Various forms of organic and inorganic P fertilizers did not negatively affect soil- and root-inhabiting AM fungi in a maize-soybean rotation system.

Arbuscular mycorrhizal (AM) fungi are key components of most agricultural ecosystems. Therefore, understanding the impact of agricultural practices on their community structure is essential to improve nutrient mobilization and reduce plant stress in the field. The effects of five different organic or mineral sources of phosphorus (P) for a maize-soybean rotation system on AM fungal diversity in roots and soil were assessed over a 3-year period. Total DNA was extracted from root and soil samples collected at three different plant growth stages. An 18S rRNA gene fragment was amplified and taxa were detected and identified using denaturing gradient gel electrophoresis followed by sequencing. AM fungal biomass was estimated by fatty acid methyl ester analysis. Soil P fertility parameters were also monitored and analyzed for possible changes related with fertilization or growth stages. Seven AM fungal ribotypes were detected. Fertilization significantly modified soil P flux, but had barely any effect on AM fungi community structure or biomass. There was no difference in the AM fungal community between plant growth stages. Specific ribotypes could not be significantly associated to P treatment. Ribotypes were associated with root or soil samples with variable detection frequencies between seasons. AM fungal biomass remained stable throughout the growing seasons. This study demonstrated that roots and soil host distinct AM fungal communities and that these are very temporally stable. The influence of contrasting forms of P fertilizers was not significant over 3 years of crop rotation.

Long-term phosphorus fertilization impacts soil fungal and bacterial diversity but not AM fungal community in alfalfa.

Soil function may be affected by cropping practices impacting the soil microbial community. The effect of different phosphorus (P) fertilization rates (0, 20, or 40 kg P(2)O(5) ha(-1)) on soil microbial diversity was studied in 8-year-old alfalfa monocultures. The hypothesis that P fertilization modifies soil microbial community was tested using denaturing gradient gel electrophoresis and phospholipids fatty acid (PLFA) profiling to describe soil bacteria, fungi, and arbuscular mycorrhizal (AM) fungi diversity. Soil parameters related to fertility (soil phosphate flux, soluble P, moisture, phosphatase and dehydrogenase assays, and carbon and nitrogen content of the light fraction of soil organic matter) were also monitored and related to soil microbial ribotype profiles. Change in soil P fertility with the application of fertilizer had no effect on crop yield in 8 years, but on the year of this study was associated with shifts in the composition of fungal and bacterial communities without affecting their richness, as evidenced by the absence of effect on the average number of ribotypes detected. However, variation in soil P level created by a history of differential fertilization did not significantly influence AM fungi ribotype assemblages nor AM fungi biomass measured with the PLFA 16:1omega5. Fertilization increased P flux and soil soluble P level but reduced soil moisture and soil microbial activity, as revealed by dehydrogenase assay. Results suggest that soil P fertility management could influence soil processes involving soil microorganisms. Seasonal variations were also recorded in microbial activity, soil soluble P level as well as in the abundance of specific bacterial and fungal PLFA indicators of soil microbial biomass.

Role of the modification in root exudation induced by arbuscular mycorrhizal colonization on the intraradical growth of Phytophthora nicotianae in tomato.

We studied the role of modification in root exudation induced by colonization with Glomus intraradices and Glomus mosseae in the growth of Phytophthora nicotianae in tomato roots. Plants were grown in a compartmentalized plant growth system and were either inoculated with the AM fungi or received exudates from mycorrhizal plants, with the corresponding controls. Three weeks after planting, the plants were inoculated or not with P. nicotianae growing from an adjacent compartment. At harvest, P. nicotianae biomass was significantly reduced in roots colonized with G. intraradices or G. mosseae in comparison to non-colonized roots. Conversely, pathogen biomass was similar in non-colonized roots supplied with exudates collected from mycorrhizal or non-mycorrhizal roots, or with water. We cannot rule out that a mycorrhiza-mediated modification in root exudation may take place, but our results did not support that a change in pathogen chemotactic responses to host root exudates may be involved in the inhibition of P. nicotianae.

Trees, fungi and bacteria: tripartite metatranscriptomics of a root microbiome responding to soil contamination.

