Induction of defence enzymes activities in grape plant treated by seaweed algae against Plasmopara viticola and Uncinula necator causing downy and powdery mildews of grapes

Plasmopara viticola and Uncinula necator are the causative agents of downy mildew and powdery mildew diseases, respectively, which become a major problem in grape crop ( Vitis vinifera L.). The use of natural products such as seaweed provides a rich source of structurally diverse and biologically active secondary metabolites; and is the ultimate way of combating these diseases. In this context, six different seaweed algae such as; Sargassum wightii , Padina gymospora , Caulerpa peltata , Halimeda gracilis , Acanthophora spicifera and Gracilaria opuntia ; were used to control the downy and powdery mildew diseases of grapes. Enzymatic studies were carried out during two growing seasons 1 and 2 on peroxidase; polyphenol oxidase, phenylalanine ammonia lyase and β-1,3-glucanase. During season 1; in grape plants treated with Sargassum wightii , the four enzymes raised the glucose concentration to 60.79µg, 3.87µg, 5.20µg and 225.5µg; respectively, and then these plants were treated with Bordeaux mixture at 1%. After seaweed application; enzymes raised the glucose level to 59.83µg, 3.57µg, 5.17µg, 221.7µg. During season 2; when plants were treated with Sargassum wightii , the enzymes raised the glucose concentration to 62.48µg, 5.93µg, 7.25µg and 226.2µg, and then these plants were treated with Bordeaux mixture at 1%. After application; the enzymes raised the glucose level to 61.92µg, 3.57µg, 7.21µg and 224.2µg, respectively. During both seasons; β-1,3-glucanase released more amounts of glucose in both normal plants and those treated with Bordeaux mixture. The present study was undertaken to evaluate whether various extracts of seaweeds algae such as; brown, red, and green seaweeds along with the defence enzymes studied, would increase the resistance of grapes to downy and powdery mildews.


