scholarly journals Novel Primers and Sampling for PCR Detection of Xylella fastidiosa in Peach

2019 ◽  
Vol 109 (2) ◽  
pp. 307-317 ◽  
Author(s):  
Chunxian Chen ◽  
Clive H. Bock ◽  
Phillip M. Brannen
Keyword(s):  
Prunus Persica ◽  
Xylella Fastidiosa ◽  
Southeastern United States ◽  
Sampling Time ◽  
Pcr Detection ◽  
Detection Sensitivity ◽  
Tissue Type ◽  
Conventional Pcr ◽  
Reliable Detection ◽  
Sampling Times

Epidemics of phony peach disease (PPD), caused by Xylella fastidiosa, are of increasing concern to peach (Prunus persica) producers in the southeastern United States. Primers suitable for both conventional PCR (cPCR) and quantitative PCR (qPCR), along with optimal tissue and sampling time, are needed for comparative and reliable detection of X. fastidiosa. In this study, we developed and assessed novel primers for X. fastidiosa and for peach and compared detection of X. fastidiosa in four peach tissue types sampled at three time points using both cPCR and qPCR. Primer C06Xf-bamA was extensively tested for reliable detection of X. fastidiosa due to the more consistent intensity of the cPCR products and the marginally lower average quantification cycle (Cq) values of the qPCR products, compared with the other primers screened. Among the four peach tissue types tested, only root samples demonstrated reliable and consistent detection of X. fastidiosa; stem, petiole, and leaf samples, regardless of source trees, primers used, sampling times, or PCR methods (cPCR or qPCR), were unreliable for detection, due to insufficient quantity of DNA of X. fastidiosa in these samples based on the relative quantification assay. The Cq means and ratios were compared and statistically analyzed, to ascertain effects of source tree, tissue type, sampling time, and primer. Differences in detection sensitivity and the Cq means among sampled trees, sampling times, tested primers, and tissues (except root) were not significant or were inconsistent precluding further exploitation. In summary, these novel primers are a useful resource for detecting X. fastidiosa, and based on our results, root is the only tissue type reliable for year-round detection of X. fastidiosa in peach. Further research on potential utilization of above-ground tissues for PCR detection of X. fastidiosa are discussed.

scholarly journals The 28 + 28 day design is an effective sampling time for analyzing mutant frequencies in rapidly proliferating tissues of MutaMouse animals

2021 ◽  
Vol 95 (3) ◽  
pp. 1103-1116
Author(s):  
Francesco Marchetti ◽  
Gu Zhou ◽  
Danielle LeBlanc ◽  
Paul A. White ◽  
Andrew Williams ◽  
...  
Keyword(s):  
Germ Cells ◽  
False Negative ◽  
Sampling Time ◽  
Male Germ Cells ◽  
Negative Results ◽  
False Negative Results ◽  
Detection Of Mutations ◽  
The Impact ◽  
Sampling Times

AbstractThe Organisation for Economic Co-Operation and Development Test Guideline 488 (TG 488) uses transgenic rodent models to generate in vivo mutagenesis data for regulatory submission. The recommended design in TG 488, 28 consecutive daily exposures with tissue sampling three days later (28 + 3d), is optimized for rapidly proliferating tissues such as bone marrow (BM). A sampling time of 28 days (28 + 28d) is considered more appropriate for slowly proliferating tissues (e.g., liver) and male germ cells. We evaluated the impact of the sampling time on mutant frequencies (MF) in the BM of MutaMouse males exposed for 28 days to benzo[a]pyrene (BaP), procarbazine (PRC), isopropyl methanesulfonate (iPMS), or triethylenemelamine (TEM) in dose–response studies. BM samples were collected + 3d, + 28d, + 42d or + 70d post exposure and MF quantified using the lacZ assay. All chemicals significantly increased MF with maximum fold increases at 28 + 3d of 162.9, 6.6, 4.7 and 2.8 for BaP, PRC, iPMS and TEM, respectively. MF were relatively stable over the time period investigated, although they were significantly increased only at 28 + 3d and 28 + 28d for TEM. Benchmark dose (BMD) modelling generated overlapping BMD confidence intervals among the four sampling times for each chemical. These results demonstrate that the sampling time does not affect the detection of mutations for strong mutagens. However, for mutagens that produce small increases in MF, sampling times greater than 28 days may produce false-negative results. Thus, the 28 + 28d protocol represents a unifying protocol for simultaneously assessing mutations in rapidly and slowly proliferating somatic tissues and male germ cells.

