Bacterial endosymbionts protect beneficial soil fungus from nematode attack.

Fungi of the genus Mortierella occur ubiquitously in soils where they play pivotal roles in carbon cycling, xenobiont degradation, and promoting plant growth. These important fungi are, however, threatened by micropredators such as fungivorous nematodes, and yet little is known about their protective tactics. We report that Mortierella verticillata NRRL 6337 harbors a bacterial endosymbiont that efficiently shields its host from nematode attacks with anthelmintic metabolites. Microscopic investigation and 16S ribosomal DNA analysis revealed that a previously overlooked bacterial symbiont belonging to the genus Mycoavidus dwells in M. verticillata hyphae. Metabolic profiling of the wild-type fungus and a symbiont-free strain obtained by antibiotic treatment as well as genome analyses revealed that highly cytotoxic macrolactones (CJ-12,950 and CJ-13,357, syn necroxime C and D), initially thought to be metabolites of the soil-inhabiting fungus, are actually biosynthesized by the endosymbiont. According to comparative genomics, the symbiont belongs to a new species (Candidatus Mycoavidus necroximicus) with 12% of its 2.2 Mb genome dedicated to natural product biosynthesis, including the modular polyketide-nonribosomal peptide synthetase for necroxime assembly. Using Caenorhabditis elegans and the fungivorous nematode Aphelenchus avenae as test strains, we show that necroximes exert highly potent anthelmintic activities. Effective host protection was demonstrated in cocultures of nematodes with symbiotic and chemically complemented aposymbiotic fungal strains. Image analysis and mathematical quantification of nematode movement enabled evaluation of the potency. Our work describes a relevant role for endofungal bacteria in protecting fungi against mycophagous nematodes.

Aposymbiosis of a Burkholderiaceae-Related Endobacterium Impacts on Sexual Reproduction of Its Fungal Host.

Bacterial endosymbionts inhabit diverse fungal lineages. Although the number of studies on bacteria is increasing, the mechanisms by which bacteria affect their fungal hosts remain unclear. We herein examined the homothallic isolate, Mortierella sugadairana YTM39, harboring a Burkholderiaceae-related endobacterium, which did not produce sexual spores. We successfully eliminated the bacterium from fungal isolates using ciprofloxacin treatment and asexual spore isolation for germinated asexual spores. Sexual spore formation by the fungus was restored by eliminating the bacterium from isolates. These results indicate that sexual reproduction by the fungus was inhibited by the bacterium. This is the first study on the sexual spore infertility of fungal hosts by endofungal bacteria.

A novel approach to determine generalist nematophagous microbes reveals Mortierella globalpina as a new biocontrol agent against Meloidogyne spp. nematodes.

Root-knot nematodes (RKN) such as Meloidogyne spp. are among the most detrimental pests in agriculture affecting several crops. New methodologies to manage RKN are needed such as efficient discovery of nematophagous microbes. In this study, we developed an in vitro high-throughput method relying on the free-living nematode Caenorhabditis elegans and the infection of those nematodes with a soil slurry containing a microbiome likely to house nematophagous microbes. Nematodes were monitored for presence of infection and sub-cultured repeatedly for the purpose of isolating pure cultures of the microbe responsible for conferring the nematicidal activity. Once soil microbes were confirmed to be antagonistic to C. elegans, they were tested for pathogenicity against Meloidogyne chitwoodi. Using this methodology, the fungal isolate Mortierella globalpina was confirmed to be pathogenic in vitro against M. chitwoodi by nematode trapping via hyphal adhesion to the cuticle layer, penetration of the cuticle layer, and subsequently digestion of its cellular contents. M. globalpina was also observed to reduce disease symptomology of RKNs in vivo via significant reduction of root-galls on tomato (Solanum lycopersicum var. Rutgers).

The Beneficial Root-Colonizing Fungus Mortierella hyalina Promotes the Aerial Growth of Arabidopsis and Activates Calcium-Dependent Responses That Restrict Alternaria brassicae-Induced Disease Development in Roots.

