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Evaluation of the Efficacy of Truffle Fungi (Tirmania nivea and Terfezia claveryi) in the Mycosynthesis of Iron Nanoparticles for Controlling Bacterial Pathogens of Oyster Mushroom Pleurotus spp.

M
Maha Talib Ibrahim1,*
https://orcid.org/0009-0009-9552-6649
A
Abdullah Abdulkareem Hassan1
1Department of Plant Protection, College of Agriculture, Tikrit University, Republic of Iraq.

ABSTRACT

Background: Oyster mushrooms are edible fungi known for their nutritional and health benefits. However, these mushrooms are susceptible to bacterial diseases that reduce their yield in both quantity and quality.

Methods: Eight isolates of bacteria associated with oyster mushrooms were isolated, molecularly characterized and the effectiveness of iron nanoparticles prepared from the fungi, T. nivea and T. claveryi in inhibiting these bacteria was tested.

Result: Based on 16S rRNA gene sequencing, the pathogenic bacteria of oyster mushroom were identified as Pantoea pleuroti strain Iraq-20 (PV679803.1), Pseudomonas reactans strain Iraq-22 (PV679806.1), Pseudomonas agarici strain Iraq-21(PV679801.1) and Pseudomonas tolaasii strain Iraq-23 (PV679807.1). This study recorded three bacterial diseases on oyster mushrooms: Bacterial yellowing caused by P. agarici and P. reactans, soft rot caused by P. pleuroti and bacterial brown blotch caused by P. tolaasii.  The results of the synthesis of iron nanoparticles (FeNPs) from extracts of the fungi T. claveryi and T. nivea showed a change in the solution’s color from yellow to brown and exhibited a peak of 270 nm using spectral curves. The dimensions of the FeNPs prepared from T. claveryi and T. nivea ranged from (15-83 nm) and (14-91 nm), respectively. The results showed that the biologically prepared iron nanoparticles (FeNP) from T. claveryi and T. nivea were superior in their antibacterial activity compared to iron sulfate (FeSO4) at all studied concentrations (5-15) mM.

INTRODUCTION

The oyster mushroom (Pleurotus) is the second most widely cultivated edible mushroom in the world after the white mushroom (Agaricus bisporus). It is distinguished by its high nutritional and medicinal value, being a rich source of proteins, vitamins and minerals. Furthermore, it contains bioactive compounds such as beta-glucans and polyphenols, which possess antioxidant, anti-tumor and anti-inflammatory properties. This has made it the focus of numerous therapeutic and nutritional studies (Ayuso et al., 2025). Oyster mushroom cultivation is susceptible to serious bacterial infections. Several types of pathogenic bacteria infect oyster mushrooms, resulting in significant economic losses. These include P. reactans, Pseudomonas agarici, Ewingella americana and P. tolaasii, which cause brown spot disease. Symptoms of this disease begin with the appearance of brown spots on the fruiting bodies, which gradually develop into deeper, more sunken brown spots, leading to a decline in mushroom quality and reduced yields (Okorley et al., 2019; Jung et al., 2026).
       
Desert truffles are symbiotic rhizomatous fungi widespread in the Middle East, North Africa and Europe. In Iraq, several species are found, including Terfezia claveryi and Tirmania nivea, which grow in desert regions in a symbiotic relationship with the roots of plants of the Cistaceae family. Studies have also demonstrated that these two species possess a wide range of biological activities, including antioxidant, antibacterial, antiviral and anti-inflammatory properties, as well as hepatoprotective properties. This makes them a promising natural resource for pharmaceutical, nutritional and technological applications (Veeraraghavan et al., 2022).
       
Nanotechnology is defined as the science concerned with the synthesis, engineering and application of materials with dimensions ranging from 1 to 100 nanometers. These materials are known as nanoparticles, which possess unique physical, chemical and biological properties that differ significantly from those of their original materials. Nanoparticles have become increasingly important in various sectors, including the environment, agriculture, food, biotechnology and medicine. The importance of iron nanoparticles in the field of agriculture lies in improving the growth and yield of plants such as wheat (Triticum aestivum) (Nazma et al., 2025) and tomatoes (Solanum lycopersicum) (Sharma et al., 2022) and improving the growth of fish (Koi Carp) (Sangeetha and Rajan, 2021). They have also found prominent applications in treating various diseases, monitoring the environment, developing food additives and producing antimicrobial agents (Altammar, 2023; Eker et al., 2024). Furthermore, truffle extracts have been shown to inhibit the growth of Staphylococcus aureus and Escherichia coli bacteria (Karaogul and Hussein, 2025). Aqueous extracts of the truffle fungi T. nivea and T. claveryi inhibit the growth of Staphylococcus aureus bacteria (Gouzi et al., 2011). The current study aims to evaluate the efficacy of extracts from the truffle fungi T. nivea and T. claveryi in the biosynthesis of iron nanoparticles and to compare the efficiency of the two species in this process, as well as to assess their antibacterial activity against oyster mushroom pathogens.

