Search

Geographical distribution and diversity of current plant species have been strongly shaped by climatic oscillations during the Quaternary. Analysing the resulting divergence among species and differentiation within species is crucial to understand the evolution of taxa like the Vitis genus, which provides very useful genetic resources for grapevine improvement and might reveal original recolonization patterns due to growth habit and dispersal mode. Here, we studied the genetic structure in natural populations of three species from eastern North America: Vitis aestivalis, V. cinerea and V. riparia using different marker types. Vitis aestivalis and V. cinerea showed higher diversity than V. riparia. The two former species are less differentiated, confirming an earlier divergence of V. riparia. V. aestivalis and V. riparia exhibited different genetic groups on both sides of the Appalachian Mountains that could mirror different recolonization routes from southern refugia. Genetic structure was stronger in V. cinerea, for which two varieties (var. berlandieri and var. cinerea) are morphologically recognized. Our results confirm this distinction and suggest the existence of three other lineages within var. cinerea. These discontinuities appear linked to adaptation of var. berlandieri to dry and limy areas of Texas and partially to the Mississippi River Valley. Rapid range expansions from refugia upon climate warming are also suggested by the low linkage disequilibrium values observed. Furthermore, large variation for downy mildew resistance was observed in the three species. Our findings appeared consistent with the vegetation history of eastern North America.

Crop breeding involves two selection steps: choosing progenitors and selecting offspring within progenies. Genomic prediction, based on genome-wide marker estimation of genetic values, could facilitate these steps. However, its potential usefulness in grapevine (Vitis viniferaL.) has only been evaluated in non-breeding contexts mainly through cross-validation within a single population. We tested across-population genomic prediction in a more realistic breeding configuration, from a diversity panel to ten bi-parental crosses connected within a half-diallel mating design. Prediction quality was evaluated over 15 traits of interest (related to yield, berry composition, phenology and vigour), for both the average genetic value of each cross (cross mean) and the genetic values of individuals within each cross (individual values). Genomic prediction in these conditions was found useful: for cross mean, average per-trait predictive ability was 0.6, while per-cross predictive ability was halved on average, but reached a maximum of 0.7. Mean predictive ability for individual values within crosses was 0.26, about half the within-half-diallel value taken as a reference. For some traits and/or crosses, these across-population predictive ability values are promising for implementing genomic selection in grapevine breeding. This study also provided key insights on variables affecting predictive ability. Per-cross predictive ability was well predicted by genetic distance between parents and when this predictive ability was below 0.6, it was improved by training set optimization. For individual values, predictive ability mostly depended on trait-related variables (magnitude of the cross effect and heritability). These results will greatly help designing grapevine breeding programs assisted by genomic prediction.

To cope with the challenges facing agriculture, speeding-up breeding programs is a worthy endeavor, especially for perennial species such as grapevine, but requires understanding the genetic architecture of target traits. To go beyond the mapping of quantitative trait loci in bi-parental crosses, we exploited a diversity panel of 279 Vitis vinifera L. cultivars planted in 5 blocks in the vineyard. This panel was phenotyped over several years for 127 traits including yield components, organic acids, aroma precursors, polyphenols, and a water stress indicator. The panel was genotyped for 63k single nucleotide polymorphisms by combining an 18K microarray and genotyping-by-sequencing. The experimental design allowed to reliably assess the genotypic values for most traits. Marker densification via genotyping-by-sequencing markedly increased the proportion of genetic variance explained by single nucleotide polymorphisms, and 2 multi-single nucleotide polymorphism models identified quantitative trait loci not found by a single nucleotide polymorphism-by-single nucleotide polymorphism model. Overall, 489 reliable quantitative trait loci were detected for 41% more response variables than by a single nucleotide polymorphism-by-single nucleotide polymorphism model with microarray-only single nucleotide polymorphisms, many new ones compared with the results from bi-parental crosses. A prediction accuracy higher than 0.42 was obtained for 50% of the response variables. Our overall approach as well as quantitative trait locus and prediction results provide insights into the genetic architecture of target traits. New candidate genes and the application into breeding are discussed.

Fomitiporia mediterranea M. Fisch. (Fmed) is a basidiomycete first described in 2002, and was considered up to then as part of Fomitiporia punctata (P. Karst) Murrill. This fungus can degrade lignocellulosic biomass, causing white rot and leaving bleached fibrous host residues. In Europe Fmed is considered the main grapevine wood rot (Esca) agent within the Esca disease complex, which includes some of the most economically important Grapevine Trunk Diseases (GTDs). This review summarises and evaluates published research on Fmed, on white rot elimination by curettage or management by treatments with specific products applied to diseased grapevines, and on the relationship between wood symptoms and Grapevine Leaf Stripe Disease (GLSD) in the Esca disease complex. Information is also reviewed on the fungus biology, mechanisms of pathogenicity, and their possible relationships with external foliar symptoms of the Esca disease complex. Information on Fmed control strategies is also reviewed.

Breeding new cultivars is an important lever to cope with the various challenges facing viticulture. As a prerequisite, it is necessary to assess how much genetic variation is available for the traits of interest, and to understand their genetic architecture. To go beyond quantitative trait locus (QTL) mapping in biparental crosses, we exploited the panel of 279 cultivars capturing most of the genetic and phenotypic diversity of Vitis vinifera L. present within the French collection of genetic resources (INRA Vassal). This panel was planted in the vineyard in five randomized complete blocks and numerous traits (berry weight, cluster weight, length and compactness, organic acids, δ13C, polyphenols, etc.) were phenotyped over several years. In parallel, the panel was genotyped with the GrapeReSeq microarray as well as with restriction-assisted DNA sequencing. This allowed us to conduct a genome-wide association study per trait, either by testing each single-nucleotide polymorphism (SNP) separately or by modeling all SNPs jointly. Focusing here on berry weight, we showed that genotyping by sequencing provided enough SNPs to explain most of the genetic variance, and that, compared with SNP-by-SNP approaches, a multi-SNP statistical model led to a gain in power. Several significant associations were detected, some being new compared with the QTLs detected in biparental crosses. Future prospects include setting up an international consortium to plant the panel on various sites to help to decipher genotype-environment interactions.

