Root‐specific expression of opine genes and opine accumulation in some cultivars of the naturally occurring genetically modified organism Nicotiana tabacum

Previous studies have shown that Nicotiana tabacum contains three Agrobacterium‐derived T‐DNA sequences inherited from its paternal ancestor Nicotiana tomentosiformis. Among these, the TB locus carries an intact mannopine synthase 2′ gene (TB‐mas2′). This gene is similar to the Agrobacterium rhizogenes A4‐mas2′ gene that encodes the synthesis of the Amadori compound deoxyfructosyl‐glutamine (DFG or santhopine). In this study we show that TB‐mas2′ is expressed at very low levels in N. tomentosiformis and in most N. tabacum cultivars; however, some cultivars show high TB‐mas2′ expression levels. The TB‐mas2′ promoter sequences of low‐ and high‐expressing cultivars are identical. The low/high level of expression segregates as a single Mendelian factor in a cross between a low‐ and a high‐expression cultivar. pTB‐mas2′‐GUS and pA4‐mas2′‐GUS reporter genes were stably introduced in N. benthamiana. Both were mainly expressed in the root expansion zone and leaf vasculature. Roots of tobacco cultivars with high TB‐mas2′ expression contain detectable levels of DFG.     

Study of rhizosphere and phyllosphere bacterial community and resistance to bacterial canker in genetically engineered phytochrome A cherry plants

The cherry rootstock 'Colt' line was transformed with a phytochrome A rice gene with the aim of altering light perception. Three transgenic events were chosen because of a modified developmental behavior. When red enriched light was supplied horizontally to stems, the PD3 transgenic line showed an increased rate of phytomer formation associated to a superior rate of plant growth compared to wild type (WT). Under the same light conditions, the PO1 and PA lines were less altered in morphology and development. When far-red enriched light was supplied, all transgenic lines had a reduced rate of growth, with the PD3 line being the most similar to the WT. The influence of the alien gene on root and leaf-associated bacteria was studied for a duration of 1 year. Significantly more culturable bacteria were recovered from PA lines than from PO1, PD3 and WT lines. On average, significantly more fluorescent pseudomonads were recovered from the rhizosphere of PA and PO1 lines than from PD3 and WT. No significant differences were detected in the number of bacteria recovered from the phyllosphere of transgenic and WT plant lines. A total of 143 Pseudomonas fluorescens strains isolated from rhizosphere of transgenic and WT lines were tested for their antagonistic activity against Phytophthora nicotianae and differences between bacteria derived from transgenic and WT were not detected. Fluorescent pseudomonads strains isolated from phyllosphere of PA and PO1 lines showed antagonistic activity against P. syringae pv. syringae, whereas no difference among the transgenic and WT lines was detected when fluorescent Pseudomonas strains were tested against P. syringae pv. mors-prunorum. Pathogenicity tests were conducted on rooted and micropropagated plants with P. s. pv. syringae and P. s. pv. mors-prunorum: in all assays, the PO1 lines were the most tolerant to P. s. pv. Syringae, and the PO1 and PD3 were tolerant to P. s. pv. mors-prunorum.     

Spatial soil heterogeneity has a greater effect on symbiotic arbuscular mycorrhizal fungal communities and plant growth than genetic modification with Bacillus thuringiensis toxin genes

Maize, genetically modified with the insect toxin genes of Bacillus thuringiensis (Bt), is widely cultivated, yet its impacts on soil organisms are poorly understood. Arbuscular mycorrhizal fungi (AMF) form symbiotic associations with plant roots and may be uniquely sensitive to genetic changes within a plant host. In this field study, the effects of nine different lines of Bt maize and their corresponding non‐Bt parental isolines were evaluated on AMF colonization and community diversity in plant roots. Plants were harvested 60 days after sowing, and data were collected on plant growth and per cent AMF colonization of roots. AMF community composition in roots was assessed using 454 pyrosequencing of the 28S rRNA genes, and spatial variation in mycorrhizal communities within replicated experimental field plots was examined. Growth responses, per cent AMF colonization of roots and AMF community diversity in roots did not differ between Bt and non‐Bt maize, but root and shoot biomass and per cent colonization by arbuscules varied by maize cultivar. Plot identity had the most significant effect on plant growth, AMF colonization and AMF community composition in roots, indicating spatial heterogeneity in the field. Mycorrhizal fungal communities in maize roots were autocorrelated within approximately 1 m, but at greater distances, AMF community composition of roots differed between plants. Our findings indicate that spatial variation and heterogeneity in the field has a greater effect on the structure of AMF communities than host plant cultivar or modification by Bt toxin genes.     