scholarly journals First Report of Xylella fastidiosa in Peach in New Mexico

Plant Disease ◽  
2011 ◽  
Vol 95 (7) ◽  
pp. 871-871 ◽  
Author(s):  
J. J. Randall ◽  
J. French ◽  
S. Yao ◽  
S. F. Hanson ◽  
N. P. Goldberg
Keyword(s):  
New Mexico ◽  
Prunus Persica ◽  
Xylella Fastidiosa ◽  
Its Region ◽  
23S Rrna ◽  
Pcr Analysis ◽  
Sequencing Analysis ◽  
First Report ◽  
Dark Green ◽  
Branch Dieback

Xylella fastidiosa is a gram-negative bacterium that causes disease in a wide variety of plants such as grapes, citrus trees, oleanders, and elm and coffee trees. This bacterium is xylem limited and causes disease symptoms such as leaf scorch, stunting of plant growth, branch dieback, and fruit loss. The presence of X. fastidiosa was previously reported in New Mexico where it was found to be infecting chitalpa plants and grapevines (3). In the summer of 2010, peach (Prunus persica (L.) Batsch) trees from two locations in northern New Mexico exhibited leaf deformity and stunting, dark green venation, slight mottling, and branch dieback. Preliminary viral diagnostic screening was performed by Agdia (Elkhart, IN) on one symptomatic tree and it was negative for all viruses tested. Three trees from two different orchards tested positive for X. fastidiosa by ELISA and PCR analysis using X. fastidiosa-specific primer sets HL (1) and RST (2). Bacterial colonies were also cultured from these samples onto periwinkle wilt media. Eight colonies obtained from these three plants tested PCR positive using the X. fastidiosa-specific primers. The 16S ribosomal and 16S-23S rRNA internal transcribed spacer (ITS) region (557 nucleotides) (GenBank Accession No. HQ292776) along with the gyrase region (400 nucleotides) (GenBank Accession No. HQ292777) was amplified from the peach total DNA samples and the bacterial colonies. Sequencing analysis of these regions indicate that the X. fastidiosa found in peach is 100% similar to other X. fastidiosa multiplex isolates including isolates from peach, pecan, sycamore, and plum trees and 99% similar to the X. fastidiosa isolates previously found in New Mexico. Further analysis of the 16S ribosomal and 16S-23S rRNA ITS sequences with maximum likelihood phylogenetic analysis using Paup also groups the peach isolates into the X. fastidiosa multiplex subspecies. The gyrase sequence could not be used to differentiate the peach isolates into a subspecies grouping because of the lack of variability within the sequence. This X. fastidiosa multiplex subspecies could possibly be a threat to the New Mexico pecan industry since pecan infecting X. fastidiosa isolates belong to the same bacterial subspecies. It is not known if X. fastidiosa subspecies multiplex isolates from peach are capable of infecting pecans but they are closely genetically related. It is interesting to note that the isolates from peach are different than previously described X. fastidiosa isolates in New Mexico that were infecting chitalpa and grapes (3). X. fastidiosa has previously been described in peach; the disease is called “phony peach”. The peach trees exhibited stunting and shortened internodes as reported for “phony peach”. They also exhibited slight mottling and branch dieback that may be due to the environment in New Mexico or perhaps they are also exhibiting mineral deficiency symptoms in association with the X. fastidiosa disease. To our knowledge, this is the first report of X. fastidiosa in peach in New Mexico. References: (1) M. H. Francis et al. Eur. J. Plant Pathol. 115:203, 2006. (2) G. V. Minsavage et al. Phytopathology 84:456, 1994. (3) J. J. Randall et al. Appl. Environ. Microbiol. 75:5631, 2009.

