ALLELOPATHIC EFFECT OF RHAZYA STRICTA PLANT RESIDUE ON RAPHANUS SATIVUS (RADISH)

This study was designed to investigate the allelopathic potential of Rhazya stricta on Raphanus sativus using laboratory bioassay and greenhouse pot experiment. In laboratory bioassay, aqueous extract of R. stricta showed inhibitory effect on R. sativus seedling growth particularly at the high concentrations. The germination percentages were not significantly affected. Results in greenhouse pot experiment showed that the residue of R. stricta showed inhibitory effect on root length, dry weight and root to shoot length ratio of R. sativus especially at the high concentrations at different ages. The Rhazya residue showed positive effects on the photosynthetic pigments of R. sativus particularly on the carotenoids and chlorophyll a/b ratio at different ages. A significant increase in nitrogen content of R. sativus including total amount of free amino acid, soluble and insoluble nitrogen and crude protein was prominent at the early growth stage especially at the high concentrations. The Rhazya residue inhibited the contents of soluble nitrogen of R. sativus in the late growth stage. The results of RAPD-DNA profiles showed significant effect of Rhazya residue on R. sativus plant from where variation in band intensity, disappearance of bands, and appearance of new PCR product.


Introduction
Plants live association groups depending upon the ecological requirements; they have generally similar structural and morphological adaptations. Whenever two or more plants occupy the same niche in nature, they compete with each other for various life support requirements (Khan et al.,2011a, p. 81). Allelopathy refers to the beneficial or harmful effects of one plant on another one, both crop and weed species, by the release of chemicals from plant parts by leaching, root exudation, volatilization, residue decomposition, and other processes in both natural and agricultural systems. In agroecosystems, allelopathic effects between living weeds and crops, crops in mixtures, plant straw residue and succeeding crops during decomposition of residue are also well documented. Allelopathy is expected to be an important mechanism in the plant invasion process because of the lack of coevolved tolerance of resistant vegetation to new chemicals produced by the invader. This phenomenon could allow the new introduced species to overlook natural plant communities (Khan et al., 2011b, p. 6392).
Rhazya stricta Decne, (Apocynaceae) is a perennial plant locally known as Harmal. It is widely distributed throughout Western Asia from Yemen to Arabia, to the North West Province of India and abundantly found in various regions of Pakistan (Baeshin et al., 2009, p. 986). R. stricta like other plants is competing with the main crops for nutrients and other resources and hamper the healthy growth of crops ultimately, reducing the yield both qualitatively and quantitatively (Mutawakil., 2012, p.11). (Al-Yahya et al.,1990 , p. 123) have reported the presences of alkaloids, glycosides, triterpenes, tannins and volatile bases in the leaves of this plant.
Raphanus sativus (radish) is a globally edible root and leaf vegetable. Radish is rich in ascorbic acid, folic acid, and potassium. It is also a good source of vitamin B6, riboflavin, magnesium, copper and calcium, Raphanus sativus contains flavonoids, saponins, tannins, glycosides, steroids and alkaloids. No previous studies reported on the tolerance of Raphanus sativus to the allelopathic effect of Rhazya plant. However, there are many studies on the effect of Rhazya stricta as tested on seed germination of some other species (Khan et al., 2011b, p. 6391). The present study was conducted to explore allelopathic potential of R. stricta on ecophysiology of R. sativus in laboratory bioassay and greenhouse pot experiment.

Material and Methods Plant materials
Plant material of Rhazya stricta was collected from its natural habitats in central Saudi Arabia. The plants were air dried, then ground into a fine powder and stored in refrigerator until used. The seeds of radish were obtained from the Agricultural Research Center, Vegetables Department, Egypt.

Preparation of Rhazya extract
Aqueous extracts of Rhazya stricta were prepared by shaking dry powdered tissue with distilled water for 24 hours at room temperature. Mixture was filtered through a suction filtration. The clear supernatant was brought to the original volume with distilled water to obtain the extract concentrations 0.05, 0.1, 0.5, 1, 2, 3, 4, 5 % (w/v). These water extracts were used in the bioassay tests.

