ACTA BOT. CROAT. 80 (2), 2021 131 Acta Bot. Croat. 80 (2), 131–139, 2021 CODEN: ABCRA 25 DOI: 10.37427/botcro-2021-013 ISSN 0365-0588 eISSN 1847-8476 Allelopathic potential of Ficus retusa L. leaf litter on understory vegetation in urban gardens Mahmoud Omar Hassan, Howida Yacoup Mohamed*, Ayman Hassan Aboellil Beni-Suef University, Faculty of Science, Department of Botany and Microbiology, 62511 Beni-Suef, Egypt Abstract – Pruning Ficus trees in urban green spaces may lead to the accumulation and spread of their leaf litter on the understory vegetation. This study was conducted to evaluate the allelopathic effect of Ficus retusa L. leaf litter on the understory species in urban gardens. A field study showed that the plant cover and species richness of litter-affected plots were lower than those of litter-free areas. The litter-affected soils had substantially lower pH and higher electrical conductivity. In a greenhouse experiment, litter-affected soil significantly inhibited the emergence and growth of un- derstory species selected for the purpose of this study: Melilotus indicus (L.) All., Trifolium resupinatum L. and Amaranthus viridis L. Osmotic potentials equivalent to those of the litter-affected soils did not affect emergence or growth of these species. A spectrophotometric analysis indicated that the litter-affected soils contained larger amounts of phenolics and flavonoids. An HPLC analysis revealed that the litter-affected soils contained higher concentrations of free phenolic and flavonoid allelochemicals. These results demonstrate that F. retusa leaf litter may reduce plant cover and species rich- ness. The significant inhibition in both field and greenhouse experiments could be attributed to phenolic and flavonoid allelochemicals released from the tree litter, as the osmotic potential of the litter had no effect on the understory species. The allelopathic potential of F. retusa leaf litter plays at least a partial role in reducing urban vegetation. Keywords: allelopathy, Ficus retusa, leaf litter, understory species, urban gardens Introduction Allelopathy is a process that occurs when a donor plant releases chemical compounds into the environment that exert an adverse or positive effect on associated species (Rice 1984). These compounds can leach from leaf litter, be exuded from roots, or arise from the decomposition of plant residues. Their release is modulated by environmental fac- tors such as temperature, soil moisture, microorganisms and nutrients (El-Khawas and Shehata 2005). Depending on their concentrations, these substances can hinder the ger- mination and growth of plant species, and their effect varies according to species (Quddus et al. 2014). Leaf litter can be found in abundance under the canopies of some trees. In urban ecosystems, leaf litter may arise naturally from de- ciduous trees (Hassan 2018) or be generated when evergreen trees undergo pruning. Several toxic compounds may be released from this leaf litter into the surrounding environ- ment, adversely affecting the cover, diversity, species rich- ness and species composition of the understory species (Chou 1999, Hassan 2018). For example, toxic compounds released from the leaf litter of Eucalyptus globulus Labill. and Acacia melanoxylon R.Br. have been shown to suppress some understory species (Souto et al. 1994). Under precipi- tation conditions, Acacia dealbata Link canopy released phytotoxic compounds that inhibited the net photosynthet- ic rate and consequently affected the distribution of under- story species (Lorenzo et al. 2011). In this article, we will highlight such phenomena. Soil is a complex medium that influences the availabil- ity of phytotoxic compounds released from plant residues, thereby affecting plant growth (El-Khatib et al. 2004). When these compounds are released into the soil, retention, trans- formation and transport processes may occur. These pro- cesses can be influenced by soil properties, chemical com- pound nature, and the physical environment (Cheng 1992, Kobayashi 2004). Substantial amounts of toxic substances that leach from litter or are released by its decay accumulate on the soil surface and interfere with plant growth (Facelli and Pickett 1991). In general, allelochemicals released from * Corresponding author e-mail: howidayacoup71@yahoo.com HASSAN M. O., MOHAMED H. Y., HASSAN ABOELLIL A. 132 ACTA BOT. CROAT. 