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Article

Inhibitory Effect of Trichoderma citrinoviride Secondary Metabolites on the Growth Kinetics and Spore Germination of Fungal Phytopathogens

by
Michał Piegza
1,*,
Aleksandra Kaliciak
1,2 and
Wojciech Łaba
1
1
Department of Biotechnology and Food Microbiology, Wroclaw University of Environmental and Life Sciences, 50-375 Wrocław, Poland
2
Student’s Scientific Circle of Biotechnology, Department of Biotechnology and Food Microbiology, Wroclaw University of Environmental and Life Sciences, 50-375 Wrocław, Poland
*
Author to whom correspondence should be addressed.
Molecules 2026, 31(5), 827; https://doi.org/10.3390/molecules31050827
Submission received: 30 January 2026 / Revised: 26 February 2026 / Accepted: 27 February 2026 / Published: 28 February 2026
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Abstract

Hyphae fungi of the Trichoderma genus are widely recognized as effective biological control factors (BCAs) due to their ability to inhibit the growth of plant pathogens through a variety of mechanisms such as mycoparasitism, antibiotics or competition for resources. Specialized secondary metabolites (SMs), including volatile organic compounds (VOCs), lytic enzymes and surfactants, play an important role in these interactions. The aim of this study was to evaluate the antagonistic activity and characterization of secondary metabolites from the aqueous phase or suspended in an organic solvent produced by three strains of Trichoderma citrinoviride. The study focused on their enzymatic properties, surfactant potential and effect on the growth kinetics of sixteen fungal species. Antagonistic activity against phytopathogens was tested using the turbidimetric method, analyzing various forms of preparations. Lytic enzyme activity and surface tension of fluids were also evaluated. The C1 strain showed the broadest spectrum of antagonistic activity. Analysis of growth kinetics revealed that the way metabolites are prepared is crucial for their efficacy. Studies have shown that the effectiveness of biocontrol depends not only on the Trichoderma strain, but also on the extraction method and form of the preparation (e.g., rehydration of lyophilizate vs. organic phase extraction). The presence of diverse metabolites, including lytic enzymes, biosurfactants and volatile organic compounds, indicates a complex mechanism of action of T. citrinoviride, making this species an ideal candidate for the production of plant protection biopreparations.

Graphical Abstract

1. Introduction

Due to the properties directed against certain fungal plant pathogens, filamentous fungi of the genus Trichoderma have gained the status of Biological Control Agents. This is associated with a number of mechanisms by which they act on phytopathogens, which entails the possibility of using living cells, their genes or substances produced by them, to reduce the negative effects of plant pathogens and to exert positive effects on plant development. Trichoderma spp. are non-virulent, opportunistic plant symbionts [1]. As endophytic fungi, they colonize plant tissues without causing disease [2]. This symbiosis is based on the fungal ability to utilize plant-derived sucrose and other metabolites, while providing multiple benefits to the host, including enhanced resistance to pathogens, improved growth and photosynthetic performance, and more efficient use of nutrients and fertilizers resulting in higher yield [1,3]. Biological control is defined as the application of living organisms or their metabolites to suppress harmful microorganisms and mitigate their negative effects [2,4]. Trichoderma fungi are considered particularly suitable as BCAs because of their ability to inhabit various ecosystems, minimal disruption of soil microbial balance, and safety for beneficial organisms involved in pathogen suppression. These attributes make them cost-effective, efficient, and environmentally sustainable BCAs [1]. Since the first registration of Trichoderma harzianum by the United States Environmental Protection Agency in 1989 for plant disease management, Trichoderma-based products have become the most widely commercialized BCAs worldwide [4]. Their biocontrol activity relies on multiple mechanisms, including mycoparasitism, antibiosis, and competition against phytopathogens [1].
Filamentous fungi are recognized as one of the major producers of specialized metabolites (SMs). These structurally diverse, low-molecular-weight compounds (typically <3 kDa) are not directly required for their growth or survival but play a crucial role in mediating ecological interactions. Acting as chemical signals, they enable fungi to communicate with their environment, establish competitive or cooperative relationships with other microorganisms, and adapt to changing external conditions [5,6]. The biosynthesis of SMs is tightly regulated by environmental and nutritional factors such as carbon, nitrogen, and metal availability, temperature, pH, light, and redox balance. It proceeds via non-canonical biochemical pathways, generating a wide spectrum of natural products derived from primary metabolism. Within fungal cells, SMs interact with proteins, nucleic acids, and membranes, initiating molecular responses that support physiological adaptation and survival [5,7].
Among filamentous fungi, the genus Trichoderma is particularly efficient in the production of biologically active SMs, including polyketides; non-ribosomal peptides (NRPs); terpenoids; extracellular volatile metabolites (VOCs, volatile organic compounds) that inhibit microbial growth, which is particularly noteworthy; and surfactants [8,9]. NRPs constitute one of the most diverse and ecologically significant classes of SMs. They are synthesized by large, multimodular enzyme complexes, i.e., non-ribosomal peptide synthetases (NRPSs), and can incorporate both proteogenic and non-proteogenic amino acids [10]. Trichoderma NRPs encompass peptaibiotics, epipolythiodioxopiperazines (ETPs), and siderophores. Peptaibiotics, especially peptaibols, are characterized by the presence of α-aminoisobutyric acid and a C-terminal alcohol. They exhibit strong antimicrobial activity against fungi, Gram-positive bacteria, and viruses, and act by forming voltage-dependent ion channels in biological membranes, thereby increasing permeability and inducing cell death [11,12]. Notably, peptaibols can also trigger plant defense responses and act synergistically with hydrolytic enzymes, enhancing fungal antagonism against phytopathogens such as Botrytis cinerea [13]. ETPs, such as gliotoxin, are another group of Trichoderma NRPs. It exhibits pleiotropic activity, has medicinal value and biocontrol potential, yet demonstrates toxicological impact on humans [11,14]. Siderophores, in turn, mediate iron acquisition and competition in the rhizosphere, contributing to plant–fungus–microbe interactions [6,15].
Beyond NRPs, Trichoderma species also produce volatile organic compounds (VOCs), including lactones such as 6-pentyl-α-pyrone, terpenoids, alcohols, ketones, and hydrocarbons. These metabolites contribute to antifungal activity, plant growth promotion, stress tolerance, and interspecies signaling [11,12,13,14,15,16]. The VOCs are isolated from cultures of Trichoderma fungi grown on solid or liquid media, by extraction from the solid phase with organic solvents or using a silica gel column, followed by characterization, e.g., with a mass spectrometer or flame ionization detector [17,18,19,20]. Given their remarkable chemical diversity and ecological importance, Trichoderma metabolites are of growing interest in agriculture, biotechnology, and medicine. The aim of this study was to analyze the antagonistic activity and determine the production levels of specific secondary metabolites in various Trichoderma citrinoviride strains. These strains were cultured under optimized conditions to promote the synthesis of surface-active compounds, followed by a comprehensive analysis of growth parameters across multiple pathogens.

