RESUMEN

Acidovorax avenae subsp. avenae (Aaa) is the causal agent of red stripe in sugarcane, a disease characterized by two forms: leaf stripe and top rot. Despite the importance of this disease, little is known about Aaa virulence factors (VFs) and their function in the infection process. Among the different array of VFs exerted by phytopathogenic bacteria, exopolysaccharides (EPSs) often confer a survival advantage by protecting the cell against abiotic and biotic stresses, including host defensive factors. They are also main components of the extracellular matrix involved in cell-cell recognition, surface adhesion, and biofilm formation. EPS composition and properties have been well studied for some plant pathogenic bacteria; nevertheless, there is no knowledge about Aaa-EPS. In this work, we describe a simple and reliable method for EPS production, precipitation, and quantification based on cold precipitation after ethanol addition, which will allow to study EPS characteristics of different Aaa strains and to evaluate the association among EPS (e.g., amount, composition, viscosity) and Aaa pathogenicity.

Asunto(s)

RESUMEN

In recent years Acidovorax avenae subsp. avenae was identified as a major cause of bacterial etiolation and decline (BED) in turfgrasses and has become a growing economical concern for the turfgrass industry. The symptoms of BED resemble those of "bakanae," or foolish seedling disease, of rice (Oryzae sativa), in which the gibberellins produced by the infecting fungus, Fusarium fujikuroi, contribute to the symptom development. Additionally, an operon coding for the enzymes necessary for bacterial gibberellin production was recently characterized in plant-pathogenic bacteria belonging to the γ-proteobacteria. We therefore investigated whether this gibberellin operon might be present in A. avenae subsp. avenae. A homolog of the operon has been identified in two turfgrass-infecting A. avenae subsp. avenae phylogenetic groups but not in closely related phylogenetic groups or strains infecting other plants. Moreover, even within these two phylogenetic groups, the operon presence is not uniform. For that reason, the functionality of the operon was examined in one strain of each turfgrass-infecting phylogenetic group (A. avenae subsp. avenae strains KL3 and MD5). All nine operon genes were functionally characterized through heterologous expression in Escherichia coli and enzymatic activities were analyzed by liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry. All enzymes were functional in both investigated strains, thus demonstrating the ability of phytopathogenic ß-proteobacteria to produce biologically active GA4. This additional gibberellin produced by A. avenae subsp. avenae could disrupt phytohormonal balance and be a leading factor contributing to the pathogenicity on turf grasses. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Asunto(s)

RESUMEN

Sugarcane (Saccharum spp.) is an important cash crop for production of sugar and bioethanol. Red stripe caused by Acidovorax avenae subsp. avenae (Aaa) is a disease that occurs in numerous sugarcane-growing regions worldwide. In this study, 17 strains of Aaa were isolated from 13 symptomatic leaf samples in China. Nine of these strains produced white-cream colonies on nutrient agar medium while the other eight produced yellow colonies. In pairwise sequence comparisons of the 16S-23S rRNA internally transcribed spacer (ITS), the 17 strains had 98.4-100% nucleotide identity among each other and 98.2-99.5% identity with the reference strain of Aaa (ATCC 19860). Three RFLP patterns based on this ITS sequence were also found among the strains of Aaa obtained in this study. Multilocus sequence typing (MLST) based on five housekeeping genes (ugpB, pilT, lepA, trpB, and gltA) revealed that the strains of Aaa from sugarcane in China and a strain of Aaa (30179) isolated from sorghum in Brazil formed a unique evolutionary subclade. Twenty-four additional strains of Aaa from sugarcane in Argentina and from other crops worldwide were distributed in two other and separate subclades, suggesting that strains of A. avenae from sugarcane are clonal populations with local specificities. Two strains of Aaa from China (CNGX08 forming white-cream colored colonies and CNGD05 forming yellow colonies) induced severe symptoms of red stripe in sugarcane varieties LC07-150 and ZZ8 but differed based on disease incidence in two separate inoculation experiments. Infected plants also exhibited increased salicylic acid (SA) content and transcript expression of gene PR-1, indicating that the SA-mediated signal pathway is involved in the response to infection by Aaa. Consequently, red stripe of sugarcane in China is caused by genetically different strains of Aaa and at least two morphological variants. The impact of these independent variations on epidemics of red stripe remains to be investigated.

