Snow mold is a severe plant disease caused by psychrophilic or psychrotolerant fungi, of which Microdochium species are the most harmful. A clear understanding of Microdochium biology has many gaps; the pathocomplex and its dynamic are poorly characterized, virulence factors are unknown, genome sequences are not available, and the criteria of plant snow mold resistance are not elucidated. Our study aimed to identify comprehensive characteristics of a local community of snow mold-causing Microdochium species colonizing a particular crop culture. By using the next-generation sequencing (NGS) technique, we characterized fungal and bacterial communities of pink snow mold-affected winter rye (Secale cereale) plants within a given geographical location shortly after snowmelt. Twenty-one strains of M. nivale were isolated, classified on the basis of internal transcribed spacer 2 (ITS2) region, and characterized by morphology, synthesis of extracellular enzymes, and virulence. Several types of extracellular enzymatic activities, the level of which had no correlations with the degree of virulence, were revealed for Microdochium species for the first time. Our study shows that genetically and phenotypically diverse M. nivale strains simultaneously colonize winter rye plants within a common area, and each strain is likely to utilize its own, unique strategy to cause the disease using “a personal” pattern of extracellular enzymes.

Winter rye is a versatile crop widely used for food and industry. Although rye is resistant to abiotic stressors and many phytopathogens, it is severely damaged by pink snow mold (SM)—a progressive disease caused by the psychrotolerant fungus Microdochium nivale under the snow cover or during prolonged periods of wet and cool conditions. Due to little use of the SM resistance sources in contemporary breeding, varieties with at least moderate resistance to SM are limited. Our study aimed to integrate field assessment under natural conditions and an artificially enriched infection background with laboratory techniques for testing rye accessions and selecting SM resistant sources for applied breeding programs and genetic research. We revealed valuable sources of SM resistance and split rye accessions, according to the level of the genetic divergence of the SM resistance phenotype. This allowed us to select the most distinct donors of the SM resistance, for their use as parental forms, to include novel variability sources in the breeding program for achieving high genetic variability, as well as enhanced and durable SM resistance, in progeny. The rye accessions analyzed here, and the suggested options for their use in breeding, are valuable tools for rye breeding.

Microdochium nivale is a progressive and devastating phytopathogen that causes different types of cereal crop and grass diseases that are poorly characterized at the molecular level. Although rye (Secale cereale L.) is one of the most resistant crops to most of the phytopathogens, it is severely damaged by M. nivale. The recent high-quality chromosome-scale assembly of rye genome has improved whole-genome studies of this crop. In the present work, the first transcriptome study of the M. nivale-infected crop plant (rye) with the detailed functional gene classification was carried out, along with the physiological verification of the RNA-Seq data. The results revealed plant reactions that contributed to their resistance or susceptibility to M. nivale. Phytohormone abscisic acid was shown to promote plant tolerance to M. nivale. Flavonoids were proposed to contribute to plant resistance to this pathogen. The upregulation of plant lipase encoding genes and the induction of lipase activity in M. nivale-infected plants revealed in our study were presumed to play an important role in plant susceptibility to the studied phytopathogen. Our work disclosed important aspects of plant-M. nivale interactions, outlined the directions for future studies on poorly characterized plant diseases caused by this phytopathogen, and provided new opportunities to improve cereals breeding and food security strategies.

To cause plant diseases, phytopathogenic fungi use numerous extracellular enzymes, among which, the phenoloxidases (POs) seem underestimated for the pathogens of non-woody plants. Our study aimed to (1) compare extracellular PO activities (lignin peroxidase, Mn peroxidase, laccase, and tyrosinase) in differentially virulent strains (inhabiting winter rye in a single field) of the phytopathogenic species, Microdochium nivale; (2) check whether these activities are responsive to host plant metabolites; and (3) search for correlations between the activities, lignin-decomposing capacity, and virulence. All strains displayed all four enzymatic activities, but their levels and dynamics depended on the particular strain. The activities displayed the hallmarks of co-regulation and responsiveness to the host plant extract. No relationships between the virulence of strains and levels of their extracellular PO activities or lignin-degrading capacity were revealed. We consider that different strains may rely on different POs for plant colonization, and that different POs contribute to the “uniqueness” of the enzymatic cocktails that are delivered into host plant tissues by different virulent strains of M. nivale. Our study supports the hypothesis of the differential behavior of closely related M. nivale strains, and discusses an important role of POs in the interactions of phytopathogens with herbaceous plants.

The aim of the study was to analyze the resistance of 21 Microdochium nivale strains (fungi that cause pink snow mold in winter crops), living within a single area and a single crop (winter rye), to fungicides that differ in chemical nature and mechanisms of action as well as to test the interconnection between levels of virulence and fungicide resistance of strains. The virulence of M. nivale strains was determined on detached leaves of rye (Ogonek variety) plants as well as on whole plants grown under sterile conditions in vitro. The resistance of strains to fungicides (Provisor (a.s. azoxystrobin) and Ferazim (a.s. carbendazim)) was determined by inhibition of mycelium growth. As a result of the experiments, more strains (13) capable of growing in the presence of Provisor were revealed than Ferazim-resistant strains (2); however, Ferazim-resistant strains had a greater level of resistance (5-16% of growth inhibition) than Provisor-resistant strains (63-94 % of growth inhibition). A negative correlation (Spearman's correlation coefficient -0.604 and -0.532) between the level of virulence of M. nivale strains and the level of their susceptibility to Provisor was found. This evidently means that the acquisition of resistance to Provisor by M. nivale strains is accompanied by an increase in their virulence. No correlations were found between the fungicide resistance of M. nivale strains and their attribution to one or another phylogenetic group, to which the strains were assigned based on the nucleotide sequence of the ITS2 (internal transcribed spacer 2) region. The study shows that for choosing the fungicide application strategy, it is necessary to analyze phytopathogen populations for the presence of strains that are simultaneously characterized by high virulence and resistance to various fungicides and also take into account that the adaptation of fungi to certain fungicides can be accompanied by an increase in their virulence, which will negatively affect the phytopathological state of agrocenosis.

Measurement of the turnover rate of proteins, different metabolites and lipids in living organisms is important for the understanding of biochemical pathways and physiology studies. Such experiments can be performed by administering isotopically labeled substances (food or water) to the organism and measuring the amount of the isotopes in the endogenous compounds. Here, we administered 20% heavy water (D₂O) to a guinea pig for 156 days and regularly measured the deuterium uptake in C-H groups in the different compounds of blood, urine and feces using high-resolution mass spectrometry. We successfully measured the time required for reaching the maximum deuteration level for several classes of compounds: 10 days for blood lipids (PC, PE, TAG); 60 days for sterol derivatives, heme B and hemoglobin; and 70 days for stercobilin. Also, for those compounds, we measured the deuterium elimination time from the organism when deuterium administration was stopped. The turnover of lipids was also studied by administering deuterated oat leaves grown at 10% D₂O to the guinea pig. The analysis of blood revealed that triglycerides demonstrate the inclusion of the deuterium after 5 h. All experiments were performed on a single guinea pig that remained alive and in good health after all experiments. The current research demonstrates the possibility of using long-term D₂O administration for the investigation of metabolism.