Evaluation of Cytotoxicity and Mould Contamination of Selected Plants from Meadows Covered by the Agri-Environmental Program.

The aim of the study was the evaluation of selected species of meadow plants obtained from the first cut from the area covered by the agri-environmental program 'Natura 2000' in terms of the presence of cytotoxic compounds detected by the MTT test and the level of fungal contamination. The research was carried out on plant species that were evaluated differently in previously used methods for quality assessment of pasture feeds according to Klapp and Filipek. Twenty-six plant species were harvested in 2014 from meadows located in the valley of the Bydgoszcz Canal. Mycological examination of meadow plant samples was carried out according to PN-ISO 7954:1999. Cytotoxicity evaluation was performed using the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] test. Selected samples were also subjected to evaluation of the endophytes occurrence in grasses using PCR. Natural meadow positions included in the study were dominated by moulds belonging to Humicola spp., Alternaria spp., Cladosporium spp., Torula spp., Fusarium spp. and Mucor spp. The highest level of fungal contamination was observed for Carex acutiformis Ehrh. The most infested grasses were Deschampsia caespitosa (L.) P.Beauv., Festuca arundinacea Schreb. and Lolium perenne L. The MTT test showed that the most cytotoxic species were Arrhenatherum elatius (L.) P.Beauv. (IC50 1.563 mg/mL) and Ranunculus repens L. (IC50 3.125 mg/mL). Epichloë endophytes were detected in one of 13 examined grass samples. Our own research suggests that previously used feed quality assessments should be verified by introducing modern methods of molecular biology and instrumental analysis. Results of this study may broaden the knowledge of the causes of problems resulting from feeding of roughage, mainly from natural meadows, and help in creating new rankings of the feed value of meadow sward components.

Is the vertical transmission of Neotyphodium lolii in perennial ryegrass the only possible way to the spread of endophytes?

Endophytic fungi live their whole life within host tissues usually without any visible symptoms. Their vertical transmission (seed-plant-seed) has been well described and documented. However, horizontal transmission (plant-plant) needs more clarification. The aim of this study was to assess the extent to which endophytes move vertically in ecotypes of perennial ryegrass and whether there is evidence for the horizontal transmission of endophytes. Ecotypes from grasslands in Poland were collected in the form of living plants and used for vertical transmission analysis. Plants, the seed collected from these plants and plants grown from this collected seed were tested for endophytic infection. Provided that all produced seeds were viable and able to germinate and produce seedlings, the vertical transmission of Neotyphodium endophytes in perennial ryegrass ecotypes was nearly complete. For the horizontal transmission experiment, endophyte-hosting plants (E+) and endophyte-free plants (E-) of four cultivars were planted in the field in close proximity on small plots that were frequently mown. These studies revealed that after 7 months of growth next to E+ plants, the characteristic Neotyphodium spp. mycelia were found in E- plants, which was especially true for plants growing in close proximity to the infected plants. The occurrence of horizontal transmission of endophytes has not been previously demonstrated.

Production of phenolics and the emission of volatile organic compounds by perennial ryegrass (Lolium perenne L.)/Neotyphodium lolii association as a response to infection by Fusarium poae.

Grasses very often form symbiotic associations with Neotyphodium/Epichloë endophytic fungi. These endophytes often allow the host grass to be protected from different pathogens. However, there is little known about the mechanisms of such endophyte influence on the host. Thus, the purpose of this research was to examine the effect of the N. lolii endophyte on the total production of phenolic compounds, VOCs emission and the resistance of three perennial ryegrass genotypes infected by pathogenic Fusarium poae. Analyses of total phenolics content were performed in control (not inoculated) and inoculated plants after 1, 2, 3, 4, 5, and 6 days (DAI) and for VOCs after 0, 3, 6 and 12 DAI. The presence of endophytes significantly reduced the disease index in two of the three genotypes relative to that in E-. Plants infected by N. lolii exhibited higher production of phenolics relative to the E- plants. The highest amounts of phenolics were observed on the second and sixth DAI. Genotype Nl22 showed the strongest effect of the endophyte on the production of phenolics, which increased by over 61%. Both the endophyte infected and non-infected plants emitted most abundantly two GLVs ((Z)-3-hexenal, (Z)-3-hexen-1-yl acetate), three terpenes (linalool, (Z)-ocimene, ß-caryophyllene) and three shikimic acid pathway derivatives (benzyl acetate, indole, and methyl salicylate). The endophyte presence and the intervals of VOCs detection were a highly significant source of variation for all emitted volatiles (P<0.001). The genotype of the perennial ryegrass significantly affected only the emission of methyl salicylate (P<0.05) and ß-caryophyllene (P<0.05). Most of the VOCs ((Z)-3-hexen-1-yl acetate, (Z)-3-hexenal, linalool and methyl salicylate) reached their highest levels of emission on the sixth DAI, when averaged over genotypes and endophyte status. The results highlight the role of Neotyphodium spp. in the mediation of quadro-trophic interactions among plants, symbiotic endophytes, invertebrate herbivores and plant pathogenic fungi. Our results also confirm the fact that symbiotic plants can activate a defense reaction faster than non-symbiotic plants after a pathogen attack. Thus, N. lolii can be involved in the defense of perennial ryegrass against pathogens and potentially could be central to the host plants' protection.

Cereal crop volatile organic compound induction after mechanical injury, beetle herbivory (Oulema spp.), or fungal infection (Fusarium spp.).

Herbivory, mechanical injury or pathogen infestation to vegetative tissues can induce volatile organic compounds (VOCs) production, which can provide defensive functions to injured and uninjured plants. In our studies with 'McNeal' wheat, 'Otana' oat, and 'Harrington' barley, plants that were mechanically injured, attacked by either of two Oulema spp. (melanopus or cyanella) beetles, or infected by one of the three Fusarium spp. (graminearum, avenaceum, or culmorum), had significant VOC induction compared to undamaged plants. Mechanical injury to the main stem or one leaf caused the induction of one green leaf volatile (GLV) - (Z)-3-hexenol, and three terpenes (ß-linalool, ß-caryophyllene, and α-pinene) with all three grasses; wheat and barley also showed ß-linalool oxide induction. The blend of induced VOCs after Fusarium spp. infestation or Oulema spp. herbivory was dominated by GLVs ((Z)-3-hexenal, (E)-2-hexenal, (E)-2-hexenol, (Z)-3-hexenyl acetate, and 1-hexenyl acetate) and ß-linalool and ß-caryophyllene; beetle herbivory also induced (E)-ß-farnesene. Different ratios of individual VOCs were induced between the two Oulema spp. for each cereal grass and different ratios across the three cereals for each beetle species. Also, different ratios of individual VOCs were induced between the three Fusarium spp. for each cereal grass and different ratios across the three cereals for each fungal pathogen species. Our results are preliminary since we could not simultaneously measure VOC induction from controls with each of the ten different injury treatments for each of the three cereals. However, the comparison of mechanical injury, insect herbivory, and fungal infection has not been previously examined with VOC responses from three different plant species within the same family. Also, our work suggests large qualitative and quantitative overlap of VOC induction from plants of all three cereals having beetle herbivory injury when compared to infection injury from necrotrophic fungal pathogens.