Phytochemical characterisation and antifungal activities of five botanicals used by subsistence farmers to manage plant diseases

Abstract

Fungal phytopathogens cause many plant diseases resulting in severe crop losses. The phytochemical profiles, antioxidant and antifungal potentials of Aloe ferox, Allium cepa, Capsicum annuum, Tagetes minuta and Tulbaghia violacea extracted using acetone and methanol were investigated. Phytochemical profiling was undertaken using spectrophotometry and Liquid Chromatography-Mass Spectrometry (LC-MS). Antioxidant screening was done using 2,2´-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays. Antifungal effect was evaluated against Pythium ultimum and Botrytis cinerea using agar well diffusion and poisoned food techniques, respectively. Total phenolic concentrations ranged from 66 to 845 mg GAE/g (acetone extracts) and 20 to 195 mg GAE/g (methanol extracts); flavonoid concentrations ranged from 4 to 65 mg QE/g (acetone extracts) and 4 to 95 mg QE/g (methanol extracts). LC-MS analysis yielded 106 compounds, with 9-[2-(piperidin-1-yl)ethyl]-9 H-purin-6-amine, azelaic acid, glutarylcarnitine and sporovexin C as the common compounds across the five plants. Aloe ferox methanol extract (EC₅₀ = 0.2732 mg/mL) was the most potent extract in the ABTS assay, while T. minuta acetone extract (EC₅₀ = 0.1199 mg/mL) was the most potent in the DPPH assay. Tagetes minuta acetone extract (zone of inhibition = 26.67 mm) was the most potent against P. ultimum, while T. violacea methanol extract (62.4%) exhibited the highest mycelial growth inhibition against B. cinerea. Overall, Tulbaghia violacea and T. minuta were considered the most potent plants with antifungal effects. To fully understand the potential of these botanicals as antifungal, especially against P. ultimum and B. cinerea, further studies focusing on the efficacy of the bioactive compounds under in vivo condition is recommended.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-35591-6.

Introduction

Agricultural production of crops is pivotal to small-scale and subsistence farmers for improving livelihoods^1–3. However, it faces enormous challenges impacted by factors such as plant pests and diseases, which contribute to major crop losses globally⁴. Often, this significant losses of crops exacerbates food insecurity^5,6. Fungal pathogens remain the most prevalent causative agents of plant diseases in many crops⁷. For instance, Pythium ultimum causes damping-off in several major crops^8,9, and its polyphagous nature makes the disease management difficult^9,10. Botrytis cinerea is mostly associated with grey mould rot on leaves and stem of fruits. Additionally, it causes damping-off, bulb, corm, fruit, root and tuber rots, leaf spots, and stem cankers in many fruits and vegetable plants^11–14.

Generally, synthetic fungicides are used in managing plant diseases¹⁵. However, they are often associated with detrimental effects on humans and the environment^16–18. Furthermore, the increasing prevalence of fungicide resistance presents significant short and long-term challenges^19–21, as evidenced by the resistance of Fusarium strains to azole treatment²². These setbacks have prompted research focused on investigating alternative ways of managing plant diseases and natural-based products including botanical extracts hold great potential^5,18. For instance, Seepe et al.²³ revealed promising in vitro antifungal effects of plants such as Combretum erythrophyllum, C. mole and Schotia brachypetala against Fusarium spp. The biological effects of many botanicals have been attributed to diverse chemicals such as phenolics, coumarins, lectins, and saponins^18,24,25. Particularly, phenolics as a class entails a wide range of chemicals that exert antioxidant^26,27 and antifungal effects^28,29, which may acts against pathogens and pests through their repellent properties or innate toxicity³⁰. These phytochemicals and their interaction may be vital in managing plant diseases^18,31–33, including their antioxidant effects which is critical in disease management in crops^34,35.

In the previous ethnobotanical survey conducted, smallholder farmers in OR Tambo Municipality, South Africa identified the plants they often use for managing plant diseases³⁶. Thus, the current study selected five of the identified plants which were phytochemically profiled and assessed for their in vitro antioxidant and antifungal effects. The five plants (Aloe ferox Mill., Allium cepa L., Capsicum annuum L., Tagetes minuta L. and Tulbaghia violacea Harv) were selected as they were well recognised and utilised among the participants involved in the ethnobotanical survey. Furthermore, the choice of plant part used for analyses was based on the ethnobotanical information from participants during the ethnobotanical survey. Based on existing review on the potential of South African botanicals in managing phytopathogens³⁷, most of these selected plants have limited empirical data on their efficacy against phytopathogens. These include the efficacies of Aloe ferox and Tagetes minuta in managing Alternaria alternaria and Aspergillus niger³⁸, as well as the potential of T. minuta in managing Fusarium verticillioides and F. proliferatum³⁹. This study is geared toward contributing to new empirical data on ethnobotanical-driven approach for exploring South African rich and diverse botanicals with potential for managing phytopathogens.

Methodology

Collection of botanicals

The five plants namely Aloe ferox, Allium cepa, Capsicum annuum, Tagetes minuta and Tulbaghia violacea were collected between March and April 2023 from their natural habitats at Alice, Amathole district (Eastern Cape Province, South Africa). To allow for identification and verification by the taxonomist (Dr M Struwig), the voucher specimens were prepared, verified, and deposited at the S.D. Phalatse Herbarium located in North-West University, Mmabatho, South Africa. The allocated voucher numbers were LMO3, LM04, LM07, LM15 and LM16 for Aloe ferox, Allium cepa, Capsicum annuum, Tagetes minuta and Tulbaghia violacea, respectively.

Extraction of plant materials

The method described by Eloff et al.⁴⁰ with some modifications was used for extracting the plant extracts. The freshly harvested plants namely A. ferox (leaves), A. cepa (bulb), C. annuum (fruit), T. minuta (leaves and stem) and T. violacea (whole plant) were dried in open air under shade with temperatures of 25 ± 2 °C until constant weight. They were ground into fine particle powder (0.5 mm particle size) using a blender (PHRX RAF R.2843) and extracted using acetone and methanol (1:10, w/v ratio). Following extraction in an ultrasonic bath for 30 min (Sonicator 5510 Branson), the extracts were filtered with the aid of Whatman No. 1 filter papers. The resultant solutions were concentrated under reduced pressure with the aid of a rotary evaporator, followed by air-drying in a fume-hood.

Phytochemical analysis

Quantification of total phenolics and flavonoids

Folin-Ciocalteu colourimetric method was used to quantify the total phenolic content (TPC) in the acetone and methanol extracts⁴¹, with slight modifications. An aliquot (1 mL of 100 µg/mL) of each plant extract was used for the assay, and the resultant mixture was measured spectrophotometrically at 760 nm.

A slightly modified aluminium chloride colorimetric method by Yang et al.⁴² was adopted for flavonoid content quantification, whereby 1 mL (2 mg/mL) of each plant extract was used to prepare the resultant mixture. The absorbance was measured spectrophotometrically at 510 nm.

Liquid chromatography-mass spectrometry analysis

A previously established procedure as described by Khoza et al.⁴³ was adopted to prepare the methanolic plant extracts. As outlined by Magangana et al.⁴⁴, the compounds in the methanol extracts were identified using the high-resolution ultra-performance liquid chromatograph mass spectrometry (Waters, Milford, MA, USA). Electrospray ionization was used with desolvation gas at 650 L/h, desolvation temperature of 275 °C, and a cone voltage of 15 V. Leucine enkephalin (Sigma-Aldrich, Darmstadt, Germany) was used as reference mass for accurate mass determination, and sodium formate was used to calibrate the instrument. Data were acquired by scanning from 150 to 1500 m/z in MSE and resolution modes. Additional details on acquisition of data and separation followed previous study⁴⁴.

