Studies on exudate-depleted sclerotial development in Sclerotium ...

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Studies on exudate-depleted sclerotial development in Sclerotium rolfsii and the effect of oxalic acid, sclerotial exudate, and culture filtrate on phenolic acid induction in chickpea (Cicer arietinum) U.P. Singh, B.K. Sarma, D.P. Singh, and Amar Bahadur

Abstract: Exudate depletion from developing sclerotia of Sclerotium rolfsii Sacc. in culture caused reduced size and weight of sclerotia. Germination of exudate-depleted sclerotia was delayed on Cyperus rotundus rhizome meal agar medium when compared with that of control sclerotia. The exudate-depleted sclerotia caused infection in chickpea (Cicer arietinum) plants in a glasshouse. Different temperatures and incubation periods had no effect on the germination ability of the exudate-depleted sclerotia. Oxalic acid, sclerotial exudate, and culture filtrate of S. rolfsii induced the synthesis of phenolic acids, including gallic, ferulic, chlorogenic, and cinnamic acids, as well as salicylic acid, in treated chickpea leaves. Gallic acid content was increased in treated leaves compared with the untreated controls. Maximum induction of gallic acid was seen in both leaves treated with oxalic acid followed by exudate and leaves treated with culture filtrate. Cinnamic and salicylic acids were not induced in exudate-treated leaves. Ethyl acetate fractionation indicated that the sclerotial exudates consisted of gallic, oxalic, ferulic, chlorogenic, and cinnamic acids, whereas the culture filtrate consisted of gallic, oxalic, and cinnamic acids along with many other unidentified compounds. Key words: oxalic acid, phenolic acid, salicylic acid, sclerotial exudate, culture filtrate, Sclerotium rolfsii, sclerotial germination. Résumé : L’appauvrissement des exsudats de sclérotes en développement de Sclerotium rolfsii Sacc. cultivé a réduit la taille et la masse de ceux-ci. La germination de sclérotes appauvris en exsudats a été retardée sur milieu agar nutritif de rhizhome de Cyperus rotundus comparativement aux sclérotes témoins. Les sclérotes appauvris en exsudats ont été capables d’infecter des plants de pois chiches (Cicer arietinum) en serre. Des températures et des périodes d’incubation diverses n’ont pas eu d’effet sur la viabilité germinative des sclérotes appauvris en exsudats. L’acide oxalique, l’exsudat scléroteux et du filtrat de culture de S. rolfsii ont induit la synthèse d’acides phénoliques, incluant l’acide gallique, férulique, chlorogénique, cinnamique de même de salicylique, dans des feuilles de pois chiches traitées. Le contenu en acide gallique était plus élevé dans les feuilles traitées que dans les témoins non traités. L’induction maximale d’acide gallique a été observé chez les feuilles traitées à l’acide oxalique, suivie par les feuilles traitées avec l’exsudat et avec le filtrat de culture. Les acides cinnamique et salicylique n’ont pas été induits chez les feuilles traitées avec l’exsudat. Une fractionnement à l’acétate d’éthyle a indiqué que les exsudats scléroteux étaient constitués d’acides gallique, oxalique, férulique, chlorogénique et cinnamique, alors que le filtrat de culture était constitué d’acides gallique, oxalique et cinnamique ainsi qu’un grand nombre d’autre composés non identifiés. Mots clés : acide oxalique, acide phénolique, acide salicylique, exsudat scléroteux, filtrat de culture, Sclerotium rolfsii, germination de sclérotes. [Traduit par la Rédaction]

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Introduction Sclerotium rolfsii Sacc. is a devastating soilborne pathoReceived 20 November 2001. Revision received 18 March 2002. Accepted 11 April 2002. Published on the NRC Research Press Web site at on 17 May 2002. U.P. Singh,1 B.K. Sarma, D.P. Singh, and A. Bahadur. Department of Mycology and Plant Pathology, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi – 221005, India. 1

Corresponding author (e-mail: [email protected]).

