In vitro nematocidal effects of medicinal plants from São Paulo state, Brazil

Abstract

Synthetic nematicides have been used broadly in past decades for the control of plant parasitic nematodes. Despite their relevant effectiveness, these compounds can cause great damage to the environment, and have a relevant impact on human and animal health. The growing search for new nematicides, particularly natural ones, may lead to higher safety and efficiency in nematode control. In this work, the in vitro nematocidal effect of ethanol extracts obtained from the following plant species was evaluated: Tithonia diversifolia (Hemsl.) A.Gray; Ecilpta alba (L.) Hassk; Mikania glomerata Sprengel (Asteraceae); Tabernaemontana catharinensis A. DC; Mandevilla velutina (Mart.) Woodson (Apocynaceae); Casearia sylvestris Sw. (Salicaceae); Zeyheria montana Mart. (Bignoniaceae); Lippia alba (Mill.) (Verbenaceae); Croton antisyphiliticus Mart. (Euphorbiaceae) and Serjania erecta Radlk. (Sapindaceae). The plant parasitic nematodes Pratylenchus zeae (Graham) (Nematoda: Pratylenchidae) and Pratylenchus jaehni (Inserra) (Nematoda: Pratylenchidae) were used for tests. Moreover, a preliminary phytochemical characterization of these plant extracts was performed in order to associate these data with those observed in nematocidal assays. Our results indicated a significant nematocidal activity of the analyzed extracts, especially those demonstrated by E. alba (DL₅₀ (ppm) = 304.08; 55.32 – P. zeae and DL₅₀ (ppm) =>1000; 212.82 – P. jaehni; 12 and 24 h, respectively), T. catharinensis (DL₅₀ (ppm) = 215.26; 60.04 – P. zeae and DL₅₀ (ppm) = 825.44; 376.60 – P. jaehni; 12 and 24 h, respectively), C. sylvestris (DL₅₀ (ppm) = 198.05; 56.94 – P. zeae and DL₅₀ (ppm) = 747.98; 322.98 – P. jaehni; 12 and 24 h, respectively), Z. montana (DL₅₀ (ppm) = 166.43; 34.08 – P. zeae and DL₅₀ (ppm) =>1000; 427.34 – P. jaehni; 12 and 24 h, respectively) and S. erecta (DL₅₀ (ppm) = 178.74; 74.12 – P. zeae e DL₅₀ (ppm) = 689.24; 249.50 – P. jaehni; 12 and 24 h, respectively). Thus, these data show that the evaluated plants present significant nematocidal effects, which are of high economic or environmental interest and may be useful for the growth of agricultural activities worldwide.

Keywords::

• Plant extracts
• plant parasitic nematodes
• Pratylenchus jaehni
• Pratylenchus zeae

Introduction

Plant parasitic nematodes are significant agricultural pests distributed worldwide and characterized by causing mechanical root destruction, leading to plant damage and decreased agricultural production. It has been postulated that an increase in world agricultural production is not realized due to the attack of different species of nematodes, causing losses of millions of dollars annually in various types of agriculture worldwide (Hussey & Grundler, 1998; De Waele & Elsen, 2007).

The nematode Pratylenchus zeae (Graham) (Nematoda: Pratylenchidae) is a widely known agricultural pest due to its broad distribution and the serious damage caused in plant tissues (Aung & Prot, 1990; Embrapa, 2006). In Brazil, the nematode Pratylenchus jaehni (Inserra) was first identified in 1995, and since then it has rapidly advanced into at least 29 cities of the state of Sao Paulo, two in regions of the state of Minas Gerais, and three in the state of Parana. P. jaehni has been considered the most harmful nematode to citrus cultures in Brazil (Inserra et al., 2001; Calzavara et al., 2007). However, little is known about the biology of this nematode species.

