Minimal impacts on the wheat microbiome when Trichoderma gamsii T6085 is applied as a biocontrol agent to manage fusarium head blight disease.

Fusarium head blight (FHB) is a major fungal disease that causes severe yield and quality loss in wheat. Biological control can be integrated with other management strategies to control FHB. For this purpose, Trichoderma gamsii strain T6085 is a potential biocontrol agent to limit the infection of F. graminearum and F. culmorum in wheat. However, the possible impacts of T. gamsii T6085 on the broader microbiome associated with the wheat plant are not currently understood. Therefore, we identified bacteria and fungi associated with different wheat tissues, including assessment of their relative abundances and dynamics in response to the application of T6085 and over time, using amplicon sequencing. Residues of the prior year's wheat crop and the current year's wheat spikes were collected at multiple time points, and kernel samples were collected at harvest. DNA was extracted from the collected wheat tissues, and amplicon sequencing was performed to profile microbiomes using 16S v4 rRNA amplicons for bacteria and ITS2 amplicons for fungi. Quantitative PCR was performed to evaluate the absolute abundances of F. graminearum and T. gamsii in different wheat tissues. Disease progression was tracked visually during the growing season, revealing that FHB severity and incidence were significantly reduced when T6085 was applied to wheat spikes at anthesis. However, treatment with T6085 did not lessen the F. graminearum abundance in wheat spikes or kernels. There were substantial changes in F. graminearum abundance over time; in crop residue, pathogen abundance was highest at the initial time point and declined over time, while in wheat spikes, pathogen abundance increased significantly over time. The predominant bacterial taxa in wheat spikes and kernels were Pseudomonas, Enterobacter, and Pantoea, while Alternaria and Fusarium were the dominant fungal groups. Although the microbiome structure changed substantially over time, there were no community-scale rearrangements due to the T6085 treatment. The work suggests several other taxa that could be explored as potential biocontrol agents to integrate with T6085 treatment. However, the timing and the type of T6085 application need to be improved to give more advantages for T6085 to colonize and reduce the F. graminearum inoculum in the field.

Achievements, Developments and Future Challenges in the Field of Bioherbicides for Weed Control: A Global Review.

The intrusion of weeds into fertile areas has resulted in significant global economic and environmental impacts on agricultural production systems and native ecosystems, hence without ongoing and repeated management actions, the maintenance or restoration of these systems will become increasingly challenging. The establishment of herbicide resistance in many species and unwanted pollution caused by synthetic herbicides has ushered in the need for alternative, eco-friendly sustainable management strategies, such as the use of bioherbicides. Of the array of bioherbicides currently available, the most successful products appear to be sourced from fungi (mycoherbicides), with at least 16 products being developed for commercial use globally. Over the last few decades, bioherbicides sourced from bacteria and plant extracts (such as allelochemicals and essential oils), together with viruses, have also shown marked success in controlling various weeds. Despite this encouraging trend, ongoing research is still required for these compounds to be economically viable and successful in the long term. It is apparent that more focused research is required for (i) the improvement of the commercialisation processes, including the cost-effectiveness and scale of production of these materials; (ii) the discovery of new production sources, such as bacteria, fungi, plants or viruses and (iii) the understanding of the environmental influence on the efficacy of these compounds, such as atmospheric CO2, humidity, soil water stress, temperature and UV radiation.

Tissue-specific mRNA profiling of the Brassica napus-Sclerotinia sclerotiorum interaction uncovers novel regulators of plant immunity.

White mold is caused by the fungal pathogen Sclerotinia sclerotiorum and leads to rapid and significant loss in plant yield. Among its many brassicaceous hosts, including Brassica napus (canola) and Arabidopsis, the response of individual tissue layers directly at the site of infection has yet to be explored. Using laser microdissection coupled with RNA sequencing, we profiled the epidermis, mesophyll, and vascular leaf tissue layers of B. napus in response to S. sclerotiorum. High-throughput tissue-specific mRNA sequencing increased the total number of detected transcripts compared with whole-leaf assessments and provided novel insight into the conserved and specific roles of ontogenetically distinct leaf tissue layers in response to infection. When subjected to pathogen infection, the epidermis, mesophyll, and vasculature activate both specific and shared gene sets. Putative defense genes identified through transcription factor network analysis were then screened for susceptibility against necrotrophic, hemi-biotrophic, and biotrophic pathogens. Arabidopsis deficient in PR5-like RECEPTOR KINASE (PR5K) mRNA levels were universally susceptible to all pathogens tested and were further characterized to identify putative interacting partners involved in the PR5K signaling pathway. Together, these data provide insight into the complexity of the plant defense response directly at the site of infection.

