Cultivable Fungal Diversity in Deep-Sea Sediment of the East Pacific Ocean

The diversity of fungi in the marine environment has been extensively studied, but their denitrification activity has rarely been reported to date. In the present study, we used six different media to isolate fungi from 10 sediment samples collected at five different locations of the East Pacific Ocean with water depths ranging from 4545 to 7068?m. Fungal identification and phylogenetic diversity analysis were conducted based on morphological characteristics and internal transcribed spacers of ribosomal DNA (ITS-rRNA) sequencing. A total of 106 fungal isolates, belonging to 12 genera, including Aspergillus (five strains), Aureobasidium (three strains), Candida (two strains), Cladosporium (56 strains), Cystobasidium (one strain), Devriesia (nine strains), Knufia (one strain), Nigrospora (three strains), Penicillium (18 strains), Rhodotorula (four strains), Sarocladium (three strains), and unclassified Xylariales (one strain) were obtained. The most dominant culturable genus was Cladosporium. One possible novel fungal strains showed less than 97% similarity to their closest matches of unclassified Xylariales in the Genbank. In addition, we used nirK gene as the molecular marker to detect denitrifying fungi among the cultivable fungal isolates. The nirK gene was detected in Aspergillus niger and Penicillium chrysogenum. Our research indicated that diverse fungi from the deep sea sediments of the East Pacific Ocean and highlighted the involvement of these fungi in denitrification process.     

Fungal colonization of automobile air conditioning systems

Air samples and swab samples of the air conditioning vents were collected from 29 automobiles in the metropolitan region of Atlanta, GA, and cultured for fungi. Among the fungi observed, species of Acremonium, Aspergillus, Alternaria, Aureobasidium, Cladosporium, and Penicillium were in the highest densities. Transparent adhesive tape imprints, SEM observations, and enrichment culture of components of five systems demonstrated fungal hyphae on the metal surfaces and within the matrix of various insulation materials. The evaporator removed from one automobile because of a series of complaints of noxious odors was densely colonized by Penicillium viridicatum. The amplification of known allergenic and odor-producing fungi occurred within the automobile air conditioning systems. Received 27 May 1997/ Accepted in revised form 16 July 1997     

Diversity and Drug Resistance of Filamentous Fungi Isolated from the Fresh Raspberries

Fungi are one of the most widely distributed microorganisms in the environment, including food such as fruits, vegetables and other crops, posing a potential threat to food safety and human health. The aim of this study was to determine the diversity, intensity and drug resistance of potentially pathogenic filamentous fungi isolated from the fresh raspberries (Rubus idaeus L.). A total of 50 strains belonging to genera Fusarium, Cladosporium, Alternaria, Penicillium, Mucor, Rhizopus, Aspergillus and Acremonium were tested for drug resistance against 11 antifungals by disc diffusion and gradient strips methods. The average mycological contamination in the examined samples of raspberries amounted to 4.34 log CFU/g. The Cladosporium was isolated from all tested samples, followed by Alternaria and Fusarium with a frequency of 61% and 34%, respectively. The highest level of drug resistance was observed for Acremonium genera and Fusarium strains recorded a wide variation in drug resistance as revealed by susceptibility with amphotericin B and voriconzole with MICs ranged from 0.5–4 µg/ml and posaconazole with MICs ranging from 3–8 µg/ml. All fungal strains showed 100% resistance to caspofungin, fluconazole and flucytosine with both the methods, and 100% resistance to micafungin and anidulafungin in the gradient strip method.     

Diverse Deep-Sea Fungi from the South China Sea and Their Antimicrobial Activity

We investigated the diversity of fungal communities in nine different deep-sea sediment samples of the South China Sea by culture-dependent methods followed by analysis of fungal internal transcribed spacer (ITS) sequences. Although 14 out of 27 identified species were reported in a previous study, 13 species were isolated from sediments of deep-sea environments for the first report. Moreover, these ITS sequences of six isolates shared 84–92 % similarity with their closest matches in GenBank, which suggested that they might be novel phylotypes of genera Ajellomyces, Podosordaria, Torula, and Xylaria. The antimicrobial activities of these fungal isolates were explored using a double-layer technique. A relatively high proportion (56 %) of fungal isolates exhibited antimicrobial activity against at least one pathogenic bacterium or fungus among four marine pathogenic microbes (Micrococcus luteus, Pseudoaltermonas piscida, Aspergerillus versicolor, and A. sydowii). Out of these antimicrobial fungi, the genera Arthrinium, Aspergillus, and Penicillium exhibited antibacterial and antifungal activities, while genus Aureobasidium displayed only antibacterial activity, and genera Acremonium, Cladosporium, Geomyces, and Phaeosphaeriopsis displayed only antifungal activity. To our knowledge, this is the first report to investigate the diversity and antimicrobial activity of culturable deep-sea-derived fungi in the South China Sea. These results suggest that diverse deep-sea fungi from the South China Sea are a potential source for antibiotics’ discovery and further increase the pool of fungi available for natural bioactive product screening.     

