Effects of Persian walnut leaf extracts on seed germination, seedling ...

Journal of Horticultural Science & Biotechnology (2013) 88 (4) 433–438

Effects of Persian walnut leaf extracts on seed germination, seedling growth, and some physiological characteristics of the basil (Ocimum basilicum L.) cultivar ‘Genovese’ By MONAD DADI*, DAVOOD BAKHSHI, GHOLAMALI PEYVAST and ZAHRA BALOUCHI Department of Horticultural Science, Faculty of Agriculture, University of Guilan, Rasht 41376, Rasht-Qazvin Road, Iran (e-mail: [email protected]) (Accepted 21 March 2013) SUMMARY This study was conducted to elucidate the allelopathic effects of a series of dilutions of Persian walnut (Juglans regia) leaf extracts [i.e., undiluted, 1:2 (v/v), 1:4 (v/v), or 1:8 (v/v) dilutions] and distilled water (control) on the characteristics of seed germination, seedling growth, and subsequent physiological changes in the basil (Ocimum basilicum L.) cultivar, ‘Genovese’. The results indicated that all aqueous leaf extracts of walnut had statistically significant inhibitory effects on the germination of basil seeds and on seedling growth characteristics. Seed germination percentages, the lengths and fresh weights (FWs) of the plumule and the radicle, the FWs and dry weights (DWs) of the shoots and roots, and leaf areas decreased significantly with increasing concentrations of aqueous walnut leaf extract. The DWs of the plumules and radicles were increased by the undiluted walnut leaf extract. Moreover, it was found that the yields of essential oils and total phenolics, and the anti-oxidant activities of basil seedlings increased with increasing concentrations of aqueous walnut leaf extract, whereas the relative water contents of basil leaves, leaf water potentials, as well as the total chlorophyll and carotenoid contents of basil leaves decreased significantly. The highest protein contents and peroxidase (POD) activities were achieved using undiluted or 1:2 (v/v) diluted aqueous walnut leaf extracts, respectively. The results of this study suggest that cultivation of the basil cultivar, ‘Genovese’ may be appropriate for walnut tree-based intercropping systems, based on the increases in anti-oxidant capacity and the accumulation of secondary metabolites.

S

ome plants can inhibit seed germination and growth in other plants by producing toxic allelochemicals (or allelopathins). The chemical interactions that occur between living organisms including plants, insects, and microorganisms are referred to as allelopathy (Rizvi and Rizvi, 1992), The organic compounds involved in allelopathy are collectively called allelochemicals (Einhellig, 1986). Such compounds are released by volatilisation, by leaching from leaves, stems, buds, flowers, fruit or seed, by exudation from roots, and by the degradation of dead plant tissues. All plant parts have been shown to contain allelochemicals, but leaves and roots are the most abundant sources (Rizvi and Rizvi, 1992). One prominent example of allelopathy occurs in walnut (Juglans regia). The main chemical responsible for allelopathy in walnut is juglone (5-hydroxy-1,4 naphthoquinone). Juglone has been isolated from many species in the walnut family (the Juglandaceae) including J. nigra and J. regia. A colourless, non-toxic, reduced form called hydrojuglone is abundant (e.g., at 750 – 1,000 µg ml–1), especially in the leaves, fruit hulls, and roots of walnut trees. When exposed to oxidising agents (air), hydrojuglone is oxidised to its toxic form, juglone. Rain then washes the juglone from the leaves to the soil. Thus, neighbouring plants can be negatively affected by *Author for correspondence.

