Physiological Optimization and Biocontrol Potential of a Rhizospheric Isolate of Victoriomyces antarcticus Against Cucumber Soil-Borne Pathogens

Prepared by the researche : Sarah Ali Umran¹ & Professor: Ahmed M. Hussein^{2 –} Department of Plant Protection, Faculty of Agriculture, University of Kufa, Najaf, Iraq

DAC Democratic Arabic Center GmbH

International Journal of Environmental and Biological Sciences : First issue – January 2026

A Periodical International Journal published by the “Democratic Arab Center” Germany – Berlin

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ORCID: https://orcid.org/0009-0009-5776-1920         https://orcid.org/0000-0002-6286-8863

Abstract

Driven by the need for sustainable agricultural alternatives to synthetic fungicides, this study investigates the physiological optimization and biocontrol potential of the fungus Victoriomyces antarcticus (isolate AF1), isolated for the first time from the cucumber rhizosphere in Iraq (GenBank: OR131302). Physiological assays revealed that the optimal conditions for mycelial growth were Potato Dextrose Agar (PDA), a temperature of 20°C, pH 6.0, and complete darkness, highlighting its psychrotolerant nature. Antagonistic assays demonstrated significant efficacy against the soil-borne pathogen Rhizoctonia solani, with a maximum growth inhibition rate of 82.22% using the fungal sterile filtrate at a 50% concentration. HPLC analysis identified key bioactive phenolic compounds, predominantly Gallic acid (40.23 µg/ml) and Caffeic acid (22.51 µg/ml), which are likely responsible for the observed antifungal activity. These findings position V. antarcticus as a novel and potent biocontrol agent, offering a viable, eco-friendly strategy for managing root-rot diseases in sustainable cucumber production.

Introduction:

              Fungi represent an ecologically and economically indispensable kingdom, playing fundamental roles in global biogeochemical cycles, particularly in carbon and nitrogen turnover, thus maintaining ecosystem sustainability (1, 2). Beyond their ecological significance, fungi are prolific producers of a vast array of primary and secondary metabolites, including antibiotics, industrial enzymes, and various bioactive compounds crucial for agriculture and medicine (3, 4). The escalating global drive towards sustainable agriculture is fueled by growing concerns over environmental pollution, human health risks, and the rapid emergence of pathogen resistance associated with chemical pesticides (5). This shift necessitates the exploration of microbial Biological Control Agents (BCAs), particularly those sourced from novel or extreme environments, as a major focus for sustainable crop protection (6). Extremotolerant microorganisms inhabiting harsh conditions, such as the psychrotolerant fungi of Antarctica, have evolved unique metabolic pathways. This physiological adaptation often results in the biosynthesis of novel, robust bioactive molecules chemically distinct from those produced by mesophilic organisms (7, 8). The Antarctic continent, therefore, represents a critical frontier for bioprospecting for specialized, cold-adapted fungi with potent biocontrol potential against agriculturally significant phytopathogens (9, 10).

The fungus V. antarcticus is a compelling example, formally described as a new genus and species isolated from soil in Victoria Land, Antarctica (11). Molecular phylogenetic analysis confirmed its distinct evolutionary lineage within the family Cephalothecaceae (Ascomycota) (11, 12). A key characteristic is its production of a diffusible, intense red pigment (11). Initial genomic analysis suggests that the pigment’s biosynthetic gene cluster (BGC) exhibits homology with the pathway for oosporein production (13). Oosporein is a recognized dibenzoquinone derivative with proven broad-spectrum antifungal efficacy against various plant pathogens (14). Furthermore, preliminary studies indicate that filtrates of V. antarcticus are effective against specific fungal pathogens, underscoring its potential as a BCA (15).  Despite this clear potential, knowledge regarding the ecophysiology of V. antarcticus is critically limited. The efficiency and yield of bioactive secondary metabolite production in filamentous fungi are highly dependent on culturing conditions. Environmental factors such as temperature, pH, light exposure, and nutrient composition can significantly modulate the expression of BGCs, directly impacting both the fungal biomass and the spectrum of antifungal activity (16, 17). Currently, the optimal physiological requirements for maximizing both the growth and, crucially, the metabolite production by V. antarcticus have not been systematically determined. Therefore, this study aims to systematically investigate and optimize the growth of Victoriomyces antarcticus and rigorously assess its resulting biocontrol capability. Specifically, the objectives are to:

1. Confirm the molecular identity of the fungus using DNA sequencing techniques.
2. Systematically determine and optimize the key physical and nutritional growth conditions (temperature, pH, light, and culture medium type) to enhance fungal biomass and metabolite production.
3. Evaluate the antagonistic and inhibitory activities of the V. antarcticus culture filtrates obtained under optimized conditions against a range of agriculturally significant phytopathogenic fungi, thus validating its promise as a novel biocontrol agent.

