AOBPreview originally published online on December 2, 2008
Annals of Botany 2009 103(3):525-532; doi:10.1093/aob/mcn238
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Mycorrhization and phosphorus nutrition affect water relations and CAM induction by drought in seedlings of Clusia minor
Centro de Botánica Tropical, Instituto de Biología Experimental, Universidad Central de Venezuela, Apartado 47829, Caracas 1041-A, Venezuela
* For correspondence. E-mail aherrera{at}ciens.ucv.ve
Received: 10 July 2008 Returned for revision: 25 September 2008 Accepted: 27 October 2008 Published electronically: 2 December 2008
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSLITERATURE CITED |
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Background and Aims: Crassulacean acid metabolism (CAM) is currently viewed as an adaptation to water deficit. In plants of Clusia minor, which grow mostly on acidic, P-deficient soils, CAM is induced by water deficit. The symbiosis between plants and mycorrhizal fungi alleviates the symptoms of P deficiency and may influence drought resistance. Therefore, the effect of P supply, modified by three different experimental treatments, on the induction of CAM by drought in C. minor was investigated to test the hypothesis that P deficiency will produce greater CAM activity and, in addition, that treatment will modify drought tolerance.
Methods: Seedlings were grown in forest soil sterilized and inoculated with Scutellospora fulgida (SF treatment), sterilized and supplemented with P (Ph treatment) or non-sterilized and containing native mycorrhizae (Nat treatment). Leaf turgor potential (
T) was determined psychrometrically, and CAM activity as nocturnal acid accumulation (
H+) by titration of dawn and dusk leaf sap.
Key Results: Plant mass and P content were higher in SF and Ph than in Nat seedlings. After 21 d of water deficit, T increased in SF, decreased in Ph and remained unchanged in Nat, and, after 7 and 14 d of water deficit, H+ in Nat was three times higher than at the beginning of drought, whereas in SF and Ph H+ was lower than on day 0.
Conclusions: P deficiency in Nat seedlings was ameliorated by inoculation or P addition. The SF and Nat seedlings showed greater tolerance of drought than Ph. P deficiency promoted the induction of CAM by drought in Nat seedlings, whereas P fertilization and mycorrhization did not. Nocturnal acid accumulation was highly and negatively correlated with plant P and N contents, indicating that P and N deficiencies are promoters of CAM in droughted plants of C. minor.
Key words: Clusia minor, crassulacean acid metabolism, CAM, mycorrhiza, drought, phosphorus deficiency, nitrogen–water relations
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSLITERATURE CITED |
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Clusia minor, considered a C3–crassulacean acid metabolism (CAM) species (Lüttge, 1999), shows an extraordinary plasticity in the expression of CAM, responding to changes in environmental factors such as light, and water and nitrogen supply (Franco et al., 1991). In plants of C. minor, nocturnal proton accumulation (
H+) did not increase under extreme drought or after frequent and heavy rains, but did during the transition from the rainy to the dry season (Borland et al., 1996) or with moderate rainfall (Herrera et al., 2008). Watered plants operate in the C3 mode, and CAM induction can take place in a matter of a few days (Schmitt et al., 1988).
Nutrient deficiency may modulate CAM expression. In C. minor, N deficiency did not stimulate CAM activity in plants grown under low photosynthetic photon flux (PPF) and diminished nocturnal CO2 fixation and chlorophyll content in high PPF plants (Franco et al., 1991), whereas in plants of Kalanchoë blossfeldiana, N deficiency caused an increase in the rate of nocturnal CO2 fixation, H+ and the activity of phosphoenolpyruvate carboxylase (PEPC) (Ota 1988), the key enzyme in malic acid synthesis in CAM plants. N and P deficiency increased CAM activity in plants of Mesembryanthemum crystallinum under salinity stress (Paul and Cockburn, 1990).
Under deficiency of P as well as other ions of low mobility in the soil, mycorrhizae can increase ion absorption by the root (Smith and Read, 1997). The occurrence of mycorrhization by Glomus spp. has been reported in four species of Clusia (Kreuzer et al., 2007); since plants of C. minor grow naturally on acid soils with little organic matter and low P and N availability, the role of mycorrhizae on mineral nutrition of this species must be important. Seedlings of C. minor inoculated with the highly effective foreign mycorrhizal fungus, Scutellospora fulgida, or fertilized with P were larger and had a larger P and N content than seedlings growing in native soil (Cáceres and Cuenca, 2006). Therefore, the increase in growth of seedlings of C. minor with inoculation may be related to the improvement of P absorption by the roots, which in turn influences N absorption.
