Botanical extracts against Phytopthora infestans and productivity of tomato -

Botanical extracts against Phytopthora infestans and productivity of tomato

Control of late blight is difficult because P. infestans has advanced and complex enzymes and effecter molecules coded by avirulence genes. Increasing concentration of the botanical extracts significantly reduced radial growth and increased percent inhibition of P. infestans. In addition, increasing botanical extract concentration significantly increased peroxidase (PPD), phenylalanine ammonia lyse (PAL), and polyphenol oxidase (PPO) activity. Disease incidence and severity were significantly reduced as the concentration of the botanical extracts increased. From the research, it can be concluded that M. oleifera ethyl acetate, E. nigra ethyl acetate, and L. camara water extracts contain antifungal compounds and can be used as biofungicides in late blight management.

Extraction of Botanical Crude Extracts from Plant Samples:

Crude extracts from plant samples were extracted from six-week-old young leaves. Plant samples were washed under running tap water and rinsed in distilled water. All the plant materials were air dried in the shade for two weeks and ground to powder using a hammer mill grinder. Sequential extractions were performed on 140 g of each plant powder by soaking them in 50% of analytical grade (AR) acetone, 50% of ethyl acetate (AR), and sterile distilled water (control) for 24 hours, respectively crude extracts were filtered using cheese cloth. Botanical extracts were shaken for 24 hours at room temperature on an orbital shaker at 100 rpm. Organic solvents in the supernatant were removed at 56°C and 77°C for acetone and ethyl acetate, respectively, using a rotary evaporator biobase. Thereafter, water plant filtrates were concentrated to powder using a freeze drier. The stock solutions of 100 mg of crude extracts per 100 ml of distilled water were prepared, and all the extracts were stored in the refrigerator at 4°C until use. Different concentrations (1.0, 2.5, and 5.0 mg/ml) of each stock solutions were added to 20 ml of potato dextrose agar (PDA) into 90mm diameter Petri dishes.

The P. infestans cultures were morphologically identified using a stereomicroscope, subcultured, and stored in a refrigerator at 4°C until use. Seedlings (cultivar rodade) from Emerald Seedling Company were grown in a green house. Red soil sterilized in an oven at 100°C for 24 hours to kill soilborne pathogens. The tomato seedlings were transplanted in perforated black polythene bags with dimensions of 21 cm height, 15 cm top diameter, and 11.5 cm bottom diameter. A total of 168 polythene bags were filled with sterilized red soil up to a height of 15 cm. The sterilized soil was thoroughly mixed with fungal inoculum adjusted to a concentration of 2.5 ×104 spores/ml using a haemocytometer. Thereafter, the different botanical extract treatments and the control were sprayed on the S. esculentum seedlings at two weeks after transplanting (WAT). Treatments were applied by spraying the extract solution on the tomato foliage to runoff, using a handheld sprayer calibrated to discharge at a spray rate of 100 litres/ha. Pots were watered daily with 500 ml of water using a perforated cup. Basal fertilizer application was done at planting at a rate of 40 g/pot using compound C (5% N: 15% P₂P₅: 12% K₂P) to achieve an application rate of 800 kg/ha. The S. esculentum plants were top-dressed with ammonium nitrate (34.5% N) which was split applied (5 g/pot) at 3 and 6 WAT to achieve an application rate of 400 kg/ha.

Effect of botanical extracts on the activity of polyphenol pxidase (PPP), phenylalanine ammonia lyse (PAL) and peroxidase (POD) on young S. esculentum leaves. Newly grown leaves of tomatoes were removed from the plant using a scalpel blade every week for six weeks and used in the enzyme assays. The young leaves were ground in liquid nitrogen using a pestle and mortar, and 2.5 g of homogenate was mixed with 5 ml of 0.05M sodium phosphate buffer (pH 6.0) containing 5% polyvinylpolypyrrolidone. The homogenate was filtered through four layers of muslin cloth, and the filtrate was centrifuged at 13,000 rpm for five minutes at 4°C.

Results

Laboratory Experiment: Effect of Botanical Extracts on Radial Growth of P. infestans.

There was a significant interaction among time × concentration × botanical extract on radial growth (cm) of P. infestans. E. nigra ethyl acetate, M. oleifera, and L. camara water extracts showed a concentration-dependent reduction of radial growth of P. infestans. There was a significant increase of radial growth as time increased from 3 to 12 days after incubation. Maximal reduction of radial growth was obtained at 2.5 and 5 mg/ml botanical extract concentration of L. camara water and M. oleifera ethyl acetate, respectively. Lantana camara water extract significantly elicited the lowest radial growth as compared to M. oleifera water, E. nigra water, and E. nigra acetone extracts. Complete inhibition of P. infestans was observed on L. camara water extracts, where 5 mg/ml of botanical concentration was used.

Effect of Crude Botanical Extracts on Percent Inhibition of P. infestans.

The interaction between botanical extract × extraction solvent × extract concentrations on the percent (%) inhibition of P. infestans was significant. The percent inhibition was significantly lower in E. nigra water extracts at 12 days of incubation compared to the other botanical extracts. The percent inhibition was significantly reduced as the days of incubation (DOI) increased. On the other hand, the extract concentration of 5 mg/ml significantly increased the percent inhibition of P. infestans.

Greenhouse Experiment

Effect of Botanical Extracts on POD

POD activity significantly increased as the time progressed from 1 to 6 WAT. There was a significant increase in POD activity as the concentration of botanical extracts increased from 1.0 to 5.0 mg/ml in M. oleifera ethyl acetate, E. nigra ethyl acetate, and all L. camara extracts. However, E. nigra and M. oleifera water extracts showed a concentration-dependent reduction in the activity of POD.

