2.1. Microbial Strains and Culture Condition

The fungal Mucor sp. MD7 was isolated and purified from diseased leaf samples collected in Da Lat, Lam Dong province, Vietnam [17]. The fungal strain was cultured at 25°C on potato dextrose agar (PDA) or potato dextrose broth (PDB), while actinomycete strains were cultured at 25°C in Gausse I medium for subsequent experiments.

2.2. Pathogenicity Test on Strawberry Leaves

Healthy strawberry leaves collected from Da Lat, Lam Dong province, Vietnam, underwent a thorough cleaning process, involving three gentle washes with sterile distilled water, surface sterilization with ethanol 70% for 2 minutes followed by five rinses with sterile distilled water, and air dried in a sterile condition. Subsequently, 20 µl of Mucor sp. MD7 culture, cultivated for five days PDB, was injected onto the leaf surface. The same procedure was performed for a control group using 20 µl of sterile PDB. The leaves were placed on moistened filter papers in sterilized Petri dishes at 25°C. Disease symptoms were observed daily and compared with those previously recorded [10].

2.3. In Vitro Screening of Actinomycete Antagonists

A total of 30 actinomycete strains systematically isolated from soil samples collected across multiple provinces in Vietnam, specifically Binh Thuan, Ninh Thuan, Tay Ninh, Tien Giang, Ben Tre, and Long An, were used in this study. These microbial strains were preserved in the microorganism culture collection maintained by the Institute of Biotechnology and Food Technology at the Industrial University of Ho Chi Minh City, Vietnam. To assess the antagonistic activity against Mucor sp. MD7, agar well diffusion method was employed. In brief, the actinomycete strains were cultured in Gause I medium at 37°C for 5 days. Subsequently, 0.1 ml of the culture was inoculated into the wells on PDA plates that had previously been inoculated with MD7. Antagonistic activity was quantified as: antifungal activity (%) = [(colony radius of the control − colony radius of the treatment)/colony radius of the control] × 100%.

2.4. Identification of the Antagonistic Actinomycete

The actinomycete strain with the strongest fungal inhibition was identified and performed through macroscopic and microscopic morphological characteristics, in addition to analysis of 16S rRNA gene region using the universal primer pair 27F-1540R. Homologous gene sequences were obtained from GenBank and a phylogenetic tree was constructed by Mega 11 software, utilizing the neighbor-joining method with 1000 bootstrap repetitions.

2.5. Extracellular Enzyme Assay of the Antagonistic Actinomycete

A 3-µl suspension of 5-day actinomycete culture was inoculated onto a modified Gause I plate (without soluble starch) containing the respective substrates colloidal chitin, casein, and carboxymethyl cellulose (CMC) for chitinase, protease, and cellulase activity. Enzymatic activity unit (AU) was assessed by calculating the ratio of the halo zone diameter to the colony diameter [14].

2.6. Effect of Actinomycete Culture Supernatant on the Fungal Mycelia

The antagonistic actinomycete was cultivated in Gause I broth for 5 days and the culture supernatant was obtained by centrifuging at 13,000 rpm for 10 minutes at 4°C. To prepare the fungal mycelia, the Mucor sp. MD7 pathogen was cultured in PDB for 5 days. Subsequently, the actinomycete culture supernatant was added to the Mucor sp. MD7 culture in equal volume followed by incubation at 25°C. A mixture in the same ratio of sterile Gausse I broth and Mucor sp. MD7 culture was used as a control. The impact on the mycelial morphology was evaluated after 24 hours of incubation under a light microscope.

2.7. Effect of Temperature and pH on Antifungal Activity of Actinomycete Culture Supernatant

To investigate the effect of temperature, 1.0 ml of the culture supernatant was subjected to different conditions 50–100 and 121°C for 30 minutes. To explore the influence of pH, 1.0 ml of the culture supernatant was adjusted to varying pH levels within the range of 3.0–12.0 using different buffers: citric acid/sodium citrate buffer (pH 3.0–5.0), phosphate buffer (pH 6.0–7.0), Tris amino methane/hydrochloric acid buffer (pH 8.0–9.0), sodium bicarbonate/sodium hydroxide buffer (pH 10.0), and sodium phosphate dibasic/sodium hydroxide buffer (pH 11.0–12.0). After adjustments, the samples were incubated for 2 hours at room temperature. Subsequently, 20 µl of the treated supernatant was loaded in the wells of PDA plates that had been inoculated with Mucor sp. MD7. Similar positive control experiments were conducted, with the negative control using nontreated culture supernatant. Thermal stability and pH stability were evaluated by determining the remaining mycelial growth inhibition percentage in each treatment relative to the control.

