Bacillus velezensis BE2 controls wheat and barley diseases by direct antagonism and induced systemic resistance.

Wheat and barley rank among the main crops cultivated on a global scale, providing the essential nutritional foundation for both humans and animals. Nevertheless, these crops are vulnerable to several fungal diseases, such as Septoria tritici blotch and net blotch, which significantly reduce yields by adversely affecting leaves and grain quality. To mitigate the effect of these diseases, chemical fungicides have proven to be genuinely effective; however, they impose a serious environmental burden. Currently, biocontrol agents have attracted attention as a sustainable alternative to fungicides, offering an eco-friendly option. The study aimed to assess the efficacy of Bacillus velezensis BE2 in reducing disease symptoms caused by Zymoseptoria tritici and Pyrenophora teres. This bacterium exhibited significant antagonistic effects in vitro by suppressing fungal development when pathogens and the beneficial strain were in direct confrontation. These findings were subsequently confirmed through microscopic analysis, which illustrated the strain's capacity to inhibit spore germination and mycelial growth in both pathogens. Additionally, the study analysed the cell-free supernatant of the bacterium using UPLC-MS (ultra-performance liquid chromatography-mass spectrometry). The results revealed that strain BE2 produces, among other metabolites, different families of cyclic lipopeptides that may be involved in biocontrol. Furthermore, the beneficial effects of strain BE2 in planta were assessed by quantifying the fungal DNA content directly at the leaf level after bacterization, using two different application methods (foliar and drenching). The results indicated that applying the beneficial bacterium at the root level significantly reduced pathogens pressure. Finally, gene expression analysis of different markers showed that BE2 application induced a priming effect within the first hours after infection. KEY POINTS: • BE2 managed Z. tritici and P. teres by direct antagonism and induced systemic resistance. • Strain BE2 produced seven metabolite families, including three cyclic lipopeptides. • Application of strain BE2 at the root level triggered plant defense mechanisms.

PGPR-Soil Microbial Communities' Interactions and Their Influence on Wheat Growth Promotion and Resistance Induction against Mycosphaerella graminicola.

The efficiency of plant-growth-promoting rhizobacteria (PGPR) may not be consistently maintained under field conditions due to the influence of soil microbial communities. The present study aims to investigate their impact on three PGPR-based biofertilizers in wheat. We used the PGPR Paenibacillus sp. strain B2 (PB2), PB2 in co-inoculation with Arthrobacter agilis 4042 (Mix 2), or with Arthrobacter sp. SSM-004 and Microbacterium sp. SSM-001 (Mix 3). Inoculation of PB2, Mix 2, and Mix 3 into non-sterile field soil had a positive effect on root and aboveground dry biomass, depending on the wheat cultivar. The efficiency of the PGPR was further confirmed by the protection they provided against Mycosphaerella graminicola, the causal agent of Septoria leaf blotch disease. PB2 exhibited protection of ≥37.8%, while Mix 2 showed ≥47.9% protection in the four cultivars tested. These results suggest that the interactions between PGPR and native soil microbial communities are crucial for promoting wheat growth and protection. Additionally, high-throughput sequencing of microbial communities conducted 7 days after PGPR inoculations revealed no negative effects of PB2, Mix 2, and Mix 3 on the soil microbial community structure. Interestingly, the presence of Arthrobacter spp. appeared to mitigate the potential negative effect of PB2 on bacterial community and foster root colonization by other beneficial bacterial strains.

Poly (safranine) modified carbon paste electrode as a sensor for the determination of fenitrothion pesticide.

