Seed treatment with aqueous extract of Viscumalbum induces resistance to pearl millet downy mildew pathogen

Abstract

Downy mildew (Sclerospora graminicola [Sacc.] Schroet.) is a serious agricultural problem for pearl millet (Pennisetum glaucum [L.] R. Br.) grain production under field conditions. Six medicinally important plant species Azadirachta indica, Argemone mexicana, Commiphora caudata, Mentha piperita, Emblica officinalis and Viscum album were evaluated for their efficacy against pearl millet downy mildew. Seeds of pearl millet were treated with different concentrations of aqueous extract of the plants to examine their efficacy in controlling downy mildew. Among the plant extracts tested, V. album treatment was found to be more effective in enhancing seed quality parameters and also in inducing resistance against downy mildew disease. Germination and seedling vigor was improved in seeds treated with V. album extracts over control. Seeds treated with 10% concentration of V. album showed maximum protection against downy mildew disease under greenhouse and field conditions. The downy mildew disease protection varied from 44–70% with different concentrations. Leaf extract of V. album did not inhibit sporulation and zoospore release from sporangia of Sclerospora graminicola, indicating that the disease-controlling effect was attributed to induced resistance. Seed treatment with V. album extract increased pearl millet grain yield considerably. In V. album, treated pearl millet seedlings increased activities of peroxidase, and phenylalanine ammonia-lyase enzyme was detected. FTIR analysis of V. album extracts showed the presence of amides and other aromatic compounds which are antimicrobial compounds involved in plant defense.

Keywords:

• plant extracts
• Viscum album
• pearl millet downy mildew
• induced resistance

Introduction

Exploitation and utilization of eco-friendly plant-based products have been the main focus of researchers and environmentalists, due to public concern over the toxicity and environmental impact of conventional synthetic pesticides. Hence, newer approaches are being explored and one important option is inducing resistance in the host by exploiting its inborn immunity. Pesticides derived from natural sources support both crop production and the environment by being effective in plant pathogen control, biodegradable, and are safer than synthetic fungicides.

Pearl millet (Pennisetum glaucum [L.] R. Br.) is the most important crop grown for food and fodder worldwide. In India, it is grown in an area of 9.8 million hectares with an annual production of 7.01 million tonnes (Khairwal et al. 2007). India, which produces more than half the world's pearl millet, has been the center of research efforts to meet this challenge since the crop yield is jeopardized by various biotic and abiotic stresses of which downy mildew disease caused by the biotrophic, oomycetous fungus S. graminicola assumes prime importance, responsible for annual grain yield loss of up to 80% (Howarth and Yadav 2002).

Apart from resistance breeding, a range of methods like chemical control and biological control are employed to manage the disease; however, they have their own shortcomings. At present in India, Metalaxyl is the chemical that is used to manage downy mildew. However, this chemical has been termed hazardous and it is also not economical to a crop like pearl millet. Moreover, the pathogen has evolved tolerance against these chemicals at various downy mildew screening centers in India. Although pearl millet downy mildew is effectively managed by systemic fungicides (Singh and Shetty 1990), growing awareness about these hazardous chemicals, their impact on the environment and human health has encouraged us to explore the effects of plant extracts, which are eco-friendly in nature and also economical (Heil and Bostock 2002).

