Trophic interactions in microbiomes influence plant host population size and ecosystem function.

Plant microbiomes that comprise diverse microorganisms, including prokaryotes, eukaryotes and viruses, are the key determinants of plant population dynamics and ecosystem function. Despite their importance, little is known about how species interactions (especially trophic interactions) between microbes from different domains modify the importance of microbiomes for plant hosts and ecosystems. Using the common duckweed Lemna minor, we experimentally examined the effects of predation (by bacterivorous protists) and parasitism (by bacteriophages) within microbiomes on plant population size and ecosystem phosphorus removal. Our results revealed that the addition of predators increased plant population size and phosphorus removal, whereas the addition of parasites showed the opposite pattern. The structural equation modelling further pointed out that predation and parasitism affected plant population size and ecosystem function via distinct mechanisms that were both mediated by microbiomes. Our results highlight the importance of understanding microbial trophic interactions for predicting the outcomes and ecosystem impacts of plant-microbiome symbiosis.

Actinomycetospora lemnae sp. nov., A Novel Actinobacterium Isolated from Lemna aequinoctialis Able to Enhance Duckweed Growth.

Duckweed-associated actinobacteria are co-existing microbes that affect duckweed growth and adaptation. In this study, we aimed to report a novel actinobacterium species and explore its ability to enhance duckweed growth. Strain DW7H6T was isolated from duckweed, Lemna aequinoctialis. Phylogenetic analysis based on its 16S rRNA gene sequence revealed that the strain was most closely related to Actinomycetospora straminea IY07-55T (99.0%), Actinomycetospora chibensis TT04-21T (98.9%), Actinomycetospora lutea TT00-04T (98.8%) and Actinomycetospora callitridis CAP 335T (98.4%). Chemotaxonomic and morphological characteristics of strain DW7H6T were consistent with members of the genus Actinomycetospora, while average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) between the draft genomes of this strain and its closely related type strains were below the proposed threshold values used for species discrimination. Based on chemotaxonomic, phylogenetic, phenotypic, and genomic evidence obtained, we describe a novel Actinomycetospora species, for which the name Actinomycetospora lemnae sp. nov. is proposed. The type strain is DW7H6T (TBRC 15165T, NBRC 115294T). Additionally, the duckweed-associated actinobacterium strain DW7H6T was able to enhance duckweed growth when compared to the control, in which the number of fronds and biomass dry weight were increased by up to 1.4 and 1.3 fold, respectively. Moreover, several plant-associated gene features in the genome of strain DW7H6T potentially involved in plant-microbe interactions were identified.

Duckweed-associated bacteria as plant growth-promotor to enhance growth of Spirodela polyrhiza in wastewater effluent from a poultry farm.

Duckweed has been highlighted as an invaluable resource because of its abilities to remove nitrogen and phosphorus from wastewater coupling with the production of high starch/protein-containing plant biomass. Duckweed recruits microbes and particularly forms a stable "core" bacterial microbiota, which greatly reduces the colonization efficiency of plant growth-promoting bacteria (PGPB). In this study, natural duckweeds were enriched in a sterilized-partially treated wastewater effluent from a poultry farm. After 24 days of cultivation, the duckweed-associated bacteria (DAB) were isolated and evaluated for their plant growth-promoting (PGP) potentials by co-cultivation with axenic Spirodela polyrhiza. Ten species were found in more than one location and could be considered candidates for the stable "core" DAB. Among them, all isolates of Acinetobacter soli, Acidovorax kalamii, Brevundimonas vesicularis, Pseudomonas toyotomiensis, and Shinella curvata increased duckweed growth in Hoagland medium. The highest PGP ability was observed in Sh. curvata W12-8 (with EPG value of 208.72%), followed by Paracoccus marcusii W7-16 (171.31%), Novosphingobium subterraneum W5-13 (156.96%), and Ac. kalamii W7-18 (156.96%). However, the highest growth promotion in the wastewater was observed when co-cultured with W7-16, which was able to increase biomass dry weight and root length of duckweed by 3.17 and 2.26 folds, respectively.

Potential of Predatory Bacteria to Colonize the Duckweed Microbiome and Change Its Structure: A Model Study Using the Obligate Predatory Bacterium, Bacteriovorax sp. HI3.

