Autoallelopathic and Allelopathic Influence of Aqueous Winter-Cereal Extracts

e present study aimed to assess the allelopathic potential of four cereals: winter wheat ( Triticum aestivum L.), triticale ( ×Triticosecale Wittm.), spelt wheat ( Triticum spelta L.) and barley ( Hordeum vulgare L.) through a completely randomized (CR) design. e allelopathic eﬀects of water extracts of diﬀerent parts of the cereal plants (stem, leaf, and spike) at diﬀerent concentrations (0.5%, 1.0%, 2.0%, and 4.0%) were evaluated on the seed germination and seedling growth. e germination rate, length, and dry weight of the shoot and root of the seedlings were measured. Ferulic acid was detected in all the cereals. e water extracts at 2.0% and 4.0% concentration had an allelopathic eﬀect on the germination rate, shoot and root length of seedlings of spelt wheat, barley, and triticale, and the stem and leaf extracts aﬀected the root and shoot length of winter wheat. e allelopathic eﬀect of the dried powder of the cereals were evaluated in pot experiments. Both spelt wheat and triticale powder treatment at elevated CO 2 levels increased the dry weight of the root, as well as the length of the shoot and root of winter wheat. Furthermore, treatment with 4.0 g of dry cereal powder combined with an elevated level of CO 2 increased the shoot length, whereas the root length of winter wheat was unaﬀected. In summary, the combination of dry cereal powder with elevated CO 2 stimulates the initial growth of winter wheat.


Introduction
e allelopathic effects of cover crop species are important during the germination and seedling growth (Cheng & Cheng, 2015;Shekoofa et al., 2020). e allelopathy phenomenon is understood as any influence of one plant on another plant through the release of chemical compounds (Jabran, 2017;Wu et al., 2020). Indeed, autoallelopathy or autotoxicity is a common problem in agriculture, which contributes to reduced yield (Singh et al., 2010). Autoallelopathic influence is intraspecific; chemical compounds of a plant inhibit or suppress the growth of another plant (Gaba et al., 2018;Oraon & Mondal, 2021;Singh et al., 2010). Allelopathy is a physiological and ecological mechanism influencing agricultural crop production . Due to the favorable climatic conditions and socioeconomic importance of cereals in the human diet, the cultivation of winter cereals is quite common (Georgieva, 2019;Jalali et al., 2020). us, it is important to understand the allelopathic effects influencing the germination and seedling growth of winter cereals (El Beheiry et al., 2019;Sołtys et al., 2010).
Germination and seedling growth are important stages of plant growth (Korotkova et al., 2021;Shahrajabian et al., 2019). New strategies for managing organic production using allelopathy and autoallelopathy are emerging (Karami & Akhzari, 2020). Additionally, there is a lack of data on the influence of elevated CO 2 levels on the autoallelopathic influence of cereals, specifically in light of the global greenhouse effect, hence this aspect was also considered in this study. e main purpose of this study was to assess the autoallelopathic potential of four cereals [winter wheat (Triticum aestivum L.), triticale (×Triticosecale Wittm.), spelt wheat (Triticum spelta L.), and barley (Hordeum vulgare L.)] under elevated CO 2 conditions.

Plant Materials
e specimens of four cereal species: winter wheat (Triticum aestivum), triticale (×Triticosecale Wittm.), spelt wheat (Triticum spelta L.), and barley (Hordeum vulgare L.) at the growth phase of spike/panicle development, were obtained from the experimental station of the University of Agriculture in Krakow (located at Mydlniki-Krakow). e plant materials were air-dried in the shade at room temperature of 24 to 25°C. e dried materials were then segregated partwise into stem, leaves and spikes/panicles. e separated parts were ground in an electric grinder and weighed on the balance (Radwag, Poland) up to two decimal places. e ground material was stored in plastic containers at room temperature until further use. e aqueous extracts having 0.5%, 1.0%, 2.0%, and 4.0% concentration of the dried plant material were prepared in glass flasks. For the preparation of extract, dry powder (0.25, 0.50, 1.00, and 2.00 g) of each type of cereal meal and 50 mL of distilled water was gently mixed in the flask covered with aluminum paper. e flasks containing the extracts were stored in a laboratory booth maintained at 21°C. Aer 24 hr, the extracts were filtered by using a regular filter paper (Ahlstrom-Munksjö, Germany).

