Nematicidal Activity of phytocompounds from Piliostigma thonningii Stem Bark Against Meloidogyne javanica

Abstract

Aim

The study investigated nematicidal activity of phytocompounds isolated from Piliostigma thonningii mature stem bark.

Methods

Compounds were extracted from the stem bark using solid–liquid extraction. Column chromatography was used to separate the phytocompounds and structures were characterized by LC–MS/MS QTRAP. Nematicidal activity of P. thonningii fractions were evaluated against second stage juveniles (J2) of Meloidogyne javanica.

Results

One tetracyclic diterpenoid (1), one glucosinolate (2), and two glycosylated flavonoids (3) and (4) that have strong nematicidal activity against second stage M. javanica juveniles from P. thonningii mature stem bark were identified. Nematicidal activity of the four compounds ranged from 59–99%. Compound 3 exhibited excellent nematicidal activity, 99 ± 1% that was comparable to that of the positive control fenamiphos at 100 mg/ml dose. Its activity increased from 79–99% as concentration was changed from 20 to 100 mg/ml.

Conclusion

The results of the present study shows that P. thonningii mature stem bark consist of phytocompounds that have potent nematicidal activity therefore, they could be associated in an integrated pest management program after further investigations.

Graphic Abstract

Introduction

Piliostigma thonningii is commonly found in Africa. In Southern Africa it is widespread in Zimbabwe, Namibia, Mozambique, Botswana and South Africa [1]. In other parts of Africa it is found in Sudan, Guinea, Senegal, Eritrea and some parts of Nigeria [2, 3]. P. thonningii is also found in selected countries in Asia and Australia [4]. Previously P. thonningii root, bark and leaf extracts exhibited antiviral activity against Herpes simplex virus type 1 and 2, HIV and different strains of influenza and syncytial viruses [5]. Extracts from leaves, stems and roots exhibited in-vitro activity against the rat tapeworm Hymenolepis diminuta. Extracts from mature bark showed antibacterial activity against Bacillus subtilis, Corynebacterium pyogenes, Escherichia coli, Proteus vulgaris, Shigella dysenteriae and Staphylococcus aureus [6,7,8]. Bark extracts have shown in vitro larvicidal activity against intestinal parasites of cattle. Ethanolic crude extracts and d-3-O-methylchiroinositol isolated from the stem bark showed anthelmintic activity against Haemonchus contortus larvae from faecal samples of infected lambs [9]. The presence of alkaloids was reported for the bark [3]. Published data of P. thonningii leaf extract revealed presence of various compounds including flavonoids, saponins, tannins, diterpenes, alkaloids and carbohydrates [10, 11]. Two new compounds isolated together with known polyphenols, 2β-methoxyclovan-9α-ol and methyl-ent-3β-hydroxylabd-8(17)-en-15-oate showed invitro antileishmanial activity [3].

The root-knot nematode, Meloidogyne spp. is a serious pest of many Solanaceous plants including, potato, tomato and green paper resulting in crop losses every year [12]. The method of control entails using synthetic nematicides, such as methyl bromide, 1,3-dichloropropene and metam sodium as fumigants. Although these pesticides are effective they have been banned because they are classified as ozone depleting agents [13]. Thus crop protection from root-knot nematodes should therefore depend upon other control strategies. Phytochemicals play an important role in controlling plant–insect interactions [14]. Aoudia et al. [15] showed that phenolic acids from Melia azedarach exhibited nematotoxic against Meloidogyne incognita. At the best of our knowledge, there are no scientific studies in the wide literature reporting nematicidal activity of phytochemicals from P. thonningii despite its use in controlling root-knot nematodes in traditional practices. Therefore in the present study nematicidal activity of extracts from P. thonningii mature stem extract was investigated and phytochemicals identities were ascertained using LC–MS/MS.

