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Induction of resistance against downy mildew pathogen in pearl millet by a synthetic jasmonate analogon
ARTICLE IN PRESS
Physiological and Molecular Plant Pathology 71 (2007) 96–105 www.elsevier.com/locate/pmpp
Induction of resistance against downy mildew pathogen in pearl millet by a synthetic jasmonate analogon Shantharaj Deepaka,b, Sathyanarayana Niranjan-Raja, Shekar Shailasreea, Ramachandra K. Kinia, Wilhelm Bolandb, Hunthrike S. Shettya, Axel Mitho¨ferb, a
Department of Studies in Applied Botany and Biotechnology, University of Mysore, Manasagangotri, Mysore 570006, Karnataka, India b Department Bioorganic Chemistry, Max Planck Institute for Chemical Ecology, Hans-Kno¨ll-StraX e 8, D-07745 Jena, Germany Accepted 13 December 2007
Abstract 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. r 2008 Elsevier Ltd. All rights reserved. Keywords: Field study; Induced resistance; Jasmonate analogon; Sclerospora
1. Introduction Pearl millet (Pennisetum glaucum (L.) R. Br.) is an important grain and forage crop, primarily in the arid and subtropical regions of many developing countries with a long-lasting tradition in cultivation. Being the staple food for 90 million people of the semiarid tropics, pearl millet is grown on 9.8 million hectares with an annual production of about 7 million tones in India. Downy mildew disease caused by the biotrophic oomycete Sclerospora graminicola (Sacc.) Schroet. is the most devastating disease, causing huge yield losses of 10–80% in various countries of Asia and Africa [1,2]. In India, the monetary loss due to a single epidemic of downy mildew is calculated to be £7.8 million [3]. The available management strategies include use of resistant cultivars and systemic fungicides. Corresponding author. Tel.: +49 3641571263; fax: +49 3641571256.
E-mail address: [email protected] (A. Mitho¨fer). 0885-5765/$ - see front matter r 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.pmpp.2007.12.003
However, lack of durable resistance, existence of pathogenic variability, and concerns about fungicide resistance have limited the potential of such strategies for managing the disease; for example Thakur et al. [4] reported on a pathogen-developed resistance to fungicides and break down of resistance in F1 hybrids. Hence, there is serious concern about the alternative approaches to explore and deploy in managing the disease and one such innovative approach might be the induction of resistance in the host. Plant treatment with appropriate biotic and abiotic agents can induce resistance to future pathogen attack including fungi, viruses, bacteria, and herbivores [5–7]. The induced state can be achieved by increasing the production of a range of defense-related products such as defenseinduced signaling compounds, pathogenesis-related proteins, and phytoalexins. Induced resistance can be triggered by a number of chemicals, plant growth-promoting rhizobacteria, avirulent pathogens, and pathogen-derived
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elicitors [5,7]. Exogenous application of chemicals such as b-aminobutyric acid (BABA), acibenzolar-S-methyl, and other benzothiadizole (BTH) derivatives, 2,6-dichloroisonicotinic acid (INA) and derivatives, and salicylic acid (SA) has been shown to induce systemic resistance in various plants [7–11]. This concept of induced resistance has already been demonstrated for the pearl millet/Sclerospora pathosystem. Previous studies demonstrated an enhanced protection of pearl millet against downy mildew disease after the application of such compounds, for example BABA [12], H2O2, and CaCl2 [13], proline [14], chitosan [15], Trichoshield [16], INA [17], cerebroside [18], and Iprovalicarb [19]. It has also been shown that microorganisms such as Pseudomonas fluorescens [20], as well as plant extracts of Datura metel [21], had the potential to control S. graminicola. The jasmonate family of plant signaling compounds comprises biologically highly active cyclopentenones (e.g. 12-oxo-phytodienoic acid) and cyclopentanones (e.g. jasmonic acid, JA) of related origin and structure. Among others, their biological activities include a broad range of defense-related reactions directed against pathogens and pests. Based on the structure of a bacterial phytotoxin with similar biological activities compared with jasmonates, coronatine, indanoyl isoleucine conjugates have been designed as functional synthetic mimics of octadecanoidderived signals [22–24]. The general activity of one particular compound, 1-oxo-indanoyl-L-isoleucine methyl ester (Ind-Ile-Me; Fig. 1), in the induction of plant stressrelated responses together with its simple and efficient synthesis suggests that this compound might serve as a valuable tool in the examination of various aspects in plant stress physiology and agriculture. The present study was conducted to evaluate the efficiency of Ind-Ile-Me in effective control of downy mildew incidence of pearl millet by seed treatment and foliar spray under greenhouse and field conditions. It represents the first example for an investigation of the protective capacity of a compound of the jasmonatesmimicking indanoyl isoleucine conjugate series beyond controlled laboratory conditions.
O
HN
O
MeOOC
Fig. 1. Structure of 1-oxo-indanoyl-L-isoleucine methyl ester (Ind-Ile-Me).
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2. Materials and methods 2.1. Plant material and inductor Pearl millet seeds cv. 7042S which is highly susceptible and IP18292 which is highly resistant to downy mildew were obtained from the International Crop Research Institute for Semi Arid Tropics (ICRISAT), Hyderabad, India. The jasmonate analogon, 1-oxo-indanoyl-L-isoleucine methyl ester (Ind-Ile-Me), was available in the laboratory (MPI for Chemical Ecology). The synthesis has been described [25]. 2.2. Pathogen and inoculum S. graminicola was insolated from pearl millet cv. 7042S grown in heavily infested field soil. The pathogen was maintained on its susceptible host prior to use. Leaves of pearl millet showing profuse sporulation of S. graminicola on the abaxial side were collected in the evening hours from the plants maintained under greenhouse conditions. The collected leaves were thoroughly washed under running tap water to remove sporangia from previous sporulation. The leaves were then blot-dried, cut into pieces, and maintained in a moist chamber for sporulation. Subsequently, sporangia were collected in sterile distilled water for use as inoculum at a concentration of 4 104 zoospores ml1 as read using a hemocytometer. 2.3. Plant analyses For assays under laboratory conditions, aqueous solutions were prepared with various concentrations (5, 10, 25, 50, 75, 100, 200 mM) of Ind-Ile-Me. Seeds of the susceptible (7042S) pearl millet cultivar were surface sterilized in 0.1% sodium hypochlorite solution for 5 min and washed thoroughly with sterile distilled water. Seeds were soaked in various Ind-Ile-Me concentrations as mentioned earlier at 26 1C for 6 h in a rotary shaker at 150 rpm. Seeds subjected to distilled water treatment under similar conditions served as control. Germination tests were carried out by the paper towel method following the seed treatment procedure [26]. Seedling vigor was analyzed after 7 days of incubation [27]. The experiment was performed with four replicates of 100 seeds each and was repeated independently three times. The length of the roots and shoots of individual seedlings was measured to determine the vigor index. The vigor index was calculated using the formula: vigor index ¼ (mean root length+mean shoot length) (% germination). For the assays under greenhouse conditions, two Ind-IleMe concentrations (50 and 75 mM) assessed from seed quality parameter were used. Here, seeds were treated as described earlier. The seeds treated with systemic fungicide metalaxyl formulation Apron 35 SD (6 g kg1) served as an additional chemical check [28]. The treated seeds were
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sown in pots filled with autoclaved soil, sand, and manure at the ratio of 2:1:1. Each treatment consisted of 4 replicates, 10 pots per replicate, and 10 seedlings per pot. ð%Þ protection ¼
For greenhouse and field data, percentage of protection was calculated using the formula:
ð%Þ disease in untreated plants ð%Þ disease in Ind-Ile-Me treated plants 100 ð%Þ downy mildew in untreated plants
Treatments were arranged in a randomized complete block design. Two-day-old seedlings were challenge inoculated by the whorl inoculation method of Singh and Gopinath [29] with a zoospore suspension of S. graminicola at a concentration of 4 104 zoospores ml1 prepared as described previously. In the whorl inoculation method, S. graminicola zoospores were dropped onto the leaf whorl formed by the emerging seedlings and allowed to flow down to the base. These pathogen-inoculated plants were maintained under greenhouse conditions (90–95% RH, 20–25 1C) and observed for disease development. The plants were rated for disease when they showed any typical downy mildew symptoms such as sporulation on the abaxial leaf surface, chlorosis, stunted growth, or malformation of the ear heads. Data was consolidated at 60 days after emergence. In a parallel set of experiments, seed treatment was performed also in combination with Ind-Ile-Me foliar spraying of 2-day-old seedlings. S. graminicola inoculation was done 1 day after Ind-Ile-Me spraying. In addition, after 7 and 14 days, plants were sprayed with Ind-Ile-Me solutions until run off from the foliage and further inoculated with S. graminicola (4 104 zoospores ml1) in each case 1 day after spraying. The plants were rated for disease when they showed typical downy mildew symptoms. For experiments under field conditions, Ind-Ile-Me treatments were carried out according to the greenhouse studies. The concentration of Ind-Ile-Me (75 mM) showed maximum protection and was considered for field trail. Field trails were carried out with a combination of seed treatment and foliar spray. Trails were conducted at the downy mildew sick plot, Department of Studies in Applied Botany, Seed Pathology and Biotechnology, University of Mysore, Mysore, India. Randomized split plots were used. Ind-Ile-Me-treated seeds and untreated control seeds were sown in sub-plots consisting of four 4-m rows, each 75 cm apart. Plants were about 15 cm apart within the rows. The field has been naturally infested with oospores of S. graminicola for three decades and these oospores served as the source of primary inoculum. Additional inoculum was provided by the infector rows, which were seeded 21 days prior to the sowing of the test rows as described by Williams and Singh [30]. There were two replicates per treatment. Normal agronomic practices were followed to raise the crop. The plants were observed for downy mildew disease development at 30 and 60 days after emergence and rated as diseased when they showed the typical downy mildew symptoms. The data were consolidated after 60 days.
