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Scientia Horticulturae 179 (2014) 25–35

Contents lists available at ScienceDirect

Scientia Horticulturae

journa l h om epage: www.elsev ier .com/ locate /sc ihor t i

romatic plants as soil amendments: Effects of spearmint and sage onoil properties, growth and physiology of tomato seedlings

alliopi Kadoglidoua, Dimitris Chalkosb, Katerina Karamanoli a, Ilias G. Eleftherohorinosc,elen-Isis A. Constantinidoua,∗, Despoina Vokoub

Laboratory of Agricultural Chemistry, Department of Crop Science, School of Agriculture, Aristotle University, GR-54124 Thessaloniki, GreeceDepartment of Ecology, School of Biology, Aristotle University, GR-54124 Thessaloniki, GreeceLaboratory of Agronomy, Department of Crop Science, School of Agriculture, Aristotle University, GR-54124 Thessaloniki, Greece

r t i c l e i n f o

rticle history:eceived 31 January 2014eceived in revised form 26 July 2014ccepted 3 September 2014

eywords:ompostsssential oil degradationrganic farmingenewable inputsoil microbesomato seedbeds

a b s t r a c t

Improvement of soil characteristics through the use of renewable inputs is fundamental to environ-mentally friendly farming systems. In the present study, the potential of improving soil properties and,consequently, growth of tomato (Lycopersicon esculentum L.) seedlings through a direct incorporation ofaromatic plant tissues into seedbeds is assessed. Dried spearmint (Mentha spicata L.) and sage (Salviafruticosa Mill.) tissues are incorporated at different rates into the soil of experimental field plots. At 0,20, 40, 60, and 90 days following incorporation, soil samples are removed from the plots and used assubstrates in tomato seedbeds. Growth and physiological parameters of tomato seedlings (emergence,size of the most robust leaf, shoot length, dry weight, net photosynthetic rate, stomatal conductance,photosynthetic yield) as well as soil attributes (pH, nitrogen and organic carbon content, organic matterdecomposition rate, microbial populations, changes in essential oil content) are monitored. Spearmintincorporation into the soil improved emergence, physiology and growth of tomato seedlings. This was notthe case with sage. Soil microbial populations and organic matter decomposition increased with increas-ing rate of incorporated aromatic plant tissues, especially in the case of spearmint which exhibited amore prominent increasing trend. Soil pH was not affected, remaining within the range for optimumtomato growth. Further, C:N ratio increased, yet it did not inhibit tomato growth. Lastly, the observed

decrease with time of the essential oil content in soil was dependent on the aromatic plant incorporated,and is discussed in relation to the beneficial effects of spearmint on tomato growth. The herein under-taken study demonstrates that incorporating intact spearmint tissues into the soil is a promising tool forimproving tomato seedling production. This practice circumvents the arduous composting process and,therefore, it can be more cost-and-time-effective compared to the currently applied techniques.

© 2014 Elsevier B.V. All rights reserved.

. Introduction

Soil amendments aim to improve soil properties. This includesncrease of the soil organic matter and of the nutrient pool,

Abbreviations: DAE, days after establishment of the field experiment (i.e. daysfter incorporating aromatic plant tissue into the soil, coinciding with the samplingimes); CFU, colony forming units.∗ Corresponding author. Tel.: +30 2310 998631/+30 2310 998639;

ax: +30 2310 998848.E-mail addresses: kkadogli@agro.auth.gr (K. Kadoglidou), dchalkos@bio.auth.gr

D. Chalkos), katkar@agro.auth.gr (K. Karamanoli), eleftero@agro.auth.grI.G. Eleftherohorinos), constad@agro.auth.gr (H.-I.A. Constantinidou),okou@bio.auth.gr (D. Vokou).

ttp://dx.doi.org/10.1016/j.scienta.2014.09.009304-4238/© 2014 Elsevier B.V. All rights reserved.

stimulation of beneficial microbial populations and/or suppressionof pathogens and weeds. The desired outcome of all these benefi-cial effects is the improvement of soil fertility and consequently ofits productivity. Soil amendments are of particular importance inorganic farming; most often, they are composted organic materialsof different origin.

The biological process of composting is the most com-monly used method for the fermentation of organic materialsprior to their incorporation into the soil. The key element ofthis process is the production of a stable and mature end

product suitable for use as soil amendment. In general, thecomposted organic material positively affects important soilfeatures and processes, and has beneficial impacts on the envi-ronment surrounding agricultural systems (Hargreaves et al.,

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6 K. Kadoglidou et al. / Scienti

008; Toumpeli et al., 2013). In particular, activity, diversity,nd structure of soil microbial communities have been shown toespond fast to such agricultural management practices. Hence,hey can provide insight into the impact of management onoil quality (Arancon et al., 2003; Diacono and Montemurro,010).

There is a plethora of composted materials that can be useds soil amendments in agricultural systems. So far the primaryaterials used for composts have been municipal and agriculturalastes. This practice is attributed to the environmental benefits

ssociated with the use of such materials contributing to pol-ution reduction and nutrient recycling, besides maintaining soilertility (Hargreaves et al., 2008; Snyman and Vorster, 2011; Tellat al., 2013). Consequently, the use of alternative sources for com-ost, such as naturally occurring or cultivated aromatic plants, haseceived relatively limited attention thus far (Chalkos et al., 2010;hima et al., 2009).

