Evaluation of physiological and molecular effect of variable virulence of Alternaria brassicae isolates in Brassica juncea, Sinapis alba and Camelina sativa.

Brassica genus comprises many prominent species valuable for human nutrition including vegetable crops and oilseed. Production of B. juncea is challenged by many abiotic and biotic stresses, Alternaria blight caused by a necrotrophic fungal pathogen Alternaria brassicae is one of the most prominent diseases of cruciferous crops including B. juncea. However, some closely related wild species like Sinapis alba and Camelina sativa exhibit a variable level of resistance towards the pathogen. Apart from the host resistance, intra-specific pathogen variability also influences disease severity to a larger extent. In this study, we identified and isolated two strains of A. brassicae viz ABS1 and ABS2 exhibiting morphological and pathological variability. These isolates were further used to artificially inoculate B. juncea and two of its wild relatives under in-vitro as well as in-vivo conditions to inspect their pathogenicity in a susceptible, a moderately resistant and a highly resistant host. virulent isolate (ABS2) was able to readily establish infection in all the three species whereas the less virulent isolate (ABS1) readily infected susceptible species B. juncea but delayed and mild infection was noticed in tolerant hosts. Variable physiological and molecular host response towards the differential level of virulence of pathogen were established with many confirmatory experiments like DAB staining study, Disease severity index and microscopic analysis. Real-time PCR results confirm that these two isolates induce a variable level of induction in genes PR1 and PDF1.2 within 48 h of the artificial inoculation in B. juncea and its wild relatives.

Gene expression, DNA damage and other stress markers in Sinapis alba L. exposed to heavy metals with special reference to sewage sludge application on contaminated sites.

Bioindicators are promising tools used to detect the long-term effects of selected biosolids on plants development and should be implemented before large-scale supplementation of sewage sludge into the soil. The presented study shows the impact of sewage sludge application on metal-sensitive toxicity biological parameters (biomarkers) in Sinapis alba including: germination, root length, the activity of guaiacol peroxidase, the chlorophyll content, the level of DNA damage and the expression level of Ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL) and metallothionein (mt). We evaluated data from selected biomarkers in order to broaden our understanding of plants defense mechanisms against heavy metal contamination and the application of sewage sludge into soils. Overall, in contaminated soil after supplementation with both municipal sewage sludges, an increase in toxicity was noticed in DNA damage, mt and rbcl expression and total chlorophyll content. The supplementation of both soils with municipal sewage sludge caused a two-time induction in the mt expression. Moreover, clean soil supplemented with sewage sludge caused an increase in DNA damage shown as the tail moment from approximately 12 µm on control to 40 µm after supplementation. Even if those biosolids increased the initial germination, roots length, and biomass in comparison to the unamended soil, the toxicity was evidenced with other stress markers. Results showed, that in order to accurately assess the influence of sewage sludge application on plants the use of several specific biomarkers is required for safe land restoration. The conducted study also confirmed, both under biochemical and genotoxic tests, that iron enrichment for biosolids or contaminated soil can significantly reduce the bioavailability and toxicity of other metals.

Evaluating resistant brassica trap crops to manage Heterodera schachtii (Schmidt) infestations in eastern England.

BACKGROUND: The beet cyst nematode (BCN), Heterodera schachtii Schmidt, is a plant parasitic nematode that causes severe yield losses in sugar beet. Resistant brassicas (radish and mustard) have been bred to be planted after harvest of the main crop, for example a cereal, and encourage BCN juvenile hatching. The resistant plants stimulate juvenile hatching but are not suitable hosts. Juveniles are unable to complete their lifecycle and thus populations are lowered. This research aimed to investigate the effectiveness of a range of brassicas in terms of BCN control when grown in infested fields in eastern England. RESULTS: Experiments used four different radish cultivars, which differed in their resistance to BCN, and one resistant mustard variety. Field experiments were sown in early September in 2016 and 2017. Significant reductions in BCN populations were found only following resistant mustard and the radish with the greatest resistance level. CONCLUSIONS: Further research is needed to understand how best to utilize brassicas and whether they are economically viable when alternative management options for BCN are available. Time of planting may be crucial to fully achieve their BCN-reducing potential. © 2018 Society of Chemical Industry.

Comprehensive analysis of fly ash induced changes in physiological/growth parameters, DNA damage and oxidative stress over the life cycle of Brassica juncea and Brassica alba.

