Anchoring alien chromosome segment substitutions bearing gene(s) for resistance to mustard aphid in Brassica juncea-B. fruticulosa introgression lines and their possible disruption through gamma irradiation.

KEY MESSAGE: Heavy doses of gamma irradiation can reduce linkage drag by disrupting large sized alien translocations and promoting exchanges between crop and wild genomes. Resistance to mustard aphid (Lipaphis erysimi) infestation was significantly improved in Brassica juncea through B. juncea-B. fruticulosa introgression. However, linkage drag caused by introgressed chromatin fragments has so far prevented the deployment of this resistance source in commercial cultivars. We investigated the patterns of donor chromatin segment substitutions in the introgression lines (ILs) through genomic in situ hybridization (GISH) coupled with B. juncea chromosome-specific oligonucleotide probes. These allowed identification of large chromosome translocations from B. fruticulosa in the terminal regions of chromosomes A05, B02, B03 and B04 in three founder ILs (AD-64, 101 and 104). Only AD-101 carried an additional translocation at the sub-terminal to intercalary position in both homologues of chromosome A01. We validated these translocations with a reciprocal blast hit analysis using shotgun sequencing of three ILs and species-specific contigs/scaffolds (kb sized) from a de novo assembly of B. fruticulosa. Alien segment substitution on chromosome A05 could not be validated. Current studies also endeavoured to break linkage drag by exposing seeds to a heavy dose (200kR) of gamma radiation. Reduction in the size of introgressed chromatin fragments was observed in many M3 plants. There was a complete loss of the alien chromosome fragment in one instance. A few M3 plants with novel patterns of chromosome segment substitutions displayed improved agronomic performance coupled with resistance to mustard aphid. SNPs in such genomic spaces should aid the development of markers to track introgressed DNA and allow application in plant breeding.

Exposure to (Z)-11-hexadecenal [(Z)-11-16:Ald] increases Brassica nigra susceptibility to subsequent herbivory.

It is well established that plants emit, detect and respond to volatile organic compounds; however, knowledge on the ability of plants to detect and respond to volatiles emitted by non-plant organisms is limited. Recent studies indicated that plants detect insect-emitted volatiles that induce defence responses; however, the mechanisms underlying this detection and defence priming is unknown. Therefore, we explored if exposure to a main component of Plutella xylostella female sex pheromone namely (Z)-11-hexadecenal [(Z)-11-16:Ald] induced detectable early and late stage defence-related plant responses in Brassica nigra. Exposure to biologically relevant levels of vapourised (Z)-11-16:Ald released from a loaded septum induced a change in volatile emissions of receiver plants after herbivore attack and increased the leaf area consumed by P. xylostella larvae. Further experiments examining the effects of the (Z)-11-16:Ald on several stages of plant defence-related responses showed that exposure to 100 ppm of (Z)-11-16:Ald in liquid state induced depolarisation of the transmembrane potential (Vm), an increase in cytosolic calcium concentration [Ca2+]cyt, production of H2O2 and an increase in expression of reactive oxygen species (ROS)-mediated genes and ROS-scavenging enzyme activity. The results suggest that exposure to volatile (Z)-11-16:Ald increases the susceptibility of B. nigra to subsequent herbivory. This unexpected finding, suggest alternative ecological effects of detecting insect pheromone to those reported earlier. Experiments conducted in vitro showed that high doses of (Z)-11-16:Ald induced defence-related responses, but further experiments should assess how specific the response is to this particular aldehyde.

Physiological change alters endophytic bacterial community in clubroot of tumorous stem mustard infected by Plasmodiophora brassicae.

BACKGROUND: Endophytic bacteria are considered as symbionts living within plants and are influenced by abiotic and biotic environments. Pathogen cause biotic stress, which may change physiology of plants and may affect the endophytic bacterial communiy. Here, we reveal how endophytic bacteria in tumorous stem mustard (Brassica juncea var. tumida) are affected by plant physiological changes caused by Plasmodiophora brassicae using 16S rRNA high-throughput sequencing. RESULTS: The results showed that Proteobacteria was the dominant group in both healthy roots and clubroots, but their abundance differed. At the genus level, Pseudomonas was dominant in clubroots, whereas Rhodanobacter was the dominant in healthy roots. Hierarchical clustering, UniFrac-weighted principal component analysis (PCA), non-metric multidimensional scaling (NMDS) and analysis of similarities (ANOSIM) indicated significant differences between the endophytic bacterial communities in healthy roots and clubroots. The physiological properties including soluble sugar, soluble protein, methanol, peroxidase (POD) and superoxide dismutase (SOD) significantly differed between healthy roots and clubroots. The distance-based redundancy analysis (db-RDA) and two-factor correlation network showed that soluble sugar, soluble protein and methanol were strongly related to the endophytic bacterial community in clubroots, whereas POD and SOD correlated with the endophytic bacterial community in healthy roots. CONCLUSIONS: Our results illustrate that physiologcial changes caused by P. brassicae infection may alter the endophytic bacterial community in clubroots of tumorous stem mustard.