MATERIALS AND METHODS

The study was conducted in the laboratories of the College of Agriculture, Department of Plant Protection and, College of Agriculture -Mushroom Farm at Tikrit university/ Iraq, during 2025/2026. Two species of Iraqi truffle fungus, T. claveryi and T. nivea, were used, isolated and characterized morphologically and molecularly according to the study (Hassan et al., 2026).
 
Isolation and identification of pathogenic bacteria from oyster mushrooms
 
The bacteria were isolated from the fruiting bodies of cultured oyster mushrooms in the mushroom farm- College of Agriculture, Tikrit University during the production cycles from 2024-2026. Fruiting bodies were collected from the cultured P. pulmonarius that showed various pathological symptoms, represented by yellow and brown spots, in the form of spots and blotches, as well as soft, watery, decomposing areas. The fruiting bodies were superficially sterilized with 3% sodium hypochlorite solution for two minutes, then washed with sterile distilled water. The infected areas were then cut at 1 cm2 intervals and cultured on nutrient agar (NA) medium and incubated at a temperature of 25°C for 24-48 hours. After growth was complete, the isolates were purified and initially classified according to colony color, texture and size for later use in subsequent tests (Hassan and Ibrahim, 2023).
 
Pathogenicity test of bacteria on oyster mushroom
 
The test was performed by treating the fruiting bodies of the oyster mushroom P. pulmonarius with a suspension of bacterial isolates. A freshly grown, active colony (24 hours old) was used for each bacterial isolate. This was done by adding 30 ml of sterile distilled water to the dish containing the colony. Using sterile cotton swabs, the colonies were harvested and the suspension concentration was adjusted using a cell counting slide to 108 colony-forming units/ml. The fruiting bodies of P. pulmonarius (intact and sterilized with 3% sodium hypochlorite for 2 minutes, then washed with sterile distilled water) were immersed in the bacterial suspension of each bacterial isolate, with 30 fruiting bodies per isolate. They were then incubated in the dark (culture conditions) at 25°C. After incubation, the infection rate was calculated using the following equation:


The fruiting bodies were considered infected through the appearance of spotting and/or areas of decay and soft rot.
 
Molecular diagnosis of bacteria
 
Extraction of genomic DNA, polymerase chain reaction (PCR) and nucleotide sequence analysis was achieved according to Hassan et al., (2026). Nucleotide sequence analysis was performed at the Korean Biotechnology Laboratory using an Applied Biosystems 3730XL DNA Sequencer. The resulting sequences were compared using software connected to the Basic Local Alignment Search (BLAST) online database with the NCBI website. BLAST enables the matching of nucleotide sequences to determine the bacterial species.
 
Preparation of the aqueous extract of iraqi truffles (T. claveryi and T. nivea)
 
The preparation process begins with a 1:4 ratio, where 100 g of truffles are mixed with 400 ml of distilled water using an electric mixer at a slow speed to ensure a homogeneous solution. This mixture is then passed through three layers of cheesecloth to filter out large impurities. Next, it is centrifuged at 4000 rpm for 15 minutes to remove any remaining fine sediment and obtain a completely pure filtrate. Finally, this filtrate is placed in a rotary evaporator for concentration, maintaining a temperature of 40°C to preserve the chemical properties of the extract and prevent heat damage. Evaporation continues until 100 ml is obtained (Karaogul and Hussein, 2025).
 
Preparation of stock solution
 
To prepare the stock solution, 278 g of hydrated ferrous sulfate (FeSO4•7H2O) was dissolved in 1000 mL of distilled water with thorough stirring until complete dissolution, yielding 1 M of hydrated ferrous sulfate solution.
 
Preparation of iron nanoparticles (FeNPs)
 
The biosynthesis of iron nanoparticles was carried out using a 1:10 mixing ratio of fungal extract to ferrous sulfate solution at concentrations of 5, 10 and 15 mM (prepared from the stock solution). Two mL of fungal extract was added to 20 mL of each concentration. The three solutions were continuously stirred for 8 hours at 60-70°C, followed by an additional 24 hours of stirring at 37°C (Batool et al., 2021).
 
Properties of prepared iron nanoparticles
 
Colorimetric analysis, wavelength determination (visible-ultraviolet) and electron microscopy were performed on the prepared iron nanoparticle solutions according to the methods described previously (Hassan and Sarhan, 2023).
 
Effect of iron nanoparticles prepared from the fungi T. claveryi and T. nivea in inhibiting oyster fungus pathogenic bacteria
 
The diffusion method was used to determine the effect of prepared iron nanoparticles at concentrations of 5, 10 and 15 mM against oyster fungus pathogenic bacteria (with the same concentrations of hydrated ferrous sulfate prepared as a control). The bacteria were dispersed in a dish (containing NA medium) and 5 mm diameter wells were made in the medium. 0.1 mL of each of these was added. The diameter of the inhibition zone around the wells was measured after incubation at 25°C for 24 hours and the area of the inhibition zone was determined.
 
Statistical analysis
 
The experiment was conducted using a completely randomized design and variance was analyzed using SPSS software. Means were compared using Duncan’s multiple range test at a significance level of 0.05 (Al-Rawi and Khalafallah, 1980).

