Marine biofilm formation on flax fibre reinforced biocomposites.

Artificial reefs represent useful tools to revitalize coastal and ocean ecosystems. Their formulation determines the biofilm formation which is the prerequisite for the colonization process by marine micro- and macroorganisms. In comparison with concrete, biobased polymers offer improved characteristics, including architecture, formulation, rugosity and recycling. This article aims to explore a new scale of artificial reef made of biocomposites reinforced with a high flax fibre (Linum utilatissimum) content (30%). Cellular adhesion and resulting biofilm formation were assessed using two marine microorganisms: Pseudoalteromonas sp. 3J6 and Cylindrotheca closterium. The influence of flax fibre leachates and plastic monomers on the growth of those marine microorganisms were also evaluated. Results indicated that the introduction of flax fibres inside the polymer matrix modified its physicochemical properties thus modulating adhesion and biofilm formation depending on the microorganism. This study gives insights for further developments of novel functionalized artificial reefs made of biocomposites.

Effect of combined bacteria on the flax dew degumming process: Substance degradation sequence and changes in functional bacteria taxa.

In this study, 16S rDNA high-throughput sequencing, Fourier transform infrared spectroscopy, and two-dimensional correlation spectroscopy techniques were used to analyze the mechanisms driving the sequence of degradation of gummy substances by the microbial community and hydrolytic enzymes during the flax dew degumming process. The results revealed that the inoculation of combined bacteria induced quorum sensing, modulated hydrolytic enzyme production, and reshaped the community structure. Lignin-degraded genera (Pseudomonas and Sphingobacterium) were enriched, and the relative abundances of pectin- and cellulose-degraded genera (Chryseobacterium) decreased in the early degumming stages. Hemicellulose-degraded genera (Brevundimonas) increased over the degumming time. Moreover, the abundance of lignin hydrolytic enzymes improved in the early stages, while the abundance of pectin hydrolytic enzymes increased at the end of degumming. Various types of functional bacteria taxa changed the sequence of substance degradation. Electron scanning microscopy and differential scanning calorimetry results indicated that the degumming, facilitated by the inoculation of combined bacteria, was nearly completed by 21 d. The fibers exhibited smoother and more intact properties, along with higher thermal stability, as indicated by a melting temperature of 71.54 °C. This study provides a reference for selecting precise degumming bacterial agents to enhance degumming efficiency.

Investigation of genetic diversity using molecular and biochemical markers associated with powdery mildew resistance in different flax (Linum usitatissimum L.) genotypes.

Under greenhouse conditions, the resistance of 18 different genotypes of flax to powdery mildew was evaluated. To investigate genetic diversity and identify the molecular and biochemical markers linked to powdery mildew resistance in the tested genotypes, two molecular marker systems-start codon targeted (SCoT) and inter-simple sequence repeat (ISSR)-as well as a biochemical marker (protein profiles, antioxidant enzyme activity, and secondary metabolites) were used. Based on the results, the genotypes were classified into four categories: highly susceptible, susceptible, moderately susceptible, and moderately resistant. The genotypes differed significantly in powdery mildew severity: Polk had a severity of 92.03% and Leona had a severity of 18.10%. Compared to the other genotypes, the moderately resistant genotypes had higher levels of flavonoids, antioxidant enzymes, phenolics, and straw yield; nevertheless, their hydrogen peroxide and malondialdehyde levels were lower. Protein profiles revealed 93.75% polymorphism, although the ISSR marker displayed more polymorphism (78.4%) than the SCoT marker (59.7%). Specific molecular and biochemical markers associated with powdery mildew resistance were identified. The 18 genotypes of flax were divided into two major clusters by the dendrogram based on the combined data of molecular markers. The first main cluster included Leona (genotype number 7), considered moderate resistance to powdery mildew and a separate phenetic line. The second main cluster included the other 17 genotypes, which are grouped together in a sub-cluster. This means that, besides SCoT, ISSR markers can be a useful supplementary technique for molecular flax characterization and for identifying genetic associations between flax genotypes under powdery mildew infection.

Changes in microRNAs expression of flax (Linum usitatissimum L.) planted in a cadmium-contaminated soil following the inoculation with root symbiotic fungi.

