Seed treatment with beta-aminobutyric acid protects Pennisetum glaucum systemically from Sclerospora graminicola

beta-Aminobutyric acid (BABA) treatment of pearl millet [Pennisetum glaucum (L) R Br] seeds influenced seedling vigour and protected the seedlings from downy mildew disease caused by the oomycetous biotropic fungus Sclerospora graminicola (Sacc) Schroet. Of the different concentrations of BABA tested, viz 25, 50, 75 and 100 mM, seeds treated with 50 mM for 6 h resulted in the maximum of 1428 seedling vigour and showed 23% disease incidence in comparison with the control which recorded a seedling vigour of 1260 and 98% disease incidence i.e. 75% protection from disease. Seeds treated with BABA when challenged for downy mildew disease using zoospores of S graminicola required 48 h after inducer treatment to develop maximum resistance. Durability of induced resistance was also tested in plants raised from seeds treated with the inducer and identified as resistant, by second challenge inoculation with the downy mildew pathogen at tillers and inflorescence axes. Reduced disease incidence of only 10 and 12% in these plants, compared with 71 and 76% disease in control plants inoculated at the tillers and inflorescence axes, respectively, suggested that resistance induced in seeds with BABA remained operative through vegetative and reproductive growth of pearl millet plants. Induction of resistance by seed treatment with BABA enhanced vegetative growth, viz height, fresh weight, leaf area and tillering, and reproductive growth, viz early flowering, number of productive ear heads and 1000 seed weight. Studies on induction of resistance in different cultivars of pearl millet with varying resistance reaction to downy mildew indicated that the protection offered by BABA is independent of the nature of cultivars used and not dependent on their constitutive resistance.     

Chitosan enhances disease resistance in pearl millet against downy mildew caused by Sclerospora graminicola and defence-related enzyme activation

BACKGROUND: The present study investigated the effect of chitosan seed priming on the induction of disease resistance in pearl millet against downy mildew disease caused by Sclerospora graminicola (Sacc.) Schroet. RESULTS: Pearl millet seeds were primed with chitosan at different concentrations: 0.5, 1.5, 2.5 and 3 g kg^?1 seed. Of the different concentrations, 2.5 g kg^?1 was found to be optimum, with enhanced seed germination of 99% and seedling vigour of 1782, whereas the untreated control recorded values of 87% and 1465 respectively. At optimum concentration, chitosan did not inhibit sporulation and release of zoospores from sporangia. Furthermore, pearl millet seedlings raised after seed treatment with chitosan showed an increased level of the defence‐related enzymes chitosanase and peroxidase as compared with the untreated pearl millet seedlings on downy mildew pathogen inoculation. The effect of chitosan in reducing downy mildew incidence was evaluated in both greenhouse and field conditions, in which respectively 79.08 and 75.8% disease protection was obtained. CONCLUSION: Chitosan was effective in protecting pearl millet plants against downy mildew under both greenhouse and field conditions by inducing resistance against the pathogen. Thus, chitosan formulation can be recommended for seed treatment in the management of downy mildew disease. Copyright © 2008 Society of Chemical Industry     

Nitric oxide is involved in chitosan‐induced systemic resistance in pearl millet against downy mildew disease

BACKGROUND: The nature and durability of resistance offered by chitosan and the involvement of nitric oxide (NO) in chitosan‐induced defence reactions in pearl millet against downy mildew disease were investigated. RESULTS: It had previously been reported that chitosan seed priming protected pearl millet plants against downy mildew disease. Further elucidation of the mechanism of resistance showed that chitosan seed priming protects the plants systemically. A minimum 4 day time gap is required between the chitosan treatment and pathogen inoculation for maximum resistance development, and it was found to be durable. Chitosan seed priming elevated NO accumulation in pearl millet seedlings, beginning from 2 h post‐inoculation, and it was found to be involved in the activation of early defence reactions such as hypersensitive reaction, callose deposition and PR‐1 protein expression. Pretreatment with NO scavenger C‐PTIO and nitric oxide synthase (NOS) inhibitor L‐NAME before pathogen inoculation reduced the disease‐protecting ability of chitosan, and defence reactions were also downregulated, which indicated a possible role for NO in chitosan‐induced resistance. CONCLUSION: Protection offered by chitosan against pearl millet downy mildew disease is systemic in nature and durable. Chitosan‐induced resistance is activated via NO signalling, as defence reactions induced by chitosan were downregulated under NO deficient conditions. Copyright © 2009 Society of Chemical Industry     

