Efficiency of Gaseous Ozone in Disinfection of Mushroom Growing Rooms

Button mushroom Agaricus bisporus Lange (Imbach) is a widely cultivated edible mushroom in the world (Sánchez 2010, Kaliyaperumal et al. 2018, Kapahi 2018). Commodity cultivation of these fungi in modern mushroom houses takes place throughout the year in halls specially adapted for running only this type of crop. This ensures optimum conditions for the development of mycelium and fruiting throughout the production stage, which allows to obtain high yields and high-quality products. On the other hand, long-term intensive cultivation in monoculture is conducive to the appearance of diseases and pests (Fletcher & Gaze 2008).

The most common pathogens in the cultivation of button mushroom are Trichoderma aggressivum, Cladobotryum dendroides, Mycogone perniciosa, and Lecanicillium fungicola (Fletcher & Gaze 2008, Berendsen et al. 2010). The losses caused by these pathogens are varied and depend on the moment of their occurrence in the mushroom crop and the number of fungal spores (Szumigaj-Tarnowska et al. 2011, Szumigaj-Tarnowska et al. 2015, Ślusarski et al. 2013).

An important element of crop protection against pathogens in mushroom houses is the “cooking-out” of production halls after each cultivation cycle, using steam to heat the air in the hall to a temperature of 65–70 °C and keeping it for at least 8 hours. This treatment allows to kill all mushroom pathogens and pests even deep inside the substrate and the peat cover (Ekman 2017). However, not all mushroom farms have technical capabilities to carry out this treatment (Ślusarski et al. 2012).

In this case, an alternative is chemical disinfection of the hall, which is a less effective but cheaper and easier method to use. The aim of disinfection is to reduce the pathogen population or to eliminate them completely. However, even a high concentration of biocides operating for a prolonged period cannot completely reduce pathogen populations in mushroom substrate. This treatment can be more effective when it is used on the surface thoroughly cleaned of mushroom debris or any other organic contamination (O’Neill et al. 2015).

Gaseous ozone was granted generally recognized as safe status by the Food and Drug Administration for direct contact with food as a sanitizing agent. Ozone is a very strong oxidant that has long been used to disinfect drinking water and wastewater (Rodríguez et al. 2008). It is also used as a disinfectant in the food industry and for removing unpleasant odors in rooms (Skalska et al. 2006, Krosowiak et al. 2007). In horticulture, it is successfully used in fruit and vegetable storage (Krosowiak et al. 2007) as well as in the disinfection of media in recirculating hydroponic systems (Vanachter et al. 1988). Ozone can also be used in the production of a substrate for cultivated mushrooms (Buntat et al. 2013) and to reduce the occurrence of disease during the course of the cultivation cycle (Toto 2010). Gaseous ozone is also suitable for reduction of microorganisms that can occur on food, including mushrooms, such as Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7 (Akata et al. 2015). There were also attempts to apply ozone to extend the shelf life of button mushrooms, but the problem was browning of mushrooms under the influence of gas (Skog & Chu 2001, Escriche et al. 2001).

Investigation on the possibility of using ozone to control fungal pathogens occurring in the mushroom cultivation has been undertaken only once so far and concerned only one species. O’Neill et al. (2015) showed the lack of efficiency of ozone gas in mycelium and spores of T. aggressivum. There is thus a need for extended efficacy evaluation of gaseous ozone for reducing the other most common fungal pathogens of white button mushrooms. However, due to the high sensitivity of mushrooms to browning under the influence of ozone, it is worth considering the possibility of using it after harvest at the end of the production cycle.

The aim of the study was to determine the efficiency of ozone gas for disinfection of empty growing rooms between production cycles in order to reduce the most important fungal diseases occurring in the mushroom cultivation.

Green mold caused by T. aggressivum has been a major disease affecting mushroom production (O’Brien et al. 2017). During our study, Trichoderma isolates proved to be the most resistant to ozone among the pathogens tested (Table 2; Fig. 2). Only one of three tested isolates of this species was damaged after 6 hours of ozonation (Table 3). These differences in ozone sensitivity among isolates of the same species were also observed by Antony-Babu and Singleton (2009). They reported that only one isolate exposed to a continuous low level of ozone did not produce spores, whereas another isolate only delayed spore production by a day compared to the control.