Cadmium is one of the most harmful heavy metals that harm agricultural products. Evaluating microRNAs expression is a new and accurate method to study plant response in various environmental conditions. So this study aimed to evaluate the contribution of two symbiotic fungi in improving flax tolerance in a Cd-polluted soil using microRNAs and their target gene expression. A factorial pot experiment in a completely randomized design was conducted with different levels of Cd (0, 20, and 40 mg kg-1) on non-inoculated and inoculated flax with Claroideoglomus etunicatum and Serendipita indica. The results presented that increasing Cd levels caused a constant decline of alkaline phosphatase of soil (from 243 to 210 and 153 µg PNP g-1 h-1), respectively, from control (Cd0) to 20 and 40 mg Cd kg-1. However, the inoculation of flax with fungi significantly enhanced these properties. A negative correlation was observed between the expression level of microRNA 167 and microRNA 398 with their corresponding target genes, auxin response factor 8 and superoxide dismutase zinc/copper 1, respectively. The expression level of both microRNAs and their targets indicated that the inoculation with symbiont fungi could diminish Cd stress and enhance the growth of flax.

Soil contamination with Cd affects plant growth.Root symbiotic fungi can improve plant growth in Cd-polluted soils.Examining microRNA expression is a new and accurate method to evaluate plant response to Cd pollution and symbiotic fungi.

Microbial communities that drive the degradation of flax pectin and hemicellulose during dew retting with Bacillus licheniformis HDYM-04 and Bacillus subtilis ZC-01 addition.

In this study, the combined addition of Bacillus licheniformis HDYM-04 and Bacillus subtilis ZC-01 to flax degradation increased the degradation rates of pectin (74.7 %) and pectinic acid (59.3 %) and increased the maximum activities of pectinase (610.66 ± 7.03 U/mL) and mannanase (656.97 ± 13.16 U/mL). 16S rRNA sequencing showed that the added bacterial agent (Bacillus) was the dominant bacterium, and its addition increased the relative abundance (RA) of Firmicutes and decreased the RA of Bacteroidetes. The core bacterial community linked to degradation (Firmicutes) was determined by RDA. Network analysis showed that the number of bacteria related to pectin and hemicellulose degradation increased with the addition of the bacteria combination. SEM analysis showed that Bacillus was positively correlated with the degradation of pectic substances. These results provide new ideas for improving the utilization of agricultural waste resources and promoting sustainable development in modern agriculture.

Predicting the effects of climate change on the cross-scale epidemiological dynamics of a fungal plant pathogen.

The potential for climate change to exacerbate the burden of human infectious diseases is increasingly recognized, but its effects on infectious diseases of plants have received less attention. Understanding the impacts of climate on the epidemiological dynamics of plant pathogens is imperative, as these organisms play central roles in natural ecosystems and also pose a serious threat to agricultural production and food security. We use the fungal 'flax rust' pathogen (Melampsora lini) and its subalpine wildflower host Lewis flax (Linum lewisii) to investigate how climate change might affect the dynamics of fungal plant pathogen epidemics using a combination of empirical and modeling approaches. Our results suggest that climate change will initially slow transmission at both the within- and between-host scales. However, moderate resurgences in disease spread are predicted as warming progresses, especially if the rate of greenhouse gas emissions continues to increase at its current pace. These findings represent an important step towards building a holistic understanding of climate effects on plant infectious disease that encompasses demographic, epidemiological, and evolutionary processes. A core result is that neglecting processes at any one scale of plant pathogen transmission may bias projections of climate effects, as climate drivers have variable and cascading impacts on processes underlying transmission that occur at different scales.

Profiling of Bacillus cereus on Canadian grain.

