Louis Bernier

Rock phosphate solubilization and colonization of maize rhizosphere by wild and genetically modified strains of Penicillium rugulosum

Maize root colonization and phosphate solubilizing activity of the fungus Penicillium rugulosum were assessed in a greenhouse trial using soil-plant microcosms. The bacterial gene hph conferring resistance to hygromicin B was introduced by electroporation in the wild-type strain IR-94MF1 of P. rugulosum and one transformant, w-T3, was selected. Maize plants were grown for 5 weeks in a P-poor soil and fertilized with a Florida apatite mineral, with Navay, an apatite rock deposit from Venezuela, or with simple superphosphate. Inoculation treatments included strain IR-94MF1, transformant w-T3 and two IR-94MF1 UV-induced mutants with enhanced (Mps++) or reduced (Mps−) in vitro mineral phosphate solubilizing activity. In the absence of P fertilization, inoculation with any P. rugulosum isolate significantly reduced the size of the total and P-solubilizing bacterial community present in maize rhizosphere. The bacterial community significantly increased in maize inoculated with IR-94MF1 and w-T3 when P was added as apatites Navay or Florida. All P. rugulosum strains were able to stimulate the growth of maize plants as indicated by 3.6 to 28.6% increases in dry matter yields. In the presence of rock phosphate, P uptake by maize plants inoculated with the two mutants Mps++ and Mps− was not always in agreement with their P-solubilizing phenotypes. Strain IR-94MF1 and transformant w-T3 increased P assimilation by the plants fertilized with Navay rock phosphate by 26 and 38%, respectively. In this treatment, w-T3 showed its highest significant maize rhizosphere colonization. With the simple superphosphate treatment, w-T3 increased P uptake in plants by 8% over the uninoculated control and also decreased significantly the community size of total bacteria, total fungi, and P-solubilizing fungi in the rhizosphere.

Molecular profiling of rhizosphere microbial communities associated with healthy and diseased black spruce (Picea mariana) seedlings grown in a nursery

ABSTRACT Bacterial and fungal populations associated with the rhizosphere of healthy black spruce (Picea mariana) seedlings and seedlings with symptoms of root rot were characterized by cloned rRNA gene sequence analysis. Triplicate bacterial and fungal rRNA gene libraries were constructed, and 600 clones were analyzed by amplified ribosomal DNA restriction analysis and grouped into operational taxonomical units (OTUs). A total of 84 different bacterial and 31 different fungal OTUs were obtained and sequenced. Phylogenetic analyses indicated that the different OTUs belonged to a wide range of bacterial and fungal taxa. For both groups, pairwise comparisons revealed that there was greater similarity between replicate libraries from each treatment than between libraries from different treatments. Significant differences between pooled triplicate samples from libraries of genes from healthy seedlings and pooled triplicate samples from libraries of genes from diseased seedlings were also obtained for both bacteria and fungi, clearly indicating that the rhizosphere-associated bacterial and fungal communities of healthy and diseased P. mariana seedlings were different. The communities associated with healthy and diseased seedlings also showed distinct ecological parameters as indicated by the calculated diversity, dominance, and evenness indices. Among the main differences observed at the community level, there was a higher proportion of Acidobacteria, Gammaproteobacteria, and Homobasidiomycetes clones associated with healthy seedlings, while the diseased-seedling rhizosphere harbored a higher proportion of Actinobacteria, Sordariomycetes, and environmental clones. The methodological approach described in this study appears promising for targeting potential rhizosphere-competent biological control agents against root rot diseases occurring in conifer nurseries.

Solubilization of phosphate rocks and minerals by a wild-type strain and two UV-induced mutants of Penicillium rugulosum

Abstract Two Venezuelan phosphate rocks (PRs), apatite deposits from Monte-Fresco and Navay areas, and two minerals, Florida apatite and Utah variscite were used to investigate phosphate solubilization by the wild type strain IR-94MF1 of Penicillium rugulosum initially selected for its high mineral phosphate activity (Mps + ) and two of its mutants Mps ++ and Mps − . In liquid cultures, the three fungal strains showed better growth on the Navay PR than on Monte Fresco PR. The Utah variscite was the best phosphorus (P) source for the growth of the wild type and the Mps ++ mutant. Solubilization of the various P sources by the wild-type IR-94MF1 and the Mps ++ mutant resulted mostly from the action of organic acids. Citric acid seemed to be more active agent for the solubilization of the Utah variscite while gluconic acid appeared to be responsible for the solubilization of the Florida apatite and the Monte Fresco PR. Both organic acids are likely involved in the solubilization of the Navay PR. The Mps - mutant did not produce any organic acid when grown on all the P sources used.

