A high diversity of mechanisms endows ALS-inhibiting herbicide resistance in the invasive common ragweed (Ambrosia artemisiifolia L.).

Ambrosia artemisiifolia L. (common ragweed) is a globally invasive, allergenic, troublesome arable weed. ALS-inhibiting herbicides are broadly used in Europe to control ragweed in agricultural fields. Recently, ineffective treatments were reported in France. Target site resistance (TSR), the only resistance mechanism described so far for ragweed, was sought using high-throughput genotyping-by-sequencing in 213 field populations randomly sampled based on ragweed presence. Additionally, non-target site resistance (NTSR) was sought and its prevalence compared with that of TSR in 43 additional field populations where ALS inhibitor failure was reported, using herbicide sensitivity bioassay coupled with ALS gene Sanger sequencing. Resistance was identified in 46 populations and multiple, independent resistance evolution demonstrated across France. We revealed an unsuspected diversity of ALS alleles underlying resistance (9 amino-acid substitutions involved in TSR detected across 24 populations). Remarkably, NTSR was ragweed major type of resistance to ALS inhibitors. NTSR was present in 70.5% of the resistant plants and 74.1% of the fields harbouring resistance. A variety of NTSR mechanisms endowing different resistance patterns evolved across populations. Our study provides novel data on ragweed resistance to herbicides, and emphasises that local resistance management is as important as mitigating gene flow from populations where resistance has arisen.

On the metabolites produced by Colletotrichum gloeosporioides a fungus proposed for the Ambrosia artemisiifolia biocontrol; spectroscopic data and absolute configuration assignment of colletochlorin A.

Ambrosia artemisiifolia L. is responsible for serious allergies induced on humans. Different approaches for its control were proposed during the COST Action FA1203 "Sustainable management of Ambrosia artemisiifolia in Europe" (SMARTER). Fungal secondary metabolites often show potential herbicidal activity. Three phytotoxins were purified from the fungal culture filtrates of Colletotrichum gloeosporioides, isolated from infected leaves of A. artemisiifolia. They were identified by spectroscopic and chemical methods as colletochlorin A, orcinol and tyrosol (1, 2 and 3). The absolute configuration 6'R to colletochlorin A was assigned for the first time applying the advanced Mosher's method. When assayed by leaf-puncture on A. artemisiifolia only 1 caused the appearance of large necrosis. The same symptoms were also induced by 1 on ambrosia plantlets associated with plant wilting. On Lemna minor, colletochlorin A caused a clear fronds browning, with a total reduction in chlorophyll content.

Glyphosate resistance in Ambrosia trifida: Part 1. Novel rapid cell death response to glyphosate.

BACKGROUND: Glyphosate-resistant (GR) Ambrosia trifida is now present in the midwestern United States and in southwestern Ontario, Canada. Two distinct GR phenotypes are known, including a rapid response (GR RR) phenotype, which exhibits cell death within hours after treatment, and a non-rapid response (GR NRR) phenotype. The mechanisms of resistance in both GR RR and GR NRR remain unknown. Here, we present a description of the RR phenotype and an investigation of target-site mechanisms on multiple A. trifida accessions. RESULTS: Glyphosate resistance was confirmed in several accessions, and whole-plant levels of resistance ranged from 2.3- to 7.5-fold compared with glyphosate-susceptible (GS) accessions. The two GR phenotypes displayed similar levels of resistance, despite having dramatically different phenotypic responses to glyphosate. Glyphosate resistance was not associated with mutations in EPSPS sequence, increased EPSPS copy number, EPSPS quantity, or EPSPS activity. CONCLUSION: These encompassing results suggest that resistance to glyphosate in these GR RR A. trifida accessions is not conferred by a target-site resistance mechanism. © 2017 Society of Chemical Industry.

Glyphosate resistance in Ambrosia trifida: Part 2. Rapid response physiology and non-target-site resistance.

BACKGROUND: The glyphosate-resistant rapid response (GR RR) resistance mechanism in Ambrosia trifida is not due to target-site resistance (TSR) mechanisms. This study explores the physiology of the rapid response and the possibility of reduced translocation and vacuolar sequestration as non-target-site resistance (NTSR) mechanisms. RESULTS: GR RR leaf discs accumulated hydrogen peroxide within minutes of glyphosate exposure, but only in mature leaf tissue. The rapid response required energy either as light or exogenous sucrose. The combination of phenylalanine and tyrosine inhibited the rapid response in a dose-dependent manner. Reduced glyphosate translocation was observed in GR RR, but only when associated with tissue death caused by the rapid response. Nuclear magnetic resonance studies indicated that glyphosate enters the cytoplasm and reaches chloroplasts, and it is not moved into the vacuole of GR RR, GR non-rapid response or glyphosate-susceptible A. trifida. CONCLUSION: The GR RR mechanism of resistance is not associated with vacuole sequestration of glyphosate, and the observed reduced translocation is likely a consequence of rapid tissue death. Rapid cell death was inhibited by exogenous application of aromatic amino acids phenylalanine and tyrosine. The mechanism by which these amino acids inhibit rapid cell death in the GR RR phenotype remains unknown, and it could involve glyphosate phytotoxicity or other agents generating reactive oxygen species. Implications of these findings are discussed. The GR RR mechanism is distinct from the currently described glyphosate TSR or NTSR mechanisms in other species. © 2017 Society of Chemical Industry.

