scholarly journals Assessment of Xanthomonas arboricola pv. juglandis Bacterial Load in Infected Walnut Fruits by Quantitative PCR

Plant Disease ◽  
2019 ◽  
Vol 103 (10) ◽  
pp. 2577-2586
Author(s):  
Leonor Martins ◽  
Camila Fernandes ◽  
Pedro Albuquerque ◽  
Fernando Tavares
Keyword(s):  
Quantitative Pcr ◽  
Juglans Regia ◽  
Bacterial Load ◽  
Etiologic Agent ◽  
Economic Losses ◽  
Rapid Diagnostics ◽  
In Planta ◽  
Chromosomal Dna ◽  
Fruit Samples ◽  
Xanthomonas Arboricola

Xanthomonas arboricola pv. juglandis is the etiologic agent of important walnut (Juglans regia L.) diseases, causing severe fruit drop and high economic losses in walnut production regions. Rapid diagnostics and knowledge of bacterial virulence fitness are key to hinder disease progression and apply timely phytosanitary measures. This work describes an X. arboricola pv. juglandis-specific real-time quantitative PCR (qPCR) using X. arboricola pv. juglandis-specific DNA markers to quantify the bacterial load in infected walnut plant tissues. Method validation was achieved using calibration curves obtained with serial dilutions of X. arboricola pv. juglandis chromosomal DNA and standard curves obtained from walnut samples spiked with X. arboricola pv. juglandis cells. High correlations (R2 > 0.990 and > 0.995) and low limits of detection (35 chromosomes/qPCR reaction and 2.7 CFU/qPCR reaction) were obtained for both markers considering the calibration and standard curves, respectively. Assessment of qPCR repeatability, reproducibility, and specificity allowed us to demonstrate the reliability and consistency of the method. Furthermore, in planta quantification of X. arboricola pv. juglandis bacterial load using infected walnut fruit samples showed a higher detection resolution compared with standard PCR detection. By allowing quantification of virulence fitness of distinct X. arboricola pv. juglandis strains in planta, the proposed qPCR method may contribute to assertive risk assessment of walnut diseases caused by X. arboricola pv. juglandis and ultimately help to improve phytosanitary practices.

scholarly journals Application of Quantitative-PCR to Monitor Netpen Sites in British Columbia (Canada) for Tenacibaculum Species

Pathogens ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 414
Author(s):  
Joseph P. Nowlan ◽  
Scott R. Britney ◽  
John S. Lumsden ◽  
Spencer Russell
Keyword(s):  
Atlantic Salmon ◽  
Quantitative Pcr ◽  
Salmo Salar ◽  
Bacterial Load ◽  
Temporal Distribution ◽  
Quality Parameters ◽  
Antimicrobial Use ◽  
Live Fish ◽  
Dead Fish ◽  
Pcr Assays

Tenacibaculum are frequently detected from fish with tenacibaculosis at aquaculture sites; however, information on the ecology of these bacteria is sparse. Quantitative-PCR assays were used to detect T. maritimum and T. dicentrarchi at commercial Atlantic salmon (Salmo salar) netpen sites throughout several tenacibaculosis outbreaks. T. dicentrarchi and T. maritimum were identified in live fish, dead fish, other organisms associated with netpens, water samples and on inanimate substrates, which indicates a ubiquitous distribution around stocked netpen sites. Before an outbreak, T. dicentrarchi was found throughout the environment and from fish, and T. maritimum was infrequently identified. During an outbreak, increases in the bacterial load in were recorded and no differences were recorded after an outbreak supporting the observed recrudescence of mouthrot. More bacteria were recorded in the summer months, with more mortality events and antibiotic treatments, indicating that seasonality may influence tenacibaculosis; however, outbreaks occurred in both seasons. Relationships were identified between fish mortalities and antimicrobial use to water quality parameters (temperature, salinity, dissolved oxygen) (p < 0.05), but with low R2 values (<0.25), other variables are also involved. Furthermore, Tenacibaculum species appear to have a ubiquitous spatial and temporal distribution around stocked netpen sites, and with the potential to induce disease in Atlantic salmon, continued research is needed.

