Molecular Techniques to Study Polymorphism between Closely Related Microorganisms in Relation to Specific Protein Phosphatase

  • Rajani Malla
  • Utprekshya Pokharel
  • Ram Prasad
  • Ajit VarmaEmail author
Part of the Soil Biology book series (SOILBIOL, volume 22)


Phylogenetically, the Sebacinaceae that include P. indica recognized a new order, the Sebacinales. The order primarily contains the genera Sebacina, Tremelloscypha, Efibulobasidium, Craterocolla, and Piriformospora. In this study, attempts were made to purify acid phosphatase from P. indica. The fungus produced only one form of intracellular acid phosphatase irrespective of the phosphate concentration. The enzyme was possibly a constitutive enzyme showing molecular mass of 66 kD as separated by SDS-PAGE. The enzyme showed the pH and temperature optima of 5.3 and 40°C, respectively. K m for p-NPP (monoester) was 0.35 mM. Antibodies raised against cytosolic acid phosphatase using gel band in Native PAGE after selective precipitation with ammonium sulfate followed by gel filtration and ion exchange chromatography gave sufficient quantities of antibodies based on immunoblot analysis.

Its reaction with native protein as well as denatured protein was significant. The antibody immunoprecipitated a single band of approximately 66 kD protein in SDS gel. The antiserum localized the enzyme on the vacuoles, cell wall, and cytoplasm of the mycelium, indicating the possible sites of phosphate metabolism.

The acid phosphatase in P. indica and S. vermifera were similar in their molecular mass. The immunoblot showed the strong reactivity of antiserum with protein of S. vermifera. P. indica antiserum blotted the bands in S. vermifera at similar location of P. indica on SDS-PAGE. The antiserum also localized the enzyme in S. vermifera by immunofluorescence technique, showing strong relationship of this fungus with P. indica. The immunogold labeling of antiserum from P. indica precisely localized the enzyme in cytoplasm and vacuoles of S. vermifera supporting the immunological link between these two fungi.

Using α-naphthyl phosphate as substrate, different isoforms of ACPase were obtained by Fast Garnet GBC staining. P. indica and S. vermifera CF and W/MF showed three distinct isoforms of ACPase each, one with higher molecular mass and two other with lower molecular masses. The patterns of isoforms were similar in both the fungi.

Piriformospora indica and Sebacina vermifera sensu showed similar morphology, functions, and isozymes. However, they show distinct genetic variation based on the random amplified polymorphic DNA (RAPD) analysis. Un-weighted Pair Group method with Arithmetic Mean (UPGMA) cluster analysis clustered the isolates into two distinct groups. Statistical analysis of the data was preformed using the NTSYS-pc (Numerical Taxonomy System, Applied Biostatistics) program. An average genetic similarity between both the fungi was 0.58 (i.e., 58%), can be considered to place in species of same roof. Results illustrated the potential value of RAPDs technique for detecting polymorphism among fungal.


Sodium Dodecyl Sulphate Sodium Acetate Buffer Water Bath Shaker Native Page Average Genetic Similarity 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



Rajani Malla is thankful to Dr. Ashok K Chauhan, Founder President, Amity-RBEF, New Delhi for his encouragement and support. The author is also thankful to Pham Giang from International Center for Genetic Engineering and Biotechnology, Ram Prasad from Amity Institute, Dr. Upendra and Sweta from All India Institute of Medical Sciences.