In vitro antioxidant tests

Antioxidant evaluation of the extracts was performed firstly using the 2,2´-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) as described by Arnao et al.⁴⁵. In addition, 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity protocol of Karioti et al.⁴⁶ was used. The absorbances were measured spectrophotometrically at 700 nm (ABTS) and 520 nm (DPPH).

In vitro antifungal activity assay

Fungal culturing and inoculum Preparation

Pythium ultimum and Botrytis cinerea pathogens were isolated from decayed strawberries. Fast growing fungal species were repeatedly isolated and identified using the method described by Campbell et al.⁴⁷ and López et al.⁴⁸. The pathogens were maintained on potato dextrose agar (PDA) (Sigma-Aldrich, Darmstadt, Germany). Working cultures were prepared by inoculating PDA agar slants with each fungal species. The inoculated agar slants were left to grow for 5 days at 25 ± 2 °C. Conidia were harvested by aseptically transferring 10 mL of potato dextrose broth with 0.2% Tween 80 (Sigma-Aldrich, Darmstadt, Germany) into the agar slants while shaking. A microscope and haemocytometer were used to determine the number of fungal spores of P. ultimum and B. cinerea in the retrieved suspension. A final working volume of the suspension comprised of 1.0 × 10⁶ spores/mL⁴⁹.

Agar well diffusion assay

Agar well diffusion method was adopted to assess the antifungal effect of the acetone and methanol extracts against Pythium ultimum, as described by Mativandlela et al.⁵⁰, with slight modifications. Aliquot (1 mL) of P. ultimum working volume was transferred onto PDA plates and evenly spread using a sterile spreader. Afterwards, wells of 9 mm diameter were punched into the PDA using a sterile cork borer. Approximately 50 µL of each plant extract (100 mg/mL for acetone and methanol) were added to the wells. Blank plates containing 2% acetone or PDA only served as controls³⁸. After 2 days of incubation at 30 °C, the zone of inhibition was measured (in millimetres).

Poisoned food method

Methanol extracts exhibited higher antioxidant activities, as well as demonstrated stronger antifungal activity against P. ultimum than acetone extracts, we opted to test methanol extracts of the plants against B. cinerea. As described by Adjou et al.⁵¹, poisoned food technique was used to assess the antifungal effect of the methanolic extracts against B. cinerea, with slight modifications. Concentrations of each plant extracts (10, 20, and 30 mg/mL) were prepared aseptically in 9 mm petri dishes by mixing with molten PDA and left to solidify. Negative control plates were prepared without any antifungal agent while positive control plates were prepared with a commercial fungicide (containing an active ingredient: propiconazole) as a positive control at 5 mg/mL. Thereafter, 6 mm agar disks containing mycelia of B. cinerea were inoculated onto the centre of each prepared petri dish and incubated at 28 °C for 3 days. The inhibition of mycelial growth (%) of B. cinerea was calculated for each extract⁵².

Inhibition of mycelial growth (%) = ((mean diameter in negative control sample - mean diameter in treated sample)/ mean diameter in negative control sample)) × 100.

Potency ranking

A ranking system was devised to establish the plant that demonstrated the highest potency among the five plants. The ranking system was based on seven factors which included the potencies of (1) acetone extracts in ABTS, (2) methanol extracts in ABTS, (3) acetone extracts in DPPH, (4) methanol extracts in DPPH (5), acetone extracts against Pythium, (6) methanol extracts against Pythium, and (7) methanol extracts against Botrytis for each plant. They were ranked from 1 to 5 based on the degree of potency demonstrated in each factor, with 1 being the most potent plant and 5 being the plant that displayed the least potency. The ranks were summed up, and the divided by the total number (5) of plants evaluated. The potency index was calculated as:

The plant with the lowest potency index was considered the most potent among the five evaluated plants.

Data analysis

A non-linear regression model was adopted to calculate the EC₅₀ values of the antioxidant activity. The data was subjected to analysis of variance (ANOVA) computed using GraphPad Prism 10.1.2 and means (triplicates) compared to each other where p < 0.05. Tukey’s Multiple Comparisons Test was used to calculate significance differences within means of the treatments.

Results and discussion

Phenolic and flavonoid contents

The concentration of total phenolics ranged from 66.88 to 845.11 mg GAE/g DW (acetone extracts) and 20 to 195.53 mg GAE/g DW (methanol extracts) (Fig. 1), while the concentration of flavonoids ranged from 4.28 to 65.14 mg QE/g DW (acetone extracts) and 4.96 to 95.48 mg QE/g DW (methanol extracts) (Fig. 2). Based on the plant-type, there was significant differences (p < 0.05) in the concentration of total phenolics. However, total phenolic concentrations for A. forex, C. annuum and T. violacea were similar for the acetone extract. In this study, acetone extract of T. minuta (845.11 mg GAE/g DW) and methanol extract of T. violacea (195.53 mg GAE/g DW) had the highest phenolic concentrations, while the highest concentrations of flavonoids were observed in methanol extract of C. annuum (95.48 mg QE/g DW), and acetone extract of T. minuta (65.14 mg QE/g DW). The antioxidant effect is inherent in the redox potential of phenolics^26,53. The antimicrobial activity of phenolics can be linked to their interference with cell wall or membrane of the microorganism resulting in the destruction of cells^54,55. Flavonoids which are subset of phenolics possess free radical scavenging capacity²⁷. Flavonoids have demonstrated antifungal effects against phytopathogens⁵⁶. Particularly, flavonoids such as catechin, epicatechin, and quercetin displayed antifungal effects against Botrytis cinerea^28,29. The varying concentrations of phenolics and flavonoids could be significant in the biological activities of medicinal plants⁵⁷. For instance, the high concentrations of phenolics and flavonoids displayed in T. minuta could be vital in its good antioxidant and antifungal activities, which may also be applicable to T. violacea.

Fig. 1.

Total phenolic content (mg GAE/g = milligrams of gallic acid equivalents per gram DW) of the five selected plants. Values are reported as means ± standard error (n = 3). For each extract type, bars with different letter case are significantly different at p < 0.05 according to Tukey’s multiple comparisons test.

Fig. 2.

Flavonoid content (mg QE/g = milligram quercetin equivalents per gram of extract) of the selected plant extracts. Values are reported as means ± standard error (n = 3). For each extract type, bars with different letter case are significantly different at p < 0.05 according to Tukey’s multiple comparisons test.

The results obtained from the spectrophotometry analysis was supported by different studies. For instance, Kumar and Kumar⁵⁸ revealed different concentrations of phenolics of A. cepa peels (167.58 mg GAE/g DW) and leaves (44.15 mg GAE/g DW) with hydroalcoholic and ethanolic extracts, respectively. The leaves of A. ferox had approximately 1.8 mg GAE/g DW for the phenolic concentration while the flavonoid concentration was 0.35 mg CE/g DW⁵⁹. According to Rikisahedew and Naidoo⁶⁰, methanol extract of T. minuta contain phenols, but absent in acetone extract. However, the current findings showed that acetone extract of T. minuta also contain phenols, which is contrary to their findings.

