Molecular detection and quantification of Xanthomonas albilineans in juice from symptomless sugarcane stalks using a real-time quantitative PCR assay

Plant Disease ◽  
2021 ◽  
Author(s):  
Yang Shi ◽  
Jian-Ying Zhao ◽  
Jing-Ru Zhou ◽  
Mbuya Sylvain Ntambo ◽  
Peng-Yuan Xu ◽  
...  
Keyword(s):  
Qpcr Assay ◽  
Taqman Probe ◽  
Bacterial Suspension ◽  
Bacterial Disease ◽  
Conventional Pcr ◽  
Pcr Assay ◽  
Leaf Scald ◽  
Sugarcane Production ◽  
Xanthomonas Albilineans ◽  
Detection And Quantification

Leaf scald, a bacterial disease caused by Xanthomonas albilineans (Ashby) Dowson, is a major limiting factor for sugarcane production worldwide. Accurate identification and quantification of X. albilineans is a prerequisite for successful management of this disease. A very sensitive and robust qPCR assay was developed in this study for detection and quantification of X. albilineans using TaqMan probe and primers targeting a putative adenosine triphosphate-binding cassette (ABC) transporter gene (abc). The novel qPCR assay was highly specific to the 43 tested X. albilineans strains belonging to different pulsed-field gel electrophoresis (PFGE) groups. The detection thresholds were 100 copies/µL of plasmid DNA, 100 fg/µL of bacterial genomic DNA, and 100 CFU/ml of bacterial suspension prepared from pure culture. This qPCR assay was 100 times more sensitive than a conventional PCR assay. The pathogen was detected by qPCR in 75.1% (410/546) symptomless stalk samples, whereas only 28.4% (155/546) samples tested positive by conventional PCR. Based on qPCR data, population densities of X. albilineans in symptomless stalks of the same varieties differed between two sugarcane production areas in China, Beihai (Guangxi province) and Zhanjiang (Guangdong province), and no significant correlation between these populations was identified. Furthermore, no relationship was found between these populations of the pathogen in asymptomatic stalks and the resistance level of the sugarcane varieties to leaf scald. The newly developed qPCR assay proved to be highly sensitive and reliable for the detection and quantification of X. albilineans in sugarcane stalks.

scholarly journals Detection and Quantification of Xanthomonas albilineans by qPCR and Potential Characterization of Sugarcane Resistance to Leaf Scald

Plant Disease ◽  
2014 ◽  
Vol 98 (1) ◽  
pp. 121-126 ◽  
Author(s):  
F. F. Garces ◽  
A. Gutierrez ◽  
J. W. Hoy
Keyword(s):  
Field Experiments ◽  
Quantitative Polymerase Chain Reaction ◽  
Sybr Green ◽  
Taqman Probe ◽  
Biosynthesis Gene Cluster ◽  
Germplasm Exchange ◽  
Bacterial Populations ◽  
Leaf Scald ◽  
Xanthomonas Albilineans ◽  
Detection And Quantification

Leaf scald is an important disease of sugarcane with erratic symptom expression. Latency represents a threat to germplasm exchange, and erratic symptom development makes accurate evaluation of disease resistance during breeding and selection problematic. Real-time quantitative polymerase chain reaction (qPCR) assays for Xanthomonas albilineans, the causal agent of leaf scald, were developed and evaluated for the sensitive, specific detection and quantification of the pathogen. Assays with SYBR Green primers and TaqMan probe and primers derived from the albicidin toxin biosynthesis gene cluster efficiently and reproducibly amplified X. albilineans. Detection was more sensitive with qPCR compared with conventional PCR. Assays were specific for X. albilineans and sap extracts did not inhibit the qPCR reaction. Leaf-scald-resistant and -susceptible cultivars were distinguished by infection incidence, disease severity, and X. albilineans population determined by SYBR Green qPCR in both greenhouse and field experiments. Populations of X. albilineans varied in different tissues. Differences were the greatest within tissues in resistant cultivars, and bacterial populations in systemically infected, young, not yet fully emerged leaves exhibited the greatest differences between resistant and susceptible cultivars. The results demonstrate that qPCR is a highly sensitive method for the detection of X. albilineans that could provide a reliable method for leaf scald resistance screening.

scholarly journals Compared with conventional PCR assay, qPCR assay greatly improves the detection efficiency of predation