Localization of a Pathogenicity Gene in Ophiostoma novo-ulmi and Evidence That It May Be Introgressed from O. ulmi

Ophiostoma novo-ulmi, the principal agent of Dutch elm disease, has recently replaced another species of Dutch elm disease pathogen, O. ulmi, across much of the Northern Hemisphere. Field inoculations of the moderately resistant elms Ulmus procera and Ulmus × Commelin were carried out with progeny of a genetic cross between AST27, a Eurasian (EAN) O. novo-ulmi isolate with an unusually low level of pathogenicity, and H327, a highly aggressive EAN isolate. These confirmed the results of a previous study that indicated that the difference in phenotype was controlled by a single nuclear gene. This pathogenicity gene, designated here Pat1, is the first putative pathogenicity gene to be identified in O. novo-ulmi. In a bulked segregant analysis, involving 80 random primers, 10 RAPD (random amplified polymorphic DNA) markers were identified linked to Pat1. Linkage distances between these markers and Pat1 were confirmed by genetic analysis of all individual progeny. Five RAPD amplicons identified in AST27 were O...

Assessment of genetic variation within Chondrostereum purpureum from Quebec by random amplified polymorphic DNA analysis

Genetic variation in Chondrostereum purpureum was assessed by random amplified polymorphic DNA (RAPD) analysis. All 43 isolates examined from 14 different host species in three forest zones, representing the eastern meridional forest ecosystems of Canada, could be differentiated using RAPD amplification profiles generated by seven randomly selected primers. Considerable genetic variation was found among isolates from both small (500 m 2 ) and large (600 000 km 2 ) geographical areas, as well as among isolates from the same and different host species. No RAPD markers specific to host species or regional origin and no groupings with similar profiles were identified. Monokaryons from five different dikaryons were fully interfertile, irrespective of their regional or host origin. Implications of the high level of genetic variation and absence of specialization within and among Quebec populations of C. purpureum are discussed in view of the possible use of this organism as a mycophytocide to control stump sprouting of various deciduous trees.

Genetic Structure of Cronartium ribicola Populations in Eastern Canada

ABSTRACT The genetic structure of populations of Cronartium ribicola was studied by sampling nine populations from five provinces in eastern Canada and generating DNA profiles using nine random amplified polymorphic DNA markers. Most of the total gene diversity (H(t) = 0.386) was present within populations (H(w) = 0.370), resulting in a low level of genetic differentiation among populations in northeastern North America (F(st) = 0.062). A hierarchical analysis of genetic structure using an analysis of molecular variance (AMOVA) revealed no statistically significant genetic differentiation among provinces or among regions. Yet, genetic differentiation among populations within regions or provinces was small (AMOVA phi(st) = 0.078) but statistically significant (P < 0.001) and was several orders of magnitude larger than differentiation among provinces. This is consistent with a scenario of subpopulations within a metapopulation, in which random drift following migration and new colonization are major evolutionary forces. A phenetic analysis using genetic distances revealed no apparent correlation between genetic distance and the province of origin of the populations. The hypothesis of isolation-by-distance in the eastern populations of C. ribicola was rejected by computing Mantel correlation coefficients between genetic and geographic distance matrices (P > 0.05). These results show that eastern Canadian provinces are part of the same white pine blister rust epidemiological unit. Nursery distribution systems are controlled provincially, with virtually no seedling movement among provinces; therefore, infected nursery material may not play an important role in the dissemination of this disease. Long-distance spore dispersal across provincial boundaries appears to be an epidemiologically important factor for this pathogen.

Fungal Diversity, Dominance, and Community Structure in the Rhizosphere of Clonal Picea mariana Plants Throughout Nursery Production Chronosequences

Fungal diversity in the rhizosphere of healthy and diseased clonal black spruce (Picea mariana) plants was analyzed with regard to nursery production chronosequences. The four key production stages were sampled: mother plants (MP), 8-week-old cuttings (B + 0), second-year cuttings (B + 1), and third-year cuttings (B + 2). A total of 45 fungal taxa were isolated and identified based on cultural, phenotypic, and molecular characters. Members of phylum Ascomycota dominated, followed by Basidiomycota and Zygomycota. Diagnosis characters and distance analysis of the internal transcribed spacer rDNA sequences allowed the identification of 39 ascomycetous taxa. Many belong to the order Hypocreales, families Hypocreaceae and Nectriaceae, which contain many clusters of potentially pathogenic taxa (Cylindrocladium, Fusarium, and Neonectria) and are also ecologically associated with antagonistic taxa (Chaetomium, Hypocrea, Microsphaeropsis, Penicillium, Paecilomyces, Verticillium, Trichoderma, and Sporothrix). This is also the first report of a Cylindrocladium canadense association with disease symptoms and relation with Pestalotiopsis, Fusarium, Exserochilum, Rhizoctonia, and Xenochalara fungal consortia. Both production chronosequence and plant health considerably influenced fungal taxa assemblages. Unweighted pair-group arithmetic average clustering showed that isolates from MP, B + 0, and B + 1 plant rhizospheres clustered together within healthy or diseased health classes, whereas isolates from healthy and diseased B + 2 plants clustered together. Canonical correspondence analysis revealed substantial alteration in community assemblages with regard to plant health and yielded a principal axis direction that regrouped taxa associated with diseased plant rhizosphere soil, whereas the opposite axis direction was associated with healthy plants. Two diversity indices were defined and applied to assess the fungal taxa contribution (Tc) and persistence (Pi) throughout the production.

