-LM-02 White 12 Fs-LM-12 White
3 Fs-LM-03 White-Light purple 13 Fs-LM-13 White
4 Fs-LM-04 White-Light yellow 14 Fs-LM-14 White
5 Fs-LM-05 White-Light purple 15 Fs-LM-15 White-Light purple
6 Fs-LM-06 White-Light purple 16 Fs-LM-16 White
7 Fs-LM-07 White-Light yellow 17 Fs-LM-17 White
8 Fs-LM-08 White-Light purple 18 Fs-LM-18 White
9 Fs-LM-09 White 19 Fs-LM-19 White
10 Fs-LM-10 White 20 Fs-LM-20 White-Light yellow

Note: Fs, Fusarium spp.; LM, Long My.


Fig. 1. Morphology of colonies at 96 h after inoculation (A) and spore shape (B) of Fusarium spp. fungi on PDA media under a microscope (40X).

Growth rate of the Fusarium spp. fungi isolated from pineapple stems: growth of the Fusarium spp. strains fluctuated roughly at values of 0.43–12.5, 5.76–25.5, 14.3–40.0, and 29.3–58.3 mm corresponding to 24, 48, 72 and 96 HAI. At 96 HAI, mycelia diameters of the Fs-LM-04, Fs-LM-10, and Fs-LM-19 strains were the biggest and ranged 57.0–58.3 mm. The diameters of the Fs-LM-07, Fs-LM-14, Fs-LM-16, and Fs-LM-18 strains were equivalent to 53.3–55.0 mm. Fungal strains that grew the strongest were as follows: Fs-LM-02, Fs-LM-09, and Fs-LM-13 (51.0–53.0 mm) (Table 2). Specifically, the four strains with the most substantial growth are demonstrated in Fig. 2. Thus, 10 Fusarium spp. strains, including Fs-LM-02, Fs-LM-04, Fs- LM-07, Fs-LM-09, Fs-LM-10, Fs-LM-13, Fs-LM-14, Fs-LM-16, Fs-LM-18, and Fs-LM-19, whose growth diameter at 96 HAI was different from the other strains (P < 0.05), were selected for the next experiment.

Growth of the Fusarium spp. fungi

No. Strain Growth rate of the strains

24 h 48 h 72 h 96 h
1 Fs-LM-01 5.50e 13.5fg 28.8d 38.0j
2 Fs-LM-02 5.50e 17.5cde 34.3c 51.3de
3 Fs-LM-03 0.50g 7.00h 14.3g 29.3l
4 Fs-LM-04 1.50f 19.8c 34.5c 58.3a
5 Fs-LM-05 1.67f 15.0ef 27.3de 40.0i
6 Fs-LM-06 1.50f 13.5fg 28.5d 46.0g
7 Fs-LM-07 9.50b 24.0ab 36.5abc 54.0bc
8 Fs-LM-08 0.46g 5.76h 21.5f 32.7k
9 Fs-LM-09 9.00b 22.5b 34.5c 53.0cd
10 Fs-LM-10 12.5a 16.5de 38.5ab 57.0a
11 Fs-LM-11 6.50d 18.5cd 30.3d 40.6i
12 Fs-LM-12 0.46g 7.00h 17.5g 33.3k
13 Fs-LM-13 9.50b 25.0a 39.0a 51.0e
14 Fs-LM-14 9.77b 23.5ab 35.0bc 53.3bc
15 Fs-LM-15 0.50g 11.0g 24.5ef 43.0h
16 Fs-LM-16 12.5a 25.5a 38.5ab 55.0b
17 Fs-LM-17 6.00de 19.7c 34.5c 49.0f
18 Fs-LM-18 7.50c 23.5ab 37.0abc 54.3bc
19 Fs-LM-19 7.50c 23.5ab 40.0a 58.3a
20 Fs-LM-20 0.43g 11.0g 22.5f 32.7k

Significance level * * * *

CV (%) 9.39 8.64 6.44 2.27

Note: Numbers in the same column followed by identical letters are insignificantly different from each other. *, significantly different at 5%; CV, Coefficient of variation.


Fig. 2. Growth of three Fusarium spp. strains on petri dishes at 24, 48, 72 and 96 h after inoculation.

