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Article

Evidence of Allelopathy among Selected Moss Species with Lettuce and Radish

by
Nikolina A. Matić
1,
Marija V. Ćosić
1,
Djordje P. Božović
1,
Silvia Poponessi
2,
Sara D. Pavkov
3,
Michal Goga
4,
Milorad M. Vujičić
1,5,
Aneta D. Sabovljević
1,5 and
Marko S. Sabovljević
1,4,5,*
1
Institute of Botany and Botanical Garden, Faculty of Biology, University of Belgrade, Takovska 43, RS-11000 Belgrade, Serbia
2
Department of Life and Environmental Sciences, Botany Section, University of Cagliari, Viale S. Ignazio, IT-09123 Cagliari, Italy
3
Department of Biology and Ecology, Faculty of Sciences, University of Novi Sad, Trg Dositeja Obradovića 3, RS-21000 Novi Sad, Serbia
4
Department of Botany, Institute of Biology and Ecology, Faculty of Science, Pavol Jozef Šafárik University in Košice, Mánesova 23, SK-040 01 Košice, Slovakia
5
Center of Plant Biotechnology and Conservation (CPBC), Takovska 43, RS-11000 Belgrade, Serbia
*
Author to whom correspondence should be addressed.
Agriculture 2024, 14(6), 812; https://doi.org/10.3390/agriculture14060812
Submission received: 8 May 2024 / Revised: 18 May 2024 / Accepted: 22 May 2024 / Published: 23 May 2024
(This article belongs to the Section Crop Production)
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Versions Notes

Abstract

:
There is limited evidence on bryophyte-tracheophyte allelopathic interactions. Even less is known about such relationships among commercially important plants and mosses. With the aim of screening such interactions, various extract concentrations of nine different mosses were tested on the seed germination and seedlings, i.e., hypocotyl elongation and total chlorophyll content of lettuce and radish. The allelopathic effects are documented to be pairwise (moss-vegetable) and species-specific. Based on the results, the extracts of Leucodon sciuroides and Dicranum polysetum are not harmful to lettuce and radish. Lower extract concentrations of Leucodon sciuroides and Ctenidium molluscum have a positive effect on lettuce development, while those of Thuidium delicatulum, Ctenidium molluscum, and Dicranum polysetum showed to be effective on radish. Further, negative effects were noticed when applying higher extract concentration of Abietinella abietina, Isothecium alopecuroides, Dicranum polysetum, and Racomitrium elongatum to lettuce and Isothecium alopecuroides to radish. The dataset presented in this study offers numerous possibilities for further target pest/vegetable type applications since some of the moss extracts are shown to be positive, negative, or indifferent to the tested features in lettuce and radish.

1. Introduction

Being sessile, plants interact with other organisms in their habitats in a specific way, primarily through secondary metabolites. Interactions through such chemical compounds between two organisms, including plants, algae, fungi, and bacteria, are defined as allelopathy [1] and may be stimulatory or inhibitory [2]. While an organism can stimulate the growth of many other organisms, it may also inhibit developmental and physiological processes in another. Thus, allelopathy is a very complex and often species-to-species-specific trait, especially in plants that are abundant in secondary metabolites. Chemical compounds that exhibit allelopathic effects are called allelopathic compounds, allelopathins, or allelochemicals [3].
A large number of plants possess secondary metabolites, which exhibit biological activity in the organisms in their environment. Bryophytes, as the second-largest group of plants (over 20,000 species worldwide), are particularly interesting due to their richness in secondary metabolites [4]. Compared to vascular plants, bryophytes are smaller in size, with reduced morphological complexity, and a lack of highly differentiated tissues and organs. On the other hand, bryophytes synthesise a wide array of different chemical compounds, some of which are unique and not synthesised by vascular plants [5]. Representatives of all three main lineages of bryophytes (mosses, hornworts, and liverworts) produce secondary metabolites [6], which mainly belong to the group of terpenoid and aromatic compounds [5,7]. Additionally, the chemical profile of liverworts is particularly important due to the presence of oil bodies in which larger amounts of terpenoid and lipid compounds are stored, some of which are exclusive to liverworts [4,5]. On the other hand, many phenolic compounds are present in mosses [8], including benzoic, cinnamic, and phthalic acid derivatives and coumarins [4,5]. Such compounds, the products of complex secondary metabolism in bryophytes, facilitate the interaction of bryophytes with other organisms, i.e., microorganisms, pathogens, vascular plants, and, to a lesser extent, herbivores, evolved over a long coevolutionary history to increase bryophyte fitness and survival [9,10]. Moreover, increased synthesis and the accumulation of certain secondary products can be induced by the effects of some abiotic or biotic stresses or their combinations [8,11,12]. Despite the large amount of information on the biological activity of secondary metabolites, their properties, and their use in pharmacy, medicine, and agriculture (e.g., [6,13,14,15]), there is still little knowledge on how bryophytes interact with the other plants present in their environment. Certain bryophyte species have an impact on the spore germination and growth of young gametophytes of other species of bryophytes through secondary metabolism products [16]. Vicherova et al. [17] documented peat-moss Sphagnum flexuosum Dozy and Molk. positively affected the shoot elongation of moss Hamatocaulis vernicosus (Mitt.) Hedenäs via volatile organic compounds, which increased the production of β-cyclocitral. This compound is commonly found in vascular plants as a signalling molecule for plant-to-plant communication during stress responses. Conversely, interactions between bryophytes and vascular plants through various chemical compounds are less studied, despite their potential ecological and functional significance. Extracts of some bryophyte species can affect and modify the seed germination process of vascular plants [18,19]. For example, Porella platyphylla (L.) Pfeiff. extracts inhibited the germination of seeds from cress (Lepidium sativum L.) and lettuce (Lactuca sativa L.) [19]. In contrast, extracts derived from the moss Abietinella abietina (Hedw.) M. Fleisch. stimulated the seed germination of spruce species Picea crassifolia Kom. when applied in low and moderate concentrations [20]. Besides the influence on seed germination, specific metabolites of bryophytes may also affect the growth and development of vascular plants by promoting or inhibiting the growth of the shoots and roots of vascular plants [21]. Thus, it is important to study the chemistry of bryophytes to test the isolates on various growth and developmental phases and physiological features of vascular plants since those metabolites can be used as potential bioherbicides or plant stimulants [10].
According to Kato-Noguchi [22], a variety of secondary metabolites produced by bryophytes are also secreted into the rhizosphere substrate and act as allelopathic agents. However, very few of those compounds have been studied. Some of the better-known ones are momilactones from Hypnum plumaeforme Wilson [23] and 3-hydroxy-β-ionene from Rhynchostegium pallidifolium (Mitt.) A. Jaeger. [24,25], both exhibiting inhibitory and phytotoxic effects on surrounding vascular plants. Kato-Noguchi [22] further speculates that these compounds may be responsible for monospecific mat formation as well as the elimination of vascular plants from their micro- to meso-habitats.
As shown above, while there is some knowledge of bryophyte allelopathy, the subject of allelopathic interactions and effects remains obscure. Therefore, the main goals of this study were to investigate how different concentrations of selected bryophyte species extracts affect the seed germination and hypocotyl length of lettuce and radish and to explore whether these extracts influence their physiological traits. The aim is to screen for non-harmful crop natural agents that may serve as alternatives in weed suppression and treatments.