Development of RNAi methods to control the harlequin bug, Murgantia histrionica.

The harlequin bug (HB), Murgantia histrionica, is a major pest of cabbage family plants throughout its range in the United States. RNA interference (RNAi) is a posttranscriptional gene silencing mechanism that is showing promise as a biopesticide due to the ability to target species-specific genes necessary for growth and/or survival with synthetic double-stranded RNA (dsRNA). In the present study, dsRNA stability assays revealed that nucleases present in the saliva of harlequin bugs did not rapidly degrade dsRNA. We tracked the movement and localization of radioactively labeled dsRNA in both mustard plant seedlings and harlequin bug nymphs that fed on treated host plants. Movement of 32 P-labeled-dsRNA from soil to plant and plant to insect was detected. The efficacy of RNAi in inducing mortality in harlequin bug adults and nymphs injected or fed with dsRNA targeting inhibitor of apoptosis (IAP), ATPase N2B (ATPase), serine/threonine-protein phosphatase PP1-ß catalytic subunit (PP1), signal recognition particle 54 kDa protein (SRP), and G protein-coupled receptor 161-like (GPCR) genes was evaluated. Injection of dsRNA targeting candidate genes into adults caused between 40% and 75% mortality and induced significant knockdown of target gene expression. Feeding dsRNA targeting the IAP gene to nymphs by plant-mediated and droplet feeding methods induced knockdown of the target gene and caused 40-55% mortality. These findings suggest that RNAi may be a viable approach for managing this pest.

The optimal mixing ratio of Brassica napus and Brassica juncea meal improve nematode Meloidogyne hapla effects.

The use of rapeseed (Brassica napus L.) or leaf mustard (Brassica juncea L. Czern) meal or both as organic fertilizer not only improves the soil environment and crop productivity by supplying nutrients but also has nematicidal effects. This study aimed to establish the optimal application levels of rapeseed and leaf mustard meal for stronger nematode control in tomato. Tomato is one of the most important solanaceous crops which is severely damaged by nematodes. At first, meal (120 g of varying mixing ratios of rapeseed and leaf mustard meal) was mixed with sterilized soil (1 kg). The optimal ratio of rapeseed:leaf mustard meal for effective nematode control was 20:100 g/kg of soil. Progoitrin and gluconapin were the most abundant glucosinolates found in rapeseed meal, while sinigrin was the most abundant in leaf mustard meal. The amount of sinigrin increased if the leaf mustard meal proportion increased in the meal mixture. Although the content of sinigrin in optimal ratio mixture of rapeseed and leaf mustard meal is lower than only leaf mustard meal, it is presumed that nematocidal effects of the mixture are better than that of the single component due to the high contents of progoitrin and gluconapin. So, we propose that rapeseed and leaf mustard meal mixture at an appropriate ratio can be used as an environmentally friendly nematocide.

Infection of Plasmodiophora brassicae changes the fungal endophyte community of tumourous stem mustard roots as revealed by high-throughput sequencing and culture-dependent methods.

Diverse fungal endophytes live in plants and are shaped by some abiotic and biotic stresses. Plant disease as particular biotic stress possibly gives an impact on the communities of fungal endophytes. In this study, clubroot disease caused by an obligate biotroph protist, Plasmodiophora brassicae, was considered to analyze its influence on the fungal endophyte community using an internal transcribed spacer (ITS) through high-throughput sequencing and culture-dependent methods. The results showed that the diversity of the endophyte community in the healthy roots was much higher than the clubroots. Ascomycota was the dominant group of endophytes (Phoma, Mortierella, Penicillium, etc.) in the healthy roots while P. brassicae was the dominant taxon in the clubroots. Hierarchical clustering, principal component analysis (PCA), principal coordinates analysis (PCoA) and analysis of similarities (ANOSIM) indicated significant differences between the endophyte communities in the healthy roots and clubroots. Linear discriminant analysis effect size (LefSe) analysis showed that the dominant genera could be regarded as potential biomarkers. The endophyte community in the healthy roots had a more complex network compared with the clubroots. Also, many plant pathogenic Fusarium were isolated from the clubroots by the culture-dependent method. The outcome of this study illustrates that P. brassicae infection may change the fungal endophyte community associated with the roots of tumourous stem mustard and facilitates the entry of soil pathogen into the roots.

