Development and evaluation of SCAR markers for a Pseudomonas brassicacearum strain used in biological control of snow mould

Holmberg, Anna-Ida Johnsson; Melin, Petter; Levenfors, Jens P.; Sundh, Ingvar

doi:10.1016/j.biocontrol.2008.10.016

Cited by 25 publications

(27 citation statements)

References 61 publications

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“…As already stated by Holmberg et al (2009), such a tiny difference is enough to distinguish the presence of a particular strain in complex environments. In this study, a specific DNA fragment has been identified by PCR fingerprinting but the first primer set designed from its sequence was not specific for Fo47.…”

Section: Discussionmentioning

confidence: 86%

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

Edel-Hermann

Aimé

Cordier

et al. 2011

FEMS Microbiology Letters

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Being able to identify specifically a biological control agent at the strain level is not the only requirement set by regulations (EC)1107/2009, it is also necessary to study the interactions of the agent with the plant and the pathogen in the rhizosphere. Fo47 is a soil-borne strain of Fusarium oxysporum which has the capacity to protect several plant species against the pathogenic formae speciales of F. oxysporum inducing wilts. A strain-specific sequence-characterized amplified region marker has been designed which makes it possible to distinguish Fo47 from other strains of F. oxysporum. In addition, a real-time PCR assay has been developed to quantify Fo47 in root tissues. The proposed assay has been validated by following the dynamics of root colonization of tomato plants grown in soil infested with Fo47. Results showed that with the method it is possible to quantify Fo47 in roots in the absence or presence of the pathogen and in the absence or in presence of the native microbial communities.

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Section: Discussionmentioning

confidence: 86%

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

Edel-Hermann

Aimé

Cordier

et al. 2011

FEMS Microbiology Letters

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“…Strain‐specific Q‐PCR methods have recently been developed for other bacteria using random amplified polymorphic DNA (RAPD), i.e. Lactobacillus rhamnosus GG (Ahlroos and Tynkkynen 2009), Pseudomonas brassicacearum MA250 (Holmberg et al. 2009), Pseudomonas fluorescens EPS62e (Pujol et al.…”

Section: Discussionmentioning

confidence: 99%

Specific detection ofLysobacter enzymogenes(Christensen and Cook 1978) strain 3.1T8 with TaqMan^®PCR

Nijhuis¹,

Pastoor

Postma³

2010

Journal of Applied Microbiology

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Aims: To develop a strain‐specific TaqMan® PCR method for detecting and quantifying the biocontrol strain Lysobacter enzymogenes 3.1T8. Methods and Results: A primer–probe combination was designed on the basis of a strain‐specific sequence selected using REP‐PCR (repetitive extragenic palindromic‐polymerase chain reaction). The specificity of this combination was demonstrated by 14 other Lysobacter strains that did not react with the selected primer–probe combination. To quantify strain 3.1T8 in cucumber root samples, a calibration curve was prepared by spiking roots with a 10‐fold dilution series of the strain. Detection of the biocontrol strain 3.1T8 with this method showed that the strain survived well for 22 days on root tips as well as on older cucumber roots. Survival was higher when the strain was inoculated to younger plants. In a cucumber production system with large volumes of substrate, strain 3.1T8 was detected in high numbers on cucumber roots 3 weeks after inoculation. Conclusions: The primer–probe combination developed was strain specific, because it did not react with other strains of the same species and genus. The TaqMan® PCR method successfully quantified the inoculated biocontrol strain on cucumber roots grown in different cropping systems. Significance and Impact of the Study: The developed TaqMan® PCR method is a strain‐specific real‐time detection method that can be used to assess the population dynamics of L. enzymogenes strain 3.1T8 for further optimization of its biocontrol efficacy.

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“…RAPD-PCR was performed as previously described (Holmberg et al 2009) with defined decamer primer sets (OPX, Eurofins MWG Operon). PCR products were separated on 1.5% agarose gels in 0.59 TBE buffer and stained with ethidium bromide.…”

Section: Rapd-pcr Amplificationmentioning

confidence: 99%

“…Another approach is to track the BCA based on sequence-characterised amplified region (SCAR) PCR methodology. This approach has been applied to monitor potential bacterial biocontrol strains of for example the Pseudomonas genus, such as Pseudomonas fluorescens (Pujol et al 2005) and Pseudomonas brassicacearum (Holmberg et al 2009) but also various fungi such as Chondrostereum purpureum (Becker et al 1999), Gliocladium catenulatum (Paavanen-Huhtala et al 2000), Colletotrichum coccodes (Dauch et al 2003), Pichia anomala (De Clercq et al 2003), Beauvaria bassiana (Castrillo et al 2003), Rhizoctonia solani (Grosch et al 2007), among them several Trichoderma spp. (Abbasi et al 1999;Hermosa et al 2001;Dodd et al 2004;Rubio et al 2005;Cordier et al 2007;Savazzini et al 2008).…”

Section: Introductionmentioning

confidence: 99%

Specific SCAR markers and multiplex real-time PCR for quantification of two Trichoderma biocontrol strains in environmental samples

et al. 2011

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Several strains from the genus Trichoderma (Ascomycetes, Hypocreales) are commercially used as biocontrol agents, e.g. in formulations containing the two Trichoderma strains IMI206039 (Hypocrea parapilulifera B.S. Lu, Druzhinina & Samuels) and IMI206040 (T. atroviride P. Karst). To quantify the presence of the two isolates after application, we developed primers for SCAR markers (Sequence-Characterised Amplified Region). In order to quantify both fungal strains simultaneously, we also designed fluorophore-labelled probes distinguishing the two strains, to be used in combination with the SCAR primers. In incubations of two different soils, artificially inoculated and maintained under controlled conditions, the quantification through amplification with the SCAR markers in qPCR and through colony-forming units from plate counting correlated well. Further tests of the markers on samples taken from a golf green treated with a product containing both strains indicated that the two biocontrol strains did not establish, either on the golf green or in the surrounding area.

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Development and evaluation of SCAR markers for a Pseudomonas brassicacearum strain used in biological control of snow mould

Cited by 25 publications

References 61 publications

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

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

Specific detection ofLysobacter enzymogenes(Christensen and Cook 1978) strain 3.1T8 with TaqMan^®PCR

Specific SCAR markers and multiplex real-time PCR for quantification of two Trichoderma biocontrol strains in environmental samples

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Development and evaluation of SCAR markers for a Pseudomonas brassicacearum strain used in biological control of snow mould

Cited by 25 publications

References 61 publications

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

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

Specific detection ofLysobacter enzymogenes(Christensen and Cook 1978) strain 3.1T8 with TaqMan®PCR

Specific SCAR markers and multiplex real-time PCR for quantification of two Trichoderma biocontrol strains in environmental samples

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Specific detection ofLysobacter enzymogenes(Christensen and Cook 1978) strain 3.1T8 with TaqMan^®PCR