2. Materials and Methods

2.1. Plant Material

The allelopathic effect on the lettuce and radish seed germination of a total of nine randomly selected moss species was tested. Namely, eight moss species, Abietinella abietina (Thuidiaceae), Ctenidium molluscum Mitt. (Hynaceae), Dicranum polysetum Sw. (Dicranaceae), Hylocomium splendens Hedw. (Hylocomiaceae), Isothecium alopecuroides Isov. (Lembophyllaceae), Leucodon sciuroides (Hedw.) Schwaegr. (Leucodontaceae), Racomitrium elongatum Ehrh. ex Frisvoll. (Grimmiaceae), and Thuidium tamariscinum Schimp. (Thuidiaceae) were collected in a wide area of Tara Mountain, W. Serbia, and one moss species (Thudium delicatulum (Hedw.) Schimp.) was collected on Zlatar Mountain, W. Serbia, in October 2020. Lettuce (Lactuca sativa L. “May Queen”) and radish seeds (Raphanus sativus L. “Saxa Treib”) were commercially obtained from (Semenarnacoop d.o.o., Petrovaradin, Serbia) with the aim of investigating the allelopathic potential of extracts from the selected mosses.
The vouchers of the bryophyte specimens were confirmed by the M. Sabovljević and S. Poponessi, and they are deposited in the University of Belgrade Bryophyte Collection (BEOU-Bryo) collection: A. abietina (BEOU-Bryo 09230), C. molluscum (BEOU-Bryo 09226), D. polysetum (BEOU-Bryo 09234), H. splendens (BEOU-Bryo 09239), I. alopecuroides (BEOU-Bryo 09225), L. sciuroides (BEOU-Bryo 09228), R. elongatum (BEOU-Bryo 09238), T. delicatulum (BEOU-Bryo 09224) and T. tamariscinum (BEOU-Bryo 09232).

2.2. Experimental Design

2.2.1. Preparation of the Moss Extracts

The collected mosses were cleaned of old parts and impurities and air-dried at room temperature for 10 days. Initially, 5 g of dried plant material for each moss species was measured, ground in liquid nitrogen, and extracted with 50 mL of 96% ethanol. The mixture was then transferred to a glass bottle and incubated for 24 h on an orbital shaker (BioSan PSU-20i, Riga, Latvia) at room temperature. After the incubation, the mixtures of ethanol and plant material were filtrated through filter paper, and the clarified extracts were evaporated to dry using a rotary vacuum evaporation apparatus (IKA RV 3 V, IKa-Werke GmbH & Co. KG, Staufen im Breisgau, Germany) set at 40 °C and 800–1000 mbar. The mass of the distillation balloons used for evaporation was measured before and after the evaporation of the ethanol from the filtrates in order to calculate the mass of the dry residue (extraction yield) for each of the tested moss species. The mass of the dry residue was used for the preparation of the primary ethanolic stock solution (10 mg/mL). The dilution series of different concentrations (10−4, 10−3, 2 × 10−3 mg/mL) was prepared by dissolving the primary stock in distilled water. These diluted extracts were used in the subsequent experiments.

2.2.2. Testing the Allelopathic Potential of the Moss Extracts

The allelopathic effects of the moss extracts were tested on lettuce and radish seed germination. Commercially obtained seeds were already washed and prepared for germination. Ten lettuce and ten radish seeds were placed in each Petri dish (Ø55 mm) and soaked in 3 mL of moss extract at different concentrations (10−4, 10−3, 2 × 10−3 mg/mL) or distilled water (as the control). Seven replications were set for each of the experimental groups, comprising a total of 70 seeds of lettuce and radish per experimental group. All the Petri dishes were placed under controlled long-day low light intensity conditions provided by fluorescent tubes (Tesla, Pančevo, Serbia, 65 W, 50 μmol m−2 s −1) at a temperature of 18 ± 2 °C (16 h: 8 h light: dark) for five days.
The percentage of germinated seeds and the hypocotyl length of the seedlings were measured after three and five days. The length of the hypocotyl was measured using a millimetre ruler and all the changes were photographed and documented using a stereomicroscope (OPTIKA Microscopes, Ponteranica (BG), Italy).

2.2.3. Analysis of the Total Chlorophyll Concentration

The photosynthetic pigments were extracted in 96% (v:v) ethanol from 20 mg of frozen lettuce and radish seedling leaves. After incubation at 70 °C for 10 min and centrifugation (Tehtnica Centric 200R, Zelenzniki, Slovenia), the supernatant was transferred to a microtiter plate, and the absorbance was measured at 648 and 664 nm using the Multiscan Sky Thermo Scientific (Waltham, MA, USA) plate reader (method modified according to Lichtenhthaler [26]). The total chlorophyll concentration in the lettuce and radish seedlings was calculated according to the equation C(a + b) = 5.24 × A664 + 22.24 × A648 and expressed as µg g−1 fresh weight.

