1520cm (as illustrated on the example of heather (Calluna vulgaris) from Langsett Moors, UK (a)), ericoid plants also thrive in soils with low organic content and their roots may penetrate deep into the mineral horizon (as illustrated on the example of Erica sp. Mycorrhiza: Definition, Types & Benefits - Collegedunia Ericaceae (Ericales, eight/nine subfamilies depending on the status of Pyroloideae, see Kron and Luteyn 2005 and Freudenstein et al. 2005), the ultrastructural details by transmission electron microscopy (see Plates 2 and 3 in Bonfante-Fasolo and Gianinazzi-Pearson 1979), and the hyphal mantle morphology by scanning electron microscopy (SEM, see Fig. I. Kochs postulatesfor a detailed background, explanation,and current views on the postulates see https://en.wikipedia.org/wiki/Koch%27s_postulates). (2012a) Novel root-fungus symbiosis in Ericaceae: sheathed ericoid mycorrhiza formed by a hitherto undescribed basidiomycete with affinities to Trechisporales. In: Smith SE, Read DJ (eds) Mycorrhizal symbiosis, 3rd edn. 2003. ERICOID MYCORRHIZAE - Smart Microbes Some studies have also shown that fungal communities colonizing ericoid roots can lack specificity for different species of ericoid plant, suggesting that at least some of these fungi have a broad host range.[13][14]. New Phytol 72:13251331, Pearson V, Read DJ (1973b) Biology of mycorrhiza in Ericaceae I: Isolation of endophyte and synthesis of mycorrhizas in aseptic culture. After many years of keeping our isolates both as slant agars and in Petri dishes, I have moved the whole collection (a few hundred of isolates) to small plastic Petri dishes (6cm in diameter), mainly because they are easier to prepare and inoculate, the colony morphology is easier to observe, and possible contaminations are easier to distinguish. 2013). Basic theoretical knowledge on the ErM symbiosis is summarized, followed by practical advices on Ericaceae root sample collection and handling, microscopic observations and photo-documentation of root fungal colonization, mycobiont isolation, maintenance and identification and resynthesis experiments with ericoid plants. At the same time, proper separation of ericoid roots from those of the surrounding vegetation is absolutely crucial to the success of all subsequent activities and, as such, should be approached with maximal caution and patience (cf. 2018). An easy protocol modified from Phillips and Hayman (1970) that works well for both field collected and in vitro grown ericoid plants is as follows: roots are autoclaved in 10% potassium hydroxide (KOH) at 121C for ca. (2009) confirmed one basidiomycete strain as a putative ErMF, because it formed coil-like mycorrhizal structures in the roots of Rhododendron fortunei in vitro and showed positive effects on the seedlings of R. fortunei, but provided no data to support these statements (in fact, a description of these effects and how the authors evaluated them is missing in that paper). Ad (7): At the time of harvest, a few small pieces of the substrate from each experimental vessel should be plated on a nutrient medium and incubated in the dark at room temperature until producing mycelium. Mol Ecol Resour 18:927939, Obase K, Matsuda Y, Ito S (2013) Enkianthus campanulatus (Ericaceae) is commonly associated with arbuscular mycorrhizal fungi. Leake and Read (1991) also proposed three requirements that must be met before any root-fungus association can be described as being mycorrhizal and in the case of ericoid mycorrhiza, the second part of the third one is indisputably the most intricate (in italics): There must be experimental evidence that infection by the fungus leads to enhancement of growth or nutrient capture by the host, and knowledge of the extent of the dependence of the heterotroph upon its host for carbon supplies should be obtained. However, although relatively mild, table salt is a desiccant so in the long term, such a treatment may dehydrate the medium also in the remaining non-contaminated compartments. Since it is not entirely possible to distinguish single deteriorated/moribund cells (that may be already taken over by parasites or saprobes) even with a good compound microscope (cf. ErM colonization occurs in the thinnest parts of the ericoid root system and while AM and EcM root colonization can be observed, counted and documented using a good stereomicroscope, such an approach is of little use when working with ericoid hair roots. 