Barbara De Coninck

Hairy Root Disease (HRD) is a significant threat to hydroponic crops such as tomato, cucumber, melon, eggplant, and bell pepper. Rhizogenic agrobacteria are the primary causative agents of HRD and infection of host plants results in excessive root growth, which leads to substantial yield losses. This study investigated infection efficiency and symptom severity across 24 rhizogenic Agrobacterium strains and explored potential correlations with specific genomospecies, opine types or host of isolation. This was done using a laboratory-scale assay, i.e. an in vitro carrot disk bioassay, and a soil-based tomato bioassay. In the carrot disk assay, substantial variation was observed in infection efficiency and symptom severity, in particular excessive root development, across strains. While infection efficiency showed no clear correlation with genomospecies, opine types or host of origin, the carrot disk assay showed that hairy root biomass as a measure for symptom severity varied depending on genomospecies, opine type, and plant host from where the isolates are originally isolated. Based on the outcomes of this carrot disk assay, six strains were selected for additional assessment under greenhouse conditions in the soil-based tomato bioassay. Similar to the carrot disk assay, large variations were observed between strains regarding infection efficiency and symptom severity. The use of these complementary bioassays not only allowed identification of strain-specific differences in virulence, but also provided valuable insight into the diversity of rhizogenic Agrobacterium and its interaction with different host plants.

Clubroot, caused by the soil‐borne protist Plasmodiophora brassicae , is a major disease of Brassica crops, resulting in severe root malformations and yield losses. While most research has centred on immune signalling and hormone dynamics, plant–pathogen interactions also dramatically reshape primary metabolism, often modifying source activity and converting infected tissues into strong metabolic sinks. The SnRK1 (SNF1‐related kinase 1) protein kinase acts as a cellular fuel gauge in plants, integrating metabolic status and environmental and developmental cues to maintain carbon and energy homoeostasis. Here, we explored SnRK1‐mediated quantitative resistance against clubroot disease in the related crucifer model Arabidopsis thaliana . Both soil‐ and hydroponics‐based disease assays revealed how especially increased nuclear SnRK1α1 activity antagonizes clubroot development, suggesting a pivotal role for transcriptional regulation. qRT‐PCR analysis and quantification of soluble sugar contents and invertase activity in roots indicate that SnRK1‐mediated resistance coincides with reduced sucrose transporter expression as well as cell wall invertase expression and activity, likely limiting clubroot development by reducing sink strength. Consistently, cellular assays indicate that the recently identified SnRK1α1‐targeting P. brassicae effector PBZF1 interferes with SnRK1α1 nuclear translocation. Our study thus corroborates that SnRK1 is a primary effector target and shows that SnRK1‐mediated reprogramming of gene expression and sink activity is an effective mechanism against clubroot disease development.

2. Hairy root disease (HRD), caused by rhizogenic Agrobacterium , is an economically important disease affecting hydroponic tomato ( Solanum lycopersicum L.) production worldwide. HRD-affected plants show extensive root proliferation, resulting in decreased energy expenditure towards fruit production. Host plant susceptibility to rhizogenic Agrobacterium is typically evaluated through artificial wounding-based infection bioassays. However, under natural infection settings, rhizogenic Agrobacterium can induce disease symptoms without deliberate, artificial wounding. We developed a soil-based, non-wounding bioassay that closely mimics natural rhizosphere interactions and permits quantitative and qualitative assessment of HRD symptoms. The assay measured root dry weight, documented agravitropic root development typical of HRD and confirmed in planta T-DNA gene expression using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). We used this bioassay to evaluate disease symptoms towards rhizogenic Agrobacterium in tomato cv. ‘Moneymaker’ and the rootstocks ‘Optifort’, ‘Maxifort’, and ‘Arnold’. ‘Optifort’ and ‘Maxifort’ exhibited significantly higher root biomass than ‘Arnold’ and ‘Moneymaker’, indicating more pronounced symptom development. The bioassay also differentiated virulence levels amongst various rhizogenic Agrobacterium strains isolated from HRD-affected plants. Together, these results show that our soil-based bioassay provides a robust and ecologically relevant platform for screening tomato genotypes and comparing virulence levels of rhizogenic Agrobacterium strains supporting resistance breeding and disease management efforts.

In seed plants, ethylene is produced from 1-aminocyclopropane-1-carboxylic acid (ACC) by the enzyme ACC oxidase (ACO). Despite the critical role of ACO in ethylene biosynthesis, the ACO gene family has not been fully characterized in Arabidopsis (Arabidopsis thaliana). This study investigated the 5 ACO genes in Arabidopsis, revealing distinct tissue-specific and developmental expression patterns. Each ACO enzyme exhibited a unique enzymatic capacity for ethylene production, facilitating isoform-specific regulation of ethylene biosynthesis. At the subcellular level, ACO localized predominantly in the cytosol, where ethylene biosynthesis likely occurs, but, unexpectedly, also in the nucleus. Through reverse genetics, including single and higher-order aco mutants, we observed a high degree of gene redundancy, sustaining ethylene biosynthesis. Disruption of all 5 ACO genes resulted in plants unable to produce ethylene but did not adversely affect seedling, vegetative, or reproductive development. However, some development processes associated with high rates of ethylene production, such as germination and petal abscission, were impaired in the aco quintuple mutant, while others, such as leaf senescence, were not. This suggests that modulation of ethylene emission rates by ACOs is key in determining specific developmental processes. Furthermore, the aco quintuple mutant showed impaired responses to abiotic (eg nutrient deficiency and metal toxicity) and biotic stress (eg Botrytis cinerea), akin to ethylene-insensitive plants. This highlights the pivotal role of ethylene in modulating stress responses. In conclusion, the ACO gene family plays a vital role in fine-tuning ethylene biosynthesis in a spatial–temporal way, thereby modulating plant development and stress resilience.