Activation of NLR-Mediated Autoimmunity in Arabidopsis Early in Short Days 4 Mutant.

From a reverse genetic screen using CRISPR/Cas9 gene editing tool, we unintentionally identified an autoimmune mutant. Map-based cloning and whole-genome sequencing revealed that it contains a deletion in SMALL UBIQUITIN-RELATED MODIFIER (SUMO) protease encoding gene EARLY IN SHORT DAYS 4 (ESD4). Previous studies reported that esd4 mutants accumulate elevated levels of plant defense hormone salicylic acid (SA). However, upregulated PATHOGENESIS-RELATED GENE 1 (PR1) expression in esd4 only partly relies on SA level. In this study, we show that plant metabolite N-hydroxypipecolic acid (NHP) biosynthetic genes are upregulated in esd4, and NHP biosynthesis mutant flavin-dependent-monooxygenase 1 (fmo1) partially suppresses the autoimmune phenotypes of esd4, suggestive of a requirement of NHP signaling for the autoimmunity in esd4. As activation of nucleotide-binding leucine-rich repeat immune receptors (NLRs) are associates with the biosynthesis of SA and NHP and lipase-like protein ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) is a key component downstream of many NLRs, we examined the relationship between EDS1 and ESD4 by analyzing the eds1 esd4 double mutant. We found that eds1 largely suppresses esd4 autoimmunity and blocks the elevated expressions of SA and NHP biosynthesis-related genes in esd4. Overall, our study provides evidence supporting the hypothesis that SUMO protease ESD4 likely targets a yet to be identified guardee of NLR by removing its SUMO modification to avoid recognition by the cognate NLR. Loss of ESD4 results in activation of NLR-mediated autoimmunity.

Naturally Occurring Fusarium Species and Mycotoxins in Oat Grains from Manitoba, Canada.

Fusarium head blight (FHB) can lead to dramatic yield losses and mycotoxin contamination in small grain cereals in Canada. To assess the extent and severity of FHB in oat, samples collected from 168 commercial oat fields in the province of Manitoba, Canada, during 2016-2018 were analyzed for the occurrence of Fusarium head blight and associated mycotoxins. Through morphological and molecular analysis, F. poae was found to be the predominant Fusarium species affecting oat, followed by F. graminearum, F. sporotrichioides, F. avenaceum, and F. culmorum. Deoxynivalenol (DON) and nivalenol (NIV), type B trichothecenes, were the two most abundant Fusarium mycotoxins detected in oat. Beauvericin (BEA) was also frequently detected, though at lower concentrations. Close clustering of F. poae and NIV/BEA, F. graminearum and DON, and F. sporotrichioides and HT2/T2 (type A trichothecenes) was detected in the principal component analysis. Sampling location and crop rotation significantly impacted the concentrations of Fusarium mycotoxins in oat. A phylogenetic analysis of 95 F. poae strains from Manitoba was conducted using the concatenated nucleotide sequences of Tef-1α, Tri1, and Tri8 genes. The results indicated that all F. poae strains belong to a monophyletic lineage. Four subgroups of F. poae strains were identified; however, no correlations were observed between the grouping of F. poae strains and sample locations/crop rotations.

Transcriptome Alterations of an in vitro-Selected, Moderately Resistant, Two-Row Malting Barley in Response to 3ADON, 15ADON, and NIV Chemotypes of Fusarium graminearum.

