Martin Filion

Direct quantification of fungal DNA from soil substrate using real-time PCR

Detection and quantification of genomic DNA from two ecologically different fungi, the plant pathogen Fusarium solani f. sp. phaseoli and the arbuscular mycorrhizal fungus Glomus intraradices, was achieved from soil substrate. Specific primers targeting a 362-bp fragment from the SSU rRNA gene region of G. intraradices and a 562-bp fragment from the F. solani f. sp. phaseoli translation elongation factor 1 alpha gene were used in real-time polymerase chain reaction (PCR) assays conjugated with the fluorescent SYBR(R) Green I dye. Standard curves showed a linear relation (r(2)=0.999) between log values of fungal genomic DNA of each species and real-time PCR threshold cycles and were quantitative over 4-5 orders of magnitude. Real-time PCR assays were applied to in vitro-produced fungal structures and sterile and non-sterile soil substrate seeded with known propagule numbers of either fungi. Detection and genomic DNA quantification was obtained from the different treatments, while no amplicon was detected from non-seeded non-sterile soil samples, confirming the absence of cross-reactivity with the soil microflora DNA. A significant correlation (P<0.0001) was obtained between the amount of genomic DNA of F. solani f. sp. phaseoli or G. intraradices detected and the number of fungal propagules present in seeded soil substrate. The DNA extraction protocol and real-time PCR quantification assay can be performed in less than 2 h and is adaptable to detect and quantify genomic DNA from other soilborne fungi.

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.

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.

Production of DAPG and HCN by Pseudomonas sp. LBUM300 Contributes to the Biological Control of Bacterial Canker of Tomato

Bacterial canker caused by Clavibacter michiganensis subsp. michiganensis is known to cause significant economic losses to tomato production worldwide. Biological control has been proposed as an alternative to current chemical containment methods, which are often inefficient and may leave adverse effects on the environment. However, only little headway has so far been made in developing biocontrol strategies against C. michiganensis subsp. michiganensis. To address this knowledge gap, we investigated the antagonistic capacity of PCA, produced by Pseudomonas sp. LBUM223, and DAPG and HCN, both produced by Pseudomonas sp. LBUM300, on C. michiganensis subsp. michiganensis under in vitro and in planta conditions. Nonsynthesizing isogenic mutants of the producer strains were also developed to further dissect the role of each individual metabolite on C. michiganensis subsp. michiganensis biological control. Novel specific quantitative polymerase chain reaction TaqMan assays allowed quantification of C. michiganensis subsp. michiganensis in tomato plants and rhizospheric soil. Pseudomonas spp. LBUM223 and LBUM300 significantly repressed C. michiganensis subsp. michiganensis growth in vitro, while their respective nonproducing mutants showed less or no significant antagonistic activity. In planta, only Pseudomonas sp. LBUM300 was capable of significantly reducing disease development and C. michiganensis subsp. michiganensis rhizospheric population, suggesting that the production of both DAPG and HCN was involved. In summary, simultaneous DAPG/HCN production by Pseudomonas sp. LBUM300 shows great potential for controlling bacterial canker of tomato.

Effects of temperatures near the freezing point on N2O emissions, denitrification and on the abundance and structure of nitrifying and denitrifying soil communities

Climate warming in temperate regions may lead to decreased soil temperatures over winter as a result of reduced snow cover. We examined the effects of temperatures near the freezing point on N(2)O emissions, denitrification, and on the abundance and structure of soil nitrifiers and denitrifiers. Soil microcosms supplemented with NO3 - and/or NO3 - plus red clover residues were incubated for 120 days at -4 °C, -1 °C, +2 °C or +5 °C. Among microcosms amended with residues, N(2)O emission and/or denitrification increased with increasing temperature on Days 2 and 14. Interestingly, N(2)O emission and/or denitrification after Day 14 were the greatest at -1 °C. Substantial N(2) O emissions were only observed on Day 2 at +2 °C and +5 °C, while at -1 °C, N(2)O emissions were consistently detected over the duration of the experiment. Abundances of ammonia oxidizing bacteria (AOB) and archaea (AOA), Nitrospira-like bacteria and nirK denitrifiers were the lowest in soils at -4 °C, while abundances of Nitrobacter-like bacteria and nirS denitrifiers did not vary among temperatures. Community structures of nirK and nirS denitrifiers and Nitrobacter-like bacteria shifted between below-zero and above-zero temperatures. Structure of AOA and AOB communities also changed but not systematically among frozen and unfrozen temperatures. Results indicated shifts in some nitrifier and denitrifier communities with freezing and a surprising stimulation of N(2)O emissions at -1 °C when NO3 - and C are present.

