RNA-Seq Bulked Segregant Analysis of an Exotic B. napus ssp. napobrassica (Rutabaga) F2 Population Reveals Novel QTLs for Breeding Clubroot-Resistant Canola.

In this study, a rutabaga (Brassica napus ssp. napobrassica) donor parent FGRA106, which exhibited broad-spectrum resistance to 17 isolates representing 16 pathotypes of Plasmodiophora brassicae, was used in genetic crosses with the susceptible spring-type canola (B. napus ssp. napus) accession FG769. The F2 plants derived from a clubroot-resistant F1 plant were screened against three P. brassicae isolates representing pathotypes 3A, 3D, and 3H. Chi-square (χ2) goodness-of-fit tests indicated that the F2 plants inherited two major clubroot resistance genes from the CR donor FGRA106. The total RNA from plants resistant (R) and susceptible (S) to each pathotype were pooled and subjected to bulked segregant RNA-sequencing (BSR-Seq). The analysis of gene expression profiles identified 431, 67, and 98 differentially expressed genes (DEGs) between the R and S bulks. The variant calling method indicated a total of 12 (7 major + 5 minor) QTLs across seven chromosomes. The seven major QTLs included: BnaA5P3A.CRX1.1, BnaC1P3H.CRX1.2, and BnaC7P3A.CRX1.1 on chromosomes A05, C01, and C07, respectively; and BnaA8P3D.CRX1.1, BnaA8P3D.RCr91.2/BnaA8P3H.RCr91.2, BnaA8P3H.Crr11.3/BnaA8P3D.Crr11.3, and BnaA8P3D.qBrCR381.4 on chromosome A08. A total of 16 of the DEGs were located in the major QTL regions, 13 of which were on chromosome C07. The molecular data suggested that clubroot resistance in FGRA106 may be controlled by major and minor genes on both the A and C genomes, which are deployed in different combinations to confer resistance to the different isolates. This study provides valuable germplasm for the breeding of clubroot-resistant B. napus cultivars in Western Canada.

Genome-wide identification of the ICS family genes and its role in resistance to Plasmodiophora brassicae in Brassica napus L.

The isochorismate synthase (ICS) proteins are essential regulators of salicylic acid (SA) synthesis, which has been reported to regulate resistance to biotic and abiotic stresses in plants. Clubroot caused by Plasmodiophora brassicae is a common disease that threatens the yield and quality of Oilseed rape (Brassica napus L.). Exogenous application of salicylic acid reduced the incidence of clubroot in oilseed rape. However, the potential importance of the ICS genes family in B. napus and its diploid progenitors has been unclear. Here, we identified 16, 9, and 10 ICS genes in the allotetraploid B. napus, diploid ancestor Brassica rapa and Brassica oleracea, respectively. These ICS genes were classified into three subfamilies (I-III), and member of the same subfamilies showed relatively conserved gene structures, motifs, and protein domains. Furthermore, many hormone-response and stress-related promoter cis-acting elements were observed in the BnaICS genes. Exogenous application of SA delayed the growth of clubroot galls, and the expression of BnaICS genes was significantly different compared to the control groups. Protein-protein interaction analysis identified 58 proteins involved in the regulation of ICS in response to P. brassicae in B. napus. These results provide new clues for understanding the resistance mechanism to P. brassicae.

A Basic Guide to the Propagation and Manipulation of the Clubroot Pathogen, Plasmodiophora brassicae.

Clubroot caused by the obligate parasite Plasmodiophora brassicae is a devastating disease affecting the canola industry worldwide. The socio-economic impact of clubroot can be significant, particularly in regions where Brassica crops are a major agricultural commodity. The disease can cause significant crop losses, leading to reduced yield and income for farmers. Extensive studies have been conducted to understand the biology and genetics of the pathogens and develop more effective management strategies. However, the basic procedures used for pathogen storage and virulence analysis have not been assembled or discussed in detail. As a result, there are discrepancies among the different protocols used today. The aim of this article is to provide a comprehensive and easily accessible resource for researchers who are interested in replicating or building upon the methods used in the study of the clubroot pathogen. Here, we discuss in detail the methods used for P. brassicae spore isolation, inoculation, quantification, propagation, and molecular techniques such as DNA extraction and PCR. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Extraction of Plasmodiophora brassicae resting spores and propagation Support Protocol 1: Evans blue staining to identify resting spore viability Support Protocol 2: Storage of Plasmodiophora brassicae Basic Protocol 2: Generation of single spore isolates from P. brassicae field isolates Basic Protocol 3: Phenotyping of Plasmodiophora brassicae isolates Basic Protocol 4: Genomic DNA extraction from Plasmodiophora brassicae resting spores Basic Protocol 5: Molecular detection of Plasmodiophora brassicae.

