Optimum Harvest Time of Cucumis africanus Fruit Using Concentration of Cucurbitacin B as a Maturity Standard

Fruit of wild watermelon (Cucumis africanus) are used to manufacture nemafric-BL phytonematicide for the management of population densities of the notorious root-knot (Meloidogyne species) nematodes on various crops. However, the concentration of the active ingredient, namely, cucurbitacin B, is dependent on the developmental stage of the fruit. A field study was, therefore, conducted to establish the optimum harvest time where C. africanus fruit would have the highest concentration of cucurbitacin B in order to ensure consistent quality of the phytonematicide. Fruit were harvested weekly from 60 to 110 days after transplating, prepared for extraction and cucurbitacin B quantified using reverse phase HPLC. Concentrations of cucurbitacin B were optimised at 5.0 weeks, which translated to a 95-day harvest time after seeding.


The Research Problem
Maturity tests are inherently used in various crop industries to establish the optimum harvest time (Lee et al., 1983;Saevels et al., 2003;Lebrun et al., 2004Lebrun et al., , 2008)).This ensures consistent quality of the produce (ISO 9000, 2000), and the quality of the products (Parasuraman et al., 1990).Generally, maturity tests could include the quantification of total soluble solids (TSS), total acidity, pH, acid/sugar ratio, tannins, volatile compounds, ascorbic acid and oil content (Abbasi et al., 2011;El-Buluk et al., 1995;Hulme, 1971;Kader, 2002).Most established maturity tests require some form of equipment or laboratory procedure (Wilson, 2013;Wilson & Baietto, 2009) and could not always be accessible to developing industries.Generally, maturity tests should not be confounded with maturity indices, which comprise the use of various shapes and colours during grading of various produce (Cristo, 1994).The introduction of alternative crops is confronted with various challenges, amongst which could be to ensure consistent quality of the product, which is associated with maturity testing.

Importance of Research Problem
Wild watermelon (Cucumis africanus L. F.), indigenous to Limpopo Province, South Africa (Kristkova et al., 2003), has widely been used in various industries, which range from nutraceutical, pharmaceutical, cosmeceutical and pesticidal industries (Lee et al., 2010;Mashela et al., 2011;Thies et al., 2010;Van Wyk & Wink, 2012;Van Wyk et al., 2002).The active ingredient had been identified as cucurbitacin B (Chen et al., 2005), which is an allelochemical compound (C 32 H 46 O 8 ) that is distributed equally in all organs of C. africanus.Nemafric-BL phytonematicide has been researched and developed from C. africanus fruit under greenhouse, microplot and field conditions (Mashela et al., 2011).This phytonematicide has the attributes of serving as an alternative to methyl bromide, which had since been withdrawn from the agro-chemical markets due to its environment-unfriendliness (Mashela et al., 2011).

Relevant Scholarship
Biological entities, including plant-parasitic nematodes, respond to increasing concentrations of allelochemicals in density-dependent growth (DDG) patterns (Pelinganga et al., 2012;Salisbury & Ross, 1992).Generally, the DDG patterns have three phases, viz; stimulation, neutral and inhibition phases (Liu et al., 2003;Pelinganga et al., 2012;Salisbury & Ross, 1992).In most cases, DDG patterns are characterized by quadratic curves (Pofu & Mashela, 2014;Salisbury & Ross, 1992), which allow for the computation of the optimum point in the neutral phase (Pofu & Mashela, 2014).Generally, the optimum point is the independent (x-axis) variable for the highest level of the dependent (y-axis) variable.Pelinganga et al. (2012) used the DDG patterns to establish the application interval of nemafric-BL 3% phytonematicide as 18 days for effective suppression of root-knot (Meloidogyne species) nematode population densities in tomato (Solanum lycopersicum L.) production.

Hypothesis and Its Correspondence to Research Design
Cucurbitacins, as secondary metabolites, accumulate in various organs of plants within the Cucurbitaceae Family (Chen et al., 2005).The accumulation of cucurbitacins in organs over time could probably follow the DDG patterns during various developmental stages of plants, which would allow for the determination of the optimum time for the highest accumulation of cucurbitacins.The objective of this study, therefore, was to use the accumulation of cucurbitacin B during different developmental stages of fruit in C. africanus to establish the optimum harvest time in order to ensure consistent quality of nemafric-BL phytonematicide.

Study Field and Treatments
The field study was conducted at the Green Technologies Research Centre, University of Limpopo, Limpopo Province, South Africa (23°53′10″S, 29°44′15″E).Seeds were prepared as described previously (Mafeo & Mashela, 2009) and sown in seedling trays containing Hygromix (Hygrotech, Pretoria North, South Africa) growing medium.At two-leaf stage, seedlings were hardened off for 5 days, selected for uniformity and transplanted at 4 weeks.Plot size was 1 m x 1 m, each containing 4 seedlings.Treatments,viz.,60,67,74,81,89,96,103and 110 harvest days after transplanting, were arranged in randomised complete block design, with 12 replications.Three days after transplanting, each plant was fertilised with 3 g 2:3:2 ( 22) to provide a total of 186 mg N, 126 mg K and 156 mg P per ml water and 2 g 2:1:2 (43) -providing 0.35 mg N, 0.32 mg K and 0.32 mg P, 0.9 mg Mg, 0.75 mg Fe, 0.075 mg Cu, 0.35 mg Zn, 1.0 mg B, 3.0 mg Mn and 0.07 mg Mo per ml water, with irrigation being every other day.

