Genome-wide association study of Verticillium longisporum resistance in Brassica genotypes.

Verticillium stripe, caused by Verticillium longisporum, presents an emerging threat to Canadian canola (Brassica napus). Initially detected in Manitoba in 2014, the presence of this pathogen has since been confirmed across western Canada. Infections by V. longisporum can result in yield losses of up to 50%, which is a cause for concern given the susceptibility of most commercial Canadian canola cultivars. The objective of this study was to screen a collection of 211 Brassica genotypes for their reactions to V. longisporum, and to use genome-wide association study (GWAS) to identify single nucleotide polymorphism (SNP) markers for resistance. The plant material consisted of 110 rutabaga (B. napus ssp. napobrassica), 35 canola, 40 Brassica rapa, and 15 Brassica oleracea accessions or cultivars, alongside 11 hosts of the European Clubroot Differential (ECD) set. These materials were screened for resistance under greenhouse conditions and were genotyped using a 19K Brassica SNP array. Three general linear models (GLM), four mixed linear models (MLM), and three GWAS methods were employed to evaluate the markers. Eleven non-commercial Brassica accessions and 9 out of 35 commercial canola cultivars displayed a low normalized area under the disease progress curve (AUDPCnorm.). The non-commercial accessions could prove valuable as potential sources of resistance against V. longisporum. Forty-five SNP markers were identified to be significantly associated with V. longisporum resistance using single-SNP based GWAS analysis. In comparison, haplotype-based GWAS analyses identified 10 to 25 haplotype blocks to be significantly associated with V. longisporum resistance. Between 20% and 56% of QTLs identified by the more conventional single-SNP based GWAS analysis were also detected by the haplotype-based GWAS analysis. The overlapping genomic regions identified by the two GWAS methods present promising hotspots for marker-assisted selection in the future development of Verticillium stripe-resistant canola.

Nitrate transporter protein NPF5.12 and major latex-like protein MLP6 are important defense factors against Verticillium longisporum.

Plant defense responses to the soil-borne fungus Verticillium longisporum causing stem stripe disease on oilseed rape (Brassica napus) are poorly understood. In this study, a population of recombinant inbred lines (RILs) using the Arabidopsis accessions Sei-0 and Can-0 was established. Composite interval mapping, transcriptome data, and T-DNA mutant screening identified the NITRATE/PEPTIDE TRANSPORTER FAMILY 5.12 (AtNPF5.12) gene as being associated with disease susceptibility in Can-0. Co-immunoprecipitation revealed interaction between AtNPF5.12 and the MAJOR LATEX PROTEIN family member AtMLP6, and fluorescence microscopy confirmed this interaction in the plasma membrane and endoplasmic reticulum. CRISPR/Cas9 technology was applied to mutate the NPF5.12 and MLP6 genes in B. napus. Elevated fungal growth in the npf5.12 mlp6 double mutant of both oilseed rape and Arabidopsis demonstrated the importance of these genes in defense against V. longisporum. Colonization of this fungus depends also on available nitrates in the host root. Accordingly, the negative effect of nitrate depletion on fungal growth was less pronounced in Atnpf5.12 plants with impaired nitrate transport. In addition, suberin staining revealed involvement of the NPF5.12 and MLP6 genes in suberin barrier formation. Together, these results demonstrate a dependency on multiple plant factors that leads to successful V. longisporum root infection.

Local Competition and Enhanced Defense: How Metarhizium brunneum Inhibits Verticillium longisporum in Oilseed Rape Plants.

