Folia Horticulturae Folia Hort. 30(1), 2018, 79-92

Published by the Polish Society DOI: 10.2478/fhort-2018-0009 for Horticultural Science since 1989

ORIGINAL ARTICLE Open access www.foliahort.ogr.ur.krakow.pl

Morpho-physiological and biochemical responses of bladder cherry ( alkekengi L.) induced by multienzymatic biostimulant, IBA, and citric acid Arefeh Rahimi Shokouh1, Ali Mehrafarin2, Vahid Abdossi1, Hassanali Naghdi Badi2*

1 Department of Horticulture, Science and Research Branch, Islamic Azad University, Tehran, Iran 2 Medicinal Research Center, Institute of Medicinal Plants, ACECR, Karaj, Iran

ABSTRACT enzymes, growth regulators and organic acids are the main groups of plant biostimulants (PBs), and their combined use in the final formulation may be important for increasing the quantitative and qualitative composition of plant products. This study aimed to determine the effects of a multienzymatic biostimulant (MB), indole-3-butyric acid (IBA), and citric acid (CA) on the morphological and phytochemical traits of bladder cherry (Physalis alkekengi L.). The treatments included different concentrations of MB (0, 0.5 and 1.0%), IBA (0, 25, and 50 ppm), and CA (0, and 500 mg dm-3), which were sprayed four times during the vegetative stage, at 12-day intervals, 35 days after planting. The results showed that the treatments had a significant effect on plant height, stem number, diameter and weight, number and weight, fruit number, diameter and weight, the amounts of total phenols, alkaloids and flavonoids, and on the radical scavenging activity. The most effective formulation for improving the fruit yield of bladder cherry was 1% MB with 50 ppm IBA and 500 ppm CA. However, the best treatment for increasing the total phenolic and alkaloid contents, and radical scavenging activity was 0.5% MB. In general, the maximum values of most traits were obtained by spraying the plants with 0.5 and 1% MB combined with IBA and CA. The concentration of alkaloids, the main pharmaceutical metabolites of bladder cherry, increased as a result of the application of the multienzymatic biostimulant.

Key words: alkaloid, indole-3-butyric acid, flavonoids, phenols, radical scavenging activity

INTRODUCTION in Iran and many other countries in the world (Kusumaningtyas et al., 2015). The genus Physalis has great economic importance Global demand for medicinal plants is rapidly not only as a food supplier but also as a source increasing in the pharmaceutical and food of important chemical compounds. The fruits of industries. This need requires the use of good Physalis contain various phenols, alkaloids, agricultural practices (GAP) to guarantee medicinal steroids, saponins, fatty acids, and flavonoids plant quality and facilitate standardization for herbal (Sharma et al., 2015). Bladder cherry (Physalis drugs (Chan et al., 2012; Muchugi et al., 2008). It alkekengi L.) is a perennial of the has been reported that the use of biostimulants in family, used in traditional medicine GAP leads to increased growth and quality of most

*Corresponding author. e-mail: [email protected] (H. Naghdi Badi). 80 Responses to biostimulant, IBA, and citric acid in bladder cherry plant species (Asad et al., 2002; Du Jardin, 2015). Organic acids are involved in a broad range Plant biostimulants, or agricultural biostimulants, of physiological processes of plant growth and include diverse substances and microorganisms development (An et al., 2014), and are also considered that enhance plant growth. Furthermore, it is to alleviate biotic stresses (Jafari and Hadavi, 2012). documented that they also promote cell respiration, Endogenous organic acids are the source of both photosynthesis, protein synthesis, water and nutrient carbon skeleton and energy for cells, and are used uptake, and enzymatic activities (Nardi et al., 2016; in the respiratory cycle and other biochemical Du Jardin, 2015). Plant biostimulants contain pathways. It has been expressed that increasing a substance/substances and/or microorganisms endogenous organic acids can influence the biomass whose function, when applied to plants or the and yield of crops (Da Silva, 2003). rhizosphere, is to stimulate the natural processes Application of biostimulants in the commercial to enhance nutrient uptake, nutrient efficiency, production of medicinal plants is a viable tolerance to abiotic stresses, and crop quality management practice for the production of these (Calvo et al., 2014). Biostimulants generally species, increasing biomass production and include plant growth regulators, organic acids, enhancing the synthesis of secondary metabolites. and enzymes (Mandal et al., 2007; Du Jardin, Studies on the effects of plant biostimulants on 2015). These formulations decrease the need for the accumulation of secondary metabolites in chemical fertilizers and have the capacity to satisfy medicinal plants have been conducted in order to the nutritional requirements of plants, which increase the medicinal and trade values of those further results in higher yields (Subbarao et al., species (Rafiee et al., 2016). Application of different 2015). Furthermore, there is strong evidence that plant biostimulants at various concentrations and foliar spraying of biostimulants enhances plant diverse mechanisms of action have resulted in growth, nutrient uptake, and improves the yield different effects on medicinal plants. Considering and production quality of many crops (El-Desuki, the biochemical and pharmacological importance of 2004). The most effective biostimulants usually bladder cherry (Physalis alkekengi L.) as a multi- contain several types of bioactive stimulants purpose plant, as well as recognizing the biological in their formulations, such as multienzymes. effects of biostimulants, particularly enzymatic Multienzymatic biostimulants (MB) are new biostimulants, bioregulators and organic acids on integrated agricultural organic products composed the production of metabolites and plant growth, of enzymes, amino acids, polysaccharides, humic this study focused on the morpho-physiological acids, phytohormones, and other components, and biochemical responses of bladder cherry to which are usually derived from natural products, different formulations containing a multienzymatic and their biological activities occur at limited doses. biostimulant (MB), indole-3-butyric acid (IBA), Auxins, especially indole-3-butyric acid and citric acid (CA). So far there has been very (IBA), are used as plant growth regulators to little information on the influence of MB, IBA, improve germination and subsequent growth and and CA on the morpho-physiological traits and development of the plant (Bertoni, 2011). Auxins concentrations of chemical compounds in bladder have an important role in major plant growth and cherry. The results of this study could be useful in development processes such as expansion, division increasing Physalis alkekengi L. crop production and differentiation of plant cells, which can lead with reduced fertilizer consumption, and also in to organogenesis, apical dominance, tropism providing practical information for the food and phenomenon, and vascular patterning (Mockaitis pharmaceutical industries. and Estelle, 2008). The normal growth of a plant requires balancing auxin biosynthesis and transport, MATERIAL AND METHODS as well as managing its storage forms. IBA is In order to evaluate the effects of some biostimulants a potential storage form for auxin in a variety of on the growth and chemical composition of bladder plants, which can be synthesized from indole acetic cherry (Physalis alkekengi L.), two field experiments acid (IAA) and converted to IBA by peroxisomal were conducted as factorial experiments based on β-oxidation (Strader et al., 2010; Enders and Strader, a randomized complete block design (RCBD) with 2015). Therefore, it is postulated that IBA has 18 treatments (Tab. 1) and 3 replications at the a crucial effect on the sensitivity, transport, and agricultural system research farm of Medicinal function of auxin (Epstein and Ludwig-Müller, Plant Institute (MPI), ACECR of Karaj in Iran (35°, 1993; Enders and Strader, 2015). 90′ N and 50°, 88′ E, 1500 m above sea level) during Arefeh Rahimi Shokouh, Ali Mehrafarin, Vahid Abdossi, Hassanali Naghdi Badi 81

