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209 Advances in Environmental Biology, 5(1): 209-215, 2011 ISSN 1995-0756 This is a refereed journal and all articles are professionally screened and reviewed ORIGINAL ARTICLE Systemic Resistance Induced in Potato Plants Against Potato Virus Y Common Strain (PVYo) by Plant Extracts in Iraq Rakib A. Al-Ani, Mustafa A. Adhab, Sabir N.H. Diwan Plant Protection Department, College of Agriculture, University of Baghdad, IRAQ. Rakib A. Al-Ani, Mustafa A. Adhab, Sabir N.H. Diwan: Systemic Resistance Induced in Potato Plants Against Potato Virus Y Common Strain (PVYo) by Plant Extracts in Iraq ABSTRACT Potato virus Y is one of the most important viruses affecting potato wherever grown in Iraq. The virus is transmitted by sap and by more than 50 species of aphids, in non–persistant manner. The present study was conducted to evaluate the efficacy of frog fruit (Lippia nodiflora L.), Datura (Datura metel L.), and Thuja (Thuja orientalis L.) extracts for inducing systemic resistance against Potato virus Y common strain (PVYO) in potato plants. Air dried of aerial parts powdered was extracted with 70% ethanol (1:3) weight: volume, then concentrated to consistence syrup in water bath at 42<C. The alcoholic extract was used as tuber-dipping before cultivation or foliage spraying after germination. The PVYO multiplication in the plants was followed serologically by Enzyme-linked immunosorbent assay (ELISA). Results showed that dipping the tubers in the extracts, at 1, 2, 3 g/L for 24 hrs., or spraying the foliage by the extracts at the same concentrations exhibited a protection period to the plants against PVYO infection up to one month in the presence of virus source with the vector Myzus persicae Sulz. Means of ELISA readings at 405 nm for samples from treated plants were found to be 0.11 and 0.125 for L. nodiflora extract, 0.13 and 0.126 for D. metel extract, 0.13 and 0.12 for T. orientalis extract for the three concentrations and the two methods of application respectively, compared with 2.52 for untreated plants. No significant differences between the concentrations or between the methods of application on virus multiplication were observed. Key words: Systemic resistance, PVYO, medicinal plant extracts, control. Introduction The potato (Solanum tuberosum L., Solanaceae) is considered among the most nutritive in the world and in Iraq. Potato plants are susceptible to many viruses that caused heavy losses in the yield of both quality and quantity [23,30,34]. Potato virus Y (PVY) the type member of the genus potyvirus, belong to the largest plant virus family Potyviridae [12,31] one of the most wide distributed and the most important viruses infected potato and some other species in the family Solanaceae [12,16,32]. PVY is occurred worldwide wherever potatoes are grown [11]. PVY is easily transmitted by sap and is transmitted by more than 50 species of aphids in non-persistant mode [14,23]. Green peach aphid Myzus persicae Sulz. was found to be the most efficient vector to PVY [18,23]. The aphid can acquire the virus from infected plants in less than 60 seconds and transmit in to a healthy plant in less than 60 seconds [40]. Several strain groups of PVY were identified on the basis of symptoms in naturally infected potatoes S. tuberosum, Nicotiana tabacum, and Physalis floridana. The common (ordinary) strain PVYO induce mild to severe mosaic often associated with crinkling, yellowing, leaf necrosis and leaf drop on potato, vein clearing and mottle in tobacco. The tobacco veinal necrosis group (PVYN) produces milder mottle than PVYO on potato and severe Corresponding Author Mustafa A. Adhab, Plant Protection Department, College of Agriculture, University of Baghdad, IRAQ. Email: [email protected] Adv. Environ. Biol., 5(1): 209-215, 2011 necrosis on leaf veins and petiols in N. tabaccum. This strain (PVYN) was identified in previous study in our laboratory in Iraq [6]. The PVYC isolates induce stipple streak symptoms on some potato cultivars and is not transmissible by aphids [12,33]. The objective of this study is to characterize the common strain of PVY (PVYO) prevailing among isolates collected from different potato production regions in Abu-Ghraib – Iraq on the basis of serology and bioassay, and to evaluate the possibility of inducing systemic resistance in potato plants against this strain by some medicinal plants extracts. Materials and Methods Virus isolates: potato leaves from plants showing mottling, leaf necrosis lesions, rugosity, yellowing, and stunting suspected to be of Potato virus Y (PVY) infection were collected from potato fields in Abu-Ghraib area (West of Baghdad). The virus was identified serologically by polyclonal antibodies to PVY provided by Bioreba Company (Switzerland) as