Microorganisms that cause foodborne illnesses challenge the food industry; however, environmental studies of these microorganisms on raw grain, prior to food processing, are uncommon. Bacillus cereus sensu lato is a diverse group of bacteria that is common in our everyday environment and occupy a wide array of niches. While some of these bacteria are beneficial to agriculture due to their entomopathogenic properties, others can cause foodborne illness; therefore, characterization of these bacteria is important from both agricultural and food safety standpoints. We performed a survey of wheat and flax grain samples in 2018 (n = 508) and 2017 (n = 636) and discovered that B. cereus was present in the majority of grain samples, as 56.3% and 85.2%, in two years respectively. Whole genome sequencing and comparative genomics of 109 presumptive B. cereus isolates indicates that most of the isolates were closely related and formed two genetically distinct groups. Comparisons to the available genomes of reference strains suggested that the members of these two groups are not closely related to strains previously reported to cause foodborne illness. From the same data set, another, genetically more diverse group of B. cereus was inferred, which had varying levels of similarity to previously reported strains that caused disease. Genomic analysis and PCR amplification of genes linked to toxin production indicated that most of the isolates carry the genes nheA and hbID, while other toxin genes and gene clusters, such as ces, were infrequent. This report of B. cereus on grain from Canada is the first of its kind and demonstrates the value of surveillance of bacteria naturally associated with raw agricultural commodities such as cereal grain and oilseeds.

Genomic Regions Associated with Fusarium Wilt Resistance in Flax.

Modern flax cultivars are susceptible to many diseases; arguably, the most economically damaging of these is the Fusarium wilt fungal disease. Over the past decades international flax breeding initiatives resulted in the development of resistant cultivars. However, much remains to be learned about the mechanisms of resistance to Fusarium infection in flax. As a first step to uncover the genetic factors associated with resistance to Fusarium wilt disease, we performed a genome-wide association study (GWAS) using 297 accessions from the collection of the Federal Research Centre of the Bast Fiber Crops, Torzhok, Russia. These genotypes were infected with a highly pathogenic Fusarium oxysporum f.sp. lini MI39 strain; the wilt symptoms were documented in the course of three successive years. Six different single-locus models implemented in GAPIT3 R package were applied to a selected subset of 72,526 SNPs. A total of 15 QTNs (Quantitative Trait Nucleotides) were detected during at least two years of observation, while eight QTNs were found during all three years of the experiment. Of these, ten QTNs occupied a region of 640 Kb at the start of chromosome 1, while the remaining QTNs mapped to chromosomes 8, 11 and 13. All stable QTNs demonstrate a statistically significant allelic effect across 3 years of the experiment. Importantly, several QTNs spanned regions that harbored genes involved in the pathogen recognition and plant immunity response, including the KIP1-like protein (Lus10025717) and NBS-LRR protein (Lus10025852). Our results provide novel insights into the genetic architecture of flax resistance to Fusarium wilt and pinpoint potential candidate genes for further in-depth studies.

A Genomic Blueprint of Flax Fungal Parasite Fusarium oxysporum f. sp. lini.

Fusarium wilt of flax is an aggressive disease caused by the soil-borne fungal pathogen Fusarium oxysporum f. sp. lini. It is a challenging pathogen presenting a constant threat to flax production industry worldwide. Previously, we reported chromosome-level assemblies of 5 highly pathogenic F. oxysporum f. sp. lini strains. We sought to characterize the genomic architecture of the fungus and outline evolutionary mechanisms shaping the pathogen genome. Here, we reveal the complex multi-compartmentalized genome organization and uncover its diverse evolutionary dynamics, which boosts genetic diversity and facilitates host adaptation. In addition, our results suggest that host of functions implicated in the life cycle of mobile genetic elements are main contributors to dissimilarity between proteomes of different Fusaria. Finally, our experiments demonstrate that mobile genetics elements are expressed in planta upon infection, alluding to their role in pathogenicity. On the whole, these results pave the way for further in-depth studies of evolutionary forces shaping the host-pathogen interaction.

Transcriptomic profiling of susceptible and resistant flax seedlings after Fusarium oxysporum lini infection.

In this study transcriptome was analyzed on two fibrous varieties of flax: the susceptible Regina and the resistant Nike. The experiment was carried out on 2-week-old seedlings, because in this phase of development flax is the most susceptible to infection. We analyzed the whole seedlings, which allowed us to recognize the systemic response of the plants to the infection. We decided to analyze two time points: 24h and 48h, because our goal was to learn the mechanisms activated in the initial stages of infection, these points were selected based on the previous analysis of chitinase gene expression, whose increase in time of Fusarium oxysporum lini infection has been repeatedly confirmed both in the case of flax and other plant species. The results show that although qualitatively the responses of the two varieties are similar, it is the degree of the response that plays the role in the differences of their resistance to F. oxysporum.

Effect of a Bacillus subtilis strain on flax protection against Fusarium oxysporum and its impact on the root and stem cell walls.