2. Results

The obtained results highlight the biocontrol potential of Trichoderma citrinoviride, as its culture fluids featured reduced surface tension, enzymatic activity, and phytopathogen growth inhibition.
A decrease in surface tension was observed in all culture fluids (Table 1), suggesting the presence of surface-active compounds. The lowest value was recorded for T. citrinoviride C1 (34 mN/m).
Enzymatic activity was assessed in fluids obtained from cultures incubated at three different temperatures (culture 1: 19–21 °C; culture 2: 22–23 °C; culture 3: 24–25 °C). In all samples, laminarinase activity predominated over lichenase activity. The highest laminarinase activity across all three culture conditions was observed for strains HL (0.918, 0.914, and 0.889 U/mL, respectively) and C1 (0.943, 0.867, and 0.833 U/mL, respectively), with maximum values obtained under culture condition 1. Activity generally decreased as culture temperature increased. In contrast, strain B3 displayed a different pattern: the highest laminarinase activity was recorded in culture 3 (0.921 U/mL), while cultures 1 (0.718 U/mL) and 2 (0.661 U/mL) showed lower values. The activity of culture 3 in strain B3 was comparable to that of HL and C1 strains (Figure S1).
The highest lichenase activity was detected in the culture fluid of T. citrinoviride C1 grown under culture condition 1 (0.245 U/mL). The activity decreased proportionally with increasing temperature, reaching 0.169 U/mL in culture 2 and 0.132 U/mL in culture 3. For the HL strain, lichenase activity varied differently, with culture 3 (0.167 U/mL) showing higher activity than the corresponding culture of the C1 strain, while culture 1 (0.166 U/mL) and culture 2 (0.145 U/mL) were characterized by lower activity. The lowest lichenase activity was consistently observed in the B3 strain, mirroring its laminarinase profile (Figure S2). The assessment of the tested Trichoderma strains’ activity level, with additional consideration of the marginal activity of chitinases under the proposed conditions, supported the decision to apply cultivation temperature lower than generally considered standard in further experiments. Thus, culture 3 was excluded from further studies.
All T. citrinoviride strains generally demonstrated antagonistic activity against the tested pathogens, with the exception of Mucor hiemalis, where both organisms grew in direct contact along a boundary line. The three strains strongly inhibited Botrytis cinerea, Rhizopus stolonifer, and Epicoccum nigrum, surrounding more than two-thirds of the pathogen colonies (score +4). In particular, each strain completely inhibited the growth of E. nigrum. The weakest inhibition was observed against Rhizopus nigricans, Fusarium poae, and Fusarium sp., with scores of +2 (Table S2).
Among the tested isolates, T. citrinoviride C1 exhibited the strongest antagonistic potential. It achieved the maximum score in interactions with 10 pathogen strains. The B3 strain reached this score in 8 interactions, while HL reached it in only 3. The C1 strain surrounded more than one-third but less than half of the colonies (+2) of Penicillium expansum, R. nigricans, Fusarium sp., and F. poae. In its interaction with P. expansum, however, the strain C1 scored lower than the other two strains. This strain scored +3 only in culture with Monilinia fructigena (Table S2). The B3 strain displayed slightly lower antagonism overall. It inhibited at least half but less than two-thirds of the colonies (+3) of F. culmorum and M. fructigena. Its interaction with Alternaria alternata, R. nigricans, Penicillium sp., F. poae, and Fusarium sp. yielded a score of +2, indicating inhibition between one-third and one-half of colony growth.
The weakest antagonistic effect was recorded for the strain HL. In interactions with R. nigricans, Penicillium sp., F. poae, F. sporotrichioides, M. fructigena, S. brevicaulis, P. janthinellum, and Fusarium sp., it scored only +2. In cultures with P. expansum, A. alternata, A. terreus, and F. culmorum, it achieved a score of +3. However, in interactions with F. sporotrichioides, S. brevicaulis, and P. janthinellum, it showed markedly weaker antagonism compared with strains B3 and C1 (Table S2).
