Evaluation of biocontrol potential of bacterial endophytes against European canker of apple

Abstract

European canker, caused by the fungal pathogen Neonectria ditissima, is one of the most significant diseases of apple worldwide. Biological control has drawn considerable attention in plant disease control, as it reduces pesticide input and occurrence of fungicide resistance in pathogens. Endophytes are a novel source of biocontrol agents as effective isolates can also extensively colonise the plant host tissue. The aim of this study was to evaluate biocontrol potential of apple endophytic bacteria against European canker. One-year old apple shoots of ‘Royal Gala’ and ‘Braeburn’ were sampled from apple orchards in Lincoln University, Nelson (South Island) and Hawke’s Bay (North Island) in New Zealand in 2015 and 2016. ‘Scifresh’ and ‘Scilate’ were also sampled from some orchards. Denaturing gradient gel electrophoresis (DGGE) was used to characterise bacterial endophyte community in apple leaves and/or stems with focus on the effect of tissue type, cultivar, site, region and season. Tissue type, cultivar and site were found to be the main factors influencing bacterial endophyte community, with the endophyte taxa in ‘Royal Gala’ more variable than that in ‘Braeburn’ and with that in leaves often differed from that in stems in ‘Royal Gala’ and ‘Braeburn’. A collection of 1004 bacterial endophytes isolated from leaves and stems of the apple shoots were tested in a dual culture plating assay for ability to inhibit the mycelial growth of N. ditissima. Eighteen isolates were antagonistic to N. ditissima (percent inhibition ≥20%). Of these, 16 isolates (9 Bacillus spp. and 7 Pseudomonas spp.) were selected for further study based on identification by rDNA sequencing showing similarity to species reported to be endophytes and/or known biocontrol agents, but not plant pathogens. The bioactivity of these selected isolates were further tested using a range of other in vitro antagonistic assays including volatile, culture filtrate and siderophore assays. Spontaneous rifampicin resistant bacterial mutants were produced from six selected isolates and used as inocula to assess their colonisation in detached apple shoots. Pseudomonas isolates P-1R, P-2R and P-6R (originated from stems), but not Bacillus isolates B-1R, B-5R and B-6R (originated from leaves), showed persistent colonisation and spread in detached shoots. Inoculation of mutant strains P-1R and P-2R 14 days before N. ditissima in attached apple shoots reduced the recovery frequency of N. ditissima from the stem sections surrounding the wounded inoculation point, indicating their potential as wound protectants. The approach taken in this study has promise to identify strains that may help protect apples against European canker.