Apr 10, 2019
Semi-quantitative measure of roots colonization by arbuscular mycorrhizal fungi using standard light microscopy
- 1Université de Montréal
- Plant Functional Ecology LabTech. support email: sabrina.demers-thibeault@umontreal.ca
Protocol Citation: Florence Blanchard, Alexis Carteron, Xavier Guilbeault-Mayers, Etienne Laliberté 2019. Semi-quantitative measure of roots colonization by arbuscular mycorrhizal fungi using standard light microscopy. protocols.io https://dx.doi.org/10.17504/protocols.io.zbgf2jw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 18, 2019
Last Modified: April 10, 2019
Protocol Integer ID: 21576
Keywords: Colonization, arbuscular, mycorrhiza, arbuscules, fungi, endophytes, sugar maple
Preparation
Preparation
Preparing slides for observation
- Prepare you slides as to put root segments parallel to one another on multiple from top to bottom of the slides, using multiple columns depending on root segment length. A 3x3 frame was used.
Note
Notes: Roots were collected from living Acer saccharum seedlings from a greenhouse experiment.
Note
For subsampling, roots were separated into 3 diameter categories, namely small, medium and large, in order to facilitate discoloration protocol, and cut into 1 cm long fragments. They were then ink stained following the ink and vinegar protocol (Vierheilig et al., 1998) and mounted in glycerol.
Start observation
Observe slides under light microscope at 200x. Start of observation is the first field to be filled by the length of a root from the left of slide, may it be the 1st or 3rd root of the column.
Note
Notes: Higher or lower magnification can be used to identify structures
Record observations
Record observations
Record the presence of fungal structures in the root.
If applicable, categorize structures observed and estimate the amount using a semi quantitative scale e.g. from 1 to 3
Note
Notes: The scale is meant to enrich the presence data by obtaining more information on the degree of colonization, without being as time-consuming as an estimate percentage colonization ( Zemunik et al. 2018).
Note
Decision criterias differ as they depend on the colonization extent and are highly subjective. Preferably, A single observer should be in charge of measuring the complete sample, or a simpler presence-absence protocol should be used. (McGonigle et al.; 1990)
Observation pattern
Observation pattern
Move field of view vertically in direction of other roots. Repeat count
Note
Notes: count only in roots segment which fill the field of view.
Shift field of view horizontally, for a distance equal to the field, and so at the direct right of field just observed.
Note
Notes: With this methods the same hyphaes can be counted more than once. However, a great number of observations ensures the objectivity of the method.(McGonigle et al.; 1990)
Repeat observation on roots moving vertically, and continue onward on the slide creating comb-like pattern over the roots.
Note
Notes: If roots have sled vertical, a complete change in the field of view moving vertically is considered a distinct observation.
Continue until end of root column. If columns stayed well separated, start again from the first field to be filled by the length of a root from the left of column. If columns overlap, continue observations in pattern.
Create a working sheet
For purpose of analysis, all observations of different structures should have their own column, and rows carry each the unique identifier of the observation, of the individual and of other categories (e.g. diameter class).
Example:
| ID | Type | Neg | Arb | Ves | Coil | Hyp.amf | Endo | Pres | |
| M-FT8-NiR-AS-215 | M | 1 | 2 | 3 | 1 | ||||
| M-FT8-NiR-AS-215 | M | 1 | 1 | 3 | 1 | ||||
| M-FT8-NiR-AS-215 | M | 2 | 1 | 1 | |||||
| M-FT8-NiR-AS-215 | M | 1 | 1 | ||||||
| M-FT8-NiR-AS-215 | M | 1 | 1 | ||||||
| M-FT8-NiR-AS-215 | M | 1 | 1 | 3 | 1 | ||||
| M-FT8-NiR-AS-215 | M | 1 | 1 | ||||||
| M-FT8-NiR-AS-215 | M | 1 | 0 | ||||||
| M-FT8-NiR-AS-215 | M | 1 | 1 |
RECORD VARIABLES
RECORD VARIABLES
go to step #3 scale and abbreviation used
ID: Specimen identifier
Type: Diameter categories, namely:
P: Small
M: Medium
G: Large
Arb: Scale from 0-3 on quantity of arbuscules observed, where
0: None
1: One arbuscule
2: Some (~2-4)
3: Many (~>4)
Coil: Scale from 0-3 on quantity of coils observed, where
0: None
1: One or two coils
2: Some (~3-6)
3: Many (~>6)
Ves: Scale from 0-3 on quantity of vesicles observed, where
0: None
1: One to three vesicles, small if many
2: Some (4-6)
3: Many (>6) and or very large vesicles
Hyp.amf: Scale from 0-3 on quantity of VAM hyphae (less than 2 µm diameter) observed, where
0: None
1: Isolated fragments
2: Many unbroken hyphae and/or at least two visible appressorium
3: Colonization of the major part of the cortex (approx 60% and over).
Endo: Scale from 0-3 on quantity of endophytes, or fungal structures other than VAM observed, where
0: None
1: Isolated fragments of hypeas (less or equal to 2 µm) or microsclerotia
2: Some fragments or microsclerotia
3: Many fragments and/or microsclerotia and/or more than 3 cells showing signs of parasitism (by chytrids fungi)
Neg: Negative, no fungal structure observed
Pres: Presence, at least one fungal structure observed
N.B.: Debris, or everything clearly outside the root, even if root broken apart, was not taking into consideration for colonization data.
Photo example
Photo example
Arbuscule 1
Arbuscules 2
coil 1
coil 2
coil 3
Vésicule 1
Vésicule 2
Vésicule 3
Hyphes 1
Hyphes 2
Hyphes 3
Endophytes 1
Endophytes 2
Endophytes 3