Identified compounds in the evaluated botanicals

The LC-MS analysis of methanolic extracts of the five botanicals yielded 106 phytochemicals, 81 of which were tentatively identified (Tables 1, 2, 3, 4 and 5, Supplementary Figures S1-S5). These included alkaloids (6), carboximidic acids and derivatives (1), carboxylic acids and derivatives (1), cinnamic acids and derivatives (3), fatty acyls (5), flavonoids (13), lignan glycosides (1), macrolides and analogues (1), peptides (3), phenolic acids (4), porphyrins (1), prenol lipids (4), pteridines (1), quinic acids and derivatives (3), saponins (2), steroids and steroid derivatives (3), terpenoids (3), and other unclassified chemicals (20). The various classes of the phytochemicals were confirmed using PubChem⁶¹, The Natural Products Magnetic⁶², and The Human Metabolome⁶³ databases. Flavonoids were the most represented phytochemicals (approximately 27%), which may have significance on the biological activities of the evaluated plants. The findings generated from the LC-MS analysis of A. ferox, A. cepa, C. annuum, T. minuta and T. violacea offer a comprehensive inventory of phytochemicals, with some of these compounds reported for the first time. This indicates the sensitivity of LC-MS in separation and identification of organic compounds⁶⁴. Four phytochemicals namely, 9-[2-(piperidin-1-yl)ethyl]-9 H-purin-6-amine, azelaic acid, glutarylcarnitine and sporovexin C were common across the evaluated five plants. Currently, 9-[2-(piperidin-1-yl)ethyl]-9 H-purin-6-amine has not yet been analysed for its biological activities. Numerous identified phytochemicals are unique to LC-MS analysis and have not been previously identified for the evaluated plant species. For instance, quercetin 3-p-coumarylsophoroside-7-glucoside, kaempferol 3-caffeylsophoroside, (-)-cilistol i, and isoorientin 2’’-[feruloyl-(->6)-glucoside] which were highly concentrated in Aloe ferox are likely the first report for this plant species⁶⁵.

Table 1.

Compounds tentatively identified in Aloe ferox extracts with the retention times (RT), detected [M-H]⁻ and M + H]⁺ ion, elemental composition, MS^E fragments and class.

No.	RT	Experimental m/z [M-H]−/[M + H]+					MSE fragmentation ions	Elemental formula	Class	Tentative identity
1	2.21	279.0590		[M-H]−			279.0596, 128.0355,183.9702, 257.0777, 261.9222, 263.0864, 264.9921, 265.999, 267.0045, 279.0601	-	-	Unknown 1
2	2.69	243.0621		[M-H]−			243.0617, 128.0344, 163.0383, 180.0664, 181.0695, 191.0196, 200.0557, 243.0617	-	-	Unknown 2
3	2.73	276.1448		[M + H]+			276.1446, 212.1284, 230.1393, 231.14244, 241.1558, 248.1491, 252.1098, 258.1339, 259.1374, 275.1630, 276.1447	-	-	Unknown 3
4	3.09	379.0699		[M-H]−			379.0696, 164.0716, 240.9754, 241.0014, 264.9833, 292.8097, 300.8153, 309.8147, 341.0850, 341.1054, 360.8284, 379.0317	C15H19NO6	Phenolic acids	N-{6,8-dihydroxy-2-phenyl-hexahydro-2 H-pyrano[3,2-d][1,3]dioxin-7-yl}acetamide
5	3.32	315.1078		[M-H]−			315.1074, 153.0551,179.0338, 241.0016, 300.8172, 309.8120	C14H20O8	Flavonoids	Hydroxytyrosol 1-O-glucoside
6	3.64	405.0497		[M-H]−			405.0497, 241.0021, 242.0078, 327.0756, 360.7964, 379.0716	-	-	Unknown 4
7	3.79	485.1696		[M-H]−			485.1696, 241.0024, 242.0058, 259.0127, 292.8107, 341.0863, 397.1030, 435.0181, 436.0621, 437.0630, 447.2157, 483.1191	-	-	Unknown 5
8	4.11	933.2297		[M-H]−			933.2297, 292.8121, 300.8164, 341.0799, 355.0788, 360.7984, 389.1616, 395.1017, 397.1122, 429.0822, 466.1117, 467.1190, 495.1873, 610.1922, 629.2114, 649.1770, 651.1837, 689.1379, 711.8766	C42H46O24	Flavonoids	Quercetin 3-p-coumarylsophoroside-7-glucoside
9	4.39	771.1776		[M-H]−			771.1775, 593.4564, 465.1424, 383.0766, 351.0495, 328.0542, 285.1322, 125.7667, 101.9799, 80.7710	C36H36O19	Flavonoids	Kaempferol 3-caffeylsophoroside
10	4.61	785.1925		[M-H]−			785.1926, 633.1832, 625.2183,615.1828, 465.1427, 429.0818, 369.0604, 341.1083, 300.8178, 241.0025	C37H38O19	Lignan glycosides	Isoorientin 2’’-[feruloyl-(->6)-glucoside]
11	4.99	453.1794		[M-H]−			453.1800, 395.1921, 389.7517, 385.0857, 360.8288, 383.1119, 353.0680, 341.0832, 323.0783, 261.1334, 254.9816, 241.0023	None	-	Unknown 6
12	5.38	187.0970		[M-H]−			187.0977, 125.0966	C9H16O4	Fatty acyls	Azelaic acid
13	5.86	815.4423		[M-H]−			815.4433, 800.4482, 799.4429, 785.4661, 741.2327, 653.4289, 637.2409, 602.2970, 601.2888, 497.2043, 507.3331, 491.1576, 481.1749, 479.1933, 477.1792, 449.1440, 407.2171, 341.1074	C41H68O16	Saponins	Steroidal glycosides
14	6.07	799.4475		[M-H]−			799.4475, 797.3741, 789.3474, 785.4668, 627.2458, 613.2928, 594.1522, 497.2451, 491.3342, 479.1591, 477.1789, 447.2558, 445.2454, 399.1878, 389.1826, 385.0567, 360.2387, 285.0400	C41H68O15	-	Arenaric acid
15	6.46	653.3892		[M-H]−			653.3893, 644.2813, 643.2396, 595.2558, 479.1545, 403.1672, 389.7792, 341.1098	C35H58O11	Macrolides and analogues	(-)-Cilistol i
16	6.65	219.1749		[M + H]+			219.1744, 203.1427, 204.1467	C15H22O	Terpenoids	Sesquiterpenoids
17	7.14	431.2281		[M-H]−			431.2280, 417.2090, 389.7454, 377.0874, 353.2005, 331.1744, 297.1696, 265.1469, 261.1338, 243.1221, 225.0516, 210.0829, 209.0789, 187.0974,125.0965	C21H36O9	Terpenoids	Glucosyl (2E,6E,10x)-10,11-dihydroxy-2,6-farnesadienoate
18	7.73	399.2391		[M + H]+			399.2514, 385.3784, 379.2475, 374.2758, 365.1359, 359.2415, 353.2311, 347.2196, 338.3407, 333.2807, 326.3774, 317.2091, 315.2694, 305.2092, 299.1618, 247.1670, 184.0742	C25H34O4	Prenol lipids	2-[3-(3,5-dimethyl-1 H-pyrazol-1-yl)-6-oxo-1,6-dihydropyridazin-1-yl]-N-[(octahydro-1 H-quinolizin-1-yl) methyl]acetamide (Quinolizines)
19	7.76	415.2338		[M-H]−			415.2331, 397.1040, 389.7785, 354.2275, 353.1987, 341.1088, 340.2124, 309.8122, 266.1462, 265.1428, 262.1367, 261.1342, 243.1594, 225.1478, 209.0784, 187.0973, 171.1023	C21H36O8	-	Aeginetoside
20	8.59	374.2448		[M-H]−			374.2441, 341.1036, 325.0716, 311.1674, 290.1864, 265.1459, 261.1323, 232.1090, 187.0970	-	-	Unknown 7
21	9.63	371.2440		[M-H]−			371.2434, 355.1957, 354.2024, 353.1993, 339.2241, 325.1853, 315.2537, 311.2227, 309.1729, 298.1561, 281.2494, 279.1624, 266.1796, 263.1628, 243.1963	C20H36O6	-	8,8a-Deoxyoleandolide
22	10.18	265.1479		[M-H]−			265.1474, 253.2177, 183.0114	-	-	Unknown 8
23	11.08	311.1686		[M-H]−			311.1686, 933.5454, 309.1156, 300.2616, 295.2632, 293.2076, 281.1612, 280.1669, 269.21207, 255.2334, 253.1889, 225.18513, 197.0283, 194.9056,183.01248	C12H7O10	-	Unknown 9
24	11.58	385.2933		[M + H]+			385.2926, 371.2279, 367.2216, 363.3103, 357.1470, 342.2690, 335.2569, 327.2015, 311.2563, 301.1389, 283.1746,223.2053	C22H40O5	-	1,3-diacylglycerols
25	11.84	555.2853		[M-H]−			555.2842, 415.2317, 409.3474, 397.2678, 323.3232, 265.1239, 255.2318, 227.2009, 225.0067, 183.0119	-	-	Unknown 10
26	12.39	457.3502		[M + H]+			457.3500, 195.1225, 290.1759, 293.1365	-	-	Unknown 12
27	14.15	248.9608		[M-H]−			248.9605, 238.9319, 230.8993, 228.9024, 192.9283, 186.9312, 170.9457, 112.9852	-	-	Unknown 13

