2024.01.02.11

Antagonistic activity of biocontrol agent Trichoderma spp. against Fusarium sp., the causal agent of Ananas comosus fruitlet rot

Lucas Martín Madrassi ^1,3*, Adriana Elizabet Alvarenga^1,3, María Celina Vedoya ^2,

¹ Universidad Nacional de Misiones. Facultad de Ciencias Exactas, Químicas y Naturales. Instituto de Biotecnología Misiones "Dra. María Ebe Reca" (INBIOMIS). Laboratorio de Biotecnología Molecular (BIOTECMOL). Ruta Nacional 12 Km 7,5, C.P. 3300, Misiones, Argentina;

² Universidad Nacional de Misiones. Facultad de Ciencias Exactas, Químicas y Naturales. Laboratorio de Micología “Dra. Martha G. Medvedeff”. Av. Mariano Moreno 1375, C.P. 3300, Misiones, Argentina;

³ Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). Godoy Cruz 2290, C.P. 1650. CABA, Argentina.

* Correspondence: lmmadrassi@hotmail.com

Available from. http://dx.doi.org/10.21931/BJ/2024.02.01.11

ABSTRACT

Pineapple (Ananas comosus) is a significant crop, with an annual production exceeding 25 million tons. However, fusariosis can severely impact its cultivation, a fungal disease that causes fruitlet rot and results in substantial yield losses. To decrease dependency on chemical control methods, biocontrol agents (BCAs) present a promising alternative. Among these, Trichoderma species are noteworthy due to their diverse antagonistic mechanisms. The efficacy of each mechanism can be assessed through fungal confrontation assays. This study aimed to isolate, identify, and evaluate in-vitro nine Trichoderma spp. strains as potential BCAs against Fusarium sp. associated with pineapple fruitlet rot. The antagonistic fungi were isolated from rhizosphere soils in both open-field and greenhouse pineapple farms in Misiones province, Argentina. Identification of the fungi required both morphologic and genetic data. In the in-vitro assays, the capabilities for direct competition for substratum, production of metabolites, and mycoparasitism were evaluated. The results indicated that isolates T. harzianum TC7, T. harzianum TC9, T. asperellum TU3, and T. asperellum TU4 had statistically superior inhibitory effects against Fusarium sp. These isolates can be potentially used in formulating natural fungicides to reduce pineapple fruitlet rot caused by Fusarium, promoting sustainable production practices.

Keywords: pineapple, confrontation, mycoparasitism, metabolites, ITS region

INTRODUCTION

The excessive or inadequate use of pesticides in tropical and subtropical crops can result in economic, social and environmental harm¹. Furthermore, pesticide-mediated control of microorganisms is often inefficient, as these organisms may develop resistance". In tropical and subtropical regions, higher frequencies of pesticide applications are required to manage microorganism-related diseases, such as fungal infections caused by Fusarium². Fusariosis in both open-field and greenhouse pineapple farms is particularly problematic, as it is associated with fruitlet rot, leading to severe yield losses and significant economic costs ^3–5.

Concerns regarding chemical-based control have driven the development of alternative approaches⁶, such as integrated crop management (ICM) strategies⁷. A key component of ICM is biocontrol agents (BCAs), also known as biopesticides⁸. These can be applied on farms as natural antagonists of pests. The use of BCAs is considered environmentally friendly and harmless to human health⁹.

Most investigations concerning BCAs have focused on Trichoderma species due to their multiple antagonistic mechanisms, which act synergistically to control plant diseases. Trichoderma species exhibit high growth and reproductive rates, survival across various environmental conditions, nutrient consumption and competition efficiency, and ability to act as necrotrophs against other fungi.

In practice, morphological classification must be complemented with molecular data, for which multiple deoxyribonucleic acid (DNA) loci analysis is required. In particular, the Internal Transcribed Spacer region (ITS), within ribosomal DNA serves as a reference locus for identifying Trichoderma species¹⁰.