scholarly journals Validation of Melampsora larici-populina reference genes for in planta RT-quantitative PCR expression profiling during time-course infection of poplar leaves

2011 ◽  
Vol 75 (3) ◽  
pp. 106-112 ◽  
Author(s):  
Stéphane Hacquard ◽  
Claire Veneault-Fourrey ◽  
Christine Delaruelle ◽  
Pascal Frey ◽  
Francis Martin ◽  
...  
Keyword(s):  
Quantitative Pcr ◽  
Expression Profiling ◽  
Reference Genes ◽  
Time Course ◽  
In Planta ◽  
Poplar Leaves

Identification and quantification of Fusarium oxysporum in planta and soil by means of an improved specific and quantitative PCR assay

2010 ◽  
Vol 46 (3) ◽  
pp. 372-382 ◽  
Author(s):  
Daniel Jiménez-Fernández ◽  
Miguel Montes-Borrego ◽  
Juan A. Navas-Cortés ◽  
Rafael M. Jiménez-Díaz ◽  
Blanca B. Landa
Keyword(s):  
Fusarium Oxysporum ◽  
Quantitative Pcr ◽  
In Planta ◽  
Pcr Assay ◽  
Quantitative Pcr Assay ◽  
Identification And Quantification

scholarly journals Tylosin injectable for the treatment of porcine proliferative enteropathy in experimentally inoculated pigs

2019 ◽  
Vol 39 (3) ◽  
pp. 168-174
Author(s):  
Luisa V.A. Otoni ◽  
Michelle P. Gabardo ◽  
Núbia R. Macêdo ◽  
Mariane M. Wagatsuma ◽  
Marina M. Pereira ◽  
...  
Keyword(s):  
Body Condition Score ◽  
Initial Period ◽  
Etiologic Agent ◽  
Economic Losses ◽  
Control Group ◽  
Lawsonia Intracellularis ◽  
Medication Group ◽  
Proliferative Enteropathy ◽  
Condition Score ◽  
And Control

ABSTRACT: Porcine proliferative enteropathy (PPE) is one of the most common enteric diseases in growing and finishing pigs. PPE is characterized by reduced growth performance, accompanied or not by diarrhea. PPE is highly prevalent in several countries of the Americas, Europe and Asia, causing high economic losses in swine herds. The most common form of PPE control in pigs is antibiotic therapy. The objective of this study was to evaluate a new product based on tylosin injectable (Eurofarma Laboratórios S.A.) to control PPE in experimentally inoculated animals. Sixty 5-week-old pigs with mean weight of 9.5kg were divided into two experimental groups of 30 animals: medication and control. All pigs were challenged with Lawsonia intracellularis, the etiologic agent of PPE, on day zero. Fecal score, body condition score, and behavior were daily evaluated. Pigs were weighted on days -2, 13 and 21 of the experiment. Pigs in the Medication Group received tylosin injectable 13 days after inoculation, in three doses with a 12-hour interval between them. Pigs in the Control Group received injectable saline solution following the same protocol. In the Control Group, 23pigs presented with diarrhea before day 13. After day 13, the number of diarrheic animals in this group was reduced to 17. In the Medication Group, 26 pigs presented with diarrhea in the initial period, and in the period after medication, only 11 animals had diarrhea. The score of gross intestinal PPE lesions in the Medication Group was lower than that in the Control Group (p=0.031). The Medication Group also showed lower score for Lawsonia intracellularis antigen-labeling by immunohistochemistry compared with that of the Control Group (p=0.032), showing lower level of infection. These results demonstrate that tylosin injectable (Eurofarma Laboratórios S.A.), administrated in three doses (1mL/20kg) every 12 hours, was effective for the control of PPE in experimentally inoculated pigs.

