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An experimental setup was devised to investigate the permeability of cocoa bean seed coat and pulp to key volatile compounds during fermentation. Four labeled compounds (ethyl acetate-d3, ethyl octanoate-d15, 2-phenylethanol-d5, linalool-d5) and 2 unlabeled (beta-damascenone, delta-decalactone) were chosen for the investigation. The beans (cotyledons), depulped beans, or pulped beans were immersed separately in a concentrated solution of these volatile compounds at 36 or 46 °C for durations ranging from 3 to 120 h. The imbibed beans were dissected, and the cotyledons were analyzed by SPME-GC/MS. The diffusion of volatile compounds from the external solution to the seed was categorized into three groups: (1) not diffusible (ethyl octanoate-d15); (2) semidiffusible (ethyl acetate); and (3) totally diffusible (2-phenylethanol-d5, linalool-d5, beta-damascenone, delta-decalactone). The impact of the yeast on volatile compound diffusion was also investigated by immerging the pulped beans into the same concentrated solution with a yeast starter. Results highlighted the positive role of yeast in the diffusion of volatile compounds. The starter positively contributed to volatile compound diffusion after a transition phase occurring at approximately 48 h of fermentation, enriching the cocoa beans with key aromatic volatile compounds.

The quality of chocolate products proven by consumer acceptability mostly depends on cocoa volatile compounds. In fact, aroma profile of cocoa or chocolate is correlated to cocoa genotype, culture conditions, postharvest practices, and chocolate manufacturing. To the best of our knowledge, no study has been published on both sugars and aromas of controlled cocoa hybrids from Cameroon. The present study aimed at analyzing biochemical and volatile composition of six hand-pollinated cocoa hybrids belonging to different traditional groups. Specifically, the sugar profile was obtained by high performance ion chromatography-electrochemical detector (HPIC-ED) and aroma composition was analyzed using headspace solid phase micro-extraction coupled with gas chromatography-mass spectrometry (HS-SPME/GC-MS) on fermented dried beans and roasted nibs. The content of sugars was variable among hybrids and fructose showed the highest concentration in all hybrids in the two matrices. Roasting decreased sugar content. However, the decrease in sugar content seemed genotype dependent. A total of 48 volatile aromas were classified in six groups according to their odor description (fruity, floral, chocolate/nutty, buttery/creamy, undesirable, and unknown). Fruity flavors were not specific to a given chemical group. However, floral volatiles were specific to terpenes, chocolate/nutty to pyrazines, buttery/creamy to lactones and undesirable to phenols and acids. Principal component analysis (PCA) showed specificity of each hybrid and the hierarchical cluster analysis (HCA) Heatmap presented four distinct groups. SCA12 × ICS40 with the highest pyrazine concentration was associated with important content of sugars. Roasting of nibs reduced total volatiles and particularly fruity volatiles whereas other flavor groups moderately increased. In fact, some volatiles such as 2,3-butanediol, 2-nonanol, linalool, β-myrcene, cis/trans-β-ocimene, 2-nonanone, 2-heptanol, methyl acetate, acetophenone could be used as markers in separating traditional cocoa groups in Cameroon.

Severe Acute Malnutrition (SAM) is still a public health issue in many low-income and middle-income countries. Its management has changed with the development of Ready-to-Use Therapeutic Foods (RUTF) that are energy-dense, lipid-based, micronutrient-enriched foods. The purpose of the present study was to define some aspects of the nutritional profile (in particular, lipid content, fatty acids profile, lipophilic vitamin content), oxidative status, and mycotoxin content of three RUTF products (of which two manufactured in Asia and one in Europe), at different time points during their storage. All RUTF displayed good lipid stability, with peroxide values < 10 meqO2/kg lipids, acidity index < 0.6 g/100 g lipids, and very low amounts of secondary oxidation volatile compounds. Only the one produced in Europe complied with the international recommendations on vitamin A and E contents. Lipid content ranged from 28.7 ± 0.5 to 41.2 ± 2.4 g/100 g. Oleic acid, palmitic acid, and linoleic acid (47.2 ± 7.4%, 26.8 ± 2.1%, and 12.4 ± 3.9% of total lipids, respectively) were the main fatty acids. Only one of the samples made in Asia exceeded the regulatory limit of mycotoxins. The RUTF lipid phase was rather stable at ambient temperature from 9 to 12 months, and appropriate packaging may have contributed to a major stability.