Exploration of cultivable fungal communities in deep coal‐bearing sediments from ∼1.3 to 2.5 km below the ocean floor

Although subseafloor sediments are known to harbour a vast number of microbial cells, the distribution, diversity, and origins of fungal populations remain largely unexplored. In this study, we cultivated fungi from 34 of 47 deep coal‐associated sediment samples collected at depths ranging from 1289 to 2457 m below the seafloor (mbsf) off the Shimokita Peninsula, Japan (1118 m water depth). We obtained a total of 69 fungal isolates under strict contamination controls, representing 61 Ascomycota (14 genera, 23 species) and 8 Basidiomycota (4 genera, 4 species). Penicillium and Aspergillus relatives were the most dominant genera within the Ascomycetes, followed by the members of genera Cladosporium, Hamigera, Chaetomium, Eutypella, Acremonium, Aureobasidium, Candida, Eurotium, Exophiala, Nigrospora, Bionectria and Pseudocercosporella. Four Basidiomycota species were identified as genera Schizophyllum, Irpex, Bjerkandera and Termitomyces. Among these isolates, Cladosporium sphaerospermum and Aspergillus sydowii relatives were isolated from a thin lignite coal‐sandstone formation at 2457 mbsf. Our results indicate that these cultivable fungal populations are indigenous, originating from past terrigenous environments, which have persisted, possibly as spores, through ～20 million years of depositional history.     

Biodiversity study and potential of fungal endophytes of peppermint and effect of their extract on chickpea rot pathogens

India is the highest producer of Cicer arietinum, however the crop is susceptible to plant fungal diseases i.e. Sclerotinia sclerotiorum, Botrytis cinerea, Fusarium oxysporum and Rhizoctonia solani. For a sustainable alternative, anti-plant pathogenic efficacy of fungal endophytes were investigated. Endophytic fungi of Mentha piperita were investigated for biodiversity, biocontrol potential towards these phytopathogens and their metabolite profiling. Sixty three fungal isolates were recovered from peppermints sampled in different seasons from distinct regions of India. Endophytic fungi were identified by ITS-rDNA sequence process. PCA divulged seasonal variability with exclusive presence of Colletotrichum sp., D. phaseolorum, Alternaria sp., Hypocrea sp. and R. oryzae in second sampling season. Shannon diversity index (H′) was found to be highest in leaf (1.253) from Mukteshwar. Acremonium sp. (MPM-2.1) extract exhibited anti-plant pathogenic activity with < 1 mg/ml IC₅₀ value towards phytopathogens. GC-MS chromatography of potent biocontrol fungus Acremonium sp. (MPHSS-2.1) confirmed presence of antifungal compounds 1-heptacosanol and 1-nonadecane.     

葡萄生单轴霉菌围可培养微生物多样性及潜在生防菌研究

【目的】揭示葡萄生单轴霉(Plasmoparaviticola)菌围可培养细菌和真菌的多样性特征,筛选对葡萄霜霉病有较强稳定防治效果的生防菌。【方法】连续两年从我国南北方具有代表性的7个葡萄产区采集葡萄霜霉病叶,镊子夹取经保湿培养获得的新鲜霉层并配制孢子囊悬浮液,采用传统分离培养法,结合形态分类、BOX-PCR指纹图谱分析以及分子鉴定结果,对葡萄生单轴霉菌围的可培养细菌和真菌进行聚类分析;采用菌株及其发酵液与病原菌孢子囊悬浮液等体积混合培养测定其对孢子囊的抑制作用,离体叶片接种法检测该菌株及其发酵液对霜霉病的防治效果。【结果】分离获得了90株细菌和110株真菌,分别归属于8个细菌属和14个真菌属,且相同地区不同葡萄品种葡萄生单轴霉菌围的细菌和真菌在同年处于同一分支。假单胞菌属(Pseudomonas)和枝孢属(Cladosporium)稳定存在于各地区不同品种葡萄霜霉病叶上葡萄生单轴霉菌围;在两年间稳定存在的菌株占比多数在80.0%以上且均具有较高的生防作用;其中,广泛分布的6株枝顶孢属(Acremonium)真菌对葡萄霜霉病的防治效果均较好,最高可达100.0%;防治效果较高的11个菌株的无菌发酵液中,黑曲霉(Aspergillusniger) NX2F、苋楔孢黑粉菌(Thecaphora amaranthi) BJ1G和匍枝根霉(Rhizopus stolonifer) BM1L的无菌发酵液防治效果均为100.0%。【结论】葡萄生单轴霉菌围的可培养细菌和真菌群落主要受地区因素影响,有较高的稳定性和生防作用,揭示了枝顶孢属真菌在我国葡萄主要产区葡萄生单轴霉菌围附生的普遍性,为葡萄霜霉病的防治提供了丰富和宝贵的资源。     