Anaerobic Soil Disinfestation to Manage Soilborne Diseases in Muck Soil Vegetable Production Systems.

Anaerobic soil disinfestation (ASD) was evaluated as a tool for managing the root-knot nematode Meloidogyne hapla in lettuce (Lactuca sativa) and clubroot disease, caused by Plasmodiophora brassicae, in mustard greens (Brassica juncea) produced on Ohio muck soils in Huron and Stark Counties. In two consecutive years of field trials, wheat bran (20.2 Mg ha-1), molasses (10.1 Mg ha-1), and wheat bran (20.2 Mg ha-1) plus molasses (10.1 Mg ha-1) were assessed as ASD carbon sources and compared with nonamended controls. Data were collected from plants grown in the field and from plants grown in field-treated soils in growth chamber-based post-ASD bioassays. Anaerobic conditions developed in ASD-treated soils in both trial years, as indicated by polyvinyl chloride pipes painted with an iron oxide paint. Soil pH did not decrease during ASD at the Huron County site of the mustard greens clubroot trials in either trial year but soil pH decreased significantly during ASD in Stark County soils treated with ASD with either wheat bran or wheat bran plus molasses compared with control soils in both trial years. Impacts of ASD on plant biomass were inconsistent in direct field measurements; however, significantly higher biomasses were observed in lettuce and mustard greens grown in bioassay soils collected from plots treated with ASD with wheat bran-based amendments compared with plants grown in soils from control plots. Based on direct field measurements and bioassays, the use of ASD with any carbon source led to significant reductions in root-knot nematode galling on lettuce compared with controls. Reductions in clubroot severity in mustard greens following ASD were less consistent; however, significant reductions in clubroot severity were observed in the field in one trial year and in both years of bioassays. The results of these studies indicate that ASD is a promising tool for managing soilborne diseases in muck soil vegetable production systems.

Expression of Colocasia esculenta tuber agglutinin in Indian mustard provides resistance against Lipaphis erysimi and the expressed protein is non-allergenic.

KEY MESSAGE: Transgenic Brassica juncea plants expressing Colocasia esculenta tuber agglutinin (CEA) shows the non-allergenic nature of the expressed protein leading to enhanced mortality and reduced fecundity of mustard aphid-Lipaphis erysimi. Lipaphis erysimi (common name: mustard aphid) is the most devastating sucking insect pest of Indian mustard (Brassica juncea L.). Colocasia esculenta tuber agglutinin (CEA), a GNA (Galanthus nivalis agglutinin)-related lectin has previously been reported by the present group to be effective against a wide array of hemipteran insects in artificial diet-based bioassays. In the present study, efficacy of CEA in controlling L. erysimi has been established through the development of transgenic B. juncea expressing this novel lectin. Southern hybridization of the transgenic plants confirmed stable integration of cea gene. Expression of CEA in T0, T1 and T2 transgenic plants was confirmed through western blot analysis. Level of expression of CEA in the T2 transgenic B. juncea ranged from 0.2 to 0.47% of the total soluble protein. In the in planta insect bioassays, the CEA expressing B. juncea lines exhibited enhanced insect mortality of 70-81.67%, whereas fecundity of L. erysimi was reduced by 49.35-62.11% compared to the control plants. Biosafety assessment of the transgenic B. juncea protein containing CEA was carried out by weight of evidence approach following the recommendations by FAO/WHO (Evaluation of the allergenicity of genetically modified foods: report of a joint FAO/WHO expert consultation, 22-25 Jan, Rome, http://www.fao.org/docrep/007/y0820e/y0820e00.HTM , 2001), Codex (Codex principles and guidelines on foods derived from biotechnology, Food and Agriculture Organization of the United Nations, Rome; Codex, Codex principles and guidelines on foods derived from biotechnology, Food and Agriculture Organization of the United Nations, Rome, 2003) and ICMR (Indian Council of Medical Research, guidelines for safety assessment of food derived from genetically engineered plants, http://www.icmr.nic.in/guide/Guidelines%20for%20Genetically%20Engineered%20Plants.pdf , 2008). Bioinformatics analysis, pepsin digestibility, thermal stability assay, immuno-screening and allergenicity assessment in BALB/c mice model demonstrated that the expressed CEA protein from transgenic B. juncea does not incite any allergenic response. The present study establishes CEA as an efficient insecticidal and non-allergenic protein to be utilized for controlling mustard aphid and similar hemipteran insects through the development of genetically modified plants.