As an initial step in evaluating a Trichoderma isolate as a BCA, qualitative in-vitro assays are necessary. These trials are predictive tools to determine mycelial growth inhibition against specific pathogens. It is possible to separately analyze the antagonistic mechanisms of direct competition for substrate, mycoparasitism, and the production of fungistatic compounds, known as metabolites^11,12. As the final steps in this evaluation, it is necessary to investigate the most promising isolates in planta assays under controlled or field conditions and develop mass production techniques for the biopesticide.

This work aimed to isolate, identify, and evaluate in-vitro the potential of native Trichoderma isolates as BCAs against Fusarium sp., associated with pineapple fruitlet rot.

MATERIALS AND METHODS

Isolation of rhizosphere Trichoderma strains

Rhizospheric soil samples were collected from 9 pineapple farms in Argentina, and two farming systems were considered: open-field and greenhouse cultivation (see Table 1). 100g of soil was collected from each farm, mixed, and homogenized manually. Fungal colonies were obtained using serial dilution and cultured on Trichoderma Selective Medium (TSM)^13,14 at 28±2ºC for 7 days. Trichoderma colonies were individually subcultured on Potato Dextrose Agar (PDA) at 28±2ºC for 7 days.

Table 1. Information on the Trichoderma isolates, including the type of farming system (open-field or greenhouse cultivation), region, and town of the pineapple farms

Morphological identification of the Trichoderma isolates

Monosporic colonies were obtained for further proposals. These were cultivated in PDA and Malt Extract Agar at 28±2°C, in darkness, for 15 days. Regular observations of macroscopic characteristics, including color, shape, pigment production or liberation, and growth rate, were recorded. Microscopic examination involves the identification of vegetative and reproductive structures according to ¹⁵ using an optical microscope (Olympus, CX23).

DNA extraction, amplification, and sequence analysis

Genomic DNA was extracted according to ¹⁶. The ITS1-5.8S-ITS2 region was amplified using the polymerase chain reaction (PCR) technique according to ¹⁷. Macrogen Inc. (Seoul, Korea) standard sequencing services purified and sequenced PCR products. The quality of DNA sequencing was verified using Chromas Lite v2.1, and sequences were manually trimmed using BioEdit v7.2¹⁸. Subsequently, the sequences underwent an essential local alignment search tool for nucleotides (BLASTn) analysis against the National Centre for Biotechnology Information (NCBI) database. Multiple sequence alignment was performed using AliView v1.28¹⁹ with the ClustalW method.

Phylogenetic analysis of ITS1-5.8S-ITS2 region

Phylogenetic inference was conducted using the Bayesian method. All characters were treated as equally weighted, with gaps considered missing data. The results from the BLASTn submission were downloaded, verified and selected for phylogenetic reconstruction (sequences are listed in Table 2). The outgroup in the inference was Clonostachys spp., with C. rosea CBS 154.27 and C. solani CBS 183.30 (NCBI database Reference Sequence, accession numbers: NR_165993 and NR_163540, respectively).

The find-best-fit-model function was performed in Mega v7²⁰ to identify the most informative phylogenetic model. With the resulting model, the Maximum Likelihood method with a bootstrap of 1000 repetitions was applied. The tree was visualized and edited with the same software.

Table 2. The taxonomic identification, isolate name, accession number in the NCBI database, and publication or reference are listed for each sequence used in the phylogenetic reconstruction of the ITS1-5.8S-ITS2 region of the native Trichoderma isolates and sequences from NCBI database

Antagonism potential of rhizosphere Trichoderma, in-vitro

In-vitro assays were made in Petri dishes with PDA at 28±2°C in darkness for 10 days. The pathogenic Fusarium sp. strain used in these assays was obtained from the culture collection of the Laboratory of Mycology "Dra. Martha G. Mevdeveff" from the "Facultad de Ciencias Exactas, Químicas y Naturales, UNaM". This strain was isolated on a diseased pineapple plant from Apostoles City, Misiones province. Negative controls consisted of identical assays but without Trichoderma being inoculated.

Mycoparasitic activity determination

The mycoparasitic activity was confirmed by directly observing cellular interactions between the Trichoderma and Fusarium isolates. The interactions between the colonies were investigated using the microculture method ²¹ with slight modifications ²², and the colonies were cultivated in 0.1ml of PDA for 5 days. During observation with an optical microscope, the mycelium was stained with lactophenol-cotton-blue to enhance visualization and examined using 40X and 100X magnifications.

