WO2011028144A2 - Composition against pathogens, the method of its preparation and the use of hemp panicle extract for production of composition against pathogens - Google Patents

Abstract

The present invention provides a composition against pathogens, a method of its preparation and the use of hemp panicle extract for production of composition against pathogens. More precisely, the invention provides a composition based on common hemp panicle extract to fight off fungal pathogens, having an effect on the vigour, germination and health of seeds of the Apiaceae family plants, preferably carrot.

Description

Composition against pathogens, the method of its preparation and the use of hemp panicle extract for production of composition against pathogens

The present invention provides a composition against pathogens, a method of its preparation and the use of hemp panicle extract for production of composition against pathogens. More precisely, the invention provides a composition based on common hemp panicle extract to fight off fungal pathogens, having an effect on the vigour, germination and health of seeds of the Apiaceae family plants, preferably carrot.

Advanced vegetable production requires the best seed material possible. Healthy seeds of high germination capacity, vigour and purity are the determinants of good harvest yields. In practice, the seeds are often colonized by fungi. Seed health can be largely improved by various seed treatment methods [1]. The most common method is the chemical method [2]. However, a trend was observed in recent years to replace the conventional plant protection agents by biological agents [3, 4]. This is associated with the increasing popularity of ecological agriculture. Ecological agriculture is based mostly on using natural agents reducing the growth of pathogens and pests in plant protection and resignation of synthetic pesticides. Only physical or natural methods are allowed, including plant-based preparations, such as extracts [5]. New plant protection methods that could be potentially used in ecological production are being sought for.

Carrot (Daucus carota L.) belongs to Apiaceae (formerly Umbellifereae) family. Crop carrot originates from wild carrot Daucus carota var. carota, which grows in Asia (as far as China), and can also be found in Europe and both Americas. The wild carrot has woody, ramified storage roots and is an annual plant. The plant has been known by ancient Romans and Greeks, but its cultivation was started probably at the end of the first millennium AD in the Middle East. Crop varieties reached Europe via Asia Minor at the turn of the 14^th century. Initially, the crop carrot had purple or yellow roots. Production of orange-colored storage root carrot was started in Holland as late as in the 17^th century. First reports of carrot cultivation on Polish terri *tory date back as far as to the 16 th century.

Carrot cultivation is most common in Asia and Europe. The largest European producers of carrot are Poland, France and the United Kingdom [10]. In Poland, carrot is the second most common crop vegetable, just after cabbage. Also in other countries with climate similar to that of Poland, carrot is a staple vegetable and is consumed all around the year. Carrot owes its popularity to its high nutritional and taste values. The presence of carotenoids (β- and a-carotene) that comprise a large source of vitamin A is most valued. Other vitamins, such as Bi, B₂, C and E, are also present in the roots, albeit in small quantities. Minerals present in large quantities include calcium, phosphorus, magnesium and iron. Carrot roots also contain hydrocarbons, mostly sugars, such as glucose and sucrose, dietary fiber, as well as organic acids (malic acid) and essential oils responsible for the pleasant taste [7,9].

Crop carrot is a biannual plant. In the first year of cultivation, carrot produces a rosette of leaves and the storage root, while in the second year it forms an umbel-topped inflorescence bundle. The storage root is formed of the hypocotyl and the proper taproot. The root system can be up to 150 cm deep and expand over the area with 60 cm radius [12]. Its formation begins about one month after sprouting. In the initial stage, the root growth is only longitudinal; at later stage, it is both longitudinal and crosswise, while at the last stage it is only crosswise. Upon maturation, roots change their colour from white to orange or yellow. The carrot fruit is of bipartite schizocarp type, splitting into two carpels that constitute the sowing material, in practice referred to as seeds. The carpels are bulged at dorsal side and flat on the bottom side. Initially, they are greenish-coloured, turning brown upon maturation. The seeds are equipped with seven fins distributed along the ridge and the sides of the seed. Four of these ribs are equipped with long, hooked processes. Therefore, the sowing material must be abraded before selling, After abrasion, the seeds are about 2-4 mm long, 1-2 mm wide and 0.8-1.0 mm thick. The characteristic smell of the seeds is determined by the presence of tubules filled with essential oils; the tubules are distributed beneath the fins. Storage materials in carrot seeds are accumulated in the endosperm, surrounded by a small plumule [13, 14].

Carrot seeds may be produced by outplanting or non-outplanting methods. In the outplanting method, plants are produced during the first year of cultivation, dug out in the autumn and subjected to selection. The roots are stored in mounds for winter and are planted back into the soil following second selection in the second year. In the non- outplanting method, the seeds are sown in August, and the resulting plants are left in the soil for the winter. There are advantages and disadvantages to both of these methods. The outplanting method offers a possibility to carefully select the material upon harvesting the roots and removing the plant-outs from mounds. However, this is work- and time consuming. On the other hand, the non-outplanting method does not allow for the selection of plant-outs; therefore, only elite-grade seeds should be sown. The advantages of this method include possibility to cultivate other plants before the carrots and labour saving. Of disadvantage is the risk of plants being frozen when wintered in soil. However, this can be avoided by heeling the plants up before winter or by sowing the seeds into furrows, which partially protects the seeds from frosts. The yield of seeds obtained by the first method is 600-1000 kg/ha. In case of the non-outplanting method, the yield is usually higher [12]. Major pathogens transferred with carrot seeds include Alternaria dauci (J.G. Kiihn) J.W. Groves & Skolko, A. radicina Meier, Drechsler & Eddy, Cercospora carotae (Pass.) Kazn. & Siem., Erysiphe heraclei DC, Sclerotinia sclerotiorum (Lib.) de Bary and the bacteria Xanthomonas hortorum pv. carotae (Kendrick) Vauterin, Hoste, Kersters & Swings [15, 2]. Pathogenic fungi of Fusarium species may also be found [16].

