Hypoxylon sp., an Endophyte of Persea indica, Producing 1,8-Cineole and Other Bioactive Volatiles with Fuel Potential

An endophytic fungus of Persea indica was identified, on the basis of its anamorphic stage, as Nodulosporium sp. by SEM. Partial sequence analysis of ITS rDNA revealed the identity of the teleomorphic stage of the fungus as Hypoxylon sp. It produces an impressive spectrum of volatile organic compounds (VOCs), most notably 1,8-cineole, 1-methyl-1,4-cyclohexadiene, and tentatively identified (+)-.alpha.-methylene-.alpha.-fenchocamphorone, among many others, most of which are unidentified. Six-day-old cultures of Hypoxylon sp. displayed maximal VOC-antimicrobial activity against Botrytis cinerea, Phytophthora cinnamomi, Cercospora beticola, and Sclerotinia sclerotiorum suggesting that the VOCs may play some role in the biology of the fungus and its survival in its host plant. Media containing starch- or sugar-related substrates best supported VOC production by the fungus. Direct on-line quantification of VOCs was measured by proton transfer mass spectrometry covering a continuous range with optimum VOC production occurred at 6 days at 145 ppmv with a rate of production of 7.65 ppmv/h. This report unequivocally demonstrates that 1,8-cineole (a monoterpene) is produced by a microorganism, which represents a novel and important source of this compound. This monoterpene is an octane derivative and has potential use as a fuel additive as do the other VOCs of this organism. Thus, fungal sourcing of this compound and other VOCs as produced by Hypoxylon sp. greatly expands their potential applications in medicine, industry, and energy production.     

An Endophytic <Emphasis Type="Italic">Phomopsis</Emphasis> sp. Possessing Bioactivity and Fuel Potential with its Volatile Organic Compounds

An unusual Phomopsis sp. was isolated as endophyte of Odontoglossum sp. (Orchidaceae), associated with a cloud forest in Northern Ecuador. This fungus produces a unique mixture of volatile organic compounds (VOCs) including sabinene (a monoterpene with a peppery odor) only previously known from higher plants. In addition, some of the other more abundant VOCs recorded by GC/MS in this organism were 1-butanol, 3-methyl; benzeneethanol; 1-propanol, 2-methyl and 2-propanone. The gases of Phomopsis sp. possess antifungal properties and an artificial mixture of the VOCs mimicked the antibiotic effects of this organism with the greatest bioactivity against a wide range of plant pathogenic test fungi including: Pythium, Phytophthora, Sclerotinia, Rhizoctonia, Fusarium, Botrytis, Verticillium, and Colletotrichum. The IC₅₀ values for the artificial gas mixture of Phomopsis sp. varied between 8 and 25.65 μl/mL. Proton transfer reaction-mass spectrometry monitored the concentration of VOCs emitted by Phomopsis sp. and yielded a total VOC concentration of ca. 18 ppmv in the head space at the seventh day of incubation at 23°C on PDA. As with many VOC-producing endophytes, this Phomopsis sp. did survive and grow in the presence of the inhibitory gases of Muscodor albus. A discussion is presented on the possible involvement of VOC production by the fungus and its role in the biology/ecology of the fungus/plant/environmental relationship.     

Induction of systemic resistance in Arabidopsis thaliana in response to a culture filtrate from a plant growth-promoting fungus, Phoma sp. GS8-3

The plant growth-promoting fungus (PGPF), Phoma sp. GS8-3, isolated from a zoysia grass rhizosphere, is capable of protecting cucumber plants against virulent pathogens. This fungus was investigated in terms of the underlying mechanisms and ability to elicit systemic resistance in Arabidopsis thaliana . Root treatment of Arabidopsis plants with a culture filtrate (CF) from Phoma sp. GS8-3 elicited systemic resistance against the bacterial speck pathogen Pseudomonas syringae pv. tomato DC3000 ( Pst ), with restricted disease development and inhibited pathogen proliferation. Pathway-specific mutant plants, such as jar1 (jasmonic acid insensitive) and ein2 (ethylene insensitive), and transgenic NahG plants (impaired in salicylate signalling) were protected after application of the CF, demonstrating that these pathways are dispensable (at least individually) in CF-mediated resistance. Similarly, NPR1 interference in npr1 mutants had no effect on CF-induced resistance. Gene expression studies revealed that CF treatment stimulated the systemic expression of both the SA-inducible PR-1 and JA/ET-inducible PDF1.2 genes. However, pathogenic challenge to CF-treated plants was associated with potentiated expression of the PR-1 gene and down-regulated expression of the PDF1.2 gene. The observed down-regulation of the PDF1.2 gene in CF-treated plants indicates that there may be cross-talk between SA- and JA/ET-dependent signalling pathways during the pathogenic infection process. In conclusion, our data suggest that CF of Phoma sp. GS8-3 induces resistance in Arabidopsis in a manner where SA and JA/ET may play a role in defence signalling.     

