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Two faba bean, Vicia faba L., cultivars , Gazira2 and Misr1, representing moderate resistant and susceptible respectively, were analyzed for peroxidase and polyphenol oxidase (PPO) activities induced by cowpea aphid, Aphis craccivora Koch. infestation. Two tissue types (whole plant and detached leaf), two infestation status (infested and uninfested), three infestation durations (1, 3 and 5 days) were considered in POD and PPO data analysis. Factorial analysis showed that only cultivar factor has a significant effect on both POD and PPO activity, especially on first day of aphid infestation (P:0.0003 and 0.001 respectively). While tissue type and infestation duration factors as main effect does not have any significant effect as well as no interaction among them. Mann Whiteny -U test 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 respectively. Repeated measure analysis showed that the POD and PPO activity on Gazira2 significantly higher as compared to Misr 1, additionally the POD activity changed significantly over the time in 1, 3 and 5 days after aphid infestation. Finally, it was suggested that higher activity of POD and PPO in cv. Gazira2 is strongly associated with their resistant characters.
Plants have an ability to respond to insects feeding by altering the state of their enzymes, by either constitutive or inducible (Hildebrand et al. 1986; Nabity et al. 2006; Chen et al. 2009; Giordanengo et al. 2010; He et al. 2010). These enzymes finally affect insect feeding success (Lukasik et al. 2012). Overproduction of harmful Reactive Oxygen Species (ROS) is the first mechanism in plants when they are injured by insect feeding, resulting in oxidative stress, which may cause cell and tissue damage (Michalak 2006; Gill and Tuteja 2010; Å½ivkoviÄ‡ et al. 2010). Plants overcome this situation by producing an efficient enzymatic antioxidant defense system which prevent and protect plant cells from oxidative damage 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 important role in plant stress caused by insect feeding (Lattanzio et al. 2006; Jaiti et al. 2009; Ramírez et al. 2009; He et al. 2010). Both enzymes are widely distributed among plant 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 oxidize a wide variety of molecules (Yoshida et al. 2003). Their activity is localized in the cytoplasm and cell wall (Heng-Moss et al. 2004; Chen et al. 2009). Peroxidase has a specific role in lignifications and strengthening the plant cell wall that is highly resistant to biodegradation (Schoemaker and Piontek 1996; Lee et al. 2007; Jaiti et al. 2009). PPO, also contributes to lignifications (Ralph et al. 2008) and together with POD it consumes oxygen and produces quinones, which may reduce plant digestibility 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 salinity (Wang et al. 1997; Nabity et al. 2006), adding jasmonic acid (Jaiti et al. 2009), soluble silicon (Gomes et al. 2005; Ranger et al. 2009), arsenate (Lin et al. 2008), or by introducing a gene to plants. Transgenic tobacco anionic peroxidase is an example which can express approximately up to 400 times higher peroxidase activity than corresponding tissues of wild-type plants (Behle et al. 2002; Dowd and Lagrimini 2006). Tomato TPX2 gene or sweet potato swpa1 gene are other examples in which over expression of the gene could confer increased salt-tolerance or oxidative-stress tolerance (Yoshida et al. 2003). The peroxidase engineered plant is possible since the peroxidase nucleotide had been sequenced (Botella et al. 1993).
The increased activities of these enzymes in a plant are considered as a resistant state of the plant to the insect pest (Wei et al. 2007; Ramírez et al. 2009; Gulsen et al. 2010). However, some other studies reported that there were no differences in the PPO activities between infested and uninfested buffalo 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 plant 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 elucidate the mechanism relies on resistant plant (Gulsen et al. 2010). This biochemical response has been used for selecting Chinch bug resistant turf grasses and black pecan aphid,Melanocallis caryaefoliae (Davis) resistance in pecan germplasm (Chen et al. 2009) also in aphid resistance of alfalfa (Wei et al. 2007).
The cowpea aphid, Aphis craccivora Koch. is one of the most important pests of Faba bean, Vicia faba L. (Laamari et al. 2008; Larocca et al. 2011). Pesticides are the most common way to control the aphids (Sadeghi et al. 2009). However, aphids have an ability to develop pesticide resistance due to the small size, high reproductive capacity and strong adversity adaptability. Non target effects of the use of pesticides are an important issue (Wei et al. 2007). The promising alternative is using plant resistance as a sustainable, safe and economical alternative (Heng-Moss et al. 2004).
