PCR Detection Assay of Fumonisin-Producing Fusarium verticillioides Strains

2004 ◽  
Vol 67 (6) ◽  
pp. 1278-1283 ◽  
Author(s):  
BELÉN PATIÑO ◽  
SALVADOR MIRETE ◽  
M. TERESA GONZÁLEZ-JAÉN ◽  
GIUSEPPINA MULÉ ◽  
M. TERESA RODRÍGUEZ ◽  
...  
Keyword(s):  
Fusarium Species ◽  
Fusarium Verticillioides ◽  
Animal Health ◽  
Intergenic Spacer ◽  
Fungal Species ◽  
Intergenic Spacer Region ◽  
Detection And Identification ◽  
Geographical Regions ◽  
Species Specific ◽  
A Minor

Fusarium verticillioides is considered to be the main source of fumonisins, a group of toxins that contaminate commodities and result in chronic and acute diseases affecting humans and animals. The detection and control of this species is crucial to prevent fumonisins from entering the food chain. The objective of the present research was to develop a specific, sensitive, and robust PCR assay to detect F. verticillioides strains using two pairs of specific primers for F. verticillioides, which have been designed on the basis of the intergenic spacer region of the rDNA units. The first pair of primers was F. verticillioides species specific, whereas the second pair of primers detected fumonisin-producing F. verticillioides strains. This second pair of primers allowed for the discrimination between the major group of F. verticillioides strains, fumonisin-producing strains that are mainly associated with crops, and a minor group of strains, non–fumonisin-producing strains that are associated with bananas. Fifty-four strains of F. verticillioides from different geographical regions and hosts were tested using both sets of primers. Sixteen additional Fusarium species were examined. The specificity of the primer sequences provides the basis for a simple, rapid, accurate, and sensitive detection and identification method of this fungal species that represents a risk for human and animal health.

scholarly journals Evaluation of Nonpathogenic Fusarium oxysporum and Pseudomonas fluorescens for Panama Disease Control

Plant Disease ◽  
2011 ◽  
Vol 95 (8) ◽  
pp. 951-959 ◽  
Author(s):  
A. Belgrove ◽  
C. Steinberg ◽  
A. Viljoen
Keyword(s):  
Fusarium Oxysporum ◽  
Pseudomonas Fluorescens ◽  
Fusarium Wilt ◽  
Intergenic Spacer ◽  
Polymorphism Analysis ◽  
Panama Disease ◽  
Intergenic Spacer Region ◽  
Soil Microbial ◽  
Fusarium Wilt Of Banana ◽  
Species Specific

Nonpathogenic Fusarium oxysporum endophytes from healthy banana roots were evaluated for their ability to reduce Fusarium wilt of banana (Panama disease). Isolates were identified morphologically and by using species-specific primers. Pathogenicity was confirmed by inoculating banana plantlets in the greenhouse. Nonpathogenic F. oxysporum isolates were grouped into 14 haplotype groups by polymerase chain reaction restriction fragment length polymorphism analysis of the intergenic spacer region, and representative isolates evaluated for biocontrol of F. oxysporum f. sp. cubense. In the greenhouse, 10 nonpathogenic F. oxysporum isolates were able to significantly reduce Fusarium wilt of banana. The isolate that protected banana plantlets best in the greenhouse, a nonpathogenic F. oxysporum from the root rhizosphere, and Pseudomonas fluorescens WCS 417 were then field tested. When the putative biological control organisms were tested in the field, neither the nonpathogenic F. oxysporum, P. fluorescens, nor combinations thereof reduced Fusarium wilt development significantly. A number of factors could contribute to the lack of field protection, including soil microbial and chemical composition and reduced survival of biocontrol organisms in banana roots. A lack of knowledge regarding the etiology of Fusarium wilt of ‘Cavendish’ banana in the subtropics and the effect of F. oxysporum f. sp. cubense race and banana cultivar in protection of banana by biocontrol organisms should be further investigated.

scholarly journals Identification of Porphyromonas gingivalis Strains by Heteroduplex Analysis and Detection of Multiple Strains