RESULTS AND DISCUSSION

Eight bacterial isolates were isolated from the fruiting bodies of P. pulmonarius fungus infected with symptoms of bacterial diseases. The pathogenicity results shown in Fig 1 indicate that the highest infection rate was recorded by isolates C2, C3, S1 and S4, with infection rates of 87.42%, 82.95%, 80.06% and 77.28% respectively. The lowest infection rate was for isolate S3 at 12.37%, while isolates S2, C4 and C1 did not show any pathogenicity (infection rate was 0). One of the reasons for the variation in bacterial infection may be attributed to the fact that they are different types and each type has a genetic structure and its genetic difference is reflected in its pathogenic factors. One of the most important virulence factors is the enzymes that break down the tissues of the oyster fungus, such as enzymes (protease, chitinase and glucanase). Such enzymes have the main role in attacking the host tissues. Another reason may be due to the production of bacterial toxins, such as the tolaasin toxin, which is an extracellular bacterial toxin that attacks the tissues of the fungus and overcomes its defense mechanisms by forming pores in the cell membranes and destroying their structure (Xu et al., 2023; Yun et al., 2023).

Fig 1: Pathogenicity of bacterial isolates associated with oyster mushroom diseases estimated by the percentage of infection.


 
Molecular diagnosis of isolated bacteria
 
According to 16S rRNA gene sequencing, the most pathogenic bacteria were identified as four strains: p. pleuroti strain Iraq-20, P. reactans strain Iraq-22, P. agarici strain Iraq-21 and P. tolaasii strain Iraq-23. (Table 1) shows the percentage of similarity of these isolates to the highest percentage of similarity with globally registered bacterial strains and their corresponding numbers in the NCBI GeneBank database, with similarity rates ranging from 99.31% to 99.50%. These bacteria are registered in the NCBI under accession numbers PV679803.1, PV679806.1, PV679801.1 and PV679807.1, respectively.

Table 1: Molecular diagnosis of pathogenic bacteria isolates on oyster mushrooms based on the percentage of 16S rRNA gene sequences matching bacterial strains in the World GeneBank.


 
Pathological symptoms of isolated bacterial species
 
Bacterial yellowing
 
Appearance of yellowish spots and blotches that become brownish and merge together as the infection progresses. The infection appears on the cap and along the entire stem (Fig 2A). Causal agent: P. agarici strain Iraq-21 and P. reactans strain Iraq-22.

Fig 2: Pathological symptoms of the isolated bacterial species.


 
Soft rot
 
Symptoms of water-soaked spots on the cap and stems. The general symptoms of this disease can be seen as light brown, water-soaked spots that darken in the early stages and develop into large, water-soaked spots, leading to a foul odor in severe infections during growth (Fig 2B). Causal agent: P. pleuroti strain Iraq-20.
 
Bacterial brown blotch
 
Appearance of light to dark brown spots that spread mainly on the mushroom cap (Fig 2C). Causal agent: P. tolaasii strain Iraq-23.
       
The pathological symptoms clearly show the variation between different bacteria and these results are consistent with previous studies on The bacterial pathogens of the fungus Pleurotus include multiple species associated with a wide range of different symptoms and each bacterial species has a specific pathogenic mechanism that is reflected in the nature of the symptoms appearing on the fruiting bodies and they differ substantially when each is individually infected (Wang et al., 2024; Wei et al., 2025). The pathological symptoms caused by the isolated bacterial species are presented in Fig 2.

Properties of iron nanoparticles
 
Color variation
 
The results in the Fig 3 show a change in the solution’s color from yellow to brown, indicating the reduction of iron ions by phenolic and flavonoid compounds in the truffle extract, which acted as both reducing and coating agents. The results also showed that the extract of the fungus T. claveryi exhibited a darker color variation than the extract of the fungus T. nivea at all concentrations. This is likely due to its higher phenolic content. The flavonoid content led to a higher reduction than that of the T. nivea mushroom extract.

Fig 3: Color variation of ferrous sulfate solution from yellow to brown due to truffle fungal extracts.


       
The browning of aqueous ferrous sulfate solutions is indicative of the formation of iron nanoparticles, consistent with previous studies, confirmed that the browning and subsequent precipitation are caused by the reaction of phytochemicals such as tannins, flavonoids and phenols with metal ions, acting as both reducing and stabilizing agents during synthesis (Mahlaule-Glory et al., 2022). This aligns with the general principle of biosynthesis, where chemical compounds such as phenols and flavonoids act as both reducing and encapsulating agents (Ali, 2026).
 
Wavelengths of iron nanoparticles
 
The nanoparticles prepared using the fungi T. claveryi and T. nivea showed similar wavelength curves for both fungi at concentrations of 5, 10 and 15. The results (Fig 4) indicate that the concentrations used produced the highest peak in the spectral curves, reaching 270 nm in the ultraviolet range. In contrast, non-nanoparticle iron at the same concentrations showed a highest peak at 390 nm. This suggests that the truffle extracts led to the formation of nanoparticles, based on wavelengths close to ultraviolet, whereas the non-nanoparticle iron exhibited an absorption peak at 390 nm, closer to the visible region of the spectrum. Visible light is one of the pieces of evidence confirming the presence of nanoparticles in the fungi when the resulting peaks are in the ultraviolet range. These results are consistent with the study by Hussain et al., (2023) and Alzubaidy (2024), who demonstrated that the biosynthesis of iron oxide nanoparticles using Beauveria bassiana extract resulted in a peak recorded at 282 nm in the ultraviolet range.

