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.

Phosphorus supply, arbuscular mycorrhizal fungal species, and plant genotype impact on the protective efficacy of mycorrhizal inoculation against wheat powdery mildew.

A potential alternative strategy to chemical control of plant diseases could be the stimulation of plant defense by arbuscular mycorrhizal fungi (AMF). In the present study, the influence of three parameters (phosphorus supply, mycorrhizal inoculation, and wheat cultivar) on AMF protective efficiency against Blumeria graminis f. sp. tritici, responsible for powdery mildew, was investigated under controlled conditions. A 5-fold reduction (P/5) in the level of phosphorus supply commonly recommended for wheat in France improved Funneliformis mosseae colonization and promoted protection against B. graminis f. sp. tritici in a more susceptible wheat cultivar. However, a further decrease in P affected plant growth, even under mycorrhizal conditions. Two commercially available AMF inocula (F. mosseae, Solrize®) and one laboratory inoculum (Rhizophagus irregularis) were tested for mycorrhizal development and protection against B. graminis f. sp. tritici of two moderately susceptible and resistant wheat cultivars at P/5. Mycorrhizal levels were the highest with F. mosseae (38 %), followed by R. irregularis (19 %) and Solrize® (SZE, 8 %). On the other hand, the highest protection level against B. graminis f. sp. tritici was obtained with F. mosseae (74 %), followed by SZE (58 %) and R. irregularis (34 %), suggesting that inoculum type rather than mycorrhizal levels determines the protection level of wheat against B. graminis f. sp. tritici. The mycorrhizal protective effect was associated with a reduction in the number of conidia with haustorium and with an accumulation of polyphenolic compounds at B. graminis f. sp. tritici infection sites. Both the moderately susceptible and the most resistant wheat cultivar were protected against B. graminis f. sp. tritici infection by F. mosseae inoculation at P/5, although the underlying mechanisms appear rather different between the two cultivars. This study emphasizes the importance of taking into account the considered parameters when considering the use of AMF as biocontrol agents.

Are ineffective defence reactions potential target for induced resistance during the compatible wheat-powdery mildew interaction?

Powdery mildew caused by Blumeria graminis f.sp. tritici, an obligate aerial biotrophic fungus, would be one of the most damaging wheat (Triticum aestivum) diseases without the extensive use of conventional fungicides. In our study, the expression levels of some basal defence-related genes were investigated during a compatible interaction in order to evaluate wheat reactions to infection, along with the different stages of the infectious process in planta. As fungal conidia initiated their germination and developed appressorial germ tube (AGT), early defence reactions involved the expression of a lipoxygenase (LOX)- and an oxalate oxidase (OXO)-encoding genes, followed by activations of corresponding LOX (EC 1.13.11.12) and OXO (EC 1.2.3.4) activities, respectively. When penetration of AGT took place, up-regulation of chitinases (CHI) and PR1-encoding genes expression occurred along with an increase of CHI (EC 3.2.1.14) activity. Meanwhile, expression of a phenylalanine ammonia-lyase-encoding gene also took place. Up-regulation of a phospholipase C- and lipid transfer proteins-encoding genes expression occurred during the latest stages of infection. Neither the phi glutathione S-transferase (GST)-encoding gene expression nor the GST (EC 2.5.1.13) activity was modified upon wheat infection by powdery mildew. Whether these defence reactions during such a compatible interaction are markers of immunity or susceptibility, and whether they have the ability to contribute to protection upon modulation of their timing and their intensity by resistance inducers are discussed.

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.

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.

MECHANISMS INVOLVED IN MYCORRHIZAL WHEAT PROTECTION AGAINST POWDERY MILDEW.