Estimation of the growth inhibition of Fusarium sp. by Trichoderma

The antagonistic activity was estimated as %I=(C-T/C)x100, where %I is the percentage of mycelial growth inhibition and T and C are the mean Fusarium colonies radius in the treatments (with Trichoderma) and the controls (without Trichoderma), respectively. Three biological replicates were performed for each treatment.

Direct confrontation (DC)

%I was measured in the DC experiments according to ²³. In this case, 5mm discs of Trichoderma and Fusarium were deposited oppositely at 5mm of the edge of a single Petri dish. We also used the antagonism scale (from 1 to 5) proposed by ²⁴.

Indirect confrontation by diffusible (ID) and volatile (IV) metabolites

This study evaluated two types of indirect confrontation assays: diffusible metabolites (ID) and volatile metabolites (IV).

The ID production experiments were carried out as per ¹¹. 5mm discs of Trichoderma were plated on sterile PDA plates, and the medium surface was covered with a layer of cellulose paper. Trichoderma strains were grown for 2 days. Afterward, the Trichoderma colonies were carefully removed, and a 5mm disc of Fusarium was plated on the medium.

The IV production experiments followed the protocol outlined in ¹². 5mm discs of Trichoderma and Fusarium were plated on separate PDA dishes. The plates were overlaid, with Trichoderma at the bottom and Fusarium at the top. The junction was sealed with parafilm. The technique was modified to visualize Fusarium sp. colonies exposed to Trichoderma volatiles as per ²⁵.

Statistical analysis of the growth inhibition

The antagonistic activities were compared with one-way ANOVA to find the statistically significant inhibitions. Tukey's test was implemented for the pairwise comparisons. Data analysis and visualization were performed using GraphPad Prism v8²⁶.

RESULTS

Isolation of rhizosphere Trichoderma strains

Nine fungal isolates with the classic Trichoderma characters were obtained from the rhizosphere soils of pineapple plants from farms located in Misiones province. Six isolates were obtained from open-field farms and named TU1, TU2, TU3, TU4, TU5, and TU6. The remaining three isolates were obtained from greenhouse farms and named TC7, TC8, and TC9. All fungal isolates were maintained and stored in PDA at 4ºC.

Morphological identification of the Trichoderma isolates

Colonies of Trichoderma spp. were initially white for the first 48-72 hours (early mycelium), later turning dark or light green (late mycelium), with scarce aerial mycelia. No diffusible pigment was observed when Trichoderma isolates were cultured alone. Conidial production was observed after 4 days of incubation, with denser conidia in the center of the colonies. Older colonies formed 1-3 concentric rings. Conidiophores were symmetrical, with a uniform central axis from which paired secondary axes emerged. Phialides were slender, hooked, and flask-shaped. Conidia were dark green and globose, appearing after 48-72 hours. Chlamydospores were green and globose, appearing after 5-7 days. We identified these isolates as members of the genus Trichoderma based on morphological characteristics.

During the late mycelium state, the growth rate and sporulation pattern were slightly different between Trichoderma isolates depending on the type of farming system (open-field or greenhouse cultivation) from which they were collected.

Phylogenetic study of the ITS1-5.8S-ITS2 region

The two main groups formed consisted of sequences from the T. harzianum and T. asperellum species complexes (see Fig. 1). These groups had significant statistical support, with bootstrap values higher than 70. The sequences of isolates TU1, TU2, TU3, TU4, TU5, and TU6 grouped with T. asperellum species-complex sequences from NCBI. The sequences of the isolates TC7, TC8, and TC9 grouped with T. harzianum species-complex.

Figure 1. Phylogenetic reconstruction of the ITS1-5.8S-ITS2 region. The tree was constructed using the Maximum Likelihood method with the Jukes-Cantor model, and bootstrap support values were generated from 1000 replicates. Only bootstrap supports equal to or higher than 70 are shown. The isolates from this work are indicated by black triangles (▲) for open-field cultivation and white triangles (△) for greenhouse cultivation. The outgroup (Clonostachys spp.) is colored in red. The vertical bars indicate sequences from the same Trichoderma species complex. These groups are supported by bootstrap values of 70 for the T. harzianum species-complex isolates and 95 for the T. asperellum species-complex isolates.

