The role of mycorrhizal fungi in growth enhancement of dune plants following burial in sand

Transcription

1 Functional Ecology 1999 ORIGINAL ARTICLE OA 000 EN The role of mycorrhizal fungi in growth enhancement of dune plants following burial in sand J. V. PERUMAL* and M. A. MAUN Department of Plant Sciences, University of Western Ontario, London, Ontario, Canada N6A 5B7 Summary 1. Burial in sand of Agropyron psammophilum and Panicum virgatum plants had a stimulating effect on carbon dioxide exchange rate, leaf area and biomass, irrespective of whether sand used for burial did or did not contain mycorrhizal fungi. 2. Plants of both A. psammophilum and P. virgatum species grown in mycorrhiza-containing sand and then buried with mycorrhiza-containing sand had the highest CO 2 exchange rate, leaf area and biomass. 3. The growth stimulation following a burial episode is probably a composite response of several factors. The major contribution of mycorrhizal fungi will possibly be the exploitation of resources in the burial deposit. Key-words: Agropyron psammophilum, arbuscular mycorrhizal fungi, burial in sand, foredunes, net CO 2 uptake, Panicum virgatum, sand dunes Functional Ecology (1999) Ecological Society Introduction Read (1989) showed that plant communities in successional sand-dune chronosequences are governed by an interaction between biotic and physico chemical components of the sandy matrix. Not only does the composition of plant species change with the seasons (Nicolson 1960) and the age of the dune systems (Koske & Gemma 1997) but also the soil microorganismal association changes with succession probably because of an increase in organic matter content, improved substrate stability and nutrient enrichment (Read 1989). In the foredune plant communities, the driftline vegetation consists of annuals that may not have a mycorrhizal association and depend primarily on detritis cast on the shoreline for their nutritional needs. However, the pioneer dune-forming species, such as Ammophila breviligulata Fern., benefit from colonization by arbuscular mycorrhizal (AM) fungi (Koske & Polson 1984). In this habitat, the major recurrent event affecting plant and microbial communities is sand deposition that may bury plants to variable depths (Maun 1996). Plant species growing in the foredunes are well adapted to stress imposed by burial. In fact, there is clear evidence that all foredune species exhibit enhanced growth following episodes of burial (Maun 1998). According to Eldred & Maun (1982), some plants have become so specialized that they require regular burial to maintain high vigour. Several explanations have emerged over the years to explain this *Present address: Southern Adventist University, PO Box 370, Collegedale, TN 37315, USA. phenomenon but little experimental evidence is available for any of the theories (Maun 1998). Possibly, the growth enhancement may be caused by their association with AM fungi. In this study, we tested the hypothesis that the enhanced performance of dune plants following burial in sand is owing to the beneficial effects of mycorrhizal fungi. We examined the effects of AM fungi inoculation on the growth and vigour of two dune grasses, Agropyron psammophilum Gillett & Senn and Panicum virgatum L., under burial and control conditions in a greenhouse. Materials and methods BURIAL EXPERIMENTS A greenhouse experiment was conducted in winter 1993 to determine the effects of mycorrhizal fungi and burial in sand on the net CO 2 uptake, leaf area and biomass of two common sand dune grasses, A. psammophilum and P. virgatum. The same experiment was repeated in summer 1996 with some modifications of methods as outlined below. The mycorrhiza inoculum was produced by using sweet-corn (Zea mays L.). In 1993 and 1996 surface-sterilized seeds were germinated in an incubator (30 C, 14 h light and 10 h dark) and the seedlings were planted in sand collected directly from the rhizosphere of P. virgatum and A. psammophilum plants growing naturally on the sand dunes of Port Burwell Provincial Park. In the 1993 experiment when corn plants were 3 weeks old a sample of roots was harvested and assessed for AM-fungi colonization. This procedure confirmed the presence 560

