The Molecular Identification of Isolated Fluoride-resistant Plant Growth-promoting Fungi

Fluoride is a naturally occurring element found in various environmental sources such as minerals, magmatic gases and industrial processes. It disperses through the atmosphere and water, leading to widespread exposure. (Prabhu et al., 2023; Singh et al., 2023). Fluoride can enter plants through water, soil, or leaves, leading to various biochemical, physiological and molecular changes. (Kumar et al., 2021). Continuous exposure to fluoride can result in stunted growth, chlorosis and necrosis of leaves, ultimately affecting the overall health and productivity of crops. (Chahine et al., 2023b). Fluoride levels in soil can negatively affect crop productivity and pose dangers to environmental health (Rizzu et al., 2021). Elevated levels of fluoride, which are prevalent in industries, artificial fertilizers (Type NPK) and or irrigation systems that utilize contaminated water, can affect soil microbiological activities and nutrients, thereby impairing soil fertility (Malik et al., 2024; Singh et al., 2023). The accumulation of fluoride in plants inhibits fundamental physiological mechanisms, including photosynthesis, respiration and enzyme activity, which restricts crop growth, leads to leaf chlorosis and reduces yield (Kamruzzaman et al., 2025). Long-term fluoride residuals can bioaccumulate into edible plant portions and cause transfer through food systems, which may be detrimental to human and animal health. To combat this issue, the implementation of sustainable agricultural practices and pollution controls is warranted. Researchers have been examining whether the molecular identification of fluoride resistant plant growth promoting fungi (PGPF) may present new opportunities for sustainable agricultural practices. The fungi contribute to plant growth via a variety of pathways and demonstrate resistance to environmental threats such as fluoride contamination which is a risk to agricultural soil quality (Boorboori et al., 2022). Fluoride from varying sources (soil, water) can affect plant health which limits agricultural productivity. The capacity of some fungi to survive and thrive in high fluoride levels marks them as potentially valuable organisms, capable of being used for bioremediation as well as in eco-friendly agricultural systems. The reported benefits of the fungi include enhancing nutrient uptake while improving plant health by suppressing plant disease (Whipps, 2001).
       
The accurate molecular identification is needed for Fungi, that resist fluoride, using  ITS region sequencing of rRNA genes because this method is common due to its high species variability according to Schoch et al., (2012). Fungi identification relies on the ITS region, which functions like a genetic barcode for precise classification (Nilsson et al., 2019; Nizamani et al., 2020).
       
Researchers have recently identified PGPF species in fluorine-contaminated areas which demonstrate both survival capabilities and the ability to boost plant growth under these adverse conditions (Kanthiya et al., 2025). The ability to perform two functions simultaneously is essential for creating biotechnological advancements that improve crop production in polluted locations. For instance, various species related to the genera Trichoderma, Penicillium and Aspergillus have shown potential for PGP and fluoride resistance. The molecular characterization of these fungi through Sanger sequencing and bioinformatic analysis helps in understanding their mechanisms of action and potential applications (Raja et al., 2017; Schoch et al., 2014). This knowledge allows the development of fungal inoculants that can be used in fluoride-affected soils, thereby promoting sustainable agriculture and food security. In the present study, we molecularly identified fungi that demonstrated positive results in various assays, including siderophore production, phosphate solubilization, ammonia production, hydrogen cyanide (HCN) production and biocontrol activity against pathogenic fungi in high-fluoride soil environments. We employed molecular techniques to accurately determine the fungal species of the most promising Plant Growth-Promoting Fungi (PGPF) isolate. 

In the present study, we have isolated the fungus species from evaluated were isolated from the rhizosphere of plants in fluoride-impacted areas of Sikar District of Rajasthan and Bhopal District of Madhya Pradesh and the work performed at Department of Bioscience at Mody University of Science and Technology, Lakshmangarh, Sikar (Rajasthan) and CMBR Biotech Pvt Ltd, Bhopal, MP during 2023 to 2024.
       