2016b). Short segments of surface-sterilized healthy-looking hair roots of Cassiope tetragona from a natural site in Longyearbyen (Svalbard) were plated on nutrient agar (MLA) and incubated for one month at 21C in the dark, yielding 16 fungal isolates (i.e., 64% success ratio; note no contaminating sporulating fungi). Ericoid mycorrhizal fungi have been shown to have enzymatic capabilities to break down complex organic molecules. Excess trypan blue can be removed by leaving the stained roots in lactoglycerol overnight at room temperature/for several days in a fridge. (2004), and Smith and Read (2008b). In addition, the recovery of the basidiomycetous strain from the Rhododendron roots was very low (only one isolate out of the 220 obtained) and BLAST searches in GenBank at NCBI (see below, accessed 7 January 2020) showed that its ITS nuc-rDNA sequence (EU888636) displays 100/100% coverage/similarity to several sequences of Trametes hirsuta, a known plant pathogen/wood decaying saprobe. Stud Mycol 53:121145, Tedersoo L, Partel K, Jairus T et al. The plant makes organic molecules such as sugars by photosynthesis and supplies them to the fungus, while the fungus supplies the plant with water and mineral nutrients, such as phosphorus, taken from the soil. "myco" refers to "fungi" while "rhiza" refers to the roots system of the plants. 2002, also see Huang and Chen 2005). New Phytologist 174:864-878. Cadophora finlandica) (Vrlstad 2004). New Phytologist 191: 515-527. This vast variety should be taken into account when selecting an appropriate sampling protocol and choosing suitable equipment. Midgley, D.J., Rosewarne, C.P., Greenfield, P., Li, D., Vockler, C.J., Hitchcock, C.J., Sawyer, N.A., Brett, R., Edwards, J., Pitt, J.I. Ann Bot 77:365374, Read DJ, Kerley S (1999) The status and function of ericoid mycorrhizal systems. Perhaps most of ErMF, and especially K. argillacea and O. maius, are in fact facultatively ericoid mycorrhizal? A detailed description of the whole process is far beyond the scope and dimensions of this guide; hence, I will focus on a simple (and in a way intuitive) workflow that works well with the great majority of the ericoid mycobionts I have so far isolated. Second, the total DNA must be isolateda cheap but relatively time-demanding option is the modified sorbitol extraction method described in torchov et al. High-order lateral roots with attached hair roots are separated from the washed root system using scissors or a scalpel and placed in a large Petri dish with tap water. New Phytol 139:589593, Midgley DJ, Chambers SM, Cairney JWG (2002) Spatial distribution of fungal endophyte genotypes in a Woollsia pungens (Ericaceae) root system. 2. Ericaceae species occupy at least some habitats on all continents except Antarctica. 2016). In mycology, it is mandatory to deposit vouchers for name-bearing types in a publicly accessible reference collection, but vouchers are necessary not only when new fungi are described, but also in connection with any scientific study, whether by taxonomists, systematists, physiologists, chemists, molecular biologists, pathologists, ecologists, clinicians, etc., dealing with organisms (Agerer et al. 1999; Villarreal-Ruiz et al. When dealing with hundreds of isolates, the 96-well microplates save working time and space and typically also reduce costs of sequencing. Acta Hortic 346:230239, Grelet GA, Johnson D, Paterson E, Anderson IC, Alexander IJ (2009) Reciprocal carbon and nitrogen transfer between an ericaceous dwarf shrub and fungi isolated from Piceirhiza bicolorata ectomycorrhizas. Ericoid mycorrhiza found to be associated with the members of family Ericaceae, which enables the plants to establish in inhospitable environments such as heathlands, bogs and evergreen forests. Sebacinales are common mycorrhizal associates of Ericaceae. b Note the thick brownish intercellular hyphae (arrows) and an intracellular microsclerotium developed at the very tip of the hair root (asterisk). 1990, see Fig. 4), saving hours of inefficient labor (e.g., cleaning hair roots from soil particles using a small brush or tweezers). 