Elsevier

Geochimica et Cosmochimica Acta

Volume 318, 1 February 2022, Pages 55-69
Geochimica et Cosmochimica Acta

Hyphal tips actively develop strong adhesion with nutrient-bearing silicate to promote mineral weathering and nutrient acquisition

https://doi.org/10.1016/j.gca.2021.11.017Get rights and content

Abstract

Fungi actively enhance the local dissolution of nutrient-bearing minerals through the combined biomechanical and biochemical actions of their hyphal tips to obtain mineral-bound inorganic nutrients (MINs). However, little is known about the dynamic processes underlying hyphal tip-mineral interactions. Here, we assess the adhesive force between a single hypha of the common fungus Talaromyces flavus and the Fe-bearing silicate lizardite and quartz (as a control), as well as hyphal tip-induced lizardite weathering and hyphal Fe uptake. We showed that T. flavus hyphae formed their maximal adhesive force with lizardite at the growing tips, reaching 6.11 ± 0.69 nN after a contact time of one minute. The adhesive forces of the tip-lizardite interface within two minutes were >2.65 times stronger than those of the tip-quartz interface. Examination of the hyphal tip-lizardite interface after 18 h indicated the formation of dissolution channels with a depth of 27.7 ± 8.0 nm. Furthermore, the hyphal tips resulted in an altered lizardite up to 46 nm. The thickness of the altered lizardite increased to ∼130 nm after contact with the mature regions of the hyphae for ∼173 min. And the altered lizardite was found to have depleted Fe levels that increased with increasing contact time. The total content of Fe in T. flavus associated with the lizardite surface after 18 h was 52.98 ± 12.20 nmol mg−1, which was 6 times greater than the total amount of Fe in quartz surface-associated T. flavus after 24 h of culture. These results demonstrate that fungi access MINs by the active development of a strong adhesive force with target minerals through their hyphal tips, effectively enabling fungi to flourish in heterogeneous environments and be major geological agents for biogeochemical transformation.

Introduction

Jongmans et al. (1997) discovered the pores in feldspar and hornblende in podzol E soil horizons and granitic bedrocks under European coniferous forests more than two decades ago. The authors suggested that these pores were formed through the exudation of organic acids from the hyphal tips of mycorrhizal or saprotrophic fungi and coined the term “rock-eating fungi”. A later work on the rate of feldspar tunneling across a north Sweden podzol chronosequence by Hoffland et al. (2002) indicated that the bioavailability of Ca and K may be a factor during the formation of these pores. Mounting evidence has indicated that fungal weathering of minerals is an active process with the purpose of obtaining mineral-bound inorganic nutrients (MINs) (Rogers et al., 1998, Gadd, 2007, Balogh-Brunstad et al., 2008, Li et al., 2016). Consequently, fungal weathering of nutrient-bearing minerals proceeding at the single-hypha scale (Bonneville et al., 2009, Li et al., 2016, Wild et al., 2021) contributes to soil formation (Mitchell et al., 2016) and the biogeochemical cycling of essential elements (Sterflinger, 2000, Hoffland et al., 2004, Gadd, 2006, Gadd, 2010).

