scholarly journals A Species-Specific PCR Assay for Detection of Diplodia pinea and D. scrobiculata in Dead Red and Jack Pines with Collar Rot Symptoms

Plant Disease ◽  
2006 ◽  
Vol 90 (3) ◽  
pp. 307-313 ◽  
Author(s):  
Denise R. Smith ◽  
Glen R. Stanosz
Keyword(s):  
Fungal Pathogens ◽  
Jack Pine ◽  
Small Subunit ◽  
Collar Rot ◽  
Specific Pcr ◽  
Rdna Sequences ◽  
Diplodia Pinea ◽  
Specific Pcr Assay ◽  
Species Specific ◽  
Forward Primer

A polymerase chain reaction (PCR)-based assay was developed for the specific detection of the fungal pathogens Diplodia pinea and D. scrobiculata from pine host tissues. Variation among mitochondrial small subunit ribosome gene (mt SSU rDNA) sequences of Botryosphaeria species and related anamorphic fungi was exploited to design primer pairs. Forward primer DpF and forward primer DsF, each when used with the nonspecific reverse primer BotR, amplified DNA of D. pinea or D. scrobiculata, respectively. Specificity was confirmed using multiple isolates of each of these two species and those of closely related fungi including Botryosphaeria obtusa. The detection limits for DNA of each pathogen in red and jack pine bark were 50 to 100 pg μl-1 and 1 pg μl-1 in red and jack pine wood. The assay was tested using naturally occurring red and jack pine seedlings and saplings exhibiting symptoms of Diplodia collar rot. Samples from lower stems/root collars of 10 dead trees of each species from each of three sites at each of two locations were tested. Results were positive for D. pinea or D. scrobiculata for the large majorities of symptomatic bark and wood samples from both locations. For positive samples, however, there were effects of location and host species on detection of D. pinea (more frequent on red pine) and D. scrobiculata (more frequent on jack pine) (P < 0.01 in both cases). These results indicate that these new primers are potentially useful for studies in areas or hosts in which both pathogens may be present.

scholarly journals Shoot Blight Caused by Diplodia pinea on Afghan and Austrian Pines in Texas

Plant Disease ◽  
2007 ◽  
Vol 91 (8) ◽  
pp. 1056-1056 ◽  
Author(s):  
K. Ong ◽  
S. Hill ◽  
D. R. Smith ◽  
G. R. Stanosz
Keyword(s):  
Gulf Coast ◽  
Pcr Assay ◽  
Specific Pcr ◽  
Diplodia Pinea ◽  
Pine Seedlings ◽  
Pure Cultures ◽  
Shoot Blight ◽  
Specific Pcr Assay ◽  
Species Specific ◽  
Hyphal Tip

Shoot blight was observed on ornamental Afghan (Pinus eldarica) and Austrian pines (P. nigra) at several sites in metropolitan Dallas, TX in the summer of 2005. Shoots were stunted, cankered, often resinous, sometimes curled or crooked at the tips, and bore brown needles that often had been killed before full elongation. Pycnidia on necrotic needles, stems, and cones of each host species yielded conidia characteristic of the fungus Diplodia pinea. Individual conidia and hyphal tip transfers produced pure cultures confirmed as D. pinea using a species-specific PCR assay (1), which allows differentiation from the similar pine shoot blight pathogen D. scrobiculata. Five isolates (three from Afghan pine and two from Austrian pine) were tested for pathogenicity by inoculation of potted 1-year-old Afghan pine seedlings obtained from the Texas Forest Service Nursery. Elongating terminal shoots were wounded by removing a needle pair approximately 2 cm below the shoot apex. A 4-mm-diameter plug cut from an actively growing culture on water agar (WA) was placed fungus side down on the wound. Noncolonized WA plugs were placed on similarly wounded control seedlings. Nonwounded control seedlings also were used. Parafilm was wrapped around the shoots to hold the agar plugs in place and was removed 2 weeks later. Each treatment was applied to four seedlings. Five weeks after inoculation, 9 of the 20 inoculated seedlings (including at least one inoculated with each isolate) exhibited dieback of shoot tips. One wounded control seedling exhibited slight tip dieback, no other nonwounded or wounded control seedlings developed symptoms. Segments of shoots were harvested, surface disinfested, and incubated on WA to determine the presence of the pathogen. The pathogen was reisolated from 11 of the 20 inoculated seedlings but not from any control seedlings. To our knowledge, this is the first report of D. pinea as a cause of shoot blight of Afghan pine and the first substantiated report of the occurrence of D. pinea in Texas. Although widely distributed in much of eastern North America, reports of the presence of D. pinea in the other southern gulf coast states of Alabama, Louisiana, and Mississippi, as well as the western states of Colorado, New Mexico, and Utah, are lacking. Reference: (1) D. R. Smith and G. R. Stanosz. Plant Dis. 90:307, 2006.

Species-specific PCR assay for L. infantum/L. donovani discrimination

Acta Tropica ◽  
2006 ◽  
Vol 100 (3) ◽  
pp. 241-245 ◽  
Author(s):  
Mallorie Hide ◽  
Anne-Laure Bañuls
Keyword(s):  
Pcr Assay ◽  
Specific Pcr ◽  
Specific Pcr Assay ◽  
Species Specific

scholarly journals Zoonotic Microsporidia in Wild Lagomorphs in Southern Spain

Animals ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 2218
Author(s):  
Anabel Martínez-Padilla ◽  
Javier Caballero-Gómez ◽  
Ángela Magnet ◽  
Félix Gómez-Guillamón ◽  
Fernando Izquierdo ◽  
...  
Keyword(s):  
Fungal Pathogens ◽  
Enterocytozoon Bieneusi ◽  
Southern Spain ◽  
Health Concern ◽  
Wild Rabbit ◽  
Specific Pcr ◽  
Encephalitozoon Intestinalis ◽  
Wild Lagomorphs ◽  
Species Specific ◽  
Iberian Hare

Microsporidia are obligate intracellular protist-like fungal pathogens that infect a broad range of animal species, including humans. This study aimed to assess the presence of zoonotic microsporidia (Enterocytozoon bieneusi, Encephalitozoon intestinalis, Encephalitozoon hellem, and Encephalitozoon cuniculi) in organ meats of European wild rabbit (Oryctolagus cuniculus) and Iberian hare (Lepus granatensis) consumed by humans in Spain. Between July 2015 and December 2018, kidney samples from 383 wild rabbits and kidney and brain tissues from 79 Iberian hares in southern Spain were tested by species-specific PCR for the detection of microsporidia DNA. Enterocytozoon bieneusi infection was confirmed in three wild rabbits (0.8%; 95% CI: 0.0–1.7%) but not in hares (0.0%; 95% CI: 0.0–4.6%), whereas E. intestinalis DNA was found in one wild rabbit (0.3%; 95% CI: 0.0–0.8%) and three Iberian hares (3.8%; 95% CI: 0.0–8.0%). Neither E. hellem nor E. cuniculi infection were detected in the 462 (0.0%; 95% CI: 0.0–0.8%) lagomorphs analyzed. The absence of E. hellem and E. cuniculi infection suggests a low risk of zoonotic foodborne transmission from these wild lagomorph species in southern Spain. To the authors’ knowledge, this is the first report of E. intestinalis infection in wild rabbits and Iberian hares. The presence of E. bieneusi and E. intestinalis in organ meats from wild lagomorphs can be of public health concern. Additional studies are required to determine the real prevalence of these parasites in European wild rabbit and Iberian hare.

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.

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