Introduction
Grape (Vitis vinifera L.) is one of the most delicious; refreshing and nourishing sub-tropical fruit, and its cultivation is one of the most remunerative farming enterprises in India. It is grown in a variety of soils. This fruit is rich in minerals and vitamins such as; A, B1, B2, C and K. Grape was cultivated in an area of about 138 thousand hectares with an annual production of 2,967.00 thousand tons of fruits (Indian Horticulture database. 2017-2018. India ranks the 9 th in grape production throughout the world (Shikamany, 2001;Gade et al., 2014). In India, Maharashtra is one of the largest grapes producing state in India with an annual productivity of 7,74,000 tons in 2015. Other major producing states are Karnataka, Tamil Nadu and Punjab with an annual production of 330.3, 53 and 27.6 thousand tons, respectively (Dethe, 2000).
Plasmopara viticola causes downy mildew of grapes; it mainly infects leaves and clusters of young berries, produces oil spot lesions on the adaxial leaf surface accompanied with massive sporulation on the abaxial surface (Perazzolli et al., 2012). Moreover, all green tissues of the grapevine are susceptible to powdery mildew disease caused by Uncinula necator. On the surface of the plant, this disease appears as whitish-grey powdery coatings on the leaves and fruits due to fungal mycelia and conidia. Late in the season, small black round reproductive structures (cleistothecia) begin to appear on the white powdery lesions. Severe leaf infections cause distortion, drying and premature drop. Infected berries covered with the fungus may turn dark brown; shrivel, split and may not ripen properly (Berkett and Cromwell, 2015).
Marine products cause plant induced systemic resistance (ISR) through fortifying the physical and mechanical strength of the cell wall; as well as chemical, physiological and biochemical reactions within host cells, leading to the synthesis of defense chemicals against pathogens. Defense reaction occurs due to accumulation of peroxidase, phenylalanine ammonia lyase and PR-protein like β-1,3-glucanase enzymes. Several authors reported the induction of defense enzymes in crop plants treated with organic products and challenged with the pathogen (Nakkeeran et al., 2006;Jayaraj et al., 2008;Flora and Rani, 2012).
According to Vidhyasekaran, (1997); peroxidase (PO) which is part of the PR-9 family has been implicated in many physiological activities within the plant cells such as; regulation of plant cell elongation, phenol oxidation, polysaccharide cross linking, Indole acetic acid oxidation, cross linking of extension monomers, oxidation of hydroxyl cinnamyl alcohols into free radical intermediates, and wound healing. Polyphenol oxidase (PPO) enzyme usually accumulates upon wounding in plants. Biochemical approaches to understand PPO function and regulation were difficult; because the quinoid reaction products covalently modify and cross link the enzyme (Constabel et al., 1995).
On the other hand; phenylalanine ammonia lyase (PAL) is a defense enzyme catalyzing the determination of L-phenylalanine to produce cinnamic acid, a substrate feeding to several biosynthetic routes to produce various classes of phenyl propanoid secondary plant products (Halbrook and Sheel, 1989).
PAL activity provides precursors for lignin biosynthesis and other phenolics that accumulate in plants in response to infection such as salicyclic acid (SA); a molecule that is essential for systemic acquired resistance (Klessig and Malamy, 1994). Later, Mauch-Mani and Slusarenko, (1996) reported that the major function of PAL is the production of SA precursors in the resistance of Arabidopsis to Peronospora parasitica. Several studies of Nayar, (1996);Jayaraj et al., (2008);Flora and Rani, (2012) reported that seed treatment and seedling root dipping with plant growth promoting rhizobacteria Novel Research in Microbiology Journal, 2018 (PGPR) induced early and enhanced levels of PAL in rice plants.
Evidence of β-1,3-glucanases activities in disease resistance was first reported by Kauffimann et al., (1987). In dicots, β-1,3-glucanase genes were considered to constitute a part of the general array of defense genes induced during pathogenesis (Mauch et al., 1988). β-1,3-glucanases especially in conjunction with chitinases were capable of hydrolyzing fungal cell walls in vitro (Mauch et al., 1988). Vogeli et al., (1988) added that both of these enzymes were co-induced in response to pathogen attack.

Survey on the occurrence of powdery and downy mildews of grapes in Theni district
A field survey was conducted to assess the extent of occurrence of grape powdery and downy mildews caused by U. necator and P. viticola, respectively, in Theni district. Villages where grape is traditionally grown were selected for assessing the prevalence of both diseases. During this survey, affected plants were recorded and their total numbers were counted. The percentage of disease incidence was checked out as per phytopathometry according to Sriram et al., (2000).

Names and sites of collections of seaweeds algae
The names and cities of collection of the various seaweeds algae tested for inducting resistance against U. necator and P. viticola are shown in Table. 1.

Preparation of seaweeds algae crude extracts
Each 1 Kg of alive, healthy and matured seaweed samples collected along the Coast of Pamban (Rameswaram (9°14`N; 79°14`E), Kanyakumari and Pondicherry were washed thoroughly in sea water followed by tap water to remove extraneous particles and epiphytes. These samples were air dried under shade in the laboratory for 3 days. The shade-dried samples were then chopped and pulverized. 50 g of each powdered sample in three replicates was separately extracted for 7 days in 500 ml of 1:1(v/v) chloroform: methanol under dark conditions. These extracts were pooled; concentrated using rotary evaporator under reduced pressure at 45°C, weighed and then stored at 0°C till use (Vallianayagam et al., 2009).

