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A Chitosan formulation Elexa™ induces downy mildew disease resistance and growth promotion in pearl millet
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Crop Protection 23 (2004) 881–888
A Chitosan formulation Elexat induces downy mildew disease resistance and growth promotion in pearl millet R.G. Sharathchandra, S. Niranjan Raj, N.P. Shetty, K.N. Amruthesh, H. Shekar Shetty* Downy Mildew Research Laboratory, Department of Studies in Applied Botany, Seed Pathology and Biotechnology, University of Mysore, Manasagangotri, Mysore 570 006, India Received 27 June 2003; received in revised form 19 December 2003; accepted 23 December 2003
Abstract A commercially developed aqueous Chitosan formulation Elexa was used in different concentrations viz., 1:5, 1:10, 1:15, 1:19 and 1:25 as seed soaking treatment to pearl millet for 3, 6 and 9 h duration to test for its effect on germination and vigor index. Among the treatments used 1:19 for 6 h soaking recorded maximum germination and seedling vigor. Seed treatment, foliar spray and combination of seed treatment and foliar spray were tested for control of downy mildew disease caused by Sclerospora graminicola in pearl millet under greenhouse and field conditions. Metalaxyl at the rate of 2.1% a.i. in the form of Apron 35 SD seed treatment was used as check. Under greenhouse conditions seed treatment offered 48% protection. Foliar spray was carried out to two, seven and 14-day-old seedlings and there was marked reduction in downy mildew disease incidence. Maximum protection of 67% was recorded with foliar spray to 2-day-old seedlings. With the combination of seed treatment and foliar spray to 2-day-old seedlings offered 71% protection. Under field conditions Elexa treatments were evaluated for their effect on incidence and severity of downy mildew disease. Seed treatment reduced downy mildew severity to 42.5% and recorded 38% protection, whereas foliar spray to 7day-old seedlings gave 67% protection and reduced severity to 25%. Combination of seed treatment and foliar spray to 7-day-old seedlings recorded 69% protection and reduced severity by 23%. The nature of disease control mechanisms has been investigated and the results indicated that it is due to induction of systemic resistance. The induction of resistance was observed as early as 24 h time gap between the inducer treatment and pathogen inoculation and the maximum resistance developed at 24–48 h time gap and maintained thereafter. Elexa treated to pearl millet seeds offered growth promoting effect under greenhouse conditions and recorded increase in plant height, earhead length and seed weight. Hence, it is inferred that Elexa is a good downy mildew disease management commercial formulation and also exhibits growth-promoting effects in pearl millet. r 2004 Published by Elsevier Ltd. Keywords: Elexa; Chitosan; Pearl millet; Downy mildew; Sclerospora graminicola; Induced resistance
1. Introduction Conventional methods of disease management have met with considerable success in controlling plant diseases in general and downy mildew in particular but have raised issues that are credible as well as controversial. Use of fungicides has raised large-scale concern for environment and health. Resistant cultivars face the problem of resistance break down. Biocontrol is restricted due to lack of commercial formulations and has not been successful under field conditions due to loss *Corresponding author. Tel.: +91-821-2515126; fax: +91-8212411467. E-mail address: hss [email protected] (H.S. Shetty). 0261-2194/$ - see front matter r 2004 Published by Elsevier Ltd. doi:10.1016/j.cropro.2003.12.008
of viability, poor competitiveness, lack of proper delivery systems and environmental factors. Hence, there is a need to explore new strategies based on activating the plant’s own immune and defense mechanism to control plant diseases (Sharma et al., 2002). Phenotypically, systemic resistance is manifested as protection, which is long lasting and active against a broad spectrum of pathogens. The induced state is corroborated by an increase in production of a range of defense related products like pathogenesis related proteins, phytoalexins and signaling compounds (Heil and Bostock, 2002). Induced systemic resistance is triggered by a number of chemicals, non-pathogenic rhizobacteria, avirulent pathogens and pathogenderived elicitors. Exogenous application of chemicals
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like beta amino butyric acid (BABA), acibenzolar-smethyl (BTH), isonicotinic acid (INA), and salicylic acid (SA) have been shown to induce systemic resistance by triggering a multitude of defense response and can protect various crops like tomato, tobacco, pea, maize, cotton, rice, potato and other vegetables against viruses, fungi and bacteria (Kuc, 2001; Oostendorp et al., 2001). A few of these chemicals, like BTH, have already been commercialized as plant defense activators (Bounaurio et al., 2002; Agostini et al., 2003; Ben-shalom et al., 2003; Decapdiville et al., 2003). Pearl millet (Pennisetum glaucum (L.) R.Br.) is the only cereal that reliably provides grain and fodder under dry land conditions. It is grown in India in an area of 11.2 mha and is the staple food for millions of poor in arid and semi arid regions of the country. However, downy mildew caused by Sclerospora graminicola (Sacc.) Schroet. is a major biotic constraint in pearl millet production. S. graminicola is an oomycete that resemble fungi both morphologically and physiologically but is phylogenetically related to diatoms and brown algae. It is highly destructive and widespread. An estimated yield loss up to 80% has been recorded in F1 hybrids and many epidemics have been recorded. The oomycetes have distinct physiology, which causes many of the most effective fungicides to fail against them (Thakur and Mathur, 2002). However, seed treatment with inducers like calcium chloride, BTH, hydrogen peroxide, plant growth promoting rhizobacteria and bio agent like cerebrosides have been reported to protect pearl millet against downy mildew disease (Geetha and Shetty, 2002; Niranjan Raj et al., 2003; Deepak et al., 2003). Oligosaccharides like N-acetylchito-oligosaccharides and b, 1–3 glucans act as elicitors of plant defense response. Chitosan is a deacetylated form of Nacetylchito-oligosaccharides containing poly D-glucosamine as its building blocks but is structurally related to cellulose. Chitosan is a very common polymer found in nature and has been reported in shells of crustaceans, insects and fungi (Hadwiger, 1999;Takechi et al., 2000). Chitosan has been extensively researched to determine its ability to elicit natural defense response and protect various crop plants from pathogenic fungi. Chitosan has been reported to protect tomato, cucumber, pea, melon, strawberry, lettuce against powdery mildews and other diseases (Bhaskarareddy et al., 1999). All the earlier work has reported Chitosan induces disease resistance in plants against different pathogens but very few commercial formulation of chitosan have been developed. Glycogenesys Inc (Boston, MA, USA) has developed a chitosan formulation known as Elexa, which contains 4% chitosan as its active component. Elexa is a plant defense booster and protects cucumber, strawberry, grape and other vegetable crops from powdery mildews and different fungal pathogens (Jones, 2000; Agostini et al., 2003).
In this investigation, experiments were carried out to test the efficiency of Elexa to control downy mildew disease incidence and severity of pearl millet by seed treatment, foliar spray and the combination of seed treatment and foliar spray. In addition the effect of treatments on vegetative, reproductive growth and yield of pearl millet was studied.
