Abstract

Media summary

Key Words

Introduction

Chemicals that originate from plants or microorganisms impact many organisms in the ecosystem, but the term allelopathy has most often referred to the activity of these chemicals on other plants or microorganisms (Enhellig 2002). Many of the phytotoxic substances suspected of causing germination and growth inhibition have been identified from plant tissues and soil. A wide array of these compounds is released into the environment in appropriate quantities via root exudation and as leachates during litter decomposition. Most of these are phenolic compounds and are implicated in allelopathy, a process which includes the direct or indirect detrimental effect of one plant on the germination, growth and development of another plant (Zaprometov 1992).

Plants produce a large variety of secondary metabolites like phenols, tannins, terpenoids, alkaloids, polyacetylenes, fatty acids and steroids, which have an allelopathic effect on the growth and development of the same plant or neighboring plants. Considerable knowledge has been obtained concerning the chemicals involved in allelopathy (Rice 1984; Narwal and Tauro 1994). Several researchers have documented the existence of allelochemicals in higher plants and microorganisms (Levin 1976; Rice 1984; Salisbury and Ross 1992; Grayer and Horborne 1994; Rauha et al. 2000). These chemicals are produced in above or below ground plant parts or in both to cause allelopathic effects in a wide range of plant communities. Plant parts like roots, rhizomes, stems, leaves, flowers/inflorescence, pollen, fruits and seeds are known to contain allelochemicals (Rice 1984). The bark, leaf and leaf litter extracts of Quercus glauca and Q. leucotrichophora significantly suppressed both the germination, plumule and radicle length and of chlorophyll content of wheat, mustard and lentil (Bhatt et al. 1994). Aqueous leachates of Eucalyptus globulus reduced the chlorophyll content in the leaves of Costus speciosus and finger millet (Konar and Kushari 1995; Padhy et al. 2000).

One of the most worked out aspects of allelopathy in manipulated ecosystems is its role in agriculture. In this, the effects of weeds on crops, crops on weeds and crops on crops have been invariably emphasised. In addition, the possibility of using allelochemicals as bioregulators and natural pesticides promoted sustainable agriculture (Pellissier and Souto 1999).

In the present work an attempt has been made to study the allelopathic effect of Acacia leucopholea aqueous leaf extracts on the growth and yield of groundnut and sorghum which commonly grow in this area.

Methods

Mature fresh leaves of Acacia leucopholea Willd. were collected from the college campus and dried in an oven at 60^oC ± 20^oC for two days, powdered (40 mesh) and used for bioassay, pot experiments and post-emergence treatment. Seeds of Arachis hypogaea var. VRI (groundnut), Sorghum vulgare var. CSHI (sorghum) were collected from Tamil Nadu Agricultural University, Coimbatore, India.

The dried leaves were ground to a fine powder in Wiley Mill 40 mesh). Using this powder aqueous extracts were prepared by the method of Heisey (1990). The aqueous extract was diluted with water to get 5, 10, 15 and 20% concentrations. The dilutions corresponded to 0.05, 0.1, 0.15 and 0.2 % of water extractable materials. The seeds of crops were surface sterilized with 0.1% mercuric chloride for 1 min. to remove the fungal spores on the seeds. Then the seeds were washed with distilled water for several times to remove the mercuric chloride.

Bioassay studies

Bioassay studies were carried out following the method of Heisey (1990). The experimental design was a randomized complete block with five replicates for each treatment and control. Germination percentage, plumule and radicle length were measured after three days.

Pot experiment studies

The seeds of A. hypogaea and S. vulgare were sown in earthern pots (24 cm x 24 cm). The treatment was started after the 3^rd leaf emerged (15^th day after sowing). The seedlings were irrigated with different concentrations of aqueous extracts of the test plants (500 ml/pot) on alternate days. Control plants were irrigated with water. Shoot length, root length, leaf area, yields and root nodules (for A. hypogaea) were determined at the end of the life-span of each crop plant.

