Broad Spectrum Antagonistic Activity of Streptomyces sp. CACIS-1.16CA Against Phytopathogenic Fungi

Abstract

The most common reason for a decrease in the quantity and quality of produced crops is microbial diseases. The aims of this study were to evaluate the antagonistic activity of Streptomyces sp. CACIS-1.16CA against plant pathogenic fungi and to assess its bioactive metabolites to inhibit fungal conidial germination. Antagonistic evaluations of fungal phytopathogens were performed using dual and multiple confrontation assays. Additionally, the inhibitory effect of the bioactive extract (BE) containing secondary metabolites produced by the CACIS-1.16CA strain on the germination of conidia from some fungi was tested. The results indicate that Streptomyces sp. CACIS-16CA inhibited the growth of all tested pathogens (16 strains) with percentages of inhibition (PIs) ranging from 43.3% to 72%, while S. lydicus inhibited 13 of the 16 fungi, with PI values from 35.6% to 68.5%. Moreover, CACIS-1.16CA exerted superior PI values (significant differences at p < 0.05) than S. lydicus against the damping-off fungi consortia with Phytophthora capsici, Fusarium oxysporum, and Rhizoctonia solani. Otherwise, an inhibitory effect was observed on the germination of conidial cells due to the interaction with the BE in Alternaria sp., Botrytis cinerea, and Colletotrichum spp. In conclusion, Streptomyces sp. CACIS-1.16CA may serve as an effective and natural alternative for managing several fungal plant diseases.

1. Introduction

Streptomycetes (Streptomycetaceae) are a group of microorganisms that has been considered a major producer of antibiotics and other metabolites with biological activities [1,2]. With more than 600 species, Streptomyces is the most studied genus in this family of bacteria. It produces more than half of all known antibiotics, compounds with antifungal properties, a variety of hydrolytic enzymes (chitinases, cellulases, glucanases, and proteinases), and other metabolites that prevent the growth of plant pathogenic microorganisms [3]. Plant growth regulators, siderophores, antivirals, insecticides, pesticides, and herbicides are among the bioactive chemicals produced by Streptomyces sp. [4].

Plant diseases induced by pathogenic microorganisms affect plant health and pose a serious obstacle to the production of food [5]. Chemical fungicides have been used for years to regulate and control fungal diseases. However, the uncontrolled use of these chemical fungicides causes serious damage to the wellbeing of humans, animals, and the environment, and has led to the development of resistant strains of pathogenic microorganisms [6]. Beneficial microorganisms are considered biological control agents (BCAs) when they comprise phytopathogenic microorganisms, since they contribute to the suppression of several fungal plant pathogens. Additionally, compared with chemical fungicides, BCAs can create complex relationships with soil microorganisms, adapt to the soil and environment, and increase their efficacy and durability [7].

The rhizospheric microbial antagonists that have been used as effective BCAs include species of genera Bacillus, Pseudomonas, Streptomyces, Trichoderma, and non-pathogenic Fusarium [8,9]. Actinomycete-based biological control products have been developed to inhibit the growth of plant pathogens in agricultural crops. Commercial products comprising Streptomyces species have been used to control the foliar and root diseases of various crops: Actinovate^® comprises S. lydicus WYEC108 spores, while Mycostop^® comprises spores of S. griseoviridis strain K61 [10,11]. Numerous studies have shown that non-commercial Streptomyces sp. can inhibit the growth of fungi. For example, Streptomyces hydrogenans DH16 inhibited Alternaria brassicicola mycelial growth, which is the cause of black leaf spot and damping-off in Raphanus sativus seedlings, and its antifungal metabolites, which were produced in the culture supernatant. In vivo studies showed the efficacy of streptomycete cells and culture supernatants as a seed dressing to control the damping-off of Raphanus sativus seedlings [12]. Additionally, the collar rot infestation caused by Sclerotium rolfsii in chickpeas was reduced by endophytic Streptomyces sp. obtained from medicinal plants [13]. S. corchorusii strain AUH-1 has also been reported to exert antagonistic activity against F. oxysporum f. sp. Niveum; to damage the structure and function of mycelial cell membranes; and to exert antagonistic effects against other fungal pathogens such as Phytophthora parasitica var. nicotianae, P. capsici, Rhizoctonia solani, Botryosphaeria dothidea, F. oxysporum f. sp. vasinfectum, Verticillium dahliae, and F. oxysporum f. sp. cucumerinum [14]. Streptomyces sp. CACIS-2.15CA was recently found to inhibit the growth of a variety of postharvest pathogenic fungal strains isolated from fruits, including grape, mango, tomato, habanero pepper, papaya, sweet orange, and banana [15].

