Newly Designed Fluorescence In Situ Hybridization Probes Reveal Previously Unknown Endophytic Abilities of Tuber magnatum in Herbaceous Plants

Abstract

Tuber magnatum Picco (the Italian white truffle) is the most valuable and widely appreciated truffle. It is an ectomycorrhizal fungus known to associate with many broadleaf tree species. However, its mycorrhizae are rarely observed in the field, suggesting possible alternative symbiotic strategies, such as endophytism with non-ectomycorrhizal plants. In order to test potential endophytic interactions of T. magnatum with wild plants, a combination of polymerase chain reaction (PCR) and Fluorescence In Situ Hybridization (FISH) approaches were used. Specific FISH probes for T. magnatum were designed, tested in vitro on hyphae and/or ectomycorrhizae, and selected for their specificity. These probes were then used on a wide variety root samples of wild plants collected from three T. magnatum production areas in Italy and previously tested for the presence of T. magnatum mycelium using PCR-specific primers. Molecular analyses detected the presence of T. magnatum in 21 of 100 plant samples analyzed. FISH analysis confirmed the extracellular presence of active T. magnatum hyphae inside the root system of Carex pendula Huds plant. This study provides the first evidence of T. magnatum acting as an endophyte in an herbaceous plant. The newly designed, highly specific T. magnatum FISH probes can be used for further investigations to confirm the endophytic tendencies of T. magnatum and to understand their influence on the life cycle and biology of this fungus.

Keywords: Endophytism; FISH; Non-ectomycorrhizal host plants; Truffle ecology.

Fig. 1

Percentual distribution of positive and negative plant samples collected in Spring 2022 and 2023 after PCR with Tuber magnatum Picco specific primers

Fig. 2

Evaluation of the Fluorescence In Situ Hybridization (FISH) probe T.mag1647, targeting the 18S ribosomal RNA of Tuber magnatum Picco, was conducted using either ascomata or pure cultures. In the figure is showed the comparison between the ascoma of T. magnatum (a–d, left panel), a pure culture of Tuber melanosporum Vittad. (e–h, left panel), and ectomycorrhizae of T. magnatum (i, l), Tuber aestivum Vittad. (m, n), and T. melanosporum (o, p) (right panel). The co-hybridizations were performed on T. magnatum ascoma (herbarium number 5377), T. aestivum (strain 5230), and T. melanosporum (strain TME2). The mycorrhizae were provided by Robin Pépinières nurseries (St. Laurent du Cros, France). Co-hybridization experiments were performed with a generalist probe targeting eukaryotic cells, Euk516 (in red), as a positive control. The positive control showed high signal intensity in the cytoplasm of fungal cells (a, c, e, g, i, m, and o), in combination with the T.mag1647-specific probe (b, d, f, h, l, n, and p). An anti-sense probe, AS-Euk516, was used as a negative control to verify the absence of non-specific binding of the probe or random fluorochrome binding. For each co-detection, 2D images from individual channels are presented. Fluorochrome Atto565 and Atto633 were excited at 561 nm and 639 nm, respectively, and detected with bandpass filters (570‒630 nm, 640‒720 nm). The cell wall dye SR2200 was excited at 405 nm and detected with a 420–477-nm filter

Fig. 3

Observation of plant roots by confocal microscopy after Fluorescence In Situ Hybridization (FISH) with the Tuber magnatum Picco specific probe T.mag1647 (b, d; green) and the eukaryotic Euk516 probe (a, c; red) as a positive control. a, b Endomycorrhizal fungi found into a Carex pendula Huds. root sample with no colocalization of Euk516 and T.mag1647 probes. c, d Colocalization of Euk516 and T.mag1647 probes into a C. pendula roots. The arrows indicate hyphae bonded just with the Euk516 probe. Fluorochrome Atto565 and Atto633 were excited at 561 nm and 639 nm, respectively