Protection Against Fusarium Head Blight: Important Defense Mechanisms Studied in Three Regenerated Egyptian Wheat Cultivars

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Journal of Stress Physiology & amp- Biochemistry, Vol. 12 No. 3 2016, pp. 18−43 ISSN 1997−0838 Original Text Copyright © 2016 by Hassanein, Hashem, Fahmy, Ibrahim, El Shihy and Qaid
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Protection Against Fusarium Head Blight: Important Defense Mechanisms Studied in Three Regenerated Egyptian Wheat Cultivars
1 12 Raifa A. Hassanein, Hanan A. Hashem, Ashraf H. Fahmy ,
Ahmed S. Ibrahim3, Osama M. El Shihy3, Ebtesam A. Qaid4
1 Department of Botany, Faculty of Science, Ain Shams University, Abbassia, Cairo, Egypt.
2 Agricultural Genetic Engineering Research Institute, 9 Gamaa St. Giza, Egypt.
3 Department of Plant Physiology, Faculty of Agriculture, Cairo University, Egypt.
4 Department of Biology, Faculty of Science, Taiz University, Yemen.
*E-Mail: Hashem. hanan@smail. com
Received June 1, 2016
Fusarium graminearum is one of the major global pathogens of cereals and is considered the main causal agent of Fusarium head blight (FHB) disease in wheat. F. graminearum leads to severe losses in grain yield and quality of wheat. In the present study, immature embryos of three Egyptian wheat cultivars (Giza 164, Sids 1 and Bani Suef 6) have been used to highlight the physiological changes in wheat plants in response to treatment with different concentrations of Fugal Culture Filtrate (5%, 10%, 20% and 40%). Samples were taken at 5, 10 and 15 days after inoculation. The three studied wheat cultivars exhibited different responses for inoculation with different concentrations of FCF. However, inoculation with 20% FCF significantly increased the activities of antioxidant enzymes (catalase, peroxidase and ascorbate peroxidase) in shoots of wheat cultivars compared with uninoculated ones. In addition, proline and secondary metabolites (total phenol and flavonoids) significantly increased throughout the experimental period. Our results also proved that in addition to the common protective mechanisms detected in all the infected cultivars there are cultivar — dependent physiological changes exhibited by wheat plant during abiotic stress.
Key words: Antioxidant enzymes, Fusarium graminearum, phenylalanine ammonia lyase, regenerated plant, secondary metabolites, wheat.
Abbreviations: APX: Ascorbate peroxidase, CAT: catalase, FCF: Fusarium culture filtrate, FHB: Fusarium head blight, LSD: Least significant difference, MS: Murashige and Skoog, PAL: Phenylalanine ammonia lyase, POX: Peroxidase, PR: Pathogenesis related proteins, PVP: Polyvinyl pyrrodine, ROS: Reactive oxygen species.
Wheat is one of the most important crops and is counted among the & quot-big three& quot- cereal crops (rice, wheat and maize). Wheat is produced on more than 18% of the arable land in the world (FAOSTAT data 2014). Wheat is one of the most abundant sources of energy and nourishment for mankind.
Wheat holds a number one position in human nutrition within the world. Therefore, sustained wheat production is necessary to make sure world food security. Wheat subjected lots of biotic and abiotic stresses- plant diseases are a major impediment to the production and quality of important foodstuffs. The International Maize and Wheat Improvement Center (CIMMYT) has identified Fusarium head blight (FHB) as the major factor limiting wheat production in many parts of the world. FHB has a great economic impact on the wheat industry due to the reduced grain yield and quality (Kazan et al., 2012)
The genus Fusarium, common soil saprophytes or plant pathogens, comprises a large number of species. They are common pathogens in small grains of wheat, barley, oats, triticale and rye and cause various disease such as: foot and root rot, crown rot, vascular wilt, seedling blight, cereal ear rot, fruit rot and Fusarium head blight (FHB) (Naz, 2007). FHB of small grains is caused primarily by Fusarium graminearum and Fusarium culumolum that are ascomycetes.
There are factors that affect FHB management such as selecting resistant wheat varieties, practicing cultivation, planting high-quality seed, and fungicide applications. Producers are encouraged to select varieties that have some level of FHB resistant.
When plants are exposed to pathogens, only small number of pathogens is able to provoke disease in a certain species (compatible response), whereas most pathogens aggressors are recognized and blocked in their penetration by plant-defenses (incompatible response) (De Gara, 2003). Plants produce various stress compounds by activating a wide variety of productive mechanisms designed to prevent pathogen replication and spread. The expression of antimicrobial proteins in plants that are not normally produced may have a greater potential to limit pathogen infection or growth. In compatible or incompatible interactions between plants and microorganisms, salicylic acid pathway, jasmonic acid pathway and shikimic acid pathway are involved in plant defense. Shikimic acid pathway is responsible for the secondary metabolites such as lignin, phenolic phytoalexins that adding mechanical rigidity and strength to cell walls and providing barriers to inhibit pathogens (Zheng et al, 2005).
The production of reactive oxygen species (ROS) is one of the earliest cellular responses following successful pathogen recognition. Generally when the level of antioxidants processes and detoxification mechanisms are lesser than the level of ROS accumulation, the cellular damage occurs through lipid peroxidation, membrane fatty acids and ruin macromolecules like pigments, proteins, nucleic acids and lipids (Torres et al., 2006). The rapid accumulation of plant ROS at the pathogen attack site, a phenomenon called oxidative burst, is toxic to pathogens directly and can lead to a hypersensitive response that results in a zone of host cell death which prevent further spread of pathogens. Plants have evolved
enzymatic and non-enzymatic defense mechanisms in order to reduce oxidative damages by detoxifying free radical (Borden and Higgin, 2002- EL-Khallal, 2007- Venkatesh et al., 2013).
Phenyl alanine ammonia lyase (PAL) is the most important enzyme in phenylpropanoid pathway catalyzing the transformation of L-phenylalanine into trans-cinnamic acid by deamination, which is the prime intermediary in the biosynthesis of phenolics, phytoalexin and lignin. A very rapid induction of PAL could be occurred during biotic and abiotic stresses. After pathogen infection, PAL activity increased leading to the formation of phenypropanoid phytoalexines (Boubakri et al., 2013).
Proline can serve as a protector of enzyme denaturation, as a stabilizer of protein synthesis machinery. Proline plays an important role in protecting the subcellular structures and mediating osmotic adjustment in plant under stressed conditions (Bagdi and Shaw, 2013). Accumulation of the amino acid proline is one of the indications to plant adaptation to unfavorable conditions. It regulates the osmotic equilibrium between the cytoplasm and vacuole. Proline can now be added to an elite list of non-enzymatic antioxidants that microbes, animals, and plants need to counteract the inhibitory effects of ROS so that proline acting as s singlet oxygen quencher (Abdel-fattah and Asrar, 2012). Moreover, proline has been shown to function as a molecular chaperone able to protect protein integrity and enhance the activities of different enzymes (Rajendrakumar et al., 1994).