Besides beneficial microbial agents and disease resistance activators, numerous studies have documented the effectiveness of plant extracts against phytopathogens. (Singh et al. 1990; Herger and Klingauf 1990; Mende et al. 1994; Daayf et al. 1995; Singh and Prithiviraj 1997; Paul and Sharma 2002). For instance, leaf extracts of Reynoutria sachalineusis and Azadirachta indica induced resistance in barley against leaf stripe disease and in cucumber against powdery mildew (Daayf et al. 1995; Paul and Sharma 2002). Ginger extract protected pea plants from powdery mildew pathogen under field conditions and Ajoene, a product of garlic, has shown significant control of powdery mildews (Singh et al. 1990). Induction of systemic resistance in pearl millet against downy mildew with Datura metel extract (Shivakumar et al. 2003), plant growth promoting rhizobacteria (PGPR) (Niranjan Raj et al. 2003), abiotic inducers like Benzothiadiazole (BTH), CaCl₂ and H₂O₂ (Geetha and Shetty 2002), and cerebrocides elicitor (Deepak et al. 2003) have been demonstrated earlier. Furthermore, the involvement of various defense enzymes such as peroxidase, phenylalanine ammonia lyase, and polyphenol oxidase have also been studied (Shivakumar et al. 2003; Geetha et al. 2005; Niranjan Raj et al. 2006). However, there are very limited reports on the use of plant extracts for plant disease management. The present study was designed to investigate the efficacy of aqueous extract of different plant species to induce resistance in pearl millet against downy mildew disease. The activity of defence enzymes peroxidase (POX) and phenylalanine ammonia-lyase (PAL) in induced pearl millet seedlings was also studied.

Materials and methods

Pearl millet seeds and pathogen

Pearl millet cultivars IP18292 (highly resistant) and 7042S (highly susceptible) to downy mildew disease procured from the International Corp Research Institute for Semi-Arid Tropics (ICRISAT), Hyderabad, India, and from All India Coordinated Pearl Millet Improvement Project (AICPMIP), Mandor, Jodhpur, India, respectively, were used. S. graminicola was isolated from pearl millet cv 7042S and maintained on the same cultivar under greenhouse conditions was used for all pathogen inoculation experiments.

Inoculum preparation

Leaves of infected pearl millet plants were collected in the evening hours and washed in running tap water to remove pre-existing sporulation and blot-dried and placed in moist chamber for sporulation. Fresh sporangia were collected the next morning and zoospores released by them was adjusted to 4×10⁴ zoospores ml⁻¹ in distilled water using a haemocytometer and used as inoculum for all the experiments (Safeeulla 1976).

Preparation of plant extracts

A. indica, A. mexicana, C. caudata, M. piperita, E. officinalis and V. album plants were collected in and around the University campus, University of Mysore, and local market, Mysore, India. The shoots and leaves were washed in running tap water, blotted dry and then homogenized with sterile distilled water in the ratio 1:2 (w/v) in a pre-chilled mortar and pestle. The leaf homogenate was filtered through four layers of cheesecloth and the filtrate was centrifuged at 10,000 rpm for 20 min at 4°C.

Inducers treatment

The aqueous plant extracts were diluted into different concentrations: 1, 2, 3, 5, 7, 10 and 15% in distilled water and used for seed treatment and seeds treated with distilled water served as control.

Effect of plant extracts on pearl millet seed germination and seedling vigor

Germination test was carried out by the paper towel method (International Seed Testing Association [ISTA] 2003). Seeds treated with aqueous plant extracts were placed on moist germination paper equidistantly. Another presoaked paper towel was placed on the first one so that the seeds were held in position. The towels were then rolled and wrapped with polythene to prevent drying and incubated at 25±2°C. After seven days, the towels were unrolled and the number of seeds germinated was counted. Seedling vigor was analyzed according to Abdul Baki and Anderson (1973). The length of the root and shoot was measured and vigor index was calculated using the formula:

Four replicates of 100 seeds were used for all the treatments.

Effect of plant extracts on pearl millet downy mildew disease under the following conditions

Greenhouse conditions

Seeds treated with different concentrations of the above mentioned plant extracts, Apron 35 SD (6 g/kg of seed) and distilled water for 6 h were sown in earthen pots containing 1:2:1 ratio of sand, soil and manure and maintained under greenhouse conditions. Leaf-whorls of two-day old seedlings were inoculated with a suspension of 4×10⁴ zoospores ml⁻¹ of S. graminicola by whorl inoculation method (Singh and Gopinath 1985). Each treatment consisted of four replicates of 25 plants each.