The order Magnaporthalescomprises about 200 species and includes the economically and scientifically important rice blast fungus and the take-all pathogen of cereals, as well as saprotrophs and endophytes. Recent advances in phylogenetic analyses of these fungi resulted in taxonomic revisions. In this paper we list the 28 currently accepted genera in Magnaporthaleswith their type species and available gene and genome resources. The polyphyletic Magnaporthe1972 is proposed for suppression, and Pyricularia1880 and Nakataea1939 are recommended for protection as the generic names for the rice blast fungus and the rice stem rot fungus, respectively. The rationale for the recommended names is also provided. These recommendations are made by the Pyricularia/MagnaportheWorking Group established under the auspices of the International Commission on the Taxonomy of Fungi (ICTF).

The present paper represents the second contribution in the Genera of Fungi series, linking type species of fungal genera to their morphology and DNA sequence data, and where possible, ecology. This paper focuses on 12 genera of microfungi, 11 of which the type species are neo- or epitypified here: Allantophomopsis (A. cytisporea, Phacidiaceae, Phacidiales, Leotiomycetes), Latoruagen. nov. (Latorua caligans, Latoruaceae, Pleosporales, Dothideomycetes), Macrodiplodiopsis (M. desmazieri, Macrodiplodiopsidaceae, Pleosporales, Dothideomycetes), Macrohilum (M. eucalypti, Macrohilaceae, Diaporthales, Sordariomycetes), Milospium (M. graphideorum, incertae sedis, Pezizomycotina), Protostegia (P. eucleae, Mycosphaerellaceae, Capnodiales, Dothideomycetes), Pyricularia (P. grisea, Pyriculariaceae, Magnaporthales, Sordariomycetes), Robillarda (R. sessilis, Robillardaceae, Xylariales, Sordariomycetes), Rutola (R. graminis, incertae sedis, Pleosporales, Dothideomycetes), Septoriella(S. phragmitis, Phaeosphaeriaceae, Pleosporales, Dothideomycetes), Torula (T. herbarum, Torulaceae, Pleosporales, Dothideomycetes)and Wojnowicia(syn. of Septoriella, S. hirta, Phaeosphaeriaceae, Pleosporales, Dothideomycetes).Novel species include Latorua grootfonteinensis, Robillarda africana, R. roystoneae, R. terrae, Torula ficus, T. hollandica, and T. masoniispp. nov, and three new families: Macrodiplodiopsisceae, Macrohilaceae, and Robillardaceae.Authors interested in contributing accounts of individual genera to larger multi-authored papers to be published in IMA Fungus, should contact the associate editors listed for the major groups of fungi on the List of Protected Generic Names for Fungi (www.generaoffungi.org).

SummaryWhat might arithmetic look like on an island that eschews carry digits? How would primes, squares and other number theoretical concepts play out on such an island?

Fungi inhabit every natural and anthropogenic environment on Earth. They have highly varied life-styles including saprobes (using only dead biomass as a nutrient source), pathogens (feeding on living biomass), and symbionts (co-existing with other organisms). These distinctions are not absolute as many species employ several life styles (e.g. saprobe and opportunistic pathogen, saprobe and mycorrhiza). To efficiently survive in these different and often changing environments, fungi need to be able to modify their physiology and in some cases will even modify their local environment. Understanding the interaction between fungi and their environments has been a topic of study for many decades. However, recently these studies have reached a new dimension. The availability of fungal genomes and development of postgenomic technologies for fungi, such as transcriptomics, proteomics and metabolomics, have enabled more detailed studies into this topic resulting in new insights. Based on a Special Interest Group session held during IMC9, this paper provides examples of the recent advances in using (post-)genomic approaches to better understand fungal interactions with their environments.

