1. Practical methods in AM fungal research
Yongjun Liu
Advisor: Prof. Huyuan Feng
Dec Lanzhou University

2. Belowground Ecosystem
De Deyn & van der Putten. Trends in Ecology & Evolution, 2005, 20:

3. Mycorrhiza
= plant roots + fungi
Arbuscular mycorrhiza (AM)
Ectomycorrhiza (ECM)
Other mycorrhizas

4. Arbuscular Mycorrhiza (AM)
Plant roots + AM fungi (Glomeromycota)
Physiological &Ecological significance

5. Rillig. Ecology Letters, 2004,7:740-754

6. Outline Experimental design Sampling strategy Working with roots
Working with soils
Other data collection

7. A Case of Experimental Design
What is the AM fungal diversity in semiarid agricultural field?
Do mulching film change the status of AM fungi (colonization; community composition; …..)?
Was there a link of AM fungi and agronomic practices?

8. M5 CK5 M4 CK4 M3 CK3 CK2 M2 M1 CK1 1*2m plots 2 treatments
5 replicates
CK2 M2 M1
CK1
Liu, 2008

9. Sampling Strategy
Roots
Rhizosphere soils
Other samples

10. Sampling strategy in each plot
Liu
roots
soils
mix
Soil cores
Whole dig out
sealed bags (transport to lab with ice)
mix
roots
soils

11. Working with Roots
Estimation of AM colonization
Molecular analysis

12. Roots staining 10% KOH (time & ℃) 2% HCl Staining (time & ℃)
Destaining
Photo: INVAM

13. Estimation of AM colonization
Using a dissecting microscope
Brundrett et al Practical methods in mycorrhiza research.
Using a compound microscope
X200 magnification

14. Magnified intersection method
McGonigle et al. New Phytologist, 1990,115:
Mounting roots on slides
Slides NO.
Time
Quantified using the magnified intersections method

15. Brundrett et al. 1994. Practical methods in mycorrhiza research.
p : no fungal structures q: arbuscules
r : mycorrhizal vesicles, s : arbuscules and
mycorrhizal vesicles t : mycorrhizal hyphae but
no arbuscules or
mycorrhizal vesicles u : hyphae not seen to be
connected to arbuscules or
mycorrhizal vesicles.
G ( = p + q + r + s + t + u)
AC= (q+s)/G*100%
VC= (r+s)/G*100%
HC= (G-p)/G*100%
RLC; root length colonization
Brundrett et al Practical methods in mycorrhiza research.

16. Total intersections (G): N+A+V+H
%RLC= (G-N)/G*100%
%AC= A/G*100%
%VC= V/G*100%
Don’t acount those hypha which not seen to be connected to arbuscules or vesicles.

17. H: 0
A: 0
V: 1
H: 1
A: 0
V: 0
H: 0
A: 1
V: 0
H: 0
A: 1
V: 0

18. Roots AM microscopic photos
Liu
Arum or Paris type
C. korshinskii

19. Roots AM colonization data
Count colonization data (RLC%,AC%,VC%)
Data analysis and make histogram
Correlation analysis
Significant Difference
SPSS or other Statistical programs
Other analyses

20. Molecular analysis Roots cleaning DNA extraction PCR
DGGE
Clone-RFLP
Separation of PCR production
Clone-Sequencing
T-RFLP

21. Genomic DNA Low signal Genomic DNA of Clover Roots
Liu
Genomic DNA of Clover Roots
(Plant DNA Extraction Kit; Tiangen, Beijing)

22. Primers choose & PCR strategy
Liu et al. unpublished figure
Helgason et al. Nature, 1998, 384:431 (JPW. Young)
Lee et al. FEMS Microbiology Ecology, 2008, 65: (JPW. Young)
Krüger et al. New Phytologist, 2009, 183: (A. Schüßler)

23. Primers used in our studies
Nested PCR
GeoA2/Geo11 (first PCR)
Schwarzott & Schüßler. Mycorrhiza,2001,10:
NS31/AM1 (c. 550bp);GC-NS31/AM1
used before. Liu et al. 2009, FEMS Microbiol Ecol, 67:81-92
NS31/AML2 (c. 560bp); GC-NS31/AML2
used recently in two experiments
AML1/AML2 as first PCR primers
Problems? Can not work well.