International audience; Key message A half-diallel population involving five elite grapevine cultivars was generated and genotyped by GBS, and highly-informative segregation data was used to construct a high-density genetic map for Vitis vinifera L. Abstract Grapevine is one of the most relevant fruit crops in the world. Deeper genetic knowledge could assist modern grapevine breeding programs to develop new wine grape varieties able to face climate change effects. To assist in the rapid identification of markers for crop yield components, grape quality traits and adaptation potential, we generated a large Vitis vinifera L. population (N = 624) by crossing five red wine cultivars in a half-diallel scheme, which was subsequently sequenced by an efficient GBS procedure. A high number of fully informative genetic variants was detected using a novel mapping approach capable of reconstructing local haplotypes from adjacent biallelic SNPs, which were subsequently used to construct the densest consensus genetic map available for the cultivated grapevine to date. This 1378.3-cM map integrates 10 bi-parental consensus maps and orders 4437 markers in 3353 unique positions on 19 chromosomes. Markers are well distributed all along the grapevine reference genome, covering up to 98.8% of its genomic sequence. Additionally, a good agreement was observed between genetic and physical orders, adding confidence in the quality of this map. Collectively, our results pave the way for future genetic studies (such as fine QTL mapping) aimed to understand the complex relationship between genotypic and phenotypic variation in the cultivated grapevine. In addition, the method used (which efficiently delivers a high number of fully informative markers) could be of interest to other outbred organisms, notably perennial fruit crops.

The major Grapevine Trunk Diseases (GTD) gather diseases associated with either one particular fungal species, e.g., eutypiose, or with fungal species complexes, e.g., Botryosphaeriae dieback. GTD-associated fungi are part of different fungal classes, depending on their substrate degradation mechanisms or on their lifestyles. They are identified in grapevine wood and they lead to wood typical symptoms. They are sometimes associated to foliar symptoms too, despite not found in aerial parts, probably acting through fungal toxins or through physiological disorders, such as non-functional wood vessels. Grapevine is a liana, and as such shows structural and chemical differences compared to forest tree wood. However, knowledge on wood-degrading fungi growing on tree wood can contribute to a better understanding of grapevine wood adaptation mechanisms to fungal attack. For example, wood density, xylem vessels size and number, or lignin content and composition can play a role in wood sensitivity to fungi. Also, some fungal detoxification mechanisms against wood extractibles are host-specific. In a first part of this chapter, structure and chemical composition of grapevine wood are described, pointing out properties that might play a role in fungal growth. In a second part, GTD main associated fungi and their known mechanisms for wood degradation or wood chemical detoxification are discussed. Extracellular and intracellular enzymatic and non-enzymatic mechanisms are considered. These mechanisms, well described for their role in fungal adaptation to tree wood, have been much less studied for GTD associated fungi. Further studies about those mechanisms would contribute to understand fungal adaptation to grapevine.

To cope with the challenges faced by agriculture, speeding-up breeding programs is a worthy endeavor, especially for perennials such as grapevine, but requires understanding the genetic architecture of target traits. To go beyond the mapping of quantitative trait locus (QTL) in bi-parental crosses, we exploited a diverse panel of 279 Vitis vinifera L. cultivars. This panel planted in five blocks in the vineyard was phenotyped over several years for 127 traits including yield components, organic acids, aroma precursors, polyphenols, and a water stress indicator. The panel was genotyped for 63k single nucleotide polymorphisms (SNPs) by combining an 18K microarray and genotyping-by-sequencing (GBS). The experimental design allowed to reliably assess the genotypic values for most traits. Marker densification via GBS markedly increased the proportion of genetic variance explained by SNPs, and two multi-SNP models identified QTLs not found by a SNP-by-SNP model. Overall, 489 reliable QTLs were detected for 41% more response variables than by a SNP-by-SNP model with microarray-only SNPs, many new ones compared to the results from bi-parental crosses. Prediction accuracy higher than 0.42 was obtained for 50% of the response variables. Our overall approach as well as QTL and prediction results provide insights into the genetic architecture of target traits. New candidate genes and the application in breeding are discussed.

Impacts of plant genotype on microbial assemblage in the phyllosphere (above-ground parts of plants, which predominantly consists of the set of photosynthetic leaves) of Vitis vinifera cultivars have been studied previously but the impact of grape species (under the grape family Vitaceae) was never investigated. Considering the fact, that the phyllosphere microbiome may have profound effects on host plant health and its performance traits, studying the impact of grape species in microbial taxa structuring in the phyllosphere could be of crucial importance. We performed 16S and ITS profiling (for bacteria and fungi respectively) to access genus level characterization of the microflora present in the leaf phyllosphere of five species within this plant family, sampled in two successive years from the repository situated in the Mediterranean. We also performed α and β-diversity analyses with robust statistical estimates to test the impacts of grape species and growing year, over a two-year period. Our results indicated the presence of complex microbial diversity and assemblages in the phyllosphere with a significant effect of both factors (grape species and growing year), the latter effect is being more pronounced. We also compared separate normalization methods for high-throughput microbiome data-sets followed by differential taxa abundance analyses. The results suggested the predominance of a particular normalization method over others. This also indicated the need for more robust normalization methods to study the differential taxa abundance among groups in microbiome research.

International audience; Plant surface or phyllosphere is the habitat of hyperdiverse microbial communities and it is always exposed to the fluctuating environmental factors, which is thought to be one of the potential drivers of microbial community structuring. Impact of grapevine genotypes in variable environmental factors (i.e., at different geographic locations) on the phyllosphere has never been studied and is the main objective of this report. Using high throughput short amplicon sequencing of 16S rRNA genes and internal transcribed spacer (ITS), we analyzed the impacts of genotypes of Vitis Vinifera (coming from three genetic pool), on the microbial (bacterial and fungal) assemblage in the phyllosphere. First, we performed the analysis of the phyllosphere microbiome while using fifteen genotypes that were chosen to maximize intra-specific diversity and grown in two Mediterranean vineyards. Then, the same analysis was performed on five commercially important varieties of Vitis vinifera that were sampled from three different French agro-climatic zones (or terroir: a combination of climate, soils, and human practices). Our study revealed that, at a particular geographic location, genotypes have an impact on microbial assemblage in the phyllosphere and carposphere of leaf and fruit (or berries), respectively, which is more prominent on the carposphere but the effect of terroir was much stronger than the genotype when the leaf phyllosphere of five grapevine varieties grown in different agro-climatic zones was compared. Impacts of the season and exterior plant organs (leaf and berries) on microbial taxa structuring in the phyllosphere was also assessed and presented in this report.

