Search Results (245)

Agrobacterium tumefaciens (A. tumefaciens), the causal agent of crown gall disease on several plant species, is responsible for substantial yield losses worldwide. The limitations of conventional pesticides in controlling this disease highlight the need for alternative antibacterial solutions. Phage biocontrol can be an option, effectively managing bacterial plant diseases, by reducing pathogen loads while driving evolutionary trade-offs, often enhancing synergy with other antibacterial strategies. In this study, we aimed to explore and develop a sustainable strategy to control A. tumefaciens, by combining Agrobacterium phage PAT1 with the natural antimicrobial peptide “Ascaphin 8” and leveraging the fitness trade-offs resulting from phage resistance. In vitro and in planta investigations showed that PAT1 in combination with Ascaphin 8 at the sublethal concentration of 3 μM could effectively eradicate A. tumefaciens in YPG broth and reduce tumor formation by 46.33% on tomato plants, unlike their individual applications, indicating that the combination was synergistic against A. tumefaciens. This synergy was attributed to the fitness trade-offs in A. tumefaciens induced by phage resistance, which led to increased sensitivity to antimicrobial peptides, slower growth rate, and an 89.96% attenuation of virulence in the PAT1-resistant mutant (AT-M1). Transmission electron microscopy analyses showed that treatment with 1 µM of Ascaphin 8 induced cytoplasmic condensation in 80% of AT-M1 cells, whereas only 16% of the wild-type CFBP 5770 cells exhibited similar alterations under identical conditions. Furthermore, proteomic analyses performed on AT-M1 and CFBP 5770 revealed that the mutant AT-M1 exhibited a loss of DNA-binding protein HupB and downregulation of SDR family oxidoreductase and superoxide dismutase. These molecular alterations are potentially associated with the reduced virulence and heightened AT-M1 sensitivity. This study investigated the fitness costs associated with phage resistance in A. tumefaciens and laid the first foundation for potential biocontrol of plant bacterial diseases, particularly A. tumefaciens infections, using phage–peptide combination. Full article

Stropharia rugosoannulata is a cultivated edible mushroom characterized by its nutritional composition and efficient cellulolytic enzymatic systems. However, the lack of genetic tools has significantly impeded the investigation of its molecular mechanisms, severely constraining the study of functional genomic and precision breeding in S. rugosoannulata. It was demonstrated in this study that the Agrobacterium-tumefaciens-mediated genetic transformation (ATMT) system is applicable for the transformation of S. rugosoannulata protoplasts. Through this proposal, we successfully achieved the expression of exogenous genes (mCherry gene encoding red fluorescent protein, hph gene encoding hygromycin B phosphotransferase, and GUS gene encoding β-glucuronidase) and the endogenous mutant gene SDI encoding the iron-sulfur protein subunit of succinate dehydrogenase in S. rugosoannulata. Furthermore, this study employed endogenous promoters of GPD encoding glyceraldehyde-3-phosphate dehydrogenase and SDI to enhance transformation efficiency and drive target gene expression. This study establishes the feasibility of ATMT in S. rugosoannulata systems, while achieving stable expression of a panel of selectable marker genes and reporter genes critical for genetic research in S. rugosoannulata. Full article

The efficient compounding of microbial agents for use in aerobic composting processes is a pressing problem that needs to be addressed. This work focused on the lack of effective oil-degrading microorganisms and the challenges in formulating microbial consortia during the composting of food waste (FW). Following the isolation of three bacteria and three fungi with high oil-degrading ability, a simplex-lattice mixture design methodology was used to conduct compounding within and between groups of bacteria and fungi. Three special cubic response models were successfully developed and validated by performing an analysis of variance. From our analysis, it was demonstrated that the three models had high R² values of 96.06%, 97.18%, and 96.27%. The global solution of the mixture optimization predicted the optimal value for a blend comprising 11.83% Agrobacterium tumefaciens, 8.10% Pseudomonas geniculata, 10.97% Luteibacter rhizovicinus, 20.9% Simplicillium cylindrosporum, 22.3% Fusarium proliferatum, and 25.9% Simplicillium lanosoniveum. Thus, these proportions were considered the optimal combination of strains for oil degradation during FW composting. Composting verification in a 60 L fermenter revealed that the composite microbial agent group had a 31.3% higher oil degradation efficiency than the control group. This work provides valuable insights for the compounding of microbial agents and the resource utilization of rural FW. Full article

