MycoRed: Betalain pigments enable in vivo real-time visualisation of arbuscular mycorrhizal colonisation

Arbuscular mycorrhiza (AM) are mutualistic interactions formed between soil fungi and plant roots. AM symbiosis is a fundamental and widespread trait in plants with the potential to sustainably enhance future crop yields. However, improving AM fungal association in crop species requires a fundamental understanding of host colonisation dynamics across varying agronomic and ecological contexts. To this end, we demonstrate the use of betalain pigments as in vivo visual markers for the occurrence and distribution of AM fungal colonisation by Rhizophagus irregularis in Medicago truncatula and Nicotiana benthamiana roots. Using established and novel AM-responsive promoters, we assembled multigene reporter constructs that enable the AM-controlled expression of the core betalain synthesis genes. We show that betalain colouration is specifically induced in root tissues and cells where fungal colonisation has occurred. In a rhizotron setup, we also demonstrate that betalain staining allows for the noninvasive tracing of fungal colonisation along the root system over time. We present MycoRed, a useful innovative method that will expand and complement currently used fungal visualisation techniques to advance knowledge in the field of AM symbiosis.

Intrinsic root morphology determines the phosphorus acquisition efficiency of five annual pasture legumes irrespective of mycorrhizal colonisation

Mycorrhizal fungi are ubiquitous in agroecosystems and form symbiotic associations that contribute to the phosphorus (P) acquisition of many plants. The impact of mycorrhizas is most pronounced in P-deficient soil and commonly involves modifications to the root morphology of colonised plants. However, the consequences of mycorrhizal colonisation on root acclimation responses to P stress are not well described. Five annual pasture legumes, with differing root morphologies, were grown to determine the effect of mycorrhizal colonisation on shoot yield, root morphology and P uptake. Micro-swards of each legume were established in pots filled with a topsoil layer that had been amended with five rates of P fertiliser. The topsoil overlaid a low-P subsoil that mimicked the stratification of P that occurs under pasture. Mycorrhizal colonisation improved P acquisition and shoot yield in the low-P soil treatments, but did not reduce the critical external P requirement of the legumes for near-maximum yield. The yield responses of the mycorrhizal plants were associated with reduced dry matter allocation to topsoil roots, which meant that the P acquisition benefit associated with mycorrhizal colonisation was not additive in the P-deficient soil. The contribution of the mycorrhizal association to P acquisition was consistent among the legumes when they were compared at an equivalent level of plant P stress, and was most pronounced below a P stress index of ~0.5. The intrinsic root morphology of the legumes determined their differences in P-acquisition efficiency irrespective of mycorrhizal colonisation.

Growth, Rhizosphere Carboxylate Exudation, and Arbuscular Mycorrhizal Colonisation in Temperate Perennial Pasture Grasses Varied with Phosphorus Application

Phosphorus (P) fertiliser is applied regularly to the nutrient-poor sandy soils in southwestern Australia to elevate and/or maintain pasture production. This study aimed to characterise differential growth, root carboxylate exudation, and mycorrhizal responses in three temperate perennial pasture grasses at variable P supply. Tall fescue (Festuca arundinacea L. cv. Prosper), veldt grass (Ehrharta calycina Sm. cv. Mission), and tall wheatgrass (Thinopyrum ponticum L. cv. Dundas) with five P rates varying from 0 to 100 mg P kg−1 soil were evaluated in a controlled environment. Rhizosphere carboxylate exudation and mycorrhizal colonisation were assessed. Veldt grass produced the maximum shoot dry weight, highest agronomic phosphorus-use efficiency at low P supply, as well as the highest specific root length and shoot P content at all P rates. Across species, the maximum shoot weight was obtained at 20 and 50 mg P kg−1 soil, which differed significantly from the two lowest P rates (0 and 5 mg P kg−1 soil). Phosphorus application influenced carboxylate exudation, with plants exuding acetate only in the zero P treatment, and citrate and malonate in the P-supplemented treatments. In all three species, acetate and malonate were the major carboxylates exuded (37–51% of the total). Only tall wheatgrass released trans-aconitate. Citrate and malonate concentrations in the rhizosphere increased with P supply, suggesting their important role in P acquisition. Phosphorus applications reduced arbuscular mycorrhizal colonisation and increased root diameter as the P rate increased. Root carboxylate exudation in low-P soil played a role in mobilisation of P via P solubilisation, but the role of exuded carboxylate in soils well supplied with P might be diminished.

Mycorrhizal colonisation in roots of Holcus lanatus (Yorkshire Fog) in a permanent pasture under conditions of reduced precipitation.

The UK climate is projected to become warmer, with more frequent hotter, drier summers. Many governments and international organisations are concerned about how climate change will affect food production and security. Mycorrhizal fungi are an essential part of agricultural systems and yet little is known about how climate change will affect mycorrhizal fungi. We investigated the effect of reduced precipitation on levels of arbuscular mycorrhizal (AM) colonisation in the top 10 cm of soil in the grass Holcus lanatus L. (Yorkshire Fog) in a permanent pasture in South Gloucestershire, UK. Incident rainfall was reduced, by approximately 50 %, using clear gutters supported on steel frames. Over three growing seasons we observed little difference in levels of AM colonisation and numbers of intra-root fungal structures between the roots of H. lanatus grown with reduced or full incident rainfall. Time of year when water stress occurred had a stronger effect on levels of colonisation than the absolute amount of precipitation received. In H. lanatus, growing in a permanent pasture, levels of AM colonisation were around 40 - 50%, across a range of precipitation, from 18% above to 36% below the long-term average. The results highlighted the complex relationship between mycorrhizal fungi, host plant and abiotic stress.