2 561 Growth stimulation by burial: role of mycorrhiza of AM fungi in the roots of corn plants. In the 1996 burial experiment, corn plants were 8 weeks old when they were harvested, confirmed for the presence of AM fungi and then used to inoculate the two test species. All corn plants in both experiments were then harvested, their roots were washed and then cut into ca. 1 cm fragments. These fragments were used for the inoculation of plants. The procedures for planting, inoculation and burial of plants for both experiments were as follows. Surface-sterilized seeds of A. psammophilum and P. virgatum, were germinated in an incubator and seedlings planted in 8 cm long and 5 cm diameter PVC tubes (Fig. 1a) filled with sand which had been sterilized at 1 mega rad of gamma radiation (Thompson 1990) prior to placing in the tubes. The tubes were fitted with a cm coupling on one side (Fig. 1d) and a Teflon net at the base for drainage (Fig. 1). For the 1993 and 1996 experiments, the seedlings of A. psammophilum and P. virgatum were planted in PVC tubes and inoculated with AM fungi in late September 1993 and May 1996, respectively. Before planting the seedlings in the PVC tubes, 5 g of corn root fragments were placed 1 cm below the roots of seedlings to serve as inoculum. During both years the roots of corn plants grown in sand collected from the rhizosphere of A. psammophilum were placed below A. psammophilum seedlings while corn roots grown in sand from the rhizosphere of P. virgatum were placed below the seedlings of P. virgatum. The tubes containing these inocula are referred to as mycorrhiza-containing sand (+) and those with mycorrhiza-free sand were called ( ). The seedlings were then placed on benches in a greenhouse. In the 1993 experiment, the greenhouse was maintained at 24 C day (14 h, with a light intensity of ca. 350 µe) and 20 C night (10 h). In the summer 1996 experiment the temperatures during the day ranged from 25 to 30 C (14 16 h with a light intensity of µe) and 20 C at night (10 8 h). Four weeks after planting in PVC tubes, the seedlings in both experiments, were given the following six burial treatments. Seedlings were grown in: (1) control, AM-containing sand (+), no burial; (2) control, AM-free sand ( ), no burial; (3) AM-containing sand and then buried with AM-containing sand (+ +); (4) AM-containing sand, buried with AM-free sand (+ ); (5) AM-free sand, buried with AM-free sand ( ); (6) AM-free sand, buried with AM-containing sand ( +). The first two treatments served as controls. To prevent the movement of AM fungi between the mycorrhiza-containing (+) and the mycorrhiza-free ( ) sand layers in treatments (4) (+ ) and (6) ( +), a 0 45 µm pore size millipore filter of 4 7 cm diameter with a hole in the centre for the stem was placed at the rim of the coupling (Fig. 1b) and sealed. The 0 45 µm pores prevented the penetration of AM fungal hyphae (Li, George & Marschner 1991). After the fitting of filters the plants were buried to 50% of their height by fitting a 3 8 cm diameter PVC tube (Fig. 1e) on the coupling. A completely assembled unit with a buried plant is shown in Fig. 1c. Eight replicates were used in the 1993 and four in the 1996 experiment. CARBON DIOXIDE EXCHANGE RATE In 1993, carbon dioxide exchange rates (CER) were measured using a LI-COR Portable Photosynthesis Gas Analyzer LI-6200 (Li-Cor Inc., Lincoln, NE, USA). The CER measurements were taken three times; 14, 20 and 26 days after the imposition of burial treatments. In all instances the second fully expanded leaf was used for this measurement and three readings were taken on each leaf. To minimize the variations in light intensity, measurements were taken between and h on sunny days. The same procedure was used in 1996 and one CER measurement was recorded 6 weeks after the imposition of burial treatments. LEAF AREA AND TOTAL PLANT BIOMASS Fig. 1. Assembly of PVC tubes and coupling used to determine the effect of mycorrhizal fungi on buried plants. (a) an unburied plant of Agropyron psammophilum growing in a PVC tube with coupling (d); (b) PVC tube, coupling (d) with 0 45 µmillipore filter and extension PVC tube (e); (c) a completely assembled unit with a buried plant. The experiments were terminated 5 and 11 weeks after the imposition of burial treatments in 1993 and 1996, respectively. At harvest, the leaves of each plant were carefully removed from the plants, their leaf areas were recorded using the LI-3000 portable leaf area meter (Li-Cor Inc., Lincoln, NE, USA), and then placed in a labelled paper bag. The stems above sand, buried portions of stems and roots of four replicates of each species were carefully excavated from the PVC tubes, washed with water to remove any sand adhering to their roots and then placed in paper bags containing the leaves of the same plants. The same procedure was used to harvest the remaining four replicates; however, the buried portions and roots of plants were used to assess AM colonization. In 1996, the root samples from each replicate were examined for AM colonization. All

