Lisa Sanchez

Grapevine crown gall is caused by the tumorigenic bacterium Allorhizobium vitis. The fight against this disease is a major challenge in viticulture since this pathogen can systemically colonize the plant host, persisting in vineyards once infected. Understanding the dynamics of the plant-pathogen relationship not only improves our knowledge of plant biology but also could lead to innovative strategies for sustainable agricultural practices. In this way, this study aimed to gain insights into metabolic changes occurring in the tumor induced by A. vitis, using two techniques, Water-Assisted Laser Desorption/Ionization Mass Spectrometry (WALDI) and Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI MSI). We highlighted compounds such as phenolics alongside specific lipids acting as stress signaling regulators. Moreover, vitopine, produced after plant cell transformation by A. vitis and exclusively found in infected plants, was successfully identified by real time detection by SpiderMass and localized for the first time through MSI. We illustrate here the effectiveness of SpiderMass and MSI in identifying biomarkers of grapevine crown gall, enhancing our understanding of plant responses to bacterial infection. These techniques allow for real-time analysis and spatial distribution mapping of metabolites, paving the way for future research and potential applications in diagnostics.

Efficient early pathogen detection, before symptom apparition, is crucial for optimizing disease management. In barley, the fungal pathogen Pyrenophora teres is the causative agent of net blotch disease, which exists in two forms: P. teres f. sp. teres (Ptt), causing net-form of net blotch (NTNB), and P. teres f. sp. maculata (Ptm), responsible for spot-form of net blotch (STNB). In this study, we developed primers and a TaqMan probe to detect both Ptt and Ptm. A comprehensive k-mer based analysis was performed across a collection of P. teres genomes to identify the conserved regions that had potential as universal genetic markers. These regions were then analyzed for their prevalence and copy number across diverse Moroccan P. teres strains, using both a k-mer analysis for sequence identification and a phylogenetic assessment to establish genetic relatedness. The designed primer-probe set was successfully validated through qPCR, and early disease detection, prior to symptom development, was achieved using ddPCR. The k-mer analysis performed across the available P. teres genomes suggests the potential for these sequences to serve as universal markers for P. teres, transcending environmental variations.

Net blotch disease caused by Drechslera teres is a major fungal disease that affects barley ( Hordeum vulgare) plants and can result in significant crop losses. In this study, we developed a deep learning model to quantify net blotch disease symptoms on different days postinfection on seedling leaves using Cascade R-CNN (region-based convolutional neural network) and U-Net (a convolutional neural network) architectures. We used a dataset of barley leaf images with annotations of net blotch disease to train and evaluate the model. The model achieved an accuracy of 95% for Cascade R-CNN in net blotch disease detection and a Jaccard index score of 0.99, indicating high accuracy in disease quantification and location. The combination of Cascade R-CNN and U-Net architectures improved the detection of small and irregularly shaped lesions in the images at 4 days postinfection, leading to better disease quantification. To validate the model developed, we compared the results obtained by automated measurement with a classical method (necrosis diameter measurement) and a pathogen detection by real-time PCR. The proposed deep learning model could be used in automated systems for disease quantification and to screen the efficacy of potential biocontrol agents to protect against disease.

Controlling crown gall in grapevine ( Vitis vinifera L.) caused by the pathogenic bacterium Allorhizobium vitis is a major challenge for global viticulture, as this pathogen is highly persistent in vineyards once infected. The bacteria can enter the plant through open wounds during pruning and then systemically colonize the plant. This study aimed at evaluating the potential of Paraburkholderia phytofirmans PsJN, a beneficial endophytic bacterium able to colonize the xylem of grapevine, the same ecological niche as A. vitis, to control grapevine crown gall disease. P. phytofirmans PsJN was root-inoculated on grapevine plantlets before infection by A. vitis S4 on shoots. A. vitis S4 level in planta, vitopine production, accumulation of lignin in tumors, and symptoms of crown gall were investigated on grapevine prebacterized or not with PsJN. The expression of 28 grapevine genes involved in defense mechanisms was also simultaneously determined by quantitative real-time PCR. Despite a direct antibacterial effect against AvS4, PsJN has no significant impact on the incidence of crown gall or disease severity. However, PsJN leads to a stronger accumulation of vitopine in tumors and significantly reduced the population level of the pathogen in planta. [ ] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Grapevine flowering is an important stage in the epidemiology of Botrytis cinerea, the causal agent of gray mold disease. To prevent infection and to minimize postharvest losses, the control of this necrotrophic fungus is mainly based on chemical fungicides application. However, there is a growing interest in other control alternatives. Among them, the use of beneficial microorganisms appears as an eco-friendly strategy. This study aims to investigate the effect of Paraburkholderia phytofirmans PsJN, root-inoculated or directly sprayed on fruiting cuttings inflorescences to control B. cinerea growth. For this purpose, quantification by real time PCR of Botrytis development, direct effect of PsJN on fungal spore germination and chemotaxis were assayed. Our results showed a significant protective effect of PsJN only by direct spraying on inflorescences. Moreover, we demonstrated an inhibition exerted by PsJN on Botrytis spore germination, effective when there was a direct contact between the two microorganisms. This study showed that PsJN is positively attracted by the pathogenic fungus B. cinerea and forms a biofilm around the fungal hyphae in liquid co-culture. Finally, microscopic observations on fruit cuttings revealed a co-localization of both beneficial and pathogenic microorganisms on grapevine receptacle and stigma that might be correlated with the protective effect induced by PsJN against B. cinerea via a direct antimicrobial effect. Taking together, our findings allowed us to propose PsJN as a biofungicide to control grapevine gray mold disease.