Unsaturated fatty acids from zoospores of <Emphasis Type="Italic">Sclerospora graminicola</Emphasis> induce resistance in pearl millet

Downy mildew of pearl millet, caused by Sclerospora graminicola, is a devastating disease, resulting in high economic losses in the semi-arid regions of the world. Recently, induction of host plant resistance using biotic and abiotic inducers are included among disease management practices as an eco-friendly approach. Unsaturated fatty acids are considered as a new generation of plant disease resistance inducers. In the present study, six unsaturated fatty acids, viz. docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), arachidonic acid (AA), linolenic acid, linoleic acid and oleic acid, all originally detected in the zoospores of S. graminicola,were applied to seeds of susceptible cultivars of pearl millet to examine their ability to protect against downy mildew under greenhouse and field conditions. In greenhouse experiments, EPA and AA induced a maximum of 78.6% and 76.5% protection, whereas linoleic acid, DHA and linolenic acid provided up to 66.3%, 61.2% and 24.5% protection, respectively. Oleic acid was not effective in protecting pearl millet (only 5.1% protection). A time interval of four days between treatment of seeds and challenge inoculation was required to obtain optimum protection. Plants raised from treated seeds and challenge inoculated at the tillering and inflorescence stages showed enhanced resistance, resulting in higher grain yield compared to untreated plants of the same cultivar. Chitinase activity was found to be higher in susceptible seedlings of pearl millet after treatment with the fatty acids and pathogen inoculation than in seedlings only inoculated with the pathogen. This indicates that host defence responses are activated and thus that induced resistance is involved in the protection observed. The role of unsaturated fatty acids as activators of resistance against downy mildew in pearl millet is discussed.     

Differential induction of superoxide dismutase in downy mildew-resistant and -susceptible genotypes of pearl millet

Differential induction of superoxide dismutase (SOD) in downy mildew-resistant and -susceptible genotypes of pearl millet ( Pennisetum glaucum ) was observed on inoculation with Sclerospora graminicola . SOD activity was studied in resistant (IP18292) and susceptible (23B) pearl millet seedlings inoculated with S. graminicola . SOD activity increased by 2·3-fold in resistant seedlings upon inoculation. SOD activity was greatest in roots, with a specific activity of 3182 U per mg protein, after inoculation. SOD activity increased in all the resistant genotypes upon inoculation with S. graminicola . Native PAGE analysis showed four isozymes of SOD, three of which (SOD-1, -2 and -4) were Cu/Zn-SOD, whereas isozyme SOD-3 was Mn-SOD. This study also revealed increased intensity of all four isozymes of SOD in the resistant genotype upon inoculation. The involvement of SOD in pearl millet (host)–downy mildew pathogen interaction is discussed.     

Serodiagnosis of pearl millet resistance to downy mildew by quantitating cell wall P/HRGP using polyclonal antiserum Pab-P/HRGP

Proline/hydroxyproline-rich glycoprotein (P/HRGP) level in pearl millet genotypes resistant to downy mildew increase after inoculation with the oomycete pathogen Sclerospora graminicola. Using purified P/HRGPs from pearl millet cell walls, polyclonal antibodies (Pab-P/HRGP) were raised in rabbit. Based on this antiserum, an enzyme immunoassay was developed that displays a linearity detection range from 0.01 to 10 μg P/HRGP. Western blot analysis, confirming the induction of three marker P/HRGPs in the infected resistant genotype, and immunocytochemical studies on P/HRGP localization either in epidermal peelings or in suspension-cultured cells demonstrated the specificity of the antiserum. Besides its characterization, Pab-P/HRGP was employed to screen various genotypes of pearl millet for fast, sensitive and specific detection of induced P/HRGPs upon infections. The results presented are discussed with presumed importance to downy mildew disease and the use of this new antiserum in pearl millet screening for disease resistance.     

