Development of an agroinfiltration-based transient hairpin RNA expression system in pak choi leaves (Brassica rapa ssp. chinensis) for RNA interference against Liriomyza sativae.

The vegetable leafminer (Liriomyza sativae) is a devastating invasive pest of many vegetable crops and horticultural plants worldwide, causing serious economic loss. Conventional control strategy against this pest mainly relies on the synthetic chemical pesticides, but widespread use of insecticides easily causes insecticide resistance development and is harmful to beneficial organisms and environment. In this context, a more environmentally friendly pest management strategy based on RNA interference (RNAi) has emerged as a powerful tool to control of insect pests. Here we report a successful oral RNAi in L. sativae after feeding on pak choi (Brassica rapa ssp. chinensis) that transiently express hairpin RNAs targeting vital genes in this pest. First, potentially lethal genes are identified by searching an L. sativae transcriptome for orthologs of the widely used V-ATPase A and actin genes, then expression levels are assessed during different life stages and in different adult tissues. Interestingly, the highest expression levels for V-ATPase A are observed in the adult heads (males and females) and for actin in the abdomens of adult females. We also assessed expression patterns of the target hairpin RNAs in pak choi leaves and found that they reach peak levels 72 h post agroinfiltration. RNAi-mediated knockdown of each target was then assessed by letting adult L. sativae feed on agroinfiltrated pak choi leaves. Relative transcript levels of each target gene exhibit significant reductions over the feeding time, and adversely affect survival of adult L. sativae at 24 h post infestation in genetically unmodified pak choi plants. These results demonstrate that the agroinfiltration-mediated RNAi system has potential for advancing innovative environmentally safe pest management strategies for the control of leaf-mining species.

MiR1885 Regulates Disease Tolerance Genes in Brassica rapa during Early Infection with Plasmodiophora brassicae.

Clubroot caused by Plasmodiophora brassicae is a severe disease of cruciferous crops that decreases crop quality and productivity. Several clubroot resistance-related quantitative trait loci and candidate genes have been identified. However, the underlying regulatory mechanism, the interrelationships among genes, and how genes are regulated remain unexplored. MicroRNAs (miRNAs) are attracting attention as regulators of gene expression, including during biotic stress responses. The main objective of this study was to understand how miRNAs regulate clubroot resistance-related genes in P. brassicae-infected Brassica rapa. Two Brassica miRNAs, Bra-miR1885a and Bra-miR1885b, were revealed to target TIR-NBS genes. In non-infected plants, both miRNAs were expressed at low levels to maintain the balance between plant development and basal immunity. However, their expression levels increased in P. brassicae-infected plants. Both miRNAs down-regulated the expression of the TIR-NBS genes Bra019412 and Bra019410, which are located at a clubroot resistance-related quantitative trait locus. The Bra-miR1885-mediated down-regulation of both genes was detected for up to 15 days post-inoculation in the clubroot-resistant line CR Shinki and in the clubroot-susceptible line 94SK. A qRT-PCR analysis revealed Bra019412 expression was negatively regulated by miR1885. Both Bra019412 and Bra019410 were more highly expressed in CR Shinki than in 94SK; the same expression pattern was detected in multiple clubroot-resistant and clubroot-susceptible inbred lines. A 5' rapid amplification of cDNA ends analysis confirmed the cleavage of Bra019412 by Bra-miR1885b. Thus, miR1885s potentially regulate TIR-NBS gene expression during P. brassicae infections of B. rapa.

Quantitative Trait Locus Mapping of Clubroot Resistance and Plasmodiophora brassicae Pathotype Banglim-Specific Marker Development in Brassica rapa.

