The effects of pH and Phosphorus concentrations on the chlorophyll content of
Salvia chamelaeagnea (Lamiaceae) is a slow growing water‐wise evergreen shrub originating from the western province of South Africa. It is an attractive landscape, and S. chamelaeagnea is a medicinal plant. It is important to develop enhanced cultivation protocols that could result in high yield and high‐quality medicinal materials. Chlorophyll is a fundamental part of the light‐dependent reactions of the photosynthesis process. This chapter investigates the effects of four phosphorus concentrations and three pH levels of supplied irrigated water on the production of chlorophyll A, chlorophyll B, total chlorophyll, leaf colour and the nutrient uptake of S. chamelaeagnea grown in hydroponics over an 8‐week period at the Cape Peninsula University of Technology. The treatments of pH 4, pH 6 and pH 8 at 31, 90, 150 and 210 ppm of phosphorus were received by 12 groups of plants and were replicated 10 times. The results indicated that at pH 4, P fertilization significantly (P < 0.05) induced a higher chlorophyll production of S. chamelaeagnea grown in hydroponics compared to other pH treatments (pH 8 and pH 6).
- chlorophyll production
- medicinal plants
- Salvia chamelaeagnea
Salvias are renowned for their variety and their many uses around the home and garden; they have beautiful flowers and attract birds . In its natural habitat,
Unfortunately, very little information has been documented on the cultivation of this species. Cultivation of medicinal plants is gaining traction worldwide; it is seen as a tool for biodiversity conservation, poverty alleviation and cultural preservation . However, good knowledge of plant physiology must be attained in order to develop enhanced cultivation protocols that could result in high yield and high‐quality medicinal materials. Effects of nutrients and nutrient ratios on many food and medicinal crop plants, such as soya bean, thyme, wheat cultivars, barley, spinach and pelargoniums, have been studied. In most cases, a positive result in growth is noticed with the addition of some macro‐nutrients such as N, P, K, Mg or Ca [13–21]. It is therefore crucial that adequate plant nutrition and soil pH levels are met for any given plant so that the cell's functioning is not impeded. Chlorophyll is a fundamental part of the light‐dependent reactions of the photosynthesis process, capturing light rays from the sun and producing energy‐storing ATP molecules that are essential for the functioning of a healthy plant [22, 23]. The effects of poor nutrition, be it through infertile soils or incorrect soil pH level, directly affect the production of chlorophyll molecules resulting in chlorosis of leaves and a reduced photosynthetic rate, thus inhibiting some biological processes and decreasing the general health of the plants [23–25]. There are plausible mechanisms through which the production of chlorophyll could be affected, for example, the pH level of a growing medium affects the uptake of P  and the P level influences the nutrient uptake by plants . The relationship between the nutrient P and chlorophyll is not fully understood. According to Nicholls and Dillon , there are substantial variations of the published phosphorus‐chlorophyll relationship, which they ascribed to variations in sampling and analytical techniques.
This chapter aims to investigate the effects of P and pH on the chlorophyll production, leaf colour and the nutrient uptake of medicinal
2. Materials and methods
2.1. Experimental process
The experiment took place in the research glasshouse at the Cape Peninsula University of Technology (CPUT), Cape Town campus, South Africa, latitude and longitude S33°55′ 58 E18°25′ 57, from June 2012 to August 2012. Inside the glasshouse was a 40%‐Aluminet shade cloth, raised 2 m above the floor, resulting in light intensities ranging from 10 to 13 Klx, determined by using a Toptronic T630 light meter. The climate was controlled between 16 and 28°C during the day while 10–20°C during the night, with an average relative humidity of 42%.
The experiment was laid out in a randomized block design with plants being spaced 30 cm apart and consisted of 12 treatments of four differing nutrient solutions offering a low concentration of P, a balanced concentration of supplementary P, a moderate concentration of supplementary P and a high concentration of supplementary P at three differing pH levels. The control treatment of 31 ppm was chosen due to the nature of fynbos soils being low in available P [29–31].
Hoagland solution, a well‐known hydroponic nutrient solution modified by Hershey [32, 33], offering all the necessary macro‐ and micro‐nutrients for healthy plant growth, was used as a base nutrient and supplemented with P.