Bioassay tests
Effects of Rhazya extracts on seed germination and seedling growth of Raphanus sativus were performed in the laboratory in covered glass Petri dish (9cm diameter) lined with one layer filter paper. In every dish10 radish seeds and 10 ml of the test extract were used. Distilled water was applied in the control treatment. The dishes were incubated in a dark growth chamber, at room temperature. Four replicates per treatment were used. Tests were terminated after 10 days. The final germination was calculated as percentage of control. The radical and plumule lengths of the seedlings were measured. The samples were dried to constant weight in an oven at 80 o C to obtain the dry weight. Root/shoot length and weight ratios were calculated.

Pot experiment
A greenhouse pot experiment was conducted to assess the possible inhibitory or stimulatory effects of Rhazya plant powder on Raphanus sativus plant. Pot experiment was carried out in plastic pots (13 cm in diameter and 14 cm in depth), each containing 2 kg of clay soil. The pots were divided into 8 groups, each was 12 pots, one was left without treatment as control and the other seven groups were treated with Rhazya residues. The fine ground shoot powder was incorporated into the upper soil layer with 2 cm depth that finally gave the percentages of 2, 4, 6, 8, 10, 12 and 16% (w/w). Ten healthy R. sativus seeds of uniform size were sown at 1 cm soil depth and the seedlings were thinned to 5 plants per pot after emergence. Plants were irrigated with tap water, and soil was kept at field capacity, along the whole experimental period, using weighing procedure. Pots were placed in an open greenhouse under natural conditions during March to April months. The plants, at the vegetative stage, were harvested after 30 days from sowing, then washed thoroughly with tap water and divided into root and shoot systems for measurement of growth criteria. Lengths of the main root and shoot, and their root/shoot length ratio were calculated. The samples were oven dried to a constant weight at 80 o C for dry weight measurements.

Extraction and Determination of Nitrogen
Total nitrogen was determined in plant powder after the acid digestion with 1 ml 50% H 2 SO 4 and 1 ml 30% perchloric acid, using Bertholet reaction (Chaney and Marbach,. 1962, p 130). Soluble nitrogen were extracted from the dried Raphanus sativus shoot tissue with 10% trichloroacetic acid (TCA) and the remaining dried residue was acid digested to obtain the insoluble components. Total amount of free amino acids was estimated in the TCA extract as amino-N (Russell, 1944). Multiplying the total organic nitrogen by 6.25 estimated the crude protein (AOAC., Detection of DNA polymorphism using RAPD technique DNA extraction DNA was isolated from 50 mg of plant material using Qiagen Kit for DNA extraction by a modified CTAB method (Doyle & Doyle., 1990, p. 13). The extracted DNA was dissolved in 100 µl of elution buffer. The concentration and purity of the obtained DNA was determined by using "Gen qunta" system-pharnacia Bio-teck. The purity of the DNA for all samples was between 90-97%. Concentration was adjusted at 6 mg/µl for all samples using TE buffer PH 8.

RAPD analysis
A total of 10 (10 mer) oligonucleotide random primers were used for RAPD analysis (Table1). DNA amplification reactions were performed in 25 µl reaction mixture consisting of 1 unit of Tag DNA polymerase, 0.2 mM dNTP, 1x PCR buffer, 3 mM MgCl 2, 10 Pmol of each primer and approximately 50 ng of the extracted genomic DNA. Amplification reactions were carried out using PCR unit II Biometra with the following thermal profile: 1 cycle of 95 ºC for 5 min (initial denaturation), followed by 45 cycles of amplification with denaturation at 95 ºC for 1 min, annealing at 36 ºC for 1 min and extension at 72 ºC for 2 min. The final extension was carried out at 72 ºC for 5 min. The amplified DNA were separated using electrophoresis unit (WIDE mini-subcell GT Bio-RAD) on 1% agarose containing ethidium bromide (0.5 µg/ml) at constant volt and determined with UV transilluminator.