Clubroot resistance is an economically important trait in Brassicaceae crops. Although many quantitative trait loci (QTLs) for clubroot resistance have been identified in Brassica, disease-related damage continues to occur owing to differences in host variety and constant pathogen variation. Here, we investigated the inheritance of clubroot resistance in a double haploid population developed by crossing clubroot resistant and susceptible lines "09CR500" and "09CR501", respectively. The resistance of "09CR500" to Plasmodiophora brassicae pathotype "Banglim" was controlled as a single dominant gene, with the segregation of resistance and susceptibility being nearly 1:1. PbBrA08Banglim was identified as having a logarithm of odds value of 7.9-74.8, and a phenotypic variance of 26.0-97.1% with flanking marker "09CR.11390652" in A08. After aligning QTL regions to the B. rapa reference genome, 11 genes were selected as candidates. PbBrA08Banglim was located near Crr1, CRs, and Rcr9 loci, but differences were validated by marker analysis, gene structural variations, and gene expression levels, as well as phenotypic responses to the pathotype. Genotyping using the "09CR.11390652" marker accurately distinguished the Banglim-resistance phenotypes in the double haploid population. Thus, the developed marker will be useful in Brassica breeding programs, marker-assisted selection, and gene pyramiding to identify and develop resistant cultivars.

Mapping of a novel clubroot resistance QTL using ddRAD-seq in Chinese cabbage (Brassica rapa L.).

BACKGROUND: Plasmodiophora brassicae is a soil-borne plant pathogen that causes clubroot disease, which results in crop yield loss in cultivated Brassica species. Here, we investigated whether a quantitative trait locus (QTL) in B. rapa might confer resistance to a Korean P. brassicae pathotype isolate, Seosan. We crossed resistant and susceptible parental lines and analyzed the segregation pattern in a F2 population of 348 lines. We identified and mapped a novel clubroot resistance QTL using the same mapping population that included susceptible Chinese cabbage and resistant turnip lines. Forty-five resistant and 45 susceptible F2 lines along with their parental lines were used for double digest restriction site-associated DNA sequencing (ddRAD-seq). High resolution melting (HRM)-based validation of SNP positions was conducted to confirm the novel locus. RESULTS: A 3:1 ratio was observed for resistant: susceptible genotypes, which is in accordance with Mendelian segregation. ddRAD-seq identified a new locus, CRs, on chromosome A08 that was different from the clubroot resistance (CR) locus, Crr1. HRM analysis validated SNP positions and constricted CRs region. Four out of seventeen single nucleotide polymorphisms (SNPs) positions were within a 0.8-Mb region that included three NBS-LRR candidate genes but not Crr1. CONCLUSION: The newly identified CRs locus is a novel clubroot resistance locus, as the cultivar Akimeki bears the previously known Crr1 locus but remains susceptible to the Seosan isolate. These results could be exploited to develop molecular markers to detect Seosan-resistant genotypes and develop resistant Chinese cabbage cultivars.

Mining of Brassica-Specific Genes (BSGs) and Their Induction in Different Developmental Stages and under Plasmodiophora brassicae Stress in Brassica rapa.

Orphan genes, also called lineage-specific genes (LSGs), are important for responses to biotic and abiotic stresses, and are associated with lineage-specific structures and biological functions. To date, there have been no studies investigating gene number, gene features, or gene expression patterns of orphan genes in Brassica rapa. In this study, 1540 Brassica-specific genes (BSGs) and 1824 Cruciferae-specific genes (CSGs) were identified based on the genome of Brassica rapa. The genic features analysis indicated that BSGs and CSGs possessed a lower percentage of multi-exon genes, higher GC content, and shorter gene length than evolutionary-conserved genes (ECGs). In addition, five types of BSGs were obtained and 145 out of 529 real A subgenome-specific BSGs were verified by PCR in 51 species. In silico and semi-qPCR, gene expression analysis of BSGs suggested that BSGs are expressed in various tissue and can be induced by Plasmodiophora brassicae. Moreover, an A/C subgenome-specific BSG, BSGs1, was specifically expressed during the heading stage, indicating that the gene might be associated with leafy head formation. Our results provide valuable biological information for studying the molecular function of BSGs for Brassica-specific phenotypes and biotic stress in B. rapa.

The tandem repeated organization of NB-LRR genes in the clubroot-resistant CRb locus in Brassica rapa L.

