Elsa Petit

Characterization of Cylindrocarpon Species, the Cause of Black Foot Disease of Grapevine in California

This study investigated phylogenetic divergence, morphological difference, and pathogenic variation among Cylindrocarpon species isolates associated with black foot disease of grapevine (Vitis sp.) in California. To assess phylogenetic divergence, we sequenced the internal transcribed spacer (ITS) of the nuclear ribosomal DNA (rDNA), partial beta-tubulin (BT) gene introns and exons, and the small subunit mitochondrial rDNA. Isolates associated with black foot disease belonged to two paraphyletic species, Cylindrocarpon destructans and C. macrodidymum. The morphology of these isolates was in agreement with published descriptions of both species. We found that C. macrodidymum isolates were reliably distinguishable from C. destructans isolates in culture by a unique orange-dark brown colony color on 2% malt extract agar and genetically by a species-specific 52-bp DNA insertion in the BT region. Selected isolates of each species inoculated onto grapevine rootstock 5C caused typical black foot disease symptoms. This is the first report of C. macrodidymum in California.

Involvement of a bacterial microcompartment in the metabolism of fucose and rhamnose by Clostridium phytofermentans.

Background Clostridium phytofermentans, an anaerobic soil bacterium, can directly convert plant biomass into biofuels. The genome of C. phytofermentans contains three loci with genes encoding shell proteins of bacterial microcompartments (BMC), organelles composed entirely of proteins. Methodology and Principal Findings One of the BMC loci has homology to a BMC-encoding locus implicated in the conversion of fucose to propanol and propionate in a human gut commensal, Roseburia inulinivorans. We hypothesized that it had a similar role in C. phytofermentans. When C. phytofermentans was grown on fucose, the major products identified were ethanol, propanol and propionate. Transmission electron microscopy of fucose- and rhamnose-grown cultures revealed polyhedral structures, presumably BMCs. Microarray analysis indicated that during growth on fucose, operons coding for the BMC locus, fucose dissimilatory enzymes, and an ATP-binding cassette transporter became the dominant transcripts. These data are consistent with fucose fermentation producing a 1,2-propanediol intermediate that is further metabolized in the microcompartment encoded in the BMC locus. Growth on another deoxyhexose sugar, rhamnose, resulted in the expression of the same BMC locus and similar fermentation products. However, a different set of dissimilatory enzymes and transport system genes were induced. Quite surprisingly, growth on fucose or rhamnose also led to the expression of a diverse array of complex plant polysaccharide-degrading enzymes. Conclusions/Significance Based on physiological, genomic, and microarray analyses, we propose a model for the fermentation of fucose and rhamnose in C. phytofermentans that includes enzymes encoded in the same BMC locus. Comparative genomic analysis suggests that this BMC may be present in other clostridial species.

High Variability of Mitochondrial Gene Order among Fungi

From their origin as an early alpha proteobacterial endosymbiont to their current state as cellular organelles, large-scale genomic reorganization has taken place in the mitochondria of all main eukaryotic lineages. So far, most studies have focused on plant and animal mitochondrial (mt) genomes (mtDNA), but fungi provide new opportunities to study highly differentiated mtDNAs. Here, we analyzed 38 complete fungal mt genomes to investigate the evolution of mtDNA gene order among fungi. In particular, we looked for evidence of nonhomologous intrachromosomal recombination and investigated the dynamics of gene rearrangements. We investigated the effect that introns, intronic open reading frames (ORFs), and repeats may have on gene order. Additionally, we asked whether the distribution of transfer RNAs (tRNAs) evolves independently to that of mt protein-coding genes. We found that fungal mt genomes display remarkable variation between and within the major fungal phyla in terms of gene order, genome size, composition of intergenic regions, and presence of repeats, introns, and associated ORFs. Our results support previous evidence for the presence of mt recombination in all fungal phyla, a process conspicuously lacking in most Metazoa. Overall, the patterns of rearrangements may be explained by the combined influences of recombination (i.e., most likely nonhomologous and intrachromosomal), accumulated repeats, especially at intergenic regions, and to a lesser extent, mobile element dynamics.

