PCR as an ecological tool to determine the establishment and persistence of Rhizobium strains introduced into the field as seed inoculant Diane

1998 ◽  
Vol 49 (6) ◽  
pp. 923 ◽  
Author(s):  
M. Hebb ◽  
Alan E. Richardson ◽  
R. Reid ◽  
John Brockwell
Keyword(s):  
Rhizobium Leguminosarum ◽  
Field Experiments ◽  
Rhizobium Meliloti ◽  
Pcr Amplification ◽  
Field Trials ◽  
Rhizobium Strains ◽  
Inoculant Strain ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Field Plots

Appraisal of the establishment and persistence of inoculant strains, and the diversity of the rhizobial populations in 2 field experiments, required the precise characterisation of isolates from individual nodules. The polymerase chain reaction (PCR) method of generating randomly amplified polymorphic DNA (RAPD) from simple bacterial cell lysates was applied to nodule isolates obtained from field plots of clover (Trifolium spp.) and medic (Medicago spp.) plants inoculated with Rhizobium leguminosarum bv. Trifolium and Rhizobium meliloti. Comparison of the PCR method with gel immune diffusion serology was applied to selected nodule isolates obtained from the clover trial. Agreement between the methods was high (97·6%). Further characterisation of nodule isolates from the 2 field trials proceeded using PCR amplification profiles only, which allowed a large number of isolates to be quickly and precisely identified. Mean recovery of inoculant strains in the first season of the clover trial was 76·5% across 10 hosts, and 45·3% in the second season sampling. Recovery of inoculant strains in the medic trial was assessed only during the second season of growth, which recorded a mean recovery of 53% across 8 hosts. In addition to providing a rapid and reliable means of identifying inoculant strains of R. leguminosarum bv. Trifolium and R. meliloti directly in association with a range of different host plants, the PCR approach also allowed inoculant strain types to be readily identified when recovered as isolates from plots in which they had not been introduced. Analysis of the non-inoculant strains by PCR also indicated that the naturalised populations of rhizobia at both sites were highly diverse.

scholarly journals PCR Detection and RFLP Differentiation of Botrytis Species Associated with Neck Rot of Onion

Plant Disease ◽  
2002 ◽  
Vol 86 (6) ◽  
pp. 682-686 ◽  
Author(s):  
Karsten Nielsen ◽  
David S. Yohalem ◽  
Dan Funck Jensen
Keyword(s):  
Pcr Amplification ◽  
Pcr Detection ◽  
Sequence Characterized Amplified Region ◽  
Specific Pcr ◽  
Isolation Techniques ◽  
Detection And Identification ◽  
Fungal Dna ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Direct Amplification

Botrytis aclada and other Botrytis spp. can cause neck rot on onions, a storage disease that normally is very difficult to detect at harvest using traditional isolation techniques. Sequence characterized amplified region primers (BA2f/BA1r) were designed based on a previously cloned and amplified DNA fragment for direct amplification of isolates of Botrytis spp. associated with neck rot of onions. Digestion of the polymerase chain reaction (PCR) amplification product with the restriction enzyme ApoI makes it possible to distinguish the five groups: Botrytis aclada types AI and AII (B. allii); B. byssoidea; B. squamosa; and B. cinerea. The detection limit was 1 to 10 pg of pure fungal DNA. It was possible to detect B. aclada with the PCR method in artificially inoculated onion bulb tissue and in mature onion leaves showing no symptoms of the disease. The availability of a sensitive and specific PCR detection and identification method for Botrytis onion neck rot pathogens should facilitate ecological studies of this group of onion pathogens.

Molecular differentiation of Trichinella spiralis, T. pseudospiralis, T. papuae and T. zimbabwensis by pyrosequencing

2013 ◽  
Vol 89 (1) ◽  
pp. 118-123 ◽  
Author(s):  
L. Sadaow ◽  
C. Tantrawatpan ◽  
P.M. Intapan ◽  
V. Lulitanond ◽  
T. Boonmars ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Trichinella Spiralis ◽  
Large Subunit ◽  
Pcr Amplification ◽  
Epidemiological Studies ◽  
Chain Reaction ◽  
Target Region ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Zoonotic Parasites

