62 ACTA BOT. CROAT. 77 (1), 2018 Acta Bot. Croat. 77 (1), 62–69, 2018 CODEN: ABCRA 25 DOI: 10.2478/botcro-2018-0005 ISSN 0365-0588 eISSN 1847-8476 Effects of carrageenan as elicitor to stimulate defense responses of basil against Cuscuta campestris Yunck Effat Ahmadi Mousavi *1, Khosrow Manochehri Kalantari1, Fatemeh Nasibi1, Hakimeh Oloumi2 1 Biology Department, Faculty of Science, Shahid Bahonar University of Kerman, Kerman, Iran 2 Ecology Department, Institute of Science and High Technology and Environmental Sciences, Graduate University of Advanced Technology, Kerman, Iran Abstract – Cuscuta campestris is a holostemparasitic plant that obtains its resources from its hosts. Sweet basil is an important commercial plant, widely cultivated in many countries. It is a common host for C. campestris. Generally, C. campestris has negative effect on the growth of infected plants and its infestation is difficult to control. Therefore, en- vironmental friendly control of C. campestris seems to be useful. In this work, the relationship between C. campestris and its host, sweet basil, and effects of κ-carrageenan on protection against C. campestris and suppression of its inva- sion were studied. Basil was sprayed with a solution of carrageenan at a final concentration of 1 g L–1, once a week, 3 times in total. Infection of basil with C. campestris was performed 2 days after the last carrageenan treatment and the plants were collected two weeks after C. campestris attachment. In this study, phenylalanine ammonia-lyase activity (PAL), phenolic, flavonoids and antioxidant content increased remarkably in the basil plants parasitized with C. campestris, and therefore it seems that the parasitic plant induced a defense response in the host plants. Treatment with carrageenan significantly increased shoot length and leaf area of basil and decreased C. campestris infestation by about 26%. Carrageenan treatment caused a significant increase in PAL activity, phenols, antioxidant and lignin con- tent in basil. Thus, the present observation suggested the phenylpropanoid pathway was activated and defense re- sponses were stimulated. Our results showed that carrageenan spraying induced beneficial effects in plants, corre- sponding to growth stimulation and defense compound synthesis. Thus carrageenan treatment is recommended as a natural biostimulator for the protection of plants against C. campestris. Keywords: carrageenan, defense against parasites, field dodder, Ocimum basilicum * Corresponding author, e-mail: effatmousavi@yahoo.com Introduction Among plants with medicinal value, plants of the genus Ocimum (Lamiaceae) are very important for their therapeu- tic potentials (Ramasubramania 2012). Sweet basil (Ocimum basilicum L.) is an important species of the genus Ocimum that grows in different parts of the world (Tewari et al. 2012). Sweet basil is an annual plant native to Asia that is cultivated as a field crop in the Mediterranean countries and is widely used in food (for flavour) and in oral care. The essential oil of the plant is additionally used in perfumery (Tewari et al. 2012). Leaves and flowers of basil are traditionally used as digestive, antispasmodic, stomachic, carminative, galacta- gogue, aromatic and tonic agents (Gülçin et al. 2007). Anti- microbial and antiviral activities of this plant have also been reported (Chiang et al. 2005). Sweet basil is a common host for the stem-parasitic plant Cuscuta campestris Yunck. with the common name of field dodder. This parasite is one of the major constraints that limit the productivity of basil (Behbahani 2014) and there- fore the parasitic interaction of C. campestris with its hosts seems interesting. C. campestris is a well known member of the Cuscuta genus, of which it is the most widespread spe- cies. It is a holostemparasitic plant without leaves and roots, but it can produce absorptive haustoria that provide physical and physiological bridges between the parasite and its host (Kushan et al. 2006). It can infect various host species (Daw- son et al. 1994), self-parasitize and hyper-parasitize (Liao et al. 2005). Upon successful creation of vascular connections with the