Metarhizium brunneum is a soil-borne fungal entomopathogen that can be associated with plant roots. Previous studies have demonstrated that root colonization by beneficial fungi can directly affect soil-borne pathogens through competition and antibiosis and can activate a systemic response in plants, resulting in a primed state for a faster and/or stronger response to stressors. However, the mechanisms by which Metarhizium inoculation ameliorates symptoms caused by plant pathogens are not well known. This study evaluated the ability of M. brunneum to protect oilseed rape (Brassica napus L.) plants against the soil-borne pathogen Verticillium longisporum and investigated whether the observed effects are a result of direct interaction and/or plant-mediated effects. In vitro and greenhouse experiments were conducted to measure fungal colonization of the rhizosphere and plant tissues, and targeted gene expression analysis was used to evaluate the plant response. The results show that M. brunneum delayed pathogen colonization of plant root tissues, resulting in decreased disease symptoms. Direct competition and antibiosis were found to be part of the mechanisms, as M. brunneum growth was stimulated by the pathogen and inhibited the in vitro growth of V. longisporum. Additionally, M. brunneum changed the plant response to the pathogen by locally activating key defense hormones in the salicylic acid (SA) and abscisic acid (ABA) pathways. Using a split-root setup, it was demonstrated that there is a plant-mediated effect, as improved plant growth and decreased disease symptoms were observed when M. brunneum was in the systemic compartment. Moreover, a stronger systemic induction of the gene PR1 suggested a priming effect, involving the SA pathway. Overall, this study sheds light on the mechanisms underlying the protective effects of M. brunneum against soil-borne pathogens in oilseed rape plants, highlighting the potential of this fungal entomopathogen as a biocontrol agent in sustainable agriculture.

Tissue-Specific Hormone Signalling and Defence Gene Induction in an In Vitro Assembly of the Rapeseed Verticillium Pathosystem.

Priming plants with beneficial microbes can establish rapid and robust resistance against numerous pathogens. Here, compelling evidence is provided that the treatment of rapeseed plants with Trichoderma harzianum OMG16 and Bacillus velezensis FZB42 induces defence activation against Verticillium longisporum infection. The relative expressions of the JA biosynthesis genes LOX2 and OPR3, the ET biosynthesis genes ACS2 and ACO4 and the SA biosynthesis and signalling genes ICS1 and PR1 were analysed separately in leaf, stem and root tissues using qRT-PCR. To successfully colonize rapeseed roots, the V. longisporum strain 43 pathogen suppressed the biosynthesis of JA, ET and SA hormones in non-primed plants. Priming led to fast and strong systemic responses of JA, ET and SA biosynthesis and signalling gene expression in each leaf, stem and root tissue. Moreover, the quantification of plant hormones via UHPLC-MS analysis revealed a 1.7- and 2.6-fold increase in endogenous JA and SA in shoots of primed plants, respectively. In roots, endogenous JA and SA levels increased up to 3.9- and 2.3-fold in Vl43-infected primed plants compared to non-primed plants, respectively. Taken together, these data indicate that microbial priming stimulates rapeseed defence responses against Verticillium infection and presumably transduces defence signals from the root to the upper parts of the plant via phytohormone signalling.

Pseudomonas Strains Induce Transcriptional and Morphological Changes and Reduce Root Colonization of Verticillium spp.

Phytopathogenic Verticillia cause Verticillium wilt on numerous economically important crops. Plant infection begins at the roots, where the fungus is confronted with rhizosphere inhabiting bacteria. The effects of different fluorescent pseudomonads, including some known biocontrol agents of other plant pathogens, on fungal growth of the haploid Verticillium dahliae and/or the amphidiploid Verticillium longisporum were compared on pectin-rich medium, in microfluidic interaction channels, allowing visualization of single hyphae, or on Arabidopsis thaliana roots. We found that the potential for formation of bacterial lipopeptide syringomycin resulted in stronger growth reduction effects on saprophytic Aspergillus nidulans compared to Verticillium spp. A more detailed analyses on bacterial-fungal co-cultivation in narrow interaction channels of microfluidic devices revealed that the strongest inhibitory potential was found for Pseudomonas protegens CHA0, with its inhibitory potential depending on the presence of the GacS/GacA system controlling several bacterial metabolites. Hyphal tip polarity was altered when V. longisporum was confronted with pseudomonads in narrow interaction channels, resulting in a curly morphology instead of straight hyphal tip growth. These results support the hypothesis that the fungus attempts to evade the bacterial confrontation. Alterations due to co-cultivation with bacteria could not only be observed in fungal morphology but also in fungal transcriptome. P. protegens CHA0 alters transcriptional profiles of V. longisporum during 2 h liquid media co-cultivation in pectin-rich medium. Genes required for degradation of and growth on the carbon source pectin were down-regulated, whereas transcripts involved in redox processes were up-regulated. Thus, the secondary metabolite mediated effect of Pseudomonas isolates on Verticillium species results in a complex transcriptional response, leading to decreased growth with precautions for self-protection combined with the initiation of a change in fungal growth direction. This interplay of bacterial effects on the pathogen can be beneficial to protect plants from infection, as shown with A. thaliana root experiments. Treatment of the roots with bacteria prior to infection with V. dahliae resulted in a significant reduction of fungal root colonization. Taken together we demonstrate how pseudomonads interfere with the growth of Verticillium spp. and show that these bacteria could serve in plant protection.