Effect of Botanical Extracts on PPP

M. oleifera ethyl acetate, E. nigra ethyl acetate, and L. camara extracts extracts at 1 mg/ml significantly reduced the activity of PPO. For most (except M. oleifera and E. nigra water) extracts, there was a concentration-dependent increase in PPO activity. Conversely, PPO activity decreased in the negative control (distilled water).

Effect of Botanical Extracts on PAL

The increasing the extract concentration from 1 to 5 mg/ml significantly increased PAL activity in plants treated with M. oleifera ethyl acetate, E. nigra ethyl acetate, and L. camara extracts extracts.

Effect of Botanical Extracts on the Leaves, Stem, and Root Biomass

Extracts from M. oleifera ethyl acetate, E. nigra ethyl acetate, and L. camara water extracts showed a concentration-dependent increase of the leaves, stem, and root biomass. Highest biomass on the leaves, stems, and roots was obtained where plants were treated with 2.5 and 5 mg/ml botanical extract concentrations of M. oleifera ethyl acetate and L. camara water extract. The leaves, stem and root biomass was significantly higher on M. oleifera ethyl acetate, E. nigra ethyl acetate, and L. camara extracts. However, L camara water, E. nigra water, L camara acetone extracts, and the negative control significantly reduced the leaves, stem, and root biomass.

Effect of Botanical Extracts on the Yield total and Yield Parameters

Extracts from M. oleifera ethyl acetate, E. nigra ethyl acetate, and L. camara showed a concentration-dependent increase on the yield of S. esculentum. Highest total and marketable yield were obtained at 2.5 and 5 mg/ml botanical extract concentration in plants treated with E. nigra ethyl acetate, E. nigra ethyl acetate, and L. camara water extract. There were no marketable S. esculentum fruits obtained from plants that were treated with distilled water (negative control).

Disease Incidence

M. oleifera ethyl acetate, E. nigra ethyl acetate, L. camara water, and L. camara acetone extracts significantly reduced disease incidence of late blight. However, disease incidence was significantly higher on tomato plants treated with M. oleifera water and E. nigra water extracts compared to the other treatments. As the number of weeks increased from 5 to 11 WAT, E. nigra ethyl acetate, M. oleifera ethyl acetate and L. camara water extracts significantly reduced disease incidence of late blight. In addition, increasing extract concentration from 1 to 2.5 mg/ml significantly reduced disease incidence. However, extract concentration of 2.5 mg/ml was not significantly different to 5.0 mg/ml on S. esculentum plants treated with M. oleifera ethyl acetate and L. camara water extracts.

Disease Severity

L. camara acetone, E. nigra ethyl acetate, M. oleifera ethyl acetate, and L. camara water extracts caused a concentration-dependent reduction on disease severity. Disease severity was significantly reduced at 2.5 and 5mg/ml botanical extract concentration on plants treated with M. oleifera ethyl acetate and L. camara water extracts. Disease severity was significantly reduced on M. oleifera ethyl acetate, E. nigra ethyl acetate, and L. camara water extracts. However, M. oleifera water, E. nigra water, L. camara acetone extracts, and the negative control significantly increased disease severity.

Gas Chromatography-Mass Spectrometric Analysis of Botaniucal Extracts of M. oleifera, A. Indica, E. nigra, and L. camara.

GC-MS analysis of M. oleifera acetone, M. oleifera ethyl acetate, and M. oleifera water extracts showed major molecular peaks at different m/z values of the possible antifungal compounds. Tentative identification of compounds shows that the major compounds in M. oleifera acetone, M. oleifera ethyl acetate, and M. oleifera water extracts were cyclopentasiloxane, decamethyl, 3,4-dihydroxyphenylglycol, 4TMS derivative, and acenaphthene, respectively. Different compounds were extracted by different compounds except cyclopentasiloxane, decamethyl- which was detected in both acetone and water extracts.  Ethyl acetate extracted furfural and eucalyptol both which had a match score of above 90% which suggest that they were the major compounds in E. nigra ethyl acetate extracts. On the other hand, 3-furaldehyde was detected in E. nigra water extract with a match score above 95%. Furfural, cyclopentasiloxane, decamethyl, and Guanidine which had a match score above 90% were detected in L. camara acetone extract. On the other hand, cyclopentasiloxane, decamethyl- were detected with a match score above 90% in both methyl acetate and water extracts. In addition, the probability that 2-pentanone, 4-hydroxy-4- methyl was present in the L. camara extract was above 95%.

Conclusion

Based on the results obtained in this study, it can be concluded that M. oleifera ethyl acetate, E. nigra ethyl acetate, and L. camara water extracts reduced radial growth and increased percent inhibition in vitro. Green house evaluation showed that M. oleifera ethyl acetate, E.nigra ethyl acetate, and L. camara acetone and water extracts increase economic and total yield and reduce disease incidence and severity in S. esculentum plants. The PPO, PAL, and POD enzymatic activity increased after the application of botanical extracts on S. esculentum plants. Overall, L. camara water, M. oleifera ethyl acetate, and E. nigra ethyl acetate extracts effectively suppressed P. infestans.

Reference:

Choga, T., Ngadze, E., Rugare, J.T., Mabasa, S., Makaza, W., Gwatidzo, V.O., Chikuta, S. and Karubanga, G., 2021. Effect of Botanical Extracts on Late Blight (Phytopthora infestans) and Productivity of Tomato (Solanum esculentum). International Journal of Agronomy, 2021.