Evaluation of Terminalia glaucescens Chromatographic Fractions on Meloidogyne incognita Infecting Chili Pepper (Capsicum chinense)

The leaves and twigs of Terminalia glaucescens were collected from Tanke area of Ilorin, Nigeria, and deposited at the herbarium of Plant Biology Department, University of Ilorin, Nigeria, as sample number HI1812. They were chopped into tiny pieces and air dried in the laboratory for two weeks. The weight of leaves and twigs after drying was 15.68 kg and this was divided into three equal parts of 5.23 kg each and extracted cold in 25 l each of methanol (MeOH), acetone (Me₂CO) and water (H₂O). Each extraction lasted five days. The organic solvents were decanted, filtered, and concentrated under reduced pressure (Fabiyi et al. 2020c), while the aqueous extract was allowed to evaporate to dryness at 37 °C. The crude extracts of T. glaucescens were coded TEMG/MeOH, TEMG/Me₂CO, and TEMG/H₂O from methanol, acetone and aqueous, respectively.

The concentrated methanol and acetone crude extracts (250 g) were eluted over silica gel column (70–220 mesh) with n-hexane as the eluent. Nineteen fractions of 2000 ml were collected. Polarity of eluting solvents was increased to hexane/dichloro-methane 10 : 3 (500 ml) and later dichloromethane (800 ml) alone. The individual fractions were tested for similarities in R_f on precoated TLC plates (G_F254, 0.25 mm Merck Germany). They were developed with appropriate solvent system and viewed under UV lamp at 366 and 254 nm. Fractions with identical R_f were combined accordingly, giving rise to twelve fractions in all. Further purification of the fractions over aluminum oxide (alumina) afforded two clean fractions each from methanol and acetone crude extracts.

The fractions were screened for the presence of important plant metabolites such as carbohydrates, terpenoids, steroids, alkaloids, polyphenols (tannins), flavonoids, anthraquinones, saponins and reducing sugars following standard methods (Furniss et al. 1989; Trease & Evans 1989; Harborne 1976; Reid & Sarker 2006). All reagents used were of analytical grade and were not further purified. Vanillin spray colour reactions with heat (60 °C) on precoated TLC plates (G_F254, 0.25 mm Merck Germany) were also used to identify and characterize the phytochemicals.

Ultra violet-visible (UV-Vis) of fractions were taken on Du 730 Life Science UV/Vis spectrophotometer Beckman Coulter. Infrared (IR) spectra were recorded on SHIMADZU 8400s FTIR (Fourier Transform) spectrophotometer and gas chromatography-mass spectrometry was carried out with Agilent 7890A GC/MS equipped with a quadrupole mass spectra detector and an auto sampler. The GC-MS system settings are as follows: 2000 °C, interfaced temperature, 2500 °C, solvent cut time; 2.50 min; relative detector mode, ACQ mode; Scan; start time–end time, 3.00–56.00 min; event time, 0.50 s; scan speed 1428. Then, the GC system was equilibrated to a stable temperature of 2500 °C, the isolates were diluted with acetone and put into micro sample vials.

Pure culture of M. incognita race 2 raised on Celosia argentea were used as a source of inoculum in the micro plots. The roots were washed under running tap water to remove soil debris, then chopped with a sharp knife into 1–2 cm pieces and packed in a container with 600 ml of 0.6% NaOCl (Hussey & Baker 1973). The contents were agitated constantly for 4 minutes. The solution was sieved to separate the eggs from root particles by passing through 73, 56, and finally, 25 μm aperture sieves. Eggs were rinsed thoroughly from the 25 μm sieve into a clean beaker (Makete et al. 2012). This was left in the laboratory for the eggs to hatch into juveniles. The modified Baermann (1917) technique was used to separate the eggs from hatched juveniles (Fabiyi et al. 2020a). The juveniles were transferred into Doncaster's (1962) counting dish for enumeration under the microscope (×40), and 1 ml was found to contain approximately 315 of M. incognita juveniles.

A portion of land of 35 m by 25 m was mapped out, ploughed, and harrowed. This was divided into 48 micro plots of 1 m by 5 m (5.0 m²) in size with an alley way of 0.5 m between them (Fabiyi et al. 2012). The selected site had been planted with tomatoes for three consecutive seasons. Soil samples were taken from four points on each of the micro plots using systemic sampling method (Coyne et al. 2007), to identify the native nematode genera in the soil and their population. 192 points were sampled in all to represent the entire plot. These were taken to the laboratory and the nematode population was estimated using Whitehead and Hemming (1965) tray method of nematode extraction. A sub sample of 250 cm³ soil was taken from 800 cm³ of each composite sample for extraction. Pepper seedlings (Capsicum chinense) local cultivar were transplanted from the nursery on to each micro plots with a spacing of 50 cm between the rows and 20 cm in the rows, thus making a total of 10 plants per plot. Two weeks after transplanting, each pepper plant was inoculated with approximately 315 juveniles per ml following the method of Fabiyi et al. (2019). After completion of the experiments, the population of nematodes in soil and roots of pepper was examined. Root nematodes were extracted for enumeration using the blending and sieving method. Briefly, the roots of harvested pepper plants were washed, cut, dried, and weighed. The roots were placed in a blender with some water to cover the roots, and blending was done at low speed for 15 seconds. Blended roots were transferred to pie-pan tray set up; this was left for 24 hours and nematodes migrated to the water in the pan. The water was decanted and nematodes were transferred to a vial until counting under the microscope. Each soil sample was sieved to get out lumps and debris, and was measured out at a quantity of 100 ml from each sample. The soil was poured in a sieve lined with two ply tissue paper placed on a tray. Water was added in the gap between tray and sieve. The sieve and tray was carefully removed and water in the tray was poured into a beaker. The samples were left overnight and later decanted into vials. This procedure was carried out twice for each soil sample. Thus, 200 ml soil was extracted for nematodes from each sample and the average nematode count was documented.

