First report of black leaf streak disease in bananas caused by Pseudocercospora fijiensis on Mauritius island.

Pseudocercospora fijiensis, the causal agent of the black leaf streak disease of bananas (plants in the genus Musa) (BLSD), is considered to be the major economic threat to export-banana cultivation (de Bellaire, Fouré, Abadie, & Carlier, 2010). The disease has a worldwide distribution throughout the humid tropical regions and has been previously reported in the Southwestndian Ocean (SWIO) area: in 1993 in Mayotte and Comoros islands (DR Jones & Mourichon, 1993), in 2000 in Madagascar (Jones, 2003; Rivas, Zapater, Abadie, & Carlier, 2004) and in 2018 in Reunion Island (Rieux et al., 2019). In Mauritius, the presence of Pseudocercospora fijiensis was suspected in 1996 (Soomary & Benimadhu, 1998) but has never been confirmed, as symptoms could have been confounded with Pseudocercospora musae or Pseudocercospora eumusae, two causal agents of others leaf spot diseases of banana which were previously described in Mauritius in 1959 (Orieux & Felix, 1968) and 2000 (Carlier, Zapater, Lapeyre, Jones, & Mourichon, 2000), respectively. In March 2022, typical BLSD symptoms were observed at relatively low prevalence in a Cavendish crop located in the "Balance John" area (site S1 on Fig. S1-A) of Mauritius island. Typical early symptoms (stages 2) were 1- to 4-mm long brown streaks at the abaxial leaf surface, and typical older streaks (stages 3 and 4) were also observed (Fig. S1-B). These symptoms were mixed with symptoms of ELSD caused by P. eumusae. Since both species cannot be clearly distinguished only on the description of symptoms, conidial sporulation on stages 2 was checked in the laboratory (Ngando et al., 2015) since P. eumusae does not produce conidia on these young stages. In April 2022, banana leaves bearing symptoms of leaf spot diseases were collected in 7 different sites (Fig. S1-A). All leaf fragments were sent to the CIRAD laboratories where molecular diagnosis was performed following the protocol developed by Arzanlou et al. (2007). In brief, genomic DNA was extracted from ground leaf fragments displaying symptoms using the DNeasy® Plant Mini Kit (Qiagen®, Courtaboeuf, France). At each site, a total of 6 lesions cut from 6 different leaves were pooled. The DNA extracts were added as templates for real-time PCR assay designed to specifically detect the presence of P. fijiensis, P. musae and P. eumusae using MFbf/MFbrtaq/MFbp, MEbf/MEbrtaq/FMep and MMbf/Mmbrtaq/FMep primers and probes, respectively (Arzanlou et al., 2007). Both positive and negative controls were included in the assay and every sample reaction was duplicated. P. fijiensis was detected from 2 out of 7 sites (S2 and S7, see Fig.S2-B). P. eumusae was detected at all sites while P. musae was found in one site only (S6). Interestingly, our results also showed coinfection by P. fijiensis - P. eumusae & P. musae - P. eumusae on several sites. The presence of P. fijiensis was further confirmed by several investigations performed on conidia isolated from S2 samples including i) morphological observations of conidia displaying P. fijiensis type description (Pérez-Vicente, Carreel, Roussel, Carlier, & Abadie (2021), Fig. S2-A), ii) DNA sequencing of 16S ribosomal gene with ITS1 & ITS4 primers (GenBank accessions Nos. OR515818-OR515810) with BLAST results displaying percentages of identity > 99.70% with type strains and iii) Koch's postulates were fulfilled by artificial inoculation of detached leaf pieces as described in Pérez-Vicente, Carreel, Roussel, Carlier, & Abadie (2021) (Fig. S2-D). In brief, for the artificial inoculation, symptoms obtained after inoculation of both a strain isolated in Mauritius (S2-MAU) and a positive control (T+) were compared and shown to be typical of P. fijiensis species for the 3 replicates. To the best of our knowledge, this is the first official report of P. fijiensis and BLSD in Mauritius Island. This revelation holds significant importance for both the agricultural and scientific communities, shedding light on the potential spread and impact of this devastating pathogen in previously unaffected regions. From a global perspective, this discovery underscores the interconnectedness of agricultural ecosystems and the need for vigilance in monitoring and responding to emerging plant diseases in an increasingly interconnected world (Vega et al. 2022). Future investigations will be required to monitor the spread of BLSD on the island, describe the genetic structure of populations and identify routes of invasion at the SWOI scale.