Elucidating the interaction between light competition and herbivore feeding patterns using functional-structural plant modelling.

Background and Aims: Plants usually compete with neighbouring plants for resources such as light as well as defend themselves against herbivorous insects. This requires investment of limiting resources, resulting in optimal resource distribution patterns and trade-offs between growth- and defence-related traits. A plant's competitive success is determined by the spatial distribution of its resources in the canopy. The spatial distribution of herbivory in the canopy in turn differs between herbivore species as the level of herbivore specialization determines their response to the distribution of resources and defences in the canopy. Here, we investigated to what extent competition for light affects plant susceptibility to herbivores with different feeding preferences. Methods: To quantify interactions between herbivory and competition, we developed and evaluated a 3-D spatially explicit functional-structural plant model for Brassica nigra that mechanistically simulates competition in a dynamic light environment, and also explicitly models leaf area removal by herbivores with different feeding preferences. With this novel approach, we can quantitatively explore the extent to which herbivore feeding location and light competition interact in their effect on plant performance. Key Results: Our results indicate that there is indeed a strong interaction between levels of plant-plant competition and herbivore feeding preference. When plants did not compete, herbivory had relatively small effects irrespective of feeding preference. Conversely, when plants competed, herbivores with a preference for young leaves had a strong negative effect on the competitiveness and subsequent performance of the plant, whereas herbivores with a preference for old leaves did not. Conclusions: Our study predicts how plant susceptibility to herbivory depends on the composition of the herbivore community and the level of plant competition, and highlights the importance of considering the full range of dynamics in plant-plant-herbivore interactions.

Overexpression of biologically safe Rorippa indica defensin enhances aphid tolerance in Brassica juncea.

MAIN CONCLUSION: Transgenic mustard plants ( Brassica juncea ) expressing non-allergenic and biologically safe RiD peptide show higher tolerance against Lipaphis erysimi. Rorippa indica defensin (RiD) has previously been reported as a novel insecticidal protein derived from a wild crucifer Rorippa indica. RiD was found to have an effective insecticidal property against mustard aphid, Lipaphis erysimi. In the present study, RiD was highly upregulated in R. indica during aphid infestation initiating a defense system mediated by jasmonic acid (JA), but not by salicylic acid (SA)/abscisic acid (ABA). RiD has also been assessed for biosafety according to the FAO/WHO guideline (allergenicity of genetically modified foods; Food And Agriculture Organisation of the United Nations, Rome, Italy, 2001) and Codex Alimentarius Guideline (Guidelines for the design and implementation of national regulatory food safety assurance programme associated with the use of veterinary drugs in food producing animals. Codex Alimentarius Commission. GL, pp 71-2009, 2009). The purified protein was used to sensitize BALB/c mice and they showed normal histopathology of lung and no elevated IgE level in their sera. As the protein was found to be biologically safe and non-allergenic, it was used to develop transgenic Brassica juncea plants with enhanced aphid tolerance, which is one of the most important oilseed crops and is mostly affected by the devastating pest-L. erysimi. The transgene integration was monitored by Southern hybridization, and the positive B. juncea lines were further analyzed by Western blot, ELISA, immunohistolocalization assays and in planta insect bioassay. Transgenic plants expressing RiD conferred a higher level of tolerance against L. erysimi. All these results demonstrated that RiD is a novel, biologically safe, effective insecticidal agent and B. juncea plants expressing RiD are important components of integrated pest management.

Combined biotic stresses trigger similar transcriptomic responses but contrasting resistance against a chewing herbivore in Brassica nigra.

