Sexual reproduction of Zymoseptoria tritici on durum wheat in Tunisia revealed by presence of airborne inoculum, fruiting bodies and high levels of genetic diversity.

Septoria tritici blotch (STB) caused by the heterothallic ascomycete Zymoseptoria tritici is currently one of the most devastating diseases of wheat worldwide. The extent of sexual reproduction of this pathogen is well documented on bread wheat, but not on durum wheat. The objective of the present study was to quantify the occurrence of Z. tritici sexual reproduction on durum wheat in the Tunisian environment. The assessment was undertaken using a triple approach combining fruiting body assessment, ascospore trapping and population genetic analyses. The results highlighted the formation of pseudothecia on leaves and stubble from the autumn until the end of the growing season. Likewise, qPCR monitoring highlighted a constant release of Z. tritici airborne inoculum during the wheat-growing season, with a peak of production at the end of the season. Genetic investigations using microsatellites revealed high levels of gene and genotypic diversities, an equal distribution of mating types, and a lack of genetic clustering within and between growing seasons. Taken together, these findings indicate that Z. tritici undergoes sexual reproduction on durum wheat in Tunisia at least to the same extent than on bread wheat in Western Europe, and that the dry and warm climate does not affect the mating process of the fungus. Frequent occurrence of sexual reproduction is a valuable knowledge to take into account in STB control strategies on durum wheat.

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.

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.

GENETIC CHARACTERIZATION OF AN ALGERIAN POPULATION OF MYCOSPHAERELLA GRAMINICOLA WITH MICROSATELLITE MARKERS.

Mycosphaerella graminicola (anamorph: Zymoseptoria tritici, formerly Septoria tritici), the responsible for Septoria tritici blotch, is the most frequently occurring disease on wheat crops worldwide. The populations of this pathogen were previously characterized in several areas around the world, but not in Algeria so far. The present study aims thus at investigating the genetic diversity and population structure of M. graminicola in this country. One hundred and twenty monoconidial isolates of this fungus (60 from bread wheat and 60 from durum wheat) were collected during the 2012 growing season from five distinct geographical locations in Algeria. They were then fingerprinted using eight microsatellite markers. The number of alleles per locus ranged from 2 to 11, with an average of 6.25 alleles per locus. We found out a moderate gene diversity, a high genotype diversity (72% of unique haplotypes) and a low population differentiation within the population. Further analyses using both UPGMA and Bayesian clustering methods confirmed the lack of genetic structuration irrespective of geographical locations and host species. These findings are likely due to the frequent occurrence of sexual reproduction in the field, leading to genetic diversification and allele homogenization via wind born ascospores within the population.

RELATIONSHIP BETWEEN PATHOGENICITY AND FUNGICIDE TOLERANCE IN THE WHEAT PATHOGEN MYCOSPHAERELLA GRAMINICOLA.

Septoria tritici blotch caused by Mycosphaerella graminicola (anamorph: Zymoseptoria tritici) is currently the most devastating disease on wheat crops worldwide, especially in regions with suitable climate conditions such as Western Europe. Pathogen control relies mainly on the use of fungicides and resistant cultivars. However, the durability of chemical and genetic control strategies is increasingly compromised in the field since the fungus frequently develops resistance to fungicides and overcomes host resistance. Here, we assessed the association between pathogenicity and fungicide tolerance in eighteen different M. graminicola strains isolated in 2009 from Northern France. These strains were chosen because they exhibited in preliminary experiments a wide range of in vitro tolerance levels against six demethylation inhibitors (epoxiconazole, cyproconazole, fluquinconazole, propiconazole, prothioconazole and pyrefenox). Inoculation of the Scorpion susceptible wheat cultivar in the greenhouse with these strains revealed a great variability in their pathogenicity levels (disease symptoms ranged from 0 to 66% of leaf area bearing pycnidia). Interestingly, strains with high fungicide tolerance levels caused overall the highest disease symptoms (45% of diseased leaf area on average) when compared to those with low fungicide tolerance levels (22% of diseased leaf area on average), thus confirming the association between pathogenicity level and fungicide tolerance in M. graminicola. The occurrence and selection in the field of fungal genotypes combining both pathogenicity and fungicide resistance should be taken into account in disease management strategies.

