Endophytic fungi have garnered immense importance in various fields of biotechnology (Kusari and Spiteller 2011). For the isolation of endophytes with desirable traits, certain strategies are employed (Strobel et al. 2002). For instance, a detailed investigation of a particular ecosystem, its plant species, their relationship with the environment, soil composition etc before selection of a plant would result in the isolation of diverse and potential endophytes (Kusari and Spiteller 2011). Plants with medicinal importance are preferably used for the isolation of endophytes (Aly et al. 2011; Dhayanithy et al. 2019; Tyagi et al. 2021; Keshri et al. 2021; Kapoor et al. 2022). Since, D. bulbifera L. has been less explored for its endophytic community, therefore, the current study was conducted to explore of fungal endobiome of D. bulbifera L. for diversity, community composition and phylogenetic analysis. A few workers have isolated fungal endophytes from D. bulbifera L., but hardly any report is available on diversity analysis. Seasonal variation among endophytic fungi associated with D. bulbifera was done by Suradkar et al (2014). Ahmed et al (2012) isolated 70 fungal isolates from 270 explants of D. bulbifera L. Furthermore, other studies on endophytes of D. bulbifera L. include actinomycetes and bacteria (Peng et al. 2015).
The current study reported maximum colonisation in the bulbils followed by other tissues which is in contrast to previous studies reporting maximum colonisation of endophytic fungal isolates of D. bulbifera L. in the root tissue (Ahmed et al. 2012). The differences in fungal assembly depend on certain physiological and environmental factors (Gond et al. 2012). The structure and substrates of the bulbils can influence infection of the endophytic fungi and increase their colonization frequency. The study conducted by Kaur et al (2015) reported that peculiar anatomy of leaves harbour myriad of endophytic fungi. The leaf provides large surface area which aids in capturing of inoculum due to its natural stomatal opening, hydathodes, and glandular entry points (Rajini et al. 2020). Also, it has been noted that the hairy surface helps with accumulation of endophytic inoculum and easy access to internal tissues. However, our study reported that leaf tissue possessed lesser number of endophytes which does not corroborate previous studies (Kumar and Hyde 2004; Katoch et al. 2017; Sharma et al. 2018). This might be due to the shorter life time of leaves as compared with the other tissues (Sadeghi et al. 2019).
The current study recorded that endophytic fungal taxa of D. bulbifera L. belonged to 2 phyla, 4 classes, 10 orders, 25 genera and 38 species showing abundant diversity. Except for one fungal species belonging to Basidiomycota, all the fungal endophytes isolated in this study belonged to the phylum Ascomycota. The predominance of Ascomycota reported in this study is typical of fungal endophytes which implies that they have co-evolved with plants (Ramos-Garza et al. 2016). Ascomycota covering approximately 8% of the Earth’s landmasses and is one of the most diverse and ubiquitous phyla of eukaryotes (Katoch et al. 2017). In our study endophytic community belonging to Sordariomycetes, Dothidiomycetes, Eurotiomycetes and Agaricomycetes was reported. Majority of the endophytic fungal isolates, obtained in our study belonged to the class, Sordariomycetes and Dothideomycetes of the phylum Ascomycota. According to various research groups fungal endophytic community of terrestrial plants mainly belong to the class Sordariomycetes, Dothideomycetes and Pezizomycetes fungi (Suryanarayanan et al, 2011). Therefore, our results were consistent with the previous study that Sordariomycetes and Dothideomycetes were the main groups of endophytic fungi from other plants (Zhang et al. 2014; Chen et al. 2019) and contradictory to studies conducted to other groups (Katoch et al. 2017; Xiang et al. 2018; Alomia et al, 2022). Among four different classes of endophytic fungal isolates species richness was abundant in the class Dothidiomycetes which is in lines with previous report (Sadeghi et al. 2019).
Some of the endophytic fungal isolates obtained in our study have already been reported as endophytes either from various species of Dioscorea or other medicinal plants (Kusari et al. 2013; Xiang et al. 2018; Bhattacharya et al. 2019; Chowdhary and Kaushik 2019; Duan et al. 2019; Gong et al. 2019; Manoharan et al. 2019; Suleiman et al. 2019; Gupta et al. 2022). However, to the best of our knowledge the endophytic fungal isolates, A. medicaginis, C. lycoperscii, T. macrospora, F. laceratum, P. formosus and M. cirrosus have not been earlier reported as endophytes while N. oryzae, F. flavus, Talaromyces sp., have been reported first time as endophytes from D. bulbifera L.
Although, reports on other species of Acrocalymma such as A. vagum, A. fici, isolated as endophytes are available but so far hardly any report is available where A. medicaginis has been reported as an endophyte (Jin et al. 2018; Maulana et al. 2018; Chen et al. 2019; Liu and Wei 2019; Radiastuti et al. 2019; Huang et al. 2020). The genus Acrocalymma was first introduced for a single novel species Acrocalymma medicaginis causing root and crown rot of Medicago sativa (Alcorn and Irwin 1987; Nikandrow 1990). Transition of fungal lifestyles from parasitic to mutualistic or vice versa has been reported in the literature. Apart from physiological stress on the host plant, it is influenced by certain parameters viz. genetic factors of both partners, imbalance in nutrient exchange and environmental variations (Kogel et al. 2006; Rodriguez and Redman 2008). A. medicaginis was described as a sexual morph of Massarina walkeri (Shoemaker et al. 1991). Although, there are only eight described species of Acrocalymma, more than 200 ITS sequences have been deposited from these species in GenBank. Endophytic strains of Acrocalymma vagum have the highest number of reported sequences, followed by A. medicaginis (Mortimer et al. 2021).