Table 1. Composition of the biostimulant formulations used in the experimental treatments

MB* IBA** CA*** Treatment (%) (ppm) (mg dm-3) T1 0 0 0 T2 0 0 500 T3 0 25 0 T4 0 25 500 T5 0 50 0 T6 0 50 500 T7 0.5 0 0 T8 0.5 0 500 T9 0.5 25 0 T10 0.5 25 500 T11 0.5 50 0 T12 0.5 50 500 T13 1 0 0 T14 1 0 500 T15 1 25 0 T16 1 25 500 T17 1 50 0 T18 1 50 500 *MB: Multienzymatic biostimulant; **IBA: Indole-3-butyric acid; ***CA: Citric acid

2015 and 2016. Monthly changes in temperature and and 6 m in length, with 1 m distance between precipitation during 2015 and 2016 are presented in each plot in order to prevent spray drift. Each Figure 1. plot had 12 planting rows spaced 50 cm apart Transplants of bladder cherry were obtained and planting intervals of 25 cm in the rows. The from the gene bank and seed collection of MPI treatments included different concentrations of (1341-MPISB), and were transferred into farm a multienzymatic biostimulant (MB) at 0, 0.5, and plots. Plot size in each replication was 5 m in width 1.0% of a commercial product called Multienzim

Figure 1. Monthly changes in temperature and precipitation between 2015 and 2016 at the experimental farm 82 Responses to biostimulant, IBA, and citric acid in bladder cherry

