Modified Melin Norkrans Research Articles

In vitro growth of nine edible ectomycorrhizal fungi under a range of pH conditions

Ectomycorrhizal fungi are considered to play an essential role in the development of forest ecosystems and can protect plant against pathogenic infections. Among other factors, soil pH may affect the successful inoculation of forest seedlings in nurseries. The effect of pH on the growth rate of strains of nine species of edible ectomycorrhizal (ECM) fungi was evaluated in vitro. In the experiments, Boletus edulis, B. aereus, B. pinophilus, B. fragrans, Amanita rubescens, Xerocomus ferrugineus, Lactarius deliciosus, Lactarius sanguifluus and Suillus luteus were grown in Petri dishes containing modified Melin Norkrans medium and adjusted at seven different pH levels. Colony area was measured at 7-day intervals for 8 weeks. Final fungal biomass and residual pH of the medium at 8th week were also measured. The optimum pH levels and pH tolerance ranges for the tested ECM fungal species are presented and discussed in the text. The results showed that the greatest growth in vitro was produced by A. rubescens and S. luteus at high pH levels (between 6.5-8.5), and by X. ferrugineus at low pH (3.5-6.5). Almost all the strains acidified the medium where they were grown after eight incubation weeks.

The unusual sclerotium of Rhizopogon roseolus reported from pure cultures

During the assessment of mycelial cords of Rhizopogon roseolus on poor nutrient of Modified Melin-Norkrans (MMN) medium, we found some sclerotia produced on the surface of extraradical mycelia. The sclerotia were 0.27 mm in average of diameter and produced after 2 months of incubation. The current knowledge defined the sclerotium as mass of hyphae and normally having no spores in or on it. However, we found and suspected the small structures like spores (1-1.5 um) inside the sclerotium. These structures were ellipsoid, hyaline, with the smooth surface. We then incubated the sclerotium and these small structures on TM7 detecting medium whether they can produced the secondary mycelia of R. Roseolus, but no germination was observed. Interestingly, the bacterial colonies which connected to hyphae of sclerotium were appeared. The colonies were transferred to Luria agar (LA) medium. The morphological observation of bacterial cells from TM7 and LA confirmed that they were the same as small structures inside the sclerotium. This is the first report on production of unusual sclerotium of R. roseolus in pure cultures. Further study is required to reveal the role of bacteria on production of sclerotium of R. Roseolus.

Influence of pH on in vitro mycelial growth in three Japanese truffle species: Tuber japonicum, T. himalayense, and T. longispinosum

Abstract Truffle species indigenous to Japan include a white-colored truffle Tuber japonicum and two black-colored truffles, T. himalayense and T. longispinosum. As the fruiting bodies of these Tuber species are promising edible, studies on the artificial cultivation for these truffles are currently underway in Japan. In the present study, we investigated the influence of pH on in vitro mycelial growth in these Tuber species to determine the optimal pH conditions for the cultivation of these truffles. Mycelia of five strains from each species were cultured in modified Melin–Norkrans liquid medium at different pH values. Tuber japonicum grew well at pH 5.0 and 6.0, whereas T. himalayense and T. longispinosum grew well at pH 7.0. This results suggest that the optimal pH for mycelial growth varies among Tuber species. The growth data collected in this study can be used to design optimal pH conditions for artificial cultivation of these Japanese truffles.

Purification and characterization of an amyloglucosidase from an ericoid mycorrhizal fungus (Leohumicola incrustata)

This study aimed to purify and characterize amyloglucosidase (AMG) from Leohumicola incrustata. AMG was purified to homogeneity from cell-free culture filtrate of an ERM fungus grown in a modified Melin–Norkrans liquid medium. The molecular mass of the AMG was estimated to be 101 kDa by combining the results of Sephadex G-100 gel filtration, sodium dodecyl sulphate–polyacrylamide gel electrophoresis, and zymography. The Km and kcat values were 0.38 mg mL−1 and 70 s−1, respectively, using soluble starch as a substrate. The enzyme was stable at 45 °C (pH 5.0), retaining over 65% activity after a pre-incubation period of 24 h. The metal inhibition profile of the AMG showed that Mn2+ and Ca2+ enhanced activity, while it was stable to metals ions, except a few (Al3+, Co2+, Hg2+ and Cd2+) that were inhibitory at a concentration higher than 5 mM. Thin layer chromatography revealed that only glucose was produced as the product of starch hydrolysis. The amylase from L. incrustata is a glucoamylase with promising characteristics such as temperature stability over an extended period, high substrate affinity and stability to a range of chemicals. Also, this study reports for the first time the possibility of using some culturable ERM fungi to produce enzymes for the bio-economy.