Attachment onto mineral surfaces is the most potent strategy for fungi to obtain access to MINs (Banfield and Nealson, 1997). To maximize the chances of acquiring MINs and minimize energy consumption, fungi selectively attach onto mineral surfaces containing growth-limiting nutrients. Their filamentous morphology and hyphal tip-direct growth allow fungi to rapidly find and colonize substrates with nutrients (Moore et al., 2011). Previous studies have revealed that fungal hyphae selectively attach to and colonize minerals bearing growth-limiting nutrients such as apatite inclusions in feldspars (Rogers et al., 1998, Smits, 2009) and Ca-plagioclase in igneous bedrocks (Jongmans et al., 1997, Berner and Cochran, 1998, Quirk et al., 2012). The attachment of microorganisms to mineral surfaces is primarily maintained by cumulative adhesive forces that are nonspecific (e.g., hydrogen bonding, hydrophobic, van der Waals, electrostatic, and macromolecular forces) and/or specific (e.g., molecular recognition between receptors and ligands) (Hermansson, 1999, Busscher et al., 2008). The much greater adhesive force of specific bonds than nonspecific bonds (e.g., ∼0.80 versus 0.25 nN for specific and nonspecific bonds, respectively (Lower et al., 2001)) causes bacterial cells to selectively attach onto Fe oxide and hydroxide surfaces (Ohmura et al., 1993). Due to their explorative lifestyle (Moore et al., 2011), fungi may form more intimate and specialized associations with minerals than bacteria. Force mapping of mineral surface-associated fungal hyphae by atomic force microscopy using nonconductive silicon nitride in air indicated that the highest adhesive forces are preferentially localized at the hyphal tips (Saccone et al., 2012). However, the adhesive force between each individual hypha and the mineral surface has never been investigated.

The colonization of fungal hyphae on mineral surfaces usually produces distinct dissolution features. Fungus-mineral interfacial dissolution is a result of a combination of biomechanical and biochemical actions (Sterflinger, 2000, Gadd, 2007, Bonneville et al., 2009). The former (as high as 10–20 MPa (Hoffland et al., 2004, Howard et al., 1991)) is rooted in the apical extension of the hyphae (Moore et al., 2011), and the latter is derived from acidolysis and complexolysis by the excreted low-molecular-weight organic compounds (LMWOCs) (van Hees et al., 2006). The combined effects of these two actions ultimately produce dissolution channels with depths ranging from 2 to 2000 nm on the surfaces of silicate minerals over the experimental period from days to months (Balogh-Brunstad et al., 2008, Gazzè et al., 2012, Saccone et al., 2012, Li et al., 2016, Balogh-Brunstad et al., 2017). These experimental results have led to the widely accepted view that fungal hyphae need to be in contact with minerals for several days, or even months, to produce dissolution channels. Fungal hyphae are mostly active near the tip, where direct biomechanical action is involved (Bonneville et al., 2009) and the metabolic activity is the highest (van Hees et al., 2006). In addition, reactive oxygen species have been found to be actively produced at hyphal tips (Yu et al., 2019), and these products may lead to the oxidation of structural Fe(II) (Bonneville et al., 2016). It is therefore reasonable to assume that hyphal tips can rapidly weather minerals. However, the rate of hyphal tip-induced mineral weathering is poorly constrained quantitatively.