The study demonstrated that secondary metabolites of Trichoderma citrinoviride strongly influence the growth dynamics of phytopathogenic fungi, with effects depending on both the fungal strain and the culture conditions. Growth inhibition of pathogenic fungal strains showed high variability depending on the T. citrinoviride strain used and the variant of the post-culture fluid. Differences were observed in lag phase duration, logarithmic growth phase, and maximum specific growth rate. In the absence of T. citrinoviride, the lag phase of most pathogens ranged from 2.5 to 6 h, with extended times for A. terreus (13.5 h) and S. brevicaulis (16 h). Individual crude culture fluids affected the pathogens differently, e.g., metabolites of the HL strain reduced the lag phase of F. poae by 1.5 h in culture 1 but extended it by 1 h in culture 2. F. culmorum lag phase was shortened by 2 h in culture 2 but remained unaffected in culture 1. The most extended lag phase was observed for A. alternata (up to 16.5 h), B. cinerea (up to 16.5 h), M. hiemalis (up to 12.5 h), and S. brevicaulis (up to 11.5 h) (Table S3A).
Freeze-dried post-culture fluids of Trichoderma exhibited the most pronounced effects. Metabolites of the HL strain completely inhibited the growth of all pathogen strains. The C1 strain metabolites did not fully inhibit the growth but significantly delayed spore germination, i.e., in eight strains the lag phase was extended by 23–38.5 h, with the strongest effect on S. brevicaulis (54.5 h). F. culmorum was least sensitive (extension by 8 h in culture 1 and only 0.5 h in culture 2). For F. poae, culture 1 extended the lag phase by 20 h, while culture 2 prolonged it by 7.5 h. The B3 strain metabolites also showed strong differentiation: culture 1 compounds completely inhibited A. terreus and extended lag phase in most other pathogens by 8.5–29 h, while culture 2 shortened lag phase of several strains (e.g., S. brevicaulis by 11 h) but significantly prolonged it in M. fructigena (18 h) (Table S3C).
Preparations from T. citrinoviride B3 devoid of proteins (after TCA precipitation) accelerated spore germination of most pathogens. In culture 1, the lag phase was prolonged only in 4 strains (Fusarium sp., up to 0.5 h; M. fructigena, F. culmorum, F. sporotrichioides, up to 4.5 h). In culture 2, the effect was stronger, as 7 strains showed lag phase prolongation (up to 0.5–6 h), no change was observed for S. brevicaulis, while others showed shortened lag phase (Table S3D).
Metabolites of the HL strain under culture 2 shortened lag in A. alternata, Penicillium sp., and B. cinerea, while under culture 1, it shortened lag in R. nigricans and F. poae. However, in both cases, there was a significant lag phase prolongation: F. sporotrichioides (up to 28–30 h), F. culmorum (27.5–31.5 h), S. brevicaulis (18–20.5 h), A. terreus (15.5–21 h), and the growth of Fusarium sp. was completely inhibited.
Metabolites of the C1 strain (culture 1) shortened lag phase in A. alternata, Penicillium sp., B. cinerea, F. poae, P. janthinellum, and Fusarium sp., while in culture 2, they shortened lag phase in P. expansum, B. cinerea, and F. poae. Lag phase of other strains was mostly unaffected, except strains with their clear prolongation: S. brevicaulis (up to 17–20.5 h), F. culmorum (28.5 h), F. sporotrichioides (28 h), and A. terreus (11.5–14.5 h) (Table S3D).
Hexane extracts of HL and B3 strains had weaker effects on pathogens’ growth: no change in P. expansum, slight lag phase reduction in Penicillium sp. (2 h) and A. terreus (1.5 h), but prolongation in S. brevicaulis (6.5 h), Fusarium sp. (6.5 h), and in other strains only moderately (0.5–2.5 h). The effect of the C1 hexane extract differed: no effect was exerted on Penicillium sp., a shortened lag phase in R. nigricans, but marked prolongation in F. culmorum (27 h), S. brevicaulis (27 h), and Fusarium sp. (16.5 h). The lag phase of other strains was extended by 0.5–4.5 h. Hexane extracts from freeze-dried post-culture fluids were mainly characterized by a higher content of metabolites, prolonging the lag phase of pathogen strains. The growth of Epicoccum nigrum was completely inhibited by HL strain compounds, which also delayed F. culmorum germination by 46.5 h and S. brevicaulis by 27 h. The smallest delays were observed for Penicillium sp. (0.5 h) and B. cinerea (1.5 h), while other strains showed extensions of 3.5–22 h (Table S3E).
Metabolites from the B3 strain caused complete inhibition of S. brevicaulis and strongly prolonged lag phase in E. nigrum (49.5 h), F. culmorum (47 h), F. poae (40.5 h), and F. sporotrichioides (38.5 h). Lesser but still notable effects were observed for Penicillium sp. (29.5 h) and A. terreus (29 h), while B. cinerea remained unaffected. Compounds of the C1 strain strongly prolonged lag phase in Fusarium sp. (50.5 h), F. culmorum (44 h), S. brevicaulis (33.4 h), E. nigrum (28.5 h), and F. sporotrichioides (21 h). For the remaining strains, lag phase elongation ranged from 1.5 to 15 h (Figure 1).