One of the advantages of a nematicide is the effectiveness at low doses, as well as low toxicity to humans, plants, domestic or raised animals, low costs, pest selectivity and easy and safe manipulation (Shaalan et al., 2005). The great interest for the search of new natural nematicide and insecticide agents has been motivated by the proposal that natural products could be in most cases advantageous when compared to synthetic nematicides (Chitwood, 2002; Isman, 2006). In fact, natural agents may work as substitutes for synthetic nematicides or insecticides on organic agriculture, representing a valuable alternative for developing countries due to their lower cost (Isman, 2006). The search for new nematicides of differing chemical structures and natural sources could represent an improvement of potency, safety, pest resistance manegement and cost in relation to synthetic nematocides presently employed against plant parasitic nematodes (Chitwood, 2002; Isman, 2006; Shaalan et al., 2005).

This work evaluated the nematocidal effects of ethanol extracts from Tithonia diversifolia (Hemsl) A. Gray, Eclipta alba (L.) Hassk, Mikania glomerata Sprengel (Asteraceae), Tabernaemontana catharinensis A.DC, Mandevilla velutina (Mart.) Woodson (Apocynaceae), Casearia sylvestris Sw. (Salicaceae), Zeyheria montana Mart. (Bignoniaceae), Lippia alba (Mill.) (Verbenaceae), Serjania erecta Radlk (Sapindaceae) and Croton antisyphiliticus Mart. (Euphorbiaceae) against P. zeae and P. jaheni, to establish their DL₅₀ values, minimum and maximal effective doses in different times of exposition (12 and 24 h), and determine preliminary phytochemical properties.

Materials and methods

Plant material and ethanol extracts

Plants were collected in the region of Ribeirão Preto, São Paulo state, Brazil by Dr. Ana Maria S. Pereira (Unaerp). The material was authenticated by authorities from the Botanical Institute of Campinas University. Voucher specimens of T. diversifolia (085), E. alba (848), M. glomerata (024), C. sylvestris (531), Z. montana (803), L. alba (461), C. antisyphiliticus (293), S. erecta (837) and Mandevilla velutina (013) were deposited at the Herbarium of Ribeirão Preto University, Brasil, and voucher specimens of T. catharinensis (02940, 02944 and 03224) were deposited at the Herbanarium of FFCLRP-USP, São Paulo University, Brazil.

Ethanol extracts were obtained from 200 g dried and pulverized plant material Tithonia diversifolia (leaves), Eclipta alba (leaves), Mikania glomerata (leaves), Casearia sylvestris (leaves), Zeyheria montana (leaves), Lippia alba (leaves), Croton antisyphiliticus (leaves) and Serjania erecta (leaves), Tabernaemontana catharinensis (stem bark), Mandevilla velutina (root) by macerating with 1 L of absolute ethanol for 24 h, three times at 25°C. After filtration, extracts were concentrated in a rotory evaporator and then lyophilized. To perform in vitro nematocidal assays, this material was dissolved in sterile Milli-Q water prior to use.

Preliminary phytochemical analyses: Thin-layer chromatography (TLC)

Phytochemical properties of plant extracts were assessed by means of silica gel thin-layer chromatographic plates (10.0 × 10.0 cm; Sigma, St. Louis, USA). A sample of each extract was dissolved in absolute methanol (0.1–0.5 mg/mL) and applied in different chromatographic plates using as mobile phases chloroform:methanol (8:2, v/v) or BAW (butanol/acetic acid/water 4:1:5, v/v). After development, the plates were sprayed with a 2-aminoethylester diphenylboric acid (NP/PEG) solution followed by UV light exposition (λ = 365 nm) or still sprayed with vanillin in sulfuric acid solution followed by heating.