scholarly journals Pathogenicity and a TaqMan Real-Time PCR for Specific Detection of Pantoea allii, a Bacterial Pathogen of Onions

Plant Disease ◽  
2019 ◽  
Vol 103 (12) ◽  
pp. 3031-3040 ◽  
Author(s):  
Shabnam Rahimi-Khameneh ◽  
Sanni Hsieh ◽  
Renlin Xu ◽  
Tyler J. Avis ◽  
Sean Li ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Phylogenetic Analyses ◽  
Similarity Index ◽  
Taqman Probe ◽  
Economic Losses ◽  
In Planta ◽  
Pcr Assay ◽  
Blind Test ◽  
Reliable Detection

Bacterial diseases of onion are reported to cause significant economic losses. Pantoea allii Brady, one of the pathogens causing the center rot on onions, has not yet been reported in Canada. We report the pathogenicity of P. allii on commercially available Canadian green onions (scallions). All P. allii-inoculated plants, irrespective of the inoculum concentration, exhibited typical leaf chlorotic discoloration on green onion leaves, which can reduce their marketability. Reisolation of P. allii from infected scallion tissues and reidentification by sequencing and phylogenetic analyses of the leuS gene suggest that the pathogen can survive in infected tissues 21 days after inoculation. This is the first report of P. allii as a potential pathogen of green onions. This study also reports the development and validation of a TaqMan real-time PCR assay targeting the leuS gene for reliable detection of P. allii in pure cultures and in planta. A 642-bp leuS gene fragment was targeted because it showed high nucleotide diversity and positively correlated with genome-based average nucleotide identity with respect to percent similarity index and identity of Pantoea species. The assay specificity was validated using 61 bacterial and fungal strains. Under optimal conditions, the selected primers and FAM-labeled TaqMan probe were specific for the detection of nine reference P. allii strains by real-time PCR. The 52 strains of other Pantoea spp. (n = 25), non-Pantoea spp. (n = 20), and fungi/oomycetes (n = 7) tested negative (no detectable fluorescence). Onion tissues spiked with P. allii, naturally infested onion bulbs, greenhouse infected green onion leaf samples, as well as an interlaboratory blind test were used to validate the assay specificity. The sensitivities of a 1-pg DNA concentration and 30 CFU are comparable to previously reported real-time PCR assays of other bacterial pathogens. The TaqMan real-time PCR assay developed in this study will facilitate reliable detection of P. allii and could be a useful tool for screening onion imports or exports for the presence of this pathogen.

scholarly journals Pepper Bacterial Spot Control by Bacillus velezensis: Bioprocess Solution

2020 ◽  
Vol 8 (10) ◽  
pp. 1463
Author(s):  
Ivana Pajčin ◽  
Vanja Vlajkov ◽  
Marcus Frohme ◽  
Sergii Grebinyk ◽  
Mila Grahovac ◽  
...  
Keyword(s):  
Biocontrol Agent ◽  
Plant Protection ◽  
Medium Composition ◽  
Plant Diseases ◽  
Mass Spectrometry Analysis ◽  
Economic Losses ◽  
In Planta ◽  
Bacterial Spot ◽  
Bacillus Velezensis ◽  
Food Isolate

