scholarly journals Detection and Quantification of Plectosphaerella cucumerina, a Potential Biological Control Agent of Potato Cyst Nematodes, by Using Conventional PCR, Real-Time PCR, Selective Media, and Baiting

2003 ◽  
Vol 69 (8) ◽  
pp. 4788-4793 ◽  
Author(s):  
S. D. Atkins ◽  
I. M. Clark ◽  
D. Sosnowska ◽  
P. R. Hirsch ◽  
B. R. Kerry
Keyword(s):  
Biological Control ◽  
Population Size ◽  
Real Time ◽  
Real Time Pcr ◽  
Biological Control Agent ◽  
Control Agent ◽  
Taqman Probe ◽  
Potato Cyst Nematodes ◽  
Cyst Nematodes ◽  
Plectosphaerella Cucumerina

ABSTRACT Potato cyst nematodes (PCN) are serious pests in commercial potato production, causing yield losses valued at approximately $300 million in the European Community. The nematophagous fungus Plectosphaerella cucumerina has demonstrated its potential as a biological control agent against PCN populations by reducing field populations by up to 60% in trials. The use of biological control agents in the field requires the development of specific techniques to monitor the release, population size, spread or decline, and pathogenicity against its host. A range of methods have therefore been developed to monitor P. cucumerina. A species-specific PCR primer set (PcCF1-PcCR1) was designed that was able to detect the presence of P. cucumerina in soil, root, and nematode samples. PCR was combined with a bait method to identify P. cucumerina from infected nematode eggs, confirming the parasitic ability of the fungus. A selective medium was adapted to isolate the fungus from root and soil samples and was used to quantify the fungus from field sites. A second P. cucumerina-specific primer set (PcRTF1-PcRTR1) and a Taqman probe (PcRTP1) were designed for real-time PCR quantification of the fungus and provided a very sensitive means of detecting the fungus from soil. PCR, bait, and culture methods were combined to investigate the presence and abundance of P. cucumerina from two field sites in the United Kingdom where PCN populations were naturally declining. All methods enabled differences in the activity of P. cucumerina to be detected, and the results demonstrated the importance of using a combination of methods to investigate population size and activity of fungi.

scholarly journals Development of a strain-specific real-time PCR assay for the detection and quantification of the biological control agent Fo47 in root tissues

2011 ◽  
Vol 322 (1) ◽  
pp. 34-40 ◽  
Author(s):  
Veronique Edel-Hermann ◽  
Sébastien Aimé ◽  
Christelle Cordier ◽  
Chantal Olivain ◽  
Christian Steinberg ◽  
...  
Keyword(s):  
Biological Control ◽  
Real Time ◽  
Real Time Pcr ◽  
Biological Control Agent ◽  
Control Agent ◽  
Pcr Assay ◽  
Root Tissues ◽  
Detection And Quantification

scholarly journals Assessment of the Environmental Fate of the Biological Control Agent of Fire Blight, Pseudomonas fluorescens EPS62e, on Apple by Culture and Real-Time PCR Methods

2006 ◽  
Vol 72 (4) ◽  
pp. 2421-2427 ◽  
Author(s):  
Marta Pujol ◽  
Esther Badosa ◽  
Charles Manceau ◽  
Emilio Montesinos
Keyword(s):  
Biological Control ◽  
Pseudomonas Fluorescens ◽  
Real Time ◽  
Real Time Pcr ◽  
Environmental Fate ◽  
Biological Control Agent ◽  
Field Trials ◽  
Control Agent ◽  
Cell Counting ◽  
Standard Curve

ABSTRACT The colonization of apple blossoms and leaves by Pseudomonas fluorescens EPS62e was monitored in greenhouse and field trials using cultivable cell counting and real-time PCR. The real-time PCR provided a specific quantitative method for the detection of strain EPS62e. The detection level was around 102 cells g (fresh weight)−1 and the standard curve was linear within a 5-log range. EPS62e actively colonized flowers reaching values from 107 to 108 cells per blossom. In apple flowers, no significant differences were observed between population levels obtained by real-time PCR and plating, suggesting that viable but nonculturable (VBNC) cells and residual nondegraded DNA were not present. In contrast, on apple leaves, where cultivable populations of EPS62e decreased with time, significant differences were observed between real-time PCR and plating. These differences indicate the presence of VBNC cells or nondegraded DNA after cell death. Therefore, the EPS62e population was under optimal conditions during the colonization of flowers but it was stressed and poorly survived on leaves. It was concluded that for monitoring this biological control agent, the combined use of cultivable cell count and real-time PCR is necessary.

Conventional and real-time PCR-based species identification and diversity of potato cyst nematodes (Globodera spp.) from Victoria, Australia

Nematology ◽  
2008 ◽  
Vol 10 (4) ◽  
pp. 471-478 ◽  
Author(s):  
Lila Nambiar ◽  
James Cunnington ◽  
Motiul Quader
Keyword(s):  
Sequence Analysis ◽  
Real Time ◽  
Real Time Pcr ◽  
Binding Sites ◽  
Sequence Variation ◽  
Control Strategies ◽  
Globodera Pallida ◽  
Pcr Analysis ◽  
Potato Cyst Nematodes ◽  
Cyst Nematodes

AbstractPCR (conventional and real-time) and DNA sequence analysis were used to identify species and genotypes of potato cyst nematodes from sites in Victoria, Australia. Only Globodera rostochiensis was detected. Sequence analyses of these isolates of PCN have confirmed the PCR-based results and have revealed the presence of genetically diverse populations in infested fields. However, the sequence variation was not in the diagnostic primer binding sites. The melting peaks, from multiplex real-time PCR analysis, for Globodera pallida and G. rostochiensis were 83.3 and 88.7, respectively. The importance of DNA extraction, PCR and sequence analysis for the molecular identification of PCN is discussed. This study has significant implications for detecting species of PCN in order to monitor/develop control strategies for the PCN of quarantine importance.

scholarly journals Enhancement of Population Size of a Biological Control Agent and Efficacy in Control of Bacterial Speck of Tomato through Salicylate and Ammonium Sulfate Amendments

2003 ◽  
Vol 69 (2) ◽  
pp. 1290-1294 ◽  
Author(s):  
Pingsheng Ji ◽  
Mark Wilson
Keyword(s):  
Biological Control ◽  
Population Size ◽  
Ammonium Sulfate ◽  
Pseudomonas Syringae ◽  
Sodium Salicylate ◽  
Biological Control Agent ◽  
Control Agent ◽  
Control Efficacy ◽  
Bacterial Speck ◽  
Leaf Surfaces

ABSTRACT Sodium salicylate and ammonium sulfate were applied to leaf surfaces along with suspensions of the biological control agents Pseudomonas syringae Cit7(pNAH7), which catabolizes salicylate, and Cit7, which does not catabolize salicylate, to determine whether enhanced biological control of bacterial speck of tomato could be achieved. Foliar amendment with salicylate alone significantly enhanced the population size and the efficacy of Cit7(pNAH7), but not of Cit7, on tomato leaves. Application of ammonium sulfate alone did not result in enhanced population size or biological control efficacy of either Cit7(pNAH7) or Cit7; however, when foliar amendments with both sodium salicylate and ammonium sulfate were applied, a trend toward further increases in population size and biological control efficacy of Cit7(pNAH7) was observed. This study demonstrates the potential of using a selective carbon source to improve the efficacy of a bacterial biological control agent in the control of a bacterial plant disease and supports previous conclusions that the growth of P. syringae in the phyllosphere is primarily carbon limited and secondarily nitrogen limited.

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