Field conditions

The treatments found promising were further evaluated under field conditions against downy mildew disease. Seeds treated with A. mexicana and V. album extracts at the concentrations, which were most promising under greenhouse conditions, were sown in the pearl millet experimental plot of Department of Studies in Biotechnology, University of Mysore, Karnataka, India. The systemic fungicide Apron 35 SD (Metalaxyl formulation) at the rate of 6 g/kg of seed was used as a standard chemical check (positive control) and distilled water treated seeds served as control. The plot size was 10×5 m and recommended agronomic practices were followed during the trial. Plant-to-plant distance of 15 cm and row-to-row distance of 50 cm was maintained. The experiment was designed as a Randomized Complete Block (Williams et al. 1981) with four replicates. Grain yield was estimated by following the procedures of Williams and Singh (1981). Yield data was calculated by collecting ear head from the central 3.8 m of two center rows after maturity.

Demonstration of systemic nature of induced resistance

Demonstration of systemic acquired resistance was carried out following the procedure described earlier by Shailasree et al. (2001). Seeds treated with V. album as described above were sown in earthen pots filled with autoclaved soil, sand and manure in the ratio 2:1:1. The emerging seedlings were challenge inoculated with the zoospore suspension of S. graminicola following the whorl inoculation procedure with a time gap of 1, 2, 3, 4, and 5 days in different sets of plants. The plants were maintained under greenhouse conditions and were observed for the downy mildew disease reaction and downy mildew disease data recorded. Each treatment consisted of 25 plants in four replicates.

Disease assessment

Plants were observed daily and any symptom of the downy mildew disease recorded. Seedlings were rated diseased when they had any one of the typical symptoms of downy mildew, i.e. sporulation or green ear, stunting and chlorosis, and protection was calculated using the formula:

where C represents percentage of downy mildew disease incidence in control, and T represents percentage of downy mildew disease in treated plants (Shetty et al. 1995).

Effect of plant extracts on asexual spores of S. graminicola

To test the efficacy of aqueous extracts of the afore-mentioned plant species on sporulation and zoospore release of S. graminicola, the sporangial suspension (40,000 zoospore/ml) of S. graminicola were mixed with 1, 3, 5, 7, 10 and 15% of the plant extract into cavity glass boxes. The boxes were incubated at 25±2°C in the dark for 30 min. The percentage of zoospores releasing was counted under the microscope. For sporulation of S. graminicola, the infected leaves were collected, washed, blot dried, cut into 4 cm strips and these leaf strips were treated with different concentrations of V. album extract for 1 h, with distilled water-treated served as control, and placed in a moist chamber for sporulation overnight. Early in the morning the next day, the treated leaf strips were observed for sporulation, intensity of sporulation and release of the zoospore.

Fourier-Transform Infra-red spectroscopy (FTIR) studies of V. album leaf extract

An FTIR spectrometer (Perkin Elmer Spectrum Bx) equipped with a deuterated triglycerine sulphate (dTGS) detector and KBr beam splitter was used for the characterization of active compounds and functional groups present in the V. album extract. The V. album extract was subjected to FTIR studies and the results analyzed.

Biochemical studies

Sampling of seedlings

Effect of V. album seed treatment on the activity of defense enzymes like peroxidase and phenylalanine ammonia-lyase activity during host pathogen interaction in pearl millet was carried out. Two-day old seedlings of susceptible (7042S) and resistant (IP18292) pearl millet cultivars were harvested at different time intervals (0, 2, 4, 8, 12, 16, 20, 24 and 48 h) after pathogen inoculation. Distilled water-treated seedlings were used as control.

Phenylalanine ammonia-lyase (PAL) (E.C. 4.1.3.5) enzyme extraction and assay

One gram of pearl millet seedlings was used for extraction of PAL enzyme with 25 mM sodium borate buffer (pH 8.8) at 4°C with 32 mM β-mercaptoethanol using a pre-chilled pestle and mortar. The homogenate was centrifuged at 20,000 g for 20 min at 4°C in a refrigerated high-speed centrifuge (Hitachi, Japan). PAL activity in the supernatant of the cell free extracts was assayed as described by Geetha et al. (2005). The reaction mixture (3 ml) consisted of 50 mM L-phenylalanine in 100 mM sodium borate buffer (pH 8.8), 500 µl of the crude extract and 25 mM sodium borate buffer was measured spectrophotometrically at 290 nm (Hitachi U 2000, Japan). The enzyme activity was expressed in terms of µ mol t-cinnamic acid mg⁻¹ protein h⁻¹.

