s2fieldsofstudy:
Agricultural And Food Sciences
Biology

text:
Ameliorating Effects of Vitamin E and Selenium on Immunological Alterations Induced by Imidacloprid Chronic Toxicity in Chickens

Copyright: © 2012 Kammon AM, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Ameliorating Effects of Vitamin E and Selenium on Immunological Alterations Induced by Imidacloprid Chronic Toxicity in Chickens

Introduction
Insecticides are being used extensively in the field of agriculture, veterinary medicine, and public health. The indiscriminate use of insecticides has led to a widespread concern over the potential adverse effects of these chemicals on human and animal health. There is ample evidence to suggest that the use of insecticides in crops, in storehouses, in poultry houses and livestock leaves behind its residue that may cause interference with some of the fundamental biological processes. Many cases of high mortality among poultry and other avian species are associated with acute exposure to insecticides [1][2][3].
Imidacloprid is the first representative of the neonicotinoid insecticides that was registered and is presently the most widely used insecticide worldwide. Imidacloprid is the insecticide with the world's fastest growing sales and is considered possible replacement for organophosphorus insecticides in agriculture [4,5].
Few studies have examined the deleterious effects of Imidacloprid on vertebrate animals. The acute oral toxicity (LD 50 ) of imidacloprid is 31 mg/kg, 152 mg/kg and 25-50 mg/kg in Japanese quail, bobwhite quail and pigeon, respectively [4], while the acute oral LD 50 of imidacloprid in chicken is 104.1 mg/kg bw [6]. However there is lack of information on chronic toxicity of imidacloprid in chickens. Among the sublethal effects of insecticides, immunotoxicity is important because immune system is the key for disease prevention in poultry. The immunotoxic effects of insecticides may include histopathologic effects in immune tissues and organs (bone marrow, thymus, spleen, and lymph nodes); cellular pathology, including abnormal proliferation of stem cells; altered maturation of immunocompetent cells; changes in B and T cell subpopulations; and functional alterations of immunocompetent cells, which are classified as altered humoral-mediated immunity, cell-mediated immunity, or nonspecific responses. Imidacloprid was found to be immunotoxic to rats inducing significant reduction of hemagglutination antibody titer, leukocytic migration inhibition and phagocytic indices [7]. Insecticides may affect the immune cells by production of free radicals. Antioxidants can increase immune responses by controlling the amount of free radicals generated by insecticides. Vitamin E and selenium functions as antioxidants in vivo as well as in vitro by scavenging active free radicals [8].
The present study was conducted to investigate the chronic immunological implications of imidacloprid in chickens and the role of vitamin E and selenium to modulate these implications.

Experimental design
Broiler chicks (n=90) aged day old were randomly segregated into three groups of 30 chicks each and were kept in separate pens (30 chicks for each pen divided into 3 replicates of 10 chicks for each replicate). Chicks were acclimatized to the place for seven days before the experiment. On day 7, chicks in all groups were weighed and vaccinated against Newcastle disease (ND) using F1 strain (Venkys, Pune, India) by ocular route. On day 8, chicks in group I were administered 1/20 LD 50 imidacloprid (5 mg/kg bw mixed with water) orally. Chicks in

Abstract
The purpose of the current study was to investigate the immunological impacts of chronic imidacloprid insecticide toxicity in broiler chickens and the protective effects of vitamin E and selenium. Broiler chicks (n=90) aged day old were randomly segregated into three groups of 30 chicks each and were kept in separate pens. Newcastle disease (ND) vaccine was given on day 7 and 28 of age. On day 8, chicks in group I were administered 5 mg/kg bw (1/20 LD 50 ) imidacloprid orally. Chicks in group II were given imidacloprid 5 mg/kg bw orally plus a mixture of vitamin E and selenium ≅ 200 mg/kg diet, while group III was given distilled water (DW) orally and served as a control. The treatments were given daily based on weekly body weight till day 45. Antibody titration, serum total immunoglobulins, circulating immune complexes, cell-mediated immunity and histopathology of bursa of Fabricius and spleen were examined. Imidacloprid produced significant decline in the titer of antibodies against ND vaccine, total immunoglobulins and circulating immune complexes in imidacloprid treated group on day 45 as compared to control group. There were no significant changes in the skin thickness between treated chickens and chickens of control group. Histopathology of the bursa of Fabricius revealed edema, lymphocytic depletion in the medulla and cortex and mild interfollicular fibrosis in imidacloprid treated group. The spleen showed mild haemorrhages and lymphocytic depletion. Supplementation of vitamin E and selenium resulted in marked improvements in humoral immunity and pathology of lymphoid organs.
group II were given imidacloprid at the dose rate of 5 mg/kg bw by the same route as in group I plus a mixture of vitamin E and selenium (Micro Labs, Ltd., Banglore, India) at the dose rate of 200 mg/kg feed, while group III was given distilled water (DW) orally and served as a control for both the groups.
On day 21, eight chicks were randomly selected from each group and humanely euthanized for collection of blood and tissue samples.
On day 28, chickens of all groups were given a booster vaccination against ND using La Sota strain (Indovax Pvt Ltd, Hisar, Haryana, India) by intraocular route. At the end of the experiment on day 45, remaining chickens from each group were humanely euthanized and representative blood and tissue samples were collected. The experiment was performed after getting the permission from Institutional Animal Ethics Committee, GADVASU, India.

Immunological examinations
The chronic effect of oral administration of imidacloprid (5 mg/kg bw) on humoral immunity in broiler chickens was assessed by measuring serum antibodies against ND vaccine strain using haemagglutination inhibition (HI) test [9]. The results were expressed as log2 [10].
The chronic effect of oral administration of imidacloprid (5 mg/kg bw) on cell-mediated immunity was assessed by contact hypersensitivity response of the chicken's skin to dinitrochlorobenzene (DNCB) [11]. The percentage of skin thickness increase was calculated as follows: Percentage of thickness increase (%) = [(skin thickness after challenge -skin thickness before challenge)/skin thickness before challenge] × 100 [12].
Total immunoglobulins and Circulating immune complexes were estimated in serum samples [13] and [14], respectively.