Preparation, Extraction and Quantifying Cucurbitacin B
Eight fruit from each replication of C. africanus were harvested weekly, cut into pieces, dried at 52 °C for 72 h in an air-forced oven and ground in a Wiley mill to pass through a 1-mm-pore sieve (Makkar, 1991).Four grams of dried crude extracts of fruit were mixed with 50:50 ml methanol and dichloromethane and allowed to run for 4 h on a waterbath at 40 °C at 45 rpm (Rotary Evaporator).After extraction, sub-samples were concentrated by reducing the volume to 30 ml and then 1 ml centrifuged at 4500 rpm for 10 minutes before filtering through 0.22 µm-pore filter (Miller, Sigma).A cucurbitacin B reference standard obtained from Wuhan ChemFaces Biochemical Co., Ltd.(Wuhan, China), was dissolved in methanol, with a range of standard concentrations (0.01-1.0 mg/ml) prepared.Concentration of cucurbitacin B was quantified using a Shimadzu Prominence HPLC system (Shimadzu Corporation, Kyoto, Japan) equipped with a Discovery® Wide Pore C18 reverse phase column (25 cm × 4.0 mm) with 5 µm particle size packing.Isocratic elution was done using methanol/water (40:60) (v/v) at flow rate of 1 ml/min.Eluents were detected using a Shimadzu CTO-20A photo diode array detector (PDA) and the peaks were quantified using Shimadzu LC solutions software.

Data Analysis
Cucurbitacin B data were subjected to analysis of variance (ANOVA) procedure using SAS software (SAS Institute Inc., 2008).When the treatments were significant at the probability level of 5%, the degrees of freedom and their associated sum of squares were partitioned to determine the percentage contribution of sources of variation to the total treatment variation (TTV) among the treatment means (Steyn et al., 2003).Mean separation was achieved using Waller-Duncan multiple range test.The variable with significant (P ≤ 0.01) treatment means were further subjected to lines of the best fit using cucurbitacin B responses to different harvest time and modelled by the regression curve estimations resulting in a quadratic equation: Y = b 2 x 2 + b 1 x + a, where Y = Cucurbitacin B concentration and x = harvest time with -b 1 /2b 2 = x being the value for the optimum cucurbitacin B concentration.Unless otherwise stated, only treatment means significant at the probability level of 5% were discussed.

Results
Harvest time had highly significant (P ≤ 0.01) effects on concentration of cucurbitacin B in C. africanus fruit and contributed 73% in total treatment variation of cucurbitacin B concentration.Cucurbitacin B (y-axis) and harvest time (x-axis) had quadratic relationships, where the model explained 87% of the observed variation in cucurbitacin B (Figure 1).The concentration of cucurbitacin B was optimised at 5.0 weeks starting from 60 days after transplanting four-week old seedlings (Table 1), which translated to harvest time of approximately 95 days after seeding.Relative to initial harvest time (60 days), all other harvest times had strong impact to the accumulation of cucurbitacin B concentration as shown by relative increases ranging from 22% to 898% (Table 2).Note.Impact z = treatment/control (0) -1) × 100.

Discussion
Generally, there are five developmental stages during the formation of fruits in Cucumis species (Shaik et al., 2011).Flowering is followed by fruit formation, which is characterised by rapid cell division of the ovary.Elongation stage, which proceeds for two to three weeks, ensues.Thereafter, fruit maturation stage occurs, characterised by fruit swelling without increases in fruit size.Colour changes from green to slightly yellow signify fruit ripening.Finally, senescence sets in, resulting in softening and thereafter, fruit shriveling (Oh et al., 2011;Shaik et al., 2011).Consequently, results of the study suggested that the optimum concentration of cucurbitacin B coincided with the maturity stage in C. africanus fruit as observed in other maturity standards (Abbasi et al., 2011;El-Buluk et al., 1995;Gry et al., 2006;Hulme, 1971;Kader, 2002).
The response of cucurbitacin B level to varying fruit developmental stages demonstrated a DDG pattern (Liu et al., 2003).Biological entities respond to extrinsic and intrinsic factors in a DDG pattern (Liu et al., 2003).Declining and increasing levels of cucurbitacin B concentration coincided with senescence and maturity stages, respectively.Results of this study agree with the finding of cucurbitacin concentration in Ecballium elaterium and Trichosanthes cucumerina L. var.cucumerina, where the concentration of cucurbitacin reached the highest peak during fruit maturation stage (Attard & Scicluna-Spiteri, 2004;Devendra et al., 2011).
The production of allelochemicals is highly dependent on the physiological and developmental stage of plants.Developmental stages have an impact on the metabolic pathways which are responsible for accumulation of secondary metabolite (Achackzai et al., 2009;Devendra et al., 2011;Ramakrishna & Ravishankar, 2011).The observed decline in the concentration of cucurbitacin B during senescence in our study was an important observation since this could also have a detrimental effect on quality of nemafric-BL.However, the mechanism involved in this decline is not yet clear, but could be suggesting the existence of reversible reactions as shown in the accumulation of other allelochemical compounds (Jilani et al., 2008).

Conclusion
In conclusion, results suggested that concentration of cucurbitacin B in C. africanus was optimised at maturation phase, which was followed by decline in the concentration of this allelochemical.Consequently, accurate timing of the harvest time could be essential in ensuring consistent quality of nemafric-BL.

Table 2 .
Relative accumulation of cucurbitacin B in fruit of Cucumis africanus over increasing developmental stages of fruit (n = 96)