immunostrip. The isolate that gives positive reaction were purified by single-spot on Chenopodium amaranticolor [5], and propagated in Nicotiana tabacum cv. Samsun plants. Virus strain characterization: leaves surfaces of test plants consist of Chenopodium amaranticolor, N. tabacum cv. Samsun, Capsicum annum, Datura stramonium, Lycopersicon esculentum, Physalis floridana and Solanum tuberosum were slightly dusted with carborandum 600 mesh and mechanically inoculated by sap from systemically PVY-infected N. tabacum cv. Samsun. The sap was prepared by grinding leaves showing virus symptoms in 0.01 M phosphate buffer containing 0.2% Diethyldithiocarbamate (DIECA) pH 7.2 at 1g/4ml. Virus transmission by vector: Apterous Myzus persicae were collected from Apricot, and placed on PVY-infected N. tabacum cv. Samsun for 15 min. to acquire the virus. Viruliferous aphids were then transferred to healthy plants (10 aphids/plant) for 30 min. for inoculation. Also, healthy plants were exposed to non-viruliferous aphids as control. The plants were maintained in an insect-free glasshouse at 25±2 <C for 4 weeks. Virus purification: Leaves from N. tabacum cv. Samsun showing virus symptoms (100g) were grounded in a warring blender with 0.5 M phosphate buffer (Na2HPO4) pH 7.0 containing 1% 2-mercaptoethanol 1:1 (g/ml). The extract was passed through 2 layers of cheese cloth, 210 and n-butanol was added to the filtrate at 7%. The mixture was agitated for one hour at 4<C and centrifuged at 5000 rpm for 45 minutes in cooling centrifuge. Polyethylene glycol (PEG) 6000 MW and NaCl were added at 4% and 2% respectively to the supernatant with agitation. The solution was maintained at 4<C for 24 hours and submitted to a centrifugation at 5000 rpm for 45 min. The pellet was solved in 1 ml of 0.01M phosphate buffer pH 7.2 and centrifuged at 5000 rpm for 20 min. The supernatant was loaded on the surface of sucrose gradient 10-50% with reverse gradient of Ammonium sulfate 50-10% in gradient tubes. The tubes were kept at 4<C for 24 hours and centrifuged at 5000 rpm in swinging rotor for 60 min in cooling centrifuge. Virus bands were collected from the gradient and dialyzed against 0.01 M phosphate buffer pH 7.2 for 24 hour with agitation. Serological identification ELISA: Leaves of infected potato plants were grounded in phosphate buffer saline (PBS) pH 7.0 at 1 g/10 ml. The homogenate was filtered through double layer of cheese cloth followed by a centrifugation at 5000 rpm for 5 min. and tested by DAS-ELISA using polyclonal antibodies as described by Clark and Adams [10]. The absorbance of ELISA reactions were read at 405 nm one hour after adding the substrate (P-nitrophenol phosphate) in ELISA-reader. Sample from healthy plants were treated in the same way and used as control. Absorbance values twice the average of uninfected plants was considered positive. Immunostrip test: Polyclonal antibodies to PVY provided by Bioreba Co. (Switzerland) as immunostrip were used. Leaves samples of 0.15 g from plants showing virus symptoms were homogenized in plastic sacs containing extraction buffer by pestle. The end of the strip was dipped in the extract to 1 cm for 15 min[1]. An extract from healthy plants was treated in the same way as control. Induced systemic resistance against PVYO: Aerial parts of medicinal plants, Thuja orientalis, Datura metel, and Lippia nodiflora were collected from the gardens of the College of Agriculture, University of Baghdad, IRAQ. Samples of air dried were ground with a mortar and pestle and 100 g of the powder obtained was added to 300 ml of ethanol 80%, in a 1000 ml flask. The mixture was agitated for 24 h at room temperature and passed through Adv. Environ. Biol., 5(1): 209-215, 2011 filter paper (whatman 2) in Buchner funnel with vacuum. The filtrate was concentrated at 42 EC in a water bath and stored at -20 EC. Three concentrations of each extract, 1, 2, 3 g/L in distilled water amended with 0.1% of tween-20 were used. Potato tubers were soaked in each concentration for 24 h and sown in plastic pots (25 * 18 * 18 cm) containing soil mix and the plants were inoculated by PVYO at 3 leaves stage. Also, potato plants generated from healthy tubers were sprayed by the extracts and inoculated by PVYO after 48 h of application. The virus in the plants was serologically detected by ELISA. Results and Discussion Virus detection: Symptoms: Leaf mottling often associated with chlorotic spots appeared on the lower leaves of potato plants mechanically inoculated by sap from leaves of N. tabacum cv. Samsun exhibited virus symptoms or transmitted to potato plants by aphids Myzus persicae. These symptoms were developed to necrosis on the upper leaves began by the main veins then to secondary ones and to leaf petiol led finally to death and leaf drop (fig. 1). Similar results were previously obtained by many workers on PVY [9,22,25]. Immunostrip test: A band specific to PVY appeared on the strip containing polyclonal antibodies to PVY treated with extract from potato plants showing virus-symptoms (fig. 2). This band is absent on the strip treated with extract from healthy plants which indicates that the virus under identification is PVY[1]. 