Effective In Vitro Control of Two Phytopathogens of Agricultural Interest Using Cell-Free Extracts of Pseudomonas fluorescens and Chitosan.

A biofungicide is a natural product that can be derived from various sources such as, among others, microorganisms, higher plants, animal products, phytochemicals, semiochemicals, and antagonist microorganisms. One of the most important approaches for the production of biofungicides is the combination of biocontrol agents. This study showed the inhibition growth of Alternaria alternata and Fusarium solani treated with cell-free extracts of P. fluorescens. Using thin-layer chromatography and plate assays it was also demonstrated that the cell-free extracts of P. fluorescens contained siderophores and derivates of 4-diacetylphloroglucinol and phenazine. Moreover, the combination of cell-free extracts of P. fluorescens and chitosan [50-1.5% (v/v)] had a synergistic effect since they notably inhibited the mycelial growth of A. altenata and F. solani. Various morphological alterations to the mycelia and conidia of the treated fungi as a result of this combination were also observed. The present study could be a starting point to control other fungal phytopathogens using different cell-free extracts and chitosan as biocontrol agents.

Phenylpropanoids Are Connected to Cell Wall Fortification and Stress Tolerance in Avocado Somatic Embryogenesis.

Somatic embryogenesis (SE) is a valuable model for understanding the mechanism of plant embryogenesis and a tool for the mass production of plants. However, establishing SE in avocado has been complicated due to the very low efficiency of embryo induction and plant regeneration. To understand the molecular foundation of the SE induction and development in avocado, we compared embryogenic (EC) and non-embryogenic (NEC) cultures of two avocado varieties using proteomic and metabolomic approaches. Although Criollo and Hass EC exhibited similarities in the proteome and metabolome profile, in general, we observed a more active phenylpropanoid pathway in EC than NEC. This pathway is associated with the tolerance of stress responses, probably through the reinforcement of the cell wall and flavonoid production. We could corroborate that particular polyphenolics compounds, including p-coumaric acid and t-ferulic acid, stimulated the production of somatic embryos in avocado. Exogen phenolic compounds were associated with the modification of the content of endogenous polyphenolic and the induction of the production of the putative auxin-a, adenosine, cellulose and 1,26-hexacosanediol-diferulate. We suggest that in EC of avocado, there is an enhanced phenylpropanoid metabolism for the production of the building blocks of lignin and flavonoid compounds having a role in cell wall reinforcement for tolerating stress response. Data are available at ProteomeXchange with the identifier PXD019705.

Flocculation and Expression of FLO Genes of a Saccharomyces cerevisiae Mezcal Strain with High Stress Tolerance.

Mezcal is a distillate produced by spontaneous fermentation of the must obtained from stalks of Agave spp. plants that are cooked and pressed. Agave must contains a high amount of fructose and phenolic compounds, and fermentation usually occurs under stressful (and uncontrolled) environmental conditions. Yeasts capable of growing under such conditions usually display advantageous biological and industrial traits for stress tolerance such as flocculation. In this study, seven Saccharomyces cerevisiae strains isolated from mezcal must were exposed to temperatures ranging between 10 and 40 °C, and to different sugar sources (fructose or glucose). Yeasts grown in fructose increased their stress tolerance, determined by colony count in a microdrop assay, under low temperature (10 °C) compared to the growth at 40 °C on solid cultures. The most stress-tolerant mezcal strain (Sc3Y8) and a commercial wine (Fermichamp) strain, used as control, were grown under fermentation conditions and exposed to long-term temperature stress to determine their performance and their potential for flocculation. Compared to glucose, fermentation on fructose increased the metabolite accumulation at the end of culture, particularly at 40 °C, with 2.3, 1.3 and 3.4 times more glycerol (8.6 g/L), ethanol (43.6 g/L) and acetic acid (7.3 g/L), respectively. Using confocal microscopy analysis, we detected morphological changes such as aggregation and wall recognition at the level of budding scars in yeast, particularly in the Sc3Y8 strain when it was exposed to 40 °C. The analysis confirmed that this mezcal strain was positive for flocculation in the presence of Ca2+ ions. Analysis of FLO1, FLO5 and FLO11 gene expression implicated in flocculation in both Saccharomyces strains showed a strong transcriptional induction, mainly of the FLO5 gene in the mezcal Sc3Y8 strain.

IAA-producing rhizobacteria from chickpea (Cicer arietinum L.) induce changes in root architecture and increase root biomass.

