Exploring Synergistic Effects of Levan and Levan-Metabolizing Bacillaceae in Promoting Growth and Enhancing Immunity of Tomato and Wheat.

Boosting plant immunity by priming agents can lower agrochemical dependency in plant production. Levan and levan-derived oligosaccharides (LOS) act as priming agents against biotic stress in several crops. Additionally, beneficial microbes can promote plant growth and protect against fungal diseases. This study assessed possible synergistic effects caused by levan, LOS and five levan- and LOS-metabolizing Bacillaceae (Bacillus and Priestia) strains in tomato and wheat. Leaf and seed defense priming assays were conducted in non-soil (semi-sterile substrate) and soil-based systems, focusing on tomato-Botrytis cinerea and wheat-Magnaporthe oryzae Triticum (MoT) pathosystems. In the non-soil system, seed defense priming with levan, the strains (especially Bacillus velezensis GA1), or their combination significantly promoted tomato growth and protection against B. cinerea. While no growth stimulatory effects were observed for wheat, disease protective effects were also observed in the wheat-MoT pathosystem. When grown in soil and subjected to leaf defense priming, tomato plants co-applied with levan and the bacterial strains showed increased resistance to B. cinerea compared with plants treated with levan or single strains, and these effects were synergistic in some cases. For seed defense priming in soil, more synergistic effects on disease tolerance were observed in a non-fertilized soil as compared to a fertilized soil, suggesting that potential prebiotic effects of levan are more prominent in poor soils. The potential of using combinations of Bacilliaceae and levan in sustainable agriculture is discussed.

The phenylpropanoid pathway inhibitor piperonylic acid induces broad-spectrum pest and disease resistance in plants.

Although many phenylpropanoid pathway-derived molecules act as physical and chemical barriers to pests and pathogens, comparatively little is known about their role in regulating plant immunity. To explore this research field, we transiently perturbed the phenylpropanoid pathway through application of the CINNAMIC ACID-4-HYDROXYLASE (C4H) inhibitor piperonylic acid (PA). Using bioassays involving diverse pests and pathogens, we show that transient C4H inhibition triggers systemic, broad-spectrum resistance in higher plants without affecting growth. PA treatment enhances tomato (Solanum lycopersicum) resistance in field and laboratory conditions, thereby illustrating the potential of phenylpropanoid pathway perturbation in crop protection. At the molecular level, transcriptome and metabolome analyses reveal that transient C4H inhibition in tomato reprograms phenylpropanoid and flavonoid metabolism, systemically induces immune signalling and pathogenesis-related genes, and locally affects reactive oxygen species metabolism. Furthermore, C4H inhibition primes cell wall modification and phenolic compound accumulation in response to root-knot nematode infection. Although PA treatment induces local accumulation of the phytohormone salicylic acid, the PA resistance phenotype is preserved in tomato plants expressing the salicylic acid-degrading NahG construct. Together, our results demonstrate that transient phenylpropanoid pathway perturbation is a conserved inducer of plant resistance and thus highlight the crucial regulatory role of this pathway in plant immunity.

Actinobacteria as Promising Biocontrol Agents for In Vitro and In Planta Degradation and Detoxification of Zearalenone.

Zearalenone (ZEN) is a prevalent mycotoxin found in grains and grain-derived products, inducing adverse health effects in both animals and humans. The in-field application of microorganisms to degrade and detoxify ZEN is a promising strategy to enhance the safety of food and feed. In this study, we investigated the potential of three actinobacterial strains to degrade and detoxify ZEN in vitro and in planta on wheat ears. The residual ZEN concentration and toxicity in the samples were analysed with UHPLC-MS/MS and a bioluminescence BLYES assay, respectively. Streptomyces rimosus subsp. rimosus LMG19352 could completely degrade and detoxify 5 mg/L ZEN in LB broth within 24 h, along with significant reductions in ZEN concentration both in a minimal medium (MM) and on wheat ears. Additionally, it was the only strain that showed a significant colonisation of these ears. Rhodococcus sp. R25614 exhibited partial but significant degradation in LB broth and MM, whereas Streptomyces sp. LMG16995 degraded and detoxified ZEN in LB broth after 72 h by 39% and 33%, respectively. Although all three actinobacterial strains demonstrated the metabolic capability to degrade and detoxify ZEN in vitro, only S. rimosus subsp. rimosus LMG19352 showed promising potential to mitigate ZEN in planta. This distinction underscores the importance of incorporating in planta screening assays for assessing the potential of mycotoxin-biotransforming microorganisms as biocontrol agents.