Proteome- and metabolome-level changes during early stages of clubroot infection in Brassica napus canola.

Clubroot is a destructive root disease of canola (Brassica napus L.) caused by Plasmodiophora brassicae Woronin. Despite extensive research into the molecular responses of B. napus to P. brassicae, there is limited information on proteome- and metabolome-level changes in response to the pathogen, especially during the initial stages of infection. In this study, we have investigated the proteome- and metabolome- level changes in the roots of clubroot-resistant (CR) and -susceptible (CS) doubled-haploid (DH) B. napus lines, in response to P. brassicae pathotype 3H at 1-, 4-, and 7-days post-inoculation (DPI). Root proteomes were analyzed using nanoflow liquid chromatography coupled with tandem mass spectrometry (nano LC-MS/MS). Comparisons of pathogen-inoculated and uninoculated root proteomes revealed 2515 and 1556 differentially abundant proteins at one or more time points (1-, 4-, and 7-DPI) in the CR and CS genotypes, respectively. Several proteins related to primary metabolites (e.g., amino acids, fatty acids, and lipids), secondary metabolites (e.g., glucosinolates), and cell wall reinforcement-related proteins [e.g., laccase, peroxidases, and plant invertase/pectin methylesterase inhibitors (PInv/PMEI)] were identified. Eleven nucleotides and nucleoside-related metabolites, and eight fatty acids and sphingolipid-related metabolites were identified in the metabolomics study. To our knowledge, this is the first report of root proteome-level changes and associated alterations in metabolites during the early stages of P. brassicae infection in B. napus.

A Hydroponic-Based Bioassay to Facilitate Plasmodiophora brassicae Phenotyping.

Clubroot, caused by the obligate parasite Plasmodiophora brassicae, is one of the most devastating diseases affecting the canola/oilseed rape (Brassica napus) industry worldwide. Currently, the planting of clubroot-resistant (CR) cultivars is the most effective strategy used to restrict the spread and the economic losses linked to the disease. However, virulent P. brassicae isolates have been able to infect many of the currently available CR cultivars, and the options to manage the disease are becoming limited. Another challenge has been achieving consistency in evaluating host reactions to P. brassicae infection, with most bioassays conducted in soil and/or potting medium, which requires significant space and can be labor intensive. Visual scoring of clubroot symptom development can also be influenced by user bias. Here, we have developed a hydroponic bioassay using well-characterized P. brassicae single-spore isolates representative of clubroot virulence in Canada, as well as field isolates from three Canadian provinces in combination with canola inbred homozygous lines carrying resistance genetics representative of CR cultivars available to growers in Canada. To improve the efficiency and consistency of disease assessment, symptom severity scores were compared with clubroot evaluations based on the scanned root area. According to the results, this bioassay offers a reliable, less expensive, and reproducible option to evaluate P. brassicae virulence, as well as to identify which canola resistance profile(s) may be effective against particular isolates. This bioassay will contribute to the breeding of new CR canola cultivars and the identification of virulence genes in P. brassicae that could trigger resistance and that have been very elusive to this day.[Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Automating insect monitoring using unsupervised near-infrared sensors.