Pls1 tetraspanins were shown for some pathogenic fungi to be essential for appressorium-mediated penetration into their host plants. We show here that Podospora anserina, a saprobic fungus lacking appressorium, contains PaPls1, a gene orthologous to known PLS1 genes. Inactivation of PaPls1 demonstrates that this gene is specifically required for the germination of ascospores in P. anserina. These ascospores are heavily melanized cells that germinate under inducing conditions through a specific pore. On the contrary, MgPLS1, which fully complements a PaPls1 ascospore germination defect, has no role in the germination of Magnaporthe grisea nonmelanized ascospores but is required for the formation of the penetration peg at the pore of its melanized appressorium. P. anserina mutants with mutation of PaNox2, which encodes the NADPH oxidase of the NOX2 family, display the same ascospore-specific germination defect as the PaPls1 mutant. Both mutant phenotypes are suppressed by the inhibition of melanin biosynthesis, suggesting that they are involved in the same cellular process required for the germination of P. anserina melanized ascospores. The analysis of the distribution of PLS1 and NOX2 genes in fungal genomes shows that they are either both present or both absent. These results indicate that the germination of P. anserina ascospores and the formation of the M. grisea appressorium penetration peg use the same molecular machinery that includes Pls1 and Nox2. This machinery is specifically required for the emergence of polarized hyphae from reinforced structures such as appressoria and ascospores. Its recurrent recruitment during fungal evolution may account for some of the morphogenetic convergence observed in fungi.

ABSTRACTPls1 tetraspanins were shown for some pathogenic fungi to be essential for appressorium-mediated penetration into their host plants. We show here that Podospora anserina, a saprobic fungus lacking appressorium, contains PaPls1, a gene orthologous to known PLS1genes. Inactivation of PaPls1demonstrates that this gene is specifically required for the germination of ascospores in P. anserina. These ascospores are heavily melanized cells that germinate under inducing conditions through a specific pore. On the contrary, MgPLS1, which fully complements a ?PaPls1ascospore germination defect, has no role in the germination of Magnaporthe griseanonmelanized ascospores but is required for the formation of the penetration peg at the pore of its melanized appressorium. P. anserinamutants with mutation of PaNox2, which encodes the NADPH oxidase of the NOX2 family, display the same ascospore-specific germination defect as the ?PaPls1mutant. Both mutant phenotypes are suppressed by the inhibition of melanin biosynthesis, suggesting that they are involved in the same cellular process required for the germination of P. anserinamelanized ascospores. The analysis of the distribution of PLS1and NOX2genes in fungal genomes shows that they are either both present or both absent. These results indicate that the germination of P. anserinaascospores and the formation of the M. griseaappressorium penetration peg use the same molecular machinery that includes Pls1 and Nox2. This machinery is specifically required for the emergence of polarized hyphae from reinforced structures such as appressoria and ascospores. Its recurrent recruitment during fungal evolution may account for some of the morphogenetic convergence observed in fungi.

