Chapter 6: Effects of some botanicals on growth and phytochemistry of nematode-infected
6.2. Material and methods
6.2.1. Study location, and plant material collection and preparation
The study was conducted under greenhouse conditions as described previously (Chapter 5;
Section 5.2.1.12).
Plant material belonging to the eight botanicals viz. Pelargonium sidoides (T1), Prunus africana (T2), Solanum aculeastrum (T3), Vernonia colorata (T4), Searsia lancea (T5), Merwilla plumbea (T6), Croton sylvaticus (T7), Cucurbita maxima (T8) were collected from the University of KwaZulu-Natal Botanical Garden. The fruit of C. maxima was purchased from a local supermarket, and only the seeds were used in this experiment. Plants were identified by the horticulturist, Mrs. Alison Young, at the Department of Life Sciences, University of KwaZulu- Natal, Pietermaritzburg, South Africa. Voucher specimens were deposited at the UKZN, Herbarium (NU), Pietermaritzburg for botanical verification and future reference and prepared as explained previously (Chapter 4; Section 3.2.1).
6.2.2. Nematodes inoculation preparation, experimental design and cultural practices Root-knot nematode (M. incognita) inoculum was prepared by extracting eggs and second-stage juveniles (J2s) from roots of the greenhouse-grown nematode-susceptible tomato cv. Roma V.F.
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in 1% NaOCl (Sodium hypochlorite) (Chapter 5; Section 5.2.2.2). Seeds of P. sidoides (African geranium) were purchased from Mountain Herb Estate Nursery, Pretoria, South Africa, and were raised under greenhouse conditions for three months. Eighteen-cm-diameter plastic pots were placed on the greenhouse bench at 0.3 m inter-row and 0.25 m intra-row spacing. Each pot was filled with 2.5 L steam-pasteurised river sand mixed with vermiculite at 2:1 (v/v). Three-month- old P. sidoides seedlings were transplanted to these pots. Seven days after transplanting, each pot was inoculated with ±1000 M. incognita J2s and eggs using a 50-mL plastic syringe by infesting approximately 10-cm-deep into the soil at cardinal points (around/all directions) of the roots of the plants.
After seven days, 10 g of each ground plant material were separately used to treat P. sidoides seedlings infested with nematodes by applying them within a 5-cm-radius around the seedlings, while 5 and 2.5 mgmL-1 crude extract was applied to the soil around the plant stem by a drenching treatment and arranged in a randomised complete block design (RCBD) with five replicates at 5-cm-radius next to the stem of the plants and mixed well with soil. The application occurred once per week for the first four weeks after inoculation. Every other day each plant was watered with 250 mL chlorine-free tap water, and half-strength Hoagland nutrient solution was applied once every other week for the duration of the experiment, while NPK was applied as previously outlined (Chapter 5; Section 5.2.2.2).
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6.2.3.1. Growth parameters
Fifty-six days after nematode inoculation, shoot height was measured from the crown to the flag leaf's top end (Sithole et al., 2016). The shoots were detached from the roots and oven-dried at 52 ºC for 72 h and weighed. Roots were removed from pots, rinsed in water to remove soil, blotted dry using filter paper, and weighed to facilitate the calculation of nematode density per plant’s total root system.
6.2.3.2. Determination of photosynthetic pigment
The photosynthetic pigments [total chlorophyll (a+b) and carotenoids] were calculated using Lichtenthaler's (1987) methodology, as described in Chapter 5; Section 5.2.1.13.
6.2.3.3. Protein and superoxide dismutase estimation
Bovine serum albumin (BSA) was used as a standard to calculate total protein (Bradford, 1976) as described in Chapter 5; Section 5.2.1.14, while superoxide dismutase (SOD) activity was estimated using the method of Beauchamp and Fridovich (1971) at 560 nm was measured against a blank using a spectrophotometer.
6.2.3.4. Carbohydrate estimation
With slight adjustments, the total carbohydrate content was calculated according to Sadasivam and Manickam (2008), following the methodology described previously (Chapter 5; Section 5.2.1.14).
155 6.2.3.5. Determination of total phenolic content
Total phenolic content was determined using the Folin-Ciocalteu assay, as described by Makkar (1999), with modifications using gallic acid as a standard according to the methodology described in Chapter 3; Section 3.2.3.
6.2.3.6. Nematode extraction and counting
Nematodes were extracted from roots using the maceration and blending method for 30 sec in 1% NaOCl (Hussey and Barker, 1973). The material was filtered through 125, 75, and 25-µm nested sieves, with nematodes collected from the75 and 25-µm mesh sieve. Soil per pot was thoroughly mixed, and a 300 mL soil sample was collected, with nematodes extracted using the sugar-floatation and centrifugation method (Jenkins, 1964). Eggs and J2s from root samples and J2s from soil samples were counted from a 10 mL aliquot of each sample using a stereomicroscope. Nematode numbers (J2s) in soil were converted to 2500 mL soil per pot and were used to determine the final nematode population densities (Pf).
6.2.3.7. Gas chromatography-mass spectroscopy (GC-MS) profile of C. maxima seeds crude extracts
One gram (1 g) of powdered C. maxima seeds were extracted with dichloromethane (DCM), ethyl acetate (EtOAc), and petroleum ether (PE). Extracts were concentrated in a vacuum at 35 ºC using a Buchi Rotary evaporator (Büchi, Flawil, Switzerland) and dried at room temperature under a stream of cold air. About 0.5 mg of each extract was re-dissolved in their respective solvents and analysed for their constituents using Gas Chromatography-Mass Spectrometry (GC- MS).
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GC-MS analysis was carried out using a Shimadzu QP-2010 SE Gas Chromatography coupled with an Agilent, 5973 Mass Selective detector driven by Agilent Chemstation software. A Zebron ZB-5MS plus capillary column with 30 m x 0.25 mm internal diameter and 0.25 μm film thickness was used. Ultra-pure helium at a flow rate of 1.0 mL/min was used as a carrier gas with a linear velocity of 37 cm/s. The samples (3 μL) were injected into the column with the injector temperature at 250 ºC. The initial oven temperature was 60 ºC, which was set to increase at a rate of 10 ºC/min to 280 ºC, with a holding time of 3 min at each increment. The mass spectrometer (MS) was operated in the electron ionization mode at 70 eV, and electron multiplier voltage at 1859 V. Other MS operating parameters was: ion source temperature 230 ºC, quadrupole temperature 150 ºC, solvent delay 4 min and scan range 50-700 amu. The compounds were identified by directly comparing the analyte’s mass spectrum at a particular retention time to that of the reference standards found in the National Institute of Standards and Technology (NIST) library. The area percentage of each component was calculated by comparing its average peak area to the total areas obtained.
6.2.3.8. Data analysis
All reported data were collected at 56-days after nematode inoculation and were subjected to two-way factorial analysis of variance (ANOVA) using SPSS version 24.00 (IBM Corporation Armonk, New York, NY, United States). Where the data were found not to be normal (1-KS), or the variances were not homogenous, discrete nematode data were transformed through log10 (x + 1) (Gomez and Gomez, 1984), but untransformed data were reported. Duncan’s multiple-range test (p<0.05) was performed to separate the means.
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