Abstract
A method to measure chitin content in fungi and ectomycorrhizal roots with high-performance liquid chromatography (HPLC) was developed. Measurements of fluorescence of 9-fluorenylmethylchloroformate (FMOC-CI) derivatives of glucosamine were made on acid hydrolysates of pure chitin, chitin-root mixtures and fungal-root mixtures. The method was applied on 5 isolates of ectomycorrhizal fungi, and ectomycorrhizal and non-mycorrhizal Pinus sylvestris roots. Interference from amino acids was removed by pre-treatment of samples with 0.2 N NaOH. This pre-treatment did not reduce the recovery of chitin, nor did plant material affect the recovery of chitin. The HPLC method was compared with a colorimetric chitin-method by measurements on root-fungal mixtures, with known fungal content. The HPLC method gave estimates of fungal biomass which were equal to the expected while the colorimetric method showed values significantly (p<0.001) lower than the expected. The present chitin method offers a sensitive and specific tool for the quantification of chitin in fungi and in ectomycorrhizal roots.
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References
Bethlenfalvay G J, Pacovsky R S and Brown M S 1981 Measurement of mycorrhizal infection in soybeans. Soil Sci. Soc. Am. J. 45, 871–875.
Ekblad A 1995 Actinorhizal and ectomycorrhizal activities in symbioses of Alnus incana and Pinus sylvestris. PhD thesis, Department of Plant Physiology, Umeå University, Umeå, Sweden.
Giovanetti M and Mosse B 1980 An evaluation of techniques for measuring vesicular arbuscular mycorrhizal infection in roots. New Phytol. 84, 489–500.
Harley J L and Smith S E 1983 Mycorrhizal Symbiosis. Academic Press, London, UK. 483p.
Hicks R E and Newell S Y 1984 A comparison of glucosamine and biovolume conversion factors for estimating fungal biomass. Oikos 42, 355–360.
Lezica R F and Quesada-Allué L 1990 Chitin. Methods Plant Biochem. 2, 443–481.
Matcham S E, Jordan B R and Wood D A 1985 Estimation of fungal biomass in a solid substrate by three independent methods. Appl. Microbiol. Biotechnol. 21, 108–112.
Modess O 1941 Zur Kenntnis der Mycorrhizabildner von Kiefer und Fichte. Symbolae Bot. Upsal. 5, 1–147.
Nylund J-E and Wallander H 1992 Ergosterol analysis as a means of quantifying mycorrhizal biomass. In Methods in Microbiology, Vol 24. Eds. J R Norris, D J Read and A K Varma. pp -88. Academic Press, London, UK.
Plassard C S, Mousain D G and Salsac L E 1982 Estimation of mycelial growth of basidiomycetes by means of chitin determination. Phytochemistry 21, 345–348.
Ride J P and Drysdale R B 1972 A rapid method for the chemical estimation of filamentous fungi in plant tissue. Physiol. Plant Pathol. 2, 7–15.
Roche N, Venague A, Desgranges C and Durand A 1993 Use of chitin measurement to estimate fungal biomass in solid state fermentation. Biotechnol. Adv. 11, 677–683.
Roseman S and Daftner I 1956 Colorimetric method for determination of glucosamine and galactosamine. Anal. Chem. 28, 1743–1746.
Salmanowicz W E and Nylund J-E 1988 High performance liquid chromatography determination of ergosterol as a measure of ectomycorrhizal infection in Scots pine. Eur. J. For. Pathol. 18, 291–298.
Sawicki E, Hauser T R, Stanley T W and Elbert W 1961 The 3-methyl-2-benzothiazolone hydrazone test. Anal. Chem. 1, 93–96.
Schmitz O, Danneberg G, Hundeshagen B, Klingner A and Bothe H 1991 Quantification of vesicular-arbuscular mycorrhiza by biochemical parameters. J. Plant Physiol. 139, 106–114.
Tsuji A, Kinoshita T and Hoshino M 169a Microdetermination of hexosamines. Chem. Pharm. Bull. 17, 217–218.
Tsuji A, Kinoshita T and Hoshino M 1969b Analytical chemical studies on amino sugars. II. Determination of hexosamines using 3-methyl-2-benzothiazolone hydrazone hydrochloride. Chem. Pharm. Bull. 17, 1505–1510.
Vignon C, Plassard C, Mousain D and Salsac L 1986 Assay of fungal chitin and estimation of mycorrhizal infection. Physiol. Veg. 24, 201–207.
Wessels J G H 1993 Tansley Review No. 45. Wall growth, protein excretion and morphogenesis in fungi. New Phytol. 123, 397–413.
Whipps J M, Haselwandter K, McGee E E M and Lewis D H 1982 Use of biochemical markers to determine growth, development and biomass of fungi in infected tissues, with particular reference to antagonistic and mutualistic biotrophs. Trans. Br. Mycol. Soc. 79, 385–400.
Zelles L 1988 The simultaneous determination of muramic acid and glucosamine in soil by high-performance liquid chromatography with precolumn fluorescence derivatization. Biol. Fert. Soils 6, 125–130.
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Ekblad, A., Näsholm, T. Determination of chitin in fungi and mycorrhizal roots by an improved HPLC analysis of glucosamine. Plant Soil 178, 29–35 (1996). https://doi.org/10.1007/BF00011160
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DOI: https://doi.org/10.1007/BF00011160