Mycoparasitic activity determination

The mycoparasitic capability of all the Trichoderma isolates was confirmed by observing the microscopic interaction of the hyphae. These interactions included cellular adhesion, coiling, and penetration toward Fusarium sp. (Fig. 2).

Figure 2. Microphotography of a microculture assay between the isolate T. asperellum TU2 (T) and Fusarium sp. (F), associated with pineapple fruitlet rot. The image shows multiple coiling of Trichoderma towards the Fusarium hyphae. Scale: 20um.

Estimation of the growth inhibition of Fusarium sp. by Trichoderma

The antagonistic strains exhibited a remarkable inhibitory effect against the Fusarium isolate after 10 days of cultivation. The percentage of mycelial growth inhibition (%I) varied among the antagonistic treatments tested. In other words, the inhibition effect against Fusarium sp. varied among the different assays involving Trichoderma isolates (see Table 3). The average colony radius for the Fusarium sp. isolates, without Trichoderma, was 39±2,4mm across three biological replicates after 10 days of cultivation.

Table 3. PICP values for nine Trichoderma isolates against Fusarium sp. in dual culture (DC) and diffusible (IV) and volatile (IV) metabolites assays. Values are shown as mean ± standard deviation. Absolute values for the Fusarium colonies are detailed in Table S1.

Direct confrontation (DC)

The T. harzianum isolates showed the highest antagonistic activity in the DC cultures. In this case, the inhibition ranged from 63 to 69.3%. The inhibition obtained by the T asperellum isolates ranged from 51.7 to 66.3%.

The Trichoderma isolates showed high scores on Bell's scale, with T. asperellum and T. harzianum isolates scoring 1 and 2, respectively. At the end of the 10^th day, they exhibited moderate to high overgrowth on the Fusarium sp. colonies.

In all the DC plates tested, the contact zone between the fungi exhibited a curved shape, with the concavity orientated towards the Fusarium isolate. The T. asperellum isolates produced inhibition halos (3-5mm wide) between both colonies, indicating the presence of diffusible metabolite production. The T. harzianum isolates emanated a profuse coconut-like smell, a classical indicator of volatile metabolite production in Trichoderma species^27–29.

Indirect confrontation by diffusible (ID) and volatile (IV) metabolites

In ID production assays, T. asperellum isolates showed the highest antagonistic activity. In this case, inhibition ranged from 43.3 to 57.6%. In contrast, inhibition for T. harzianum isolates was weaker for this assay and ranged from 43.7 to 48.7%.

The T. harzianum isolates obtained the strongest inhibitions against Fusarium sp in IV cultures. The %I values ranged from 51.3 to 60.3%. The inhibition for T. asperellum isolates was lower and ranged from 39 to 49.7%.

Remarkably, in the IV assays, the Trichoderma colonies had a yellow coloration after 10 days of cultivation, and the Fusarium isolate did not produce any pigments in the presence of the volatile metabolites produced by Trichoderma spp., so the late mycelia of the colonies remained white (Fig. 3).

Figure 3. Illustrative picture of macro-morphological changes of the Fusarium colonies in controls and treatments within the IV assays. In (a), the Fusarium colony exhibits the typical red pigment produced during cultivation on potato dextrose agar. In (b), the Fusarium colony is exposed to the Trichoderma volatile compounds and does not produce any observable pigments, resulting in a white appearance after 10 days of cultivation. Scale: (a, b): 5mm

Statistical analysis of the growth inhibition

The one-way ANOVA test revealed statistically significant differences in the inhibition values among some of the Trichoderma isolates for each type of the antagonism mechanisms tested (see Fig. 4). Particularly, in the DC experiments (F= 133.3, df= 26, P <0.0001), T. harzianum TC7 was statistically superior to other isolates (Tukey, P<0.05). In the ID assays (F= 59.2, df= 26, P <0.0001), T. asperellum TU3 and T. asperellum TU4 had a statistically different inhibitory effect on Fusarium sp. (Tukey, P<0.05) but not between each other (Tukey, P≥0.5). In the IV assays (F= 147.3, df= 26, P <0.0001), T. harzianum TC9 produced a statistically higher inhibition than any other isolate (Tukey, P<0.05).