However, our understanding of these defensive mechanisms is still limited. The present work aimed to identify the induced defense mechanisms in wheat plants
in response to inoculation with Fusarium graminearum. Induced resistance could be exploited as an important tool for the pathogen control. The elicitors of induced responses can be sprayed on crop plants to build up the natural defense system against damage caused by that pathogen. The induced responses can also be engineered genetically, so that the defensive compounds are constitutively produced in plants those are challenged by that pathogen.
MATERIALS AND METHODS
Plant materials. Seeds of two bread wheat (Triticium aestivium L.) cultivars- Giza 164, Sids 1 and one durum wheat (Triticium durum L.) cultivar- Bani Suef 6 were kindly provided by Field Crop Research Institute (FCRI), Agricultural Research Center (ARC), Giza, Egypt.
Fusarium graminearum was obtained from Mycological Center, Faculty of Science, Assiut University, Egypt.
Growth conditions. This experiment was carried out in the Plant Cellular and Molecular Genetics Laboratory (PCMG), Agricultural Genetic Engineering Research Institute (AGERI), Agricultural Research Center (ARC), Giza, Egypt. Immature caryopsis of the three Egyptian wheat cultivars Giza 164, Sids 1 and Bani Suef 6 were collected approximately two weeks post anthesis. Immature grains for each cultivar were surface sterilized (by soaking for 1 min in 70% (v/v) ethanol followed by 20% commercial Clorox (5. 25% Sodium hypochlorite) supplemented with few drops of Tween 20, and washed five times with sterile ddH2O). Immature embryos were aseptically isolated and cultured with the scutellum side up onto the callus induction medium. Consequently, immature embryos were cultured
in the dark at 25 °C on callus induction medium for two weeks then subculture for another two weeks. Four week-old calli of different cultivars were transferred onto plant regeneration medium at 24 °C under 16 h /8 h light/ dark cycle for a month with subculture of two weeks interval.
The regenerated plantlets of each wheat cultivar were transferred into treated-rooting medium inoculated with F. graminearum extract at different concentrations (5, 10, 20 and 40% v/v) and kept at the same condition of regeneration phase. Physiological parameters were determined including: total soluble proteins- the activity of antioxidant enzymes (catalase- CAT, Ascorbate peroxidase- APX and peroxidase- POX) — PAL activity, proline content- phenols content and flavonoids content at 5, 10 and 15 days after inoculation. Statistical analysis of physiological parameters (5 replicates each) was analyzed using Duncan test at 5% of probabilities according to SPSS program (version 17).
Wheat Regeneration
Isolation and culturing of the explant. Immature embryos for each cultivar were aseptically isolated with forceps under sterile conditions in the laminar air — flow hood. Fifty immature embryos were cultured with the scutellum side up onto the callus induction medium modified for wheat cell culture (Weeks et al. 1993- Fahmy et al. 2006). Basically, it contains MS (Murashige and Skoog, 1962) medium salt, supplemented with 2 mg/L 2,4-D as a source of auxin, 0. 15 g of L-Asparagine, 0. 1g of myo-inositol, 50 mg thiamine-HCL, 20 g sucrose and adjusted to 5.8 pH and solidified by 2.5 g/L phytagel. Immature embryos were cultured in the dark at 25 °C on the callus induction medium for two weeks for infection.
Regeneration Two week-old calli were transferred onto plant regeneration medium in Petri dishes containing 50 ml of MS medium (MS salts with vitamins, 30 g / L sucrose and 3 g / L phytagel) supplemented with 1.5 mg / L TDZ at 24 °C and kept under 16 h / 8 h light / dark cycle. Calli of different cultivars were subcultured twice times with two weeks interval, then the regenerated plantlets of each calli were transferred onto rooting medium which was half strength MS medium (MS salts with vitamins, 30 g / L: sucrose and 2.5 g / L phytagel), and kept in the growth chamber under the same light and growth conditions.
Fungus maintenance medium. The Fusarium graminearum was cultured and maintained on Czapek'-s medium and incubated at 28 ± 1 °C in the dark. The Czapek'-s medium contained for each liter: 2 g NaN03- 1 g K2HPO4- 0.5 g MgSO4. 7H2O- 0.5 g KCl- 0. 01 g Fe-EDTA- 5 g yeast extract- 30 g sucrose and solidified with 15 g agar (Jiao et al. 2008).
Culture of fungus inoculum. The fungus was sub-cultured on Petri-dishes containing sterilized, solidified Czapek'-s medium and kept for 7 days in the dark growth chamber at 28 ± 1 °C. Sterile inoculation loop were used to transfer a F. graminearum colonies from the Petri-dish into sterile flasks, each with 250 ml of liquid Czapek'-s medium without antibiotics and phytohormones. All flasks (12 flasks) were incubated in dark for two weeks at 28 ± 1 °C in shaking orbital incubator at 100 rpm to provide aeration.
Fungus extract. At the end of the incubation period, the culture was filtered through Whatman number one filter paper to remove conidia and mycelia. The pH of filtrate was adjusted to 5.8 and then passed through
micro-Millipore filter to sterilize the filtrate, yielding of F. graminearum extract which was used as stock for the inoculation.
Inoculation and incubation. Rooting medium was prepared by replacing part of ddH2O with equivalent volume of culture filtrate. The pH of the medium was adjusted to 5.8 before autoclaving, cooled (60 ± 4°C) then supplemented with different concentrations of F. graminearum extract (5. 0, 10. 0, 20.0 and 40.0% v/v), under aseptic conditions. Four weeks-old regenerated plants from each cultivar were transferred into treated-rooting medium (with F. graminearum culture filtrate) or into non-treated rooting medium (without F. graminearum culture filtrate) as control and kept at the same condition of regeneration phase. Effect of fungal filtrate was studied by collecting the samples at 5, 10 and 15 days after inoculation.
Protein determination. Total soluble proteins were extracted from the samples. The method described by Guy et al. (1992), the shoot tissue was ground in liquid nitrogen and extracted with buffer containing ice-cold 50 mM Tris-HCl, pH 7. 5- 2 mM EDTA and a 0. 04% (v/v) 2-?-mercaptoethanol. The homogenate was centrifuged at 14 000 rpm for 20 min at 4 °C. Total protein amount was determined using the dye-binding assay of Bradford (1976) with bovine serum albumin as a standard.
Determination of antioxidant enzymes activities
Extraction of enzymes. Samples (0.1 g of shoots) were ground and homogenized in 4 ml ice-cold extraction buffer (100 mM KH2PO4 / K2HPO4 (pH 7. 8), 60 mg polyvinyl-pyrrolidone (PVP) and 5 mM ascorbate. The homogenate was centrifuged at 12,000 x g for 15 min at 4 °C. The supernatant was used for the determination of
peroxidase (POX), ascorbate peroxidase (APX) and catalase (CAT) activities.
