Precise Detection and Tracing of Trichoderma hamatum 382 in Compost-Amended Potting Mixes by Using Molecular Markers

1999 ◽  
Vol 65 (12) ◽  
pp. 5421-5426 ◽  
Author(s):  
Pervaiz A. Abbasi ◽  
Sally A. Miller ◽  
Tea Meulia ◽  
Harry A. J. Hoitink ◽  
Jin-Man Kim
Keyword(s):  
Genomic Dna ◽  
Biocontrol Agent ◽  
Rapd Analysis ◽  
Simple Procedure ◽  
Low Molecular Weight ◽  
Dna Fragments ◽  
Pcr Assay ◽  
Sequence Characterized Amplified Region ◽  
Random Primers ◽  
Trichoderma Hamatum

ABSTRACT Randomly amplified polymorphic DNA (RAPD) analysis and the PCR assay were used in combination with dilution plating on a semiselective medium to detect and enumerate propagules of Trichoderma hamatum 382, a biocontrol agent utilized in compost-amended mixes. Distinct and reproducible fingerprints were obtained upon amplification of purified genomic DNA of T. hamatum 382 with the random primers OPE-16, OPH-19, and OPH-20. Three amplified DNA fragments of 0.35 (OPE-160.35), 0.6 (OPH-190.6), and 0.65 (OPH-200.65) kb were diagnostic for T. hamatum 382, clearly distinguishing it from 53 isolates of four other Trichoderma spp. tested. Some isolates of T. hamatum shared these low-molecular-weight fragments with T. hamatum 382. However, RAPD analysis of isolates of T. hamatum with all three random primers used in consecutive PCR tests distinguishedT. hamatum 382 from other isolates of T. hamatum. These three RAPD amplicons were cloned and sequenced, and pairs of oligonucleotide primers for each cloned fragment were designed. Use of the primers in the PCR assay resulted in the amplification of DNA fragments of the same size as the cloned RAPD fragments from genomic DNA of T. hamatum 382. A combination of dilution plating on a semiselective medium forTrichoderma spp. and PCR, with the RAPD primers OPH-19, OPE-16, and OPH-20 or the three sequence-characterized primers, was used successfully to verify the presence of T. hamatum 382 propagules in nine different soil, compost, and potting mix samples. All 23 Trichoderma isolates recovered on semiselective medium from commercial potting mixes fortified with T. hamatum 382 were identified as T. hamatum 382, whereas 274 Trichoderma isolates recovered from the other nine samples were negative in the PCR assay. Thus, this highly specific combination of techniques allowed detection and enumeration of propagules of T. hamatum 382 in fortified compost-amended potting mixes. Sequence-characterized amplified region markers also facilitated the development of a very simple procedure to amplify DNA of T. hamatum 382 directly from fortified compost-amended potting mixes.

scholarly journals Diversitatea moleculară a două ecotipuri de Datura inoxia provenite din vestul şi estul României

2020 ◽  
Author(s):  
Cristina Chelu ◽  
◽  
Carmen Varlam ◽  
Gheorghe Titescu ◽  
Gallia Butnaru ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Genetic Similarity ◽  
Molecular Diversity ◽  
Rapd Analysis ◽  
Genomic Variation ◽  
Origin And Evolution ◽  
Random Primers ◽  
Datura Inoxia

Molecular Diversity of two Ecotypes of Datura inoxia Originating from Western and Eastern Romania. To characterize genomic variation among genotypes, we have performed RAPD analysis using ten random primers. The results yielded 88 bands out of which 39 were polymorphic. The primers US1 and US7 showed 87.71% and 72.72% polymorphism respectively. The least polymorphism was shown by primer US9 (12.50%). The primer US15 did not produce any bands suggesting the absence of matching sequences in the genomic DNA. The dendrogram classified ecotypes into two clusters (A and B); cluster B possess three sub-clusters: B1 - Socodor 2; B2 - Flamura 1 and Flamura 2, and B3 - Flamura 3. Overall, the values of genetic similarity between ecotypes were low pointing out their particular origin and “evolution”.

scholarly journals Application of Random Amplified Polymorphic DNA Analysis in Identifying Phellinus Igniarius Strains

2007 ◽  
Vol 26 (4) ◽  
pp. 289-293
Author(s):  
Ping Du ◽  
Yan-Qiu Chen ◽  
Chang-Tian Li ◽  
Yu Li
Keyword(s):  
Cluster Analysis ◽  
Dna Analysis ◽  
Rapd Analysis ◽  
Random Amplified Polymorphic Dna ◽  
Dna Fragments ◽  
Base Pairs ◽  
Phellinus Igniarius ◽  
Random Primers ◽  
Upgma Cluster Analysis ◽  
The Difference

