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Alcohol Dehydrogenase in Plant Response to Drought

1. Introduction Plant growth and productivity is adversely affected by nature’s wrath in the form of various abiotic and biotic stress factors (e.g. salinity, low temperature, drought, and flooding heat, oxidative stress and heavy metal toxicity). All these stress factors are a menace for plants and prevent them from reaching their full genetic potential and limit the crop productivity worldwide. Abiotic stress is the principal cause of crop failure, decrease average yields for most major crops by more than 50% (Bray, 2000) and causes losses worth hundreds of million dollars each year. In fact these stresses, threaten the sustainability of agricultural industry (Shilpi, 2005).
Environmental degradation and climate change have become severe global problems because of the explosive population increases and industrialization in developing countries. To solve this problem, one of the keys is plant biotechnology based on physiology of crop, plant biochemistry, genomics and transgenic technology. This is becoming more and more important for molecular breeding of crops that can tolerate droughts. For this technology, we need to understand plant responses to drought stress at the molecular level.
For agricultural and environmental sustainability, it is important to breed or genetically engineer crops with improved stress tolerance. The identification of key genes and that gene can be used directly for engineering transgenic crops with improved drought tolerance. Although a number of candidate genes have been identified in recent years, only very few have been tested in functional assays for a beneficial effect on drought tolerance. In order to assess gene function directly in plant suffering from abiotic stress caused by the drought, proved to be useful. Analysing the functions of these genes is critical for understanding of the molecular mechanisms governing plant stress response and tolerance, ultimately leading to enhancement of stress tolerance in crops through genetic manipulation. In this study, this will be used for overexpression of genes as well as for induced gene silencing, by using GATEWAY technology. A comprehensive investigation of Adh and Pdc induction and the determination of ethanol production during stress treatments would provide valuable information on how ethanol involved in the response to limited water condition.
2. Literature review 2.1. What is stress?
Stress in physical terms is defined as mechanical force per unit area applied to an object. In response to the applied stress, an object undergoes a change in the dimension. Biological term is difficult to define in the plant stress. A biological condition, which may be stress for one plant may be optimum for another plant. The most practical definition of a biological stress is an adverse force or a condition, which inhibits the normal functioning and well being of a biological system such as plants (Jones et al., 1989 )
2.2. Stress signalling pathways
The stress is first perceived by the receptors present on the membrane of the plant cells , the signal is then transduced downstream and this results in the generation of second messengers including calcium, reactive oxygen species (ROS) and inositol phosphates. These second messengers, further modulate the intracellular calcium level. This Ca2 level is sensed by calcium binding proteins, Ca2 sensors. These sensory proteins then interact with their respective interacting partners often initiating a phosphorylation cascade and target the major stress responsive genes or the transcription factors controlling these genes.
The products of these stress genes ultimately lead to plant adaptation and help the plant to survive the unfavourable conditions. Thus, plant responds to stresses as individual cells and synergistically as a whole organism. Stress induced changes in gene expression in turn may participate in the generation of hormones like ABA, salicylic acid and ethylene. The various stress responsive genes can be broadly categorized as early and late induced genes. Early genes are induced within minutes of stress signal perception and often express transiently. In contrast, most of the other genes, which are activated by stress more slowly, i.e. after hours of stress perception are included in the late induced category. These genes include the major stress responsive genes such as RD (responsive to dehydration)/ KIN (cold induced)/COR (cold responsive), which encodes and modulate the LEA-like proteins (late embryogenesis abundant), antioxidants, membrane stabilizing proteins and synthesis of osmolytes.
2.3. Drought stress
Among all abiotic stresses, drought is one of the most serious problems for sustainable agriculture worldwide. The adverse effect of drought stress is reductions in yield as reported in crops such as rice (Oryza sativa) (Brevedan and Egli, 2003), wheat (Triticum aestivum) (Cabuslay et al., 2002), soybean (Glycine max) (Kirigwi et al., 2004), and chickpea (Cicer aerietum) (Khanna-Chopra and Khanna-Chopra, 2004).
