Specific genes from the potato brown rot strains of Ralstonia solanacearum and their potential use for strain detection.

Ralstonia solanacearum is the agent of bacterial wilt infecting >200 different plant species covering >50 botanical families. The genus R. solanacearum can be classified into four phylotypes and each phylotype can be further subdivided into sequevars. The potato brown rot strains of R. solanacearum from phylotype IIB, sequevar 1 (IIB1), historically known as race 3, biovar 2 strains, are responsible for important economic losses to the potato industry and threaten ornamental crop production worldwide. Sensitive and specific detection methods are required to control this pathogen. This article provides a list of 70 genes and 15 intergenes specific to the potato brown rot strains of R. solanacearum from phylotype IIB1. This list was identified by comparative genomic hybridization on microarray and subsequent polymerase chain reaction validation with 14 IIB1 strains against 45 non-IIB1 strains that covered the known genetic diversity in R. solanacearum. The microarray used consisted of the previously described microarray representative of the phylotype I strain GMI1000, to which were added 660 70-mer oligonucleotides representative of new genomic islands detected in the phylotype IIB1 strain IPO1609. The brown rot strain-specific genes thus identified were organized in nine clusters covering 2 to 29 genes within the IPO1609 genome and 6 genes isolated along the genome. Of these specific genes, 29 were parts of mobile genetic elements. Considering the known instability of the R. solanacearum genome, we believe that multiple probes are required to consistently detect all IIB1 strains and we recommend the use of probes which are not part of genetic mobile elements.

Microsatellite markers for the ectomycorrhizal basidiomycete Lactarius mammosus.

We report the characterization of polymorphic microsatellite markers in the ectomycorrhizal fungus Lactarius mammosus Fr. Two enrichment protocols were used to isolate microsatellite loci and polymorphism was explored within 31 sporocarps originating from a forest site in northern Sweden. We found nine variable microsatellite loci with the number of alleles per locus varying between 2 and 5, and expected heterozygosities ranging from 0 to 0.84. These loci are available for the analysis of genetic structure and gene flow in L. mammosus populations.

Hebeloma cylindrosporum- a model species to study ectomycorrhizal symbiosis from gene to ecosystem.

The basidiomycete Hebeloma cylindrosporum has been extensively studied with respect to mycorrhiza differentiation and metabolism and also to population dynamics. Its life cycle can be reproduced in vitro and it can be genetically transformed. Combined biochemical, cytological, genetical and molecular approaches led to the characterisation of mutant strains affected in mycorrhiza formation. These studies demonstrated the role of fungal auxin as a signal molecule in mycorrhiza formation and should allow the characterisation of essential fungal genes necessary to achieve a compatible symbiotic interaction. Random sequencing of cDNAs has identified numerous key functional genes which allowed dissection of essential nitrogen assimilation pathways. H. cylindrosporum also proved to be a remarkable model species to uncover the dynamics of natural populations of ectomycorrhizal fungi and the way in which they respond and adapt to anthropogenic disturbance of the forest ecosystem. Although studies on mycorrhiza differentiation and functioning and those on the population dynamics of H. cylindrosporum have been carried out independently, they are likely to converge in a renewed molecular ecophysiology which will envisage how ectomycorrhizal symbiosis functions under varying field conditions. Contents Summary 481 I. Introduction 482 II. Taxonomy, distribution, autecology, and host range of H. cylindrosporum 482 III. The Hebeloma cylindrosporum toolbox 483 IV. Mycorrhiza differentiation 486 V. Nutritional interactions 488 VI. Genetic diversity and dynamics of H. cylindrosporum populations in P. pinaster forest ecosystems 491 VII. Future directions 494 Acknowledgements 494 References 494.

Parental tracking in the postfire wood decay ascomycete Daldinia loculata using highly variable nuclear gene loci.

The origin of the male and female gametes involved in fertilization events within a local population of the postfire wood decay ascomycete Daldinia loculata was investigated by genotyping the mycelia growing in the wood and the sexual ascospores, using three highly variable nuclear gene loci. The study was conducted in a geographically isolated burned forest site in southern Sweden. An intensive sampling was performed by collecting stromata containing ascospores and wood samples containing mycelia. In total, from 32 mapped burned birches, cultures of 22 haploid genets from decayed wood and six ascospores from each of 19 stromata were isolated and analysed. In 80% of the investigated burned branches, only one genet was found. From the analysis of the ascospore genotypes, we detected 30 fertilization events and 60% of them were the result of mating between conidia (clonal propagules) acting as male gametes and the genets in the branches representing the female gametes. The male parents producing the conidia were detected within the same local population as the female parents in 27% of the fertilization events and originated either from the same branch or from different trees located at 0.5-36 m away from the female parents. In 33% of the fertilization events, conidia originated from three male parents that were not found within the local population sampled. These parents could be anywhere inside or outside the sampled area. For the remaining fertilization events, we could not rule out the ascospores or the conidia as fertilizing propagules. No strong evidence for fertilization by recombinant propagules (ascospores) was detected in this study. The pyrophilous insect species associated with conidia of D. loculata are suggested to be essential vectors for the realization of the sexual cycle of this fungal species. By feeding on the conidia and flying between nearby trees inhabiting wood decay mycelia, these insects allow the transfer of conidia and therefore the opposite mating types to meet within a localized burned forest site.