MATERIALS AND METHODS

Fungi under study, reactivation and diagnosis confirmation

The pathogenic fungi R. solani and F. solani were obtained from infected plants in  a preliminary study (19). as these two fungi recorded a higher dominance in appearance and frequency than other fungi in plants infected with root and crown rot of Cucumbers. An isolate of the fungus V. antarcticus was also obtained from the same study, as it appeared to accompany healthy plants very close to infected plants. The fungi were purified and reactivated in the graduate Phytopathology laboratory in the Dept. of Plant Protection, Faculty of Agriculture, University of Kufa. Fungal isolates were cultured in Petri dishes containing autoclave sterile PDA (Potato Dextrose Agar) treated with chlorophenol before being poured in the Petri dishes. The petri dishes were incubated at 25 ± 2 °C, and fungus growth was monitored for each Petri dishes after 72 h.  Fungi diagnosis was confirmed under 40X light compound microscope based on the sexual and asexual spores and structures formed by the fungus according to the taxonomic keys (20,21).

Preservation of fungal isolates

Three fungi V. antarcticus from healthy appearing plants, and R. solani and F. solani from infected plant parts were purified using the mycelial tip technique to obtain pure and homogeneous cultures of the fungal isolates (22). The pure isolates were preserved on sterile PDA, and kept in the refrigerator at 4°C for use in subsequent tests.

Molecular diagnosis of V. antarcticus

 The pure fungal isolate underwent molecular diagnosis at Al-Amin Centre for biotechnology and molecular technologies, where the complete sequences of the nitrogenous bases of the amplified DNA of V. antarcticus genome were obtained using the ITS4 and ITS2 primers. Data were processed using Ltd chromas program 6.6.2, and partial matching was done with all the registered sequences in the NCBI using the basic local alignment search Tool (BLAST). The maximum similarity score and the query cover score in determining the diagnostic identity of the fungus was used at 97% or higher. Phylogenetic tree was drawn based on a number of available sequences in the NCBI. (18).

Pathogenicity of isolated fungi on Cucumber (Cucumis sativus) seeds

1. solani, F. solani, and V. antarcticus, isolated from 7 days old cultures, were grown in PDA petri dishes with three replicates for each fungus and incubated at 25 ± 2°C for three days, then 10 superficially sterilized seeds were planted in each plate and three control plates left without fungus. After 14 days, the percentage of germinated and rotten seeds and the percentage of infected and healthy seedlings were calculated.

Effect of V. antarcticus fungal filtrate on growth of the pathogenic fungi

1. antarcticus filtrate was prepared by growing a 0.5 cm disc of 7-day-old fungal culture in 250 ml conical flasks containing 100 ml/flask of sterile liquid potato sucrose broth PSB. The flasks were incubated at 25 ± 2 °C, and shaked every 2-3 days. After 28 days of incubation, two sets of filtrates were made. The first set of flasks was autoclaved., while the second group was filtered with a Millipore filter of 0.22 µm. V. antarcticus filtrate from both group was added to PDA petri dishes at an amount of 0, 2, 4 or 6 ml/plate with three replications before solidification, then the centre of the solid medium was inoculated with a 0.5 cm disk of a culture of the 7 days old R. solani or F. solani. The plates were incubated at 25 ± 2 °C for seven days. Then, the radial growth was measured to estimate the percentage of inhibition in fungal growth at the highest fungal growth in the control treatment. (23).

Effect of V. antarcticus filtrate on Cucumber seed germination

Effect of V. antarcticus fungal filtrate prepared by both methods as previously mentioned on the germination test of Cucumber seeds. Where 6 ml of either type of filtrate was added to Petri dishes containing sterile filter paper, on which ten sterile seeds were planted, with three replicates for each treatment, and for the control, treated with 6 ml of sterile distilled water. The plates were incubated at 27 ± 2°C for ten days, after which the percentage of germination of seeds and rotten and healthy seeds was calculated.

Detection of active substances in V. antarcticus filtrate

Chemical analysis of V. antarcticus filtrate prepared by both methods diagnosed and the percentage of each compound was estimated using a high-performance liquid chromatography device HPLC (22).