The plants for the experiment were rooted tip cuttings sourced from healthy mother stock plants at the CPUT Glass House Nursery. The rooted cuttings were gently rinsed in deionized water to remove any rooting media from the root's zone. They were then weighed and planted into 25‐cm plastic pots filled with leca clay and placed into a recirculating closed hydroponics system at a spacing of 30 cm, where their heights were recorded (Figure 1).
The plants were irrigated with the treatments 15 times per day at equal timed intervals for the duration of the experiment. For each treatment, there were 10 plants. The treatments were as follows:
Hoagland hydroponic nutrient solution with 31 ppm of P at a pH of 4.
Hoagland hydroponic nutrient solution with 31 ppm of P at a pH of 6.
Hoagland hydroponic nutrient solution with 31 ppm of P at a pH of 8.
Hoagland hydroponic nutrient solution supplemented with 90 ppm of P at a pH of 4.
Hoagland hydroponic nutrient solution supplemented with 90 ppm of P at a pH of 6.
Hoagland hydroponic nutrient solution supplemented with 90 ppm of P at a pH of 8.
Hoagland hydroponic nutrient solution supplemented with 150 ppm of P at a pH of 4.
Hoagland hydroponic nutrient solution supplemented with 150 ppm of P at a pH of 6.
Hoagland hydroponic nutrient solution supplemented with 150 ppm of P at a pH of 8.
Hoagland hydroponic nutrient solution supplemented with 210 ppm of P at a pH of 4.
Hoagland hydroponic nutrient solution supplemented with 210 ppm of P at a pH of 6.
Hoagland hydroponic nutrient solution supplemented with 210 ppm of P at a pH of 8.
2.2. pH level
The pH levels of the nutrient solutions were monitored using a Martini Instrument PH55 pH probe and were adjusted accordingly using either hydrochloric acid (HCl) to lower the pH or sodium hydroxide (NaOH) to raise the pH.
The treatments were set to irrigate 15 times daily for a duration of 15 min using a 1350 L/h Boyu submersible pump and a Tedelex analogue timer to regulate irrigation frequencies.
2.4. Data collection
2.4.1. Measurement of leaf colour
Green leaf colour intensity was measured using a hand‐held, dual‐wavelength SPAD meter (SPAD 502, chlorophyll meter, Minolta Camera Co., Ltd., Japan). Readings were taken from the top three fully developed leaves of each plant. For each treatment, 30 fully developed leaves were used weekly. The SPAD meter stored and automatically averaged the recordings to generate one reading per plant.
2.4.2. Measurement of chlorophyll content in leaves
The extraction of leaf chlorophyll using dimethylsulphoxide (DMSO) was carried out as described in Hiscox and Israelsta . A third of plant leaves from the tip were collected from each plant. About 100 mg of the middle portion of the fresh leaf slices was placed in a 15‐mL vial containing 7 mL DMSO and incubated at 4°C for 72 h. After the incubation, the extract was diluted to 10 mL with DMSO. A 3‐mL sample of chlorophyll extract was then transferred into curvets for absorbance determination. A spectrophotometer (UV/Visible Spectrophotometer, Pharmacia LKB. Ultrospec II E) was used to determine absorbance values at 645 and 663 nm, which were then used in the equation proposed by Arnon  to determine the total leaf chlorophyll content against DMSO blank, expressed as mg L-1 as follows: Chl
2.4.3. Measurement of the levels of macro‐ and micro‐nutrients in dry plant material
The measurements of macronutrients (N, P, K, Ca and Mg) and micronutrients (Cu, Zn, Mn, Fe and B) were determined by ashing a 1 g ground sample in a porcelain crucible at 500°C overnight. This was followed by dissolving the ash in 5 mL of 6 M HCl and putting it in an oven at 50°C for 30 min; 35 mL of deionized water was added, and the extract was filtered through Whatman no. 1 filter paper. Nutrient concentrations in plant extracts were determined using an inductively coupled plasma (ICP) emission spectrophotometer (IRIS/AP HR DUO Thermo Electron Corporation, Franklin, Massachusetts, USA) .