The endophytic fungus Mortierella hyalina colonizes the roots of Arabidopsis thaliana and stimulates growth and biomass production of the aerial parts but not of roots. An exudate fraction from the fungus induces rapid and transient cytoplasmic Ca2+elevation in the roots. The Ca2+ response does not require the well-characterized (co)receptors BAK1, CERK1, and FLS2 for pathogen-associated molecular patterns, and the Ca2+ channels GLR-2.4, GLR-2.5, and GLR-3.3 or the vacuolar TWO PORE CHANNEL1, which might be involved in cytoplasmic Ca2+ elevation. We isolated an ethyl-methane-sulfonate-induced Arabidopsis mutant that is impaired in this Ca2+ response. The roots of the mutant are impaired in M. hyalina-mediated suppression of immune responses after Alternaria brassicae infection, i.e., jasmonate accumulation, generation of reactive oxygen species, as well as the activation of jasmonate-related defense genes. Furthermore, they are more colonized by M. hyalina than wild-type roots. We propose that the mutant gene product is involved in a Ca2+-dependent signaling pathway activated by M. hyalina to suppress immune responses in Arabidopsis roots.

Prevalence and Intra-Family Phylogenetic Divergence of Burkholderiaceae-Related Endobacteria Associated with Species of Mortierella.

Endofungal bacteria are widespread within the phylum Mucoromycota, and these include Burkholderiaceae-related endobacteria (BRE). However, the prevalence of BRE in Mortierellomycotinan fungi and their phylogenetic divergence remain unclear. Therefore, we examined the prevalence of BRE in diverse species of Mortierella. We surveyed 238 isolates of Mortierella spp. mainly obtained in Japan that were phylogenetically classified into 59 species. BRE were found in 53 isolates consisting of 22 species of Mortierella. Among them, 20 species of Mortierella were newly reported as the fungal hosts of BRE. BRE in a Glomeribacter-Mycoavidus clade in the family Burkholderiaceae were separated phylogenetically into three groups. These groups consisted of a group containing Mycoavidus cysteinexigens, which is known to be associated with M. elongata, and two other newly distinguishable groups. Our results demonstrated that BRE were harbored by many species of Mortierella and those that associated with isolates of Mortierella spp. were more phylogenetically divergent than previously reported.

Synthesis of Indoleacetic Acid, Gibberellic Acid and ACC-Deaminase by Mortierella Strains Promote Winter Wheat Seedlings Growth under Different Conditions.

The endogenous pool of phytoregulators in plant tissues supplied with microbial secondary metabolites may be crucial for the development of winter wheat seedlings during cool springs. The phytohormones may be synthesized by psychrotrophic microorganisms in lower temperatures occurring in a temperate climate. Two fungal isolates from the Spitzbergen soils after the microscopic observations and "the internal transcribed spacer" (ITS) region molecular characterization were identified as Mortierella antarctica (MA DEM7) and Mortierella verticillata (MV DEM32). In order to study the synthesis of indoleacetic acid (IAA) and gibberellic acid (GA), Mortierella strains were grown on media supplemented with precursor of phytohormones tryptophan at 9, 15 °C, and 20 °C for nine days. The highest amount of IAA synthesis was identified in MV DEM32 nine-day-culture at 15 °C with 1.5 mM of tryptophan. At the same temperature (15 °C), the significant promoting effect (about 40% root and shoot fresh weight) of this strain on seedlings was observed. However, only MA DEM-7 had the ACC (1-aminocyclopropane-1-carboxylate) deaminase activity with the highest efficiency at 9 °C and synthesized IAA without tryptophan. Moreover, at the same conditions, the strain was confirmed to possess the strong promoting effect (about 40% root and 24% shoot fresh weight) on seedlings. Both strains synthesized GA in all tested terms and temperatures. The studied Mortierella strains had some important traits that led them to be considered as microbial biofertilizers components, improving plant growth in difficult temperate climates.

Fungal Communities and Functional Guilds Shift Along an Elevational Gradient in the Southern Appalachian Mountains.

Nitrogen deposition alters forest ecosystems particularly in high elevation, montane habitats where nitrogen deposition is greatest and continues to increase. We collected soils across an elevational (788-1940 m) gradient, encompassing both abiotic (soil chemistry) and biotic (vegetation community) gradients, at eight locations in the southern Appalachian Mountains of southwestern North Carolina and eastern Tennessee. We measured soil chemistry (total N, C, extractable PO4, soil pH, cation exchange capacity [ECEC], percent base saturation [% BS]) and dissected soil fungal communities using ITS2 metabarcode Illumina MiSeq sequencing. Total soil N, C, PO4, % BS, and pH increased with elevation and plateaued at approximately 1400 m, whereas ECEC linearly increased and C/N decreased with elevation. Fungal communities differed among locations and were correlated with all chemical variables, except PO4, whereas OTU richness increased with total N. Several ecological guilds (i.e., ectomycorrhizae, saprotrophs, plant pathogens) differed in abundance among locations; specifically, saprotroph abundance, primarily attributable to genus Mortierella, was positively correlated with elevation. Ectomycorrhizae declined with total N and soil pH and increased with total C and PO4 where plant pathogens increased with total N and decreased with total C. Our results demonstrate significant turnover in taxonomic and functional fungal groups across elevational gradients which facilitate future predictions on forest ecosystem change in the southern Appalachians as nitrogen deposition rates increase and regional temperature and precipitation regimes shift.

