2. Methods for Seed Treatment
2.1. Seed Immersion
Seed immersion methods are those in which seeds are soaked in aqueous or solvent-based liquid for a certain length of time, depending on the nature of the seed coat and the substance used. The soaking results in partial or full hydration of both the host and pathogen and produces microscopic ruptures, making them more susceptible to the penetration of active substances compared to the dry state
[12]. Not all the substances are soluble in water, so in some cases (e.g., essential oil, chitosan), it is necessary to use an emulsifier to allow for mixing and emulsion homogeneity
[18]. Besides the direct antimicrobial effects that depend on the type of substance used, immersion treatment can have the following priming effects: increased germination rate and seedling vigor; induced diverse range of morphophysiological, biochemical, and molecular responses in plants; and thus improved abiotic and biotic stress tolerance and increased crop yields
[19]. Immersion represents the most widely used method for treatment with elicitors for resistance induction, such as chitosan and methyl salicylate
[20][21]. Timing of the treatment plays a key role in phytotoxicity, negatively influencing seed vitality
[20]. Moreover, excessive imbibition during seed submersion can damage the outer seed coats, especially in the case of seeds with softer teguments such as legume seeds
[22]. The challenge is to find the right combination of treatment durations for different seed types to ensure efficacy without causing phytotoxicity. Primed seeds are known to have low storage longevity, which can be partially remedied via post-storage treatments such as dehydration, heat shock, or post-storage humidification
[23]. The soaking process is considered cumbersome and time-consuming when treating large quantities of seeds at a large scale, because it requires a large volume of liquid and needs subsequent drying
[18].
2.2. Seed Dressing and Coating
Innovative seed coating and dressing technologies are useful as delivery systems for the application of active ingredients on the seed surface. The technique of seed dressing involves the application on the seed surface of a thin layer of the active product, such as pesticides, fertilizers, or growth promoters which can be applied both as dry or liquid formulations
[12]. Seed dressing is the most widely used method for low dosages of active components onto seeds
[24] and although there are many types of equipment used for coating, the most commonly used device is performed with a rotary coater
[18]. Seed coating is a technique in which an external material is applied to the surface of the seed using a binder which acts as an adhesive to improve the adhesion of the active ingredients to the seed. The role of the binder is also to ensure coating integrity during and after drying and to prevent cracking and dusting off during handling and sowing
[18]. The layer is applied to the seed typically from 2 to 5% of the seed weight
[25]. In this context, nanotechnology could represent an innovative tool exploitable in agriculture, since nanoparticles (materials with a size ranging from 1 to 100 nm)
[26][27] can be effective carriers of seed health-promoting compounds when applied as seed coatings or seed dressing material
[27]. Nanoagroproducts are an upcoming technology that might be beneficial for the development of future generations of formulations for seed treatment to enhance the sustainability of agricultural systems. Among them, a wide selection of organic and natural compounds can be loaded into these nanoparticles, including essential oils, cellulose, and chitosan, making this technology suitable for sustainable and ecofriendly farming. Basic substances can take advantage of this technology to take place in adapted formulations of seed coating products. Seed coating allows for a controlled release of the substance reducing the active ingredient dosage needed, thus reducing their release into the ecosystem and soils, the possible toxicity for plants and the environment, and the treatment cost. Nanoscale materials used in seed coating technologies such as nanocapsules, nanogels, nanofibers, nanoclays, and nanosuspensions are supposed to increase the accuracy and efficiency of seed protection products, allowing for a reduction in pesticides in the field
[27]. On the other hand, specific machines and equipment are required for seed dressing and coating techniques which are performed with a dry power applicator, rotary or drum machine, motor, or hand driving
[18].
3. Seed Treatment with Approved Basic Substances
3.1. Activity of Approved Basic Substances against Fungi and Oomycetes
Chitosan is a naturally occurring biopolymer with antimicrobial properties explored in agriculture for many uses as a plant defense inducer, growth promoter, and carrier for delivery systems of biocontrol agents
[28]. In 2014, chitosan hydrochloride was approved by the EU as one of the first basic substances for plant protection
[29], and a second chitosan formulation was approved in 2022
[30]. Chitosan has been shown to be effective against several species of seedborne pathogens
[31][32][33][34][35].
Besides chitosan, other compounds like mustard seed power, vinegar, and hydrogen peroxide were approved as basic substances by the European Union between 2015 and 2017
[15] and allowed for agricultural uses. Kowalska et al.
