Rhizospheric-Derived Nocardiopsis alba BH35 as an Effective Biocontrol Agent Actinobacterium with Antifungal and Plant Growth-Promoting Effects: In Vitro Studies

Abstract

The biocontrol approach using beneficial microorganisms to control crop diseases is becoming an essential alternative to chemical fungicides. Therefore, new and efficient biocontrol agents (BCA) are needed. In this study, a rhizospheric actinomycete isolate showed unique and promising antagonistic activity against three of the most common phytopathogenic fungi, Fusarium oxysporum MH105, Rhizoctonia solani To18, and Alternaria brassicicola CBS107. Identification of the antagonistic strain, which was performed according to spore morphology and cell wall chemotype, suggested that it belongs to the Nocardiopsaceae. Furthermore, cultural, physiological, and biochemical characteristics, together with phylogenetic analysis of the 16S rRNA gene (OP869859.1), indicated the identity of this strain to Nocardiopsis alba. The cell-free filtrate (CFF) of the strain was evaluated for its antifungal potency, and the resultant inhibition zone diameters ranged from 17.0 ± 0.92 to 19.5 ± 0.28 mm for the tested fungal species. Additionally, the CFF was evaluated in vitro to control Fusarium wilt disease in Vicia faba using the spraying method under greenhouse conditions, and the results showed marked differences in virulence between the control and treatment plants, indicating the biocontrol efficacy of this actinomycete. A promising plant-growth promoting (PGP) ability in seed germination and seedling growth of V. faba was also recorded in vitro for the CFF, which displayed PGP traits of phosphate solubilization (48 mg/100 ml) as well as production of indole acetic acid (34 μg/ml) and ammonia (20 μg/ml). This study provided scientific validation that the new rhizobacterium Nocardiopsis alba strain BH35 could be further utilized in bioformulation and possesses biocontrol and plant growth-promoting capabilities.

Keywords: Nocardiopsis alba BH35; Rhizosphere; antifungal; biocontrol; plant growth-promoting.

Fig. 1. Antagonistic activity of actinomycete isolate BH35 against phytopathogenic fungal species using dual culture assay on PDA plates.

(A) Rhizoctonia solani To18. (B) Fusarium oxysporum MH105. (C) Alternaria brassicicola CBS107.

Fig. 2. Cultural and morphological characteristics of actinomycete isolate BH35.

(A) White aerial mycelium formed on ISP–4 medium. (B) Strong brown-melanin pigment produced on ISP–6 medium. (C) Aerial hyphae bearing zigzag-shaped spore chains under light microscopy (400×). (D) SEM micrograph (16,000×) showing recti-flexible chains of rod-shaped spores with a smooth surface.

Fig. 3. Phylogenetic tree of Nocardiopsis alba strain BH35 inferred using the neighbor-joining method in MEGA 11.0 software.

This tree shows the relationships between isolate BH35 and closely linked species of the genus Nocardiopsis. The bootstrap consensus tree inferred from 1,000 replicates is taken to represent the evolutionary history of the taxa analyzed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown next to the branches. This analysis involved 13 nucleotide sequences. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There were a total of 1,752 positions in the final dataset.

Fig. 4. Inhibition ability of the different concentrations of N. alba BH35 CFF against phytopathogenic fungal species.

The values represent the mean ± SD of triplicate experiments (n = 3, p < 0.05).

Fig. 5. Antifungal effects of CFF of N. alba BH35 against phytopathogenic F. oxysporum.

(A) Antagonistic effect using a dual culture assay on PDA plate. In this plate, the actinomycete was inoculated with gradual inoculum sizes in two different forms, where (a) is a linear very heavy inoculum, and (b, c, d) are circular moderate, mild, and very mild inoculums, respectively. (B) Inhibitory effect of CFF (100.0% conc.) against radial growth on Czapek-Dox plate. In this plate, (c) is the control (uninoculated SNB medium), and (a) is the CFF of actinomycete. (C) SEM micrograph (1200×) of untreated (control) mycelium. (D) SEM micrograph (1200×) of treated mycelium at 70% CFF. (E) SEM micrograph (2500×) of treated mycelium at a CFF concentration of 100%.

Fig. 6. In vitro biocontrol capability of CFF of N. alba BH35 against phytopathogenic wilt-mold F. oxysporum in V. faba under greenhouse conditions.

(A) Pathogenicity and treatment. (B) Suppression effect of the pathogen on the shoot and root length of the infected plants. The values represent the mean ± SD of triplicate experiments (n = 3, p < 0.05).

Fig. 7. Promoting capability of the different concentrations of N. alba BH35 CFF for growth of V. faba.

(A) Seed germination. (B) Seedling growth. (C) Promotion percentages in seed, shoot, and root length. In this experiment, the control was uninoculated SNB medium. Different concentrations of CFF (25-100%) were prepared by dilution with double distilled water. The values represent the mean ± SD of triplicate experiments (n = 3, p < 0.05).