The Effect of Phosphorus on Growth and Cluster-Root Formation in the Chilean Proteaceae: Embothrium Coccineum (R

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Plant Soil (2010) 334:113–121 DOI 10.1007/s11104-010-0419-x

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The effect of phosphorus on growth and cluster-root formation in the Chilean Proteaceae: Embothrium coccineum (R. et J. Forst.)

Alejandra Zúñiga-Feest & Mabel Delgado & Miren Alberdi

Received: 10 July 2009 /Accepted: 3 May 2010 /Published online: 26 May 2010 # Springer Science+Business Media B.V. 2010

Abstract One of the main factors that favours the were measured. Also acid exudation of CR was formation of cluster roots is a low supply of phosphorus assayed using bromocresol purple on sterile agar (P). The soils of southern Chile are mainly formed from plates. Treatments significantly affected growth and volcanic ash, characterized by low levels of available P. proportion of CR, the highest growth was observed Embothrium coccineum, a Chilean Proteaceae species with H. Under all treatments plants produced a similar produces cluster roots (CR). The factors that control number of CR. However, the proportion of CR CR formation in Chilean Proteaceae have not been biomass was higher with W and H-P than with H. extensively studied. The objective of this work was to Plants under W exhibited the lowest growth and low assess the effects of P on the growth and cluster-root shoot/root ratio. Acid exudation of CR was not formation of E. coccineum. Plants were produced from detectable in our experiment. These results are dis- seeds collected at two different locations: Valdivia and cussed comparing CR formation in low P conditions Pichicolo both at 39ºS. They were cultured under on Lupinus albus and other Proteaceae species, and the similar greenhouse conditions, from June to Septem- possible role of CR formation in E. coccineum ber, watered twice a week using: distilled water (W), considering its wide geographical distribution. full strength Hoagland’s nutrient solution (H) or Hoagland without P (H-P). At the end of the Keywords Cluster roots . Embothrium coccineum . experiment, height, total dry biomass, number of Growth rate . Phosphorus . Acid exudation cluster roots (CR) per plant, CR /total root weight,