1999 ◽  
Vol 37 (12) ◽  
pp. 3906-3911 ◽  
Author(s):  
Eugene J. Leys ◽  
James H. Smith ◽  
Sharon R. Lyons ◽  
Ann L. Griffen
Keyword(s):  
Porphyromonas Gingivalis ◽  
Dna Sequences ◽  
Allelic Variation ◽  
Intergenic Spacer ◽  
Heteroduplex Analysis ◽  
Clinical Samples ◽  
Specific Primers ◽  
Intergenic Spacer Region ◽  
Species Specific ◽  
Multiple Strains

Heteroduplex analysis has been used extensively to identify allelic variation among mammalian genes. It provides a rapid and reliable method for determining and cataloging minor differences between two closely related DNA sequences. We have adapted this technique to distinguish among strains or clonal types of Porphyromonas gingivalis. The ribosomal intergenic spacer region (ISR) was amplified directly from a subgingival plaque sample by PCR with species-specific primers, avoiding the need for culturing the bacteria. The PCR products were then directly compared by heteroduplex analysis with known strains of P. gingivalis for identification. We identified 22 distinct but closely related heteroduplex types ofP. gingivalis in 1,183 clinical samples. Multiple strains were found in 34% of the samples in which P. gingivaliswas detected. Heteroduplex types were identified from these multistrain samples without separating them by culturing or molecular cloning. PCR with species-specific primers and heteroduplex analysis makes it possible to reliably and sensitively detect and identify strains ofP. gingivalis in large numbers of samples.

scholarly journals Fusarium Species and Mycotoxins Contaminating Veterinary Diets for Dogs and Cats

2019 ◽  
Vol 7 (1) ◽  
pp. 26 ◽  
Author(s):  
Natalia Witaszak ◽  
Łukasz Stępień ◽  
Jan Bocianowski ◽  
Agnieszka Waśkiewicz
Keyword(s):  
Animal Feed ◽  
Fusarium Species ◽  
Fusarium Verticillioides ◽  
Animal Health ◽  
Companion Animals ◽  
Fumonisin B1 ◽  
Additional Risk ◽  
Two Samples ◽  
The Mean ◽  
The Eu

Veterinary diets are intended for diseased animals and may contain cereal grains, mainly maize and/or wheat. These, in turn, are often infected with pathogens of the Fusarium genus, which are able to produce numerous harmful mycotoxins. Forty-two samples of veterinary diets for dogs and cats were analyzed for the presence of Fusarium species and mycotoxins. Species were identified using molecular methods and the ergosterol and mycotoxins (fumonisin B1, deoxynivalenol, nivalenol and zearalenone) were quantified using HPLC methods. Two Fusarium species were identified: Fusarium proliferatum and Fusarium verticillioides. The highest concentrations of fumonisin B1, deoxynivalenol, nivalenol and zearalenone were 74.83, 2318.05, 190.90, and 45.84 ng/g, respectively. Only 9.5% of the samples were free from Fusarium mycotoxins. The acceptable limits of mycotoxin content in animal feed, specified by the EU regulations, were not exceeded in any of the samples tested. The mean mycotoxin content in veterinary diets for cats was lower than for dogs. Thus, it is recommended that veterinary diets are examined, since the mycotoxin contamination pose additional risk to animal health. The knowledge on Fusarium occurrence in veterinary diets is scarce and as far as we are aware this is the first report concerning the occurrence of Fusarium spp. and their important secondary metabolites—mycotoxins—in different types of veterinary diets for companion animals in Poland.

scholarly journals Simultaneous Detection and Identification of Common Cell Culture Contaminant and Pathogenic Mollicutes Strains by Reverse Line Blot Hybridization

2004 ◽  
Vol 70 (3) ◽  
pp. 1483-1486 ◽  
Author(s):  
Hui Wang ◽  
Fanrong Kong ◽  
Peter Jelfs ◽  
Gregory James ◽  
Gwendolyn L. Gilbert
Keyword(s):  
Cell Culture ◽  
16S Rrna ◽  
Cell Line ◽  
Intergenic Spacer ◽  
23S Rrna ◽  
Pcr Assay ◽  
Intergenic Spacer Region ◽  
Specific Pcr ◽  
Specific Pcr Assay ◽  
Species Specific

