He Liu

Identification of pesticide targets is of great significance for the development of new pesticides. The new compound GLY‐15, containing a pyrimidine heterocycle and a moroxydine skeleton structure, has good anti‐TMV activity, but the underlying molecular targets and mechanism of action remain elusive. Here, host malate dehydrogenase (MDH), glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), and tobacco mosaic virus (TMV) coat protein (CP) were identified as potential targets of GLY‐15 using activity‐based protein profiling (ABPP) and drug affinity responsive target stability (DARTS), and their interactions with GLY‐15 were validated by microscale thermophoresis (MST) and pull‐down analysis. Functional analyses demonstrate that MDH silencing significantly reduces TMV accumulation, while transient overexpression of MDH results in elevated viral infection. Meanwhile, yeast two‐hybrid (Y2H), co‐immunoprecipitation (Co‐IP), and bimolecular fluorescence complementation (BiFC) analysis uncover that MDH interacts with CP, and their interaction is effectively inhibited by GLY‐15. Site‐directed mutagenesis identifies E225 as a critical residue for both GLY‐15/MDH binding and MDH/CP interaction. Further investigations reveal that GLY‐15 functions as an MDH inhibitor and affects its interaction with CP. Meanwhile, we showed that GLY‐15 targeting MDH indicates broad antiviral activity against pepper mild mottle virus (PMMoV) and potato virus Y (PVY). This investigation systematically reveals novel insights into the anti‐TMV mechanisms of GLY‐15, establishing a valuable theoretical basis for antiviral target discovery and plant disease resistance breeding.

BACKGROUND: Our laboratory participated in the development of a 10.5 billion colony‐forming units (CFU) g ^–1 Paenibacillus polymyxa and Bacillus subtilis wettable powder (PBM), which has been registered in China as a novel microbial fungicide. Subsequent research has demonstrated that PBM exhibits significant inhibitory effects on tobacco target spot disease caused by Rhizoctonia solani , yet the underlying mechanisms remain unclear. RESULTS: PBM effectively inhibits the mycelial growth of R. solani and induces morphological distortions in the fungal hyphae. Field trials demonstrated that PBM provid a control efficiency of 86.03 ± 1.76% against R. solani . Additionally, PBM enhanced agronomic traits of the plants, including height, leaf length and leaf width. Analysis of the phyllosphere microbial community revealed that application of PBM to the abaxial surface of leaves under high humidity conditions at 28 °C facilitates the colonization of biocontrol bacteria. Moreover, PBM not only significantly reducedthe abundance of pathogenic microorganisms including R. solani , but also promote the enrichment of beneficial microbial communities on the leaf surface, thereby restoring the microbial community structure to a state resembling that of healthy plants. Furthermore, PBM treatment stimulated host resistance responses by inducing the expression of defense‐related genes and upregulating the activity of defense‐related enzymes. CONCLUSION: This study elucidates the functions and mechanisms of the composite microbial pesticide PBM, underscoring its potential as an environmentally friendly biological control agent against plant diseases caused by R. solani . © 2025 Society of Chemical Industry.

The necrotrophic fungal pathogen Rhizoctonia solani anastomosis group 3 (AG3‐TB) is a major cause of global tobacco crop yield losses. Secreted proteins produced by filamentous fungi, as important virulence factors, play a core role in the interaction between plants and pathogens. In this study, we identified a secretory protein, RsDN3377, which localised to the intercellular space and induced cell death in Nicotiana benthamiana . Heterologous expression in Escherichia coli coupled with matrix‐assisted laser desorption/ionisation time‐of‐flight mass spectrometry analysis confirmed RsDN3377 possessed deacetylase activity. In addition, RsDN3377 was an essential pathogenicity factor for mycelial development by double‐stranded RNA‐mediated gene silencing. Through yeast two‐hybrid and bimolecular fluorescence complementation assays, we demonstrated that RsDN3377 interacted with the calcium‐binding protein NtCML19. In addition, transgenic Yunyan 87 overexpressing NtCML19 exhibited enhanced resistance to R. solani AG3‐TB infection. Microscale thermophoresis analysis verified the calcium‐binding activity of NtCML19. These lines of evidence indicate that the deacetylase RsDN3377 is secreted by R. solani AG3‐TB, and this protein, critical for promoting fungal mycelial development and pathogenicity, was disrupted by its resistance‐related interaction with NtCML19.

