Antagonistic potential of Streptomyces sp. VSMKU1027
Numerous bacterial and fungal diseases that affect both flowering and nonflowering plants result in large losses in food production globally, with major economic consequences. Plant pathogenic fungi are the primary cause of agricultural losses among the organisms responsible for these illnesses,³⁷ as they infect a diverse variety of cultivated plants. In light of this, the current investigation examined the antifungal activity of an indigenous isolate of Streptomyces sp. VSMKU1027, which demonstrated varying degrees of zone of inhibition in comparison to the control against two economically significant soil-borne fungal pathogens, R. solani and F. oxysporum (Table 1). Both soil fungi are notable for being extensively dispersed diseases that can seriously harm economically important crops by producing, sheath blight, vascular wilt, root rot, fruit and flower rot, and finally plant death.⁷ Various Streptomyces spp. have been recognized as bio-control agents against numerous plant pathogens, demonstrating their capability to produce bioactive compounds that suppress or inhibit the mycelial growth of several fungi.³⁸ Secondary metabolites may be used in addition to or instead of chemical pesticides, and their microbiological application in crop protection has gained significance in recent years.¹⁵

Table (1):
Antagonistic activity of Streptomyces sp. VSMKU1027 against soil-borne fungal plant pathogens

Research has concentrated on employing Streptomyces spp. as biocontrol agents to manage various bacterial and fungal-induced plant diseases.^33,37 Bacteria of the genus Streptomyces spp. can control a variety of bacterial and fungal plant diseases through diverse mechanisms. Their action mechanism has been described as the synthesis of volatile and non-volatile antibiotics, enzymes that break down cell walls, hyperparasitism on pathogenic organisms, growth stimulation in plants, and the creation of systemic resistance in the host plant.³⁹

Interaction between Pathogens and Streptomyces sp. VSMKU1027
Given this context, the present study has investigated the antifungal activity of indigenous isolate Streptomyces sp. VSMKU1027, which exhibited significant inhibition against two economically important soil-borne fungal pathogens, R. solani and F. oxysporum, as evidenced by varying levels of zone of inhibition compared to the control (Table 1). Various Streptomyces spp. have been recognized as bio-control agents against numerous plant pathogens, demonstrating their capability to produce bioactive compounds that suppress or inhibit the mycelial growth of several fungi.³⁹ Secondary metabolites may be used in addition to or instead of chemical pesticides, and their microbiological application in crop protection has gained significance in recent years.¹⁵

Mycelia of R. solani were collected from the outermost zone of a three-day-old dual culture. Light microscopy observations revealed that, unlike the control, the mycelia of R. solani had disintegrated, the mycelium exhibited swelling, and no fruiting bodies were formed on the R. solani test plant (Figure 1a, 1b, 1c and  1d). Moreover, the isolate VSMKU1027 significantly altered the morphology of F. oxysporum, encapsulated hyphae causing it to become extensively branched and coiled. Additionally, the control mycelium displayed a different shape, with the mycelial tips expanding to form a spherical structure (Figure 2).

The secretion of exogenous compounds, such as cell-wall degrading enzymes and antibiotics, which visibly harm and disintegrate R. solani hyphae under light microscopy, is believed to be responsible for inhibiting R. solani mycelial growth in the presence of VSMGT1014. Compared to the control, interactions between VSMGT1014 and the pathogen resulted in abnormalities such as deformation and swelling of hyphae.^3,40 In contrast to the control, interactions between VSMGT1014 and the pathogen resulted in abnormalities such as morphological deformities and hyphal swelling. This observation aligns closely with previous research.³⁷ In the current study, pathogens like R. solani and F. oxysporum were observed under the light microscope exhibiting dissolution along with some swelling.⁴¹ This phenomenon may be attributed to the action of lytic enzymes such as protease and chitinase, or to secondary metabolites produced by VSMGT1027.