ABSTRACTMagnaporthe griseais responsible for a devastating fungal disease of rice called blast. Current control of this disease relies on resistant rice cultivars that recognize M. griseasignals corresponding to specific secreted proteins encoded by avirulence genes. The M. grisea ACE1avirulence gene differs from others, since it controls the biosynthesis of a secondary metabolite likely recognized by rice cultivars carrying the Pi33resistance gene. Using a transcriptional fusion between ACE1promoter and eGFP, we showed that ACE1is only expressed in appressoria during fungal penetration into rice and barley leaves, onion skin, and cellophane membranes. ACE1is almost not expressed in appressoria differentiated on Teflon and Mylar artificial membranes. ACE1expression is not induced by cellophane and plant cell wall components, demonstrating that it does not require typical host plant compounds. Cyclic AMP (cAMP) signaling mutants ?cpkAand ?mac1 sum1-99and tetraspanin mutant ?pls1::hphdifferentiate melanized appressoria with normal turgor but are unable to penetrate host plant leaves. ACE1is normally expressed in these mutants, suggesting that it does not require cAMP signaling or a successful penetration event. ACE1is not expressed in appressoria of the buf1::hphmutant defective for melanin biosynthesis and appressorial turgor. The addition of hyperosmotic solutes to buf1::hphappressoria restores appressorial development and ACE1expression. Treatments of young wild-type appressoria with actin and tubulin inhibitors reduce both fungal penetration and ACE1expression. These experiments suggest that ACE1appressorium-specific expression does not depend on host plant signals but is connected to the onset of appressorium-mediated penetration.

Magnaporthe grisea is responsible for a devastating fungal disease of rice called blast. Current control of this disease relies on resistant rice cultivars that recognize M. grisea signals corresponding to specific secreted proteins encoded by avirulence genes. The M. grisea ACE1 avirulence gene differs from others, since it controls the biosynthesis of a secondary metabolite likely recognized by rice cultivars carrying the Pi33 resistance gene. Using a transcriptional fusion between ACE1 promoter and eGFP, we showed that ACE1 is only expressed in appressoria during fungal penetration into rice and barley leaves, onion skin, and cellophane membranes. ACE1 is almost not expressed in appressoria differentiated on Teflon and Mylar artificial membranes. ACE1 expression is not induced by cellophane and plant cell wall components, demonstrating that it does not require typical host plant compounds. Cyclic AMP (cAMP) signaling mutants cpkA and mac1 sum1-99 and tetraspanin mutant pls1::hph differentiate melanized appressoria with normal turgor but are unable to penetrate host plant leaves. ACE1 is normally expressed in these mutants, suggesting that it does not require cAMP signaling or a successful penetration event. ACE1 is not expressed in appressoria of the buf1::hph mutant defective for melanin biosynthesis and appressorial turgor. The addition of hyperosmotic solutes to buf1::hph appressoria restores appressorial development and ACE1 expression. Treatments of young wild-type appressoria with actin and tubulin inhibitors reduce both fungal penetration and ACE1 expression. These experiments suggest that ACE1 appressorium-specific expression does not depend on host plant signals but is connected to the onset of appressorium-mediated penetration.