Organik Gübre (MOG), which in Turkish means was used for determining the total concentration of multienzymatic organic liquid fertilizer, indole-3- phenolic and flavonoid compounds as well as radical butyric acid (IBA) at 0, 25 and 50 ppm, and citric scavenging activity. acid (CA) at 0, and 500 mg dm-3. The details of the Total phenolic content formulations used in the experimental treatments are given in Table 1. MOG is a liquid multienzymatic Total phenolic content was determined by the biostimulant for use as an organic growth promoter; Singleton and Rossi (1965) method with some it is manufactured from fruit juice and crop residues, modifications. Briefly, 500 μL of diluted extract and contains 18 enzymes (such as alkali proteinase, was mixed with 5 mL of the Folin-Ciocalteu reagent amylosine, glucamylase, xylanase, lipase, lyase, and 4 mL of a sodium carbonate solution (7%). rubisco, transglutaminase, catalase, lipoxygenase, The reaction mixture was brought to 25 mL with isomerase, urease, etc.), natural forms of micro- and distilled water and was incubated for 15 min. in the macronutrients, and vegetable-based vitamins. The dark. Then the absorbance was measured at 765 nm. product was provided by Azarabadegan Company Total phenolic content was calculated according to (West Azerbaijan province, Iran). The MOG liquid a gallic acid standard curve. biostimulant contained 25% total organic carbon, Total flavonoids 4% total nitrogen, 4% K O, 1.06% P, 0.42% Fe, 2 Flavonoid content was estimated by the Ordonez 0.16% Cu, and 13% enzymes, with a pH of 6.1. The et al. (2006) method. Accurately measured 0.5 ml solutions for all the treatments contained surfactant of the extract was mixed with 1.5 ml of methanol, “Tween 60” at 0.5% (v/v) to improve adhesion. 0.1 mL of 10% aluminum chloride, 0.1 mL of All the chemicals used in the experiment, except 1 M potassium acetate, and 2.8 mL of distilled the biostimulant, were purchased from Merck water. The mixture was allowed to stand at room (Germany). All of the treatments were performed temperature for 15 min. The absorbance of the by spraying four times during the vegetative plant reaction mixture was read at 415 nm with a double growth, at 12-day intervals, starting on the 35th day beam spectrophotometer (Human crop, Xma-2000). after transplanting the bladder cherry plants. The The total flavonoid content was calculated based on spraying was carried out in such a way that all of a quercetin standard curve. the aboveground parts of the bladder cherry plants were covered with the liquid. Motorized backpack- Radical scavenging activity 3 type sprayer with a capacity of 14 dm was used for DPPH (1,1-diphenyl-2-picrylhydrazyl) scavenging spraying, and the sprayer nozzle was held 40 cm was used for an assessment of the antioxidant activity above the plants. of the extract. The reaction mixture consisted of After the four spray applications, 83 days 2925 μl of a freshly prepared DPPH solution (25 mg after transplanting to the farm, fruits at the stage dm-3) in methanol and 75 μl diluted extract. After of full maturity (golden yellow ripe fruit) were 30 minutes, the absorbance of the reaction mixture collected manually. To record data on the morpho- was read at 517 nm with a spectrophotometer physiological and biochemical traits, six plants (Human crop, Xma-2000) against the blank and from each experimental unit (plot) were randomly control (the mixture of methanol and DPPH) (Blois, selected, and then average values of the traits were 1958). calculated for the two years. In order to measure total dry matter, the plants were divided into stems, Total alkaloid content and fruits. Then they were placed in an oven The fruit powder was subjected to extraction with at 75°C until constant weight was obtained. methanol for 24 h in a Soxhlet apparatus. After filtering, the methanolic extract was evaporated to Phytochemical analysis dryness, and the residue was dissolved in 2 N HCl Extraction and discoloured with diethyl ether. Then the solution Shade-dried fruit was powdered with an electric pH was adjusted to 10 with 0.1 N NaOH and washed mill and stored at room temperature in darkness 3 times with 10 ml chloroform. The chloroform before it was subjected to the extraction process. phase was evaporated and the residue was dissolved The fruit powder (10 g) was macerated with 200 in methanol. For alkaloid measurements, 1 ml of the ml of methanol (85%) at ambient temperature for extract was mixed with 5 ml of a bromocresol green 48 h. The extract was concentrated after filtering solution and 5 ml of phosphate buffer. The mixture through a Whatman filter paper no.1. This extract was shaken and the formed complex was extracted Arefeh Rahimi Shokouh, Ali Mehrafarin, Vahid Abdossi, Hassanali Naghdi Badi 83 with chloroform. The volume of the collected together with IBA or MB was often ineffective on complex was brought to 10 ml with chloroform. these features. However, the addition of CA to the The absorbance of the complex was read at 470 mixture of MB and IBA was effective only at the nm. The alkaloid content was measured against an higher concentration of IBA. The highest number atropine standard curve (Sharief et al., 2014). of stems (20.2) was recorded for the combination -3 Statistical analysis of 1% MB with 50 ppm IBA and 500 mg dm CA. The greatest number of nodes and stem diameter Analysis of variance (A NOVA) was performed for were obtained with the mixture of 0.5% MB and each trait included in the field experiments in 2015 25 ppm IBA (49.1 and 10.05, respectively). However, and 2016. Bartlett’s test was used to assess the the lowest values of these traits were observed in the homogeneity of variance over the two years. SAS control (Tab. 2 – part 1). statistical analysis software (ver. 9.2, SAS Institute) The interaction of MB, IBA, and CA had was used for ANOVA. Significant differences significantly (p ≤ 0.01) impacted on the dry and between the treatment means were determined fresh weight of stems and on fresh weight. using Duncan’s Multiple Range Test at the 0.05 The results of means comparison showed that probability level. spraying with CA did not produce a significant difference relative to the control. In contrast, the RESULTS two concentrations of both IBA and MB improved Morphology and growth parameters the fresh and dry weight of stems and root fresh The results of means comparisons for each trait weight compared to the control. However, the were presented based on the overall average for the values of stem and root weight were reduced by years, and if the effect of the years was significant increasing the concentration of IBA, which was then each year was analyzed separately. The effect ameliorated by adding CA to 50 ppm IBA. Adding of the treatments on the traits is shown in Table 2 CA to MB reduced stem number, stem fresh and and 3. The results show that only stem and leaf fresh dry weight, and root fresh weight. Thus, the greatest weight, radical scavenging activity, and flavonoid stem fresh weight, stem dry weight, and root fresh content were significantly different in the two years weight were obtained by spraying with a mixture of as a result of using the various formulations of 0.5% MB with 25 ppm IBA (129.6, 21.66 and the biostimulant. MB and IBA had a significant 13.82 g, respectively). Their lowest values were (p ≤ 0.01) effect on plant height, whereas CA had no obtained in the control (18.9, 4.24 and 2.98 g, significant influence on it. However, their interaction respectively) (Tab. 2 – part 2). had a significant effect (p ≤ 0.01) on plant height in The treatments had a significant effect (p ≤ 0.01) both years. Spraying IBA at both concentrations on fruit number, fruit diameter, fruit fresh weight improved plant height compared to the control and fruit dry weight. Fruit diameter in the plants (71.4 to 75.5% increase), although increasing IBA treated with IBA and CA was not significantly concentration had led to a lower increase in plant different from the control (27.14 mm). However, height in 2015. Spraying CA, either alone or in although 0.5% MB had no significant effect on fruit combination with both IBA concentrations and MB, diameter compared to the control, its combination had no significant effect on plant height. The two with 500 ppm CA and 50 ppm IBA increased fruit MB concentrations strongly increased plant height diameter. The use of 1.0% MB increased fruit compared to the control; however, the highest plant diameter, but combining it with 500 ppm CA and height was observed for 1% MB (135.6 cm). Overall, 50 ppm IBA was more effective. Thus, the the highest plant height was obtained by spraying greatest fruit diameter was obtained with the with a mixture of 0.5% MB and 25 ppm IBA (141.1 formulation of 500 ppm CA and 50 ppm IBA with cm) (Tab. 2). both concentration of MB (33.9 and 34.3 mm, Spraying IBA at both concentrations significantly respectively) (Tab. 2 – part 3). (p ≤ 0.01) increased stem number, node number, and Spraying with CA increased the number of fruits the main stem diameter compared to the control. and fruit fresh weight compared to the control However, the lower concentration of IBA was more in 2015 and 2016 (an average increase of 40 and effective than the higher concentration. Also, the 41.4%, respectively) (Tab. 2 – part 3). The use of two concentrations of MB increased these traits 25 ppm IBA increased the number of fruits and fruit compared to the control. Spraying with CA increased fresh weight, but the higher concentration had an only the number of stems (43.4%). Spraying CA adverse effect. In addition, CA combined with IBA 84 Responses to biostimulant, IBA, and citric acid in bladder cherry Mean 4.39 j 4.69 i 7.32 e 5.18 h 6.68 f 6.32 g 6.41 g 6.72 f 3.82 k 7.64 cd 7.55 d 7.82 c 6.35 g 7.61 cd 7.74 cd 7.58 d 9.22 b 10.05 a 2016 (mm) 4.66 ij 4.99 i 7.23 de 6.16 h 7.05 e 6.63 fg 6.31 gh 6.93 ef 9.9 a 4.31 j 7.76 c 7.89 c 7.71 c 6.40 gh 7.70 c 7.55 cd 7.56 cd 9.13 b Stem diameter 2015 4.13 i 4.4 h 7.4 de 4.2 hi 6.31 f 6.0g 6.5 f 6.5 f 3.32 j 7.51 d 7.2 e 7.93 c 6.3 f 7.51 d 7.93 c 7.6 d 9.3 b 10.2 a Mean 29.6 j 31.0 j 38.7 efg 41.9 bcd 35.7 hi 35.6 hi 35.7 hi 36.8 ghi 49.1 a 34.2 i 41.3 fgh 43.9 cde 42.2 bc 40.0 bcd 36.2 def 43.5 ghi 44.3 bc 44.4 b 2016 30.2 g 31.0 g 40.4 cd 41.7 bc 35.4 f 37.2 def 39.5 c-f 36.6 def 49.2 a 36.4 def 35.6 f 39.6 c-f 39.9 cde 40.4 cd 36.1 ef 36.4 def 44.9 ab 43.6 bc Node number h