Multiplication and maintenance of sub-populations of P. zeae and P. jaehni

P. zeae and P. jaehni nematodes in vitro multiplication was performed in carrot cylinders (4.0 × 2.0 cm) prepared from fresh and pesticide-free carrots, which were previously disinfected by washing with soap under running water, followed by a 0.05% (v/v) sodium hypochlorite solution for 30 min. Under sterile conditions, the carrots were washed with absolute ethanol followed by rapid flame exposure, and centrally hollowed by cutting out a cylander of approximately 4 × 2 cm, which was stored in sterile and closed flasks for 3-4 days at 25°C and 40% humidity. The nematodes, either raised in our laboratory or kindly provided by Jaime Maia dos Santos (Phytosanitary Department-UNESP-Jaboticabal-SP), were grouped and submitted to repeated baths in ampicillin solution (0.1%, w/v) for 10 min followed by sterile Milli-Q water-bath for 5 min. Under sterile conditions, the nematodes suspended in sterile Milli-Q water were applied on the upper extremity of the previously prepared carrot cylinders and incubated for approximately 3 months, after which a new sub-population corresponding to the fourth-stadium of the nematode development was obtained.

Extraction, axenization and inoculation of nematodes

The carrot cylinders, incubated for a period of 90-100 days with P. zeae or P. jaheni nematodes, were homogenized in sterile Milli-Q water and filtered by an overlayed aggregate of sieves (60 and 500 mesh, respectively). To the collected nematodes, aqueous suspensions, 0.1% (w/v) aqueous solution of kaolin, were added, and the suspensions were submitted to centrifugation at 1750 rpm for 5 min at 25°C (Universal 32R Centrifuge, Hettich, Tuttlingen, Germany). The pellet was homogenized in a 53.3% (w/v) aqueous sucrose solution, followed by centrifugation at 1750 rpm for 1 min (25°C) and filtration through a 500-mesh sieve. Collected nematodes were used either for nematocidal bioassays or for the inoculation of new carrot cylinders for nematode culture maintenance.

Nematocidal bioassays

Twenty nematodes per transparent glass plate were used on each control or experimental group. Lyophilized plant extracts were dissolved in sterile Milli-Q water and used in geometrical doses at final concentrations of 1000, 500, 250, 125, and 62.5 ppm. Nematocidal action was evaluated on a Leica MZ 6 stereomicroscope after time exposures of 12 and 24 h, and non-motile nematodes were considered as dead. DL₅₀ values were graphically calculated (Microsoft Office Excel for Windows-2003). A solution of 700 ppm of Carbofuran (Furandan^®) and sterile Milli-Q water were used for positive and negative controls, respectively. Each experiment was repeated three times and performed according to a double-blind trial. Statistical analysis was performed using Student’s t-test (p < 0.05 and p < 0.01).

Results and discussion

P. zeae and P. jaehni species were chosen over other nematodes due to their enhanced agricultural interest, easy manipulation, availability and culture maintenance simplicity, as well as by their rapid multiplication under controlled conditions (Aung & Prot, 1990; Embrapa, 2006; Calzavara et al., 2007).

Plants were chosen based on their nematocidal/insecticide potential, scientific originality, availability and wide geographical distribution in Brazil. Moreover, these species belong to botanical families/genera where the presence of phytochemical classes of nematocidal/insecticidal compounds have been identified, which suggests the plants used in the present study may be considered a potential source of new nematocidal agents (Chang et al., 1995; Kuo & Chen,1998; Begum et al., 2000; Carbonell et al., 2000; Hitmi et al., 2000; Maciel et al., 2000; Do Vale et al., 2002; Saravanan et al., 2004; Qamar et al., 2005). As expected, we confirmed the presence of compounds such as flavanols, tannins, and alkaloids, usually found in several nematocidal plants (Table 1).

Table 1.  Phytochemical analysis of ethanol extracts from selected species of plants.