2.8. In Situ Test of Fungal Antagonistic Activity

Healthy young strawberry leaves, without any mechanical damage or signs of disease, were carefully collected based on their uniform color and size, from a planting farm in Da Lat. A rigorous sterilization process was employed, which involved two rounds of treatment with 70% ethanol, a 2-minute exposure to 2.0% sodium hypochlorite, followed by five times rinsing with sterilized distilled water.

In addition, suspensions of the fungal pathogen and the expected actinomycete were prepared as described in the previous section. Strawberry leaves were placed in sterilized Petri dishes and subjected to four conditions: spraying with 500 µl of sterilized mixture of PDB and Gause I in equal ratio (negative control), spraying with 500 µl of the actinomycete suspension (healthy control), spraying with 500 µl of mixture of the actinomycete suspension and the fungal pathogen suspension in equal ratio, and spraying with 500 µl of the fungal pathogen suspension (infected control). For each condition, five leaves were used as replicates. The treated leaves were then incubated for 3 days at 25°C ± 2°C with disease progression being monitored daily [10].

2.9. Data Analysis

All the experiment was done in triplicate except indicated elsewhere. The data were visualized using Microsoft Excel and ANOVA tests (α = 0.05) were done using Statgraphics Centurion 18 software (Statgraphics Technologies, Inc., USA).

3.1. Confirmation of the Strawberry Leaf Spot Pathogen Mucor sp. MD7

The fungal strain Mucor sp. MD7 was isolated from infected strawberry leaves originating from farms in Da Lat, Lam Dong province, Vietnam. Macroscopic and microscopic characteristics are depicted in Figure 1A. Following Koch’s postulates method, it was confirmed that Mucor sp. MD7 causes disease to healthy strawberry leaves after 6 days of infection with typical leaf spot symptoms. The leaf tissues of both sides became deep brown, exhibited necrosis, and developed disease manifestations rapidly during a 6-day incubation period at 25°C ± 2°C (Fig. 1C). In contrast, the control sample, injected with sterilized PDB, showed no disease symptoms (Fig. 1B).

The disease induced by Mucor sp. MD7 exhibits similarities with previously reported cases of other strawberry leaf spot fungal pathogens such as G. fragariae [18], E. rostratum [3], and C. cassiicola [4], suggesting a diversity of strawberry leaf pathogens. In addition to documented instances of Mucor sp. causing fruit rot, this marks the initial report of a Mucor sp. strain responsible for leaf spots in strawberries in Vietnam.

Figure 1. Strawberry leaf spot caused by Mucor sp. MD7. (A) Fungal pathogen with macromorphology (a) and micromorphology (b, c). (B) Healthy and (C) infected leaf with upper side (a) and lower side (b). DAI: day after infection. Scale bar = 50 µm. [Click here to view]

3.2. In Vitro Screening of Antifungal Actinomycetes

The findings revealed that 18 out of 30 actinomycetes strains exhibited antifungal activity against Mucor sp. MD7, causing varied mycelial inhibition ranging from 4.55% to 36.4% (Fig. 2A). Notably, the strain CNXK31.2 demonstrated significant antifungal activity with the highest inhibition rate of 36.4% (Fig. 2B). This achieved fungal inhibition percentage surpasses the inhibitory rates of 24 yeast strains against B. cinerea on strawberry fruit, as determined by the agar diffusion method [14]. In addition, it is on par with the inhibitory effect on C. sojina growth observed in Streptomyces sp. MM140, as determined by the dual assay method [19]. Given robust and consistent mycelial inhibition activity against Mucor sp. MD7, CNXK31.2 was chosen for further investigation.

