Muscodor sutura, a novel endophytic fungus with volatile antibiotic activities

Muscodor sutura is described as a novel species that is also an endophyte of Prestonia trifidi. Uniquely, this fungus produces a reddish pigment, on potato dextrose agar (PDA), when grown in the dark. In addition, the organism makes some volatile organic compounds that have not been previously reported from this genus, namely, thujopsene, chamigrene, isocaryophyllene, and butanoic acid, 2-methyl. These and other volatile compounds in the mixture possess wide-spectrum antifungal activity and no observable antibacterial activity. Most unusually, on PDA, the newly developing hyphae of this fungus grow in a perfect stitching pattern, in and out of the agar surface. The partial ITS–DNA sequence of this organism is identical to that of Muscodor vitigenus but it differs from all other Muscodor spp. Justification for a new species, as Muscodor sutura, is collectively based on morphological, cultural, chemical, and bioactivity properties.     

Muscodor fengyangensis sp. nov. from southeast China: morphology, physiology and production of volatile compounds

The fungal genus Muscodor was erected on the basis of Muscodor albus, an endophytic fungus originally isolated from Cinnamomum zeylanicum. It produces a mixture of volatile organic compounds (VOCs) with antimicrobial activity that can be used as mycofumigants. The genus currently comprises five species. Here we describe the isolation and characterization of a new species of Muscodor on the basis of five endophytic fungal strains from leaves of Actinidia chinensis, Pseudotaxus chienii and an unidentified broad leaf tree in the Fengyangshan Nature Reserve, Zhejiang Province, Southeast of China. They exhibit white colonies on potato dextrose agar (PDA) media, rope-like mycelial strands, but did not sporulate. The optimum growth temperature is 25°C. The results of a phylogenetic analysis based on four loci (ITS1-5.8S-ITS2, 28S rRNA, rpb2 and tub1) are consistent with the hypothesis that these five strains belong to a single taxon. All five strains also produce volatile chemical components with antimicrobial activity in vitro, which were different from those previously described for other Muscodor species.     

Metulocladosporiella gen. nov. for the causal organism of Cladosporium speckle disease of banana

Cladosporium musae, a widespread leaf-spotting hyphomycete on Musa spp., is genetically and morphologically distinct from Cladosporium s. str. (Davidiella anamorphs, Mycosphaerellaceae, Dothideales). DNA sequence data derived from the ITS and LSU gene regions of C. musae isolates show that this species is part of a large group of hyphomycetes in the Chaetothyriales with dematiaceous blastoconidia in acropetal chains. Cladosporium adianticola, a foliicolous hyphomycete known from leaf litter in Cuba is also a member of this clade and is closely related to C. musae. A comparison with other genera in the Cladosporium complex revealed that C. musae belongs to a lineage for which no generic name is currently available, and for which the genus Metulocladosporiella gen. nov. is proposed. Two species of Metulocladosporiella are currently known, namely M. musae, which is widely distributed, and M. musicola sp. nov., which is currently known from Africa.     

Muscodor albus MOW12 an Endophyte of Piper nigrum L. (Piperaceae) Collected from North East India Produces Volatile Antimicrobials

Muscodor albus MOW12, an endophytic fungus isolated from Piper nigrum in Mawlong, Meghalaya, India, resembles some cultural and hyphal characteristics of previous isolates of Muscodor sp. In addition, it possesses about 99 % similarity in its ITS rDNA with other M. albus isolates and thus is nicely centered within the genetic tree to other Muscodor spp. This xylariaceae fungus effectively inhibits and kills certain plant pathogenic fungi by virtue of a mixture of volatile compounds that it produces. The majority of these compounds were identified by gas chromatography/mass spectrometry as small molecular weight esters, alcohols, and acids. The main ester components of this isolate of M. albus in its volatile mixture are acetic acid, ethyl ester; propanoic acid, 2-methyl-, methyl ester and acetic acid, 2-methylpropyl ester. This appears to be the first report of any M. albus strain from India.     

