Two faba bean, Vicia faba L. , cultivars, Gazira2 and Misr1, stand foring moderate resistant and susceptible severally, were analyzed for peroxidase and polyphenol oxidase ( PPO ) activities induced by black-eyed pea aphid, Aphis craccivora Koch. infestation. Two tissue types ( whole works and detached foliage ) , two infestation position ( infested and uninfested ) , three infestation continuances ( 1, 3 and 5 yearss ) were considered in POD and PPO informations analysis. Factorial analysis showed that merely cultivar factor has a important consequence on both POD and PPO activity, particularly on first twenty-four hours of aphid infestation ( P:0.0003 and 0.001 severally ) . While tissue type and infestation continuance factors as chief consequence does non hold any important consequence every bit good as no interaction among them. Mann Whiteny -U trial indicated that POD and PPO activities in Gazira2 was higher than Misr 1 with P-value 0.0006 and 0.0015 for POD and PPO severally. Repeated step analysis showed that the POD and PPO activity on Gazira2 significantly higher as compared to Misr 1, to boot the POD activity changed significantly over the clip in 1, 3 and 5 yearss after aphid infestation. Finally, it was suggested that higher activity of POD and PPO in curriculum vitae. Gazira2 is strongly associated with their immune characters.
Plants have an ability to react to insects feeding by changing the province of their enzymes, by either constituent or inducible ( Hildebrand et al. 1986 ; Nabity et Al. 2006 ; Chen et al. 2009 ; Giordanengo et Al. 2010 ; He et al. 2010 ) . These enzymes eventually affect insect eating success ( Lukasik et al. 2012 ) . Overproduction of harmful Reactive Oxygen Species ( ROS ) is the first mechanism in workss when they are injured by insect eating, ensuing in oxidative emphasis, which may do cell and tissue harm ( Michalak 2006 ; Gill and Tuteja 2010 ; A?ivkoviA‡ et Al. 2010 ) . Plants get the better of this state of affairs by bring forthing an efficient enzymatic antioxidant defence system which prevent and protect works cells from oxidative harm by scavenging the ROS ( Mittler et al. 2004 ; Gill and Tuteja 2010 ) . Peroxidase ( POD ) and polyphenol oxidase ( PPO ) are among the antioxidative enzymes which play an of import function in works emphasis caused by insect eating ( Lattanzio et al. 2006 ; Jaiti et Al. 2009 ; Ramirez et Al. 2009 ; He et al. 2010 ) . Both enzymes are widely distributed among works Kingdom ( Lattanzio et al. 2006 ; Dogan et Al. 2007 ; Zhang et Al. 2008 ) .
Peroxidase is heme-containing monomeric glycoproteins that utilize either H2O2 or O2 to oxidise a broad assortment of molecules ( Yoshida et al. 2003 ) . Their activity is localized in the cytol and cell wall ( Heng-Moss et al. 2004 ; Chen et al. 2009 ) . Peroxidase has a specific function in lignifications and beef uping the works cell wall that is extremely immune to biodegradation ( Schoemaker and Piontek 1996 ; Lee et Al. 2007 ; Jaiti et Al. 2009 ) . PPO, besides contributes to lignifications ( Ralph et al. 2008 ) and together with POD it consumes O and produces quinones, which may cut down works digestibleness for the insect ( Duffey and Stout 1996 ; Lattanzio et Al. 2006 ; Jaiti et Al. 2009 ; Ranger et Al. 2009 ) .
Artificially, PPO and POD activities can be elevated by increasing salt ( Wang et al. 1997 ; Nabity et Al. 2006 ) , adding jasmonic acid ( Jaiti et al. 2009 ) , soluble Si ( Gomes et al. 2005 ; Ranger et Al. 2009 ) , arsenate ( Lin et al. 2008 ) , or by presenting a cistron to workss. Transgenic baccy anionic peroxidase is an illustration which can show about up to 400 times higher peroxidase activity than matching tissues of wild-type workss ( Behle et al. 2002 ; Dowd and Lagrimini 2006 ) . Tomato TPX2 cistron or sweet murphy swpa1 cistron are other illustrations in which over look of the cistron could confabulate increased salt-tolerance or oxidative-stress tolerance ( Yoshida et al. 2003 ) . The peroxidase engineered works is possible since the peroxidase base had been sequenced ( Botella et al. 1993 ) .
