Sooty mould populations on kiwifruit: post harvest and seasonal variation : A thesis submitted in partial fulfilment of the requirements for the Degree of Master at Lincoln University

Sooty mould is a universal term used to describe the complex of superficial, Ascomycota fungi that grow upon the honeydew exudates of sap-sucking insects. These organisms form an extensive network of dense, black mycelia on the surfaces of affected leaves, fruit and other plant material. Scolypopa australis (passionvine hopper) are Hemiptera that feed from the petiole of kiwifruit and subsequently excrete copious amounts of honeydew onto fruit surfaces. In the 2018/19 kiwifruit season, S. australis was responsible for $44 million in economic loss due to sooty mould. This study identified the fungal organisms constituting sooty mould on green-fleshed Actinidia chinensis var. deliciosa ‘Hayward’ and A. chinensis var. chinensis ‘Zesy002’ (informally referred to as Gold3) during cool storage and on ‘Zesy002’ throughout a single, growing season. ‘Hayward’ and ‘Zesy002’ fruit with sooty mould symptoms were harvested from the Plant and Food Research orchards in June 2021. Fruit were assessed at three sampling periods throughout six months in cool storage (1°C). Isolations were taken from the flesh, lenticel and skin tissue on non-sooty mould and sooty mould areas of the same fruit, followed by incubation (20°C) on ½ strength potato dextrose agar for seven days, with a 12 h photoperiod. Sub-culturing and morphological identification on representative morphotypes was conducted, followed by amplification of the ITS region of rDNA, with primer pairs ITS1-F/ITS4. Amplified PCR products were then SANGER sequenced and the consensus contig analysed using NCBI BLAST®. This study identified a total of 42 morphotypes within 17 fungal genera. There was no difference in fungal communities recovered from non-sooty mould and sooty mould areas of fruit surfaces. The number of isolates recovered from non-sooty mould and sooty mould areas decreased over time in cool storage however, the diversity of genera increased. No difference was observed between the fungal communities recovered from ‘Hayward’ and ‘Zesy002’. Fungi were also recovered from all tissue types. The most frequently recovered isolates belonged to Cladosporium, Epicoccum, Alternaria, Phoma, Diaporthe and Pestalotiopsis genera, with the greatest diversity of morphotypes within the Alternaria genus. The sooty mould communities on ‘Zesy002’ were also analysed throughout a growing season from a Bay of Plenty orchard in December 2021, January 2022 and February 2022. An artificial honeydew solution was applied weekly to 24 fruit and sampled after four weeks of applications. Fruit with naturally infected sooty mould growth were also sampled. Subsequent sooty mould growth on the shoulder of fruit was analysed using the previous chapter’s methodology. This study identified a total of 52 morphotypes and 17 fungal genera. A greater number of isolates were recovered from sooty mould areas however, a similar fungal diversity was recovered between areas. The number of isolates recovered from non-sooty mould and sooty mould areas increased throughout the growing season however the fungal diversity remained similar. Fungi were recovered from all tissue types. The most frequently recovered fungi belonged to the genus Phoma followed by isolates from Cladosporium, Pestalotiopsis, Epicoccum, Didymella and Fusarium genera with the greatest diversity of morphotypes within the Fusarium genus. A greater number of isolates were recovered from fruit directly adjacent to a source of S. australis, compared to spatially removed. Only four naturally infected sooty mould fruit were observed in the orchard, therefore comprehensive analyses’ were not conducted. A total of 19 morphotypes and 12 genera were identified from these fruit, with no difference in fungal diversity between non-sooty mould and sooty mould areas. The Aureobasidium genus was only isolated from ‘Zesy002’ with naturally infected sooty mould, compared to the artificially applied honeydew. A background diversity of fungi are present on the surfaces of ‘Zesy002’ and ‘Hayward’ fruit, irrespective of honeydew association. The sooty mould complex also constitutes organisms that are both epiphytic and endophytic in nature. Future studies should investigate the fungal microbiome of kiwifruit and whether these organisms could develop into pathogens. Furthermore, assessing fungal communities from a greater number of orchards, including organically grown fruit should be conducted and compared to the current study. Control must focus on reducing S. australis populations and developing a method that prevents fungal infection during fruit development.