AbstractNematodes of the genus Trichinella which infect wildlife and domestic animals show a cosmopolitan distribution. These zoonotic parasites are the aetiological agents of a severe human disease, trichinellosis. Twelve taxa are recognized in the Trichinella genus, but they cannot be identified by morphology since they are sibling species/genotypes. For epidemiological studies, it is extremely important to identify each taxon since they have different distribution areas and host ranges. In the present study, polymerase chain reaction (PCR) amplification of the mitochondrial large subunit ribosomal RNA (lsu-RNA) gene coupled with a pyrosequencing technique was developed to distinguish among four Trichinella species: Trichinella spiralis, T. pseudospiralis, T. papuae and T. zimbabwensis. A PCR method was used to amplify the lsu-RNA of Trichinella sp. larvae in mouse muscles and single larvae collected from infected muscles by digestion. The results show that the four Trichinella species can be distinguished by using 26 nucleotides in the target region and the method is sensitive enough to identify individual larvae. The pyrosequencing provides a simple, rapid and high-throughput tool for the differentiation of Trichinella species.

scholarly journals Differential Cultivars and Criteria for Evaluating Resistance to Rhizoctonia solani in Seedling Brassica oleracea

Plant Disease ◽  
1997 ◽  
Vol 81 (8) ◽  
pp. 946-952 ◽  
Author(s):  
Anthony P. Keinath ◽  
Mark W. Farnham
Keyword(s):  
Rhizoctonia Solani ◽  
Brassica Oleracea ◽  
Field Experiments ◽  
Field Trials ◽  
Plant Weight ◽  
Progress Curve ◽  
Growth Room ◽  
Differential Cultivars ◽  
Field Plots ◽  
Sand Cultures

Growth-room and field experiments were conducted to develop methods of studying resistance in Brassica oleracea crops to Rhizoctonia solani anastomosis groups (AG) 2-1 and 4, causal agents of wirestem. Seedlings of 12 cultivars (3 each of broccoli, cauliflower, cabbage, and collard) at the four- to five-leaf stage were transplanted to trays in a growth room and covered with steamed soil infested with cornmeal-sand cultures or sclerotia of R. solani or to fumigated field plots infested with sclerotia. The percent healthy, diseased, and dead plants was assessed every 3 to 5 days for 2 weeks in the growth room and for 3 weeks in field trials. At harvest, plants were dug out with roots intact and rated for wirestem severity. In most experiments, wirestem incidence (percent diseased and dead plants) stabilized within 10 to 14 days after inoculation. Inoculation with cornmeal-sand cultures of both AGs and sclerotia of AG-4 resulted in severe wirestem in all experiments, whereas sclerotia of AG-2-1 were less effective in the growth room and not effective in the field. Percent healthy and surviving (healthy plus diseased) plants, area under the disease progress curve (AUDPC), and wirestem severity all separated the most susceptible from the partially resistant cultivars more consistently than fresh weight of inoculated plants expressed as a percentage of noninoculated plant weight. Wirestem severity and AUDPC were always negatively and significantly (P ≤ 0.01) correlated with percent healthy plants. Although genotype by environment interactions were observed, the cauliflower cvs. Snowcone and Snow Crown were severely diseased in all experiments, whereas collard cv. Blue Max was consistently and significantly (P ≤ 0.05) less diseased.

Characterization of trypanosome infections by polymerase chain reaction (PCR) amplification in wild tsetse flies in Cameroon

Parasitology ◽  
1998 ◽  
Vol 116 (6) ◽  
pp. 547-554 ◽  
Author(s):  
I. MORLAIS ◽  
P. GREBAUT ◽  
J. M. BODO ◽  
S. DJOHA ◽  
G. CUNY
Keyword(s):  
Polymerase Chain Reaction ◽  
Infection Rate ◽  
Forest Type ◽  
Pcr Amplification ◽  
Tsetse Flies ◽  
Microscopical Examination ◽  
Chain Reaction ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Primer Sets