Fusarium head blight caused by Fusarium graminearum is a devastating disease of malting barley. Mycotoxins associated with contaminated grain can be transferred from malt to beer and pose a health risk to consumers. In western Canada, F. graminearum has undergone an adaptive shift from 15ADON constituency to dominance by virulent 3ADON-producers; likewise, NIV-producers have established in regions of southern United States. Lack of adapted resistance sources with adequate malting quality has promoted the use of alternative breeding methodologies, such as in vitro selection. We studied the low-deoxynivalenol characteristic of in vitro selected, two-row malting barley variety "Norman" by RNAseq in contrast to its parental line "CDC Kendall," when infected by 15ADON-, 3ADON-, and NIV-producing isolates of F. graminearum. The current study documents higher mycotoxin accumulation by 3ADON isolates, thereby representing increased threat to barley production. At 72-96-h post infection, significant alterations in transcription patterns were observed in both varieties with pronounced upregulation of the phenylpropanoid pathway and detoxification gene categories (UGT, GST, CyP450, and ABC), particularly in 3ADON treatment. Defense response was multitiered, where differential expression in "Norman" associated with antimicrobial peptides (thionin 2.1, defensing, non-specific lipid-transfer protein) and stress-related proteins, such as late embryogenesis abundant proteins, heat-shock, desiccation related, and a peroxidase (HvPrx5). Several gene targets identified in "Norman" would be useful for application of breeding varieties with reduced deoxynivalenol content.

Validating the Strategic Deployment of Blackleg Resistance Gene Groups in Commercial Canola Fields on the Canadian Prairies.

Blackleg, caused by the fungal pathogen Leptosphaeria maculans, is a serious threat to canola (Brassica napus L.) production in western Canada. Crop scouting and extended crop rotation, along with the use of effective genetic resistance, have been key management practices available to mitigate the impact of the disease. In recent years, new pathogen races have reduced the effectiveness of some of the resistant cultivars deployed. Strategic deployment and rotation of major resistance (R) genes in cultivars have been used in France and Australia to help increase the longevity of blackleg resistance. Canada also introduced a grouping system in 2017 to identify blackleg R genes in canola cultivars. The main objective of this study was to examine and validate the concept of R gene deployment through monitoring the avirulence (Avr) profile of L. maculans population and disease levels in commercial canola fields within the Canadian prairies. Blackleg disease incidence and severity was collected from 146 cultivars from 53 sites across Manitoba, Saskatchewan, and Alberta in 2018 and 2019, and the results varied significantly between gene groups, which is likely influenced by the pathogen population. Isolates collected from spring and fall stubble residues were examined for the presence of Avr alleles AvrLm1, AvrLm2, AvrLm3, AvrLm4, AvrLm5, AvrLm6, AvrLm7, AvrLm9, AvrLm10, AvrLm11, AvrLepR1, AvrLepR2, AvrLep3, and AvrLmS using a set of differential host genotypes carrying known resistance genes or PCR-based markers. The Simpson's evenness index was very low, due to two dominant L. maculans races (AvrLm2-4-5-6-7-10-11 and AvrLm2-5-6-7-10-11) representing 49% of the population, but diversity of the population was high from the 35 L. maculans races isolated in Manitoba. AvrLm6 and AvrLm11 were found in all 254 L. maculans isolates collected in Manitoba. Knowledge of the blackleg disease levels in relation to the R genes deployed, along with the L. maculans Avr profile, helps to measure the effectiveness of genetic resistance.

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.

Hormonal Responses to Susceptible, Intermediate, and Resistant Interactions in the Brassica napus-Leptosphaeria maculans Pathosystem.

Hormone signaling plays a pivotal role in plant-microbe interactions. There are three major phytohormones in plant defense: salicylic acid (SA), jasmonic acid (JA), and ethylene (ET). The activation and trade-off of signaling between these three hormones likely determines the strength of plant defense in response to pathogens. Here, we describe the allocation of hormonal signaling in Brassica napus against the fungal pathogen Leptosphaeria maculans. Three B. napus genotypes (Westar, Surpass400, and 01-23-2-1) were inoculated with two L. maculans isolates (H75 8-1 and H77 7-2), subsequently exhibiting three levels of resistance: susceptible, intermediate, and resistant. Quantitative analyses suggest that the early activation of some SA-responsive genes, including WRKY70 and NPR1, contribute to an effective defense against L. maculans. The co-expression among factors responding to SA/ET/JA was also observed in the late stage of infection. The results of conjugated SA measurement also support that early SA activation plays a crucial role in durable resistance. Our results demonstrate the relationship between the onset patterns of certain hormone regulators and the effectiveness of the defense of B. napus against L. maculans.