Identification of selected isolates VSMKU1027
The isolate VSMKU1027’s peach color and rough surface on different ISP mediums. On the ISP2 medium, the isolate displayed a punch of aerial substrate mycelium, short-looped chain spores, and aerial mycelium. Under a light microscope (20X), morphological features of the isolate VSMKU1027 spore chains were observed to be short chains resembling the flexuous sporophores group of Streptomyces (Figure 2).

In comparison to the control, the isolate VSMKU1027 displayed favorable results for the Gram’s reaction, catalase, and oxidase. In addition, urease synthesis, citrate utilization, casein, gelatin liquefaction, and gelatin hydrolysis were all significantly higher in the isolate VSMKU1027 than in the control. Regarding H2S, Voges Proskauer, methyl red, indole, nitrate reduction, arabinose, and mannitol utilization, the isolate VSMKU1027 exhibited a negative reaction. In comparison to the control, the strain VSMKU1027 used the majority of simple sugars, including lactose, glucose, fructose, and sucrose, as the only carbon source, excluding arabinose and mannitol. VSMKU1027 grew to its maximum on the ISP2 medium that contained glycerol and starch (Table 2). According to our findings, certain researchers discovered. The findings showed that the isolates is capable of using a wide range of substrates, including glycerol, starch, lactose, inositol, rhamnose, raffinose, and maltose.^3,42-43 The actinobacterium’s smooth rough surface of the colony nature, form of the spores, and colony and mycelial nature were extremely consistent with other research findings.^40,44

Table (2):
Streptomyces sp. VSMKU1027 morphological, physiological, and biochemical characteristics

Given their ability to produce spores, these filamentous, Gram-positive rhizobacteria offer an added advantage for their potential as formulation-based agents to enhance plant growth.^45-47 As reported by Harikrishnan et al.³ and Al-Askar et al.,⁴⁸ specific Streptomyces species have been successfully developed as potential biocontrol agents and formulated to combat fungal phytopathogens across various crops, For instance, Mycostop, a commercially available wettable formulation containing spores and mycelium of Streptomyces griseoviridis, has been extensively used in North America and Europe to protect vegetables and ornamental crops.⁴⁹

Production of antimicrobial traits and Hydrolytic enzyme by Streptomyces sp. VSMKU1027
Compared to the control, except for hydrogen cyanide, the isolate VSMKU1027 was much more capable of producing siderophores and phosphate solubilization. Analogously, isolate VSMKU1027 demonstrated exceptional hydrolytic enzyme synthesis, including chitinase, protease, gelatinase, amylase, and pectinase, in their respective substrate-amended mediums. In contrast to the control, the isolate VSMKU1027 did not produce cellulase (Table 2). The production of antibiotics, lytic enzymes, volatile compounds, and siderophores constitutes a primary mechanism through which plant growth-promoting rhizobacteria (PGPR) inhibit pathogens.⁵⁰ Strain VSMGT010143 was found to produce IAA, siderophores, volatile compounds, and hydrolytic enzymes including chitinase, cellulase, protease, gelatinase, amylase, pectinase, and solubilized phosphorus. Moreover, rhizospheric Streptomyces spp. exhibit plant growth-promoting traits (PGPT), including enzyme secretion, organic acid production, phosphate solubilization, and siderophore synthesis. These PGPTs contribute to enhanced plant growth and the management of soil-borne bacterial and phytofungal diseases through mechanisms such as systemic acquired resistance and induced systemic resistance, providing protection against diverse biotic and abiotic stresses (Table 3). Previous studies have demonstrated that Streptomyces strains with these attributes can enhance plant growth. For instance, S. violaceusniger AC12AB was found capable of producing siderophores, nitrogen fixation, phosphate solubilisation, and intermediate amino acid production.⁵¹ In field trials, it significantly increased potato yields by up to 26.8%. Additionally, Chouyia et al.⁵² found that barley plants infected with S. roseocinereus MS1B15, which produces IAA, solubilize phosphate, and fixes nitrogen, exhibited substantial increases in shoot and spike length.

