Productivity of hydrolytic enzymes by mycorrhizal mushrooms

To survey the potential for production of extracellular hydrolytic enzymes by mycorrhizal mushrooms, productivities of these exo-enzymes from mycelia on potato-dextrose liquid medium were determined.Tricholoma matsutake produced relatively high levels of CM-cellulase and avicelase activities in all test strains. It also produced higher activity of acid proteinase than neutral proteinase. Its xylanase activities seemed to be higher than those of the other carbohydrases. The productivities ofLyophyllum shimeji strains were at similar levels to those ofT. matsutake strains. CM-cellulase and avicelase activities ofL. shimeji were higher than those ofT. matsutake. Neutral proteinase inL. shimeji strains showed higher activity levels than acid proteinase. The relative productivities of hydrolytic enzymes between the groups of mycorrhizal mushrooms and wood-rotting mushrooms were also examined.T. matsutake andL. shimeji both produce many kinds of hydrolytic enzymes in their culture broth, and the potential for production of hydrolytic enzymes by mycorrhizal mushrooms was judged to be relatively high.     

Production of fruit-bodies of a mycorrhizal fungus,Lyophyllum shimeji,in pure culture

Cultivation of mycorrhizal fungus,Lyophyllum shimeji, was examined using selected strains capable of forming primordia in pure culture. Mycelia grew fastest on barley grains containing synthetic liquid medium. The primordia readily formed in test-tubes after lowering the incubation temperature from 23°C to 15°C. The co-existence of pine seedlings had no promotive effect on primordium formation. Fruit-bodies formed on a medium consisting of barley, beech sawdust, and liquid synthetic nutrients in 500-ml glass bottles. Mature fruit-bodies produced basidiospores. The spores thus produced could germinate on an agar medium and formed mycelial colonies. Thereby, the life cycle inL. shimeji was accomplished in pure culture without using the host plant.     

Detection of the edible ectomycorrhizal fungus Lyophyllum shimeji colonising seedlings of cultivated conifer species in New Zealand

Lyophyllum shimeji is an edible ectomycorrhizal fungus that is widely distributed in East Asia and also present in the northern regions of Europe. In Japan, L. shimeji is a culinary delicacy, considered amongst all edible mushrooms to have the best taste and to be second only to Tricholoma matsutake in price. Traditionally, fruiting bodies of L. shimeji have been collected from the wild but fruiting of L. shimeji is now relatively uncommon and cannot keep up with increasing consumer demand. As a result, methods for its cultivation are being developed for commercial production in Japan and other countries. In this work, techniques were developed to cultivate L. shimeji on coniferous seedlings using a pure culture inoculum. They resulted in successful mycorrhization of Pinus pinaster and Picea abies in only 8 to 10 months. As ectomycorrhizae of L. shimeji are difficult to identify morphologically, mycorrhization was confirmed using an L. shimeji-specific PCR diagnostic, which was designed following a phylogenetic analysis of the Lyophyllum section Difformia using DNA sequences of the internal transcribed spacer (ITS), intergenic spacer (IGS) and elongation factor 1-α (EF1-α) gene. L. shimeji is a member of the Lyophyllum decastes complex in section Difformia, which also includes Lyophyllum fumosum and L. decastes. This analysis confirmed the separation of L. shimeji from closely related Lyophyllum spp. and enabled its unambiguous detection using an IGS-based PCR diagnostic. This is the first report of successful mycorrhization of L. shimeji on P. pinaster and P. abies and provides an opportunity for its commercial cultivation on conifers in New Zealand.     

