Genetic structure and population diversity of Phytophthora infestans strains in Pacific western Canada.

Late blight caused by Phytophthora infestans is an economically important disease of potato and tomato worldwide. In Canada, an increase in late blight incidence and severity coincided with changes in genetic composition of P. infestans. We monitored late blight incidence on tomato and potato in Pacific western and eastern Canada between 2019 and 2022, identified genotypes of P. infestans, and examined their population genetic diversity. We identified four major existing genotypes US11, US17, US8, and US23 as well as 25 new genotypes. The US11 genotype was dominant in Pacific western Canada, accounting for 59% of the total population. We discovered the US17 genotype for the first time in Canada. We revealed a higher incidence of late blight and quite diverse genotypes of P. infestans in Pacific western Canada than in eastern Canada. We found high genetic diversity of P. infestans population from Pacific western Canada, as evidenced by the high number of multilocus genotypes, high values of genetic diversity indices, and emergence of 25 new genotypes. Considering the number of disease incidence, the detection of diverse known genotypes, the emergence of novel genotypes, and the high number of isolates resistant to metalaxyl-m (95%) from Pacific western Canada, the region could play a role in establishing sexual recombination and diverse populations, which could ultimately pose challenges for late blight management. Therefore, continuous monitoring of P. infestans populations in Pacific western region and across Canada is warranted. KEY POINTS: • Genotypes of P. infestans in Pacific western were quite diverse than in eastern Canada. • We discovered US17 genotype for the first time in Canada and identified 26 novel genotypes. • Approximately 95% of P. infestans isolates were resistant to metalaxyl-m.

Seasonal Dynamics of Various Scab-Causing Streptomyces Genotypes Among Potato Fields on Prince Edward Island.

Potato common scab is an important bacterial plant disease caused by numerous Streptomyces species and strains. A better understanding of the genetic diversity and population dynamics of these microorganisms in the field is crucial to develop effective control methods. Our research group previously studied the genetic diversity of scab-causing Streptomyces spp. in Prince Edward Island, one of Canada's most important potato-growing provinces. Fourteen distinct Streptomyces genotypes were identified and displayed contrasting aggressiveness toward potato tubers. To better understand the distribution and occurrence of these genotypes over time under field conditions, the population dynamics were studied in nine commercial potato fields throughout a growing season. A comparative genomic-driven approach was used to design genotype-specific primers and probes, allowing us to quantify, using quantitative polymerase chain reaction, the abundance of each of the 14 genotypes in field soil. Thirteen of the previously identified genotypes were detected in at least one soil sample, with various frequencies and population sizes across the different fields under study. Interestingly, weakly virulent genotypes dominated, independent of time or location. Among them, three genotypes accounted for more than 80% of the genotypes' combined population. Although the highly virulent genotypes were detected in lower relative abundance than the weakly virulent ones, an increase in the highly virulent genotypes' population size was observed over the growing season in most fields. The results will ultimately be useful for the development of targeted common scab control strategies.

Interplay between Arabidopsis thaliana Genotype, Plant Growth and Rhizosphere Colonization by Phytobeneficial Phenazine-Producing Pseudomonas chlororaphis.

Rhizosphere colonization by phytobeneficial Pseudomonas spp. is pivotal in triggering their positive effects on plant health. Many Pseudomonas spp. Determinants, involved in rhizosphere colonization, have already been deciphered. However, few studies have explored the role played by specific plant genes in rhizosphere colonization by these bacteria. Using isogenic Arabidopsis thaliana mutants, we studied the effect of 20 distinct plant genes on rhizosphere colonization by two phenazine-producing P. chlororaphis strains of biocontrol interest, differing in their colonization abilities: DTR133, a strong rhizosphere colonizer and ToZa7, which displays lower rhizocompetence. The investigated plant mutations were related to root exudation, immunity, and root system architecture. Mutations in smb and shv3, both involved in root architecture, were shown to positively affect rhizosphere colonization by ToZa7, but not DTR133. While these strains were not promoting plant growth in wild-type plants, increased plant biomass was measured in inoculated plants lacking fez, wrky70, cbp60g, pft1 and rlp30, genes mostly involved in plant immunity. These results point to an interplay between plant genotype, plant growth and rhizosphere colonization by phytobeneficial Pseudomonas spp. Some of the studied genes could become targets for plant breeding programs to improve plant-beneficial Pseudomonas rhizocompetence and biocontrol efficiency in the field.