In France, the Ecophyto 2018 national action plan will set out to reduce the use of pesticides by 50% by 2018, if possible. To achieve this goal, the use of arbuscular mycorrhizal (AM) fungi could be a potential alternative method allowing the control of crop diseases. The inoculation by AM fungi has been demonstrated to protect plants against soil-borne pathogens, but little is known about their effectiveness against aerial pathogens, such as the biotrophic fungus Blumeria graminis f.sp. tritici (Bgt) causing wheat (Triticum aestivum) powdery mildew. In the present study, wheat plants were grown in pots, under controlled conditions. Using various phosphorus (P) concentrations, the effectiveness of three AM inocula (Rhizophagus irregularis (Ri), Funneliformis mosseae (Fm)) and Solrize, a mixture of Ri and Fm) in Orvantis wheat cultivar, were tested. After 42 days of culture, mycorrhizal (M) and non-mycorrhizal (NM) wheat plants were infected by Bgt. A satisfactory mycorrhizal rate was obtained with the phosphorus concentration P/5 (P corresponding to the dose used in wheat fields in = 62 mg/L). Our work shows, for the first time, (i) a protective effect of AM inoculation against wheat powdery mildew, reaching up to 73% with Fm inocula, and (ii) its ability to induce a systemic resistance in wheat. Thereafter, we investigated mechanisms involved in this protection. Control plants, M plants, infected plants by Bgt, and M-infected plants were compared at: (i) cytological level, our results revealed that papillae and whole-fluorescent cells presence was induced, conversely fungal haustorium formation in epidermal cells was reduced within M plants leaves (ii) enzymatic level-by assessing defense enzyme activities (lipoxygenase, peroxidase) known as defense markers were measured 24, 48, 72 and 96 hours after infection (hai). The importance of these activities in the defense pathways induced in wheat by AM fungi will be discussed.

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.

Lipids as markers of induced resistance in wheat: a biochemical and molecular approach.

Our work aimed at a global investigation of the lipid metabolism during the induction of resistance in wheat (Triticum aestivum) against powdery mildew (Blumeria graminis f.sp. tritici). More specifically, the effect of salicylic acid, known as playing a key role in the activation of defence reactions against pathogens in plants, has been investigated. After salicylic acid infiltration, accumulation of phosphatidic acid was observed that could be due to the phospholipase C pathway since an up-regulation of a phospholipase C-encoding gene expression as well as an accumulation of diacylglycerol were observed. The phosphatidic acid accumulation could also result from the phospholipase D pathway since a reduction of phosphatidylethanolamine content occurred. The response to salicylic acid at the octadecanoid pathway level was also investigated: both a lipoxygenase-encoding gene expression and lipoxygenase enzymatic activity were induced by salicylic acid simultaneously with a decrease of the linolenic acid content. Finally, a lipid transfer protein-encoding gene expression was also up-regulated upon salicylic acid infiltration. These observations indicate that lipid metabolism could be considered as a marker of elicitation in wheat.

Towards a more sustainable agriculture: wheat mycorrhization to protect against powdery mildew.

One of the means to reduce the use of pesticides, which are harmful for humans and the environment, is the development of alternative methods to control crop diseases. In this context, arbuscular mycorrhizal inoculation possesses a great potential for crop production by a more sustainable agriculture. Our work aims to (i) determine the optimal conditions for wheat mycorrhization (ii) study the impact of arbuscular mycorrhizal inoculation on a foliar disease of wheat, powdery mildew (Blumeria graminis f.sp. tritici, Bgt), (iii) evaluate the stimulation of natural defences of wheat (Triticuma estivum). Therefore, this work consisted firstly of defining the parameters, affecting the establishment of wheat mycorrhization, such as: phosphorus concentration (62, 12.5, 6.2 mg/L), culture time (4, 5, 6, 7 weeks), arbuscular mycorrhizal species used as an inoculum (Rhizophagus irregularis (Ri), Glomus masseae (Gm) and the mixture of (Ri+Gm)) and wheat cultivars (Orvantis and Lord, sensitive and moderately resistant to Bgt, respectively). Secondly, the protective effect of mycorrhizal inoculation against Bgt was estimated by comparing infection rates of wheat seedlings subjected and non-subjected to AMF. Finally, to better understand the biochemical mechanisms involved in the protection, two enzymatic activities described as defense markers [lipoxygenase (LOX) and peroxidase (POX)] were also assessed. Extensive mycorrhization (about 31%) was obtained at P/5 concentration (12.5 mg/L) when wheat plants were 6 weeks old. The highest colonization rate was obtained when wheat was inoculated with Gm compared to SZE and Ri. The higher resistance level of Lord wheat cultivar against Bgt did not affect the mycorrhizal rate compared to the more susceptible cultivar Orvantis. Our work showed a significant protection level in mycorrhizal (M) wheat plants against Bgt, estimated to about 25 and 43% with Ri and SZE respectively compared to non-mycorrhizal (NM) Orvantis plants. The protection levels percent's were about 30 and 64% for Lord plants. The protection was higher for Lord than Orvantis and seems to depend on the resistance degree. These results suggest the induction of a systemic resistance by mycorrhizal inoculation. Our results showed an increase of both activities (LOX and POX) in wheat infected by Bgt for both (M) and (NM) plants by the inoculum SZE (Ri+Gm) at P/5 phosphorus concentration.

