Arabinoxylans Release from Brewers' Spent Grain Using Extrusion and Solid-State Fermentation with Fusarium oxysporum and the Antioxidant Capacity of the Extracts.

Brewers' spent grain (BSG) is the most abundant byproduct generated from the beer-brewing process. BSG is a material rich in hemicellulose, composed of arabinoxylans (AX). However, the high crosslinking of this material causes low availability of AX, for which it is necessary to apply different treatments. The objective of this research is to increase the release of arabinoxylans through solid-state fermentation with Fusarium oxysporum f. sp. lycopersici using extruded brewery spent grain. First, the BSG is subjected to two types of physical treatments: extrusion at 20% moisture, 200 rpm and 50 °C (BSGe), and blade milling (BSGm). The chemical composition is determined for each sample (BSG, BSGe and BSGm). Subsequently, the solid-state fermentation process (SSF) is carried out on each sample. The fermentation kinetics at 30 °C are monitored for 7 days. Once the SSF concludes, AX are extracted, and the purity of AX is determined by the phloroglucinol colorimetric assay. Finally, the total phenolic compounds, phenolic acids and antioxidant capacity by DPPH are quantified. No significant differences (p ≥ 0.05) in the protein, lipid, ash or total dietary fiber contents are found among the samples. No significant difference (p ≥ 0.05) in the content of soluble fiber is found, although BSGe and BSGm have higher values than BSG. On the other hand, the yields of soluble AX exhibit significant differences (p ≤ 0.05) among nonfermented samples (BSG, 0.03%; BSGm, 0.53%; BSGe, 0.70%) and with SSF (BSG, 2.95%; BSGm, 6.24%; and BSGe, 9.58%). In addition, the contents of free phenolic compounds and free phenolic acids and the percent inhibition of free extracts by 2,2-diphenyl-1-picrylhydrazyl (DPPH) differ significantly (p ≤ 0.05) between samples subjected to SSF and nonfermented samples. Therefore, extrusion and SSF treatment increase AX release from BSG as well as the antioxidant capacity of the extracts.

Toward Understanding the Molecular Recognition of Fungal Chitin and Activation of the Plant Defense Mechanism in Horticultural Crops.

Large volumes of fruit and vegetable production are lost during postharvest handling due to attacks by necrotrophic fungi. One of the promising alternatives proposed for the control of postharvest diseases is the induction of natural defense responses, which can be activated by recognizing molecules present in pathogens, such as chitin. Chitin is one of the most important components of the fungal cell wall and is recognized through plant membrane receptors. These receptors belong to the receptor-like kinase (RLK) family, which possesses a transmembrane domain and/or receptor-like protein (RLP) that requires binding to another RLK receptor to recognize chitin. In addition, these receptors have extracellular LysM motifs that participate in the perception of chitin oligosaccharides. These receptors have been widely studied in Arabidopsis thaliana (A. thaliana) and Oryza sativa (O. sativa); however, it is not clear how the molecular recognition and plant defense mechanisms of chitin oligosaccharides occur in other plant species or fruits. This review includes recent findings on the molecular recognition of chitin oligosaccharides and how they activate defense mechanisms in plants. In addition, we highlight some of the current advances in chitin perception in horticultural crops.

Functional analysis of tomato rhamnogalacturonan lyase gene Solyc11g011300 during fruit development and ripening.

Rhamnogalacturonan I (RG-I) is a domain of plant cell wall pectin. The rhamnogalacturonan lyase (RGL) enzyme (EC 4.2.2.23) degrades RG-I by cleaving the α-1,4 glycosidic bonds located between the l-rhamnose and d-galacturonic residues of the main chain. While RGL's biochemical mode of action is well known, its effects on plant physiology remain unclear. To investigate the role of the RGL enzyme in plants, we have expressed the Solyc11g011300 gene under a constitutive promoter (CaMV35S) in tomato cv. 'Ohio 8245' and evaluated the expression of this and other RGL genes, enzymatic activity and alterations in vegetative tissue, and tomato physiology in transformed lines compared to the positive control (plants harboring the pCAMBIA2301 vector) and the isogenic line. The highest expression levels of the Solyc11g011300, Solyc04g076630, and Solyc04g076660 genes were observed in leaves and roots and at 10 and 20 days after anthesis (DAA). Transgenic lines exhibited lower RGL activity in leaves and roots and during fruit ripening, whereas higher activity was observed at 10, 20, and 30 DAA than in the isogenic line and positive control. Both transgenic lines showed a lower number of seeds and fruits, higher root length, and less pollen germination percentage and viability. In red ripe tomatoes, transgenic fruits showed greater firmness, longer shelf life, and reduced shriveling than did the isogenic line. Additionally, a delay of one week in fruit ripening in transgenic fruits was also recorded. Altogether, our data demonstrate that the Solyc11g011300 gene participates in pollen tube germination, fruit firmness, and the fruit senescence phenomena that impact postharvest shelf life.