Application of Random Amplified Polymorphic DNA Analysis in Identifying Phellinus Igniarius Strains Described in this paper, the random amplified polymorphic DNA (RAPD) analysis was conducted with 20 random primers in various strains of Phellinus igniarius collected from different localities. The results showed that 17 of the 20 random primers were polymorphic ones. The DNA bands derived from each primer amplifying in tested strains ranged from 10 to 33. The size of the amplified DNA fragments ranged from 250 to 2000 base pairs. Of each test primer, a wide variation in banding profiles was observed among the 7 strains of P. igniarius. A total of 377 band positions were scored for all of the tested strains, which differed significantly among the bands from different primers. UPGMA cluster analysis subdivided the tested strains into two groups, which was helpful to find out the difference among the tested strains and to distinguish them directly.

scholarly journals Molecular record for the first authentication of Isaria cicadae from Vietnam

2021 ◽  
Vol 16 (1) ◽  
pp. 711-718
Author(s):  
Thuan Duc Lao ◽  
Hanh Van Trinh ◽  
Loi Vuong ◽  
Luyen Tien Vu ◽  
Thuy Ai Huyen Le ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Genomic Dna ◽  
Morphological Analysis ◽  
Phylogenetic Analyses ◽  
Neighbor Joining ◽  
Pcr Assay ◽  
Parsimony Method ◽  
Maximum Parsimony Method ◽  
Cordyceps Cicadae ◽  
High Bootstrap

Abstract The entomopathogenic fungus T011, parasitizing on nymph of Cicada, collected in the coffee garden in Dak Lak Province, Vietnam, was preliminarily morphologically identified as Isaria cicadae, belonged to order Hypocreales and family Clavicipitaceae. To ensure the authenticity of T011, phylogenetic analysis of the concatenated set of multiple genes including ITS, nrLSU, nrSSU, Rpb1, and Tef1 was applied to support the identification. Genomic DNA was isolated from dried sample T011. The PCR assay sequencing was applied to amplify ITS, nrLSU, nrSSU, Rpb1, and Tef1 gene. For phylogenetic analysis, the concatenated data of both target gens were constructed with MEGAX with a 1,000 replicate bootstrap based on the neighbor-joining, maximum likelihood, maximum parsimony method. As the result, the concatenated data containing 62 sequences belonged to order Hypocreales, families Clavicipitaceae, and 2 outgroup sequences belonged to order Hypocreales, genus Verticillium. The phylogenetic analysis results indicated that T011 was accepted at subclade Cordyceps and significantly formed the monophyletic group with referent Cordyceps cicadae (Telemorph of Isaria cicadae) with high bootstrap value. The phylogenetically analyzed result was strongly supported by our morphological analysis described as the Isaria cicadae. In summary, phylogenetic analyses based on the concatenated dataset were successfully applied to strengthen the identification of T011 as Isaria cicadae.

Enzymatic amplification and characterization of large DNA fragments from genomic DNA

Gene ◽  
1988 ◽  
Vol 71 (1) ◽  
pp. 211-216 ◽  
Author(s):  
Phouthone Keohavong ◽  
Cindy C. Wang ◽  
Rita S. Cha ◽  
William G. Thilly
Keyword(s):  
Genomic Dna ◽  
Dna Fragments ◽  
Enzymatic Amplification

scholarly journals The use of random amplified polymorphic DNA to evaluate the genetic variability of Ponkan mandarin (Citrus reticulata Blanco) accessions

2000 ◽  
Vol 23 (1) ◽  
pp. 169-172 ◽  
Author(s):  
Helvécio Della Coletta Filho ◽  
Marcos Antonio Machado ◽  
M. Luiza P.N. Targon ◽  
Jorgino Pompeu Jr.
Keyword(s):  
Genetic Variability ◽  
Rapd Analysis ◽  
Random Amplified Polymorphic Dna ◽  
Point Mutations ◽  
The Other ◽  
Citrus Reticulata ◽  
Random Primers ◽  
Citrus Reticulata Blanco ◽  
Ponkan Mandarin

RAPD analysis of 19 Ponkan mandarin accessions was performed using 25 random primers. Of 112 amplification products selected, only 32 were polymorphic across five accessions. The absence of genetic variability among the other 14 accessions suggested that they were either clonal propagations with different local names, or that they had undetectable genetic variability, such as point mutations which cannot be detected by RAPD.

scholarly journals Detection of Small Numbers of Campylobacter jejuni and Campylobacter coli Cells in Environmental Water, Sewage, and Food Samples by a Seminested PCR Assay

1999 ◽  
Vol 65 (4) ◽  
pp. 1636-1643 ◽  
Author(s):  
Astrid S. Waage ◽  
Traute Vardund ◽  
Vidar Lund ◽  
Georg Kapperud
Keyword(s):  
Campylobacter Jejuni ◽  
Simple Procedure ◽  
Environmental Water ◽  
Food Samples ◽  
Proteinase K ◽  
Coliform Bacteria ◽  
Intergenic Sequence ◽  
Campylobacter Coli ◽  
Pcr Assay ◽  
Seminested Pcr

