A Plant Nutrient- and Microbial Protein-Based Resistance Inducer Elicits Wheat Cultivar-Dependent Resistance Against Zymoseptoria tritici.

The induction of plant defense mechanisms by resistance inducers is an attractive and innovative alternative to reduce the use of fungicides on wheat against Zymoseptoria tritici, the responsible agent of Septoria tritici blotch (STB). Under controlled conditions, we investigated the resistance induction in three wheat cultivars with different susceptible levels to STB as a response to a treatment with a sulfur, manganese sulfate, and protein-based resistance inducer (NECTAR Céréales). While no direct antigermination effect of the product was observed in planta, more than 50% reduction of both symptoms and sporulation were recorded on the three tested cultivars. However, an impact of the wheat genotype on resistance induction was highlighted, which affects host penetration, cell colonization, and the production of cell-wall degrading enzymes by the fungus. Moreover, in the most susceptible cultivar Alixan, the product upregulated POX2, PAL, PR1, and GLUC gene expression in both noninoculated and inoculated plants and CHIT2 in noninoculated plants only. In contrast, defense responses induced in Altigo, the most resistant cultivar, seem to be more specifically mediated by the phenylpropanoid pathway in noninoculated as well as inoculated plants, since PAL and CHS were most specifically upregulated in this cultivar. In Premio, the moderate resistant cultivar, NECTAR Céréales elicits mainly the octadecanoid pathway, via LOX and AOS induction in noninoculated plants. We concluded that this complex resistance-inducing product protects wheat against Z. tritici by stimulating the cultivar-dependent plant defense mechanisms.

QoI Resistance and Mitochondrial Genetic Structure of Zymoseptoria tritici in Morocco.

In total, 230 single-conidial isolates of the fungal wheat pathogen Zymoseptoria tritici (formerly Septoria tritici, teleomorph: Mycosphaerella graminicola) were sampled in Morocco in 2008 and 2010 to assess resistance against quinone outside inhibitors (QoIs), a widely used group of fungicides in wheat pest management. All 134 isolates sampled in 2008 were QoI sensitive. In contrast, 9 of the 96 isolates from the 2010 collection were resistant, suggesting a recent emergence of the resistance. Mitochondrial (mt)DNA-sequence analyses identified four haplotypes among the resistant isolates. Wright's F statistics (FST) analyses from mtDNA sequences revealed a shallow population structure of Z. tritici within Morocco and a substantial asymmetric gene flow from Europe into Morocco. A phylogenetic reconstruction including Moroccan and European isolates clustered the haplotypes regardless of their geographic origin. The four Moroccan QoI-resistant mitochondrial haplotypes clustered in two distinct clades in the tree topology, suggesting at least two independent origins of the resistance. This study reported, for the first time, the occurrence of QoI-resistant genotypes of Z. tritici in Morocco. Our findings are consistent with the hypothesis that QoI resistance emerged very recently through parallel genetic adaptation in Morocco, although gene flow from Europe cannot be excluded.

Detection of strobilurin-resistant isolates of Mycosphaerella graminicola in Morocco.

Septoria tritici blotch caused by Mycosphaerella graminicola (anamorph: Septoria tritici) is nowadays one of the most frequently occurring diseases on both bread and durum wheat crops. Two hundred and thirty isolates of the fungus were sampled from six distinct wheat-producing regions of Morocco in order to investigate the resistance of M. graminicola to strobilurins in this country, where this fungicide class is increasingly used in wheat-pest management. A subset of 134 isolates was first collected in 2008 from Meknes-Tafilalet, Tadla-Azilal, Gharb and Chaouia. Furthermore, 96 additional isolates were sampled in 2010 from the fourth regions investigated in 2008 plus Fes-Boulmane and Doukkala-Abda. Sensitivity or resistance within the isolates were determined by screening the G143A cytochrome b substitution conferring resistance. We used a mismatch amplification mutation assay allowing the amplification of either G143 (sensitive) or A143 (resistant) allele. All the 2008 isolates were found to be sensitive since they carry the wild-type allele G143. However, 9 (9%) out of the 2010 isolates were found to contain the resistant allele A143 and therefore to be resistant. Four of them were from Gharb and five from Fes-Boulmane. This study highlighted for the first time the occurrence of strobilurin-resistant isolates of M. graminicola in Morocco. Further genetic investigations should determine if the resistant isolates emerged independently in Morocco or traveled by wind-migration from Europe.

Two Mycosphaerella graminicola French isolates differ in symptoms, in planta sporulation and cell wall degrading enzymes in vitro production.