Plants	Botanical family	Popular nomenclature (Brazil)	Flavonols	Tanins	Alkaloids	Cumarins
Casearia sylvestris (leaves)	Salicaceae	“Guaçatonga”	1	1	2	2
Croton antisyphiliticus (leaves)	Euphorbiaceae	“Pé-de-perdiz”	1	1	2	2
Eclipta alba (leaves)	Asteraceae	“Agrião-do-brejo”	1	2	2	2
Lippia alba (leaves)	Verbenaceae	“Erva-cidreira”	2	2	2	2
Mandevilla velutina (root)	Apocynaceae	“Infali´vel”	2	1	2	1
Mikania glomerata (leaves)	Asteraceae	“Guaco”	2	2	2	2
Serjania erecta (leaves)	Sapindaceae	“Cinco-folhas”	1	1	2	2
Tabernaemontana catharinensis (stem bark)	Apocynaceae	“Leiteiro-de-vaca”	2	2	1	2
Tithonia diversifolia (leaves)	Asteraceae	“Girassol mexicano”	1	2	2	2
Zeyheria montana (leaves)	Bignoniaceae	“Bolsa-de-pastor”	1	2	2	2

In Tables 2, 3, 4, and 5 we demonstrate the plant extracts with nematocidal activity against P. zeae and P. jaehni. The low and high mortality induced by the negative and positive controls, respectively, demonstrated the bioassay reliance and reproducibility.

Table 2.  Mortality percentage (± SEM) of the nematode Pratylenchus zeae on nematocidal assays (12 h).

Plant extracts	Carbofuran 700 ppm	Sterile Milli-Q Water	62.5 ppm	125 ppm	250 ppm	500 ppm	1000 ppm	DL50 (ppm)
Casearia sylvestris	73.33 ± 10.4	8.33 ± 2.89	26.67 ± 10.41	36.67 ± 10.41*	53.33 ± 7.64**	73.33 ± 2.89**	86.67 ± 2.89**	198.05
Tabernaemontana catharinensis	81.67 ± 17.5	10.00 ± 5.00	31.67 ± 10.4*	35.00±15.00*	48.33 ± 5.77**	66.67 ± 5.77**	83.33 ± 2.89**	215.26
Eclipta alba	76.67 ± 2.89	8.33 ± 2.89	23.33 ± 7.64*	31.67±10.41*	38.33 ± 11.55*	53.33 ± 16.07*	81.67 ± 2.89**	304.08
Serjania erecta	76.67 ± 5.77	6.67 ± 2.89	20.00 ± 8.66	35.00±5.00*	66.67 ± 7.64**	81.67 ± 2.89**	96.67 ± 2.89**	178.74
Zeyheria montana	76.67 ± 2.89	11.67 ± 2.89	30.00 ± 5.00**	41.67±5.77**	56.67 ± 5.77**	78.33 ± 2.89**	90.00 ± 0.00**	166.43
Lippia alba	80.00 ± 0.00	5.00 ± 5.00	13.33 ± 7.64*	16.67±5.77*	25.00 ± 5.00*	28.33 ± 5.77**	31.67 ± 7.64**	> 1000
Mandevilla velutina	78.33 ± 2.89	5.00 ± 5.00	30.00 ± 0.00*	35.00±0.00**	43.33 ± 2.89**	51.67 ± 2.89**	61.67 ± 2.89**	406.01
Tithonia diversifolia	78.33 ± 2.89	5.00 ± 5.00	36.67 ± 2.89*	43.33±2.89**	53.33 ± 7.64*	61.67 ± 2.89**	68.33 ± 2.89**	199.69
Mikania glomerata	76.67 ± 2.89	1.67 ± 2.89	0.00 ± 0.00	15.00±5.00*	15.00 ± 5.00*	25.00 ± 10.00*	41.67 ± 2.89**	>1000
Croton antisyphiliticus	80.00 ± 0.00	3.33 ± 2.89	20.00 ± 5.00	23.33±7.64	31.67 ± 2.89*	41.67 ± 2.89**	50.00 ± 10.00*	>1000

*p < 0.05, **p < 0.01.

Table 3.  Mortality percentage (± SEM) of the nematode Pratylenchus zeae on nematocidal assays (24 h).