International audience; The aerial surface of the plant (phyllosphere) is the habitat of complex microbial communities and the structure of this microbiome may be dependent on plant genetic factors, local environment or interactions between them. In this study, we explored the microbial diversity present in the phyllosphere of a very diverse set of grapevine cultivars representing the three genetic pools of the species, grown on an experimental plot at Montpellier (French Mediterranean region). We assessed microbiome variation in the phyllosphere using amplicon sequencing of the 16S rRNA gene and of the internal transcribed spacer (ITS), according to the grapevine genetic pools or cultivars, and organs (i.e. leaves and grape berries). The observed microbiome was complex; out of 542 bacterial genera; Pseudomonas, Pantoea, Sphingomonas, and Acinetobacter were the most abundant and almost ubiquitously present across the samples, and out of 267 fungal genera; Aureobasidium, Alternaria, Mycosphaerella and Aspergillus were most represented. Our results illustrated that the microbial taxa were almost uniformly distributed among the genetic pools and only a few cultivar or genetic pool level differences were found, but a very clear differential taxa abundance was found between the leaf and berry samples. Some genus level associations were also observed with certain genetic pools.

The MybA1 gene in the genus Vitis encodes a transcription factor, belonging to the R2R3 Myb family, that controls the last steps in the anthocyanins biosynthesis pathway. Polymorphism within MybA1 has been associated with color variation in berries of V. vinifera and other Vitis species. In this work, we analyzed the sequence variation in MybA1 both in the subg. Muscadinia and in an extended set of Asian, American and European genotypes of subg. Vitis. Our aims were to infer the evolution of this gene during the speciation process and to identify polymorphisms that could potentially generate changes in gene regulation. The results show that MybA1 experienced many insertions and deletions in non-coding regions but also in the third exon sequence. Owing to the larger set of Vitis species compared here, new indels were identified and the origin of previously described indels was reconsidered. A large number of single nucleotide polymorphisms were found in non-coding regions but also in the sequence coding for the R2R3 domain and the C terminal part of the protein. Some of these changes led to amino acid substitutions and therefore could have modified MybA1 protein activity. Bayesian phylogenetic analysis of all polymorphisms did not provide a consensus tree depicting the geographical partitioning of the species but allowed highlighting several species relationships within subgenus Vitis. Finally, the evolutionary events described could be useful to gain more insight into the role of MybA1 for anthocyanin biosynthesis in grapevine.

UMR AGAP Equipe Diversité, adaptation et amélioration de la vigne (DAAV); Eutypa lata is a fungal pathogen causing severe dieback in vineyards worldwide. This fungus colonizes vines through pruning wounds, eventually causing a brown sectorial necrosis in wood as well as stunted vegetative growth. Several years may pass between infection and the expression of external symptoms, hindering the rapid evaluation of both grapevine cultivars susceptibility and E. lata variation in aggressiveness. We aimed to develop a rapid quantitative method for the assessment of wood colonization after inoculation of cuttings in controlled conditions. We used several grape cultivars varying in susceptibility in the vineyard and fungal isolates with different levels of aggressiveness to monitor wood colonization during a maximum period of 2 months. Re-isolation allowed demonstration of the effects of both cultivars and fungal isolates on the rate of wood colonization. We also developed a real-time PCR method that was efficient in measuring fungal biomass, which was found to be correlated with isolate aggressiveness based on foliar symptom severity. The real-time PCR approach appears to be a useful technology to evaluate grapevine susceptibility to E. lata, and could be adapted to other pathogens associated with grapevine trunk diseases.

We produced a unique large data set of reference transcriptomes to obtain new knowledge about the evolution of plant genomes and crop domestication. For this purpose, we validated a RNA-Seq data assembly protocol to perform comparative population genomics. For the validation, we assessed and compared the quality of de novo Illumina short-read assemblies using data from two crops for which an annotated reference genome was available, namely grapevine and sorghum. We used the same protocol for the release of 26 new transcriptomes of crop plants and wild relatives, including still understudied crops such as yam, pearl millet and fonio. The species list has a wide taxonomic representation with the inclusion of 15 monocots and 11 eudicots. All contigs were annotated using BLAST, prot4EST and Blast2GO. A strong originality of the data set is that each crop is associated with close relative species, which will permit whole-genome comparative evolutionary studies between crops and their wild-related species. This large resource will thus serve research communities working on both crops and model organisms. All the data are available at http://arcad-bioinformatics.southgreen.fr/.

Aim: In the long term, genetic improvement is one of the major strategies to support sustainable wine production in a changing climate. Over the past 5 years, we have developed an interdisciplinary research program that aimed to: i) characterize the impact of temperature increase sensed by the entire plant or individual bunches on the development and functioning of the plant, ii) identify the physiological and molecular mechanisms regulating the response of vegetative and reproductive development to heat stress and iii) develop tools to map quantitative trait loci (QTLs) of plant and berry development in duly controlled, stable, and contrasting environmental conditions.Methods and results: Performing high-throughput genomic analyses combined with the use of innovative experimental designs (fruiting cuttings, microvines, single berry sampling) was critical to decipher the ecophysiological and molecular mechanisms involved in the vine response to high temperature.Conclusion: Warming promotes vegetative growth and hampers plant carbon balance, disturbing flower set and young berry development. High temperatures modify primary and secondary fruit metabolisms, desynchronizing sugar and organic acid metabolisms and delaying sugar and polyphenol accumulation during ripening. The study of day and night transcriptomic and proteomic signatures associated with heat highlighted key players of the response to temperature in the fruit. Significance and impact of the study: Capitalizing on this knowledge, a new program is being proposed for the selection of cultivars limiting the accumulation of sugars in the berry while maintaining other qualitative compounds.