Tobacco root rot caused by Fusarium spp. infection results in significant losses to tobacco production. The disease is primarily prevalent in southern and central China, particularly in Fujian, Shandong, and Shanxi (Qiu et al. 2018). In July 2023, tobacco samples (n=10) exhibiting root rot symptoms were collected from Maodianzi Town (40.67°N, 124.62°E; cv. Liaoyan 21) and Qingyishan Town (40.68°N, 124.62°E; cv. Liaoyan 21) in Kuandian, Liaoning Province, China. The disease incidence in the surveyed fields reached approximately 60% to 70% based on the five-point sampling method. The symptoms exhibited blackening and rotting of the main root, stem base, and sparse fibrous roots. To isolate and identify the pathogen, the diseased tissues (2 × 2 mm) were cut from the edge of root lesions, treated with 75% alcohol rinsed with sterile water, and then incubated on potato dextrose agar (PDA) medium at 28 ℃. A total of 21 isolations were obtained through the single-spore isolation method. The experiment was conducted in a laminar flow cabinet and replicated three times. Two morphological types were observed among the colonies and spores of these strains. One type exhibited white, fluffy colonies. Large conidia were sickle-shaped, slightly rounded at both ends, and mostly had two to five septa (16.2×2.1 μm). Small conidia were spindle-shaped to ovate (8.2×2.3 μm) and generally had one septum (Qiu et al. 2024). Another type showed colonies with pink and slightly purple hues, and the spores were sickle-shaped, slightly curved spores with pointed somewhat ends, small conidia were mostly without septa or one septum (15.1×2.0 μm), and large conidia were unequally septate or sickle-shaped (39.4×4.2 μm), mostly with three to five septa (Sang et al. 1998). The mycelia of both fungal isolates had a small number of branches. Two isolates (GF11, GF12) were selected from two different morphologies, and subjected to DNA extraction using the CTAB method (Sagar et al. 2014). Sequencing analysis of the two strains was performed using the nuclear ribosomal internal transcribed spacer region (ITS, GenBank accession no. PQ608542 and PQ608543), translation elongation factor (TEF-1a, PQ671287 and PQ671288), and the second largest subunit of RNA polymerase II (RPB2, PQ671289 and PQ671290) (O'Donnell et al. 2000, 2015). Upon comparison with NCBI database sequences, the ITS and EF-1α sequences of strain GF11 were found to exhibit a maximum homology of 100% with the corresponding sequences of F. oxysporum, while the RPB2 sequence showed a maximum homology of 99.66%. For strain GF12, its ITS, EF-1α, and RPB2 sequences were determined to have maximum homologies of 100%, 99.72%, and 99.55% respectively with the corresponding sequences of F. solani. According to Koch's postulates, GF11 and GF12 were used for pathogenicity testing. Nicotiana tabacum cv. K326 were cultivated at the 26°C growth chamber and used at the four-leaf stage. The base of the stem of ten plants was wrapped in sterile cotton treated with a spore suspension (1×10⁶ conidia/mL) of GF11 and GF12. The control group was treated with sterile water. After 10 days, the GF11 and GF12 treated groups showed symptoms consistent with those observed in the field, with the fibrous roots becoming sparse and the leaves turning yellow, and the F. oxysporum and F. solani were successfully re-isolated and identified by sequencing. This is the first report of tobacco root rot caused by Fusarium spp. in northeast China.

