Toxicity of Leaf and Stem Extracts to Tylenchorhynchus Dubius
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Ultrastructure Changes in Alfalfa: Chang et aL 173 high pH as an electron-opaque stain in electron Histochemistry of soybean varieties resistant or microscopy. J. Cell Biol. 17:208-212. susceptible to Meloidogyne incognita acrita. 16. ROBARDS, A. W. and P. G. HYMPHERSON. Phytopathology 56:586 (Abstr.). 1967. Phytoferritin in plastids of the cambial 20.SHERWOOD, R. T., J. W. DUDLEY, T. H. zone of willow. Planta 76:169-178. BUSBICE and C. H. HANSON. 1967. Breeding 17.ROBARDS, A. W. and C. L. ROBINSON. 1968. alfalfa for resistance to the stem nematode, Further studies on phytoferritin. Planta Ditylenchus dipsaci. Crop Sci. 7:382-384. 82:179-188. 21.THORNE, G. 1961. Principles of Nematoiogy. 18.SHAW, M. and M. S. MANOCHA. 1965. Fine McGraw-HiU BookCo., Inc., N.Y. 131 p. structure in detached, senescing wheat leaves. 22.WATSON, M. L. 1958. Staining of tissue sections Can. J. Bot. 43:747-755. for electron microscopy with heavy metals. J. 19.SHEETZ, R. W. and H. W. CR1TTENDEN. 1966. Biophys. Biochem. Cytol. 4:475-478. Toxicity of Leaf and Stem Extracts to Tylenchorhynchus dubius P. M. MILLER, N. C. TURNER and H. TOMLINSON 1 Abstract: Plant extracts, made by grinding 2 g of fresh tissue in 5 ml of water, were toxic to Tylen~horhynchus dubius and Hoplolaimus spp. Such extracts from leaves and stems of bean (Phaseolus vulgaris L.) and leaves of tobacco (Nicotiana tabacum L.) were most toxic; those from leaves of corn (Zea mays L.), tomato (Lycopersicon esculentum Mill.) and rhododendron (Rhododendron catawbiense L.) were less toxic; and extracts of bean roots were nontoxic. Nematode movement slowed markedly within 1 hr in tobacco leaf extract, and within 4 hr in bean leaf extract; both extracts completely inactivated or killed 95% of the nematodes in 24 hr. Heating leaf extract 10 min at 80 C eliminated toxicity. Absorption of fusicoccin, a phytotoxin produced by Fusicoccum amygdali Del., increased the toxicity of tomato leaf extracts, whereas water extracts of acetone-extracted powder preparations of leaves were about 15-fold more toxic than water extracts of fresh tissue. Addition of homogenized leaves of bean, tobacco and tomato to soil significantly reduced nematode populations within 3 days. Some plant roots are toxic to nematodes (7, MATERIALS AND METHODS 8, 10). Although residues of decomposing rye Plant extracts were obtained by grinding 2 g (Secale cereale L.) and timothy (Phleum of fresh whole leaves of corn (Zea mays L. pratense L.) have nematicidal properties (9, 'Pa602A'), beans (Phaseolus vulgaris L. 'Pinto'), 11), the authors are not aware of any report tobacco (Nicotiana tabacum L. 'Bel. W3'), that leaf extracts are toxic to nematodes. In tomato (Lycopersicon esculentum Mill. 'Bonny this study, we report nematicidal activity in Best') and rhododendron (Rhododendron extracts of leaves and stems of bean, the leaves catawbiense L.), in 5 ml of water with a mortar of tobacco, and to a lesser extent in leaves of and pestle and filtering each sample through corn, tomato and rhododendron. The effect of cheesecloth to remove debris. Corn and bean fusicoccin [a fungal toxin produced by the plants were 2 weeks old and tobacco and actively growing mycelium of Fusicoccum tomato plants were 25-cm tall. The plants had amygdali Del. (2)] on the nematicidal activity not been treated with nematicides, fungicides of leaf extracts of tomato and bean plants that or insecticides before extraction. Two ml of had absorbed the toxin, was also investigated. each extract was mixed with 2 ml of a Finally, we added aqueous homogenates of leaf nematode suspension containing 60-80 material to soil to determine whether its Tylenchorhynchus dubius and 15-25 nematicidal activity was retained in the soil. Hoplolaimus spp. A control, containing 2 ml of water plus an equal volume of the nematode Received for publication 14 July 1972. suspension, also was prepared. Nematodes were 1 Plant Pathologist, Assistant Plant Physiologist and obtained from untreated Windsor fine sandy Assistant Plant Pathologist, respectively. The Connecticut Agricultural Experiment Station, P.O. loam around roots of bluegrass (Poa pratensis Box 1106, New Haven 06504. L.) and native bentgrass (Agrostis stolonifera 174 Journal of Nematology, VoL 5, No. 3, July 1973 L.) by decantation, sieving (44-/a screen) and discarded. Thirty grams of fresh tissue yielded sugar flotation (5). Nematodes in plant extracts 2.7 g of acetone powder. To prepare a mixture were kept at room temperature (23 C) and the for test, 80 mg of acetone powder was soaked active nematodes were counted