Thyme oil and thyme oil hydrosol as alternative fungicides against Phyllosticta citricarpa (causative agent of citrus black spot)

Abstract

The South African citrus industry is one of the most important agricultural industries economically, as it contributes considerably to the country’s Gross Domestic Product (GDP) (Dlikilili and Van Rooyen, 2018; Venter, 2018). The citrus sector in the country also contributes greatly to employment opportunities, particularly for disadvantaged communities, as it currently employs over 125 000 people across the country (Genis, 2018). Over the years and despite the Covid 19 pandemic, the number of citrus trees planted in hectares (ha) has continued to increase. This is due to the high vitamin C content of citrus fruits, the increase in investments and aggressive new plantings of soft citrus, lemons, and new varieties of oranges (Cramer and Chisoro-Dube, 2021). Citrus cultivation for commercial purposes is reported mainly in Limpopo, Western Cape, Mpumalanga, Eastern Cape, KwaZulu-Natal, and Northern Cape. Within the Southern African Development Community (SADC), countries such as Zimbabwe, Swaziland and Mozambique also produce citrus fruits in much smaller quantities (Department of Agriculture and Fisheries, 2017). Most citrus fruits produced in South Africa are exported to countries such as Europe and the United States of America (USA) because of far greater returns from the export market than the domestic market. However, the spread of citrus black spot (CBS) has resulted in fears of a possible ban on South African citrus exports to other countries (Kau et al., 2018; Van Dyk and Maspero, 2004). Evidence that CBS affects the agricultural industry negatively regarding food production, security and trade is available.

CBS is a citrus disease caused by the fungus Phyllosticta citricarpa, which affects almost all commercial citrus species externally but does not cause internal decay (Yonow et al., 2013; Roberts et al., 2012; Fialho et al., 2010). Almost all commercially sold citrus species are susceptible to Phyllosticta citricarpa infection, with lemons and Valencia oranges known to be highly susceptible. CBS was first discovered, recorded and described by Benson in Australia and was observed on Valencia oranges over 120 years ago (Paul, 2006; Kotzé, 2000; Kiely, 1948;). Phyllosticta citricarpa produces sexual and asexual spores, with each stage producing different spores responsible for spreading CBS using various modes of dispersion (Truter, 2010). Synthetic fungicides are currently used to minimize the spread of CBS; however, their use has raised health and environmental pollution concerns, including resistance to pathogens (Du Plooy et al., 2009). Hence, research into developing alternative agents for replacing synthetic fungicides for fungal disease control in agriculture continues, such as using essential oils (EOs) and their hydrosol. EOs are complex mixtures of secondary plant metabolites with relatively high vapour pressure and are poorly soluble in water. The EOs reported in various studies have shown to exhibit antifungal properties by targeting structures responsible for the life cycle of fungal organisms such as ascospores and conidia in vitro and in vivo in different fresh produce. The overall aim of the study was to investigate the in vivo effect of thyme oil and thyme oil hydrosol against citrus fungal pathogen Phyllosticta citricarpa (Feyaerts et al., 2018). Thyme oil characterization was done using gas chromatography-mass spectrometry GC-MS and GC × GC-TOFMS. Their effectiveness, together with that of hydrosol, was tested against Phyllosticta citricarpa (CBS causative agent) using methods such as broth microdilution assay (minimum inhibitory concentration (MIC) determination) and fungicidal or fungistatic activity. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to evaluate morphological changes that occurred due to EOs, while the inhibition of pathogen growth on leaves and fruits was also determined. The results of the study revealed that the pathogenic fungi P. citricarpa depend on increased mitochondrion activity, such as conidia, and these structures are sensitive to thyme oil used in the study. Furthermore, thyme oil hydrosol used in the study has indicated a moderate antifungal activity against this fungal pathogen.