Comparison of aniline blue staining and immunoblotting methods for detecting endophytic fungi in grass seeds
Barbara Wiewióra
b.wiewiora@ihar.edu.plZakład Nasiennictwa i Nasionoznawstwa, Instytut Hodowli i Aklimatyzacji Roślin, Radzików (Poland)
Maria Prończuk
Samodzielna Pracownia Traw i Roślin Motylkowych, Instytut Hodowli i Aklimatyzacji Roślin, Radzików (Poland)
Abstract
The objective of the study was to compare the efficiency of and the input of labour and money in aniline blue staining and immunoblotting assays used to identify endophytic fungi infesting grass seed. Seeds of 17 cultivars, strains and ecotypes of meadow fescue (Festuca pratensis L.), red fescue (F. rubra L.), perennial ryegrass (Lolium perenne L.) and tufted hairgrass (Deschampsia caespitosa (L.) P. Beauv.) were tested. Detectability of endophytes using the two methods was comparable. However, the aniline blue staining was found to be a very laborious and time-consuming method. Moreover, great experience of the researcher is needed to recognize the endophyte mycelium under the microscope. Furthermore, the method requires additional laboratory equipment to protect the staff against the harmful effects of some reagents. The immunoblotting method is comparatively more expensive, but it is evidently easier and less labour- and time-consuming.
Keywords:
aniline blue, endophytic fungi, grass seed, immunoblot method, staining methodReferences
Bacon C. W., White J. F. Jr. 1994. Stains, media and procedure for analyzing endophytes. In: Biotechnology of endophytic fungi of grasses. Bacon C. W., White J.F.Jr. (eds.) CRC Press, Boca Raton: 47 — 56.
DOI: https://doi.org/10.1201/9781351070324-4
Google Scholar
Belesky D. P., Robbins J. D., Stuedemann J. A., Wilkinson S. R., Devine O. J. 1987. Fungal endophyte infection-loline derivative alkaloid concentration of grazed tall fescue. Agron. J. 79: 217 — 220.
DOI: https://doi.org/10.2134/agronj1987.00021962007900020009x
Google Scholar
Dapprich P., Paul V. H., Krohn K. 1994. A novel and rapid staining method for the detection of vital endophytes in seeds and leaf sheaths of Lolium perenne. IOBC wprs Bulletin 17/1: 139 — 146.
Google Scholar
Doss R. P., Welty R. E. 1995. A polymerase chain reaction-based procedure for detection of Acremonium coenophialum in tall fescue. Phytopathology 85: 913 — 917.
DOI: https://doi.org/10.1094/Phyto-85-913
Google Scholar
Guillaumin J. J., Frain M., Pichon N., Ravel C. 2000. Survey of fungal endophytes in wild grass species in the Auvergne region (central France). Proc. of the 4th International Neotyphodium. Grass Interactions Symposium, Soest, Germany: 85 — 92.
Google Scholar
Hahn H., Huth W., Schöberlein W. 2000 a. Tissue print immunoassay — a rapid and sensitive method also for detection of Neotyphodium endophytes. Proc. of the 4th International Neotyphodium/ Grass Interactions Symposium, Soest, Germany: 139 — 143.
Google Scholar
Hahn H., Huth W., Schöberlein W. 2000 b. A rapid and sensitive method also for detection of Neotyphodium endophytes. 4th International Neotyphodium. Grass Interactions Symposium, Soest, Germany. Book of Abstracts: 40.
Google Scholar
Hill N. S., Hiatt E. E., III, De Battista J. P., Costa M. C., Griffiths C. H., Klap J., Thorogood D., Reeves J. H. 2002. Seed testing for endophytes by microscopic and immunoblot procedures. Seed Sci. & Technol. 30: 347 — 355.
Google Scholar
ISTA. 2005. International Rules for Seed Testing. Annexe to Chapter 7 Seed Health Testing Methods. Edition 2005. Published by The International Seed Testing Association (ISTA), P. O. BOX 308, 8303 Bassersdorf, CH-Switzerland.
Google Scholar
Liu D., Van Heeswijck R., Latch G., Leonforte T., Panaccio M., Langford C., Cunninham P., Reed K. 1995. Rapid identification of Acremonium coenophialum endophytes through arbitrarily primed PCR. FEMS Microbiology Letter 133: 95 — 98.
DOI: https://doi.org/10.1111/j.1574-6968.1995.tb07867.x
Google Scholar
Majewska-Sawka A., Nakashima H. 2004. Endophyte transmission via seeds of Lolium perenne L.: immunodetection of fungal antigens. Fung. Genet. Biol. 41: 534 — 541.
DOI: https://doi.org/10.1016/j.fgb.2004.01.005
Google Scholar
Pańka D., Podkówka L., Lamparski R. 2004. Preliminary observations on the resistance of meadow fescue (Festuca pratensis Huds.) infected by Neotyphodium uncinatum to diseases and pests and native value. In: Proc. of the 5th International Symposium on Neotyphodium Grass Interactions. Kallenbach R et al. (eds.). Fayetteville, AR USA, May, 2004, 401: 88 — 90.
Google Scholar
Petroni O. 1986. Taxonomy of endophytic fungi of aerial plant tissues. In: Microbiology of Phyllosphere. Cambridge. Cambridge University Press: 175 — 187.
Google Scholar
Prończuk M. 2005. Endofity traw — znaczenie, występowanie i metody wykrywania. Przegląd literatury. Biul. IHAR 235: 297 — 309.
Google Scholar
Saha D. C., Jackson M. A., Jonson-Cicalese J. M. 1988. A rapid staining method for detection of endophytic fungi in turf and forage grasses. Phytopathology 78: 237 — 239.
DOI: https://doi.org/10.1094/Phyto-78-237
Google Scholar
Siegel M. R., Latch G. C. M., Johnes M. C. 1985. Acremonium fungal endophytes of tall fescue and perennial ryegrass: significance and control. Plant Dis. 69/2: 179183.
Google Scholar
Welty R. E., Rennie W. J. 1985. ISTA Handbook on Seed Health Testing, Working sheet No. 55: Grasses, Endophyte. International Seed Testing Association, Zurich, Switzerland: 1 — 4.
Google Scholar
Welty R. E., Azevedo M. D., Cook K. L. 1986. Detecting viable Acremonium endophytes in leaf sheaths and meristems of tall fescue and perennial ryegrass. Plant Dis. 70: 431 — 435.
DOI: https://doi.org/10.1094/PD-70-431
Google Scholar
White J. F. Jr. 1987. Widespread distribution of endophytes in the Poaceae. Plant Dis. 71: 340 — 342.
DOI: https://doi.org/10.1094/PD-71-0340
Google Scholar
Wiewióra B., Prończuk M., Ostrowska A. 2006. Infekcja nasion traw przez endofity w kolejnych latach użytkowania plantacji. Biul. IHAR 242: 285 — 293.
Google Scholar
Authors
Barbara Wiewiórab.wiewiora@ihar.edu.pl
Zakład Nasiennictwa i Nasionoznawstwa, Instytut Hodowli i Aklimatyzacji Roślin, Radzików Poland
Authors
Maria PrończukSamodzielna Pracownia Traw i Roślin Motylkowych, Instytut Hodowli i Aklimatyzacji Roślin, Radzików Poland
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