Wheat rust fungi can cause significant damage to wheat crops, leading to reduced yields and economic losses. To combat disease, certain plant varieties can trigger defense responses upon recognition of specific pathogen effector proteins, thereby conferring avirulence. Identifying such avirulence (Avr) genes is crucial for developing strategies to protect crops from devastating losses, ranging from identifying matching resistance genes to designing diagnostic assays for monitoring pathogen populations. Puccinia triticina (Pt) causes wheat leaf rust and is an obligate biotrophic fungus, and because of its life cycle and mode of reproduction, it is difficult to study genetically. To identify Avr genes in Pt, a F2 population of fifty-seven progeny was generated from a sexual cross of race 9 (SBDG) and race 161 (FBDJ) on the alternate host Thalictrum speciosissimum under controlled conditions. The population segregated for avirulent/virulent traits screened at the seedling stage on thirteen single gene resistance lines in the wheat host cultivar Thatcher background. The genomes of the parents, F1, and progeny were sequenced and mapped onto an assembled parental race 9 phased haplotype B genome, resulting in the generation of 21,154 high-quality SNP markers suitable for genetic mapping of the F2 population. A genetic map composed of 61 linkage groups was obtained, containing a total of 10,923 markers, and spanning 10,730.5 centimorgans. Effector loci correlating with avirulence to specific leaf rust resistance (Lr) genes, PtAvrLr14a, PtAvrLr11 and PtAvrLr2a, were mapped to chromosome 1, chromosome 3 and chromosome 4, respectively. To strengthen the identification of candidate Avr genes, a region-specific association study was done on a natural population of fifty-nine Pt isolates that were collected in Canada and whose genomes were sequenced using Illumina. Significant markers and corresponding candidate effector genes were identified for these mapped Avr loci. The identification of these candidate genes is an essential step towards cloning Avr and subsequentially their matching host resistance genes, and for studying the molecular mechanisms underlying pathogen-host interactions and host defense.

The different steps of alternative splicing (AS) in plants and its regulatory mechanisms have already been studied extensively. Its broader impact on cell identity, plant immunity-related genes, and their study as a whole remains to be investigated. Using transgenic plants, we sorted 11 different Arabidopsis thaliana cell types ranging from root to aerial organs using fluorescence-activated cell sorting. RNA-seq data were analyzed with vast-tools and enabled us to generate a high-resolution AS landscape across multiple cell types, all collected through the same experimental procedure. The analysis of cell type-specific gene expression and alternative splicing events highlights the importance of AS on transcription and AS regulation itself. AS is also shown to be tightly linked to cell identity. By using closely related cell types, we captured alternative splicing events involved in specific stages of plant development. The columella cells, among others, show intensified AS regulation and an interesting splicing profile, especially regarding immunity-related genes. Overall, our analysis brings a valuable tool in the study of cell type identity, plant immunity, and AS.

Due to its previously illicit nature, Cannabis sativa had not fully reaped the benefits of recent innovations in genomics and plant sciences. However, Canada’s legalization of C. sativa and products derived from its flower in 2018 triggered significant new demand for robust genotyping tools to assist breeders in meeting consumer demands. Early molecular marker-based research on C. sativa focused on screening for plant sex and chemotype, and more recent research has sought to use molecular markers to target traits of agronomic interest, to study populations and to differentiate between C. sativa cultivars. In this study, we have conducted whole genome sequencing of 32 cultivars, mined the sequencing data for SNPs, developed a reduced SNP genotyping panel to discriminate between sequenced cultivars, then validated the 20-SNP panel using DNA from the sequenced cultivars and tested the assays on commercially available dried flower. The assay conversion rate was higher in DNA extracted from fresh plant material than in DNA extracted from dried flower samples. However, called genotypes were internally consistent, highlighting discrepancies between genotypes detected using sequencing data and observed using genotyping assays. The primary contributions of this work are to clearly document the process used to develop minimal SNP genotyping panels, the feasibility of using such panels to differentiate between C. sativa cultivars, and outline improvements and goals for future iterations of PCR-based, minimal SNP panels to enable efficient development genotyping tools to identify and screen C. sativa cultivars. Our key recommendations are to increase sampling density to account for intra-cultivar variability; leverage higher read length paired-end short-read technology; conduct in-depth pre- and post-processing of reads, mapping, and variant calling data; integrate trait-associated loci to develop multi-purpose panels; and use iterative approaches for in vitro validation to ensure that only the most discriminant and performant SNPs are retained.

