Name: mycorrhizal maps as a tool to explore colonization patterns and fungal strategies in the roots of festuca rubra and zea mays
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Description: Watch this Scientific Journal Video about Mycorrhizal Maps as a Tool to Explore Colonization Patterns and Fungal Strategies in the Roots of Festuca rubra and Zea mays at JoVE.com

This paper uses data resulting from two Ph.D. studies in the thematic area of &#34;Corn Mycorrhizal Patterns Driven by Agronomic Inputs&#34;, conducted by Victoria Pop-Moldovan, and &#34;Mycorrhizal Status and Development of Colonization in Mountain Grassland Dominant Species&#34;, conducted by Larisa Corcoz, under the coordination of Prof. Dr. Roxana Vidican.

1. Selection of biological material, root sampling, and storage
<ol>
	<li>Collect the entire root of plants with a shovel (Figure 1A) separately for each variant and replicate. Remove gently, by hand, the large soil aggregates from the roots. Wash the entire root system and measure it on a scale with 1 cm x 1 cm cells (Figure 1B). Cut the roots separately for each plant, and place them into a plastic bag.</li>
	<li>Collect all the clean roots ...

<ol>
	<li>Trivedi, P., Leach, J. E., Tringe, S. G., Sa, T., Singh, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant-microbiome+interactions:+From+community+assembly+to+plant+health.">Plant-microbiome interactions: From community assembly to plant health.</a> Nature Reviews Microbiology. 18 (11), 607-621 (2020).</li><li>Jeffries, P., Barea, J. M., Hock, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=4+Arbuscular+Mycorrhiza:+A+Key+Component+of+Sustainable+Plant-Soil+Ecosystems.">4 Arbuscular Mycorrhiza: A Key Component of Sustainable Plant-Soil Ecosystems.</a> The Mycota. IX Fungal Associations. , 51-75 (2012).</li><li>Parniske, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arbuscular+mycorrhiza:+the+mother+of+plant+root+endosymbioses.">Arbuscular mycorrhiza: the mother of plant root endosymbioses.</a> Nature Reviews Microbiology. 6 (10), 763-775 (2008).</li><li>Gianinazzi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agroecology:+The+key+role+of+arbuscular+mycorrhizas+in+ecosystem+services.">Agroecology: The key role of arbuscular mycorrhizas in ecosystem services.</a> Mycorrhiza. 20 (8), 519-530 (2010).</li><li>Lee, E. -. H., Eo, J. -. K., Ka, K. -. H., Eom, A. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+of+arbuscular+mycorrhizal+fungi+and+their+roles+in+ecosystems.">Diversity of arbuscular mycorrhizal fungi and their roles in ecosystems.</a> Mycobiology. 41 (3), 121-125 (2013).</li><li>Zhang, Y., Zeng, M., Xiong, B., Yang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecological+significance+of+arbuscular+mycorrhiza+biotechnology+in+modern+agricultural+system.">Ecological significance of arbuscular mycorrhiza biotechnology in modern agricultural system.</a> Ying Yong Sheng Tai Xue Bao = The Journal of Applied Ecology. 14 (4), 613-617 (2003).</li><li>Shah, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+endophytic+Bacillus+megaterium+colonization+on+structure+strengthening,+microbial+community,+chemical+composition+and+stabilization+properties+of+Hybrid+Pennisetum.">Effect of endophytic Bacillus megaterium colonization on structure strengthening, microbial community, chemical composition and stabilization properties of Hybrid Pennisetum.</a> Journal of the Science of Food and Agriculture. 100 (3), 1164-1173 (2020).</li><li>Souza, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Arbuscular Mycorrhizal Fungi. , (2015).</li><li>Sun, X. -. G., Tang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+four+routinely+used+methods+for+assessing+root+colonization+by+arbuscular+mycorrhizal+fungi.">Comparison of four routinely used methods for assessing root colonization by arbuscular mycorrhizal fungi.</a> Botany. 90 (11), 1073-1083 (2012).</li><li>Smith, S., Read, D., Smith, S. E., Read, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colonization+of+Roots+and+Anatomy+of+Arbuscular+Mycorrhiza.">Colonization of Roots and Anatomy of Arbuscular Mycorrhiza.