host, Cuscuta becomes a strong sink, withdrawing water, ions, sugars, amino acids and other nutrients. It ob- tains its resources entirely from its host plants (Dawson et al. 1994). Cuscuta-infested plants gradually become weak, resulting in decline in growth and yield (Aly 2013). In addi- tion to its impact on ornamentals, native plants and forage crops, it has been reported that Cuscuta can suppress or kill https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&cad=rja&uact=8&sqi=2&ved=0ahUKEwidleOL7onOAhVkOMAKHQObDJAQFggcMAA&url=https%3A%2F%2Fen.wikipedia.org%2Fwiki%2FPhenylalanine_ammonia-lyase&usg=AFQjCNFN3bYpkIs3RtL9bwITQbgkm6Ydeg&sig2=4s6hTXdxWzZQ53HPj4fgGA&bvm=bv.127984354,d.bGg mailto:effatmousavi@yahoo.com CARRAGEENAN STIMULATES DEFENSE OF BASIL AGAINST CUSCUTA ACTA BOT. CROAT. 77 (1), 2018 63 vegetables and weeds (Fathoulla and Duhoky 2008). Con- trol of Cuscuta spp. is extremely difficult, thus it causes huge losses in agriculture (Shen et al. 2007). Natural extracts from plants and other organism for pro- tecting crops from pathogens and pests have recently been reported. They protect plants without negatively affecting the growth and yield (Kapooria et al. 2007, Aly 2013).An ef- fective method of achieving crop protection is inducing re- sistance by activating natural protection system with elici- tors that are environmentally safe. It has been reported that this strategy is effective and was used against a vast range of pathogens, pests and parasites, including fungi, bacteria, vi- ruses, insects and herbivores (Halim et al. 2004), but there is no report of such method being used for the control of parasitic plants, especially C. campestris. The major elicitors described in literature are varied in nature and include oligo- saccharides, polysaccharides, glycopeptides, lipids, peptides and proteins and are derived from cell wall, culture filtrate and cytoplasm of different parasitic and nonparasitic plant pathogens (Rao et al. 1996, Halim et al. 2004). In most cas- es natural elicitor activity is related with the polysaccharide fraction of the varied preparations. Other elicitors derived from plants can be linear or ramified oligosaccharides and polysaccharides (Creelman and Mullet 1997). It is well doc- umented that polysaccharides purified from algae as well as derived oligosaccharides have the capacity to trigger plant defense responses (Potin et al. 1999, Khan et al. 2009). Liquid extracts obtained from seaweeds have been re- ported to stimulate the growth of plants, improve resistance to diseases and pests, amend resistance to abiotic stresses (e.g., temperature extremes, salinity, drought and heavy met- al stress), increase yield and quality, enhance mineral uptake from soil and antioxidant properties (Zhang et al. 2003, Bi et al. 2008, Khan et al. 2009, Craigie 2011, Naeem et al. 2012, Hashmi et al. 2012). The best-characterized seaweed elici- tors that have been reported are laminarin (from the brown seaweeds Laminaria digitata) and carrageenans (from spe- cies of red seaweeds of the class Rhodophyceae) that have the potential to activate disease resistance in plants and an- imals (Gonzàlez et al. 2013b). Carrageenans are the major polysaccharides existing in many red macroalgae (Jatinder et al. 2011). These gel-forming polysaccharides have a lin- ear structure of D-galactose residues joined with alternat- ing α-(1,3) and ß-(1,4) linkages which are substituted for by one (kappa (κ)-carrageenan), two (iota (ɩ)-carrageenan), or three (lambda (λ)-carrageenan) ester-sulphonic groups per di-galactose repeating unit (Mercier et al. 2001, Gonzàlez et al. 2013a). Mercier et al. (2001) reported that ĸ-carrageenan elicited numerous plant defense responses, probably through the effect of its high sulphate content, and induced signaling and defense gene expression in tobacco leaves. Since carra- geenans have been shown to activate plant defense respons- es against pathogens (Bi et al. 2011, Shukla et al. 2016), it is plausible that carrageenans might stimulate resistance in plants to parasitic plants like Cuscuta. However, the effect of carrageenans on plant resistance in the face of C. camp- estris is not