A long-amplicon viability-qPCR test for quantifying living pathogens that cause bacterial spot in tomato seed

Plant Disease ◽  
2021 ◽  
Author(s):  
Hehe Wang ◽  
Rieanna Wagnon ◽  
Daniela Negrete ◽  
Sujan Timilsina ◽  
Jeffrey B. Jones ◽  
...  
Keyword(s):  
Management Practices ◽  
Latent Class ◽  
Qpcr Assay ◽  
Bacterial Spot ◽  
Conventional Pcr ◽  
Tomato Seed ◽  
Infected Seed ◽  
Seed Testing ◽  
Plating Method ◽  
Seed Extracts

Bacterial spot is one of the most serious diseases of tomato. It is caused by four species of Xanthomonas: X. euvesicatoria, X. gardneri, X. perforans, and X. vesicatoria. Contaminated and/or infected seed can serve as a major source of inoculum for this disease. The use of certified pathogen-free seed is one of the primary management practices to reduce the inoculum load in commercial production. Current seed testing protocols rely mainly on plating the seed extract and conventional PCR, however, the plating method cannot detect viable but non-culturable cells and the conventional PCR assay has limited capability to differentiate DNA extracted from viable versus dead bacterial cells. To improve the sensitivity and specificity of the tomato seed testing method for the bacterial spot pathogens, a long-amplicon qPCR assay coupled with propidium monoazide (PMA-qPCR) was developed to quantify selectively the four pathogenic Xanthomonas species in tomato seed. The optimized PMA-qPCR procedure was evaluated on pure bacterial suspensions, bacteria-spiked seed extracts, and seed extracts of inoculated and naturally-infected seed. A crude DNA extraction protocol also was developed and PMA-qPCR with crude bacterial DNA extracts resulted in accurate quantification of 104-108 CFU/ml of viable bacteria when mixed with dead cells at concentrations as high as 107 CFU/ml in the seed extracts. With DNA purified from concentrated seed extracts, the PMA-qPCR assay was able to detect DNA of the target pathogens in seed samples spiked with ≥75 CFU/ml (~0.5 CFU/seed) of the viable pathogens. Latent class analysis of the inoculated and naturally-infected seed samples showed that the PMA-qPCR assay had greater sensitivity than plating the seed extracts on the semi-selective MTMB and CKTM media for all four target species. Being much faster and more sensitive than dilution plating, the PMA-qPCR assay has a promising potential to serve as a standalone tool or used in combination with the plating method to improve tomato seed testing and advance the production of clean seed.

A COMPARISON OF SEED TESTING METHODS IN RELATION TO THE STEM-BREAK- AND BROWNING DISEASE OF FLAX, CAUSED BY POLYSPORA LINI LAFF.

1951 ◽  
Vol 29 (2) ◽  
pp. 138-142 ◽  
Author(s):  
V. R. Wallen ◽  
A. J. Skolko
Keyword(s):  
New Zealand ◽  
Health Condition ◽  
Centrifuge Test ◽  
Flax Seed ◽  
Testing Methods ◽  
Saprophytic Fungi ◽  
Superficial Infection ◽  
Running Water ◽  
Seed Testing ◽  
Plating Method

A comparison of the Ulster, New Zealand, and Ottawa methods of seed examination for the presence of Polyspora lini Laff., the cause of stem-break and browning disease of flax, showed great variation in the percentage of infection obtained, depending upon the method used. The Ulster method, in which the seed is not treated before it is plated, does not differentiate deep-seated infection from superficial infection of the seed, but has proved satisfactory in actual practice with seed samples that are not contaminated seriously by saprophytic fungi. The New Zealand method, by which surface contamination is supposedly removed by washing the seed in running water, has not been found reliable. The Ottawa method, by which the seed is surface sterilized before it is plated, does not indicate the degree of superficial infection, but does give the percentage of deep-seated infection, and hence it indicates the suitability of the seed for planting purposes if the seed is treated with a fungicide. It is suggested that a centrifuge test combined with the Ottawa plating method would provide a better index of the health condition of flax seed as far as disease caused by. P. lini is concerned.

scholarly journals Quantification of Viable Cells of Pseudomonas syringae pv. tomato in Tomato Seed Using Propidium Monoazide and a Real-Time PCR Assay