2.4. Effect of Ind-Ile-Me treatment on induction of defense enzymes Pearl millet seeds were germinated on moist filter paper under aseptic conditions at 2572 1C in the dark. Two-dayold seedlings of resistant (IP18296) untreated, susceptible (7042S) untreated, and susceptible treated (75 mM Ind-IleMe; 26 1C, 6 h, 150 rpm) seeds were subjected to inoculation by root dip technique with 4 104 zoospores ml1 suspension of S. graminicola [31]. For non-inoculated controls, seedlings soaked in sterile distilled water were used. The seedlings were harvested at the particular time for biochemical analyses: 4 h post-infection (h.p.i.) for phenylalanine-ammonia-lyase (PAL) activity, 8 h.p.i. for peroxidase and catalase activity, 24 h.p.i. for b-1,3-glucanase and chitinase activity, and 8 h.p.i. for hydroxyproline (Hyp) estimation. The samples were stored at 45 1C until further use. All enzyme analyses were repeated three times, taking three replicates at a time. 2.4.1. Phenylalanine-ammonia-lyase (PAL) enzyme assay Seedlings (1 g fresh weight) were extracted with 25 mM sodium borate buffer (pH 8.8) and 32 mM b-mercaptoethanol at 4 1C with a pre-chilled pestle and mortar. The homogenate was centrifuged at 20,000g for 20 min at 4 1C in a refrigerated centrifuge. PAL activity in the supernatant of the cell-free extracts was assayed as described by Lisker et al. [32] with slight modifications. The reaction mixture (3 ml) consisted of 50 mM L-phenylalanine in 100 mM sodium borate buffer (pH 8.8) and 100 ml of the crude extract. The reaction was measured with a spectrophotometer at 290 nm. The enzyme activity was expressed in terms of mmol trans-cinnamic acid (tCA) min1 mg1 protein. 2.4.2. Peroxidase activity Seedlings (1 g fresh weight) were homogenized in 2 ml of 0.2 M Tris/HCl buffer pH 8.0 at 4 1C. The homogenate was filtered through cheesecloth and the filtrate centrifuged at 12,000g for 15 min. The supernatant was dialyzed against distilled water for 48 h at 4 1C, lyophilized, and stored. This crude enzyme extract was used for peroxidase and catalase spectrophotometric assay. Peroxidase enzyme assay was carried out as described by Hammerschmidt et al. [33]. The reaction mixture (3 ml) consisted of 0.25% (v/v) guaiacol and 10 mM hydrogen peroxide in 10 mM potassium phosphate buffer (pH 6.9). Enzyme reaction was initiated with 25 ml of crude enzyme extract and measured spectrophotometrically at 470 nm. One unit of peroxidase enzyme
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activity is defined as the increase in absorbance recorded at one OD value of A470 min1. Peroxidase activity is expressed in terms of the change in absorbance at 470 nm (A470 min1 mg1 protein). 2.4.3. Catalase activity Catalase activity was measured spectrophotometrically by monitoring the consumption of H2O2 at 240 nm for 1 min after adding a known amount of enzyme extract to the reaction mixture (3 ml, 10 mM hydrogen peroxide in 10 mM potassium phosphate buffer, pH 6.9). Catalase activity was expressed in terms of the change in absorbance at 240 nm (A240 min1 mg1 protein), according to Luck [34].
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5 vol of acetone and then air-dried. The hydrolysis of the cell walls with 6 N HCl for 18 h at 110 1C was carried out in sealed tubes. To remove HCl, the hydrolysates were evaporated to dryness. Hyp was then extracted in minimum amount of distilled water from the dried hydrolyzed samples, derivatized, and spectrophotometrically estimated at 560 nm [38]. Hyp content was expressed as mg Hyp mg1 cell wall (dry weight). 2.5. Protein determination Protein concentration was determined according to Bradford [39] using bovine serum albumin as standard. 3. Results
2.4.4. b-1,3-Glucanase assay Fresh seedlings (1 g) were homogenized using 0.05 M sodium acetate buffer pH 5.2 in a pre-chilled pestle and mortar at 4 1C. The homogenate was centrifuged at 10,000g for 20 min at 4 1C and supernatant was used as crude extract. According to Isaac and Gokhale [35], b-1,3glucanase activity was determined using 0.1% laminarin in 0.05 M sodium acetate buffer (pH 5.2) as substrate. Released glucose was calculated based on a glucose standard curve. Products released after incubation were estimated for reducing groups at 540 nm using the dinitrosalicylic acid reagent. Enzyme activity was expressed in terms of mmol min1 mg1 protein.
3.1. Effect of Ind-Ile-Me seed treatment on seed germination and seedling vigor To analyze whether or not Ind-Ile-Me had any negative effects on seed germination and/or seedling vigor per se, both parameters were tested in a concentration range from 5 to 200 mM Ind-Ile-Me (Table 1). Compared to the watertreated control, up to a concentration of 75 mM, no impairment on germination could be observed. Higher concentration showed slight decrease of the germination rate. The vigor index revealed positive effects for all tested concentrations (Table 1).
2.4.5. Chitinase activity Fresh seedling tissue (1 g) was macerated using 0.05 M sodium acetate buffer, pH 5.2 and acid-washed glass beads at 4 1C. The samples were centrifuged at 12,100g for 30 min and the supernatant used as crude extract. Chitinase was assayed following the method of Isaac and Gokhale [35] with N-acetyl glucosamine as standard. Colloidal chitin in 0.05 M sodium acetate buffer (pH 5.2), purified from chitin following the method of Skujins et al. [36] was used as substrate. The concentration of N-acetyl glucosamine released after incubation was measured spectrophotometrically at 585 nm, using dimethylamino benzaldehyde reagent [37]. Enzyme activity was expressed in mmol N-acetyl glucosamine min1 mg1 protein.
3.2. Effect of Ind-Ile-Me on downy mildew disease incidence under greenhouse conditions
2.4.6. Hydroxyproline estimation Cell walls from the coleoptile of pearl millet seedlings were prepared and further processed as described Deepak et al. [38]. Briefly, roots and coleoptiles of the seedlings were separated and homogenized using pestle and mortar at 4 1C in 0.5 M potassium phosphate buffer, pH 7.0. The paste was observed under microscope for complete disruption of cells. The suspension of broken cells was centrifuged at 2000g for 10 min. Cell walls were repeatedly washed with the above buffer followed by distilled water. Washed cell walls were suspended by vigorous stirring in 5 vol of 1:1 chloroform–methanol. The organic solvent was carefully removed. Cell walls were repeatedly washed with
Table 1 Effects of Ind-Ile-Me concentration on pearl millet seed germination and seedling vigor
Based on the results obtained, concentrations which showed both good seed germination and seedling vigor were used for further studies. In the greenhouse, seed treatment with Ind-Ile-Me followed by challenge inoculation with S. graminicola zoospores showed anti-mildew activity and resulted in protection rates of 42% and 51% at 50 and 75 mM Ind-Ile-Me, respectively (Table 2). Seed treatment followed by additional foliar spray on 7-day-old and 14-day-old seedlings resulted in elevated resistance of pearl millet with protection rates of 58% and 60% at 50 and 75 mM Ind-Ile-Me, respectively. In a parallel
Treatment (mM)
Seed germination (%)7S.E.
Seedling vigor7S.E.
Water 5 10 25 50 75 100 200
92.570.35 93.570.35 92.570.35 94.570.35 93.571.06 94.570.35 90.570.35 82.571.77
136371.5 151774.5 155873.5 156774.5 157976.0 161976.5 142877.0 140775.5
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Table 2 Effects of Ind-Ile-Me treatments on downy mildew disease incidence in pearl millet under greenhouse conditions Treatment
Seed treatment
Water Ind-Ile-Me (75 mM) Metalaxyl (6 g kg1 seeds) Ind-Ile-Me (50 mM)
Seed treatment+foliar spray
Plants with downy mildew symptoms (%)7S.E.
Protection (%)7S.E.
Plants with downy mildew symptoms (%)7S.E.
Protection (%)7S.E.
9870.0 4973.1 870.7 5872.5
270.7 5173.1 9271.0 4272.5
9870.0 4072.1 971.2 4271.2
270.7 6071.5 9171.4 5871.8
Table 3 Effects of Ind-Ile-Me treatments on downy mildew disease incidence in pearl millet under field conditions Treatment
Plants with downy mildew symptoms (%)7S.E.