Development,persistence and regeneration of foraging ectomycorrhizal mycelial systems in soil microcosms

Development of extraradical mycelia of two strains each of Paxillus involutus and Suillus bovinus in ectomycorrhizal association with Pinus sylvestris seedlings was studied in two dimensions in non-sterile soil microcosms. There were significant inter- and intra-specific differences in extraradical mycelial growth and morphology. The mycelial systems of both strains of P. involutus were diffuse and extended more rapidly than those of S. bovinus. Depending on the strain, P. involutus mycelia were either highly plane filled, with high mass fractal dimension (a measure of space filling) or sparse, low mass fractal dimension systems. Older mycelial systems persisted as linear cords interlinking ectomycorrhizal tips. S. bovinus produced either a mycelium with a mixture of mycelial cords and diffuse fans that rapidly filled explorable area, or a predominately corded mycelium of minimal area cover. In the soil microcosms, mass fractal dimension and mycelial cover tended to increase with time, mycelia encountering litter having significantly greater values. Results are discussed in terms of the ecology of these fungi, their foraging activities and functional importance in forest ecosystems.     

Production and properties of xylanases from thermophilic actinomycetes

30 strains of xylanolytic thermophilic actinomycetes were isolated from composted grass and cattle manure and identified as members of the generaThermomonospora, Saccharomonospora, Microbispora, Streptomyces andActinomadura. Screening of these strains for extracellular xylanase indicated that strains ofSaccharomonospora andMicrobispora generally were poor xylanase producers (0.5–1.5 U/ml) whereas relatively high activities were observed in cultures ofStreptomyces andActionomadura (4–12 U/ml).A preliminary characterization of the enzymes of strains of the latter genera suggested that xylanases of all the strains ofActinomadura exhibited higher thermostabilities than those ofStreptomyces. To evaluate the potential of thermophilicActinomadura for industrial applications, xylanases of three strains were studied in more detail. The highest activity levels for xylanases were observed in cultures grown on xylan and wheat bran. The optimal pH and temperature for xylanase activities ranged from 6.0 to 7.0 and 70 to 80°C. The enzymes exhibited considerable thermostability at their optimum temperature. The half-lives at 75°C were in the range from 6.5 to 17h. Hydrolysis of xylan by extracellular xylanases yielded xylobiose, xylose and arabinose as principal products. Estimated by the amount of reducing sugars liberated the degree of hydrolysis was 55 to 65%. Complete utilization of xylan is presumably achieved by -xylosidase activities which could be shown to be largely cell-associated in the 3Actinomadura strains.     

Mycelium growth kinetics and optimal temperature conditions for the cultivation of edible mushroom species on lignocellulosic substrates

The influence of environmental parameters on mycelial linear growth ofPleurotus ostreatus, P. eryngii, P. pulmonarius, Agrocybe aegerita, Lentinula edodes, Volvariella volvacea andAuricularia auricula-judae was determined in two different nutrient media in a wide range of temperature, forming the basis for the assessment of their temperature optimaV. volvacea grew faster at 35°C,P. eryngii at 25°C,P. ostreatus andP. pulmonarius at 30°C,A. aegerita at 25 or 30°C andA. auricula-judae at 20 or 25°C depending on the nutrient medium used andL. edodes at 20 or 30°C depending on the strain examined. The mycelium extension rates were evaluated on seven mushroom cultivation substrates: wheat straw, cotton gin-trash, peanut shells, poplar sawdust, oak sawdust, corn cobs and olive press-cake. The mycelium extension rates (linear growth and colonization rates) were determined by the ‘race-tube’ technique, and were found to be the highest on cotton gin-trash, peanut shells and poplar sawdust forPleurotus spp. andA. aegerita. Wheat straw, peanut shells and particularly cotton gin-trash supported fast growth ofV. volvacea, whereas wheat straw was the most suitable substrate forL. edodes andA. auricula-judae. Supplemented oak sawdust and olive press-cake were poor substrates for most species examined, white almost all strains performed adequately on corn cobs.     

Influence of growth medium on antifungal activity of neem oil (<Emphasis Type="Italic">Azadirachta indica</Emphasis>) against <Emphasis Type="Italic">Lagenidium giganteum</Emphasis> and <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis>

The inhibition of mycelial growth of Lagenidium giganteum by neem oil was lower than that of Metarhizium anisopliae in PYG and Emerson’s YpSs agar media. However, neem oil did not inhibit the mycelial growth of L. giganteum in sunflower seed extract agar medium, but did it inhibit the mycelial growth of M. anisopliae. The minimum inhibitory concentration of neem oil for L. giganteum was higher than that for M. anisopliae. The minimum fungicidal concentration of neem oil in PYG medium was lower than in YpSs for both fungi. The spores of L. giganteum grown in SFE medium could be used with neem oil for vector control.     