ASSOCIATION BETWEEN SPORULATION AND CELL-WALL DEGRADING ENZYMES IN THE WHEAT PATHOGEN MYCOSPHAERELLA GRAMINICOLA.

Mycosphaerella graminicola is a hemibiotrophic fungus that causes Septoria tritici blotch (STB), one of the most serious foliar diseases of wheat. STB can occur with a wide range of disease levels on the host, which depend not only on the pathogenicity of fungal strains, but also on the resistance of host cultivars. Here, we investigated the association between the disease level and fungal cell-wall degrading enzyme and protease activities in three wheat cultivars differing in their resistance levels against M. graminicola. The experiments were carried out in the greenhouse using artificial inoculations with the M. graminicola strain T01193. Disease symptoms scored at 21-day post-inoculation (dpi) were significantly higher on the susceptible and moderately resistant cultivars, Alixan and Premio (48% and 42% of diseased leaf area, respectively), than in the resistant one, Altigo (28% of diseased leaf area). Regarding sporulation, the rate of pycnidial density was significantly higher on Alixan (2.9) compared to Premio and Altigo (1.1 and 1.0, respectively). Further biochemical investigations revealed, by 17 dpi, significant fungal beta-1,4-endoxylanase, beta-1,4-endoglucanase and protease activities, whose amounts increased according to the pycnidial density recorded on the infected leaves. At 21 dpi, the amounts of these activities were significantly higher on Alixan compared to Premio and Altigo (0.36 U/mg, 0.63 U/mg and 2.70 mU/mg total proteins on Alixan, 0.09 U/mg, 0.19 U/mg and 0.72 mU/mg total proteins on Premio and 0.05 U/mg, 0.15 U/mg and 0.52 mU/mg total proteins on Altigo for beta-1,4-endoxylanase, beta-1,4-endoglucanase and protease activities, respectively). These results confirm the importance of CWDE and protease activities in the process of fungal sporulation during the necrotrophic phase of M. graminicola.

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.

CHARACTERIZATION OF A TUNISIAN POPULATION OF MYCOSPHAERELLA GRAMINICOLA USING MITOCHONDRIAL DNA MARKERS.

The causal agent of Septoria tritici blotch, Mycosphaerella graminicola, is currently the most frequently reported pathogen on wheat crops worldwide. The populations of this fungus were previously studied in Tunisia using microsatellite markers, but no information is available on the mitochondrial (mt) genetic structure of the fungus in this country. The objective of this study was thus to examine the genetic diversity and population structure of M. graminicola in Tunisia using mtDNA markers. A set 105 monoconidial isolates of the pathogen were sampled in 2012 from four geographical locations in Tunisia (Bizerte, Béja, Kef and Jendouba) and assessed for diversity and polymorphism of three mtDNA sequences (Mg1: 388 bp, Mg2: 570 bp, Mg3: 929 bp) using PCR-RFLP-SSCP. Mg2 and Mg3 were the more polymorphic sequences with 12 and 9 observed alleles, respectively, while Mg1 was the less polymorphic one with only two alleles. Data analyses revealed a moderate mt gene diversity and a low mt haplotype diversity within the population. Further analyses showed a low population differentiation (G(ST) = 0.16) and a moderate gene flow (Nm = 2.61) within the population. Both UPGMA and Bayesian clustering methods confirmed this lack of mt genetic structuration. Our study allowed us to characterize for the first time mt genetic structure of M. graminicola in Tunisia and to report a moderate mt gene diversity, a low mt genotype diversity and a lack of mt genetic structure of the parasite in this country.

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.

PROTECTION EFFICACY AND MODES OF ACTION OF TWO RESISTANCE INDUCERS ON WHEAT AGAINST SEPTORIA TRITICI BLOTCH.

