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?

M5 CK5 M4 CK4 M3 CK3 CK2 M2 M1 CK1 1*2m plots 2 treatments 5 replicates CK2 M2 M1 CK1 Liu, 2008

Sampling Strategy Roots Rhizosphere soils Other samples

Sampling strategy in each plot Liu roots soils mix Soil cores Whole dig out sealed bags (transport to lab with ice) mix 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

Brundrett et al. 1994. Practical methods in mycorrhiza research. 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. 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: 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 Correlation analysis Significant Difference SPSS or other Statistical programs Other analyses

Molecular analysis Roots cleaning DNA extraction PCR DGGE Clone-RFLP Separation of PCR production Clone-Sequencing T-RFLP

Genomic DNA Low signal Genomic DNA of Clover Roots 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 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) DNA mixture similar size but 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

NS31 40bp GC AM1(AML2) GC-NS31 DGGE DGGE pattern (GC-NS31/AM1) 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

Proportion of total signal sample1 sample7 sample3 sample5 DGGE pattern analysis 0/1 Proportion of total signal Bandscan

Need more accurate data (sequencing) 1-1 1-2 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 4-1 4-2 4-3 4-4 4-5 4-6 4-7 4-8 4-9 3-2 3-1 5-1 1 2 3 4 5 4-6 2-3 Overnight at 4℃ PCR DGGE 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 *Molar ratio of PCR product:vector may require optimization Promega *Molar ratio of PCR product:vector may require optimization

Clone library Liu

RFLP Typing Liu, 2008 508bp 509bp

Liu 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 Liu Hin1II(Hsp92II) : 1U HinfI: 1U 37℃, 4h; 2.5% agrose, 140V c. 50min A: 1 B: 5 C: 1 D: 8 E: 1 F: 6 G: 1 H: 1 No. of clones of each RFLP types

Sequencing Sequencing primer T7/SP6; M13 F/R M13 F (c.60bp) M13 R (c. 200bp) M13 F (c.60bp)

Sequences analyses Sequences edit (ContigExpress) Liu et al. unpublished data 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

Why do we study on AMF spores? 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

Three types of hyphal attachment in Glomus genus Straight Funnel Recurved Liu Liu INVAM Three types of hyphal attachment in Glomus genus Most of them are very difficult to separate

Globose swelling ---Bulbous sporogenous cell Germination shield Scutellospora Gigaspora Liu 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)

X100 X200 X400 X1000

INVAM Globose swelling ---Bulbous sporogenous cell Germination shield Liu S. calospora INVAM Gigaspora Liu

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

single-spore pot culture 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 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.