Modeling pollen-mediated gene flow from glyphosate-resistant to -susceptible giant ragweed (Ambrosia trifida L.) under field conditions.

A field experiment was conducted to quantify pollen mediated gene flow (PMGF) from glyphosate-resistant (GR) to glyphosate-susceptible (GS) giant ragweed under simulated field conditions using glyphosate resistance as a selective marker. Field experiments were conducted in a concentric design with the GR giant ragweed pollen source planted in the center and GS giant ragweed pollen receptors surrounding the center in eight directional blocks at specified distances (between 0.1 and 35 m in cardinal and ordinal directions; and additional 50 m for ordinal directions). Seeds of GS giant ragweed were harvested from the pollen receptor blocks and a total of 100,938 giant ragweed plants were screened with glyphosate applied at 2,520 g ae ha-1 and 16,813 plants confirmed resistant. The frequency of PMGF was fit to a double exponential decay model selected by information-theoretic criteria. The highest frequency of gene flow (0.43 to 0.60) was observed at ≤0.5 m from the pollen source and reduced rapidly with increasing distances; however, gene flow (0.03 to 0.04) was detected up to 50 m. The correlation between PMGF and wind parameters was inconsistent in magnitude, direction, and years.

Pollen of common ragweed (Ambrosia artemisiifolia L.): Illumina-based de novo sequencing and differential transcript expression upon elevated NO2/O3.

Common ragweed (Ambrosia artemisiifolia L.) is a highly allergenic annual ruderal plant and native to Northern America, but now also spreading across Europe. Air pollution and climate change will not only affect plant growth, pollen production and duration of the whole pollen season, but also the amount of allergenic encoding transcripts and proteins of the pollen. The objective of this study was to get a better understanding of transcriptional changes in ragweed pollen upon NO2 and O3 fumigation. This will also contribute to a systems biology approach to understand the reaction of the allergenic pollen to air pollution and climate change. Ragweed plants were grown in climate chambers under controlled conditions and fumigated with enhanced levels of NO2 and O3. Illumina sequencing and de novo assembly revealed significant differentially expressed transcripts, belonging to different gene ontology (GO) terms that were grouped into biological process and molecular function. Transcript levels of the known Amb a ragweed encoding allergens were clearly up-regulated under elevated NO2, whereas the amount of allergen encoding transcripts was more variable under elevated O3 conditions. Moreover transcripts encoding allergen known from other plants could be identified. The transcriptional changes in ragweed pollen upon elevated NO2 fumigation indicates that air pollution will alter the transcriptome of the pollen. The changed levels of allergenic encoding transcripts may have an influence on the total allergenic potential of ragweed pollen.

Common ragweed (Ambrosia artemisiifolia L.): allergenicity and molecular characterization of pollen after plant exposure to elevated NO2.

Ragweed pollen is the main cause of allergenic diseases in Northern America, and the weed has become a spreading neophyte in Europe. Climate change and air pollution are speculated to affect the allergenic potential of pollen. The objective of this study was to investigate the effects of NO2 , a major air pollutant, under controlled conditions, on the allergenicity of ragweed pollen. Ragweed was exposed to different levels of NO2 throughout the entire growing season, and its pollen further analysed. Spectroscopic analysis showed increased outer cell wall polymers and decreased amounts of pectin. Proteome studies using two-dimensional difference gel electrophoresis and liquid chromatography-tandem mass spectrometry indicated increased amounts of several Amb a 1 isoforms and of another allergen with great homology to enolase Hev b 9 from rubber tree. Analysis of protein S-nitrosylation identified nitrosylated proteins in pollen from both conditions, including Amb a 1 isoforms. However, elevated NO2 significantly enhanced the overall nitrosylation. Finally, we demonstrated increased overall pollen allergenicity by immunoblotting using ragweed antisera, showing a significantly higher allergenicity for Amb a 1. The data highlight a direct influence of elevated NO2 on the increased allergenicity of ragweed pollen and a direct correlation with an increased risk for human health.