Septoria tritici blotch caused by Mycosphaerella graminicola is one of the most devastating foliar diseases of wheat. Disease control relies heavily on fungicides, but frequent development of fungal resistance and the negative impact of their extensive use on the environment and human health increasingly compromise this control strategy. Plant resistance inducers could be an alternative to conventional fungicides to control in a more durable manner this pathogen. Here, we tested in the greenhouse two resistance inducers (FSOV7 and FSOV10) on two wheat cultivars, Alixan (susceptible) and Altigo (resistant), against M. graminicola. FSOV7 conferred a significant protection level on both cultivars, while FSOV10 conferred a significant protection level on the resistant cv. Altigo only. Furthermore, the modes of action of the two inducers were examined using cytological, biochemical and molecular approaches. In planta, investigation of the infection process showed that FSOV10 significantly reduced fungal spore germination, whereas FSOV7 did not. An association of protection efficacy with a decrease of fungal biomass and fungal ß-1, 4-endoxylanase and protease activities was observed in both cultivars. However, no association of plant peroxidase activity with protection efficacy was recorded, whatever the cultivar and the resistance inducer. A RT-qPCR assay revealed significant inductions of the expression of genes involved in different defence pathways; further statistical analyses should determine which genes are associated with the observed protection efficacies. This study showed significant inducer-cultivar interactions on wheat against M. graminicola and allowed us to investigate the modes of action on wheat of the two studied resistance inducers.

BOTH MAT1-1 AND MAT1-2 MATING TYPES OF MYCOSPHAERELLA GRAMINICOLA OCCUR AT EQUAL FREQUENCIES IN ALGERIA.

Septoria tritici blotch caused by Mycosphaerella graminicola is currently the most devastating disease on wheat crops worldwide. Mycosphaerella graminicola sexual reproduction involves two mating type idiomorphs that were previously studied in several areas around the world, but not in Algeria so far. The objective of this study was thus to determine the frequencies and distribution of M. graminicola mating types in this country. One hundred and twenty monoconidial isolates of this fungus (60 from bread wheat and 60 from durum wheat) were collected during the 2012 growing season from five distinct geographical locations in Algeria. The mating type of each isolate was identified using a multiplex PCR that amplifies either MAT1-1 or MAT1-2 fragment from mating type loci. Both idiomorphs were found at equal frequencies according to the chi-square test at the whole country level (46% MAT1-1 and 54% MAT1-2) and in each of the sampled locations. The two mating types were also detected at equal frequencies on both host species (47% MAT1-1 vs 53% MAT1-2 on bread wheat and 45% MAT1-1 vs 55% MAT1-2 on durum wheat). Our study showed that the two mating types of M. graminicola occur at equal proportions in Algeria and suggests a strong potential for sexual reproduction of the pathogen in this country that may eventually lead to either adaptation to local conditions, plant resistance overcoming or the emergence of resistance to fungicides.

Genetic diversity of Mycosphaerella graminicola isolates from a single field.

Septoria tritici blotch caused by Mycosphaerella graminicola is currently one of the most economically damaging diseases on wheat crops worldwide. Two hundred and sixty single-conidial isolates of this fungus were sampled in April 2012 in the Nord-Pas de Calais region (France). They have all been collected from 13 distinct plots in a single field. The corresponding isolates were then fingerprinted using 8 microsatellite markers in order to assess the genetic diversity and population structure of M. graminicola at the single field scale. The results revealed a high genotypic diversity within the collected population, with the detection of 83% of unique haplotypes among the isolates tested (clonal fraction = 17%). A high genic diversity was also found as indicated by the Nei's index value (0.50) and strong allele diversity obtained (number of alleles per locus ranged from 7 to 17, with an average of 10 alleles per locus). Further analyses showed a low population differentiation (G(ST) = 0.08) and a high gene flow (Nm = 5.64) between the 13 sampled plots. Our study suggests that sexual reproduction, by its frequency, plays a major role in the genetic diversification of M. graminicola at the field level and in the distribution and homogenization of this diversity in the field via wind-born ascospores.

In vitro evaluation of dill seed essential oil antifungal activities to control Zymoseptoria tritici.

