Search Results (59)

Investigations of endogenous plant circular RNAs (circRNAs) in several plant species have revealed changes in their circular RNA profiles in response to biotic and abiotic stresses. Recently, circRNAs have emerged as critical regulators of gene expression. The destructive impacts on agriculture due to plant viral infections necessitate better discernment of the involvement of plant circRNAs during viral infection. However, few such studies have been conducted hitherto. Sobemoviruses cause great economic impacts on important crops such as rice, turnip, alfalfa, and wheat. Our current study investigates the dynamics of plant circRNA profiles in the host Arabidopsis thaliana (A. thaliana) during infections with the sobemoviruses Turnip rosette virus (TRoV) and Rice yellow mottle virus (RYMV), as well as the small circular satellite RNA of the Lucerne transient streak virus (scLTSV), focusing on circRNA dysregulation in the host plants and its potential implications in triggering plant cellular defense responses. Towards this, two rounds of deep sequencing were conducted on the RNA samples obtained from infected and uninfected plants followed by the analysis of circular RNA profiles using RNA-seq and extensive bioinformatic analyses. We identified 760 circRNAs, predominantly encoded in exonic regions and enriched in the chloroplast chromosome, suggesting them as key sites for circRNA generation during viral stress. Gene ontology (GO) analysis indicated that these circRNAs are mostly associated with plant development and protein binding, potentially influencing the expression of their host genes. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed photosynthesis as the most affected pathway. Interestingly, the non-coding exogenous scLTSV specifically induced several circRNAs, some of which contain open reading frames (ORFs) capable of encoding proteins. Our biochemical assays demonstrated that transgenic expression of scLTSV in A. thaliana enhanced resistance to TRoV, suggesting a novel strategy for improving plant viral resistance. Our results highlight the complexity of circRNA dynamics in plant–virus interactions and offer novel insights into potential circRNA-based strategies for enhancing plant disease resistance by modulating the differential expression of circRNAs. Full article

Alfalfa mosaic virus (AMV) is one of the most widely distributed viruses. It often exhibits combined infection with white clover mosaic virus (WCMV) and occurs with a synergistic effect at 3:1 (AMV: WCMV). This study sought to clarify whether this synergistic effect is related to the molecular mutation of the coat protein (CP) sequences of the two viruses and their interactions, as well as the effect of the WCMV CP concentration on infection with AMV. This study identified and analyzed the CP sequences of two viruses after the co-infection of AMV and WCMV in Nicotiana benthamiana and found that the CP sequences of the two viruses mutated after co-infection with AMV and WCMV compared with the sequences from separate single infections with each virus. The mutation rate of the nucleotide bases was 7.66% and 3.37% in the Co-AMV CP and Co-WCMV CP, respectively, and 9.05% and 5.77% in the amino acid, respectively. The effect of WCMV CP and AMV at different proportions antagonistically affected infection with AMV when the proportion of WCMV CP: AMV was 3:1, 2:1, and 1:1. These proportions of treatment alleviated the symptoms caused by infection with N. benthamiana and reduced the relative expression of the AMV CP by 0.56, 0.47, and 0.76-fold, respectively, compared with single infection by AMV. Thus, the CP sequences of both viruses mutated after the co-infection of AMV and WCMV, and a proportion of WCMV CP: AMV of 3:1, 2:1, and 1:1 inhibited infection by AMV. 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

Increasing soil salinity is threatening agricultural productivity which implies the development of new sustainable strategies to deal with this challenge. The main objective here is to assess the potential for improving the tolerance of alfalfa to salinity by combining inoculations with rhizobia and AMF. However, the distinguishing feature of this study is the comparison of two alfalfa varieties’ microbial response to salinity. The greenhouse trial was conducted on an Australian variety Siriver and an indigenous Demnate population, which were inoculated with Rhizoglomus irregulare and/or native AMF, and/or a RhOL1 rhizobial strain. The RhOL1 strain was selected from nine rhizobia tested for their plant growth promoting rhizobacteria (PGPR) activities. In addition to its ability to tolerate high salinity levels (769 mM) and solubilize insoluble phosphate as well as potassium, it can also synthesize auxins such as IAA. The application of these biofertilizers was carried out in the absence and the presence of the saline stress (0 and 120 mM NaCl). The double inoculations of native AMF and RhOL1 significantly improve the shoot and root dry biomass, plant elongation, number of formed leaves, and mineral nutrition, as well as the number of nodules and the rate of mycorrhizal root colonization. The synergistic effects between the native AMF and RhOL1 strain have been demonstrated in this study. However, the behavior of alfalfa genotypes towards microbial inoculation was significantly different. The ability to react to the double indigenous RhOL1 + AMF inoculation is more important in the Siriver than in the Demnate population. Thus, the possibility of formulating biofertilizers is based on the AMF–rhizobia–hote tripartite combination for alfalfa production in saline areas. Full article

