Final SPH -JHS Coverpage 17-1 Jan 2022 single 209 J. Hortl. Sci. Vol. 17(1) : 209-219, 2022 This is an open access article d istributed under the terms of Creative Commons Attribution-NonCommer cial-ShareAl ike 4.0 International License, which permits unrestricted non-commercial use, d istribution, and reproduction in any med ium, provide d the original author and source are credited. Original Research Paper INTRODUCTION Black spot, caused by Diplocarpon rosae is the major destructive a nd dominant disease among different fungal diseases in rose. Owing to the demand and popularity of roses in current flower trade and landscape gardening, breeding for the resistant varieties or developing the varieties that require less care in terms of management against the destructive diseases is the need of the hour. Plants protect themselves through various means of defenses through accumulation of several defense related biochemical compounds following infection by pathogens. Reactive Oxygen Species (ROS) that are produced as initial cellular responses following s u cc es sf u l pa t hogen r ec ognit ion ( Ashr y a nd Mohamed, 2011) have major roles in cell signaling a nd t hes e a r e t he s econda r y mes s enger s f or a ctiva tion of genes tha t encode for pr otective proteins (Lamb and Dixon, 1997 and Mendoza, 2011). However, the increased ROS production causes cellular damage through peroxidation of membrane fatty acids (Lamb and Dixon, 1997) and plants defend against this with up regulation of antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT) a nd peroxida se (POX) (Mittler et al. , 2004). Phenolics a r e toxic to microbes in nature and increase the physical and mechanica l str ength of the host cell wa ll. T he oxidation of these toxic phenolic compounds by polyphenol oxida se (PPO) pr oduces quinones (antimicrobial compounds) that are highly toxic to invading fungi thereby offering resistance against a wide range of pathogens (Cahill and Mccomb, 1992). Phenylalanine ammonia lyase (PAL), one of the key enzymes in the phenyl propanoid pathway, has a role in synthesis of phytoalexin and salicylic acid. Increase in the PAL activity subsequently increases the phenolic contents offering disease resistance to plants (Klessig and Malamy, 1994). Biochemical characterization of defense responses in rose genotypes in response to artificial inoculation with black spot pathogen Diplocarpon rosae Saidulu Y.1*, Tejaswini P., Upreti K.K.2, Sriram S.2, Seetharamu G.K.1, Devappa V.1 and Mythili J.B.2 1College of Horticulture, Bengaluru-65, University of Horticultural Sciences, Bagalkote, Karnataka, 587 104, India. 2 ICAR-Indian Institute of Horticultural Research, Hessaraghatta Lake Post, Bengaluru, Karnataka, 560 089, India. *Corresponding author Email : saidulugowd08@gmail.com ABSTRACT Resistance responses in the leaves of eight rose genotypes, Knock Out (highly resistant), Arka Nishkant (moderately resistant), R. multiflora (highly susceptible), Arka Swadesh (highly susceptible), IIHRR 13-4 (susceptible), Arka Parimala (susceptible), R. indica (susceptible) and IIHRR 4-15-12 (moderately susceptible), exhibiting varied levels of resistance against black spot were investigated post artificial inoculation with black spot pathogen, Diplocarpon rosae. There was consistent increase in the activities of defense related enzymes such as catalase, peroxidase, polyphenol oxidase, superoxide dismutase and phenylalanine ammonia lyase and other defense related secondary metabolites like phenols and flavonoids at different phases of black spot progression and increase was high in resistant genotypes Knock Out and Arka Nishkant. The peak activity of defense enzymes and high concentration of other metabolites was witnessed during early stages of infection in the resistant genotypes while it was during later phase in the susceptible genotypes. These results suggested that the faster and stronger activation of defense system is associated with the