The adaptive responses to drought must be coordinated at the molecular, cellular, and whole-plant levels. These conditions induce dehydration of plant cells, which may trigger physiological, biochemical and molecular responses against such stresses (Shinozaki and Yamaguchi, 1996). Water deficit is a complex of responses, which depends upon severity and duration of the stress, plant genotype, developmental stage, and environmental factors providing the stress.
Yield losses due to drought are highly variable in nature depending on the stress timing, intensity, and duration. Although, different plant species have variable thresholds for stress tolerance, and some of them can successfully tolerate severe stresses and still complete their life cycles, most cultivated crop plant species are highly sensitive and either die or suffer from productivity loss after they are exposed to long periods of stress. It has been estimated that two-thirds of the yield potential of major crops are routinely lost due to unfavourable growing environments ( Shilpi, 2005 ).
Plants have evolved a number of strategies to severe drought. These include escape strategies such as avoidance (flowering, deep rooting, enhanced water uptake efficiency, or reduced water loss) as well as tolerance mechanisms. Reduced shoot growth and increased root development could result in increased water absorption and reduced transpiration, thereby maintaining plant tissue water status. In addition to such avoidance mechanisms, plant responses to water shortages can involve changes in biochemical pathways and expression of genes encoding proteins that contribute to drought adaptation. The proteins could be enzymes involved in the synthesis of osmolytes, antioxidants, or hormones such as ABA and others. Such changes can bring about drought tolerance, whereby plants continue to function at the low water potentials caused by water deficit (Hall, 1993). A central response to water deficit is often increased synthesis of ABA, which in turn induces a range of developmental (avoidance) and physiological or biochemical (tolerance) mechanisms. There is an ongoing debate as to whether the exploitation of avoidance or tolerance mechanisms should be the focus of plant breeding programmes. However, it appears likely that the exploitation of tolerance mechanisms may be more promising for the stabilization of crop yield under severe drought conditions (Araus et al, 2002).
An assortment of genes with diverse functions are induced or repressed by these drought stresses (Bartels and Sunkar, 2005; Yamaguchi and Shinozaki, 2005). Drought tolerance has been shown to be a highly complex trait, regulated expression of multiple genes that may be induced during drought stress and thus more difficult to control and engineer. Plant engineering strategies for abiotic stress tolerance rely on the expression of genes that are involved in signaling and regulatory pathways (Seki and Shinozaki, 2003) or genes that encode proteins conferring stress tolerance (Wang, 2004) or enzymes present in pathways leading to the synthesis of functional and structural metabolites. Current efforts to improve plant stress tolerance by genetic transformation have resulted in several important achievements; however, the genetically complex mechanisms of abiotic stress tolerance make the task extremely difficult.
2.3.1 Physiological and biochemical responses of drought
Physiological and biochemical changes at the cellular level that are associated with drought stress include turgor loss, changes in membrane fluidity and composition, changes in solute concentration, and protein and protein-lipid interactions (Chaves et al,2003) .
Other physiological effects of drought on plants are the reduction in vegetative growth, in particular shoot growth. Leaf growth is generally more sensitive than the root growth. Reduced leaf expansion is beneficial to plants under water deficit condition, as less leaf area is exposed resulting in reduced transpiration. Many mature plants, for example cotton subjected to drought respond by accelerating senescence and abscission of the older leaves. This process is also known as leaf area adjustment. Regarding root, the relative root growth may undergo enhancement, which facilitates the capacity of the root system to extract more water from deeper soil layers.
Plant tissues can maintain turgor during drought by avoiding dehydration, tolerating dehydration or both (Kramer,1995). These forms of stress resistance are controlled by developmental and morphological traits such as root thickness, the ability of roots to penetrate compacted soil layers, and root depth and mass (Pathan, 2004). By contrast, adaptive traits, such as osmotic adjustment and dehydration tolerance, arise in response to water deficit . Reduction of photosynthetic activity, accumulation of organic acids and osmolytes, and changes in carbohydrate metabolism, are typical physiological and biochemical responses to stress.
Synthesis of osmoprotectants, osmolytes or compatible solutes is one of the mechanisms of adaptation to water deficit. These molecules, which act as osmotic balancing agents, are accumulated in plant cells in response to drought stress and are subsequently degraded after stress relief (Tabaeizadeh ,1998).
