The use of rifampicin mutants and ERIC-PCR to track plant colonization and in planta efficacy of bacterial biocontrol agents against Neonectria ditissima.

AIMS: Endophytic colonization of apple shoots by bacterial endophytes with in vitro antagonism against Neonectria ditissima was evaluated. Their biocontrol activity against N. ditissima was assessed.

METHODS AND

RESULTS

Spontaneous mutants resistant to 125 ppm rifampicin produced from three Pseudomonas sp. and three Bacillus sp. strains were used to assess endophytic colonization of detached 'Royal Gala' apple shoots. Re-isolation on rifampicin amended agar followed by enterobacterial repetitive intergenic consensus (ERIC)-polymerase chain reaction (PCR) verified endophytic colonization by three Pseudomonas sp. rifampicin mutants up to 4-5 cm above and below the inoculation point. Colonization ability was not found for the three Bacillus rifampicin mutants. Recovery frequency and total length of detached shoots colonized by N. ditissima was not reduced. In attached shoots, length of shoot tissue colonized by Pseudomonas mutant strains did not differ between treatments at either assessment time. Pseudomonas sp. 1RIF inoculated 14 days before N. ditissima reduced length of shoot colonized by N. ditissima. The other treatments did not reduce length of shoot colonized by N. ditissima indicating no in planta biocontrol activity.

CONCLUSION

Combination of spontaneous rifampicin resistant bacterial mutants and ERIC-PCR reliably tracked bacteria in planta. Lack of in planta biocontrol activity was not due to absence of endophytic colonization.