MS^E fragments in bold typeface refer to the base peak (the highest peak) (82).

Table 2.

Compounds tentatively identified in Allium Cepa extracts with the retention times (RT), detected [M-H]⁻ and M + H]⁺ ion, elemental composition, MS^E fragments and class.

No.	RT		Experimental m/z [M-H]−/[M + H]+		MSE fragmentation ions	Elemental formula	Class	Tentative identity
1	0.87		290.8478	[M + H]+	291.0987, 275.8765,207.8123	-	-	Unknown1
2	1.18		342.1403	[M + H]+	325.6767, 365.6677, 276.1213	-	-	Unknown 2
3	2.66		289.1396	[M + H]+	289.4545, 432.676, 280.1185, 271.0987, 238.0707, 229.1554, 215.7654, 182.0813, 147.0454, 136.06282, 128.3434	C12H20N2O6	Dipeptides	N-γ-Glutamyl-S-allyl cysteine
4	2.96		303.0719	[M-H]−	303.1534, 315.4343, 323.1433, 341.1544, 390.4333	C12H16O9	Flavonoids	O-glycosyl compounds
5	4.28		627.1570	[M-H]+	303.6655, 292.5454, 465.5656,627.1115, 645.676	C27H30O17	Flavonoids	Quercetin 3-glucosyl-(1->2)-galactoside
6	4.41		441.1978	[M-H]−	395.1020, 441.0001, 463.1411, 431.1114, 313.0111, 301.0001	C18H33O12	-	Unknown 3
7	5.17		465.1039	[M + H]+	303.4343, 411.4334, 315.0503, 275.0548, 269.1926, 266.7454, 247.0603, 201.0552	C21H20O12	Flavonoids	Quercetin 3-galactoside
8	5.37		447.1038	[M-H]−	477.0009, 463.0101, 409.1200, 314.0121, 301.0010	C22H21O12	-	Unknown 4
9	5.92		314.1389	[M + H]+	314.4343, 411.4545, 314.1387, 303.1413, 301.1081, 286.1809, 257.1183, 216.1033, 177.0549	C18H19NO4	Phenols	Moupinamide
10	6.08		344.1498	[M-H]+	344.1121, 329.1115, 314.1367, 311.1028, 303.6555, 293.0937, 279.0949, 186.7676	C19H21NO5	Cinnamic acids and derivatives	N-trans-Sinapoyltyramine
11	7.13		415.2341	[M + H]+	455.3434, 437.5454, 415.3434; 365.4545, 293.3434	C20H27N2O10	Peptides and amino acids	N–Hexosyl-glutamyl phenylalanine
12	7.73		399.2394	[M + H]+	439.6555, 399.5645, 383.2230, 345.2436, 318.2774, 309.1675, 219.1747, 203.1433	C21H34O7	Prenol lipids	Hypochoeroside K; (-)-Hypochoeroside K
13	7.94		1043.5396	[M + H]−	1044.1535, 915.3535, 869.3545, 507.1111, 299.5353	C52H84O21	-	Unknown 5
14	8.51		739.4282	[M + H]+	739.1654, 761.4666, 423.1755	C39H62O13	Steroids and steroid derivatives	Isonuatigenin 3-[rhamnosyl-(1->2)-glucoside]
15	9.15		709.4166	[M-H]+	709.4160, 731.2101, 674.3981, 413.3988, 395.4641, 279.2131, 271.2026, 270.1934, 265. 2251, 258. 2691, 253.1955, 251.1799, 241.1951, 227.1296, 211.1457	C38H60O12	Steroids and steroid derivatives	Alliospiroside A
16	10.42		399.2519	[M + H]+	421.7878, 373.0987, 335.8766, 399.9876, 783.0009	C21H30N6O2	-	2-[3-(3,5-dimethyl-1 H-pyrazol-1-yl)-6-oxo-1,6-dihydropyridazin-1-yl]-N-[(octahydro-1 H-quinolizin-1-yl)methyl]acetamide
17	11.57		341.2670	[M + H]+	429.4343, 385.9876, 341.9898, 473.7878, 517.7770	C19H45N2O8	-	Unknown 6
18	11.97		526.4326	[M + H]+	443.1245, 487.1542, 399.1245, 531.1200, 575.1444, 619.1111	C21H43N6O4	-	Unknown 7
19	12.39		457.3502	[M + H]+	457.0202, 501.1111, 413.1919, 746.1233, 762.1919, 1045.1010	C22H45N6O4	-	Unknown 8
20	12.65		1036.6926	[M + H]+	1059.1010, 1037.9292, 819.5981, 778.6994, 746.2020, 762.0002, 229.1429, 213.1114	C53H93N7O13	Peptides	Surfactin C
21	14.14		247.1673	[M + H]+	431.7676, 746.5656, 365.7777	C12H18N6	-	9-[2-(piperidin-1-yl)ethyl]-9 H-purin-6-amine

MS^E fragments in bold typeface refer to the base peak (the highest peak) (82).

Table 3.

Compounds tentatively identified in Capsicum annuum extracts with the retention times (RT), detected [M-H]⁻ and M + H]⁺ ion, elemental composition, MS^E fragments and class.