2013) are of only limited ecophysiological significance, mainly because it seems (at leastso far) thatthese fungi typically do not occur in ericoid roots (to my knowledge only two reports are available on their isolation, see Vrlstad et al. No matter how obviously it sounds, before setting up a resynthesis experiment, one should firstly decide what is the main aim/hypothesis and how it is to be achieved/tested, including appropriate control/reference treatments. This is commonly done by ethanol (>95%), solutions containing active chlorine like sodium hypochlorite (NaClO) or calcium hypochlorite [Ca(ClO)2], solutions containing hydrogen peroxide (H2O2, usually >30%) and consecutive combinations of these (cf. In: Norris JR, Read DJ, Varma AK (eds) Techniques for the study of mycorrhiza. Google Scholar, Freudenstein JV, Broe MB, Feldenkris ER (2016) Phylogenetic relationships at the base of Ericaceae: Implications for vegetative and mycorrhizal evolution. Can J Bot 74:337346, Xu L, Li S, Shabala S, Jian T, Zhang W (2019) Plants grown in Parafilm-wrapped Petri dishes are stressed and possess altered gene expression profile. When contaminated, individual compartments can be covered with ~3-mm layer of table salt carefully dispersed over the surface of the medium, a procedure much more effective in stopping the spread of the contamination than any attempts to clean them, e.g., by removing the contaminated medium (personal observation). 2016) form a characteristic mutualistic (but seethe section Conclusionswhat it takes to assign a mycobiont as ericoid mycorrhizal?) root-fungus association that is called the ErM symbiosis (coined by Harley 1959). As a mycorrhizal type, ErM are restricted to a few families within the Ericales (Straker 1996), but there appears to be a lack of preference by ErM fungi for specific hosts within this group. Using updated taxonomy is important, e.g., because the mycobionts genus/family assignment is often used for extrapolation of its lifestylewhile Pezizella (typified by P. sordida)belongs to Thelebolaceae, Hymenoscyphus (H. fructigenus), Rhizoscyphus (now monotypic withR. monotropae) and Scytalidium (S. lignicola)to Helotiaceae, andPezoloma (P. griseum) to Leotiaceae (MycoBank, https://www.mycobank.org, accessed 6 October2020), the actual placement of the prominent ErMF is in Hyaloscypha/Hyaloscyphaceae (Fehrer et al. Vohnk and Albrechtov 2011). Mycorrhizal Plants Types & Function | What are Mycorrhiza? - Video This symbiosis represents an important adaptation to acidic and nutrient poor soils that species in the Ericaceae typically inhabit,[1] including boreal forests, bogs, and heathlands. NRC Research Press, Ottawa, Peterson RL, Wagg C, Pautler M (2008) Associations between microfungal endophytes and roots: do structural features indicate function? A. Meharg. str., see Fehrer et al. Trans Br Mycol Soc 55:158161, Porras-Alfaro A, Liu K-L, Kuske CR, Xie G (2014) From genus to phylum: large-subunit and internal transcribed spacer rRNA operon regions show similar classification accuracies influenced by database composition. 2003). 2011; Vohnk et al. In 2005, Hambleton and Sigler wrote that standard (or optimal) conditions for ericoid mycorrhizal synthesis have not been established and that a thorough assessment of experimental conditions is needed to develop a more uniform protocol for ericoid mycorrhizal synthesis experiments. Ericoid Mycorrhiza - an overview | ScienceDirect Topics a Empetrum nigrum growing intermingled with Betula nana (Betulaceae), which forms ectomycorrhizae. Note that in some cells, both patterns are combined (arrows), meaning eitherthey are formed by the same mycobiont or the cells are colonized by at least two different mycobionts. Cullings, K. W. 1996. (2006). Mycorrhiza - an overview | ScienceDirect Topics The extent of fungal colonization is an important variable that should be assessed in field-collected as well as in vitro grown host roots and that is typically omitted in recent ErM studies. Mycorrhiza is one of the best examples of symbiotic1 association between plants and fungi. 