Recently, some resistant 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 resistant to A. craccivora (Laamari et al. 2008), but caution should be addressed for resistant-breaking biotypes, which have occurred in several plants-aphid systems (Dogimont et al. 2010).
The availability of insect-resistant crops is still rare (Klingler et al. 2001). One of the problems is the undesirable large scale insect bioassays which have to be incorporated in breeding programs. Therefore, developing new means such as a biochemical marker for evaluating insect résistance efficiently is a challenge (Schoonhoven et al. 1998).
This study was conducted to establish a baseline biochemical information regarding 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 moderately resistant and susceptible cultivars, respectively. Colony development study for some faba bean cultivars showed that Gazira2 was less preferred for A. craccivora, indicated by significantly fewer numbers of aphids after 14-day infestation. Feeding behavior studies using DC-EPG, suggested that the longer duration of waveform F, which means more stylet penetration difficulties into the cell, was the possible resistant mechanism in Gazira2 (Soffan and Aldawood 2012ab, in review). The relations of the above results with antioxidative enzyme activity were expected to get an understanding of resistant mechanism in Gazira2.
Materials and methods
Peroxidase (POD) and Polyphenol oxidase (PPO) analysis were conducted in the Microbiology laboratory of Plant Protection Department, College of Food and Agriculture Sciences, King Saud University, Riyadh, Kingdom of Saudi Arabia. Faba bean cultivars were maintained in growth chambers with environment setting 26 ± 0.03ËšC, 44 ± 0.12 % RH (Means ± S.E), photoperiod of 16:8 L:D (recorded by HOBO data loggers, ONSET Co. USA).
Faba bean cv. Gazira2 and cv. Misr1 were used for experiments, representing the moderately resistant and susceptible cultivars, respectively. 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) growth medium after soaking in water for 48 h. After one week, seedlings were transplanted to plastic pots (d: 11 cm, h: 14 cm). Four granules per pot of complete fertilizer (N:12%, P:12%, K:17%; BASF-Asoco Agro) was applied in the growth medium once at seedling stage (nineteen days old plant). Watering was by drenching the pot for 150 ml once at every two days.
Both cultivars were prepared for POD and PPO analysis using 23-28 days old plants having five true leaves. Only three true leaves, consisted of two leaf blade, laid between the most base and upper part of the plant were used.
Cowpea aphids were used to induce the activity of Peroxidase and polyphenol oxidase in the plant. They were obtained from a colony which was collected from alfalfa plants grown in Al Amaria, Riyadh, Kingdom of Saudi Arabia (N46° 31' 5.5518" E24° 48' 40.179"). Single mother of an apterous adult aphid was used for the initiation of cowpea aphid culture on the faba bean cv. Misr. Before the beginning of the experiment, the cowpea aphid culture had been running for eight months. New faba bean seedlings were provided continuously to replace the old plant for the maintenance and continuous growth of aphid cultures.
Apterous nymphs were collected from the pure culture above. One hundred of the apterous nymphs were introduced on each true leave, distributed equally in each leaf blade. A 60 ml transparent plastic cups (d: 7 cm, H: 2.5 cm) were used as a leaf cage only in whole plant to prevent aphid escaping from the chosen leaf. Aphid infestation on detached leaf following the same procedure as in whole plant. The same 60 ml transparent plastic cup was used to accommodate the detached leaf. Detached leaf was kept its freshness by wrapping the leaf base with cotton. Water was filled into a plastic cup to keep the cotton wet. Uninfested plants were prepared with the same procedure as mentioned above without aphid infestation.
Three true leaves from each plant were sampled on 1, 3 and 5 days, respectively, to indicate the possible difference in PPO and POD activities across the infestation duration period. One leaf blade in each true leave was considered as a single sample for both whole plant and detached leaf. The sampled leaves were collected on a certain day according to infestation duration, and it was used directly for enzyme analysis. Each sample had six replications.
Fresh leaf blade having about 0.1-0.2 g were used for enzyme extraction. Each fresh leaf was grounded into powder using a mortar and pestle by immersing it first with liquid nitrogen. Phosphate buffer (100 mM, pH 6.0) 1.2 ml was mixed with the sample powder in a reaction tube, followed by incubation for overnight at 4ËšC. Centrifugation was conducted after incubation at 12000 rpm for 15 min at 4ËšC. Supernatant obtained after centrifugation was used directly for enzyme analysis (Kumar et al. 2011; Kavitha and Umesha 2008).