Figure 4. The values of %I (vertical axis) of the nine Trichoderma isolates (horizontal axis) against Fusarium sp., associated with pineapple fruitlet rot, in DC (dark bars), ID (grey bars), and IV (white bars) assays. For each %I value, the 95% confidence intervals are shown. The asterisks (*) indicate statistically higher inhibitions (Tukey, P<0,05) for the dual culture (*DC), diffusible metabolites (*ID), and volatile metabolites (*IV) assays.

DISCUSSION

Isolation of rhizosphere Trichoderma strains

The reports suggest that Trichoderma³⁰ and Fusarium⁵ are prevalent fungal genera in pineapple farms. Specifically, T. harzianum and T. asperellum are frequently isolated in tropical and subtropical environments³¹, which comprise the primary geographic zones for pineapple cultivation.

Morphological and molecular identification of the Trichoderma isolates

In the present assay, two groups were delineated based on Trichoderma morphological characters. Each group presented the typical morphotypes reported for the T. asperellum species-complex³² and the T. harzianum species-complex isolates³³. However, it should be noted that these morphotypes serve as broad descriptors.

On average, the analysis of the ITS1-5.8S-ITS2 region leads to an accurate taxonomic fungal species identification, particularly within the ITS1 and the ITS2 introns³⁴. The formation of phylogenetically significant subgroups within specific species is standard, as observed in ³⁵ and ¹⁷, where the T. harzianum isolates were grouped in two or more "clades." Similarly, in the experiments by ^32,the T. asperellum isolates formed various subgroups. The two Trichoderma morphotypes in this assay corresponded to distinct ITS1-5.8S-ITS2 phylogenetic groups. The sequences of the T. harzianum species-complex isolates formed subgroups with statistically significant bootstrap values.

However, taxonomic identification at the species level in Trichoderma necessitates the utilization of additional DNA markers³⁶. For example, the Translational Elongation Factor 1α (TEF1α)³⁷ and the RNA Polymerase II Second Largest Subunit (RPB2)³⁸ have been used to distinguish species within the T. asperellum³⁹ and T. harzianum complexes⁴⁰.

Mycoparasitic activity determination

The ability to parasitize other fungi is widespread among species of the genus Trichoderma. T. asperellum and T. harzianum are no exceptions, as their mycoparasitic capacity has been validated against numerous phytopathogens³⁵.

In this research, the ability of native T. asperellum and T. harzianum isolates to mycoparasitize Fusarium sp. associated with pineapple fruitlet rot was demonstrated. These results are similar to those obtained by ⁴¹, where most Trichoderma isolates exhibited the capacity to mycoparasitic Fusarium spp., showing a range of antagonist-pathogen cellular interactions, including adhesion, coiling, and penetration of host hyphae.

Direct confrontation (DC)

The autochthonous T. asperellum and T. harzianum isolates inhibited the mycelial growth of Fusarium sp. by at least 50 and 60%, with maximum %I values of 66.3% (TU2) and 69.3% (TC7), respectively. These %I values are similar to those previously reported by ⁷ for T. asperellum ^33,42 for T. harzianum, with most isolates exhibiting medium to high antagonistic activity in dual cultures. Nevertheless, %I values documented in the literature range from 20% to 70% for T. asperellum and 30% to 90% for T. harzianum. Consistent with the findings in this study, the highest dual culture %I values for T. harzianum isolates were also reported by ⁴².

The highest %I values were primarily obtained in the DC tests, compared to other assays. This is logical, considering that under these conditions, multiple antagonistic mechanisms could be present simultaneously, such as producing diffusible and volatile metabolites, competition for the substrate, and mycoparasitism, etc⁴³.