Water 9870.0 Ind-Ile-Me (75 mM) 3872.5 water Metalaxyl (6 g kg1 870.7 seeds)
Protection (%)7S.E. 271.6 6274.2 9270.1
experiment, seed treatment with the systemic fungicide metalaxyl (6 g kg1 seeds) showed a protection of more than 90% when challenge inoculated with S. graminicola (Table 2). 3.3. Effect of Ind-Ile-Me on downy mildew disease incidence under field conditions
oxidative stress responses such as peroxidase and catalase, hydrolytic enzymes (b-1,3-glucanase, chitinase) able to cleave fungal cell walls, and hydroxyproline-rich glycoproteins (HRGPs) as inducible components of the plant cell wall strengthening [40]. 3.4.1. Phenylalanine-ammonia-lyase (PAL) The activity of PAL was determined to increase slightly (from 4.2 to 5.3 mmol tCA min1 mg1 protein) upon inoculation with S. graminicola in the susceptible cultivar 7042S. Ind-Ile-Me treatment of seeds of cultivar 7042S enhanced PAL activity (7.9 mmol tCA min1 mg1 protein) compared to the non-treated control and reached a level of PAL activity that was also determined for the resistant cultivar IP18292. In the latter cultivar, a higher basic PAL activity compared to the susceptible cultivar was recorded, which even slightly increased upon infection with S. graminicola (Fig. 2).
In order to test the robustness of the induced resistance provided by Ind-Ile-Me, further studies were conducted by seed treatment followed by foliar spraying under field conditions. 7042S seeds treated with 75 mM Ind-Ile-Me clearly showed protection against downy mildew disease (Table 3). The better protection was observed when the foliar spraying compared with seed treatment with a dose of 75 mM Ind-Ile-Me was repeated at days 7, 14, and 30. Data analysis after 60 days revealed that the protection rate imparted by Ind-Ile-Me to downy mildew was 62%. Single seed treatment with metalaxyl again resulted in more than 90% protection (Table 3).
3.4.2. Peroxidase In susceptible pearl millet 7042S seedlings, peroxidase activity only slightly increased when challenge inoculated with S. graminicola from 16 to 21 units (A470 min1 mg1 protein). This activity strongly increased upon treatment with Ind-Ile-Me and no further rise in activity was induced by S. graminicola infection. The resistance cultivar IP18296 showed a 75% higher basic peroxidase activity compared to the susceptible cultivar and more than doubling of this enzyme activity upon S. graminicola infection from 29 to 65 units (A470 min1 mg1 protein) (Fig. 3A).
3.4. Effect of Ind-Ile-Me treatment on the activities of defense-related enzymes
3.4.3. Catalase A significant decrease in catalase activity was recorded in the resistant cultivar upon S. graminicola infection relative to the control from 7.1 to 4.5 units (A240 min1 mg1 protein). A similar trend was observed for the susceptible seedlings where the catalase activity decreased from 1.9 to 1.4 units (A240 min1 mg1 protein) upon S. graminicola infection. When Ind-Ile-Me-treated 7042S plants were analyzed, no enhancement of catalase activity was observed but the infection-mediated decrease in activity was suppressed (Fig. 3B).
The observed effects of Ind-Ile-Me treatment on the induced resistance of pearl millet against S. graminicola infection was further investigated with respect to the activities of defense-related proteins including pathogenesis-related proteins (PR) or enzymes, which are known to have a role in plant defense [40]. This included the PAL, the key enzyme of the phenylpropanoid pathway involved, for example, in flavanoids and lignin biosyntheses, enzymes of
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3.4.4. b-1,3-Glucanase A higher level of b-1,3-glucanase activity was observed in resistant over susceptible seedlings. In both cases, S. graminicola infection caused an increase of b-1,3-glucanase activity from 1.0 to 1.5 mmol glucose min1 mg1 protein in the resistant and from 0.5 to 0.9 mmol glucose min1 mg1 protein in the susceptible cultivar, respectively. However, in the susceptible cultivar treated with Ind-Ile-Me, the enzyme activity only marginally increased upon infection compared to the non-infected plants from 0.6 to 1.2 mmol glucose min1 mg1 protein (Fig. 4A).
Fig. 2. Phenylalanine-ammonia-lyase activity in susceptible 7042S and resistant IP18296 pearl millet seedlings and the impact of Ind-Ile-Me on S. graminicola infection. PAL activity was assayed in seedlings in controls (&) and after inoculation with S. graminicola (’) at 4 h. Enzyme activity is expressed as mmol t-cinnamic acid (tCA) min1 mg1 protein. Values represent the mean of three independent experiments. Bars indicate 7S.E. RU, resistant uninoculated; RI, resistance inoculated; SU, susceptible uninoculated; SI, susceptible inoculated; ST, susceptible Ind-Ile-Me treatment; STI, susceptible Ind-Ile-Me treated followed by inoculation.
3.4.5. Chitinase Chitinase activity was not or only slightly increased after downy mildew infection both in the susceptible (3.4 nmol N-acetyl glucosamine min1 mg1 protein) and the resistant (4.4 nmol N-acetyl glucosamine min1 mg1 protein) cultivar. A strong increase in chitinase activity up to 5.0 nmol N-acetyl glucosamine min1 mg1 protein was observed in the Ind-Ile-Me-treated 7042S seedlings only after inoculation with the pathogen (Fig. 4B). 3.4.6. Hydroxyproline Hydroxyproline (Hyp) estimation gives a measure of HRGPs in planta. In the IP18296-resistant cultivar, 0.35 mg Hyp was recorded in non-infected controls which strongly increased to 0.74 mg Hyp after S. graminicola inoculation. Ind-Ile-Me treatment of the 7042S cultivar resulted in enhanced accumulation of HRGPs, recording 0.43 mg
Fig. 3. Peroxidase and catalase activities in susceptible 7042S and resistant IP18296 pearl millet seedlings and the impact of Ind-Ile-Me on S. graminicola infection. (A) Peroxidase activity was assayed in control (&) and inoculated (’) seedlings at 8 h. Enzyme activity was expressed as units at A470 nm min1 mg1 protein. (B) Catalase activity was assayed in control (&) and inoculated (’) seedlings at 8 h. Enzyme activity was expressed as units at A240 nm min1 mg1 protein. Values represent the mean of three independent experiments. Bars indicate 7S.E. RU, resistant uninoculated; RI, resistance inoculated; SU, susceptible uninoculated; SI, susceptible inoculated; ST, susceptible Ind-Ile-Me treatment; STI, susceptible Ind-Ile-Me treated followed by inoculation.
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Fig. 4. b-1,3-Glucanase and chitinase activities in susceptible 7042S and resistant IP18296 pearl millet seedlings and the impact of Ind-Ile-Me on S. graminicola infection. b-1,3-Glucanase activity was assayed in control (&) and inoculated (’) seedlings at 24 h. Enzyme activity was expressed as mmol of glucose released min1 mg1 protein. (B) Chitinase activity was assayed in seedlings in control (&) and after inoculation with S. graminicola (’) at 24 h. Enzyme activity is expressed in nmol min1 mg1 protein. Values represent the mean of three independent experiments. Bars indicate 7S.E. RU, resistant uninoculated; RI, resistance inoculated; SU, susceptible uninoculated; SI, susceptible inoculated; ST, susceptible Ind-Ile-Me treatment; STI, susceptible Ind-Ile-Me treated followed by inoculation.
4. Discussion
Fig. 5. Hydroxyproline-rich glycoprotein content in susceptible 7042S and resistant IP18296 pearl millet seedlings and the impact of Ind-Ile-Me on S. graminicola infection. RI, resistance inoculated; SU, susceptible uninoculated; SI, susceptible inoculated; ST, susceptible Ind-Ile-Me treatment; STI, susceptible Ind-Ile-Me treated followed with inoculation. The data are means of three independent experiments. Bars indicate 7S.E. The samples are control (&) and inoculated (’) samples of pearl millet seedlings.
Hyp and 0.13 mg Hyp in the non-treated plants. This further increased to 0.72 mg in Ind-Ile-Me samples upon S. graminicola infection and 0.3 mg Hyp in the respective control seedlings (Fig. 5).
Induced resistance is defined as an enhancement of plants defensive capacity against a broad spectrum of pathogens after appropriate stimulation. Various inducers are known to initiate such processes leading to increased levels of defense-related compounds and signals, for example salicylic acid and jasmonates [5,7]. Octadecanoids such as OPDA and JA are widely distributed phytohormones in higher plants with a broad spectrum of biological effects in plant development, disease resistance, and stress physiology. However, there are only very few reports on jasmonates used as inducer of resistance in field studies: Exogenous JA sprayed on tomato plants in agricultural plots increased levels of polyphenol oxidase and proteinase inhibitors [41] and JA also increased the parasitization of induced plants two-fold under field conditions [42]. Because application of JA induces these defensive compounds at low concentrations, it may prove to be a useful tool for stimulating plant resistance to insects in the field [41]. A series of jasmonate-mimicking compounds have recently been synthesized [24,25] among which the 6-ethyl indanoyl isoleucine conjugate, coronalon, has been proven to act as elicitor in syntheses of many secondary metabolites including phytoalexins in cell cultures as well as in differentiated plants [23,24,43–45]. The efficiency of this synthetic conjugate as well as that of chemically related compounds added advantage in providing protection to various biotic stresses and, hence, bears agrochemical benefit.