Peroxidase. POX (EC 1. 11.1. 7) activity was assayed by the method of Velikova et al. (2000). The reaction mixture contained 50 mM potassium phosphate buffer (pH 7. 0), 0. 2% of guaiacol (w/v) and 0. 04 ml of enzymes extract. The reaction mixture was incubated at room temperature for 5 min after the addition of 3 mM H2O2. The absorbance was then measured at 470 nm. The activity of POX was calculated from the rate of formation of guaiacol dehydrogenation product (GDHP) using the extinction coefficient of 26.6 mm-1 cm-1, and the activity was expressed as pM GDHP / min / g fresh weight.
Catalase. CAT (EC 1. 11.1. 6) activity was measured according to Aebi (1984). The reaction mixture contained 50 mM potassium phosphate buffer (pH 7. 0), 0.1 ml enzyme extract and 0. 035% H2O2. The activity of CAT was calculated based on the decomposition of H2O2 measured as a decline in the absorbance at 240 nm. The activity was calculated using the extinction coefficient of 40. 84 mm-1 cm-1, and expressed as pM H2O2 reduced / min / g fresh weight.
Ascorbate peroxidase. For APX (EC 1. 11.1. 11) assay, the reaction mixture contained 0.5 mM ascorbic acid, 0.1 mM EDTA and 0.1 mL of enzyme extract. The reaction was initiated when adding 1.5 mM H2O2. The absorbance of the reaction mixture was measured at 290 nm 2 min after H2O2 was added. The activity of APX was calculated using the extinction coefficient of 3. 98 mm-1cm-1and the activity was expressed as pM ascorbate oxidized / min / g fresh weight (Asada, 1992).
Assay of Phenylalanine Ammonia Lyase. To
determine Phenylalanine ammonia lyase (PAL) activity (EC 4.3.1. 5) — 0. 15 g of shoot was homogenized in 1.5 ml of ice-cold 0. 1M sodium borate buffer, pH 7.0 containing 1.4 mM of 2-mercaptoethanol and 0. 1g of insoluble PVP. The homogenate was centrifuged at 12,000 x g for 30 min and the supernatant was collected for enzyme assay. PAL activity was determined based on the rate of conversion of L-phenylalanine to frans-cinnamic acid at 290 nm (Dickerson ef al., 1984). Sample containing 0.4 ml of enzyme extract was incubated with 0. 5ml of 0. 1M borate buffer, pH 8.8 and 0.5 ml of 12 mM L-phenylalanine in the same buffer for 30 minutes at 30 °C. The activity of PAL was expressed as pM frans-cinnamic acid synthesized / min / g fresh weight.
Measurement of free proline. Proline content in shoots and roots of the three wheat cultivars was determined according to the method of Bates et al. (1973). Fresh weight of tissue was ground with 10 ml of 3% sulphosalicylic acid then the homogenate was filtered through two sheets of 3 mm Whatman paper. Two ml of the filtrate was mixed with 2 ml of ninhydrin reagent (1. 25 g ninhydrine in 3 ml glacial acetic acid and 20 ml 6 M phosphoric acid with agitation, until dissolved) and 2 ml of glacial acetic acid. The reaction mixture was incubated in water bath for 1 h at 100 °C then tube was immediately transferred to ice bath. Four ml of the toluene was added to the mixture, mixed vigorously with a test tube stirrer for 15−20 sec. The chromophore containing toluene was aspirated from the aqueous phase, warmed to room temperature and absorbance read at 520 nm using toluene as a blank. The results were expressed as pg /g fresh weight.
Determination of phenol content. Shoot tissue (0. 1
g) was extracted with chilled 1.5 ml 80% methanol and 1% HCl at room temperature with shaking for 2 h. The extract was centrifuged at 3000 g for 10 min. Supernatant was used to determination phenol content.
Free phenol content in the extract of three wheat cultivars were immediately determined using the Folin-Ciocalteu'-s method by Hung et al. (2009), with a modification. To the 150 pL of the extract 750 pL of distilled water were added. This solution was oxidized with 300 pL of Folin-Ciocalteu'-s reagent and then neutralized with 300 pL of 20% sodium carbonate solution (20% w/v). Thoroughly mixed samples were allowed to stand for 120 min at dark place at ambient temperature. The solution was centrifuged for 10 min at 12 000 rpm and the absorbance of the clear supernatants was measured at 765 nm using a spectrophotometer (Spectro. DioRAD Smartspec™ 3000). The content of phenolics in extracts was expressed in terms of gallic acid equivalent (mg of GA / g of extract).
Determination of Flavonoids. Flavonoids were extracted according to the method reported by (Abeysinghe et al., 2007). Shoot tissue (0.1 g) was ground with liquid nitrogen, 1 ml of methanol-water (80: 20, v/v) was added, then covered with aluminium foils and finally placed into water bath for extraction at 80 °C for 1 h. The solution was filtered through Whatman filter paper and filtrate was evaporated in an oven at 60 ° C. The residue was dissolved in the 1 ml of methanol (80%).
The content of flavonoids in the examined plant extracts was determined using spectrophotometric method (Quettier et al., 2000). The sample contained 1 ml of methanol solution of the extract in the concentration of 1 mg / ml and 1 ml of 2% AlCb solution dissolved in
methanol. The samples were incubated for an hour at room temperature. The absorbance was determined using spectrophotometer at 415 nm. The content of flavonoids in extracts was expressed in terms of rutin equivalent (mg of RU / g of extract).
RESULTS
Total soluble proteins. Table (1) showed that inoculation wheat plants cv. Giza 164 with F. graminearum extract (5, 10, 20 and 40% v/v) caused, in general, a highly significant reduction in total soluble protein during the experimental period. The low concentration of fungal culture filtrate (10%) recorded the highest reduction at the three ages. The highest reduction was 41. 14% at five days from inoculation. But the 20% of fungal culture filtrate showed different behavior, the total soluble protein was slightly increased at the second and third ages of experiment reaching 6. 83% and 19. 23% over the control plants, respectively.
On the other hand, inoculation with F. graminearum extract at different concentrations caused significantly increases in total soluble protein in the shoots of wheat plants cv. Sids1. The highest amount of soluble protein was observed at 20% of fungal culture filtrate at the third age recording 74. 36% over the control plants.
In the case of Triticium durum L. cv. Bani Suef 6, total soluble protein increased gradually in the shoots of wheat cultivar that inoculated with different concentrations of F. graminearum extract. The highest increment was at 20% of F. graminearum extract at 5, 10 and 15 days after inoculation reaching 66. 75%, 82. 02% and 146. 26% over those of the uninoculated plants, respectively. At 40% of F. graminearum extract, shoots of wheat cultivar showed increment in the soluble protein but this
increment was markedly lower compared with 20% of F. graminearum extract at the three ages.
Antioxidant enzymes
Catalase. Catalase activity was significantly increased in shoots of the three studied wheat cultivars Giza 164, Sids 1 and Beni suef 6 in response to inoculation. The maximum catalase activity was recorded in most cases at 20% of F. graminearum extract (Figure 1). The increase was evaluated by 49. 63% and 102. 48% in Giza 164 after 10 and 15 days of inoculation, respectively, 132. 86% in Sids 1 after 15 days of inoculation and 50. 26%, 77. 23% and 112. 02% at 5, 10 and 15 days from inoculation, respectively in cultivar Bani Suef 6.
