Identification of members of the apple endomicrobiome with the potential to protect against European canker : A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Lincoln University

Abstract

Apple (Malus domestica Borkh.) is a widely cultivated and important economic fruit crop in New Zealand (NZ). European canker, caused by the fungal pathogen Neonectria ditissima, is one of the most significant diseases of apple. Application of chemical fungicides is the main control strategy for European canker, which can cause fungicide resistance in pathogens, environmental pollution and also chemical residues on fruit. Endophytes have drawn considerable attention as a novel source of biocontrol agents as effective isolates can also extensively colonise the plant host tissue. The aim of this study was to identify apple endophytes as biocontrol agents of European canker. It was achieved by i) identification of the effect of different factors on the complete endophyte community of apple shoots, ii) investigation of in vitro biocontrol activity of culturable endophytic bacteria and fungi against N. ditissima and their mode of action, and iii) determination of whether endophytic bacteria with in vitro biocontrol activity against N. ditissima could colonise and show activity against N. ditissima in apple shoots. Denaturing gradient gel electrophoresis (DGGE) was used to characterise bacterial and fungal endophyte communities in apple leaves and/or stems with specific focus on the effect of different factors. This study used propidium monoazide (PMA) to enrich amplification of DNA from endophytes. The 2nd leaf, 3rd leaf, green stem and woody stem of ‘Royal Gala’ from three sites and ‘Braeburn’ from site 2 were analysed by DGGE and this showed that tissue type, cultivar and site were the main factors influencing bacterial and fungal endophyte communities, with the endophyte taxa in ‘Royal Gala’ more variable than that in ‘Braeburn’. Bacterial and fungal endophyte communities in leaves often differed from those in stems in ‘Royal Gala’ and ‘Braeburn’. Bacterial and fungal endophyte communities in woody stem were less influenced by season and not affected by region. A representative collection of 1004 bacterial and 87 fungal endophytes were isolated from apple leaves and stems from nine sites in Nelson and Hawke’s Bay in the spring main sampling and three sites in Nelson in the autumn main sampling, with a small proportion of those bacterial isolates from samplings conducted in Lincoln University Research Orchard and Plant & Food Research Orchard in Hawke’s Bay. A dual culture plating assay was used to test the inhibition effect of bacterial and fungal endophytes on mycelial growth of N. ditissima, with 18 bacterial and 17 fungal isolates found to be antagonistic to N. ditissima. Sixteen bacterial (9 Bacillus spp. and 7 Pseudomonas spp.) and 13 fungal isolates (3 Chaetomium sp., 5 Epicoccum sp., 3 Biscogniauxia sp., 1 Penicillium sp. and 1 Dothideomycetes sp.) were identified by rDNA sequencing and showed to be potential biocontrol agents due to their similarity to reported endophytes and/or known biocontrol agents, but not plant pathogens. Mechanisms of action were investigated using a series of in vitro bioactivity assays for bacterial and fungal antagonism, and by detection of antibiotic encoding genes for bacteria. A cell-free culture filtrate assay showed two Bacillus isolates 42-1206(19)b and R1GS-12b and one Pseudomonas isolate 27-801(89)b inhibited mycelial growth of N. ditissima by producing diffusible antifungal compounds. Apart from Bacillus sp. 41-1182(4)b, Chaetomium sp. 2-57f and Biscogniauxia sp. 2-51f, all the bacterial and fungal isolates identified as potential biocontrol candidates produced siderophores. Bioactive volatile compounds were not observed for any of the bacterial or fungal isolates. Antibiotic encoding genes were detected in three Bacillus isolates (R1GS-12b and 42-1206(19)b were positive for Bam C and ItuD, and R3L-1b positive for sfp) showing their potential to synthesise antibiotics (bacillomycin D encoded by Bam C, iturin A encoded by ItuD or surfactin encoded by sfp) to suppress N. ditissima, but no antibiotic encoding genes were detected in the Pseudomonas isolates. Spontaneous rifampicin resistant bacterial mutants (Rif125ppm) were produced from six selected bacterial isolates and used as inocula to assess their colonisation in detached apple shoots. Pseudomonas isolates (20-579(18)b125ppmRif+, 7-208(18)b125ppmRif+ and 31b3125ppmRif+) but not Bacillus isolates (42-1206(19)b125ppmRif+, 21-606(28)b125ppmRif+ and R3L-6b125ppmRif+) showed persistent colonisation and spread in detached shoots. Mutant strains 20-579(18)b125ppmRif+ and 7-208(18)b125ppmRif+ had potential as wound protectants when they were inoculated 14 days before N. ditissima in attached apple shoots, as shown by the reduced recovery frequency of N. ditissima from the stem sections surrounding the wounded inoculation point. The approach taken in this study has promise to identify strains that may help protect apples against European canker.