2. Materials and methods 2.1. Host Seeds of pearl millet cultivar HB3 highly susceptible to downy mildew pathogen were obtained from All India Coordinated Pearl Millet Improvement Project (AICPMIP), Mandor, Jodhpur, India and were used throughout the study. 2.2. Pathogen and inoculum preparation Sclerospora graminicola was isolated from its susceptible host pearl millet HB3 cv and maintained under greenhouse conditions. Infected leaves were collected from the diseased plants in the evenings and washed thoroughly with tap water to remove the remnants of earlier sporulation, then leaf surfaces were blot dried. The leaves were placed on petri dishes lined by moist blotters and incubated in humidity chamber(s) overnight. The sporangia were harvested into sterile distilled water for zoospore release. The concentration of zoospores in the suspension was adjusted to 4 104 zoospores/ml with sterile distilled water using a haemocytometer (Safeeulla, 1976). 2.3. Disease resistance inducer An aqueous commercial formulation containing 4% chitosan as its active ingredient and having the trade name Elexa was obtained from Glycogenesys Inc. (Boston, MA, USA, Courtesy of Dr. Richard Daoust). 2.4. Effect of seed treatment with Elexa on pearl millet growth promotion 2.4.1. Effect of Elexa on pearl millet seed germination and seedling vigor Pearl millet seeds of HB3 cultivar were surface sterilized with 0.02% mercuric chloride for 5 min and rinsed thoroughly in sterile distilled water before treatment with Elexa. Two grams of seeds were then soaked with 20 ml of different Elexa dilutions: 1:5, 1:10, 1:15, 1:19 and 1:25, prepared in distilled water and incubated at 24–26 C in a rotary shaker at 100 rpm for varying time intervals like 3, 6 and 9 h and later blot dried. Seeds treated with distilled water served as
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control. Treated seeds were subjected to germination tests carried out by paper towel method as described in ISTA (1993) and seedling vigor analyzed for the 7-dayold seedlings using the method of Abdul Baki and Anderson (1973). The experiment was carried out with four replicates of 100 seeds each and was repeated three times. 2.4.2. Effect of Elexa on pearl millet vegetative and reproductive growth parameters For the evaluation of growth promotion effect of Elexa under greenhouse conditions, pearl millet seeds of HB3 cultivar were treated by soaking seeds in 1:19 dilution of Elexa for 6 h and sown in earthen pots filled with soil, sand and manure in the ratio of 2:1:1. Seedlings were watered and maintained under greenhouse conditions at 25–30 C and >95% relative humidity. When the plants were 30-day-old, number of tillers, plant height, fresh weight and dry weight were recorded. Numbers of productive ear heads, ear head length and girth, 1000 seed weight were recorded at grain maturity time. The experiment was carried out in five replicates of 20 plants each and repeated twice. 2.5. Effect of Elexa on downy mildew disease incidence under green house conditions Elexa was applied in the form of seed treatment, foliar spray and combination of seed treatment and foliar spray. 2.5.1. Seed treatment Pearl millet seeds of HB3 cv soaked in aqueous Elexa at 1:19 dilution for 6 h were sown in earthen pots. Twoday-old seedlings were inoculated to the whorl region with the zoospore suspension of S. graminicola at a concentration of 4 104 zoospores/ml by following the procedure of Singh and Gopinath (1985). Seeds treated with sterile distilled water served as control and Metalaxyl at the rate of 2.1% a.i. in the form of Apron 35 SD (6 g/kg of seed) seed treatment was used as standard chemical control. The experiment was carried out in four replicates of 100 seedlings each and repeated twice. 2.5.2. Foliar sprays Foliar sprays were carried out in the experiments designed to determine the optimum number and exact stage of Elexa spray required to obtain the maximum reduction in downy mildew disease. In this experiment, Elexa was sprayed to 2-day-old seedlings using portable hand sprayer till run off. Inoculation with S. graminicola was done at 1, 4 and 11 days after Elexa spray. The experiment was carried out in four replicates of 100 seedlings each and repeated twice.
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2.5.3. Seed treatment followed by foliar spray Seeds soaked in 1:19 dilution of Elexa for 6 h were sown in earthen pots. The same set of plants was also given foliar spray treatment and inoculated with S. graminicola as described in the foliar spray section. The experiments in this study was carried out in four replicates of 100 seedlings each and repeated twice. 2.5.4. Optimization of time required for Elexa to reduce downy mildew disease incidence Nature of protection offered was studied by maintaining spatial and temporal separation of the inducer and the pathogen inoculation and observing for the downy mildew disease reaction. Seeds of pearl millet cultivar HB3 soaked in 1:19 dilution of aqueous Elexa for 6 h were sown in earthen pots filled with soil, sand and manure in the ratio of 2:1:1.The emerging seedlings were inoculated to the whorl region with zoospore suspension of S. graminicola in the concentration of 4 104 ml with time gaps of 1, 2, 3, 4, 5 days after emergence in different sets of plants. The same conditions were followed for distilled water treated seeds, which served as controls. The experiment was carried out in four replicates of 100 seedlings each and repeated twice. 2.6. Effect of Elexa on downy mildew disease incidence and severity under field conditions Field trials were conducted with seed treatment, foliar spray and combination of seed treatment and foliar spray. Elexa treatments are same as described under greenhouse studies. Trials were conducted at the Downy Mildew Sick Plot, Department of Studies in Applied Botany, Seed Pathology and Biotechnology, University of Mysore, Mysore, India. The plot 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 (1984). There were two replications in each treatment. These were arranged as a randomized complete block design. Normal agronomic practices were followed to raise the crop. Thinning was done after 15 days to maintain uniform number of plants per row and uniform distance between the plants. The crop was irrigated when required. The plants were observed for downy mildew disease development on 30 and 60 days after sowing and rated as diseased when they showed the typical downy mildew symptoms: sporulation on the abaxial leaf surface, chlorosis, stunted growth and malformation of the earheads. The data was consolidated at 60 days after emergence. Percentage of
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protection was calculated using the formula Percent protection ¼
Percent downy mildew in untreated plants Percent downy mildew in Elexa treated plants 100 Percent downy mildew in untreated plants
In addition to disease incidence, disease severity was recorded on a 1–5 rating scale according to Singh et al. (1997). 2.7. Statistical analysis Data for greenhouse and field experiments were analyzed separately for each experiment and were subjected to Arcsine transformation and analysis of variance (ANOVA) (JMP Software; SAS Institute, Cary, NC, US). Significant effects of treatments were determined by the magnitude of P value (P ¼ 0:05). Treatment means were separated by Tukey’s HSD test.
3. Results
3.2. Growth promoting effect of Elexa on the vegetative and reproductive parameters of pearl millet Elexa treatment increased the vegetative growth parameters of pearl millet. Elexa treatment significantly increased the height of pearl millet plants at the vegetative phase. The treated plants recorded a height of 115.7 cm while the control plants showed a height of 81.6 cm. Elexa treatment produced more number of tillers with an average of 5.5 whereas in the untreated plants an average of 3 tillers were recorded. At the seed maturation stage there was a significant enhancement in the number, length and girth of earheads over that of the untreated plants. It was also found that there was 19% increase in 1000 seed weight, which was significantly higher than that of the untreated plants (Table 1).
3.1. Effect of Elexa seed treatment on seed germination and seedling vigor
3.3. Effect of Elexa treatments on downy mildew disease incidence under green house conditions
Germination was observed in all the pearl millet seeds soaked in different dilutions of Elexa for different time durations. Among the different dilutions of Elexa tested 1:19 recorded maximum germination and the optimum soaking period was 6 h. Seed germination was 91% and the vigor index 1698. Distilled water treated seeds showed 83% germination and vigor index was 1345 (Fig. 1).
3.3.1. Seed treatment Seedlings of pearl millet showed decreased incidence of downy mildew disease compared to the check when inoculated with S. graminicola zoospores. Elexa treatment recorded 46% downy mildew disease incidence and 48% protection. Apron 35SD used as a standard check recorded 20% downy mildew disease incidence and 78% protection. The untreated plants recorded 91% downy mildew disease incidence (Table 2).