Watershed-Scale Fungal Community Characterization along a pH Gradient in a Subsurface Environment Cocontaminated with Uranium and Nitrate

The objective of this study was to characterize fungal communities in a subsurface environment cocontaminated with uranium and nitrate at the watershed scale and to determine the potential contribution of fungi to contaminant transformation (nitrate attenuation). The abundance, distribution, and diversity of fungi in subsurface groundwater samples were determined using quantitative and semiquantitative molecular techniques, including quantitative PCR of eukaryotic small-subunit rRNA genes and pyrosequencing of fungal internal transcribed spacer (ITS) regions. Potential bacterial and fungal denitrification was assessed in sediment-groundwater slurries amended with antimicrobial compounds and in fungal pure cultures isolated from the subsurface. Our results demonstrate that subsurface fungal communities are dominated by members of the phylum Ascomycota, and a pronounced shift in fungal community composition occurs across the groundwater pH gradient at the field site, with lower diversity observed under acidic (pH <4.5) conditions. Fungal isolates recovered from subsurface sediments, including cultures of the genus Coniochaeta, which were detected in abundance in pyrosequence libraries of site groundwater samples, were shown to reduce nitrate to nitrous oxide. Denitrifying fungal isolates recovered from the site were classified and found to be distributed broadly within the phylum Ascomycota and within a single genus of the Basidiomycota. Potential denitrification rate assays with sediment-groundwater slurries showed the potential for subsurface fungi to reduce nitrate to nitrous oxide under in situ acidic pH conditions.     

Antimicrobial potentials of endophytic fungi residing in <Emphasis Type="Italic">Quercus variabilis</Emphasis> and brefeldin A obtained from <Emphasis Type="Italic">Cladosporium</Emphasis> sp.

Among 67 endophytic fungi isolated from Quercus variabilis, 53.7% of endophytic fungal fermentation broths displayed growth inhibition on at least one test microorganism, such as pathogenic fungi (Trichophyton rubrum, Candida albicans, Aspergillus niger, Epidermophyton floccosum, Microsporum canis) and bacteria (Escherichia coli, Bacillus subtilis, Pseudomonas fluorescens). Moreover, 19.4% of strains showed a broader antimicrobial spectrum, such as Aspergillus sp., Penicillium sp., Alternaria sp., 20.9% of strains showed strong inhibition (+++) to pathogenic bacteria, while only 7.5% displayed that to test fungi. The most active antifungal strain I(R)9-2, Cladosporium sp. was selected and fermented. From the broth, a secondary metabolite, brefeldin A was obtained. This is the first report on the antimicrobial potentials of endophytic fungi residing in Q. variabilis and isolation of brefeldin A produced by Cladosporium sp.     

Anaerobic denitrification in fungi from the coastal marine sediments off Goa, India

Denitrification is a microbial process during which nitrate or nitrite is reduced under anaerobic condition to gaseous nitrogen. The Arabian Sea contains one of the major pelagic denitrification zones and in addition to this, denitrification also takes places along the continental shelf. Prokaryotic microorganisms were considered to be the only players in this process. However recent studies have shown that higher microeukaryotes such as fungi can also adapt to anaerobic mode of respiration and reduce nitrate to harmful green house gases such as NO and N₂O. In this study we examined the distribution and biomass of fungi in the sediments of the seasonal anoxic region off Goa from two stations. The sampling was carried out in five different periods from October 2005, when dissolved oxygen levels were near zero in bottom waters to March 2006. We isolated mycelial fungi, thraustochytrids and yeasts. Species of Aspergillus and thraustochytrids were dominant. Fungi were isolated under aerobic, as well as anaerobic conditions from different seasons. Four isolates were examined for their denitrification activity. Two cultures obtained from the anoxic sediments showed better growth under anaerobic condition than the other two cultures that were isolated from oxic sediments. Our preliminary results suggest that several species of fungi can grow under oxygen deficient conditions and participate in denitrification processes.     

