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Plant Disease Apr 2021Sweet potato stem and root rot is an important bacterial disease and often causes serious economic losses to sweet potato. Development of rapid and sensitive detection...
Sweet potato stem and root rot is an important bacterial disease and often causes serious economic losses to sweet potato. Development of rapid and sensitive detection methods is crucial for diagnosis and management of this disease in field. Here, we report the production of four hybridoma cell lines (25C4, 16C10, 9B1, and 9H10) using strain FY1710 as an immunogen. Monoclonal antibodies (MAbs) produced by these four hybridoma cell lines were highly specific and sensitive for detection. Indirect enzyme-linked immunosorbent assay (indirect-ELISA) results showed that the four MAbs 25C4, 16C10, 9B1, and 9H10 could detect in suspensions diluted to 4.89 × 10, 4.89 × 10, 9.78 × 10, and 9.78 × 10 CFU/ml, respectively. Furthermore, all four MAbs can react strongly and specifically with all four strains used in this study, not with the other seven tested bacterial strains. Using these four MAbs, three different serological approaches, triple-antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA), dot-ELISA, and tissue-print-ELISA, were developed for detection of in crude extracts prepared from field-collected sweet potato plants. Among these three methods, TAS-ELISA and dot-ELISA were used to detect in suspensions diluted up to 1.23 × 10 and 1.17 × 10 CFU/ml, respectively, or in sweet potato crude extracts diluted up to 1:3,840 and 1:1,920 (wt/vol, grams per milliliter), respectively. Surprisingly, both TAS-ELISA and dot-ELISA serological approaches were more sensitive than the conventional PCR. Analyses using field-collected sweet potato samples showed that the newly developed TAS-ELISA, dot-ELISA, or tissue-print-ELISA were reliable in detecting in sweet potato tissues. Thus, the three serological approaches were highly valuable for diagnosis of stem and root rot in sweet potato production.
Topics: Dickeya; Enterobacteriaceae; Enzyme-Linked Immunosorbent Assay; Ipomoea batatas; Plant Diseases
PubMed: 33689450
DOI: 10.1094/PDIS-07-20-1551-RE -
Brazilian Journal of Microbiology :... 2015One of the most economically important bacterial pathogens of plants and plant products is Dickeya dadantii. This bacterium causes soft rot disease in tubers and other...
One of the most economically important bacterial pathogens of plants and plant products is Dickeya dadantii. This bacterium causes soft rot disease in tubers and other parts of the potato and other plants of the Solanaceae family. The application of restricted host range bacteriophages as biocontrol agents has recently gained widespread interest. This study purposed to isolate the infectious agent of the potato and evaluate its biocontrol by bacteriophages. Two phytopathogenic strains were isolated from infected potatoes, identified based on biochemical and 16S rRNA gene sequencing, and submitted to GenBank as D. dadantii strain pis3 (accession no. HQ423668) and D. dadantii strain sip4 (accession no. HQ423669). Their bacteriophages were isolated from Caspian Sea water by enriching the water filtrate with D. dadantii strains as hosts using spot or overlay methods. On the basis of morphotypes, the isolated bacteriophages were identified as members of the Myoviridae and Siphoviridae families and could inhibit the growth of antibiotic resistant D. dadantii strains in culture medium. Moreover, in Dickeya infected plants treated with bacteriophage, no disease progression was detected. No significant difference was seen between phage-treated and control plants. Thus, isolated bacteriophages can be suggested for the biocontrol of plant disease caused by Dickeya strains.