Peroxidase (POX) (E.C. 1.11.1.7) extraction and assay

Seedlings (1 g fresh weight) were homogenized in 2 ml of 0.2M Tris/HCl buffer pH 8.0 at 4°C. The homogenate was filtered through cheesecloth and the filtrate centrifuged at 12,000 g for 15 min. The supernatant was dialyzed against distilled water for 48 h at 4°C, lyophilized, and stored. This crude enzyme extract was used for peroxidase spectrophotometric assay and peroxidase activity was carried out following the method of Hammerschmidt et al. (1982) by monitoring the increase in absorbance at 470 nm for 60 sec. One unit of enzyme activity was defined as increase in one OD value at A470 nm/min.

Protein estimation

The protein content of each of the test sample was estimated following the procedure of Bradford (1976) using BSA (Sigma, St Louis, MO, USA) as a standard.

Statistical analysis

All the experimental results were subjected to Duncan's Multiple Range test (DMRT). The means were compared for significance at p ≤0.05. All the results are based on two independent experiments.

Results

Efficacy of seed treatment with plant extracts on seed germination and seedling vigor

In general, all the concentrations of the different plant extracts treated to seeds except for C. caudata significantly enhanced the seed germination and seedling vigor of pearl millet. However, the enhancement of germination and seedling vigor varied with concentrations. It was observed that, the highest germination (85%) and seedling vigor (1866) was recorded at 10% V. album extract-treated seeds (Table 1).

Table 1. Effect of seed treatment with aqueous extract of different plants on seed germination, seeding vigor, pathogen sporulation, zoospore release and downy mildew disease incidence.

Plant species	Parts used	Conc. (%)	Germ. (%)	Vigor index (MRL + MSL) ×% germination	Downy mildew disease protection Greenhouse	Sporulation of asexual spores	Zoospore release from sporangia
Argemone mexicana	Shoot	2	85^a	1809	29^d	+ + +	+ + +
		3	87^a	1901	44^c	+ + +	+ + +
Emblica officinalis	Fruits	2	80^b	1420	18^g	+ + +	+ + +
		3	80^b	1499	24^e	+ + +	+ + +
Commiphora caudata	Leaves	7	74^d	1608	16^g	+ + +	+ + +
		10	78^c	1666	19^ef	+ + +	+ + +
Viscum album	Leaves	5	84^ab	1728	50^b	+ + +	+ + +
		7	84^ab	1784	54^b	+ + +	+ + +
		10	85^a	1866	61^a	+ + +	+ + +
Mentha piperita	Leaves	1	80^b	1449	29^d	+ + +	+ + +
		2	80^b	1479	30^d	+ + +	+ + +
Azadirachta indica	Leaves	1	82^b	1206	16^g	+ + +	+ + +
		2	84^ab	1361	20^e	+ + +	+ + +
Distilled water – Control			80^b	1332		+ + +	+ + +
Apron 35 SD at 6 g/kg of seeds					92^a	No sporulation	No zoospore
+ = 25%,+ + = 50%,+ + + = 100% sporulation and zoospore release.
Vigor index was calculated based on percent germination and mean root length and mean shoot length of the seedlings. Means followed by the same letter are not significantly different according to Duncan's Multiple Range test (DMRT).
(P≤0.05). MRL, mean root length; MSL, mean shoot length.

Efficacy of seed treatment with plant extracts on pearl millet downy mildew disease under greenhouse conditions

The plant extracts and their optimized concentrations based on their effect on seed quality parameters were further selected for evaluation against downy mildew disease under greenhouse studies. Generally, all the plant extracts and concentrations selected for greenhouse were significantly effective in reducing downy mildew disease incidence compared to the distilled water control and the degree of protection varied with type and concentration of the plant extract. The downy mildew disease protection offered due to seed treatment with V. album extract, at concentrations 5, 7 and 10% was 50, 54 and 61%, respectively. Apron 35SD-treated seeds recorded 92% protection and none of the plant extract concentrations were on a par with Apron treatment (Table 1).