The maximum specific growth rate in the control cultures varied depending on the strain of pathogenic fungi (Table S4A). The use of broth as a medium resulted in a reduction in this parameter for most strains, with the exception of E. nigrum, F. poae, and F. sporotrichioides. In contrast, the mixed YM + PDB medium increased the specific growth rate in B. cinerea, M. hiemalis, E. nigrum, F.poae, F. sporotrichioides, F. culmorum, and P. janthinellum. The lyophilized medium caused a decrease in the specific growth rate across all strains, except for E. nigrum.
The presence of crude culture fluids exerted a differential effect on the specific growth rate of pathogenic strains, depending on both the T. citrinoviride strain and the culture conditions. The maximum specific growth rate of P. expansum, B. cinerea, and F. culmorum was reduced compared with the control only in the presence of metabolites produced by the T. citrinoviride C1 strain under culture 1. A consistent reduction in growth rate, induced by metabolites present in the supernatant from both culture conditions of each strain, was observed in R. nigricans, R. stolonifer, and M. fructigena (Table S4B, Figure 2).
For Penicillium sp., Scopulariopsis brevicaulis and Penicillium janthinellum strains, the exceptions that increased the specific growth rate were compounds from cultures of T. citrinoviride HL (cultures 1 and 2) and T. citrinoviride B3. Conversely, the exceptions that did not affect the growth of Alternaria alternata and Aspergillus terreus strains were compounds from culture 2 of the T. citrinoviride C1 and culture 2 of T. citrinoviride B3, respectively. The specific growth rate of Mucor hiemalis was reduced by the presence of metabolites biosynthesized by T. citrinoviride HL and C1 strains under culture conditions 1, as well as by B3 strains and slightly by C1 strains under culture conditions 2. The compounds present in each culture fluid caused an increase in the specific growth rate of Epicoccum nigrum, Fusarium poae, Fusarium sp. and Fusarium sporotrichioides.
The presence of compounds in freeze-dried fluids after cultures of Trichoderma citrinoviride HL strain caused complete inhibition of the growth of all strains of pathogenic fungi. A significant reduction in the specific growth rate of each strain was also observed in the presence of metabolites secreted by Trichoderma citrinoviride B3 and C1 (Table S4C, Figure 2).
Residual secondary metabolites remaining in TCA-treated culture fluids, and consequently following protein precipitation, led to a decrease in the specific growth rate of all tested strains, with the exception of E. nigrum. Among them, metabolites biosynthesized by T. citrinoviride HL, which completely inhibited the growth of Fusarium sp., were associated with the strongest reduction in this parameter. The specific growth rate of pathogen strains in the presence of metabolites from the organic phase of both raw and freeze-dried culture fluids showed variability. Compounds extracted with hexane from freeze-dried culture fluids of T. citrinoviride HL and B3 strains generally reduced the specific growth rate of pathogen strains more effectively than those obtained from crude liquid extracts (Table S4D, Figure 2).
Extracellular metabolites of T. citrinoviride HL, present in hexane extracts from crude culture fluids, reduced the specific growth rate of A. alternata, P. expansum, R. nigricans, Penicillium sp., M. hiemalis, R. stolonifer, A. terreus, Fusarium sp., and M. fructigena, while in other strains they exerted the opposite effect. Metabolites from the B3 culture fluid displayed similar effects, except in B. cinerea, where they reduced the specific growth rate. Similarly, metabolites from the C1 culture fluid produced comparable effects, although they lowered this parameter in B. cinerea, F. culmorum, and S. brevicaulis (Table S4E, Figure 2).
Notably, compounds present in hexane extracts of freeze-dried culture fluids from T. citrinoviride HL completely inhibited the growth of E. nigrum and reduced the specific growth rate of all other pathogens except F. culmorum. In the case of T. citrinoviride B3, these extracts increased the specific growth rate of E. nigrum, F. sporotrichioides, F. culmorum, and Penicillium janthinellum, while reducing it in the remaining strains; in addition, they completely inhibited the growth of S. brevicaulis. Conversely, metabolites of T. citrinoviride C1 increased the specific growth rate of P. expansum, E. nigrum, Fusarium sp., P. janthinellum, and F. sporotrichioides (Figure 2).
The SPME and GC/MS spectrometry were selected, as it allows for the detailed analysis of volatile compounds synthesized by the Trichoderma strains, including the detection of compounds with confirmed toxic activity and especially antimicrobial activity. The detected compounds were 1-methyl-3 phenylindole, 2(4 methylphenyl)-indolysine, 5-methyl-2-phenylindolysine, bis(2-ethylhexyl) hexanedioic acid ester.