scholarly journals Impact of Exposure to Low Temperature Degrees in Field Conditions on Leaf Pigments and Chlorophyll Fluorescence in Leaves of Mango Trees

2020 ◽  
Vol 10 ◽  
pp. 30-45
Author(s):  
Ali A.S. Sayed ◽  
Farouk M. Gadallah ◽  
Mohamed A. Seif El-Yazal ◽  
Gamal A. Abdel-Samad
Keyword(s):  
Chlorophyll Fluorescence ◽  
Low Temperature ◽  
Chlorophyll Concentration ◽  
Ambient Air ◽  
Sampling Time ◽  
Chlorophyll B ◽  
Physiological And Biochemical ◽  
Sampling Times ◽  
Fayoum Governorate

This experiment was conducted to found the connection between low temperature stress in vivo conditions (ambient-air temperature) and the changes in some physiological and biochemical events (leaf pigments and chlorophyll fluorescence) of mango trees in response to exposure to natural low temperature (cold). To verify this objective, 12 popular commonly mango cultivars (25 years old) which grown in private orchard in Fayoum Governorate, Egypt were selected for this study which carried out during the period from November to March of years; 2012 and 2013. The selected cultivars were: Alphonso, Baladi, Bullock's Heart, Helmand, Hindi Besennara, Mabrouka, Mestekawy, Nabeeh, Oweisi, Spates, Taimour and Zebda. Based on the obtained results, it can be stated that, chlorophyll (a) concentration in the leaves was significantly differed among the cultivars throughout the whole sampling times, in this respect, Helmand one gave the highest one while, and the highest one by sampling times was November one. The concentration of chlorophyll (b) was significant as effected by the effect of cultivars and sampling time recorded the highest value by the cultivar of Spates and December sample, respectively. Total chlorophyll concentration in the leaves reached its peak by the cultivar of Nabeeh and sampling time of December as compared to others. The both of Ewais cultivar and the sample of March showed the highest values of carotenoids concentration in the leaves. The levels of anthocyanin in leaves were significantly differed as affected by the cultivars and sampling times, indicating that the cultivar of Helmand and November sample recorded the highest values of anthocyanin in leaves. The greatest reductions in Fv/Fmratio were recorded at month of November and indicated that the reductions were in the order of Alphonso˃ Mabrouka˃Taimour˃ others. The effect of sampling time, cultivars and their interaction on Fv/Fm were significant, but small between some values of Fv/Fm.

Xylella fastidiosa subsp. multiplex. [Distribution map].

2018 ◽  
Author(s):  
Keyword(s):  
North Carolina ◽  
Prunus Persica ◽  
Xylella Fastidiosa ◽  
District Of Columbia ◽  
Balearic Islands ◽  
Rio Grande Do Sul ◽  
Distribution Map ◽  
Polygala Myrtifolia ◽  
New Distribution ◽  
Distribution In Europe

Abstract A new distribution map is provided for Xylella fastidiosa subsp. multiplex Schaad et al. Gammaproteobacteria: Xanthomonadales: Xanthomonadaceae. Hosts: olive (Olea europaea), Prunus persica, coffee (Coffea spp.), Citrus, Polygala myrtifolia, many tree species. Information is given on the geographical distribution in Europe (France, Mainland France, Corsica, Spain, Mainland Spain, Balearic Islands), North America (USA, Alabama, California, District of Columbia, Florida, Georgia, Indiana, Kentucky Louisiana, North Carolina, Tennessee, Texas, Virginia), South America (Argentina, Brazil, Minas Gerais, Parana, Rio Grande do Sul, Santa Catarina, Sao Paulo, Paraguay).

Xylella fastidiosa. [Distribution map].