Streptomyces sp. CACIS-1.16CA produces a yellow diffusible metabolite that exhibits inhibitory activity on mycelium growth against Phytophthora capsici, Fusarium sp., Rhizoctonia sp., Curvularia sp., Aspergillus niger, Helminthosporium sp., Alternaria sp., and Colletotrichum sp. [16]. Recently, the antibacterial activity of a thermally stable cell-free supernatant produced by the bacterial strain Streptomyces sp. CACIS-1.16CA against bacterial pathogens in plants and humans was evaluated. The supernatant of this bacterial strain showed inhibitory activity against Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, Bacillus pumilus, and Pectobacterium carotovorum [17]. To suppress fungal diseases, antagonistic microorganisms or their byproducts have been used as natural alternatives for crop disease management. This study aims to evaluate the antagonistic activity of Streptomyces sp. CACIS-1.16CA against plant pathogenic fungi and to assess the inhibitory effect of its BE on the conidial germination of some fungal pathogens.

2. Materials and Methods

2.1. Fungi and Culture Conditions

The fungi used in this study were Bipolaris sp., Phomopsis sp., Corynespora casiicolla, Fusarium equisetti, F. oxysporum f. sp. lycopersici, Fusarium sp. CDBB 1172, Fusarium solani, Fusarium oxysporum, Phytophthora capsici, Rhizoctonia solani, Sclerotium sp., Colletotrichum siamense M1.2, Colletotrichum musae Cm6, Alternaria sp., Aspergillus sp., and Botrytis cinerea [15,18]. The strains were maintained on potato dextrose agar (PDA BD-Difco^TM) for 7–12 days at 29 °C. Fungal conidial suspensions (FCSs) were prepared from 12-day cultures inoculated on PDA, and the final concentration of the suspensions was adjusted to 1 × 10⁶ cells/mL (Oliveira et al. 2015) [19].

2.2. Growth Conditions of Streptomyces

The strains of Streptomyces were obtained from the Actinomycetes Germplasm Bank preserved at CIATEJ. Streptomyces sp. CACIS-1.16CA and Streptomyces lydicus WYEC108 were grown on International Streptomyces Project agar medium no. 2 (ISP 2) at 29 °C for 14 days [20]. For the antagonism experiment, a general inoculum (GI) of spores of each fungal strain was prepared with a concentration of 10⁸ CFU/mL [16]. S. lydicus WYEC108 was used as the reference strain.

2.3. Antagonistic Activity

The antagonistic activity of Streptomyces sp. CACIS-1.16CA was determined in two phases. Initially, one-to-one experiments were performed to focus on the antagonism against the phytopathogenic fungus using a dual confrontation method [16]. Thereafter, a multiple confrontation assay, modified from the square streak confrontation method, was performed to evaluate the antagonism of Streptomyces sp. CACIS-1.16CA on fungal consortia linked to the wilting of tomato and chili plants (Fusarium oxysporum, Phytophthora capsici, and Rhizoctonia solani) [21]. For dual confrontation experiments, 2 µL of the GI was added to Petri plates with ISP 2 agar medium, 1 cm away from the edge. A 6 mm diameter agar disk coated with fungal mycelium that had been growing for 10 days was then placed in the middle of the plates. These cultures were maintained at 29 °C until the fungal growth reached the edge of the control plates. To evaluate the antagonism over fungi consortia, 2 µL of the GI was deposited into the middle of Petri plates with ISP 2 media maintained at 29 °C and, after 5 days, a 0.6 cm diameter agar disk covered in fungal mycelium was placed 1 cm from the edge of the plates. All cultures were maintained at 29 °C until the growth of fungi reached the center of the control plates. Then, using a caliper, the growth of the fungal mycelium in the direction of the Streptomyces strain colony’s boundary was measured. The PI was measured using the following formula: PI (%) = [(RHC − RHS)/RHC × 100], where RHC represents the radius growth of the fungus in the control culture (mm), and RHS is the radius growth of the fungus in the direction of the streptomycete (mm).