The increased activities of these enzymes in a works are considered as a immune province of the works to the insect plague ( Wei et al. 2007 ; Ramirez et Al. 2009 ; Gulsen et Al. 2010 ) . However, some other surveies reported that there were no differences in the PPO activities between infested and uninfested American bison grasses challenged to Chinch bug, Blissus occiduus ( Heng-Moss et al. 2004 ) . Similarly, no differences were reported in POD activities between some non-infested and infested cereal genotype to Russian wheat aphid Diuraphis noxia ( Kurdjumov ) ( Ni et al. 2001 ) . Therefore, the biochemical response during plant-insect interaction might be specific, either increased and/or decreased, depending on the works or insect species ( Chen et al. 2009 ) .
Once the specific POD and PPO responses have been revealed, they can be used as biomarker ( Heng-Moss et al. 2004 ) and to clarify the mechanism relies on resistant works ( Gulsen et al. 2010 ) . This biochemical response has been used for choosing Chinch bug immune sod grasses and black pecan aphid, Melanocallis caryaefoliae ( Davis ) opposition in pecan germplasm ( Chen et al. 2009 ) besides in aphid opposition of lucerne ( Wei et al. 2007 ) .
The black-eyed pea aphid, Aphis craccivora Koch. is one of the most of import plagues of Faba bean, Vicia faba L. ( Laamari et al. 2008 ; Larocca et Al. 2011 ) . Pesticides are the most common manner to command the aphids ( Sadeghi et al. 2009 ) . However, aphids have an ability to develop pesticide opposition due to the little size, high generative capacity and strong hardship adaptability. Non mark effects of the usage of pesticides are an of import issue ( Wei et al. 2007 ) . The promising option is utilizing works opposition as a sustainable, safe and economical option ( Heng-Moss et al. 2004 ) .
Recently, some immune faba bean cultivars to aphids have been found, such as V. faba Minor, which is tolerant to A. fabae ( Shannag and Ja’far 2007 ) and V. faba landrace V51 which is immune to A. craccivora ( Laamari et al. 2008 ) , but cautiousness should be addressed for resistant-breaking biotypes, which have occurred in several plants-aphid systems ( Dogimont et al. 2010 ) .
The handiness of insect-resistant harvests is still rare ( Klingler et al. 2001 ) . One of the jobs is the unwanted big graduated table insect bio-assaies which have to be incorporated in engendering plans. Therefore, developing new agencies such as a biochemical marker for measuring insect resistance expeditiously is a challenge ( Schoonhoven et al. 1998 ) .
This survey was conducted to set up a baseline biochemical information sing the response of two antioxidative enzymes ( POD and PPO ) in two faba bean cultivars ( Gazira2 and Misr1 ) infested by A. craccivora. Both cultivars have been reported as reasonably immune and susceptible cultivars, severally. Colony development survey for some faba bean cultivars showed that Gazira2 was less preferable for A. craccivora, indicated by significantly fewer Numberss of aphids after 14-day infestation. Feeding behavior surveies utilizing DC-EPG, suggested that the longer continuance of wave form F, which means more stylet incursion troubles into the cell, was the possible resistant mechanism in Gazira2 ( Soffan and Aldawood 2012ab, in reappraisal ) . The dealingss of the above consequences with antioxidative enzyme activity were expected to acquire an apprehension of immune mechanism in Gazira2.
Materials and methods
Peroxidase ( POD ) and Polyphenol oxidase ( PPO ) analysis were conducted in the Microbiology research lab of Plant Protection Department, College of Food and Agriculture Sciences, King Saud University, Riyadh, Kingdom of Saudi Arabia. Faba bean cultivars were maintained in growing Chamberss with environment puting 26 A± 0.03EsC, 44 A± 0.12 % RH ( Means A± S.E ) , photoperiod of 16:8 Liter: D ( recorded by HOBO information lumbermans, ONSET Co. USA ) .
