It's time to shed some light on the importance of fungi in neonatal intensive care units: what do we know about the neonatal mycobiome?

The 21st century, thanks to the development of molecular methods, including DNA barcoding, using Sanger sequencing, and DNA metabarcoding, based on next-generation sequencing (NGS), is characterized by flourishing research on the human microbiome. Microbial dysbiosis is perceived as a new pathogenetic factor for neonatal diseases. Fungi are crucial, but neglected, components of the neonatal microbiome, which, despite their low abundance, significantly impact morbidity and mortality rates of premature infants hospitalized in Neonatal Intensive Care Units (NICUs). The neonatal mycobiome's composition and effect on health remain poorly studied research areas. Our knowledge about neonatal mycobiome, composed of limited genera, is mainly based on research on the bacterial microbiome. We presume it is influenced by clinical factors, including prematurity, antibiotic therapy, and type of delivery. Understanding these risk factors may be useful in prevention strategies against dysbiosis and invasive fungal infections. Despite the methodological challenges resulting from the biology of the fungal cell, this topic is an attractive area of research that may contribute to more effective treatment, especially of newborns from risk groups. In this mini review, we discuss the current state of knowledge, research gaps, study difficulties, and future research directions on the neonatal mycobiome, concerning potential future clinical applications.

Constellation of the endophytic mycobiome in spring and winter wheat cultivars grown under various conditions.

The mycobiome is an integral component of every living organism. Among other fungi associated with plants, endophytes are an interesting and favorable group of microorganisms, but information regarding them is still largely unknown. Wheat is the most economically significant and essential crop for global food security, which is exposed to a range of abiotic and biotic stresses. Profiling plants' mycobiomes can help in sustainable, chemical-reducing wheat production. The main objective of this work is to understand the structure of endogenous fungal communities in winter and spring wheat cultivars growing under different growth conditions. Further, the study attempted to investigate the effect of host genotype, host organs and plant growth conditions on the composition and distribution of fungi in wheat plant tissues. Comprehensive, high throughput analyzes of the diversity and community structure of the wheat mycobiome were performed, complemented by the simultaneous isolation of endophytic fungi, resulting in candidate strains for future research. The findings of the study revealed that the type of plant organs and growth conditions influence the wheat mycobiome. It was also assessed that fungi representing the genera Cladosporium, Penicillium, and Sarocladium form the core mycobiome of Polish spring and winter wheat cultivars. The coexistence of both symbiotic and pathogenic species in the internal tissues of wheat was also observed. Those commonly considered beneficial for plants can be used in further research as a valuable source of potential biological control factors and/or biostimulators of wheat plant growth.

Expression Patterns of miR398, miR167, and miR159 in the Interaction between Bread Wheat (Triticum aestivum L.) and Pathogenic Fusarium culmorum and Beneficial Trichoderma Fungi.

Bread wheat (Triticum aestivum L.) is an agronomically significant cereal cultivated worldwide. Wheat breeding is limited by numerous abiotic and biotic stresses. One of the most deleterious factors is biotic stress provoked by the Fusarium culmorum fungus. This pathogen is a causative agent of Fusarium root rot and Fusarium head blight. Beneficial fungi Trichoderma atroviride and T. cremeum are strong antagonists of mycotoxigenic Fusarium spp. These fungi promote plant growth and enhance their tolerance of negative environmental conditions. The aim of the study was to determine and compare the spatial (in above- and underground organs) and temporal (early: 6 and 22 hpi; and late: 5 and 7 dpi reactions) expression profiles of three mature miRNAs (miR398, miR167, and miR159) in wheat plants inoculated with two strains of F. culmorum (KF846 and EW49). Moreover, the spatial expression patterns in wheat response between plants inoculated with beneficial T. atroviride (AN35) and T. cremeum (AN392) were assessed. Understanding the sophisticated role of miRNAs in wheat-fungal interactions may initiate a discussion concerning the use of this knowledge to protect wheat plants from the harmful effects of fungal pathogens. With the use of droplet digital PCR (ddPCR), the absolute quantification of the selected miRNAs in the tested material was carried out. The differential accumulation of miR398, miR167, and miR159 in the studied groups was observed. The abundance of all analyzed miRNAs in the roots demonstrated an increase in the early and reduction in late wheat response to F. culmorum inoculation, suggesting the role of these particles in the initial wheat reaction to the studied fungal pathogen. The diverse expression patterns of the studied miRNAs between Trichoderma-inoculated or F. culmorum-inoculated plants and control wheat, as well as between Trichoderma-inoculated and F. culmorum-inoculated plants, were noticed, indicating the need for further analysis.

Fungi Inhabiting the Wheat Endosphere.