Seasonal changes in the abundance and activity of bacterial and fungal denitrifying communities associated with different compost amendments.

Composts can be efficient organic amendments in potato culture as they can supply carbon and nutrients to the soil. However, more information is required on the effects of composts on denitrification and nitrous oxide emissions (N2O) and emission-producing denitrifying communities. The effects of three compost amendments (municipal source separated organic waste compost (SSOC), forestry waste mixed with poultry manure compost (FPMC), and forestry residues compost (FRC)) on fungal and bacterial denitrifying communities and activity was examined in an agricultural field cropped to potatoes during the fall, spring, and summer seasons. The denitrification enzyme activity (DEA), N2O emissions, and respiration were measured in parallel. N2O emission rates were greater in FRC-amended soils in the fall and summer, whereas soil respiration was highest in the SSOC-amended soil in the fall. A large number of nirK denitrifying fungal transcripts were detected in the fall, coinciding with compost application, while the greatest nirK bacterial transcripts were measured in the summer when plants were actively growing. Denitrifying community and transcript levels were poor predictors of DEA, N2O emissions, or respiration rates in compost-amended soil. Overall, the sampling date was driving the population and activity levels of the three denitrifying communities under study.

Biocontrol Activity of Bacillus spp. and Pseudomonas spp. Against Botrytis cinerea and Other Cannabis Fungal Pathogens.

Gray mold caused by Botrytis cinerea is one of the most widespread and damaging diseases in cannabis crops worldwide. With challenging restrictions on pesticide use and few effective control measures, biocontrol agents are needed to manage this disease. The aim of this study was to identify bacterial biocontrol agents with wide-spectrum activity against B. cinerea and other cannabis fungal pathogens. Twelve Bacillus and Pseudomonas strains were first screened with in vitro confrontational assays against 10 culturable cannabis pathogens, namely B. cinerea, Sclerotinia sclerotiorum, Fusarium culmorum, F. sporotrichoides, F. oxysporum, Nigrospora sphaerica, N. oryzae, Alternaria alternata, Phoma sp., and Cercospora sp. Six strains displaying the highest inhibitory activity, namely Bacillus velezensis LBUM279, FZB42, LBUM1082, Bacillus subtilis LBUM979, P. synxantha LBUM223, and P. protegens Pf-5, were further assessed in planta where all, except LBUM223, significantly controlled gray mold development on cannabis leaves. Notably, LBUM279 and FZB42 reduced disease severity by at least half compared with water-treated plants and prevented lesion development and/or sporulation up to 9 days after pathogen inoculation. Genomes of LBUM279, LBUM1082, and LBUM979 were sequenced de novo and taxonomic affiliations were determined to ensure nonrelatedness with pathogenic strains. Moreover, the genomes were exempt of detrimental genes encoding major toxins and virulence factors that could otherwise pose a biosafety risk when used on crops. Eighteen gene clusters of potential biocontrol interest were also identified. To our knowledge, this is the first reported attempt to control cannabis fungal diseases in planta by direct antagonism with beneficial bacteria.

In Tuber Biocontrol of Potato Late Blight by a Collection of Phenazine-1-Carboxylic Acid-Producing Pseudomonas spp.

Phenazine-1-carboxylic acid (PCA) produced by plant-beneficial Pseudomonas spp. is an antibiotic with antagonistic activities against Phytophthora infestans, the causal agent of potato late blight. In this study, a collection of 23 different PCA-producing Pseudomonas spp. was confronted with P. infestans in potato tuber bioassays to further understand the interaction existing between biocontrol activity and PCA production. Overall, the 23 strains exhibited different levels of biocontrol activity. In general, P. orientalis and P. yamanorum strains showed strong disease reduction, while P. synxantha strains could not effectively inhibit the pathogen's growth. No correlation was found between the quantities of PCA produced and biocontrol activity, suggesting that PCA cannot alone explain P. infestans' growth inhibition by phenazine-producing pseudomonads. Other genetic determinants potentially involved in the biocontrol of P. infestans were identified through genome mining in strains displaying strong biocontrol activity, including siderophores, cyclic lipopeptides and non-ribosomal peptide synthase and polyketide synthase hybrid clusters. This study represents a step forward towards better understanding the biocontrol mechanisms of phenazine-producing Pseudomonas spp. against potato late blight.