红托竹荪菌种快速分离培养基优化

通过对红托竹荪快速分离培养基优化,提高红托竹荪菌种分离与评价效率。采用响应面分析法,以菌种生长速度为响应值拟合二次多元回归方程,确定培养基配方;测定优化培养基与PDA对照培养基菌丝生长速度和菌丝直径,以菌丝形态、锁状联合和菌落形态等指标评价优化培养基;测定优化培养基与PDA培养基培养菌丝在木屑培养基中菌丝生长速度,验证应用效果。通过试验,筛选出快速分离培养基配方为葡萄糖20.71 g/L、全麦粉8.36 g/L、玉米粉8.07 g/L、琼脂粉18.00 g/L、木屑水1.06 L。快速分离培养基与PDA培养基对比,培养的菌落直径平均增加66.25%,快速分离培养基菌丝日平均生长速度增加33.33%,木屑培养基菌丝日平均生长速度增加44.22%。由于优化培养基中含有淀粉、纤维素等有效成分,其刺激了菌种分泌淀粉酶、纤维素酶等,维持了胞外酶系的完整性。还可根据菌丝培养基过程形成的透明圈大小判定菌种胞外酶产生能力,达到快速评价菌种质量,保障菌种质量的目的。     

Armillaria jezoensis,a new symbiont ofGaleola septentrionalis (Orchidaceae) in Hokkaido

NineArmillaria isolates obtained from the roots ofGaleola septentrionalis in Hokkaido were identified asA. jezoensis by means of mating tests. Cultures of these isolates were similar in colony morphology, mycelial growth and rhizomorph formation on each of malt extract-dextrose agar (MDA), potato-dextrose agar (PDA), andG. septentrionalis root extractdextrose agar (GDA) media, showing better mycelial growth and rhizomorph formation on GDA medium.     

Effects of temperature,water activity and gas atmosphere on mycelial growth of tempe fungi Rhizopus microsporus var. microsporus and R. microsporus var. oligosporus

Rhizopus microsporus var. microsporus and var. oligosporus are used in the manufacture of various Asian fermented foods (tempe, black oncom, sufu). In view of solid-substrate fermentation (SSF) control, mycelial growth of strains of both varieties was tested for sensitivity to fluctuations of temperature, water activity and interstitial gas composition. This was achieved by measuring radial growth as well as biomass dry weight of pre-germinated microcolonies on defined media. The optimum conditions were temperature 40 °C, a _w 0.995 and a gas composition of air for the growth of both strains on a model medium. Whereas radial growth rates of var. microsporus and var. oligosporus were similar, biomass growth rates of var. oligosporus were higher than those of var. microsporus under optimum conditions. The temperature-dependent growth of Rhizopus spp. at a _w > 0.98 could be described by the Ratkowsky Equation. Carbon dioxide (5–10% v/v) inhibited the growth of Rhizopus spp. at non-limiting levels of oxygen. The two strains were able to grow at low (0.5% v/v) oxygen levels, but the mycelial density was rather low. No interrelation of water activity and gas composition was observed, but at high water activity the fungi were more sensitive to changes of temperature. The implications for process control are discussed. This revised version was published online in November 2006 with corrections to the Cover Date.     

Bacterization of peanut withPseudomonas fluorescens for biological control ofRhizoctonia solani and for enhanced yield

Severity of stem-rot disease of peanut caused byRhizoctonia solani was reduced by 54.9 and 68% in plants of two cultivars treated in the greenhouse with antagonistic strains ofPseudomonas fluorescens. These strains were selected based on theirin vitro toxicity to mycelial growth and sclerotial germination ofR. solani. In field experiments, bacterization of peanuts withP. fluorescens resulted in taller plants (by 25.7%) and increased yields (by 59.0%).     