known. Most elicitors derived from plants or al- gae stimulate plant defense responses, mainly through the activation of the phenylpropanoid pathway (La Caméra et al. 2004). The stimulation of the phenylpropanoid pathway produces phenolic compounds such as phytoalexins, lignins and salicylic acid, with potential defensive activities (Lattan- zio et al. 2006). Thus, the purpose of this study was to inves- tigate how carrageenan acts as elicitor to stimulate growth and defense responses to C. campestris infestation in sweet basil plants. Materials and methods Plant material and treatments Sweet basil (Ocimum basilicum L.) seeds were steril- ized by immersing them in 0.1% (w/v) sodium hypochlo- rite solution for 5 min and then washed extensively with dis- tilled water and finally rinsed with sterile water. Pots (14 cm diameter × 12 cm height) were filled with a homogenous mixture of soil and organic manure (4:1). Physicochemical characteristics of the soil were: texture – sandy loam, pH (1:2) (soil:water, v:v) 7.2 and electrical conductivity (1:2) (soil:water, v:v) 0.43 dS m−1 (Hanlon 2015). Basil and Cus- cuta campestris grow best under conditions of high light, moisture and temperature, these conditions are provided in a natural environment, and therefore the pot experiment was conducted in natural conditions on June, 2015. All experi- ments were repeated twice. The repetition of cultivation and experiments was conducted in the following year (2016). Ba- sil seeds were planted at 2 cm depth in the center of each pot. Most seedlings emerged above the soil surface 4 days after planting and were kept then thinned to 8 plants per pot and the pots were watered when required. Pure κ-carrageenan (Sigma–Aldrich, USA) was dissolved in hot deionized water. A concentration of carrageenan solu- tion (1 g L−1) was finally prepared for foliar spray treatments. The first carrageenan treatments were applied to the basil plants 14 days after sowing, when the plants were at the 2 true leaf stages. Carrageenan solution was sprayed on the up- per and lower surface of basil leaves and on the stem, while the 2nd and 3rd sprays were done one and two weeks after the first treatment, respectively. Control plants were sprayed with deionized water. C. campestris is simply transferable to host plants by veg- etative propagation. Significant changes in the amount of metabolites at the apical region of filament indicate that this region is most active during haustorium development for parasitization (Furuhashi et al. 2012). Placement of actively growing Cuscuta spp. stems on host plants usually results in the formation of haustoria and thus causes a new C. camp- estris plant (Hong et al. 2011). Therefore, threadlike stems (120 mm from tip) of C. campestris without haustorium were separated and placed on the top of 30-day-old basil stem. The basil plants were treated two days in advance with car- rageenan before C. campestris inoculation. The C. campes- tris was allowed to attach to plants. Usually, C. campestris stem attaches to basil shoot one day after inoculation. The threadlike stem of C. campestris and parasitized basil leaves MOUSAVI E. A., MANOCHEHRI KALANTARI K., NASIBI F., OLOUMI H. 64 ACTA BOT. CROAT. 77 (1), 2018 (third row of opposite leaves counted from the cotyledon node) were collected 2 weeks after C. campestris attachment, the carrageenan treated and un-treated healthy basil leaves (third row of opposite leaves counted from cotyledon) were harvested 44 days after sowing. Performance of the plant was assessed in terms of growth attributes (infestation percent- age, the plant height and leaf area), and biochemical param- eters (with three biological replications). Infestation percentage of C. campestris For analysis of defense against C. campestris invasion and measuring of infestation of basil plants by C. campestris, the percentage of C. campestris tight coupling (its haustorium penetration) to basil shoot was determined 14 days after the infection. The experiments comprised twenty replications and were repeated twice. Plant growth parameters The height of a plant is the vertical distance from the soil at its base to the highest