Histopathology
The detailed post mortem of chickens was conducted on day 21 and 45 and the representative tissue samples of bursa of Fabricius and spleen were collected in 10% neutral formalin. After overnight washing in running water and dehydration in ascending grades of alcohol, the tissues were embedded in paraffin and 5 μm thick sections were cut and stained with haematoxylin and eosin [15].

Statistical analyses
Data are expressed as means ± SE. Analysis of variance (ANOVA), with Tukey HSD post hoc test was performed using the SPSS 11.5 computer program (SPSS Inc. Chicago, Illions, USA). P-values less than 0.05 were considered statistically significant.

Immunological examinations
The effect of chronic oral administration of imidacloprid at the dose rate of 5 mg/kg bw on humoral immunity in broiler chickens and the protective effect of a mixture of vitamin E and selenium ≅ 200 mg/ kg feed are summarized in Table 1. Imidacloprid produced significant (P<0.05) decline in the titer of antibodies against ND vaccine from 8.25 ± 0.42 log2 in control group to 6.5 ± 0.15 log2 in imidacloprid treated group on day 45. There was no significant difference between imidacloprid plus vitamin E and selenium group and control on day 45, suggesting the protective role of vitamin E and selenium to enhance the response of immune system in the presence of insecticide's immune suppression.
The skin revealed infiltration of heterophils and mononuclear cells in dermis and hypodermis layers. There were no significant differences in the skin thickness between imidacloprid treated chickens ( Figure  1A) and chickens of control group. However, skin thicknesses of chickens belong to imidacloprid plus vitamin E and selenium group was slightly increased but was not statistically significant ( Figure 1B).
The results of daily oral administration of imidacloprid on serum total immunoglobulins and circulating immune complexes are presented in Table 2, which revealed significant changes on day 45. Serum levels of total immunoglobulins and circulating immune complexes were significantly decreased from 0.72 ± 0.02 g/dl in control chickens to 0.59 ± 0.02 g/dl in imidacloprid treated chickens and from 0.07 ± 0.003 g/dl in control to 0.04 ± 0.006 g/dl in imidacloprid treated chickens, respectively. There were no significant changes in serum total immunoglobulins and circulating immune complexes between chickens given imidacloprid plus vitamin E and selenium and chickens in control group indicating the protective role of vitamin E and selenium. Values indicate mean ± S.E Means ± S.E. within a row lacking a common superscript differ at P<0.05

Histopathology of lymphoid organs
Daily administration of Imidacloprid at the dose rate of 5 mg/kg bw produced edema and severe depletion of lymphoid cells in cortex and medulla of bursa of Fabricius, mild vacuolation in epithelial cells and mild interfollicular fibrosis on day 45 (Figure 2A). Spleen showed mild haemorrhage and lymphocytic depletion on day 45 ( Figure 3A). However, chickens given imidacloprid plus vitamin E and selenium showed mild depletion of lymphoid cells in bursa ( Figure 2B) and spleen ( Figure 3B).

Discussion
The immune system integrity is the key to effectively preventing poultry diseases. Many poultry diseases are now controlled by properly planned biosecurity and vaccination. Immunosuppressive effects of xenobiotics are very important causes of vaccination failure thus predisposing animals to diseases. Alterations of the normal immune response can result in increased susceptibility to viral, bacterial, or parasitic infections [16].
Our data showed that daily administration of imidacloprid at the dose rate of 5 mg/kg bw is immunotoxic to chickens, altering mainly their humoral immunity measured by assessing the titer of antibodies against ND vaccine on day 45. Histopathological investigation of lymphoid organs showed some alterations in bursa of Fabricius and spleen. The lesions observed in bursa of Fabricius may explain the significant decline in antibodies measured against ND vaccine on day 45. Moreover, the serum levels of total immunoglobulins and circulating immune complexes were significantly declined on day 45 in chickens administered imidacloprid. There were no significant changes in the skin thickness between imidacloprid treated group, imidacloprid plus vitamin E and selenium group and control group. However, it is not clear whether the reason is due to compensatory responses. The presence of both enhanced and suppressed immune responses following exposure to pesticides suggests that the immune alterations are not a generalized cytotoxic effect and specific immunomodulation involving immuno-compensation may be present, although the extent, exact nature, and environmental significance of the compensating action are unknown [24].
Similarly significant progressive decrease in haemagglutinating antibody titer was reported in rats administered imidacloprid for 28 consecutive days [7]. The spleen exhibited depopulation of lymphocytes, mild fibrous tissue proliferation, thickening of capsule      and disintegration of white pulp and focal necrosis in all treated groups of rats as compared to control. The thymus showed mild congestion, depopulation of leukocytes, hemorrhages, edema and fibrous tissue proliferation. However, cell-mediated immunity which was assessed by subcutaneous administration of sheep red blood cells (SRBC) suspension into footpad showed insignificant difference between different groups of rats [7].
In a recent study, spleen tissues of the imidacloprid-treated rats displayed low numbers of lymphocytes and thymus tissues showed lymphocytic depletion with pyknotic nuclei [25].
In the present study, supplementation of vitamin E and selenium resulted in marked improvements in humoral immunity and pathology of lymphoid organs. This is in agreement with significant increase of antibody titres against SRBC antigen in growing Japanese quail fed two selenium supplemented diets (0.5 and 1.0 mg/kg) suggesting that supplementing the diet with selenium has a beneficial effect on immune responses [26]. Moreover, significant decrease in the concentration of total immunoglobulins and circulating immune complexes in chicks following administration of malathion were successfully counteracted by a combination of vitamin E and selenium [22]. Addition of vitamin E and selenium to the poultry ration was found to significantly increase antibody titres (HI and ELISA) against ND Virus at 10 days post inoculation in broilers [27].
Antioxidants can increase immune responses by controlling the amount of free radicals generated by exposure to insecticides in a cell. Vitamin E functions as an antioxidant in vivo as well as in vitro by scavenging active free radicals. α-Tochopherol is biologically the most active form of vitamin E and the major lipid-soluble antioxidant present in cells and blood [8]. Selenium activates an antioxidant enzyme called glutathione peroxidase. It forms selenocystine, part of the active site of glutathione peroxidase [28].
It is concluded that imidacloprid insecticide has immunological deleterious effects in chickens targeting the humoral immune responses with vitamin E and selenium supplementation decreasing the ill effects of imidacloprid. In view of the scarcity of data on imidacloprid immunotoxicity in poultry and other animals, the mechanism of action of imidacloprid on immune system is not known and needs to be elucidated. Thus further research may focus on the effect of imidacloprid on inhibition of non-target serine hydrolases, inhibition of esterases or production of cytokines etc.