3 562 J. V. Perumal & M. A. Maun harvested plant materials were then dried in an oven at 70 C for 48 h and weighed. ASSESSMENT OF AM COLONIZATION OF ROOTS In both experiments, the roots of plants were examined for mycorrhizal root colonization. In the buried treatments (+ ) and ( +) where both the mycorrhizafree and mycorrhiza-containing sand was used for burial, care was taken to keep plant parts above and below the burial surface separated to avoid contamination. The assessment for mycorrhizal colonization of roots was carried out by thoroughly washing them with deionized water and then soaking them for h in FAA (formyl acetic alcohol containing 1050 ml water, 1500 ml 95% ethanol, 150 ml glacial acetic acid and 300 ml 37 40% formalin). The roots were taken out of FAA, cut into 1 cm long pieces and then dispersed in a 2 litre beaker full of water. The water was stirred vigorously and a subsample of 100 ml was collected using a beaker (Brundrett, Piche & Peterson 1984). These subsamples were then cleared by immersing them in 10% potassium hydroxide and placing them in an autoclave at 121 C for 7 12 min (depending on the texture of roots). The roots were then rinsed in deionized water and submerged in Chlorazol Black-E stain and heated in an oven set at C for 45 min to 3 h (Brundrett et al. 1984). The stained roots were then rinsed thoroughly in deionized water, mounted on slides and examined under a compound microscope for the assessment of root colonization using the magnified intersections method (McGonigle et al. 1990). STATISTICAL ANALYSIS A one-way analysis of variance (ANOVA) was performed on the data for carbon dioxide exchange rate (CER), leaf area and biomass to determine differences between the six treatments in both experiments. If the ANOVA produced significant F-values, Tukey s multiple comparison test was applied to find differences between treatment means. Results CARBON DIOXIDE EXCHANGE RATE Fig. 2. Mean (± 1 SE) carbon dioxide exchange rate (CER) of A. psammophilium and Panicum virgatum plants buried or unburied with sterilized sand without AM fungi or sterilized sand containing AM fungi. For this experiment sterilized sand was used for all treatments. Plants were grown in: ; control, AM-containing sand, no burial later (+); ; control, AM-free sand, no burial later ( ); ; AM-containing sand, buried later with AM-containing sand (+ +); ; AMcontaining sand buried later with AM-free sand (+ ); ; AM-free sand, buried later with AM-free sand ( ); ; AMfree sand, buried later with AM-containing sand ( +). In 1993, there were no significant differences in CERs between control plants of both species growing in sterile mycorrhiza-containing sand (+) and those growing in sterile sand with no mycorrhizal ( ) fungi (Fig. 2). Burial in sand had a stimulating effect on CER irrespective of whether sand used for burial did or did not contain mycorrhizal fungi and became evident after 14 days of burial. In general, burial of plants with sand containing mycorrhizal fungi was beneficial but the results were not always clear cut. The (+ +) treatment in both species had the highest CER reading. There was a significant (P < 0 001) increase in the final (26 days after burial) CER readings between both unburied controls, (+) and ( ), and the buried plants of both species except that the ( +) treatment in A. psammophilum was not significantly different from control (+). After 26 days of burial, the (+ +) treatment of A. psammophilum plants had significantly higher CER than the (+ ) treatment. The other burial treatments did not differ. In P. virgatum there were no significant differences in CER readings among all four burial treatments after 26 days of burial in sand (Fig. 2). In 1996, the control plants of both species grown in mycorrhiza-containing sand (+) showed significantly higher CER (Table 1) than control plants grown in