absorbing juglone through their roots (Segura-Aguilar et al., 1992). The allelopathic effects of juglone on plants are generally toxic, but may be beneficial in some species. In previous studies, seedling growth in tomato, cucumber, garden cress, and alfalfa was strongly inhibited by juglone or walnut leaf extracts (Terzi and Kocaçalis¸kan, 2010); however, muskmelon seedling growth increased following juglone treatment (Kocaçaliflkan and Terzi, 2001). Basil (Ocimum basilicum L.) is an economically important crop that is grown in several Mediterranean countries. It is a popular culinary, medicinal, and ornamental herb. Basil is marketed fresh or dry, and is also cultivated on an agro-industrial scale for extraction of its essential oils. Basil oil is rich in phenolic compounds (Simon et al., 1990) and is used in the pharmaceutical and perfume industries throughout the World. Other members of the family Lamiaceae are also valuable due to their pharmaceutical properties. For example, the volatile oils produced in their leaves are used as anti-oxidants (Harsh et al., 2002). Recent interest in “Mediterranean cuisine” has led to a significant increase in the consumption of basil, world-wide. The concentrations of secondary products in some medicinal and aromatic plants such as basil depend on the growing conditions. Abiotic stresses have a strong positive impact on secondary metabolic pathways,

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Effects of Persian walnut leaf extracts on basil seed

including those responsible for the accumulation of natural products such as essential oils (Selmar, 2008). Intercropping with walnut is a commonly-used cultivation system in several countries. Its high value, aesthetic qualities, capacity for nut production, rapid growth potential, and adaptability to management, make walnut suitable to be intercropped (Thevathasan et al., 1999). However, the agronomic yields of some sensitive species can be reduced significantly by the deleterious effects of juglone (Terzi and Kocaçalis¸kan, 2010). There is little or no reliable information on the physiological effects of walnut leaf extracts or juglone during seed germination and seedling growth in medicinal or aromatic plants. Therefore, the aim of this work was to evaluate whether basil was a susceptible or resistant species for intercropping with walnut, and its physiological responses to juglone.

MATERIALS AND METHODS Two studies were undertaken. In the first, several variables of basil seed germination were investigated in response to different dilutions of Persian walnut leaf extract [i.e., undiluted, 1:2 (v/v), 1:4 (v/v), or 1:8 (v/v)]. In the second study, aqueous extracts of walnut leaves were applied to the growing media used for basil seedlings, to evaluate the physiological effects of walnut leaf extracts. Preparation of aqueous walnut leaf extracts Studies were carried out using Persian walnut leaves (J. regia) grown in Iran. Fresh leaves (20 kg in total) were collected from 20 trees at the beginning of September, towards the end of the vegetative growth period (Cosmulescu et al., 2011) from an orchard in Shiraz, in the South of Iran. Trees were planted at a spacing of 7 m 7 m (204 trees ha–1) and all trees were 20 years old. Walnut trees younger than 7 years do not contain sufficient juglone to cause toxic allelopathic effects (Ercisli et al., 2005). Leaves (approx. 1 kg per tree) were collected from the middle-third of those branches exposed to sunlight. Walnut leaf extracts were prepared from leaves by drying them at 70°C in an oven for 48 h. The dried leaves were then powdered and 10 g was homogenised in 100 ml distilled water in a Waring blender and filtered through a Whatman No.1 filter paper. The filtrate was centrifuged at 850 g and the supernatant was decanted. Different dilutions were prepared by diluting this extract 1:8 (v/v), 1:4 (v/v), or 1:2 (v/v). Distilled water was used as a control treatment. Effect of aqueous walnut leaf extracts on basil seed germination characteristics Seeds were surface-sterilised with 1% (v/v) sodium hypochlorite for 15 min at 24°C, followed by a sterile water rinses. At least 20 seeds were placed in each 9-cm Petri dish on two sheets of filter paper moistened with the different dilutions of aqueous walnut leaf extract, undiluted extract, or water (control). The Petri dishes were then placed in a growth chamber at 22° ± 1°C and 70 ± 5% relative humidity (RH). Each treatment consisted of three Petri dishes. After 7 d, germination percentages, the lengths of the radicles and plumules, and the fresh weights (FWs) and dry weights (DWs) of the radicles and plumules were measured.

