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Practical methods in

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

= 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

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

Whole dig out

roots

mix

soils

mix

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

RLC; root length colonization

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%

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%
Dont 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)
Krger et al. New Phytologist, 2009, 183:212-223 (A. Schler)

Primers used in our studies


Nested PCR
GeoA2/Geo11 (first PCR)
Schwarzott & Schler. 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

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

4-2

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

How to make a clone library

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
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

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

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+Melzers 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.

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

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
yjliu@lzu.edu.cn. Thank you.