Know your enemy: Colletotrichum, the upcoming postharvest disease on apple (Malus x domestica)

A major challenge in fruit production nowadays is to limit losses due to postharvest diseases. Despite optimized storage conditions and the application of fungicides, postharvest fungal pathogens are still responsible for considerable losses. The postharvest pathogen Colletotrichum is a recent problem on apple fruits and information is still limited. This pathogen is not visible at harvest due to a quiescent phase. It is only during storage and due to ripening that the pathogen becomes active when fruits become more susceptible and necrotic lesions appear on the fruits. The main objective of our research is to get insights into the specific plant-pathogen interaction Malus x domestica – Colletotrichum. Fruits have passive and active defense to overcome the multiple attacks by fungal pathogens. The focus in this manuscript is mainly towards the passive defense, more specifically the role of the physiological status of fruits and some putative defense metabolites in the susceptibility changes to Colletotrichum.

Since Colletotrichum is a relatively new and unknown postharvest pathogen on apple fruit in Belgium (and Europe), we firstly monitored the presence of this pathogen on apple fruits and executed an identification of the different species present by different methods. Briefly we could conclude that the postharvest pathogen Colletotrichum is an important threat for apple fruits in Belgium. Based on sequence analysis of six different genetic regions six different Colletotrichum spp. were identified on 21 different apple cultivars from Belgian orchards: C. fioriniae, probably C. kahawae, C. salicis, C. rhombiforme, C. acutatum and C. godetiae. Colletotrichum fioriniae was found to be the most present and pathogenic species in Belgian orchards. Reliable morphological discrimination between all species, seems not possible. Inoculation assays on two apple cultivars, the more susceptible ‘Pinova’ and the less susceptible ‘Nicoter’, revealed a significant difference in pathogenicity among isolates and among Colletotrichum species. The pathogenicity tests indicated that isolates coming from another host species, e.g. strawberry, are also pathogenic on apple fruits.

Since Colletotrichum is a postharvest pathogen characterized by a quiescent phase in the apple fruit, we investigated fruit susceptibility of nine apple cultivars at harvest and after storage for Colletotrichum fioriniae, and studied the relationship of the susceptibility with some fruit ripening characteristics. Two artificial inoculation techniques were compared and the wound inoculating method had more advantages compared to the nebulization method. Fruits revealed a higher susceptibility after storage compared to harvest and the nine apple cultivars had clear differences in susceptibility to C. fioriniae. The ripening parameters respiration and Brix had a positive and the firmness a negative correlation with the lesion expansion growth rate of C. fioriniae after artificial wound inoculations of the studied cultivars.

Based on these findings, more detailed experiments indicated indeed a link between the ripening of apple fruits and the susceptibility to the postharvest pathogen Colletotrichum. Fruits are ripening during the storage time and the changes being cultivar dependent. Cultivar ‘Nicoter’ again was the less susceptible cultivar compared to ‘Pinova’, with each cultivar having their specific ripening characteristics. A stepwise regression pointed out that firmness and ethylene production are key parameters correlating to the lesion expansion growth rate (LEGR) of C. fioriniae on apple fruits. Still, it is not correct to make firm conclusions about physiological fruit changes and susceptibility issues. It is expected that a combination of parameters underlying these physiological changes will make apple fruits more or less susceptible. Besides ripening aspects, the preformed antifungal polyphenols that were studied didn’t shown a clear link with the susceptibility for Colletotrichum in apple fruits, while this was often expected in literature.

Next to the passive, the induced defense is also very important. In this manuscript a selection of seven induced antifungal polyphenolic compounds and the polyphenol oxidase (PPO) activity was studied on artificial inoculated fruits. The differences in susceptibility, based on symptom expression, were confirmed by qPCR of fungal DNA in the fruit. Next to that, limited significant changes in specific induced polyphenol concentrations in time due to inoculations with C. fioriniae were found. Chlorogenic acid was the most important polyphenol being induced in apple fruits, which is in agreement with previous research on other pathosystems. The induced response based on PPO activity measurements was not visible in our experiments. PPO activity increased in mock wound-inoculated and intact tissue, but not in the pathogen wound-inoculated tissues. For apples that were stored up to 20 weeks, no induction of polyphenols nor significant PPO activities were present, probably due to the fruits being too ripe. We concluded that the induction of polyphenolics and PPO activities alone cannot explain the differences in susceptibility of the cultivars studied.

The RNA sequencing analysis executed was exploratory. We focused on the role of ethylene production and cell wall metabolism, the latter related to firmness, since both ripening parameters had a considerable correlation with the changes in fruit susceptibility. The ethylene production measurements during storage and between apple cultivars could partially be explained by the differential expression of transcripts encoding for ACC synthase and ACC oxidase. It is clear that during storage several cell wall degrading enzymes in apple fruits were upregulated and these changes in gene expression, involved in cell wall metabolism, were correlated to the softening of the fruits during storage times and ripening.