Results

Chapter 6: Effects of some botanicals on growth and phytochemistry of nematode-infected

6.3. Results

157

158

2.5 mgmL^-1 and 10 g/pot treatments over control plants. All growth parameters were significantly increased in nematode-infected plants that were treated with C. maxima (T8) when compared with nematode-infected control plants (Table 6.1).

159

Table 6.1. Effects of application of different botanicals on growth of Pelargonium sidoides 56-days after nematode application.

Treatments Plant species No. of leaves Leaf area

(cm²) Shoot length

(cm) Fresh root

weight (g) Fresh shoot

weight (g) Dry root

weight (g) Dry shoot weight (g) 10 g T1 104±4.01^a 49.14±0.78^e-i 32.43±0.57^b-d 12.39±0.49^h-j 89.42±2.52^a 2.86±0.18^c 12.54±0.29^a

T2 34±0.27^i-m 57.75±0.57^b-f 24.33±1.37^g-k 13.78±0.84^g-j 34.99±2.69^e-i 3.18±0.26^g-i 4.96±0.85^f-i T3 43±2.13^d-h 68.66±1.37^b-d 33.33±1.02^bc 16.29±4.15^d-i 39.66±1.93^d-g 3.76±0.33^d-i 6.01±0.19^d-g T4 60±7.31^b 49.60±1.02^e-i 29.53±0.79^c-f 15.04±2.38^f-j 42.09±2.99^de 3.47±0.11^e-i 6.56±0.13^d T5 42±1.25^e-i 55.32±0.79^c-f 21.50±0.29^jk 22.98±2.09^b-d 39.62±1.07^d-h 5.30±0.11^a-d 4.69±0.39^g-j T6 33±0.94^j-m 41.94±0.29^f-j 30.17±1.03^c-e 13.66±3.27^g-j 27.38±5.08^i-k 3.15±0.47^g-i 5.35±0.18^d-h T7 40±0.72^f-j 71.28±1.03^a-c 38.13±0.33^a 30.04±3.35^a 54.95±1.28^bc 6.93±0.54^a 8.44±0.56^c T8 49±4.97^c-e 86.62±0.33^a 33.47±0.35^bc 15.20±0.99^e-j 54.57±2.23^bc 3.51±0.71^e-i 8.97±0.34^bc 5.0 mgmL^-1 T1 35.00±2.83^h-m 49.67±3.93^e-i 22.43±0.42^i-k 18.53±0.97^c-h 34.99±3.24^e-i 4.28±0.22^c-h 5.73±0.23^d-h

T2 38.00±3.30^g-l 38.90±4.66^g-j 25.73±1.43^f-i 20.64±3.17^b-g 39.36±1.71^d-h 4.76±0.73^b-g 4.92±0.35^f-i T3 36.00±0.94^h-m 48.39±1.82^f-i 22.23±0.43^i-k 30.20±3.93^a 41.98d±2.03^e 6.97±0.50^a 5.37±0.91^d-h T4 38.00±0.94^g-l 35.29±1.59^h-k 23.33±±0.97^h-k 11.03±0.78^ij 30.87±0.50^h-j 2.54±0.09ⁱ 3.76±0.18^ij T5 28.67±3.07^m 42.28±4.22^f-t 26.63±0.57^e-h 14.80±1.93^f-j 36.41±4.51^d-h 3.42±0.11^e-i 4.91±0.45^f-i T6 42.00 ±1.41^e-i 48.42±1.25^f-i 21.37±1.51^j-l 22.11±0.76^b-e 38.41±1.52^d-h 5.10±0.26^b-e 4.82±0.18^g-i T7 48.67±3.54^c-e 34.88±0.85^i-k 21.87±1.48^i-k 14.35±2.35^f-j 40.82±3.78^d-f 3.54±0.54^e-i 5.08±0.73^e-i T8 52.00±1.70^bc 47.81±4.04^f-i 31.90±0.38^cd 21.38±2.57^b-f 42.06±2.3^de 4.93±0.59^b-f 6.21±0.59^d-f 2.5 mgmL^-1 T1 45.67±2.13^c-g 51.48±4.32^e-h 33.27±0.43^bc 25.47±0.78^a-c 53.72±2.05^c 5.87±0.18^a-c 8.41±0.29^c

