The Effect of Trichoderma spp. on the Composition of Volatile Secondary Metabolites and Biometric Parameters of Coriander (Coriandrum sativum L.)

Laboratory of Agricultural Microbioloy, Department of Plant Protection, Wrocław University of Environmental and Life Sciences, Grunwaldzka 53, 50-357 Wrocław, Poland Department of Crop Production, Wroclaw University of Environmental and Life Sciences, Grunwaldzka 24a, 53-363 Wrocław, Poland Department of Chemistry, Wroclaw University of Environmental and Life Sciences, Norwida 25, 53-375 Wrocław, Poland Institute of Animal Breeding, Wroclaw University of Environmental and Life Sciences, Chełmońskiego 38C, 51-630 Wrocław, Poland


Introduction
In recent years, there has been a continuous increase in sales on the pharmaceutical market, or more broadly understood as the market of food, dietary supplements, natural substances, and herbs. eir usefulness in most cases is determined by pharmacopoeial standards (Polish or European Pharmacopoeia). Research shows that the quality of pharmacopoeial raw materials is in uenced by many factors, such as (1) the cultivar used in cultivation, (2) type of substrate, (3) time and period of harvesting, or (4) the drying method [1]. e in uence of the plant's condition on the metabolites of secondary herbal raw materials is also known, which can be positively in uenced by the use of living microorganisms. Microorganisms that support growth and protect plants from pathogens are a subject of growing interest [2][3][4]. e possibility of using living microorganisms to increase soil fertility and plant production e ciency has already been used in the middle of the last century [4,5]. Among soil microorganisms, antagonistic fungi from genus Trichoderma are the most commonly studied and used in the biological plant protection and/or integrated pest management (IPM) programs [6]. ese fungi are capable not only for stimulation of plant growth but also for induction of its defense mechanisms (i.e., ISR, induced systemic resistance; SAR, systemic acquired resistance) [7][8][9]. Trichoderma fungi produce a number of substances with antibiotic properties and hydrolytic enzymes (cellulases, chitinases, xylanases, pectinases, β-1,3-glucanases, and proteases among others), thanks to which they can quickly colonize plant roots and compete with phytopathogens for the place of infection or nutrients. Numerous reports show that several strains of Trichoderma had a significant reducing effect on plant diseases caused by soilborne and foliar pathogens (such as Rhizoctonia solani, Phytophthora spp., Pythium ultimum, Fusarium spp., Alternaria alternata, Sclerotinia spp., Gaeumannomyces graminis, ielaviopsis basicola, Verticillium dahliae, and Botrytis cinerea) under greenhouse and field conditions [6,[9][10][11][12][13]. e use of Trichoderma fungi to protect herbal plants or organic crops can be an alternative to synthetic pesticides. e mechanisms of activity of the Trichoderma genus towards pathogens (e.g., antibiosis, micoparasitism, and competition) or stimulation of plant growth or induction of defense mechanisms is indicated widely in the literature [6][7][8][9][10][11]. However, there are very few papers describing the use of these antagonists in the cultivation of herbs or their impact on the content of aromatic compounds [14,15].
One of the world's oldest herbs and species plants is coriander (Coriandrum sativum L.). Coriander is an annual, herbal, spicy, and melliferous plant, belonging to the celery family (Apiaceae). e main herbal raw materials are fruits containing large amounts of essential oil (0.2-2.6%) and polyphenolic acids (e.g., linoleic, oleic, palmitic, and stearic acids) as well as protein compounds, cellulose, pectins, mineral salts, and vitamins [14,16]. Moreover, the coriander essential oils also have antibacterial properties against Gram-positive and Gram-negative bacteria (Staphylococcus aureus, Streptococcus haemolyticus, Bacillus subtilis, Pseudomonas aeruginosa, Escherichia coli, and Proteus vulgaris) [14,16]. e data presented in the FEMA report (Flavor and Extract Manufacturers Association) indicate that the global annual production of coriander fruits is about 600,000 tonnes per year (http://www.femaflavor.org). Fruits of coriander are used mainly as a flavoring and aromatic additive in the production of food products (meat, snack, alcohol, and confectionery products). Moreover, coriander oils are included in the group of so-called safe (nontoxic) oils that can be used as a fragrance and supplement (in alcoholic beverages, cosmetics, soaps, tobacco, and medicines) [17][18][19]. e aim of the present research was to determine the effect of coriander fruits treatment with Trichoderma fungi on the composition and content of volatile secondary metabolites. is study also investigated the influence of Trichoderma fungi on the biometric features of coriander and the degree of root colonization with these fungi. e degree of colonization of roots by toxicogenic fungi of the Fusarium genus was also examined.