Cultural properties of a luminous mushroom,Mycena chlorophos

Cultural conditions on mycelial growth and fruit-body formation ofMycena chlorophos were studied. The optimum temperature of the mycelial growth was 27°C and the optimum initial pH of medium was 4.0. Peptone agar medium was suitable for the spawn culture. Compost medium containing rice bran at 10% (fw/fw) was appropriate for fruitbody formation in the Petri dish. Light was essential for initiation of primordia, and low-temperature treatment induced fruit-body formation effectively. The optimum conditions for fruit-body formation were found to be the cultivation at 27°C for 4 wk and continued cultivation for 3 wk under illumination at an intensity above 0.2 lx and at 21°C after casing with moist compost powder. In the fruit-bodies obtained, the maximum photosensitive wavelength of luminescence was 522 nm and the optimum temperature for emission was 27°C. The luminescence of a fruit-body was observed for about 3 d consecutively at 21°C.     

Bacterial and fungal metabolites of rhizospheric microflora of cotton antagonistic to Rhizoctonia solani Kühn

Summary Growth response ofRhizoctonia solani Kühn, the damping-off fungus, to metabolites of selected antagonistic rhizospheric bacteria and fungi of some Egyptian cotton varieties, namely, two strains ofBacillus subtilis Cohn,Aspergillus terreus Thom, andAspergillus flavus Link produced in culture media containing nitrate- or ammonium-nitrogen sources, proved the potency ofB. subtilis metabolites in inhibitingR. solani mycelial growth whether from nitrate- or ammonium-nitrogen culture media. Metabolite filtrates ofB. subtilis (II) are more potent than those ofB. subtilis (I). Increasing concentration of bacterial metabolite filtrates resulted in a decreased mycelial dry weight ofR. solani. The bacterial inhibitory factor forR. solani mycelial growth is partially affected by heat. Metabolite filtrates ofA. terreus from nitrate-nitrogen are slightly more potent than from ammonium-nitrogen culture media while an opposite relation is evident withA. flavus metabolites. Growth responses ofR. solani to different experimental dilutions of metabolite filtrates ofA. terreus andA. flavus proved the intervention of the nutritive factor in witholding growth of the damping-off fungus.     

Biological species ofArmillaria and their mycoparasitic associations withRhodophyllus abortivus in Hokkaido

Specimens of basidiomes and/or rhizomorphs ofArmillaria mellea complex and basidiomes ofRhodophyllus abortivus, developing on the same decaying stumps or stems of forest trees, were collected in three forests in Hokkaido. Normal basidiomes ofR. abortivus were found near to, but free from, the rhizomorphs and/or basidiomes ofArmillaria, while abnormal basidiomes, as carpophoroid forms, were developed on the rhizomorphs ofArmillaria. Of three mycoparasiticArmillaria isolates found withR. abortivus, one was identified asA. gallica and two asA. jezoensis. The isolates ofR. abortivus showed excellent mycelial growth and rhizomorph formation on PDA. However, on MDA, RMDA and BMDA, they showed poor aerial mycelia growth and no rhizomorphs. In the contrapositional cultures, the growth ofA. gallica was completely inhibited byR. abortivus on PDA but only slightly inhibited on MDA and RMDA. On the other hand, mutual inhibition at a distance was observed on BMDA. The mycelial growth and rhizomorph formation inA. jezoensis were severely inhibited by the colony ofR. abortivus on PDA, but only slightly inhibited on MDA. On RMDA and BMDA, the colonies of twoArmillaria species andR. abortivus showed mutual inhibition at a distance and apparent rhizomorph formation by bothArmillaria species.     