4 563 Growth stimulation by burial: role of mycorrhiza Table 1. Mean (± 1 SE) for carbon dioxide exchange rate (µ mol m 2 s 1 ) of Agropyron psammophilum and Panicum virgatum recorded 6 weeks after burial in August For this experiment sterilized sand was used for all treatments. Control (MCS), AM-containing sand (+), no burial; control (MFS), AM-free sand ( ), no burial; MCS/MCS, AM-containing sand, buried with AM-containing sand (+ +); MCS/MFS, AM-containing sand, buried with AM-free sand (+ ); MFS/MFS, AM-free sand, buried with AM-free sand ( ); MFS/MCS, AM-free sand, buried with AM-containing sand ( +). Means in the same column followed by the same letters are not significantly different at P < 0 05 according to Tukey s test In the 1993 experiment, for both species there was no significant difference in biomass per plant between control plants grown in AM-free sand ( ) and control plants grown in AM-containing sand (+) (Fig. 3). The A. psammophilum plants grown in mycorrhizae-containing sand and then buried with AM-containing (+ +) or AM-free sand (+ ) produced significantly higher biomass than both controls, ( ) and (+) (Fig. 3). In P. virgatum there was a significant difference between the unburied controls, (+) and ( ), and the burial treatments, (+ +), (+ ) and ( ), but there was no significant difference between AM-fungi containing control (+) and ( +) burial treatment (Fig. 3). In the 1996 experiment, control plants of A. psammophilum grown in AM-containing sand (+) produced significantly greater biomass than control plants grown in AM-free sand ( ) but not in P. virgatum (Fig. 3). The A. psammophilum plants grown in AMcontaining sand and then buried in AM-containing (+ +) or AM-free sand (+ ) produced significantly higher biomass than all other treatments. There was also a significant increase in biomass of plants grown in AM-free sand and then buried in AM-free ( ) or AM-containing sand ( +) as compared to control ( ), however, their total biomass was not significantly higher than mycorrhiza-containing (+) control (Fig. 3). In P. virgatum, there was a significant increase in total biomass in all burial treatments as compared to both controls, (+) and ( ). Agropyron psammophilum Panicum virgatum LEAF AREA MCS (+) ± 2 54 a ± 1 14 a MFS ( ) ± 1 00 b ± 0 77 b MCS/MCS (+ +) ± 4 04 c ± 1 47 c MCS/MFS (+ ) ± 3 09 c ± 1 11 c MFS/MFS ( ) ± 1 03 d ± 1 45 b MFS/MCS ( +) ± 1 58 d ± 2 00 b sterile sand without AM fungi ( ). Burial of A. psammophilum plants in sand, irrespective of whether sand used for burial did or did not contain AM fungi, significantly increased CER values (Table 1) over controls grown in mycorrhiza-free sand ( ) but not in mycorrhiza-containing sand (+). However, plants grown in AM-containing sand and then buried with AM-containing (+ +) or AM-free sand (+ ), had significantly higher CER values than those grown in AM-free sand and then buried in AM-free ( ) or AM-containing sand ( +). Results for P. virgatum were identical except that plants grown in AM-free sand and then buried with AM-free sand ( ) or AM-containing sand ( +) were significantly lower (Table 1) than AM-containing control (+) but not different from AMfree control ( ). In 1993 there was no significant difference between the leaf area of the mycorrhizal (+) and non-mycorrhizal ( ) controls of A. psammophilum. However, there was a significant difference between P. virgatum plants growing in AM-free ( ) or AM-containing (+) sand (Table 2). Burial in sand stimulated the growth of leaves of both plant species as shown by a significant increase in leaf area (Table 2). In A. psammophilum a significant difference was also seen between plants grown in AM-containing sand and then buried with AM-containing (+ +) or AM-free TOTAL BIOMASS Fig. 3. Mean (± 1 SE) for total dry biomass (g) per plant of A. psammophilum and P. virgatum recorded 5 weeks after burial in 1993 and 11 weeks after burial in For this experiment sterilized sand was used for all treatments. Plants were grown in: (+) control, AM-containing sand, no burial later; ( ) control, AM-free sand, no burial later; (+ +) AM-containing sand, buried later with AM-containing sand; (+ ) AM-containing sand buried later with AM-free sand; ( ) AM-free sand, buried later with AM-free sand; ( +) AM-free sand, buried later with AM-containing sand. Bars within each species and each year with different superscript letters are significantly (P < 0 05) different according to Tukey s test.