2015 29.0 31.0 gh 37.0 ef 42.0 cd 36.0 ef 34.0 fg 32.0 gh 37.0 ef 49.0 a 32.0 gh 39.0 de 43.0 c 48.0 ab 44.0 c 44.0 c 36.0 ef 42.0 cd 45.0 bc . L. – part 1 7.6 j 7.3 j 9.7 i 7.5 j 8.1 j Mean 11.5 fgh 11.5 fgh 11.6 fg 11.7 gh 11.01 10.9 gh 13.8 d 13.8 d 17.5 c 12.9 de 10.6 hi 12.2 ef 19.1 b 20.2 a 2016 8.2 l 8.8 kl 9.5 ijk 9.1 jkl 8.5 kl 9.9 hij 11.6 fg 11.6 fg 11.2 gh 11.0 13.5 d 20.8 a 12.9 de 14.9 c 13.3 de 12.3 ef 10.4 ghi 18.9 b 19.5 a Stem number Physalis alkekengi 7.0 i 5.0 j 5.0 j 3.0 k 9.0 h 2015 11.0 g 11.0 g 11.0 g 11.0 13.0 ef 16.0 c 14.0 de 12.0 fg 14.2 de 15.0 cd 10.6 gh 14.0 d 19.3.0 b 21.0 a Mean 65.8 h 68.1 h 112.8 fg 112.8 def 119.4 efg 115.5 def 119.1 efg 114.3 120.7 c-f 129.3 bcd 128.6 bcd 141.1 a 107.6 g 137.3 ab 135.6 ab 131.1 abc 121.1 c-f 124.3 cde 129.6 bcd (cm) 2016 62.5 c 71.6 c 113.9 ab 113.9 ab 119.1 ab 115.2 ab 117.5 ab 119.1 102.6 b 121.7 ab 122.3 ab 128.5 a 120.1 ab 133.1 a 128.6 a 129.2 a 129.7 a 132.6 a 132.1 a Plant height efg 0 2015 69.0 j 64.5 j 96.0 i 111.0 h 111.0 117.0 gh 117.0 gh 117.0 fgh 119.0 gh 116.0 123. 130.0 cde 137.0 bc 149.0 a 123.0 efg 146.0 a 142.0 ab 132.0 cd 123.0 efg 127.0 def Effect of the treatments on morpho-physiological traits Treatment* T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 T16 T17 T18 Table 2. Table Means with the same letter in each column are not significantly different at 5% level of probability 1 Table *For abbreviations see Arefeh Rahimi Shokouh, Ali Mehrafarin, Vahid Abdossi, Hassanali Naghdi Badi 85