Transportability of non‐target arthropod field data for the use in environmental risk assessment of genetically modified maize in Northern Mexico

In country, non‐target arthropod (NTA) field evaluations are required to comply with the regulatory process for cultivation of genetically modified (GM) maize in Mexico. Two sets of field trials, Experimental Phase and Pilot Phase, were conducted to identify any potential harm of insect‐protected and glyphosate‐tolerant maize (MON‐89Ø34‐3 × MON‐88Ø17‐3 and MON‐89Ø34‐3 × MON‐ØØ6Ø3‐6) and glyphosate‐tolerant maize (MON‐ØØ6Ø3‐6) to local NTAs compared to conventional maize. NTA abundance data were collected at 32 sites, providing high geographic and environmental diversity within maize production areas from four ecological regions (ecoregions) in northern Mexico. The most abundant herbivorous taxa collected included field crickets, corn flea beetles, rootworm beetles, cornsilk flies, aphids, leafhoppers, plant bugs and thrips while the most abundant beneficial taxa captured were soil mites, spiders, predatory ground beetles, rove beetles, springtails (Collembola), predatory earwigs, ladybird beetles, syrphid flies, tachinid flies, minute pirate bugs, parasitic wasps and lacewings. Across the taxa analysed, no statistically significant differences in abundance were detected between GM maize and the conventional maize control for 69 of the 74 comparisons (93.2%) indicating that the single or stacked insect‐protected and herbicide‐tolerant GM traits generally exert no marked adverse effects on the arthropod populations compared with conventional maize. The distribution of taxa observed in this study provides evidence that irrespective of variations in overall biodiversity of a given ecoregion, important herbivore, predatory and parasitic arthropod taxa within the commercial maize agroecosystem are highly similar indicating that relevant data generated in one ecoregion can be transportable for the risk assessment of the same or similar GM crop in another ecoregion.     

Atmospheric CO2 enrichment and drought stress modify root exudation of barley

Rising CO₂ concentrations associated with drought stress is likely to influence not only aboveground growth, but also belowground plant processes. Little is known about root exudation being influenced by elements of climate change. Therefore, this study wanted to clarify whether barley root exudation responds to drought and CO₂ enrichment and whether this reaction differs between an old and a recently released malting barley cultivar. Barley plants were grown in pots filled with sand in controlled climate chambers at ambient (380 ppm) or elevated (550 ppm) atmospheric [CO₂] and a normal or reduced water supply. Root exudation patterns were examined at the stem elongation growth stage and when the inflorescences emerged. At both dates, root exudates were analyzed for different compounds such as total free amino acids, proline, potassium, and some phytohormones. Elevated [CO₂] decreased the concentrations in root exudates of some compounds such as total free amino acids, proline, and abscisic acid. Moreover, reduced water supply increased proline, potassium, electric conductivity, and hormone concentrations. In general, the modern cultivar showed higher concentrations of proline and abscisic acid than the old one, but the cultivars responded differentially under elevated CO₂. Plant developmental stage had also an impact on the root exudation patterns of barley. Generally, we observed significant effects of CO₂ enrichment, watering levels, and, to a lesser extent, cultivar on root exudation. However, we did not find any mitigation of the adverse effects of drought by elevated CO₂. Understanding the multitude of relationships within the rhizosphere is an important aspect that has to be taken into consideration in the context of crop performance and carbon balance under conditions of climate change.     