2020 ◽  
Vol 10 (14) ◽  
pp. 7713-7722 ◽  
Author(s):  
Ting‐bang Yang ◽  
Jie Liu ◽  
Jian Chen
Keyword(s):  
Detection Efficiency ◽  
Qpcr Assay ◽  
Conventional Pcr ◽  
Pcr Assay

scholarly journals Rapid and Quantitative Detection ofLeifsonia xylisubsp.xyliin Sugarcane Stalk Juice Using a Real-Time Fluorescent (TaqMan) PCR Assay

2016 ◽  
Vol 2016 ◽  
pp. 1-8 ◽  
Author(s):  
Hua-Ying Fu ◽  
Sheng-Ren Sun ◽  
Jin-Da Wang ◽  
Kashif Ahmad ◽  
Heng-Bo Wang ◽  
...  
Keyword(s):  
Real Time ◽  
Disease Incidence ◽  
Quantitative Polymerase Chain Reaction ◽  
Field Trials ◽  
Diagnostic Methods ◽  
Qpcr Assay ◽  
Taqman Probe ◽  
Quantitative Detection ◽  
Conventional Pcr ◽  
Sugarcane Stalk

Ratoon stunting disease (RSD) of sugarcane, one of the most important diseases seriously affecting the productivity of sugarcane crops, was caused by the bacterial agentLeifsonia xylisubsp.xyli(Lxx). A TaqMan probe-based real-time quantitative polymerase chain reaction (qPCR) assay was established in this study for the quantification ofLxxdetection in sugarcane stalk juice. A pair of PCR primers (Pat1-QF/Pat1-QR) and a fluorogenic probe (Pat1-QP) targeting thePart1gene ofLxxwere used for the qPCR assay. The assay had a detection limit of 100 copies of plasmid DNA and 100 fg ofLxxgenomic DNA, which was 100-fold more sensitive than the conventional PCR. Fifty (28.7%) of 174 stalk juice samples from two field trials were tested to be positive by qPCR assay, whereas, by conventional PCR, only 12.1% (21/174) were tested to be positive with a published primer pair CxxITSf#5/CxxITSr#5 and 15.5% (27/174) were tested to be positive with a newly designed primer pair Pat1-F2/Pat1-R2. The new qPCR assay can be used as an alternative to current diagnostic methods forLxx, especially when dealing with certificating a large number of healthy cane seedlings and determining disease incidence accurately in commercial fields.

The β-1,4-endoglucanase gene is suitable for the molecular quantification of the root-lesion nematode, Pratylenchus thornei

Nematology ◽  
2014 ◽  
Vol 16 (7) ◽  
pp. 789-796 ◽  
Author(s):  
Fouad Mokrini ◽  
Lieven Waeyenberge ◽  
Nicole Viaene ◽  
Fouad Abbad Andaloussi ◽  
Maurice Moens
Keyword(s):  
Qpcr Assay ◽  
Single Individual ◽  
Root Lesion Nematode ◽  
Pcr Assay ◽  
Standard Curve ◽  
Real Time Quantitative Pcr ◽  
Pratylenchus Thornei ◽  
Lesion Nematode ◽  
Quantitative Pcr Assay ◽  
Detection And Quantification

A real-time quantitative PCR assay was developed for the accurate detection and quantification of the root-lesion nematode, Pratylenchus thornei. A qPCR primer set, including two primers and a probe, was designed based on the sequence of the β-1,4-endoglucanase gene. The assay was optimised by using the primers with SYBR green I dye and setting the qPCR program to different annealing temperatures ranging from 62 to 69°C. Based on the Ct values, we retained the program with an annealing temperature of 69°C. The specificity of the qPCR assay including the probe was confirmed by the lack of amplification of DNA from 47 populations belonging to 15 other Pratylenchus species and nine isolates from P. thornei. The assay was very sensitive as it was able to detect a single individual of P. thornei, even when mixed with up to 80 individuals of P. penetrans. DNA was extracted from exactly 80 P. thornei individuals. A dilution series from this DNA resulted in a standard curve showing a highly significant linearity between the Ct values and the dilution rates (; slope = −3.38; ). The qPCR assay developed in this study proved to be specific and sensitive, thus providing a fast and accurate tool for detection and quantification of this pathogen during research, as well as for diagnostic labs.

scholarly journals Comparative Transcriptome Profiling of Resistant and Susceptible Sugarcane Cultivars in Response to Infection by Xanthomonas albilineans

