Molecular identification and phylogenetic characterization of banana-associated fungal pathogens and GRAS salt-based management

Introduction

Banana (Musa spp.) is an important fruit crop cultivated throughout tropical and subtropical regions and contributes substantially to food, nutrition and farm income. The crop is highly perishable because ripening is rapid and fruit tissues become increasingly susceptible to microbial infection. Mechanical injury, unsuitable handling, temperature and humidity fluctuations and pathogen invasion can accelerate deterioration during harvesting, transport, storage and marketing. Among fungal diseases, anthracnose caused by Colletotrichum Musae is an important postharvest constraint. The pathogen can establish latent infections during fruit development and symptoms become prominent as fruits ripen. In addition, Curvularia species have been associated with banana leaf spot. Because closely related fungal taxa may show overlapping morphological characters, molecular characterization is increasingly useful for reliable identification and phylogenetic placement. Post-harvest disease management has traditionally relied heavily on synthetic fungicides. Concerns related to residues, environmental effects and resistance development have encouraged the evaluation of safer alternatives. GRAS salts are food-compatible compounds with documented antifungal potential. Sodium bicarbonate, carbonate salts, sodium benzoate and potassium sorbate have been investigated against post-harvest fungi, although their effectiveness can vary with pathogen and treatment conditions. The present investigation was therefore undertaken to (i) isolate and identify fungal pathogens associated with banana diseases using morphological and molecular approaches, (ii) characterize the isolates through phylogenetic analysis and (iii) evaluate the in vitro efficacy of selected GRAS salts against C. musae and C. lunata.

Materials and Methods

Study site and sample collection

The investigation was conducted during 2025-2026 in the Department of Plant Pathology, School of Agricultural Sciences, Dhanalakshmi Srinivasan University, Tiruchirappalli, Tamil Nadu, India. Banana samples showing typical symptoms of anthracnose and leaf spot were collected from the university garden land. Symptomatic fruits and leaves showing dark sunken lesions, necrotic tissues or crown-region rotting were selected for isolation.

Isolation and maintenance of fungal cultures

Infected tissue pieces of approximately 1-2 mm² were cut from the lesion margins, surface sterilized with 1% sodium hypochlorite for 1 min, rinsed three times in sterile distilled water, blot dried and placed on Potato Dextrose Agar (PDA) media. Plates were incubated at 27 ± 1 °C for 2-4 d. Developing colonies were sub-cultured by the hyphal tip culture method and maintained on PDA slants at 4°C.

Pathogenicity testing

Healthy surface-sterilized banana fruits were inoculated with mycelial discs of pure cultures of C. musae and healthy leaf bits were inoculated with mycelial discs of pure cultures of C. lunata. Inoculated fruits were incubated at 27 ± 1°C under humid conditions. Characteristic disease symptoms were observed within 5-7 d and the associated fungi were re-isolated and compared with the original cultures.

Morphological identification

Microscopic preparations were examined for mycelial and conidial characteristics. The isolates were identified based on the observed cultural and microscopic features.

Genomic DNA extraction and ITS amplification

Mycelial mass from 5-d-old cultures grown in potato broth was harvested using sterile muslin cloth, gently dried and ground to a fine powder. Genomic DNA was extracted using the HiPurA Fungal DNA Purification Kit (HiMedia, India) following the manufacturer's protocol. DNA quality was assessed by electrophoresis on 2% agarose gel and by the A260/A280 ratio. The ITS region was amplified using ITS1 (5′-TCC GTA GGT GAA CCT GCG-3′) and ITS4 (5′-TCC TCC GCT TAT TGA TAT GC-3′) primers. PCR was performed in a 25 µL reaction containing 20 ng template DNA, 100 µM each dNTP, 20 ng each primer, 1.5 mM MgCl₂, 1× Taq buffer and 1 U Taq DNA polymerase. The thermal profile comprised initial denaturation at 94 °C for 3 min; 35 cycles of 94 °C for 30 s, 55 °C for 1 min and 72 °C for 1 min; and final extension at 72 °C for 10 min. Amplicons were resolved on 1.5% agarose gel containing ethidium bromide and visualized under UV illumination.