Rhizobacteria promote and have beneficial effects on plant growth, making them useful to agriculture. Nevertheless, the rhizosphere of the chickpea plant has not been extensively examined. The aim of the present study was to select indole-3-acetic acid (IAA) producing rhizobacteria from the rhizosphere of chickpea plants for their potential use as biofertilizers. After obtaining a collection of 864 bacterial isolates, we performed a screen using the Salkowski reaction for the presence of auxin compounds (such as IAA) in bacterial Luria-Bertani supernatant (BLBS). Our results demonstrate that the Salkowski reaction has a greater specificity for detecting IAA than other tested auxins. Ten bacterial isolates displaying a wide range of auxin accumulation were selected, producing IAA levels of 5 to 90 µmol/L (according to the Salkowski reaction). Bacterial isolates were identified on the basis of 16S rDNA partial sequences: 9 isolates belonged to Enterobacter, and 1 isolate was classified as Serratia. The effect of BLBS on root morphology was evaluated in Arabidopsis thaliana. IAA production by rhizobacteria was confirmed by means of a DR5::GFP construct that is responsive to IAA, and also by HPLC-GC/MS. Finally, we observed that IAA secreted by rhizobacteria (i) modified the root architecture of A. thaliana, (ii) caused an increase in chickpea root biomass, and (iii) activated the green fluorescent protein (GFP) reporter gene driven by the DR5 promoter. These findings provide evidence that these novel bacterial isolates may be considered as putative plant-growth-promoting rhizobacteria modifying root architecture and increasing root biomass.

A high-throughput screening assay to identify bacterial antagonists against Fusarium verticillioides.

A high-throughput antagonistic assay was developed to screen for bacterial isolates capable of controlling the maize fungal phytopathogen Fusarium verticillioides. This assay combines a straightforward methodology, in which the fungus is challenged with bacterial isolates in liquid medium, with a novel approach that uses the plant lectin wheat germ agglutinin (WGA) coupled to a fluorophore (Alexa-Fluor® 488) under the commercial name of WGA, Alexa Fluor® 488 conjugate. The assay is performed in a 96-well plate format, which reduces the required laboratory space and streamlines quantitation and automation of the process, making it fast and accurate. The basis of our assay is that fungal biomass can be assessed by WGA, Alexa Fluor® 488 conjugate staining, which recognizes the chitin in the fungal cell wall and thus permits the identification of potential antagonistic bacteria that inhibit fungal growth. This principle was validated by chitin-competition binding assays against WGA, Alexa Fluor® 488 conjugate; confocal laser microscopy confirmed that the fluorescent WGA, Alexa Fluor® 488 conjugate binds to the chitin of the fungal cell wall. The majority of bacterial isolates did not bind to the WGA, Alexa Fluor® 488 conjugate. Furthermore, including washing steps significantly reduced any bacterial staining to background levels, even in the rare cases where bacterial isolates were capable of binding to WGA. Confirmatory conventional agar plate antagonistic assays were also conducted to validate our technique. We are now successfully employing this large-scale antagonistic assay as a pre-screening step for potential fungal antagonists in extensive bacteria collections (on the order of thousands of isolates).

Combining Ability and Reciprocal Effects for the Yield of Elite Blue Corn Lines from the Central Highlands of Mexico.

The development of hybrid plants can increase the production and quality of blue corn, and, thus, satisfy its high demand. For this development, it is essential to understand the heterotic relationships of the germplasm. The objectives of this study were to determine the effects of general (GCA) and specific (SCA) combining ability, as well as the reciprocal effects (REs) on the yields of 10 blue corn lines, and to select the outstanding lines. Diallel crosses were generated with 10 lines and evaluated at the Valle de México Experimental Station in Chapingo, Mexico, and Calpulalpan, Tlaxcala, Mexico. There were differences (p ≤ 0.01) in the hybrids, Loc, effects of GCA, SCA, and REs, and in the following interactions: hybrids × Loc, GCA × Loc, SCA × Loc, and RE × Loc. For GCA, lines Ll, L4, L6, and L9 stood out, with significant values of 3.4, 2.9, 2.9, and 3.1, respectively. For SCA, the hybrids featured were L4 × L10, L2 × L10, L1 × L10, L7 × L8, and L2 × L6, with values of 3.0, 2.5, 2.3, 2.3, and 2.2, and yields of 11.2, 10.2, 10.4, 10.4, and 10.5 t ha-l, respectively. There were no significant REs in these lines. Considerable effects of GCA and SCA were detected; therefore, we concluded that native populations had favorable dominance and additive genetic effects that could be used to support the development of high-yielding lines and hybrids.