Genetic variability and structure of Canadian populations of Chondrostereum purpureum, a potential biophytocide

Genetic diversity was studied in four Canadian ecological populations, each corresponding to a Canadian ecozone, of Chondrostereum purpureum, including 93 isolates of various host origin. Pseudo‐allelic frequencies were estimated at each of 22 putative RAPD loci by scoring for presence or absence of amplicons in haploid mycelial cultures. The analysis of the hierarchical population structure did not reveal any trend with regard to ecological or host origin. Total gene diversity (HT† = 0.288) was mostly attributable to diversity within populations (HS† = 0.269). In addition, the AMOVA analysis detected most of the molecular variability within subpopulations (89.3%; P < 0.001), whereas a significant (7.3%; P = 0.001) proportion of the gene diversity was found among subpopulations, within ecozones. The results indicate that C. purpureum is a highly heterogeneous pathogen with a continuously distributed population across Canada (GST† = 0.048), and underscore the importance of considering the population structure in the process of its registration as a microbial control agent. A genotype isolated in either central or eastern Canada (ecozones 2, 4 and 5 populations) and selected for its potential as a biophytocide should be considered indigenous in any of these regions of intended use.

Phylogenetic species recognition reveals host-specific lineages among poplar rust fungi.

Fungal species belonging to the genus Melampsora (Basidiomycota, Pucciniales) comprise rust pathogens that alternate between Salicaceae and other plant hosts. Species delineation and identification are difficult within this group due to the paucity of observable morphological features. Several Melampsora rusts are highly host-specific and this feature has been used for identification at the species level. However, this criterion is not always reliable since different Melampsora rust species can overlap on one host but specialize on a different one. To date, two different species recognition methods are used to recognize and define species within the Melampsora genus: (i) morphological species recognition, which is based solely on morphological criteria; and (ii) ecological species recognition, which combines morphological criteria with host range to recognize and define species. In order to clarify species recognition within the Melampsora genus, we applied phylogenetic species recognition to Melampsora poplar rusts by conducting molecular phylogenetic analyses on 15 Melampsora taxa using six nuclear and mitochondrial loci. By assessing the genealogical concordance between phylogenies, we identified 12 lineages that evolved independently, corresponding to distinct phylogenetic species. All 12 lineages were concordant with host specialization, but only three belonged to strictly defined morphological species. The estimation of the species tree obtained with Bayesian concordance analysis highlighted a potential co-evolutionary history between Melampsora species and their reciprocal aecial host plants. Within the Melampsora speciation process, aecial host may have had a strong effect on ancestral evolution, whereas telial host specificity seems to have evolved more recently. The morphological characters initially used to define species boundaries in the Melampsora genus are not reflective of the evolutionary and genetic relationships among poplar rusts. In order to construct a more meaningful taxonomy, host specificity must be considered an important criterion for delineating and describing species within the genus Melampsora as previously suggested by ecological species recognition.

Fungal colonization and host defense reactions in Ulmus americana callus cultures inoculated with Ophiostoma novo-ulmi.

The host-pathogen interaction leading to Dutch elm disease was analyzed using histo- and cyto-chemical tests in an in vitro system. Friable and hard susceptible Ulmus americana callus cultures were inoculated with the highly aggressive pathogen Ophiostoma novo-ulmi. Inoculated callus tissues were compared with water-treated callus tissues and studied with light microscopy (LM), transmission-electron microscopy (TEM), and scanning-electron microscopy (SEM). New aspects of this interaction are described. These include the histological observation, for the first time in plant callus cultures, of suberin with its typical lamellar structure in TEM and the intracellular presence of O. novo-ulmi. Expression of the phenylalanine ammonia lyase gene, monitored by real-time quantitative polymerase chain reaction, was correlated with the accumulation of suberin, phenols, and lignin in infected callus cultures. This study validates the potential use of the in vitro system for genomic analyses aimed at identifying genes expressed during the interaction in the Dutch elm disease pathosystem.