However, there is important scope in examining aromaticlants as potential soil amendment due to their abundance in theediterranean cultivated and natural ecosystem and due to theirultifaceted biological activity. In particular, their essential oils

ave been shown to selectively affect various organisms induc-ng both stimulatory and inhibitory effects on weed germinationnd growth (Angelini et al., 2003; Dudai et al., 1999; Vasilakoglout al., 2007), inhibiting several plant pathogens (Daferera et al.,003; Kadoglidou et al., 2011; Karamanoli et al., 2000), and enhanc-

ng soil metabolism and microbial activity (Broudiscou et al.,007; Owen et al., 2007; Vokou and Liotiri, 1999; Vokou et al.,002). Use of these plants and of their metabolites in agricultureould have many advantages because of their (i) natural origin,aking them less harmful than synthetic chemicals, (ii) volatil-

ty, implying less residue on the produce or in the environmentfter application, and (iii) composite nature, implying multipleechanisms of action that prevent pathogens from developing

esistance to all participating compounds (Chalkos et al., 2010).evertheless, we are dealing with costly materials, therefore thisractice might be advisable only in small scale applications, e.g.

n seedbeds for production of profitable vegetables or ornamentalrops.

In this respect, the authors previously assessed (Chalkos et al.,010) the potential of using composts derived from spearmintnd sage as soil amendments in tomato cultivation. To this end,his paper considers the incorporation of intact (above grownry biomass) material from aromatic plants directly to the soil,hat is, with no prior composting, to improve productivity inomato (Lycopersicon esculentum L.) seedlings. In this manner, theoil amendment practice becomes easier, faster and more cost-ffective. To this aim, dried spearmint (Mentha spicata L.) and sageSalvia fruticosa Mill.) tissues are considered for the purpose andheir effectiveness is gauged by monitoring a comprehensive listf plant and soil parameters. It is noted that the choice of tomatos a case-study has been motivated by the fact that it is amonghe ten most important crops in South Eastern Europe (FAO, 2012)nd by the fact that it is often used in rotations (Poudel et al., 2001)nd, therefore, it holds an important role in alternative farming sys-ems. Our interest was focused on tomato seedbed management,pecifically on the requirement of producing healthy and rapidlyrowing seedlings in nurseries. It is estimated that a reduction ofhe transplanting period by only 3–5 days provides surplus ben-fit on the income of the tomato seedling suppliers. In addition,rowers aim at producing robust seedlings as early in the growingeason as possible, so as to minimize infections and subsequently

aintain productivity in the greenhouse. Therefore, any manip-

lation that might satisfy the above requirements is of outmostmportance.

iculturae 179 (2014) 25–35

2. Materials and methods

2.1. Plant material

The aromatic plants used in this study are the same as the onesused by Chalkos et al. (2010) in the form of composts, i.e. spearmintand sage. The two species differ in both their habit and essentialoil. Spearmint is a herbaceous perennial species, whereas sage is ashrub. They both grow abundantly in the wild and are also culti-vated in Greece. Spearmint, in particular, is the commonest mintspecies in the country forming large populations at an altitudebetween sea level and 1500 m (occasionally up to 2000 m) (Kokkiniand Vokou, 1989).

The plant tissues applied here are the whole abovegroundbiomass, for spearmint, and the upper part of shoots, for sage. Thesewere purchased from a commercial supplier. Plant tissues were cutinto small pieces, air-dried in the dark till moisture content wasapproximately 5–7%, and stored in a cool (12 ◦C), dark and dry placeuntil use. Storage duration varied from a few days to a few months,given that the whole experiment had to be repeated in time.

To estimate the essential oil content of the two aromatic plants, aquantity of the plant material purchased was water-distilled whendry (100 g each time) for 3 h in a Clevenger apparatus. The essentialoils thus extracted from each species were analyzed by gas chro-matography as described by Vokou et al. (1993) and by GC–MS asdescribed by Karamanoli et al. (2008). The major constituents ofthese essential oils were identified on the basis of the retentiontimes and according to the chemical profiles found by Karousouet al. (1998) and Kokkini and Vokou (1989). The essential oil yield ofspearmint was 1.6 ml 100 g−1 d.w., that of sage 1.5 ml 100 g−1 d.w.Carvone (>50%) and 1,8-cineol (>40%) were the major constituentsof the essential oils of spearmint and sage, respectively.

The above aromatic plants were applied as soil amendmentsin tomato seedbeds. Seeds (84% germination) of Carla F1 hybridtomato (Lycopersicon esculentum L.) were used in the experiments.

2.2. Experimental design, methods and conditions

A field experiment was established at the farm of the AristotleUniversity of Thessaloniki (40◦ 32′ 08.74′ ′N and 22◦ 59′17.76′ ′E).The soil consisted of 32% clay, 56% silt, 12% sand, 1.5% organic matterand 7.5% CaCO3 (analysis performed at the Soil Science Institute ofthe National Agricultural Research Foundation, Thessaloniki). Soilcation exchange capacity was 28.6 meq 100 g−1. The experimen-tal field, left in fallow for a 10-year period, was subdivided into24 plots, 50 × 50 cm in size, separated from each other by a 50 cmwide alley. The top soil (upper 15 cm) from each plot was removed,weighed and mixed with plant material of either spearmint or sage,at rates of 0, 2, 4 and 8% (w/w, plant material:soil). Following this,the soil–aromatic plant mixture was put back to the plots (15 cmdepth), at the bottom of which a plastic mesh had been placed. Themesh served in avoiding both an uneven incorporation of the mix-ture into the remaining soil, and a removal of samples from deeperuntreated soil layers. The experimental field area was weekly irri-gated and left without any further intervention. The examined soiltreatments along with their abbreviations are shown in Table 1.A 2 × 4 (two types of plant material × four rates) factorial experi-ment was used in a randomized complete block design with threereplications (plots) per treatment. The entire field experiment wasrepeated in time.

field experiment (DAE), i.e. the replacement of the top soil by thesoil–aromatic plant mixture (hence, the initiation of the decompo-sition process of the plant material), soil samples from each treated

K. Kadoglidou et al. / Scientia Hort

Table 1Soil treatments applied and their abbreviations.