An effective electroanalytical method was developed for the quantitative evaluation of fenitrothion pesticide. The electrochemically modified carbon paste electrode CPE was constructed by applying a safranine layer on its surface. Safranine monomer is easily oxidized. So, a safranine layer was applied via electropolymerization using cyclic voltammetry in (2 × 10-5 M) safranine buffered solution with phosphate buffer BPS at pH.6, potential window (- 1.6: + 2 V), scan rate 100 mV/s. The morphology of the modified electrode was characterized using SEM images. The electropolymerization process was characterized by observing the gradual increases of the peak current with the subsequent scanning cycles. This modified CPE electrode showed an obvious sensitivity by cyclic voltammetry towards the cathodic peak of the fenitrothion nitro group at approximately - 0.73V with good sensitivity by enhancing it to be approximately 10 times more sensitive than on a bare carbon paste electrode CPE. The number of cycles was optimized for the electropolymerization process to be 12 cycles. Where, the peak current at - 0.73 V was gradually enhanced until 12 cycles when it is obviously decreased before slightly increasing again. The reproducibility of the modified electrode was ensured by repeating the sweep cycles using LSV for determining the fenitrothion at 5 µM where it was found that the peak current was unchanged for 10 sweeps before it starts to drop gradually. LSV voltammetry at previously optimized conditions of the potential window (- 0.4: - 1 V), sweep rate 100 mV/s, phosphate buffer at pH.6 was used for the quantitative studies. Where, the pHs of the determining medium were varied from pH 5.5 to pH 8 using phosphate buffer. It was observed that the most identified peak current was at pH.6 which is then decreased gradually until it completely disappeared at pH 8. The optimal accumulation time by adsorption of 140 s for the fenitrothion pesticide was confirmed in the range of (20 s-170 s). Where, the peak current was increased gradually with time up to 140 s then a plateau with a constant response was observed. The developed method showed an excellent linearity range of (1 µM:15 µM) with R2 parameter equal to 0.99906. LOD and LOQ were calculated to be 0.1 µM, and 0.34 µM respectively. Satisfactory levels have been reached for the calculated recovery, accuracy. Precision limits not exceed 1% for both repeatability and reproducibility measurements. F-value and t-value were measured for the suggested LSV method versus the standard HPLC method for the concentration of 8 µM fenitrothion and were found to be 1.482 and 0.123, respectively which didn't exceed the tabulated values. The ruggedness of the suggested method was examined toward deliberate safranine concentration variations in the concentration range of (0.01 mM-0.03 mM). Insignificant differences for the mean recovery at (98.33-98.93%) and precision at (1.39-2.6%) were observed. Hence, the reliability and validity of the developed LSV method were achieved and it was considered as rigid method.

Systemic anti-commensal response to fungi analyzed by flow cytometry is related to gut mycobiome ecology.

BACKGROUND: Interest for the study of gut mycobiota in relation with human health and immune homeostasis has increased in the last years. From this perspective, new tools to study the immune/fungal interface are warranted. Systemic humoral immune responses could reflect the dynamic relationships between gut mycobiota and immunity. Using a novel flow cytometry technology (Fungi-Flow) to determine immunoglobulin (Ig) responses to fungi, we studied the relationships between gut mycobiota and systemic humoral anti-commensal immunity. RESULTS: The Fungi-Flow method allows a sensitive and specific measurement of systemic IgG responses against 17 commensal and environmental fungi from the two main divisions; Ascomycota and Basidiomycota. IgG responses exhibited a high inter-individual variability. Anti-commensal IgG responses were contrasted with the relative abundance, alpha-diversity, and intra-genus richness of fungal species in gut mycobiota of twenty healthy donors. Categorization of gut mycobiota composition revealed two differentiated fungal ecosystems. Significant difference of anti-Saccharomyces systemic IgG responses were observed in healthy donors stratified according to the fungal ecosystem colonizing their gut. A positive and significant correlation was observed between the variety of IgG responses against fungal commensals and intestinal alpha-diversity. At the level of intra-genus species richness, intense IgG responses were associated with a low intra-genus richness for known pathobionts, but not commensals. CONCLUSIONS: Fungi-Flow allows an easy and reliable measure of personalized humoral responses against commensal fungi. Combining sequencing technology with our novel Fungi-Flow immunological method, we propose that there are at least two defined ecosystems in the human gut mycobiome associated with systemic humoral responses. Fungi-Flow opens new opportunities to improve our knowledge about the impact of mycobiota in humoral anti-commensal immunity and homeostasis. Video Abstract.

The Effect of Plant Genotype, Growth Stage, and Mycosphaerella graminicola Strains on the Efficiency and Durability of Wheat-Induced Resistance by Paenibacillus sp. Strain B2.