Clubroot, caused by the soil-borne protist Plasmodiophora brassicae , is a major disease of Brassica crops, resulting in severe root malformations and yield losses. While most research has centered on immune signaling and hormone dynamics, plant-pathogen interactions also dramatically reshape primary metabolism, often modifying source activity and converting infecting tissues into strong metabolic sinks. The SnRK1 (SNF1-related kinase 1) protein kinase acts as a cellular fuel gauge in plants, integrating metabolic status and environmental and developmental cues to maintain carbon and energy homeostasis. Here, we explored SnRK1-mediated quantitative resistance against clubroot disease in the related crucifer model Arabidopsis thaliana . Both soil and hydroponic-based disease bioassays revealed how especially increased nuclear SnRK1α1 activity antagonizes clubroot development, suggesting a pivotal role for transcriptional regulation. qRT-PCR analysis and quantification of soluble sugar contents and invertase activity in roots indicate that SnRK1 represses sucrose transporter expression as well as cell wall invertase (CWINV) expression and activity, likely limiting clubroot development by reducing sink strength. Consistently, cellular assays indicate that the recently identified SnRK1α1-targeting P. brassicae effector PBZF1 interferes with SnRK1α1 nuclear translocation. Our study thus corroborates that SnRK1 is a primary effector target and shows that SnRK1-mediated reprogramming of gene expression and sink activity is an effective mechanism against clubroot disease development.

Microalgae are increasingly recognized as promising biostimulants for sustainable agriculture, yet their potential remains underexplored due to the complexity of biostimulant activity and the vast diversity of species. Efficient standardized screening approaches are therefore needed. In this study, a high-throughput screening platform assessed the biostimulant activity of five microalgal species (Limnospira platensis, Chlorella vulgaris, Dunaliella salina, Microchloropsis gaditana, and Isochrysis galbana) in Arabidopsis thaliana. The system enabled full life-cycle assessment of A. thaliana under optimal and drought stress conditions, incorporating three application methods (soil amendment, irrigation, foliar spray) and a wide concentration range of 0.01–0.5 g/L. Biostimulant efficacy depended strongly on concentration and application method. Irrigation-based applications generally enhanced drought tolerance but delayed bolting and flowering. The highest concentration inhibited germination and root elongation, likely due to bioactive compound toxicity rather than salinity or pH. L. platensis exhibited broad activity across environmental conditions, while I. galbana likewise showed wide-ranging effects, including enhanced generative growth. In contrast, D. salina and M. gaditana primarily improved drought tolerance, and C. vulgaris acted mainly under optimal conditions. These findings highlight the value of A. thaliana to accommodate rapid biostimulant screening and identify both novel and established microalgae for further validation in crops.

Climate change poses a major threat to crop production, resulting in the emergence of new pests and diseases. Phytophthora cryptogea has recently emerged as a major concern in hydroponic lettuce cultivation, causing substantial yield and economic losses. This oomycete pathogen thrives in elevated water temperatures induced by warmer weather conditions (e.g., heatwaves), facilitating rapid pathogen propagation. Although the disease is already present for several decades in chicory cultivation, where it originates from the field, its origin in lettuce cultivation remains unclear. To get a better understanding of its origin, we conducted a multilocus sequence analysis using five reference genes (ITS, β-tub, COI, EF1α, and HSP90) and 33 P. cryptogea isolates from various hosts, including chicory and lettuce. Results revealed a clear separation between lettuce and chicory isolates. Furthermore, we developed and implemented a robust disease bioassay and qPCR assay to investigate the interaction between P. cryptogea strains and lettuce. Our findings revealed that while lettuce isolates exhibited the highest virulence, some chicory isolates also caused disease in lettuce, suggesting a potential evolutionary link between P. cryptogea in lettuce and chicory. Our experiments also revealed that even a low concentration of zoospores (100 zoospores/liter) can elicit severe symptoms, underscoring the pathogen’s high virulence. Therefore, effective disease management strategies are needed for controlling (the spread of) the disease. Together, this research provides several tools that can be used to enhance our understanding of the interaction between P. cryptogea and its host plants, including the development of proper disease management strategies.

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.

Understanding the complex interactions between plants and their associated microorganisms is crucial for optimizing plant health and productivity. While microbiomes of soil-bound cultivated crops are extensively studied, microbiomes of hydroponically cultivated crops have received limited attention. To address this knowledge gap, we investigated the rhizosphere and root endosphere of hydroponically cultivated lettuce. Additionally, we sought to explore the potential impact of the oomycete pathogen Phytophthora cryptogea on these microbiomes. Root samples were collected from symptomatic and nonsymptomatic plants in three different greenhouses. Amplicon sequencing of the bacterial 16S rRNA gene revealed significant alterations in the bacterial community upon P. cryptogea infection, particularly in the rhizosphere. Permutational multivariate analysis of variance (perMANOVA) revealed significant differences in microbial communities between plants from the three greenhouses, and between symptomatic and nonsymptomatic plants. Further analysis uncovered differentially abundant zero-radius operational taxonomic units (zOTUs) between symptomatic and nonsymptomatic plants. Interestingly, members of Pseudomonas and Flavobacterium were positively associated with symptomatic plants. Overall, this study provides valuable insights into the microbiome of hydroponically cultivated plants and highlights the influence of pathogen invasion on plant-associated microbial communities. Further research is required to elucidate the potential role of Pseudomonas and Flavobacterium spp. in controlling P. cryptogea infections within hydroponically cultivated lettuce greenhouses.