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 Evaluation of Resistance to Leaf Scald by Quantitative PCR of Xanthomonas albilineans in Sugarcane

Plant Disease ◽  
2016 ◽  
Vol 100 (7) ◽  
pp. 1331-1338 ◽  
Author(s):  
A. Gutierrez ◽  
F. F. Garces ◽  
J. W. Hoy
Keyword(s):  
Field Experiments ◽  
Quantitative Polymerase Chain Reaction ◽  
Screening Method ◽  
Resistance Screening ◽  
Visual Symptom ◽  
Visual Evaluation ◽  
Bacterial Populations ◽  
Leaf Scald ◽  
Symptom Rating ◽  
Xanthomonas Albilineans

Leaf scald, caused by Xanthomonas albilineans, is a major sugarcane disease controlled primarily with host resistance. Because visual evaluation can be uncertain due to erratic symptom expression, a reliable resistance screening method is needed. A quantitative polymerase chain reaction (qPCR) with potential for resistance screening was used to compare bacterial populations in 31 clones at different times after inoculation, and the correlation with the visual symptom rating method was determined. Comparisons of bacterial populations quantified by qPCR and visual symptom severity ratings in systemically infected leaves showed variable results, with the highest correlation at 8 weeks after inoculation. To measure consistency, the correlation was determined among three different field experiments for data obtained with the same method at different times after inoculation. The qPCR assay was more consistent among experiments compared with visual symptom rating at 8 weeks after inoculation. Susceptible check cultivars always had high bacterial populations but the severe inoculation resulted in moderate to high bacterial populations in two of three resistant checks in some experiments. The results suggest that qPCR can provide an improved method to evaluate resistance to leaf scald in sugarcane; however, multiple experiments will be needed to accurately determine clone resistance levels.

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 Leaf Surface Colonization of Sugarcane by Xanthomonas albilineans and Subsequent Disease Progress Vary According to the Host Cultivar

Plant Disease ◽  
2014 ◽  
Vol 98 (2) ◽  
pp. 191-196 ◽  
Author(s):  
Jean Heinrich Daugrois ◽  
Rosiane Boisne-Noc ◽  
Philippe Rott
Keyword(s):  
Vascular System ◽  
Leaf Tissue ◽  
Host Genotype ◽  
Bacterial Populations ◽  
Leaf Scald ◽  
Foliar Symptoms ◽  
Infection Resistance ◽  
Inoculum Pressure ◽  
Xanthomonas Albilineans ◽  
Leaf Symptoms

Spread of leaf scald in modern sugarcane cultivars in Guadeloupe occurs through aerial dissemination of Xanthomonas albilineans. However, the importance of host genotype on the foliar spread of leaf scald has never been investigated. To explore this, we followed two trials used to screen sugarcane cultivars for resistance to leaf scald under natural inoculum pressure. Leaf scald epidemic characteristics were studied by measuring epiphytic populations of X. albilineans, leaf symptom incidence and severity, and the number of infected stalks. In both trials, epiphytic X. albilineans populations and incidence of foliar symptoms varied between sugarcane cultivars (P < 0.001 in each trial for both traits) and differences in stalk infection between cultivars was also observed (P < 0.002 and P < 0.07 for trials A and B, respectively). Part of the cultivar resistance that minimizes epiphytic bacterial populations is correlated to resistance to internal leaf tissue infection as expressed by leaf symptoms. No correlation was found between epiphytic X. albilineans populations of cultivar and the incidence of stalk infection. However, foliar symptom incidence was inconsistently correlated with stalk infection. Resistance of sugarcane to leaf scald appears to involve several traits, including limiting size of epiphytic X. albilineans populations and limiting the capacity of the pathogen to produce leaf necrotic symptoms by invading the leaf vascular system or to move from the leaf into the stalk.

scholarly journals Variation in Albicidin Biosynthesis Genes and in Pathogenicity of Xanthomonas albilineans, the Sugarcane Leaf Scald Pathogen