Pepper bacterial spot is one of the most severe plant diseases in terms of infection persistence and economic losses when it comes to fresh pepper fruits used in nutrition and industrial processing. In this study, Bacillus velezensis IP22 isolated from fresh cheese was used as a biocontrol agent of pepper bacterial spot, whose main causal agent is the cosmopolitan pathogen Xanthomonas euvesicatoria. After optimization of the cultivation medium composition aimed at maximizing of the antimicrobial activity against X. euvesicatoria and validation of the optimized medium at the scale of a laboratory bioreactor, in planta tests were performed. The results have showed significant suppression of bacterial spot symptoms in pepper plants by the produced biocontrol agent, as well as reduction of disease spreading on the healthy (uninoculated) pepper leaves. Furthermore, HPLC-MS (high pressure liquid chromatography–mass spectrometry) analysis was employed to examine antimicrobial metabolites produced by B. velezensis IP22, where lipopeptides were found with similar m/z values compared to lipopeptides from fengycin and locillomycin families. The bioprocess solution developed at the laboratory scale investigated in this study represents a promising strategy for production of pepper bacterial spot biocontrol agent based on B. velezensis IP22, a food isolate with a great perspective for application in plant protection.

scholarly journals Multiple DNA Markers for Identification of Xanthomonas arboricola pv. juglandis Isolates and its Direct Detection in Plant Samples

Plant Disease ◽  
2017 ◽  
Vol 101 (6) ◽  
pp. 858-865 ◽  
Author(s):  
Camila Fernandes ◽  
Pedro Albuquerque ◽  
Rui Sousa ◽  
Leonor Cruz ◽  
Fernando Tavares
Keyword(s):  
Dna Markers ◽  
Plant Material ◽  
Juglans Regia ◽  
Infected Plant ◽  
Geographic Origin ◽  
Large Set ◽  
Dot Blot ◽  
Plant Samples ◽  
Culture Independent ◽  
Xanthomonas Arboricola

Xanthomonas arboricola pv. juglandis (Xaj) is the etiological agent of walnut (Juglans regia L.) bacterial blight (WBB), and has been associated to other walnut emerging diseases, namely brown apical necrosis (BAN) and vertical oozing canker (VOC), altogether severely affecting the walnut production worldwide. Despite the research efforts carried out to disclose Xaj genetic diversity, reliable molecular methods for rapid identification of Xaj isolates and culture-independent detection of Xaj in infected plant samples are still missing. In this work, we propose nine novel specific DNA markers (XAJ1 to XAJ9) selected by dedicated in silico approaches to identify Xaj isolates and detect these bacteria in infected plant material. To confirm the efficacy and specificity of these markers, dot blot hybridization was carried out across a large set of xanthomonads. This analysis, which confirmed the pathovar specificity of these markers, allowed to identify four broad-range markers (XAJ1, XAJ4, XAJ6, and XAJ8) and five narrow-range markers (XAJ2, XAJ3, XAJ5, XAJ7, and XAJ9), originating 12 hybridization patterns (HP1 to HP12). No evident relatedness was observed between these hybridization patterns and the geographic origin from which the isolates were obtained. Interestingly, four isolates that clustered together according the gyrB phylogenetic analysis (CPBF 1507, 1508, 1514, and 1522) presented the same hybridization pattern (HP11), suggesting that these nine markers might be informative to rapidly discriminate and identify different Xaj lineages. Taking into account that a culture-independent detection of Xaj in plant material has never been described, a multiplex PCR was optimized using markers XAJ1, XAJ6, and XAJ8. This triplex PCR, besides confirming the dot blot data for each of the 52 Xaj, was able to detect Xaj in field infected walnut leaves and fruits. Altogether, these nine Xaj-specific markers allow conciliating the specificity of DNA-detection assays with typing resolution, contributing to rapid detection and identification of potential emergent and acutely virulent Xaj genotypes, infer their distribution, disclose the presence of this phytopathogen on potential alternative host species and improve phytosanitary control.

scholarly journals Identification of Loci of Pseudomonas syringae pv. actinidiae Involved in Lipolytic Activity and Their Role in Colonization of Kiwifruit Leaves

2017 ◽  
Vol 107 (6) ◽  
pp. 645-653 ◽  
Author(s):  
Hitendra Kumar Patel ◽  
Patrizia Ferrante ◽  
Meng Xianfa ◽  
Sree Gowrinadh Javvadi ◽  
Sujatha Subramoni ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Plant Pathogens ◽  
Lipid A ◽  
Lipolytic Activity ◽  
Genetic Screen ◽  
Economic Losses ◽  
In Planta ◽  
Tn5 Transposon ◽  
Tn5 Mutants ◽  
Transposon Mutants