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In the current study, Ind-Ile-Me treatment did negatively affect neither seed germination nor seedling vigor of pearl millet but provided protection against the pathogen S. graminicola. The evaluation of Ind-Ile-Me in combination with seed treatment and foliar spray gave promising disease prevention both under greenhouse and field conditions. Under greenhouse conditions, 58% at 50 mM and 60% at 75 mM protection was obtained. Field condition results even showed protection of 62% using a combination of seed treatment and foliar spray. Thus, the protection imparted by Ind-Ile-Me treatment in pearl millet to downy mildew in greenhouse and field data has proven its efficiency in managing disease at mM concentrations. Agricultural practices such as the usage of pesticide as seed treatment agents against pests could be avoided by replacing those compounds with the jasmonate mimetics. In a more detailed analysis concerning the involvement and behavior of typical defense-related enzymes, Bowles [40] showed that most of them have been up-regulated simply upon treatment with Ind-Ile-Me. The PAL is involved in biosynthesis of phytoalexins and lignin, thus contributing to the production of physical and chemical barriers against infections. In the present study, a primed increase of PAL in Ind-Ile-Me-treated seedlings was observed. Thus, an incompatible environment was generated by Ind-Ile-Me in a previously susceptible pearl millet cultivar. This suggested that in Ind-Ile-Me-induced protection of pearl millet, PAL may be involved in triggering phenylpropanoid pathway resulting in the release of toxic phytoalexins at the site of S. graminicola penetration [46]. Peroxidases are implicated in multiple functions of plant disease resistance against pathogens. The various physiological functions of peroxidases contributing to resistance include oxidation of hydroxy-cinnamyl alcohols into free radical intermediates, phenol oxidation, polysaccharide cross-linking, cross-linking of extensin monomers, and lignification. Increased level of peroxidase activity is reported in a number of resistant interactions involving plant-pathogenic and plant–pest interactions [47,48]; in pearl millet, peroxidase activity has already been described to be associated with reduction in the rate of pathogen multiplication and spread [21]. Elevated levels of peroxidases recorded in Ind-Ile-Me-protected pearl millet seedlings indicated that seed treatment of 7042S with Ind-Ile-Me again generated a partial incompatibility for S. graminicola. In contrast, catalase activity did increase neither on S. graminicola infection nor on IndIle-Me treatment. Expression of higher levels of hydrolases such as b-1,3glucanases and chitinases has been shown to provide enhanced resistance to fungal pathogens [49,50]. Their induction after pathogen infection supplies protection by directly degrading fungal cell wall components and indirectly by releasing some elicitors from the decaying fungal cell wall that might stimulate other plant defense mechanisms like phytoalexin accumulation in the host plant [51]. Seed treatment with Ind-Ile-Me resulted in
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increased levels of b-1,3-glucanase activity following the pattern of induction similar to that observed in resistant cultivar [52]. By contrast, Ind-Ile-Me treatment per se did not result in increase of chitinase activity; however, a significant enhancement of chitinase activity was recorded upon S. graminicola infection in these samples. Hydroxyproline-rich glycoproteins (HRGPs) are important plant cell wall structural components that are induced in response to pathogen attack [53]. Accumulation of HRGPs inferred from Hyp-measurements in pearl millet seedlings in response to S. graminicola as early as 2 h.p.i. and their further rapid deposition with a maximum occurring at 9 h.p.i. in plant cell walls offer effective restriction for the downy mildew pathogen [38,53]. Elevated levels of this glycoprotein in Ind-Ile-Me-treated pearl millet seedlings support a role for HRGPs in the IndIle-Me-offered protection of pearl millet to downy mildew pathogen. Increased level of HRGPs represented by hydroxyproline has been recorded in the present study in Ind-Ile-Me-treated seedlings, which further increased upon subsequent S. graminicola infection. Thus, Ind-Ile-Me treatment may bring about faster and stronger reinforcement of the pearl millet cell wall thereby contributing to disease resistance. Taken together, the efficacy of Ind-Ile-Me to induce plant disease resistance has been demonstrated in pearl millet downy mildew interaction under laboratory, greenhouse, and field conditions. The low concentration necessary, the simple and fast synthesis procedure, and the phytohormone character justify to rank Ind-Ile-Me as a valuable compound with a high agricultural potential. Acknowledgments The authors are grateful to the DANIDA-ENRECA for the facilities made available for the research programme. We also thank the Department of Science and Technology, Government of India, and Indian Council of Agricultural Research, New Delhi, and the Max-Planck-Society for financial support. S.D. would like to thank German Academic Exchange Service (DAAD) for financial support to carry out research work at the Max Planck Institute for Chemical Ecology, Jena, Germany. S.S. acknowledges the financial support and research grant received from Department of Science and Technology, New Delhi, India, under SERC Fast Track Young Scientist Programme. References [1] Nene YL, Singh SD. Downy mildew and ergot of pearl millet. Present News Search 1976;22:366–85. [2] Shetty SA, Shetty HS, Mathur SB. Down mildew of pearl millet. Technical bulletin. India: Downy Mildew Research Laboratory, University of Mysore; 1995. [3] Hash CT, Yadav RS, Sharma A, Bidinger FR, Devos KM, Gale MD, et al. Pearl millet molecular marker research. PSP annual report, vol. 35, 2003.
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[4] Thakur RP, Rao VP, Sastry JG, Sivaramkrishnan S, Amruthesh KN, Barbind LD. Evidence for a new virulent pathotype of Sclerospora graminicola on pearl millet. J Mycol Plant Pathol 1999;29:61–9. [5] Kuc J. Concepts and direction of induced systemic resistance in plants and its applications. Eur J Plant Pathol 2001;128:271–81. [6] Heil M, Bostock RM. Induced systemic resistance against pathogens in the context of induced plant defense responses. Ann Botany 2002;89:503–12. [7] Vallard GE, Goodman RM. Systemic acquired resistance and induced systemic resistance in conventional agriculture. Crop Sci 2004;44:1920–34. [8] Oostendorp M, Kunz W, Dietrich B, Staub T. Induced disease resistance in plants by chemicals. Eur J Plant Pathol 2001;107:19–28. [9] Agostoni JP, Bushong PM, Timmer LW. Green house evaluation of products that induce host resistance for control of scab melanose and Alternaria brown spot of citrus. Plant Dis 2003;87:69–74. [10] Ben-Shalom N, Ardi R, Pinto R, Aki C, Fallik E. Controlling gray mold caused by Botrytis cinerea in cucumber plants by means of chitosan. Crop Prot 2003;22:285–90. [11] de Capdiville G, Beer SV, Watkins CB, Wilson CL, Luis O, Tedeschi LO, et al. Pre- and post-harvest harpin treatment of apples induce resistance to blue mold. Plant Dis 2003;87:39–44. [12] Shailasree S, Sarosh BR, Vasanthi NS, Shetty HS. Seed treatment with b-aminobutyric acid protects Pennisetum glaucum systemically from Sclerospora graminicola. Pest Manage Sci 2001;57:721–8. [13] Geetha HM, Shetty HS. Induction of resistance in pearl millet against downy mildew disease caused by Sclerospora graminicola using benzothiadiazole, calcium chloride and hydrogen peroxide— a comparative evaluation. Crop Prot 2002;21:601–10. [14] Raj SN, Shetty NP, Shetty HS. Proline—an inducer of resistance against pearl millet downy mildew disease caused by Sclerospora graminicola. Phytoparasitica 2004;32:523–7. [15] Sharathchandra RG, Raj NS, Shetty NP, Amruthesh KN, Shetty HS. A chitosan formulation ElexaTM induces downy mildew disease resistance and growth promotion in pearl millet. Crop Prot 2004;23:881–8. [16] Raj SN, Shetty NP, Shetty HS. Synergistic effects of Trichoshield on enhancement of growth and resistance to downy mildew in pearl millet. Biocontrol 2005;50:493–509. [17] Shivakumar PD, Vasanthi NS, Shetty HS, Smedegaard-Petersen V. Ribonucleases in the seedlings of pearl millet and their involvement in resistance against downy mildew disease. Eur J Plant Pathol 2000;106:825–36. [18] Deepak SA, Raj NS, Umemura K, Kono T, Shetty HS. Cerebroside as an elicitor for induced resistance against the downy mildew pathogen in pearl millet. Ann Appl Biol 2003;143:169–73. [19] Deepak SA, Oros G, Raj NS, Shetty NP, Shetty HS. Iprovalicarb has potential for the control of downy mildew of pearl millet. Acta Phytopathol Entomol Hung 2004;39:55–69. [20] Raj SN, Shetty NP, Shetty HS. Seed bio priming with Pseudomonas fluorescens isolates enhances growth of pearl millet plants and induces resistance against downy mildew. Int J Pest Manage 2004;50:41–8. [21] Shivakumar PD, Geetha HM, Shetty HS. Peroxidase activity and isozyme analysis of pearl millet seedlings and their implications in downy mildew disease resistance. Plant Sci 2003;164:85–93. [22] Krumm T, Bandemer K, Boland W. Induction of volatile biosynthesis in the Lima bean (Phaseolus lunatus) by leucine- and isoleucine conjugates of 1-oxo- and 1-hydroxyindan-4-carboxylic acid: evidence for amino acid conjugates of jasmonic acid as intermediates in the octadecanoid signalling pathway. FEBS Lett 1995;377:523–9. [23] Schu¨ler G, Mitho¨fer A, Baldwin IT, Berger S, Ebel J, Santos JG, et al. Coronalon: a powerful tool in plant stress physiology. FEBS Lett 2004;563:17–22. [24] Mitho¨fer A, Maitrejean M, Boland W. Structural and biological diversity of cyclic octadecanoids, jasmonates and mimetics. J Plant Growth Regul 2005;23:170–8. [25] Lauchli R, Boland W. Indanoyl amino acid conjugates: tunable elicitors of plant secondary metabolism. Chem Rec 2003;3:12–21.
