The fungus Acremonium alternatum enhances salt stress tolerance by regulating host redox homeostasis and phytohormone signaling.

While endophytic fungi offer promising avenues for bolstering plant resilience against abiotic stressors, the molecular mechanisms behind this biofortification remain largely unknown. This study employed a multifaceted approach, combining plant physiology, proteomic, metabolomic, and targeted hormonal analyses to illuminate the early response of Brassica napus to Acremonium alternatum during the nascent stages of their interaction. Notably, under optimal growth conditions, the initial reaction to fungus was relatively subtle, with no visible alterations in plant phenotype and only minor impacts on the proteome and metabolome. Interestingly, the identified proteins associated with the Acremonium response included TUDOR 1, Annexin D4, and a plastidic K+ efflux antiporter, hinting at potential processes that could counter abiotic stressors, particularly salt stress. Subsequent experiments validated this hypothesis, showcasing significantly enhanced growth in Acremonium-inoculated plants under salt stress. Molecular analyses revealed a profound impact on the plant's proteome, with over 50% of salt stress response proteins remaining unaffected in inoculated plants. Acremonium modulated ribosomal proteins, increased abundance of photosynthetic proteins, enhanced ROS metabolism, accumulation of V-ATPase, altered abundances of various metabolic enzymes, and possibly promoted abscisic acid signaling. Subsequent analyses validated the accumulation of this hormone and its enhanced signaling. Collectively, these findings indicate that Acremonium promotes salt tolerance by orchestrating abscisic acid signaling, priming the plant's antioxidant system, as evidenced by the accumulation of ROS-scavenging metabolites and alterations in ROS metabolism, leading to lowered ROS levels and enhanced photosynthesis. Additionally, it modulates ion sequestration through V-ATPase accumulation, potentially contributing to the observed decrease in chloride content.

Pathogen lifestyle determines host genetic signature of quantitative disease resistance loci in oilseed rape (Brassica napus).

KEY MESSAGE: Using associative transcriptomics, our study identifies genes conferring resistance to four diverse fungal pathogens in crops, emphasizing key genetic determinants of multi-pathogen resistance. Crops are affected by several pathogens, but these are rarely studied in parallel to identify common and unique genetic factors controlling diseases. Broad-spectrum quantitative disease resistance (QDR) is desirable for crop breeding as it confers resistance to several pathogen species. Here, we use associative transcriptomics (AT) to identify candidate gene loci associated with Brassica napus constitutive QDR to four contrasting fungal pathogens: Alternaria brassicicola, Botrytis cinerea, Pyrenopeziza brassicae, and Verticillium longisporum. We did not identify any shared loci associated with broad-spectrum QDR to fungal pathogens with contrasting lifestyles. Instead, we observed QDR dependent on the lifestyle of the pathogen-hemibiotrophic and necrotrophic pathogens had distinct QDR responses and associated loci, including some loci associated with early immunity. Furthermore, we identify a genomic deletion associated with resistance to V. longisporum and potentially broad-spectrum QDR. This is the first time AT has been used for several pathosystems simultaneously to identify host genetic loci involved in broad-spectrum QDR. We highlight constitutive expressed candidate loci for broad-spectrum QDR with no antagonistic effects on susceptibility to the other pathogens studies as candidates for crop breeding. In conclusion, this study represents an advancement in our understanding of broad-spectrum QDR in B. napus and is a significant resource for the scientific community.

Location and timing govern tripartite interactions of fungal phytopathogens and host in the stem canker species complex.

