Rice-arsenate interactions in hydroponics:whole genome transcriptional analysis

Rice (Oryza sativa) varieties that are arsenate-tolerant (Bala) and -sensitive (Azucena) were used to conduct a transcriptome analysis of the response of rice seedlings to sodium arsenate (AsV) in hydroponic solution. RNA extracted from the roots of three replicate experiments of plants grown for 1 week in phosphate-free nutrient with or without 13.3 muM AsV was used to challenge the Affymetrix (52K) GeneChip Rice Genome array. A total of 576 probe sets were significantly up-regulated at least 2-fold in both varieties, whereas 622 were down-regulated. Ontological classification is presented. As expected, a large number of transcription factors, stress proteins, and transporters demonstrated differential expression. Striking is the lack of response of classic oxidative stress-responsive genes or phytochelatin synthases/synthatases. However, the large number of responses from genes involved in glutathione synthesis, metabolism, and transport suggests that glutathione conjugation and arsenate methylation may be important biochemical responses to arsenate challenge. In this report, no attempt is made to dissect differences in the response of the tolerant and sensitive variety, but analysis in a companion article will link gene expression to the known tolerance loci available in the BalaxAzucena mapping population.

Rice-arsenate interactions in hydroponics: a three-gene model for tolerance

In this study, the genetic mapping of the tolerance of root growth to 13.3 muM arsenate [As(V)] using the BalaxAzucena population is improved, and candidate genes for further study are identified. A remarkable three-gene model of tolerance is advanced, which appears to involve epistatic interaction between three major genes, two on chromosome 6 and one on chromosome 10. Any combination of two of these genes inherited from the tolerant parent leads to the plant having tolerance. Lists of potential positional candidate genes are presented. These are then refined using whole genome transcriptomics data and bioinformatics. Physiological evidence is also provided that genes related to phosphate transport are unlikely to be behind the genetic loci conferring tolerance. These results offer testable hypotheses for genes related to As(V) tolerance that might offer strategies for mitigating arsenic (As) accumulation in consumed rice.

The mechanistic basis of interactions between mycorrhizal associations and toxic metal cations

Mycorrhizal associations, including ericoid, arbuscular and ecto-mycorrhizas, are found colonising highly metal contaminated soils. How do mycorrhizal fungi achieve metal resistance, and does this metal resistance confer enhanced metal resistance to plant symbionts? These are the questions explored in this review by considering the mechanistic basis of mycorrhizal adaptation to metal cations. Recent molecular and physiological studies are discussed. The review reappraises what constitutes metal resistance in the context of mycorrhizal associations and sets out the constitutive and adaptive mechanisms available for mycorrhizas to adapt to contaminated sites. The only direct evidence of mycorrhizal adaptation to metal cation pollutants is the exudation of organic acids to alter pollutant availability in the rhizosphere. This is not to say that other mechanism of adaptation do not exist, but conclusive evidence of adaptive mechanisms of tolerance are lacking. For constitutive mechanisms of resistance, there is much more evidence, and mycorrhizas possess the same constitutive mechanisms for dealing with metal contaminants as other organisms. Rhizosphere chemistry is critical to understanding the interactions of mycorrhizas with polluted soils. Soil pH, mineral weathering, pollutant precipitation with plant excreted organic acids all may have a key role in constitutive and adaptive tolerance of mycorrhizal associations present on contaminated sites. The responses of mycorrhizal fungi to toxic metal cations are diverse. This, linked to the fact that mycorrhizal diversity is normally high, even on highly contaminated sites, suggests that this diversity may have a significant role in colonisation of contaminated sites by mycorrhizas. That is, the environment selects for the fungal community that can best cope with the environment, so having diverse physiological attributes will enable colonisation of a wide range of metal contaminated micro-habitats.