No.	RT	Experimental m/z [M-H]−/[M + H]+		MSE fragmentation ions		Elemental formula	Class	Tentative identity
1	0.89	274.8739	[M + H]+	274.9223,311.3622,123.0202		-	-	Unknown 1
2	1.18	360.0786	[M + H]+	360.8282,651.3722,121.7272,128.011		-	-	Unknown 2
3	1.90	262.1293	[M + H]+	262.9922,262.0192,123.9282,111.0011		-	-	Unknown 3
4	1.95	358.0638	[M-H]−	358.9191,234.2627,182.1911, 111.0191		-	-	Unknown 4
5	2.73	276.1444	[M + H]+	276.1447, 259.1374, 252.1098, 248.1491, 241.1558, 231.1424, 230.1393, 212.1284		C12H21NO6	Fatty acyls	Glutarylcarnitine
6	3.08	310.1289	[M + H]+	310.1293, 293.1211, 282.1336, 279.0952, 265.1280, 264.1233, 246.1133		C15H19NO6	Alkaloids	Sporovexin C; (-)-Sporovexin C
7	3.74	355.1027	[M-H]−	355.1026, 341.1096, 309.8125, 300.8155, 194.0519, 193.0500, 178.0258		C16H20O9	Cinnamic acids	1-O-Feruloylglucose
8	4.23	757.2222	[M + H]+	757.2202, 654.2708, 611.1615, 595.1679, 555.2431, 539.1730, 292.1905, 289.0608, 278.1753, 260.1647		C33H40O20	Flavonoids	Kaempferol 3-gentiobioside 7-rhamnoside
9	4.71	595.1674	[M + H]+	595.1673, 561.2954, 543.2853, 539.2697, 534.3130, 525.2903, 505.0317, 287.0557, 259.0614, 241.0496, 234.1125, 229.0963, 213.0550,156.0819, 153.0190		C27H30O15	Flavonoids	Astragalin 7-rhamnoside
10	5.05	304.1912	[M + H]+	304.1911, 295.1527, 290.1381, 287.0553, 272.1651, 265.0979, 247.1334, 232.1333, 219.1260, 215.1063, 200.1073, 186.0905		C18H25NO3	Alkaloids	Camporidine A
11	5.39	187.0972	[M-H]−	187.0977, 125.0966		C9H16O4	Fatty acyls	Azelaic acid
12	6.66	219.1750	[M + H]+	219.1744, 203.1427, 204.1467		C15H22O	Prenol lipids	Zerumbone
13	7.14	415.2338	[M + H]+	415.2333, 171.1492, 203.1210, 757.9876, 219.1742, 227.1281, 245.1384, 267.1210, 279.0902, 293.0219, 355.1751, 367.1377, 381.1863, 397.2225, 398.2258		C21H34O8	Terpenoids	Dendromoniliside A
14	7.73	399.2386	[M + H]+	399.2380, 203.1433, 219.1747, 227.1281, 229.1435, 245.1388, 309.1675, 318.2774, 345.2436, 383.2230		C21H34O7	-	Neorustmicin B
15	8.58	376.2601	[M + H]+	376.2595, 227.1280, 279.0927, 292.2012, 302.1859, 321.2037, 365.1057, 376.2592		C21H33N3O3	-	1-(2-methoxyethyl)-4,6-dimethyl-N-[(octahydro-1 H-quinolizin-1-yl)methyl]-2-oxo-1,2-dihydropyridine-3-carboxamide
16	8.99	304.3004	[M + H]+	304.1900, 295.1500, 290.1381, 287.0553, 272.1651, 265.0979, 247.1334, 232.1333, 219.1260, 215.1063, 200.1073, 186.0905		C18H25NO3	Alkaloids	Camporidine A
17	10.49	399.2521	[M + H]+	399.2321, 203.9993, 219.1747, 227.1281, 229.1435, 245.1388, 309.1675, 318.2774, 345.2436, 383.2230		C21H34O7	-	Neorustmicin B
18	11.12	328.2640	[M + H]+	328.2636, 315.1791, 313.2153, 309.1500, 291.2312, 279.0932, 273.2201, 271.1546, 247.2048		C22H33NO	-	Carboximidic acids
19	11.58	385.2931	[M + H]+	385.2926, 377.2695, 373.2707, 367.2216, 363.2515, 357.1470, 342.2690, 335.2569, 327.2015, 311.2563, 309.2013, 304.2976, 301.1389, 283.1746, 223.2053		C22H40O5	-	2-(1,4-dihydroxybutyl)-7-(6-hydroxy-4-pentylcyclohex-2-en-1-yl)heptanoic acid
20	11.97	526.4324	[M + H]+	526.4310, 355.0721, 351.2484, 184.0736, 195.1231, 227.1271, 229.1411, 239.1489, 245.1384, 247.1658, 255.2306, 256.2631, 279.0954, 281.0496, 339.2902,343.1659		-	-	Unknown 5
21	12.64	540.4475	[M + H]+	540.4472, 283.0481, 251.2361, 247.1676, 239.1495, 227.1286, 225.0416, 195.1228		-	-	Unknown 7
22	14.15	247.1672	[M + H]+	247.1670, 124.0872, 148.0880, 158.9645, 165.1141, 166.0986, 166.1170, 174.9318, 186.9112, 189.1136, 206.1400, 207.1251		C12H18N6	Pteridines	2,4-diamino-6,7-diisopropylpteridine

MS^E fragments in bold typeface refer to the base peak (the highest peak) (82).

Table 4.

Compounds tentatively identified in Tagetes Minuta extracts with the retention times (RT), detected [M-H]⁻ and M + H]⁺ ion, elemental composition, MS^E fragments and class.

