Lab Report

Report one for BIO3318 Plant Microbe Interactions will be focussed on arbuscular mycorrhizal colonisation in plants. Given the timing of the residential school, you will be provided with data with which to write this report. You will conduct the actual exercise of harvesting plants and processing roots at the residential school so that you gain experience of this important technique.

EXERCISE 4 – DEVELOPMENT OF ARBUSCULAR MYCORRHIZAS IN PLANTS

Background

Mycorrhizas refer to the structures formed when a mutually beneficial symbiotic association occurs between mycorrhizal fungi and the plant root system. Formation of mycorrhizas usually facilitates mineral (particularly phosphate) and water uptake by the plant while the fungus receives all the nutrients and growth factors it requires for reproduction and growth without competition from other organisms. There are four main types of mycorrhizas:

(a) Arbuscular mycorrhizas (AM) are the most common association found in plants (including many crop species) and are typified by a large branching mycelium which is attached to root cells, forming vesicles and arbuscules within these cells. The fungi that form these associations belong to the phylum Mucoromycota, subphylum Glomeromycotina.

(b) Ectomycorrhizal associations are found in many tree species (eg. Eucalyptus spp, Callitris spp.) and consist of a Hartig net (seen in appropriately-stained root cross-sections) formed by a complex of intercellular fungal hyphae which ramify throughout the root cortex, between the cells and a sheath of extraradical hyphae known as a mantle. Ectomycorrhizal fungi are typically members of the phylum Basidiomycota.

(c) Ericoid mycorrhizas are found in members of the Epacridaceae (Australian heaths) and Ericaceae (European heathers). Structurally, this mycorrhizal type consists of a loose mantle surrounding the plant root with coils of intracellular hyphae within the plant. The fungal associates of ericoid mycorrhizas are members of the phylum Ascomycota.

(d) Orchid mycorrhizas are found in the largest plant family, the Orchidaceae. This mycorrhizal association, formed largely with Basidiomycetes, is fairly simple in morphology with fungal coils present in root cortical cells.

References

Moore D., Robson G.D. & Trinci A.P.J. (2019). 21st Century Guidebook to Fungi (online)

Petersen J.H. (2013). The Kingdom Fungi (online)

Smith S.E. & Read D.J. (2008). Mycorrhizal Symbiosis, 3rd edn, Academic Press

Exercise summary

This practical aims to examine the effect of two different soils and two different hosts on the development of arbuscular mycorrhizal associations. Roots will be cleared and stained to highlight AM colonisation. Statistical analyses will be used to draw conclusions about soil and plant host type with regards AM colonisation. The data obtained in this exercise will be used as a basis for one of the assignments in the course.

Laboratory activities

1.1 Growth of plants (to be done for you)

1. Soil was collected from near the surface and from underneath vegetation to ensure that there will be plenty of AM spores in it. Soil was sieved to produce a fine consistency for planting and to minimise heterogeneity caused by stones, pieces of debris or large clods of soil. The soils used included red soil (lateritic krasnozem; Toowoomba) and sandy soil (Flagstone Creek-Preston). Two different plant hosts will be used namely oats (Avena sativa) and mungbean (Vigna radiata).

2. Twelve 10cm pots were filled with soil and eight seeds planted in each as appropriate, that is, 2 soils x 2 hosts x 3 reps x 1 sampling time = 12 pots with at least 8 seeds in each.

1.2 Harvesting plants and processing roots

1. Samples are to be taken at 12 weeks after planting. At this time, 3 pots from each treatment are taken (12 pots) and the soil washed from the root system. Then the root systems are placed in the empty pots containing the appropriate treatment label and taken up to the laboratory. The roots are then cleared and examined for mycorrhizal colonisation. Four slides per pot are prepared with 10 root segments per slide.

2. Select pieces of root system about 5cm long and no greater than 1mm thick. Wash gently under the tap taking care not to damage the cortex. Cut into 1cm segments. Four slides will be prepared, each with 10x 1 cm segments for each pot. At least 40 segments are needed for each pot (it is a good idea to prepare some extra ones to allow for losses during clearing and staining).

3. Place the root segments into a separate labelled microfuge tube and add enough 10% KOH to cover the root segments. Place the tubes into a 90C hotplate for 20 minutes. Remove the tubes and decant off the KOH. Rinse once with tap water.

4. Rinse with 0.1M HCl and then distilled water. Cover root segments in the microfuge tube with the stain, 0.5% trypan blue in lactoglycerol (1:1:1 lactic acid:glycerol:water) and incubate at 95C for 5 minutes.

5. Remove tubes from the incubator, decant stain from segments and transfer them to a watch glass. Add clear 50% glycerol-water and shake segments gently to remove excess stain.

6. Mount ten segments per slide in clear 50% glycerol-water and place a long coverslip over the segments. Squash gently and examine under the microscope. Record the number of colonised root segments per slide. Take photomicrographs of clear views of AM structures within roots eg. arbuscules, intercellular hyphae.

7. Pool all the class results and compare the data obtained for the two species grown in each of the soils for twelve weeks. Present this data in an appropriate table and perform suitable statistical analyses to help define any differences in the extent of AM development in the different treatments. Discuss the class results in terms of what is known about the nature of AM associations.

NOTE: THE DATA BELOW SHOULD BE USED TO COMPILE THE LAB REPORT

Figure X. Arbuscular mycorrhizal colonisation of Avena sativa (oats)

Width of cortical cell is 20µm

Figure Y. Arbuscular mycorrhizal colonisation of Vigna radiata (mungbean)

Width of cortical cell is 20µm

Table X. Raw data for arbuscular mycorrhizal colonisation of plants

Soil type	Oats	Oats	Oats	Mung bean	Mung bean	Mung bean
Red soil	3, 2, 3, 0	3, 3, 1, 0	4, 2, 3, 3	9, 9, 7, 9	10, 9, 7, 9	7, 8, 7, 9
Sandy soil	0, 1, 1, 2	3, 0, 1, 2	2, 1, 0, 1	5, 4, 5, 6	6, 5, 0, 8	2, 5, 5, 6

BIO3318 – PLANT MICROBE INTERACTIONS 2020

DEVELOPMENT OF ARBUSCULAR MYCORRHIZAS IN PLANTS

This report will be marked out of 100 and is worth 20% of your total assessment for this course. It should be written as a brief report (max 5 typed pages plus tables, figures etc.). Proper report format for scientific writing must be used. This report is to be submitted via the Study Desk by 5:00pm on Friday September 11th. A late penalty of 5% per day will apply.

Attach this form to your report

Criteria	Weighting
Introduction: Provide a brief background to arbuscular mycorrhizal fungi.	10
Objectives: Provide a clear statement of the objectives of the exercise.	4
Methods: Briefly outline the experimental approach used in the exercise.	8
Results: Correctly labelled photomicrographs, with appropriate scale bars and descriptions. Summarised bar graph of class colonisation results. Appropriate statistical analysis of the data. Brief description of the key results.	9 19
Discussion: Explanation of the main results. Possible sources of error. Improvement(s).	32 2 2
General conclusion	4
General presentation: correct format, spelling, grammar, expression, correct use of references.	10
Total	100