Mechanisms of arsenic hyperaccumulation in Pteris vittata. Uptake kinetics, interactions with phosphate, and arsenic speciation

The mechanisms of arsenic (As) hyperaccumulation in Pteris vittata, the first identified As hyperaccumulator, are unknown. We investigated the interactions of arsenate and phosphate on the uptake and distribution of As and phosphorus (P), and As speciation in P. vittata. In an 18-d hydroponic experiment with varying concentrations of arsenate and phosphate, P. vittata accumulated As in the fronds up to 27,000 mg As kg(-1) dry weight, and the frond As to root As concentration ratio varied between 1.3 and 6.7. Increasing phosphate supply decreased As uptake markedly, with the effect being greater on root As concentration than on shoot As concentration. Increasing arsenate supply decreased the P concentration in the roots, but not in the fronds. Presence of phosphate in the uptake solution decreased arsenate influx markedly, whereas P starvation for 8 d increased the maximum net influx by 2.5-fold. The rate of arsenite uptake was 10% of that for arsenate in the absence of phosphate. Neither P starvation nor the presence of phosphate affected arsenite uptake. Within 8 h, 50% to 78% of the As taken up was distributed to the fronds, with a higher translocation efficiency for arsenite than for arsenate. In fronds, 49% to 94% of the As was extracted with a phosphate buffer (pH 5.6). Speciation analysis using high-performance liquid chromatography-inductively coupled plasma mass spectroscopy showed that >85% of the extracted As was in the form of arsenite, and the remaining mostly as arsenate. We conclude that arsenate is taken up by P. vittata via the phosphate transporters, reduced to arsenite, and sequestered in the fronds primarily as As(III).

Choosing and using diversity indices: Insights for ecological applications from the German Biodiversity Exploratories

Biodiversity, a multidimensional property of natural systems, is difficult to quantify partly because of the multitude of indices proposed for this purpose. Indices aim to describe general properties of communities that allow us to compare different regions, taxa, and trophic levels. Therefore, they are of fundamental importance for environmental monitoring and conservation, although there is no consensus about which indices are more appropriate and informative. We tested several common diversity indices in a range of simple to complex statistical analyses in order to determine whether some were better suited for certain analyses than others. We used data collected around the focal plant Plantago lanceolata on 60 temperate grassland plots embedded in an agricultural landscape to explore relationships between the common diversity indices of species richness (S), Shannon's diversity (H'), Simpson's diversity (D1), Simpson's dominance (D2), Simpson's evenness (E), and Berger–Parker dominance (BP). We calculated each of these indices for herbaceous plants, arbuscular mycorrhizal fungi, aboveground arthropods, belowground insect larvae, and P. lanceolata molecular and chemical diversity. Including these trait-based measures of diversity allowed us to test whether or not they behaved similarly to the better studied species diversity. We used path analysis to determine whether compound indices detected more relationships between diversities of different organisms and traits than more basic indices. In the path models, more paths were significant when using H', even though all models except that with E were equally reliable. This demonstrates that while common diversity indices may appear interchangeable in simple analyses, when considering complex interactions, the choice of index can profoundly alter the interpretation of results. Data mining in order to identify the index producing the most significant results should be avoided, but simultaneously considering analyses using multiple indices can provide greater insight into the interactions in a system.

A simple inhibition coefficient for quantifying potency of biocontrol agents against plant-pathogenic fungi

Microbial interactions depend on a range of biotic and environmental variables, and are both dynamic and unpredictable. For some purposes, and under defined conditions, it is nevertheless imperative to evaluate the inhibitory efficacy of microbes, such as those with potential as biocontrol agents. We selected six, phylogenetically diverse microbes to determine their ability to inhibit the ascomycete Fusarium
coeruleum, a soil-dwelling pathogen of potato tubers that causes the storage disease dry rot. Interaction assays, where colony development was quantified (for both fungal pathogen and potential control agents), were therefore carried out on solid media. The key parameters that contributed to, and were indicative of, inhibitory efficacy were identified as: fungal growth-rates (i) prior to contact with the biocontrol
agent and (ii) if/once contact with the biocontrol agent was established (i.e. in the zone of mixed
culture), and (iii) the ultimate distance traveled by the fungal mycelium. It was clear that there was no correlation between zones of fungal inhibition and the overall reduction in the extent of fungal colony development. An inhibition coefficient was devised which incorporated the potential contributions of distal inhibition of fungal growth-rate; prevention of mycelium development in the vicinity of the biocontrol
agent; and ability to inhibit plant-pathogen growth-rate in the zone of mixed culture (in a ratio of 2:2:1). The values derived were 84.2 for Bacillus subtilis (QST 713), 74.0 for Bacillus sp. (JC12GB42), 30.7 for Pichia anomala (J121), 19.3 for Pantoea agglomerans (JC12GB34), 13.9 for Pantoea sp. (S09:T:12), and
21.9 (indicating a promotion of fungal growth) for bacterial strain (JC12GB54). This inhibition coefficient, with a theoretical maximum of 100, was consistent with the extent of F. coeruleum-colony development (i.e. area, in cm2) and assays of these biocontrol agents carried out previously against Fusarium
spp., and other fungi. These findings are discussed in relation to the dynamics and inherent complexity of natural ecosystems, and the need to adapt models for use under specific sets of conditions.