[36] recommended the dose of 15 g mustard meal per 1 kg common wheat grain (
Triticum aestivum ssp.
vulgare) as a seed dressing applied with 45 mL of water, to significantly reduce disease caused by
F. culmorum on wheat during the early stage of growth. The authors reported a stimulating effect of mustard meal seed dressing on seedling development without perceiving any negative influence on the germination and development of seedlings, accompanied by a reduction in the number of infected seeds and by a 43–78% disease incidence reduction in the field, according to the type of seed dressing applied, respectively, wet or dry. Berbegal et al.
[37] evaluated
Pinus radiata seed treatments using hydrogen peroxide (33%
w/
v, disinfectant conc. 30%) to control
Fusarium circinatum. Seeds artificially inoculated and treated by soaking in hydrogen peroxide were sown in peat moss and then maintained in a forest nursery. The reduction of disease incidence in seedlings from seeds treated with hydrogen peroxide ranged from 98.2% to 100% but the germination rate was also reduced compared to inoculated untreated seeds. Differently, hydrogen peroxide stabilized with silver ions applied to
Daucus carota seeds had no phytotoxic effects, and it caused a significant decrease in the percentage of seeds infested with
Alternaria radicina [38].
Table vinegar (pH = 3, acetic acid 5%) was also tested in order to reduce
Colletotrichum lupini seed infection on lupin (
Lupinus albus)
[39]. Anthracnose-infected seeds from highly infected plots were soaked in vinegar and grown under field conditions. The authors reported that vinegar treatment successfully reduced disease severity (16.9%) and increased yield to levels similar to those observed for certified seeds, without significantly affecting germination rate
[39].
3.2. Activity of Approved Basic Substances against Bacteria
The bactericidal action of oxygen released from peroxides is well known, and the possibility of direct horticultural benefits plus bactericidal activity make hydrogen peroxide attractive in agriculture for seed disinfection. However, there are only a few recent reports on in vivo or field applications. Since 2002, hydrogen peroxide was investigated as a seed treatment for the control of bacterial leaf spot of lettuce (
Lactuca sativa) caused by the seedborne bacterium
Xanthomonas campestris pv.
vitians. Bacteria were not detected when seeds were treated with 3 or 5% hydrogen peroxide, even if the treatments at 5% concentration reduced seed germination up to 28% compared with controls
[40]. More recent works about seed treatment with hydrogen peroxide against bacterial diseases have only come after years of research: hydrogen peroxide at 3% was investigated as a seed treatment against
Xanthomonas campestris pv.
campestris in cabbage (
Brassica oleracea) seeds
[41]. The treatment for 30 min was the most effective, both in terms of disinfection rate and of seed viability, but the side effects on the seed coat observed when the procedure was carried out at the company facilities suggested 15 min as the maximum time of immersion without losing effectiveness.
4. Seed Treatment with Potential Basic Substances against Pathogens
4.1. Activity of Potential Basic Substances against Fungi and Oomycetes
Essential oils (EOs) are secondary metabolites accumulated by aromatics or medical plants and extracted from leaves, flowers, roots, and barks. They exhibit antifungal activity due to the presence of different bioactive ingredients (alkaloids, phenols, monoterpenes and sesquiterpenes, isoprenoids) in different concentrations, their composition of which may vary even within the same species, affecting antimicrobial activity
[42][43]. EOs have widely demonstrated over the years their efficacy against various fungal pathogens in vitro
[44][45] and in recent years, the scientific research in this field has focused primarily on in vivo and field applications. Immersion seed treatment with clove (
Syzygium aromaticum) EO was able to reduce
Fusarium spp. infection on maize and wheat seeds at different doses, but the effective rates (5 × 10
3 and 5 × 10
4 ppm, respectively, for maize and wheat) had a high phytotoxicity effect
[46]. Clove oil has also been tested in field trials, both as a seed soak and as coating (spray) on wheat and field peas against, respectively,
Tilletia laevis [47] and
Ascochyta blight complex
[22], artificially inoculated on seeds, with good effectiveness, which varied from year to year. Submersion application has demonstrated a more reliable effectiveness over the years, compared to coating application. In the tomato, eucalyptus (
Eucalyptus grandis), caraway (
Cuminum cyminum), and citrus (
Citrus sinensis), EOs have been tested as seed treatments against
Fusarium oxysporum [48], and oregano EO (
Origanum vulgare), against
F. oxysporum f.sp.
lycopersici [49] artificially inoculated in soil, with a reduction in disease incidence and severity. Tomato seedlings showed no phytotoxic effects after soaking treatment at the applied rates.
