Philippe Reignault

Distinct Defenses Induced in Wheat Against Powdery Mildew by Acetylated and Nonacetylated Oligogalacturonides

In wheat, little is known about disease resistance inducers and, more specifically, about the biological activities from those derived from endogenous elicitors, such as oligogalacturonides (OGAs). Therefore, we tested the ability of two fractions of OGAs, with polymerization degrees (DPs) of 2-25, to induce resistance to Blumeria graminis f. sp. tritici and defense responses in wheat. One fraction was unacetylated (OGAs-Ac) whereas the second one was 30% chemically acetylated (OGAs+Ac). Infection level was reduced to 57 and 58% relative to controls when OGAs-Ac and OGAs+Ac, respectively, were sprayed 48 h before inoculation. Activities of various defense-related enzymes were then assayed in noninoculated wheat leaves infiltrated with OGAs. Oxalate oxidase, peroxidase, and lipoxygenase were responsive to both OGAs-Ac and OGAs+Ac, which suggests involvement of reactive oxygen species and oxilipins in OGAs-mediated responses in wheat. In inoculated leaves, both fractions induced a similar increase in H₂O₂ accumulation at the site of fungal penetration. However, only OGAs+Ac led to an increase in papilla-associated fluorescence and to a reduction of formed fungal haustoria. Our work provides the first evidence for elicitation and protection effects of preventive treatments with OGAs in wheat and for new properties of acetylated OGAs.

Inhibition of Blumeria graminis f. sp. tritici Germination and Partial Enhancement of Wheat Defenses by Milsana

ABSTRACT The prophylactic efficiency of Milsana against powdery mildew was evaluated on wheat (Triticum aestivum). A single short spraying on 10-day-old plantlets reduced the infection level by 85% and two long sprayings led to the total restriction of the disease. Although microscopic studies showed that Milsana treatments enhance hydrogen peroxide accumulation at the fungal penetration site, biochemical analysis did not allow us to correlate this accumulation with the activation of several enzyme activities involved in active oxygen species (AOS) metabolism. Only lipoxygenase activity, which is involved in both AOS metabolism and lipid peroxidation, showed a 26 to 32% increase 48-h posttreatment in leaves infiltrated with Milsana. This weak effect of Milsana on wheat lipid metabolism was confirmed at the lipid peroxidation level, which surprisingly, was shown to decrease in treated plants. In order to explain the high efficacy of Milsana, the fungistatic effect on conidia germination was also examined. In planta, we showed that a Milsana treatment resulted in a higher proportion of abnormally long appressorial germ tubes, whereas in vitro, it dramatically inhibited fungal conidia germination. The partial activity of Milsana in terms of defense response induction in the wheat/powdery mildew pathosystem and its newly described direct fungistatic activity are discussed.

Exogenous Trehalose Induces Defenses in Wheat Before and During a Biotic Stress Caused by Powdery Mildew

Powdery mildew would be one of the most damaging wheat diseases without the extensive use of conventional fungicides. Some of the alternative control strategies currently emerging are based on the use of resistance inducers. The disacharride trehalose (TR) is classically described as an inducer of defenses in plants to abiotic stress. In this work, the elicitor or priming effect of TR was investigated in wheat both before and during a compatible wheat-powdery mildew interaction through molecular, biochemical, and cytological approaches. In noninoculated conditions, TR elicited the expression of genes encoding chitinase (chi, chi1, and chi4 precursor), pathogenesis-related protein 1, as well as oxalate oxidase (oxo). Moreover, lipid metabolism was shown to be altered by TR spraying via the upregulation of lipoxygenase (lox) and lipid-transfer protein (ltp)-encoding gene expression. On the other hand, the protection conferred by TR to wheat against powdery mildew is associated with the induction of two specific defense markers. Indeed, in infectious conditions following TR spraying, upregulations of chi4 precursor and lox gene expression as well as an induction of the LOX activity were observed. These results are also discussed with regard to the impact of TR on the fungal infectious process, which was shown to be stopped at the appressorial germ tube stage. Our findings strongly suggest that TR is a true inducer of wheat defense and resistance, at least toward powdery mildew.