Plant community assembly at small scales: spatial versus environmental factors in a central European grassland

Dispersal limitation and environmental conditions are crucial drivers of plant species distribution and establishment. As these factors operate at different spatial scales, we asked: Do the environmental factors known to determine community assembly at broad scales operate at fine scales (few meters)? How much do these factors account for community variation at fine scales? In which way do biotic and abiotic interactions drive changes in species composition? We surveyed the plant community within a dry grassland along a very steep gradient of soil characteristics like pH and nutrients. We used a spatially explicit sampling design, based on three replicated macroplots of 15x15, 12x12 and 12x12 meters in extent. Soil samples were taken to quantify several soil properties (carbon, nitrogen, plant available phosphorus, pH, water content and dehydrogenase activity as a proxy for overall microbial activity). We performed variance partitioning to assess the effect of these variables on plant composition and statistically controlled for spatial autocorrelation via eigenvector mapping. We also applied null model analysis to test for non-random patterns in species co-occurrence using randomization schemes that account for patterns expected under species interactions. At a fine spatial scale, environmental factors explained 18% of variation when controlling for spatial autocorrelation in the distribution of plant species, whereas purely spatial processes accounted for 14% variation. Null model analysis showed that species spatially segregated in a non-random way and these spatial patterns could be due to a combination of environmental filtering and biotic interactions. Our grassland study suggests that environmental factors found to be directly relevant in broad scale studies are present also at small scales, but are supplemented by spatial processes and more direct interactions like competition.

Toxic interactions of metal ions (Cd2+, Pb2+, Zn2+ and Sb3- on in vitro biomass production of ectomycorrhizal fungi

A number of ectomycorrhizal (ECM) fungi, from sites uncontaminated by toxic metals, were investigated to determine their sensitivity to Cd^2-, Pb²⁺, Zn²⁺ and Sb^3-, measured as an inhibition of fungal biomass production. Isolates were grown in liquid media amended with the metals, individually (over a range of concentrations) and in combination (at single concentrations) to determine any significant interactions between the metals. Significant interspecific variation in sensitivity to Cd²⁺ and Zn²⁺ was recorded, while Pb²⁺ and Sb^3- individually had little effect. The presence of Pb²⁺ and Sb^3- in the media did however, ameliorate Cd²⁺ and Zn²⁺ toxicity in some circumstances. Interactions between Cd²⁺ and Zn²⁺ were investigated further over a range of concentrations. Zn²⁺ was found to significantly ameliorate the toxicity of Cd²⁺ to three of the four isolates tested. The influence of Zn²⁺ varied between ECM species and with the concentrations of metals tested.

Promise for plant pest control: root-associated pseudomonads with insecticidal activities.

Insects are an important and probably the most challenging pest to control in agriculture, in particular when they feed on belowground parts of plants. The application of synthetic pesticides is problematic owing to side effects on the environment, concerns for public health and the rapid development of resistance. Entomopathogenic bacteria, notably Bacillus thuringiensis and Photorhabdus/Xenorhabdus species, are promising alternatives to chemical insecticides, for they are able to efficiently kill insects and are considered to be environmentally sound and harmless to mammals. However, they have the handicap of showing limited environmental persistence or of depending on a nematode vector for insect infection. Intriguingly, certain strains of plant root-colonizing Pseudomonas bacteria display insect pathogenicity and thus could be formulated to extend the present range of bioinsecticides for protection of plants against root-feeding insects. These entomopathogenic pseudomonads belong to a group of plant-beneficial rhizobacteria that have the remarkable ability to suppress soil-borne plant pathogens, promote plant growth, and induce systemic plant defenses. Here we review for the first time the current knowledge about the occurrence and the molecular basis of insecticidal activity in pseudomonads with an emphasis on plant-beneficial and prominent pathogenic species. We discuss how this fascinating Pseudomonas trait may be exploited for novel root-based approaches to insect control in an integrated pest management framework.

