Development of high-resolution melting PCR (HRM-PCR) assay to identify native fungal species associated with the wheat endosphere

Abstract

Understanding the complexity and biodiversity of fungal communities associated with the wheat endosphere can facilitate the identification of novel strains that might be beneficial to the host plant. However, the differentiation and taxonomic classification of the endosphere-associated fungi with respect to various cultivars and plant organs are challenging, time-consuming, and expensive, even with the use of molecular techniques. In the present work, we describe a fast, simple, and low-cost method based on high-resolution melting PCR (HRM-PCR) for the identification and differentiation of wheat endogenous fungal isolates. Using this approach, we differentiated 28 fungal isolates, which belonged to five different genera, namely Alternaria , Penicillium , Epicoccum , Fusarium , and Trichoderma. Furthermore, the results of the study revealed that this method can allow large-scale screening of cultured samples.

Fungi Inhabiting the Wheat Endosphere

“…In research on wheat endophytes, before surface sterilization, plant samples are usually washed under running water [ 46 , 47 ], and soil residues are removed from the roots, for example by brushing or scraping [ 48 , 49 ]. Ethyl alcohol and sodium hypochlorite are usually used to remove microorganisms from the surface of plant fragments.…”

“…Separated for this purpose, the glumes, lemmas, paleas, and kernels were surface sterilized according to Comby et al [ 38 ]; however, they sterilized flower tissues in 96% ethanol for 1 min, 2% sodium hypochlorite for 3 min, and 96% ethanol for 30 s, and finally rinsed the tissues twice with sterile MilliQ water. In contrast, Cłapa et al [ 48 ] and Salamon et al [ 49 ] developed a protocol for wheat in which plant tissue fragments were rinsed in 70% ethyl alcohol for 30 s and then in 0.5% sodium hypochlorite for 2 min. In order to remove the reagents, the wheat tissues were rinsed several times with distilled water.…”

“…After surface sterilization, the plant material was aseptically cut into smaller pieces 5–10 mm in length [ 48 , 49 ], re-sterilized, and then placed on the agar medium. Many different media can be used, but the most common ones are potato dextrose agar (PDA), malt extract agar (MEA), corn meal agar (CMA), yeast extract peptone dextrose agar, as well as minimal plant tissues or extract media [ 51 , 52 , 53 , 54 ].…”

“…This region of DNA was used only by Larran et al [ 47 ] to identify endophytic fungi isolated from wheat leaves, stalks, chaff, and grains; by Comby et al [ 38 ] for the classification of endoffites from aerial roots and organs, including leaves, stems, anthers, chaff, sediments, and nuclei; and by Bouzouina et al [ 41 ] to determine the species of endophytic fungi isolated from wheat roots. Using high-resolution melting (HRM) techniques and the differences in the melting points of ITS sequences, distinguishing fungi isolated from the inner tissues of wheat plants at the genus level has become possible [ 48 ]. However, the multilocus DNA barcode was used by Llorens et al [ 78 ] for correct classification of 2 isolates from the ancestor of wheat, namely Aegilops sharonensis ; by Salamon et al [ 49 ] for identifying 54 isolates from the root endosphere of common wheat and spelt wheat; and by Rojas et al [ 50 ] to determine the species for 163 fungal isolates from healthy wheat spikes.…”

Rapid Identification of Four Fusarium spp. Complex by High-Resolution Melting Curve Analysis and their Antifungal Susceptibility Profiles

“…High-resolution melting curve (HRM) analysis can be performed in a closed tube right after real-time quantitative PCR (qPCR). Different melting pro les results from various GC contents, DNA sequences, and lengths, facilitating the identi cation of small differences in the target sequences [21,22]. HRM analysis has been used to successfully identify species from the following: Trichinella (8 species) [23], Candida spp.…”