resistance against black spot in the rose genotypes. Keywords: Artificial inoculation, diplocarpon rosae, enzymes, flavonoids, phenols, resistance and rose 210 J. Hortl. Sci. Vol. 17(1) : 209-219, 2022 Understanding of plant resistance mechanism against pathogens at various levels provides new opportunities to breed improved cultivars with better resistance to the diseases. There were not many studies in this line on black spot disease infection in rose. Thus, the study was conducted to investigate the role of various defense related enzymes and compounds at different progression periods post infection by black spot pathogen in different rose genotypes possessing differential resistance. MATERIAL AND METHODS Location and climate of experimental site The present investigation was carried out during 2016 at ICAR-IIHR, Bengaluru, which is geographically located at 130 58’ N Latitude, 780 E Longitude and at an elevation of 890 m above mean sea level with an average annual rainfall of about 890 mm. Plant material The rose genotypes evaluated in the present study were part of rose breeding program at ICAR-Indian Institute of Horticultural Research, Bengaluru. A total of eight rose genotypes were used for the study in completely randamized design (CRD). The plants were provided with all inputs as per the package of practices for rose cultivation except for fungicidal sprays during the period of investigation. Young and healthy leaves from 4th to 6th node from apex of the shoot were collected (Dong, 2014) ran damly from three plants of the selectedfrom the selected genotypes viz., R. multiflora (highly susceptible), Ar ka Swa desh (highly susceptible), IIHRR 13-4 (susceptible), Arka Parimala (susceptible), R. indica (susceptible), IIHRR 4-15-12 (moderately susceptible), Arka Nishkant (moderately resistant) and Knock Out (highly resistant) in three replications. The collected leaves were cleaned with deionized sterile water and wiped with sterile tissue paper. Preparation of conidia suspension Rose leaves, severely infected with black spot, preferably with yellow halo around the spots were collected and surface cleaned with sterile tissue paper. Later, the infected leaf portions were cut and submerged in deionized sterile distilled water in the sterile tubes. The tubes were kept in orbital shaker for one minute after adding two drops of Tween- 2 0 . T he s u s p ens ion wa s f ilt er ed a nd t he concentration of conidia in the filtrate was adjusted t o 2 × 1 0 4 c oni dia / ml ( L e u s , 2 0 0 5 ) u s ing ha emoc yt ome t er. T his f ilt r a t e w a s u s ed f or inoculation of leaves. The spores from pure culture of the pathogen maintained on detached leaves was used for further inoculation of healthy leaves in the present study. Artificial inoculation of leaves The inoculation of excised leaves with D. rosae was performed as described by Debener et al. (1998). The cleaned healthy leaves were placed on moist blotting paper, with their petioles wrapped in moist cotton plugs in glass petri plates to maintain 100 per cent humidity. On each leaflet surface, 4-6 dr op lets of 10µ L c onidia l suspension (2 ×10 4 conidia/ml) was pipetted out in laminar air flow to avoid contamination. The inoculated leaves were then incubated at ~25ºC under 10 h photoperiod for two weeks (Dong, 2014). Enzyme assays: The infected and control leaves were analyzed for the activities of following defense related biochemical compounds on every third day i.e., on 0, 3, 6, 9, 12 and 15 days after inoculation (DAI). POX activity (EC 1.11.1.7) (Enzyme units/ g fresh weight -EU/g FW): The POX activity was determined by following same method described by Chander (1990) and enzyme activity was expressed as enzyme units g/ FW. PPO activity (EC 1.10.3.1) (EU/g FW): The PPO activity was determined following the method of Selvaraj and Kumar (1989) without any modifications and the enzyme activity was expressed as EU/g FW. CAT activity (EC 1.11.1.6) (EU/mg FW): The CAT