2.3. Statistical Analysis

Complete statistical analysis was carried out using R (v. 4.3.1) [27]. The data gathered in this research underwent two different types of statistical analysis. Considering that the seed germination data has two responses, the seeds either germinated or did not, a binomial logistic regression model [28] was used in order to predict the probability of germination using two categorical predictors and their interaction (extract concentration and treatment duration) in nine different moss species. Subsequently, the estimated marginal means (EMMs) were computed using the emmeans() function of the emmeans R package (v. 1.8.9; http://cran.r-project.org/package=emmeans, accessed on 7 January 2024), followed by contrast tests for pairwise comparisons with the Benjamini-Hochberg p-value adjustment, using the contrast() function of the same R package. The second type of analysis was done for the other two parameters (hypocotyl length and total chlorophyll content). Firstly, a preliminary exploration of the data was carried out using Lavene’s test in order to test the homogeneity of variance and the Shapiro-Wilk normality test to check whether the data followed a normal distribution. It was found that not all the experimental groups followed a normal distribution and that the homogeneity of variance was violated across the groups, therefore a nonparametric factorial ANOVA was carried out using the Aligned Rank Transform (ART) procedure [29,30] employing the ARTool R package (v0.11.1) [31]. The factorial models were created for both parameters and nine bryophyte species using the art() function. Subsequently, the significance of these effects was assessed using the anova() function, and ultimately contrast tests were conducted using the art.con() function.

3. Results

3.1. The Effects of the Moss Extracts on the Lettuce Seed Germination

Different concentrations of the extracts of all the investigated moss species as well as the treatment duration did not affect the germination of the lettuce seeds (Table 1). No statistically significant differences were observed in the germination of the lettuce seeds between the third and fifth days within the same treatment or between different treatments (Figure 1A–I).

3.2. The Effects of the Moss Extracts on the Radish Seed Germination

The extract concentration of 10−3 mg/mL of four moss species (A. abietina, C. molluscum, H. splendens, and L. sciuroides), affected the radish seed germination (p < 0.05, Table 2, predictor C [II]). Also, the highest extract concentrations (2 × 10−3 mg/mL) of four moss species (C. molluscum, D. polysetum, H. splendens and I. alopecuroides) inhibited the germination of the radish seeds (p < 0.01, Table 2, predictor C [III]). However, neither the treatment duration nor the interaction between the extract concentrations and the treatment duration impacted the germination of the radish seeds (Table 2).
The extracts derived from four moss species (A. abietina, R. elongatum, T. delicatulum, and T. tamariscinum) did not affect the germination of the radish seeds (Figure 2A,G–I). No statistically significant differences were detected in germination, either between the different treatments or within the same experimental group, between the third and fifth days from the beginning of the experiments.
The treatment with the extract concentration of 10−3 mg/mL from moss L. sciuroides resulted in a decrease in the germination of the radish seeds compared to the control only after three days, i.e., short-term treatment had a slight inhibitory effect on the seed germination (Figure 2F). On the other hand, the highest applied concentration (2 × 10−3 mg/mL) of the two moss species extracts (namely D. polysetum and I. alopecuroides) had a significant negative effect on the germination of the radish seeds on the third and the fifth days (Figure 2B,D).
A significant decrease in the germination of the radish seeds occurred when the seeds were treated with the extract concentrations 10−3 and 2 × 10−3 mg/mL derived from C. molluscum and H. splendens after three and five days compared to the control group (Figure 2B,D).
No statistically significant differences were documented within the same experimental groups between the predicted germination percentage of the lettuce and radish seeds after three and five days (Figure 1 and Figure 2). This contrasts with the findings for the other measured parameters in this study, where statistically significant differences were observed within the same treatments between the third and fifth days (Figure 3, Figure 4, Figure 5 and Figure 6).

3.3. The Effect of the Moss Extracts on the Lettuce Seedling Hypocotyl Length

The extract concentrations of eight out of nine tested moss species (namely A. abietina, C. molluscum, D. polysetum, H. splendens, I. alopecuroides, R. elongatum, T. delicatulum, and T. tamariscinum) significantly affected the lettuce seedling hypocotyl length (p < 0.01, Table 3). The treatment duration of all the tested moss extracts affected the lettuce seedling hypocotyl length. (p < 0.001, Table 3). Additionally, the interaction between the extract concentrations and the treatment duration significantly impacted the lettuce seedling hypocotyl length in the treatments of three moss species (A. abietina, H. splendens, and I. alopecuroides) (p < 0.05, Table 3). Conversely, the extracts derived from L. sciuroides exhibited no effects on the lettuce hypocotyl length, irrespective of treatment duration (Figure 3F). In addition, no statistically significant differences were observed between the treatments of different extract concentrations of L. sciuroides (Figure 3F). Furthermore, different extract concentrations of two tested mosses (R. elongatum and T. delicalutum) did not show any clear pattern in the effects on the hypocotyl length (Figure 3H,G).
Three tested species of mosses (namely, C. molluscum, H. splendens and T. tamariscinum) exhibited a mild inhibitory effect on the lettuce hypocotyl length (Figure 3B,D,I). The highest concentration extracts (2 × 10−3 mg/mL) derived from C. molluscum, H. splendens and T. tamariscinum resulted in a reduction of the lettuce seedling hypocotyl length both after three and five days, i.e., statistically significant differences were noted between the treatments with the highest extract concentrations of these mosses and the control group (Figure 3B,D,I). Also, a decrease in the lettuce seedling hypocotyl length was detected when the lettuce seedlings were treated with extracts of C. molluscum and H. splendens at a concentration of 10−3 mg/mL after five days. The long-term treatment with extracts of this concentration from two moss species (C. molluscum and H. splendens) negatively affected the lettuce seedling hypocotyl length (Figure 3B,D).
However, the extracts of three tested moss species (A. abietina, D. polysetum, and I. alopecuroides) exhibited a strong allelopathic effect on the length of the lettuce hypocotyl (Figure 3A,C,E). Each of the used concentrations of the A. abietina extract demonstrated an inhibitory effect on the lettuce hypocotyl length compared to the control group (Figure 3A). Additionally, statistically significant differences were detected between the treatments with lower concentrations of the extracts (10−4 and 10−3 mg/mL) as well as the highest concentration of the extract (2 × 10−3 mg/mL) (Figure 3A). Similar effects on the length of the lettuce hypocotyl were detected when the seeds were treated with the I. alopecuroides extracts (Figure 3E). All concentrations of the extract derived from D. polysetum resulted in a significant decrease in the lettuce hypocotyl length compared to the control group. Statistically significant differences were detected between treatments of lower extract concentrations (10−4 and 10−3 mg/mL) obtained from D. polysetum after five days (Figure 3C).
Among all the tested extracts on the lettuce hypocotyl length, the strongest allelopathic effects were documented for the A. abietina, D. polysetum and I. alopecuroides extracts, while the extracts of L. sciuroides, R. elongatum, and T. delicatulum exhibited no clear allelopathic patterns.