Nitric oxide donor seed priming enhances defense responses and induces resistance against pearl millet downy mildew disease

Nitric oxide (NO) donors Nitroso-R-Salt, 2-Nitroso-1-Naphthol and Sodium Nitro Prusside (SNP) were evaluated for their effectiveness in protecting pearl millet [(Pennisetum glaucum L.) R. Br.] plants against downy mildew disease caused by Sclerospora graminicola [(Sacc). Schroet]. Optimization experiments with NO donors showed no adverse effect either on the host or pathogen. Aqueous SNP seed treatment with or without polyethylene glycol (PEG) priming was the most effective in inducing the host resistance against downy mildew both under greenhouse and field conditions. Potassium Ferrocyanide, a structural analog of NO donor lacking NO moiety failed to protect the pearl millet plants from downy mildew indicating a role for NO in induced host resistance. Spatio-temporal studies corroborated that the protection offered by NO donor treatment was systemic in nature and a minimum of 3-day time gap between the inducer treatment and subsequent pathogen inoculation was necessary for maximum resistance development. Disease protection ability of NO donors was also validated as durable in nature. Conversely, prior-treatment with NO scavenger 2-4-carboxyphenyl-4,4,5,5 tetrazoline-1-oxyl-3-oxide potassium salt (C-PTIO) rendered the pearl millet plants relatively susceptible for pathogen infection. Expression of primary defense responses like hypersensitive response, lignin deposition and defense related enzyme phenylalanine ammonialyase −EC 4.3.1.5 (PAL) were enhanced by NO donor treatments.     

Note: Proline — An inducer of resistance against pearl millet downy mildew disease caused by<Emphasis Type="Italic">Sclerospora graminicola</Emphasis>

In an attempt to find a suitable alternative to the otherwise perilous chemical control strategy of disease management, the amino acid proline was evaluated for its efficiency to elicit resistance in pearl millet (Pennisetum glaucum (L.) R. Br.) against downy mildew disease caused bySclerospora graminicola (Sacc.) Schroet both under greenhouse and field conditions. Proline treatment to seeds at 50 mM concentration for 3 h, significantly enhanced the seed germination and seedling vigor of pearl millet in comparison with the control. The same concentration and duration of seed treatment protected the pearl millet plants from downy mildew by offering 58% protection under greenhouse and 67% protection under field conditions. Studies revealed that 3 days were required for proline-treated plants to develop resistance, which was systemic and was sustained throughout the life of the plants. Apart from disease protection, proline was also found effective in enhancing vegetative and reproductive growth of the plants, as evidenced by the increase in height, fresh weight, leaf area, tillering capacity, 1000-seed weight and grain yield in comparison with the control plants. http://www.phytoparasitica.org posting Oct. 3, 2004.     

Screening pearl millet cultivars by ELISA for resistance to downy mildew disease

In an earlier study, we described identification of a protein from a virulent pathotype of Sclerospora graminicola , the binding reaction of which differentiated susceptible and resistant cultivars of pearl millet to downy mildew disease. This protein and corresponding antibody were used in an enzyme-linked immunosorbent assay (ELISA) to screen suspension cells of pearl millet cultivars for their resistance to the downy mildew pathogen. Screening results for 31 pearl millet cultivars correlated positively with the established field screening method.     

The epidemiology, variability and control of the downy mildews of pearl millet and sorghum, with particular reference to Africa

Sorghum downy mildew ( Peronosclerospora sorghi ) infecting sorghum and maize, and pearl millet downy mildew ( Sclerospora graminicola ) infecting pearl millet can cause considerable yield loss in Africa. The last 15 years have witnessed an increase in knowledge of the biology, epidemiology and control of these two pathogens. Much information has been obtained on the effect of environmental factors on disease epidemiology, spore production and dispersal. Molecular techniques applied to study pathogenic variability have aided in defining relationships among these pathogens, although scope of the work is limited. Knowledge of the genetics and inheritance of resistance, and of resistance mechanisms, has also increased. This review presents the current state of knowledge of both downy mildew pathogens, with focus on their status on sorghum and pearl millet in Africa. Despite the advances in knowledge over the last 15 years, these downy mildews remain important constraints to sustainable crop production in the semi-arid regions of Africa. In some cases information obtained in Asia and the Americas can be extrapolated to Africa but care must be taken in ensuring its applicability. Priorities for future research relevant for Africa are proposed and discussed.     