Potato late blight is a devastating disease caused by Phytophthora infestans that affects organs, such as leaves and tubers. Previous studies of organ specificity in this interaction have mainly addressed the plant side (i.e., screening for resistance to late blight in potato leaves versus tubers). However, the extent of the organ specificity of P. infestans virulence mechanisms remains understudied. Here, we investigated the extent of organ-specific expression of effector genes using RNA-seq by contrasting the infection of leaves and tubers of potato (cultivar Russet Burbank) by the P. infestans strain 1306. We focused on RxLR and Crinkler (CRN) effectors to obtain insight on genes putatively involved in P. infestans virulence exhibiting differential expression during leaf versus tuber infection. Our results indicated that (i) the proportion of differentially expressed genes (DEGs) increased over time; (ii) a high proportion of effector genes was differentially expressed at one or more time points (34% of RxLRs and 58% of CRNs); and (iii) some RxLR and CRN families appeared to host more DEGs than others. Quantitative reverse transcription PCR (RT-qPCR) was also used to assess the expression of seven RxLR effectors in a time-course experiment, allowing us to validate their organ-biased profiles. Organ-specific effector genes may perform distinct virulence roles at the organ level, which should be considered when performing effectoromics or any infection study. These results bring new insights into the organ specificity of the potato- P. infestans interaction that could lead to improved potato-breeding programs using effectoromics-based approaches. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Hemp and marijuana, both Cannabis sativa L., are revitalized crops to U.S. agricultural and horticultural industries. Hemp (Δ⁹-Tetrahydrocannabinol content < 0.3%) was reintroduced in 2014 under a pilot research program and legalized in 2018. Hemp can now be grown in all 50 states. Marijuana (Δ⁹-THC content > 0.3%), although classified as a Schedule I narcotic by the U.S. Drug Enforcement Administration, is legal in 37 states for medical and/or recreational use. Although C. sativa is often promoted as a pest-free crop, multiple diseases and arthropod pests have been identified and confirmed in recent years. There are limited options for control of diseases and pests affecting hemp. A survey of diagnosticians, researchers, and industry leaders conducted from 2021 to 2022 sought to determine the distribution and occurrence of 76 common diseases and pests on C. sativa across the United States. A total of 148 responses were collected and grouped by U.S. region: Western, Great Plains, North Central, Northeastern, and Southern. Survey results suggest that whereas some pathogens and pests are widely distributed across the United States, others occur more frequently in specific regions. This finding may indicate variations in economic importance by region. Results from this survey provide a foundation for regional and national prioritization of research and regulatory activities.

Organ-specificity of plant defence is an under-represented axis of research in plant biology. However, recent publications point out the importance of this concept in plant-pathogen interactions. Indeed, assuming the resistance status of a whole plant against a given pathogen following studies conducted on a single organ might lead to erroneous conclusions. This study aims at understanding the extent of potato (Solanum tuberosum) organ-specific defence mechanisms after leaf and tuber treatments with pathogen-associated molecular patterns (PAMPs). Our results show that potato leaves and tubers activate contrasting defence responses, in terms of production of reactive oxygen species (ROS) and activation of defence-related genes, following treatments with the Phytophthora PAMP Pep-25 or the bacterial PAMP flg22. Interestingly, flg22 induced ROS production in both leaves and tubers, indicating that this peptide is recognized in these organs. By contrast, ROS production was induced in the leaf but not in the tuber after Pep-25 treatment. Analysis of changes in expression of defence-related genes showed that at least six genes exhibited organ-specific expression patterns. In addition to those early responses, inhibition of potato seedling growth was also found to affect both the aboveground and belowground organs following treatment with Pep-25 and with flg22. Taken together, these experiments will help further the investigation of potato defence against Phytophthora spp.

