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Practical methods in AM fungal research

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

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Practical methods in AM fungal research

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  1. Practical methods in AM fungal research Yongjun Liu yjliu@lzu.edu.cn Advisor: Prof.Huyuan Feng Dec. 2009Lanzhou University

  2. Belowground Ecosystem De Deyn & van der Putten. Trends in Ecology & Evolution, 2005, 20:625-633

  3. Mycorrhiza • =plant roots + fungi • Arbuscular mycorrhiza (AM) • Ectomycorrhiza (ECM) • Other mycorrhizas

  4. Arbuscular Mycorrhiza (AM) • Plant roots + AM fungi (Glomeromycota) • Physiological &Ecological significance

  5. Rillig. Ecology Letters, 2004,7:740-754

  6. Outline • Experimental design • Sampling strategy • Working with roots • Working with soils • Other data collection

  7. 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?

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

  9. Sampling Strategy • Roots • Rhizosphere soils • Other samples

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

  11. Working with Roots • Estimation of AM colonization • Molecular analysis

  12. 10% KOH (time & ℃) • Staining (time & ℃) Roots staining • 2% HCl • Destaining Photo: INVAM

  13. Estimation of AM colonization Using a dissecting microscope Brundrett et al. 1994. Practical methods in mycorrhiza research. Using a compound microscope X200 magnification

  14. Slides NO. Time Magnified intersection method McGonigle et al. New Phytologist, 1990,115:495-501 • Mounting roots on slides • Quantified using the magnified intersections method

  15. p : no fungal structuresq: arbuscules r : 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.

  16. 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.

  17. H: 0 A: 0 V: 1 H: 0 A: 1 V: 0 H: 1 A: 0 V: 0 H: 0 A: 1 V: 0

  18. Roots AM microscopic photos Liu Arum or Paris type C. korshinskii

  19. SPSS or other Statistical programs Correlation analysis Significant Difference Other analyses Roots AM colonization data • Count colonization data (RLC%,AC%,VC%) • Data analysis and make histogram

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

  21. Genomic DNA Low signal Liu Genomic DNA of Clover Roots (Plant DNA Extraction Kit; Tiangen, Beijing)

  22. 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)

  23. Primers used in our studies • Nested PCR • NS31/AM1 (c. 550bp);GC-NS31/AM1 • GeoA2/Geo11 (first PCR) Schwarzott & Schüßler. Mycorrhiza,2001,10:203-207 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.

  24. 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

  25. Purification of DNA • PCR purification Kit or Gel Excised Kit Liu

  26. ? Nested-PCR strategy Genomic DNA 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

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

  28. 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

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

  30. 1-4 1-5 1-6 1-7 1-8 1-9 1-2 1-3 1 2 4 1-1 2-4 2-5 2-6 2-7 2-8 2-9 2-3 2-2 2-1 3-1 3-2 3 4-4 4-5 4-6 4-8 4-9 4-7 4-2 4-3 4-1 5 5-1 2-3 4-6 DGGE Cloning& Sequencing Overnight at 4℃ PCR RFLP Need more accurate data (sequencing)

  31. How to make a clone library • DNA • clone vector • ligation • competent cell • transform • plate transform culture onto plates

  32. clone vector (Promega)

  33. ligation Promega *Molar ratio of PCR product:vector may require optimization

  34. Clone library Liu

  35. Liu, 2008 508bp 508bp 508bp 509bp RFLP Typing

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

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

  38. No. of clones of each RFLP types or DGGE DNA bands 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)

  39. Working with Soils Why do we study on AMF spores? • Soil AM fungal spores • Soil characteristics • Moisture, TN, TC, OC, TP, AP…….

  40. Spores extraction wet-sieving and sucrose centrifugation method Brundrett et al. 1994. Practical methods in mycorrhiza research.

  41. Photo: INVAM

  42. INVAM INVAM Liu INVAM

  43. 60-100 X

  44. 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

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

  46. Globose swelling ---Bulbous sporogenous cell Germination shield Scutellospora Gigaspora Liu INVAM

  47. Sporiferous saccule Scar Entrophospora Schenck & Perez. 1989.Manual for the identification of VA mycorrhizal fungi

  48. Scar Acaulospora & Archaeospora Schenck & Perez. 1989.Manual for the identification of VA mycorrhizal fungi

  49. 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 ofJanusz Blaszkowski (Poland) (http://www.agro.ar.szczecin.pl/~jblaszkowski/Species%20descriptions%20of%20AMF.html)

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