Can. J. Microbiol. 48: 443–448 (2002)

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gen of many dicotyledonous and monocotyledonous plant species (Amin 1976; Aycock 1966; Lucas 1976; Odvody and Madden 1984; Punja et al. 1982; Singh and Pavgi 1965). The pathogen causes crown (collar) rot in susceptible crops, thereby killing infected plants. Tissue death precedes hyphal penetration of infected plants because of the production of oxalic acid and polygalacturonases (Bateman 1972), which act in concert (Bateman and Beer 1965; Punja et al. 1985). Oxalic acid sequesters calcium from the cell walls to form calcium oxalate (Punja and Jenkins 1984) and lowers the tissue pH to the optimum for endopolygalacturonase and cellulase activities (Bateman 1969, 1972; Bateman and Beer 1965; Punja et al. 1985). Oxalic acid is also reported to be

DOI: 10.1139/W02-040

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directly toxic to plant tissue (Bateman and Beer 1965; Franceschi and Horner 1980; Hodgkinson 1977). However, according to some recent reports, synthetic oxalic acid can induce resistance in plant tissues against pathogen attack (Zhang and Liu 1992; Wang et al. 1995; Toal and Jones 1999; Nabila 1999). Toal and Jones (1999) reported successful protection of oilseed rape against Sclerotinia sclerotiorum by application of oxalic acid that induced resistance. Similarly, Nabila (1999) reported protection of squash plants from cucumber mosaic virus by application of oxalic acid that induced resistance in host plants. The objectives of the present investigation were to determine the impact of exudate depletion on sclerotial development, survival, and pathogenicity. The effect of synthetic oxalic acid, sclerotial exudate, and culture filtrates of S. rolfsii, known to contain oxalic acid (Punja 1985), on the profiles of phenolic compounds and salicylic acid, after application to chickpea (Cicer arietinum) plants was also studied.

Materials and methods Exudate depletion experiment Mycelium of an actively growing isolate of S. rolfsii from chickpea grown on potato dextrose agar (PDA; peeled potato 200 g, dextrose 20 g, agar 15 g, distilled water 1 L) was cut with a 5 mm diameter cork borer, transferred to the center of 100 × 15 mm petri dishes containing PDA, and incubated at 25 ± 2°C. The plates were observed regularly for sclerotium formation as well as the presence of exudation on the sclerotial primordia (Christias 1980), which was typically formed after 7–10 days. Exudate droplets were removed with a sterilized capillary tube and stored at 4°C in screw-capped glass tubes. After exudate removal, sclerotial development was observed in relation to fresh weight and sclerotial diameter of 100 matured exudate-depleted sclerotia and compared with 100 nondepleted sclerotia of the same age. The experiment was repeated three times and the data were pooled before being subjected to ANOVA for statistical significance by the Student’s t test at P = 0.01. Exudate-depleted sclerotial germination on PDA and Cyperus rotundus rhizome meal agar medium and their pathogenicity Fifty matured sclerotia (21-day-old), following removal of exudate, were placed singly on either PDA or Cyperus rotundus rhizome meal agar (CRMA) (Prithiviraj et al. 2000) in sterilized petri plates and incubated at 25 ± 2°C to assess germination. Nondepleted sclerotia of the same age were also kept similarly on both media. Germination of exudate-depleted sclerotia was assessed visually every 24 h with a hand lens, and germination percentage was compared with the nondepleted sclerotia. The exudate-depleted and nondepleted sclerotia were inoculated (10 sclerotia/10 g soil) separately onto 15-day-old C. arietinum plants grown in plastic pots (20 cm diameter) containing field soil (sandy loam, pH 7.6) in a glasshouse regulated alternatively with 12 h light and 12 h darkness at a temperature of 25–28°C for testing pathogenicity of these sclerotia. Percent seedling mortality was recorded after 10 days of sclerotial inoculation. Five pots for each treatment served as one replication,

