Heat Treatment of Seeds to Control Invasive Common Ragweed (Ambrosia artemisiifolia), Narrow-Leaved Ragwort (Senecio inaequidens) and Giant Hogweed (Heracleum mantegazzianum).

The intended or unintentional transport of soil material contaminated with weed seeds is one of the most important drivers in the spreading dynamics of invasive alien plants (IAPs). This phenomenon can be observed at any kind of construction site. Typical transfer of soil contaminated with IAP seeds can be observed along with road construction (soil translocation) or road maintenance services (deposit of mown plant biomass). Thus, an effective inactivation of these seeds by heating can avoid the spread of IAPs substantially. In the present study, the effects of various thermal control techniques (dry air heating and wet heating with hot steam, hot water, and hot foam) on seed survival of the widespread European IAPs common ragweed (Ambrosia artemisiifolia), narrow-leaved ragwort (Senecio inaequidens), and giant hogweed (Heracleum mantegazzianum) are discussed. Dry and wet seeds which were either uncovered or covered with soil were tested for survival at different treatment temperatures and different exposure times. Results revealed that particularly dry seeds of all three species could withstand temperatures of 100 °C for at least 6 h in climate chambers. Dry seeds of common ragweed and narrow-leaved ragwort survived exposure times of up to 48 h. Wet seeds were significantly more susceptible to heat treatments. Giant hogweed seeds were completely killed after 12 h at 70 °C. The exposure of IAP seeds to hot water was generally more effective than the treatment with hot steam. The treatment with hot foam was only effective when seeds were lying unprotected on the soil surface. Dry seeds of all the three species survived hot foam application in the field when they were covered by vegetation and leaf litter or soil. Due to the robustness of the seeds, a preventive management of IAPs by an efficient control before seeds formation is substantial to avoid their further dispersal.

Extracts and Residues of Common Ragweed (Ambrosia artemisiifolia L.) Cause Alterations in Root and Shoot Growth of Crops.

Following the novel weapon hypothesis, the invasiveness of non-native species, such as common ragweed (Ambrosia artemisiifolia L.) can result from a loss of natural competitors due to the production of chemical compounds, which negatively affect native communities. Particularly the genus Ambrosia produces several types of organic compounds, which have the potential to inhibit germination and growth of other plants. Subsequent to an assessment of the chemical content of three different ragweed extracts (aqueous shoot and root extracts, as well as essential oil), two different trials on the effects of different concentrations of these extracts, as well as ragweed residues, were conducted on two different mediums (Petri dish vs. soil). In addition, we investigated the impact on the infection potential of Bradyrhizobium japonicum on soybean roots in three different soil types (arable soil, potting soil, and sand). The results showed that the exposure to common ragweed extracts and residues induced changes in the biomass and root production of crops and ragweed itself. Even though crops and ragweed differed in their response behavior, the strongest negative impact on all crops and ragweed was observed with ragweed residues, leading to reductions in biomass and root growth of up to 90%. Furthermore, we found a decrease in the number of rhizobial nodules of up to 48% when soybean was exposed to ragweed root extract.

Talking Different Languages: The Role of Plant-Plant Communication When an Invader Beats up a Strange Neighborhood.

Communication through airborne volatile organic compounds (VOCs) and root exudates plays a vital role in the multifarious interactions of plants. Common ragweed (Ambrosia artemesiifolia L.) is one of the most troublesome invasive alien species in agriculture. Below- and aboveground chemical interactions of ragweed with crops might be an important factor in the invasive species' success in agriculture. In laboratory experiments, we investigated the contribution of intra- and interspecific airborne VOCs and root exudates of ragweed to its competitiveness. Wheat, soybean, and maize were exposed to VOCs emitted from ragweed and vice versa, and the adaptation response was measured through plant morphological and physiological traits. We observed significant changes in plant traits of crops in response to ragweed VOCs, characterized by lower biomass production, lower specific leaf area, or higher chlorophyll contents. After exposure to ragweed VOCs, soybean and wheat produced significantly less aboveground dry mass, whereas maize did not. Ragweed remained unaffected when exposed to VOCs from the crops or a conspecific. All crops and ragweed significantly avoided root growth toward the root exudates of ragweed. The study shows that the plant response to either above- or belowground chemical cues is highly dependent on the identity of the neighbor, pointing out the complexity of plant-plant communication in plant communities.

