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

Date Published: April 9, 2019

Publisher: Public Library of Science

Author(s): Natesan Sundaresan, Enthai Ganeshan Jagan, GokulRaj Kathamuthu, Mohan Pandi, Tamás Papp.


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.

Partial Text

Foliar fungal endophytes are asymptomatic inhabitants of healthy plant leaves for all or part of their life cycle [1–3]. Such co-evolution of endophytic fungi and host likely existed when plants colonized land (since 400MYr), thus contributing a long and significant role in steering the evolution of fungal life on earth [4,5]. Endophytes, transmitted from one generation to another through infected host tissues (seeds, vegetative propagules) or fungal spores [5,6], may confer the host with physiological/fitness benefits over its competitors [7]. They have been proven as a fascinating source of structurally novel and biologically active secondary metabolites [8–12]. Also, the fungal endophytes have been catalogued to produce plant bioactive compounds and their analogs, thus making them a promising source of novel compounds with great potentials in medicinal, agricultural and industrial arenas [12,13]. Scientific communities also have renewed their interests in bio-prospecting these microorganisms to avoid large-scale harvesting of medicinal plants [12,14].

A total of 392 foliar endophytic fungi were isolated and characterized from 900 segments of healthy leaf tissues of Aglaia elaeagnoidea, Flacourtia inermis and Premna serratifolia found in the ABS Botanical garden. These hosts and this geographical location (Fig 2) had not been explored erstwhile for FEF diversity.

In the past four decades, numerous investigations have reported the FEF population to be abundant and multifarious in most plants [1,3,59–61]. The stomatal openings in the foliages offer passage to the entry of fungal propagules and diverse fungal lineages have been observed to colonise the extensive surface area of the foliar cover [62]. FEF have been touted as the reservoir of valuable metabolites and their bioprospecting could meet the pharmaceutical demands in a cost-effective, easily accessible and reproducible way [12,15,63]. Also, in several cases the medicinal property of compounds derived from these FEF resembles that of their host plants; hence they could potentially serve as an alternative source of the host metabolites [64,65].




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