Bacterial canker disease caused by Pseudomonas syringae pv. actinidiae, an emerging pathogen of kiwifruit plants, has recently brought about major economic losses worldwide. Genetic studies on virulence functions of P. syringae pv. actinidiae have not yet been reported and there is little experimental data regarding bacterial genes involved in pathogenesis. In this study, we performed a genetic screen in order to identify transposon mutants altered in the lipolytic activity because it is known that mechanisms of regulation, production, and secretion of enzymes often play crucial roles in virulence of plant pathogens. We aimed to identify the set of secretion and global regulatory loci that control lipolytic activity and also play important roles in in planta fitness. Our screen for altered lipolytic activity phenotype identified a total of 58 Tn5 transposon mutants. Mapping all these Tn5 mutants revealed that the transposons were inserted in genes that play roles in cell division, chemotaxis, metabolism, movement, recombination, regulation, signal transduction, and transport as well as a few unknown functions. Several of these identified P. syringae pv. actinidiae Tn5 mutants, notably the functions affected in phosphomannomutase AlgC, lipid A biosynthesis acyltransferase, glutamate–cysteine ligase, and the type IV pilus protein PilI, were also found affected in in planta survival and/or growth in kiwifruit plants. The results of the genetic screen and identification of novel loci involved in in planta fitness of P. syringae pv. actinidiae are presented and discussed.

scholarly journals Variable Lipoprotein Hemagglutinin A Gene (vlhA) Expression in VariantMycoplasmagallisepticumStrainsIn Vivo

2018 ◽  
Vol 86 (11) ◽  
Author(s):  
K. Pflaum ◽  
E. R. Tulman ◽  
J. Beaudet ◽  
J. Canter ◽  
S. J. Geary
Keyword(s):  
Gene Family ◽  
No Reduction ◽  
Phase Variation ◽  
Chronic Respiratory Disease ◽  
Etiologic Agent ◽  
Economic Losses ◽  
Wild Type ◽  
Exact Role ◽  
Content Type ◽  
Host Interaction

ABSTRACTMycoplasma gallisepticum, the primary etiologic agent of chronic respiratory disease, is a significant poultry pathogen, causing severe inflammation and leading to economic losses worldwide. Immunodominant proteins encoded by the variable lipoprotein and hemagglutinin (vlhA) gene family are thought to be important forM. gallisepticum-host interaction, pathogenesis, and immune evasion, but their exact role remains unknown. Previous work has demonstrated thatvlhAphase variation is dynamic throughout the earliest stages of infection, withvlhA3.03 being the predominantvlhAexpressed during the initial infection, and that the pattern of dominantvlhAexpression may be nonrandom and regulated by previously unrecognized mechanisms. To further investigate this gene family, we assessed thevlhAprofile of two well-characterized vaccine strains, GT5 and Mg7, avlhA3.03 mutant strain, and anM. gallisepticumpopulation expressing an alternative immunodominantvlhA. Here, we report that twoM. gallisepticumvaccine strains show differentvlhAprofiles over the first 2 days of infection compared to that of wild-type Rlow, while the population expressing an alternative immunodominantvlhAgene reverted to a profile indistinguishable from that of wild-type Rlow. Additionally, we observed a slight shift in thevlhAgene expression profile but no reduction in virulence in avlhA3.03 mutant. Taken together, these data further support the hypothesis thatM. gallisepticum vlhAgenes change in a nonstochastic temporal progression of expression and thatvlhA3.03, while preferred, is not required for virulence. Collectively, these data may be important in elucidating mechanisms of colonization and overall pathogenesis ofM. gallisepticum.

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