Antimicrobial activity of some macroalgae of the Veracruzano Reef System (SAV), Mexico

The study of macroalgae antimicrobial agents is limited to Mexico and scarce in the Veracruzano Reef System (SAV). It is necessary to devote efforts towards this field of applied phycology. The aim was to evaluate the antimicrobial activity of some phyla of Rhodophyta, Chlorophyta and Ochrophyta from SAV. Methanolic extracts from 23 marine macroalgae species (7 Chlorophyta, 4 Phaeophyta and 12 Rhodophyta) from the Veracruzano Reef System (SAV) (Mexico) were evaluated for antimicrobial activity. Antibacterial and antifungal activity were assessed by agar diffusion and agar dilution methods. The differences between mean values obtained for experimental groups was done by analysis of variance (ANOVA multifactorial model), p-values of 0.001 or less were considered statistically significant. Two new records are recognized for SAV (Laurencia gracilis and Sebdenia flabellata) and Compsothamnion thuioides for the Gulf of Mexico coasts. 16 species showed antibacterial activity, of which Caulerpa sertularioides, Ulva lactuca and Laurencia obtuse had significant activity on Gram-positive bacteria. 43.7% belong to the phyla Chlorophyta (7 species), 50% Rhodophyta (8 species) and 6.25% Ochrophyta (1 species). This indicates that the extracts of the algae of the Rhodophyta and Chlorophyta are the ones that showed the greatest activity. Regarding the yeasts, 16.6% of the total algae collected were active in the different yeast strains. 43.7% belongs to Chlorophyta species and for Rhodophyta were 60%. The macroalgae with the highest antifungal activity were: Cymopolia barbata, Ulva lactuca and Laurencia gracilis. The macroalgae of the Veracruzano Reef System present antimicrobial activity. This study is the first investigation of macroalgae's bioactive components from SAV, where they could be sources for future medical applications.     

Three New Species of Cyphellophora (Chaetothyriales) Associated with Sooty Blotch and Flyspeck

The genus Cyphellophora includes human- and plant-related species from mammal skin and nails, plant materials, and food. On the basis of analysis of ITS, LSU, TUB2 and RPB1 data and morphological characters, three new species, Cyphellophora phyllostachysdis, C. artocarpi and C. musae, associated with sooty blotch and flyspeck disease, were added to this genus. The 2D structure of ITS1 and ITS2 confirmed this taxonomic status. Pathogenicity tests on apple fruit indicated that C. artocarpi could be a sooty blotch and flyspeck pathogen of apple.     

Species Delimitation in the Genus Moschus (Ruminantia: Moschidae) and Its High-Plateau Origin

The authenticity of controversial species is a significant challenge for systematic biologists. Moschidae is a small family of musk deer in the Artiodactyla, composing only one genus, Moschus. Historically, the number of species in the Moschidae family has been debated. Presently, most musk deer species were restricted in the Tibetan Plateau and surrounding/adjacent areas, which implied that the evolution of Moschus might have been punctuated by the uplift of the Tibetan Plateau. In this study, we aimed to determine the evolutionary history and delimit the species in Moschus by exploring the complete mitochondrial genome (mtDNA) and other mitochondrial gene. Our study demonstrated that six species, M. leucogaster, M. fuscus, M. moschiferus, M. berezovskii, M. chrysogaster and M. anhuiensis, were authentic species in the genus Moschus. Phylogenetic analysis and molecular dating showed that the ancestor of the present Moschidae originates from Tibetan Plateau which suggested that the evolution of Moschus was prompted by the most intense orogenic movement of the Tibetan Plateau during the Pliocene age, and alternating glacial-interglacial geological eras.     

The Diversity and Antimicrobial Activity of Preussia sp. Endophytes Isolated from Australian Dry Rainforests

Limited knowledge currently exists regarding species diversity and antimicrobial activity of endophytic isolates of Preussia within Australia. This report describes endophytic Preussia species that were identified through molecular analysis of the internal transcribed spacer region. Screening for antimicrobial secondary metabolites was determined by testing crude ethyl acetate (EtOAc) extracts derived from fungal mycelia against a panel of ATCC type strains which included Bacillus cereus, Escherichia coli, Enterococcus faecalis, Pseudomonas aeruginosa, Serratia marcescens, methicillin-resistant Staphylococcus aureus (MRSA) and the opportunist yeast pathogen Candida albicans. Subsequently, high-performance liquid chromatography generated fractions of bioactive EtOAc extracts which were subject to confirmatory testing using the Clinical and Laboratory Standards Institute reference microdilution antimicrobial activity assay. A total of 18 Preussia were isolated from nine host plants with 6/18 having a <97 % sequence similarity to other known species in Genbank, suggesting that they are new species. In preliminary screening, 13/18 Preussia isolates revealed antimicrobial activity against at least one of the microbes tested, whilst 6/18 isolates, including 4/6 putative new species showed specific antimicrobial activity against MRSA and C. albicans. These results highlight the antimicrobial potential of Australian Preussia spp. and also the importance of Australian dry rainforests as an untapped repository of potentially significant bioactive compounds.     