First of the listed pathogens is responsible for carrot leaf blight (alternariosis). The disease caused pre- and post-sprouting gangrene in field crops. The affected seedlings show symptoms of canker on the root and the hypocotyl. The seedlings wither within 2-3 weeks from sprouting [17, 18]. Leaves and peduncles are covered by small, overlapping red- brown spots. In strongly affected plants, the leaves dry up prematurely. This may hamper harvesting the carrots by mechanical methods. The disease may cause a reduction in harvest yields and root quality. In the second year of carrot cultivation A. dauci affects the flowering shoots, umbels and seeds [20, 21].

A common pathogen fount on carrot roots is A. radicina, responsible for black rot [22]. Carrot is prone to this disease at all stages of the development [23]. The pathogen is one of the causes of seedling withering. In high air humidity conditions, seedlings and peduncle of older leaved may become infected. The pathogen spreads from the affected peduncles onto the leaf laminae and the root head. Black rot develops during the storage of infected roots. The disease is manifested by dry, depressed spots covered by fungal spores. Symptoms of the disease may also be observed on carrot seedlings. Initially, brown streaks can be observed in lower parts of the stems. Over time, entire stems decay or dry up; affected plants may not form seeds [24, 25, 7]. Saprotrophic fungi may also be found on carrot seeds, such as: Alternaria alternata (Fr) Keissler, Cladosporium spp. Link ex Fr., Epicoccum nigrum Link, Stemphylium botryosum Wallr. (perfect stage of Pleospora tarda E. Simmons) and others [16]. Some researchers consider Alternaria alternata to be a weak carrot pathogen [16].

The presence of pathogenic fungi on the seeds is one of the reasons for poor seed germination and the development of field diseases [26]. Therefore, the sowing material should be free of pathogens [27]. Seed dressing is both the cheapest and the most effective method to eliminate pathogens and pests posing danger to plants at the initial stages of the development [28]. The efficacy of this procedure depends on several factors, including the choice of the dressing, the dosage and the dressing technique [29]. Chemical, physical and biological methods of seed dressing are distinguished. Chemical method is most common; however, popularity of ecological agriculture is increasing [2]. This is associated, among other factors, with excess chemisation of the environment [5]. Criteria for registration of chemical plant protection agents have been sharpened. In recent years many synthetic fungicides were withdrawn from the market due to their persistence, high toxicity and mutagenic or teratogenic effects, including those used for the protection of carrot [5, 30]. Excessive research is conducted with regard to the potential use of materials of biological origin for plant protection, including natural essential oils and extracts. Plants produce essential oils for protection against pathogens and parasites [31]. Oxenham et al. [31] studied the fungicidal properties of the essential oil obtained from common basil (Ocimum basilicum L.). They observed that both the essential oil and its major components, such as methyl chavicol, linalol, eugenol and eucalyptol significantly reduced the growth of Botrytis fabae fungi in vitro. Other components, present in trace amounts, were less important in inhibiting the fungal growth process.

The effect of basil (Ocimum basilicum L.) and thyme (Thymus vulgaris L.) oils on the health of carrot seeds was also studied by Dorna et al. [32]. Treatment of the seeds with thyme oil solutions at concentrations of 0.05 and 0.1% was most effective in reduction of Alternaria spp. and Epicoccum nigrum colonization.

Wilson et al. [33] studied the effect of extracts and essential oils of various plants on the growth of Botrytis cinerea. 13 of 49 essential oils used in the experiments inhibited the fungal growth. Of highest efficacy were the essential oils of palmarosa (Cymbopogon martini) and Thymus zygis. Extracts of Allium and Capsicum species were the most efficient of all plant extracts. Jamiolkowska and Wagner [34] studied the use of thyme oil in the protection of field-cultivated red pepper from pathogenic fungi. The thyme oil was used at two concentrations of 0.02 and 0.03%. The researchers have observed that the oil has led to an increase in the number of saprobiotic fungi with simultaneous reduction in the number of pathogenic fungi. The oil was found to have an inhibiting effect on A. alternata in the overground parts of the plants. The higher concentration of thyme oil led to higher reduction in fungal growth.

Wolf et al. [35] studied the antifungal properties of essential oils from basil, cinnamon, clove, manuca, oregano, peppermint and thyme, organic acids - acetic acid, ascorbic acid, citric acid, lactic acid, propionic acid and Biosept 33 SL (grapefruit extract). In vitro tests showed that thyme oil, oregano oil, cinnamon oil, clove oil and Biosept were most effective against: Xanthomonas campestris pv. campestris, Clavibacter michiganensis subsp. michiganensis, A. dauci and Botrytis aclada. Organic acids had little effect on the reduction of pathogenic fungi. When used at concentrations of above 1%, cinnamon oil and Biosept had negative effect on germination of the tested plant species. Soaking cabbage seeds in the solutions of the essential oils or Biosept at concentrations of 0.1 to 1% for 30 minutes led to reduction of fungal colonization by 10-70% in the filter paper test and eliminated more than 99% of the bacteria.

Soto-Mendivil et al. [36] studied the effects of thyme (Thymus vulgaris) oil on the citrus tree pathogens Alternaria citri. When used at concentrations above 1000 ppm, the oil inhibited the growth of the test fungi in vitro.

Sas-Piotrowska and Piotrowski [37] assessed the efficacy of aqueous extracts of various plants against pea pathogens. The studies included the use of: common hogweed, lingonberry, common hop, common garlic, Chinese lantern plant, forking larkspur, sweet scented geranium, common nettle, wild strawberry, black nightshade, meadow bistort, common horehound and globe candytuft. The efficacy of extracts was compared to two antibiotics and three fungicides. Most of the tested compounds, particularly common nettle and wild strawberry root extracts, significantly reduced the fungal colonization of non- disinfected seeds, including Ascochyta and Fusarium species. After preliminary decontamination of seeds, increase in colonization was observed following application of most compounds, including the antibiotic nystatin and black nightshade extract. Nettle root extract and Kaptan had the most beneficial effect on the seed health.