Among the oldest domesticated crops, cannabis plants ( Cannabis sativa L., marijuana and hemp) have been used to produce food, fiber, and drugs for thousands of years. With the ongoing legalization of cannabis in several jurisdictions worldwide, a new high-value market is emerging for the supply of marijuana and hemp products. This creates unprecedented challenges to achieve better yields and environmental sustainability, while lowering production costs. In this review, we discuss the opportunities and challenges pertaining to the use of beneficial Pseudomonas spp. bacteria as crop inoculants to improve productivity. The prevalence and diversity of naturally occurring Pseudomonas strains within the cannabis microbiome is overviewed, followed by their potential mechanisms involved in plant growth promotion and tolerance to abiotic and biotic stresses. Emphasis is placed on specific aspects relevant for hemp and marijuana crops in various production systems. Finally, factors likely to influence inoculant efficacy are provided, along with strategies to identify promising strains, overcome commercialization bottlenecks, and design adapted formulations. This work aims at supporting the development of the cannabis industry in a sustainable way, by exploiting the many beneficial attributes of Pseudomonas spp.

Pseudomonas protegens Pf-5 is an effective biocontrol agent that protects many crops against pathogens, including the fungal pathogen Botrytis cinerea causing gray mold disease in Cannabis sativa crops. Previous studies have demonstrated the important role of antibiotics pyoluteorin (PLT) and 2,4-diacetylphloroglucinol (DAPG) in Pf-5-mediated biocontrol. To assess the potential involvement of PLT and DAPG in the biocontrol exerted by Pf-5 against B. cinerea in the phyllosphere of C. sativa, two knockout Pf-5 mutants were generated by in-frame deletion of genes pltD or phlA, required for the synthesis of PLT or DAPG respectively, using a two-step allelic exchange method. Additionally, two complemented mutants were constructed by introducing a multicopy plasmid carrying the deleted gene into each deletion mutant. In vitro confrontation assays revealed that deletion mutant ∆pltD inhibited B. cinerea growth significantly less than wild-type Pf-5, supporting antifungal activity of PLT. However, deletion mutant ∆phlA inhibited mycelial growth significantly more than the wild-type, hypothetically due to a co-regulation of PLT and DAPG biosynthesis pathways. Both complemented mutants recovered in vitro inhibition levels similar to that of the wild-type. In subsequent growth chamber inoculation trials, characterization of gray mold disease symptoms on infected cannabis plants revealed that both ∆pltD and ∆phlA significantly lost a part of their biocontrol capabilities, achieving only 10 and 19% disease reduction respectively, compared to 40% achieved by inoculation with the wild-type. Finally, both complemented mutants recovered biocontrol capabilities in planta similar to that of the wild-type. These results indicate that intact biosynthesis pathways for production of PLT and DAPG are required for the optimal antagonistic activity of P. protegens Pf-5 against B. cinerea in the cannabis phyllosphere.

Gray mold caused by Botrytis cinerea is one of the most widespread and damaging diseases in cannabis crops worldwide. With challenging restrictions on pesticide use and few effective control measures, biocontrol agents are needed to manage this disease. The aim of this study was to identify bacterial biocontrol agents with wide-spectrum activity against B. cinerea and other cannabis fungal pathogens. Twelve Bacillus and Pseudomonas strains were first screened with in vitro confrontational assays against 10 culturable cannabis pathogens, namely B. cinerea, Sclerotinia sclerotiorum, Fusarium culmorum, F. sporotrichoides, F. oxysporum, Nigrospora sphaerica, N. oryzae, Alternaria alternata, Phoma sp., and Cercospora sp. Six strains displaying the highest inhibitory activity, namely Bacillus velezensis LBUM279, FZB42, LBUM1082, Bacillus subtilis LBUM979, P. synxantha LBUM223, and P. protegens Pf-5, were further assessed in planta where all, except LBUM223, significantly controlled gray mold development on cannabis leaves. Notably, LBUM279 and FZB42 reduced disease severity by at least half compared with water-treated plants and prevented lesion development and/or sporulation up to 9 days after pathogen inoculation. Genomes of LBUM279, LBUM1082, and LBUM979 were sequenced de novo and taxonomic affiliations were determined to ensure nonrelatedness with pathogenic strains. Moreover, the genomes were exempt of detrimental genes encoding major toxins and virulence factors that could otherwise pose a biosafety risk when used on crops. Eighteen gene clusters of potential biocontrol interest were also identified. To our knowledge, this is the first reported attempt to control cannabis fungal diseases in planta by direct antagonism with beneficial bacteria.