211 Das-elisa: The isolates showing symptoms similar to those exhibited by the ordinary strain were reacted positively with monoclonal antibodies specific to PVYO purchased from sigma chemical company USA. The absorbance of ELISA reactions at 405 nm were found to be 3.11, 3.04, and 2.07 for extracts from N. tabacum cv. Samsun, Physalis floridana, and S. tuberosum showing virus symptoms respectively compared with 0.059 for extract from healthy plants. The PVY-strain under identification in this study is considered as PVY-common strain (PVYO), because it induced typical mild mosaic on the leaves of N. tabacum cv. Samsun without any indication of veinal necrosis, while PVYN cause severe veinal necrosis on this plant [13], and reacted positively with monoclonal antibodies PVYO. It was found that the present strain is easily transmissible by aphids M. persicae in non persistant manner, while PVYC is not transmissible by aphids [12,33]. Induced systemic resistance against PVYO: Lippia nodiflora extract: Results obtained showed that the concentrations 1, 2, 3 g/l of Lippia extract were highly effective in inducing systemic resistance in potato plants against PVYO infection. The means of absorbance values of ELISA reactions at 405 nm were found to be 0.11 for extracts from plants generated from tubers treated with the extract, compared with 2.55 for extract from PVY-infected plants (control). Similar results were obtained from plants sprayed by the extracts with absorbance values 0.125 compared with 2.55 for the control (virus infected plants) (Table 1). It was shown in previous work that the alcoholic extract of Lippia contain phenolic compounds; flavonoids and terpenoids such Lippacin acts as antioxidants [8,29]. Datura metel extract: Virus strain characterization: Symptoms: The virus induced systemic mild mottling on N. tabacum cv. Samsun, Capsicum annum, and Lycopersicon esculentum mechanically inoculated by sap from PVY-infected N. tabacum cv. Samsun leaves. Chlorotic local lesions were developed on the inoculated leaves of Ch. amaranticolor, vein clearing associated with mottle symptoms induced on the leaves of Physalis floridana upon inoculation by extract from virus infected N. tabacum cv. Samsun (fig. 3). No symptoms were observed on sap inoculated Datura stramonium and no virus was detected from this plant. Similar results were previously reported concerning PVYO strain [9,11,18,25]. It has been found that the alcoholic extract of D. metel induced systemic resistance in treated potato plants. No virus was detected in the treated plants for a period up to one month of PVY inoculation. The means of ELISA readings at 405 nm were 0.13 for samples from plants generated from treated tubers, 0.126 for samples from plants sprayed by the extracts compared with 2.53 for the control (Table 1). It has been reported that D. metel contain alkaloids, tannins, flavonoids, and carbohydrates. The medical effect of this plant is often attributed to its content scopolamine (an alkaloid) [38]. Thuja orientalis extract: The treatment of potato tubers with the alcoholic Adv. Environ. Biol., 5(1): 209-215, 2011 extract of Thuja at 1, 2, 3 g/L induced systemic resistance in the plant against PVYO for the same period mentioned for Lippia and Datura extract. The means of absorbance values at 405 nm were 0.13 for samples from plants generated from treated tubers, 0.12 for samples from plants sprayed by the extracts compared with 2.52 for the control (Table 1). The effect of Thuja extracts may be due to the presence of tannins as shown by previous study in our laboratory [2,4]. The results obtained showed that the three extracts from Lippia, Datura, and Thuja, used in this study induced resistance against PVY. High significant differences were found in ELISA readings between the plants treated and non-treated as control. No significant differences were observed between the extracts or methods of application. These results indicate that the use of substances derived from higher plant as biological control against viruses appears to be promising. 