Peroxidase activity measurement
Peroxidase activity was assayed according to Ramanathan et al. (2001) with modification. Enzyme extracts for each sample were taken (0.4 ml) then reacted with 3.2 ml 50 mM pyrogallol in 50 mM phosphate buffer (pH 6) and 0.4 ml of 3% hydrogen peroxide as an initiator. Directly, after adding the initiator, enzyme activity was measured as change of absorbance at wave length 430 nm for 1 minute using a spectrophotometer (Groppa et al. 1999). Peroxidase activity was expressed as change of absorbance/min/gram fresh leaves (Kavitha and Umesha 2008).
Polyphenol oxidase activity measurement
Ninety six well micro plates were used as a reaction container. Each well contained the reaction mixture of 150 µl 50 mM cathecol in 50 mM phosphate buffer (pH 6.5) and 50 µl of enzyme extract. Catechol was used as a substrate for the enzyme. First reading was done before incubation in a micro plate reader at 490 nm (BIOTEK) to get an initial absorbance. Second reading was done after one-hour incubation at 38ËšC. Each reading was repeated twice for the means value. Polyphenol oxidase activity units per leaf gram were expressed as change of absorbance at 490 nm (A490/gr leaf) from the first and second reading (Heng-Moss et al. 2004; Ni et al. 2001).
Experimental design and statistical analysis
The experimental design was an entirely randomized factorial model (2 x 2 x 2). The two faba bean cultivars (Gazira2 and Misr1) were combined with two infestation status (Infested and uninfested), and two tissue type (detached leaf and whole plant). All the data was collected on three levels of infestation duration (1, 3 and 5 day after infestation). Six sample data were collected as the replication number.
Data analysis was conducted using SAS ver. 9.2 (SAS 2008). Each parameter in the experiment was tested for normality distribution using PROC UNIVARIATE with Shapiro-Wilk method. PROC GLM was used to evaluate the factorial ANOVA 2x2x2 (cultivar factor, infestation factor, and tissue factor) followed by Mann Whitney-U for means separation using PROC NPAR1WAY. Repeated measures analysis was conducted with PROC MIX to evaluate effects of cultivars across the infestation duration day (Madden et al. 1982), square root (x) transformation was applied prior analysis (Osborne 2010, Wilkinson and Douglas 1998).
Peroxidase (POD) and Polyphenol oxidase (PPO) activity of two cultivars were analysed firstly using factorial analysis 2x2x2, considering three main effects, which were the cultivars (Gazira2 and Misr1), Infestation status (Infested and Non infested) and tissue type (Detached and whole plant) (Table 1.). This test was conducted in order to get the idea on the presence of interaction among the treatments.
In overall ANOVA result, the model showed that only first day of infestation both POD and PPO have a significant P-value (0.014 and 0.005 respectively), it means that the means weights of the eight groups were significantly different. While, in 3rd and 5th days of infestation, it didn't show the same result as in the day 1.
More detail interaction effect was shown in table 2. This analysis measures whether or not three factors (cultivar, infestation status and tissue type) react differently. It showed that most of the main factor, and its interaction has P-value than 0.05 on both POD and PPO. P-value less than 0.005 only shown in the day POD and PPO (0.0003 and 0.001) and in the day3 for PPO (P: 0.03). Therefore, it can be concluded that only cultivar effect is an important factor for consideration, and there is no assumption of interaction. Further analysis then concern about the main Mean comparison was conducted to reveal which cultivars have a more POD and PPO activity (Table 3).
Mann Whitney-U analysis as showed in table 3 confirming the factorial analysis that only cultivars factor gave a significantly different regarding POD and PPO activity, especially in one day and five days after aphid infestation. On one-day after aphid infestation, the P-value was 0.0006 and 0.002 for POD and PPO respectively, in which Gazira2 has higher activity. While on five days after aphid infestation, the P-value was 0.16 and 0.009 for POD and PPO respectively. Using alpha 0.1, we may conclude that Gazira2 had significantly higher POD and PPO activity on five-day after aphid infestation. However, it was noticed in the table 2. that numerically All POD and PPO value for Gazira2 was higher compared to Misr1.
Different tissue type and infestation status did not give any effect on the POD and PPO activity most of P-value in day 1, 3 and 5 have no significant different. However, it is important to note that in tissue type Detached leaf numerically has higher POD and PPO activity as compared to whole plant tissue. While infestation status has a slight effect on the POD and PPO activity. Numerically aphid induced the elevation of POD and PPO activity as shown in the day 5 after aphid infestation.