Abbreviations Treatments

0 Untreated soilM2 Soil + 2% (w/w) spearmint plant materialM4 Soil + 4% (w/w) spearmint plant materialM8 Soil + 8% (w/w) spearmint plant materialS2 Soil + 2% (w/w) sage plant material

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lot were taken and sieved through a 3 mm sieve. Sieved samplesere placed in plastic pots (150 g per pot, 9 pots per treatment)

nd seeded with six tomato seeds per pot. Pots were then placedn a controlled temperature greenhouse where no other cultivationreatment was applied except watering (once daily) and thinningf the emerged seedlings to three per pot. Greenhouse conditionsere the following: 20 ± 4 ◦C day temperature, 15 ± 3 ◦C night tem-eratures, 70% relative humidity, 10–12 h day-duration at ambient

ight, 12–14 h night-duration. Growth and physiological parame-ers of emerging seedlings were evaluated when the plants became0 days old.

At each sampling time, which coincided with DAE (0, 20, 40,0, 90 DAE), pH, organic C:N ratio, changes in essential oil contentf the aromatic plant incorporated into the soil, as well as bacte-ial and fungal population sizes of the soil–aromatic plant mixturesere evaluated per experimental field plot. Decomposition rate of

rganic material was also recorded in the field plots at 40 and 90AE (at approximately the middle and at the completion of thexperimental period).

.3. Growth characteristics

In each pot experiment conducted with soil samples takent 0, 20, 40, 60 and 90 DAE, percent emergence of the 20-dayld tomato seedlings, as well as their agronomic characteristics,uch as shoot length, size of the most robust leaf (based on leafength × width), and dry weight per seedling, were determined. For

eight determination, plants were removed from the pots, gentlyinsed with water to eliminate the soil from the roots, dried at0 ◦C for 48 h and weighed. For length and weight measurements,7 seedlings of each experiment/treatment (three seedlings perot × three pots × three replicate field plots) were used, whereasine seedlings (i.e. one seedling per pot × three pots × three repli-ate plots) were used for the size of the most robust leaf.

.4. Physiological parameters

Net photosynthetic rate (Anet, �mol m−2 s−1) and photosyn-hetic yield, as well as stomatal conductance (gs, mol m−2 s−1) wereecorded on 20-day old tomato seedlings grown in soil samplesaken from the field plots at 40 and 90 DAE. Nine measurementsere taken per treatment, per physiological parameter and per

xperiment. Measurements for all physiological parameters wereonducted on the terminal leaflet of the second composite leaf,ounting from the plant apex. Photosynthetic rate and stomatalonductance were monitored using the portable photosynthesisystem LI-6400 (LI-COR, Inc. Lincon NE, USA). The CO2 concentra-ion inside the photosynthesis system cuvette was at 340 ppm, their temperature at 22 ◦C with a relative humidity of 70–75% and aapour pressure deficit of 1.1 KPa. Chlorophyll fluorescence param-ters were recorded on leaves (chosen as above) of completely

dapted to light seedlings using a direct portable fluorometer (Pho-osynthesis yield analyzer MINI–PAM, Walz, Effeltrich, Germany).hotosynthetic yield (effective quantum yield of photochemicalnergy conversion in photosystem II) was computed in terms of

iculturae 179 (2014) 25–35 27

the efficiency of the energy harvesting by open PSII reaction cen-ters in the light as follows: Photosynthetic Yield (Y) = (Fm

′ − F0′)/Fm

′,where Fm

′ is the maximal chlorophyll fluorescence yield under lightconditions and F′

0 the steady state fluorescence prior to the lightflash (Thwe et al., 2014). Procedures for measuring and computingall physiological parameters tested were described in previous pub-lications (Kadoglidou et al., 2008; Ouzounidou and Constantinidou,1999; Samartzidis et al., 2005).

2.5. Soil bacterial populations

At each sampling time (0, 20, 40, 60, 90 DAE), total bacterialpopulations of the soil–aromatic plant mixtures were estimatedper field plot using a standard plating procedure based on serialdilutions of the initial sample (Karamanoli et al., 2000). At eachsampling time (DAE), nine measurements per treatment wererecorded, as following: Three samples (replications), each con-sisting of 5 g of soil, were taken per plot. Each sample was thensuspended into a 100-ml flask containing 50 ml of phosphate buffer(0.1 M potassium phosphate, pH 7.0, amended with 0.1% bacto-peptone). Flasks were shaken for 30 min at 150 rpm, then serialdilutions of each suspension were carried out. Final dilutions (10−3

to 10−4) were spread on plates filled with non-selective solid Nutri-ent Agar (10 g tryptone peptone, 5 g yeast extract, 5 g NaCl, 15 gBacto agar, 1 L dd H2O). Three agar plates per dilution were pre-pared and incubated in the dark at 24 ± 2 ◦C for 72 h. At the end ofthe incubation period, bacterial colonies were counted and popula-tions were expressed as colony forming units (CFU) g−1 of soil dryweight.

2.6. Soil fungal populations

For determining the total fungal population, the soil-dilutionplate technique was applied (Kadoglidou et al., 2011). Three sam-ples (replications) of each soil–aromatic plant mixture were dilutedin 0.2% Water Agar and plated on non-selective solid Czapek DoxAgar (2 g NaNO3, 1 g K2HPO4, 0.5 g MgSO4·7H2O, 0.5 g KCl, 0.01 gFeSO4, 30 g sucrose, 20 g agar, 1 L dd H2O) supplied with antibiotics(streptomycin sulfate, 150 mg L−1) and galactic acid (0.8 ml L−1).Three agar plates per dilution were prepared and incubated at24 ± 2 ◦C for 5–6 days. The fungal colonies were then countedand populations were expressed as CFU g−1 of soil dry weight. Ateach sampling time (DAE), nine measurements per treatment weretaken.

2.7. Soil parameters

Soil pH was measured with an electronic pH meter at a 1:1water–soil ratio using 5 g of each soil sample and adding water untilthe soil had reached its maximum plasticity. The organic carboncontent was determined by a wet oxidation method, as following:Soil samples (0.1 g) were oxidized using a K2Cr2O7 H2SO4 solu-tion (1:1 v/v) and organic carbon in them was estimated throughferrous ammonium sulphate titration. The total nitrogen in the soilsamples was quantified by applying the Kjeldahl method.