2.4. Susceptibility Evaluation of Diffusible Secondary Metabolites from Streptomyces sp. CACIS-1.16CA on Fungal Mycelia Growth

The inhibitory effect of the secondary metabolites on fungal mycelia growth was evaluated via the agar disk diffusion method. Initially, 100 µL of the GI spore suspension from CACIS-1.16CA was spread across the Petri plates with ISP 2 medium with a Drigalski loop. These plates were then incubated at 29 °C for 14 days. Subsequently, the cultures were exposed to chloroform vapors for 30 min inside a sealed container to inactivate the growth of bacteria [22]. An agar disk (9 mm in diameter) that had been removed from the inactivated cultures were placed in the middle of the Petri plates with PDA agar media, and three agar blocks (6 mm in diameter) with actively developing mycelium of fungus were seeded 1 cm from the edge of the plate. The 9 mm agar disks obtained from the Petri plate containing the ISP2 medium were used as controls. All plates were incubated at 29 °C for 10 days. Assays were carried out in duplicate, and PI was determined as described previously.

2.5. Extraction of Secondary Metabolites and Preparation of the Bioactive Extract (BE)

The extraction of the metabolites was carried out according to the procedure described by Córdova-Dávalos et al. [23]. In brief, five Petri plates with ISP2 agar media were inoculated with 100 μL of the Streptomyces sp. CACIS 1.16CA GI and maintained for 15 days of incubation at 29 °C. Then, the agar medium was removed from each plate, crushed, and placed in a sterile bottle with 100 mL of methanol overnight at 4 °C, after which the compounds were extracted. This polar solvent with high solubility is effective for extracting various polar compounds, phenolic compounds, lipids, fatty acids, proteins, terpenoids, etc. [24]. The methanolic solution was filtered using Whatman no. 1 and centrifuged at 2500× g for 10 min. The bioactive extract (BE) was concentrated at 45 °C for 24 h. The precipitate obtained from the extract was dissolved in sterile distilled water, filtered with 0.22 µM filters, and stored at −20 °C until further use.

2.6. Inhibition of Conidial Germination by the BE

The inhibitory effect of the BE on conidial germination was implemented in sterile concave microscopic slides. The experimental assay consisted of a mixture with the BE at 15% of the final concentration, 2500 cells from FCS, and sterile distilled water to obtain a final volume of 30 μL. Then, 15 µL of pre-warmed (40 °C) PDA agar medium was added to the mixture and allowed to solidify for 5 min. The mixture was then covered with sterile coverslips and incubated at 29 °C for 12 hrs in a humid environment. The conidia were examined under a light microscope, and for each well, the number of germinated conidia was counted twice. The conidia were considered to have germinated if the germination tube reached a diameter twice that of the conidium. Utilizing at least two images taken with the microscope camera, conidium counting was carried out. One-hundred conidia were randomly selected, and the germinated conidia were separated from the non-germinated ones; then, the effect on germination was assessed. The results are expressed as the inhibition rate of conidia germination (IG) and were determined as follows: IG (%) = (Number of germinating spores/Total number of spores) × 100 [25]. The effects on conidia germination were documented on a Eclipse Ti Nikon inverted microscope (Nikon^®, Tokyo, Japan).

2.7. Scanning Electron Microscopy

An agar block of 14 × 14 mm with a 14-day active culture of CACIS-1.16CA from ISP2 media maintained at 29 °C was placed in 2% glutaraldehyde in a 0.2 M phosphate buffer (PBS, pH 7.2) and left for 48 h (24 h at 25 °C and 24 h at 4 °C). The next day, the agar pieces were washed three times with a fresh PBS buffer for 30 min, followed by a 60 min change in 30% ethanol, a 60 min change in 50% ethanol, and a 60 min change in 70% ethanol. The samples were analyzed with a scanning electronic microscope EVO^® 50 (Carl Zeiss AG, Oberkochen, Germany)at the Science Faculty of the Autonomous University of Querétaro, México.

