Novel and highly diverse fungal endophytes in soybean revealed by the consortium of two different techniques

Fungal endophytes were isolated from the leaves of soybean cultivars in Brazil using two different isolation techniques — fragment plating and the innovative dilution-to-extinction culturing — to increase the species richness, frequency of isolates and diversity. A total of 241 morphospecies were obtained corresponding to 62 taxa that were identified by analysis of the internal transcribed spacer (ITS) of the ribosomal DNA (rDNA). The Phylum Ascomycota predominated, representing 99% and 95.2% of isolates in the Monsoy and Conquista cultivars, respectively, whereas the Phylum Basidiomycota represented 1% and 4.8% of isolates, respectively. The genera Ampelomyces, Annulohypoxylon, Guignardia, Leptospora, Magnaporthe, Ophiognomonia, Paraconiothyrium, Phaeosphaeriopsis, Rhodotorula, Sporobolomyces, and Xylaria for the first time were isolated from soybean; this suggests that soybean harbours novel and highly diverse fungi. The yeasts genera Rhodotorula and Sporobolomyces (subphylum Pucciniomycotina) represent the Phylum Basidiomycota. The species richness was greater when both isolation techniques were used. The diversity of fungal endophytes was similar in both cultivars when the same isolation technique was used except for Hill’s index, N₁. The use of ITS region sequences allowed the isolates to be grouped according to Order, Class and Phylum. Ampelomyces, Chaetomium, and Phoma glomerata are endophytic species that may play potential roles in the biological control of soybean pathogens. This study is one of the first to apply extinction-culturing to isolate fungal endophytes in plant leaves, thus contributing to the development and improvement of this technique for future studies.     

Isolation of sulfate‐reducing bacteria from deep sediment layers of the pacific ocean

Bacterial populations exist at great depths in marine sediments, but little is known about the type and characteristics of organisms in this unique bacterial environment. Cascadia Margin sediments from the Pacific Ocean have deep bacterial activity and bacterial populations, which are stimulated around a gas hydrate zone (215–225 m below sea floor [mbsf]). Bacterial sulfate reduction is the dominant anaerobic process within these sediments, and the depth distribution of sulfate‐reducing activity corresponds with distributions of viable sulfate‐reducing bacteria (SRB). Anaerobically stored sediments from this site were used to isolate sulfate‐reducing bacteria using a temperature‐gradient system, elevated pressure and temperatures, different media, and a range of growth substrates. A variety of enrichments on lactate were obtained from 0.5 and 222 mbsf, with surprisingly more rapid growth from the deeper sediments. The temperature range of enrichments producing strong growth from 222 mbsf was markedly wider than those from the near surface sediment (15–45°C and 9–19°C, respectively). This presumably reflects a temperature increase in deeper sediments. Only a few of these enrichments were successfully isolated due to very slow or no growth on subculture, despite the use of a wide range of different media and growth conditions. Psychrophilic and mesophilic sulfate‐reducing isolates were obtained from 0.5 m depth. As the minimum growth temperature of the mesophile (probably a Desulfotomaculum sp.) was above the in situ temperature of 3°C, it must have been present in the sediment as spores. A larger number of isolates (23) was obtained from 222 mbsf, and these barophilic SRB were closely related (based on 16S rRNA gene analysis), but not identical to, Desulfovibrio profundus, recently isolated from deep sediments from the Japan Sea. Bacteria related to D. profundus may be widespread in deep marine sediments.     

Microbial stratification in deeply buried marine sediment reflects changes in sulfate/methane profiles

We examined sediments collected at Ocean Drilling Program (ODP) Leg 201 Site 1229 on the Peru Margin for microbial populations throughout the sediment column. Heterotrophic cultivation from these sediments yielded numerous colonies from various depths, including 49 bacterial isolates. At ODP Site 1229, there are significant interfaces of sulfate and methane, across which microbial cell numbers increase substantially. At these sulfate/methane transition zones (SMTZs), however, we observed a decrease in the success rate for the cultivation of bacterial colonies. Utilizing both direct plating and enrichment in different media, we cultivated isolates from the upper SMTZ around 30 m below seafloor (mbsf); however, similar attempts yielded no colonies from within the lower zone at 85 mbsf. The phylogenetic relationships of the 16S rRNA gene sequences for the isolates were determined and most were related to other organisms and sequences previously found in the subsurface belonging to the γ‐Proteobacteria, cytophaga–flavobacterium–bacteroides, high G + C Gram‐positives, and Firmicutes groups. The most diverse group of isolates from Site 1229 was found between the SMTZs at 50 mbsf. ODP Leg 201 Site 1228 was examined for comparison and yielded an additional 18 isolates from 16 to 179 mbsf that were similar to those found at Site 1229. Direct plating at Site 1228 also showed decreased colony formation in the area of sulfate/methane transition. Our results suggest that heterotrophic bacterial populations are affected by SMTZs in deeply buried sediment.     