In-depth evaluation of root infection systems using the vascular fungus Verticillium longisporum as soil-borne model pathogen.

BACKGROUND: While leaves are far more accessible for analysing plant defences, roots are hidden in the soil, leading to difficulties in studying soil-borne interactions. Inoculation strategies for infecting model plants with model root pathogens are described in the literature, but it remains demanding to obtain a methodological overview. To address this challenge, this study uses the model root pathogen Verticillium longisporum on Arabidopsis thaliana host plants and provides recommendations for selecting appropriate infection systems to investigate how plants cope with root pathogens. RESULTS: A novel root infection system is introduced, while two existing ones are precisely described and optimized. Step-by-step protocols are presented and accompanied by pathogenicity tests, transcriptional analyses of indole-glucosinolate marker genes and independent confirmations using reporter constructs. Advantages and disadvantages of each infection system are assessed. Overall, the results validate the importance of indole-glucosinolates as secondary metabolites that limit the Verticillium propagation in its host plant. CONCLUSION: Detailed assistances on studying host defence strategies and responses against V. longisporum is provided. Furthermore, other soil-borne microorganisms (e.g., V. dahliae) or model plants, such as economically important oilseed rape and tomato, can be introduced in the infection systems described. Hence, these proven manuals can support finding a root infection system for your specific research questions to further decipher root-microbe interactions.

A 20-kb lineage-specific genomic region tames virulence in pathogenic amphidiploid Verticillium longisporum.

Amphidiploid fungal Verticillium longisporum strains Vl43 and Vl32 colonize the plant host Brassica napus but differ in their ability to cause disease symptoms. These strains represent two V. longisporum lineages derived from different hybridization events of haploid parental Verticillium strains. Vl32 and Vl43 carry same-sex mating-type genes derived from both parental lineages. Vl32 and Vl43 similarly colonize and penetrate plant roots, but asymptomatic Vl32 proliferation in planta is lower than virulent Vl43. The highly conserved Vl43 and Vl32 genomes include less than 1% unique genes, and the karyotypes of 15 or 16 chromosomes display changed genetic synteny due to substantial genomic reshuffling. A 20 kb Vl43 lineage-specific (LS) region apparently originating from the Verticillium dahliae-related ancestor is specific for symptomatic Vl43 and encodes seven genes, including two putative transcription factors. Either partial or complete deletion of this LS region in Vl43 did not reduce virulence but led to induction of even more severe disease symptoms in rapeseed. This suggests that the LS insertion in the genome of symptomatic V. longisporum Vl43 mediates virulence-reducing functions, limits damage on the host plant, and therefore tames Vl43 from being even more virulent.

Plant roots employ cell-layer-specific programs to respond to pathogenic and beneficial microbes.

