Michelle R. Lum

Rhizobium common nod genes are required for biofilm formation.

In legume nitrogen‐fixing symbioses, rhizobial nod genes are obligatory for initiating infection thread formation and root nodule development. Here we show that the common nod genes, nodD1ABC, whose products synthesize core Nod factor, a chitin‐like oligomer, are also required for the establishment of the three‐dimensional architecture of the biofilm of Sinorhizobium meliloti. Common nod gene mutants form a biofilm that is a monolayer. Moreover, adding Nod Factor antibody to S. meliloti cells inhibits biofilm formation, while chitinase treatment disrupts pre‐formed biofilms. These results attest to the involvement of core Nod factor in rhizobial biofilm establishment. However, luteolin, the plant‐derived inducer of S. melilotis nod genes, is not required for mature biofilm formation, although biofilm establishment is enhanced in the presence of this flavonoid inducer. Because biofilm formation is plant‐inducer‐independent and because all nodulating rhizobia, both alpha‐ and beta‐proteobacteria have common nod genes, the role of core Nod factor in biofilm formation is likely to be an ancestral and evolutionarily conserved function of these genes.

Roots and Their Symbiotic Microbes: Strategies to Obtain Nitrogen and Phosphorus in a Nutrient-Limiting Environment

The association between Rhizobium and legumes and that between arbuscular mycorrhizal (AM) fungi and most land plants display a remarkable degree of similarity. Both events involve the recognition of, entrance into, and coexistence within the plant root, with the development of a specialized interface that always separates the two partners and at which nutrient exchange occurs. Molecules produced by rhizobia during the early stages of the symbiosis are related to fungal chitin, and the plant responds to both microbes with an increase in the production of flavonoids, which may assist in recognition and development of the symbioses. Many of the same plant genes are up-regulated in the two symbiotic pathways, and notably plants that are Nod− are often defective in the AM association as well. However, there are a number of differences between the associations, and these are important for understanding the relationship between the two symbioses. The Rhizobium and AM symbioses will be compared and the question of whether the nitrogen-fixing association evolved from the much more ancient AM symbiosis will be discussed.

Nodulation and effective nitrogen fixation of Macroptilium atropurpureum (siratro) by Burkholderia tuberum, a nodulating and plant growth promoting beta-proteobacterium, are influenced by environmental factors

Background and aimsBurkholderia tuberum STM678T was isolated from a South African legume, but did not renodulate this plant. Until a reliable host is found, studies on this and other interesting beta-rhizobia cannot advance. We investigated B. tuberum STM678T’s ability to induce Fix+ nodules on a small-seeded, easy-to-propagate legume (Macroptilium atropurpureum). Previous studies demonstrated that B. tuberum elicited either Fix- or Fix+ nodules on siratro, but the reasons for this difference were unexplored.MethodsExperiments to promote effective siratro nodule formation under different environmental conditions were performed. B. tuberum STM678T’s ability to withstand high temperatures and desiccation was checked as well as its potential for promoting plant growth via mechanisms in addition to nitrogen fixation, e.g., phosphate solubilization and siderophore production. Potential genes for these activities were found in the sequenced genomes.ResultsHigher temperatures and reduced watering resulted in reliable, effective nodulation on siratro. Burkholderia spp. solubilize phosphate and produce siderophores. Genes encoding proteins potentially involved in these growth-promoting activities were detected and are described.ConclusionsSiratro is an excellent model plant for B. tuberum STM678T. We identified genes that might be involved in the ability of diazotrophic Burkholderia species to survive harsh conditions, solubilize phosphate, and produce siderophores.

DNA-Based Authentication of Botanicals and Plant-Derived Dietary Supplements: Where Have We Been and Where Are We Going?