activity was determined by following the same procedure as per Masia (1998) and activity was expressed as EU/mg FW. PAL activity (EC 4.3.1.24) (EU/g FW): The PAL activity was estimated as per the same procedure followed by Hodgins (1971) and activity wa s expressed as EU/g FW. SOD activity (EC 1.15.1.1) (EU/mg FW): The SOD activity was estimated as per the same procedure followed by Du and Bramlage (1994) and activity was expressed as EU/mg FW. Saidulu et al 211 Total phenols (mg/ g fresh weight): Total phenol content was estimated by same procedure followed by Singleton and Rossi (1965) by spectrophotometric method using Folin-Ciocalteau’s Phenol Reagent (FCR) and the phenol content was expressed as mg/g FW. Total flavonoids (mg/ g fresh weight): Total flavonoid content was estimated by the spectrophotometric method using same procedure as followed by Chun et al. (2003) and total flavonoid content was expressed as mg/g FW. RESULTS AND DISCUSSION a) POX activity Analysis of data revealed that POX activity increased significantly in inoculated leaves of all genotypes after infection with the pathogen (Fig S1, S2). Among the inoculated leaves (I2) of all genotypes, highest peroxidase activity (1.84 EU/ g FW) was observed in highly resistant variety Knock Out, on 6th day after inoculation (G8D3) which was almost 1.4 times to the maximum a ctivity found in highly susceptible genotype, R. multiflora (1.29 EU/ g FW) that was observed on 9th day after inoculation (G1D4) (Table 1). In the va r iety Ar ka Nishka nt, which wa s moderately resistant, peak enzyme activity was observed (1.76 EU/ g FW) on 12th day (G7D5). No significant changes in enzyme activity were found in un-inoculated leaves (I1) of all genotypes during entire observation period (Fig. S1, S2) (data not presented). Peroxidase is involved in biosynthesis of lignin and other oxidized phenols (Bruce and West, 1989). Peroxidase mediates the oxidation of phenols to oxidized phenols that are highly toxic to the pathogen (Sequeira, 1983). Thus, the increased activity of peroxidase in infected tissues contributes to resistance by inhibiting the pathogen growth. In the present study, resistant genotypes have recorded quick and high peroxidase activity compared to susceptible ones. Due to the increased activity of peroxidase at early stages of infection, the pathogen growth was hindered and thus offered resistance against black spot. Similar increased activity of peroxidase in resistant genotypes in response to pathogen infection has been reported in Fusarium infection in melon (Hanifei et al., 2013), Botrytis infection in faba bean (El-Komy, 2014) and brown rust infection in wheat (Riaz et al., 2014). b) PPO activity PPO activity increased in inoculated leaves of all genotypes in response to the pathogen inoculation (Fig. S3, S4). At a given time period on 3rd (D2) and 6th (D3), highest PPO activity (2.62 EU/ g FW and 3.13 EU/ g FW respectively) was found in highly resistant variety Knock Out (G8D2 and G8D3) respectively) whereas the lowest activity (0.85 EU/ g FW and 1.84 Table 1. POX activity (EU/g FW) in D. rosae inoculated leaves (I2) of rose genotypes (G) at different intervals after inoculation (D) POX activity (EU/g FW) in D. rosae inoculated leaves Sl.No. Genotypes (G) (I2) at different days interval after inoculation (D) Day 0 Day 3 Day 6 Day 9 Day 12 Day 15 (D1) (D2) (D3) (D4) (D5) (D6) 1 R. multiflora (G1) 0.47 0.68 1.22 1.29 1.18 0.71 2 Arka Swadesh (G2) 0.38 0.69 1.25 1.38 1.23 0.8 3 IIHRR 13-4 (G3) 0.46 0.71 1.3 1.38 1.22 1.02 4 Arka Parimala (G4) 0.55 0.93 1.32 1.56 1.39 1.04 5 R. indica (G5) 0.47 1.01 1.44 1.52 1.62 1.65 6 IIHRR 4-15-12 (G6) 0.49 0.78 1.31 1.7 1.59 1.65 7 Arka Nishkant (G7) 0.44 1.48 1.69 1.68 1.76 1.71 8 Knockout (G8) 0.46 1.81 1.84 1.74 1.71 1.64 S.Em ± 0.06 - - - - - C.D. @ 5% 0.18 - - - - - J. Hortl. Sci. Vol. 17(1) : 209-219, 2022 Biochemical changes in rose genotypes in respons to black spot 212 EU/ g FW respectively) was recorded in highly