In Normandy, flax is a plant of important economic interest because of its fibres. Fusarium oxysporum, a telluric fungus, is responsible for the major losses in crop yield and fibre quality. Several methods are currently used to limit the use of phytochemicals on crops. One of them is the use of plant growth promoting rhizobacteria (PGPR) occurring naturally in the rhizosphere. PGPR are known to act as local antagonists to soil-borne pathogens and to enhance plant resistance by eliciting the induced systemic resistance (ISR). In this study, we first investigated the cell wall modifications occurring in roots and stems after inoculation with the fungus in two flax varieties. First, we showed that both varieties displayed different cell wall organization and that rapid modifications occurred in roots and stems after inoculation. Then, we demonstrated the efficiency of a Bacillus subtilis strain to limit Fusarium wilt on both varieties with a better efficiency for one of them. Finally, thermo-gravimetry was used to highlight that B. subtilis induced modifications of the stem properties, supporting a reinforcement of the cell walls. Our findings suggest that the efficiency and the mode of action of the PGPR B. subtilis is likely to be flax variety dependent.

Bacterial inoculation positively affects the quality and quantity of flax under deficit irrigation regimes.

AIM: The present research was conducted to investigate the effect of plant growth-promoting rhizobacteria (PGPR) and deficit irrigation on quality and quantity of flax under field and pot conditions to determine bacterial efficiency and to decrease water deficit effects. METHODS AND RESULTS: Initially, in vitro experiments were performed to determine the growth-promoting characteristics of bacteria. Then in the field, the effects of bacterial inoculation (control, Azotobacter chroococcum, Azospirillum lipoferum, Bacillus amyloliquefaciens, Bacillus sp. strain1 and Pseudomonas putida) on flax traits were evaluated at different irrigation levels (100, 75 and 50% crop water requirement). Bacterial treatments in the pot experiment were selected based on the field experiment results. The irrigation regimes in the pot and field experiments were the same and bacterial treatments included single, doublet and triplet applications of the bacteria. All the bacterial strains could solubilize phosphate, produce ammonia (except for Bacillus sp. strain1), indole acetic acid and siderophore (except P. putida). Field results indicated that the bacteria significantly mitigated the effects of water deficit. Compared with control plants, bacterial treatments increased the oil, linolenic acid, protein and sulphur content; the number of shoots and capsules; and the harvest index in the flax plants. Pot experimental results revealed that the combined inoculations were more effective than single inoculum treatments. CONCLUSIONS: Bacterial inoculation alleviates deficit irrigation effects in flax plants. SIGNIFICANCE AND IMPACT OF THE STUDY: The effectiveness of applying A. chroococcum, B. amyloliquefaciens and Bacillus sp. strain1 was confirmed, especially as a combination to protect flax against water deficit and to improve its nutritional quality and growth.

Identification of two compounds able to improve flax resistance towards Fusarium oxysporum infection.

Plants are surrounded by a diverse range of microorganisms that causes serious crop losses and requires the use of pesticides. Flax is a major crop in Normandy used for its fibres and is regularly challenged by the pathogenic fungus Fusarium oxysporum (Fo) f. sp. lini. To protect themselves, plants use "innate immunity" as a first line of defense level against pathogens. Activation of plant defense with elicitors could be an alternative for crop plant protection. A previous work was conducted by screening a chemical library and led to the identification of compounds able to activate defense responses in Arabidopsis thaliana. Four compounds were tested for their abilities to improve resistance of two flax varieties against Fo. Two of them, one natural (holaphyllamine or HPA) and one synthetic (M4), neither affected flax nor Fo growth. HPA and M4 induced oxidative burst and callose deposition. Furthermore, HPA and M4 caused changes in the expression patterns of defense-related genes coding a glucanase and a chitinase-like. Finally, plants pre-treated with HPA or M4 exhibited a significant decrease in the disease symptoms. Together, these findings demonstrate that HPA and M4 are able to activate defense responses in flax and improve its resistance against Fo infection.

Genetic analysis reveals long-standing population differentiation and high diversity in the rust pathogen Melampsora lini.