Antimicrobial activity of marine organisms collected off the coast of South East India

In vitro antimicrobial screening of nine marine sponges (Porifera) and two seaweeds, collected from south east coast of India, against selected clinical isolates of bacteria and fungi was conducted in this study. Methanolic extracts of all the marine organisms demonstrated activity against one or more of microbes tested. Sigmadocia carnosa was the most active exhibiting a broad spectrum antimicrobial activity against each of the microbe tested with the exception of Fusarium species. Contrary to this, the genus Echinogorgia did not show any detectable bactericidal activity but, Echninogorgia reticulata was weakly fungicidal against Rhodotorula species and E. compecta against Fusarium and Nocardia species. Considerable antibacterial activity was exhibited by Haliclona cribricutis and Chrotella australiensis against Klebsiella species and Vibrio cholerae, respectively. Petrocia testudinaria showed equally good activity against the bacterium V. chlorae and the fungus Cryptococcus neoformans. The sponges Callyspongia fibrosa, Ircinia species and the seaweed Stoecheospermum margilatum are totally inactive against fungi. The extracts showing good antimicrobial activity are undergoing further analysis to identify the active constituents.     

Antifungal properties of essential oil and crude extracts of Hypericum linarioides Bosse

The chemical composition of the essential oil isolated from the aerial parts of Hypericum linarioides Bosse by hydrodistillation was analysed by GC–MS. It was determined that 74 compounds, which represent 84.1% of total oil, were present in the oil. The oil contains mainly δ-cadinene (6.9%), (Z)-β-farnesene (5.2%), γ-muurolene (5.5%), spathulenol (4.8%), hexahydrofarnesyl acetone (4.5%) and α-selinene (4.0%). The oil was also characterized by high content of sesquiterpenes (64.2% of total oil). The oil was tested for antifungal activity using mycelial growth inhibition assays (in vitro) against 11 agricultural pathogenic fungi, which consisted of six Fusarium species (Fusarium acuminatum, Fusarium culmorum, Fusarium equiseti, Fusarium oxysporum, Fusarium sambucinum and Fusarium solani) and three anastomosis groups of Rhizoctonia solani (AG-5, AG-9 and AG-11), Alternaria solani and Verticillium albo-atrum. The oil of H. linarioides showed antifungal activity against AG-9 and V. albo-atrum. In addition, petroleum ether, chloroform, acetone and methanol extracts of H. linarioides were tested against species of 11 fungi. The extracts showed moderate inhibition effects on the growth of A. solani, F. culmorum, F. equiseti and all anastomosis groups of R. solani.     

Comparative and Evolutionary Analyses of Meloidogyne spp. Based on Mitochondrial Genome Sequences

Molecular taxonomy and evolution of nematodes have been recently the focus of several studies. Mitochondrial sequences were proposed as an alternative for precise identification of Meloidogyne species, to study intraspecific variability and to follow maternal lineages. We characterized the mitochondrial genomes (mtDNAs) of the root knot nematodes M. floridensis, M. hapla and M. incognita. These were AT rich (81–83%) and highly compact, encoding 12 proteins, 2 rRNAs, and 22 tRNAs. Comparisons with published mtDNAs of M. chitwoodi, M. incognita (another strain) and M. graminicola revealed that they share protein and rRNA gene order but differ in the order of tRNAs. The mtDNAs of M. floridensis and M. incognita were strikingly similar (97–100% identity for all coding regions). In contrast, M. floridensis, M. chitwoodi, M. hapla and M. graminicola showed 65–84% nucleotide identity for coding regions. Variable mitochondrial sequences are potentially useful for evolutionary and taxonomic studies. We developed a molecular taxonomic marker by sequencing a highly-variable ~2 kb mitochondrial region, nad5-cox1, from 36 populations of root-knot nematodes to elucidate relationships within the genus Meloidogyne. Isolates of five species formed monophyletic groups and showed little intraspecific variability. We also present a thorough analysis of the mitochondrial region cox2-rrnS. Phylogenies based on either mitochondrial region had good discrimination power but could not discriminate between M. arenaria, M. incognita and M. floridensis.     