Complex adaptive architecture underlies adaptation to quantitative host resistance in a fungal plant pathogen.

Plant pathogens often adapt to plant genetic resistance so characterization of the architecture underlying such an adaptation is required to understand the adaptive potential of pathogen populations. Erosion of banana quantitative resistance to a major leaf disease caused by polygenic adaptation of the causal agent, the fungus Pseudocercospora fijiensis, was recently identified in the northern Caribbean region. Genome scan and quantitative genetics approaches were combined to investigate the adaptive architecture underlying this adaptation. Thirty-two genomic regions showing host selection footprints were identified by pool sequencing of isolates collected from seven plantation pairs of two cultivars with different levels of quantitative resistance. Individual sequencing and phenotyping of isolates from one pair revealed significant and variable levels of correlation between haplotypes in 17 of these regions with a quantitative trait of pathogenicity (the diseased leaf area). The multilocus pattern of haplotypes detected in the 17 regions was found to be highly variable across all the population pairs studied. These results suggest complex adaptive architecture underlying plant pathogen adaptation to quantitative resistance with a polygenic basis, redundancy, and a low level of parallel evolution between pathogen populations. Candidate genes involved in quantitative pathogenicity and host adaptation of P. fijiensis were identified in genomic regions by combining annotation analysis with available biological data.

A world-wide analysis of reduced sensitivity to DMI fungicides in the banana pathogen Pseudocercospora fijiensis.

BACKGROUND: Pseudocercospora fijiensis is the causal agent of the black leaf streak disease (BLSD) of banana. Bananas are important global export commodities and a major staple food. Their susceptibility to BLSD pushes disease management towards excessive fungicide use, largely relying on multisite inhibitors and sterol demethylation inhibitors (DMIs). These fungicides are ubiquitous in plant disease control, targeting the CYP51 enzyme. We examined sensitivity to DMIs in P. fijiensis field isolates collected from various major banana production zones in Colombia, Costa Rica, Dominican Republic, Ecuador, the Philippines, Guadalupe, Martinique and Cameroon and determined the underlying genetic reasons for the observed phenotypes. RESULTS: We observed a continuous range of sensitivity towards the DMI fungicides difenoconazole, epoxiconazole and propiconazole with clear cross-sensitivity. Sequence analyses of PfCYP51 in 266 isolates showed 28 independent amino acid substitutions, nine of which correlated with reduced sensitivity to DMIs. In addition to the mutations, we observed up to six insertions in the Pfcyp51 promoter. Such promoter insertions contain repeated elements with a palindromic core and correlate with the enhanced expression of Pfcyp51 and hence with reduced DMI sensitivity. Wild-type isolates from unsprayed bananas fields did not contain any promoter insertions. CONCLUSION: The presented data significantly contribute to understanding of the evolution and global distribution of DMI resistance mechanisms in P. fijiensis field populations and facilitate the prediction of different DMI efficacy. The overall reduced DMI sensitivity calls for the deployment of a wider range of solutions for sustainable control of this major banana disease. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Convergent Adaptation to Quantitative Host Resistance in a Major Plant Pathogen.

Plant pathogens can adapt to quantitative resistance, eroding its effectiveness. The aim of this work was to reveal the genomic basis of adaptation to such a resistance in populations of the fungus Pseudocercospora fijiensis, a major devastating pathogen of banana, by studying convergent adaptation on different cultivars. Samples from P. fijiensis populations showing a local adaptation pattern on new banana hybrids with quantitative resistance were compared, based on a genome scan approach, with samples from traditional and more susceptible cultivars in Cuba and the Dominican Republic. Whole-genome sequencing of pools of P. fijiensis isolates (pool-seq) sampled from three locations per country was conducted according to a paired population design. The findings of different combined analyses highly supported the existence of convergent adaptation on the study cultivars between locations within but not between countries. Five to six genomic regions involved in this adaptation were detected in each country. An annotation analysis and available biological data supported the hypothesis that some genes within the detected genomic regions may play a role in quantitative pathogenicity, including gene regulation. The results suggested that the genetic basis of fungal adaptation to quantitative plant resistance is at least oligogenic, while highlighting the existence of specific host-pathogen interactions for this kind of resistance.IMPORTANCE Understanding the genetic basis of pathogen adaptation to quantitative resistance in plants has a key role to play in establishing durable strategies for resistance deployment. In this context, a population genomic approach was developed for a major plant pathogen (the fungus Pseudocercospora fijiensis causing black leaf streak disease of banana) whereby samples from new resistant banana hybrids were compared with samples from more susceptible conventional cultivars in two countries. A total of 11 genomic regions for which there was strong evidence of selection by quantitative resistance were detected. An annotation analysis and available biological data supported the hypothesis that some of the genes within these regions may play a role in quantitative pathogenicity. These results suggested a polygenic basis of quantitative pathogenicity in this fungal pathogen and complex molecular plant-pathogen interactions in quantitative disease development involving several genes on both sides.