Denitrification by Actinomycetes and Purification of Dissimilatory Nitrite Reductase and Azurin from Streptomyces thioluteus

Many actinomycete strains are able to convert nitrate or nitrite to nitrous oxide (N₂O). As a representative of actinomycete denitrification systems, the system of Streptomyces thioluteus was investigated in detail. S. thioluteus attained distinct cell growth upon anaerobic incubation with nitrate or nitrite with concomitant and stoichiometric conversion of nitrate or nitrite to N₂O, suggesting that the denitrification acts as anaerobic respiration. Furthermore, a copper-containing, dissimilatory nitrite reductase (CuNir) and its physiological electron donor, azurin, were isolated. This is the first report to show that denitrification generally occurs among actinomycetes.     

Nitrous Oxide Formation in the Colne Estuary, England: the Central Role of Nitrite

Nitrate and nitrite concentrations in the water and nitrous oxide and nitrite fluxes across the sediment-water interface were measured monthly in the River Colne estuary, England, from December 1996 to March 1998. Water column concentrations of N₂O in the Colne were supersaturated with respect to air, indicating that the estuary was a source of N₂O for the atmosphere. At the freshwater end of the estuary, nitrous oxide effluxes from the sediment were closely correlated with the nitrite concentrations in the overlying water and with the nitrite influx into the sediment. Increases in N₂O production from sediments were about 10 times greater with the addition of nitrite than with the addition of nitrate. Rates of denitrification were stimulated to a larger extent by enhanced nitrite than by nitrate concentrations. At 550 μM nitrite or nitrate (the highest concentration used), the rates of denitrification were 600 μmol N · m⁻² · h⁻¹ with nitrite but only 180 μmol N · m⁻² · h⁻¹ with nitrate. The ratios of rates of nitrous oxide production and denitrification (N₂O/N₂ × 100) were significantly higher with the addition of nitrite (7 to 13% of denitrification) than with nitrate (2 to 4% of denitrification). The results suggested that in addition to anaerobic bacteria, which possess the complete denitrification pathway for N₂ formation in the estuarine sediments, there may be two other groups of bacteria: nitrite denitrifiers, which reduce nitrite to N₂ via N₂O, and obligate nitrite-denitrifying bacteria, which reduce nitrite to N₂O as the end product. Consideration of free-energy changes during N₂O formation led to the conclusion that N₂O formation using nitrite as the electron acceptor is favored in the Colne estuary and may be a critical factor regulating the formation of N₂O in high-nutrient-load estuaries.     

Diversity of epiphytic fungi on the surface of Kyoho grape berries during ripening process in summer and winter at Nanning region,Guangxi, China

The two-harvest-per-year farming system allow table grape to be harvested a year both in summer and winter in southern China. Herein, we used high-throughput sequencing to investigate the diversity of fungi on grape fruits surface during the ripening process in summer and winter at subtropical Nanning region, Guangxi, China. The results showed that 23 fungal species existed in all samples. Among them, the five most dominant species were Cladosporium ramotenellum, Pseudozyma aphidis, Gyrothrix spp., Gibberella intricans and Acremonium alternatum, with abundance from 61.62 % to 91.26 %. Analysis using the student's t-test for Shannon index indicated that components of fungal community varied significantly between the two ripening seasons. The dominant genera of core fungal community were Cladosporium, Gyrothrix, Paramycosphaerella, Acremonium, Penicillium and Tilletiopsis in the summer and Cladosporium, Pseudozyma, Gibberella, Colletotrichum, Sporobolomyces, Rhodosporidium, Alternaria and Aspergillus in the winter. Overall, fungi diversity on grape fruits surface at Nanning showed significantly differences between different ripening seasons. Our results ennrich the understanding of epiphytic communities of grape fruits in subtropics.     

Nitrite reduction to nitrous oxide by propionibacteria: Detoxication mechanism

Characteristics of dissimilatory nitrate reduction by Propionibacterium acidi-propionici, P. freudenreichii, P. jensenii, P. shermanii and P. thoenii were studied. All strains reduced nitrate to nitrite and further to N₂O. Recovery of added nitrite-N as N₂O-N approached 100%, so that no other end product existed in a significant quantity. Specific rates of N₂O production were 3 to 6 orders of magnitude lower than specific rates of N₂ production by common denitrifiers. Oxygen but not acetylene inhibited N₂O production in P. acidi-propionici and P. thoenii. Nitrite reduction rates were generally higher than nitrate reduction rates. The enzymes involved in nitrate and nitrite reduction were either constitutive or derepressed by anacrobiosis. Nitrate stimulated synthesis of nitrate reductase in P. acidi-propionici. Specific growth rates and growth yields were increased by nitrate. At 10 mM, nitrite was toxic to all strains, and at 1 mM its effect ranged from none to total inhibition. No distinction was obvious between incomplete forms of denitrification and dissimilatory nitrate reduction to ammonia. N₂O production from nitrite by propionibacteria may represent a detoxication mechanism rather than a part of an energy transformation system.     