Relative and Absolute Quantitative Real-Time PCR-Based Quantifications of hcnC and phlD Gene Transcripts in Natural Soil Spiked with Pseudomonas sp. Strain LBUM300

ABSTRACT Transcriptional analysis of microbial gene expression using relative quantitative real-time PCR (qRT-PCR) has been hampered by various technical problems. One such problem is the unavailability of an exogenous standard robust enough for use in a complex matrix like soil. To circumvent this technical issue, we made use of a recently developed artificial RNA (myIC) as an exogenous “spike-in” control. Nonsterile field soil was inoculated with various concentrations of the test bacterium Pseudomonas sp. strain LBUM300, ranging from 4.3- to 8.3-log bacterial cells per gram of soil. Total soil RNA was extracted at days 0, 7, and 14 postinoculation, and using two-step TaqMan assays, phlD (encoding the production of 2,4-diacetylphloroglucinol) and hcnC (encoding the production of hydrogen cyanide) gene expression was monitored. For relative quantification, a defined quantity of in vitro-synthesized myIC RNA was spiked during the RNA extraction procedure. Absolute qRT-PCR was also performed in parallel. Both the absolute and relative quantifications showed similar transcriptional trends. Overall, the transcriptional activity of phlD and hcnC changed over time and with respect to the bacterial concentrations used. Transcripts of the phlD and hcnC genes were detected for all five bacterial concentrations, but the phlD transcript copy numbers detected were lower than those detected for hcnC, regardless of the initial bacterial concentration or sampling date. For quantifying a low number of transcripts, the relative method was more reliable than the absolute method. This study demonstrates for the first time the use of a relative quantification approach to quantifying microbial gene transcripts from field soil using an exogenous spike-in control.

Genetic diversity of Streptomyces spp. causing common scab of potato in eastern Canada

Common scab is an important disease of potato caused by Streptomyces scabies and other closely related species. In this study, the genetic diversity of Streptomyces spp. causing common scab of potato in eastern Canada was for the first time investigated. Forty-one Streptomyces spp. isolates were retrieved from necrotic lesions of potato tubers harvested from different regions of the Canadian provinces New-Brunswick, Nova Scotia and Prince-Edward-Island. Most isolates were closely related to known pathogenic S. scabies strains on the basis of partial 16S ribosomal (r) RNA and rpoB gene sequence analyses. Two isolates were identified as pathogenic species of Streptomyces acidiscabies. To our knowledge, this species has never been previously isolated in these areas. Genome fingerprinting studies using repetitive elements (rep) polymerase chain reactions (PCR) revealed 10 distinct genetic groups in eastern Canada. The geographical distribution of the genetic groups was region-dependant. Pathogenicity- and virulence-related genes (txtA, txtC, and tomA) were PCR-amplified from each isolate, and nucleotide sequence analysis of partial gene fragments revealed slight polymorphisms in both txtA and txtC genes. No genetic variation was noted in the partial tomA gene sequences.