Modifying the duckweed microbiome is a major challenge for enhancing the effectiveness of duckweed-based wastewater treatment and biomass production technologies. We herein examined the potential of the exogenous introduction of predatory bacteria to change the duckweed microbiome. Bacteriovorax sp. HI3, a model predatory bacterium, colonized the core of the Lemna microbiome, and its predatory behavior changed the microbiome structure, which correlated with colonization density. These results reveal that bacterial predatory interactions may be important drivers that shape the duckweed microbiome, suggesting their potential usefulness in modifying the microbiome.

Environment and Host Genetics Influence the Biogeography of Plant Microbiome Structure.

To understand how microbiota influence plant populations in nature, it is important to examine the biogeographic distribution of plant-associated microbiomes and the underlying mechanisms. However, we currently lack a fundamental understanding of the biogeography of plant microbiomes across populations and the environmental and host genetic factors that shape their distribution. Leveraging the broad distribution and extensive genetic variation in duckweeds (the Lemna species complex), we identified key factors that governed plant microbiome diversity and compositional variation geographically. In line with the microbial biogeography of free-living microbiomes, we observed higher bacterial richness in temperate regions relative to lower latitudes in duckweed microbiomes (with 10% higher in temperate populations). Our analyses revealed that higher temperature and sodium concentration in aquatic environments showed a negative impact on duckweed bacterial richness, whereas temperature, precipitation, pH, and concentrations of phosphorus and calcium, along with duckweed genetic variation, influenced the biogeographic variation of duckweed bacterial community composition. Analyses of plant microbiome assembly processes further revealed that niche-based selection played an important role (26%) in driving the biogeographic variation of duckweed bacterial communities, alongside the contributions of dispersal limitation (33%) and drift (39%). These findings add significantly to our understanding of host-associated microbial biogeography and provide important insights for predicting plant microbiome vulnerability and resilience under changing climates and intensifying anthropogenic activities.

Microbial Population Dynamics in Lemnaceae (Duckweed)-Based Wastewater Treatment System.

The dynamic microflora associated within, and in the surrounding aquatic environment, has been found to be responsible for the functional properties of many aquatic plants. The aim of the current work was to evaluate the effectiveness of Lemnaceae-based wastewater treatment system under tropical conditions and investigate the changes in the aquatic microflora upon plant growth. A biological wastewater treatment system was designed and investigated using mixed Lemnaceae culture comprising Lemna minor and Spirodela polyrhiza in a batch mode. A significant reduction in total solids (31.8%), biochemical oxygen demand (93.5%), and chemical oxygen demand (73.2%) was observed after seven days of duckweed growth using a low inoculum. A preliminary study on the change in the microbial population diversity and functionality, in the wastewater before and after treatment, revealed an increase in the denitrifying microflora in wastewater post-Lemnaceae treatment. Dominance of 10 bacterial phyla, contributing for 98.3% of the total bacterial communities, was recorded, and ~ 50.6% loss of diversity post-treatment of wastewater was revealed by the Shannon Index. Among 16 bacterial families showing relative abundance of ≥ 1% in untreated wastewater, Methylobacteriaceae, Pseudomonadaceae, Brucellaceae, Rhodobacteraceae, and Acetobacteraceae prevailed in the water post-treatment by duckweeds. This is a novel work done on the dynamics of aquatic microflora associated with Lemnaceae under tropical Indian conditions. It confirms the application of Lemnaceae-based wastewater treatment system as effective biofilter and calls for further studies on the active involvement of the endophytic and aquatic microflora in the functions of these plant.

Genome-wide identification of bacterial colonization and fitness determinants on the floating macrophyte, duckweed.

Bacterial communities associated with aquatic macrophytes largely influence host primary production and nutrient cycling in freshwater environments; however, little is known about how specific bacteria migrate to and proliferate at this unique habitat. Here, we separately identified bacterial genes involved in the initial colonization and overall fitness on plant surface, using the genome-wide transposon sequencing (Tn-seq) of Aquitalea magnusonii H3, a plant growth-promoting bacterium of the floating macrophyte, duckweed. Functional annotation of identified genes indicated that initial colonization efficiency might be simply explained by motility and cell surface structure, while overall fitness was associated with diverse metabolic and regulatory functions. Genes involved in lipopolysaccharides and type-IV pili biosynthesis showed different contributions to colonization and fitness, reflecting their metabolic cost and profound roles in host association. These results provide a comprehensive genetic perspective on aquatic-plant-bacterial interactions, and highlight the potential trade-off between bacterial colonization and proliferation abilities on plant surface.

Streptomyces epipremni sp. nov., an endophytic actinomycete isolated from the root of Epipremnum aureum.