Interactions Between Bacillus Spp., Pseudomonas Spp. and Cannabis sativa Promote Plant Growth.

Plant growth-promoting rhizobacteria (PGPR) deploy several mechanisms to improve plant health, growth and yield. The aim of this study was to evaluate the efficacy of two Pseudomonas spp. strains and three Bacillus spp. strains used as single treatments and in consortia to improve the yield of Cannabis sativa and characterize the impact of these treatments on the diversity, structure and functions of the rhizosphere microbiome. Herein, we demonstrate a significant C. sativa yield increase up to 70% when inoculated with three different Pseudomonas spp./Bacillus spp. consortia but not with single inoculation treatments. This growth-promoting effect was observed in two different commercial soil substrates commonly used to grow cannabis: Promix and Canna coco. Marker-based genomic analysis highlighted Bacillus spp. as the main modulator of the rhizosphere microbiome diversity and Pseudomonas spp. as being strongly associated with plant growth promotion. We describe an increase abundance of predicted PGPR metabolic pathways linked with growth-promoting interactions in C. sativa.

Comparative Genomics of Potato Common Scab-Causing Streptomyces spp. Displaying Varying Virulence.

Common scab of potato causes important economic losses worldwide following the development of necrotic lesions on tubers. In this study, the genomes of 14 prevalent scab-causing Streptomyces spp. isolated from Prince Edward Island, one of the most important Canadian potato production areas, were sequenced and annotated. Their phylogenomic affiliation was determined, their pan-genome was characterized, and pathogenic determinants involved in their virulence, ranging from weak to aggressive, were compared. 13 out of 14 strains clustered with Streptomyces scabiei, while the last strain clustered with Streptomyces acidiscabies. The toxicogenic and colonization genomic regions were compared, and while some atypical gene organizations were observed, no clear correlation with virulence was observed. The production of the phytotoxin thaxtomin A was also quantified and again, contrary to previous reports in the literature, no clear correlation was found between the amount of thaxtomin A secreted, and the virulence observed. Although no significant differences were observed when comparing the presence/absence of the main virulence factors among the strains of S. scabiei, a distinct profile was observed for S. acidiscabies. Several mutations predicted to affect the functionality of some virulence factors were identified, including one in the bldA gene that correlates with the absence of thaxtomin A production despite the presence of the corresponding biosynthetic gene cluster in S. scabiei LBUM 1485. These novel findings obtained using a large number of scab-causing Streptomyces strains are challenging some assumptions made so far on Streptomyces' virulence and suggest that other factors, yet to be characterized, are also key contributors.

Inhibition of Three Potato Pathogens by Phenazine-Producing Pseudomonas spp. Is Associated with Multiple Biocontrol-Related Traits.