60 50 40
c
c b
b a
a
cde
e c
abc
1600 f
a
abc
c
a
e
c
abcde
de abcde a
a de
h
g
1800
d
1400 1200
c b
a
1000 800
30
600
20
400
10
200
0
Vigor index
% Germination
70
2000
abc
a
ab
bcde
% germination Vigour index
abc
80
abcd
90
abcde
100
3.3.2. Foliar sprays Foliar application of Elexa was carried out to 2-dayold seedlings of pearl millet. The same seedlings were inoculated with downy mildew pathogen S. graminicola after 1, 4 and 11 days after Elexa spray. The results indicated that foliar-sprayed seedlings inoculated after 1
3 T2 6 T3 9 T4 3 T5 6 T6 9 T7 3 T8 6 T9 9 T1 30 T11 6 T1 92 T13 3 T1 6 4 T15 9 C31 C62 C93 T1 1:5 1:10 1:15 1:19 1:25 Control Elexa dilution and duration of treatment
Fig. 1. Effect of Elexa on pearl millet seed germination and seedling vigor. The experiment was carried out with four replicates of 100 seeds each and was repeated three times and the bars indicate standard error. Means designated with the same letter are not significantly different at po0:05.
Table 1 Effect of Elexa seed treatment on pearl millet growth parameters Growth parameter
Control b
Elexa seed treatment bc
Plant height (cm) Number of tillers Number of ear heads Ear head length (cm) Ear head girth (cm) 1000 seed weight (g)
81.670.49 e 370.0 e 370.0 e 10.670.47 e 5.670.14 e 6.470.34 e
115.772.28 f 5.570.28 f 4.570.28 f 11.470.30 f 6.770.0 f 7.670.11 f
The experiment was carried out in five replicates of 20 plants each and repeated twice. Values are expressed as mean7standard error. Means designated with the same letter are not significantly different at po0:05:
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Table 2 Effect of Elexa on pearl millet downy mildew disease incidence and protection under green house condition Elexa treatment
Percentage
Seed treatment Foliar spray to 2-day-old seedlings and inoculation 1 day after Elexa spray Foliar spray to 2-day-old seedlings and inoculation 4 days after Elexa spray Foliar spray to 2-day-old seedlings and inoculation 7 days after Elexa spray Seed treatment and Foliar spray to 2-day-old seedlings and inoculation 1 day after Elexa spray Seed treatment and Foliar spray to 2-day-old seedlings and inoculation 4 days after Elexa spray Seed treatment and Foliar spray to 2-day-old seedlings inoculation 7 days after Elexa spray Apron Control
DMDI
DMDP
46 31 25 26 26 31 28 20 91
48 67 72 71 71 67 68 78 —
c b ab ab ab b ab a d
b cd cd cd cd cd cd d
The experiments in this study was carried out in four replicates of 100 seedlings each and repeated twice. Means designated with the same letter are not significantly different at po0:05: DMDI=downy mildew disease incidence. DMDP=downy mildew disease resistance.
3.3.3. Seed treatment followed by foliar spray Seed treatment and Elexa spray to 2-day-old seedlings and inoculated 1 day after foliar spray offered a maximum of 71% protection reducing the disease incidence to 26% (Table 1). Seed treatment and Elexa spray to 2-day-old seedlings followed by inoculation 4 and 11 days after spray recorded 31% and 28% downy mildew resulting in 67% and 68% protection, respectively (Table 2). However seed treatment with Apron 35SD was better than all the above-mentioned combinations in protecting pearl millet against downy mildew showing a 20% disease incidence and offered 78% protection. In the untreated check 91% downy mildew disease incidence was recorded. 3.4. Optimization of time required for Elexa to reduce downy mildew disease incidence Protection increased from 48.53% on first day to 58.01% on the fifth day after inoculation. It was found that the protection increased throughout the 5-day-time gap period as 47%, 57% and 55% protection was observed on second, third and fourth day time gap, respectively (Fig. 2). The results indicated that the Elexa seed treatment offered maximum reduction of downy mildew disease incidence when inoculated 3 days after emergence. The resistances build up started as early as 24 h and the maximum resistance was developed when the time gap between Elexa seed treatment and pathogen inoculation was 24–48 h.
80 70
% protection over control
day recorded 31% disease incidence resulting in 67% protection and foliar sprayed seedlings inoculated after 4 and 11 days recorded 25% and 26% downy mildew which resulted in 72% and 71% protection, respectively (Table 2).
ab
60
c 50
abc
a
c
40 30 20 10 0
day1
day2
day3
day4
day5
Time gap in days
Fig. 2. Optimization of time required for Elexa to reduce downy mildew disease incidence. The experiments in this study was carried out in four replicates of 100 seedlings each and repeated twice and the bars indicate standard errors. Means designated with the same letter are not significantly different at po0:05.
3.5. Effect of Elexa on downy mildew disease incidence and severity under field conditions 3.5.1. Seed treatment Seed treatment with Elexa at 1:19 dilution for 6 h recorded 60% downy mildew incidence, which corresponds to 38% protection under epiphytotic conditions. When the disease was evaluated it was found to be 43%. Control plants recorded 90% downy mildew incidence and 99.5% severity. Apron treatment recorded 12% disease incidence and offered 87% protection showing a disease severity of 7.5% (Table 3). 3.5.2. Foliar spray Foliar spray to 2-day-old seedlings recorded 30% downy mildew incidence and 62% protection with
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Table 3 Effect of Elexa on pearl millet downy mildew disease incidence, severity and protection under field condition Treatment
Control Apron 35SD Seed treatment Foliar spray to 2-day-old seedlings Foliar spray to 7-day-old seedlings Foliar spray to 14-day-old seedlings Seed treatment and Foliar spray to 2-day-old seedlings Seed treatment and Foliar spray to 7-day-old seedlings Seed treatment and Foliar spray to 14-day-old seedlings
Percentage DMDI
DMDP
DMDS
90 12 60 30 33 32 28 30 32
0 87 38 62 67 67 67 69 67
99.5 7.5 42.5 27.1 25.0 24.3 23.2 23.0 24.4
a d b c c c c c c
a c b b b b b b
The experiments in this study was carried out in four replicates of 100 seedlings each and repeated twice. Means designated with the same letter are not significantly different at p ¼ 0:05: DMDI=downy mildew disease incidence. DMDP=downy mildew disease resistance. DMDS=downy mildew disease severity.
27.1% severity. Foliar spray to 7 and 14-day-old seedlings recorded 33% and 32% disease incidence, the disease protection was 67% and 67% respectively. The disease severity was also evaluated and it was found to be 25% and 24%, respectively (Table 3). 3.5.3. Seed treatment followed by foliar spray In seed treatment with Elexa and followed by foliar spray to 2-day-old seedlings recorded 28% disease incidence with 23% severity and 67% protection. Seed treatment followed by foliar spray to 7-day-old seedlings recorded 30% disease with 23% severity and 69% protection. Seed treatment followed by foliar spray to 14-day-old seedlings recorded 32% downy mildew incidence with 67% protection and 24% severity (Table 3).
4. Discussion Elexa is a commercial formulation of 4% Chitosan used in different vegetable and fruit crops like cucumber, pumpkin, zucchini, grapes and strawberries to control downy mildew, powdery mildew and gray mold diseases. In all these cases Elexa was used as foliar spray and the disease control was effective (Jones, 2000). In the present study Elexa at 1:19 dilution was used in pearl millet as seed treatment, foliar spray and also combination of seed treatment and foliar spray against S. graminicola, the incitant of downy mildew disease. Pearl millet is an important cereal crop of semi-arid tropics of India and Africa susceptible to downy mildew disease. Pearl millet is considered as a poor man’s crop in low input rain fed agricultural system. The farmers adopt only seed treatment pesticides against pests and diseases. The present study demonstrated that seed treatment alone reduced downy mildew disease and offered
acceptable levels of protection. Although foliar spray gives better downy mildew disease protection and seed treatment followed by foliar sprays is still better in protecting pearl millet farmers may not be able to practice the foliar spray. Currently recommended downy mildew disease control is by Metalaxyl a.i 2.1 g/kg of seeds in the form of Apron 35SD. However, metalaxyl is expensive and not yet made available to poor farmers in India. It has already been reported that oomycete pathogens are showing metalaxyl resistance hence search for cheaper commercial formulations of effective compounds are required (Thakur and Mathur, 2002). The effectiveness of Elexa indicates that such formulations may be cheaper and easy to produce and recommended to the farmers. However, much research is needed to increase the effectiveness of Elexa by seed treatment to achieve higher protection rates in pearl millet. Elexa has additional advantages when compared to metalaxyl treatment. Elexa treatment significantly enhanced seed germination and seedling vigor of pearl millet. Pearl millet seedlings showed increased height and more number of tillers. The number of productive ear heads, length and girth and the yield also increased, as it is evident from the 1000 seed weight. In the current study Elexa was evaluated in various methods like seed treatment, foliar spray and the combination of seed treatment and foliar spray both under green house and epiphytotic field conditions. The disease protection obtained was greater in the green house conditions than that in the field conditions. This may be due to the controlled environmental conditions in the green house and many environmental factors such as soil and continuous inoculum shower at all stages of plant growth operating in the field conditions which are not available in the green house conditions. Under green house conditions Elexa seed treatment protected pearl millet against downy mildew disease by up to 48%.