An endophytic Streptomyces-like micro-organism, designated strain PRB2-1T was isolated from root tissue of Epipremnum aureum (Linden and André) G.S. Bunting. The typical morphological and chemotaxonomic characteristics, i.e. the ability to produce straight spore chains directly on aerial mycelium and the presence of ll-diaminopimelic acid in cell-wall peptidoglycan, were consistent with its assignment to the genus Streptomyces. 16S rRNA gene analysis showed that strain PRB2-1T is a member of the genus Streptomyces with the highest similarity to Streptomyces bryophytorum DSM 42183T (98.4 %). Moreover, the draft genome sequence of strain PRB2-1T exhibited low average nucleotide identity by blast (79.9-83.8 %) and digital DNA-DNA hybridization (24.9-28.3 %) values to the reference strains, which were well below the species circumscription threshold. The DNA G+C content of genomic DNA was 73.6 mol%. Comparison of phenotypic characteristics and whole-genome sequence between strain PRB2-1T and its close relatives indicated that strain PRB2-1T could be classified as a novel species of the genus Streptomyces. Thus the name, Streptomyces epipremni sp. nov. is proposed for the strain. The type strain is PRB2-1T (=TBRC 7642T=NBRC 113169T).

Streptomyces spirodelae sp. nov., isolated from duckweed.

A novel actinobacterium, designated strain DW4-2T, was isolated from duckweed (Spirodela sp.) collected from an agricultural pond in Kasetsart University, Bangkok, Thailand. The morphological, chemotaxonomic and phylogenetic characteristics were consistent with its classification in the genus Streptomyces. Strain DW4-2T showed the highest 16S rRNA gene sequence similarity values to Streptomyces qinglanensis DSM 42035T (98.5 %), Streptomyces smyrnaeus DSM 42105T (98.4 %) and Streptomyces oryzae S16-07T (98.4 %). Digital DNA-DNA hydridization and average nucleotide identity values between the genome sequences of strain DW4-2T with S. qinglanensis DSM 42035T (29.8 and 87.8 %), S. smyrnaeus DSM 42105T (33.1 and 89.0 %) and S. oryzae S16-07T (33.0 and 88.9 %) were below the thresholds of 70 and 95-96 % for prokaryotic conspecific assignation. Chemotaxonomic data revealed that strain DW4-2T possessed MK-9(H6) and MK-9(H8) as the predominant menaquinones. It contained ll -diaminopimelic acid as the diagnostic diamino acid and glucose, ribose and trace amount of madurose in whole-cell sugars. The polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside, an unidentified aminolipid, an unidentified lipid and an unidentified phospholipid. The predominant cellular fatty acids were anteiso-C17 : 0, anteiso-C15 : 0 and iso-C16 : 0. The genomic DNA size of the strain DW4-2T was 7 310 765 bp with DNA G+C content 71.0 mol%. Genomic analysis of the genome indicated that the strain DW4-2T had the potential to produce bioactive compounds. On the basis of these genotypic and phenotypic data, it is supported that strain DW4-2T represents a novel species of the genus Streptomyces, for which the name Streptomyces spirodelae sp. nov. is proposed. The type strain is strain DW4-2T (=TBRC 13095T=NBRC 114803T).

The Metabolomic-Gut-Clinical Axis of Mankai Plant-Derived Dietary Polyphenols.

BACKGROUND: Polyphenols are secondary metabolites produced by plants to defend themselves from environmental stressors. We explored the effect of Wolffia globosa 'Mankai', a novel cultivated strain of a polyphenol-rich aquatic plant, on the metabolomic-gut clinical axis in vitro, in-vivo and in a clinical trial. METHODS: We used mass-spectrometry-based metabolomics methods from three laboratories to detect Mankai phenolic metabolites and examined predicted functional pathways in a Mankai artificial-gut bioreactor. Plasma and urine polyphenols were assessed among the 294 DIRECT-PLUS 18-month trial participants, comparing the effect of a polyphenol-rich green-Mediterranean diet (+1240 mg/polyphenols/day, provided by Mankai, green tea and walnuts) to a walnuts-enriched (+440 mg/polyphenols/day) Mediterranean diet and a healthy controlled diet. RESULTS: Approximately 200 different phenolic compounds were specifically detected in the Mankai plant. The Mankai-supplemented bioreactor artificial gut displayed a significantly higher relative-abundance of 16S-rRNA bacterial gene sequences encoding for enzymes involved in phenolic compound degradation. In humans, several Mankai-related plasma and urine polyphenols were differentially elevated in the green Mediterranean group compared with the other groups (p < 0.05) after six and 18 months of intervention (e.g., urine hydroxy-phenyl-acetic-acid and urolithin-A; plasma Naringenin and 2,5-diOH-benzoic-acid). Specific polyphenols, such as urolithin-A and 4-ethylphenol, were directly involved with clinical weight-related changes. CONCLUSIONS: The Mankai new plant is rich in various unique potent polyphenols, potentially affecting the metabolomic-gut-clinical axis.

