Direct nitrogen, phosphorus and carbon exchanges between Mucoromycotina 'fine root endophyte' fungi and a flowering plant in novel monoxenic cultures

Abstract

Most plants form mycorrhizal associations with mutualistic soil fungi. Through these partnerships, resources are exchanged including photosynthetically fixed carbon for fungal-acquired nutrients. Recently, it was shown that the diversity of associated fungi is greater than previously assumed, extending to Mucoromycotina fungi. These Mucoromycotina 'fine root endophytes' (MFRE) are widespread and generally co-colonise plant roots together with Glomeromycotina 'coarse' arbuscular mycorrhizal fungi (AMF). Until now, this co-occurrence has hindered the determination of the direct function of MFRE symbiosis. To overcome this major barrier, we developed new techniques for fungal isolation and culture and established the first monoxenic in vitro cultures of MFRE colonising a flowering plant, clover. Using radio- and stable-isotope tracers in these in vitro systems, we measured the transfer of ³³ P, ¹⁵ N and ¹⁴ C between MFRE hyphae and the host plant. Our results provide the first unequivocal evidence that MFRE fungi are nutritional mutualists with a flowering plant by showing that clover gained both ¹⁵ N and ³³ P tracers directly from fungus in exchange for plant-fixed C in the absence of other micro-organisms. Our findings and methods pave the way for a new era in mycorrhizal research, firmly establishing MFRE as both mycorrhizal and functionally important in terrestrial ecosystems.

Keywords: Mucoromycotina; arbuscular mycorrhizal fungi; carbon; clover; fine root endophytes; flowering plants; soil nutrients; symbiosis.

Fig. 1

In vitro isolation of Mucoromycotina fine root endophytes (MFRE) from Lycopodiella inundata (a–c) and colonisation of white clover (Trifolium repens) by isolated MFRE (d–i). (a) L. inundata gametophyte (*) with copious fungal outgrowth, magnified in (b) and imaged under a scanning electron microscope (c); note the fine hyphae with numerous swellings (*). (d, e) Monoxenic culture of T. repens and isolated MFRE after 12 wk of culture; (e) note the abundant mycelium extending from plugs of pure MFRE cultures (*) and enveloping the roots (arrowed), enlarged in (f) (see also Fig. 4). (g–i) Trypan blue/ink‐stained roots of Trifolium showing fine, irregularly branching hyphae with larger vesicles or spores (g, arrowed), forming tightly wound intracellular coils with small intercalary and terminal swellings (h, arrowed); note also the subtending intracellular hyphal ropes (*). (i) Young, arbuscule‐like structure (arrowed) forming inside a clover root cell. Bars: (d) 50 mm; (e) 2 mm; (a) 500 μm; (b, f) 300 μm; (g) 50 μm; (c, h, i) 20 μm.

Fig. 2

Set‐up of in vitro monoxenic experimental system used to quantify fluxes of nutrients exchanged between Mucoromycotina fine root endophytes (MFRE) and white clover (Trifolium repens). (a) Monoxenic cultures were developed in sterile conditions in 140 mm Petri dishes half‐filled with modified Strullu–Romand (MSR) media, which lacked vitamins and sucrose, solidified with 0.4% Phytagel and poured at a slant. Three plugs of axenic MFRE culture were placed on the media and allowed to establish before the introduction of a T. repens seedling. Plates were sealed for 12 wk. One hundred microlitres of an aqueous solution containing ³³P or ¹⁵N was added to a well in each plate (see top‐left detail panel). In the control plates, a trench was cut and subsequently filled with sterile medium to sever MFRE and to ensure there was no direct fungal access to radio‐ and stable‐isotope tracers (see bottom‐left detail panel). Purple colour on hyphae denotes the flow of fungal‐acquired isotope tracers. (b) The MSR media and its contents were sealed off from the aboveground plant tissue, and each plate was placed in a gas‐tight chamber before ¹⁴C‐labelling. Lactic acid was added to ¹⁴C‐labelled sodium bicarbonate to release a 0.5 Mbq pulse of ¹⁴CO₂ for the plant to fix. At the end of the 24‐h labelling period, KOH was added to the system to capture any remaining headspace ¹⁴CO₂. Red circles in (a, b) denote regions that were sampled for the analysis of ³³P, ¹⁵N and ¹⁴C.

Fig. 3

Nutrient fluxes between white clover (Trifolium repens) and Mucoromycotina fine root endophytes (MFRE). (a, b) Total shoot tissue phosphorus (³³P) in nanograms (ng) in sterile monoxenic cultures with intact fungi, trenched fungi and with no fungi present (a) and shoot concentration (ng g⁻¹) of fungal‐acquired ³³P in T. repens (b). (c, d) Total shoot tissue (¹⁵N) in nanograms (ng) in sterile monoxenic cultures with intact MFRE fungi (c) and shoot concentrations (ng g⁻¹) of fungal‐acquired ¹⁵N in T. repens (d). In (a, b), n = 9 for monoxenic cultures with intact fungi and n = 5 for those with trenched fungi and n = 3 for monoxenic cultures with no fungi present. In (c, d), n = 16 for monoxenic cultures with intact fungi and n = 5 for those with trenched fungi and n = 6 for monoxenic cultures with no fungi present (n indicates the number of biological replicates used during radio‐ and stable‐isotope tracing experiments). Letters denote significant differences where P < 0.05, Mann–Whitney U and Kruskal–Wallis tests. Error bars represent the standard error of the mean, with all data points shown (minimum to maximum) collected during the experiments.

Fig. 4

Total plant‐derived carbon present in extraradical Mucoromycotina fine root endophytes (MFRE) fungal hyphae. (a) Total plant‐derived carbon present in Phytagel with MFRE fungi present or plant‐only microcosms where no MFRE were present after a 24‐h labelling period (ng) and concentrations (ng g⁻¹) (b). For both (a) and (b), n = 17 for microcosms with MFRE present and n = 7 for microcosms with no MFRE present. Letters denote significant differences where P < 0.05, Mann–Whitney U and Kruskal–Wallis tests. Error bars represent the standard error of the mean, with all data points shown (minimum to maximum) collected during the experiments.