2013, https://www.ncbi.nlm.nih.gov/genbank) and in UNITE (Abarenkov et al. Provided by the Springer Nature SharedIt content-sharing initiative, Over 10 million scientific documents at your fingertips, Not logged in Method Microbiol 23:413422, Hopple JS, Vilgalys R (1999) Phylogenetic relationships in the mushroom genus Coprinus and dark-spored allies based on sequence data from the nuclear gene coding for the large ribosomal subunit RNA: divergent domains, outgroups, and monophyly. High magnification photos of longitudinal and transversal sections warrant the best illustration of the root colonization pattern. New Phytol 94:95102, Duddridge J, Read DJ (1982) An ultrastructural analysis of the development of mycorrhizas in Rhododendron ponticum. [16], The ericoid mycorrhizal symbiosis is widespread. Hence, the maceration method could be especially efficient for recovery of slow-growing ericoid root mycobionts (see below). Phytopathology 27:11821183, Agerer R, Ammirati J, Blanz P et al. Investigations into functional aspects of other mycobionts associating with ericoid roots (cf. Fast-growing and sporulating mycelia are typically discarded as contaminants; they may produce submerged hyaline hyphae and grow unnoticed until contaminating most of the dish while others may immediately sporulate that typically leads to a loss of the whole dish. 3mm in diam.) For explanation of basic mycorrhizal terminology, see Brundrett et al. In addition, some lines of possible future research are indicated. The ericoid mycorrhiza is a mutualistic relationship formed between members of the plant family Ericaceae and several lineages of mycorrhizal fungi. (2018) Comparative genomics and transcriptomics depict ericoid mycorrhizal fungi as versatile saprotrophs and plant mutualists. Dishes with plated segments are sealed by an air permeable foil/tape and incubated in the dark at room temperature. Example: Peat is passed through a 3mm sieve, mixed 1: 1 (v/v) with perlite, moistened with tap water and autoclaved 2-times after 24h (60min at 121C). 3F in Villarreal-Ruiz et al. 1995; Fujimura and Egger 2012) symbioses in the roots of ericoid plants, but ecophysiological significance of these observations remains unknown (also see the sectionRoot sample collection and handling and item 3 under Things to considerin the section "Microscopic observations and photo-documentation") and further research on this topic is needed and encouraged. Ericoid - an overview | ScienceDirect Topics Pre-selected (preferably using a stereomicroscope) washed healthy-looking hair roots have to be surface-sterilized to avoid contamination by surface dwelling organisms and their propagules. Additionally, minuscule alpine and (sub-)arctic ericoid species often grow intermingled with AM/EcM vegetation (Fig. MycoKeys 4:3763, Nichols RV, Vollmers C, Newsom LA et al. 2018). New Phytol 129:7381, Smith SE, Read DJ (2008a) Ericoid mycorrhizas. If resources are limited, several isolates per each conservatively established morphotype must be identified to confirm ingroup conspecificity. The effects of ErMF on host plant growth and nutrient uptake may change in varying degrees with timewhile overall impressions of the effect of specific treatments can be gained from inspection of the data over the whole experiment, comparisons made at single harvests can give different and conflicting results (Stribley et al. In the INSD, there are plethora of sequences labelled as if they originated from ericoid mycorrhizal or endophytic isolates in spite of the fact that this has rarely been rigorously tested (cf. Evolutionary history of mycorrhizal symbioses and global host plant This work was supported by the Czech Science Foundation (GAR 18-05886S) and the Institute of Botany, Czech Academy of Sciences (RVO 67985939), Department of Mycorrhizal Symbioses, Institute of Botany, Czech Academy of Sciences, Prhonice, 252 43, Czech Republic, You can also search for this author in The basic rule is the shorter the betterwhile the minimal root segment length suitable for handling with fine forceps/tweezers is ~2mm, using segments longer than 3mm significantly increases the chance of obtaining fast growing endophytes (e.g., those belonging to PAC) or casual saprobes at the expense of the relatively slow growing ErMF. 2006). Use the Previous and Next buttons to navigate the slides or the slide controller buttons at the end to navigate through each slide. They can be simultaneously amplified, e.g., by the V9G+LR8 primer pair (de Hoog and van den Ende 1998; Hopple and Vilgalys 1999) under the following PCR protocol: initial denaturation at 98C for 30s, 40 cycles of denaturation at 98C for 10s, annealing at 62C for 30s, and elongation at 72C for 90s, and a final extension step at 72C for 5min. 10a). item 6 under Things to considerin the section "Resynthesis experiments"). 