Plant Disease ◽  
2020 ◽  
Vol 104 (8) ◽  
pp. 2225-2232
Author(s):  
A-li Chai ◽  
Hai-yan Ben ◽  
Wei-tao Guo ◽  
Yan-xia Shi ◽  
Xue-wen Xie ◽  
...  
Keyword(s):  
Real Time ◽  
Pseudomonas Syringae ◽  
Real Time Pcr ◽  
Light Exposure ◽  
Qpcr Assay ◽  
Bacterial Suspension ◽  
Propidium Monoazide ◽  
Chromosomal Dna ◽  
Tomato Seed ◽  
Viable Cells

Pseudomonas syringae pv. tomato is a seedborne pathogen that causes bacterial speck disease in tomato. P. syringae pv. tomato is typically detected in tomato seed using quantitative real-time PCR (qPCR) but the inability of qPCR to distinguish between viable and nonviable cells might lead to an overestimation of viable P. syringae pv. tomato cells. In the present study, a strategy involving a propidium monoazide (PMA) pretreatment followed by a qPCR (PMA-qPCR) assay was developed for quantifying viable P. syringae pv. tomato cells in contaminated tomato seed. PMA could selectively bind to the chromosomal DNA of dead bacterial cells and, therefore, block DNA amplification of qPCR. The primer pair Pst3F/Pst3R was designed based on gene hrpZ to specifically amplify and quantify P. syringae pv. tomato by qPCR. The PMA pretreatment protocol was optimized for selectively detecting viable P. syringae pv. tomato cells, and the optimal PMA concentration and light exposure time were 10 μmol liter−1 and 10 min, respectively. In the sensitivity test, the detection limit of PMA-qPCR for detecting viable cells in bacterial suspension and artificially contaminated tomato seed was 102 CFU ml−1 and 11.86 CFU g−1, respectively. For naturally contaminated tomato seed, viable P. syringae pv. tomato cells were quantified in 6 of the 19 samples, with infestation levels of approximately 102 to 104 CFU g−1. The results indicated that the PMA-qPCR assay is a suitable tool for quantifying viable P. syringae pv. tomato cells in tomato seed, which could be useful for avoiding the potential risks of primary inoculum sources from contaminated seed.

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.

Antifungal activity of tomato seed extracts

Fitoterapia ◽  
1999 ◽  
Vol 70 (2) ◽  
pp. 181-183 ◽  
Author(s):  
E.S.J Nidiry
Keyword(s):  
Antifungal Activity ◽  
Tomato Seed ◽  
Seed Extracts

scholarly journals Conventional PCR for detection of Corynespora cassiicola in soybean seeds

2016 ◽  
Vol 38 (2) ◽  
pp. 85-91 ◽  
Author(s):  
Marcella Viana de Sousa ◽  
Carolina da Silva Siqueira ◽  
José da Cruz Machado
Keyword(s):  
Pure Culture ◽  
Early Stage ◽  
Soybean Seeds ◽  
Inoculum Potential ◽  
Corynespora Cassiicola ◽  
Severe Damage ◽  
Conventional Pcr ◽  
Seed Testing ◽  
Target Spot ◽  
Specificity And Sensitivity

Abstract The fungus Corynespora cassiicola, causal agent of target spot in soybeans, can be transmitted by soybean seeds and as of that point cause severe damage. This disease may be diagnosed at an early stage by seed testing, but knowledge in this area is insufficient. Because of that and increased attack by the disease in soybean areas in Brazil, further studies are required. The aim of this study was to evaluate the use of conventional PCR in detecting C. cassiicola in soybean seeds. The GA4-F/GA4-R primers described in the literature were tested for their specificity and sensitivity for detection of C. cassiicola in pure culture and in soybean seeds. Uninoculated and inoculated seed samples were used with different incidence levels - 100%, 10%, 1%, 0.5%, 0.25%, and 0% of preestablished inoculum potentials, P0, P1, P2, and P3. Detection of C. cassiicola in P1 inoculum potential was observed in samples with incidence levels of 10% to 100%. In the P3 potential, detection of the pathogen was successful in samples at the low level of 0.25%.