Ability of the selected ten Fusarium spp. strains to cause the stem rot disease in pineapples: at 1–5 WAI, the Fs-LM-09 strain caused the most severe disease, with 40.7, 39.1, 41.2, 41.9, and 41.5%, respectively. However, at 6 WAI, four Fusarium spp. strains with high and equivalent infection rates were Fs-LM-07 (37.2%), Fs-LM-09 (42.2%), Fs-LM-13 (40.0%), and Fs-LM-19 (38.9%) (Table 3). All of the Fusarium spp. strains performed pathogenic activities on pineapple leaves, leading to infected pineapple plants (Fig. 3). Among the strains, the Fs-LM-09 strain maintained a strong infection rate from 1 to 6 WAI.

Infection rate of the Fusarium spp. strains

No. Strain Infection rate (%)

Week 1 Week 2 Week 3 Week 4 Week 5 Week 6
1 Control 1.67f 6.43f 6.43e 6.43d 6.73f 1.57g
2 Fs-LM-02 26.6c 26.5c 28.8c 29.2b 30.4bc 31.4cd
3 Fs-LM-04 24.0c 24.3c 28.6c 29.0b 33.1bc 28.6def
4 Fs-LM-07 26.8c 27.3c 31.2bc 31.8b 32.7bc 37.2abc
5 Fs-LM-09 40.7a 39.1a 41.2a 41.9a 41.5a 42.2a
6 Fs-LM-10 26.9c 27.1c 27.1c 28.4b 28.4cd 29.3de
7 Fs-LM-13 25.3c 22.8cd 30.0bc 33.2b 35.1b 40.0ab
8 Fs-LM-14 18.1d 18.7de 20.7d 20.9c 21.2e 22.7f
9 Fs-LM-16 14.0e 16.5e 16.9d 18.6c 24.2de 24.3ef
10 Fs-LM-18 33.9b 32.9b 34.0b 33.8b 34.7bc 33.7bcd
11 Fs-LM-19 25.4c 27.3c 33.7b 33.6b 35.3b 38.9ab

Significance level * * * * * *

CV (%) 9.60 12.1 10.5 12.7 13.9 13.8

Note: Numbers in the same column followed by identical letters are insignificantly different from each other. *, significantly different at 5%; CV, Coefficient of variation.


Fig. 3. Symptoms of the pineapple stem rot disease after treated with the Fusarium equisetti Fs-LM-02.

Identification of the selected Fusarium spp. fungi: strains of Fusarium spp., Fs-LM-02, Fs-LM-04, Fs-LM-07, Fs-LM-09, Fs-LM-10, Fs-LM-13, Fs-LM-14, Fs-LM-16, Fs-LM-18, and Fs-LM-19, were 99% similar to Fusarium equiseti strains, Fs-LM-02 (PP702415), Fs-LM-04 (PP702416), Fs-LM-07 (PP702417), Fs-LM-10 (PP702419), Fs-LM-13 (PP702420), and Fs-LM-14 (PP702421); F. incarnatum strain, Fs-LM-09 (PP702418); F. oxysporum strains, Fs-LM-16 (PP702422), and Fs-LM-18 (PP702423); and F. solani strain, Fs-LM-19 (PP702424) (Fig. 4).

Fig. 4. Neighbor-joining phylogenetic trees based on ITS sequences of the 10 selected Fusarium spp., Fs-LM-02, Fs-LM-04, Fs-LM-07, Fs-LM-09, Fs-LM-10, Fs-LM-13, Fs-LM-14, Fs-LM-16, Fs-LM-18, and Fs-LM-19 compared to the closely related strains in the GenBank database. The percentage levels of bootstrap analysis of 1,000 replicates are indicated at each node. Bar, 0.1 substitutions per nucleotide position. Ceratocystis paradoxa isolate B125 was used as the outgroup strain. Access numbers of GenBank sequences are implied in brackets.

Isolation, selection, and identification of Trichoderma spp. from acid sulfate soil for pineapples

Isolation of Trichoderma spp. fungi: then, 50 strains of Trichoderma spp. were separated from 30 soil samples in 30 farms in Long My district, Hau Giang province. The antagonistic fungal strains were labeled from T-LM-01 to T-LM-50. Morphologies of colonies, hyphae, and spores are shown in Fig. 5.

Fig. 5. Morphology of colonies, hyphae, and spores of Trichoderma spp. on PDA media under a microscope (40X).

Selection for Trichoderma spp. fungi antagonizing the Fusarium spp. fungi causing the stem rot disease in pineapples: fungi of Trichoderma spp. covered a dish at 4 DAI. Thus, the diameters of Trichoderma spp. mycelia were not considered as a selecting criterion for the next experiment. Because endo-chitinase was an enzyme that hydrolyzed the cell walls of fungal pathogens, high chitinase production by Trichoderma fungi could be inferred as high antagonism against the Fusarium spp. fungi. The ability to secrete chitinase of Trichoderma spp. strains varied at 5% significance. In particular, three strains with high amounts of chitinase were T-LM-12, T-LM-22, and T-LM-42, whose results were 208.1, 243.9, and 147.2 U/ml, respectively. Therefore, the three strains were potent to highly antagonize the Fusarium spp. strains (Table 4).

