The effect of plant extracts from Solanum nigrum L. and Avena fatua L. on the growth of some weed species and agropathogenic fungi

Experiments to evaluate 40% and 80% methanol extracts prepared from the aboveground parts of two Solanum nigrum biotypes and from the roots of two Avena sativa biotypes were used to test the growth and development of some weed species and agropathogens. The species collected from different crop fields were Viola arvensis, Chenopodium album, Stellaria media, Papaver rhoeas, and Thlaspi arvense together with the pathogenic fungi Fusarium avenaceum, F. culmorum, and F. oxysporum. Plant morphological changes and significant fresh weight reduction of Viola arvensis were observed when treated with both 40% and 80% extracts. A similar response was found in C. album, but only when plants were treated with an 80% extract obtained from plants of S. nigrum collected from a maize field. The most susceptible to both extracts from A. fatua roots growing in a spring wheat field were C. album and S. media, whereas V. arvensis was only susceptible to the 80% extract. The addition of the extracts obtained from A. fatua to PDA medium at concentrations of 0.1% and 1% contributed to significant reductions in the mycelium growth of all three Fusarium species. The extract obtained from S. nigrum added to PDA medium at 1% concentration slightly stimulated mycelium growth of F. oxysporum.


Digital signature
This PDF has been certified using digital signature with a trusted timestamp to assure its origin and integrity. A verification trust dialog appears on the PDF document when it is opened in a compatible PDF reader. Certificate properties provide further details such as certification time and a signing reason in case any alterations made to the final content. If the certificate is missing or invalid it is recommended to verify the article on the journal website.
to other plant species as well as to soil microorganisms and, therefore, can be a threat to the ecological balance in agrobiocenoses [5]. They easily establish on fields with a zero-ploughing system characterized by the abundance of crop plant residues in the upper layers of soil. They prefer acidic soils from which uptake and accumulation of heavy metals from fertilizers and pesticides is easier. Rapid adaptation to different habitat conditions is one of the factors affecting the concentration of bioactive substances and enzymatic activity [6], as well as their influence on agrophages, e.g., Fusarium fungi, pests, or segetal weeds [14]. Previous studies have reported that intensified production of the secondary metabolites reflected plant reaction to oxidative stress provoked by water or nutrient deficit, too high or too low a temperature, pathogen invasion, mechanical injury, etc. [15][16][17]. An example of plants with high bioactivity is species from Solanum genus, notably Solanum nigrum L. The most toxic plant parts are its immature fruits containing glycoalkaloids, which are harmful for human health, and others such as solasodine. Steroidal saponins, solanigrosides, and degalactotigonin have also been extracted from whole plants of S. nigrum. They have properties similar to cortisone and activity similar to natural insecticides [18]. Highly bioactive are also species from the genus Avena, both wild forms, such as A. strigosa, A. abyssinica, A. bizantina, and A. fatua L., as well as a cultivated form, namely A. sativa . Bioactivity of these species results from the presence of some valuable substances such as flavonoids, nicotinamide, water-soluble silicates, amino acids, peptides, and triterpene saponin -avenacin (A-1, A-2, B-1, B-2) that shows fungicidal activity. Avenacin A-1 exists only in the epidermal cells of roots and, among all avenacins, is the most toxic to fungi [11: p. 213-223] [19]. An example can be the use of the toxic activity of saponin present in alfalfa (Medicago sativa ssp. sativa L.) to protect hops from some pathogens and insects.
There is no knowledge as to whether the bioactivity of these species is a result of their high propagation and invasiveness or it is derived from the presence of specific substances with synergetic, additive, or inhibitory action. The aim of this present research was to investigate the differences in the effects of extracts from plants of S. nigrum and A. fatua on some segetal weed species and pathogenic fungi.

Material and methods
Plant material of S. nigrum was collected from maize and potato fields of A. sativa collected from spring wheat and maize fields and was used to investigate the bioactivity for some segetal weed species and plant pathogenic fungi. It was obtained from fields with different soil conditions. Both maize fields were located on brown soil with a high organic matter (3.4% and 3.0%) and nutrient contents. The potato and spring wheat fields were located on podzolic soil also with low organic matter (1.2% and 1.3%) and nutrient contents. The experiment was carried out under glasshouse and laboratory conditions.