Wheat production is influenced by changing environmental conditions, including climatic conditions, which results in the changing composition of microorganisms interacting with this cereal. The group of these microorganisms includes not only endophytic fungi associated with the wheat endosphere, both pathogenic and symbiotic, but also those with yet unrecognized functions and consequences for wheat. This paper reviews the literature in the context of the general characteristics of endophytic fungi inhabiting the internal tissues of wheat. In addition, the importance of epigenetic regulation in wheat-fungus interactions is recognized and the current state of knowledge is demonstrated. The possibilities of using symbiotic endophytic fungi in modern agronomy and wheat cultivation are also proposed. The fact that the current understanding of fungal endophytes in wheat is based on a rather small set of experimental conditions, including wheat genotypes, plant organs, plant tissues, plant development stage, or environmental conditions, is recognized. In addition, most of the research to date has been based on culture-dependent methods that exclude biotrophic and slow-growing species and favor the detection of fast-growing fungi. Additionally, only a few reports of studies on the entire wheat microbiome using high-throughput sequencing techniques exist. Conducting comprehensive research on the mycobiome of the endosphere of wheat, mainly in the context of the possibility of using this knowledge to improve the methods of wheat management, mainly the productivity and health of this cereal, is needed.

Changes in root-associated fungal communities in Triticum aestivum ssp. spelta L. and Triticum aestivum ssp. vulgare L. under drought stress and in various soil processing.

Plant roots are inhabited by an enormous variety of microorganisms, including fungi, which can control the growth as well as regulate the health of the host plants. The mycobiome composition of the roots of wheat plants, especially spelt, under drought stress has been rarely investigated. Therefore, the aim of the present study was to examine the composition of fungal communities in the root endosphere and rhizosphere of three Triticum aestivum ssp. spelta L. cultivars and one Triticum aestivum ssp. vulgare L. cultivar, grown under drought and controlled conditions in different soil preparations. Culture-dependent fungal community profiling was performed to examine the impact of rhizocompartments (endosphere, rhizosphere), host genotype, watering status and different soil preparation on roots mycobiome structure. A total of 117 fungal strains, belonging to 22 genera, were found to colonize the internal and external parts of roots in T. aestivum ssp. spelta L. and T. aestivum ssp. vulgare L. cultivars. The results showed that the part of root and soil preparation type significantly determined the mycobiome composition of wheat roots.

Growth and Photosynthetic Activity of Selected Spelt Varieties (Triticum aestivum ssp. spelta L.) Cultivated under Drought Conditions with Different Endophytic Core Microbiomes.

The role of the microbiome in the root zone is critically important for plants. However, the mechanism by which plants can adapt to environmental constraints, especially water deficit, has not been fully investigated to date, while the endophytic core microbiome of the roots of spelt (Triticum aestivum ssp. spelta L.) grown under drought conditions has received little attention. In this study, we hypothesize that differences in the endophytic core of spelt and common wheat root microbiomes can explain the variations in the growth and photosynthetic activity of those plants, especially under drought conditions. Our greenhouse experimental design was completely randomized in a 2 × 4 × 3 factorial scheme: two water regime levels (well-watered and drought), three spelt varieties (T. aestivum ssp. spelta L.: 'Badenstern', 'Badenkrone' and 'Zollernspelz' and one wheat variety: T. aestivum ssp. vulgare L: 'Dakotana') and three mycorrhizal levels (autoclaved soil inoculation with Rhizophagus irregularis, control (autoclaved soil) and natural inoculation (non-autoclaved soil-microorganisms from the field). During the imposed stress period, relative water content (RWC), leaf chlorophyll fluorescence, gas exchange and water use efficiency (WUE) were measured. Microscopic observations of the root surface through fungi isolation and identification were conducted. Our results indicate that 'Badenstern' was the most drought tolerant variety, followed by 'Zollernspelz' and 'Badenkrone,' while the common wheat variety 'Dakotana' was the most drought sensitive. Inoculation of 'Badenstern' with the mycorrhizal fungi R. irregularis contributed to better growth performance as evidenced by increased whole plant and stalk dry matter accumulation, as well as greater root length and volume. Inoculation of 'Zollernspelz' with arbuscular mycorrhizal fungi (AMF) enhanced the photochemical efficiency of Photosystem II and significantly improved root growth under drought conditions, which was confirmed by enhanced aboveground biomass, root dry weight and length. This study provides evidence that AMF have the potential to be beneficial for plant growth and dry matter accumulation in spelt varieties grown under drought conditions.

Methylation Patterns of SOX3, SOX9, and WNT4 Genes in Gonads of Dogs with XX (SRY-Negative) Disorder of Sexual Development.