In many vineyards around the world, Botrytis cinerea (B. cinerea) causes one of the most serious diseases of aerial grapevine (Vitis vinifera L.) organs. The control of the disease relies mainly on the use of chemical products whose use is increasingly challenged. To develop new sustainable methods to better resist B. cinerea, beneficial bacteria were isolated from vineyard soil. Once screened based on their antimicrobial effect through an in vivo test, two bacterial strains, S3 and S6, were able to restrict the development of the pathogen and significantly reduced the Botrytis-related necrosis. The photosynthesis analysis showed that the antagonistic strains also prevent grapevines from considerable irreversible PSII photo-inhibition four days after infection with B. cinerea. The 16S rRNA gene sequences of S3 exhibited 100% similarity to Bacillus velezensis, whereas S6 had 98.5% similarity to Enterobacter cloacae. On the other hand, the in silico analysis of the whole genome of isolated strains has revealed the presence of “biocontrol-related” genes supporting their plant growth and biocontrol activities. The study concludes that those bacteria could be potentially useful as a suitable biocontrol agent in harvested grapevine.

Allorhizobium vitis is the primary causal pathogen of grapevine crown gall disease. Because this endophytic bacterium can survive as a systemic latent (symptomless) infection in grapevine, detecting and monitoring its development in planta is of great importance. In plant bacteria studies, plate counting is routinely used as a simple and reliable method to evaluate the bacterial population level in planta. However, isolation techniques are time-consuming and present some disadvantages such as the risk of contamination and the need for fresh samples for research. In this study, we developed a DNA-based real-time PCR assay that can replace the classical method to monitor the development of Allorhizobium vitis in grapevine plantlets. Primers targeting Allorhizobium vitis chromosomic genes and the virulent tumor-inducing plasmid were validated. The proposed quantitative real-time PCR technique is highly reliable and reproducible to assess Allorhizobium vitis numeration at the earliest stage of infection until tumor development in grapevine plantlets. Moreover, this low-cost technique provides rapid and robust in planta quantification of the pathogen and is suitable for fundamental research to monitor bacterial development over time.

Natural rhamnolipids are potential biocontrol agents for plant protection against bacterial and fungal diseases. In this work, we synthetized new synthetic mono-rhamnolipids (smRLs) consisting in a rhamnose connected to a simple acyl chain and differing by the nature of the link and the length of the lipid tail. We then investigated the effects of these ether, ester, carbamate or succinate smRL derivatives on Botrytis cinerea development, symptoms spreading on tomato leaves and immune responses in tomato plants. Our results demonstrate that synthetic smRLs are able to trigger early and late immunity-related plant defense responses in tomato and increase plant resistance against B. cinerea in controlled conditions. Structure-function analysis showed that chain length of the lipidic part and type of acyl chain were critical to smRLs immune activity and to the extent of symptoms caused by the fungus on tomato leaves.

Phenolic compounds are implied in plant-microorganisms interaction and may be induced in response to plant growth-promoting rhizobacteria (PGPRs). Among PGPR, the beneficial bacterium Paraburkholderia phytofirmans PsJN was previously described to stimulate the growth of plants and to induce a better adaptation to both abiotic and biotic stresses. This study aimed to investigate the impact of PsJN on grapevine secondary metabolism. For this purpose, gene expression (qRT-PCR) and profiling of plant secondary metabolites (UHPLC-UV/DAD-MS QTOF) from both grapevine root and leaves were compared between non-bacterized and PsJN-bacterized grapevine plantlets. Our results showed that PsJN induced locally (roots) and systemically (leaves) an overexpression of PAL and STS and specifically in leaves the overexpression of all the genes implied in phenylpropanoid and flavonoid pathways. Moreover, the metabolomic approach revealed that relative amounts of 32 and 17 compounds in roots and leaves, respectively, were significantly modified by PsJN. Once identified to be accumulated in response to PsJN by the metabolomic approach, antifungal properties of purified molecules were validated in vitro for their antifungal effect on Botrytis cinerea spore germination. Taking together, our findings on the impact of PsJN on phenolic metabolism allowed us to identify a supplementary biocontrol mechanism developed by this PGPR to induce plant resistance against pathogens.