T2 48.00±1.41^c-f 65.13±5.22^b-e 36.27±3.04^ab 26.97±1.12^ab 62.87±4.43^b 6.23±0.26^ab 9.84±0.85^b T3 35.00±3.30^h-m 51.38±0.77^e-h 27.43±0.63^e-g 11.82±1.42^h-j 35.39±3.06^e-i 2.73±0.33^hi 6.42±0.19^d-e T4 46.67±1.66^c-g 35.55±2.06^h-k 24.73±0.93^g-k 12.23±0.47^h-j 42.80±1.39^de 2.82±0.11^hi 5.84±0.13^d-g T5 51.00±3.29^b-d 42.62±0.37^f-j 24.43±2.30^g-k 21.38±0.48^b-f 46.27±4.36^cd 4.93±0.11^b-f 6.33±0.39^de T6 29.67±0.72^lm 72.67±3.07^ab 23.97±0.79^g-k 12.45±2.03^h-j 31.30±1.05^g-j 2.88±0.47^hi 4.42±0.18^h-j T7 39.67±1.19^f-j 47.12±8.24^f-i 23.63±0.1.63^g-k 14.04±2.5^g-j 33.16±0.98^f-i 3.24±0.54^f-i 5.51±56^d-h T8 30.00±2.16^j-m 54.91±4.02^d-g 29.07±1.06^d-e 19.90±3.08^c-g 34.061.85^e-i 4.59±0.71^b-g 5.49±0.34^d-h Control N 19.00±1.24ⁿ 21.90±2.09^k 17.47±0.59^l 8.84±1.08^j 19.15±1.15^k 2.78±0.21^hi 1.28±0.15^k Control UNI 38.67±1.91^g-k 29.94±3.57^jk 20.63±0.78^kl 16.70±1.43^d-i 23.96±1.47^jk 3.85±0.33^d-i 3.32±0.27^ij Columns represent mean ±standard error, n = 5. Different letter(s) indicate significant differences (p<0.05) between treatments as separated by Duncan’s Multiple Range Test. T1 = P. sidoides, T2 = P. africana, T3 = S. aculeastrum, T4 = V. colorata, T5 = S. lancea, T6 = M. plumbea, T7 = C. sylvaticus, T8 = C. maxima, N = nematode-infested control (Untreated control) and UNI = uninoculated control (nematode free plants).

160

6.3.2. Effect of botanicals in chlorophyll and carotenoid contents

Chlorophyll and carotenoid contents were enhanced in all treated plants (Table 6.2). In the inoculated plants, reduction in Chlorophyll a+b and carotenoid contents were 70 and 51%, respectively, when compared to uninoculated (Table 6.2). Furthermore, there was a significant enhancement in plant pigmentation (chlorophyll a, b, and a+b and carotenoid) of P. sidoides plants treated with either C. maxima crude seed extracts or powdered treatments.

6.3.3. Effect of botanicals on biochemical attributes of African geranium

The protein, superoxide dismutase (SOD) and total phenolics content was increased in nematode-infected control by 1.45, and 1.45-fold and 331%, respectively, compared to the nematode free plants, with a reduction in carbohydrate content by 1.25-fold (Table 6.3). Relative to the untreated control, all tested botanicals yielded low phenolic levels. However, a slight increase was produced in plants treated with 5.0 mgmL^-1 of C. sylvaticus (T7). On the other hand, seedlings treated with C. maxima (T8) at 5.0 mgmL^-1 and 10 g significantly reduced the biochemical content, however, stimulated protein, SOD, and carbohydrate contents were observed on plants treated with 2.5 mgmL^-1 (Table 6.3).

161

Table 6.2. Effect on Chlorophyll a, b, and a + b and carotenoid contents when plants were treated with different powered plant material against root-knot nematodes.