2.8. Data Analysis

Each experiment in this study used a completely random design with three replicates, and the data were analyzed using the two-ways analysis of variance and Tukey’s test (p ≤ 0.05) to compare means. The Minitab 18 software was used for the analysis.

3. Results

3.1. Morphological Characterization of Streptomyces sp. CACIS-1.16CA

The phenotypic characterization of Streptomyces sp. CACIS-1.16CA has been reported previously [16]. Its colonies exhibit a powdery and dry morphology, and the vegetative mycelium changes into a fragmented white aerial hypha after 10–12 days of development. This hypha displays an abundance of branching straight-type hyphae grouped into chains with 10–20 spores, and it is doliform in shape with a smooth surface [26]. Yellow secondary metabolites are evidently produced, diffusing throughout the agar and accumulating as droplets on the surface (Figure 1).

3.2. Antagonistic Activity of Streptomyces sp. CACIS-1.16CA

The experimental evidence showed that Streptomyces sp. CACIS-1.16CA antagonizes mycelial growth for all fungal pathogens, with PIs ranging from 43% to 72%; the lowest value corresponds to Bipolaris sp., and the highest PI value matches C. siamense. In contrast, S. lydicus WYEC108 did not inhibit the mycelium growth of F. oxysporum f. sp. Lycopersici, F. oxysporum, and Aspergillus sp.; the reference strain significantly inhibited both Phytophthora capsici and Corynespora casiicola. In general, significant differences were observed for PI between Streptomyces sp. CACIS-1.16CA and S. lydicus WYEC108 (p < 0.5), wherein CACIS-1.16CA showed superior antagonism against all the fungal pathogens (

Table 1. Percentage of inhibition (PI) of fungal growth by Streptomyces sp. CACIS-1.16CA.

Fungi	Hosts	Streptomyces sp. CACIS-1.16CA A	S. lydicus WYEC 108 B
Bipolaris sp.	Habanero pepper	43.3 ± 1.7 a	42.2 ± 1.2 a
Phomopsis sp.	Jatropha curcas	57.6 ± 2.6 a	38.0 ± 0.7 b
Corynespora casiicolla	Jatropha curcas	60.2 ± 0.9 a	53.2 ± 0.4 b
Fusarium equisetti	Habanero pepper	51.5 ± 3.0 a	37.6 ± 1.6 b
F. oxysporum f. sp. lycopersici	Tomato	44.6 ± 2.5 a	5.9 ± 1.7 b
Fusarium sp. CDBB 1172 1	CDBB	52.9 ± 1.5 a	36.1 ± 1.5 b
Fusarium solani	Potato	46.8 ± 0.1 a	35.9 ± 0.7 b
Fusarium oxysporum	Agave	47.3 ± 1.1 a	1.9 ± 0.6 b
Phytophthora capsici	Serrano pepper	60.2 ± 3.6 b	68.5 ± 2.2 a
Rhizoctonia solani	Serrano pepper	46.8 ± 2.8 a	46.7 ± 0.8 a
Sclerotium sp.	Aloe	47.5 ± 2.8 a	34.8 ± 4.9 b
Colletotrichum siamense M1.2	Habanero pepper	72.0 ± 2.9 a	34.7 ± 2.8 b
Colletotrichum musae Cm6	Banana fruit	47.5 ± 1.7 a	41.4 ± 4.8 b
Alternaria sp.	Tomato	64.9 ± 5.6 a	49.9 ± 5.1 b
Aspergillus sp.	Sweet citrus	49.3 ± 2.4 a	4.7 ± 1.4 b
Botrytis cinerea	Tomato	44.4 ± 5.6 a	45.5 ± 1.9 a

¹ National Collection of Microbial Strains and Cell Cultures, CINVESTAV https://cdbb.cinvestav.mx/cdbb/index.html (accessed on 28 august 2025). Media values with the same letter showed non-statistical differences (p < 0.05). Uppercase letters indicate statistical differences between both Streptomyces species for the complete set of fungi; lowercase letters indicate statistical differences between both Streptomyces species for an individual fungus species.