BACKGROUND: Leptosphaeria maculans "brassicae" (Lmb) and Leptosphaeria biglobosa "brassicae" (Lbb) make up a species complex involved in the stem canker (blackleg) disease of rapeseed (Brassica napus). They coinfect rapeseed together, from the early stage of infection on leaves to the final necrotic stage at the stem base, and both perform sexual crossings on plant residues. L. biglobosa is suggested to be a potential biocontrol agent against Lmb, but there has been no mechanistic investigation of the different types of interactions that may occur between the plant and the two fungal species. RESULTS: We investigated the bi- or tripartite interaction mechanisms by (i) confronting Lmb and Lbb in culture conditions or during cotyledon infection, with different timing and/or spore concentration regimes, (ii) performing RNA-Seq experiments in vitro or on the kinetics of infection of cotyledons infected by Lmb and/or Lbb to evaluate the transcriptomic activity and the plant response when both fungal species are inoculated together. Lbb infection of B. napus cotyledons was typical of a necrotrophic behavior, with a very early setup of one pathogenicity program and very limited colonization of tissues. This contrasted with the complex succession of pathogenicity programs of the hemibiotroph Lmb. During simultaneous co-infection by both species, Lmb was strongly impacted in its growth and transcriptomic dynamics both in vitro and in planta, while Lbb was unaffected by the presence of Lmb. However, the drastic inhibition of Lmb growth by Lbb was ineffective in the case of delayed inoculation with Lbb or a lower amount of spores of Lbb compared to Lmb. CONCLUSIONS: Our data suggest that Lmb growth inhibition by Lbb is the result of a combination of factors that may include competition for trophic resources, the generation by Lbb of an environment unsuitable for the lifecycle of Lmb or/and the effect on Lmb of plant defense responses induced by Lbb. It indicates that growth inhibition occurs in very specific conditions (i.e., co-inoculation at the same place of an equal amount of inoculum) that are unlikely to occur in the field where their coexistence does not prevent any species from completing their life cycle.

The neighbouring genes AvrLm10A and AvrLm10B are part of a large multigene family of cooperating effector genes conserved in Dothideomycetes and Sordariomycetes.

Fungal effectors (small-secreted proteins) have long been considered as species or even subpopulation-specific. The increasing availability of high-quality fungal genomes and annotations has allowed the identification of trans-species or trans-genera families of effectors. Two avirulence effectors, AvrLm10A and AvrLm10B, of Leptosphaeria maculans, the fungus causing stem canker of oilseed rape, are members of such a large family of effectors. AvrLm10A and AvrLm10B are neighbouring genes, organized in divergent transcriptional orientation. Sequence searches within the L. maculans genome showed that AvrLm10A/AvrLm10B belong to a multigene family comprising five pairs of genes with a similar tail-to-tail organization. The two genes, in a pair, always had the same expression pattern and two expression profiles were distinguished, associated with the biotrophic colonization of cotyledons and/or petioles and stems. Of the two protein pairs further investigated, AvrLm10A_like1/AvrLm10B_like1 and AvrLm10A_like2/AvrLm10B_like2, the second one had the ability to physically interact, similarly to what was previously described for the AvrLm10A/AvrLm10B pair, and cross-interactions were also detected for two pairs. AvrLm10A homologues were identified in more than 30 Dothideomycete and Sordariomycete plant-pathogenic fungi. One of them, SIX5, is an effector from Fusarium oxysporum f. sp. lycopersici physically interacting with the avirulence effector Avr2. We found that AvrLm10A/SIX5 homologues were associated with at least eight distinct putative effector families, suggesting that AvrLm10A/SIX5 is able to cooperate with different effectors. These results point to a general role of the AvrLm10A/SIX5 proteins as "cooperating proteins", able to interact with diverse families of effectors whose encoding gene is co-regulated with the neighbouring AvrLm10A homologue.

Association Mapping Combined with Whole Genome Sequencing Data Reveals Candidate Causal Variants for Sclerotinia Stem Rot Resistance in Brassica napus.

Canola (Brassica napus) yield can be significantly reduced by the disease sclerotinia stem rot (SSR), which is caused by Sclerotinia sclerotiorum, a necrotrophic fungal pathogen with an unusually large host range. Breeding cultivars that are physiologically resistant to SSR is desirable to enhance crop productivity. However, the development of resistant varieties has proved challenging due to the highly polygenic nature of S. sclerotiorum resistance. Here, we identified regions of the B. napus genome associated with SSR resistance using data from a previous study by association mapping. We then validated their contribution to resistance in a follow-up screen. This follow-up screen also confirmed high levels of SSR resistance in several genotypes from the previous study. Using publicly available whole genome sequencing data for a panel of 83 B. napus genotypes, we identified nonsynonymous polymorphisms linked to the SSR resistance loci. A qPCR analysis showed that two of the genes containing these polymorphisms were transcriptionally responsive to S. sclerotiorum infection. In addition, we provide evidence that homologues of three of the candidate genes contribute to resistance in the model Brassicaceae species Arabidopsis thaliana. The identification of resistant germplasm and candidate genomic loci associated with resistance are important findings that can be exploited by breeders to improve the genetic resistance of canola varieties.