Deep sequencing of mycovirus‐derived small RNAs from Botrytis species

RNA silencing is an ancient regulatory mechanism operating in all eukaryotic cells. In fungi, it was first discovered in Neurospora crassa, although its potential as a defence mechanism against mycoviruses was first reported in Cryphonectria parasitica and, later, in several fungal species. There is little evidence of the antiviral potential of RNA silencing in the phytopathogenic species of the fungal genus Botrytis. Moreover, little is known about the RNA silencing components in these fungi, although the analysis of public genome databases identified two Dicer‐like genes in B. cinerea, as in most of the ascomycetes sequenced to date. In this work, we used deep sequencing to study the virus‐derived small RNA (vsiRNA) populations from different mycoviruses infecting field isolates of Botrytis spp. The mycoviruses under study belong to different genera and species, and have different types of genome [double‐stranded RNA (dsRNA), (+)single‐stranded RNA (ssRNA) and (–)ssRNA]. In general, vsiRNAs derived from mycoviruses are mostly of 21, 20 and 22 nucleotides in length, possess sense or antisense orientation, either in a similar ratio or with a predominance of sense polarity depending on the virus species, have predominantly U at their 5′ end, and are unevenly distributed along the viral genome, showing conspicuous hotspots of vsiRNA accumulation. These characteristics reveal striking similarities with vsiRNAs produced by plant viruses, suggesting similar pathways of viral targeting in plants and fungi. We have shown that the fungal RNA silencing machinery acts against the mycoviruses used in this work in a similar manner independent of their viral or fungal origin.     

Succession of fungi colonizing porous and compact limestone exposed to subtropical environments

Little is known about the dynamics of succession of fungi on limestone exposed in subtropical environments. In this study, the colonization of experimental blocks of compact and porous limestone by a fungal community derived from natural biofilms occurring on Structure X from the archaeological site of Becán (México), was studied using a cultivation-dependent approach after short-term (9 m) exposure in order to provide a preliminary insight of the colonization process under seminatural conditions. Microbial growth seen as the change of colour of stone surfaces to black/dark green was more abundant on the porous limestone. There was a fairly clear difference in microbial colonization between the onset of the experiment and the 6th month for both limestone types, but no significant increase in the colonization of coupons occurred between months 6 and 9. This could be related to the low rainfall expected for this period, corresponding to the dry season. A total of 977 isolates were obtained. From these, 138 sterile fungi were unidentified, 380 could only be assigned to the order Sphaeropsidales; the remaining isolates (459) were grouped into 27 genera and 99 different species. Nearly all detected fungal species belonged to the Ascomycota (90 %). Rare taxa (species represented by one to three isolates) included the recently described genus Elasticomyces, several species of genera Hyalodendron, Monodyctis, Papulospora, Curvularia, and Septoria. Other taxa were Minimedusa and Gliomastix luzulae, which have not been previously described for stone environments. Abundant fungi included several species of the common genera Cladosporium, Alternaria, and Taeniolella typical for a range of habitats. Succession of populations was observed for certain taxa, this shift in the composition of fungal communities was more evident in porous limestone. After 6 m of exposure, species of the genera Scolecobasidium, Hyalodendron, and Taeniolella were predominant, while after 9 m, the predominant species belonged to the genera Curvularia and Alternaria, particularly on porous stone. These results suggest that Curvularia and Alternaria replaced other fungi, due to a higher tolerance towards low levels of available water during the dry season. Higher levels of water within the porous stone, keep longer periods of microbial activity, minimizing the impact of desiccation. This study contributes to understand the diversity of fungal communities in stone surfaces in subtropical settings and the dynamics of colonization on limestone.     