Fig 4: Wavelengths of nanoparticles prepared using the fungi T. claveryi and T. nivea.


 
Electron microscopy
 
The shapes and dimensions of the iron nanoparticles were determined using electron microscopy (Fig 5). The particles prepared from the fungus T. claveryi ranged in size from 15 to 83 nm, mostly spherical. The particles prepared from the fungus T. nivea ranged in size from 14 to 91 nm, also spherical, with some exhibiting cubical shapes. Overall, extracts from both fungi resulted in the formation of nanoparticles with dimensions within the nanoscale range. These results are consistent with those of Bhargava et al. (2014), who demonstrated that extracellular fungal synthesis of iron nanoparticles using Aspergillus japonicus produced cubic particles ranging in size from 60 to 70 nm with a crystalline structure, confirming the role of fungal proteins in nanoparticle synthesis and stabilization. The properties of iron nanoparticles are determined by chemical and physical factors, including size, shape, surface charge and paramagnetic behavior. These properties collectively influence their biological distribution and antibacterial activity (Hetta et al., 2021).

Fig 5: Electron microscopy images of nanoparticles prepared using the fungi T. claveryi and T. nivea (Ti.n: T. nivea, Te.c: T. claveryi).


 
Effect of FeNPs from the fungus T. claveryi on inhibiting pathogenic bacteria of the oyster mushroom
 
The results in Fig 6 showed that all concentrations of iron nanoparticles were more effective against all tested bacteria than non-nanoparticle iron sulfate. At 15 mM, the inhibition zones ranged from 2.76 to 3.33 cm for FeNPs and from 1.07 to 1.64 cm for FeSO4. Fig. 8 illustrates the inhibitory effect of T. claveryi-derived FeNPs at 5-15 mM against P. agarici, compared with 15 mM FeSO4.

Fig 6: Effect of iron nanoparticles prepared from the truffle fungus T. claveryi in inhibiting four types of bacteria pathogenic to the oyster mushroom Pleurotus.


 
Effect of FeNPs from the fungus T. nivea on inhibiting pathogenic bacteria of the oyster mushroom
 
As shown in Fig 7, FeNPs prepared from T. nivea were more effective than FeSO4 against all tested bacteria. At 15 mM, the inhibition zones ranged from 2.45 to 3.02 cm for FeNPs and from 0.93 to 1.50 cm for FeSO4.

Fig 7: Effect of iron nanoparticles prepared from the truffle fungus T. nivea in inhibiting four types of bacteria pathogenic to the oyster mushroom Pleurotus.


       
Fig 8 illustrates the effect of concentrations (5-15 mM) of iron nanoparticles prepared from the fungus T. claveryi, which showed the highest efficacy against the bacterium P. agarici, compared to a concentration of 15 mM of non-nanoparticle iron sulfate (FeSO4).

Fig 8: The effect of iron nanoparticles prepared from the fungus T. claveryi was superior against the bacterium P. agarici.


       
Furthermore, biosynthesis using T. claveryi may impart additional properties to these particles due to the associated bioactive compounds (Ahmadi et al., 2021; Mohamed-Benkada et al., 2024). The variation in susceptibility among the four bacterial species may be attributed to differences in cell wall structure and resistance mechanisms (Zúñiga-Miranda et al., 2023).
       
This superiority of iron nanoparticles can be explained by their nanoscale dimensions and high surface area to volume ratio, which increases the area of interaction with bacterial cells and enhances surface interactions with them. This can lead to disruption of cell membrane integrity and increased generation of reactive oxygen species (ROS), which trigger a cascade of cellular damage including lipid peroxidation in the cell membrane, DNA damage and impaired replication, protein oxidation and disruption of several cellular systems and enzymes (Natan and Banin, 2017; Shoudho et al., 2024). Direct interaction between nanoparticles and the cell surface can also contribute to cell membrane disruption and some particles may penetrate the cell, leading to cellular homeostasis and metabolic disturbances, thereby inhibiting bacterial growth (Li et al., 2018).

CONCLUSION

We conclude from the current study that the bacteria P. pleuroti, Pseudomonas reactans, P. agarici and P. tolaasii are responsible for diseases in oyster mushroom. Additionally, iron nanoparticles were successfully biosynthesized using extracts from the truffle fungi T. claveryi and T. nivea, yielding stable, uniform nanoscale particles. These bio-synthesized nanoparticles demonstrated superior inhibitory activity against oyster mushroom pathogenic bacteria compared to the conventional ferrous sulfate source, highlighting their strong potential for biological applications and disease control.

CONFLICT OF INTEREST

There is no conflict of interest.