Effect of Culture Medium Type on V. antarcticus Radial Growth

To comprehensively evaluate the metabolic flexibility of V. antarcticus and its capacity to utilize diverse nutrient sources, eight distinct solid agar media with varied nutritional compositions were selected: Potato Dextrose Agar (PDA), Malt Extract Agar (MEA), MacConkey Agar (MA), MR-VP Agar, Blood Agar (BA), Eosin Methylene Blue Agar (EMB Agar), Chocolate Agar (CA), and Simmons Citrate Agar (SCA). These media were specifically chosen to represent standard growth media, enriched media, and selective/differential media for assessing the fungus’s ability to utilize complex chemical compounds and sole carbon sources. All media were prepared strictly according to the manufacturers’ instructions and subsequently sterilized by autoclaving at 121 C for 15 minutes. The sterilized media were then aseptically poured into sterile Petri dishes under a Laminar Flow Hood. The experiment was designed using a Completely Randomized Design (CRD) with three replicates for each medium. For inoculation, a standard 5   mm diameter mycelial plug was excised from the actively growing edge of a 5-day-old V. antarcticus colony and centrally placed onto each agar plate. The plates were then incubated under the predetermined optimal conditions of 20 C and a 12/12-hour light/dark cycle. Radial growth (colony diameter) was measured after seven days of incubation to assess the fungal growth rate across the different nutritional environments (24).

Effect of Salinity-Induced Osmotic Stress on Fungal Radial Growth

The impact of osmotic stress on the radial growth dynamics of V. antarcticus was investigated using Potato Dextrose Agar (PDA) as a basal medium, supplemented with varying concentrations of sodium chloride (NaCl). Eleven distinct salinity levels were established, ranging from 0 to 20 dS/m with increments of 2 dS/m. To ensure high precision in osmotic potentials, the specific mass of NaCl required for each salinity treatment was determined based on a preliminary calibration curve generated using an Electrical Conductivity (EC) meter. The precisely calculated amounts of NaCl were thoroughly incorporated into the PDA basal medium prior to sterilization. This pre-sterilization adjustment was critical to achieving a homogenous saline environment and maintaining consistent electrolytic stability across all treatments after autoclaving.

             The salt-adjusted PDA was sterilized by autoclaving at 121 °C and 15 psi for 15 minutes. After cooling to approximately 40 °C, the media were aseptically poured into sterile 90 mm Petri dishes (20 mL per plate). The experiment followed a Completely Randomized Design (CRD) with three independent replicates for each salinity level. Inoculation was performed by centrally placing a standardized 5 mm agar disc, excised from the periphery of an actively growing 7-day-old V. antarcticus colony. All cultures were incubated at the optimal temperature of 20 °C in total darkness. The total colony diameter (mm) was recorded once the growth in the control group reached the edge of the Petri dish (90 mm). Growth was quantified by averaging two orthogonal diameter readings following established protocols (25).

 Determination of the Optimal Thermal Conditions for Mycelial Growth of V. antarcticus 

The effect of temperature on the radial growth of the fungus V. antarcticus was evaluated to determine its optimum growth conditions. Six different temperatures were selected for the experiment:  5, 10, 15, 20, 25, and 30 C. The experiment was set up using a triplicate design for each temperature treatment, resulting in a total of 18 inoculated plates. Sterile Potato Dextrose Agar (PDA) plates were inoculated by placing a single 5 mm Mycelial plugtaken from the edge of actively growing, 7-day-old fungal colonies, using a sterile cork borer. The inoculated plates were immediately transferred to calibrated incubators set at the designated experimental temperatures. Incubation was conducted in complete darkness to prevent any potential photoperiodic effect on colony growth or pigmentation. Colony diameter was measured daily or every two days (depending on the growth rate) using a sterile ruler. For each colony, two perpendicular measurements were taken, and the average diameter was recorded. Measurements were continued until the colony diameter in one of the treatments (the fastest-growing) reached the edge of the plate (90 mm), marking the conclusion of the experiment. (26).

Impact of Hydrogen Ion Concentration (pH) on Mycelial Radial Growth of V. Antarcticus

      To evaluate the influence of pH on the radial growth dynamics of V. antarcticus, five distinct pH levels (5.0, 6.0, 7.0, 8.0, and 9.0) were evaluated. Potato Dextrose Agar (PDA) was prepared as the basal medium and sterilized via autoclaving at 121C and 15psi for 15 minutes. Following sterilization, the medium was allowed to equilibrate to approximately 40 C in a water bath.

     The medium was then aseptically partitioned into five equal volumes to facilitate pH adjustment. The pH of each portion was precisely modified to the target values using sterile 1 M Sodium Hydroxide (NaOH) or 1 M Hydrochloric Acid (HCl) solutions, with measurements verified using a digital, pre-calibrated pH meter. Once adjusted, the media were poured into sterile 90 mm Petri dishes at a standardized volume of 20mL per plate.  Inoculation was performed by placing a standardized 5 mm mycelial plug—excised from the leading edge of an actively growing 7-day-old culture—at the center of each plate using a sterile cork borer. To eliminate confounding variables, all plates were incubated at a constant temperature of 20 C (previously identified as the optimal thermal condition for this species (27)) under continuous darkness to prevent photo-induced growth variations.