3. Discussion

Soil-borne pathogens cause significant losses in both natural and productive ecosystems. Fungi, which constitute the most numerous group of plant pathogens, are responsible for destroying up to a third of all crops annually [2,16]. Conventional plant protection techniques primarily rely on synthetic pesticides to manage these undesirable microorganisms. However, the chemical control of plant diseases poses numerous environmental risks, including soil and groundwater contamination, and endangers the health of farmers, consumers, and nearby communities. Pesticides also exert detrimental effects on non-target organisms, such as beneficial insects. Furthermore, the development of resistance in the targeted phytopathogens is a significant concern [2,21,22].
Among the filamentous fungi studied, species within the genus Trichoderma are particularly noteworthy [2]. Martínez-Medina et al. [23] demonstrated that the endophytic fungus T. harzianum T-78 modulates plant interactions with the root-knot nematode Meloidogyne incognita throughout the infection cycle, enhancing specific host defenses. A study by Khan et al. [24] indicated that soil application of T. harzianum or T. viride can be an effective agent for controlling root nematodes in tomato, particularly the K-21 cultivar. Similarly, Abbas et al. [25] and Asad et al. [26] demonstrated that Trichoderma spp. plays a key role as a biocontrol agent against diseases caused by Rhizoctonia solani. Daryaei et al. [27] showed that T. atroviride LU132 could colonize ryegrass root systems, increase plant dry matter, and protect them from R. solani.
Błaszczyk et al. [28] identified T. atroviride AN240 as an effective biological control agent against toxicogenic Fusarium species. This strain acts by reducing Fusarium and preventing mycotoxin accumulation in plant tissues. Schöneberg et al. [29] investigated the potential of antagonistic fungi to control F. graminearum and F. crookwellense. Results indicated that T. gamsii, T. koningiopsis, and T. viride exhibited strong inhibitory potential and were fast-growing antagonists against these pathogens. Zhang et al. [30] isolated a T. harzianum strain, designated T-soybean, and demonstrated production of substances that stimulate root growth and development. The strain also exhibited significant antagonism against F. oxysporum.
Gezgin et al. [31] demonstrated high efficacy of T. atroviride against R. solani, as well as strong antagonistic effects of T. citrinoviride against F. oxysporum. Zhao et al. [32] found that T. citrinoviride strains of varying virulence are significant in the biological control of nematode diseases and that their modes of action may differ. Fan et al. [33] showed that T. citrinoviride Snef 1910 significantly inhibited egg hatching and exhibited virulence against Meloidogyne incognita. This strain also demonstrated significant in vitro antagonistic activity against other pathogens that cause diseases in wheat, cotton, melon, and other plants. Chen et al. [34] investigated the efficacy of T. citrinoviride HT-1, isolated from Rheum palmatum root, for root rot control and its mechanisms of induced systemic resistance. They found that the inhibition rate of F. oxysporum was 71.85% in dual culture. Metabolites from the T. citrinoviride HT-1 strain inhibited F. oxysporum mycelial growth by 79.07%. Furthermore, the activity of defense-related enzymes in the plants was significantly increased.
Surface tension is a significant factor in microbial interactions. According to Aparna et al. [35], effective biosurfactant producers lower the surface tension to 30 mN/m or less. Therefore, the results from the present study can be considered significant, especially when compared to values reported for other microorganisms. For instance, Meneses et al. [36] investigated the ability of Aureobasidium thailandense LB01 to produce biosurfactants on waste materials, such as wastewater from olive oil mills. After partial purification, these biosurfactants were able to reduce surface tension to 31.2 mN/m. In comparison, high-strength bacterial biosurfactants are capable of reducing surface tension to 22–25 mN/m [37].
To our knowledge, no reports were found investigating how metabolite interactions vary depending on the method used to prepare the metabolite extracts. It is therefore important to conclude that metabolites present in the rehydrated post-culture fluids of Trichoderma citrinoviride strains HL, B3, and C1 exhibit strong antifungal activity. Notably, metabolites from the HL strain in this variant completely inhibited the growth of all tested pathogen strains.
Metabolites biosynthesized by T. citrinoviride strains HL, B3, and C1, when present in either the rehydrated freeze-dried culture fluid or the organic phase, limited the growth of Epicoccum nigrum. Conversely, metabolites in other variants of the culture fluid stimulated the growth of this fungus. The supernatant remaining after precipitation of proteinaceous compounds from T. citrinoviride post-culture fluids reduced the specific growth rate of most pathogens but did not delay the spore germination. Metabolites that partitioned into the organic phase from freeze-dried culture fluids showed greater inhibitory properties against pathogens compared to metabolites extracted into the organic phase from raw (unprocessed) culture fluids.
According to literature data, Trichoderma fungi release multiple volatile substances into the environment during growth, which play an important role in antibiosis interactions, inhibiting the growth of competitors [38] and contributing to the induction of plant resistance against phytopathogens [39,40,41]. These compounds belong to numerous classes, including: monoterpenes, sesquiterpenes, lactones, alcohols, ketones, esters [42,43], hydrocarbons, aldehydes, phenols, thioalcohols, thioesters and their derivatives [44]. The most thoroughly characterized volatile metabolite synthesized by Trichoderma is 6-pentyl-α-pyrone (6PAP), of which biological activity has been well documented against fungi of the genus Chaetomium, Curvularia, Aspergillus, Fusarium, Rhizoctonia, Pseudocercosporella, Botrytis, Pythium, Pyrenochaeta, and Verticillium [45,46,47]. In turn, Stracuadanio et al. [48] confirmed a high proportion of phenylethyl alcohol and 3-methyl-1-butanol and a high proportion of tetramethyl pyrazine in cultures of T. asperellum and T. atroviride.
The presence of 1-methyl-3 phenylindole was observed in T. citrinoviride HL culture. This compound, which belongs to the group of 3-phenylindoles, has been studied as a growth-limiting factor of such fungi as Botrytis allii, Penicillium italicum, Aspergillus niger and Cladosporium cucumerinum [49]. Moreover, it exhibits notable antibacterial activity [50,51]. Further compounds detected in the T. citrinoviride HL culture were 8-chloro-5-quinolinecarboxylic acid, 2-chloro-4-quinolinecarboxylic acid and 3-acetonitrile-pyrrol-2,3-quinoline. According to literature data, these compounds, being derivatives of quinoline-carboxylic acid and quinoline, have antimicrobial activity, which was confirmed by Kumada and Neu [52], as well as Eswaran et al. [53]. In addition, the analysis of volatile compounds in T. citrinoviride C1 culture also showed the presence of quinoline derivatives; however, a low content of 2(4 methylphenyl)-indolysine and 5-methyl-2-phenylindolysine, both being derivatives of indolysine, that could exhibit antifungal and antibacterial activity [54,55].

4. Materials and Methods

4.1. Microorganisms

The research material consisted of three strains of Trichoderma citrinoviride designated C1, B3 and HL, and 16 phytopathogenic fungal strains: A. Penicillium expansum, B. Alternaria alternata IOR 1526, C. Rhizopus nigricans, D. Penicillium sp., E. Botrytis cinerea, F. Mucor hiemalis, G. Rhizopus stolonifer CBS 347.69, H. Epicoccum nigrum DMK1, I. Aspergillus terreus DSM 1958, J. Fusarium poae, K. Fusarium sporotrichioides KF 196, L. Fusarium culmorum, M. Monilinia fructigena IOR 2139, N. Scopulariopsis brevicaulis, O. Penicillium janthinellum, and P. Fusarium sp. All strains originated from the culture collection of the Department of Biotechnology and Food Microbiology, Wrocław University of Environmental and Life Sciences.