Cell wall-related genes and lignin accumulation contribute to the root resistance in different maize (Zea mays L.) genotypes to Fusarium verticillioides (Sacc.) Nirenberg infection.

Introduction: The fungal pathogen Fusarium verticillioides (Sacc.) Nirenberg (Fv) causes considerable agricultural and economic losses and is harmful to animal and human health. Fv can infect maize throughout its long agricultural cycle, and root infection drastically affects maize growth and yield. Methods: The root cell wall is the first physical and defensive barrier against soilborne pathogens such as Fv. This study compares two contrasting genotypes of maize (Zea mays L.) roots that are resistant (RES) or susceptible (SUS) to Fv infection by using transcriptomics, fluorescence, scanning electron microscopy analyses, and ddPCR. Results: Seeds were infected with a highly virulent local Fv isolate. Although Fv infected both the RES and SUS genotypes, infection occurred faster in SUS, notably showing a difference of three to four days. In addition, root infections in RES were less severe in comparison to SUS infections. Comparative transcriptomics (rate +Fv/control) were performed seven days after inoculation (DAI). The analysis of differentially expressed genes (DEGs) in each rate revealed 733 and 559 unique transcripts that were significantly (P ≤0.05) up and downregulated in RES (+Fv/C) and SUS (+Fv/C), respectively. KEGG pathway enrichment analysis identified coumarin and furanocoumarin biosynthesis, phenylpropanoid biosynthesis, and plant-pathogen interaction pathways as being highly enriched with specific genes involved in cell wall modifications in the RES genotype, whereas the SUS genotype mainly displayed a repressed plant-pathogen interaction pathway and did not show any enriched cell wall genes. In particular, cell wall-related gene expression showed a higher level in RES than in SUS under Fv infection. Analysis of DEG abundance made it possible to identify transcripts involved in response to abiotic and biotic stresses, biosynthetic and catabolic processes, pectin biosynthesis, phenylpropanoid metabolism, and cell wall biosynthesis and organization. Root histological analysis in RES showed an increase in lignified cells in the sclerenchymatous hypodermis zone during Fv infection. Discussion: These differences in the cell wall and lignification could be related to an enhanced degradation of the root hairs and the epidermis cell wall in SUS, as was visualized by SEM. These findings reveal that components of the root cell wall are important against Fv infection and possibly other soilborne phytopathogens.

Production of indole-3-acetic acid by Bacillus circulans E9 in a low-cost medium in a bioreactor.

Bacillus circulans E9 (now known as Niallia circulans) promotes plant growth-producing indole-3-acetic acid (IAA), showing potential for use as a biofertilizer. In this work, the use of a low-cost medium containing industrial substrates, soybean, pea flour, Solulys, Pharmamedia, yeast extract, and sodium chloride (NaCl), was evaluated as a substitute for microbiological Luria Broth (LB) medium for the growth of B. circulans E9 and the production of IAA. In Erlenmeyer flasks with pea fluor medium (PYM), the maximum production of IAA was 7.81 ± 0.16 µg mL-1, while in microbiological LB medium, it was 3.73 ± 0.15 µg mL-1. In addition, an oxygen transfer rate (OTR) of 1.04 kg O2 m-3 d-1 allowed the highest bacterial growth (19.3 ± 2.18 × 1010 CFU mL-1) and IAA production (10.7 µg mL-1). Consequently, the OTR value from the flask experiments was used to define the conditions for the operation of a 1 L stirred tank bioreactor. The growth and IAA production of B. circulans cultured in a bioreactor with PYM medium were higher (8 and 1.6 times, respectively) than those of bacteria cultured in Erlenmeyer flasks. IAA produced in a bioreactor by B. circulans was shown to induce the root system in Arabidopsis thaliana, similar to synthetic IAA. The results of this study demonstrate that PYM medium may be able to be used for the mass production of B. circulans E9 in bioreactors, increasing both bacterial growth and IAA production. This low-cost medium has the potential to be employed to grow other IAA-producing bacterial species.

Proteometabolomic Analysis Reveals Molecular Features Associated with Grain Size and Antioxidant Properties amongst Chickpea (Cicer arietinum L.) Seeds Genotypes.