Fly ash (FA) being a heterogeneous mixture of heavy metal affects plant system in various ways. Previous studies have shown bioaccumulation of toxic metals in the plants and disturbance in cellular activities. Here, we have studied the impacts of FA treatment through the life cycle of economically important, annual crop plant mustard (Brassica juncea and Brassica alba). Result revealed that FA did not alter germination rate and photosynthetic pigment levels. Tolerance index of B. juncea was higher compared to B. alba. Seed setting was significantly affected by FA in B. alba. Significant increase in DNA damage was observed in both B. alba and B. juncea. Proline accumulation was significantly higher in B. alba. In B. juncea catalase activity and reduced glutathione content declined in initial days which were restored at the end of experimental period. Significant decrease in non-enzymatic antioxidants was noted in B. alba. Higher accumulation of Pb and As was noted in shoot of B. juncea and in B. alba Cu, Pb, Cr and As accumulated in shoots. As observed from these results, both plants could translocate certain toxic heavy metals from roots to the shoot which affected the physiological and biochemical balance and induced genotoxic response.

Genetic and environmental determinants of unreduced gamete production in Brassica napus, Sinapis arvensis and their hybrids.

Unreduced gametes, sperm or egg cells with the somatic chromosome number, are an important mechanism of polyploid formation and gene flow between heteroploid plants. The meiotic processes leading to unreduced gamete formation are well documented, but the relative influence of environmental and genetic factors on the frequency of unreduced gametes remain largely untested. Furthermore, direct estimates of unreduced gametes based on DNA content are technically challenging and, hence, uncommon. Here, we use flow cytometry to measure the contribution of genetic (hybridization) and environmental (nutrient limitation, wounding) changes to unreduced male gamete production in Brassica napus, Sinapis arvensis and two hybrid lines. Treatments were applied to greenhouse grown plants in a random factorial design, with pollen sampled at two time intervals. Overall, the frequency of unreduced gametes averaged 0.59% (range 0.06-2.17%), plus a single outlier with 27%. Backcrossed hybrids had 39 to 75% higher unreduced gamete production than parental genotypes, averaged across all treatments, although the statistical significance of these differences depended on sampling period and wounding treatment. Unreduced gamete frequencies were higher for the second sampling period than the first. There were no direct effects of wounding or nutrient regime. Our results indicate that both genetic and environmental factors can induce increased unreduced gametes, highlighting the potential importance of environmental heterogeneity and genetic composition of populations in driving polyploid evolution.

Time-invariant differences between plant individuals in interactions with arthropods correlate with intraspecific variation in plant phenology, morphology and floral scent.

The basic units of ecological and evolutionary processes are individuals. Network studies aiming to infer mechanisms from complex systems, however, usually focus on interactions between species, not individuals. Accordingly, the structure and underlying mechanisms of individual-based interaction networks remain largely unknown. In a common garden, we recorded all interactions on flowers and leaves of 97 Sinapis arvensis individuals from seedling stage to fruit set and related interindividual differences in interactions to the plant individuals' phenotypes. The plant individuals significantly differed in their quantitative and qualitative interactions with arthropods on flowers and leaves. These differences remained stable over the entire season and thus were time-invariant. Variation in interacting arthropod communities could be explained by a pronounced intraspecific variability in flowering phenology, morphology and flower scent, and translated into variation in reproductive success. Interestingly, plant individuals with a similar composition of flower visitors were also visited by a similar assemblage of interaction partners at leaves. Our results show that the nonuniformity of plant species has pronounced effects in community ecology, potentially with implications for the persistence of communities and populations, and their ability to withstand environmental fluctuations.

Bidirectional but asymmetrical sexual hybridization between Brassica carinata and Sinapis arvensis (Brassicaceae).

With transgenic crop development it is important to evaluate the potential for transgenes to escape into populations of wild, weedy relatives. Ethiopian mustard (Brassica carinata, BBCC) is easily transformed and is being investigated for uses from biodiesel fuels to biopharmaceuticals. However, little work has been done evaluating its ability to cross with relatives such as wild mustard (Sinapsis arvensis, SrSr), an abundant, cosmopolitan weedy relative. Here we conducted bidirectional crosses with Ethiopian mustard as a maternal parent in 997 crosses and paternal parent in 1,109 crosses. Hybrids were confirmed using flow cytometry and species-specific ITS molecular markers and indicate a high hybridization rate of 6.43 % between Ethiopian mustard (♀) and wild mustard (♂) and a lower, but not insignificant, hybridization rate of 0.01 % in the reverse direction. The majority of the hybrids were homoploid (BCSr) with less than 1 % of pollen production of their parents and low seed production (0.26 seeds/pollination) in crosses and backcrosses indicating a potential for advanced generation hybrids. The accession used had a significant effect on hybrid seed production with different accessions of Ethopian mustard varying in their production of hybrid offspring from 2.69 to 16.34 % and one accession of wild mustard siring almost twice as many hybrid offspring per flower as the other. One pentaploid (BBCCSr) and one hexaploid (BBCCSrSr) hybrid were produced and had higher pollen viability, though no and low seed production, respectively. As wild mustard is self-incompatible and the outcrossing rate of Ethiopian mustard has been estimated as 30 % potential for hybrid production in the wild appears to be high, though the hybridization rate found here represents a worst case scenario as it does not incorporate pre-pollination barriers. Hybridization in the wild needs to be directly evaluated as does the propensity of Ethiopian mustard to volunteer.