Plant extracts	Carbofuran 700 ppm	Sterile Milli-Q Water	62.5 ppm	125 ppm	250 ppm	500 ppm	1000 ppm	DL50 (ppm)
Casearia sylvestris	100.00 ± 0.00	18.33 ± 2.89	51.67 ± 10.41*	63.3 ± 16.07*	83.33 ± 2.89**	96.67 ± 2.89**	100.00 ± 0.00**	56.94
Tabernaemontana catharinensis	100.00 ± 0.00	16.67 ± 2.89	53.33 ± 16.07	60.00 ± 17.32*	75.00 ± 0.00**	85.00 ± 5.00**	100.0 ± 0.00**	60.04
Eclipta alba	100.00 ± 0.00	11.67 ± 2.89	51.67 ± 5.77**	63.33 ± 7.64**	76.67 ± 5.77**	90.00 ± 0.00**	96.67 ± 2.89**	55.32
Serjania erecta	100.00 ± 0.00	11.67 ± 2.89	40.00 ± 10.00*	75.00 ± 0.00**	96.67 ± 2.89**	98.33 ± 2.89**	100.00 ± 0.00**	74.12
Zeyheria montana	100.00 ± 0.00	13.33 ± 2.89	55.00 ± 5.00**	75.00 ± 0.00**	83.33 ± 2.89**	98.33 ± 2.89**	100.00 ± 0.00**	34.08
Lippia alba	100.00 ± 0.00	6.67 ± 2.89	21.67 ± 10.41	30.00 ± 13.23	36.67 ± 10.41*	40.00 ± 13.2*	45.00 ± 13.23*	> 1000
Mandevilla velutina	100.00 ± 0.00	6.67 ± 2.89	40.00 ± 5.00*	50.00 ± 5.00*	56.67 ± 2.89**	61.67 ± 2.89**	81.67 ± 2.89**	145.02
Tithonia diversifolia	100.00 ± 0.00	6.67 ± 2.89	65.00 ± 0.00**	66.67 ± 2.89**	71.67 ± 2.89**	76.67 ± 2.89**	81.67 ± 2.89**	7.68
Mikania glomerata	100.00 ± 0.00	5.00 ± 0.00	10.00 ± 8.66	20.00 ± 5.00*	23.33 ± 7.64	35.00 ± 8.66*	70.00 ± 10.00**	553.78
Croton antisyphiliticus	100.00 ± 0.00	6.67 ± 2.89	28.33 ± 2.89*	38.33 ± 2.89*	50.00 ± 10.00*	58.33 ± 12.5*	65.00 ± 10.00*	289.37

*p < 0.05, **p < 0.01.

Table 4.  Mortality percentage (± SEM) of the nematode Pratylenchus jaehni on nematocidal assays (12 h).