Herbal medicines and botanicals have long been used as sole or additional medical aids worldwide. Currently, billions of dollars are spent on botanicals and related products, but minimal regulation exists regarding their purity, integrity, and efficacy. Cases of adulteration and contamination have led to severe illness and even death in some cases. Identifying the plant material in botanicals and phytomedicines using organoleptic means or through microscopic observation of plant parts is not trivial, and plants are often misidentified. Recently, DNA-based methods have been applied to these products because DNA is not changed by growth conditions unlike the chemical constituents of many active pharmaceutical agents. In recent years, DNA barcoding methods, which are used to identify species diversity in the Tree of Life, have been also applied to botanicals and plant-derived dietary supplements. In this review, we recount the history of DNA-based methods for identification of botanicals and discuss some of the difficulties in defining a specific bar code or codes to use. In addition, we describe how next generation sequencing technologies have enabled new techniques that can be applied to identifying these products with greater authority and resolution. Lastly, we present case histories where dietary supplements, decoctions, and other products have been shown to contain materials other than the main ingredient stipulated on the label. We conclude that there is a fundamental need for greater quality control in this industry, which if not self-imposed, that may result from legislation.

Investigation of Four Classes of Non-nodulating White Sweetclover (Melilotus alba annua Desr.) Mutants and Their Responses to Arbuscular-Mycorrhizal Fungi.

Abstract The nitrogen-fixing symbiosis between Rhizobiaceae and legumes is one of the best-studied interactions established between prokaryotes and eukaryotes. The plant develops root nodules in which the bacteria are housed, and atmospheric nitrogen is fixed into ammonia by the rhizobia and made available to the plant in exchange for carbon compounds. It has been hypothesized that this symbiosis evolved from the more ancient arbuscular mycorrhizal (AM) symbiosis, in which the fungus associates with roots and aids the plant in the absorption of mineral nutrients, particularly phosphate. Support comes from several fronts: 1) legume mutants where Nod− and Myc− co-segregate, and 2) the fact that various early nodulin (ENOD) genes are expressed in legume AM. Both strongly argue for the idea that the signal transduction pathways between the two symbioses are conserved. We have analyzed the responses of four classes of non-nodulating Melilotus alba (white sweetclover) mutants to Glomus intraradices (the mycorrhizal symbiont) to investigate how Nod− mutations affect the establishment of this symbiosis. We also re-examined the root hair responses of the non-nodulating mutants to Sinorhizobium meliloti (the nitrogen-fixing symbiont). Of the four classes, several sweetclover sym mutants are both Nod− and Myc−. In an attempt to decipher the relationship between nodulation and mycorrhiza formation, we also performed co-inoculation experiments with mutant rhizobia and Glomus intraradices on Medicago sativa, a close relative of M. alba. Even though sulfated Nod factor was supplied by some of the bacterial mutants, the fungus did not complement symbiotically defective rhizobia for nodulation.

Complete Genome Sequence of Micromonospora Strain L5, a Potential Plant-Growth-Regulating Actinomycete, Originally Isolated from Casuarina equisetifolia Root Nodules

ABSTRACT Micromonospora species live in diverse environments and exhibit a broad range of functions, including antibiotic production, biocontrol, and degradation of complex polysaccharides. To learn more about these versatile actinomycetes, we sequenced the genome of strain L5, originally isolated from root nodules of an actinorhizal plant growing in Mexico.

ENOD40 Gene Expression and Cytokinin Responses in the Nonnodulating, Nonmycorrhizal (Nod−Myc−) Mutant, Masym3, of Melilotus alba Desr.