Experimental

Plant Material Collection and Preparation

Piliostigma thonningii stem bark samples were collected from Shurugwi and lower Gweru districts of Zimbabwe. The plant was further authenticated by taxonomists at the Harare National Herbarium and voucher specimen 017/8 was kept in the Bindura University of Science Education natural product herbal library for future reference. The samples were air dried for 3 weeks, ground and sieved through a 1 mm mesh sieve. A mass of 1000 g each of the ground plant material was extracted with water/methanol, 20:80 ratio) by maceration with 500 mL solvent mixture and allowed to stand for 24 h [10]. The plant extract was filtered over 50 g of sodium sulphate into a 1000 mL flat bottomed flask and concentrated to dryness on a rotary evaporator and stored under refrigeration until used. This stock sample continued to exhibit nematicidal activity even after being stored in the fridge at a temperature of 5 °C for 3 months.

Chromatography Analysis

The column was prepared by weighing 60 g of silica gel; particle size 0.063–0.2 mm/70–230 mesh (Merck, Germany) as stationary phase into a beaker and adding adequate amount of hexane and swirling until it forms slurry. The silica gel was then transferred into a glass column clamped on a stand. The silica gel was allowed to settle and a small amount of sand was added to form a bed onto the silica gel in the glass column. Extracts were transferred into the glass column and analytes were eluted with different solvents as shown in Table 1. The purity of the fractions which showed significant nematicidal activity was tested using aluminium silica gel coated TLC strips (1.5 × 10 cm) which were activated by heating gently in an oven and then each spotted. Separation was allowed through a distance of 8 cm in an air tight development chamber using different mobile phase systems as reported by Christinah et al. [16]. The developed plates were air dried and observed on a UV chamber at 275, 302 and 365 nm. Fractions were also analyzed by scanning on a UV spectrophotometer to obtain UV spectra in the range of 200–1000 nm.

Table 1 Eluting solvent ratios

Nematicidal Activity Against Juveniles of Meloidogyne javanica

Nematicidal efficacies of P. thonningii fractions were assessed following a slightly modified previous method by Ok et al. [13] against second stage juveniles (J2) of M. javanica with different concentrations doses, 20, 50, and 100 mg/mL. The concentrations were varied by increasing the sample weight before extraction. The final extract was reconstituted in 9 mL of ethanol. The second stage juveniles of M. javanica suspension concentration were adjusted to 100 J2 per mL. 9 mL of each reconstituted plant extract were added to 1 mL of nematode suspension in separate petri dishes. Ethanol was used as a control and 9 mL of distilled water were added to the petri dish. The petri dishes were kept in a dark cupboard at 20 °C. The mortality of the nematodes was monitored using an Eclipse 80i, Nikon, Tokyo light microscope after 24, 48 and 72 h [17]. From these counts, the percentages of nematode mortality were calculated for each treatment. Nematodes were considered alive if they moved or assumed a winding shape, and they were considered dead if they had adopted a straight shape and were immobile. To avoid incorrect classification, the nematodes in each petri dish were then transferred to distilled water for 48 h to check whether dead nematodes regained motility or not [17]. The corrected nematode mortality percentages were calculated according to Mureya [17].

$$\mathrm{M}\mathrm{o}\mathrm{r}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y} \left(\mathrm{\%}\right)=\frac{\left(m-n\right)\times 100}{100-n}$$

where m and n indicate the mortality (%) in treatments and control, respectively. This experiments were replicated three times and means and standard deviation computed.