Specificity of root microbiomes in native‐grown Nicotiana attenuata and plant responses to UVB increase Deinococcus colonization

Plants recruit microbial communities from the soil in which they germinate. Our understanding of the recruitment process and the factors affecting it is still limited for most microbial taxa. We analysed several factors potentially affecting root microbiome structure – the importance of geographic location of natural populations, the microbiome of native seeds as putative source of colonization and the effect of a plant's response to UVB exposure on root colonization of highly abundant species. The microbiome of Nicotiana attenuata seeds was determined by a culture‐dependent and culture‐independent approach, and the root microbiome of natural N. attenuata populations from five different locations was analysed using 454‐pyrosequencing. To specifically address the influence of UVB light on root colonization by Deinococcus, a genus abundant and consistently present in N. attenuata roots, transgenic lines impaired in UVB perception (irUVR8) and response (irCHAL) were investigated in a microcosm experiment with/without UVB supplementation using a synthetic bacterial community. The seed microbiome analysis indicated that N. attenuata seeds are sterile. Alpha and beta diversities of native root bacterial communities differed significantly between soil and root, while location had only a significant effect on the fungal but not the bacterial root communities. With UVB supplementation, root colonization of Deinococcus increased in wild type, but decreased in irUVR8 and irCHAL plants compared to nontreated plants. Our results suggest that N. attenuata recruits a core root microbiome exclusively from soil, with fungal root colonization being less selective than bacterial colonization. Root colonization by Deinococcus depends on the plant's response to UVB.     

Plant host and soil origin influence fungal and bacterial assemblages in the roots of woody plants

Microbial communities in plant roots provide critical links between above‐ and belowground processes in terrestrial ecosystems. Variation in root communities has been attributed to plant host effects and microbial host preferences, as well as to factors pertaining to soil conditions, microbial biogeography and the presence of viable microbial propagules. To address hypotheses regarding the influence of plant host and soil biogeography on root fungal and bacterial communities, we designed a trap‐plant bioassay experiment. Replicate Populus, Quercus and Pinus plants were grown in three soils originating from alternate field sites. Fungal and bacterial community profiles in the root of each replicate were assessed through multiplex 454 amplicon sequencing of four loci (i.e., 16S, SSU, ITS, LSU rDNA). Soil origin had a larger effect on fungal community composition than did host species, but the opposite was true for bacterial communities. Populus hosted the highest diversity of rhizospheric fungi and bacteria. Root communities on Quercus and Pinus were more similar to each other than to Populus. Overall, fungal root symbionts appear to be more constrained by dispersal and biogeography than by host availability.     

The devil is in the detail: Nonadditive and context‐dependent plant population responses to increasing temperature and precipitation

In climate change ecology, simplistic research approaches may yield unrealistically simplistic answers to often more complicated problems. In particular, the complexity of vegetation responses to global climate change begs a better understanding of the impacts of concomitant changes in several climatic drivers, how these impacts vary across different climatic contexts, and of the demographic processes underlying population changes. Using a replicated, factorial, whole‐community transplant experiment, we investigated regional variation in demographic responses of plant populations to increased temperature and/or precipitation. Across four perennial forb species and 12 sites, we found strong responses to both temperature and precipitation change. Changes in population growth rates were mainly due to changes in survival and clonality. In three of the four study species, the combined increase in temperature and precipitation reflected nonadditive, antagonistic interactions of the single climatic changes for population growth rate and survival, while the interactions were additive and synergistic for clonality. This disparity affects the persistence of genotypes, but also suggests that the mechanisms behind the responses of the vital rates differ. In addition, survival effects varied systematically with climatic context, with wetter and warmer + wetter transplants showing less positive or more negative responses at warmer sites. The detailed demographic approach yields important mechanistic insights into how concomitant changes in temperature and precipitation affect plants, which makes our results generalizable beyond the four study species. Our comprehensive study design illustrates the power of replicated field experiments in disentangling the complex relationships and patterns that govern climate change impacts across real‐world species and landscapes.     

Responses of stream fish populations to farming intensity and water abstraction in an agricultural catchment

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Carbon cost of plant nitrogen acquisition: global carbon cycle impact from an improved plant nitrogen cycle in the Community Land Model