Nesting biology of three Megachile (Hymenoptera: Megachilidae) species from Eastern Amazonia,Brazil

Megachile Latreille is a conspicuous genus of solitary bees distributed worldwide. However, the biology of tropical species is still little known. We present data on biology of Megachile brasiliensis Data Torre, Megachile sejuncta Cockerell and Megachile stilbonotaspis Moure found in two remnants of eastern Amazonian forest in northeastern Brazil. The study was conducted using the trap-nest methodology in two different areas during four periods. We collected a total of 24 nests of M. brasiliensis, 26 of M. sejuncta and 28 of M. stilbonotaspis. The differential abundance of collected nests may reflect the population size in each sampled place. The nesting activity was concentrated mainly between July and January and species presented a multivoltine pattern, except for M. sejuncta, which was partly univoltine. Assessed pollen use showed a predominant use of Attalea sp. (Arecaceae) and, for M. stilbonotaspis, Tylesia sp. and Lepidaploa sp. (Asteraceae). Babassu is a very common palm in the studied areas and the studied species seem to have a strong link with it. We also reported change of pollen use by M. sejuncta, probably due to competition with M. brasiliensis, which may have influenced the biased sex ratio observed in M. sejuncta toward males. Parasites reported here were also recorded for other Megachile species, such as Coelioxys, Brachymeria, Meloidae and Pyralidae species. Mites were observed in association with M. stilbonotaspis. The data presented here set up a background that encourages new studies on the ecology of these three Amazonian species, providing tools for proper biodiversity management and conservation.     

Bacterial Species-Specific Activity of a Fluoroquinolone against Two Closely Related Pasteurellaceae with Similar MICs: Differential In Vitro Inoculum Effects and In Vivo Efficacies

We investigated the antimicrobial activity of a fluoroquinolone against two genetically close bacterial species belonging to the Pasteurellaceae family. Time-kill experiments were used to measure the in vitro activity of marbofloxacin against two strains of Mannheimia haemolytica and Pasteurella multocida with similar MICs. We observed that marbofloxacin was equally potent against 10⁵ CFU/mL inocula M. haemolytica and P. multocida. However, an inoculum effect was observed with P. multocida, meaning that marbofloxacin activity was decreased against a 10⁸ CFU/mL inoculum, whereas no inoculum effect was observed with M. haemolytica. Marbofloxacin activity was also tested in a lung infection model with immunocompromised mice intratracheally infected with 10⁹ CFU of each bacteria. At the same dose, the clinical and bacteriological outcomes were much better for mice infected with M. haemolytica than for those infected with P. multocida. Moreover, bacteriological eradication was obtained with a lower marbofloxacin dose for mice infected with M. haemolytica. Our results suggest that the differential in vivo marbofloxacin efficacy observed with the two bacterial species of similar MIC could be explained by a differential inoculum effect. Consequently, MICs determined on 10⁵ CFU inocula were not predictive of the differences in antibiotic efficacies against high bacterial inocula of closely related bacterial strains. These results could stimulate further investigations on bacterial species-specific antibiotic doses in a clinical setting.     