QTL mapping and transcriptome analysis identify novel QTLs and candidate genes in Brassica villosa for quantitative resistance against Sclerotinia sclerotiorum.

KEY MESSAGE: Novel QTLs and candidate genes for Sclerotinia-resistance were identified in B. villosa, a wild Brassica species, which represents a new genetic source for improving oilseed rape resistance to SSR. Sclerotinia stem rot (SSR), caused by Sclerotinia sclerotiorum, is one of the most destructive diseases in oilseed rape growing regions. To date, there is no effective genetic resistance against S. sclerotiorum in the B. napus germplasm and knowledge of the molecular plant-fungal interaction is also limited. To identify new resistance resources, we screened a set of wild Brassica species and identified B. villosa (BRA1896) with a high level of Sclerotinia-resistance. Two segregating F2 populations for Sclerotinia-resistance, generated by interspecific crosses between the resistant B. villosa (BRA1896) and the wild susceptible B. oleracea (BRA1909) were assessed for Sclerotinia-resistance. Genetic mapping using a 15-k Illumina Infinium SNP-array resulted in a high-density genetic map containing 1,118 SNP markers and spanning a total genetic length of 792.2 cM. QTL analysis revealed seven QTLs explaining 3.8% to 16.5% of phenotypic variance. Intriguingly, RNAseq-based transcriptome analysis identified genes and pathways specific to B. villosa, of which a cluster of five genes encoding putative receptor-like kinases (RLKs) and two pathogenesis-related (PR) proteins are co-localized within a QTL on chromosome C07. Furthermore, transcriptomic analysis revealed enhanced ethylene (ET)-activated signaling in the resistant B. villosa, which is associated with a stronger plant immune response, depressed cell death, and enhanced phytoalexin biosynthesis compared to the susceptible B. oleracea. Our data demonstrates that B. villosa represents a novel and unique genetic source for improving oilseed rape resistance against SSR.

Development of a Duplex qPCR System for Detection and Quantification of the Two Canola Blackleg Pathogens Leptosphaeria biglobosa and L. maculans.

Two probe-based qPCR systems, namely P-Lb and P-Lm, specific to the canola blackleg pathogens Leptosphaeria biglobosa and L. maculans, respectively, were developed, and their efficiencies were tested. Each of the two systems targets a single-copy gene exclusively present in the corresponding species. The specificities of the two systems on the species level and their ubiquities on the subspecies level were confirmed by in silico sequence analyses and testing on L. biglobosa (17 strains), L. maculans (10 strains), and other plant pathogens (31 species). For sensitivities, the two systems were tested on synthesized DNA fragments (gBlock) of the targeted regions, from which a standard curve was generated for each system. In addition, standard curves were also generated on gBlocks for duplex qPCR in which the two systems were used in the same reaction. The two systems were further tested in both singleplex and duplex qPCR on DNA samples extracted from fungal spores, inoculated canola cotyledons, and naturally infected canola stubble samples collected from commercial fields. Our data indicated that the two systems are specific to L. biglobosa and L. maculans, respectively, and one reaction could detect as few as 200 spores of either species. When used in duplex qPCR on DNA samples with various origins, the two systems generated similar results as in singleplex qPCR. The duplex qPCR system, along with the sample preparation and DNA extraction specified in this study, constituted a first-reported duplex qPCR protocol for detection and quantification of the two blackleg pathogens from field samples.

Polymorphism of Avirulence Genes and Adaptation to Brassica Resistance Genes Is Gene-Dependent in the Phytopathogenic Fungus Leptosphaeria maculans.