Central American and Caribbean population history of the Pseudocercospora fijiensis fungus responsible for the latest worldwide pandemics on banana.

Among the emerging fungal diseases threatening food security, the Pseudocercospora fijiensis fungus causing black leaf streak disease of banana is one of the most marked examples of a recent worldwide pandemic on a major crop. We assessed how this pathogen spread throughout the latest invaded region, i.e. Central America and the Caribbean. We retraced its population history combining detailed monitoring information on disease outbreaks and population genetic analyses based on large-scale sampling of P. fijiensis isolates from 121 locations throughout the region. The results first suggested that sexual reproduction was not lost during the P. fijiensis expansion, even in the insular Caribbean context, and a high level of genotypic diversity was maintained in all the populations studied. The population genetic structure of P. fijiensis and historical data showed that two disease waves swept northward and southward in all banana-producing countries in the study area from an initial entry point in Honduras, probably mainly through gradual stepwise spore dispersal. Serial founder events accompanying the northern and southern waves led to the establishment of two different genetic groups. A different population structure was detected on the latest invaded islands (Martinique, Dominica and Guadeloupe), revealing multiple introductions and admixture events that may have been partly due to human activities. The results of this study highlight the need to step up surveillance to limit the spread of other known emerging diseases of banana spread mainly by humans, but also to curb gene flow between established pathogen populations which could increase their evolutionary potential.

Pattern of local adaptation to quantitative host resistance in a major pathogen of a perennial crop.

Understanding the mechanisms involved in pathogen adaptation to quantitative resistance in plants has a key role to play in establishing durable strategies for resistance deployment, especially in perennial crops. The erosion of quantitative resistance has been recently suspected in Cuba and the Dominican Republic for a major fungal pathogen of such a crop: Pseudocercospora fijiensis, causing black leaf streak disease on banana. This study set out to test whether such erosion has resulted from an adaptation of P. fijiensis populations, and to determine whether or not the adaptation is local. Almost 600 P. fijiensis isolates from Cuba and the Dominican Republic were sampled using a paired-population sampling design on resistant and susceptible banana varieties. A low genetic structure of the P. fijiensis populations was detected in each country using 16 microsatellite markers. Cross-inoculation experiments using isolates from susceptible and resistant cultivars were carried out, measuring a quantitative trait (the diseased leaf area) related to pathogen fitness on three varieties. A further analysis based on those data suggested the existence of a local pattern of adaptation to resistant cultivars in both of the study countries, due to the existence of specific (or genotype by genotype) host-pathogen interactions. However, neither cost nor benefit effects for adapted populations were found on the widely used "Cavendish" banana group. These results highlight the need to study specific host-pathogen interactions and pathogen adaptation on a wide range of quantitative resistance phenotypes in banana, in order to develop durable strategies for resistance deployment.

Combating a Global Threat to a Clonal Crop: Banana Black Sigatoka Pathogen Pseudocercospora fijiensis (Synonym Mycosphaerella fijiensis) Genomes Reveal Clues for Disease Control.