In vitro GROWTH OF ECTOMYCORRHIZAL FUNGI ASSOCIATED WITH Pinus radiata PLANTATIONS IN CHILE

A comparative study of in vitro growth of three species of ectomycorrhizal fungi (ECMF) (Rhizopogon luteolus, Suillus granulatus and Suillus luteus) was performed. Fungal material was collected in adult Pinus radiata plantations. Isolation and purification of the strains were performed on potato-dextrose-agar medium and the evaluation of the radial growth rate and the increase in mycelial biomass, under different culture conditions, was performed on the Modified Melin Norkrans growth medium. The effects of temperature (24, 28 and 32 ºC) and pH (4.8, 5.3, 5.8, 6.3 and 6.8) of the growth medium were tested for the three fungal species in two independent assays. The results indicate that the temperature had a significant effect on the radial growth rate (RG) and mycelial biomass increase (MB) in all of the evaluated fungal species. The highest RGR and MBI were recorded in R. luteolus, and the lowest values for these variables were registered in S. luteus. Rhizopogon luteolus had the highest sensitivity to pH changes. Meanwhile, there was no pattern in S. granulatus and S. luteus growth response under different pH conditions. When cultivated in vitro, the three studied species of ECMF presented adaptation, exponential, declining and stationary growth phases. The in vitro growth responses recorded in the present study showed the great potential of R. luteolus to be used in future programs using mycorrhizal inoculation in the production of Pinus radiata trees in nurseries in Chile.

Development of growth media for solid substrate propagation of ectomycorrhizal fungi for inoculation of Norway spruce (Picea abies) seedlings.

A silica-based propagation medium was developed for large-scale production of ectomycorrhizal (ECM) fungal inoculum by solid state fermentation. Development of the medium was started by screening for an optimal growth medium among six different semisynthetic agar media traditionally used in cultivation of ECM fungi. The majority (65 %) of the twenty tested ECM fungal strains that typically colonize Norway spruce (Picea abies) seedlings grew best on modified Melin-Norkrans (MMN) medium with reduced sugar content (½MMN). In order to develop a nutritionally similar medium for large-scale cultivation of the ECM fungi, we chose silica to form a solid matrix and light brewery malt extract to provide nutrients. The medium was supplemented with a commercial humic acid product that was shown to boost fungal growth. The optimal concentration of the constituents was screened for in two assays by determining the growth rates of seven potential inoculant ECM fungal strains (Amphinema sp., Cenococcum geophilum, Hebeloma sp., Meliniomyces bicolor, Paxillus involutus, Piloderma byssinum, and Tylospora asterophora). As a result, we composed a silica-based mass propagation medium (pH 5.8) containing 2.5 % brewery malt extract and 0.5 g/l humic acid product Lignohumate AM. This medium is easily produced and supported good growth of even the slowly growing and rarely studied Athelioid ECM strains. Furthermore, root systems of Norway spruce nursery seedlings were colonized by the tested ECM fungi by using solid inoculum formulated from the silica medium.

Carbohydrate oxidases in ericoid and ectomycorrhizal fungi: a possible source of Fenton radicals during the degradation of lignocellulose