9) and can be efficiently used for the paper-sandwich resynthesis technique (see Chilvers et al. 2011, but see Vohnk et al. Molecular clock estimates suggest that the symbiosis originated . 20min (depending on their origin and age), then washed with tap water, acidified by brief washing in 3% hydrochloric acid (HCl), washed with tap water, autoclaved in a 0.05% solution of trypan blue in lactoglycerol (glycerol/lactic acid/water 2:1:2) at 121C for ca. 2003). Ericoid Mycorrhiza Isolation and Identification | SpringerLink (2011) Ericaceous dwarf shrubs affect ectomycorrhizal fungal community of the invasive Pinus strobus and native Pinus sylvestris in a pot experiment. Ericoid mycorrhizal fungi are characterized by the formation of distinctive hyphal coils within root cells during establishment of mycorrhizal associations with plants in the Ericaceae. Bot Rev 68:335423, Kron KA, Luteyn JL (2005) Origins and biogeographic patterns in Ericaceae: New insights from recent phylogenetic analyses. Read, D. J., J. R. Leake, and J. Perez-Moreno. From the practical point of view, factors like easy assembly/setup, inoculation and access to the plants at harvest, the space occupied in the growth chamber and reusability/price typically influence the final decision. Mol Ecol 20:30013013, Gemma JN, Koske RE (1990) Mycorrhizae in recent volcanic substrates in Hawaii. Slider with three articles shown per slide. Hence, the following text is an attempt to briefly describe and discuss various approaches to resynthesis experiments pertinent to the ErM research. [3][8][9][10] The application of DNA sequencing to fungal isolates and clones from environmental PCR has uncovered diverse fungal communities in ericoid roots, however, the ability of these fungi to form typical ericoid mycorrhizal coils has not been verified and some may be non-mycorrhizal endophytes, saprobes or parasites. Smith, S. E. and D. J. The Structure and Function of the Ericoid Mycorrhizal Root - JSTOR 2016a); and modified Leonians agar (MLA, Malloch 1981) that seems to be the best medium for long term storage and subculturing of all fungal isolates I have so far tested, including the serendipitoid fungi. Allen, T. R., T. Millar, S. M. Berch, and M. L. Berbee. Ericoid mycorrhiza is a mutualistic relationship between several lineages of plants in the family Ericaceae (Cassiopoideae, Ericoideae, Harrimanelloideae, Styphelioideae, and Vaccinioideae subfamilies) and a diverse group of soil fungi. Mycorrhiza 23:199208, Obase K, Matsuda Y (2014) Culturable fungal endophytes in roots of Enkianthus campanulatus (Ericaceae). Example: Ripe undamaged European blueberry (V. myrtillus) fruits are collected in nature when available, dried at room temperature for a few days and stored in a fridge at ca. ( 1992) comprehensively synthesized concepts, phenomena, and ecological implications of mycorrhiza specificity . (2019)). This obviously applies also for species reclassificationsimproper taxonomy leads to unnecessary confusion that may take years to resolve (e.g., the unnecessary inter-family transfer of the prominent ErMF from Rhizoscyphus to Pezoloma based solely on a morphological observation of a single apothecium, see Baral and Krieglsteiner 2006). During past 3years (20172019), Mycorrhiza had received only nine manuscripts focused on this intriguing type of root-fungus association (four short notes including one re-submission and five original articles) and two thirds of them had to be declined. (2017) Does warming by open-top chambers induce change in the root-associated fungal community of the arctic dwarf shrub Cassiope tetragona (Ericaceae)? Research on the diversity and identity of mycobionts associated with ericoid roots especially outside the already explored areas (see Fig. New Phytol 83:739744, Brown W (1924a) Two mycological methods. 57 leaves. Fungal Ecology 3:205-214. In general, priority should be given to sequences derived from reliably identified cultures with deposited vouchers and the queries can be limited, e.g., to ITS or LSU sequences from fungal type and other reference material.