Sugar beet (Beta vulgaris L.), a biennial sugar crop, provides about 16% of the world’s sucrose production. PEG and Agrobacterium tumefaciens-mediated transformation have been established for sugar beet. However, the traditional transformation of sugar beet is time-consuming, low efficiency, and dependent on tissue regeneration. Recently, the use of Agrobacterium rhizogenes for genetic transformation without tissue culture has become a new possibility. Here, we describe an optimized A. rhizogenes-mediated transformation for the generation of composite sugar beet without tissue culture. By dipping A. rhizogenes K599 colonies onto a wound of hypocotyl and petiole, about 81.7% and 51.1% of shoots and leaves could be induced to produce adventitious roots. Of these, more than 60% of the explants contained transformed adventitious roots. Specifically, we discovered that the transformation efficiency was significantly improved when the MAS promoter was employed instead of the CaMV35S promoter. The transformation in adventitious roots was also validated by qRT-PCR and Western blot at the transcriptional and translational levels. The transformed adventitious roots have great potential for the study of taproot development, sugar accumulation, and resistance to root diseases, which is closely related to sugar beet yield and quality. Full article

The success of truffle cultivation is especially dependent on the quality of truffle-mycorrhized seedlings, which are typically produced in nurseries under aseptic conditions to avoid root colonization by undesired ectomycorrhizal fungi. However, such practices may also eliminate beneficial microorganisms that could support truffle symbiosis and improve seedling quality. In this study, twelve endophytic bacterial and fungal strains, isolated from the Tuber melanosporum environment (gleba tissue, mycorrhizae and truffle brûlé), were tested for their effect on T. melanosporum mycorrhization levels in inoculated Quercus ilex seedlings under nursery conditions. Co-inoculation with a strain of Agrobacterium tumefaciens significantly enhanced root colonization by T. melanosporum, supporting its potential role as mycorrhizal helper bacterium. In contrast, a strain of Trichoderma harzianum negatively affected mycorrhization. The remaining strains did not show significant effects on seedling mycorrhization or seedling growth. Our findings support the hypothesis that specific bacterial strains associated with truffles can act as mycorrhizal helper bacteria, highlighting the potential for co-inoculation strategies to enhance quality of truffle-inoculated seedlings in nurseries. However, further research is needed to gain a deeper understanding of the interactions within the mycorrhizosphere that could contribute to improving nursery seedling quality. Full article

Villosiclava virens (anamorph: Ustilaginoidea virens), the causal fungal pathogen of rice false smut, has been found to produce various secondary metabolites. The albino strain LN02 is a natural albino phenotype mutant of V. virens due to its inability to produce ustilaginoidins. The fermentation of V. virens LN02 was performed in solid rice medium to obtain fungal cultures, which were chemically investigated. After removing the known metabolites, two new dimeric sorbicillinoids, namely ustisorbicillinols G (1) and H (2), were isolated from the ethyl acetate extract. Their structures were elucidated using spectroscopic data analyses and quantum chemical calculations. Compounds 1 and 2 displayed antibacterial activity towards Ralstonia solanacearum, Agrobacterium tumefaciens and Bacillus subtilis, with median inhibitory concentration (IC₅₀) values of 19.76–25.43 μg/mL for 1 and 25.35–45.48 μg/mL for 2. The discovery of new sorbicillinoids will increase the diversity of the secondary metabolites of V. virens and provide candidates for the creation of new antimicrobials as well. Full article