Effect of Two Seeding Rates on Nitrogen Yield and Nitrogen Fixation of Winter and Spring Faba Bean.

Faba bean (Vicia faba L. minor) is an important grain legume and is widely used as food and feed. It is traditionally used as a spring crop in Central European cropping systems. There is increasing interest in winter faba bean due to a higher yield potential, but limited knowledge of nitrogen (N) yields and nitrogen fixation (NFIX) exists. Therefore, the purpose of this study was to compare N concentrations, N yield of plant fractions, soil mineral N (SMN) and SMN sparing in the soil after harvest, NFIX and N balance of two winter faba bean varieties (Diva and Hiverna) to those of a spring faba bean (Alexia) using two seeding rates (25 versus 50 germinable seeds m-2) in a two-year field experiment under Pannonian climate conditions in eastern Austria. The winter faba bean varieties had higher N yields and NFIX, not only due to higher biomass yields, but also due to higher N concentrations and a higher percentage of N derived from atmosphere in the biomass. Conversely, the soil mineral N after harvest was lower compared to the spring faba bean. All treatments had a negative N balance due to higher grain N yield than NFIX. Winter faba beans left higher amounts of biologically-fixed N in residues for the subsequent crop, whereas spring faba bean left more SMN. Winter faba bean varieties obtained good results with both seeding rates, whereas the grain yield and the grain N yield of Alexia tended to higher with the higher seeding rate.

The management success of the invasive late goldenrod (Solidago gigantea Aiton.) in a nature conservation area is strongly related to site, control measures and environmental factors.

The late goldenrod (Soldiago gigantea Aiton; Asteraceae) is one of the most abundant invasive species in various types of habitats. Its long-creeping plagiotropic rhizomes enable the plant to build up dense, monospecific stands within a short time. Particularly in nature conservation areas, the invasion of goldenrod can cause severe disruptions in the naturally occuring mutualims between plants, insects and higher trophic levels, subsequently impeding the achievement of nature conservation goals. As management options of goldenrod in nature conservation areas are limited, this three-year study aimed to test the effectiveness of three management treatments (two-time mowing, triticale cultivation, and reverse rotary cutting) on four different sites in the Austrian Donau-Auen National Park. The number and height of goldenrod shoots were recorded three times a year on twelve permanent trial plots on each site to test for the effectiveness of the treatments. In addition, vegetation surveys were performed to observe the recovery potential of native plant species. Even though the three-years mowing and the triticale cultivation reduced goldenrod by 95.6% and 97.2% resp., we could find no relation between the effectiveness of the treatment and the intensity of disturbance created by the control option. On the contrary, with a reduction of only 5.4% in goldenrod density the most intensive treatment, the rotary cutting, showed the lowest efficiency. The highest positive effect on the re-establishment of native plant species was recorded with two mowing events per year. Even though the study revealed that certain management options have the potential to effectively reduce goldenrod and to simultaneously increase the establishment success of native species, results can only be seen as so-called snapshots. For example, as shown on site EJW one unforeseeable wild boar digging event transformed a 84.5% reduction into a 4.7% increase in goldenrod density. Therefore, a proper and regular monitoring is essential to be able to react to the effects of unpredictable events that can have severe impact on vegetation dynamics.

Exchangeable and Plant-Available Macronutrients in a Long-Term Tillage and Crop Rotation Experiment after 15 Years.

The status of macronutrients phosphorus (P), potassium (K), sulphur (S), calcium (Ca) and magnesium (Mg) was assessed 15 years after the establishment of a long-term crop rotation and soil tillage trial with mouldboard ploughing (MP), no-till (NT), deep conservation tillage (CTd) and shallow conservation tillage (CTs). The mobile proportions of macronutrients in an Austrian Chernozem soil were determined to a depth of 50 cm with the single reagent extractant acetic acid (AA) and Mehlich 3 (M3), which uses several reagents as extractants. AA revealed less P and K, but more Ca and Mg compared to M3. Both extractants could capture the distribution pattern of the nutrients in the soil profile, but M3 showed higher differences among the soil layers. In the first 5 cm in NT, the P concentration was higher than in MP, CTd and CTs. The concentration of K was higher in NT, CTd and CTs than in MP in the first 10 cm of the soil. Phosphorus and K concentrations did not differ between tillage treatments below these soil layers, and S, Ca and Mg were similar in all soil layers. As none of the analysed elements except for Ca were fertilized and no accumulation of S, Ca and Mg was observed in the upper soil layer, the higher concentrations are attributed to accumulation through crop residues and then less leaching of P and K. Crop rotation did not affect the distribution of the analysed macronutrients in the soil but affected the nutrient uptake by winter wheat mostly due to the yield differences of winter wheat in the two crop rotations.