To facilitate prevention of clubroot disease, a major threat to the successful cultivation of Chinese cabbage (Brassica rapa L.), we bred clubroot-resistant (CR) cultivars by introducing resistance genes from CR turnips via conventional breeding. Among 11 CR loci found in B. rapa, we identified CRb in Chinese cabbage cultivar 'CR Shinki' as a single dominant gene for resistance against Plasmodiophora brassicae pathotype group 3, against which the stacking of Crr1 and Crr2 loci was not effective. However, the precise location and pathotype specificity of CRb have been controversial, because CRa and Rcr1 also map near this locus. Previously, our fine-mapping study revealed that CRb is located in a 140-kb genomic region on chromosome A03. Here, we determined the nucleotide sequence of an approximately 64-kb candidate region in the resistant line; this region contains six open reading frames (ORFs) similar to NB-LRR encoding genes that are predicted to occur in tandem with the same orientation. Among the six ORFs present, only four on the genome of the resistant line showed a strong DNA sequence identity with each other, and only one of those four could confer resistance to P. brassicae isolate No. 14 of the pathotype group 3. These results suggest that these genes evolved through recent gene duplication and uneven crossover events that could lead to the acquisition of clubroot resistance. The DNA sequence of the functional ORF was identical to that of the previously cloned CRa gene; thus, we showed that the independently identified CRb and CRa are one and the same clubroot-resistance gene.

Detection of Ribosomal DNA Sequence Polymorphisms in the Protist Plasmodiophora brassicae for the Identification of Geographical Isolates.

Clubroot is a soil-borne disease caused by the protist Plasmodiophora brassicae (P. brassicae). It is one of the most economically important diseases of Brassica rapa and other cruciferous crops as it can cause remarkable yield reductions. Understanding P. brassicae genetics, and developing efficient molecular markers, is essential for effective detection of harmful races of this pathogen. Samples from 11 Korean field populations of P. brassicae (geographic isolates), collected from nine different locations in South Korea, were used in this study. Genomic DNA was extracted from the clubroot-infected samples to sequence the ribosomal DNA. Primers and probes for P. brassicae were designed using a ribosomal DNA gene sequence from a Japanese strain available in GenBank (accession number AB526843; isolate NGY). The nuclear ribosomal DNA (rDNA) sequence of P. brassicae, comprising 6932 base pairs (bp), was cloned and sequenced and found to include the small subunits (SSUs) and a large subunit (LSU), internal transcribed spacers (ITS1 and ITS2), and a 5.8s. Sequence variation was observed in both the SSU and LSU. Four markers showed useful differences in high-resolution melting analysis to identify nucleotide polymorphisms including single- nucleotide polymorphisms (SNPs), oligonucleotide polymorphisms, and insertions/deletions (InDels). A combination of three markers was able to distinguish the geographical isolates into two groups.

Assessing climate change impacts on the rape stem weevil, Ceutorhynchus napi Gyll., based on bias- and non-bias-corrected regional climate change projections.

Agricultural production is directly affected by projected increases in air temperature and changes in precipitation. A multi-model ensemble of regional climate change projections indicated shifts towards higher air temperatures and changing precipitation patterns during the summer and winter seasons up to the year 2100 for the region of Goettingen (Lower Saxony, Germany). A second major controlling factor of the agricultural production is the infestation level by pests. Based on long-term field surveys and meteorological observations, a calibration of an existing model describing the migration of the pest insect Ceutorhynchus napi was possible. To assess the impacts of climate on pests under projected changing environmental conditions, we combined the results of regional climate models with the phenological model to describe the crop invasion of this species. In order to reduce systematic differences between the output of the regional climate models and observational data sets, two different bias correction methods were applied: a linear correction for air temperature and a quantile mapping approach for precipitation. Only the results derived from the bias-corrected output of the regional climate models showed satisfying results. An earlier onset, as well as a prolongation of the possible time window for the immigration of Ceutorhynchus napi, was projected by the majority of the ensemble members.

Disarming the jasmonate-dependent plant defense makes nonhost Arabidopsis plants accessible to the American serpentine leafminer.