SYAUP-491 is a novel alkyl sulfonamide. In this study, in vivo and in vitro tests were performed along with a proteomic analysis to determine the effects and underlying mechanisms of the antibacterial activity of SYAUP-491 against the causative agent of bacterial leaf blight in rice. The antibacterial test results suggested that SYAUP-491 exhibited significant activities against Xanthomonas oryzae pv. oryzae (Xoo) in vitro and in vivo. The minimal EC₅₀ values reached 6.96 μg/mL and the curative activity reached 74.1%. Detailed studies demonstrated that SYAUP-491 altered membrane permeability and caused morphological changes. Based on proteomics results, SYAUP-491 might inhibit bacterial protein synthesis. SYAUP-491 may disrupt and alter cell membrane permeability and could further act on ribosomes in the bacterial body. Given the above results, SYAUP-491 could serve as a new lead compound in the research of antibacterial control of plant pathogenic bacterial disease.

Cyetpyrafen is a compound that lacks inherent uptake and systemic translocation activity. If mites do not come into direct contact with the pesticide solution on leaves, the efficacy cannot be achieved. Controlling the particle size can potentially play a crucial role in the manifestation of efficacy. In this study, high-throughput formulation technology was used to systematically screen a large number of adjuvants to obtain cyetpyrafen formulations. The particle size of the active ingredient in the formulation was measured. By examining the dynamic light scattering and contact angle, we simulated the actual process of the efficacy transmission of cyetpyrafen formulations against Tetranychus cinnabarinus. Our results showed that the activity of cyetpyrafen increases as the particle size decreases, suggesting that reducing the particle size can enhance the coverage and deposition on crop leaves, and further improve the dispersion efficiency and enhance spreading capabilities. Furthermore, controlling the particle size at 160 nm resulted in an LC₅₀ value of 0.2026, which is approximately double than that of the commercial product. As a novel pesticide for mites, our study presents the most effective cyetpyrafen formulation in practice. Our findings provide valuable insights into controlling other mite species that pose a threat to agricultural products.

Novel anti-viral natural product ε-poly-l-lysine (ε-PL) produced by Streptomyces is a homopolymer of l-lysine, of which the underlying molecular mode of action remains to be further elucidated. In this study, ε-PL induced significant fragmentation of tobacco mosaic virus (TMV) virions and delayed the systemic infection of TMV-GFP as well as wild-type TMV in plants. ε-PL treatment also markedly inhibited RNA accumulation of TMV in tobacco BY-2 protoplasts. The results of RNA-seq indicated that the agent induced significantly differential expression of genes that are associated with defense response, stress response, autophagy, and ubiquitination. Among them, 15 critical differential expressed genes were selected for real-time quantitative PCR validation. We further demonstrated that ε-PL can induce host defense responses by assessing the activity of several defense-related enzymes in plants. Our results provided valuable insights into molecular anti-viral mode of action for ε-PL, which is expected to be applied as a novel microbial natural product against plant virus diseases.

Microbial secondary metabolites produced by actinomycetes are important natural products widely applied to control plant diseases. A variety of actinomycetes were isolated from soil samples collected from Tianzhu Mountain in Shenyang, China. A Streptomyces strain Shenyang Tianzhu (STZ) exhibits effective antiviral activity against Tobacco mosaic virus (TMV). The isolate was identified as Streptomyces ahygroscopicus based on its cultural, morphological, physiological, biochemical characteristics as well as the phylogenetic analysis using 16S rRNA sequences. To obtain the pure anti-TMV compound from Streptomyces STZ, the culture broth was subjected to Amberlite IRC-50 ion-exchange resin, SX-8 macroporous adsorption resin and Sephadex G-25 gel column chromatography. The purified active compound was confirmed to be ε-poly-l-lysine (ε-PL), with molecular mass in the range of 3454–4352 Da by structural analysis with infrared (IR), matrix-assisted laser desorption ionization-time-of-flight MS (MALDI-TOF), thin-layer chromatography (TLC) and high-resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR). The protective and curative effects of the purified compound ε-PL were tested and the results showed that the compound exhibited significant protective and curative activity against TMV. The potential application of ε-PL as an efficient anti-plant virus agent was expected.