A priority for research on infectious disease is to understand how epidemiological and evolutionary processes interact to influence pathogen population dynamics and disease outcomes. However, little is understood about how population adaptation changes across time, how sexual vs. asexual reproduction contribute to the spread of pathogens in wild populations and how diversity measured with neutral and selectively important markers correlates across years. Here, we report results from a long-term study of epidemiological and genetic dynamics within several natural populations of the Linum marginale-Melampsora lini plant-pathogen interaction. Using pathogen isolates collected from three populations of wild flax (L. marginale) spanning 16 annual epidemics, we probe links between pathogen population dynamics, phenotypic variation for infectivity and genomic polymorphism. Pathogen genotyping was performed using 1567 genome-wide SNP loci and sequence data from two infectivity loci (AvrP123, AvrP4). Pathogen isolates were phenotyped for infectivity using a differential set. Patterns of epidemic development were assessed by conducting surveys of infection prevalence in one population (Kiandra) annually. Bayesian clustering analyses revealed host population and ecotype as key predictors of pathogen genetic structure. Despite strong fluctuations in pathogen population size and severe annual bottlenecks, analysis of molecular variance revealed that pathogen population differentiation was relatively stable over time. Annually, varying levels of clonal spread (0-44.8%) contributed to epidemics. However, within populations, temporal genetic composition was dynamic with rapid turnover of pathogen genotypes, despite the dominance of only four infectivity phenotypes across the entire study period. Furthermore, in the presence of strong fluctuations in population size and migration, spatial selection may maintain pathogen populations that, despite being phenotypically stable, are genetically highly dynamic.

The Genome Sequence of Five Highly Pathogenic Isolates of Fusarium oxysporum f. sp. lini.

Fusarium wilt is the most destructive fungal disease in flax, limiting flax cultivation in all the main flax and linseed growing countries. The causative agent is seedborne and soilborne fungus F. oxysporum f. sp. lini. Here, we report, for the first time, genome assemblies of five highly pathogenic isolates of Fusarium oxysporum f. sp. lini, namely monoisolate 39 and strains F329, F324, F282, F287. In addition, syntenic analysis provided a powerful approach to distinguish between core and lineage-specific parts of the genome. These results lay a solid foundation for comparative genomics studies of plant fungal pathogens, evolution of pathogenicity, and virulence factors underlying the dynamics of host-pathogen interactions, thus eventually offering solutions to Fusarium disease control.

Survival and thermal resistance among four Salmonella serovars inoculated onto flaxseeds.

Thermal resistance among Salmonella serovars has been shown to vary, however, such data are minimal for Salmonella inoculated onto low moisture foods. We evaluated survival and subsequent thermal resistance for 32 strains of Salmonella from four serovars (Agona, Enteritidis, Montevideo, and Tennessee) on flaxseed over 24 weeks. After inoculation, flaxseeds were adjusted to aw = 0.5 and stored at 22 °C. After 24 weeks at 22 °C, strains of serovar Agona had a significantly slower rate of reduction compared to those of Enteritidis and Montevideo (adj. p < 0.05). Inoculated flaxseeds were processed at 71 °C with vacuum steam pasteurization at 4 time points during storage. Average initial D71°C values ranging from 1.0 to 1.5 min were similar across serovars. Over 24 weeks, D71°C varied in a serovar-dependent manner. D71°C at 8, 16, and 24 weeks did not change significantly for Enteritidis and Montevideo but did for Tennessee and Agona. While significant, the differences in D71°C over time were less than 1 min, indicating that storage time prior to heat treatment would have a minimal effect on the processing time required to inactivate Salmonella on flaxseed.

Fusarium oxysporum infection activates the plastidial branch of the terpenoid biosynthesis pathway in flax, leading to increased ABA synthesis.

MAIN CONCLUSION: Upregulation of the terpenoid pathway and increased ABA content in flax upon Fusarium infection leads to activation of the early plant's response (PR genes, cell wall remodeling, and redox status). Plants have developed a number of defense strategies against the adverse effects of fungi such as Fusarium oxysporum. One such defense is the production of antioxidant secondary metabolites, which fall into two main groups: the phenylpropanoids and the terpenoids. While functions and biosynthesis of phenylpropanoids have been extensively studied, very little is known about the genes controlling the terpenoid synthesis pathway in flax. They can serve as antioxidants, but are also substrates for a plethora of different compounds, including those of regulatory functions, like ABA. ABA's function during pathogen attack remains obscure and often depends on the specific plant-pathogen interactions. In our study we showed that in flax the non-mevalonate pathway is strongly activated in the early hours of pathogen infection and that there is a redirection of metabolites towards ABA synthesis. The elevated synthesis of ABA correlates with flax resistance to F. oxysporum, thus we suggest ABA to be a positive regulator of the plant's early response to the infection.