2.3.2 Molecular responses
Studies on the molecular responses to water deficit have identified multiple changes in gene expression. Functions for many of these genè products have been predicted from the deduced amino acid sequence of the genes. Genes expressed during stress are anticipated to promote cellular tolerance of dehydration through protective functions in the cytoplasm, alteration of cellular water potentia1 to promote water uptake, control of ion accumulation, and further regulation of gene expression.
Expression of a gene during stress does not guarantee that a gene product promotes the ability of the plant to survive stress. The expression of some genes may result from injury or damage that occurred during stress. Other genes may be induced, but their expression does not alter stress tolerance. Yet others are required for stress tolerance and the accumulation of these gene products is an adaptive response.
Complex regulatory and signaling processes, most of which are not understood, control the expression of genes during water deficit. In addition to induction by stress, the expression of water-deficit-associated genes is controlled with respect to tissue, organ, and developmental stage and may be expressed independently of the stress conditions. The regulation of specific processes will also depend upon the experimental conditions of stress application. Stress conditions that are applied in the laboratory may not accurately represent those that occur in the field. Frequently, laboratory stresses are rapid and severe, whereas stress in the field often develops over an extended period of time ( Radin, 1993). These differences must also be evaluated when studying the adaptive value of certain responses. The function of the gene products and the mechanisms of gene expression are intertwined, and both must be understood to fully comprehend the molecular response to water deficit.
2.4. Function of water-stress inducible genes
Genes induced during water-stress conditions are thought to function not only in protecting cells from water deficit by the production of important metabolic proteins but also in the regulation of genes for signal transduction in the water-stress response .
Thus, these gene products are classified into two groups. The first group includes proteins that probably function in stress tolerance: water channel proteins involved in the movement of water through membranes, the enzymes required for the biosynthesis of various osmoprotectants (sugars, Pro, and Gly-betaine), proteins that may protect macromolecules and membranes (LEA protein, osmotin, antifreeze protein, chaperon, and mRNA binding proteins), proteases for protein turn over (thiol proteases, Clp protease, and ubiquitin), the detoxification enzymes (glutathione S-transferase, soluble epoxide hydrolase, catalase, superoxide dismutase, and ascorbate peroxidase). Some of the stress-inducible genes that encode proteins, such as a key enzyme for Pro biosynthesis, were over expressed in transgenic plants to produce a stress tolerant phenotype of the plants; this indicates that the gene products really function in stress tolerance ( Shinozaki ,1996 ).
The second group contains protein factors involved in further regulation of signal transduction and gene expression that probably function in stress response: Most of the regulatory proteins are involved in signal transduction. Now it becomes more important to elucidate the role of these regulatory proteins for further understanding of plant responses to water deficit. Many transcription factor genes were stress inducible, and various transcriptional regulatory mechanisms may function in regulating drought, cold, or high salinity stress signal transduction pathways. These transcription factors could govern expression of stress-inducible genes either cooperatively or independently, and may constitute gene networks in Arabidopsis ( Pathan.2004 ),
2.5. Model plant for studying the drought tolerant
Arabidopsis thaliana is a small weed in the mustard family. It has been a convenient for studies in classical genetics for over forty years ( Redei,1975). This flowering plant also has a genome size and genomic organization that recommend it for certain experiments in molecular genetics and it is coming to be widely used as a model organism in plant molecular genetics, development, physiology, and biochemistry. Arabidopsis thaliana provides an excellent experimental plant system for molecular genetics because of its remarkably small genome size and short life cycle. Arabidopsis thaliana, a genetic model plant, has been extensively used for unravelling the molecular basis of stress tolerance. Arabidopsis also proved to be extremely important for assessing functions for individual stress associated genes due to the availability of knock-out mutants and its amenability for genetic transformation. It has been collected or reported in many different regions and climates, ranging from high elevations in the tropics to the cold climate of northern Scandinavia and including locations in Europe, Asia, Africa, Australia, and North America (Kirchheim,1981).
