Reviewer response for version 1

Abstract

The spread of Citrus black spot (CBS) is a major concern in the citrus industry because the disease threatens fruit marketability and citrus tree health. Furthermore, there is a public concern about the safety and side effects of synthetic fungicides currently being used to control citrus black spot. Synthetic fungicides are reported to have carcinogenic effects on humans and are also toxic to the environment, however this depends on the structure of the active ingredient and the dose present. Furthermore, microorganisms tend to develop resistance to most synthetic fungicides. This problem has prompted research into the identification of new ways with broad activity in treatment of microbial disease in plants such as the use of essential oils.The purpose of the study was to investigate the use of Thyme oil (Thymus vulgaris) as an alternative antifungal against Citrus Black Spot (CBS). Thyme oil was characterised using GC/MS. Thymol (32.1%) and ρ-Cymene (20.4%), were identified, as major compounds. Furthermore, the minimum inhibitory concentration (MIC) of essential oils against the test organism was 25 (µg/ml). The antifungal activity of Thyme oil, Thyme hydrosol and antifungal anti-mitochondrials was tested in vitro against Phyllosticta citricarpa using the agar diffusion bioassay. Comparative antifungal activity was observed between antifungal anti-mitochondrial, Thyme oil and hydrosol as indicated by an inhibition zone, minimal growth and maximum growth zone towards the edge of the plate. We propose that Thyme oil, like antifungal anti-mitochondrials, inhibits fungal growth by targeting structures with increased mitochondrial activity. This was further confirmed by Scanning Electron Microscopy (SEM) and XTT colorimetric assay. This indicates that Thyme oil and hydrosol can be used as potential alternative antifungal agents against Citrus Black Spot (CBS).