Fungal infestation
All grape vines variety Muscat (panneer) was cultivated under field conditions for powdery and downy mildews fungal infestation; three replicates were used for each fungal pathogen. For infestation of plants with powdery mildew pathogen; conidia of U. necator were collected from infected leaves, washed with 78% glucose solution to imitate the osmotic pressure of powdery mildew conidia, and then suspended in sterile water. Vine leaves were sprayed at their upper surfaces with the conidial suspension at 2×10 5 conidia/ ml. Inoculated leaves were immediately covered with thin plastic bags for 6 h according to Wang et al., (1995).
The methods of Rumbolz et al., (2002);Boso et al., (2006) were used to propagate sporangial inoculum for downy mildew fungal infestation. P. viticola was obtained from naturally infected grape plants in the vineyards. Plants were sprayed with a water suspension of fungal sporangia (40,000 sporangia/ ml) on the abaxial leaf side; and then were covered with a polyethylene cover overnight. On the following day, the polyethylene covers were removed and plants were incubated at 25ºC for 5-6 days. Artificial infestation was carried out 20 days after pruning.

Seaweeds algae extract application
The seaweed extracts dosage were sprayed on all aerial parts of the infested vine using a high volume Knapsack sprayer. Sprayings were carried Novel Research in Microbiology Journal, 2018 out when the mildew diseases were first noticed and subsequently two sprays were undertaken at the 30 th and 50 th days after pruning. Field trials were conducted with eight treatments were applied during the first and second seasons (February-May, July-October, 2017; respectively). During field trails, the cultivation practices such as; pruning, irrigation, weeding and other cultural operations have been performed following standard procedures as and when required uniformly in all the experimental plots. Three replicates were used for each treatment.

. Application of seaweeds algae extracts on grape plants in the field
Field trials were conducted using the Muscat (panneer) variety of grape plants with eight treatments during the first and second seasons (February-May, July-October, 2017, respectively); to assess the induction of defence enzymes by seaweed algae formulations after challenge inoculation with U. necator and P. viticola as described previously. The following eight treatments were adopted; Treatment 1: spraying of Hydroclathrus clathratus (brown seaweed) at 10%, T2: spraying of Liagora ceranoides (Red seaweed), T3: spraying of Jania rubens (Red seaweed) at 10%, T4: spraying of Dictyota dichotoma (brown seaweed) at 10%, T5: spraying of Ulva intestinalis (green seaweed) at 10%, T6: Sspraying of Ulva reticulate (green seaweed) at 10%, T7: spraying of Bordeaux mixture at 1% (positive control), and T8: as control. All treatments were applied after 30 and 50 days from pruning of grape plants. The treated plant samples were collected at different time intervals (1, 3, 5, 7 and 9 days) after powdery and downy mildews infestation. Three replications were maintained for each treatment, and fresh plant samples were used for enzymatic analysis.

Enzyme extraction from treated grape plants
The collected plant tissues were immediately homogenized with liquid nitrogen. One gram of each powdered sample was extracted with 2 ml of sodium phosphate buffer, 0.1 M (pH 7.0) at 4°C. The homogenate was centrifuged for 20 min. at 10,000 rpm. Plighting enzyme extracts prepared from leaves were used for the estimation of peroxidase (PO), polyphenol oxidase (PPO), L-phenylalanine ammonia lyase (PAL) and β-1,3-glucanase enzymes.

Evaluation of enzymes level in treated grape plants using spectrophotometric assays
Novel Research in Microbiology Journal, 2018

Peroxidase (PO)
Peroxidase activity was assayed spectrophotometrically (Hartee, 1955). The reaction mixture consisted of 1.5 ml of 0.05 M pyrogallol; 0.5 ml of enzyme extract and 0.5 ml of 1 % H 2 O 2 which was incubated at room temperature (28 ± 1°C). The change in absorbance at 420 nm was recorded at 30 sec. intervals for 3 min., the boiled enzyme preparation served as blank. Enzyme activity was expressed as change in the absorbance of the reaction mixture at 420 nm min -1 g -1 fresh weight of tissue as described by Hammerschmidt et al., (1982).

Polyphenol oxidase (PPO)
The method of Mayer et al., (1965) was adopted. The reaction mixture consisted of 1.5 ml of 0.1M sodium phosphate buffer (pH 6.5) and 200 µl of the enzyme extract. To start the reaction, 200 µl of 0.01 M catechol was added, and the activity was expressed as changes in absorbance at 495 nm min -1 g -1 fresh weight of tissue.