Arabidopsis has the smallest known genome among the higher plants. The reasons for a small genome include little repetitive DNA and, in some cases, simpler gene families. Leutwiler et al. (1984) reported that the haploid genome from Arabidopsis (n = 5 chromosomes) contains only roughly 70,000 kilobase pairs (kb). The contrast of the Arabidopsis genome with that of other plants frequently used in molecular genetic work is striking: tobacco, for example, has a haploid nuclear genome of 1,600,000 kb; the pea haploid genome is 4,500,000 kb; and the wheat haploid genome is 5,900,000 kb . The significance of this small DNA content for molecular genetics is that a genomic library of Arabidopsis chromosomal fragments is easy to make, and simple and economical to screen. It is thus rapid and inexpensive to repeatedly screen Arabidopsis genomic libraries. In addition to its remarkably low content of nuclear DNA, Arabidopsis has a genomic organization that makes it uniquely suited to certain types of molecular cloning experiments.
All of the properties of the plant —small, short generation time, high seed set, ease of growth, self- or cross-fertilization at will–make Arabidopsis a convenient subject for studies in classical genetics.
2.6. Drought related gene
Alcohol dehydrogenase and pyruvate decarboxylase are enzyme whose activity has been observed in numerous higher plants including Arabidopsis, maize, pearl millet, sunflower, wheat, and pea (Gottlieb, 1982). In a number of plants, different ADH genes are expressed in various organs, at specific times during development, or in re-sponse to environmental signals. High levels of ADH activity are found in dry seeds and in anaerobically treated seeds (Freeling, 1973. Banuett-Bourrillon .1979), roots (Freeling .1973), and shoots (App, 1958).
During periods of anaerobic stress, the enzyme is presumably required by plants for NADH metabolism, via reduction of acetaldehyde to ethanol. With respect to secondary metabolites, ADH is involved in the inter conversion of volatile compounds such as aldehydes and alcohols (Bicsak et al., 1982; Molina et al., 1986; Longhurst et al., 1990).
The ethanolic fermentation pathway branches off the main glycolytic pathway at pyruvate. In the first step, pyruvate is the substrate of pyruvate decarboxylase (PDC), yielding CO2 and acetaldehyde. Subsequently, acetaldehyde is reduced to ethanol with the concomitant oxidation of NADH to NAD by alcohol dehydrogenase (ADH).
Although PDC and ADH gene induction has been demonstrated, ethanol and acetaldehyde production as a result of stress treatment has only been reported for red pine (Pinus resinosa) and birch (Betula spp.) seedlings exposed to sulfur dioxide, water deficiency, freezing, and ozone(Kimmerer and Kozolowski. 1982).
Many plants contain more than one ADH gene (Gottlieb, 1982 ), resulting in the expression of different ADH proteins (i.e. ADH isozymes, often designated ADH 1, ADH2, etc. ). The most extensive study of maize Adh genes, AdhI and Adh2, have been cloned and sequenced. The coding sequences of these genes are 82% homologous, interrupted by nine identically positioned introns that differ in sequence and length.
The expression of the Arabidopsis Adh gene (Chang and Meyerowitz, 1986; Dolferus et al., 1990) has many features in common with maize Adhl gene (Walker et al., 1987). The two genes have comparable developmental expression pattens, and both have tissue-specific responses to hypoxic stress. In both maize and Arabidopsis, the gene is expressed in seeds, roots, and pollen grains, whereas green aerial plant parts are devoid of detectable levels of ADH activity. In both species, hypoxic induction of the gene occurs in cells of the root system (reviewed by Freeling and Bennett, 1985; Dolferus and Jacobs, 1991; Okimoto et al., 1980;). ADH is induced anaerobically in Arabidopsis (Dolferus, 1985) as in maize. ADH is also induced in both maize root and Arabidopsis callus by the synthetic auxin 2,4-dichlorophenoxyacetic acid (Dolferus,1985. Feeling, 1973).