Functional analysis of a tomato (Solanum lycopersicum L.) rhamnogalacturonan lyase promoter.

The enzyme rhamnogalacturonan lyase (RGL) cleaves α-1,4 glycosidic bonds located between rhamnose and galacturonic acid residues in the main chain of rhamnogalacturonan-I (RG-I), a component of the plant cell wall polymer pectin. Although the mode of action of RGL is well known, its physiological functions associated with fruit biology are less understood. Here, we generated transgenic tomato plants expressing the ß-glucuronidase (GUS) reporter gene under the control of a -504 bp or a -776 bp fragment of the promoter of a tomato RGL gene, Solyc11g011300. GUS enzymatic activity and the expression levels of GUS and Solyc11g011300 were measured in a range of organs and fruit developmental stages. GUS staining was undetectable in leaves and roots, but high GUS enzymatic activity was detected in flowers and red ripe (RR) fruits. Maximal expression levels of Solyc11g011300 were detected at the RR developmental stage. GUS activity was 5-fold higher in flowers expressing GUS driven by the -504 bp RGL promoter fragment (RGFL3::GUS) than in the isogenic line, and 1.7-fold higher when GUS gene was driven by the -776 bp RGL promoter fragment (RGLF2::GUS) or the constitutive CaMV35S promoter. Quantitative real-time polymerase chain reaction analysis showed that the highest expression of GUS was in fruits at 40 days after anthesis, for both promoter fragments. The promoter of Solyc11g011300 is predicted to contain cis-acting elements, and to be active in pollen grains, pollen tubes, flowers and during tomato fruit ripening, suggesting that the Solyc11g011300 promoter is transcriptionally active and organ-specific.

Changes in the phenylalanine ammonia lyase activity, total phenolic compounds, and flavonoids in Prosopis glandulosa treated with cadmium and copper.

The aim of the present work is to evaluate the changes on the phenylalanine ammonia lyase (PAL) activity, phenolic compounds accumulation and photochemical efficiency in leaves of P. glandulosa treated with Cd2+ (0.001 M) and Cu2+ (0.52 M) concentrations for 96 h under hydroponic conditions. The results showed that only leaves treated with copper had a decrease in photochemical efficiency and leaf epidermal polyphenols in P. glandulosa leaves after 96 h of exposure. On the other hand the reverse-phase HPLC analysis revealed higher levels of phenolic compound (gallic, vanillic and caffeic acids) and flavonoids (rutin and kaempferol-3-O-glucosides) in plant leaves from Cu and Cd-treatments with respect to control plants. Finally, highest increments in PAL activity was observed in extracts of leaves treated with Cu and Cd (about 205 and 284%), respectively, with respect to control plants after 96 h treatment. These suggest that activation of phenylpropanoid pathway represent a source of nonenzymatic antioxidants that protect at P. glandulosa against oxidative stress when exposed to cadmium and copper. Hence future studies are necessary to elucidate the participation of phenylpropanoid pathway in the reduction of metal toxicity in Prosopis species.

Poly(lactic-co-glycolic acid) nanoparticles for sustained release of allyl isothiocyanate: characterization, in vitro release and biological activity.