The fungal phytopathogen Leptosphaeria maculans, which causes stem canker (blackleg) of rapeseed (Brassica napus), is mainly controlled worldwide by genetic resistance, which includes major resistance genes (Rlm). This model is one of those for which the highest number of avirulence genes (AvrLm) has been cloned. In many systems, including the L. maculans-B. napus interaction, intense use of resistance genes exerts strong selection pressure on the corresponding avirulent isolates, and the fungi may rapidly escape resistance through various molecular events which modify the avirulence genes. In the literature, the study of polymorphism at avirulence loci is often focused on single genes under selection pressure. In this study, we investigate allelic polymorphism at 11 avirulence loci in a French population of 89 L. maculans isolates collected on a trap cultivar in four geographic locations in the 2017-2018 cropping season. The corresponding Rlm genes have been (i) used for a long time, (ii) recently used, or (iii) unused in agricultural practice. The sequence data generated indicate an extreme diversity of situations. For example, genes submitted to an ancient selection may have either been deleted in populations (AvrLm1) or replaced by a single-nucleotide mutated virulent version (AvrLm2, AvrLm5-9). Genes that have never been under selection may either be nearly invariant (AvrLm6, AvrLm10A, AvrLm10B), exhibit rare deletions (AvrLm11, AvrLm14), or display a high diversity of alleles and isoforms (AvrLmS-Lep2). These data suggest that the evolutionary trajectory of avirulence/virulence alleles is gene-dependent and independent of selection pressure in L. maculans. [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.

Genome-Wide Identification of GYF-Domain Encoding Genes in Three Brassica Species and Their Expression Responding to Sclerotinia sclerotiorum in Brassica napus.

GYF (glycine-tyrosine-phenylalanine)-domain-containing proteins, which were reported to participate in many aspects of biological processes in yeast and animals, are highly conserved adaptor proteins existing in almost all eukaryotes. Our previous study revealed that GYF protein MUSE11/EXA1 is involved in nucleotide-binding leucine-rich repeat (NLR) receptor-mediated defense in Arabidopsis thaliana. However, the GYF-domain encoding homologous genes are still not clear in other plants. Here, we performed genome-wide identification of GYF-domain encoding genes (GYFs) from Brassica napus and its parental species, Brassica rapa and Brassica oleracea. As a result, 26 GYFs of B. napus (BnaGYFs), 11 GYFs of B. rapa (BraGYFs), and 14 GYFs of B. oleracea (BolGYFs) together with 10 A. thaliana (AtGYFs) were identified, respectively. We, then, conducted gene structure, motif, cis-acting elements, duplication, chromosome localization, and phylogenetic analysis of these genes. Gene structure analysis indicated the diversity of the exon numbers of these genes. We found that the defense and stress responsiveness element existed in 23 genes and also identified 10 motifs in these GYF proteins. Chromosome localization exhibited a similar distribution of BnaGYFs with BraGYFs or BolGYFs in their respective genomes. The phylogenetic and gene collinearity analysis showed the evolutionary conservation of GYFs among B. napus and its parental species as well as Arabidopsis. These 61 identified GYF domain proteins can be classified into seven groups according to their sequence similarity. Expression of BnaGYFs induced by Sclerotinia sclerotiorum provided five highly upregulated genes and five highly downregulated genes, which might be candidates for further research of plant-fungal interaction in B. napus.

Large-Scale Surveys of Blackleg of Oilseed Rape (Leptosphaeria biglobosa) Revealed New Insights into Epidemics of This Disease in China.

Blackleg of oilseed rape caused by Leptosphaeria maculans/L. biglobosa is a worldwide important disease. L. maculans is more virulent than L. biglobosa, so it causes a great concern for oilseed rape production. In China, blackleg (L. biglobosa) of oilseed rape was reported in the 2000s, but epidemiological features of blackleg have not been well elucidated. Moreover, whether L. maculans exists in China is still an open question. Therefore, a 5-year survey was done in China to collect blackleg-occurrence data for characterizing the features of blackleg epidemics and to identify the blackleg pathogens for assessing the risk of L. maculans invasion. The results showed that all the 19 surveyed provinces had blackleg on oilseed rape, and the most frequently occurring provinces are Gansu, Qinghai, Shaanxi, and Hubei. Phoma stem canker was the most common symptom, which was associated with stem cracks on winter oilseed rape and with stem-weevil activities on spring oilseed rape. Temperature and rainfall were the main factors for blackleg epidemics on winter oilseed rape, whereas rainfall was the main factor for blackleg epidemics on spring oilseed rape. Brassica campestris and B. juncea oilseed rapes were more susceptible than B. napus to blackleg. Oilseed rapes cultivated under the continuous dry land-cropping pattern were more prone to blackleg than those cultivated under the paddy land/dry land-cropping pattern. All 6,015 fungal isolates from blackleg plant tissues belonged to L. biglobosa. These results are helpful for understanding the blackleg epidemics of oilseed rapes and for management of this disease in China.