Plant-growth-promoting rhizobacteria are known as potential biofertilizers and plant-resistance inducers. The current work aims to study the durability of the resistance induced as a response to the inoculation of wheat grains with Paenibacillus sp. strain B2 (PB2) and its influence by plant genotype, growth stage, and Mycosphaerella graminicola strain (the causal agent of Septoria tritici blotch or STB). The results of the plate-counting method showed that PB2 has high potential for wheat-root external colonization [>106 colony-forming unit (CFU)/g of root], and the quantitative real-time polymerase chain reaction (qPCR) analysis demonstrated its internal root-colonization capacity on all tested cultivars. However, the colonization seems to be dependent on wheat-growth stage. The durability of PB2-induced resistance (PB2-IR) was tested at the 3-leaf, tillering, and flag-leaf-growth stages. Additionally, the results showed that the PB2-IR is durable and able to protect the flag leaf, the most important leaf layer during grain fill. It conferred a high protection efficiency (55-94%) against four virulent strains of M. graminicola and over 11 wheat cultivars with different resistance levels to STB. Although, PB2-IR is dependent on M. graminicola strains, wheat genotypes and growth stages, its efficiency, under field conditions, at protecting the last wheat-leaf layers was not an influence. However, it showed 71-79% of protection and reached 81-94% in association with half of the recommended dose of Cherokee® fungicide. This may be explained using laboratory results by its direct impact on M. graminicola strains in these leaf layers and by the indirect reduction of the inoculum coming from leaves infected during the earlier growth stages. Gene expression results showed that PB2-IR is correlated to upregulation of genes involved in defense and cell rescue and a priming effect in the basal defense, jasmonic acid signaling, phenylpropanoids and phytoalexins, and reactive oxygen species gene markers. To conclude, PB2 induces a high and durable resistance against M. graminicola under controlled and field conditions. The PB2-IR is a pathogen strain and is plant-growth-stage and genotype dependent. These results highlight the importance of taking into consideration these factors so as to avoid losing the effectiveness of induced resistance under field conditions.

Systemic Induction of the Defensin and Phytoalexin Pisatin Pathways in Pea (Pisum sativum) against Aphanomyces euteiches by Acetylated and Nonacetylated Oligogalacturonides.

Oligogalacturonides (OGs) are known for their powerful ability to stimulate the plant immune system but little is known about their mode of action in pea (Pisum sativum). In the present study, we investigated the elicitor activity of two fractions of OGs, with polymerization degrees (DPs) of 2-25, in pea against Aphanomyces euteiches. One fraction was nonacetylated (OGs - Ac) whereas the second one was 30% acetylated (OGs + Ac). OGs were applied by injecting the upper two rachises of the plants at three- and/or four-weeks-old. Five-week-old roots were inoculated with 105 zoospores of A. euteiches. The root infection level was determined at 7, 10 and 14 days after inoculation using the quantitative real-time polymerase chain reaction (qPCR). Results showed significant root infection reductions namely 58, 45 and 48% in the plants treated with 80 µg OGs + Ac and 59, 56 and 65% with 200 µg of OGs - Ac. Gene expression results showed the upregulation of genes involved in the antifungal defensins, lignans and the phytoalexin pisatin pathways and a priming effect in the basal defense, SA and ROS gene markers as a response to OGs. The reduction of the efficient dose in OGs + Ac is suggesting that acetylation is necessary for some specific responses. Our work provides the first evidence for the potential of OGs in the defense induction in pea against Aphanomyces root rot.

Real-time PCR to study the effect of timing and persistence of fungicide application and wheat varietal resistance on Mycosphaerella graminicola and its sterol 14α-demethylation-inhibitor-resistant genotypes.

BACKGROUND: Sterol 14α-demethylase inhibitors (DMIs) have been widely used for more than 20 years against wheat Septoria leaf blotch. However, resistance towards DMIs has increased in recent years. The objective of this study was to evaluate the effect of fungicide timing and persistence and wheat resistance varietal on Mycosphaerella graminicola and its DMI-resistant genotypes. RESULTS: Using qPCR, M. graminicola was detected 2 weeks later in the resistant cultivar than in the susceptible cultivar. A high proportion of DMI-moderate resistant genotypes (≥94%) was found in all samples, with an average of 74.2, 0.1 and 19.7% for R6, R7- and R7+ genotypes, respectively. Distribution of DMI-resistant genotypes was neither affected by different wheat cultivars nor by analysis dates. Electron microscopy coupled with qPCR analysis showed that the DMI fungicide prothioconazole had a significant inhibitive effect against spore germination and post-germination. However, the preventive treatment was the most effective, but it was affected strongly by fungicide persistence. CONCLUSION: Preventive fungicide applications are more effective against Septoria leaf blotch than the curative treatments, so persistence and wheat varietal resistance should be taken into account in the management of this disease. It would seem that none of the studied factors affect the frequency of DMI-resistant genotypes.