Topics: Bacteriophages; Base Sequence; Biological Control Agents; DNA, Bacterial; Dickeya chrysanthemi; Microbial Sensitivity Tests; Molecular Sequence Data; Myoviridae; Plant Diseases; RNA, Ribosomal, 16S; Sequence Analysis, DNA; Siphoviridae; Solanum tuberosum
PubMed: 26413062
DOI: 10.1590/S1517-838246320140498 -
Frontiers in Microbiology 2022species are causal agents of soft rot diseases in many economically important crops, including soft rot disease of potato (). Using random barcode transposon-site...
species are causal agents of soft rot diseases in many economically important crops, including soft rot disease of potato (). Using random barcode transposon-site sequencing (RB-TnSeq), we generated genome-wide mutant fitness profiles of 3937, ME23, and 67-19 isolates collected after passage through several and conditions. Though all three strains are pathogenic on potato, 3937 is a well-characterized model while strains ME23 and 67-19 are recent isolates. Strain ME23 specifically was identified as a representative strain from a 2014 outbreak on potato. This study generated comparable gene fitness measurements across ecologically relevant conditions for both model and non-model strains. Tubers from the potato cultivars "Atlantic," "Dark Red Norland," and "Upstate Abundance" provided highly similar conditions for bacterial growth. Using the homolog detection software PyParanoid, we matched fitness values for orthologous genes in the three bacterial strains. Direct comparison of fitness among the strains highlighted shared and variable traits important for growth. Bacterial growth in minimal medium required many metabolic traits that were also essential for competitive growth , such as amino acid, carbohydrate, and nucleotide biosynthesis. Growth in tubers specifically required the pectin degradation gene . Disruption in three putative DNA-binding proteins had strain-specific effects on competitive fitness in tubers. Though the Soft Rot can cause disease with little host specificity, it remains to be seen the extent to which strain-level variation impacts virulence.
PubMed: 35145503
DOI: 10.3389/fmicb.2022.778927 -
Molecular Plant Pathology Aug 2022Bacteria use signal transduction systems to sense and respond to their external environment. The two-component system CpxA/CpxR senses misfolded envelope protein stress...
Bacteria use signal transduction systems to sense and respond to their external environment. The two-component system CpxA/CpxR senses misfolded envelope protein stress and responds by up-regulating envelope protein factors and down-regulating virulence factors in several animal pathogens. Dickeya dadantii is a phytopathogen equipped with a type III secretion system (T3SS) for manipulating the host immune response. We found that deletion of cpxR enhanced the expression of the T3SS marker gene hrpA in a designated T3SS-inducing minimal medium (MM). In the ∆cpxR mutant, multiple T3SS and c-di-GMP regulators were also up-regulated. Subsequent analysis revealed that deletion of the phosphodiesterase gene egcpB in ∆cpxR abolished the enhanced T3SS expression. This suggested that CpxR suppresses EGcpB levels, causing low T3SS expression in MM. Furthermore, we found that the ∆cpxR mutant displayed low c-di-GMP phenotypes in biofilm formation and swimming. Increased production of cellular c-di-GMP by in trans expression of the diguanylate cyclase gene gcpA was negated in the ∆cpxR mutant. Here, we propose that CpxA/CpxR regulates T3SS expression by manipulating the c-di-GMP network, in turn modifying the multiple physiological activities involved in the response to environmental stresses in D. dadantii.
Topics: Bacterial Proteins; Cyclic GMP; Dickeya; Enterobacteriaceae; Gene Expression Regulation, Bacterial; Virulence
PubMed: 35460168
DOI: 10.1111/mpp.13219 -
MBio Jun 2022The phytopathogenic proteobacterium Dickeya dadantii secretes an array of plant cell wall-degrading enzymes and other virulence factors via the type 2 secretion system...
Scaffolding Protein GspB/OutB Facilitates Assembly of the Dickeya dadantii Type 2 Secretion System by Anchoring the Outer Membrane Secretin Pore to the Inner Membrane and to the Peptidoglycan Cell Wall.