To determine decomposition of organic matter under fieldconditions, 0.22 g of coarse filter papers (100% cellulose, Fisher 09-795C) were placed in a plastic mesh and then buried in the soil plotsamended with spearmint and sage. The filter papers were sampledat approximately the middle (40 DAE) and the end (90 DAE) of theexperimental period [(9 filter papers per sampling time (DAE), pertreatment)]. The data obtained were expressed as percent loss of

the filter paper initial weight.

At 0, 20, 40, 60 and 90 DAE the content of the essential oils inthe soil in which aromatic plant tissues had been incorporated, wasdetermined. Samples of 2 kg each were taken per experimental plot

2 a Horticulturae 179 (2014) 25–35

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nd subjected to a 3-h hydrodistillation in a Clevenger apparatus.he amount of essential oils obtained was expressed in �l per00 g of soil d.w. and compared to that of the initial plant material

ncorporated into the soil.

.8. Statistical analysis

A combined over the two field experiments (ET, experimentsepeated in time) analysis of variance was performed to test the

× 4 (two tested plant materials × four rates of incorporation)actorial arrangement, DAE being a sub-plot factor (repeated meas-res). Since the combined over the two field experiments analysisf variance had indicated no differences between them, the pre-ented treatment means were averaged over the two experiments.onferroni test procedures were used to detect and separate meanreatment differences at P < 0.05. All statistical analyses were per-ormed using the STATISTICA software (ver. 7.06, Statsoft Inc.).

. Results

.1. Tomato seedling emergence and growth parameters

Analysis of variance applied on data obtained from tomatoeedlings emergence and from the determined growth parametershowed significant effects due to sampling time (DAE), incorpo-ated aromatic plant, rate of incorporation, and some of theirnteractions (Table 2).

In particular, decomposition spearmint tissues into the soil sig-ificantly improved tomato seedling emergence at all samplingimes (20, 40, 60 and 90 DAE) and at all rates applied. At all ratesnd sampling times, seedling emergence was greater by at least0% compared to controls (Fig. 1).

For shoot length and dry weight, the effect of spearmint compostas significant at all rates leading to an average increase of 70% and

5%, respectively, compared to control (Fig. 2a and b). The size of theost robust leaf was 2–3 fold greater than that of control (Fig. 2c).Incorporation of sage into the soil did not have any stimula-

ory or inhibitory effect on tomato seedling emergence (Fig. 1) asompared to controls. However, sage tissues at 20 and 40 DAEnhibited tomato seedling elongation by more than 20%, whereast 90 DAE there was a significant increase as compared to controlFig. 2a). Similarly, sage decomposing tissues brought about a sig-ificant decrease on the size of the most robust leaf in almost allates of incorporation and sampling times as compared to controllants (Fig. 2b). Regarding dry weight, tomato seedlings grown inreated soil samples were significantly lighter than seedlings grownn untreated soil for almost all rates at which sage tissues werencorporated into the soil (Fig. 2c).

.2. Physiological parameters

The analysis of variance applied on net photosynthetic rate, sto-atal conductance and photosynthetic yield data obtained from

omato seedlings grown in soil samples taken at 40 and 90 DAEhowed that these parameters were in most cases significantlyffected by sampling time (DAE), incorporated aromatic plant, ratef incorporation, and the interaction between incorporated aro-atic plant × rate of incorporation (Table 2).At 40 DAE, net photosynthetic rate of seedlings grown in soil

mended with 4 and 8% of decomposing spearmint tissues was sig-ificantly different from control seedlings, being 18 and 34% higher,espectively. Regarding the sage treatments, a significant effect was

ecorded and the respective increases were 13 and 16% (Fig. 3a). At0 DAE, the net photosynthetic rate of seedlings grown in amendedoil did not differ from control seedlings by more than 8%, whateverhe aromatic plant applied. Specifically, there was a non-significant Ta

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K. Kadoglidou et al. / Scientia Horticulturae 179 (2014) 25–35 29

Fig. 1. Percent emergence (means ± se) of tomato seedlings grown in soil amended with 0, 2, 4 or 8% (w/w) of spearmint (M) or sage (S) tissues, determined at 0, 20, 40, 60a mparea

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nd 90 days after the establishment of the field experiments (DAE). Means were cond graphically.

–4% increase in net photosynthesis of sage and 1–8% increase inpearmint, as compared to control.

Stomatal conductance of tomato seedlings exhibited a compa-able tendency to the photosynthetic rate. In seedlings grown inoil samples amended with 4 and 8% of spearmint, stomatal con-uctance was significantly higher than that of control seedlings atoth 40 and 90 DAE, by 24% at 40 DAE and 18% at 90 DAE (Fig. 3b).

n tomato seedlings grown in soil amended with 4 and 8% of sage,tomatal conductance was significantly higher as well, but only upo 10%.

Photosynthetic yield of tomato seedlings was significantlyigher at 40 than at 90 DAE. At both sampling times, seedlingsrown in soils amended with 4 and 8% of decomposing spearmintissues had a 10 up to 20% increase in photosynthetic yield com-ared to control seedlings. Seedlings grown in soil amended withage were not significantly affected, except at the 8% rate at 90AE, where photosynthetic yield showed a 10% increase compared

o control seedlings (Fig. 3c).In all non-mentioned cases, there were no significant differ-

nces in the physiological parameters examined between controlnd treated plants.

.3. Microbial populations and soil environment

The soil bacterial population analysis of variance showed thathis parameter was significantly affected by sampling time, incor-orated aromatic plant, rate of incorporation and the interactions ofampling time × incorporated aromatic plant, sampling time × ratef incorporation, incorporated aromatic plant × rate of incorpo-ation, and sampling time × incorporated aromatic plant × rate ofncorporation (Table 2). The size of bacterial populations was sig-ificantly higher in soils treated with spearmint or sage than inontrol soils in all cases and for the whole duration of the exper-ment (Fig. 4). The recorded increase was proportional to the ratet which spearmint or sage material was incorporated into the soil.omparing the effect of the two aromatic plants, it becomes evidenthat the increase induced by spearmint far exceeded that inducedy sage at all sampling times, except at 0 DAE, at which time allamples had similarly sized bacterial populations.