Faba bean curriculum vitae. Gazira2 and curriculum vitae. Misr1 were used for experiments, stand foring the reasonably immune and susceptible cultivars, severally. The seeds were obtained from the Legume Research Unit ( LRU ) Plant Production Department, College of Food and Agriculture Science, King Saud University. Seeds were germinated in a mixture of sand and peat moss ( 1:1 ) growing medium after soaking in H2O for 48 h. After one hebdomad, seedlings were transplanted to plastic pots ( vitamin D: 11 centimeter, H: 14 centimeter ) . Four granules per pot of complete fertiliser ( N:12 % , P:12 % , K:17 % ; BASF-Asoco Agro ) was applied in the growing medium one time at seedling phase ( 19 yearss old works ) . Watering was by swamping the pot for 150 milliliter one time at every two yearss.
Both cultivars were prepared for POD and PPO analysis utilizing 23-28 yearss old workss holding five true foliages. Merely three true foliages, consisted of two foliage blade, laid between the most basal and upper portion of the works were used.
Cowpea aphids were used to bring on the activity of Peroxidase and polyphenol oxidase in the works. They were obtained from a settlement which was collected from lucerne workss grown in Al Amaria, Riyadh, Kingdom of Saudi Arabia ( N46A° 31 ‘ 5.5518 ” E24A° 48 ‘ 40.179 ” ) . Single female parent of an apteral grownup aphid was used for the induction of black-eyed pea aphid civilization on the faba bean curriculum vitae. Misr. Before the beginning of the experiment, the black-eyed pea aphid civilization had been running for eight months. New faba bean seedlings were provided continuously to replace the old works for the care and uninterrupted growing of aphid civilizations.
Apterous nymphs were collected from the pure civilization above. One hundred of the apteral nymphs were introduced on each true leave, distributed every bit in each foliage blade. A 60 milliliter transparent plastic cups ( vitamin D: 7 centimeter, H: 2.5 centimeter ) were used as a foliage coop merely in whole works to forestall aphid get awaying from the chosen foliage. Aphid infestation on degage foliage following the same process as in whole works. The same 60 milliliter transparent fictile cup was used to suit the degage foliage. Detached foliage was kept its freshness by wrapping the leaf base with cotton. Water was filled into a fictile cup to maintain the cotton moisture. Uninfested workss were prepared with the same process as mentioned above without aphid infestation.
Three true foliages from each works were sampled on 1, 3 and 5 yearss, severally, to bespeak the possible difference in PPO and POD activities across the infestation continuance period. One foliage blade in each true leave was considered as a individual sample for both whole works and detached foliage. The sampled foliages were collected on a certain twenty-four hours harmonizing to infestation continuance, and it was used straight for enzyme analysis. Each sample had six reproductions.
Fresh foliage blade holding about 0.1-0.2 g were used for enzyme extraction. Each fresh foliage was grounded into pulverization utilizing a howitzer and stamp by plunging it foremost with liquid N. Phosphate buffer ( 100 millimeter, pH 6.0 ) 1.2 milliliter was assorted with the sample pulverization in a reaction tubing, followed by incubation for nightlong at 4EsC. Centrifugation was conducted after incubation at 12000 revolutions per minute for 15 min at 4EsC. Supernatant obtained after centrifugation was used straight for enzyme analysis ( Kumar et al. 2011 ; Kavitha and Umesha 2008 ) .
Peroxidase activity measuring
Peroxidase activity was assayed harmonizing to Ramanathan et Al. ( 2001 ) with alteration. Enzyme extracts for each sample were taken ( 0.4 milliliter ) so reacted with 3.2 milliliters 50 millimeters pyrogallic acid in 50 millimeter phosphate buffer ( pH 6 ) and 0.4 milliliter of 3 % H peroxide as an instigator. Directly, after adding the instigator, enzyme activity was measured as alteration of optical density at moving ridge length 430 nanometer for 1 minute utilizing a spectrophotometer ( Groppa et al. 1999 ) . Peroxidase activity was expressed as alteration of absorbance/min/gram fresh foliages ( Kavitha and Umesha 2008 ) .
Polyphenol oxidase activity measuring
Ninety six good micro home bases were used as a reaction container. Each well contained the reaction mixture of 150 Aµl 50 millimeter cathecol in 50 millimeter phosphate buffer ( pH 6.5 ) and 50 Aµl of enzyme infusion. Catechol was used as a substrate for the enzyme. First reading was done before incubation in a micro home base reader at 490 nanometer ( BIOTEK ) to acquire an initial optical density. Second reading was done after one-hour incubation at 38EsC. Each reading was repeated twice for the agencies value. Polyphenol oxidase activity units per foliage gm were expressed as alteration of optical density at 490 nanometer ( A490/gr foliage ) from the first and 2nd reading ( Heng-Moss et al. 2004 ; Ni et Al. 2001 ) .