susceptible genotype R. multiflora (G1D2 and G1D3 respectively) (Table 2). This revealed that the enzyme activity in resistant genotype increased immediately in response to the pathogen infection whereas the enzyme a ctivity increased slowly a nd gr adua lly in the susceptible genotype. Among all genotypes, highest activity of PPO in inoculated leaves (I2) (3.64 EU/ g FW) was recorded in moderately resistant genotype Arka Nishkant on 9th day after inoculation (G7D4). No significant changes in enzyme activities were found in un-inoculated leaves (I1) of all genotypes during entire observation period (Fig. S3, S4) (data not presented). PPO catalyzes the oxidation of phenols released due to membrane damage (Siddique et al., 2014) during microbial invasion into oxidized phenols i.e., quinones that are more reactive and highly toxic (Batsa, 2004) which creates toxic environment for pathogen development (Jockusch, 1966 and Mohamed et al., 2012). Thus increase in PPO activity is associated with resistance. In present study, PPO activity increased quickly with pathogen inoculation in resistant genotypes wher ea s the incr ea se in susceptible genotypes was slow and less. This early increase in activity of PPO in resistant genotypes inhibited the fungal growth and thereby contributed for resistance in resistant genotype. These results are in conformity with the findings of Khatun et al. (2009) who reported increased activity of PPO in black spot (Alternaria tenuis) infected resistant rose leaf tissues during progression of disease. Highest activity of PPO was also reported in Fusarium infected resistant melon genotypes compared to susceptible ones (Hanifei et al., 2013). c) CAT activity The inoculated lea ves of all genotypes showed significantly higher levels of CAT activity during the period of observation than those of un-inoculated controls (Fig. S5, S6). In highly resistant genotype Knock Out, the CAT activity increased sharply and reached its peak (19.83 EU/ mg FW) on 12th day after inoculation (G8D5), whereas in R. multiflora which was highly susceptible, the CAT activity increased comparatively at a slower pace and reached its peak (8.71 EU/ mg FW) on 9th day (G1D4), followed by a decrease by 15th day (7.15 EU/ mg FW) (G1D6). When the CAT activity of all genotypes was compared on third day (D2) immediately after pathogen inoculation, the highest activity (16.06 EU/ mg FW) was observed in highly r esistant genotype Knock Out (G 8D2) whereas lowest activity (7.64 EU/ mg FW) was found in Arka Swadesh (G2D2) which was highly susceptible to the disease. No significant changes were detected in control leaves (I1) throughout the observation period (Fig. S5, S6) (data not presented). CAT is one of the important H2O2 scavenging enzymes that eliminate the toxic effects of H2O2 through a mechanism known as Halliwell–Asada–Foyer pathway J. Hortl. Sci. Vol. 17(1) : 209-219, 2022 Saidulu et al Table 2. PPO activity (EU/g FW) in D. rosae inoculated leaves (I2) of rose genotypes (G) at different intervals after inoculation (D) PPO activity (EU/g FW) in D. rosae inoculated leaves Sl.No. Genotypes (G) (I2) at different days interval after inoculation (D) Day 0 Day 3 Day 6 Day 9 Day 12 Day 15 (D1) (D2) (D3) (D4) (D5) (D6) 1 R. multiflora (G1) 0.59 0.85 1.84 2.65 2.45 2.19 2 Arka Swadesh (G2) 0.53 0.99 2.05 2.82 2.68 2.35 3 IIHRR 13-4 (G3) 0.57 1.02 2.16 2.30 2.52 2.44 4 Arka Parimala (G4) 0.57 1.53 2.41 3.09 3.24 3.33 5 R. indica (G5) 0.69 1.13 2.32 2.88 2.60 2.46 6 IIHRR 4-15-12 (G6) 0.74 2.11 2.82 3.35 3.47 3.54 7 Arka Nishkant (G7) 0.74 2.27 2.94 3.64 3.44 3.40 8 Knockout (G8) 0.70 2.62 3.13 3.29 3.39 3.42 S.Em ± 0.07 - - - - - C.D. @ 5% 0.18 - - - - - 213 (Hanifei et al., 2013). It protects the plant cells from oxidative damage caused by ROS (Gill and Tuteja, 2010). The results of present study revealed that inocula ted lea ves of a ll genotypes showed sig­nificantly higher levels of CAT activity than those of un-inoculated controls. In