Several approaches have been undertaken to assess the functional role of Adh in development, stress response, and metabolite synthesis. The expression of the alcohol dehydrogenase (Adh) gene is known to be regulated developmentally and to be induced by environmental stresses (Christie et al., 1991; Bucher et al., 1995). Alcohol dehydrogenase (ADH) plays a key enzymatic function in the response to anaerobic conditions in plants (Sachs, Subbaiah, and Saab 1996). A new and exciting aspect of ethanolic fermentation is the suggested involvement in stress signaling and response to environmental stresses other than low oxygen (Tadege et al., 1999). Furthermore, specific analysis of the ADH gene from rice (Oryza sativa), maize, and Arabidopsis showed ADH to be induced by cold (Christie et al., 1991), wounding (Kato-Noguchi, 2001), dehydration (Dolferus et al., 1994), and the phytohormone abscisic acid (ABA; de Bruxelles et al., 1996), in line with the observation from the micro-array experiments.
In Arabidopsis thaliana, Adh overexpression improved the tolerance of hairy roots to low oxygen conditions and was effective in improving root growth (Dennis et al., 2000; Shiao et al., 2002). However, it had no effect on flooding survival (Ismond et al., 2003). Adh over expression in tomato has been shown to modify the balance between C₆, Adh overexpression in tomato aldehydes and alcohols in ripe fruits (Speirs et al., 1998). Grapevine plants overexpressing Adh displayed a lower sucrose content, a higher degree of polymerization of proanthocyanidins, and a generally increased content of volatile compounds, mainly in carotenoid- and shikimate-derived volatiles (Catherine et al., 2006).

Effect of Greywater on Plant Growth

Water availability in South Africa is integral to the economy, but South Africa is a water scarce nation. An alternate solution for household waste water, excluding toilet waste commonly known as greywater is to use it for irrigation in rural community gardens. This is likely to decrease the stress on the current potable water supply and simultaneously improve food security. Indigenous African leafy vegetables are a staple diet throughout Africa. A viability trial highlighted three out of six African leafy vegetable species; Amaranthus terere, Corchorus olitorius and Cloeme gynandra. Two treatments were used as suitable for trials of germination and growth under irrigation of tap water and greywater. Greywater treatments throughout the species decreased germination and seedling height was diminished. A. terere was the most robust to both the treatments as well as weather variability. Continuous investigation is needed to address the water scarcity and subsequent food insecurity.
Keywords: greywater, irrigation trial, African leafy vegetables, germination and growth
Introduction Water scarcity in South Africa is an issue that requires robust discussion and debate. If not addressed, it is likely to have serious consequences for both economic growth and the country’s population (Momba et al., 2006). Agricultural industry constitutes 12% of South Africa’s GDP. Even though this sector is decreasing, it is still water intensive. Without aviable water source, economic input in this sector is likely to have ramifications on the country’s health (Morel and Diener, 2006). Statistics show 65% of the country receives less than 500mm of rainfall per annum (Schulze, 1997). The level of water insecurity places pressure on the existing water resource for irrigation. This shortage is felt disproportionally by small-scale subsistence farmers and community gardeners.
South Africa is one of twelve countries that have safe drinking in the world and is ranked third in this group. However, there are many rural communities with under-developed water supply systems or these communities lack access to potable water sources (Momba et al., 2006 and Mackintosh and Colvin, 2002). While the need is great these small communities, they account a small percentage of the customer base. This then fuels the vicious cycle of supply and demand. As a result alternative water sources need to be acquired to satisfy the demand.
Grey water is likely to be a viable prospect to efficiently mitigate this deficiency (Alcamo et al., 2000). It consists of domestic waste water excluding toilet waste. The use of potable water is not needed for all consumptive practices, example irrigation (Alfiya et al., 2011). The main objective of finding alternative and sustainable water usage is to attain water security. Water insecurity is highly interlinked with food insecurity (Al-Jayyousi, 2002 and Blaine, 2012). Thus the use of greywater for small scale agriculture has the potential to address both water insecurity and food insecurity (Rodda et al., 2011). They are most usually harvested from the wild. This practice is a threat to the continued survival of these plants Cultivating African leafy vegetables would also address their conservation need (Momba et al., 2006).
Indigenous African leafy vegetables are a part of the staple diet in South Africa (Momba et al., 2006). The challenge is to continue production of these vegetables without jeopardizing potable water supplies, but by utilizing alternate water source such as grey water as a means of irrigation.