The objective of this study is to establish the ability of entrap allyl isothiocyanate (AITC) into polymeric nanoparticles to extend its shelf life and enhance its antiproliferative properties. Natural compounds, such as AITC, have showed multi-targeting activity resulting in a wide-range spectrum of therapeutic properties in chronic and degenerative diseases, conversely with most current pharmaceutical drugs showing single targeting activity and often result in drug resistance after extended administration periods. Apparently, AITC-loaded poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs) reduced AITC degradation and volatility and were able to extend AITC shelf life compared with free AITC (65% vs. 20% in 24 h, respectively). Cell viability and uptake of AITC-loaded nanoparticles were studied in vitro, showing that the protection and sustained release of AITC from polymeric NPs involved a larger toxicity of tumoral cells. These nanoparticles could be used as protective systems for enhancing a biological activity.

Antifungical Activity of Autochthonous Bacillus subtilis Isolated from Prosopis juliflora against Phytopathogenic Fungi.

The ability of Bacillus subtilis, strain ALICA to produce three mycolytic enzymes (chitinase, ß-1,3-glucanase, and protease), was carried out by the chemical standard methods. Bacillus subtilis ALICA was screened based on their antifungal activity in dual plate assay and cell-free culture filtrate (25%) against five different phytopathogenic fungi Alternaria alternata, Macrophomina sp., Colletotrichum gloeosporioides, Botrytis cinerea, and Sclerotium rolfesii. The B. subtilis ALICA detected positive for chitinase, ß-1,3-glucanase and protease enzymes. Fungal growth inhibition by both strain ALICA and its cell-free culture filtrate ranged from 51.36% to 86.3% and 38.43% to 68.6%, respectively. Moreover, hyphal morphological changes like damage, broken, swelling, distortions abnormal morphology were observed. Genes expression of protease, ß-1,3-glucanase, and lipopeptides (subtilosin and subtilisin) were confirmed their presence in the supernatant of strain ALICA. Our findings indicated that strain ALICA provided a broad spectrum of antifungal activities against various phytopathogenic fungi and may be a potential effective alternative to chemical fungicides.

Analysis of a suppressive subtractive hybridization library of Alternaria alternata resistant to 2-propenyl isothiocyanate

Background Isothiocyanates (ITCs) are natural products obtained from plants of the Brassicas family. They represent an environmentally friendly alternative for the control of phytopathogenic fungi. However, as it has been observed with synthetic fungicides, the possibility of inducing ITC-resistant strains is a major concern. It is, therefore, essential to understanding the molecular mechanisms of fungal resistance to ITCs. We analyzed a subtractive library containing 180 clones of an Alternaria alternata strain resistant to 2-propenyl ITC (2-pITC). After their sequencing, 141 expressed sequence tags (ESTs) were identified using the BlastX algorithm. The sequence assembly was carried out using CAP3 software; the functional annotation and metabolic pathways identification were performed using the Blast2GO program. Results The bioinformatics analysis revealed 124 reads with similarities to proteins involved in transcriptional control, defense and stress pathways, cell wall integrity maintenance, detoxification, organization and cytoskeleton destabilization; exocytosis, transport, DNA damage control, ribosome maintenance, and RNA processing. In addition, transcripts corresponding to enzymes as oxidoreductases, transferases, hydrolases, lyases, and ligases, were detected. Degradation pathways for styrene, aminobenzoate, and toluene were induced, as well as the biosynthesis of phenylpropanoid and several types of N-glycan. Conclusions The fungal response showed that natural compounds could induce tolerance/resistance mechanisms in organisms in the same manner as synthetic chemical products. The response of A. alternata to the toxicity of 2-pITC is a sophisticated phenomenon including the induction of signaling cascades targeting a broad set of cellular processes. Whole-transcriptome approaches are needed to elucidate completely the fungal response to 2-pITC.

Efecto antifúngico de extractos fenólicos y de carotenoides de chiltepín (Capsicum annum var. glabriusculum) en Alternaria alternata y Fusarium oxysporum / Antifungal effect of phenolic and carotenoids extracts from chiltepin (Capsicum annum var. glabriusculum) on Alternaria alternata and Fusarium oxysporum