4.2. Culture Conditions

Trichoderma citrinoviride strains were cultivated in 250 mL Erlenmeyer flasks containing 50 mL of Mineral-Glucose-Peptone (MGP) medium supplemented with yeast biomass (6 g/L) and fungal biomass (10 g/L), serving as both inducers and carbon sources [56]. Cultures were incubated for 6 days at 25 °C with shaking at 160 rpm. Pathogenic strains were cultured under the same conditions in 50 mL of Potato Dextrose Broth (PDB) supplemented with 1% Malt Extract Broth (MEB) for 7 days. Subsequently, they were transferred to Potato Dextrose Agar (PDA) plates supplemented with 1% glucose and incubated for an additional 7 days.

4.3. Surface Tension Measurement

Post-culture fluids of T. citrinoviride were centrifuged at 5000 rpm for 6 min at 6 °C using an Centrifuge 5430 R (Eppendorf, Hamburg, Germany). The supernatants were filtered through 0.22 μm syringe filters. Surface tension was measured immediately using the ring detachment method with the K6 force tensiometer (Krüss, Europe, Hamburg, Germany), at room temperature [57]. Deionized water and uninoculated medium served as controls.

4.4. Enzymatic Activity Assays

Enzymatic activity of post-culture fluids (culture 1: 19–21 °C (H1); culture 2: 22–23 °C (H2); culture 3: 24–25 °C) was determined for chitinases, laminarinases, and lichenases using the 3,5-dinitrosalicylic acid (DNS) method [9]. The substrates were 4% chitin suspension, 0.5% aqueous laminarin solution, and 0.5% aqueous lichenan solution, respectively.

4.5. Determination of Volatile Compounds

Determination of volatile compounds was performed with SPME (Solid Phase Microextraction) and GC/MS (Gas chromatograph with mass detector model: 7890A/5975MSD with software OL II (Agilent, Santa Clara, CA, USA)).
Flask cultures were carried out in medium, from which samples were withdrawn on days 2, 4, and 6, and further analyzed by SPME and GC-MS. This method is widely used in the determination of fungal volatile compounds due to its accuracy and sensitivity [58,59,60], which was used by scientists in the study of VOCs produced by molds of the genera Aspergillus, Fusarium, Penicilium and Trichoderma [20,60].
Volatiles were extracted from samples for 3 min by microextraction to the solid phase. The stationary phase of the fiber was vinylbenzene/carboxen/poly(dimethylsiloxane) (DVB/CAR/PDMS) with a length of 2 cm and 50/30 μm diameter. For thermal desorption of the analyzed compounds, the microsyringe fiber was exposed for 3 min to 250 °C in a gas chromatograph dispenser (with a mass detector, at the following conditions: HP—88 capillary column (Agilent, CA, USA) with high polarity 100 m · 250 μm · 0.25 μm ((88%—cyanoproplyl)aryl-polysiloxane). The temperature program was as follows: 40 °C (5 min.) to 240 °C (4 °C/min.), carrier gas: helium (20 cm3/s), split 1:1. The substance identification was performed on the basis of comparative analysis of mass spectra with the commercial NIST spectra library.

4.6. Antagonistic Test

Trichoderma citrinoviride HL, B3 and C1 strains and pathogenic fungal strains were grown on PDA slant and incubated at 25 °C for 7 days. With 5 mL of sterile H2O, the biomass was washed off the slants. The resulting spore suspension was standardized to a density of 107 in the thoma chamber. In the plates containing the PDA substrate, two wells with a diameter of 10 mm were cut with a sterile cork borer, at a distance of 2 cm from each other. One was filled with 100 μL of a suspension of an appropriate pathogenic strain, and the other with 100 μL of an appropriate Trichoderma citrinoviride strain. The plates were incubated for 7 days at 25 °C. The test was performed in two repetitions. After incubation, the effect of Trichoderma citrinoviride fungi on pathogens growth dynamics was determined according to the scale: The colonies of the test strain T and test strain X contact along a straight line, The colony of strain T contacts the colony of strain X along the curve line, surrounding less than 1/3 of the colony of strain X, The colony of strain T contacts the colony of strain X along the line of the curve, surrounding at least 1/3, but less than 1/2 of the X strain colony, The T strain colony contacts the X strain colony along the curve line, surrounding at least 1/2 but less than 2/3 of the X strain colony, The T strain colony contacts the X strain colony along the curve line, surrounding at least 2/3 and more of the X strain colony.

4.7. Microculture Test

The turbidimetric assay was performed using a microplate reader (TECAN Spark, Swizerland), which measured microbial growth by recording optical density (OD) at 30 min intervals. Data were processed with SparkControl software V2.3 (TECAN Spark, Männedorf, Swizerland), and each variant of culture fluid was tested in five replicates. The following preparations were analyzed: crude culture fluid, freeze-dried culture fluid, trichloroacetic acid (TCA) precipitate, hexane extract of crude culture fluid, and hexane extract of freeze-dried culture fluid.
Freeze-dried products were obtained by lyophilizing 10 mL of post-culture liquid in the LABCONCO (Kansas City, MO, USA) freeze dryer. The dried material was stored at −18 °C and reconstituted in 10 mL of sterile Potato Dextrose Broth (PDB) before analysis. TCA precipitates were prepared by mixing 20 mL of crude culture fluid with 5 mL of 30% TCA (final concentration 6%) and incubating the mixture at 4 °C for 24 h. The precipitate was collected after centrifugation (2 × 7 min, 8000 rpm), the supernatant was discarded, and the pellet was stored at −18 °C until use. Before testing, the pellet was resuspended in 10 mL of PDB. Hexane extracts were prepared by mixing 20 mL of crude culture fluid with 20 mL of hexane in an Erlenmeyer flask and shaking the mixture for 30 min at 160 rpm. The upper hexane fraction (15 mL) was collected, evaporated to dryness, and the residue was stored at −18 °C, then dissolved in 10 mL of PDB prior to analysis. Extracts of freeze-dried products were prepared in the same way, using pooled lyophilized material from cultures 1 and 2.
For the assay, 200 μL of each preparation was combined with 50 μL of a suspension of pathogenic fungal spores (prepared as in the standard biotic test) in the wells of a 96-well microplate. The control consisted of 200 μL of PDB and 50 μL of the spore suspension. Plates were incubated at 25 °C with continuous shaking at 162 rpm. Growth curves were generated from OD measurements, from which the duration of the lag phase was taken, and the maximum specific growth rate (μmax) was calculated according to the equation:
μ = l n   O D max − l n   O D min t max − t min
where
ODmax—the highest value of the optical density in the logarithmic growth phase;
ODmin—the lowest value of the optical density in the logarithmic growth phase;
tmax—time corresponding to ODmax;
tmin—time corresponding to ODmin.