Secondary structure prediction of ITS rRNA region and molecular phylogeny: an integrated approach for the precise speciation of <Emphasis Type="Italic">Muscodor</Emphasis> species

Muscodor is a non-sporulating, volatile organic compounds producing endophytic fungi that has been extensively explored as a bio-fumigant and bio-preservative. Novel species of this genus have been mainly identified using ITS sequences. However, the ITS hyper-variability hinders the creation of reproducible alignments and stable phylogenetic trees. Conserved structural data of the ITS region represents as a vital auxiliary information for accurate speciation of fungi. In the present study, secondary structural data of ITS1, 5.8S, and ITS2 region of all Muscodor species were generated using LocaRNA web server. The predicted secondary structural data displayed greater variability in ITS1 region in comparison to ITS2. The structural data of all sequences exhibited characteristic conserved features of eukaryotic rRNA. Evolutionary conserved motifs were found among all 5.8S and ITS2 sequences. Profile neighbor joining (PNJ) tree based on combined sequence-structural information of ITS region was generated in ProfDists. The PNJ tree resolved into four major groups whereby M. fengyangenesis and M. albus species formed monophyletic clades. However, three M. albus species along with other Muscodor species emerged as sister branches to the existing clades, thereby, improving the precision of phylogenetic analysis for identification of novel species of Muscodor genus. Hence, the results indicated that structural analysis along with primary sequence information can provide new insights for precise identification of Muscodor species.     

Pathogenic Nontuberculous Mycobacteria Resist and Inactivate Cathelicidin: Implication of a Novel Role for Polar Mycobacterial Lipids

Nontuberculous mycobacteria (NTM) are a large group of environmental organisms with worldwide distribution, but only a relatively few are known to be pathogenic. Chronic, debilitating lung disease is the most common manifestation of NTM infection, which is often refractory to treatment. The incidence and prevalence of NTM lung disease are increasing in the United States and in many parts of the world. Hence, a more complete understanding of NTM pathogenesis will provide the foundation to develop innovative approaches to treat this recalcitrant disease. Herein, we demonstrate that several species of NTM show broad resistance to the antimicrobial peptide, cathelicidin (LL-37). Resistance to LL-37 was not significantly different between M. avium that contain serovar-specific glycopeptidolipid (GPL, M. avium^ssGPL) and M. avium that do not (M. avium^ΔssGPL). Similarly, M. abscessus containing non-specific GPL (M. abscessus^nsGPL(+)) or lacking nsGPL (M. abscessus^nsGPL(-)) remained equally resistant to LL-37. These findings would support the notion that GPL are not the components responsible for NTM resistance to LL-37. Unexpectedly, the growth of M. abscessus^nsGPL(-) increased with LL-37 or scrambled LL-37 peptide in a dose-dependent fashion. We also discovered that LL-37 exposed to NTM had reduced antimicrobial activity, and initial work indicates that this is likely due to inactivation of LL-37 by lipid component(s) of the NTM cell envelope. We conclude that pathogenic NTM resist and inactivate LL-37. The mechanism by which NTM circumvent the antimicrobial activity of LL-37 remains to be determined.     

Muscodor species- endophytes with biological promise

A novel fungal genus is described that produces extremely bioactive volatile organic compounds (VOC’s). The initial fungal isolate was discovered as an endophyte in Cinnamomum zeylanicum in a botanical garden in Honduras. This endophytic fungus was named Muscodor albus because of its odor and its white color. This fungus produces a mixture of VOC’s that are lethal to a wide variety of plant and human pathogenic fungi and bacteria. It also is effective against nematodes and certain insects. The mixture of VOC’s has been analyzed using GC/MS and consists primarily of various alcohols, acids, esters, ketones, and lipids. Final verification of the identity of the VOC’s 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 available VOC’s mimicked some but not all of the biological effects of the fungal VOC’s when tested against a wide range of fungal and bacterial pathogens. Other species and isolates of this genus have been found in various tropical forests in Australia, Bolivia, Ecuador, and Thailand. The most recent discovery is Muscodor crispans whose VOCs are active against many plant and human pathogens. Potential applications for “mycofumigation” by members of the Muscodor genus are currently being investigated and include uses for treating plant diseases, buildings, soils, agricultural produce and many more. This report will describe how the fungus was discovered, identified, and found potentially useful to agriculture, medicine and industry.     