Sequencing and phylogenetic analysis PCR products were purified using a DNA/PCR Purification Mini Kit and sequenced by Barcode Biosciences Private Limited, Bangalore, India. Raw sequences were edited using BioEdit and compared with sequences in the National Center for Biotechnology Information database by BLAST. Representative sequences were deposited in GenBank. Sequences of the study isolates and related taxa retrieved from NCBI were aligned using CLUSTALW. Phylogenetic trees were constructed in MEGA 11.0 using the Neighbor-Joining method.

In vitro evaluation of GRAS salts

Sodium benzoate, sodium carbonate, sodium bicarbonate and potassium sorbate were evaluated at 0.2% against C. musae and C. lunata. The food-poison technique was used. PDA was melted and cooled to a suitable temperature before incorporation of the test salts at the required concentration. The amended medium was dispensed into sterile Petri plates; untreated PDA served as the control. A 5 mm mycelial disc from a 4-d-old culture was placed at the centre of each plate. Plates were incubated at 27 ± 1 °C for 5 d. Five replications were maintained for each treatment. Radial growth was recorded when the control plates reached maximum growth. Percentage inhibition of mycelial growth was calculated using the standard relationship:

Where C represents radial growth in the untreated control and T represents radial growth in the treatment. The experiment was analysed using a Completely Randomized Design (CRD) in R software.

Results

Isolation and morphological characterization

Three fungal pathogens were recovered from symptomatic banana samples collected at the study site and were identified morphologically as C. musae and C. lunata. C. musae produced fast-growing white to light-grey colonies that became darker with age. The mycelium was hyaline, septate and branched, while conidia were hyaline, aseptate and cylindrical in C. musae. C. lunata produced rapidly growing white to greyish colonies that became dark grey to black, with dark brown septate hyphae and characteristically curved, multicelled brown conidia. The disease symptoms observed in the field and laboratory were consistent with the respective disease associations. Anthracnose was characterized by dark, sunken lesions and progressive peel deterioration and Curvularia-associated leaf spot was characterized by brown to reddish-brown lesions that enlarged and coalesced.

Molecular

identification and phylogenetic characterization: PCR amplification using ITS1 and ITS4 generated an amplicon of approximately 850 bp. BLAST analysis showed 97-99% nucleotide similarity between the study sequences and related fungal sequences available in GenBank. The sequences were deposited in NCBI under accession numbers PZ819402-PZ819403 for Colletotrichum and PZ675716 for Curvularia. CLUSTALW alignment and Neighbor-Joining analysis in MEGA 11.0 supported the molecular placement of the isolates among their respective taxonomic groups.

In vitro efficacy of GRAS salts against C. musae and C. lunata:

All four GRAS salts inhibited the mycelial growth of C. musae and C. lunata at 0.2%, although their efficacy varied among the pathogens. Against C. musae, sodium benzoate recorded the highest inhibition (76.46%), followed by potassium sorbate (56.46%), sodium carbonate (48.62%) and sodium bicarbonate (46.27%). In contrast, potassium sorbate was the most effective against C. lunata, with 78.54% inhibition, followed by sodium benzoate (66.66%), sodium carbonate (25.29%) and sodium bicarbonate (14.59%). Thus, sodium benzoate was the most effective GRAS salt against C. musae, whereas potassium sorbate showed the highest inhibitory activity against C. lunata at the tested concentration.

Discussion

The These results identify sodium benzoate and potassium sorbate as promising candidates for further evaluation. In vivo and post-harvest studies are required before recommending these treatments for practical banana disease management.

Acknowledgement

Acknowledgments:The authors acknowledge the School of Agricultural Sciences, Dhanalakshmi Srinivasan University, Tiruchirappalli, for providing facilities and support for the study. The authors also acknowledge the assistance and guidance received from Director-NRCB, scientists and staff associated with the National Research Centre for Banana, Tiruchirappalli, during the course of the project.• Conflict of interest: The authors declare that there is no conflict of interest associated with this study. • Funding: No external funding was reported for this study.

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