24. PCR condition DNA polymerase Taq or Pfu? Templates concentration
Optimization of anneal temperature
high or low?
Elongation time
the expected DNA size;
Taq: c.1kb/min; Pfu: c. 600bp/min

25. Purification of DNA
PCR purification Kit or Gel Excised Kit
Liu

26. ? Genomic DNA (rDNA or other genes) DNA mixture similar size but
Nested-PCR strategy
Specific AMF primers:
NS31/AM1(AML2), AML1/AML2, et al.
(rDNA or other genes)
DNA
mixture
similar size but
different sequence
separate these sequences
sequencing

27. NS31
40bp GC
AM1(AML2)
GC-NS31
DGGE
DGGE pattern (GC-NS31/AM1)
Liu

28. 6% or 8%(w/v) PAGE
Denaturing Gradient
20-35% ? or other optimized gradient
Voltage & Time
V; 5-6h or V; 14-16h

29. Proportion of total signal
sample1
sample7
sample3
sample5
DGGE pattern analysis
0/1
Proportion of total signal
Bandscan

30. Need more accurate data (sequencing)
1-1
1-2
1-3
1-4
1-5
1-6
1-7
1-8
1-9
2-1
2-2
2-3
2-4
2-5
2-6
2-7
2-8
2-9
4-1
4-2
4-3
4-4
4-5
4-6
4-7
4-8
4-9
3-2
3-1
5-1 1 2 3 4 5
4-6
2-3
Overnight at 4℃
PCR
DGGE
RFLP
Cloning& Sequencing

31. How to make a clone library
DNA
clone vector
ligation
competent cell
transform
plate transform culture onto plates

32. clone vector (Promega)

33. ligation *Molar ratio of PCR product:vector may require optimization
Promega
*Molar ratio of PCR product:vector may require optimization

34. Clone library
Liu

35. RFLP Typing
Liu, 2008
508bp
509bp

36. Liu Hin1II(Hsp92II) : 1U HinfI: 1U 37℃, 4h; 2.5% agrose, 140V c. 50min
A B C D B E D B F D D G F D H B F D D B F F F D
Liu
Hin1II(Hsp92II) : 1U
HinfI: 1U
37℃, 4h; 2.5% agrose, 140V c. 50min
A: 1
B: 5
C: 1
D: 8
E: 1
F: 6
G: 1
H: 1
No. of clones of each RFLP types

37. Sequencing Sequencing primer T7/SP6; M13 F/R M13 F (c.60bp)
M13 R (c. 200bp)
M13 F (c.60bp)

38. Sequences analyses Sequences edit (ContigExpress)
Liu et al. unpublished data
Sequences analyses
Sequences edit (ContigExpress)
BLAST (NCBI Genbank; online)
Chimera check (RDP release 9; online)
Phylogenetic analysis (ClustalX; Mega4.0)
Delimit phylotypes
(bootstrap value, %identity, tree topology)
No. of clones of each RFLP types or DGGE DNA bands

39. Why do we study on AMF spores?
Working with Soils
Soil AM fungal spores
Soil characteristics
Moisture, TN, TC, OC, TP, AP…….
Why do we study on AMF spores?

40. Spores extraction wet-sieving and sucrose centrifugation method
Brundrett et al Practical methods in mycorrhiza research.

41. Photo: INVAM

42. INVAM
INVAM
Liu
INVAM

43. X

44. Primarily distinguishing the genera
No stalk
Acaulospora; Archaeospora; Entrophospora
Have stalk
Glomus; Paraglomus; Scutellospora; Gigaspora
Most of AM fungal species are belonging to the Glomus genus

45. Three types of hyphal attachment in Glomus genus
Straight
Funnel
Recurved
Liu
Liu
INVAM
Three types of hyphal attachment in Glomus genus
Most of them are very difficult to separate

46. Globose swelling ---Bulbous sporogenous cell
Germination shield
Scutellospora
Gigaspora
Liu
INVAM

47. Entrophospora Sporiferous saccule Scar
Schenck & Perez Manual for the identification of VA mycorrhizal fungi

48. Acaulospora & Archaeospora
Scar
Schenck & Perez Manual for the identification of VA mycorrhizal fungi

49. Working with spores Permanent slides
PVLG & PVLG+Melzer’s reagent (1 : 1, v/v)
Classified using current taxonomic criteria and information published by:
INVAM ( or
by the website of Janusz Blaszkowski (Poland) (

54. X100
X200
X400
X1000

55. INVAM
Globose swelling
---Bulbous sporogenous cell
Germination shield
Liu
S. calospora
INVAM
Gigaspora
Liu

56. Glomus mosseae
INVAM
INVAM
Glomus intraradices

57. Spore density number of spores per 100 gram or 1 gram soil.
Spore density can partly reflect the AMF response to the environmental variation and further to the variation of ecosystem.

58. C. korshinskii platations
Liu
farmland
Liu

59. Trap culture
Trapping is necessary to obtain many healthy spores of colonizing fungi for identification and as inoculum to establish monospecific cultures.

60. coarse sand Harvest after 3 or 4 months later
store at 4℃ and use within 30days
Photo: INVAM

61. Establishment of monospecific culture
Photo: INVAM

62. single-spore pot culture
Spore germination and
single-spore pot culture
Brundrett et al Practical methods in mycorrhiza research.

63. I hope this lecture will facilitate your researches of AM fungi
I hope this lecture will facilitate your researches of AM fungi. If you have any suggestion about the AM fungal research protocols, especially the methods of spores identification and culture (we are poor about these), please send mail to Thank you.