Plant roots are built of concentric cell layers that are thought to respond to microbial infections by employing specific, genetically defined programs. Yet, the functional impact of this radial organization remains elusive, particularly due to the lack of genome-wide techniques for monitoring expression at a cell-layer resolution. Here, cell-type-specific expression of tagged ribosomes enabled the isolation of ribosome-bound mRNA to obtain cell-layer translatomes (TRAP-seq, translating ribosome affinity purification and RNA sequencing). After inoculation with the vascular pathogen Verticillium longisporum, pathogenic oomycete Phytophthora parasitica, or mutualistic endophyte Serendipita indica, root cell-layer responses reflected the fundamentally different colonization strategies of these microbes. Notably, V. longisporum specifically suppressed the endodermal barrier, which restricts fungal progression, allowing microbial access to the root central cylinder. Moreover, localized biosynthesis of antimicrobial compounds and ethylene differed in response to pathogens and mutualists. These examples highlight the power of this resource to gain insights into root-microbe interactions and to develop strategies in crop improvement.

Towards Practical Application of Verticillium isaacii Vt305 to Control Verticillium Wilt of Cauliflower: Exploring Complementary Biocontrol Strategies.

Verticillium wilt is one of the most important diseases of cauliflower and can lead to serious economic losses. In this study, two complementary strategies were explored to employ the antagonistic capacity of Verticillium isaacii towards Verticillium wilt of cauliflower. The first strategy focused on introducing V. isaacii Vt305 by artificial inoculation of cauliflower plantlets at the nursery stage. Two inoculum types (spores and microsclerotia of V. isaacii Vt305) and different concentrations of microsclerotia were tested in greenhouse and field trials. Seed treatment with 500 microsclerotia seed-1 led to a satisfying biocontrol level of Verticillium wilt. In addition, the PHYTO-DRIP® system was successful in delivering the microsclerotia to cauliflower seeds. The second strategy relied on the stimulation of the natural V. isaacii populations by rotating cauliflower with green manures and potato. Four green manure crops and potato were tested during multiple field experiments. Although these crops seemed to stimulate the V. isaacii soil population, this increase did not result in a control effect on Verticillium wilt of cauliflower in the short term. Importantly, our results indicate that the use of green manures is compatible with the application of V. isaacii Vt305 as biocontrol agent of Verticillium wilt in cauliflower.

Verticillium longisporum Elicits Media-Dependent Secretome Responses With Capacity to Distinguish Between Plant-Related Environments.

Verticillia cause a vascular wilt disease affecting a broad range of economically valuable crops. The fungus enters its host plants through the roots and colonizes the vascular system. It requires extracellular proteins for a successful plant colonization. The exoproteomes of the allodiploid Verticillium longisporum upon cultivation in different media or xylem sap extracted from its host plant Brassica napus were compared. Secreted fungal proteins were identified by label free liquid chromatography-tandem mass spectrometry screening. V. longisporum induced two main secretion patterns. One response pattern was elicited in various non-plant related environments. The second pattern includes the exoprotein responses to the plant-related media, pectin-rich simulated xylem medium and pure xylem sap, which exhibited similar but additional distinct features. These exoproteomes include a shared core set of 221 secreted and similarly enriched fungal proteins. The pectin-rich medium significantly induced the secretion of 143 proteins including a number of pectin degrading enzymes, whereas xylem sap triggered a smaller but unique fungal exoproteome pattern with 32 enriched proteins. The latter pattern included proteins with domains of known pathogenicity factors, metallopeptidases and carbohydrate-active enzymes. The most abundant proteins of these different groups are the necrosis and ethylene inducing-like proteins Nlp2 and Nlp3, the cerato-platanin proteins Cp1 and Cp2, the metallopeptidases Mep1 and Mep2 and the carbohydrate-active enzymes Gla1, Amy1 and Cbd1. Their pathogenicity contribution was analyzed in the haploid parental strain V. dahliae. Deletion of the majority of the corresponding genes caused no phenotypic changes during ex planta growth or invasion and colonization of tomato plants. However, we discovered that the MEP1, NLP2, and NLP3 deletion strains were compromised in plant infections. Overall, our exoproteome approach revealed that the fungus induces specific secretion responses in different environments. The fungus has a general response to non-plant related media whereas it is able to fine-tune its exoproteome in the presence of plant material. Importantly, the xylem sap-specific exoproteome pinpointed Nlp2 and Nlp3 as single effectors required for successful V. dahliae colonization.