80 (2), 2021 plant residues can influence the germination and growth of tested species when they are present in sufficient concentra- tions in the associated soil (Inderjit et al. 1996, El-Khatib 2000, El-Khatib et al. 2004). Therefore to assess the allelo- pathic effect of a particular plant, it is necessary to study its associated soil. Several studies have been undertaken to as- sess the potential release of allelochemicals and their effects on the understory vegetation (Molina et al. 1991, Espinosa- Garcia et al. 2008). However, knowledge of the allelopathic potential of the leaf litter of some trees is still lacking. Ficus is a genus of about 750 woody species belonging to the Moraceae family (Semwal et al. 2013). Ficus species are highly distributed all over the world, widely used in medici- nal purposes and are native to India, Southwest China and Nepal (Rawat et al. 2012). Ficus retusa is an evergreen tree that can grow to a height of 15 m with a wide-spread canopy ( Semwal et al. 2013, Khan 2017). It was introduced to Egypt for ornamental purposes. Trees dominate the urban ecosys- tem of the new city of Beni-Suef governorate, and most of them are F. retusa trees. They are widely distributed in gar- dens, streets and along roadsides. Previous studies revealed the presence of a variety of phenolic compounds and flavo- noids in the stem and leaves of F. retusa (Takahashi et al. 2002, Khan et al. 2011, Rawat et al. 2012, Aly et al. 2013, Singhal et al. 2017). Additionally, several compounds such as luteolin, ß-sitosterol acetate, ß-amyrin, friedelenol and new polyphenolic compounds called retusaphenol and (+)-retusa afzelechin were first isolated from the aerial parts of F. retusa (Sarg et al. 2011). Many of these compounds were shown to be allelochemicals (Rice 1984). Nevertheless, the allelopathic activity of F. retusa leaves has not been investigated. The goal of this study was to fill this gap in scientific knowledge. The annual pruning of ornamental trees, such as F. retusa, and the residents’ lack of interest in removing the falling leaves cause the accumulation of substantial amounts of leaf litter underneath their canopies. There is a lower cover of some species under these canopies than in adjacent re- gions. In the light of litter effects, two main hypotheses were tested. The first hypothesis was that potential allelochemi- cals can be released from Ficus litter, accumulate in the soil, and affect the cover, richness and diversity of the understo- ry vegetation. To test this hypothesis, a field study was con- ducted to measure the cover, richness and diversity indices of species under F. retusa canopies and compared them with those in neighboring areas away from the canopies. Addi- tional testing in a greenhouse assessed the effects of litter- affected soils on the germination and growth of selected co- occurring species detected in the field study by comparing them with the effects of soils unaffected by litter. To confirm the allelopathic effect of field soil, HPLC analysis was used to investigate the putative allelochemicals (phenolics and flavonoids) present in litter-affected and unaffected soils. This analysis reflected the presence of allelochemicals re- leased from leaf litter into the soil under natural conditions. The second hypothesis stated that the litter altered the chemical properties of the soil in a way that led to adverse effects on the cover and diversity of understory plants. In other words, the litter may interfere with soil pH, electrical conductivity (EC), organic matter (OM) and nutrient avail- ability. To test this hypothesis, soil samples collected from under and outside the tree canopy were analyzed to study the potential changes in these properties. Since changes in the EC of field soil may exert an osmotic effect on the un- derstory vegetation, a polyethylene glycol (PEG) 6000-based bioassay was used to assess this possibility. The