</a> Mycorrhizal Symbiosis. , 42-90 (2008).</li><li>Stoian, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+approach+and+future+perspectives+in+microscopic+patterns+of+mycorrhizal+roots.">Sensitive approach and future perspectives in microscopic patterns of mycorrhizal roots.</a> Scientific Reports. 9 (1), 10233 (2019).</li><li>Pop-Moldovan, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Divergence+in+corn+mycorrhizal+colonization+patterns+due+to+organic+treatment.">Divergence in corn mycorrhizal colonization patterns due to organic treatment.</a> Plants. 10 (12), 2760 (2021).</li><li>Corcoz, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycorrhizal+patterns+in+the+roots+of+dominant+Festuca+rubra+in+a+high-natural-value+grassland.">Mycorrhizal patterns in the roots of dominant Festuca rubra in a high-natural-value grassland.</a> Plants. 11 (1), 112 (2021).</li><li>Corcoz, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deciphering+the+colonization+strategies+in+roots+of+long-term+fertilized+Festuca+rubra.">Deciphering the colonization strategies in roots of long-term fertilized Festuca rubra.</a> Agronomy. 12 (3), 650 (2022).</li><li>Stoian, V., Florian, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycorrhiza+-+Benefits,+influence,+diagnostic+method.">Mycorrhiza - Benefits, influence, diagnostic method.</a> Bulletin of University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca. Agriculture. 66 (1), 2009 (2009).</li><li>Prates J&#250;nior, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agroecological+coffee+management+increases+arbuscular+mycorrhizal+fungi+diversity.">Agroecological coffee management increases arbuscular mycorrhizal fungi diversity.</a> PLoS One. 14 (1), 0209093 (2019).</li><li>Rillig, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+farmers+should+manage+the+arbuscular+mycorrhizal+symbiosis.">Why farmers should manage the arbuscular mycorrhizal symbiosis.</a> New Phytologist. 222 (3), 1171-1175 (2019).</li><li>Rillig, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+an+integrated+mycorrhizal+technology:+Harnessing+mycorrhiza+for+sustainable+intensification+in+agriculture.">Towards an integrated mycorrhizal technology: Harnessing mycorrhiza for sustainable intensification in agriculture.</a> Frontiers in Plant Science. 7, 1625 (2016).</li><li>Bhale, U. N., Bansode, S. A., Singh, S., Gehlot, P., Singh, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multifactorial+Role+of+Arbuscular+Mycorrhizae+in+Agroecosystem.">Multifactorial Role of Arbuscular Mycorrhizae in Agroecosystem.</a> Fungi and their Role in Sustainable Development: Current Perspectives. , 205-220 (2018).</li><li>Khaliq, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+control+of+dry+root+rot+of+chickpea+caused+by+Rhizoctonia+bataticola+under+the+natural+field+condition.">Integrated control of dry root rot of chickpea caused by Rhizoctonia bataticola under the natural field condition.</a> Biotechnology Reports. 25, 00423 (2020).</li><li>Vaida, I., P&#259;curar, F., Rotar, I., Tomo&#537;, L., Stoian, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+diversity+due+to+long-term+management+in+a+high+natural+value+grassland.">Changes in diversity due to long-term management in a high natural value grassland.</a> Plants. 10 (4), 739 (2021).</li><li>. The International Collection of (Vesicular) Arbuscular Mycorrhizal Fungi Available from: <a target="_blank" href="https://invam.wvu.edu/collection">https://invam.wvu.edu/collection</a> (2022)</li><li>. The International Bank for the Glomeromycota Available from: <a target="_blank" href="https://www.i-beg.eu/">https://www.i-beg.eu/</a> (2022)</li></ol>

Studies on mycorrhizal colonization are vital for new strategy development in the agronomic field. The potential of multiple cultivated plants to form a symbiotic association with arbuscular mycorrhizas made them an important component of the agroecosystem&#39;s sustainable development and the maintenance of its health16,17,18,19,20. Thus, there is a need for ...