Chitinase producing capacity of the Trichoderma spp. fungi

No. Strain Chitinase content (U/ml) No. Strain Chitinase content (U/ml)
1 T-LM-01 44.9x 26 T-LM-26 58.8s
2 T-LM-02 65.0pqr 27 T-LM-27 62.5r
3 T-LM-03 72.3mn 28 T-LM-28 78.0i
4 T-LM-04 54.1uv 29 T-LM-29 11.6y
5 T-LM-05 53.9v 30 T-LM-30 77.1ijk
6 T-LM-06 73.2lmn 31 T-LM-31 77.4ịj
7 T-LM-07 76.0i–l 32 T-LM-32 74.2j–m
8 T-LM-08 66.8p 33 T-LM-33 75.3i–n
9 T-LM-09 57.5s–v 34 T-LM-34 68.2op
10 T-LM-10 94.6g 35 T-LM-35 76.2i–l
11 T-LM-11 73.6k–n 36 T-LM-36 65.7pqr
12 T-LM-12 208.1b 37 T-LM-37 70.6no
13 T-LM-13 49.5w 38 T-LM-38 62.8qr
14 T-LM-14 56.8s–v 39 T-LM-39 74.4j-m
15 T-LM-15 55.2tuv 40 T-LM-40 82.1h
16 T-LM-16 138.8d 41 T-LM-41 94.3g
17 T-LM-17 55.7s–v 42 T-LM-42 147.2c
18 T-LM-18 66.0pq 43 T-LM-43 73.5lmn
19 T-LM-19 120.6e 44 T-LM-44 68.2op
20 T-LM-20 112.4f 45 T-LM-45 49.5w
21 T-LM-21 121.1e 46 T-LM-46 57.7st
22 T-LM-22 243.9a 47 T-LM-47 54.7tuv
23 T-LM-23 85.1h 48 T-LM-48 55.4s-v
24 T-LM-24 75.0i–m 49 T-LM-49 43.1x
25 T-LM-25 68.1op 50 T-LM-50 57.5stu

Significance level *

CV (%) 2.40

Note: Numbers in the same column followed by identical letters are insignificantly different from each other. *, significantly different at 5%; CV, Coefficient of variation.



Identification of Trichoderma spp. potent in controlling the Fusarium spp. fungi: based on the ITS regions, Trichoderma T-LM-22, T-LM-12, and T-LM-42 strains belonged to Trichoderma asperellum T-LM-12 and T-LM-22 strains (PP770478 and PP770479) and T. hamatum T-LM-42 strain (PP770480), with 99% similarity (Fig. 6).

Fig. 6. Neighbor-joining phylogenetic trees based on ITS sequences of the three selected Trichoderma spp. strains T-LM-22, T-LM-12 and T-LM-42 compared to the closely related strains in the GenBank database. The percentage levels of bootstrap analysis of 1,000 replicates are indicated at each node. Bar, 0.1 substitutions per nucleotide position. Alternaria alli isolate Alt35 was used as the outgroup strain. Access numbers of GenBank sequences are implied in brackets.

The ability of the Trichoderma spp. fungi to restrict the growth of the Fusarium spp. fungi causing the stem rot disease

The T-LM-12 Strain: at 48 and 72 HAI, IPMG varied between strains (P < 0.05), with 14.0%–33.9% and 22.8%–45.1%, respectively (Table 5). Additionally, at 96 and 120 HAI, IPMG value peaked when against the Fs-LM-13 strains (49.2 and 56.3%, respectively). At 144 HAI, IPMG values of the T-LM-12 were the highest against the Fs-LM-13, Fs-LM-14, Fs-LM-16, and Fs-LM-18 strains (57.0%–61.3%). In general, the IPMG values of the T-LM-12 strains against the Fusarium spp. strains were high at the above points of time (Supplementary data Fig. S1).

