A PIVOTAL ROLE OF REACTIVE OXYGEN SPECIES IN NON-HOST RESISTANCE MECHANISMS IN LEGUME AND CEREAL PLANTS TO THE INCOMPATIBLE PATHOGENS

Most of plants under normal conditions are resistant to most of the incompatible pathogens (viral, fungal and bacterial infections). This is called ״non-host resistance (NHR) phenomenon״. Till now it is not clear the non-host resistance mechanisms. As a result of inoculation of legume (pea and soybean) and cereal (barley and wheat) plants with compatible and incompatible pathogens, strong resistance symptoms were observed in the nonhost/incompatible pathogen combinations as compared with host/compatible pathogen combinations which showed severe infection (susceptibility). Levels of reactive oxygen species (ROS) mainly hydrogen peroxide (H2O2) and superoxide (O2.-) were significantly increased early 6, 12, 24 and 36 hours after inoculation (hai) in the non-host plants as compared with host plants. Interestingly enough that the activities of the antioxidant enzymes such as catalase (CAT), dehydroascorbate reductase (DHAR) and peroxidase (POX) were not significantly increased at the same early time 6 36 hai in the non-host plants. However, these enzymes were significantly increased later on 48, 72 and 96 dai in the non-host plants as compared with host plants. It seems that early accumulation of H2O2 and O2.could have a dual roles, first role is inhibiting or killing the pathogens early in the non-host plants, second immunization of the non-host plants by stimulating the activities of the antioxidant enzymes later on which thereby, neutralize the harmful effect of ROS and consequently suppressing disease symptoms. The author recommends giving more attention to these new mechanisms of non-host resistance particularly in relation to ROS levels and antioxidant activities which are very important for plant breeders and useful for finding alternative control strategies as well.


INTRODUCTION
Non-host resistance (NHR) is a resistance showed by an entire plant species to all genetic variants of a non-adapted pathogen species. NHR refers to plant species immunity against the majority of microbial pathogens and represents the most healthy and strong form of plant resistance in nature (Yulin et al., 2012;Lipka et al., 2010). NHR phenomenon explains why most of plants are immune to the majority of pathogens and normally healthy. Mechanisms supporting NHR remain relatively unknown compared with the well-studied host resistance mediated by the products of plant resistance (R) genes, which establish pathogen race-or plant cultivar-specific resistance (Schulze-Lefert and Bieri 2005; Dangl and Jones 2001). NHR plants to pathogens mainly fungal, bacterial and viral infections can be defined as an innate non-specific resistance which is effective against all known isolates of several species of the pathogens (Király et al., 2007;Thordal-Christensen, 2003). This NHR resistance is a strong and very effective type of plant immunity (Heath, 2000). On the other hand, appropriate pathogens escape defense reactions of the host by avoiding recognition or suppressing resistance of non-host or host but resistant plants (Schulze-Lefert and Panstruga, 2003). Researchers showed some experiments in relation to genetics of nonhost type of resistance. However, only a few biochemical results are available as regards the formation of host cell wall appositions (papillae), local accumulation of autofluorogens and reactive oxygen species (ROS), such as hydrogen peroxide (H2O2) (Trujillo et al., 2004;Hückelhoven et al., 2001;Carver et al., 1992). Pea (Pisum sativum L.) is one of the most important legume vegetable crops grown in Egypt and many other countries all over the world. It has many nutritional values such as high content of protein, carbohydrates, phosphorus, iron, calcium and vitamins A and B (Watt and Merrill, 1963). Soybean (Glycine max L.) is one of the world's most important sources of oil and protein. It has the highest protein content among leguminous crops (Abdel-Monaim et al., 2011). Barley (Hordeum vulgare L.) ranks the fourth among the major cereal grians in terms of World and Egyptian production after maize, wheat and rice crops . Wheat (Triticum aestivum L.) is one of the most important cereal crops in the world for both human food and animal feed Chen et al., 2003 Botrytis cinerea Pers., Bc-1 and Alternaria solani isolates were kindly supplied by Prof. László Vajna, Plant Protection Institute, Hungarian Academy of Sciences, Hungary. These pathogens were maintained on potato dextrose agar medium (PDA) under continuous fluorescent light. For inoculation, agar discs 5 mm in diameter were cut from 7-day-old cultures of the fungus Botrytis cinerea and 15-days old culture of the fungus Alternaria solani then, placed on the surface of pea and soybean leaves, respectively. Leaves were cut and put on wet filter paper in a Petri dish and held at 20°C in continuous light for at least 3 days for pea and 2-5 days for soybean (Hafez et al., 2012). Barley powdery mildew (Blumeria graminis f. sp. hordei) and wheat powdery mildew (Blumeria graminis f. sp. tritici) Egyptian specimens were maintained under greenhouse conditions and were used for all inoculation experiments. Powdery mildew inocula were dispersed in the greenhouse atmosphere by placing plants of barley and wheat bearing sporulating colonies of both pathogens beneath ventilation fans of the greenhouse (Hafez and Kiraly, 2003). Disease Severity Assessments: Disease severity percentage (%) of pea inoculated with B. cinerea (host) and PRSV (non-host) as wel as soybean inoculated with A. solani (host) and B. cinerea (non-host) were measured as lesion diameter (cm 2 ) according to Hafez et al., 2004. However, the disease severity percentage (%) of barley inoculated with barely powdery mildew (host) and inoculated with wheat powdery mildew (non-host) as well as wheat inoculated with wheat powdery mildew (host) and inoculated with barley powdery mildew (no-host) were determined as follows: Ten plants of each replicate were scored visually for percentage of leaf area covered by powdery mildew on a 0 (resistant) to 10 (susceptible) scale three times 3, 6 and 9 days after inoculation (dai) in each experiment. For analysis, disease scores were converted using the modified logarithmic scale of Horsfall-Barrett (Horsfall and Cowling, 1978) and Hafez et al., 2014. The scale was 0 = 0 %, 1 = 0-3 %, 2 = +3-6 %, 3 = +6-12 %, 4 = +12-25 %, 5 = +25-50 %, 6 = +50-75 %, 7 = +75-88 %, 8 = +88-94 %, 9 = +94-97 % and 10 = +97-100 %. Disease severity index (DSI) was calculated according to Kim et al., 2000 using the following formula: ∑ Histochemical Analysis of ROS: Histochemical staining for O2 .-production in leaf tissue was based on the ability of O2  -to reduce nitro blue tetrazolium (NBT). O2 .-was visualised as a purple coloration of NBT. Leaf discs (2 cm) of pea and soybean plants as well as hall barley and wheat leaves were vacuum infiltrated or injected (Hagborg, 1970) with 10 mM potassium phosphate buffer (pH 7.8) containing 0.1 w/v % NBT (Sigma-Aldrich, Germany) according to Ádám et al., (1989). NBT-treated samples were incubated under daylight for 20 min and subsequently cleared in 0.15 % trichloroacetic acid (wt/vol) in ethanol: chloroform 4:1 (vol/vol). The solution was exchanged once during the next 48 h of incubation (Hückelhoven et al., 1999). Subsequently, leaf discs and leaves were stored in 50% glycerol for evaluation. For histochemical analysis of hydrogen peroxide (H2O2), leaf discs and hall leaves were infiltrated with 0.1% 3, 3diaminobenzidine (DAB) in 10 mM Tris buffer (pH 7.8). Samples were incubated under daylight for two hours after the vacuum infiltration. Following staining, leaves were cleared as described above and the intensity of brown color was estimated (Hückelhoven et al., 1999). Levels of O2 .-and H2O2 were estimated 6, 12, 24, 36, and 48 hours after inoculation. These tests were repeated five times during six independent experiments.