2.5. Statistical analysis
Data collected was analysed for statistical significance using the two‐way analysis of variance (ANOVA), with the computations being done using the software program STATISTICA. Fisher's least significance difference (LSD) was used to compare treatment means at
3.1. Effects of pH and phosphorus concentrations on the chlorophyll content of
S. chamelaeagneagrown in hydroponics
Treatment significantly (
|Treatments||Chlorophyll A||Chlorophyll B||Total chlorophyll|
|pH 4, P 31 ppm||12.242 ± 1.7a||3.446 ± 0.5a||15.684 ± 2.2a|
|pH 6, P 31 ppm|
|10.384 ± 1.0cd||2.848 ± 0.3cde||13.229 ± 1.3cd|
|pH 8, P 31 ppm||10.173 ± 1.1cde||2.784 ± 0.3ef||12.954 ± 1.5cde|
|pH 4, P 90 ppm||11.419 ± 0.5ab||3.233 ± 0.2ab||14.649 ± 0.6ab|
|pH 6, P 90 ppm||8.348 ± 1.1g||2.227 ± 0.3hi||10.574 ± 1.4g|
|pH 8, P 90 ppm||9.327 ± 1.3ef||2.600 ± 0.4g||11.924 ± 1.7ef|
|pH 4, P 150 ppm||10.929 ± 0.7bc||3.014 ± 0.3bcd||13.941 ± 0.9bc|
|pH 6, P 150 ppm||8.463 ± 1.4fg||2.282 ± 0.4h||10.744 ± 1.8fg|
|pH 8, P 150 ppm||7.063 ± 0.6h||1.988 ± 0.2i||9.049 ± 0.7h|
|pH 4, P 210 ppm||10.900 ± 0.7bc||3.108 ± 0.3bc||14.005 ± 0.9bc|
|pH 6, P 210 ppm||9.817 ± 1.0de||2.650 ± 0.3g||12.465 ± 1.3de|
|pH 8, P 210 ppm||3.547 ± 0.5i||0.910 ± 0.2j||4.456 ± 0.7i|
3.2. Effects of pH and phosphorus concentrations on the leaf colour of
S. chamelaeagneagrown in hydroponics
Effects of various P treatments at differed pH levels induced varied colour intensities, ranging from 16 to 31.7 from week 1 to week 8 on the leaf colour of
|pH 4, P 31 ppm||30.156 ± 3.6ab||31.667 ± 4.1a||31.433 ± 3.0a||30.878 ± 1.3a||30.189 ± 3.1ab||30.011 ± 2.2a||31.078 ± 1.8a||28.467 ± 1.9ab|
|pH 6, P 31 ppm (Control)||32.800 ± 2.5a||31.644 ± 4.0a||30.533 ± 1.3ab||29.933 ± 2.0a||30.122 ± 2.4ab||28.822 ± 2.6ab||28.644 ± 1.6bcd||28.922 ± 1.4a|
|pH 8, P 31 ppm||30.033 ± 4.5ab||30.567 ± 3.8ab||28.933 ± 1.4b||29.944 ± 2.1a||29.189 ± 1.6ab||30.111 ± 2.4a||29.589 ± 1.7abc||28.278 ± 2.3ab|
|pH4, P 90 ppm||29.689 ± 3.8ab||29.911 ± 5.3ab||30.867 ± 2.2ab||31.111 ± 1.5a||30.711 ± 2.1ab||29.656 ± 2.4a||30.567 ± 2.1a||28.122 ± 2.0ab|
|pH 6, P 90 ppm||31.789 ± 3.6ab||31.156 ± 4.6ab||31.078 ± 2.8ab||30.578 ± 2.0a||29.444 ± 1.6ab||27.000 ± 1.4b||28.067 ± 2.2cde||27.056 ± 1.0bc|
|pH 8, P 90 ppm||29.411 ± 3.3b||27.356 ± 3.0bc||22.756 ± 3.0c||20.067 ± 2.3b||20.278 ± 1.2c||24.489 ± 1.6c||27.000 ± 1.9e||27.800 ± 1.7abc|
|pH 4, P 150 ppm||30.289 ± 3.6ab||30.044 ± 3.7ab||31.233 ± 2.7ab||30.622 ± 1.5a||29.189 ± 2.6ab||29.989 ± 4.0a||30.178 ± 1.8ab||27.956 ± 1.1ab|
|pH 6, P 150 ppm||29.333 ± 3.5b||30.944 ± 3.6ab||29.933 ± 2.1ab||29.356 ± 2.7a||28.633 ± 2.3b||28.233 ± 2.2ab||26.456 ± 1.5e||25.100 ± 2.1de|
