scholarly journals PCR-Based Detection of Cephalosporium gramineum in Winter Wheat

Plant Disease ◽  
2012 ◽  
Vol 96 (3) ◽  
pp. 437-442 ◽  
Author(s):  
K. L. E. Klos ◽  
L. M. Vásquez-Siller ◽  
H. C. Wetzel ◽  
T. D. Murray
Keyword(s):  
Winter Wheat ◽  
Internal Transcribed Spacer ◽  
Seed Infection ◽  
Wheat Seed ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Internal Transcribed Spacer Region ◽  
Polymerase Chain ◽  
Cephalosporium Gramineum ◽  
Field Plots

A polymerase chain reaction (PCR) assay was developed amplifying a 496-bp fragment of the internal transcribed spacer region of Cephalosporium gramineum genomic DNA at concentrations of 100 fg/μl. Winter wheat seed and seedlings were collected from field plots where C. gramineum was present. Seed was tested by PCR using 20-seed samples bulked for DNA extraction. Estimates of seed infection, based on isolation of the pathogen on semiselective medium and PCR, were comparable at 0.18 and 0.13% of winter wheat ‘Stephens’ (P = 0.6042), and 0.45 and 0.58% of experimental line WA7970 (P = 0.5636), respectively. PCR differentiated between plants with well-developed symptoms of Cephalosporium stripe and noninoculated plants. Positive PCR was obtained from 22% of asymptomatic leaf blades from inoculated plants. We found no false positives when PCR and C. gramineum isolation on a semiselective medium were performed using tissue from the same leaf. The PCR assay has potential to diagnose Cephalosporium stripe disease prior to the appearance of symptoms. Negative PCR for some samples from which C. gramineum was isolated suggests that C. gramineum may be present below the level of detection in some asymptomatic leaves. This PCR assay may be useful for investigations into C. gramineum infection of wheat.

Minimal intraspecific variation in the sequence of the transcribed spacer regions of the ribosomal DNA of lake trout (Salvelinus namaycush)

Genome ◽  
1994 ◽  
Vol 37 (4) ◽  
pp. 664-671 ◽  
Author(s):  
Lang Zhuo ◽  
S. L. Sajdak ◽  
R. B. Phillips
Keyword(s):  
Polymerase Chain Reaction ◽  
Intraspecific Variation ◽  
Ribosomal Dna ◽  
Internal Transcribed Spacer ◽  
Lake Trout ◽  
Intergenic Spacer ◽  
Coding Region ◽  
Chain Reaction ◽  
Internal Transcribed Spacer Region ◽  
Polymerase Chain

Intraspecific variation in the sequence of the transcribed spacer regions of the ribosomal DNA (rDNA) in lake trout was examined by restriction mapping and sequencing of these regions amplified by the polymerase chain reaction. The length of the first internal transcribed spacer region (ITS-1) was 566 bases and the second internal transcribed spacer region (ITS-2) was 368 bases in lake trout. When the 1.4-kb region including the ITS-1, the 5.8S coding region, and the ITS-2 was amplified from 12 individuals from four populations and digested with eight different enzymes only one intraindividual polymorphism was found that occurred in each population. When the amplified ITS-1 region was sequenced from an additional 10 individuals from five populations, no interindividual variation was found in the sequence. A 6-kb portion of the rDNA repeat unit including 1.6 kb of the 18S coding region, the 5′ external spacer region (5′ ETS), and part of the adjacent intergenic spacer was cloned and a restriction map was prepared for these regions in lake trout. No intraspecific variation was found in the region adjacent to the 18S rDNA, which includes the 5′ ETS, although intraspecific and intraindividual length variation was found in the intergenic spacer region 3–6 kb from the 18S. Sequencing of a 609-b segment of the 5′ ETS adjacent to the 18S coding region revealed the presence of two 41-b repeats. The 198-b sequence between the repeats had some similarity to the 18S coding region of other fishes. Primers were designed for amplification of 559 b of the 5′ ETS using the polymerase chain reaction. No intraspecific variation in this region in lake trout was found when the DNA amplified from this region in 12 individuals from four populations was digested with eight restriction enzymes.Key words: ribosomal DNA, internal transcribed spacer regions, 5′ external spacer region, transcribed spacer, lake trout.