Black Sigatoka or black leaf streak disease, caused by the Dothideomycete fungus Pseudocercospora fijiensis (previously: Mycosphaerella fijiensis), is the most significant foliar disease of banana worldwide. Due to the lack of effective host resistance, management of this disease requires frequent fungicide applications, which greatly increase the economic and environmental costs to produce banana. Weekly applications in most banana plantations lead to rapid evolution of fungicide-resistant strains within populations causing disease-control failures throughout the world. Given its extremely high economic importance, two strains of P. fijiensis were sequenced and assembled with the aid of a new genetic linkage map. The 74-Mb genome of P. fijiensis is massively expanded by LTR retrotransposons, making it the largest genome within the Dothideomycetes. Melting-curve assays suggest that the genomes of two closely related members of the Sigatoka disease complex, P. eumusae and P. musae, also are expanded. Electrophoretic karyotyping and analyses of molecular markers in P. fijiensis field populations showed chromosome-length polymorphisms and high genetic diversity. Genetic differentiation was also detected using neutral markers, suggesting strong selection with limited gene flow at the studied geographic scale. Frequencies of fungicide resistance in fungicide-treated plantations were much higher than those in untreated wild-type P. fijiensis populations. A homologue of the Cladosporium fulvum Avr4 effector, PfAvr4, was identified in the P. fijiensis genome. Infiltration of the purified PfAVR4 protein into leaves of the resistant banana variety Calcutta 4 resulted in a hypersensitive-like response. This result suggests that Calcutta 4 could carry an unknown resistance gene recognizing PfAVR4. Besides adding to our understanding of the overall Dothideomycete genome structures, the P. fijiensis genome will aid in developing fungicide treatment schedules to combat this pathogen and in improving the efficiency of banana breeding programs.

A novel bioassay to monitor fungicide sensitivity in Mycosphaerella fijiensis.

BACKGROUND: Black leaf streak disease (BLSD) is the most important disease of bananas for export. The successful control of BLSD requires an intensive use of systemic fungicides, leading to the build-up of resistance and failure of control. Early detection of fungicide resistance is crucial to drive rational chemical strategies. Present methods relying on ascospore germination bioassays have several drawbacks that could be overcome using conidia. RESULTS: Generally, a single genotype is present on the conidial population derived from one lesion. Conidial germination tests with thiabendazole (5 mg L(-1)) enable a clear detection of strains resistant to methyl benzimidazole carbamates. Germination bioassays on azoxystrobin (10 mg L(-1)) enable the detection of most QoI-resistant strains, but their proportion might be underestimated with cut-off limits of germ tube length (L > 120 µm) or growth inhibition (GI < 50%). The level of fungicide resistance differs at different canopy levels of a banana tree, which should be considered for sampling. The ascospore germination bioassay provided more variable estimations of the level of resistance by comparison with the new conidial germination bioassay. CONCLUSION: Germination bioassays performed with conidia obtained from young lesions overcome most drawbacks encountered with ascospore germination bioassays and could be considered as a new reference method for fungicide resistance monitoring in this species. Different steps are proposed, from sampling to microscopic examinations, for the implementation of this technique.

Long-distance wind-dispersal of spores in a fungal plant pathogen: estimation of anisotropic dispersal kernels from an extensive field experiment.

Given its biological significance, determining the dispersal kernel (i.e., the distribution of dispersal distances) of spore-producing pathogens is essential. Here, we report two field experiments designed to measure disease gradients caused by sexually- and asexually-produced spores of the wind-dispersed banana plant fungus Mycosphaerella fijiensis. Gradients were measured during a single generation and over 272 traps installed up to 1000 m along eight directions radiating from a traceable source of inoculum composed of fungicide-resistant strains. We adjusted several kernels differing in the shape of their tail and tested for two types of anisotropy. Contrasting dispersal kernels were observed between the two types of spores. For sexual spores (ascospores), we characterized both a steep gradient in the first few metres in all directions and rare long-distance dispersal (LDD) events up to 1000 m from the source in two directions. A heavy-tailed kernel best fitted the disease gradient. Although ascospores distributed evenly in all directions, average dispersal distance was greater in two different directions without obvious correlation with wind patterns. For asexual spores (conidia), few dispersal events occurred outside of the source plot. A gradient up to 12.5 m from the source was observed in one direction only. Accordingly, a thin-tailed kernel best fitted the disease gradient, and anisotropy in both density and distance was correlated with averaged daily wind gust. We discuss the validity of our results as well as their implications in terms of disease diffusion and management strategy.

Optimized genotyping with microsatellite markers in the fungal banana pathogen Mycosphaerella fijiensis (Mycosphaerellaceae).

PREMISE OF THE STUDY: Large-scale population genetics studies are required to investigate the dispersal processes underlying the emergence of Mycosphaerella fijiensis, a fungal pathogen of banana. To this end, we have developed an optimized genotyping procedure combining novel microsatellite markers and a modified DNA extraction protocol. • METHODS AND RESULTS: Primers for tetranucleotide loci were designed directly from the recently published genome sequence of M. fijiensis. A total of 19 new polymorphic and easy-to-score markers were developed. Their use was combined with an adapted protocol for total DNA extraction starting from young lesions collected from banana leaves, thus avoiding a pathogen isolation step. • CONCLUSIONS: The combination of the two technical developments presented here will permit the expansion of genotyping capacity in M. fijiensis, allowing large-scale analysis of samples from various geographic locations.

