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

Abstract

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.

Indigenous Populations of Three Closely Related Lysobacter spp. in Agricultural Soils Using Real-Time PCR

“…An extensive search for antagonistic bacteria, using an agar medium together with a fungus, showed the presence of antagonistic Lysobacter species in several soils [24]. Population densities of introduced Lysobacter enzymogenes strains were determined on plant roots in biocontrol experiments with plate counts and immunofluorescent colony staining [4, 13] and more recently by real-time PCR detection [19]. In several other studies, reviewed by Hayward et al, only one or few Lysobacter isolates were found in various niches (soil, rhizosphere, compost water, sludge, salamander skin) of different continents (Europe, Asia, and North America) [7], showing the general appearance of Lysobacter but not giving any quantitative information.…”

“…Currently, quantitative molecular detection techniques are increasingly applied to specifically quantify bacterial or fungal strains, species, genera, or larger taxonomic entities [15, 16, 19, 20, 27]. Meanwhile, recent improvements in extraction methods for DNA or RNA and the sufficient removal of inhibitory compounds with easy-to-use commercial kits has also made these techniques suitable for application on complex substrates such as soil.…”

Biocontrol of Phytophthora Blight and Anthracnose in Pepper by Sequentially Selected Antagonistic Rhizobacteria against Phytophthora capsici

“…These variable results might be due to the limitations of the required populations of the treated strains for inducing effective ISR against anthracnose in pepper fruit. Recently, Nijhuis et al (2010) suggested that a sufficient population of Lysobacter enzymogenes 3.1T8 on cucumber roots was required for the biocontrol of Pythium root rot. Consistency and improvement in the microbial biocontrol activity can be gained by the use of certain additional compounds (Shaukat and Siddiqui, 2003).…”

Pseudomonas chlororaphis and organic amendments controlling Pythium infection in tomato

“…aureofaciens DSM 6698, and strains 10.2.5 and 11.4.2 appeared to be identical in REP-PCR (repetitive extragenic palindromic-PCR) (Rademaker et al 1998) fingerprint analysis. Consequently, no suitable specific band allowing the design of primers and probes based on a strain specific sequence as described by Nijhuis et al (2010) was available.…”