The pathogenicity level of two French Mycosphaerella grominicola field isolates (T0414 and T0251) was evaluated on Soissons wheat cultivar using two methods: detached wheat leafs assay in a climatic chamber and wheat seedlings assay in a greenhouse. Both methods revealed that chlorosis and necrosis caused by the T0414 isolate were larger than those caused by the T0251 isolate. Indeed, in the detached leaf assay, the first symptoms on leaves inoculated by T0414 were observed 12 days post inoculation and 50% of the leaves were infected on day 18, with a maximum of 84% of leaves on day 22. On the other hand, the first symptoms were observed on day 18 on leaves inoculated with T0251 with a maximum of only 10% of leaves that were infected on day 22. In addition, both methods showed that necrosis surfaces caused by T0414 were covered by large number of pycnidia, while no pycnidia were observed on the reduced necrosis caused by the T0251 isolate. To understand the pathogenicity variation between these two isolates, their ability to produce cell wall degrading enzymes, xylanases and polygalacturonases was investigated in vitro every 2 days for 20 days. The results showed similar time course for production of polygalacturonases for the two isolates, with non significant higher production for T0414. However, a peak of maximum production of xylanases by T0414 (343 +/- 52 mU mL(-1)) was observed on day 12 post inoculation, while the maximum production (265 +/- 72 mU mL(-1)) by T0251 was observed only on day 20 post inoculation. This result shows a relationship between the beginning of the appearance of symptoms on detached leaves inoculated by T0414 on day 12 and the maximum of xylanases production on the same day in enzymes assays. In conclusion, this study suggests pathogenicity variability between M. graminicola isolates and the role of xylanases in the pathogenicity of this fungus.

Trehalose induces resistance to powdery mildew in wheat.

• Reduction in the degree of powdery mildew infection of wheat leaves is observed after treatments with trehalose, a nonreducing disaccharide commonly found in a wide variety of organisms, including fungi. • Wheat (Triticum aestivum) cv. Sideral plants grown in phytotrons were inoculated with Blumeria graminis f.sp. tritici. In addition to degree of infection, the effect of trehalose solution was further investigated using light and fluorescence microscopy and enzyme assays. • Infection in wheat leaves was reduced by 50 and 95% with trehalose solution (15 g l-1 ) following a single spraying and three sprayings, respectively; in a detached leaf assay, trehalose was effective at concentrations as low as 0.01 g l-1 . Trehalose did not inhibit conidial germination and differentiation of appressoria (in vitro or on the leaf epidermis), but enhanced papilla deposition in epidermal cells. Trehalose also enhanced phenylalanine ammonia-lyase (PAL) and peroxidase (PO) activities; both markers of plant defence responses. However, the level of three cinnamyl alcohol dehydrogenase (CAD) activities (conyferyl, p-coumaryl and sinapyl alcohol dehydrogenase) was unchanged. • Trehalose treatment of wheat confers resistance to B. graminis infection by activating plant defence responses (e.g. papilla deposition, PAL and PO activities).

Four Arabidopsis RPP loci controlling resistance to the Noco2 isolate of Peronospora parasitica map to regions known to contain other RPP recognition specificities.

Interactions between Arabidopsis thaliana and the downy mildew fungus Peronospora parasitica provide a model system to study the genetic and molecular basis of plant-pathogen recognition. With the use of the Noco2 isolate of P. parasitica, the reaction phenotypes of 46 accessions of Arabidopsis were examined and 31 accessions exhibited resistance. Resistance phenotypes examined ranged from distinct necrotic pits or flecks to a weak necrosis accompanied by late and sparse fungal sporulation. Segregating populations generated from crosses between the susceptible accession Col-0 and the resistant accessions Ws-0, Pr-0, Oy-0, Po-1, Bch-1, Ge-1, Di-1, Ji-1, and Te-0 were also screened with Noco2. The genetic data were consistent with the presence of single resistance (RPP) loci in all of these accessions except Oy-0, in which resistance was inherited as a digenic trait. As a first step to molecular cloning, the map positions of four resistance loci were determined. These have been designated RPP14.1 from Ws-0, RPP14.2 from Pr-O, and RPP14.3 and RPP5.2 from Oy-0. RPP14.1 was mapped to a 3.2-cM interval on chromosome 3 that is linked to a region between the markers Gl-1 and m249 known to contain other P. parasitica resistance specificities. RPP14.2 from Pr-0 and RPP14.3 from Oy-0 were also positioned in this interval. Moreover, RPP14.1 and RPP14.2 showed linkage of < 0.05 cM, suggesting possible allelism. The second RPP locus from Oy-0, RPP5.2, was located on chromosome 4 and exhibited strong linkage (< 2 cM) to RRP5.1, a locus previously identified in the Arabidopsis accession Landsberg-erecta. The results reinforce evidence for RPP gene clustering in the Arabidopsis genome and provide new targets for cloning and examination of RPP gene structure, function, allelic variation, and organization within defined loci.

Transformation of Botrytis cinerea with the hygromycin B resistance gene, hph.

A transformation method has been developed for the phytopathogenic fungus Botrytis cinerea. Protoplasts were transformed with pAN7-1 plasmid carrying the Escherichia coli hygromycin phosphotransferase gene (hph), conferring hygromycin B resistance, downstream from an Aspergillus nidulans promoter. Molecular analysis, showed that transformation resulted in an integration of the plasmid into different regions of the B. cinerea genome and occurred through non-homologous recombination. The frequency was 2-10 transformants per micrograms of DNA. Transformants expressed phosphotransferase activity confirming that the hph gene conferred the hygromycin-resistance phenotype. All transformants analysed so far proved to be stable after several subcultures without any selective pressure.