primary goal of the present study was to assess the allelopathic ef- fects of F. retusa leaf litter on the cover, composition and floristic diversity of the understory vegetation. Material and methods Study area This study was conducted in the second district of the city New Beni-Suef (29°01.50′ to 29°02.50′ N, 31°06′ to 31°07.30′ E). This region is considered one of the common new urban areas in Egypt. It is found about 124 km south of Cairo and located east of the Nile Valley at an elevation of 32 to 42 m above sea level. It has been completely built since 1990 and has an area of about 1.42 km2. The climate of this city is characterized by mild winters with low pre- cipitation and hot, dry summers (Hassan and Hassan 2019). The average annual precipitation, which falls from Novem- ber through April, is about 11.98 mm. The texture of the soil in this region is sandy, sandy loam or sandy clay loam. Oth- er properties of the soil in the gardens of this area, such as pH, EC, organic matter and some available nutrients, were measured by Hassan (2018). Many tree species have been introduced into this area for ornamental purposes. F. retusa trees are among the most common trees in this territory. Field study To assess the effect of F. retusa tree litter on the under- story vegetation, a total of 62 plots, each of 3 × 0.1 m2, were set randomly under and just outside the tree canopies. Thir- ty-one plots located under tree canopies were designated as treatment areas and the remaining plots outside those can- opies (about 2 m away) were set as controls. These plots were selected in different seasons (mid-winter and mid-summer of 2018) to include the total species richness present in this area through the entire year. In each plot, the vegetative pa- rameters measured in this study were the cover of each spe- cies, total plant cover of all species, species richness, bare length (measured by the area not occupied by plant cover), relative cover of species [(cover of species i/cover of all spe- cies) × 100] and diversity indices (Simpson’s index (D), Shannon-Weaver index (H’) and Evenness index (E)) as shown by Hassan (2018). The species detected were identi- fied using Boulos (2000, 2002, 2005): ′ − ×H = pi ln pi i=1 s S( ) E = pi ln pi S i=1 s − ×  S( ) / ln D =1/ pi i=1 s 2S where pi is the relative cover of species i. ALLELOPATHIC POTENTIAL OF FICUS RETUSA L. LEAF LITTER ACTA BOT. CROAT. 80 (2), 2021 133 Soil samples taken from 0-20 cm depth of each plot were air-dried, passed through a 2 mm sieve and stored in plastic bags prior to analysis. The measured parameters in soil samples were pH, EC, available nutrients (N, P, K, Cu and Zn) and organic carbon. Soil pH was measured in a soil-water extract (1:2.5, w/v) using a pH meter (AD 3000), while soil EC was measured in a soil-water extract (1:5, w/v) using a conductivity meter (Jenway 3305). Available nitrogen was determined as de- scribed by Allen (1989). Briefly, 5 g of soil was added to 50 mL of 2N KCl, shaken for 30 min and then filtered. Ammo- nium nitrogen and nitrate nitrogen in the filtrate were mea- sured with a Technician Auto Analyzer. Total available ni- trogen is expressed as the sum of ammonium and nitrate nitrogen. Available P, K, Cu and Zn were determined as de- scribed by Soltanpour (1991). Briefly, 20 g of soil sample was added to 40 mL of a solution containing diethylenetri- aminepentaacetic acid (DTPA, 97%) and ammonium bicar- bonate at pH 7.6 and then mixed well. After 15 minutes of shaking, the extract was filtered and the filtrate used for further analyses. P, Cu and Zn were measured using induc- tively coupled plasma (ICP) spectrometry (Ultima 2 JY Plas- ma), while K was measured using a flame photometer. Soil organic carbon was determined using the method described by Walkley and Black (1934). Allelopathic potential of litter-affected soils under Ficus canopy To assess the allelopathic effect of F. retusa leaf litter, its residual toxicity in field soil collected under tree canopies was determined using selected understory species, Melilotus indicus (L.) All., Trifolium resupinatum L. and Amaranthus viridis L. These species showed appreciably