ABSTRACT We have developed a reverse line blot (RLB) hybridization assay to detect and identify the commonest mollicutes causing cell line contamination (Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma hyorhinis, Mycoplasma orale, and Acholeplasma laidlawii) and human infection (Mycoplasma pneumoniae, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma parvum, and Ureaplasma urealyticum). We developed a nested PCR assay with “universal” primers targeting the mollicute 16S-23S rRNA intergenic spacer region. Amplified biotin-labeled PCR products were hybridized to membrane-bound species-specific oligonucleotide probes. The assay correctly identified reference strains of 10 mollicute species. Cell cultures submitted for detection of mollicute contamination, clinical specimens, and clinical isolates were initially tested by PCR assay targeting a presumed mollicute-specific sequence of the 16S rRNA gene. Any that were positive were assessed by the RLB assay, with species-specific PCR assay as the reference method. Initially, 100 clinical and 88 of 92 cell culture specimens gave concordant results, including 18 in which two or more mollicute species were detected by both methods. PCR and sequencing of the 16S-23S rRNA intergenic spacer region and subsequent retesting by species-specific PCR assay of the four cell culture specimens for which results were initially discrepant confirmed the original RLB results. Sequencing of amplicons from 12 cell culture specimens that were positive in the 16S rRNA PCR assay but negative by both the RLB and species-specific PCR assays failed to identify any mollicute species. The RLB hybridization assay is sensitive and specific and able to rapidly detect and identify mollicute species from clinical and cell line specimens.

scholarly journals Investigation of virulence factors of Enterococcus faecalis strains isolated in secondary/ persistent infections

2014 ◽  
Vol 17 (1) ◽  
pp. 32
Author(s):  
Ana Claudia C. Xavier ◽  
Frederico Canato Martinho ◽  
Izabel C.G. Camões ◽  
Lilian F. Freitas
Keyword(s):  
Enterococcus Faecalis ◽  
Virulence Factor ◽  
Intergenic Spacer ◽  
Virulence Determinants ◽  
Test Species ◽  
Intergenic Spacer Region ◽  
Culture Techniques ◽  
Endodontic Infection ◽  
Species Specific ◽  
Virulence Factor Genes

<p><strong>Objective</strong>: More virulent strains may result from  the acquisition of genes by genetic exchange,  pathogenicity islands in several species encoding  toxins, adhesion factors and other factors associated  with virulence. The aim of this study was to investigate  the prevalence of E. faecalis strains in secondary  endodontic/ persistent using endodontic infection by  culture and PCR technqiues; and to investigate for  the presence of virulence factor genes of gelatinase  (gelE), cytolysin activator (Cyla), surface adhesin  of Enterococcus (ESP) and collagen adhesin of  Enterococcus (ACE). <strong>Material and methods</strong>: Microbial samples were obtained from 12 teeth with  secondary/ persistent endodontic infection showing  apical periodontitis. Culture techniques were used  including serial dilution, plating, incubation, and  biochemical identification. For PCR detection, samples  were analyzed using a species-specific primer of the  16S rDNA and the downstream intergenic spacer  region. <strong>Results</strong>: Culture and PCR detected the test  species in 3/12 (25%) and 5/12 (41.6%) of teeth,  respectively. A total of 38 Enterococcus faecalis strains  were isolated and submitted to the virulence factor  genes analysis. PCR products consistent with genes  encoding surface adhesion (ESP), gelatinase (gelE)  and collagen binding antigen (ACE) were found in  26/38 (68%), 31/38 (81%) and 38/38 (100%) of  the isolates. The Cytolysin activator (Cyla) gene was  not recovered from E. faecalis isolates. <strong>Conclusions</strong>:  In conclusion, the present study revealed by culture  and molecular methods revealed a high prevalence  of E. faecalis in teeth with secondary/ persistent  endodontic infection. Moreover, of a clinical  relevance, we found different E. faecalis strains  carrying different virulence determinants.</p><p><strong>KEYWORDS</strong> Bacteria; E. faecalis; Root canal, virulence.    </p>

Regional Differences in Plant Development, Water Relations, and Chloroplast DNA of Hard Maples

HortScience ◽  
1998 ◽  
Vol 33 (3) ◽  
pp. 456d-456
Author(s):  
Rolston St. Hilaire ◽  
William R. Graves
Keyword(s):  
Chloroplast Dna ◽  
Water Relations ◽  
Plant Development ◽  
Regional Differences ◽  
Intergenic Spacer ◽  
Principal Component ◽  
Western Region ◽  
Intergenic Spacer Region ◽  
Geographical Regions ◽  
Leaf Osmotic Potential

