Effects of different combinations of Hoagland’s solution and Azolla filiculoides on photosynthesis and chlorophyll content in Beta vulgaris subsp. Cycla ‘fordhook giant’ grown in hydroponic cultures

The assessments of photosynthetic rate, stomatal conductance, evapotranspiration, intercellular CO 2 concentration and chlorophyll content in Beta vulgaris subsp. cycla ‘Fordhook Giant’ grown in hydroponic cultures containing different compositions of hydroponic solutions were evaluated in this study. The aim of the study was to quantify the effects of different combinations of Hoagland’s solution and Azolla filiculoides on photosynthesis processes and chlorophyll content in B. vulgaris grown in hydroponic cultures. The following treatments were evaluated in four replications: (1) Control (Hoagland’s solution minus N solution excluding Azolla ; (2) Hoagland’s minus N solution including Azolla ; (3) full Hoagland’s solution plus Azolla ; and (4) full Hoagland’s solution excluding Azolla . Results show that photosynthetic rate, evapotranspiration, intercellular CO 2 concentration and chlorophyll were generally higher in full Hoagland’s solution. This was closely followed by full Hoagland’s solution plus Azolla , and Hoagland’s minus N solution plus Azolla treatments. The lowest photosynthetic rates and chlorophyll contents were found in the control (Hoagland’s minus N solution) treatment.


INTRODUCTION
Photosynthesis and chlorophyll concentration are directly related to N inputs in plants (Schepers et al., 1992;Blackmer and Schepers, 1995;Guo, 2001). In the photosynthesis process, nitrogen plays an important function in electron transport and photophosphorylation (Terashima and Evans, 1988). Chlorophyll is also constituted by nitrogen as it forms an important part in the formation *Corresponding author. E-mail: laubscherc@cput.ac.za.
Abbreviations: DMSO, Dimethyl sulphoxide; DWC, deep water channel; PVC, polyvinylchloride. Kumarasinghe and Eskew, 1995). Chemical nitrogen fertili-zers can be applied in different forms such as nitrate and ammonium nitrogen (Cox and Reisenauer, 1973;Reddy et al., 1989;Pahlsson, 1992;Lu et al., 2005). These substances are a main component in agrarian applications as they are able to ensure sufficient crop harvests (Watanabe and Liu, 1992;Kumarasinghe and Eskew, 1995). Biological nitrogen systems such as those involving Azolla filiculoides are able to fix nitrogen from the atmosphere into the surrounding aqueous environment (Roger and Ladha, 1992). A. filiculoides achieves this by having a symbiotic relationship with cyanobacteria Anabaena azollae (Shi and Hall, 1988). Anabaena occurs extracellularly and fixes nitrogen in the leaf fronds of A. filiculoides (Peters, 1977(Peters, , 1978. Azolla provides suitable surroundings for the Anabaena to survive in. A. filiculoides and A. azollae affiliation has been used as a biological source of N in rice cultivation (Fogg et al., 1973;Tran and Dao, 1973;Becking, 1976;Talley and Rains, 1980;Roger and Reynaud, 1982;Watanabe, 1984;Watanabe and Liu, 1992;Wagner, 1997).
The production of N from biological sources namely that of the Azolla-Anabaena symbiosis has shown promising potential to be used as a source N in crop cultivation. In this study, an experiment was conducted to evaluate the effect of this symbiosis on other food crops such as B. vulgaris in hydroponic cultures. This study was conducted with the objective of quantifying the effects of different combinations of Hoagland's solution and A. filiculoides on photosynthesis processes and chlorophyll content in B. vulgaris grown in hydroponic cultures.