Analysis of variance applied on fungal population data obtainedrom soil samples indicated significant effect due to sampling time,ncorporated aromatic plant, rate of incorporation and their inter-

ctions (Table 2). Specifically, the size of fungal populations inoil samples taken at 0 DAE did not differ among treatments.t increased significantly in soil samples amended with eitherpearmint or sage in all cases and for the whole duration of the

d by the Bonferroni adjusted LSD value at P < 0.05. LSDB is given both numerically

experiment (Fig. 5). In general, regardless of the aromatic plantapplied, fungal density tended to increase proportionally with theincreasing rate of incorporation as well as with the time havingelapsed after the establishment of the experiment, up to 40 DAE.After this sampling time, values tended to drop, though not sig-nificantly most of the times. The highest fungal population wasrecorded in soil treated with 8% spearmint tissues at 40 DAE.

The cellulose decomposition analysis of variance showed sig-nificant effects due to sampling time, incorporated aromaticplant, rate of incorporation and the interactions of samplingtime × incorporated aromatic plant, as well as of incorporated aro-matic plant × rate of incorporation (Table 2). Decomposition rateswere significantly higher in soil samples amended with spearmintor sage compared to unamended samples (Table 3).

Losses in cellulose weight were higher in soils amended withspearmint than with sage and increased with increasing rate ofincorporated plant material. In particular, at 40 DAE, the weight ofcellulose pieces in untreated soil samples was reduced by 7%, whenthe respective reduction in soil–aromatic plant mixtures rangedbetween approximately 16 and 48%, depending on the aromaticplant and the rate at which it was applied. Similarly, at 90 DAE, cel-lulose lost only 29% of its weight in untreated soil samples, whereasin those amended with 4 and 8% of either spearmint or sage it wasalmost fully decomposed (weight loss ranging between 85 to 99%).

Analysis of variance performed on soil pH data indicated sig-nificant effects only due to sampling time (Table 2). Namely,incorporation of either spearmint or sage into the soil decreased soilpH from 7.8 at 0 DAE to 7.5 at 90 DAE (averaged over incorporatedaromatic plant and rate, data not shown).

The C:N ratio was affected by sampling time, incorporated aro-matic plant, rate of incorporation and the interactions of samplingtime × incorporated aromatic plant, sampling time × rate of incor-poration and incorporated aromatic plant × rate of incorporation(Table 2). In all samples treated with either spearmint or sage, theC:N ratio was higher than in control samples with values beinggreater in those treated with sage for the whole duration of theexperiment. In general, soil C:N ratio values tended to increase withincreasing rate of incorporated aromatic plant and decrease withtime. In particular, at 0 DAE, the C:N ratio ranged from 7.8 (controlsoil) to 42.4 (soil amended with sage at 8%), whereas at 90 DAE therespective values were 6.6 and 12.9 (Table 4).

The amount of essential oil obtained by hydrodistillation from

plots where the soil was replaced by a soil–aromatic plant mixturewas affected by the incorporated aromatic plant, the rate of incor-poration and the sampling time. Specifically, analysis of samplestaken at 0 DAE showed that the essential oil yield was proportional

30 K. Kadoglidou et al. / Scientia Horticulturae 179 (2014) 25–35

Fig. 2. Means (±se) of (a) shoot length (b) size of the most robust leaf and (c) total dry weight of tomato seedlings grown in soil amended with 0, 2, 4 or 8% (w/w) of spearmint(M) or sage (S) tissues, determined at 0, 20, 40, 60 and 90 days after the establishment of the field experiments (DAE). All parameters were recorded in 20-day old tomatoseedlings. Means were compared by the Bonferroni adjusted LSD value at P < 0.05. LSDB is given both numerically and graphically.

Table 3Decomposition rate expressed as percent loss of dry weight (means ± se) of cellulose pieces buried in soils amended with spearmint or sage tissues at incorporation rates of0, 2, 4 and 8% (w/w). Cellulose pieces were recovered and weighed at different sampling times, viz. at 40 and 90 days after the establishment of the field experiment (DAE).Pairwise comparisons were performed between aromatic species within each application rate and at each DAE. Within each DAE, means followed by the same letter are notstatistically significant different at P < 0.05, according to Bonferroni adjusted LSD value (LSDB = 5.6).

DAE Plant material % Loss of initial cellulose weight

Incorporation rate (%)

0 2 4 8

40 Spearmint 7.2 ± 0.8 e 35.2 ± 1.8 bc 34.0 ± 1.4 bc 47.8 ± 2.5 aSage 7.2 ± 0.8 e 15.5 ± 0.4 d 33.5 ± 2.1 c 39.4 ± 3.8 b

90 Spearmint 28.5 ± 2.2 e 58.7 ± 3.6 c 95.9 ± 3.4 a 99.0 ± 0.4 aSage 28.5 ± 2.2 e 50.3 ± 2.6 d 84.5 ± 4.5 b 93.7 ± 3.8 a

K. Kadoglidou et al. / Scientia Horticulturae 179 (2014) 25–35 31

F photoo ments SDB is

tesr6dcoo

ro4

ig. 3. Means (±se) of (a) net photosynthetic rate (b) stomatal conductance and (c)f spearmint (M) or sage (S) tissues, determined at 40 and 90 days after the establisheedlings. Means were compared by the Bonferroni adjusted LSD value at P < 0.05. L

o the incorporation rate (Table 5). For spearmint, the amounts ofssential oil obtained at 0 DAE were 30.5, 62.5 and 123.5 �l 100 g−1

oil for the rates 2, 4 and 8% (w/w, aromatic plant material:soil)espectively. These values are very close to the expected ones (32,4 and 128 �l 100 g−1)1, considering that distillation of 100 g ofried spearmint tissues yielded 1.6 ml of essential oil. Similar is the

ase for sage yielding 1.5 ml per 100 g d.w. The amount of essentialil decreased with the time having elapsed after the establishmentf the experiment for all soil treatments. For instance, after a period