[26] ISTA. International rules for seed testing. Seed Sci Technol 1999;27(Suppl.). [27] Abdul-Baki A, Anderson JD. Vigor analysis in soybean seed by multiple criteria. Crop Sci 1973;13:630–3. [28] Singh SD, Shetty HS. Efficacy of systemic fungicide metalyaxyl for the control of downy mildew (Sclerospora graminicola) of pearl millet (Pennisetum glaucum). Indian J Agric Sci 1990;60:575–81. [29] Singh SD, Gopinath R. A seedling inoculation technique for detecting downy mildew resistance in pearl millet. Plant Dis 1985;69:582–4. [30] Williams RJ, Singh SD. Control of pearl millet downy mildew by seed treatment with metalaxyl. Ann Appl Biol 1981;97:263–8. [31] Safeeulla KM. Biology and control of the downy mildew of pearl millet, sorghum and finger millet. Mysore, India: Wesely Press; 1976. [32] Lisker N, Corn L, Chalutz E, Fucus Y. Fungal infections suppress ethylene-induced phenylalanine ammonia lyase activity in grape fruits. Physiol Mol Plant Pathol 1983;22:331–8. [33] Hammerschmidt R, Nuckles E, Kuc J. Association of enhanced peroxidase activity with induced systemic resistance of cucumber to Colletotrichum lagenarium. Physiol Mol Plant Pathol 1982;20: 73–82. [34] Luck H. Catalase. In: Bergmeryer HU, editor. Methods of enzymatic analysis. New York: Academic Press; 1963. p. 885–94. [35] Isaac S, Gokhale AV. Autolysis: a tool for protoplast production from Aspergillus nidulans. Trans Br Mycol Soc 1982;78:389–94. [36] Skujins JJ, Potgieter HJ, Alexander M. Dissolution of fungal cell walls by a streptomycete chitinase and b-(1,3)-glucanase. Arch Biochem Biophys 1965;111:358–64. [37] Reissig JL, Stominger JL, Leloir LF. A modified colorometric method for the estimation of N-acetylamino sugars. J Biol Chem 1955;217:959–66. [38] Deepak S, Shailasree S, Kini RK, Hause B, Shetty SH, Mitho¨fer A. Role of hydroxyproline rich glycoproteins in resistance of pearl millet against downy mildew pathogen Sclerospora graminicola. Planta 2007;226:323. [39] Bradford MM. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248–54. [40] Bowles DJ. Defense-related proteins in higher plants. Annu Rev Biochem 1990;59:873–907. [41] Thaler JS, Stout MJ, Karban R, Duffey SS. Exogenous jasmonates simulate insect wounding in tomato plants (Lycopersicon esculentum) in the laboratory and field. J Chem Ecol 1996;22:1767–81. [42] Thaler JS. Jasmonate-inducible plant defenses cause increased parasitism of herbivores. Nature 1999;399:686–8. [43] Fliegmann J, Schu¨ler G, Boland W, Ebel J, Mitho¨fer A. The role of octadecanoids and functional mimics in soybean defence response. Biol Chem 2003;384:437–46. [44] Berim A, Spring O, Conrad J, Maitrejean M, Boland W, Petersen M. Enhancement of lignan biosynthesis in suspension cultures of Linum nodiflorum by coronalon, indanoyl-isoleucine and methyl jasmonate. Planta 2005;222:769–76. [45] Hu Q, Boland W, Liu J. 6-Substituted indanoyl isoleucine conjugate induces tobacco plant responses in secondary metabolites. Z Naturforsch C 2005;60:1–4. [46] Geetha NP, Amruthesh KN, Sharatchandra RG, Shetty HS. Resistance to downy mildew in pearl millet is associated with increased phenylalanine ammonia lyase activity. Funct Plant Biol 2005;32:267–75. [47] Young SA, Guo A, Guikema JA, White FF, Leach JE. Rice cationic peroxidase accumulates in xylem vessels during incompatible interactions with Xanthomonas oryzae pv. oryzae. Plant Physiol 1995;107:1333–41. [48] Maffei ME, Mitho¨fer A, Arimura G-I, Uchtenhagen H, Bossi S, Bertea CM, et al. Effects of feeding Spodoptera littoralis on Lima beans leaves. III. Membrane depolarization and involvement of hydrogen peroxide. Plant Physiol 2006;140:1022–35.
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Physiological and Molecular Plant Pathology 71 (2007) 96–105 www.elsevier.com/locate/pmpp
Induction of resistance against downy mildew pathogen in pearl millet by a synthetic jasmonate analogon Shantharaj Deepaka,b, Sathyanarayana Niranjan-Raja, Shekar Shailasreea, Ramachandra K. Kinia, Wilhelm Bolandb, Hunthrike S. Shettya, Axel Mitho¨ferb, a
Department of Studies in Applied Botany and Biotechnology, University of Mysore, Manasagangotri, Mysore 570006, Karnataka, India b Department Bioorganic Chemistry, Max Planck Institute for Chemical Ecology, Hans-Kno¨ll-StraX e 8, D-07745 Jena, Germany Accepted 13 December 2007
Abstract 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. r 2008 Elsevier Ltd. All rights reserved. Keywords: Field study; Induced resistance; Jasmonate analogon; Sclerospora
1. Introduction Pearl millet (Pennisetum glaucum (L.) R. Br.) is an important grain and forage crop, primarily in the arid and subtropical regions of many developing countries with a long-lasting tradition in cultivation. Being the staple food for 90 million people of the semiarid tropics, pearl millet is grown on 9.8 million hectares with an annual production of about 7 million tones in India. Downy mildew disease caused by the biotrophic oomycete Sclerospora graminicola (Sacc.) Schroet. is the most devastating disease, causing huge yield losses of 10–80% in various countries of Asia and Africa [1,2]. In India, the monetary loss due to a single epidemic of downy mildew is calculated to be £7.8 million [3]. The available management strategies include use of resistant cultivars and systemic fungicides. Corresponding author. Tel.: +49 3641571263; fax: +49 3641571256.
E-mail address: [email protected] (A. Mitho¨fer). 0885-5765/$ - see front matter r 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.pmpp.2007.12.003
However, lack of durable resistance, existence of pathogenic variability, and concerns about fungicide resistance have limited the potential of such strategies for managing the disease; for example Thakur et al. [4] reported on a pathogen-developed resistance to fungicides and break down of resistance in F1 hybrids. Hence, there is serious concern about the alternative approaches to explore and deploy in managing the disease and one such innovative approach might be the induction of resistance in the host. Plant treatment with appropriate biotic and abiotic agents can induce resistance to future pathogen attack including fungi, viruses, bacteria, and herbivores [5–7]. The induced state can be achieved by increasing the production of a range of defense-related products such as defenseinduced signaling compounds, pathogenesis-related proteins, and phytoalexins. Induced resistance can be triggered by a number of chemicals, plant growth-promoting rhizobacteria, avirulent pathogens, and pathogen-derived
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elicitors [5,7]. Exogenous application of chemicals such as b-aminobutyric acid (BABA), acibenzolar-S-methyl, and other benzothiadizole (BTH) derivatives, 2,6-dichloroisonicotinic acid (INA) and derivatives, and salicylic acid (SA) has been shown to induce systemic resistance in various plants [7–11]. This concept of induced resistance has already been demonstrated for the pearl millet/Sclerospora pathosystem. Previous studies demonstrated an enhanced protection of pearl millet against downy mildew disease after the application of such compounds, for example BABA [12], H2O2, and CaCl2 [13], proline [14], chitosan [15], Trichoshield [16], INA [17], cerebroside [18], and Iprovalicarb [19]. It has also been shown that microorganisms such as Pseudomonas fluorescens [20], as well as plant extracts of Datura metel [21], had the potential to control S. graminicola. The jasmonate family of plant signaling compounds comprises biologically highly active cyclopentenones (e.g. 12-oxo-phytodienoic acid) and cyclopentanones (e.g. jasmonic acid, JA) of related origin and structure. Among others, their biological activities include a broad range of defense-related reactions directed against pathogens and pests. Based on the structure of a bacterial phytotoxin with similar biological activities compared with jasmonates, coronatine, indanoyl isoleucine conjugates have been designed as functional synthetic mimics of octadecanoidderived signals [22–24]. The general activity of one particular compound, 1-oxo-indanoyl-L-isoleucine methyl ester (Ind-Ile-Me; Fig. 1), in the induction of plant stressrelated responses together with its simple and efficient synthesis suggests that this compound might serve as a valuable tool in the examination of various aspects in plant stress physiology and agriculture. The present study was conducted to evaluate the efficiency of Ind-Ile-Me in effective control of downy mildew incidence of pearl millet by seed treatment and foliar spray under greenhouse and field conditions. It represents the first example for an investigation of the protective capacity of a compound of the jasmonatesmimicking indanoyl isoleucine conjugate series beyond controlled laboratory conditions.
O
HN
O
MeOOC
Fig. 1. Structure of 1-oxo-indanoyl-L-isoleucine methyl ester (Ind-Ile-Me).