Cannabis sativa is increasingly being grown around the world for medicinal, industrial, and recreational purposes. As in all cultivated plants, cannabis is exposed to a wide range of pathogens, including powdery mildew (PM). This fungal disease stresses cannabis plants and reduces flower bud quality, resulting in significant economic losses for licensed producers. The Mildew Locus O (MLO) gene family encodes plant-specific proteins distributed among conserved clades, of which clades IV and V are known to be involved in susceptibility to PM in monocots and dicots, respectively. In several studies, the inactivation of those genes resulted in durable resistance to the disease. In this study, we identified and characterized the MLO gene family members in five different cannabis genomes. Fifteen Cannabis sativa MLO (CsMLO) genes were manually curated in cannabis, with numbers varying between 14, 17, 19, 18, and 18 for CBDRx, Jamaican Lion female, Jamaican Lion male, Purple Kush, and Finola, respectively (when considering paralogs and incomplete genes). Further analysis of the CsMLO genes and their deduced protein sequences revealed that many characteristics of the gene family, such as the presence of seven transmembrane domains, the MLO functional domain, and particular amino acid positions, were present and well conserved. Phylogenetic analysis of the MLO protein sequences from all five cannabis genomes and other plant species indicated seven distinct clades (I through VII), as reported in other crops. Expression analysis revealed that the CsMLOs from clade V, CsMLO1 and CsMLO4, were significantly upregulated following Golovinomyces ambrosiae infection, providing preliminary evidence that they could be involved in PM susceptibility. Finally, the examination of variation within CsMLO1 and CsMLO4 in 32 cannabis cultivars revealed several amino acid changes, which could affect their function. Altogether, cannabis MLO genes were identified and characterized, among which candidates potentially involved in PM susceptibility were noted. The results of this study will lay the foundation for further investigations, such as the functional characterization of clade V MLOs as well as the potential impact of the amino acid changes reported. Those will be useful for breeding purposes in order to develop resistant cultivars.

Plants get their food from the soil they grow in. But this food is only available thanks to a great diversity of microbes, especially bacteria and fungi, that can chemically and mechanically transform materials in the soil into nutrients. These soil microbes have received a lot of attention from scientists all around the world, because these organisms could improve the way we grow our food and could make agriculture more sustainable. In this article, we explore some of the main mechanisms used by these microbes to feed plants.

Industrial hemp is increasingly grown and harvested for its cannabinoids of pharmaceutical interest. These compounds are generally obtained from plants harvested at maturity but not all cannabinoids are present or abundant during the last stage of hemp development. This study examined intraspecific cannabinoid variability during ontogenic development of hemp to identify growth stages and cultivars that optimize production of specific compounds. The cannabinoid content of twelve commercial industrial hemp (Cannabis sativa L. subsp. sativa) cultivars at three growth stages (i.e., flowering, grain filling and maturity) was determined by high performance liquid chromatography in an experimental field in Cocagne, New Brunswick, Canada. Most cannabinoids in acidic or neutral form were more abundant at maturity. However, cannabigerolic acid, a precursor to all acidic and neutral forms of cannabinoids mentioned in our study, was more abundant during the grain-filling stage. In contrast, cannabichromene was associated with the flowering stage and found in greater abundances in grain cultivars than in dual-purpose cultivars. The cultivar Katani exhibited higher concentrations of most cannabinoids while the cultivars Ferimon, Altair and Anka exhibited higher concentrations of cannabinoid acidic precursors. The current study could help optimize the targeted production of cannabinoids at specific growth stages and to identify the chemical phenotype of different hemp cultivars.

Plant growth-promoting rhizobacteria (PGPR) deploy several mechanisms to improve plant health, growth and yield. The aim of this study was to evaluate the efficacy of two Pseudomonas spp. strains and three Bacillus spp. strains used as single treatments and in consortia to improve the yield of Cannabis sativa and characterize the impact of these treatments on the diversity, structure and functions of the rhizosphere microbiome. Herein, we demonstrate a significant C. sativa yield increase up to 70% when inoculated with three different Pseudomonas spp./Bacillus spp. consortia but not with single inoculation treatments. This growth-promoting effect was observed in two different commercial soil substrates commonly used to grow cannabis: Promix and Canna coco. Marker-based genomic analysis highlighted Bacillus spp. as the main modulator of the rhizosphere microbiome diversity and Pseudomonas spp. as being strongly associated with plant growth promotion. We describe an increase abundance of predicted PGPR metabolic pathways linked with growth-promoting interactions in C. sativa.