Post- emergence treatment

Post-emergence treatments were given following the method of Heisey (1990). Five crop seeds were sown in plastic pots (7 cm x 6.5 cm). The treatments were started 8 days after sowing. Extracts (12 ml) were sprayed on each set of seedlings using an automizer. Before post-emergence treatments the seedlings were irrigated thoroughly. After the treatment the seedlings were irrigated thoroughly again until 4 days to allow time for foliar absorption. Post-emergence treatments were subsequently watered from above as needed. Shoot length, root length, biomass and mortality rate of the seedlings were recorded on the 15^th day after sowing.

The extraction and identification of phenolic compounds

The extraction of phenolic compounds for Gas Chromatography (GC) analysis was carried out by the method of Kil and Yim (1983). The resulting extract was used fro the analysis of free phenolic acids by GC. Phenolic acids were identified by GC (Hewlett Packard-5890, USA) using J & W fused silica capillary and SE-54 column. The detector was FID. Identification of each peak was made by comparing the retention times of the peaks with those of commercial Sigma phenolic acid samples.

IR spectrophotometric analysis of tannins

For the IR spectrophotometric analysis, tannins were extracted following the method of Streit and Fengel (1994). Tannins were identified by IR spectrophotometer (Nicolet Magna IR 550, series II, USA) using beam splitter KBr and DTGS/KBr.

Results and Discussion

Bioassay

Leaf aqueous extracts of A. leucopholea on bioassay study of A. hypogaea and S. vulgare showed a gradual reduction in all parameters. The seed germination, plumule and radicle length was inhibited in all concentrations (Table 1). The decrease was concentration dependent. At the highest concentration studied, a maximum of 37% and 44% of reduction in seed germination was observed in leaf extracts on A. hypogaea and S. vulgare respectively. Similar trend was followed in plumule and radicle length. In A. hypogaea a maximum of 50% and 28% reduction was recorded in plumule and radicle length respectively. But in S. vulgare the inhibition in plumule and radicle length brought about by the 20% leaf extract was 37% and 23% respectively.

Table 1. Bioassay studies of Acacia leucopholea leaf aqueous extracts on seed germination, plumule and radicle growth of Arachis hypogaae and Sorghum vulgare. Values are mean ± SE of 5 samples.

Arachis hypogaea				Sorghum vulgare
Concentration (%)	Germination (%)	Plumule length (cm)	Radicle length (cm)	Germination (%)	Plumule length (cm)	Radicle length (cm)
Control	80 ± 8.0	2.6 ±0.2	7.0 ± 0.6	90 ± 8.5	8.0 ±0.7	6.0 ± 0.5
5	70 ± 6.0	2.3 ±0.2	6.8 ± 0.6	85 ± 8.0	7.0 ±0.6	5.5 ± 0.4
10	60 ± 6.0	2.0 ±0.2	6.0 ± 0.5	75± 7.0	6.0 ±0.5	5.0 ± 0.5
15	55 ± 5.0	1.8 ±0.1	5.5 ± 0.4	70± 5.0	5.5 ±0.5	5.0 ± 0.3
20	50 ± 5.0	1.3 ±0.1	5.0 ± 0.5	50 ± 4.0	5.0 ±0.4	4.6 ± 0.4

The results of our study showed that the leaf extracts of A. leucopholea was inhibitory in both A. hypogaea and in S. vulgare. Similar results have been reported by Heisey (1990). He observed that the leaflets of among the various plant parts of Ailanthus altissima showed the highest inhibitory effect on seed germination of several weeds and crops viz. Pisum sativum and Zea mays. Results similar to our study have been cited by Peters and Zam (1981) who reported significant decreases in germination of red clover seed in leaf extracts of tall fescue plants. The leaf extracts of A. leucopholea affected the plumular length of the crop plants more than the radicle length. Inhibition of seedling growth by the extracts of many plants has been reported in a few crops and weed species by Acacia tortilis in pearl millet, cluster bean and in sesame (Sundramoorthy and Katra 1991). Allelochemical activity of plants is measured by the sensitivity of radicles in the bioassay (Heisey 1990). Hence A. hypogaea crops are very sensitivity than the other crop.

