Mating systems inOmphalotus (Paxillaceae, Agaricales)

Pairings of monokaryon cultures representingOmphalotus illudens (eastern North America),O. nidiformis (southeastern Australia),O. olearius (southern Europe),O. olivascens (North American Pacific coast), andO. subilludens (southern North America) showed widely variable compatibility patterns.Omphalotus olearius ×O. subilludens ×O. olivascens showed high compatibility, whileO. illudens was significantly less compatible with all other taxa. Isolates ofOmphalotus nidiformis represented an almost genetically isolated biological species. The role of partial compatibility in nomenclatural ranking is discussed.     

Control of the bean rust fungus <Emphasis Type="Italic">Uromyces appendiculatus</Emphasis> by means of <Emphasis Type="Italic">Trichoderma harzianum</Emphasis>: leaf disc assays on the antibiotic effect of spore suspensions and culture filtrates

Several species of the fungal genus Trichoderma act as antagonists of other fungi. A number of strains from the Trichoderma species T. harzianum Rifai are used as biological control agents for the control of soilborne as well as foliar plant pathogens. Six T. harzianum strains, five of them isolated from commercial preparations, were evaluated for their capability to control the bean rust fungus Uromyces appendiculatus (Pers. ex Pers.) Unger. Different kinds of leaf disc assays were performed with conidial spore suspensions and sterile culture filtrates of the T. harzianum strains. Great differences were observed concerning the efficacy of the Trichoderma strains to reduce the number of the uredial pustules developing after rust inoculation which followed the application of the particular Trichoderma strains. Efficacy values ranged from 1 to over 50%. Increasing spore or culture filtrate concentrations of the two most effective isolates T12 and T_U led to decreases in the number of developing uredial pustules. Culture filtrate applications had a protective but no curative effect. T12 spore suspensions maintained their disease reducing activity even when autoclaved. This and some other evidence for an antibiotic interaction between T. harzianum and U. appendiculatus are discussed. Handling Editor: Reijo Karjalainen.     

In vitro evaluation of combination of Trichoderma harzianum and chitosan for the control of sapstain fungi

In vitro assays were undertaken to evaluate the control of two sapstain fungi, Leptographium procerum and Sphaeropsis sapinea by a combination of chitosan or chitosan oligomer and an albino strain of Trichoderma harzianum. Spore germination and hyphal growth of the test fungi were assessed on media amended with chitosan or chitosan oligomer with and without T. harzianum using either simultaneous inoculation with test fungus or inoculation 1, 2, or 3 days after pre-infection with test fungus.There was no mycelial growth of the test fungi regardless of chitosan concentrations used when either L. procerum or S. sapinea was simultaneously inoculated with T. harzianum. However, the dose–response of chitosan or chitosan oligomer on the test fungi was apparent when T. harzianum was not simultaneously inoculated with test fungus but introduced later. There was a greater growth reduction at higher concentrations (0.075–0.1% v/v) of chitosan, and overall chitosan oligomer was more effective than chitosan aqueous solution.Chitosan alone was able to restrict or delay the germination of spores but the combination of chitosan and T. harzianum inhibited spore germination and hence colony formation of test fungi regardless of time delay.     

Fungi bioluminescence revisited

A review of the research conducted during the past 30 years on the distribution, taxonomy, phylogeny, ecology, physiology and bioluminescence mechanisms of luminescent fungi is presented. We recognize 64 species of bioluminescent fungi belonging to at least three distinct evolutionary lineages, termed Omphalotus, Armillaria and mycenoid. An accounting of their currently accepted names, distributions, citations reporting luminescence and whether their mycelium and/or basidiomes emit light are provided. We address the physiological and ecological aspects of fungal bioluminescence and provide data on the mechanisms responsible for bioluminescence in the fungi.     

