Internal transcribed spacer 2 (ITS2) molecular morphometric analysis based species delimitation of foliar endophytic fungi from Aglaia elaeagnoidea, Flacourtia inermis and Premna serratifolia

Abstract

Molecular morphometrics is an emerging third dimensional aspect of fungal species delimitation. They have been demonstrated to be more informative than conventional barcoding methods. Hence in this study, foliar endophytic fungal (FEF) assemblages in three Magnoliopsida plants were delimited using nuclear ribosomal internal transcribed spacer 2 (ITS2) sequence—secondary structural features based phylogenetic analysis, also known as molecular morphometrics. A total of 392 FEF isolates were obtained from the Aglaia elaeagnoidea , Flacourtia inermis , and Premna serratifolia leaves and grouped into 98 morphotypes. Among these host plants, P . serratifolia showed the maximum percentage of colonization frequency. Representatives of each morphotype was sequenced and subjected to further molecular characterization. The results revealed that morphotypes were belonged to the phylum of Ascomycota, distributed over two classes (Sordariomycetes (68.59%) and Dothideomycetes (31.41%)), 6 orders and 19 genera. Based on compensatory base changes (CBC) analysis and absolute identity of ITS2 structure, 21, 20 and 23 species were recognized from A . elaeagnoidea , F . inermis , and P . serratifolia respectively. Diversity indices were higher in A . elaeagnoidea , despite it accounted for a modest 16.8% of total isolates recorded in this study. The genus Colletotrichum was predominant in A . elaeagnoidea (39%) and P . serratifolia (48%). Similarly, Diaporthe (43%) was dominant in F . inermis . Several host-specific species were also observed. This study concludes that these plants host diverse species of Ascomycota. To the best of our knowledge, this is the first detailed report on FEF diversity from these plants. Also, the inclusion of ITS2 secondary structure information along with the sequence provides a further dimension to resolve the inherent problems in identification of fungal species.

Phylogenetic Utility of rRNA ITS2 Sequence-Structure under Functional Constraint

“…Although CBC exhibits great potential use for species distinction, some authors caution that CBC should not be expected to distinguish all species from each other. For example, only 25 out of 98 morphological species of foliar endophytic fungi were differentiated by CBC characters [ 58 ]. Likewise, only two CBCs are present among ten closely related species of Corydalis [ 43 ].…”

Detection and Molecular Phylogenetic-Morphometric Characterization of Rhizoctonia tuliparum, Causal Agent of Gray Bulb Rot of Tulips and Bulbous Iris

“…On the other hand, if there is no CBC, then individuals may belong to the same species with 76% confidence [45]; Wolf et al [31]. Molecular morphometric analyses utilize the ITS2 primary sequence as well as secondary structural constraints, homologous locations, indels and substitution to distinguish closely related fungal and other species [21,22]. As a proof of concept for Rhizoctonia species, Ahvenniemi et al [46] showed that the potato (PT) and tobacco (TB) subgroups of R. solani AG 3 differ in the ITS2 secondary structure helix I by two CBCs.…”

“…Therefore, we determined the phylogenetic status of Rtul within the Ceratobasidiaceae using the ribosomal internal transcribed spacer (ITS1-5.8S-ITS2). To gain information on the evolutionary relationships and genomic diversity of Rtul, we also characterized the ITS2-primary and secondary structure-based molecular morphometrics (MMC) of isolates currently available in GenBank [21,22].…”

Plant associated fungal endophytes as a source of natural bioactive compounds

“…The application of the molecular approach along with bioinformatics (such as phylogenetic studies) can resolve the problem of identification of fungal strains at species level. The identification of fungal endophytes at the level of species and genus could be assisted by using various DNA barcodes including internal transcribed spacer (ITS) regions, COX2 (partner DNA barcode along with ITS), β-actin, glyceraldehyde 3-phosphate dehydrogenase (house-keeping gene), and region of translational elongation factor 1α (TEF1α) (Schoch et al 2012 ; Hoang et al 2019 ; Sundaresan et al 2019 ). The advantages of approaching DNA barcode based on ITS are the availability of a number of databases, appropriate length of the fragment, and increased amplification of all lineages of fungal endophytes using universal primers (Schoch et al 2012 ).…”