First Cryo-Scanning Electron Microscopy Images and X-Ray Microanalyses of Mucoromycotinian Fine Root Endophytes in Vascular Plants

Abstract

Aims: Arbuscule-producing fine root endophytes (FRE) (previously incorrectly Glomus tenue) were recently placed within subphylum Mucoromycotina; the first report of arbuscules outside subphylum Glomeromycotina. Here, we aimed to estimate nutrient concentrations in plant and fungal structures of FRE and to test the utility of cryoscanning electron microscopy (cryoSEM) for studying these fungi. Methods: We used replicated cryoSEM and X-ray microanalysis of heavily colonized roots of Trifolium subterraneum. Results: Intercellular hyphae and hyphae in developed arbuscules were consistently very thin; 1.35 ± 0.03 µm and 0.99 ± 0.03 µm in diameter, respectively (mean ± SE). Several intercellular hyphae were often adjacent to each other forming "hyphal ropes." Developed arbuscules showed higher phosphorus concentrations than senesced arbuscules and non-colonized structures. Senesced arbuscules showed greatly elevated concentrations of calcium and magnesium. Conclusion: While uniformly thin hyphae and hyphal ropes are distinct features of FRE, the morphology of fully developed arbuscules, elevated phosphorus in fungal structures, and accumulation of calcium with loss of structural integrity in senesced arbuscules are similar to glomeromycotinian fungi. Thus, we provide evidence that FRE may respond to similar host-plant signals or that the host plant may employ a similar mechanism of association with FRE and AMF.

Carbon for nutrient exchange between Lycopodiella inundata and Mucoromycotina fine root endophytes is unresponsive to high atmospheric CO2

“…Such functional complementarity is further supported by the observation that MFRE transfer significant amounts of 15 N but relatively little 33 P tracers to a host lycophyte, Lycopodiella inundata, in the first experimental demonstration of MFRE nutritional mutualism in a vascular plant (Hoysted et al 2019). These results contrast with the majority of studies on MFRE and fine root endophytes (FRE) which have, to date, focussed on the role of the fungi in mediating plant phosphorus (P) acquisition (Orchard et al 2017b, and literature within;Albornoz et al 2020).…”

Critical research challenges facing Mucoromycotina ‘fine root endophytes’

“…MFRE, within Endogonales (Mucoromycotina, Mucoromycota), are recognized as phylogenetically (Bidartondo et al ., 2011; Spatafora et al ., 2016; Orchard et al ., 2017b) and functionally (Field et al ., 2015, 2019; Hoysted et al ., 2019) distinct from the more commonly studied arbuscular mycorrhizal fungi (AMF), which belong to the Glomeromycotina (or Glomeromycota) (Spatafora et al ., 2016). Research using isotope tracers has shown that MFRE exchange both phosphorus and nitrogen for plant‐fixed carbon when in association with liverworts (Field et al ., 2015, 2016, 2019) and with the vascular plant Lycopodiella inundata (Hoysted et al ., 2019, 2021b), while a cryo‐scanning electron microscopy (SEM) and X‐ray microanalysis study suggests MFRE may play a role in phosphorus assimilation in Trifolium subterraneum (Albornoz et al ., 2020). Where it has been measured, MFRE have been shown to transfer a significant amount of nitrogen to their host plant (Field et al ., 2016, 2019; Hoysted et al ., 2019, 2021a), suggesting that there may be a complementary role for these fungal symbionts alongside AMF.…”

“…Inoculum production through soil sieving (Gerdemann & Nicolson, 1963; An et al ., 1990; Orchard et al ., 2017b) is currently the only published technique for colonizing experimental vascular plants with MFRE, while excluding the other arbuscule‐forming symbionts, AMF (Albornoz et al ., 2020). This method, based on the observation that AMF spores appear to be much larger than those produced by MFRE, involves wet‐sieving soil collected from a site known to contain MFRE to obtain the material that accumulates between sieves of pore sizes 200 µm and 50 µm.…”

“…An improvement to this method, as used in Albornoz et al . (2020), is to first use the soil inoculum to produce an MFRE pot culture. Plants are grown in pots containing diluted soil inoculum, after which the substrate within pots containing the highest amount of MFRE and lowest amount of AMF is sieved again to produce soil inoculum to grow more plants.…”

“…This is then dried and used as a soil inoculum enriched in MFRE. The inoculum is mixed with autoclaved sand or soil at a ratio of 1 : 81 (Orchard et al ., 2017b) or 1 : 162 (Albornoz et al ., 2020) to produce a substrate for plant growth (Fig. 1).…”