Mycorrhizae can modify host water relations, and the variety of responses is vast and not consistent (Augé, 2001, and references therein). In mycorrhizal (M) compared with non-mycorrhizal (NM) plants, transpiration rate and leaf conductance to water vapour are rarely higher, frequently similar or different only under drought, and differences between M and NM plants depend on the host and mycorrhiza species. Also, leaf water potential () is not usually affected by mycorrhizae (Augé, 2001); was slightly higher in droughted M than NM onion plants (Nelsen and Safir, 1982). Osmotic potential (s) and turgor potential (T) did not differ between M and NM plants (Augé, 2001) but s at saturation was lower in watered M than NM plants (Augé et al., 1986).
The combined effects of mycorrhizae on P and N nutrition may, therefore, modify the water status and the induction of CAM by drought in seedlings of C. minor.
The main objective of the present investigation was to determine the effects of mycorrhizae and P nutrition on water relations and the operation of CAM in seedlings of C. minor. The rationale is that since it is known that drought induces CAM in C. minor, P deficiency induces CAM in salted plants, N deficiency induces CAM in C. minor and K. blossfeldiana, and fertilization and mycorrhizae improve plant P and N status, drought will promote CAM induction in P/N-deficient plants of C. minor relative to non-P/N-deficient seedlings. In addition, seedlings will differ in their tolerance of water deficit depending on nutrient status and the way in which this is altered.
In this work, seedlings of C. minor collected in a low mountain forest were subjected to three treatments: SF, sterilized native soil inoculated with S. fulgida; Ph, sterilized native soil supplied with triple superphosphate; and Nat, non-sterilized native soil containing native mycorrhizae. Once the effects on growth of these treatments were assessed, seedlings were subjected to water deficit in order to induce CAM. Plant water status and CAM activity, determined as H+, were assessed during the water deficit treatment. Upon re-watering, organ and whole-plant mass and N and P contents were determined.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSLITERATURE CITED |
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Plant material and treatments
Seedlings of Clusia minor L. approx. 2 years old were collected in a low cloudy mountain forest adjacent to the Center of Ecology of the Instituto Venezolano de Investigaciones Científicas (IVIC), Altos de Pipe, Venezuela, at 10°23'N, 66°59'W and an altitude of 1600 m. Forest soil, containing 3·05 µg g–1 available P (Cáceres and Cuenca 2006), was collected at a depth of approx. 20 cm and sterilized with 8 kGy
-radiation. Mycorrhizal inoculation was done with a concentrated mix of spores (1000 spores g–1), mycelium and mycorrhizal root fragments produced in the Soil Laboratory of the Center of Ecology, IVIC, from pure cultures of S. fulgida, a species previously shown to stimulate growth, using Vigna luteola as a host plant. Seedlings were planted in plastic bags of approx. 3·5 L volume containing 4 kg of soil and grown under a transparent roof in the gardens of the Instituto de Biología Experimental under three treatments, 16 seedlings in each: SF, sterilized soil inoculated with 20 g of S. fulgida inoculum; Ph, sterilized soil fertilized with 375 mg P kg–1 as triple superphosphate at the beginning of the experiment and after 4 months; and Nat, non-sterilized native soil. Seedlings in sterile, non-mycorrhizal soil were not included as control, since they die shortly after planting (Cáceres and Cuenca, 2006). In all treatments, a soil extract filtered through Whatman No. 1 paper was added to reintroduce the microbial population except for mycorrhizal propagules. Seedlings of all the treatments were watered every other day to field capacity with tap water. After 10 months of growth, seedlings were subjected to water deficit by interruption of irrigation for 21 d. Then, they were watered frequently for 2 months.
Microclimatic conditions
PPF was measured with a LI-250 radiometer (LI-COR Inc., Lincoln, NE, USA), air temperature with YSI 405 thermistors connected to a YSI 400 mod. 43TD telethermometer (Yellow Springs Instruments, Co., Yellow Springs, OH, USA), and relative humidity with a hygrometer (Abbeon, Santa Barbara, CA, USA).