Ovotesticular or testicular disorder of sexual development in dogs with female karyotype and lack of SRY (XX DSD) is a common sexual anomaly diagnosed in numerous breeds. The molecular background, however, remains unclear, and epigenetic mechanisms, including DNA methylation, have not been studied. The aim of our study was comparative methylation analysis of CpG islands in promoters of candidate genes for XX DSD: SOX9, SOX3, and WNT4. Methylation studies were performed on DNA extracted from formalin-fixed/paraffin-embedded or frozen gonads from 2 dogs with ovotesticular and 2 dogs with testicular XX DSD as well as control females (n = 4) and males (n = 2). Bisulfite-converted DNA was used for CpG methylation analysis using quantitative pyrosequencing. Promoter regions of SOX9 and WNT4 showed similar CpG methylation in each group, ranging from 0 to 5.5% and from 39 to 74%, respectively. The SOX3 promoter showed significantly higher methylation in the ovotesticular XX DSD cases and the testicular XX DSD and control males, suggesting that SOX3 methylation may play a role in canine XX DSD pathogenesis.

Polymorphism of the CTNNB1 and FOXL2 Genes is not Associated with Canine XX Testicular/Ovotesticular Disorder of Sex Development.

78,XX testicular or ovotesticular disorder of sex development (DSD) is the most common sex anomaly in dogs, but its molecular background remains unknown. It was hypothesized that the causative mutation may reside in canine chromosome 23 (CFA23), where two genes playing a pivotal role in ovarian development (CTNNB1 and FOXL2) are located. The aim of our study was to search for polymorphism in both candidate genes in 15 DSD dogs (78,XX and a lack of the SRYgene) and 29 normal females. Altogether, 7 novel polymorphic variants were identified: 5 SNPs in CTNNB1 and 2 indels in the FOXL2 gene. The distribution of the identified variants was similar in the DSD and control dogs. Therefore, we concluded that the conducted research did not prove an association between these polymorphisms and canine testicular or ovotesticular XX DSD.

Testicular disorder of sex development in four cats with a male karyotype (38,XY; SRY-positive).

The molecular background of disorders of sex development (DSD) in cats is poorly recognized. In this study we present cytogenetic, molecular and histological analyses of four cats subjected for the analysis due to ambiguous external genitalia. Three cases, with rudimentary penises and an abnormal position of the urethral orifice, represented different types of hypospadias. The fourth case had a normal penis, a blind vulva and spermatogenetically active testes. Histological studies showed structures typical of testes, but spermatogenic activity was observed in two cats only. All the cats had a normal male chromosome complement (38,XY) and the Y-chromosome linked genes (SRY and ZFY) were also detected. Fluorescent in situ hybridization (FISH), with the use of the feline BAC probe harboring the SRY gene, excluded the possibility of chromosome translocation of the Y chromosome fragment carrying the SRY gene onto another chromosome. Sequencing of four candidate genes (SRY--sex determining region Y; AR--androgen receptor; SRD5A2--steroid-5-alfa reductase 2 and MAMLD1--mastermind-like domain containing (1) revealed one SNP in the SRY gene, one common polymorphism in exon 1 of the AR gene (tandem repeat of a tri-nucleotide motif--CAG), six polymorphisms (5 SNPs and 1 indel) in the SRD5A2 gene and one SNP in the MAMLD1 gene. Molecular studies of the candidate genes showed no association with the identified polymorphisms, thus molecular background of the studied DSD phenotypes remains unknown.

A lack of association between polymorphisms of three positional candidate genes (CLASP2 , UBP1, and FBXL2) and canine disorder of sexual development (78,XX; SRY -negative).

A disorder of sexual development (DSD) of dogs with a female karyotype, missing SRY gene, and presence of testicles or ovotestes is quite commonly diagnosed. It is suggested that this disorder is caused by an autosomal recessive mutation; however, other models of inheritance have not been definitely ruled out. In an earlier study it was hypothesized that the mutation may reside in a pericentromeric region of canine chromosome 23 (CFA23). Three positional candidate genes (CLASP2, UBP1, and FBXL2) were selected in silico in the search for polymorphisms in 7 testicular or ovotesticular XX DSD dogs, 8 XX DSD dogs of unknown cause (SRY-negative, with enlarged clitoris and unknown histology of gonads), and 29 normal female dogs as a control group. Among the 15 molecularly studied dogs with enlarged clitoris there were 3 new cases of testicular or ovotesticular XX DSD and 4 new cases of XX DSD with unknown cause (histology of the gonads unknown). Altogether, 11 (including 10 novel) polymorphisms in 5'- and 3'-flanking regions of the studied genes were found. The distribution analysis of these polymorphisms showed no association with the DSD phenotypes. Thus, it was concluded that the presence of the causative mutation for testicular or ovotesticular XX DSD in the pericentromeric region of CFA23 is unlikely.

Non-CpG hypermethylation in placenta of mutation-induced intrauterine growth restricted bovine foetuses.

The existence of non-CpG methylation in mammalian DNA has mainly been observed in embryonic stem cells, but its functional significance is uncertain. We found an age-dependent non-CpG hypermethylation in DMR at the 3' end of the MIMT1 in the placenta of intrauterine growth restricted foetuses in cattle. Data suggest that this DMR play a role in epigenetic regulation of the PEG3 domain.