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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. 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. 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. 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.

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.

Key messageHeavy 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.

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.

Background Clubroot disease, caused by the soil-borne protist Plasmodiophora brassicae , is a major threat to Brassica crops worldwide, leading to significant yield losses. Genetic resistance is the most effective and sustainable management strategy; however, the identification and characterization of clubroot resistance (CR) genes remain a challenge, particularly in Brassica nigra . Despite its abundant CR resources, only one CR gene, Rcr6 , has been identified in the B genome of B. nigra , leaving much of its genetic potential unexplored. Understanding the genomic distribution and diversity of CR genes in B. nigra is crucial for expanding resistance breeding options, especially for canola ( B. napus ). Results This study identified Rcr12 , a single dominant CR gene on chromosome B3 of the highly resistant B. nigra line BRA19278. Using bulked segregant RNA sequencing (BSR-seq) and fine mapping in segregating populations derived from a cross between CR2748 (a susceptible B. nigra line) and BRA19278, together with s ingle-root p rotoplast-derived i solates (SPIs) of P. brassicae and comparative analysis across multiple reference genomes, we established that Rcr12 is distinct from Rcr6 despite their close physical proximity. Evidence supporting its distinctiveness includes differential resistance patterns against various SPIs, unique SNP marker associations, and pangenomic analyses. Fine mapping refined the Rcr12 locus to a 0.33 Mb region on chromosome B3, containing multiple resistance gene candidates, in contrast to the single candidate identified for Rcr6 . This study is the first to report an nucleotide-binding leucine-rich repeat (NLR) cluster-type CR locus near an NLR singleton in Brassica crops, underscoring the evolutionary and functional significance of this gene arrangement. Conclusion The discovery of Rcr12 expands our understanding of NLR gene organization and its role in host resistance evolution. Beyond advancing clubroot resistance breeding, this discovery lays the groundwork for studying functional interactions between NLR singletons and clusters in plant immunity. Additionally, the use of purified SPIs as a pathogen differentiation tool offers a novel approach to resolving ambiguities in clubroot research, addressing the complexity of host-pathogen interactions and facilitating future investigations, especially with the anticipated release of a new pathogen classification system.

Oilseed Brassica, an important edible oilseed crop of the world, is facing severe threat from the parasitic weed ‘Egyptian broomrape’ [ Phelipanche aegyptiaca (Pers.) Pomel] with yield penalty of 0-100%. The problem is more severe due to its physico-biochemical connection with the hosts, where, general weed control measures are not effective to control this parasitic weed. Since, there is no absolute measure to control Egyptian broomrape. The present study was aimed to identify and develop the most effective herbicide formulation and deliver the herbicide to the targeted underground parasite weed for effective and safe control in Indian mustard. Simple (single) and complex (combinational) smart herbicide formulations (NE, ME, SE, SC & ZC) were developed and evaluated under hot-spot conditions. Among all the smart formulations, metsulfuron methyl 5% SC @ 4 ml a.i./ha applied at pre-emergence stage was found most effective in controlling Egyptian broomrape with no phytotoxicity, and recorded higher seed yield of Indian mustard (48–74%) over weedy check plots. Residue analysis using LC-MS/MS revealed that metsulfuron methyl was not detected in soil as well as plant samples thus, it was environmentally safe also. This research successfully demonstrated that metsulfuron methyl-based smart formulations (5% SC) can safely and effectively control Egyptian broomrape in Indian mustard.

In response to insect herbivory, plants emit volatile organic compounds which may act as indirect plant defenses by attracting natural enemies of the attacking herbivore. In nature, plants are often attacked by multiple herbivores, but the majority of studies which have investigated indirect plant defenses to date have focused on the recruitment of different parasitoid species in a single-herbivore context. Here, we report our investigation on the attraction of egg parasitoids of lepidopteran hosts (Trichogramma brassicae and T. evanescens) toward plant volatiles induced by different insect herbivores in olfactometer bioassays. We used a system consisting of a native crucifer, Brassica nigra, two naturally associated herbivores [the butterfly Pieris brassicae (eggs and caterpillars) and the aphid Brevicoryne brassicae] and an alien invasive herbivore (eggs and caterpillars of the moth Spodoptera exigua). We found that Trichogramma wasps were attracted by volatiles induced in the plants by P. brassicae eggs, but not by those induced in the plants by S. exigua eggs, indicating the specificity of the plant responses toward lepidopteran herbivores. The results of the chemical analysis revealed significant differences between the volatile blends emitted by plants in response to attack by P. brassicae and S. exigua eggs which were in agreement with the behavioural observations. We investigated the attraction of Trichogramma wasps toward P. brassicae egg-induced volatiles in plants simultaneously attacked by larvae and nymphs of different non-hosts. The two chewing caterpillars P. brassicae and S. exigua, but not the phloem-feeding aphid B. brassicae, can disrupt the attraction of Trichogramma species toward P. brassicae egg-induced volatiles. Indirect plant defenses are discussed in the context of multiple herbivory by evaluating the importance of origin, dietary specialization and feeding guild of different attackers on the recruitment of egg-killing parasitoids.

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.

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.

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.