Efficacy of seed treatment with V. album extract on pearl millet under field conditions

Since 10% V. album treatment recorded significantly high downy mildew disease protection under greenhouse conditions, it was further tested under field conditions. Despite heavy inoculum pressure, V. album extract-treated pearl millet plants recorded protection of 62% over distilled water treated control plants. However, V. album extract treatments were not on a par with Apron in showing downy mildew protection, and Apron 35SD treatment showed 92% protection against downy mildew disease (Figure 1).

Figure 1.  Efficacy of seed treatment with Viscum album extract (10%) on downy mildew disease of pearl millet under field conditions. Percentage of downy mildew incidence (DMI) and downy mildew disease protection (DMP) are the means of two experiments. Bars indicate the standard error of the mean value. DH₂O, distilled water control.

Effect of seed treatment with V. album extract on grain yield of pearl millet

The efficacy of V. album extract was further assessed on pearl millet grain yield in comparison with Apron 35 SD and distilled water control. Seed treatment using 10% of V. album extract resulted in a yield of 1390 kg/ha as against control (distilled water)-treated pearl millet seeds which showed 1016 kg/ha and Apron 35 SD recording 1597 kg/ha (Figure 2).

Figure 2.  Efficacy of seed treatment with Viscum album extract on yield of pearl millet. Yield (kg/ha) was calculated by the means of two experiments following the procedure of Williams and Singh (1981). Bars indicate the standard error of the mean value.

Demonstration of induction of resistance in time gap inoculation with S. graminicola in pearl millet

Since 10% V. album extract showed significant downy mildew disease both under greenhouse and field conditions, it was further evaluated for demonstrating the nature of resistance induction by using spatio-temporal studies. The downy mildew disease protection varied from 58–70.5% at different day interval inoculations. The maximum protection obtained was 70.5% on the fifth day-inoculated plants (Figure 3). The downy mildew protection was 58, 59, 64 and 68, on the 1st, 2nd, 3rd and 4th day inoculated plants, respectively.

Figure 3.  Effect of seed treatment with Viscum album aqueous extract and time gap inoculation with Sclerospora graminicola. Percentage of downy mildew incidence (DMI) and downy mildew disease protection (DMP) are the means of two experiments. Bars indicate the standard error of the mean value.

Effect of V. album extract on sporangia and zoospore release

All the concentrations such as 1, 2, 3, 5, 7 and 10% of the extract tested against S. graminicola did not inhibit the growth of sporangia and zoospore release when compared to control. However, 15, 20 and 25% of V. album extract inhibited the sporangia and zoospore discharge from the sporangia. Complete inhibition was observed at 25% V. album extract (Table 1).

FTIR studies of V. album leaf extract

The FTIR spectrum of the bioactive region showed absorption bands at 3647 (Free O-H), 2970 (C-H), 2555 (Free S-H), 1646 (C = O most probable amides), 1170 (C-O). The bioactive substance showed strong absorption between 3100 and 2850 cm⁻¹ and weak absorption above 3000 cm⁻¹ which indicate that C = C, which may be an alkene or aromatic compound. Finding peaks at 1600–1500 cm⁻¹ are confirms the aromatic ring. Strong C-H absorption at 2970 cm⁻¹ is due to the aliphatic hydrogen (Figure 4).

Figure 4.  Efficacy of Viscum album extract on peroxidase activity in pearl millet seedlings. Peroxidase activity was calculated by taking the means of two experiments. Bars indicate the standard error of the mean value. IR, induced resistant; R, resistant.