Isolates of the ericoid mycorrhizal fungus Hymenoscyphus ericae (Read) Korf et Kernan, and the ectomycorrhizal fungi Suillus variegatus (Swartz ex Fr.) and Pisolithus tinctorius (Pers.) Coker & Couch, along with a Cortinarius sp. and the white rot Phanerochaete chrysosporium Burdsall were examined for the ability to oxidize carbohydrates to their corresponding lactones and to excrete the H2O2 produced thereby. All except Phanerochaete chrysosporium were found to express cellobiose oxidase (cellobiose dehydrogenase, EC 1.1.19.88) and glucose oxidase (β‐d‐glucose∶oxygen 1‐oxidoreductase, EC 1.1.3.4) when grown on cellobiose and glucose respectively. Production of extracellular H2O2 was visualized during growth on both substrates using ABTS as the chromogen. According to the Fenton reaction, H2O2 will react with hydrated or chelated Fe(II) in the environment to produce hydroxyl (Fenton) radicals, HO·. Mycelial extracts from each of the mycorrhizal fungi produced HO· in the presence of cellobiose and Fe(II), presumably mediated by H2O2 produced by cellobiose oxidase activity in the extracts. Conditions favourable to HO· production were shown to exist in Modified Melin–Norkrans medium, and the data discussed in relation to previously observed lignin degradation by mycorrhizal fungi.

Effect of temperature on sclerotium induction in Paxillus involutus

Paxillus involutus (Batsch.: Fr.) Fr., an ectomycorrhizal fungus, is capable of forming sclerotia, resistant vegetative propagules. Sclerotia were induced in vitro on agar-solidified modified Melin Norkrans medium by exposure of the mycelium to temperatures between 6 and 14 °C. The number and size of sclerotia varied greatly with temperature. Mycelium exposed to either 6 or 8 °C produced many smaller sclerotia (less than 1.0 mm in diameter) lacking droplets of exudate, whereas mycelium exposed to temperatures between 10 and 14 °C formed fewer, larger sclerotia (1.0 mm or more in diameter) with abundant exudate. Sclerotia were not induced from mycelium grown in liquid medium at 10 or 25 °C or from mycelium on agar-solidified medium maintained at 25 °C under a variety of conditions, including changes in medium composition and volume. Key words: sclerotia, Paxillus involutus, temperature, ectomycorrhiza, in vitro.

Interaction between the Ectomycorrhizal Fungus Paxillus involutus, Damping‐off Fungi and Pinus resinosa Seedlings

Abstract Paxillus involutus, an ectomycorrhizal fungus, had an inhibitory effect on the root pathogenic fungus Fusarium moniliforme and two isolates of F. oxysporum when grown in paired cultures on modified Melin Norkrans’ medium. In contrast, one isolate of F. oxysporum was not inhibited and another damping‐off fungus, Cylindrocarpon destructans inhibited growth of Pax. involutus in similar paried cultures. Survival of Pinus resinosa (red pine) seedlings was increased significantly when they were grown in vitro concomitantly with either Pax. involutus and F. moniliforme or Pax. involutus and the three isolates of F. oxysporum, compared with seedlings inoculated with either F. moniliforme or F. oxysporum isolates alone. pax. involutus showed no protective effect against C. destructans. The number of colony forming units of Fusarium spp. was reduced significantly in the root extract and rhizosphere substrate of P. resinosa seedlings inoculated with Pax. involutus. Spore germination of Fusarium spp. was reduced significantly when treated with culture filtrate of Pax. involutus and root extract of P. resinosa seedlings inoculated with Pax. involutus. Neither colony forming units nor spore germination of C. destructans was affected either by culture filtrate of Pax. involutus or root extract of P. resinosa seedlings inoculated with Pax. involutus.

Studies on the systematics of ectomycorrhizal fungi in axenic culture. V. Linear growth response to standard extreme temperatures used as a taxonomic character

Ectomycorrhizal fungi showed characteristic differences in linear growth rate reduction when grown on modified Melin–Norkrans agar at 7 and 30 °C. The two standard temperature extremes differentiated ectomycorrhizal fungi into three growth response categories which reflected taxonomic affinity: sensitive, semitolerant, and tolerant. At 7 °C, species of Laccaria and Hebeloma generally exhibited tolerant or semitolerant growth, whereas at 30 °C growth tended to be restricted to species of Amanita, Laccaria, Suillus, and a few gasteromycetous fungi (e.g., Rhizopogon, Scleroderma, and Pisolithus). The results reveal the potential value of standard extreme temperatures as a taxonomic character for differentiating species or genera of ectomycorrhizal fungi in vitro. Tolerance or sensitivity to 30 °C by ectomycorrhizal fungi may reflect their association with specific host trees. Key words: ectomycorrhizal fungi, temperature, sensitivity, tolerance, cultures, identification.