Mortierella alpina CS10E4, an oleaginous fungal endophyte of Crocus sativus L. enhances apocarotenoid biosynthesis and stress tolerance in the host plant.

Crocus sativus is the only plant species which produces apocarotenoids like crocin, picrocrocin and safranal in significant amounts. These compounds impart organoleptic properties to saffron (dried stigmas of Crocus flower) making it world's costliest spice. Crocus apocarotenoids have tremendous medicinal properties as well. Effect of endophytes on Crocus apocarotenoid production and the molecular mechanism involved has not been reported so far. Here we studied the effect of an oleaginous fungal endophyte, Mortierella alpina CS10E4 on Crocus growth, apocarotenoid metabolism and tolerance to corm rot disease. The results demonstrated that there was a significant improvement in many morphological and physiological traits in endophyte treated Crocus plants including total biomass and size of corms, stigma biomass, number of apical sprouting buds, and number of adventitious roots. The endophyte also shifted metabolic flux towards enhanced production of apocarotenoids by modulating the expression of key pathway genes. Further, M. alpina CS10E4 enhanced tolerance to corm rot disease by releasing arachidonic acid which acts as conserved defense signal and induces jasmonic acid production in endophyte treated Crocus corms. This is first report on effect of a fungal endophyte on Crocus apocarotenoid metabolism and stress tolerance.

Comparative genomics of Mortierella elongata and its bacterial endosymbiont Mycoavidus cysteinexigens.

Endosymbiosis of bacteria by eukaryotes is a defining feature of cellular evolution. In addition to well-known bacterial origins for mitochondria and chloroplasts, multiple origins of bacterial endosymbiosis are known within the cells of diverse animals, plants and fungi. Early-diverging lineages of terrestrial fungi harbor endosymbiotic bacteria belonging to the Burkholderiaceae. We sequenced the metagenome of the soil-inhabiting fungus Mortierella elongata and assembled the complete circular chromosome of its endosymbiont, Mycoavidus cysteinexigens, which we place within a lineage of endofungal symbionts that are sister clade to Burkholderia. The genome of M. elongata strain AG77 features a core set of primary metabolic pathways for degradation of simple carbohydrates and lipid biosynthesis, while the M. cysteinexigens (AG77) genome is reduced in size and function. Experiments using antibiotics to cure the endobacterium from the host demonstrate that the fungal host metabolism is highly modulated by presence/absence of M. cysteinexigens. Independent comparative phylogenomic analyses of fungal and bacterial genomes are consistent with an ancient origin for M. elongata - M. cysteinexigens symbiosis, most likely over 350 million years ago and concomitant with the terrestrialization of Earth and diversification of land fungi and plants.

Proteomic analysis of Mortierella isabellina M6-22 during cold stress.

We aimed to gain a better understanding of cold adaption in Mortierella isabellina M6-22 by using proteomics approaches. The temperature range and optimal temperature for M6-22 growth were investigated, and composition changes in fatty acids were analyzed. Accompanied with the 2-D gel electrophoresis, MALDI-TOF/TOF-MS analysis was conducted to characterize alterations in protein profiling in M6-22 cultured at 30 °C for 24 h and 15 °C for another 24 h when compared with those cultured at 30 °C for 48 h. Gene Ontology (GO) cluster analysis was finally conducted for successfully identified proteins. M6-22 cells could grow well at temperatures ranging from 15 to 30 °C. As temperature decreased from 30 to 15 °C, LA and GLA significantly increased from 11.63 to 17.85 % and from 9.12 to 13.19 %, respectively, while oleic acid significantly decreased from 47.25 to 36.53 %. Proteomics analyses revealed 111 differentially expressed protein spots, among which 5 unique proteins (A38, A40, A47, A49 and A58), 29 up-regulated proteins and 10 down-regulated proteins were identified by MALDI-TOF/TOF-MS. GO enrichment analysis demonstrated that these proteins mainly involved in glycolytic pathway (A34 and A50), electron transport (A28), ATP production (A35 and B39) and protein modification (A38). A total of 44 differentially expressed proteins have been successfully identified in M. isabellina M6-22 cultured at 15 °C. These proteins may play important roles in cold adaption via regulation of ATP synthesis, activation of cold-adaptive proteins, degradation of needless protein, accumulation of PUFAs, etc.