Background As for many crops, new high-quality grapevine varieties requiring less pesticide and adapted to climate change are needed. In perennial species, breeding is a long process which can be speeded up by gaining knowledge about quantitative trait loci linked to agronomic traits variation. However, due to the long juvenile period of these species, establishing numerous highly recombinant populations for high resolution mapping is both costly and time-consuming. Genome wide association studies in germplasm panels is an alternative method of choice, since it allows identifying the main quantitative trait loci with high resolution by exploiting past recombination events between cultivars. Such studies require adequate panel design to represent most of the available genetic and phenotypic diversity. Assessing linkage disequilibrium extent and panel power is also needed to determine the marker density required for association studies. Results Starting from the largest grapevine collection worldwide maintained in Vassal (France), we designed a diversity panel of 279 cultivars with limited relatedness, reflecting the low structuration in three genetic pools resulting from different uses (table vs wine) and geographical origin (East vs West), and including the major founders of modern cultivars. With 20 simple sequence repeat markers and five quantitative traits, we showed that our panel adequately captured most of the genetic and phenotypic diversity existing within the entire Vassal collection. To assess linkage disequilibrium extent and panel power, we genotyped single nucleotide polymorphisms: 372 over four genomic regions and 129 distributed over the whole genome. Linkage disequilibrium, measured by correlation corrected for kinship, reached 0.2 for a physical distance between 9 and 458 Kb depending on genetic pool and genomic region, with varying size of linkage disequilibrium blocks. This panel achieved reasonable power to detect associations between traits with high broad-sense heritability (> 0.7) and causal loci with intermediate allelic frequency and strong effect (explaining > 10 % of total variance). Conclusions Our association panel constitutes a new, highly valuable resource for genetic association studies in grapevine, and deserves dissemination to diverse field and greenhouse trials to gain more insight into the genetic control of many agronomic traits and their interaction with the environment. Electronic supplementary material The online version of this article (doi:10.1186/s12870-016-0754-z) contains supplementary material, which is available to authorized users.

Transcriptome sequences of the grape powdery mildew fungus Erysiphe necator were used to develop microsatellite markers (EST-SSRs) to study its relatively unexplored population structure in its centre of diversity in eastern North America. Screening the transcriptome sequences revealed 116 contigs with candidate microsatellites, from which 11 polymorphic microsatellite markers were developed from 31 markers tested. Eight of these markers were used to genotype isolates from different regions and hosts in the eastern USA and compare them to samples from southern France and Italy. Genetic diversity in the eastern USA is much greater than in Europe. Bayesian cluster analyses showed that 10 isolates from North America have high affinities with, but differ from, European group A; these are referred to as A-like isolates. No isolates with close affinity to European group B were found in the eastern USA. Bayesian analyses also detected genetic differentiation between isolates from Vitis rotundifolia and isolates from other Vitis hosts. Genetic differentiation detected between the northeastern and southeastern USA was mostly attributable to the A-like isolates in the southeast, which are significantly more aggressive than the other populations. This research demonstrates that transcriptome sequencing of fungal pathogens is useful for developing genetic markers in protein-coding regions and highlights the role of these markers in population biology studies of E. necator.

The generalist ascomycete fungus Eutypa lata causes Eutypa dieback of grapevine (Vitis vinifera) worldwide. To decipher the cosmopolitan distribution of this fungus, the population genetic structure of 17 geographic samples was investigated from four continental regions (Australia, California, Europe and South Africa), based on analysis of 293 isolates genotyped with nine microsatellite markers. High levels of haplotypic richness (R = 0·91–1) and absence of multilocus linkage disequilibrium among loci supported the preponderance of sexual reproduction in all regions examined. Nonetheless, the identification of identical multilocus haplotypes with identical vegetative compatibility groups, in some vineyards in California and South Africa, suggests that asexual dispersal of the fungus among neighbouring plants could be a rare means of disease spread. The greatest levels of allelic richness (A = 4·89–4·97) and gene diversity (H = 0·66–0·69) were found in Europe among geographic samples from coastal areas surrounding the Mediterranean Sea, whereas the lowest genetic diversity was found in South Africa and Australia (A = 2·78–3·74; H = 0·49–0·57). Samples from California, Australia and South Africa, which had lower genetic diversity than those of Europe, were also characterized by demographic disequilibrium and, thus, may represent founding populations of the pathogen. Low but significant levels of genetic differentiation among all samples (DEST = 0·12, P = 0·001; FST = 0·03, P = 0·001) are consistent with historical gene flow preventing differentiation at continental scales. These findings suggest that global, human-mediated spread of the fungus may have resulted in its current global distribution.

Aim : Vitis subg. Vitis provides an example of a plant disjunction occurring in the Northern Hemisphere. It shows broad morphological variation but is assumed to be a species complex with limited genetic differentiation. Based on a comprehensive sampling of taxa and polymorphism in both chloroplast and nuclear DNA, we assessed genetic variation within this subgenus. Our aims were to clarify the relationships among species and to examine their historical biogeography. Location: Asia, Europe, North America. Methods: We analysed a total of 30 species and putative hybrids from subgenus Vitis and examined the infra-specific variation in some species. Polymorphism in chloroplast DNA was assessed in trnL and trnH-psbA-trnK sequences (c. 2170 bp) and in 15 microsatellite loci. We also obtained nuclear data for size variation at 24 microsatellite loci. Phylogenetic inference was performed with Bayesian analyses. A maximum parsimony network was constructed to depict the evolutionary relationships among haplotypes, and microsatellite data were also subjected to hierarchical clustering analysis using the Ward distance. In addition, we assessed size homoplasy by sequencing both chloroplast and nuclear microsatellite loci. Results Chloroplast polymorphisms resolved subgenus Vitis as a monophyletic group with limited genetic variation. The ancestral haplotypes were found in Eurasia. American taxa all harboured derived haplotypes. Most of them formed a monophyletic group that did not include Vitis californica. The four main haplotypes in Vitis vinifera corresponded to two different origins. Nuclear microsatellites indicated that genetic variation was especially large in North America. Asian species exhibited a lower level of nuclear divergence and the European V. vinifera corresponded to a differentiated nuclear lineage. Main conclusions: We obtained some evidence that subgenus Vitis has an Asian origin and then dispersed to Europe and North America. Geographic separation was followed by diversification, presumably during the Pleistocene, resulting in phylogeographic patterns similar to other biota. In contrast to chloroplast DNA, nuclear DNA shows a larger than expected genetic variation. Our molecular data also highlight the need to re-examine certain aspects of the current subgeneric classification

In this study, 18 microsatellite loci were used to examine the genetic structure and mode of reproduction of Phaeomoniella chlamydospora, the fungus that causes Petri disease of grapevine and is involved in another grapevine disease, esca. A total of 60 southeastern Australian isolates were tested and compared with 64 isolates from southern France. The French population possessed relatively high genotypic diversity (G = 29·2) whilst the Australian population showed low genotypic diversity (G = 11·1), consistent with a limited founder population. Haplotypes from four different grapevine cultivars were not significantly differentiated (Φpt values effectively zero). Likewise, genetic differentiation of haplotypes from different regions within each country was not significant, although small but significant genetic differentiation (9%) was identified between Australia and France. Based on bootstrapped cluster analysis, there did not appear to be any genetic groups within the overall sample of isolates. Significant linkage disequilibrium identified in both countries, together with overrepresented, widespread identical haplotypes, indicated limited genetic recombination and a largely clonal structure consistent with the absence of an observed sexual cycle in this species.