|pH 8, P 150 ppm||29.267 ± 3.0b||22.422 ± 4.9d||15.978 ± 2.8d||15.756 ± 3.5c||15.156 ± 2.7d||15.267 ± 3.1e||21.756 ± 1.6f||24.278 ± 2.3e|
|pH 4, P 210 ppm||30.944 ± 2.8ab||30.456 ± 3.1ab||28.956 ± 2.4b||31.033 ± 2.0a||30.911 ± 3.2a||28.767 ± 2.0ab||29.533 ± 0.7abc||29.278 ± 1.4a|
|pH 6, P 210 ppm||31.756 ± 3.9ab||28.489 ± 3.2abc||29.478 ± 2.4ab||29.444 ± 1.2a||29.722 ± 1.0ab||28.622 ± 1.7ab||27.756 ± 2.0de||26.278 ± 1.7cd|
|pH 8, P 210 ppm||31.411 ± 2.6ab||24.922 ± 5.7cd||16.167 ± 3.6d||13.900 ± 2.7c||14.511 ± 1.8d||18.044 ± 1.7d||18.567 ± 1.6g||16.067 ± 1.8f|
3.3. Effects of pH and phosphorus concentrations on the uptake of macro‐nutrients in
S. chamelaeagneagrown in hydroponics
Macro‐nutrient uptake of P, K and Mg was significantly (
|Treatments||N (%)||P (%)||K (%)||Ca (%)||Mg (%)|
|pH 4, P 31 ppm||4.18 ± 0.29bcd||0.64 ± 0.06g||4.23 ± 0.29g||1.13 ± 0.07a||0.28 ± 0.01h|
|pH 6, P 31 ppm||4.24 ± 0.55abc||0.73 ± 0.06f||4.41 ± 0.23fg||1.12 ± 0.10a||0.36 ± 0.03e|
|pH 8, P 31 ppm||4.19 ± 0.18abcd||0.62 ± 0.08g||4.47 ± 0.26efg||1.10 ± 0.11ab||0.43 ± 0.04c|
|pH 4, P 90 ppm||4.27 ± 0.26abc||0.77 ± 0.08ef||4.64 ± 0.36cdef||1.10 ± 0.10ab||0.31 ± 0.02g|
|pH 6, P 90 ppm||4.41 ± 0.20a||0.82 ± 0.08cde||4.53 ± 0.13defg||1.08 ± 0.07ab||0.38 ± 0.03d|
|pH 8, P 90 ppm||4.20 ± 0.12abcd||0.80 ± 0.07def||4.45 ± 0.24efg||1.14 ± 0.05a||0.48 ± 0.03b|
|pH 4, P 150 ppm||4.37 ± 0.19ab||0.82 ± 0.04cde||4.79 ± 0.58cd||1.07 ± 0.06abc||0.32 ± 0.03fg|
|pH 6, P 150 ppm||4.09 ± 0.22cd||0.88 ± 0.07bc||4.87 ± 0.19c||1.01 ± 0.07cd||0.36 ± 0.02de|
|pH 8, P 150 ppm||4.00 ± 0.08de||1.07 ± 0.08a||6.29 ± 0.39a||0.77 ± 0.03e||0.55 ± 0.02a|
|pH 4, P 210 ppm||4.13 ± 0.18cd||0.84 ± 0.06bcd||4.73 ± 0.32cde||1.05 ± 0.06bc||0.31 ± 0.02g|
|pH 6, P 210 ppm||4.05 ± 0.18cd||0.87 ± 0.08bcd||4.23 ± 0.35g||0.97 ± 0.06d||0.35 ± 0.02ef|
|pH 8, P 210 ppm||3.77 ± 0.16e||0.91 ± 0.13b||5.71 ± 0.38b||0.46 ± 0.03f||0.48 ± 0.03b|
3.4. Effects of pH and phosphorus concentrations on the uptake of micro‐nutrients in
S. chamelaeagneagrown in hydroponics
The micro‐nutrient uptake of Na, Mn, Fe, Cu, Zn and B was significantly (
|Treatments||Na (mg/kg)||Mn (mg/kg)||Fe (mg/kg)||Cu (mg/kg)||Zn (mg/kg)||B (mg/kg)|
|pH 4, P 31 ppm||477.89 ± 36.27fg||84.67 ± 7.48efg||151.56 ± 7.32cde||5.22 ± 1.99a||39.56 ± 2.88bc||37.78 ± 3.63ab|
|pH 6, P 31 ppm|
|479.78 ± 57.99fg||105.89 ± 11.40c||139.11 ± 10.17def||2.89 ± 0.60d||38.00 ± 3.20c||38.56 ± 3.09a|
|pH 8, P 31 ppm||472.89 ± 58.58g||156.78 ± 9.11a||137.11 ± 8.25ef||2.89 ± 0.33d||37.89 ± 3.44c||37.33 ± 2.29ab|
|pH4, P 90 ppm||548.44 ± 74.72ef||84.00 ± 8.19fg||144.11 ± 10.59def||4.11 ± 0.60bc||40.11 ± 4.31bc||38.67 ± 3.67a|