inoculated leaves of both resistant and susceptible genotypes, the CAT activity increased during the progress of infection. However, induced levels of CAT were significantly higher during progression of infection in the inoculated leaves of resistant genotypes compared to those of susceptible genotypes. These differences in CAT activity in present study suggested that the low enzyme activity in susceptible genotypes made them less efficient in reducing the high levels of H2O2 produced during D. rosae infection. These results are in agreement with the findings of El-Komy (2014) who r epor ted increased activity of CAT in resistant genotypes over susceptible ones after inoculation with chocolate spot pathogen of faba bean. Mandal et al. (2008) have also r epor ted tha t a less efficient enzyma tic ROS scavenging system, mainly a decrease in CAT activity caused high level of damage caused by F. oxysporum f. sp. Lycopersici, in tomato. (El-Komy, 2014). d) PAL activity The inoculated lea ves of all genotypes showed significantly higher levels of PAL activity than those of un-inoculated controls (Fig S7, S8). PAL activity changed significantly in inoculated leaves (I2) of all genotypes with progression of time after inoculation. In inoculated leaves of all genotypes, PAL activity increased in response to pathogen infection and reached peak by 9th day in all genotypes except in highly resistant variety Knock Out where the peak activity was observed on 6th day itself. Further, the enzyme activity got decreased slightly by 15th day in all genotypes after reaching peak (Fig. S7). In highly resistant variety Knock Out, maximum activity that was recorded on 6th day was 2.95 EU/ g FW (G8D3) whereas in R. multiflora which was highly susceptible, peak PAL activity was recorded as 1.48 EU/ g FW (G1D4) which was observed on 9 th day. No significant changes were detected in control leaves (data not pr esented) thr oughout the obser va tion per iod (Fig. S7, S8). PAL is primary enzyme in the phenylpropanoid metabolism and plays a significant role in the synthesis of several defense-related secondary compounds such J. Hortl. Sci. Vol. 17(1) : 209-219, 2022 Biochemical changes in rose genotypes in respons to black spot as phenols and lignin (Hemm et al. 2004; Tahsili et al. 2014). The activation of PAL and subsequent increase in phenolic content in plants is a general response associated with disease resistance (Siddique et al. 2014). Results of present study revealed that PAL activity was high in resistant genotype compared to susceptible genotypes. This increased activity of PAL in r esista nt genotypes have lea d to mor e production of defense related secondary compounds which conferred protection against disease. The increased activity of PAL in defense against fungal pathogens in resistant genotypes was also reported in case of brown rust interactions in wheat (Riaz et al., 2014) e) SOD activity The SOD activity changed significantly in inoculated leaves of all genotypes with progression in days after inoculation (Fig S9, S10). At a given time period on third day (D2) immediately after inoculation, highest SOD activity (2.99 EU/ mg FW) was found in moderately resistant genotype Arka Nishkant (G7D2) wher e the lowest a ctivity (1. 50 EU/ mg FW respectively) was recorded in highly susceptible genotype R. multiflora (G1D2). The highly resistant genotype Knock Out (G8) recorded SOD activity equivalent to 2.91 EU/ mg FW on third day (G8D2). On sixth day (D3) after inoculation, highest SOD activity among all genotypes (3.76 EU/ mg FW) was found in highly resistant genotype Knock Out (G8D3) where the lowest activity (2.08 EU/ mg FW) was recorded in highly susceptible genotype R. multiflora (G1D3). This revealed that the enzyme activity in resistant genotype increased immediately in response to the pathogen infection whereas the enzyme activity increased gradually at a slower pace in susceptible genotype. The SOD activity in highly r esistant