Plant extract	Carbofuran 700 ppm	Sterile Milli-Q Water	62.5 ppm	125 ppm	250 ppm	500 ppm	1000 ppm	DL50 (ppm)
Casearia sylvestris	81.67 ± 2.89	11.67 ± 2.89	20.00 ± 5.00	28.33 ± 2.89	40.00 ± 5.00*	43.33 ± 2.89**	55.00 ± 5.00*	747.98
Tabernaemontana catharinensis	66.67 ± 23.09	20.0 ± 10.00	18.33 ± 10.41	25.00 ± 13.23	30.00 ± 8.66	30.00 ± 0.00	56.67 ± 2.89*	825.44
Eclipta alba	83.33 ± 2.89	10.00 ± 0.00	16.67 ± 2.89	28.33 ± 5.77*	35.00 ± 5.00*	40.00 ± 5.00**	50.00 ± 5.00**	>1000
Serjania erecta	75.00 ± 5.00	10.00 ± 5.00	20.00 ± 5.00	20.00 ± 5.00*	35.00 ± 0.00*	38.33 ± 2.89**	56.67 ± 14.43*	689.24
Zeyheria montana	63.33 ± 16.07	6.67 ± 2.89	21.67 ± 7.64	21.67 ± 5.77*	20.00 ± 0.00*	36.67 ± 2.89**	45.00 ± 15.00*	>1000
Lippia alba	75 ± 5.00	13.33 ± 2.89	11.67 ± 2.89	21.67 ± 2.89	23.33 ± 2.89	26.67 ± 2.89*	33.33 ± 2.89*	>1000
Mandevilla velutina	73.33 ± 5.77	13.33 ± 2.89	26.67 ± 2.89*	30.00 ± 8.66*	35.00 ± 5.00**	45.00 ± 5.00*	50.00 ± 5.00**	995.15
Tithonia diversifolia	83.33 ± 2.89	11.67 ± 2.89	23.33 ± 2.89	30.00 ± 0.00**	36.67 ± 2.89*	45.00 ± 5.00*	55.00 ± 5.00*	715.25
Mikania glomerata	85.00 ± 5.00	8.33 ± 2.89	15.00 ± 5.00	23.33 ± 2.89*	31.67 ± 2.89*	38.33 ± 2.89**	50.00 ± 5.00**	>1000
Croton antisyphiliticus	73.33 ± 5.77	13.33 ± 2.89	16.67 ± 2.89**	26.67 ± 2.89*	36.67 ± 2.89 **	43.33 ± 2.89**	50.00 ± 5.00**	884.42

*p < 0.05, **p < 0.01.

Table 5.  Mortality percentage (± SEM) of the nematode Pratylenchus jaehni on nematocidal assays (24 h).

Plant extracts	Carbofuran 700 ppm	Sterile Milli-Q Water	62.5 ppm	125 ppm	250 ppm	500 ppm	1000 ppm	DL50 (ppm)
Casearia sylvestris	100.00 ± 0.00	21.67 ± 2.89	33.33 ± 2.89	38.33 ± 2.89	50.00 ± 5.00	55.00 ± 5.00	73.33 ± 5.77*	322.98
Tabernaemontana catharinensis	100.00 ± 0.00	20.00 ± 10.0	31.67 ± 7.64	35.00 ± 13.23	38.33 ± 7.64	41.67 ± 11.55	70.00 ± 5.00*	376.60
Eclipta alba	100.00 ± 0.00	20.00 ± 5.00	28.33 ± 2.89*	45.00 ± 5.00*	51.67 ± 5.00*	61.67 ± 2.89**	76.67 ± 2.89**	212.82
Serjania erecta	100.00 ± 0.00	23.3± 1 2.58	30.00 ± 0.00	28.33 ± 7.64*	41.67 ± 2.89*	48.33 ± 7.64**	76.67 ± 2.89**	249.50
Zeyheria montana	100.00 ± 0.00	18.33 ± 2.89	25.00 ± 10.00	28.33 ± 8.16	26.67 ± 7.64*	56.67 ± 10.41*	63.33 ± 17.56*	427.34
Lippia alba	100.00 ± 0.00	18.33 ± 2.89	23.33 ± 2.89	28.33 ± 2.89	30.00 ± 00.00*	36.67 ± 2.89*	41.67 ± 2.89**	>1000
Mandevilla velutina	100.00 ± 0.00	21.67 ± 2.89	33.33 ± 2.89	36.67 ± 5.77	43.33 ± 2.89*	56.67 ± 2.89**	63.33 ± 2.89**	330.24
Tithonia diversifolia	100.00 ± 0.00	17.50 ± 5.00	30.00 ± 2.89	42.50 ± 2.89*	47.50 ± 5.00*	62.50 ± 2.89**	75.00 ± 5.77**	228.27
Mikania glomerata	100.00 ± 0.00	18.33 ± 5.77	21.67 ± 5.77	33.33 ± 2.89*	41.67 ± 2.89*	48.33 ± 2.89**	61.67 ± 2.89**	467.05
Croton antisyphiliticus	100.00 ± 0.00	16.67 ± 2.89	26.67 ± 2.89	36.67 ± 2.89*	45.00 ± 0.00**	51.67 ± 2.89**	61.67 ± 2.89**	396.01

*p < 0.05, **p < 0.01.

