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Lysobacter antibioticus is an important biocontrol bacteria against phytopathogens in soil, and with the ability to produce nonvolatile antimicrobial metabolites has been extensively characterised. It is important to establish applicable techniques to detect and monitor L. antibioticus directly and accurately in soil samples. We developed and tested 13 primer sets according to phenazine gene (phzA, phzB, phzD, phzF, phzS) and the cyclohexanone monooxygenase gene (phzNO1); a pair of primer phzNO1 F1/phzNO1 R1 based on the cyclohexanone monooxygenase (phzNO1) gene of L. antibioticus strain OH13 was selected and optimized polymerase chain reaction (PCR) amplification conditions for rapid and accurate detection. After screening eight strains of L. antibioticus, two strains of Lysobacter enzymogenes, one strain of Lysobacter capsici, Arthrobacterium, Bacillus, Microbacterium, Burkholderia, Pseudomonas and other bacterial strains isolated from different agricultural soils, the phzNO1 F1/phzNO1 R1 primers amplified a single PCR band of about 229 bp from L. antibioticus. The detection sensitivity with primers phzNO1 F1/phzNO1 R1 was 5.14 × 104 fg/25μL of genomic DNA and 2.254 × 1010 to 2.254 × 1011 colony-forming units/mL for the soil samples. Quantitative PCR assays were to develope as a specific method to monitor the L. antibioticus population in soil as well as guide soil micro-ecological management.

Survival in the phyllosphere is a critical feature for biofungicides based on non-spore forming bacteria. Moreover, knowledge of their persistence on plants is important to design effective formulations and application techniques. With this scope, the aim of this work was to develop a specific method to monitor the fate in the environment of Lysobacter capsici AZ78, a biocontrol agent of Plasmopara viticola, and to evaluate the contribution of formulation in its persistence on grapevine leaves. A strain-specific primer pair derived from REP-PCR fingerprinting was used in quantitative PCR experiments to track the evolution of L. capsici AZ78 population in vineyards. The population reached between 5 and 6 log10 cells gram of leaf(-1) after application and decreased by more than 100 times in one week. Multiple regression analysis showed that unfavourable temperature was the main environmental factor correlating with the decrease of L. capsici AZ78 persistence on grapevine leaves. Importantly, the use of formulation additives protected L. capsici AZ78 against environmental factors and improved its persistence on the leaves by more than 10 times compared to nude cells. Formulation and the knowledge about the persistence of L. capsici AZ78 in vineyards will be useful to develop commercial biofungicides for foliar application., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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.

The exploration of new source materials and the use of alternative isolation and identification methods have led to rapid expansion in the knowledge of diversity; in Lysobacter, 11 new species having been described since 2005, and in Stenotrophomonas with six new species since 2000. The new species of Lysobacter, isolated by dilution and direct plating on standard media, differ in several key phenotypic properties from those obtained by enrichment on complex polysaccharides in the original description of the genus. Revision of the definition of the genus will be required. Both culture-dependent and culture-independent methods to assess community structure, in a variety of host and nonhost environments, have established that some species of Lysobacter are a dominant component of the microflora, where previously their presence had not been suspected. Culture-independent studies have generally not added new information on the occurrence and distribution of Stenotrophomonas maltophilia and other members of the genus, which are readily isolated on standard media from source materials. Lysobacter enzymogenes and Sten. maltophilia produce similar antibiotics and share some enzyme activities which, subject to safety considerations, may make them attractive candidates for use in biological control of plant diseases and of nematodes.