|pH 6, P 90 ppm||505.78 ± 39.02fg||101.00 ± 4.69cd||153.33 ± 13.87bcd||2.56 ± 0.53de||41.33 ± 6.12bc||37.44 ± 2.40ab|
|pH 8, P 90 ppm||532.56 ± 70.06efg||150.33 ± 12.56a||167.33 ± 13.27abc||3.22 ± 0.44cd||39.78 ± 6.28bc||36.56 ± 1.24abc|
|pH 4, P 150 ppm||604.22 ± 102.07de||82.67 ± 9.84g||168.56 ± 23.51ab||4.67 ± 1ab||41.33 ± 6.24bc||37.67 ± 2.29ab|
|pH 6, P 150 ppm||680.33 ± 55.08bc||94.00 ± 19.68de||151.00 ± 34.86de||3.11 ± 1.90d||38.00 ± 6.75c||35.22 ± 3.03bcd|
|pH 8, P 150 ppm||716.00 ± 117.06b||131.44 ± 7.32b||152.78 ± 16.20bcde||4.33 ± 0.5ab||39.44 ± 4.48bc||31.67 ± 3.35e|
|pH 4, P 210 ppm||696.78 ± 69.99bc||82.11 ± 4.43g||175.00 ± 14.42a,||5.11 ± 0.60a||44.00 ± 2.92ab||34.56 ± 2.51cd|
|pH 6, P 210 ppm||640.89 ± 36.55cd||90.78 ± 9.38efg||145.11 ± 14.16def||2.44 ± 0.53de||43.89 ± 5.69ab||35.33 ± 2.45bcd|
|pH 8, P 210 ppm||867.67 ± 131.72a||93.44 ± 8.14def||129.33 ± 21.17f||1.89 ± 0.60e||46.78 ± 7.31a||33.33 ± 2.40de|
In this chapter, the significantly (
Despite the relatively high nutrient uptake values in plants receiving a nutrient solution with a pH 8, chlorosis of their leaves was apparent during the growth period. This suggests that the uptake of some essential nutrients responsible for chlorophyll development was affected at this pH level, namely the mineral nutrients Cu, B, N and Fe which are directly involved in photosynthesis, respiration, cell division and protein formation [23, 40]. In soil‐less media, the affinity of soluble nutrients to negatively charged surfaces and the interactions between charged cations can have a profound effect on nutrient availability and subsequently, the uptake of nutrients by plants. For example, fertilization with phosphorous increases the soil's nitrogen absorption in young plants of
In conclusion, this chapter gives insight into the unknown cultivation requirements of the leaf's chlorophyll development of
This study was funded by Cape Peninsula University of Technology through CPUT Bursary and University Research Funds.
Gali‐Muhtasib, H., Hilan, C. & Khater, C. 2000. Traditional uses of Salvia libanotica(East Mediterranean sage) and the effects of its essential oils. Journal of Ethnopharmacology. 71(3):513–520.
Kamatou, G.P.P., van Zyl, R.L., Davids, H., van Heerden, F.R., Lourens, A.C.U. & Viljoen. A.M. 2008. Antimalarial and anticancer activities of selected South African Salvia species and isolated compounds from S. radula. South African Journal of Botany. 74:238–243.
Cornara, L., La Rocca, A., Marsili, S. & Mariotti, M.G. 2009. Traditional uses of plants in the Eastern Riviera (Liguria, Italy). Journal of Ethnopharmacology. 125(1):16–30.
Clebsch, B. 2003. The new book of Salvias, 2nd ed. Port Land: Timber Press Inc., pp. 1–80.
