4.1. Effect of Incorporation of Aromatic Plants at Soil Environment (C/N Ratio and Volatiles)
A good indicator for the maturity of composts is a C/N range between 15 and 20 [
36]. In our work, C/N ratio was lower than 20 in the soil amended with spearmint or oregano at 60 DAI. Differences in C/N ratio values between soil amended with spearmint and oregano mainly at 0–30 DAI might be due to differences in C and N content of incorporated plant material, due to the decomposability of organic substrates [
37], as well as due to the anatomical features of spearmint and oregano. The short stabilization of C/N ratio in soil amended with spearmint and oregano indicates that the direct incorporation of the plant material into the soil—without the preposterous time-consuming process of composting the aromatic plants—is possible. In tomato cultivation, the C/N values 38.5 and 27.2, recorded at 0 DAI, in soil amended with spearmint and oregano respectively, are much lower than those reported for wood-residue composts [
38].
Essential oil composition of either spearmint or oregano changed dramatically with time. Oregano volatiles persist at high concentrations for a longer period than spearmint volatiles. This could possibly explain the inhibitory effect of oregano on tomato height (observed mainly up to 24 DAT), on stem thickness (at 12 DAT), on CCI (at 12 DAT) and on photosynthetic rate (at 15 DAT). The presence of monoterpenes in oregano oil–especially of oxygenated like carvacrol–has been previously related with plant growth inhibition [
39]. Generally, monoterpenes decreased in time and almost all of them were not detected at 90 DAI and this is in agreement with results of Karamanoli et al. [
40,
41]. Based on the herein reported outcomes, further research is needed to shed light on whether the primary reason for the antifungal effects of spearmint and oregano into the soil is due to the presence of terpenes. The current results confirmed in vivo the findings of previous study published by Kadoglidou et al. [
2], which carried out in vitro and in soil fungal cultures. In that study,
Vd was the most sensitive fungi (both
Vd’s growth and sporulation were inhibited) among the four fungi tested, whereas fungicidal effects were exerted of the presence of either spearmint or oregano essential oils, as well as of the presence of their main components, carvone and carvacrol. In the current study, the low ADI values observed for
Vd (
Table 6) may be partially attributed to the presence of carvone and carvacrol into the soil environment maintained until 15 and 60 DAI, respectively (
Table 3 and
Table 4). Nevertheless, the high ADI value for
Fol under oregano treatment in the current study is in contrast with the findings of Kadoglidou et al. [
2], who concluded that oregano essential oil was very drastic against Fol. These differences between two studies could partially attributed to the type of the tested material of aromatic plant (dried biomass/essential oil/components of essential oil) and consequently their differences in corresponding rates. Concerning the mechanism of action of terpenes against pathogenic fungi (bacteria and yeasts also) this is not fully understood, but it is speculated to involve membrane disruption by the lipophilic compounds. Several studies have shown that monoterpenes exert membrane-damaging effects to microbial strains and also stimulates leakage of cellular potassium ions which provides evidence of a lethal action related to cytoplasmic membrane damage [
42]. According to Trombetta et al. [
43] monoterpenes perturb the lipid fraction of microorganism plasma membrane, resulting in alterations of membrane permeability and in leakage of intracellular materials, whereas Cox et al. [
44] refers that they inactivate essential enzymes for physiological functions of microbes like respiration.
Regarding sesquiterpenes, they were detected in volatiles obtained from soil after spearmint and oregano incorporation, and in most cases at higher concentrations than in the initial oils. Monoterpenes and sesquiterpenes are found to act as allelochemicals which enhanced the plant defense, and several studies are focused on the isolation, introduction and overexpression of the responsible genes in genetically modified plants, to produce these terpenes [
45]. Several researchers have related the occurrence of sesquiterpenes such as
β-caryophyllene,
β-elemene and
α-humulene–which were detected in the current study—with root signalling on rhizosphere of cultivated plants [
46,
47], or as herbivore-induced plant volatiles, that attract natural enemies of the herbivores [
48]. Sesquiterpenes, apart from their supporting role in plant defense, act as plant growth promoters in vegetables (representing D, E and F and 1,2-dehydrolactarolide A at lettuce seedlings) [
49,
50]. In the present study, it should be pointed out the long persistence of sesquiterpenes in the soil environment (mainly
β-caryophyllene,
β-bourbonene and germacrene-D), which could possibly explain the positive effects on tomato growth, on physiological characteristics, and on yield as well.