BACKGROUND: In nature, plants are frequently exposed to simultaneous biotic stresses that activate distinct and often antagonistic defense signaling pathways. How plants integrate this information and whether they prioritize one stress over the other is not well understood. RESULTS: We investigated the transcriptome signature of the wild annual crucifer, Brassica nigra, in response to eggs and caterpillars of Pieris brassicae butterflies, Brevicoryne brassicae aphids and the bacterial phytopathogen Xanthomonas campestris pv. raphani (Xcr). Pretreatment with egg extract, aphids, or Xcr had a weak impact on the subsequent transcriptome profile of plants challenged with caterpillars, suggesting that the second stress dominates the transcriptional response. Nevertheless, P. brassicae larval performance was strongly affected by egg extract or Xcr pretreatment and depended on the site where the initial stress was applied. Although egg extract and Xcr pretreatments inhibited insect-induced defense gene expression, suggesting salicylic acid (SA)/jasmonic acid (JA) pathway cross talk, this was not strictly correlated with larval performance. CONCLUSION: These results emphasize the need to better integrate plant responses at different levels of biological organization and to consider localized effects in order to predict the consequence of multiple stresses on plant resistance.

Feeding behaviour of generalist pests on Brassica juncea: implication for manipulation of glucosinolate biosynthesis pathway for enhanced resistance.

Differential accumulation of plant defence metabolites has been suggested to have important ecological consequence in the context of plant-insect interactions. Feeding of generalist pests on Brassica juncea showed a distinct pattern with selective exclusion of leaf margins which are high in glucosinolates. Molecular basis of this differential accumulation of glucosinolates could be explained based on differential expression profile of BjuMYB28 homologues, the major biosynthetic regulators of aliphatic glucosinolates, as evident from quantitative real-time PCR and promoter:GUS fusion studies in allotetraploid B. juncea. Constitutive overexpression of selected BjuMYB28 homologues enhanced accumulation of aliphatic glucosinolates in B. juncea. Performance of two generalist pests, Helicoverpa armigera and Spodoptera litura larvae, on transgenic B. juncea plants were poor compared to wild-type plants in a no-choice experiment. Correlation coefficient analysis suggested that weight gain of H. armigera larvae was negatively correlated with gluconapin (GNA) and glucobrassicanapin (GBN), whereas that of S. litura larvae was negatively correlated with GNA, GBN and sinigrin (SIN). Our study explains the significance and possible molecular basis of differential distribution of glucosinolates in B. juncea leaves and shows the potential of overexpressing BjuMYB28 for enhanced resistance of Brassica crops against the tested generalist pests.

Identification and comprehensive evaluation of reference genes for RT-qPCR analysis of host gene-expression in Brassica juncea-aphid interaction using microarray data.

Brassica juncea is a chief oil yielding crop in many parts of the world including India. With advancement of molecular techniques, RT-qPCR based study of gene-expression has become an integral part of experimentations in crop breeding. In RT-qPCR, use of appropriate reference gene(s) is pivotal. The virtue of the reference genes, being constant in expression throughout the experimental treatments, needs to be validated case by case. Appropriate reference gene(s) for normalization of gene-expression data in B. juncea during the biotic stress of aphid infestation is not known. In the present investigation, 11 reference genes identified from microarray database of Arabidopsis-aphid interaction at a cut off FDR ≤0.1, along with two known reference genes of B. juncea, were analyzed for their expression stability upon aphid infestation. These included 6 frequently used and 5 newly identified reference genes. Ranking orders of the reference genes in terms of expression stability were calculated using advanced statistical approaches such as geNorm, NormFinder, delta Ct and BestKeeper. The analysis suggested CAC, TUA and DUF179 as the most suitable reference genes. Further, normalization of the gene-expression data of STP4 and PR1 by the most and the least stable reference gene, respectively has demonstrated importance and applicability of the recommended reference genes in aphid infested samples of B. juncea.

Dual herbivore attack and herbivore density affect metabolic profiles of Brassica nigra leaves.

Plant responses to dual herbivore attack are increasingly studied, but effects on the metabolome have largely been restricted to volatile metabolites and defence-related non-volatile metabolites. However, plants subjected to stress, such as herbivory, undergo major changes in both primary and secondary metabolism. Using a naturally occurring system, we investigated metabolome-wide effects of single or dual herbivory on Brassica nigra plants by Brevicoryne brassicae aphids and Pieris brassicae caterpillars, while also considering the effect of aphid density. Metabolomic analysis of leaf material showed that single and dual herbivory had strong effects on the plant metabolome, with caterpillar feeding having the strongest influence. Additionally, aphid-density-dependent effects were found in both the single and dual infestation scenarios. Multivariate analysis revealed treatment-specific metabolomic profiles, and effects were largely driven by alterations in the glucosinolate and sugar pools. Our work shows that analysing the plant metabolome as a single entity rather than as individual metabolites provides new insights into the subcellular processes underlying plant defence against multiple herbivore attackers. These processes appear to be importantly influenced by insect density.