Arbuscular mycorrhiza (AM) fungi are a category of soil-borne endophytes that are constantly an area of interest for researchers. Their presence in the roots of most plants and their involvement in nutrient cycles makes them vital components in the stability of every ecosystem where herbaceous plants are present1,2. Through their extra-radicular mycelium, AM act as a fungal extension for plant roots, especially in hard-to-reach areas3. The main activity is in host plant roots, where AM develops large hyphae networks and specific intracellular structures called arbuscules. The lack of host specificity allows the symbiont to colonize multiple species at the same time. This ability provides AM with the role of resource allocation and nutrient regulation in the ecosystem; the fungus also provides support in plant survival and aids in plant performance4,5,6,7. The reaction of AM species to host roots is visible in the extension and location of the intra-radicular mycelium and the presence and shape of the arbuscules developed intracellularly. The intracellular arbuscules act as an interchange point between the two symbionts and represent areas characterized by fast transfer processes. The structures that the AM produce are species-dependent, and, in addition to arbuscules, in the roots, they also develop vesicles, spores, and auxiliary cells.
There are many challenges in the assessment of AM symbionts in plant roots8,9. The first one is their constant development during the entire vegetation period of hosts, which leads to multiple changes in the hyphal arbuscular structure. The different stages of arbuscular growth, up to their collapse, are clearly present in roots, but the senescent AM structures are sometimes digested, which makes them only partially visible10. The second challenge is represented by the staining method and protocol, the large diversity of root systems, the dimension of their cells, and the differences in thickness, which make it hard to propose a unified method. The last challenge is represented by the assessment and scoring of AM colonization. There are numerous methods that score AM with different degrees of objectivity, and most of them are still restricted to microscopy techniques. The simple ones are based on the presence/absence of structures in the root cortex, while the more complex ones are based on visual scoring and the use of colonization classes, with the integration of the frequency and intensity of the colonization phenomenon. A lot of data have been produced in the last decades on the mycorrhizal status of multiple species, but most of the methods are restricted to the observed value of colonization without pointing to the real position of each structure in the root cortex. As a response to the necessity of more accurate results on AM colonization, a method based on microscopic analysis of mycorrhizal patterns (MycoPatt) in roots was developed to assemble, in a digital form, the detailed mycorrhizal maps11. Also, the method allows the objective calculation of colonization parameters and the determination of the actual position of each structure in the root.
The position of the AM fungal structures can be important in answering the following two questions. The first one is related to the analysis of the colonization in one specific moment from the vegetation cycle of a plant. In this context, it is very useful to observe the arbuscular/vesicle abundance, report how are they located in the root, and provide a very clear colonization image and parameters. The second one is related to the detection of fungal strategy and its orientation and even the forecast of its future development. One application of the MycoPatt can be for plants analyzed daily, every 2-3 days, weekly, or during various growth stages. In this context, the location of the vesicles/arbuscules is important to better understand the biological mechanism of AM colonization. These parameters and observations are very useful to supplement the mathematical parameters.
The aim of this article is to demonstrate the ability of the MycoPatt system to explore the native AM fungi colonization potential and strategy in Zea mays (corn) roots during different development stages and in Festuca rubra (red fescue) roots under different long-term fertilization conditions. To fulfill the aim, two large databases from two experiments were analyzed. The corn experiment was established at Cojocna (46&#176;44&#8242;56&#8243; lat. N and 23&#176;50&#8242;0&#8243; long. E), in the Experimental Didactic Farm of the University of Agricultural Sciences and Veterinary Medicine Cluj on a phaeoziom with a loamy texture soil12. The red fescue experiment is a part of a larger experimental site established in 2001 in Ghe&#539;ari, Apuseni Mountains (46&#176;49&#39;064&#34; lat. N and 22&#176;81&#39;418&#39;&#39; long. E), on a preluvosol (terra rossa) soil type13,14. Corn was collected in five different growth phenophases12: B1 = 2-4 leaves (as a control point for the start of mycorrhizal colonization); B2 = 6 leaves; B3 = 8-10 leaves; B4 = cob formation; B5 = physiological maturity. Starting from the 2-4 leaves stage (A0), an organic treatment was applied, which resulted in a two-graduation factor (A1 = control and A2 = treated). Roots of red fescue were collected at flowering from an experiment with five long-term fertilizations13,14: V1 = control, non-fertilized; V2 = 10 t&#183;ha-1 manure; V3 = 10 t&#183;ha-1 manure + N&#160;50 kg&#183;ha-1, P2O5 25 kg&#183;ha-1, K2O 25 kg&#183;ha-1; V4 = N 100 kg&#183;ha-1, P2O5 50 kg&#183;ha-1, K2O 50 kg&#183;ha-1; V5 = 10 t&#183;ha-1 manure + N 100 kg&#183;ha-1, P2O5 50 kg&#183;ha-1, K2O 50 kg&#183;ha-1. Five plants were collected in each development stage from every fertilization variant. The staining protocols and their performance in terms of sample processing time and quality of staining were analyzed. The relation between AM hyphae development and the presence of its structures in roots was analyzed separately for each species and continued with the identification of the most permissive roots for colonization. The specific colonization patterns of each root system were analyzed based on colonization maps and the value of AM parameters.
Corn is an annual plant, which implies continuous growth of the roots, and that was the main reason to apply the MycoPatt in the growing stages. Red fescue is a perennial plant from a grassland treated for a long time with different fertilizers. Its roots have a shorter development of 1 year, and the anthesis is considered as the vegetation point when the plant changes its metabolism from vegetative to generative. To catch these plants during these intense activity periods, the abovementioned time points were chosen. Sampling during the vegetation period is difficult for this species when grown in natural grasslands.