Plastid expression of a double-pentameric vaccine candidate containing human papillomavirus-16 L1 antigen fused with LTB as adjuvant: transplastomic plants show pleiotropic phenotypes.

Human papillomavirus (HPV) causes cervical cancer in women worldwide, which is currently prevented by vaccines based on virus-like particles (VLPs). However, these vaccines have certain limitations in their availability to developing countries, largely due to elevated costs. Concerning the highest burden of disease in resource-poor countries, development of an improved mucosal and cost-effective vaccine is a necessity. As an alternative to VLPs, capsomeres have been shown to be highly immunogenic and can be used as vaccine candidate. Furthermore, coupling of an adjuvant like Escherichia coli heat-labile enterotoxin subunit B (LTB) to an antigen can increase its immunogenicity and reduce the costs related to separate co-administration of adjuvants. Our study demonstrates the expression of two pentameric proteins: the modified HPV-16 L1 (L1_2xCysM) and LTB as a fusion protein in tobacco chloroplasts. Homoplasmy of the transplastomic plants was confirmed by Southern blotting. Western blot analysis showed that the LTB-L1 fusion protein was properly expressed in the plastids and the recombinant protein was estimated to accumulate up to 2% of total soluble protein. Proper folding and display of conformational epitopes for both LTB and L1 in the fusion protein was confirmed by GM1-ganglioside binding assay and antigen capture ELISA, respectively. However, all transplastomic lines showed chlorosis, male sterility and growth retardation, which persisted in the ensuing four generations studied. Nevertheless, plants reached maturity and produced seeds by pollination with wild-type plants. Taken together, these results pave the way for the possible development of a low-cost adjuvant-coupled vaccine with potentially improved immunogenicity against cervical cancer.

Transplastomic expression of a modified human papillomavirus L1 protein leading to the assembly of capsomeres in tobacco: a step towards cost-effective second-generation vaccines.

Certain types of human papillomaviruses (HPV) are causatively associated with cervical carcinoma, the second most common cancer in women worldwide. Due to limitations in the availability of currently used virus-like particle (VLP)-based vaccines against HPV to women of developing countries, where most cases of cervical cancer occur, the development of a cost-effective second-generation vaccine is a necessity. Capsomeres have recently been demonstrated to be highly immunogenic and to have a number of advantages as a potential cost-effective alternative to VLP-based HPV vaccines. We have expressed a mutated HPV-16 L1 (L1_2xCysM) gene that retained the ability to assemble L1 protein to capsomeres in tobacco chloroplasts. The recombinant protein yielded up to 1.5% of total soluble protein. The assembly of capsomeres was examined and verified by cesium chloride density gradient centrifugation and sucrose sedimentation analysis. An antigen capture enzyme-linked immunosorbent assay confirmed the formation of capsomeres by using a conformation-specific monoclonal antibody which recognized the conformational epitopes. Transplastomic tobacco plants exhibited normal growth and morphology, but all such lines showed male sterility in the T0, T1 and T2 generations. Taken together, these results indicate the possibility of producing a low-cost capsomere-based vaccine by plastids.

Seed Metabolism and Pathogen Resistance Enhancement in Pisum sativum During Colonization of Arbuscular Mycorrhizal Fungi: An Integrative Metabolomics-Proteomics Approach.