Phylogenetic diversity of culturable fungi associated with the Hawaiian Sponges Suberites zeteki and Gelliodes fibrosa

Sponges are well documented to harbor large amounts of microbes. Both culture-dependent and molecular approaches have revealed remarkable bacterial diversity in marine sponges. Fungi are commonly isolated from marine sponges, yet no reports on phylogenetic diversity of sponge-inhabiting fungi exist. In this report, we investigated the phylogenetic diversity of culturable fungi from the Hawaiian alien marine sponges Suberites zeteki and Gelliodes fibrosa. A total of 44 independent isolates were recovered from these two sponge species, representing 7 orders and 22 genera of Ascomycota. The majority (58%) of fungal isolates from S. zeteki resided in the Pleosporales group, while the predominant isolates (52%) from G. fibrosa were members of the Hypocreales group. Though differing in fungal species composition and structure, culturable communities of these two sponges displayed similar phylogenetic diversity. At the genus level, only two genera Penicillium and Trichoderma in the Eurotiales and Hypocreales orders, respectively, were present in both sponge species. The other genera of the fungal isolates were associated with either S. zeteki or G. fibrosa. Some of these fungal genera had been isolated from sponges collected in other marine habitats, but more than half of these genera were identified for the first time in these two marine sponges. Overall, the diversity of culturable fungal communities from these two sponge species is much higher than that observed in studies of marine sponges from other areas. This is the first report of phylogenetic diversity of marine sponge-associated fungi and adds one more dimension to our current understanding of the phylogenetic diversity of sponge-symbiotic microbes.     

中国南海部分深海沉积物真菌多样性及其抗菌活性

【目的】从南海4个站位的深海沉积物中分离真菌,揭示其多样性并测定抗菌活性。【方法】使用4种培养方法和8种培养基,从12个深海沉积物样本中分离培养真菌,通过菌落形态观察和ITS序列系统发育分析进行鉴定。采用滤纸片扩散法和生长速率法分别测试真菌小量发酵液粗浸膏的抗细菌和抗真菌活性。【结果】共分离到125株纯培养真菌,基于形态和ITS序列分析,排重后得到18个种类型,这些真菌可以划分到12个属,大多数属于子囊菌门(Ascomycota),只有2株属于担子菌门(Basidiomycota)。4个站位可培养真菌多样性具有差异性。抑菌活性筛选显示,大多数真菌具有较好的抑菌活性;链格孢属(Alternaria)、青霉属(Penicillium)、匐柄霉属(Stemphylium)这几个属的真菌表现出对多种指示细菌有抑制作用,尤其是Alternaria tenuissima DN09、Alternaria alternata DN14和Penicillium chrysogenum DN16对G~+和G~–细菌均表现出抑制作用。【结论】本研究揭示了南海深海沉积物可培养真菌多样性和抑菌活性,为进一步利用深海沉积物来源真菌奠定了基础。     

Species‐Specific PCR‐Based Assay for Identification and Detection of Phomopsis (Diaporthe) azadirachtae Causing Die‐Back Disease in Azadirachta indica

Die‐back disease caused by Phomopsis (Diaporthe) azadirachtae is the devastating disease of Azadirachta indica. Accurate identification of P. azadirachtae is always problematic due to morphological plasticity and delayed appearance of conidia. A species‐specific PCR‐based assay was developed for rapid and reliable identification of P. azadirachtae by designing a species‐specific primer‐targeting ITS region of P. azadirachtae isolates. The assay was validated with DNA isolated from different Phomopsis species and other fungal isolates. The PCR assay amplified 313‐bp product from all the isolates of P. azadirachtae and not from any other Phomopsis species or any genera indicating its specificity. The assay successfully detected the pathogen DNA in naturally and artificially infected neem seeds and twigs indicating its applicability in seed quarantine and seed health testing. The sensitivity of the assay was 100 fg when genomic DNA of all isolates was analysed. The PCR‐based assay was 92% effective in comparison with seed plating technique in detecting the pathogen. This is the first report on the development of species‐specific PCR assay for identification and detection of P. azadirachtae. Thus, PCR‐based assay developed is very specific, rapid, confirmatory and sensitive tool for detection of pathogen P. azadirachtae at early stages.     