Introduction Responsible Editor: Michael Denis Cramer. : A. Zúñiga-Feest (*) M. Delgado Embothrium coccineum is an endemic species of the Laboratorio de Fisiología Vegetal, Instituto de Geociencias, temperate rainforests of the south of South America; Universidad Austral de Chile, in Chile it occurs between 35 º S and 56 º S Casilla 567, Valdivia, Chile (Rodriguez et al. 1983). It is a shade-intolerant e-mail: [email protected] pioneer tree that can develop from seedlings on sites of bare volcanic slag and it can also resist low M. Alberdi temperatures and in some cases summer drought Instituto de Agroindustrias, Universidad de la Frontera, Casilla 54-D, (Alberdi 1995). E.coccineum can grow under different Temuco, Chile climatic and soils conditions and has a high coloniz- 114 Plant Soil (2010) 334:113–121 ing capacity (Donoso 2006). This species, like most Table 1. Seeds were stratified in wet sand as described of the members of the family of Proteaceae, exhibits by Donoso and Escobar (1986) and subsequently an interesting root adaptation, namely cluster roots treated with gibberellic acid at 250 ppm to stimulate (CR). These are dense clusters of fine roots with germination. This process was carried out in a abundant hairy rootlets attached to an axis (Purnell temperature controlled growth chamber at 20°C, 1960). Such structures have been frequently reported photoperiod 12/12 h light/dark. in species that grow on poor soils (Lamont 2003; When the radicle had emerged, the seeds were Lambers et al. 2006). The CR exudes organic acids transferred to vermiculite and irrigated with the nutrient that allow these species to solubilise phosphate solution Phostrogen® (pbi Ltd, Enfield, Middlesex, associated with iron and aluminium in the rhizosphere EN1 1SP, UK), and maintained in a growth chamber and take it up through their roots. Moreover, these for 15 days under constant conditions with 150± organic acids chelate aluminium, thus detoxifying it 10 μmol photons m−2s−1, temperature of 18±5°C and (Jones 1998, Peñaloza et al. 2004). c. 80% relative humidity. Subsequently, the plants were One of the main factors that favours the formation moved to 1 L containers with clean sand and kept in a of cluster roots is a low supply of P (Skene et al. greenhouse for 4 months (June to September). Clean 1996; Gilbert et al. 2000; Neumann and Römheld sand was obtained from a river, washed ten times with 2000; Shane et al. 2004a; Shane and Lambers 2005) distilled water until constant conductivity readings or iron (Zaid et al. 2003). The factors that induce the were obtained. development of CR in Chilean Proteaceae have been The temperature and relative humidity conditions of poorly studied, however some authors proposed the greenhouse were measured with a data logger (LI- interaction with microorganisms within the rhizo- 1400) using LI-1400-104 and LI-1400-106 sensors, sphere (Grinbergs et al. 1987; Ramirez et al. 1990, respectively (Li-Cor, Lincoln, NE). In addition, photon 2004). Other factors proposed as inducers of CR flux density (PFD) at the top of the plants was recorded growth in G. avellana are organic matter content and using a quantum sensor (Li-Cor 250; Lincoln, NE). soil texture (Pozo 1989; Krause 1996) However, During the trial, noon maximum temperatures were several of these reports showed contrasting results between 13 and 30°C, while the humidity was between and the factors that control the induction of CR in E. coccineum remain unknown. Because the soils of southern Chile are mainly formed from volcanic ash, Table 1 Climatic, geological and physiochemical properties of characterized by low levels of available P and low pH soils at the locations where seeds of Embothrium coccineum were collected. Sources: DGAC (Dirección general de aguas) (Borie and Rubio 2003) the objective of this investi- (2007), Covarrubias and Contreras (2004), CIREN (Centro de gation was to study the effect of P on growth, biomass investigaciones de recursos naturales) (2003) and Huber (1970). distribution and cluster-root formation in seedlings Nutrients (P, Al, Fe) and pH were measured at first 30 cm of of E. coccineum grown from seed under nursery soil conditions. Characteristics Locations

Valdivia Pichicolo Geological origin Metamorphic Modern volcanic Materials and methods rock ash

Seed collection, processing and maintenance of plant Annual rainfall (mm) 2000 1500 material Mean annual temperature °C 12.1 14.2 Soil depth (cm) 80 40 Seeds of E. coccineum were collected during the Extractable aluminium (ppm) 2.69 5 summer at two locations: Valdivia and Pichicolo, both Extractable Iron (ppm) 0.52 1.2 located in the region of Los Rios, central Chile. Nitrogen (%) 0.83 0.31 Valdivia is located on the coast at 39 º 38 ′S and 73 º 5 P Olsen (ppm) 4.1 6.8 ′ W, while Pichicolo is 20 km from Los Lagos and pH 5.14 4.78 ′ ′ located at 39° 51 S and 72° 48 W. The climatic and Aluminium saturation (%) 0.8 1.18 soil characteristics of both locations are shown in Plant Soil (2010) 334:113–121 115

48 and 86%. The photon flux density at noon varied change was provided by adding drops of diluted between 622 and 1 428 μmol m−2s−1. hydrochloric acid (0.01 N) to the plate to verify colour change from purple to yellow. Also CR from Experimental design Gevuina avellana and Lupinus albus were assessed using the same methodology to provide another Plants were separated into three groups in the green- positive control. Photographs of the plates were taken house and each group watered with distilled water (W), after 24 hours of exposure to the pH indicator. full strength Hoagland’s nutrient solution (H) or Hoagland’s without phosphorus (H-P) (Strasburger et Statistical analysis al. 1986). Nutrient concentrations of full strength Hoagland solution (H) in mM are follows: 5 Ca To determine if there were significant differences