Saniewska and Orlowski [38] studied the effect of garlic homogenate on the development of phytophtorosis in Gerbera daisies and the effect of garlic and onion used as forecrops on the development of vascular fusariosis in asters and the number of Fusarium oxysporum f. sp. callistephi fungi. Pre-vegetational use of the homogenate for disinfection of substrate artificially infected with Phytophthora cryptogea reduced the development of phytophtorosis in Gerbera daisies cultivated on this substrate. Positive effects were observed regardless of the amount of homogenate used for decontamination. Garlic, when used as forecrop and planted one month before asters contributed to inhibition of vascular fusariosis in the substrate artificially infected WithFusarium oxysporum f. sp. callistephi. Also in case of asters cultivated after had been onion used as forecrop resulted in significant reduction in the disease prevalence.

Szopinska et al. [39] studied the effect of grapefruit extract (Biosept 33 SL) on the health of cabbage, onion and zinnia seeds. Biosept 22 SL was superior than the fungicide in reducing the prevalence of A. alternata and A. brassicicola on cabbage seeds. Comparable efficacies of the extract and the fungicide were achieved when fighting A. zinniae on zinnia seeds and Botrytis cinerea and B. aclada on onion seeds. The grapefruit extract was also found to significantly improve the germination capacity of cabbage and onion seeds at 10°C and accelerate the germination of cabbage seeds at 10 and 20°C.

Another potential species to deliver extracts or essential oils for the protection of plants against pathogens is common hemp {Cannabis sativa L.). The species belongs to genus Cannabis, family Cannabaceae. There are many types, forms and varieties of this plants, different in their biological and morphological features and in their economic value [40]. There are three species within the Cannabis genus: Indian hemp {Cannabis indica Lam.), common hemp {Cannabis sativa L.), wild hemp {Cannabis rude alis Janisch.) [41].

Due to different environmental conditions hemp had to become adapted to when spreading across different climate zones, plants are differentiated into Northern hemp, Southern hemp and Central European (transitional) hemp Cannabis species contain a narcotic substance, tetrahydrocannabinol (Δ⁹ THC). Due to the THC content, hemp is differentiated into hashish hemp (5 to 20% THC) and fibrous hemp (less than 0.2% THC) [42, 43]. Tetrahydrocannabinol (THC) is an isomer of cannabidiol. It is practically insoluble in water and well soluble in organic compounds such as alcohol or fats fl ttp://pl.wildpedia.org/wild/Tetrahvdrokannabinol).

Hemp and hemp extracts were studied as repellents against Cabbage White {Pieris brassicae L.), mosquito larvae {Heterotermes indicola) and termites {Coptotermes heimi) [44, 45, 46]. The essential oil of hemp has bacteriostatic effect on Gram-positive bacteria {Staphylococcus, Streptococcus, Bacillus cereus, Bacillus subtilis, Staphylococcus aureus), as well as Gram-negative bacteria {Escherichia coli, Pseudomonas aeruginosa, Salmonella paratyphi, Shigella species) [47, 48]. The oil contains 58 monoterpenes and 38 sesquiterpenes. It is obtained by steam distillation of inflorescens and young leaves (http://en.wikipedia.org/wiki/Cannabis flower essential oil). Mediavilla and Steinemann [49] claim that 1 ha of the crop may yield as much as 10 L of the essential oil.

Patent application No. P-378852 (publication date 2007/08/06) described the use of carotol as an antifungal agent. The invention relates to the use of a sesquiterpenic cyclic alcohol - carotol - as a fungistatic agent, active against several strains of lower fungi, including especially phytopatogens, saprophytes and dermatophytes. The source of carotol is the essential oil obtained from carrot seeds. The main ingredient of the oil is a sesquiterpenic cyclic alcohol active against various genera of lower fungi at concentrations not lower than 150 mg/L. Carotol is applied onto surfaces as a protective coating to protect the materials, raw materials and products against the fungi of Alternaria, Fusarium and Penicillium species. Carotol is introduced into the entire volume of the protected materials in the form of a solution, suspension of emulsion.

Patent No. PL 176497 (publication date 1996/10/14) describes a preparation method of a bactericidal and fungicidal emulsion characterized in that 5-15% of alkyldimethylbenzylammonium chloride is combined with 15-40% of ethoxylated castor oil and 1-8% of propylene glycol. Next, 20-40% of aromatic oil is dissolved in 10-50% of diethyl phthalate. Thus prepared intermediates are then combined to form a concentrate which forms a disinfecting, bactericidal and fungicidal emulsion after reconstitution in the ratio of 5-15% of the concentrate per 85-95% of water and addition of 0.5-1% of acetic acid. Combination of individual components and intermediates are performed at 18-30°C.

Patent application No. P-327658 (publication date 1998/12/21) describes disinfectant compositions and methods of surface disinfection. The invention relates to disinfection of surfaces using a disinfectant composition containing 0.1 to 15% (w/w) of hydrogen peroxide and an antimicrobial essential oil of a mixture of essential oils as main ingredients.

Patent application No. P-327834 (publication date 1999/01/04) describes disinfectant compositions containing peroxide bleach, amphoteric surfactant, glutaraldehyde and an antimicrobial essential oil or an active ingredient thereof, as well as methods of surface disinfection.

Patent No. PL 186883 (publication date 1999/05/24) describes a slow-release gel preparation and a method of fighting acarid, butterfly, fungal and bacterial invasions on honeybee colonies. The invention relates to a method of fighting various beehive diseases by charging the beehives with efficient amounts of essential oils in slow-release preparations, wherein the term "oil" includes, but is not limited to, oils extracted from plants and their essential ingredients, such as monoterpenes, e.g. menthol, geraniol, thymol, myrcene, citral, limonene, carene, camphor, eugenol or cineol (eucalyptol); natural oils, such as lemon oil, eucalyptus oils, neem oil; or organic acids, such as formic acid, ascetic acid or oxalic acid. Monoterpenes, such as thymol or menthol, are preferable.