For several decades, recurring outbreaks of human gastrointestinal infections associated with contaminated sprouts have posed an enduring challenge, highlighting the necessity of controlling the etiological agents on contaminated sprout seeds. This study investigated the efficacy of calcium hypochlorite and peroxyacetic acid treatments in inactivating the cells of four enterohemorrhagic Escherichia coli (EHEC) isolates—viz. E. coli O157:H7 K4492, F4546, and H1730, as well as E. coli O104:H4 BAA-2326—on alfalfa seeds and sprouts. The 2–3 log CFU/g of EHEC cells inoculated to sprout seeds became undetectable (≤1.40 log CFU/g) after treatment with the two sanitizers, even with the enrichment steps. Sprouts grown from calcium hypochlorite- and peroxyacetic acid-treated seeds had mean EHEC populations that were 4.54–4.60 log CFU/g and 1.25–1.52 log CFU/g lower, respectively, compared to those on sprouts grown from the untreated control seeds. Significantly (p ≤ 0.05) different from one another, the mean populations of the four EHEC isolates on harvested sprout samples were in the descending order of E. coli O157:H7 K4492, F4546, H1730, and E. coli O104:H4 BAA-2326. The results suggest that both sanitizing treatments effectively suppressed EHEC growth on alfalfa seeds and sprouts, but their effectiveness was bacterial-isolate-dependent. Full article

In this study, we selected Sitona callosus, one of the primary insect pests of alfalfa, as the experimental insect and infected it with Beauveria bassiana. Transcriptomic and metabolomic analyses were conducted to explore alterations in gene expression and metabolic processes in S. callosus at 48, 96, and 144 h post infection with B. bassiana. The transcriptomic analysis unveiled that B. bassiana infection boosted immune responses in tubercula, affecting carbohydrate metabolism, cytochrome P450 activity, lysosome function, apoptosis regulation, phagosome formation, glutathione metabolism, amino acid metabolism, and pathogen response pathways. Subsequent metabolomics analysis confirmed that glycerophospholipids, carboxylic acids and derivatives, organooxygen compounds, keto acids and derivatives, and azane immune metabolites were significantly upregulated in response to B. bassiana infection. Additionally, we utilized the Pearson correlation coefficient method to examine the relationships between differentially expressed immune-related genes and metabolites, revealing notably strong correlations between these two sets of variables. By leveraging the WGCNA method to analyze immune metabolite data for immune-related genes, we identified hub genes crucial at various stages of immune activation. These central genes predominantly included C-type lectin receptors for pattern recognition, cytochrome P450 enzymes linked to detoxification processes, and cathepsin proteases. By combining transcriptome and metabolome analyses, it was determined that autophagy and arachidonic acid metabolism play significant roles in the response of S. callosus to infection by B. bassiana. This research will facilitate the understanding of the immune response to B. bassiana infection in adult S. callosus, laying a theoretical groundwork for future biological control strategies targeting S. callosus. Full article

To investigate the precise and efficient symbiosis between Sinorhizobium meliloti LL2 and different alfalfa varieties, we conducted experiments using eight alfalfa varieties along with the S. meliloti LL2. Our objective was to identify highly effective symbiotic combinations by analyzing differences in nodulation, nitrogen fixation, and biomass accumulation. The results revealed that Gannong NO.9 had higher values for single effective root nodule weight (1.30 mg) and the number of infected cells in root nodules (2795) compared to other varieties (p < 0.05). Additionally, Gannong NO.9 exhibited the highest nitrogenase activity (0.91 μmol·g⁻¹·h⁻¹), nitrogen fixation percentage (67.16%), and amount of nitrogen fixation (18.80 mg/pot). Moreover, there was a significant 26.50% increase in aboveground tissue nitrogen accumulation compared to the control check (CK) (p < 0.05). Furthermore, underground tissue showed excellent values for nitrogen accumulation (35.68 mg/plant) and crude protein content (17.75%) when compared with other treatments. The growth of plants was demonstrated by the combined impact of nodulation and nitrogen fixation. The distribution of biomass after nitrogen fixation was compared to the control group (p < 0.05) to investigate accumulation. The eight combinations of symbiotic nitrogen fixation (SNF) were classified into six distinct types based on their significantly different biomass growth rates compared to CK. ① Aboveground accumulation type: Gannong NO.9 (there was a 24.31% increase in aboveground dry weight); ② aboveground and underground accumulation type: Qingshui (the aboveground dry weight increased by 135.94%, while the underground dry weight grew by 35.26%); ③ aboveground accumulation, underground depletion type: Gannong NO.5 ( ); ④ zero-growth type (there was no significant difference in dry weights, both above and below ground, compared to CK): WL168HQ, WL319HQ and Longzhong; ⑤ aboveground and underground depletion type: WL298HQ (the aboveground dry weight decreased by 29.29%, while the underground dry weight decreased by 20.23%); ⑥ underground depletion type: Gannong NO.3 (the underground dry weight showed a decrease of 34.49%); no type with aboveground consumption and underground accumulation was found. The study clarified the optimal combination of LL2 and Gannong NO.9, finding that biomass accumulation after symbiotic nitrogen fixation is variety-dependent. Full article