Statistical analysis
The data obtained were analyzed with (SPSS) one-way ANOVA.

Effect of plant extracts on germination and seedling growth
The effects of Rhazya extract concentrations on the germination of R. sativus, calculated as a percentage of their controls, are shown in (Table2). Generally no significant differences in the germination percentages occurred at low concentrations of Rhazya extract, but at 5% concentration a significant reduction was observed. Growth of R. sativus seedling treated with Rhazya aqueous extract, during the germination period, are shown in Table (2). The treatments at low concentrations of Rhazya extract increased the length of R. sativus radicle and plumule over the control, while the high concentrations produced significant growth reduction. The highest radicle and plumule lengths inhibition reached 0.21cm and 3.36 cm at concentrations 5% and 4%, respectively. Alternatively, the dry weight of radicle and plumule did not show significant difference except at 4% and 5% extract concentration which showed significant decrease in dry weight of radicle of treated plants. Concerning root/shoot length and weight ratios, the results suggest that stimulatory and inhibitory effects of the plant extract of concentration ( Figures 1&2). The low concentrations of Rhazya extract increased root/shoot length ratio at concentrations up to 0.1% then decreased at higher concentrations ( Figure 1). Reduced root/shoot weight ratio of R. sativus was significant above concentration 0.5% of Rhazya extract (Figure2). .

Effects on plant growth
In the pot experiment, the growth response of R. sativus at different concentrations as affected by Rhazya residue is shown in Tables (3-1&2). At 30 days old R. sativus shoot length decreased with the decrease of Rhazya residue compared to control, whereas at 60 days old, shoot length generally showed no constant trend with the increase of Rhazya residue concentration. At age of 30days, the increase of Rhazya residue caused a significant decline in R. sativus root length at high concentration, while at 60 days old a significant decline was observed in all Rhazya residue concentrations. The 4 grams concentration of the residue caused the highest root length. Reduced root/shoot length ratio of R. sativus at ages of 30 and 60 days was significant in all Rhazya residue concentrations. The highest root/shoot length ratio inhibition at age of 30 days reached 51.40% at residue concentration of 16 grams, while at age of 60 days, the root/shoot length ratio reached 71.7% at residue concentration of 10 grams. The dry weight of R. sativus at age of 30 days did not show any positive or negative variation from the control treatment except at 10-12 and 16 grams residue concentration which showed a significant decrease in dry weight of treated plant. At age of 60days, the increased Rhazya residue caused a significant decline in R. sativus dry weight particularly at 12 and 16 grams residue concentrations.

Effects on photosynthetic pigments content
Changes in the various photosynthetic pigments in the shoot system of R. sativus for 30 and 60 days with different rates of Rhazya plant residue are shown in Figure (3). At age 30 and 60 days, Chlorophyll a doesn't show any significant difference except at 16 grams residue concentrations which shows significant decrease in chlorophyll a content after 30 days, while at age 60 days, chlorophyll a content increased by 12 grams residue concentration ( Figure 3-a). Significant reduction in chlorophyll b content of R. sativus was detected by the effect of 10 -12 and 16 grams residue after 30 day and at 60 days age, the chlorophyll b content increased by 6 grams residue concentration, while at 4 and 8 grams residue concentration, the chlorophyll b content of R. sativus significantly decreased compared with the control (Figure  3-b).
Concerning chlorophyll a/b ratio of R. sativus at 30 and 60days age generally increased with increasing the concentrations of Rhazya residue (Figure 3-c). At 30 days age carotenoid of R. sativus showed a significant increase with increasing residue concentration, while at 60 days age an increase in carotenoid content was is highly marked at both 4 and 10 grams residue concentration (Figure 3-d).