Antagonistic ability of the three selected Trichoderma spp. to inhibit the growth of the 10 Fusarium spp. fungi

No. Strain T-LM-22 T-LM-12 T-LM-42



IPMG 48 IPMG72 IPMG 96 IPMG 120 IPMG 144 IPMG 48 IPMG72 IPMG 96 IPMG 120 IPMG 144 IPMG 48 IPMG72 IPMG 96 IPMG 120 IPMG 144
1 Fs-LM-02 33.0d 22.2c 27.9d 38.9c 42.6de 33.1ab 34.7bc 35.9cd 41.4cde 50.0b 29.4a 28.8a 37.0a 43.9a 51.2ab
2 Fs-LM-04 20.3e 38.1b 42.2bc 46.6b 51.7bc 19.4de 33.7bc 36.0cd 42.4cd 49.3c 22.6b 27.3ab 36.0ab 44.8a 49.3abc
3 Fs-LM-07 49.0c 40.5b 43.5bc 52.8ab 55.7bc 14.0e 24.9e 33.2de 44.5bc 50.8b 19.0b 18.5cd 20.7e 31.9b 37.9e
4 Fs-LM-09 72.2a 39.4b 38.2c 46.9b 50.0c 17.2e 26.0de 31.1de 38.8de 47.3c 17.7bc 20.1bcd 23.2de 33.5b 37.0e
5 Fs-LM-10 12.8f 16.5c 23.9de 31.6d 43.9d 25.5cd 22.8e 23.0f 36.4e 46.0c 12.0cd 24.4abc 27.8cd 36.1b 45.2cd
6 Fs-LM-13 59.4b 52.2a 50.6a 54.2a 61.9a 32.8ab 45.1a 49.2a 56.3a 61.3a 20.9b 30.4a 37.1a 47.1a 49.4abc
7 Fs-LM-14 26.7de 41.5b 43.5bc 55.3a 61.9a 29.2abc 33.0bcd 39.4bc 49.2b 57.3a 18.4bc 26.8ab 35.6ab 48.9a 54.5a
8 Fs-LM-16 24.7e 33.0b 40.7bc 48.7ab 59.1ab 15.6e 27.6cde 37.0cd 48.4b 57.0a 19.8b 18.6cd 31.0bc 44.0a 50.7abc
9 Fs-LM-18 59.6b 53.1a 44.4b 52.3ab 59.6ab 33.9a 42.7a 42.9b 45.4bc 57.7a 23.4ab 25.0abc 30.1c 36.8b 46.3bc
10 Fs-LM-19 20.3e 14.3c 20.6e 30.5d 37.5e 26.1bcd 38.4ab 28.5e 37.9de 43.9c 10.9d 14.9d 23.0de 34.7b 40.5de

Significance level * * * * * * * * * * * * * * *

CV (%) 13.0 15.6 9.85 9.60 7.17 18.7 14.3 10.4 8.16 6.67 21.7 19.2 11.6 9.76 7.71

Note: Numbers in the same column followed by identical letters are insignificantly different from each other. *, significantly different at 5%; CV, Coefficient of variation.



The T-LM-22 Strain: at 48, 72, and 96 HAI, the T-LM-22 strain promoted the highest growth inhibition for the Fs-LM-09, Fs-LM-13, and Fs-LM-18 strains (Table 5). However, at 120 HAI, heavily repressed strains were Fs-LM-13 and Fs-LM-14. Meanwhile, at 144 HAI, IPMG values of the T-LM-22 strains against the Fs-LM-13, Fs-LM-14, Fs-LM-16, and Fs-LM-18 strains were equivalently high (59.1%–61.9%). Therein, the T-LM-22 strain inhibited the growth of the Fs-LM-13 strain the most, from 72 to 144 HAI (Supplementary data Fig. S2).

The T-LM-42 Strain: at 48, 72, and 96 HAI, IPMG values of the T-LM-42 strain against the Fusarium spp. strains fluctuated roughly at 10.9–29.4, 14.9–30.4, and 20.7%–37.1%, respectively (Table 5). At 120 HAI, the T-LM-42 strain had the best IPMG against the Fs-LM-02, Fs-LM-04, Fs-LM-13, Fs-LM-14, and Fs-LM-16 strains (43.9%–48.9%). At 144 HAI, IPMG values of the T-LM-42 strain went on the same pattern as that at 120 HAI, but the antagonistic performance was not as remarkably as the others (Supplementary data Fig. S3).

T-LM-12, T-LM 22, and T-LM-42 strains restricted the growth of Fusarium spp. strains at rates above 37% at 144 HAI. The antagonistic performance of the T-LM 22 strain against the Fs-LM-13 strain is illustrated in Fig. 7.