The phytopathogenic proteobacterium Dickeya dadantii secretes an array of plant cell wall-degrading enzymes and other virulence factors via the type 2 secretion system (T2SS). T2SSs are widespread among important plant, animal, and human bacterial pathogens. This multiprotein complex spans the double membrane cell envelope and secretes fully folded proteins through a large outer membrane pore formed by 15 subunits of the secretin GspD. Secretins are also found in the type 3 secretion system and the type 4 pili. Usually, specialized lipoproteins termed pilotins assist the targeting and assembly of secretins into the outer membrane. Here, we show that in , the pilotin acts in concert with the scaffolding protein GspB. Deletion of profoundly impacts secretin assembly, pectinase secretion, and virulence. Structural studies reveal that GspB possesses a conserved periplasmic homology region domain that interacts directly with the N-terminal secretin domain. Site-specific photo-cross-linking unravels molecular details of the GspB-GspD complex . We show that GspB facilitates outer membrane targeting and assembly of the secretin pores and anchors them to the inner membrane while the C-terminal extension of GspB provides a scaffold for the secretin channel in the peptidoglycan cell wall. Phylogenetic analysis shows that in other bacteria, GspB homologs vary in length and domain composition and act in concert with either a cognate ATPase GspA or the pilotin GspS. Gram-negative bacteria have two cell membranes sandwiching a peptidoglycan net that together form a robust protective cell envelope. To translocate effector proteins across this multilayer envelope, bacteria have evolved several specialized secretion systems. In the type 2 secretion system and some other bacterial machineries, secretins form large multimeric pores that allow transport of effector proteins or filaments across the outer membrane. The secretins are essential for nutrient acquisition and pathogenicity and constitute a target for development of new antibacterials. Targeting of secretin subunits into the outer membrane is often facilitated by a special class of lipoproteins called pilotins. Here, we show that in and some other bacteria, the scaffolding protein GspB acts in concert with pilotin, facilitating the assembly of the secretin pore and its anchoring to both the inner membrane and the bacterial cell wall. GspB homologs of varied domain composition are present in many other T2SSs.
Topics: Bacterial Outer Membrane Proteins; Bacterial Proteins; Cell Wall; Dickeya; Enterobacteriaceae; Lipoproteins; Peptidoglycan; Phylogeny; Secretin; Type II Secretion Systems
PubMed: 35546537
DOI: 10.1128/mbio.00253-22 -
Plants (Basel, Switzerland) Apr 2023Nanomaterials are increasingly being used for crop growth, especially as a new paradigm for plant disease management. Among the other nanomaterials, silver nanoparticles...
Nanomaterials are increasingly being used for crop growth, especially as a new paradigm for plant disease management. Among the other nanomaterials, silver nanoparticles (AgNPs) draw a great deal of attention because of their unique features and multiple usages. Rapid expansion in nanotechnology and utilization of AgNPs in a large range of areas resulted in the substantial release of these nanoparticles into the soil and water environment, causing concern for the safety of ecosystems and phytosanitary. In an attempt to find an effective control measure for sweet potato soft rot disease, the pathogen was exposed to AgNPs, the cell-free culture supernatant (CFCS) of alone, and both in combination. AgNPs were synthesized using CFCS of strain A3. The green synthesized AgNPs exhibited a characteristic surface plasmon resonance peak at 410-420 nm. Electron microscopy and X-ray diffraction spectroscopy determined the nanocrystalline nature and 20-100 nm diameters of AgNPs. Release of metal Ag ion from biosynthesized AgNPs increases with time. AgNPs and CFCS of alone exhibited antibacterial activity against the growth, biofilm formation, swimming motility, and virulence of strain A3. The antibacterial activities elevated with the elevation in AgNPs and CFCS concentration. Similar antibacterial activities against were obtained with AgNPs at 50 µg·mL, 50% CFCS alone, and the combination of AgNPs at 12 µg·mL and 12% CFCS of . In planta experiments indicated that all the treatments reduced infection and increased plant growth. These findings suggest that AgNPs along with CFCS of can be applied to minimize this bacterial disease by controlling pathogen-contaminated sweet potato tuber with minimum Ag nano-pollutant in the environment.