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2. Materials and methods 2.1. Plant material and inductor Pearl millet seeds cv. 7042S which is highly susceptible and IP18292 which is highly resistant to downy mildew were obtained from the International Crop Research Institute for Semi Arid Tropics (ICRISAT), Hyderabad, India. The jasmonate analogon, 1-oxo-indanoyl-L-isoleucine methyl ester (Ind-Ile-Me), was available in the laboratory (MPI for Chemical Ecology). The synthesis has been described [25]. 2.2. Pathogen and inoculum S. graminicola was insolated from pearl millet cv. 7042S grown in heavily infested field soil. The pathogen was maintained on its susceptible host prior to use. Leaves of pearl millet showing profuse sporulation of S. graminicola on the abaxial side were collected in the evening hours from the plants maintained under greenhouse conditions. The collected leaves were thoroughly washed under running tap water to remove sporangia from previous sporulation. The leaves were then blot-dried, cut into pieces, and maintained in a moist chamber for sporulation. Subsequently, sporangia were collected in sterile distilled water for use as inoculum at a concentration of 4 104 zoospores ml1 as read using a hemocytometer. 2.3. Plant analyses For assays under laboratory conditions, aqueous solutions were prepared with various concentrations (5, 10, 25, 50, 75, 100, 200 mM) of Ind-Ile-Me. Seeds of the susceptible (7042S) pearl millet cultivar were surface sterilized in 0.1% sodium hypochlorite solution for 5 min and washed thoroughly with sterile distilled water. Seeds were soaked in various Ind-Ile-Me concentrations as mentioned earlier at 26 1C for 6 h in a rotary shaker at 150 rpm. Seeds subjected to distilled water treatment under similar conditions served as control. Germination tests were carried out by the paper towel method following the seed treatment procedure [26]. Seedling vigor was analyzed after 7 days of incubation [27]. The experiment was performed with four replicates of 100 seeds each and was repeated independently three times. The length of the roots and shoots of individual seedlings was measured to determine the vigor index. The vigor index was calculated using the formula: vigor index ¼ (mean root length+mean shoot length) (% germination). For the assays under greenhouse conditions, two Ind-IleMe concentrations (50 and 75 mM) assessed from seed quality parameter were used. Here, seeds were treated as described earlier. The seeds treated with systemic fungicide metalaxyl formulation Apron 35 SD (6 g kg1) served as an additional chemical check [28]. The treated seeds were
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sown in pots filled with autoclaved soil, sand, and manure at the ratio of 2:1:1. Each treatment consisted of 4 replicates, 10 pots per replicate, and 10 seedlings per pot. ð%Þ protection ¼
For greenhouse and field data, percentage of protection was calculated using the formula:
ð%Þ disease in untreated plants ð%Þ disease in Ind-Ile-Me treated plants 100 ð%Þ downy mildew in untreated plants
Treatments were arranged in a randomized complete block design. Two-day-old seedlings were challenge inoculated by the whorl inoculation method of Singh and Gopinath [29] with a zoospore suspension of S. graminicola at a concentration of 4 104 zoospores ml1 prepared as described previously. In the whorl inoculation method, S. graminicola zoospores were dropped onto the leaf whorl formed by the emerging seedlings and allowed to flow down to the base. These pathogen-inoculated plants were maintained under greenhouse conditions (90–95% RH, 20–25 1C) and observed for disease development. The plants were rated for disease when they showed any typical downy mildew symptoms such as sporulation on the abaxial leaf surface, chlorosis, stunted growth, or malformation of the ear heads. Data was consolidated at 60 days after emergence. In a parallel set of experiments, seed treatment was performed also in combination with Ind-Ile-Me foliar spraying of 2-day-old seedlings. S. graminicola inoculation was done 1 day after Ind-Ile-Me spraying. In addition, after 7 and 14 days, plants were sprayed with Ind-Ile-Me solutions until run off from the foliage and further inoculated with S. graminicola (4 104 zoospores ml1) in each case 1 day after spraying. The plants were rated for disease when they showed typical downy mildew symptoms. For experiments under field conditions, Ind-Ile-Me treatments were carried out according to the greenhouse studies. The concentration of Ind-Ile-Me (75 mM) showed maximum protection and was considered for field trail. Field trails were carried out with a combination of seed treatment and foliar spray. Trails were conducted at the downy mildew sick plot, Department of Studies in Applied Botany, Seed Pathology and Biotechnology, University of Mysore, Mysore, India. Randomized split plots were used. Ind-Ile-Me-treated seeds and untreated control seeds were sown in sub-plots consisting of four 4-m rows, each 75 cm apart. Plants were about 15 cm apart within the rows. The field has been naturally infested with oospores of S. graminicola for three decades and these oospores served as the source of primary inoculum. Additional inoculum was provided by the infector rows, which were seeded 21 days prior to the sowing of the test rows as described by Williams and Singh [30]. There were two replicates per treatment. Normal agronomic practices were followed to raise the crop. The plants were observed for downy mildew disease development at 30 and 60 days after emergence and rated as diseased when they showed the typical downy mildew symptoms. The data were consolidated after 60 days.
2.4. Effect of Ind-Ile-Me treatment on induction of defense enzymes Pearl millet seeds were germinated on moist filter paper under aseptic conditions at 2572 1C in the dark. Two-dayold seedlings of resistant (IP18296) untreated, susceptible (7042S) untreated, and susceptible treated (75 mM Ind-IleMe; 26 1C, 6 h, 150 rpm) seeds were subjected to inoculation by root dip technique with 4 104 zoospores ml1 suspension of S. graminicola [31]. For non-inoculated controls, seedlings soaked in sterile distilled water were used. The seedlings were harvested at the particular time for biochemical analyses: 4 h post-infection (h.p.i.) for phenylalanine-ammonia-lyase (PAL) activity, 8 h.p.i. for peroxidase and catalase activity, 24 h.p.i. for b-1,3-glucanase and chitinase activity, and 8 h.p.i. for hydroxyproline (Hyp) estimation. The samples were stored at 45 1C until further use. All enzyme analyses were repeated three times, taking three replicates at a time. 2.4.1. Phenylalanine-ammonia-lyase (PAL) enzyme assay Seedlings (1 g fresh weight) were extracted with 25 mM sodium borate buffer (pH 8.8) and 32 mM b-mercaptoethanol at 4 1C with a pre-chilled pestle and mortar. The homogenate was centrifuged at 20,000g for 20 min at 4 1C in a refrigerated centrifuge. PAL activity in the supernatant of the cell-free extracts was assayed as described by Lisker et al. [32] with slight modifications. The reaction mixture (3 ml) consisted of 50 mM L-phenylalanine in 100 mM sodium borate buffer (pH 8.8) and 100 ml of the crude extract. The reaction was measured with a spectrophotometer at 290 nm. The enzyme activity was expressed in terms of mmol trans-cinnamic acid (tCA) min1 mg1 protein. 2.4.2. Peroxidase activity Seedlings (1 g fresh weight) were homogenized in 2 ml of 0.2 M Tris/HCl buffer pH 8.0 at 4 1C. The homogenate was filtered through cheesecloth and the filtrate centrifuged at 12,000g for 15 min. The supernatant was dialyzed against distilled water for 48 h at 4 1C, lyophilized, and stored. This crude enzyme extract was used for peroxidase and catalase spectrophotometric assay. Peroxidase enzyme assay was carried out as described by Hammerschmidt et al. [33]. The reaction mixture (3 ml) consisted of 0.25% (v/v) guaiacol and 10 mM hydrogen peroxide in 10 mM potassium phosphate buffer (pH 6.9). Enzyme reaction was initiated with 25 ml of crude enzyme extract and measured spectrophotometrically at 470 nm. One unit of peroxidase enzyme
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activity is defined as the increase in absorbance recorded at one OD value of A470 min1. Peroxidase activity is expressed in terms of the change in absorbance at 470 nm (A470 min1 mg1 protein). 2.4.3. Catalase activity Catalase activity was measured spectrophotometrically by monitoring the consumption of H2O2 at 240 nm for 1 min after adding a known amount of enzyme extract to the reaction mixture (3 ml, 10 mM hydrogen peroxide in 10 mM potassium phosphate buffer, pH 6.9). Catalase activity was expressed in terms of the change in absorbance at 240 nm (A240 min1 mg1 protein), according to Luck [34].
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5 vol of acetone and then air-dried. The hydrolysis of the cell walls with 6 N HCl for 18 h at 110 1C was carried out in sealed tubes. To remove HCl, the hydrolysates were evaporated to dryness. Hyp was then extracted in minimum amount of distilled water from the dried hydrolyzed samples, derivatized, and spectrophotometrically estimated at 560 nm [38]. Hyp content was expressed as mg Hyp mg1 cell wall (dry weight). 2.5. Protein determination Protein concentration was determined according to Bradford [39] using bovine serum albumin as standard. 3. Results
2.4.4. b-1,3-Glucanase assay Fresh seedlings (1 g) were homogenized using 0.05 M sodium acetate buffer pH 5.2 in a pre-chilled pestle and mortar at 4 1C. The homogenate was centrifuged at 10,000g for 20 min at 4 1C and supernatant was used as crude extract. According to Isaac and Gokhale [35], b-1,3glucanase activity was determined using 0.1% laminarin in 0.05 M sodium acetate buffer (pH 5.2) as substrate. Released glucose was calculated based on a glucose standard curve. Products released after incubation were estimated for reducing groups at 540 nm using the dinitrosalicylic acid reagent. Enzyme activity was expressed in terms of mmol min1 mg1 protein.