The present study involved the molecular identification of fungi that showed positive results in various assays, such as siderophore production, phosphate solubilization, ammonia production, hydrogen cyanide (HCN) production and biocontrol activity against pathogenic fungi in high-fluoride soil environments. The following methods we used for the molecular validation of the most promising Plant Growth-Promoting Fungi (PGPF) isolates:
 
Pure culture preparation
 
The pure fungal cultures were prepared by inoculating fungal isolates in Potato Dextrose (PD) broth, a nutrient-rich medium commonly used for cultivating fungi. This medium encourages fast fungal growth, which is needed for the development of sufficient biomass for subsequent genomic analyses. The cultures were incubated under controlled environmental conditions, including appropriate temperature and humidity levels, to facilitate robust fungal growth and minimize contamination risks. Care was taken to monitor the growth phase of the fungi, ensuring maximum yield before proceeding to DNA extraction (Kanthiya et al., 2025).
 
Genomic DNA extraction
 
The extraction of genomic DNA from the fungal biomass was accomplished using the CTAB (cetyltrimeth-ylammonium bromide) method (Prakash et al., 2019; Choudhary et al., 2024), a reliable and widely used technique for isolating DNA from fungi and plants. This process began with the lysis of fungal cell walls and membranes using a combination of chemical reagents and physical disruption. The CTAB solution effectively bound contaminants such as polysaccharides, proteins and other cellular debris, allowing for the separation and purification of DNA. The final product was high-quality DNA that was free from impurities and suitable for downstream applications like amplification and sequencing.
 
PCR amplification
 
The polymerase chain reaction (PCR) was employed to amplify the Internal Transcribed Spacer (ITS) region of the fungal rRNA gene a region widely recognized for its utility in fungal identification and phylogenetic studies (Jadhav et al., 2021). Universal fungal primers (listed in Table 1) were utilized to target this conserved region. The PCR process included three main stages: denaturation of the DNA double helix at high temperatures to produce single-stranded templates, annealing of primers to complementary sequences and extension of primers by DNA polymerase to synthesize new DNA strands. This resulted in specific and reproducible amplification of the ITS region, which serves as a genetic marker for fungal identification.


 
Sequencing and analysis
 
 The amplified PCR products were then purified to remove residual primers, dNTPs and other reaction components. The purified samples were sequenced to determine the nucleotide composition of the ITS region (Nuratika et al., 2020). This sequencing data was subsequently analyzed using the Basic Local Alignment Search Tool (BLAST) database-a powerful resource for comparing genetic sequences. The fungal ITS sequences obtained in the study were matched against known sequences in the database, facilitating accurate identification of the fungal species. This step provided valuable insights into the genetic diversity and phylogenetic relationships of the fungi under investigation.

The molecular analysis conducted in this study validated the species identification of fluoride-resistant fungal organisms that may contribute significantly to sustainable agriculture in fluoride-affected areas. In the present study, we molecularly characterized Trichoderma harzianum, which proved to be the most effective biocontrol agent due to its ability to enhance plant resistance against fungal pathogens through the production of secondary metabolites and enzymes with antipathogenic properties (Haque et al., 2025).
       
Other key representatives were also validated, including Cladosporium cladosporioides and Penicillium chrysogenum, both recognized for their capabilities in phosphate solubilization and production of auxin-type plant growth hormones, particularly indole acetic acid (IAA) (Seibold et al., 2024). These molecular identifications confirm the taxonomic classification of these fungal species and provide essential information regarding their functional roles in improving plant growth under fluoride stress conditions.
       
All of these fungi demonstrated robust growth in high fluoride environments, indicating their effective stress management capabilities. Moreover, these fungal taxa function as competitive inhibitors and produce antifungal metabolites. Specifically, Cladosporium cladosporioides could be classified as an antifungal pathogen suppressor, while Penicillium chrysogenum and Aspergillus niger emerged as significant isolates that solubilized phosphate and generated bioactive compounds supporting plant flexibility and productivity (Hasan et al., 2025).
       
The findings for all isolated fungi were successfully validated through molecular techniques that amplified the Internal Transcribed Spacer (ITS) region of fungal DNA. This sequencing confirmed both the identification and taxonomic classification of these fungal species, legitimizing their traits and contributing to our understanding of their functional characteristics as crucial contributors to plant growth, particularly in stressful conditions (Fig 1-3).






       
Collectively, these results demonstrated the functional diversity associated with the identified PGPF and provide a foundation for applying these fungi to enhance crop productivity, supporting sustainable and resilient agricultural practices in fluoride-contaminated conditions.
               