Evidence of the production of silver nanoparticles via pretreatment of Phoma sp.3.2883 with silver nitrate

AIMS: The objective was to demonstrate the size of silver particles produced by the filamentous fungus Phoma sp.3.2883 via adsorption and accumulation, and to confirm that this silver was in a reduced state. METHODS AND RESULTS: Mycelium was freeze-dried and then shake-cultured in a silver nitrate solution. It was found that up to 13.4 mg of silver was produced per gram of dry mycelium via atomic absorption spectrometer (AAS) analysis. The silver particles adsorbed on the mycelium were observed and measured under transmission electron microscope and their estimated size was 71.06 +/- 3.46 nm. Further examination of the particles via X-ray photoelectron spectroscope confirmed that the adsorbed silver particle had been reduced. CONCLUSION: The frozen mycelium of Phoma sp3.2883 has the potential for use in silver nanoparticle production. SIGNIFICANCE AND IMPACT OF THE STUDY: Silver nanoparticles could be used in the oil industry as an important catalyst and in the field of human medicine as a bactericide. The fungus Phoma sp3.2883 is a potential biosorbent that could be used for the production of these silver nanoparticles, and may also be useful in waste detoxification and in silver recovery programmes.     

Endophytic fungi from the root tubers of medicinal plant Stephania dielsiana and their antimicrobial activity

The adverse effects of chemical synthetic fungicides on agricultural fields and the environment are driving a need to search for safer and less environmentally harmful plant protectants to move toward more sustainable development of agriculture. The endophytic fungal community associated with the medicinal plant Stephania dielsiana, and its potential for providing antimicrobial secondary metabolites were investigated. A total of 26 isolates of endophytic fungi were obtained, and 21 isolates were identified and classified into eight different genera, including Briansuttonomyces, Glomerella, Pleosporales, Diaporthe, Phoma, Penicillium, Periconia and Colletotrichum, and the most frequent endophytic species obtained were Diaporthe phaseolorum, Penicillium sp., Periconia igniari and Colletotrichum sp. The ethyl acetate (EtOAc) extract of the endophytic fungus Diaporthe phaseolorum Stdif6 displayed the most significant antifungal activity against all tested phytopathogens, with EC₅₀ values ranging from 0.0138 to 0.3103 mg/mL. While the EtOAc extract of the endophytic fungus Penicillium sp. Stdif9 exhibited greater potential for antibacterial activity, with the minimum inhibitory concentration (MIC) values against seven bacteria ranging from 1.25 to 6 mg/mL. The remarkable antimicrobial activity of fungal endophytes suggests that fungal endophytes harbored inside the root tubers of S. dielsiana hold great promise as biocontrol agents against a broad spectrum of economically significant pathogens.     

Harnessing endophytes for industrial microbiology

Endophytic microorganisms exist within the living tissues of most plant species. They are most abundant in rainforest plants. Novel endophytes usually have associated with them novel secondary natural products and/or processes. Muscodor is a novel endophytic fungal genus that produces bioactive volatile organic compounds (VOCs). This fungus, as well as its VOCs, has enormous potential for uses in agriculture, industry and medicine. Muscodor albus produces a mixture of VOCs that act synergistically to kill a wide variety of plant and human pathogenic fungi and bacteria. This mixture of gases consists primarily of various alcohols, acids, esters, ketones and lipids. Artificial mixtures of the VOCs mimic the biological effects of the fungal VOCs when tested against a wide range of fungal and bacterial pathogens. Many practical applications for 'mycofumigation' by M. albus have been investigated and the fungus is now in the market place.     