Carbofuran 3G traded as worm force (2,3-dihydro-2,2-dimethylbenzofuran-7-yl-N-methylcarbamate) was applied at 0.5, 1.0, and 1.5 kg a.i.·ha⁻¹. The chromatographic fractions of T. glaucescens in concentrations 100, 75, and 50 mg·ml⁻¹ at 100 ml each were dissolved in 200 ml of nonionic surfactant to achieve total solubility and provide homogeneous solution of the fractions. They were then applied in a small 3 cm deep trench made at the base of each plant at 300 ml per plant.

There were four main treatments in the experiment: T. glaucescens methanol extract fraction (TEMG/MeOH), T. glaucescens acetone extract fraction (TEMG/Me₂CO), T. glaucescens aqueous extract (TEMG/H₂O), and carbofuran – a standard synthetic nematicide (CBFN). Each treatment had three concentrations (0, 100, 75, and 50 mg·ml⁻¹) and for carbofuran 0, 0.5, 1.0, and 1.5 kg a.i.·ha⁻¹ was applied. All the treatments were replicated three times in a randomized complete block design (RCBD). The experiment was conducted over a two-year period. Altogether, there were 48 micro plots in both years. The following data were collected: population of M. incognita in 200 ml soil and 10 g root sample, number of days to 50% flowering, number of fruits per plant, fruit weight per plant, height of pepper plants, numbers of leaves and branches. A two-way analysis of variance (Anova) was used to evaluate the data obtained using GenStat 5.32. Duncan's multiple range test was employed to establish different means at alpha level <0.05.

Phytochemical screening of the chromatographic fractions has revealed that they contained alkaloids, flavonoids, terpenoids, saponins, anthraquinones, phenols, and carbohydrates in the methanol fraction, while anthraquinone was absent in acetone fraction. Colour reaction with vanillin spray and heating also confirmed the presence of terpenes and alkaloids in all the T. glaucescens fractions. The UV-visible spectra of the fractions in dichloromethane showed that they contained a wide range of compounds, which absorbed in different ultraviolet regions, while GCMS analysis of the fractions revealed twenty-six compounds belonging to triterpenes, terpene hydrocarbons and fatty acid esters of which (E)-3-penten-2-one, pyridine, isovaleric acid, p-cymene, 2-ethylfuran, 2-propylfuran, 2-methyltetrahydrofuran-3-one, α-phellandrene, α-terpinene and salicylaldehyde are more than 5%. From the infrared analysis, the fractions exhibited, hydroxyl, aliphatic hydrocarbons, aldehydes, phenol, carboxylic acid, ester, and alkyl functional groups.

The initial native nematode population in the soil samples before inoculation was as follow: Meloidogyne spp. 534, Pratylenchus spp 105, Scutellonema spp. 22 and Helicotylenchus 9. M. incognita population in 200 ml soil samples at harvest was at the similar level in treatments of CBFN, TEMG Me₂CO, and TEMG MeOH (161–170) and significantly higher when water extract of TEMG was applied (234). In the nontreated soil, the number of M. incognita was 656. The effect of the treatments depended on the concentration of T. glaucescens extracts lowering from 656 in nontreated to 36 at 50 mg·l⁻¹ to 17 at 100 mg·l⁻¹. The populations of M. incognita scored in roots was three times higher in the nontreated roots than in the treated ones, but it did not differ between CBFN and different TEMG extracts. The lowest number of nematodes was found at the highest concentration of extracts (Table 1).

Vegetative growth and yielding attributes depended on the applied protective treatments against M. incognita. An earlier flowering was recorded in pepper plants protected with CBFN and Terminalia methanol and acetone extracts, especially with the highest concentration. The 100 mg·ml⁻¹ treated plants needed an average of 44 days to 50% of flowering, while an average of 77 days was recorded in the untreated pepper plants and 52 days for plants protected with CBFN. The flowering was delayed (63 days) in plants treated with aqueous extracts. The numbers and weights of harvested pepper fruits had similar values distribution – more and heavier fruits in protected plants with methanol and acetone extracts and CBFN, especially at the highest concentration, except water extract that gave similar results as was recorded for unprotected plants. The most detrimental effect caused by a lack of protection was low number and low fruit weight resulting from a weak vegetative growth. Likewise, the growth of the plants treated with the methanol and acetone extracts and carbofuran fractions improved gradually and consistently; the plants were significantly taller, had more leaves and side shoots compared to the plants treated with the aqueous extract (Table 1), which were characterized by growth inhibition and fewer leaves and branches.