Thorsen, M.A. & Hildebrandt, K.S. 2003. Quantitative determination of phenolic diterpenes in rosemary extracts, aspects of accurate quantification. Journal of Chromatography. 995:119–125.
Kamatou, G.P.P., Viljoen, A.M. & Steenkamp, P. 2010. Antioxidant, anti‐inflammatory activities and HPLC analysis of South African Salvia species. Food Chemistry. 119(2):684–688.
Kamatou, G.P.P., van Vuuren, S.F., van Heerden, F.R., Seaman, T. & Viljoen A.M. 2007. Antibacterial and antimycobacterial activities of South African Salvia species and isolated compounds from S. chamelaeagnea. South African Journal of Botany. 73(4):552–557.
Western, P. & Claßen‐Bockhoff, R. 2006. Bird pollination in South African Salvia species. Flora. 201:396–406.
Pienaar, K. 2000. What flower is that? 2nd ed. Cape Town: Struik, pp. 297–298.
Ulubelen, A. 2003. Cardio active and antibacterial terpenoids from some Salvia species. Phytochemistry. 64:395–399.
Nenadis, N., Zafiropoulou, I. & Tsimidou, M. 2003. Commonly used food antioxidants: a comparative study in dispersed systems. Food Chemistry. 82:403–407.
Wiersum, K.F., Dold, A.P., Husselman, M. & Cocks, M. 2006. Cultivation of medicinal plants as a tool for biodiversity conservation and poverty alleviation in the amatola region, South Africa. In R.J. Bogers (ed.), Medicinal and aromatic plants. The Netherlands: Springer, pp. xx–xxx. http://bioculturaldiversity.co.za/publications/CULTIVATION%20OF%20MEDICINAL%20PLANTS%20AS%20A%20TOOL.pdf
Gan, Y., Stulen, I., van Keulen, H. & Kuiper, P.J.C. 2003. Effect of N fertilizer top‐dressing at various reproductive stages on growth, N2 fixation and yield of three Soybean ( Glycine max(L.) Merr.) genotypes. Field Crops Research. 80:147–155.
Khazaie, H.R., Nadjafi, F. & Bannayan, M. 2008. Effect of irrigation frequency and planting density on herbage biomass and oil production of thyme ( Thymus vulgaris) and hyssop ( Hyssopus officinalis). Industrial Crops and Products. 27:315–321.
Ichir, L.L., Ismaili, M. & van Cleemput, O. 2003. Effect of organic and mineral fertilizers on N‐use by wheat under different irrigation frequencies. Competes Rendus Biologies. 326:391–399.
Maidl, F.X., Panse, A., Dennert, J., Ruser, R. & Fischbeck, G. 1996. Effect of varied N rates and N timings on yield, N uptake and fertilizer N use efficiency of a six‐row and a two‐row winter barley. European Journal of Agronomy. 5:247–257.
Stagnari, F., Bitetto, V.D. & Pisante, M. 2007. Effects of N fertilizers and rates on yield, safety and nutrients in processing spinach genotypes. Scientia Horticulturae. 114:225–233.
Manzanares, M., Tenorio, J.L. & Ayerbe, L. 1997. Sowing time, cultivar, plant population and application of N fertilizer on Kenaf in Spain's central plateau. Biomass and Bioenergy. 12(4):263–271.
Liu, X., Ren, G. & Shi, Y. 2011. The effect of organic manure and chemical fertilizer on growth and development of Stevia rebaudianaBertoni. Energy Procedia. 5:1200–1204.
Ram, M., Ram, D. & Roy, S.K. 2003. Influence of an organic mulching on fertilizer nitrogen use efficiency and herb and essential oil yields in geranium ( Pelargonium graveolens). Bioresource Technology. 87:273–278.
Zhu, Z., Liang, Z., Han, R. & Dong, J. 2009. Growth and saikosaponin production of the medicinal herb Bupleurum chinenseDC. Under different levels of nitrogen and phosphorus. Industrial Crops and Products. 29:96–101.
Glass, A.D.M. 1989. Plant nutrition. An introduction to current concepts. Boston: Jones and Bartlett Publishers, Inc., pp. 30–50, 140–230.
Stern, K. 2006. Introductory plant biology, 10th ed. New York: McGraw‐Hill, pp. 81, 193–201.