genotype Knock Out reached its peak on 6th day (3.76 EU/ mg FW) (G8D3) after inoculation and thereafter decreased by 15th day (2.21 EU/ mg FW) (G8D6) whereas in highly susceptible genotype R. multiflora, the activity remained increasing throughout the observation period and reached peak on 15th day (2.60 EU/ mg FW) (G1D6). No significant changes in enzyme activity were detected in control leaves throughout the observation period (Fig S9, S10) (data not presented). SOD is one of the important reactive oxygen species scavenging enzymes which catalyzes the dismutation of superoxide anion radicals (O2-) into H2O2 and O2 214 (Smirnoff, 1993; Khan and Panda, 2008). H 2O2 generation in infected plants is considered one of the important defense strategies of plants against the invading necrotrophic pathogen (Hanifei et al., 2013). Results of present study revealed that increased SOD activity was observed in both resistant and susceptible genotypes but the increase was more and quick in resistant ones, in response to pathogen inoculation. In case of susceptible genotypes, though there was increase in enzyme activity, it may not be adequate and quick enough to counter pathogen development, making them susceptible to the disease. Similar results of higher SOD activity in resistant cultivar over susceptible cultivar, after pathogen inoculation were reported in case of chocolate spot disease of faba bean (El-Komy, 2014) and Mycosphaerella fragariae infection in strawberry (Ehsani-Moghaddam et al. 2006). f) Total phenols The inoculated lea ves of all genotypes showed significantly higher levels of total phenols during t he p er iod of ob ser va t ion t ha n t hose of u n- inoculated controls (Fig S11, S12). In inoculated leaves (I2) of all genotypes, total phenols changed significantly with progression in time period after inocula tion a nd reached their peak on 9th day inoculation (D4) and thereby decreased by 15th day (D6) (Table 6). In highly resistant genotype Knock Out, the total phenols increased sharply and reached peak (81.94 mg/g FW) on 9th day (G8D4) whereas in R. multiflora which was highly susceptible, total phenols increased comparatively at a slower rate and reached its peak (49.84 mg/g FW) on 9th day (G 1D4). When the tota l phenols content of all genot yp es wa s c omp a r ed on t hi r d da y ( D 2 ) immediately after pathogen inoculation, highest accumulation (71.94mg/g FW) was observed in highly r es is ta nt genotype K nock O ut ( G 8 D 2 ) whereas lowest accumulation (31.59 mg/g FW) was found in IIHRR 13-4 (G3D2) which was susceptible to the disease. No significant changes in enzyme activity were detected in control leaves throughout the observation period (Fig S11& S12) (data not presented). Phenols enhance the mechanical strength of host cell walls by synthesis of lignin and suberin which are involved in the formation of physical barriers that can block the spread of pathogens (Ngadze et al. 2012; Singh et al. 2014). Further, Khatun et al., 2009 reported that the phenols are fungitoxic in nature. In the present study, the amount of total phenols was significantly higher in inoculated leaves of resistant genotypes, while it wa s significa ntly lower in susceptible genotypes. Thus, high accumulation of phenols in resistant genotypes may be playing role in eliciting resistance response against black spot pathogen. The increased phenolic content in resistant genotypes after pathogen inoculation was also reported in case of chocolate spot disease of faba bean (El- Komy, 2014) and in cotton interaction with cotton leaf curl Burewala virus (Siddique et al., 2014). g) Total flavonoids The results revealed that inoculated leaves of all genotypes showed significantly higher levels of total flavonoids during the period of observation than those of un-inoculated controls (Fig. S13, S14). Total flavonoids changed significantly in inoculated leaves (I2) of all genotypes with increase in number of days after inoculation and showing