Naturally contaminated
Colletotrichum lindemuthianum beans were treated with basil (
Ocimum gratissimum) and clove EOs, and the treatment caused a significant reduction in anthracnose incidence without affecting the germination and the emergence speed index
[50]. Lemongrass (
Cymbopogon citratus), lavender (
Lavandula dentata), lavandin (
Lavandula hybrida), tea tree (
Melaleuca alternifolia), bay laurel (
Laurus nobilis), and two different marjoram (
Origanum majorana) EOs were tested as seed treatments against the main
Cucurbita maxima seedborne fungal pathogens:
Stagonosporiopsis cucurbitacearum,
Alternaria alternata, and
F. solani [51]. The seed immersion treatments were carried out at a concentration of 0.5 mg mL
−1 for 6 h, with mixing every 30 min, and the results showed that the incidence of multiple seedborne fungal pathogens was significantly reduced on squash seeds, with no negative effect on germination. In addition, the
C. citratus EO increased seedling emergence and reduced the incidence of
S. cucurbitacearum in plantlets.
Waureck et al.
[52] found that the main fungi observed in organic and untreated lettuce seeds were
Cladosporium sp. and
Alternaria sp. seed, and treatments with clove, lemongrass, and rosemary EOs at a dose of 0.5% (
v/
v) significantly reduced their presence on seeds, but with negative effects on germination, suggesting that the application dose of these essential oils should be modulated for lettuce seeds
[52].
Exogenous application of specific plant extracts can induce resistance in the host plant via higher levels of host defense enzymes and PR protein stimulation. An absinthium (
Artemisia absinthium) EO seed coating was tested on tomato seeds and was able to protect seed germination and seedling growth, priming tolerance in tomato seedlings previously infected with
F. oxysporum f.sp.
lycopersici by the induction of metabolic changes responsible for the long-term tolerance of the tomato
[53]. An extract of
Jacaranda mimosifolia (1.2%) applied to maize seeds provided significant protective effects on plants compared to the inoculated control, by also inducing a systemic resistance in the host plants
[54].
Silver fir (
Abies alba), pine (
Pinus sylvestris), and thyme EOs were tested as seed treatments on onion by immersion for 6 h, and seed health test on potato dextrose agar showed that all the oil treatments effectively controlled
Fusarium spp. on the onion seeds and frequently reduced their infestation with
Botrytis spp. The lowest dose tested with antifungal activity and without phytotoxic effects was 0.2 µL cm
−3, while increasing the dose led to increased phytotoxicity
[55].
Commercial EOs obtained from different parts of black cumin (
Nigella sativa), mustard (
Sambucus nigra), St. John’s wort (
Hypericum perforatum), garlic (
Allium sativum), grape (
Vitis vinifera), and ginger (
Zingiber officinale) plants were evaluated in vivo against the oomycete
Plasmopara halstedii. The application of the above oils as a spray seed treatment was shown to provide protection against mildew in sunflower plants under in vivo conditions, assessed as a percentage reduction in the sporangium count ranging from 70.1% to 90.5%
[56].
In order to obtain the best advantages from the volatile nature of active compounds, oregano, thyme (
Thymus vulgaris), and coriander (
Coriandrum sativum) EOs were tested in vapor form for their antifungal potential against
Alternaria spp.,
Fusarium spp. and
Drechslera spp. infection on wheat seeds
[57]. Wheat seeds were stored in an atmosphere enriched with essential oil vapors and a selective antifungal effect was highlighted as the following: oregano EO and thyme EO significantly inhibited
Alternaria, Fusarium, and
Drechslera (that was the most sensitive). Regarding the phytotoxic effects of EO vapors on the germination of the seeds, thyme EO and oregano EO had an inhibitory effect, especially at 0.4%. This effect was cumulative over time. The EOs inhibited deoxynivalenol (DON) occurrence, and the maximum percentage of inhibition was obtained after 21 days of vapor exposure, with the most effective timing being when applied at 0.2%.
Ozone has been declared as a generally recognized as safe (GRAS) substance and its application in agriculture has increased in recent years
[58]. Ozone gas was applied on maize and wheat seeds for fungal decontamination: ozone gas application for 300 min at a rate of 60 mg L
−1 was able to reduce the incidence of
Aspergillus spp. and
Penicillium spp. (both ~ 54%) on artificially infected wheat seeds
[59], while 50 h application at a rate of 2.14 mg L
−1 reduced
Aspergillus spp. (78.5%) and
Penicillium spp. (98.0%) incidence on naturally infected maize seeds
[60]. Thanks to its oxidizing properties, ozonation can also represent an effective method for the remediation of cereals contaminated by mycotoxins, where gaseous ozone application for 480 min at the rate of 60 mg L
−1 reduced aflatoxins and microbial contamination in corn artificially infected with
Aspergillus spp. and
Penicillium spp.