Insect monitoring is critical to improve our understanding and ability to preserve and restore biodiversity, sustainably produce crops, and reduce vectors of human and livestock disease. Conventional monitoring methods of trapping and identification are time consuming and thus expensive. Automation would significantly improve the state of the art. Here, we present a network of distributed wireless sensors that moves the field towards automation by recording backscattered near-infrared modulation signatures from insects. The instrument is a compact sensor based on dual-wavelength infrared light emitting diodes and is capable of unsupervised, autonomous long-term insect monitoring over weather and seasons. The sensor records the backscattered light at kHz pace from each insect transiting the measurement volume. Insect observations are automatically extracted and transmitted with environmental metadata over cellular connection to a cloud-based database. The recorded features include wing beat harmonics, melanisation and flight direction. To validate the sensor's capabilities, we tested the correlation between daily insect counts from an oil seed rape field measured with six yellow water traps and six sensors during a 4-week period. A comparison of the methods found a Spearman's rank correlation coefficient of 0.61 and a p-value = 0.0065, with the sensors recording approximately 19 times more insect observations and demonstrating a larger temporal dynamic than conventional yellow water trap monitoring.

Effect of Pathogen Virulence on Pathogenicity, Host Range, and Reproduction of Plasmodiophora brassicae, the Causal Agent of Clubroot Disease.

A series of greenhouse experiments was conducted to evaluate the effect of Plasmodiophora brassicae virulence on clubroot development and propagation of resting spores in 86 plant species from 19 botanical families. Plants were artificially inoculated with two isolates of P. brassicae, which were virulent on clubroot-resistant oilseed rape cultivar Mendel [pathotype 1; P1 (+)] or avirulent on this cultivar (P1). Clubroot severity and the number of resting spores inside the roots were assessed 35 days post inoculation. Typical clubroot symptoms were observed only in the Brassicaceae family. P1 (+)-inoculated species exhibited more severe symptoms (two- to 10-fold more severe), bigger galls (1.1- to 5.8-fold heavier), and greater numbers of resting spores than the P1-inoculated plants. Among all Brassica species, Bunias orientalis, Coronopus squamatus, and Raphanus sativus were fully resistant against both isolates, whereas Camelina sativa, Capsella bursa-pastoris, Coincya monensis, Descurainia sophia, Diplotaxis muralis, Erucastrum gallicum, Neslia paniculata, Sinapis alba, Sinapis arvensis, Sisymbrium altissimum, Sisymbrium loeselii, and Thlaspi arvense were highly susceptible. Conringia orientalis, Diplotaxis tenuifolia, Hirschfeldia incana, Iberis amara, Lepidium campestre, and N. paniculata were completely or partially resistant to P1 isolate but highly susceptible to P1 (+). These results suggest that the basis for resistance in these species may be similar to that found in some commercial cultivars, and that these species could contribute to the buildup of inoculum of virulent pathotypes. Furthermore, the pathogen DNA was detected in Alopecurus myosuroides, Phacelia tanacetifolia, Papaver rhoeas, and Pisum sativum. It can be concluded that the number and diversity of hosts for P. brassicae are greater than previously reported.

Comparison of Gut Bacterial Communities of Fall Armyworm (Spodoptera frugiperda) Reared on Different Host Plants.

Spodoptera frugiperda is a highly polyphagous and invasive agricultural pest that can harm more than 300 plants and cause huge economic losses to crops. Symbiotic bacteria play an important role in the host biology and ecology of herbivores, and have a wide range of effects on host growth and adaptation. In this study, high-throughput sequencing technology was used to investigate the effects of different hosts (corn, wild oat, oilseed rape, pepper, and artificial diet) on gut microbial community structure and diversity. Corn is one of the most favored plants of S. frugiperda. We compared the gut microbiota on corn with and without a seed coating agent. The results showed that Firmicutes and Bacteroidetes dominated the gut microbial community. The microbial abundance on oilseed rape was the highest, the microbial diversity on wild oat was the lowest, and the microbial diversity on corn without a seed coating agent was significantly higher than that with such an agent. PCoA analysis showed that there were significant differences in the gut microbial community among different hosts. PICRUSt analysis showed that most of the functional prediction categories were related to metabolic and cellular processes. The results showed that the gut microbial community of S. frugiperda was affected not only by the host species, but also by different host treatments, which played an important role in host adaptation. It is important to deepen our understanding of the symbiotic relationships between invasive organisms and microorganisms. The study of the adaptability of host insects contributes to the development of more effective and environmentally friendly pest management strategies.