RESUMEN

The production of bioethanol and sugar from sugarcane is an important economic activity in several countries. Sugarcane is susceptible to different phytopathogens. Over the last years, the red stripe disease caused by the bacterium Acidovorax avenae subsp. avenae produced significant losses in sugarcane crops. Bio-nanotechnology emerged as an eco-friendly alternative to the biosynthesis of antimicrobial molecules. The aims of this study were to (a) produce extracellular silver nanoparticles using the heavy metal resistant strain Amycolatopsis tucumanensis, (b) evaluate their antibacterial in vitro effect and (c) determine the potential of silver nanoparticles to protect sugarcane against red stripe disease. Amycolatopsis tucumanensis synthesized spherical silver nanoparticles with an average size of 35 nm. Nanoparticles were able to control the growth of A. avenae subsp. avenae in in vitro assays. In addition, in vivo assays in sugarcane showed a control upon the red stripe disease when silver nanoparticles were applied as preventive treatment. The Disease Severity Index was 28.94% when silver nanoparticles were applied 3 days before inoculation with A. avenae subsp. a venae. To our knowledge, this is the first report of silver nanoparticles extracellularly synthesized by an Amycolatopsis strain that were able to inhibited the growth of A. avenae subsp. avenae and control the red stripe disease in sugarcane.

RESUMEN

Acidovorax avenae is a flagellated, pathogenic bacterium to various plant crops that has also been found in human patients with haematological malignancy, fever, and sepsis; however, the exact mechanism for infection in humans is not known. We hypothesized that the human innate immune system could be responsive to the purified flagellin isolated from A. avenae, named FLA-AA. We observed the secretion of inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, and IL-8 by treating FLA-AA to human dermal fibroblasts, as well as macrophages. This response was exclusively through TLR5, which was confirmed by using TLR5-overexpression cell line, 293/hTLR5, as well as TLR5-specific inhibitor, TH1020. We also observed the secretion of inflammatory cytokine, IL-1ß, by the activation of NLRC4 with FLA-AA. Overall, our results provide a molecular basis for the inflammatory response caused by FLA-AA in cell-based assays.

Asunto(s)

RESUMEN

In 2018, a bacterial disease complex composed of bleached spots and soft rot-blight on onion seedlings was observed in nursery beds in Changnyeong, a major onion-producing county in South Korea. Four bacteria isolated from the diseased lesions were identified: Pseudomonas viridiflava, Acidovorax avenae subsp. avenae, Pantoea ananatis, and Xanthomonas axonopodis, respectively. We referred to the four strains as a "bacterial disease complex" because they were isolated from the same sample with multiple symptoms. We examined the synergistic activity among the four strains to understand their relationships and roles. We monitored in vivo bacterial population density and disease progression after artificially inoculating the bacteria on onion seedlings at a temperature of 22 or 28°C. The disease pattern progressed sooner at 28 than at 22°C (by an average of 4 to 6 days). The rate of disease progression induced by inoculation of P. ananatis alone was consistent with that induced by coinoculation of P. ananatis with the other strains, regardless of the temperature (22 or 28°C). The in vivo growth of P. ananatis on onion seedlings was not different after inoculation alone versus together with the other strains. The rate of disease progression induced by P. viridiflava was similar when inoculated alone and when inoculated with other tree strains at 28°C, but disease progression induced by inoculation alone was slower at 22°C. The in vivo growth of P. viridiflava or X. axonopodis on onion seedlings decreased rapidly or gradually, respectively, when inoculated with the other strains. Coinfection with the other three strains had repression effects on the growth of P. viridiflava, a slight effect on X. axonopodis, and no effect on P. or A. avenae subsp. avenae in vivo. These results indicate that the strains coexist or interact antagonistically, rather than synergistically, depending on the conditions. These results were consistent with the results of the in vitro growth inhibition assay, in which P. viridiflava growth was inhibited by X. axonopodis or P. ananatis. These results also confirmed that X. axonopodis is present on bleached spots and P. viridiflava on soft rot-blight lesions, and that P. viridiflava and P. ananatis cause soft rot-blight but do not coexist. A. avenae subsp. avenae is a minor causative pathogen of bleached spots on onion seedlings, but it is not significantly affected by temperature and has no antagonistic or synergistic interactions with X. axonopodis.