their peak on 9th day after inoculation (D4) and further decreased by 15th day (D6) (Ta ble 7). In highly r esista nt Knock Out genotype, total flavonoids increased sharply and reached peak (35.11 mg/g FW) on 9th da y a f t er ino c u la t ion ( G 8 D 4) wher e a s in R . multiflora which was highly susceptible, total flavonoids increased comparatively at a slower rate and reached peak (18.33 mg/g FW) on 9 th day (G1D4). When the total flavonoids of all genotypes were compared on third day (D2) immediately after pathogen inoculation, highest accumulation (28.73 mg/ g F W ) wa s obs er ved in highly r esis t a nt genotyp e K noc k O ut (G 8 D 2 ) wher ea s lowes t a ccumula tion (10. 57 mg/g FW) wa s found in IIHRR 13-4 (G3D2) which is susceptible to the disease. No significant changes were detected in control leaves throughout the observation period (Fig. S13, S14) (data not presented). Flavonoids are very important in plant resistance against pathogenic bacteria and fungi. Antipathogenic properties of flavonoids can be non-specific in nature and partly could be the result of their antioxidative properties. Flavonoid compounds are transported to the site of infection and induce the hypersensitivity reaction, which is the earliest defense mechanism employed by the infected plants and programmed cell death (Mierziak et al., 2014) thus restrict the spread J. Hortl. Sci. Vol. 17(1) : 209-219, 2022 Saidulu et al 215 J. Hortl. Sci. Vol. 17(1) : 209-219, 2022 Biochemical changes in rose genotypes in respons to black spot PAL activity (EU/g FW) in D. rosae inoculated leaves (I2) Sl.No. Genotypes (G) at different days interval after inoculation (D) Day 0 Day 3 Day 6 Day 9 Day 12 Day 15 (D1) (D2) (D3) (D4) (D5) (D6) 1 R. multiflora (G1) 0.60 0.86 1.36 1.48 1.41 1.33 2 Arka Swadesh (G2) 0.71 1.18 1.51 1.80 1.68 1.48 3 IIHRR 13-4 (G3) 0.88 1.41 1.60 1.91 1.73 1.46 4 Arka Parimala (G4) 0.85 1.51 1.79 2.07 1.98 1.68 5 R. indica (G5) 0.85 1.40 1.76 1.86 1.70 1.59 6 IIHRR 4-15-12 (G6) 0.57 1.87 2.03 2.22 2.14 2.10 7 Arka Nishkant (G7) 0.63 1.94 2.25 2.36 2.24 2.21 8 Knockout (G8) 0.69 2.48 2.95 2.91 2.71 2.51 S.Em ± 0.02 - - - - - C.D. @ 5% 0.06 - - - - - Table 4. PAL activity (EU/g FW) in D. rosae inoculated leaves (I2) of rose genotypes (G) at different intervals after inoculation (D) Table 3. CAT activity (EU/mg FW) in D. rosae inoculated leaves (I2) of rose genotypes (G) at different intervals after inoculation (D) CAT activity (EU/mg FW) in D. rosae inoculated leaves (I2) Sl.No. Genotypes (G) at different days interval after inoculation (D) Day 0 Day 3 Day 6 Day 9 Day 12 Day 15 (D1) (D2) (D3) (D4) (D5) (D6) 1 R. multiflora (G1) 6.78 8.10 8.10 8.71 7.73 7.15 2 Arka Swadesh (G2) 5.08 7.64 9.65 10.71 9.44 8.91 3 IIHRR 13-4 (G3) 5.38 8.65 7.87 6.54 5.68 5.88 4 Arka Parimala (G4) 6.60 9.40 10.18 10.63 9.61 8.70 5 R. indica (G5) 5.58 9.83 8.39 7.65 6.10 5.83 6 IIHRR 4-15-12 (G6) 6.69 12.08 15.02 16.77 17.44 18.28 7 Arka Nishkant (G7) 6.55 13.12 16.27 17.86 18.88 17.39 8 Knockout (G8) 4.50 16.06 18.01 18.48 19.83 17.21 S.Em ± 0.18 - - - - - C.D. @ 5% 0.51 - - - - - 216 J. Hortl. Sci. Vol. 17(1) : 209-219, 2022 Saidulu et al Table 5. SOD activity (EU/mg FW) in D. rosae inoculated leaves (I2) of rose genotypes (G) at different intervals after inoculation (D) SOD activity (EU/mg FW) in D. rosae inoculated leaves (I2) Sl.No. Genotypes (G) at different days interval after inoculation (D) Day 0 Day 3 Day 6 Day 9 Day 12 Day 15 (D1) (D2) (D3) (D4) (D5) (D6) 1 R. multiflora (G1) 1.18 1.50 2.08 2.31 2.41 2.60 2 Arka Swadesh (G2) 1.20 1.75 2.35 2.27 2.08 1.62 3 IIHRR 13-4 (G3) 1.23 1.86 2.44 2.53 2.60 2.57 4 Arka Parimala (G4) 1.18 1.97 2.53 2.76 2.59 2.63 5 R. indica (G5) 1.09 2.02 2.67 2.82 2.81 2.68 6 IIHRR 4-15-12 (G6) 1.32 2.80 3.30 3.48 3.55 3.58 7 Arka Nishkant (G7) 1.17 2.99 3.42 3.60 3.30 3.43 8 Knockout (G8) 1.19 2.91 3.76 3.10 2.61 2.21 S.Em ± 0.03 - - - - - C.D. @ 5% 0.07 - - - - - Table 6. Total phenols (mg/g FW) in D. rosae inoculated