1 They are calculated as follows: Spearmint was incorporated in the soil at theates of 2, 4, and 8% (w/w, plant material:soil). As the plant’s content in essentialils was 16 �l g−1, in the 100 g of the soil-spearmint mixture, there should be 2 × 16,

× 16, and 8 × 16 �l of essential oil, respectively.

synthetic yield of tomato seedlings grown in soil amended with 0, 2, 4 or 8% (w/w) of the field experiments (DAE). All parameters were recorded in 20-day old tomato

given both numerically and graphically.

of 60 days, the essential oil content in the soil amended with 4% ofspearmint or sage tissues was reduced by 94% and 76% respectively,as compared to that determined at 0 DAE.

4. Discussion

The beneficial seedling responses to the addition of spearminttissues in the growth substrate presently reported are in agree-ment with those of other studies evaluating the effects of compostsof different origin on tomato growth including that of spearmint

compost (Chalkos et al., 2010). Ribeiro et al. (2007) reported thatroot dry weight and leaf number of tomato seedlings grown onsubstrates containing compost from forest wastes (mixed withsolid phase of pig slurry) increased by 60 and 63%, respectively,

32 K. Kadoglidou et al. / Scientia Horticulturae 179 (2014) 25–35

Fig. 4. Bacterial populations (means ± se) in field soil amended with 0, 2, 4 or 8% (w/w) of spearmint (M) or sage (S) tissues, counted at 0, 20, 40, 60 and 90 days after theestablishment of the field experiments (DAE). Means were compared by the Bonferroni adjusted LSD value at P < 0.05. LSDB is given both numerically and graphically.

F /w) oe roni a

aea(sawcdtc

TCwa

ig. 5. Fungal populations (means ± se) in field soil amended with 0, 2, 4 or 8% (wstablishment of the field experiments (DAE). Means were compared by the Bonfer

s compared to those of plants grown on soil. Similarly, Atiyeht al. (2002) reported that plants performed best when grown in

medium containing organic composts at 10–40% (v/v). Manios2004) showed that vegetables (including tomato) grew better inoil amended with 30% compost (v/v) from olive or vine tissues,nd Ali et al. (2003) found that tomato fruit increased in weight,hen plants were grown in media amended with 30% of rice-straw

ompost (w/w). Kostov et al. (1996b) reported that composterived from vine, rice and flax tissues increased the yield ofomatoes by 24–61% compared to control and suggested thatomposts from these materials increase water and nutrient uptake

able 4:N ratio (means ± se) in soil samples taken at different sampling times, viz. at 0, 20, 40,

ere each amended with 0, 2, 4 or 8% (w/w) of spearmint or sage tissues. Pairwise compat each DAE. Within each DAE, means followed by the same letter are not statistically sign

DAE Plant material C:N

Incorporation rate (%)

0

0 Spearmint 9.1 ± 0.4 d

Sage 7.8 ± 0.4 d

20 Spearmint 9.0 ± 0.7 e

Sage 7.9 ± 1.5 e

40 Spearmint 7.9 ± 0.5 b

Sage 7.6 ± 0.9 b

60 Spearmint 7.3 ± 0.3 b

Sage 8.1 ± 0.4 b

90 Spearmint 7.6 ± 0.3 a

Sage 6.6 ± 0.6 a

f spearmint (M) or sage (S) tissues, counted at 0, 20, 40, 60 and 90 days after thedjusted LSD value at P < 0.05. LSDB is given both numerically and graphically.

efficiency. Along the same line, Kostov et al. (1996a) reported thattomato seedlings grown on a mixture of compost substrates fromvine branches, grape prunings, husks and seeds were ready to betransplanted four to five days earlier than plants grown on peatmixtures. Bletsos and Gantidis (2004) found that transplantedtomato plants grew faster in a medium containing at least 75%of municipal sewage sludge compost. Finally, Toumpeli et al.

(2013) reported that physicochemical characteristics of soil andgrowth of tomato plants were improved following addition at a 4%application rate of compost consisting of 70% mature Phragmitesaustralis plant material plus 30% animal manure.

60 and 90 days after the establishment of the field experiment (DAE). Soil samplesrisons were performed between aromatic species within each application rate andificant different at P < 0.05, according to Bonferroni adjusted LSD value (LSDB = 6.6).

2 4 8

19.4 ± 0.9 c 23.2 ± 1.6 c 39.7 ± 1.1 a24.7 ± 1.3 bc 30.1 ± 0.7 b 42.4 ± 1.0 a18.1 ± 1.5 d 21.1 ± 1.0 d 35.0 ± 2.3 ab22.7 ± 2.1 cd 28.8 ± 2.0 bc 37.4 ± 1.5 a15.1 ± 0.9 a 14.1 ± 0.2 ab 15.8 ± 0.9 a16.2 ± 0.9 a 17.1 ± 0.4 a 17.5 ± 0.7 a10.6 ± 0.4 ab 12.5 ± 0.5 ab 10.7 ± 1.2 ab12.9 ± 0.3 ab 12.3 ±0.4 ab 14.3 ± 1.1 a8.4 ± 0.6 a 8.5 ± 0.4 a 9.5 ± 1.1 a10.0 ± 0.4 a 10.8 ± 0.8 a 12.9 ± 1.5 a

K. Kadoglidou et al. / Scientia Hort

Table 5Essential oil yield of the soil mixed with spearmint or sage plant material at incor-poration rates of 0, 2, 4 and 8% (w/w), at the different sampling times, viz. at 0, 20,40, 60 and 90 days after the establishment of the field experiment (DAE).

