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

Abstract

Aims: Endophytic colonisation 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 colonisation of detached 'Royal Gala' apple shoots. Re-isolation on rifampicin amended agar followed by enterobacterial repetitive intergenic consensus (ERIC)-PCR verified endophytic colonisation by three Pseudomonas sp. rifampicin mutants up to 4-5 cm above and below the inoculation point. Colonisation ability was not found for the three Bacillus rifampicin mutants. Recovery frequency and total length of detached shoots colonised by N. ditissima was not reduced. In attached shoots, length of shoot tissue colonised 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 colonised by N. ditissima. The other treatments did not reduce length of shoot colonised 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 colonisation.