5 564 J. V. Perumal & M. A. Maun Table 2. Mean (± 1 SE) for leaf area (cm 2 ) of A. psammophilum and P. virgatum recorded 11 weeks after burial. For this experiment sterilized sand was used for all treatments. Control (MCS), AM-containing sand, no burial (+); control (MFS), AM-free sand, no burial ( ); MCS/MCS, AM-containing sand, buried with AM-containing sand (+ +); MCS/MFS, AM-containing sand, buried with AM-free sand (+ ); MFS/MFS, AM-free sand, buried with AM-free sand ( ); MFS/MCS, AM-free sand, buried with AM-containing sand ( +). Means in each column followed by the same letters are not significantly different at P < 0 05 according to Tukey s test Treatments A. psammophilum P. virgatum A. psammophilum P. virgatum MCS (+) 7 69 ± 0 48 a 5 77 ± 0 14 b 7 90 ± 1 55 a 7 66 ± 1 15 a MFS ( ) 6 53 ± 0 53 a 3 75 ± 0 17 a 7 45 ± 1 73 a 6 76 ± 0 77 a MCS/MCS (+ +) ± 0 75 c 7 56 ± 0 21 c ± 4 50 c ± 2 30 c MCS/MFS (+ ) ± 0 64 c 7 14 ± 0 55 c ± 1 96 c ± 3 25 c MFS/MFS ( ) ± 0 70 b 3 88 ± 0 16 a ± 3 45 b 7 94 ± 1 19 a MFS/MCS ( +) ± 0 88 b 4 11 ± 0 25 a ± 2 13 b ± 1 89 b sand (+ ) and those grown in AM-free sand and then buried with AM-free ( ) and AM-containing sand ( +) (Table 2). In P. virgatum all plants grown in AM-containing sand produced significantly greater leaf area than all those grown in AM-free sand (Table 2). Sand used for burial (AM-free or AM-containing) did not make any difference. In 1996, the mycorrhizal control (+) of both species did not significantly differ in its leaf area from nonmycorrhizal ( ) control (Table 2). Plants of both species grown in AM-containing sand or AM-free sand and then buried in mycorrhizal (+ +) or nonmycorrhizal (+ ) sand produced significantly higher leaf area than both controls (Table 2). The presence of mycorrhiza in sand used for planting, (+ +) or (+ ), significantly increased the leaf area as compared to ( ) and ( +) treatments in both species. The ( ) treatment in P. virgatum produced leaf area similar to that of controls (Table 2). ROOT COLONIZATION BY AM FUNGI In both the 1993 and 1996 experiments, no AM fungal colonization was observed in the ( ) and ( ) treatments confirming that sterilization of soil had killed AM fungi. Use of a millipore filter was effective because it did not allow hyphae to move from the lower to the upper horizon in (+ ) treatments (Table 3). In 1996, the plants had produced roots even in the burial deposits and they had been colonized by AM fungi only in treatments (+ +) and ( +) in which the plants were buried in soil containing AM fungi. Table 3. Mean (%) (± 1 SE) for arbuscular (AC), vesicular (VC) and hyphal colonization (HC) of A. psammophilum and P. virgatum after 11 weeks of burial in For this experiment sterilized sand was used for all treatments. Plants were grown in: control (MCS), AM-containing sand, no burial later (+); control (MFS), AM-free sand, no burial later ( ); MCS/MCS, AMcontaining sand, buried later with AM-containing sand (+ +); MCS/MFS, AM-containing sand buried later with AM-free sand (+ ); MFS/MFS, AM-free sand, buried later with AM-free sand ( ); MFS/MCS, AM-free sand, buried later with AMcontaining sand ( +) % Colonization of roots AC VC HC Original Burial Original Burial Original Burial Treatments soil deposit soil deposit soil deposit Agropyron psammophilum MCS (+) 23 5 ± ± ± 3 7 MFS ( ) MCS/MCS (+ +) 25 0 ± ± ± ± ± ± 3 1 MCS/MFS (+ ) 24 0 ± ± ± MFS/MFS ( ) MFS/MCS ( +) ± ± ± 3 0 Panicum virgatum MCS (+) 34 0 ± ± ± 4 3 MFS ( ) MCS/MCS (+ +) 32 5 ± ± ± ± ± ± 1 9 MCS/MFS (+ ) 31 3 ± ± ± MFS/MFS ( ) MFS/MCS ( +) ± ± ± 4 9

6 565 Growth stimulation by burial: role of mycorrhiza However, the percentage hyphal colonization in ( +) plants was not as high as the (+ +) treatment. Discussion The data from both experiments clearly showed that burial of plants in sand, irrespective of whether sand used for burial contained or did not contain AM fungi, increased all growth parameters of both A. psammophilum and P. virgatum species. For example, there was an increase (not always significant) in net CO 2 uptake, biomass and leaf area per plant in all buried treatments as compared to control. Why does plant growth increase following a burial episode? The growth stimulation may be owing to a number of factors (Maun 1998). For example, sand burial may (1) protect the root system of plants from drying out (Zhang & Maun 1991), (2) provide increased nutrient input and more soil surface area for the expansion of roots (Maun 1998), (3) exhibit reactive growth response to burial (Danin 1996) and (4) decrease interspecific competition through the