Fig. 7. Efficacy of the Trichoderma asperellum strain T-LM-22 fungi (below) in antagonizing the Fusarium equiseti strain Fs-LM-13 fungi (above) causing the pineapple stem rot disease.
Discussion

Pineapple is an important fruit crop in tropical regions, but various fungi, such as Fusarium spp., can infect it (Ibrahim et al., 2020; Silva et al., 2023). Stem rot disease possesses signs of withering crops (Blanco-Meneses et al., 2022). Ibrahim et al. (2016) reported eight strains of Fusarium spp. causing fusariosis disease on pineapples in Malaysia had colonies turning from white to dark purple. However, in this study, hyphae changed from light yellow to light purple (Fs-LM-03, Fs-LM-06, Fs-LM-08, Fs-LM-11, and Fs-LM-15) (Table 1). Among the 20 Fusarium spp. strains collected, 10 strains were causing the disease the strongest, with growth rates of 51.0–58.3 mm at 96 HAI (Table 2). Because the study focused on the antagonism of Trichoderma spp., their IPMG was intensively described instead of their growth. However, colonies of Trichoderma on PDA media grew fast, were white or became green from white, and were foamy. Their hyphae were colorless, and their sporophore was highly branched. Their spores were oval, round, or oblong. At 48 HAI, their mycelia became green, light blackish green, then covered a dish and turned completely greenish black at 72–96 HAI (Fig. 4). These morphological features were consistent with Zheng et al.’s description (2022). However, the spore shapes and mycelia colors were different. Therefore, these Trichoderma spp. strains were sequenced based on the ITS region and identified as Trichoderma asperellum T-LM-12 and T-LM-22 strains and T. hamatum T-LM-42 strain. As can be seen in Fig. 6, the three strains belonged to 2 different Trichoderma species. This could be due to the identification in the current study was based on the ITS region which is considered inaccurate in some circumstances (Japanis et al., 2022). Therefore, the two species were distinguished by morphology as described in the studies by Pandian et al. (2016) and Siddiquee (2017). Thus, some other regions, such as tef1 and rpb2 genes, should be investigated to precisely identify the species of these three strains (Japanis et al., 2022).

The 40 selected pathogenic fungi of Fusarium spp. genus from infected pineapples belonged to 10 different species, including F. ananatum, F. concentricum, F. fujikuroi, F. guttiforme, F. incnatum, F. oxysporum, F. immunatum, F. temperatum, F. verticillioides, and F. subglutinans. Thus, many species of the Fusarium spp. genus have been detected from infected pineapples, but most appear at a low rate and are not considered pathogens to pineapples (Stępień et al., 2013). Consequently, the ability to infect or damage the strains on pineapples needed to follow the Koch’s postulates. The Fusarium spp. strains could spread the disease at a rate of > 20% and up to 42.2% (Table 3).

The Trichoderma spp. fungi control pathogens via degradation of the walls of pathogen cells (Wang et al., 2023), where chitin is the main component of fungal cells (Tyśkiewicz et al., 2022). Concurrently, plant chitinase is an enzyme stimulating the hydrolysis of fungal pathogenic chitin (Alsalman et al., 2022). Urbina-Salazar et al. (2019) and Mohiddin et al. (2021) also assumed that T. harzianum produced the highest chitinase content among the Trichoderma spp. genus.

Based on the chitinase production, the antagonistic ability of the strains was ranked as follows: T-LM-22 (243.9 U/ml) > T-LM-12 (208.1 U/ml) > T-LM-42 (147.2 U/ml) (Table 4). Thus, the T-LM-12 and T-LM-22 strains had a better average antagonistic rate than the T-LM-42 strain, with 52.1–52.4% compared to 46.2%, respectively (Table 5). Sharma et al. (2023) demonstrated that antagonistic fungi produce chitinase that degrades the cell walls of pathogenic fungi, so chitinase produced by Trichoderma spp. was considered the primary criterion for the biological control. Moreover, Trichoderma spp. fungi have been proven to control the infection of Fusarium spp. fungi on many crops (Abdelmoteleb et al., 2023; Aris et al., 2023; Bellini et al., 2023; Liu et al., 2023).