Ectomycorrhizal fungi of Pinus pinea L. in northeastern Spain

 Although Pinus pinea L. is an important forest species in the Mediterranean region, few reports exist on its ectomycorrhizal associates. Sixty isolates, obtained from fungal sporocarps collected in mixed forests of P. pinea in Catalonia (northeastern Spain), were tested for ectomycorrhiza formation on containerized P. pinea seedlings when applied as mycelial inoculum produced in peat-vermiculite. A total of 17 isolates, in 8 genera (Amanita, Hebeloma, Laccaria, Lactarius, Pisolithus, Rhizopogon, Scleroderma and Suillus), formed ectomycorrhizas and the percentages of mycorrhizal short roots varied among isolates and species from 13% to 89%. Some of these fungi are cited for the first time in association with P. pinea. The results indicate further fungal candidates for controlled inoculation of P. pinea seedlings in the nursery. Accepted: 29 October 1998     

Assessment of bioaerosols at a concentrated dairy operation

Increased bioaerosol loadings in downwind plumes from concentrated animal feeding operations (CAFOs) may increase the risk for allergy and infection in humans. In this study, we monitored airborne concentrations of culturable bacteria and fungi at upwind (background) and downwind sites at a 10,000 milking cow dairy over the course of a year. The average bacterial concentrations at the upwind site were 8.4 × 10³ colony forming units (CFU) m⁻³ and increased to 9.9 × 10⁵ CFU m⁻³ at the downwind edge of the cattle lots, decreasing to 6.3 × 10⁴ CFU m⁻³ 200 m farther downwind. At the same sites, the average fungal concentrations were 515, 945, and 1,010 CFU m⁻³, respectively. Significant correlations between the ambient weather conditions and airborne fungal and bacterial concentrations were identified. Sequence analysis of PCR-amplified DNA from bacterial clones and fungal isolates revealed genus and species level differences between upwind and downwind sites. Although we could not cultivate gram-negative bacteria, bacterial clones at downwind sites identified as being gram-negative matched with the following genera: Acinetobacter, Bradyrhizobium, Escherichia, Idiomarina, Methylobacterium, Ralstonia, and Novosphingobium. Fungal isolates from downwind matched with the following genera: Acremonium, Alternaria, Ascomycte, Aspergillus, Basidiomycete, Cladosporium, Davidiella, Doratomyces, Emericella, Lewia, Onygenales, Penicillium, Rhizopus, and Ulocladium. None of the bacterial and fungal sequence matches were affiliated with genera and species known to be pathogenic to humans. Overall, the data suggest that exposure to bioaerosols in the downwind environment decreases with increasing distance from the open-lot dairy.     

Evaluation of reported sediment samples from 20 Ma using a molecular phylogenetic approach: comment on Liu et al. (2017)

Liu et al. reported the cultivation and DNA sequencing of 69 fungal isolates (Ascomycota and Basidiomycota) from ancient subseafloor sediments, suggesting that they represent living fungal populations that have persisted for over 20 million years. Because these findings could bring about a paradigm shift in our understanding of the spatial breadth of the deep subsurface biosphere as well as the longevity of ancient DNA, it is extremely important to verify that their samples represent pure ancient fungi from 20 million years ago without contamination by modern species. For this purpose, we estimated the divergence times of Dikarya (Ascomycota + Basidiomycota) and Mucoromycota fungi assuming that the fungal isolates were actually sampled from 20 Ma (mega-annum) sediments and evaluated the validity of the sample ages. Using this approach, we estimate that the age of the last common ancestor of Dikarya and Mucoromycota fungi greatly exceeds the age of the Earth. Our finding emphasizes the importance of using reliable approaches to confirm the dating of ancient samples.     