Trt Chlorophyll a µg g^-1 FW

Chlorophyll b µg g^-1 FW

Chlorophyll a+b µg g^-1 FW

Carotenoid µg g^-1 FW

Chlorophyll a µg g^-1 FW

Chlorophyll b µg g^-1 FW

Chlorophyll a+b µg g^-1 FW

Carotenoid µg g^-1 FW

Chlorophyll a µg g^-1 FW

Chlorophyll b µg g^-1 FW

Chlorophyll a+b µg g^-1 FW

Carotenoid µg g^-1 FW

GPM (10 g) 2.5 mgmL^-1 5.0 mgmL^-1

N 353±0.6^f 108±0.2^c 461±0.5^f 105±0.3^d 353±0.6^h 108±0 2^d 461±0.5^f 105±0.3^h 353±0.6^h 108±0.2^f 461±0.5^f 105±0.3^d U 920±22^d 598±29^ab 1518±50^de 215±1.4^e 920±22^d 598±29^a 1518±50^bc 215±1.4^f 920± 22^f 598±29^bc 1518±50^cd 215±1.4^c T1 1029 ±23^b 571±35^ab 1601±58^cd 314 +2.2^ab 944±27^cd 613±61^a 1557±87^abc 245±1.4^d 1057±3^cd 418±2^de 1475±4^d 263±0.6^b T2 1178±19^a 706±51^a 1884±70^a 338±11.7^a 1104±15^a 594±37^a 1698±52^a 273±0.2^b 770±21^g 501±48^cd 1271±70^e 230±0.8^c T3 974±8^c 551±22^b 1525±29^de 293±0.7^b 1005±24^bc 611±54^a 1617±78^ab 264±0.3^c 1219±7^a 777±73^a 1996±65^a 318±19^a T4 1012±5^b 697±84^a 1710±83^bc 243±21 5^cd 725±36^g 301±13^c 1027±23^e 227±3.7^e 1126±10^b 687±24^ab 1814±34^b 317±1.5^a T5 1057±11^b 673±29^ab 1731±39^abc 322±1.1^a 889±1^de 535±2^a 1424±2^c 233±0.1^e 982±23^e 625±54^bc 1607±76^cd 259±2.7^b

T6 814±8^e 585±18^ab 1399±26^e 222±0.4^de 819±10^f 416±19^b 1236±28^d 244±5.4^d 878±1^f 345±1^e 1223±1^e 271±0.1^b

T7 877±9^d 571±27^ab 1448±36^de 251±1.4^c 849±18^ef 571±43^a 1421±61^c 207±1.7^g 1072±10 ^c 564±29^c 1637±39^c 310±0.6^a T8 1165±18^a 686±49^ab 1852±67^ab 336±3 1^a 1018±9^b 649±22^a 1667±30^ab 282±0.9^a 1015±10^de 537±18^cd 1552±26^cd 267±0.9^b

Columns represent mean ±standard error, n = 3. Different letter(s) indicate significant differences (p<0.05) between treatments as separated by Duncan’s Multiple Range Test. T1 = P. sidoides, T2 = P. africana, T3 = S. aculeastrum, T4 = V. colorata, T5 = S. lancea, T6 = M. plumbea, T7 = C. sylvaticus, T8 = C. maxima, N = nematode-infested control (Untreated control) and U = uninoculated control (Nematode free plants).

162

Table 6.3. Effects of different botanical application on activity of phenols, SOD, protein and carbohydrate contents in Pelargonium sidoides roots.

Trt

Protein µg g^-I FW

Carbohydrate µg g^-I FW

SOD Unit/mg FW

Total phenolic (mg g−1

Protein µg g^-I FW

Carbohydrate µg g^-I FW

SOD Unit/mg FW

Total phenolic (μg mg−1

Protein µg g^-I FW

Carbohydrate µg g^-I FW

SOD Unit/mg FW

Total phenolic (μg mg⁻¹

2.5 mgmL^-1 5 mgmL^-1 GPM (10 g)