Negativicoccus succinicivorans gen. nov., sp. nov., isolated from human clinical samples, emended description of the family Veillonellaceae and description of Negativicutes classis nov., Selenomonadales ord. nov. and Acidaminococcaceae fam. nov. in the bacterial phylum Firmicutes.

Three strains of a hitherto unknown, Gram-negative, tiny, anaerobic coccus were collected from human clinical samples originating from skin and soft tissues. The three isolates displayed at least 99.9 % identity in their 16S rRNA gene sequences and more than 99.8 % identity in their dnaK gene sequences. The isolates were affiliated to the family Veillonellaceae, the coccobacillus Dialister micraerophilus being the most closely related species, but there was no more than 91.1 % identity in the 16S rRNA gene sequence between this species and the three isolates. Phylogeny based on the 16S rRNA gene confirmed that the three strains represent a novel and robust lineage within the current family Veillonellaceae. A similar genomic structure was demonstrated for the three isolates by PFGE-based analysis. Morphology and metabolic end products, as well as genotypic and phylogenetic data supported the proposal of the novel genus Negativicoccus gen. nov., with the novel species Negativicoccus succinicivorans sp. nov. [type strain ADV 07/08/06-B-1388(T) (=AIP 149.07(T)=CIP 109806(T)=DSM 21255(T)=CCUG 56017(T)) as type species]. Phylogenetic analyses based on the 16S rRNA gene sequences of members of the phylum Firmicutes and other phyla indicated that the family Veillonellaceae forms a robust lineage clearly separated from those of the classes 'Bacilli', 'Clostridia', Thermolithobacteria and 'Erysipelotrichi' in the phylum Firmicutes. Therefore, we propose that this family is a class-level taxon in the phylum Firmicutes, for which the name Negativicutes classis nov. is proposed, based on the Gram-negative type of cell wall of its members, with the type order Selenomonadales ord. nov. In this order, a novel family, Acidaminococcaceae fam. nov., is proposed and description of the family Veillonellaceae is emended.

Proposal to unify Clostridium orbiscindens Winter et al. 1991 and Eubacterium plautii (Séguin 1928) Hofstad and Aasjord 1982, with description of Flavonifractor plautii gen. nov., comb. nov., and reassignment of Bacteroides capillosus to Pseudoflavonifractor capillosus gen. nov., comb. nov.

We isolated several strains from various clinical samples (five samples of blood, four of intra-abdominal pus and one of infected soft tissue) that were anaerobic, motile or non-motile and Gram-positive rods. Some of the strains formed spores. Phylogenetic analysis of the 16S rRNA gene sequence showed that these organisms could be placed within clostridial cluster IV as defined by Collins et al. [(1994). Int J Syst Bacteriol 44, 812-826] and shared more than 99 % sequence similarity with Clostridium orbiscindens DSM 6740(T) and Eubacterium plautii DSM 4000(T). Together, they formed a distinct cluster, with Bacteroides capillosus ATCC 29799(T) branching off from this line of descent with sequence similarities of 97.1-97.4 %. The next nearest neighbours of these organisms were Clostridium viride, Oscillibacter valericigenes, Papillibacter cinnamivorans and Sporobacter termitidis, with sequence similarities to the respective type strains of 93.1-93.4, 91.2-91.4, 89.8-90 and 88.7-89.3 %. On the basis of biochemical properties, phylogenetic position, DNA G+C content and DNA-DNA hybridization, it is proposed to unify Clostridium orbiscindens and Eubacterium plautii in a new genus as Flavonifractor plautii gen. nov., comb. nov., with the type strain Prévot S1(T) (=ATCC 29863(T) =VPI 0310(T) =DSM 4000(T)), and to reassign Bacteroides capillosus to Pseudoflavonifractor capillosus gen. nov., comb. nov., with the type strain CCUG 15402A(T) (=ATCC 29799(T) =VPI R2-29-1(T)).

Construction of a genetic linkage map of the fungal pathogen of banana Mycosphaerella fijiensis, causal agent of black leaf streak disease.

