Alejandro Pérez-García

The rise of fungicide-resistant pathogens and increasingly strict regulatory frameworks have heightened the demand for sustainable alternatives in plant disease control. This study assesses the potential of spray-induced gene silencing (SIGS) targeting SdhC, a critical subunit of the mitochondrial succinate dehydrogenase (SDH) complex and determinant of SDHI fungicide resistance, for managing Podosphaera xanthii, the causal agent of cucurbit powdery mildew. To evaluate this approach, PxSdhC double-stranded RNA (dsRNA) was initially infiltrated into melon cotyledons, resulting in a 65% reduction in fungal biomass and a 60% decrease in SdhC gene expression, validating its effectiveness for SIGS applications. A key achievement of this study was the demonstration that strong disease control can be achieved with substantially reduced doses of SDHI fungicides when combined with PxSdhC-dsRNA, providing the first direct evidence of the synergistic potential of this strategy. Building on these results, greenhouse trials were conducted in which dsRNA was applied in both naked and carbon dot (CD)-nanoencapsulated forms, alone or in combination with low doses of the SDHI fungicides boscalid and fluopyram. Nanoencapsulation enhanced dsRNA stability and extended gene silencing effects, achieving up to 69 and 53% disease suppression at 14 and 21 days postinoculation, respectively. Moreover, combined treatments with PxSdhC-dsRNA-CD and low-dose fungicides exhibited synergistic effects, with disease suppression ranging from 33 to 91%, depending on isolate resistance levels. Sensitive and low-resistance isolates responded effectively, while efficacy declined in highly resistant ones. In vitro specificity assays confirmed that PxSdhC-dsRNA selectively inhibited Botrytis cinerea, a species with high SdhC sequence homology, without affecting unrelated fungi. Altogether, these results highlight the promise of gene-targeted, nanoformulated dsRNA as an effective, precise, and environmentally friendly strategy for integrated disease management, capable of boosting the performance of low-dose fungicide applications and reducing chemical input. [ ] Copyright © 2026 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

Cucurbit powdery mildew, predominantly caused by Podosphaera xanthii, poses a major threat to global cucurbit production due to the pathogen’s rapid adaptability and resistance to conventional fungicides. This growing challenge highlights the urgent need for alternative, sustainable disease management strategies. As the primary interface between the fungus, host plant, and environment, the fungal cell wall emerges as a strategic target for development of innovative control approaches. This study focuses on ECM33, a glycophosphatidylinositol (GPI)-anchored protein believed to play a role in cell wall architecture and integrity, although its specific biochemical function remains undefined. In silico structural modeling revealed that PxECM33 resembles leucine-rich repeat proteins and contains potential carbohydrate-binding motifs. Recombinant PxECM33 exhibited binding affinity for chitin, β-glucans, and mannans, the main glycosidic components of the P. xanthii cell wall, supporting these structural predictions. Molecular docking analyses uncovered distinct ligand-specific interactions with these carbohydrates, further implicating PxECM33 in cell wall dynamics. Silencing PxECM33 via RNA interference significantly impaired fungal growth and caused pronounced cell wall disorganization. Notably, dual silencing with the melon immune receptor gene CmCERK1 mitigated these defects, suggesting that PxECM33 may function in masking immunogenic oligosaccharides to evade host detection. Furthermore, spray-induced gene silencing (SIGS) targeting PxECM33 effectively reduced disease symptoms in melon plants, highlighting its potential as a sustainable and nontoxic biocontrol strategy. Given the high-sequence conservation of ECM33 among ascomycete fungi, these findings support its candidacy as a broad-spectrum molecular target for managing powdery mildew in cucurbits and potentially other crops.

