ABSTRACT
Pectobacterium brasiliense (Pbr) has caused significant economic losses in major vegetable production areas in Northern China by causing bacterial soft rot in cash crops such as potatoes and cucumbers. This study aimed to establish a PMA-qPCR detection method for Pbr by screening specific and sensitive primers based on the glu gene and the conserved region of the 23S rRNA gene. Based on the optimized PMA pretreatment conditions, a standard curve was designed and constructed for PMA-qPCR detection (y = -3.391x + 36.28; R2 = 0.99). The amplification efficiency reached 97%, and the lowest detection limit of viable cells was approximately 2 × 102 CFU·mL-1. The feasibility of the PMA-qPCR method was confirmed through a manually simulated viable/dead cell assay under various concentrations. The analysis of potato tubers and cucumber seeds revealed that nine naturally collected seed samples contained a range from 102 to 104 CFU·g-1 viable Pbr bacteria. Furthermore, the system effectively identified changes in the number of pathogenic bacteria in cucumber and potato leaves affected by soft rot throughout the disease period. Overall, the detection and prevention of bacterial soft rot caused by Pbr is crucial.
ABSTRACT
AIMS: Bacterial soft rot caused by Pectobacterium brasiliense (Pbr) has resulted in severe economic losses of cucumber production in northern China. Quantitative reverse transcription PCR (RT-qPCR) is widely used to determine the fold change in the expression of genes of interest, and an appropriate reference gene played a critical role in the evaluation of genes expression. However, the suitable reference genes for transcript normalization during the interaction between cucumber and Pbr have not yet been systematically validated. In this study, we aimed to identify the suitable reference genes for accurate and reliable normalization of cucumber and Pbr RT-qPCR data. METHODS AND RESULTS: We selected 14 candidate reference genes for cucumber and 10 candidate reference genes for Pbr were analysed by using four algorithms (the deltaCt method, BestKeeper, NormFinder and geNorm). Furthermore, five genes in cucumber involved in plant resistance and five genes in Pbr related to the virulence were selected to confirm the reliability of the reference genes by RT-qPCR. CsARF (ADP-ribosylation factor 1) and pgi (glucose-6-phosphate isomerase) were suggested as the most suitable reference genes for cucumber and Pbr respectively. CONCLUSION: Our results suggested that CsARF (ADP-ribosylation factor 1) and pgi (glucose-6-phosphate isomerase) could be as the reference genes to normalize expression data for cucumber and Pbr during the process of pathogen-host interaction respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first systematic study of the optimal reference genes specific to cucumber and Pbr, which could help advance the molecular interactions research in Cucurbitaceae vegetables and Pectobacterium species pathosystems.
Subject(s)
Cucumis sativus , Pectobacterium , ADP-Ribosylation Factor 1 , Cucumis sativus/genetics , Gene Expression , Gene Expression Profiling/methods , Glucose-6-Phosphate Isomerase , Real-Time Polymerase Chain Reaction/methods , Reference Standards , Reproducibility of ResultsABSTRACT
PURPOSE: To investigate dynamically the X-ray appearance of cervical degenerative disease induced by unbalanced dynamic and static forces in rats. METHODS: A total of 60 Sprague Dawley rats were randomized into test (n=45) and control (n=15) groups, which were randomly subdivided into the one-, three- and six-month post-operative groups. The test group included 10, 15 and 20 rats at the respective corresponding post-operative stage and the control group included five rats at each time-point. By excising cervicodorsal muscles, interspinous ligaments and supraspinous ligament of rats in the test group, the balance of dynamic and static forces on cervical vertebrae was disrupted to establish a rat model of cervical degeneration. Spinal X-ray images were acquired, and intervertebral disc space and intervertebral foramen size were measured at one, three and six months post-operation. The results were analyzed and compared among groups. RESULTS: Cervical dynamic and static imbalance accelerated the appearance of cervical degenerative disease on X-ray. CONCLUSION: Cervical degenerative disease may be induced by unbalanced dynamic and static forces in rats.
Subject(s)
Intervertebral Disc Degeneration/diagnostic imaging , Animals , Biomechanical Phenomena , Disease Models, Animal , Intervertebral Disc Degeneration/etiology , Male , Random Allocation , Rats , Rats, Sprague-DawleyABSTRACT
Abstract Purpose: To investigate dynamically the X-ray appearance of cervical degenerative disease induced by unbalanced dynamic and static forces in rats. Methods: A total of 60 Sprague Dawley rats were randomized into test (n=45) and control (n=15) groups, which were randomly subdivided into the one-, three- and six-month post-operative groups. The test group included 10, 15 and 20 rats at the respective corresponding post-operative stage and the control group included five rats at each time-point. By excising cervicodorsal muscles, interspinous ligaments and supraspinous ligament of rats in the test group, the balance of dynamic and static forces on cervical vertebrae was disrupted to establish a rat model of cervical degeneration. Spinal X-ray images were acquired, and intervertebral disc space and intervertebral foramen size were measured at one, three and six months post-operation. The results were analyzed and compared among groups. Results: Cervical dynamic and static imbalance accelerated the appearance of cervical degenerative disease on X-ray. Conclusion: Cervical degenerative disease may be induced by unbalanced dynamic and static forces in rats.
Subject(s)
Animals , Male , Rats , Intervertebral Disc Degeneration/diagnostic imaging , Biomechanical Phenomena , Random Allocation , Rats, Sprague-Dawley , Disease Models, Animal , Intervertebral Disc Degeneration/etiologyABSTRACT
Purpose: To investigate dynamically the X-ray appearance of cervical degenerative disease induced by unbalanced dynamic and static forces in rats. Methods: A total of 60 Sprague Dawley rats were randomized into test (n=45) and control (n=15) groups, which were randomly subdivided into the one-, three- and six-month post-operative groups. The test group included 10, 15 and 20 rats at the respective corresponding post-operative stage and the control group included five rats at each time-point. By excising cervicodorsal muscles, interspinous ligaments and supraspinous ligament of rats in the test group, the balance of dynamic and static forces on cervical vertebrae was disrupted to establish a rat model of cervical degeneration. Spinal X-ray images were acquired, and intervertebral disc space and intervertebral foramen size were measured at one, three and six months post-operation. The results were analyzed and compared among groups. Results: Cervical dynamic and static imbalance accelerated the appearance of cervical degenerative disease on X-ray. Conclusion: Cervical degenerative disease may be induced by unbalanced dynamic and static forces in rats.(AU)