Patent application No. P-386340 (publication date 2009/04/14) describes a method of preparation of an essential oil by means of steam distillation of the macerate. The solution is characterized in that the distillation process is conducted in two stages. In the first stage, distillation flask containing the macerate and connected to the distillation apparatus is immersed in ultrasonic bath filled with high-boiling liquid and heated at the boiling point for 30 hours by heating the high-boiling liquid, preferably oil, and maintaining the oil temperature at 120°C; the distilled oil is obtained after condensation of the oil-containing steam. After the macerate is cooled down, the second stage of distillation is performed, consisting in subjecting the macerate-containing flask to ultrasounds by turning on the ultrasound system and mamtaining the distillation conditions as in the first stage.

Patent application No. CN101185446 (publication date 2008/05/28) describes a pesticidal sterilizing emulsifier in the form of a complex of gadolinium nitrate with berberine and the method of preparation of said complex. The pesticidal sterilizing emulsifier consists of a berberine and gadolinium complex, emulsifier, plant oil and water. The pesticidal sterilizing emulsifier is characterized by high efficiency, low toxicity, handy application etc. The prepared formulation is a suspension agent; in addition, the invention is characterized by strong sterilizing effect against bacterial tomato rot disease pathogen, carrot black speck pathogen, sunflower head and stem rot disease, and melon leaf speck disease pathogen.

Patent application No. CN101185445 (publication date 2008/05/28) describes a similar preparation, wherein the pesticidal sterilizing emulsifier is in the form of a complex of samarium nitrate an berberine, while patent application no. CN101185444 (publication date 2008/05/28) describes a pesticidal sterilizing emulsifier in the form of a complex of neodymium nitrate with berberine and the method of preparation of said complex.

Despite the continued research on preparation effective against pathogens, there is a constant need for a solution that would allow to efficiently fight off fungal pathogens without simultaneous eradication of useful organisms, without any hazards for humans and the environment, as well as without negative effects on the useful soil flora and fauna. The goal of this invention is to prepare a composition containing biologically active compounds present in hemp, which are environmentally safer that the commonly used chemicals and could be used for the protection of crop plants.

This invention achieves the goal defined above while solving the problems described in current art and associated with preparation of efficient plant protection agents, including natural extracts and essential oils that would protect seeds from pathogens.

The subject matter of invention is a composition directed against pathogens, particularly fungal pathogens, wherein said composition contains aqueous hemp panicle extract with low cannabinoid content, wherein the content of A⁹-tetrahydrocannabinol (Δ⁹- THC) is below 0.2% per dry plant mass and wherein said extract contains monoterpenes and sesquiterpenes.

Preferably, the composition contains a-pinene and/or β-pinene and/or Δ 3-carene and/or β- myrcene and/or limonene and/or β-felandrene and/or cis-ocimene and/or trans-ocimene and/or a-terpinene and/or trans-a-bergamotene and/or β-caryophyllene and/or β-humulene and/or β-farnesene and/or β-selinene and/or selina-3,7(l l)-diene.

Preferably, the aqueous hemp panicle extract is diluted in distilled water to achieve the concentration of at least 0.1%.

Preferably, the values of Ti, Ti₀, T₂₅, T₅₀, T₇₅, T₉₀ and MGT are reduced along with the increase in aqueous extract concentration from 0.1 to 0,05% compared to seeds not treated with the composition.

Preferably, the increase in the aqueous extract concentration reduces the number of dead seeds compared to the untreated seeds and increases the number of healthy, non- germinating seeds compared to the untreated seeds.

Preferably, the said composition reduces the prevalence of Alternaria dauci and Alternaria radicina fungi.

Preferably, the said composition improves the vigour, germination and health of the seeds of plants of Apiaceae family, preferably carrot.

The next subject of invention is a method of preparation of composition directed against pathogens, wherein hemp panicles are dried and ground, the ground plant matter is soaked in water in the ratio of 1 :14, heated to 100°C and extracted for at least 6 hours; next, the solution is filtered and washed with water to obtain a colourless filtrate, and the solution is evaporated to concentration of at least 4% of the aqueous extract, and wherein said method allows to obtain composition described above. The next subject of invention is the use of the aqueous hemp panicle extract to produce composition described above, wherein the seeds are soaked in the extract for 30 minutes.

The solution of the invention is presented in example embodiments as shown in the drawings, where:

Figure 1 presents the prevalence of Alternaria alternata on carrot seeds.

K - untreated seeds (control); KW - seeds macerated in water (control); WW - seeds macerated in the aqueous solution; KM - seeds macerated in methanol (control); WM - seeds macerated in the methanolic extract; KE - seeds macerated in the emulsifier (control); OE - seeds macerated in the essential oil; F - seeds dressed with fungicide;

Figure 2 presents the prevalence of Alternaria dauci on carrot seeds; explanation of acronyms as in Fig. 1 ;

Figure 3 presents the prevalence of Alternaria radicina on carrot seeds; explanation of acronyms as in Fig. 1 ;

Figure 4 presents the prevalence of Fusarium spp. on carrot seeds; explanation of acronyms as in Fig. 1 ;

Figure 5 presents the prevalence of fungus-free seeds; explanation of acronyms as in Fig. 1 ; For all figures, values followed by identical letters are not significantly different at a=0.05.

Below are example embodiments of the invention defined above.

Examples

The aim of the research was to evaluate the effect of aqueous and methanolic extracts as well as the essential oil of common hemp (Cannabis sativa L.) on the vigour, germination and health of carrot seeds.

The experiments were carried out using Berlikumer 2 type carrot seeds (previously Perfekcja GOW), batch no. 230196/033, obtained from the horticultural seed and nursery farm CNOS in Poznan.

Materials and methods

Preparation of extracts

Common hemp (Cannabis sativa L.) panicle extracts

Aqueous extract

Panicles of "Bialobrzeskie" variety hemp were dried outdoors. 105 g of ground plant material was placed in a 2000 mL round-bottom flask equipped with a reflux condenser. The dried plant material was soaked in 1500 mL of water, heated to 100°C and extracted for 6 hours. Next, the solution was filtered through a porcelain Buchner funnel and washed with water, yielding a colourless filtrate. The solution was evaporated to the volume of 1000 mL. A 4.07% aqueous solution was obtained.