In conclusion, 10 strains of Fusarium spp., that is, Fs-LM-19, Fs-LM-04, Fs-LM-10, Fs-LM-18, Fs-LM-16, Fs-LM-07, Fs-LM-09, Fs-LM-13, Fs-LM-14, and Fs-LM-02, were selected for being highly virulent on pineapple plants. They belonged to Fusarium equiseti strains, Fs-LM-02, Fs-LM-04, Fs-LM-07, Fs-LM-10, Fs-LM-13, and Fs-LM-14; F. incarnatum Fs-LM-09 strain; F. oxysporum strains, Fs-LM-16, and Fs-LM-18; and F. solani Fs-LM-19 strain. The Trichoderma asperellum T-LM-22 and T-LM-12 strains and T. hamatum T-LM-42 strain were selected for highly producing endo-chitinase. The T-LM-22 strain exhibited stronger control against the Fusarium spp. strains (51.7%–61.9%) than the other two, T-LM-12 (57.0%–61.3%) and T-LM-42 (46.3%–54.5%), at 144 HAI. The selected Trichoderma spp. should be applied to pineapple to control Fusarium spp. in the field.

References
  1. Abdelmoteleb A, Gonzalez-Mendoza D, and Zayed O. 2023. Cell-free culture filtrate of Trichoderma longibrachiatum AD-1 as alternative approach to control Fusarium solani and induce defense response Phaseolus vulgaris L. plants. Rhizosphere 25, 100648.
    CrossRef
  2. Alsalman AJ, Farid A, Al Mohaini M, Al Hawaj MA, Muzammal M, Khan MH, Dadrasnia A, Alhashem YN, Ghazanfar S, and Almusalami EMAlmusalami EM, et al. 2022. Analysis and characterization of chitinase in Bacillus salmalaya strain 139SI. Int. J. Curr. Res. Rev. 14, 31-36.
    CrossRef
  3. Aris A, Mohd Zainudin NAI, and Ibrahim MH. 2023. Growth and photosynthetic performance of Fusarium solani infected Cucumis sativus L. treated with Trichoderma asperellum. J. Taibah Univ. Sci. 17, 2161292.
  4. Arshad A, Mushtaq N, Sajjad M, Ahad A, Ilyas M, and Gul A. Phytohormones and Stress Responsive Secondary Metabolites, In . Academic Press, San Diego, California, USA.
    CrossRef
  5. Barral B, Chillet M, Doizy A, Grassi M, Ragot L, Léchaudel M, Durand N, Rose LJ, Viljoen A, and Schorr-Galindo S. 2020. Diversity and toxigenicity of fungi that cause pineapple fruitlet core rot. Toxins 12, 339.
    Pubmed KoreaMed CrossRef
  6. Bellini A, Gilardi G, Idbella M, Zotti M, Pugliese M, Bonanomi G, and Gullino ML. 2023. Trichoderma enriched compost, BCAs and potassium phosphite control Fusarium wilt of lettuce without affecting soil microbiome at genus level. Appl. Soil Ecol. 182, 104678.
    CrossRef
  7. Bezerra GDA, Mussi-Dias V, Santos PHDD, Aredes FAS, and Silveira SFD. 2019. Identification and selection of Trichoderma spp. endophytic to bromeliacea from "restingas" as biocontrol agents of fusariosis in pineapples. Summa Phytopathol. 45, 172-178.
  8. Blanco-Meneses M, Castro-Zúñiga O, and Umaña-Rojas G. 2022. Preliminary study of Fusarium species in pineapple crop (Ananas comosus) in Costa Rica. Agron. Costarricense 46, 47-64.
  9. Caracciolo R, Sella L, De Zotti M, Bolzonello A, Armellin M, Trainotti L, Favaron F, and Tundo S. 2023. Efficacy of Trichoderma longibrachiatum Trichogin GA IV peptaibol analogs against the black rot pathogen Xanthomonas campestris pv. campestris and other phytopathogenic bacteria. Microorganisms 11, 480.
    Pubmed KoreaMed CrossRef
  10. Edlinger A, Garland G, Hartman K, Banerjee S, Degrune F, García-Palacios P, Hallin S, Valzano-Held A, Herzog C, and Jansa JJansa J, et al. 2022. Agricultural management and pesticide use reduce the functioning of beneficial plant symbionts. Nat. Ecol. Evol. 6, 1145-1154.
    Pubmed KoreaMed CrossRef
  11. Go WZ, Chin KL, H'ng PS, Wong MY, Lee CL, and Khoo PS. 2023. Exploring the biocontrol efficacy of Trichoderma spp. against Rigidoporus microporus, the causal agent of white root rot disease in rubber trees (Hevea brasiliensis). Plants 12, 1066.
    Pubmed KoreaMed CrossRef
