Query:  isolate hitherto uncultured fungi

title: State of the Art in the Culture of the Human Microbiota: New Interests and Strategies
abstract: The last 5 years have seen a turning point in the study of the gut microbiota with a rebirth of culture dependent approaches to study the gut microbiota. High throughput methods have been developed to study bacterial diversity with culture conditions aimed at mimicking the gut environment by using rich media such as YCFA (yeast extract, casein hydrolysate, fatty acids) and Gifu anaerobic medium in an anaerobic workstation, as well as media enriched with rumen and blood and coculture, to mimic the symbiosis of the gut microbiota.  The last 5 years have seen a turning point in the study of the gut microbiota with a rebirth of culture dependent approaches to study the gut microbiota. High throughput methods have been developed to study bacterial diversity with culture conditions aimed at mimicking the gut environment by using rich media such as YCFA (yeast extract, casein hydrolysate, fatty acids) and Gifu anaerobic medium in an anaerobic workstation, as well as media enriched with rumen and blood and coculture, to mimic the symbiosis of the gut microbiota. Other culture conditions target phenotypic and metabolic features of bacterial species to facilitate their isolation. Preexisting technologies such as next generation sequencing and flow cytometry have also been utilized to develop innovative methods to isolate previously uncultured bacteria or explore viability in samples of interest. These techniques have been applied to isolate CPR (Candidate Phyla Radiation) among other, more classic approaches. Methanogenic archaeal and fungal cultures present different challenges than bacterial cultures. Efforts to improve the available systems to grow archaea have been successful through coculture systems. For fungi that are more easily isolated from the human microbiota, the challenge resides in the identification of the isolates, which has been approached by applying matrix assisted laser desorption ionization time of flight mass spectrometry technology to fungi. Bacteriotherapy represents a nonnegligible avenue in the future of medicine to correct dysbiosis and improve health or response to therapy. Although great strides have been achieved in the last 5 years, efforts in bacterial culture need to be sustained to continue deciphering the dark matter of metagenomics, particularly CPR, and extend these methods to archaea and fungi.

title: Purpureocillium roseum sp. nov. A new ocular pathogen for humans and mice resistant to antifungals
abstract: Infectious keratitis is the main cause of preventable blindness worldwide, with about 1.5 2.0 million new cases occurring per year. This inflammatory response may be due to infections caused by bacteria, fungi, viruses or parasites. Fungal keratitis is a poorly studied health problem.

title: Discovery of divided RdRp sequences and a hitherto unknown genomic complexity in fungal viruses
abstract: By identifying variations in viral RNA genomes, cutting edge metagenome technology has potential to reshape current concepts about the evolution of RNA viruses. This technology, however, cannot process low homology genomic regions properly, leaving the true diversity of RNA viruses unappreciated. To overcome this technological limitation we applied an advanced method, Fragmented and Primer Ligated Double stranded (ds) RNA Sequencing (FLDS) to screen RNA viruses from 155 fungal isolates, which allowed us to obtain complete viral genomes in a homology independent manner. We created a high quality catalog of 19 RNA viruses (12 viral species) that infect Aspergillus isolates. Among them, nine viruses were not detectable by the conventional methodology involving agarose gel electrophoresis of dsRNA, a hallmark of RNA virus infections. Segmented genome structures were determined in 42% of the viruses. Some RNA viruses had novel genome architectures; one contained a dual methyltransferase domain and another had a separated RNA dependent RNA polymerase (RdRp) gene. A virus from a different fungal taxon (Pyricularia) had an RdRp sequence that was separated on different segments, suggesting that a divided RdRp is widely present among fungal viruses, despite the belief that all RNA viruses encode RdRp as a single gene. These findings illustrate the previously hidden diversity and evolution of RNA viruses, and prompt reconsideration of the structural plasticity of RdRp. By highlighting the limitations of conventional surveillance methods for RNA viruses, we showcase the potential of FLDS technology to broaden current knowledge about these viruses.  The development of RNA seq technology has facilitated the discovery of RNA viruses in all types of biological samples. However, it is technically difficult to detect highly novel viruses using RNA seq. We successfully reconstructed the genomes of multiple novel fungal RNA viruses by screening host fungi using a new technology, FLDS. Surprisingly, we identified two viral species whose RNA dependent RNA polymerase (RdRp) proteins were separately encoded on different genome segments, overturning the commonly accepted view of the positional unity of RdRp proteins in viral genomes. This new perspective on divided RdRp proteins should hasten the discovery of viruses with unique RdRp structures that have been overlooked, and further advance current knowledge and understanding of the diversity and evolution of RNA viruses.

