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Allelopathic investigations of Quercus conferta and Quercus cerris domination in oak forest at Avala Mt. (Serbia)

Lola Djurdjević, Miroslava Mitrović, Anka Dinić, Pavle Pavlović, Srdjan Bojović, Gordana Gajić and Olga Kostić

Department of Ecology, Institute for Biological Research “Siniša Stanković”, Bulevar Despota Stefana 142, 11060 Belgrade,

Serbia and Montenegro, kalac@ibiss.bg.ac.yu

Abstract

Xerophyte oaks Quercus conferta and Quercus cerris form a climatogenous forest community widely distributed in hill regions in Serbia. As dominant species they decrease the number and abundance of herbaceous as well as woody plants. The phenolics of oaks are the main allelochemicals, which regulate the composition and structure of plant communities. The aim of this study was the quantification of total phenolics and phenolic acids of fallen leaves of oaks as a main source of secondary metabolites, than in partially decomposed plant litter and soil of forest ecosystem. The inhibitory capacity of fallen leaves, plant litter and soil under oak trees was tested using two grass species: Poa nemoralis and Dactylis glomerata. The fallen leaves of Q. conferta and Q. cerris were found to contain 44.16 mg/g and 42.86 mg/g of total phenolics. The litter contained 11.79%, and soil only 0.35% of total phenolics measured in leaves of both oaks. p-Coumaric, ferulic, syringic and vanillic acids were found as the main acids in free and bound forms in leaves. Their total content was 1245.66 μg/g in Q. conferta and 2219.73 μg/g in Q. cerris leaves. The litter contained 13.27%, and soil only 2.08% of total phenolic acids content measured in both oak leaves. The soil and aqueous leachates of fallen leaves and litter were found to be strong inhibitors of seed germination and seedlings growth of P. nemoralis and D. glomerata. Results indicated the inhibitory allelopathic effects of oak trees on other plants in the community.

Media summary

Xerophyte oaks Quercus conferta and Quercus cerris are dominant trees of climatogenous forest communities. Their inhibitory effects are connected to a high phenolic content in their leaves, litter and soil.

Key Words

allelochemicals, phenolic acids, phenolics, Quercus conferta, Quercus cerris, seed germination and seedlings growth inhibitors.

Introduction

Autochthonous oaks Q. conferta and Q. cerris form climatogenous forest community (Quercetum confertae-cerris Rudski 1940) wide distributed in hill regions in Serbia. As dominant trees species they decrease the number and abundance of herbaceous as well as woody plants, pointing out to their inhibitory allelopathic effects. The phenolics of oaks are the main allelochemicals, which regulate the composition and structure of plant community. The allelopathic effects of dominant plants on other plants in phytocoenosis are caused by phenolic phytotoxins present in all parts of plants, but the highest amount of these compounds is accumulated in leaves. Fallen leaves of dominant trees represent the main components of the litter in the forest. Phenolic compounds are also presented in the majority of the soils associated with forest trees. (Lodhi 1975, 1978; Kögel 1986; Kutiers and Denneman 1987; Souto et al. 1995; Djurdjevic and Oberan 1998; Djurdjevic et al. 1998; Djurdjevic et al 2004). Phenolic acids represent the main allelopathic compounds that inhibit seed germination, plant growth and other physiological processes what results in the changes of floristic composition within a phytocoenosis and dominance of one plant species over the others (Muller 1966; Lodhi and Rice 1971; Rice 1974, 1979; Chou and Muller 1972; Lodhi, 1975; Li et al. 1992). The aim of this study was the quantification of total phenolics and phenolic acids in fallen leaves, in partially decomposed plant litter and in soil under oak trees. Poa nemoralis and Dactylis glomerata which grow in examined oak forest were used as test plants.

Methods

Description of forest community

Experimental oak community is situated at Avala Mt., near Belgrade city (lat. 44°48' N long. 20°28' E), at altitude of 511 m. The forest is middle-aged with expressed floors. The floor of high trees (up to 22 m high) consists of Quercus conferta, Quercus cerris, and Quercus petraea. The floor of lower trees (5-12 m high) includes: Fraxinus ornus, Crataegus monogyna, Q. conferta, Tilia argentea, Q. cerris and Q. petraea. In the floor of herbaceous plants, a low number of other species (Orobus niger, Galium pseudoaristatum, Chrysanthemun corymbosum, Melica uniflora, Dactylis glomerata, Lathyrus vernus, Thlaspi avalanum, Poa nemoralis and Danaa cornubiensis) are present.