Effect of aqueous walnut leaf extracts on the morphological features of basil seedlings The experiment was conducted in a greenhouse at the Agricultural Faculty, University of Guilan, Rasht, Iran (37°16 N) in 2010. Seeds of basil (O. basilicum L.) ‘Genovese’ were sown in plastic pots (25 cm in diameter) filled with a 5:2:2:1(v/v/v/v) mix of peat moss:composted bark:pomix:clay with a pH of 6.5 that had been steam sterilised at 100°C for 30 min. Pomix is a highly porous, peat-based growing medium that is ideal for watersensitive crops, rooting cuttings, or low-light growing conditions. After germination, the seedlings were thinned to three plants per pot. Conditions within the greenhouse were as follows: day/night temperatures of 27°C/20°C (± 2°C) and 80%/75% (± 5%) RH with a 16 h photoperiod at a light intensity of approx. 150 µmol photons m–2 s–1 provided by cool-white fluorescent lights, controlled automatically. Prior to each treatment, the field capacity (FC) of the medium in the pot was determined. Three pots were saturated with water, covered with plastic to avoid evaporation and allowed to drain for 24 h. Soil samples from each pot were then taken to determine their moisture content. An average moisture content was calculated according to Kramer and Boyer (1995). When the plants reached the three-to-four true-leaf stage, 150 ml of walnut leaf extract (estimated according to the FC and soil water content) was applied directly by hand onto the substrate, twice a month, using a graduated glass beaker to avoid direct contact with the foliage. This was continued until the full-flowering stage. Plants were harvested, and selected morphological and physiological characteristics of each seedling were determined. Effect of aqueous walnut leaf extracts on basil leaf water relations Leaf tissue is most commonly used to determine relative water contents (RWC). RWC was analysed at full-flowering in three leaf samples from the third node from the top of the shoot (three replications per treatment). Leaf discs (n = 3; diameter = 12.7 mm) were cut from each leaf. After determining their FW, the discs were immersed in distilled water for 6 h to estimate their turgid weight (TW), then the discs were dried at 70°C for 24 h to measure their DW. RWC was calculated using the following equation (Reigosa and Oonzález, 2001): RWC (%) = [(FW - DW)/(TW - DW)] 100 The water potential of plants was determined using leaves at the second and third nodes from the top of the shoots. The measurements were carried out at the fullflowering stage using a pressure chamber (Model 600; PMS Instruments, Corvallis, OR, USA) with three replications per treatment. CO2 was used as the gas for the chamber (Turner, 1988). Determination of juglone concentrations in walnut leaf extracts by HPLC Juglone was measured using high-performance liquid chromatography (HPLC), as described by Girzu et al. (1998) with three replications of each extract. Each leaf extract was filtered through a 0.45-µm syringe filter, then 50 µl was injected into the HPLC system (Waters,

M. DADI, D. BAKHSHI, G. PEYVAST and Z. BALOUCHI Milford, CT, USA) equipped with a UV-visible detector (Waters Dual-Absorbance 2487), a column (Waters Symmetry C18; 5 µm pore size; 4.6 mm 150 mm; Waters, Dublin, Ireland). The mobile phase was a mixture of solvent A [95 : 5 (v/v) water : acetic acid] and solvent B [90 : 10 (v/v) acetonitrile : water]. Samples and the column were held at 25°C, the eluent flow was at 1.0 ml min–1, and the run time was 40 min. A linear gradient was programmed as follows: t0–10 min: 80% A plus 20% B; t10–20 min: 60% A plus 40% B; t20–40 min: 20% A plus 80% B (isocratic). Juglone was identified by comparing its retention time with that of a corresponding standard and by spiking samples with the standard. Its concentration was calculated from the absorbance at 420 nm using its molar extinction coefficient ( = 3,811 M–1 cm–1). Juglone concentrations were expressed in mg 100 g–1 FW of leaf. The juglone standard was purchased from Sigma-Aldrich (Oakville, ON, Canada).