[61].
4.2. Activity of Potential Basic Substances against Bacteria
Several studies have investigated the effects of potential basic substances to control bacterial seedborne pathogens. Kotan et al.
[62] revealed the antibacterial effects of different extracts of
Origanum onites (hexane, acetone, and chloroform) on tomato and lettuce seeds inoculated with
Clavibacter michiganensis ssp.
michiganensis,
Xanthomonas axonopodies pv.
vesicatoria, and
X. campestris pv.
zinniae. Extracts were applied by seed soaking after inoculation. The hexane extract was the most effective against
C. michiganensis ssp.
michiganensis, with a 75% disease severity reduction at 15 mg mL
−1, whereas the chloroform extract was more effective against
X. axonopodies pv.
vesicatoria and
X. campestris pv.
vitians, with a 77% reduction at 20 mg ml
−1 and a 74% reduction at 15 mg mL
−1, respectively. The authors attributed this strong antibacterial activity to the presence of carvacrol and thymol, two of EO’s major constituents. No phytotoxicity was found on seeds treated with all the extracts tested; indeed, different extracts even increased seed germination and plant height in tomato seedlings at concentrations of 5 and 10 mg mL
−1.
A study by Karabüyük and Aysan
[63] on the reduction in bacterial speck disease caused by
Pseudomonas syringae pv.
tomato demonstrated that immersion treatments of tomato seeds with aqueous extracts of
Zingiber officinale and
Origanum vulgare (Istanbul thyme) reduced 100% of bacterial speck disease incidence and severity on tomato seedlings. The antimicrobial activity of thyme
EO on soybean seeds infected with
P. savastanoi pv
. glycinea B076 and
P. syringae M7-C1, causal agents of bacterial blight in soybean, was investigated at a greenhouse scale by Sotelo et al.
[64]. Another study
[65] focused on the plant pathogenic bacteria
Burkholderia glumae showed that immersion treatment of rice seeds for 10 min with clove EO at 2% and 5%
v/
v and citronella (
Cymbopogon nardus) EO at 1% and 3% v/v reduced by 50% the disease incidence in plants, with the 5% clove oil treatment giving the highest rice grain production. However, no phytotoxicity data were provided.
Cistus ladaniferus subsp.
ladanifer EO
, together with its methanolic and ethanolic extracts, and
Mentha suaveolens EO, were used for the treatment of tomato seeds infected with the phytopathogenic bacterium
C. michiganensis subsp.
michiganensis [66]. The results evidenced that C.
ladaniferus subsp.
ladanifer oil and extracts and
Mentha suaveolens EO inhibited in vitro the growth of
C. michiganensis with a minimal inhibitory concentration (MIC) of 0.78 mg mL
1, but the in vivo treatment with such EOs at MIC and 4 × MIC showed a negative effect on tomato seed germination. In another study on the tomato, two other EOs (cinnamon and oregano) were tested in vivo for their antibacterial activity against
C. michiganensis subsp.
michiganensis [67].
Artificially infected tomato seeds were treated by immersion with these two oils at a concentration of 0.4% and their efficacy in controlling the pathogen was evaluated using a real-time PCR molecular assay for in planta bacterial quantification at the very first stage of development: both oils significantly reduced the bacterial presence in seedlings compared to controls (untreated and water-treated), with oregano being the most effective. Oregano EO showed no phytotoxicity at the concentrations tested up to 0.4%, while cinnamon EO had little effect on germination, reducing it by one or two percentage points.
4.3. Activity of Potential Basic Substances against Viruses and Phytoplasma
Basic substances or potential basic substances having a direct action on viruses or phytoplasma inside plant cells are nowadays quite unknown. Research directly targeting these pathogens inside the plant host cells by applying sustainable means of control is useful and highly recommended. Stommel and colleagues
[68] demonstrated that exposure of pepper mild mottle virus to ozone resulted in viral inactivation, but at insufficient levels to prevent viral transmission from highly contaminated pepper (
Capsicum annuum) seeds. Viruses and phytoplasma are non-culturable organisms; therefore, it is not easy to verify their direct effects on pathogens and just in vivo trials can be used. However, in vivo trials are much more complex and require infected materials with a high load of the pathogen to gain significant results. Virus and phytoplasma can be controlled by physical treatments such as thermotherapy or by controlling their insect vectors. Basic substances or potentially basic substances can also effectively be used against the vectors to reduce the spread of viruses and phytoplasma.