Introduction. Among pineapple varieties (Ananas comosus [L.] Merr.), variability in sensory characteristics is low between clones but it is marked between varieties. Ten Cayenne ? Perolera hybrids were selected by Cirad for commercial purposes. They presented aromatic profiles distinct from the parents. The pineapple must be collected at the green-ripe stage to be able to arrive in good condition on the consuming markets. Since the flavor released can influence the consumer to buy the fresh fruit, we studied, in different varieties and hybrids, the impact of the maturation stage of the fruit harvested and of the storage conditions on the volatile compound emission. Materials and methods. The differences in volatile compound content between the pineapple varieties were measured by extraction in headspace by SPME (PDMS 100 ?m fiber), followed by CPG/FID analysis on a Stabilwax column. The analyses were related to the fruits of four or five clones of the varieties Cayenne, Perolera, Queen and Rondon, as well as five selected hybrids by Cirad and another commercial hybrid , MD2. The impact of the maturity stage (soluble solids and acidity) and of the fruit storage conditions on the volatile compounds was assessed. Results and discussion. The aromatic profiles obtained for the clones of the same variety showed a certain similarity and the differences between varieties appeared repeatable. During maturation, an increase in the volatile compound emission occurred before full maturation. Storage in cold conditions between 13??C and 24??C caused a significant increase in the aromatic emission. Conclusion. To differentiate the aromatic profiles by SPME /GC-FID from different varieties of pineapple seems possible in spite of their great similarity. Analysis by GC-MS would allow the better characterization of the volatile pineapple compounds of fruits of various origins.Introduction. Chez Ananas comosus, les variations entre clones des caract?ristiques organoleptiques sont faibles? celles entre vari?t?s sont plus marqu?es. Une dizaine d?hybrides Cayenne ? Perolera a ?t? s?lectionn?e par le Cirad pour ?tre d?velopp?e commercialement. Ils pr?sentent des profils aromatiques distincts des parents. L?ananas doit ?tre r?colt? relativement vert pour pouvoir parvenir en bon ?tat sur les march?s consommateurs. L?ar?me d?gag? pouvant inciter le consommateur ? acheter le fruit frais, nous avons ?tudi?, sur diff?rentes vari?t?s et hybrides, l?impact du stade de maturation du fruit ? la r?colte et de la dur?e de conservation sur l??mission des compos?s volatils. Mat?riel et m?thodes. Les diff?rences de teneurs en compos?s volatils entre les vari?t?s d?ananas ont pu ?tre mesur?es par extraction en espace de t?te par SPME (fibre PDMS 100??m), suivie d?une analyse CPG/FID sur colonne Stabilwax. Les analyses ont port? sur les fruits de quatre ou cinq clones de chacune des vari?t?s Cayenne, Perolera, Queen, Rondon, ainsi que de cinq hybrides Cirad et d?un hybride MD2. L?impact du stade de maturit? (extrait sec et acidit? libre) et des conditions de conservation sur les compos?s volatils a ?t? ?valu?. R?sultats et discussion. Les profils aromatiques obtenus pour les clones d?une m?me vari?t? ont ?t? tr?s semblables et les diff?rences intervari?tales se sont r?v?l?es r?p?tables. Au cours de la maturation, une augmentation de l??mission des compos?s volatils s?est produite avant la pleine maturation. La conservation au froid entre 13??C et 24??C a provoqu? une forte augmentation de l??mission aromatique. Conclusion. Diff?rencier les profils aromatiques par SPME/GC-FID de diff?rentes vari?t?s d?ananas semble possible malgr? leur grande similitude. Une identification plus pouss?e, par GC-MS, des compos?s volatils d?ananas de diff?rentes origines pourrait permettre de mieux pr?ciser leur caract?risation.

Introduction. Chez Ananas comosus, les variations entre clones des caractéristiques organoleptiques sont faibles ; celles entre variétés sont plus marquées. Une dizaine d’hybrides Cayenne × Perolera a été sélectionnée par le Cirad pour être développée commercialement. Ils présentent des profils aromatiques distincts des parents. L’ananas doit être récolté relativement vert pour pouvoir parvenir en bon état sur les marchés consommateurs. L’arôme dégagé pouvant inciter le consommateur à acheter le fruit frais, nous avons étudié, sur différentes variétés et hybrides, l’impact du stade de maturation du fruit à la récolte et de la durée de conservation sur l’émission des composés volatils. Matériel et méthodes. Les différences de teneurs en composés volatils entre les variétés d’ananas ont pu être mesurées par extraction en espace de tête par SPME (fibre PDMS 100 µm), suivie d’une analyse CPG/FID sur colonne Stabilwax. Les analyses ont porté sur les fruits de quatre ou cinq clones de chacune des variétés Cayenne, Perolera, Queen, Rondon, ainsi que de cinq hybrides Cirad et d’un hybride MD2. L’impact du stade de maturité (extrait sec et acidité libre) et des conditions de conservation sur les composés volatils a été évalué. Résultats et discussion. Les profils aromatiques obtenus pour les clones d’une même variété ont été très semblables et les différences intervariétales se sont révélées répétables. Au cours de la maturation, une augmentation de l’émission des composés volatils s’est produite avant la pleine maturation. La conservation au froid entre 13 °C et 24 °C a provoqué une forte augmentation de l’émission aromatique. Conclusion. Différencier les profils aromatiques par SPME/GC-FID de différentes variétés d’ananas semble possible malgré leur grande similitude. Une identification plus poussée, par GC-MS, des composés volatils d’ananas de différentes origines pourrait permettre de mieux préciser leur caractérisation.