Analysis of the Oxidative Burst and Its Relevant Signaling Pathways in Leptosphaeria maculans-Brassica napus Pathosystem.

An oxidative burst is an early response of plants to various biotic/abiotic stresses. In plant-microbe interactions, the plant body can induce oxidative burst to activate various defense mechanisms to combat phytopathogens. A localized oxidative burst is also one of the typical behaviors during hypersensitive response (HR) caused by gene-for-gene interaction. In this study, the occurrence of oxidative burst and its signaling pathways was studied from different levels of disease severity (i.e., susceptible, intermediate, and resistant) in the B. napus-L. maculans pathosystem. Canola cotyledons with distinct levels of resistance exhibited differential regulation of the genes involved in reactive oxygen species (ROS) accumulation and responses. Histochemical assays were carried out to understand the patterns of H2O2 accumulation and cell death. Intermediate and resistant genotypes exhibited earlier accumulation of H2O2 and emergence of cell death around the inoculation origins. The observations also suggested that the cotyledons with stronger resistance were able to form a protective region of intensive oxidative bursts between the areas with and without hyphal intrusions to block further fungal advancement to the uninfected regions. The qPCR analysis suggested that different onset patterns of some marker genes in ROS accumulation/programmed cell death (PCD) such as RBOHD, MPK3 were associated with distinct levels of resistance from B. napus cultivars against L. maculans. The observations and datasets from this article indicated the distinct differences in ROS-related cellular behaviors and signaling between compatible and incompatible interactions.

Genome-wide transcriptome reveals mechanisms underlying Rlm1-mediated blackleg resistance on canola.

Genetic resistance to blackleg (Leptosphaeria maculans, Lm) of canola (Brassica napus, Bn) has been extensively studied, but the mechanisms underlying the host-pathogen interaction are still not well understood. Here, a comparative transcriptome analysis was performed on a resistant doubled haploid Bn line carrying the resistance gene Rlm1 following inoculation with a virulent (avrLm1) or avirulent (AvrLm1) Lm isolate on cotyledons. A total of 6999 and 3015 differentially expressed genes (DEGs) were identified, respectively, in inoculated local tissues with compatible (susceptible) and incompatible (resistant) interactions. Functional enrichment analysis found several biological processes, including protein targeting to membrane, ribosome and negative regulation of programmed cell death, were over-represented exclusively among up-regulated DEGs in the resistant reaction, whereas significant enrichment of salicylic acid (SA) and jasmonic acid (JA) pathways observed for down-regulated DEGs occurred only in the susceptible reaction. A heat-map analysis showed that both biosynthesis and signaling of SA and JA were induced more significantly in the resistant reaction, implying that a threshold level of SA and JA signaling is required for the activation of Rlm1-mediated resistance. Co-expression network analysis revealed close correlation of a gene module with the resistance, involving DEGs regulating pathogen-associated molecular pattern recognition, JA signaling and transcriptional reprogramming. Substantially fewer DEGs were identified in mock-inoculated (control) cotyledons, relative to those in inoculated local tissues, including those involved in SA pathways potentially contributing to systemic acquired resistance (SAR). Pre-inoculation of cotyledon with either an avirulent or virulent Lm isolate, however, failed to induce SAR on remote tissues of same plant despite elevated SA and PR1 protein. This study provides insights into the molecular mechanism of Rlm1-mediated resistance to blackleg.

Genetic Structure of Plasmodiophora brassicae Populations Virulent on Clubroot Resistant Canola (Brassica napus).