Asunto(s)

RESUMEN

Microbial pathogens deliver effectors into plant cells to suppress plant immune responses and modulate host metabolism in order to support infection processes. We sought to determine if the Acidovorax avenae rice-virulent K1 strain can suppress pathogen-associated molecular pattern-triggered immunity (PTI) induced by flagellin isolated from the rice-avirulent N1141 strain. The flagellin-triggered PTI, including H2O2 generation, callose deposition, and expression of several immune-related genes were strongly suppressed in K1 preinoculated cultured rice cells in a type III secretion system (T3SS)-dependent manner. By screening 4,562 transposon-tagged mutants based on their suppression ability, we found that 156 transposon-tagged K1 mutants lost the ability to suppress PTI induction. Mutant sequence analysis, comprehensive expression analysis using RNA sequencing, and the prediction of secretion through T3SS showed that a protein named A. avenae K1 suppression factor 1 (AKSF1) suppresses flagellin-triggered PTI in rice. Translocation of AKSF1 protein into rice cells is dependent on the T3SS during infection, an AKSF1-disruption mutant lost the ability to suppress PTI responses, and expression of AKSF1 in the AKSF1-disruption mutant complemented the suppression activity. When AKSF1-disruption mutants were inoculated into the host rice plant, reduction of the disease symptoms and suppression of bacterial growth were observed. Taken together, our results demonstrate that AKSF1 is a novel effector that can suppress the PTI in a host rice plant.[Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law. 2021.

Asunto(s)

RESUMEN

Red stripe disease in sugarcane caused by Acidovorax avenae subsp. avenae (Aaa) is related to serious global losses in yield. However, the underlying molecular mechanisms associated with responses of sugarcane plants to infection by this pathogen remain largely unknown. Here, we used Illumina RNA-sequencing (RNA-seq) to perform large-scale transcriptome sequencing of two sugarcane cultivars to contrast gene expression patterns of plants between Aaa and mock inoculations, and identify key genes and pathways involved in sugarcane defense responses to Aaa infection. At 0-72 hours post-inoculation (hpi) of the red stripe disease-resistant cultivar ROC22, a total of 18,689 genes were differentially expressed between Aaa-inoculated and mock-inoculated samples. Of these, 8498 and 10,196 genes were up- and downregulated, respectively. In MT11-610, which is susceptible to red stripe disease, 15,782 genes were differentially expressed between Aaa-inoculated and mock-inoculated samples and 8807 and 6984 genes were up- and downregulated, respectively. The genes that were differentially expressed following Aaa inoculation were mainly involved in photosynthesis and carbon metabolism, phenylpropanoid biosynthesis, plant hormone signal transduction, and plant-pathogen interaction pathways. Further, qRT-PCR and RNA-seq used for additional validation of 12 differentially expressed genes (DEGs) showed that eight genes in particular were highly expressed in ROC22. These eight genes participated in the biosynthesis of lignin and coumarin, as well as signal transduction by salicylic acid, jasmonic acid, ethylene, and mitogen-activated protein kinase (MAPK), suggesting that they play essential roles in sugarcane resistance to Aaa. Collectively, our results characterized the sugarcane transcriptome during early infection with Aaa, thereby providing insights into the molecular mechanisms responsible for bacterial tolerance.