No.	RT	Experimental m/z [M-H]−/[M + H]+		MSE fragmentation ions	Elemental formula	Class	Tentative identity
1	0.87	242.9258	[M + H]+	243.9876, 259.9754, 463.3255, 447.8533, 479.1222	-	-	Unknown 1
2	1.18	341.1091	[M + H]−	341.9898, 683.0147, 503.0120, 377.0999, 191.0120	C12H21O11	-	Unknown 2
3	3.70	366.1558	[M + H]+	366.1245, 367.7666, 426.6121, 366.1202, 323.0744, 318.1326, 303.0499, 290.1387, 268.1538, 225.1103, 163.0396	C18H23NO7	Alkaloids and derivatives	Sennecicannabine
4	3.87	353.0875	[M + H]−	191.1311, 353.2444, 707.1322	C7H12O6	Quinic acids and derivatives	Quinic acid
5	4.24	350.1607	[M + H]+	370.1221, 350.1211	C19H27NO5	-	Unknown 3
6	4.58	352.1765	[M-H]+	352.3434, 402.1111, 386.2544	C19H2N3O5	-	Unknown 4
7	4.85	368.2073	[M + H]+	400.2323, 402.1111, 384.2222, 417.1111, 409.0099	C18H25NO7	Alkaloids	Isatidine
8	5.02	515.1193	[M + H]−	353.9855, 191.1213, 179.0543, 173.8777, 161.8765	C25H24O12	Quinic acids and derivatives	3,5 or 4,5 di-caffeoylquinic acid (CQA)
9	5.17	515.1196	[M + H]−	353.8754, 191.7654, 179.8755, 173.8444, 135.2222	C25H24O12	Quinic acids and derivatives	3,5 or 4,5 di-caffeoylquinic acid (CQA)
10	5.46	392.1714	[M + H]+	392.1010, 393.1111, 411.6168, 423.1818	C21H29NO6	-	6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-2-benzofuran-5-yl)-N-(3-methoxypropyl)-4-methylhex-4-enamide
11	5.75	169.0867	[M + H]−	595.0212, 529.1452, 971.0121, 353.0121, 255.0121	C9H14O3	-	Aspinonediol; (+)-Aspinonediol
12	5.88	466.2450	[M-H]+	392.6565, 417.1211, 422.1222, 435.1111, 439.0009	C24H35NO8	Flavonoids	O-glucuronides
13	6.02	466.2448	[M + H]+	392.1111, 393.2422, 417.1545, 422.1211, 439.7575	C24H35NO8	Flavonoids	O-glucuronides
14	6.72	327.2175	[M + H]−	327.4333, 595.6566, 456.6667, 520.8889, 785.2555	C18H31O5	-	Unknown 5
15	7.40	564.3306	[M + H]−	504.7676, 279.5575, 224.6766	C30H42N7O4	-	Unknown 6
16	9.06	721.3645	[M + H]−	675.5655, 397.3533, 415.1111, 277.5656	C30H53N6O14	-	Unknown 7
17	9.61	540.3293	[M + H]−	504.7655, 564.7654, 279.8754, 187.4333	C36H2N3O4	-	Unknown 8
18	10.24	522.3566	[M + H]+	415.5454, 419.3232, 405.1145, 439.2556	C28H47N3O6	-	3-(acetyloxy)-1-{4-[(4-hydroxy-3-methoxyphenyl)methyl]piperidin-4-yl}octyl 2,3-bis(methylamino)propanoate
19	12.16	609.2715	[M + H]+	609.3232, 625.2323, 976.4343, 399.1424, 413.5445, 415.4343, 421.6565, 429.43334, 441.6565, 443.4333	C35H36N4O6	Porphyrins	Harderoporphyrin
20	12.54	593.2770	[M-H]+	593.3233, 413.3222, 399.2121, 443.2323	C33H52O9	Saponins	Agavoside A
21	12.64	540.4479	[M + H]+	540.1212, 342.2929, 300.0222,111.2122	C12H14N1O23	-	Unknown 9
22	14.14	247.1674	[M + H]+	431.8282, 421.1211, 411.3232, 423.2323, 443.2323	C12H18N6	-	9-[2-(piperidin-1-yl)ethyl]-9 H-purin-6-amine

MS^E fragments in bold typeface refer to the base peak (the highest peak) (82).

Table 5.

Compounds tentatively identified in Tulbaghia violacea extracts with the retention times (RT), detected [M-H]⁻ and M + H]⁺ ion, elemental composition, MS^E fragments and class.

No.		RT		Experimental m/z [M-H]−/[M + H]+		MSE fragmentation ions	Elemental formula	Class	Tentative identity
1		2.67		243.0621	[M-H]−	180.2355, 330.0053, 342.3122, 191.3211	C12H22O6	-	Unknown 1
2		2.75		276.1448	[M + H]+	276.0009, 258.2100, 230.1111	C12H21NO6	Fatty acyls	Glutarylcarnitine
3		3.09		310.1293	[M + H]+	310.0098, 292.3211, 268.8754, 328.3210	C15H19NO6	Carboxylic acid and derivatives	Sporovexin C; (-)-Sporovexin C
4		3.85		325.0924	[M-H]−	349.0087, 651.0086, 673.0087, 163.4322	C15H18O8	Cinnamic acids and derivatives	1-O-p-Coumaroyl-beta-D-glucose
5		4.57		471.2079	[M-H]−	425.0002, 471.0754, 853.2213	C36H36O19	Flavonoids	Quercetin 3-O-beta-(6’’-O-E-p-coumaroylglucoside)-7-O-beta-glucoside
6		5.08		565.1564	[M + H]+	271.0876, 565.0219	C26H28O14	Flavonoids	Flavonoid-7-O-glycosides
7		5.17		1405.6519	[M + H]+	1406.4322, 1244.0531, 757.0987, 595.1211	C61H93N14O24	-	Unknown 2
8		6.37		1085.5391	[M + H]+	1103.0211, 417.3211, 271.2345, 671.0099	C50H84O25	-	Unknown 3
9		6.89		395.1381	[M-H]−	1260.0654, 1043.0421, 1126.3332	-	-	Unknown 4
10		7.44		643.3320	[M-H]−	643.0322, 787.3222, 1084.0333	C32H52O13	Steroids and steroid derivatives	Virescenoside R; (+)-Virescenoside R
11		7.79		778.4598	[M + H]+	431.0543, 577.3222, 783.0444, 413.4321, 217.0432	C51H59N3O4	-	4-(3-aminopropyl)-5’,33’-dimethyl-3 H-29’-oxa-1’,34’-diazaspiro[2-benzofuran-1,12’-decacyclo-derivative
12		8.60		306.2069	[M + H]+	306.0233, 328.0433, 376.5333, 398.1112, 633.0004	C17H23NO4	Alkaloids	Anisodamine
13		9.09		308.2225	[M + H]+	330.0043, 308.3222, 699.0543, 537.3222, 615.0766	C18H29NO3	Phenols	Dihydrocapsaicin
14		9.66		322.2382	[M + H]+	496.5411, 537.0654, 640.0865, 701.3334, 344.4311	C19H31NO3	Phenols	Homodihydrocapsaicin
15		9.84		353.2700	[M + H]+	537.3433, 261.0222, 1052.4322	C23H28O3	Prenol lipids	Methyl (9Z)-8’-oxo-6,8’-diapo-6-carotenoate
16		10.11		476.2780	[M-H]−	593.3222, 577.0233, 285.2222, 614.0532	C25H38N3O6	-	Unknown 5
17		11.27		695.3856	[M-H]−	695.2322, 688.1222, 649.2222, 504.2233	C33H59O15	-	Unknown 6
18		11.85		555.2839	[M-H]−	555.0433, 325.0555	C16H27O24	-	Unknown 7
19		12.24		282.2794	[M + H]+	282.0654, 240.0007	C18H35NO	Fatty acyls	Oleamide
20		12.38		270.2797	[M + H]+	270.6211, 111.0098	C17H36NO	-	Unknown 8
21		12.89		284.2953	[M + H]+	284.2323, 661.6544, 577.0009	C18H37NO	Carboximidic acids and derivatives	Octadecanamide
22		14.15		247.1673	[M + H]+	227.0098, 431.0977, 746.0088	C12H18N6	-	9-[2-(piperidin-1-yl)ethyl]-9 H-purin-6-amine

MS^E fragments in bold typeface refer to the base peak (the highest peak) (82).