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Foliar spray with Elexa to 2-day-old seedlings offered 72% protection. Combination of Elexa spray and seed treatment offered more protection than either treatment alone. Results also showed that Elexa spray at seedling emergence stage was sufficient for reduction of disease incidence both under green house and field conditions. Under field conditions Elexa treatments considerably reduced the severity as well as the incidence of downy mildew. Elexa seed treatment offered 37% protection. Foliar spray to seedlings at 2, 7 and 14 days offered wide-ranging protection from 64% to 69%. The combination of seed treatment and foliar spray offered 71% protection. Field trials with Elexa in the United States, has shown that the disease severity of powdery mildew of grapes, cucumber, strawberries, pumpkins, zucchini was reduced very effectively when compared to other available bioactive products (Jones, 2000). The active ingredient of Elexa is Chitosan, which is reported to promote growth and elicit plant defense response in various crops. Chitosan seed treatment enhanced seed germination and seedling vigor in wheat. Chitosan treatment also increased the growth of Vitis vinifera L. plantlets (Bhaskarareddy et al., 1999; Barka et al., 2003). Chitosan seed treatment has been reported to protect tomato plants from crown rot and root rot pathogens upon seed treatment (Benhmaou et al., 1994). Chitosan was able to protect susceptible tomato seedlings from Fusarium oxysporum when applied as a foliar spray (Benhmaou et al., 1998). Chitosan spray protected cucumber plants by reducing the incidence of the gray mold (Ben-shalom et al., 2003). Chitosan has been shown to induce resistance effectively in a number of crops like cucumber, wheat, pea, celery, and groundnut against Pythium aphanidermatum, Alternaria alternata, Fusarium gramenareum, Fusarium solani f.sp. pisi and Fusarium oxysporum f.sp. apii. (Ghouth and Ahmad, 1994; Sathiyabama and Balasubramaniam, 1998; Bhaskarareddy et al., 1999). Corroborating with these earlier reports the present study also indicated that possible mechanism of downy mildew control by Elexa is through induction of systemic resistance in the host. This was demonstrated by spatio-temporal separation of the inducer and the pathogen inoculation. ISR normally requires a time-lapse period between inducer treatment and pathogen inoculation and gene activation for defense response to take place (Ryals et al., 1996). The present study also identified an optimal time interval of 24–48 h between inducer treatment and inoculation for the resistance to build up under green house conditions. In vitro studies recorded no fungitoxic effect of Elexa on sporulation, zoospore release and viability of S. graminicola (data not shown). Chitosan’s role as a plant defense elicitor and the mechanism of induction of resistance is one that has been well studied. Chitosan induces reduction of the stomatal aperture, lignin biosynthesis and plant cell wall
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lignification, ultra structural changes in leaf cells, production of antifungal hydrolases such as chitinase and beta-1,3 glucanase. Chitosan stimulates the accumulation of signal molecule jasmonic acid, hydrogen peroxide, reactive oxygen species and protein kinases all of which play a crucial role in intracellular signaling pathways (Chang and Mei, 1995; Pospieszny and Zilienska, 1997; Peter, 1998; Hadwiger, 1999; Sumin, 1999; Link et al., 2002; Rakwal et al., 2002; Barka et al., 2003). Considering all the aspects presented above Elexa is an active inducer of defense responses in pearl millet against downy mildew disease and thus has the potential of becoming an alternative means of disease control.
Acknowledgements The authors thank Dr. Richard Daoust Programme Manager, Agriculture Glycogenesys Inc. formerly Safe Science Inc., Boston, MA, USA for the kind supply of Elexa and for his support throughout the investigation. This work was carried out in the Project on Systemic Acquired Resistance funded by Danish International Agency under the Enhancement of Research Capacity Programme (DANIDA ENRECA). The authors are grateful to Dr. Eigil de Neergard, the Principal Responsible leader of the DANIDA ENRECA Project for his co-operation during this study. The facilities provided by Indian Council of Agricultural Research, Government of India through All India Coordinated Pearl Millet Improvement Project is also gratefully acknowledged. References Abdul Baki, A., Anderson, J.D., 1973. Vigor analysis in soybean seed by multiple criteria. Crop Sci. 13, 630–633. Agostini, J.P., Bushong, P.M., Timmer, L.W., 2003. Green house evaluation of products that induce host resistance for control of Scab, Melanose and Alternaria brown spot of Citrus. Plant Dis. 87, 69–74. Barka, A., Eullaffroy, P., Clement, C., Vernet, G., 2003. Chitosan improves development, and protects Vitis vinifera L. against Botrytis cinerea. Plant Cell Rep., in press. Benhmaou, N., Kloepper, J.W., Tuzun, S., 1994. Induction of systemic resistance to Fusarium crown rot and root rot in tomato plants by seed treatment with chitosan. Phytopathology 84, 1432–1444. Benhmaou, N., Kloepper, J.W., Tuzun, S., 1998. Induction of resistance to Fusarium wilt of tomato by combination of chitosan and endophytic bacterial strain: ultra structure and cytochemistry of host response. Planta 204, 153–168. Ben-shalom, N., Ardi, R., Pinto, R., Aki, C., Fallik, E., 2003. Controlling gray mold caused by Botrytis cinerea in cucumber plants by means of chitosan. Crop Prot 22, 285–290. Bhaskarareddy, M.V., Arul, J., Angers, P., Couture, L., 1999. Chitosan treatment of wheat seeds induces resistance to Fusarium graminearum and improves seed quality. J. Agric. Food Chem. 47, 1208–1216.