RESUMEN

The Type VI secretion system (T6SS) is a class of macromolecular machine that is required for the virulence of gram-negative bacteria. However, it is still not clear what the role of T6SS in the virulence of rice bacterial brown stripe pathogen Acidovorax avenae subsp. avenae (Aaa) is. The aim of the current study was to investigate the contribution of T6SS in Aaa strain RS2 virulence using insertional deletion mutation and complementation approaches. This strain produced weak virulence but contains a complete T6SS gene cluster based on a genome-wide analysis. Here we compared the virulence-related phenotypes between the wild-type (RS-2) and 25 T6SS mutants, which were constructed using homologous recombination methods. The mutation of 15 T6SS genes significantly reduced bacterial virulence and the secretion of Hcp protein. Additionally, the complemented 7 mutations ΔpppA, ΔclpB, Δhcp, ΔdotU, ΔicmF, ΔimpJ, and ΔimpM caused similar virulence characteristics as RS-2. Moreover, the mutant ΔpppA, ΔclpB, ΔicmF, ΔimpJ and ΔimpM genes caused by a 38.3~56.4% reduction in biofilm formation while the mutants ΔpppA, ΔclpB, ΔicmF and Δhcp resulted in a 37.5~44.6% reduction in motility. All together, these results demonstrate that T6SS play vital roles in the virulence of strain RS-2, which may be partially attributed to the reductions in Hcp secretion, biofilm formation and motility. However, differences in virulence between strain RS-1 and RS-2 suggest that other factors may also be involved in the virulence of Aaa.

Asunto(s)

RESUMEN

BACKGROUND: There is a need to minimize economic damage by sorting infected seeds from healthy seeds before seeding. However, current methods of detecting infected seeds, such as seedling grow-out, enzyme-linked immunosorbent assays, the polymerase chain reaction (PCR) and the real-time PCR have a critical drawbacks in that they are time-consuming, labor-intensive and destructive procedures. The present study aimed to evaluate the potential of visible/near-infrared (Vis/NIR) hyperspectral imaging system for detecting bacteria-infected watermelon seeds. RESULTS: A hyperspectral Vis/NIR reflectance imaging system (spectral region of 400-1000 nm) was constructed to obtain hyperspectral reflectance images for 336 bacteria-infected watermelon seeds, which were then subjected to partial least square discriminant analysis (PLS-DA) and a least-squares support vector machine (LS-SVM) to classify bacteria-infected watermelon seeds from healthy watermelon seeds. The developed system detected bacteria-infected watermelon seeds with an accuracy > 90% (PLS-DA: 91.7%, LS-SVM: 90.5%), suggesting that the Vis/NIR hyperspectral imaging system is effective for quarantining bacteria-infected watermelon seeds. CONCLUSION: The results of the present study show that it is possible to use the Vis/NIR hyperspectral imaging system for detecting bacteria-infected watermelon seeds. © 2016 Society of Chemical Industry.

Asunto(s)

RESUMEN

Studies have highlighted the importance of non-coding RNA regulation in plant-microbe interaction. However, the roles of sugarcane microRNAs (miRNAs) in the regulation of disease responses have not been investigated. Firstly, we screened the sRNA transcriptome of sugarcane infected with Acidovorax avenae. Conserved and novel miRNAs were identified. Additionally, small interfering RNAs (siRNAs) were aligned to differentially expressed sequences from the sugarcane transcriptome. Interestingly, many siRNAs aligned to a transcript encoding a copper-transporter gene whose expression was induced in the presence of A. avenae, while the siRNAs were repressed in the presence of A. avenae. Moreover, a long intergenic non-coding RNA was identified as a potential target or decoy of miR408. To extend the bioinformatics analysis, we carried out independent inoculations and the expression patterns of six miRNAs were validated by quantitative reverse transcription-PCR (qRT-PCR). Among these miRNAs, miR408-a copper-microRNA-was downregulated. The cleavage of a putative miR408 target, a laccase, was confirmed by a modified 5'RACE (rapid amplification of cDNA ends) assay. MiR408 was also downregulated in samples infected with other pathogens, but it was upregulated in the presence of a beneficial diazotrophic bacteria. Our results suggest that regulation by miR408 is important in sugarcane sensing whether microorganisms are either pathogenic or beneficial, triggering specific miRNA-mediated regulatory mechanisms accordingly.