Russulaceous ectomycorrhizae of Abies lasiocarpa and Picea engelmannii

1997 ◽  
Vol 75 (11) ◽  
pp. 1843-1850 ◽  
Author(s):  
G. Kernaghan ◽  
R. S. Currah ◽  
R. J. Bayer
Keyword(s):  
Internal Transcribed Spacer ◽  
Fragment Length Polymorphism ◽  
Picea Engelmannii ◽  
Polymorphism Analysis ◽  
Abies Lasiocarpa ◽  
Tree Line ◽  
Chain Reaction ◽  
Internal Transcribed Spacer Region ◽  
Lactarius Deliciosus ◽  
Polymerase Chain

During a 3-year study of the ectomycorrhizal fungi of subalpine forests in the Front Ranges of the Canadian Rockies, species of Russula and Lactarius were conspicuous mycobionts of both erect and krummholz forms of Abies lasiocarpa (Hook.) Nutt. and Picea engelmannii Parry. Morphological identifications of Russulaceous mycorrhizae were confirmed by comparing polymerase chain reaction amplified ribosomal DNA (internal transcribed spacer region) with that of sporocarp tissue. Restriction fragment length polymorphism analysis using AluI, HhaI, HinfI, and RsaI gave a distinctive profile for each of 14 Russulaceous sporocarps and facilitated the identification of five mycorrhizae. Mantles formed by Lactarii (Lactarius alnicola, Lactarins caespitosus, and Lactarius deliciosus var. areolatus) exhibit characteristic laticifers and pigments comparable to the associated sporocarp. Those formed by species of Russula (R. brevipes and R. silvicola) bear distinctive cystidia or sulphovanillin-reactive cells. Key words: ITS, Lactarius, RFLP, Russula, subalpine, tree line.

scholarly journals Seed Transmission of Cephalosporium gramineum in Winter Wheat

Plant Disease ◽  
2006 ◽  
Vol 90 (6) ◽  
pp. 803-806 ◽  
Author(s):  
Timothy D. Murray
Keyword(s):  
Winter Wheat ◽  
Field Experiment ◽  
Seed Transmission ◽  
Selective Medium ◽  
Controlled Environment ◽  
Wheat Seed ◽  
Surface Disinfection ◽  
Infected Seed ◽  
Cephalosporium Gramineum ◽  
Field Plots

Although isolation of Cephalosporium gramineum from wheat (Triticum aestivum) seed has been reported, development of Cephalosporium stripe in plants from infected seed has not been demonstrated experimentally. Winter wheat seed was collected from three experimental field plots where Cephalosporium stripe was present, and C. gramineum was isolated from the seed following surface-disinfection and incubation on a semi-selective medium. C. gramineum was isolated from 0.10 to 0.88% of seed from 11 of 12 cultivars in a field experiment at Pullman, WA, and from 0.10 to 0.30% of seed from 3 of 4 genotypes in a field experiment at Fort Hall, ID; differences among cultivars were not significant in either experiment. C. gramineum was isolated from 0.35 and 0.55% of cv. Stephens plants with no symptoms and severe symptoms, respectively, from a uniform seeding in Pullman. Seed of the four genotypes from Fort Hall and Stephens from Pullman were grown under controlled environment in a soilless potting mix with no added inoculum and in which C. gramineum was not detected. Symptoms of Cephalosporium stripe developed in 0.08 and 0.17% of Stephens and breeding line 87-00314A plants, respectively, from Fort Hall, and from 0.18 and 0.55% of Stephens plants with no symptoms and severe symptoms, respectively. Although development of Cephalosporium stripe in plants grown from seed lots harvested from diseased plants was low, infected seed can provide an important source of inoculum for introducing the pathogen and initiating epidemics in areas where the pathogen did not occur previously.

scholarly journals Detection of the Pinewood Nematode, Bursaphelenchus xylophilus, Using a Real-Time Polymerase Chain Reaction Assay

2005 ◽  
Vol 95 (5) ◽  
pp. 566-571 ◽  
Author(s):  
A. X. Cao ◽  
X. Z. Liu ◽  
S. F. Zhu ◽  
B. S. Lu
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Bursaphelenchus Xylophilus ◽  
Pinewood Nematode ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Internal Transcribed Spacer Region ◽  
Significant Damage ◽  
Field Samples ◽  
Polymerase Chain

The pinewood nematode, Bursaphelenchus xylophilus, has caused significant damage to pine plantations both in East Asia and North America and is an important quarantine organism. A real-time polymerase chain reaction (PCR) assay was developed to detect B. xylophilus. A set of primers and probe specific for B. xylophilus was designed to target the ribosomal DNA internal transcribed spacer region. Optimal primer concentration, Mg2+ concentration, and extension temperature were 400 nM, 3.0 mM, and 60°C, respectively. The assay was highly specific and sensitive, detecting as little as 0.01 ng of B. xylophilus DNA. The real-time PCR assay also successfully detected B. xylophilus in field samples, and it should be very useful for quarantine purposes.

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