Endophytic Bacteria From the Roots of the Medicinal Plant Alkanna tinctoria Tausch (Boraginaceae): Exploration of Plant Growth Promoting Properties and Potential Role in the Production of Plant Secondary Metabolites

Alkannin and shikonin (A/S) are enantiomeric naphthoquinones produced in the roots of certain plants from the Boraginaceae family such as Lithospermum spp. and Alkanna spp. They possess antimicrobial, anti-tumoral and wound healing properties. The production of secondary metabolites by Alkanna tinctoria might be influenced by its endomicrobiome. To study the interaction between this medicinal plant and its bacterial endophytes, we isolated bacteria from the roots of wild growing Alkanna tinctoria collected near to Athens and Thessaloniki in Greece. Representative strains selected by MALDI-TOF mass spectrometry were identified by partial 16S rRNA gene sequence analysis. In total, 197 distinct phylotypes of endophytic bacteria were detected. The most abundant genera recovered were Pseudomonas, Xanthomonas, Variovorax, Bacillus, Inquilinus, Pantoea, and Stenotrophomonas. Several bacteria were then tested in vitro for their plant growth promoting activity and the production of cell-wall degrading enzymes. Strains of Pseudomonas, Pantoea, Bacillus and Inquilinus showed positive plant growth properties whereas those of Bacteroidetes and Rhizobiaceae showed pectinase and cellulase activity in vitro. In addition, bacterial responses to alkannin and shikonin were investigated through resistance assays. Gram negative bacteria were found to be resistant to the antimicrobial properties of A/S, whereas the Gram positives were sensitive. A selection of bacteria was then tested for the ability to induce A/S production in hairy roots culture of A. tinctoria. Four strains belonging to Chitinophaga sp., Allorhizobium sp., Duganella sp., and Micromonospora sp., resulted in significantly more A/S in the hairy roots than the uninoculated control. As these bacteria can produce cell-wall degrading enzymes, we hypothesize that the A/S induction may be related with the plant-bacteria interaction during colonization

Advanced backcross QTL analysis and comparative mapping with RIL QTL studies and GWAS provide an overview of QTL and marker haplotype diversity for resistance to Aphanomyces root rot in pea (Pisum sativum)

Aphanomyces euteiches is the most damaging soilborne pea pathogen in France. Breeding of pea resistant varieties combining a diversity of quantitative trait loci (QTL) is a promising strategy considering previous research achievements in dissecting polygenic resistance to A. euteiches. The objective of this study was to provide an overview of the diversity of QTL and marker haplotypes for resistance to A. euteiches, by integrating a novel QTL mapping study in advanced backcross (AB) populations with previous QTL analyses and genome-wide association study (GWAS) using common markers. QTL analysis was performed in two AB populations derived from the cross between the susceptible spring pea variety “Eden” and the two new sources of partial resistance “E11” and “LISA”. The two AB populations were genotyped using 993 and 478 single nucleotide polymorphism (SNP) markers, respectively, and phenotyped for resistance to A. euteiches in controlled conditions and in infested fields at two locations. GWAS and QTL mapping previously reported in the pea-Aphanomyces collection and from four recombinant inbred line (RIL) populations, respectively, were updated using a total of 1,850 additional markers, including the markers used in the Eden x E11 and Eden x LISA populations analysis. A total of 29 resistance-associated SNPs and 171 resistance QTL were identified by GWAS and RIL or AB QTL analyses, respectively, which highlighted 10 consistent genetic regions confirming the previously reported QTL. No new consistent resistance QTL was detected from both Eden x E11 and Eden x LISA AB populations. However, a high diversity of resistance haplotypes was identified at 11 linkage disequilibrium (LD) blocks underlying consistent genetic regions, especially in 14 new sources of resistance from the pea-Aphanomyces collection. An accumulation of favorable haplotypes at these 11 blocks was confirmed in the most resistant pea lines of the collection. This study provides new SNP markers and rare haplotypes associated with the diversity of Aphanomyces root rot resistance QTL investigated, which will be useful for QTL pyramiding strategies to increase resistance levels in future pea varieties

Bacterial responses to plant antimicrobials : the case of alkannin and shikonin derivatives