(g) 2.21 ij 1.27 l 9.29 a 1.26 l ijk 2.11 2.87 gh 3.32 f 2.47 ih 3.15 fg 6.43 c 1.82 jk 4.70 de 5.07 d 4.64 e 2.21 ij 1.72 k 7.91 b 9.29 a Leaf dry weight (g) 8.3 j 8.9 j 14.7 hi 71.7 a 14.6 hi 19.0 g 22.2 f 15.3 h 22.6 f 45.1 c 12.2 i 35.8 e 47.0 c 40.7 d 15.8 h 13.3 hi 60.2 b 72.2 a Leaf fresh weight )

2 LA (cm 171.7 efg 165.4 g 185.7 ab 168.0 g 172.8 d-g 167.6 g 166.4 g 165.4 g 167.1 g 181.1 a-d 165.2 g 165.7 g 179.2 b-e 176.9 cdef 168.7 f 165.6 g 188.8 a 183.8 abc 48.7 l 55.1 l 62.2 k Mean 114.9 ij 114.9 i 117.4 200.8 c 125.2 h 139.5 fg 141.9 fg 149.4 e 156.7 d 144.3 ef 199.3 c 225.4 b 135.8 g 109.5 j 267.1 a 225.7 b 2016 51.4 j 56.1 j 65.3 i 118.8 h 118.8 h 118.1 201.5 c 120.7 h 139.3 g 152.9 e 143.6 fg 153.8 e 165.0 d 149.6 ef 204.6 c 209.8 c 138.6 g 280.1 a 230.5 b . For significant effect of year, the data for each year were analyzed separately. . For significant effect of year, Leaf number L. – part 2 46.0 l 54.0 kl 59.0 k 2015 111.2 ij 111.2 116.0 hi 116.0 200.0 d 109.0 ij 126.0 gh 140.2 ef 145.0 e 148.3 e 139.0 ef 194.0 d 241.0 b 133.0 fg 101.0 j 254.0 a 221.0 c Physalis alkekengi (g) 2.98 i 3.51 ghi 4.36 efg 3.46 hi 3.59 f-i 4.44 ef 5.95 d 3.18 hi 6.09 d 3.99 fgh 7.12 c 6.02 d 4.96 e 4.42 ef 4.39 ef 13.82 a 12.33 b 12.98 ab Root fresh weight (g) Stem 4.24 i 4.75 i 9.32 h 11.54 e-h 11.54 13.73 def 14.39 cde 13.21 d-g 10.24 h 21.66 a 10.98 fgh 10.12 h 15.13 cd 14.05 de 13.28 d-g 10.4 gh 10.98 fgh 18.56 b 17.23 bc dry weight h 0 (g) Stem 18.9 i 22.23 i 99.6 bc 45. 54.4 f 67.5 e 51.4 fg 53.2 fg 47.7 gh 68.7 e 68.2 e 76.1 d 48.8 gh 48.2 gh 96.2 c 95.5 c 102.4 b 129.6 a fresh weight Effect of the treatments on morpho-physiological traits Treatment* T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 T16 T17 T18 Means with the same letter in each column are not significantly different at 5% level of probability 1 Table *For abbreviations see Table 2. Table 86 Responses to biostimulant, IBA, and citric acid in bladder cherry (g) 18.8 f 27.01 de 31.86 c 28.29 de 13.69 h 19.05 f 19.84 f 13.23 h 31.74 c 17.83 f 25.71 e 35.43 b 29.77 cd 14.26 gh 17.58 f 17.26 fg 55.66 a 57.52 a Fruit dry weight Mean 193.6 hi 273.8 ef 319.7 c 283.9 de 146.8 k 198.5 gh 205.8 g 142.8 k 321.1 c 184.9 ij 263.3 f 355.7 b 288.0 d 151.9 k 183.2 ij 179.9 j 544.8 a 542.2 a (g) 2016 196.9 gh 272.2 ef 316.3 d 279.6 e 147.5 i 197.9 gh 205.5 g 142.6 i 312.7 d 188.9 gh 258.2 f 346.4 c 309.0 d 153.4 i 185.4 h 182.5 h 526.4 b 561.1 a Fruit fresh weight 2015 190.3 hi 275.4 ef 323.1 d 288.3 e 146.1 j 199.2 gh 206.0 g 143.0 j 329.5 d 181.6 i 268.4 f 365.0 c 267.0 f 150.4 j 180.9 i 177.2 i 563.1 a 523.2 b b a