Root diseases of grapevines in California and their control

Root rots of grapevine in California are caused by several soilborne pathogens. While root rot in vineyards in general has been considered to be a relative minor problem, nearly all root diseases have increased in incidence and severity since the introduction of rootstocks resistant to Phylloxera. Pathogens such as Phytophthora spp. and Armillaria mellea have been known to occur for many years. However, seldom did root rot become a serious problem in vineyards. New diseases such as Petri disease caused by Phaeomoniella chlamydospora, vine decline caused by Phaeoacremonium spp. and black foot caused by Cylindrocarpon destructans have only recently been shown to occur in California. Wilt caused by Verticillium dahliae has become more prevalent in nearly all grape growing regions in California. Though losses due to actual root disease have been minor over the past 10 years, losses due to vineyard reestablishment have been large.

Patterns of Repeat-Induced Point Mutation in Transposable Elements of Basidiomycete Fungi

Transposable elements (TEs) are ubiquitous genomic parasites that have prompted the evolution of genome defense systems that restrict their activity. Repeat-induced point mutation (RIP) is a homology-dependent genome defense that introduces C-to-T transition mutations in duplicated DNA sequences and is thought to control the proliferation of selfish repetitive DNA. Here, we determine the taxonomic distribution of hypermutation patterns indicative of RIP among basidiomycetes. We quantify C-to-T transition mutations in particular di- and trinucleotide target sites for TE-like sequences from nine fungal genomes. We find evidence of RIP-like patterns of hypermutation at TpCpG trinucleotide sites in repetitive sequences from all species of the Pucciniomycotina subphylum of the Basidiomycota, Microbotryum lychnidis-dioicae, Puccinia graminis, Melampsora laricis-populina, and Rhodotorula graminis. In contrast, we do not find evidence for RIP-like hypermutation in four species of the Agaricomycotina and Ustilaginomycotina subphyla of the Basidiomycota. Our results suggest that a RIP-like process and the specific nucleotide context for mutations are conserved within the Pucciniomycotina subphylum. These findings imply that coevolutionary interactions between TEs and a hypermutating genome defense are stable over long evolutionary timescales.

Extensive Divergence Between Mating-Type Chromosomes of the Anther-Smut Fungus

Genomic regions that determine mating compatibility are subject to distinct evolutionary forces that can lead to a cessation of meiotic recombination and the accumulation of structural changes between members of the homologous chromosome pair. The relatively recent discovery of dimorphic mating-type chromosomes in fungi can aid the understanding of sex chromosome evolution that is common to dioecious plants and animals. For the anther-smut fungus, Microbotryum lychnidis-dioicae (= M. violaceum isolated from Silene latifolia), the extent of recombination cessation on the dimorphic mating-type chromosomes has been conflictingly reported. Comparison of restriction digest optical maps for the two mating-type chromosomes shows that divergence extends over 90% of the chromosome lengths, flanked at either end by two pseudoautosomal regions. Evidence to support the expansion of recombination cessation in stages from the mating-type locus toward the pseudoautosomal regions was not found, but evidence of such expansion could be obscured by ongoing processes that affect genome structure. This study encourages the comparison of forces that may drive large-scale recombination suppression in fungi and other eukaryotes characterized by dimorphic chromosome pairs associated with sexual life cycles.

Degeneration of the Nonrecombining Regions in the Mating-Type Chromosomes of the Anther-Smut Fungi

Dimorphic mating-type chromosomes in fungi are excellent models for understanding the genomic consequences of recombination suppression. Their suppressed recombination and reduced effective population size are expected to limit the efficacy of natural selection, leading to genomic degeneration. Our aim was to identify the sequences of the mating-type chromosomes (a1 and a2) of the anther-smut fungi and to investigate degeneration in their nonrecombining regions. We used the haploid a1 Microbotryum lychnidis-dioicae reference genome sequence. The a1 and a2 mating-type chromosomes were both isolated electrophoretically and sequenced. Integration with restriction-digest optical maps identified regions of recombination and nonrecombination in the mating-type chromosomes. Genome sequence data were also obtained for 12 other Microbotryum species. We found strong evidence of degeneration across the genus in the nonrecombining regions of the mating-type chromosomes, with significantly higher rates of nonsynonymous substitution (dN/dS) than in nonmating-type chromosomes or in recombining regions of the mating-type chromosomes. The nonrecombining regions of the mating-type chromosomes also showed high transposable element content, weak gene expression, and gene losses. The levels of degeneration did not differ between the a1 and a2 mating-type chromosomes, consistent with the lack of homogametic/heterogametic asymmetry between them, and contrasting with X/Y or Z/W sex chromosomes.