We analysed symptoms, wood lesions and fungi associated with esca in mature organic vineyards in the Languedoc-Roussillon region. Most previous surveys concerning esca syndrome were conducted in conventionally managed vineyards in other regions. We first found that esca may be present at a very low level in vineyards that were not treated with sodium arsenite. Affected vines displayed three types of symptoms: leaves with interveinal necrosis, wilt of entire branches and these two symptoms in combination. During the 3-year survey, not all affected vines displayed symptoms every year and the same vine rarely displayed the same symptom or combination of symptoms in successive years. The incidence of esca appeared to be correlated with the percentage of vines that died during the survey but no correlation was found with either mortality before the survey or with the age of the vineyard. Observation of cross-sections of a total of 210 vines with esca symptoms and isolation of fungi from the wood lesions led to similar results to those obtained in conventionally managed vineyards. Four different lesions were identified: a white rot lesion, a brown lesion in a central position, a brown lesion in a sectorial position and a scattered black spotting in healthy wood. The most frequently observed fungal species were Fomitiporia mediterranea (Fom), Phaeomoniella chlamydospora, Phaeoacremonium aleophilum and Eutypa lata. The white rot lesion caused by Fom was generally accompanied by one or more other lesions in the same vine. Similarly, Fom was generally isolated with one or several other fungi. No relationship was detected between the expression of external symptoms and any specific lesion or fungus. Our study also showed that esca and Eutypa dieback are often superimposed in our region of sampling.

Eutypa lata is an ascomycete fungus causing a severe dieback in grapevine. The genetic structure of populations of E. lata from seven regions in Australia, France, Italy and Spain was examined using 20 random amplified polymorphic DNA (RAPD) markers. In some regions, populations were subdivided and a total of 14 samples were analysed. A total of 231 RAPD haplotypes were found among the 240 isolates. Vegetative compatibility testing further demonstrated that isolates of the same haplotype were genetically distinct. Gene diversity was the highest in the population from northern Italy and lowest in the Alsace region in France. Linkage disequilibrium between pairs of putative loci was very low and most of the multilocus analyses were consistent with the hypothesis of random association of the loci. This suggests that random mating occurred in every population and that the sexual stage shapes the genetic structure of E. lata populations in the regions sampled. Only 6% of the total variability was attributable to differences between populations. Nevertheless, significant differences in allele frequency appeared with respect to six RAPD markers indicating some genetic differentiation between populations. This differentiation appeared attributable to differences between the Italian and Spanish populations and the other populations. We thus hypothesize that a restriction of gene flow exists within Europe. The population from Australia was genetically closer to the French and Spanish populations than to that from Italy. Genetic diversity is associated with considerable variation in aggressiveness, which was assessed on cuttings in the greenhouse in six populations. All populations included a range of isolates differing in aggressiveness, but the Italian population seemed to have more isolates with low aggressiveness.

The aphid relative Daktulosphaira vitifoliae (Fitch) (Homoptera : Phylloxeridae) feeds on the leaves and roots of grapevine (Vitis spp.). The insect was introduced into France from North America around 140 years ago and caused severe damage until control of the root-infesting form was initiated by grafting European cultivars onto resistant rootstocks. The objective of our research was to analyze both the variation in pathogenicity and the genetic diversity of the insect in southern France. Twenty samples of phylloxera were collected from leaf galls throughout the region. Their aggressiveness was monitored by measuring the reduction in root dry mass of artificially inoculated potted plants. One trial was performed with three rootstocks (41B, 3309C and 140R) and another with the highly susceptible Vitis vinifera cv. Carignan. No evidence for preference was shown, but aggressiveness varied widely among phylloxera samples, especially in the Carignan trial. Genetic diversity was assessed using 30 Random Amplified Polymorphic DNA (RAPD) markers obtained with 15 primers. Wide diversity was found and all samples corresponded to distinct RAPD phenotypes. The genetic structure of phylloxera population in Southern France was not showed to be influenced either by the location or by the host.

Isolates of Phaeomoniella chlamydospora (Phc) and Phaeoacremonium aleophilum (Pha), two haploid, deuteromycetous fungi, were obtained from vines showing symptoms of esca disease in different localities in two French regions, and within a single vineyard in one of these regions. The population genetic structure was determined in both fungi using random amplified polymorphic DNA (RAPD) analysis. Populations of Phc showed similar levels of diversity at local and regional levels. The most frequent Phc haplotypes were found in every population, and the frequencies of positive alleles of markers were similar across populations. The hypothesis that recombination had occurred was rejected for the full set of samples, but not for the samples reduced to haplotypes, indicating that Phc may be a recombining species. Different features were identified in Pha populations. First, the southern population of Pha appeared more diverse than the south-western populations. Second, genetic differentiation was identified between Pha populations from southern and south-western regions for several RAPDs. Finally, in the southern population of Pha no evidence for recombination was obtained, even by reducing the sample to haplotypes. Within the single vineyard surveyed, several haplotypes of both fungi were recovered and randomly distributed. Thus different infection events appeared to have occurred on a low spatial scale. Data from this study showed that haplotypes of both fungi were distributed over long distances geographically, and that most of the vineyards surveyed were infested by more than one haplotype of Phc and Pha.