2006 ◽  
Author(s):  
Keyword(s):  
New York ◽  
South Carolina ◽  
Prunus Persica ◽  
Xylella Fastidiosa ◽  
Acer Rubrum ◽  
Prunus Dulcis ◽  
Wild Plants ◽  
Distribution Map ◽  
Ulmus Americana ◽  
Platanus Occidentalis

Abstract A new distribution map is provided for Xylella fastidiosa Wells et al. Bacteria. Hosts: Grapevine (Vitis vinifera and others), peach (Prunus persica), Citrus, almond (Prunus dulcis), lucerne (Medicago sativa), some wild trees (including Acer rubrum, Platanus occidentalis, Quercus rubra, Ulmus americana), other wild plants and weeds. Information is given on the geographical distribution in Europe (France, Italy), Asia (Taiwan), North America (Canada (Ontario), Mexico, USA (Alabama, Arizona, Arkansas, California, Delaware, District of Columbia, Florida, Georgia, Indiana, Kentucky, Louisiana, Maryland, Mississippi, Missouri, Montana, Nebraska, New Jersey, New Mexico, New York, North Carolina, Pennsylvania, South Carolina, Tennessee, Texas, Virginia, Washington, West Virginia)), Central America and Caribbean (Costa Rica), and South America (Argentina, Brazil (Bahia, Goias, Minas Gerais, Parana, Rio de Janeiro, Rio Grande do Sul, Santa Catarina, Sao Paulo, Sergipe), Paraguay, Venezuela).

Xylella fastidiosa subsp. fastidiosa. [Distribution map].

2018 ◽  
Author(s):  
Keyword(s):  
North Carolina ◽  
North America ◽  
Central America ◽  
Prunus Persica ◽  
Xylella Fastidiosa ◽  
District Of Columbia ◽  
Balearic Islands ◽  
Distribution Map ◽  
New Distribution ◽  
Distribution In Europe

Abstract A new distribution map is provided for Xylella fastidiosa subsp. fastidiosa Schaad et al. Gammaproteobacteria: Xanthomonadales: Xanthomonadaceae. Hosts: grapevine (Vitis vinifera), coffee (Coffea spp.), Prunus persica. Information is given on the geographical distribution in Europe (Germany, Spain, Balearic Islands), Asia (Taiwan, North America, Mexico, USA, California, District of Columbia, Florida, Georgia, Louisiana, Maryland, North Carolina, Texas), Central America & Caribbean (Costa Rica).

Comparison of Two Sampling Methods for Escherichia coli O157:H7 Detection in Feedlot Cattle

2005 ◽  
Vol 68 (8) ◽  
pp. 1724-1728 ◽  
Author(s):  
M. L. KHAITSA ◽  
M. L. BAUER ◽  
P. S. GIBBS ◽  
G. P. LARDY ◽  
D. DOETKOTT ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Sampling Methods ◽  
Feedlot Cattle ◽  
Sampling Time ◽  
Escherichia Coli O157 ◽  
Confounding Factors ◽  
Rank Tests ◽  
E Coli ◽  
Signed Rank ◽  
Sampling Times

Two sampling methods (rectoanal swabs and rectal fecal grabs) were compared for their recovery of Escherichia coli O157:H7 from feedlot cattle. Samples were collected from 144 steers four times during the finishing period by swabbing the rectoanal mucosa with cotton swabs and immediately obtaining feces from the rectum of each individual steer. The number of steers with detectable E. coli O157:H7 increased from 2 of 144 (1.4%) cattle on arrival at the feedlot to 10 of 144 (6.9%) after 1 month, 76 of 143 (52.8%) after 7 months, and 30 of 143 (20.8%) at the last sampling time before slaughter. Wilcoxon signed-rank tests indicated that the two sampling methods gave different results for sampling times 3 and 4 (P < 0.05) but not for sampling time 2 (P = 0.16). Agreement between the two sampling methods was poor (kappa < 0.2) for three of the four sampling times and moderate (kappa = 0.6) for one sampling time, an indication that in this study rectoanal swabs usually were less sensitive than rectal fecal grabs for detection of E. coli O157:H7 in cattle. Overall, the herd of origin was not significantly associated with E. coli O157:H7 results, but the weight of the steers was. Further investigation is needed to determine the effects of potential confounding factors (e.g., size and type of swab, consistency of feces, site sampled, and swabbing technique) that might influence the sensitivity of swabs in recovering E. coli O157:H7 from the rectoanal mucosa of cattle.