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and the experiment was conducted in triplicate. The experiments on sclerotial germination and their pathogenicity was repeated twice and thrice, respectively. The means of repeated experiments were pooled for comparison. Effect of temperature on exudate-depleted sclerotial germination Twenty-one-day-old mature sclerotia (after depletion of exudate) or nondepleted mature sclerotia (100 each) of the same age were mixed with 25 g of field soil (sandy loam, pH 7.6) and incubated at 25, 30, 35, 40, and 45°C for 24, 48, and 96 h. The sclerotia were extracted, washed three times in sterilized distilled water, and transferred to PDA (five per plate) containing 100 µg/mL streptomycin. At least six plates were kept for each type of sclerotia per treatment. The plates were incubated at 25 ± 2°C and sclerotial germination was regularly observed with a hand lens. Moisture depletion from the soil was also recorded for the different time intervals by assessing moisture content of 25 g of soil kept separately at the above temperatures. The objective of assessing moisture depletion was to eliminate the probable effect of the dried soil on sclerotial germination. The analysis was conducted in triplicate, and the whole experiment was repeated twice. Application of oxalic acid, exudate, and culture filtrate of S. rolfsii on C. arietinum and extraction of phenolic compounds A solution of oxalic acid (4 and 8 mM), sclerotial exudate, and culture filtrate (both diluted by 50% and undiluted (100% of the collected form)) were applied to 21-dayold seedlings of C. arietinum (cv. Avrodhi) grown in 20 cm diameter plastic pots containing the same field soil described earlier. The culture filtrate was prepared by transferring singly a 5-mm disc of actively growing mycelia of S. rolfsii on PDA into a 150-mL Erlenmeyer flask containing 50 mL sterilized potato dextrose broth and incubated at 25 ± 2°C for 10 days. The filtrate was aseptically passed through Whatman No. 1 filter paper (W. & R. Balston Ltd., England) and stored at 4°C for further use. Plants sprayed only with distilled water served as control. Each pot contained six to eight plants and at least five pots received a single treatment. Leaves from six randomly selected plants from each pot were mixed and a sample of 1 g was taken out. Leaves were macerated with a pestle and mortar followed by suspension of the fine-crushed samples in 5 mL of ethanol–water (80:20, v/v). Samples were collected in screw-capped tubes, and the suspension was subjected to ultrasonication (Branson Sonifier, U.S.A.) at 60% duty cycles for 15 min at 4°C followed by centrifugation at 13 200 × g for 15 min. The clear greenish supernatant was then subjected to charcoal treatment to remove pigments in each sample and transferred to glass tubes after filtering through Whatman No. 1 filter paper. The residue was re-extracted twice, and the supernatant was pooled prior to evaporation under vacuum (Buchi Rotavapor Re Type, Marco Scientific Works (R), India). Dried samples were resuspended in 1.0 mL HPLCgrade methanol by vortexing and were stored at 4°C for high performance liquid chromatography (HPLC) analysis. The © 2002 NRC Canada



Singh et al.

analysis was conducted in triplicate and the whole experiment was repeated twice. Ethyl acetate fractionation of exudate and culture filtrates of S. rolfsii An equal volume of ethyl acetate was mixed with sclerotial exudate and culture filtrates separately. After vigorous shaking in a separatory funnel, the ethyl acetate fractions of the exudate and culture filtrate were collected. The residue was then re-extracted for a second time and the ethyl acetate fractions were pooled with the previous extract. The fractions were then evaporated under vacuum. Dried samples were resuspended in 1.0 mL HPLC grade methanol by vortexing and stored at 4°C for HPLC analysis. All samples were prepared in triplicate and the experiment was repeated twice. HPLC analysis HPLC was performed according to Singh et al. (2002). The HPLC system (Shimadzu Corporation, Kyoto, Japan) was equipped with two Shimadzu LC-10 ATVP reciprocating pumps, a variable UV-VIS detector (Shimadzu SPD-10 AVP), and a Winchrom integrator (Winchrom). Reversephase chromatographic analysis was carried out using a C18 reverse-phase HPLC column (250 × 4.6 mm id, particle size 5 µm Luna 5µ C-18 (2), Phenomenex, U.S.A.) at 25°C under isocratic conditions where the concentration of the mobile phase was constant throughout the run. Running conditions included a 5-µL injection volume of mobile phase methanol – 0.4% acetic acid (80:20, v/v), flow rate 1 mL/min, attenuation 0.03, and detection at 290 nm. Samples were filtered through a membrane filter (pore size 0.45 µm, Merck) prior to injection in a sample loop. Tannic, gallic, chlorogenic, ferulic, cinnamic, vanillic, caffeic, oxalic, and salicylic acids were used as internal and external standards. Phenolic compounds and salicylic acid present in the samples were identified by comparing the retention time (Rt) of standards and by co-injection. Concentrations were calculated by comparing peak areas of reference compounds with those in the samples run under the same elution conditions. The data from repeated HPLC analyses were pooled and subjected to ANOVA for statistical significance by least significance difference (LSD) test at P = 0.01.