suppressed cov- er under canopies in field conditions. Litter-affected soil samples were collected under F. retusa canopies and mixed well to form a composite treatment soil, while litter-free soil samples collected outside those cano- pies were used as a control. These samples were used for germination and growth of the three tested species. Specif- ically, the soil samples were shade-dried, passed through a 2-mm sieve, and then put in plastic pots (11 cm diameter × 11 cm deep) that received about 0.5 kg soil each. In each pot, ten seeds of each tested species were sown at a depth of 0.2 cm. Regular irrigation was carried out by spraying when needed. This experiment was kept with four replicates in a protected area for 30 days under prevailing environmental conditions (12 h light and 12 h dark photoperiod, 24 to 34 °C daytime temperature, 14 to 22 °C nighttime temperature, and 27 to 28% relative humidity). The germination rate (in %) was calculated after the emergence of the tested species ceased. Three growth criteria were measured at harvest: shoot length, root length and biomass. Individuals from each target species were dried in an oven at 70 °C for 72 h then weighed to determine the dry mass. To assess the osmotic potential of the litter-affected soil and separate its osmotic effect from its allelopathic effect, another experiment was conducted using PEG 6000. In this experiment, solutions with osmotic potentials equivalent to those of the field soils (litter-affected and litter-free) were prepared. From soil analysis, EC values of the field soil were converted to osmotic potentials (Osm·kg–1 H2O) by using the following equation (Gomaa et al. 2014): MPa = Osm · kg–1 H2O / 0.407 The osmotic potentials of litter-affected and unaffected soil were -0.0097 and -0.0039 Osm·kg–1 H2O, respectively. To obtain a PEG solution with osmotic potential equal to that of litter-affected soil, 32.3 g of PEG 6000 was dissolved in 1 liter of H2O at room temperature (28 °C) (Michel and Kaufmann 1973). By diluting the previous PEG solution, we prepared a solution of osmotic potential value equivalent to that of the unaffected soil. To show the effect of the osmotic potential of field soil on the target species, a germination test was performed us- ing the prepared PEG solutions. Due to the hard seed coat and dormancy characteristics of the legume and Amaranthus species, scarification treatments were done (Ates 2011, Assad et al. 2017). Seeds of the legume species were soaked in con- centrated H2SO4 (98%, v/v) for 10 min while Amaranthus seeds were soaked for 2 min. After soaking, the seeds were washed with distilled H2O, sterilized with 5% (w/v) sodium hypochlorite for 2 min, and rinsed three times with distilled H2O. Twenty-five seeds of each tested species were put on fil- ter paper in sterile petri dishes (9 cm diameter) and then sup- plied with 5 mL of the prepared PEG solutions. All the experi- mental samples were kept in a dark chamber at 23 ± 2 °C in a completely randomized design with four replicates. After seven days, germination (in %) was assessed by counting the number of germinated seeds. Root lengths, shoot lengths and seedling biomasses were determined seven days after seeding by measuring similar seedlings in each dish. Root and shoot lengths were estimated as described by Hussain et al. (2011). Determination of phenols and flavonoids in litter-affected and unaffected soils Polyphenols were extracted from the field soil samples with aqueous methanol (80%) in a 250 mL Erlenmeyer flask using an ultrasound-assisted method (Kim and Lee 2002). Briefly, aqueous methanol (80%, 100 mL) was added to soil (10 g) and the mixture was subjected to continuous sonica- tion for 60 min. After filtration, the filtrate was evaporated in a vacuum evaporator at 40 °C. The residue was dissolved in 50 mL methanol then diluted to a final volume of 100 mL with distilled H2O. The resulting solution was centrifuged for 15 min at 12,000 rpm and stored at −20 °C until analysis. The total phenolic content was determined according to Kim et al. (2003) protocol. One mL of the stock extract, 9 mL of distilled H2O and then 1 mL of Folin-Ciocalteu phe- nol reagent were added to a 25-mL volumetric flask and mixed well. After 5 min, 10 mL of 7% Na2CO3 solution were added to the mixture with shaking. Finally, distilled H2O HASSAN M. O., MOHAMED H. Y., HASSAN ABOELLIL A. 134 ACTA BOT. CROAT. 