Biochemical Assays of Antioxidant Enzymes:
The tested antioxidant enzyme activities were measured on 5 weeks-old pea and soybean plants as well as 2 weeksold barley and wheat plants. For enzyme assays in plants, 0.5 g detached leaves material from 5 and 2 weeks old plants, respectively, was homogenized at 0-4˚C in 3 ml of 50 mM TRIS buffer (pH 7.8), containing 1 mM EDTA-Na2 and 7.5% polyvinylpyrrolidone 1, 2, 3 and 4 days after inoculation. The homogenates were centrifuged (12,000 rpm, 20 min, 4˚C), and the total soluble enzyme activities were measured spectrophotometrically in the supernatant (Hafez, 2010. All measurements were carried out at 25˚C, using the model UV-160A spectrophotometer (Shimadzu, Japan). Activity of catalase (CAT) was determined spectrophotometrically according to Aebi (1984). Decomposition of H2O2 by catalase results in the decrease of the ultraviolet absorption of hydrogen peroxide at 240 nm. Enzyme activity can be calculated from this decrease. The reaction mixture contained, in a final volume of 2.15 ml, 2 ml 0.1 M Na-phosphate buffer (pH 6.5), 100 μl hydrogen peroxide and 50 μl leaf extract supernatant. The solution is mixed, and then the absorption change is registered for 3 min at 240 nm using a quartz cuvette. Activity of dehydroascorbate reductase (DHAR) was determined spectrophotometrically according to Asada (1984). The reaction mixture contained, in a final volume of 2.3 ml, 50 mM sodium phosphate buffer (pH 6.5), 0.5 mM dehydroascorbate (DHA), 1.0 mM reduced glutathione (GSH), 0.1 mM EDTA and 0.1 ml supernatant. The assay was carried out in quartz cuvettes following the increase in absorbance at 265 nm due to the formation of ascorbate with extinction coefficient 14 mM -1 cm -1 (Klapheck et al., 1990). The reaction rate was corrected for the non-enzymatic reduction of dehydroascorbate by GSH. Activity of peroxidase (POX) was directly determined of the crude enzyme extract according to a typical procedure proposed by Hammerschmidt et al., (1982). Changes in absorbance at 470 nm were recorded every 30 sec intervals for 3min. Enzyme activity was expressed as increase in absorbance min -1 g -1 fresh weight. Statistical Analysis: Six experiments were conducted in a completely randomized design with five replicates for each treatment. Data represent the mean ± SD. Student's t-test was used to determine whether significant difference (P<0.05) existed between mean values according to O'Mahony (1986).