point reached with all parts in their natural position. The height of the basil plant in its natural position was measured. The experiments comprised twenty replications and were repeated. The methods for leaf area measurement include weighing, copying the shape of the 5th leaf counted from cotyledon on a fragment of paper and weighing the copy. So leaf area is estimated by the fol- lowing equation: LA = W / c, where LA is the leaf area, W is the weight of the paper and c is the coefficient of the pa- per (weight of unit area). The experiments comprised twenty replications and were repeated twice. Phenolic content analysis In order to measure total phenolic content, fifty milli- grams of plant material of each sample (the 5th basil leaf counted from the cotyledon node and threadlike stem of C. campestris) were ground and dissolved in 1 mL of 80% eth- anol, using pestle and mortar . The homogenate was kept at 25° C for 24 h in the dark. Then it was brought to a volume of 5 mL with ethanol and centrifuged at 2000 g for 10 min. Soluble phenolic content was analyzed by the method of Gao et al. (2000), using the Folin-Ciocalteu reagent. One hundred µL of extract was mixed with Folin-Ciocalteu reagent (200 µL) and distilled water (2 mL) and incubated at room tem- perature for 3 min. After incubation, a sample of aqueous so- dium carbonate (20% w/w, 1 mL) was added to the mixture. The phenolics were measured after one hour of incubation at room temperature. The absorbance of the resulting blue color was determined at 765 nm. Gallic acid was used as a standard and the results were expressed in milligrams of gal- lic acid equivalent per gram fresh weight of leaf. The experi- ments comprised three replications and were repeated twice. In order to measure total flavonoids, nine hundred mil- ligrams of plant material of each sample (the 5th basil leaf counted from the cotyledon node and the threadlike stem of C. campestris) were ground and dissolved in 3 mL of 80% methanol, using mortar and pestle. The homogenate was kept at 25 °C in the dark. Then it was brought to a volume of 3 mL with methanol and centrifuged at 2000 g for 10 min. Total flavonoid content was determined according to the al- uminium chloride colorimetric method (Chang et al. 2002). Two mL of extracts (0.3 g mL–1) in methanol were mixed with 0.1 mL of 10% aluminium chloride hexahydrate, 0.1 mL of potassium acetate (1 M) and 2.8 mL of distilled wa- ter. After 40 min incubation at room temperature, the absor- bance of the reaction solution was determined spectropho- tometrically at 415 nm. Quercetin was chosen as a standard (standard concentration range: 12 to 200 mg L–1) and the results were expressed in milligrams of quercetin equivalent per gram fresh weight of leaf. Anthocyanin content was determined according to a modified Wagner (1979) method. The plant material of each sample (0.1 g of the 5th basil leaf counted from the cotyledon node and the threadlike stem of C. campestris) was crushed in 10 mL acidified methanol [methanol:HCl (99:1, v:v)]. The tissues were soaked and incubated at room temperature for 24 h in the dark. The extracts were then centrifuged at 4000 g for 10 min at 25 °C. The absorption of the supernatant was read spectrophotometrically at 550 nm. To calculate the amount of anthocyanins, the extinction coefficient 33000 L mol–1 cm–1 was used and it was expressed as μmol per gram fresh weight of leaf. Antioxidant activity The antioxidant activity of plant material of each sample was assessed by a modified Girennavar method (2007), using the stable free radical 2, 2-diphenyl-1-picrylhydrazyl radical (DPPH) which forms a violet solution and reacts with anti- oxidants by losing color. Ten µL of each extract was mixed with 100 µM DPPH in methanol in a final volume of 1 mL. The changes were monitored over 20 min. A control was prepared as described above without samples or standards. Methanol was used for the baseline correction. The chang- es in the absorbance of all the samples and standards were measured at 517 nm. Radical scavenging activity was ex- pressed as the inhibition percentage and was calculated using the following formula: percent