3.3. Identification of Antagonistic Actinomycete CNXK 31.2

The identification of actinomycete strain CNXK31.2 involved an examination of its microscopic, macroscopic, and 16S rRNA characteristics. After 5 days of cultivation on Gause I medium at 37°C, colonies were round with a diameter of 3–4 mm, brownish-gray color, regular edges, raised centers, and numerous small gaps on the surface along with reddish-brown pigment production (Fig. 3A-a). Microscopic observation revealed branching mycelia and spore-bearing aerial hyphae. The retinaculiaperti spore chain was spiraled, short, and cleaved into conidia as it matured (Fig. 3A-b).

The 16S rRNA sequence, consisting of 1,306 bp, was amplified and sequenced. The BLAST results revealed that CNXK31.2 exhibited a high sequence identity with S. fungicidicus NBRC 13848 and S. fungicidicus A1 (100%). Furthermore, the phylogenetic tree analysis positioned CNXK31.2 in the genus Streptomyces and in the same clade as S. fungicidicus NBRC 13848 with 71% similarity (Fig. 3B). Streptomyces fungicidicus strains have been previously reported to produce an antibiotic, enduracidin, which acts against methicillin-resistant Staphylococcus aureus [20]. It is worth noting that S. fungicidicus was also found to exhibit biocontrol effects on pathogenic fungi such as Alternaria solani and Sclerotium rolfsii [21,22]. Thus, actinomycete strain CNXK31.2 was named Streptomyces sp. CNXK31.2 and 16S rRNA sequence data were deposited at NCBI GenBank with accession numbers PQ146974.1.

Figure 2. Mucor sp. MD7 mycelial inhibition of actinomycetes. Different numbers represent different strains, (−) negative control. [Click here to view]

3.4. Extracellular Enzyme Activity of Streptomyces sp. CNXK31.2

The results presented in Figure 4 and Table 1 demonstrated that CNXK31.2 exhibited the production of extracellular hydrolytic enzymes, including 1.4 ± 0.2 AU of chitinase activity, 3.2 ± 0.3 AU of protease activity, and 1.7 ± 0.2 AU of CMCase. The biosynthesis of chitinase, protease, and cellulase, known for their role in breaking down fungal cell walls, has been observed in various fungal antagonists, such as actinomycete strains isolated from the rhizosphere of strawberry, rosemary, acacia, and olive plants for controlling S. sclerotiorum on tomato plants [15], Bacillus velezensis CE 100 combating anthracnose caused by Colletotrichum gloeosporioides [23], and Bacillus safensis B3 for controlling B. cinerea gray mold in strawberry fruits [24]. In addition, yeast antagonists have also shown similar hydrolytic enzyme production when combating B. cinerea on strawberry fruits [14]. Thus, these results suggest that Streptomyces sp. CNXK31.2 possesses a robust capacity for hydrolytic enzyme production, potentially contributing to its ability to inhibit fungal pathogens. This offers an eco-friendly alternative to chemical fungicides, avoiding environmental toxicity and resistance development associated with chemical use.

Figure 3. Identification of actinomycete CNXK31.2. (A-a) Colony morphology and (A-b) vegetative hyphae (black arrows), spore-bearing aerial hyphae (white arrows). (B) Phylogenetic tree of CNXK31.2 based on 16S rRNA sequence. Numbers on branch nodes are bootstrap values (expressed in percentage). Scale bar = 10 µm. [Click here to view]

Figure 4. Hydrolytic activity of CNXK31.2. (A) Chitinase. (B) Caseinase. (C) CMCase. [Click here to view]

Table 1. Hydrolytic enzyme activity of CNXK31.2. [Click here to view]

3.5. Effect of Streptomyces sp. CNXK31.2 Culture Supernatant on the Fungal Mycelia

After a 24-hour inoculation, in the control group without the Streptomyces sp. CNXK31.2 culture supernatant, Mucor sp. MD7 mycelia exhibited normal growth with long, thick, and branched mycelia (Fig. 5A). On the contrary, in the presence of culture supernatant, the fungal mycelia displayed aberrant morphology, including deformed, thinning, or swollen of hyphae, and even hyphae degradation (Fig. 5B). Irregularity observed in the pathogenic MD7 mycelia when treated with the CNXK31.2 culture supernatant can be attributed to cell wall degradation facilitated by hydrolytic enzymes in the culture. These findings are in line with the study of Lee et al. [25], where the culture solution of Streptomyces cavourensis SY224 had a similar influence on the mycelium of C. gloeosporioides after a 72-hour inoculation. In addition, compounds produced by Streptoverticillium morookaense demonstrated comparable effects on the mycelial morphology of phytopathogenic fungi, including Ustilaginoidea virens, Rhizoctonia solani, and Bipolaris maydis [26].