Bioluminescence expression during the transition from mycelium to mushroom in three North American Armillaria and Desarmillaria species

Unlike most bioluminescent fungi, mycelia of Armillaria and Desarmillaria are constitutively bioluminescent while mature mushrooms are not. The absence of the luciferin, 3-hydroxyhispidin, and its precursor hispidin in mature mushrooms have been proposed to explain the lack of bioluminescence from Armillaria mushrooms. Using three North American species, A. gallica, A. mellea and D. tabescens (syn., Armillaria tabescens), we documented a decline in luminescence of ten fold during the transition from mycelia to, immature mushrooms (i.e., pins) for the two Armillaria species. As pins matured, luminescence declined by an additional two or three orders of magnitude. Lower initial luminescence of D. tabescens mycelia declined to negligible levels during mushroom development. Further, light production was localized in the gills and lower stipe of A. mellea mushrooms. The decline in luminescence during mushroom formation was reversed by addition of hispidin to stipe or gills which significantly enhanced luminescence by one and three orders of magnitude, respectively. We conclude that the modulation of Armillaria and Desarmillaria luminescence is achieved by luciferin availability early in mushroom development. However, since the temporal regulation of bioluminescence differs between Armillaria species and other genera, we conclude that bioluminescence in Armillaria is under unique selective pressures.     

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.     

Characteristics of a newly created bioluminescent Pseudomonas putida harboring TOL plasmid for use in analysis of a bioaugmentation system

Insertion of a bacterial lux operon into the chromosome of Pseudomonas putida mt-2 holding TOL plasmid, yielded a new bioluminescent strain of P. putida BLU. Both in the cultures containing toluene and m-toluic acid as the sole carbon sources, P. putida BLU showed the same specific growth rate and cell yield as those of the wild strain. The bioluminescence output in the cell growth phases correlated with the cell concentration, indicating that the bioluminescent P. putida BLU can be monitored and quantified in a mixed culture in real time by the luminescence detection.     

Potential of genes and gene products fromTrichoderma sp. andGliocladium sp. for the development of biological pesticides

Fungal cell wall degrading enzymes produced by the biocontrol fungiTrichoderma harzianum andGliocladium virens are strong inhibitors of spore germination and hyphal elongation of a number of phytopathogenic fungi. The purified enzymes include chitinolytic enzymes with different modes of action or different substrate specificity and glucanolytic enzymes with exo-activity. A variety of synergistic interactions were found when different enzymes were combined or associated with biotic or abiotic antifungal agents. The levels of inhibition obtained by using enzyme combinations were, in some cases, comparable with commercial fungicides. Moreover, the antifungal interaction between enzymes and common fungicides allowed the reduction of the chemical doses up to 200-fold. Chitinolytic and glucanolytic enzymes fromT. harzianum were able to improve substantially the antifungal ability of a biocontrol strain ofEnterobacter cloacae. DNA fragments containing genes encoding for different chitinolytic enzymes were isolated from a cDNA library ofT. harzianum and cloned for mechanistic studies and biocontrol purposes. Our results provide additional information on the role of lytic enzymes in processes of biocontrol and strongly suggest the use of lytic enzymes and their genes for biological control of plant diseases.     

Effect of chitosan on physiological, morphological, and ultrastructural characteristics of wood-degrading fungi

An investigation was undertaken to understand the mechanism(s) by which chitosan exerts its antifungal effects against the wood-degrading fungi Sphaeropsis sapinea and Trichoderma harzianum. Exposure to increasing concentrations of chitosan caused an increase in the amount of hydrogen peroxide accumulation in cultures of S. sapinea, which was accompanied by a decrease in superoxide formation. The same effect was not observed in T. harzianum. Potassium ion leakage was an early event for both test fungi, leakage being more pronounced for S. sapinea than T. harzianum for the first 5 min, particularly at higher concentrations of chitosan treatment. Fluorescence microscopy provided evidence that the effect of chitosan on fungal hyphae was mediated through alterations in the plasma membrane properties. Chitosan also severely affected fungal morphology. Increasing concentrations of chitosan induced excessive branching, vacuolation, and a reduction in hyphal diameter. Transmission electron microscopy, which showed more severe ultrastructural changes in S. sapinea hyphae from chitosan treatment as compared to T. harzianum, provided valuable complementary information. The data suggest that the plasma membrane may be the primary target of chitosan action, and that the two fungi differ in the extent to which they are affected.     