Indirect confrontation by diffusible (ID) and volatile metabolites (IV)

The production of diffusible metabolites has been demonstrated in several Trichoderma species, including T. asperellum⁴⁴and T. harzianum⁴⁵. In the present work, the native T. asperellum and T. harzianum isolates produced diffusible substances with fungistatic action against Fusarium sp. in-vitro. This was especially remarkable for the T. asperellum isolates, which exhibited %I values greater than 50%. Moreover, the presence of inhibition halos during the dual cultures may be attributed to these isolates' high production of diffusible compounds.

Similar to the previous case, the ability to produce volatile metabolites has been demonstrated for T. asperellum⁴⁶and T. harzianum⁴⁷. The %I values obtained by the Trichoderma isolates in this assay suggest the production of volatile compounds capable of inhibiting Fusarium sp. mycelial growth. This was particularly notable for T. harzianum isolates, which achieved %I values greater than 50% in this assay. Remarkably, these fungi emitted an intense coconut-like smell during cultivation with Fusarium spp., a scent previously associated with a Trichoderma volatile antibiotic^12,27–29.

Utilization of Trichoderma spp. as BCAs in pineapple farms

Our study provides preliminary insights into the presence of T. harzianum and T. asperellum species complexes and their mechanism for controlling Fusarium sp., the causal agent of pineapple fruitlet rot. In the present work, the antagonistic effects of the Trichoderma isolates varied within isolates from the same species complex. The interactions of fungal isolates are considered strain-specific ^48,49. In this context, the interactions of novel Trichoderma isolates against multiple pathogenic Fusarium strains associated with pineapple fruitlet rot could improve our understanding of the behavior of BCAs under different conditions.

While promising Trichoderma isolates were identified and evaluated in this work, further research on the formulations and applications of these BCAs for field treatments is necessary⁵⁰. The effectiveness of Trichoderma isolates in protecting pineapple plants from Fusarium infection should be evaluated in planta to represent a more realistic scenario⁵¹. Future research in these areas will enhance the development of robust biocontrol strategies using Trichoderma species against Fusarium-induced fruitlet rot in pineapples under both open-field and greenhouse cultivation in Misiones farms using ICM strategies.

CONCLUSIONS

This work has studied the biocontrol mechanisms of Trichoderma strains belonging to different species groups. Furthermore, the Trichoderma isolates from healthy pineapple rhizosphere soil displayed robust antagonistic activity against Fusarium sp. These results showed that native Trichoderma spp. could reduce the mycelial growth of Fusarium sp., associated with pineapple fruitlet rot.

This is the first report on the isolation, identification, and characterization of antagonistic Trichoderma species from rhizosphere soil in open-field and greenhouse-cultivated pineapple farms in Misiones, Argentina. The antagonistic activity of T. asperellum and T. harzianum species complexes highlights the potential of using these BCAs to formulate natural and highly effective fungicides. Different formulation and application methods must be studied to gain further insight into the utility of Trichoderma as BCA. These isolates might be integrated with other management strategies to reduce pineapple fruitlet rot caused by Fusarium under a sustainable production practice.

Supplementary Materials: Table S1

Table S1. Values of the radius (mm) of the Fusarium sp. colonies during in vitro antagonism assays against nine Trichoderma isolates.

Author Contributions: LMM made the taxonomic identification, the in-vitro characterization of the fungal isolates, the analysis of the data, and the writing of the manuscript. AEA made a significant contribution in the manuscript's interpretation and writing. MCV designed and conducted the experiments and isolated the fungal material. All authors read and approved the final manuscript.

Funding: Not applicable

Institutional Review Board Statement: Not applicable

Informed Consent Statement: Not applicable

Data Availability Statement: All data generated or analyzed during this study are included in this published article.

Acknowledgments: Not applicable

Conflicts of Interest: The authors declare no conflict of interest

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Received: April 20, 2023/ Accepted: May 28, 2024 / Published: June 15, 2024

Citation: Madrassi, L.M.; Alvarenga, A.E.; and Vedoya, M.C. Antagonistic activity of biocontrol agent Trichoderma spp. against Fusarium sp., the causal agent of Ananas comosus fruitlet rot. Bionatura journal 2024; 1 (2) 11. http://dx.doi.org/10.21931/BJ/2024.01.02.11

Additional information Correspondence should be addressed to lmmadrassi@hotmail.com

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