of radical scavenging activ- ity = (control optical density-sample optical density/control optical density) × 100. Ascorbic acid was chosen as a stan- dard antioxidant and extract activity is expressed in µmol of ascorbic acid equivalents per gram fresh weight of leaf. Lignin content analysis Protein and other UV-absorbing materials removal pro- tocols were essential to avoid the measurement of these con- stituents together with lignin at 280 nm. Five hundred mil- ligrams of the plant material of each sample (the same age stem of basils based on leaf number and threadlike stem of the dodders) were homogenized in 2 mL water using a pestle and mortar and then transferred into a screwcap centrifuge tube and centrifuged at 1000 g for 10 min and the superna- tant was removed. The pellet was washed two times with ethanol, then the pellet was soaked and incubated at room https://www.google.com/search?q=quercetin&start=0&spell=1&biw=1366&bih=610 https://www.google.com/search?q=quercetin&start=0&spell=1&biw=1366&bih=610 CARRAGEENAN STIMULATES DEFENSE OF BASIL AGAINST CUSCUTA ACTA BOT. CROAT. 77 (1), 2018 65 temperature for 24 h in methanol: chloroform (1:2), the su- pernatant was removed and rinsed with acetone. The pellet was dried in an oven (60 °C, 24 h) and cooled in a vacuum desiccator. The dry matter obtained was defined as the pro- tein-free cell wall fraction (Hatfield et al. 1999). Lignin content was determined according to a modified Iiyama and Wallis method (1990). Protein-free cell wall sam- ple (20 mg) was placed into a screwcap centrifuge tube con- taining 0.5 mL of 25% acetyl bromide (v/v in glacial acetic acid) and incubated at 70 °C for 30 min. Samples also con- tained 100 µL of perchloric acid to aid in the total dissolution of the wall material. After complete digestion, the sample was quickly cooled in an ice bath, and mixed with 0.9 mL of 2 M NaOH and 6 mL of glacial acetic acid sufficient for com- plete solubilization of the lignin extract, and then samples were diluted to 25 mL with acetic acid. After centrifugation at 1000 g for 5 min, the absorbance of the supernatant was measured at 280 nm. Guaiacol was used as a standard and the results were expressed in milligrams of guaiacol equiva- lent per gram fresh weight of basil stem. Phenylalanine ammonia lyase (EC 4.3.1.5) enzyme assay Three hundred milligrams of plant material of each sample (the 5th basil leaf counted from the cotyledon and threadlike stem of C. campestris) was homogenized in an ice cold mortar using 50 mM Tris-HCl buffer (pH=8.8) contain- ing 10 mM 2-β-mercaptoethanol, 1 mM ethylenediaminetet- raacetic acid (EDTA) and 2.5% polyvinylpyrrolidone (PVP). The mixture was centrifuged at 20000 g for 20 min and the clear supernatant was desalted in aliquots using an Amicon Ultra-15 Centrifugal Filter Units with Ultracel-50 membrane (Merck Millipore, Germany) and assayed for PAL activity. PAL activity was determined according to the method of Şirin et al. (2016). The enzyme reaction mixture contained 400 µL of reaction buffer (100 mM Tris-HCl, pH 8.8) and 200 µL of substrate (40 mM L-phenylalanine, 100 mM Tris-HCl, pH 8.8) and a 200 µL aliquot of the sample filtrate (or 200 µL of deionised water used as a blank). The reaction was carried out at 37 °C for 30 min and terminated by the addition of 50 μL of 4 M HCl, and the cinnamic acid concentration was measured spectrophotometrically by the absorbance at 290 nm. One unit of PAL activity is equal to 1 μmol of cinnamic acid produced per min. The enzyme activity was expressed in U per milligram protein. Protein content was determined according to the method of Bradford (1976) using bovine se- rum albumin as standard. Data analysis The experiments were performed by a factorial arrange- ment, based on complete randomized design. Obtained data were the average of the replications and two repetitions of each experiment, because there was no interaction. Results were determined using analysis of variance (ANOVA) via statistical analysis software (SAS, Version 9.4, SAS Institute Inc., Cary, NC, USA). The univariate procedure of SAS was used to test for normality of residuals. Each column value in figures