Meanwhile, 40% FCF caused the maximum increase in catalase activity (89. 64% and 38. 12%) in cultivar Sids 1 after 5 and 10 days of inoculation and in Giza 164 after 5 days of inoculation (58. 52% over the control value.
Peroxidase. Data in Figure (2) showed changes in the guaiacol peroxidase activity in uninoculated wheat plants of the three cultivars during the experimental period.
The inoculation with F. graminearum extract induced significant increase in the activity of guaiacol peroxidase in shoots of wheat cultivar Giza 164. The maximum value of guaiacol peroxidase activity was observed at 40% in the first age (12. 19%), at 20% in the second and third ages (20. 24% and 42. 98%, respectively) as compared with untreated plants. However, shoots of wheat cultivar Giza 164 inoculated with 5% of F. graminearum extract exhibited decreases in the guaiacol peroxidase activity at first and second
ages evaluated by 6. 49% and 13. 65%, respectively below the control value.
In case of Trificium aesfivium L. cv. Sids 1, the shoots that inoculated with different concentrations of F. graminearum extract showed general increases in the activity of guaiacol peroxidase. The high activity of guaiacol peroxidase was shown at 40% of F. graminearum extract at the 5 and 10 days after inoculation reaching 76. 22% and 85. 49%, over the control, respectively. However, in the third age, the high activity of guaiacol peroxidase recorded at 20% of F. graminearum extract 97. 06% increase over that of the uninoculated plants.
In contrast, Trificium durum L. cv. Bani Suef 6, shoots that inoculated with different concentration of F. graminearum extract (5%, 10%, 20% and 40%) showed reduction in the activity of guaiacol peroxidase at the first age followed by increases at the second and third ages.
Ascorbate peroxidase. From results presented in Figure (3) it is obvious that treatment with different concentrations of F. graminearum extract caused significant marked increases in the activity of ascorbate peroxidase in the shoots of wheat cultivar Giza 164 reaching the maximum level (247. 19%) at 20% FCF at the third age.
The inoculation with different concentration of F. graminearum extract stimulated ascorbate peroxidase activity in the shoot of wheat cultivar Sids 1 as compared with uninoculated plants. Data in Figure (3) showed that the activity of ascorbate peroxidase gradually increased in the shoot inoculated with F. graminearum extract at 5%, 10% and 20% and decreased in response to
treatment with 40% of the fungal extract at 5, 10 and 15 days after inoculation.
In case of Beni Suef 6 cultivar, the results showed that the inoculation with F. graminearum extract at different concentrations caused a gradual increase in the ascorbate peroxidase activity in the inoculated shoots along the experimental time. The highest value of enzyme activity observed at 20% of F. graminearum extract recording 26. 31%, 101. 96% and 144. 23% at the first, second and third age, respectively above that of the control plants.
Phenylalanine ammonia lyase. Figure (4) showed that there was a significant increase in the PAL activity in the uninoculated shoots of the three wheat cultivars at the three ages (5, 10 and 15 days). The inoculation with F. graminearum extract significantly increased the activity of PAL in the shoots of wheat Giza 164 cultivar from the 5th day up to 15th day after inoculation. The concentration (10%) of F. graminearum extract exhibited a high enhancement (55. 16%) in the PAL activity at the first age, while higher increases were observed in the PAL activity at the second (63. 32%) and third ages (134. 89%) using 20% of F. graminearum extract.
In the case of inoculated shoots of wheat cultivar Sids 1, the maximum PAL activity was detected in plant infected with 10% FCF at 5 days after inoculation, 40% FCF at 10 DAI and 20% at 15 DAI and was estimated by 83. 62%, 89. 89% and 227%, respectively over the control value.
In contrast inoculation of Trificium durum L. cv. Bani Suef 6, with 5, 10 and 40% FCF caused, in most cases,
a significant reduction in PAL activity. Whereas, 20% of F. graminearum extract caused a significant increase in PAL activity by 11. 21%, 8. 32% and 29. 95% at 5th, 10th and 15th days, respectively as compared with the control plants.
Proline content. The data presented in Table (2) showed that proline content significantly increased in inoculated shoots of the three cultivars of wheat plants throughout the experimental period. At 5 DAI the maximum proline content was detected in wheat shoots treated with 40% FCF and was calculated by 245% in Giza 164, 203% in Sids 1 and 227% in Bani Suef 6 compared to the corresponding control value. Whereas, at the end of the experimental period (15 DAI) the highest proline content was obtained in response to treatment with 20% FCF and was estimated by 349%, 313% and 217% compared to corresponding control in cultivars Giza 164, Sids 1 and Bani Suef 6, respectively.
Phenol content. It is clear from Table (3) there was slight change in the phenol content in uninoculated shoots of three wheat cultivars. Interesting, At 5th day after inoculation in bread wheat cultivars, Giza 164 and Sids 1 the phenol content decreased showing the highest reduction in the phenol content at 40% of F. graminearum extract (29. 71% in Giza 164 and 22. 35% in Sids1). At 10th day in the bread wheat, the phenol content significantly increased in inoculated shoots and reached its maximum level (49. 70%) at 20% of F. graminearum extract in case of cultivar Giza 164, in this meantime, the highest level of phenol in case of cultivar Sids 1 was 26. 76% at 40% of F. graminearum extract as compared with uninoculated plants. At 15th day after inoculation with different concentrations of F.
graminearum extract in Giza 164 and Sids 1 cultivars, the maximum level of phenol content was shown at 20% of F. graminearum extract in both cultivars reaching 189. 43% in Giza 164 and 123. 22% in Sids 1.
In case of Triticium durum L. cv. Bani Suef 6, inoculated shoots showed different behavior at 5th day after inoculation with F. graminearum extract. The highest level of phenol was 9. 38% at 10% of F. graminearum extract and the reduction of phenol content exhibited 3. 13% at 40% of F. graminearum extract as compared with control plants. At 10th day and 15th day, the inoculated shoots exhibited slight increase in the phenol content at 20% of F. graminearum extract with a maximum of 22. 86% and 40. 04%, respectively over the control level.
Flavonoid content. The data presented in Table (4) showed that in case of Triticum aestivum L. cv. Giza164 and Sids 1, inoculated shoots of both cultivars exhibited reduction in the flavonoid content with different concentration of F. graminearum extract at 5th day after inoculation reaching 43. 64% at 20% of F. graminearum extract in Giza 164 and 29. 45% at 10% of F. graminearum extract in Sids 1 as compared with control plants. At 10th and 15th days after inoculation, the flavonoid content increased in the shoots of both wheat cultivars. The highest level of flavonoid content in both wheat cultivars was shown with 20% of F. graminearum extract recording 36. 94% at 10th day & amp- 75. 84% at 15th day after inoculation for cv. Giza 164, and 22. 41% at 10th day and 127. 93% at 15th day after inoculation in cv. Sids 1 as compared with uninoculated plants.