Methanolic extract

The methanolic extract was obtained from the panicles of "Bialobrzeskie" variety hemp. The plant material (105 g) was placed in a flask , soaked in 1500 ml of 100% methanol, heated to 65 °C and extracted for 6 hours. The resulting solution was filtered through a porcelain Buchner funnel and washed with 100% methanol, yielding a colourless filtrate. The solution was evaporated to the volume of 1000 mL. A 2.09% methanolic solution was obtained.

Common hemp (Cannabis sativa L.) panicle essential oil

The essential oil was obtained by steam distillation from fresh panicles of "Bialobrzeskie" variety hemp. The oil is characterized by low cannabinoid content, including Δ⁹ tetrahydrocannabinol (Δ⁹ THC) (below 0.2% per dry plant mass). It is a clear, light-yellow to dark-green liquid. The relative density at 20°C is 0.890 to 0.10 g cm . Hemp oil contains 58 monoterpenes and 38 sesquiterpenes. These most important include: a-pinene, β-pinene, Δ 3- carene, β-myrcene, limonene, β-felandrene, cis-ocimene, trans-ocimene, a-terpinene, trans-a- bergamotene, β-caryophyllene, β-humulene, β-farnesene, β-selinene, selina-3,7(l l)-diene.

Fungicide

Seeds dressing was performed using Penncozeb 80 WP preparation, recommended by the Institute of Plant Protection (2008), containing 80% mancozeb (a dithiocarbamate class compound) The preparation is manufactured by Cerexagri S.A.

Treatment of seeds with the aqueous and methanolic extracts

The seeds were macerated for 30 minutes in aqueous and methanolic extract solutions at concentrations of 0.1, 0.3, 0.5, 1.0, 2.0 and 3.0%. Both extracts were diluted in distilled water. After the predefined time, the seeds were strained with a sieve and the seed surface was dried on filter paper. The control sample in case of seeds treated with the aqueous extract solutions consisted of seeds macerated for 30 minutes in distilled water. The control sample in case of seeds treated with the aqueous extract solutions consisted of seeds macerated for 30 minutes in methanol solutions at concentrations of 0.1, 0.3, 0.5, 1.0, 2.0 and 3.0%, respectively.

Treatment with the essential oil The seeds were macerated in the essential oil solutions at concentrations of 0.025, 0.05 and 0.1% for 30 minutes. The oil was dissolved in emulsifier solutions at respective concentrations of 0.003, 0.006 and 0.013%. After maceration, seeds were handled as in the case of treatment with aqueous and methanolic extracts. The control sample in case of seeds treated with the essential oil solutions consisted of seeds macerated in emulsifier solutions at concentrations of 0.003, 0.006,^'and 0.013%, respectively.

Dressing the seeds with fungicide

The seeds were macerated for 30 minutes in 0.5% fungicide solution. The fungicide was dissolved in distilled water.

Experimental combinations

The experiments studied the vigour, germination and health of carrot seeds at 20°C for the following combinations:

K - control (untreated seeds);

WW - seeds macerated for 30 minutes in aqueous hemp panicle extract solutions at concentrations of: 0.1% (WW 0.1%), 0.3% (WW 0.3%), 0.5% (WW 0.5%), 1.0%

(WW 1.0%), 2.0% (WW 2.0%), 3.0% (WW 3.0%);

KW - seeds macerated for 30 minutes in distilled water (control sample for seeds treated with the aqueous extract solutions);

WM - seeds macerated for 30 minutes in methanolic hemp panicle extract solutions at concentrations of: 0.1% (WM 0.1%), 0.3% (WM 0.3%), 0.5% (WM 0.5%), 1.0% (WM 1.0%), 2.0% (WM 2.0%), 3.0% (WM 3.0%);

KM - seeds macerated for 30 minutes in methanol solutions (control sample for seeds treated with the methanolic extract solutions) at concentrations of: 0.1% (KM 0.1%)→ control for WM 0.1%, 0.3% (KM 0.3%)→ control for WM 0.3%, 0.5% (KM 0.5%)→ control for WM 0.5%, 1.0% (KM 1.0%)→ control for WM 1.0%, 2.0% (KM 2.0%)→ control for WM 2.0%, 3.0% (KM 3.0%)→ control for WM 3.0%;

OE - seeds macerated for 30 minutes in hemp panicle essential oil solutions at concentrations of: 0.025% (OE 0.025%), 0.05% (OE 0.05%), 0.1% (OE 0.1%);

KE - seeds macerated for 30 minutes in emulsifier solutions (control sample for seeds treated with the essential oil solutions) at concentrations of: 0.003% (KE 0.003%)→ control for OE 0.025%, 0.006% (KE 0.006%)→ control for OE 0.05%, 0.013% (KE 0.013%)→ control for OE 0.1%;

F - seeds macerated for 30 min in 0,5% Penncozeb 80 WP fungicidal solution. Evaluation of seed vigour

Seed vigour was determined on samples of 300 seeds in each combination. Six repetitions of 60 seeds were performed. The seeds were distributed over Petri dishes (9 cm in diameter) onto six layers of filter paper previously moistened with distilled water. The seeds were incubated for 14 days in darkness at 20°C. Every 24 hours, seeds with radicles that have pierced the seed coats and were at least 1 mm long were counted and removed. This allowed for calculation of parameters defining the rate and synchronicity of seed germination.

Parameters defining the rate of germination:

- Ti - time required for germination of 1 % of the total of germinating seeds,

- Tio— time required for germination of 10% of the total of germinating seeds,

- T25 - time required for germination of 25% of the total of germinating seeds,

- T50 - time required for germination of 50% of the total of germinating seeds,

- T₇5 - time required for germination of 75% of the total of germinating seeds,

- T90 - time required for germination of 90% of the total of germinating seeds,

- MGT - mean germination time for a single seed.