ABSTRACT A rapid and sensitive assay was developed for detection of small numbers of Campylobacter jejuni and Campylobacter coli cells in environmental water, sewage, and food samples. Water and sewage samples were filtered, and the filters were enriched overnight in a nonselective medium. The enrichment cultures were prepared for PCR by a rapid and simple procedure consisting of centrifugation, proteinase K treatment, and boiling. A seminested PCR based on specific amplification of the intergenic sequence between the two Campylobacter flagellin genes, flaA andflaB, was performed, and the PCR products were visualized by agarose gel electrophoresis. The assay allowed us to detect 3 to 15 CFU of C. jejuni per 100 ml in water samples containing a background flora consisting of up to 8,700 heterotrophic organisms per ml and 10,000 CFU of coliform bacteria per 100 ml. Dilution of the enriched cultures 1:10 with sterile broth prior to the PCR was sometimes necessary to obtain positive results. The assay was also conducted with food samples analyzed with or without overnight enrichment. As few as ≤3 CFU per g of food could be detected with samples subjected to overnight enrichment, while variable results were obtained for samples analyzed without prior enrichment. This rapid and sensitive nested PCR assay provides a useful tool for specific detection of C. jejuni or C. coli in drinking water, as well as environmental water, sewage, and food samples containing high levels of background organisms.

scholarly journals Genetic profiling of mistletoe (Viscum album L.) using RAPD-analysis

2020 ◽  
Vol 26 ◽  
pp. 82-86
Author(s):  
Yu. O. Bilonozhko ◽  
A. N. Rabokon ◽  
A. S. PostovoitovA ◽  
L. O. Kalafat ◽  
S. M. PrivAlikhin ◽  
...  
Keyword(s):  
Rapd Markers ◽  
Rapd Analysis ◽  
Molecular Genetic ◽  
Viscum Album ◽  
Dna Fragments ◽  
Coniferous Species ◽  
Genetic Profiling ◽  
Random Amplification ◽  
Polymerase Chain ◽  
Genetic Profiles

Aim. The aim of this research was genetic profiling and identification of genetic differences between V. album speciments, growing on deciduous and coniferous species of woody plants using RAPD markers. Methods. The method of polymerase chain reaction (PCR) with random primers (Random Amplification of Polymorphic DNA - RAPD) was used. Amplified DNA fragments were fractionated by electrophoresis in non-denaturing polyacrylamide gel. DNA bands were detected using the staining with silver nitrate. Results. All the studied mistletoe samples were differentiated from each other, and their unique molecular genetic profiles were obtained. 241 amplified DNA fragments were detected in the range from 200 to 2000 bp, 152 fragments (63%) were polymorphic. The samples were divided into two separate groups depending on the type of host plant. Conclusions. The fact that the samples formed two separate clades confirms the assumption that mistletoe, which grow on pine and grow on maple, represents two separate subspecies of V. album. Keywords: Viscum album L., molecular genetic markers, polymorphism, RAPD.

Development of a fungal transformation system based on selection of sequences with promoter activity

1987 ◽  
Vol 7 (9) ◽  
pp. 3297-3305
Author(s):  
B G Turgeon ◽  
R C Garber ◽  
O C Yoder
Keyword(s):  
Genomic Dna ◽  
Pathogenic Fungus ◽  
Low Frequency ◽  
Hygromycin B ◽  
Fungal Transformation ◽  
Dna Fragments ◽  
Transformation System ◽  
Chromosomal Dna ◽  
General Utility ◽  
Novel Strategy

A novel strategy was used to develop a transformation system for the plant pathogenic fungus Cochliobolus heterostrophus. Sequences capable of driving the expression of a gene conferring resistance to the antibiotic hygromycin B in C. heterostrophus were selected from a library of genomic DNA fragments and used, with the selectable marker, as the basis for transformation. The library of random 0.5- to 2.0-kilobase-pair fragments of C. heterostrophus genomic DNA was inserted at the 5' end of a truncated, promoterless Escherichia coli hygromycin B phosphotransferase gene (hygB) whose product confers resistance to hygromycin B. C. heterostrophus protoplasts were transformed with the library and selected for resistance. Resistant colonies arose at low frequency. Each colony contained a transformation vector stably integrated into chromosomal DNA. When the transforming DNA was recovered from the genome and introduced into C. heterostrophus, resistant colonies appeared at higher frequency. We determined the sequences of two of the C. heterostrophus DNA fragments which had been inserted at the 5' end of hygB in the promoter library and found that both made translational fusions with hygB. One of the two fusions apparently adds 65 and the other at least 86 amino acids to the N-terminus of the hygB product. Plasmids containing hygB-C. heterostrophus promoter fusions can be used unaltered to drive hygB expression in several other filamentous ascomycetes. This approach to achieving transformation may have general utility, especially for organisms with relatively undeveloped genetics.

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