Induction of protoplast formation in the ectomycorrhizal fungus Paxillus involutus by the root rot pathogen Fusarium oxysporum

SUMMARYCo‐culture of the pathogenic fungus Fusarium oxysporum f. sp. pini Schlecht. emend Snyd. & Hans, with the ectomycorrhizal fungus Paxillus involutus Fr. on potato dextrose agar caused no visible morphological changes in the fungi. Co‐culture on modified Melin Norkrans medium in petri plates yielded, however, protoplast formation at or near tips of P. involutus hyphae. No changes were visible in F. oxysporum as a result of co‐culture and the two fungi grew in close association with each other without inhibition. Staining with Calcofluor White M2R New and fluorescein diacetate displayed that, at this stage in development, a wall surrounding the protoplast was absent but a plasma membrane sheathed by an extracellular mucilage was present. Protoplasts lacking definite form adhered closely to adjoining hyphae while those with the characteristic spherical form were easily dislodged from hyphae. Average size of spherical protoplasts was 17–20 μm. Breaks were present in the hyphal wall in regions adjacent to protoplast formation and these may have been pathways for cytoplasmic leakage. Transmission electron microscopy revealed these breaks more clearly, and provided additional evidence of a plasma membrane around protoplast cytoplasm. Protoplasts contained few organelles except vesicles, larger electron dense bodies and occasional nuclei. Hyphae forming protoplasts often lacked cytoplasmic integrity although those not forming protoplasts appeared healthy.

Early effects of the ectomycorrhizal fungus Paxillus involutus on the root rot organism Fusarium associated with Pinus resinosa

Infection of Pinus resinosa Ait. seedlings in a sterile test tube culture system by Fusarium oxysporum f.sp. pini Schlecht. emend. Snyd. & Hans., which was determined microscopically by the presence of the fungus within root tissues, was significantly reduced within the first 6 days of infection after prior inoculation with the ectomycorrhizal fungus Paxillus involutus (Batsch) Fr. Hyphae and conidia of the pathogen were swollen and filled with osmiophilic material when P. involutus was also present in the rhizosphere of P. resinosa seedlings. Germination of F. oxysporum conidia was suppressed when they were placed in holes in P. involutus cultures on modified Melin Norkrans medium in Petri plates but was restored with removal of conidia from plates. Suppressed, ungerminated conidia had thick cell walls and large cytoplasmic inclusions. Staining using Calcofluor White M2R New revealed changes in the binding properties of septal wall material. Key words: Paxillus, Fusarium, ectomycorrhiza, disease suppression, anatomy.

Studies on the systematics of ectomycorrhizal fungi in axenic culture. II. The enzymatic degradation of selected carbon and nitrogen compounds

Ninety-six species of ectomycorrhizal fungi from 30 genera were grown on modified Melin–Norkrans agar where the carbon (glucose) or nitrogen (ammonium phosphate dibasic) supply was replaced by cellulose, lignin, pectin, lipid, amylose, gelatin, casamino acids, or urea. Ectomycorrhizal fungi did not appreciably degrade cellulose, lignin, or pectin. The remaining compounds were broken down by representatives of certain taxonomic groups. Lipase was produced by Amanita species, amylase by species of Amanita and Cortinarius, gelatinase by Piloderma, Thelephora, species of Lactarius section Dapetes, and some species of both Amanita and Cortinarius. Casamino acids were degraded by Laccaria, Hebeloma, and some Tricholoma species. Urease was detected in species of Hebeloma and Laccaria. The ability to enzymatically degrade selected carbon and nitrogen compounds have potential as taxonomic characters for the identification of isolates of ectomycorrhizal fungi. The significance of these results are also discussed in relation to the ecology of these fungi. Key words: ectomycorrhizal fungi, enzyme activity, cultures, identification, systematics.

The time-course of disease suppression and antibiosis by the ectomycorrhizal fungus Paxillus involutus.