Biomass production
Seedlings (n = 4 seedlings per treatment) were harvested after 12 months of treatment and the dry mass (72 h at 60 °C) of plant compartments determined; the length and thickness (with a precision caliper) of roots of first, second and third order of branching were measured.
Chlorophyll content
Chlorophyll content was determined in extracts of 0·28 cm2 leaf disks of four seedlings per treatment taken at 1600 h (to avoid damage to chlorophylls caused by acids) and macerated in cold 80 % acetone and a small amount of CaCO3, according to Bruinsma (1963).
Phosphorus and nitrogen contents
For the estimation of P and N contents, 50 mg of dried plant material from the harvest done after 10 months of treatment was digested with a binary H2SO4–HClO4 mixture. Total P concentration was determined by the method of Murphy and Riley (1962) and total N concentration by microkjeldahl.
Colonization with mycorrhizae
Root samples of eight plants per treatment were collected after 12 months of growth and fixed in commercial isopropanol until processing. Segments were submerged in 10 % KOH and tubes placed in a boiling water bath for 1 h; segments were thoroughly washed with tap water, acidified for 15 min with HCl, stained with 0·05 % trypan blue in lactoglycerin for 5 min in a boiling water bath, rinsed with tap water and observed under the microscope. Thirty 1 cm long segments were randomly taken and the presence of mycorrhiza, arbuscules and vesicles recorded. Frequency of colonization (F), intensity of colonization (I), arbuscule presence (A) and vesicle presence (V) were determined after Trouvelot et al. (1986).
Water relations
Leaf was determined in 0·28 cm2 leaf discs of four seedlings per treatment collected at dawn and placed in C-52 chambers connected to an HR33T microvoltmeter (Wescor Inc., Logan, UT, USA) operated in the dew point mode. Leaf s was determined likewise in the same discs after freezing in liquid N2. Turgor potential was calculated as the difference between and s. Leaf water content was determined as LWC = (fresh mass – dry mass)/area in discs collected at dawn from four seedlings per treatment.
Nocturnal acid accumulation
Leaf discs (area = 0·28 cm2) of four seedlings per treatment collected at dawn and dusk were placed in plastic syringes that were stored in liquid N2, then placed in centrifuge tubes and centrifuged at 6000 rpm for 10 min in a centrifuge model Multex (MSE, London, UK). The discs were rinsed with 20 mL of distilled water and the extracts thus obtained titrated using a model 601 A meter (Orion, Cambridge, UK) with 1 mM KOH to pH 8·4 after Franco et al. (1990) for the determination of protons corresponding to malic and citric acid. The H+ was calculated as the difference in H+ content between dawn and dusk.
Natural carbon isotope ratio
Dried leaf samples (n = 3 per treatment) collected after 12 months of treatment were ground in a mill and analysed by mass spectrometry for 
13C.
Statistical analysis
One-way analysis of variance (ANOVA) was performed using the Statistica 5·5 program. Significance was assessed at P < 0·05, and the post hoc Duncan's multiple range test applied to denote significant differences.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSLITERATURE CITED |
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Average values of microclimatic variables during growth were 18/32 °C minimum/maximum air temperature, 75/30 % maximum/minimum and a maximum of 350 ± 50 µmol m–2 s–1 PPF at noon, similar to those measured in the understorey of the forest where seedlings were collected (A. Cáceres, unpubl. res.).
Organ mass per plant, root characteristics and chlorophyll content after 12 months were significantly different among treatments (Fig. 1). Leaf and stem mass were highest in SF, whereas root mass in SF was similar to that in Ph and higher than in Nat. The ratio root mass:leaf area followed the order SF > Nat > Ph. Roots were longer in SF than in Ph or Nat, and thicker in SF and Nat than in Ph; the ratio root length:mass followed the trend Ph > Nat >> SF. Chlorophyll content and the ratio chlorophyll a : b (not shown) were significantly lower in Nat than in SF and Ph, no significant differences being found between SF and Ph.
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Mycorrhizal colonization was as frequent in SF as in Nat but more intense, and the proportion of arbuscules and vesicles was greater (Fig. 2); no mycorrhizal colonization was observed in Ph seedlings.
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Differences among treatments in whole-plant nutrient content showed the trend SF = Ph > Nat for P, and SF > Ph > Nat for N (Fig. 3). The ratio N : P was 45 (SF), 31 (Ph) and 35 (Nat).