extfieldsofstudy: Chemistry Medicine

provenance: AgriculturalAndFoodSciences,1970-2015-Agricultural And Food Sciences-2012.jsonl.gz.gz

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Thermostability of xylanolytic enzymes produced by Lentinula edodes UFV70

Xylanolytic enzymes produced by Lentinula edodes UFV70, cultivated in eucalyptus sawdust/rice bran medium, were stable at 50, 60 and 65°C for 21 hours, losing only 15–25% activity. Fungus incubation at 50°C for 12 hours and at 65°C for 24 hours increased the amount of xylose produced.

The increased expansion of agro-industrial activity has led to heavy accumulations of lignocellulosic residues that often contaminate the soil. Lignocellulose is the most abundant renewable biomass available on the planet, which consists of three major groups: cellulose, hemicellulose, and lignin (14).
Hemicellulose classification is based on the main sugar unit, where a hydrolyzed polymer that yields xylose is considered a xylan (12).
Lentinula edodes is a white rot fungus that grows on lignocellulosic residues, such as Eucalyptus sawdust and rice bran (3) . Its enzymes can be used in biotechnological applications, i.e., to create xylose for xylitol production (15), lignolytic enzyme production (2,4), and applications in other biotechnological processes, such as bioethanol production (6), and to increase the final brightness ceiling in bleached pulp (13).
The thermostability of xylanolytic enzymes is an important characteristic that allows for their application in industrial processes (13). These enzymes must be stable at high temperatures for use in pulp bleaching, for example (13). It is known that thermophilic fungi produce thermostablexylanase, which retains its catalytic activity for many hours at high temperatures ranging from 80-100 ˚C (5,9,16).
This study aimed to evaluate agro-industrial residue incubation conditions to maximize the amount of xylose produced, which is released by the enzymes from Lentinula edodes grown in Eucalyptus sawdust/rice bran medium. It was hypothesized that if lignocellulosic material, colonized by the fungus, was incubated at a temperature at which the fungus was unable to grow, but its xylanolytic enzymes were still able to hydrolyze the polysaccharides in the growth medium, then xylose concentrations would increase in the hydrolysates. The apparent specific activity of xylanolytic enzymes was detected after the second week, varying from 0.5 to 1.5 U mg -1 of protein (data not shown). The temperature for maximum apparent specific xylanolytic activity, 70 ºC (data not shown), was similar to thermophilic fungi, such as Thermomyces lanuginosus (16), but higher than that previously found in other L. edodes strains (50-60 ºC) (7,8,11). Xylanolytic enzymes were stable in a temperature range of 35-65 ºC for 3 hours. At 70 ºC, the activity was 11% lower than that of the control (data not shown). When the activity was measured after pre-incubation, it was 25, 20, and 15% lower after 21 hours of pre-incubation at 65, 60, and 50 ºC, respectively ( Figure 1). The maximum apparent activity occurred at a higher temperature (70 ºC) than previously reported (50-60 ºC) (7,8,11). Furthermore, these enzymes were considerably stable when pre-incubated up to 65 ºC, for up to 21 hours (Figure 1).
Compared to the reported 50% loss of -xylanase activity at 60 ºC after 6 hours for this same species (7), this current study found these enzymes to be much more stable. These differences may be due to the use of crude enzymatic extracts in this study, instead of purified enzymes (7). Some substances present in the crude extracts may have had an influence on the thermostability of these enzymes. Also, the current study used a different strain, L. edodes UFV70. These results are promising, because the use of crude extracts is a less expensive process than using enzymes.  -Control (xylose concentration in samples that were collected before thermal treatment); • -12 hours at 65 ºC; ♦ -24 hours at 65 ºC; -12 hours at 50 ºC; -24 hours at 50 ºC.


extfieldsofstudy: Biology Medicine

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Evaluation of antioxidant activity of Malus domestica fruit extract from Kashan area.

Objective: Antioxidants are considered as the main factors in the inhibition of unwanted oxidation reactions. Materials and Methods: In this research the antioxidant potential of the fresh fruits of 4 cultivars (A to D) of Malus domestica (M. domestica) cultivated in the Kashan, Qamsar area was evaluated. The antioxidant activity of the samples were evaluated using two complementary antioxidant assays: 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and β-carotene/linoleic acid tests and the results were compared with the synthetic standard antioxidant butylated hydroxytoluene (BHT). Results: Total phenolic contents of the samples are also estimated by Folin-Ciocalteu's phenol test. In both DPPH β-carotene/linoleic acid tests in the concentration of 2 mg/ml, only samples from cultivar A showed moderate antioxidant activity with 63.92±0.42 and 6.02±0.03 inhibition percentages, respectively and other samples were weakly active. Conclusion: The Folin-Ciocalteu's phenol test was also showed very little phenolic compounds for the fruits. In conclusion, weak antioxidant activity was estimated for the studied apple cultivars.

Introduction
Free radicals are present in biological systems and may oxidize all the biological molecules present in our body, such as nucleic acids, proteins, lipids, initiating degenerative diseases (Cook et al., 1996;Harborne et al., 2000;Heim et al., 2002). Antioxidants are substances that neutralize free radicals and their negative effects. Antioxidants can inhibit or delay the oxidation of oxidizable substrates and this appears to be very important in the prevention of oxidative stress which is suggested as the leading cause of many oxidation related diseases (Bamoniri et al., 2010).
Also antioxidants are substances that are able to prevent or retard the oxidation of lipids, proteins and DNA; and to protect the compounds or tissues from damage caused by oxygen or free radicals. Therefore, their health promoting effects reduce the risk of various diseases (Manach et al., 2004).Recently, antioxidant activity has been determined in many species of fruits, vegetables, herbs, cereals, sprouts and seeds (Kahkonen et al., 1999;Velioglu et al., 1998). Especial attention is paid to fruits, as rich sources of phenolic compounds (Kalt et al., 1999;Robards et al., 1999;Wang & Lin., 2000). Among others, the antioxidant properties of apple polyphenols have been extensively examined (Ju & Bramlage., 1999;Lu & Foo., 2000;Robards et al., 1999). Apples have the highest levels of antioxidant activity (Chinnici et al., 2004). Activity and concentration of antioxidants in fruit differ among cultivars, the part of the fruit, the growth stage and environmental conditions (Awad et al., 2001a, b, c;Addie et al., 2001).
Apple fruit contain several health and sensory related constituents including dietary fiber, sugars, vitamins and phenolic compounds (Hagen et al., 2007).The antioxidant capacity of apple is mostly attributed to phenolic compounds such as flavonoids and phenolic acids (Eberhardtet al., 2000;Lee et al., 2003).