Brassica napus Bacterial Assembly Processes Vary with Plant Compartment and Growth Stage but Not between Lines.

Holobiont bacterial community assembly processes are an essential element to understanding the plant microbiome. To elucidate these processes, leaf, root, and rhizosphere samples were collected from eight lines of Brassica napus in Saskatchewan over the course of 10 weeks. We then used ecological null modeling to disentangle the community assembly processes over the growing season in each plant part. The root was primarily dominated by stochastic community assembly processes, which is inconsistent with previous studies that suggest of a highly selective root environment. Leaf assembly processes were primarily stochastic as well. In contrast, the rhizosphere was a highly selective environment. The dominant rhizosphere selection process leads to more similar communities. Assembly processes in all plant compartments were dependent on plant growth stage with little line effect on community assembly. The foundations of assembly in the leaf were due to the harsh environment, leading to dominance of stochastic effects, whereas the stochastic effects in the root interior likely arise due to competitive exclusion or priority effects. Engineering canola microbiomes should occur during periods of strong selection assuming strong selection could promote beneficial bacteria. For example, engineering the microbiome to resist pathogens, which are typically aerially born, should focus on the flowering period, whereas microbiomes to enhance yield should likely be engineered postflowering as the rhizosphere is undergoing strong selection. IMPORTANCE In order to harness the microbiome for more sustainable crop production, we must first have a better understanding of microbial community assembly processes that occurring during plant development. This study examines the bacterial community assembly processes of the leaf, root, and rhizosphere of eight different lines of Brassica napus over the growing season. The influence of growth stage and B. napus line were examined in conjunction with the assembly processes. Understanding what influences the assembly processes of crops might allow for more targeted breeding efforts by working with the plant to manipulate the microbiome when it is undergoing the strongest selection pressure.

Two independent approaches converge to the cloning of a new Leptosphaeria maculans avirulence effector gene, AvrLmS-Lep2.

Brassica napus (oilseed rape, canola) seedling resistance to Leptosphaeria maculans, the causal agent of blackleg (stem canker) disease, follows a gene-for-gene relationship. The avirulence genes AvrLmS and AvrLep2 were described to be perceived by the resistance genes RlmS and LepR2, respectively, present in B. napus 'Surpass 400'. Here we report cloning of AvrLmS and AvrLep2 using two independent methods. AvrLmS was cloned using combined in vitro crossing between avirulent and virulent isolates with sequencing of DNA bulks from avirulent or virulent progeny (bulked segregant sequencing). AvrLep2 was cloned using a biparental cross of avirulent and virulent L. maculans isolates and a classical map-based cloning approach. Taking these two approaches independently, we found that AvrLmS and AvrLep2 are the same gene. Complementation of virulent isolates with this gene confirmed its role in inducing resistance on Surpass 400, Topas-LepR2, and an RlmS-line. The gene, renamed AvrLmS-Lep2, encodes a small cysteine-rich protein of unknown function with an N-terminal secretory signal peptide, which is a common feature of the majority of effectors from extracellular fungal plant pathogens. The AvrLmS-Lep2/LepR2 interaction phenotype was found to vary from a typical hypersensitive response through intermediate resistance sometimes towards susceptibility, depending on the inoculation conditions. AvrLmS-Lep2 was nevertheless sufficient to significantly slow the systemic growth of the pathogen and reduce the stem lesion size on plant genotypes with LepR2, indicating the potential efficiency of this resistance to control the disease in the field.

Modeling risk of Sclerotinia sclerotiorum-induced disease development on canola and dry bean using machine learning algorithms.