Integrating temperature and nutrition--environmental impacts on an insect immune system.

Globally increasing temperatures may strongly affect insect herbivore performance. In contrast to direct effects of temperature on herbivores, indirect effects mediated via thermal effects on host-plant quality are only poorly understood, despite having the potential to substantially impact the herbivores' performance. Part of this performance is the organisms' immune system which may be of pivotal importance for local survival. We here use a full-factorial design to explore the direct (larvae were reared at 17°C or 25°C) and indirect effects (host plants were reared at 17°C or 25°C) of temperature on immune function of the temperate-zone butterfly Pieris napi. At the higher rearing temperature haemocyte numbers and prophenoloxidase activity were reduced. Plant temperature, in contrast, did not affect immune competence despite clear effects on insect growth patterns. Overall, thermal and dietary impacts on the insects' immune responses were weak and trait-specific.

An evolutionarily conserved signaling mechanism mediates far-red light responses in land plants.

Phytochromes are plant photoreceptors important for development and adaptation to the environment. Phytochrome A (PHYA) is essential for the far-red (FR) high-irradiance responses (HIRs), which are of particular ecological relevance as they enable plants to establish under shade conditions. PHYA and HIRs have been considered unique to seed plants because the divergence of seed plants and cryptogams (e.g., ferns and mosses) preceded the evolution of PHYA. Seed plant phytochromes translocate into the nucleus and regulate gene expression. By contrast, there has been little evidence of a nuclear localization and function of cryptogam phytochromes. Here, we identified responses to FR light in cryptogams, which are highly reminiscent of PHYA signaling in seed plants. In the moss Physcomitrella patens and the fern Adiantum capillus-veneris, phytochromes accumulate in the nucleus in response to light. Although P. patens phytochromes evolved independently of PHYA, we have found that one clade of P. patens phytochromes exhibits the molecular properties of PHYA. We suggest that HIR-like responses had evolved in the last common ancestor of modern seed plants and cryptogams and that HIR signaling is more ancient than PHYA. Thus, other phytochromes in seed plants may have lost the capacity to mediate HIRs during evolution, rather than that PHYA acquired it.

Conspecific flowers of Sinapis arvensis are stronger competitors for pollinators than those of the invasive weed Bunias orientalis.

Biological invasions can affect the structure and function of ecosystems and threaten native plant species. Since most weeds rely on mutualistic relationships in their new environment, they may act as new competitors for pollinators. Pollinator competition is likely to be density dependent, but it is often difficult to disentangle competition caused by flower quality from effects caused by flower quantity. In order to test the effects of the presence and number of flowers of the invasive weed Bunias orientalis on the insect visitation rates in a native species (Sinapis arvensis), we performed two replacement experiments using plants with standardised flower numbers. The visitation rates in S. arvensis were significantly higher than in B. orientalis and the number of insect visits dropped significantly with increasing density of S. arvensis flowers. These results suggest that intraspecific competition among flowers of S. arvensis is stronger than the competitive effect of alien flowers. As flowers of B. orientalis do not seem to distract visitors from S. arvensis, it is unlikely that pollinator competition between these two plant species plays a crucial role. However, it cannot be excluded that mass blossom stands of B. orientalis may distract flower visitors from nativespecies.

Larval performance of the mustard leaf beetle (Phaedon cochleariae, Coleoptera, Chrysomelidae) on white mustard (Sinapis alba) and watercress (Nasturtium officinale) leaves in dependence of plant exposure to ultraviolet radiation.

