Ozonated Water on Tomato Seedlings: Irrigation Versus Spraying in a 2021 Controlled Trial

Background

Ozone dissolved in water is used commercially as a disinfectant, and there has been longstanding interest in whether it has a place in growing plants as well as cleaning what is harvested from them. The question this study set out to answer is narrower and more practical than most: if you are going to use ozonated water on young plants, does it work better watered into the ground or sprayed onto the leaves, and at what strength?

That matters because the two routes are not interchangeable. Ozone is highly reactive and short-lived, so how long it stays in contact with living tissue - and what it meets on the way - changes what it can do. It also matters because ozone at higher concentrations is known to damage plants rather than help them, so any useful answer has to include an upper limit as well as a lower one.

The researchers looked at four indicators of how a seedling is coping. Malondialdehyde, usually shortened to MDA, is a by-product of damaged cell membranes and is used as a marker of oxidative stress - more MDA means more cell damage. Superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) are the plant's own antioxidant enzymes, which neutralise reactive oxygen. The SPAD value is a non-destructive measure of leaf greenness that stands in for chlorophyll content. Leaf nitrogen content reflects how well the plant is taking up and using nitrogen from the soil.

Study design

The work was carried out on tomato seedlings of the cultivar 'Little Tom', transplanted at 10 cm tall and 10 to 15 days old into sandy loam soil with an organic matter content of 3.82 mg/g and a pH of 6.93. Plants were grown under natural light at 22 to 25 degrees Celsius with relative humidity between 40 and 50%.

Each treatment group consisted of five plants, with three biological replicates.

Irrigation treatments used ozonated water at 1.0, 2.0, 3.0 and 4.0 mg/L, applied to the soil before transplanting and again after 20 days of growth. Control plants received ordinary water.

Spray treatments used ozonated water at 0.2, 0.4, 0.6 and 0.8 mg/L, applied after 15 days of growth and repeated five days later. Control plants were sprayed with ordinary water.

The ozonated water was produced with a commercial ozone water machine, its concentration confirmed with a dissolved ozone meter, and applied within five minutes of preparation - a detail that matters, because dissolved ozone begins decaying immediately.

Physiological measurements were taken five days after the final treatment. For the gene expression work, leaf and root samples were collected at 0, 6, 12, 24 and 48 hours after treatment at the best-performing concentration for each method. Results were analysed by t-test at P less than 0.05, with Tukey's test used to compare treatment means.

Findings

Irrigation at 3.0 mg/L was the best-performing treatment in the study. Compared with plants watered normally, MDA content fell by 53%, indicating less oxidative damage to cell membranes. Antioxidant enzyme activity rose sharply: SOD by 197%, POD by 286% and CAT by 149%. SPAD values and leaf nitrogen content both increased.

Spraying also produced measurable effects, at a far lower concentration. The best spray result came at 0.6 mg/L, where MDA fell by 32% and SOD, POD and CAT rose by 117%, 16% and 74% respectively.

The two optima are worth putting side by side. The concentration that worked best watered into the soil was five times the concentration that worked best on the leaves.

Higher was not better. Irrigation at 4.0 mg/L performed less well than at 3.0 mg/L, which the authors read as evidence of a ceiling above which ozonated water starts to work against the plant rather than for it.

In the gene expression work, expression of the antioxidant enzyme genes, the chlorophyll synthesis genes SlGLK1, SlGLK2 and SlDCL, and the nitrogen absorption genes SlAMT1-1 and SlNRT2.3 all peaked 12 hours after treatment. Irrigation promoted this gene expression more strongly than spraying did.

What the authors concluded

The authors recommend 3.0 mg/L for irrigation and 0.6 mg/L for foliar spray as the optimal concentrations, and state that the effect of ozone water irrigation on improving the physiological characteristics of tomato seedlings was better than that of spraying.

They also state plainly that high-concentration ozone water treatment has certain harmful effects on plants - a limit rather than a recommendation, and one of the more useful sentences in the paper.

Their proposed explanation is that ozonated water at moderate concentrations acts as a mild stressor, prompting the seedling to raise its own antioxidant defences, with knock-on improvements in chlorophyll synthesis and nitrogen uptake.

Limitations of this study

This is a small study. Each treatment group contained five plants with three biological replicates, which is a modest basis for the percentage changes reported.

It examined a single tomato cultivar, 'Little Tom', in a single soil type - sandy loam with a low organic matter content of 3.82 mg/g. Ozone reacts with organic matter, so results in a soil richer in organic material, such as a well-composted home garden bed, may differ. Nothing here establishes what happens in other species, other soils or other growing conditions.

The authors acknowledge that the specific molecular mechanism is still unclear, and say further work is needed to understand how ozonated water improves disease tolerance through gene upregulation.

The study measured plant physiology and gene expression. It did not measure yield, fruit quality, or disease incidence in the field, so it does not tell you whether healthier-looking seedlings go on to produce a better crop.

It also did not examine effects on soil biology. Ozone is not selective between organisms, and this study was not designed to detect what repeated ozonated irrigation might do to the beneficial microorganisms in a growing medium.

The concentrations here were produced by a laboratory-verified ozone water machine and used within five minutes. Dissolved ozone decays quickly, and the concentration actually delivered by any given device will depend on the equipment, the water and the time between making it and using it.