The concept of grey water had both advantages and disadvantages (Rodda et al., 2011). Reducing stress on the potable supply is a main benefit but there are drawbacks to using waste material to grow plants, households have different proportions of additives, thus may effect plant growth (Roesner et al., 2006). Whereas the risks are divided into three main categories; possible detrimental effects on the environment which decreases the ability for soil to provide plant growth, subsequent effects on plant growth and yield, and risk to human health (Rodda et al., 2011).
The aim of this study was to determine whether irrigation with grey water had an effect on seed germination and seedling growth, and whether this effect differed with detergent formulation. The objective was to assess if grey water can replace potable water for irrigation of indigenous plants. It was predicted that seedlings under tap water-irrigated conditions would have a greater growth rate than under greywater conditions. It was further predicted that rate of germination would not be affected by the grey water.
Materials and methods This investigation took place in 2 parts. This first was to assess the viability of the seeds and to select the species for further investigation. In the second, seed germination and seedling growth under grey water and tap water treatments irrigation were evaluated.
Germination trials
Germination trials were performed in the laboratory in the Biology Building at UKZN (Westville Campus).
An initial experiment was conducted with six species (Solanum nigrum, Amarathus terere, Corchorus olitorius, Solanum villosum, Amarathus dubois and Cloeme gynandra). Germination was tested. The most viable 3 were chosen to determine the germinability of the three selected species of African leafy vegetables. Only viability was tested for as this was pertinent to the success of the actual trial. The viability criteria were the speed at which germination took place. This indicated the viability of the seeds and validates the ability to germinate under controlled conditions.
The germination viability trial was conducted in the laboratory. Each of the three species (Cloeme gynandra; Amaranthus terere; Cochorus olitorius) had six replicates of ten seeds each. Seeds were placed randomly on filter paper in a Petri dish and a smaller piece of filter paper was placed over. They were watered with deionised water until moist. An equal number of seeds were placed under illuminated and dark conditions. These were then monitored every 24 hours and replenished with deionised water as necessary. Once germination had occurred and the radicle was greater than 1 cm, seedlings were moved to the left side of the Petri dish. This prevented recounting and recording. Percentage germination was recorded.
Description of Species
The initial viability trial revealed that the following three Kenyan species were the most viable. C. gynandra is commonly known as spider plant. It is used as a component of a high fibre diet and, from indigenous knowledge, has medicinal properties (Mauyo et al., 2008). A. terere is another widely grown consumable in East Africa (Nabulo et al., 2011). The final species used was Corchorus olitorius, Jew’s mallow, a dark green leafy vegetable high in protein which is consumed in most African communities.
Irrigation Trial
Trails of irrigation with greywater and tap water were then performed in the Biology greenhouse at UKZN (Westville campus).
Synthetic greywater (10 l) was made up freshly weekly (Table 1). It was stored in the cold storage to impede bacterial and algal growth.
Detergent products used to generate the greywater were representative of solid or powder detergent products typically used in lower income households, which are those most likely to benefit from the use of greywater for irrigation of subsistence crops. The flour, nutrient broth and cooking oil were used to represent carbohydrates, salts and proteins, and greases respectively in the synthetic greywater.

Seedling trays (6) were filled with Berea red soil. For three days prior to planting, the seedling trays were watered with tap water and greywater respectively until they were saturated to field capacity. The seeds were then planted into seedling trays. Species were randomized per tray. Sixty seeds of each species were watered with tap water and the other sixty seedlings were watered with the synthetic grey water. For the first 14 days, trays were watered every 24 hours. Each seed was hydrated with 0.25 ml of either synthetic grey water or tap water. Thereafter, trays were watered every second day for the remainder of the trial. The experiment was repeated three time under three treatment groups; the first treatment group was tap water for germination and subsequent growth, tap water for germination and then greywater for growth and the final treatment of grey water throughout the lifespan of the plants. Height was measured weekly. Productivity was measured by destructive harvesting (dry mass production) at end of experiment. However plant height was gauged growth during the experiment.
On two occasions there was death of seedlings due to severe weather conditions and this restricted the growth period. Since this investigation was over a short time span. The weather impacted the progress of experiment. Weather variability such as intense heat, humidity and berg winds, and strong rains affected the seedlings. Even though they were protected in the shade house, the extreme elements could have inhibited their germination and growth.