Plants typically expend a significant portion of their available carbon (C) on nutrient acquisition – C that could otherwise support growth. However, given that most global terrestrial biosphere models (TBMs) do not include the C cost of nutrient acquisition, these models fail to represent current and future constraints to the land C sink. Here, we integrated a plant productivity‐optimized nutrient acquisition model – the Fixation and Uptake of Nitrogen Model – into one of the most widely used TBMs, the Community Land Model. Global plant nitrogen (N) uptake is dynamically simulated in the coupled model based on the C costs of N acquisition from mycorrhizal roots, nonmycorrhizal roots, N‐fixing microbes, and retranslocation (from senescing leaves). We find that at the global scale, plants spend 2.4 Pg C yr^?1 to acquire 1.0 Pg N yr^?1, and that the C cost of N acquisition leads to a downregulation of global net primary production (NPP) by 13%. Mycorrhizal uptake represented the dominant pathway by which N is acquired, accounting for ~66% of the N uptake by plants. Notably, roots associating with arbuscular mycorrhizal (AM) fungi – generally considered for their role in phosphorus (P) acquisition – are estimated to be the primary source of global plant N uptake owing to the dominance of AM‐associated plants in mid‐ and low‐latitude biomes. Overall, our coupled model improves the representations of NPP downregulation globally and generates spatially explicit patterns of belowground C allocation, soil N uptake, and N retranslocation at the global scale. Such model improvements are critical for predicting how plant responses to altered N availability (owing to N deposition, rising atmospheric CO₂, and warming temperatures) may impact the land C sink.     

Similarities and spatial variations of bacterial and fungal communities in field rice planthopper (Hemiptera: Delphacidae) populations

Rice planthoppers are notorious plant sap‐feeding pests which cause serious damage. While several microbes in rice planthoppers have been broadly characterized, the abundance and diversity of bacteria and fungi in field planthoppers are largely unknown. This study investigated the bacterial and fungal community compositions of Chinese wild rice planthoppers Laodelphax striatellus and Sogatella furcifera using parallel 16S rRNA gene amplicon and internal transcribed space region sequencing. The bacteria varied significantly between the species and were partitioned significantly by sex, tissues and host environments in each species. The majority of bacteria were affiliated with the genera Wolbachia, Cardinium, Rickettsia and Pantoea. The abundance of Wolbachia was negatively correlated with that of Cardinium in both planthopper species. Compared with bacteria, the abundance and diversity of fungi did not differ between sexes but both were enriched in the gut. The bacterial community as a whole showed no significant correlation with the fungal community. The majority of fungi were related to Sarocladium, Alternaria, Malassezia, Aspergillus and Curvularia. A phylogenetic analysis revealed that these fungi were closely related to botanic symbionts or pathogens. Our results provide novel insights into the bacteria and fungi of rice planthoppers.     

Glyphosate applied to genetically modified herbicide-tolerant sugar beet and 'volunteer' potatoes reduces populations of potato cyst nematodes and the number and size of daughter tubers

Glyphosate, applied early or later or twice to genetically modified glyphosate‐tolerant sugar beet, gave excellent control of planted ‘volunteer’ potatoes growing within the crop compared to conventional herbicide programmes with or without clopyralid. In three out of four trials, this resulted in significant reductions in the numbers of eggs and cysts of potato cyst nematodes (Globodera rostochiensis and G. pallida) where infestations were moderate (23–89 eggs g^?1 soil). In the fourth trial, which had very high initial populations (130 eggs ^?1 soil), none of the herbicide treatments had any significant effect on numbers of nematode eggs or cysts. This was probably due to competition for feeding sites, and the early death of the potatoes in all treatments caused by feeding damage by the nematodes and infection by blight, which prevented the nematodes from completing their life cycle. Glyphosate also significantly reduced the number and size of daughter tubers produced, thus helping to prevent a further volunteer problem in the next crop in the rotation. This was achieved by one or two applications of one chemical compared to 2–5 applications of cocktails of conventional herbicides.     

An untargeted global metabolomic analysis reveals the biochemical changes underlying basal resistance and priming in Solanum lycopersicum,and identifies 1‐methyltryptophan as a metabolite involved in plant responses to Botrytis cinerea and Pseudomonas syringae

In this study, we have used untargeted global metabolomic analysis to determine and compare the chemical nature of the metabolites altered during the infection of tomato plants (cv. Ailsa Craig) with Botrytis cinerea (Bot) or Pseudomonas syringae pv. tomato DC3000 (Pst), pathogens that have different invasion mechanisms and lifestyles. We also obtained the metabolome of tomato plants primed using the natural resistance inducer hexanoic acid and then infected with these pathogens. By contrasting the metabolomic profiles of infected, primed, and primed + infected plants, we determined not only the processes or components related directly to plant defense responses, but also inferred the metabolic mechanisms by which pathogen resistance is primed. The data show that basal resistance and hexanoic acid‐induced resistance to Bot and Pst are associated with a marked metabolic reprogramming. This includes significant changes in amino acids, sugars and free fatty acids, and in primary and secondary metabolism. Comparison of the metabolic profiles of the infections indicated clear differences, reflecting the fact that the plant's chemical responses are highly adapted to specific attackers. The data also indicate involvement of signaling molecules, including pipecolic and azelaic acids, in response to Pst and, interestingly, to Bot. The compound 1‐methyltryptophan was shown to be associated with the tomato–Pst and tomato–Bot interactions as well as with hexanoic acid‐induced resistance. Root application of this Trp‐derived metabolite also demonstrated its ability to protect tomato plants against both pathogens.     