En el presente estudio se evaluó el efecto de extractos fenólicos y de carotenoides procedentes de frutos de chiltepín sobre el crecimiento micelial y la germinación de conidios de Alternaria alternata y Fusarium oxysporum, 2 importantes hongos causantes de podredumbres en frutas y hortalizas. Los extractos fenólicos presentaron una inhibición en el crecimiento micelial de A. alternata del 38,46 %, y redujeron significativamente la germinación de conidios al quinto día después del tratamiento al 92 % en relación al control. No se observaron cambios significativos en el crecimiento micelial de F. oxysporum, pero sí se redujo significativamente al 85 % en relación al control, el número de conidios germinados a los 5 días de tratamiento. Los extractos de carotenoides mostraron una inhibición del 38,5 % en el crecimiento micelial y del 85,3 % en la germinación de conidios de A. alternata, 5 días después del tratamiento. Frente a F. oxysporum, dichos extractos presentaron menor inhibición del crecimiento micelial (20,3 %), mientras que hubo una mayor inhibición en la germinación de conidios (96 %). Los extractos fenólicos y de carotenoides de chiltepín pueden ser una alternativa promisoria de importancia agrícola como fungicidas naturales.

The effect of phenolic and carotenoid extracts from chiltepin fruits on mycelial growth and the inhibition of conidial germination of Alternaria alternata and Fusarium oxysporum were investigated in the present work. Phenolic extracts inhibited mycelial growth of A. alternata by 38.46%, and significantly reduced conidial germination on the fifth day after treatment to 92% in relation to control. No significant changes were observed in the inhibition of mycelial growth in Fusarium oxysporum; however, the number of germinated conidia was reduced, showing 85% inhibition five days after treatment in relation to control. Moreover, carotenoid extracts showed 38.5% inhibition of mycelial growth and 85.3% inhibition of conidial germination of A. alternata, five days after treatment. Carotenoid extracts showed less inhibition of mycelial growth (20.3%) in F. oxysporum, with respect to A. alternata; while there was greater inhibition of conidial germination (96%) on the fifth day after treatment. Phenolic and carotenoid extracts from chiltepin may be a promising alternative as a natural fungicide against fungi of agricultural importance.

Efecto antifúngico de extractos fenólicos y de carotenoides de chiltepín (Capsicum annum var. glabriusculum) en Alternaria alternata y Fusarium oxysporum / Antifungal effect of phenolic and carotenoids extracts from chiltepin (Capsicum annum var. glabriusculum) on Alternaria alternata and Fusarium oxysporum

En el presente estudio se evaluó el efecto de extractos fenólicos y de carotenoides procedentes de frutos de chiltepín sobre el crecimiento micelial y la germinación de conidios de Alternaria alternata y Fusarium oxysporum, 2 importantes hongos causantes de podredumbres en frutas y hortalizas. Los extractos fenólicos presentaron una inhibición en el crecimiento micelial de A. alternata del 38,46 %, y redujeron significativamente la germinación de conidios al quinto día después del tratamiento al 92 % en relación al control. No se observaron cambios significativos en el crecimiento micelial de F. oxysporum, pero sí se redujo significativamente al 85 % en relación al control, el número de conidios germinados a los 5 días de tratamiento. Los extractos de carotenoides mostraron una inhibición del 38,5 % en el crecimiento micelial y del 85,3 % en la germinación de conidios de A. alternata, 5 días después del tratamiento. Frente a F. oxysporum, dichos extractos presentaron menor inhibición del crecimiento micelial (20,3 %), mientras que hubo una mayor inhibición en la germinación de conidios (96 %). Los extractos fenólicos y de carotenoides de chiltepín pueden ser una alternativa promisoria de importancia agrícola como fungicidas naturales.(AU)

The effect of phenolic and carotenoid extracts from chiltepin fruits on mycelial growth and the inhibition of conidial germination of Alternaria alternata and Fusarium oxysporum were investigated in the present work. Phenolic extracts inhibited mycelial growth of A. alternata by 38.46%, and significantly reduced conidial germination on the fifth day after treatment to 92% in relation to control. No significant changes were observed in the inhibition of mycelial growth in Fusarium oxysporum; however, the number of germinated conidia was reduced, showing 85% inhibition five days after treatment in relation to control. Moreover, carotenoid extracts showed 38.5% inhibition of mycelial growth and 85.3% inhibition of conidial germination of A. alternata, five days after treatment. Carotenoid extracts showed less inhibition of mycelial growth (20.3%) in F. oxysporum, with respect to A. alternata; while there was greater inhibition of conidial germination (96%) on the fifth day after treatment. Phenolic and carotenoid extracts from chiltepin may be a promising alternative as a natural fungicide against fungi of agricultural importance.(AU)

[Antifungal effect of phenolic and carotenoids extracts from chiltepin (Capsicum annum var. glabriusculum) on Alternaria alternata and Fusarium oxysporum]. / Efecto antifúngico de extractos fenólicos y de carotenoides de chiltepín (Capsicum annum var. glabriusculum) en Alternaria alternata y Fusarium oxysporum.