represents mean ± standard deviation (SD). Means were compared using Fisher’s protected least significant dif- ferences (LSD) test. Differences at P ≤ 0.05 were considered to be significant. Results In the present study, we observed that the infestation per- centage of C. campestris in basil that was not treated with carrageenan was 60.48%, but in carrageenan-treated ba- sil plants a significantly lower attachment of C. campestris, 33.93%, was recorded. These findings showed that the infes- tation of basil plant treated with carrageenan by C. campes- tris decreased by about 26% (Tab. 1), and therefore spraying with carrageenan significantly increased the resistance of ba- sil plants to C. campestris invasion. C. campestris attachment occurred along the shoot of control plants (Fig. 1), but in basils which were sprayed with carrageenan, it was observed only at shoot apical part with newly formed organs (stem, leaves and petioles). Tab. 1. Effect of foliar application of carrageenan (1 g L–1) and Cuscuta campestris invasion on the growth parameters of Ocimum basilicum (basil) and percentage of Cuscuta infestation. B0 – basil sprayed with water (control); B1 – basil sprayed with carrageenan; B0+D – basil parasitized by C. campestris; B1+D – basil sprayed with carrageenan and parasitized by C. campestris. Values are mean ± SD of twenty replications. The different letters in the same col- umn indicate significant difference at P ≤ 0.05. Treatments Shoot length (cm) Leaf area (cm2) Cuscuta infestation (%) B0 22.05b±1.56 5.91b±0.43 – B1 24.325a±0.99 8.43a±0.95 – B0+D 13.625d±0.54 4.06c±0.73 60.48a±2.69 B1+D 16.375c±0.60 6.09b±0.71 33.93b±2.23 Fig. 1. Infestation of basil plants Ocimum basilicum by Cuscuta campestris: a) control and b) carrageenan treated basil plant. MOUSAVI E. A., MANOCHEHRI KALANTARI K., NASIBI F., OLOUMI H. 66 ACTA BOT. CROAT. 77 (1), 2018 Infestation of C. campestris significantly decreased the shoot length and leaf area of basil, and the carrageenan treat- ment significantly increased the growth parameters in com- parison with the carrageenan-untreated set of basil (Tab. 1). Therefore, it seems that the foliar application of carrageenan significantly alleviated the negative effect of the parasite (C. campestris) on the growth of infested host plants (Tab. 1). The carrageenan treatment alone caused a significant in- crease of PAL activity and phenolic and antioxidant content of basil. Moreover, basil plants parasitized by C. campestris showed significantly higher PAL activity, phenolic, flavo- noids and antioxidant content (Fig. 2). The lignin content of the carrageenan treated-basil did not show significant changes compared to the control set but the carrageenan treatment significantly increased the lignin content of basil plants that were parasitized by C. campes- tris (Fig. 2). In this study, we did not observe any significant change in phenolics, flavonoids, antioxidant, anthocyanin and lignin content of the C. campestris attached to the control group of basil plants and C. campestris from basil hosts treated with carrageenan (Fig. 2). Discussion Besides microbial pathogens and herbivorous arthro- pods, plants can also be parasitized by other plants. Cus- cuta species is recognized to cause serious economic losses in crop plants. In this study, infestation by C. campestris sig- nificantly decreased the shoot length and leaf area of basil. Generally, with regards to the infected plant species, Cuscuta invasion has been reported severely to affect the growth and reproduction of its host (Lanini and Kogan 2005). Some re- searchers have shown that parasitic flowering plants of the genus Cuscuta, most especially C. campestris Yunck., C. australis R. Br. and C. chinensis Lam., can significantly re- duce their host’s growth (Zan et al. 2003, Lanini and Kogan 2005). Also Farah and Al-Abdulsalam (2004) showed that Fig. 2. Effect of foliar application of carrageenan and Cuscuta campestris invasion on the biochemical parameters of basil leaves and Cus- cuta stems: a) phenol content, b) total flavonoid content c) antioxidant content, d) phenylalanine ammonia-lyase activity (PAL), e) total anthocyanin content, f ) lignin content. Values represent mean ± standard deviation (n=3). Different letters above bars indicate significant difference at P ≤ 0.05. B – basil; B+D – basil parasitized by C. campestris; D – C. campestris CARRAGEENAN STIMULATES DEFENSE OF BASIL AGAINST CUSCUTA ACTA BOT. CROAT. 