Evaluation of in-vitro methods to select effective streptomycetes against toxigenic fusaria

Biocontrol microorganisms are emerging as an effective alternative to pesticides. Ideally, biocontrol agents (BCAs) for the control of fungal plant pathogens should be selected by an in vitro method that is high-throughput and is predictive of in planta efficacy, possibly considering environmental factors, and the natural diversity of the pathogen. The purpose of our study was (1) to assess the effects of Fusarium strain diversity (N = 5) and culture media (N = 6) on the identification of biological control activity of Streptomyces strains (N = 20) against Fusarium pathogens of wheat in vitro and (2) to verify the ability of our in vitro screening methods to simulate the activity in planta. Our results indicate that culture media, Fusarium strain diversity, and their interactions affect the results of an in vitro selection by dual culture assay. The results obtained on the wheat-based culture media resulted in the highest correlation score (r = 0.5) with the in planta root rot (RR) inhibition, suggesting that this in vitro method was the best predictor of in planta performance of streptomycetes against Fusarium RR of wheat assessed as extension of the necrosis on the root. Contrarily, none of the in vitro plate assays using the media tested could appropriately predict the activity of the streptomycetes against Fusarium foot rot symptoms estimated as the necrosis at the crown level. Considering overall data of correlation, the activity in planta cannot be effectively predicted by dual culture plate studies, therefore improved in vitro methods are needed to better mimic the activity of biocontrol strains in natural conditions. This work contributes to setting up laboratory standards for preliminary screening assays of Streptomyces BCAs against fungal pathogens

Detection of Quantitative Trait Loci Controlling the Content of Phenolic Compounds in an Asian Plum (Prunus salicina L.) F1 Population

Consumption of fresh fruit is known to protect against non-communicable diseases due to the fruit's content in compounds with an antioxidant capacity, among them is polyphenols. Asian plums (Prunus salicina L.) accumulate more than 40 phenolic compounds, with a remarkable diversity in their profiles, depending on the variety and environmental conditions. Although candidate genes have been indicated to control this trait, the loci controlling its phenotypic variation have not yet been defined in this species. The aim of this work was to identify the quantitative trait Loci (QTL) controlling the phenolic compounds content in the Asian plum skin and flesh. Using UHPLC-DAD-Orbitrap-MS, we determined that cyanidin-3-glucoside and cyanidin-3-rutinoside are the main anthocyanins in Asian plums. Other anthocyanins found to a lesser extent were tentatively identified as cyanidin bound to different sugar and procyanidin moieties. Then we phenotyped fruits of 92 and 80 F1 seedlings from the cross (98 Ang) for two harvest seasons. We used HPLC-DAD to quantify single anthocyanin and spectrophotometric techniques to determine the total content of phenols, flavonoids, procyanidins, and antioxidant activity (DPPH and FRAP). To determine the phenotype-genotype association of phenolic compounds content, phenotypic values (adjusted by linear mixed-effects models), genotypic data and linkage maps were analyzed with the multiple QTL model (MQM) approach. We found a total of 21 significant trait-marker associations: 13 QTLs segregating from “98.99” and 8 QTLs from “Angeleno.” From these associations, 8 corresponded to phenolic compound content in the flesh and 13 in the skin. Phenotype variance was explained by the detected loci, ranging from 12.4 to 27.1%. The identified loci are related to the content of cyanidin-3-glucoside (LG4), cyanidin-3-rutinoside (LG4), total flavonoids and procyanidins (LG5 and LG8), and minor anthocyanin compounds (LG3 and LG4). These results will help improve the efficiency of breeding programs for the generation of Asian plum varieties with high phenolic compound content.This work has been funded by the National Agency of Research and Development (ANID), Chile: Fondecyt start into Research No. 11150662, Fondecyt Regular No. 1191446, and FONDEF Project IT17I0069 Sweet Pekeetah: un modelo tecnológico-comercial para una nueva variedad chilena de fruta; BB and CS-A were supported by BECAS DE DOCTORADO NACIONAL/2020 No. 21200330 and 21191605, respectively. The work of JS was supported by the Ministry of Science and Innovation of Spain through Juan de la Cierva incorporation contract (IJC2018-036623-I