BACKGROUND: One method for rejuvenating land polluted with anthropogenic contaminants is through phytoremediation, the reclamation of land through the cultivation of specific crops. The capacity for phytoremediation crops, such as Salix spp., to tolerate and even flourish in contaminated soils relies on a highly complex and predominantly cryptic interacting community of microbial life. METHODS: Here, Illumina HiSeq 2500 sequencing and de novo transcriptome assembly were used to observe gene expression in washed Salix purpurea cv. 'Fish Creek' roots from trees pot grown in petroleum hydrocarbon-contaminated or non-contaminated soil. All 189,849 assembled contigs were annotated without a priori assumption as to sequence origin and differential expression was assessed. RESULTS: The 839 contigs differentially expressed (DE) and annotated from S. purpurea revealed substantial increases in transcripts encoding abiotic stress response equipment, such as glutathione S-transferases, in roots of contaminated trees as well as the hallmarks of fungal interaction, such as SWEET2 (Sugars Will Eventually Be Exported Transporter). A total of 8252 DE transcripts were fungal in origin, with contamination conditions resulting in a community shift from Ascomycota to Basidiomycota genera. In response to contamination, 1745 Basidiomycota transcripts increased in abundance (the majority uniquely expressed in contaminated soil) including major monosaccharide transporter MST1, primary cell wall and lamella CAZy enzymes, and an ectomycorrhiza-upregulated exo-ß-1,3-glucanase (GH5). Additionally, 639 DE polycistronic transcripts from an uncharacterised Enterobacteriaceae species were uniformly in higher abundance in contamination conditions and comprised a wide spectrum of genes cryptic under laboratory conditions but considered putatively involved in eukaryotic interaction, biofilm formation and dioxygenase hydrocarbon degradation. CONCLUSIONS: Fungal gene expression, representing the majority of contigs assembled, suggests out-competition of white rot Ascomycota genera (dominated by Pyronema), a sometimes ectomycorrhizal (ECM) Ascomycota (Tuber) and ECM Basidiomycota (Hebeloma) by a poorly characterised putative ECM Basidiomycota due to contamination. Root and fungal expression involved transcripts encoding carbohydrate/amino acid (C/N) dialogue whereas bacterial gene expression included the apparatus necessary for biofilm interaction and direct reduction of contamination stress, a potential bacterial currency for a role in tripartite mutualism. Unmistakable within the metatranscriptome is the degree to which the landscape of rhizospheric biology, particularly the important but predominantly uncharacterised fungal genetics, is yet to be discovered.

A PCR-denaturing gradient gel electrophoresis approach to assess Fusarium diversity in asparagus.

In North America, asparagus (Asparagus officinalis) production suffers from a crown and root rot disease mainly caused by Fusarium oxysporum f. sp. asparagi and F. proliferatum. Many other Fusarium species are also found in asparagus fields, whereas accurate detection and identification of these organisms, especially when processing numerous samples, is usually difficult and time consuming. In this study, a PCR-denaturing gradient gel electrophoresis (DGGE) method was developed to assess Fusarium species diversity in asparagus plant samples. Fusarium-specific PCR primers targeting a partial region of the translation elongation factor-1 alpha (EF-1 alpha) gene were designed, and their specificity was tested against genomic DNA extracted from a large collection of closely and distantly related organisms isolated from multiple environments. Amplicons of 450 bp were obtained from all Fusarium isolates, while no PCR product was obtained from non-Fusarium organisms. The ability of DGGE to discriminate between Fusarium taxa was tested over 19 different Fusarium species represented by 39 isolates, including most species previously reported from asparagus fields worldwide. The technique was effective to visually discriminate between the majority of Fusarium species and/or isolates tested in pure culture, while a further sequencing step permitted to distinguish between the few species showing similar migration patterns. Total genomic DNA was extracted from field-grown asparagus plants naturally infested with different Fusarium species, submitted to PCR amplification, DGGE analysis and sequencing. The two to four bands observed for each plant sample were all affiliated with F. oxysporum, F. proliferatum or F. solani, clearly supporting the reliability, sensitivity and specificity of this approach for the study of Fusarium diversity from asparagus plants samples.

Root Rot of Black Spruce Caused by Cylindrocladium canadense in Eastern North America.