Diseases caused by the fungus Sclerotinia sclerotiorum are managed mainly through fungicide applications in canola and dry bean. Accurate estimation of the risk of disease development on these crops could help farmers make spraying decisions. Five machine learning (ML) models were evaluated in classification and regression modes for predicting disease establishment under different air temperatures and leaf wetness duration conditions. Model algorithms were trained and tested using 20-fold cross validation. Correspondence between predicted and observed values were measured using Cohen's Kappa (classification) and Lin's concordance coefficients (regression). The artificial neural network (ANN) algorithms had average accuracies ≥ 89% (classification) and R2 ≥ 88% (regression) on canola and dry bean and their correspondence agreements were ≥ 0.83, which is considered substantial to almost perfect. In contrast, logistic regression algorithms had accuracies of 88% for dry bean and 78% for canola; other models were similarly inconsistent. Implementation of ANN models in disease warning systems could help farmers with spraying decisions. At the same time, these models provide insights on temperature and leaf wetness requirements for development of S. sclerotiorum diseases in these crops. Results of this study show the potential of ML models as tools for epidemiological studies on other pathosystems.

Characterization of non-specific lipid transfer protein (nsLtp) gene families in the Brassica napus pangenome reveals abundance variation.

BACKGROUND: Brassica napus is an important agricultural species, improving stress resistance was one of the main breeding goals at present. Non-specific lipid transfer proteins (nsLTPs) are small, basic proteins which are involved in some biotic or abiotic stress responses. B. napus is susceptible to a variety of fungal diseases, so identify the BnLTPs and their expression in disease responses is very important. The common reference genome of B. napus does not contain all B. napus genes because of gene presence/absence variations between individuals. Therefore, it was necessary to search for candidate BnLTP genes in the B. napus pangenome. RESULTS: In the present study, the BnLTP genes were identified throughout the pangenome, and different BnLTP genes were presented among varieties. Totally, 246 BnLTP genes were identified and could be divided into five types (1, 2, C, D, and G). The classification, phylogenetic reconstruction, chromosome distribution, functional annotation, and gene expression were analyzed. We also identified potential cis-elements that respond to biotic and abiotic stresses in the 2 kb upstream regions of all BnLTP genes. RNA sequencing analysis showed that the BnLTP genes were involved in the response to Sclerotinia sclerotiorum infection. We identified 32 BnLTPs linked to blackleg resistance quantitative trait locus (QTL). CONCLUSION: The identification and analysis of LTP genes in the B. napus pangenome could help to elucidate the function of BnLTP family members and provide new information for future molecular breeding in B. napus.

Knockout of the lignin pathway gene BnF5H decreases the S/G lignin compositional ratio and improves Sclerotinia sclerotiorum resistance in Brassica napus.

Ferulate-5-hydroxylase is a key enzyme involved in the conversion of the guaiacyl monolignol to the syringyl monolignol in angiosperms. The monolignol ratio has been proposed to affect biomass recalcitrance and the resistance to plant disease. Stem rot caused by the fungus Sclerotinia sclerotiorum in Brassica napus causes severe losses in its production. To date, there is no information about the effect of the lignin monomer ratio on the resistance to S. sclerotiorum in B. napus. Four dominantly expressed ferulate-5-hydroxylase genes were concertedly knocked out by CRISPR/Cas9 in B. napus, and three mutant lines were generated. The S/G lignin compositional ratio was decreased compared to that of the wild type based on the results of Mӓule staining and 2D-NMR profiling in KO-7. The resistance to S. sclerotiorum in stems and leaves increased for the three f5h mutant lines compared with WT. Furthermore, we found that the stem strength of f5h mutant lines was significantly increased. Overall, we demonstrate for the first time that decreasing the S/G ratio by knocking out of the F5H gene improves S. sclerotiorum resistance in B. napus and increases stem strength.

Role of beneficial microbes with nitrogen and phosphorous levels on canola productivity / Papel de microbes benéficos em associação com níveis de nitrogênio e fósforo na produtividade de canola