Legumes are an essential source of nutrients that complement energy and protein requirements in the human diet. They also contribute to the intake of bioactive compounds such as polyphenols, whose content can vary depending on cultivars and genotypes. We conducted a comparative proteomics and metabolomics study to determine if there were significant variations in relevant nutraceutical compounds in the five genotypes of Kabuli-type chickpea grains. We performed an isobaric tandem mass tag (TMT) couple to synchronous precursor selection (SPS)-MS3 method along with a targeted and untargeted metabolomics approach based on accurate mass spectrometry. We observed an association between the overproduction of proteins involved in starch, lipid, and amino acid metabolism with gibberellin accumulation in large grains. In contrast, we visualized the over-accumulation of proteins associated with water deprivation in small grains. It was possible to visualize in small grains the over-accumulation of some phenolics such as vanillin, salicylic acid, protocatechuic acid, 4-coumaric acid, 4-hydroxybenzoic acid, vanillic acid, ferulic acid, and kaempferol 3-O-glucoside as well as the amino acid l-phenylalanine. The activated phenolic pathway was associated with the higher antioxidant capacity of small grains. Small grains consumption could be advantageous due to their nutraceutical properties.

Recent Developments in Enzymatic Antioxidant Defence Mechanism in Plants with Special Reference to Abiotic Stress.

The stationary life of plants has led to the evolution of a complex gridded antioxidant defence system constituting numerous enzymatic components, playing a crucial role in overcoming various stress conditions. Mainly, these plant enzymes are superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), glutathione peroxidase (GPX), glutathione reductase (GR), glutathione S-transferases (GST), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), and dehydroascorbate reductase (DHAR), which work as part of the antioxidant defence system. These enzymes together form a complex set of mechanisms to minimise, buffer, and scavenge the reactive oxygen species (ROS) efficiently. The present review is aimed at articulating the current understanding of each of these enzymatic components, with special attention on the role of each enzyme in response to the various environmental, especially abiotic stresses, their molecular characterisation, and reaction mechanisms. The role of the enzymatic defence system for plant health and development, their significance, and cross-talk mechanisms are discussed in detail. Additionally, the application of antioxidant enzymes in developing stress-tolerant transgenic plants are also discussed.

Advances in Entomopathogen Isolation: A Case of Bacteria and Fungi.

Entomopathogenic bacteria and fungi are quite frequently found in soils and insect cadavers. The first step in utilizing these microbes as biopesticides is to isolate them, and several culture media and insect baiting procedures have been tested in this direction. In this work, the authors review the current techniques that have been developed so far, in the last five decades, and display brief protocols which can be adopted for the isolations of these entomopathogens. Among bacteria, this review focuses on Serratia spp. and bacteria from the class Bacilli. Among fungi, the review focuses those from the order Hypocreales, for example, genera Beauveria, Clonostachys, Lecanicillium, Metarhizium, and Purpureocillium. The authors chose these groups of entomopathogenic bacteria and fungi based on their importance in the microbial biopesticide market.

Ergosterol Peroxide Isolated from Oyster Medicinal Mushroom, Pleurotus ostreatus (Agaricomycetes), Potentially Induces Radiosensitivity in Cervical Cancer.

Every year, more than 500,000 new cases of cervical cancer are reported, making it the fourth leading cause of cancer globally. Although human papillomavirus (HPV) vaccines show promise as a protective measure, HPV-related cancers remain a public health problem since the vaccines, which are only specific to certain viral types, are unavailable for mass distribution. Furthermore, the effects of toxicity following ionizing radiation therapy have reoriented views toward the search for radiosensitizers that can reduce toxicity as a consequence of decreased radiation doses. Here, we isolated ergosterol peroxide (EP) from Pleurotus ostreatus and purified it to test its potential effects in vitro. We thus observed that a gradual increase in EP dose correlates with a loss of viability in HeLa and CaSki cervical cell lines. Dose/response curves were constructed using cervical cancer cell lines, as well as normal human peripheral blood mononuclear cells. The selectivity of EP in human lymphocytes and cervical cancer cell lines was tested, and no toxicity was found in normal cells. A combination of treatments revealed a radiosensitizer effect in HeLa cells, when measuring the exposure to EP followed by irradiation with 137Cs. Our findings suggest that EP may be effective as a radiosensitizer in treating cervical cancer.

First Demonstration of Clinical Fusarium Strains Causing Cross-Kingdom Infections from Humans to Plants.

Mycotoxins from the Fusarium genus are widely known to cause economic losses in crops, as well as high mortalities rates among immunocompromised humans. However, to date, no correlation has been established for the ability of Fusarium to cause cross-kingdom infection between plants and humans. The present investigation aims to fill this gap in the literature by examining cross-kingdom infection caused by Furasium strains isolated from non-immunocompromised or non-immunosuppressed humans, which were subsequently reinfected in plants and on human tissue. The findings document for the first time cross-kingdom infective events in Fusarium species, thus enhancing our existing knowledge of how mycopathogens continue to thrive in different hosts.