Nitrogen and Potassium Fertilisation Influences Growth, Rhizosphere Carboxylate Exudation and Mycorrhizal Colonisation in Temperate Perennial Pasture Grasses

Optimisation of potassium (K) use efficiency in pastures on sandy soil is challenging. We characterised growth response, root carboxylate exudation and mycorrhizal colonisation in three perennial pasture grasses: tall fescue (Festuca arundinacea L.), veldt grass (Ehrharta calycina Sm.) and tall wheatgrass (Thinopyrum ponticum L.) in two glasshouse experiments with: (1) four K rates (0, 40, 80 and 120 mg K kg-1 soil), and (2) four N and K treatments (no N and K (–N–K), 81 mg N kg-1 soil but no K, 80 mg K kg-1 soil but no N, and N at 81 and K at 80 mg kg-1 soil (+N+K)) in low-K sandy soil. Veldt grass had the highest shoot dry weight and shoot P content, but the lowest mycorrhizal colonisation. Potassium fertilisation had no significant impact on exudation of citrate and oxalate. The K0 plants had significantly lower exudation of acetate and total carboxylates than K40 plants. The +N+K plants had maximum shoot growth at both harvests (30 and 60 days after sowing (DAS)) and highest N and K shoot contents at 60 DAS. The –N–K plants exuded maximum amounts of citrate and malate at 30 DAS, but at 60 DAS tall fescue had the highest rhizosphere concentrations of citrate and malate in the +N+K treatment. At 60 DAS, mycorrhizal colonisation was significantly lower with than without N and K fertilisation. We concluded that pasture grasses could yield well even in inherently low-K soil without external K fertilisation and mycorrhizal symbiosis. However, the +N+K plants had the highest yield and root carboxylate exudation.

An improved clearing and staining protocol for evaluation of arbuscular mycorrhizal colonisation in darkly pigmented woody roots

Arbuscular mycorrhizal fungi (AMF) establish symbiotic interactions with the roots of vascular plants, including grapevines. Verifying AMF colonisation routinely requires establishing the presence of hyphae, arbuscules and vesicles. Clearing roots with potassium hydroxide (KOH) followed by staining with trypan blue has been used previously to visualise fungal structures, however visualisation is difficult with darkly pigmented roots, such as those of grapevines so additional steps are required to ensure clear visualisation. Three fixing and clearing processes were evaluated prior to staining with trypan blue: 1) fixing grapevine roots in 70% v/v ethanol overnight; 2) clearing by heating the roots in either 2% or 10% w/v KOH; and 3) clearing the roots in 3% v/v hydrogen peroxide for 10 min. Roots were examined under a compound light microscope for the presence of AMF. A combination of fixing grapevine roots in 70% ethanol overnight and clearing by autoclaving in 10% KOH produced the greatest enhancement in subsequent staining of grapevine roots with trypan blue overnight. The best method tested enabled the discrimination of arbuscular mycorrhizal structures in fresh roots of grapevines without the use of toxic chemical fixatives.

The role of CLV signalling in the negative regulation of mycorrhizal colonisation and nitrogen response of tomato

Plants form mutualistic nutrient acquiring symbioses with microbes, including arbuscular mycorrhizal fungi. The formation of these symbioses is costly and plants employ a negative feedback loop termed autoregulation of mycorrhizae (AOM) to limit arbuscular mycorrhizae (AM) formation. We provide evidence for the role of one leucine-rich-repeat receptor like kinase (FAB), a hydroxyproline O-arabinosyltransferase enzyme (FIN) and additional evidence for one receptor like protein (SlCLV2) in the negative regulation of AM formation in tomato. Reciprocal grafting experiments suggest that the FAB gene acts locally in the root, while the SlCLV2 gene may act in both the root and the shoot. External nutrients including phosphate and nitrate can also strongly suppress AM formation. We found that FAB and FIN are required for nitrate suppression of AM but are not required for the powerful suppression of AM colonisation by phosphate. This parallels some of the roles of legume homologs in the autoregulation of the more recently evolved symbioses with nitrogen-fixing bacteria leading to nodulation. This deep homology in the symbiotic role of these genes suggests that in addition to the early signalling events that lead to the establishment of AM and nodulation, the autoregulation pathway might also be considered part of the common symbiotic toolkit that enabled plants to form beneficial symbioses.

The role of CLV signalling in the negative regulation of mycorrhizal colonisation and nitrogen response of tomato

Plants form mutualistic nutrient acquiring symbioses with microbes, including arbuscular mycorrhizal fungi. The formation of these symbioses is costly and plants employ a negative feedback loop termed autoregulation of mycorrhizae (AOM) to limit arbuscular mycorrhizae (AM) formation. We provide evidence for the role of one leucine-rich-repeat receptor like kinase (FAB), a hydroxyproline O-arabinosyltransferase enzyme (FIN) and additional evidence for one receptor like protein (SlCLV2) in the negative regulation of AM formation in tomato. Reciprocal grafting experiments suggest that the FAB gene acts locally in the root, while the SlCLV2 gene may act in both the root and the shoot. External nutrients including phosphate and nitrate can also strongly suppress AM formation. We found that FAB and FIN are required for nitrate suppression of AM but are not required for the powerful suppression of AM colonisation by phosphate. This parallels some of the roles of legume homologs in the autoregulation of the more recently evolved symbioses with nitrogen-fixing bacteria leading to nodulation. This deep homology in the symbiotic role of these genes suggests that in addition to the early signalling events that lead to the establishment of AM and nodulation, the autoregulation pathway might also be considered part of the common symbiotic toolkit that enabled plants to form beneficial symbioses.HighlightWe describe the role of CLV signalling elements in the negative regulation of arbuscular mycorrhizal symbioses of tomato, including influencing nitrate but not phosphate suppression of mycorrhizal colonisation.