Efficacy of selected plant species against Meloidogyne incognita and phytopathogenic microbes infecting tomatoes

Abstract

Soilborne pathogens are economically important, causing great losses in agricultural production globally. Nematodes, fungi, bacteria and viruses are microscopic, destructive pathogens which are extremely difficult to control. Farmers depend on synthetic pesticides for quick and effective control of pests and diseases. However, the majority of nematicides have been banned due to environmental problems resulting from their use and this has awakened interest in finding alternative methods of nematode and other plant pathogen control. Several indigenous plants have been identified as having potential anthelmintic efficacy in managing gastrointestinal infections in small ruminants and some of these have also been found to be effective in killing nematodes infecting crops.

In the present study, extracts of plants previously reported to have anthelmintic activity against free-living Caenorhabditis elegans and animal parasitic Haemonchus contortus nematodes were screened for their efficacy against the root-knot nematode species Meloidogyne incognita. Activity of the selected plants was confirmed against C. elegans and H. contortus. For in vitro safety determination, cytotoxicity tests on Leonotis leonurus, Clausena anisata and Lantana rugosa was conducted against Vero African green monkey kidney cells. Chemical profiling was performed using gas chromatography-mass spectrometry (GC-MS). The plants were further investigated for their ability to inhibit the growth of interrelated phytopathogens infecting tomatoes, including five pathogenic bacterial species: Clavibacter michiganensis subsp. michiganensis, Xanthomonas vesicatoria, X. perforans, Ralstonia solanacearum and R. pseudosolanacearum and one fungal strain (Fusarium oxysporum f.sp. lycopersici). Bioactive compounds were isolated from the acetone leaf extract of L. leonurus using bioassay-guided fractionation. Clausena anisata, L. rugosa and L. leonurus water extracts were subjected to in vitro phytotoxicity tests on tomato seedlings, and an in vivo glasshouse trial against M. incognita was also conducted with the powdered material (dried leaves) of L. leonurus and C. anisata.

All plant extracts and fractions investigated had good anthelmintic activity against free-living and animal-parasitic nematodes. Activity of the plant species Acokanthera oppositifolia, Searsia lancea, Cotyledon orbiculata, Hippobromus pauciflorus and Lantana rugosa was reported for the first time against Haemonchus contortus. The phytochemicals detected in the extracts contribute to the activity of the plants reported, as some of these compounds were previously reported to have antibacterial, insecticidal and nematicidal properties. Cytotoxicity results indicated that C. anisata and L. leonurus extracts were relatively non-toxic to Vero cells compared to the positive control doxorubicin (LC50 = 0.0133 mg/mL). Lantana rugosa extracts were highly toxic with LC50 values less than 0.0075 mg/mL, the lowest concentration tested.

When screening the selected plants for activity against M. incognita second-stage juveniles (J2) at the highest concentration of 1 mg/ml, results indicated that all selected non-crop plant species had potential in managing root-knot nematodes, but the most promising activity was observed with C. anisata, L. rugosa and L. leonurus extracts. The three plants were further evaluated in terms of motility against J2s as well as J2 egg hatch inhibition. Only the L. leonurus water extract showed good dose-related activity in inhibiting motility of M. incognita J2s. Clausena anisata extracts had weak activity in the motility assay but both plants had good inhibitory activities against J2 hatching. In the antimicrobial assay, acetone extracts of Leucosidea sericea and Searsia lancea demonstrated interesting antibacterial activity against a broad range of tested bacteria with MIC values ranging from 19.5 to 97.5 µg/mL. None of the extracts was active against Fusarium oxysporum f. sp. lycopersici except for the acetone extract of Cotyledon orbiculata and L. leonurus water extract, which inhibited the growth of F. oxysporum f.sp. lycopersici at 39 µg/mL and 97.5 µg/mL after 24 h respectively. Clausena anisata and L. leonurus fractions obtained from liquid/liquid partitioning indicated poor activity against all tested phytopathogens, except for the L. rugosa fractions which had good to moderate activity against most bacterial pathogens with MIC values ranging from 78 to 156 µg/mL.

Bioassay-guided fractionation was used to isolate nematicidal compounds from the dichloromethane fraction of Leonotis leonurus using Caenorhabditis elegans as the test organism. This led to isolation of leoleorin C, and other compounds which could not be identified due to insufficient time and purity. Leoleorin C was previously isolated from the plant, but activity against selected organisms and the in vitro lack of cytotoxicity is reported for the first time in this study. Leoleorin C had moderate activity against C. elegans but was not active against M. incognita. The compound was not active against the tested bacterial phytopathogens, but promising activity was observed in the bioautography assay against Clavibacter michiganensis subsp. michiganensis, causal organism of bacterial canker in tomatoes. The plant was not toxic to the Vero cells at the highest concentration of 1 mg/mL.

Leonotis leonurus, C. anisata and L. rugosa water extracts were not phytotoxic to the growth of tomato seedlings at all tested concentrations (highest concentration of 10 mg/mL) under in vitro conditions. Lower extract concentrations of all the selected non-crop plant species stimulated germination, whereas high concentrations had inhibitory effects, except for the C. anisata extract. In the treatment of tomato plants under in vivo conditions, M. incognita infection did not negatively affect the growth parameters assessed, namely stem height, wet shoot mass, dry shoot mass, number of flowers, stem diameter, fruit number and fruit mass. The plants had the ability to reduce gall index, number of eggs and J2 when compared to the untreated control of tomato plants. There was a lack of phytotoxic and fertilizer effect on tomato plants at the applied rates on growth parameters since activity was comparable to the untreated control. The low effect demonstrated by the plants might be due to low quantities of active phytochemicals present. Leonotis leonurus and C. anisata were not effective in the form of powder as nematicidal preparations in the glasshouse trials.