M. domestica
Borkh. is one of the most commonly consumed fruit worldwide (Shoji et al., 2004) and we collected samples named Hossain, Sayyed Babaeei, Shekareh and Golab, were randomly named as A, B, C and D, from Iran.
These samples have been cultivated since most past times are medium in size with a circular shape. The yellow-pink skins are thin, rather wax-like, and the white fleshes are soft, juicy, aromatic and sweet. Because of staying on the tree, the skin color of these 4 apple cultivars changes gradually and becomes red. Thus, the present research reports (1) the in vitro profile of the antioxidant activity of the fruit extracts using two complementary assays: DPPH radical and β-carotene linoleic acid tests; (2) the total phenolic content of the fruit extracts, expressed as gallic acid equivalents.

Fruit collection
Fresh fruit samples from Hossain, Sayyed Babaeei, Shekareh and Golab apple cultivars were collected in the Kashan, Qamsar area in the June 2008 when the fruit had just been harvested.

Extraction procedure
Apples characterized by plant taxonomist, immediately transported to the laboratory, washed, dried, cut manually with a knife into small pieces, whole fruit except seeds extracts were obtained using a kitchen-type blender (Moulinex, France) and concentrated with a rotary evaporator.

Antioxidant activity DPPH radical scavenging
The 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical assay usually involves hydrogen atom transfer reaction but, based on kinetic data, an electron transfer mechanism has also been suggested for this assay (Huang et al., 2005;Foti et al., 2004). Radical scavenging activities of the plant essential oil and extract were determined using a published DPPH radical scavenging activity assay method (Sarker et al., 2006) with minor modifications.
Briefly, stock solutions (10 mg/ml each) of the extracts and the synthetic standard antioxidant BHT were prepared in methanol. Dilutions are made to obtain concentrations ranging from 1 to 510 10 mg/ml. Diluted solutions (1 ml each) were mixed with 1 ml of a freshly prepared 80 µg/ml DPPH radical methanol solution and allowed to stand for 30 min in the dark at room temperature for any reaction to take place. Absorbance values of these solutions were recorded on an ultraviolet and visible (UV-Vis) spectrometer (Cintra 6, GBC, Dandenong, Australia) at 517 nm using a blank containing the same concentration of DPPH radicals. Inhibitions of DPPH radical in percent (I%) were calculated as follow (Gholivand et al., 2010): In this research, dilution wasn't performed due to low concentration of extracts and low inhibitory percentage. Where A blank is the absorbance value of the control reaction (containing all reagents except the test compound) and A sample is the absorbance values of the test compounds. The sample concentration providing 50% inhibition (half-maximal inhibitory concentration, IC 50 ) was calculated by plotting inhibition percentages against concentrations of the sample.

β-Carotene/linoleic acid bleaching
The β-carotene/linoleic acid test evaluates the inhibitory effect of a compound or a mixture on the oxidation of β-carotene in the presence of molecular oxygen (O 2 ). Assay of the remained βcarotene gives an estimation of the antioxidant potential of the sample. The method described by Miraliakbari and Shahidi (2008), was used with slight modifications. A mixture of β -carotene and linoleic acid was prepared by adding together of 0.5 mg β-carotene in 1 ml chloroform (HPLC grade), 25 µl linoleic acid and 200 mg Tween 40. The chloroform was then completely evaporated under vacuum and 100 ml of oxygenated distilled water was subsequently added to the residue and mixed gently to form a clear yellowish emulsion. The essential oil, extract and BHT (positive control) were individually dissolved in methanol (2 g/l) and 350 µl volumes of each of them were added to 2.5 ml of the above emulsion in test tubes and mixed thoroughly. The test tubes were incubated in a water bath at 50°C for 2 h together with a negative control (blank) contained the same volume of methanol instead of the extracts. The absorbance values were measured at 470 nm on an ultraviolet and visible (UV-Vis) spectrometer (Cintra 6, GBC, Dandenong, Australia).
Antioxidant activities (inhibitions percentage, I%) of the samples were calculated using the following equation (Bamoniri et al., 2010): Where Aβ carotene after 2-h assay is the absorbance values of β-carotene after 2 h assay remaining in the samples and A initial βcarotene is the absorbance value of β-carotene at the beginning of the experiments. All tests were carried out in triplicate and inhibition percentages were reported.

Total phenolics
Total phenolic constituents of extracts of 4 apple cultivars were determined by literature methods involving Folin-Ciocalteu's phenol reagent and gallic acid standard (Slinkard & Singleton., 1977). A solution of the extract (0.1 ml) containing 1000 µg of the extract was pipetted into a 50 ml volumetric flask, 46 ml distilled water and 1 ml Folin-Ciocalteu's phenol reagent were added, and the flask was thoroughly shaken. After 3 min, 3 ml of 2% Na 2 CO 3 solution was added and the mixture was allowed to stand for 2 h with intermittent shaking. Absorbance values were measured at 760 nm. The same procedure was repeated for all the standard gallic acid solutions (0-1000 lg/0.1 ml) and a standard curve obtained with the following equation ( Total phenols of the extract, as gallic acid equivalent, was determined by using the absorbance value of the extract measured at 760 nm as input to the standard curve and the equation. Test was carried out in triplicate and gallic acid equivalent value was reported.