Correspondence between genet diversity and spatial distribution of above- and below-ground populations of the ectomycorrhizal fungus Hebeloma cylindrosporum.

Population studies of ectomycorrhizal fungal species have largely relied upon fruit body (the reproductive organ) sampling. Analysis of the fruit bodies alone supposes that they reflect the present and spatial organization of all below-ground genets (mycorrhizas and extramatrical mycelia). The relation between fruit bodies and ectomycorrhizas was investigated for the basidiomycete agaric Hebeloma cylindrosporum in four Pinus pinaster stands in south-west France. Genet identification was based on the comparison of polymorphisms within a hypervariable segment of the ribosomal intergenic spacer amplified by polymerase chain reaction (PCR) using a H. cylindrosporum species-specific primer. Mycorrhizas were sorted from soil samples collected underneath patches of fruit bodies or patches where fruit bodies had or had not been observed during the years prior to mycorrhiza collection. On average 65% of the 1026 mycorrhizas collected underneath fruit bodies were formed by H. cylindrosporum, whereas only 2% of the 954 collected in places from where fruit bodies were absent were formed by this species. All genotypes identified above ground were also identified below ground. In patches where one genotype formed all or more than 90% of the fruit bodies, the same genotype formed all or a large majority of the mycorrhizas. In patches occupied by several different fruiting genotypes, additional nonfruiting ones could be present on the root systems. In all cases, the mycorrhizas of one genotype were found no more than 10-20 cm away from its corresponding fruit bodies, and fruit body disappearance at a given place was associated with the disappearance of the corresponding mycorrhizas within 1 year. Although there was not a strict coincidence between the total numbers of genets present below ground and of those forming fruit bodies, fruit body analysis for H. cylindrosporum appears to reflect both the genetic diversity and the spatial structure of its below-ground populations. The results obtained also illustrate the rapid turnover of ectomycorrhizal fungal species on the root systems in the absence of any obvious major disturbance of the ecosystem.

The nuclear ribosomal DNA intergenic spacer as a target sequence to study intraspecific diversity of the ectomycorrhizal basidiomycete Hebeloma cylindrosporum directly on pinus root systems.

Polymorphism of the nuclear ribosomal DNA intergenic spacer (IGS) of the ectomycorrhizal basidiomycete Hebeloma cylindrosporum was studied to evaluate whether this sequence could be used in field studies to estimate the diversity of strains forming mycorrhizas on individual Pinus pinaster root systems. This sequence was amplified by PCR from 125 haploid homokaryotic strains collected in 14 P. pinaster stands along the Atlantic coast of France by using conserved oligonucleotide primers. Restriction enzyme digestion of the amplified 3.4-kbp-long IGS allowed us to characterize 24 alleles whose frequencies differed. Nine of these alleles were found only once, whereas about 60% of the strains contained four of the alleles. Local populations could be almost as diverse as the entire population along a 150-km stretch of coastline that was examined; for example, 13 alleles were found in a single forest stand. The IGS from one strain was partially sequenced, and the sequence data were used to design oligonucleotides which allowed separate PCR amplification of three different segments of the IGS. Most polymorphisms observed among the full-length IGS regions resulted from polymorphisms in an internal ca. 1,500-bp-long sequence characterized by length variations that may have resulted from variable numbers of a T2AG3 motif. This internal polymorphic sequence could not be amplified from the genomes of nine other Hebeloma species. Analysis of this internal sequence amplified from the haploid progenies of 10 fruiting bodies collected in a 70-m2 area resulted in identification of six allelic forms and seven distinct diplotypes out of the 21 possible different combinations. Moreover, optimization of the PCR conditions resulted in amplification of this sequence from more than 80% of the DNA samples extracted from individual H. cylindrosporum infected P. pinaster mycorrhizal root tips, thus demonstrating the usefulness of this sequence for studying the below-ground diversity of mycorrhizas formed by genets belonging to the same fungal species.