Molecular techniques, particularly ITS region sequencing, have revolutionised fungal species identification, providing crucial insights into their functional roles and agricultural applications. Fungal species can be identified with high precision using ITS region sequencing, which remains the “gold standard” for fungal identification (Wu et al., 2023). Recent advancements in metabarcoding approaches have further enhanced the resolution and throughput of this method (Abarenkov et al., 2022). This technique has proven especially valuable in differentiating cryptic fungal species that traditional culturing methods struggle to identify (Raja et al., 2022). Characterized plant growth-promoting fungi (PGPF) demonstrate multiple functional traits including phosphatase production, phosphate solubilization, enhanced iron availability through siderophore production and biosynthesis of ammonia and hydrogen cyanide (HCN), while also suppressing pathogenic fungi. These capabilities are particularly valuable in fluoride-contaminated environments where soil fertility and plant growth are severely compromised. For instance, recent studies have demonstrated that Trichoderma harzianum not only exhibits strong biocontrol properties against soil-borne pathogens but also significantly enhances nutrient acquisition pathways, effectively improving overall plant health and stress tolerance (Sharma et al., 2023). Similarly, the phosphate solubilization capabilities of Aspergillus flavus and Aspergillus niger have been shown to mitigate nutrient deficiencies commonly associated with fluoride-contaminated soils. These fungi play a critical role in converting insoluble phosphate forms into bioavailable forms that plants can readily utilize (Verma et al., 2024). Recent metabolomic analyses have revealed the specific organic acids responsible for this solubilization process, providing new insights into optimization strategies (Liu et al., 2023). The antifungal activity of Cladosporium clados-porioides and the bioactive compounds produced by Penicillium chrysogenum further complement these functions by suppressing detrimental pathogens and enhancing plant stress tolerance (González-Rodríguez et al., 2024). Meta-analysis of field trials has demonstrated that these antifungal properties remain effective across diverse soil conditions, including those with moderate fluoride contamination (Kumar et al., 2023). Integration of these fungi into agricultural systems presents a viable approach for rehabilitating soil and addressing nutrient deficiencies resulting from fluoride contamination. Implementation of PGPF inoculants can potentially reduce dependence on chemical fertilizers and pesticides, thereby promoting alternative and sustainable agricultural practices with reduced synthetic inputs. While laboratory studies consistently demonstrate the effectiveness of these fungi, comprehensive field evaluations are essential to ensure that fungicide formulations and bioactive compounds can be practically implemented and their efficacy optimized under real-world conditions. Addressing the challenges associated with fluoride contamination, recent research emphasizes the significant potential of molecularly identified PGPF in contributing to sustainable agricultural practices. A 2024 meta-analysis by Patel and colleagues demonstrated that PGPF applications resulted in average yield increases of 18-27% in crops grown in moderately contaminated soils (Patel et al., 2024). The deployment of PGPF in agricultural systems creates opportunities to enhance soil health while simultaneously improving crop yields, ultimately supporting environmental sustainability, promoting food security and building resilience against pollution challenges. Future research should focus on understanding the genetic basis of fluoride tolerance in these fungi and exploring the potential for developing improved strains through traditional selection or genetic approaches. Additionally, field trials investigating the performance of these molecularly characterized fungi under various fluoride contamination levels would provide practical validation of their effectiveness in real agricultural settings.

This molecular study successfully validates several key fluoride-resistant plant growth-promoting fungi (PGPF), including Trichoderma harzianum, Aspergillus flavus, Aspergillus niger, Cladosporium cladosporioides and Penicillium chrysogenum. These species demonstrate significant potential for enhancing sustainable agricultural practices in fluoride-stressed environments through their roles in nutrient cycling, pathogen suppression and growth promotion.

The authors are thankful to the Department of Biosciences at Mody University of Science and Technology, Lakshmangarh, Sikar (Rajasthan) and CMBR Biotech Pvt Ltd, Bhopal, MP, for providing laboratory facilities.

The authors state that there are no conflicts of interest concerning the publication of this article. Additionally, no funding or sponsorship affected the study’s design, data collection, analysis, publication decision, or manuscript preparation. Additionally, no ethical approval was required for this work, as the research did not involve human or animal subjects and did not entail any procedures subject to ethical review.