leaves (I2) of rose genotypes (G) at different intervals after inoculation (D) Total phenols (mg/g FW) in D. rosae inoculated leaves (I2) Sl.No. Genotypes (G) at different days interval after inoculation (D) Day 0 Day 3 Day 6 Day 9 Day 12 Day 15 (D1) (D2) (D3) (D4) (D5) (D6) 1 R. multiflora (G1) 35.78 42.27 47.07 49.84 45.72 39.14 2 Arka Swadesh (G2) 28.45 36.40 40.48 43.36 41.33 36.17 3 IIHRR 13-4 (G3) 26.84 31.59 37.27 46.66 41.25 37.03 4 Arka Parimala (G4) 34.31 44.79 52.81 56.63 51.49 45.72 5 R. indica (G5) 28.38 41.27 46.46 52.84 49.78 41.42 6 IIHRR 4-15-12 (G6) 35.20 54.72 67.07 74.04 68.35 57.41 7 Arka Nishkant (G7) 26.99 54.27 58.09 62.84 59.61 49.93 8 Knockout (G8) 33.15 71.94 76.77 81.94 71.11 65.91 S.Em ± 0.66 - - - - - C.D. @ 5% 1.84 - - - - - 217 J. Hortl. Sci. Vol. 17(1) : 209-219, 2022 Biochemical changes in rose genotypes in respons to black spot of pathogen. In the present study, the amount of total flavonoids was significantly higher in inoculated leaves of resistant genotypes, while it was significantly lower in susceptible genotypes. This high accumulation of fla vonoids in r esista nt genotypes might ha ve contributed for the resistance. Resistance against the fungal infection due to increased accumulation of flavonoids in leaves was also reported in interaction of cedar-apple rust pathogen and apple trees (Lu et al., 2017). CONCLUSION The changes in activity of defense related enzymes like CAT, POX, PPO, SOD and PAL and accumulation of plant defense related secondary compounds like phenols and flavonoids were distinguished clearly in inoculated leaves compared to un-inoculated leaves. Further, the trend of either increase or decrease in activity of defense related biochemical c omp ou nds wa s mor e p r ominent a nd va r ied significantly among the studied genotypes with progression in time period of black spot disease. All studied defense related biochemical compounds increased drastically faster in higher quantities in r es ist a nt genot yp es compa r ed to su sc ept ible genotypes during disease progression contributing for resistance. Table 7. Total flavonoids (mg/g FW) in D. rosae inoculated leaves (I2) of rose genotypes (G) at different intervals after inoculation (D) Total flavonoids (mg/g FW) in D. rosae inoculated leaves (I2) Sl.No. Genotypes (G) at different days interval after inoculation (D) Day 0 Day 3 Day 6 Day 9 Day 12 Day 15 (D1) (D2) (D3) (D4) (D5) (D6) 1 R. multiflora (G1) 11.86 14.48 17.10 18.33 17.62 15.41 2 Arka Swadesh (G2) 7.45 12.93 14.11 16.34 14.19 12.96 3 IIHRR 13-4 (G3) 4.46 10.57 14.46 15.36 12.82 11.11 4 Arka Parimala (G4) 13.92 20.73 24.89 26.38 24.91 19.48 5 R. indica (G5) 10.85 18.81 24.63 27.45 24.12 19.41 6 IIHRR 4-15-12 (G6) 13.13 22.87 27.10 29.55 28.89 21.14 7 Arka Nishkant (G7) 4.60 18.45 21.57 23.85 20.94 18.85 8 Knockout (G8) 12.38 28.73 32.63 35.11 29.54 19.95 S.Em ± 0.25 - - - - - C.D. @ 5% 0.68 - - - - - REFERENCES Ashry, N.A and Mohamed, H.I., 2011, Impact of secondary metabolites and related enzymes in flax resistance and or susceptibility to powdery mildew. Afr. J. 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Vol. 17(1) : 209-219, 2022 Biochemical changes in rose genotypes in respons to black spot (Received:14.07.2021; Revised: 17.02.202; Accepted: 15.03.2022) 00 A Final SPH -JHS Coverpage First 2 pages.pdf 00 Content and in this issue.pdf 01 Mohan Kumar G N.pdf 02 Meera Pandey.pdf 03 Biradar C.pdf 04 Varalakshmi B.pdf 05 Vijayakumari N.pdf 06 Barik S.pdf 07 Sajid M B.pdf 08 Ranga D.pdf 09 Usha S.pdf 10 Manisha.pdf 11 Amulya R N.pdf 12 Akshatha H J.pdf 13 Adak T.pdf 14 Sujatha S.pdf 15 Gowda P P.pdf 16 Subba S.pdf 17 Dhayalan V.pdf 19 Ahmed S.pdf 20 Vishwakarma P K.pdf 21 Deep Lata.pdf 22 Udaykumar K P.pdf 23 Nayaka V S K.pdf 24 Sahel N A.pdf 25 Bayogan E R V.pdf 26 Rathinakumari A C.pdf 27 Yella Swami C.pdf 28 Saidulu Y.pdf 29 Sindhu S.pdf 30 Neeraj.pdf 31 Sivaranjani R.pdf 32 Rashied Tetteh.pdf 34 Sangeetha G.pdf 35 Shareefa M.pdf 36 Last Pages.pdf