3.4. The Effect of the Moss Extracts on the Total Chlorophyll Concentration of the Lettuce Seedlings

Regarding the effect of extract concentration on the total chlorophyll concentration of the lettuce seedlings, all of the tested moss extracts affected the total chlorophyll concentration except that of A. abietina (p < 0.05, Table 3). However, the treatment duration of all the tested moss extracts did affect the total chlorophyll concentration of the lettuce seedlings (p < 0.001, Table 3). When considered multifactorially, the interaction, extract concentrations, and time affected the total chlorophyll concentration of the lettuce seedlings in treatments of six moss species (C. molluscum, D. polysetum, I. alopecuroides, L. sciuroides, R. elongatum and T. delicatulum) (p < 0.01, Table 3).
The total chlorophyll concentration of the lettuce seedlings differed significantly after three and five days within the same treatment. In the first three days, the young seedlings started the process of biosynthesis of photosynthetic pigments, so variations in the total chlorophyll concentration were observed between the third and fifth days. Extracts from two moss species (namely, A. abietina and T. tamriscinum) did not affect the total chlorophyll concentration of the lettuce seedlings either after three or five days i.e., this moss species did not have any significant effects on chlorophyll biosynthesis (Figure 4A,I). On the other hand, the extract concentrations of three moss species (C. molluscum, H. splendens, and T. delicatulum) did not show a clear effect on the total chlorophyll concentration of the lettuce seedlings (Figure 4B,D,H). However, extract concentrations of 10−3 and 2 × 10−3 mg/mL derived from T. delicatulum significantly decreased the total chlorophyll concentration of the lettuce seedlings, but only after three days (Figure 4H).
Nevertheless, the extracts obtained from D. polysetum, I. alopecuroides, L. sciuroides, as well as R. elongatum affected the total chlorophyll concentration of the lettuce seedlings (Figure 4C,E–G). The highest used concentration (2 × 10−3 mg/mL) of the D. polysetum moss extract had a significant effect on the reduction of photosynthetic pigment synthesis at both three and five days—long exposure (Figure 4C). Lower chlorophyll concentration was noticed in the lettuce seedlings treated with extract concentrations of 10−3 and 2 × 10−3 mg/mL derived from I. alopecuroides after three days. In contrast, on the fifth day, none of the used extract concentrations influenced the expected developmental increase in the total chlorophyll concentration of the lettuce seedlings. Statistically significant differences were observed in the total chlorophyll concentration of the lettuce seedlings for all the experimental groups after three and five days compared to the control (Figure 4E). Two moss species (L. sciuroides and R. elongatum) had a similar effect on the total chlorophyll concentration of the lettuce seedlings. No statistically significant differences regarding the total chlorophyll concentration were observed between the treatments and the control group or between different treatments after three days. However, those moss species affected a decrease in the total chlorophyll concentration of the lettuce seedlings when treated with somewhat higher extract concentrations of 10−3 and 2 × 10−3 mg/mL after five days (Figure 4F,G).

3.5. The Effect of the Moss Extracts on the Radish Seedling Hypocotyl Length

The extract concentrations from eight out of nine moss species (A. abietina, C. molluscum, D. polysetum, H. splendens, I. alopecuroides, L. sciuroides, R. elongatum, and T. tamariscinum) also had a significant impact on the hypocotyl length of the radish seedlings (p < 0.05, Table 4). The treatment duration with the extracts of all the tested moss species affected the radish seedlings’ hypocotyl length (p < 0.001, Table 4).
Again, only one species out of nine exhibited no effect. However, it was not the same one as in the case of the lettuce. The extract obtained from T. delicatulum did not affect the hypocotyl length of the radish seedlings, either after three or five days (Figure 5H), i.e., this extract did not affect the hypocotyl elongation. Moreover, no statistically significant differences regarding the radish hypocotyl length were observed between treatments and the control group or between different treatments (Figure 5H). However, the elongation of the radish seedlings’ hypocotyl length did not correlate with increased extract concentrations in two of the used moss species (L. sciuroides and R. elongatum) (Figure 5F,G).
Three moss species (D. polysetum, I. alopecuroides, and T. tamariscinum) exhibited a mild inhibitory effect on the radish hypocotyl length when the seeds were treated with the highest extract concentration (2 × 10−3 mg/mL) (Figure 5C,E,I). Lower extract concentrations (10−4 and 10−3 mg/mL) did not affect the length of the radish hypocotyl at all when compared with the control group (Figure 5C,E,I). The highest extract concentration of D. polysetum (2 × 10−3 mg/mL) showed an inhibitory effect on the radish hypocotyl length only after three days (Figure 5C). The same effect on the elongation of the radish hypocotyl was observed in the treatment with the highest extract concentration of I. alopuroides, but only after five days (Figure 5E). However, the highest extract concentration of 2 × 10−3 mg/mL obtained from T. tamariscinum resulted in a decrease in the radish hypocotyl length both after three and five days, compared to the control group (Figure 5I).
On the other hand, the extracts obtained from A. abietina, C. molluscum, and H. splendens significantly affected the decrement of the radish hypocotyl length, especially after three days (Figure 5A,B,D). Increased concentrations of the A. abietina moss extracts gradually inhibited the formation of the radish hypocotyl, which was statistically significant (Figure 5A). Additionally, the extracts derived from C. molluscum and H. splendens exhibited a similar pattern regarding the inhibition of the radish hypocotyl growth (Figure 5B,D).
In general, a strong allelopathic effect on the radish hypocotyl formation was documented for the extracts derived from A. abietina, C. molluscum, and H. splendens while the extract of T. delicatulum had no effect.