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mycorrhizal maps as a tool to explore colonization patterns and fungal strategies in the roots of festuca rubra and zea mays

Arbuscular mycorrhizal fungi are symbionts in the roots of plants. Their role is to sustain host development and maintain the nutritional equilibrium in the ecosystems. The colonization process is dependent on several factors like soil ecology, the genetic diversity of the fungi and host, and agronomic practices. Their synchronized action leads to the development of a complex hyphal network and leads to the secondary development of vesicles and arbuscules in the root cells. The aim of this research was to analyze the efficiency of the mycorrhizal patterns (MycoPatt) method for the positioning of fungal structures in the roots of Festuca rubra and Zea mays. Another objective was to explore the fungal colonization strategy as revealed by mycorrhizal maps of each species. The acquisition and assemblage of multiple microscopic images allow mycorrhizal colonization assessment in both corn and red fescue plants to provide information on the realistic position of the developed structures. The observed mycorrhizal patterns highlight the variable efficiency of each plant in terms of developing connections with soil symbiotic fungi, caused by applied treatments and growth stage. Mycorrhizal detailed maps obtained through the MycoPatt method are useful for the early detection of plant efficiency in symbiotic acquisition from the soil.

The correct use of the gentle crushing method of the roots after the staining procedures provides good details of mycorrhizal structures, both for Zea mays (Figure 8A-C) and Festuca rubra (Figure 9A-E), good contrast between mycorrhizal structures and root cells, and a confirmation of the stele due to the blue color. If the clearing and staining procedures fail to succeed, root...

Watch this Scientific Journal Video about Mycorrhizal Maps as a Tool to Explore Colonization Patterns and Fungal Strategies in the Roots of Festuca rubra and Zea mays at JoVE.com

Mycorrhizal Maps as a Tool to Explore Colonization Patterns and Fungal Strategies in the Roots of Festuca rubra and Zea mays

The protocol here describes the methods for the assessment of the arbuscular mycorrhizal colonization patterns and strategy in roots for two species: Zea mays and Festuca rubra. The use of the MycoPatt method permits the calculation of parameters, the conversion of mycorrhizal structures into digital data, and the mapping of their real position in roots.

Mycorrhizal Maps as a Tool to Explore Colonization Patterns and Fungal Strategies in the Roots of Festuca rubra and Zea mays

Arbuscular mycorrhizal fungi are symbionts in the roots of plants. Their role is to sustain host development and maintain the nutritional equilibrium in ...

