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.

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.

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.

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.

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.

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.

Both mating types of the wheat pathogen Mycosphaerella graminicola are present in Morocco.

Septoria tritici blotch caused by the heterothallic ascomycete Mycosphaerella graminicola is one of the most currently damaging diseases on wheat crops worldwide. So far, no information was reported about the status of sexual reproduction of this pathogen under Moroccan conditions. We investigated here for the first time the occurrence of the two mating types (MAT1-1 and MAT1-2) of M. graminicola in Morocco by sampling 141 single-conidial isolates from 4 important wheat producing regions (Gharb, Saïs, Chaouia and Tadla). The mating type of each isolate was determined by amplification with multiplex PCR of a partial sequence from the corresponding idiomorph. Overall, 43% out of the assessed isolates were MAT1-1 and 57 % were MAT1-2. Both mating types were identified within the 3 sampled regions Gharb, Saïs and Chaouia, but not in Tadla, where only MAT1-2 isolates were found. The presence of the two mating types highlighted here offers a suitable genetic condition for M. graminicola to occur sexual reproduction in Morocco. The potential of sexual recombination will be examined by the study of mating type frequencies using a large sample size as well as by searching and quantification of pseudothecia in the field.

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.

Assessment of Mycosphaerella graminicola resistance to azoxystrobin.

Azoxystrobin resistance levels of twenty two strains sampled from ten French locations and two reference isolates (IPO323 and IPO94269) of the wheat pathogen Mycosphaerella graminicola were investigated in vitro. French strains assayed were selected from twenty two genetic groups determined from three hundred sixty three strains previously characterised using microsatellites, actine and beta-tubuline markers. For the first time, the evaluation was carried out using four distinct methods: spotting on PDA medium, spore germination on PDA medium and using microtitre plates with and without Alamar blue, a growth indicator. From dose-response curve, half maximal inhibitory concentration (IC50) was determined for each strain. The results obtained using microtitre plates with the addition of Alamar blue displayed high standard deviations from the growth averages observed. Therefore, we suggest that this method is inadequate to assess M. graminicolo resistance to fungicides. However, a good correlation was observed between the rankings of strains according to their IC50 values with the three other methods used. The two reference isolates, as expected, were inhibited by low azoxystrobin concentrations. On the other hand, the IC50 values obtained showed presence of a threshold between sensitive and resistant strains that corroborates the disruptive resistance of M. graminicola against strobilurin fungicides. In addition, the strains showing resistance were those sampled mainly from northern France, where a high frequency of strobilurin resistant isolates among M. graminicola populations was reported by several studies.

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.