Alterations in the Transcriptome of Rye Plants Following the Microdochium nivale Infection: Identification of Resistance/Susceptibility-Related Reactions Based on RNA-Seq Analysis

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.

Quantitative Trait Loci and Candidate Genes Associated with Cold-Acclimation and Microdochium nivale Tolerance/Susceptibility in Winter Triticale (x Triticosecale)

Tolerance to pink snow mold caused by Microdochium nivale appears after a cold-hardening period and it is an essential, genotype-dependent, complex quantitative trait for the wintering of triticale (x Triticosecale) and other cereals. Despite long-term studies, a marker for the selection of the tolerant genotypes is still insufficiently recognized. Chlorophyll fluorescence has been reported as a sensitive indicator of stress effects on photosynthesis and can be used to predict plant tolerance. In this study, the genomic regions (QTLs) associated with the level of winter triticale seedlings damage caused by M. nivale infection as well as photosynthesis quantum efficiency and chlorophyll a fluorescence parameters were identified in seedlings of mapping population of 89 doubled haploids lines (DHs) derived from F1 hybrid of cv. ‘Hewo’ and cv. ‘Magnat’ accompanied with the genetic map consisting of 20 linkage groups with a total map length 4997.4 cm. Independent experiments performed in controlled conditions revealed 13 regions identified by a composite interval mapping, located on 7A, 1B, 2B, 6B, 7B, 3R, 5R, and 6R linkage groups and related to the PI, PIABS, TRo/CS, ABS/CS, ABS/CSm, ABS/RC, and Qy values as well as M. nivale tolerance T and susceptibility level P expressed by the seedling damage index. Additionally, candidate genes were in silico identified with the sequence position on wheat (2B and 7B) and rye (5R) chromosomes, where relevant QTL regions were found. The most important candidate genes indicated for M. nivale tolerance of cold-hardened triticale seedlings include those coding: sterol 3-beta-glucosyltransferase UGT80A2-like, transcription factor NAI1-like, and flavonol3-sulfotransferase-like proteins on chromosomes 2B and 5R.

First report of Microdochium nivale and M. majus associated with brown foot rot of wheat in China

Microdochium nivale and M. majus not only cause seedling blight of wheat (Triticum aestivum L.) in cold dry soils, but also cause foot rot and ear blight of wheat under favorable conditions (Haigh et al. 2009). In May 2017, 2019, and 2020, a serious foot rot of wheat with an incidence of 92%, 45%, and 51% was observed in the field in Xiangcheng County (33.43° N, 114.84° E), Tanghe County (32.43° N, 112.66° E), and Linzhou City (36.13° N, 113.75° E), Henan Province, respectively. The serious brown lesions of the lower leaf sheaths is visible. The pathogens were isolated from brown leaf sheaths on potato dextrose agar (PDA) after being surface-sterilized (70% EtOH for 30 s followed by 3% NaClO for 1.5 min) and rinsed three times in sterile distilled water. After 5 d, mycelia were transferred to fresh PDA, and nine representative isolates (G17ZK2-1, G17ZK2-2, G17ZK2-3, g19TH10-4, g19TH10-5, g19TH10-6, G20LZ1-6, G20LZ1-7, and G20LZ1-8) were further purified by hyphal tipping. Species were identified based on morphological characteristics, and sequence analysis of partial sequences of the translation elongation factor-1α (TEF), the RNA polymerase II subunit (RPB2) gene and β-tubulin gene (Abdelhalim et al. 2020). Among the nine isolates, six isolates belonged to M. majus, three isolates belonged to M. nivale. Sequences of six isolates M. majus and three isolates M. nivale were deposited in GenBank with accession numbers MW428296-MW428298, MZ734119-MZ734121and MZ734139-MZ734141(TEF), MW384889, MW428291, MW428292, MZ734203-MZ734205 and MZ734161-MZ734163(RPB2), MW428293-MW428295, MZ501004-MZ501006 and MZ501024-MZ501026 (β-tubulin). For all the genes, isolates revealed 98-100% similarity to M. majus and M. nivale accessions, respectively. Microscopy of the six M. majus isolates showed: the conidia were falcate, straight to curved, apex pointed or obtuse to subacute, lacking basal differentiation, with 1 to 6 septa, 3.6 to 5.0 × 15.0 to 30.5 μm (av.= 4.5 × 23.2; n = 60). The three M. nivale isolates showed: the conidia were hyaline, 1 to 3 septa, 2.4 to 4.4 × 11.9 to 26.0 μm (av.= 3.5 × 14.7; n = 60). Perithecia of M. majus are dark brown, globose, and 95.2 to 190.5 × 95.2 to 228.6 μm (av.= 144.4 ×152.5; n = 30). Asci are clavate, and 6.8 to 11.0 ×68.2 to 77.3 μm (av.= 8.6×72.0; n = 30), contain eight ascospores. Mature ascospores are ellipsoidal, and 3.8 to 4.9 ×11.5 to 19.2 μm (av.= 4.0 ×15.2; n = 30), with 1 to 3 septa. These morphological characteristics were consistent with previous descriptions of these two species (Glynn et al. 2005). For pathogenicity tests, mycelia of M. nivale and M. majus was prepared using the modified procedure of Zhang et al. (2015). Two-week-old healthy wheat seedlings (cv. Aikang 58) were inoculated using 1 mL of prepared mycelia to one seedling, which was sprayied on soil. Control seedlings were inoculated with 1 mL distilled water containing 0.2% gelatin. After 10 days under 15/10℃, 16h/8h, all the inoculated plants had developed brown spots; while control plants remained healthy. The pathogens were reisolated from inoculated plants and identified as M. nivale and M. majus based on morphological characteristics and molecular methods described above. Although there are reports of M. majus associated with brown foot rot of wheat in Anhui Province and M. nivale associated with seedling blight of oat in Gansu Province (Chen et al. 2021; Tai et al. 2019). To our knowledge, this is the first report of brown foot rot of wheat caused by M. nivale and M. majus in China.