During October 2002, symptoms of root rot of black spruce, Picea mariana (Mill.) B.S.P., were observed in the St-Modeste (47°46'N, 69°36'W) conifer nursery (400 km northeast of Montreal, Quebec, Canada). Disease severity was low in the greenhouse-produced mother plants and 1-year-old seedlings and moderate in field-grown 2- and 3-year-old seedlings. A species of Cylindrocladium was isolated on potato dextrose agar from 12 symptomatic seedlings from the greenhouse and 12 from the field. The isolates produced chestnut-colored colonies and chlamydospores, both of which were typical of C. canadense Kang, Crous & Schoch (2). DNA was extracted from representative isolates (MTF 101, MTF 102), and the internal transcribed spacer (ITS) of the rDNA gene was amplified and sequenced (GenBank Accession Nos. AY705980 and AY705981). There was a 99% match with a sequence of C. canadense (GenBank Accession No. AF348256). However, there was approximately 10% divergence with the ITS sequence of C. floridanum (GenBank Accession No AF307343). MTF101 and MTF102 were pathogenic on black spruce seedlings when fungal suspension (106 CFU/ml) was added to germinating seeds in petri plates or infiltrated into roots of 2-week-old seedlings growing in sterilized, moist, sandy soil in the greenhouse. Within 3 weeks, inoculated seedlings exhibited typical root necrosis, while control seedlings were symptomless. C. canadense was reisolated only from symptomatic seedlings. The occurrence of C. canadense in eastern North America has significant implications for forestry regeneration. Previously, only C. floridanum had been reported as pathogenic in the St-Modeste nursery and in eastern North America(1). References: (1) R. C. Hamelin et al. Appl. Environ. Microbiol. 62:4026, 1996. (2) J. C. Kang et al. Syst. Appl. Microbiol. 24:206, 2001.

Quantification of Fusarium solani f. sp. phaseoli in Mycorrhizal Bean Plants and Surrounding Mycorrhizosphere Soil Using Real-Time Polymerase Chain Reaction and Direct Isolations on Selective Media.

ABSTRACT The capacity of the arbuscular mycorrhizal fungus Glomus intraradices in reducing the presence of Fusarium solani f. sp. phaseoli in bean plants and the surrounding mycorrhizosphere soil was evaluated in a compartmentalized experimental system. Quantification of the pathogen and the symbiont in plant tissues, the soil regions of the mycorrhizosphere (rhizosphere and mycosphere), and the bulk soil was accomplished using specific polymerase chain reaction (PCR) primers in real-time PCR assays, culture-dependant methods, and microscopic determination techniques. Nonmycorrhizal bean plants infected with the pathogen had distinctive Fusarium root rot symptoms, while infected plants previously colonized by G. intraradices remained healthy. The amount of F. solani f. sp. phaseoli genomic DNA was significantly reduced in mycorrhizal bean plants and in each mycorrhizosphere soil compartment. The presence of G. intraradices in the mycorrhizosphere was not significantly modified, although the mycorrhizal colonization of roots was slightly increased in the presence of the pathogen. The results suggest that the reduced presence of Fusarium as well as root rot symptoms are caused by biotic and/or abiotic modifications of the mycorrhizosphere as a result of colonization with G. intraradices.

First Report of Root Rot on Asparagus Caused by Phytophthora megasperma in Canada.

In August 2002, Phytophthora megasperma Drechs. was isolated from wilted plants of Asparagus officinalis L. cv. Guelph Millenium displaying spear and crown rot. Six affected plants were sampled in a commercial asparagus field located in the Saguenay-Lac-Saint-Jean Region (300 km northeast of Montreal, Quebec, Canada). The fungus was isolated from asparagus fern stalks, crown tissue, and spears after a rainy period and identified using morphological and cultural characteristics (2). In pinkish 4-week-old cultures, unbranched stalks bore abundant sporangia, which were ovoid to obpyriform in shape, 15 to 45 m long, and 10 to 30 m in diameter. Characteristic circular oospores >30 m in diameter were produced on V8 juice agar at 25°C in darkness after 1 month. Pathogenicity was tested on asparagus cvs. Guelph Millenium and Jersey Knight. A mycelium suspension (3 ml at 106 CFU/ml) prepared from 1-week-old shaken potato dextrose (PD) broth was sprayed on 30 1-week-old seedlings grown in petri plates filled with sterilized, moist, sandy soil and held at 20°C (day/night). Controls received sterile PD broth. Within 3 weeks of incubation in the dark, inoculated seedlings exhibited necrotic symptoms similar to those observed initially, while controls remained healthy. The pathogen was isolated from 75% of the 'Guelph Millenium' and 98% of the 'Jersey Knight' symptomatic seedlings, but not isolated from the control seedlings. In North America, disease caused by P. megasperma resulting in yield loss has been reported in California and New York (1,3). In Canada, the etiology of asparagus diseases is not well characterized. To our knowledge, this is the first report of P. megasperma on asparagus plants in Canada. References: (1) P. A. Ark and J. T. Barrett. Phytopathology 28:754, 1938. (2) D. C. Erwin et al. Phytophthora: Its Biology, Taxonomy, Ecology, and Pathology. The American Phytopathological Society, St Paul, MN, 1983. (3) T-L. Kuan and D. C. Erwin. Phytopathology 70:333, 1980.

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.

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

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.