Enrichment,isolation and characterization of fungi tolerant to 1‐ethyl‐3‐methylimidazolium acetate

Aims: This work aimed to characterize microbial tolerance to 1‐ethyl‐3‐methylimidazolium acetate ([C2mim][OAc]), an ionic liquid that has emerged as a novel biomass pretreatment for lignocellulosic biomass. Methods and Results: Enrichment experiments performed using inocula treated with [C2mim][OAc] under solid and liquid cultivation yielded fungal populations dominated by Aspergilli. Ionic liquid‐tolerant Aspergillus isolates from these enrichments were capable of growing in a radial plate growth assay in the presence of 10% [C2mim][OAc]. When a [C2mim][OAc]‐tolerant Aspergillus fumigatus strain was grown in the presence of switchgrass, endoglucanases and xylanases were secreted that retained residual enzymatic activity in the presence of 20% [C2mim][OAc]. Conclusions: The results of the study suggest that tolerance to ionic liquids is a general property of the Aspergilli. Significance and Impact of the Study: Tolerance to an industrially important ionic liquid was discovered in a fungal genera that is widely used in biotechnology, including biomass deconstruction.     

Characterization of <Emphasis Type="Italic">Penicillium</Emphasis> Species by Ribosomal DNA Sequencing and BOX,ERIC and REP-PCR Analysis

The genus Penicillium is one of the largest and widely distributed fungal genera described to date. As a result, its taxonomic classification and species discrimination within this genus has become complicated. In this study, 52 isolates that belonged to the Penicillum genus and other related genera were characterized using two DNA-based methods: (i) analysis of the nucleotide sequences of internal transcribed spacers in ribosomal DNA and (ii) analysis of DNA fingerprints that were generated by polymerase chain reactions with specific primers for enterobacterial repetitive intergenic consensus (ERIC) and repetitive extragenic palindromic (REP) sequences, and BOX elements. Using both methods, Penicillium species were discriminated from other fungal genera. Furthermore, Penicillium species that include strains which are used as biocontrol agents, such as P. glabrum, P. purpurogenum, and P. oxalicum, could be distinguished from other Penicillium species using these techniques. Based on our findings, we propose that a polyphasic approach that includes analysis of the nucleotide sequences of ribosomal DNA and detecting the presence of highly conserved, repeated nucleotide sequences can be used to determine the genetic relationships between different Penicillium species. Furthermore, we propose that our results can be used as a start point to develop a strategy to monitor the environmental presence of particular strains of Penicillium species when they are used as biocontrol agents.     

Mycobiota and molecular detection of Aspergillus flavus and A. parasiticus aflatoxin contamination of Strawberry (Fragaria ananassa Duch.) fruits

Strawberry fungi were isolated from fresh fruits and juice on the two types of media (Sabouraud dextrose agar, SDA and potato-dextrose agar, PDA) at 28 °C. Nineteen fungal species belong to 12 genera were isolated from fruits and juice on both isolation media. The most common fungal genera and species were Aspergillus flavus, A. niger, Mucor racemosus, Neurospora crassa, Penicillium chrysogenum, Rhizopus stolonifer and Trichoderma harzianum. Twenty A. flavus and A. parasitics isolates were assayed for their abilities to produce aflatoxins. The concentration of aflatoxins ranged between 25.8–75.2 and 23.6–71.1 ng/ml at 350 and 365 nm, respectively. Among A. flavus and A. parasiticus strains tested, aflatoxin B contributed 30–60% of total isolates. However, G type contributed 85–90%. The R_f values of B₁, B₂, G₁ and G₂ were 0.79, 0.61, 0.44 and 0.32, respectively. High-performance liquid chromatography analysis of extracts revealed the presence of aflatoxins with variable levels.     

Mycotoxin-producing potential of fungi associated with qat (Catha edulis) leaves in yemen

Forty-four fungal species belonging to 20 genera were isolated from 30 samples of qat leaves. The most frequent genera wereAspergillus, Alternaria, Penicillium, andCladosporium followed byFusarium, Drechslera, Chœtomium, andMucor. The most prevalent species in above genera wereAspergillus niger, A. flavus, A fumigatus, Alternaria alternata, Penicillium chrysogenum, P. citrinum, Cladosporium cladosporioides, andFusarium verticillioides. From these fungi, 17 species (39%) related to 7 genera (35%) proved to be true endophytes. Eleven out of 75 isolates were mycotoxigenic.A. alternata produced alternariol and alternariol monomethyl ether whereasA. flavus produced aflatoxins B₁ and B₂. Ochratoxin A, sterigmatocystin, citrinin and T-2 toxin were produced byA. ochraceus, A. versicolor, P. citrinum andF. oxysporum, respectively. The presence of such toxigenic fungi associated with qat leaves is considered to be a threat to public health.     