(NO3)2, 5 KNO3, 2 MgSO4,1KH2PO4, 1 FeEDTA, 1 between treatments and seedling origin, a two-way Micronutrients (NaNO3, MgCl2, NaSO4 ,NaH2PO4, ANOVA was applied. A Tukey test was used to CaCl2). For Hoagland without P solution (H-P) identify those values with significant differences. KH2PO4 was not added. The pH of the nutrient Both analyses were performed with Sigma Stat 2.0 solutions was 5.2 (H) and 5.5 (H-P), also soils was software (SPSS, Chicago, IL). Differences between monitored throughout the experiment, and pH ranged the values were considered significant at P≤0.05. from 5.1 to 5.8. The solutions were added once a week; plants were in addition watered with distilled water depending on their requirements. Plants were Results grown for three months from July to September. At the beginning and end of the experiment the height of P effect on the growth of E. coccineum the stem was recorded. Also at the end of the experiment tissue was harvested from the: stems, Nutrient treatments significantly affected the growth of leaves, non-cluster and cluster roots; and was weighed seedlings from both locations. The greatest height using an analytical balance. Tissue was dried at 70°C increase was observed in plants irrigated with full using oven until a constant weight was obtained. nutrient solution (35±4.7 cm) and the smallest in plants Biomass distribution was expressed as relative values irrigated with distilled water (1.12±0.28 cm) (Fig. 1). of total dry weight of the plants. In addition, number The greatest dry mass, 4±0.2 cm and 1.82±0.4 cm of CR in each treatment was determined at the end of from Pichicolo and Valdivia respectively, was observed the experiment and CR/total root weight was calculated. Seedlings were distributed randomly in blocks in the greenhouse, with 20 seedlings for each nutrient 50 Valdivia treatment. CR obtained from 5 seedlings of each 40 A Pichicolo treatment was used to evaluate acid exudation on agar plates with bromocresol purple as indicator at the end 30 of the experiment. 20

Acid exudation of CR Increment height (cm) 10 B C 0 Cluster roots were separated and severed from each W H H-P plant, with 5 individuals per treatment. Each CR was Treatment gently washed in distilled water and then air-dried for Fig. 1 Effect of P on growth of Embothrium coccineum 5 minutes. The CR were placed on sterile agar plates seedlings. Height increment of seedlings which were cultured containing a pH indicator (Bromocresol purple, 0.1% from seeds from Valdivia and Pichicolo. Plants were main- w/v) with the pH adjusted to 7, following the tained under greenhouse conditions and watered using different methodology described by Neumann et al. (2000). nutrient solutions. Lower-case letters indicate significant differences between different locations (P≤0,05). Capital letters The exudation of acid was detected by a change in indicate significant differences among different treatments colour from purple to yellow. A positive control of pH (P≤0,05) 116 Plant Soil (2010) 334:113–121 for plants irrigated with full nutrient solution (Fig. 2). treatments. Relative CR biomass increased under Plants irrigated with distilled water or without P did water irrigation treatment and was significantly higher not exhibit significant differences in total dry biomass for both locations; the highest value was 29.9±2.6 % (Fig. 2). Pichicolo plants had significantly higher total of total plant biomass for plants from Valdivia dry biomass than Valdivia plants, both in full nutrient (Table 2). and without P solutions (Fig. 2). However significant differences were not observed between locations when Cluster root formation and acid exudation height increase was compared. All plants had CR under all treatments and for both P effect on biomass distribution locations; the nutrient treatment did not significantly affect the number of cluster roots. The average Irrigation treatments significantly affected the relative number of CR per plant was higher for Pichicolo biomass distribution, of plants from both locations. plants than for those from Valdivia, with values of Plants watered with full strength Hoagland’snutrient 35.5 and 22.5 respectively (Fig. 3a). However, the solution, showed the highest shoot/root ratios (3.7 to values of CR/total root biomass, exhibited significant 3.2) but without significant differences between plant differences between nutrient treatments, full nutrient locations (Table 2). Plants H-P exhibited intermediate solution had the lowest values for both locations values c. 2 and plants irrigated with distilled water (0.06–0.15) and the highest values were observed showed the lowest values, c. 1. Under nutritional under W and H-P solution (0.4 and 1.3). Plants from deficiencies plants from Pichicolo had the higher Valdivia had significantly higher values of CR/Total values of shoot/root compared to plants from Valdivia. root dry biomass than those from Pichicolo in all In general, shoots had the highest biomass alloca- nutrient treatments (Fig. 3b). In general, plants in all tion for all treatments and locations, with values of treatments had mainly young white CR, at a similar 42.6 and 82.4 % of total plant biomass, under water stage of development (Fig. 4b and c). Additionally, and full nutrient solution respectively (Table 2). the average weight of each cluster root did not differ Plants irrigated with water or without P exhibited a between treatments, ranging from 0.01 to 0.02 g DW significant decrease in shoot relative biomass (Table 2, (Fig. 4b). Acid exudation of the CR of the harvested Fig. 4a). Plants irrigated with distilled water had little plants was only evaluated at the end of the experiment above-ground growth, and had significantly higher (September), but no acid exudation was detected non cluster roots (NCR) and CR biomass than other (Fig. 5).