The polymerase chain reaction (PCR) method was used to characterize trypanosome infections in tsetse flies from 3 sleeping sickness foci in Cameroon. The predominant tsetse species found was Glossina palpalis palpalis. An average infection rate of 12·1% was revealed by microscopical examination of 888 non-teneral tsetse flies. PCR amplification analyses for trypanosome identification were carried out on 467 flies, with primer sets specific for Trypanosoma (Trypanozoon) brucei s.l., T. (Duttonella) vivax, T. (Nannomonas) simiae and forest type T. (Nannomonas) congolense. Of 467 flies 93 were positive by microscopical analysis while PCR succeeded in identifying 89 positive flies. Of the PCR-positive flies 34 (38·2%) were negative by microscopical examination. PCR amplification, when compared to the parasitological technique, gave a higher estimate of infection rate of trypanosomes in natural tsetse populations. The PCR technique did, however, fail to identify 40·9% (38/93) of the parasitologically positive flies. The reasons for this failure are discussed. The overall prevalence of mixed infections, assessed by PCR, was 37·1%; the majority (72·7%) involved T. brucei and forest type T. congolense.

scholarly journals A Set of Plasmid-Based Modules for Easy Switching of C-Terminal Epitope Tags in Saccharomyces cerevisiae

2021 ◽  
Vol 9 (12) ◽  
pp. 2505
Author(s):  
Hiroki Hayashi ◽  
Tsutomu Kishi
Keyword(s):  
Dna Sequences ◽  
Affinity Purification ◽  
Pcr Amplification ◽  
Restriction Enzymes ◽  
Epitope Tagging ◽  
One Step ◽  
Pcr Method ◽  
Polymerase Chain ◽  
The One ◽  
Step Polymerase Chain Reaction

Epitope tagging is a powerful strategy for analyzing the functions of targeted proteins. The use of this strategy has become more convenient with the development of the epitope switch, which is another type of epitope tagging designed to convert the previously tagged epitopes on the chromosome to other epitopes of interest. Various modules for C-terminal epitope switching have been developed and amplified using the one-step polymerase chain reaction (PCR) method before transformation. However, PCR amplification occasionally generates mutations that affect the fidelity of epitope switching. Here, we constructed several plasmids to isolate modules for epitope switching through digestion by restriction enzymes. The isolated modules contained DNA sequences for homologous recombination, various epitopes (13×Myc, 6×HA, GFP, Venus, YFP, mCherry, and CFP), and a transformation marker (Candida glabrata LEU2). The restriction enzyme-digested plasmids were used to directly transform the cells for epitope switching. We demonstrate the efficient and accurate switching of the MX6 module-based C-terminal tandem affinity purification tags to each aforementioned epitope. We believe that our plasmids can serve as powerful tools for the functional analysis of yeast proteins.

scholarly journals Identification and Prevalence of Botrytis spp. from Blackberry and Strawberry Fields of the Carolinas

Plant Disease ◽  
2012 ◽  
Vol 96 (11) ◽  
pp. 1634-1637 ◽  
Author(s):  
Xingpeng Li ◽  
Dolores Fernández-Ortuño ◽  
Wenxuan Chai ◽  
Fei Wang ◽  
Guido Schnabel
Keyword(s):  
North Carolina ◽  
South Carolina ◽  
Pcr Amplification ◽  
Gray Mold ◽  
Chain Reaction ◽  
Reverse Primer ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Prevalent Species ◽  
Reference Strains

Gray mold disease of blackberry and strawberry is caused by Botrytis cinerea and B. caroliniana in the southeastern United States. In this study, methods to distinguish both species were established and their prevalence was determined in commercial blackberry and strawberry fields. Using DNA from B. cinerea and B. caroliniana reference strains, a species-differentiating polymerase chain reaction (PCR) amplification was developed that amplified G3PDH gene fragments of two different sizes depending on the species. The PCR is performed with three primers (two species-differentiating forward primers and one universal reverse primer) and amplified a 238-bp product from B. cinerea and a 536-bp fragment from B. caroliniana reference isolates. A total of 400 Botrytis isolates were collected from 6 commercial blackberry and 11 strawberry fields of the Carolinas and identified to the species level by the new PCR method. Both Botrytis spp. were identified in blackberry and strawberry fields, but B. caroliniana was less common than B. cinerea. Only 33 of 202 isolates from blackberry fields were identified as B. caroliniana, and the majority of these isolates came from two fields in South Carolina. Only 1 of 198 isolates from strawberries was identified as B. caroliniana, and this isolate was found in central North Carolina. B. cinerea but not B. caroliniana isolates sporulated on potato dextrose agar and Kings medium B. Our results show that B. cinerea and B. caroliniana coexist in at least some commercial blackberry and strawberry fields of the Carolinas, with B. cinerea being the more prevalent species.