Fig. 2

What is more, the results obtained confirm earlier information on the high resistance of green molds to ozone. O’Neill et al. (2015) reported that gaseous ozone is completely ineffective in relation to the spores and mycelium of T. aggressivum. Conditions in which the researchers conducted experiments were different than those in the presented work. The exposure time was definitely shorter (maximum 80 minutes), and higher ozone concentrations were obtained (up to 100 ppm). The experiment was also carried out at a lower air humidity of around 70%. In such conditions, however, ozone gas showed quite good effectiveness with respect to the mushroom mycelium. A comparison of ozone with other chemical disinfectants used in mushroom houses shows that gaseous ozone is the least effective of them. The high tolerance of Trichoderma to ozone was demonstrated also by Hudson and Sharma (2009). What is important, Baars and Rutjens (2016) also confirm the lack of efficiency of ozone as a disinfectant, while suggesting that it can stimulate spore germination. In our research, however, we did not confirm this statement. The growing rate as well as the time of appearance of the first colonies of Trichoderma isolates on ozone plates was at a similar level (Table 3). This finding also could have been applied to other tested pathogens (Tables 4 & 5). Slightly different results regarding the efficiency of ozone in relation to molds occurring on button mushrooms have been obtained by Akata et al. (2015). Exposure to gaseous ozone at a concentration of 5.3 mg·dm⁻³ for 45 minutes resulted in a decrease in mold count below the detection limit (<2 log CFU per mushroom) with a reduction of more than 1.43 log. Unfortunately, the authors did not provide the exact mold species, which makes it difficult to compare both results. Molds that occur on mushrooms can belong to different species, such as Trichoderma, Penicillium, Pythium, and others (Fletcher & Gaze 2008), and as our result suggests, differences in response to ozone occur even between isolates of the same species (Fig. 3).

Fig. 3

The phenomenon of diversity of isolates within one species was also observed by Szumigaj-Tarnowska et al. (2015). The authors showed that individual isolates of M. perniciosa differ in pathogenicity in relation to the button mushroom. For example, the isolate M. perniciosa M9 was the most tolerant to ozonation among all the tested isolates and also the most virulent to A. bisporus (Szumigaj-Tarnowska et al. 2015).

Unfortunately, there are no published scientific data on the effect of ozone on spores of M. perniciosa, C. dendroides, and L. fungicola. In our study, all these species showed high susceptibility to ozone gas. In order to achieve 100% efficacy against Mycogone isolates, a minimum of 6 hours of ozonation had to be applied, whereas for Cladobotryum strains, a minimum of 8 hours had to be applied (Table 2). Individual isolates of Cladobotryum differed in their sensitivity to ozonation. Two isolates were completely inhibited after 6 hours of ozone, whereas for inhibition of isolate Cladobotryum spp. D30 growth, a minimum of 8 hours of ozonation was required (Fig. 3).

The Lecanicillium isolates were particularly sensitive to ozonation as only 30 minutes was sufficient to gain total inhibition of its mycelium growth. That is why the diameter of its colonies after ozonation was not recorded on Fig. 3, where results concern exposure ranging from 2 to 8 hours.

However, the experiments presented by Luković et al. (2018) showed also the high sensitivity of L. fungicola to some essential oils. Inhibitory and fungicidal activity of the selected essential oils was assayed using microdilution and macrodilution fumigant methods. And what is more, Lecanicillium isolate was more sensitive to tested oils than Cladobotryum isolate. These results indicated that Lecanicillium isolates can be highly sensitive to various biological substances and non-synthetic factors. Galvão and Bettiol (2014) showed the high susceptibility of Lecanicillium genera to UV-B radiation, while Mehrparvar et al. (2016) determined the antifungal activity of two essential oils at low concentrations for the growth of L. fungicola.

According to the findings of Antony-Babu and Singleton (2009), gas ozone is more effective in reducing spore germination than in reducing mycelial growth. In our research, fungal spores were exposed to gaseous ozone. Probably, this is the reason why ozonation efficiency was so high in relation to the tested pathogens.

The time of ozone exposure was highly correlated with inhibition of the growth of Trichoderma, Mycogone, and Cladobotryum isolates (r = 0.969, r = 0.960, and r = 0.908, respectively) (Fig. 2).

Therefore, we conclude that short-term (0.5 hour) ozonation can be suitable for disinfection of growing rooms at the end of the cultivation cycle only for infection caused by L. fungicola. Long-term (6–8 hours) ozonation may be useful for disinfection of empty growing rooms for M. perniciosa and C. dendroides, whereas ozonation with a concentration of 50 ppm may be ineffective in relation to the pathogenic isolates of Trichoderma.