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    Full Research Article

    Evaluation of the Efficacy of Truffle Fungi (Tirmania nivea and Terfezia claveryi) in the Mycosynthesis of Iron Nanoparticles for Controlling Bacterial Pathogens of Oyster Mushroom Pleurotus spp.

    M
    Maha Talib Ibrahim1,*
    https://orcid.org/0009-0009-9552-6649
    A
    Abdullah Abdulkareem Hassan1
    1Department of Plant Protection, College of Agriculture, Tikrit University, Republic of Iraq.

    ABSTRACT

    Background: Oyster mushrooms are edible fungi known for their nutritional and health benefits. However, these mushrooms are susceptible to bacterial diseases that reduce their yield in both quantity and quality.

    Methods: Eight isolates of bacteria associated with oyster mushrooms were isolated, molecularly characterized and the effectiveness of iron nanoparticles prepared from the fungi, T. nivea and T. claveryi in inhibiting these bacteria was tested.

    Result: Based on 16S rRNA gene sequencing, the pathogenic bacteria of oyster mushroom were identified as Pantoea pleuroti strain Iraq-20 (PV679803.1), Pseudomonas reactans strain Iraq-22 (PV679806.1), Pseudomonas agarici strain Iraq-21(PV679801.1) and Pseudomonas tolaasii strain Iraq-23 (PV679807.1). This study recorded three bacterial diseases on oyster mushrooms: Bacterial yellowing caused by P. agarici and P. reactans, soft rot caused by P. pleuroti and bacterial brown blotch caused by P. tolaasii.  The results of the synthesis of iron nanoparticles (FeNPs) from extracts of the fungi T. claveryi and T. nivea showed a change in the solution’s color from yellow to brown and exhibited a peak of 270 nm using spectral curves. The dimensions of the FeNPs prepared from T. claveryi and T. nivea ranged from (15-83 nm) and (14-91 nm), respectively. The results showed that the biologically prepared iron nanoparticles (FeNP) from T. claveryi and T. nivea were superior in their antibacterial activity compared to iron sulfate (FeSO4) at all studied concentrations (5-15) mM.

    INTRODUCTION

    The oyster mushroom (Pleurotus) is the second most widely cultivated edible mushroom in the world after the white mushroom (Agaricus bisporus). It is distinguished by its high nutritional and medicinal value, being a rich source of proteins, vitamins and minerals. Furthermore, it contains bioactive compounds such as beta-glucans and polyphenols, which possess antioxidant, anti-tumor and anti-inflammatory properties. This has made it the focus of numerous therapeutic and nutritional studies (Ayuso et al., 2025). Oyster mushroom cultivation is susceptible to serious bacterial infections. Several types of pathogenic bacteria infect oyster mushrooms, resulting in significant economic losses. These include P. reactans, Pseudomonas agarici, Ewingella americana and P. tolaasii, which cause brown spot disease. Symptoms of this disease begin with the appearance of brown spots on the fruiting bodies, which gradually develop into deeper, more sunken brown spots, leading to a decline in mushroom quality and reduced yields (Okorley et al., 2019; Jung et al., 2026).
           
    Desert truffles are symbiotic rhizomatous fungi widespread in the Middle East, North Africa and Europe. In Iraq, several species are found, including Terfezia claveryi and Tirmania nivea, which grow in desert regions in a symbiotic relationship with the roots of plants of the Cistaceae family. Studies have also demonstrated that these two species possess a wide range of biological activities, including antioxidant, antibacterial, antiviral and anti-inflammatory properties, as well as hepatoprotective properties. This makes them a promising natural resource for pharmaceutical, nutritional and technological applications (Veeraraghavan et al., 2022).
           
    Nanotechnology is defined as the science concerned with the synthesis, engineering and application of materials with dimensions ranging from 1 to 100 nanometers. These materials are known as nanoparticles, which possess unique physical, chemical and biological properties that differ significantly from those of their original materials. Nanoparticles have become increasingly important in various sectors, including the environment, agriculture, food, biotechnology and medicine. The importance of iron nanoparticles in the field of agriculture lies in improving the growth and yield of plants such as wheat (Triticum aestivum) (Nazma et al., 2025) and tomatoes (Solanum lycopersicum) (Sharma et al., 2022) and improving the growth of fish (Koi Carp) (Sangeetha and Rajan, 2021). They have also found prominent applications in treating various diseases, monitoring the environment, developing food additives and producing antimicrobial agents (Altammar, 2023; Eker et al., 2024). Furthermore, truffle extracts have been shown to inhibit the growth of Staphylococcus aureus and Escherichia coli bacteria (Karaogul and Hussein, 2025). Aqueous extracts of the truffle fungi T. nivea and T. claveryi inhibit the growth of Staphylococcus aureus bacteria (Gouzi et al., 2011). The current study aims to evaluate the efficacy of extracts from the truffle fungi T. nivea and T. claveryi in the biosynthesis of iron nanoparticles and to compare the efficiency of the two species in this process, as well as to assess their antibacterial activity against oyster mushroom pathogens.

