Characterization of vesicular-arbuscular mycorrhizal fungi (Glomus spp) by selective enzyme staining following polyacrylamide gel electrophoresis

Isozyme expression of nutrient-mobilizing and defence-related enzymes were examined in different functional components of intact Scots pine (Pinus sylvestris L.) ectomycorrhizal root systems developed in natural unsterilized forest humus. Scots pine seedlings colonized by Suillus bovinus (L. ex Fr.) O. Kuntze or Paxillus involutus (Batsch ex Fr.) strains were grown on humus in two-dimensional Perspex2 microcosms to allow development of complete ectomycorrhizal root systems. Soluble proteins from uncolonized short roots, whole mycorrhizal root tips or dissected mantle and core fractions, fungal strands and the outermost soil colonizing fine hyphae were subjected to native polyacrylamide gel electrophoresis and stained to detect esterase, acid phosphatase, peroxidase and different polyphenoloxidase isozyme activities. Tissue-specific esterase isozymes were detected in all components and most, including unique Hartig net isozymes, were of fungal origin. High fungal acid phosphatase activities were detected in mycorrhiza and fine hyphae of S. bovinus, supporting earlier findings of active phosphatase activity at the fungal interface in the Hartig net region and in the fine hyphal margins of extramatrical mycelium actively colonizing the humus. All compartments in the P. involutus mycorrhizosphere had weaker acid-phosphatase activities. Peroxidases formed a large part of the soluble-protein content in non-mycorrhizal short roots. The amount of peroxidases, on a tissue f. wt basis, was similar in mycorrhizal and non-mycorrhizal roots, but differential isozyme expression was detected, indicating a change in root peroxidase activities following mycorrhiza formation. The expression of -3,4-dihydroxyphenylalanine metabolizing polyphenol oxidase enzymes was reduced in mycorrhizas compared with non-mycorrhizal short roots. In the extramatrical mycelial components, fine hyphae expressed the highest polyphenoloxidase activity. P. involutus displayed some polyphenol-oxidizing isoenzymes not detected in S. bovinus systems. Laccase activity was not detected in the plant and fungal components examined. It is concluded that enzyme activities and isozyme expression are differentially regulated in the different functional components of Scots pine mycorrhizospheres.

mentioning

confidence: 99%

Heterogeneity of fungal and plant enzyme expression in intact Scots pine–Suillus bovinus and –Paxillus involutus mycorrhizospheres developed in natural forest humus

Timonen

Sen

1998

“…Isozyme analysis is also an efficient method for determining variation in fungi (Micales, Bonde & Peterson, 1986) and has been widely used in the identification of species and isolates and population structures of a large number of genera including Neurospora (Spieth, 1975), Heterobasidium (Stenlid, 1985;Orotsina et al, 1993), Pleurotus (Magae et al, 1990), Phytophthora (Oudeman & CofTey, 1991), Lentinus (Ohmasa & Furukawa, 1986), Schizophyllum (Lily & Charvat, 1987), Tuber (Dupre et al, 1993) and several ecto and endomycorrhizal fungi (Sen & Hepper, 1986;Sen, 1990;Keller, 1992). Isozyme patterns has already been used to characterize Armillaria species both in Europe (Wahlstrom et al, 1991) and in North America (Morrison et al, 1985;Lin, Dumas & Hubbes, 1989).…”

mentioning

confidence: 99%

Discrimination of some African Armillaria species by isozyme electrophoretic analysis

