Professional Documents
Culture Documents
AM fungal research
Yongjun Liu
yjliu@lzu.edu.cn
Advisor: Prof. Huyuan Feng
Dec. 2009
Lanzhou University
Belowground Ecosystem
De Deyn & van der Putten. Trends in Ecology & Evolution, 2005, 20:625-633
Mycorrhiza
Outline
Experimental design
Sampling strategy
Working with roots
Working with soils
Other data collection
1*2m plots
2 treatments
5 replicates
M2
M1
Liu, 2008
M5
M4
M3
CK5
CK4
CK3
CK2
CK1
Sampling Strategy
Roots
Rhizosphere soils
Other samples
Soil cores
roots
mix
soils
mix
mix
sealed bags (transport to lab with ice)
roots soils
Molecular analysis
Roots staining
10% KOH (time & )
2% HCl
Staining (time & )
Destaining
Photo: INVAM
Estimation of AM colonization
Using a
dissecting
microscope
Brundrett et al. 1994.
Practical methods in
mycorrhiza research.
Using a
compound
microscope
X200
magnification
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
G (connected
= p + q to
+r+s+t
AC=
(q+s)/G*100%
arbuscules
or
mycorrhizal
VC=
(r+s)/G*100%
vesicles.
HC=
(G-p)/G*100%
H: 0
A: 0
V: 1
H: 1
A: 0
V: 0
H: 0
A: 1
V: 0
H: 0
A: 1
V: 0
Liu
C. korshinskii
Correlation analysis
Significant Difference
Other analyses
Molecular analysis
Roots cleaning
DNA extraction
PCR
Separation of PCR production
DGGE
Clone-RFLP
Clone-Sequencing
T-RFLP
Genomic DNA
Low signal
Liu
PCR condition
DNA polymerase
Taq or Pfu?
Templates concentration
1:10; 1:50; 1:100 or 1:1000?
Optimization of anneal temperature
high or low?
Elongation time
the expected DNA size;
Taq: c.1kb/min; Pfu: c. 600bp/min
Purification of DNA
PCR purification Kit or Gel Excised Kit
Liu
Genomic DNA
Nested-PCR strategy
AM1(AML2)
DGGE
NS31
40bp GC
GC-NS31
Liu
6% or 8%(w/v) PAGE
Denaturing Gradient
20-35% ? or other optimized gradient
Voltage & Time
150-160V; 5-6h or 60-80V; 14-16h
sample1
sample3
sample5
0/1
sample7
1-2
1-3
1-4
1-5
1-6
1-7
1-8
1-9
2-2
2-3
2-4
2-5
2-6
2-7
2-8
2-9
4-2
4-3
4-7
3-2
4-8
4-9
3-1
4-4
4-5
4-6
5-1
1
2
3
4
5
2-3
4-6
Overnight at 4
DGGE
PCR
RFLP
Cloning& Sequencing
DNA
clone vector
ligation
competent cell
transform
plate transform culture onto plates
ligation
Promega
Clone library
Liu
RFLP Typing
Liu, 2008
508bp
508bp
508bp
509bp
A B
D B E
B F D D
F D H
B F
D D B
F F
F D
Liu
Hin1II(Hsp92II) : 1U
A: 1 HinfI: 1U
B: 5
C: 1
D: 8 No. of clones of each RFLP types
E: 1
F: 6
G: 1
H: 1
Sequencing
Sequencing primer
T7/SP6; M13 F/R
M13 F (c.60bp)
M13 R (c. 200bp)
Sequences analyses
Spores extraction
Photo: INVAM
INVAM
INVAM
Liu
INVAM
60-100 X
Have stalk
Glomus; Paraglomus; Scutellospora; Gigaspora
Most of AM fungal species are belonging to the Glomus genus
Liu
Liu
INVAM
Recurved
Funnel
Straight
Germination shield
Liu
Scutellospora
INVAM
Gigaspora
Entrophospora
Sporiferous saccule
Scar
Scar
X100
X200
X400
X1000
INVAM
Germination shield
Globose swelling
---Bulbous sporogenous cell
Liu
INVAM
S. calospora
Liu
Gigaspora
Glomus mosseae
INVAM
INVAM
Glomus intraradices
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.
C. korshinskii
platations
Liu
farmland
Liu
Trap culture
Trapping is necessary to obtain many
healthy spores of colonizing fungi for
identification and as inoculum to establish
monospecific cultures.
coarse sand
Harvest after 3 or
4 months later
Establishment
of monospecific
culture
Photo: INVAM