    MATERIALS AND METHODS

    The study was conducted in the laboratories of the College of Agriculture, Department of Plant Protection and, College of Agriculture -Mushroom Farm at Tikrit university/ Iraq, during 2025/2026. Two species of Iraqi truffle fungus, T. claveryi and T. nivea, were used, isolated and characterized morphologically and molecularly according to the study (Hassan et al., 2026).
     
    Isolation and identification of pathogenic bacteria from oyster mushrooms
     
    The bacteria were isolated from the fruiting bodies of cultured oyster mushrooms in the mushroom farm- College of Agriculture, Tikrit University during the production cycles from 2024-2026. Fruiting bodies were collected from the cultured P. pulmonarius that showed various pathological symptoms, represented by yellow and brown spots, in the form of spots and blotches, as well as soft, watery, decomposing areas. The fruiting bodies were superficially sterilized with 3% sodium hypochlorite solution for two minutes, then washed with sterile distilled water. The infected areas were then cut at 1 cm2 intervals and cultured on nutrient agar (NA) medium and incubated at a temperature of 25°C for 24-48 hours. After growth was complete, the isolates were purified and initially classified according to colony color, texture and size for later use in subsequent tests (Hassan and Ibrahim, 2023).
     
    Pathogenicity test of bacteria on oyster mushroom
     
    The test was performed by treating the fruiting bodies of the oyster mushroom P. pulmonarius with a suspension of bacterial isolates. A freshly grown, active colony (24 hours old) was used for each bacterial isolate. This was done by adding 30 ml of sterile distilled water to the dish containing the colony. Using sterile cotton swabs, the colonies were harvested and the suspension concentration was adjusted using a cell counting slide to 108 colony-forming units/ml. The fruiting bodies of P. pulmonarius (intact and sterilized with 3% sodium hypochlorite for 2 minutes, then washed with sterile distilled water) were immersed in the bacterial suspension of each bacterial isolate, with 30 fruiting bodies per isolate. They were then incubated in the dark (culture conditions) at 25°C. After incubation, the infection rate was calculated using the following equation:


    The fruiting bodies were considered infected through the appearance of spotting and/or areas of decay and soft rot.
     
    Molecular diagnosis of bacteria
     
    Extraction of genomic DNA, polymerase chain reaction (PCR) and nucleotide sequence analysis was achieved according to Hassan et al., (2026). Nucleotide sequence analysis was performed at the Korean Biotechnology Laboratory using an Applied Biosystems 3730XL DNA Sequencer. The resulting sequences were compared using software connected to the Basic Local Alignment Search (BLAST) online database with the NCBI website. BLAST enables the matching of nucleotide sequences to determine the bacterial species.
     
    Preparation of the aqueous extract of iraqi truffles (T. claveryi and T. nivea)
     
    The preparation process begins with a 1:4 ratio, where 100 g of truffles are mixed with 400 ml of distilled water using an electric mixer at a slow speed to ensure a homogeneous solution. This mixture is then passed through three layers of cheesecloth to filter out large impurities. Next, it is centrifuged at 4000 rpm for 15 minutes to remove any remaining fine sediment and obtain a completely pure filtrate. Finally, this filtrate is placed in a rotary evaporator for concentration, maintaining a temperature of 40°C to preserve the chemical properties of the extract and prevent heat damage. Evaporation continues until 100 ml is obtained (Karaogul and Hussein, 2025).
     
    Preparation of stock solution
     
    To prepare the stock solution, 278 g of hydrated ferrous sulfate (FeSO4•7H2O) was dissolved in 1000 mL of distilled water with thorough stirring until complete dissolution, yielding 1 M of hydrated ferrous sulfate solution.
     
    Preparation of iron nanoparticles (FeNPs)
     
    The biosynthesis of iron nanoparticles was carried out using a 1:10 mixing ratio of fungal extract to ferrous sulfate solution at concentrations of 5, 10 and 15 mM (prepared from the stock solution). Two mL of fungal extract was added to 20 mL of each concentration. The three solutions were continuously stirred for 8 hours at 60-70°C, followed by an additional 24 hours of stirring at 37°C (Batool et al., 2021).
     
    Properties of prepared iron nanoparticles
     
    Colorimetric analysis, wavelength determination (visible-ultraviolet) and electron microscopy were performed on the prepared iron nanoparticle solutions according to the methods described previously (Hassan and Sarhan, 2023).
     
    Effect of iron nanoparticles prepared from the fungi T. claveryi and T. nivea in inhibiting oyster fungus pathogenic bacteria
     
    The diffusion method was used to determine the effect of prepared iron nanoparticles at concentrations of 5, 10 and 15 mM against oyster fungus pathogenic bacteria (with the same concentrations of hydrated ferrous sulfate prepared as a control). The bacteria were dispersed in a dish (containing NA medium) and 5 mm diameter wells were made in the medium. 0.1 mL of each of these was added. The diameter of the inhibition zone around the wells was measured after incubation at 25°C for 24 hours and the area of the inhibition zone was determined.
     
    Statistical analysis
     
    The experiment was conducted using a completely randomized design and variance was analyzed using SPSS software. Means were compared using Duncan’s multiple range test at a significance level of 0.05 (Al-Rawi and Khalafallah, 1980).