80 (2), 2021 was added to reach 25 mL and then the solution was left standing for 90 min. At this point, the absorbance of the so- lution at 750 nm was measured versus a prepared blank. Gallic acid was used to prepare a calibration curve. Total phenolic content was expressed as mg gallic acid equivalent (GAE) per gram of soil sample (mg·g–1 soil sample). The total flavonoid content was performed using the method described by Chun et al. (2003). One mL of the stock extract and 4 mL of distilled H2O were added to a 10- mL volumetric flask. After 5 min, 300 μL of 5% NaNO2 fol- lowed by 300 μL 10% AlCl3 were added to the mixture. This mixture was allowed to stand for 6 min, and then 2 mL of 1 M NaOH was added and the final volume was adjusted to 10 mL using distilled H2O. The absorbance at 510 nm was measured versus a prepared blank. Rutin was used to pre- pare a calibration curve. Total flavonoids in the soil sample were expressed as mg of rutin equivalents (RE) g–1 soil (mg·g–1 soil sample). HPLC analysis Phenolics and flavonoids were analyzed according to Hassan (2018). This analysis was carried out using a Shi- madzu HPLC system equipped with an LC 1110 pump, a Kromasil C8 column (4.6 mm × 250 mm; particle size, 4.6 μm; pore size, 100 Å) and a diode-array UV detector and run using WinCrome Chromatography software (version 1.3). Phenolic compounds were analyzed using a solvent gradient consisting of acetonitrile: 0.05% H3PO4 (99:1; sol- vent A) and water: H3PO4 (99:1; solvent B) and a flow rate of 1 mL·min–1. The elution program consisted of 90% A from 0 to 30 min, a linear decline to 50% A from 30.01–40 min, and a further decline to 0% A from 40.01 to 55 min. Flavo- noid compounds were analyzed using a solvent gradient composed of methanol:H3PO4 (99:1; solvent A) and water:H3PO4 (99:1; solvent B) and a flow rate of 1 mL·min–1. Flavonoid compounds were detected using UV (250–400 nm) absorbance. Phenolic and flavonoid compounds were identified by comparison of their retention time with those of phenolic standards including trans-cinnamic, vanillic, p-coumaric acids, catechol, sinapic, protocatechuic, syrin- gic, caffeic, p-hydroxybenzoic acids, vanillin and resorcinol; and flavonoid standards including hesperidin, luteolin, ru- tin, apigenin, catechin, kaempferol and quercetin. The con- centrations, expressed as mg g–1, were estimated according to knowledge of the heights and areas under peaks of de- tected compounds in soil samples. Values reported are the average of three replicates. Statistical analysis Kolmogorov–Smirnov and Levene’s tests were used to check the normality and homogeneity, respectively, of the data obtained from field, greenhouse and laboratory exper- iments. When the data were normally distributed, they were analyzed using the independent Samples T test. Data show- ing abnormality and heterogeneity were analyzed using the nonparametric Mann-Whitney U test. All data in this study were analyzed with the use of the SPSS Statistics software package, version 20.0 (IBM Corporation, USA) at probabil- ity levels *P < 0.05 and **P < 0.01. Results Field study A total of 12 species belonging to 11 genera and five families were detected throughout the study area (Tab. 1). Poaceae had the highest number of species (five) followed by Fabaceae (three), Plantaginaceae (two), Amaranthaceae and Euphorbiaceae (in each one). Nine species were detect- ed as annuals and three species were detected as perennials (Tab. 1). Sites heavily covered with F. retusa leaf litter attained lower cover of some understory species. Among the annuals, the covers of Amaranthus viridis L., Medicago polymorpha L., Melilotus indicus (L.) All. and Trifolium resupinatum L. Tab. 1. Plant cover of each plant species detected in plots with and without Ficus retusa leaf litter. Values are given as means ± standard error from 31 replicates. An asterisk means there is a significant