Direct somatic embryogenesis in Coffea canephora.

Somatic embryogenesis (SE) provides a useful model to study embryo development in plants. In contrast to zygotic embryogenesis, SE can easily be observed, the culture conditions can be controlled, and large quantities of embryos can be easily obtained. In Coffea spp several model systems have been reported for in vitro SE induction. SE for coffee was first reported in Coffea canephora. Several systems have been developed since then, including SE from callus cultures derived from leaf explants; a two-phase experimental protocol for SE from leaves of Coffea arabica; and from leaf explants of Arabusta or C. arabica using a medium with cytokinins. Here we report a protocol using young leaves from in vitro seedling pre-conditioned with growth regulators. This is a simplified method to obtain a faster and more efficient protocol to produce direct somatic embryos in C. canephora.

Culture-induced variation in plants of Coffea arabica cv. caturra rojo, regenerated by direct and indirect somatic embryogenesis.

Amplified fragment-length polymorphism (AFLP) was used to evaluate the stability of DNA in regenerated plantlets of Coffea arabica obtained by direct (DSE) and indirect somatic embryogenesis (ISE). Cluster analysis using the unweighted pair-group method (UPGMA), showed no specific grouping pattern related to the type of embryogenesis. These results suggest that the somatic embryogenesis (SE) process has a mechanism for the selection of normal and competent cells. Bulked DNA from regenerated plants obtained by DSE and ISE, and from the mother plants, was used to characterize specific AFLP fragments associated with each SE process. Twenty-three primer combinations were tested. A total of 1446 bands were analyzed, with 11.4% being polymorphic and 84% being specific for regenerated plants. Furthermore, specific bands were detected for DSE, ISE, and the mother plants. These results indicate that the SE process induces rearrangements at the DNA level and demonstrates discrepancies between the mechanisms involved in each SE process. Coffea arabica breeding programs that involve DSE and ISE can use AFLP as an additional tool for assessing DNA stability.

PvLOX2 silencing in common bean roots impairs arbuscular mycorrhiza-induced resistance without affecting symbiosis establishment.

The arbuscular mycorrhizal (AM) symbiosis is an intimate association between specific soil-borne fungi and the roots of most land plants. AM colonisation elicits an enhanced defence resistance against pathogens, known as mycorrhizal-induced resistance (MIR). This mechanism locally and systemically sensitises plant tissues to boost their basal defence response. Although a role for oxylipins in MIR has been proposed, it has not yet been experimentally confirmed. In this study, when the common bean (Phaseolus vulgaris L.) lipoxygenase PvLOX2 was silenced in roots of composite plants, leaves of silenced plants lost their capacity to exhibit MIR against the foliar pathogen Sclerotinia sclerotiorum, even though they were colonised normally. PvLOX6, a LOX gene family member, is involved in JA biosynthesis in the common bean. Downregulation of PvLOX2 and PvLOX6 in leaves of PvLOX2 root-silenced plants coincides with the loss of MIR, suggesting that these genes could be involved in the onset and spreading of the mycorrhiza-induced defence response.

Maize cyclin D2 expression, associated kinase activity and effect of phytohormones during germination.

A cDNA corresponding to 16 kDa of the maize cyclin D2 N-terminus was cloned and this polypeptide was overexpressed to produce homologous antibodies. This antibody recognized a 38 kDa protein in extracts from maize embryonic axes which corresponds to the predicted size for cyclin D2 protein. Expression of cyclin D2 was followed at the transcriptional and protein levels, and the effect of cytokinins and abscisic acid (ABA) was followed during maize germination. Cytokinins importantly stimulated cyclin D2 gene expression at late germination times and sucrose was necessary for stimulation, whereas the effect of ABA was not different from that in controls. However, cyclin D2 protein levels in control axes reached a peak at 6 h germination, declining thereafter, and neither cytokinins nor ABA modified this behavior. Two cyclic-dependent kinase A (Cdk-A)-type proteins and proliferating cell nuclear antigen (PCNA) were found co-immunoprecipitating with cyclin D2, and these immunoprecipitates were able to phosphorylate both histone H1 and the maize retinoblastoma-related protein (RBR). This protein kinase activity differed from the pattern of protein accumulation during germination, and the activity was not modified by either cytokinins or ABA. We discuss these findings in terms of the importance of the cell cycle for the germination process.