  12. Haridas N and Prathap SK. Fifth World Congress on Disaster Management: Volume V: Proceedings of the international conference on disaster management, November 24-27, 2021, New Delhi, India, In . Taylor & Francis, London, the United Kingdom.
    CrossRef
  13. Ibrahim NF, Mohd MH, Mohamed Nor NMI, and Zakaria L. 2016. Fusarium fujikuroi causing fusariosis of pineapple in peninsular Malaysia. Australasian Plant Dis. Notes 11, 21.
    CrossRef
  14. Ibrahim NF, Mohd MH, Mohamed Nor NMI, and Zakaria L. 2020. Mycotoxigenic potential of Fusarium species associated with pineapple diseases. Arch. Phytopathol. Plant Prot. 53, 217-229.
  15. Imtiaj A and Lee TS. 2008. Antagonistic effect of three Trichoderma species on the Alternaria porri pathogen of onion blotch. World J. Agric. Sci. 4, 13-17.
  16. Iqbal S, Ashfaq M, Malik AH, and Haq MI; Khan, KS. 2022. Antagonistic screening and confronting potential of Trichoderma viride against Pakistani and American soil borne-pathogens (Pythium aphenidermatum, Fusarium oxysporum and Phytophthora capsici) in controlled conditions. Pak. J. Phytopathol. 34, 81-91.
    CrossRef
  17. Japanis FG, Vetaryan S, Raja NKK, Mokhtar MAA, and Fishal EMM. 2022. The impact of Trichoderma spp. on agriculture and their identification. Malays. Appl. Biol. 51, 1-15.
    CrossRef
  18. Jeevalatha A, Zumaila F, Biju CN, and Peeran MF. 2022. Multiplex PCR assay for simultaneous detection of Phytophthora, Pythium and Fusarium associated with foot rot and yellowing diseases of black pepper. J. Spices Aromat. Crops 31, 92-96.
    CrossRef
  19. Kaur R, Mavi GK, and Raghav S. 2019. Pesticides classification and its impact on environment. Int. J. Curr. Microbiol. Appl. Sci. 8, 1889-1897.
    CrossRef
  20. Khuong QN, Thuy TTC, Xuan TD, Quang TL, Huu NT, Tranh XNL, Sakagmi J, and Thuc VL. 2023. Evaluation of the antagonistic potential of Trichoderma spp. against Fusarium oxysporum F.28.1A. J. Plant Prot. Res. 63, 13-26.
    CrossRef
  21. Kumar K, Amaresan N, Bhagat S, Madhuri K, and Srivastava RC. 2012. Isolation and characterization of Trichoderma spp. for antagonistic activity against root rot and foliar pathogens. Ind. J. Microbiol. 52, 137-144.
    Pubmed KoreaMed CrossRef
  22. Lal M, Kumar A, Chaudhary S, Singh RK, Sharma S, and Kumar M. 2022. Antagonistic and growth enhancement activities of native Pseudomonas spp. against soil and tuber-borne diseases of potato (Solanum tuberosum L.). Egypt. J. Biol. Pest Control 32, 22.
    CrossRef
  23. Leiva S, Oliva M, Hernández E, Chuquibala B, Rubio K, and García F; Torres de la Cruz M. 2020. Assessment of the potential of Trichoderma spp. strains native to bagua (Amazonas, Peru) in the biocontrol of frosty pod rot (Moniliophthora roreri). Agronomy 10, 1376.
    CrossRef
  24. Liu Z, Xu N, Pang Q, Khan RAA, Xu Q, Wu C, and Liu T. 2023. A salt-tolerant strain of Trichoderma longibrachiatum HL167 is effective in alleviating salt stress, promoting plant growth, and managing fusarium wilt disease in cowpea. J. Fungi 9, 304.
    Pubmed KoreaMed CrossRef
  25. Mohiddin FA, Padder SA, Bhat AH, Ahanger MA, Shikari AB, Wani SH, Bhat FA, Nabi SU, Hamid A, and Bhat FA; et al. 2021. Phylogeny and optimization of Trichoderma harzianum for chitinase production: evaluation of their antifungal behaviour against the prominent soil borne phyto-pathogens of temperate India. Microorganisms 9, 1962.
    Pubmed KoreaMed CrossRef
  26. Mulk S, Wahab A, Yasmin H, Mumtaz S, El-Serehy HA, Khan N, and Hassan MN. 2022. Prevalence of wheat associated Bacillus spp. and their bio-control efficacy against Fusarium root rot. Front. Microbiol. 12, 798619.
    Pubmed KoreaMed CrossRef
  27. Nelson N. 1944. A photometric adaptation of the Somogyi method for the determination of glucose. J. Biol. Chem. 153, 375-380.