Drug discovery is limited by target identification, a major bottleneck in antimicrobial development and in combating emerging fungicide resistance. We present APEX (Attention-based Protein EXplainer), an explainable AI framework for cross-species, proteome-scale target discovery and pocket-guided molecular design. APEX combines ESM-2 embeddings, GAT, and a MLP to train pathogen-specific essentiality predictors (APEX-Tar) alongside a universal druggability model (APEX-Drug). Attention maps and GNNExplainer-derived subgraphs highlight residues driving predictions, enabling direct conditioning of structure-based diffusion models for inhibitor generation. APEX-Tar identifies key residues in known fungal targets and nominates new candidates, including adenylosuccinate lyase (ADSL) in fungi and the bacterial adhesin YadV. APEX-Drug recapitulates established fungicide binding sites, guides the design of ADSL inhibitors exploiting a fungal-specific active-site residue, and uncovers in YadV a previously undescribed pocket distinct from known pilicide sites. Together, APEX provides a kingdom-agnostic, explainable pipeline for target prioritization and guided molecular design, accelerating the search for next-generation antimicrobials.

Botrytis cinerea, the causal agent of gray mold, represents a serious threat to horticultural crops worldwide and is considered a high-risk pathogen for fungicide resistance. Among the mechanisms conferring fungicide resistance in B. cinerea, the most prominent are point mutations in fungicide target genes and the overexpression of drug efflux transporters, which result in multidrug resistance (MDR). In this study, spray-induced gene silencing (SIGS) targeting Bcmrr1, a transcriptional regulator of the ABC transporter BcatrB, was evaluated as a novel strategy to control fungicide-sensitive and MDR B. cinerea isolates. Double-stranded RNA (dsRNA) targeting Bcmrr1 and BcatrB were initially tested in vitro and in detached-leaves assays. Both dsRNAs effectively inhibited spore germination and reduced lesion development, fungal biomass, and target gene expression. However, Bcmrr1-dsRNA consistently provided superior and more stable protection and was therefore selected for further analyses. Foliar application of Bcmrr1-dsRNA conferred effective disease suppression for up to 14 days post-treatment, associated with decreased fungal colonization and transient Bcmrr1 repression. Comparable protective effects were observed in apple fruit, confirming cross-host efficacy. Notably, co-application of Bcmrr1-dsRNA with sublethal doses of fludioxonil enhanced disease control and partially restored fungicide sensitivity in MDR isolates, demonstrating a synergistic interaction between RNAi-based and chemical control strategies. Cross-species analyses revealed high sequence conservation of Bcmrr1 within the Botrytis genus and significant inhibition of B. fragariae and B. pseudocinerea, highlighting the broad-spectrum potential of this approach. Collectively, these findings identify Bcmrr1 as a promising RNAi target for overcoming multidrug resistance in and establish SIGS as a selective and environmentally sustainable tool for gray mold management in horticultural systems.

Phytopathogenic fungi pose an escalating threat to global food security and ecosystem stability, as resistance and environmental concerns diminish the effectiveness of conventional fungicides. Double‐stranded RNA (dsRNA)‐based fungicides offer a species‐specific, eco‐friendly alternative. We introduce LUCID (Locating Uncovered, conserved, and Indispensable for pathogenicity Determinants), a computational pipeline that accelerates the development of RNAi‐based biofungicides by integrating target identification with dsRNA design and off‐target prediction. LUCID employs a dual‐branch strategy to identify both Conserved Essential Proteins (CEPs) and Conserved Non‐Annotated Proteins (CNAPs), leveraging transcriptomic data and comparative genomics across diverse fungal species. Validation in Botrytis cinerea demonstrated high efficacy, with 67% of proposed targets successfully silenced and an average silencing efficiency of 96%. Additionally, coupling LUCID with advanced protein language models (PLMs) revealed a novel pathogenicity determinant in B. cinerea : a putative mediator complex protein. LUCID offers a scalable, species‐agnostic framework for designing sustainable fungicides, enabling rapid, targeted control of fungal diseases with minimal ecological impact.

