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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.
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.
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°44′56″ lat. N and 23°50′0″ 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 Ghețari, Apuseni Mountains (46°49'064" lat. N and 22°81'418'' 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·ha-1 manure; V3 = 10 t·ha-1 manure + N 50 kg·ha-1, P2O5 25 kg·ha-1, K2O 25 kg·ha-1; V4 = N 100 kg·ha-1, P2O5 50 kg·ha-1, K2O 50 kg·ha-1; V5 = 10 t·ha-1 manure + N 100 kg·ha-1, P2O5 50 kg·ha-1, K2O 50 kg·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.
1. Selection of biological material, root sampling, and storage
2. Root processing, clearing, and staining for microscopy
NOTE: Use gloves, a mask, and a microbiological/chemical hood for this step of the protocol.
3. Root processing for microscopy
4. Microscopic analysis of the root samples
5. Post-microscopy image assemblage
6. Scoring of mycorrhizal colonization
7. Raw data analysis and result extraction
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...
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's sustainable development and the maintenance of its health16,17,18,19,20. Thus, there is a need for ...
The authors declare no conflicts of interest.
This paper uses data resulting from two Ph.D. studies in the thematic area of "Corn Mycorrhizal Patterns Driven by Agronomic Inputs", conducted by Victoria Pop-Moldovan, and "Mycorrhizal Status and Development of Colonization in Mountain Grassland Dominant Species", conducted by Larisa Corcoz, under the coordination of Prof. Dr. Roxana Vidican.
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