Phenazine-producing Pseudomonas spp. are effective biocontrol agents that aggressively colonize the rhizosphere and suppress numerous plant diseases. In this study, we compared the ability of 63 plant-beneficial phenazine-producing Pseudomonas strains representative of the worldwide diversity to inhibit the growth of three major potato pathogens: the oomycete Phytophthora infestans, the Gram-positive bacterium Streptomyces scabies, and the ascomycete Verticillium dahliae. The 63 Pseudomonas strains are distributed among four different subgroups within the P. fluorescens species complex and produce different phenazine compounds, namely, phenazine-1-carboxylic acid (PCA), phenazine-1-carboxamide (PCN), 2-hydroxyphenazine-1-carboxylic acid, and 2-hydroxphenazine. Overall, the 63 strains exhibited contrasted levels of pathogen inhibition. Strains from the P. chlororaphis subgroup inhibited the growth of P. infestans more effectively than strains from the P. fluorescens subgroup. Higher inhibition was not associated with differential levels of phenazine production nor with specific phenazine compounds. The presence of additional biocontrol-related traits found in P. chlororaphis was instead associated with higher P. infestans inhibition. Inhibition of S. scabies by the 63 strains was more variable, with no clear taxonomic segregation pattern. Inhibition values did not correlate with phenazine production nor with specific phenazine compounds. No additional synergistic biocontrol-related traits were found. Against V. dahliae, PCN producers from the P. chlororaphis subgroup and PCA producers from the P. fluorescens subgroup exhibited greater inhibition. Additional biocontrol-related traits potentially involved in V. dahliae inhibition were identified. This study represents a first step toward harnessing the vast genomic diversity of phenazine-producing Pseudomonas spp. to achieve better biological control of potato pathogens. IMPORTANCE Plant-beneficial phenazine-producing Pseudomonas spp. are effective biocontrol agents, thanks to the broad-spectrum antibiotic activity of the phenazine antibiotics they produce. These bacteria have received considerable attention over the last 20 years, but most studies have focused only on the ability of a few genotypes to inhibit the growth of a limited number of plant pathogens. In this study, we investigated the ability of 63 phenazine-producing strains, isolated from a wide diversity of host plants on four continents, to inhibit the growth of three major potato pathogens: Phytophthora infestans, Streptomyces scabies, and Verticillium dahliae. We found that the 63 strains differentially inhibited the three potato pathogens. These differences are in part associated with the nature and the quantity of the phenazine compounds being produced but also with the presence of additional biocontrol-related traits. These results will facilitate the selection of versatile biocontrol agents against pathogens.

Diversity and Virulence of Streptomyces spp. Causing Potato Common Scab in Prince Edward Island, Canada.

Common scab (CS) is a potato disease that significantly decreases the market value of potato tubers after the development of necrotic lesions on their surface. Streptomyces scabiei is the main causal agent of CS; however, other closely related species, including S. acidiscabies and S. turgidiscabies, have also been shown to cause the disease. In this study, we characterized the genetic and phenotypic diversity of Streptomyces spp. causing CS in Prince Edward Island, the main potato-producing province in Canada. Two hundred and ninety-six pathogenic Streptomyces spp. isolates were retrieved from diseased tubers harvested from six fields located across a longitudinal geographical gradient. Genome fingerprinting analyses using repetitive elements PCR (ERIC- and BOX-PCR) revealed 14 distinct genetic groups. Thirteen groups were taxonomically affiliated with S. scabiei, whereas the fourteenth group was affiliated with S. acidiscabies. Their geographical distribution was characterized and revealed that on average between six and eight different genetic groups were detected per field, with variable abundance. Virulence assays showed strong differences in virulence between the genetic groups, ranging from low to highly virulent. Interestingly, pathogenic Streptomyces spp. populations in each field seem to be dominated by the most virulent genetic groups. The results obtained will contribute to better understanding of the population dynamic of pathogenic Streptomyces spp. causing CS of potato and promoting the development of more efficient detection and intervention tools to manage this important potato disease.

Expression of Putative Defense Responses in Cannabis Primed by Pseudomonas and/or Bacillus Strains and Infected by Botrytis cinerea.

Cannabis (Cannabis sativa L.) offers many industrial, agricultural, and medicinal applications, but is commonly threatened by the gray mold disease caused by the fungus Botrytis cinerea. With few effective control measures currently available, the use of beneficial rhizobacteria represents a promising biocontrol avenue for cannabis. To counter disease development, plants rely on a complex network of inducible defense pathways, allowing them to respond locally and systemically to pathogens attacks. In this study, we present the first attempt to control gray mold in cannabis using beneficial rhizobacteria, and the first investigation of cannabis defense responses at the molecular level. Four promising Pseudomonas (LBUM223 and WCS417r) and Bacillus strains (LBUM279 and LBUM979) were applied as single or combined root treatments to cannabis seedlings, which were subsequently infected by B. cinerea. Symptoms were recorded and the expression of eight putative defense genes was monitored in leaves by reverse transcription quantitative polymerase chain reaction. The rhizobacteria did not significantly control gray mold and all infected leaves were necrotic after a week, regardless of the treatment. Similarly, no systemic activation of putative cannabis defense genes was reported, neither triggered by the pathogen nor by the rhizobacteria. However, this work identified five putative defense genes (ERF1, HEL, PAL, PR1, and PR2) that were strongly and sustainably induced locally at B. cinerea's infection sites, as well as two stably expressed reference genes (TIP41 and APT1) in cannabis. These markers will be useful in future researches exploring cannabis defense pathways.