Introduction. L’identification des composés d’arôme des dattes permet d’apprécier leur qualité organoleptique ; elle revêt en outre un intérêt technologique en guidant les industriels dans certains processus de transformation du fruit et de production d’extraits d’arômes à partir des variétés de faible qualité, augmentant ainsi leur valeur marchande. Notre étude a eu pour objectif d’identifier des composés volatils responsables de l'arôme de huit variétés de dattes marocaines. Une classification statistique des variétés étudiées, basée sur leurs profils aromatiques, et une comparaison avec les résultats de travaux antérieurs effectués sur les composés d’arôme des dattes ont été également réalisées. Matériel et méthodes. Les composés volatils ont été analysés par la technique de piégeage dynamique de l’espace de tête et par désorption au micro-onde, puis ils ont été conjointement identifiés par chromatographie en phase gazeuse-spectrométrie de masse. L’étude statistique des données a été réalisée par analyse factorielle des correspondances et par classification hiérarchique ascendante. Résultats et discussion. Quarante-sept composés ont été identifiés dont vingt-trois non identifiés auparavant dans la datte. Cinq composés : la 2,3-pentanedione, le 2-méthyl-butanal, l’hexanal, le n-pentanol et le limonène se sont révélés être communs à toutes les variétés, tandis que certains composés ont été caractéristiques d’une variété particulière. La variété ‘Aziza’ s’est distinguée par la présence de cyclobutanol, de p-cymène et de 1,3-diméthyl-benzène. Le cyclopentadécadièn-1-one, l’éthanol et le géraniol ont été identifiés respectivement dans les variétés ‘Najda’, ‘Mejhoul’ et ‘Boufeggous’. En revanche, les variétés ‘Bouskri’ et ‘Iklane’ ont présenté une grande similarité de leurs profils aromatiques. Conclusion. Des différences de profils aromatiques entre les huit variétés étudiées ont été mises en évidence et des composés volatils « spécifiques » ont pu être identifiés.

Introduction. As a living organism, fruit is a site of intense gas exchange due to respiration. Carbon dioxide, oxygen and ethylene are the main components involved in this biological activity; they influence the fruit maturity and ripening phenomena. To help studies on pineapple ripening, this paper presents an easy technique to recover and measure the internal atmosphere of pineapple. Materials methods. Experiments were performed with 30 pineapples (two varieties) at the maturity stage. After a procedure validation with standard gas, the sampling technique was applied to pineapple pieces. The fruit’s internal atmosphere was released after scratching the fruit into a calcium chloride-saturated brine. The recovered gas was transferred into an air-tight flask equipped with a septum and a bladder. The headspace was analysed with a gas chromatograph or an IR analyser, at atmospheric pressure. Results. Twenty complete procedure cycles were done with three different standard gases to test the reproducibility of the technique. Each pineapple of each variety was sampled once according to the procedure. All of the results were situated in the best standardisation range for reproducibility. Conclusion. The main advantages and drawbacks of the technique are presented.

Introduction. As a living organism, fruit is a site of intense gas exchange due to respiration. Carbon dioxide, oxygen and ethylene are the main components involved in this biological activity; they influence the fruit maturity and ripening phenomena. To help studies on pineapple ripening, this paper presents an easy technique to recover and measure the internal atmosphere of pineapple. Materials methods. Experiments were performed with 30 pineapples (two varieties) at the maturity stage. After a procedure validation with standard gas, the sampling technique was applied to pineapple pieces. The fruit’s internal atmosphere was released after scratching the fruit into a calcium chloride-saturated brine. The recovered gas was transferred into an air-tight flask equipped with a septum and a bladder. The headspace was analysed with a gas chromatograph or an IR analyser, at atmospheric pressure. Results. Twenty complete procedure cycles were done with three different standard gases to test the reproducibility of the technique. Each pineapple of each variety was sampled once according to the procedure. All of the results were situated in the best standardisation range for reproducibility. Conclusion. The main advantages and drawbacks of the technique are presented.