Biological efficacy of <Emphasis Type="Italic">Trichoderma harzianum</Emphasis> isolate to control some fungal pathogens of wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis>) in Turkey

Gaeumannomyces graminis var. tritici, Fusarium culmorum and F. moniliforme are highly important and widespread pathogens of wheat in Turkey. Trichoderma isolates have been used as biocontrol agents to protect plants against soilborne diseases in several crops. The present work was carried out to evaluate the potential of Trichoderma harzianum isolate T1 as biocontrol agents for G. graminis, F. culmorum and F. moniliforme under field conditions in 2001 and 2002. Quantitative differences were found in microbial number in soil. T. harzianum T1 had considerable effect on population densities of the tested pathogens. The total number of G. graminis, F. culmorum and F. moniliforme were lower in the T. harzianum T1 application made to seed. T. harzianum T1 application to seed had increasing affect on the yield components of wheat through better control over pathogens. The greatest counts of T. harzianum T1 were detected on root segments. Seed application by T. harzianum T1 had increasing effect on yield components of wheat.     

嗜热毁丝霉高通量培养技术及其应用

【背景】丝状真菌是一类重要的工业发酵生产宿主菌,如何进行高通量纯菌培养和高效检测筛选性能优异的菌株是工业丝状真菌研究的重要方向。【目的】研究建立丝状真菌的高通量培养技术并测试应用效果。【方法】通过对丝状真菌培养过程中的制种、接种、培养和检测研究,建立基于孔板的高通量培养技术,并以嗜热毁丝霉为例对该技术进行验证。【结果】与传统的平板制种和摇瓶接种培养方式相比,高通量孔板的培养方式将制种通量提高24倍,单位面积产孢子能力提高350%,液体培养转接效率提高10-40倍,并建立96孔板测定乙醇含量的高通量检测技术。【结论】将丝状真菌的培养和检测通量提高1-2个数量级,为快速检测丝状真菌改造过程产生的大量性状不同菌株并获得目标菌株奠定基础,为丝状真菌高通量筛选研究提供应用指导价值。     

A combination of NADHP and hispidin is not essential for bioluminescence in luminous fungal living gills of Mycena chlorophos

The chemical mechanisms underlying visible bioluminescence in the fungus Mycena chlorophos are not clear. A combination of dihydronicotinamide adenine dinucleotide phosphate (NADPH) and hispidin, which has been reported to increase the intensity of in vitro luminescence in crude cold‐water extracts prepared from the bioluminescent fruiting bodies of M. chlorophos, exhibited potential bioluminescence activation in the early bioluminescence stages, in which the bioluminescence was ultra‐weak, for living gills and luminescence activation for non‐bioluminescent gills, which was collapsed by freezing and subsequent thawing, at all bioluminescence stages. These abilities were not evident in considerably bioluminescent gills. These abilities were blocked by trans‐4‐hydroxycinnamic acid and trans‐3,4‐dihydroxycinnamic acid, which were identified as in vivo bioluminescence‐activating components. Original bioluminescence and bioluminescence produced from the addition of trans‐4‐hydroxycinnamic acid and trans‐3,4‐dihydroxycinnamic acid in living gills were almost completely inhibited by 10 mM NaN₃, whereas the luminescence produced form the combination of NADPH and hispidin in thawed non‐bioluminescent and living gills at the early weak bioluminescence stages was not inhibited by 10 mM NaN₃. Thus, the combination of NADPH and hispidin plays different roles in luminescence systems compared with essential bioluminescence systems, and the combination of NADPH and hispidin was not essential for visible bioluminescence in living gills.     

Interaction between pathogenic and saprobic fungi isolated from soybean roots and seeds

A variety of interactions was recorded in culture between 11 saprobic fungi isolated from soybean (Glycine max) roots and seeds and the soybean pathogens Cercospora sojina, Colletotrichum truncatum, Macrophomina phaseolina, Phomopsis sojae, and Septoria glycines. The most active saprobes were Aspergillus terreus, Chaetomium cupreum, Epicoccum nigrum, Gliocladium roseum, Myrothecium roridum, Penicillium thomii, and Trichothecium roseum. Hyphal lysis of several fungal pathogens by Acremonium sp., C. cupreum and P. thomii was recorded perhaps because of parasitism by G. roseum and T. roseum. In greenhouse studies, seeds coated with G. roseum, P. thomii, and T. harzianum emerged significantly (P=0.05) more than those coated with A. terreus and the control. In field studies, seeds coated with a conidial suspension of A. terreus, G. roseum, P. thomii or Trichoderma harzianum produced a significantly greater stand than the control. The area of cotyledons covered with lesions caused by C. truncatum was significantly less on seeds coated with G. roseum, P. thomii and T. harzianum than the control.     