    RESULTS AND DISCUSSION

    Eight bacterial isolates were isolated from the fruiting bodies of P. pulmonarius fungus infected with symptoms of bacterial diseases. The pathogenicity results shown in Fig 1 indicate that the highest infection rate was recorded by isolates C2, C3, S1 and S4, with infection rates of 87.42%, 82.95%, 80.06% and 77.28% respectively. The lowest infection rate was for isolate S3 at 12.37%, while isolates S2, C4 and C1 did not show any pathogenicity (infection rate was 0). One of the reasons for the variation in bacterial infection may be attributed to the fact that they are different types and each type has a genetic structure and its genetic difference is reflected in its pathogenic factors. One of the most important virulence factors is the enzymes that break down the tissues of the oyster fungus, such as enzymes (protease, chitinase and glucanase). Such enzymes have the main role in attacking the host tissues. Another reason may be due to the production of bacterial toxins, such as the tolaasin toxin, which is an extracellular bacterial toxin that attacks the tissues of the fungus and overcomes its defense mechanisms by forming pores in the cell membranes and destroying their structure (Xu et al., 2023; Yun et al., 2023).

    Fig 1: Pathogenicity of bacterial isolates associated with oyster mushroom diseases estimated by the percentage of infection.


     
    Molecular diagnosis of isolated bacteria
     
    According to 16S rRNA gene sequencing, the most pathogenic bacteria were identified as four strains: p. pleuroti strain Iraq-20, P. reactans strain Iraq-22, P. agarici strain Iraq-21 and P. tolaasii strain Iraq-23. (Table 1) shows the percentage of similarity of these isolates to the highest percentage of similarity with globally registered bacterial strains and their corresponding numbers in the NCBI GeneBank database, with similarity rates ranging from 99.31% to 99.50%. These bacteria are registered in the NCBI under accession numbers PV679803.1, PV679806.1, PV679801.1 and PV679807.1, respectively.

    Table 1: Molecular diagnosis of pathogenic bacteria isolates on oyster mushrooms based on the percentage of 16S rRNA gene sequences matching bacterial strains in the World GeneBank.


     
    Pathological symptoms of isolated bacterial species
     
    Bacterial yellowing
     
    Appearance of yellowish spots and blotches that become brownish and merge together as the infection progresses. The infection appears on the cap and along the entire stem (Fig 2A). Causal agent: P. agarici strain Iraq-21 and P. reactans strain Iraq-22.

    Fig 2: Pathological symptoms of the isolated bacterial species.


     
    Soft rot
     
    Symptoms of water-soaked spots on the cap and stems. The general symptoms of this disease can be seen as light brown, water-soaked spots that darken in the early stages and develop into large, water-soaked spots, leading to a foul odor in severe infections during growth (Fig 2B). Causal agent: P. pleuroti strain Iraq-20.
     
    Bacterial brown blotch
     
    Appearance of light to dark brown spots that spread mainly on the mushroom cap (Fig 2C). Causal agent: P. tolaasii strain Iraq-23.
           
    The pathological symptoms clearly show the variation between different bacteria and these results are consistent with previous studies on The bacterial pathogens of the fungus Pleurotus include multiple species associated with a wide range of different symptoms and each bacterial species has a specific pathogenic mechanism that is reflected in the nature of the symptoms appearing on the fruiting bodies and they differ substantially when each is individually infected (Wang et al., 2024; Wei et al., 2025). The pathological symptoms caused by the isolated bacterial species are presented in Fig 2.

    Properties of iron nanoparticles
     
    Color variation
     
    The results in the Fig 3 show a change in the solution’s color from yellow to brown, indicating the reduction of iron ions by phenolic and flavonoid compounds in the truffle extract, which acted as both reducing and coating agents. The results also showed that the extract of the fungus T. claveryi exhibited a darker color variation than the extract of the fungus T. nivea at all concentrations. This is likely due to its higher phenolic content. The flavonoid content led to a higher reduction than that of the T. nivea mushroom extract.

    Fig 3: Color variation of ferrous sulfate solution from yellow to brown due to truffle fungal extracts.


           
    The browning of aqueous ferrous sulfate solutions is indicative of the formation of iron nanoparticles, consistent with previous studies, confirmed that the browning and subsequent precipitation are caused by the reaction of phytochemicals such as tannins, flavonoids and phenols with metal ions, acting as both reducing and stabilizing agents during synthesis (Mahlaule-Glory et al., 2022). This aligns with the general principle of biosynthesis, where chemical compounds such as phenols and flavonoids act as both reducing and encapsulating agents (Ali, 2026).
     
    Wavelengths of iron nanoparticles
     
    The nanoparticles prepared using the fungi T. claveryi and T. nivea showed similar wavelength curves for both fungi at concentrations of 5, 10 and 15. The results (Fig 4) indicate that the concentrations used produced the highest peak in the spectral curves, reaching 270 nm in the ultraviolet range. In contrast, non-nanoparticle iron at the same concentrations showed a highest peak at 390 nm. This suggests that the truffle extracts led to the formation of nanoparticles, based on wavelengths close to ultraviolet, whereas the non-nanoparticle iron exhibited an absorption peak at 390 nm, closer to the visible region of the spectrum. Visible light is one of the pieces of evidence confirming the presence of nanoparticles in the fungi when the resulting peaks are in the ultraviolet range. These results are consistent with the study by Hussain et al., (2023) and Alzubaidy (2024), who demonstrated that the biosynthesis of iron oxide nanoparticles using Beauveria bassiana extract resulted in a peak recorded at 282 nm in the ultraviolet range.