Germination Test
e glass Petri dishes (11 cm diameter) were cleaned with ethanol (99.5%) and sterilized in an oven at 105°C for 1 hr to remove the dust and other impurities. A 5 mL volume of the aqueous extract concentration (0.5%, 1.0%, 2.0%, and 4.0%) of cereal was dispensed in a Petri dish in three replicates per concentration. Distilled water was used as control. Seeds of the four cereal species were surface sterilized with 5% ethanol and washed with 100 mL of tap water for 2 min, rinsed under tap water, and then 15 seeds were placed in each Petri dish on two layers of filter paper as seedbeds. e seeds of the cereal species used in the test were from the same sample batch as those used for the extract. To prevent dehydration, the Petri dishes were sealed with parafilm and then incubated at 21°C in a laboratory booth, avoiding direct sunlight.
e potential allelopathic effects of the aqueous extracts were evaluated using the following growth parameters: germination rate, length of the shoot and root of the seedlings. e germination rate was assessed on the third day aer seeding in the Petri dishes by counting the number of germinated seeds (having at least 1 mm of shoot) divided by the total number of seeds used per Petri dish and multiplying by 100. e shoot and root length of the seedlings was measured aer 7 days of growth by using a millimeter scale. e length of the shoots was measured from the apex of the shoot to the bottom of the shoot, whereas the length of the roots was obtained by measuring the meristematic apex of the main root up to the lap region.

Pot Experiment in the CO 2 Chamber
Pot experiment was conducted in a CO 2 chamber at two concentrations of CO 2 in the atmosphere, ambient and elevated (2,000 ppm). is experiment was performed in the laboratory of the Department of Agroecology and Crop Production, University of Agriculture in Krakow during the winter season of 2019/2020. e CO 2 chamber was a 400-L top-closed container, equipped with an LED GX 300 (PhytoLite, Italy) lamp (288/192 W) and a CO 2 source with a CO 2 flow sensor NDIR (Omni, UK), operating during the light (day) phase. e photoperiod was 16 hr/8 hr, and the temperature range was 18-25°C. Water was continuously provided in a closed cycle. Pots of 1 L volume were lined at the bottom with two layers of filter paper and filled with an adequate amount of sand (445 g) of 0.2 mm grain size (BIOVITA, Poland). Dry powder, a mixture of different parts of winter wheat (T. aestivum L.), triticale (×Triticosecale Wittm.), or spelt wheat (T. spelta L.), such as spikes, leaves, and stems, mixed in a 1:1:1 ratio, was added to the upper layer of sand (2.0 or 4.0 g of dry powder) per pot. Next, 15 seeds of cereal species were seeded approximately 2 cm deep into each pot. e control pots contained only sand. e setup was a completely randomized (CR) design with three replicates (pots) per treatment. Plant biomass was determined as the dry weight recorded up to two decimal places by using a balance (Radwag, Poland).

Analyses of Chemical Composition of the Cereals
Fluorescence analysis of phenols of the dried leaves, stem, and spikes from cereal plants was performed according to the method of Lichtenthaler and Babani (2004), by evaluating the fluorescence emission spectra of each sample recorded by using a spectrofluorimeter (Perkin-Elmer LS55B, UK), equipped with an attachment for measuring solids (the so-called Front Surface Accessory -52123130). Grounded plant sample (2.0 g) was placed on one of the two quartz slides (1 cm in diameter) and covered with the second slide. e slides loaded with the sample were placed in the metal ring of the attachment 52123130. Fluorescence intensity measurements were performed in the range of blue-green light (430-650 nm), with 390 nm excitation, and near-and far-red light (650-800 nm), with 430 nm blue excitation. e gap for the excitation ray was 15 nm, and that for the emitted ray was 20 nm. e measurements were performed in replicates of five, and the results were collected and analyzed using the FL WinLab ver. 3.00.

Statistical Analyses
e data were subjected to statistical analysis, which included one-way ANOVA and two-way ANOVA for a completely randomized design. Means were compared using Tukey's post hoc test at p < 0.05. e analyses were performed using the RUDNICKI STAT and STATISTICA ver. 13 soware.

Chemical Composition of the Plant Materials for Extracts
e highest concentration of chlorophyll was observed in the leaf extract and the lowest in the spike and the stem. e barley leaves and spelt wheat leaves had the highest chlorophyll content, and the chlorophyll content of winter wheat spikes and spelt wheat spikes was the least (Figure 1). Chemical analysis revealed that phenolic acids were the most predominant chemical compounds in all the cereals. Samples of barley, especially barley spikes were the richest in phenolic compounds. Interestingly, there were phenolic acids were not detected in the spike samples of both wheat and triticale ( Figure 2).