Impact of biocontrol agents Pseudomonas fluorescens CHA0 and its genetically modified derivatives on the diversity of culturable fungi in the rhizosphere of mungbean

AIMS: To assess whether Pseudomonas fluorescens strain CHA0 and its genetically modified derivatives, CHA0/pME3424 (antibiotic over-producer) and CHA89 (antibiotic-deficient) could have an impact on the fungal community structure and composition in the rhizosphere of mungbean. METHODS AND RESULTS: Under glasshouse conditions, mungbean was grown repeatedly in the same soil, which was inoculated with CHA0, CHA0/pME3424, CHA89 or was left untreated. Treatments were applied to soil at the start of each 36-day mungbean growth cycle, and their effects on the diversity of the rhizosphere populations of culturable fungi were assessed at the end of the first, second and third cycles. The effects of CHA0 and CHA0/pME3424 did differ from the controls while CHA89 did not. Whereas all major fungal species were frequently isolated from both bacterized and nonbacterized rhizospheres, certain fungal species were exclusively promoted or specifically suppressed from Pseudomonas-treated soils. In general, fungal diversity and equitability tended to decrease with time while species richness slightly increased. Whilst a total of 29 fungal species were isolated from the mungbean rhizosphere, only eight species colonized the root tissues. CONCLUSIONS: Soil inoculation with Ps. fluorescens CHA0 or CHA0/pME3424 altered fungal community structure in mungbean rhizosphere but strain CHA89 failed to produce such effect. SIGNIFICANCE AND IMPACT OF THE STUDY: Pseudomonas fluorescens-mediated alteration in the composition and structure of fungal communities might have acute or lasting effects on ecosystem functioning. Furthermore, the study provides useful data pertinent to characterization of the fate of genetically modified inoculants (e.g. antibiotic-overproducing Pseudomonas strains) released into the environment.     

An AGAMOUS intron‐driven cytotoxin leads to flowerless tobacco and produces no detrimental effects on vegetative growth of either tobacco or poplar

Flowerless trait is highly desirable for poplar because it can prevent pollen‐ and seed‐mediated transgene flow. We have isolated the second intron of PTAG2, an AGAMOUS (AG) orthologue from Populus trichocarpa. By fusing this intron sequence to a minimal 35S promoter sequence, we created two artificial promoters, fPTAG2I (forward orientation of the PTAG2 intron sequence) and rPTAG2I (reverse orientation of the PTAG2 intron sequence). In tobacco, expression of the β‐glucuronidase gene (uidA) demonstrates that the fPTAG2I promoter is non‐floral‐specific, while the rPTAG2I promoter is active in floral buds but with no detectable vegetative activity. Under glasshouse conditions, transgenic tobacco plants expressing the Diphtheria toxin A (DT‐A) gene driven by the rPTAG2I promoter produced three floral ablation phenotypes: flowerless, neuter (stamenless and carpel‐less) and carpel‐less. Further, the vegetative growth of these transgenic lines was similar to that of the wild‐type plants. In field trials during 2014 and 2015, the flowerless transgenic tobacco stably maintained its flowerless phenotype, and also produced more shoot and root biomass when compared to wild‐type plants. In poplar, the rPTAG2I::GUS gene exhibited no detectable activity in vegetative organs. Under field conditions over two growing seasons (2014 to the end of 2015), vegetative growth of the rPTAG2I::DT‐A transgenic poplar plants was similar to that of the wild‐type plants. Our results demonstrate that the rPTAG2I artificial promoter has no detectable activities in vegetative tissues and organs, and the rPTAG2I::DT‐A gene may be useful for producing flowerless poplar that retains normal vegetative growth.