77 (1), 2018 67 C. campestris caused variable reductions in the vegetative (plant height, dry weights of shoot and root systems, number of leaves per plant) and reproductive (number of pods per plant and number of flowers per plant) traits of numerous legume crops. Although Cuscuta contains a few chloroplasts and slight chlorophylls, and thus possesses an extremely low photosynthetic activity, it is believed to be absolutely dependent on its host for nutrient sources (Birschwilks et al. 2006, Furuhashi et al. 2014) and will severely inhibit the growth of the host by removing its resources for photosyn- thesis, growth and leaf production. In addition, Cuscuta spp. forms a dense, thick mat over the host, which decreases the sun’s radiation and consequently photosynthesis and growth (Dawson et al. 1994). The specific pathways involved in defense against para- sitic plants are unknown. In this research we showed that C. campestris induced PAL activity and increased phenolic and flavonoid content in the basil plants. It has been well docu- mented that responses of host plants to attack by C. reflexa include a hypersensitive-like response and phytoalexin pro- duction (Borsics and Lados 2002). Best studied among host plant defenses against Cuscuta sp. is the resistance responses of tomato plants to C. reflexa, in which elongation of hypo- dermal host cells, accumulation of phenolics and peroxidas- es at binding site create a mechanical barrier that can block haustorial formation (Sahm et al. 1995). Our results con- firmed that C. campestris infestation increased the flavonoid levels in host plants. The application of herbicides to manage weed popula- tions is very common but is not nature friendly and is not suitable for all situations. Therefore, other ways of control, such as biological control, more friendly to the environment and humans, are necessary (Rosskopf et al. 1999). Carra- geenans are recognized to elicit defense responses in plants and animals against various plant pathogens and mamma- lian viruses (Khan et al. 2009). The effect of carrageenans on plant resistance to weeds is not known, and in this research we investigated the effects of ĸ-carrageenan on sweet basil resistance to Cuscuta campestris infestation. In this study, the application of external carrageenan enhanced shoot length and leaf area of basil and alleviated the negative effect of the parasite on the growth of infested plants. It is known that κ, λ and ɩ-carrageenans at a concentration of 1 mg mL–1 increased shoot height and leaf biomass in tobacco plants by increas- ing the net photosynthesis, rubisco activity, the glutamate dehydrogenase activity involved in nitrogen assimilation, basal metabolism, and cell proliferation (Castro et al. 2012, Gonzàlez et al. 2013a). Also, there is a report showing that seaweed extracts improved nutrient uptake by roots, thereby causing enhanced plant growth (Mancuso et al. 2006). In this research, the anti-infestation activities of carrageenan were observed. Infestation by C. campestris of basil plants treat- ed with carrageenan decreased by about 26%. Carrageenan- treated basil plants attracted fewer C. campestris as compared to the control. It has been reported that the chemical reac- tivity of carrageenans is mostly due to their half-ester sulfate groups that are extremely anionic (Necas and Bartosikova 2013). These negatively charged compounds perform their inhibitory effect by interacting with the positive charges on the cell wall surface of parasite plants and thereby prevent the prehaustorium attachment and entrance into the host cells. In addition, the negative charge and antioxidant properties (Yuan et al. 2006) of carrageenan probably cause inhibition of hydrolysis enzymes inside the cell wall of C. campestris, causing the inhibition of prehaustorium penetration in the host cell wall. Moreover, it seems likely that