Nutrient Supply and Simulated Herbivory Differentially Alter the Metabolite Pools and the Efficacy of the Glucosinolate-Based Defense System in Brassica Species.

Environmental stress hinders growth of plants and commonly results in the accumulation of carbon-based defense compounds. However, the dynamics of nitrogen (N)-containing defense compounds are less predictable under environmental stress. The impact of nutrient deficiency on plant defenses that require the metabolic conversion of a less toxic compound to a more potent toxin is even more poorly understood. We evaluated the effects of nitrogen (N) and potassium (K) deficiency and simulated herbivory on the concentration of metabolites including glucosinolates (GSLs), on the conversion of GSLs to more toxic isothiocyanates (ITCs), and on the activity of myrosinase (MYR) in leaves of Brassica juncea and Brassica nigra. Both species contained GSLs, predominantly sinigrin, but also derivatives of glucobrassicin. Compared to the control, N deficiency increased the sinigrin concentration in both species. Methyl jasmonate (MeJA) application increased sinigrin production in B. junceae, whereas in B. nigra MeJA increased sinigrin only under K-deficiency. Compared to the aliphatic-glucosinolates, MeJA application produced a greater compositional change in the profiles of indolic-glucosinolates. In both species the increase in sinigrin content of the tissue was associated with a decrease in its overall nutritive value as assessed by the content of sugars and amino acids. In B. juncea, application of MeJA decreased the conversion of sinigrin to allyl isothiocyanate (AITC) under both N and K deficiency. The potential activity of MYR decreased in both species under N deficiency. The reduced conversion of sinigrin to AITC and the lower activity of MYR suggest that the GSL-ITC defense system might have a limited efficiency in deterring generalist herbivores under environmental stress.

Central Metabolic Responses to Ozone and Herbivory Affect Photosynthesis and Stomatal Closure.

Plants have evolved adaptive mechanisms that allow them to tolerate a continuous range of abiotic and biotic stressors. Tropospheric ozone (O3), a global anthropogenic pollutant, directly affects living organisms and ecosystems, including plant-herbivore interactions. In this study, we investigate the stress responses of Brassica nigra (wild black mustard) exposed consecutively to O3 and the specialist herbivore Pieris brassicae Transcriptomics and metabolomics data were evaluated using multivariate, correlation, and network analyses for the O3 and herbivory responses. O3 stress symptoms resembled those of senescence and phosphate starvation, while a sequential shift from O3 to herbivory induced characteristic plant defense responses, including a decrease in central metabolism, induction of the jasmonic acid/ethylene pathways, and emission of volatiles. Omics network and pathway analyses predicted a link between glycerol and central energy metabolism that influences the osmotic stress response and stomatal closure. Further physiological measurements confirmed that while O3 stress inhibited photosynthesis and carbon assimilation, sequential herbivory counteracted the initial responses induced by O3, resulting in a phenotype similar to that observed after herbivory alone. This study clarifies the consequences of multiple stress interactions on a plant metabolic system and also illustrates how omics data can be integrated to generate new hypotheses in ecology and plant physiology.

Comprehensive evaluation of candidate reference genes for qRT-PCR studies of gene expression in mustard aphid, Lipaphis erysimi (Kalt).

Mustard aphid, also known as turnip aphid (Lipaphis erysimi) is a major insect pest of rapeseed-mustard group of crops. Tremendous economic significance has led to substantial basic research involving gene-expression studies in this insect species. In qRT-PCR analysis of gene-expression, normalization of data against RNA variation by using appropriate reference gene is fundamental. However, appropriate reference genes are not known in case of L. erysimi. We evaluated 11 candidate reference genes for their expression stability in 21 samples of L. erysimi subjected to various regimes of experimental treatments. Unlike other studies, we validated true effects of the treatments on the samples either by gene-expression study of an associated marker gene or by biochemical tests. In the validated samples, expression stability of the reference genes was analysed by employing four different statistical softwares geNorm, NormFinder, BestKeeper and deltaCt. Drawing consensus on the results from different softwares, we recommend three best reference genes 16S, RPS18 and RPL13 for normalization of qRT-PCR data in L. erysimi. This study provides for the first time a comprehensive list of suitable reference genes for mustard aphid and demonstrates the advantage of using more than one reference gene in combination for certain experimental conditions.