Phytohormone crosstalk in the host-Verticillium interaction.

Functional genomics can be applied to shed light on the Brassica napus - Verticillium interaction. RNAseq data indicated already that abscisic acid (ABA) is apparently involved in the early oilseed rape response to fungal infection with Verticillium longisporum isolate 43 (Vl43). A calreticulin (CRT1a) was identified as novel susceptibility factor for Vl43 infecting both Arabidopsis and oilseed rape. CRT1a is involved in calcium homeostasis and contributes in the endoplasmatic reticulum to the unfolded protein response. The latter function could either affect the correct folding of other susceptibility factors or of negative regulators in ethylene (ET) signaling. Which CRT1a function is affected in the mutants is currently unknown, but both hypotheses can explain that crt1a loss-of-function mutants display increased resistance to V. longisporum and enhanced expression of ethylene signaling related genes. This indicates that besides other phytohormones such as ABA or salicylic acid (SA) also ET plays a critical role in the plant-Verticillium interaction, which might be exploited to improve plant resistance.

Loss of function of CRT1a (calreticulin) reduces plant susceptibility to Verticillium longisporum in both Arabidopsis thaliana and oilseed rape (Brassica napus).

Brassica napus is highly susceptible towards Verticillium longisporum (Vl43) with no effective genetic resistance. It is believed that the fungus reprogrammes plant physiological processes by up-regulation of so-called susceptibility factors to establish a compatible interaction. By transcriptome analysis, we identified genes, which were activated/up-regulated in rapeseed after Vl43 infection. To test whether one of these genes is functionally involved in the infection process and loss of function would lead to decreased susceptibility, we firstly challenged KO lines of corresponding Arabidopsis orthologs with Vl43 and compared them with wild-type plants. Here, we report that the KO of AtCRT1a results in drastically reduced susceptibility of plants to Vl43. To prove crt1a mutation also decreases susceptibility in B. napus, we identified 10 mutations in a TILLING population. Three T3 mutants displayed increased resistance as compared to the wild type. To validate the results, we generated CRISPR/Cas-induced BnCRT1a mutants, challenged T2 plants with Vl43 and observed an overall reduced susceptibility in 3 out of 4 independent lines. Genotyping by allele-specific sequencing suggests a major effect of mutations in the CRT1a A-genome copy, while the C-genome copy appears to have no significant impact on plant susceptibility when challenged with Vl43. As revealed by transcript analysis, the loss of function of CRT1a results in activation of the ethylene signalling pathway, which may contribute to reduced susceptibility. Furthermore, this study demonstrates a novel strategy with great potential to improve plant disease resistance.

Identification and Characterization of Verticillium longisporum Lineage A1/D1 from Brassica Crops in Manitoba, Canada.

Verticillium stripe in canola (Brassica napus L.) caused by Verticillium longisporum was first reported in Manitoba in 2014. In this study, Brassica crops including canola, mustard (Brassica juncea) and radish (Raphanus sativus) with visible symptoms of Verticillium stripe were collected from Portage La Prairie, Manitoba, and the pathogens were isolated. Isolates from canola and radish were identified to V. longisporum, which produced longer conidia (7.92-12.00 µm) than Verticillium dahliae (4.32-7.04 µm). An isolate derived from mustard was characterized as V. dahliae. Molecular diagnostics with 18S rDNA, 5.8S rDNA and mating-type marker primers were used to confirm the identification of Verticillium isolates. PCR-RFLP of the mitochondrial small subunit rDNA and the cytochrome b gene were also employed to distinguish V. longisporum isolates from V. dahliae. The multi-gene characterization approach allowed for lineage determination, and V. longisporum isolates from canola and radish were in the A1/D1 group. Isolates of Verticillium longisporum from canola inoculated onto the canola cultivar 'Westar' caused symptoms of stem striping, stunting and short plants. Re-isolated fungal strains from infected stems were again inoculated onto canola plants, in order to confirm that V. longisporum was the causal agent of Verticillium stripe disease in the pathogenicity test.

Endophytic bacterial communities of oilseed rape associate with genotype-specific resistance against Verticillium longisporum.