κ-carrageenan induces several biochemical pathways in the development of plant resistance. In this work, C. campestris attachment occurred along the stem of control group of basil plants, but in basil with foliar spray of carrageenan few infections oc- curred in shoot apical parts not affected by carrageenan, be- cause these organs were formed after carrageenan treatment. A recent study has shown that seaweed extracts can be used as an important source of plant defense elicitors (Khan et al. 2009). Besides influencing the physiology and metabo- lism of plants, it has been reported that ĸ-carrageenan is very effective and induces maximum browning and high level of secondary metabolites (pathogen resistance compounds) in various crop plants like chickpea, carrots and potatoes (Bi et al. 2008). In addition ĸ-carrageenan is an effective substance for induction of defensive genes in tobacco leaves (Mercier et al. 2001). We demonstrated that plant defense response was induced by ĸ-carrageenan. Carrageenan treatment caused a significant increase in the PAL activity and phenolic com- pound of basil, but flavonoid and anthocyanin content did not show considerable changes. Induction of PAL activity, the first and key enzyme of the phenylpropanoid pathway (Vera et al. 2012) induces the accumulation of some phe- nyl propanoid compounds (PPCs) in tobacco plants. It has been reported that κ-, λ- and ɩ-carrageenans increase protec- tion against fungal, viral, and bacterial infection in tobacco plants, which was due, at least in part, to the accumulation of PPCs (Vera et al. 2011). Also, it has been reported that oligo-carrageenans increased growth and activated defensive mechanisms against pathogens by enhancing the amount of some PPCs in eucalyptus trees (Gonzàlez et al. 2013a, b). We observed that in basil, ĸ-carrageenans elicited PAL en- zyme activities, with the consequence of a higher pheno- lic content. The phenolics are implicated in plant defense through scavenging reactive oxygen species (Cho and Lee 2015). Phenolic compounds are also involved in the defense response through reinforcement of the cell wall by biopoly- mer deposition near parasite infestation sites. In accordance with this explanation, the present study showed carrageenan treatment increased the lignin content of a basil plant para- sitized by C. campestris, which is a desirable trait that may be associated with resistance of basil against C. campestris in- vasion. Deposition of lignin has been hypothesized to inter- fere with the enzymatic hydrolysis and mechanical penetra- tion of host plant tissue by Cuscuta and may also weaken the movement of water and diffusible molecules between host and Cuscuta and help to starve the parasite (Lattanzio et al. MOUSAVI E. A., MANOCHEHRI KALANTARI K., NASIBI F., OLOUMI H. 68 ACTA BOT. CROAT. 77 (1), 2018 References 2006). As pointed out, the ability of C. campestris haustori- um to penetrate from a looser site of the basil stem was the cause of the lower lignin content in the parasitized basil plant than in the control. This research demonstrates the ability of carrageenan to modulate the resistance of basil to C. campes- tris. The mechanism of this suppression is not clearly known, but we believe that the activation of natural plant resistance mechanisms and the ability of carrageenan to stimulate the synthesis of compounds (secondary metabolites) with anti- infestation activities will be the cause. Importantly, this study is the first indication that carrageenan treatment can directly suppress infestation of the basil plant by C. campestris. In this study, antioxidant content increased remarkably in basil plants with C. campestris infestation and carrageen- an treatment. The antioxidative effect is mainly due to phe- nolic components, which can act as reducing agents, hydro- gen donors, and singlet oxygen quenchers. They may also have a metal chelating potential (Niccholson 1992). Basil plants possess valuable antioxidant properties that may be associated with lower incidence and lower mortality rates of cancer in several human populations (Gülçin et al. 2007). 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