. For significant effect of year, the data for each year were analyzed separately. . For significant effect of year, Mean 19.07 h 26.71 e 28.68 d 22.96 g 18.2 hi 17.6 hi 23.88 fg 14.0 k 26.06 e 16.75 i 23.24 fg 31.65 c 24.94 ef 14.93 jk 18.14 hi 16.54 ij 38.58 43.07 L. – part 3 19.1 e 24.4 cd 27.3 bc 24.9 cd 15.4 gh 19.2 e 19.7 e 15.0 h 27.1 bc 18.5 ef 23.4 d 29.3 b 26.8 bc 15.8 fgh 18.3 efg 18.1 efg 40.2 a 42.1 a 2016 Physalis alkekengi Fruit number 19.0 gh 29.0 d 30.0 d 21.0 fg 21.0 fg 16.0 ij 28.0 d 13.0 k 25.0 e 15.0 jk 23.0 ef 34.0 c 23.0 ef 14.0 jk 18.0 hi 15.0 jk 37.0 b 44.0 a 2015 (mm) 27.14 de 27.74 de 26.69 de 25.86 def 27.08 de 27.89 d 23.56 d 25.05 ef 33.62 ab 27.74 de 28.53 cd 33.9 a 30.77 c 31.09 bc 27.3 de 27.67 de 33.73 ab 34.35 a Fruit diameter Effect of the treatments on morpho-physiological traits Treatment* T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 T16 T17 T18 Table 2. Table Means with the same letter in each column are not significantly different at 5% level of probability 1 Table *For abbreviations see Arefeh Rahimi Shokouh, Ali Mehrafarin, Vahid Abdossi, Hassanali Naghdi Badi 87 Mean 245.2 efg 198.0 j 250.2 de 236.6 gh 258.1 cd 267.9 ab 234.3 h 259.3 bc 233.4 h 236.6 gh 232.9 h 217.3 i 239.8 fgh 273.4 a 233.2 h 246.7 ef 198.2 j 151.5 k ) -1 L. fruit 2016 (mg g 211.1 ef 211.1 239.1 c 219.1 de 208.6 ef 216.3 de 253.1 a 239.2 c 233.8 c 252.7 a 213.3 def 241.4 bc 222.6 d 202.5 f 251.9 ab 242.3 abc 243.1 abc 163.9 g 156.4 g Total flavonoids Total Physalis alkekengi 2015 251.2 de 176.8 h 291.8 a 256.8 b-e 263.1 bcd 296.5 a 234.8 fg 265.9 bc 253.5 cde 231.8 fg 243.1 ef 232.1 fg 268.5 b 294.9 a 224.1 g 250.4 de 232.3 fg 146.5 i (%) 25.05 g 26.14 fg 27.70 d-g 35.01 c 26.77 efg 27.06 efg 59.02 a 38.67 b 35.54 bc 35.3 c 28.76 def 28.61 def 26.46 efg 28.32 d-g 29.66 de 28.63 edf 30.62 d 34.45 c . For significant effect of year, the data for each year were analyzed separately. . For significant effect of year, DPPH scavenging ) -1 2.06 h 2.24 gh 3.1 ef 3.45 de 3.29 ef 3.28 ef 7.14 a 5.83 b 4.04 cd 4.02 cd 3.40 de 3.07 ef 3.23 ef 4.48 c 3.56 de 3.55 de 3.01 ef 2.74 fg (mg g Total alkaloids Total ) -1 73.6 i 76.4 ih 87.8 cde 83.9 efg 89.8 abc 89.3 bcd 93.5 a 92.6 ab 89.8 abc 87.9 cde 88.9 bcd 90.2 abc 85.2 def 86.2 c-f 82.8 fg 84.5 ef 80.0 gh 84.7 ef (mg g Total phenolics Total Effect of the treatments on amounts phenolics, flavonoids and alkaloids, radical scavenging activity in Treatment* T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 T16 T17 T18 Means with the same letter in each column are not significantly different at 5% level of probability 1 Table *For abbreviations see Table 3. Table 88 Responses to biostimulant, IBA, and citric acid in bladder cherry reduced their positive effect on fruit number and fresh increased the alkaloid content as compared to the weight (Tab. 2 – part 3). Both concentrations of MB control. Similar to the phenolic content, the greatest improved fruit number and fresh weight compared amount of alkaloids (7.14 mg g-1) was recorded for to the control. However, the addition of CA to MB 0.5% MB. CA and both concentrations of IBA strongly diminished this advantageous effect. The produced no significant difference relative to the combinations of 25 ppm IBA with 0.5% MB and control with respect to the radical scavenging 50 ppm IBA with 1% MB were more beneficial than activity. However, the combination of 500 ppm CA spraying each of them alone. The highest number of with 25 ppm IBA significantly increased the radical fruits and fruit fresh weight were recorded for the scavenging activity (a 39.8% increase). Spraying combination of 500 ppm CA, 50 ppm IBA, and 1.0% with 0.5% MB induced a higher radical scavenging MB (43.07 and 542.2, respectively). activity in comparison with 1.0% MB. Thus, the Fruit dry weight was increased by spraying with highest radical scavenging activity (59.02%) was CA (27.01 g) and 25 ppm IBA (31.86 g), while it achieved when 0.5% MB was used alone. The was reduced by using 50 ppm IBA compared to lowest activity was found in the control (25.05%). the control (27.2% reduction). Moreover, spraying Spraying with 500 ppm CA reduced the total with the lower concentration of MB produced no flavonoid content relative to the control in 2015 significant difference relative to the control, but and 2016 (29.6 and 8.4% reduction, respectively). increasing the MB concentration to 1.0% caused an Spraying with IBA increased the flavonoid content, improvement in fruit dry weight. Similar to fruit but only the 50 ppm IBA produced a significant -1 fresh weight, the use of MB with CA diminished difference in relation to the control (245.2 mg g ). fruit dry weight. While this trait increased by adding In contrast, the use of MB decreased the amount IBA to MB, the combination of 25 ppm IBA with of flavonoids, although 1.0% MB produced no 1.0% MB was an exception. The highest fruit dry significant difference relative to the control -1 weight was recorded for the combination of 