A genetic linkage map of the fungal plant pathogen Mycosphaerella fijiensis, causal agent of black leaf streak disease of banana was developed. A cross between the isolates CIRAD86 (from Cameroon) and CIRAD139A (from Colombia) was analyzed using molecular markers and the MAT locus. The genetic linkage map consists of 298 AFLP and 16 SSR markers with 23 linkage groups, containing five or more markers, covering 1,879 cM. Markers are separated on average by around 5.9 cM. The MAT locus was shown to segregate in a 1:1 ratio but could not be successfully mapped. An estimate of the relation between physical size and genetic distance was approximately 39.0 kb/cM. The estimated total haploid genome size was calculated using the genetic mapping data at 4,298.2 cM. This is the first genetic linkage map reported for this important foliar pathogen of banana. The great utility of the map will be for anchoring contigs in the genome sequence, evolutionary studies in comparison with other fungi, to identify quantitative trait loci (QTLs) associated with aggressiveness or oxidative stress resistance and with the recently available genome sequence, for positional cloning.

Jonquetella anthropi gen. nov., sp. nov., the first member of the candidate phylum 'Synergistetes' isolated from man.

Six clinical isolates of a hitherto unknown, strictly anaerobic, Gram-negative rod showing fastidious growth were subjected to a polyphasic taxonomic study, including phenotypic, genomic and phylogenetic feature analyses. 16S rRNA gene sequenced-based phylogeny revealed that the novel strains represent a homogeneous group distant from any recognized species in the candidate phylum 'Synergistetes'. The novel isolates were most closely related to species of the genus Dethiosulfovibrio, with 88.2-88.7 % 16S rRNA gene sequence similarity. Large-scale chromosome structure and DNA G+C content also differentiated the novel strains from members of the genus Dethiosulfovibrio. The novel strains were asaccharolytic. Major metabolic end products in trypticase/glucose/yeast extract broth were acetic, lactic, succinic and isovaleric acids and the major cellular fatty acids iso-C(15 : 0) and C(16 : 0). Based on the data presented here, a new genus, Jonquetella gen. nov., is proposed with one novel species, Jonquetella anthropi sp. nov. J. anthropi is the first characterized species of the candidate phylum 'Synergistetes' that includes human isolates. The G+C content of the DNA of the type strain of J. anthropi ADV 126(T) (=AIP 136.05(T)=CIP 109408(T)=CCUG 53819(T)) is 59.4 mol%.

Veillonella denticariosi sp. nov., isolated from human carious dentine.

Selective culture of human carious dentine for Veillonella strains resulted in the isolation of two strains of a Gram-negative, coccus-shaped bacterium that has not been described previously. Comparative 16S rRNA and dnaK gene sequence analysis indicated that the two strains were homogeneous and comprised a distinct lineage within the genus Veillonella, phylogenetically most closely related to Veillonella rodentium. This was supported by DNA-DNA hybridization, which showed clearly that the two strains were similar and distinct from other Veillonella species, and the production of major cellular fatty acids (C(13 : 0) and C(17 : 1)omega8), which is consistent with other members of the genus Veillonella. Based on these observations, strains RBV81 and RBV106(T) represent a novel species, for which the name Veillonella denticariosi sp. nov. is proposed, with the type strain RBV106(T) (=CIP 109448(T) =CCUG 54362(T) =DSM 19009(T)).

Acidaminococcus intestini sp. nov., isolated from human clinical samples.

Eleven strains of a hitherto unknown, Gram-negative, anaerobic coccus were recovered from various human clinical samples of patients hospitalized in two geographically distant French hospitals. These strains displayed the morphology and growth characteristics of those related to the genus Acidaminococcus. The clinical isolates shared at least 99.9 and 99.7 % of their nucleotide positions in the 16S and 23S rRNA gene sequences, respectively. They displayed 95.6 and 88.9 % 16S and 23S rRNA gene sequence similarities, respectively, with Acidaminococcus fermentans. The 16S rRNA-based phylogeny revealed that all the clinical isolates grouped in a statistically well supported cluster separate from A. fermentans. Enzymic activity profiles as well as metabolic end product patterns, including propionic acid production, differentiated the novel bacteria from A. fermentans. Finally, phenotypic, genotypic and phylogenetic data, including large-scale chromosome structure and DNA G+C content, supported the proposal of a novel species of the genus Acidaminococcus, for which the name Acidaminococcus intestini sp. nov. is proposed. The type strain is ADV 255.99(T) (=AIP 283.01(T)=CIP 108586(T)=CCUG 50930(T)).