The bioassay presented in this work may be applied as an important experimental methodology for obtaining potential nematocidal compounds active against various species of nematodes. Only a small amount of compound or of crude extract is required for each assay. Results are obtained quickly, which renders the assay adequate for guiding the purification of chromatographic fractions toward active principles. In this work, the plant extract concentration range was fixed to be as low as possible, based on previous assays and scientific reports (Zia et. al., 2001).

As demonstrated in Tables 2, 3, 4, and 5, the nematocidal effects observed for each plant extract were dose- and time-dependent. In assays using C. sylvestris, S. erecta, and Z. montana extracts against P. zeae (Table 2), after exposure for 12 h, the mortality percentage induced at the dose of 1000 ppm was 10-20% higher than those obtained with carbofuran. Remarkably, the plant extracts from T. diversifolia, Z. montana, E. alba, T. catharinensis and C. sylvestris showed a significant nematocidal effect against P. zeae even when used at the lowest doses (62.5 ppm) (Table 3).

All ten analyzed extracts presented significant nematocidal action. Those presented by E. alba (DL₅₀ (ppm) = 304.08; 55.32 – P. zeae; DL₅₀ (ppm) =>1000; 212.82 – P. jaehni, 12 and 24 h, respectively), T. catharinensis (DL₅₀ (ppm) = 215.26; 60.04 – P. zeae; DL₅₀ (ppm) = 825.44; 376.60 – P. jaehni, 12 and 24 h respectively), C. sylvestris (DL₅₀ (ppm) = 198.05; 56.94 – P. zeae; DL₅₀ (ppm) = 747.98; 322.98 – P. jaehni, for 12 and 24 h respectively), Z. montana (DL₅₀ (ppm) = 166.43; 34.08 – P. zeae; DL₅₀ (ppm) =>1000; 427.34 – P. jaehni, for 12 and 24 h, respectively) and S. erecta (DL₅₀ (ppm) = 178.74; 74.12 – P. zeae; DL₅₀ (ppm) = 689.24; 249.50 – P. jaehni, for 12 and 24 h respectively) were notable (Tables 2, 3, 4 and 5). With the exception of E. alba, those extracts induced 100% of mortality of P. zeae after 24 h of exposure and at dose of 1000 ppm (Table 3).

Z. montana and S. erecta plant extracts presented better DL₅₀ values as nematocidal agents against P. zeae, after a 12 h exposure (166.43 ppm and 178.74 ppm, respectively). On the other hand, S. erecta and T. diversifolia displayed better action against P. jaehni (689.24 and 715.25, respectively) (Tables 2 and 4). E. alba, C. sylvestris, T. catharinensis and S. erecta also presented meaningful values of DL₅₀ against P. zeae, after a 24 h exposure (55.32, 56.93, 60.04 and 74.12 ppm, respectively), and T. diversifolia presented the lowest DL₅₀ value following 24 h exposure against P. zeae (7.68 ppm) (Table 3). After a 24 h exposure against P. jaehni, the best values were observed with E. alba, T. diversifolia, S. erecta, C. silvestris and M. velutina extracts (Table 5). M. glomerata (against P. zeae and P. jaehni), C. antisyphiliticus (against P. zeae), E. alba (against P. jaehni), Z. montana (against P. jaehni) and L. alba (against P. zeae and P. jaehni) presented DL₅₀ values above 1000 ppm after 12 h. However, L. alba (against P. zeae and P. jaehni) was not effective (DL₅₀ > 1000 ppm) after 24 h (Tables 2, 3, 4, and 5). Finally, in our assays, all plant extracts displayed a better nematocidal activity against P. zeae when compared to P. jaehni. On the other hand, results for carbofuran were similar against both nematode species (Tables 2, 3, 4, and 5).