Induction of resistance in wheat by bacterial cyclic lipopeptides.

Three families of lipopeptides (LPs), surfactin, iturin (including mycosutilin) and fengycin, produced by the rhizabacterium Bacillus subtilis have received considerable attention for their antimicrobial, cytotoxic, antitumor, immunosuppressant and surfactant properties. Among them, iturins and fengycins generally display a strong in vitro antifungal activity. In addition, surfactins are powerful bio surfactants, and although they don't show any direct fungitoxicity, they exhibit some synergistic effect with the antifungal activity of iturins. The aim of our work is to characterize mycosubtilin, surfactin and fengycin, for their ability to protect wheat against two pathogens, Blumeria graminis f.sp. tritici (Bgt), an obligate parasitic fungus responsible for powdery mildew and Mycosphaerella graminicola (Mg), an hemibiotrophic fungus causing Septoria leaf blotch, two of the most important diseases of this crop. In a first step, we measured the protection efficacy of a preventive treatment with LPs against powdery mildew on wheat leaves. At least 41% and 44% protection levels were obtained when surfactin and mycosubtilin, respectively, were sprayed on wheat leaves at doses corresponding to 4g.ha(-1) in the field. We also tested in vitro the impact of LPs on germination of spores. No direct antifungal effect of LPs was observed on Bgt conidia germination, irrespective of the tested concentrations. However, fengycin strongly decreased the germination of Mg conidia and also considerably delayed its growth. Mycasubtilin completely inhibited conidial germination and therefore also its growth while surfactin did not remarkably affect either germination or growth of this fungus. In a second step, RT-qPCR was used to investigate elicitor and priming effects of surfactin and mycosubtilin on the expression levels of defence-related genes. In non-infectious conditions, both surfactin and mycosubtilin induced the expression of two peroxidase encoding genes (POX2, POX381), however, the expression of LOX (lipoxygenase), AOS (allene oxide synthase) genes involved in the octadecanoid pathway, as well as of the pathogenesis-related genes PR1 was only induced by mycosubtilin. In infectious conditions, only surfactin induced the expression of these genes in response to pBgt, mycosubtilin did not exhibit any priming effect. Depending on the targeted pathogen and on the type of LP, these molecules could exhibit direct antifungal effect and/or defence induction, through either elicitation or potentialisation.

Induction of resistance in wheat against powdery mildew by bacterial cyclic lipopeptides.

In conventional agricultural practices, fungicides are extensively used to control the development of many fungal plant pathogens. However, the reduction of pesticide applications in the field, which is recommended by authorities and approved by public opinion, may lead to a re-emergence of diseases. Alternative strategies have therefore to be developed in order to control fungal infection; one of them is based on plants natural resistance reinforcement caused by elicitors. Our project aims at characterizing mycosubtilin, surfactin and fengycin, three bacterial cyclic non-ribosomal lipopeptides produced by the rhizobacterium Bacillus subtilis, for their ability to induce resistance in wheat against powdery mildew (Blumeria graminis f.sp. tritici). RT-qPCR approach was used to investigate effect of lipopeptides on expression pattern of defence-related genes, such as PR protein-encoding genes or genes involved in plant defence pathways (reactive oxygen species (ROS) metabolism, lipid peroxidation, phenylpropanoid and octadecanoid pathways) in response to fungus and revealed that expression of LOX and PR1 was strongly induced by surfactin at 24h and 18h after inoculation, respectively. Mycosubtilin seemed to be more efficace to induce expression of OXO and Ltp. Surfactin could also induce expression of chitinase encoding genes but neither surfactin nor mycosubtilin could induce the expression of GSTphi and PAL genes in response to the fungus.

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.

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.

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.