              The growth rate was monitored daily by measuring the colony diameter along two pre-marked perpendicular axes. The mean diameter for each treatment was recorded until the mycelium in the fastest-growing treatment reached the periphery of the Petri dish (90 mm) (28). The experiment was conducted using a Completely Randomized Design (CRD) with three independent replicates per pH treatment.

Effect of Light and Dark Conditions on the Radial Growth Rate of V. Antarcticus

            To investigate the effect of light and dark conditions (photoperiod) on the fungal growth    rate, three experimental treatments were established, each replicated in triplicate. Inoculation was performed by taking a  0.5   cm Mycelial plugfrom the edge of a fresh, pure fungal colony using a sterile cork borer, and placing it in the center of a Petri dish containing Potato Dextrose Agar (PDA) medium. The plates were divided into the following treatments (29):

Complete Darkness (Darkness), where three plates were wrapped in black carbon paper and enclosed in a clear plastic bag to totally exclude light;

Continuous Illumination (Continuous Light), where three plates were wrapped only in a clear plastic bag and exposed to a consistent white light source providing 200 – 300 lux.

 Alternating Photoperiod (Light/Dark Cycle), where three plates were subjected to a cyclic light regime of 12 hours of light followed by 12 hours of darkness. All inoculated plates were incubated at a constant temperature of 20 C. The mycelial radial growth was measured in centimeters after seven days of incubation. This setup specifically investigates the potential inhibitory effect of light on the radial expansion process, which is often observed during the vegetative growth phase (mycelium) of filamentous fungi.

Effect of V. antarcticus Cell-Free Filtrate on the Growth of Phytopathogenic Fungi

The Cell-Free Filtrate (CFF) of Victoriomyces antarcticus was prepared by inoculating 0.5   cm agar plugs (from 7-day-old cultures) into 250   mL conical flasks containing 100   mL of sterile Potato Sucrose Broth (PSB). The flasks were incubated at 25 C for 28 days, with manual shaking performed periodically (e.g., every 2-3 days). After incubation, the fungal biomass was removed via sequential filtration. The liquid filtrate was then divided into two sets: the first set was sterilized by autoclaving at 121 C for 15 minutes (heat-treated control), and the second set was sterilized by membrane filtration through a 0.22 mu m syringe filter to obtain the active CFF. (30) The CFF from both sets was subsequently incorporated into molten Potato Dextrose Agar (PDA) medium (cooled to 45 C) using the Poisoned Food Technique at final concentrations corresponding to 0, 2, 4, and 6   mL per Petri dish, representing concentrations of approximately 0 %, 10 %, 20 %, and 30 % (v/v). The plates were inoculated at the center with a 0.5    cm agar disc from a 7-day-old culture of one of the five tested phytopathogenic fungi: Rhizoctonia solani, and Fusarium solani, the experiment utilized three replications for each treatment and was incubated at 20 C in darkness for seven days. The radial growth of the colonies was measured by taking two perpendicular readings, and the percentage of growth inhibition (PGI) was calculated relative to the growth in the control treatment (0 mL filtrate).

Results and Discussion

Molecular diagnosis of V. antarcticus

The molecular diagnosis of   V. antarcticu f isolated from Cucumberwas confirmed. After completing the sequences of nitrogenous bases and matching the results using BLAST for nucleotides, the fungus was confirmed to be V. antarcticus ‘isolate AF1 internal transcribed spacer1, partial sequence; 5.8S ribosomal RNA gene and internal transcribed spacer2, complete sequence; and large subunit ribosomal RNA gene, partial sequence’. Which was identical 95% to the formerly registered isolates in the NCBI and was registered under accession No. OR131302.

Effect of Culture Medium Composition on the Radial Growth of V. antarcticus

The evaluation of V. antarcticus growth across eight distinct culture media revealed significant variability in colony morphology and radial expansion rates. These variations reflect the organism’s physiological plasticity and specific nutritional requirements. The radial growth measurements, recorded after seven days of incubation, demonstrated a statistically significant response to the varying substrate compositions (L.S.D. _0.05 = 1.573; Figure 1).