An Internet‐based platform for the estimation of outcrossing potential between cultivated and Chilean vascular plants

A national‐scale study of outcrossing potential within Chilean vascular flora was conducted using an upgraded algorithm, which adds parameters such as pollinator agents, climate, and geographic conditions. Datasets were organized and linked in a Web platform ( www.flujogenico.cl ), in which the development of a total outcrossing potential (TOP) predictor was formulated. The TOP predictor is the engine in the Web platform, which models the effect of a type of agricultural practice on others (coexistence calculation mode) and on the environment (biodiversity calculation mode). The scale for TOP results uses quintiles in order to define outcrossing potential between species as “very low,” “low,” “medium,” “high,” or “very high.” In a coexistence analysis considering 256 species (207 genera), the 10 highest TOP values were for genera Citrus, Prunus, Trifolium, Brassica, Allium, Eucalyptus, Cucurbita, Solanum, Lollium, and Lotus. The highest TOP for species in this analysis fell at “high” potential, 4.9% of the determined values. In biodiversity mode, seven out of 256 cultivated species (2.7%) were native, and 249 (97.3%) corresponded to introduced species. The highest TOP was obtained in the genera Senecio, Calceolaria, Viola, Solanum, Poa, Alstroemeria, Valeriana, Vicia, Atriplex, and Campanula, showing “high” potential in 4.9% of the values. On the other hand, 137 genetically modified species, including the commercial and pre‐commercial developments, were included and represented 100 genera. Among these, 22 genera had relatives (i.e., members of the same genus) in the native/introduced group. The genera with the highest number of native/introduced relatives ranged from one (Ipomea, Limonium, Carica, Potentilla, Lotus, Castanea, and Daucus) to 66 species (Solanum). The highest TOP was obtained when the same species were coincident in both groups, such as for Carica chilensis, Prosopis tamarugo, and Solanum tuberosum. Results are discussed from the perspective of assessing the possible impact of cultivated species on Chilean flora biodiversity. The TOP predictor ( http://epc.agroinformatica.cl/ ) is useful in the context of environmental risk assessment.     

Antimicrobial Activity and Biodiversity of Endophytic Fungi in <Emphasis Type="Italic">Dendrobium</Emphasis><Emphasis Type="Italic">devonianum</Emphasis> and <Emphasis Type="Italic">Dendrobium thyrsiflorum</Emphasis> from Vietman

Endophytic fungi are rich in orchids and have great impacts on their host plants. 53 endophytes (30 isolates from Dendrobium devonianum and 23 endophytic fungi from D. thyrsiflorum) were isolated, respectively, from roots and stems of Dendrobium species. All the fungi were identified by way of morphological and/or molecular biological methods. 30 endophytic fungi in D. devonianum were categorized into 11 taxa and 23 fungal endophytes in D. thyrsiflorum were grouped into 11 genera, respectively. Fusarium was the dominant species of the two Dendrobium species in common. Antimicrobial activity of ethanol extract of fermentation broth of these fungi was explored using agar diffusion test. 10 endophytic fungi in D. devonianum and 11 in D. thyrsiflorum exhibited antimicrobial activity against at least one pathogenic bacterium or fungus among 6 pathogenic microbes (Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus). Out of the fungal endophytes isolated from D. devonianum and D. thyrsiflorum, Phoma displayed strong inhibitory activity (inhibition zones in diameter >20 mm) against pathogens. Epicoccum nigrum from D. thyrsiflorum exhibited antibacterial activity even stronger than ampicillin sodium. Fusarium isolated from the two Dendrobium species was effective against the pathogenic bacterial as well as fungal pathogens. The study reinforced the assumption that endophytic fungi isolated from different Dendrobium species could be of potential antibacterial or antifungal resource.     