Fusarium acuminatum is a major pathogenic fungus causing root rot in alfalfa (Medicago sativa). DNA polymerase theta is known to play a crucial role in repairing DNA double-strand breaks. However, its biological function in F. acuminatum remains unknown. In this study, the POLQ gene was deleted by homologous recombination using Agrobacterium tumefaciens-mediated transformation. Compared to the wild type (with the POLQ gene), the mutants (without the POLQ gene) showed significant phenotypic changes: they produced brown-yellow pigments instead of pink, slowed mycelial growth, and exhibited changes in macroconidia size and shape. The virulence of the mutants was greatly reduced, inducing only mild symptoms in alfalfa. In addition, FITC-WGA staining showed impaired spore germination and hyphal growth. These results suggest that POLQ is a key gene regulating growth and development of F. acuminatum, indicating that DNA repair may play an essential role in the pathogenicity of the pathogen in alfalfa. The POLQ gene could thus be a promising target for limiting F. acuminatum infections in alfalfa. Full article

Mango is a vital fruit crop in tropical and subtropical regions, yet pests and diseases cause 30–70% production losses. Developing disease-resistant cultivars through transgenic methods could mitigate these issues. Agrobacterium-mediated callus transformation is a common genetic engineering approach, but successful transgenic mango plants from callus remain unreported due to severe browning and necrosis post-infection. We hypothesized that Agrobacterium-induced immune responses trigger callus death, hindering transformation. To improve efficiency, we engineered an Agrobacterium strain carrying the type III secretion system (T3SS) and effector gene AvrPto. Compared to controls, infected calluses exhibited elevated reactive oxygen species (ROS), along with up-regulated ROS-related, gallic acid biosynthesis, and defense genes. Calluses infected with T3SS-AvrPto-harboring Agrobacterium showed delayed browning and necrosis versus those infected with the empty vector (NV). The transformation rate with Agrobacterium (T3SS-AvrPto-EGFP) reached 1.6%, while Agrobacterium (NV-EGFP) failed entirely. These findings demonstrate that T3SS and AvrPto enhance mango transformation efficiency, offering a promising strategy for breeding multi-resistant varieties. Full article

Soybean (Glycine max L.) is a vital grain and oil crop, serving as a primary source of edible oil, plant-based protein, and livestock feed. Its production is crucial for ensuring global food security. However, soybean yields are severely impacted by various diseases, and the development of disease-resistant cultivars remains the most sustainable strategy for mitigating these losses. While stable genetic transformation is a common approach for studying gene function, virus-induced gene silencing (VIGS) offers a rapid and powerful alternative for functional genomics, enabling efficient screening of candidate genes. Nevertheless, the application of VIGS in soybean has been relatively limited. In this study, we established a tobacco rattle virus (TRV)-based VIGS system for soybean, utilizing Agrobacterium tumefaciens-mediated infection. The TRV vector was delivered through cotyledon nodes, facilitating systemic spread and effective silencing of endogenous genes. Our results demonstrate that this TRV–VIGS system efficiently silences target genes in soybean, inducing significant phenotypic changes with a silencing efficiency ranging from 65% to 95%. Key genes, including phytoene desaturase (GmPDS), the rust resistance gene GmRpp6907, and the defense-related gene GmRPT4, were successfully silenced, confirming the system’s robustness. This work establishes a highly efficient TRV–VIGS platform for rapid gene function validation in soybean, providing a valuable tool for future genetic and disease resistance research. Full article