Phylogeny of Morella rubra and Its Relatives (Myricaceae) and Genetic Resources of Chinese Bayberry Using RAD Sequencing

Phylogenetic relationships among Chinese species of Morella (Myricaceae) are unresolved. Here, we use restriction site-associated DNA sequencing (RAD-seq) to identify candidate loci that will help in determining phylogenetic relationships among Morella rubra, M. adenophora, M. nana and M. esculenta. Three methods for inferring phylogeny, maximum parsimony (MP), maximum likelihood (ML) and Bayesian concordance, were applied to data sets including as many as 4253 RAD loci with 8360 parsimony informative variable sites. All three methods significantly favored the topology of (((M. rubra, M. adenophora), M. nana), M. esculenta). Two species from North America (M. cerifera and M. pensylvanica) were placed as sister to the four Chinese species. According to BEAST analysis, we deduced speciation of M. rubra to be at about the Miocene-Pliocene boundary (5.28 Ma). Intraspecific divergence in M. rubra occurred in the late Pliocene (3.39 Ma). From pooled data, we assembled 29378, 21902 and 23552 de novo contigs with an average length of 229, 234 and 234 bp for M. rubra, M. nana and M. esculenta respectively. The contigs were used to investigate functional classification of RAD tags in a BLASTX search. Additionally, we identified 3808 unlinked SNP sites across the four populations of M. rubra and discovered genes associated with fruit ripening and senescence, fruit quality and disease/defense metabolism based on KEGG database.     

Selection shapes the patterns of codon usage in three closely related species of genus Misgurnus

Neutrality plots revealed that selection probably dominates codon bias, whereas mutation plays only a minor role, in shaping the codon bias in three loaches, Misgurnus anguillicaudatus, M. mohoity, and M. bipartitus. These three species also clearly showed similar tendencies in the preferential usage of codons. Nineteen, nine, and 14 preferred codon pairs and 179, 182, and 173 avoided codon pairs were also detected in M. anguillicaudatus, M. bipartitus, and M. mohoity, respectively, and the most frequently avoided type of cP3-cA1 dinucleotide in these species was nnUAnn. The expression-linked patterns of codon usage revealed that higher expression was associated with higher GC₃, lower ENC, and a smaller proportion of amino acids with high size/complexity (S/C) scores in these three species. These results elucidate selectively driven codon bias in Misgurnus species, and reveal the potential importance of expression-mediated selection in shaping the genome evolution of fish.     

A new species of Scolecobasidium associated with the sooty blotch and flyspeck complex on banana from China

Sooty blotch and flyspeck is caused by a complex of epiphytic fungi on diverse hosts. A fungus morphologically similar to Scolecobasidium humicola was isolated from a banana fruit exhibiting SBFS signs in Hainan, China. ITS and LSU sequences of rDNA corroborated that it was a Scolecobasidium species. The results of LSU rDNA analysis support the interpretation that Ochroconis is synonymous with Scolecobasidium. Five new combinations, S. anomalum, S. calidifluminale, S. gallopavum, S. gamsii, and S. lascauxense, were proposed. A new species, S. musae, was described based on morphological characteristics and sequence comparison with previous species.     

Some Like It Fat: Comparative Ultrastructure of the Embryo in Two Demosponges of the Genus Mycale (Order Poecilosclerida) from Antarctica and the Caribbean

During embryogenesis, organisms with lecithotrophic indirect development usually accumulate large quantities of energetic reserves in the form of yolk that are necessary for larval survival. Since all sponges have lecithotrophic development, yolk formation is an ineludible step of their embryogenesis. Sponge yolk platelets have a wide range of morphological forms, from entirely lipid or protein platelets to a combined platelet showing both lipids and proteins and even glycogen. So far, there are no comparative studies on the nature and content of yolk in congeneric species of sponges inhabiting contrasting environments, which could have putative effects on the larval adaptation to environmental conditions. Here, we have taken advantage of the worldwide distribution of the sponge genus Mycale, in order to compare the embryogenesis and yolk formation in two species inhabiting contrasting latitudinal areas: M. acerata from Antarctic waters and M. laevis from the Caribbean. We have compared their brooded embryos and larvae using scanning and transmission electron microscopy, and calculated their energetic signatures based on the nature of their yolk. While the general morphological feature of embryos and larvae of both species were very similar, the main difference resided in the yolk nature. The Antarctic species, M. acerata, showed exclusively lipid yolk, whereas the Caribbean species, M. laevis, showed combined platelets of lipids and proteins and less frequently protein yolk platelets. The larvae of M. acerata were estimated to possess a two-fold energetic signature compared to that of M. laevis, which may have important ecological implications for their survival and for maintaining large population densities in the cold waters of the Southern Ocean.