Microbial ecology in a future climate: effects of temperature and moisture on microbial communities of two boreal fens.

Impacts of warming with open-top chambers on microbial communities in wet conditions and in conditions resulting from moderate water-level drawdown (WLD) were studied across 0-50 cm depth in northern and southern boreal sedge fens. Warming alone decreased microbial biomass especially in the northern fen. Impact of warming on microbial PLFA and fungal ITS composition was more obvious in the northern fen and linked to moisture regime and sample depth. Fungal-specific PLFA increased in the surface peat in the drier regime and decreased in layers below 10 cm in the wet regime after warming. OTUs representing Tomentella and Lactarius were observed in drier regime and Mortierella in wet regime after warming in the northern fen. The ectomycorrhizal fungi responded only to WLD. Interestingly, warming together with WLD decreased archaeal 16S rRNA copy numbers in general, and fungal ITS copy numbers in the northern fen. Expectedly, many results indicated that microbial response on warming may be linked to the moisture regime. Results indicated that microbial community in the northern fen representing Arctic soils would be more sensitive to environmental changes. The response to future climate change clearly may vary even within a habitat type, exemplified here by boreal sedge fen.

Efficient arachidonic acid-rich oil production by Mortierella alpina through a repeated fed-batch fermentation strategy.

Arachidonic acid (ARA)-rich oil production by Mortierella alpina is a long fermentation period needed process due to the low growth rate of the filamentous fungus used. This causes the low productivity of ARA-rich oil and hinders its industrial mass scale production. In the present study, different fed-batch strategies were conducted to shorten the fermentation period. The result showed that compared with the batch culture, the fermentation period was shortened from 7days to 5days with the productivity of ARA-rich oil increased from 0.9g/(L·d) to 1.3g/(L·d) by using the fed-batch fermentation strategy. Furthermore, repeated fed-batch fermentation strategy was adopted to achieve the purpose of continuous production. By using this strategy, the fermentation period was shortened from 40days to 26days in a four cycle repeated fed-batch fermentation. This strategy proved to be convenient and economical for ARA-rich oil commercial production process.

Improved lipid accumulation by morphology engineering of oleaginous fungus Mortierella isabellina.

Oleaginous fungi capable of accumulating a considerable amount of lipids are promising sources for lipid-based biofuel production. The specific productivities of filamentous fungi in submerged fermentation are often correlated with morphological forms. However, the relationship between morphological development and lipid accumulation is not known. In this study, distinct morphological forms of oleaginous fungus Mortierella isabellina including pellets of different sizes, free dispersed mycelia, and broken hyphal fragments were developed by additions of different concentrations of magnesium silicate microparticles. Different morphological forms led to different levels of lipid accumulation as well as different spatial patterns of lipid distribution within pellets/mycelial aggregates. Significant higher lipid content (0.75 g lipid/g cell biomass) and lipid yield (0.18 g lipid/g glucose consumed) were achieved in free dispersed mycelia than in pellets. Moreover, extracellular metabolite analysis showed that production of undesirable by-product malate was repressed in free dispersed mycelium form. Unveiling the desired morphological form of M. isabellina for lipid accumulation provided insights into molecular mechanism of lipid biosynthesis linked with morphological development, as well as design and optimization of bioprocess to produce lipid-based biofuels.

Production of the quorum-sensing molecules N-acylhomoserine lactones by endobacteria associated with Mortierella alpina A-178.

Gram-negative bacteria communicate with one another using N-acylhomoserine lactones (AHLs) as signaling molecules. This mechanism, known as quorum sensing (QS), is needed to develop pathogenicity, as well as symbiotic interactions with eukaryotic hosts, such as animals and plants. Increasing evidence indicates that certain bacteria, namely endobacteria, also inhabit fungal cells and establish symbiotic relationships with their hosts. However, it has not been clear whether bacterial QS acts in developing the relationships. Here we describe the isolation and identification of N-heptanoylhomoserine lactone and N-octanoylhomoserine lactone from the culture broth of the zygomycete fungus Mortierella alpina A-178. This suggested the presence of endobacteria in the fungus, as was confirmed by PCR, fluorescence in situ hybridization, and transmission electron microscopy. Two major bands obtained by PCR-denaturing gradient gel electrophoresis showed sequence identity to genes in the ß-proteobacterium Castellaniella defragrans (100 %) and the Gram-positive bacterium Cryobacterium sp. (99.8 %). The production of AHLs depended on the presence of endobacteria and was induced in response to the increase in the concentration of AHLs, suggesting that the bacterium conducts AHL-mediated QS in the fungus. This paper is the first to report the production of AHLs by endofungal bacteria and raises the possibility that QS plays roles in the development of fungus-endobacterium symbiosis.

