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

Summary

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.

Abstract

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.

Introduction

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 present^1,2. 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 ho....

Protocol

1. Selection of biological material, root sampling, and storage

1. 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.
2. Collect all the clean roots from each plant in a plastic bag, and collect all samples from one variant in one bigger bag. Write on each bag the stage/variant name and the samplin....

Results

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.......

Discussion

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 health^{16,17,18,19,20}. Thus, there is a need for .......

Materials

Name	Company	Catalog Number	Comments
Apple vinegar 5%	FABRICA DE CONSERVE RAURENI S.R.L.	OȚET DE MERE	https://www.raureni.ro/ro-ro/produs/otet-de-mere
Blue Ink	Pelikan	4001	https://www.pelikan.com/pulse/Pulsar/ro_RO.Store.displayStore.224848./cerneal%C4%83-4001-de-la-pelikan
Cover slips	Menzel-Glaser	D 263 M	https://si.vwr.com/store/product/20545757/cover-glasses-menzel-glaser
Forceps, PMP	Vitalab	9.171 411	http://shop.llg.de/info881_Forceps_PMP_lang_UK. htm?UID=55005bf838d8000000000000 &OFS=33
Glass jar 47 mL	Indigo Cards	BORCAN 47 ML HEXAGONAL	https://indigo.com.ro/borcan-47-ml-hexagonal
Laminating Pouches	Peach	PP525-08	Business Card (60x90mm) / https://supremoffice.ro/folie-laminare-60x90mm-125mic-carte-vizita-100-top-peach-pp525-08-510328
Microflow Class II ABS Cabinet	Bioquell UK Ltd	Microflow Class II ABS Cabinet	http://www.laboratoryanalysis.co.uk/graphics/products/034_11%20CLASS%202BSC%20(STD).pdf
Microscope slides	Deltalab	D100001	https://distrimed.ro/lame-microscop-matuite-la-un-capat-26x76-mm-deltalab/?utm_source=Google%20Shopping&utm_campaign= google%20shopping%20distrimed&utm_medium=cpc& utm_term=1647&gclid=CjwKCAjwu YWSBhByEiwAKd_n_odzr8CaCXQ hl9VQkAB3p-ODo2Ssuou9cnoRtz1Gb xsjqPY7F05HmhoCj6oQAvD_BwE
Microsoft Office 365	Microsoft	Office 365	Excel and Powerpoint; spreadsheet and presentation
NaOH	Oltchim	01-2119457892-27-0065	http://www.sodacaustica.com.ro/pdf/fisa-tehnica-soda-caustica.pdf
Nitrile gloves	SemperGuard	816780637	https://www.sigmaaldrich.com/RO/en/product/aldrich/816780637?gclid=CjwKCAjwuYWSBhByEiwAKd _n_rmo4RRt8zBql7ul8ox AAYhwhxuXHWZcw4hlR x0Iro_4IyVt69aFHRoCmd wQAvD_BwE
Optika camera	OPTIKA	CP-8; P8 Pro Camera, 8.3 MP CMOS, USB 3.0	https://www.optikamicroscopes.com/optikamicroscopes/product/c-p-series/
Optika Microscope	OPTIKA	B383pL	https://www.optikamicroscopes.com/optikamicroscopes/product/b-380-series/
Protective mask FFP3	Hermes Gift	HERMES000100	EN 149-2001+A1:2009 / https://www.emag.ro/set-10-masti-de-protectie-respiratorie-hermes-gift-ffp3-5-straturi-albe-hermes000100/pd/DTZ8CXMBM/#specification-section
Scalpel	Cutfix	9409814	https://shop.thgeyer-lab.com/erp/catalog/search/search.action;jsessionid=C258CA 663588CD1CBE65BF 100F85241B?model.query=9409809
White wine vinegar 9%	FABRICA DE CONSERVE RAURENI S.R.L.	OȚET DE VIN ALB	https://www.raureni.ro/ro-ro/produs/otet-de-vin-alb