Protective and curative efficacy of prothioconazole against isolates of Mycosphaerella graminicola differing in their in vitro sensitivity to DMI fungicides.

BACKGROUND: Septoria leaf blotch is the most important disease of wheat in Europe. To control this disease, fungicides of the 14α-demethylase inhibitor group (DMIs) have been widely used for more than 20 years. However, resistance towards DMIs has increased rather quickly in recent years. The objective of this study was to evaluate, on plants and under controlled conditions, the protective and curative efficacy of the DMI fungicide prothioconazole against three current isolates of M. graminicola, chosen to belong to different DMI-resistant phenotypes. Fungicide efficacy was assessed by visual symptoms and by quantitative real-time polymerase chain reaction (PCR). RESULTS: With a protective fungicide application, prothioconazole was always effective against each isolate. This was in accordance with the EC50 results. However, curative efficacy differed between the isolates. It remained at a good level, between 60 and 70% against one isolate, whereas it was strongly affected by late applications from 7 days post-inoculation with the two other isolates. CONCLUSION: A protective application of prothioconazole in wheat crops could be the best strategy to keep a high efficacy against Septoria leaf blotch.

Stimulation of defense reactions in Medicago truncatula by antagonistic lipopeptides from Paenibacillus sp. strain B2.

With the aim of obtaining new strategies to control plant diseases, we investigated the ability of antagonistic lipopolypeptides (paenimyxin) from Paenibacillus sp. strain B2 to elicit hydrogen peroxide (H2O2) production and several defense-related genes in the model legume Medicago truncatula. For this purpose, M. truncatula cell suspensions were used and a pathosystem between M. truncatula and Fusarium acuminatum was established. In M. truncatula cell cultures, the induction of H2O2 reached a maximum 20 min after elicitation with paenimyxin, whereas concentrations higher than 20 µM inhibited H2O2 induction and this was correlated with a lethal effect. In plant roots incubated with different concentrations of paenimyxin for 24 h before inoculation with F. acuminatum, paenimyxin at a low concentration (ca. 1 µM) had a protective effect and suppressed 95% of the necrotic symptoms, whereas a concentration higher than 10 µM had an inhibitory effect on plant growth. Gene responses were quantified in M. truncatula by semiquantitative reverse transcription-PCR (RT-PCR). Genes involved in the biosynthesis of phytoalexins (phenylalanine ammonia-lyase, chalcone synthase, chalcone reductase), antifungal activity (pathogenesis-related proteins, chitinase), or cell wall (invertase) were highly upregulated in roots or cells after paenimyxin treatment. The mechanisms potentially involved in plant protection are discussed.

Impact of a new biopesticide produced by Paenibacillus sp. strain B2 on the genetic structure and density of soil bacterial communities.

The effect of paenimyxin, a new biopesticide produced by Paenibacillus sp. strain B2, on the density of soil bacterial communities was assessed by colony counting and by 16S rDNA and nirK quantitative polymerase chain reaction (PCR). Paenimyxin had a negative effect on the bacterial colony-forming unit (CFU) number, which was significantly reduced 2 and 4 days after treatment. The effect of paenimyxin on cultivatable bacteria was negligible 7 days after treatment. Approximately 10(7) 16S rDNA sequences per gram of soil (dry weight) were detected by quantitative PCR in all samples. Paenimyxin did not affect the quantification of 16S rDNA or of the denitrifying bacterial community. In addition, RISA fingerprinting showed that the genetic structure of the bacterial communities was significantly modified 2 days after paenimyxin application at 50 microM and 4 days after treatment at lower concentrations (0.5 and 5 microM). The impact of paenimyxin treatment on the genetic structure of soil bacterial communities was transient, as no effect could be observed after 7, 14 and 28 days when compared with the untreated control.