Estimation of immunity of the winter grain varieties sown in the south of Russia to the pink snow mold pathogen (Microdochium nivale)

The purpose of the current study was to estimate immunity of the winter grain varieties sown in the south of Russia to the pink snow mold pathogen (Microdochium nivale (Fr.) Samuels & I.C. Hallett) in the sprouting phase. For resistance to M. nivale there have been studied 35 winter wheat varieties sown in the south of the Russian Federation, 19 winter barley varieties and 4 variety samples and 13 winter triticale varieties developed in the LLC “Agrostandart”, FSBSI RCG named after P.P. Lukyanenko, FSBSI FRC Kabardino-Balkarian Research Center of the RAS, FSBSI “ARC Donskoy”, FSBSI “North Caucasian FRSC”, FSBSI “FRAC”, NPO “KUBANZERNO”, FSBEI HE “KubSAU”. There has been substantiated a methodological approach to conducting research on immunological estimation of winter grain varieties in the laboratory conditions. The optimal temperature for the cultivation of the pathogen was +10/+15 °C (with a photoperiod of 12 hours). There was found that the required temperature to stimulate sporulation was +5 °C. The optimum temperature for the incubation period was +5 °C at 85% humidity. There has been established that the only winter wheat variety ‘Dolya’ had a very high resistance degree to pink snow mold; the varieties ‘Antonina’ and ‘Brigada’ had a high resistance degree; 21 varieties were classified as resistant. M. nivale resistance was demonstrated by 9 winter barley varieties and 3 variety samples (‘Versal’, ‘Iosif’, ‘KA-12’, ‘KA-5/KA-3’, ‘KA-5/KA-1’, ‘Karrera’, ‘Kondrat’, ‘Kubagro-1’, ‘Lazar’, ‘Master’, ‘Romans’, ‘Sarmat’). Among the studied winter triticale varieties, 4 varieties had a very high resistance degree (‘Argus’, ‘Slon’, ‘Tikhon’, ‘Ullubiy’) and 9 varieties had a high resistance degree to pink snow mold pathogen (‘Aznavur’, ‘Argo’, ‘Arioso’, ‘Valentin 90’, ‘Iliya’, ‘Sotnik’,’ Styuard’, ‘Forte’, ‘Khleborob’).

An Apoplastic Defensin of Wheat Elicits the Production of Extracellular Polysaccharides in Snow Mold

TAD1 (Triticum aestivum defensin 1) is a plant defensin specifically induced by low temperature in winter wheat. In this study, we demonstrated that TAD1 accumulated in the apoplast during cold acclimation and displayed antifungal activity against the pink snow mold fungi Microdochium nivale. When M. nivale was treated with TAD1, Congo red-stainable extracellular polysaccharides (EPS) were produced. The EPS were degradable by cellulase treatment, suggesting the involvement of β-1,4 glucans. Interestingly, when the fungus was treated with FITC-labeled TAD1, fluorescent signals were observed within the EPS layer. Taken together, these results support the hypothesis that the EPS plays a role as a physical barrier against antimicrobial proteins secreted by plants. We anticipate that the findings from our study will have broad impact and will increase our understanding of plant–snow mold interactions under snow.