Experimental design and statistical analysis
The experimental design was an wholly randomized factorial theoretical account ( 2 x 2 ten 2 ) . The two faba bean cultivars ( Gazira2 and Misr1 ) were combined with two infestation position ( Infested and uninfested ) , and two tissue type ( detached foliage and whole works ) . All the information was collected on three degrees of infestation continuance ( 1, 3 and 5 twenty-four hours after infestation ) . Six sample informations were collected as the reproduction figure.
Data analysis was conducted utilizing SAS ver. 9.2 ( SAS 2008 ) . Each parametric quantity in the experiment was tested for normalcy distribution utilizing PROC UNIVARIATE with Shapiro-Wilk method. PROC GLM was used to measure the factorial ANOVA 2x2x2 ( cultivar factor, infestation factor, and tissue factor ) followed by Mann Whitney-U for means separation utilizing PROC NPAR1WAY. Repeated steps analysis was conducted with PROC MIX to measure effects of cultivars across the infestation continuance twenty-four hours ( Madden et al. 1982 ) , square root ( x ) transmutation was applied anterior analysis ( Osborne 2010, Wilkinson and Douglas 1998 ) .
Peroxidase ( POD ) and Polyphenol oxidase ( PPO ) activity of two cultivars were analysed foremost utilizing factorial analysis 2x2x2, sing three chief effects, which were the cultivars ( Gazira2 and Misr1 ) , Infestation position ( Infested and Non infested ) and tissue type ( Detached and whole works ) ( Table 1. ) . This trial was conducted in order to acquire the thought on the presence of interaction among the interventions.
In overall ANOVA consequence, the theoretical account showed that merely first twenty-four hours of infestation both POD and PPO have a important P-value ( 0.014 and 0.005 severally ) , it means that the means weights of the eight groups were significantly different. While, in 3rd and 5th yearss of infestation, it did n’t demo the same consequence as in the twenty-four hours 1.
More detail interaction consequence was shown in table 2. This analysis measures whether or non three factors ( cultivar, infestation position and tissue type ) react otherwise. It showed that most of the chief factor, and its interaction has P-value than 0.05 on both POD and PPO. P-value less than 0.005 merely shown in the twenty-four hours POD and PPO ( 0.0003 and 0.001 ) and in the day3 for PPO ( P: 0.03 ) . Therefore, it can be concluded that merely cultivar consequence is an of import factor for consideration, and there is no premise of interaction. Further analysis so concern about the chief Mean comparing was conducted to uncover which cultivars have a more POD and PPO activity ( Table 3 ) .
Mann Whitney-U analysis as showed in table 3 corroborating the factorial analysis that merely cultivars factor gave a significantly different sing POD and PPO activity, particularly in one twenty-four hours and five yearss after aphid infestation. On one-day after aphid infestation, the P-value was 0.0006 and 0.002 for POD and PPO severally, in which Gazira2 has higher activity. While on five yearss after aphid infestation, the P-value was 0.16 and 0.009 for POD and PPO severally. Using alpha 0.1, we may reason that Gazira2 had significantly higher POD and PPO activity on five-day after aphid infestation. However, it was noticed in the tabular array 2. that numerically All POD and PPO value for Gazira2 was higher compared to Misr1.
Different tissue type and infestation position did non give any consequence on the POD and PPO activity most of P-value in twenty-four hours 1, 3 and 5 have no important different. However, it is of import to observe that in tissue type Detached foliage numerically has higher POD and PPO activity as compared to whole works tissue. While infestation position has a little consequence on the POD and PPO activity. Numerically aphid induced the lift of POD and PPO activity as shown in the twenty-four hours 5 after aphid infestation.
In order to acquire the overall position of the POD and PPO analysis over the clip, the perennial measuring analysis was conducted ( Table 4 ) .
For POD, there was two interesting fact that the P-value in all within capable ( Time ) trial and three between groups trial ( Cultivar, infestation and tissue ) was less than 0.005, bespeaking the enzyme activity for POD do alter over clip. Second, among the between groups test merely cultivars factor, which has P-value less than 0.005. For cultivar factor, the between groups ( Cultivars ) and within capable ( Time ) , P-value was 0.01 and & lt ; 0.0001 severally and the interaction between so was non significantly different. Both important value of between group and within capable trial indicating that the cultivar factor has important different over the clip ; accordingly, in the graph, the lines for the two groups were far apart and do alter over clip. as a consequence of no important interaction.