Induced proteome of Trichoderma harzianum by Botrytis cinerea

As a notable biocontrol agent, Trichoderma harzianum can antagonize a diverse array of phytopathogenic fungi, including Botrytis cinerea, Rhizoctonia solani and Fusarium oxysporum. Elucidating the biocontrol mechanism of T. harzianum in response to the pathogens enables it to be exploited in the control of plant diseases. Two-dimensional gel electrophoresis (2-DE) was performed to obtain secreted protein patterns of T. harzianum ETS 323, grown in media that contained glucose, a mixture of glucose and deactivated B. cinerea mycelia, deactivated B. cinerea mycelia or deactivated T. harzianum mycelia. Selected protein spots were identified using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Ninety one out of 100 excised protein spots were analyzed and some proteins were sequence identified. Of these, one l-amino acid oxidase (LAAO) and two endochitinases were uniquely induced in the media that contained deactivated B. cinerea mycelia as the sole carbon source. Activities of the cell wall-degrading enzymes (CWDEs), including β-1,3-glucanases, β-1,6-glucanases, chitinases, proteases and xylanases, were significantly higher in media with deactivated B. cinerea mycelia than in other media. This finding suggests that the cell wall of B. cinerea is indeed the primary target of T. harzianum ETS 323 in the biocontrol mechanism. The possible roles of LAAO and xylanase were also discussed.     

Fungitoxicity of the essential oil of Citrus sinensis on post-harvest pathogens

Summary The essential oil extracted from the epicarp of Citrus sinensis exhibited absolute fungitoxicity against the 10 post-harvest pathogens. GC–MS studies of the oil revealed the presence of 10 chemical constituents, of which limonene was found to be the major component (84.2%). The activity of the oil was tested by the poisoned food technique (PF) and the volatile activity (VA) assay and the oils showed greater toxicity in the VA assay than in the poisoned food assay. The nature of the toxicity was studied in the VA assay and it was observed that the oil was fungicidal for the 10 pathogens in the 700 ppm (mg/l) to 1000 ppm range. The oil was extremely toxic for spore germination and it was found that at 700 ppm, spore germination was inhibited in the 10 test fungi out of the 12 tested. Treatment at 300 ppm concentration exhibited 70–100% inhibition of spore germination in most of the fungi tested. Scanning electron microscopy (SEM) was done to study the mode of action of the oil in Aspergillus niger and it was observed that treatment with the oil leads to distortion and thinning of the hyphal wall and the reduction in hyphal diameter and absence of conidiophores.     

Statistical optimization of lovastatin production by Omphalotus olearius (DC.) singer in submerged fermentation

In this study, culture conditions were optimized to improve lovastatin production by Omphalotus olearius, isolate OBCC 2002, using statistical experimental designs. The Plackett–Burman design was used to select important variables affecting lovastatin production. Accordingly, glucose, peptone, and agitation speed were determined as the variables that have influence on lovastatin production. In a further experiment, these variables were optimized with a Box–Behnken design and applied in a submerged process; this resulted in 12.51 mg/L lovastatin production on a medium containing glucose (10 g/L), peptone (5 g/L), thiamine (1 mg/L), and NaCl (0.4 g/L) under static conditions. This level of lovastatin production is eight times higher than that produced under unoptimized media and growth conditions by Omphalotus olearius. To the best of our knowledge, this is the first attempt to optimize submerged fermentation process for lovastatin production by Omphalotus olearius.     