Uncovering Genomic Features and Biosynthetic Gene Clusters in Endophytic Bacteria from Roots of the Medicinal Plant Alkanna tinctoria Tausch as a Strategy To Identify Novel Biocontrol Bacteria.

The world's population is increasing at a rate not seen in the past. Agriculture, providing food for this increasing population, is reaching its boundaries of space and natural resources. In addition, changing legislation and increased ecological awareness are forcing agriculture to reduce its environmental impact. This entails the replacement of agrochemicals with nature-based solutions. In this regard, the search for effective biocontrol agents that protect crops from pathogens is in the spotlight. In this study, we have investigated the biocontrol activity of endophytic bacteria isolated from the medicinal plant Alkanna tinctoria Tausch. To do so, an extensive collection of bacterial strains was initially genome sequenced and in silico screened for features related to plant stimulation and biocontrol. Based on this information, a selection of bacteria was tested in vitro for antifungal activity using direct antagonism in a plate assay and in planta with a detached-leaf assay. Bacterial strains were tested individually and in combinations to assess the best-performing treatments. The results revealed that many bacteria could produce metabolites that efficiently inhibit the proliferation of several fungi, especially Fusarium graminearum. Among these, Pseudomonas sp. strain R-71838 showed a strong antifungal effect, in both dual-culture and in planta assays, making it the most promising candidate for biocontrol application. Using microbes from medicinal plants, this study highlights the opportunities of using genomic information to speed up the screening of a taxonomically diverse set of bacteria with biocontrol properties. IMPORTANCE Phytopathogenic fungi are a major threat to global food production. The most common management practice to prevent plant infections involves the intensive use of fungicides. However, with the growing awareness of the ecological and human impacts of chemicals, there is a need for alternative strategies, such as the use of bacterial biocontrol agents. Limitations in the design of bacterial biocontrol included the need for labor-intensive and time-consuming experiments to test a wide diversity of strains and the lack of reproducibility of their activity against pathogens. Here, we show that genomic information is an effective tool to select bacteria of interest quickly. Also, we highlight that the strain Pseudomonas sp. R-71838 produced a reproducible antifungal effect both in vitro and in planta. These findings build a foundation for designing a biocontrol strategy based on Pseudomonas sp. R-71838.

Cytotoxic Effects of Alternariol, Alternariol Monomethyl-Ether, and Tenuazonic Acid and Their Relevant Combined Mixtures on Human Enterocytes and Hepatocytes.

Alternariol (AOH), alternariol monomethyl-ether (AME), and tenuazonic acid (TeA) are major mycotoxins produced by fungi of the genus Alternaria and are common contaminants of food products such as fruits, vegetables, cereals and grains. Alternaria mycotoxins are known to cause relevant economic losses and to have a negative impact on human and animal health. EFSA stated in its scientific opinion that data on the toxicity of Alternaria mycotoxins in humans and livestock are generally lacking, precluding proper hazard characterization. This study aimed to fill some knowledge gaps by studying the in vitro cytotoxicity toward human intestinal epithelial cells (Caco-2) and hepatocytes (HepG2). Cytotoxic properties were assessed by flow cytometric analyses of remaining viable cells (i.e., propidium iodide negative) after mycotoxin exposure for 24-48 h versus solvent control. Treatment of cells with single doses of AOH, AME, and TeA resulted in a dose-dependent loss of cell viability for both cell lines. Half maximal effective concentrations (EC50) of the different mycotoxins were comparable for the two cell lines. On HepG2 cells, EC50 values varying between 8 and 16, 4 and 5, and 40 and 95 µg/mL were calculated for AOH, AME, and TeA, respectively. On Caco-2 cells, EC50 values of 19 µg/mL and varying between 6 and 23, and 60 and 90 µg/mL were calculated for AOH, AME, and TeA, respectively. A general relative cytotoxicity ranking of about 1 = 1 >>> 3 was obtained for AOH, AME, and TeA, respectively. Treatment of both cell lines with combined binary and ternary mixtures of AOH, AME, and TeA in a 1:1:3 ratio, also showed a dose-dependent decrease in cell viability. For both cell lines, the binary combination of especially AME and TeA (1:3 ratio) but also of AOH and AME (1:1 ratio) significantly increased the cytotoxicity compared to the single compound toxicity, although mainly at the highest concentrations tested. The ternary combinations of AOH, AME, and TeA induced only a slight increase in cytotoxicity compared to the single mycotoxins, again at the highest concentrations tested.