title: Sequence based Detection and Identification Biodiversity of Uncultivated Fungi in Soils.
abstract: Detection and identification biodiversity of uncultured agriculture soil is not an easy one of the major problems that participate to this difficulty is the fastidious nature of the majority of unculturable soil fungal species .In this study we have need for identification of uncultivated soil groups, fundamentally rely on isolation of DNA from soil with a polymerase chain reaction and sequencing to diagnose and classify uncultivated soil fungal isolates .In the present study, two fungal specific primer sets (NSIF/LRIFr and NSIF/ITS4r) were used to amplify partial sequences of fungal rRNA gene included ITS sequences. Five partial sequences of five fungal species were aligned through the BLASTN phylogenetic analysis against NCBI database which revealed higher identities with Fungal sp. uncultured fungus clone, Fusarium oxysporum, Clonostachys rosea and uncultured fungal clone. The blast tree showed that fungal species are neighbor joined, these results suggest that the uncultivated soil fungi is complex and diverse. Distinctly beneficial in ways and integration of various molecular methods (DNA based) has shed light on these facets but a synthesis of both techniques is necessary for a finer image and a deeper understanding. Therefore, the taxonomic complexity of unculturable communities of fungal organisms needs to be unraveled.

title: The Role of Non Mycorrhizal Fungi in Germination of the Mycoheterotrophic Orchid Pogoniopsis schenckii Cogn.
abstract: Endophytic fungi are those that inhabit within organs and tissues without causing damage, while mycorrhizal fungi develop hyphal complexes called pelotons within cortical cells of orchid roots. Although abundant and frequent in all plant organs, the role of endophytic fungi has been neglected in relation to orchid's early development. Pogoniopsis schenckii Cogn. is an aclorophyllated and mycoheterotrophic (MH) orchid. This study aimed at i) investigating the endophytic fungal community in organs of P. schenckii and its mycorrhizal fungi associated; ii) evaluating the ability of isolated fungus in the in vitro germination of the seeds of the species, and iii) describing the development of P. schenckii protocorm, analyzing the ultrastructure of the infected cells. Six genera of fungi were isolated and identified through the partial sequencing of the internal transcribed spacer region, all belonging to the phylum Ascomycota. Also, Tulasnellaceae was identified through uncultured technique as potentially mycorrhizal in this MH orchid. Some isolates of the genera Trichoderma, Fusarium, and especially Clonostachys presented germinative potential on P. schenckii seeds, causing rupture of the external tegument. The protocorms showed complete absence of peloton formation, but fungal hyphae were clearly observed within living cells. This is the first report of germination of a MH and aclorophyllated orchid species stimulated by the presence of non mycorrhizal endophytic fungi isolated from fruits and roots of the same species.

title: Neo clerodane diterpenoids from Conyza pyrrhopappa Sch.Bip. ex A.Rich
abstract: Two hitherto unknown neo clerodane type diterpenoids along with twelve known compounds have been isolated from Conyza pyrrhopappa Sch.Bip. ex A.Rich, a medicinal plant traditionally used across tropical Africa to relieve fever. The structures of isolates have been elucidated by a combination of spectroscopic techniques. The crude extract and the isolated compounds were evaluated in the Hela S3 cell line and in a panel of microorganisms (bacteria and fungi) at concentrations up to 50 ug/mL. The new compounds were inactive while the pentamethylated flavonoids showed low to significant activity against the cancer cell line used. However, none of the samples showed any activity against the tested microorganisms at this concentration. The present manuscript is the first investigation of the cytotoxicity of phytochemicals and extract from C. pyrrhopappa.

title: Culturable plant pathogenic fungi associated with sugarcane in southern China
abstract: Sugarcane (Saccharum officinarum) is one of the oldest crops cultivated by mankind. Numerous fungal taxa have been reported from this host, although most were not identified beyond genus level. In this study, we explored 31 sampling sites in Guangxi and Guangdong Provinces (China) and collected 370 diseased samples from leaves and roots of sugarcane, from which 762 strains were isolated. Our preliminary analysis based on internal transcribed spacer (ITS) sequences suggested that these isolates belonged to 143 species in 51 genera, but we could not assign 129 strains. Bipolaris, Chaetomium, Curvularia, Phoma and Nigrospora represented the top five most common genera identified, while 27 rare genera comprised only one identified species. In this study, we chose above mentioned common genera for in depth morphological observations and multi locus analyses, in order to identify these strains to species level. In this paper, we described one new genus, 32 new species, and reported 19 new records for China and the asexual morph of Chaetomium olivaceum. Hitherto, this is the most comprehensive study with molecular identification and illustration of fungi associated with sugarcane, which greatly improves our understanding of culturable mycota associated with this host.