Phenylalanine ammonia lyase (PAL)
The assay mixture contained 100 µl of enzyme extract; 500 µl of 50 mM Tris HCl (pH 8.8) and 600 µl of 1mM L-phenylalanine were then incubated for 60 min. The reaction was arrested by adding 2 N HCl. Later, 1.5 ml of toluene was added and vortexed for 30 sec. The centrifuged (1000 rpm, 5 min.) toluene fraction containing trans-cinnamic acid was separated. Absorbance of the toluene phase was measured at 290 nm against the blank of toluene. Standard curve was drawn with graded amounts of cinnamic acid in toluene as described earlier.
Enzyme activity was expressed as µmoles of cinnamic acid min -1 g -1 fresh tissue according to Ross and Sederoff, (1992).

β-1, 3-glucanase
Crude enzyme extract of 62.5 µl was added to 62.5 µl of 4 % laminarin and incubated at 40°C for 10 min. The reaction was stopped by adding 375 µl of dinitrosalicylic acid (DNS), and then heated for 5 min. on boiling water bath (DNS prepared by adding 300 ml of 4.5 % NaOH to 880 ml containing 8.8 g of DNS and 22.5g potassium sodium tartarate). The resulting colored solution was diluted with dist. water; vortexed and then absorbance was measured at 500 nm. Crude enzyme extract preparation mixed with laminarin at 0 time incubation served as blank. The enzyme activity was expressed as µg equivalents of glucose min -1 g -1 fresh weight of tissue (Pan et al., 1991).

Peroxidase (PO)
Plants treated with seaweed Sargassum wightii (10%) recorded a maximum induction of peroxidase activity (60.79 µg changes in absorbance min -1 g -1 of Novel Research in Microbiology Journal, 2018  fresh tissue) on the 5 th day after pathogen inoculation. This was followed by plants treated with Bordeaux mixture (1%); 30 and 50 days after pruning (T7), which recorded 59.83 µg changes in absorbance min -1 g -1 of fresh tissue on the 5 th day after pathogen inoculation. Enzyme activity then declined slowly in all the treatments (Fig. 1).

Phenylalanine ammonia lyase (PAL)
PAL activity was found to increase significantly in plants treated with Sargassum wightii (10%) which recorded a maximum induction of PAL activity (5.20 µg changes in absorbance min -1 g -1 of fresh tissue) on the 7 th day, and then it decreased. Spraying of Bordeaux mixture (1%) 30 and 50 days after pruning (T7) recorded a maximum induction on the 7 th day about 5.17 µg changes in absorbance min -1 g -1 of fresh tissue. The enzyme activity was then declined slowly in all the treatments (Fig. 3).

Induction of defence enzymes (season 2) 3.3.1. Peroxidase (PO)
Plants treated with seaweed Sargassum wightii (10%) 30 and 50 days after pruning followed by artificial infestation with U. necator and P. viticola (T1) recorded maximum induction of peroxidase activity (62.48 µg changes in absorbance min -1 g -1 of fresh tissue) on the 5 th day after pathogen inoculation. It was followed by the plants treated with Bordeaux mixture (1%) (Fig. 5).

Polyphenol oxidase (PPO)
Spraying of Sargassum wightii (10%) 30 and 50 days after pruning followed by artificial inoculated with pathogens (T1) recorded a maximum induction of PPO activity (5.93 µg changes in absorbance min -1 g -1 of fresh tissue) on the 5 th day, which then decreased further (Fig. 6).

Phenylalanine ammonia lyase (PAL)
PAL activity was found to increase significantly in plants treated with Sargassum wightii (10%) 30 and 50 days after pruning, which recorded a maximum induction of PAL activity (7.25 µg changes in absorbance min -1 g -1 of fresh tissue) on the 5 th day and there after it decreased. It was followed by the spraying of Bordeaux mixture (1%) which recorded a maximum induction of 7.21 µg changes in absorbance min -1 g -1 of fresh tissue at the 5 th day (Fig. 7).