Plant material
Plants were collected from untreated plots of arable fields differing in their cultivated crop species. Complete shoots and roots of plants obtained from the field were chopped into small pieces and dried in a cabinet dryer at a temperature of 25-30°C for 60 h. The final water content of the dried material was <10%.
Preparation of extracts for weed testing and for the antifungal assay Fifty g of plant dry matter was treated with 250 mL of 80% methanol at room temperature for 24 h. This was then centrifuged in a rotary centrifuge at 125 rpm for 1 hour. The next step was fractionation using the method of extraction to solid phase (SPE) with a C18 filler. Fractionation was initiated with the addition of water to remove sugars from the extract, then 40% methanol was added to obtain the flavonoid fraction, or 80% methanol was added to separate the saponin fraction. The methanol fractions so obtained were placed in a rotary evaporator to evaporate off the solvent. The dry residue of each fraction was dissolved in 100 mL of water for testing on the weed species or 100 mL of dimethylsulfoxide (DMSO) for the antifungal assay. The extract was stored at 4°C.

Examination of extract effect on weeds
In order to evaluate the inhibitory effects of the plant fractions extracted (saponins and flavonoids), a glasshouse experiment was set up to test on the selected weed species. The test species were: Viola arvensis Murr., Chenopodium album L., Stellaria media L., Papaver rhoeas L., and Thlaspi arvense L. The experimental design was a completely randomized blocks pattern with three replications. Seeds of the weed species were sown onto 15-cm diameter pots filled with a mixture of 2:1 peat:sand (v/v). Soon after seedling emergence, they were thinned to leave five plants per pot. When they had reached the four-five-leaves growth stage, they were sprayed with the prepared extracts. The treatment was carried out using a laboratory sprayer (Aporo) fitted with a beam equipped with flat fan nozzles (TeeJet XR 11003-VS). The nozzles were operated at a pressure of 200 kPa and a speed of 2.5 km h −1 producing a spray volume of 250 L ha −1 .
Phytotoxicity evaluation, including the degree and type of injury, was carried out 2 weeks after treatment. It was recorded using a 9-point scale, where: 1 -no injury, 2 -very slight symptoms, 3 -slight symptoms, 4 -strong symptoms, 5 -slight injuries, 6 -pronounced injuries, 7 -strong injuries, 8 -very strong injuries, 9 -total plant destruction. Four weeks after spraying, plants were harvested and the fresh weights of weed shoots was recorded. The efficacy of the extracts was determined based on fresh weight loss as a result of the extract spraying by comparison with untreated plants. The data were subjected to analysis of variance using Statgraphics (Microsoft).

Examination of extract effects on Fusarium fungi
The effect of the plant extracts on fungal growth was performed under laboratory conditions using the standard "poisoned food technique" (see [20]). The pathogenic fungi F. oxysporum Schlecht, F. culmorum (W. G. Sm.), and F. avenaceum (Fr.) Sacc. were isolated in 2016 from maize plants exhibiting the typical symptoms of fusariosis. Fragments of infected plants were disinfected in 0.5% sodium hypochlorite for 1 minute and then cut to produce inocula that were plated onto PDA medium (potato dextrose agar). After incubation at room temperature, growing colonies of fungi were transferred to standard tubes with PDA medium. They were identified taxonomically using monographic keys. Plant extracts were tested at concentrations of 0.1% and 1%. The extracts dissolved in DMSO were introduced into cooled PDA medium at given concentrations and, after thorough mixing, they were poured onto Petri dishes. In order to eliminate bacteria from the PDA medium, an antibiotic (tetracycline, 20 µg mL −1 ) was added. After medium solidification, a 10-mm diameter disc of the fungi was placed in the center of each Petri dish. Discs were cut from the edges of 10-day cultures of the fungi growing on PDA medium. Radial growth of mycelium was measured every day until the control plates were completely overgrown. The experiment was terminated after 9 days from inoculation. All tests were performed in triplicate, in two series.
The controls were fungal colonies growing on PDA supplied with solvent (DMSO) alone at the same concentrations of 0.1% and 1%. Plates were incubated at 27°C.
Coefficients of radial growth (T) and coefficients of linear growth inhibition (H) were calculated according to the equations below [20]: where: T -the coefficient of radial growth, A -average diameter for colonies (mm), D -number of days from the set-up of colonies to the final measurement, b 1 , b x -the increment in colony diameter up to the final measurement (mm), d 1 , d x -number of days from the final measurement.
The coefficient of linear growth inhibition (H) was calculated according to Abbot's equation [20] at the ninth day of the experiment: where: H -the coefficient of linear growth inhibition, K 0 -diameter of colonies on control plate, F -diameter of colonies growing on dishes containing the extract at a particular concentration.
Statistical analysis was performed by analysis of variance at p ≥ 0.05 using Statistica 8.0 (StatSoft).