Population Genetic Structure and Chemotype Diversity of Fusarium graminearum Populations from Wheat in Canada and North Eastern United States.

Fusarium head blight (FHB) is a major disease in wheat causing severe economic losses globally by reducing yield and contaminating grain with mycotoxins. In Canada, Fusarium graminearum is the principal etiological agent of FHB in wheat, producing mainly the trichothecene mycotoxin, deoxynivalenol (DON) and its acetyl derivatives (15-acetyl deoxynivalenol (15ADON) and 3-acetyl deoxynivalenol (3ADON)). Understanding the population biology of F. graminearum such as the genetic variability, as well as mycotoxin chemotype diversity among isolates is important in developing sustainable disease management tools. In this study, 570 F. graminearum isolates collected from commercial wheat crops in five geographic regions in three provinces in Canada in 2018 and 2019 were analyzed for population diversity and structure using 10 variable number of tandem repeats (VNTR) markers. A subset of isolates collected from the north-eastern United States was also included for comparative analysis. About 75% of the isolates collected in the Canadian provinces of Saskatchewan and Manitoba were 3ADON indicating a 6-fold increase in Saskatchewan and a 2.5-fold increase in Manitoba within the past 15 years. All isolates from Ontario and those collected from the United States were 15ADON and isolates had a similar population structure. There was high gene diversity (H = 0.803-0.893) in the F. graminearum populations in all regions. Gene flow was high between Saskatchewan and Manitoba (Nm = 4.971-21.750), indicating no genetic differentiation between these regions. In contrast, less gene flow was observed among the western provinces and Ontario (Nm = 3.829-9.756) and USA isolates ((Nm = 2.803-6.150). However, Bayesian clustering model analyses of trichothecene chemotype subpopulations divided the populations into two clusters, which was correlated with trichothecene types. Additionally, population cluster analysis revealed there was more admixture of isolates among isolates of the 3ADON chemotypes than among the 15ADON chemotype, an observation that could play a role in the increased virulence of F. graminearum. Understanding the population genetic structure and mycotoxin chemotype variations of the pathogen will assist in developing FHB resistant wheat cultivars and in mycotoxin risk assessment in Canada.

Microbial Community Dynamics of Soybean (Glycine max) Is Affected by Cropping Sequence.

The microbial composition of the rhizosphere soil could be an important determinant of crop yield, pathogen resistance, and other beneficial attributes in plants. However, little is known about the impact of cropping sequences on microbial community dynamics, especially in economically important species like soybean. Using 2-year crop sequences of corn-soybean, canola-soybean, and soybean-soybean, we investigated how crops from the previous growing season influenced the structure of the microbiome in both the bulk soil and soybean rhizosphere. A combination of marker-based Illumina sequencing and bioinformatics analyses was used to show that bacterial species richness and evenness in the soybean rhizosphere soil were similar following canola and soybean compared to a previous corn sequence. However, fungal species richness and evenness remained unaffected by crop sequence. In addition, bacterial and fungal species diversity in both the bulk and soybean rhizosphere soil were not influenced by crop sequence. Lastly, the corn-soybean sequence significantly differed in the relative abundance of certain bacterial and fungal classes in both the soybean rhizosphere and bulk soil. While canola-soybean and a continuous soybean sequence did not, suggesting that a preceding corn sequence may reduce the occurrence of overall bacterial and fungal community members. For the present study, crop sequence impacts bacterial diversity and richness in both the bulk soil and soybean rhizosphere soil whereas fungal diversity and richness are resilient to crop sequence practices. Together, these findings could help drive decision making for annual crop and soil management practices.

GPTransformer: A Transformer-Based Deep Learning Method for Predicting Fusarium Related Traits in Barley.