MATERIALS AND METHODS
The physiological responses photosynthesis and chlorophyll concentration of B. vulgaris was measured throughout an 8 week period. An actively ventilated greenhouse sited at the Cape Peninsula University of Technology (CPUT) in Cape Town, South Africa was used to facilitate the experiment. A polyvinyl chloride (PVC) pipe deep water channel (DWC) hydroponic system on a four block experiential design was employed (Roberto, 2000). Each treatment comprised of four PVC pipes containing 20 plants resulting in 80 plants for the experiment. A 3500 L/h submersible pump circulated the nutrient solutions contained within a 70 L reservoir.
The PVC systems discussed by Roberto (2000) were suitable to cultivate B. vulgaris and A. filiculoides. B. vulgaris was positioned in the channels and allowed to establish while being supplied with flowing nutrient solution within the gully. A. filiculoides, a floating water fern located in nutrient reservoir, was allowed to drift on the nutrient solution. Both plants species were exposed to the nutrient solution within each treatment. The plant identification section at CPUT rendered the botanical material of A. filiculoides and a garden centre located in Cape Town provided B. vulgaris seedlings. A total of 70 g of A. filiculoides was introduced to two of the systems one week earlier to that of the B. vulgaris. A one week period allowed for the fixation of N into the nutrient solution while A. de Bever et al. 2007 filiculoides stabilised. B. vulgaris was placed at a plant spacing of 40 cm. Two compositions were utilized: a full Hoagland's solution and a Hoagland's minus nitrogen nutrient solution (Hershey, 1994(Hershey, , 1995

Measurement of photosynthesis
A portable photosynthesis system LCpro + 1.0 ADC, (Bioscientific Limited, 12 Spurling Works, Pinder Road, Hoddesdon, Hertfordshire, EN11 ODB, UK) was used to take photosynthesis readings. Measurements were taken on a healthy fully functional third leaf on the apical growth point of each plant. Readings taken between 09.00 to 11.00 a.m from the apparatus included: photosynthetic rate, stomatal conductance, intercellular carbon dioxide concentration and where water in the nutrient solution is absorbed and expelled as the water vapour or evapotranspiration rate.

Determination of chlorophyll concentrations in leaves
A method determined by Hiscox and Israelstam (1979) was used to compare chlorophyll concentrations. Dimethylsulphoxide (DMSO) was added to the plant matter to extract the chlorophyll pigment. Plant material collected from B. vulgaris comprised of a small section removed from the tip of the leaf. From this, a 100 mg sample was removed and placed into 50 ml container. 7 ml of DMSO was added and incubated for 72 h at 4°C. 3 ml of DMSO was added to dilute the extraction to 10 ml. Absorbance values were then determined by adding 3 ml of the extracted samples to curvets and measuring them with a spectrophotometer (UV/Visible Spectrophotometer, Pharmacia LKB. Ultrospec II E). Values were determined at 645 and 663 nanometres (nm) by comparing the extracted samples to pure DMSO. Total leaf chlorophyll, chlorophyll a and chlorophyll b were calculated by using the formulae developed by Arnon (1949).

Statistical analysis
The collected data was analysed using a One-way analysis of variance (ANOVA). The analysis was performed using STATISTICA Software Programme 2010 (StatSoft Inc., Tulsa OK, USA). Where F-value was found to be significant, Fisher's least significant difference (LSD) was used to compare the means at P ≤ 0.05 level of significance (Steel and Torrie, 1980).

Treatment
Week 1

Effects of different combinations of Hoagland's solution and A. filiculoides on evapotranspiration of B. vulgaris
The evapotranspiration rate of B. vulgaris is documented de Bever et al.