elimination of species intolerant of sand accretion (Huiskes 1979). It is now possible to address the question posed in this study: can the growth enhancement following a burial episode be attributed to mycorrhizal fungi? This study provided a partial answer. First, in both years, the treatments in which the plants were grown in soil containing AM fungi and then buried with AMcontaining (+ +) or AM-free sand (+ ) always produced significantly higher biomass, leaf area and CER than control for both species. This increase may be the result of three factors: burial, AM fungi, or most probably a combination of both. Little & Maun (1996) showed that an increase in root biomass and root:shoot ratio of A. breviligulata was caused by an interaction between burial and AM fungi. As explained earlier and consistent with several other studies (Eldred & Maun 1982; Maun & Lapierre 1984; van der Putten 1989; Yuan, Maun & Hopkins 1993), burial by itself definitely increases all growth parameters. However, when plants were grown in sand containing AM fungi and then buried in sand with (+ +) or without AM fungi (+ ), a combination effect was produced in which the plants always produced the highest biomass. Second, if we compare plants grown in mycorrhizacontaining sand, (+ +) and (+ ), with those without AM fungi, ( ) and ( +), the former produced a significantly greater leaf area in both species, however, the increased leaf area did not always translate into increased biomass per plant. For example, in both A. psammophilum and P. virgatum, the (+ +) and (+ ) treatments did not show a significant increase in biomass during 1993 and 1996, respectively. One may ask though, why did ( +) plants not show an enhancement in vigour as compared to ( ) plants when they were buried in sand containing AM fungi. The most probable reason was the absence of AM fungi in the original planting and the availability of all other benefits of burial even to ( ) plants. Apparently, following a burial episode, the plants show reactive growth and exhibit enhanced vigour under buried conditions (Danin 1996). Another factor may be the length of time required by a plant to develop new roots on the stem within the sand deposit. Maun (1985) showed that depending on the species, it may take a long time for the plants to develop new roots. Even though AM fungi were present in the deposited substrate, they could not colonize these roots. In the 1996 experiment, the plants had produced new roots in the burial deposit after 11 weeks of burial in sand and these roots had been colonized by AM fungi but the expected increase in vigour did not materialize. ECOLOGICAL IMPLICATIONS We have shown that mycorrhizal fungi alone were not responsible for the enhanced growth shown by plants in the burial treatments. The growth stimulation following a burial episode is a composite response of numerous factors. The major contribution of mycorrhizal fungi to the plant would be the exploitation of the newly created soil volume. Because the levels of root colonization differed among species (Perumal 1994), the benefits of mycorrhizal fungi would be variable for different species (Anderson & Liberta 1987). Thus as suggested by Read (1993) and Francis & Read (1994), the overall development of plant communities in a chronosequence may be closely related to this fungal mutualism. Colonization by AM fungi increases CO 2 uptake as compared to non-mycorrhizal plants (Allen et al. 1981). The foredunes along the Great Lakes mark the first stage in plant succession where several perennial dune species, such as A. breviligulata, A. psammophilum and P. virgatum, had rather high levels of AM-fungal colonization (Perumal 1994). This mutualism would not only protect the roots from soil pathogens (Newsham et al. 1995; Little & Maun 1996), enhance nutrient uptake, improve translocation of water, and increase rootlet size and longevity (Allen 1991, 1996), but also in conjunction with burial episodes would allow the growth and buildup of dune systems. However, as soon as sand accretion ceases, the plants start to deteriorate in vigour (Eldred & Maun 1982), soil organic matter and nitrogen begin to build up (Baldwin & Maun 1983), soil flora and fauna are altered (Killam 1995) and a transition occurs from a herbaceous type to woody vegetation. Acknowledgements We thank Irene Krajnyk for drawing Figs 2 and 3, and Richard Little and three anonymous referees for constructive suggestions on earlier drafts of the manuscript. This study was supported by a grant from

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