5 Discussion a Valdivia 4 A Pichicolo In our experiments E. coccineum seedlings from both locations and with all treatments produced CR, these 3 structures in our experiment were abundant young b 2 small white shape, reaching up to 40 per plant under full nutrient treatment. However, net acid exudation Total dry mass (g) B 1 a B b was not detected using pH indicator (bromocresol purple), in parallel we used Lupinus albus CR to 0 W H H - P confirm exudation detected by the yellow colour on Treatments the agar plates. This unexpected result using CR from Fig. 2 Effect of P on growth of Embothrium coccineum E. coccineum could be related to seasonal variation seedlings. Total dry mass at the end of watering treatments, exhibited by this species, because recent results show plants were cultured from seeds from Valdivia and Pichicolo, that CR net acid exudation was detected only during and maintained under greenhouse conditions. Lower-case autumn. Recently Zúñiga-Feest et al. (2009) showed letters indicate significant differences between different locations (P≤0,05). Capital letters indicate significant differences that the abundance of CR was highest during spring among different treatments (P≤0,05) and summer in two year old E. coccineum seedlings Plant Soil (2010) 334:113–121 117

Table 2 Effect of P on biomass distribution and shoot/root ±standard error. Lowercase letters indicate average values with ratio of Embothrium coccineum seedlings from two locations significant differences between watering treatments at similar (Valdivia and Pichicolo). Values are expressed in relation to tissues. Capital letters indicate significant differences between total dry biomass of each treatment and correspond to mean different locations (P≤0,05)

Shoot Root CR Shoot/Root

Valdivia W 45.94±1.80 c 24.16±1.34 a 29.90±2.06 a 0.52±0.05 Bc H 82.44±0.62 a 15.34±0.79 b 2.22±0.77 c 3.71±0.30 a H-P 66.80±2.62 b 18.47±2.44 ab 14.73±1.72 b 1.58±0.19 Bb Pichicolo W 42.60±4.80 b 38.15±5.32 a 19.25±2.32 a 0.93±0.12 Ac H 73.79±1.10 a 24.55±0.94 b 1.65±0.28 c 3.17±0.19 a H-P 71.68±0.55 a 19.61±0.08 c 8.71±0.64 b 2.55±0.10 Ab under nursery conditions, without fertilization (39ºS). summer. There are known seasonal variations in Also, Donoso-Ñanculao et al. (2009) sorting plants growth rate for Chilean Proteaceae species; E. under field conditions found young CR mainly during coccineum and G. avellana grows mainly during spring (41ºS) and non CR were observed during spring and summer (Donoso 2006), however seasonal nutrient acquisition is not completely understood. 60 Preliminary measurements in our laboratory, using Valdivia a a mature CR collected from E.coccineum plants and 50 Pichicolo evaluated by HPLC, showed citrate and malate