Flax rust infection transcriptomics reveals a transcriptional profile that may be indicative for rust Avr genes.

Secreted effectors of fungal pathogens are essential elements for disease development. However, lack of sequence conservation among identified effectors has long been a problem for predicting effector complements in fungi. Here we have explored the expression characteristics of avirulence (Avr) genes and candidate effectors of the flax rust fungus, Melampsora lini. We performed transcriptome sequencing and real-time quantitative PCR (qPCR) on RNA extracted from ungerminated spores, germinated spores, isolated haustoria and flax seedlings inoculated with M. lini isolate CH5 during plant infection. Genes encoding two categories of M. lini proteins, namely Avr proteins and plant cell wall degrading enzymes (CWDEs), were investigated in detail. Analysis of the expression profiles of 623 genes encoding predicted secreted proteins in the M. lini transcriptome shows that the six known Avr genes (i.e. AvrM (avrM), AvrM14, AvrL2, AvrL567, AvrP123 (AvrP) and AvrP4) fall within a group of 64 similarly expressed genes that are induced in planta and show a peak of expression early in infection with a subsequent decline towards sporulation. Other genes within this group include two paralogues of AvrL2, an AvrL567 virulence allele, and a number of genes encoding putative effector proteins. By contrast, M. lini genes encoding CWDEs fall into different expression clusters with their distribution often unrelated to their catalytic activity or substrate targets. These results suggest that synthesis of M. lini Avr proteins may be regulated in a coordinated fashion and that the expression profiling-based analysis has significant predictive power for the identification of candidate Avr genes.

Contrasting common measures of arbuscular mycorrhizal fungal root colonization.

Estimating the abundance of arbuscular mycorrhizal fungi relies entirely on indirect methods, meaning all measures are associated with some variability. The most common methods use microscopic estimates of the relative proportion of root length colonized by fungal structures. These methods typically examine root subsamples. While such methods are inexpensive and relatively simple, significant variation within single root system means there is opportunity for sampling bias. We evaluated the two most common methods of percent root length colonization for AM fungi both as a subsample and for the entire root system of flax plants. We compared these measures to a novel technique that returns projected fungal surface area (fungal coverage), by using microphotography and imaging analysis. Both microscopic methods overestimated the colonization intensity compared to image analysis. Among the microscopic methods, the method which incorporated colonization intensity (Trouvelot) was significantly more similar to imaging method results, than the method that is based on the presence/absence of the fungus (McGonigle).

Antimicrobial Carvacrol Incorporated in Flaxseed Gum-Sodium Alginate Active Films to Improve the Quality Attributes of Chinese Sea bass (Lateolabrax maculatus) during Cold Storage.

The objective of this research was to explore the antimicrobial activity and mechanism of carvacrol against Vibrio Parahemolyticus, Shewanella putrefaciens, Staphylococcus aureus and Pseudomonas fluorescens and evaluate the effect of the addition of carvacrol/ß-cyclodextrin emulsions to flaxseed gum (FSG)-sodium alginate (SA) edible films on the preservation of Chinese sea bass (Lateolabrax maculatus) fillets during refrigerated storage. The minimum inhibitory concentration (MIC) of carvacrol against V. parahemolyticus, S. putrefaciens, S. aureus and P. fluorescens were 0.5, 0.5, 0.125, and 0.5 mg/mL, respectively. Alkaline phosphatase activity assay, nucleotide and protein leakage, and scanning electron microscope demonstrated that carvacrol damaged the external structure of the tested bacterial cells causing leakage of cytoplasmic components. At the same time, when FSG-SA films containing carvacrol used as coating agents for Chinese sea bass fillets cold storage, FSG-SA films containing 1.0 or 2.0 mg/mL carvacrol could significantly reduce TVB-N content, K-value, the degree of microbial deterioration and maintain quality of sea bass fillets according to organoleptic evaluation results.