DPPH
DPPH radical scavenging activity potentials of fruit extract were evaluated for the assessment of their antioxidant capacities and compared with BHT (the standard commercial synthetic antioxidant). Among the above mentioned extracts, the best radical scavenging activity against DPPH was observed in cultivar A (63.92±0.42%) in the concentration of 2 mg/ml. The results obtained from 4 apple cultivars and BHT are presented in Table 1.

β-Carotene/linoleic acid
The potential of the plant to inhibit lipid peroxidation was evaluated using the βcarotene/linoleic acid bleaching test. In βcarotene/linoleic acid tests in the concentration of 2 mg/ml, only samples from cultivar A showed 6/015 ± 0/003 inhibition percentages. The results of 4 apple cultivars and standard (BHT) are presented in Table 2. Table 2. Antioxidant activity of β-carotene/linoleic acid bleaching assay method (percentage ± SD) of 4 apple cultivars in the concentration of 2 mg/ml.

Total phenolic constituents
Total phenolic content of the plant extracts were determined using a colorimetric assay method based on Folin-Ciocalteu reagent reduction.
The Folin-Ciocalteu's phenol test was also showed very little phenolic compounds for the fruits. The amounts of total phenols found in the fruit extracts are shown in Table 3. Table 3. The contents of total phenol of 4 apple cultivars.

Discussion
The measurement of the antioxidant capacity of food extracts and pure compounds is commonly performed using several methods. Each method relates to the generation or use of a different radical that is directly involved in the oxidative process, acting through a variety of mechanisms.Among the various assays, we selected the DPPH and β-Carotene/linoleic acid assays to determine the antioxidant activity of fruit extracts.
During DPPH radical test, the capacity of the samples to donate hydrogen atom and/or electron to this blue/purple stable radical and converting it to yellow diphenylpicrylhydrazine molecule was measured (Tepe et al., 2005). This reaction is used for measuring the ability of the extracts or pure molecules (such as BHT) to scavenge free radicals. Our results estimate a mild antioxidant potential for the cultivar A while other samples were weakly active.
Results of antioxidant test of 4 apple cultivars showed that none of 4 samples did not have high antioxidant properties at 2 mg/ml concentration but only cultivar A showed %64 inhibitory power.It is to be noted that the extracts were prepared with low concentration, therefore samples were not diluted.
These findings are in agreement with measured total phenolic contents of the samples (Drogoudi et al., 2008;Lata, 2007;D΄Abrosca et al., 2007;Tsao et al., 2005;Vieira et al., 2009). β-Carotene/linoleic acid test of 4 apple cultivars showed the same results as antioxidant test with the exception that cultivar A showed greater inhibitory power (%6) compared to DPPH procedure.
This finding is in contradiction with findings of Garcia et al., 2009;Lata et al., 2009;Lee et al., 2003;Bandoniene and Murkovic., 2002;Kondo et al., 2002; which might be due to different cultivars they have selected under different climatic conditions.
The basic structure of the phenols and other structural factors play a fundamental role in the mechanism by which these compounds are able to scavenge free radicals (Sadeghipour et al., 2005). As underlined also by others (Lata et al., 2009;Lata, 2008), it is difficult to compare the content of apple phenolic among different studies, as many variations can be principally caused by different growth period, geographic location, storage type, genetic diversity and many other factors.
The results, expressed as gallic acid equivalents, were 0 µg/mg and 1.5±0.6 for the extracts of apples, respectively.
These values are comparable to the values reported in the literature for other apple cultivars, such as Golden Delicious, Stark Delicious, Mora, Nesta, Panaia-red and Ruggine (Iacopini et al., 2009). Phenolic compounds normally play main role in the antioxidant activity of the plant extracts, thus, low DPPH antioxidant activity of our samples may be related to their negligible total phenolic compounds contents. Folin-Ciocalteu test showed that there is low percentage of phenolic compounds in all samples which is in accordance with antioxidant tests.
Overall conclusions was that all samples did not show high antioxidant power and only cultivar A showed higher antioxidant power, which might be due to the presence of phenolic compounds.

Conclusions
Natural products, especially those produced by edible and medicinal plant species, are currently under special interest

Sample
Total phenol contents (µg/mg) as food additive due to their safety, usefulness and accessibility. In this study, we have focused on antioxidant activity and total phenolic compounds of apples. Our results conclude that the phenolic content, the radical-scavenging and antioxidant properties of old local apple varieties demonstrate that these new cultivars could be a good source of phytochemicals, bioactive compounds with important protective properties. These local apple cultivars could be also considered as an important source of genes for apple breeding program and for the production of value added apple cultivar. Thus, further studies on local and ancient varieties for determining of their biological potentials have are highly recommended.


s2fieldsofstudy:
Agricultural And Food Sciences
Environmental Science

text:
Mature Pecan Orchard Floor Vegetation Management: Impacts on Tree Water Status, Nutrient Content, and Nut Production

P ecan (Carya illinoinensis (Wangenh.) K. Koch) growers are advised to control orchard floor vegetation when establishing new orchards, but there is not a set recommen- dation for vegetation control in mature orchards. The objective of this study was to measure the effect of orchard floor vegetation on water and nitrogen (N) status of flood-irrigated mature pecan trees. Four treatments studied were: completely vegetated orchard floor, vegetation-free inner area directly under the tree canopy with vegetation in the outer area, completely vegetation-free, and vegetated inner area under the canopy with a vegetation-free outer area. Treatments were organized as a 2 · 2 factorial structure with inner and outer treatment factors, both with levels vegetated and vegetation-free. Soil moisture and tree middaystemwaterpotential(MSWP)weremeasuredduringirrigationcyclestoevaluatethe development of water stress in the pecan trees. Soil moisture data showed a significant outer main effect when the soil in the entire orchard was the driest, that is, just before irrigation events.Areas with vegetation cover thatwereexposed to fullsun were significantly drier than shaded vegetated areas and vegetation-free areas in the orchardfloor. However, this was not correlated with differences in tree water status as indicated by MSWP. Leaf tissue and soil analyses showed no significant differences in N concentrations among treatments in either year. Treatments with orchard floor vegetation in the outer area had significantly higher yield efficiency and marginally significant improvements in percent kernelfill and number of nuts per kilogram. Our findings suggest that there may be more benefits to maintaining orchard floor vegetation in mature orchards than were previously acknowledged.