Agustian¹,

Mohammed

Guillaumin

et al. 1994

SUMMARYTen Armillaria isolates, collected from various host plants and widespread geographical origins in tropical Africa, were cultivated on orange fragments in the presence of water and two different culture media in order to optimize enzyme and mycelial cord production. Seven enzymes involved in the primary metabolism of nitrogen and carbon (glutamate dehydrogenases, aspartate aminotransferase, malate dehydrogenase, glucose 6-phosphate dehydrogenase, 6-phospbogluconatt dehydrogenase and isocitrate dehydrogenase) were extracted from the mycelial cords and analyzed by polyacrylamide gel electrophoresis. Cluster analy.sis based on calculated similarity i-alues derived from isozyme banding patterns separated the isolates into fi\'e groups. Two isolates considered as belonging to A. metlea ssp. africana (an African species closely related to European A. metlea) were present in a clearly separated cluster when compared to the other isolate groups. Two Kenyan isolates, belonging to an as yet unnamed biological species, which w ere characterized by the production of few slow growing mycelial cords, were also found in a separate cluster with slightly greater similarit\' coefficients to the other isolates. The six other isolates, referred to as isolates of A. heimii (a highly variable species with different sexual systems) fell into three sub^clusters of variable homology. The two bomothallic heimii isolates from Tanzania and Malawi, which were \ery closely related, displaying 100 "" isozyme similarity, exhibited a higher degree of similarit>' with the two other homothalhc heimii isolates from Zimbabwe and Congo, than with the two heterothailic unifactorial heimti isolates from Cameroon and Gabon. The value of isozymes in the classification of African Armtllaria spp. is discussed.

“…Variations in isozyme broad range of physiological traits which include the mobilities of six enzymes from the resting spores of utilisation of carbon and nitrogen compounds, vesicular-arbuscular mycorrhizal fungi has enabled production of hydrolytic enzymes and temperature the characterization of six Glomus species (Sen & optima for axenic growth (e,g. Ferry & Das, 1968;Hepper, 1986) and the detection of intraspecific Laiho, 1970;Lundeberg, 1970;Theodorou & variation in morphologically similar isolates of Bowen, 1971), Although these types of studies have Glomus clarum, Glomus monosporum and Glomus been of little value in explaining differences in the mosseae (Hepper et al, 1988), In ectomycorrhizal mycorrhizal status of related strains and species studies Ho & Trappe (1987) showed that isolates (Harley & Smith, 1983), they do reflect the con-representing six species and three host-related siderable genetic diversity present within species sections of i^/izVopo^OK had acid phosphatase isozyme which clearly needs further investigation, mobilities that differed greatly between sections but The eleetrophoretie analysis of isozymes, exten-were more conserved within species, A similar sively used in studies of plant genetics (Tanksley & analysis of Laccaria laccata enabled the division of isolates into three host-related groups (Ho, 1987). In the most detailed study to date, using genetic interpretations of the isozyme mobilities of eight enzymes (May, Roberts & Soper, 1979), intraspecific variation of Suillus tomentosus was shown to be greater among than within four distinct forest regions containing either pure or mixed conifer stands (Zhu et aL, 1988).…”

mentioning

confidence: 99%

Iǹtraspecific variation in two species of Suillus from Scots pine (Pinus sylvestris L.) forests based on somatic incompatibility and isozyme analyses

Sen

1990

Self Cite

SUMMARYIsolates of Suillus variegatus (Swartz ex Fr.) O, Kuntze and Suillus bovinus (Fr.) O. Kuntze cultured from basidiocarps collected in different geographically located Scots pine {Pinus sylvestris L.) forests were subjected to an analysis of axenic growth rates, somatic incompatibility and eleetrophoretie isozyme expression. Intraspecific variation in the radial growth rates and at eertain putative enzyme loci was detected in these fungal populations which, after tests for somatic incompatibility, were identified as being composed mainly of different clones. Following a numerical analysis of the isozyme data of seven enzyme systems, considerable interspecific dissimilarity (11 % similarity) and intraspecific similarity (> 65 % similarity) were detected in these fungi. Isozymes of esterase, peptidase and acid phosphatase were conserved within species but differed between these species enabling their unambiguous identification. Isolates of S. variegatus were found in two main clusters that represented the two forest locations from which they were collected. A single putative locus of glutamate oxaloacetate transaminase was found to display isozyme polymorphism directly related to the forest location of the isolates. No habitat-related isozyme activities could be detected in the isolates of S. bovinus which, unlike those of S. variegatus, exhibited isozyme expression that was strongly dependent upon the medium. Isolates of S. variegatus and S. bovinus identified as originating from the same vegetative elone by somatic incompatibility testing were found to exhibit 100 and 93-97% similarity in their respective isozyme activities. These studies indicate that isozyme analyses in conjunction with somatic incompatibility testing will provide powerful tools for tbe genetic analysis of ectomycorrhizal communities.