Francisco R. Quiroz-Figueroa

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).

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.

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.

The current status of proteomic studies in somatic embryogenesis

Somatic embryogenesis includes the genetic reprogramming of somatic cells to acquire the embryogenic potency necessary to generate an embryo, which can develop into a whole plant. Acquisition of embryogenic capacity requires rigorous biochemical coordination that includes several metabolic and signal transduction pathways. Recent genomic and epigenetic studies in somatic embryogenesis have shown interconnection among signals associated with growth regulators, stress factors, and modulation of the genome structure. A broad range of key proteins, posttranslational modifications, protein turnover, and protein–protein interactions are common factors associated with the establishment of the necessary biochemical status of cells during the acquisition of the embryogenic potential. Recent proteomic studies have begun describing the molecular basis of somatic embryogenesis. However, the diversity of the embryogenic response among plant species makes it difficult to define key protein factors associated with embryogenic cultures or specific stages during the transdifferentiation of somatic embryos. In this chapter, we review the most prominent proteomic studies carried out in the past decade and discuss the contributions of proteomics studies to elucidating the molecular basis of somatic embryogenesis.

Somatic Embryogenesis in Jatropha curcas

Jatropha curcas is an economically important member of the Euphorbiaceae family with numerous uses as a food source or fertilizer, as well as in the production of bioactive compounds and biodiesel. Propagation by seeds results in variation in the biochemistry of the plant, including oil productivity and other important compounds. In contrast, plant tissue culture offers the alternative approach of clonal propagation, which yields numerous genetically homogeneous plants. Although several studies associated with tissue culture in J. curcas have been published, the extensive genetic diversity of this semidomesticated plant makes it necessary to reevaluate and improve the established protocols with several genotypes. The application of herbicides with plant growth regulator activity could be useful for inducing somaclonal variation, which could then result in the addition of new agronomical traits. However, continuing studies in genetic diversity, molecular marker-assisted breeding, the production of secondary metabolites, and oils in in vitro cultures such as calli, suspension culture, and hairy roots are necessary to exploit the full potential of J. curcas. In this chapter, we will discuss recent studies of J. curcas plant tissue culture, as well as new research topics that will improve the efficiency of somatic embryogenesis.

Microscopy Applied In Biomass Characterization

This chapter serves as an introduction to the major types of microscopy that are applied to the characterization of lignocellulosic biomasses. The covered techniques include optical microscopies (light, Raman, and confocal microscopy), scanning probe microscopy, and electron microscopy. This chapter provides a general description of the principles, advantages and drawbacks, type of information that can be obtained using the different microscopic techniques, and includes a wide range of examples on the use of such techniques to characterize lignocellulosic biomass samples before and after pretreatments. Finally, some of the reviewed microscopic techniques were used to visualize samples of wheat straw nodes before and after acid and alkali pretreatments. This chapter is designed to help scientists select the best microscopic technique to study biomass feedstocks with recalcitrant natures.