Biochemical studies

Estimation of peroxidase (POX) activity in induced resistance

Spectroscopic analysis showed maximum POX activity of 43 units at 8 hpi time interval harvested induced resistant seedlings, whereas in control seedlings, the activity was 27 units. In the distilled water-treated seedlings, the maximum activity of 38.5 units was obtained at 48 h. However, the POX activity in highly resistant seedlings was higher compared to induced resistant and control seedlings (Figure 5).

Figure 5.  Estimation of phenylalanine ammonia-lyase activity by spectroscopic method by seed treatment with Viscum album on pearl millet resistant, induced resistant and susceptible seedlings. Bars indicate the standard error of the mean value. R, Resistant; IR, induced resistant.

Estimation of PAL activity in induced resistance

Irrespective of the time intervals, PAL activity recorded sequential and significant increase in treated seedlings when compared with control seedlings. Maximum PAL activity was recorded in resistant at 4 hpi, which were 6-fold higher than the control seedlings. In induced resistance, seedlings treated with V. album extract recorded maximum activity at 4 hpi, which was 1.6-fold higher than the control. The resistant seedlings followed this at the same time interval. A minimum activity of PAL was recorded at 72 hpi in induced resistant seedlings. In the control seedlings, the least PAL activity was recorded at 24 and 48 hpi, which was lower than any treatment (Figure 6).

Figure 6.  Fourier-Transform Infra-red (FTIR) spectroscopy profile of V. album extract.

Discussion

Continuous and indiscriminate use of chemical pesticides has posed a serious threat to human and environmental health. Therefore eco-friendly approaches for plant disease management have been exploited worldwide. There are many known abiotic and biotic inducers of resistance against various phytopathogens; however, the use of plant extracts for disease management is limited and presently management of plant diseases by plant extracts is gaining worldwide importance and acceptance. It has been demonstrated that many plants and plant products have been reported to possess pest control properties and plant extracts and plant essential oils are effective antimicrobials against various foliar and soil-borne phytopathogens (Awuah 1994; Grayer and Harborne 1994; Lawson and Kennedy 1998). Medicinally important plants have an almost limitless ability to synthesize aromatic substances, most of which are phenols or their oxygen-substituted derivatives. In many cases these substances enhance plant defense mechanism against microorganisms (Cowan 1999). In the present study, different plant extracts like Azadirachta indica, Argemone mexicana, Commiphora caudata, Mentha piperita, Emblica officinalis and V. album were evaluated for their efficacy against pearl millet downy mildew disease both under greenhouse and field conditions. The results indicated that although all the extracts were able to reduce downy mildew incidence, V. album extract at 10% concentration considerably enhanced seed quality parameters and also significantly reduced downy mildew both under greenhouse and field conditions by inducing durable resistance in the host.

Among the tested plant extracts, C. caudata failed to enhance seed germination and seedling vigor. However, the maximum enhancement of germination and seedling vigor was due to the treatment with 10% V. album extract. SAR is a method to trigger the host defense mechanism rather than act directly on the pathogen and therefore any compound which is an inducer of systemic resistance should not have inherent antimicrobial activity (Kessmann et al. 1994).

Since zoospores of S. graminicola were not inhibited by V. album extracts at concentrations up to 15%, it is evident that the disease control mechanisms may be through induction of resistance in pearl millet by V. album.

V. album extract at 10% concentration recorded a protection of 61% under greenhouse conditions; therefore, it was further evaluated under field conditions where it recorded 62% protection against downy mildew disease. Apart from protection against downy mildew, V. album extract treatment was also effective in promoting the growth of pearl millet which was evident by the significant enhancement of pearl millet grain yield compared to the controls.

Furthermore, the nature of resistance induction by V. album studied in our experiment was found to be systemic, which we demonstrated by maintaining spatial and temporal separation of the inducer and the challenger. Necessity of a time interval between application of the inducer and the onset of protection is important since the plant requires time to reach the induced state. Many studies have shown that, generally, it takes from a few days to a week for SAR or ISR to develop. In line with these observations, our study revealed that resistance build-up due to V. album treatment in pearl millet requires a minimum period of three days. The requirement of an appropriate time interval for induction of resistance has been reported in pearl millet plants against S. graminicola with β-aminobutyric acid (BABA) treatment, where maximum protection was observed two days after treatment (Shailasree et al. 2001). The difference in the time intervals required for resistance to build up may be dependent on the nature of the inducer used. The argument that the protection is due to induction of resistance is further supported by the fact that V. album treatment did not have any direct effect on the pathogen propagules and also the treatment induced and enhanced the activities of important defense enzymes of systemic acquired resistance like POX and PAL.

