scholarly journals Detecting Heterobasidion irregulare in Minnesota and Assessment of Indigenous Fungi on Pines

Forests ◽  
2021 ◽  
Vol 12 (1) ◽  
pp. 57
Author(s):  
Eric Otto ◽  
Benjamin Held ◽  
Samuel Redford ◽  
Robert A. Blanchette

Heterobasidion irregulare is one of the most problematic forest pathogens in the northern hemisphere, but has only been found relatively recently in the north central United States. Discovered in Wisconsin in 1993, but probably established sometime before that, it quickly spread throughout the state. In November 2014, it was found in southeastern Minnesota. Field surveys were then conducted throughout Minnesota with the focus in the southeast near the initial discovery. To find additional infection sites, surveys were conducted with accompanying aerial imagery of red pine (Pinus resinosa Aiton) stands that were previously thinned. Samples were collected from selected sites with dead and dying trees as well as samples from stumps in recently thinned pine stands. These samples were processed first with a nested polymerase chain reaction (PCR) protocol, which was replaced by a real-time PCR assay after its development. No samples tested positive for H. irregulare using these methods and no cultures from isolations were obtained outside the original infection area. Other indigenous fungi were also identified. The majority were wood decay fungi in the Basidiomycota. A spore collection study was also conducted after field surveys. Automated rotary arm spore collectors were used and assayed with an ITS TaqMan real-time PCR assay. Collectors were placed strategically in different areas of Minnesota. A positive control was used in an infected red pine plantation in Wisconsin and this location had the highest number of spores trapped, with 63,776 over a week period. Spores of H. irregulare were detected at several sites in Minnesota, with the highest spore total observed in traps at 413 over a week period. All other locations sampled also had some spores collected except Itasca State Park located in northwestern Minnesota. The weekly deposition of spores ranged from 0 to 1.26 m−2 h−1. Low spore levels occurring in Minnesota indicate that some spores are present, but they are currently being detected in amounts that may not be sufficient for colonization to be successful.

2016 ◽  
Vol 29 (4) ◽  
pp. 499-507 ◽  
Author(s):  
Amaresh Das ◽  
Gordon Ward ◽  
Andre Lowe ◽  
Lizhe Xu ◽  
Karen Moran ◽  
...  

Parapoxviruses (PaPVs) cause widespread infections in ruminants worldwide. All PaPVs are zoonotic and may infect humans after direct or indirect contact with infected animals. Herein we report the development and validation of a highly sensitive real-time PCR assay for rapid detection of PaPVs. The new assay (referred to as the RVSS assay) was specific for PaPVs only and had no cross-reactivity against other pox viruses. Using a recombinant plasmid as positive control, the analytical sensitivity of the assay was determined to be 16 genome copies of PaPV per assay. The amplification efficiency estimate (91–99%), the intra- and interassay variability estimate (standard deviation [SD]: 0.28–1.06 and 0.01–0.14, respectively), and the operator variability estimate (SD: 0.78 between laboratories and 0.28 between operators within a laboratory) were within the acceptable range. The diagnostic specificity was assessed on 100 specimens from healthy normal animals and all but 1 tested negative (99%). The diagnostic sensitivity (DSe) was assessed on 77 clinical specimens (skin/scab) from infected sheep, goats, and cattle, and all tested positive (100%). The assay was multiplexed with beta-actin as an internal positive control, and the multiplex assay exhibited the same DSe as the singleplex assay. Further characterization of the PaPV specimens by species-specific real-time PCR and nucleotide sequencing of the PCR products following conventional PCR showed the presence of Orf virus not only in sheep and goats but also in 1 bovid. The validated RVSS assay demonstrated high specificity, sensitivity, reproducibility, and ruggedness, which are critical for laboratory detection of PaPVs.


2004 ◽  
Vol 50 (4) ◽  
pp. 269-278 ◽  
Author(s):  
Ann C Grimm ◽  
Jennifer L Cashdollar ◽  
Frederick P Williams ◽  
G Shay Fout

Astrovirus is a common cause of gastroenteritis in humans that has been determined to be responsible for outbreaks of illness in several countries. Since astrovirus can be waterborne, it is important to be able to identify this virus in environmental water. We have developed and optimized a reverse transcription – polymerase chain reaction (RT–PCR) method that was able to amplify all eight astrovirus serotypes in a single reaction. In addition, a positive control construct was designed so that any inhibitors of this astrovirus assay could be detected. The assay was adapted for use in a real-time PCR assay and the sensitivity of these two methods was compared. The real-time assay was then combined with CaCo2 cell culture to produce an integrated cell culture/RT–PCR (ICC/RT–PCR) assay that was able to detect low levels of astrovirus after an incubation of 7 days or less. Also, the sensitivity of the ICC/RT–PCR assay was compared with RT–PCR alone. The methods were used to detect astrovirus in acute phase illness stool samples as well as in a water sample spiked with astrovirus.Key words: astrovirus, RT–PCR, real-time PCR, ICC/RT–PCR, environmental water.


2016 ◽  
Vol 55 (2) ◽  
pp. 180-184 ◽  
Author(s):  
Solène Le Gal ◽  
Florence Robert-Gangneux ◽  
Yann Pépino ◽  
Sorya Belaz ◽  
Céline Damiani ◽  
...  

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