UNI 8.6±0.03^f 20.2±0.15^cd 0.011±0.001ⁱ 6.05±0.29^i-k 8.6±0.03^f 20.2±0.15^d 0.011±0.001ⁱ 6.05±0.29^i-k 8.6 ± 0.03^f 20.2±0.15^a 0.011±0.001ⁱ 6.05±0.29^i-k N 12.5±0.03^abc 16.2±0.09^d 0.016±0.001^d 26.07±2.11^b 12.5±0.03^a 16.2±0.01^e 0.016±0.001^g 26.07±2.11^b 12.5±0.03^b 16.2±0.09^a 0.016±0.001^f 26.07±2.11^b T1 8.1±0.05^f 21.1±1.09^cd 0.032±0.001^a 6.22±0.26^ij 9.5±0.05^e 35.3±0.46^ab 0.017±0.001^f 25.75±5.26^b 6.9±0.05^h 14.0±1.30^a 0.024±0.001^e 9.64±5.64^gh T2 13.2±0.05^ab 21.8±0.10^cd 0.020±0.001^c 4.37±0.06^jk 12.0±0.03^b 39.1±4.52^a 0.039±0.001^b 8.07±0.99^hi 10.8±0.05^f 16.7±0.09^a 0.044±0.001^a 5.89±0.54^i-k T3 11.9±0.05^bcd 63.2±0.59^a 0.011±0.001^e 4.08±0.09^jk 12.6±0.03^a 23.2±0.85^c 0.042±0.001^a 19.82±3.77^de 10.2±0.05^g 14.8±0.56^a 0.029±0.001^c 11.92±2.28^fg T4 10.5±0.03^de 29.1±8.40^bc 0.011±0.001^e 6.12±0.33^i-k 9.7±0.03^e 12.7±0.63^e 0.035±0.001^c 8 22±0.89^hi 11.0±0.03^e 13.3±0.64^a 0.036±0.001^b 13.20±2.34^f T5 11.2±0.03^cde 14.7±0.70^d 0.001±0.001^h 11.51±0.56^fg 11.2±0.03^d 38.9±1.79^a 0.032±0.001^d 12.98±0.86^f 12.7±0.1^a 15.7±2.00^a 0.025±0.001^e 13.17±0.24^f T6 9.6±0.03^ef 19.6±0.46^d 0.003±0.001^f 4.87±0.20^jk 9.7±0.07^e 31.1±0.78^b 0.020±0.001^e 12.24±3.32^f 12.3±0.03^c 17.6±4.24^a 0.025±0.001^e 21.54±2.83^cd T7 10.3±0.03^de 34.5±0.78^b 0.002±0.001^g 3.64±0.21^l 11.4±0.03^d 15.3±1.02^e 0.016±0.001^g 29.98±4.12^a 11.5±0.01^d 13.1±3.64^a 0.027±0.001^d 17.82±3.89^e T8 13.9±1.70^a 16.7±0.09^d 0.021±0.001^b 11.28±0.27^fg 11.8±0.03^c 15.2±0.09^e 0.012±0.001^h 8 31±0.15^hi 12.7±0.03^a 14.0±6.12^a 0.011±0.001^g 9.49±0.60^gh

Columns represent mean ±standard error, n = 3. Different letter(s) indicate significant differences (p<0.05) between treatments as separated by Duncan’s Multiple Range Test. T1 = P. sidoides, T2 = P. africana, T3 = S. aculeastrum, T4 = V. colorata, T5 = S. lancea, T6 = M. plumbea, T7 = C.

sylvaticus, T8 = C. maxima, N = nematode-infested control (Untreated control), UNI = uninoculated control (nematode free plants) and SOD = superoxide dismutase.

163 6.3.4. Effect of botanicals on nematode infection

Effects of the botanicals and application methods were highly significant on the second-stage juveniles (J2s) in soil, eggs and J2s in roots and reproduction factor (Rf), contributing 17, 2, 1 and 5% in total treatment variation (TTV) of the four variables, respectively (Appendix 6.2).

The treatments had highly significant effects on number J2s in soil, number of eggs and J2s in roots and reproduction factor (Rf), with botanicals contributing 63, 96, 97 and 88% of TTV, respectively, while the application methods contributed 20, 1, 1 and 6%, respectively, in TTV of the four variables. Hence, Table 6.4 shows that treatments significantly reduced nematode infection in P. sidoides after 56-days of infestation. The application of C. maxima seeds as either crude extract (drenching) or ground meal reduced the number of eggs and juveniles by 97 and 90%, and 97 and 96%, respectively, at 5 mgmL^-1 and 10 g compared to the untreated control.

The numbers of second-stage juveniles (J2s) and eggs on the root system of P. sidoides were less than that of untreated control; hence, similar trends were noted on the numbers of J2s in soil.