Eight coating formulations were evaluated for beneficial effects on the shelf‐life of mangoes (cvs. Kent, Tommy Atkins, and Lirfa) harvested at different maturity stages. Four coatings were then selected for further study under various conditions of storage including ambient (19–22C and56–60% RH) and simulated commercial (12C and 80% RH) storage. The four coating formulations contained carnauba wax, shellac, zein, and/or cellulose derivatives. All coatings reduced the respiratory rate, the development of external and internal color, and all but carnauba wax retarded the loss of firmness. Changes in acids were also delayed in all coated mangoes. Based on these parameters, fruit maturation was estimated to have been delayed by a few days compared with uncoated fruit. Shellac and cellulose‐based coatings, however, caused elevated levels of ethanol, although this did not lead to significant flavor differences from the control (uncoated fruit) in sensory tests. Only the carnauba wax coating was an effective water loss barrier under conditions of high RH.

Pasteurized pineapple juice was concentrated by osmotic evaporation to produce a 51 d̀Brix concentrate that was reconstituted to single strength juice for evaluation. Headspace gas chromatography (HSGC) showed that the concentrate retained an average of 62% of the volatile components present in the initial juice. A sensory panel preferred initial juice over reconstituted concentrate, and noted a decrease in desirable flavor top notes as well as development of some processed flavor in the concentrate. Similar HSGC analysis of four other commercial juice samples showed a wide range of quantitative values for volatile components, with the initial juice being similar to the weakest of these commercial juices. Analysis of concentrated juice extracts permitted identification of additional less‐volatile components not monitored directly by HSGC of the juice. Relatively low levels of these components were also present in the initial juice. Although this nonthermally produced concentrate retains more volatile components than when traditional thermal processing methods are used, addition of aqueous aroma to the concentrate may be required for satisfactory flavor.

We characterized two spontaneous developmental mutants of the heterothallic AscomyceteMagnaporthe grisea.The first mutant, isolated from finger millet isolate OG2, sporulated abundantly when grown on artificial medium. Compared to wild type, the sporulation rate of OG2-HSP was 10 times higher on artificial medium and 3 times lower from infected leaves. OG2-HSP was unable to develop perithecia in crosses (female sterile) and behaved only as a male. These two characters always cosegregated as a single gene named HSP1 (high sporulation on plates). A second spontaneous mutant, isolated fromM. griseafinger millet isolate OG5, differentiated perithecia in homokaryotic culture. These perithecia did not contain asci or ascospores, whatever their developmental stage. This mutant was still hermaphroditic in crosses with strains of opposite mating type. Genetic analysis showed that this mutant character was controlled by a single gene named SFP1 (self-formation of sterile perithecia). In crosses heterozygous for bothsfp1andhsp1,all progeny sporulating abundantly on plates (Hsp) was female sterile and unable to develop sterile perithecia in homokaryotic culture, showing thathsp1-1was epistatic tosfp1-1.These mutants will be particularly useful to study the control of sporulation and perithecium development inM. grisea.