Synthesis of 4-hydroxycinnamic amides of di-, tri-, and tetraamines : potential insect toxins /

The monoconjugates of phenolic acids (i.e. coumaric acid) with polyamines such as spermidine and spermine are strikingly similar to some toxins from spiders and predatory wasps. Many plants contain phenolic acid polyamine conjugates and there is some reliable information supporting their roles as plant defense chemicals. Eleven monoacylated compounds of diamines, triamines, tetraamines and oxa-polyamine amines were prepared in three to seven steps: 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 and 32. The synthesis proceeds through stepwise construction of the polyamine backbone (as in 62 and 72), followed by protection and deprotection steps of the amino functions. Desymmetrization of readily available and prepared symmetrical polyamines is a key step in the synthesis. The protecting groups employed were tert-butoxycarbonyl (BOC) and trifluoroacetyl (TFA) group which were removed under different conditions: acid and base respectively. Deprotection and refunctionalization of the polyamine reagent demonstrated the versatility of these systems for N-acylation.

Starvation Induces Expression of the Plant-Adhesin Gene, Mad2, of the Entomopathogenic Fungus Metarhizium robertsii.

Metarhizium robertsii is an entomopathogenic fungus that is additionally plant rhizosphere competent. Two adhesin-encoding gens, Mad1 and Mad2, are involved in insect pathogenesis or plant root colonization, respectively. This study examined differential expression of the Mad genes for M robertsii grown on a variety of insectand plant-related substrates. Mad1 was up regulated in response to insect cuticles and up regulation of Mad2 resulted from root exudates, tomato stems and non-preferred carbohydrates. A time course analysis that compared water, minimal media, and nutrient rich broth revealed Mad2 gene expression increased as nutrient availability decreased. The regulation of Mad2 compared to known stress-related genes (Hsp30, Hsp70 and ssgA) under various stresses (nutrient, pH, osmotic, oxidative, temperature) revealed Mad2 to be generally up regulated by nutrient starvation only. Examination of the Mad2 promoter region revealed two copies of a stress-response element (S TRE) known to be regulated under the general stress response pathway.

The analysis of Metarhizium robertsii potential as endophytic plant root coloniser, plant growth enhancer and antagonist to bean root pathogen Fusarium solani f. sp. phaseolis.

The soil-inhabiting insect-pathogenic fungus Metarhizium robertsii also colonizes plant roots endophytically, thus showing potential as a plant symbiont. M robertsii is not randomly distributed in soils but preferentially associates with the plant rhizosphere when applied in agricultural settings. Root surface and endophytic colonization of switchgrass (Panicum virgatum) and haricot beans (Phaseolus vulgaris) by M robertsii were examined after inoculation with fungal conidia. Light and confocal microscopies were used to ascertain this rhizosphere association. Root lengths, root hair density and emergence of lateral roots were also measured. Initially, M robertsii conidia adhered to, germinated on, and colonized, roots. Furthermore, plant roots treated with Metarhizium grew faster and the density of plant root hairs increased when compared with control plants. The onset of plant root hair proliferation was initiated before germination of M robertsii on the root (within 1-2 days). Plants inoculated with M robertsii AMAD2 (plant adhesin gene) took significantly longer to show root hair proliferation than the wild type. Cell free extracts of M robertsii did not stimulate root hair proliferation. Longer term (60 days) associations showed that M robertsii endophytically colonized individual cortical cells within bean roots. Metarhizium appeared as an amorphous mycelial aggregate within root cortical cells as well as between the intercellular spaces with no apparent damage to the plant. These results suggested that not only is M robertsii rhizosphere competent but displays a beneficial endophytic association with plant roots that results in the proliferation of root hairs. The biocontrol of bean (Phaseolis vulgaris) root rot fungus Fusarium solani f. sp. phaseolis by Metarhizium robertsii was investigated in vitro and in vivo. Dual cultures on Petri dishes showed antagonism of M robertsii against F. solani. A relative inhibition of ca. 60% of F. solani growth was observed in these assays. Cell free culture filtrates of M robertsii inhibited the germination of F. solani conidia by 83% and the inhibitory metabolite was heat stable. Beans plants colonized by M robertsii then exposed to F. solani showed healthier plant profiles and lower disease indices compared to plants not colonized by M robertsii. These results suggested that the insect pathogenic/endophytic fungus M robertsii could also be utilized as a biocontrol agent against certain plant pathogens occurring in the rhizosphere.