Innovative Rhizosphere-Based Enrichment under P-Limitation Selects for Bacterial Isolates with High-Performance P-Solubilizing Traits.

The use of phosphate solubilizing bacteria (PSB) as inoculants for the rhizosphere is a well-known strategy to mitigate P-deficiency in plants. However, despite the multiple modes of action to render P available for plants, PSB often fail to deliver in the field as their selection is often based on a single P-solubilizing trait assessed in vitro. Anticipating these shortcomings, we screened 250 isolates originating from rhizosphere-based enriched consortia for the main in vitro P-solubilizing traits, and subsequently grouped the isolates through trait-based HCPC (hierarchical clustering on principal components). Representative isolates of each cluster were tested in an in planta experiment to compare their in vitro P-solubilizing traits with their in planta performance under conditions of P-deprivation. Our data convincingly show that bacterial consortia capable to mitigate P-deficiency in planta were enriched in bacterial isolates that had multiple P-solubilizing traits in vitro and that had the capacity to mitigate plant P-stress in planta under P-deprived conditions. Furthermore, although it was assumed that bacteria that looked promising in vitro would also have a positive effect in planta, our data show that this was not always the case. Opposite, lack of performance in vitro did not automatically result in a lack of performance in planta. These results corroborate the strength of the previously described in planta-based enrichment and selection technique for the isolation of highly efficient rhizosphere competent PSB. IMPORTANCE With the growing awareness on the ecological impact of chemical phosphate fertilizers, research concerning the use of phosphate solubilizing bacteria (PSB) as a sustainable alternative for, or addition to these fertilizers is of paramount importance. In previous research, we successfully implemented a plant-based enrichment technique for PSB, which simultaneously selected for the rhizosphere competence and phosphate solubilizing characteristics of bacterial suspensions. Current research follows up on our previous findings, whereas we screened 250 rhizobacteria for their P-solubilizing traits and were able to substantiate the results obtained from the enriched suspensions at a single-isolate level. With this research, we aim for a paradigm shift toward the plant-based selection of PSB, which is a more holistic approach compared to the plate-based methods. We emphasize the strength of the previously described plant-based enrichment and selection technique for the isolation of highly efficient and diverse PSB.

Uncovering New Insights and Misconceptions on the Effectiveness of Phosphate Solubilizing Rhizobacteria in Plants: A Meta-Analysis.

As the awareness on the ecological impact of chemical phosphate fertilizers grows, research turns to sustainable alternatives such as the implementation of phosphate solubilizing bacteria (PSB), which make largely immobile phosphorous reserves in soils available for uptake by plants. In this review, we introduce the mechanisms by which plants facilitate P-uptake and illustrate how PSB improve the bioavailability of this nutrient. Next, the effectiveness of PSB on increasing plant biomass and P-uptake is assessed using a meta-analysis approach. Our review demonstrates that improved P-uptake does not always translate in improved plant height and biomass. We show that the effect of PSB on plants does not provide an added benefit when using bacterial consortia compared to single strains. Moreover, the commonly reported species for P-solubilization, Bacillus spp. and Pseudomonas spp., are outperformed by the scarcely implemented Burkholderia spp. Despite the similar responses to PSB in monocots and eudicots, species responsiveness to PSB varies within both clades. Remarkably, the meta-analysis challenges the common belief that PSB are less effective under field conditions compared to greenhouse conditions. This review provides innovative insights and identifies key questions for future research on PSB to promote their implementation in agriculture.

Shifts in the rhizobiome during consecutive in planta enrichment for phosphate-solubilizing bacteria differentially affect maize P status.

