Methods for Determining Extracellular and Surface-Bound Phosphatase Activities in Ectomycorrhizal Fungi

Tibbett, Mark; Chambers, Susan M.; Cairney, John

doi:10.1007/978-3-642-60268-9_14

Cited by 10 publications

(3 citation statements)

References 24 publications

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“…The fluorescence method has already been shown to allow the detection of enzymes such as acid phosphatase, chitinase and β‐glucosidase after incubation times as short as 5, 10 and 20 min, respectively (Pritsch et al ., 2004). For phosphatase detection, the fluorescence method therefore allowed a six‐ to over ten‐fold reduction of incubation times (30–60 min) compared with the previously used photometric method using pNPP (Bartlett & Lewis, 1973; Tibett et al ., 1998; Buée et al ., 2004). In the present study, enzymes with lower activities could also be easily detected after 40 min (cellobiohydrolase) or 60 min incubation (xylosidase, glucuronidase and leucine aminopeptidase).…”

Section: Resultsmentioning

confidence: 99%

Activity profiling of ectomycorrhiza communities in two forest soils using multiple enzymatic tests

et al. 2005

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Summary• Data on the diversity and distribution of enzyme activities in native ectomycorrhizal (ECM) communities are inadequate.• A microplate multiple enzymatic test was developed which makes it possible to measure eight enzyme activities on 14 individual, excised ECM root tips. Hydrolytic and oxidative enzymes are involved in the decomposition of lignocellulose, chitin and phosphorus-containing organic compounds. This test system was used to describe the functional diversity of ECM communities in two forest sites.• This set of tests proved to be accurate and sensitive enough to reveal a high diversity of activity profiles, depending on the fungal symbiont and the soil horizon. Ectomycorrhizas can be classified into specialists and generalists, and appear to complement each other in the same horizon to collectively perform all eight activities studied.• By including a higher number of different assays for more detailed analyses, ECM activity profiling will provide a valuable tool for studying the functional diversity of ECM communities.

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Section: Resultsmentioning

confidence: 99%

Activity profiling of ectomycorrhiza communities in two forest soils using multiple enzymatic tests

et al. 2005

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“…Our newly developed method leads to a precise anatomic-physiologic description of SBPA in transversal cuts of roots or mycorrhizae in vivo. None of the existing methods for the determination of SBPA (Antibus et al 1992, Tibbett et al 1998, Tisserant et al 1993 can offer comparable features. Through interactive segmentation of root, mantle, and PA, our method distinguished between SBPA expressed by the root and by the fungi.…”

Section: Discussionmentioning

confidence: 99%

New Perspectives on the Determination of Phosphatase Activity in Ectomycorrhizae of Nothofagus Obliqua Forests in Southern Chile