Here, we analyzed the interaction between Arabidopsis (Arabidopsis thaliana) and the American serpentine leafminer (Liriomyza trifolii), an important and intractable herbivore of many cultivated plants. We examined the role of the immunity-related plant hormone jasmonate (JA) in the plant response and resistance to leafminer feeding to determine whether JA affects host suitability for leafminers. The expression of marker genes for the JA-dependent plant defense was induced by leafminer feeding on Arabidopsis wild-type plants. Analyses of JA-insensitive coi1-1 mutants suggested the importance of JA in the plant response to leafminer feeding. The JA content of wild-type plants significantly increased after leafminer feeding. Moreover, coi1-1 mutants showed lower feeding resistance against leafminer attack than did wild-type plants. The number of feeding scars caused by inoculated adult leafminers in JA-insensitive coi1-1 mutants was higher than that in wild-type plants. In addition, adults of the following generation appeared only from coi1-1 mutants and not from wild-type plants, suggesting that the loss of the JA-dependent plant defense converted nonhost plants to accessible host plants. Interestingly, the glucosinolate-myrosinase defense system may play at most a minor role in this conversion, indicating that this major antiherbivore defense of Brassica species plants probably does not have a major function in plant resistance to leafminer. Application of JA to wild-type plants before leafminer feeding enhanced feeding resistance in Chinese cabbage (Brassica rapa), tomato (Solanum lycopersicum), and garland chrysanthemum (Chrysanthemum coronarium). Our results indicate that JA plays an important role in the plant response and resistance to leafminers and, in so doing, affects host plant suitability for leafminers.

Functional analysis of the key BrSWEET genes for sugar transport involved in the Brassica rapa-Plasmodiophora brassicae interaction.

Plasmodiophora brassicae, the causative agent of clubroot disease, establishes a long-lasting parasitic relationship with its host by inducing the expression of sugar transporters. Previous studies have indicated that most BrSWEET genes in Chinese cabbage are up-regulated upon infection with P. brassicae. However, the key BrSWEET genes responsive to P. brassicae have not been definitively identified. In this study, we selected five BrSWEET genes and conducted a functional analysis of them. These five BrSWEET genes showed a notable up-regulation in roots after P. brassicae inoculation. Furthermore, these BrSWEET proteins were localized to the plasma membrane. Yeast functional complementation assays confirmed transport activity for glucose, fructose, or sucrose in four BrSWEETs, with the exception of BrSWEET2a. Mutants and silenced plants of BrSWEET1a, -11a, and -12a showed lower clubroot disease severity compared to wild-type plants, while gain-of-function Arabidopsis thaliana plants overexpressing these three BrSWEET genes exhibited significantly higher disease incidence and severity. Our findings suggested that BrSWEET1a, BrSWEET11a, and BrSWEET12a play pivotal roles in P. brassicae-induced gall formation, shedding light on the role of sugar transporters in host-pathogen interactions.

Melatonin and copper oxide nanoparticles synergistically mitigate clubroot disease and enhance growth dynamics in Brassica rapa.

Clubroot, a devastating soil borne disease affecting 30%∼50% of Brassicaceae crops worldwide, lacks effective control measures. In the present study, we explored the potential of melatonin (MT) and copper oxide nanoparticle (CuO-NPs) in mitigating clubroot severity in the Brassica rapa ssp. pekinensis. Following 18 h priming with MT, CuO-NPs, or both seeds were grown in controlled environment using synthetic potting mix. Inoculated with Plasmodiophora brassicae spores on 5th day, followed by a soil drench phyto-nano treatment with a week interval. Plants were assessed for various health and growth indices including disease, biometrics, photosynthesis, reactive oxygen species (ROS), antioxidant enzyme activity, hormones and genes expression at onset of secondary clubroot infection using established protocols. Statistical analysis employed ANOVA with Fisher's LSD for significance assessment (P < 0.05). Our results revealed that seed priming with both MT (50 µMol/L) and CuO-NPs (200 mg/L), followed by soil drenching significantly reduced clubroot incidence (38%) and disease index (57%), compared to control treatments. This synergistic effect was associated with enhanced plant growth (shoots: 48% and roots: 59%). Plants treated with both MT and CuO-NPs showed robust antioxidant defenses, significantly increased superoxide dismutase (SOD (25/29%)), catalase (CAT (83/55%)), and ascorbate peroxidase (APX (83/46%)) activity in both shoots/roots, respectively, compared to infected control. Notably, salicylic acid and jasmonic acid levels doubled in treated plants, while stress hormone abscisic acid (ABA) decreased by 80% in roots and 21% in shoots. Gene expression analysis corroborated these findings, showing that the combined treatment activated antioxidant defense genes (SOD, APX and CAT) by 1.9-7.2-fold and upregulated hormone signaling genes JAZ1 (7.8-fold), MYC2 (3.9-fold) and SABP2 (36-fold). Conversely, ABA biosynthesis genes (ABA1 and NCED1) were downregulated up to 7.2-fold, while plant resistance genes NPR1, PRB1 and PDF1.2 were dramatically increased by up to 6.3-fold compared to infected plants. Overall, our combined treatment approach significantly reduces clubroot severity in B. rapa via enhanced antioxidant defenses, improved ROS scavenging, coordinated hormonal regulation and increased pathogen response genes. This study offers promising strategy for developing effective control measures against clubroot in susceptible cruciferous crops.