3.6. The Effect of the Moss Extracts on the Total Chlorophyll Concentration of the Radish Seedlings

The extract concentrations of all the tested moss species significantly affected the total chlorophyll concentration of the radish seedlings (p < 0.01, Table 4). Also, the treatment duration with the extracts of all the tested moss species affected the total chlorophyll concentration of the radish seedlings (p < 0.001, Table 4). The interaction between the treatment duration and the extract concentration of all the tested moss species (with the exception of A. abietina) showed a significant effect on the total chlorophyll concentration of the radish seedlings (p < 0.001, Table 4).
Our results indicated that radish seedlings start the process of chlorophyll biosynthesis slightly later than lettuce under the laboratory conditions provided. Therefore, an extremely low total chlorophyll concentration was observed after the first three days. Thus, statistically significant differences in the total chlorophyll concentration were detected between the third and fifth days within the same experimental groups, which may be regarded as an artefact (Figure 6).
Abietinella abietina did not affect the synthesis of the photosynthetic pigments of the radish seedlings; moreover, no statistically significant differences were noticed between the control and all the experimental groups (Figure 6A). Increasing extract concentrations from two moss species (R. elongatum and T. delicatulum) did not show a clear trend regarding the total chlorophyll content of the radish seedlings after five days (Figure 6G,H). Nevertheless, increasing extract concentrations from R. elongatum exhibited a slight stimulatory effect on the chlorophyll biosynthesis of the radish seedlings after three days (Figure 6G). In contrast, with increasing concentrations of the T. delicatulum extracts, a gradual decrease in chlorophyll concentration was detected in the radish seedlings after three days (Figure 6H).
The extract concentration of 10−4 mg/mL derived from H. splendens influenced a slight increase in the chlorophyll concentration, while the highest extract concentration (2 × 10−3 mg/mL) of H. splendens resulted in a decrease in the total chlorophyll concentration after five days (Figure 6D).
On the other hand, gradually increased concentrations of the extract of five moss species (C. molluscum, I. alopecuroides, L. sciuroides, and T. tamariscinum) caused a gradual decrease in the total chlorophyll concentration of the radish seedlings after five days (Figure 6B,E,F,I). No statistically significant differences were noticed between the experimental groups of two extract concentrations 10−3 and 2 × 10−3 mg/mL, indicating that these extract concentrations had a similar effect on the chlorophyll synthesis of the radish seedlings (Figure 6B,E,F,I).
The total chlorophyll concentration increased when the radish seedlings were treated with lower extract concentrations (10−4 and 10−3 mg/mL) of the moss D. polysetum after five days (Figure 6C). Those two extract concentrations of D. polysetum had a significant stimulatory impact on the chlorophyll synthesis of the radish seedlings. However, the highest extract concentration (2 × 10−3 mg/mL) of D. polysetum did not affect the total chlorophyll concentration of the radish seedlings (Figure 6C).
A summary of the effects of various moss extracts for better visibility is presented as a heat map in Figure 7.

4. Discussion

As allelopathy is a very complex phenomenon, it is not surprising to encounter contrasting results regarding the extract’s effects on seed germination and seedling development. The results obtained in this study clearly show the presence of chemical communication among moss species with lettuce and radish. These interactions can be regarded as stimulatory, inhibitory, or indifferent, depending on the tested features in lettuce and radish. In similar studies conducted to date, both inhibitory and promoting effects have been documented [10,32]. These different allelopathic interactions are mostly driven by the chemical composition of the moss and the concentration of the extracts [32]. Moreover, seed germination is likely to be dosage-dependent, i.e., higher extract concentrations usually exhibit stronger effects than lower concentrations. Such results were also documented for the undiluted aqueous extracts of the liverwort Bazzania trilobata (L.) Gray which inhibited the seed germination of cress [10]. Moreover, the inhibition of seed germination is likely to occur when ethanolic, methanolic, and acetone extracts are applied rather than aqueous because of the chemical nature of allelochemicals [10,33,34].

4.1. The Effect of the Moss Extracts on Tested Vegetable Seed Germination

In our study, none of the tested bryophyte extracts affected the germination of the lettuce seeds (Figure 1). On the other hand, only four extracts, those from the mosses C. molluscum, D. polysetum, H. splendens, and I. alopecuroides inhibited the germination of the radish seeds (Figure 2). However, the effects on the seedling growth phase, i.e., the hypocotyl length were species-specific and somewhat dependent on the concentrations (Figure 3 and Figure 5). Although some extracts did not affect seed germination, their effects were noticeable in the later developmental stages. Those results are not surprising since allelopathic effects can act primarily at the seedling phase rather than on germination [32,35].

4.2. The Effect of the Moss Extracts on Tested Vegetable Hypocotyl Length

In this study, we found that the interaction is species-specific and depends on the tested counterparts (Figure 7). Despite being unable to infer very much from the hypocotyl length and total chlorophyll content after 3 day-long treatments with various concentrations of different moss extracts, it is noticeable that hypocotyl elongation was slightly suppressed in both lettuce and radish. Although there are certain dissimilarities between all the tested moss extracts concerning the lettuce and radish hypocotyl growth, the extracts of L. sciuroides and R. elongatum did not affect the elongation of the hypocotyl length either in lettuce or in radish. On the other hand, the extracts of A. abietina inhibited the elongation of the hypocotyl in both lettuce and radish, indicating a strong allelopathic effect. The other tested extracts manifested variable effects depending on concentration, the duration of the experiment, and the species tested. Therefore, the allelopathic effects can be described as species-to-species specific for most of the tested taxa.
Moreover, the results also show that the extracts of L. sciuroides do not affect the hypocotyl length in either lettuce or radish. These patterns are also confirmed after 5 days for the same moss species extracts, which makes this species a good candidate for testing among lettuce pests as a nature-friendly treatment, i.e., for potential biohericides. The extracts of the moss T. tamariscinum also showed promising potential for use in lettuce treatments. Previous studies have indicated that some isolated compounds from bryophytes such as those from the liverwort Porella obtusata (Tayl.) Trevis [36] and moss Dicranum scoparium Hedw Ref. [37] display repellent potential on slugs during the consumption of lettuce leaves. Regarding the radish, the pattern shown by the T. delicatulum extracts suggests that this species could be a potential source for testing as a radish pest-effective agent.

4.3. The Total Chlorophyll Content Is Rather Unstable under Moss Extract Treatments

On the other hand, the total chlorophyll content measured after three days of treatments may not serve as an adequate reference point since there were no significant differences between the development of the plantlets and the control group. Also, the short period of underdeveloped primarily leaves during which energy is derived from the cotyledons should be considered when interpreting these effects rather than after the fifth day of treatments. Indeed, non-harmful or even slightly stimulative effects of the extracts applied to lettuce as indicated by the total chlorophyll content compared to the control plant group under the same laboratory conditions were recorded in the extracts of most of the moss species applied. However, this was not the case in the radish tests, where the total chlorophyll content decreased compared to the control plant group, except in low extract concentrations of D. polysetum and H. splendens. The extract of D. polysetum at a concentration of 10−3 mg/mL even showed a stimulatory effect on the total chlorophyll content in radish.