While for PPO, the between groups trial which has P-value less than 0.05 were merely Cultivars factor. The within capable trial ( Time ) and the interaction between cultivar and clip was non significantly different. This consequence bespeaking that PPO activity was different in different cultivars, but it di n’t reflects a strong alteration over the clip as indicated by the P-value for clip, which is 0.05.
In this survey, a Factorial analysis showed that the difference POD activity was related to cultivars factor, peculiarly in one twenty-four hours after aphid infestation. Mann Whitney-U trial confirmed that Gazira2 well has higher POD activity as compared to Misr1, particularly on one twenty-four hours after aphid infestation, while on 3 and 5 yearss after infestation they did non differ significantly. H numerically Gazira2 has the same tendency as on twenty-four hours 1. Across the twenty-four hours of infestation, repeated measuring analysis showed that the higher activity in Gazira2 over Misr1 was occurred over the clip ( 1, 3, 5 yearss of infestation continuance ) , particularly on POD. Upper ordinance of POD activity for immune cultivar was in understanding with the survey of A. medicaginis Koch on immune lucerne ( Wei et al. 2007 ) and chinch bug, Blissus occiduus Barber on American bison grasses Buchloe dactyloides ( Gulsen et al. 2010 ) .
Higher POD activity might explicate the possible mechanism of immune Gazira2 against aphid. As antecedently reported in feeding behaviour and biological surveies, aphid population figure was less with curriculum vitae. Gazira2 after 14 yearss feeding compared to Misr1. Feeding behaviour survey showed that this may be due to longer continuance of wave form F nowadays in Gazirea2, which bespeaking that aphids have more troubles when perforating their stylet into cells ( Soffan and Aldawood 2012ab, in reappraisal ) . Therefore, it was suggested that longer wave form F continuance had strong relationship with increased activity of POD, which act to beef up the cell walls. The immune character of curriculum vitae. Gazira2 seems a constituent non inducible, supported by statistically undistinguished value of the infestation position factors ( Table 2. ) .
Non inducible POD activities by infestation position were in contrary with Zhang et al. , ( 2008 ) which reported that Bemisia tabaci infestation could bring on PPO and POD of Cucumis sativus seedling. It was besides reported the addition of POD by Spilosoma virginica on the halophyte, Atriplex subspicata ( Nabity et al. 2006 ) . In our consequence, it was suggested that the degree of aphid infestation or the feeding behaviour of A. craccivora did non range degrees where it could bring on the overrun of ROS, which eventually may ensue from oxidative emphasis or cell harm, indicated by no symptom appeared in the faba bean after A. craccivora infestation. This fact, along with the determination that for symptomless aphid, feeding ( Rhopalosiphum padi ) did non arouse any alterations of POD activity in cereals compared to the control. While Russian Wheat aphid, D. noxia, which caused greensickness during eating, could arouse a nonuple addition of POD on susceptible “ morex ” barley and a threefold on immune “ Halt ” in comparing with control leaves. It was suggested that D. noxia feeding likely consequence in oxidative emphasis to the wheat works ( Ni et al. 2001 ) . Sing the tissue type, there were no important differences between whole works ( WP ) and detached foliage ( DL ) assays as showed in Table 1. Which means both types of tissue can be used to mensurate the POD activity..
PPO had similar activity as POD sing the response to aphid eating. Factorial analysis and Mann Withney-U analysis showed that cultivar factor was the lone factor impacting the PPO activity in which Gazira2 has higher PPO activity as compared to Misr1 ( Table 1. & A ; 2. ) . This consequence was in understanding with the increasing response of PPO activity in immune intercrossed poplar against the aphid Chaitophorus leucomelas Koch. ( Ramirez et al. 2009 ) besides in Macrosiphoniella sanbourni Gillette aphids on immune chrysanthemum works ( He et al. 2010 ) .
Through this survey, it might be concluded that Peroxidase ( POD ) and Polyphenol oxidase ( PPO ) enzymes have a possible to be used as a biochemical marker to bespeak the immune cultivars among faba bean cultivars.