40 a exudation during the autumn. However further analyses are needed in order to understand CR exudation 30 b b in E. coccineum and the possible effects of seasonal 20 variation on: growth rate, CR formation and acid Number cluster roots exudation. 10 Nutrient treatments significantly affected the growth 0 of E. coccineum plants from both locations. Full a 1,4 b strength Hoagland’ssolutionfavouredgrowthin A 1,2 comparison to the treatments supplied with water, in a A which the plants were chlorotic. Although E. cocci- 1,0 neum can grow in soils with low P availability, a high 0,8 nutrient supply led to enhanced growth, especially of 0,6 bb aerial tissues (Donoso and Escobar 1986;Donoso 0,4 B 2006). Toxicity using full strength Hoagland’ssolution a 0,2 b was not detected during the experiment. However in

CR weight dry/g total root dry 0,0 other Chilean Proteaceae, like Gevuina avellana the W H H-P Treatment effects of fertilization on soil were only significant when the fertilizer was added at the beginning of the Fig. 3 Effect of P of cluster root formation on Embothrium growing season (Morales 2004). Toxicity was reported coccineum seedlings. a Number of cluster roots (CR) per plant; in plants of G. avellana under field soil culture b CR/total root biomass ratio calculated on dry weight value. Each value corresponds to a mean of six independent samples conditions at 40º S, when fertilization included 2000 ±standard error. Seedlings were cultured from seeds from Kg/ha of complete macronutrient supply (N, P, K; 16- Valdivia and Pichicolo, maintained under greenhouse condi- 14-5) and 500 Kg/ha was reported as being likely to be tions and watered using different nutrient solutions. Lower-case adequate (Medel 2001). The amounts used for nursery letters indicate significant differences between different locations (P≤0,05). Capital letters indicate significant differences production of E. coccineum are provided by Osmo- among different treatments (P≤0,05) cote® (N, P, K: 14-14-14) at the beginning of growing 118 Plant Soil (2010) 334:113–121

Fig. 4 General view of Embothrium coccineum seedlings at the end of the watering experiment. a: Representative individuals watered with different solutions; b representative CR of one seedling watered with Hoagland solution and c): representative young white CR of E. coccineum with some dark sand particles

season with an amount of 0.032 g per plant. Optimal Hakea prostrata (R. Br.) can grow well at low levels fertilization ranges are not yet clear for Chilean of P, compared to other species (Shane et al. 2004b). Proteaceae and our experiments represent the first It has been reported that for some Australian approach to studying nutritional effects on E. cocci- Proteaceae species symptoms of toxicity, resulting neum growth and CR formation. from an excess of P, can develop even in soils with Apparently, the P requirements of Proteaceae are relatively low concentrations of this nutrient (Shane lower than for other tree species (Denton et al. 2007). and Lambers 2005). In our experiments we used a full