Germination Test
e germination rate was significantly influenced by the treatments of the water extracts. e highest germination rate was observed in the control and the lowest in presence of 4.0% extract. e germination rate of spelt wheat treated with the highest concentration of spelt wheat extract was 98% less than that of the control ( Figure 3A). e shoot and root lengths of spelt wheat were significantly influenced by the water extracts. e maximum reduction in the shoot length of spelt wheat seedlings (98.7% less than the control) was observed in the presence of the highest concentration of the spelt wheat extract ( Figure 3B). e maximum reduction in the root length of spelt wheat seedlings (98.2% less than the control) was observed in presence of the highest concentration of spelt wheat extract ( Figure 3C).
Shoot and root lengths of winter wheat were significantly influenced by the type of water extract used. Winter wheat seedlings germinated poorly in presence of the stem extracts ( Figure 4A). In contrast, significantly longer shoots were observed in the presence of the leaf extracts ( Figure 4B), and the root length was reduced in the presence of the stem extracts ( Figure 4C). e highest concentration of barley extract reduced the germination rate of barley by 31.4% as compared to the control ( Figure 5A). e shoot and root lengths of barley were significantly influenced by the water extracts. e maximum reduction in the shoot length of barley seedlings was seen in presence of the highest concentration of the barley extracts, 48.5% less as compared to the control ( Figure 5B), while the maximum reduction in the root length of barley seedlings observed in presence of the highest concentration of barley extracts was 51.2% less than that of the control ( Figure 5C). e shoot length of triticale was significantly influenced by the water extract. e maximum reduction in the shoot length of triticale seedlings observed at the highest concentration of triticale extract was 69.7% less than the control ( Figure 6A). Root length was affected by the water extracts and the maximum reduction of the root (B) Mean length of shoot for concentration. (C) Mean length of root for concentration, the differences between treatments are significant. Legend: 0% -control; 0.5% -0.25 g; 1% -0.5 g; 2% -1 g; 4% -2 g.
* Mean values are shown with whiskers that represent standard error (SE). Differences between means are shown with asterisks in comparison to control (p < 0.05), according to Tukey test (α = 0.05). length of triticale seedlings was observed at the highest concentration of triticale extracts, which was 23% lower than that of the control ( Figure 6B).

Effect of Elevated CO 2 and Powdered Winter Wheat on Winter Wheat
On an average, the mean dry weight of the shoot of winter wheat was lower at an elevated level of CO 2 compared to the ambient level. At the same time, the mean dry weight of root was the same for both the CO 2 treatments, whereas the mean length of the shoot and root was greater in the elevated CO 2 treatment (Table 1). A detailed analysis of the effect of dry powders on the growth of winter wheat revealed that, in comparison to the control, at the ambient CO 2 level, the addition of powders * Mean values are shown with whiskers that represent standard error (SE). Differences between means are shown with asterisks in comparison to control (p < 0.05), according to Tukey test (α = 0.05).
significantly stimulated biomass accumulation in both shoots and the roots, without any effect on plant height (Table 1). At elevated CO 2 concentration, the effects of the powders were unclear. At a lower dose of powder, the biomass of both the shoot and root of winter wheat was either higher or same as the control; however, the length of the shoot and root was significantly lower than that of the control. In the case of higher powder dose (4.0 g), the biomass accumulation was significantly lower but increase in plant height was significant (Table 1).

Effect of Elevated CO 2 and Powdered Spelt Wheat on Winter Wheat
On an average, at elevated CO 2 concentration, a significant increase in dry weight of root, and shoot and root lengths of winter wheat was observed compared to the values at ambient CO 2 concentration (  root for concentration, the differences between treatments are significant. Legend: 0% -control; 0.5% -0.25 g; 1% -0.5 g; 2% -1 g; 4% -2 g. * Mean values are shown with whiskers that represent standard error (SE). Differences between means are shown with asterisks in comparison to control (p < 0.05), according to Tukey test (α = 0.05). the addition of spelt wheat powder to the sand caused a significant increase in shoot and root dry weight compared to the control plants. e height of the plants was unaffected by change in CO 2 . In the elevated CO 2 condition, addition of spelt wheat powders caused a significant increase only in the winter wheat root dry weight. All other traits were unaffected (Table 2).