The isolate exhibited maximal radial growth on Potato Dextrose Agar (PDA), achieving a diameter of 8.50 cm. This was followed by robust expansion on Malt Extract Agar (MEA), which reached 6.30 cm. This rapid proliferation underscores the fungus’s strong preference for simple carbohydrates, such as glucose and dextrose, as primary carbon sources. These findings align with the mechanism of Carbon Catabolite Repression (CCR), a regulatory phenomenon where the presence of preferred carbon sources supports rapid vegetative growth while suppressing the metabolic pathways required for less favorable substrates (3).

Intriguingly, V. antarcticus demonstrated a remarkable adaptive capacity to selective environments. Growth on MacConkey Agar (MA) reached 4.50 cm despite the presence of bile salts and crystal violet, which are typically inhibitory to many fungal species. This expansion suggests that V. antarcticus possesses inherent detoxification mechanisms or specialized cell wall properties that confer resistance to these selective agents (37).

A significant contrast was observed between the growth on Chocolate Agar (CA) (3.50 cm) and the minimal development on Blood Agar (BA) (0.50 cm). This difference (3.0 cm) is statistically significant as it exceeds the L.S.D. value. This indicates that while the fungus can utilize hydrolyzed proteins and growth factors available in lysed blood (CA), it likely lacks the potent hemolysins required to access nutrients in fresh, non-lysed blood, or its growth is inhibited by active serum factors present in the BA (38).

Fungal growth was notably restricted on other specialized media. On MR-VP medium, the colony diameter was limited to 2.50 cm, reflecting reduced efficiency in the specific glucose fermentation pathways favored by this formulation. Growth on Eosin Methylene Blue (EMB) Agar was further suppressed to 2.00 cm, likely due to the fungistatic effect of the methylene blue dye. Furthermore, the isolate showed poor development on Simmons Citrate Agar (SCA) (1.50 cm). Since SCA tests the ability to utilize citrate as the sole carbon source, the limited growth indicates that V. antarcticus lacks efficient transport systems, such as citrate permease, or the metabolic pathways necessary to rely exclusively on citrate. This further confirms the isolate’s primary reliance on saccharolytic nutritional sources.

Figure 1. Radial growth of Victoriomyces antarcticus on eight different culture media after seven days of incubation.

Pathogenicity of isolated fungi on Cucumber (Cucumis sativus)

Results in table (1) showed that F. solani significantly surpassed other treatments in reducing Cucumber seeds germination rate to 0.66 %, followed by R. solani with a germination rate of   0.00%. while significant effect was detected in V. antarcticus treatment. F. solani also resulted in 86.67% seedlings Mortality rate followed by R. solani with a seedlings rate of 100%, while V. antarcticus resulted in complete healthy seedlings. They may be due to the ability of V. antarcticus to produce phenols, saponin, tannins, flavonoids, and some organic acids such as p-coumaric acid, Ferulic acid, and Caffeic acid. At the same time, the study showed the negative effect of F. solani and R. solani fungi on the plant due to the ability of these fungi to secrete hydrolytic enzymes or secrete metabolites with a toxic effect in plants (31,32). the experiment results, V. antarcticus was studied for its moleculary diagnosed, and its l filtrate effect on F. solani and R. solani were studied.

Table (1) Pathogenicity of some Fungi on Seeds germination of Cucumber (Cucumis sativus) seeds on Water agar (W.A).

Effect of V. antarcticus filtrate on Cucumber seed germination

As for V. antarcticus filtrate effect on Cucumber seed germination, Findings (Figure1) showed that the percentage of seed germination differed between heat sterile and micro-filtered using Millipore filter (0.22 µm) filtrate V. antarcticus, and compared to the control treatment (distilled water DW). The Cucumber seeds treated with heat-sterile fungus filtrate gave the highest germination rate of 86.66% but did not differ from 96.66% and 100% seed germination resulted from seed treated with micro-filtered filtrate and DW control respectively. Perhaps the reason is that the seeds of the plant exploited the spores and units of the fungus V. antarcticus after breaking them with sterilization to encourage its growth, as it was found that the spores of fungi contain types of complex sugars, including mannan(s), which is a long chain of mannose sugar (33) as well as glucan, a long chain of glucose sugar and is one of the components of fungal spores. The third multiple sugar is chitin, where there is between 3-60% of the dry weight of the cell wall, there is a complex compound associated with the Glucan, which added nutrient to the medium that encouraged the germination rate of Cucumber (34).