Fungal community associated with marine macroalgae from Antarctica

Filamentous fungi and yeasts associated with the marine algae Adenocystis utricularis, Desmarestia anceps, and Palmaria decipiens from Antarctica were studied. A total of 75 fungal isolates, represented by 27 filamentous fungi and 48 yeasts, were isolated from the three algal species and identified by morphological, physiological, and sequence analyses of the internal transcribed spacer region and D1/D2 variable domains of the large-subunit rRNA gene. The filamentous fungi and yeasts obtained were identified as belonging to the genera Geomyces, Antarctomyces, Oidiodendron, Penicillium, Phaeosphaeria, Aureobasidium, Cryptococcus, Leucosporidium, Metschnikowia, and Rhodotorula. The prevalent species were the filamentous fungus Geomyces pannorum and the yeast Metschnikowia australis. Two fungal species isolated in our study, Antarctomyces psychrotrophicus and M. australis, are endemic to Antarctica. This work is the first study of fungi associated with Antarctic marine macroalgae, and contributes to the taxonomy and ecology of the marine fungi living in polar environments. These fungal species may have an important role in the ecosystem and in organic matter recycling.     

Phylogenetics of Darwiniothamnus (Asteraceae: Astereae) – molecular evidence for multiple origins in the endemic flora of the Galápagos Islands

Aim The aims of this study were (1) to investigate whether the two growth forms of Darwiniothamnus Harling (Asteraceae) originated from the colonization of a single ancestor, (2) to identify the closest relative(s) of Darwiniothamnus, and (3) to review molecular phylogenies from other plant groups to infer the origin of Galápagos endemics. Location Darwiniothamnus is endemic to the Galápagos Islands. Methods All putative relatives of Darwiniothamnus plus 38 additional species were included. Nucleotide sequences of the internal transcribed spacers of the nuclear ribosomal DNA were used for Bayesian and parsimony analyses. Results Darwiniothamnus is polyphyletic. Two species (D. lancifolius (Hook. f.) Harling and D. tenuifolius (Hook. f.) Harling) are woody shrubs that usually grow to 1–2 m in height; they belong to a clade composed of species otherwise restricted to the Caribbean. These two species are sister to Erigeron bellidiastroides Griseb., a herbaceous species endemic to Cuba. The third species (D. alternifolius Lawesson & Adsersen) is a perennial herbaceous plant, woody at the base and reaching only up to 50 cm in height. It is sister to two Chilean (Coquimbo–Valparaiso region) species that also have a perennial herbaceous habit: E. fasciculatus Colla and E. luxurians (Skottsb.) Solbrig. They are placed in an assemblage restricted to South America. The review of previous molecular phylogenetic studies revealed that two of the endemic genera and endemic species of three non‐endemic genera have their closest relatives in South America. Endemic species belonging to three non‐endemic genera have sister species in North America or the West Indies. One endemic genus and endemic species in three non‐endemic genera have sister taxa with a widespread continental distribution, or their molecular phylogenies yielded equivocal results. Main conclusions The flora of Galápagos has affinities with both North America (including the Antilles) and South America. Darwiniothamnus exhibits both patterns: two species of this genus are sister to a taxon endemic to Cuba, supporting a connection between the Cocos plate and the West Indies; the third species, D. alternifolius, provides a link with the Coquimbo–Valparaiso region, suggesting a biogeographical connection between the Nazca plate and southern South America.     

Isolation of Bacterial Antagonists of Aspergillus flavus from Almonds

Bacteria were isolated from California almond orchard samples to evaluate their potential antifungal activity against aflatoxin-producing Aspergillus flavus. Fungal populations from the same samples were examined to determine the incidence of aflatoxigenic Aspergillus species. Antagonistic activities of the isolated bacterial strains were screened against a nonaflatoxigenic nor mutant of A. flavus, which accumulates the pigmented aflatoxin precursor norsolorinic acid (NOR) under conditions conducive to aflatoxin production. Using solid and liquid media in coculture assays, 171 bacteria isolated from almond flowers, immature nut fruits, and mature nut fruits showed inhibition of A. flavus growth and/or inhibition of NOR accumulation. Bacterial isolates were further characterized for production of extracellular enzymes capable of hydrolyzing chitin or yeast cell walls. Molecular and physiological identification of the bacterial strains indicated that the predominant genera isolated were Bacillus, Pseudomonas, Ralstonia, and Burkholderia, as well as several plant-associated enteric and nonenteric bacteria. A set of 20 isolates was selected for further study based on their species identification, antifungal phenotypes, and extracellular enzyme production. Quantitative assays using these isolates in liquid coculture with a wild-type, aflatoxin-producing A. flavus strain showed that a number of strains completely inhibited fungal growth in three different media. These results indicate the potential for development of bacterial antagonists as biological control agents against aflatoxigenic aspergilli on almonds.