  1. Alves JM, Siachakr CD, Allot M, Tizroutine S, Mussio I, Servaes A (1994) Isozyme modifications and plant regeneration through somatic embryogenesis in sweet potato. Plant Cell Rep 13:437–441Google Scholar
  2. Cox G, Moran KG, Sanders F, Nokolds C, Tinker PB (1980) Translocation and transfer of nutrients in vesicular arbuscular mycorrhizas. III. Polyphosphate granules and phosphorus translocation. New Phytol 84:649–659CrossRefGoogle Scholar
  3. Fries LLM, Pacovsky RS, Safir GR, Kaminski J (1998) Phosphorus effect on phosphatase activity in endomycorrhizal maize. Physiol Plantarum 103:162–171CrossRefGoogle Scholar
  4. Gianinazzi-Pearson V, Gianinazzi S (1978) Enzymatic studies on the metabolism of vesiculararbuscular mycorrhiza. II. Soluble alkaline phosphatase specific to mycorrhizalinfection in onion roots. Physiol Plant Pathol 12:45–53CrossRefGoogle Scholar
  5. Gravel P, Golaz G (1996) Two-dimensional PAGE using carrier ampholyte pH gradients in the first dimension. In: Walker JM (ed) The protein protocols handbook. Humana, Totowa, New Jersey, pp 127–32CrossRefGoogle Scholar
  6. Harrison MJ, Dewbre GR, Liu JY (2002) A phosphate transporter from Medicago truncatula involved in the acquisition of phosphate released by arbuscular mycorrhizal fungi. Plant Cell 14:2413–2429PubMedCrossRefGoogle Scholar
  7. Horst MN (2000) Molecular approaches to glycobiology. pp 2–30Google Scholar
  8. Hill TW, Kaefer E (2001) Improved protocols for aspergillus medium: trace elements and minimum medium salt stock solutions. Fungal Genet Newslett 48:20–21Google Scholar
  9. van Aarle IM (2001) Microscopic detection of phosphatase activity of saprophytic and arbuscular mycorrhizal fungi using a fluorogenic substrate. Mycologia 93:17–24CrossRefGoogle Scholar
  10. Kaldorf M, Koch B, Rexer K-H, Kost G, Varma A (2005) Patterns of interaction between populus Esch5 and Piriformospora indica: a transition from mutualism to antagonism. Plant Biol 7:210–218PubMedCrossRefGoogle Scholar
  11. Malla R, Md Z, Yadav V, Suniti, Verma A, Rai M, A Varma A (2002) Piriformospora indica and plant growth promoting Rhizobacteria: An Appraisal. In: Rao GP, Bhat DJ, Lakhanpal TN, Manoharichari C (Eds). Frontiers of fungal diversity in India, pp 401–419Google Scholar
  12. Malla R, Prasad R, Giang PH, Pokharel U, Oelmueller R, Varma A (2004) Characteristic features of symbiotic fungus Piriformospora indica. Endocytobiosis Cell Res 15:579–600Google Scholar
  13. Malla R, Pokharel U, Varma A (2005) Random amplified polymorphic DNA of the two fungi from Sebacinales. Journal of Institute of Science and Technology 14:34–43Google Scholar
  14. Malla R, Pokharel U, Varma A (2006a) Random amplified polymorphic DNA of the two fungi from members of Sebacinales. J Inst Sci Technol 14:34–43Google Scholar
  15. Malla R, Pokharel U, Varma A (2006b) Immunological characterization of acid phosphatase in a endophytic fungus. Nepal J Sci Technol 7:77–84Google Scholar
  16. Malla R, Pokharel U, Prasad R, Varma A (2007a) Proteomics and genomics approach to study plant-microbe cross communication. In: Chauhan AK, Harsha K, Varma A (eds) Microbes for human life, vol 4. IK International, India, pp 609–636Google Scholar
  17. Malla R, Varma A (2005) Phosphatase (s) in microorganisms. Biotechnological applications of microbes. IK International, India, New York/Kluwer Academic, Holland, pp 125–50Google Scholar
  18. Malla R, Varma A (2007) Use of short oligonucleotide primers in random amplified polymorphic DNA techniques for species identification. In: Oelmüller R, Varma A (eds) Advanced techniques in soil microbiology, vol 11. Springer, Germany, pp 235–244CrossRefGoogle Scholar
  19. Malla R, Pokharel U, Prasad R, Oelmüller R, Varma A (2007b) Immuno-technology for the localization of acid phosphatase using native gel bands in Piriformospora indica and other soil microorganism. In: Oelmüller R, Varma A (eds) Modern tools and techniques, vol 11. Springer-Verlag, Germany, pp 211–234Google Scholar
  20. McCormick MK, Whigham DF, O’Neill J (2004) Mycorrhizal diversity in photosynthetic terrestrial orchid. New Phytol 163:425–438CrossRefGoogle Scholar
  21. Moller EM, Bahnweg G, Sandermann H, Geiger HH (1992) A simple and efficient protocol for isolation of high molecular weight DNA from filament fungi, fruit bodies and infected plant tissue. Nucleic Acids Res 20:6115–6116PubMedCrossRefGoogle Scholar