The Effect of Heat-Sterile and Micro-Filtered Filtrates of the Fungus V. antarcticus on the Percentage of Cucumber Seed Germination

Detection of active substances in V. antarcticus filtrate

Based on the HPLC analysis, the concentration of secondary metabolite compounds of V. antarcticus  filterate was calculated and estimated, as the results of table (3)  indicated that the  heat-sterilized of  V. antarcticus filtrate was higher concentration of the compounds Phenols, Sugars, Tannins, Glycosides, Flavonoids, Saponins, Ferulic acid, Caffeic acid and phenolic acids than in  V. antarcticus filtrate sterilized  by Millipore filter and since these compounds are known for their high biological effectiveness, as  the reason for the effectiveness  of the heat-sterilized  V. antarcticus f filtrate  is attributed to the effectiveness of these  compounds,(34)  stated that Glycosides,  Alkaloids, Flavonoids, Terpenes, Amines, and Saponins are released to the environment by a number of microorganisms in the soil, and these compounds may suffer from chemical transformations as they may suffer from oxidation, decomposition, and polymerization that changes their nature and concentration, and some of the compounds can be associated with soil elements clay or humus, which may increase soil fertility, and some compounds such as phenolic acids can accumulate  in the soil to reach the level of toxicity (inhibitory effect) has shown a study(34), that the high concentrations of these compounds negatively affect the activity of some pathogenic soil organisms  , while the Saponins, which showed the results of their presence in   V. antarcticus filtrate sterilized by heat and/or Millipore filter, have an inhibitory effect. These compounds have been reported to affect division of chromosomes with clear impact on the plant physiological processes including germination. Glycosides and have a role in the physiological changes in the stage of germination as it is one of the materials necessary for metabolism. Alkaloids also found to affect cell division, while flavonoids affect hormones to reach the effective concentration to show their impact on the physiological and biochemical qualities necessary for the germination process including water absorption and stimulate seeds embryo, and enzymes activity, effect on osmotic potential and mitochondrial activity and cell membrane permeability (35, 36).

Table (3) HPLC Detection of active substances in V. antarcticus filtrate sterilized by Millipore filter or heat

Effect of Light and Dark Conditions on the Radial Growth of V. antarcticus

The investigation into the influence of photoperiod on the growth of V. antarcticus revealed a statistically significant gradient in mycelial radial expansion, directly correlated with light exposure time (P < 0.05). The radial growth measurements and   the corresponding morphological variations   recorded after seven days are summarized in Table 4   and illustrated in Figure 1, respectively.  The colonies incubated in Complete Darkness (Darkness) demonstrated the most robust proliferation, achieving a mean radial diameter of 9.0 cm (reaching the edge of the Petri dish), as clearly observed in the vigorous growth shown in Figure 2 (Darkness treatment).   This finding strongly suggests that complete darkness provides the optimal conditions for V. antarcticus vegetative mycelial growth. This rapid linear extension requires the full allocation of metabolic resources towards cell wall synthesis and hyphal extension. In the absence of photic cues, the stored energy is fully dedicated to radial growth, allowing the fungus to expand at its maximum possible rate. This result is consistent with studies on similar filamentous fungi, such as Ganoderma species, which often show maximal growth rates in the dark (39).  Conversely, Continuous Illumination (Continuous Light) at a density of 200-300 lux served as a potent inhibitory factor, resulting in the lowest recorded growth (4.5 cm). Statistically, the difference between the Dark and Continuous Light treatments was highly significant (P < 0.05), a disparity that is visually prominent when comparing the plates in Figure 2.   Light acts as an environmental signal perceived by fungi through photoreceptors. Typically, light promotes a switch from the vegetative phase to the reproductive phase (sporulation or fruiting body formation). Even when full sporulation is not achieved in Petri plates, the light cues result in the diversion of energy away from linear mycelial extension and suppress the growth rate. Studies have indicated that light may inhibit key enzymes related to nutrient assimilation and processing within the fungus, collectively leading to a significant reduction in the colony’s radial expansion (40)

The Alternating Photoperiod (12 h Light / 12 h Dark) treatment yielded an intermediate radial growth of 7.5 cm.   This intermediate growth pattern, shown in Figure 2, demonstrates the fungus’s capacity to partially recover its metabolic activity and vegetative expansion during the 12-hour dark cycle, compensating for the inhibition incurred during the 12-hour light period. This improved rate represents a functional average between the inhibitory and optimal conditions; a pattern observed in research on other fungal species where alternating cycles ranked second to continuous darkness (4). The results confirm that V. antarcticus adheres to the general physiological pattern of cold-tolerant filamentous fungi in its vegetative stage, where complete darkness is crucial for achieving the highest efficiency of mycelial growth. Continuous light, even at low intensity, significantly disrupts growth mechanisms and drastically reduces the growth rate (42)

Table 4: Influence of Different Photoperiod Regimes on the Radial Mycelial Growth of Victoriomyces antarcticus on Potato Dextrose Agar (PDA)

*Interpretation of Superscripts: Means followed by the same lowercase letter (a, b, c) are not significantly different at the P > 0.05 level according to Duncan’s Multiple Range Test.

