Correlation of cytological and biochemical parameters with resistance and tolerance to Mycosphaerella graminicola in wheat.

This study investigated the infection process of Mycosphaerella graminicola and enzyme activities related to reactive oxygen species (ROS) or oxylipin biosynthesis in four French wheat cultivars with variable resistance to M. graminicola infection. At field level, cultivars Caphorn, Maxyl and Gen11 were susceptible, whereas Capnor showed high levels of quantitative resistance. Moreover, Capnor and Gen11 were tolerant, i.e., their yield was less affected by infection compared to non-tolerant Maxyl and Caphorn. These four cultivars were inoculated under laboratory conditions with the M. graminicola IPO323 reference strain. Cytological and biochemical responses were studied on collected first plantlet leaves and several features discriminated between cultivars. However, resistance and tolerance had no impact on the fungal infection process. Levels of lipoxygenase (LOX), peroxidase (PO) and glutathione-S-transferase (GST) activities were also compared with regard to cultivar resistance or tolerance to M. graminicola. LOX, PO and GST activities did not discriminate resistance and tolerance profiles, although a low level of PO in inoculated and non-inoculated plants could be associated with tolerance. In addition, cell necrosis correlated positively with LOX in non-tolerant cultivars, while mycelia surrounding stomata were positively correlated with PO in the resistant cultivar. GST activity presented correlations between cytological and biochemical parameters only for susceptible cultivars. Stomatal and direct penetration were positively correlated with GST activity in the susceptible non-tolerant cultivars, while these correlations were negative in the tolerant cultivar. When combining cytological and biochemical observations with resistance and tolerance profiles, for each cultivar and at each time point, cultivars could be classified in tight accordance with their previous field characterisation. Moreover, tolerance allowed us to distinguish susceptible cultivars when both biochemical and cytological parameters were considered together.

Single strand conformation polymorphism is a sensitive method for screening nucleotide variations in Mycosphaerella graminicola.

Single Strand Conformation Polymorphism (SSCP) and sequencing were performed in order to assess molecular polymorphism of mating type sequences in the heterothallic ascomycete Mycosphaerella graminicola, the causal agent of Septoria tritici blotch of wheat. The screening was undertaken on mat1-1 and mat1-2 partial sequences of 341 and 657 bp, respectively, amplified with multiplex PCR from 510 French single-conidial strains plus the two reference isolates IPO323 and IPO94269 from The Netherlands. After restriction with Taq1 in order to reduce the fragment sizes, all digested amplicons were subjected to SSCP. Sequencing was then performed when a SSCP pattern deviates from the most frequently occurring profile. Among the assessed strains, 228 ones plus IPO323 were MAT1-1 and 282 ones plus IPO94269 were MAT1-2. Among the MAT1-1 strains, only a single one exhibited a SSCP profile distinct to the other MAT1-1 strains, whereas 10 MAT1-2 strains (among which 2 and 4 with same profiles, respectively) showed a SSCP profile differing to the other MAT1-2 strains. Sequencing revealed that all polymorphisms observed on SSCP gels were single nucleotide variations and all strains displaying the same SSCP profiles showed identical nucleotide sequences. Among the seven disclosed nucleotide variations, only two were non-synonymous and both were non-conservative. This study reports a high sensitivity of SSCP allowing detection of single point mutations in M. graminicola, shows a conservation of mating type idiomorphs in the fungus at both sequence and population scales, but also suggests a difference in polymorphism level between the two mating type sequences.

Oligogalacturonides induce resistance in wheat against powdery mildew.