Some of the identified phytochemicals possess antioxidant effects including 1-O-feruloylglucose⁶⁶, 3,4 or 4,5 di-caffeoylquinic acid²⁶, chlorogenic acid⁶⁷, hydroxytyrosol-1-O-glucoside⁶⁸, and quinic acid^26,69. Additionally, the evaluated plant extracts had different classes of phytochemicals which are considered as good sources of antioxidants, with the ability to protect against oxidative stress. Examples of such classes include coumarins⁷⁰ and phenolics²⁶. Neorustmicin B inhibited stem rust fungus on wheat and prevented its occurrence caused by Puccinia graminis f. sp. tritici at 1 µg/mL⁷¹. Bais et al.⁷² highlighted the efficacy of surfactin C as a biocontrol agent of Bacillus subtilis against Pseudomonas syringae pv tomato causing Arabidopsis root infections. Findings by Seepe et al.⁷³ revealed that 5-hydroxy-7,4′-dimethoxyflavone had promising antifungal effects against Fusarium species. In the study by Neto et al.⁷⁴, varying concentrations of 3-O-p-coumaroylquinic acid and quercetin-3-O-galactoside exerted promising biological effects by preventing pest invasion. High concentrations of quercetin glycosides observed in ‘Early Black’ cranberry leaves may deter pests. In that regards, the different classes of phytochemicals identified in the tested botanicals suggest their use as antifungal against phytopathogens^24,25, and may serve as potential alternative to synthetic fungicides^18,31–33.

Antioxidant activity of the investigated plant extracts

The use of multiple assays for determining antioxidant activity is well known, which necessitated the use of ABTS and DPPH in this study. The ABTS radical can react rapidly with both natural (i.e. amino acids, peptides, phenols, vitamin C and vitamin E) and synthetic antioxidant substances, allowing the determination of a variety of antioxidant substances⁷⁵. Additionally, ABTS antioxidant test may be used over a wide range of pH⁷⁶. The DPPH test was used due to its reproducibility and applicability at room temperature. This method also allows the understanding of various chemical phenomena⁷⁷. Based on the ABTS assay, the EC₅₀ values ranged from 0.297 to 0.510 mg/mL and 0.273 to 0.540 mg/mL for acetone and methanol extracts, respectively. The most potent effect was exerted by A. ferox methanol extract (0.273 mg/mL) while the methanol extract of C. annuum (0.540 mg/mL) had the least antioxidant potency (Table 6). This agrees with studies by Wintola and Afolayan⁷⁸, whereby a high ABTS radical scavenging activity was exerted by Aloe ferox methanol extract (EC₅₀ = 0.02 mg/mL). As a cationic radical, 2,2´-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) decreases in absorbance when scavenged with an extract that has antioxidant effect⁷⁹.

Table 6.

Antioxidant activity based on EC₅₀ values (mg/mL) using 2,2´-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) models of acetone and methanol extracts of the selected five plants.

Plant species	Plant part	ABTS		DPPH
		Acetone	Methanol	Acetone	Methanol
Aloe ferox Mill.	Leaves	0.297 ± 0.0053c	0.273 ± 0.0061c	0.321 ± 0.0093ab	0.358 ± 0.0353ab
Allium cepa L.	Bulb	0.389 ± 0.0232bc	0.314 ± 0.0089bc	0.411 ± 0.0379a	0.399 ± 0.0015a
Capsicum annuum L.	Fruit	0.402 ± 0.1227b	0.540 ± 0.1301a	0.458 ± 0.1717a	0.356 ± 0.0405ab
Tagetes minuta L.	Leaves and stem	0.510 ± 0.0994a	0.356 ± 0.0611b	0.120 ± 0.0109c	0.149 ± 0.0012c
Tulbaghia violacea Harv.	Whole plant	0.339 ± 0.1592c	0.303 ± 0.0042c	0.192 ± 0.0456c	0.185 ± 0.0094c

Standard positive control (Trolox) = 0.044 ± 0.0271 mg/mL (ABTS); = 0.355 ± 0.0058 mg/mL (DPPH). In each column, values presented as mean ± standard error (n = 3) with different letter(s) are significantly (p ≤ 0.05) different according to Tukey’s Multiple Comparisons Test.

Based on DPPH assay, the EC₅₀ values ranged from 0.120 to 0.458 mg/mL and 0.149 to 0.399 mg/mL for acetone and methanol extracts, respectively. Tagetes minuta acetone extract (0.1199 mg/mL) displayed the highest antioxidant activity, while C. annuum acetone extract (0.458 mg/mL) was the least potent among the tested plants (Table 6). Tagetes minuta demonstrated a higher antioxidant activity denoted with a lower EC₅₀ value in the DPPH model, this could be attributed to the presence of 3,5 or 4,5 di-caffeoylquinic and quinic acids which have been associated with antioxidant activity^26,69. Both T. minuta and T. violacea generally exerted a noteworthy antioxidant effect relative to the standard positive control in the DPPH model. The results indicated the electron scavenging ability of the phenolic antioxidants which rapidly quench the radicals⁸⁰.

The ABTS and DPPH models indicate electron scavenging⁷⁹ and hydrogen-donating⁸¹ abilities of antioxidants, respectively which are essential in neutralizing free radicals. The antioxidant effect could be attributed to the quantified phenolics and associated chemicals identified in the plant extracts²⁶. Likewise, the flavonoids in the evaluated plant extracts could contribute to their antioxidant potential. Existing evident suggest that flavonoids in plants may be responsible for their antioxidant activity²⁷. In the current study, the type of extracting solvents had a significant influence on the resultant antioxidant. The effects of acetone and methanol extracts were remarkably evident in the ABTS and DPPH models. The antioxidant activity results revealed that methanol extracts generally demonstrated higher antioxidant potential indicated with the lower EC₅₀ values particularly in ABTS model (Table 6). This is an indication that methanol extracts were more potent than acetone extracts for most of the investigated plants, especially for T. minuta and T. violacea.

Phytopathogen invasion causes oxidative stress in plants leading to the development and progression of diseases. To counter act oxidative stress, plants have a developed antioxidant system which includes enzymatic and non-enzymatic antioxidants for rapid scavenging of reactive oxygen species (ROS). The scavenging activity of antioxidants on ROS is key to plant disease management^82,83.

Antifungal effect of the evaluated botanicals

All the evaluated plants demonstrated statistically significant differences (p < 0.05) in concentrations with various potency levels of antifungal activities against P. ultimum. This was indicated by varying degrees of zones of inhibition, ranging from 11.67 to 22.33 mm and 14.67 to 22.67 mm for acetone and methanol extracts, respectively (Table 7). The highest zone of inhibition was exerted by T. minuta (22.67 mm) and A. cepa (22.33 mm) for acetone and methanol extracts, respectively. The antifungal activity of T. minuta was linked to its phenolic and flavonoid concentrations, as it displayed the highest concentrations for the phenolic and flavonoid contents in its acetone extracts. However, acetone extracts of A. ferox and A. cepa, as well as C. annuum methanol extracts did not display any inhibition against P. ultimum. Notably, that methanol extracts were more potent against the tested pathogen, this observation was similar to the pattern shown in the antioxidant test.

Table 7.

Antifungal activity (growth Inhibition in millimetres, mm) of methanol and acetone extracts of the five selected plants at 100 mg/mL against Pythium ultimum.

Plant species	Plant part	Methanol	Acetone
Aloe ferox Mill.	Leaves	11.67 ± 0.33d	0c
Allium cepa L.	Bulb	22.33 ± 2.03a	0c
Capsicum annuum L.	Fruit	0e	14.67 ± 1.76b
Tagetes minuta L.	Leaves and stem	14.67 ± 1.76c	22.67 ± 1.45a
Tulbaghia violacea Harv.	Whole plant	16 ± 1.53b	0c

In each column, values presented as mean ± standard error (n = 3) for growth inhibition (mm) followed by the different letter(s) are significantly (p < 0.05) different according to Tukey’s Multiple Comparisons Test.