et al. 2003

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We present a method to localise and quantify surfacebound phosphatase activity (SBPA) in mycorrhizal fungi and mycorrhizae of Nothofagus obliqua (Mirb.) Oerst., using image processed confocal fluorescent microscopy. ELF-97 is a hydrophilic substrate which turns into a strongly fluorescent precipitate upon activation by phosphomonoesterases. In fungal mycelium, this technique has recently been approved by comparison with a standard method (p-nitrophenyl-phosphate). The microscopic technique based on ELF-97 revealed that Paxillus involutus (Batsch: Fr.) Sing. and Austropaxillus boletinoides (Sing.) Brsky. & Jarosch provide different adaptive strategies to changing phosphate concentrations and different pH (3-7). We also analysed SBPA in four mycorrhizal associations of N. obliqua and found that the organisation of the mantle played an essential role regarding the SBPA. In general, mycorrhizal roots shifted SBPA from the root to the mantle. In this context, Pisolithus tinctorius (Pers.) Coker & Couch proved to be the most relevant mycorrhiza partner for N. obliqua by increasing significantly the overall SBPA of the mycorrhiza in respect to non-mycorrhizal roots. In conclusion, ELF-97 fluorescence microscopy in combination with image processing routines determined SBPA and revealed phosphorus (P) adaptation strategies of mycorrhizal fungi and mycorrhizae on a structuralphysiological level.KEYWORDS: Confocal microscopy, ectomycorrhizae of Nothofagus obliqua, ELF-97, phosphatase activity, quantitative fluorescence microscopy. RESUMENNosotros presentamos un método basado en la microscopía confocal de fluorescencia y en el análisis de imágenes, para localizar y cuantificar la actividad de la fosfatasa superficial (SBPA) en hongos ectomicorrícicos y en ectomicorrizas de Nothofagus obliqua (Mirb.) Oerst. El sustrato ELF-97 es hidrofílico, el cual se desarrolla en un precipitado altamente fluorescente bajo su activación a través de fosfomonoesterasas. Recientemente esta técnica ha sido aprobada mediante comparaciones con un método estándar (fosfato de pnitrofenilo) realizadas en micelio fúngico. La técnica microscópica con ELF-97 reveló que Paxillus involutus (Batsch: Fr.) Sing. y Austropaxillus boletinoides (Sing.) Brsky. & Jarosch poseen diferentes estrategias de adaptación frente a distintas concentraciones de fosfatos y pH (3-7). Nosotros analizamos la SBPA en cuatro asociaciones micorrícicas de N. obliqua y encontramos que la organización del manto juega un rol esencial con respecto a la SBPA. En general, las raíces micorrizadas transfieren la SBPA de la raíz hacia el manto. En este contexto, Pisolithus tinctorius (Pers.) Coker & Couch demostró ser el hongo ectomicorrícico más relevante para N. obliqua, al incrementar significativamente el 42 Gayana Bot. 60(1), 2003 total de la SBPA de la micorriza en relación a la raíz sin micorrizar. En conclusión, a través de la microscopía confocal de fluorescencia basada en el sustrato ELF-97 y su combinación con el procesamiento de imágenes es posible, tanto en hongos mi...

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“…Deficiency or absence of phosphate in the culture medium leads to a significant increase in activity of acid phosphatases in ectomycorrhizal fungi (Tibbett et al, 1998;Tran et al, 2010;Bechem, 2013). This increase in activity results from the normal secretion of surface phosphatases and liberation of extracellular phosphatases (Alvarez et al, 2005;Tran et al, 2010) leading to a more efficient utilization of different forms of organic phosphorous (Wang et al, 2006;Khade et al, 2010), thereby contributing to the nutrition of infected plants (Stancheva et al, 2008;Wu et al, 2011;Li et al, 2011).…”

Section: Introductionmentioning

confidence: 99%

Activity of Acid Phosphatases in Ectomycorrhizal Fungi

Costa¹,

Souza²,

Rondon³

et al. 2016

JAS

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The colonization of plant roots by mycorrhizal fungi, generally increases P content in the host plant when in soils with low levels of P. The objective of this work was to evaluate the activity of acid phosphatases in two isolates of Pisolithus microcarpus, grown in different sources and concentrations of phosphate and to characterize the phosphatase isoenzymes produced by isolates. Both isolates were grown on Melin-Norkrans modified (MNM) medium enriched with organic (Po) or inorganic (Pi) sources of phosphorus, at five different concentrations, in order to study the activity of mycelial surface acid phosphatases during incubation for up to 96 h. Activity of acid phosphatases increased when the fungi were grown without or at low concentrations of Pi. Intraspecific differences were observed between the isolates with regard to acid phosphatase production. A greater decrease in phosphatase activity was observed when incubation time was increased than when Pi concentration was increased. At an incubation time of 96 h, activity of acid phosphatases in isolate 90A increased with increasing Po concentration, while for isolate RV82 remained constant over the different incubation periods and Po concentrations tested. When grown in media without Pi, an additional band appeared in the isoenzyme pattern of RV82, while isoenzyme production was not altered in isolate 90A when grown in media without Pi or with 2 mM Pi, showing differences in their isoenzyme patterns. Isolate 90A possesses a potential competitive advantage when utilized in mycorrhizal association with Eucalyptus because of its ability to utilize Po.

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Methods for Determining Extracellular and Surface-Bound Phosphatase Activities in Ectomycorrhizal Fungi

Cited by 10 publications

References 24 publications

Activity profiling of ectomycorrhiza communities in two forest soils using multiple enzymatic tests

Activity profiling of ectomycorrhiza communities in two forest soils using multiple enzymatic tests

New Perspectives on the Determination of Phosphatase Activity in Ectomycorrhizae of Nothofagus Obliqua Forests in Southern Chile

Activity of Acid Phosphatases in Ectomycorrhizal Fungi

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