Thyme essential oil as an alternative mechanism: biofungicide-causing sensitivity of Mycosphaerella graminicola.

AIMS: To understand the mode of action of thyme essential oil as an alternative biofungicide. METHODS AND RESULTS: The chemical composition of thyme essential oil isolated by hydrodistillation from the aerial parts of Thymus vulgaris was analyzed. The main constituents of thyme essential oil were thymol (76·96%), ρ-cymene (9·89%), γ-terpinene (1·92%) and caryophyllene oxide (1·69%). The antifungal activity of the oil and its pure major component (thymol) was assessed by the in vitro assay against Mycosphaerella graminicola. Thyme oil exhibited higher antifungal activity than thymol. The expression pattern of genes involved in fungal development and detoxification acting in M. graminicola under thyme oil and thymol treatment was analyzed. Thyme oil overexpressed, more than thymol, the genes encoding for the efflux pump (MgMfs1, MgAtr4), the regulatory subunit of protein kinase A (PKA) (MgBcy1) and the MAPK MgHog1. Thyme oil repressed the expression of the genes encoding for the efflux pump MgAtr4, the MAPK (MgSlt2) and the regulatory subunit of PKA (MgBcy1). However, thymol repressed only MgAtr4 and MgSlt2 expression. CONCLUSIONS: These data highlight the ability of thyme oil to target genes involved in fungal development and virulence of the yeast-like fungi M. graminicola, which explain its higher antifungal activity. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings will probably be useful to design an alternative biofungicide which will not lead to pathogen multidrug resistance.

PLANT OLIGOSACCHARIDES ENHANCE WHEAT DEFENCE RESPONSE AGAINST SEPTORIA LEAF BLOTCH.

Our work provides the first evidence for elicitation and protection effects of preventive treatments with oligosaccharides (20%)-based new formulation (Oligos) against Mycosphaerella graminicola, a major pathogen of bread wheat (BW) and durum wheat (DW). In planta Oligos treatment led to strongly reduced hyphal growth, penetration, mesophyll colonization and fructification. During the necrotrophic phase, Oligos also drastically decreased the production of M. graminicola CWDE activities, such as xylanase and glucanase as well as protease activity in both wheat species, suggesting their correlation with disease severity. Concerning plant defence markers, PR2, Chi 4 precursor-, Per- and LOX-1-encoding genes were up-regulated, while glucanase (GLUC), catalase (CAT) and lipoxygenase (LOX) activities and total phenolic compound (PC) accumulation were induced in both (non-inoculated and inoculated contexts. In inoculated context, a localized accumulation of H2O2 and PC at fungal penetration sites and a specific induction of phenylalanine ammonia-Lyase (PAL) enzymatic activity were observed. Moreover, our experiment exhibited some similarities and differences in both wheat species responses. GLUC and CAT activities and H2O2 accumulation were more responsive in DW leaves, while LOX and PAL activities and PC accumulation occurred earlier and to a stronger extent in BW leaves. The tested Oligos formulation showed an interesting resistance induction activity characterized by a high and stable efficiency whatever the wheat species, suggesting it integration in common control strategies against STB on both DW and BW.

ASCORBIC ACID CONTROLS MYCOSPHAERELLA GRAMINICOLA IN BREAD AND DURUM WHEAT THROUGH DIRECT EFFECT ON THE PATHOGEN AND INDIRECT ACTION VIA PLANT DEFENCE.

Septoria tritici blotch (STB) caused by Mycosphaerella graminicola is one of the most devastating foliar diseases on wheat. Due to the emergence of fungicide-resistant M. graminicola strains and in an effort to reduce the impact of pesticides on the environment, considerable interest has been devoted to alternative control strategies. The use of natural products, especially through a defense-activating effect on the host, could be considered. Acid ascorbic (AA) is synthesized by plants and most animal cells with antioxidant properties. This study aimed at: (i) assessing the protective effect of an AA-based product on bread (BW) and durum (DW) wheat (Triticum aestivum and T. durum, respectively) susceptible cultivars against M. graminicola and (ii) investigating the mechanisms involved in wheat protection. Therefore, the foliar application of a formulated AA-based product (50 mg L-) on 3-week-old wheat plants reduced the infection level by more than 75% for both BW and DW. In vitro experiments revealed that AA induced a strong inhibition of spore germination (at 50 mg L.(-1)) and hyphal growth (at 16 mg L.(-1)) for both M. graminicola strains, infecting either BW or DW. Used as a preventive foliar spray on wheat leaves, microscopic observations revealed that AA inhibits in planta spore germination, hyphal growth, leaf penetration, substomatal colonization and eventually sporulation. Moreover, AA treatment also decreased fungal protease and cell wall degrading enzyme activities, putative pathogenicity determinants of M. graminicola. In addition to these effects on the fungus, AA induced defence reactions in both BW and DW. Indeed, in non-inoculated context, eliciting effect was observed on (i) stimulation of enzymatic activities such as lipoxygenase, peroxydase and catalase and (ii) transcript accumulation of genes encoding for pathogenesis-related (PR) proteins (chitinase class IV, peroxidase). In inoculated condition, accumulation of H2O2 and phenolic compounds increased at the penetration site in AA-treated leaves. In addition, AA treatment impacted the phenylpropanoid pathway through the induction of phenylalanine ammonia lyase activity. These results show that, in our conditions, AA both presents an antifungal activity and triggers several plant defences in wheat and suggest its use to control M. graminicola on both DW and BW.

EFFICACY AND MODES OF ACTION OF RESISTANCE INDUCERS ON TWO WHEAT SPECIES AGAINST MYCOSPHAERELLA GRAMINICOLA.

Plant resistance inducers could be an alternative to conventional fungicides to control in a more durable and environmentally friendly manner fungal pathogens. Here, we tested the protection efficacy and the modes of action of four resistance inducers (R1, R2, R3 and R4) against the causal agent of Septoria tritici blotch, Mycosphaerella graminicola, the most frequently occurring pathogen on wheat crops worldwide. The four inducers were tested on two wheat cultivars, Premio (a French bread wheat cultivar) and Karim (a Tunisian durum wheat cultivar), each inoculated with a bread-wheat or a durum-wheat adapted isolate; respectively. All inducers exhibited in the greenhouse a significant protection level on both cultivars regarding disease symptoms (necrosis and chlorosis) and sporulation (pycnidium density). The most efficient inducer was R3 which showed 84% symptom reduction, while the less efficient one was R2 with only a 39% reduction. None of the studied inducers showed direct biocide effect against the fungus, except R4 which displayed a significant in planta inhibition of spore germination. Further investigations revealed that all inducers elicited the plant defence enzymes peroxidase and lipoxygenase, but the activity levels varied depending on the considered inducer. In addition, the effect of resistance inducers on the infection process and the fungal cell-wall degrading enzymes xylanases and glucanases was also investigated. Our study allowed us to find out four efficient resistance inducers on wheat against M. graminicola and to establish data about the modes of action of these inducers.