The Effect of Foliar Application of an Amino Acid-Based Biostimulant on Lawn Functional Value

The purpose of this research was to assess the functional value of the “Super Trawnik” lawn mixture. The studies were carried out between 2017 and 2019 at the Experimental Station of the University of Agriculture in Krakow (50°07′ N, 20°05′ E), and the experimental factor was the AGRO-SORB® Folium, a biostimulant containing amino acids and applied at three doses: 1, 2, and 3 L·ha−1. Lawn visual quality was assessed on a 9-point scale, with 10–11 mowings at 4 cm during the growing period. An increase in the concentration of the stimulant applied as a spray resulted in a significant increase in its effectiveness; plants in plots with the highest dose of amino acid solution (Variant III) had the highest aesthetic and functional values. The AGRO-SORB® Folium reduced the occurrence of fungal diseases; compared to control plants, there was a 16% reduction of Fusarium patch (Microdochium nivale) infection and a 20% reduction of Dreschlera leaf spot (Drechslera siccans). Satisfactory effects were also recorded on plots where the product was applied at a dose of 2 L·ha−1 (Variant II). Those plots had more favourably rated turf, with higher resistance of plants to Fusarium patch by 12% and to Dreschlera leaf spot by 20% compared to control.

First Report of Seedling Blight of Oat (Avena sativa) Caused by Microdochium nivale in China

Oat (Avena sativa) is extensively planted as a fodder crop on the vast ranges of northern and northwestern China, and it has become an important supplementary feed for grazing livestock (Yang et al. 2010). Microdochium nivale has been reported associated with seedling blight in many temperate regions (Imathiu et al. 2010) and the damage can result in serious loss of oat production. In August 2018, a serious seedling blight of oat (cv. Baiyan 7; about 30-day-old) was observed in the field in Shandan County, Zhangye City, Gansu Province (38.22° N, 101.22° E). More than 20% of oat plants were severely affected. Symptoms included leaf chlorosis and wilt. The root systems of infected plants were black and severely rotted, often with only a small amount of fine root remaining after removal from the soil. Twenty isolations were made from blackened roots on potato dextrose agar (PDA) and five isolations (TM-1, TM-2, TM-3, TM-4 and TM-5) were further purified by a single-spore method (Choi et al. 1999). Each isolate was identical based on preliminary molecular analyses of their DNA sequences of ITS by blast in the NCBI GenBank. The representative isolate TM-2 was selected for sequencing of the RNA polymerase II subunit (RPB2) gene. The isolated colonies were grown on PDA and formed colonies of approximately 62 mm (diameter) in 5 days at 25 ± 1 °C. Colonies exhibited entire margins, the color varied from white to pale yellow, and the sparse aerial mycelium were villous-floccose and cottony. The conidia were falcate, straight to curved, apex pointed or obtuse to subacute, lacking basal differentiation, 0-3-septate, most one-septate, 2.2 to 3.1 × 12.3 to 22.6μm (av.= 2.8 ×17.6; n=50). These morphological characteristics were consistent with previous descriptions of Microdochium (Zhang et al. 2010). Molecular identity was confirmed by sequencing partial sequences of ITS gene (ITS1 and ITS4 primers) (White et al. 1990) and RPB2 regions (RPB2-5f2 and RPB2-7cr) (O’Donnell et al. 2010). Sequences were deposited in GenBank under accessions MN428647 (RPB2) and MN428646 (ITS). Blast search revealed that both of the ITS and RPB2 sequences to be 99% similar to the corresponding sequences of M. nivale(CBS 116205) accession numbers KP859008.1 and KP859117.1. For pathogenicity tests, millet seed-based inoculum of M. nivale was prepared using a modified procedure of Fang et al. (2011). Three-week-old healthy oat seedlings of cv. Baiyan 7 were transplanted into potting mix containing millet seed-based inoculum of M. nivale at a rate of 3%. Control seedlings for comparison were transplanted into pots containing uninoculated potting mix. After 10 days, all the inoculated plants had developed seedling blight symptoms and that were similar to those observed in the field; while control plants remained healthy. The pathogen was reisolated from inoculated plants and identified as M. nivale based on morphological characteristics and the molecular methods described above. To our knowledge, this is the first report of seedling blight of oat caused by M. nivale in China.