The interaction of viral proteins with host factors represents a crucial aspect of the infection process in plants. In this work, we developed a strategy to identify host factors in Nicotiana tabacum that interact with movement proteins (MPs) of the 30K family, a group of viral proteins around 30 kDa related to the MP of tobacco mosaic virus, which enables virus movement between plant cells. Using the alfalfa mosaic virus (AMV) MP as a model, we incorporated tags into its coding sequence, without affecting its functionality, enabling the identification of 121 potential interactors through in vivo immunoprecipitation of the tagged MP. Further analysis of five selected candidates (histone 2B (H2B), actin, 14-3-3A protein, eukaryotic initiation factor 4A (elF4A), and a peroxidase-POX-) were conducted using bimolecular fluorescence complementation (BiFC). The interactions between these factors were also studied, revealing that some form part of protein complexes associated with AMV MP. Moreover, H2B, actin, 14-3-3, and eIF4A interacted with other MPs of the 30K family. This observation suggests that, beyond functional and structural features, 30K family MPs may share common interactors. Our results demonstrate that tagging 30K family MPs is an effective strategy to identify host factors associated with these proteins during viral infection. Full article

In agriculture, new and sustainable strategies are increasingly demanded to integrate the traditional management of viral diseases based on the use of virus-free propagation materials and resistant or tolerant cultivars and on the control of insect vectors. Among the possible Integrated Pest Management (IPM) approaches, organic biostimulants have shown promising results in enhancing plant tolerance to virus infections by improving plant fitness and productivity and modulating metabolic functions. In this study, the combination of two organic biostimulants, Alert D-Max and Resil EVO Q, composed of seaweed and alfalfa extracts, enzymatic hydrolysates, and micronized zeolite, was applied on the leaves and roots of zucchini squashes, both healthy and infected by zucchini yellow mosaic virus (ZYMV). Four applications were scheduled based on ZYMV inoculation timing, and plant vegetative and reproductive parameters were recorded along with the virus titre and symptom severity. The modulation of the expression of specific genes potentially involved in pattern-triggered immunity (PTI), systemic acquired resistance (SAR), and oxidative stress defence pathways was also investigated. Besides increasing the general fitness of the healthy plants, the biostimulants significantly improved the production of flowers and fruits of the infected plants, with a potential positive impact on their productivity. The repeated biostimulant applications also led to a one-tenth reduction in ZYMV titre over time and induced a progressive slowdown of symptom severity. Genes associated with SAR and PTI were up-regulated after biostimulant applications, suggesting the biostimulant-based priming of plant defence mechanisms. Due to the observed beneficial effects, the tested biostimulant mix can be an effective component of the IPM of cucurbit crops, acting as a sustainable practice for enhancing plant fitness and tolerance to potyviruses. Full article

In grassland agroecosystems, some plant pathogenic bacteria can cause disease in animals. These strains are known as plant and animal cross-kingdom pathogenic bacteria. In this study, we established an alfalfa root infection model and a mouse model via the gavage administration of the Pantoea alfalfae CQ10 (CQ10) bacterial suspension. It was confirmed that the CQ10 strain caused bacterial leaf blight of alfalfa. Mice inoculated with 0.4 mL of 10⁹ cfu/mL bacterial suspension developed clinical symptoms 48 h later, such as diminished vitality, tendencies to huddle, and lack of appetite, including severe lesions in stomach, liver, kidney, and spleen tissues. CQ10 strains were isolated from mouse feces at different time points of inoculation. Thus, CQ10 is a plant and animal cross-kingdom pathogenic bacterium. Transcriptome and proteome analyses showed that biofilm and iron uptake are important virulence factors of the pathogen CQ10, among which Bap and Lpp regulating biofilm are the key cross-kingdom virulence genes of CQ10. From an evolutionary perspective, insights gained from this dual animal–plant pathogen system may help to elucidate the molecular basis underlying the host specificity of bacterial pathogens. The result provides a theoretical basis for the risk assessment, prevention, and control strategies of new pathogenic bacteria entering a new region. Full article