Effects of inoculation method and size of Rhizobium meliloti population in the soil on nodulation of alfalfa

1992 ◽  
Vol 72 (1) ◽  
pp. 57-67 ◽  
Author(s):  
Wendell A. Rice ◽  
Perry E. Olsen
Keyword(s):  
Field Experiments ◽  
Indigenous Population ◽  
Rhizobium Meliloti ◽  
Enzyme Linked Immunosorbent Assay ◽  
Root Weight ◽  
Nodule Occupancy ◽  
Ph Tolerance ◽  
Inoculant Strain ◽  
Shoot Weight ◽  
Inoculation Treatment

Field experiments were conducted with alfalfa (Medicago sativa L. ’Peace’) planted on a Black Solod (Landry CL) with pH 5.8 in 3 consecutive years. The same experiment was conducted at two adjacent sites in each year. The population of Rhizobium meliloti in the soil was modified at one site by applying and incorporating a suspension of an indigenous isolate to increase the size to 105 g−1. The other site was on unamended soil with a normal indigenous population of less than 102 g−1. Granular inoculant was applied with the seed or below the seed, or, granular or liquid inoculant was applied beside the row after stand establishment. The soil-placed inoculant treatments were compared with an uninoculated control and a standard seed-applied inoculation treatment. All inoculants were prepared with Rhizobium meliloti NRG-61 which had been selected for low-pH tolerance. Root weight, shoot weight, nodule weight, nodule numbers and strain occupancy of nodules were measured in September of the establishment year and again the following June. Granular inoculant applied with or below the seed resulted in greater nodule weights, nodule numbers and percent nodule occupancy at both sites. Granular inoculant applied with or below the seed was more effective at the site with the normal indigenous population of R. meliloti than at the site with the modified population. When granular inoculants were applied, nodule weights were lower on the site with the modified indigenous population than on the soil with the normal indigenous population. Granular inoculants resulted in significant yield increases over the standard seed-applied inoculant only at the site with the normal indigenous population. There were no substantial differences between the sites in the proportion of nodules occupied by the inoculant strain. These results show that strain occupancy measurements do not necessarily reflect the beneficial effect of inoculation. Key words: Enzyme-linked immunosorbent assay (ELISA), granular inoculant, Rhizobium inoculant, seed inoculant, soil inoculant

scholarly journals Gaeumannomyces graminis vars. avenae, graminis, and tritici Identified Using PCR Amplification of Avenacinase-like Genes

Plant Disease ◽  
2002 ◽  
Vol 86 (6) ◽  
pp. 652-660 ◽  
Author(s):  
Sansanalak Rachdawong ◽  
Carole L. Cramer ◽  
Elizabeth A. Grabau ◽  
Verlyn K. Stromberg ◽  
George H. Lacy ◽  
...  
Keyword(s):  
Pcr Amplification ◽  
Rapid Identification ◽  
Gaeumannomyces Graminis ◽  
Specific Primers ◽  
Single Tube ◽  
Pcr Products ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Cereal Fields ◽  
Take All

Identifying take-all pathogens, Gaeumannomyces graminis varieties avenae (Gga), graminis (Ggg), and tritici (Ggt), is difficult. Rapid identification is important for development of disease thresholds. We developed a single-tube, polymerase chain reaction (PCR) method differentiating among Gga, Ggg, and Ggt. Nucleotide base sequence analyses of avenacinase-like genes from Gga, Ggg, and Ggt isolates provided the basis for designing variety-specific primers. Sequences from Ggg and Ggt were highly related (99% identity), but Gga sequences were <95% identical to Ggg and Ggt sequences. Three 5′ primers specific for Gga, Ggt, and Ggg and a single 3′ common primer allowed amplification of variety-specific fragments of 617, 870, and 1,086 bp, respectively. Each 5′ primer was specific in mixed populations of primers and templates. No PCR products were amplified from related fungi including Gaeumannomyces cylindrosporus and Phialophora spp. We surveyed 16 putative Ggt isolates using our assay; nine produced Ggt-specific fragments and seven produced Ggg-specific fragments. Five Gga isolates produced Gga-specific fragments. However, Gga- and Ggt-specific fragments were observed from a sixth Gga isolate, RB-W, which indicates a mixed culture or a heterokaryon. Our single-tube, PCR method rapidly differentiates among the important take-all pathogens commonly encountered together in cereal fields.