Figure 2.  Radial mycelial growth and colony morphology of V. antarcticus under different photoperiod regimes (Complete Darkness, Complete Light, 12:12 h, and 8:16 h light/dark) on PDA at 20°C.

Impact of NaCl-Induced Osmotic Stress on the Radial Growth Dynamics of V. antarcticus

The experimental findings elucidated the physiological nature of V. antarcticus as a psychrotolerant, non-halophilic fungus. Mycelial radial growth exhibited a significant, dose-dependent inhibitory response to increasing concentrations of NaCl ($P < 0.05$), where salinity levels were inversely correlated with the colony expansion rate. Maximal mycelial growth was recorded in the control group (0 dS/m), with the colony diameter reaching the full plate capacity of 90.0 mm within a 10-day incubation period. This optimal performance underscores that dilute osmotic conditions are most conducive to rapid cellular metabolism and vegetative proliferation. A progressive decline in growth was observed at low salinity levels of 2 dS/m (83.33 mm) and 4 dS/m (75.66 mm), indicating that the isolate begins to perceive and respond to shifts in external osmotic potential even at low concentrations (43).

As salinity progressed to moderate levels (8–10 dS/m), a pronounced deceleration in radial expansion was observed. This retardation is scientifically attributed to a metabolic “energy trade-off”; the fungus is forced to divert its metabolic currency from hyphal elongation toward homeostatic defense mechanisms. Specifically, the isolate prioritizes the synthesis and intracellular accumulation of compatible solutes, such as glycerol, to sustain the turgor pressure required for apical growth and to prevent osmotic dehydration (44). Since metabolic resources are finite, this shift from biomass production to cellular osmoregulation inevitably results in reduced radial expansion.

The upper limit of halotolerance was reached at high salinity concentrations. Radial growth was severely restricted at 16 dS/m and reached a point of complete physiological arrest at 18–20 dS/m. These concentrations represent the critical osmotic threshold for the isolate. At this juncture, the extreme negative external osmotic potential surpasses the fungus’s compensatory capacity, leading to plasmolysis, acute cytoplasmic dehydration, and the total cessation of metabolic activity (43, 44). These results demonstrate that while V. antarcticus possesses a degree of resilience to mild salinity, its ecological fitness is strictly constrained by high saline gradients.

Table 1. Effect of increasing salinity levels on the radial growth and inhibition percentage of V. antarcticus after 10 days of incubation.

Impact of Hydrogen Ion Concentration (pH) on Mycelial Radial Growth

The experimental data unequivocally demonstrate that the hydrogen ion concentration (pH) of the growth medium exerts a profound influence on the vegetative development of V. antarcticus. The fungus exhibited a clear preference for slightly acidic to neutral environments, with the optimal growth range identified between pH 6.0 and 7.0. The maximum radial expansion was recorded at pH 6.0, where the colony reached its full plate capacity of 90.0 mm, followed closely by significant growth at pH 7.0 (85.0 mm), a trend that aligns with the established physiological profile of most fungal species typically flourishing within a pH range of 5.0 to 7.0 (45). This superior performance in the 6.0–7.0 range is scientifically attributed to optimal enzymatic activity, where key extracellular enzymes and membrane-bound transport proteins reach peak catalytic efficiency to ensure rapid nutrient uptake and metabolic flux, as well as enhanced nutrient bioavailability, as slightly acidic conditions facilitate the solubility and absorption of essential micronutrients, such as iron and zinc, which often precipitate and become less available in alkaline environments (45). Conversely, a notable decline in growth was observed as the medium shifted toward more extreme values. At pH 5.0, the radial growth was reduced to 70.0 mm, suggesting that while the isolate is acid-tolerant, it does not achieve its full metabolic potential compared to the slightly more moderate pH 6.0 (45).

The most significant inhibitory effects were observed under alkaline conditions. A sharp reduction in growth occurred at pH 8.0 (45.0 mm), culminating in the lowest recorded expansion at pH 9.0 (10.0 mm). This pronounced sensitivity to alkaline stress indicates that high pH levels act as a major limiting factor for V. antarcticus, as elevated pH levels trigger a cascade of detrimental effects, including enzyme inhibition through conformational changes in vital proteins that disrupt essential metabolic pathways, membrane instability caused by alkalinity-induced alterations in electrical charge and lipid composition that compromise permeability and transmembrane transport, and an ecological shift that favors bacterial dominance over fungal proliferation, further explaining the evolutionary preference of most fungi for acidic substrates (46).