ABSTRACTDelineating species and epidemic lineages in fungal plant pathogens is critical to our understanding of disease emergence and the structure of fungal biodiversity and also informs international regulatory decisions. Pyricularia oryzae(syn. Magnaporthe oryzae) is a multihost pathogen that infects multiple grasses and cereals, is responsible for the most damaging rice disease (rice blast), and is of growing concern due to the recent introduction of wheat blast to Bangladesh from South America. However, the genetic structure and evolutionary history of M. oryzae, including the possible existence of cryptic phylogenetic species, remain poorly defined. Here, we use whole-genome sequence information for 76 M. oryzaeisolates sampled from 12 grass and cereal genera to infer the population structure of M. oryzaeand to reassess the species status of wheat-infecting populations of the fungus. Species recognition based on genealogical concordance, using published data or extracting previously used loci from genome assemblies, failed to confirm a prior assignment of wheat blast isolates to a new species (Pyricularia graminis-tritici). Inference of population subdivisions revealed multiple divergent lineages within M. oryzae, each preferentially associated with one host genus, suggesting incipient speciation following host shift or host range expansion. Analyses of gene flow, taking into account the possibility of incomplete lineage sorting, revealed that genetic exchanges have contributed to the makeup of multiple lineages within M. oryzae. These findings provide greater understanding of the ecoevolutionary factors that underlie the diversification of M. oryzaeand highlight the practicality of genomic data for epidemiological surveillance in this important multihost pathogen.IMPORTANCEInfection of novel hosts is a major route for disease emergence by pathogenic microorganisms. Understanding the evolutionary history of multihost pathogens is therefore important to better predict the likely spread and emergence of new diseases. Magnaporthe oryzaeis a multihost fungus that causes serious cereal diseases, including the devastating rice blast disease and wheat blast, a cause of growing concern due to its recent spread from South America to Asia. Using whole-genome analysis of 76 fungal strains from different hosts, we have documented the divergence of M. oryzaeinto numerous lineages, each infecting a limited number of host species. Our analyses provide evidence that interlineage gene flow has contributed to the genetic makeup of multiple M. oryzaelineages within the same species. Plant health surveillance is therefore warranted to safeguard against disease emergence in regions where multiple lineages of the fungus are in contact with one another.

Background Filamentous fungi synthesize many secondary metabolites and are rich in genes encoding proteins involved in their biosynthesis. Genes from the same pathway are often clustered and co-expressed in particular conditions. Such secondary metabolism gene clusters evolve rapidly through multiple rearrangements, duplications and losses. It has long been suspected that clusters can be transferred horizontally between species, but few concrete examples have been described so far.Results In the rice blast fungus Magnaporthe grisea, the avirulence gene ACE1 that codes for a hybrid polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) belongs to a cluster of 15 genes involved in secondary metabolism. Additional related clusters were detected in the ascomycetes Chaetomium globosum, Stagonospora nodorum and Aspergillus clavatus. Gene-by-gene phylogenetic analysis showed that in C. globosum and M. grisea, the evolution of these ACE1-like clusters is characterized by successive complex duplication events including tandem duplication within the M. grisea cluster. The phylogenetic trees also present evidence that at least five of the six genes in the homologous ACE1 gene cluster in A. clavatus originated by horizontal transfer from a donor closely related to M. grisea.Conclusion The ACE1 cluster originally identified in M. grisea is shared by only few fungal species. Its sporadic distribution within euascomycetes is mainly explained by multiple events of duplication and losses. However, because A. clavatus contains an ACE1 cluster of only six genes, we propose that horizontal transfer from a relative of M. grisea into an ancestor of A. clavatus provides a much simpler explanation of the observed data than the alternative of multiple events of duplication and losses of parts of the cluster.

RAPD markers are frequently used for positional cloning. However, RAPD markers often contain repeated sequences which prevent genomic library screening by hybridisation. We have developed a simple RAPD analysis of genomic libraries based on the identification of cosmid pools and clones amplifying the RAPD marker of interest. Our method does not require the cloning or characterisation of the RAPD marker as it relies on the analysis of cosmid pools or clones using a simple RAPD protocol. We applied this strategy using four RAPD markers composed of single copy or repeated sequences linked to avirulence genes of the rice blast fungus Magnaporthe grisea. Cosmids containing these RAPD markers were easily and rapidly identified allowing the construction of physical contigs at these loci.