The bacterial and fungal communities associated with Anthurium ssp. leaves: Insights into plant endemism and microbe association.

It is known that different plant species select specific microbes to live inside their tissues in a process determined by the host genotype, phenotype and geographic location, which can introduce discussion on plant endemism and the assembly of specific microbial communities. Herein, we report the results of an investigation relating the geographic distribution of plant species and the composition of microbial communities associated with plant hosts. The bacterial and fungal community associated with Anthurium plant leaves was mapped to assess the diversity and ecology of the endophytic community associated with Anthurium spp. collected on islands and on the Brazilian mainland. Twenty-six Anthurium specimens were surveyed, distributed throughout the São Paulo state coastline, including Alcatrazes Island, some coastal islands and distinct mainland environments. Bacterial and fungal endophytes were obtained from the leaves of A. alcatrazense, A. loefgrenii, A. penthaphyllum, A. urvellianum and A. intermedium and subjected to massive bacterial 16S rRNA and fungal ITS sequencing. The results indicated that A. alcatrazense, endemic to Alcatrazes Island, hosted a specific bacterial community structure, while its fungal community was similar to that of Anthurium species from other locations. Betaproteobacteria showed a high differential occurrence in A. alcatrazense. Some groups of fungi were found mainly inhabiting A. loefgrenii plants. While Alphaproteobacteria, Gammaproteobacteria, Actinobacteria and Sordariomycetes, Dothiodeomycetes and Tremellomycetes composed the core microbial community among Anthurium plants. The results suggest crucial role for the bacterial communities to endemic plants, while endophytic fungal diversity is less specifically distributed among endemic and nonendemic plant species.

Synthetic Bacterial Community of Duckweed: A Simple and Stable System to Study Plant-microbe Interactions.

A complete understanding of the plant microbiome has not yet been achieved due to its complexity and temporal shifts in the community structure. To overcome these issues, we created a synthetic bacterial community of the aquatic plant, duckweed. The synthetic community established with six bacterial strains showed a stable composition for 50 days, which may have been because duckweed maintains a similar physiological status through its clonal reproduction. Additionally, the synthetic community reflected the taxonomic structure of the natural duckweed microbiome at the family level. These results suggest the potential of a duckweed-based synthetic community as a useful model system for examining the community assembly mechanisms of the plant microbiome.

Comparative Analysis of Microbial Communities in Fronds and Roots of Three Duckweed Species: Spirodela polyrhiza, Lemna minor, and Lemna aequinoctialis.

The microbial communities inhabiting the fronds of duckweeds have not been investigated in as much detail as those on the roots. We herein examined the microbial communities in three duckweed species using 16S rRNA amplicon sequencing and compared them to those on the roots. The microbial compositions of the fronds were distinct from those of the roots in the three species. Various types of taxonomic bacteria, including rarely cultivated phyla, Acidobacteria, Armatimonadetes, and Verrucomicrobia, were also isolated from the fronds, but at a slightly lower abundance than those from the roots. These results suggest that duckweed fronds are an alternative source for isolating rare and novel microbes, which may otherwise be recalcitrant to cultivation using conventional strategies.

Novel chitosan based smart cathode electrocatalysts for high power generation in plant based-sediment microbial fuel cells.

Smart electrocatalysts are synthesized from chitosan polymer and magnetic particles to enhance power by plant based sediment microbial fuel cell (P-SMFC). Cross-linked procedure is performed gelatinous microspheres as supporting metals (Cu, Pd, Mn, Pt, and Ni) and magnetic particles which create a porous structure on smart catalysts for increase ORR activity. A high and quick OCV rising is achieved with addition of Mag-Pd-Ch in reactor, and OCV value immediately increase from 0.408 V to 0.819 V within 10 minutes. The highest power density is also obtained as 1298 mW m-2 for reactor with Mag-Pd-Ch, which was 15 times higher than control. Significant metal leaching is observed using plant growth for smart catalyst containing Cu. Consequently, high power production, good stabilization, easy separation from water environment due to magnetic property, and relatively low cost make use of Mag-Pd-Ch both economic and environment friendly tools to enhance power generation in P-SMFC.