Materials and Methods
In the study, coriander fruits (Coriandrum sativum var. microcarpum) of the Pallas variety belonging to the plants from the oil group (Legutko, Poland) were used. e study examined two strains of antagonistic fungi: T. harzianum strain T22 (commercial biological preparation Trianum, Koppert B.V., the Netherlands) and the T. asperellum strain B35. e T. asperellum B35 strain comes from the collection of the Agricultural Microbiology Lab, Department of Plant Protection, Wrocław University of Environmental and Life Sciences, Poland. is strain stimulates the growth of plants and effectively limits a number of diseases of the root system of onions, cabbage, cucumbers, peppers, tomatoes, leek, and celery [20][21][22]. e following treatments were applied: (1) control (dry fruit); (2) soaking fruits in sterile water for 10 min; and soaking fruits for 10 min in a conidia spores suspension of (3) T. harzianum T22 and (4) T. asperellum B35. e suspension of Trichoderma fungi spores was prepared by mixing the preparation with distilled water in an amount to obtain 1 × 10 7 spores 1 mL −1 . Conidial densities in the suspension were determined by use of a hemocytometer under a light microscope. e coriander fruits were poured into a suspension and soaked for 10 min, mixing it constantly; then, the seeds were dried at room temperature and sown.
Germination energy and capacity were determined before sowing. According to ISTA [23] regulations, germination energy was determined as the percentage of seeds that have been produced by seedlings classified as normal after 7 days of germination under optimal conditions. e germination capacity was expressed as a percentage of seeds that have produced normal seedlings after 14 days of germination under optimal conditions. e tests were performed on 100 fruits from each combination.
After the growing season, 25 randomly selected plants were collected from each plot and biometric analyses were carried out. e number of fruits, the mass of about 1000 pieces (MTS), and the weight of fruits were calculated. e biomass increase of the aboveground parts and roots was also measured.

Root Colonization. Root colonization with
Trichoderma fungi was examined in the period of full vegetation-before the harvest of the raw material for analyses on the content of active compounds. Roots were harvested from representative plants.
e roots after mechanical soil removal were thoroughly washed with running tap water to remove adhering soil particles and then were rinsed with a sterile solution of 0.1 M MgSO 4 × 7H 2 O. e roots were aseptically cut into ∼5-mm fragments and transferred on a PDA medium (Potato Dextrose Agar, Emapol, Poland) with 30 μg mL −1 streptomycin (to inhibit growth of bacteria colony). Eight fragments were placed per plate and incubated at 28°C. Growing colonies were observed daily. After incubation, the percent degree of roots colonization with Trichoderma and Fusarium fungi was determined [21,24].

Analysis of the Content of Essential Oils.
Determination of the composition and content of volatile metabolites was carried out by the method of hydrodistillation in the Deryng apparatus [25]. In a 250 mL round-bottom flask, 30 g of fresh coriander fruits was placed, 100 mL of redistilled water was added, and hydrodistillation was carried out for 3 hours. e collected distillate (extracted into 1 mL of cyclohexane and dried with Na 2 SO 4 ) was stored at a temperature of −15°C until the GC-MS analysis was performed. e determination of the composition of volatile fractions was performed according to the chromatographic method developed by Szumny et al. [26]. About 50 μL of the previously prepared solution of the essential oils was diluted ten times in cyclohexane and subjected to the GC-MS analysis (injection of 1 μL).
A gas chromatograph of the PerkinElmer Clarus 680 coupled to a mass spectrometer was used to identify volatile compounds. e separation was performed using an Elite-5MS column (30 m × 0.25 mm × 0.25 μm film thickness). e identification of the compounds was carried out by (1) comparing the EI-MS spectrum of the compound with the spectrum in the NIST14 data library; (2) comparison of Kovats retention index of the identified molecules with data included in the NIST14, NIST Webbook database, and (3) comparison of retention times with commercially available standards isolated from plants.
In addition, chromatographic analysis (GC-MS) of the main components of the obtained essential oils was confirmed by performing nuclear magnetic resonance spectra. e structures of compounds were confirmed on the basis of the comparison of signals obtained from NMR analysis with literature data. e spectra 1 H NMR, 13 C NMR, and HSQC were performed on the Bruker Avance DRX-500 apparatus, using solutions in CDCl 3 .
Specific rotation was measured on the Autopol IV Automatic Polarimeter (Rudolph) equipped with a thermostatic system. e analysis was performed in ethanol at a concentration expressed in g 100 mL −1 . e enantiomeric excess of linalool was determined based on the GC analysis using a chiral column, separation on cyclodextrin associated with dimethyl polysiloxane (CP-Chirasil-DEX CB), with the 25 m, 0.25 mm, 0.25 μm film thickness.