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determine statistical differences between treatments, variance analysis, and least significant difference (LSD) tests were performed (P ≤ 0.01). RESULTS AND DISCUSSION Juglone concentrations Juglone is a well-known component of walnut and occurs at high concentrations in all green and growing parts of walnut trees. The juglone concentration in fresh leaves was quantified by HPLC. Based on its spectral characteristics, the average juglone concentration was 8.63 mg 100 g–1 FW. This was in agreement with Cosmulescu et al. (2011), who reported 5.42 – 22.82 mg juglone 100 g–1 FW in leaves from different walnut cultivars.

Evaluation of the physiological characteristics of basil plants To measure essential oil concentrations, fresh basil plants were collected at the full-flowering stage. The yield of essential oil obtained was expressed as a percentage of the absolute DW of herbage (Khalid, 2006). The concentrations of chlorophyll a, chlorophyll b, total chlorophyll, and total carotenoids were determined by UV-visible spectrophotometry (PG Instruments Ltd., Leicester, UK), as described by Wellburn (1994). Peroxidase (POD) activity was assayed by measuring the formation of guaiacol, spectro-photometrically, according to In et al. (2007). The increase in absorbance at 470 nm was recorded for 2 min following the addition of H2O2. POD activity was expressed in µmol g–1 FW min–1. The total protein contents of plants were estimated according to Bradford (1976). Total phenolics contents were measured using the Folin-Ciocalteau method, as described by Singleton et al. (1999) and were expressed in mg gallic acid equivalents (mg GAE) 100 g–1 FW of herbage. Anti-oxidant capacities were determined by measuring the scavenging of 2, 2-diphenyl-2picrylhydrazyl hydrate (DPPH) radicals, according to Brand-Williams et al. (1995).

Basil seed germination and the lengths, FWs and DWs of radicles and plumules Basil seed germination was significantly inhibited by all walnut leaf extracts (Table I). Juglone significantly decreased the germination percentages, from 95% in the water control to 67.5% in the undiluted leaf extract. The lengths and FWs of plumules and radicles were affected by all walnut leaf extracts. The maximum plumule and radicle lengths were obtained in the water controls, while the minimum lengths were observed using the undiluted walnut leaf extract. All aqueous extracts decreased the FWs of plumules and radicles. However, plumule DWs were enhanced by the undiluted leaf extract. A significant difference was also found between the undiluted leaf extract and all other treatments. Radicle DWs increased significantly from 16 mg per seedling in the controls to 20 mg with the undiluted leaf extract. These results agree with those of Kocaçalis¸kan and Terzi (2001), who reported that the germination of cucumber seed was inhibited by juglone or walnut leaf extracts, whereas Terzi et al. (2004) found that juglone had no significant effect on seed germination in muskmelon. In a previous study, Segura-Aguilar et al. (1992) observed that juglone caused oxidative stress during seed germination. During seed germination, the most important event for embryo growth is protein synthesis. Juglone blocks protein synthesis at the transcription stage by inhibiting RNA polymerases (Chao et al., 2001).

Statistical analysis Both experiments were carried out according to a completely randomised design. For the seed germination experiment, each treatment contained four replications of each walnut leaf extract preparation. For physiological analysis, there were three replications of each treatment. Before analysis of variance (ANOVA), data were tested for normality and homoscedasticity using the Kolmogorov-Smirnov and Cochran tests, respectively. To

Changes in the morphological characteristics of basil seedlings The FWs and DWs of basil shoots and roots decreased following treatment with all walnut leaf extracts. Also, the leaf areas of seedlings decreased markedly following all walnut leaf extract treatments (Table II). Kocaçalis¸kan and Terzi (2001) demonstrated that both juglone and walnut leaf extracts inhibited seed germination and seedling growth in several plant species

TABLE I Influence of dilutions of Persian walnut leaf extract on the mean values of selected seed germination characteristics in basil (Ocimum basilicum L. ‘Genovese’) Proportion of leaf extract (v/v) 0 1:8 1:4 1:2 Undiluted †

Percentage germination

Plumule length (mm)

Radicle length (mm)

Plumule fresh weight (g)