Treatment
Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8  in

Effects of different combinations of Hoagland's solution and A. filiculoides on intercellular CO 2 concentration of B. vulgaris
The intercellular CO 2 concentration of B. vulgaris in Table 4 shows no significant results in weeks 1, 3, 4, 5, 7 and 8. Significant differences are noted in weeks 2 and 6 (P ≤ 0.01). In week 2, the Azolla plus Hoagland's minus N solution treatment and the control had significantly higher intercellular CO 2 concentration compared with the Azolla plus full Hoagland's solution and the treatment containing full Hoagland's solution. In week 6, the highest intercellular CO 2 concentrations were recorded in the Azolla plus Hoagland's minus N solution and full Hoagland's solution treatments.   These results were noted in total chlorophyll (P ≤ 0.001), chlorophyll a (P ≤ 0.001) and chlorophyll b (P ≤ 0.01) concentrations. The full Hoagland's solution treatment achieved the highest concentrations succeeded by the Azolla plus full Hoagland's, Azolla plus Hoagland's minus N solution and control (Hoagland's minus N solution) treatments. Similarities were noted between the full Hoagland's solution and Azolla plus full Hoagland's solution treatments in the chlorophyll a concentrations. The Azolla plus full Hoagland's, and Azolla plus Hoagland's minus N solution treatments also produced results that were similar in the chlorophyll b concentration. Tables 1 to 5 show that photosynthetic rate, evapotranspiration, intercellular CO 2 concentration and chlorophyll were generally higher in treatments with nitrogen which was readily available to plants. This was specifically evident in the treatment composed of the full Hoagland's solution. This was followed by Azolla sp. plus full Hoagland's solution and Azolla sp. plus Hoagland's minus N solution treatment. The control (Hoagland's minus N solution) had lowest values for these para-meters. It is widely reported that nitrogen plays a significant role in the function of physiological responses of plants namely that of photosynthesis (Bottrill et al., 1970;Evans and Terashima, 1987;Terashima and Evans, 1988;Evans, 1989;Paul and Driscoll, 2008), chlorophyll formation (Evans, 1989;Schepers et al., 1992;Blackmer and Schepers, 1995) and proteins such as RubisCO represent 30% of the part of soluble proteins and in relation to nitrogen utilised in the synthesis of proteins in plant organs such as seeds (Dalling et al., 1976;Ellis, 1979).

Results in
In photosynthesis, nitrogen has crucial function in electron transport and photophosphorylation (Terashima and Evans, 1988). Several other studies have shown that chlorophyll is directly linked to nitrogen, and the amount available will determine the chlorophyll concentration in leaf tissues (Evans, 1989;Schepers et al., 1992;Blackmer and Schepers, 1995;Tadahiko, 1997;Zhouping et al., 2000). An adequate supply of nitrogen will promote these functions, and an insufficiency will result in a disruption of the processes. Therefore, nitrogen in sufficient amounts will encourage a higher rate of photosynthesis, evaporative transpiration, intercellular CO 2 concentration and chlorophyll formation, a phenomenon which was also observed in our study.
The Azolla sp. plus Hoagland's solution minus N treat-ment alone in this study displayed improved photo-synthesis, evaporative transpiration, intercellular CO 2 concentration and chlorophyll content. Azolla sp. has the capability of fixing atmospheric nitrogen into usable forms (Lumpkin and Plucknett, 1982;Peters et al., 1982;Lambers and Poorter, 1992). Based on the settings of the hydroponic system in this study, it is possible that N was released from the Azolla into the hydroponic solution and then absorbed by B. vulgaris hence contributing to improving the photosynthesis, evaporative transpiration, intercellular CO 2 concentration and chlorophyll content at different measurements dates.
The growth and development of A. filiculoides in the experiment showed various differences. The Azolla sp. was exposed to light in its nutrient reservoir on a continual basis, so that the Anabaena sp. was able to use this light for Nfixation. From visual observation, it was noted that the surface area covered by A. filiculoides was noticeably larger in the Hoagland's minus N solution, even though the surface areas had increased in both solutions after inoculation.
Another visible factor was that the leaf fronds  were larger and darker green in the Hoagland's minus N solution as opposed to the full Hoagland's solution. These observations suggest that A. filiculoides in the Hoagland's minus N solution thrived and excreted N-rich substances into the solution which aided B. vulgaris to thrive. In comparison to the full Hoagland's solution, A. filiculoides competed with B. vulgaris for the nitrogen that was readily available in the solution, and thus resulted in poorer growth of both A. filiculoides and B. vulgaris. These results are of valuable agricultural importance in using A. filiculoides successfully as a possible nitrogen fertilizer in the hydroponic cultivation of B. vulgaris and other food crops. To the best of our knowledge, this is the only study in literature to evaluate the effect of Azolla on the physiological responses of B. vulgaris in hydroponic cultures. Further insight must be gathered on the execution of more efficient hydroponic cultivation method, where a more prominent amount, growth and nitrogen fixation of Azolla can be achieved for effective crop production in hydroponic cultures.

ACKNOWLEDGEMENT
This study was supported by Cape Peninsula University of Technology (CPUT) through University Research Fund (RP 03).