4.8. Statistical Analysis and Data Visualization

Experimental data for growth kinetics (maximum growth rate μmax and lag phase) were analyzed using Python v3.11, including pandas, matplotlib, and scipy.stats libraries.
For each strain and treatment variant, mean values were calculated and compared to the corresponding pathogen-specific control group. The resulting differences (Δ values) were visualized as heatmaps. Statistical significance between each treatment and its control was evaluated using Welch’s two-tailed t-test (for unequal variance assumption). Significance levels were indicated directly on the heatmaps as: * p < 0.05, ** p < 0.01, *** p < 0.001.
All heatmaps were generated using a diverging, non-linear color normalization centered at Δ = 0 (TwoSlopeNorm function in matplotlib) to enhance contrast near zero while compressing extreme values.

5. Conclusions

Soil-borne phytopathogens, particularly fungi, are a primary cause of significant crop losses worldwide. The conventional reliance on synthetic pesticides to manage these pathogens poses substantial environmental risks and leads to the development of pathogen resistance. To counteract these drawbacks, biological control, utilizing antagonistic microorganisms, represents a promising and sustainable alternative. Among these, species of the genus Trichoderma are widely recognized for their biocontrol capabilities. This study investigated the inhibitory effects of metabolites produced by three T. citrinoviride strains against various phytopathogenic fungi. The findings indicated that metabolites from these strains, particularly strain HL, exhibit strong antifungal activity, completely inhibiting the growth of several tested pathogens. The study also demonstrates that the preparation method of the post-culture fluid (e.g., rehydrated, freeze-dried, or organic phase extraction) significantly influences the antagonistic efficacy of the metabolites. These results underscore the wide spectrum of T. citrinoviride metabolites’ potential as effective biocontrol agents for the management of fungal plant diseases.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules31050827/s1, Figure S1: Enzymatic activity of laminarinases under culture 1 (H1), culture 2 (H2) and culture 3 (H3) of Trichoderma citrinoviride HL, B3 and C1 strains; Figure S2: Enzymatic activity of lichenases under culture 1 (H1), culture 2 (H2) and culture 3 (H3) of Trichoderma citrinoviride HL, B3 and C1 strains; Table S2: The degree of Trichoderma citrinoviride HL, B3 and C1 strains competitiveness against pathogenic fungal strains in basic antagonistic test; Table S3A: Duration of the lag phase of pathogen strains depending on the substrate in turbidimetric test; Table S3B: Duration of lag phase of pathogen strains in the presence of crude fluid of Trichoderma citrinoviride strains in turbidimetric test; Table S3C: Duration of the lag phase of pathogen strains in the presence of lyophilized lean fluid of Trichoderma citrinoviride strains in turbidimetric test; No growth at all (–); Table S3D: Duration of lag phase of pathogen strains in the presence of the preparation after precipitation of the leash fluid of Trichoderma citrinoviride strains with TCA in turbidimetric test; No growth at all (–); Table S3E: Duration of the lag phase of pathogen strains in the presence of crude fluid extract and from lyophilized relaxant fluid of Trichoderma citrinoviride strains using hexane in turbidimetric test; No growth at all (–); Table S4A: Maximum specific growth rate of pathogen strains depending on the substrate in turbidimetric test; Table S4B: Maximum specific growth rate of pathogen strains in the presence of crude lean fluid of Trichoderma citrinoviride strains in turbidimetric test; Table S4C: Maximum specific growth rate of pathogen strains in the presence of freezedried leaching fluid of Trichoderma citrinoviride strains in turbidimetric test; No growth at all (–); Table S4D: Maximum specific growth rate of pathogen strains in the presence of the preparation after precipitation of the leaching fluid of Trichoderma citrinoviride strains with TCA in turbidimetric test; No growth at all (–); Table S4E: Maximum specific growth rate of pathogen strains in the presence of raw fluid extract and from lyophilized pohoda fluid of Trichoderma citrinoviride strains using hexane in turbidimetric test; No growth at all (–).

Author Contributions

Conceptualization, M.P.; methodology, M.P.; software, M.P.; validation, M.P.; formal analysis, M.P. and A.K.; investigation, M.P. and A.K.; visualization, M.P.; writing, review and editing, M.P. and W.Ł.; statistical analysis, W.Ł.; supervision, W.Ł. All authors have read and agreed to the published version of the manuscript.

Funding

The APC/BPC is financed/co-financed by Wrocław University of Environmental and Life Sciences.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data and materials that support the findings of this study are available from the corresponding author on request.

Conflicts of Interest

The authors declare no conflicts of interest.