2006 ◽  
Vol 96 (1) ◽  
pp. 33-45 ◽  
Author(s):  
P. Champoiseau ◽  
J.-H. Daugrois ◽  
J.-C. Girard ◽  
M. Royer ◽  
P. C. Rott
Keyword(s):  
Genetic Diversity ◽  
Fragment Length Polymorphism ◽  
Pathogen Population ◽  
Biosynthesis Gene Cluster ◽  
Leaf Scald ◽  
Genetic Groups ◽  
Biosynthesis Gene ◽  
Genomic Regions ◽  
Xanthomonas Albilineans

Total genomic DNA from 137 strains of Xanthomonas albilineans from worldwide locations was hybridized with two DNA probes that together harbor the entire 49-kb albicidin biosynthesis gene cluster and two additional 3-kb genomic regions required for albicidin production. Fourteen haplotypes and two major genetic groups (albicidin [ALB]-restriction fragment length polymorphism [RFLP] A and ALB-RFLP B) were identified, and strains that were isolated after recent outbreaks of leaf scald disease belonged to group ALB-RFLP B. Albicidin genetic diversity was very similar to the previously described genetic diversity of the pathogen based on the whole genome. No relationship was found between variability of albicidin biosynthesis genes and the amount of albicidin produced in vitro by X. albilineans. Leaf scald-susceptible sugarcane cv. H70-144 was inoculated with 20 strains of the pathogen belonging to different ALB-RFLP haplotypes. Among them, 10 strains from Guadeloupe belonged to the same ALB-RFLP group but differed in the amount of albicidin produced in vitro. Strains were distributed in at least three different pathogenicity groups based on symptom severity and pathogen population density in the stalk. These two pathogenicity factors varied concurrently; however, no relationship between variation in albicidin biosynthesis genes, variation in the amount of albicidin produced in vitro, and variation in pathogenicity of X. albilineans was found. Further investigation is necessary to identify other genes involved in pathogenicity of X. albilineans.

scholarly journals Detection and Quantification of Bremia lactucae by Spore Trapping and Quantitative PCR

2016 ◽  
Vol 106 (11) ◽  
pp. 1426-1437 ◽  
Author(s):  
Sridhara G. Kunjeti ◽  
Amy Anchieta ◽  
Frank N. Martin ◽  
Young-Joon Choi ◽  
Marco Thines ◽  
...  
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
Amplicon Sequencing ◽  
Coefficient Of Determination ◽  
Target Sequence ◽  
Bremia Lactucae ◽  
Cropping Season ◽  
Leaf Chlorosis ◽  
Salinas Valley ◽  
Species Specific ◽  
Detection And Quantification

Bremia lactucae is an obligate, oomycete pathogen of lettuce that causes leaf chlorosis and necrosis and adversely affects marketability. The disease has been managed with a combination of host resistance and fungicide applications with success over the years. Fungicide applications are routinely made under the assumption that inoculum is always present during favorable environmental conditions. This approach often leads to fungicide resistance in B. lactucae populations. Detection and quantification of airborne B. lactucae near lettuce crops provides an estimation of the inoculum load, enabling more judicious timing of fungicide applications. We developed a quantitative polymerase chain reaction (qPCR)-based assay using a target sequence in mitochondrial DNA for specific detection of B. lactucae. Validation using amplicon sequencing of DNA from 83 geographically diverse isolates, representing 14 Bremia spp., confirmed that the primers developed for the TaqMan assays are species specific and only amplify templates from B. lactucae. DNA from a single sporangium could be detected at a quantification cycle (Cq) value of 32, and Cq values >35 were considered to be nonspecific. The coefficient of determination (R2) for regression between sporangial density derived from flow cytometry and Cq values derived from the qPCR was 0.86. The assay was deployed using spore traps in the Salinas Valley, where nearly half of U.S. lettuce is produced. The deployment of this sensitive B. lactucae-specific assay resulted in the detection of the pathogen during the 2-week lettuce-free period as well as during the cropping season. These results demonstrate that this assay will be useful for quantifying inoculum load in and around the lettuce fields for the purpose of timing fungicide applications based on inoculum load.

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