Metabotyping: a new approach to investigate rapeseed (Brassica napus L.) genetic diversity in the metabolic response to clubroot infection.

Clubroot disease affects all Brassicaceae spp. and is caused by the obligate biotroph pathogen Plasmodiophora brassicae. The development of galls on the root system is associated with the establishment of a new carbon metabolic sink. Here, we aimed to deepen our knowledge of the involvement of primary metabolism in the Brassica napus response to clubroot infection. We studied the dynamics and the diversity of the metabolic responses to the infection. Root system metabotyping was carried out for 18 rapeseed genotypes displaying different degrees of symptom severity, under inoculated and noninoculated conditions at 42 days postinoculation (dpi). Clubroot susceptibility was positively correlated with clubroot-induced accumulation of several amino acids. Although glucose and fructose accumulated in some genotypes with minor symptoms, their levels were negatively correlated to the disease index across the whole set of genotypes. The dynamics of the metabolic response were studied for the susceptible genotype 'Yudal,' which allowed an "early" metabolic response (established from 14 to 28 dpi) to be differentiated from a "late" response (from 35 dpi). We discuss the early accumulation of amino acids in the context of the establishment of a nitrogen metabolic sink and the hypothetical biological role of the accumulation of glutathione and S-methylcysteine.

Molecular characterization of the CRa gene conferring clubroot resistance in Brassica rapa.

Clubroot disease is one of the major diseases affecting Brassicaceae crops, and a number of these crops grown commercially, such as Chinese cabbage (Brassica rapa L. ssp. pekinensis), are known to be highly susceptible to clubroot disease. To provide protection from this disease, plant breeders have introduced genes for resistance to clubroot from the European turnip into susceptible lines. The CRa gene confers specific resistance to the clubroot pathogen Plasmodiophora brassicae isolate M85. Fine mapping of the CRa locus using synteny to the Arabidopsis thaliana genome and partial genome sequences of B. rapa revealed a candidate gene encoding a TIR-NBS-LRR protein. Several structural differences in this candidate gene were found between susceptible and resistant lines, and CRa expression was observed only in the resistant line. Four mutant lines lacking clubroot resistance were obtained by the UV irradiation of pollen from a resistant line, and all of these mutant lines carried independent mutations in the candidate TIR-NBS-LRR gene. This genetic and molecular evidence strongly suggests that the identified gene is CRa. This is the first report on the molecular characterization of a clubroot Resistance gene in Brassicaceae and of the disease resistance gene in B. rapa.

Developmental responses of the diamondback moth parasitoid Diadegma semiclausum (Hellén) (Hymenoptera: Ichneumonidae) to temperature and host plant species.

Effects of constant rearing temperature and the plant species fed upon by its hosts were investigated for several developmental parameters of Diadegma semiclausum (Hellén), an important parasitoid of the diamondback moth, Plutella xylostella (L.). Temperature had highly significant effects on all developmental parameters measured, and effects were usually both linear and quadratic with increasing temperature. Host plant species, comprising Brassica napus L., Brassica rapa L. ssp. pekinensis and Brassica oleracea L. var. capitata, also affected development of the parasitoid, and significant interactions were observed between plant species and rearing temperature for all developmental parameters measured. Development of D. semiclausum occurred successfully on all host plant species tested for the temperature range of 10 to 25°C. However, when its P. xylostella hosts consumed leaf tissue of B. napus, no specimens survived to pupate at 30°C, whilst pupation and adult eclosion occurred at 30°C on B. rapa ssp. pekinensis and B. oleracea var. capitata. At high ambient temperatures, such as those characteristic of tropical or subtropical regions (especially at low elevations) or regions that undergo temperature increases due to climate change, P. xylostella is predicted to occur at a higher range of temperatures than its biocontrol agent, D. semiclausum. Effects of high temperatures are expected to be more profound on the parasitoid for some host plants than others, with greater developmental limitations for the parasitoid on B. napus than on B. rapa or B. oleracea.

