Chapter 5: Phenolic acids and antioxidant regulation in Vigna unguiculata L. (Walp) growing
5.2 Materials and methods
5.2.1 Seed collection and soil analysis
Seeds of four V. unguiculata varieties commonly grown by local farmers of South Africa (Brown mix, Dr Saunders, IT18 and Batch white) were obtained from AGT foods Africa, in KwaZulu-Natal, South Africa. Soils were collected from four geographically discrete areas in KwaZulu-Natal (KZN) province, South Africa. These sites included Izingolweni, Southern KZN (30°43′32″S 30°6′10″E, altitude 450 masl); Hluhluwe, Northern KZN (28°0′58″S
104
32°12′4″E, altitude 100 masl); Ashburton/Pietermaritzburg, Midlands KZN (29°38′55″S 30°26′42″E, altitude 670 masl) and Bergville, Mountainous KZN (28°34′14″S 29°4′17″E, altitude 1040 masl). Twenty soil samples were collected at random from each site. These were 0-30 cm deep and 2 m away from each other. Thereafter, the soil samples were pooled together into composite samples for each site. The composite soil samples were air-dried at room temperature before they were crushed in a soil crusher. Concentration of macronutrients (N, P, K) and pH were measured in the soil samples. The Automated Dumas dry combustion method was used to determine total nitrogen using an LECO CNS 2000 (Leco Corporation, Michigan, USA). Atomic absorption method was used to determine the amount of P and K in the soil according to reported methods by Manson and Roberts (2000).
The procedure consisted of extracting 2.5 ml of soil solution with 25 mL of ambic-2 solution at a pH of 8. The liquid was then agitated at 400 rpm for 10 min with a multiple stirrer before being filtered through Whatman No.1 paper. A KCL solution was used to determine the pH of the soil. The geochemical assessment revealed differences in soil nutrient contents and relative acidity in different KZN soils as shown by (Matiwane et al. 2019). The soils were acidic with low levels of macronutrients as described in Chapter 3 (Table 3.1).
5.2.2 Experimental design and seedling growth
Vigna unguiculata seeds were surface sterilized for 15 min in a bleach solution (30%
commercial bleach + 0.02% Triton X-100), rinsed 10 times in sterile water, and air dried in a sterile laminar flow. Prior to germination, the seeds of the four V. unguiculata varieties were soaked overnight in distilled water. Seeds were then germinated and cultivated in 20 cm diameter pots filled with soils from the four sites at a greenhouse on the University of KwaZulu- Natal, Pietermaritzburg campus in South Africa.
Each cowpea variety had 30 seedlings for every soil treatment and the experiment was arranged in a randomized complete block design. The greenhouse conditions were as follows; humidity (70 to 80%), the irradiance was approximately 35% of full sunlight, night temperatures reached 12 to 14 °C while day temperatures spanned from 30 to 35 °C. Before seedling emergence, irrigation was done every day with 200 ml of distilled water applied to each pot and thereafter the seedlings were irrigated after every two days. The experiment was carried out from September to December 2018.
5.2.3 Plant harvesting and nutrient analysis
Ten plants per treatment were harvested 12 weeks after seedling emergence and these were separated into stems, leaves, nodules and roots after rinsing with distilled water. Thereafter the
105
separated plant parts were dried in an oven at 80 °C until all moisture was removed. Recording of dry weights of the samples was then done. A pre-chilled pestle and mortar was used to pulverize the plant material to fine powder. The ground plant material was then sent for plant isotope N and P concentration analysis at commercial laboratories using the LECO-nitrogen analyzer and inductively coupled mass spectrometry (ICP-MS) at the Archaeometry Department, University of Cape Town, and Central Analytical Facilities (CAF), Stellenbosch University, South Africa, respectively.
There were differences in plant biomass and nutrition across all varieties in the four soil types.
In terms of the biomass accumulation of the different varieties, the order was as follows; IT18, Batch white, Brown mix and Dr Saunders; from highest to lowest across the four soil treatments. Total plant N concentrations differed statistically among varieties with IT18 recording the highest total plant N concentration. Variety IT18 also had the highest total plant P across the four soil types (Table 4.3).
5.2.4 Determination of arbuscular mycorrhizal fungi colonization percentage and identification of V. unguiculata root nodulating bacteria
To determine the AMF root colonization percentage, six roots which had nodules were taken per treatment and these were put in glass vials which contained 50% ethanol. The procedure which involved cleaning and dyeing of the roots and thereafter, microscopic evaluations of colonization structures were done following experimental methods described by Smith and Dickson (1991). There were significant differences in AMF colonization percentages across the four varieties in the different soil treatments and this ranged from 58.5% to 100% (Figure 4.1).