Leopold, A.C. & Kriedemann, P.E. 1975. Plant growth and development, 2nd ed. USA: McGraw‐Hill, pp. 133, 417, 439–442.
Marschner, H. 1995. Mineral nutrition of higher plants. London: Academic Press Limited, pp. 605–609, 641–645.
Hipps, N.A., Davies, M.J., Dodds, P. & Buckley, G.P. The effects of phosphorous nutrition and soil pH on growth of some ancient woodland indicator plants and their interaction with competitor species. Plant and Soil. 271:131–141.
Li, Y., Wang, T., Li, J. & Ao, Y. 2010. Effects of phosphorus on celery growth and nutrient uptake under different calcium and magnesium levels in substrate culture. Horticultural Science. 37:99–108.
Nicholls, K.H. & Dillon, P.J. 1978. An evaluation of phosphorus‐chlorophyll‐phytoplankton relationships for lakes. International Review of Hydrology63:141–154.
Hawkins, H.K., Hettasch, H., Mesjasz‐Przybylowicz, J., Przybylowicz, W. & Cramer, M.D. 2008. Phosphorus toxicity in the Proteaceae: a problem in post‐agricultural lands. Scientia Horticulturae. 117:357–365.
Rebelo, A.G. 2001. Proteas. A field guide to the Proteas of Southern Africa. South Africa: Fernwood Press, pp. 180–235.
Witkowski, E.T.F. & Mitchell, D.T. 1987. Variations in soil phosphorus in the Fynbos biome. South African Journal of Ecology. 72–76:1100–1200.
Hershey, D.R. 1994. Solution culture hydroponics: history and inexpensive equipment. American Biology Teacher. 56:111–118.
Hershey, D.R. 1995. Plant biology science projects. New York: Wiley, pp. 20–41.
Hiscox, J.D. & Israelstam, G.F. 1979. A method for the extraction of chlorophyll from leaf tissue without maceration. Canadian Journal of Botany. 57:1332–1334.
Arnon, D.I. 1949. Copper enzymes in chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiology. 24:1–15.
Giron, H.C. 1973. Comparison between dry ashing and wet digestion in the preparation of plant materials for atomic absorption analysis. Atomic Absorption Newsletter.12:28–29.
Steel, R.G.D. & Torrie, J.H. 1980. Principles and procedures of statistics: a biometrical approach, 2nd edition. New York: McGraw‐Hill Inc.
Wood C.W., Reeves, D.W. & Hilmerick, D.G. 1993. Relationships between chlorophyll meter readings and leaf chlorophyll concentration, N status, and crop yield: a review. Proceedings Agronomy Society of New Zealand. 23: 1-9
Graciano C., Goya, J.F., Frangi J.L. & Guiamet, J.J. 2006. Fertilization with phosphorus increases soil nitrogen absorption in young plants of Eucalyptus grandis. Forest Ecology and Management. 236:202–210.
Molassiotis, A., Tanou, G., Diamantidis, G., Patakas, A. & Therios, I. 2006. Effects of 4‐month Fe deficiency exposure on Fe reduction mechanism, photosynthetic gas exchange, chlorophyll fluorescence and antioxidant defense in two peach rootstocks differing in Fe deficiency tolerance. Journal of Plant Physiology. 163:176–185.
Silber, A. 2008. Chemical characteristic of soilless media. In: Michael Raviv and J. Heinrich Lieth, editors. Soilless culture: theory and practice. San Diego: Academic Press, 2008, pp. 209–244.
Shen, J., Yuan, L., Zhang, L., Li, H., Bai, Z., Chen, X., Zhang, W. & Zhang, F. 2011. Phosphorus dynamics: From soil to plant. Plant Physiology. 156:997–1005.
Wu C., Fan Z. & Wang, Z. 2004. Effect of phosphorus stress on chlorophyll biosynthesis, photosynthesis and biomass partitioning pattern of Fraxinus mandchuricaseedlings]. The Journal of Applied Ecology. 15(6):935–940.
Naeem, M., Khan, M.M.A., Idrees, M. & Aftab, T. 2010. Phosphorus ameliorates crop productivity, photosynthetic efficiency, nitrogen‐fixation, activities of the enzymes and content of nutraceuticals of Lablab purpureusL. Scientia Horticulturae. 126:205–214.