Statistical Analyses.
e data from field experiment, biometric analyses, and root colonization were subjected to the analysis of variance using Tukey's test (p < 0.05) using STATISTICA 13.3 software for Windows.

Results and Discussion
In the first stage of the study, the influence of Trichoderma fungi on the germination energy and capacity of the coriander seeds was evaluated (Table 1). e germination energy of fruits depended on the used treatment. Soaking coriander fruits in water and spore suspension of Trichoderma spp. increased the germination energy in the range from 18% to 47%. Treatment of fruits with T. harzianum strain B35 increased the germination energy by 20% in comparison with water and Trianum treatment. e experimental combinations used did not affect the germination capacity of the coriander fruits. e development of coriander during the growing season depended on the method of seed treatment before sowing ( Table 2). Coriander growing on control objects had a slower development during the entire growing season, but it was fruiting earlier and had a slightly shorter growing season. A similar rate of coriander development was observed on objects with fruits soaked in water before sowing. In turn, biological treatment of coriander fruit with T22 and B35 strains accelerated the development of plants from 4 days during emergence to 6 days during flowering. It also extended the flowering period, fruit settling, and the growing season. Trichoderma species also had a positive influence on the increase in yield and fruit number in the range from 55% to 64% (Table 3). Newman et al. [13] had obtained similar effects in their study, while applying T. harzianum spores during the tomato cultivation. In our research, after inoculation fruits with T. asperellum strain B35, an ∼2-fold increase in plant biomass was obtained compared with the remaining experimental combinations. At the same time, an increased colonization of the roots by Trichoderma species was found in the treatment of fruits with T22 and B35 strains in comparison with objects a and b. Sobolewski et al. [27] investigating carrots had also observed significant increase in height and mass of the plants after applying Trichoderma spp. preparation instead of standard T 75 DS/WS (2012) preparation.
Moreover, from the coriander roots, phytopathogenic fungi from the Fusarium genus (F. culmorum, F. oxysporum, and F. avenaceum) were isolated. e roots were colonized by these fungi most numerously, whose fruits before sowing was not biologically treated. is may be related to the faster colonization of these specific microniches by antagonistic fungi Trichoderma and their parasitic activity against these phytopathogens [12,[20][21][22]. e obtained results unambiguously indicate the effect of coriander fruits treatment with Trichoderma strains tested on the content of essential oils (Table 4). e amount of volatile fraction for fruits treated with the Trianum preparation increased by ∼36%. A similar effect was obtained by Ratnakumari et al. (2014) [15], observing the increase in the content of the mint essential oil (Mentha arvensis) grown on the substrate inoculated with the T. harzianum strain NFCCI 2241 and T. ovosporum strain NFCCI 2689. e authors isolated 40 to 50% more volatile fractions from plants compared with controls. Singh et al. (2002) [14] observed an increase in the content of a dozen or so percent Journal of Food Quality of the essential oil contained in Pogostemon cablin, in the soil inoculated with T. harzianum strain ATCC PTA-3701.
Studies carried out with the GC-MS technique allowed to identify 26 compounds (Table 4). In plants from the control objects a and b, monoterpenoid alcohol, linalool (77%), was the component with the highest content. In contrast, plants from the objects c and d, received 81.8% on average. e next components of the obtained oil were the monoterpenoids: camphor (∼5% to 7%) and geraniol (∼4%) as well as monoterpene α-pinene (∼2%) and γ-terpinene (∼4%). Monoterpenoids and monoterpenes accounted for 85.11% and 14.63% of the total amount of oil, respectively. e aliphatic aldehydes were found in trace amounts (less than 0.3%). With regard to the scientific literature, a number of studies on the composition and content of coriander oil were carried out. e literature data indicate that the composition and content of the oil