2019 ◽  
Vol 20 (24) ◽  
pp. 6138 ◽  
Author(s):  
Mbuya Sylvain Ntambo ◽  
Jian-Yu Meng ◽  
Philippe C. Rott ◽  
Robert J. Henry ◽  
Hui-Li Zhang ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Molecular Mechanisms ◽  
Plant Hormone ◽  
Glutathione Metabolism ◽  
Bacterial Disease ◽  
Post Inoculation ◽  
Comparative Transcriptome ◽  
Rna Seq ◽  
Leaf Scald ◽  
Xanthomonas Albilineans

Sugarcane (Saccharum spp. hybrids) is a major source of sugar and renewable bioenergy crop worldwide and suffers serious yield losses due to many pathogen infections. Leaf scald caused by Xanthomonas albilineans is a major bacterial disease of sugarcane in most sugarcane-planting countries. The molecular mechanisms of resistance to leaf scald in this plant are, however, still unclear. We performed a comparative transcriptome analysis between resistant (LCP 85-384) and susceptible (ROC20) sugarcane cultivars infected by X. albilineans using the RNA-seq platform. 24 cDNA libraries were generated with RNA isolated at four time points (0, 24, 48, and 72 h post inoculation) from the two cultivars with three biological replicates. A total of 105,783 differentially expressed genes (DEGs) were identified in both cultivars and the most upregulated and downregulated DEGs were annotated for the processes of the metabolic and single-organism categories, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the 7612 DEGs showed that plant–pathogen interaction, spliceosome, glutathione metabolism, protein processing in endoplasmic reticulum, and plant hormone signal transduction contributed to sugarcane’s response to X. albilineans infection. Subsequently, relative expression levels of ten DEGs determined by quantitative reverse transcription-PCR (qRT-PCR), in addition to RNA-Seq data, indicated that different plant hormone (auxin and ethylene) signal transduction pathways play essential roles in sugarcane infected by X. albilineans. In conclusion, our results provide, for the first time, valuable information regarding the transcriptome changes in sugarcane in response to infection by X. albilineans, which contribute to the understanding of the molecular mechanisms underlying the interactions between sugarcane and this pathogen and provide important clues for further characterization of leaf scald resistance in sugarcane.

A Diagnostic TaqMan Real-Time PCR Assay for in planta Detection and Quantification of Colletotrichum theobromicola, Causal Agent of Boxwood Dieback

Plant Disease ◽  
2021 ◽  
Author(s):  
Harleen Kaur ◽  
Raghuwinder Singh ◽  
Vinson P. Doyle ◽  
Rodrigo Valverde
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Species Complex ◽  
The United States ◽  
Taqman Probe ◽  
In Planta ◽  
Pcr Assay ◽  
Phytophthora Root Rot ◽  
Specificity And Sensitivity ◽  
Detection And Quantification

Boxwood dieback, caused by Colletotrichum theobromicola, is spreading at an alarming rate in the boxwood industry in the United States. Although C. theobromicola has been accepted as a distinct species within the C. gloeosporioides species complex, it is difficult to distinguish it from other closely related species based on morphology. Moreover, molecular identification of C. theobromicola requires amplification and sequencing of multiple loci, which can be expensive and time consuming. Therefore, a diagnostic TaqMan real-time PCR assay was developed for early and accurate detection and quantification of C. theobromicola in boxwood. The study involved the design of species-specific primers and a TaqMan probe to differentiate C. theobromicola from other closely related Colletotrichum species. The primers and probe discriminate between C. theobromicola and other species in the C. gloeosporioides species complex and can detect C. theobromicola at very low concentrations, illustrating the high specificity and sensitivity of the assay. This TaqMan real-time PCR assay accurately and rapidly distinguishes boxwood dieback from other diseases with similar symptomatology including, Macrophoma blight, Phytophthora root rot, and Volutella blight, as well as some disorders produced by abiotic agents.

scholarly journals Complete Genome Sequence Reveals Evolutionary and Comparative Genomic Features of Xanthomonas albilineans Causing Sugarcane Leaf Scald

2020 ◽  
Vol 8 (2) ◽  
pp. 182
Author(s):  
Hui-Li Zhang ◽  
Mbuya Sylvain Ntambo ◽  
Philippe C. Rott ◽  
Gongyou Chen ◽  
Li-Lan Chen ◽  
...  
Keyword(s):  
Complete Genome ◽  
Genomic Analysis ◽  
Dna Methyltransferases ◽  
Comparative Genomic ◽  
Bacterial Disease ◽  
High Genetic Diversity ◽  
Circular Chromosome ◽  
Leaf Scald ◽  
Genomic Features ◽  
Xanthomonas Albilineans