DAE Plant material Essential oil yield (�l/100 g of soil)

Incorporation rate (%)

0 2 4 8

0 Spearmint 0 30.5 62.5 123.5Sage 0 29.0 57.5 118.0

20 Spearmint 0 18.0 42.6 80.0Sage 0 28.0 47.5 105.0

40 Spearmint 0 2.8 6.4 6.5Sage 0 14.5 29.5 55.0

60 Spearmint 0 1.9 3.6 4.2Sage 0 6.0 14.0 29.5

90 Spearmint 0 tra tr trSage 0 2.5 6.3 13.4

ttptsgsPtstsetsssssvtco

ypeipmipdpreatawnssro

which is consistently observed at all rates applied even at 60 DAE,

a Trace.

The rates of spearmint (2–8%) used in this study are far lowerhan the rates of the different composted materials reported inhe above mentioned literature. It should also be noted that incor-oration of spearmint tissues into the soil resulted in remarkablyaller (70% average increase) and heavier (75% average increase)eedlings compared to control, as early as 20 days after emer-ence. The positive effect was evident even at the lowest rate of 2%pearmint, with 59% increase in height and 47% in dry weight. Diaz-erez and Camacho-Ferre (2010) also reported significant effect ofen different composts (i.e. mixtures of blond peat and composts ofolid urban waste, vegetable waste or vine pomace) on 34-days oldomato seedlings. The height attained by the 20-day old tomatoeedlings grown on the 4%-spearmint soil mixture was actuallyither the same or only 25% lower compared to the 14-days olderomato seedlings grown on the different composts. In the presenttudy, the positive effect on shoot length of the 20-days old tomatoeedlings grown in soil amended with 2, 4, or 8% spearmint tis-ues is comparable to that reported by Chalkos et al. (2010), whotudied 20-days old tomato seedlings grown in soil amended withpearmint compost (at the same rates as above), fortified with con-entional synthetic fertilizers. Thus, these findings clearly indicatehat the proposed method is comparable with techniques involvingomposting of aromatic plants, as well as with methods applyingrganic composts of different origins.

Photosynthetic rate, stomatal conductance and photosyntheticield of tomato also responded very favourably to spearmint incor-oration treatments, providing the physiological basis for theffects observed on growth. Photosynthetic rate of seedlings grow-ng on substrate taken after 90 days of incubation of either aromaticlant into the soil was lower than that of 40 DAE seedlings. Thisight be the outcome of decomposition processes and alteration

n the essential oil quantity and quality in the soil which might takelace when the incubation of aromatic tissues lasts longer than 40ays. It should also be noted that the beneficial effect on tomatohysiology and growth as well as on the soil’s biological activityesulted from direct incorporation of plant tissues and that suchffects are not the rule when raw organic material is used (Levynd Taylor, 2003). These results clearly demonstrate that produc-ion of tomato seedlings can be considerably improved in soil mediamended not only with compost (Chalkos et al., 2010) but alsoith crude material of spearmint. In contrast, incorporation of sageegatively affected growth of tomato seedlings, though improvingoil parameters. The latter, in conjunction with the positive impact

age had on physiological responses of tomato (e.g. photosyntheticate at 40 days) might be the cause of the lag in the positive effectbserved on tomato growth characteristics (e.g. shoot length).

iculturae 179 (2014) 25–35 33

Several studies indicate that some aromatic plants, the essentialoils they produce (spearmint oil included) and individual essential-oil constituents inhibit germination of several species includingweeds (Argyropoulos et al., 2008; Azirak and Karaman, 2008;Vokou, 2007). This consideration combined with the results of thepresent study suggests selectivity of essential oil (e.g. spearmint oil)action on plant germination. In fact, our previous research efforts(Chalkos et al., 2010) demonstrated that spearmint compost hadan inhibitory effect on weed emergence, especially of broadleafweeds. However, results of the current study show that decompo-sing spearmint tissues promoted emergence of tomato seedlings atall rates applied. This feature is highly desirable, if spearmint is tofind use as soil amendment in tomato cultivation, particularly inseedbeds.

C:N ratio values for soils amended with sage were in generalhigher, though not significant in most cases, than the respectiveones for soils amended with spearmint. At the early stages, therate of decomposition may be affected by the higher sage lignincontent as compared to the herbaceous spearmint, as well as bythe anatomical features common to the genus Salvia, such as thickcuticle and multiple layers of palisade parenchyma under the upperepidermis (Anackov et al., 2009; Baran et al., 2008). The decreasingover time C:N ratio values in amended soils and the absence of sig-nificant differences between control and amended soils at 60 and90 DAE indicate that decomposition of aromatic plant material isachieved within 60 days following incorporation into the soil. C:Nvalues (19.4–42.4) recorded in amended soils at 0 DAE are muchlower than those reported by Levy and Taylor (2003) for wood-residue composts. The latter values, ranging between 36 and 100,led to microbial immobilization and nitrogen deficiency. The higherC:N ratio in soils amended with aromatic plants as compared tountreated ones at 0, as well as at 20 and 40 DAE, although asso-ciated with reduced nitrogen availability, did not negatively affectthe growth of young seedlings.

The absence of significant pH changes after incorporating intothe soil material of the two aromatic plants, even at the highestrate, was a desirable outcome, as soil pH remained within the rangerequired for optimum tomato growth (Heuvelink, 2005).

The enhancement of seedling emergence and growth could be,at least partially, attributed to the higher soil microbial activityassociated with the decomposition of the incorporated aromaticplant tissues and the concomitant nutrient release (Vokou andMargaris, 1988; Vokou et al., 1984). Arancon et al. (2003) found thatcompost incorporation into the soil resulted in an increase of themicrobial biomass and, hence, into nutrient mineralization. Nev-ertheless, the plant growth and yield improvement they recordedcould not be explained on the basis of macronutrient availability, asall compost treatments had been supplemented with inorganic fer-tilizers to equalize availability at transplanting time. These authorsfurther conclude that other factors might have a role in induc-ing positive effects on plant growth and yield, namely growthregulators produced by microorganisms (Atiyeh et al., 2002) orhumates produced during the decomposition process (Canellaset al., 2002). Hormones or humates deriving from the decompo-sing spearmint material might similarly account for the beneficialeffects observed on tomato growth, independently of nutrient sup-ply.