The pecan industry is thriving in the western United States (west Texas, New Mexico, Arizona, and California) totaling almost 27,500 ha with over 16,000 ha in New Mexico alone (USDA, National Agricultural Statistics Service, 2009). However, water resources are limited and expensive in this semiarid or arid region. Consumptive water use requirements of pecan orchards are high, at more than 1.5 m annually (Miyamoto, 1983;Sammis et al., 2004). Increases in pecan acreage are placing new demands on irrigation districts and groundwater supplies throughout the Southwest.
Orchard floor management practices of pecan growers in the southwestern United States are varied, ranging from only periodic mowing of vegetation to complete vegetation control through herbicides or cultivation. The presence of orchard floor vegetation has benefits including reduced soil erosion, reduced surface crusting, which leads to improved water infiltration, and improved orchard access by heavy machinery (Elmore, 1989;Folorunso et al., 1992;MacRae and Mehuys, 1985). However, pecan harvest operations are hindered by excess orchard floor vegetation and vegetation may also compete with trees for nutrients and water (Merwin and Ray, 1997;Radosevich et al., 2007).
Many studies demonstrated substantial tree growth and yield reductions associated with floor vegetation of young pecan orchards in the humid southeastern United States (Faircloth et al., 2007;Foshee et al., 1995;Patterson and Goff, 1994;Patterson et al., 1988Patterson et al., , 1990Smith, 2011;Smith et al., 2002Smith et al., , 2005. In Patterson and Goff's 1994 study that examined newly planted trees for 8 consecutive years, trees subjected to total chemical vegetation control produced over five times more pecans in the last 3 years of the study than trees competing with vegetation. This difference was attributed to higher potassium levels in total vegetation control treatments. Goff et al. (1991) also showed that orchard floor vegetation can affect nutrient status of immature trees as a result of competition. However, there have been no studies investigating the effects of vegetation management on mature pecan orchard productivity under the arid growing conditions of the southwestern United States, where water stress and N deficiency are likely to be primary factors limiting pecan orchard productivity.
The optimization of irrigation water use and fertilizer efficiency in mature orchards requires a better understanding of the impact of orchard floor vegetation on trees. Therefore, the objective of this study was to assess the effect of orchard floor vegetation on mature pecan trees by monitoring soil moisture, leaf and soil N concentrations, tree water status, and orchard productivity in plots with different amounts of vegetative cover. The null hypothesis of this study was that floor vegetation would have no effect on N status, water status, nut yield, or nut quality of mature pecan trees.