An Upgraded, Highly Saturated Linkage Map of Japanese Plum (Prunus salicina Lindl.), and Identification of a New Major Locus Controlling the Flavan-3-ol Composition in Fruits

Japanese plum fruits are rich in phenolic compounds, such as anthocyanins and flavan-3-ols, whose contents vary significantly among cultivars. Catechin (C) and epicatechin (EC) are flavan-3-ol monomers described in the fruits of this species and are associated with bitterness, astringency, antioxidant capacity, and susceptibility to enzymatic mesocarp browning. In this study, we aimed to identify quantitative trait loci (QTL) associated with the content of flavan-3-ol in Japanese plum fruits. We evaluated the content of C and EC in the mesocarp and exocarp of samples from 79 and 64 seedlings of an F1 progeny () in the first and second seasons, respectively. We also constructed improved versions of linkage maps from ‘98–99’ and ‘Angeleno,’ presently called single-nucleotide polymorphisms (SNPs) after mapping the already available GBS reads to Prunus salicina Lindl. cv. ‘Sanyueli’ v2.0 reference genome. These data allowed for describing a cluster of QTLs in the cultivar, ‘Angeleno,’ associated with the flavan-3-ol composition of mesocarp and exocarp, which explain up to 100% of the C/EC ratio. Additionally, we developed a C/EC metabolic marker, which was mapped between the markers with the highest log of odds (LOD) scores detected by the QTL analysis. The C/EC locus was located in the LG1, at an interval spanning 0.70 cM at 108.30–108.90 cM. Our results suggest the presence of a novel major gene controlling the preferential synthesis of C or EC in the Japanese plum fruits. This study is a significant advance in understanding the regulation of synthesizing compounds associated with fruit quality, postharvest, and human health promotion.This study has been funded by the National Agency of Research and Development (ANID)/the Scholarship Program/BECAS DE DOCTORADO NACIONAL/2020 – 21200330; Fondecyt Regular No. 1191446; Fondecyt Iniciación No. 11150662; Fondecyt Regular No. 1200718; FONDEF Project IT17I0069 Sweet Pekeetah: “un modelo tecnológico- comercial para una nueva variedad chilena de fruta”. JS was supported by the Ministry of Science and Innovation of Spain through the Juan de la Cierva incorporation contract (IJC2018-036623-I

Evaluation of the Degree of Polymerization of the Proanthocyanidins in Cranberry by Molecular Sieving and Characterization of the Low Molecular Weight Fractions by UHPLC-Orbitrap Mass Spectrometry

4-dimethylammino-cinnamaldehyde (DMAC) assays quantify total proanthocyanidins (PACs) but do not provide qualitative PAC molecular weight distribution information and cannot discriminate between A- and B-type PACs. We developed an efficient method for assessing PAC molecular weight distributions. The PACs from three commercial cranberry extracts (A1–A3) were fractionated by molecular sieves with cut-offs of 3, 10, 30, 50, and 100 kDa, and each fraction was analyzed by DMAC assays. A1, A2, and A3 contained 27%, 33%, and 15% PACs, respectively. Approximately 28 PACs, 20 flavonols, and 15 phenolic acids were identified by UHPLC-DAD-Orbitrap MS in A1 and A3, while A2 contained only flavan-3-ols. Epicatechin was the main monomer in A1 and A3, and catechin was the main in A2. Procyanidin A2 was the main dimer in A1 and A3, representing more than 85% of the total dimers, while it constituted approximately only 24% of A2. A1 and A3 contained quercetin, isorhamnetin, myricetin, and their glycosides, which were totally absent in A2. In A1 and A3 the PACs were mainly distributed in the fractions 30–3 and <3 kDa, while in A2 more than 70% were present in the fraction less than 3 kDa. Overall, obtained data strongly suggests that A2 is not cranberry-derived, or is adulterated with another source of PACs

Evaluation of the Degree of Polymerization of the Proanthocyanidins in Cranberry by Molecular Sieving and Characterization of the Low Molecular Weight Fractions by UHPLC-Orbitrap Mass Spectrometry