Genetic diversity and population structure in French populations of Mycosphaerella graminicola

Mycosphaerella graminicola populations were examined in France with microsatellite markers and PCR-SSCP analysis of partial actin and β-tubulin encoding sequences. A total of 363 isolates was sampled in 2005 from 17 provinces, and genotypes from corresponding strains were characterized. Unique haplotypes comprised 84% of the population, and gene diversity was high nationwide (0.70) and locally. A moderate genetic differentiation (GST = 0.18) was found and indicated that in France the M. graminicola population was more structured than in other previously studied European countries. Bayesian structure analysis identified three genetic clusters distributed among the 17 provinces. Our results highlighted the potential for the adaptation of the fungus to local conditions, leading to genetic clusters among the French population of the fungus as well as genotype flow between regional clusters.

Mating type idiomorphs from a French population of the wheat pathogen Mycosphaerella graminicola: widespread equal distribution and low but distinct levels of molecular polymorphism.

Septoria tritici blotch caused by the heterothallic ascomycete Mycosphaerella graminicola is currently the most frequent and the most economically damaging disease on wheat worldwide. Five hundred and ten strains of this fungus were sampled from 16 geographical locations representing the major wheat producing areas in France. Multiplex PCR amplification, PCR-RFLP-SSCP screening and sequencing of parts of mating type encoding sequences were performed in order to assess the distribution and molecular polymorphism of the mating type idiomorphs. The two idiomorphs were scored at similar frequencies within all sampled locations. Both mating types were also identified at the leaf spatial scale, on 42% of leaves from which two or three strains were isolated. No correlation was found between distribution of mating types and either host cultivars from which the sampling was carried out or in vitro colony phenotypes observed during the culture of strains on potato dextrose agar (PDA) medium. PCR-RFLP-SSCP assay highlighted only one MAT1-1 strain exhibiting a profile distinct from all other MAT1-1 strains, whereas ten MAT1-2 strains (among which two and four with same profiles, respectively) showed profiles differing from the other MAT1-2 strains. Sequencing revealed that all polymorphisms corresponded to single nucleotide variations and all strains displaying the same single strand conformation polymorphism (SSCP) profiles showed identical nucleotide sequences, thereby confirming the high sensitivity of SSCP. Only two out of the disclosed nucleotide variations were nonsynonymous. This study strongly suggests a large potential for sexual reproduction in the French population of M. graminicola and reports a high conservation of mating type sequences in the fungus at both nucleotide and population levels, with a great difference in molecular variability between the two idiomorphs.

Lipid metabolism is differentially modulated by salicylic acid and heptanoyl salicylic acid during the induction of resistance in wheat against powdery mildew

Heptanoyl salicylic acid (HSA) is a salicylic acid (SA) derivative obtained by esterification of 2-OH benzoic acid with heptanoic acid. In wheat, the protection levels obtained against Blumeria graminis f. sp. tritici (Bgt) increased from 50% with SA to 95% with HSA. Using molecular, biochemical and cytological approaches, we investigated here how wheat lipid metabolism is differentially activated by SA and HSA in both infectious and non-infectious conditions, and how Bgt infectious process is altered by both inducers. First, in the absence of Bgt, continuous lipoxygenase (LOX)-encoding gene expression and corresponding activity were specifically induced by HSA. Moreover, compared to SA, HSA treatment resulted in earlier up-regulations of the phospholipase C2-encoding gene expression and it specifically affected the expression of a lipid transfer protein-encoding gene. In infectious context, both HSA and SA sprayings impaired penetration events and therefore haustorium formation, leading to less frequent fungal colonies. While this alteration only slowed down the evolution of Bgt infectious process in SA-sprayed leaves, it completely impaired the establishment of successful infectious events in HSA-sprayed leaves. In addition, HSA induced continuous increases of a LOX-encoding gene expression and of the corresponding LOX activity when compared to SA-sprayed leaves. Lipid metabolism is therefore overall highly responsive to HSA spraying and could represent effective defence mechanism triggered during the induction of resistance in wheat toward Bgt. The concepts of priming and energy costs of the defences induced by SA and HSA are also discussed.

Banning of methyl bromide for seed treatment: could Ditylenchus dipsaci again become a major threat to alfalfa production in Europe?