Several nonnodulating, nonmycorrhizal (Nod−Myc−) mutants of Melilotus alba Desr. (white sweetclover) have been described. However, the details of their responses to Sinorhizobium meliloti have not been fully elucidated. We investigated rhizobial entry and colonization using Confocal Scanning Laser Microscopy on the Masym1–5 mutants and isolated an early nodulin (ENOD40) gene from wild-type M. alba. We focused on Masym3, the least responsive of the mutants to S. meliloti and VA-fungi, to determine its response to cytokinin. Cytokinin appears to be a downstream signal in the nodule developmental pathway based not only on our previous observations whereby Nod−Myc− alfalfa roots treated with cytokinin accumulated several ENOD gene transcripts, but also on recent reports showing the importance of cytokinin receptors for nodulation. Here we show that applying 10−6 M 6-benzylaminopurine to uninoculated Masym3 roots elicited ENOD40 transcript accumulation. In addition, Masym3 root hairs inoculated with either wild-type S. meliloti or Nod− S. meliloti expressing the trans-zeatin synthase gene of Agrobacterium tumefaciens exhibited tip swelling, suggesting that cytokinin mediated this response. However, Masym3 root hair tips swelled following inoculation with Nod− S. meliloti or after mock-inoculation, a response resembling the phenotype of root hairs, after handling, of the Medicago truncatula mutant, dmi2. Mtdmi2 is Nod−Myc− due to a defect in a gene encoding a Nodule Receptor Kinase (NORK). Like Mtdmi2, the root hair swelling response appears in part to be mediated by touch because Masym3 root hairs not contacted by either bacteria or drops of water or buffer remain elongated and do not exhibit tip swelling.

The prevalence of antibiotic-resistant bacteria (ARB) in waters of the Lower Ballona Creek Watershed, Los Angeles County, California.

Screening for the prevalence of antibiotic-resistant bacteria (ARB) was done at the Ballona Creek and Wetlands, an urban-impacted wetland system in Los Angeles, California. The goals were (1) to assess the overall prevalence of ARB, and (2) compare differences in ARB abundance and the types of antibiotic resistance (AR) among the following sample types: lagoon water from Del Rey Lagoon, urban runoff from Ballona Creek, and water from the Ballona Wetlands (tidal water flooding in from the adjacent estuary, and ebbing out from the salt marsh). Antibiotic resistance distributions were analyzed using the Kolmogorov-Smirnov test to develop the cumulative frequency of bacteria having resistance of up to eight antibiotics. Distributions from the environmental water samples were compared to unchlorinated secondary effluent from the Hyperion Water Reclamation Plant that was used as comparator samples likely to have an abundance of ARB. As expected, densities of total and ARB were highest in secondary effluent, followed by urban runoff. Samples of water flooding into the wetlands showed similar results to urban runoff; however, a reduction in densities of total and ARB occurred in water ebbing out of the wetlands. During preliminary work to identify ARB species, several bacterial species of relevance to human illness (e.g., Staphylococcus aureus, Enterococcus hirae, Pseudomonas aeruginosa, Aeromonas veronii, Enterobacter cancerogenus, Serratia marcescens, Pseudomonas stutzeri, and Staphylococcus intermedius) were isolated from sampled waters. If wetlands are a sink for ARB, construction and restoration of wetlands can help in the mediation of this human and environmental health concern.

A reliable method for the selection and confirmation of transconjugants of plant growth-promoting bacteria especially plant-associated Burkholderia spp.

Selectable markers, e.g., antibiotic resistance, for conjugation experiments are not always effective for slow-growing plant growth promoting bacteria such as Burkholderia. We used PCAT medium containing Congo Red for selecting Burkholderia transconjugants. This method allows for the reliable selection of transconjugants of these novel plant growth-promoting bacteria.

An Engineering Approach Investigating the Uptake and Phytotoxicity of One Type of Engineered Nanoparticle (CdSe/ZnS Quantum Dots) by Solanum Lycopersicum