Results Exudate depletion from the sclerotial primordia caused a significant reduction in size of sclerotia of S. rolfsii. In comparison with control sclerotia, which were 1.8–2.1 mm in diameter (mean = 1.81 mm, standard deviation (SD) = 0.26 mm), the exudate-depleted sclerotia were 0.6–1.2 mm in diameter (mean = 0.78 mm, SD = 0.43 mm). Similarly, the fresh weight of 100 normal sclerotia also differed significantly. In comparison with 107 mg (mean; SD = 3.7 mg) fresh weight of 100 nondepleted sclerotia, 100 exudatedepleted sclerotia were 47 mg (mean; SD = 8.9 mg). Colour differences in exudate-depleted versus nondepleted sclerotia were not observed. Exudate-depleted and nondepleted sclerotia germinated within 24 h on PDA. However, there was a marked delay in germination of the exudate-depleted

445 Table 1. Quantification of gallic acid (µg/g fresh weight) in leaves of Cicer arietinum following treatments with oxalic acid, exudate, and culture filtrate of Sclerotium rolfsii. Treatment Substance Oxalic acid Exudate Culture filtrate Control

Time (h) Concn. 8 mM 4 mM 100% 50% 100% 50%

24 131.00a 164.06b 157.34c 118.85d 133.01a 195.35e 75.26f

48 125.37a 119.18b 81.20c 144.74d 71.18e 99.13f 76.91g

96 456.46a 317.00b 301.36c 206.04d 144.26e 172.59f 75.84g

Note: Data represent the mean of triplicate samples of repeated experiments. Column data with different letters are significantly different by the LSD test at P = 0.01.

sclerotia on CRMA medium, as nondepleted sclerotia germinated (100%) within 24 h while exudate-depleted sclerotia germinated only after 72 h of incubation (mean = 84%, SD = 6.4%), with some sclerotia only germinating after 96 h (mean = 7.2%, SD = 4.3%). Subsequent colony growth rates were similar for both types of sclerotia after germination. Exudate-depleted sclerotia were able to cause infection in C. arietinum plants similar to control sclerotia as 100% seedling mortality was caused by both types (depleted and nondepleted) of sclerotia. Sclerotial germination on PDA was not affected for both sources of sclerotia exposed to different temperatures for different time intervals, as all the sclerotia germinated (100%) within 24 h. However, moisture depletion from the soil (up to 94.95% after 96 h at 45°C) also did not affect sclerotial germination until 96 h. From the HPLC analysis, six to eight peaks appeared almost consistently in the samples from treated leaves. Among them, five were identified as gallic (Rt = 2.92 min), ferulic (Rt = 3.36 min), chlorogenic (Rt = 4.16 min), cinnamic (Rt = 4.42 min), and salicylic acids (SA) (Rt = 8.56 min) based on internal and external standards co-injected for their confirmation (Fig. 1a). Gallic acid (GA) was discerned in all samples with significantly high amounts in treated leaves compared with the control (Table 1). GA did not increase uniformly, but varied with treatments at different time intervals. After 24 h of treatment, a maximum increase of GA was observed in leaves sprayed with the 50% diluted culture filtrate of S. rolfsii followed by 4 mM of oxalic acid and the undiluted exudate (Table 1). The level of GA decreased by approximately 40% after 48 h compared with the amount found at 24 h in the leaves treated with exudate and culture filtrate, whereas the level in the leaves treated with oxalic acid only decreased by 12%. Interestingly, the level of GA was the highest after 96 h, with leaves treated with oxalic acid having the highest amount followed by treatments with the exudate. In leaves treated with the culture filtrate, the amount of GA was almost at the same level at 96 h compared with that at 24 h. In all treatments, the accumulation of GA was greater than in the control except for the undiluted culture filtrate at 48 h (Table 1). Table 2 shows that the occurrence of other compounds (e.g., ferulic, chlorogenic, cinnamic, and salicylic acids) was infrequent. Ferulic acid was detected in leaves treated with © 2002 NRC Canada