95.0 a† 82.5 b 77.5 bc 70.0 c 67.5 c

32.69 a 31.84 a 25.16 b 14.19 c 7.70 d

49.32 a 44.01 b 31.07 c 14.71 d 3.83 e

0.240 a 0.023 b 0.097 c 0.022 c 0.019 c

Radicle fresh weight (g) 0.261 a 0.229 ab 0.201 b 0.125 c 0.084 c

Plumule dry weight (g)

Radicle dry weight (g)

0.0022 b 0.0022 b 0.0026 b 0.0022 b 0.0052 a

0.016 b 0.016 b 0.017 ab 0.017 ab 0.020 a

Mean values (n = 4) in each column followed by the same lower-case letters are not significantly different at P ≤ 0.01 according to the LSD test.

Effects of Persian walnut leaf extracts on basil seed

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TABLE II Influence of dilutions of Persian walnut leaf extract on the mean values of selected seedling growth characteristics in basil (Ocimum basilicum L. ‘Genovese’) Proportion of leaf extract (v/v) 0 1:8 1:4 1:2 Undiluted †

Shoot fresh weight (g) 3.76 a† 3.73 a 2.32 ab 1.79 b 1.76 b

Shoot dry weight (g)

Root fresh weight (g)

Root dry weight (g)

Leaf area (cm2)

Leaf RWC (%)

Leaf water potential (MPa)

2.18 a 2.08 ab 1.43 bc 1.143 c 0.76 c

0.26 a 0.18 b 0.13 bc 0.13 bc 0.08 c

14.02 a 9.72 b 9.22 bc 7.04 dc 6.59 d

87.20 a 72.14 b 55.56 c 42.48 cd 36.73 d

–0.43 a –0.58 b –0.64 b –0.70 bc –0.93 d

0.60 a 0.54 ab 0.27 bc 0.22 c 0.17 c


such as watermelon, tomato, garden cress, and alfalfa. In later studies, similar results were reported with walnut leaf extracts on strawberry (Ercisli et al., 2005), although seedling growth in muskmelon increased significantly following treatment with walnut leaf extracts (Kocaçalis¸kan and Terzi, 2001). Juglone inhibited plant growth by reducing the rates of photosynthesis and respiration (Jose and Gillespie, 1998) and increasing oxidative stress (Segura-Aguilar et al., 1992). In addition, in cucumber, it was shown that juglone decreased seedling growth by stimulating the synthesis of abscisic acid (ABA), a growth-inhibiting hormone, or by preventing the synthesis of a growth-promoting hormone such as gibberellic acid (GA; Terzi, 2008). Effects of aqueous walnut leaf extracts on leaf water relations in basil Table III shows the influence of walnut leaf extracts on basil leaf water relations. Leaf RWC values and water potentials decreased significantly with increasing concentrations of walnut leaf extract. The minimum values were found using undiluted walnut leaf extract. Juglone-mediated reductions in growth arose from the decreased ability of roots to translocate water, by the inhibition of plasma membrane H+-ATPase activity, and by causing drought stress (Hejl and Koster, 2004; ElHadrami et al., 2005). A number of studies have reported allelochemicalinduced disruptions of plant water balance. Some have suggested that this is due to interference with normal membrane function and the disruption of active transport (Glass and Dunlap, 1974; Barkosky et al., 1999; or 2000). Barkosky et al. (1999) reported that disruptions in water relations appeared to be the primary mode of action that led to an overall reduction in growth in leafy spurge (Euphorbia esula L.) in the presence of the phytotoxin, hydroquinone. Changes in the physiological characteristics of basil plants Essential oil yields: As shown in Figure 1, essential oil yields increased significantly (P ≤ 0.01) with an increase in walnut leaf extract concentration. As mentioned above, Persian walnut leaf extracts reduced leaf water