4-dimethylammino-cinnamaldehyde (DMAC) assays quantify total proanthocyanidins (PACs) but do not provide qualitative PAC molecular weight distribution information and cannot discriminate between A- and B-type PACs. We developed an efficient method for assessing PAC molecular weight distributions. The PACs from three commercial cranberry extracts (A1–A3) were fractionated by molecular sieves with cut-offs of 3, 10, 30, 50, and 100 kDa, and each fraction was analyzed by DMAC assays. A1, A2, and A3 contained 27%, 33%, and 15% PACs, respectively. Approximately 28 PACs, 20 flavonols, and 15 phenolic acids were identified by UHPLC-DAD-Orbitrap MS in A1 and A3, while A2 contained only flavan-3-ols. Epicatechin was the main monomer in A1 and A3, and catechin was the main in A2. Procyanidin A2 was the main dimer in A1 and A3, representing more than 85% of the total dimers, while it constituted approximately only 24% of A2. A1 and A3 contained quercetin, isorhamnetin, myricetin, and their glycosides, which were totally absent in A2. In A1 and A3 the PACs were mainly distributed in the fractions 30–3 and <3 kDa, while in A2 more than 70% were present in the fraction less than 3 kDa. Overall, obtained data strongly suggests that A2 is not cranberry-derived, or is adulterated with another source of PACs

Long-term kinetics of daidzein and its main metabolites in human equol-producers after soymilk intake: identification of equol-conjugates by UPLC-orbitrap-MS and influence of the number of transforming bacteria on plasma kinetics

<p>The main aim of the study was to establish <i>in vivo</i> a correlation between equol (EQU) production and a number of intestinal bacteria able to perform the transformation. Thus, healthy female volunteers were selected for their ability to convert slowly (<i>n</i> = 6, 10⁵–10⁹ cells/g wet feces) or quickly (<i>n</i> = 6, 10¹⁰–10¹² cells/g wet feces) daidzein (DAI) in EQU. After oral administration of 100 mg DAI in soymilk, plasma (0–99 h) and urine (0–96 h) samples were collected. DAI and its metabolites were determined by LC-MS/MS and EQU -conjugates by UPLC-High Resolution-MS. Only for EQU a direct correlation was found between the number of transforming microorganisms and parameters such as <i>t</i>_max and <i>t</i>_1/2 (<i>p</i> = 0.027). Peak serum concentration time, <i>C</i>_max, AUC_0–72 h and <i>t</i>_1/2 for total EQU (<i>n</i> = 12) were 36 ± 10 h, 89 ± 78 nM, 2.4 ± 1.7 (μmol × h/L) and 15.6 ± 3.3 h, respectively. In plasma and urine EQU was found mainly as 7-<i>O</i>-glucuronide.</p

Evaluation of Flavonoids and Furanocoumarins from Citrus bergamia (Bergamot) Juice and Identification of New Compounds

molecule

Radical scavenger activity of three flavonoid metabolites studied by inhibition of chemiluminescence in human PMNs

3,4-Dihydroxyphenylacetic acid (I), 3-hydroxyphenylacetic acid (II), and 3-(4-hydroxyphenyl)propionic acid (III), metabolites which arise from quercetin glycosides, resp., from flavones and probably from procyanidins by the human intestinal microflora, have been tested for their effects on oxygen radical prodn. by human PNMs stimulated with FMLP or opsonized zymosan. Oxygen radicals were detected by luminol-augmented chemiluminescence measurements. Furthermore free radical scavenging activity of these metabolites was investigated in a cell-free system in which oxygen radicals were generated by horseradish peroxidase with H2O2 as substrate. I reduced considerably chemiluminescence in PMNs in an amt. which was much more pronounced than those of the two metabolites. Concns. of 1 micro mol/l showed an inhibition by 84% with FMLP as stimulant and by 15% with opsonized zymosan, indicating that different signal transduction pathways are influenced in PMNs. Using the same conditions the unmetabolized quercetin showed an inhibition of chemiluminescence by 74% (FMLP), resp. 20% (opsonized zymosan). In the cell-free system I suppressed much more effectively chemiluminescence than II. In contrast, III led to an increase of chemiluminescence generated in the cell-free system (FMLP and zymosan), i.e. by 30% and by 25%, at the highest concn. of 4 micro mol/l. In conclusion, flavonoid metabolites differ in their effects on free radical prodn. of PMNs and their radical scavenging potencies