Activity of apple endophytic fungi to control Neonectria ditissima infection of apple : A thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy at Lincoln University

Apple (Malus domestica) is one of the most important fruit crops in New Zealand. European canker, caused by Neonectria ditissima, is an economically important disease in apple-producing regions worldwide. Currently, the disease is managed by the application of fungicides and the removal of canker-infected branches. There is growing concern regarding the potential health risks of chemical residues on fruit for human consumption and environmental pollution. Also, the development of fungicide-resistant strains of N. ditissima is a risk for apple production. In addition, canker pruning is labour-intensive and costly. Biological control of plant pathogens utilising endophytic microorganisms has received increased attention in recent years as an eco-friendly and effective control mechanism for many plant diseases. This study aimed to identify apple fungal endophytes with biocontrol efficacy against N. ditissima. It was achieved by i) investigation of the in vitro activity of fungal isolates against N. ditissima using plate-based bioassays, ii) investigation of the in vivo colonisation potential and pathogenicity of endophytes, and iii) evaluation of the in planta biocontrol activity of selected isolates using shoot assays. The three hundred and eleven fungal endophytes used in this research project were isolated from apple stem and leaf tissues. A preliminary dual-plate assay was conducted to investigate the activity of these fungal isolates to reduce the in vitro growth of N. ditissima, with 37 isolates exhibiting antagonism to N. ditissima. Based on 16S rRNA gene sequencing, 37 isolates were identified into 12 genera: Biscogniauxia (n=8), Diaporthe (n=8), Trichoderma (n=5), Chaetomium (n=4), Paraphaeosphaeria (n=1), Epicoccum (n=2), Neosetophoma (n=2), Xylaria (n=1), Hansfordia (n=1), Penicillium (n=1), Fusarium (n=1), Colletotrichum (n=1), and an additional class, Sordariomycetes, and an unidentified fungal endophyte isolate. These isolates were tested further for the production of volatile and non-volatile inhibitory metabolites and siderophores related to their potential bioactivity in vitro and in planta. The volatile assay revealed that 24 isolates produced volatile compounds that inhibited the mycelium growth of N. ditissima compared to the untreated control. Additionally, agar amended with a 50% culture filtrate of six isolates inhibited the in vitro growth of N. ditissima by over 20%. Further, 15 isolates were shown to produce siderophores in modified chrom-azurol S agar (CAS) assay. The colonisation and pathogenicity ability in planta of 37 isolates was examined in ‘Royal Gala’ detached shoots and additionally ‘Royal Gala’ and ‘Braeburn’ fruit. Results indicated that 24 isolates were non-pathogenic to apple shoots and fruit. Of these 24 isolates, 22 were reisolated from the inoculated detached shoots. Almost all the isolates (36 out of 37) produced at least one of the enzymes assayed; amylase (20/37 isolates), cellulase (15/37 isolates), pectinase (25/37 isolates), protease (26/37 isolates) and xylanase (13/37 isolates). The in planta biocontrol activity of four isolates (one each Biscogniauxia sp., Neosetophoma sp., Trichoderma sp. and Paraphaeosphaeria sp.) that showed in vitro antagonism against N. ditissima with good colonisation capability were evaluated using in vivo detached shoot assays. The results of the autumn experiment indicated that inoculation of 7 days prior to the N. ditissima with either of the two isolates (Trichoderma sp. and Paraphaeosphaeria sp.) effectively reduced N. ditissima colonisation in the detached shoot tissues. The presence and absence of N. ditissima in the shoot tissue, with or without endophytes, were assessed using isolation media and PCR. The colony of N. ditissima was recovered from tissue up to 50 mm from the inoculation point in both the upward and downward directions. In PCR analysis, N. ditissima was detected up to 30 mm beyond the inoculation point in asymptomatic tissues harvested 30 days after inoculation. The two selected endophytes, T. atroviride isolate FE3 and P. neglecta isolate FE9, were further tested for their ability to reduce N. ditissima colonisation of intact shoots on potted ‘Royal Gala’ and ‘Braeburn’ trees under shade house conditions. The results indicated that P. neglecta isolate FE9 and T. atroviride sp. isolate FE3 did not significantly reduce the colonisation of N. ditissima in the shoot tissues. Overall, this is the first New Zealand study that focused on investigating the biocontrol capabilities of endophytic fungal isolates against N. ditissima. This study provides information on the assessment of potential antagonistic fungal endophytes, their enzymatic activity and colonisation ability in asymptomatic apple shoot tissues