Pot experiment studies

The leaf aqueous extracts of A. leucopholea showed inhibitory effects on all growth parameters in A. hypogaea and S. vulgare plants. Decrease in shoot length, root length, leaf area, yield and root nodule (only in A. hypogaea) was concentration dependent (Table 2). A maximum of 72% and 45% reduction in shoot length was observed in Arachis and Sorghum plants respectively at 20% leaf extract concentration. The reduction in the root length was 43% in both the plants at the highest concentration. The leaf area was much affected in the Arachis plants (50%) than the Sorghum plants (37%). The reduction in root nodule number was 56% at 20% leaf extracts of Acacia. The leaf extracts of A. leucopholea drastically reduced the yield in S. vulgare plants (86%) than the A. hypogaea (80%).

Table 2. Effect of Acacia leucopholea leaf aqueous extracts on the shoot, root length and leaf area of Arachis hypogaae and Sorghum vulgare plants. Values are mean ± SE of 5 samples.

Arachis hypogaae				Sorghum vulgare
Concentration (%)	Shoot length (cm)	Root length (cm)	Leaf area (cm2)	Shoot length (cm)	Root length (cm)	Leaf area (cm2)
Control	51.5± 5.0	16.6 ±1.2	6.3 ± 0.5	115.0 ± 10.5	18.0 ±1.4	196.0 ± 19.5
5	48.5± 4.0	16.0 ±1.3	5.2 ± 0.4	100.0 ± 10.0	15.5 ±1.0	190.5 ± 10.4
10	45.0± 4.0	14.5 ±1.0	4.1 ± 0.3	99.5± 7.0	15.0 ±1.5	185.5 ± 16.5
15	35.8± 2.0	10.4 ±1.0	3.8 ± 0.2	99.0± 8.0	15.0 ±1.5	154.0 ± 12.3
20	22.5± 2.0	9.5 ±0.8	3.5 ± 0.2	78.5 ± 6.0	10.5 ±1.1	120.6 ± 11.0

In the pot experiments the aqueous extracts decreased the growth of the crop plants considerably only at higher concentration. Similar inhibition of shoot length and root length of crop plants by allelopathic extracts have been reported in Oryza sativa, Zea mays by Rhizophora apiculata (Rajangam 1984), in ground nut by bamboo (Eyini et al. 1989), in groundnut and corn by Eucalyptus (Jayakumar et al., 1990). Reduction in leaf area of the crop plants by aqueous leaf extracts have been reported in few crop species viz., by bamboo in groundnut (Eyini et al. 1989) by Eucalyptus globulus in groundnut and maize (Jayakumar et al. 1990), by Acacia holosericea in cowpea, sesame, horse gram (Palani et al. 1998). Inhibition of yield in several other crops, by aqueous leaf extracts was reported by only few researchers. Sundramoorthy and Kalra (1991) reported a reduction in yield of pearl millet, sesame and cluster bean by the aqueous leaf extracts of Acacia fortilis. Palani and Dasthagir (1998) observed a significant yield reduction in cowpea, sesame, horse gram and sorghum by aqueous leaf extracts of Acacia holosericea. These reports on the inhibitory effect of allelochemicals on the yield of crop plants corroborate our results. The inhibition of nodulation by aqueous leaf extracts of Arisdia adscensionis on rhizobium (Rice et al. 1981) by decomposed rice straw on rhizobium was reported. Reduced nodulation similar to our study occurred in white clover and red kidney bean after exposure to extracts of Bromus and Euphorbia species. Putnam and Weston (1986) explained that allelochemics were inhibitory to root hair formation which subsequently prevented infection by Rhizobia.

Post-emergence studies

In post-emergence treatment of A. leucopholea leaf aqueous extracts on ground nut and sorghum shoot length, root length, and biomass was decreased in all concentrations. No mortality was observed in both the plants in any of the concentrations. In ground nut, at 20% concentration a maximum of 10% reduction in shoot length, 6 % reduction in root length, 7% reduction in biomass was observed. But in the case of sorghum the reduction in shoot length, root length and biomass was 25%, 11% and 29% respectively. Inhibition of seed germination, reduction in shoot, root length, leaf area and yield of seedlings of crop species tested showed a linear relationship with increasing concentration of aqueous leaf extracts of A. leucopholea (Tables 3 and 4).