International audience; BACKGROUND: In grapevine, as in other fruit crops, fruit size and seed content are key components of yield and quality; however, very few Quantitative Trait Loci (QTLs) for berry weight and seed content (number, weight, and dry matter percentage) have been discovered so far. To identify new stable QTLs for marker-assisted selection and candidate gene identification, we performed simultaneous QTL detection in four mapping populations (seeded or seedless) with various genetic backgrounds. RESULTS: For berry weight, we identified five new QTLs, on linkage groups (LGs) 1, 8, 11, 17 and 18, in addition to the known major QTL on LG 18. The QTL with the largest effect explained up to 31% of total variance and was found in two genetically distant populations on LG 17, where it colocalized with a published putative domestication locus. For seed traits, besides the major QTLs on LG 18 previously reported, we found four new QTLs explaining up to 51% of total variance, on LGs 4, 5, 12 and 14. The previously published QTL for seed number on LG 2 was found related in fact to sex. We found colocalizations between seed and berry weight QTLs only for the major QTL on LG 18 in a seedless background, and on LGs 1 and 13 in a seeded background. Candidate genes belonging to the cell number regulator CNR or cytochrome P450 families were found under the berry weight QTLs on LGs 1, 8, and 17. The involvement of these gene families in fruit weight was first described in tomato using a QTL-cloning approach. Several other interesting candidate genes related to cell wall modifications, water import, auxin and ethylene signalling, transcription control, or organ identity were also found under berry weight QTLs. CONCLUSION: We discovered a total of nine new QTLs for berry weight or seed traits in grapevine, thereby increasing more than twofold the number of reliable QTLs for these traits available for marker assisted selection or candidate gene studies. The lack of colocalization between berry and seed QTLs suggests that these traits may be partly dissociated.

Background - Grapevine (Vitis vinifera subsp. vinifera) is one of the most important and ancient horticultural plants in the world. Domesticated about 8--10,000 years ago in the Eurasian region, grapevine evolved from its wild relative (V. vinifera subsp. sylvestris) into very diverse and heterozygous cultivated forms. In this work we study grapevine genetic structure in a large sample of cultivated varieties, to interpret the wide diversity at morphological and molecular levels and link it to cultivars utilization, putative geographic origin and historical events. Results - We analyzed the genetic structure of cultivated grapevine using a dataset of 2,096 multi-locus genotypes defined by 20 microsatellite markers. We used the Bayesian approach implemented in the STRUCTURE program and a hierarchical clustering procedure based on Ward's method to assign individuals to sub-groups. The analysis revealed three main genetic groups defined by human use and geographic origin: a) wine cultivars from western regions, b) wine cultivars from the Balkans and East Europe, and c) a group mainly composed of table grape cultivars from Eastern Mediterranean, Caucasus, Middle and Far East countries. A second structure level revealed two additional groups, a geographic group from the Iberian Peninsula and Maghreb, and a group comprising table grapes of recent origins from Italy and Central Europe. A large number of admixed genotypes were also identified. Structure clusters regrouped together a large proportion of family-related genotypes. In addition, Ward's method revealed a third level of structure, corresponding either to limited geographic areas, to particular grape use or to family groups created through artificial selection and breeding. Conclusions - This study provides evidence that the cultivated compartment of Vitis vinifera L. is genetically structured. Genetic relatedness of cultivars has been shaped mostly by human uses, in combination with a geographical effect. The finding of a large portion of admixed genotypes may be the trace of both large human-mediated exchanges between grape-growing regions throughout history and recent breeding.

Algerian grapevines showing symptoms of Eutypa dieback and esca were examined for the presence of Phaeoacremonium species. Species were identified on the basis of morphological and cultural characteristics as well as DNA sequence data (β-tubulin and actin). From a total of 200 vines sampled, 61 Phaeoacremonium isolates were obtained corresponding to four different species. Pm. aleophilum was the most frequently isolated (n=42) followed by Pm. parasiticum (n=10) and Pm. venezuelense (n=8). Pm. hispanicum was also found but only once. Phaeoacremonium species were more frequently associated with Eutypa dieback than esca symptoms. This correlates with their frequent association with sectorial brown necrosis (V-shaped necrosis). Algerian grapevines showing symptoms of Eutypa dieback and esca were examined for the presence of Phaeoacremonium species. Species were identified on the basis of morphological and cultural characteristics as well as DNA sequence data (β-tubulin and actin). From a total of 200 vines sampled, 61 Phaeoacremonium isolates were obtained corresponding to four different species. Pm. aleophilum was the most frequently isolated (n=42) followed by Pm. parasiticum (n=10) and Pm. venezuelense (n=8). Pm. hispanicum was also found but only once. Phaeoacremonium species were more frequently associated with Eutypa dieback than esca symptoms. This correlates with their frequent association with sectorial brown necrosis (V-shaped necrosis).

Contact: valerie.laucou@supagro.inra.fr; Using 20 SSR markers well scattered across the 19 grape chromosomes, we analyzed 4,370 accessions of the INRA grape repository at Vassal, mostly cultivars of Vitis vinifera subsp. sativa (3,727), but also accessions of V. vinifera subsp. sylvestris (80), interspecific hybrids (364), and rootstocks (199). The analysis revealed 2,836 SSR single profiles: 2,323 sativa cultivars, 72 wild individuals (sylvestris), 306 interspecific hybrids, and 135 rootstocks, corresponding to 2,739 different cultivars in all. A total of 524 alleles were detected, with a mean of 26.20 alleles per locus. For the 2,323 cultivars of V. vinifera, 338 alleles were detected with a mean of 16.9 alleles per locus. The mean genetic diversity (GDI) was 0.797 and the level of heterozygosity was 0.76, with broad variation from 0.20 to 1. Interspecific hybrids and rootstocks were more heterozygous and more diverse (GDI = 0.839 and 0.865, respectively) than V. vinifera cultivars (GDI = 0.769), Vitis vinifera subsp. sylvestris being the least divergent with GDI = 0.708. Principal coordinates analysis distinguished the four groups. Slight clonal polymorphism was detected. The limit between clonal variation and cultivar polymorphism was set at four allelic differences out of 40. SSR markers were useful as a complementary tool to traditional ampelography for cultivar identification. Finally, a set of nine SSR markers was defined that was sufficient to distinguish 99.8% of the analyzed accessions. This set is suitable for routine characterization and will be valuable for germplasm management