Potted soil treated with 10 g of P. africana, S. aculeastrum, V. colorata, M. plumbea, C.

sylvaticus and C. maxima had the lowest numbers of juveniles.

164

Table 6.4. Effect of botanical applications on nematode parameters after 56-days of inoculation with an initial population of 1 000 ± eggs and juveniles.

Trt 5 mgmL^-1 2.5 mgmL^-1 10 g

Eggs J2s in roots J2s in soil Eggs J2s in roots J2s in soil Eggs J2s in roots J2s in soil

N 413±2.96^a 967±4.16^a 1060±4.62^b 413±2.95^a 967±544.33^a 1060±161.19^b 413±73.63^a 967±4.33^a 1060±161.19^b T1 47±2.35^c 133±2.19^c-f 40±1.77^c 20±1.35^c 120±3.10^c-g 80±2.33^e 47 ±14.40^c 193±15.85^b-d 353 ±23.72^c T2 53±2.36^c 73±1.66^e-g 47±1.67^c 27±1.67^c 20±1.35^fg 167±2.13^cd 26 ±5.44^c 27 ±18.85^fg 48 ±14.40^e T3 47±1.45^c 27±1.67^fg 60±1.77^c 33±1.45^c 7±1.02^g 47±1.67^e 7 ±5.44^c 153±28.28^c-e 60±5,44^e T4 47±2.33^c 100±2.13^d-g 33±1.45^e 47±2.42^c 40±1.91^e-g 87±2.52^e 27±5.34^c 27±3.99^fg 80 ±19.63^e T5 43±2.52^b 227±1.66^bc 100±2.81^d 27±1.46^c 67±1.65^e-g 120±1.77^d 193±23.73^b 300±12.29^b 1607 ±24.94^a T6 47±1.94^c 93±1.78^d-g 40±1.35^e 27±2.14^c 87±2.35^d-g 33±1.66^e 33 ±14.40^c 13 ±28.80^g 53 ±5.44^e T7 7±1.67^c 47±1.02^e-g 113±1.94^d 40±2.28^c 107±2.05^d-g 80±1.91^e 47 ±21.77^c 67 ±18.86^e-g 60 ±23.73^e T8 13±1.23^c 27±1.21^fg 126±3.90^d 53±2.19^c 40±21.35^e-g 73±1.05^e 40 ±24.94^c 48 ±21.77^e-g 27 ±10.89^e Columns represent mean ±standard error, n = 5. Different letter(s) indicate significant differences (p<0.05) between treatments as separated by Duncan’s Multiple Range Test. T1 = P. sidoides, T2 = P. africana, T3 = S. aculeastrum, T4 = V. colorata, T5 = S. lancea, T6 = M. plumbea, T7 = C. sylvaticus, T8 = C. maxima and N = nematode-infested control (Untreated control).

165

6.3.5. Gas chromatography-mass spectroscopy (GC-MS) profile of C. maxima seeds crude extracts

The gas chromatography-mass spectroscopy (GC-MS) of the C. maxima seeds confirmed the presence of various bioactive compounds with different peak area percentages and retention time (Table 6.5; Figure 6.2a-c). The GC-MS chromatograms of the extract further revealed the presence of 141 compounds collectively in crude extracts of EtOAc, DCM, and PE, which manifest the presence of several classes of compounds like alkenes, aromatic hydrocarbon, fatty acids, fatty alcohols, esters, alkenes, aldehydes among others, of which 42, 49 and 50 of the compounds were found in EtOAc, DCM, and PE, respectively, with 13 compounds previously reported with nematicidal attributes (Table 6.6; Figure 6.3).

Figure 6.2a.Chromatogram of Petroleum ether (PE) Cucurbita maxima seed extracts obtained from GC-MS.

166

Figure 6.2b.Chromatogram of Dichloromethane (DCM) Cucurbita maxima seed extracts obtained from GC-MS.

167

Figure 6.2c. Chromatogram of Ethyl acetate (EtOAc) Cucurbita maxima seed extracts obtained from GC-MS.