Associations of endophytic bacterial community composition of oilseed rape (Brassica napus L.) with quantitative resistance against the soil-borne fungal pathogen Verticillium longisporum was assessed by 16S rRNA gene amplicon sequencing in roots and hypocotyls of four plant lines with contrasting genetic composition in regard to quantitative resistance reactions. The plant compartment was found to be the dominating driving factor for the specificity of bacterial communities in healthy plants. Furthermore, V. longisporum infection triggered a stabilization of phylogenetic group abundance in replicated samples suggesting a host genotype-specific selection. Genotype-specific associations with bacterial phylogenetic group abundance were identified by comparison of plant genotype groups (resistant versus susceptible) and treatment groups (healthy versus V. longisporum-infected) allowing dissection into constitutive and induced directional association patterns. Relative abundance of Flavobacteria, Pseudomonas, Rhizobium and Cellvibrio was associated with resistance/susceptibility. Relative abundance of Flavobacteria and Cellvibrio was increased in resistant genotypes according to their known ecological functions. In contrast, a higher relative abundance of Pseudomonas and Rhizobium, which are known to harbor many species with antagonistic properties to fungal pathogens, was found to be associated with susceptibility, indicating that these groups do not play a major role in genetically controlled resistance of oilseed rape against V. longisporum.

Suppression of abscisic acid biosynthesis at the early infection stage of Verticillium longisporum in oilseed rape (Brassica napus).

Verticillium longisporum infects oilseed rape (Brassica napus) and Arabidopsis thaliana. To investigate the early response of oilseed rape to the fungal infection, we determined transcriptomic changes in oilseed rape roots at 6 days post-inoculation (dpi) by RNA-Seq analysis, in which non-infected roots served as a control. Strikingly, a subset of genes involved in abscisic acid (ABA) biosynthesis was found to be down-regulated and the ABA level was accordingly attenuated in 6 dpi oilseed rape as compared with the control. Gene expression analysis revealed that this was mainly attributed to the suppression of BnNCED3-mediated ABA biosynthesis, involving, for example, BnWRKY57. However, this down-regulation of ABA biosynthesis could not be observed in infected Arabidopsis roots. Arabidopsis ABA- defective mutants nced3 and aao3 displayed pronounced tolerance to the fungal infection with delayed and impeded symptom development, even though fungal colonization was not affected in both mutants. These data suggest that ABA appears to be required for full susceptibility of Arabidopsis to the fungal infection. Furthermore, we found that in both 6 dpi oilseed rape and the Arabidopsis nced3 mutant, the salicylic acid (SA) signalling pathway was induced while the jasmonic acid (JA)/ethylene (ET) signalling pathway was concomitantly mitigated. Following these data, we conclude that in oilseed rape the V. longisporum infection triggers a host-specific suppression of the NCED3-mediated ABA biosynthesis, consequently increasing plant tolerance to the fungal infection. We believe that this might be part of the virulence strategy of V. longisporum to initiate/establish a long-lasting compatible interaction with oilseed rape (coexistence), which appears to be different from the infection process in Arabidopsis.

Analysis of the hybrid genomes of two field isolates of the soil-borne fungal species Verticillium longisporum.