Parameters defining the synchronicity of germination:

- U75-25 - difference between the times required for germination of 75 and 25% of the total of germinating seeds,

- U75-25 - difference between the times required for germination of 90 and 10% of the total of germinating seeds,

Evaluation of seed germination

Seed germination was evaluated on samples of 300 seeds in each combination. Six repetitions of 60 seeds were performed. The seeds were distributed over Petri dishes (9 cm in diameter) onto six layers of filter paper previously moistened with distilled water. The seeds were incubated for 14 days in darkness at 20°C. Germination energy was assessed after 7 days and germination capacity (percentage of normal seedlings), percentage of seedlings with pathological symptoms, percentage of deformed seedlings and percentages of dead and healthy, non-germinating seedlings were determined after 14 days of incubation.

Gmax, or the total percentage of germinating seeds, was also established. This parameter was determined using the seeds used for the assessment of the rate and synchronicity of germination. Evaluation of seed health

Evaluation of seed health was performed on samples of 200 seeds, in 5 repetitions of 40 seeds. The seeds were distributed over Petri dishes (9 cm in diameter), 20 seeds per dish, onto six layers of filter paper previously moistened with distilled water. Plates with seeds were placed in darkness at 20°C for three days, and then in darkness at -20°C for 24 hours. After freezing, plates with seeds were incubated for 7 days at 20°C under alternating lighting conditions (12 hours of darkness and 12 hours of light at the wavelength of 320- 400 nm). Fungi were identified based on sporulation and spore appearance. Evaluation was performed using a stereomicroscope at magnification of 50-60x; microscopic slides were prepared if necessary.

Statistical calculations

Germination rate (T1-T90 i MGT), synchronicity (U75-25, U90-10) and total number of germinating seeds (Gmax) were calculated using SeedCalculator 2.1 software [50].

All results were compared by single factor analysis of variance, and the significant differences were determined by Duncan test at the significance level of a=0.05, after converting the percentage values to Bliss angular degrees. Calculations were performed with help of STAT software.

RESULTS

The effect of the aqueous and methanolic extracts and the essential oil on the rate and synchronicity of seed germination

Maceration of seeds in aqueous extract solutions at concentrations of 0.1, 0.3 and 0.5% accelerated seed germination. When the aqueous extract was used at the lowest concentration, T₅₀, T₇₅, T₉₀ and MGT values were lower than the respective values for untreated seeds, soaked in distilled water and dressed with fungicide. Treating the seeds with the aqueous solution at concentration of 0.3% reduced the values of Ti, T]₀, T₂₅, T₅₀ and MGT compared to untreated seeds. However, no significant differences were observed in comparison to seeds soaked in distilled water or fungicide solution. Only MGT was significantly lower compared to the control combination, where the seeds were treated with water. In case of seeds treated with the aqueous solution at concentration of 0.5%, the values of T₂5, T₅o, T₇5, T₉o and MGT were significantly lower compared to untreated seeds. T₇₅, T₉₀ and MGT were also significantly lower compared to seeds soaked in water (Table 1).

Methanolic extracts at concentrations of 0.1, 0.3, 0.5, 1.0 and 3.0% reduced the time required for seed germination. After treating the seeds with methanolic extracts at two lowest concentrations, reduction in Ti, Tio, Τ₂₅, T50 and MGT was observed compared to all control combinations, i.e. to untreated seeds, seeds soaked in methanolic extract solutions at concentrations of 0.1 or 0.3% and in the fungicide solution. Only in case of T50 following the treatment of seeds with 0.3% methanolic extract, no significant difference was found compared to the fungicide-dressed control. At concentration of 0.5%, the methanolic extract reduced the Tio, T₂₅, T50 and MGT compared to untreated seeds and seeds soaked in 0.5% methanol. However, no significant differences were observed compared to the fungicide solution. Treatment of seeds with 1.0 and 3.0% methanolic extract solutions accelerated the genriination of seeds at the initial stage, as Ti, Tio and T₂₅ were significantly lower than in case of untreated seeds or seeds treated with 1.0 or 3.0% methanol. Treatment of seeds with 1.0% methanolic extract solution has also led to reduction in mean germination time (MGT) of a single seed compared to untreated seeds or seeds treated with 1.0 methanol (Table 1).

The essential oil accelerated the germination of seeds at the initial stage when used at the highest concentration of 0,1%. Ti, Tio, T₂₅ were significantly lower from the respective values observed in untreated seeds. However, no significant differences were observed compared to control combinations where the seeds were treated with 0.013% emulsifier solution or fungicide solution. In case of seeds treated with 0.025% essential oil solution, T_\ , T₂5 and T50 were significantly higher than in case of seeds treated with 0.003% emulsifier solution or fungicide solution. However, these values were not significantly different compared to those of the untreated seeds (Table 1).

Treating carrot seeds with aqueous and methanolic extracts and essential oil solution did not improve germination synchronicity compared to the control combinations, i.e. the untreated seeds, seeds treated in water, methanol or emulsifier solutions and seeds dressed with fungicide. Only the 0.1 and 0.5% aqueous extracts improved the U_75-25 values compared to seeds treated with distilled water or fungicide. Poorer values of this parameter compared to untreated seeds were observed after soaking the seeds in 0.3 and 3.0% methanolic extract solutions (Table 2).

The effect of the aqueous and methanolic extracts and the essential oil on seed germination

No significant differences in total number of germinating seeds (Gmax) were observed between the particular experimental combinations (Table 3).

Germination energy of the untreated seeds was 14%, while the germination capacity was 14,7% (Table 3). Soaking the seeds in aqueous extract solutions at concentrations of 0.3, 0.5, 2.0 and 3.0% and in methanolic extract solutions at concentrations of 0.1, 0.3, 0.5, 1.0 and 3.0% improved the seed germination energy and capacity compared to untreated seeds. No significant differences were observed compared to the combination involving fungicide dressing. Germination energy was significantly higher compared to seeds soaked in water only in case of seeds treated with 2.0 and 3.0% aqueous extract solutions. Following the treatment with 0.1% methanolic extract solution, the germination capacity was higher than following the treatment with 0.1% methanol solution. Highest germination energy was observed in seeds treated with 1.0% methanolic extract solution and 2.0 and 3.0 aqueous extract solutions. Seed germination capacity was best increased by treatment with 2.0% aqueous extract solution and 1.0% methanolic extract solution (Table 3).