Lindsey et al.⁸⁴ reported a promising antifungal activity of T. violacea extract against P. ultimum, demonstrating 41 and 10% inhibition of the fungal growth at 50 and 10 mg/mL, respectively. The antifungal activity of many plants in South Africa have been tested against P. ultimum³⁷. However, most of the plants lacked promising antifungal activity. For instance, the study by Eloff et al.⁴⁰ evaluated Melianthus comosus extract against P. ultimum, and the methanol extract of the plant had weak antifungal activity with minimum inhibitory concentration (MIC) of 1.25 mg/mL. Furthermore, Mdee et al.⁵ investigated the antifungal effect of seven plants including Campuloclinium macrocephalum, Cestrum laevigatum, Datura stramonium, Lantana camara, Nicotiana glauca, Ricinus communis and Solanum mauritianum against P. ultimum. The findings revealed moderate to weak antifungal activity with MIC ranging from 0.63 to 2.5 mg/mL.

In the poisoned food method, the methanolic extracts displayed various potency levels of antifungal activity against B. cinerea, indicated by varying degrees of mycelium growth inhibition which ranged from 5 to 62.4% (Table 8). Among the evaluated plants, T. violacea had the highest inhibition (62.4%) of mycelial growth at 30 mg/mL (Fig. 3). This agrees with findings by Lindsey et al.⁸⁴ who evaluated the antifungal effect of T. violacea extract. The antifungal activity against B. cinerea revealed 51 and 28% mycelial growth inhibition at 50 and 10 mg/mL, respectively. The high concentrations of phenolics and flavonoids observed in T. violacea and T. minuta methanol extracts could be responsible for the promising antifungal activity^28,57.

Table 8.

Antifungal activity (mycelial growth inhibition, %) of methanol extracts of the five selected plants at three (10, 20 and 30 mg/mL) concentrations against Botrytis cinerea.

Plant species	Plant part	10 mg/mL	20 mg/mL	30 mg/mL
Aloe ferox Mill.	Leaves	5 ± 1.30c	7.5 ± 0.50c	12.5 ± 1.32d
Allium cepa L.	Bulb	0d	0d	9.7 ± 0.61d
Capsicum annuum L.	Fruit	0d	10.3 ± 1.21c	27.3 ± 1.05c
Tagetes minuta L.	Leaves and stem	25.5 ± 1.70a	33 ± 0.50b	42.5 ± 3.28b
Tulbaghia violacea Harv.	Whole plant	15.2 ± 1.21b	44.8 ± 0.61a	62.4 ± 1.60a

Positive control (Efeckto lawn fungicide) = 100% mycelial growth inhibition at 5 mg/mL. In each column, values (n = 3) for mycelial growth inhibition (%) followed by different letter(s) are significantly different (p < 0.05) according to Tukey’s Multiple Comparisons Test.

Fig. 3.

Examples of mycelial growth inhibitions for Botrytis cinerea with Tagetes minuta methanol extracts at (A) 10 mg/mL (25.5%), (B) 20 mg/mL (33%), and (C) 30 mg/mL (42.5%). Examples of mycelial growth inhibition for Botrytis cinerea on (D) negative control (0%).

All the evaluated plant extracts contain coumarins and saponins which have been linked with antifungal effects against phytopathogenic fungi^85,86. In addition, neorustmicin B, surfactin C, 5-hydroxy-7,4′-dimethoxyflavone, 3-O-p-coumaroylquinic acid and quercetin-3-O-galactoside which are present in the evaluated plant extracts are associated with antifungal activity against phytopathogens such as Puccinia graminis f. sp. tritici, Pseudomonas syringae pv tomato and Fusarium species^71–74.

Potency ranking

Based on the antioxidant and antifungal effects demonstrated by the evaluated plants in this study, T. violacea was ranked the most potent followed by T. minuta (Table 9). This is likely linked to the high concentrations of phenolics and flavonoids in these different extracts^56,57, as well as 3, 4 or 4, 5 di-caffeoylquinic acid (T. minuta) and quinic acid (T. minuta) which are known for their antioxidant and antimicrobial effects^26,69. Capsicum annuum displayed the highest flavonoid concentration, however, it was the least potent among the five evaluated botanicals. This may be due to C. annuum having less concentrations of the relevant bioactive compounds to produce significant antioxidant and antifungal properties, thereby affecting its ranking in this study. Additionally, stage of maturity of the plant/plant part as well as its harvesting season may be a factor influencing the resultant biological effects^87–89. The high potency ranking of T. violacea agrees with its wide applications in folk medicine. For instance, it has been associated with treating earache, fever and high blood pressure⁹⁰, its leaves are used as culinary herbs⁹¹, as well as having insecticidal⁹², and antifungal properties against some plant pathogenic fungi⁸⁴.

Table 9.

Potency ranking of the evaluated five plants based on seven (7) factors (acetone ABTS, methanol ABTS, acetone DPPH, methanol DPPH, acetone Pythium, methanol Pythium, and methanol Botrytis). ABTS = 2,2´-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid); DPPH = 2,2-diphenyl-1-picrylhydrazyl.

Plant species	*Potency index	Potency ranking
Tulbaghia violacea Harv.	2.8	1
Tagetes minuta L.	3.4	2
Aloe ferox L.	4.0	3
Allium cepa L.	4.8	4
Capsicum annuum L.	5.4	5

*Potency index = sum of ranking/total number of plants (5).

Conclusion

The comprehensive phytochemical profiles, antioxidant and antifungal effect of five tested botanicals were generated. The varying concentrations of phenolics and flavonoids likely accounted for the observed biological effects. Neorustmicin B (C. annuum), surfactin C (A. cepa) and quinic acid (T. minuta), 1-O-feruloylglucose (C. annuum), 3, 4 or 4, 5 di-caffeoylquinic acid (T. minuta), identified through LC-MS are noteworthy due to their diverse antioxidant and antimicrobial activities. Overall, T. violacea and T. minuta were considered as the most potent among the five evaluated plants. The current findings indicate the great potential of the evaluated botanicals as an alternative source of bio-fungicides for managing P. ultimum and B. cinerea related plant diseases. However, to fully understand their potential, in vivo evaluation and application of their phytochemical compounds are required. As in vitro effect does not necessarily translate to in vivo efficacy, the need for further experimentations cannot overemphasised. Even though in vivo studies are costly, it remains essential to establish and generate real-life empirical data on ethnobotanical-driven approach and the increasing efforts aimed at developing botanical-based antifungal for plant protection.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

AOA, OAF, BPK and WO-M assisted JLM in conceptualizing the research. JLM and TMP performed the experiments. The data was analyzed by JLM as well as preparing the manuscript. AOA, WO-M, OAF and BPK supervised the project and edited the final manuscript.

Funding

Research funding was provided by the National Research Foundation (NRF) of South Africa, with grant numbers 135452 (WO-M), SPAR231013155231 (OAF), CPRR23033088376 (OAF) and SRUG2204224395 (AOA). In addition, AOA received partial funding from the South African Research Chairs Initiative of the Department of Science, Technology and Innovation (DSTI)-National Research Foundation (NRF) of South Africa (Grant No: RCHDI2411105279212) to support the project.

Data availability

Data used for this study have been included as part of the article.

Declarations

Competing interests

The authors declare no competing interests.

Institutional review board statement

This study was approved with reference number NWU-00530-21-A9 by the Faculty of Natural and Agricultural Sciences Research Ethics Committee (FNASREC), North-West University, South Africa.