446 Fig. 1. HPLC analysis of the ethyl acetate fraction of sclerotial exudate and culture filtrate of Sclerotium rolfsii. (a) Standards: tannic acid (1), gallic acid (2), oxalic acid (3), ferulic acid (4), chlorogenic acid (5), and cinnamic acid (6). (b) Ethyl acetate fraction of sclerotial exudate of S. rolfsii. (c) Ethyl acetate fraction of culture filtrate of S. rolfsii.

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leaves treated with oxalic acid and culture filtrate 24 h after application. It was not detected in the leaves treated with the exudate or in the controls (Table 2). The ethyl acetate fraction of the sclerotial exudate and culture filtrates analysed by HPLC showed that both contained oxalic acid (Rt = 3.01 min) along with many other compounds. The other compounds identified in sclerotial exudate were some phenolics (e.g., gallic (Rt = 2.95 min), ferulic (Rt = 3.48 min), chlorogenic (Rt = 4.18 min), and cinnamic acids (Rt = 4.42 min)) (Fig. 1b). Similarly, the phenolic compounds identified in the culture filtrates were gallic acid (Rt = 2.92 min) and cinnamic acid (Rt = 4.38 min) (Fig. 1c).

Discussion

oxalic acid (8 mM at 24 h; 4 mM at 48 h), diluted exudate, and undiluted culture filtrate at 48 h. It was not detected in controls. Similarly, chlorogenic acid was detected in leaves treated with oxalic acid (8 mM at 48 h; 4 mM at 24 and 48 h), exudate (undiluted at 48 h), and culture filtrate (undiluted at 96 h and diluted at 48 and 96 h, respectively) applied at different concentrations and time intervals. It was not detected in controls. Cinnamic acid was detected only in the samples prepared from leaves treated with the undiluted culture filtrate collected 48 h after application, as well as in the control. The occurrence of salicylic acid was only in the

Results from the present study indicate that the exudates formed on the surface of developing sclerotia of S. rolfsii have some relationship with their subsequent development, as depletion of exudate affected the size and mass of the sclerotia. According to Punja (1985), excess nutrients, water, and other materials (e.g., cations, proteins, carbohydrates, amino acids, enzymes, oxalic acid) that translocate to the developing sclerotia to sustain increased metabolic activity may contribute to exudate formation. From our observation, it is possible that sclerotia may be using the nutrients present in their exudates for growth and development, since their depletion affected sclerotial development. Moreover, the delay in germination of exudate-depleted sclerotia on CRMA, a nutritionally deficient medium with very low carbon sources compared with PDA, also suggested an alteration of physiological processes during germination of these sclerotia. The pathogenic ability of the exudate-depleted sclerotia indicated that the basic functions of the pathogen were not affected by depletion of their exudates, although it affected other aspects, such as sclerotial size, weight, and germination. Oxalic acid is a constituent of the exudate present on the surface of sclerotia (Punja 1985) and also of culture filtrates of S. rolfsii (Punja et al. 1985), and this was confirmed in the present investigation. According to Punja (1985), oxalic acid is important for the pathogenicity of the fungus, while other authors demonstrated that synthetic oxalic acid played a role in the induction of resistance in plants against pathogen invasion (Zhang and Liu 1992; Wang et al. 1995; Toal and Jones 1999; Nabila 1999). From the results of the present investigation, it was observed that synthetic oxalic acid as well as exudate and culture filtrates of S. rolfsii were capable of inducing some phenolic compounds and salicylic acid in chickpea plants. Phenolic compounds are known to impart resistance in plants against pathogen attack. Accumulation of substantial concentrations of ferulic acid in cell walls of parsley leaves following inoculation with Phytophthora megasperma f.sp. glycinea has been observed (Nicholson and Hammerschmidt 1992; Matern and Kneusel 1988; Tietjen et al. 1983). Similarly, chlorogenic acid accumulation varies depending on potato genotypes following inoculation with Phytophthora infestans (Friend 1981; Gans 1978; Henderson and Friend 1979). Friend (1981) suggested that chlorogenic acid, not a particularly toxic compound, may act as an activated phenylpropanoid and could be shunted to the synthesis of other phenolics possibly involved in contain© 2002 NRC Canada



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447 Table 2. Effect of foliar application of oxalic acid, exudate, and culture filtrate of Sclerotium rolfsii on phenolic and salicylic acid content (µg/g fresh weight) in leaves of Cicer arietinum. Treatment

Phenolic acid

Substance

Concn.