RESUMEN

We screened a highly specific monoclonal antibody (McAb), named 6D, against Acidovorax avenae subsp. citrulli (Aac). Single McAb 6D was used as both nanogold-labeled antibody and test antibody to develop a single self-paired colloidal gold immunochromatographic test strip (Sa-GICS). The detection limit achieved using the Sa-GICS approach was 10(5) CFU/mL, with a result that can be observed by the naked eye within 10 min. Moreover, Sa-GICS can detect eight strains of Aac and display no cross-reactions with other pathogenic plant microorganisms. Artificial contamination experiments demonstrated that Sa-GICS would not be affected by impurities in the leaves or stems of the plants and were consistent with the PCR results. This is the first report on the development of a colloidal gold immunoassay strip with self-paired single McAb for the rapid detection of Aac. Graphical Abstract Schematic representation of the test strip.

Asunto(s)

RESUMEN

The hypersensitive response (HR), a type of programmed cell death that is accompanied by DNA degradation and loss of plasma membrane integrity, is a common feature of plant immune responses. We previously reported that transcription of IREN which encodes a novel EF-hand containing plant nuclease is controlled by OsNAC4, a key positive regulator of HR cell death. Transient overexpression of IREN in rice protoplasts also led to rapid DNA fragmentation, while suppression of IREN using RNA interference showed remarkable decrease of DNA fragmentation during HR cell death. Maximum DNA degradation associated with the recombinant IREN was observed in the presence of Ca(2+) and Mg(2+) or Ca(2+) and Mn(2+). Interestingly, DNA degradation mediated by the recombinant IREN was completely abolished by Zn(2+), even when Ca(2+), Mg(2+), or Mn(2+) were present in the reaction buffer. These data indicate that IREN functions in the degradation of nuclear DNA during HR cell death.

Asunto(s)

RESUMEN

The aim of this study was to develop a SYBR Green-based real-time PCR assay for the rapid, specific, and sensitive detection of Acidovorax avenae subsp. citrulli, which causes bacterial fruit blotch (BFB), a serious disease of cucurbit plants. The molecular and serological methods currently available for the detection of this pathogen are insufficiently sensitive and specific. Thus, a novel SYBR Green-based real-time PCR assay targeting the YD-repeat protein gene of A. avenae subsp. citrulli was developed. The specificity of the primer set was evaluated using DNA purified from 6 isolates of A. avenae subsp. citrulli, 7 other Acidovorax species, and 22 of non-targeted strains, including pathogens and non-pathogens. The AC158F/R primer set amplified a single band of the expected size from genomic DNA obtained from the A. avenae subsp. citrulli strains but not from the genomic DNA of other Acidovorax species, including that of other bacterial genera. Using this assay, it was possible to detect at least one genomeequivalents of the cloned amplified target DNA using 5 × 10(0) fg/µl of purified genomic DNA per reaction or using a calibrated cell suspension, with 6.5 colony-forming units per reaction being employed. In addition, this assay is a highly sensitive and reliable method for identifying and quantifying the target pathogen in infected samples that does not require DNA extraction. Therefore, we suggest that this approach is suitable for the rapid and efficient diagnosis of A. avenae subsp. citrulli contaminations of seed lots and plants.

Asunto(s)