    CrossRef
  28. Pandian RTP, Raja M, Kumar A, and Sharma P. 2016. Morphological and molecular characterization of Trichoderma asperellum strain Ta13. Indian Phytopath. 69, 297-303.
  29. Olowe OM, Nicola L, Asemoloye MD, Akanmu AO, and Babalola OO. 2022. Trichoderma: potential bio-resource for the management of tomato root rot diseases in Africa. Microbiol. Res. 257, 126978.
    Pubmed CrossRef
  30. Sallam N, Abd Elrazik AA, Hassan M, and Koch E. 2009. Powder formulations of Bacillus subtilis, Trichoderma spp. and Coniothyrium minitans for biocontrol of Onion White Rot. Arch. Phytopathol. Plant Prot. 42, 142-147.
  31. Sharma A, Arya SK, Singh J, Kapoor B, Bhatti JS, Suttee A, and Singh G. 2023. Prospects of chitinase in sustainable farming and modern biotechnology: an update on recent progress and challenges. Biotechnol. Genet. Eng. Rev. 2023, 1-31.
    Pubmed CrossRef
  32. Siddiquee S. In Practical Handbook of the Biology and Molecular Diversity of Trichoderma Species from Tropical Regions. Springer, Cham, Switzerland.
  33. Silva CDFBD, de Medeiros SC, Sousa AJS, da Costa RH, Xavier JG, Santos JEDÁ, Pastori PL, and Grangeiro TB. 2023. Identification of pathogenic fungal isolates of the Fusarium oxysporum and the Fusarium fujikuroi species complex, causing fusariosis in ornamental pineapple, and antifungal activity of elicitors. Eur. J. Plant Pathol. 165, 125-137.
    CrossRef
  34. Singh G, Tiwari A, Choudhir G, Kumar A, and Sharma S. 2022. Unraveling the potential role of bioactive molecules produced by Trichoderma spp. as inhibitors of tomatinase enzyme having an important role in wilting disease: an in-silico approach. J. Biomol. Struct. Dyn. 40, 7535-7544.
    Pubmed CrossRef
  35. Sirikamonsathien T, Kenji M, and Dethoup T. 2023. Potential of endophytic Trichoderma in controlling Phytophthora leaf fall disease in rubber (Hevea brasiliensis). Biol. Control 179, 105175.
    CrossRef
  36. Sood M, Kapoor D, Kumar V, Sheteiwy MS, Ramakrishnan M, Landi M, Araniti F, and Sharma A. 2020. Trichoderma: the "secrets" of a multitalented biocontrol agent. Plants 9, 762.
    Pubmed KoreaMed CrossRef
  37. Stępień Ł, Koczyk G, and Waśkiewicz A. 2013. Diversity of Fusarium species and mycotoxins contaminating pineapple. J. Appl. Genet. 54, 367-380.
    Pubmed KoreaMed CrossRef
  38. Tyśkiewicz R, Nowak A, Ozimek E, and Jaroszuk-Ściseł J. 2022. Trichoderma: the current status of its application in agriculture for the biocontrol of fungal phytopathogens and stimulation of plant growth. Int. J. Mol. Sci. 23, 2329.
    Pubmed KoreaMed CrossRef
  39. Urbina-Salazar ADR, Inca-Torres AR, Falcón-García G, Carbonero-Aguilar P, Rodríguez-Morgado B, del Campo JA, Parrado J, and Bautista J. 2019. Chitinase production by Trichoderma harzianum grown on a chitin-rich mushroom by product formulated medium. Waste Biomass Valor. 10, 2915-2923.
    CrossRef
  40. Wang R, An X, Lv Y, Khan RAA, Xue M, Chen J, and Liu T. 2023. Trichoderma asperellum GD040 upregulates defense-related genes and reduces lesion size in Coffea canephora leaves inoculated with Colletotrichum cairnsense. Biol. Control 181, 105213.
    CrossRef
  41. White TJ, Bruns T, Lee SB, and Taylor JW. PCR protocols: A Guide to Methods and Applications. Academic Press, San Diego, California, USA.
    CrossRef
  42. Yassin MT, Mostafa AAF, and Al-Askar AA. 2022. In vitro antagonistic activity of Trichoderma spp. against fungal pathogens causing black point disease of wheat. J. Taibah Univ. Sci. 16, 68-65.
    CrossRef
  43. Zheng XL, Xia H, Ayra‐Pardo C, and Huang SL. 2022. First report of Fusarium wilt caused by species of the Fusarium solani complex on sweet cherry in Henan province, China. New Dis. Rep. 46, e12108.
    CrossRef