Buckwheat is a promising crop with grains that are rich in nutrients and bioactive compounds. Genome sequence data for common and Tartary buckwheat have recently become available. Currently, there is a critical need for the development of a simple and reliable transient gene expression protocol, as well as a stable genetic transformation method, to facilitate metabolic engineering of bioactive compounds, functional analysis of genes, targeted editing, and, in a long-term perspective, to accelerate the breeding process in buckwheat. In this paper, we report optimized methods for Agrobacterium-mediated transient and stable transformation of Fagopyrum esculentum and F. tartaricum. Leaf and cotyledon tissues were infiltrated with an A. tumefaciens-bearing construct containing eGFP and GUS reporter genes. Histochemical staining and Western blotting were used to confirm the expression of reporter proteins. We also demonstrate the usefulness of the developed method for engineering the gramine biosynthetic pathway in buckwheat. HvAMIS and HvNMT genes were transiently expressed in buckwheat leaves, and the de novo production of gramine was confirmed by LC-MS. Moreover, in planta genetic transformation of common and Tartary buckwheat with a reporter gene (eGFP) and selectable marker gene (NptII) was achieved by Agrobacterium-mediated vacuum infiltration. Genomic integration of the construct was confirmed by polymerase chain reaction (PCR), whereas the production of eGFP was confirmed by fluorescence microscopy. Full article

Grapevine crown gall (GCG) is a major disease caused by the Allorhizobium vitis that causes significant losses in the grape industry. In this study, a strain of Agrobacterium tumefaciens (Ag-8) from soil was found to have a significant effect on grapevine crown gall. The present study was conducted to investigate the potential impact of the AraC family transcription factor ata (Agrobacterium tumefaciens AraC) on the biocontrol capacity of Ag-8 and to elucidate the potential mechanisms of its action. To this end, an ata deletion mutant (Δata) was constructed. It was found that the biofilm, motility, and colonization of the ata deletion mutant strain were significantly lower than those of the wild strain Ag-8. By analyzing the Δata transcriptome data, we focused our attention on the ABC transporter protein araH, and RT-qPCR showed that it was significantly down-regulated in the Δata strain. In a follow-up study, the candidate gene araH deletion strain (ΔaraH) was found to have significantly lower biofilm formation, motility, and biocontrol ability than the wild-type strain Ag-8. Therefore, araH may be implicated as a pivotal target gene of ata in modulating the biocontrol capability of Ag-8. This study supports developing biocontrol strategies targeting ata-regulated colonization in Agrobacterium to enhance the sustainable management of grapevine crown gall. Full article

Hemerocallis middendorffii is widely used in the landscaping of Northern China for its exceptional ornamental and ecological attributes. It is also the focus of a substantial body of germplasm development and stress tolerance research. However, the absence of an efficient regeneration and genetic transformation system has been a critical barrier to conducting gene function studies on this species. In this research, the aerial parts of seed-derived H. middendorffii plantlets were used as explants, and the callus induction, proliferation, subculture, differentiation, and rooting conditions in the in vitro regeneration process were optimized. A callus induction rate of 95.6% was achieved, with a regeneration rate of 84.4%. Based on this procedure, a simple and effective Agrobacterium-mediated genetic transformation system was preliminarily developed using a hygromycin-based selection system. The system comprised an Agrobacterium tumefaciens culture solution optical density at 600 nm (OD₆₀₀) of 0.6, an acetosyringone concentration of 100 μmol·L⁻¹ in both the A. tumefaciens infection solution and the co-cultivation medium, a sterilization culture with Timentin at 300 mg·L⁻¹, and a selection culture with hygromycin at 9 mg·L⁻¹. Transgenic H. middendorffii T0 rooted plants were produced within a 5-month period, with a transformation rate of 11.9% and positive rate of 32.8%. The regeneration and genetic transformation system established in this study should help advance functional gene research and genetic improvement in H. middendorffii. However, the genetic transformation was only validated in the T0 plants. To confirm stable integration and long-term transgene stability, future research on the phenotypic and molecular characterization of T1 progeny, including segregation analysis and Southern blot verification, will be conducted. Full article