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Oostendorp, M., Kunz, W., Dietrich, B., Staub, T., 2001. Induced disease resistance in plants by chemicals. Eur. J. Plant Pathol. 107, 19–28. Peter, V., 1998. Comparison of the ability of partially N-acetylated chitosan and chtitooligosaccharides to elicit resistance reactions in wheat leaves. Plant Physiol. 118, 1353–1359. Pospieszny, H., Zilienska, L., 1997. Ultra structure of leaves treated with chitosan. Adv. Chitin Sci. 2, 139–144. Rakwal, R., Tamogami, S., Agrawal, G.K., Iwahashi, H., 2002. Octadecanoid signaling component burst in rice (Oryza sativa L.) seedling leaves upon wounding by cut and treatment with fungal elicitor chitosan. Biochem. Biophys. Res. Commun. 295, 1041–1045. Ryals, J.A., Neuschwander, U.M., Willitis, M.G., Molina, A., Steiner, H., Hunt, M.O., 1996. Systemic acquired resistance. Plant Cell 8, 1809–1819. Safeeulla, K.M., 1976. Biology and Control of the Downy Mildews of Pearl Millet, Sorghum and Finger Millet. Wesley Press, Mysore, India. Sathiyabama, M., Balasubramaniam, R., 1998. Chitosan induces resistance components in Arachis hypogea against leaf rust caused by Puccinia arachidis speg. Crop Prot. 17, 307–313. Sharma, H.C., Crouch, J.H., Sharma, K.K., Seetharama, N., Hash, C.T., 2002. Applications of biotechnology for crop improvement: prospects and constraints. Plant Sci. 163, 381–395. Singh, S.D., Gopinath, R., 1985. A seedling inoculation technique for detecting downy mildew resistance in pearl millet. Plant Dis. 69, 582–584. Singh, S.D., Wilson, J.P., Navi, S.S., Talukdar, B.S., Hess, D.E., Reddy, K.N., 1997. Screening techniques and sources of resistance to downy mildew and rust in pearl millet. Information Bulletin No. 48. International Crops Research Institute for the Semi-Arid Tropics, Patancheru, Andhra Pradesh, India. Sumin, L., 1999. Oligogalacturonic acid and chitosan reduce the stomatal aperture by inducing the evolution of reactive oxygen species from guard cells of tomato and Commelina communis. Plant Physiol. 121, 147–215. Takechi, Y., Yoki, I., Naoto, S., 2000. Oligosaccharide elicitors and their receptors in plant defense. Trends Glycosci. Glycotechnol. 12, 113–120. Thakur, R.P., Mathur, K., 2002. Downy mildews of India. Crop Prot. 21, 333–345. Williams, R.J., 1984. Downy mildew of tropical cereals. In: Ingram, D.S., Williams, P.H. (Eds.), Advances in Plant Pathology, Vol. 2. Academic Press, London, pp. 1–103.
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A Chitosan formulation Elexat induces downy mildew disease resistance and growth promotion in pearl millet R.G. Sharathchandra, S. Niranjan Raj, N.P. Shetty, K.N. Amruthesh, H. Shekar Shetty* Downy Mildew Research Laboratory, Department of Studies in Applied Botany, Seed Pathology and Biotechnology, University of Mysore, Manasagangotri, Mysore 570 006, India Received 27 June 2003; received in revised form 19 December 2003; accepted 23 December 2003
Abstract A commercially developed aqueous Chitosan formulation Elexa was used in different concentrations viz., 1:5, 1:10, 1:15, 1:19 and 1:25 as seed soaking treatment to pearl millet for 3, 6 and 9 h duration to test for its effect on germination and vigor index. Among the treatments used 1:19 for 6 h soaking recorded maximum germination and seedling vigor. Seed treatment, foliar spray and combination of seed treatment and foliar spray were tested for control of downy mildew disease caused by Sclerospora graminicola in pearl millet under greenhouse and field conditions. Metalaxyl at the rate of 2.1% a.i. in the form of Apron 35 SD seed treatment was used as check. Under greenhouse conditions seed treatment offered 48% protection. Foliar spray was carried out to two, seven and 14-day-old seedlings and there was marked reduction in downy mildew disease incidence. Maximum protection of 67% was recorded with foliar spray to 2-day-old seedlings. With the combination of seed treatment and foliar spray to 2-day-old seedlings offered 71% protection. Under field conditions Elexa treatments were evaluated for their effect on incidence and severity of downy mildew disease. Seed treatment reduced downy mildew severity to 42.5% and recorded 38% protection, whereas foliar spray to 7day-old seedlings gave 67% protection and reduced severity to 25%. Combination of seed treatment and foliar spray to 7-day-old seedlings recorded 69% protection and reduced severity by 23%. The nature of disease control mechanisms has been investigated and the results indicated that it is due to induction of systemic resistance. The induction of resistance was observed as early as 24 h time gap between the inducer treatment and pathogen inoculation and the maximum resistance developed at 24–48 h time gap and maintained thereafter. Elexa treated to pearl millet seeds offered growth promoting effect under greenhouse conditions and recorded increase in plant height, earhead length and seed weight. Hence, it is inferred that Elexa is a good downy mildew disease management commercial formulation and also exhibits growth-promoting effects in pearl millet. r 2004 Published by Elsevier Ltd. Keywords: Elexa; Chitosan; Pearl millet; Downy mildew; Sclerospora graminicola; Induced resistance
1. Introduction Conventional methods of disease management have met with considerable success in controlling plant diseases in general and downy mildew in particular but have raised issues that are credible as well as controversial. Use of fungicides has raised large-scale concern for environment and health. Resistant cultivars face the problem of resistance break down. Biocontrol is restricted due to lack of commercial formulations and has not been successful under field conditions due to loss *Corresponding author. Tel.: +91-821-2515126; fax: +91-8212411467. E-mail address: hss [email protected] (H.S. Shetty). 0261-2194/$ - see front matter r 2004 Published by Elsevier Ltd. doi:10.1016/j.cropro.2003.12.008
of viability, poor competitiveness, lack of proper delivery systems and environmental factors. Hence, there is a need to explore new strategies based on activating the plant’s own immune and defense mechanism to control plant diseases (Sharma et al., 2002). Phenotypically, systemic resistance is manifested as protection, which is long lasting and active against a broad spectrum of pathogens. The induced state is corroborated by an increase in production of a range of defense related products like pathogenesis related proteins, phytoalexins and signaling compounds (Heil and Bostock, 2002). Induced systemic resistance is triggered by a number of chemicals, non-pathogenic rhizobacteria, avirulent pathogens and pathogenderived elicitors. Exogenous application of chemicals
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like beta amino butyric acid (BABA), acibenzolar-smethyl (BTH), isonicotinic acid (INA), and salicylic acid (SA) have been shown to induce systemic resistance by triggering a multitude of defense response and can protect various crops like tomato, tobacco, pea, maize, cotton, rice, potato and other vegetables against viruses, fungi and bacteria (Kuc, 2001; Oostendorp et al., 2001). A few of these chemicals, like BTH, have already been commercialized as plant defense activators (Bounaurio et al., 2002; Agostini et al., 2003; Ben-shalom et al., 2003; Decapdiville et al., 2003). Pearl millet (Pennisetum glaucum (L.) R.Br.) is the only cereal that reliably provides grain and fodder under dry land conditions. It is grown in India in an area of 11.2 mha and is the staple food for millions of poor in arid and semi arid regions of the country. However, downy mildew caused by Sclerospora graminicola (Sacc.) Schroet. is a major biotic constraint in pearl millet production. S. graminicola is an oomycete that resemble fungi both morphologically and physiologically but is phylogenetically related to diatoms and brown algae. It is highly destructive and widespread. An estimated yield loss up to 80% has been recorded in F1 hybrids and many epidemics have been recorded. The oomycetes have distinct physiology, which causes many of the most effective fungicides to fail against them (Thakur and Mathur, 2002). However, seed treatment with inducers like calcium chloride, BTH, hydrogen peroxide, plant growth promoting rhizobacteria and bio agent like cerebrosides have been reported to protect pearl millet against downy mildew disease (Geetha and Shetty, 2002; Niranjan Raj et al., 2003; Deepak et al., 2003). Oligosaccharides like N-acetylchito-oligosaccharides and b, 1–3 glucans act as elicitors of plant defense response. Chitosan is a deacetylated form of Nacetylchito-oligosaccharides containing poly D-glucosamine as its building blocks but is structurally related to cellulose. Chitosan is a very common polymer found in nature and has been reported in shells of crustaceans, insects and fungi (Hadwiger, 1999;Takechi et al., 2000). Chitosan has been extensively researched to determine its ability to elicit natural defense response and protect various crop plants from pathogenic fungi. Chitosan has been reported to protect tomato, cucumber, pea, melon, strawberry, lettuce against powdery mildews and other diseases (Bhaskarareddy et al., 1999). All the earlier work has reported Chitosan induces disease resistance in plants against different pathogens but very few commercial formulation of chitosan have been developed. Glycogenesys Inc (Boston, MA, USA) has developed a chitosan formulation known as Elexa, which contains 4% chitosan as its active component. Elexa is a plant defense booster and protects cucumber, strawberry, grape and other vegetable crops from powdery mildews and different fungal pathogens (Jones, 2000; Agostini et al., 2003).