Crop sequence effects on root maggot (Diptera: Anthomyiidae: Delia spp.) infestations in canola.

Strong market demand for canola, Brassica napus L., has prompted some western Canadian producers to increase the frequency of this crop in rotations with other crop species, but the impact of this practice on canola insect pests has not been determined. Here, we investigate 12 cropping sequences involving canola over a 3-yr period (2008-2010 inclusive) at five locations across western Canada. Cropping sequences varied from continuous production of two herbicide-tolerant canola varieties, to production in two of 3 yr, to canola production in one of the 3 yr. Treatments analyzed were the frequency and timing of canola within the rotational sequence. Damage by larvae of root maggots (Diptera: Anthomyiidae: Delia spp.) to canola taproots increased as the study progressed, particularly in 2010 after canola had been grown continuously for 3 yr. Yield declined with continuous canola production, and differences were greatest in 2010. At mean canola crop prices for 2010, the yield reduction from continuous production amounted to economic losses of approximately Can$282-$377/ha. Crop quality, in terms of oil and protein concentrations of harvested seed, was affected more by crop variety than cropping sequence. Crop sequence effects for root maggot damage, yield, and seed quality were relatively stable in the presence of environmental (location) variation. Results of our study suggest that continuous canola production could be unsustainable over the long-term even though market forces currently provide incentive for this practice.

[Isolation and amplification cDNA of 8H07 gene conservative region of nematode Heterodera schachtii with high relationship to its rape homolog].

Original method of small regulatory si/miRNA isolation from plant cells was elaborated. PCR amplification of fragment cDNA 8H07 nematode Heterodera schachtii gene was carried out. Using Northern-blot method hybridization of plant si/miRNA with cDNA fragment of conservative region 8H07 gene the presence of their high homology is found out. The amplified cDNA fragment of nematode 8H07 gene in future will be used for creation recombinant gene with complementary antisense dsRNA sequence for increasing resistance of rape plants to parasitic nematodes.

Identification of expressed genes during infection of Chinese cabbage (Brassica rapa subsp. pekinensis) by Plasmodiophora brassicae.

Plasmodiophora brassicae is an obligate, biotrophic pathogen causing the club-root disease of crucifers. Despite its importance as a plant pathogen, little is known about P. brassicae at the molecular level as most of its life cycle takes place inside the plant host, and axenic culturing is impossible. Discovery of genes expressed during infection and gene organization are the first steps toward a better understanding of the pathogen-host interaction. Here, suppression subtractive hybridization was used to search for the P. brassicae genes expressed during plant infection. One-hundred and forty ESTs were found of which 49% proved to be P. brassicae genes. Ten novel P. brassicae genes were identified, and the genomic sequences surrounding four of the ESTs were acquired using genome walking. Alignment of the ESTs and the genomic DNA sequences confirmed that P. brassicae genes are intron rich and that the introns are small. These results show that it is possible to discover new P. brassicae genes from a mixed pool of both plant and pathogen cDNA. The results also revealed that some of the P. brassicae genes expressed in Chinese cabbage (Brassica rapa subsp. pekinensis) were identical to the genes expressed in the infection of Arabidopsis plants, indicating that these genes play an important role in P. brassicae infection.

Controlling Myzus persicae with recombinant endophytic fungi Chaetomium globosum expressing Pinellia ternata agglutinin: using recombinant endophytic fungi to control aphids.