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; International audience; Background: Unlike in tomato, little is known about the genetic and molecular control of fleshy fruit development of perennial fruit trees like grapevine (Vitis vinifera L.). Here we present the study of the sequence polymorphism in a 1 Mb grapevine genome region at the top of chromosome 18 carrying the fleshless berry mutation (flb) in order, first to identify SNP markers closely linked to the gene and second to search for possible signatures of domestication. Results: In total, 62 regions (17 SSR, 3 SNP, 1 CAPS and 41 re-sequenced gene fragments) were scanned for polymorphism along a 3.4 Mb interval (85,127-3,506,060 bp) at the top of the chromosome 18, in both V. vinifera cv. Chardonnay and a genotype carrying the flb mutation, V. vinifera cv. Ugni Blanc mutant. A nearly complete homozygosity in Ugni Blanc (wild and mutant forms) and an expected high level of heterozygosity in Chardonnay were revealed. Experiments using qPCR and BAC FISH confirmed the observed homozygosity. Under the assumption that flb could be one of the genes involved into the domestication syndrome of grapevine, we sequenced 69 gene fragments, spread over the flb region, representing 48,874 bp in a highly diverse set of cultivated and wild V. vinifera genotypes, to identify possible signatures of domestication in the cultivated V. vinifera compartment. We identified eight gene fragments presenting a significant deviation from neutrality of the Tajima's D parameter in the cultivated pool. One of these also showed higher nucleotide diversity in the wild compartments than in the cultivated compartments. In addition, SNPs significantly associated to berry weight variation were identified in the flb region. Conclusions: We observed the occurrence of a large homozygous region in a non-repetitive region of the grapevine otherwise highly-heterozygous genome and propose a hypothesis for its formation. We demonstrated the feasibility to apply BAC FISH on the very small grapevine chromosomes and provided a specific probe for the identification of chromosome 18 on a cytogenetic map. We evidenced genes showing putative signatures of selection and SNPs significantly associated with berry weight variation in the flb region. In addition, we provided to the community 554 SNPs at the top of chromosome 18 for the development of a genotyping chip for future fine mapping of the flb gene in a F2 population when available.

Young buds and leaf tissues of sugarcane cv. R 574 (a Saccharum hybrid), healthy or infected with Maize Streak Virus (MSV) were cultured in vitro. The success rate of the cultures from infected material is lower than that from healthy material. The symptoms of the disease were displayed on most of the plantlets produced from buds but only on 6% of the plantlets regenerated from calli. The indirect double-antibody sandwich ELISA technique using a monoclonal antibody makes it possible to identify virus-infected plantlets. In vitro, the infection results in the reduced growth of shoots. (Résumé d'auteur)

International audience; Erysiphe necator, the causative agent of powdery mildew in grapevine, was introduced into Europe from North America during the middle of the 19th century. Our objective was to analyze the genetic variation and the population structure of the fungus in southern France. The sample comprised 101 isolates and was mainly of flag shoot origin, i.e., infection of sprouting shoots after overwintering of mycelium in buds. RAPD analysis identified different haplotypes that clustered in two genetic groups (A and B). The most frequent haplotypes of each group were found in several different locations in two areas separated by 100 km and throughout the 3 year period. Several haplotypes of both groups originated from flag shoots and were recovered over successive years indicating that there is no correlation between genetic group and overwintering mode. All isolates of group A were of mating type +, but those in group B could be either + or -. Lower genotypic diversity was detected within group A than within group B. These results were consistent with the hypothesis that group A reproduces only asexually.

National audience; Les premières observations réalisées en 2003 dans différentes régions viticoles de l'Algérie révèlent une mortalité élevée et de forts pourcentages de ceps exprimant les symptômes d'eutypiose et d'esca. La gravité de l'eutypiose apparaît supérieure à celle de l'esca. Les coupes transversales dans les ceps atteints montrent l'existence de différents types de nécroses dont la fréquence ne semble pas liée à la nature des symptômes externes: des ponctuations noires dispersées dans le bois sain correspondant à des vaisseaux obstrués, une nécrose centrale brune dure ou molle, une nécrose claire et tendre caractéristique de la pourriture blanche (amadou) et une nécrose dure et sectorielle. Les principaux champignons isolés des nécroses sont: Phaeomoniella chlamydospora, Phaeoacremonium aleophilum, Botryosphaeria spp., Fomitiporia mediterranea, Phomopsis viticola et Eutypa lata. Dans un même cep, différents types de nécroses et plusieurs champignons peuvent coexister. L'association des champignons avec les nécroses et leurs rôles respectifs dans le fonctionnement de la communauté fongique sont discutés.

Eutypa latais the causal fungal agent ofEutypadieback, a serious grapevine necrotic disease. The erratic and delayed (1 to 2 months) appearance of characteristic conidia on culture media and the presence of numerous microorganisms in decaying wood make it difficult either to identify or to detectE. latain grapevine wood samples. We designed six pairs of PCR primers for diagnosis ofE. lata. Three primer pairs were derived from ribosomal DNA internal transcribed spacer sequences, and three pairs were derived from randomly amplified polymorphic DNA fragments. The six primer pairs could be used to amplify DNAs extracted from all of theE. lataisolates tested. They did not amplify DNAs from fungi and bacteria representing more than 50 different species of microorganisms associated with grapevine. We developed a simple protocol, leading to a rapid release of DNA, that enabled us to identifyE. latafrom pure or mixed cultures as well as from grapevine wood samples. Identification ofE. latain wood was achieved within a few hours, instead of the several weeks required for classical cultures on agar medium. We believe that the procedure described here can be adapted to detect other microorganisms involved in woody plant diseases.

Susceptibility to numerous diseases, pests and abiotic stresses make it necessary to use a high level of chemical In vitro culture of 32 Vitis vinifera cultivars and intras- control and to adopt specific cultural practices. Genetic pecific hybrids was initiated from axillary buds. The improvement through conventional methods, i.e. hybriddevelopment of roots and shoots was followed during ization, does not appear to be practical in order to obtain 14 subcultures on two hormone-free micropropagation less susceptible scion cultivars with similar wine qualities media. One medium (M64) was used until the 8th sub- to those of classic cultivars. Non-conventional approaches culture, after which it was replaced by G90 medium have therefore been proposed (Bouquet, 1989; Meredith, which was found more suitable for plantlet growth and 1996). A prerequisite to develop such approaches for permitted an increase in the time between subcul- grapevine is the ability to perform eYcient in vitro techtures. Large differences in plantlet growth between niques (Monette, 1988), but genetic variability of the cultivars were demonstrated on both media. The material has to be taken into account (Bouquet et al., number of roots had greatest variability between culti- 1982; Stamp et al., 1990; Torregrosa and Bouquet, 1996). vars (CV=39%) as compared with stem length (CV= Micropropagation in grapevine was first performed 21‐22%) and number of nodes (CV=12‐14%). The using entire plantlets obtained from microcuttings (Galzy, number of nodes was positively correlated with shoot 1961). More recently, the induction of bud proliferation length whereas root number appeared to be poorly has been shown to provide an alternate pathway to positively correlated with shoot development. grapevine micropropagation (Jona and Webb, 1978). Adventitious bud regeneration from leaves was studied Micropropagation produces suitable tissues for regenerafor 20 cultivars and averaged 36.7% of regenerative tion through organogenesis and embryogenesis (Stamp explants with large differences between cultivars and Meredith, 1988). In addition, in vitro culture of (CV=47%). However, organogenic competence was plantlets permits the rapid propagation of the genotypes not correlated with micropropagation ability. High obtained by non-conventional methods. sensitivity of Vitis vinifera plantlets to kanamycin and Regeneration of whole plants by somatic embryogenesis hygromycin was demonstrated with a strong inter- and organogenesis has been intensively studied (for action between cultivar and antibiotic. At 0.8 mg dm’ review, Torregrosa, 1995). The genetic engineering of 3, hygromycin was lethal to plantlets. This effect was somatic embryos was first reported by Mullins et al. only observed at 4 mg dm’3 for kanamycin, whereas (1990) and has been used to transfer genes of potential 1m g dm’3 stimulated the development of plantlets. interest through Agrobacterium-mediated transformation (Le Gall et al., 1994; Krastonova et al., 1995). Colby