Histone post-translational modifications (HPTMs) can affect gene expression by rearranging chromatin structure. Between these, histone methylation is one of the most studied in filamentous fungi, and different conserved domains coding for methyltransferase were found in Aspergillus spp. genomes. In this work, the role of the histone methyltransferases AcDot1 and AcRmtA in the mycotoxigenic fungus Aspergillus carbonarius was investigated, obtaining knockout or overexpression mutants through Agrobacterium tumefaciens-mediated transformation (ATMT). A. carbonarius is responsible for grape-bunch rot, representing the major source of ochratoxin A (OTA) contamination on grapes. In vivo conditions, the deletion of Acdot1 or AcrmtA resulted in upregulation of growth when the isolates were cultivated on a minimal medium. The influence of Acdot1 on the OTA biosynthesis was differently affected by culture conditions. On rich media, an increase in OTA accumulation was observed, while on minimal medium, lower OTA concentrations were reported. The deletion of AcrmtA always resulted in lower OTA accumulation. However, the expression of OTA biosynthesis genes was regulated by both histone methyltransferases. Of the six analyzed OTA genes, three of them showed altered expression in the knockout mutants, and otaB and otaR1 were common between both mutants. Furthermore, both AcDot1 and AcRmtA play a role in oxidative stress response, induced by 1 mM hydrogen peroxide, by modulating growth, conidiation and OTA biosynthesis. Neither the deletion nor the overexpression of the Acdot1 or AcrmtA affected virulence, while both the sporulation and OTA production were negatively affected in vivo by the deletion of AcrmtA. Full article

Rice false smut, caused by Ustilaginoidea virens, is one of the three major rice diseases in China. It not only seriously affects the rice yield and quality but also endangers human and animal health. Studying the pathogenic mechanism of U. virens has important theoretical significance and application value for clarifying the infection characteristics of the pathogen and cultivating disease-resistant varieties. Plant pathogenic fungi utilize secreted effectors to suppress plant immune responses, which can function in the apoplast or within host cells and are likely glycosylated. However, the posttranslational regulation of these effectors remains unexplored. Deletion of ΔUvALG led to the cessation of secondary infection hyphae growth and a notable decrease in virulence. We observed that ΔUvALG mutants triggered a significant increase in reactive species production within host cells, akin to ALG mutants, which plays a crucial role in halting the growth of infection hyphae in the mutants. ALG functions by sequestering chitin oligosaccharides to prevent their recognition by the rice chitin elicitor, thereby inhibiting the activation of innate immune responses, including reactive species production. Our findings reveal that ALG3 possesses three N-glycosylation sites, and the simultaneous Alg-mediated N-glycosylation of each site is essential for maintaining protein stability and chitin-binding activity, both of which are critical for its effector function. These outcomes underscore the necessity of the Alg-mediated N-glycosylation of ALG to evade host innate immunity. Full article

Fluroxypyr (4-amino-3,5-dichloro-6-fluoro-2-pyridyloxyacetic acid) is a widely used herbicide sprayed on crops worldwide. The effects of fluroxypyr on maize growth and the soil microbial community structure have not been reported. In this study, the impacts of fluroxypyr on maize growth and the bacterial community structure in endophytes and rhizospheric/non-rhizospheric soils were evaluated. We found that the community structures of the non-rhizospheric and rhizospheric soils were similar. The alpha diversity showed that the richness of the endophytic communities in the mature maize roots was reduced after herbicide application. No statistically significant differences were observed between the fluroxypyr-treated and control soils in either the non-rhizospheric or rhizospheric soils. However, the composition of the endophytic bacterial community structure suggested that fluroxypyr led to a 59.1% reduction in the abundance of Acinetobacter and a 75.6% reduction in Agrobacterium, both of which are considered growth-promoting bacteria. In addition, we observed a negative effect of fluroxypyr on maize growth, including a decreased ear length and root size and a reduction in the 100-grain weight. In summary, our study suggests that fluroxypyr may negatively impact the mature growth of maize by reducing the abundance of Bacillus kineticus and Agrobacterium tumefaciens in the endophytic community of the mature root system. Full article