In this investigation, experiments were carried out to test the efficiency of Elexa to control downy mildew disease incidence and severity of pearl millet by seed treatment, foliar spray and the combination of seed treatment and foliar spray. In addition the effect of treatments on vegetative, reproductive growth and yield of pearl millet was studied.
2. Materials and methods 2.1. Host Seeds of pearl millet cultivar HB3 highly susceptible to downy mildew pathogen were obtained from All India Coordinated Pearl Millet Improvement Project (AICPMIP), Mandor, Jodhpur, India and were used throughout the study. 2.2. Pathogen and inoculum preparation Sclerospora graminicola was isolated from its susceptible host pearl millet HB3 cv and maintained under greenhouse conditions. Infected leaves were collected from the diseased plants in the evenings and washed thoroughly with tap water to remove the remnants of earlier sporulation, then leaf surfaces were blot dried. The leaves were placed on petri dishes lined by moist blotters and incubated in humidity chamber(s) overnight. The sporangia were harvested into sterile distilled water for zoospore release. The concentration of zoospores in the suspension was adjusted to 4 104 zoospores/ml with sterile distilled water using a haemocytometer (Safeeulla, 1976). 2.3. Disease resistance inducer An aqueous commercial formulation containing 4% chitosan as its active ingredient and having the trade name Elexa was obtained from Glycogenesys Inc. (Boston, MA, USA, Courtesy of Dr. Richard Daoust). 2.4. Effect of seed treatment with Elexa on pearl millet growth promotion 2.4.1. Effect of Elexa on pearl millet seed germination and seedling vigor Pearl millet seeds of HB3 cultivar were surface sterilized with 0.02% mercuric chloride for 5 min and rinsed thoroughly in sterile distilled water before treatment with Elexa. Two grams of seeds were then soaked with 20 ml of different Elexa dilutions: 1:5, 1:10, 1:15, 1:19 and 1:25, prepared in distilled water and incubated at 24–26 C in a rotary shaker at 100 rpm for varying time intervals like 3, 6 and 9 h and later blot dried. Seeds treated with distilled water served as
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control. Treated seeds were subjected to germination tests carried out by paper towel method as described in ISTA (1993) and seedling vigor analyzed for the 7-dayold seedlings using the method of Abdul Baki and Anderson (1973). The experiment was carried out with four replicates of 100 seeds each and was repeated three times. 2.4.2. Effect of Elexa on pearl millet vegetative and reproductive growth parameters For the evaluation of growth promotion effect of Elexa under greenhouse conditions, pearl millet seeds of HB3 cultivar were treated by soaking seeds in 1:19 dilution of Elexa for 6 h and sown in earthen pots filled with soil, sand and manure in the ratio of 2:1:1. Seedlings were watered and maintained under greenhouse conditions at 25–30 C and >95% relative humidity. When the plants were 30-day-old, number of tillers, plant height, fresh weight and dry weight were recorded. Numbers of productive ear heads, ear head length and girth, 1000 seed weight were recorded at grain maturity time. The experiment was carried out in five replicates of 20 plants each and repeated twice. 2.5. Effect of Elexa on downy mildew disease incidence under green house conditions Elexa was applied in the form of seed treatment, foliar spray and combination of seed treatment and foliar spray. 2.5.1. Seed treatment Pearl millet seeds of HB3 cv soaked in aqueous Elexa at 1:19 dilution for 6 h were sown in earthen pots. Twoday-old seedlings were inoculated to the whorl region with the zoospore suspension of S. graminicola at a concentration of 4 104 zoospores/ml by following the procedure of Singh and Gopinath (1985). Seeds treated with sterile distilled water served as control and Metalaxyl at the rate of 2.1% a.i. in the form of Apron 35 SD (6 g/kg of seed) seed treatment was used as standard chemical control. The experiment was carried out in four replicates of 100 seedlings each and repeated twice. 2.5.2. Foliar sprays Foliar sprays were carried out in the experiments designed to determine the optimum number and exact stage of Elexa spray required to obtain the maximum reduction in downy mildew disease. In this experiment, Elexa was sprayed to 2-day-old seedlings using portable hand sprayer till run off. Inoculation with S. graminicola was done at 1, 4 and 11 days after Elexa spray. The experiment was carried out in four replicates of 100 seedlings each and repeated twice.
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2.5.3. Seed treatment followed by foliar spray Seeds soaked in 1:19 dilution of Elexa for 6 h were sown in earthen pots. The same set of plants was also given foliar spray treatment and inoculated with S. graminicola as described in the foliar spray section. The experiments in this study was carried out in four replicates of 100 seedlings each and repeated twice. 2.5.4. Optimization of time required for Elexa to reduce downy mildew disease incidence Nature of protection offered was studied by maintaining spatial and temporal separation of the inducer and the pathogen inoculation and observing for the downy mildew disease reaction. Seeds of pearl millet cultivar HB3 soaked in 1:19 dilution of aqueous Elexa for 6 h were sown in earthen pots filled with soil, sand and manure in the ratio of 2:1:1.The emerging seedlings were inoculated to the whorl region with zoospore suspension of S. graminicola in the concentration of 4 104 ml with time gaps of 1, 2, 3, 4, 5 days after emergence in different sets of plants. The same conditions were followed for distilled water treated seeds, which served as controls. The experiment was carried out in four replicates of 100 seedlings each and repeated twice. 2.6. Effect of Elexa on downy mildew disease incidence and severity under field conditions Field trials were conducted with seed treatment, foliar spray and combination of seed treatment and foliar spray. Elexa treatments are same as described under greenhouse studies. Trials were conducted at the Downy Mildew Sick Plot, Department of Studies in Applied Botany, Seed Pathology and Biotechnology, University of Mysore, Mysore, India. The plot 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 (1984). There were two replications in each treatment. These were arranged as a randomized complete block design. Normal agronomic practices were followed to raise the crop. Thinning was done after 15 days to maintain uniform number of plants per row and uniform distance between the plants. The crop was irrigated when required. The plants were observed for downy mildew disease development on 30 and 60 days after sowing and rated as diseased when they showed the typical downy mildew symptoms: sporulation on the abaxial leaf surface, chlorosis, stunted growth and malformation of the earheads. The data was consolidated at 60 days after emergence. Percentage of
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protection was calculated using the formula Percent protection ¼
Percent downy mildew in untreated plants Percent downy mildew in Elexa treated plants 100 Percent downy mildew in untreated plants
In addition to disease incidence, disease severity was recorded on a 1–5 rating scale according to Singh et al. (1997). 2.7. Statistical analysis Data for greenhouse and field experiments were analyzed separately for each experiment and were subjected to Arcsine transformation and analysis of variance (ANOVA) (JMP Software; SAS Institute, Cary, NC, US). Significant effects of treatments were determined by the magnitude of P value (P ¼ 0:05). Treatment means were separated by Tukey’s HSD test.
3. Results
3.2. Growth promoting effect of Elexa on the vegetative and reproductive parameters of pearl millet Elexa treatment increased the vegetative growth parameters of pearl millet. Elexa treatment significantly increased the height of pearl millet plants at the vegetative phase. The treated plants recorded a height of 115.7 cm while the control plants showed a height of 81.6 cm. Elexa treatment produced more number of tillers with an average of 5.5 whereas in the untreated plants an average of 3 tillers were recorded. At the seed maturation stage there was a significant enhancement in the number, length and girth of earheads over that of the untreated plants. It was also found that there was 19% increase in 1000 seed weight, which was significantly higher than that of the untreated plants (Table 1).