Construction of a Tn5 derivative encoding bioluminescence and its introduction in Pseudomonas, Agrobacterium and Rhizobium

Summary A simple method based upon the use of a Tn5 derivative, Tn5-Lux, has been devised for the introduction and stable expression of the character of bioluminescence in a variety of gram-negative bacteria. In Tn5-Lux, the luxAB genes of Vibrio harveyi encoding luciferase are inserted on a SalI-BglII fragment between the kanamycin resistance (Km^r) gene and the right insertion sequence. The transposon derivative was placed on a transposition suicide vehicle by in situ recombination with the Tn5 suicide vector pGS9, to yield pDB30. Mating between Escherichia coli WA803 (pDB30) and a strain from our laboratory, Pseudomonas sp. RB100C, gave a Km^r transfer frequency of 10^-6 per recipient, a value 10 times lower than that obtained with the original suicide vehicle pGS9. Tn5-Lux was also introduced by insertion mutagenesis in other strains of gram-negative soil bacteria. The bioluminescence marker was expressed in the presence of n-decanal, and was monitored as chemiluminescence in a liquid scintillation counter. The recorded light intensities were fairly comparable among the strains, and ranged between 0.2 to 1.8x10⁶ cpm for a cell density of 10³ colony forming units/ml. Nodules initiated by bioluminescent strains of Rhizobium leguminosarum on two different hosts were compared for intensity of the bioluminescence they produced.     

Type studies of <Emphasis Type="Italic">Pleurotus</Emphasis> reported from Japan

Eight type specimens of Pleurotus reported from Japan were examined. Four new combinations, Marasmius alopecius, Omphalotus guepiniformis, Marasmiellus leiophyllus, and Hohenbuehelia squamula, are proposed. Pleurotus cyatheae is accepted in the original genus. The following species are synonyms: Pleurotus harmandii, a synonym of Omphalotus guepiniformis; P. minutoniger, a synonym of Resupinatus striatulus; and P. pulchellus, a synonym of Hohenbuehelia tremula. Omphalotus japonicus (= Lampteromyces japonicus) is a synonum of O. guepiniformis.     

Biological and Integrated Control of Botrytis Bunch Rot of Grape UsingTrichodermaspp.

Field trials were carried out in upstate New York in 1990, 1992, 1993, and 1994 and in Chile in 1992–1993 and 1993–1994 in order to evaluate the ability of various strains ofTrichodermaspp. to control bunch rot of grape, to assess the compatibility and possible additive effects of selected biocontrol fungi and dicarboximide fungicides, and to determine factors affecting biocontrol efficacy. In 1990, three strains ofTrichodermaspp. were evaluated for their biocontrol ability, and all provided significant control ofBotrytis cinerea.As few as two late applications of the biocontrol fungi were nearly as effective as up to five applications throughout bloom and fruit development. Trials in New York in 1992 and in Chile in 1992–1993 indicated thatTrichoderma harzianumcould replace some applications of iprodione or vinclozolin with little reduction in efficacy. In New York in 1993, we found that applications ofT. harzianumat bloom and early fruit development followed by a tank-mix application ofT. harzianumand half rates of iprodione gave extremely effective control of bunch rot. In 1994, less effective control was obtained than in earlier years. Addition of a nutritive adhesive (Pelgel, a mixture of carboxymethyl cellulose and gum arabic) applied with the biocontrol agent tended to improve results. Thus, biological control of bunch rot of grape withT. harzianumcan be an effective method of management of this disease.     

Effect of root exudates on the spore germiantion of rhizosphere and rhizoplane mycoflora of chilli (Capsicum annuum L.) cultivars

Summary From root exudates of three cultivars of chilli (Capsicum annuum L.) 12 amino acids and 7 sugars were detected. Methionine, d-1- phenylalanine, citrulline and d-xylose were detected only from the root exudates of resdistant cultivars. The root exudates of resistant variety inhibited spore germination of the pathogen (Fusarium oxysporum f. sp.capsici), but that of susceptible variety enhanced spore germiantion of the same. Spore germiantion of antagonistic fungi (Trichderma viride andAspergillus sydowi) was also influenced by the root exudates of resistant and susceptible varieties, but the influence was different.Spore germiantion of a number of rhizosphere fungi was studied and in general root exudate of susceptible cultivar enhanced spore germiantion of majority of fungi, but spore germination of antagonistic fungi against the pathogen was inhibited. However, root exudate of resistant cultivar stimulated spore germination of antagonistic fungi.