Preparation of phenolic-containing extracts from oak leaves and litter

Both phenolic acids and total phenolic were extracted from dry leaves and litter (5x2 g) with distilled water during the 24 h period. Water extracts were evaporated, the residues dissolved in water adjusting pH to 2.0 with 2N HCl and transferred to ethylacetate. The mixture was evaporated to dryness and the residue dissolved in 4 mL of 80% (v/v) MeOH (free phenolics) and used for HPLC analysis or stored at -20 °C until use. Bound phenolics were prepared by boiling the residue in 2N HCl for 60 min and transferred to ethylacetate.

Extraction of phenolics from soil sample

Free forms of phenolics were extracted from 5x30 g of dried soil with distilled water during the 24 h period. The extracts were evaporated, adjusting pH to 2.0 with 2N HCl and transferred to ethylacetate. The mixture was evaporated to dryness and the residue dissolved in 4 mL of 80% (v/v) MeOH (free phenolics). Residual soil was treated with 15 ml of 2N NaOH and after boiling for 24 h the mixture was acidified with concentrated HCl to pH 2.0, phenolic compounds were transferred into ethyl acetate (3x50ml) and were evaporated to dryness. Dry residue was dissolved in 4 ml 80% MeOH (bound phenolics), (Hennequin and Juste 1967; Katase 1981a,b).

Determination of total phenolics and phenolic acids

Total phenolics (free and bound) were measured by spectrophotometry (a Shimadzu UV 160 spectrophotometer) using the Folin-Ciocalteu reagent (Feldman and Hanks, 1968). Phenolic acids were detected between 210 and 360 nm using a Hewlett Packard diode array detector (HP 1100 HPLC system).

Plant growth tests with soil

Test plants were grown in 300 mL plastic dishes. Surface samples (0-10 cm) of soil under Q.conferta and Q.cerris as dominant plants were collected during middle of October. Two-day-old etiolated seedling of P. nemoralis and D. glomerata were planted into experimental soil-containing pots and grown in a green-house for 15 days. (14 h light, white fluorescent lamp 3.6h103 erg/cm2/sec, 25°C and 10 h dark, 20°C). The controls were grown under the same conditions in the pots containing forest soil taken from the spots without plants. Measurements were performed using 500 individuals of each test plant species.

Seedling growth tests Test plant seeds sterilized with 5% sodium hypochlorite for 10 min and thoroughly rinsed with distilled water germinated at 25°C in the dark for 36 h. Uniform seedlings approximately 5 mm long were used. They were grown in Petri dishes (diameter of 10 cm) containing 5 mL of aqueous extracts: 1. fallen leaves of Q. conferta and Q. cerris and 2. plant litter (0.25 g/ml) or 3. distilled water (controls). After 48 h growth at 25°C the length of the radicle or coleoptiles was measured. In each experiment 500 seedlings were used.

Seed germination tests

Test plant seeds (5x100 seeds of each) germinated at 25 °C in Petri dishes (diameter of 10 cm) containing 5 ml of aqueous extracts of: 1. fallen leaves of Q. conferta and Q. cerris and 2. plant litter (0.25 g/ml) or 3. distilled water (controls). In order to examine the soil impact to seed germination test plant seeds (5x100 seeds of each) germinated at 25 °C in Petri dishes (diameter of 10 cm) containing 50 g of surface soil (0-10 cm) under oak trees. Soil without of plants served as control. After 96 h, the number of germinated seeds was counted.

Results

The fallen Q. conferta and Q. cerris leaves were found to contain 44.16 mg/g and 42.86 mg/g of total phenolics. Leaves of Q. conferta contained higher amounts of free in comparison to bound phenolics whereas in leaves of Q. cerris there were no significant differences between of them. The litter contained 11.79%, and soil only 0.35% of total phenolics measured in leaves of both oaks. p-Coumaric, ferulic, syringic and vanillic acids were found as the main acids in free and bound forms in oak leaves. Their total content was 1243.55 μg/g in Q. conferta and 2219.74 μg/g in Q. cerris leaves. In leaves of both oaks bound forms of phenolic acids prevailed in relation to free forms. Derivatives of cinnamic acid (ferulic and p-coumaric) prevailed in both species comparing to the benzoic acid derivatives (p-hydroxybenzoic, vanillic and syringic). Phenolic acids represented only 2.82-5.18% of total phenolics present in the leaves of both oaks and plant litter and 23.36% in the upper soil layer. Because of leaching and microbiological degradation of plant remains decreasing of phenolic acids content in plant litter and soil in comparison to fallen leaves was occurred. So, the litter contained 13.27%, and soil only 2.08% of total phenolic acids content measured in both oak leaves (Table 1). Bound phenolic acids in relation to free prevailed in both litter and soil.