Assessing and Modelling the Efficacy of Lemna paucicostata for the Phytoremediation of Petroleum Hydrocarbons in Crude Oil-Contaminated Wetlands.

The potentials of the invasive duckweed species, Lemna paucicostata to remove pollutants from aquatic environment was tested in a constructed wetlands as an ecological based system for the phytoremediation of petroleum hydrocarbons in crude oil-contaminated waters within 120 days. Total petroleum hydrocarbons in wetlands and tissues of duckweed were analyzed using gas chromatography with flame ionization detector following established methods while the experimental data were subjected to the first-order kinetic rate model to understand the remediation rate of duckweed in wetlands. L. paucicostata effected a significant (F = 253.405, P < 0.05) removal of hydrocarbons from wetlands reaching 97.91% after 120 days. Assessment on the transport and fate of hydrocarbons in duckweed indicated that L. paucicostata bioaccumulated less than 1% and significantly biodegraded 97.74% of hydrocarbons in wetlands at the end of the study. The experimental data reasonably fitted (r2 = 0.938) into the first-order kinetic rate model. From the result of the study, it is reasonable to infer that L. paucicostata is an effective aquatic macrophyte for the removal of petroleum hydrocarbons in moderately polluted waters.

Mutualistic Outcomes Across Plant Populations, Microbes, and Environments in the Duckweed Lemna minor.

The picture emerging from the rapidly growing literature on host-associated microbiota is that host traits and fitness often depend on interactive effects of host genotype, microbiota, and abiotic environment. However, testing interactive effects typically requires large, multi-factorial experiments and thus remains challenging in many systems. Furthermore, most studies of plant microbiomes focus on terrestrial hosts and microbes. Aquatic habitats may confer unique properties to microbiomes. We grew different populations of duckweed (Lemna minor), a floating aquatic plant, in three microbial treatments (adding no, "home", or "away" microbes) at two levels of zinc, a common water contaminant in urban areas, and measured both plant and microbial performance. Thus, we simultaneously manipulated plant source population, microbial community, and abiotic environment. We found strong effects of plant source, microbial treatment, and zinc on duckweed and microbial growth, with significant variation among duckweed genotypes and microbial communities. However, we found little evidence of interactive effects: zinc did not alter effects of host genotype or microbial community, and host genotype did not alter effects of microbial communities. Despite strong positive correlations between duckweed and microbe growth, zinc consistently decreased plant growth, but increased microbial growth. Furthermore, as in recent studies of terrestrial plants, microbial interactions altered a duckweed phenotype (frond aggregation). Our results suggest that duckweed source population, associated microbiome, and contaminant environment should all be considered for duckweed applications, such as phytoremediation. Lastly, we propose that duckweed microbes offer a robust experimental system for study of host-microbiota interactions under a range of environmental stresses.

Duckweed hosts a taxonomically similar bacterial assemblage as the terrestrial leaf microbiome.

Culture-independent characterization of microbial communities associated with popular plant model systems have increased our understanding of the plant microbiome. However, the integration of other model systems, such as duckweed, could facilitate our understanding of plant microbiota assembly and evolution. Duckweeds are floating aquatic plants with many characteristics, including small size and reduced plant architecture, that suggest their use as a facile model system for plant microbiome studies. Here, we investigated the structure and assembly of the duckweed bacterial microbiome. First, a culture-independent survey of the duckweed bacterial microbiome from different locations in New Jersey revealed similar phylogenetic profiles. These studies showed that Proteobacteria is a dominant phylum in the duckweed bacterial microbiome. To observe the assembly dynamics of the duckweed bacterial community, we inoculated quasi-gnotobiotic duckweed with wastewater effluent from a municipal wastewater treatment plant. Our results revealed that duckweed strongly shapes its bacterial microbiome and forms distinct associations with bacterial community members from the initial inoculum. Additionally, these inoculation studies showed the bacterial communities of different duckweed species were similar in taxa composition and abundance. Analysis across the different duckweed bacterial communities collected in this study identified a set of "core" bacterial taxa consistently present on duckweed irrespective of the locale and context. Furthermore, comparison of the duckweed bacterial community to that of rice and Arabidopsis revealed a conserved taxonomic structure between the duckweed microbiome and the terrestrial leaf microbiome. Our results suggest that duckweeds utilize similar bacterial community assembly principles as those found in terrestrial plants and indicate a highly conserved structuring effect of leaf tissue on the plant microbiome.