Inoculation With the Plant-Growth-Promoting Rhizobacterium Pseudomonas fluorescens LBUM677 Impacts the Rhizosphere Microbiome of Three Oilseed Crops.

The bacterial communities inhabiting the rhizosphere play an important role in plant development and health. Here we studied the effect of inoculation with Pseudomonas fluorescens LBUM677, a plant growth promoting rhizobacterium that promotes seed oil accumulation, on the rhizosphere microbiome of three oilseed crops (Brassica napus, Buglossoides arvensis, and Glycine max) over time. Next-Generation high-throughput sequencing targeting the V4 region of 16S rDNA was used to characterize the microbial communities associated with the three different crops, inoculated or not with LBUM677, over a time period of up to 90 days post-inoculation. A total of 1,627,231 amplicon sequence variants were obtained and were taxonomically grouped into 39 different phyla. LBUM677 inoculation and sampling date were found to significantly influence the rhizosphere microbiome of the three oil-producing crops under study. Specifically, inoculation with LBUM677 and sampling date, but not the plant species, were found to significantly alter the alpha- and the beta-diversity of the rhizosphere microbial communities. Differential abundance analyses found that 29 taxonomical bacterial groups were significantly more abundant in the LBUM677 treatments while 30 were significantly more abundant in the control treatments. Predicted functions of the microorganisms were also enriched, including 47 enzymatic pathways in LBUM677 treatments. These non-targeted effects on rhizosphere bacterial communities are discussed in the context of oilseed crops.

Spatio-Temporal and Cultivar-Dependent Variations in the Cannabis Microbiome.

The incipient legalization and commercialization of Cannabis sativa in Canada have promulgated research into characterizing the plant's microbiome as it promotes many facets of plant growth and health. The emblematic production of commercially important secondary metabolites, namely tetrahydrocannabinol (THC), cannabidiol (CBD) and terpenes, has warranted investigating the modulating capacity of these molecules on the plant microbiome. C. sativa cultivars can be classified into chemotypes depending on the relative levels of THC and CBD they produce; their biosynthesis also varies spatially and temporally during the life cycle of the plant. To study the differential microbiome structure and diversity between cultivars in a spatio-temporal manner, we extracted microbial DNA from the rhizosphere, endorhizosphere, and phyllosphere during the entire life cycle of three different chemotypes; CBD Yummy (<1% THC/13% CBD), CBD shark (6% THC/10% CBD) and Hash (14% THC/ < 1% CBD). Illumina marker gene sequencing of bacterial (16S) and fungal (ITS) communities were coupled to the QIIME2, PICRUSt, and LEfSe pipelines for analysis. Our study describes spatio-temporal and cultivar-dependent variations in the fungal and bacterial microbiome of C. sativa, and details strong cultivar-dependent variance in the belowground microbiome. Furthermore, the predicted pathway abundance of the bacterial microbiome is concomitantly subject to spatio-temporal variations; pathways related to lipid, amino acid, glucose and pentose metabolism were noteworthy. These results describe, for the first time, spatio-temporal and cultivar-dependent variations in the microbiome of C. sativa produced under strict commercial settings. Describing the microbiome is the first step in discoveries that could help in engineering a plant growth and health promoting microbiome in future works.

Metabolic and Genomic Traits of Phytobeneficial Phenazine-Producing Pseudomonas spp. Are Linked to Rhizosphere Colonization in Arabidopsis thaliana and Solanum tuberosum.

