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.
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.
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).
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 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.
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.
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%
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.
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.
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.
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.
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).
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.
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.
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).
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.
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).
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).
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).
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.
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.