Pulses are one of the most important categories of food plants, and Pea (Pisum sativum L.) as a member of pulses is considered a key crop for food and feed and sustainable agriculture. Integrative multi-omics and microsymbiont impact studies on the plant's immune system are important steps toward more productive and tolerant food plants and thus will help to find solutions against food poverty. Didymella pinodes is a main fungal pathogen of pea plants. Arbuscular mycorrhizal fungi (AMF) promote plant growth and alleviate various stresses. However, it remained unclear as to how the AMF effect on seed metabolism and how this influences resistance against the pathogen. This study assesses the AMF impacts on yield components and seed quality upon D. pinodes infection on two different P. sativum cultivars, susceptible versus tolerant, grown in pots through phenotypic and seed molecular analyses. We found that AMF symbiosis affects the majority of all tested yield components as well as a reduction of disease severity in both cultivars. Seeds of mycorrhizal pea plants showed strong responses of secondary metabolites with nutritional, medicinal, and pharmaceutical attributes, also involved in pathogen response. This is further supported by proteomic data, functionally determining those primary and secondary metabolic pathways, involved in pathogen response and induced upon AMF-colonization. The data also revealed cultivar specific effects of AMF symbiosis that increase understanding of genotype related differences. Additionally, a suite of proteins and secondary metabolites are presented, induced in seeds of P. sativum upon AMF-colonization and pathogen attack, and possibly involved in induced systemic resistance against D. pinodes, useful for modern breeding strategies implementing microsymbionts toward increased pathogen resistance.

Key metabolic traits of Pisum sativum maintain cell vitality during Didymella pinodes infection: cultivar resistance and the microsymbionts' influence.

Ascochyta blight causes severe losses in field pea production and the search for resistance traits towards the causal agent Didymella pinodes is of particular importance for farmers. Various microsymbionts have been reported to shape the plants' immune response. However, regardless their contribution to resistance, they are hardly included in experimental designs. We delineate the effect of symbionts (rhizobia, mycorrhiza) on the leaf proteome and metabolome of two field pea cultivars with varying resistance levels against D. pinodes and, furthermore, show cultivar specific symbiont colonisation efficiency. The pathogen infection showed a stronger influence on the interaction with the microsymbionts in the susceptible cultivar, which was reflected in decreased nodule weight and root mycorrhiza colonisation. Vice versa, symbionts induced variation of the host's infection response which, however, was overruled by genotypic resistance associated traits of the tolerant cultivar such as maintenance of photosynthesis and provision of sugars and carbon back bones to fuel secondary metabolism. Moreover, resistance appears to be linked to sulphur metabolism, a functional glutathione-ascorbate hub and fine adjustment of jasmonate and ethylene synthesis to suppress induced cell death. We conclude that these metabolic traits are essential for sustainment of cell vitality and thus, a more efficient infection response. SIGNIFICANCE: The infection response of two Pisum sativum cultivars with varying resistance levels towards Didymella pinodes was analysed most comprehensively at proteomic and metabolomic levels. Enhanced tolerance was linked to newly discovered cultivar specific metabolic traits such as hormone synthesis and presumably suppression of cell death.

Rhizobium Impacts on Seed Productivity, Quality, and Protection of Pisum sativum upon Disease Stress Caused by Didymella pinodes: Phenotypic, Proteomic, and Metabolomic Traits.

In field peas, ascochyta blight is one of the most common fungal diseases caused by Didymella pinodes. Despite the high diversity of pea cultivars, only little resistance has been developed until to date, still leading to significant losses in grain yield. Rhizobia as plant growth promoting endosymbionts are the main partners for establishment of symbiosis with pea plants. The key role of Rhizobium as an effective nitrogen source for legumes seed quality and quantity improvement is in line with sustainable agriculture and food security programs. Besides these growth promoting effects, Rhizobium symbiosis has been shown to have a priming impact on the plants immune system that enhances resistance against environmental perturbations. This is the first integrative study that investigates the effect of Rhizobium leguminosarum bv. viceae (Rlv) on phenotypic seed quality, quantity and fungal disease in pot grown pea (Pisum sativum) cultivars with two different resistance levels against D. pinodes through metabolomics and proteomics analyses. In addition, the pathogen effects on seed quantity components and quality are assessed at morphological and molecular level. Rhizobium inoculation decreased disease severity by significant reduction of seed infection level. Rhizobium symbiont enhanced yield through increased seed fresh and dry weights based on better seed filling. Rhizobium inoculation also induced changes in seed proteome and metabolome involved in enhanced P. sativum resistance level against D. pinodes. Besides increased redox and cell wall adjustments light is shed on the role of late embryogenesis abundant proteins and metabolites such as the seed triterpenoid Soyasapogenol. The results of this study open new insights into the significance of symbiotic Rhizobium interactions for crop yield, health and seed quality enhancement and reveal new metabolite candidates involved in pathogen resistance.