Induction of β-1,3-glucanase in Seedlings of Pearl Millet in Response to Infection by Sclerospora graminicola

Differential resistance of pearl millet cultivars to downy mildew disease was correlated with the levels of -1,3-glucanase in their seeds. Higher activity of the enzyme in highly resistant cultivars and lower activity in the highly susceptible ones suggested the possible use of -1,3-glucanase as a biochemical marker for screening pearl millet cultivars for downy mildew disease. Inoculation of seedlings with the downy mildew pathogen Sclerospora graminicola resulted in increased enzyme levels in resistant cultivars. Mesocotyl and shoot regions of seedlings recorded higher levels of enzyme than the root. Isoelectric focusing revealed four basic isoforms with pI 9.6, 9.0, 8.9 and 8.2 and two acidic isoforms with pI 4.9 and 6.2 of -1,3-glucanase in pearl millet. The pI 9.6 isoform was a major isoform of the enzyme in the pearl millet seedlings with a probable developmental function. Isoforms pI 6.2 and pI 8.2 appeared to be involved in resistance and pI 4.9 isoform seemed to be involved in pathogenesis of pearl millet-downy mildew.     

Purification of an infection-related acidic peroxidase from pearl millet seedlings

Higher basal level of peroxidase activity was observed in highly resistant pearl millet cultivar IP 18292. Upon inoculation with downy mildew pathogen, Sclerospora graminicola, up to 60% increase in peroxidase activity was observed in highly resistant seedlings over the period of time. Iso-electric focusing analysis revealed that, two acidic isozymes of peroxidase with the pI of 5.9 and 5.1 present only in IP 18292 pearl millet seedlings. Upon inoculation with downy mildew pathogen, accumulation of these to isozymes was increased. These results indicated the possible involvement of acidic peroxidase in pearl millet defense. To study the nature of the acidic peroxidase which increases upon inoculation was purified from seedlings of highly resistant pearl millet cultivar using DEAE–Sepharose and Sephadex G-100 columns. The purified enzyme has a molecular weight of 21.8 kDa on SDS–PAGE and has a pI of 5.1. The optimum pH for maximum peroxidase activity was found to be at pH 7.0 and was resistant to high temperature (27–60 °C). The K_m for H₂O₂ and V_max of the enzyme reaction were 5.26 mM and 322.58 units, respectively. Purified peroxidase enzyme was found to be CaCl₂ dependent and both MgCl₂ and ZnCl₂ showed inhibitory effect on enzyme activity. Sodium azide and EDTA inhibited the enzyme and EGTA found to be specific inhibitor of peroxidase.     

Influence of dosage,storage time and temperature on efficacy of metalaxyl-treated seed for the control of pearl millet downy mildew

Metalaxyl (Apron 35WS) as a seed treatment has been used extensively to control downy mildew (caused by Sclerospora graminicola) in pearl millet in India. However, the extent of disease control has varied across cultivars, years and locations. We investigated the effects of fungicide dosage, storage time and storage temperature of metalaxyl-treated seed on disease incidence in four pearl millet lines having varying levels of resistance. A linear relationship was found between fungicide dosage (0.5, 1.5 and 2 g a.i. kg⁻¹ seed) and reduction in disease incidence up to 40 days after emergence in all the lines. The normal fungicide dose (2 g a.i. kg⁻¹ seed) protected the crop for up to 20, 40 and 50 days after emergence in highly susceptible (7042S), moderately susceptible (4042R), and moderately resistant (ICMP 451) lines, respectively. However, the quarter and half the normal dosage of fungicide provided protection only up to 20 days after emergence in 7042R and 40 days after emergence in ICMP 451. Storage duration of metalaxyl-treated seed (2 g a.i. kg⁻¹) up to 9 months at 25 ± 2°C did not affect fungicide efficacy. Storage temperatures (5, 25 and 40°C) and duration (30, 60 and 90 days) of metalaxyl-treated seed (2 g a.i. kg⁻¹) showed differential effects in two pearl millet lines 7042S and 843B with downy mildew incidence being significantly lower in 7042S than in 843B. Metalaxyl-treated seed of 7042S and 843B stored at 40°C for different durations showed phytotoxic effects and it was more pronounced in 843B stored for 60 and 90 days where seed germination was inhibited in pot soil.     