Species delimitation in the genusLipomyces by nuclear genome comparison

Species delimitation inLipomyces was attempted by nuclear genome comparison in conjuction with the re-evaluation of 48 physiological characters of 65 strains.High intraspecific (>75%) and low interspecific (<28%) similarity values established thatL. japonicus, L. lipofer andL. tetrasporus are genetically isolated, and also distinct fromL. kononenkoae andL. starkeyi.Ambiguous similarity values were obtained withL. kononenkoae andL. starkeyi. Strains previously assigned toL. kononenkoae constitute two related clusters. While similarity values within each cluster range from 76–99%, representatives of the two clusters reassociate for only 47%. Since these clusters are differentiated by their ecologically relevant maximum growth temperature,L. kononenkoae is subdivided. Strains previously assigned toL. starkeyi resolve into four closely related clusters. While similarity values within each cluster range from 78–100%, representatives of the four clusters reassociate for only 59–69%. Since these four clusters are poorly differentiated, the subdivision ofL. starkeyi does not appear possible without recourse to other criteria.Four unassigned strains constitute a further two clusters. Reassociation within these clusters is of the order of 91–100%, while reassociation between them occurs only at 59%. Reassociation of representatives of these clusters with those of theL. kononenkoae andL. starkeyi complexes is around 40% and 31%, respectively. These two clusters consequently appear to be intermediate betweenL. kononenkoae andL. starkeyi, and will, as such, have to be considered in any delimitation of these two species. A key to the taxa ofLipomyces and related genera of the Lipomycetaceae is given.     

Rhizobitoxine: A phytotoxin of unknown function which is commonly produced by bradyrhizobia

Summary Fifty-six percent of 93 strains ofBradyrhizobium japonicum andBradyrhizobium sp. (various hosts) from diverse geographical areas were found to produce a chlorosis-inducing toxin. Toxin production was common among bradyrhizobia originating from the USA, Africa, Central America, and South America. Toxin produced by West African strains was compared with rhizobitoxine by cation exchange chromatography, paper chromatography, and soybean (Glycine max (L.) Merr.) bioassay. The comparison suggested that the chlorosis-inducing toxin produced by West African bradyrhizobia is rhizobitoxine. Purified toxin from a West AfricanBradyrhizobium sp. (Vigna) strain inhibited the growth ofBacillus subtilis on minimal medium. The growth inhibition was reduced by addition of yeast-extract or casamino acids but not by any of 21 individual amino acids, including methionine. The same toxin did not inhibit the growth of 14 Bradyrhizobium strains, including eight strains that did not produce toxin. Mixed inoculum experiments revealed that a toxin-producing West African strain could not assist toxin non-producingB. japonicum strains in nodulating non-nodulating (rj₁ rj₁) soybeans.     

The selective allelopathic interaction of a pasture-forest intercropping in Taiwan

An allelopathic interaction of a pasture-forest intercropping system was evaluated by field and greenhouse experiments and by laboratory assays. A study site was situated in the farm of Hoshe Forestry Experiment Station at Nantou County, Taiwan. After deforestation of Chinese fir (Cunninghamia lanceolata), a split plot design of 8 treatments was set up: open ground without planting as control, planted with kikuyu grass, planted with kikuyu grass andAlnus formosana, planted with kikuyu grass andZelkova formosana, planted with kikuyu grass andCinnamomum camphora, planted withA. formosana, planted withZ. formosana, and planted withC. camphora. Field measurements showed that weeds grew luxuriantly six months after treatment in plots which had not been planted with kikuyu grass. However, the growth of weeds was significantly retarded but that of woody plants was not affected when the plots were planted with kikuyu grass. As compared with the tap water control, the aqueous leachate of kikuyu grass stimulated the seedling growth ofC. camphora andA. formosana, but the extract stimulated the growth ofC. camphora and inhibited that ofA. formosana. In contrast, the aqueous extracts of three hardwood plants exhibited variable inhibition on the root initiation of kikuyu grass, and the extract ofZ. formosana revealed the highest phytotoxic effect. The aforementioned extracts and leachates were bioassayed against seed germination and radicle growth of four test plants, namely annual rye grass, perennial rye gras, tall fescue, and Chinese cabbage and against seedling growth ofMiscanthus floridulus. The extract ofZ. formosana revealed the highest phytotoxic effect on the test species while that of kikuyu grass showed the least effect. By means of paper chromatography and high performance liquid chromatography, phytotoxic phenolics were identified and the amount of phytotoxins present was highest in the extract ofZ. formosana but was lowest in that of kikuyu grass. The degree of phytotoxicity and amount of phytotoxins was in good correlation, indicating that a selective allelopathic effect was involved. The findings suggest that allelopathy may contribute benefits in the intercropping system to reduce the need for herbicides and to lessen the labour cost for weed control.Paper No. 346 of the Scientific Journal Series of the Institute of Botany, Academia Sinica, Taipei, Taiwan, Republic of China. This study was supported in part by grants of Academia Sinica, Taipei, and Council of Agriculture, Executive Yuan of the Republic of China.     