Fig. 5 Acid exudation assay of cluster roots from Embothrium coccineum seedlings. Picture shows representative cluster roots (CR) on the agar plate with bromocresol purple 0,1 %, acid exudation indicated by a change in colour (from purple 7.0 – 6.5 to yellow 4 - 5). E. coccineum (a) and G. avellana (b) cluster roots collected during the autumn from seedlings under nursery conditions, c CR of E. coccineum seedlings cultured with different watering treatments: without acid exudation (left side) and agar plate with H Cl added showing change in the colour of bromocresol purple under similar exposure time and assay conditions Plant Soil (2010) 334:113–121 119 strength Hoagland’s nutrient solution that contained induced by P deficiency. Additionally, it has been 1 mM of P, however our plants were in sand not in a reported that a deficiency of N and Fe can enhance the permanent contact with this solution as happens under formation of CR in Australian Proteaceae (Shane and hydroponic culture. Ranges of P concentrations using Lambers 2005). Our results showed that CR produc- hydroponic cultures and other Proteacea species are: tion on E. coccineum was enhanced to low supply of P, 0–100 µM on the extremely P sensitive Hakea however critical tissue levels of P that led CR prostrata (Shane et al. 2004a), or low winter ranges formation on Chilean Proteaceae remain unknown. It from 0–6 µmol Pi/ day and high summer ranges from seems that the P content of tissues is relevant to CR 30–300 µmol Pi/day (Shane et al. 2004b), or 0–200 µ production for G.avellana, because Morales (2004) µmol Pi/ day on Grevillea crithmifolia (Shane and showed that fertilization added directly to leaves, using Lambers 2006). Symptoms of P toxicity occur in Bayfolan® solution (N,P,K: 11-8-6 m/v) produced Banksia ericifolia (L.), B. grandis (Willd.) and Hakea plants with a lower number of CR than control plants, prostrata, when winter P accumulation beyond their without fertilization. storage capacity (Parks et al. 2000; Lambers et al. Complete nutrition significantly favoured growth and 2002). In Hakea prostrata toxicity is due to a reduced the shoot/root ratio of Embothrium coccineum.Plants capacity for down-regulating net P-uptake rates from Valdivia and Pichicolo did not show significant (Shane and Lambers 2006). E. coccineum had a high differences in the mean number of CR comparing all seasonal variation in growth rate that mainly occurred treatments. However, if we compare CR/total root during spring and summer and could present P weight for plants watered with and without P (W and toxicity during winter dormant period, when could H-P) we observe a higher ratio for Valdivia plants accumulate P. However the P storage capacity of E. supplied with nutrients. In general, the response to coccineum has not been explored yet. different nutritional treatments was larger in plants from When plants were fertilized, biomass was mainly Valdivia than in those from Pichicolo. These differ- allocated to shoot growth and only a minor fraction ences exhibited under common garden conditions could was invested in CR formation. The opposite was be related to the genetic features of each population. observed in the distilled water treatment; above- Different responses in growth rate and CR production ground biomass was small and a large proportion of were also reported by Ramirez et al. (1990)and the biomass was associated with both NCR and CR. Morales (2004), using G. avellana plants, produced In our experiment E. coccineum plants watered with from seeds collected at different locations. Studies that full strength Hoagland’s nutrient solution had unex- correlate physiological features (CR formation, growth pected CR close to that exhibited with H-P or W rate, photosynthetic performance) and genetics using treatments (20 to 40 per plant), these structures were seedlings of different populations of E. coccineum from present in all the plants evaluated from the two contrasting habitats (from 38 to 56 º S in Chile) are locations. Other Proteaceae species like Grevillea under way in our laboratory. These results could robusta did not present CR when grown hydroponi- improve our understanding of the role of CR in Chilean cally using full strength Hoagland’s nutrient solution Proteaceae and the factors that induce these structures. (Skene et al. 1996). In general, recently emerged E. coccineum seedlings watered with distilled water had a high frequency of individuals with CR (64 to 100%) Acknowledgements The authors thank Fondecyt 11086162 for financial support and Vivero Bosques del Sur from Universidad (Gonzalez et al. 2007). E. coccineum seedlings also Austral de Chile, for greenhouse maintenance. Also, Dr. Hans had a high number of CR per plant (up to ca. 130 per Lambers, Dra. Frida Piper and three anonymous reviewers for plant) under greenhouse conditions with normal critical and helpful comments that improved this manuscript. complete fertilization (N; P; K) at the beginning of growing season (Zúñiga-Feest et al. 2009). Several species like: Lupinus albus (Keerthisinghe et References al. 1998; Gilbert et al. 2000; Neumann and Römheld 2000), Hakea prostata (Roelofs et al. 2001), Grevillea Alberdi M (1995) Ecofisiología de especies leñosas de los crithmifolia (Skene et al. 1996), Casuarina glauca and bosques hidrófilos templados de Chile: Resistencia a la C. cunninghamiana (Diem et al. 2000), CR are sequía y bajas temperaturas. In: Armesto J, Villagrán C, 120 Plant Soil (2010) 334:113–121

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