The current study investigated 17 bacterial strains for their ability to synthesize gold nanoparticles (AuNPs) from the aryldiazonium gold(III) salt (DS-AuCl ₄ ). The study aims to investigate the ability of bacterial cell biomass in the stationary phase of growth to synthesize AuNPs at 28 °C and 37 °C. Eleven bacterial strains were isolated from soil and identified using the VITEK® 2 system and 16S rRNA sequencing. An additional six strains were obtained from the American Type Culture Collection (ATCC). The investigated Gram-positive and Gram-negative bacterial strains successfully produced anisotropic AuNPs at a cell density of 2.0 McFarland (6.0 × 10 ⁸ CFU/mL). Nanoparticle formation was faster when samples were incubated at 37 °C than at 28 °C across all bacterial strains. The results of UV-vis spectroscopy confirmed the presence of AuNPs, with peaks observed centered at 550 nm. High-resolution transmission electron microscopy (HR-TEM) revealed a variety of morphologies, including spheres, rods, triangles, pentagons, hexagons, irregular shapes, and flower-like structures. Gram-positive and Gram-negative bacteria synthesized AuNPs of sizes 38.7 ± 26.0 and 34.0 ± 18.6 nm, respectively. Lattice-spacing analysis confirmed the formation of metallic AuNPs. Energy-dispersed X-ray spectroscopy (EDS) validated the presence of gold in the samples, and X-ray photoelectron spectroscopy (XPS) confirmed the elemental composition of AuNPs at 84.0 eV. These nanoparticles have potential applications in cancer therapy and diagnosis, antibacterial treatments, and drug delivery. Key points: • The AuNPs were synthesized using various bacterial strains • The gold precursor is aryldiazonium gold(III) salt • Various anisotropic morphologies were obtained Graphical Created in BioRender. Ahmady, I. (2025)

The widespread emergence of fungicide-resistant Botrytis cinerea populations, together with increasingly strict regulatory constraints, has intensified the need for alternative and environmentally sustainable strategies for gray mold management. This study evaluates spray-induced gene silencing (SIGS) targeting mitochondrial respiration through double-stranded RNAs (dsRNAs) directed against sdhB and cytB genes. These genes encode key subunits of respiratory complexes II and III, key targets and determinants of resistance to SDHI and QoI fungicides, respectively. Exogenous application of these dsRNAs significantly reduced B. cinerea conidial germination (~50%), lesion development in tomato leaves and apple fruits (~45%), fungal biomass (~50%), and transcript levels of sdhB (3.2-fold) and cytB (2.0-fold). When combined with sublethal doses of boscalid (SDHI) or azoxystrobin (QoI), dsRNA treatments markedly decreased lesion severity in fungicide-sensitive isolates, achieving substantial but not full equivalent to field-dose fungicide efficacy. In resistant isolates, dsRNA alone consistently reduced disease symptoms, and its combination with sublethal-dose fungicides further enhanced control. When dsRNA was applied together with full-rate fungicide, lesion development declined sharply across all resistant isolates, and in some cases gray mold symptoms were completely eradicated, indicating the strong biological effect achieved by the RNAi-fungicide combination. Sequence identity analyses revealed strong cross-species activity among closely related Botrytis and Sclerotiniaceae species, with no predicted effects on unrelated fungi, plants, or humans. This work provides the first demonstration of a dual-target SIGS strategy acting on mitochondrial respiration, highlighting its potential as a precise, eco-compatible, and effective tool for managing B. cinerea resistance within integrated pest management frameworks.