RESULTS AND DISCUSSION Disease Severity and Disease Symptoms of Host and Non-host/Pathogen Combinations:
In the "non-host" plants pea, soybean, barley and wheat showed resistance against Papaya ringspot virus, B. cinerea, Blumeria graminis f. sp. tritici and Blumeria graminis f. sp. hordei, respectively compared to the "host" plants pea, soybean, barley and wheat inoculated with Botrytis cinerea, Alternaria solani, Blumeria graminis f. sp. hordei and Blumeria graminis f. sp. tritici, respectively showed strong susceptibility. Disease severity percentage was significantly severe strongly in all the host/pathogen combinations as compared to the non-host/pathogen combinations (Figure 1 and Table 1  Disease symptoms were also significantly appeared and visible in the host/pathogen combinations compared to the non-host which no symptoms appeared (Figure 2). Similar results have been obtained by Fabro et al. (2011) in which found that in the "non-host" plant Brassica rapa (turnip) which was more effectors of Hyaloperonospora arabidopsidis are recognized than in Arabidopsis thaliana which is a "host" of this oomycete pathogen. This could be a possible cause of the inability of H. arabidopsidis to grow in turnip. In other words, the host plant cannot recognize a subset of effectors of its own pathogen which are recognized, and therefore induce an immune reaction in the non-host. However, it is still an unanswered question, how this immune reaction can inhibit pathogens in non-host plants? (Kiraly et al., 2013).

Levels of Reactive Oxygen Species in Host and Nonhost/Pathogen Combinations:
Purple discoloration of superoxide (O2 .-) and brown discoloration of hydrogen peroxide (H2O2) reflect the intensivity of ROS levels in the leaves which cleared from chlorophyll ( Figure 2). Levels of ROS mainly O2 ·-and H2O2 significantly accumulated early 6, 12, 24 and 36 hours after inoculation (hai) in all non-host/pathogen combinations in pea, soybean, barley and wheat plants (Figure 3, 4 and 5).

Activities of Antioxidant Enzymes in Host and Nonhost/Pathogen Combinations:
Activities of antioxidant enzymes such as catalase (CAT), dehydroascorbate reductase (DHAR) and peroxidase (POX) were not changed or even increased early 6, 12, 24 and 36 hours after inoculation (hai) in all non-host/pathogen combinations in pea, soybean, barley and wheat plants, however, these enzymes were increased significantly a little bit later at 48, 72 and 96 hai (Figure 6 and 7).
One can say that in these non-host resistant pea, soybean, barley and wheat plants the early accumulation of O2 ·-and H2O2 not only inhibit or kill the incompatible pathogens 6-36 hai but also stimulate the activities of antioxidants CAT, DHAR and POX alter on 24-96 hai. Our results are supported by our previous results in which pointed out that under natural conditions the up-regulation of antioxidant defense systems seems to be a general response to oxidative stress (Hafez et al., 2012). The early accumulation of ROS in the non-host resistant plants up-regulate the antioxidants go along with the results which proved that H2O2 as one of the most important reactive oxygen species associated with oxidative stress, can up-regulate antioxidant systems even at very low concentrations against abiotic stress (Gechev et al., 2002) and also immunize plants, therefore, induces resistance to symptom development by suppressing pathogen-induced host cell and tissue necroses but not pathogen multiplication, while enhancing activities of at least three antioxidant enzymes such as catalase, guaiacol peroxidase and ascorbate peroxidase ( Hafez et al., 2012). It was pointed out that often the only evidence that oxidative stress has occurred in vivo may be it is the cause of upregulation of antioxidant defense systems (Halliwell and Gutteridge, 1999). The high activities of enzymatic and nonenzymatic antioxidants neutralized the harmful effects of ROS (oxidative stress). Particularly, H2O2 seems to play a dual role by eliciting localized death of plant and pathogen cells and as a diffusible signal for the induction of antioxidant and pathogenesis-related genes in adjacent plant tissues (Hafez et al., 2012;Wu et al., 1997;Levine et al., 1994). It worth mentioning that the author use for this research four model important plants, two from legumes (pea and soybean) and two from cereals (barley and wheat) which support his new findings. It can be concluded that new histochemical and biochemical mechanisms of non-host resistance mechanisms were found by the author in this research article. ROS mainly superoxide and hydrogen peroxide accumulated early after inoculation which may be killing or inhibiting the pathogens in these non-host plants. This early accumulation of ROS stimulated the antioxidant enzymes activities later on which thereby could immunize plants by suppressing disease symptoms and neutralize the harmful effect of ROS against these incompatible pathogens. It is recommended to the researchers and plant breeders to give more attention to these interesting new results to find new strategies for future integrated control pest management practices and sustainable crop protection. AKNOWLEDGEMENT