Increased enzyme activities and the onset of systemic acquired resistance have been observed in a number of plant species, including cucumber, tobacco, melons, rice and lima beans (Hammerschmidt et al. 1982; Rasmussen et al. 1995; Young et al. 1995; Maffei et al. 2006). In pearl millet, POX and PAL activities have already been shown to be associated with reduction in the rate of pathogen multiplication and spread (Shivakumar et al. 2003; Geetha et al. 2005).

The importance of peroxidase enzyme in plant defense mechanism has been reported in a number of plant species against invading pathogens (Levine et al. 1994). Pearl millet seedlings raised with the treatment of D. metel leaf extract showed increased peroxidase activity over control (Shivakumar et al. 2003). In this study, the activity of peroxidase enzyme was significantly enhanced due to the treatment with V. album extract. Paul and Sharma (2002) demonstrated that the leaf extracts of neem (A. indica) significantly controlled the leaf stripe disease of barley and also increased activity of phenylalanine ammonia-lyase and tyrosine ammonia-lyase and these are the key enzymes in phenol biosynthesis and induced by treatment with neem products also demonstrated earlier by Singh and Prithiviraj (1997). Mende et al. (1994) reported that when the extracts from Alchemilla vulgaris, Hedera helix, R. sachalinensis and V. album were applied to leaves of two susceptible to Erwinia amylovora, all the four plant extracts induced resistance to the pathogen as evidenced by a marked reduction in bacterial development and inhibition of disease symptoms.

Extracts of R. sachalinensis led to an increase in activities of chitinase, peroxidase, β-1,3-glucanase, polyphenol oxidase, phenylalanine ammonia-lyase in cucumber and tobacco leaf tissues (Schneider and Ullrich 1994).

Biochemical studies of Milsana-treated plants showed increased activities of defense such as peroxidase, β-1,3-glucanase as well as in ethylene production (Herger and Klingauf 1990). The efficacy of neemzal, a natural product from neem (Azadirachta indica) against pea (Pisum sativum) powdery mildew caused by Erysiphe pisi was studied in detached leaf and intact plant. Neemazal significantly retarded several growth parameters of pathogens. Neemazal induced hypersensitive reaction (HR) and also the effect of compound on disease development was correlated with increased phenylalanine ammonia-lyase (PAL) activity in pea leaves following treatment with neemazal (Singh and Prithiviraj 1997). In this research, the time course study of peroxidase enzyme activity was different in control, induced resistant and resistant seedlings, and maximum activity was obtained in induced resistant over control. This indicates susceptible seedlings are unable to trigger early defense reaction against pathogen like in induced and resistant seedlings.

Fourier-Transform Infra-red spectroscopy (FTIR) studies with V. album extracts showed peaks which corresponded to compounds like amides, O-H groups, alkenes, aromatic compounds which are all well known to possess antimicrobial properties and they are also known to be involved in plant defense against various pathogens.

In conclusion, the resistance induced by seed treatment with V. album extract was significant under both greenhouse and field conditions. In addition, V. album treatment had a good growth promotional effect on pearl millet and enhanced grain yield. The resistance offered by V. album was systemic in nature and showed the involvement of defense enzymes like POX and PAL.

Therefore, botanicals/plant-based products, particularly V. album, are very effective in plant disease management and are environmentally safe compared to chemical agents and make an important component of integrated disease management (IDM) for agriculture.

Acknowledgements

The authors would like to thank the Danish International Development Agency (DANIDA) and to the Indian Council of Agricultural Research (ICAR), Government of India through All India Coordinated Pearl Millet Improvement Programme (AICPMIP).