Zymoseptoria tritici (teleomorph: Mycosphaerella graminicola) is the causal agent of Septoria Leaf Blotch of wheat (up to 40% yield loss). The study aims to evaluate the antifungal activities of dill seed Essential Oil (EO) on this pathogen to investigate an alternative solution to decrease the use of synthetic fungicides in the context of sustainable agriculture. Thus, two strains of Zymoseptoria tritici in relation to their sensitivity to DOMIs were tested in microplates (S6, sensitive strain; R1187, resistant strain). The essays were repeatedly carried out with dill seed EO crude, with Tween 80 (5% v/v) or with DMSO (1% v/v). A range of nine EO dilutions were tried out in comparison to two fungicides: a DMI (metconazole) and a SDHI (Boscalid). A Gas Chromatography - Mass Spectrometry (GC-MS) analysis reveals that dill seed EO is mostly composed of Carvone (45%) and Limonene (25%). Consequently, D-Carvone/L-Carvone and D-Limonene/L-Limonene were tested as well to determine the origins of the EO effectiveness observed. The IC50 (half maximal inhibitory concentration) are calculated and then statistically analysed to find significant differences between each product tested. The comparison of the IC50 shows that S6 is more sensitive to metconazole than R1187 but they both have the same sensitivity to Boscalid. Dill seed EO, D-Carvone/L-Carvone have the same effectiveness on S6 whatever the preparation tested. D-Limonene/L-Limonene are less efficient unless they are supplemented with Tween 80. On R1187, Dill seed EO is more efficient when it is prepared with Tween 80. This efficiency is also observed for D-Carvone/L-Carvone whatever the preparation tested. D-Limonene/L-Limonene are generally less efficient than Carvone even if L-limonene is as efficient as L-Carvone when these products are prepared with Tween 80. Dill seed EO used crude or with DMSO is more efficient on S6 (350 mg/L) than on R1187 (1000 mg/L) but with Tween 80, the EO effectiveness is the same on both strains (300 mg/L). So DMSO doesn't have any effects on both strains while Tween 80 seemsto improve the EO efficiency on the resistant strain. Carvone appears to be more efficient than Limonene, which indicates that the major compound is largely responsible for the observed EO efficiencies. In Addition, the EO preparation seems to play a role in the antifungal activities, especially on the resistant strain than the sensitive strain. The two fungicides tested are still the most efficient molecules in the control of Zymoseptoria tritici.

Tunisian population of Mycosphaerella graminicola is still sensitive to strobilurin fungicides.

Septoria tritici blotch caused by the fungal pathogen Mycosphaerella graminicola (anamorph: Zymoseptoria tritici) is one of the most frequently occurring diseases on both bread and durum wheat crops worldwide. One hundred and sixty four durum wheat-adapted isolates of this fungus were sampled during the 2012 growing season from five distinct geographical locations of Tunisia (Bizerte, Béja, Kef, Jendouba and Siliana) in order to examine the status of strobilurin resistance of M. graminicola in this country. Resistance was assessed by screening the G143A substitution (Cytochrome b) which confers resistance to this class of fungicides. We used a PCR-based mismatch mutation assay allowing the amplification of either G143 (sensitive) or A143 (resistant) allele. All isolates were found to contain the sensitive wild-type G143 allele and therefore to be sensitive. Our study confirms recent reports on M. graminicola in Tunisia and shows that the Tunisian population of the fungus remains fully sensitive to strobilurins. A durability-oriented management of strobilurin applications in Tunisia is thereby recommended to prevent the development and widespread of the corresponding resistance such as in Europe, where pathogen populations are nowadays fully resistant to strobilurins.

Assessment of the cytochrome B substitution G143A in the Algerian population of Mycosphaerella graminicola.

Mycosphoerella graminicola (anamorph: Zymoseptoria tritici), causal agent of Septoria tritici blotch, is currently one of the most damaging diseases on both bread and durum wheat crops worldwide. Since wheat resistance against this pathogen is always partial at various extents in most cultivars, disease control relies mainly on the use of fungicides. However, management of fungicide applications is necessary in order to avoid the emergence and widespread of fungicide resistant genotypes within populations of the pathogen. In the present study, we investigated for the first time the resistance of M. graminicola toward strobilurin fungicides in Algeria. This was performed by identifying the G143A substitution of the cytochrome b encoding sequence (which confers resistance to strobilurins) in a collection of 120 single-conidial isolates. These isolates have been sampled during the 2012 growing season from five distinct geographical locations (Guelma, Annaba, Constantine, Skikda and Oran). We used a PCR-based mismatch mutation assay allowing the amplification of either G143 (sensitive) or A143 (resistant) allele in each isolate. This study should give valuable information regarding the management of strobilurin use in order to control in a durable manner M. graminicola epidemics in Algeria.

Evolution of Mycosphaerella graminicola resistance to epoxiconazole and pyrifenox in northern France.