Pseudomonas aeruginosa  was used to synthesize anisotropic gold nanoparticles from the unusually reducible aryldiazonium gold (III) salt of the chemical formula [HOOC-4-C ₆ H ₄ N≡N]AuCl ₄ (abbreviated as DS-AuCl ₄ ). We investigated the effect of bacterial cell density, temperature, and pH on the AuNP synthesis. The bacterial cell density of 6.0 × 10 ⁸ CFU/mL successfully reduced 0.5 mM DS-AuCl ₄ salt to AuNPs after incubation at 37 °C (24 h), 42 °C (24 h), and 25 °C (48 h). Transmission electron microscopy (TEM) images revealed the formation of spherical, triangle, star, hexagon, and truncated triangular morphologies for the AuNPs synthesized using P. aeruginosa bacteria. The average size of AuNPs synthesized at 25 °C (48 h), 37 °C (24 h), and 42 °C (24 h) was 39.0 ± 9.1 nm, 26.0 ± 8.1 nm, and 36.7 ± 7.7 nm, respectively. The average size of AuNPs synthesized at pH 3.7, 7.0, and 12.7 was 36.7 ± 7.7 nm, 14.7 ± 3.8 nm, and 7.3 ± 2.5 nm, respectively, with the average size decreasing at a pH of 12.7. The reduction of the DS-AuCl ₄ salt was confirmed using X-ray photoelectron spectroscopy (XPS). The significant peaks for C1s, Au4f doublet, N1s, and O1s are centered at 285, 84–88, 400, and 532 eV. The ability of inactivated bacteria (autoclave-dead and mechanically lysed bacteria), peptidoglycan, and lipopolysaccharides to reduce the DS-AuCl ₄ salt to AuNPs was also investigated. Anisotropic AuNPs were synthesized using inactivated bacteria and peptidoglycan but not using lipopolysaccharides. The AuNPs demonstrated biocompatibility with human RBCs and were safe, with no antibacterial activities against Escherichia coli and Staphylococcus aureus . This is the first report demonstrating the synthesis of AuNPs using aryldiazonium gold(III) salts with P. aeruginosa . These AuNPs are promising candidates for exploring potential applications in nanomedicine and drug delivery. Key points: • Anisotropic AuNPs were synthesized using P. aeruginosa bacteria. • Dead and lysed bacterial residues synthesized anisotropic AuNPs. • AuNPs are hemocompatible. Graphical

β-glucanases play a critical role in degrading β-glucans, which are essential components of fungal cell walls that maintain fungal structure and physiology. This study characterizes BvlzGluc, a novel β-glucanase produced by Bacillus velezensis CECT 8237, which has potent antifungal properties. BvlzGluc specifically targets the β-glucan layer of Botrytis cinerea , causing significant structural malformations, impaired fungal development, and reduced virulence. These effects weaken the fungal cell wall, increasing its susceptibility to environmental stress and reducing its capacity to infect host plants. Notably, BvlzGluc is a noncanonical β-glucanase belonging to glycoside hydrolase family 16 (GH16) but presents unique structural features that distinguish it from typical members of this family, suggesting potential reclassification. Its physicochemical stability and simple structural design increase its suitability for scalable production, positioning it as a promising candidate for agricultural and industrial applications requiring efficient β-glucan degradation. This work builds on previous studies of microbial antifungal mechanisms, providing new insights into bacterial‒fungal interactions and reinforcing the potential of bacterial enzymes in sustainable pathogen management and advancing biotechnological innovations.

Bioactive metabolites play a crucial role in shaping interactions among diverse organisms. In this study, we identified cyclo(Pro-Tyr), a metabolite produced by Bacillus velezensis, as a potent inhibitor of Botrytis cinerea and Caenorhabditis elegans, two potential cohabitant eukaryotic organisms. Based on our investigation, cyclo(Pro-Tyr) disrupts plasma membrane polarization, induces oxidative stress and increases membrane fluidity, which compromises fungal membrane integrity. These cytological and physiological changes induced by cyclo(Pro-Tyr) may be triggered by the destabilization of membrane microdomains containing the [H⁺]ATPase Pma1. In response to cyclo(Pro-Tyr) stress, fungal cells activate a transcriptomic and metabolomic response, which primarily involves lipid metabolism and Reactive Oxygen Species (ROS) detoxification, to mitigate membrane damage. This similar response occurs in the nematode C. elegans, indicating that cyclo(Pro-Tyr) targets eukaryotic cellular membranes.