In case of Triticium durum L. cv. Bani Suef 6, the
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Table 1. Effect of different concentrations ot F. graminearum culture filtrate on (Trificium aestivium L.) cv. Giza 164, cv. Sids 1 and (Trificium durum L) inoculation. Values listed are expressed as mg / g fresh weight.
the total soluble protein of shoots ol wheat cv. Bani Suef 6 at 5,10 and 15 days after
Cultivar Treatment 5 DAI % ot control 10 DAI % of control 15 DAI % of control
Giza 164 Control 5. 022 ± 0. 076 a 100 5. 241 ± 0. 047 b 100 5. 354 ± 0. 084 b 100
FCF 5% 3. 954 ± 0. 196 c 78. 73 4. 344 ± 0. 039 d 82. 88 4. 821 ± 0. 067 d 90. 04
FCF 10% 2. 956 ±0. 112 d 58. 86 3. 339 ± 0. 084 e 63. 70 4. 080 ± 0. 089 e 76. 20
FCF 20% 4. 570 ± 0. 108 b 90. 99 5. 599 ±0. 196 a 106. 83 6. 384 ±0. 104 a 119. 23
FCF 40% 3. 898 ± 0. 154 c 77. 61 4,582 ±0. 113 c 87,42 5. 165 ± 0. 076 c 96. 46
LSD 5% 0. 175 0. 139 0. 106
Sids 1 Control 2. 304 ± 0. 057 d 100 2. 178 ± 0. 132c 100 2. 329 ± 0. 039 c 100
FCF 5% 3. 245 ± 0. 113 b 140. 84 3. 308 ±0. 164 a 151. 88 3. 452 ± 0. 089 b 148. 21
FCF 10% 3. 446 ± 0. 067 a 140. 56 3. 321 ±0. 174 a 152. 47 3−980 ± 0. 047 a 170. 88
FCF 20% 2. 913± 0. 054 c 126. 43 2. 975 ± 0. 094 b 136. 59 4. 061 ± 0. 057 a 174. 36
FCF 40% 3. 270 ± 0. 060 b 141. 92 3. 333 ± 0. 049 a 153. 03 3. 547 ± 0. 039 b 152. 29
LSD 5% 0. 089 0. 165 0. 069
Bani Suef G Control 1. 167 ± 0. 037 e 100 1,224 ± 0. 018 e 100 1. 193 ± 0. 086 d 100
FCF 5% 1. 450 ± 0. 049 d 124. 25 1,556 ±0. 021 d 127. 12 2. 090 ± 0. 049 c 175. 18
FCF 10% 1. 782 ± 0. 047 b 152. 69 1,921 ± 0. 037 b 156. 94 2. 693 ±0. 117 b 225. 73
FCF 20% 1. 946 ±0. 060 a 166. 75 2,228 ± 0. 029 a 182. 02 2. 938 ± 0. 086 a 246. 26
FCF 40% 1. 638 ± 0. 049 c 140. 35 1. 852 ± 0. 028 c 151. 30 1. 933 ± 0. 092 c 162. 02
LSD 5% 0. 056 0. 040 0. 113
FC F: Fu n gal cu I ture f i Itrate DA I: Day s after inocu lation Values are mean of three samples ± SD.
Different letters indicate significant variation between vertical readings for each cultivar at P = 0. 05 according to Duncan'-s multiple range test
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Table 2. Effect of different concentrations of F. graminearum culture filtrate on proline contentof shoots of wheat (Triticium aestivium L.) cv. Giza 164, cv. Sids 1 and (Triticium durum L) cv. Bani Suef 6 at 5,10 and 15 days after inoculation. Values listed are expressed asig / g fresh weight,
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Cultivar Treatment 5 DAI % of control 10 DAI % of control IS DAI % of control
Giza 164 Control 0. 946±0,039 e 100 1. 3 810. 035 e 100 1,165±0,040d 100
FCF 5% 1 142±0. Q34d 120. 71 1. 53 410. 053 d 147. 78 0. 969±0. 035 c 83. 17
FCF 10% 1. 661 ±0. 067 c 175. 58 2. 099±0. 072 c 202. 21 3. 229± 0. 072 b 277. 16
FCF20% 2. 134±0. 053b 225. 58 2. 918±0. 087a 281. 11 4. 071± 0. 106 a 349. 44
FCF 40% 2. 318±0,069 a 245. 03 2. 480±0. 052 b 238. 92 2. 814± 0,105 c 241. 54
LSD 5% 0. 069 0. 080 0. 098
Sids 1 Control 1. 545±0. 052e 100 1. 543±0. 055e 100 1,684±0. 072 e 100
FCF 5% 2. 318±0,034 c 150. 03 2. 313±0. 036c 149. 90 3, l83±0. 103d 189. 01
FCF 10% 1. 995±0. 052d 129. 12 1. 985±0. 069d 128. 64 3. 806±0. 069 b 226. 00
FCF20% 2. 768±0,069 b 179,15 2. 738±0. 117 b 177. 44 5,271 ±0. 053 a 313. 00
FCF 40% 3. 137+0. 072 a 203. 04 3. 143+0. 083 a 203. 43 3. 425+0. 060 c 203. 38
LSD 5% 0. 069 0. 098 0. 089
Bani Suef 6 Control 2. 468±0,053 d 100 2. 676±0. 040 e 100 2. 837±0. 103e 100
FCF 5% 3. 898±0,122 c 157,94 3. 402±0. 053d 127. 13 4. 175±0. 121 d 147. 16
FCF 10% 4. 763±0. 106b 192. 99 5. 386±0. 072 b 201. 27 6. 447±0. 052 b 227. 24
FCF20% 3. 783±0,144 C 153. 28 5. 032±0. 052a 225. 41 7,704±0. 087a 271. 55
FCF 40% 5. 617±0. 105 a 227. 59 4. 959+0,087 C 185. 31 5. 790±0. 072 c 204. 08
LSD 5% 0. 139 0. 080 0. 113
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Values are mean of three samples ± SD, Different letters indicate significant variation between vertical readings for each cultivar at P multiple range test.
FCF: Fungal culture filtrate, DAI: Days after inoculation.
= 0. 05 according to Duncan'-s
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Table 3. Effect ot different concentrations of F. graminearum culture filtrate on phenol contentof shoots of wheat [Triticium aestivium L.) cv, Giza 164, cv. Sids 1 and (Triticium durum L.) cv. Bani Suef 6 at 5, 10 and 15 days after inoculation. Values listed are expressed as mg / g fresh weight.