Iron (Fe) is an essential micronutrient for all eukaryotes (Kappler and Straub, 2005). Due to the extremely slow dissolution rates of Fe-bearing silicates and oxides in aerobic environments (Brady and Walther, 1989), Fe is often the limiting nutrient for fungal growth (Guerinot, 1994). Therefore, fungi are obliged to acquire mineral Fe through active processes such as the selective attachment onto Fe-bearing silicates (Roberts, 2004). Here, we cultured the common fungus Talaromyces flavus on the surfaces of the Fe-bearing silicate lizardite [(Mg, Fe)3(Si, Fe)2O5(OH)4] and quartz in Mg- and Fe-free Czapek medium for 24 h. T. flavus was found to effectively weather lizardite at the hyphal-mineral interface (Li et al., 2016). The goal of this study was to assess the adhesive force between a single T. flavus hypha and lizardite and quartz, as well as hyphal tip-induced lizardite weathering and Fe uptake of the fungus. We observed the growth of T. flavus on the surfaces of the polished mineral flakes using optical microscopy over the course of culture. After 18 h of culture, the adhesive forces of these two individual minerals along the length of T. flavus hyphae with different contact times were measured under physiological conditions using atomic force microscopy (AFM)-based force spectroscopy (FS). Additionally, hyphal-induced lizardite weathering was characterized by AFM and transmission electron microscopy (TEM). Fe uptake was semiquantified using laser ablation-multiple collector-inductively coupled plasma-mass spectrometry (LA-MC-ICP-MS). AFM-FS has emerged as a key platform to quantitatively measure pico- to nano-scale Newton forces (10−9–10−12 N) as a function of nanoscale distance between living cells and nonliving objects/other living cells in aqueous solutions. Thus, it has a unique advantage in measuring the adhesive force between microorganisms and minerals (Lower et al., 2001, Lower et al., 2007). The LA system was used to mark the position of the surface-associated hyphae so we could accurately locate the positions that had been in contact with the hyphae after hyphae chemical removal. AFM is a powerful technique for detecting and measuring the morphology of solid surfaces in three dimensions down to the nanoscale, and it is therefore widely used to examine the local weathering on mineral surfaces induced by fungi (Balogh-Brunstad et al., 2008, Gazzè et al., 2012, Li et al., 2016). The site-specific nature of focused ion beam (FIB) milling renders it indispensable during the preparation of lamellae for high-resolution (HR)-TEM imaging as well as TEM-selected area electron diffraction (SAED) and energy dispersive X-ray spectroscopy (EDS) analyses to probe the mineral alterations beneath microorganism-mineral interfaces (Benzerara et al., 2005, Obst et al., 2005, Bonneville et al., 2009, Ward et al., 2013). LA-MC-ICP-MS can be used to perform in situ analysis of the elemental content in a substance without any pretreatment.

Section snippets

Fungal strain and minerals

The common fungus T. flavus used in this study was isolated from serpentine soil (Li et al., 2015). To obtain the spores, T. flavus was first cultured on 2% malt extract agar medium (MEA: 20 g L−1 malt extract and 18 g L−1 agar) (Daghino et al., 2009) for four weeks, and then the mycelia were rinsed gently with 0.05 wt% Tween 20 in a sterile cabinet. The obtained dormant spores were further rinsed three times with 0.05 wt% Tween 20 and five times with ultrapure water to remove the spore

T. flavus spore germination and hyphal elongation

T. flavus spores on lizardite germinated earlier and had a higher germination rate than those on quartz within a culture period of 24 h (Fig. 1A). T. flavus spores on quartz did not germinate until 15 h, and only ∼24% of the spores germinated within 18 h; then, the germination rate increased to ∼53% at 24 h. However, T. flavus spores on lizardite germinated at a rate of ∼11% within 8 h, and nearly all of the spores (∼97%) germinated after 18 h. Moreover, the elongation rate of the hyphae on

Discussion

We probed the interactions between T. flavus and lizardite and quartz (as a control) using state-of-the-art techniques on the single hypha scale. The experimental results revealed rapid feedback between the hyphal tips of T. flavus and lizardite manifested by the development of a much stronger adhesive force with lizardite than that with quartz within one minute and lizardite weathering mediated by the hyphal tips. We also explored the dynamic mechanisms underlying the interactions and the

Summary and conclusions

A detailed investigation of the interactions between the T. flavus hyphae and nutrient-bearing silicate lizardite at the single-hyphal scale using AFM-FS, AFM, FIB-TEM, and LA-MC-ICP-MS revealed rapid feedback at the hyphal tip-mineral interface. We found the following.

  • (1)

    The germination of spores and the elongation of T. flavus hyphae were significantly enhanced by lizardite. This enhancement was primarily due to the essential nutrients provided by lizardite during weathering, as manifested by

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

We are grateful to Tianyu Chen and Maoyu Wang for technical support in LA-MC-ICP-MS analysis, Yu Wang and Renduo Liu from the Analysis and Testing Center for Nuclear Science, Shanghai Institute of Applied Physics, for technical support in FIB milling and HR-TEM-SAED/EDS analyses, respectively, and Weishuai Di from the Department of Physics of NJU for his help with AFM data analysis. We would like to thank the anonymous reviewers for thorough comments which help to improve the manuscript. This

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