Isolation, structure elucidation and biological activities of trichofumins A, B, C and D, new 11 and 13mer peptaibols from Trichoderma sp. HKI 0276W.

Trichofumins A-D were isolated from cultures of Trichoderma sp. HKI 0276 as new 11 and 13mer peptaibols. Similar to 15mer peptaibols they promote morphogenesis of the fungus Phoma destructiva and cause hypothermia in mice as a characteristic of neuroleptic activity. Membrane measurements using a synthetic BLM model showed that A, B, C and D increased membrane permeability for cations in a similar manner as was shown for larger peptaibols but with comparably lower efficiency.     

Diversity and antimicrobial activities of the fungal endophyte community associated with the traditional Brazilian medicinal plant Solanum cernuum Vell. (Solanaceae)

The diversity and antimicrobial activity of endophytic fungi associated with the Brazilian medicinal plant Solanum cernuum Vell. were studied during summer and winter seasons. A total of 246 fungal isolates were obtained, including 225 filamentous fungi and 21 yeasts. They were identified by morphological, physiological, and molecular methods. Fifty-five different taxa represented by the phyla Ascomycota (33 taxa), Basidiomycota (21 taxa), and Zygomycota (one taxon) were identified. The most abundant taxa were closely related to Arthrobotrys foliicola , Colletotrichum gloeosporioides , Coprinellus radians , Glomerella acutata , Diatrypella frostii , Phoma glomerata , Mucor sp., Phlebia subserialis , Phoma moricola , Phanerochaete sordida , and Colletotrichum sp. A total of 265 fungal extracts were screened and 64 (26.01%) displayed antimicrobial activities. Among these extracts, 18 (28.12%) presented antibacterial and antifungal activities, 42 (65.62%) displayed selective antibacterial activity, and four (6.25%) exhibited only antifungal activity. The best values of minimum inhibitory concentration were obtained from extracts of Cryptococcus rajasthanensis , Glomerella acutata, Leptosphaeria sp., and Phoma glomerata ranging from 7.8 to 15.62?μg/mL. This study is the first survey of the endophytic fungi community associated with S. cernuum, and our results show that they can represent a promising source of bioactive compounds.     

Muscodor albus and its biological promise

We have found a novel fungal genus that produces extremely bioactive volatile organic compounds (VOCs). This fungal isolate was initially discovered as an endophyte in Cinnamomum zeylanicum in a botanical garden in Honduras. This endophytic fungus, Muscodor albus, produces a mixture of VOCs that are lethal to a wide variety of plant and human pathogenic fungi and bacteria. It is also effective against nematodes and certain insects. The mixture of VOCs has been analyzed using GC/MS and consists primarily of various alcohols, acids, esters, ketones, and lipids. Final verification of the identity of the VOCs was carried out by using artificial mixtures of the putatively identified compounds and showing that the artificial mixture possessed the identical retention times and mass spectral qualities as those of the fungal derived substances. Artificial mixtures of the VOCs nicely mimicked the biological effects of the fungal VOCs when tested against a wide range of fungal and bacterial pathogens. Potential applications for “mycofumigation” by M. albus are currently being investigated and include uses for treating various plant parts, and human wastes. Another promising option includes its use to replace methyl bromide fumigation as a means to control soil-borne plant diseases.     

Synergism among volatile organic compounds resulting in increased antibiosis in Oidium sp

Oidium sp. has been recovered as an endophyte in Terminalia catappa (tropical chestnut) in Costa Rica. The volatile organic compounds (VOCs) of this organism uniquely and primarily consist of esters of propanoic acid, 2-methyl-, butanoic acid, 2-methyl-, and butanoic acid, 3-methyl-. The VOCs of Oidium sp. are slightly inhibitory to many plant pathogenic fungi. Previous work on the VOCs of Muscodor albus demonstrated that besides esters of small organic acids, a small organic acid and a naphthalene derivative were needed to obtain maximum antibiotic activity. Thus, the addition of exogenous volatile compounds such as isobutyric acid and naphthalene, 1,1'-oxybis caused a dramatic synergistic increase in the antibiotic activity of the VOCs of Oidium sp. against Pythium ultimum. In fact, at elevated concentrations, there was not only 100% inhibition of P. ultimum but killing as well. In addition, a coculture of Muscodor vitigenus (making only naphthalene) and Oidium sp. plus isobutyric acid produced an additive antibiosis effect against P. ultimum. The biological implications of multiple volatile compounds acting to bring about antibiosis in nature are discussed.     