1.0% (239.8 mg g ). The positive effect of CA combined MB with 50 ppm IBA and CA (57.5 g), whereas the with IBA depended on the concentration of IBA. lowest was obtained with the mixture of 0.5 % MB The flavonoid content was increased by mixing with 500 ppm citric acid (13.23 g). the higher concentration of IBA with CA. The Using 500 ppm CA reduced leaf fresh and combination of MB with CA also improved the dry weight compared to the control, but it had no flavonoid content. In contrast, the mixture of MB significant effect on leaf number and leaf area (LA). with IBA reduced the flavonoid content. Thus, the -1 At the lower concentration (25 ppm), IBA increased maximum flavonoid content (273.4 mg g ) was recorded for the combination of 1.0% MB with the number of leaves, LA, and leaf fresh and dry -1 weight, whereas the higher concentration (50 ppm) 500 ppm CA, and its minimum (151.5 mg g ) was had an adverse effect on these features except the obtained with the formulation of 1.0% MB with 500 ppm CA and 50 ppm IBA (Tab. 3). number of leaves. The number of leaves, LA, and leaf fresh and dry weight were improved after DISCUSSION spraying with MB at both concentrations, especially at the higher concentration. The greatest number of In recent decades, customer interest in more leaves and LA were obtained in the combination advantageous nourishment and government of 1.0% MB with 50 ppm IBA (267.1 and 188.8, strategies concentrated on environmentally respectively). Also, the greatest leaf fresh and dry sustainable agricultural systems have both weight was obtained in the combination of 1.0% MB advanced a fast extension of organic farming with 50 ppm IBA and 500 ppm CA (72.2 and 9.29 g, (Rigby and Caceres, 2001). One of the recently respectively) (Tab. 2 – part 2). introduced organic biostimulants is MB (MOG), which is manufactured by using fruit juice and crop Amounts of phenolics, flavonoids and alkaloids, residues, and contains various types of enzymes and radical scavenging activity such as alkali proteinase, amylosine, glucamylase, The effect of the treatments on the total phenolic xylanase, lipase, lyase, rubisco, transglutaminase, content of bladder cherry fruits is shown in catalase, lipoxygenase, isomerase, urease, etc. Table 3. It shows that all of the formulations In spite of the fact that the advantageous impact increased the total phenolic content in fruit compared of biostimulants has been demonstrated on a few to the control. The highest total phenolic content plants, it appears that in semi-arid areas utilization (93.5 mg g-1) was found for 0.5% MB. All treatments of biostimulants alone will not be adequate, and Arefeh Rahimi Shokouh, Ali Mehrafarin, Vahid Abdossi, Hassanali Naghdi Badi 89 concurrent use of other supplementary resources of stems. CA, when combined with IBA and MB, is unavoidable (Janmohammadi et al., 2014, 2016). could improve stem weight, but the highest value of This finding substantiates the thoughts of Rayan et stem weight was obtained with the formulation of al. (2012), who have stated that biofertilizers alone 25 ppm IBA and 5% MB. Moreover, these results are not very effective on the dry lands of the West are consistent with some reports for other plants Asia-North Africa area. The best biostimulants (Karima and Abdel Wahed, 2005; Abou Dahab are those containing multiple stimulants and and Abd El-Aziz, 2006). Spraying with CA, both enzymes (Janmohammadi et al., 2016), such as concentrations of IBA and with MB alone helped the formulations of biostimulants used in this to increase the number of leaves and their weight, experiment. but the right combination of these compounds had Analysis of the quantitative traits (plant height, the greatest impact. main stem diameter, fresh and dry weight of aerial MB combined with IBA and CA had distinct parts and ) revealed that using the combination effects on fruit setting (fruit number) and fruit of MB with IBA and CA increased all measured development (fruit diameter and fruit fresh weight). parameters. The same trend was also observed for These results confirmed the study by Nahed et the bladder cherry fruit and its components (number al. (2010) on the use of multiple amino acids and of fruits per plant, fruit diameter, fruit fresh and biostimulant compounds on Thuja orientalis L. dry weight). Plant height in bladder cherry showed Likewise, enhancement of fruit dry weight may be no significant response to the spraying with CA, ascribed to the increase in fruit diameter. Moreover, but the use of both concentrations of IBA and MB it has been reported that the positive effect of significantly increased plant height. The increment biostimulants on plant yield might be due to the in plant height produced by spraying with MB might stimulating effect of amino acids and enzymes on have been due to the positive effect of the enzymes the growth of plant cells. Also, a positive correlation on the increase in the number of nodes. Moreover, has been reported between the spraying with