Denitrification in lucerne nodules is not involved in nitrite detoxification

Dissimilatory reduction of ionic nitrogen oxides to gaseous forms such as nitrous oxide or nitrogen can be carried out by free living or symbiotic forms of some strains of Rhizobium meliloti. In this paper we investigate whether bacteroid denitrification plays a role in the alleviation of the inhibitory effects of nitrate on nitrogen fixation both in bacteroid incubations as in whole nodules. The presence of a constitutive nitrate reductase (NR) activity in isolated bacteroids caused nitrite accumulation in the incubation medium, and acetylene reduction activity in these bacteroids was progressively inhibited, since nitrite reductase (NiR) activity was unable to reduce all the nitrite produced by NR and denitrification occurred slowly. Even nodules infiltrated with nitrate and nitrite failed to increase gaseous forms of nitrogen substantially, indicating that nitrite availability was not limiting denitrification by bacteroids. In spite of the low rates of bacteroidal denitrification, the effect of nodule denitrification on the inhibition of nitrogen fixation by nitrate in whole plants was tested. For that purpose, lucerne plants (Medicago sativa L. cv. Aragon) were inoculated with two Rhizobium meliloti strains: 102-F-65 (non denitrifying) and 102-F-51 (a highly denitrifying strain). After a seven days nitrate treatment, both strains showed the same pattern of inhibition, and it occurred before any nitrate or nitrite accumulation within the nodules could be detected. This observation, together with the lack of alleviation of the ARA inhibition in the denitrifying strain, and the limited activity of dissimilatory nitrogen reduction present in these bacteroids, indicate a role other than nitrite detoxification for denitrification in nodules under natural conditions.     

<Emphasis Type="Italic">Acr</Emphasis><Emphasis Type="Italic">emonium</Emphasis> Species: A Review of the Etiological Agents of Emerging Hyalohyphomycosis

Unusual fungal agents that exist environmentally as saprophytes can often lead to opportunistic infections. Hyalohyphomycosis is a group of fungal infections caused by fungi characterized by hyaline septate hyphae and can infect both immunocompetent as well as immunocompromised patients. Many a times it becomes difficult to distinguish a pathogenic and a contaminant fungus, because many such agents can assume clinical significance depending on circumstances. Subcutaneous and invasive fungal infection due to the emerging hyalohyphomycotic fungus, Acremonium, has drawn the attention of clinicians and microbiologists, as a potential pathogen in patients with and without underlying risk factors. Generally considered to be minimally invasive in the past, genus Acremonium has been responsible for eumycotic mycetomas and focal infections in otherwise healthy individuals. It has also been increasingly implicated in systemic fungal diseases. The management with different antifungals in various clinical situations has been very conflicting and hence needs to be carefully evaluated. This overview is an endeavor to consolidate the available clinical infections due to Acremonium and the recommendations on treatment.     

Mixed Herbs Drugs: Inhibitory Effect on Growth of the Endogenous Mycoflora and Aflatoxin Production

Twenty commercial mixed herbal drugs were examined for mycological profile. Aspergillus species were the predominant fungi found in the drugs. Other fungi harboured in the drugs with less frequency were Paecilomyces species, Eurotium species, Monascus species, Acremonium species, Penicillium species, Cladosporium species, Scopulariopsis species, Phialophora species and Fonseceae species. Fungal count was between 1.0 log₁₀ CFU and 2.4 log₁₀ CFU per gram of sample. When the drugs were incubated in 85% humidity at 25°C, fungal colonies grew on only two of the drugs. The mixed herbal drugs were extracted with water and the extracts were used to grow Aspergillus parasiticus. All extracts reduced aflatoxin B₁ and aflatoxin G₁ production by 62–97%. All but two of the extracts reduced aflatoxin B₂ and aflatoxin G₂ production by 39–95%. It can be concluded that the commercial powdered mixed herbal drugs contained low number of endogenous fungi, and these drugs are inhibitory to the growth of its endogenous fungi and aflatoxins production by aflatoxigenic fungi.