Ragweed (Ambrosia artemisiifolia) pollen allergenicity: SuperSAGE transcriptomic analysis upon elevated CO2 and drought stress.

BACKGROUND: Pollen of common ragweed (Ambrosia artemisiifolia) is a main cause of allergic diseases in Northern America. The weed has recently become spreading as a neophyte in Europe, while climate change may also affect the growth of the plant and additionally may also influence pollen allergenicity. To gain better insight in the molecular mechanisms in the development of ragweed pollen and its allergenic proteins under global change scenarios, we generated SuperSAGE libraries to identify differentially expressed transcripts. RESULTS: Ragweed plants were grown in a greenhouse under 380 ppm CO2 and under elevated level of CO2 (700 ppm). In addition, drought experiments under both CO2 concentrations were performed. The pollen viability was not altered under elevated CO2, whereas drought stress decreased its viability. Increased levels of individual flavonoid metabolites were found under elevated CO2 and/or drought. Total RNA was isolated from ragweed pollen, exposed to the four mentioned scenarios and four SuperSAGE libraries were constructed. The library dataset included 236,942 unique sequences, showing overlapping as well as clear differently expressed sequence tags (ESTs). The analysis targeted ESTs known in Ambrosia, as well as in pollen of other plants. Among the identified ESTs, those encoding allergenic ragweed proteins (Amb a) increased under elevated CO2 and drought stress. In addition, ESTs encoding allergenic proteins in other plants were also identified. CONCLUSIONS: The analysis of changes in the transcriptome of ragweed pollen upon CO2 and drought stress using SuperSAGE indicates that under global change scenarios the pollen transcriptome was altered, and impacts the allergenic potential of ragweed pollen.

Development of a simple PCR-based assay for the identification of triazine resistance in the noxious plant common ragweed (Ambrosia artemisiifolia) and its applicability in higher plants.

Bidirectional allele-specific PCR (Bi-PASA) was used to detect a point mutation causing triazine resistance in common ragweed (Ambrosia artemisiifolia). The external primers amplified a 278 bp standard DNA fragment in all genotypes. In the susceptible S264S genotypes, a 208 bp fragment was expected while in resistant S264G common ragweed genotypes a 109 bp band was expected. In resistant plants, both the wild and mutant type fragments were detected, indicating that the original triazine sensitive cpDNA is maintained in a heteroplasmic state in the resistant S264G genotypes. Additionally, in silico analysis confirmed the potential applicability of our diagnostic assay for other plant species. In 24 out of 74 taxa (32%), the assay could be used without any change, while in the others some of the primers should be redesigned before use.

Ozone affects pollen viability and NAD(P)H oxidase release from Ambrosia artemisiifolia pollen.

Air pollution is frequently proposed as a cause of the increased incidence of allergy in industrialised countries. We investigated the impact of ozone (O(3)) on reactive oxygen species (ROS) and allergen content of ragweed pollen (Ambrosia artemisiifolia). Pollen was exposed to acute O(3) fumigation, with analysis of pollen viability, ROS and nitric oxide (NO) content, activity of nicotinamide adenine dinucleotide phosphate (NAD[P]H) oxidase, and expression of major allergens. There was decreased pollen viability after O(3) fumigation, which indicates damage to the pollen membrane system, although the ROS and NO contents were not changed or were only slightly induced, respectively. Ozone exposure induced a significant enhancement of the ROS-generating enzyme NAD(P)H oxidase. The expression of the allergen Amb a 1 was not affected by O(3), determined from the mRNA levels of the major allergens. We conclude that O(3) can increase ragweed pollen allergenicity through stimulation of ROS-generating NAD(P)H oxidase.

[Effects of nitrogen and carbon addition and arbuscular mycorrhiza on alien invasive plant Ambrosia artemisiifolia].

A greenhouse control experiment was conducted to explore the effects of nitrogen and carbon addition and arbuscular mycorrhiza (AM) on the growth of alien invasive plant Ambrosia artemisiifolia (common ragweed). Nitrogen addition had no significant effects on the morphological indices, biomass and its allocation, and absolute growth rate of A. artemisiifolia, but increased the nitrogen content in the aboveground and underground parts of the plant significantly. Carbon addition increased the content of soil available nitrogen. In this case, the biomass allocation in root system for nutrient (nitrogen) absorption promoted, resulting in a remarkable decrease of branch number, total leaf area, specific leaf area (SLA), and leaf mass ratio. As a result, the total biomass decreased significantly. The symbiosis of A. artemisiifolia and AM fungi had great influence on the common ragweed's soil nitrogen acclimation, which enhanced its resource-capture by the increase of SLA, and this effect was more significant when the soil nitrogen content was low. AM fungi played an important role in the growth of A. artemisiifolia in low-nitrogen environment.