To have an effect under field conditions, essential oils mustpersist in the soil environment. Hydrodistillation results from thesoil–aromatic plant mixtures showed that sage oil persists at highconcentrations for a longer period than spearmint oil. This can pos-sibly explain the inhibitory effect of sage on tomato dry weight,

but not at 90 DAE, when there does not seem to be much essen-tial oil left in the soil. Such an inhibitory effect of sage essentialoil on tomato growth has been reported by Argyropoulos et al.

3 a Hort

(o

cmtsigct2obsbsi22

pv2sLbacftcSsotts

asa(aiHchf2ippiso

eGbwagboe

4 K. Kadoglidou et al. / Scienti

2008), who also observed an inhibitory effect of 1,8 cineol, a majorxygen-containing constituent of this oil (Chalkos et al., 2010).

The major constituent of spearmint is carvone, a very activeompound against seedling growth (Chalkos et al., 2010). In fact,ost oxygenated constituents of essential oils are far more active

han non-oxygenated ones (Vokou et al., 2003). Despite this,pearmint had no inhibitory effect in the present study. Therefore,t should be assumed that the very positive effects seen on tomatorowth were mediated by another factor. In this regard, the authorsonducted a GC–MS analysis of soil samples treated with spearmintissues at the same rates as in the present study (Karamanoli et al.,008). The latter analysis revealed not only a considerable decreasef essential oil content at 35 DAE (as also observed in this study),ut a dramatic reduction of carvone accompanied by an increase ofesquiterpenes like caryophyllene and calamelene as well. It shoulde pointed out that several researchers have related the presence ofuch sesquiterpenes in growth media with plant growth promot-ng activity in vegetables (Kashiwabara et al., 2006; Nakajima et al.,005) and with root signalling (Cheng et al., 2007; Rasmann et al.,005).

It is reported that different composts stimulate the growth oflant growth-promoting rhizobacteria (PGPR) on different culti-ations such as tomato (Alvarez et al., 1995) and maize (Viti et al.,010). Other researchers indicated that essential oils and their con-tituents activate soil respiration (Vokou et al., 1984; Vokou andiotiri, 1999; Vokou and Margaris, 1988) and shift the microbialalance in soil favouring bacterial strains that are tolerant and/orble to catabolize them (Vokou et al., 2002). Given this, it can be con-luded that the biologically active secondary metabolites releasedrom the decomposing aromatic plants (namely spearmint) are ableo modify the soil microbial communities and to favour the benefi-ial microbes needed for a rapid colonization of the rhizosphere.uch soil microbes may prevent invasion of other detrimentaltrains near the root surface and get involved in the productionf metabolites which positively influence plant growth. Althoughhis is a hypothesis to be tested, results of the present study allowo be said that at least spearmint has the potential for use in tomatoeedling production as a soil amendment.

For all rates and irrespective of the aromatic plant incorporated,t 0 DAE, the values of the examined soil microbial and tomatoeedling attributes were similar to those of controls. However,t this time, the essential oil concentrations of the soil–aromaticspearmint or sage) plant mixtures attained their peak values. Thebsence of any inhibitory or promoting effect at 0 DAE can be eas-ly explained for microbial populations (determined in soil plots).owever, this is not the case for plant attributes, since seedlingsorresponding to this sampling time (like to any sampling time)ad grown in the soil–aromatic (spearmint or sage) plant mixtures

or 20 days before measurements were taken. This suggests that a0-day period is not sufficient to allow processes initiated by the

ncorporation of spearmint or sage tissues to be expressed in thelant parameters studied. It also supports the hypothesis that theromoting effect of spearmint is related to the transformations of

ts essential oil taking place in the soil environment. If so, this woulduggest that there are no similar transformations of sage essentialil, hence inhibitory effects were observed.

As a final note, we should consider the fact that spearmint is anconomically important herb of high value. Although abundant inreece and in other Mediterranean-type climate areas, the feasi-ility of its application in vast field crops (including tomato) in theay that we recommend might be questionable, if production costs

re also taken into consideration. However, as its effect on tomato

rowth is expressed very early, even 20 days after seeding, it coulde applied very efficiently in seedbeds to produce healthy and vig-rous seedlings. In addition, further research is needed not only tolucidate the mode of action of spearmint compounds as a tomato

iculturae 179 (2014) 25–35

growth stimulant, but also to examine its effectiveness under fieldconditions and particularly its potential use in mixed, rotational orcover crop systems.

5. Conclusions

This study demonstrates that enhanced tomato seedling growthcan be achieved in a cost and time effective manner by incorporat-ing into the soil intact spearmint tissues without undertaking anyarduous composting step first. In particular, it has been demon-strated that this novel amendment practice improves seedlingphysiology, as well as soil microbial activity and soil physicochem-ical properties, leading to improved growth of tomato in seedbeds.To this end, the time required for tomato seedlings to be readyfor transplantation is reduced. Based on the herein reported out-comes, further research is undertaken by the authors to shedlight on whether the primary reason for the beneficial effects ofspearmint on tomato growth is the shift from a carvone-rich toa sesquiterpene-rich oil. Arguably, due to the selective and multi-faceted biological activity of essential oils, there exists considerablepotential on the use of certain aromatic plants for novel applica-tions in sustainable agriculture. This study contributes a furtherstep along these lines.

Acknowledgements

This study was funded by the General Secretariat for Researchand Technology, Ministry of Development, Greece (Programme2001 ED317). We express our thanks to Professor K. Radoglou forher contribution in carrying out the photosynthetic measurementsand to Ms V. Porfyridou for technical assistance.

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.scienta.2014.09.009.

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