difference between litter-unaffected plots and litter-affected plots by Independent samples T test (*P < 0.05, **P < 0.01). Species/Family Life span Litter-unaffected plots Litter-affected plots Amaranthus viridis L. (Amaranthaceae) Annual 1.40 ± 0.32 0.13 ± 0.13** Cenchrus echinatus L. (Poaceae) Annual 1.27 ± 0.62 0.37 ± 0.15 Cynodon dactylon (L.) Pers. (Poaceae) Perennial 1.35 ± 0.17 0.86 ± 0.09* Digitaria sanguinalis (L.) Scop. (Poaceae) Annual 1.00 ± 0.17 0.70 ± 0.20 Eragrostis pilosa (L.) P.Beauv. (Poaceae) Annual 0.84 ± 0.34 0.30 ± 0.04 Euphorbia peplus L. (Euphorbiaceae) Annual 0.16 ± 0.11 0.42 ± 0.20 Medicago polymorpha L. (Fabaceae) Annual 0.55 ± 0.13 0.10 ± 0.05* Melilotus indicus (L.) All. (Fabaceae) Annual 1.20 ± 0.22 0.07 ± 0.04** Paspalum dilatatum Poir. (Poaceae) Perennial 0.67 ± 0.31 0.27 ± 0.13 Plantago amplexicaulis Cav. (Plantaginaceae) Annual 0.15 ± 0.03 0.00 ± 0.00** Plantago major L. (Plantaginaceae) Perennial 0.74 ± 0.22 0.16 ± 0.08 Trifolium resupinatum L. (Fabaceae) Annual 1.66 ± 0.29 0.05 ± 0.05** ALLELOPATHIC POTENTIAL OF FICUS RETUSA L. LEAF LITTER ACTA BOT. CROAT. 80 (2), 2021 135 under tree canopies were significantly lower than those outside the canopies. In addition, Plantago amplexicaulis Cav. was completely absent from the infested sites (Tab. 1). For perennials, only the cover of Cynodon dactylon (L.) Pers. was significantly reduced at the litter-affected sites, whereas the remaining perennial species were not affected. Significant reductions in vegetation cover, and species richness were also recorded in the plots affected by litter, compared with those free from litter (Tab. 2). Also, there was a significant increase in bare length in litter affected plots. However diversity, as measured using Shannon-Weaver, evenness and Simpson’s indices, was not influenced (Tab. 2). Most of the soil analysis criteria were unaffected by the presence of litter. However, the litter-affected soils exhibited lower pH and higher EC values (Tab. 3). Greenhouse experiment Generally, litter-affected soil collected from under F. retusa canopies drastically reduced germination and some of the growth variables of studied species. Litter-affected soils sig- nificantly decreased seed germination of the selected target species (P < 0.01) (Fig. 1A). Litter-affected soil significantly decreased the root lengths of A. viridis and T. resupinatum (Fig. 1B), while the shoot length was significantly reduced Tab. 2. Vegetation cover, bare length, species richness, and diver- sity indices (mean ± standard error; n = 31) in the stands with and without Ficus retusa leaf litter. An asterisk means there is a sig- nificant difference between plots without litter and plots with lit- ter by Mann-Whitney U test (*P < 0.05, **P < 0.01). Parameters Plots without litter Plots with litter Vegetation cover (%) 83.33 ± 4.63 36.58 ± 2.81** Bare length (%) 16.67 ± 4.63 63.42 ± 2.81** Species richness (S) 2.68 ± 0.23 1.9 ± 0.19* Shannon-Weaver index (H’) 0.60 ± 0.077 0.41 ± 0.075 Evenness index (E) 0.55 ± 0.06 0.45 ± 0.07 Simpson’s index (D) 1.81 ± 0.14 1.51 ± 0.12 Tab. 3. Soil properties in stands with and without Ficus retusa lit- ter. Values are given as means ± standard error from 31 replicates. An asterisk means there is a significant difference in the parame- ters between stands without litter and stands with litter by inde- pendent samples T test (*P < 0.05, **P < 0.01). Soil parameter Stand without litter Stand with litter pH 7.9 ± 0.03 7.63 ± 0.06** Electrical conductivity (µs cm–1) 263.75 ± 36.85 663.8 ± 146.55* Organic carbon (%) 2.18 ± 0.58 2.21 ± 0.4 Available nutrient concentrations N (mg kg–1) 73.75 ± 13.24 85.50 ± 3.52 P (mg kg–1) 6.29 ± 1.82 9.8 ± 2.52 K (mg kg–1) 102.82 ± 31.08 182.64 ± 31.74 Cu (mg kg–1) 0.91 ± 0.16 1.85 ± 0.54 Zn (mg kg–1) 3.59 ± 0.60 5.35 ± 0.78 Fig. 1. Effect of the residual toxicity of Ficus retusa leaf litter in soil on selected understory species Melilotus indicus, Trifolium resupinatum and Amaranthus viridis (n = 4). A – germination percent (%), B – root length (cm), C – shoot length (cm), D – bio- mass (g). Test refers to litter-affected soil and control refers to unaffected soil. The bars on each column represent the standard error. Significant difference were at *P < 0.05 and **P < 0.01 (in- dependent samples T test). HASSAN M. O., MOHAMED H. Y., HASSAN ABOELLIL A. 136 ACTA BOT. CROAT. 