BACKGROUND: Brassica plant species are attacked by a number of pathogens; among them, the ones with a soil-borne lifestyle have become increasingly important. Verticillium stem stripe caused by Verticillium longisporum is one example. This fungal species is thought to be of a hybrid origin, having a genome composed of combinations of lineages denominated A and D. In this study we report the draft genomes of 2 V. longisporum field isolates sequenced using the Illumina technology. Genomic characterization and lineage composition, followed by selected gene analysis to facilitate the comprehension of its genomic features and potential effector categories were performed. RESULTS: The draft genomes of 2 Verticillium longisporum single spore isolates (VL1 and VL2) have an estimated ungapped size of about 70 Mb. The total number of protein encoding genes identified in VL1 was 20,793, whereas 21,072 gene models were predicted in VL2. The predicted genome size, gene contents, including the gene families coding for carbohydrate active enzymes were almost double the numbers found in V. dahliae and V. albo-atrum. Single nucleotide polymorphisms (SNPs) were frequently distributed in the two genomes but the distribution of heterozygosity and depth was not independent. Further analysis of potential parental lineages suggests that the V. longisporum genome is composed of two parts, A1 and D1, where A1 is more ancient than the parental lineage genome D1, the latter being more closer related to V. dahliae. Presence of the mating-type genes MAT1-1-1 and MAT1-2-1 in the V. longisporum genomes were confirmed. However, the MAT genes in V. dahliae, V. albo-atrum and V. longisporum have experienced extensive nucleotide changes at least partly explaining the present asexual nature of these fungal species. CONCLUSIONS: The established draft genome of V. longisporum is comparatively large compared to other studied ascomycete fungi. Consequently, high numbers of genes were predicted in the two V. longisporum genomes, among them many secreted proteins and carbohydrate active enzyme (CAZy) encoding genes. The genome is composed of two parts, where one lineage is more ancient than the part being more closely related to V. dahliae. Dissimilar mating-type sequences were identified indicating possible ancient hybridization events.

Aerial Warfare: A Volatile Dialogue between the Plant Pathogen Verticillium longisporum and Its Antagonist Paenibacillus polymyxa.

Verticillium wilt caused by Verticillium spp. results in severe yield losses in a broad range of crops. Verticillium outbreaks are challenging to control, and exacerbated by increases in soil temperatures and drought associated with global warming. Employing natural antagonists as biocontrol agents offers a promising approach to addressing this challenge. Paenibacillus polymyxa Sb3-1 was proven to reduce the growth of Verticillium longisporum during in vitro experiments and was shown to promote the growth of oilseed rape seedlings infested with V. longisporum. Our novel approach combined in vitro and in planta methods with the study of the mode of interaction between Sb3-1 and V. longisporum EVL43 via their volatile organic compounds (VOCs). Volatile and soluble substances, produced by both microorganisms as a reaction to one another's VOCs, were detected by using both gas and liquid chromatography-mass spectrometry. P. polymyxa Sb3-1 continually produced antimicrobial and plant growth promoting VOCs, such as 2-nonanone and 3-hydroxy-2-butanone. Several other antimicrobial volatile substances, such as isoamyl acetate and durenol, were downregulated. The general metabolic activity of Sb3-1, including protein and DNA biotransformations, was upregulated upon contact with EVL43 VOCs. V. longisporum increased its production of antimicrobial substances, such as 1-butanol, and downregulated its metabolic activities upon exposure to Sb3-1 VOCs. Additionally, several stress response substances such as arabitol and protein breakdown products (e.g., L-Isoleucyl-L-glutamic acid), were increased in the co-incubated samples. The results obtained depict an ongoing dialog between these microorganisms resulting in growth inhibition, the slowing down of metabolism, and the cell death of V. longisporum due to contact with the P. polymyxa Sb3-1 VOCs. Moreover, the results indicate that VOCs make a substantial contribution to the interaction between pathogens and their natural antagonists and have the potential to control pathogens in a novel, environmentally friendly manner.

Brassica napus L. cultivars show a broad variability in their morphology, physiology and metabolite levels in response to sulfur limitations and to pathogen attack.