after 48 hr. In in 20 ml of water for 2 min, centrifuged for 10 this and successive experiments, treatments min and the supernatant retained. Bean and were replicated four times. tobacco acetone powders contained 14 mg and In another experiment, leaf extracts from 6.5 mg of dry matter/ml, respectively. These bean and tobacco were diluted with water 2-, extracts were diluted 2-, 6-, 12-, 20- and 6-, 20- and 60-fold. Each ml of the undiluted 60-fold. Two-ml samples of undiluted extracts extracts from leaves of bean and tobacco and of each extract dilution were mixed with contained 33 mg and 25 mg of dry matter, equal volumes of nematode suspension and respectively. A 2-ml sample of the undiluted active and inactive nematodes were counted extracts and of each dilution was mixed with an after 24 and 48 hr. equal volume of nematode suspension. The To determine whether fusicoccin would numbers of active and inactive nematodes were increase the toxicity of plant extracts, tomato counted after 1, 4, 7, 24 and 48 hr. This and bean plants grown in sand culture were experiment was repeated twice. washed free of sand and placed in a greenhouse The effect of heat on the toxicity of leaf with their roots in a solution of 5 X 10-6 M extracts to nematodes was tested by heating fusicoccin or in water. After 24 hours, the bean and tobacco leaf extracts in a water bath leaves of plants in fusicoccin had wilted. Leaf at 80 C for 10 min and then cooling them in samples from three plants were removed after cold water. Two-ml samples of unheated and 8, 24 and 48 hr, frozen and the leaf extracts heated extracts were mixed separately with were made as previously described except that equal volumes of the nematode suspension and the desiccated leaves of the plants treated with the number of active nematodes counted after fusicoccin were prepared in 6.8 ml of water to 24 hr. replace the water lost by desiccation of the Acetone powders (a residue of tissue. A two-ml volume of each extract was acetone-extracted homogenized tissues)were mixed with an equal volume of the nematode made by grinding 30 g of bean or tobacco suspension and the number of active nematodes leaves in 300 ml of cold acetone followed by counted after 24 hr. vacuum filtration. The residue was washed Finally, 5-, 10- and 20-g samples of leaf further with 200 ml of acetone, dried and tissue from bean, tobacco and tomato plants stored at -20 C until used. The acetone was were homogenized separately for 10 min in 100 ml of water; then each was mixed with 2 kg of soil and shaken for 5 min. A control was also TABLE 1. Mean percent inactive Tylenchorhynchus prepared by mixing 100 ml of water with 2 kg dubius and Hoplolaimus spp. after 48 hours in of soil and shaking it for 5 min. After 3, 8 and plant extracts. 21 days, a 100-g sample from each soil mixture was suspended in water, allowed to settle for 3 Extract Mean % inactive min and decanted through a 44-~t screen. source T. dubius Hoplolaimus spp. Nematodes in the material retained by this screen were washed onto a cellulose tissue (6) Control (no extract) 9 12 and the tissue was left in contact with water Bean leaves 100 100 overnight. By morning, the active nematodes Bean leaves - had moved into the water and could be heated a 30 32 counted. Bean stems 100 100 Bean roots 33 25 RESULTS Tobacco leaves 100 100 After 48 hr, all T. dubius and Hoplolaimus Tobacco leaves- spp. in bean and tobacco leaf extracts and bean heated a 36 38 Tomato leaves 31 22 stem extracts were inactive, 49% and 55% in Corn leaves 49 52 leaf extracts of corn and thododendron Rhododendron (respectively) were inactive and 31% in tomato leaves 55 38 leaf extract were inactive (Table 1). Bean and aExtracts heated to 80 C for 10 min and then cooled tobacco leaf extracts heated at 80 C for 10 min before nematode suspension was added. were much less toxic (Table 1). Toxicity of Plant Extracts to Tylenchorhynchus dubius: Miller et aL 175 TABLE 2. Mean percent inactive 7;'ylenchorhynchus dubius after 1 to 48 hours in various dilutions of water extracts of tobacco or bean leaves. The equivalent concentration of extract (dry weight) per ml of suspension for each dilution is given. Mean % inactive T. dubius after Equivalent extract 1 hr 4 hr 7 hr 24 hr 48 hr 24 hr 48 hr cohen. Extract Dilution (mg/ml) Test A Test B Bean 0 16.5 4 61 75 96 95 100 100 2 8.2 10 27 66 86 84 63 66 6 2.8 5 18 27 49 31 47 34 12 1.4 11 7 13 24 14 27 21 20 0.8 4 0 4 14 12 19 15 60 0.3 18 24 Tobacco 0 12.5 44 77 100 96 100 100 100 2 6.2 49 76 85 94 92 99 80 6 2.1 33 39 68 85 60 72 a 67 a 12 1.0 6 5 12 13 48 11 19 20 0.6 1 2 15 4 21 7 5 60 0.2 3 3 None(ck) 0 0 4 2 15 21 25 17 20 aActive nematodes very sluggish.