The novel and extraordinary physiochemical properties of engineered nanoparticles (ENPs) is certain, yet, at the same time, their unique characteristics raise growing concerns regarding potentially adverse effects on biological and ecological systems. It is becoming increasingly evident, that before the full potential of nanotechnology can be realized, standardized characterization of ENPs behavior, fate, and their effects in the ecosystem are essential, to ensure the safe manufacturing and use of ENP products. Otherwise, the promise of such extraordinary advancements may find itself limited to applications such as electronics, and sporting equipments, industries in which it currently resides. The current toxicity profile of engineered nanomaterials is not only preliminary, but highly variable amongst researchers. Emphasizing the great need to develop a highly organized, efficient, and precise approach to assess the hazardous potential ENPs may pose, and address the safety concerns surrounding and limiting nanotechnology. In response to such concerns, the present study took an engineering approach, in an otherwise traditionally viewed discipline, to assess the potential impact of engineered nanoparticles on tomato (Solanum lycopersicum) seedlings, by implementing a full factorial design of experiment (FDOE) in an effort to identify what factors, and their interactions, have a significant (p ≤ 0.05) effect on root and shoot elongation, and if any observed effects are a result of particle uptake, evaluated via fluorescence microscopy imaging. Therefore, the goal of our study was to design and implement an efficient, effective, and precise method to assess the effect of one type of ENP, water-soluble CdSe/ZnS quantum dots, using Solanum lycopersicum as our model organism, one of 10 species recommended by the Unites States Environmental Protection Agency (US EPA) for use in phytotoxicity studies, via a methodology we believe novel to nanotechnology. By implementing factorial experimental design methodologies, not only are we efficiently identifying the factors that affect phytotoxicity, we are providing, for the first time to our knowledge, the first scientific data to report the significant interaction effects between the factors responsible for ENP toxicity.Water soluble (MUA) CdSe/ZnS quantum dots used in our study had a negative influence on root and shoot lengths of tomato seeds exposed for 3 and 6 days. The observed influence depended on (MUA) CdSe/ZnS concentration and QD exposure time. The importance of the factor effects were examined via analysis of variance (ANOVA), t-tests, confidence intervals, and normal plot statistical analyses. The findings concluded that factors B, C, and the BC-interaction (CdSe/ZnS: Exposure time, concentration, and exposure time–concentration interaction) significantly (p ≤ 0.05) affected root and shoot lengths of tomato seedlings. Thus, factors A, AB, AC, and ABC (CdSe/ZnS QD: Size, size–concentration, size-exposure time, and size-concentration-exposure time interactions) were not found to have a significant effect on root and shoot lengths of tomato seedlings, and ultimately eliminated from our model. After analyzing the interaction plots, it became evident that low percentages of root reduction are obtained at low concentration levels for short lengths of time; thus, to obtain the least amount of phytotoxic effects one would set factors B (concentration) and C (exposure time) to their low levels, 125 mg/L for 3 days, respectively. Alternatively, high percentages of root reduction are obtained at high concentration levels for long lengths of time; thus, to obtain the greatest phytotoxic effect one would set factors B and C to their high levels, 1000 mg/L for 6 days, respectively. This indicates that as exposure time increases, root reduction increases; thus, phytotoxicity increases. Since our study attempted to realize which factors minimize phytotoxicity effects of one type of ENP, these findings suggest that to minimize phytotoxicity effects (i.e. maximize root length or minimize percent of root reduction) of (MUA) CdSe/ZnS QDs on tomato seeds, set factors B and C (QD concentration and exposure time) to their low levels; that is, expose tomato seeds to 125 mg/L of QD solution for a maximum of 3 days. These settings will yield the least amount of root reduction (5.15%) and; thus, phytotoxicity effects will be minimized.With regard to tomato roots ability to uptake MUA QDs, our results contribute to the literature by reporting uptake possible. Although we did see particles inside the root, it was sporadic and difficult to quantify. As to whether it was intracellular (within the cell) or intercellular (in the spaces between the cells, i.e., outside the cells) we could not conclude with certainty, although we suspect the QDs were intercellular. Thus, we highly recommend future experiments involving cross sections and more in-depth microscopy imaging. Additionally, although the results of our experiment failed to support that particle size (t = 2.13; d.f. = 1; p = 0.065) or the particle size-exposure time interaction (t = 2.17; d.f. = 1; p = 0.062) had a significant effect on root and shoot lengths of tomato seedlings, due to the small p-value associated with both test statistics, it is our belief that particle size and the particle size-exposure time interaction may, in fact, be a real effect; thus, further investigation is recommended.Copyright