Bacterial rhizosphere colonization is critical for phytobeneficial rhizobacteria such as phenazine-producing Pseudomonas spp. To better understand this colonization process, potential metabolic and genomic determinants required for rhizosphere colonization were identified using a collection of 60 phenazine-producing Pseudomonas strains isolated from multiple plant species and representative of the worldwide diversity. Arabidopsis thaliana and Solanum tuberosum (potato) were used as host plants. Bacterial rhizosphere colonization was measured by quantitative PCR using a newly designed primer pair and TaqMan probe targeting a conserved region of the phenazine biosynthetic operon. The metabolic abilities of the strains were assessed on 758 substrates using Biolog phenotype microarray technology. These data, along with available genomic sequences for all strains, were analyzed in light of rhizosphere colonization. Strains belonging to the P. chlororaphis subgroup colonized the rhizospheres of both plants more efficiently than strains belonging to the P. fluorescens subgroup. Metabolic results indicated that the ability to use amines and amino acids was associated with an increase in rhizosphere colonization capability in A. thaliana and/or in S. tuberosum The presence of multiple genetic determinants in the genomes of the different strains involved in catabolic pathways and plant-microbe and microbe-microbe interactions correlated with increased or decreased rhizosphere colonization capabilities in both plants. These results suggest that the metabolic and genomic traits found in different phenazine-producing Pseudomonas strains reflect their rhizosphere competence in A. thaliana and S. tuberosum Interestingly, most of these traits are associated with similar rhizosphere colonizing capabilities in both plant species.IMPORTANCE Rhizosphere colonization is crucial for plant growth promotion and biocontrol by antibiotic-producing Pseudomonas spp. This colonization process relies on different bacterial determinants which partly remain to be uncovered. In this study, we combined a metabolic and a genomic approach to decipher new rhizosphere colonization determinants which could improve our understanding of this process in Pseudomonas spp. Using 60 distinct strains of phenazine-producing Pseudomonas spp., we show that rhizosphere colonization abilities correlated with both metabolic and genomic traits when these bacteria were inoculated on two distant plants, Arabidopsis thaliana and Solanum tuberosum Key metabolic and genomic determinants presumably required for efficient colonization of both plant species were identified. Upon further validation, these targets could lead to the development of simple screening tests to rapidly identify efficient rhizosphere colonizers.

Complete Genome Sequence of the Plant Growth-Promoting Rhizobacterium Pseudomonas fluorescens LBUM677.

Pseudomonas fluorescens LBUM677 has shown the ability to increase plant biomass and seed oil yield in soybean, canola, and Buglossoides arvensis (corn gromwell) when inoculated in the rhizosphere. Here, we report a draft genome sequence of P. fluorescens LBUM677, with an estimated size of 6.14 Mb.

Diversity of phytobeneficial traits revealed by whole-genome analysis of worldwide-isolated phenazine-producing Pseudomonas spp.

Plant-beneficial Pseudomonas spp. competitively colonize the rhizosphere and display plant-growth promotion and/or disease-suppression activities. Some strains within the P. fluorescens species complex produce phenazine derivatives, such as phenazine-1-carboxylic acid. These antimicrobial compounds are broadly inhibitory to numerous soil-dwelling plant pathogens and play a role in the ecological competence of phenazine-producing Pseudomonas spp. We assembled a collection encompassing 63 strains representative of the worldwide diversity of plant-beneficial phenazine-producing Pseudomonas spp. In this study, we report the sequencing of 58 complete genomes using PacBio RS II sequencing technology. Distributed among four subgroups within the P. fluorescens species complex, the diversity of our collection is reflected by the large pangenome which accounts for 25 413 protein-coding genes. We identified genes and clusters encoding for numerous phytobeneficial traits, including antibiotics, siderophores and cyclic lipopeptides biosynthesis, some of which were previously unknown in these microorganisms. Finally, we gained insight into the evolutionary history of the phenazine biosynthetic operon. Given its diverse genomic context, it is likely that this operon was relocated several times during Pseudomonas evolution. Our findings acknowledge the tremendous diversity of plant-beneficial phenazine-producing Pseudomonas spp., paving the way for comparative analyses to identify new genetic determinants involved in biocontrol, plant-growth promotion and rhizosphere competence.

Transcriptome alteration in Phytophthora infestans in response to phenazine-1-carboxylic acid production by Pseudomonas fluorescens strain LBUM223.