). Representative antagonistic activity is shown in Figure 2; a difference is observed between the antagonisms of Streptomyces sp. CACIS-1.16CA and S. lydicus WYEC108.

An evaluation of the antagonistic activity against fungal pathogens associated with damping-off disease revealed an inhibitory effect on mycelia growth for the fungal consortia by Streptomyces sp. CACIS-1.16CA, highlighting an inhibition of mycelia growth at all fungi, with the highest antagonisms over P. capsici and F. oxysporum. Conversely, S. lydicus WYEC 108 did not inhibit the mycelia growth for F. oxysporum and R. solani (Figure 3a). The in vitro multiple confrontation assay revealed that both Streptomyces species inhibited the mycelial growth of P. capsici by at least 87% (non-statistical difference p > 0.05). Nevertheless, CACIS-1.16CA displayed higher inhibition of F. oxysporum and R. solani compared with that for WYEC108 strain, which was statistically significant (p < 0.05) (Figure 3b). Ultimately, a comparative analysis of the antagonism of strains indicates a higher inhibitory activity of CACIS-1.16CA compared with the reference strain WYEC108 against three fungal pathogens (p < 0.05), which strongly suggests that Streptomyces sp. CACIS-1.16CA could be considered a potential biocontrol agent to prevent and/or reduce damping-off disease in plants.

3.3. Antifungal Activity of Diffusible Compounds Produced by Streptomyces sp. CACIS-1.16CA

The effects of secondary metabolites produced by the CACIS-1.16CA strain on growth focused on the inhibition of mycelia growth and conidia germination. The inhibitory activity of antifungal metabolites accumulated in the agar of the culture media on mycelia growth was established via a simple diffusion assay, wherein the diffusible metabolites reduce the growth of Bipolaris sp., Alternaria sp., Corynespora casiicola, C. siamense, Fusarium sp., and F. oxysporum. The PI values showed that the fungus most susceptible to diffusible metabolites was C. siamense (Figure 4). This assay confirmed the antifungal action of diffusible compounds produced by Streptomyces sp. CACIS-1.16CA.

The exposure of conidia to 15% of the BE produced by Streptomyces sp. CACIS-1.16A was determined previously with an in vivo test to obtain the maximum concentration at which seeds of different vegetables maintain their viability, considering the potential of the BE to be used in fields. Thus, after exposure to the BE (15%), the IG percentages for conidia from Alternaria sp., B. cinerea, C. siamense, and C. musae were observed to be above 87%, which represents a lethal effect on cell viability (Figure 5a). Exposure to the BE affected the conidia, which includes lysis, disruption of the conidia’s transition phase toward the formation of the germination tube, a reduction in size, a disruption of the cell membrane, and restricted expansion of the hypha cells (Figure 5b).

4. Discussion

Streptomyces sp. CACIS-1.16CA has demonstrated a wide range of growth inhibition activities against fungi belonging to different soil-borne pathogens affecting roots, leaves, stems, flowers, and fruits, including P. capsici, Rhizoctonia sp., Fusarium sp., Colletotrichum sp., Alternaria sp., Aspergillus niger., Helminthosporium sp., and Curvularia sp. [16]. In addition, considering the results of the present study, the strain CACIS-1.16CA antagonizes the growth of other fungi species such as Phomopsis sp., Sclerotium sp., and Botrytis cinerea and inhibits the growth of novel species from the Fusarium, Aspergillus, Colletotrichum, and Rhizoctonia genera. The relevance of the CACIS-1.16CA strain as a potential biocontrol agent lies in the fact that some of its extracellular metabolites are thermostable and maintain antibacterial activity against Pectobacterium carotovorum, a broad host-range pathogen that causes soft rot of vegetables and ornamental plants worldwide [17,27].

