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?

M1

M2

M3M4

M5

CK1

CK2

CK3CK4

CK51*2m plots

5 replicates2 treatments

Liu, 2008

Sampling Strategy

• Roots• Rhizosphere soils• Other samples

Sampling strategy in each plot

Soil cores Whole dig out

sealed bags (transport to lab with ice)mix

roots soils

roots

soils

mix

mix

Liu

Working with Roots

• Estimation of AM colonization

• Molecular analysis

Roots staining• 10% KOH (time & )℃

• 2% HCl

• Destaining

• Staining (time & )℃

Photo: INVAM

Using a dissecting microscope

Using a compound microscope

X200 magnification

Estimation of AM colonization

Brundrett et al. 1994. Practical methods in mycorrhiza research.

Magnified intersection method

• Mounting roots on slides

Slides NO.

Time

McGonigle et al. New Phytologist, 1990,115:495-501

• Quantified using the magnified intersections method

p : no fungal structuresq:  arbusculesr : mycorrhizal vesicles,s : arbuscules and             mycorrhizal vesiclest : mycorrhizal hyphae but     no arbuscules or     mycorrhizal vesiclesu : 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. 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: 1A: 0V: 0

H: 0A: 1V: 0

H: 0A: 1V: 0

H: 0A: 0V: 1

Roots AM microscopic photos

Arum or Paris typeC. korshinskii

Liu

• Count colonization data (RLC%,AC%,VC%)• Data analysis and make histogram

Roots AM colonization data

Correlation analysis Significant Difference

SPSS or other Statistical programs

Other analyses80

85

90

95

100

5-years 13-years 20-years 42-years

% R

LC

April July Octoberc

ab

a

b

ca

a

b

c

b

a

Molecular analysis• Roots cleaning

• DNA extraction

• PCR

• Separation of PCR production

DGGEClone-RFLP

T-RFLPClone-Sequencing

Genomic DNA

Low signal

Genomic DNA of Clover Roots(Plant DNA Extraction Kit; Tiangen, Beijing)

Liu

Primers choose & PCR strategy

Lee et al. FEMS Microbiology Ecology, 2008, 65:339-349 (JPW. Young)Krüger et al. New Phytologist, 2009, 183:212-223 (A. Schüßler)

Helgason et al. Nature, 1998, 384:431 (JPW. Young)

Liu et al. unpublished figure

• NS31/AM1 (c. 550bp);GC-NS31/AM1

Primers used in our studies• Nested PCR

• GeoA2/Geo11 (first PCR)Schwarzott & Schüßler. Mycorrhiza,2001,10:203-207

• NS31/AML2 (c. 560bp); GC-NS31/AML2

used before. Liu et al. 2009, FEMS Microbiol Ecol, 67:81-92

used recently in two experiments

Problems? Can not work well.

• AML1/AML2 as first PCR primers

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

(rDNA or other genes)

Specific AMF primers: NS31/AM1(AML2), AML1/AML2, et al.

Nested-PCR strategy

DNA mixture

similar size but different sequence

separate these sequences

sequencing

?

NS31

40bp GC

AM1(AML2)

GC-NS31

DGGE

DGGE pattern (GC-NS31/AM1)

Liu

• Denaturing Gradient

20-35% ? or other optimized gradient

• 6% or 8%(w/v) PAGE

• Voltage & Time

150-160V; 5-6h or 60-80V; 14-16h

DGGE pattern analysis

0/1 Proportion of total signal

Bandscan

sample1 sample7sample3 sample5

1-11-2 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

4-14-2 4-3 4-4 4-5 4-6 4-7 4-8 4-9

3-23-1

5-1

1

2 3

4 5

4-6

2-3

Overnight at 4 ℃ PCRDGGE

RFLP

Need more accurate data (sequencing)

Cloning& Sequencing

How to make a clone library

• DNA• clone vector• ligation• competent cell• transform• plate transform culture onto plates

clone vector (Promega)

ligation

*Molar ratio of PCR product:vector may require optimization

Promega

Clone library

Liu

RFLP Typing

508bp

508bp

508bp

509bp

Liu, 2008

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

A: 1B: 5C: 1D: 8E: 1F: 6G: 1H: 1

No. of clones of each RFLP types

Liu

Sequencing

• Sequencing primer T7/SP6; M13 F/R

M13 R (c. 200bp)

M13 F (c.60bp)

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 bandsLiu et al. unpublished data

Why do we study on AMF spores?

• Soil AM fungal spores

• Soil characteristics Moisture, TN, TC, OC, TP, AP…….

Working with Soils

wet-sieving and sucrose centrifugation method

Spores extraction

Brundrett et al. 1994. Practical methods in mycorrhiza research.

Photo: INVAM

INVAM

INVAM

INVAM

Liu

60-100 X

• No stalk Acaulospora; Archaeospora; Entrophospora

• Have stalk Glomus; Paraglomus; Scutellospora; Gigaspora

Primarily distinguishing the genera

Most of AM fungal species are belonging to the Glomus genus

Straight

Funnel

Recurved

Three types of hyphal attachment in Glomus genusMost of them are very difficult to separate

INVAM

Liu Liu

Globose swelling ---Bulbous sporogenous cell

Germination shield

Scutellospora GigasporaLiu INVAM

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) (htt

p://www.agro.ar.szczecin.pl/~jblaszkowski/Species%20descriptions%20of%20AMF.html)

X100X200X400X1000

Gigaspora

Globose swelling---Bulbous sporogenous cell

Germination shield

S. calospora

INVAM

INVAM

Liu

Liu

Glomus mosseae

Glomus intraradices 

INVAMINVAM

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

farmland

Liu

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 c

ulture

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.