title: The Arabidopsis leucine rich repeat receptor kinase MIK2 is a crucial component of pattern triggered immunity responses to Fusarium fungi
abstract: Fusarium is a genus of fungi causing severe economic damage in many crop species exemplified by Fusarium Head Blight of wheat or Panama Disease of banana. Plants sense immunogenic patterns (termed elicitors) at the cell surface contributing to disease resistance via the activation of pattern triggered immunity (PTI) Knowledge of such elicitors or corresponding plant immunity components is largely lacking for Fusarium species. We describe a new peptide elicitor fraction present in several Fusarium spp. which elicits canonical PTI responses in Arabidopsis thaliana but depends on a currently unknown perception mechanism. We therefore employed a forward genetics screen using Arabidopsis plants containing a cytosolic calcium reporter (apoaequorin) to isolate fere (Fusarium Elicitor Reduced Elicitation) mutants. The fere1 mutant showed impaired PTI marker responses to an enriched elicitor fraction derived from Fusarium oxysporum but normal responses to other fungal elicitors. We mapped the causal mutation to the receptor like kinase MIK2 (MALE DISCOVERER1 INTERACTING RECEPTOR LIKE KINASE 2) with a hitherto undescribed role in PTI pathways but documented functions in other cell surface signalling pathways. The strength of the phenotype in fere1 and independent mik2 mutants supports that MIK2 is a new key component in sensing Fusarium. Fusarium elicitor responses also partially depend on PTI signalling components known for other cell surface elicitor responses such as BAK1, BIK1, PBL1, FERONIA, LLG1 and RBOHD. This shows that Arabidopsis senses Fusarium by a novel receptor complex at the cell surface that feeds into common PTI pathways and positions MIK2 as a central player that potentially integrates plant endogenous signals with biotic and abiotic stress responses.

title: Management of root rotting fungi and root knot nematode with endophytic fluorescent Pseudomonas associated with Salvadora species
abstract: Root diseases caused by soilborne plant pathogens are responsible for the losses of billions of dollars in agricultural crops annually. The biological control of soilborne pathogens with microbial antagonists is gaining popularity in the crop protection system, due to the adverse effects of chemicals. In this study 40 isolates of fluorescent Pseudomonas were isolated from roots, shoots and leaves of Salvadora persica L. and S. oleoides Decne. Most of them showed strong suppressive effect on root rotting fungi Macrophomina phaseolina, Rhizoctonia solani, Fusarium solani and F. oxysporum In vitro. Significant nematicidal activity against root knot nematode (Meloidogyne javanica) was also observed by the cellfree culture filtrates of these isolates. Identification of six potential isolates was confirmed by using molecular biology tools. Out of which 3 were identified as Pseudomonas aeruginosa, one each as P. putida and P. monteilii, while one appeared as uncultured Pseudomonas. Experiments conducted on sunflower both in clay pots and field plots, test isolates of endophytic Pseudomonas were found effective in suppressing the root rot disease with improved plant growth as compared to untreated control. Efficacy of some isolates was found comparable with commercial fungicide carbendazim. Some isolates were also found effective in reducing the infestation of root knot nematode under field condition with improved flower weight.

title: Antagonistic yeasts from a salt lake region in Egypt: identification of a taxonomically distinct group of phylloplane strains related to Sporisorium
abstract: Non pathogenic yeasts antagonising microorganisms that cause pre and postharvest diseases of plants have been found in diverse habitats. Their practical applicability as biocontrol agents (BCAs) depends on the strength of their antagonistic activity and/or spectrum of sensitive target microorganisms. In this study, yeasts were isolated from the phylloplane and fruits of plants growing in the alkaline water lake region Wadi El Natrun, Egypt, and tested for antifungal and antibacterial activity. All phylloplane yeast isolates belonged to the Basidiomycota and most of them could antagonise at least certain test organisms. One group of isolates showing strong antagonism against almost all fungi and yeasts appears to represent a hitherto undescribed species distantly related to the smut genus Sporisorium. This is the first report of antagonistic activity in Sporisorium. The isolates assigned to Naganishia and Papiliotrema were more effective against bacteria. The broadest range and intensity of antagonism was observed in the fruit associated strains belonging to the ascomycetous species Wickerhamomyces subpelliculosus. The Wickerhamomyces strains are good broad spectrum BCA candidates, the Sporisorium strains could be used as efficient antifungal BCAs, whereas the Papiliotrema isolate can be exploited as an antibacterial biocontrol agent.