Statistical Analysis
The data were analyzed using SSPS version 19. Two sets of statistical analyses were performed. The first test was to show the difference between greywater and tap water in terms of growth (height). A Kolmogorov-Smirnov test was performed to test for normality. Levenes test for Equality of Variances was performed, the assumption homoscedasticity was violated but all other assumptions were satisfied. Since the data was not normally distributed a more robust Mann-Whitney U test was done, to evaluate the differences in germination for each species under the two conditions (greywater-irrigated and tap water-irrigated). An excel graph was then used to show the rate at which the all three species comparatively germinated in terms of the two treatments (greywater-irrigated and tap water-irrigated).
Results Seed germination and seedling growth are gauged by the germination totality and seedling height measured weekly. Initial germination was 70% in tap water-irrigated seeds whereas as 45% in greywater-irrigated seeds.
Figure 1 shows the totality of germinated seeds present over time for each of the three species. A. terere and C. olitorius had the highest totality under controlled laboratory conditions; C. gynandra seeds had the lowest survival percentage >40%. A. terere has the highest standard deviation, indicating the data is wide spread.

Figure 2 the initial and final number of seedlings present per species and the treatment. Co. olitorius under the grey water treatment had the least number of seedlings that survived. This species also had the greatest difference between the treatments. A. terere had the greatest number of individuals that survived in both the treatments.

The results of the Mann-Whitney U test rejected the H0 that there will be no difference between the two treatments, there is a significant difference between height of the three species per treatment. Therefore the distributions of height for each species across the treatments are different. Plant height differed significantly among the treatment (p < 0.05). Greywater -irrigated seedlings consistently attained a lesser height than tap water-irrigated seedlings across all species (Figure 3).
Figures 3 indicate the difference in height between tap water irrigated and greywater-irrigated plants for each species. The standard deviation is shown as an error bar. Seedling height 18days after germination was lower in grey-water irrigated plants than in tap water-irrigated plants for all 3 species. A. terere had the largest standard deviation of tap water-irrigation with 10.197 whereas the greywater-irrigation treatment was 9.1197. C. olitorius which had a visibly lower standard deviation than A. terere tap water-irrigated treatment was 5.753186 and the greywater-irrigated treatment was 1.558646. Finally with the lowest standard deviation, C. gynandra tap water-irrigated treatment was 0.588196 and the greywater-irrigated treatment was 0.316563. C. olitorius had the greatest discrepancy for tap water-irrigated and greywater-irrigated.
Discussion and Conclusion Africa, according to Morel and Diener (2006) is known as a water insecure continent. As adjustments are discussed on the efficient use of potable water, reusing waste water is seen as a possible solution. Alternative irrigation methods are needed for progress.
Greywater is a possible alternative water source, however contrasting evidence in Morel and Diener (2006) indicates that the potential drawbacks, even though greywater is less contaminated than other waste water. Untreated greywater contains solid particles, pathogens, grease and oils, salts, and chemicals. According to Rodda et al. (2011) these impurities could have negative effects on soil quality, ground water supply and human health.
With such strong findings there are studies that have shown greywater reuse as a viable alternative to 100% potable water. Greywater has been implemented a cost-effective means to reduce domestic water levels. According to Morel and Diener (2006) greywater reuse produced average yield, with decrease in water usage and fertilizer requirements. In both Cyprus and Israel domestic water used was reduced by effective greywater management schemes (Moral and Diener, 2006).
In this study, greywater-irrigated seeds and plants consistently yielded poorer germination (Figure 1 and 2) and growth (Figure 3) of three species of African leafy vegetables. The germination trial (Figure1) yielded a higher percentage of germinated seeds than the outdoor irrigation trial. This is possibly due to the controlled, pathogen-free environment in the laboratory. Cited by Pinto (2010) experiment alternate watering regimes of potable water and greywater resulted in the growth of the plants very similar to 100% potable water. This is a means to mitigate the soil health risks related with greywater reuse. Even though Pinto (2010) had no significant change of plant biomass in the control and treatment, it differed in this investigation.
