Diversity of Endophytic Fungi in Plant Species: Traditional vs. High‐Throughput Sequencing  Approaches

The plant microbiome significantly impacts plant life, with fungi playing a crucial role in shaping interactions and classifications. Advances in cultivation technologies have refined fungal classification, and research highlights the vital connection between endophytic fungi and their plant hosts. The present study employs morphological and phylogenetic techniques, predicting the Internal Transcribed Spacer 2 (ITS2) secondary structure and using next-generation sequencing (NGS) data to detect fungal endophytes in plant leaves via both traditional and conventional approaches. The research area, with its hot semi-arid environment and red and black soils, supports drought-resistant plants like Senna auriculata, Ziziphus mauritiana, and Catunaregam spinosa, known for their medicinal properties. These plants, rich in antioxidants, play a vital role in traditional medicine and highlight the region's rich ethno-botanical heritage. The culture-dependent study on the foliage yielded a total of 17 isolates from S. auriculata and 16 each from both C. spinosa and Z. mauritiana. The most common genera, Alternaria and Nigrospora, account for 18.3% of all isolated endophytic fungi. Three plants were colonized with Nigrospora and Lasiodiplodia, and their morphotypes were determined using ITS2 secondary structure prediction. Recent ecological studies highlight unculturable taxa, or dark taxa, where many species cannot sporulate or be cultured, emphasizing the need for High - Throughput Sequencing (HTS) approaches. The study gathered 68,791 reads from S. auriculata with 101 operational taxonomic units (OTUs), 58,620 from C. spinosa with 219 OTUs, and 66,087 from Z. mauritiana with 193 OTUs, with the majority of OTUs related to Colletotrichum (69%) and a minimum of Myrmaecium (2%). A total of 49 fungal isolates were obtained from traditional methods, whereas 513 fungal OTUs were retrieved through HTS methods, confirming the presence of a highly abundant fungus population in plant samples. The study reveals that using the ITS short amplicon sequencing technique provides distinct insights into endophytic fungal communities in three plant samples. In conclusion, analyzing plant fungal components using a combination of culture-dependent and culture-independent techniques may be a novel strategy.