Time (h)

Ferulic

Chlorogenic

Cinnamic

Salicylic acid

Oxalic acid

8 mM

24 48 96 24 48 96 24 48 96 24 48 96 24 48 96 24 48 96 24 48 96

2.72a ND ND ND 4.94a ND ND 2.80a ND ND 3.33a ND ND 1.32a ND ND 2.06a ND ND ND ND

ND 1.6a ND 0.96b 1.77a ND ND 1.38ac ND ND ND ND ND ND 0.79b ND 1.02bc 0.88b ND ND ND

ND ND ND ND ND ND ND ND ND ND ND ND ND 0.2a ND ND ND ND ND 0.1a ND

2.35a ND ND 2.26a ND ND ND ND ND ND ND ND 2.21a ND ND 2.51a ND ND ND ND ND

4 mM

Exudate

100%

50%

Culture filtrate

100%

50%

Control

Note: Data represent the mean of triplicate samples of repeated experiments. Column data with different letters are significantly different by the LSD test at P = 0.01. ND, not detected, as determined by HPLC analysis.

ment of the pathogen. Thus, the accumulation of chlorogenic acid may represent a general rise in phenolic biosynthesis (Nicholson and Hammerschmidt 1992). Ferulic and chlorogenic acids are suspected to be involved in the detoxification of piricularin, a toxin produced by the rice blast pathogen Pyricularia oryzae (Tamari and Kaji 1954). Harborne (1988) has also correlated chlorogenic acid with active postinfection defence responses in potato. Based on the association of these two phenolic compounds in host defence mechanisms for other host–pathogen systems, their accumulation in chickpea observed in the present investigation is noteworthy. Gallic acid is also reported to have antimicrobial activity either directly (Binutu and Cordell 2000) or indirectly through conversion into gallotannins (Swain 1979 (as cited in Salisbury and Ross 1986); Haslam 1981 (as cited in Salisbury and Ross 1986)). Rapid accumulation of gallic acid at 24 h followed by a sharp decrease at 48 h suggests that it may serve as a pool of phenols required for diversion to other toxic products. Similar activities for some phenolic compounds have also been reported (Nicholson and Hammerschmidt 1992). Similarly, salicylic acid, a plant metabolite, is known to act as a signal molecule in induced resistance in plants (Gaffney et al. 1993). Induction of phenolic compounds in C. arietinum in the present investigation following exogenous application of oxalic acid and culture filtrates of S. rolfsii further suggests their potential for application to crop plants to protect against pathogen attack, although this was not tested here. According to some studies (Bateman and Beer 1965; Franceschi and Horner 1980; Hodgkinson 1977), oxalic acid is toxic to plant tissue at concentrations above 10 mM. However, other studies have suc-

cessfully used synthetic oxalic acid to induce resistance in plants against phytopathogens without phytotoxicity at 5 mM (Zhang and Liu 1992; Wang et al. 1995; Toal and Jones 1999; Nabila 1999). The induction of phenolics and salicylic acid upon application of oxalic acid, sclerotial exudates, and culture filtrates may be involved in the expression of resistance in C. arietinum, although this was not tested here. More studies are needed to establish the multidimensional activity of oxalic acid in plants for various biological functions. The presence of phenolic compounds in the sclerotial rind was reported by Punja and Damiani (1996). However, the detection of phenolics in the sclerotial exudate is reported here for the first time.

Acknowledgements B.K. Sarma is grateful to the Council of Scientific and Industrial Research, New Delhi, India, for the award of Senior Research Fellowship. The authors are also grateful to the two unknown reviewers of this manuscript for their excellent comments for improving the manuscript.

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