5. Conclusions

Mosses communicate with vascular plants chemically, and the type of interaction and its effect are species-to-species-specific. Apart from species, the targeted effect depends on the type of extract, concentration, and duration of extract applications. Among the tested species, L. sciuroides exhibited the least harmful effect on lettuce overall, while for radish, that was D. polysetum. The most negative effect on both vegetable species was documented for I. alopecuroides, although higher concentrations of its extracts were stimulative for lettuce as indicated by the chlorophyll content. A stimulatory effect was also documented for lower concentrations of D. polysetum extracts in radish. Although the tested bryophyte extracts showed bryophyte-crop interaction, it is clear that more pairwise tests are needed along with additional approaches to clarify the mechanisms of these interactions.

Author Contributions

Conceptualization, M.S.S. and A.D.S.; methodology, M.S.S. and A.D.S.; software, D.P.B.; validation, M.G., M.M.V., A.D.S. and M.S.S.; formal analysis, N.A.M., M.V.Ć., D.P.B., S.D.P. and S.P.; investigation, N.A.M., M.V.Ć. and D.P.B.; resources, A.D.S. and M.S.S.; data curation, N.A.M. and D.P.B.; writing—original draft preparation, N.A.M., M.V.Ć., D.P.B., S.D.P. and S.P.; writing—review and editing, M.S.S. and A.D.S.; visualization, D.P.B.; supervision, M.S.S. and A.D.S.; project administration, A.D.S.; funding acquisition, A.D.S. and M.S.S. All authors have read and agreed to the published version of the manuscript.