potentials and caused water stress in plants. Drought stress increased essential oils yields in other medicinal and aromatic plants as these compounds can prevent oxidation in plant cells (Aliabadi et al., 2008). In addition, the stimulation of essential oil production under water stress could be due to the fact that plants produce high concentrations of terpenes under environmental stress conditions because of the reduced allocation of carbon to growth, suggesting a trade-off between plant growth and defence (Turtola et al., 2003). Pigment contents: Total chlorophyll and chlorophyll a concentrations decreased significantly following undiluted walnut leaf extract treatment; while the maximum level of chlorophyll b was obtained in the 1:2 (v/v) leaf extract treatment (Table III). Total carotenoid contents decreased gradually with increasing concentrations of walnut leaf extract. The maximum and minimum carotenoid contents were found in the water control and undiluted leaf extracts, respectively (Table III). These results agree with Hejl et al. (1993), who reported that juglone inhibited plant growth by reducing the rate of photosynthesis and chlorophyll contents in Lemma minor. Bioassays indicated that the inhibitory effect of walnut leaf extracts was proportional to its concentration. The maximum concentration had the strongest inhibitory effect, whereas in some cases lower concentrations showed no, or only slight effects. Einhellig (1986) showed that juglone acted directly on the photosynthetic pathway and not by indirect effects such as stomatal interference or affecting water relations. POD activity: To verify whether the allelochemical stress caused by walnut leaf extracts induced oxidative stress in basil leaves, we measured the activity of the key antioxidant enzyme, POD, in basil. The results showed that POD activity was significantly increased (P ≤ 0.01) in the 1:2 (v/v) extract treatment (Table III). Increased POD activity may contribute to the protection of basil from oxidative stress. Similar increases in POD activity have been reported in soybean roots treated with juglone. Bohm et al. (2006) showed that, at low juglone concentrations (≤ 1.0 µM), POD activity increased, while

TABLE III Influence of dilutions of Persian walnut leaf extract on the mean values of some physiological characteristics of basil (Ocimum basilicum L. ‘Genovese’) Proportion of leaf extract (v/v) 0 1:8 1:4 1:2 Undiluted †

Chlorophyll (mg g–1 FW) a 0.88 a† 0.85 ab 0.76 bc 0.68 c 0.65 c

b 0.51 b 0.49 b 0.54 ab 0.58 a 0.49 b

Total 1.39 a 1.34 a 1.30 ab 1.26 b 1.14 c

POD activity (µmol g–1 FW min–1) 0.015 c 0.016 bc 0.022 b 0.034 a 0.022 b

Total protein (mg g–1 FW)

Total carotenoids (mg 100 g–1 FW)

40.22 b 41.22 b 39.22 b 40.88 b 53.60 a

23.63 a 21.41 ab 17.39 ab 16.90 ab 13.67 b


0.12

b

0.10

c

0.08

d

d

0.04 0.02 0.00

0

1:8

1:4

1:2

Proportion of walnut leaf extract (v/v)

100 80

at high concentrations (≥ 10 µM) it was inhibited. POD activity tended to be higher in juglone-tolerant plant species, enabling the plants to protect themselves against oxidative stress, whereas such activity was not observed in juglone-sensitive plants (Scalet et al., 1995). Peroxidases are widely distributed in all higher plants. One of their main functions is as part of the defence complex in cells, ensuring detoxification of reactive oxygen species (ROS). This function is important during plant metabolic responses to different stress factors. Peroxidases protect cells against the harmful effects of peroxides and hydroperoxides (Castillo, 1992). Since POD activity was enhanced following treatment with the 1:2 (v/v) walnut leaf extract, and decreased at the maximum extract concentration, it is possible that this ROS-scavenging system was impaired by the undiluted extract. However, the capacity of the cells to scavenge ROS may be exceeded by the undiluted extract and they subsequently decrease POD activity (Bohm et al., 2006). Proteins: As shown in Table III, total leaf protein contents were significantly increased by the undiluted walnut leaf extract. An increase in protein contents by juglone treatment was indicated in a previous study (Terzi et al., 2004). Increases in protein contents may include increases in some enzyme proteins. In a previous study, phenol oxidases increased significantly following juglone treatment (Kocaçalis¸kan et al., 2008). These activities may be increased by the oxidative defence mechanisms induced by allelochemical (juglone) stress (Segura-Aguilar et al., 1992). Total phenolics: Changes in total phenolics contents are shown in Figure 2. Total phenolics concentrations increased slightly with increasing walnut leaf extract concentration, and the maximum content of phenolic compounds was observed following the undiluted walnut leaf extract treatment. Phenolic compounds are important for the prevention of stress-induced oxidative damage as they play important roles in removal of toxic substances (Di Carlo et al., 1999). Coder (2011) reported that phenolic compounds can act as anti-oxidants in walnut. . . The production of ROS such as H2O2, O2–, and OH due to non-host specific toxins such as juglone is responsible for tissue damage in many plant species (Daub and Ehrenshaft, 2000). The oxidative burst is an early event in