80 (2), 2021 for the later (Fig. 1C). Moreover, the biomass of both species was significantly suppressed (Fig. 1D). The PEG solution with an osmotic potential equivalent to that of the litter-affected soil did not significantly affect the germination (Fig. 2A) or growth parameters (Fig. 2B-D) of the tested species, compared with the control. Biochemical analysis The concentrations of the phenolic and flavonoid com- pounds detected in litter-affected and unaffected soils are summarized in Tab. 4. Among the free compounds, quer- cetin and resorcinol were completely absent in control soils (Tab. 4). Litter-affected soils also contained significantly more quercetin, resorcinol, caffeic acid, coumaric acid and ellagic acid than unaffected soils. Furthermore, the total phenolics and f lavonoids were significantly higher (by 65.84% and 47.54%, respectively) in litter-affected than in control soils (Tab. 4). Discussion The results of this study clearly demonstrate that F. retusa leaf litter has an inhibitory effect on the selected understory species. Significant reductions in the cover of many species, total plant cover and species richness were observed in plots under tree canopies. These observations are similar to those of previous studies, which illustrated considerable inhibition of plant cover beneath the tree canopy, compared with areas outside the canopy, due to the presence of leaf litter (Ahmed et al. 2008, Souza et al. 2010, Hassan 2018). Many tree spe- cies have been shown to negatively affect the cover, diversity and composition of some understory herbaceous species (Barbier et al. 2008). Moreover, Loydi et al. (2013) indicated that a high amount of oak tree litter reduced the cover, com- position, species richness and biomass of some associated Fig. 2. Effects of polyethylene glycol (PEG) 6000 solutions with osmotic potentials equivalent to those of field soil under and out- side the tree canopy on selected target understory species Melilo- tus indicus, Trifolium resupinatum and Amaranthus viridis (n = 4). A – germination (%), B – root length (cm), C – shoot length (cm), D – biomass (g). The bars on each column represent stan- dard error. Data were analyzed using the independent samples T test. Tab. 4. Free and total concentrations of phenolic and flavonoid compounds (mean ± standard error, mg·g-1 soil, n = 4) detected in the field soil affected and unaffected by Ficus retusa leaf litter using HPLC and spectrophotometric analyses, respectively. Significant difference at *P < 0.05 and **P < 0.01 according to Independent samples T test, –: not detected. Compounds Litter-unaffected soil Litter-affected soil Phenolics Caffeic acid 1.28 ± 0.15 2.10 ± 0.07** Coumaric acid 0.40 ± 0.23 2.01 ± 0.00** Ellagic acid 1.10 ± 0.07 1.66 ± 0.03** Gallic acid 2.52 ± 0.10 2.56 ± 0.04 Resorcinol – 1.39 ± 0.05** Total phenolics 16.42 ± 0.72 48.07 ± 3.28** Flavonoids Quercetin – 1.56 ± 0.02** Total flavonoids 22.79 ± 3.07 43.44 ± 6.54* ALLELOPATHIC POTENTIAL OF FICUS RETUSA L. LEAF LITTER ACTA BOT. CROAT. 80 (2), 2021 137 species. This effect may be due to toxic compounds leaching from litter residues (Batish et al. 2007). The soil analysis revealed that the differences in organ- ic matter and available nutrients between litter-affected and unaffected soils were not significant. This result suggests that the litter does not interfere with these soil criteria. It also indicates that Ficus trees do not have a competitive ef- fect on associated weed species. Therefore, the reduction in plant cover and species richness could not be attributed to decreased soil organic matter or nutrient availability. These results are similar to those obtained by Hassan (2018). In contrast, there was a considerable reduction in pH and a significant increase in the EC of litter-affected soils. These results may be due to phenolic compounds and minerals liberated from litter residue, as mentioned by Hassan et al. (2014). In this study, the