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Molecules 31 00827 g001aMolecules 31 00827 g001b
Figure 1. Heatmap illustrating the relative differences (Δ) in lag phase duration between experimental strains and pathogen-specific controls across all tested variants. Cells represent mean Δ values (strain–control), and color intensity indicates the magnitude and direction of the change. Statistical significance of differences was assessed using Welch’s two-tailed t-test, with significance levels denoted as: * p < 0.05, ** p < 0.01, *** p < 0.001. A. Penicillium expansum, B. Alternaria alternata IOR 1526, C. Rhizopus nigricans, D. Penicillium sp., E. Botrytis cinerea, F. Mucor hiemalis, G. Rhizopus stolonifer CBS 347.69, H. Epicoccum nigrum DMK1, I. Aspergillus terreus DSM 1958, J. Fusarium poae, K. Fusarium sporotrichioides KF 196, L. Fusarium culmorum, M. Monilinia fructigena IOR 2139, N. Scopulariopsis brevicaulis, O. Penicillium janthinellum, and P. Fusarium sp.
Figure 1. Heatmap illustrating the relative differences (Δ) in lag phase duration between experimental strains and pathogen-specific controls across all tested variants. Cells represent mean Δ values (strain–control), and color intensity indicates the magnitude and direction of the change. Statistical significance of differences was assessed using Welch’s two-tailed t-test, with significance levels denoted as: * p < 0.05, ** p < 0.01, *** p < 0.001. A. Penicillium expansum, B. Alternaria alternata IOR 1526, C. Rhizopus nigricans, D. Penicillium sp., E. Botrytis cinerea, F. Mucor hiemalis, G. Rhizopus stolonifer CBS 347.69, H. Epicoccum nigrum DMK1, I. Aspergillus terreus DSM 1958, J. Fusarium poae, K. Fusarium sporotrichioides KF 196, L. Fusarium culmorum, M. Monilinia fructigena IOR 2139, N. Scopulariopsis brevicaulis, O. Penicillium janthinellum, and P. Fusarium sp.
Molecules 31 00827 g001aMolecules 31 00827 g001b
Molecules 31 00827 g002aMolecules 31 00827 g002b
Figure 2. Heatmap depicting the relative changes (Δ) in maximum growth rate between experimental strains and pathogen-specific controls across tested variants. Each cell represents the mean difference (strain–control), with color intensity corresponding to the magnitude and direction of Δ. Statistical significance of differences was assessed using Welch’s two-tailed t-test, with significance levels denoted as: * p < 0.05, ** p < 0.01, *** p < 0.001. A. Penicillium expansum, B. Alternaria alternata IOR 1526, C. Rhizopus nigricans, D. Penicillium sp., E. Botrytis cinerea, F. Mucor hiemalis, G. Rhizopus stolonifer CBS 347.69, H. Epicoccum nigrum DMK1, I. Aspergillus terreus DSM 1958, J. Fusarium poae, K. Fusarium sporotrichioides KF 196, L. Fusarium culmorum, M. Monilinia fructigena IOR 2139, N. Scopulariopsis brevicaulis, O. Penicillium janthinellum, and P. Fusarium sp.
Figure 2. Heatmap depicting the relative changes (Δ) in maximum growth rate between experimental strains and pathogen-specific controls across tested variants. Each cell represents the mean difference (strain–control), with color intensity corresponding to the magnitude and direction of Δ. Statistical significance of differences was assessed using Welch’s two-tailed t-test, with significance levels denoted as: * p < 0.05, ** p < 0.01, *** p < 0.001. A. Penicillium expansum, B. Alternaria alternata IOR 1526, C. Rhizopus nigricans, D. Penicillium sp., E. Botrytis cinerea, F. Mucor hiemalis, G. Rhizopus stolonifer CBS 347.69, H. Epicoccum nigrum DMK1, I. Aspergillus terreus DSM 1958, J. Fusarium poae, K. Fusarium sporotrichioides KF 196, L. Fusarium culmorum, M. Monilinia fructigena IOR 2139, N. Scopulariopsis brevicaulis, O. Penicillium janthinellum, and P. Fusarium sp.
Molecules 31 00827 g002aMolecules 31 00827 g002b
Table 1. Measurements of surface tension (mN/m) in raw fluid of Trichoderma citrinoviride HL, B3 and C1 strains.
Table 1. Measurements of surface tension (mN/m) in raw fluid of Trichoderma citrinoviride HL, B3 and C1 strains.
Trichoderma citrinovirideSurface Tension [mN/m]
HL35
B336
C134
H2O68
edium64
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Piegza, M.; Kaliciak, A.; Łaba, W. Inhibitory Effect of Trichoderma citrinoviride Secondary Metabolites on the Growth Kinetics and Spore Germination of Fungal Phytopathogens. Molecules 2026, 31, 827. https://doi.org/10.3390/molecules31050827

AMA Style

Piegza M, Kaliciak A, Łaba W. Inhibitory Effect of Trichoderma citrinoviride Secondary Metabolites on the Growth Kinetics and Spore Germination of Fungal Phytopathogens. Molecules. 2026; 31(5):827. https://doi.org/10.3390/molecules31050827

Chicago/Turabian Style

Piegza, Michał, Aleksandra Kaliciak, and Wojciech Łaba. 2026. "Inhibitory Effect of Trichoderma citrinoviride Secondary Metabolites on the Growth Kinetics and Spore Germination of Fungal Phytopathogens" Molecules 31, no. 5: 827. https://doi.org/10.3390/molecules31050827

APA Style

Piegza, M., Kaliciak, A., & Łaba, W. (2026). Inhibitory Effect of Trichoderma citrinoviride Secondary Metabolites on the Growth Kinetics and Spore Germination of Fungal Phytopathogens. Molecules, 31(5), 827. https://doi.org/10.3390/molecules31050827

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