A research was conducted to evaluate the impact of various nitrogen and phosphorus levels along with beneficial microbes to enhance canola productivity. The research was carried out at Agronomy Research Farm, The University of Agriculture Peshawar in winter 2016-2017. The experiment was conducted in randomized complete block factorial design. The study was comprised of three factors including nitrogen (60, 120 and 180 kg ha-¹), phosphorous (70, 100 and 130 kg ha-¹) and beneficial microbes (with and without BM). A control treatment with no N, P and BM was also kept for comparison. Application of beneficial microbes significantly increased pods plant, seed pod, seed filling duration, 1000 seed weight, biological yield and seed yield as compared to control plots. Nitrogen applied at the rate of 180 kg ha-¹ increased pods plant-¹, seed pod, seed filling duration, seed weight, biological yield and seed yield. Maximum pods plant-¹, seed pod, early seed filling, heavier seed weight, biological yield, seed yield, and harvest index were observed in plots treated with 130 kg.ha-¹ phosphorous. As comparison, the combine treated plots have more pods plant-¹, seeds pod-¹, seed filling duration, heaviest seeds, biological yield, seed yield and harvest index as compared to control plots. It is concluded that application of beneficial microbes with N and P at the rate of 180 kg ha-¹ and 130 kg ha-¹, respectively, increased yield and its attributes for canola.

Uma pesquisa foi realizada para avaliar o impacto de vários níveis de nitrogênio e fósforo, juntamente com micróbios benéficos, para aumentar a produtividade da canola. A pesquisa foi realizada no inverno de 2016-17 no Agronomy Research Farm, Universidade de Agricultura do Peshawar. O experimento foi conduzido por planejamento fatorial aleatorizado em blocos. O estudo focou-se em três fatores, incluindo o teor de nitrogênio, N, (60, 120 e 180 kg.ha-¹), o teor de fósforo, P, (70, 100 e 130 kg ha-¹) e a presença de micróbios benéficos (com BM e sem BM). Para fins de comparação, um tratamento controle sem N, P e BM também foi incluído no estudo. A aplicação de micróbios benéficos aumentou significativamente as vagens das plantas e de sementes, a duração do enchimento das sementes, o peso de 1000 sementes, o rendimento biológico e o rendimento de sementes em comparação com os resultados do controle. O nitrogênio aplicado na taxa de 180 kg ha-¹ aumentou as vagens por planta, vagem, duração do enchimento, peso da semente, rendimento biológico e rendimento de sementes. Vagens máximas por planta, vagem, enchimento precoce de sementes, peso maior de semente, rendimento biológico, rendimento de sementes e índice de colheita foram observados em parcelas tratadas com 130 kg.ha-¹ de fósforo. Em comparação aos blocos cultivados de controle, os blocos cultivados tratados combinados têm mais vagens por planta e sementes por vagem, maior duração do enchimento das sementes, maior número de sementes mais pesadas e maior rendimento biológico, rendimento de sementes e índice de colheita. Conclui-se que a aplicação de micróbios benéficos junto com N e P nas doses de 180 kg ha-¹ e 130 kg ha-¹, respectivamente, aumentou a produtividade e atributos de produtividade para a canola.

Comparative Analysis of Herbaceous and Woody Cell Wall Digestibility by Pathogenic Fungi.

Fungal pathogens have evolved combinations of plant cell-wall-degrading enzymes (PCWDEs) to deconstruct host plant cell walls (PCWs). An understanding of this process is hoped to create a basis for improving plant biomass conversion efficiency into sustainable biofuels and bioproducts. Here, an approach integrating enzyme activity assay, biomass pretreatment, field emission scanning electron microscopy (FESEM), and genomic analysis of PCWDEs were applied to examine digestibility or degradability of selected woody and herbaceous biomass by pathogenic fungi. Preferred hydrolysis of apple tree branch, rapeseed straw, or wheat straw were observed by the apple-tree-specific pathogen Valsa mali, the rapeseed pathogen Sclerotinia sclerotiorum, and the wheat pathogen Rhizoctonia cerealis, respectively. Delignification by peracetic acid (PAA) pretreatment increased PCW digestibility, and the increase was generally more profound with non-host than host PCW substrates. Hemicellulase pretreatment slightly reduced or had no effect on hemicellulose content in the PCW substrates tested; however, the pretreatment significantly changed hydrolytic preferences of the selected pathogens, indicating a role of hemicellulose branching in PCW digestibility. Cellulose organization appears to also impact digestibility of host PCWs, as reflected by differences in cellulose microfibril organization in woody and herbaceous PCWs and variation in cellulose-binding domain organization in cellulases of pathogenic fungi, which is known to influence enzyme access to cellulose. Taken together, this study highlighted the importance of chemical structure of both hemicelluloses and cellulose in host PCW digestibility by fungal pathogens.