The effect of phenolic and carotenoid extracts from chiltepin fruits on mycelial growth and the inhibition of conidial germination of Alternaria alternata and Fusarium oxysporum were investigated in the present work. Phenolic extracts inhibited mycelial growth of A.alternata by 38.46%, and significantly reduced conidial germination on the fifth day after treatment to 92% in relation to control. No significant changes were observed in the inhibition of mycelial growth in Fusarium oxysporum; however, the number of germinated conidia was reduced, showing 85% inhibition five days after treatment in relation to control. Moreover, carotenoid extracts showed 38.5% inhibition of mycelial growth and 85.3% inhibition of conidial germination of A.alternata, five days after treatment. Carotenoid extracts showed less inhibition of mycelial growth (20.3%) in F.oxysporum, with respect to A.alternata; while there was greater inhibition of conidial germination (96%) on the fifth day after treatment. Phenolic and carotenoid extracts from chiltepin may be a promising alternative as a natural fungicide against fungi of agricultural importance.

Efecto antifúngico de extractos fenólicos y de carotenoides de chiltepín (Capsicum annum var. glabriusculum) en Alternaria alternata y Fusarium oxysporum. / [Antifungal effect of phenolic and carotenoids extracts from chiltepin (Capsicum annum var. glabriusculum) on Alternaria alternata and Fusarium oxysporum].

The effect of phenolic and carotenoid extracts from chiltepin fruits on mycelial growth and the inhibition of conidial germination of Alternaria alternata and Fusarium oxysporum were investigated in the present work. Phenolic extracts inhibited mycelial growth of A.alternata by 38.46

, and significantly reduced conidial germination on the fifth day after treatment to 92

in relation to control. No significant changes were observed in the inhibition of mycelial growth in Fusarium oxysporum; however, the number of germinated conidia was reduced, showing 85

inhibition five days after treatment in relation to control. Moreover, carotenoid extracts showed 38.5

inhibition of mycelial growth and 85.3

inhibition of conidial germination of A.alternata, five days after treatment. Carotenoid extracts showed less inhibition of mycelial growth (20.3

) in F.oxysporum, with respect to A.alternata; while there was greater inhibition of conidial germination (96

) on the fifth day after treatment. Phenolic and carotenoid extracts from chiltepin may be a promising alternative as a natural fungicide against fungi of agricultural importance.

Differentially expressed cDNAs in Alternaria alternata treated with 2-propenyl isothiocyanate.

The molecular mechanism of the fungal tolerance phenotype to fungicides is not completely understood. This knowledge would allow for the development of environmentally friendly strategies for the control of fungal infection. With the goal of determining genes induced by 2p-ITC, a forward suppressive subtractive hybridization was performed using cDNAs from ITC-treated Alternaria alternata as a "tester" and from untreated fungus as a "driver." Using this approach, a library containing 102 ESTs was generated that resulted in 50 sequences after sequence assembly (17 contigs and 33 singletons). Blastx analysis revealed that 38% and 40% of the sequences showed significant similarity with known and hypothetical proteins, respectively, whereas 18% were not similar to known genes. These last sequences could represent novel genes that play an unknown role in the molecular responses of fungi during adaptation to 2p-ITC. Clones similar to opsins, ABC transporters, calmodulin, ATPases and SNOG proteins were identified. Using real-time RT-PCR analysis, significant inductions of an ABC transporter and a Ca(++) ATPase during 2p-ITC treatment were discovered. These results suggest that the fungal resistance phenotype to 2p-ITC involves calcium ions and 2p-ITC efflux via an ABC transporter.