Clubroot, caused by Plasmodiophora brassicae Woronin, is a significant threat to the canola (Brassica napus L.) industry in Canada. Clubroot resistance has been overcome in more than 200 fields since 2013, representing one of the biggest challenges to sustainable canola production. The genetic structure of 36 single-spore isolates derived from 12 field isolates of P. brassicae collected before and after the introduction of clubroot resistant (CR) canola cultivars (2005-2014) was evaluated by simple sequence repeat (SSR) marker analysis. Polymorphisms were detected in 32 loci with the identification of 93 distinct alleles. A low level of genetic diversity was found among the single-spore isolates. Haploid linkage disequilibrium and number of migrants suggested that recombination and migration were rare or almost absent in the tested P. brassicae population. A relatively clear relationship was found between the genetic structure and virulence phenotypes of the pathogen as defined on the differential hosts of Somé et al., Williams, and the Canadian Clubroot Differential (CCD) set. Although genetic variability within each pathotype group, as classified on each differential system, was low, significant genetic differentiation was observed among the pathotypes. The highest correlation between genetic structure and virulence was found among matrices produced with genetic data and the hosts of the CCD set, with a threshold index of disease of 50% to distinguish susceptible from resistant reactions. Genetically homogeneous single-spore isolates provided a more complete and clearer picture of the population genetic structure of P. brassicae, and the results suggest some promise for the development of pathotype-specific primers.

Chemical defence in Brassicaceae against pollen beetles revealed by metabolomics and flower bud manipulation approaches.

Divergence of chemical plant defence mechanisms within the Brassicaceae can be utilized to identify means against specialized pest insects. Using a bioassay-driven approach, we (a) screened 24 different Brassica napus cultivars, B. napus resyntheses and related brassicaceous species for natural plant resistance against feeding adults of the pollen beetle (Brassicogethes aeneus), (b) tested for gender-specific feeding resistance, (c) analysed the flower bud metabolomes by a non-targeted approach and (d) tested single candidate compounds for their antifeedant activity. (a) In no-choice assays, beetles were allowed to feed on intact plants. Reduced feeding activity was mainly observed on Sinapis alba and Barbarea vulgaris but not on B. napus cultivars. (b) Males fed less and discriminated more in feeding than females. (c) Correlation of the metabolite abundances with the beetles' feeding activity revealed several glucosinolates, phenylpropanoids, flavonoids and saponins as potential antifeedants. (d) These were tested in dual-bud-choice assays developed for medium-throughput compound screening. Application of standard compounds on single oilseed rape flower buds revealed highly deterrent effects of glucobarbarin, oleanolic acid and hederagenin. These results help to understand chemical plant defence in the Brassicaceae and are of key importance for further breeding strategies for insect-resistant oilseed rape cultivars.

Identification of lncRNAs in response to infection by Plasmodiophora brassicae in Brassica napus and development of lncRNA-based SSR markers.

Clubroot resistance in spring canola has been introgressed from different Brassica sources; however, molecular mechanism underlying this resistance, especially the involvement of long non-coding RNAs (lncRNAs), is yet to be understood. We identified 464 differentially expressed (DE) lncRNAs from the roots of clubroot-resistant canola, carrying resistance on chromosome BnaA03, and susceptible canola lines challenged with Plasmodiophora brassicae pathotype 3. Pathway enrichment analysis showed that most of the target genes regulated by these DE lncRNAs belonged to plant-pathogen interaction and hormone signaling, as well as primary and secondary metabolic pathways. Comparative analysis of these lncRNAs with 530 previously reported DE lncRNAs, identified using resistance located on BnaA08, detected 12 lncRNAs that showed a similar trend of upregulation in both types of resistant lines; these lncRNAs probably play a fundamental role in clubroot resistance. We identified SSR markers within 196 DE lncRNAs. Genotyping of two DH populations carrying resistance on BnaA03 identified a marker capable of detecting the resistance in 98% of the DH lines. To our knowledge, this is the first report of the identification of SSRs within lncRNAs responsive to P. brassicae infection, demonstrating the potential use of lncRNAs in the breeding of Brassica crops.

Comparative Transcriptome Analysis of Rutabaga (Brassica napus) Cultivars Indicates Activation of Salicylic Acid and Ethylene-Mediated Defenses in Response to Plasmodiophora brassicae.