AIMS: Sap-sucking insect pests have become the major threats to many crops in recent years; however, only a few biopesticides have been developed for controlling those pests. Here, we developed a novel pest management strategy, which uses endophytes to express anti-pest plant lectins. METHODS AND RESULTS: The fungal endophyte of Chaetomium globosum YY-11 with anti-fungal activities was isolated from rape seedlings. Pinellia ternata agglutinin (pta) gene was cloned into YY-11 mediated by Agrobacterium tumefaciens. The positive transformants, as selected by antibiotic resistance, were evaluated using PCR and Western blot assay. We found that the recombinant endophytes colonized most of the crops, and the resistance of rape inoculated with recombinant endophytic fungi significantly inhibited the growth and reproduction of Myzus persicae. CONCLUSIONS: Our results showed that the recombinant endophytes expressing Pinellia ernata agglutinin (PTA) may endow hosts with resistance against sap-sucking pests. SIGNIFICANCE AND IMPACT OF THE STUDY: This research may have important implications for using endophytes to deliver insecticidal plant lectin proteins to control sap-sucking pests for crop protection.

Effect of temperature on cortical infection by Plasmodiophora brassicae and clubroot severity.

A study was conducted to assess the effect of temperature on infection and development of Plasmodiophora brassicae in the root cortex of Shanghai pak choy (Brassica rapa subsp. chinensis) and on subsequent clubroot severity. Ten-day-old seedlings were grown individually, inoculated with resting spores, and maintained in growth cabinets at 10, 15, 20, 25, and 30?C. Seedlings were harvested at 2-day intervals, starting 8 days after inoculation (DAI) and continuing until 42 DAI. Roots were assessed at 4-day intervals for the incidence of cortical infection and stage of infection (young plasmodia, mature plasmodia, and resting spores), at 2-day intervals for symptom development and clubroot severity, and at 8-day intervals for the number of spores per gram of gall. Temperature affected every stage of clubroot development. Cortical infection was highest and symptoms were observed earliest at 25?C, intermediate at 20 and 30?C, and lowest and latest at 15?C. No cortical infection or symptoms were observed at 42 DAI in plants grown at 10?C. A substantial delay in the development of the pathogen was observed at 15?C. Resting spores were first observed at 38 DAI in plants at 15?C, 26 DAI at 20 and 30?C, and 22 DAI at 25?C. The yield of resting spores from galls was higher in galls that developed at 20 to 30?C than those that developed at 15?C over 42 days of assessment. These results support the observation in companion studies that cool temperatures result in slower development of clubroot symptoms in brassica crops, and demonstrate that the temperature has a consistent pattern of effect throughout the life cycle of the pathogen.

[Development of molecular markers linked to the resistant QTL for downy mildew in Brassica rapa L. ssp. pekinensis].

Downy mildew, caused by the oomycete Hyaloperonospora parasitica Constant. (Pers. ex Fr.), is one of the most severe diseases in Chinese cabbage, leading to reduction of yield and quality of the harvested products. Therefore, identifying molecular markers linked to the major QTL for downy mildew resistance will be helpful in breeding resistant varieties of Chinese cabbage. Here, one highly susceptible line 91-112, one highly resistant line T12-19, and the derived DH population were employed to develop linked molecular markers for the major QTL, BrDW, for downy mildew. With BLAST and IMap analysis, the RAPD marker K14-1030 linked to BrDW was anchored on KBrB058M10 (on Contig214). On the basis of the BAC and BAC-end sequences around KBrB058M10, a set of PCR primers were designed, and the methods of restriction analysis and HRM analysis were used to develop molecular makers. Finally, five polymorphism markers were developed, containing one Indel marker named Brb062-Indel230, three CAPS markers named Brb094-DraⅠ787, Brb094-AatⅡ666 and Brb043-BglⅡ715, and one SNP marker named Brh019-SNP137. In addition, one SSR marker from Unigene sequence homologous with KBrB058M10 (known as bru1209) was developed. The map distances between the six markers and RAPD marker K14-1030 were 4.3 cM, 1.7 cM, 5.9 cM, 5.9 cM, 4.6 cM, and 0.8 cM, respectively. The percentage of accuracy in selecting for downy mildew-resistant lines from the DH population were 69.7%, 70.9%, 72.4%, 72.4%, 58.3%, and 74.2%. These markers could be used in marker assisted selection to improve downy mildew resistance in Chinese cabbage.