Challenges of microsatellite isolation in fungi.

Although they represent powerful genetic markers in many fields of biology, microsatellites have been isolated in few fungal species. The aim of this study was to assess whether obtaining microsatellite markers with an acceptable level of polymorphism is generally harder from fungi than in other organisms. We therefore surveyed the number, nature and polymorphism level of published microsatellite markers in fungi from the literature and from our own data on seventeen fungal microsatellite-enriched libraries, and in five other phylogroups (angiosperms, insects, fishes, birds and mammals). Fungal microsatellites indeed appeared both harder to isolate and to exhibit lower polymorphism than in other organisms. This appeared to be due, at least in part, to genomic specificities, such as scarcity and shortness of fungal microsatellite loci. A correlation was observed between mean repeat number and mean allele number in the published fungal microsatellite loci. The cross-species transferability of fungal microsatellites also appeared lower than in other phylogroups. However, microsatellites have been useful in some fungal species. Thus, the considerable advantages of these markers make their development worthwhile, and this study provides some guidelines for their isolation.

Clonality and host selection in the wheat pathogenic fungus Puccinia triticina.

Clonal reproduction in Puccinia triticina, the cause of wheat leaf rust, has long been hypothesized but has never been demonstrated. Using a population genetics approach and microsatellite markers, we analysed genetic diversity of this fungus at each level of genome organisation. Sampling included isolates from two field populations growing on two cultivars carrying specific resistance genes, completed with isolates representing the main pathotypes identified from a national survey. For the two cultivars, populations differentiated according to the distribution of their genotypes and pathotypes. There was a high proportion of repeated genotypes, combined with a significant linkage disequilibrium and a strong negative value for FIS. These three factors, especially heterozygote excess, strongly support the hypothesis of a high rate of clonal reproduction. Each pathotype matched a unique multilocus genotype, except for a few isolates, which were taken to be mutants of the dominant genotype. We discussed the strong relationship between pathotypes and genotypes as the consequence of clonal reproduction combined with a strong selection exerted by host cultivars.

Moryella indoligenes gen. nov., sp. nov., an anaerobic bacterium isolated from clinical specimens.

Three Gram-positive, anaerobic, non-spore-forming, rod-shaped bacteria with pointed ends were isolated from clinical specimens. The organisms were weakly saccharolytic and produced indole, acetate, butyrate and lactate as major metabolic end products. 16S rRNA gene sequence analysis indicated that the isolates had no known close relatives among recognized bacteria but that they exhibited a phylogenetic association with Clostridium rRNA cluster XIVa [as defined by Collins, M. D. et al. (1994). Int J Syst Bacteriol 44, 812-826]. The closest recognized relatives were the type strains of Clostridium clostridioforme, Clostridium bolteae and Clostridium asparagiforme (16S rRNA gene sequence similarity values of 90.2-91.4 %). These results suggest that these three clinical isolates represent a novel species of a new genus, for which the name Moryella indoligenes gen. nov., sp. nov. is proposed. The type strain of Moryella indoligenes is AIP 220.04(T) (=CIP 109174(T)=CCUG 52648(T)).

Evidence for the dispersal of a unique lineage from Asia to America and Africa in the sugarcane fungal pathogen Ustilago scitaminea.

The basidiomycete Ustilago scitaminea Sydow, which causes sugarcane smut disease, has been spreading throughout Africa and America since the 1940s. The genetic diversity and structure of different populations of this fungus worldwide was investigated using microsatellites. A total of 142 single-teliospore were isolated from 77 distinct whips (sori) collected in 15 countries worldwide. Mycelium culture derived from on generation of selfing of these single teliospores were analysed for their polymorphisms at 17 microsatellite loci. All these strains but one were homozygous at all loci, indicating that selfing is likely the predominant reproductive mode of U. scitaminea. The genetic diversity of either American or African U. scitaminea populations was found to be extremely low and all strains belong to a single lineage. This lineage was also found in some populations of Asia, where most U. scitaminea genetic diversity was detected, suggesting that this fungal species originated from this region. The strong founder effect observed in U. scitaminea African and American populations suggests that the fungus migrated from Asia to other continents on rare occasions through movement of infected plant material.