Short-term exposure to ambient or attenuated ultraviolet (UV) radiation resulted in shifts in plant metabolite concentrations of the Brassicaceae Sinapis alba and Nasturtium officinale. Leaf quality also varied between plant species and within species due to age. Larvae of the oligophagous leaf beetle Phaedon cochleariae were raised on these different host leaves, in order to investigate the effects of variable plant chemistry on this herbivore. The performance of P. cochleariae was influenced by chemical differences between and within plant species but it responded with high plasticity to plants stressed by ultraviolet radiation. Body mass increase and developmental times of larvae were exclusively affected by plant species and leaf-age. However, developmental differences were fully compensated in the pupal stage. We suggest that the plasticity of herbivores may depend on the degree of specialisation, and insect performance may not necessarily be altered by stress-induced host plants.

Vernalization-induced repression of FLOWERING LOCUS C stimulates flowering in Sinapis alba and enhances plant responsiveness to photoperiod.

* Of the Brassicaceae, Sinapis alba has been intensively studied as a physiological model of induction of flowering by a single long day (LD), while molecular-genetic analyses of Arabidopsis thaliana have disclosed complex interactions between pathways controlling flowering in response to different environmental cues, such as photoperiod and vernalization. The vernalization process in S. alba was therefore analysed here. * The coding sequence of S. alba SaFLC, which is orthologous to the A. thaliana floral repressor FLOWERING LOCUS C, was isolated and the transcript levels quantified in different conditions. * Two-week-old seedlings grown in noninductive short days (SDs) were vernalized for 1-6 wk. Down-regulation of SaFLC was already marked after 1 wk of cold but 2 wk was needed for a significant acceleration of flowering. Flower buds were initiated during vernalization. When vernalization was stopped after 1 wk, repression of SaFLC was not stable but a significant increase in plant responsiveness to 16-h LDs was observed when LDs followed immediately after the cold treatment. * These results suggest that vernalization does not only work when plants experience long exposure to cold during the winter: shorter cold periods might stimulate flowering of LD plants if they occur when photoperiod is increasing, such as in spring.

Olfactory responses to aphid and host plant volatile releases: (E)-beta-farnesene an effective kairomone for the predator Adalia bipunctata.

The volatiles released from several aphid and host plant species, alone or associated, were studied for their infochemical role in prey location. Using a four-arm olfactometer, the attraction of several combinations of three aphid (Myzus persicae, Acyrthosiphon pisum, and Brevicoryne brassicae) and three plant (Vicia faba, Brassica napus, and Sinapis alba) species toward Adalia bipunctata larvae and adults was observed. Both predatory larvae and adults were attracted only by A. pisum and M. persicae when they were crushed, whatever the host plant. (E)-beta-farnesene, the aphid alarm pheromone, was the effective kairomone for the ladybird. Plant leaves alone (V. faba, B. napus, and S. alba) or in association with nonstressed whole aphids (the three species) did not have any attraction for the predator. The B. brassicae specialist aphid is the only prey that was not attracted to A. bipunctata larvae and adults, even if they were crushed. Release of B. brassicae molecules similar to the host plant allelochemicals was demonstrated by GC-MS analysis. The lack of behavioral response of the ladybird at short distance toward the cruciferous specialist aphid was related only to the absence of (E)-beta-farnesene in the aphid prey volatile pattern.

Evolution in stressful environments II: adaptive value and costs of plasticity in response to low light in Sinapis arvensis.

Plants possess a remarkable capacity to alter their phenotype in response to the highly heterogeneous light conditions they commonly encounter in natural environments. In the present study with the weedy annual plant Sinapis arvensis, we (a) tested for the adaptive value of phenotypic plasticity in morphological and life history traits in response to low light and (b) explored possible fitness costs of plasticity. Replicates of 31 half-sib families were grown individually in the greenhouse under full light and under low light (40% of ambient) imposed by neutral shade cloth. Low light resulted in a large increase in hypocotyl length and specific leaf area (SLA), a reduction in juvenile biomass and a delayed onset of flowering. Phenotypic selection analysis within each light environment revealed that selection favoured large SLA under low light, but not under high light, suggesting that the observed increase in SLA was adaptive. In contrast, plasticity in the other traits measured was maladaptive (i.e. in the opposite direction to that favoured by selection in the low light environment). We detected significant additive genetic variance in plasticity in most phenotypic traits and in fitness (number of seeds). Using genotypic selection gradient analysis, we found that families with high plasticity in SLA had a lower fitness than families with low plasticity, when the effect of SLA on fitness was statistically kept constant. This indicates that plasticity in SLA incurred a direct fitness cost. However, a cost of plasticity was only expressed under low light, but not under high light. Thus, models on the evolution of phenotypic plasticity will need to incorporate plasticity costs that vary in magnitude depending on environmental conditions.