In addition, Paecilomyces formosus (Khan et al. 2012; Bilal et al. 2017) and Microascus cirrosus (Chaudhary and Arya 2012) that are known human pathogens have also been reported as endophytes in this study. Flavodon flavus, belonging to the phylum Basidiomycetes has previously been reported as endophytes from other plants (Brum et al. 2012; Katoch et al. 2014). Likewise, N. oryzae and Talaromyces sp. have not been earlier reported as endophytes from D. bulbifera L.
Based on colonisation frequency (%), it can be depicted that Fusarium was the most prevalent species. Higher colonisation frequency of F. equiseti (BD4) isolated from bulbils, C. lunata (ALW1), obtained from stem tissue and C. gleospoiriodes (ALG8) isolated from leaf tissue was found. Also, the endophytic fungal isolate F. equiseti (TD1) was found to have maximum colonisation frequency in the tuber tissue. It can be inferred from the data for colonization frequency that different endophytic fungi inhabit different plant tissues and to different extent. These microbiotas might have adapted to different tissue microenvironments, thereby, resulting in tissue specificity. The variation in assemblage of endophytic fungal isolates inside various tissues of the host plant can be attributed to certain factors such as the physiology of the tissue, its chemical composition and anatomy etc (Gond et al. 2012; Sharma et al. 2018). However, certain other factors such as soil conditions, climate and the dynamics of soil microflora also play a vital role in the colonization of endophytes in the plant tissues (Qin et al. 2009; Xu et al. 2010).
The genus Fusarium has been isolated as endophyte from innumerable plants genera and it shows tremendous variability in terms of genetics, biology, ecology etc (Toghueo 2020). Due to their unique biosynthetic ability, they are capable of colonising diverse hosts. Therefore, any species belonging to Fusarium genus, can be isolated as an endophyte. It is considered as an important group among fungi (Stepien et al. 2018). In the current study, Fusarium was found to be the dominant genera. Similar to the current study, various species of this genera have been reported to be in abundance in root/tuber and aerial tissues (Tan et al. 2018). On contrary, the study conducted by Hamzah et al (2018) reported that endophytic fungal isolates of the genera Pestalotiopsis are dominant followed by Alternaria and Cladosporium. Moreover, the study conducted by Gupta et al (2022) is in contrast to our study. It reported Fusarium oxysporum as most frequently isolated fungal endophyte from Zingiber officinale, whereas our study reported Fusarium equiseti as most frequently isolated fungal isolate.
Colletotrichum is considered as an important and widespread genus. Some of its species are serious plant pathogens, and some are frequent endophytes (Rodrigues and Petrini 1997; Farr and Rossman 2013). Although, there have been numerous studies on the genus Colletotrichum, but understanding of endophytic species is very limited (Vieira et al. 2014). Previously, several endophytic species of Colletotrichum have been reported from a single host. Tao et al (2013) recorded, 17 Colletotrichum species from Bletilla ochracea, out of which 7 new species were reported. Similarly, Zheng et al (2022) isolated 13 Colletotrichum spp. from aquatic plants in South China. Recently, fungal endophytic community analysis revealed predominance of the genera Colletotrichum in Hevea spp. (Amaral et al. 2022). Tan et al (2018) isolated C. gleosporiodes from leaf tissue of Dysosma versipellis in abundance, which is in consensus with our study. Similarly, Prasher and Kumar (2021) reported Colletotrichum gloeosporioides to be the dominant species from leaf tissue of Dillenia indica. In contrast, Mao et al (2021) reported that members of the genus Phyllosticta isolated from Eucalyptus excerta were the primary isolates, with high colonization frequency.
The genus Curvularia is cosmopolitan and ubiquitous. It is comprised of species associated with plant and human as pathogens (Khiralla et al. 2019). However, its species have also been isolated as endophytes from various plants (Khiralla et al. 2016; Sharma et al. 2018; Kalimuthu et al. 2022). A previous study conducted by Kharwar et al (2011) reported endophytic Curvularia lunata isolated from leaf tissue of Adenocalymma alliaceum as dominant species which well corroborates the present study.
The diversity indices of endophytic taxa colonizing various tissues of D. bulbifera L. varied. The value of the Evenness index was higher in stem tissue. High Simpson index was observed in leaves which is well supported by previous reports (Prasher and Kumar 2022). Species richness of the endophytic fungal isolates was higher in bulbil tissues which is in contrast to earlier findings (Sahani and Hemalatha 2018; Bhattacharya et al. 2020).
Overall, the current study provides insight into the diversity of culturable endophytic fungal population associated with D. bulbifera L. The endophytic fungal colonization rate, diversity, and community composition is highly affected by host species, tissue types, and abiotic factors. Although this is the first work on community composition of fungal endophytes, more research is required to identify the functional and ecological significance of these fungal endophytes. Further, exploration of non-culturable fungal endophytes can be done using Next-generation sequencing and diverse omics techniques might provide us improved understanding of complex microbiome compositions and their interactions with host plants. Moreover, systematic bio guided investigations are required to further explore the potential of these endophytes and their bioactive metabolites. Investigation of potential metabolites produced by these organisms may lead to the discovery of novel natural products.