Funding

The project is supported by the Serbian Ministry of Science, Technological Development and Innovations, contract no: 451-03-65/2024-03/200178 and 451-03-66/2024-03/200178.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors on request.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. The predicted probability of the germination of the lettuce seeds. C—control groups; I–III—applications of 10−4, 10−3, 2 × 10−3 mg/mL concentrations of the selected moss species extract (respectively). The bars represent the predicted probability of germination (0–1) with the error bars denoting the 95% confidence intervals (CI). Statistically significant differences (p < 0.05) among the experimental groups are indicated by the letters above the bars.
Figure 1. The predicted probability of the germination of the lettuce seeds. C—control groups; I–III—applications of 10−4, 10−3, 2 × 10−3 mg/mL concentrations of the selected moss species extract (respectively). The bars represent the predicted probability of germination (0–1) with the error bars denoting the 95% confidence intervals (CI). Statistically significant differences (p < 0.05) among the experimental groups are indicated by the letters above the bars.
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Figure 2. The predicted probability of the germination of the radish seeds. C—control groups; I–III—applications of 10−4, 10−3, 2 × 10−3 mg/mL concentrations of the selected moss species extract (respectively). The bars represent the predicted probability of germination (0–1) with the error bars denoting the 95% confidence intervals (CI). Statistically significant differences (p < 0.05) among the experimental groups are identified by the letters above the bars.
Figure 2. The predicted probability of the germination of the radish seeds. C—control groups; I–III—applications of 10−4, 10−3, 2 × 10−3 mg/mL concentrations of the selected moss species extract (respectively). The bars represent the predicted probability of germination (0–1) with the error bars denoting the 95% confidence intervals (CI). Statistically significant differences (p < 0.05) among the experimental groups are identified by the letters above the bars.
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Figure 3. The effects of the ethanolic moss extracts on the lettuce seedling hypocotyl length. C—control groups; I–III—applications of 10−4, 10−3, 2 × 10−3 mg/mL concentrations of the selected moss species extract (respectively). Black horizontal lines represent the median, while the box ranges from the first (Q1) to the third (Q3) quartile, with the whiskers extending to the 1.5 × IQR (interquartile range) from the edge of the boxes. The black dots represent observations outside the 1.5 × IQR (interquartile range). Statistically significant differences (p < 0.05) among the experimental groups are identified by the letters above the boxplots.
Figure 3. The effects of the ethanolic moss extracts on the lettuce seedling hypocotyl length. C—control groups; I–III—applications of 10−4, 10−3, 2 × 10−3 mg/mL concentrations of the selected moss species extract (respectively). Black horizontal lines represent the median, while the box ranges from the first (Q1) to the third (Q3) quartile, with the whiskers extending to the 1.5 × IQR (interquartile range) from the edge of the boxes. The black dots represent observations outside the 1.5 × IQR (interquartile range). Statistically significant differences (p < 0.05) among the experimental groups are identified by the letters above the boxplots.
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Figure 4. The total chlorophyll concentration of the lettuce seedlings. C—control groups; I–III—applications of 10−4, 10−3, 2 × 10−3 mg/mL concentrations of the selected moss species extract (respectively). Black horizontal lines represent the median, while the box ranges from the first (Q1) to the third (Q3) quartile, with the whiskers extending to the 1.5 × IQR (interquartile range) from the edge of the boxes. The black dots represent observations outside the 1.5 × IQR (interquartile range). Statistically significant differences (p < 0.05) among the experimental groups are indicated by the letters above the boxplots.
Figure 4. The total chlorophyll concentration of the lettuce seedlings. C—control groups; I–III—applications of 10−4, 10−3, 2 × 10−3 mg/mL concentrations of the selected moss species extract (respectively). Black horizontal lines represent the median, while the box ranges from the first (Q1) to the third (Q3) quartile, with the whiskers extending to the 1.5 × IQR (interquartile range) from the edge of the boxes. The black dots represent observations outside the 1.5 × IQR (interquartile range). Statistically significant differences (p < 0.05) among the experimental groups are indicated by the letters above the boxplots.
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Figure 5. The effect of the ethanolic moss extracts on the radish seedling hypocotyl length. C—control groups; I–III—applications of 10−4, 10−3, 2 × 10−3 mg/mL concentrations of the selected moss species extract (respectively). Black horizontal lines represent the median, while the box ranges from the first (Q1) to the third (Q3) quartile, with the whiskers extending to the 1.5 × IQR (interquartile range) from the edge of the boxes. The black dots represent observations outside the 1.5 × IQR (interquartile range). Statistically significant differences (p < 0.05) among the experimental groups are indicated by the letters above the boxplots.
Figure 5. The effect of the ethanolic moss extracts on the radish seedling hypocotyl length. C—control groups; I–III—applications of 10−4, 10−3, 2 × 10−3 mg/mL concentrations of the selected moss species extract (respectively). Black horizontal lines represent the median, while the box ranges from the first (Q1) to the third (Q3) quartile, with the whiskers extending to the 1.5 × IQR (interquartile range) from the edge of the boxes. The black dots represent observations outside the 1.5 × IQR (interquartile range). Statistically significant differences (p < 0.05) among the experimental groups are indicated by the letters above the boxplots.
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Figure 6. The total chlorophyll concentration of the radish seedlings. C—control groups; I–III—applications of 10−4, 10−3, 2 × 10−3 mg/mL concentrations of the selected moss species extract (respectively). Black horizontal lines represent the median, while the box ranges from the first (Q1) to the third (Q3) quartile, with the whiskers extending to the 1.5 × IQR (interquartile range) from the edge of the boxes. The black dots represent observations outside the 1.5 × IQR (interquartile range). Statistically significant differences (p < 0.05) among the experimental groups are indicated by the letters above the boxplots.
Figure 6. The total chlorophyll concentration of the radish seedlings. C—control groups; I–III—applications of 10−4, 10−3, 2 × 10−3 mg/mL concentrations of the selected moss species extract (respectively). Black horizontal lines represent the median, while the box ranges from the first (Q1) to the third (Q3) quartile, with the whiskers extending to the 1.5 × IQR (interquartile range) from the edge of the boxes. The black dots represent observations outside the 1.5 × IQR (interquartile range). Statistically significant differences (p < 0.05) among the experimental groups are indicated by the letters above the boxplots.
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Figure 7. The effects of various concentrations of the selected moss ethanolic extracts on the total chlorophyll content (mg/g FW) and hypocotyl length (mm) in radish and lettuce. C—control groups; I–III—applications of 10−4, 10−3, 2 × 10−3 mg/mL concentrations of the selected moss species extract (respectively).
Figure 7. The effects of various concentrations of the selected moss ethanolic extracts on the total chlorophyll content (mg/g FW) and hypocotyl length (mm) in radish and lettuce. C—control groups; I–III—applications of 10−4, 10−3, 2 × 10−3 mg/mL concentrations of the selected moss species extract (respectively).