Fungal degradation of aflatoxin B1

A number of fungal cultures were screened to select an organism suitable to be used in the detoxification of aflatoxin B1. They were co-cultured in Czapek-Dox-Casamino acid medium with aflatoxin B1 producing Aspergillus flavus. Several fungal cultures were found to prevent synthesis of aflatoxin B1 in liquid culture medium. Among these Phoma sp., Mucor sp., Trichoderma harzianum, Trichoderma sp. 639, Rhizopus sp. 663, Rhizopus sp. 710, Rhizopus sp. 668, Alternaria sp. and some strains belonging to the Sporotrichum group (ADA IV B14(a), ADA SF VI BF (9), strain 720) could inhibit aflatoxin synthesis by > or =90%. A few fungi, namely ADA IV B1, ADA F1, ADA F8, also belonging to the Sporotrichum group, were less efficient than the Phoma sp. The Cladosporium sp. and A. terreus sp. were by far the least efficient, registering <10% inhibition. The cultures which prevent aflatoxin biosynthesis are also capable of degrading the preformed toxin. Among these, Phoma sp. was the most efficient destroying about 99% of aflatoxin B1. The cell free extract of Phoma sp. destroyed nearly 50 microg aflatoxin B1 100 ml(-1) culture medium (90% of the added toxin), and this was more effective than its own culture filtrate over 5 days incubation at 28+/-2 degrees C. The degradation was gradual: 35% at 24 h, 58% at 48 h, 65% at 72 h, 85% at 96 h and 90% at 120 h. The possibility of a heat stable enzymatic activity in the cell free extract of Phoma is proposed.     

Antimicrobial volatile organic compounds affect morphogenesis-related enzymes in Guignardia citricarpa,causal agent of citrus black spot

Although non-volatile substances toxic to plant pathogenic microorganisms have been extensively studied over the years, few studies have focused on microbial volatile organic compounds (VOCs). The VOCs produced by the yeast Saccharomyces cerevisiae strain CR-1, used in fermentative processes for fuel ethanol production, are able to inhibit the vegetative development of the fungus Guignardia citricarpa, causal agent of the disease citrus black spot. How microbial VOCs affect the development of fungi is not known. Thus, the objective of the present work was to study the effect of the artificial mixture of VOCs identified from S. cerevisiae on intracellular enzymes involved in the mycelial morphogenesis in G. citricarpa. The phytopathogenic fungus was exposed to artificial mixture of VOCs constituted by alcohols (ethanol, 3-methyl-1-butanol, 2-methyl-1-butanol and phenylethyl alcohol) and esters (ethyl acetate and ethyl octanoate) in the proportions naturally found in the atmosphere produced by the yeast. The VOCs inhibited considerably the mycelial development and interfered negatively with the production of the morphogenesis-related enzymes. After 72 h of exposure to the VOCs the laccase and tyrosinase activities decreased 46 and 32%, respectively, however, the effect on the chitinase and β-1,3-glucanase activities was lower, 17 and 13% of inhibition, respectively. Therefore, the exposure of the fungus to the antimicrobial volatiles can influence both fungal mycelial growth rate and activity of enzymes implicated in morphogenesis. This knowledge is important to understand the microbial interactions mediated by VOCs in nature and to develop new strategies to control plant pathogens as G. citricarpa in postharvest.     

