The rare sugar D-tagatose protects plants from downy mildews and is a safe fungicidal agrochemical

Rare sugars, defined as monosaccharides rarely found in nature, represent a significant area of research due to their potential applications in various fields, including medicine and agriculture. The Izumoring concept, developed in 2002, provided a blueprint for the enzymatic synthesis of all hexoses, including rare sugars. Studies have shown that certain rare sugars like D-allulose and D-allose exhibit anti-aging effects and disease tolerance induction in plants. These effects are often linked to the phosphorylation of the rare sugars by plant hexose kinases and subsequent signal transduction pathways, triggering plant defense responses such as the expression of pathogenesis-related (PR) proteins. Given the enzymatic accessibility of various rare sugars thanks to Izumoring, the current study investigates the potential of D-tagatose, a C-4 epimer of D-fructose, as a plant disease control agent, focusing on downy mildews. D-tagatose is already recognized as safe for consumption by the FDA and WHO, making it an attractive candidate for eco-friendly agricultural applications. This study aims to evaluate the efficacy of D-tagatose against plant diseases and elucidate its mode of action, exploring its potential as an innovative and safe agrochemical.

Previous research has highlighted the diverse biological activities of rare sugars. D-Allulose, a C-3 epimer of D-fructose, has shown promising anti-hyperlipidemic and anti-hyperglycemic effects. Similarly, D-allose, a C-3 epimer of D-glucose, demonstrates anti-aging properties. In the context of plant pathology, both D-allulose and D-allose have been observed to induce disease tolerance, an effect linked to the activation of plant defense mechanisms involving reactive oxygen species (ROS) generation and PR protein expression. The phosphorylation of these rare sugars by plant hexose kinases is crucial for these effects. However, the investigation into the disease control potential of other rare sugars, particularly D-tagatose, remains limited. This gap in knowledge underscores the importance of exploring the potential of D-tagatose as a novel and safe plant protectant. The safety profile of D-tagatose, coupled with its potential for sustainable production, makes it a compelling candidate for developing eco-friendly agrochemicals.

The study employed both pot and field trials to assess the efficacy of D-tagatose against a range of plant diseases caused by various pathogens, including downy mildews and powdery mildews. Effective concentrations of D-tagatose were determined as those reducing disease severity to less than 50% compared to untreated controls. The timing of D-tagatose application (before and after pathogen inoculation) was varied to determine whether it acts preventively or curatively. The model system *Arabidopsis thaliana* and *Hyaloperonospora arabidopsidis* (isolate Noco2) was used to explore D-tagatose's mode of action. Hyphal growth, conidiation, conidiophore formation, and oospore formation were examined microscopically. The effects of D-tagatose on the expression of plant defense-related genes were evaluated using RT-qPCR, microarray analysis (in rice), and RNA sequencing (*Arabidopsis*). To investigate D-tagatose's impact on the pathogen's metabolism, the authors analyzed sugar metabolism in *H. arabidopsidis* isolate Noco2. They identified and characterized key enzymes involved in mannose metabolism through heterologous expression in *E. coli*. Enzyme activity assays, including kinetic analysis and competitive inhibition studies, were conducted to elucidate D-tagatose's role in inhibiting these metabolic pathways. Techniques such as HPLC and spectrophotometry were employed to quantify sugar metabolites and enzyme activity.

D-tagatose demonstrated significant efficacy in reducing disease severity across a wide range of plant diseases caused by various pathogens (oomycetes, ascomycetes, and basidiomycetes) in both pot and field trials. Effective concentrations were low (0.5–10% w/v), with particularly strong effects against downy mildews. D-tagatose exhibited both preventive and curative effects. In the *Arabidopsis*-*H. arabidopsidis* model system, D-tagatose (≥25 mM) significantly inhibited hyphal growth, conidiation, conidiophore formation, and oospore formation. Importantly, D-tagatose treatment did not induce the expression of plant defense-related genes in *Arabidopsis*, cucumber, or rice, suggesting a direct effect on the pathogen rather than plant defense activation. Analysis revealed that D-tagatose is phosphorylated at the C-6 position by a pathogen fructokinase (LC500344), leading to the production of D-tagatose 6-phosphate. This phosphorylated product competitively inhibits the pathogen's fructokinase, reducing the phosphorylation of D-fructose to D-fructose 6-phosphate. Furthermore, D-tagatose 6-phosphate also competitively inhibits phosphomannose isomerase (LC500563), disrupting the interconversion of D-fructose 6-phosphate and D-mannose 6-phosphate. This dual inhibition leads to reduced glycolysis and mannan biosynthesis, which is essential for fungal cell wall integrity and pathogen development. The K_m for D-fructose was 0.197 ± 0.044 mM, and V_max was 3.48 ± 0.89 µmol mg protein⁻¹ min⁻¹. For D-tagatose, K_m = 52.67 ± 22.75 mM and V_max = 0.15 ± 0.04 µmol mg protein⁻¹ min⁻¹. The K_i for D-tagatose was 70.05 ± 5.14 mM. D-Tagatose 6-phosphate (12.5–37.5 mM) inhibited mannose 6-phosphate production by 51.6–60.8% and glucose 6-phosphate production by 19.3–38.2%.

This study demonstrates the remarkable efficacy of D-tagatose as a plant disease control agent, particularly against downy mildews. The findings strongly suggest that D-tagatose acts directly on the pathogen by inhibiting key enzymes in its mannose metabolism pathway, rather than triggering host defense responses. This unique mode of action, involving competitive inhibition at multiple metabolic steps, offers a novel approach to plant disease control and may reduce the risk of developing pathogen resistance. The absence of phytotoxicity, combined with its existing GRAS status, makes D-tagatose a highly promising eco-friendly alternative to synthetic fungicides. The detailed understanding of its mode of action provides valuable insights for developing improved formulations and strategies for optimizing its application. The discovery of D-tagatose in plant root exudates suggests its potential role in natural plant defense mechanisms, which warrants further investigation.

This research establishes D-tagatose as a highly effective and safe fungicidal agrochemical, particularly against downy mildews. Its unique mode of action, targeting crucial enzymes in the pathogen's mannose metabolism, makes it a promising alternative to synthetic fungicides. The lack of phytotoxicity and its GRAS status further enhance its appeal for sustainable agriculture. Future research should focus on optimizing D-tagatose formulations, exploring its efficacy against a broader range of pathogens, and investigating its potential role in natural plant defense mechanisms.

While the study demonstrates strong efficacy of D-tagatose against various pathogens, further research is needed to fully characterize its mode of action across different pathogen species. The study primarily focuses on downy mildews; investigation into its mechanisms against other pathogens is needed. Long-term field trials are also necessary to assess its long-term effects and potential environmental impacts. Finally, while the current research points toward a direct effect on the pathogen, the possibility of indirect effects via plant-mediated mechanisms cannot be completely excluded.