A Proteomic Workflow Using High-Throughput De Novo Sequencing Towards Complementation of Genome Information for Improved Comparative Crop Science.

The proteomic study of non-model organisms, such as many crop plants, is challenging due to the lack of comprehensive genome information. Changing environmental conditions require the study and selection of adapted cultivars. Mutations, inherent to cultivars, hamper protein identification and thus considerably complicate the qualitative and quantitative comparison in large-scale systems biology approaches. With this workflow, cultivar-specific mutations are detected from high-throughput comparative MS analyses, by extracting sequence polymorphisms with de novo sequencing. Stringent criteria are suggested to filter for confidential mutations. Subsequently, these polymorphisms complement the initially used database, which is ready to use with any preferred database search algorithm. In our example, we thereby identified 26 specific mutations in two cultivars of Pisum sativum and achieved an increased number (17 %) of peptide spectrum matches.

Dissection of drought response of modern and underutilized wheat varieties according to Passioura's yield-water framework.

Trait-based breeding is essential to improve wheat yield, particularly when stress adaptation is targeted. A set of modern and underutilized wheat genotypes was examined in a 2-year field experiment with distinct seasonal water supply. Yield formation and drought response strategies were analyzed in relation to components of Passioura's yield-water framework based on phenological, morphological, physiological, and root characteristics. Limited water supply resulted in 60% yield loss and substantially lower water use (37%), water use efficiency (32.6%), and harvest index (14%). Phenology and root length density were key determinants of water use. Late flowering underutilized wheat species with large root system and swift ground coverage showed greatest water use. Leaf chlorophyll concentration and stomata conductance were higher in modern cultivars, supporting their high biomass growth and superior water use efficiency. While, lower chlorophyll concentration and stomata conductance of underutilized wheats indicated a water saving strategy with an intrinsic limitation of potential growth. Harvest index was strongly dependent on phenology and yield components. Optimized flowering time, reduced tillering, and strong grain sink of modern cultivars explained higher harvest index compared to underutilized wheats. Cluster analysis revealed the consistent differentiation of underutilized and modern wheats based on traits underlying Passioura's yield-water framework. We identified physiological and root traits within modern cultivars to be targeted for trait-based crop improvement under water-limited conditions. High capacity of water use in underutilized genetic resources is related to yield-limiting phenological and morphological traits, constraining their potential role for better drought resistance. Still some genetic resources provide adaptive features for stress resistance compatible with high yield as revealed by high harvest index under drought of Khorasan wheat.

A statistical approach to root system classification.

Plant root systems have a key role in ecology and agronomy. In spite of fast increase in root studies, still there is no classification that allows distinguishing among distinctive characteristics within the diversity of rooting strategies. Our hypothesis is that a multivariate approach for "plant functional type" identification in ecology can be applied to the classification of root systems. The classification method presented is based on a data-defined statistical procedure without a priori decision on the classifiers. The study demonstrates that principal component based rooting types provide efficient and meaningful multi-trait classifiers. The classification method is exemplified with simulated root architectures and morphological field data. Simulated root architectures showed that morphological attributes with spatial distribution parameters capture most distinctive features within root system diversity. While developmental type (tap vs. shoot-borne systems) is a strong, but coarse classifier, topological traits provide the most detailed differentiation among distinctive groups. Adequacy of commonly available morphologic traits for classification is supported by field data. Rooting types emerging from measured data, mainly distinguished by diameter/weight and density dominated types. Similarity of root systems within distinctive groups was the joint result of phylogenetic relation and environmental as well as human selection pressure. We concluded that the data-define classification is appropriate for integration of knowledge obtained with different root measurement methods and at various scales. Currently root morphology is the most promising basis for classification due to widely used common measurement protocols. To capture details of root diversity efforts in architectural measurement techniques are essential.