Alteration of Soil Rhizosphere Communities following Genetic Transformation of White Spruce

ABSTRACT The application of plant genetic manipulations to agriculture and forestry with the aim of alleviating insect damage through Bacillus thuringiensis transformation could lead to a significant reduction in the release of pesticides into the environment. However, many groups have come forward with very valid and important questions related to potentially adverse effects, and it is crucial to assess and better understand the impact that this technology might have on ecosystems. In this study, we analyzed rhizosphere soil samples collected from the first B. thuringiensis-transformed trees [with insertion of the CryIA(b) toxin-encoding gene] grown in Canada (Val-Cartier, QC, Canada) as part of an ecological impact assessment project. Using a robust amplified rRNA gene restriction analysis approach coupled with 16S rRNA gene sequencing, the rhizosphere-inhabiting microbial communities of white spruce (Picea glauca) genetically modified by biolistic insertion of the cryIA(b), uidA (beta-glucuronidase), and nptII genes were compared with the microbial communities associated with non-genetically modified counterparts and with trees in which only the genetic marker genes uidA and nptII have been inserted. Analysis of 1,728 rhizosphere bacterial clones (576 clones per treatment) using a Cramér-von Mises statistic analysis combined with a Monte Carlo comparison clearly indicated that there was a statistically significant difference (P < 0.05) between the microbial communities inhabiting the rhizospheres of trees carrying the cryIA(b), uidA, and nptII transgenes, trees carrying only the uidA and nptII transgenes, and control trees. Clear rhizosphere microbial community alterations due to B. thuringiensis tree genetic modification have to our knowledge never been described previously and open the door to interesting questions related to B. thuringiensis genetic transformation and also to the impact of commonly used uidA and nptII genetic marker genes.

Transcriptional activity of antifungal metabolite-encoding genes phlD and hcnBC in Pseudomonas spp. using qRT-PCR.

Production of 2,4-diacetylphloroglucinol (2,4-DAPG) and hydrogen cyanide (HCN) by Pseudomonas spp. shows great potential for controlling soilborne plant pathogens. However, little is known about the transcriptional activity of phl and hcn genes encoding 2,4-DAPG and HCN, respectively. To progress toward a better understanding of what triggers phl and hcn expression under rhizosphere conditions, novel PCR primers and TaqMan probes were designed to monitor relative phlD and hcnBC expression in quantitative real time-PCR assays. Transcriptional activity of phlD and hcnBC was studied in time-course confrontational assays using combinations of Pseudomonas spp. isolated in this study: LBUM300 (producing 2,4-DAPG and HCN) and LBUM647 (producing HCN only); pathogens Phytophthora cactorum and Verticillium dahliae; and solid growth media Kings B medium and potato dextrose agar. In correlation with the antagonistic activity observed, expression of phlD and hcnBC and production of 2,4-DAPG was detected throughout the 14-day course of the experiment in LBUM300 on both media, while hcnBC expression diminished to undetectable levels in LBUM647. In LBUM300 expression of phlD and hcnBC significantly changed over time and was also influenced by the presence of pathogen and growth media following time-dependent responses.

The ability of Pseudomonas sp. LBUM 223 to produce phenazine-1-carboxylic acid affects the growth of Streptomyces scabies, the expression of thaxtomin biosynthesis genes and the biological control potential against common scab of potato.

Streptomyces scabies causes common scab, an economical disease affecting potato crops world-wide, for which no effective control measure exists. This pathogen produces the plant toxin thaxtomin A, which is involved in symptom development on potato tubers. A biological control approach that can limit S. scabies growth and repress thaxtomin production represents an attractive alternative to classical control strategies. Pseudomonas sp. LBUM 223 produces phenazine-1-carboxylic acid (PCA), an antibiotic that inhibits the growth of plant pathogens and contributes to the biological control of plant diseases. In this study, the involvement of LBUM 223s PCA-producing ability in the growth inhibition of S. scabies, repression of thaxtomin biosynthesis genes (txtA and txtC) and the biological control of common scab of potato was investigated using a mutant defective in PCA production (LBUM 223phzC(-) ). Streptomyces scabies growth was inhibited to a significantly lesser degree by LBUM 223phzC(-) than by the wild type. LBUM 223 also significantly repressed txtA and txtC expression in S. scabies and protected potato against disease, whereas LBUM 223phzC(-) did not. These results suggest that PCA production is central to the ability of LBUM 223 to limit pathogen growth, repress the expression of key pathogenicity genes and control common scab of potato.