Medicago sativa L. (Alfalfa) is a globally recognized forage legume that has recently gained attention for its high protein content, making it suitable for both human and animal consumption. However, due to its perennial nature and autotetraploid genetics, conventional plant breeding requires a longer timeframe compared to other crops. Therefore, genetic engineering offers a faster route for trait modification and improvement. Here, we describe a protocol for achieving efficient transient gene expression in alfalfa through genetic transformation with the Agrobacterium tumefaciens pCAMBIA1304 vector. This vector contains the reporter genes β-glucuronidase (GUS) and green fluorescent protein (GFP), along with a selectable hygromycin B phosphotransferase gene, all driven by the CaMV 35s promoter. Various transformation parameters—such as different explant types, leaf ages, leaf sizes, wounding types, bacterial concentrations (OD_600nm), tissue preculture periods, infection periods, co-cultivation periods, and different concentrations of acetosyringone, silver nitrate, and calcium chloride—were optimized using 3-week-old in vitro-grown plantlets. Results were attained from data based on the semi-quantitative observation of the percentage and number of GUS spots on different days of agro-infection in alfalfa explants. The highest percentage of GUS positivity (76.2%) was observed in 3-week-old, scalpel-wounded, segmented alfalfa leaf explants after 3 days of agro-infection at a bacterial concentration of 0.6, with 2 days of preculture, 30 min of co-cultivation, and the addition of 150 µM acetosyringone, 4 mM calcium chloride, and 75 µM silver nitrate. The transient expression of genes of interest was confirmed via histochemical GUS and GFP assays. The results based on transient reporter gene expression suggest that various factors influence T-DNA delivery in the Agrobacterium-mediated transformation of alfalfa. The improved protocol can be used in stable transformation techniques for alfalfa. Full article

Finding safe and reliable alternatives to fungicides is currently one of the biggest challenges in agriculture. In this regard, this experiment investigated the effectiveness of different elicitors, botanical extracts and essential oils to control grey mold (Botrytis cinerea) and powdery mildew (Podosphaera aphanis) on strawberry plants. This trial was conducted in field conditions under a plastic tunnel with strawberry plants ‘Elsanta’. A first group of strawberry plants was treated before flowering with elicitors [acibenzolar-S-Methyl–(BTH), chitosan], botanical extracts (seaweed extract, alfalfa hydrolysate) and essential oils (thyme and juniper), and grey mold incidence on flowers was evaluated (Experiment 1). Furthermore, a second group of plants was treated before (Experiment 2) and after (Experiment 3) controlled inoculation with P. aphanis. The results indicated that the incidence of flower infected by B. cinerea was reduced by approximately 50% with thyme and juniper essential oils’ applications compared to the untreated control, with no significant difference observed compared to the commercial fungicide penconazole (positive control). As a consequence, the final yield of essential-oil-treated plants was +27% higher than that of non-treated plants. No significant differences emerged for other tested products against grey mold. However, gene expression analysis showed an up-regulation (>2 ÷ 5 folds as compared to control 4 days after application) of FaEDS1, FaLOX and PR gene expression (FaPR1, FaPR5, FaPR10) in leaves treated with BTH. The other natural substances tested also induced defense-related genes, albeit at a lower level than BTH. In Experiment 2, all treatments applied prior to inoculation significantly reduced the incidence and severity of powdery mildew as compared to control. At 28 days after inoculation, chitosan and thyme essential oil applications performed similarly to their positive controls (BTH and penconazole, respectively), showing a significant reduction in disease incidence (by −84 and −92%) as compared to control. Post-inoculum application of essential oils (Experiment 3) showed an efficacy similar to that of penconazole against powdery mildew. These results indicated that the tested substances could be used as alternatives to fungicides for the control of grey mold and powdery mildew in strawberry, therefore representing a valuable tool for the control of these fungal diseases under the framework of sustainable agriculture. Full article