Results
All the weed species tested are highly responsive to herbicides with different modes of actions, and, as such, have a rapid ability to adapt to various environments and preferences in this habitat. They also easily adapt to intensive agricultural management on arable lands.
Both the fractions extracted from shoots of S. nigrum collected from the potato fields were highly bioactive towards V. arvensis (Tab. 1). However, this species exhibited the strongest reaction to the 80% extract that was reflected in growth inhibition, leaf chlorosis, plant deformation, and consequently, biomass reduction. A similar plant response to the 80% extract obtained from S. nigrum from both the maize and potato fields was observed for Ch. album. The remaining weed species did not show any susceptibility to the extracts of S. nigrum, regardless of the origin of the plant material.
Considering the effect of all the extracts obtained from A. fatua, only the 80% extract prepared from the roots of plants collected from the spring wheat field showed high bioactivity for Ch. album, S. media, V. arvensis, and P. rhoeas (Tab. 2). The most susceptible to both the extracts were Ch. album and S. media, whereas V. arvensis was highly sensitive only to the 80% extract. The other weed species tested (P. rhoeas, T. arvense) did not show any susceptibility to the extract obtained from plants of A. fatua.
The results of this research allowed us to confirm the high bioactivity and inhibitory effect of the extracts obtained from both shoots and roots of A. fatua against three Fusarium species, i.e., F. oxysporum, F. culmorum, F. avenaceum (Tab. 3-Tab. 5). However, the root extract obtained from plants grown on the spring wheat field used at a concentration of 1% showed the greatest bioactivity. The coefficient of linear growth inhibition for F. avenaceum was as high as 72% (Tab. 5). The extract obtained from A. fatua was the only one which showed similar activity, but at a tenfold lower concentration (0.1%). The coefficients of linear growth inhibition for F. oxysporum and F. culmorum treated with both shoot and root extracts were 12% and 14%, respectively (Tab. 3, Tab. 4).  F -phytotoxicity in scale 1-9 (scale explanation is given in "Material and methods"); GI -growth inhibition; CH -chlorosis; D -deformations.

Discussion
Wild plants including segetal weeds exude different chemicals into their rhizosphere environments during their growth and at the time of their decomposition. These chemicals that possess bioactivity are allelochemicals as they influence germination, growth, development, and crop yield of other species [21]. During decomposition of plant residues resulting from tissue destruction, the allelochemicals exuded are quickly translocated into soil where decomposition is continued. Their bioactivity is therefore much affected by prevailing soil conditions, including pH and organic matter content. This was a reason for the investigation of individuals within species existing in different habitats [22,23]. The allelopathic potential of these compounds is higher in the early stages of plant growth and it decreases with ageing. For this reason, our study included individuals at different growth stages. The results we obtained confirm the greater phytotoxic effect for extracts obtained from young, aboveground parts of S. nigrum. Similar diversification in allelopathic potential was observed in laboratory and field experiments by other researchers [24,25]. They observed higher inhibitory effects of phenolics obtained from young aboveground parts of cereals and oil-seed rape as compared to those obtained from plants during the harvest period. In a natural habitat, the species examined are the most frequent on humus-rich soil with a neutral pH [22]. Under field conditions, they prefer zero-tillage environments with an acidic soil that features an abundance of crop residues in the top layer of soil, and potentially uptake and accumulation of heavy metals derived from fertilizers and pesticides [2]. In this study, the plant material was collected from a maize field growing on brown soil with a neutral pH and high organic matter content, as well as from potato and spring wheat fields growing on podzolic soil with a slightly acidic pH and low organic matter content. The extracts obtained from both S. nigrum and A. fatua growing on podzolic soil resulted in a greater phytotoxic effect on the weed species examined.
Agroecosystems considerably alter the production of specific secondary metabolites, especially in plants that aggressively establish themselves in new habitats. The level of secondary metabolites in such plants is strongly affected by nitrogen availability in the soil. Plants growing in habitats poor in assimilable nitrogen produce compounds containing carbon sugars and other compounds not containing nitrogen, including terpenoids, phenolics, some pigments, and vitamins [26,27]. However, in intensively-managed arable habitats that are rich in assimilable nitrogen, plant metabolism is altered towards the production of nitrogen compounds such as free amino acids, proteins and alkaloids [14].
Plant metabolite production can be also affected by abiotic factors including temperature, water status, pesticides use, or heavy metal contamination. Our Tab F -phytotoxicity in scale 1-9 (scale explanation is given in "Material and methods"); GI -growth inhibition; CH -chlorosis; D -deformations; N -necrosis.

Tab. 3
The coefficient of radial growth (T) and the coefficient of linear growth inhibition (H) for F. oxysporum growing on PDA medium with addition of S. nigrum (maize field) and A. fatua (spring wheat field) extracts. The negative values means the stimulation of linear growth. * Average colony diameter measured at the ninth day of the experiment.