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Cultivar Treatment 5 DAI % of control 10 DAI % of control 15 DAI % of control
Giza 164 Control 0. 771 ±0. 014 b 100 0. 845 ±0. 004 e 100 0. 852 ±0. 002 e 100
FCF 5% 0. 833 ±0. 013 a 108,04 1. 140 ±0. 003 c 134. 91 1,471 ± 0. 004 d 172. 65
FCF10% 0. 632 ±0. 004 d 81,97 1. 011 ±0. 001 d 119. 64 1. 847 ±0. 003 b 216. 78
FCF20% 0,653 ±0. 006 c 84. 69 1. 265 ± 0. 002 a 149. 70 2. 466 ±0. 002 a 289. 43
FCF40% 0. 542 ±0. 011 e 70. 29 1. 193 ± 0. 004 b 141. 10 1. 560 ±0. 007 c 183. 09
LSD 5% 0. 010 0. 0U3 0. 004
Sids 1 Control 0. 537 ±0. 003 a 100 0. 553 ±0. 002 c 100 0. 564 ±0. 004 e 100
FCF 5% 0. 496 ±0. 004 b 92. 36 0. 525 ±0. 003 d 94. 93 0. 661 ±0. 003 d 117. 19
FCF10% 0. 430 ±0. 001 d 80,07 0. 475 ±0. 004 e 85. 89 0. 934 ±0. 003 o 165. 60
FCF20% 0. 446 ±0. 007 c 83. 05 0. 657 ±0. 005 b 118. 80 1. 259 ±0. 004 a 223. 22
FCF40% 0. 417 ±0. 010 e 77. 65 0. 701 ±0. 004 a 126. 76 0. 987 ±0. 003 b 175. 00
LSD 5% 0. 005 0. 004 0. 003
Bani Suef 6 Control 0. 767 ±0. 011 b 100 0. 783 ±0. 003 d 100 0,804 ±0. 003 e 100
FCF 5% 0. 775 ±0. 002 b 101,04 0. 832 ±0. 002 c 106. 25 0,883 ±0. 004 c 109. 82
FCF10% 0. 839 ±0. 004 a 109. 38 0. 865 ±0. 002 b 110. 47 0. 932 ±0. 005 b 115. 92
FCF20% 0. 750 ±0. 003 c 97. 78 0. 962 ±0. 004 a 122. 86 1. 126 ±0. 004 a 140. 04
FCF40% 0. 743 ±0. 004 c 96. 87 0. 772 ±0. 006 e 98. 59 0. 823 ±0. 003 d 102. 36
LSD 5% 0. 006 0. 003 0. 004
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FCF: Fungal culture filtrate DAI: Days after inoculation. Values are mean of three samples ± SD. Different letters indicate significant variation between vertical readings for each cultivar at P = 0. 05 according to Duncan'-s multiple range test.
Table 4. Effect of different concentrations of F. graminearum culture filtrate on flavonoid content of shoots of wheat (Triticium aestivium L.) cv. Giza 164, cv. Sids 1 and (Triticium durum L.) cv, Bani Suef 6 at 5,10 and 15 days after inoculation. Values listed are expressed as mg / g fresh weight.
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Cultivar Treatment S DAI % of control 1UDAI % of control 15 DAI % of control
Giza 164 Control 0. 220 ±0. 004 a 100 0. 268±0. 004e 100 0. 278 ±0. 003 e 100
FCF 5% 0. 131 ±0. 002 c 59. 54 0. 308+0. 004 c 114. 92 0. 356 ±0. 004 d 128. 05
FCF10% Q. l32±G. 005c 60. 00 0. 321 ±0. 003 b 119. 77 0. 426 ±0. 003 b 153. 23
FCF20% 0. 124+0. 001 d 56. 36 0. 367 ±0. 006 a 136. 94 0. 489±0. 006 a 175. 89
FCF40% 0. 208±0. 004 b 94. 54 0. 292 ±0. 002 d 108. 95 0. 375±0. 003 c 134. 89
LSD 5% 0. 003 0. 004 0. 004
Sids 1 Control 0. 163 ±0. 003 b 100 0. 174±0. 001 d 100 0. 179+0. 002 d 100
FCF 5% 0. 159 ±0. 004 c 97. 54 0. 134 ±0. 000 C 105. 74 0. 159 ±0. 003 e 83,82
FCF10% 0. 115 ±0. 002 e 70. 55 0. 192±0. 001 b 110. 34 0. 318 ±0. 003 c 177. 65
FCF20% 0. 176 ±0. 004 a 107. 97 0. 213 ±0. 001 a 122. 41 0. 408 ±0. 004 a 227. 93
FCF40% 0. 143 ±0. 002 d 90. 79 0. 171 ±0. 00 1e 98. 27 0. 348 ±0. 002 b 194. 41
LSD 5% 0,004 0. 001 0. 003
Bani Suef 6 Control 0. 199 ±0. 002 e 100 0. 207±0. 004e 100 0. 212±0. 003 e 100
FCF 5% 0. 274 ±0. 001 6 137. 68 0. 317+0. 004 c 153. 14 0. 418±0. 003c 197. 16
FCF10% 0. 308 ±0. 004 c 154. 77 0. 354±0. 002 b 171. 01 0. 497±0. 007 b 234. 43
FCF20% 0. 335 ±0. 010 b 168. 34 0. 447±0. 004a 21 594 0. 563±0. 005 a 265. 56
FCF40% 0. 379 ±0. 002 a 190. 45 0. 231 ±0. 003 d 111. 59 0. 338±0. 008 d 159. 43
LSD 5% 0. 004 0. 003 0. 005
S
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FCF: Fungal culture filtrate DAI: Days alter Inoculation. Values are mean of three samples ± SD, Different letters Indicate significant variation between vertical readings for each cultivar at P = 0,05 according to Duncan'-s multiple range test.
o
Figure 1. Effect of different concentrations of F. graminearum culture filtrate on catalase activity of shoots of wheat (Triticium aestivium L.) cv. Giza 164, cv. Sids 1 and (Triticium durum L.) cv. Bani Suef 6 at 5, 10 and 15 days after inoculation. Data are means of three samples ± SD. Within different time, different letters indicate significant variation according to Duncan'-s multiple range test at P = 0. 05. FCF: Fungal cultures filtrate.
Figure 2. Effect of different concentrations of F. graminearum culture filtrate on peroxidase activity of shoots of wheat (Triticium aestivium L.) cv. Giza 164, cv. Sids 1 and (Triticium durum L.) cv. Bani Suef 6 at 5, 10 and 15 days after inoculation. Data are means of three samples ± SD. Within different time, different letters indicate significant variation according to Duncan'-s multiple range test at P = 0. 05. FCF: Fungal cultures filtrate.
Figure 3. Effect of different concentrations of F. graminearum culture filtrate on ascorbate peroxidases activity of shoots of wheat (Triticium aestivium L.) cv. Giza 164, cv. Sids 1 and (Triticium durum L.) cv. Bani Suef 6 at 5, 10 and15 days after inoculation. Data are means of three samples ± SD. Within different time, different letters indicate significant variation according to Duncan'-s multiple range test at P = 0. 05. FCF: Fungal cultures filtrate.