Characterization of a cDNA for Rhesus Monkey Protein C Inhibitor – Evidence for N-Terminal Involvement in Heparin Stimulation

1995 ◽  
Vol 74 (04) ◽  
pp. 1079-1087 ◽  
Author(s):  
Klaus-P Radtke ◽  
José A Fernández ◽  
Bruno O Villoutreix ◽  
Judith S Greengard ◽  
John H Griffin
Keyword(s):  
Rhesus Monkey ◽  
Protein C ◽  
Activated Protein C ◽  
Pcr Amplification ◽  
Amino Acid Sequences ◽  
Heparin Binding ◽  
Reactive Center ◽  
Protein C Inhibitor ◽  
Polymerase Chain

SummarycDNAs for protein C inhibitor (PCI) were cloned from human and rhesus monkey 1 liver RNAs by reverse transcription and polymerase chain reaction (PCR) amplification. Sequencing showed that rhesus monkey and human PCI cDNAs were 93% identical. Predicted amino acid sequences differed at 26 of 387 residues. Pour of these differences (T352M, N359S, R362K, L3631) were in the reactive center loop that is important for inhibitory specificity, and two were in the N-terminal helix (M8T, E13K) that is implicated in glycosaminoglycan binding. PCI in human or rhesus monkey plasma showed comparable inhibitory activity towards human activated protein C in the presence of 10 U/ml heparin. However, maximal acceleration of the inhibition of activated protein C required 5-fold lower heparin concentration for rhesus monkey than for human plasma, consistent with the interpretation that the additional positive charge (E13K) in a putative-heparin binding region increased the affinity for heparin.

Investigation of duck plague virus in hoar areas of Bangladesh

2020 ◽  
Vol 26 (1-2) ◽  
pp. 73-78
Author(s):  
A Hossen ◽  
MH Rahman ◽  
MZ Ali ◽  
MA Yousuf ◽  
MZ Hassan ◽  
...  
Keyword(s):  
Cytopathic Effect ◽  
Clinical Sample ◽  
Chorioallantoic Membrane ◽  
Clinical Samples ◽  
Isolation And Identification ◽  
Duck Plague Virus ◽  
Pcr Method ◽  
Polymerase Chain ◽  
The Individual ◽  
Free Ranging

Duck plague (DP) is the most important infectious disease of geese, ducks and free-ranging water birds. The present study was conducted to determine the prevalence of duck plague virus followed by isolation and identification. For these purposes, a total of 155 cloacal swabs samples were collected randomly from duck of different haor areas of Bangladesh including 45 (41 surveillance and 4 clinical) samples from Netrokona; 42 (40 surveillance and 2 clinical) samples from Kishoregonj; 30 samples from Brahmanbaria and 38 samples from Sunamganj. The samples were processed and pooled (1:5 ratio) for initial screening of target polymerase gene of duck plague virus by polymerase chain reaction (PCR) method. All the samples of a positive pool were then tested individually for identifying the individual positive samples. The result showed that out of 155 samples, 41 (26.45%) were found positive in which 17 were from Netrokona, where 15 (36.58%) were from surveillance samples and 2 (50%) were from clinical sample; 16 were from Kishoregonj, where 14 (35%) were from surveillance samples and 2 (100%) were from clinical sample; 2 (6.6%) were from Brahmanbaria and 5 (13.15%) were from Sunamganj. These positive samples were inoculated into 9-10 days embryonated duck eggs (EDE) through chorioallantoic membrane (CAM) route for the isolation of virus. The EDE died earlier was also chilled, and in a similar way, the CAMs were collected and again performed PCR for id entification of virus. Out of 41 PCR positive samples, 26 samples were isolated and reconfirmed by PCR. Subsequently, DPV was isolated in primary duck embryo fibroblasts cell culture and confirmed by observing cytopathic effect (CPE). Bang. J. Livs. Res. Vol. 26 (1&2), 2019: P. 73-78

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