The percentage of mortality of the plant parasitic nematode Meloidogyne javanica in the presence of chloroform extract of Trigonella foegun-graceum seeds, at 1000 ppm, following 24 h of exposure, was 38% (Zia et al., 2001). The nematocidal action of the terpene picrodendrin P from the Euphorbiaceae plant Picrodendron baccatum was measured against a species of Diplogastridae (Nematoda) and the minimum lethal concentration of this compound was 4.4 mM (Watanabe et al., 1999). Begum et al. (2000) demonstrated that the nematocidal activity of the lantanoside fraction isolated from the aerial parts of the plant Lantana camara against the Meloidogyne incognita plant parasitic nematode, after 24 h exposure and at 10.000 ppm, was 90%. Williams et al. (2003) verified that the crude hexane extract from Cleome viscosa stem and leaves induced a percentage mortality by 72.69% against Meloidogyne incognita, at a dose of 500 ppm following 48 h exposure. Finally, exposing the oil fraction (SA-V) of the ethanol extract from Spatoglossum asperum algae, at 1000 ppm following a 24 h exposure, against Meloidogyne javanica, caused a perceptual mortality of 58% (Ara et al., 2005). Although the comparison between absolute potencies of different plant extracts against different nematode species in similar bioassay models is always complex, a relative comparison can be done. Taking into account the low DL₅₀ values presented in this work, as well as the nematocidal action evidenced even after relatively short periods of exposure, we assume that tested plant extracts, in particular E. alba, T. catharinensis, C. sylvestris, Z. montana, and S. erecta extracts, have high potency against plant parasitic nematodes.

Plant parasitic nematodes, including P. zeae and P. jaehni, are relevant agricultural pests, whose control depends essentially on biotechnological development that includes the search for natural alternatives. Agricultural upgrading added to food production, the use of agricultural equipment and processes, besides fertilizers and chemical pesticides, has led to a production system that is highly dependent on agricultural apparatus foreign to rural conditions. Technological application, especially employment of synthetic pesticides, leads to increased agricultural production costs with following increased food prices and decreased agriculture production (Chitwood, 2002; Epstein & Bassein, 2003; Isman, 2006). The use of synthetic nematocides or insecticide also leads to cases of worker intoxication, increased mortality of domestic and sylvester animals, soil, water, and food contamination, as well as the appearance of resistant pests (Chitwood, 2002; Epstein & Bassein, 2003; Isman, 2006). The large social, environmental and economic costs of synthetic nematocides utilization has stimulated multidisciplinary research on new alternatives for plant parasitic nematode control, like the prospect of new botanical nematocides and techniques for biological control (Prakash & Rao, 1996; De Waele & Elsen, 2007).

Botanical nematocides are potentially less dangerous than their synthetic equivalents due to their rapid biodegradation. In addition, they are usually less costly, showing complementary mode of actions, particularly when used as phytocomplexes or in extract forms. It is therefore reasonable to assume that botanical nematocides are less disposed to cause the development of nematode resistance when compared to synthetic ones (Quarles, 1992). Thus, the prospect for finding new nematocidal compounds, such as alkaloids, flavonols, or tannins, as found in plants studied in this work (Table 1), associated with progress on phytocomplex development, is of great economical interest by the lowering of agricultural costs, especially on organic production. These studies also show promise when environmental conservation of ecosystems and attention to the care of human and animal health are taken in account.

Acknowledgments

The authors are grateful to Jaime Maia dos Santos, Vilmar Gonzaga, Sérgio Ademir Calzavara (UNESP-Jaboticabal-SP) as well as to Guilherme Dagrava, Sarazete Pereira (UNAERP) and Andrea Baldocchi Pizzo (Columbia University, NY, USA), for technical assistance, and to CAPES for financial support.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.