Seedlings of Pinus resinosa Ait. grown in test tubes were inoculated with the ectomycorrhizal fungus Paxillus involutus Fr. and, one day later, with a spore suspension of the root pathogenic fungus Fusarium oxysporum Schlecht emend, Sny. & Hans f.sp. pini. Controls consisted of seedlings inoculated with plugs of sterile modified Melin Norkrans medium and F. oxysporum. Seedling survival was counted, sporulation of F. oxysporum was measured, and ethanol extractions of the rhizosphere were made, 1, 2, 3, 4, 5, 6, 8, 10 and 14 d after inoculation with the pathogen. The ethanol extractives were concentrated and assayed for fungitoxic activity by measuring the germination of F. oxysporum microconidia. Dead seedlings were first observed 3 d after inoculation with the pathogen. Sporulation of F. oxysporum was reduced significantly 3 d after seedling inoculation with P. involutus when compared with controls lacking P. involutus. The fungitoxic activity of the rhizosphere of the seedlings inoculated with P. involutus was greater than the fungitoxic activity of the rhizosphere of control seedlings, increasing strongly 2.4 d after inoculation with the pathogen. Suppression of Fusarium root rot by P. involutus may, therefore, be the result of antibiosis by this ectomycorrhizal fungus.

Protein synthesis inPinus resinosa and the ectomycorrhizal fungusPaxillus involutus prior to ectomycorrhiza formation

Seedlings ofPinus resinosa Ait. in test tubes were inoculated with the ectomycorrhizal fungusPaxillus involutus Fr. or with discs of sterile modified Melin-Norkrans (MMN) medium.Paxillus involutus was also inoculated to control tubes in the absence ofPinus resinosa seedlings. In vivo labelling of proteins inPinus resinosa roots and inPaxillus involutus mycelium was carried out using35Sl-methionine 1, 2, 3, 4, 5 and 7 days after inoculation. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDSPAGE) of the protein extracts from the four treatments and autoradiography demonstrated that the presence of root exudates altered protein synthesis inPaxillus involutus as three major bands disappeared whenPaxillus involutus was exposed to root exudates. Protein synthesis inPinus resinosa was also altered whenPaxillus involutus was introduced into the tubes, since at least two bands were more intense when seedlings were inoculated withPaxillus involutus, as compared to control roots. No difference was observed in the growth and the label incorporation ofPaxillus involutus growing with or without root exudates. Ectomycorrhizal roots were not formed during this experiment. Gene regulation in this ectomycorrhizal association occurs, therefore, prior to the formation of ectomycorrhizal roots.

Pine root exudate stimulates the synthesis of antifungal compounds by the ectomycorrhizal fungus Paxillus involutus

SUMMARYThe ectomycorrhizal fungus Paxillus involutus Fr. was grown in the presence of root exudate of Pinus resinosa Ait, for two weeks. The effect of root exudate on the synthesis of ethanol‐soluble antifungal compounds by P. involutus was determined by a bioassay based on microconidial germination of Fusarium oxysporum Schlecht emend, Sny. & Hans f. sp. pini. Cultivation of P. involutus on root exudate led to a greater than two‐fold increase in fungitoxic activity when compared to cultivation on modified Melin Norkrans medium in Petri dishes. Inoculation of P. resinosa root exudate with both P. involutus and a spore suspension of F. oxysporum led to an 80 % suppression of the sporulation of F. oxysporum when compared to controls lacking P. involutus. The increased resistance of P. resinosa P. involutus associations to Fusarium root rot may, therefore, be the result of biosynthesis of antifungal compounds by P. involutus which is stimulated by P. resinosa root exudate.

Use of mycelial slurries of mycorrhizal fungi as inoculum for commercial tree seedling nurseries

Blended mycelial slurries of a variety of ectomycorrhizal fungi were assessed for their suitability as inoculum for containerized tree seedlings. Mycelium of most fungi tested, with the exceptions of Pisolithustinctorius (Pers.) Coker & Couch and Paxillusinvolutus (Batsch.: Fr.) Fr., withstood blending well. Viability remained high after storage in modified Melin–Norkrans agar medium, water, or dilute saline at either 4 °C or room temperature, indicating that slurries are robust enough to tolerate conditions that would be encountered in a commercial setting. Experiments investigating methods for applying slurries to containerized seedlings were conducted using Hebelomalongicaudum (Pers.: Fr.) Kummer. Slurry infectivity dropped after it was mixed into a peat–vermiculite growing medium, particularly in the presence of high levels of fertilizer. This indicated that incoculum should not be added to multipots until short roots capable of becoming mycorrhizal are present and that contact of the inoculum with fertilizer should be avoided. Injection of slurry into the root zone resulted in the most consistently high colonization, but application of the slurry to the surface of the growing medium was also effective, the latter being more feasible in a commercial setting. Mycorrhizae developed with as little as 1 mg mycelium per seedling, although 100 mg gave more consistent results. In inoculation trials in which slurries of nine representative fungi were injected into the root zones of 8-week-old black spruce or jack pine seedlings, five of the fungi consistently formed mycorrhizae. For these fungi, slurries could be an effective inoculum.

