Arbuscular mycorrhizal inoculum sources influence bacterial, archaeal, and fungal communities' structures of historically dioxin/furan-contaminated soil but not the pollutant dissipation rate.

Little is known about the influence of arbuscular mycorrhizal fungi (AMF) inoculum sources on phytoremediation efficiency. Therefore, the aim of this study was to compare the effects of two mycorrhizal inocula (indigenous and commercial inocula) in association with alfalfa and tall fescue on the plant growth, the bacterial, fungal, and archaeal communities, and on the removal of dioxin/furan (PCDD/F) from a historically polluted soil after 24 weeks of culture in microcosms. Our results showed that both mycorrhizal indigenous and commercial inocula were able to colonize plant roots, and the growth response depends on the AMF inoculum. Nevertheless, the improvement of root dry weight in inoculated alfalfa with indigenous inoculum and in inoculated tall fescue with commercial inoculum was clearly correlated with the highest mycorrhizal colonization of the roots in both plant species. The highest shoot dry weight was obtained in inoculated alfalfa and tall fescue with the commercial inoculum. AMF inoculation differently affected the number of bacterial and archaeal OTUs and bacterial diversity, with elevated bacterial and archaeal OTUs and bacterial diversity observed with indigenous inoculum. Mycorrhizal inoculation increases the abundance of bacterial OTUs (in particular with indigenous inoculum) and microbial richness but it does not improve PCDD/F dissipation. Vegetation had no effect on the abundance of microbial OTUs nor on richness but stimulated specific communities (Planctomycetia and Gammaproteobacteria) likely to be involved in the dissipation of PCDD/F. The reduction of toxic equivalency PCDD/F concentration also could be explained by the stimulation of soil microbial activities estimated with dehydrogenase and fluorescein diacetate hydrolase.

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.

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.

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.

Field application of mycorrhizal bio-inoculants affects the mineral uptake of a forage legume (Hedysarum coronarium L.) on a highly calcareous soil.

The efficiency of two mycorrhizal bio-inoculants on the mineral uptake during the growth stages of a Mediterranean forage legume sulla (Hedysarum coronarium L.) was studied in the field on a highly calcareous soil. The first inoculum (Mm) was made up of a mixture of native arbuscular mycorrhizal fungi (AMF) isolated from calcareous soils: Septoglomus constrictum, Funneliformis geosporum, Glomus fuegianum, Rhizophagus irregularis and Glomus sp. The second was a commercial inoculum (Mi) containing one AMF species: R. irregularis. Both mycorrhizal inoculants increased total and arbuscular colonization of sulla roots. Inoculation with Mm showed a positive effect on sulla shoot dry weight (SDW) when compared to Mi and non-inoculated plants (control). Mineral contents (P, Mg, Mn, Fe) were higher in the shoots of sulla plants cultivated on mycorrhiza-inoculated plots compared to non-inoculated ones. This enhancement was observed during the flowering stage for P, Mg and Mn and during the rosette stage for Fe. An increase in P content of 50 % in plants inoculated with Mm compared to non-inoculated ones may be explained by the induction of root alkaline and acid phosphatase activities. Higher efficiency of native AMF species adapted to calcareous soils opens the way towards the development of mycorrhiza bio-fertilizers targeted to improve sustainable fertilization management in such soils.

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.

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.

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.

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.

Evolution of Mycosphaerella graminicola at the wheat leaf level and at the field level.

Evolution of M. graminicola wheat field populations from a given French county (Morbihan, 56) between years 2005 and 2006 was investigated for thirty seven strains using molecular fingerprinting by microsatellite markers (ST1A4, ST1E3, ST1E7, and ST1D7), and SSCP analysis study of partial actin and beta-tubulin encoding sequences. In addition, twenty nine strains collected from 3 distinct lesions on a same wheat leaf in 2006 in another French county (Nord, 59) were also investigated for genetic diversity. At the field level, we observed similar gene diversity in the 2005 and in the 2006 populations, with no common clones between the two years. This indicates frequent sexual recombination undergone by the fungus. When considering each marker independently and comparing genetic variability of the two populations, we noticed a decrease in genetic variability of the 2006 strains for three of them (ST1A4, ST1D7 and the partial sequence of actin) and an increase for ST1E3, ST1E7 and the partial sequence of beta-tubulin, revealing the importance of the chosen markers. At the lesion level, 69% of the studied strains were haplotypes with 31% of the clonal population found in 2 lesions out of 3. This suggests that at least parts of the lesions were formed after asexual reproduction and dissemination of pycnidiospores by splashing. We also confirmed the exploitative competition that exists between the strains at the lesion level.