Effect of Elevated CO 2 and Powdered Triticale on Winter Wheat
On average, elevated levels of CO 2 increased the root dry weight in winter wheat, as well as the length of roots and shoots, compared to the ambient conditions. On an average, the control plants of winter wheat were taller and with higher biomass of shoots and roots at elevated levels of CO 2 than the control plants under ambient CO 2 conditions (Table 3). Under ambient CO 2 conditions, the addition of triticale * Mean values are shown with whiskers that represent standard error (SE). Differences between means are shown with asterisks in comparison to control (p < 0.05), according to Tukey test (α = 0.05). Table 1 e effect of elevated CO 2 on growth and dry mass accumulation of winter wheat growing in sand mixed with a 2 or 4 g of a powdered winter wheat in the growth phase of earing. powder to the sand caused a significant increase in winter wheat shoots only in comparison to the untreated control (Table 3). Under elevated CO 2 conditions, in the presence of 2.0 g of triticale powder, a significant increase in winter wheat shoot length was noted, with shoot dry weight remaining same as that of the control plants. Addition of 4.0 g of powder resulted in a significant increase in root dry weight and root length compared to the untreated control plants (Table 3).

Chemical Composition of the Plant Material for Extracts
e identification of chemical components of the plant material in the water extracts is important to understand the effect on the target plants (Ahmad et al., 2020). Chemical analysis revealed that the chlorophyll contents in the cereal (donors of allelopathic compounds) were different. Leaves of the cereals contained the highest concentration of chlorophyll, and spikes and stems the lowest. Barley and spelt wheat had higher chlorophyll content than the other cereals. Physiological factors affect the chemical composition of plants, such as the chlorophyll content of crops as reported by Sidʹko et al. (2017). Chemical analysis also showed that ferulic acid was the most predominant type of phenolic compound in all the four cereal species. Boutigny et al.
(2008) mentioned that in cereals, phenolic compounds represent an important group of secondary metabolites besides carotenoids and tocopherols. However, research on the presence of phenolic compounds in cereals has shown that their composition depends on the cereal species or varieties (Ndolo & Beta, 2014;Pihlava et al., 2015).

Germination Rate
Drought enhances allelopathic effects on the seed germination and seedling growth (Wu et al., 2020). e allelopathic evaluation in our study showed that the four cereal species responded to the application of different concentrations of water extracts, as seen by the considerable effects on the seed germination. e different concentrations of aqueous water extracts significantly suppressed the germination rate of spelt wheat, followed by barley; though the germination rates of triticale and winter wheat were also reduced, the difference from the control was not significant (p > 0.05). erefore, spelt wheat and barley appear to be more sensitive to the allelopathic effects of water extracts. In general, the concentrations of 1.0 and 2.0 g caused the maximum inhibition of the germination rate for all cereals studied. e effect of several concentrations of water extracts on plant growth has been explored, and higher concentrations are known to inhibit sprouting (Ahmed et al., 2008).
e extent of inhibitory effects of water extracts from the tissues of spelt wheat, winter wheat, barley, and triticale on the germination rate depended on the type of extract and the target seed species. e germination rate of winter wheat was found to be the most sensitive among the cereals when subjected to autoallelopathy stress, and differences in the effects may be due to the different crop characteristics. e water extract from the stems of winter wheat showed a greater inhibitory effect on winter wheat seed germination than did the extract from leaves and spikes. Moreover, the water extracts of the other cereals showed an inhibitory effect on their seed germination, excluding the differences among treatments, which were not statistically significant (p > 0.05).

Shoot and Root Length
Allelopathic interactions accounting for the inhibitory effect of shoots on wheat growth in dense clay subsoils have been recorded (Wang et al., 2019). e plant parts showed an inhibitory effect on root and shoot growth. Statistical analyses showed that the maximum reduction observed at the highest concentration had a significant effect on the shoot and root length of spelt wheat, barley, and triticale. However, in winter wheat, we did not record significant differences, even though the shoot and root lengths of the seedlings were inhibited. From the study on cereals in relation to the source of the extracts, the statistical analysis revealed that the different parts (leaf and stem) had a significant effect on the shoot and root length of winter wheat (p < 0.05). e effects on the other cereal species were not statistically different (p > 0.05). Shoot and root length of cereals spelt wheat, barley, and triticale were more sensitive to the highest concentration of aqueous extracts. While for the source of extracts, the results suggest that winter wheat leaf extract might improve shoot length and stem extracts might reduce root length.
In general, the effects of different concentrations and types of extracts are indispensable factors affecting the shoot and root length of winter cereals. e findings demonstrated that such effects might be associated with autoallelopathic influence due to the presence of secondary metabolites (ferulic acids) in the crops used in the study. us, the results support the explanation that the negative effect of autoallelopathy phenomenon due to secondary metabolites released from maternal plants could hinder and inhibit directly or indirectly the growth of their seedlings (Chomel et al., 2016;Fernandez et al., 2016). Furthermore, reports provided by Amb and Ahluwalia (2016) explained that the inhibitory effect of autoallelopathy in some cereals is caused by both living and dead plants, which directly or indirectly affect the growth of the plants.