Figure 2 indicates that A. terere were unable to acquire a high germination percentage in grey-water irrigated treatments but acquired the highest tap water-irrigated germination percentage. Hence the treatment of greywater-irrigated seeds affected their ability to germinate, with initial germination at 70% in tapwater-irrigated seedlings whereas as 45% in greywater-irrigated seedlings. The best germination in greywater-irrigation was observed by A. terere, possibly reflecting its resilience under a wide range of conditions as mentioned by Nabulo et al. (2011). Cl. gynandra had an average of ±7% greater tapwater-irrigated seedling germination than greywater- irrigated seedling germination. Conversely Co.olitorius had the greatest variability between tapwater-irrigated seedling germination than greywater- irrigated seedling germination. Since a significant difference was calculated, greywater does effect the germination of seeds and subsequently the amount of germinated seeds able to grow.
A possible factor in poor survival of both tap water- and greywater-irrigated seedlings, in addition to weather conditions, is nutrient depletion. Berea red sand had a composition of 62.68% SiO2 which is generally used and is nutrient poor (Okonta and Manciya, 2010)
Since a watering regime observed, nutrients to the plant was not considered. Other nutrients found in soil are needed for healthy growth. Seedling trays were used to separate species and keep difference treatment uncontaminated but after the 2 week germination period, nutrients are need for plant growth. Each seed had ± 18cm2 of Berea red soil, this soil consists of 12-64% and 15-57% of fine and medium sand respectively (Hamel, 2006). Water holding capacity of the soil is thus diminished due to porosity. This could have exacerbated the depletion of nutrients in the volume of sand thus leading to their inability to withstand weather variability.
Soapy residue may have contributed to poor performance of the greywater-irrigated seeds and seedlings. Mataix-Solera et al. (2011) point out that the detergents in greywater cause soil water repellency of soil. It can be argued that greywater might be an interim solution, but posed long-term effects that might not be easily remedied. Soapy soil could cause hydrophobic soil properties which have poor water hold capacity. This could have hampered the germinated seed’s shoot from emerging through the soil due to the coagulated surface. An alternate solution can be found according to Pinto (2010), where altering water regimes between grey water and potable resembled the results observes in 100% potable water. The pH levels remains similar between water regimes. In household greywater system the proposal ceramic pot filter is used this eradicates the large particles.
Another caveat of this investigation is changing the watering regime. Initially seeds are watered every day until germination which is ± 10days and then changed to every alternate day. Since plants are sensitive to change, the watering regime should be carefully monitored in conjunction with weather patterns. This ensures a smooth transition for the seedlings.
According to Roesner et al. (2006) household waste contains 2500-5000 chemicals which if used as greywater could cause coagulation at the soil surface. More organic products could be used to reduce the amount of chemicals in the greywater (Al-Jayyousi, 2002). Pre-treatment of greywater and limiting its used only to salt-tolerant crops could allow wide use of greywater for irrigation (Al-Jayyousi, 2002). In this investigation germination of all three species was diminished under greywater-irrigated conditions, this being said with calculated changes to the experiment, greywater could possibly be a viable option in the future.
An observation was made during the experiment, refer to appendix image 1 and 2 of A. terere, the leaf colour in greywater-irrigated treatment was lighter than the tap water-irrigated treatment. Image 3 and 4 also exhibit the same phenomenon in C. gynandra. Cultivation in Jordan of different crops yielded a similar observation, this was attributed to the solids and increased salinity of the greywater (Al-Jayyousi, 2002).
Although the results obtained conclusively show that greywater does effect the both the germinability of the seeds and subsequent growth. It is recommended that seeds should not be irrigated with grey water, possibly increasing the percentage of seed germination. Organizations such as the Water research council are investigating innovative ideas to alleviate the pressure on South Africa’s stressed water system.
Primary greywater systems in community gardens should be not be implement immediately rather as in Pinto et al. (2010) a combination of greywater and tap water should be used. This will relieve the possibility of failing crops. Social and environmental sustainability are interlinked which fuels the economy. Water is an integral part life and therefore should be continuously well-managed. Further research is necessary as water scarcity and availability still threatens food security around Africa.

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