212 Discussion: The symptoms manifested on, potato, Nicotiana tabacum cv. Samsun, serological characteristic and transmissibility by Myzus persicae, indicates that the current isolate of PVY represent the ordinary strain (PVYO). This isolate was found to be more prevalent in all the fields submitted to the study, and causing heavy losses in potato yields. The prevalence of PVYO can be attributed to PVY asymptomatic hosts, identified in previous studies in our department [20], which may acts as virus reservoir to hasten virus spread by the aphids in the same field and to those in the vicinity. Fig. 1: Symptoms of necrosis on blades (A), leaf dropping and death (B) on PVY-infected plants, compared with healthy plant (C). Fig. 2: Polyclonal antibodies to PVY on immunostrip, reacted with extracts from potato plant showing virus-symptoms (arrow) indicates the presence of PVY in the extract (A), compared with a healthy plant (control) (B). Fig. 3: Symptoms of vein clearing and mild mottling on N. tabacum cv. Samsun (A), chlorotic local lesions on Ch. amaranticolor, (B) and mild mottling on Capsicum annum (C) sap inoculated by PVY. Adv. Environ. Biol., 5(1): 209-215, 2011 213 Table 1: Elisa readings of samples from virus-infected plants generated from infected tubers and treated with extracts with two application methods Date Lippia nodiflora Datura metel Thuja oreintalis --------------------------------------------------- ------------------------------------------------ -----------------------------------------------Tuber dipped Foliage spray Control Tuber dipped Foliage spray Control Tuber dipped Foliage spray Control 26 March 0.9 0.11 2.56 0.12 0.13 2.53 0.11 0.11 2.48 29 March 0.11 0.12 2.57 0.13 0.12 2.55 0.13 0.11 2.51 1 April 0.12 0.13 2.53 0.13 0.13 2.54 0.14 0.11 2.53 3 April 0.12 0.13 2.56 0.13 0.12 2.56 0.13 0.12 2.55 6 April 0.10 0.13 2.55 0.12 0.12 2.54 0.14 0.12 2.53 9 April 0.10 0.12 2.55 0.13 0.14 2.53 0.13 0.12 2.55 12 April 0.11 0.12 2.56 0.12 0.12 2.53 0.13 0.12 2.52 15 April 0.11 0.13 2.57 0.13 0.14 2.51 0.12 0.12 2.51 18 April 0.12 0.13 2.55 0.13 0.13 2.53 0.12 0.12 2.53 21 April 0.11 0.13 2.56 0.14 0.13 2.55 0.14 0.12 2.55 24 April 0.11 0.13 2.55 0.13 0.11 2.53 0.15 0.12 2.51 26 April 0.12 0.12 2.56 0.13 0.13 2.53 0.12 0.13 2.51 Mean 0.11 0.125 2.55 0.13 0.126 2.53 0.13 0.12 2.52 Each value in the table represents the mean of 3 replicates. The values in the table represent the absorbance at 405 nm. Several strategies have been adopted to manage the virus diseases on potato through using insecticides without success especially for viruses transmitted by insects in non-persistant manner [30]. Recently the efforts were directed toward searching for substances more effective including chemicals, biological or natural plants products that can induce resistance in the treated plants against viruses [21]. This idea came from fact that when a plant infected by a pathogen, the reaction of the plant toward this pathogen is developing systemic resistance to subsequent infection [19,26]. These observation indicates that plants possess inducible defense system against pathogen attack, and this system can be triggered prior infection by certain elements may be biotic or abiotic [28,35,37]. The induce resistance render the plant resist to broad spectrum of pathogens included virus, bacteria, and fungi [26,39]. This study was focused on the use of extracts from some medicinal plants (Lippia, Datura, Thuja) in attempt to induce resistance against PVYO. The idea came from indication that these plants contain phenolic and alkaloids, substance had inhibition effects towards different pathogens [1,28,7,39]. These substances in addition to proteinacious compound present in the extracts may acts directly on the virus or indirectly through inducing host-resistance genes leading to produce proteins have the capacity to inhibit virus multiplication (antiviral proteins). The results obtained reveal effectively the capacity of these substances to induce systemic resistance in potato plants against PVY as was shown by DASELISA using polyclonal antibodies to PVYO. This resistance may enhance and activate by subsequent application of the extracts. Several previous studies reported that extracts from, Phytolacca americana, Mirabilis jalaba, Eucalyptus camalduliensis, Glycyrriza glabra, Euphorbia tinctoria, Chenopodium spp., and Datura spp. contains substance can induce systemic resistance in treated plants [3,7,15,36]. Some other studies found that the onset of induced resistance correlates with the accumulation of Salisylic acid (SA) and pathogenesis related proteins (PR-Proteins) [27]. Exogenous application of SA found to induce SAR and activates the same set of PR-Proteins genes in several plant species [24,17]. This mechanism is not excluded in this study and suggests that these proteins may play an essential role in the defense capacity of induced tissue. The use of substances derived from plants as biological elements to induce systemic resistance in the plants against virus appear to be promising in virus management strategy. 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