  22. Oakley BR, Kirsch DR, Morris NR (1980) A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal Biochem 105:361–363PubMedCrossRefGoogle Scholar
  23. Pasteur N, Pasteur G, Bonhomme F, Catalan J, Davidson Brotton (1988) Practical isozyme genetics. Ellis-Horwood, ChichesterGoogle Scholar
  24. Paszkowski U, Kroken S, Roux C, Briggs SP (2002) Rice phosphate transporters include an evolutionarily divergent gene specifically activated in arbuscular mycorrhizal symbiosis. Proc Natl Acad Sci 99:13324–13329PubMedCrossRefGoogle Scholar
  25. Peškan-Berghöfer T, Shahollari B, Pham HG, Hehl S, Markent C, Blank V, Kost G, Varma A, Oelmueller R (2004) Association of Piriformospora indica with Arabidopsis thaliana roots represent a novel system to study beneficial plant-microbe interactions and involve in early plant protein modifications in the endocytoplasmic reticulum and in the plasma membrane. Physiol Plant 122:465–71CrossRefGoogle Scholar
  26. Rausch C, Daram P, Brunner S, Jansa J, Laloi M, Leggewie G, Amrhein N, Bucher M (2001) A phosphate transporter expressed in arbuscule containing cells in potato. Nature 414:462–466PubMedCrossRefGoogle Scholar
  27. Rohlf FJ, NTSYS-pc (1992) Numerical taxonomy and multivariate analysis system. Vers. 1.70. Exeter Software, Setauket, New YorkGoogle Scholar
  28. Rosendahl S (1994) Isozyme analysis of mycorrhizal fungi and their mycorrhiza. In: Norris JR, Read D, Varma A (eds) Techniques for mycorrhizal research. Academic, London, pp 629–654Google Scholar
  29. Shahollari B, Varma A, Oelmüller R (2005) Expression of a receptor kinase in Arabidopsis roots is stimulated by the basidiomycete Piriformospora indica andthe protein accumulates in Triton X-100 insoluble plasma membrane microdomains. J Plant Physiol (Holland) 162:945–958CrossRefGoogle Scholar
  30. Straker CJ, Mitchell DT (1986) The activity and characterization of acid phosphatases in endomycorrhizal fungi of the Ericaceae. New Phytol 104:243–256CrossRefGoogle Scholar
  31. Summers DF, Szewczyk B (1996) Elution of SDS-PAGE separated proteins from immobilon membranes for use as antigens. In: Walker JM (ed) The protein protocols handbook. Humana, Totowa, New Jersey, pp 699–702CrossRefGoogle Scholar
  32. Tarafdar JC, Rao AV (1996) Contribution of Aspergillus strains to acquisition of phosphorus by wheat (Triticum aestivum L.) and chick pea (Cicer arietinum L.) grown in a loamy soil. Appl Soil Ecol 3:190–214CrossRefGoogle Scholar
  33. Tarafdar JC, Yadav RS, Meena SC (2001) Comparative efficiency of acid phosphatase originated from plant and fungal sources. J Plant Nutr Soil Sci 164:279–282CrossRefGoogle Scholar
  34. Tisserant B, Gianinazzi-Pearson V, Gianinazzi S, Gollotte A (1993) In planta histochemical staining of fungal alkaline phosphatase activity for analysis of efficient arbuscular mycorrhizal infections. Mycol Res 97:245–250CrossRefGoogle Scholar
  35. Varma A, Verma S, Sudha, Sahay N, Britta B, Franken P (1999) Piriformospora indica – a cultivable plant growth promoting root endophyte with similarities to arbuscular mycorrhizal fungi. Appl and Environ Microbiol 65:2741–2744Google Scholar
  36. Verma S, Varma A, Rexer KH, Hassel A, Kost G, Sarabhoy A, Bisen P, Bütenhorn B, Franken P (1998) Piriformospora indica, a new root colonizing fungus. Mycologia 90:896–903CrossRefGoogle Scholar
  37. Walker JM (1994) Basic protein and peptide protocols. Humana, Totowa, NJ, pp 86–187CrossRefGoogle Scholar
  38. Walker JM (1996) Non-denaturing polyacrylamide gel electrophoresis of protein. In: Walker JM (ed) The protein protocols handbook. Humana, NJ, pp 51–54CrossRefGoogle Scholar
  39. Weiß M, Selosse MA, Rexer KH, Urban A, Oberwinkler F (2004) Sebacinales: a hitherto overlooked cosm of heterobasidiomycetes with abroad mycorrhizal potential. Mycol Res 108:1–8Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2010

Authors and Affiliations

  • Rajani Malla
    • 1
  • Utprekshya Pokharel
    • 2
  • Ram Prasad
    • 3
  • Ajit Varma
    • 3
    Email author
  1. 1.Central Department of BiotechnologyTribhuvan UniversityKathmanduNepal
  2. 2.Department of MicrobiologyPunjab UniversityChandigarhIndia
  3. 3.Amity Institute of Microbial TechnologyAmity University Uttar PradeshNoidaIndia

Personalised recommendations