Rapid, specific and quantitative polymerase chain reaction (PCR) detection of pathogenic protozoa Entamoeba histolytica for drinking water supply

2011 ◽  
Vol 11 (4) ◽  
pp. 418-425 ◽  
Author(s):  
S. W. Lam ◽  
H. B. Zhang ◽  
L. Yu ◽  
C. H. Woo ◽  
K. N. Tiew ◽  
...  
Keyword(s):  
Real Time ◽  
Water Samples ◽  
Real Time Pcr ◽  
Genomic Dna ◽  
Tap Water ◽  
Pcr Detection ◽  
Raw Water ◽  
Conventional Pcr ◽  
Polymerase Chain ◽  
Species Specific

In this study, a quantitative species-specific polymerase chain reaction (PCR) method to rapidly detect E. histolytica in water is developed. First, the specificity of E. histolytica PCR detection was verified by using species-specific primers of 16S-like rRNA genes to clearly differentiate it from the closely related amoebae species E. dispar and E. moshkovskii. The sensitivity of this method was subsequently determined using purified E. histolytica genomic DNA and culture cells as PCR reaction templates. Results indicated that conventional PCR visualized on 1% agarose gel was able to detect as low as 0.02 pg genomic DNA and 5 cells, while real-time PCR could detect 0.01 pg genomic DNA and 2 cells of E. histolytica. The protocols for E. histolytica PCR detection in real water samples were then optimized by spiking E. histolytica cells into tap water and reservoir raw water samples. A two-round centrifugation treatment to concentrate amoeba cells directly as a PCR template was the most effective way to detect E. histolytica in spiked tap water samples, while DNA extraction after concentrating amoeba cells was required for spiked reservoir raw water samples. The detection limit of 50 E. histolytica cells in 100 ml tap water was achieved in 2 h from sample collection to real-time PCR data readout. With these established protocols, 78 tap water samples, 11 reservoir raw water samples and 4 feed water samples from Singapore water supply systems were analyzed by both conventional PCR and real-time PCR methods. No E. histolytica cell was detected in tested samples.

316 Does sampling time matter? Relationships of circulating metabolites at various neonatal sampling times in beef calves

2016 ◽  
Vol 94 (suppl_2) ◽  
pp. 148-149
Author(s):  
J. M. Larson ◽  
B. L. Vander Ley ◽  
S. M. Bolen ◽  
N. B. Duncan ◽  
A. M. Meyer
Keyword(s):  
Sampling Time ◽  
Beef Calves ◽  
Sampling Times

scholarly journals Quantitative LAMP and PCR Detection of Salmonella in Chicken Samples Collected from Local Markets around Pathum Thani Province, Thailand

2020 ◽  
Vol 2020 ◽  
pp. 1-6 ◽  
Author(s):  
Virun Vichaibun ◽  
Panan Kanchanaphum
Keyword(s):  
Efficient Method ◽  
Food Chain ◽  
Dna Detection ◽  
Pcr Detection ◽  
Isothermal Amplification ◽  
Artificial Infection ◽  
Conventional Pcr ◽  
Loop Mediated Isothermal Amplification ◽  
Contaminated Food ◽  
Pcr Method

Salmonella is a bacterium that infects people when they consume contaminated food or liquids. To prevent humans from becoming ill, it is useful to have an efficient method of detecting Salmonella before the disease is passed on through the food chain. In this research, the efficiency of Salmonella detection was compared using the following four methods: conventional loop-mediated isothermal amplification (LAMP), PCR, quantitative LAMP (qLAMP), and qPCR. The artificial infection of chicken samples started with incubating of 10 mL of 108 CFU of S. typhimurium for 6 hr. and enriching for 2 hr. to represent real contamination of the samples. The results show that the sensitivity of Salmonella DNA detection in PCR, qPCR, LAMP, and qLAMP were 50 ng, 5 ng, 50 pg, and and 500 fg, respectively. Thirty samples of 10 g chicken were collected from 10 markets in Pathum Thani, Thailand; then, the infection was detected. The conventional LAMP, qLAMP, and qPCR methods detected Salmonella in all the chicken samples. However, the conventional PCR method detected Salmonella infection in only eight of the samples. Overall, the qLAMP method had the highest sensitivity of Salmonella DNA detection.

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