Patrice Halama

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.

Mycosphaerella graminicola produces a range of cell wall-degrading enzyme activities in vitro that vary with the carbon source

In this study, two Mycosphaerella graminicola isolates produced a range of cell wall-degrading enzymes (CWDE) in vitro that can potentially degrade wheat cell walls. The influence of three carbon sources on CWDE in vitro production was tested: 1) 1 % galactose (w/v), 2) 1% wheat cell walls (w/v) and 3) a mixture of 1% galactose (w/v) and 1% wheat cell walls (w/v). Six major activities produced by both isolates were detected: xylanase, β-1,3-glucanase, polygalacturonase, cellulase, β-xylosidase and β-galactosidase. Time-course experiments showed that different levels of enzyme activities were obtained with isolates 323 and 94269. These activities levels varied also with the type of carbon source used. Principal Component Analysis showed that the enzyme activities are gathered into two groups. None of the activities of the first group was correlated to the activities of the second group. It also showed that the optimal medium that allowed the production of most of the major activities contained both galactose and wheat cell walls.

Protective and curative efficacy of prothioconazole against isolates of Mycosphaerella graminicola differing in their in vitro sensitivity to DMI fungicides

BACKGROUND Septoria leaf blotch is the most important disease of wheat in Europe. To control this disease, fungicides of the 14α-demethylase inhibitor group (DMIs) have been widely used for more than 20 years. However, resistance towards DMIs has increased rather quickly in recent years. The objective of this study was to evaluate, on plants and under controlled conditions, the protective and curative efficacy of the DMI fungicide prothioconazole against three current isolates of M. graminicola, chosen to belong to different DMI-resistant phenotypes. Fungicide efficacy was assessed by visual symptoms and by quantitative real-time polymerase chain reaction (PCR). RESULTS With a protective fungicide application, prothioconazole was always effective against each isolate. This was in accordance with the EC50 results. However, curative efficacy differed between the isolates. It remained at a good level, between 60 and 70% against one isolate, whereas it was strongly affected by late applications from 7 days post-inoculation with the two other isolates. CONCLUSION A protective application of prothioconazole in wheat crops could be the best strategy to keep a high efficacy against Septoria leaf blotch.

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.

Natural Agents Inducing Plant Resistance Against Pests and Diseases

Plant resistance inducers, also referred to as elicitors, are agents that confer improved protection to pathogen or pest attacks by inducing host defense mechanisms. Such products are effective against a wide range of crop enemies, including viruses, bacteria, fungi, oomycetes, nematodes, and herbivores. The mode of action of these products differs from that of traditional pesticides because they do not target directly the bio-aggressor through antifungal activity, but they inhibit its development indirectly via the elicitation of plant defense reactions. In the current context of sustainable agriculture and growing demand for healthy food, plant resistance inducers are considered as an eco-friendly and promising alternative to conventional pesticides, and their implementation in integrated pest management strategy is strongly encouraged. Plant resistance inducers can be of synthetic or natural origin. This chapter will focus on resistance inducers of natural origin including living microorganisms, plant extracts, microbial cell-wall extracts, microbial metabolites, minerals, and ions. An overview on the market and recent advances on the regulation of these products as well as future challenges to promote their development and wide use in disease management programs will be described.

New salicylic acid and pyroglutamic acid conjugated derivatives confer protection to bread wheat against Zymoseptoria tritici : New salicylic acid and pyroglutamic acid conjugated derivatives confer protection to bread wheat against Zymoseptoria tritici

BACKGROUND To promote sustainable agriculture and healthy food, research that contributes towards a new generation of eco-friendly phytosanitary compounds is increasingly encouraged. The plant hormone salicylic acid (SA) is known for its ability to induce resistance in plants against a wide range of pathogens, whereas pyroglutamic acid (PGA), a constrained analogue of γ-aminobutyric acid, has never been studied in the context of plant protection. RESULTS The present study investigated for the first time the protection efficacy of SA and PGA and five new conjugated derivatives against Zymoseptoria tritici, the main pathogen in wheat crops. SA and four derivatives showed significant disease severity reductions in planta (up to 49%). In vitro assays revealed that some molecules, including SA, displayed a small direct antifungal activity, whereas others, such as PGA, showed no effect. This finding suggests that, especially for molecules without any direct activity, the mode of action relies mainly on the induction of plant resistance. CONCLUSION Further investigations are needed to identify the defence pathways involved in plant resistance mechanisms elicited or primed by the molecules. The manufacture of these products was easily achieved on a scale of tens of grams of raw materials, and is easily scalable. The synthetic pathway is simple, short and inexpensive. For all of these reasons, the production of the target molecules is attractive for producers, whereas the prospect of a generation of non-polluting compounds with lasting efficiency against Z. tritici in wheat comes at a key moment for the sustainability of agriculture.

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.