Alkannin, shikonin and their derivatives (A/S) are secondary metabolites produced in the roots of certain plants of the Boraginaceae family such as Lithospermum erythrorhizon Siebold & Zucc. and Alkanna tinctoria (L.) Tausch. These naphthoquinones express anti-cancer, wound healing, and antimicrobial activities. To study the interactions between endophytic bacteria isolated from A. tinctoria and the antimicrobials A/S, endophytic bacteria known to be resistant to the compounds were screened for their effect on A/S in liquid medium. Thereafter, the strain Pseudomonas sp. R-72008, was selected and tested for its ability to modify A/S in nutrient medium and minimal medium with A/S as sole carbon source. Bacterial growth was recorded, and high performance liquid chromatography-diode array and ultra-high performance liquid chromatography-electrospray ionization-mass spectrometry analyses were performed to detect and quantify metabolites. In nutrient medium inoculated with R-72008, a decrease in the amount of A/S monomers initially present was observed and correlated with an increase of A/S oligomers. Moreover, a significant decrease of initial A/S monomers in minimal medium was correlated with bacterial growth, showing for the first time that a bacterial strain, Pseudomonas sp. R-72008, was able to use the naphthoquinones A/S as sole carbon source. This study opens new perspectives on the interactions between bacteria and plant antimicrobials

Uncovering Genomic Features and Biosynthetic Gene Clusters in Endophytic Bacteria from Roots of the Medicinal Plant Alkanna tinctoria Tausch as a Strategy To Identify Novel Biocontrol Bacteria

ABSTRACT The world’s population is increasing at a rate not seen in the past. Agriculture, providing food for this increasing population, is reaching its boundaries of space and natural resources. In addition, changing legislation and increased ecological awareness are forcing agriculture to reduce its environmental impact. This entails the replacement of agrochemicals with nature-based solutions. In this regard, the search for effective biocontrol agents that protect crops from pathogens is in the spotlight. In this study, we have investigated the biocontrol activity of endophytic bacteria isolated from the medicinal plant Alkanna tinctoria Tausch. To do so, an extensive collection of bacterial strains was initially genome sequenced and in silico screened for features related to plant stimulation and biocontrol. Based on this information, a selection of bacteria was tested in vitro for antifungal activity using direct antagonism in a plate assay and in planta with a detached-leaf assay. Bacterial strains were tested individually and in combinations to assess the best-performing treatments. The results revealed that many bacteria could produce metabolites that efficiently inhibit the proliferation of several fungi, especially Fusarium graminearum. Among these, Pseudomonas sp. strain R-71838 showed a strong antifungal effect, in both dual-culture and in planta assays, making it the most promising candidate for biocontrol application. Using microbes from medicinal plants, this study highlights the opportunities of using genomic information to speed up the screening of a taxonomically diverse set of bacteria with biocontrol properties. IMPORTANCE Phytopathogenic fungi are a major threat to global food production. The most common management practice to prevent plant infections involves the intensive use of fungicides. However, with the growing awareness of the ecological and human impacts of chemicals, there is a need for alternative strategies, such as the use of bacterial biocontrol agents. Limitations in the design of bacterial biocontrol included the need for labor-intensive and time-consuming experiments to test a wide diversity of strains and the lack of reproducibility of their activity against pathogens. Here, we show that genomic information is an effective tool to select bacteria of interest quickly. Also, we highlight that the strain Pseudomonas sp. R-71838 produced a reproducible antifungal effect both in vitro and in planta. These findings build a foundation for designing a biocontrol strategy based on Pseudomonas sp. R-71838

Roseomonas hellenica sp. nov., isolated from roots of wild-growing Alkanna tinctoria

Two Gram-negative, aerobic, rod-shaped and yellow-orange pigmented bacterial strains (LMG 31523(T) and LMG 31524) were isolated from roots of wild-growing Alkanna tinctoria plants collected near Thessaloniki, Greece. Analysis of their 16S rRNA gene sequences revealed that they form a separate cluster related to the genus Roseomonas. A comparative whole genome analysis of the two strains and the type strains of related Roseomonas species revealed average nucleotide identity values from 78.84 and 80.32%. The G + C contents of the genomic DNA of strains LMG 31523(T) and LMG 31524 were 69.69% and 69.74%, respectively. Combined data from phenotypic, phylogenetic and chemotaxonomic studies indicated that the strains LMG 31523(T) and LMG 31524 represent a novel species of the genus Roseomonas. Genome analysis of the new strains showed a number of genes involved in survival in the rhizosphere environment and in plant colonization and confirmed the endophytic characteristics of LMG 31523(T) and LMG 31524. Since the strains LMG 31523(T) and LMG 31524 were isolated from a plant collected in Greece the name Roseomonas hellenica sp. nov. is proposed. The type strain is LMG 31523(T) (=CECT 30032(T)). (C) 2021 The Author(s). Published by Elsevier GmbH

Food restriction but not fish oil increases fertility in hens: role of RARRES2?