Cette etude consiste a comparer la sensibilite a la rouille (Pucciuia melanocephala) et les caracteristiques de rendement des plants de canne a sucre issus de culture de cals ou de bourgeons avec celles des plants obtenus par la methode classique de miltiplication vegetative. Les plants regeneres a partir de culture de cals (somaclones) ont generalement une plus grande sensibilite a la rouille des tiges plus fines et des feuilles plus petites que le temoin. L'amplitude de ces effets depend de la methode employee pour obtenir les somaclones. Les plants issus de culture de bourgeons ont aussi montre une resistance a la rouille alteree mais des composantes de rendement ameliorees dans deux essais. L'effet sur la resistance a la rouille a persiste chez les plants issus de culture in vitro mene apres deux multiplications vegetatives. Il est donc necessaire de tenir compte de ces effets lorsque l'on utilise les techniques de culture in vitro pour l'amelioration varietale de la canne a sucre ou pour sa multiplication en masse

Strains presumed to be of Xanthomonas campestris pv. vasculorum (Cobb.) Dye, obtained from sugarcane and other gramineae in Reunion Island, were compared in terms of cultural aspects, pathogenic and physiological reactions, fatty acid profiles and restriction fragment length polymorphism (RFLP) of genomic DNA. The strains could be divided into two separate groups (G1 and G2). The G1 strains were identical to strains described as X. campestris pv. vasculorum; they showed an important variability in their cultural characteristics and in their aggressiveness. The G2 strains did not induce the usual symptoms of gumming disease on sugarcane cultivars infected under natural conditions or inoculated in the greenhouse. The G2 strains grew faster on agar medium, their colonies were more pigmented and less fluidal and had a different morphology on agar slant. Unlike the G1 strains, G2 strains hydrolyzed starch weakly and casein strongly; they utilized L-fucose and, to a lesser extent, melibioze. The fatty acid and genomic DNA profiles differed between the groups. Differences seemed large enough to support separation of G1 and G2 strains into distinct taxonomic entities, namely G1 as Xanthomonas campestris pv. vasculorum and G2 as a different pathovar of X. campestris. The taxonomic position of G2 strains is discussed. Zusammenfassung Variabilitat von Xanthomonas campestris pv. vasculorum aus Zuckerrohr und anderen Gramineae in Reunion. Charakterisierung eines anderen Xanthomonaden Verglichen wurden Stamme von Xanthomonas campestris pv. vasculorum (Cobb.) Dye., die von Zuckerrohrpflanzen und anderen Gramineae in Reunion isoliert wurden, in Bezug auf ihre Charakteristika in Kultur, ihre pathogene und physiologische Reaktionen, ihre Fettsaureprofile sowie restriction fragment length polymorphism (RFLP) der genomen DNS. Die Stamme konnten in zwei Gruppen (G1 und G2) getrennt werden. Die G1-Stamme waren mit den als X. campestris pv. vasculorum beschriebenen Stammen identisch: sie zeigten eine wichtige Variabilitat ihrer Charakteristika in Kultur sowie in ihrer Aggressivitat. Die G2-Stamme dagegen riefen die normalen Symptome der gumming-Krankheit bei Zuckerrohr nicht hervor, weder nach einer Infektion unter naturlichen Bedingungen noch bei Pflanzen die im Gewachshaus inokuliert worden waren. Die G2-Stamme hatten eine schnellere Wachstumsrate auf Agarnahrmedien, ihre Kolonien zeigten eine starkere Pigmentbildung und sie waren in ihrer Konsistenz flussiger und hatten eine andere Morphologie in Schragagarkultur als die G1-Stamme. Die G2-Stamme konnten Starke nur schwach hydrolysieren, Casein dagegen stark; L-Fucose und Melibiose, obwohl in reduziertem Umfang, wurden metabolisiert. Die Fettsaure und genomen DNS-Profile waren zwischen den Gruppen unterschiedlich. Die Unterschiede waren so gros, das eine Trennung der G1- und G2-Stamme in zwei taxonomisch getrennte Einheiten fur sinnvoll erschien, und zwar die G1-Stamme als Xanthomonas campestris pv. vasculorum und die G2-Stamme als ein anderes X. campestris-Pathovar. Die taxonomische Stellung der G2-Stammen wird diskutiert.

Sugarcane plantlets were sectioned halfway between the base and the youngest ligule and then inoculated by soaking the wound in a suspension of Xanthomonas campestris pv. vasculorum. The infection caused rapid necrosis of the inoculated leaves, chlorosis of uninoculated leaves, or death of the inoculated plantlet. New tillers sometimes showed chlorosis or white streaks. The effects of the inoculum concentration, the cultivar, and the bacterial strain on symptom severity were determined. The ranking of cultivars depended on the inoculum concentration, and strains were found to differ with regard to aggressiveness. However, cultivars and strains were more effectively classified in greenhouse trials. The poor expression of leaf resistance appeared to limit the use of the in vitro test.

Ustilago scitaminea synthesizes specific carbohydrates, mainly erythritol and mannitol, plus smaller quantities of arabitol, inositol, and trehalose. Intensive secretion of erythritol is obtained when the fungus is grown in liquid culture. Glucose, fructose, or sucrose can be used interchangeably as carbon sources by the fungus, sucrose being quickly inverted.