Maceration of seeds in both essential oil and emulsifier solutions improved the seed germination energy and capacity compared to untreated seeds. The values of these parameters did not differ significantly between the seeds treated with oil, emulsifier and fungicide solutions. 3).

A reduction in number of seedlings with pathological symptoms compared to untreated seeds was observed only in case of fungicide dressing (Table 3).

Fungicide dressing resulted in highest increase in the number of deformed seedlings; however, the difference was not statistically significant compared to the untreated seeds. No deformed seedlings were observed after treating the seeds with 0.5% aqueous and methanolic extracts, as well as with 1.0% methanolic extract (Table 3). Aqueous and methanolic extracts at concentrations of 0.1, 0.3, 0.5, 2.0 and 3.0% reduced the numbers of dead seeds compared to the untreated seeds. However, no significant differences were observed compared to seeds soaked in water and methanol solutions, respectively, as well as to the fungicide-dressed seeds. The essential oils reduced the number of dead seeds compared to the untreated control at all concentrations studied. No significant differences were observed in comparison to the remaining control combinations, i.e. seeds soaked in the emulsifier solutions and in the fungicide solution (Table 3).

The aqueous and methanolic extracts as well as the essential oil solution increased the number of healthy, non-germinating seeds compared to the untreated control at all concentrations studied. No significant differences were observed compared to control combinations where the seeds were treated with water, methanol solutions or emulsifier solutions. The largest rate of healthy, non-germinating seeds (13%) was observed following the treatment with 0.5% methanolic extract solution and fungicide solution (Table 3). The effect of the aqueous and methanolic extracts and the essential oil on seed health The untreated carrot seeds (control) were commonly colonized by pathogenic fungi Alternaria dauci Groves et Skolko (Kiihn) - 54.5%, Alternaria radicina Meier, Drechsler et Eddy (Neerg.) - 66,0% and Fusarium spp. - 1,0% (Fig. 1, 2, 3, 4, 5).

The percentage of seeds colonized by A. alternata was reduced compared to the untreated seeds following the treatment with 0.013% emulsifier solution and the fungicide solution. However, the number of seeds affected by these fungi after soaking in 0.013% emulsifier solution was significantly higher than after fungicide dressing (Fig. 1).

Treating the seeds with aqueous extract solutions at highest concentrations (2.0 and 3.0%) reduced the A. dauci colonization compared to untreated seeds and water-soaked seeds. No significant differences were observed in comparison to fungicide-dressed seeds. Methanolic extract solutions at concentrations of 1.0, 2.0 and 3.0%, as well as methanol solution at concentration of 3.0%, reduced the number of seeds colonized by this species compared to the untreated control. However, the methanolic extract solution at the highest concentration was less efficient than the fungicide solution and 3.0% methanol solution. Methanolic extract solutions at concentrations of 1.0 and 2.0% worked better than the control, where the seeds were treated with 1.0 and 2.0% methanol solutions. Only the 2.0% methanolic extract was found to be superior to the fungicide. Methanolic extract solution at concentration of 1.0% and methanol solution at concentration of 3.0% were characterized by similar effect as fungicide, which reduced the percentage of affected seeds compared to the untreated control (Fig. 2).

A. radicina colonization was reduced compared to the untreated after treatment with aqueous extract solutions at all concentrations studied. However, the effect of the aqueous extract at the two lowest concentrations (0.1 and 0.3%) was insignificant compared to seeds treated with water, and inferior to that of the fungicide solution. At the remaining solutions (0.5, 1.0, 2.0 and 3.0%), the aqueous extract had the effect comparable to that of the fungicide, and reduced the degree of colonization by this pathogen compared to the untreated seeds. Only the 1.0% aqueous extract solution did not reduce the prevalence of A. radicina compared to the control that involved water-soaked seeds. The methanolic extract solutions at concentrations of 0.1, 1.0, 2.0 and 3.0% and the methanol solutions at concentrations of 0.1, 0.3, 0.5 and 3.0% reduced the colonization of seeds by A. radicina. The methanolic extract solution at the lowest concentration and the methanol solutions at the two lowest concentrations were inferior to the fungicide solution. At the remaining concentrations, the effects of the methanolic extract and methanol solutions were comparable to the effect of the fungicide solution. Only the 2.0% methanolic extract was significantly higher than the control combination where the seeds were soaked in 2.0% methanol.

Essential oil solution at concentration of 0.025% and emulsifier solutions at concentrations of 0.003 and 0.013%, also reduced the number of seeds affected by A.radicina. The effects of the essential oil and the 0.013% emulsifier solution were comparable to that of the fungicide. 3).

The prevalence of Fusarium spp. increased compared to untreated and fungicide-dressed seeds following treatment with 0.3% aqueous extract and 2.0% methanol solutions. No significant differences were observed in the remaining combinations compared to these controls. The fungi of Fusarium spp. were eradicated only after the fungicide dressing. (Fig. 4).

An increase in the number of healthy seeds compared to the untreated control was observed only in case of fungicide dressing (26.5%), 3.0% methanolic extract solution treatment (1.5%) and 3.0% methanol solution (1.0%). The percentage of healthy seeds after treatment with methanolic extract was not significantly different compared to the control, i.e. seeds soaked in 3.0% methanol (Fig. 5).

Summary of the results

Following conclusions may be drawn from the studies performed:

1. Maceration of seeds in aqueous and methanolic extract solutions at concentrations of 0.1, 0.3 and 0.5% accelerated seed germination.

2. In most cases, treating seeds with aqueous and methanolic extract solutions, essential oil and fungicide increased seed germination energy and capacity.