Phosphorus (P) is despite its omnipresence in soils often unavailable for plants. Rhizobacteria able to solubilize P are therefore crucial to avoid P deficiency. Selection for phosphate-solubilizing bacteria (PSB) is frequently done in vitro; however, rhizosphere competence is herein overlooked. Therefore, we developed an in planta enrichment concept enabling simultaneous microbial selection for P-solubilization and rhizosphere competence. We used an ecologically relevant combination of iron- and aluminium phosphate to select for PSB in maize (Zea mays L.). In each consecutive enrichment, plant roots were inoculated with rhizobacterial suspensions from plants that had grown in substrate with insoluble P. To assess the plants' P statuses, non-destructive multispectral imaging was used for quantifying anthocyanins, a proxy for maize's P status. After the third consecutive enrichment, plants supplied with insoluble P and inoculated with rhizobacterial suspensions showed a P status similar to plants supplied with soluble P. A parallel metabarcoding approach uncovered that the improved P status in the third enrichment coincided with a shift in the rhizobiome towards bacteria with plant growth-promoting and P-solubilizing capacities. Finally, further consecutive enrichment led to a functional relapse hallmarked by plants with a low P status and a second shift in the rhizobiome at the level of Azospirillaceae and Rhizobiaceae.

Presence of the Weakly Pathogenic Fusarium poae in the Fusarium Head Blight Disease Complex Hampers Biocontrol and Chemical Control of the Virulent Fusarium graminearum Pathogen.

Fusarium head blight (FHB) in wheat (Triticum aestivum L.) is caused by a consortium of mutually interacting Fusarium species. In the field, the weakly pathogenic F. poae often thrives on the infection sites of the virulent F. graminearum. In this ecological context, we investigated the efficacy of chemical and biocontrol agents against F. graminearum in wheat ears. For this purpose, one fungicide comprising prothioconazole + spiroxamine and two bacterial biocontrol strains, Streptomyces rimosus LMG 19352 and Rhodococcus sp. R-43120 were tested for their efficacy to reduce FHB symptoms and mycotoxin (deoxynivalenol, DON) production by F. graminearum in presence or absence of F. poae. Results showed that the fungicide and both actinobacterial strains reduced FHB symptoms and concomitant DON levels in wheat ears inoculated with F. graminearum. Where Streptomyces rimosus appeared to have direct antagonistic effects, Rhodococcus and the fungicide mediated suppression of F. graminearum was linked to the archetypal salicylic acid and jasmonic acid defense pathways that involve the activation of LOX1, LOX2 and ICS. Remarkably, this chemical- and biocontrol efficacy was significantly reduced when F. poae was co-inoculated with F. graminearum. This reduced efficacy was linked to a suppression of the plant's intrinsic defense system and increased levels of DON. In conclusion, our study shows that control strategies against the virulent F. graminearum in the disease complex causing FHB are hampered by the presence of the weakly pathogenic F. poae. This study provides generic insights in the complexity of control strategies against plant diseases caused by multiple pathogens.

At the scene of the crime: New insights into the role of weakly pathogenic members of the fusarium head blight disease complex.

Plant diseases are often caused by a consortium of pathogens competing with one another to gain a foothold in the infection niche. Nevertheless, studies are often limited to a single pathogen on its host. In Europe, fusarium head blight (FHB) of wheat is caused by multiple Fusarium species, including Fusarium graminearum and F. poae. Here, we combined a time series of (co)inoculations, monitored by multispectral imaging, transcriptional, and mycotoxin analyses, to study the temporal interaction between both species and wheat. Our results showed coinoculation of F. graminearum and F. poae inhibited symptom development but did not alter mycotoxin accumulation compared to a single inoculation with F. graminearum. In contrast, preinoculation of F. poae reduced both FHB symptoms and mycotoxin levels compared to a single F. graminearum infection. Interestingly, F. poae exhibited increased growth in dual infections, demonstrating that this weak pathogen takes advantage of its co-occurrence with F. graminearum. Quantitative reverse transcription PCR revealed that F. poae induces LOX and ICS gene expression in wheat. We hypothesize that the early induction of salicylic and jasmonic acid-related defences by F. poae hampers a subsequent F. graminearum infection. This study is the first to report on the defence mechanisms of the plant involved in a tripartite interaction between two species of a disease complex and their host.