Effects on nitrogen content
The change in the nitrogenous components of 30 and 60-daysold R. sativus in response to different Rhazya treatments are shown in Figure (4). At 30-days old, R. sativus soluble nitrogen decreased by 4 grams, while at higher Rhazya residue, the soluble nitrogen of R. sativus significantly increased to (o.60mg/g dry weight) compared with control treatment. In addition after 60 days the plant showed significant reduction in soluble nitrogen which is highly marked at 10 grams Rhazya residue concentration ( Figure  4-a).
With respect to insoluble nitrogen 30 days age R. sativus plant generally increased with increasing the concentrations of Rhazya residue. After 60 days age, plants showed significant increase in insoluble nitrogen except at 16 grams residue concentration which showed a marked decrease in insoluble nitrogen of the treated plant as shown in (Figure 4-b). In contrast, R. sativus at age of 30 days showed a decrease in free amino acid at lower residue concentration while a significant increase was remarked with increasing residue concentration. After 60 days, plants did not show any significant difference except at 6 and 10 grams residue concentration which attained significant increase in free amino acid (Figure 4-c).
Crude protein of R. sativus at 30 days age increased with the increase of Rhazya residue. In addition at 60 days aged plant, crude protein showed significant increase that is highly marked at both 2 and 4 grams residue concentration, while 16 grams residue concentration showed significant decrease in crude protein ( Rhazya residue and also by primer 9 one new bands was indicated at concentration 6 grams Rhazya residue ( Figure 5). The increase in bands intensity were obvious at concentrations 6, 10 and 12 grams Rhazya residue for primer 1, 2, 6, 7, 9 and 10. In contrast, a decrease in bands intensity occurred in all concentrations but, were particularly obvious at concentration 8 grams Rhazya residue for primer 2, 3, 5 and 9 ( Figure 5).

Discussion
The results of the present study showed that the aqueous extracts of Rhazya stricta differed in their effects on seedling and adult plant growth, photosynthetic pigments, nitrogen content and RAPD-DNA profiles of Radish (Raphanus sativus) plant. The Rhazya extract was not significantly affecting on germination percentage of R. sativus. The results showed that allelochemicals in the extract of Rhazya could have harmless effect on seed germination of R. sativus. This result agrees with the earlier study of (Moosavi et al,. 2011,p 115) who demonstrated that allelopathic effect of different concentrations of water extract of sorghum was not significant for germination percentage of Vigna radiata L.
The extracts of Rhazya stimulated significantly the lengths, weight and root/shoot length and weight ratios of R. sativus particularly at the low concentrations, whereas at high concentrations produced inhibitory effect. This indicated that allelochemicals in the extract of Rhazya may have stimulating effect on seedling growth of R. sativus. On the other hand, the inhibition was correlated to the concentration of the inhibitory Similarly, (Mutlu & Atici,. 2009, p 90) demonstrated, both root and leaf extracts significantly increased the seedling growth of wheat, especially at the lower concentrations. The biological activity of the identified allelochemicals from Vulpia myuros toward test plant was stimulatory at low concentrations, and inhibitory at high concentrations (An et al,. 2001, p 383).
The effect on growth suggests that leaves and stem of Rhazya can act as a source of allelochemicals after decomposition that inturn negatively affects the neighboring or successional plants. The observed phytotoxicity difference of Rhazya may be attributed to the presence of variable amount of phytotoxic substances in different parts that leach out under natural conditions. Some modern investigations indicating the allelopathic/ phytotoxic determine of aqueous extracts of weeds contain include Raphanus raphanistrum (Norsworthy,. 2003, p 307), Andrographis paniculata (Alagesaboopathi,. 2011, p 147). These studies strongly showed the release of phototoxic chemicals during the preparation of aqueous extracts.
In pot experiment, incorporation of Rhazya residue into the soil at the low concentrations, the dry weight of R. sativus was stimulated particularly at early stages of growth. However, the inhibition of root to shoot length ratio and dry weight was more pronounced at late age stages than at the early stage. Conversely, increasing the rate of Rhazya residue caused an inhibition in growth of radish at different ages. The inhibition of cell elongation may be related to the direct action of allelochemicals by interfering with cell division either directly or through interaction with hormones. These results are in accordance with (Al-Wakeel et al,. 2007, p 413) who demonstrated the stimulation in root and shoot lengths of 45-day-old pea irrigated with Acacia nilotica leaf extract, while the higher concentration were inhibitory. On the contrary with (Abu-Romman,. 2011, p 948) showed that the growth of pepper was significantly inhibited with increasing of Achilliea leachate concentration.
The residue of Rhazya showed both inhibitory and stimulatory effects on photosynthetic pigments of R. sativus at different ages.

