Exploring the role of callose depositions in the resistance of Persea americana against root rot

Abstract

Phytophthora root rot caused by Phytophthora cinnamomi threatens sustainable avocado production in South Africa. One of the most effective control methods for this disease is the use of partially resistant avocado rootstocks. To date, no fully resistant rootstocks have been developed, so the selection process for rootstocks that are more resilient against root rot diseases remains ongoing. It takes approximately 15-20 years for a new rootstock to be commercially released thus, to improve selection efficiency, it is crucial to identify key characteristics of partially resistant rootstocks that can be exploited to allow for a more targeted selection approach. Callose is a β-1,3 glucan deposited in cell walls, to create a physical barrier preventing pathogen invasion into the cell. Previous studies provide qualitative evidence, through confocal microscopy, that callose deposition may be a hallmark of partial resistance in response to P. cinnamomi infection. The aim of this study was to optimise a fluorometric assay to quantify callose production in avocado roots and then utilise this assay to determine if callose production is induced in response to P. cinnamomi infection. The study also sought to determine if callose production is a key characteristic of avocado rootstocks that are partially resistant to P. cinnamomi. This was achieved through chemical inhibition using 2-deoxy-D-glucose (2-DDG) prior to P. cinnamomi inoculation and assessing the resulting disease severity. This project comprised of two research chapters working towards elucidating the role of callose in avocado defence against P. cinnamomi. In the first chapter, the conditions for 2-DDG treatment were optimised, as this was the first study to use this inhibitor on avocado roots. The main objectives were to determine the optimum concentration and duration of 2-DDG treatment required to 1) successfully inhibit defence-related callose production and 2) ensure that 2-DDG did not negatively affect plant health. A secondary objective of this chapter was to optimise a callose extraction protocol from avocado roots to allow for fluorometric callose quantification with minimum background interference. The protocols developed were utilised in a subsequent research chapter, which tested the effect of callose inhibition on avocado rootstocks of varying tolerance levels, namely, one susceptible (R0.12) and two partially resistant (Dusa® and LeolaTM) rootstocks. The objectives of this chapter were to 1) evaluate if callose is produced in partially resistant avocado rootstocks in response to P. cinnamomi, and 2) determine the effect of callose inhibition in these rootstocks. The study yielded evidence for the induction of callose production in avocado roots in response to P. cinnamomi infection. Another significant contribution of the study was the development of a 2-DDG treatment regimen optimised for callose inhibition in avocado roots. Surprisingly, no differences in callose production were observed among different rootstocks with varying levels of tolerance to P. cinnamomi between 1) inoculated and uninoculated plants and 2) 2-DDG treated and untreated plants. The detection of a second avocado root pathogen, Phytopythium vexans, in the roots of the trial plants contributed to these confounding results. Pp. vexans infection was confirmed morphologically and molecularly using a TaqMan qPCR probe assay optimised for avocado roots. The impact of Pp. vexans on disease severity and callose measurements were subsequently assessed. While some results from this study were inconclusive, the findings highlighted the complexity of avocado defence, suggesting that callose production could be a general defence pathway that is activated in response to infection by root rot pathogens. It may become increasingly important to understand the interplay between various pathogens within a root rot complex rather than infection by P. cinnamomi alone. Future research should consider host-pathogen interactions in the context of the avocado microbiome to provide a holistic understanding of the different factors that contribute to plant defence in the field.