The Distribution of Polyhedral Bacterial Microcompartments Suggests Frequent Horizontal Transfer and Operon Reassembly

Bacterial microcompartments (BMCs) are proteinaceous organelles that carry out specific metabolic reactions. Using domain representations of the BMC shell proteins, we identified BMCs in genomes of 358 bacterial species including human gut microbes, bioremediation agents, cellulosic ethanol producers, and pathogens. Multiple BMCs of different metabolic types are present in 40% of the BMC-containing genomes. BMC genes frequently clustered at a single locus that includes enzymes related to the compartment’s metabolic function. The distribution of BMC-containin species was mapped onto a phylogenetic tree constructed from 16S rRNA sequences. The presence of BMCs was sporadically distributed across the phylogenetic tree. All bacterial families that contained species with BMCs also had species without them. Even within a species, BMC number varied, indicative of frequent horizontal transfer and gene loss. Similarly, phylogenetic trees constructed from individual BMC genes indicated that horizontal gene transfer of the BMC loci is a common occurrence.

Influence of Glomus intraradices on Black Foot Disease Caused by Cylindrocarpon macrodidymum on Vitis rupestris Under Controlled Conditions

We examined the influence of an arbuscular-mycorrhizal fungus, Glomus intraradices (INVAM CA 501), on black foot disease caused by the fungus Cylindrocarpon macrodidymum on Vitis rupestris cv. St. George under controlled conditions. Mycorrhizal or nonmycorrhizal grape rootings were inoculated with the pathogen. Eight months following inoculation with the pathogen, we evaluated disease severity, vine growth, and mycorrhizal colonization. Mycorrhizal plants developed significantly less leaf and root symptoms than nonmycorrhizal plants (P = 0.04 and P < 0.0001, respectively). Only nonmycorrhizal grape rootings inoculated with the pathogen had significantly less dry root and leaf weights compared with the noninoculated control (P = 0.0021 and P = 0.0017, respectively). Mycorrhizal colonization was high (48.3% for the noninfected control and 54.5% for plants infected with C. macrodidymum) and not significantly affected by inoculation with C. macrodidymum (P = 0.2256). Thus, V. rupestris preinoculated with G. intraradices were less susceptible to black foot disease than nonmycorrhizal plants. Results from this study suggest that preplant applications of G. intraradices may help prevent black foot disease in the nursery and in the vineyard.

Evolution of uni- and bifactorial sexual compatibility systems in fungi

Mating systems, that is, whether organisms give rise to progeny by selfing, inbreeding or outcrossing, strongly affect important ecological and evolutionary processes. Large variations in mating systems exist in fungi, allowing the study of their origin and consequences. In fungi, sexual incompatibility is determined by molecular recognition mechanisms, controlled by a single mating-type locus in most unifactorial fungi. In Basidiomycete fungi, however, which include rusts, smuts and mushrooms, a system has evolved in which incompatibility is controlled by two unlinked loci. This bifactorial system probably evolved from a unifactorial system. Multiple independent transitions back to a unifactorial system occurred. It is still unclear what force drove evolution and maintenance of these contrasting inheritance patterns that determine mating compatibility. Here, we give an overview of the evolutionary factors that might have driven the evolution of bifactoriality from a unifactorial system and the transitions back to unifactoriality. Bifactoriality most likely evolved for selfing avoidance. Subsequently, multiallelism at mating-type loci evolved through negative frequency-dependent selection by increasing the chance to find a compatible mate. Unifactoriality then evolved back in some species, possibly because either selfing was favoured or for increasing the chance to find a compatible mate in species with few alleles. Owing to the existence of closely related unifactorial and bifactorial species and the increasing knowledge of the genetic systems of the different mechanisms, Basidiomycetes provide an excellent model for studying the different forces that shape breeding systems.