Cannabis (Cannabis sativa L.) offers many industrial, agricultural, and medicinal applications, but is commonly threatened by the gray mold disease caused by the fungus Botrytis cinerea. With few effective control measures currently available, the use of beneficial rhizobacteria represents a promising biocontrol avenue for cannabis. To counter disease development, plants rely on a complex network of inducible defense pathways, allowing them to respond locally and systemically to pathogens attacks. In this study, we present the first attempt to control gray mold in cannabis using beneficial rhizobacteria, and the first investigation of cannabis defense responses at the molecular level. Four promising Pseudomonas (LBUM223 and WCS417r) and Bacillus strains (LBUM279 and LBUM979) were applied as single or combined root treatments to cannabis seedlings, which were subsequently infected by B. cinerea. Symptoms were recorded and the expression of eight putative defense genes was monitored in leaves by reverse transcription quantitative polymerase chain reaction. The rhizobacteria did not significantly control gray mold and all infected leaves were necrotic after a week, regardless of the treatment. Similarly, no systemic activation of putative cannabis defense genes was reported, neither triggered by the pathogen nor by the rhizobacteria. However, this work identified five putative defense genes (ERF1, HEL, PAL, PR1, and PR2) that were strongly and sustainably induced locally at B. cinerea’s infection sites, as well as two stably expressed reference genes (TIP41 and APT1) in cannabis. These markers will be useful in future researches exploring cannabis defense pathways.

The incipient legalization and commercialization of Cannabis sativa in Canada have promulgated research into characterizing the plant's microbiome as it promotes many facets of plant growth and health. The emblematic production of commercially important secondary metabolites, namely tetrahydrocannabinol (THC), cannabidiol (CBD) and terpenes, has warranted investigating the modulating capacity of these molecules on the plant microbiome. C. sativa cultivars can be classified into chemotypes depending on the relative levels of THC and CBD they produce; their biosynthesis also varies spatially and temporally during the life cycle of the plant. To study the differential microbiome structure and diversity between cultivars in a spatio-temporal manner, we extracted microbial DNA from the rhizosphere, endorhizosphere, and phyllosphere during the entire life cycle of three different chemotypes; CBD Yummy (<1% THC/13% CBD), CBD shark (6% THC/10% CBD) and Hash (14% THC/ < 1% CBD). Illumina marker gene sequencing of bacterial (16S) and fungal (ITS) communities were coupled to the QIIME2, PICRUSt, and LEfSe pipelines for analysis. Our study describes spatio-temporal and cultivar-dependent variations in the fungal and bacterial microbiome of C. sativa, and details strong cultivar-dependent variance in the belowground microbiome. Furthermore, the predicted pathway abundance of the bacterial microbiome is concomitantly subject to spatio-temporal variations; pathways related to lipid, amino acid, glucose and pentose metabolism were noteworthy. These results describe, for the first time, spatio-temporal and cultivar-dependent variations in the microbiome of C. sativa produced under strict commercial settings. Describing the microbiome is the first step in discoveries that could help in engineering a plant growth and health promoting microbiome in future works.

Additional file of Gene discovery in EST sequences from the wheat leaf rust fungus Puccinia triticina sexual spores, asexual spores and haustoria, compared to other rust and corn smut fungi

Plant diseases bear names such as leaf blights, root rots, sheath blights, tuber scabs, and stem cankers, indicating that symptoms occur preferentially on specific parts of host plants. Accordingly, many plant pathogens are specialized to infect and cause disease in specific tissues and organs. Conversely, others are able to infect a range of tissues, albeit often disease symptoms fluctuate in different organs infected by the same pathogen. The structural specificity of a pathogen defines the degree to which it is reliant on a given tissue, organ, or host developmental stage. It is influenced by both the microbe and the host but the processes shaping it are not well established. Here we review the current status on structural specificity of plant-filamentous pathogen interactions and highlight important research questions. Notably, this review addresses how constitutive defence and induced immunity as well as virulence processes vary across plant organs, tissues, and even cells. A better understanding of the mechanisms underlying structural specificity will aid targeted approaches for plant health, for instance by considering the variation in the nature and the amplitude of defence responses across distinct plant organs and tissues when performing selective breeding.