Table 3. Post-emergence treatment of Acacia leucopholea leaf aqueous extracts on shoot, root length and biomass weight of Arachis hypogaae and Sorghum vulgare. Values are mean ± SE of 5 samples.

Arachis hypogaea				Sorghum vulgare
Concentration (%)	Shoot length (cm)	Root length (cm)	Biomass wt. (mg)	Shoot length (cm)	Root length (cm)	Biomass wt. (mg)
Control	4.20± 0.3	14.5± 1.4	419.0 ± 40.6	6.0 ± 0.5	16.1 ±1.4	4.81 ± 0.4
5	4.20± 0.4	14.0± 1.1	414.0 ± 30.3	5.9 ± 0.4	16.1 ±1.5	4.55 ± 0.4
10	3.90± 0.3	14.0 ± 1.0	410.0 ± 30.5	5.7 ± 0.3	15.4 ±1.5	4.05 ± 0.3
15	3.90± 0.4	13.8 ± 1.2	407.5 ± 38.0	5.1 ± 0.4	15.0 ±1.0	3.97 ± 0.4
20	3.75± 0.3	13.6 ± 1.2	387.0 ± 34.5	4.5 ± 0.4	14.3 ±1.1	3.38 ± 0.2

Table 4. Effect of Acacia leucopholea leaf aqueous extracts on the yield parameters of Arachis hypogaae and Sorghum vulgare plants. Values are mean ± SE of 5 samples.

Arachis hypogaea				Sorghum vulgare
Concentration (%)	Seed wt./plant (g)	Pods/ plant (number)	Root nodules/ plant (number)	Seed wt./plant (g)	Grains/ ear (number)
Control	6.5 ± 0.5	5	58	8.6 ± 0.7	175
5	4.4 ± 0.4	4	50	3.6 ± 0.3	120
10	3.8 ± 0.3	3	41	3.3 ± 0.3	92
15	3.8 ± 0.2	3	36	1.9 ± 0.1	88
20	1.2 ± 0.1	1	22	1.6 ± 0.1	75

Rice (1984) while discussing the chemical nature and biosynthetic pathways of allelochemicals, reported that phenolic acids and tannins have greater potential fore allelopathic activity. The GC analysis of phenolic acids in A. leucopholea leaf showed the presence of phenolic acids (Figure 1). The content of the individual phenolic acids is mentioned in dry weight basis with in the brackets.

	1. Hydroquinone 2. t-cinnamic acid 3. Gestisic acid 4. Vanillic acid 5. Protocatechuic acid 6. p-coumaric acid 7. Ferulic acid
Retention time (min)

Figure 1.Gas chromatogram of the phenolic acids of Acacia leucopholea leaf

Hydroquinone (63.8 µg/g), trans-cinnamic acid (3.1 µg/g), gentisic acid (13.0 µg/g), vanillic acid (13.9 µg/g), protocatechuic acid (16.4 µg/g), p-coumaric acid (395.2 µg/g), and trans- ferulic acid (84.9 µg/g). This indicates that the allelopathic potential is determined by the interaction of several different allelochemics and not by an individual compound. The total phenolic acid content in the extracts of A. leucopholea leaves could be attributed to the defense mechanism of the plants against plant pathogens, insects and browsers (Waller 1989) and as evidenced by the release of secondary metabolites during the degradation of litter. Similar studies is agreement with our results indicate that not only the nature of phenolics but also their concentration decides the inhibitory effect (Sivagurunathan et al. 1997).

IR spectrophotometric analysis of tannins in A. leucopholea leaf showed three bands. They are weak band of alcohol and phenol, O-H stretching vibrations (2969 cm^-1). Carbonyl chromophore, ketone stretching vibrations. Saturated cyclic strong band (1711 cm^-1), amines, C-N vibrations aromatic, secondary strong band (1358 cm^-1). IR spectrum of crude tannin concentrate of the leaves of A. leucopholea showed only the functional groups of the various tannins and not their types. Further studies on fractionation and purified tannins will shed more light on their role as allelochemics.

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