Leaf scald (caused by Xanthomonas albilineans) is an important bacterial disease affecting sugarcane in most sugarcane growing countries, including China. High genetic diversity exists among strains of X. albilineans from diverse geographic regions. To highlight the genomic features associated with X. albilineans from China, we sequenced the complete genome of a representative strain (Xa-FJ1) of this pathogen using the PacBio and Illumina platforms. The complete genome of strain Xa-FJ1 consists of a circular chromosome of 3,724,581 bp and a plasmid of 31,536 bp. Average nucleotide identity analysis revealed that Xa-FJ1 was closest to five strains from the French West Indies and the USA, particularly to the strain GPE PC73 from Guadeloupe. Comparative genomic analysis between Xa-FJ1 and GPE PC73 revealed prophage integration, homologous recombination, transposable elements, and a clustered regulatory interspaced short palindromic repeats (CRISPR) system that were linked with 16 insertions/deletions (InDels). Ten and 82 specific genes were found in Xa-FJ1 and GPE PC73, respectively, and some of these genes were subjected to phage-related proteins, zona occludens toxin, and DNA methyltransferases. Our findings highlight intra-species genetic variability of the leaf scald pathogen and provide additional genomic resources to investigate its fitness and virulence.

scholarly journals Detection and Quantification of Phialophora gregata in Soybean and Soil Samples with a Quantitative, Real-Time PCR Assay

Plant Disease ◽  
2007 ◽  
Vol 91 (6) ◽  
pp. 736-742 ◽  
Author(s):  
D. K. Malvick ◽  
A. E. Impullitti
Keyword(s):  
Real Time ◽  
Single Step ◽  
Soil Samples ◽  
Qpcr Assay ◽  
Taqman Probe ◽  
Brown Stem Rot ◽  
Phialophora Gregata ◽  
Target Dna ◽  
Infested Field ◽  
Detection And Quantification

Brown stem rot of soybean, caused by the soilborne fungus Phialophora gregata, is a common and widespread disease of soybean (Glycine max) in the midwestern United States. This pathogen is challenging to study due to a long latent period and slow growth. A TaqMan probe-based quantitative, real-time polymerase chain reaction (qPCR) assay was developed for sensitive and specific detection and quantification of genotypes A and B of P. gregata in plant and soil samples. It is sensitive with detection limits of 50 fg of pure genomic DNA, 100 copies of the target DNA sequence, and approximately 400 conidia. The qPCR assay is approximately 1,000 times more sensitive in detecting DNA and conidia of P. gregata, and is more rapid and less sensitive to PCR inhibitors from soybean stems than a standard PCR (sPCR) assay. Using this single-step qPCR assay, low levels of infection were detected in soybean stems at least 1 to 2 weeks prior to symptom development and before P. gregata was detected with sPCR. This assay also was used to detect the pathogen in field-grown plants and in naturally infested field soils. This new qPCR assay is a powerful tool for rapid, specific, and sensitive detection, diagnosis, and quantification of P. gregata in plants and soil, and for advancing studies of the ecology of P. gregata and its interactions with host plants.

A TaqMan real-time PCR assay for detection of Meloidogyne hapla in root galls and in soil

Nematology ◽  
2016 ◽  
Vol 18 (2) ◽  
pp. 147-154 ◽  
Author(s):  
Rumakanta Sapkota ◽  
Andrea M. Skantar ◽  
Mogens Nicolaisen
Keyword(s):  
Disease Management ◽  
Early Detection ◽  
Real Time ◽  
Real Time Pcr ◽  
Taqman Probe ◽  
Meloidogyne Hapla ◽  
Pcr Assay ◽  
Soil Dna ◽  
Dilution Series ◽  
Detection And Quantification

Early detection and quantification of Meloidogyne hapla in soil is essential for effective disease management. The purpose of this study was to develop a real-time PCR assay for detection of M. hapla in soil. Primers and a TaqMan probe were designed for M. hapla detection. The assay detected M. hapla and showed no significant amplification of DNA from non-target nematodes. The assay was able to detect M. hapla in a background of plant and soil DNA. A dilution series of M. hapla eggs in soil showed a high correlation (, ) with Ct values. The assay could predict root-knot development in carrots by testing soils before planting. The assay could be useful for management decisions in carrot cultivation.

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