4.2. Effect of Incorporation of Aromatic Plants at Tomato Cultivation
There is a plethora of organic composted or uncomposted materials that can be used as soil amendments on tomato seedlings production or on transplanted tomatoes. In general, literature demonstrated the enhancement of tomato seedlings growth by using compost from forestry wastes [
51], vine branches, grape prunings, husks and seeds [
52], spearmint compost [
17] and intact spearmint material [
19]. In most cases, literature revealed that the stimulation effect on tomato seedlings growth is accompanied by an increase in soil fungal and bacterial population and an inhibition of weed emergence.
Concerning the effect of composts on transplanted tomato, literature generally demonstrated the enhancement of growth, yield and quality of tomato, as well as the improvement of soil properties. More precisely, these composts were vermicompost produced from cattle manure, market food waste and recycled paper waste [
53], olive press cake, olive tree leaves and branches, vine branches and pressed grape skins [
54], rice straw [
55], vine branches, rice husks and straw and flax residues [
56] and
Phragmites australis plant material with animal manure [
57]. Moreover, De Carvalho et al. [
22] studied the allelopathic interactions in tomato intercropped with aromatic plants like basil, rue, fennel and mint and he found increases in yield per unit area. In current work, spearmint and oregano were incorporated into the soil without prior composting. Our findings indicate, that in contrast to high ratios of compost used in the most of the abovementioned studies, the spearmint and oregano material, incorporated into the soil at rate of 4% (w:w), enhanced significantly indices of growth and physiology of tomato as well as yield and soluble solids content of fruits. Specifically, compared to the untreated controls, the stem of tomato plants grown in soil amended with spearmint or oregano plant material was up to 58% taller and 59% thicker, whereas CCI was up to 142% higher, photosynthetic rate greater up to 81%, the yield higher up to 116%, and the soluble solids content greater up to 1.07 units. It should be noted that at 12 and 24 DAT, the incorporation of oregano into the soil caused a significant but transient inhibition in most of the above parameters, probably due to the higher carvacrol concentration. These increases of values of the abovementioned parameters probably related to the high organic matter content in soil treated with spearmint and oregano. According to the literature, the enhancement of the abovementioned parameters, could be, at least partially, attributed to the higher soil microbial activity associated with the decomposition of the incorporated aromatic plants and the concomitant nutrient release [
19,
53,
58]. In addition, increases in growth and yield could be attributed to plant growth regulators produced by microorganisms during the enhanced decomposition process [
59]. Canellas et al. [
60] and Scaglia et al. [
61] referred to the occurrence of humates or hormones into vermicompost, since exchangeable auxin groups in humic acids were detected. An interesting point in the current study are the increases in total soluble solids in tomato fruits, which are rather related to the incorporation of spearmint into the soil than the disease pressure in both fungi (
Table 7). These finding are in contrast to those reported of Papadaki et al. [
32], who reported high total soluble solids in the fruits of “Early Pack” cultivar due to the high Vd pressure. Nevertheless, all these assumptions require further research and validation, especially in the case of decomposition of aromatic plants. Τhe abundance of these aromatic plants—spearmint and oregano—in the Mediterranean ecosystem and their strong biological activity, combined with the abovementioned results in the current study, supports their potential use in soil management.