In vitro-Untersuchungen und mikroskopische Studien zur Entwicklung von Phoma chrysanthemicola Hollos f. sp. chrysanthemicola, dem Erreger einer Wurzel- und Stengelgrundfäule der Chrysantheme

In vitro investigations and microscopic studies about the development of Phoma chrysanthemicola Hollos f. sp. chrysanthemicola, the causal pathogen of a collar and root rot of chrysanthemums Different media were tested for cultivation of Phoma chrysanthemicola. The pathogen grew best on PDA and carrot-Agar at a temperature of 25°C. The three strains used showed many variations, concerning the structure and colour of the mycelium. Fructification has been promoted by irradiation (ultra-violer ray). The morphogenesis, especially the formation of pycnidia, conidia and sclerotial structures has been examined with help of the scanning electrone microscope (SEM), results are presented., So far unknown organs probably belonging to the fungus were observed, their identity is still not clear.     

Volatile organic compounds: a potential direct long-distance mechanism for antagonistic action of Fusarium oxysporum strain MSA 35

Fusarium oxysporum MSA 35 [wild-type (WT) strain] is an antagonistic Fusarium that lives in association with a consortium of bacteria belonging to the genera Serratia, Achromobacter , Bacillus and Stenotrophomonas in an Italian soil suppressive to Fusarium wilt. Typing experiments and virulence tests provided evidence that the F. oxysporum isolate when cured of the bacterial symbionts [the cured (CU) form], is pathogenic, causing wilt symptoms identical to those caused by F. oxysporum f. sp. lactucae . Here, we demonstrate that small volatile organic compounds (VOCs) emitted from the WT strain negatively influence the mycelial growth of different formae speciales of F. oxysporum. Furthermore, these VOCs repress gene expression of two putative virulence genes in F. oxysporum lactucae strain Fuslat10, a fungus against which the WT strain MSA 35 has antagonistic activity. The VOC profile of the WT and CU fungus shows different compositions. Sesquiterpenes, mainly caryophyllene, were present in the headspace only of WT MSA 35. No sesquiterpenes were found in the volatiles of ectosymbiotic Serratia sp. strain DM1 and Achromobacte r sp. strain MM1. Bacterial volatiles had no effects on the growth of the different ff. spp. of F. oxysporum examined. Hyphae grown with VOC from WT F. oxysporum f. sp. lactucae strain MSA 35 were hydrophobic whereas those grown without VOCs were not, suggesting a correlation between the presence of volatiles in the atmosphere and the phenotype of the mycelium. This is the first report of VOC production by antagonistic F. oxysporum MSA 35 and their effects on pathogenic F. oxysporum. The results obtained in this work led us to propose a new potential direct long-distance mechanism for antagonism by F. oxysporum MSA 35 mediated by VOCs . Antagonism could be the consequence of both reduction of pathogen mycelial growth and inhibition of pathogen virulence gene expression.     

Wasalexins A and B, new phytoalexins from wasabi: isolation, synthesis, and antifungal activity

The chemical structure determination of two phytoalexins from wasabi (Wasabia japonica, syn. Eutrema wasabi), a plant resistant to virulent isolates of the blackleg fungus [Leptosphaeria maculans (Desm.) Ces. et de Not., asexual stage Phoma lingam (Tode ex Fr.) Desm.], as well as their synthesis and antifungal activity towards isolates of P. lingam and P. wasabiae is reported.     

Construction and characterization of a cloned probe for the detection of Phoma tracheiphila in plant tissues

Summary The construction and characterization of the recombinant clone, pPhoB25 is described. This clone contains an highly repeated sequence of DNA from the fungus Phoma tracheiphila, a pathogen of the lemon. The specificity of the probe was determined by molecular hybridization with the DNA of the host plant and with those of several fungi which can be found associated with the lemon plants. The clone was used successfully as a specific and sensitive probe for the detection of the pathogen in infected plant tissues by dotblot hybridization under laboratory and field conditions.     

茅苍术内生真菌的分离鉴定及在组培苗中的回接

从茅苍术的叶、茎和根中分离鉴定内生真菌,并观测内生菌回接对茅苍术组培苗生长的影响。共获得茅苍术内生菌16株,其中叶片中14株,根茎中各获得一株。分别为交链孢霉、镰刀霉、小克银汉霉、青霉、茎点霉、小菌核菌、孔球孢霉和不产孢类。经内生菌和茅苍术快繁苗实验初筛,得到两株对植物无害的内生菌:小菌核菌和孔球孢霉,将它们回接到茅苍术组培苗中,可以从苗叶片中染色观察到菌丝。其中小菌核菌能提高茅苍术炼苗的存活率,接入内生菌的茅苍术叶片超氧化物歧化酶和过氧化物酶活性均高于未接菌的对照,脂肪酸不饱和指数基本不变,说明分离自茅苍术的内生菌回接后可以与宿主建立共生关系。     