BACKGROUND: Phytophthora infestans is responsible for late blight, one of the most important potato diseases. Phenazine-1-carboxylic acid (PCA)-producing Pseudomonas fluorescens strain LBUM223 isolated in our laboratory shows biocontrol potential against various plant pathogens. To characterize the effect of LBUM223 on the transcriptome of P. infestans, we conducted an in vitro time-course study. Confrontational assay was performed using P. infestans inoculated alone (control) or with LBUM223, its phzC- isogenic mutant (not producing PCA), or exogenically applied PCA. Destructive sampling was performed at 6, 9 and 12 days and the transcriptome of P. infestans was analysed using RNA-Seq. The expression of a subset of differentially expressed genes was validated by RT-qPCR. RESULTS: Both LBUM223 and exogenically applied PCA significantly repressed P. infestans' growth at all times. Compared to the control treatment, transcriptomic analyses showed that the percentages of all P. infestans' genes significantly altered by LBUM223 and exogenically applied PCA increased as time progressed, from 50 to 61% and from to 32 to 46%, respectively. When applying an absolute cut-off value of 3 fold change or more for all three harvesting times, 207 genes were found significantly differentially expressed by PCA, either produced by LBUM223 or exogenically applied. Gene ontology analysis revealed that both treatments altered the expression of key functional genes involved in major functions like phosphorylation mechanisms, transmembrane transport and oxidoreduction activities. Interestingly, even though no host plant tissue was present in the in vitro system, PCA also led to the overexpression of several genes encoding effectors. The mutant only slightly repressed P. infestans' growth and barely altered its transcriptome. CONCLUSIONS: Our study suggests that PCA is involved in P. infestans' growth repression and led to important transcriptomic changes by both up- and down-regulating gene expression in P. infestans over time. Different metabolic functions were altered and many effectors were found to be upregulated, suggesting their implication in biocontrol.

Interaction between 2,4-Diacetylphloroglucinol- and Hydrogen Cyanide-Producing Pseudomonas brassicacearum LBUM300 and Clavibacter michiganensis subsp. michiganensis in the Tomato Rhizosphere.

We have previously demonstrated that inoculation of tomato plants with 2,4-diacetylphloroglucinol (DAPG)- and hydrogen cyanide (HCN)-producing Pseudomonas brassicacearum LBUM300 could significantly reduce bacterial canker symptoms caused by Clavibacter michiganensis subsp. michiganensis In this study, in order to better characterize the population dynamics of LBUM300 in the rhizosphere of tomato plants, we characterized the role played by DAPG and HCN production by LBUM300 on rhizosphere colonization of healthy and C. michiganensis subsp. michiganensis-infected tomato plants. The impact of C. michiganensis subsp. michiganensis presence on the expression of DAPG and HCN biosynthetic genes in the rhizosphere was also examined. In planta assays were performed using combinations of C. michiganensis subsp. michiganensis and wild-type LBUM300 or DAPG (LBUM300ΔphlD) or HCN (LBUM300ΔhcnC) isogenic mutant strains. Populations of LBUM300 and phlD and hcnC gene expression levels were quantified in rhizosphere soil at several time points up to 264 h postinoculation using culture-independent quantitative PCR (qPCR) and reverse transcriptase quantitative PCR (RT-qPCR) TaqMan assays, respectively. The presence of C. michiganensis subsp. michiganensis significantly increased rhizospheric populations of LBUM300. In C. michiganensis subsp. michiganensis-infected tomato rhizospheres, the populations of wild-type LBUM300 and strain LBUM300ΔhcnC, both producing DAPG, were significantly higher than the population of strain LBUM300ΔphlD A significant upregulation of phlD expression was observed in the presence of C. michiganensis subsp. michiganensis, while hcnC expression was only slightly increased in the mutant strain LBUM300ΔphlD when C. michiganensis subsp. michiganensis was present. Additionally, biofilm production was found to be significantly reduced in strain LBUM300ΔphlD compared to the wild-type and LBUM300ΔhcnC strains.IMPORTANCE The results of this study suggest that C. michiganensis subsp. michiganensis infection of tomato plants contributes to increasing rhizospheric populations of LBUM300, a biocontrol agent, as well as the overexpression of the DAPG biosynthetic operon in this bacterium. The increasing rhizospheric populations of LBUM300 represent one of the key factors in controlling C. michiganensis subsp. michiganensis in tomato plants, as DAPG-producing bacteria have shown the ability to decrease bacterial canker symptoms in tomato plants.