Comparative efficacy of strobilurin fungicides against downy mildew disease of pearl millet

Three commercial formulations of strobilurins, viz., azoxystrobin, kresoxim-methyl, and trifloxystrobin were evaluated for their efficacy against pearl millet downy mildew disease caused by Sclerospora graminicola. In vitro studies revealed inhibition of S. graminicola sporulation, zoospore release, and zoospore motility at 0.1-2 μg ml⁻¹ of all the three fungicides. The fungicides were evaluated for phytotoxic effects on seed quality parameters and for their effectiveness against downy mildew disease by treating pearl millet by: (1) seed dressing, (2) seed dressing followed by foliar spray, and (3) also by foliar spray alone. The highest non-phytotoxic concentrations of 5, 10, and 10 μg ml⁻¹ for azoxystrobin, trifloxystrobin, and kresoxim-methyl, respectively, were selected for further studies. Under greenhouse conditions, these fungicides showed varying degrees of protection against downy mildew disease. Among the three fungicides, azoxystrobin proved to be the best by offering disease protection of 66%. Further, seed treatment along with foliar application of these fungicides to diseased plants showed enhanced protection against the disease to 93, 82, and 62% in treatments of azoxystrobin, kresoxim-methyl and trifloxystrobin respectively. Foliar spray alone provided significant increase in disease protection levels of 91, 79, and 59% in treatments of azoxystrobin, kresoxim-methyl, and trifloxystrobin, respectively. Disease curative activity of azoxystrobin was higher compared to trifloxystrobin and kresoxim-methyl. Tested fungicides showed weaker translaminar activity, as the disease inhibition was marginal when applied on adaxial leaf surface. Partial systemic activity of azoxystrobin was evident by root uptake, while trifloxystrobin and kresoxim-methyl showed lack of systemic action in pearl millet. A trend in protection against downy mildew disease similar to greenhouse results was evident in the field trials. Grain yield was significantly increased in all strobilurin fungicide treatments over control and maximum increase in yield of 1673 kg ha⁻¹ was observed in combination treatments of seed treatment and foliar spray with azoxystrobin.     

Spore cell wall components ofAspergillus niger elicit downy mildew disease resistance in pearl millet

Elicitors derived from the cell wall of fungi are shown to be active in eliciting resistance in plants against a wide range of pathogens. In the present study carbohydrate components from the autoclaved spore cell wall ofAspergillus niger were prepared as aqueous suspensions and tested for defense response in pearl millet (Pennisetum glaucum (L.) R.Br.) against the oomycetous downy mildew pathogenSclerospora graminicola (Sacc.) Schroet. The aqueous suspension derived from the spore cell wall ofA. niger was used as a seed soak treatment at concentrations of 0.25, 0.5, 1.0, 1.5 and 2.0 mg ml⁻¹ for time intervals of 3, 6, 9 and 12 h. The concentration of 0.5 mg ml⁻¹ for a 6 h soaking period offered 94% seed germination and seedling vigor index increased to 1526. The seed germination and the seedling vigor were significantly higher than the untreated check. Spore cell wall suspension as seed treatment at a concentration of 0.5 mg ml⁻¹ required a 3-day time interval to provide 67% protection against downy mildew. Histological and biochemical studies were conducted to elucidate the mechanism of defense response in treated seedlings uponS. graminicola infection. Resistance host response was detected in the form of lignin and callose deposition in the epidermal cell wall of pearl millet seedlings, which is the site ofS. graminicola infection. A time course study showed rapid and localized deposition of lignin and callose in epidermal cell wall of carbohydrate components-treated pearl millet seedling coleoptiles. Increased levels of the defense-related enzyme peroxidase were detected in the treated seedlings. Peroxidase activity in elicitor-treated samples reached a peak at 8 h post-infection, which was 45% more than in their respective uninoculated control. Characterization of peroxidase isoforms by isoelectric focusing revealed 16 different isoforms, of which pI 6.8, 7.2 and 8.7 increased in elicitor-treated samples uponS. graminicola infection. http://www.phytoparasitica.org posting Nov. 14, 2005.     