Fusarium spp. can cause root rot in alfalfa, leading to the death of the whole plant, which seriously affects the yield and quality of alfalfa. This study used a Fusarium acuminatum strain labeled with green fluorescent protein (GFP) to observe the infection process of F. acuminatum on alfalfa by confocal fluorescence microscopy. The aim of this study was to reveal the infection mechanism of alfalfa Fusarium root rot at the cellular histological level. The results showed that conidia of F. acuminatum attached to the surface of the root and germinated at one day post-inoculation, the mycelium then entered the vascular bundle tissue of the alfalfa root at 5 days post-inoculation, reached the base of the plant stem at 14 days post-inoculation, and colonized the stem of the first and second compound leaf at 28 and 49 days post-inoculation, respectively. Moreover, the experiment, which sprayed a spore suspension, showed that the conidia of F. acuminatum could spread through the air to infect the pericarp and seed coat tissue of the pod. For the first time, we report the infection process of alfalfa Fusarium root rot caused by F. acuminatum and clarify that F. acuminatum can initially infect the root tissue of alfalfa, colonize the bottom stem of the plant through systematic infection, and eventually cause the plant to wilt and die. The results reveal the infection mechanism of F. acuminatum at the cell level via histology and provide theoretical support for the development of control strategies and key control technologies for alfalfa root rot. Full article

Alfalfa mosaic virus (AMV) is one of the most widely distributed viruses; it often exhibits combined infection with white clover mosaic virus (WCMV). Even so, little is known about the effects of co-infection with AMV and WCMV on plants. To determine whether there is a synergistic effect of AMV and WCMV co-infection, virus co-infection was studied by electron microscopy, the double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), and real-time fluorescence quantitative PCR (RT-qPCR) of AMV and WCMV co-infection in Nicotiana benthamiana. Meanwhile, measurements were carried out on the photosynthetic pigments, photosynthetic gas exchange parameters, and chlorophyll fluorescence parameters. The results showed that the most severe disease development was induced by AMV and WCMV co-infection, and the disease grade was scale 7. N. benthamiana leaves induced mottled yellow-green alternating patterns, leaf wrinkling, and chlorosis, and chloroplasts were observed to be on the verge of disintegration. The relative accumulation of AMV CP and WCMV CP was significantly increased by 15.44-fold and 10.04-fold upon co-infection compared to that with AMV and WCMV single infection at 21 dpi. In addition, chlorophyll a, chlorophyll b, total chlorophyll, the net photosynthetic rate, the water use efficiency, the apparent electron transport rate, the PSII maximum photochemical efficiency, the actual photochemical quantum yield, and photochemical quenching were significantly reduced in leaves co-infected with AMV and WCMV compared to AMV- or WCMV-infected leaves and CK. On the contrary, the carotenoid content, transpiration rate, stomatal conductance, intercellular CO₂ concentration, minimal fluorescence value, and non-photochemical quenching were significantly increased. These findings suggest that there was a synergistic effect between AMV and WCMV, and AMV and WCMV co-infection severely impacted the normal function of photosynthesis in N. benthamiana. Full article

Alfalfa spring black stem and leaf spot disease (ASBS) is a cosmopolitan soil-borne and seed-borne disease caused by Phoma medicaginis, which adversely affects the yield, and nutritive value and can stimulate production of phyto-oestrogenic compounds at levels that may adversely affect ovulation rates in animals. This review summarizes the host range, damage, and symptoms of this disease, and general features of the infection cycle, epidemic occurrence, and disease management. ASBS has been reported from over 40 countries, and often causes severe yield loss. Under greenhouse conditions, reported yield loss was 31–82% for roots, 32–80% for leaves, 21% for stems and 26–28% for seedlings. In field conditions, the forage yield loss is up to 56%, indicating that a single-cut yield of 5302 kg/ha would be reduced to 2347 kg/ha. P. medicaginis can infect up to 50 species of plants, including the genera Medicago, Trifolium, Melilotus, and Vicia. ASBS is more severe during warm spring conditions before the first harvest than in hot summer and cooler winter conditions, and can infect alfalfa roots, stems, leaves, flowers, pods, and seeds, with leaf spot and/or black stem being the most typical symptoms. The primary infection is caused by the overwintering spores and mycelia in the soil, and on seeds and the cortex of dead and dry stems. The use of resistant cultivars is the most economical and effective strategy for the control of ASBS. Although biological control has been studied in the glasshouse and is promising, chemical control is the main control method in agriculture. Full article