Article body

Plant-parasitic nematodes are cosmopolitan parasites of many crops worldwide and are responsible for losses of 10 to 50% within a single crop yield (Barker et al. 1998). The sugar beet cyst nematode, Heterodera schachtii Schmidt, occurs in many parts of the world including Canada and the US (Baldwin and Mundo-Ocampo 1991). This nematode infects numerous cruciferous plants including sugar beet, Beta vulgaris L. Up to 90% of damage to sugar beets has been reported as a result of infection with H. schachtii (Steele 1984). The root-knot nematode, Meloidogyne hapla Chitwood, and the lesion nematode, Pratylenchus penetrans Cobb, occur in temperate regions, infect a wide range of plants worldwide, and cause varying degrees of economic losses (Barker et al. 1998; Loof 1991). Until recently, control of these nematodes was achieved by synthetic nematicides, some of which are environmentally undesirable (Dunn and Noling 1997). Recently, the need for novel plant-derived nematicides that are non-persistent, biodegradable and non-toxic to non-target organisms has been increasing.

Marigold, Tagetes spp., has been known to produce nematicidal compounds (Kimpinski et al. 2000; 1999; Sipes and Arakaki 1997). One of the main marigold compounds with nematicidal properties is a thiophene photoactivated compound called α-terthienyl (Bakker et al. 1979; Hasan 1992).

The effect of marigold seed exudates on nematodes has not been investigated. The purpose of this project was to investigate the nematicidal properties of marigold seed exudates and to identify potential sources that will lead to novel nematicidal plant-derived compounds.

Seeds of T. patula L. (var. polynema) and T. erecta L. (cv. Crackerjack) (Stokes Seeds Ltd., St. Catharines, ON, Canada) (0.2 g each) were washed and wrapped in a nylon-permeable filter and placed in individual 6-cm diame Petri dishes filled with 10 mL dechlorinated tap water for 72 h at 25°C. Seeds of radish, Raphanus sativus L., corn, Zea mays L., and tomato, Lycopersicon esculentum Mill., were used as controls for H. schachtii, P. penetrans and M. hapla, respectively. At the end of the incubation period, 20 juveniles of each of H. schachtii, P. penetrans and M. hapla were added to each marigold seed exudates and control and incubated for 48 h. Nematode mortality rates were assessed using a compound microscope after 48 h. Nematodes that did not respond to physical probing using a metal probe were considered either dead or paralyzed. To determine whether marigold seed exudates were nematicidal (i.e. nematodes are dead) or nematostatic (i.e. nematodes are temporarily paralyzed), 20 nematodes that were incubated for 48 h in exudates were transferred to 5 mL dechlorinated tap water at 25°C for 24 h. The nematodes were then observed under a compound microscope for signs of recovery and nematode mortality. Ten Petri dishes were used per exudate from either marigold or control for each nematode species. All assays were repeated three times.

Exudates from marigold seeds that were incubated in dechlorinated tap water for 72 h were collected, filtered using filter paper (#5 qualitative filter paper, Whatman International Ltd., England) and placed into 200-mL scintillation vials. Each set of exudates was analyzed using a Hewlett Packard 1090 high-performance liquid chromatography (HPLC) apparatus with a U.V. detector (Hewlett-Packard, Waldbronn, Germany), and a DuPont Zorbax PSM 60 6.22X 250 column (Wilmington, DE, USA) at 40°C. The solvents used were acetonitrile and tap water that had been prepared using millipore filtration. Exudates of each marigold variety (250 μL) were injected into the column at a flow rate of 1 mL min-1 and each HPLC run lasted for 20 min. Four runs per sample were performed. Dechlorinated tap water was used as control. The derived fractions were collected and 250 μL of each fraction was added to a microcavity slide (16 mm x 3 mm) (Clay Adams, New York, USA) with 20 H. schachtii juveniles. The slides were sealed to prevent evaporation and incubated for 72 h at 25°C, after which recovery and nematode mortality rates were assessed. For control treatment, nematodes were put in a slide containing water. Due to the limited HPLC fraction quantities extracted from marigold seeds, only H. schachtii was tested in the consecutive assays since this nematode appeared more sensitive to the effects of marigold seed exudates. HPLC fractionation of marigold seed extracts and nematode assays were repeated three times. Data from the three repetitions were combined. Prior to statistical analysis, the combined data were tested for homogeneity of variances (Devore 1987). Statistical differences (P < 0.05) were tested using the Kruskal-Wallis test and Tukey’s multiple comparisons method (Devore 1987) among exudates for each nematode in the mortality assays and among the HPLC fractions for each marigold species.

Seed exudates of T. erecta (cv. Crackerjack) and T. patula (var. polynema) caused significantly higher mortality to H. schachtii, M. hapla and P. penetrans than did their respective controls (P < 0.05) (Fig. 1a and 1b).