differ depending on the climatic conditions of the region, growth conditions, and plant species. Msaada et al. [28] proved the presence of linalool (87.54%) and cis-dihydrocarvone (2.36%) as two main components of the fruits of the Tunisian coriander. Telci et al. [29] examined the chemical composition of two fruits varieties and observed differences in the content of the main component, linalool between the two varieties. According to literature data, coriander oil, isolated by hydrodistillation, contains from 58 to 81% of linalool [29]. In studies of coriander oils carried out by Orav et al. [30], the content of linalool also ranged from 58 to 80%. Other characteristic components were γ-terpinene, α-pinene, p-cymene, camphor, geranyl acetate, and geraniol. e highest amount of linalool was found in the oil isolated from Jantar coriander growing in Estonia. Furthermore, in obtained oil, higher contents of linalool and geranyl aceate were observed, than in oils obtained from coriander fruits in Netherlands, Russia, and France. In general, the fruits of European coriander in comparison with Asian ones are characterized by a higher    Values in individual lines marked with the same letters differ significantly from the control objects according to Tukey's test (p � 0.05).
In previous studies, the presence of (S)-(+)-linalool (also called coriandrol) was found in the coriander essential oils. In fact, linalool is not a pure (S) isomer and contains from 12 to 15% of the (R) isomer [34,35]. Oliver [36] in his work studied the samples of coriander essential oils for the presence of isomeric (S)-(+)-linalool. By means of gas chromatography (GC) and the use of a chiral column, he found the presence of 88% of (S)-(+)-linalool and 12% of (R)-(-)-linalool. e GC analysis we conducted on a column with a chiral filling and a proper rotation test confirmed the presence of (S)-(+)-linalool in the tested material. e presence of (S)-(+)-linalool in the essential oils was also found by Gaydou et al. [37] and Sadowski et al. [38]. Our research yielded very similar contents of (S)-(+)-linalool −82%.
According to the European Pharmacopoeia, the fruits of coriander should contain no less than 0.3% of essential oils. e percentage content of components was also characterized: limonene (1.5-5%), geraniol (0.5-3%), geranyl acetate (0.5-4%), camphor (3-6%), linalool (65-78%), p-cymene (0.5-4%), γ-terpinene (1.5-8%), α-terpineol (0.1-1.5 %), α-pinene (3-7%). Comparing the data in the Pharmacopoeia with the data obtained in our work, it was found that the contents of camphor, α-pinene, and limonene slightly deviate from the acceptable standards. e conducted research is becoming particularly interesting in the context of recent reports that indicate the use of coriander in animal nutrition, which improves their wellbeing. ese results were demonstrated by Abou-Elkhair et al. [39] in the broiler studies and Mohammed et al. [40] in research on the use of Awassi sheep and rams. In addition, as emphasized by Bahat et al. [41], Sriti et al. [32], Rezaei et al., [42] and Prachayasittkul et al. [43], the interest in coriander due to its antibacterial, anti-inflammatory, anticancerous, or antifungal activity and its richness of bioactive components (phytosterols, monoterpenes, monoterpenoids, etc.) is constantly growing. erefore, it is a good premise for efforts such as those undertaken in our research to intensify its growth and improve the quantitative chemical composition.

Summary
e obtained results indicate the purposefulness of using biological control agents based on Trichoderma species in the cultivation of coriander as a source of essential oils. e comparison of efficiency of the distillates obtained with steam proves the positive effect of T. asperellum B35 on coriander. And most importantly, increasing the yield of essential oils obtained does not mean a change in composition. is oil is in the upper ranges of pharmacopoeial standards. At the same time, the use of antagonistic fungi affects the improvement of biometric parameters of the plant. An increased yield and the number of coriander fruits were found to be at the level of ∼60%. In the experimental combination with T. asperellum B35, the biomass of the aerial parts of the plant was twice as large.
Data Availability e NMR and GC-MS data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.