3.6. Effect of Temperature and pH on Antifungal Activity of Streptomyces sp. CNXK31.2

The culture supernatant of Streptomyces sp. CNXK31.2 demonstrated thermal stability concerning its antifungal activity within the temperature range of 50°C–80°C, with a remaining antagonistic activity of 75.0%–95.8% compared to the untreated control (Fig. 6A). Exposure to temperatures exceeding 90°C resulted in the complete loss of antifungal activity, suggesting a marginally less stable than in the case of Streptomyces albulus Z1-04-02 culture supernatant with ~34% remaining activity at 100°C [27]. In addition, the culture supernatant displayed robust stability in neutral and alkaline environments, with antifungal activity exceeding 80% at pH levels between 7.0 and 10.0. At acidic pH levels of 3.0–6.0, the activity considerably remained at 39.6%–66.7% (Fig. 6B). Similar observation in antifungal activity of Streptomyces sp. TQR12-4 culture supernatant against Geotrichum candidum, F. oxysporum, and F. udum [28]. Consequently, Streptomyces sp. CNXK31.2 demonstrated both broad-spectrum pH stability (pH 3.0–10.0) and thermal stability (50°C–80°C), making it a potential candidate for the development of a safe bio-fungicide.

Figure 5. Effect of CNXK31.2 culture supernatant on MD7 mycelia. (A) Control and (B) treatment. Black arrows indicate sites of mycelial abnormality. Scale bar = 10 µm. [Click here to view]

Figure 6. Effect of temperature (A) and pH (B) on stability of antifungal agents against Mucor sp. MD7. Means with different letters above columns statistically differ from each other (p < 0.05). [Click here to view]

3.7. In Situ Test of Fungal Antagonistic Activity on Strawberry Leaf Model

After 1 day of inoculation, disease symptoms, characterized by black spots on strawberry leaves injected with Mucor sp. MD7, were visible compared to no observable symptom on leaves of other conditions (Fig. 7A). After 3 days of inoculation, the black spots began to spread, and the symptoms gradually extended as the leaf tissues exhibited necrosis as the mold prominently proliferated. In contrast, strawberry leaves treated with a mixture of CNXK31.2 and MD7 displayed no differences in leaf surface compared to the control with sterilized media. Furthermore, leaves treated with Streptomyces sp. CNXK31.2 exhibited no signs of disease symptoms but mycelial proliferation (Fig. 7B). This suggests that Streptomyces sp. CNXK31.2 is not pathogenic to strawberries and is capable of inhibiting leaf spot caused by Mucor sp. MD7.

The utilization of microbial strains as biocontrol agents for the protection and management of strawberry leaf diseases has been getting more attention. For instance, the application of Trichoderma asperellum has been found to inhibit mycelial growth and reduce the appearance of leaf blight caused by Neopestalotiopsis clavispora in strawberries [9]. Similarly, reports indicate the effectiveness of Aureobasidium pullulans in decreasing the disease severity index associated with Mycosphaerella fragariae, a leaf spot pathogen [29]. Furthermore, Streptomyces sp. 3–10 crude extract has demonstrated the suppression of various fungal pathogens affecting strawberries, including B. cinerea, Mucor hiemails, Saxifraga stolonifera, and S. sclerotiorum [30]. These findings underscore the potential application of the actinomycete strains, including the one under study in the prevention of strawberry diseases.

Figure 7. In situ test of Mucor sp. MD7 antagonistic activity of Streptomyces sp. CNXK31.2 on strawberry leaves. (A) After 1 day and (B) after 3 days of infection. [Click here to view]