b

ab

ab

100 80

40 20

140 120

b

60

Undiluted

FIG. 1 Changes in the essential oil yields of basil (Ocimum basilicum L. ‘Genovese’) plants treated with different concentrations of Persian walnut leaf extract. Columns with the same lower-case letter were not significantly different at P ≤ 0.01 according to the LSD test. Vertical bars indicate ± standard errors (n = 3).

a

r = 0.78 **

a b

b

0

1:8

a

a

60 40 20

Total phenolic compounds (mg GAE 100 g–1 FW)

Total phenolic compounds

a

0.14

0.06

437

Anti-oxidant activity

0.16

Anti-oxidant activity

Essential oil yields [% (v/w)]

M. DADI, D. BAKHSHI, G. PEYVAST and Z. BALOUCHI

0

0 1:4

1:2

Undiluted

Proportion of walnut leaf extract (v/v) FIG. 2 Changes in the concentration of total phenolic compounds and in antioxidant activity (DPPH radical-scavenging activity) in basil (Ocimum basilicum L. ‘Genovese’) plants treated with different concentrations of Persian walnut leaf extract. r = correlation coefficient between total phenolic compounds concentration and anti-oxidant activity. Columns or datum points with the same lower-case letters are not significantly different at P ≤ 0.01 according to the LSD test. Vertical bars indicate ± standard errors (n = 3).

the plant defence response to different stresses and acts as a secondary messenger to signal subsequent defence reactions in plants (Low and Merida, 1996). Anti-oxidant activity: Anti-oxidant activity increased significantly (P ≤ 0.01) in the 1:4 (v/v), 1:2 (v/v), and undiluted walnut leaf extract treatments compared with the controls and the 1:8 (v/v) extract (Figure 2). This result may be due to the oxidative defence mechanisms induced by juglone stress (Segura-Aguilar et al., 1992). This result agrees with El-Hadrami et al. (2005) who reported that, under juglone stress, the activities of many anti-oxidant enzymes such as catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPO), and ascorbate peroxidase (APO) were stimulated. This condition could disturb the balance between ROSgenerating factors and ROS-scavenging systems. As shown in Figure 2, there was a significant (P ≤ 0.01) positive relationship between total phenolics concentration and anti-oxidant activity (r = 0.78). The mechanisms by which phenolic compounds scavenge free radicals is yet to be fully established. Polyphenols in plants contribute to the anti-oxidant and pharmacological activities of plants. Anti-oxidants such as phenolic acids, polyphenols, and flavonoids have a beneficial role in scavenging free radicals and thus reduce oxidative stress (Shetgiri et al., 2010). In conclusion, Persian walnut leaf extracts induced oxidative and water stress, resulting in increased enzymatic and non-enzymatic ROS-scavenging capacity. Concentrations of secondary metabolites such as essential oils and phenolic compounds increased in basil leaves exposed to all walnut leaf extract treatments. Therefore, the application of walnut leaf extracts could be a way to increase the concentrations of secondary metabolites. The cultivation of basil as a medicinal plant in a walnut-based intercropping system might be a profitable area of research, given recent trends towards the greater commercialisation of intercropping. We thank the University of Guilan, Iran for financial support.

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