In order to get the overall view of the POD and PPO analysis over the time, the repeated measurement analysis was conducted (Table 4).
For POD, there was two interesting fact that the P-value in all within subject (Time) test and three between groups test (Cultivar, infestation and tissue) was less than 0.005, indicating the enzyme activity for POD do change over time. Secondly, among the between groups test only cultivars factor, which has P-value less than 0.005. For cultivar factor, the between groups (Cultivars) and within subject (Time), P-value was 0.01 and <0.0001 respectively and the interaction between then was not significantly different. Both significant value of between group and within subject test indicating that the cultivar factor has significant different over the time; consequently, in the graph, the lines for the two groups were far apart and do change over time. as a result of no significant interaction.
While for PPO, the between groups test which has P-value less than 0.05 were only Cultivars factor. The within subject test (Time) and the interaction between cultivar and time was not significantly different. This result indicating that PPO activity was different in different cultivars, but it din't reflects a strong change over the time as indicated by the P-value for time, which is 0.05.
In this study, a Factorial analysis showed that the difference POD activity was related to cultivars factor, particularly in one day after aphid infestation. Mann Whitney-U test confirmed that Gazira2 considerably has higher POD activity as compared to Misr1, especially on one day after aphid infestation, while on 3 and 5 days after infestation they did not differ significantly. H numerically Gazira2 has the same trend as on day 1. Across the day of infestation, repeated measurement analysis showed that the higher activity in Gazira2 over Misr1 was occurred over the time (1, 3, 5 days of infestation duration), especially on POD. Upper regulation of POD activity for resistant cultivar was in agreement with the study of A. medicaginis Koch on resistant alfalfa (Wei et al. 2007) and chinch bug, Blissus occiduus Barber on buffalo grasses Buchloe dactyloides (Gulsen et al. 2010).
Higher POD activity might explain the possible mechanism of resistant Gazira2 against aphid. As previously reported in feeding behavior and biological studies, aphid population number was less with cv. Gazira2 after 14 days feeding compared to Misr1. Feeding behavior study showed that this may be due to longer duration of waveform F present in Gazirea2, which indicating that aphids have more difficulties when penetrating their stylet into cells (Soffan and Aldawood 2012ab, in review). Therefore, it was suggested that longer waveform F duration had strong relationship with increased activity of POD, which act to strengthen the cell walls. The resistant character of cv. Gazira2 seems a constitutive not inducible, supported by statistically insignificant value of the infestation status factors (Table 2.).
Non inducible POD activities by infestation status were in contrary with Zhang et al., (2008) which reported that Bemisia tabaci infestation could induce PPO and POD of cucumber seedling. It was also reported the increase of POD by Spilosoma virginica on the halophyte, Atriplex subspicata (Nabity et al. 2006). In our result, it was suggested that the level of aphid infestation or the feeding behavior of A. craccivora did not reach levels where it could induce the overproduction of ROS, which finally may result from oxidative stress or cell damage, indicated by no symptom appeared in the faba bean after A. craccivora infestation. This fact, along with the finding that for symptomless aphid, feeding (Rhopalosiphum padi) did not elicit any changes of POD activity in cereals compared to the control. While Russian Wheat aphid, D. noxia, which caused chlorosis during feeding, could elicit a nine-fold increase of POD on susceptible "morex" barley and a threefold on resistant "Halt" in comparison with control leaves. It was suggested that D. noxia feeding probably result in oxidative stress to the wheat plant (Ni et al. 2001). Regarding the tissue type, there were no significant differences between whole plant (WP) and detached leaf (DL) assays as showed in Table 1. Which means both types of tissue can be used to measure the POD activity..
PPO had similar activity as POD regarding the response to aphid feeding. Factorial analysis and Mann Withney-U analysis showed that cultivar factor was the only factor affecting the PPO activity in which Gazira2 has higher PPO activity as compared to Misr1 (Table 1.&2.). This result was in agreement with the increasing response of PPO activity in resistant hybrid poplar against the aphid Chaitophorus leucomelas Koch. (Ramírez et al. 2009) also in Macrosiphoniella sanbourni Gillette aphids on resistant chrysanthemum plant (He et al. 2010).
Through this study, it might be concluded that Peroxidase (POD) and Polyphenol oxidase (PPO) enzymes have a potential to be used as a biochemical marker to indicate the resistant cultivars among faba bean cultivars.