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When seedlings of all three treatments were subjected to water deficit, significant changes due to treatment were observed (Fig. 4). Leaf
was slightly but significantly higher in SF than in Ph or Nat at the beginning of the water deficit experiment; after 21 d increased in SF, remained unchanged in Ph and decreased in Nat 1·6 times relative to day 0. Leaf s was also higher in SF on day 0 and similar in SF and Ph on day 21; in both SF and Ph, s remained unchanged by drought, while it decreased in Nat nearly 2-fold relative to day 0 and more than twice relative to SF or Ph. Turgor potential at the beginning was higher in SF than in Ph or Nat, increasing 4-fold after 21 d in SF, remaining unchanged in Ph and increasing in Nat. Leaf water content at the beginning was higher in SF and Ph than in Nat, decreasing after 21 d in Ph and Nat and increasing in SF.
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Relatively high dawn and dusk H+ contents occurred in watered seedlings of all three treatments and
H+ did not differ significantly among treatments; as early as 7 d after the beginning of drought, dawn H+ content was greater than dusk H+ content in Nat and Ph, but after 7 and 14 d H+ was significantly different from zero, and >3-fold higher than on day 0 only in Nat seedlings, whereas in SF and Ph H+ was lower than at the beginning of the experiment (Fig. 5). After 21 d of drought in all three treatments, dawn and dusk H+ contents were still high but H+ was 
0 in SF and Ph and similar to values on day 0 in Nat. The estimated dawn malate concentration in the leaf sap after 21 d of water deficit was on average 1·4 times higher in Nat than in SF or Ph.
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Decreasing curvilinear correlations with high determination coefficients were obtained when values of
H+ for 7 and 14 d of drought were pooled and plotted against whole-plant N and leaf and stem P contents (Fig. 6). P and N CAM use efficiency, calculated as H+ on day 14 divided by plant P or N content, were approx. 13 (P) and four times (N) higher in Nat than in SF or Ph seedlings.
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Values of leaf
13C were not statistically different among treatment, averaging –23·2 ± 0·7 
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSLITERATURE CITED |
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Seedlings of Clusia minor cultivated in the native forest soil grew less and showed a higher
H+ under drought than seedlings inoculated with a foreign mycorrhiza or fertilized with P. Differences among treatments in the growth response, water relations and CAM operation under drought were related to differences in P, N and chlorophyll contents and pattern of biomass allocation, especially to roots. P deficiency promoted an increase in H+ with drought, whereas sufficient P supply, either by mycorrhization with a highly effective fungus or by fertilization, inhibited CAM operation under drought.
The increase with drought in nocturnal acid accumulation of Nat and, to a lesser degree, Ph seedlings resembled that which took place in plants of C. minor, C. rosea and C. lanceolata subjected to mild drought under controlled conditions (Franco et al., 1992). The relatively high values of dawn and dusk H+ contents found in all three treatments throughout the drought experiment, together with observations on the above-mentioned species (Franco et al., 1992), suggest that malate decreased with drought whereas citrate remained similar to pre-drought values; in contrast, in plants of M. crystallinum subjected to salinity, P deficiency also increased background malate content (Paul and Cockburn, 1990).
The decrease with time under drought in H+ of SF and Ph seedlings resembled the reduction in nocturnal CO2 fixation suffered by plants of O. robusta highly colonized by native mycorrhizae, as opposed to seedlings to which the fungicide benomyl was added (Pimienta-Barrios et al., 2003). In contrast, plants of the obligate CAM plant Agave victoria-reginae inoculated with a non-pathogenic strain of Fusarium oxysporum showed enhanced growth and malate accumulation (Obledo et al., 2003), drawing attention to the possibly large diversity of response of CAM to inoculation.
The present observations and those made on O. robusta by Pimienta-Barrios et al. (2003) support the hypothesis that P deficiency increased CAM in seedlings of C. minor. The effect of P deficiency on H+ in the present work may have been exerted through its activation of PEPC, as found in P-deprived seedlings of Nicotiana sylvestris, in which shoot C3 PEPC activity and amount increased, and this activation was transcriptionally regulated (Toyota et al., 2003). The higher N CAM use efficiency in Nat seedlings indicates that N deficiency, possibly caused by P deficiency, stimulated nocturnal acid accumulation, as shown before in C. minor (Franco et al., 1992).