Elevated Ozone Modulates Herbivore-Induced Volatile Emissions of Brassica nigra and Alters a Tritrophic Interaction.

Plants damaged by herbivores emit volatile organic compounds (VOCs) that are used by parasitoids for host location. In nature, however, plants are exposed to multiple abiotic and biotic stresses of varying intensities, which may affect tritrophic interactions. Here, we studied the effects of ozone exposure and feeding by Pieris brassicae larvae on the VOCs emitted by Brassica nigra and the effects on oriented flight of the parasitoid Cotesia glomerata. We also investigated the oriented flight of C. glomerata in a wind-tunnel with elevated ozone levels. Herbivore-feeding induced the emission of several VOCs, while ozone alone had no significant effect. However, exposure to 120 ppb ozone, followed by 24 hr of herbivore-feeding, induced higher emissions of all VOCs as compared to herbivore-feeding alone. In accordance, herbivore-damaged plants elicited more oriented flights than undamaged plants, whereas plants exposed to 120 ppb ozone and 24 hr of herbivore-feeding elicited more oriented flights than plants subjected to herbivore-feeding alone. Ozone enrichment of the wind-tunnel air appeared to negatively affect orientation of parasitoids at 70 ppb, but not at 120 ppb. These results suggest that the combination of ozone and P. brassicae-feeding modulates VOC emissions, which significantly influence foraging efficiency of C. glomerata.

Plant Quantity Affects Development and Survival of a Gregarious Insect Herbivore and Its Endoparasitoid Wasp.

Virtually all studies of plant-herbivore-natural enemy interactions focus on plant quality as the major constraint on development and survival. However, for many gregarious feeding insect herbivores that feed on small or ephemeral plants, the quantity of resources is much more limiting, yet this area has received virtually no attention. Here, in both lab and semi-field experiments using tents containing variably sized clusters of food plants, we studied the effects of periodic food deprivation in a tri-trophic system where quantitative constraints are profoundly important on insect performance. The large cabbage white Pieris brassicae, is a specialist herbivore of relatively small wild brassicaceous plants that grow in variable densities, with black mustard (Brassica nigra) being one of the most important. Larvae of P. brassicae are in turn attacked by a specialist endoparasitoid wasp, Cotesia glomerata. Increasing the length of food deprivation of newly molted final instar caterpillars significantly decreased herbivore and parasitoid survival and biomass, but shortened their development time. Moreover, the ability of caterpillars to recover when provided with food again was correlated with the length of the food deprivation period. In outdoor tents with natural vegetation, we created conditions similar to those faced by P. brassicae in nature by manipulating plant density. Low densities of B. nigra lead to potential starvation of P. brassicae broods and their parasitoids, replicating nutritional conditions of the lab experiments. The ability of both unparasitized and parasitized caterpillars to find corner plants was similar but decreased with central plant density. Survival of both the herbivore and parasitoid increased with plant density and was higher for unparasitized than for parasitized caterpillars. Our results, in comparison with previous studies, reveal that quantitative constraints are far more important that qualitative constraints on the performance of gregarious insect herbivores and their gregarious parasitoids in nature.

(E)-ß-farnesene gene reduces Lipaphis erysimi colonization in transgenic Brassica juncea lines.

Aphids are the major concern that significantly reduces the yield of crops. (E)-ß-farnesene (Eßf) is the principal component of the alarm pheromone of many aphids. The results of current research support the direct defense response of (E)-ß-farnesene (Eßf) against aphid Lipaphis erysimi (L.) Kaltenbach in Brassica juncea. Eßf gene was isolated from Mentha arvensis and transformed into B. juncea, showed direct repellent against aphid colonization. The seasonal mean population (SMP) recorded under field condition showed significantly higher aphid colonization in wild type in comparison to most of the transgenic lines, and shows positive correlation with the repellency of transgenic plant expressing (E)-ß-farnesene. The current research investigation provides direct evidence for aphid control in B. juncea using Eßf, a non-toxic mode of action.