In Europe, the stem and bulb nematode Ditylenchus dipsaci has been listed as a quarantine pest by EPPO: without any control, it may cause complete failure of alfalfa crops. Movement of nematodes associated with seeds is considered to be the highest-risk pathway for the spread of this pest. Since the 2010 official withdrawal of methyl bromide in Europe, and in the absence of any alternative chemical, fumigation of contaminated seed batches is no longer possible, which makes the production of nematode-free alfalfa seeds difficult to achieve and leads to unmarketable seed batches. Thermotherapy is being considered as a realistic alternative strategy, but its efficiency still remains to be validated. The combination of the currently available methods (i.e. use of resistant cultivars, seed production according to a certification scheme, mechanical sieving, seed batch inspection) could significantly reduce the likelihood of seed contamination. However, it does not guarantee a total eradication of the nematode. Although it is already widely distributed all over Europe, reclassification of D. dipsaci as a regulated non-quarantine pest to reduce the possibility of further introductions and the rate of spread of this pest appears to be a risky strategy because of the lack of up-to-date documented data to evaluate damage thresholds and determine acceptable tolerance levels.

Mating type distribution provides evidence for sexual reproduction of Mycosphaerella graminicola in Algeria

Abstract Septoria tritici blotch caused by Mycosphaerella graminicola is currently the most frequently occurring and economically damaging disease on wheat crops worldwide. A total of 120 single-conidial isolates of this fungus (60 from bread wheat and 60 from durum wheat) were sampled in 2012 from five distinct geographical locations of Algeria and analyzed for mating type distribution to provide insight into the potential of sexual reproduction. The mating type of each isolate was identified using a multiplex PCR that amplifies either a MAT1-1 or a MAT1-2 fragment from mating type loci. Both idiomorphs were scored at equal frequencies according to the χ2 test at different scales. They were found to occur at equal proportions at the whole country level (46 % MAT1-1 vs. 54 % MAT1-2) and at the level of each sampled location. The two mating types were also found at equal frequencies on both host species at the country scale (47 % MAT1-1 vs. 53 % MAT1-2 on bread wheat and 45 % MAT1-1 vs. 55 % MAT1-2 on durum wheat) and irrespective of the sampled locations. This equal mating type distribution at both geographic and host species levels suggests a large potential for sexual reproduction of M. graminicola in Algeria and indicates a lack of specificity between mating types and host species in the case of the wheat-M. graminicola pathosystem.

Lipids as markers of induced resistance in wheat: a biochemical and molecular approach.

Our work aimed at a global investigation of the lipid metabolism during the induction of resistance in wheat (Triticum aestivum) against powdery mildew (Blumeria graminis f.sp. tritici). More specifically, the effect of salicylic acid, known as playing a key role in the activation of defence reactions against pathogens in plants, has been investigated. After salicylic acid infiltration, accumulation of phosphatidic acid was observed that could be due to the phospholipase C pathway since an up-regulation of a phospholipase C-encoding gene expression as well as an accumulation of diacylglycerol were observed. The phosphatidic acid accumulation could also result from the phospholipase D pathway since a reduction of phosphatidylethanolamine content occurred. The response to salicylic acid at the octadecanoid pathway level was also investigated: both a lipoxygenase-encoding gene expression and lipoxygenase enzymatic activity were induced by salicylic acid simultaneously with a decrease of the linolenic acid content. Finally, a lipid transfer protein-encoding gene expression was also up-regulated upon salicylic acid infiltration. These observations indicate that lipid metabolism could be considered as a marker of elicitation in wheat.

Similar infection process and induced defense patterns during compatible interactions between Zymoseptoria tritici and both bread and durum wheat species

Bread wheat (BW) and durum wheat (DW) are both strongly affected by Septoria tritici blotch caused by the hemibiotrophic fungus Zymoseptoria tritici. However, only the BW-Z. tritici pathosystem has been well studied so far. Here, we compared compatible interactions between Z. tritici and both BW and DW species at the cytological, biochemical and molecular levels. Fungal infection process investigations showed close spore germination and leaf penetration features in both interactions, although differences in the patterns of these events were observed. During the necrotrophic phase, disease severity and sporulation levels were associated in both interactions with increases of the two cell-wall degrading enzyme activities endo-β-1,4-xylanase and endo-β-1,3-glucanase as well as protease. An analysis of plant defense responses during the first five days post inoculation revealed inductions of GLUC, Chi4, POX and PAL and a repression of LOX gene expressions in both wheat species, although differences in kinetics and levels of induction or repression were observed. In addition, peroxidase, catalase, glucanase, phenylalanine ammonia-lyase and lipoxygenase activities were induced in both wheat species, while only weak accumulations of hydrogen peroxide and polyphenols were detected at the fungal penetration sites. Our study revealed overall a similarity in Z. tritici infection process and triggered wheat defense pathways on both pathosystems.