Clubroot, caused by Plasmodiophora brassicae Woronin, is an important soilborne disease of Brassica napus L. and other crucifers. To improve understanding of the mechanisms of resistance and pathogenesis in the clubroot pathosystem, the rutabaga (B. napus subsp. rapifera Metzg) cultivars 'Wilhelmsburger' (resistant) and 'Laurentian' (susceptible) were inoculated with P. brassicae pathotype 3A and their transcriptomes were analyzed at 7, 14, and 21 days after inoculation (dai) by RNA sequencing (RNA-seq). Thousands of transcripts with significant changes in expression were identified in each host at each time-point in inoculated vs. non-inoculated plants. Molecular responses at 7 and 14 dai supported clear differences in the clubroot response mechanisms of the two genotypes. Both the resistant and the susceptible cultivars activated receptor-like protein (RLP) genes, resistance (R) genes, and genes involved in salicylic acid (SA) signaling as clubroot defense mechanisms. In addition, genes related to calcium signaling and genes encoding leucine-rich repeat (LRR) receptor kinases, the respiratory burst oxidase homolog (RBOH) protein, and transcription factors such as WRKYs, ethylene responsive factors, and basic leucine zippers (bZIPs), appeared to be upregulated in 'Wilhelmsburger' to restrict P. brassicae development. Some of these genes are essential components of molecular defenses, including ethylene (ET) signaling and the oxidative burst. Our study highlights the importance of activation of genes associated with SA- and ET-mediated responses in the resistant cultivar. A set of candidate genes showing contrasting patterns of expression between the resistant and susceptible cultivars was identified and includes potential targets for further study and validation through approaches such as gene editing.

Infection of canola by the root pathogen Plasmodiophora brassicae increases resistance to aboveground herbivory by bertha armyworm, Mamestra configurata Walker (Lepidoptera: Noctuidae).

Infection of plants by pathogens can result in the upregulation of induced defenses; plants may be more or less susceptible to attack by insect herbivores following infection. We investigated the interaction between canola, Brassica napus L., plants infected with clubroot, Plasmodiophora brassicae Woronin, and a generalist herbivore the bertha armyworm (BAW) Mamestra configurata Walker using two canola cultivars that varied in susceptibility to clubroot disease. Volatile organic compounds released from experimental plants differed with infection and female adult BAW could discriminate between canola plants inoculated with P. brassicae and disease-free plants. Adult female moths preferentially laid eggs on disease-free plants of the susceptible cultivar to P. brassicae. Inoculation of resistant canola with P. brassicae, however, did not influence oviposition by female BAW. The fitness of BAW larvae was reduced when they were reared on susceptible canola inoculated with P. brassicae. Salicylic acid and its conjugates in susceptible canola plants were induced following P. brassicae inoculation as compared to disease-free susceptible plants. We conclude that suppression of BAW oviposition and offspring fitness may result in part from a change in the volatile profile of the plant as a result of inoculation and the induction of defenses in inoculated susceptible canola.

Flowering plant composition shapes pathogen infection intensity and reproduction in bumble bee colonies.

Pathogens pose significant threats to pollinator health and food security. Pollinators can transmit diseases during foraging, but the consequences of plant species composition for infection is unknown. In agroecosystems, flowering strips or hedgerows are often used to augment pollinator habitat. We used canola as a focal crop in tents and manipulated flowering strip composition using plant species we had previously shown to result in higher or lower bee infection in short-term trials. We also manipulated initial colony infection to assess impacts on foraging behavior. Flowering strips using high-infection plant species nearly doubled bumble bee colony infection intensity compared to low-infection plant species, with intermediate infection in canola-only tents. Both infection treatment and flowering strips reduced visits to canola, but we saw no evidence that infection treatment shifted foraging preferences. Although high-infection flowering strips increased colony infection intensity, colony reproduction was improved with any flowering strips compared to canola alone. Effects of flowering strips on colony reproduction were explained by nectar availability, but effects of flowering strips on infection intensity were not. Thus, flowering strips benefited colony reproduction by adding floral resources, but certain plant species also come with a risk of increased pathogen infection intensity.

CRb and PbBa8.1 Synergically Increases Resistant Genes Expression upon Infection of Plasmodiophora brassicae in Brassica napus.