Neither nutritional status nor CAM activity affected the value of 13C, which remained within the range reported for C. minor under different growth conditions and is characteristic of C3–CAM species (Winter and Holtum, 2002).
The smaller biomass of Nat seedlings compared with seedlings inoculated with a foreign mycorrhiza or fertilized with P is in agreement with reports on C. minor (Cáceres and Cuenca, 2006) and C. multiflora (Cáceres, 2001), and the increase in root thickness of mycorrhizal plants (SF and Nat) relative to fertilized plants coincides with a report on the CAM species, Aloe vera (Fuentes-Carvajal et al., 2006).
A similar P content in plant but smaller values of growth parameters and N content in Ph than in SF seedlings suggests that the doses of P applied were insufficient, unlike results reported by Cáceres and Cuenca (2006), and that there was luxury consumption and plants were unable to make use of the applied P. In plants of Opuntia ficus-indica to which a single dose of P fertilizer was applied before planting, root length and mass were lower at the end of the second month of treatment, and increased afterward together with whole-plant growth; this delay in the effect of P on growth was attributed to slow P temporary mobilization from the soil towards the roots (García de Cortázar et al., 2001). Fertilizing for a third time was not considered because such small seedlings could be very sensitive to a high P content in the soil. A positive effect of natural mycorrhizae 12 months after the beginning of treatments compared with P fertilization was shown by thicker roots in Nat, indicating that native mycorrhizae play an important role in the mineral nutrition of C. minor in its natural environment.
The N : P ratio suggests that Nat seedlings suffered from P deficiency, given that values of N : P >16 are thought to indicate P limitation, at least at the community level (Koerselman and Meuleman, 1996). Nevertheless, it was evident that Nat seedlings were deprived of N; therefore, N deficiency is not ruled out as a factor inducing CAM.
The occurrence of higher N and total chlorophyll contents in SF and Ph than in Nat seedlings agrees with previously published work in which N deficiency was related to a significant diminution of the chlorophyll content in plants of C. minor (Franco et al., 1991), A. vera (Fuentes-Carvajal et al., 2006) and K. lateritia (Santos and Salema, 1991). N deficiency causes a reduction in chlorophyll content and photosynthetic rate (Schulze et al., 2002), thus causing reduced growth.
Treatment determined significant differences in seedling response to water deficit. In SF seedlings, the increase in T under water deficit, similar to the increase in T under drought reported for M rose plants (Augé et al., 1986), suggests that longer roots allowed exploration of a larger soil volume; also, a role for the extraradical mycelium in the improvement of water absorption is not precluded (Augé, 2001). In contrast, in Nat seedlings, a reduction in and s, together with the maintenance of T, suggests the occurrence of osmotic adjustment, similarly to reports in the CAM species Tillandsia ionantha (Nowak and Martin, 1997) and in M relative to NM bean plants (Augé et al., 2004). Osmotic adjustment in Nat seedlings may have been effected by acids accumulated through CAM, as suggested for a fully grown tree of C. minor by Herrera et al. (2008). The possible reason why T was lower in Ph than in Nat seedlings, in spite of having a root mass similar to that of SF and higher than Nat, is that Nat seedlings had a higher root mass to leaf area ratio than Ph seedlings, therefore losing less water from the pot by transpiration and maintaining turgor.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSLITERATURE CITED |
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Seedlings of C. minor grew better, relative to those with native mycorrhizae, when inoculated with S. fulgida but less so with P fertilization. Inoculation and P fertilization increased biomass and P and N contents. In seedlings colonized by native mycorrhizae, P deficiency, which possibly induced N deficiency, stimulated the operation of CAM under drought, which, in turn, possibly improved the water status of seedlings by contributing to turgor maintenance. Mycorrhizal infection with S. fulgida improved water status possibly because of longer roots that favoured water absorption.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSLITERATURE CITED |
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Determinations of
13C by J. P. Ometto and L. Martinelli at the Centro de Energia Nuclear na Agricultura, Universidad de Sâo Paulo, Laboratório de Ecología Isotópica, Piracicaba, Brasil, and of N and P content by S. Flores at the Instituto Venezolano de Investigaciones Científicas (IVIC), Venezuela, are gratefully acknowledged. G. Cuenca, at IVIC, kindly donated the S. fulgida inoculum. |
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