Comparison of four media for the isolation ofAspergillus flavus group fungi

Four agar media used to isolate aflatoxin producing fungi were compared for utility in isolating fungi in theAspergillus flavus group from agricultural soils collected in 15 fields and four states in the southern United States. The four media wereAspergillus flavus andparasiticus Agar (AFPA, 14), the rose bengal agar described by Bell and Crawford (BCRB; 3), a modified rose bengal agar (M-RB), and Czapek's-Dox Agar supplemented with the antibiotics in BC-RB (CZ-RB). M-RB was the most useful for studying the population biology of this group because it permitted both identification of the greatest number ofA. flavus group strains and growth of the fewest competing fungi. M-RB supported an average of 12% moreA. flavus group colonies than the original rose bengal medium while reducing the number of mucorales colonies and the number of total fungi by 99% and 70%, respectively. M-RB was successfully employed to isolate all three aflatoxin producing species,A. flavus, A. parasiticus andA. nomius, and both the S and L strains ofA. flavus. M-RB is a defined medium without complex nitrogen and carbon sources (e.g. peptone and yeast extract) present in BC-RB. M-RB should be useful for studies on the population biology of theA. flavus group.Abbreviations M-RB Modified Rose Bengal Agar - CZ-RB Czapeks Rose Bengal Agar - BC-RB Bell and Crawford's Rose Bengal Agar - AFPA Aspergillus flavus andparasiticus agar     

Antagonistic interactions between fungal pathogens and phylloplane fungi of guava

Dominant phylloplane fungi of guava (Psidium guajava L.) were screened for their antagonistic activities against the two pathogens,Colletotrichum gloeosporioides andPestalotia psidii, bothin vitro andin vivo. Culture filtrates ofAspergillus niger, Fusarium oxysporum andPenicillium citrinum caused more than 50% growth inhibition ofC. gloeosporioides. Filtrates ofCephalosporium roseo-griseum andF. oxysporum were most effective in reducing the growth ofP. psidii. Volatiles produced from the cultures ofA. niger, F. oxysporum, P. citrinum andP. oxalicum inhibited the growth ofC. gloeosporioides, whereas volatiles fromC. roseo-griseum, F. oxysporum andTrichoderma harzianum inhibited the growth ofP. psidii. The inhibitory effect of volatiles decreased with increase in incubation time. In general, the maximum effect of volatiles was noticed after 48 h incubation. Different grades of colony interactions in dual cultures were recognised between the two pathogens and the phylloplane fungi examined. Maximum inhibition ofC. gloeosporioides was caused byAureobasidium pullulans, Cladosporium cladosporioides, epicoccum purpurascens, F. oxysporum andMyrothecium roridum, whereasAspergillus terreus, C. roseo-griseum andP. oxalicum significantly reduced the growth ofP. psidii. Application of a spore suspension of each test fungus inhibited lesion development of guava leaves caused by the test pathogensin vitro. Inhibition was more pronounced when the spore concentration was increased.A. pullulans, C. cladosporioides, E. purpurascens, F. oxysporum, andT. harzianum were found to be strongly antagonistic toC. gloeosporioides. A. niger, A. terreus, C. roseo-grisem andT. harzianum were strongly antagonistic toP. psidii.