Fusarium head blight (FHB) incited by Fusarium graminearum Schwabe is a devastating disease of barley and other cereal crops worldwide. Fusarium head blight is associated with trichothecene mycotoxins such as deoxynivalenol (DON), which contaminates grains, making them unfit for malting or animal feed industries. While genetically resistant cultivars offer the best economic and environmentally responsible means to mitigate disease, parent lines with adequate resistance are limited in barley. Resistance breeding based upon quantitative genetic gains has been slow to date, due to intensive labor requirements of disease nurseries. The production of a high-throughput genome-wide molecular marker assembly for barley permits use in development of genomic prediction models for traits of economic importance to this crop. A diverse panel consisting of 400 two-row spring barley lines was assembled to focus on Canadian barley breeding programs. The panel was evaluated for FHB and DON content in three environments and over 2 years. Moreover, it was genotyped using an Illumina Infinium High-Throughput Screening (HTS) iSelect custom beadchip array of single nucleotide polymorphic molecular markers (50 K SNP), where over 23 K molecular markers were polymorphic. Genomic prediction has been demonstrated to successfully reduce FHB and DON content in cereals using various statistical models. Herein, we have studied an alternative method based on machine learning and compare it with a statistical approach. The bi-allelic SNPs represented pairs of alleles and were encoded in two ways: as categorical (-1, 0, 1) or using Hardy-Weinberg probability frequencies. This was followed by selecting essential genomic markers for phenotype prediction. Subsequently, a Transformer-based deep learning algorithm was applied to predict FHB and DON. Apart from the Transformer method, a Residual Fully Connected Neural Network (RFCNN) was also applied. Pearson correlation coefficients were calculated to compare true vs. predicted outputs. Models which included all markers generally showed marginal improvement in prediction. Hardy-Weinberg encoding generally improved correlation for FHB (6.9%) and DON (9.6%) for the Transformer network. This study suggests the potential of the Transformer based method as an alternative to the popular BLUP model for genomic prediction of complex traits such as FHB or DON, having performed equally or better than existing machine learning and statistical methods.

Genome-Wide Identification and Analysis of the Valine-Glutamine Motif-Containing Gene Family in Brassica napus and Functional Characterization of BnMKS1 in Response to Leptosphaeria maculans.

Proteins containing valine-glutamine (VQ) motifs play important roles in plant growth and development as well as in defense responses to both abiotic and biotic stresses. Blackleg disease, which is caused by Leptosphaeria maculans, is the most important disease in canola (Brassica napus) worldwide; however, the identification of Brassica napus VQs and their functions in response to blackleg disease have not yet been reported. In this study, we conducted a genome-wide identification and characterization of the VQ gene family in Brassica napus, including chromosome location, phylogenetic relations, gene structure, motif domain, synteny analysis, and cis-elements categorization of their promoter regions. To understand Brassica napus VQ gene function in response to blackleg disease, we overexpressed BnVQ7 (BnaA01g36880D, also known as the mitogen-activated protein kinase 4 substrate 1 [MKS1] gene) in a blackleg-susceptible canola variety, Westar. Overexpression of BnMKS1 in canola did not improve its resistance to blackleg disease at the seedling stage; however, transgenic canola plants overexpressing BnMKS1 displayed an enhanced resistance to L. maculans infection at the adult plant stage. Expression levels of downstream and defense marker genes in cotyledons increased significantly at the necrotrophic stage of L. maculans infection in the overexpression line of BnMKS1, suggesting that the salicylic acid- and jasmonic acid-mediated signaling pathways were both involved in the defense responses. Together, these results suggest that BnMKS1 might play an important role in defense against L. maculans.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Directing Trophic Divergence in Plant-Pathogen Interactions: Antagonistic Phytohormones With NO Doubt?

A fundamental process culminating in the mechanisms of plant-pathogen interactions is the regulation of trophic divergence into biotrophic, hemibiotrophic, and necrotrophic interactions. Plant hormones, of almost all types, play significant roles in this regulatory apparatus. In plant-pathogen interactions, two classical mechanisms underlying hormone-dependent trophic divergence are long recognized. While salicylic acid dominates in the execution of host defense response against biotrophic and early-stage hemibiotrophic pathogens, jasmonic acid, and ethylene are key players facilitating host defense response against necrotrophic and later-stage hemibiotrophic pathogens. Evidence increasingly suggests that trophic divergence appears to be modulated by more complex signaling networks. Acting antagonistically or agonistically, other hormones such as auxins, cytokinins, abscisic acid, gibberellins, brassinosteroids, and strigolactones, as well as nitric oxide, are emerging candidates in the regulation of trophic divergence. In this review, the latest advances in the dynamic regulation of trophic divergence are summarized, emphasizing common and contrasting hormonal and nitric oxide signaling strategies deployed in plant-pathogen interactions.

