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Practical methods in AM fungal research
Yongjun Liu [email protected]
Advisor: Prof. Huyuan Feng Dec. 2009 Lanzhou University
Mycorrhiza
• = plant roots + fungi
• Arbuscular mycorrhiza (AM)
• Ectomycorrhiza (ECM)
• Other mycorrhizas
Arbuscular Mycorrhiza (AM)
• Plant roots + AM fungi (Glomeromycota)
• Physiological &Ecological significance
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?
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
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.
• 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
a
b
a
b
ca
a
b
c
b
a
Molecular analysis
• Roots cleaning
• DNA extraction
• PCR
• Separation of PCR production
DGGEClone-RFLP
T-RFLPClone-Sequencing
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 unpublished figure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
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
?
• 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
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
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
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
• 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) (http://www.agro.ar.szczecin.pl/~jblaszkowski/Species%20descriptions%20of%20AMF.html)
Gigaspora
Globose swelling---Bulbous sporogenous cell
Germination shield
S. calospora
INVAM
INVAM
Liu
Liu
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.
Trap culture
• Trapping is necessary to obtain many healthy spores of colonizing fungi for identification and as inoculum to establish monospecific cultures.
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.