Biological activities, priming and protective effects of two oligogalacturonides fractions (OGAs) were assayed during a compatible wheat/Blumeria graminis f. sp. tritici interaction. These fractions were obtained from commercial polygalacturonic acid. They both consisted of oligogalacturonides with polymerisation degrees (DP) ranging from 2 to 25, and one of them was a 30% chemically acetylated fraction. A 5 g x L(-1) solution of each fraction was infiltrated in the first leave of ten-days-old plantlets, and activities of defence-related enzymes were measured 48H post-treatment. Among them, oxalate oxidase and peroxidase activities increased, suggesting an elicitation due to both fractions of oligogalacturonides. Some of the pre-treated plantlets were subsequently submitted to powdery mildew infection. As revealed by 3,3'-diaminobenzidine (DAB) staining, the accumulation of hydrogen peroxide (H2O2) at the penetration site of the fungus increased 21H after inoculation to the same extent in areas of plantlets infiltrated by both fractions. On the other hand, the intensity of fluorescence associated with papillae was higher when plantlets were pre-infiltrated with the acetylated fraction, whereas no difference was observed between control plantlets and those treated with the non-acetylated fraction. Moreover, microscopic assessment of the number of haustoria occurring 40H post-inoculation showed it was only reduced when acetylated fraction was used. Despite different modes of action of these molecules, a similar 45% protective effect occurred in both cases when the oligogalacturonides fractions were sprayed on ten-days-old plantlets.

Mechanisms of resistance and tolerance to Mycosphaerella graminicola in wheat.

The aim of this study was to investigate the infection process of M. graminicola and the defence mechanisms related to active oxygen species (AOS) in five French wheat cultivars. These cultivars exhibited various resistant levels to M. graminicola infection: Maxyl, Caphorn and Gen11 are susceptible cultivars, whereas Capnor and Gen23 show high levels of quantitative resistances. In addition, Capnor, Gen23 and Gen11 are tolerant cultivars, i.e., their yield performance was less affected by infection compared to non-tolerant cultivars. Cultivars were inoculated with the IPO323 reference M. graminicola strain. First wheat leaves were collected 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21 days after inoculation. The cytological and antioxidant response of the cultivars were both studied over the whole time course. Although infection occurred mainly through stomata, direct penetration attempts were also scored. Moreover, papilla formation turned out to be very rare. Assays for changes in peroxydase (PO), glutathione-S-transferase (GST) and lipoxygenase (LOX) activities allowed us to compare their levels in the five French wheat cultivars regarding to their resistance and/or tolerance towards M. graminicola infection. PO and GST were correlated to necrosis probably as a consequence of detoxification and LOX was related to some of the germination process steps. We also showed that significant differences for several biochemical parameters exist between the studied cultivars in non inoculated conditions but these differences were less important in the presence of the fungus.

In planta xylanase activity and pathogenicity on wheat-Mycosphaerella graminicola pathosystem.

A total of twenty four French strains and two reference strains IPO323 and IPO94269 of the hemibiotrophic fungus Mycosphoerella graminicola were investigated in planta to examine the association of the cell-wall degrading enzyme endo-beta-1,4-xylanase (EC 3.2.1.8) with pathogenicity. The French strains were selected from a collection of 363 strains previously genotyped using microsatellites, actine and beta-tubutine markers. Disease level assessments as well as enzyme quantifications were carried out at 20 days post inoculation from the third leaves of inoculated whole plants of the susceptible wheat cv. Scorpion. Great variability of both pathogenicity levels and endo-beta-1,4-xylanase activity patterns was obtained among strains. Only 15 out of the 26 assessed strains including the reference strain IPO323 were able to induce lesions bearing pycnidia. The percentages of diseased leaf areas bearing pycnidia ranged from 6.2% to 77%, while amounts of endo-beta-1,4-xylanase activity ranged from 0 to 399.15 mU/microg of total proteins. A Pearson correlation test revealed very high linkage between endo-beta-1,4-xylanase activity level and lesions bearing pycnidia production within strains (r = 0.94). Additional cytological and enzymatic investigations on two strains exhibiting different pathogenicity levels highlighted that successful disease induction by M. graminicola is not explained by either spore germination or direct and stomatal penetration rates of the host, but by the ability of the fungus to colonize the mesophyll and to secrete the endo-beta-1,4-xylanase activity during necrotrophic phase. This study strongly suggests the importance of both mesophyll colonisation and endo-beta-1,4-xylanase activity during the infection process of M. graminicola.