3. Carrot seeds were commonly colonized by Alter naria alternata, A. dauci and A. radicina fungi.

4. Only the fungicide reduced A. alternata colonization.

5. Treatment with aqueous extract solutions at concentrations of 2.0 and 3.0% and methanolic extract solutions at concentrations of 1.0 and 2.0% reduced the prevalence of A. dauci and A. radicina as effectively as fungicide dressing.

6. The highest rates of fungi-free seeds were observed following fungicide dressing.

Discussion

The study evaluates the effect of aqueous and methanolic extracts as well as the essential oil of hemp {Cannabis sativa L.) on the vigour, germination and health of Berlikumer 2 carrot seeds. The seed batch used for the experiment was largely colonized by Alternaria alternata, A. dauci and A. radicina fungi. Neither any of the tested extracts, nor the essential oil could reduce the prevalence of A. alternata. At the same time, treatment with aqueous extract solutions at concentrations of 2.0 and 3.0% and methanolic extract solutions at concentrations of 1.0 and 2.0% largely reduced the prevalence of A. dauci and A. radicina. At the above concentrations, both extracts were as effective as the fungicide. Maceration of carrot seeds in extract solutions at lowest concentrations (0.1-0.5%), in particular in methanolic extract solutions, had significant impact on the reduction of seed germination time. No information regarding the effect of hemp extracts and essential oil on seed vigour.

Our own research showed that the treatment of seeds with hemp extract and essential oil solutions at most concentrations used led to improvements in seed germination energy and capacity compared to the untreated seeds. Seed treatment had no effect on the number of seedlings with pathological symptoms, but the number of dead seeds was reduced compared to the untreated seeds. The reason for the reduction in the number of dead seeds following the treatment was the reduction in the prevalence of pathogens. One may suspect that the reduction of the inoculum led to germination of part of the dead seeds following their maceration in extract or essential oil solutions, yielding seedlings with pathological symptoms.

Table 1. The effect of hemp extracts and essential oil on carrot seed germination rate

Table 1 cont.

Table 2. The effect of hemp extracts and essential oil on the synchronicity of carrot seed germination

Seed treatment Synchronicity of seed germination (days)

method U75-25 U90-10

K 0.76 cde 1.72 abed

KW 0.86 bed 1.93 abc

WW 0.1% 0.49 e 1.29 cd

WW 0.3% 0.72 de 1.49 bed

WW 0.5% 0.55 e 1.24 d

WW 1.0% 0.84 bed 1.82 abed

WW 2.0% 0.90 abed 1.87 abc

WW 3.0% 0.82 bed 1.88 abc

KM 0.1% 0.89 abed 1.84 abed

WM 0.1% 1.01 abc 2.05 ab

KM 0.3% 0.89 abed 1.81 abed

WM 0.3% 1.08 ab 2.09 ab

KM 0.5% 0.86 bed 1.88 abc

WM 0.5% 0.70 de 1.45 bed

KM 1.0% 0.97 abed 2.00 abc

WM 1.0% 0.94 abed 1.94 abc

KM 2.0% 0.94 abed 1.95 abc

WM 2.0% 0.99 abc 2.03 ab

KM 3.0% 0.94 abed 2.05 ab

WM 3.0% 1.12 a 2.18 a

KE 0.003% 0.83 bed 1.70 abed

OE 0.025% 0.76 de 1.56 bed

KE 0.006% 0.71 de 1.52 bed

OE 0.05% 0.72 de 1.53 bed

KE 0.0125% 0.72 cde 1.47 bed

OE 0.1% 0.89 abed 1.83 abed

F 0.91 abed 1.87 abed

Values in columns followed by identical letters are not significantly different at a = 0.05

Explanation of U75-25 and U90-10 provided in Section 3.2.5.

Explanation of acronyms provided below Table 1. ZJ

Table 3. The effect of hemp extracts and essential oil on seed germination

Table 3 cont.

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Claims

Claims

1. Composition directed against pathogens, particularly fungal pathogens, wherein said composition contains aqueous hemp panicle extract with low cannabinoid content, wherein the content of A⁹-tetrahydrocannabinol (A⁹-THC) is below 0.2% per dry plant mass and wherein said extract contains monoterpenes and sesquiterpenes.

2. Composition according to claim 1, wherein the composition preferably contains a- pinene and or β-pinene and/or Δ 3-carene and/or β-myrcene and/or limonene and/or β- felandrene and/or cis-ocimene and/or trans-ocimene and/or a-terpinene and/or trans-a- bergamotene and/or β-caryophyllene and/or β-humulene and/or β-farnesene and/or β- selinene and/or selina-3,7(l l)-diene.

3. Composition according to claim 1, wherein the aqueous hemp panicle extract is diluted in distilled water to achieve the concentration of at least 0.1%.

4. Composition according to claim 1, wherein the values of Ti, Tio, T₂5, T50, T75, T90 and MGT are reduced along with the increase in aqueous extract concentration from 0.1 to 0,05% compared to seeds not treated with the composition.

5. Composition according to claim 1, wherein the increase in the aqueous extract concentration reduces the number of dead seeds compared to the untreated seeds and increases the number of healthy, non-germinating seeds compared to the untreated seeds.

6. Composition according to claim 1, wherein the said composition reduces the prevalence of Alternaria dauci and Alternaria radicina fungi.

7. Composition according to claim 1, wherein the said composition improves the vigour, germination and health of the seeds of plants of Apiaceae family, preferably carrot.

8. Method of preparation of composition directed against pathogens, wherein hemp panicles are dried and ground, the ground plant matter is soaked in water in the ratio of 1 :14, heated to 100°C and extracted for at least 6 hours; next, the solution is filtered and washed with water to obtain a colourless filtrate, and the solution is evaporated to concentration of at least 4% of the aqueous extract, and wherein said method allows to obtain composition according to claims 1-7.

9. The use of the aqueous hemp panicle extract to produce composition according to claims 1-7, wherein the seeds are soaked in the extract for 30 minutes.