Table 2. Influence of different pH levels on the mycelial radial growth and inhibition percentage of V. antarcticus after the incubation period.

Thermal Growth Dynamics and Optimization of V. Antarcticus

Statistical analysis via analysis of variance (ANOVA) indicated that temperature had a highly significant effect on the radial growth of V. antarcticus (P < 0.05). Given that all differences between the treatment means exceeded the Least Significant Difference value (L.S.D. _0.05 = 4.68), each thermal level induced a statistically distinct developmental response. The experimental results, summarized in Table 3, identify 20 C as the optimal temperature for the radial growth of V. antarcticus, where the colony reached its maximum diameter of   90.0 mm. This was followed by robust expansion at15 C (75.0 mm, a pattern that clearly classifies the isolate as a psychrotolerant fungus (47).

At lower thermal regimes of 10 C and 5 C, although growth was expectedly slower (30.0 mm and 15.0 mm respectively), the fungus maintained its developmental continuity. This persistence underscores the significant metabolic flexibility of the isolate. Such adaptation to cold stress is biologically achieved through the modification of cellular membrane lipid composition—specifically by increasing the proportion of unsaturated fatty acids to sustain membrane fluidity and ensure efficient nutrient transport. Furthermore, the fungus likely possesses enzymes with high structural flexibility, allowing them to remain catalytically active despite the low kinetic energy characteristic of 5 C environments (48).

Conversely, a sharp decline in growth was observed as temperatures increased beyond the optimum. Growth was significantly reduced at 25 C 50.0 mm and reached its minimum at 30 C (5.0 mm). This upper limit represents the Maximum Growth Temperature (MGT) or the thermal breakdown point for V. antarcticus. At 30 C, the fungus suffers from the denaturation of vital proteins and irreversible damage to cellular membranes. The severe growth inhibition at this temperature serves as a physiological indicator that the isolate’s thermal defense mechanisms have been overwhelmed, leading to metabolic failure (49).

Table 3. Effect of different incubation temperatures on the radial growth and thermal response of V. antarcticus.

The experimental data regarding the thermal requirements for V. antarcticus growth (Table 3) revealed a distinct psychrotolerant pattern. As visually evidenced in Figure (3), the radial growth exhibited a significant upward trend starting from 5°C, reaching its peak (8.50 cm) at 20°C. This optimal growth, clearly demonstrated by the dense and uniform mycelial expansion in the culture plates (Figure 2), suggests that the isolate is well-adapted to moderate and cool temperatures. Conversely, a marked decline was observed at 25°C, followed by complete cessation of growth at 30°C (Table 3, Figure 3). This thermal sensitivity at higher temperatures aligns with the physiological nature of this species, which is typically associated with cold environments, yet shows remarkable ecological plasticity in adapting to the tested conditions.

Figure 3. Effect of different incubation temperatures on the radial growth of V. antarcticus after 14 days of incubation.

Conclusions:

This study highlights the first successful isolation and characterization of Victoriomyces antarcticus (AF1) from the cucumber rhizosphere in Iraq, revealing its significant potential as a sustainable biocontrol agent. Our findings demonstrate that this psychrotolerant isolate achieves optimal mycelial growth at 20°C and pH 6.0, while exhibiting a robust antagonistic capacity against Rhizoctonia solani with an inhibition rate of 82.22%. The integration of HPLC analysis further elucidates the biochemical mechanism of this efficacy, identifying key phenolic metabolites, specifically Gallic and Caffeic acids, as the primary bioactive constituents. Consequently, V. antarcticus emerges as a novel, eco-friendly alternative to synthetic fungicides, offering a strategic tool for integrated pest management and the enhancement of sustainable cucumber production in temperate climates.

Recommendations:

Based on the promising findings of this study, it is highly recommended to transition from in vitro assays to greenhouse and open-field trials to evaluate the practical efficacy of V. antarcticus (AF1) in suppressing cucumber root-rot diseases under complex environmental conditions. Future research should prioritize the development of stable bio-fungicide formulations, such as granules or liquid concentrates, while exploring the synergistic potential of combining this isolate with other beneficial microbes or organic amendments to enhance its biocontrol performance. Furthermore, given the significant phenolic profile identified via HPLC, investigations into scaling up the production of Gallic and Caffeic acids in bioreactors are warranted. Finally, expanding the host range testing to include other economically vital crops and a broader spectrum of soil-borne pathogens will be essential to establish the versatility of V. antarcticus as a comprehensive, eco-friendly alternative to synthetic chemical fungicides in sustainable agriculture.

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