Materials and Methods
Orchard characteristics and weed composition. Irrigations were scheduled at 3-to 5-week intervals depending on rainfall, temperature, and tree growth stage at the discretion of the orchard owner. Insecticide application, fertilization, and periodic mowing were all controlled by the orchard owner. Nitrogen was applied in the irrigation water during the growing season as urea-ammonium nitrate (UAN, 32% N) solution at a rate of 54.3 kg N/ha divided into three and four applications in 2009 and 2010, respectively. Weed composition consisted of mainly annual species. Palmer amaranth (Amaranthus palmeri S. Wats.), barnyardgrass (Echinochloa crus-galli L.), and green foxtail (Setaria viridis L.) dominated the orchard with many other annual species present in lower densities. Hare barley (Hordeum leporinum L.) and London rocket (Sisymbrium irio L.) were the predominant species during the winter season. The perennial species present in the orchard were field bindweed (Convolvulus arvensis L.), purple nutsedge (Cyperus rotundus L.), broadleaf plantain (Plantago major L.), curly dock (Rumex crispus L.), silverleaf nightshade (Solanum elaeagnifolium Cav.), perennial sowthistle (Sonchus arvensis L.), and dandelion (Taraxacum officinale F. H. Wigg.). Broadleaf plantain was the only perennial weed species present throughout the orchard with all of the other populations of perennial weed species being found in small localized patches within the orchard.
Weed species did not provide a consistent amount of vegetative groundcover throughout the study. The orchard owner mowed the vegetation periodically (six times in 2009 and four times in 2010) and there were areas, especially in May 2010, when some of the vegetated plots did not have complete vegetation cover. Sometimes the cut vegetation after mowing acted as a mulch, suppressing emergence and regrowth of vegetation. Nevertheless, at all times throughout both growing seasons, weedy vegetation provided greater than 50% groundcover and clear visual distinction could be made between vegetated and vegetation-free treatment areas.
Experimental design. Four replications of four vegetation management treatments organized as a 2 · 2 factorial treatment structure ( Fig. 3) were arranged as a completely randomized design. Each experimental unit consisted of two sample trees surrounded by 10 buffer trees (buffer trees underwent the same treatment as the sample trees in each unit). Treatment factors were inner and outer, both with vegetated and vegetation-free levels. Treatment 1 (vegetated control) had vegetation (i.e., weeds) covering the entire treatment area (i.e., both the inner and outer factors were vegetated). Treatment 2 (vegetation-free inner) had a vegetation-free area 6 · 6 m around the trees and vegetation was allowed to grow outside of that vegetationfree square (i.e., inner factor was vegetationfree and outer factor was vegetated). In Treatment 3 (vegetation-free control), vegetation was controlled in the entire treatment area (i.e., both inner and outer factors were vegetation-free). Treatment 4 (vegetationfree outer) had a vegetated area 6 · 6 m directly under the trees and vegetation was controlled outside of that square (i.e., inner factor was vegetated and outer factor was vegetation-free).
Vegetation-free areas were maintained during the growing seasons of 2009 and 2010 using a combination of chemical and manual weeding. The post-emergent herbicide, glyphosate [Makaze Ò , N-(phosphonomethyl)glycine; Loveland Products, Greeley, CO] was applied at 2.24 kg a.i./ha three times in 2009 (15 Apr., 29 July, 21 Sept.) and three times in  Data collection and statistical analysis. Soil moisture and midday stem water potential were measured at weekly intervals after each irrigation event for a total of 21 times in 2009 and 17 times in 2010. Soil moisture was measured as volumetric water content and reported as percent soil moisture using a time domain reflectometry system (TRASE System I; ICT International, Armidale, Australia). Twenty-centimeter-long waveguide probes were buried vertically 30.5 cm deep to measure volumetric soil moisture content over the range of 10.5 cm to 30.5 cm deep. Both the pecan roots and the weed roots were expected to be most abundant in that part of the soil profile (Miyamoto, 1983;Ross and Lembi, 2009). Sixty-four waveguide probes were placed throughout the orchard, four per experimental unit. One sensor was placed outside of the 6 · 6-m area immediately surrounding the tree so that it received maximum sun exposure (4.8 m from tree trunk) and one sensor was placed inside of that area where it was predominantly shady (2 m from tree trunk). Within each experimental unit, the probe readings were averaged based on location: inner or outer.
Midday stem water potential (i.e., water potential of dark-bagged leaves at midday) was measured at 1-week intervals after each irrigation using a Scholander Pressure Chamber (Model 615; Plant Moisture Stress, Albany, OR) to directly assess tree water status. Measurement of water potential at midday is a reliable method to detect tree water status (Shackel et al., 1997). MSWP data were collected between 1130 HR and 1500 HR using subsamples of two measurements per sample tree, giving a total of four subsamples per experimental unit. Sample leaves were selected from the middle of fruit-bearing, shaded branches in the lower canopy. Each sampled leaf was first placed in an aluminum bag and left on the tree for a minimum of 45 min to allow leaf water potential to equilibrate with that of the stem, thus estimating tree water status.
To assess the impact of orchard floor vegetation on N availability, leaf and soil samples were analyzed for total N and nitrate-N concentration, respectively, using a LECO FP528 Nitrogen Analyzer (St. Joseph, MI) and a HACH sensION4 ISE meter with aluminum sulfate extract (Loveland, CO). Leaf samples from each sample tree (Aug. 2009 andSept. 2010) and soil samples from each plot (June 2009 and Oct. 2010), 0-15 cm deep, were collected following New Mexico State University Cooperative Extension Service guidelines (Glover and Baker, 2000;Herrera, 2000).
A 13 · 25-m rectangular area was marked out on the ground surrounding each set of sample trees to measure yield. After shaking the two sample trees within each experimental unit, the nuts were harvested and weighed. In 2009, the 13 · 25-m rectangle was divided in half so that yield was measured per individual tree but pooled by experimental unit for analysis. In 2010, nut yield data were collected from each experimental unit (i.e., with yield from the two sample trees in each plot pooled) using a pecan harvester (Savage Model 8042, Madill, OK). To account for varying levels of debris and leaf litter in the piles of nuts after they had been mostly cleaned by blowers and rakes, 1-to 1.5-kg samples from the piles of nuts and debris were removed from the orchard. These samples were later cleaned, reweighed, and the percentage of clean weight vs. the original sample weight was used to estimate total clean nut weight from each experimental tree. These same samples were then counted, cracked, and the nut meat weight was determined for harvest quality data. Nut quality factors included percent kernel in both 2009 and 2010 and number of nuts per kilogram in 2010. Data were analyzed using SAS PROC MIXED software Version 9.2 (SAS Institute, Cary, NC). Significance was defined as P # 0.05 for all analyses. However, differences were acknowledged as ''marginally significant'' when P # 0.10. Soil moisture and soil N data were analyzed by respective sample date in a split-plot experimental design with the whole plot factor being the treatments in a 2 · 2 factorial structure in which the inner and outer measurements were both either vegetated or vegetation-free. Soil moisture probe location (inner or outer) was the splitplot factor. Leaf N and MSWP were also analyzed by date using analysis of variance for a 2 · 2 factorial structure. For MSWP, there were two layers of subsampling: two trees within each experimental unit and two leaves sampled per tree per collection date. Subsampling was recognized by including appropriate random effects in the model. For yield parameters, experimental unit averages were analyzed as a 2 · 2 factorial treatment structure with year as a repeated factor (except for number of nuts per kilogram, which was only recorded in 2010). An unstructured covariance was fit to account for covariances and possible nonconstant variance between the repeated measures (years). When an effect was significant, least significant difference was used for post hoc pairwise comparisons applied to the highest order significant effect.