Figure 4. Effect of different concentrations of F. graminearum culture filtrate on phenylalanine ammonia lyase activity of shoots of wheat (Triticium aestivium L.) cv. Giza 164, cv. Sids 1 and (Triticium durum L.) cv. Bani Suef 6 at 5, 10 and 15 days after inoculation. Data are means of three samples ± SD. Within different time, different letters indicate significant variation according to Duncan'-s multiple range test at P = 0. 05. FCF: Fungal cultures filtrate.
DISCUSSION
Changes in protein. The present results showed that total soluble protein contents in wheat shoots were decreased in cv. Giza 164, but increased in cv. Sids 1 and cv. Bani Suef 6 plants inoculated with different concentrations of Fusarium graminearum compared with non-inoculated plants. The concentration 10% FCF caused the highest reduction in soluble protein content (41. 14%) in shoot of cv. Giza 164 after five days from inoculation as shown in Table (1). Some authors worked on plants subjected to stress also recorded these results. Vafaii et al. (2013) stated that Fusarium culmorum exhibited significant reduction in protein content of wheat leaf tissues as a result of pathogen infection. They explained this reduction to some activities related to a hypersensitive response. Arad and Rechimond (1976) attributed the decrease in protein synthesis to an increase in RNase under abiotic stress. While Egorov (1985) ascribed it to the inhibition of amino acid incorporation into protein at the final stages of the process without affecting the initial stage of activation of amino acids.
On the other hand, all concentrations of fungal culture filtrate (FCF) significantly increased the protein content in two wheat cultivars (cv. Sids 1 and cv. Bani Suef 6) reaching the maximum value at 20% of FCF, thereafter, it was decrease gradually at 40% of FCF as shown in Tables (1). The increased protein in treated plants might be responsible for defense response in plants. After the contact with the pathogen, a series of peptides and proteins are expressed and many of these compounds present have direct antimicrobial properties. Plant defense-related genes are known to be activated by the generation of endogenous plant signal molecules
following the primary recognition of pathogen-encoded elicitors (Maffei et al., 2007). Boller (1985) mentioned that proteins are associated with the defense of plants against fungi and bacteria by their action on the cell walls during invasion. These proteins are referred to as pathogenesis related (PR) proteins- some of these have enzymatic activities, while others have no clearly defined functions (Van Loon, 1999- Mishra et al., 2011). In this respect, the current results suggested that wheat cultivars Sids 1 and Bani Suef 6 are more tolerant to biotic stress than cultivar Giza 164 due to their higher protein contents.
Changes in antioxidant enzymes. In the present study, catalase activity was increased gradually in shoots of the three wheat cultivars Giza 164, Sids 1 and Bani Suef 6 inoculated with different concentrations of F. graminearum extract. The maximum value of catalase activity was recorded at 20% of F. graminearum extract after 15 days from inoculation reaching 102. 48%, 132. 86% and 112. 02% in cv. Giza 164, cv. Sids 1 and cv. Bani Suef 6, respectively as shown in Figure (1). These results are in agreement with those of Kwon and Anderson (2001), who mentioned that superoxide dismutase and catalase isozymes activity were enhanced in the wheat leaves infected with Fusarium. Anand et al. (2009) confirmed that phenylalanine ammonia lyase (PAL), peroxidase (PO), polyphenol oxidase (PPO) and catalase (CAT) activities contributed to higher resistance in green chilli fruits against Colletotrichum capsici and Alternaria alternate. Aslo, Hanfei et al. (2013) stated that the level antioxidant defensive enzymes (POX, PPO, SOD and CAT) increased in melon seedlings in response to infection by Fusarium oxysporum f. sp. Melonis Race 1.2. In the present study cultivar Sids 1 was found to has
the highest CAT value in inoculated plants compared to control.
The host cell under pathogenesis might accelerate the terminal respiratory pathway, which may lead to increase in CAT activity. Moreover, Anand et al. (2009) reported that an increase of CAT activity in the inoculated fruits were due to an increased H2O2 in the host tissues. Catalase in defensive mechanisms against pathogen can prevent the oxidative damage through rapid removal of H2O2.
In the present study, POX activity also was higher in inoculated shoots of the three wheat cultivars (Giza 164, Sids 1 and Bani Suef 6) compared to the corresponding non-inoculated ones with Sids 1 cultivar with the highest increase in POX compared to control (Figure 2). Such results are in consistent with the findings of Mohammadi and Kazemi (2002) who observed an increase in POX activity in susceptible wheat plants (cv. Falat) after inoculation with F. graminearium in the milk stage. Arfaoui et al. (2005) found that pretreatment of chickpea with Rhizobium increased significantly the level of peroxidase, polyphenol oxidase and total phenolics. El- Khallal (2007) concluded that POX activity was significantly increased in tomato plants in response to infection with F. oxysporum. The observed increase in both CAT and POX activities in cultivar Sids 1 might contribute to increased tolerance in response to inoculation with F. graminearium compared to the other cultivars.
In addition, Cherif et al. (2007) reported that a significant increase in POX specific activity in resistant and susceptible wheat cultivars following the inoculation with F. graminearium conidia. Ojha and Chatterjee (2012) stated that both polyphenol oxidase and peroxidase
activities were found to be much higher in tomato plants infected with F. oxysporum f. sp. Lycopersici and gradually increased as the infection period progressed.
Peroxidase activity was implicated in a number of diverse phenomena observed in plants such as lignification, suberization, cell elongation, growth and regulation of cell wall biosynthesis and plasticity, which diversified during disease period (Anjum et al. 2012).
The results obtained from this study revealed that ascorbate peroxidase plays a key role in eradicating hydrogen peroxidase molecules. From Figure (3), APX activity was significantly increased in the shoots of wheat cultivars that inoculated with different concentrations of FCF reached the maximum at 20%, thereafter, it decreased in the three wheat cultivars at 40% of FCF through the experimental period. This study was in consistent with that obtained by Mandal et al. (2009) who studied the biochemical characteristics of the oxidative burst during interaction between Solanum lycopersicum and F. oxysporum f. sp lycopersici. They reported that activities of antioxidative enzyme (SOD, CAT, POX and APX) increased in response to pathogen inoculation. A major hydrogen peroxide detoxifying system in plant cells under stress is the ascorbate-glutathione cycle, in which ascorbate peroxidase (APX) isoenzymes convert H2O2 into H2O, using ascorbate as a specific electron donor, particularly in the chloroplast (Gherbawy et al., 2012). The maximum increase in APX activity, in response to treatment with FCF, was detected in cultivar Giza 164 while the lowest increase was detected in cultivar Sids 1 indicating that the defense mechanism in wheat cultivars differ where Sids 1 depends on CAT and POX enzymes as a detoxification agents Giza 164 depends on APX
enzyme.