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Table 1. A summary of the results of nine logistic regression models that predict the probability of germination of the lettuce seeds treated with the ethanolic extracts from nine bryophyte species. The predictors include the concentration of the ethanolic extracts (C), treatment duration (T), and their interaction (C × T).
Table 1. A summary of the results of nine logistic regression models that predict the probability of germination of the lettuce seeds treated with the ethanolic extracts from nine bryophyte species. The predictors include the concentration of the ethanolic extracts (C), treatment duration (T), and their interaction (C × T).
SpeciesParameter(Intercept)C [I]C [II] C [III]T [5]C [I] × T [5]C [II] × T [5]C [III] × T [5]ObservationR2 Tjur
Abietinella abietinaOdds ratios6.00 ***1.781.001.001.001.221.131.135600.007
p<0.0010.2931.001.001.000.8030.8600.860
Ctenidium molluscumOdds ratios6.78 ***1.570.890.591.330.750.970.835600.015
p<0.0010.4150.8050.2570.5960.7250.9700.780
Dicranum polysetumOdds ratios10.67 ***0.560.380.561.550.840.710.655600.017
p<0.0010.2930.0600.2930.5140.8300.6650.597
Hylocomnium splendensOdds ratios6.78 ***0.590.790.711.330.750.750.755600.007
p<0.0010.2570.6300.4790.5960.6770.6890.685
Isothecium alopecuroidesOdds ratios4.38 ***1.551.773.76 *1.001.001.381.005600.021
p<0.0010.3550.2410.0271.0001.0000.6551.000
Leucodon sciuroidesOdds ratios10.67 ***1.001.000.731.551.000.650.895600.004
p<0.0011.0001.0000.5740.5141.0000.6280.894
Racomitrium elongatumOdds ratios9.00 ***0.671.441.001.190.840.841.005600.008
p<0.0010.4400.5481.0000.7710.8230.8471.000
Thuidium delicatulumOdds ratios7.75 ***1.000.871.381.680.600.790.925600.006
p<0.0011.0000.7960.5740.3860.5180.7710.928
Thuidium tamariscinumOdds ratios4.83 ***1.001.401.401.240.890.920.815600.003
p<0.0011.0000.4790.4790.6430.8640.9060.753
* p < 0.05, *** p < 0.001.
Table 2. A summary of the results of nine logistic regression models that predict the probability of the germination of the radish seeds treated with the ethanolic extracts from nine bryophyte species. The predictors include the concentration of the ethanolic extracts (C), treatment duration (T), and their interaction (C × T).
Table 2. A summary of the results of nine logistic regression models that predict the probability of the germination of the radish seeds treated with the ethanolic extracts from nine bryophyte species. The predictors include the concentration of the ethanolic extracts (C), treatment duration (T), and their interaction (C × T).
SpeciesParameter(Intercept)C [I]C [II] C [III]T [5]C [I] × T [5]C [II] × T [5]C [III] × T [5]ObservationR2 Tjur
Abietinella abietinaOdds ratios1.260.940.39 **0.601.430.881.350.835600.035
p0.3400.8650.0070.1290.3010.7980.5430.703
Ctenidium molluscumOdds ratios1.191.430.44 *0.36 **1.431.041.421.165600.076
p0.4740.3040.0180.0040.3040.9430.4700.767
Dicranum polysetumOdds ratios1.260.890.530.30 ***1.121.191.121.415600.048
p0.3400.7340.0640.0010.7330.7180.8090.491
Hylocomnium splendensOdds ratios1.80 *0.740.22 ***0.25 ***1.300.921.010.775600.107
p0.0180.387<0.001<0.0010.4720.8700.9900.613
Isothecium alopecuroidesOdds ratios1.191.120.600.34 **1.520.841.321.105600.057
p0.4740.7340.1290.0020.2290.7170.5700.849
Leucodon sciuroidesOdds ratios2.89 ***0.710.35 **0.661.520.701.050.995600.036
p<0.0010.3540.0030.2700.3110.5210.9280.990
Racomitrium elongatumOdds ratios2.50 ***0.680.820.821.251.031.221.055600.009
p0.0010.2810.5830.5830.5660.9490.7080.922
Thuidium delicatulumOdds ratios1.80 *1.391.211.731.211.111.010.895600.009
p0.0180.3660.5920.1420.5920.8390.9770.833
Thuidium tamariscinumOdds ratios2.04 **0.621.410.731.141.741.111.365600.022
p0.0050.1660.3540.3800.7160.2780.8450.545
* p < 0.05, ** p < 0.01, *** p < 0.001.
Table 3. The results of the factorial analysis which examined the influence of the ethanolic extracts from nine tested bryophyte species on the hypocotyl length and total chlorophyll content of the lettuce seedlings by assessing the main effects of concentration (C), treatment duration (T), and their interaction (C × T).
Table 3. The results of the factorial analysis which examined the influence of the ethanolic extracts from nine tested bryophyte species on the hypocotyl length and total chlorophyll content of the lettuce seedlings by assessing the main effects of concentration (C), treatment duration (T), and their interaction (C × T).
SpeciesParameterCTC × T
Abietinella abietinaHypocotyl length51.995 ***131.77 ***3.6807 *
Total Chl2.5974194.98 ***1.6675
Ctenidium molluscumHypocotyl length10.428 ***91.365 ***0.3476
Total Chl5.9260 **197.28 ***4.6742 **
Dicranum polysetumHypocotyl length31.86 ***109.3 ***0.152
Total Chl9.8232 ***193.99 ***10.399 ***
Hylocomium splendensHypocotyl length34.266 ***193.94 ***3.5096 *
Total Chl3.594 *193.1 ***1.703
Isothecium alopecuroidesHypocotyl length66.155 ***174.001 ***4.3349 **
Total Chl9.7412 ***193.69 ***21.769 ***
Leucodon sciuroidesHypocotyl length0.5698225.72 ***0.9065
Total Chl11.596 ***193.25 ***9.8766 ***
Racomitrium elongatumHypocotyl length4.2257 **251.05 ***1.1268
Total Chl29.512 ***201.79 ***30.297 ***
Thudium delicatulumHypocotyl length5.7759 ***203.78 ***1.1136
Total Chl8.875 ***193.8 ***8.136 ***
Thudium tamariscinumHypocotyl length15.065 ***119.10 ***0.3762
Total Chl0.41353192.87 ***2.3583
The values represent the F values with the asterisks denoting the corresponding level of statistical significance * p < 0.05, ** p < 0.01, *** p < 0.001.
Table 4. The results of the factorial analysis examining the influence of the ethanolic extracts from nine tested bryophyte species on the hypocotyl length and total chlorophyll content of the radish seedlings by assessing the main effects of concentration (C), treatment duration (T), and their interaction (C × T).
Table 4. The results of the factorial analysis examining the influence of the ethanolic extracts from nine tested bryophyte species on the hypocotyl length and total chlorophyll content of the radish seedlings by assessing the main effects of concentration (C), treatment duration (T), and their interaction (C × T).
SpeciesParameterCTC × T
Abietinella abietinaHypocotyl length11.729 ***27.274 ***0.547
Total Chl4.1820 ***194.21 ***1.3239
Ctenidium molluscumHypocotyl length13.789 ***58.425 ***2.4708
Total Chl23.895 ***198.63 ***13.959 ***
Dicranum polysetumHypocotyl length3.561 *37.84 ***0.232
Total Chl43.586 ***200.45 ***65.753 ***
Hylocomnium splendensHypocotyl length9.8567 ***60.798 ***0.3352
Total Chl18.55 ***200.0 ***16.7 ***
Isothecium alopecuroidesHypocotyl length6.9103 ***29.532 ***0.8748
Total Chl37.950 ***193.96 ***51.563 ***
Leucodon sciuroidesHypocotyl length4.3754 **90.711 ***0.4317
Total Chl17.531 ***197.61 ***9.1641 ***
Racomitrium elongatumHypocotyl length3.7890 *60.649 ***0.4399
Total Chl19.070 ***192.75 ***15.849 ***
Thuidium delicatulumHypocotyl length0.559891.984 ***0.3833
Total Chl9.615 ***193.6 ***7.518 ***
Thuidium tamariscinumHypocotyl length7.2198 ***87.760 ***0.2007
Total Chl30.479 ***198.20 ***25.062 ***
The values represent the F values with the asterisks indicating the corresponding level of statistical significance * p < 0.05, ** p < 0.01, *** p < 0.001.
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Matić, N.A.; Ćosić, M.V.; Božović, D.P.; Poponessi, S.; Pavkov, S.D.; Goga, M.; Vujičić, M.M.; Sabovljević, A.D.; Sabovljević, M.S. Evidence of Allelopathy among Selected Moss Species with Lettuce and Radish. Agriculture 2024, 14, 812. https://doi.org/10.3390/agriculture14060812

AMA Style

Matić NA, Ćosić MV, Božović DP, Poponessi S, Pavkov SD, Goga M, Vujičić MM, Sabovljević AD, Sabovljević MS. Evidence of Allelopathy among Selected Moss Species with Lettuce and Radish. Agriculture. 2024; 14(6):812. https://doi.org/10.3390/agriculture14060812

Chicago/Turabian Style

Matić, Nikolina A., Marija V. Ćosić, Djordje P. Božović, Silvia Poponessi, Sara D. Pavkov, Michal Goga, Milorad M. Vujičić, Aneta D. Sabovljević, and Marko S. Sabovljević. 2024. "Evidence of Allelopathy among Selected Moss Species with Lettuce and Radish" Agriculture 14, no. 6: 812. https://doi.org/10.3390/agriculture14060812

APA Style

Matić, N. A., Ćosić, M. V., Božović, D. P., Poponessi, S., Pavkov, S. D., Goga, M., Vujičić, M. M., Sabovljević, A. D., & Sabovljević, M. S. (2024). Evidence of Allelopathy among Selected Moss Species with Lettuce and Radish. Agriculture, 14(6), 812. https://doi.org/10.3390/agriculture14060812

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