Phenazine-1-Carboxylic Acid Production by Pseudomonas fluorescens LBUM636 Alters Phytophthora infestans Growth and Late Blight Development.

Phytophthora infestans causes late blight of potato, one of the most devastating diseases affecting potato production. Alternative approaches for controlling late blight are being increasingly sought due to increasing environmental concerns over the use of chemical pesticides and the increasing resistance of P. infestans to fungicides. Our research group has isolated a new strain of Pseudomonas fluorescens (LBUM636) of biocontrol interest producing the antibiotic phenazine-1-carboxylic acid (PCA). Wild-type LBUM636 was shown to significantly inhibit the growth of Phytophthora infestans in in vitro confrontational assays whereas its isogenic mutant (phzC-; not producing PCA) only slightly altered the pathogen's growth. Wild-type LBUM636 but not the phzC- mutant also completely repressed disease symptom development on tubers. A pot experiment revealed that wild-type LBUM636 can significantly reduce P. infestans populations in the rhizosphere and in the roots of potato plants, as well as reduce in planta disease symptoms due to PCA production. The expression of eight common plant defense-related genes (ChtA, PR-1b, PR-2, PR-5, LOX, PIN2, PAL-2, and ERF3) was quantified in tubers, roots, and leaves by reverse-transcription quantitative polymerase chain reaction and revealed that the biocontrol observed was not associated with the induction of a plant defense response by LBUM636. Instead, a direct interaction between P. infestans and LBUM636 is required and PCA production appears to be a key factor for LBUM636's biocontrol ability.

Novel P450nor Gene Detection Assay Used To Characterize the Prevalence and Diversity of Soil Fungal Denitrifiers.

UNLABELLED: Denitrifying fungi produce nitrous oxide (N2O), a potent greenhouse gas, as they generally lack the ability to convert N2O to dinitrogen. Contrary to the case for bacterial denitrifiers, the prevalence and diversity of denitrifying fungi found in the environment are not well characterized. In this study, denitrifying fungi were isolated from various soil ecosystems, and novel PCR primers targeting the P450nor gene, encoding the enzyme responsible for the conversion of nitric oxide to N2O, were developed, validated, and used to study the diversity of cultivable fungal denitrifiers. This PCR assay was also used to detect P450nor genes directly from environmental soil samples. Fungal denitrification capabilities were further validated using an N2O gas detection assay and a PCR assay targeting the nirK gene. A collection of 492 facultative anaerobic fungi was isolated from 15 soil ecosystems and taxonomically identified by sequencing the internal transcribed spacer sequence. Twenty-seven fungal denitrifiers belonging to 10 genera had the P450nor and the nirK genes and produced N2O from nitrite. N2O production is reported in strains not commonly known as denitrifiers, such as Byssochlamys nivea, Volutella ciliata, Chloridium spp., and Trichocladium spp. The prevalence of fungal denitrifiers did not follow a soil ecosystem distribution; however, a higher diversity was observed in compost and agricultural soils. The phylogenetic trees constructed using partial P450nor and nirK gene sequences revealed that both genes clustered taxonomically closely related strains together. IMPORTANCE: A PCR assay targeting the P450nor gene involved in fungal denitrification was developed and validated. The newly developed P450nor primers were used on fungal DNA extracted from a collection of fungi isolated from various soil environments and on DNA directly extracted from soil. The results indicated that approximatively 25% of all isolated fungi possessed this gene and were able to convert nitrite to N2O. All soil samples from which denitrifying fungi were isolated also tested positive for the presence of P450nor The P450nor gene detection assay was reliable in detecting a large diversity of fungal denitrifiers. Due to the lack of homology existing between P450nor and bacterial denitrification genes, it is expected that this assay will become a tool of choice for studying fungal denitrifiers.