Principal component analysis of foliar traits of hard maples (Acer saccharum Marsh. and Acer nigrum Michx. f.) near 43°N latitude clustered data into two populations composed of trees from different geographical regions. Seedlings from these two regions, and a third, geographically intermediate region, were grown in a greenhouse for 2 years with two irrigation frequencies to assess regional differences in plant development and water relations. Leaves from the most western region (west of 93°W longitude) had the highest specific mass (5.97 mg/cm2), trichome frequency (531/cm2), and stomate frequency (628/cm2). Across regions, plants irrigated frequently had more stomates (596/cm2) than plants irrigated sparsely (483/cm2). Traits similar across regions but higher with frequent irrigation included surface area and mass of lamina, shoot-to-root ratio, the ratio of lamina area to stem xylem diameter, and leaf water potential. Sparse irrigation caused a comparatively large decrease in stomatal conductance of plants from the most western region, and pressure-volume analysis showed no regional or irrigation effects on leaf osmotic potential at full turgor. Identical banding patterns resulted when Hinf I digested the PCR-amplified trnL-trnF intergenic spacer region of chloroplast DNA from each region; work with the rpL16 and ndhA introns is proceeding. Trichome frequency on abaxial leaf surfaces, which differs regionally both in nature and in controlled environments, is the most consistent character we have measured for discerning populations.

scholarly journals Species-Specific Detection and Identification of Fusarium Species Complex, the Causal Agent of Sugarcane Pokkah Boeng in China

PLoS ONE ◽  
2014 ◽  
Vol 9 (8) ◽  
pp. e104195 ◽  
Author(s):  
Zhenyue Lin ◽  
Shiqiang Xu ◽  
Youxiong Que ◽  
Jihua Wang ◽  
Jack C. Comstock ◽  
...  
Keyword(s):  
Species Complex ◽  
Fusarium Species ◽  
Causal Agent ◽  
Specific Detection ◽  
Pokkah Boeng ◽  
Detection And Identification ◽  
Species Specific

scholarly journals Survey for toxigenic Fusarium species on maize kernels in China

2020 ◽  
Vol 13 (2) ◽  
pp. 213-224 ◽  
Author(s):  
P.W. Qin ◽  
J. Xu ◽  
Y. Jiang ◽  
L. Hu ◽  
T. van der Lee ◽  
...  
Keyword(s):  
Seed Quality ◽  
Fusarium Species ◽  
Fusarium Verticillioides ◽  
Morphological Characteristics ◽  
Ear Rot ◽  
Maize Production ◽  
Fusarium Asiaticum ◽  
Maize Varieties ◽  
Species Specific ◽  
Maize Kernels

Maize is currently the most important crop in China. A major concern in maize production is maize ear rot caused by Fusarium spp., which results in yield losses, reduction of seed quality and the accumulation of mycotoxins in the harvested grains. To identify the importance of the different Fusarium species in maize infection, we performed a comprehensive survey on 9,000 asymptomatic and randomly collected maize kernels. Seeds were collected from 12 different provinces covering all major maize growing areas in China and included five maize varieties. In total 1,022 Fusarium isolates were retrieved that were identified based on morphological characteristics, by species specific diagnostic PCRs and by EF1-α gene sequencing. Eight different species were identified: Fusarium verticillioides (75.34%), Fusarium graminearum (8.32%), Fusarium proliferatum (7.14%), Fusarium subglutinans (4.11%), Fusarium meridionale (1.57%), Fusarium oxysporum (1.37%), Fusarium semitectum (1.17%), and Fusarium asiaticum (0.98%). The distribution of Fusarium species was found to be different in different regions with the largest diversity observed in Hubei province, where all eight Fusarium species were isolated. Genetic chemotyping within the F. graminearum species complex indicated that all of the 85 F. graminearum isolates showed the 15-acetyldeoxynivalenol chemotype, whereas all F. asiaticum (n=10) and F. meridionale (n=16) isolates had the nivalenol chemotype even when isolated from the same maize field. To our knowledge this is the largest collection of Fusarium isolates from maize and further exploitations of this collection are discussed.

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