PubMed: 37176882
DOI: 10.3390/plants12091817 -
Frontiers in Microbiology 2021is an important pathogenic bacterium that infects a number of crops including potato and chicory. While extensive works have been carried out on the control of the...
is an important pathogenic bacterium that infects a number of crops including potato and chicory. While extensive works have been carried out on the control of the transcription of its genes encoding the main virulence functions, little information is available on the post-transcriptional regulation of these functions. We investigated the involvement of the RNA chaperones Hfq and ProQ in the production of the main virulence functions. Phenotypic assays on the and mutants showed that inactivation of resulted in a growth defect, a modified capacity for biofilm formation and strongly reduced motility, and in the production of degradative extracellular enzymes (proteases, cellulase, and pectate lyases). Accordingly, the mutant failed to cause soft rot on chicory leaves. The mutant had reduced resistance to osmotic stress, reduced extracellular pectate lyase activity compared to the wild-type strain, and reduced virulence on chicory leaves. Most of the phenotypes of the and mutants were related to the low amounts of mRNA of the corresponding virulence factors. Complementation of the double mutant by each individual protein and cross-complementation of each chaperone suggested that they might exert their effects via partially overlapping but different sets of targets. Overall, it clearly appeared that the two Hfq and ProQ RNA chaperones are important regulators of pathogenicity in This underscores that virulence genes are regulated post-transcriptionally by non-coding RNAs.
PubMed: 34248909
DOI: 10.3389/fmicb.2021.687484 -
Phytopathology May 2024Bacteria belonging to the genus cause blackleg and soft rot symptoms on many plant hosts, including potato. Although there is considerable knowledge about the genetic...
Bacteria belonging to the genus cause blackleg and soft rot symptoms on many plant hosts, including potato. Although there is considerable knowledge about the genetic determinants that allow to colonize host plants, as well as the genes that contribute to virulence, much is still unknown. To identify the genes important for fitness in potato stems, we constructed and evaluated randomly barcoded transposon mutant (RB-TnSeq) libraries of and . We identified 169 and 157 genes important for growth in and a in stems, respectively. This included genes related to metabolic pathways, chemotaxis and motility, transcriptional regulation, transport across membranes, membrane biogenesis, detoxification mechanisms, and virulence-related genes, including a potential virulence cluster , c-di-GMP modulating genes, and pectin degradation genes. When we compared the results of the stem assay with other datasets, we identified genes important for growth in stems versus tubers and in vitro conditions. Additionally, our data showed differences in fitness determinants for and . These data provide important insights into the mechanisms used by when interacting with and colonizing plants and thus might provide targets for management.
Topics: Solanum tuberosum; Plant Diseases; Dickeya; Plant Stems; Virulence; Genes, Bacterial; Genetic Fitness
PubMed: 38170668
DOI: 10.1094/PHYTO-09-23-0351-KC -
Plant Disease Jun 2022Philodendrons are important foliage ornamentals planted worldwide (Chen et al. 2010). In November 2021, soft rot symptoms were observed on (now known as ; Sakuragui et...