Feeding and oviposition preferences of the diamondback moth Plutella xylostella (Lepidoptera: Plutellidae) on six Brassicaceae host plant species

Plutella xylostella (diamondback moth, DBM) is a globally distributed Lepidopteran that feeds and oviposits almost exclusively on plants in the Brassicaceae family. DBM disperses from the southern United States and Mexico into Canada in the spring and summer. Establishment of DBM in Ontario is partially dependent upon the quantity and quality of host plants available and the preference of DBM for different hosts. Host plants include many crops such as broccoli, canola and cabbage, as well as landscape ornamentals and wild plants. It has previously been established that DBM are attracted to host plants by chemicals, specifically glucosinolates. I examined the preference of DBM among crop, wild and ornamental host plant species and how preference varies with insect life stage (3rd and 4th instar larvae and adults). Experiments included exposing DBM larvae from five populations coming from different locations in Canada to six Brassicaceae species and evaluating the preferences and weight gain over one hour. Then adult females were exposed to these same plant species and their oviposition preferences were examined. Populations from Alberta, Saskatchewan and Ontario were compared to assess differences in preference associated with geographic region or species of host plant. The ultimate goal of my study was to understand the potential of various Brassicaceae species to act as reservoirs to sustain and promote population growth of DBM, as well as sinks that may decrease DBM abundance. Results showed that garden cress (Lepidium sativum) was highly preferred over other species (wintercress, black mustard, aubretia, broccoli and ornamental kale) for both food and oviposition sources. Previous studies report that garden cress contains saponins, chemicals shown to be toxic to developing DBM larvae, however no studies have yet shown a preference for garden cress. These results provide information on a novel host plant with the potential to control DBM population growth. No difference in preferences was found among populations of DBM from various sources in Canada.

Interactions of onion (Allium cepa) and yellow wax bean (Phaseolus vulgaris) in monoculture and intercropping with weeds, Chenopodium album and Amaranthus hybridus

Intercropping systems are seen as advantageous as they can provide higher crop yield and diversity along with fewer issues related to pests and weeds than monocultures. However, plant interactions in intercropped crop species and between crops and weeds in these systems are still not well understood. The main objective of this study was to investigate interactions between onion (Allium cepa) and yellow wax bean (Phaseolus vulgaris) in monocultures and intercropping with and without the presence of a weed species, either Chenopodium album or Amaranthus hybridus. Another objective of this study was to compare morphological traits of C. album from two different populations (conventional vs. organic farms). Using a factorial randomized block design, both crop species were planted either in monoculture or intercropped with or without the presence of one of the two weeds. The results showed that intercropping onion with yellow wax bean increased the growth of onion but decreased the growth of yellow wax bean when compared to monocultures. The relative yield total (RYT) value was 1.3. Individual aboveground dry weight of both weed species under intercropping was reduced about 5 times when compared to the control. The poor growth of weeds in intercropping might suggest that crop diversification can help resist weed infestations. A common garden experiment indicated that C. album plants from the conventional farm had larger leaf area and were taller than those from the organic farm. This might be associated with specific evolutionary adaptation of weeds to different farming practices. These findings contribute to the fundamental knowledge of crop-crop interactions, crop-weed competition and adaptation of weeds to various conditions. They provide insights for the management of diversified cropping systems and integrated weed management as practices in sustainable agriculture.

Effet de la température sur les interactions trophiques et intraguildes au sein d’un système plante-herbivore-ennemis naturels : modélisation et approches expérimentales

Doctorat réalisé en cotutelle entre l'Université de Montréal et l'Université Paul Sabatier-Toulouse III