169

23 8.318 Benzene, 1,2,4,5-

tetramethyl- 0.03 - - Aromatic

hydrocarbon

24 8.592 Benzene, 1,3-diethyl-5-

methyl- 0.01 - - Aromatic

hydrocarbon

25 8.738 1,3-Cyclopentadiene,

1,2,3,4-tetramethyl-5- methylene-

0.04 - - Alkene

26 8.806 Hexadecane, 1-chloro- 0.03 - - Aliphatics,

halogenated

27 8.889 Benzene, (1,2,2-

trimethylpropyl)-

0.03 - - Tricyclic

aromatic compound

28 9.263 Dodecane 0.08 - - Alkane

29 9.360 9.264 9.391 Octanoic acid 0.07 0.93 0.02 Medium-chain fatty acid 30 10.097 10.119 10.122 2-Decenal, (E)- 0.22 15.20 0.18 Medium-chain

aldehyde

31

11.223 11.296 12.320 13.755 14.735

2-methyltetracosane 3.85 - - Branched alkane

32 11.646 Tetradecane 0.79 - Alkane

33

12.167 12.214 12.830 13.566 13.795 13.944 13.981 14.038 14.146 14.195 14.351 14.427 14537 14.591 14.679 14.803 14.906 14.967 15.017 15.051 15.211 15.280 15.303 15.425 15.586 15.642 15.687 15.762

11.551 12.420 12.964 13.021 13.739 13.971 14.183 14.390 14.897 15.118 15.380 15.650 15.874 15.950 16.585 17.421 24.153

9,12-Octadecadienoic

acid (Z,Z)- 34.66 - 73.54 Lineolic acids and derivatives;

long-chain fatty acid

34 12.355 Naphthalene, decahydro-

1,5-dimethyl- 0.03 - - Aromatic

hydrocarbon

170 35

12.405 12.870 12.940 13.525 13.827 13.913 15.258

11.636 12.651 12.720 13.280 14.582 15.250 16.218

cis-13,16-Docasadienoic

acid 4 - 13.67 Long-chain

fatty acid

36 12.469 Z,E-3,13-Octadecadien-

1-ol

0.11 - - Fatty alcohol

37 12.525 Methyl 2-

octylcyclopropene-1- heptanoate

0.10 - -

38 12.599 16.717 cis-9-Hexadecenal 0.19 5.93 Monounsaturated

fatty aldehydes

39 12.728 Heptadecane, 2,6,10,15-

tetramethyl- 1.12 - - Long-chain

alkane

40 12.770 9,17-Octadecadienal,

(Z)- 0.08 - - Unsaturated

aldehyde

41 12.800 Isopropyl linoleate 0.13 - - Fatty acid ester

and an isopropyl ester

42 13.005

13.051 23.999 Oxacycloheptadec-8-en-

2-one, (8Z) 1.33 - 0.06 Alcohol

43 13.060 E,E,Z-1,3,12-

Nonadecatriene-5,14-diol

3.10 - - Fatty alcohol

44 13.180 23.140

23.200 23.468

Z,Z-8,10-Hexadecadien-

1-ol 3.86 - 0.34 Fatty alcohol

45 13.439 cis-7-Tetradecen-1-ol 0.26 - - Fatty alcohol

46 13.667 Oleyl alcohol,

heptafluorobutyrate 0.97 - - Ester

47 13.755 cis-11,14-Eicosadienoic

acid, methyl ester 0.58 - - Fatty acid methyl ester

48 13.685 (R)-(-)-14-Methyl-8-

hexadecyn-1-ol 0.44 - -

49 15.150 17-Octadecynoic acid 3.81 - - Acetylenic

fatty acid

50 15.797 n-Pentadecanol 0.87 - - Long-chain fatty

alcohol

51 15.930 6-Octadecenoic acid, (Z)- 1.37 - - Long-chain fatty

acid 52 16.125

17.377 Heptadecanoic acid,

heptadecyl ester 0.39 - - Fatty acid ester

53

16.161 16.200 16.225 16.257 16.345

17.173 Octadecanoic acid 2.10 2.69 Long-chain fatty acid

54

19.020 19.165 19.235 19.556 19.630 19.761

19.055 l-(+)-Ascorbic acid 2,6-

dihexadecanoate 13.32 13.85 - Ester