3.1. Effect of Elexa seed treatment on seed germination and seedling vigor
3.3. Effect of Elexa treatments on downy mildew disease incidence under green house conditions
Germination was observed in all the pearl millet seeds soaked in different dilutions of Elexa for different time durations. Among the different dilutions of Elexa tested 1:19 recorded maximum germination and the optimum soaking period was 6 h. Seed germination was 91% and the vigor index 1698. Distilled water treated seeds showed 83% germination and vigor index was 1345 (Fig. 1).
3.3.1. Seed treatment Seedlings of pearl millet showed decreased incidence of downy mildew disease compared to the check when inoculated with S. graminicola zoospores. Elexa treatment recorded 46% downy mildew disease incidence and 48% protection. Apron 35SD used as a standard check recorded 20% downy mildew disease incidence and 78% protection. The untreated plants recorded 91% downy mildew disease incidence (Table 2).
60 50 40
c
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% Germination
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2000
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100
3.3.2. Foliar sprays Foliar application of Elexa was carried out to 2-dayold seedlings of pearl millet. The same seedlings were inoculated with downy mildew pathogen S. graminicola after 1, 4 and 11 days after Elexa spray. The results indicated that foliar-sprayed seedlings inoculated after 1
3 T2 6 T3 9 T4 3 T5 6 T6 9 T7 3 T8 6 T9 9 T1 30 T11 6 T1 92 T13 3 T1 6 4 T15 9 C31 C62 C93 T1 1:5 1:10 1:15 1:19 1:25 Control Elexa dilution and duration of treatment
Fig. 1. Effect of Elexa on pearl millet seed germination and seedling vigor. The experiment was carried out with four replicates of 100 seeds each and was repeated three times and the bars indicate standard error. Means designated with the same letter are not significantly different at po0:05.
Table 1 Effect of Elexa seed treatment on pearl millet growth parameters Growth parameter
Control b
Elexa seed treatment bc
Plant height (cm) Number of tillers Number of ear heads Ear head length (cm) Ear head girth (cm) 1000 seed weight (g)
81.670.49 e 370.0 e 370.0 e 10.670.47 e 5.670.14 e 6.470.34 e
115.772.28 f 5.570.28 f 4.570.28 f 11.470.30 f 6.770.0 f 7.670.11 f
The experiment was carried out in five replicates of 20 plants each and repeated twice. Values are expressed as mean7standard error. Means designated with the same letter are not significantly different at po0:05:
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Table 2 Effect of Elexa on pearl millet downy mildew disease incidence and protection under green house condition Elexa treatment
Percentage
Seed treatment Foliar spray to 2-day-old seedlings and inoculation 1 day after Elexa spray Foliar spray to 2-day-old seedlings and inoculation 4 days after Elexa spray Foliar spray to 2-day-old seedlings and inoculation 7 days after Elexa spray Seed treatment and Foliar spray to 2-day-old seedlings and inoculation 1 day after Elexa spray Seed treatment and Foliar spray to 2-day-old seedlings and inoculation 4 days after Elexa spray Seed treatment and Foliar spray to 2-day-old seedlings inoculation 7 days after Elexa spray Apron Control
DMDI
DMDP
46 31 25 26 26 31 28 20 91
48 67 72 71 71 67 68 78 —
c b ab ab ab b ab a d
b cd cd cd cd cd cd d
The experiments in this study was carried out in four replicates of 100 seedlings each and repeated twice. Means designated with the same letter are not significantly different at po0:05: DMDI=downy mildew disease incidence. DMDP=downy mildew disease resistance.
3.3.3. Seed treatment followed by foliar spray Seed treatment and Elexa spray to 2-day-old seedlings and inoculated 1 day after foliar spray offered a maximum of 71% protection reducing the disease incidence to 26% (Table 1). Seed treatment and Elexa spray to 2-day-old seedlings followed by inoculation 4 and 11 days after spray recorded 31% and 28% downy mildew resulting in 67% and 68% protection, respectively (Table 2). However seed treatment with Apron 35SD was better than all the above-mentioned combinations in protecting pearl millet against downy mildew showing a 20% disease incidence and offered 78% protection. In the untreated check 91% downy mildew disease incidence was recorded. 3.4. Optimization of time required for Elexa to reduce downy mildew disease incidence Protection increased from 48.53% on first day to 58.01% on the fifth day after inoculation. It was found that the protection increased throughout the 5-day-time gap period as 47%, 57% and 55% protection was observed on second, third and fourth day time gap, respectively (Fig. 2). The results indicated that the Elexa seed treatment offered maximum reduction of downy mildew disease incidence when inoculated 3 days after emergence. The resistances build up started as early as 24 h and the maximum resistance was developed when the time gap between Elexa seed treatment and pathogen inoculation was 24–48 h.
80 70
% protection over control
day recorded 31% disease incidence resulting in 67% protection and foliar sprayed seedlings inoculated after 4 and 11 days recorded 25% and 26% downy mildew which resulted in 72% and 71% protection, respectively (Table 2).
ab
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day1
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Fig. 2. Optimization of time required for Elexa to reduce downy mildew disease incidence. The experiments in this study was carried out in four replicates of 100 seedlings each and repeated twice and the bars indicate standard errors. Means designated with the same letter are not significantly different at po0:05.
3.5. Effect of Elexa on downy mildew disease incidence and severity under field conditions 3.5.1. Seed treatment Seed treatment with Elexa at 1:19 dilution for 6 h recorded 60% downy mildew incidence, which corresponds to 38% protection under epiphytotic conditions. When the disease was evaluated it was found to be 43%. Control plants recorded 90% downy mildew incidence and 99.5% severity. Apron treatment recorded 12% disease incidence and offered 87% protection showing a disease severity of 7.5% (Table 3). 3.5.2. Foliar spray Foliar spray to 2-day-old seedlings recorded 30% downy mildew incidence and 62% protection with
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Table 3 Effect of Elexa on pearl millet downy mildew disease incidence, severity and protection under field condition Treatment
Control Apron 35SD Seed treatment Foliar spray to 2-day-old seedlings Foliar spray to 7-day-old seedlings Foliar spray to 14-day-old seedlings Seed treatment and Foliar spray to 2-day-old seedlings Seed treatment and Foliar spray to 7-day-old seedlings Seed treatment and Foliar spray to 14-day-old seedlings
Percentage DMDI
DMDP
DMDS
90 12 60 30 33 32 28 30 32
0 87 38 62 67 67 67 69 67
99.5 7.5 42.5 27.1 25.0 24.3 23.2 23.0 24.4
a d b c c c c c c
a c b b b b b b
The experiments in this study was carried out in four replicates of 100 seedlings each and repeated twice. Means designated with the same letter are not significantly different at p ¼ 0:05: DMDI=downy mildew disease incidence. DMDP=downy mildew disease resistance. DMDS=downy mildew disease severity.
27.1% severity. Foliar spray to 7 and 14-day-old seedlings recorded 33% and 32% disease incidence, the disease protection was 67% and 67% respectively. The disease severity was also evaluated and it was found to be 25% and 24%, respectively (Table 3). 3.5.3. Seed treatment followed by foliar spray In seed treatment with Elexa and followed by foliar spray to 2-day-old seedlings recorded 28% disease incidence with 23% severity and 67% protection. Seed treatment followed by foliar spray to 7-day-old seedlings recorded 30% disease with 23% severity and 69% protection. Seed treatment followed by foliar spray to 14-day-old seedlings recorded 32% downy mildew incidence with 67% protection and 24% severity (Table 3).