Organic soil amendments play important roles in the reduction of plant diseases caused by soilborne plant pathogens. With respect to the use of composts as soil amendment, it seems that composts may have highly suppressive effects against diseases caused by a variety of soilborne pathogens such as
Phytophthora spp. [
62],
Pythium spp. [
63],
Rhizoctonia spp. [
64] and
Fusarium spp. [
65]. Compost may suppress diseases in plants via a number of mechanisms, including antibiosis, competition, hyperparasitism and induction of systematic acquired resistance (SAR) in some host plants [
66]. The antagonistic interactions with other fungi (like
Trichoderma spp.) typically have been classified as based on antibiosis, mycoparasitism and competition for nutrients [
67]. Moreover, the effectiveness of disease suppression is affected by parameters like moisture, pH and carbon to nitrogen ratio in compost [
66].
In more detail, concerning
Fusarium and
Verticillium suppression into the soil using composts, Cotxarrera et al. [
65] found that compost from vegetable and animal market wastes, sewage sludge and yard wastes showed a high ability to suppress
Fusarium wild in tomato caused by
Fol race 1, whereas Cheuk et al. [
68] reported that
F. oxysporum f.sp.
radicis-lycopersici reduced when compost from yellow cedar sawdust used in greenhouse cultivated tomato, whose yield was improved by 74%. The co-compost of olive mill waste and leaves successfully suppressed Verticillium wilt in cotton and olive [
69]. Giotis et al. [
70] found that fresh Brassica tissue, household waste compost and composted cow manure significantly reduced soilborne disease severity (among them was
Verticillium albo-atrum) and increased also tomato fruit yield. Additionally, Davis et al. [
71] reported the suppression of
Verticillium wilt of potato using maize as green manure crop, due to change in the activity and composition of the soil microflora. As far as we know, there are a few studies in the literature which investigate the effect of aromatic plants, as compost, green manure or co-culture plant, in order to reduce soilborne diseases. Among them, Chouliaras et al. [
18] found that basil could be used as a co-culture plant, which can reduce the weed population and pathogenic soil organisms in organic farming, while exerting a positive effect on soil productivity. Kadoglidou et al. [
19] and Chalkos et al. [
17] reported that the incorporation of composted or uncomposted plant material of spearmint (intact dry material) has a positive effect in the soil environment (microbial density, action of nitrifying bacteria, soil respiration), whereas in vitro experiments [
2] revealed that essential oils of spearmint and oregano as well as their main components carvone and carvacrol had inhibitory effect against
Vd (fungicidal activity) and
Fusarium oxysporum (fungistatic activity). Towards to the same direction, recently rosemary, clove and thyme oils were able to reduce
Fusarium wilt in tomato [
26]. In current study, tomato grown in soil amended with 4% spearmint or oregano and inoculated with
Fol or
Vd had no macroscopic symptoms of disease after 50 DAT. Exception was the case of plants that were grown in soil amended with oregano and inoculated with
Fol, as occurred symptoms of chlorosis and loss of leaf area up to 50%. The remaining inoculated plants were not only seemed healthy, but also showed significantly higher values of physiological indices (plant height and stem thickness) compared to healthy untreated controls (increases about 29%–53% after 50 DAT). The abovementioned tendency strongly reflected in measurements of CCI and photosynthetic rate of plants, wherein the inoculated plants showed significantly higher values in the above indices compared to healthy untreated controls (among 36–65 DAT increases of 50%–66% in CCI and 41%–80% in photosynthetic rate). Our findings show that plants were grown in soil amended with oregano and inoculated with
Vd presented a time delay about 15 days to recovery, since, in comparison with the other treatments, they hold almost the lowest values concerning the evaluated parameters. Based on the above, we can assume that this phenomenon could be partially attributed to the long persistence of carvacrol into the soil environment (even after 60 DAI). Besides these impressive effects of spearmint and oregano incorporation into the soil, the yield of plants grown in soil amended with abovementioned aromatic plants and inoculated with
Fol or
Vd was increased by 77%–95% compared to healthy untreated controls, revealing that plants not only recovered from the initial inoculation but that they also benefited.