Plant Biomass
e effect of CO 2 levels and the source of cereal powder used influenced the growth parameters of winter wheat, such as the shoot and root dry weight, as well as shoot and root length. Statistical analysis of the results revealed significant differences among the treatments (p < 0.05). e dry weight of the shoots and roots was significantly altered by elevated CO 2 in presence of the cereal powders, with a higher biomass accumulation in the roots at 2.0 and 4.0 g of powdered spelt wheat and triticale. e longest shoots and roots of winter wheat were observed under elevated CO 2 at 2.0 g of powered triticale. e results of the study showed that the effect of elevated CO 2 combined with winter wheat dry powder on the initial growth of winter wheat resulted in an increase in shoot and root length and decrease in shoot and root dry weight. Elevated CO 2 combined with spelt wheat powder decreased shoot dry weight and increased root dry weight in winter wheat. Elevated CO 2 associated with triticale powder increased the shoot and root length, and root dry weight but decreased the dry weight of the shoot of winter wheat.
ese results are consistent with previous studies (Geiger et al., 1999), which reported that the pattern of growth and development of plants subjected to different CO 2 concentrations are different among species. Indeed, some studies have reported the effects of elevated CO 2 on plant growth and development, and several studies demonstrated that, in general, elevated CO 2 increases root dry mass (Reddy et al., 2010;Seneweera, 2011). Furthermore, Rogers et al. (1995), based on 264 observations, reported that elevated CO 2 causes several responses, where the positive responses on shoots and roots are 59.5% higher, as compared to 37.5% negative response, and 3.0% without any response. Interestingly, our results showed significant statistical differences in the biomass accumulation in the shoots and roots of powdered winter wheat grown under elevated CO 2 . Effects on shoot and root length were observed and the shortest length was recorded in presence of 2.0 g of powdered winter wheat. ese results might be related to the autoallelopathy influence of powdered cereals, as this phenomenon is observed among plants of the same species that release autotoxins from sources such as plant residues, root exudates, leachates of plants, and volatile oils (Yu, 2001).
According to Fatholahi et al. (2020), seed germination and root elongation are the primary effects of autoallelopathic influence. Furthermore, the influence on germination and roots is manifested as dwarf plants and lesions on the tap and lateral roots. In addition, the presented results are supported by studies that have demonstrated that autoallelopathy phenomenon inhibits plant growth in two main ways: direct (affecting photosynthesis and plant defense systems) and indirect (affecting nutrient absorption) (Ahmed et al., 2008;Feng et al., 2019;Mitić et al., 2018;Wardle et al., 2004;Yang et al., 2010).
In the present study, winter wheat responded to the allelopathic effects when elevated CO 2 was combined with dry cereal powder in pot experiments. Our results confirm higher reduction in shoot dry weight than in root dry weight, while beneficial effects (e.g., increased shoot and root length) were also observed.

Conclusions
Ferulic acid was identified as the main phenolic compound in all the four cereals selected for this study. e highest concentration of chlorophyll was observed in the leaves of barley and spelt wheat. e autoallelopathic effects of the cereals are confirmed by the results of the experimental studies. e highest concentrations of water extracts (2.0% and 4.0%) reduced the germination rate of spelt wheat and barley, as well as the shoot and root lengths of spelt wheat, barley, and triticale seedlings. Among the sources of extracts, the stem extracts negatively affected root length, and the leaf extracts positively affected shoot length; both effects were observed in winter wheat. e combination of elevated CO 2 level with spelt wheat powder or triticale powder decreased shoot dry weight but increased the root dry weight and shoot and root length of winter wheat. e elevated level of CO 2 combined with winter wheat powder decreased shoot and root dry weight; however, the shoot and root length in winter wheat was increased.