3.1. Effect of Ind-Ile-Me seed treatment on seed germination and seedling vigor To analyze whether or not Ind-Ile-Me had any negative effects on seed germination and/or seedling vigor per se, both parameters were tested in a concentration range from 5 to 200 mM Ind-Ile-Me (Table 1). Compared to the watertreated control, up to a concentration of 75 mM, no impairment on germination could be observed. Higher concentration showed slight decrease of the germination rate. The vigor index revealed positive effects for all tested concentrations (Table 1).
2.4.5. Chitinase activity Fresh seedling tissue (1 g) was macerated using 0.05 M sodium acetate buffer, pH 5.2 and acid-washed glass beads at 4 1C. The samples were centrifuged at 12,100g for 30 min and the supernatant used as crude extract. Chitinase was assayed following the method of Isaac and Gokhale [35] with N-acetyl glucosamine as standard. Colloidal chitin in 0.05 M sodium acetate buffer (pH 5.2), purified from chitin following the method of Skujins et al. [36] was used as substrate. The concentration of N-acetyl glucosamine released after incubation was measured spectrophotometrically at 585 nm, using dimethylamino benzaldehyde reagent [37]. Enzyme activity was expressed in mmol N-acetyl glucosamine min1 mg1 protein.
3.2. Effect of Ind-Ile-Me on downy mildew disease incidence under greenhouse conditions
2.4.6. Hydroxyproline estimation Cell walls from the coleoptile of pearl millet seedlings were prepared and further processed as described Deepak et al. [38]. Briefly, roots and coleoptiles of the seedlings were separated and homogenized using pestle and mortar at 4 1C in 0.5 M potassium phosphate buffer, pH 7.0. The paste was observed under microscope for complete disruption of cells. The suspension of broken cells was centrifuged at 2000g for 10 min. Cell walls were repeatedly washed with the above buffer followed by distilled water. Washed cell walls were suspended by vigorous stirring in 5 vol of 1:1 chloroform–methanol. The organic solvent was carefully removed. Cell walls were repeatedly washed with
Table 1 Effects of Ind-Ile-Me concentration on pearl millet seed germination and seedling vigor
Based on the results obtained, concentrations which showed both good seed germination and seedling vigor were used for further studies. In the greenhouse, seed treatment with Ind-Ile-Me followed by challenge inoculation with S. graminicola zoospores showed anti-mildew activity and resulted in protection rates of 42% and 51% at 50 and 75 mM Ind-Ile-Me, respectively (Table 2). Seed treatment followed by additional foliar spray on 7-day-old and 14-day-old seedlings resulted in elevated resistance of pearl millet with protection rates of 58% and 60% at 50 and 75 mM Ind-Ile-Me, respectively. In a parallel
Treatment (mM)
Seed germination (%)7S.E.
Seedling vigor7S.E.
Water 5 10 25 50 75 100 200
92.570.35 93.570.35 92.570.35 94.570.35 93.571.06 94.570.35 90.570.35 82.571.77
136371.5 151774.5 155873.5 156774.5 157976.0 161976.5 142877.0 140775.5
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Table 2 Effects of Ind-Ile-Me treatments on downy mildew disease incidence in pearl millet under greenhouse conditions Treatment
Seed treatment
Water Ind-Ile-Me (75 mM) Metalaxyl (6 g kg1 seeds) Ind-Ile-Me (50 mM)
Seed treatment+foliar spray
Plants with downy mildew symptoms (%)7S.E.
Protection (%)7S.E.
Plants with downy mildew symptoms (%)7S.E.
Protection (%)7S.E.
9870.0 4973.1 870.7 5872.5
270.7 5173.1 9271.0 4272.5
9870.0 4072.1 971.2 4271.2
270.7 6071.5 9171.4 5871.8
Table 3 Effects of Ind-Ile-Me treatments on downy mildew disease incidence in pearl millet under field conditions Treatment
Plants with downy mildew symptoms (%)7S.E.
Water 9870.0 Ind-Ile-Me (75 mM) 3872.5 water Metalaxyl (6 g kg1 870.7 seeds)
Protection (%)7S.E. 271.6 6274.2 9270.1
experiment, seed treatment with the systemic fungicide metalaxyl (6 g kg1 seeds) showed a protection of more than 90% when challenge inoculated with S. graminicola (Table 2). 3.3. Effect of Ind-Ile-Me on downy mildew disease incidence under field conditions
oxidative stress responses such as peroxidase and catalase, hydrolytic enzymes (b-1,3-glucanase, chitinase) able to cleave fungal cell walls, and hydroxyproline-rich glycoproteins (HRGPs) as inducible components of the plant cell wall strengthening [40]. 3.4.1. Phenylalanine-ammonia-lyase (PAL) The activity of PAL was determined to increase slightly (from 4.2 to 5.3 mmol tCA min1 mg1 protein) upon inoculation with S. graminicola in the susceptible cultivar 7042S. Ind-Ile-Me treatment of seeds of cultivar 7042S enhanced PAL activity (7.9 mmol tCA min1 mg1 protein) compared to the non-treated control and reached a level of PAL activity that was also determined for the resistant cultivar IP18292. In the latter cultivar, a higher basic PAL activity compared to the susceptible cultivar was recorded, which even slightly increased upon infection with S. graminicola (Fig. 2).
In order to test the robustness of the induced resistance provided by Ind-Ile-Me, further studies were conducted by seed treatment followed by foliar spraying under field conditions. 7042S seeds treated with 75 mM Ind-Ile-Me clearly showed protection against downy mildew disease (Table 3). The better protection was observed when the foliar spraying compared with seed treatment with a dose of 75 mM Ind-Ile-Me was repeated at days 7, 14, and 30. Data analysis after 60 days revealed that the protection rate imparted by Ind-Ile-Me to downy mildew was 62%. Single seed treatment with metalaxyl again resulted in more than 90% protection (Table 3).
3.4.2. Peroxidase In susceptible pearl millet 7042S seedlings, peroxidase activity only slightly increased when challenge inoculated with S. graminicola from 16 to 21 units (A470 min1 mg1 protein). This activity strongly increased upon treatment with Ind-Ile-Me and no further rise in activity was induced by S. graminicola infection. The resistance cultivar IP18296 showed a 75% higher basic peroxidase activity compared to the susceptible cultivar and more than doubling of this enzyme activity upon S. graminicola infection from 29 to 65 units (A470 min1 mg1 protein) (Fig. 3A).
3.4. Effect of Ind-Ile-Me treatment on the activities of defense-related enzymes
3.4.3. Catalase A significant decrease in catalase activity was recorded in the resistant cultivar upon S. graminicola infection relative to the control from 7.1 to 4.5 units (A240 min1 mg1 protein). A similar trend was observed for the susceptible seedlings where the catalase activity decreased from 1.9 to 1.4 units (A240 min1 mg1 protein) upon S. graminicola infection. When Ind-Ile-Me-treated 7042S plants were analyzed, no enhancement of catalase activity was observed but the infection-mediated decrease in activity was suppressed (Fig. 3B).
The observed effects of Ind-Ile-Me treatment on the induced resistance of pearl millet against S. graminicola infection was further investigated with respect to the activities of defense-related proteins including pathogenesis-related proteins (PR) or enzymes, which are known to have a role in plant defense [40]. This included the PAL, the key enzyme of the phenylpropanoid pathway involved, for example, in flavanoids and lignin biosyntheses, enzymes of
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3.4.4. b-1,3-Glucanase A higher level of b-1,3-glucanase activity was observed in resistant over susceptible seedlings. In both cases, S. graminicola infection caused an increase of b-1,3-glucanase activity from 1.0 to 1.5 mmol glucose min1 mg1 protein in the resistant and from 0.5 to 0.9 mmol glucose min1 mg1 protein in the susceptible cultivar, respectively. However, in the susceptible cultivar treated with Ind-Ile-Me, the enzyme activity only marginally increased upon infection compared to the non-infected plants from 0.6 to 1.2 mmol glucose min1 mg1 protein (Fig. 4A).
Fig. 2. Phenylalanine-ammonia-lyase activity in susceptible 7042S and resistant IP18296 pearl millet seedlings and the impact of Ind-Ile-Me on S. graminicola infection. PAL activity was assayed in seedlings in controls (&) and after inoculation with S. graminicola (’) at 4 h. Enzyme activity is expressed as mmol t-cinnamic acid (tCA) min1 mg1 protein. Values represent the mean of three independent experiments. Bars indicate 7S.E. RU, resistant uninoculated; RI, resistance inoculated; SU, susceptible uninoculated; SI, susceptible inoculated; ST, susceptible Ind-Ile-Me treatment; STI, susceptible Ind-Ile-Me treated followed by inoculation.