Enhanced biomass production and nutrient removal capacity of duckweed via two-step cultivation process with a plant growth-promoting bacterium, Acinetobacter calcoaceticus P23.

Plant growth-promoting bacteria (PGPB) are considered a promising tool to improve biomass production and water remediation by the aquatic plant, duckweed; however, no effective methodology is available to utilize PGPB in large hydroponic systems. In this study, we proposed a two-step cultivation process, which comprised of a "colonization step" and a "mass cultivation step," and examined its efficacy in both bucket-scale and flask-scale cultivation experiments. We showed that in the outdoor bucket-scale experiments using three kinds of environmental water, plants cultured through the two-step cultivation method with the PGPB strain, Acinetobacter calcoaceticus P23, yielded 1.9 to 2.3 times more biomass than the control (without PGPB inoculation). The greater nitrogen and phosphorus removals compared to control were also attained, indicating that this strategy is useful for accelerating nutrient removal by duckweed. Flask-scale experiments using non-sterile pond water revealed that inoculation of strain P23 altered duckweed surface microbial community structures, and the beneficial effects of the inoculated strain P23 could last for 5-10 d. The loss of the duckweed growth-promoting effect was noticeable when the colonization of strain P23 decreased in the plant. These observations suggest that the stable colonization of the plant with PGPB is the key for maintaining the accelerated duckweed growth and nutrient removal in this cultivation method. Overall, our results suggest the possibility of an improved duckweed production using a two-step cultivation process with PGPB.

Sphingomonas aracearum sp. nov., isolated from rhizospheric soil of Araceae plants.

A Gram-stain-negative, single polar flagellum bacterium, WZY27T, was isolated from rhizospheric soil of Araceae plants. The results of phylogenetic analysis based on 16S rRNA gene sequences showed that this strain is closely related to Sphingomonas adhaesiva DSM 7418T (97.2 % similarity), Sphingomonaskoreensis KCTC 2883 (97.1 %) and Sphingomonas ginsenosidimutans JCM 17074T (97.0 %). The genomic average nucleotide identity values between strain WZY27T and the above three strains were 75.3, 73.2 and 75.4 %, and the in silico DNA-DNA hybridization values were 19.1 , 20.1 and 20.9 %, respectively. The major fatty acids (>5 %) of strain WZY27T were summed feature 8 (C18 : 1 ω7c/C18 : 1 ω6c), C16 : 0, C14 : 0 2-OH and C18 : 1 ω7c 11-methyl. The predominant respiratory quinone and polyamine were ubiquinone Q-10 and homospermidine, respectively. The polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phospholipids, glycolipids, phosphatidylcholine and sphingoglycolipid. The G+C content of the genomic DNA was 68.4 mol%. Based on the results of genotypic, chemotaxonomic and phenotypic characterization, strain WZY27T represents a novel species of the genus Sphingomonas, for which the name Sphingomonas aracearum sp. nov. is proposed. The type strain is WZY27T (=KCTC 62523T=CCTCC AB 2018056T).

A duplex quantitative real-time PCR assay for the detection and quantification of Xanthomonas phaseoli pv. dieffenbachiae from diseased and latently infected anthurium tissue.

Anthurium bacterial blight caused by Xanthomonas phaseoli pv. dieffenbachiae (formerly Xanthomonas axonopodis pv. dieffenbachiae) is the major phytosanitary threat in many anthurium growing areas worldwide. Reliable and sensitive diagnostic tools are required for surveillance and certification programs. A duplex real-time quantitative PCR assay was developed for the detection and quantification of X. phaseoli pv. dieffenbachiae from anthurium tissue. This PCR assay targeted a X. phaseoli pv. dieffenbachiae-specific gene encoding an ABC transporter and an internal control encoding for chalcone synthase in Anthurium andreanum. A cycle threshold (Ct), using a receiver-operating characteristic approach (ROC), was implemented to ensure that the declaration of a positive sample was reliable. The duplex real-time assay displayed very high performance with regards to analytical specificity (100% inclusivity, 98.9% exclusivity), analytical sensitivity (LOD95% = 894 bacteria/ml corresponding to 18 bacteria per reaction) and repeatability. We demonstrated the pertinence of this real-time quantitative PCR assay for detecting X. phaseoli pv. dieffenbachiae from diseased leaf tissue (collected from outbreaks on anthurium) and from asymptomatic, latently infected anthurium plants. This assay could be useful for surveillance, as well as for indexing propagative plant material for the presence of X. phaseoli pv. dieffenbachiae.