Assessment of alcohol percentage test for fungal surface hydrophobicity measurement.

AIM: To determine whether assessing the penetration of solutions with different concentrations of ethanol (alcohol percentage test: APT) on fungal surfaces is effective in characterization of hydrophobicity on fungal surfaces. METHODS AND RESULTS: APT and contact angle (CA) measurements were conducted on nine hydrophobic and two hydrophilic fungal strains from the phyla of Ascomycota, Basidiomycota and Zygomycota. There was a strong positive correlation (R(2) = 0.95) between the APT and CA measurements from eight of the nine hydrophobic stains (four pathogenic and mycotoxigenic Fusarium taxa, one melanosporaceous biotrophic taxon, Alternaria sp, Penicillium aurantiogriseum and Cladosporium cladosporioides). Hydrophilic control strains, Mortierella hyalina and Laccaria laccata, had CAs <90 degrees and no measurable degree of hydrophobicity using the APT method. CONCLUSIONS: The APT method was effective in measuring the degree of hydrophobicity and can be conducted on different zones of fungal growth. SIGNIFICANCE AND IMPACT OF THE STUDY: Characterization of fungal surface hydrophobicity is important for understanding of its particular role and function in fungal morphogenesis and pathogenesis. APT is a simple method that can be utilized for fungal hydrophobicity measurements when CA cannot be measured because of obscured view from aerial mycelia growth.

Detection of betaproteobacteria inside the mycelium of the fungus Mortierella elongata.

Microscopic and molecular analyses showed the presence of endobacteria inside the mycelia of four out of twelve nitrous oxide (N(2)O)-producing fungal isolates identified as Mortierella elongata. The 16S rRNA gene was successfully amplified with DNA extracted directly from the endobacterium-containing fungal strains and all sequences were related to that of Candidatus Glomeribacter gigasporarum in the family Burkholderiaceae. Bacterial endotoxin was detected in the endobacterium-positive fungal strains but only trace levels were found in endobacterium-negative strains. No significant relationship was found between the fungal N(2)O-producing activity and the presence of endobacteria.

Single cell oil (SCO) production by Mortierella isabellina grown on high-sugar content media.

Mortierella isabellina cultivated in nitrogen-limited media presented remarkable cell growth (up to 35.9 g/l) and high glucose uptake even with high initial sugar concentrations (e.g. 100 g/l) in media. After nitrogen depletion, significant fat quantities were accumulated inside the fungal mycelia (50-55%, wt/wt oil in dry biomass), resulting in a notable single cell oil production of 18.1 g/l of culture medium. Total dry biomass and lipid yields presented greatly increased values (0.34 and 0.17 g respectively per gram of glucose consumed). The microbial lipid produced contained gamma-linolenic acid (GLA) at a concentration of 3.5+/-1.0%, wt/wt, which corresponded to 16-19 mg GLA per gram of dry microbial mass and a maximum concentration of 0.801 g GLA per liter of culture medium.

Sexual reactions in Mortierellales are mediated by the trisporic acid system.

Several species of Mortierella (Mortierellales, Zygomycota) were examined for substances regulating their sexual reactions. Compounds isolated from both mated and single growing Mortierella strains were purified by thin layer chromatography. Some of these compounds showed UV absorbance-characteristics similar to those of trisporoids, a group of compounds involved in sexual regulation in Mucorales. A compound with a 4-dihydromethyltrisporate-like absorbance spectrum was detected. To test for the interspecific sexual responses typically induced by trisporoids, the compounds extracted from Mortierella spp. were tested against the Mucorales Mucor mucedo and Phycomyces blakesleeanus and were found to induce sexual reactions in both tester strains. A gene encoding 4-dihydromethyltrisporate dehydrogenase was identified in several Mortierella species and the activity of the gene product was shown using a histochemical assay. We suggest that the regulation of sexual processes by trisporoids is common to both Mucorales and Mortierellales and may be more widespread within the Zygomycota.