3.4.5. Chitinase Chitinase activity was not or only slightly increased after downy mildew infection both in the susceptible (3.4 nmol N-acetyl glucosamine min1 mg1 protein) and the resistant (4.4 nmol N-acetyl glucosamine min1 mg1 protein) cultivar. A strong increase in chitinase activity up to 5.0 nmol N-acetyl glucosamine min1 mg1 protein was observed in the Ind-Ile-Me-treated 7042S seedlings only after inoculation with the pathogen (Fig. 4B). 3.4.6. Hydroxyproline Hydroxyproline (Hyp) estimation gives a measure of HRGPs in planta. In the IP18296-resistant cultivar, 0.35 mg Hyp was recorded in non-infected controls which strongly increased to 0.74 mg Hyp after S. graminicola inoculation. Ind-Ile-Me treatment of the 7042S cultivar resulted in enhanced accumulation of HRGPs, recording 0.43 mg
Fig. 3. Peroxidase and catalase activities in susceptible 7042S and resistant IP18296 pearl millet seedlings and the impact of Ind-Ile-Me on S. graminicola infection. (A) Peroxidase activity was assayed in control (&) and inoculated (’) seedlings at 8 h. Enzyme activity was expressed as units at A470 nm min1 mg1 protein. (B) Catalase activity was assayed in control (&) and inoculated (’) seedlings at 8 h. Enzyme activity was expressed as units at A240 nm min1 mg1 protein. Values represent the mean of three independent experiments. Bars indicate 7S.E. RU, resistant uninoculated; RI, resistance inoculated; SU, susceptible uninoculated; SI, susceptible inoculated; ST, susceptible Ind-Ile-Me treatment; STI, susceptible Ind-Ile-Me treated followed by inoculation.
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Fig. 4. b-1,3-Glucanase and chitinase activities in susceptible 7042S and resistant IP18296 pearl millet seedlings and the impact of Ind-Ile-Me on S. graminicola infection. b-1,3-Glucanase activity was assayed in control (&) and inoculated (’) seedlings at 24 h. Enzyme activity was expressed as mmol of glucose released min1 mg1 protein. (B) Chitinase activity was assayed in seedlings in control (&) and after inoculation with S. graminicola (’) at 24 h. Enzyme activity is expressed in nmol min1 mg1 protein. Values represent the mean of three independent experiments. Bars indicate 7S.E. RU, resistant uninoculated; RI, resistance inoculated; SU, susceptible uninoculated; SI, susceptible inoculated; ST, susceptible Ind-Ile-Me treatment; STI, susceptible Ind-Ile-Me treated followed by inoculation.
4. Discussion
Fig. 5. Hydroxyproline-rich glycoprotein content in susceptible 7042S and resistant IP18296 pearl millet seedlings and the impact of Ind-Ile-Me on S. graminicola infection. RI, resistance inoculated; SU, susceptible uninoculated; SI, susceptible inoculated; ST, susceptible Ind-Ile-Me treatment; STI, susceptible Ind-Ile-Me treated followed with inoculation. The data are means of three independent experiments. Bars indicate 7S.E. The samples are control (&) and inoculated (’) samples of pearl millet seedlings.
Hyp and 0.13 mg Hyp in the non-treated plants. This further increased to 0.72 mg in Ind-Ile-Me samples upon S. graminicola infection and 0.3 mg Hyp in the respective control seedlings (Fig. 5).
Induced resistance is defined as an enhancement of plants defensive capacity against a broad spectrum of pathogens after appropriate stimulation. Various inducers are known to initiate such processes leading to increased levels of defense-related compounds and signals, for example salicylic acid and jasmonates [5,7]. Octadecanoids such as OPDA and JA are widely distributed phytohormones in higher plants with a broad spectrum of biological effects in plant development, disease resistance, and stress physiology. However, there are only very few reports on jasmonates used as inducer of resistance in field studies: Exogenous JA sprayed on tomato plants in agricultural plots increased levels of polyphenol oxidase and proteinase inhibitors [41] and JA also increased the parasitization of induced plants two-fold under field conditions [42]. Because application of JA induces these defensive compounds at low concentrations, it may prove to be a useful tool for stimulating plant resistance to insects in the field [41]. A series of jasmonate-mimicking compounds have recently been synthesized [24,25] among which the 6-ethyl indanoyl isoleucine conjugate, coronalon, has been proven to act as elicitor in syntheses of many secondary metabolites including phytoalexins in cell cultures as well as in differentiated plants [23,24,43–45]. The efficiency of this synthetic conjugate as well as that of chemically related compounds added advantage in providing protection to various biotic stresses and, hence, bears agrochemical benefit.
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In the current study, Ind-Ile-Me treatment did negatively affect neither seed germination nor seedling vigor of pearl millet but provided protection against the pathogen S. graminicola. The evaluation of Ind-Ile-Me in combination with seed treatment and foliar spray gave promising disease prevention both under greenhouse and field conditions. Under greenhouse conditions, 58% at 50 mM and 60% at 75 mM protection was obtained. Field condition results even showed protection of 62% using a combination of seed treatment and foliar spray. Thus, the protection imparted by Ind-Ile-Me treatment in pearl millet to downy mildew in greenhouse and field data has proven its efficiency in managing disease at mM concentrations. Agricultural practices such as the usage of pesticide as seed treatment agents against pests could be avoided by replacing those compounds with the jasmonate mimetics. In a more detailed analysis concerning the involvement and behavior of typical defense-related enzymes, Bowles [40] showed that most of them have been up-regulated simply upon treatment with Ind-Ile-Me. The PAL is involved in biosynthesis of phytoalexins and lignin, thus contributing to the production of physical and chemical barriers against infections. In the present study, a primed increase of PAL in Ind-Ile-Me-treated seedlings was observed. Thus, an incompatible environment was generated by Ind-Ile-Me in a previously susceptible pearl millet cultivar. This suggested that in Ind-Ile-Me-induced protection of pearl millet, PAL may be involved in triggering phenylpropanoid pathway resulting in the release of toxic phytoalexins at the site of S. graminicola penetration [46]. Peroxidases are implicated in multiple functions of plant disease resistance against pathogens. The various physiological functions of peroxidases contributing to resistance include oxidation of hydroxy-cinnamyl alcohols into free radical intermediates, phenol oxidation, polysaccharide cross-linking, cross-linking of extensin monomers, and lignification. Increased level of peroxidase activity is reported in a number of resistant interactions involving plant-pathogenic and plant–pest interactions [47,48]; in pearl millet, peroxidase activity has already been described to be associated with reduction in the rate of pathogen multiplication and spread [21]. Elevated levels of peroxidases recorded in Ind-Ile-Me-protected pearl millet seedlings indicated that seed treatment of 7042S with Ind-Ile-Me again generated a partial incompatibility for S. graminicola. In contrast, catalase activity did increase neither on S. graminicola infection nor on IndIle-Me treatment. Expression of higher levels of hydrolases such as b-1,3glucanases and chitinases has been shown to provide enhanced resistance to fungal pathogens [49,50]. Their induction after pathogen infection supplies protection by directly degrading fungal cell wall components and indirectly by releasing some elicitors from the decaying fungal cell wall that might stimulate other plant defense mechanisms like phytoalexin accumulation in the host plant [51]. Seed treatment with Ind-Ile-Me resulted in
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increased levels of b-1,3-glucanase activity following the pattern of induction similar to that observed in resistant cultivar [52]. By contrast, Ind-Ile-Me treatment per se did not result in increase of chitinase activity; however, a significant enhancement of chitinase activity was recorded upon S. graminicola infection in these samples. Hydroxyproline-rich glycoproteins (HRGPs) are important plant cell wall structural components that are induced in response to pathogen attack [53]. Accumulation of HRGPs inferred from Hyp-measurements in pearl millet seedlings in response to S. graminicola as early as 2 h.p.i. and their further rapid deposition with a maximum occurring at 9 h.p.i. in plant cell walls offer effective restriction for the downy mildew pathogen [38,53]. Elevated levels of this glycoprotein in Ind-Ile-Me-treated pearl millet seedlings support a role for HRGPs in the IndIle-Me-offered protection of pearl millet to downy mildew pathogen. Increased level of HRGPs represented by hydroxyproline has been recorded in the present study in Ind-Ile-Me-treated seedlings, which further increased upon subsequent S. graminicola infection. Thus, Ind-Ile-Me treatment may bring about faster and stronger reinforcement of the pearl millet cell wall thereby contributing to disease resistance. Taken together, the efficacy of Ind-Ile-Me to induce plant disease resistance has been demonstrated in pearl millet downy mildew interaction under laboratory, greenhouse, and field conditions. The low concentration necessary, the simple and fast synthesis procedure, and the phytohormone character justify to rank Ind-Ile-Me as a valuable compound with a high agricultural potential. Acknowledgments The authors are grateful to the DANIDA-ENRECA for the facilities made available for the research programme. We also thank the Department of Science and Technology, Government of India, and Indian Council of Agricultural Research, New Delhi, and the Max-Planck-Society for financial support. S.D. would like to thank German Academic Exchange Service (DAAD) for financial support to carry out research work at the Max Planck Institute for Chemical Ecology, Jena, Germany. S.S. acknowledges the financial support and research grant received from Department of Science and Technology, New Delhi, India, under SERC Fast Track Young Scientist Programme. References [1] Nene YL, Singh SD. Downy mildew and ergot of pearl millet. Present News Search 1976;22:366–85. [2] Shetty SA, Shetty HS, Mathur SB. Down mildew of pearl millet. Technical bulletin. India: Downy Mildew Research Laboratory, University of Mysore; 1995. [3] Hash CT, Yadav RS, Sharma A, Bidinger FR, Devos KM, Gale MD, et al. Pearl millet molecular marker research. PSP annual report, vol. 35, 2003.
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