The cultivation of chili (Capsicum annuum L.) is restricted by several fungal diseases, which have a significant impact on its yield globally. Wilting in plants has been found to be induced by a complex of soil phytopathogens, including Phytophthora capsici Leo, Rhizoctonia solani Kühn, and Fusarium oxysporum [28,29]. The consortia of these species added to Pythium spp. are the most frequent complex of pathogens associated with damping-off and, individually, are considered important causal agents of other plant diseases [30]. An evaluation of antagonistic activity against fungal pathogens associated with damping-off disease and comparison with the reference strain S. lydicus WYEC 108 revealed the potential of Streptomyces sp. CACIS-1.16CA to control wilting disease in the cultivation of chili plants. To our knowledge, this study is the first report to evaluate multiple antagonisms in a single agar plate with a Streptomyces strain against the causal agents of damping-off in chili plants.

The antifungal effects of diffusible compounds produced by Streptomyces sp. CACIS-1.16CA on mycelia growth in several fungal species were obtained. Diffusion of secondary metabolites from the disc of agar obtained from a culture of the CACIA 1.16CA strain suggests that the extracellular production of a mixture of potential antifungal compounds of polar nature inhibited mycelia growth. The presence of an extracellular chitinase enzyme and thermostable antifungal compounds has already been reported to be involved in the antifungal action on different phytopathogenic fungi. The culture filtrate of Streptomyces hygroscopicus SRA14 induced morphological changes such as hyphal swelling and abnormal shapes [31].

Methanolic extraction, as a preliminary strategy to detect antimicrobial compounds, was selected for its efficiency in extracting a wide range of polar compounds and in dissolving both lipophilic and hydrophilic molecules. A previous study showed that a diluted methanolic extract of Streptomyces amritsarensis V31 inhibited the growth of R. solani (23.3%), A. flavus (7.7%), F. oxysporum (22.2%), Sarocladium oryzae (16.7%), and Sclerotinia sclerotiorum (19.0%) [32]. Considering the above, the inhibitory compounds accumulated in the culture media during the growth of the bacteria Streptomyces sp. CACIS-1.16CA were extracted with methanol; a watered-down solution of the BE was prepared after methanol evaporation. Thus, the BE contained diverse polar compounds that exerted inhibitory activities on the germination of conidia from Alternaria sp., B. cinerea, C. siamense, and C. musae. The damaging effects on conidia integrity after exposure to the BE included deformation, shrinkage, collapse, and broken cells. Similar adverse effects were reported with a BE of Streptomyces sp. CB-75, which inhibited fungal spore germination and hyphal growth on F. oxysporum Race 4, revealing morphological alterations, including deformation, shrinkage, collapse, tortuosity, and broken cells [33]. Distinguishable changes were observed in membrane permeability, morphology, and presence of a wrinkled surface in conidia and hyphae of C. acutatum and F. oxysporum after exposure to extracellular compounds from Streptomyces blastmyceticus [34].

To further understand the antifungal mechanism of the secondary metabolites with antifungal activity produced by the CACIS-1.16CA strain and their effects on some cellular processes, the bioactive compounds will need to be identified and purified in detail. Potential targets in cells include (a) the cell wall, the disruption of which makes fungi vulnerable to rupture and death, (b) the cell membrane, the integrity of which is disrupted by the binding of some compounds (e.g., polyenes) to ergosterol, causing leakage of cellular contents, (c) the initiation of intracellular processes that affect microtubule formation, and (d) disruptions to cell division, nucleic acid synthesis, or the electron transport chain [35]. For instance, Streptomyces corchorusii AUH-1 could damage the cell membranes of pathogens by inhibiting ergosterol formation and increasing malondialdehyde levels [14].

The production of bioactive extracts derived from Streptomyces is a fundamental key to guide further studies focused on the potential application of these extracts. The extracts could be used to control post-harvest diseases on fruits and vegetables, protect bulbs or corm flowers in ornamental plants, and control pests (insects, worms, and spider mites).

5. Conclusions

Streptomyces sp. CACIS-1.16CA has been demonstrated to be a potential biological control agent (BCA) that inhibits diverse fungal plant pathogens. It was able to reduce the growth of a wilting fungal consortium including P. capsici, F. oxysporum, and R. solani. This strain may serve as an effective and natural alternative for managing several fungal plant diseases.

This bacterium produces secondary metabolites that exert inhibitory activities on fungal cells. Considering its morphological effects on cells, exploring metabolomic and omics approaches will be fundamental in efficiently enhancing the depth and mechanistic understanding of Streptomyces sp. CACIS-1.16CA.