To identify the V. unguiculata root nodulating bacteria, five plants were harvested, and nodules were excised from them. Thereafter, surface sterilization of the nodules was done using ethanol and sodium hypochlorite solution. The root nodules were then thoroughly washed using sterilized distilled water (dH2O) before they were squashed. This was followed by the addition of 100 µl of 15% glycerol to the suspensions. Thereafter, streaking of the suspensions was done on yeast extract mannitol agar plates and incubated at 28 °C which was checked daily for growth of colonies. Repeated streaking was done on yeast extract mannitol agar plates to get pure cultures. Identification of bacteria present in the pure cultures was done by amplifying part of the 16S rDNA gene using PCR reactions. DNA amplification was done, and the acquired amplicons were viewed on 1% agarose gel and they met the expected size (1500 base pairs). Sequencing of the amplicons was done per treatment at the Central Analytical Facilities,
106
Sequencing Facilities (Stellenbosch University, South Africa). Bacterial strains were identified using BLASTN (National Centre for Biotechnology Information, NCBI (https://www.ncbi.nlm.nih.gov/genbank/). Several bacterial strains were identified in the V.
unguiculata nodules and these included Bacillus, Paenibacillus, Delftia, Nitrobacter, Rhizobium and Bradyrhizobium (Table 4.1).
5.2.5 Quantification of phenolic acids using Ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC–MS/MS)
For each of the four V. unguiculata varieties in each soil treatment, 9 plants were harvested and divided into above-ground (stems and leaves) and below ground (roots and nodules) parts. The above-ground and below ground parts were each pooled into 3 biological samples, and these were immediately freeze-dried and lyophilized to prevent further physiological changes.
Phenolic acids extraction was done using 30 mg of the freeze-dried material. Firstly, there was preparation of standard solutions in methanol at a concentration of 10-3 molL-1, thereafter, these were serially diluted to working concentrations. Internal standards of deuterium labelled 4- hydroxybenzoic acid (2,3,5,6 -D4) and salicylic acid (3,4,5,6 -D4) were then added at a final concentration of 10-5 molL-1. UPLC–MS/MS analyses were done using an ACQUITY Ultra Performance LCTM system (Waters, Milford, MA, USA) linked simultaneously to a PDA 2996 photo diode array detector (Waters, Milford, MA, USA) and a Micromass Quattro microTM API benchtop triple quadrupole mass spectrometer (Waters MS Technologies, Manchester, UK), equipped with a Z-spray electrospray ionisation (ESI) source operating in negative mode. To control the instruments and to acquire and process data, MassLynxTM software (version 4.0, Waters, Milford, MA, USA) was used. Thereafter, identification and quantification of the phenolic acids was done using UHPLC-MS/MS according to the method described by Gruz et al. (2008).
5.2.6 Determination of Oxygen Radical Absorbance Capacity of the different Vigna unguiculata varieties grown in different soil types of KZN
Determination of oxygen radical absorbance capacity (ORAC) was done using an existing protocol (Ou et al. 2001). Firstly, the above-ground (leaves and stem) and below ground (roots and nodules) cowpea extracts (25 µl) and fluorescein (150 µl, 250 nM) were added in triplicates to the 96-well microplate before shaking. Thereafter, 2,2'-Azobis (2-amidinopropane) dihydrochloride (AAPH) was added and shaken for 30s before it was subjected to pre- incubation at a temperature of 37 °C. The fluorescence was incubated at 40 °C, thereafter it
107
was read every 2 min and this was done for 30 cycles in a microplate reader. Antioxidant capacity (µmol TE/g) was then determined by using the net area under the curve.
5.2.7 Data analysis
Data on plant biomass, plant and soil nutrition, concentrations of phenolic acids and ORAC were analyzed using a two-way analysis of variance (ANOVA) using IBM SPSS Statistics 25 software. For statistical significance (p ≤ 0.05), the mean values were separated using Tuckey’s post hoc test. Phenolic acids data was processed using MassLynxTM software (version 4.0, Waters, Milford, MA, USA).
Relations among the measured values of phenolic acids, the experimental variants of the four cowpea varieties (plant biomass, plant nutrition) and soil nutrition were examined by Principal Component Analysis (PCA). For the PCA statistical procedures PAST version 4.02 was used.
Correlations matrix of the variables were determined by Pearson coefficients (p <0.05 and p
<0.01).