Results and Discussion
Soil moisture. On three dates in 2009 and nine dates in 2010 the probe location main effect was statistically significant (inner vs. outer probes). All were drier at the inner probe location except one date in 2009. The three-way interaction (outer vegetation factor, inner vegetation factor, and probe location) was statistically significant on four occasions in 2009 and two in 2010. Post hoc comparisons showed little consistency in the pattern of differences, although most differences could be characterized as simple probe location effects.
For soil moisture, there was never an inner vegetation factor main effect (Figs. 1A-2A). However, the outer vegetation factor main effect was significant twice in 2010 with outer vegetated plots being significantly drier. Also, there was a significant two-way interaction between the outer vegetation factor and probe location on six occasions across both years (Figs. 1B-2B). In these cases, the vegetated outer location was consistently drier than the vegetation-free outer location. Outer vegetation main effects and outer vegetation factorby-probe-location interactions tended to occur at the end of an irrigation dry-down cycle and in the hottest, driest parts of the growing season. Furthermore, when the interaction was significant, soil moisture differences were detected at the outer probe location where the moisture probes were placed outside of the tree canopy and exposed to full sun. For example, on 18 June 2010, volumetric soil moisture in vegetated outer areas was 13.2% vs. 16.5% volumetric soil moisture in vegetation-free outer areas (Fig. 2B). Similar results of reduced soil moisture resulting from grass competition were found in a study of mature peaches in West Virginia (Tworkoski and Glenn, 2001).
Tree water status. In the present study, treatment differences in soil moisture did not correlate with differences in tree water status as indicated by MSWP (Fig. 4). Regardless of treatment, the water status of the all of the sampled trees in this study did drop through each dry-down cycle, showing that the trees were not receiving adequate water from roots reaching the water table to maintain tree water status. Therefore, although the transpiration of orchard floor vegetation reduced available water in the soil at the outer probe location at the end of a dry-down cycle, the level of competition for water with the mature trees was not sufficient to detect significant differences in the water status of the mature pecan trees in this study.
Interestingly, although there were no significant interactions in either year, at the end of the 2010 experimental season, there were significant outer vegetation main effects in Fig. 3. Four treatments of varying vegetated area surrounding mature pecan tree. Smaller square that designates inner area is 6 · 6 m. Tree image adapted with author permission from Welker and Glenn (1991). MSWP. Trees with vegetation in the outer area had a greater water potential (less stress) than the trees without vegetation in the outer area (Fig. 4). On 29 Sept. and 3 Oct. 2010, MSWP means for trees with vegetation in the outer area were -0.62 MPa and -0.82 MPa, respectively, compared with -0.75 MPa and -0.98 MPa, respectively, for trees without vegetation in the outer area. The cause for this difference is unknown, but it is possible that at the end of the growing season, annual vegetation transpiration slowed, acting as a living mulch, thereby preserving soil moisture and increasing permeability that favorably affected tree water status (Folorunso et al., 1992). This suggests that pecan growers may see benefits by allowing some vegetation cover in their orchards at the end of the summer season. In a similar study on both immature and mature almond (Prunus dulcis Mill.) trees in California (Pritchard et al., 1990), cover crops as an orchard floor management option resulted in greater water infiltration rates in the soil compared with a clean orchard floor. Additionally, Pritchard et al. (1990) suggested that increased shading with a larger root area in the mature orchard as compared with the immature orchard was the reason for greater orchard floor management treatment differences in the young orchard than in the mature orchard. Nitrogen competition. There were no significant differences among treatments in amounts of nitrate in soil samples or N concentration in leaf samples (data not shown). Similarly, Wright et al. (2003) found no effect of orchard floor vegetation in a young citrus (Citrus limon L., Citrus sinensis L.) orchard on citrus leaf nutrient concentration. In the same study, however, the cover crop and weedy treatments did have higher soil N concentration. It should be noted that the orchard in the current experiment was a commercial orchard where the orchardist applied fertilizer as needed (including N in the form of liquid UAN mixed in the irrigation water several times in 2009 and 2010). At a range of 2.5% to 2.7%, leaf N levels remained within the normal range for pecan leaves in New Mexico. To detect N competition between orchard floor vegetation and pecan trees, the N supply would have to be low enough to affect the N concentration in the leaves. It appears that, at the applied rates, there was sufficient N in the soil to supply both the orchard floor vegetation and the trees.
Nut production and quality. The most productive orchards in the Mesilla Valley have average annual in-shell nut yields of 2500-3000 kgÁha -1 . Average yield for this experimental orchard during the study exceeded 3000 kgÁha -1 . No significant differences for year-by-factor interaction between treatments or as a result of location of vegetated areas were found in harvest yield data (Table 1). This measure of harvest yield did not take into account the overall size of the tree, so yield efficiency rating was also calculated per experimental unit. Yield efficiency (gÁcm -2 ) is a measure of tree nut weight per square centimeter of trunk cross-sectional area. For yield efficiency, outer main effects were significant (Table 1). Vegetated outer areas had higher yield efficiency (lsmeans ± SE: 54.39 ± 2.58 gÁcm -2 for vegetated vs. 46.37 ± 2.58 gÁcm -2 for vegetation-free). These results differ from results of a study of mature sour cherry (Prunus cerasus L.) tree yield efficiency in which alternative orchard floor managements did not affect yield efficiency ratings (Sanchez et al., 2003).
Percent kernel fill, a measure of the amount of nut meat in the shell, demonstrated a marginally higher (P = 0.0861) percentage for the treatments with vegetation in the outer area (55% kernel) than for the treatments that were vegetation-free in the outer area (54% kernel) ( Table 2). Furthermore, in 2010, number of nuts per kilogram had marginally significant inner and outer main effects (Table 2). Inner vegetated had 6.9 ± 3.7 fewer nuts per kilogram and outer vegetated had 7.35 ± 3.7 fewer nuts per kilogram, meaning that the nuts were bigger and therefore more desirable with vegetation. Similar results were found in a 15-year study of orchard floor management systems in apples (Malus domestica Borkh.). Atucha et al. (2011) examined effects of four apple orchard floor management practices: vegetation-free with the use of pre-emergent herbicides, vegetation-free with the use of post-emergent herbicides, a sod cover crop, and hardwood bark mulch. In the first years of orchard establishment, sod groundcover significantly reduced growth and yield of the young trees. However, as the trees matured, the effect of the cover crop became less remarkable. At the end of the study, when the apple trees were mature, there was no significant effect of orchard floor management treatment on tree growth or yield. These results were attributed to the ability of the trees to adapt to or avoid sod competition over time.
Despite reducing the soil moisture at the end of dry-down cycles in the hottest time of the summer in the sunniest parts of the orchard, orchard floor vegetation, particularly outer vegetation, had a slightly beneficial effect on yield efficiency, percent nut fill, and decreased nuts per kilogram. Several studies suggest that high root-zone temperature can cause heat injury to the roots and/or increase sensitivity of root tissues to other stresses. A study of maple (Acer sp.) suggested that heat injury to root tips may alter hormone relations and cause leaf epinasty (Wilkins et al., 1995). In Tromp's (1996) study on young apples, there was a definite temporal effect of high soil temperatures on shoot growth, suggesting that depending on the stage of fruit development, the tree may be more or less susceptible to root heat damage. It is possible that decreased temperature of the tree root-zones was a direct result of the shade provided by orchard floor vegetation and that this positively affected tree water status to the extent of increased harvest parameters. We did not measure soil temperature in our study, but the outer main effects we observed would be consistent with this mechanism because inner areas were already shaded by the tree and subject to less radiant heat.
Other possible considerations for the improved harvest parameters in plots with vegetation in the outer area could be soil condition (i.e., greater water infiltration, reduced compaction) or mulching effect of mowed vegetation. Further research is needed to fully understand the role of different types of vegetative groundcover, specifically non-weedy vegetation, on soil temperature and other factors in mature pecan orchards and how this affects pecan harvest over longer periods of time.
This study was unique in its focus on mature pecan trees and these findings suggest that there may be more benefits to maintaining orchard floor vegetation than were previously recognized. Although the term ''vegetative ground cover'' is broad and does include weedy groundcover, weedy orchard floors pose a detriment in other ways (e.g., interference with harvest), and, as such, cover crops other than weeds are recommended.