pH value decreased from 7.9 to 7.63. These values seem to be in the normal range for the germi- nation and growth of plants (Xuan et al. 2005). On the oth- er hand, the EC value of litter-affected soil in this study was 0.664 mS·cm–1. Xuan et al. (2005) reported that EC values < 1 mS·cm–1 did not affect the growth of tested species. Simi- larly, Hassan et al. (2014) confirmed that EC had a signifi- cant role in the inhibition of target species growth when it was >1.5 mS·cm–1. Therefore, since the EC value observed in this study was within a range appropriate for plants, the decline in vegetation cover and richness is not attributable to a high EC value in the field soil. Under greenhouse conditions, the litter-affected soil collected under the canopies of F. retusa trees substantially reduced the emergence and growth of the tested species. Moreover, HPLC and spectroscopic analyses showed that the litter-affected soil contained greater amounts of phenols and flavonoids. These findings suggest that the litter-affect- ed soil collected under the Ficus canopy had an inhibitory effect on the understory species. This inhibition could be due to the presence of phenolic and flavonoid compounds released into the soil during Ficus leaf litter decay or leached from Ficus leaf litter during irrigation or rainfall. Among different classes of secondary metabolites, phenolic com- pounds are the major group that inhibits plant growth ( Appel 1993). The compounds detected by HPLC are potent allelochemicals that frequently inhibit the seed germina- tion, growth and productivity of some species (Qasem and Foy 2001). For example, Golisz et al. (2007) indicated that some phenolics, such as ferulic acid, gallic acid and chloro- genic acid, reduced the germination and seedling growth of lettuce to differing degrees. Likewise, quercetin, apigenin, rutin and kaempferol are considered inhibitory substances that suppress the emergence and performance of many spe- cies (Basile et al. 2000, Sadeghi and Bazdar 2018). These compounds harmfully affect cell division, the ability to ab- sorb nutrients, membrane permeability, protein creation and the activities of enzymes, eventually reducing the growth of target species (Li et al. 2010). Additional studies have demonstrated that the cover and diversity of plant spe- cies could be changed by allelopathic compounds such as those detected during the HPLC analysis conducted in this study (Chou 1999, Hassan 2018, Hassan and Mohamed 2020). The inhibitions observed under field conditions and in greenhouse experiments may be equivalent. In this study, litter-affected soils attained substantially higher values of EC than those of litter-free soils. Although the values observed were normal for plant growth, the effect of the osmotic potential of soil solutes on seed germination and seedling growth had to be assessed to exclude potential osmotic interference with the allelopathic effect in soil. The results of our work showed that PEG solutions with osmot- ic potentials equivalent to those of litter-affected soils did not significantly reduce the germination and growth of the target species. Therefore, osmotic potential did not play a significant role in the inhibition of target species under greenhouse conditions or in the understory vegetation. Ac- cordingly, the inhibitory impact of field soil collected under the tree canopy on the germination and growth of tested species under greenhouse conditions was due to the allelo- chemicals present in the field soil. Conclusion The field study indicated that the cover of selected under- story species and species richness were reduced under the canopy of F. retusa trees. The greenhouse experiments showed that litter-affected soil suppressed the germination and growth parameters of the selected species. The biochem- ical analysis of the litter-affected soils confirmed the presence of elevated levels of many phenolic and flavonoid compounds, as compared with unaffected soils. These compounds have been shown to be phytotoxins that negatively affect the ger- mination and growth of some understory species. 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