    Fig 4: Wavelengths of nanoparticles prepared using the fungi T. claveryi and T. nivea.


     
    Electron microscopy
     
    The shapes and dimensions of the iron nanoparticles were determined using electron microscopy (Fig 5). The particles prepared from the fungus T. claveryi ranged in size from 15 to 83 nm, mostly spherical. The particles prepared from the fungus T. nivea ranged in size from 14 to 91 nm, also spherical, with some exhibiting cubical shapes. Overall, extracts from both fungi resulted in the formation of nanoparticles with dimensions within the nanoscale range. These results are consistent with those of Bhargava et al. (2014), who demonstrated that extracellular fungal synthesis of iron nanoparticles using Aspergillus japonicus produced cubic particles ranging in size from 60 to 70 nm with a crystalline structure, confirming the role of fungal proteins in nanoparticle synthesis and stabilization. The properties of iron nanoparticles are determined by chemical and physical factors, including size, shape, surface charge and paramagnetic behavior. These properties collectively influence their biological distribution and antibacterial activity (Hetta et al., 2021).

    Fig 5: Electron microscopy images of nanoparticles prepared using the fungi T. claveryi and T. nivea (Ti.n: T. nivea, Te.c: T. claveryi).


     
    Effect of FeNPs from the fungus T. claveryi on inhibiting pathogenic bacteria of the oyster mushroom
     
    The results in Fig 6 showed that all concentrations of iron nanoparticles were more effective against all tested bacteria than non-nanoparticle iron sulfate. At 15 mM, the inhibition zones ranged from 2.76 to 3.33 cm for FeNPs and from 1.07 to 1.64 cm for FeSO4. Fig. 8 illustrates the inhibitory effect of T. claveryi-derived FeNPs at 5-15 mM against P. agarici, compared with 15 mM FeSO4.

    Fig 6: Effect of iron nanoparticles prepared from the truffle fungus T. claveryi in inhibiting four types of bacteria pathogenic to the oyster mushroom Pleurotus.


     
    Effect of FeNPs from the fungus T. nivea on inhibiting pathogenic bacteria of the oyster mushroom
     
    As shown in Fig 7, FeNPs prepared from T. nivea were more effective than FeSO4 against all tested bacteria. At 15 mM, the inhibition zones ranged from 2.45 to 3.02 cm for FeNPs and from 0.93 to 1.50 cm for FeSO4.

    Fig 7: Effect of iron nanoparticles prepared from the truffle fungus T. nivea in inhibiting four types of bacteria pathogenic to the oyster mushroom Pleurotus.


           
    Fig 8 illustrates the effect of concentrations (5-15 mM) of iron nanoparticles prepared from the fungus T. claveryi, which showed the highest efficacy against the bacterium P. agarici, compared to a concentration of 15 mM of non-nanoparticle iron sulfate (FeSO4).

    Fig 8: The effect of iron nanoparticles prepared from the fungus T. claveryi was superior against the bacterium P. agarici.


           
    Furthermore, biosynthesis using T. claveryi may impart additional properties to these particles due to the associated bioactive compounds (Ahmadi et al., 2021; Mohamed-Benkada et al., 2024). The variation in susceptibility among the four bacterial species may be attributed to differences in cell wall structure and resistance mechanisms (Zúñiga-Miranda et al., 2023).
           
    This superiority of iron nanoparticles can be explained by their nanoscale dimensions and high surface area to volume ratio, which increases the area of interaction with bacterial cells and enhances surface interactions with them. This can lead to disruption of cell membrane integrity and increased generation of reactive oxygen species (ROS), which trigger a cascade of cellular damage including lipid peroxidation in the cell membrane, DNA damage and impaired replication, protein oxidation and disruption of several cellular systems and enzymes (Natan and Banin, 2017; Shoudho et al., 2024). Direct interaction between nanoparticles and the cell surface can also contribute to cell membrane disruption and some particles may penetrate the cell, leading to cellular homeostasis and metabolic disturbances, thereby inhibiting bacterial growth (Li et al., 2018).

    CONCLUSION

    We conclude from the current study that the bacteria P. pleuroti, Pseudomonas reactans, P. agarici and P. tolaasii are responsible for diseases in oyster mushroom. Additionally, iron nanoparticles were successfully biosynthesized using extracts from the truffle fungi T. claveryi and T. nivea, yielding stable, uniform nanoscale particles. These bio-synthesized nanoparticles demonstrated superior inhibitory activity against oyster mushroom pathogenic bacteria compared to the conventional ferrous sulfate source, highlighting their strong potential for biological applications and disease control.

    CONFLICT OF INTEREST

    There is no conflict of interest.

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