β-1, 3-glucanase
Among the various treatments, plants treated with seaweed Sargassum wightii (10%) 30 and 50 days after pruning followed by the artificial infestation with pathogens (T1) recorded a maximum induction of β -1,3-glucanase activity (226.2 µg of glucose released min -1 g -1 of fresh tissue) on the 5 th day. This was followed by the plants sprayed of Bordeaux mixture (1%) under the same conditions (T7) which recorded 224.2 µg of glucose released min -1 g -1 of fresh tissue on the 5 th day after pathogen inoculation. The enzyme activity significantly increased at the 5 th day from the pathogen inoculation, and then it declined slowly in all treatments (Fig. 8).
In the present study, grape plants pretreated with Sargassum wightii (brown seaweed) (at 10%) 30 and 50 days after pruning (T1) significantly induced the synthesis and accumulation of β -1,3-glucanase; PO, PPO and PAL enzymes against pathogenic U. necator and P. viticola, when compared to all the other treatments.
Enzymes activities significantly increased from the 5 th day after powdery and downy mildews pathogens infestation and growth formation; and then declined in all treatments. Similar results were recorded by Jayaraj et al., (2008), who reported that Ascophyllum nodosum + chlorothalonil treatment recorded maximum induction of PO, PPO, PAL, β-1,3-glucanase enzymes activities and total phenols; more than the application of A. nodosum alone in carrot against Alternaria leaf spot disease.
In Lizzi et al., (1998) study, application of seaweeds extracts at 0.8 or 1.6 l ha -1 stimulated PO enzyme activity; however, two applications caused an eight-fold increase in PO activity. Moreover, treated leaves accumulated a highest capsidiol (a phytoalexin in peppers) concentration when compared to the control. Garcia-Mina et al., (2004) demonstrated that incorporation of A. nodosum extract into the planting medium caused delayed and reduced incidence of Verticillium wilt of pepper plants. These plants also contained higher levels and had early accumulation of phenolics. In accordance with our findings, Jayaraj et al., (2008); Solanki et al., (2012) revealed that the activity of certain defence related enzymes including; peroxidase, polyphenyl oxidase, phenylalanine ammonia lyase, chitinase and β -1,3-glucanase significantly increased in plants treated with seaweeds.
Increase in PO and PPO enzymes activities at late stage may contribute to cross linking of hydroxyproline rich glycoproteins (HRGPs) lignification's; which act as barriers against pathogen entry. Chen et al., (2000) added that PO generated hydrogen peroxide may function as an antifungal agent in disease resistance. Hydrogen peroxide inhibits pathogens directly or it may generate other free radicals which have antimicrobial activities. PO is a key enzyme in the biosynthesis of lignin and other oxidized phenols (Bruce and West, 1989). Meanwhile, PAL played an important role in the biosynthesis of phenolics and phytoalexins (Daayf et al., 1997). In the current study, an increase in PAL activity indicated the activation of the phenylpropanoid metabolism which leads to the synthesis of phenols as confirmed earlier by Massala et al., (1980). Klessig and Malamy, (1994) later reported that the product of PAL activity is transcinnamic acid; which was an immediate precursor for the biosynthesis of salicylic acid, a signal molecule in systemic acquired resistance (SAR). These enzymes act on the fungal cell wall, resulting in degradation and loss of inner contents of these pathogen cells (Benhamou et al., 1996). Enzymatic degradation of the fungal cell wall may release nonspecific elicitors (Hammerschmidt et al., 1982); which in turn elicits various defence reactions in the plant cells. Ramanathan et al., (2000) reported that fungal cell wall elicitors have been reported to elicit various defense reactions in green gram.

Conclusion
In conclusion, prior treatment and combined application of Sargassum wightii (brown seaweed) (at 10%) 30 and 50 days after pruning (T1) followed by challenge inoculation with U. necator and P. viticola, triggered plant mediated defence mechanism that in turn reduced the powdery and downy mildews incidences in grape.