DMIs have been for many years the most used antifungal compounds against Septoria tritici blotch caused by Mycosphaerella graminicola, the most important foliar disease on winter wheat worldwide. Their use leads to a development and widespread of isolates with reduced sensitivity in several populations around Europe, especially in France. Here, we investigated the resistance level to epoxiconazole and pyrifenox of 48 M. graminicola isolates (24 from 2009 and 24 from 2011) sampled in Nord-Pas de Calais (France) from two untreated plots naturally infected by the fungus. The resistance levels were determined by microplate assays at 10 different concentrations for each fungicide (geometric progression x 3.5). Overall, IC50 (half maximal inhibitory concentration) values were slightly higher in the 2011-isolates compared to the 2009-isolates. For the 2009-isolates, IC50 values ranged from 0.14 mg/L to 2.25 mg/L and from 0.10 mg/L to 2.19 mg/L for epoxiconazole and pyrifenox, respectively, while IC50 values for 2011-isolates ranged from 0.10 mg/L to 22.13 mg/L and from 0.07 mg/L to 11.63 mg/L for epoxiconazole and pyrifenox, respectively. The determination of resistant factors (IC50 resistant isolate/IC50 sensitive isolate) revealed an increase of rates of isolates showing a resistant factor higher than 100 from 21% to 62 % for epoxiconazole and from 8% to 42% for pyrifenox between 2009 and 2011. Furthermore, statistical analyses revealed a positive correlation between resistances to epoxiconazole and pyrifenox in both years, indicating that most of isolates resistant for epoxiconazole are also resistant for pyrifenox. This study shows a shift in sensitivity to epoxiconazole and pyrifenox in northern France between 2009 and 2011 and suggests a relationship between resistance to epoxiconazole and pyrifenox in M. graminicola.

Evaluation of plant resistance inducers on different winter soft wheat cultivars against Septoria leaf blotch.

Septoria tritici blotch (STB) caused by Mycosphaerella graminicola (anamorph: Zymoseptoria tritici) is one of the most devastating foliar diseases on bread wheat (Triticum aestivum L.). Because of the emergence of fungal strains highly resistant to mainly used fungicides and the deleterious impacts of these fungicides on the environment, development of alternative control strategies to protect wheat crops against STB is needed. The induction of plant resistance by elicitors is likely to be a helpful alternative. Our study aims at characterizing the efficiency of potential resistance inducers towards STB in three bread wheat cultivars differing in their resistance levels to the pathogen: Alixan (susceptible), Premio (moderately resistant) and Altigo (resistant). These cultivars were inoculated under controlled and semi-controlled conditions with the pathogenic M. graminicolo strain T01193 in order to assess the protective effect of three potential resistance inducers against the disease. Moreover, the direct antifungal effect bf these products was evaluated in vitro at different concentrations in order to verify their potential biocide activity. Furthermore, cytological analyses were performed in order to determine the effects of these products on the fungal infection process and to compare these effects among the three wheat cultivars. Finally, reactive oxygen species metabolism was investigated in the three cultivars during their interaction with T01193 by measuring peroxidase activity.

Wheat Mycosphaerella graminicola interactions in Morocco.

Mycosphaerella graminicola is nowadays one of the most important foliar pathogens on wheat crops worldwide and more specifically in Morocco. The interactions of eight monoconidial isolates of this fungus, sampled in different regions of Morocco, with 3 Moroccan wheat cultivars (Massira, Amal and Arrihan) differing in their resistance level to the pathogen, were studied using artificial inoculations in the greenhouse. Disease notations (percentage of third leaf areas covered by lesions bearing pycnidia) at 21 days post inoculation revealed significant differences among the different isolate-cultivar combinations. Most isolates expressed pathogenicity profiles in accordance with the resistance levels of the cultivars studied. However, T01701 showed a similar pathogenicity level on the three cultivars and T01718 induced more disease on the moderately resistant cv. Amal compared to the susceptible cv. Massira and the resistant cv. Arrihan, respectively. Furthermore, the infection process of the isolate T01757 was investigated at 4, 8, 12, 16 and 21 days post-inoculation. This isolate exhibited a disease gradient (65%, 24% and 5%) which negatively correlated with the resistance levels of the cultivars. No significant differences were obtained regarding the rates of spore germination and leaf penetration events. However, rates of mesophyll colonization positively correlated with the disease levels scored on the cultivars. This study supports the presence of strain-cultivar interactions between wheat and M. graminicola in Morocco and confirms the importance of mesophyll colonization in disease establishment and extension.

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.