International audienceOverfed hens selected for their rapid growth become fatter and develop reproductive disorders. Herein, we aimed to demonstrate that food restriction leading to a weight reduction and/or a supplementation with fish oil may be effective in preventing reproductive disorders through the regulation of adipokine expression in broiler hens. This study included four groups of food restricted (Rt) or ad libitum hens (Ad, feeding at a rate 1.7 times greater than Rt hens) supplemented or unsupplemented with fish oil (1%). The Rt diet significantly increased plasma chemerin (RARRES2) levels during the laying period, delayed sexual maturity by one week and improved egg quality and fertility. These effects were associated with higher progesterone production in response to IGF1 (or LH) in cultured granulosa cells and in vivo egg yolk, as compared with Ad hens. Fish oil supplementation had similar effects to the Rt diet on progesterone (P < 0.05), but without any effect on fertility. Using RT-PCR, we found that RARRES2 levels were lower in theca cells of Rt hens and NAMPT levels were increased by the fish oil supplementation. A significant positive correlation between RARRES2 expression in granulosa cells and the weight of F1 preovulatory follicle was observed, as well as a negative correlation of plasma RARRES2 levels with hatchability. Thus, food restriction but not fish oil supplementation improved fertility, and this was associated with variations in RARRES2 plasma and ovarian expression in hens

Electrospun wound dressings containing bioactive natural products : physico-chemical characterization and biological assessment

Background Current research on skin tissue engineering has been focusing on novel therapies for the effective management of chronic wounds. A critical aspect is to develop matrices that promote growth and uniform distribution of cells across the wound area, and at the same time offer protection, as well as deliver drugs that help wound healing and tissue regeneration. In this context, we aimed at developing electrospun scaffolds that could serve as carriers for the bioactive natural products alkannin and shikonin (A/S). Methods A series of polymeric nanofibers composed of cellulose acetate (CA) or poly(epsilon-caprolactone) (PCL) and varying ratios of a mixture of A/S derivatives, has been successfully fabricated and their physico-chemical and biological properties have been explored. Results Scanning electron microscopy revealed a uniform and bead-free morphology for CA scaffolds, while for PCL beads along the fibers were observed. The average diameters for all nanofibers ranged between 361 +/- 47 and 487 +/- 88 nm. During the assessment of physicochemical characteristics, CA fiber mats exhibited a more favored profile, while the assessment of the biological properties of the scaffolds showed that CA samples containing A/S mixture up to 1 wt.% achieved to facilitate attachment, survival and migration of Hs27 fibroblasts. With respect to the antimicrobial properties of the scaffolds, higher drug-loaded (1 and 5 wt.%) samples succeeded in inhibiting the growth of Staphylococcus epidermidis and S. aureus around the edges of the fiber mats. Finally, carrying out a structure-activity relationship study regarding the biological activities (fibroblast toxicity/proliferation and antibacterial activity) of pure A/S compounds - present in the A/S mixture - we concluded that A/S ester derivatives and the dimeric A/S augmented cell proliferation after 3 days, whereas shikonin proved to be toxic at 500 nM and 1 mu M and alkannin only at 1 mu M. Additionally, alkannin, shikonin and acetyl-shikonin showed more pronounced antibacterial properties than the other esters, the dimeric derivative and the A/S mixture itself. Conclusions Taken together, these findings indicate that embedding A/S derivatives into CA nanofibers might be an advantageous drug delivery system that could also serve as a potential candidate for biomedical applications in the field of skin tissue engineering

Early carbohydrate metabolism in chicken lines divergently selected on ultimate pH, a proxy for muscle glycogen reserves

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Effect of food restriction and n-3 fatty acid supplementation on reproductive parameters in broiler hens

Effect of food restriction and n-3 fatty acid supplementation on reproductive parameters in broiler hens. 2. Journées du GdR 3606 Repr