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

Effect of acidic electrolyzed water on the viability of bacterial and fungal plant pathogens and on bacterial spot disease of tomato

2006 ◽  
Vol 52 (10) ◽  
pp. 915-923 ◽  
Author(s):  
P A Abbasi ◽  
G Lazarovits
Keyword(s):  
Xanthomonas Campestris ◽  
Plant Pathogens ◽  
Disease Incidence ◽  
Bacterial Spot ◽  
Tomato Seed ◽  
Electrolyzed Water ◽  
Spot Disease ◽  
Foliar Sprays ◽  
Fungal Plant Pathogens ◽  
Bacterial Spot Disease

Acidic electrolyzed water (AEW), known to have germicidal activity, was obtained after electrolysis of 0.045% aqueous solution of sodium chloride. Freshly prepared AEW (pH 2.3–2.6, oxidation–reduction potential 1007–1025 mV, and free active chlorine concentration 27–35 ppm) was tested in vitro and (or) on tomato foliage and seed surfaces for its effects on the viability of plant pathogen propagules that could be potential seed contaminants. Foliar sprays of AEW were tested against bacterial spot disease of tomato under greenhouse and field conditions. The viability of propagules of Xanthomonas campestris pv. vesicatoria (bacterial spot pathogen), Streptomyces scabies (potato scab pathogen), and Fusarium oxysporum f.sp. lycopersici (root rot pathogen) was significantly reduced 4–8 log units within 2 min of exposure to AEW. Immersion of tomato seed from infected fruit in AEW for 1 and 3 min significantly reduced the populations of X. campestris pv. vesicatoria from the surface of the seed without affecting seed germination. Foliar sprays of AEW reduced X. campestris pv. vesicatoria populations and leaf spot severity on tomato foliage in the greenhouse. In the field, multiple sprays of AEW consistently reduced bacterial spot severity on tomato foliage. Disease incidence and severity was also reduced on fruit, but only in 2003. Fruit yield was either enhanced or not affected by the AEW sprays. These results indicate a potential use of AEW as a seed surface disinfectant or contact bactericide.Key words: electrolyzed oxidizing water, seed disinfectant, foliar sprays, bacterial spot control.

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.

Copper tolerance in Xanthomonas arboricola pv. pruni in South Carolina peach orchards

Plant Disease ◽  
2021 ◽  
Author(s):  
Brodie Cox ◽  
Hehe Wang ◽  
Guido Schnabel
Keyword(s):  
United States ◽  
South Carolina ◽  
Management Practices ◽  
Southeastern United States ◽  
The United States ◽  
Copper Tolerance ◽  
Bacterial Spot ◽  
Organic Farm ◽  
Xanthomonas Arboricola ◽  
Tolerance To Copper

Bacterial spot of peach, caused by Xanthomonas arboricola pv. pruni (Xap), causes yield loss every year in southeastern United States peach orchards. Management is mainly driven by season-long applications of copper-based products, site location, and choice of cultivar. Although tolerance to copper has not been reported in Xap in the United States, adaptation of populations due to frequent use is a concern. We collected Xap from shoot cankers, leaves, and fruit of cv. O'Henry over two years from three conventional farms and one organic farm in South Carolina, one orchard per farm. The four farms had been using copper extensively for years to control bacterial spot. Xap was isolated from four canker types (bud canker, tip canker, non-concentric canker, and concentric canker) in early spring (‘bud break’), as well as from leaf and fruit tissues later in the season at phenological stages ‘pit hardening’ and ‘final swell’. Xap was most frequently isolated from cankers of the organic farm (24% of the cankers) and most isolates (45%) came from bud cankers. Xap isolates were assessed for sensitivity to copper using minimal glucose yeast agar and nutrient agar amended with 38 µg/ml or 51 µg/ml of Cu2+. Two phenotypes of copper-tolerance in Xap were discovered: low copper tolerance (LCT: growth up to 38 µg/ml Cu2+) and high copper tolerance (HCT: growth up to 51 µg/ml Cu2+). A total of 26 (23 LCT and 3 HCT) out of 165 isolates in 2018 and 32 (20 LCT and 12 HCT) out of 133 isolates in 2019 were tolerant to copper. Peach leaves on potted trees were sprayed with copper rates typically applied at ‘delayed dormancy’ (high rate; 2,397 µg/ml Cu2+), at ‘shuck split’ (medium rate; 599 µg/ml Cu2+), and during ‘summer cover sprays’ (low rate; 120 µg/ml Cu2+) and subsequently inoculated with sensitive, LCT and HCT strains. Results indicated that the low and medium rates of copper reduced bacterial spot incidence caused by the sensitive strain but not by the LCT and HCT strains. This study confirms existence of Xap tolerance to copper in commercial peach orchards in the southeastern United States and suggests its contribution to bacterial spot development under current management practices.

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