Figure 1a

The effect of water extract from seed exudates of Tagetes erecta (cv. Crackerjack), and radish, corn, and tomato controls on three species of plant-parasitic nematodes. Data for each column are averages ± standard deviations of 30 replications. The same letter between treatments and control indicates no difference at P < 0.05.

The effect of water extract from seed exudates of Tagetes erecta (cv. Crackerjack), and radish, corn, and tomato controls on three species of plant-parasitic nematodes. Data for each column are averages ± standard deviations of 30 replications. The same letter between treatments and control indicates no difference at P < 0.05.

-> See the list of figures

Figure 1b

The effect of water extract from seed exudates of T. patula (var. polynema), and radish, corn, and tomato controls on three species of plant-parasitic nematodes. Data for each column are averages ± standard deviations of 30 replications. The same letter between treatments and control indicates no difference at P < 0.05.

The effect of water extract from seed exudates of T. patula (var. polynema), and radish, corn, and tomato controls on three species of plant-parasitic nematodes. Data for each column are averages ± standard deviations of 30 replications. The same letter between treatments and control indicates no difference at P < 0.05.

-> See the list of figures

There was no significant difference between the mortality rates caused by the two marigold varieties. In addition, there was no significant difference between the mortality rates caused by each of the marigold seed extracts in each individual nematode species (Fig. 1c), although the trend suggests that T. patula (var. polynema) caused the highest mortality to H. schachtii. No significant nematode recovery was observed in comparison with the controls when the nematodes were transferred from the marigold seed exudates to water for 24 h (data not shown).

Figure 1c

The effect of water extract from seed exudates of T. patula (var. polynema) and T. erecta (cv. Crackerjack) on three species of plant-parasitic nematodes. Data for each column are averages ± standard deviations of 30 replications. The same letter between treatments of the same species indicates no difference at P < 0.05.

The effect of water extract from seed exudates of T. patula (var. polynema) and T. erecta (cv. Crackerjack) on three species of plant-parasitic nematodes. Data for each column are averages ± standard deviations of 30 replications. The same letter between treatments of the same species indicates no difference at P < 0.05.

-> See the list of figures

The HPLC fractions F2 and F3, derived from T. erecta, caused the highest mortality to H. schachtii juveniles in comparison with the water control and the rest of the fractions (Fig. 2).

Figure 2

The effect of HPLC fractions derived from water extract from seed exudates of Tagetes erecta (cv. Crackerjack) and water control on Heterodera schachtii. Data for each column are averages ± standard deviations of 3 replications. The same letter between treatments indicates no difference at P < 0.05.

The effect of HPLC fractions derived from water extract from seed exudates of Tagetes erecta (cv. Crackerjack) and water control on Heterodera schachtii. Data for each column are averages ± standard deviations of 3 replications. The same letter between treatments indicates no difference at P < 0.05.

-> See the list of figures

HPLC fraction F4, derived from T. patula, caused significantly (P < 0.05) higher mortality to H. schachtii juveniles than the control and the rest of the HPLC fractions (Fig. 3).

Figure 3

The effect of HPLC fractions derived from water extract from seed exudates of Tagetes patula (var. polynema) and water control on Heterodera schachtii. Data for each column are averages ± standard deviations of 3 replications. The same letter between treatments indicates no difference at P < 0.05.

The effect of HPLC fractions derived from water extract from seed exudates of Tagetes patula (var. polynema) and water control on Heterodera schachtii. Data for each column are averages ± standard deviations of 3 replications. The same letter between treatments indicates no difference at P < 0.05.

-> See the list of figures

No nematode recovery was observed in comparison with the control when nematodes were transferred from HPLC fractions to water for 24 h (data not shown).

Seed exudates from both Tagetes erecta (cv. Crackerjack) and T. patula (var. polynema) caused significantly higher mortality to H. schachtii, M. hapla and P. penetrans compared with control exudates from radish, corn and tomato seeds. Several reports exist on the nematicidal properties of marigolds from different parts of the plant (Alexander and Waldenmaier 2002; Kimpinski et al. 2000; Ploeg 1999; Siddiqui and Alam 1988). However, the nematicidal properties of marigold seed exudates in the laboratory or field have not been thoroughly investigated. Two HPLC fractions derived from T. erecta cv. Crackerjack and one fraction from T. patula var. polynema caused the highest H. schachtii mortality in comparison with the water control and the rest of the HPLC fractions. Our study shows that seed exudates contain nematicidal compounds. No nematostatic compounds were found in these exudates since nematode recovery was not observed.

Additional work is needed to isolate and characterize compounds derived from marigold seed exudate fractions. Identifying new compounds may potentially lead to the discovery of novel plant-derived nematicides.