Chemical Identification of Extracts by LC–MS/MS

Structures of fractions showing significant nematicidal activity were determined using an AB Sciex LC–MS/MS QTRAP 5500. It had a Turbo V source with an Electrospray ionisation probe operating in positive polarity. The HPLC was an Agilent 1260 LC system consisting of a vacuum degasser G4225A binary pump G1312B, auto sampler G1329B, an analytical column Phenomenex Synergi 4 μm Fusion-RP 100 Å, 50 × 2.0 mm and a guard column, Phenomenex Security Guard Cartridge Kit with Fusion-RP 4 × 2.0 mm cartridge. The mobile phase was A: water/methanol (90:10) + 5 mM ammonium formate, B: methanol/water (90:10) + 5 mM ammonium formate operated through gradient elution. The program of elution which was used ranged from 0–15 min. Source/gas parameters were CUR: 30 psi, CAD: High, IS: 5500 V, TEM: 400 °C, GS1: 60 psi GS2: 60 psi, Q1 Scan rate = 200 Da/s, enhance Product Ion (EPI) scan DP 75 V and EP 10 V. The volume injected was 20 µL at a flow rate of 1 mL/min. Compounds were characterized by means of high-resolution MS data using databases (i.e., Analyst 1.6.2, Peak View 2.2, Library View 1.0.1 and Master View) which had an online library searching capability. The compounds together with their retention times, molecular ions, probability errors and their suggested names are summarized in Table 5.

Statistical Analysis

Results of the nematicidal activity are expressed as the mean ± standard deviation (SD). Comparison was done using ANOVA analysis using SPSS version 20 and p values < 0.05 were considered to be significant different.

Results and Discussion

Column Chromatography and Nematicidal Activity

Root knot nematodes are important group of pest which cause devastating loss of yield. M. javanica has a host of more than 3000 plant species [18], thus its management reduces plant loss to a great extent. A number of practices are carried out to overcome these plant parasitic nematodes. Studies on plant phytochemicals as potential nematicidal agents are ongoing [19]. The current study investigated nematicidal activity of phytochemicals against root note M. javanica. The aqueous/methanol extract of P. thonningii was subjected to column chromatography. Different fractions 1–13 were obtained using various ratios of solvent eluent systems Table 1. While fractions 1–5 were light yellow, fractions 6–8 were deep yellow and fractions 9–13 were deep brown colour. The rotary evaporator dried fractions were weighed and then redissolved in ethanol and further tested for nematicidal activity against second stage juvenile M. javanica. Juveniles showing no movement or appearing straight were considered dead [18] The results shows that fractions 10, 11, 12 and 13 exhibited significant nematicidal activity which was lower or comparable to that of the positive commercial standard fenamiphos p < 0.05 Table 2, 3, 4. The activity increased as the concentration was increased from 20 to 100 mg/mL.

Table 2 Evaluation of the activity of 20 mg/mL plant extracts on mortality of the 2nd stage juveniles of M. javanica (mean ± standard deviation, SD)
Table 3 Evaluation of the activity of 50 mg/mL plant extracts on mortality of the 2nd stage juveniles of M. javanica (mean ± standard deviation, SD)
Table 4 Evaluation of the activity of 100 mg/mL plant extracts on mortality of the 2nd stage juveniles of M. javanica (mean ± standard deviation, SD)

Chemical Identification of Extracts by LC–MS/MS

Fractions 10–13 which showed strong nematicidal activity were subjected to structure identification Fig 1. Further TLC analysis showed that fractions 10–13 consist of similar components as only one spot was visible under UV light even after changing the eluting mobile phase several times. The UV analysis showed a single band for all the fractions with maximum wavelength, fraction 10 (226 nm), fraction 11 (229 nm), fraction, 12 (402 nm) and fraction 13 (403 nm). Compounds identification was achieved basing on the match between mass spectra and retention indices with those of mass spectra library search software; Analyst 1.6.2, Peak View 2.2, Library View 1.0.1 and Master View Table 5. A typical chromatogram is shown in Fig. 2. The weak peak at 17.2 min needs further fractionation to identify it however the mass spectrum results of the major components which probably exerted the most influence on the nematicidal activity were highly reliable with probability of 99% and low discrepancy, < 5 ppm between the calculated and observed molecular weight for all compounds. We further checked the quantities of glucosinolates in extract 11, flavonoids in, extracts 12 and 13 using HPLC and the results were, 276 ± 0.17 μg of sinigrin equivalent/mg of extract 2, 254.36 ± 3.15 μg of quercetin equivalent/mg of extract 12 and 225.66 ± 0.72 μg of quercetin equivalent/mg of extract 13. This implies that the high quantities of the compounds could contribute to the nematicidal activity. Quantities of diterpenes in extract 10 could not be determined due to lack of diterpenoids standards in our laboratory.