Changes in PAL activity. The results in Table (2) showed an increase in PAL activity in bread wheat cultivars (Giza 164 and Sids 1) at all FCF concentrations, while the PAL activity was decreased in durum wheat cultivar (Bani Suef 6) in all FCF concentrations except at 20% of FCF. In line with our results, Anand et al. (2009) demonstrated that PAL activity increased both in ripe and green chilli fruits in response to inoculation with the fruit rot pathogen. Stadnik and Buchenauer (2000) also reported the enhancement of PAL activity and accumulation of cell wall-bound phenolic compounds in wheat plants in response to powdery mildew infection. In contrast, Vafaii et al. (2013) stated that PAL activity significantly reduced in wheat plants after inoculated with F. culmorum compared with uninoculated plants. However, with prolonged pathogenic stress, the PAL levels were lower compared with other treatments or control. The chickpea seedlings exposed to cell wall protein of F. oxysporum f. sp. ciceri, showed enhanced synthesis of phenols, pathogenesis-related proteins and activities of PAL and peroxidase relative to water treated controls (Saikia et al. 2006).
PAL activity could be induced in plant pathogen interactions. PAL, one of the key enzymes in the phenylpropanoid pathway and the flavonoid pathway, was increased in both incompatible and compatible interaction between plants and pathogens (Harllen et al., 2004). This enzyme has been correlated with defense against pathogen in several plants including tomato (Borden and Higgins, 2002), pepper (Jung et al., 2004) and wheat (Mohammadi and Kazami, 2002).
Changes in proline content. The data presented in Table (3) demonstrated that proline content in the three wheat cultivars significantly increased in response to inoculation with different concentrations of FCF. In this respect, Gherbawy et al. (2012) indicated that wheat plants inoculated with F. culmorum, F. graminearum and F. sambucinum as well as a mixture of all the Fusarium species exhibited an increase in proline content. Meanwhile, Ashry and Mohamed (2011) observed that proline markedly increased in resistant and susceptible flax lines during infection with powdery mildew as compared to their parents. Moreover, Vafaii et al. (2013) mentioned that the proline content in wheat plants that inoculated with F. culmorum was higher than non-inoculated ones.
Free proline accumulates in plants in response to the imposition of a wide range of biotic and abiotic stresses (Hare and Cress, 1997). Accumulation of the amino acid proline in the present work is one of the indications to plant adaptation to unfavorable conditions. This increment might be attributed to the cell defense reactions. The physiological significance of proline accumulation has been assigned a role as a cyto-solute, as a convenient source of energy, as a protective agent for cytoplasmic enzymes. Proline regulates the osmotic equilibrium between the cytoplasm and vacuole. Proline can serve as a protector of enzyme denaturation, as a reservoir of nitrogen and carbon or as stabilizer of the machinery for protein synthesis (Maiti et al., 2009).
Changes in phenol content. The results (Table 4) indicated that phenol content slightly changed in the three wheat cultivars inoculated with different concentrations of FCF after five days from inoculation, while at ten and
fifteen days, the phenol content significantly increased in inoculated three wheat cultivars with different concentrations of FCF. The results obtained in the present work agree with those of Anjum et al. (2012) who found that infection induced accumulation of phenolic acids in various parts of wheat plant infected with Ustilago tritici and total phenolic content following the progress of the disease.
The first stage of defense mechanism involve rapid accumulation of phenols at the infection site which resistance or slowing the growth of the pathogens. Ojha and Chatterjee (2012) observed that the phenol content was significantly higher in F. oxysporum infected tomato plants treated with either salicylic acid or Trichoderma harzianum. The accumulation of phenols in the infected tissue might come from the surrounding healthy leaves in order to resist the advancement of the pathogen towards the other healthy cells. Giza 164 was the cultivar with the highest phenol content compared to control. Several changes in the metabolism of the diseased plant accompany the increase in respiration after inoculation. These increases accompanied by increased activities of the pentose phosphate pathway, which is the main source of phenolic compounds (Agrios, 1997).
Worth to mention that there was a lag time of 10 days (at least) between the increase in PAL activities in inoculated cultivars Giza 164 and Sids 1 and the increase in phenolic content of these cultivars. PAL is a key enzyme in the biosynthesis of phenyl propane unit, which is a component of phenolic acids, flavonoids and lignins. The metabolism of phenolic compounds is regulated by the activity of various enzymes. The first step in biosynthesis of the phenylpropanoid skeleton in higher
plants is the deamination of L-phenylalanine to yield trans-cinnmamic acid and ammonia, a reaction catalyzed by PAL (Rosler et al, 1997).
Changes in Flavonoids content. The data of the present study showed that the highest level of flavonoid content was recorded at 20% of FCF in the three wheat cultivars (Giza 164, Sids 1 and Bani Suef 6) as shown in Table (4). This study was consistent with that reported by Busko et al. (2014) who recorded that the highest concentration of flavonoid was found in winter wheat cultivars inoculated with F. culmorum while the level of flavonoid in control and fungicide-treated plants were significantly lower than inoculated ones.
Ma et al. (2014) stated also that drought stress significantly increased the total phenolic, total flavonoids, anthocyanin and schaftoside contents in wheat leaves. These results indicate that flavonoids have a protective role due to the special structure of flavonoids, e.g. hydroxyl group, double carbon bonds and modifications like glycosylation and methylation. In several plant pathogen interactions, the defense response has been associated with accumulation of flavonoids, phenolic compounds and phytoalexin. Flavonoids, a class of low molecular weight phenolic compounds, play important roles in plants, such as UV protection, as defense against pathogens and pests, as signaling with microorganisms, as auxin transport regulation and pigmentation (Winkel-Shirley, 2001).
Strong difference in expression between two maize genotypes were observed in secondary metabolism category pathway related to shikimate, lignin, flavonoid and terpenoid biosynthesis were strongly represented and induced in the resistant genotype, indicating that selection
to enhance these trait is an additional strategy for improving resistance against F. verticillioids infection. Taken together, the current results suggested that wheat cultivar Sids 1 is more tolerant to F. graminearum inoculation as evidenced by both increased activities of CAT, POX and PAL and elevated levels of proteins and flavonoids content compared to other cultivars under investigation.
CONCLUSION
In conclusion our results highlight the induced defense mechanisms in three wheat cultivars in response to F. graminearum infection. Infected cultivars showed increased activities of antioxidant enzymes (catalase, peroxidase and ascorbate peroxidase) and elevated levels of proline, total phenols and flavonoids content as compared with non-infected plants. On the other hand, PAL activity increased only in infected Giza 164 and Sids 1 cultivars whereas total soluble proteins increased only in infected cultivars Sids 1 and Banisuef 6. In addition, cultivar Sids 1 depends mainly on CAT and POX enzymes as detoxification agents while cultivar Giza 164 depends mainly on APX enzyme. These indicated that in addition to the common protective mechanisms detected in all the infected cultivars there are cultivar — dependent physiological changes exhibited by wheat plant during abiotic stress. In addition, our results showed that sids 1 cultivar are more tolerant to FHB than Giza 164 and Bani Suef 6 cultivars as evidenced by qualitative differences at the physiological level. These results are expected to be valuable in breeding program to produce wheat cultivars with increase biotic tolerance.
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