Transcript Document

Practical methods in
AM fungal research
Yongjun Liu
[email protected]
Advisor: Prof. Huyuan Feng
Dec. 2009
Lanzhou University
Belowground Ecosystem
De Deyn & van der Putten. Trends in Ecology & Evolution, 2005, 20:625-633
Mycorrhiza
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= plant roots + fungi
Arbuscular mycorrhiza (AM)
Ectomycorrhiza (ECM)
Other mycorrhizas
Arbuscular Mycorrhiza (AM)
• Plant roots + AM fungi (Glomeromycota)
• Physiological &Ecological significance
Rillig. Ecology Letters, 2004,7:740-754
Outline
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Experimental design
Sampling strategy
Working with roots
Working with soils
Other data collection
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?
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
Sampling strategy in each plot
Liu
Soil cores
roots
mix
soils
mix
Whole dig out
mix
sealed bags (transport to lab with ice)
roots soils
Working with Roots
• Estimation of AM colonization
• 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
Magnified intersection method
McGonigle et al. New Phytologist, 1990,115:495-501
• Mounting roots on slides
Slides NO.
Time
• Quantified using the magnified
intersections method
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.
RLC; root length colonization
G ( = p + q + r + s + t + u)
AC= (q+s)/G*100%
VC= (r+s)/G*100%
HC= (G-p)/G*100%
Brundrett et al. 1994. Practical methods in mycorrhiza research.
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.
H: 0
A: 0
V: 1
H: 1
A: 0
V: 0
H: 0
A: 1
V: 0
H: 0
A: 1
V: 0
Roots AM microscopic photos
Liu
Arum or Paris type
C. korshinskii
Roots AM colonization data
• Count colonization data (RLC%,AC%,VC%)
• Data analysis and make histogram
SPSS or other Statistical programs
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
Genomic DNA of Clover Roots
(Plant DNA Extraction Kit; Tiangen, Beijing)
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, 65:339-349 (JPW. Young)
Krüger et al. New Phytologist, 2009, 183:212-223 (A. Schüßler)
Primers used in our studies
• Nested PCR
• GeoA2/Geo11 (first PCR)
Schwarzott & Schüßler. Mycorrhiza,2001,10:203-207
• 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.
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
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Genomic DNA
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
AM1(AML2)
DGGE
NS31
40bp GC
GC-NS31
Liu
DGGE pattern (GC-NS31/AM1)
• 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
DGGE pattern analysis
Bandscan
0/1
Proportion of total signal
sample7
Need more accurate data (sequencing)
1-1
2-1
4-1
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
4-7
3-2
4-8
4-9
3-1
4-2
4-3
4-4
4-5
4-6
5-1
1
2
3
4
5
2-3
4-6
DGGE
Overnight at 4℃
PCR
RFLP
Cloning& Sequencing
How to make a clone library
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DNA
clone vector
ligation
competent cell
transform
plate transform culture onto plates
clone vector (Promega)
ligation
Promega
*Molar ratio of PCR product:vector may require optimization
Clone library
Liu
RFLP Typing
Liu, 2008
H sp 9H
2 in
II f I
H sp 9 2 II
H in f I
508bp
1
1
1
1
H in
f I i accessio
H sp 9 2nIIn u mb er .tx t
n cb
50bp
50bp
50bp
50bp
H in fI H in fI H sp 9 2 II
n cb i accessio n n u mb er .tx t
H sp 9 2 II
H sp 9H2in
IIf I
n cb i accessio n n u mb er.tx t
n cb i accessio n n u mb er .tx t
H5in0f8I
508bp
0 8fI
H5 in
508bp
H5in0 f8I
509bp
509
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
A: 1 HinfI: 1U
37℃, 4h; 2.5% agrose, 140V c. 50min
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
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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
Liu et al. unpublished data
Working with Soils
• Soil AM fungal spores
• Soil characteristics
Moisture, TN, TC, OC, TP, AP…….
Why do we study on AMF spores?
Spores extraction
wet-sieving and sucrose
centrifugation method
Brundrett et al. 1994. Practical
methods in mycorrhiza research.
Photo: INVAM
INVAM
INVAM
Liu
INVAM
60-100 X
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
Liu
Liu
INVAM
Recurved
Funnel
Straight
Three types of hyphal attachment in Glomus genus
Most of them are very difficult to separate
Globose swelling ---Bulbous sporogenous cell
Germination shield
Liu
Scutellospora
INVAM
Gigaspora
Entrophospora
Sporiferous saccule
Scar
Schenck & Perez. 1989.Manual for the identification of VA mycorrhizal fungi
Acaulospora & Archaeospora
Scar
Schenck & Perez. 1989.Manual for the identification of VA mycorrhizal fungi
Working with spores
• Permanent slides
PVLG & PVLG+Melzer’s reagent (1 : 1, v/v)
• Classified using current taxonomic criteria and
information published by:
INVAM (http://invam.caf.wvu.edu) or
by the website of Janusz Blaszkowski (Poland)
(http://www.agro.ar.szczecin.pl/~jblaszkowski/Speci
es%20descriptions%20of%20AMF.html)
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.
spore density (spores/g soil)
35
c
April
30
July
October
b
25
20
15
c
b
b
10
a
b
b
a
a
13-years
20-years
C. korshinskii
a
platations
a
5
Liu
0
5-years
42-years
spore density(spores per 100g
soil)
2500
Liu
farmland
2000
2006-Jun
2006-Sep
2007-May
2008-Apr
2008-May
1500
1000
500
0
N
NP
SNP
M
MNP
CK
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
store at 4℃ and use within 30days
Photo: INVAM
Establishment
of monospecific
culture
Photo: INVAM
Spore germination and
single-spore pot culture
Brundrett et al. 1994. Practical
methods in mycorrhiza research.
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 [email protected]. Thank
you.