Staining and microscopy of mycorrhizal fungal colonization in preserved ericoid plant roots

BACKGROUND: Visualization of ericoid mycorrhizal colonization using traditional methods relies on either fresh or KOH stored samples. Increasing interest in studying ericoid mycorrhization has highlighted a need for methods which can be used for preserved samples and are simple to implement with commonly available equipment. OBJECTIVE: The aim of this study was to improve on traditional techniques for staining ericoid mycorrhizal fungi and microscopically visualizing ericoid mycorrhizal roots which have been preserved. METHODS: Ericoid mycorrhizal roots were placed in KOH or frozen at -20 degrees C for long-term storage. Traditional Trypan Blue staining methods were modified to reduce damage to fine mycorrhizal hyphae and cortical cells. A high light-intensity dark-field microscopy technique was applied to clearly visualize stained mycorrhizae. The novel application was compared to other commonly used practices. RESULTS: Trypan Blue staining without KOH storage or clearing allowed for successful staining of ericoid mycorrhizal roots stored at -20 degrees C. The application of high light-intensity dark-field microscopy provided high contrast visualization of mycorrhizal structures. CONCLUSIONS: The modified Trypan Blue staining method was effective on frozen root samples, with dark-field microscopy being particularly effective at visualizing dark colored roots. Advantages to this method are lowcost and relatively fast application time. Therefore, this method is a realistic option for large scale analyses with many samples which require long-term preservation.Peer reviewe

Fungal colonization patterns and enzymatic activities of peatland ericaceous plants following long-term nutrient addition

Northern peatlands are often dominated by ericaceous shrub species which rely on ericoid mycorrhizal fungi (ERM) for access to organic sources of nutrients, such as nitrogen (N) and phosphorus (P), and host abundant dark septate endophytes (DSE). Relationships between hosts and fungal symbionts may change during deposition of anthropogenic N and P. We studied the long-term effects of N and P addition on two ericaceous shrubs, Calluna vulgaris and Erica tetralix, at Whim Bog, Scotland by analyzing fungal colonization of roots, enzymatic activity, and fungal species composition. Unexpectedly, the frequency of typical ERM intracellular colonization did not change while the occurrence of ERM hyphae tended to increase and DSE hyphae to decrease. Our findings indicate that altered nutrient limitations shift root associated fungal colonization patterns as well as affecting ericaceous root enzyme activity and thereby decomposition potential. Reduction of recalcitrant fungal biomass in melanized DSE may have implications for peatland C sequestration under nutrient addition.Peer reviewe

The Fungi and Bacteria in Ericoid Roots and Surrounding Soil

This thesis discusses the concept of mycorrhization in the roots of land plants, their reliance on symbiotic fungi and the diversity of these relationships. Specifically, the focus of this study is the mycorrhization of ericaceous plants in boreal forest ecosystems and the role this plays in aiding host survival of harsh conditions. This study presents the first comparison of the colonization patterns of both fungal and bacterial consortia in the roots and mycospheres of different boreal forest ericoid species. Experimentally, the mycorrhizal fungal and bacterial communities of roots of the Ericaceous species Vaccinium myrtillus, Vaccinium vitis-idaea, and Calluna vulgaris were characterized and quantified. To simulate natural conditions, the host species were grown in forest soil microcosms under controlled conditions with mycorrhization occurring naturally. The morphology and intensity of root colonization by ericoid mycorrhizal fungi was determined through light microscopy. Quantification of fungal and bacterial abundances was performed using quantitative Polymerase Chain Reaction for the roots and soil of each species. Microscopic analysis revealed differences in both morphology and intensity of ericoid mycorrhization for the species studied. The fungal and bacterial abundances for the roots of each host species were found to be significantly different, while the soil abundances of each species showed considerable variation and were not found to be significantly different. An intriguing connection between the fungal and bacterial abundances colonizing the root systems of the different plant species was indicated by comparison. These findings begin to clarify the unique relationships each ericoid species has with the highly diverse fungi and bacteria of their environment. Further refinement of techniques and more in depth analyses are suggested for confirming the differences indicated by these results. The ecological significance of such work is also discussed in regards to fully understanding forest ecosystems as well as the potential for novel discoveries that may benefit many aspects of humanity

Atmospheric nitrogen enrichment changes nutrient stoichiometry and reduces fungal N supply to peatland ericoid mycorrhizal shrubs

Peatlands store one third of global soil carbon (C) and up to 15% of global soil nitrogen (N) but often have low plant nutrient availability owing to slow organic matter decomposition under acidic and waterlogged conditions. In rainwater-fed ombrotrophic peatlands, elevated atmospheric N deposition has increased N availability with potential consequences to ecosystem nutrient cycling. Here, we studied how 14 years of continuous N addition with either nitrate or ammonium had affected ericoid mycorrhizal (ERM) shrubs at Whim Bog, Scotland. We examined whether enrichment has influenced foliar nutrient stoichiometry and assessed using N stable isotopes whether potential changes in plant nutrient constraints are linked with plant N uptake through ERM fungi versus direct plant uptake. High doses of ammonium alleviated N deficiency in Calluna vulgaris and Erica tetralix, whereas low doses of ammonium and nitrate improved plant phosphorus (P) nutrition, indicated by the lowered foliar N:P ratios. Root acid phosphatase activities correlated positively with foliar N:P ratios, suggesting enhanced P uptake as a result of improved N nutrition. Elevated foliar delta N-15 of fertilized shrubs suggested that ERM fungi were less important for N supply with N fertilization. Increases in N availability in peat porewater and in direct nonmycorrhizal N uptake likely have reduced plant nitrogen uptake via mycorrhizal pathways. As the mycorrhizal N uptake correlates with the reciprocal C supply from host plants to the soil, such reduction in ERM activity may affect peat microbial communities and even accelerate C loss via decreased ERM activity and enhanced saprotrophic activity. Our results thus introduce a previously unrecognized mechanism for how anthropogenic N pollution may affect nutrient and carbon cycling within peatland ecosystems. (C) 2021 The Authors. Published by Elsevier B.V.Peer reviewe

Do foliar 15N patterns indicate shifts in mycorrhizal abundance and function under nutrient load?

Abstrakti P128201

Do foliar δ15N patterns indicate shifts in the mycorrhizal abundance and function under nutrient load?

Abstrakti201

The effect of ericoid plants to soil chemistry and to fungal and bacterial communities in boreal forest humus

201

Effects of 15 Years of Nutrient Addition on Ericoid Mycorrhizal Colonization at Whim Bog

Abstrakti P115201

Pitkäaikaisen typpilannoituksen vaikutukset varpujen sienijuuriin karulla Whim-suolla

Abstrakti201

Plant-mycorrhizal feedbacks of nitrogen deposition to peatland carbon and nutrient cycling

201