Under adequate sulfur supply, plants accumulate sulfate in the vacuoles and use sulfur-containing metabolites as storage compounds. Under sulfur-limiting conditions, these pools of stored sulfur-compounds are depleted in order to balance the nitrogen to sulfur ratio for protein synthesis. Stress conditions like sulfur limitation and/or pathogen attack induce changes in the sulfate pool and the levels of sulfur-containing metabolites, which often depend on the ecotypes or cultivars. We are interested in investigating the influence of the genetic background of canola (Brassica napus) cultivars in sulfur-limiting conditions on the resistance against Verticillium longisporum. Therefore, four commercially available B. napus cultivars were analyzed. These high-performing cultivars differ in some characteristics described in their cultivar pass, such as several agronomic traits, differences in the size of the root system, and resistance to certain pathogens, such as Phoma and Verticillium. The objectives of the study were to examine and explore the patterns of morphological, physiological and metabolic diversity in these B. napus cultivars at different sulfur concentrations and in the context of plant defense. Results indicate that the root systems are influenced differently by sulfur deficiency in the cultivars. Total root dry mass and length of root hairs differ not only among the cultivars but also vary in their reaction to sulfur limitation and pathogen attack. As a sensitive indicator of stress, several parameters of photosynthetic activity determined by PAM imaging showed a broad variability among the treatments. These results were supported by thermographic analysis. Levels of sulfur-containing metabolites also showed large variations. The data were interrelated to predict the specific behavior during sulfur limitation and/or pathogen attack. Advice for farming are discussed.

Susceptibility to Verticillium longisporum is linked to monoterpene production by TPS23/27 in Arabidopsis.

The fungus Verticillium longisporum is a soil-borne plant pathogen of increasing economic importance, and information on plant responses to it is limited. To identify the genes and components involved in the early stages of infection, transcripts in roots of V. longisporum-challenged Arabidopsis Col-0 and the susceptible NON-RACE SPECIFIC DISEASE RESISTANCE 1 (ndr1-1) mutant were compared using ATH1 gene chips. The analysis revealed altered transcript levels of several terpene biosynthesis genes, including the monoterpene synthase TPS23/27. When transgenic 35S:TPS23/27 and TPS23/27-amiRNA plants were monitored the over-expresser line showed enhanced fungal colonization whereas the silenced genotype was indistinguishable from Col-0. Transcript analysis of terpene biosynthesis genes suggested that only the TPS23/27 pathway is affected in the two transgenic genotypes. To confirm changes in monoterpene production, emitted volatiles were determined using solid-phase microextraction and gas chromatography-mass spectrometry. Levels of all identified TPS23/27 monoterpene products were significantly altered in the transgenic plants. A stimulatory effect on conidial germination and hyphal growth of V. longisporum was also seen in co-cultivation with 35S:TPS23/27 plants and upon exposure to 1,8-cineole, the main product of TPS23/27. Methyl jasmonate treatments of myc2-1 and myc2-2 mutants and analysis of TPS23/27:uidA in the myc2-2 background suggested a dependence on jasmonic acid mediated by the transcription factor MYC2. Taken together, our results show that TPS23/27-produced monoterpenes stimulate germination and subsequent invasion of V. longisporum in Arabidopsis roots.

Identification and characterization of microRNAs in oilseed rape (Brassica napus) responsive to infection with the pathogenic fungus Verticillium longisporum using Brassica AA (Brassica rapa) and CC (Brassica oleracea) as reference genomes.

Verticillium longisporum, a soil-borne pathogenic fungus, causes vascular disease in oilseed rape (Brassica napus). We proposed that plant microRNAs (miRNAs) are involved in the plant-V. longisporum interaction. To identify oilseed rape miRNAs, we deep-sequenced two small RNA libraries made from V. longisporum infected/noninfected roots and employed Brassica rapa and Brassica oleracea genomes as references for miRNA prediction and characterization. We identified 893 B. napus miRNAs representing 360 conserved and 533 novel miRNAs, and mapped 429 and 464 miRNAs to the AA and CC genomes, respectively. Microsynteny analysis with the conserved miRNAs and their flanking protein coding sequences revealed 137 AA-CC genome syntenic miRNA pairs and 61 AA and 42 CC genome-unique miRNAs. Sixty-two miRNAs were responsive to the V. longisporum infection. We present data for specific interactions and simultaneously reciprocal changes in the expression levels of the miRNAs and their targets in the infected roots. We demonstrate that miRNAs are involved in the plant-fungus interaction and that miRNA168-Argonaute 1 (AGO1) expression modulation might act as a key regulatory module in a compatible plant-V. longisporum interaction. Our results suggest that V. longisporum may have evolved a virulence mechanism by interference with plant miRNAs to reprogram plant gene expression and achieve infection.