Pure Culture Synthesis of Pinus Resinosa Ectomycorrhizae with Scleroderma Aurantium

Numerous techniques and many variations on them have been reported for the synthesis of ec? tomycorrhizae in vitro (e.g., Chu-Chou, 1979; Danielson, 1984; Duddridge and Read, 1984a; Fortin, 1966; Molina, 1979; Palm and Stewart, 1984; Yang and Wilcox, 1984). Many of these techniques involve elaborate manipulations and exacting cultural conditions. In our study, lab? oratory procedures for the pure culture forma? tion of ectomycorrhizae were simplified as much as possible. We chose the gasteromycete Scleroderma aurantium (Vaill.) Pers. for testing because of its association with red pine (Pinus resinosa Ait.) on a variety of sites that we sampled in Michi? gan's Upper Peninsula, and because of its vigorous growth in culture. The agarics, Cantharellula umbonata (Gmel.: Fr.) Sing. and Hygrophoropsis aurantiaca (Wulf.: Fr.) Maire in Martin-Sans, were chosen for similar reasons. Red pine was selected as test host because of its importance in the Lake States forest industry. Wide-mouth quart Mason jars, about one liter capacity, were used as synthesis vessels. The in? ner lids ofthe jars were inverted within the outer ring to prevent sealing and to allow gas exchange. The outer ring was screwed on until firm, and then loosened about one-quarter turn. As a rooting substrate, 250 ml of a vermiculite-peat mix? ture (10:1, v/v) were added to each jar. The peat used in this mixture was ground sphagnum peat moss. One hundred twenty-five ml of modified Melin-Norkrans (MMN) nutrient medium (Marx, 1969) were then added and the jars were auto? claved at 121 C for 45 min. The autoclaved jars were then stored for 2-7 da before planting. Red pine seeds were surface-sterilized in 30% H202 for 30 min, rinsed in sterile distilled water several times and plated on water agar. Three axenically germinated seedlings, 2-3 wk old, were planted in each jar. Jars were placed on a northeast-facing wi dow sill and seedlings were allowed to be? come established for 2-3 wk.

Ethylene production by ectomycorrhizal fungi, Fusarium oxysporum f. sp. pini, and by aseptically synthesized ectomycorrhizae and Fusarium-infected Douglas-fir roots.

The ectomycorrhizal fungi Cenococcum geophilum, Hebeloma crustuliniforme, and Laccaria laccata produced ethylene in vitro in modified Melin-Norkrans liquid medium only if amended with 2.5-10mM methionine; Pisolithus tinctorius failed to produce ethylene unless the cultures were renewed with fresh methionine-amended medium before ethylene assay. An additional 19 ectomycorrhizal fungi, plus five isolates of Fusarium oxysporum f. sp. pini, all produced ethylene in renewed and (or) nonrenewed media. Although the rates varied, ethylene production by many ectomycorrhizal fungi equaled that of Fusarium. Culture filtrates of H. crustuliniforme and L. laccata also evolved ethylene that was apparently of nonenzymatic origin. Ethylene was produced by aseptically grown Douglas-fir seedlings inoculated with C. geophilum, H. crustuliniforme, and L. laccata and appearance of ethylene coincided with the formation of mycorrhizae; production by P. tinctorius inoculated seedlings was inconsistent. Lateral root formation of Douglas-fir was stimulated by inoculation with C. geophilum, H. crustuliniforme, and L. laccata but was inhibited by P. tinctorius. Fusarium-inoculated seedlings produced more ethylene sooner than seedlings inoculated with mycorrhizal fungi. The disparity in the levels of ethylene associated with ectomycorrhiza formation compared with Fusarium infection suggests a possible differential role for ethylene in symbiotic and pathogenic fungus-host interactions.