Ribonucleases in the Seedlings of Pearl Millet and their Involvement in Resistance Against Downy Mildew Disease

Tissue homogenates of pearl millet seedlings (cultivars HB 3, 843 B, ICMP 451 and IP 18292), with varying degree of resistance to downy mildew disease were tested for ribonuclease (RNase) enzyme activity and the profile of major RNase isozymes by substrate based gel assay. Polyacrylamide gel electrophoresis (PAGE) of the four pearl millet homogenates revealed 15–20 isozymes, varying in size from 6.5 to 121.0kDa. Most of the RNases were highly active between pH 6 and 8 with maximum activity at pH 7. Tissue specific expression of RNase was observed with more activity in the root, i.e., 38.84, 59.61, 39.90 and 49.23 units in HB 3, 843 B, ICMP 451 and IP 18292, respectively than in shoot 11.54, 9.95, 9.46 and 9.49 units in HB 3, 843 B, ICMP 451 and IP 18292, respectively. Effect of metal ions on the RNase profile indicates Zn⁺⁺ at 2, 20 and 200M concentrations to be inhibitory. Ca⁺⁺ and Mg⁺⁺ at 1mM concentration enhanced the enzyme activity while at 10mM inhibition of enzyme activity was observed. Inoculation with the downy mildew pathogen Sclerospora graminicola reduced RNase activity by 4–13% in compatible interactions while in incompatible combinations, the enzyme activity increased by 10–27%. The significance of RNase in pearl millet-downy mildew interaction and its involvement of in systemic acquired resistance of pearl millet against the downy mildew pathogen are discussed.     

谷子白发病菌Sclerospora graminicola (sacc.) Schröt分生孢子系统侵染研究初报

谷子白发病菌分生孢子在一定湿度条件下,能从谷子的分生組織分蘖分枝幼芽、幼穗、幼叶、幼苗和胚芽上侵入而形成系統侵染发病;所产生的分生孢子与卵孢子侵染产生的分生孢子,在侵染致病特性上并无差異。在谷子各个生育阶段的病征也与卵孢子侵染完全一致,并丰产卵孢子。由于分生孢子的系統侵染,因此陕西省陕北和关中地区谷子白发病在多雨年份比少雨年份、低湿田块比高燥田块田間发病显著严重。白发病菌还可能通过分生孢子系統侵染而进行杂交产生新生理小种。     

Induction of resistance against downy mildew pathogen in pearl millet by a synthetic jasmonate analogon

The synthetic 1-oxo-indanoyl-l-isoleucine methyl ester (Ind-Ile-Me) represents a highly active mimic of octadecanoic phytohormones, which are involved in plant defenses against pathogens and pests. Seed treatments and foliar spray with Ind-Ile-Me were tested for induced resistance against downy mildew disease caused by the phytopathogenic oomycete Sclerospora graminicola in pearl millet (Pennisetum glaucum) under greenhouse and field conditions. Under greenhouse conditions, a 50% protection level was achieved after seed treatment. Seed treatment in combination with foliar spray resulted in 60% protection. The induction of resistance was correlated with the enhanced activities of defense-related proteins such as phenylalanine-ammonia-lyase, peroxidase, and enhanced level of hydroxyproline-rich glycoproteins. Under field conditions, a maximum protection of 62% was recorded upon seed treatment along with foliar spray. Hence, it infers that Ind-Ile-Me can be used as a valuable protection compound at least in downy mildew disease management.     

番茄白粉病苗期抗病性鉴定方法及抗病种质资源筛选

采用不同接种方法、不同接种苗龄、不同接种浓度三因素三水平正交试验设计对番茄白粉病苗期抗性鉴定方法进行筛选。结果表明,番茄苗期接种白粉病最好的方法是喷雾法,接种苗龄为四叶期,接种的最适浓度为106孢子/mL。在此基础上,对东北农业大学番茄研究所提供的54份番茄材料进行白粉病苗期接种鉴定,结果表明,54份材料中,有抗病品种4份、中抗品种16份、感病品种17份、极感病品种17份。     

Hypersensitive reaction and P/HRGP accumulation is modulated by nitric oxide through hydrogen peroxide in pearl millet during Sclerospora graminicola infection

Priming of pearl millet seedlings with nitric oxide (NO) donors sodium nitroprusside (SNP) and S-nitrosoglutathione (SNOG) induced hypersensitive reactions (HR) and accumulation of Proline/Hydroxyproline-rich glycoprotein (P/HRGP) during infection by downy mildew pathogen Sclerospora graminicola. Such defense responses were specifically altered by concentration of NO donors resulting in the modulation of endogenous NO in seedling tissues. The stoichiometric interactions of NO and hydrogen peroxide (H₂O₂) when followed in relation to HR and P/HRGP accumulation, the degree of defense response varied with H₂O₂ level, the latter being largely influenced by NO concentration. Therefore, balancing NO and H₂O₂ is vital for optimum expression of defense responses for imparting disease resistance.