Development of a specific marker for detection of a functional AvrLm9 allele and validating the interaction between AvrLm7 and AvrLm9 in Leptosphaeria maculans.

Blackleg, which is caused by the fungus Leptosphaeria maculans (L. maculans), is a major disease of canola in western Canada and worldwide. Long-term use of one source of resistance could cause the breakdown of its effectiveness. Therefore, appropriate use of R genes is very important, and knowledge about the distribution of avirulence genes is a prerequisite for effectively deploying resistance. Of the 14 avirulence genes identified in L. maculans, AvrLm5 and AvrLm9 were recognized as the two alleles of the same gene based on two single nucleotide polymorphisms, C85T and G164A/C. In this study, a specific marker was developed to identify AvrLm5 and AvrLm9 based on two single nucleotide polymorphisms, C85T and G164A/C, which are responsible for the function of AvrLm9. The specific marker can be used to discriminate the AvrLm9 from avrLm9 accurately in L. maculans isolates, which is consistent with inoculation tests in isolates without AvrLm4-7. This specific marker was used to screen 1229 isolates collected from fields in the years 2014 through 2016 in Manitoba. From 68 to 84% of the isolates were found to contain the AvrLm9 allele; while 4-7% of them were avirulent on the variety Goéland with Rlm9 loci. Furthermore, no isolates having both AvrLm9 and AvrLm7 were detected using a cotyledon test, while 67% to 84% of isolates contained both avirulence genes via PCR detection, implying suppression of AvrLm9 by AvrLm7. In addition, avirulence gene profiles of the other 10 avirulence alleles were examined with the 1229 isolates using cotyledon tests or PCR amplifications. Taken together, this research enables the fast identification of AvrLm5/9, provides the Avr genes' landscape of western Canada and elaborates the relationship between AvrLm9 and AvrLm7 using isolates from grower fields.

Bacterial Blight Induced Shifts in Endophytic Microbiome of Rice Leaves and the Enrichment of Specific Bacterial Strains With Pathogen Antagonism.

The endophytic microbiome plays an important role in plant health and pathogenesis. However, little is known about its relationship with bacterial blight (BB) of rice caused by Xanthomonas oryzae pv. oryzae (Xoo). The current study compared the community compositional structure of the endophytic microbiota in healthy and BB symptomatic leaves of rice through a metabarcoding approach, which revealed BB induced a decrease in the alpha-diversity of the fungal communities and an increase in the bacterial communities. BB-diseased rice leaves were enriched with saprophytic fungi that are capable of decomposing plant cell walls (e.g. Khuskia spp. and Leptosphaerulina spp.), while healthy rice leaves were found to be significantly more abundant with plant pathogens or mycotoxin-producing fungi (e.g. Fusarium, Magnaporthe, and Aspergillus). The endophytic bacterial communities of BB-diseased leaves were significantly enriched with Pantoea, Pseudomonas, and Curtobacterium, strains. Pantoea sp. isolates from BB leaves are identified as promising candidates for the biocontrol of BB for their ability to inhibit in vitro growth of Xoo, suppress the development of rice BB disease, and possess multiple PGP characteristics. Our study revealed BB-induced complexed changes in the endophytic fungal and bacterial communities of rice leaves and demonstrated that BB-associated enrichment of some endophytic bacterial taxa, e.g. Pantoea sp. isolates, may play important roles in suppressing the development of BB disease in rice.

Generation and Characterization of a Virulent Leptosphaeria maculans Isolate Carrying a Mutated AvrLm7 Gene Using the CRISPR/Cas9 System.