Early Secretory Pathway-Associated Proteins SsEmp24 and SsErv25 Are Involved in Morphogenesis and Pathogenicity in a Filamentous Phytopathogenic Fungus.

Proper protein secretion is critical for fungal development and pathogenesis. However, the potential roles of proteins involved in the early secretory pathway are largely undescribed in filamentous fungi. p24 proteins are cargo receptors that cycle between the endoplasmic reticulum (ER) and Golgi apparatus in the early secretory pathway and recruit cargo proteins to nascent vesicles. This study characterized the function of two p24 family proteins (SsEmp24 and SsErv25) in a phytopathogenic fungus, Sclerotinia sclerotiorum. Both SsEmp24 and SsErv25 were upregulated during the early stages of S. sclerotiorum infection. ΔSsEmp24 mutant and ΔSsErv25 mutant displayed abnormal vegetative growth and sclerotium formation, were defective in infection cushion formation, and showed lower virulence on host plants. ΔSsEmp24 mutant had a more severe abnormal phenotype than ΔSsErv25 mutant, implying that SsEmp24 could play a central role in the early secretory pathway. Similar to their Saccharomyces cerevisiae counterparts, SsEmp24 interacted with SsErv25 and predominantly colocalized in the ER or nuclear envelope. The absence of SsEmp24 or SsErv25 led to defective in protein secretion in S. sclerotiorum, including the pathogenicity-related extracellular hydrolytic enzymes and effectors. It is proposed that SsEmp24 and SsErv25, components in the early secretory pathway, are involved in modulating morphogenesis and pathogenicity in S. sclerotiorum by mediating protein secretion. IMPORTANCE Understanding the reproduction and pathogenesis mechanism of phytopathogens could provide new opinions to effectively control fungal diseases. Although it has been known that effectors and extracellular hydrolytic enzymes secreted by phytopathogenic fungi play important roles in fungus-host interactions, the secretion system for the delivery of virulence factors to the host is still largely undescribed. Although the role of the early secretory pathway-associated p24 proteins in S. cerevisiae has been well characterized, the function of these proteins in filamentous fungi was scarcely known prior to this study. The present research provides evidence that p24 proteins participate in the reproduction and pathogenesis of phytopathogenic fungi through the mediation of protein secretion. This research advances our understanding of p24 proteins in filamentous phytopathogenic fungi. In addition, the candidate cargos of the two p24 proteins, SsEmp24 and SsErv25, were screened out by comparative proteomics, which could aid the identification of novel development and virulence-associated factors in phytopathogenic fungi.

Genome-wide association mapping and genomic prediction for adult stage sclerotinia stem rot resistance in Brassica napus (L) under field environments.

Sclerotinia stem rot (SSR) is a fungal disease of rapeseed/canola that causes significant seed yield losses and reduces its oil content and quality. In the present study, the reaction of 187 diverse canola genotypes to SSR was characterized at full flowering stage using the agar plug to stem inoculation method in four environments. Genome-wide association study (GWAS) using three different algorithms identified 133 significant SNPs corresponding with 123 loci for disease traits like stem lesion length (LL), lesion width (LW), and plant mortality at 14 (PM_14D) and 21 (PM_21D) days. The explained phenotypic variation of these SNPs ranged from 3.6 to 12.1%. Nineteen significant SNPs were detected in two or more environments, disease traits with at least two GWAS algorithms. The strong correlations observed between LL and other three disease traits evaluated, suggest they could be used as proxies for SSR resistance phenotyping. Sixty-nine candidate genes associated with disease resistance mechanisms were identified. Genomic prediction (GP) analysis with all the four traits employing genome-wide markers resulted in 0.41-0.64 predictive ability depending on the model specifications. The highest predictive ability for PM_21D with three models was about 0.64. From our study, the identified resistant genotypes and stable significant SNP markers will serve as a valuable resource for future SSR resistance breeding. Our study also suggests that genomic selection holds promise for accelerating canola breeding progress by enabling breeders to select SSR resistance genotypes at the early stage by reducing the need to phenotype large numbers of genotypes.