Atradidymella muscivora gen. et sp. nov. (Pleosporales) and its anamorph Phoma muscivora sp. nov.: A new pleomorphic pathogen of boreal bryophytes

During a survey of bryophilous fungi from boreal and montane habitats, 12 isolates of a hitherto unknown plant pathogenic member of the Pleosporales were recovered from Aulacomnium palustre, Hylocomium splendens, and Polytrichum juniperinum, and described as Atradidymella muscivora gen. et sp. nov. Atradidymella is characterized by minute, unilocular, setose pseudothecia having 2-3 wall layers; brown, fusiform, 1-septate ascospores; and a Phoma anamorph. The genus is distinguished from all other pleosporalean genera with brown, fusiform ascospores on the basis of ascospore and pseudothecium morphology and a highly reduced stroma that is localized within a single host cell. Atradidymella muscivora is distinguished by its minute pseudothecia (<115 μm) and ascospores that are slightly allantoid and constricted at the septum with the upper cell often wider than the lower. Its anamorph, Phoma muscivora sp. nov., is morphologically distinguishable from P. herbarum in having smaller conidia. Parsimony analysis of the ITS rDNA region indicates A. muscivora has affinities to the Phoma-Ascochyta-Didymella clade that is sister to the Phaeosphaeriaceae within the Pleosporales.     

Analysis of the mating-type loci of co-occurring and phylogenetically related species of Ascochyta and Phoma

Ascochyta and Phoma are fungal genera containing several important plant pathogenic species. These genera are morphologically similar, and recent molecular studies performed to unravel their phylogeny have resulted in the establishment of several new genera within the newly erected Didymellaceae family. An analysis of the structure of fungal mating-type genes can contribute to a better understanding of the taxonomic relationships of these plant pathogens, and may shed some light on their evolution and on differences in sexual strategy and pathogenicity. We analysed the mating-type loci of phylogenetically closely related Ascochyta and Phoma species (Phoma clematidina, Didymella vitalbina, Didymella clematidis, Peyronellaea pinodes and Peyronellaea pinodella) that co-occur on the same hosts, either on Clematis or Pisum. The results confirm that the mating-type genes provide the information to distinguish between the homothallic Pey. pinodes (formerly Ascochyta pinodes) and the heterothallic Pey. pinodella (formerly Phoma pinodella), and indicate the close phylogenetic relationship between these two species that are part of the disease complex responsible for Ascochyta blight on pea. Furthermore, our analysis of the mating-type genes of the fungal species responsible for causing wilt of Clematis sp. revealed that the heterothallic D. vitalbina (Phoma anamorph) is more closely related to the homothallic D. clematidis (Ascochyta anamorph) than to the heterothallic P. clematidina. Finally, our results indicate that homothallism in D. clematidis resulted from a single crossover between MAT1-1 and MAT1-2 sequences of heterothallic ancestors, whereas a single crossover event followed by an inversion of a fused MAT1/2 locus resulted in homothallism in Pey. pinodes.     

Colonization of barley roots by endophytic fungi and their reduction of take-all caused by Gaeumannomyces graminis var. tritici

Fungal root endophytes obtained from natural vegetation were tested for antifungal activity in dual culture tests against the root pathogen Gaeumannomyces graminis var. tritici. Fifteen isolates, including Acremonium blochii, Acremonium furcatum, Aspergillus fumigatus, Cylindrocarpon sp., Cylindrocarpon destructans, Dactylaria sp., Fusarium equiseti, Phoma herbarum, Phoma leveillei, and a sterile mycelium, selected based on the dual culture test, were inoculated on barley roots in growth tubes under axenic conditions, both in the absence and presence of G. graminis var. tritici. All isolates colonized the rhizosphere and very often the root cortex without causing disease symptoms and without affecting plant growth. Eight isolates significantly reduced the symptoms caused by G. graminis var. tritici, and 6 of them reduced its presence in the roots.