PbBa8.1 and CRb are two clubroot-resistant genes that are important for canola breeding in China. Previously, we combined these resistant genes and developed a pyramid-based, homozygous recurrent inbred line (618R), the results of which showed strong resistance to Plasmodiophora brassicae field isolates; however, the genetic mechanisms of resistance were unclear. In the present work, we conducted comparative RNA sequencing (RNA-Seq) analysis between 618R and its parental lines (305R and 409R) in order to uncover the transcriptomic response of the superior defense mechanisms of 618R and to determine how these two different resistant genes coordinate with each other. Here, we elucidated that the number and expression of differentially expressed genes (DEGs) in 618R are significantly higher than in the parental lines, and PbBa8.1 shares more DEGs and plays a dominant role in the pyramided line. The common DEGs among the lines largely exhibit non-additive expression patterns and enrichment in resistance pathways. Among the enriched pathways, plant-pathogen interaction, plant hormone signaling transduction, and secondary metabolites are the key observation. However, the expressions of the salicylic acid (SA) signaling pathway and reactive oxygen species (ROS) appear to be crucial regulatory components in defense response. Our findings provide comprehensive transcriptomic insight into understanding the interactions of resistance gene pyramids in single lines and can facilitate the breeding of improved resistance in Brassica napus.

Genome-wide association study reveals new genes involved in leaf trichome formation in polyploid oilseed rape (Brassica napus L.).

Leaf trichomes protect against various biotic and abiotic stresses in plants. However, there is little knowledge about this trait in oilseed rape (Brassica napus). Here, we demonstrated that hairy leaves were less attractive to Plutella xylostella larvae than glabrous leaves. We established a core germplasm collection with 290 accessions for a genome-wide association study (GWAS) of the leaf trichome trait in oilseed rape. We compared the transcriptomes of the shoot apical meristem (SAM) between hairy- and glabrous-leaf genotypes to narrow down the candidate genes identified by GWAS. The single nucleotide polymorphisms and the different transcript levels of BnaA.GL1.a, BnaC.SWEET4.a, BnaC.WAT1.a and BnaC.WAT1.b corresponded to the divergence of the hairy- and glabrous-leaf phenotypes, indicating the role of sugar and/or auxin signalling in leaf trichome initiation. The hairy-leaf SAMs had lower glucose and sucrose contents but higher expression of putative auxin responsive factors than the glabrous-leaf SAMs. Spraying of exogenous auxin (8 µm) increased leaf trichome number in certain genotypes, whereas spraying of sucrose (1%) plus glucose (6%) slightly repressed leaf trichome initiation. These data contribute to the existing knowledge about the genetic control of leaf trichomes and would assist breeding towards the desired leaf surface type in oilseed rape.

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.

Whole-genome DNA similarity and population structure of Plasmodiophora brassicae strains from Canada.

BACKGROUND: Clubroot is an important disease of brassica crops world-wide. The causal agent, Plasmodiophora brassicae, has been present in Canada for over a century but was first identified on canola (Brassica napus) in Alberta, Canada in 2003. Genetic resistance to clubroot in an adapted canola cultivar has been available since 2009, but resistance breakdown was detected in 2013 and new pathotypes are increasing rapidly. Information on genetic similarity among pathogen populations across Canada could be useful in estimating the genetic variation in pathogen populations, predicting the effect of subsequent selection pressure on changes in the pathogen population over time, and even in identifying the origin of the initial pathogen introduction to canola in Alberta. RESULTS: The genomic sequences of 43 strains (34 field collections, 9 single-spore isolates) of P. brassicae from Canada, the United States, and China clustered into five clades based on SNP similarity. The strains from Canada separated into four clades, with two containing mostly strains from the Prairies (provinces of Alberta, Saskatchewan, and Manitoba) and two that were mostly from the rest of Canada or the USA. Several strains from China formed a separate clade. More than one pathotype and host were present in all four Canadian clades. The initial pathotypes from canola on the Prairies clustered separately from the pathotypes on canola that could overcome resistance to the initial pathotypes. Similarly, at one site in central Canada where resistance had broken down, about half of the genes differed (based on SNPs) between strains before and after the breakdown. CONCLUSION: Clustering based on genome-wide DNA sequencing demonstrated that the initial pathotypes on canola on the Prairies clustered separately from the new virulent pathotypes on the Prairies. Analysis indicated that these 'new' pathotypes were likely present in the pathogen population at very low frequency, maintained through balancing selection, and increased rapidly in response to selection from repeated exposure to host resistance.