4. Discussion Elexa is a commercial formulation of 4% Chitosan used in different vegetable and fruit crops like cucumber, pumpkin, zucchini, grapes and strawberries to control downy mildew, powdery mildew and gray mold diseases. In all these cases Elexa was used as foliar spray and the disease control was effective (Jones, 2000). In the present study Elexa at 1:19 dilution was used in pearl millet as seed treatment, foliar spray and also combination of seed treatment and foliar spray against S. graminicola, the incitant of downy mildew disease. Pearl millet is an important cereal crop of semi-arid tropics of India and Africa susceptible to downy mildew disease. Pearl millet is considered as a poor man’s crop in low input rain fed agricultural system. The farmers adopt only seed treatment pesticides against pests and diseases. The present study demonstrated that seed treatment alone reduced downy mildew disease and offered
acceptable levels of protection. Although foliar spray gives better downy mildew disease protection and seed treatment followed by foliar sprays is still better in protecting pearl millet farmers may not be able to practice the foliar spray. Currently recommended downy mildew disease control is by Metalaxyl a.i 2.1 g/kg of seeds in the form of Apron 35SD. However, metalaxyl is expensive and not yet made available to poor farmers in India. It has already been reported that oomycete pathogens are showing metalaxyl resistance hence search for cheaper commercial formulations of effective compounds are required (Thakur and Mathur, 2002). The effectiveness of Elexa indicates that such formulations may be cheaper and easy to produce and recommended to the farmers. However, much research is needed to increase the effectiveness of Elexa by seed treatment to achieve higher protection rates in pearl millet. Elexa has additional advantages when compared to metalaxyl treatment. Elexa treatment significantly enhanced seed germination and seedling vigor of pearl millet. Pearl millet seedlings showed increased height and more number of tillers. The number of productive ear heads, length and girth and the yield also increased, as it is evident from the 1000 seed weight. In the current study Elexa was evaluated in various methods like seed treatment, foliar spray and the combination of seed treatment and foliar spray both under green house and epiphytotic field conditions. The disease protection obtained was greater in the green house conditions than that in the field conditions. This may be due to the controlled environmental conditions in the green house and many environmental factors such as soil and continuous inoculum shower at all stages of plant growth operating in the field conditions which are not available in the green house conditions. Under green house conditions Elexa seed treatment protected pearl millet against downy mildew disease by up to 48%.
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Foliar spray with Elexa to 2-day-old seedlings offered 72% protection. Combination of Elexa spray and seed treatment offered more protection than either treatment alone. Results also showed that Elexa spray at seedling emergence stage was sufficient for reduction of disease incidence both under green house and field conditions. Under field conditions Elexa treatments considerably reduced the severity as well as the incidence of downy mildew. Elexa seed treatment offered 37% protection. Foliar spray to seedlings at 2, 7 and 14 days offered wide-ranging protection from 64% to 69%. The combination of seed treatment and foliar spray offered 71% protection. Field trials with Elexa in the United States, has shown that the disease severity of powdery mildew of grapes, cucumber, strawberries, pumpkins, zucchini was reduced very effectively when compared to other available bioactive products (Jones, 2000). The active ingredient of Elexa is Chitosan, which is reported to promote growth and elicit plant defense response in various crops. Chitosan seed treatment enhanced seed germination and seedling vigor in wheat. Chitosan treatment also increased the growth of Vitis vinifera L. plantlets (Bhaskarareddy et al., 1999; Barka et al., 2003). Chitosan seed treatment has been reported to protect tomato plants from crown rot and root rot pathogens upon seed treatment (Benhmaou et al., 1994). Chitosan was able to protect susceptible tomato seedlings from Fusarium oxysporum when applied as a foliar spray (Benhmaou et al., 1998). Chitosan spray protected cucumber plants by reducing the incidence of the gray mold (Ben-shalom et al., 2003). Chitosan has been shown to induce resistance effectively in a number of crops like cucumber, wheat, pea, celery, and groundnut against Pythium aphanidermatum, Alternaria alternata, Fusarium gramenareum, Fusarium solani f.sp. pisi and Fusarium oxysporum f.sp. apii. (Ghouth and Ahmad, 1994; Sathiyabama and Balasubramaniam, 1998; Bhaskarareddy et al., 1999). Corroborating with these earlier reports the present study also indicated that possible mechanism of downy mildew control by Elexa is through induction of systemic resistance in the host. This was demonstrated by spatio-temporal separation of the inducer and the pathogen inoculation. ISR normally requires a time-lapse period between inducer treatment and pathogen inoculation and gene activation for defense response to take place (Ryals et al., 1996). The present study also identified an optimal time interval of 24–48 h between inducer treatment and inoculation for the resistance to build up under green house conditions. In vitro studies recorded no fungitoxic effect of Elexa on sporulation, zoospore release and viability of S. graminicola (data not shown). Chitosan’s role as a plant defense elicitor and the mechanism of induction of resistance is one that has been well studied. Chitosan induces reduction of the stomatal aperture, lignin biosynthesis and plant cell wall
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lignification, ultra structural changes in leaf cells, production of antifungal hydrolases such as chitinase and beta-1,3 glucanase. Chitosan stimulates the accumulation of signal molecule jasmonic acid, hydrogen peroxide, reactive oxygen species and protein kinases all of which play a crucial role in intracellular signaling pathways (Chang and Mei, 1995; Pospieszny and Zilienska, 1997; Peter, 1998; Hadwiger, 1999; Sumin, 1999; Link et al., 2002; Rakwal et al., 2002; Barka et al., 2003). Considering all the aspects presented above Elexa is an active inducer of defense responses in pearl millet against downy mildew disease and thus has the potential of becoming an alternative means of disease control.
Acknowledgements The authors thank Dr. Richard Daoust Programme Manager, Agriculture Glycogenesys Inc. formerly Safe Science Inc., Boston, MA, USA for the kind supply of Elexa and for his support throughout the investigation. This work was carried out in the Project on Systemic Acquired Resistance funded by Danish International Agency under the Enhancement of Research Capacity Programme (DANIDA ENRECA). The authors are grateful to Dr. Eigil de Neergard, the Principal Responsible leader of the DANIDA ENRECA Project for his co-operation during this study. The facilities provided by Indian Council of Agricultural Research, Government of India through All India Coordinated Pearl Millet Improvement Project is also gratefully acknowledged. References Abdul Baki, A., Anderson, J.D., 1973. Vigor analysis in soybean seed by multiple criteria. Crop Sci. 13, 630–633. Agostini, J.P., Bushong, P.M., Timmer, L.W., 2003. Green house evaluation of products that induce host resistance for control of Scab, Melanose and Alternaria brown spot of Citrus. Plant Dis. 87, 69–74. Barka, A., Eullaffroy, P., Clement, C., Vernet, G., 2003. Chitosan improves development, and protects Vitis vinifera L. against Botrytis cinerea. Plant Cell Rep., in press. Benhmaou, N., Kloepper, J.W., Tuzun, S., 1994. Induction of systemic resistance to Fusarium crown rot and root rot in tomato plants by seed treatment with chitosan. Phytopathology 84, 1432–1444. Benhmaou, N., Kloepper, J.W., Tuzun, S., 1998. Induction of resistance to Fusarium wilt of tomato by combination of chitosan and endophytic bacterial strain: ultra structure and cytochemistry of host response. Planta 204, 153–168. Ben-shalom, N., Ardi, R., Pinto, R., Aki, C., Fallik, E., 2003. Controlling gray mold caused by Botrytis cinerea in cucumber plants by means of chitosan. Crop Prot 22, 285–290. Bhaskarareddy, M.V., Arul, J., Angers, P., Couture, L., 1999. Chitosan treatment of wheat seeds induces resistance to Fusarium graminearum and improves seed quality. J. Agric. Food Chem. 47, 1208–1216.
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