Fig. 1
figure1

Structures of nematicidal active compounds 1–4 from Piliostigma thonningii elucidated by LC–MS/MS

Table 5 Compounds annotated by LC–MSMS library analysis
Fig. 2
figure2

LC–MS/MS chromatography profile of (Compound 1)

The compounds were identified as follows see Fig. 1, fraction 10 (Compound 1), a diterpenoid, (2β, 5α, 7α, 10α, 13β)-4, 10-diacetoxy-1, 7, 13-trihydroxy-9-oxo-5, 20-epoxytax-11-en-2-yl benzoate, probability 98.9%, another name, baccatin III [20]. While nematicidal activity of baccatin III is reported here for the first time this compound has been isolated before from yew tree and is used as a precursor for synthesis of taxol an anticancer agent [20]. Fraction 11 (compound 2) a glucosinolate named 6-((E)-N-hydroxy-6-(methylthio) hexanethioyl)-tetrahydro-2H-pyran-2, 3, 4-triol, probability 99.2%. In previous studies glucosinolates portrayed nematicidal activity against the most common phytoparasitic nematodes affecting tomatoes, potatoes and grapes [21]. Fraction 12 (Compound 3) a glycosylated flavonoid, 3-({6-O-[(2E)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]-β-d-glucopyranosyl}oxy)-7-hydroxy-2-(4-hydroxyphenyl)chromenium-5-yl6-O-(carboxyacetyl)-β-d-glucopyranoside probability 98.6% and Fraction 13 (Compound 4) also a glycosylated flavonoid 3-{[(2S,3R,4S,5S,6R)-4,5-dihydroxy-6-(hydroxymethyl)-3-{[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}-5,7-dihydroxy-2-(3,4,5-trihdroxyphenyl-1λ4-chromen-1-ylium probability 99.3%. The order of nematicidal activity was Compound 3 > compound 2 > compound 4 > compound 1. There was no significant difference between nematicidal activities of compound 3 with that of the commercial standard fenamiphos at 100 mg/mL while the activities for the other compounds were significantly lower p < 0.05. Previously flavonoids showed potent nematicidal activity and were reported to participate in both root development and defence mechanism [19]. Very low doses of flavonoids such as quercetin and myricetin reduced the movement of M. incognita in a previous study [22]. Saroj et al. [23] discusses that phytochemicals derived from plants can be novel green approaches for mitigating pests.

Conclusion

The present work investigated phytochemicals with nematicidal activity isolated from P. thonningii. The results show that P. thonningii is a potential source of potent nematicidal agents. Column extraction using different solvents yielded 13 fractions and fractions 10–13 exhibited strong nematicidal activity. Chemical characterization of the potent fractions gave 4 known compounds, a tetracyclic diterpenoid, a glucosinolate, and two glycosylated flavonoids. The activity was dose dependent. The glycosylated flavonoid exhibited the strongest nematicidal activity.

Data Availability

Most of the data obtained have been included in this paper and remaining data can be obtained from the corresponding author upon request.

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Acknowledgements

The authors thank The Tobacco Research Center for LC-MSMS analysis and Bindura University of Science Education for the nematicidal activity assays and laboratory equipment and reagents.

Funding

The work was partially funded by a block grant from the research board of the Bindura University of Science Education.

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PD and CM designed the study and carried out the experimental work. PD and LG wrote the first draft and final draft. LG managed literature and corrected the first draft. All authors read and approved the final draft.

Corresponding author

Correspondence to Pamhidzai Dzomba.

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Dzomba, P., Mureya, C. & Gwatidzo, L. Nematicidal Activity of phytocompounds from Piliostigma thonningii Stem Bark Against Meloidogyne javanica. Chemistry Africa (2020). https://doi.org/10.1007/s42250-020-00158-z

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Keywords

  • Biopesticides
  • Glycosylated polyphenols
  • Glucosinolates
  • Root knot nematode