Blackleg, caused by the fungal pathogen Leptosphaeria maculans, is the most important disease affecting canola (Brassica napus) crops worldwide. We employed the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system to generate the mutant isolate umavr7 from a point mutation of the AvrLm7 coding region in a L. maculans isolate (UMAvr7). Reverse transcription PCR and transcriptome data confirmed that the AvrLm7 gene was knocked out in the mutant isolate. Pathogenicity tests indicated that umavr7 can cause large lesions on a set of Brassica differential genotypes that express different resistance (R) genes. Comparative pathogenicity tests between UMAvr7 (wild type) and umavr7 on the corresponding B. napus genotype 01-23-2-1 (with Rlm7) showed that umavr7 is a mutant isolate, producing large gray/green lesions on cotyledons. The pathogenicity of the mutant isolate was shifted from avirulent to virulent on the B. napus Rlm7 genotype. Therefore, this mutant is virulence on the identified resistant genes to blackleg disease in B. napus genotypes. Superoxide accumulated differently in cotyledons in response to infection with UMAvr7 and umavr7, especially in resistant B. napus genotype 01-23-2-1. Resistance/susceptibility was further evaluated on 123 B. napus genotypes with the mutant isolate, umavr7. Only 6 of the 123 genotypes showed resistance to umavr7. The identification of these six resistant B. napus genotypes will lead to further studies on the development of blackleg disease resistance through breeding and the identification of novel R genes.

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.

Investigation of the quorum-sensing regulon of the biocontrol bacterium Pseudomonas chlororaphis strain PA23.

Pseudomonas chlororaphis strain PA23 is a biocontrol agent capable of protecting canola from stem rot disease caused by the fungal pathogen Sclerotinia sclerotiorum. PA23 produces several inhibitory compounds that are under control of a complex regulatory network. Included in this cascade is the PhzRI quorum sensing (QS) system, which plays an essential role in PA23 biocontrol, as well as CsaRI and AurRI, which have not yet been characterized in PA23. The focus of the current study was to employ RNA sequencing to explore the spectrum of PA23 genes under QS control. In this work, we investigated genes under the control of the main QS transcriptional regulator, PhzR, as well as those differentially expressed in an AHL-deficient strain, PA23-6863, which constitutively expresses an AiiA lactonase, rendering the strain QS defective. Transcriptomic profiling revealed 545 differentially expressed genes (365 downregulated; 180 upregulated) in the phzR mutant and 534 genes (382 downregulated; 152 upregulated) in the AHL-deficient PA23-6863. In both strains, decreased expression of phenazine, pyrrolnitrin, and exoprotease biosynthetic genes was observed. We have previously reported that QS activates expression of these genes and their encoded products. In addition, elevated siderophore and decreased chitinase gene expression was observed in the QS-deficient stains, which was confirmed by phenotypic analysis. Inspection of the promoter regions revealed the presence of "phz-box" sequences in only 58 of the 807 differentially expressed genes, suggesting that much of the QS regulon is indirectly regulated. Consistent with this notion, 41 transcriptional regulators displayed altered expression in one or both of the QS-deficient strains. Collectively, our findings indicate that QS governs expression of approximately 13% of the PA23 genome affecting diverse functions ranging from secondary metabolite production to general metabolism.

Effect of Elevated CO2 Concentration on the Disease Severity of Compatible and Incompatible Interactions of Brassica napus-Leptosphaeria maculans Pathosystem.

Global warming by increased atmospheric CO2 concentration has been widely accepted. Yet, there has not been any consistent conclusion on the doubled CO2 concentration that in the future will affect plant disease incidence and severity. Blackleg disease, mainly caused by Leptosphaeria maculans, is a major disease on canola production globally. Brassica napus and L. maculans have a gene-for-gene interaction, which causes an incompatible reaction between canola plants carrying resistance genes and L. maculans isolates carrying corresponding avirulence genes. In this study, B. napus varieties and lines inoculated with different Leptosphaeria isolates were subjected to simulated growth conditions, namely, growth chambers with normal environments and with controlled CO2 concentrations of 400, 600, and 800 ppm. The results indicated that the elevated CO2 concentrations have no noticeable effect on the inferred phenotypes of the canola-blackleg interactions. However, the disease severity decreased in most of the B. napus-L. maculans interactions at extremely high CO2 concentration (800 ppm). The varied pathogenicity changes of the B. napus-L. maculans pathosystem under elevated CO2 concentrations at 400 or 600 ppm may be due to the genetic background or physiological differences in plants and pathogenicity differences in L. maculans isolates having different Avr gene profiles. The mechanisms by which elevated CO2 concentrations affect the B. napus-L. maculans pathosystem will help us understand how climate change will impact crops and diseases.