Molecular characterization of atrazine resistance in common ragweed (Ambrosia artemisiifolia L.).

Common ragweed (Ambrosia artemisiifolia L.) is the most frequent weed in the Carpathian Basin and is spreading fast in other parts of Europe. In recent years, besides the wild type, a mutant genotype resistant to atrazine herbicides has evolved and is now widespread in many areas. The present study demonstrates that the atrazine resistance of ragweed is maternally inherited, and is caused by a point mutation in the psbA chloroplast gene. The promoter 5'-untranslated region and the open reading frame regions of the gene were analysed, and a homology search was performed. Both the atrazine-resistant and susceptible types of cpDNA were present in atrazine-resistant plants, while the mixed presence of both genotypes in the same plant, known as heteroplasmy, was not unequivocally detectable in susceptible plants.

[Comprehensive assessment on control measures of Ambrosia artemisiifolia L].

Three control measures of Ambrosia artemisiifolia, including biological control, chemical control, and CK without any treatment, were evaluated by analytic hierarchy process (AHP). Corresponding contributions of the three control measures to comprehensive profit (CP) and comprehensive cost (CC) were calculated and ranked, which were regarded as the assessment criteria of the control measures. The results showed that among the three control measures, biological control had the highest CP and the lowest CC, CK was in adverse; and chemical control was in intervenient. Biological control had the highest ratio of profit to cost, and suggesting that this control measure is an optimal and recommendable measure in controlling A. artemisiifolia.

Chemical control of ambrosia Artemisiifolia on non-crop areas: are there alternatives to glyphosate?

We compared glyphosate, glufosinate and metsulfuron-methyl to control Ambrosia artemisiifolia under non-crop conditions. A laboratory study showed that A. artemisiifolia is an easy-to-wet species and that glufosinate and glyphosate are quickly absorbed by its leaves (nearly 100% in 24 h). Metsulfuron-methyl absorption was slower (about 50% in 24 h) but was strongly promoted by terpenic alcohol and esterified rapeseed oil. In the greenhouse, all three herbicides were efficacious against A. artemisiifolia, with ED50s of <23, 23 and 0.8 g ha(-1) for glufosinate, glyphosate and metsulfuron-methyl, respectively. These results were confirmed on a non-crop area for glufosinate and glyphosate, which at half the registered dose reached high efficacies at both the 4 to 6-node and flowering stages of A. artemisiifolia. By contrast, metsulfuron-methyl showed no efficacy. However, after treatment at the 4- to 6-node stage, new emergence of A. artemisiifolia led to the presence of vigorous plants that bore numerous flowers and produced high levels of pollen. After treatment at the flowering stage, flower production by A. artemisiifolia was not significantly affected, but achene weight was decreased by 60 to 70% and seed viability was only 8 to 13% for the treated plants, as compared to 85% for the control. No significant difference was observed between the two herbicides and between the doses. It is concluded that glufosinate can be an alternative to glyphosate for the chemical control of A. artemisiifolia on non-crop areas. However, with both herbicides, it is difficult to attain the two objectives of reducing seed production and pollen production by means of only one treatment.

Weed control in sunflower (helianthus annuus L.) with post-emergent herbicides.

Sunflower is the most important oil crop in Hungary, is the base of the production of cooking oil and moreover takes an important part in production of margarine too. Extracted sunflower groats as a secondary product origining from the mentioned procedure can be used in forage successfully. The amount of harvested sunflower reaches the 20-25% of the EU's yield. The sowing area approaches 500 thousand hectares. The essential condition of successful crop production is the perfect weed control. Sowing areas are infected with monocotyledon and dicotyledonous weeds too. Annual dicotyledonous weeds are the most troublesome. The worst species is the Ambrosia artemisiifolia L. Many other weed species as Abutilon theophrasti MEDIC., Datura stramonium L. and Xanthium strumarium L. can cause serious damages. In our model experiments we examined the herbicide sensibility of two commercial sunflower cultivars as "Iregi szürke csikos", "Marica II" and a sulfonylurea-urea tolerant new hybrid "PR63E82". The experiment was set up under greenhouse conditions with the use of four important weed and different post-emergent herbicide as Modown 4F (bifenox), Pledge 50 WP (flumioxazin) and Granstar 75 DF (tribenuron-methyl). We applied normal and double doses too. Sunflower was cultivated to 4-6 leaf stage. Post-emergent herbicides were sprayed out when weeds were in 2, 2-4 and 4-6 leaf stage. Weed killer and phytotoxic effects of post-emergent herbicides were examined. We declared that development of weeds had significally effect on the effectiveness of different herbicides.