Table 1. Content of phenolic acids in Q. conferta and Q. cerris fallen leaves, plant litter and soil.

μg/g

 

leaves

litter

soil

   

Q. conferta

Q. cerris

   

p-coumaric

free

38.77±5.10

228.28±25.11

23.43±2.71

1.49±0.18

 

bound

62.47±7.41

393.81±43.13

79.10±8.82

13.28±1.92

ferulic

free

137.46±15.89

291.88±35.03

32.13±4.16

2.93±0.39

 

bound

546.87±57.31

464.69±48.25

88.94±9.88

21.51±3.40

p-hy.benz.

free

0.11±0.01

0.21±0.02

5.06±0.62

0.46±0.05

 

bound

-

-

30.28±3.98

-

vanillic

free

115.81±13.04

111.52±13.23

23.05±2.97

3.57±0.41

 

bound

140.59±15.44

193.02±21.16

54.21±6.00

16.92±1.91

syringic

free

183.15±19.91

235.20±24.41

29.39±3.81

1.69±0.18

 

bound

17.43±1.98

301.12±32.55

93.84±10.32

10.10±1.58

total

free

475.19±50.23

867.10±88.41

113.06±13.14

10.14±1.89

bound

770.36±78.85

1352.64±138.55

346.38±36.10

61.81±6.98

Table 2. Effects of water extracts from Q. conferta and Q. cerris fallen leaves and plant litter on seeds germination and seedlings growth of Poa nemoralis and Dactylis glomerata.

Plant species

Treatment

Seed germination %

Length of aerial part %

Length of root %

 

Control

81.45 (±4.89)a

100 (±7.23)

100 (±7.93)

P. nemoralis

Q. conferta
Q. cerris

3.11 (±1.20)b
4.29 (±0.49)b

37 (±3.96)b
34 (±4.69)b

29 (±4.22)b
32 (±4.69)b

 

Litter

16.10 (±4.42)b

45 (±6.10)b

48 (±5.11)b

 

Control

78.85 (±4.54)

100 (±8.86)

100 (±8.68)

D. glomerata

Q. conferta
Q. cerris

1.90 (±0.86)b
3.61(±0.40)b

28 (±3.29)b
31 (±5.11)b

27 (±4.09)b
36 (±4.91)b

 

Litter

19.65 (±1.83)b

60 (±6.90)b

58 (±6.33)b

aNumbers in parentheses are standard deviations. Control=distilled water.
b
Statistically significant difference, p<0.001 (t-test), n=500.

Aqueous extract of Q. conferta and Q. cerris leaves acted as a stronger inhibitor on seeds germination of both test plants in comparison to extract of litter (Table 2). The leachates of fallen leaves and litter acted inhibitory on growth of both aboveground parts and roots of P. nemoralis and D. glomerata. Litter less inhibited of roots and aboveground parts elongation of test plants in comparison to fallen leaves because of lower content of total phenolics and phenolic acids. Surface soil layer under the oak trees inhibited seed germination of test plants but percentage of germinated seeds were higher when compared with water extract of leaves and litter (Table 3). Plant growth was also inhibited by soil under oak trees.

Table 3. Effects of soil (0-10 cm) under oak trees on seed germination and seedling growth of Poa nemoralis and Dactylis glomerata.

Plan species

Treatment

Seed germination %

Length of aerial part %

Length of root %

 

Control

76.97 (±6.00)a

100 (±5.81)

100 (±6.86)

P. nemoralis

Soil

28.98 (±3.91)b

56 (±4.59)b

49 (±5.73)b

 

Control

78.51 (±4.24)

100 (±4.77)

100 (±6.63)

D. glomerata

Soil

34.75 (±3.97)b

63 (±5.88)b

61 (±4.83)b

aNumbers in parentheses are standard deviations. Control=soil without plants.
b
Statistically significant difference, p<0.001 (t-test), n=500.

Conclusion

Results indicated the inhibitory allelopathic effects of oak trees on other plants in the oak forest community. Their inhibitory effects are connected to high total phenolics and phenolic acids content in fallen leaves, plant litter and associated soil.

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