IBA belongs to a group of auxins which have an biostimulants and enhancements in CO2 absorption, inductive effect on cell growth (Takatsuka and stomata conductance, and yield production in the Umeda, 2014). Thus, the combination of 0.5% MB tea plant (Kumar and Thomas, 2004; Calvo et al., with 25 ppm IBA was the most effective formulation 2014). These data are in accordance with what was for plant height enhancement. The findings of the obtained by Neri et al. (2002) and El-Desuki (2004). current study are consistent with those of Mafakheri Enzymes are involved in several physiological et al. (2012), who found that concurrent application and biochemical processes such as cell growth and of organic fertilizer and biostimulants could expansion, differentiation and development, auxin significantly improve the height of dragonhead catabolism, lignification, as well as abiotic and biotic (Dracocephalum moldavica L.). Stem fresh and stress responses (Siavoshi and Laware, 2013). For dry weights were increased in response to spraying several physiological processes the action of enzymes with both IBA concentrations and MB, because the is essential, e.g. in nutrient uptake, protein turnover, height, diameter, and the number of nodes of the stem cell division, signal transduction, processing of had been enhanced. Applying exogenous enzymes polypeptide hormones, apoptosis and also in the in a liquid form as bioactive stimulants can have biological cycle of plants. The biostimulant MB a positive effect on plant performance. In this regard, is a mixture of enzymes, therefore it can enhance some studies have also been conducted on the use of plant growth, nutrient uptake, and the quantity and several enzymatic compounds on Dracocephalum quality of yield in many crops (Nardi et al., 2016; moldavica L. (Janmohammadi et al., 2014) and Ordonez et al., 2006). Multienzyme complexes offer Solanum tuberosum L. (Janmohammadi et al., distinct metabolic advantages to the plant cell, and it 2016). Alam et al. (2012) showed that spraying seems that the natural function of the cellular plant is IAA, IBA, and naphthalene acetic acid (NAA) on obtained from a specific enzyme-enzyme interaction. Catharanthus roseus L. increased plant dry weight, In addition, organic acids are used in the respiration photosynthetic rate, nitrate reductase and carbonic and other biochemical pathways as a source of anhydrase activities, leaf nitrogen and potassium carbon skeleton and energy for the plant cells. Thus, content, seed yield, leaf and root alkaloid content, it has been expressed that increasing endogenous and the amount of vinblastine. However, it seemed organic acids can influence the biomass and yield that plant height and stem diameter contributed more of crops (Da Silva, 2003). Jafari and Hadavi (2012) to the increment in stem weight than the number showed that foliar-applied CA (0.1%, w/v) as 90 Responses to biostimulant, IBA, and citric acid in bladder cherry a biostimulant increased growth, biomass production, diseases and cancers, affecting human health and essential oil yield in basil (Ocimum basilicum (Kumar and Thomas, 2004; Calvo et al., 2014). Thus, L.). IBA is a potential storage form for auxin in the application of MB is important for increasing a variety of plants. It has been found that spraying phenolic compounds and the radical scavenging with various auxin forms can increase plant dry activity of bladder cherry. Finally, it is possible that weight, photosynthetic rate, nitrate reductase and the application of MB has no direct effect on the carbonic anhydrase activities, and leaf nitrogen and production of some plant metabolites, but it can potassium content (Strader et al., 2010; Enders and cause plant growth with an indirect effect on other Strader, 2015). Therefore, it is postulated that IBA metabolic pathways and growth characteristics. has a crucial effect on the sensitivity, transport, and Each enzyme in MB will undoubtedly be able to function of auxin (Enders and Strader, 2015). Our stimulate a particular metabolic pathway. Therefore, results show that MB, IBA, and CA induced growth it will be important to make use of their multiple acceleration in various plant organs such as the leaf, application in the cultivation and production of stem, root, and fruit. This phenomenon may have medicinal plant metabolites. occurred due to improved sink and source relations, increased water absorption and nutrient uptake, CONCLUSIONS abiotic and biotic stress alleviation, hormonal This study revealed a positive effect of MB, IBA, and balancing, etc. (Kauffman et al., 2007; An et al., CA on the growth traits of the stem, leaf, and fruit. 2014; Enders and Strader, 2015). The results of this Also, the optimum combination of these compounds study are in agreement with a study by Kamari et can improve the growth parameters of bladder cherry al. (2013) on tomato, which showed that the use of (Physalis alkekengi L.) as a medicinal plant. The humic acid improved the height, crown diameter, effectiveness of MB can be due to existence of several and fruit number of the tomato plant. In addition, major enzymes that can promote the metabolic the results of this study correspond with those of activities, especially the production of amino acids. the experiment on Vicia faba L. by El-Ghamry et al. MB had the greatest effect on increasing phenolic (2009). They reported a positive effect of a mixture of and alkaloid compounds, and radical scavenging enzymes, humic acid and amino acids on enhancing activity. However, the formulation of 1% MB with yield and growth parameters. Also, it has been 50 ppm IBA and 500 ppm CA is recommended for reported that crop yield can be increased by spraying improving the fruit yield of bladder cherry. The amino acids at different stages of plant development multienzymatic biostimulant had the greatest effect (Liu and Lee, 2012). Plant enzymes with plant on increasing the amounts of alkaloid compounds growth regulators as biostimulants are a necessary as the main pharmaceutical metabolites of bladder component for the production of various amino acids cherry. in protein synthesis, energy production, maintenance of the structural integrity of bio-membranes, and ACKNOWLEDGEMENT growth regulation. Moreover, several amino acids serve as precursors for the synthesis of secondary We thank the research group of the Department of metabolites. Cultivation and Development of Medicinal Plants The present study has revealed that MB (0.5%) for making their laboratory facilities and equipment could enhance the radical scavenging activity and available to us. total content of phenolic and alkaloid compounds in bladder cherry fruit. By comparison, CA and the FUNDING two IBA concentrations increased the total phenolic The research was supported by the Institute of and alkaloid content. It has frequently been reported Medicinal Plants, ACECR. that phenolic compounds play a major role in radical scavenging activity (Kauffman et al., 2007; Calvo AUTHOR CONTRIBUTIONS et al., 2014). The present results are evidence that the change in radical scavenging activity under the All of the authors contributed to all aspects of this various formulations was similar to the change in manuscript, including the development of the ideas, the total phenolic content. Thus, one can conclude writing, and revisions of the content. that the radical scavenging activity in bladder cherry is attributed to the total phenolic content. Oxidative CONFLICT OF INTEREST stress can cause some diseases, such as chronic Authors declare no conflict of interest. Arefeh Rahimi Shokouh, Ali Mehrafarin, Vahid Abdossi, Hassanali Naghdi Badi 91

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