Tab. 4
The coefficient of radial growth (T) and the coefficient of linear growth inhibition (H) for F. culmorum growing on PDA medium with addition of S. nigrum (maize field) and A. fatua (spring wheat field) extracts. The negative values means the stimulation of linear growth. * Average colony diameter measured at the ninth day of the experiment.
Tab. 5 The coefficient of radial growth (T) and the coefficient of linear growth inhibition (H) for F. avenaceum growing on PDA medium with addition of S. nigrum (maize field) and A. fatua (spring wheat field) extracts. The negative values means the stimulation of linear growth. * Average colony diameter measured at the ninth day of the experiment.
literature review suggests that changes in metabolism of plant cells, tissues, or whole plants can be induced by addition some substances, such as dichlorophenoxyacetic acid (2.4-D), 6-benzilaminopurine (6-BAP), kinetin, α-napthylacetic acid (NAA), indolyl-3-acetic acid (IAA), methyl jasmonate, or even by UV-B radiation [15,16,21]. Plants exposed to environmental stresses or infested by pathogens produce chemical defences such as preinfection compounds. However, these substances are constantly present in the plant regardless of whether the plant is infected or not. This group includes the metabolites which limit or totally inhibit growth of microorganisms and also the metabolites whose concentration increases after infection, e.g., terpenoids, quinones, and phenolics [28,29]. The other metabolite group involved in resistance to pathogenic fungi is the hydroxystilbenes. This includes five-cyclic triterpenic glycosides represented by avenacin A1 extracted from oats. It has shown a moderate inhibitory effect against the fungus Candida albicans [30][31][32][33]. This finding was partly confirmed in the present research, in which the growth inhibition for F. oxysporum, F. culmorum, and some of the weed species treated with the root extract obtained from A. fatua was proven.
Bioactive compounds extracted from plants belonging to the genus Solanum can be also used in the pharmaceutical industry. One example is the alkaloid solasodine, which is extracted from these plants. Another reported in the literature is important pharmaceutically, the sapogenin streroid diosgenin. It occurs in plants of the Dioscoreaceae family and also in some species in the Solanaceae and Fabaceae. The presence of tigogenin, diosgenin and yamogenin has been noted in S. dulcamara [9]. However, S. nigrum is highly toxic because of the glycoalkaloids content (solanine, solamargine, solasonine) in fruits [18]. In some European countries, solasonine is used as starting material for half-synthesis of corticosteroids and sex hormones, and it also exhibits traits similar to cortisone [34,35]. Solanine, solamargine, and the plant extract obtained from S. nigrum at a concentration of 10 −5 M induced a negative chronotropic effect on the activity of the shrinkage heart beetle T. molitor. Among these substances, the strongest activity has been shown by solasonine. Some studies have also reported highly cytostatic effects of this compound [36,37].
Environmental conditions on arable fields affect both seed germination and plant morphological traits, as well as determine the concentration of bioactive compounds, enzymatic activity, and rates of plant decomposition [16,17,38]. Many plant species infected by microorganisms start to accumulate metabolites to reach a toxic level. They are mainly aromatic compounds and especially coumarin derivatives. Coumarins and phenolic acids are highly toxic to numerous microorganisms. As an example, scopolamine and chlorogenic acid are phenolic compounds that are accumulated in plants belonging to the Solanaceae due to their infection by the fungus Phytophthora infestans. Phenolic acids are also recognized as substances able to inhibit seed germination [10].
The results of our studies on the effects of plant extracts obtained from commonly occurring weed species and invasive plants encourage investigation in this area. Further research can be useful in the development new plant protection products that are proecological and neutral to the natural environment.

Conclusions
■ The most sensitive weed species to the extracts tested was V. arvensis. Its growth was significantly reduced when plants were treated with 80% methanol extracts of S. nigrum. A similar reaction to the 80% methanol extract was recorded for Ch. album. ■ Methanol extracts at concentrations of 40% and 80% obtained from A. sativa roots growing on a spring wheat field caused growth inhibition, plant injuries (chlorosis, deformation), and significant biomass reduction of Ch. album and S. media. A similar response was observed for V. arvensis treated with this extract at a concentration of 80%. ■ The addition of methanol extracts from A. fatua to PDA medium at a concentration of 1% and also as low as 0.1% caused significant growth inhibition of F. avenaceum, F. culmorum, and F. oxysporum mycelium. ■ The extract from S. nigrum at a concentration of 1% slightly stimulated the growth of F. oxysporum mycelium.