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.

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.

The detection of Glomus spp. (arbuscular mycorrhizal fungi) forming mycorrhizas in three plants, at different stages of seedling development, using mycorrhiza-specific isozymes.

A series of glasshouse experiments was used to determine mycorrhiza-specific isozymes (MSIs) produced by five species of Glomus colonizing roots of a desert shrub legume (Anthyllis cytisoides L.), Thymus vulgaris L. and Allium porrum L. over time. Extracts of colonized roots were electrophoresed on non-denaturing polyacrylamide gels (PAGE) and stained for 10 different enzymes. Staining protocols for esterase, glutamate oxaloacetate transaminase, alkaline phosphatase and malate dehydrogenase provided MSIs for the mycorrhizas formed by different arbuscular mycorrhizal (AM) fungi that had colonized roots of the three host plants. There was no apparent correlation between levels of colonization or arbuscular intensities, at or between each sampling, and the presence of MSIs. The development of colonization by the AM fungi differed little between the three plants when assessed with two methods of estimating fungal biomass. The variety of MSIs detected might reflect the diversity of metabolic activities of these Glomus species and, possibly, differing ecological functions. The high-level induction of two alkaline phosphatase MSIs in the mycorrhizas of Anthyllis cytisoides colonized by Glomus microaggregatum BEG56 was used to track the fate of this fungus when the same plant was inoculated and transplanted into a semi-arid site in south-east Spain. The probable fungal origin of the isozyme was indicated by detection of the same isozyme in the extraradical mycelium formed by Glomus microaggregatum BEG56 on Allium porrum. The use of MSIs to detect the mycorrhizas of species of Glomus in colonized roots is discussed.

Arbuscular mycorrhizal induced changes to plant growth and root system morphology in Prunus cerasifera.

We compared root system morphogenesis of micropropogated transplants of Prunus cerasifera L. inoculated with either of the arbuscular mycorrhizal (AM) fungi Glomus mosseae or Glomus intraradices or with the ericoid mycorrhizal species Hymenoscyphus ericae. All plants were grown in sand culture, irrigated with a nutrient solution that included a soluble source of phosphorus, for 75 days after transplanting. Arbuscular mycorrhizal colonization increased both the survival and growth (by over 100%) of transplants compared with either uninoculated controls or transplants inoculated with H. ericae. Arbuscular mycorrhizal colonization increased root, stem and leaf weights, leaf area, root length and specific leaf area, and it decreased root length/leaf area ratio, root/shoot weight ratio and specific root length. Both uptake of phosphorus and its concentration in leaves were increased by AM infection, although the time course of the relationships between intensity of AM infection and P nutrition were complex and suggested a role for factors other than nutrition. The time course for the development of infection varied. It was most rapid with G. mosseae, but it was ultimately higher with G. intraradices. None of the treatments significantly affected the lengths of adventitious roots or the first-, second- or third-order laterals that developed from them. Arbuscular mycorrhizal colonization increased the intensity of branching in all root orders with the effect being most obvious on first-order lateral roots where the number of branches increased from under 100 to over 300 brances m(-1). As a result, although first-order laterals made up 55% of the root systems of control plants, the comparable value was 36% in AM-infected plants. In contrast, second-order laterals represented 25% of control root systems, but 50% of AM-colonized root systems. Glomus intraradices but not G. mosseae increased root diameter. Anatomical studies revealed no changes in the overall form of the root tip, although there were changes in the diameter of the root cap, cell numbers and cell size. Hymenoscyphus ericae increased the duration of the metaphase index. Both AM fungal treatments increased the concentrations of soluble proteins in root extracts and modified the protein profiles by the elimination and addition of protein bands detected by PAGE analysis. We conclude that AM fungal inoculation influenced processes in the root system at different levels, but not all effects were due to improved P nutrition or increased physiological age.