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Chlorophylls are the core component of pigment protein complexes embedded in the photosynthetic membranes and play a major role in the photosynthesis. Any changes in chlorophyll content are expected to bring about change in photosynthesis (Reigosa,. 2006, p 315). The inhibition in Chl a and Chl b were previously reported as a result of allelochemical stress (Singh et al,. 2009, p 163) or may be due to the inhibition of chlorophyll biosynthesis or stimulation of chlorophyll degradation or both processes (Yang et al,. 2002, p 303). Moreover, (Siddiqui,. 2007, p 306) reported a reduction in chlorophyll content of Vigna mungo due to the allelochemicals present in leachates of black pepper which possibly target enzymes responsible for the conversion of porphyrin precursors.
Based on the results, a significant decrease in nitrogen content of R. sativus including total amount of free amino acid, soluble and insoluble nitrogen and crude protein were related to the age. This could be due to the higher levels of Rhazya allelochemicals, which have harmful effect on nitrogen metabolism (Reigosa,. 2006, p 320). According to the allelopathy definition, it is so evident that allelochemicals could affect all phases of nitrogen cycle involved in plant or microorganisms. When plants take up nitrate, they must use energy to convert it to ammonium form before it can be used (Reigosa,. 2006, p 321). The growth reduction due to missing energy could be an argument for nitrogen reduction in seedlings which treated by allelochemicals, also loosing of nitrogen content in some seedling, may be occurred by limiting or reducing some key factors in nitrogen metabolism such as nitrate reductase and glutamine synthetase (Nie,. 2005 Understanding the mechanisms by which higher plants perceive environmental stimuli is of vital importance to modern molecular biology. In practice, the key point to agriculture is how to regulate the harmonious relationship between soil-environment and crops and make the best of physiological potential of crops (Gang et al,. 2007, p 117). To some extent, plants could overcome environmental stress by developing efficient and specific physiobiochemical mechanisms (Sandalio et al,. 2001, p 2122). In ''genetic-ecotoxicology'' or ''eco-genotoxicology'', the effective evaluation and proper environmental monitoring of potentially genotoxic pollutions will be improved with development of sensitive and selective methods to detect toxicant-induced alterations in the genomes of a wide range of biota (Liu et al ,. 2007(Liu et al ,. , p 1160. As reported by many researchers (Atienzar and Jha,. 2006, p 98) the alteration in DNA fingerprinting is a useful biomarker in eco-genotoxicology.
In the present study, DNA damage induced by Rhazya residue treated R. sativus seedling was reflected by changes in RAPD profiles: variation in band intensity, disappearance of bands, and appearance of new PCR products occurred in the profiles. These results indicated that genomic stability in R. sativus seedling was significantly affected by Rhazya residue stress. Modifications of band intensity and lost bands are likely to be due to one or a combination of the events (changes in oligonucleotide priming sites due to genomic rearrangements and less likely to point mutation and DNA damage in the primer binding sites, interactions of DNA polymerase in R. sativus seedling with damaged DNA). The disappearance of normal band (band loss) may be related to the events such as DNA damage (e.g. single-and double-strand breaks, modified bases, abasic sites, oxidized bases, bulky adducts, DNA protein cross links), point mutation and/or complex chromosomal rearrangements induced by genotoxins (Atienzar et al,. 2002, p 160). The new bands could be attributed to mutations while the disappeared bands could be attributed to DNA damage (Atienzar & Jha,, 2006, p 99).

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The allelopathic activity of Rhazya stricta is depending on the amount and type of allelochemicals released from the decomposed shoot, as well as the uptake of these compounds by plant roots of the target species