MycoPatt method permits an in depth exploration of arbuscular mycorrhizal colonization in roots. Mycorrhizal maps point to fungal expansion and present the real position of each structure as a specific pattern. Mycorrhizal colonization is objectively analyzed at 1%resolution.Each root segment is automatically converted to a digital matrix and generates an extensive database of colonization parameters. To begin, remove the roots from the refrigerator and place them on a working platform for thawing at room temperature. To perform root clearing, place all roots from one plant in a 30 to 50 milliliter jar, then prepare a 10%sodium hydroxide solution with tap water and pour it into the jar until it completely covers the roots.Shake the jar vigorously for one to two minutes for homogenous dispersion of clearing solution in the roots. Allow the roots to remain in the clearing solution for 48 hours. For root rinsing, pass the content of the jar through a sieve.Rinse the roots several times in tap water until the clearing solution is completely removed. Next place the rinsed roots in a clean jar for staining. Then prepare a five to five percent ink vinegar solution with tap water.Pour it into the jar until it covers the roots and shake it for one to two minutes. After 48 hours for partial destaining, rinse the stained roots in tap water for one to two minutes. Shake the jar vigorously to remove the extra staining solution.Repeat the procedure until the roots are clear for microscopic evaluation. For root segmentation, place the stained roots on a scaled cutting board. Cut the roots into one centimeter segments.Choose 15 segments for each variant. Next, spread the roots on a slide for segment preparation, use a laminating pouch to cover the roots and gently crush them, starting from an edge. Use a soft plastic tool to display the roots on the slide.Carefully remove the laminating pouch and cover the sample with a cover slip. Add water to a corner of the slide with a pipette, and let the water slowly spread on the slide. Remove the extra water with a paper towel.For microscopic analysis, use a microscope equipped with a good resolution camera. Analyze the slides starting from an extremity. Capture each microscopic field.Use 10 or 40x magnification for thick roots and 40x for thin roots. After capturing, rename each image with a code that will permit the real post-assemblage of root parts. Use presentation software to design a drawing board for image assemblage, set the width two to three centimeters wider than the image width.After capturing 15 pictures, add all captured images from one segment in the order of their capture, starting with one to 15, and reconstruct the entire length of the root segment. Next, use the vertical alignment to align the images in the center and ensure that each image follows the previous one. Place a grid of 10 by 150 cells on all pictures to cover the entire root segment.Additionally, place a 10 by 10 grid on each picture and in every cell of this grid, insert a number from one to six if an arbuscular mycorrhizal or AM structure is visible, or leave blank if no AM structure is present. Add a table for a grid of 10 cells in width and 150 cells in length. Change the table with dimensions to the width of the images, then change the length of the table to comprise all the images.For scoring mycorrhizal colonization, use the unique number to score each type of structure as described in the mycorrhizal pattern method. Use one for hyphae, two for arbuscules, three for vesicles, four for spores, five for auxiliary cells, and six for entry points. Score each observed mycorrhizal structure from each cell of the previously applied grids.After scoring, insert all the obtained scores into the MycoPatt spreadsheet. Use the copy paste function to transfer all the scores from the presentation into the first sheet named Raw Data. For the primary analysis, use the third sheet named Parameters to visualize the results as percentages separately in three forms.Use columns A to K to analyze the horizontal image of colonization, columns M to W for the vertical image of colonization, and columns Y to AI to analyze the transversal colonization for each of the 15 10 by 10 squares and the final average colonization. Next, apply the definitions and formulas specific to each parameter. For production and extraction of mycorrhizal maps, visualize the image obtained from the conversion of mycorrhizal structures code into colors in the second sheet named Graphs.Then export the resultant image in the graph sheet as an image and use the color code in the legend to analyze mycorrhizal patterns. For mycorrhizal map analysis, identify the most important structures and their assemblage. Then describe the mycorrhizal colonization pattern observed in the analyzed roots, followed by a colonization strategy based on observed structural development in the root, the branching pattern, and arbuscular vesicle development.This protocol provides the details of mycorrhizal structures with good contrast between mycorrhizal structures and root cells for Zea mays and Festal rubra. The unsuccessful clearing and staining procedures resulted in unclear microscopic images of mycorrhizal structures in Festal rubra and Zea mays. Mycorrhizal colonization patterns allowed a complete exploration of the colonization mechanism, this method provides a deep, small scale exploration of colonization patterns and strategies for Zea mays and Festuca rubra with an additional visual expression of colonization parameters.In Zea mays highly fluctuating colonization potential depending on the plant's developmental stage was observed, however, in Festuca rubra, the colonization processes occur inside the roots and the development of hyphal networks was correlated with a low development speed of the roots. MycoPatt is suitable for mapping the arbuscular mycorrhizal colonization in any plant. It is also helpful to fully explore the colonization mechanism and assess fungal strategy along the roots.

Research

JoVE Journal

Biology

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