Table (3):
Production of Antimicrobial metabolites and hydrolytic enzymes by the isolate VSMKU1027

Production of IAA in different L-tryptophan concentrations by Streptomyces sp. VSMKU1027
IAA is a significant component of the auxin family of plant hormones. Streptomyces species are the predominant IAA-producing actinobacteria that also enhance plant development.^53-55 Actinomycetes and fungi are among the genera of bacteria that promote plant growth; they enhance plant growth by producing IAA through processes that rely on L-tryptophan. According to certain research, Streptomyces sp. is the predominant genus among actinomycetes that produce IAA. IAA influences various physiological and developmental processes in plants, including embryogenesis, organogenesis, vascular differentiation, root and shoot development, trophic growth, and fruit development. The availability of substrate(s), growth phase, and varied physicochemical circumstances impact IAA production, which varies greatly throughout species. The availability of substrate(s), growth phase, and varied physicochemical circumstances impact IAA production, which varies greatly among the various rhizobacterial species. All the habitat microbes were secreted IAA through a tryptophan-dependent biosynthesis pathway. Various concentrations of L- tryptophan ranging from 0.1 to 1% were tested for the production of IAA. Spectrophotometric analysis revealed a gradual increase in the IAA production with increasing L-tryptophan concentration. The highest IAA production, reaching 132.36 µg/mL, was observed when the medium contained 0.4% in ISP2 medium, surpassing other media and the control (Figure 3). This observation aligns with the understanding that actinomycetes can produce the phytohormone auxin IAA when provided with an appropriate precursor like L-tryptophan.⁵⁵ In addition to that our results highly coincides the previous report. In our study, high concentrations of L-tryptophan supplementation adversely affected the level of IAA production. However, at low concentrations of L-tryptophan supplementation, IAA production could reach up to 27.3 µg/mL compared to the control.

Production of IAA on different days, medium, temperature, and pH by Streptomyces sp. VSMKU1027
The effect of IAA production was monitored over a period of 10 days, with the highest production observed on the 6^th day of incubation (93.4 µg/mL) (Figure 4). Kaur and Manhas⁵⁶ reported that almost coincides of our results, because the maximum production IAA was 80.06 µg/mL by S. hydrogenans DH16 on 5^th day. According to previous findings, the oxidase and peroxidase synthesis that degrades IAA may be the cause of the attenuation in the IAA levels.⁵⁷ Likewise, among the five different media tested, ISP2 medium yielded the highest amount of IAA production (68.13 µg/mL) (Figure 5). In contrast, the lowest amount of IAA production, at 19.8 µg/mL, was observed in Bennett’s broth medium compared to the control. The maximum production of IAA by Streptomyces sp. VSMKU1027, reaching 66.5 µg/mL, was observed at pH 6.5. The results indicated a gradual increase in IAA production by VSMKU1027 from pH 4 to pH 6.5, whereas a significant decrease in production was noted at pH 8-10 compared to the control (Figure 6). Similarly, the highest IAA production (81.4 µg/mL) was recorded at 35°C. However the IAA production was slowly reduced at 40°C compared to control (Figure 7). The influence of pH on IAA synthesis demonstrated that S. hydrogenans DH16 produced the highest amount of IAA at a neutral pH, consistent with previous studies.^54,58 Production decreases at acidic or highly alkaline pH levels because Streptomyces spp. are unable to thrive in such extreme pH conditions. The optimal temperature for IAA formation was found to be
30°C, aligning with Aldesuquy et al.⁵⁹ study indicating maximal IAA production by Streptomyces spp. at 25-30°C. IAA produced by rhizobacteria primarily influences the root system, enhancing its size, weight, number of lateral roots, and soil contact area. This mechanism not only enhances plant development and yield but also facilitates nutrient uptake and acquisition in the soil.