Philodendrons are important foliage ornamentals planted worldwide (Chen et al. 2010). In November 2021, soft rot symptoms were observed on (now known as ; Sakuragui et al. 2018) grown in a nursery in Taichung, Taiwan. On symptomatic plants, the petioles were macerated; leaf lesions were also found on some plants (Figure S1). About 60% of the plants on site were symptomatic; these plants tended to cluster together. Four plants were sampled. Infected tissues were soaked and cut into pieces in 10 mM MgCl (using scalpels); undiluted samples were streak-plated onto nutrient agar (NA) and grown for 24 h at 28°C. Translucent, creamy-white colonies were isolated from all of the tissues examined, and 4 isolates, PHIL1 to PHIL4, were obtained (each from a different plant). All isolates exhibited typical phenotypes of bacteria belonging to ; they could cause maceration symptoms on potato slices, ferment glucose and produce phosphatase (Schaad et al. 2001); they could also produce indigoidine on NGM medium (NA added with glycerol and MnCl; Lee and Yu. 2006). Polymerase chain reactions using -specific primers 5A and 5B (Chao et al. 2006) amplified the expected amplicon in all 4 isolates. The 16S rDNA of PHIL1 to PHIL4 were amplified using primer pair 27f/1492r (Lane 1991) and the amplicons were sequenced; all 4 isolates shared the same 1,395-bp sequence (accession nos. ON203122, ON479664-ON479666). Among the strains belonging to known species (in GenBank), PHIL1 to PHIL4 shared the highest sequence identity (99.93%) with . 3937; they also shared 98.78% sequence identity with . CFBP 1269. Multilocus sequence analysis (MLSA) targeting fragments of PHIL1 to PHIL4's A (720 bp), J (672 bp), X (450 bp), B (822 bp), and N (762 bp) genes (Marrero et al. 2013) were conducted. The five-gene concatenated sequences (3,426 bp) of the 4 isolates (accession nos. ON227444-ON227448, ON494509-ON494523) were identical. A maximum-likelihood phylogenetic analysis including these sequences and those of type strains of other known species revealed that PHIL1 to PHIL4 clustered with strains belonging to . (Figure S2). Koch's postulates were fulfilled with an inoculation test conducted on . (17 cm in aboveground height; 7-months-old). Stab inoculation using sterile toothpicks was conducted on petioles. Three plants were tested for each isolate and 2 petioles were inoculated for each plant; all 4 isolates were included in the assay. The pathogen loads inoculated were quantified by the spread plate method and were 3.22 - 4.81 x 10 colony forming units. Three plants were stabbed with bacteria-free toothpicks, serving as controls. All plants were bagged post inoculation and kept in a growth chamber (28°C; 14 h light). After 72 h, all of the inoculated petioles exhibited symptoms resembling those observed in the nursery. Bacteria were re-isolated from the symptomatic tissues (one isolate from each treatment), and all of their five-gene concatenated sequences were the same as those of PHIL1 to PHIL4. This is the first formal report of the occurrence of . infecting . in Taiwan. Studies have shown that . could affect other Araceae plants in Taiwan (Lee and Chen 2021). Since different Araceae ornamentals are often planted together in gardens and nurseries, growers should be aware of potential transmission of . among them.
PubMed: 35771110
DOI: 10.1094/PDIS-04-22-0924-PDN -
PloS One 2022Dickeya are plant pathogenic bacteria able to provoke disease on a wide range of plants. A type 2 secretion system (T2SS) named Out is necessary for Dickeya virulence....
Dickeya are plant pathogenic bacteria able to provoke disease on a wide range of plants. A type 2 secretion system (T2SS) named Out is necessary for Dickeya virulence. Previous studies showed that the D. dadantii T2SS secretes a wide range of plant cell wall degrading enzymes, including pectinases and a cellulase. However, the full repertoire of exoproteins it can secrete has probably not yet been identified. Secreted proteins possess a signal peptide and are first addressed to the periplasm before their recruitment by Out. T2SS-specific secretion signals remain unknown which prevents in silico identification of T2SS substrates. To identify new Out substrates, we analyzed D. dadantii transcriptome data obtained in plant infection condition and searched for genes strongly induced and encoding proteins with a signal sequence. We identified four new Out-secreted proteins: the expansin YoaJ, the putative virulence factor VirK and two proteins of the DUF 4879 family, SvfA and SvfB. We showed that SvfA and SvfB are required for full virulence of D. dadantii and that svf genes are present in a variable number of copies in other Pectobacteriaceae, up to three in D. fanghzongdai. This work opens the way to the study of the role of non-pectinolytic proteins secreted by the Out pathway in Pectobacteriaceae.
Topics: Bacterial Proteins; Dickeya; Enterobacteriaceae; Gammaproteobacteria; Plant Diseases; Type II Secretion Systems; Virulence Factors
PubMed: 35417462
DOI: 10.1371/journal.pone.0265075