Anthelmintic activity of plant aqueous extracts against Panagrellus redivivus in vitro Atividade anti-helmíntica de extratos aquosos de plantas contra Panagrellus redivivis in vitro

Arq. Inst. Biol., v.86, 1-11, e0672018, 2019 RESUMO: O controle de fitonematoides é muito difícil e requer uma combinação de técnicas para ter sucesso. O controle alternativo via extrato vegetal pode resultar na descoberta de substâncias nematicidas. Esta pesquisa objetivou avaliar o efeito de 33 plantas submetidas à extração aquosa contra Panagrellus redivivus in vitro. As concentrações foram preparadas a 1,25; 2,5; 5; 10; e 20%. O monitoramento ocorreu em 0, 6, 12, 24 e 30 horas após a preparação. Para a contagem, foram considerados nematoides mortos subtraídos dos vivos. Nematoides jovens também foram contados, e a eficiência dos extratos foi expressa em porcentagem de controle ou de estímulo. Os dados foram submetidos à análise de variância. Resultados significativos foram analisados pelos testes de Scott-Knott (5%) e análise de regressão múltipla. Foram observados extratos agindo como controladores, bem como estimuladores da reprodução de nematoides. A melhor performance de controle foi obtida por Carica papaya (-66% a 20%; -33,7% a 10%), Euphorbia milii (-37% a 20%), Psychotria carthagenensis (-25,5% a 2,5%), Clusia variegate (-22 a 20%) e Zamioculcas zamiifolia (-21,5% a 20%). Os extratos estimuladores foram Mentha villosa a 10% (+148%) e 2,5% (+131,5%), seguido por Aloe vera (+123% a 5%), Schinus molle (+112.5% a 10%), Schefflera arboricola (+93.5% a 5%), C. variegate (+89% a 5%) e S. molle (+88% a 5%). Alguns extratos mantiveram a população estável durante todo o experimento, apresentando menores índices de controle. Além do efeito aditivo houve uma influência individual da concentração e do tempo no controle.


INTRODUCTION
Plants are affected by biotic and abiotic factors, which leads to reduced productivity levels. Among biotic factors, diseases caused by phytoparasitic nematodes compromise root functions in soil, as water and nutrients uptake and plant support (CAMPOS et al., 2011).
Meloidogyne is the most important genus followed by Heterodera, Globodera, and Pratylenchus (JONES et al., 2013). Nematoda phylum contains over 27,000 species already described (QUIST et al., 2015), including free-living nematodes and those that affect animals. Related to phytonematodes, over 4,100 species were registered, causing damage in crops, corresponding to roughly 80 billion dollars per year (DE ALMEIDA ENGLER;FAVERY, 2011).
There are few viable techniques to control nematodes in croplands, resorting on chemical method, although it presents a high toxicity level (NTALLI; CABONI, 2017). In this context, alternative methods have been deeply studied as alternative methods, especially considering environmental concerns and human health, focused on decreasing nematicides use (DIAS et al., 2016).
Plant extract is practiced since 1972 (NAKASHIMA; SHIMIZU, 1972), and stands out because of its potential molecules with nematicidal activity, derived from secondary metabolism. Both aqueous and alcoholic plant extract were studied against nematodes (TARIQ et al., 2009). Many oils not only repeal plagues, but also present contact and fumigant action on nematodes (ISMAN, 2000). Nematicide oils are especially important in small areas (GARDIANO et al., 2009).
Given the easy handling of Panagrellus redivivus in laboratory, research aimed at testing nematicidal activity of aqueous extract from 33 plant species against P. redivivus. We considered cultural knowledge to hypothesize that new plants may contain nematicidal substances.

MATERIAL AND METHODS
Samples of 33 plant species were collected during spring season. Some plants have their athelmintic activity already described in literature (Table 1).
Plants were sent to the Phytopathology Laboratory of Universidade Federal do Paraná -Setor Palotina. Leaves were cut off for extract preparing. In the case of Carica papaya, we used seeds. Plant parts were weighted (40 g) and mixed in 60 mL of distilled water in a blender for three minutes, then filtrated on gauze. The resulting liquid was stored into test tubes and frozen at -20°C for two weeks. For evaluation, such tubes were defrosted naturally for 24 hours. Next, the liquid was centrifuged for five minutes at 4,000 rpm applied for decanting substances elimination.
Dilutions were prepared just before adding nematodes, obeying a serial dilution composed by 1 mL directly pipetted from each extract placed into small Petri plates (60 × 15 mm), jointly with 1 mL of distilled water, representing the first dilution of 20% of extract concentration. The following dilutions were performed in the same pattern, at 10, 5, 2.5, and 1.25% (w/v), with four repetitions each. Control treatment was composed of distilled water. The experimental design was in a completely randomized set in room temperature (25 ± 2°C).
Panagrellus redivivus were maintained in a creamy mixture of oat flour and distilled water. Their collection comprised nematodes climbing the boundaries of the container they were into. In sequence, nematodes were washed to be free from flour particles. Population was calibrated with water into a Bequer to enable the medium collection of 10 individuals in 20 µL of water drew up by electronic micropipette. This water volume was completed with 80 µL of distilled water, the final volume.
For nematode death (non-motile) evaluation, the initial number of nematodes added into Petri plates was identified on the top of each lid to allow a precise following counting in a stereo microscope. In addition, newborn nematodes (juveniles) were also counted to determine whether extracts would stimulate P. redivivus reproduction.
Data were registered in spreadsheets for posterior percentage calculation to set how effective or which stimulator each extract it was. Evaluation criterion considered the balance of alive nematodes at all evaluation times, of which values were subtracted from the initial ones.
The first evaluation occurred after adding nematodes to check any possible immediate effect of extracts on their behavior, considering the reaction pattern, especially as to how fast they moved. Next counting was conducted after 6, 12, 24, and 30 hours of interaction with each extract.
Data were submitted to variance analysis (ANOVA). In cases of significant results, Scott-Knott media test was applied at 5% of error probability (p = 0.05) to group extracts, according to their similarity. Extracts presenting distinguished effects were analyzed with the linear regression test (p = 0.05), which was applied individually to time and dose factors to determine their particular influence on nematodes. As the dependent variable (alive nematodes) was explained by dose and time combined, the multiple regression test (p = 0.05) was applied to all extracts to describe, mathematically, the relation between independent and dependent variables. The usefulness of regression prediction with the equation was set by determination coefficient. Tests were performed with SISVAR 5.6 ® program (FERREIRA, 2011).

RESULTS
ANOVA presented significance for time, dose, extract, extract* dose and extract* time (data not presented). To these parameters, Scott-Knott media test was applied at 5% of probability.
Concerning data presentation, negative signal before percentage values determines nematodes control, whereas the absence of signal represents stimulation on nematode reproduction.
At 0 hour, a maximum stimulation and reduction of 10 and -14%, respectively, was found (Table 2). However, the number expressed at this time refers to the real number of nematodes added to each extract; therefore, it is possible for eight nematodes to have been counted for Agave angustifolia, for example. Regardless of that, there was no significant difference at 0 hour, revealing no readily effects of any extract. Fluctuations as from six hours reveals the real effects of extracts. The highest control percentage was -17.08%, reached for Peschiera fuchsiaefolia at 30 hours; the highest stimulation, 170.42%, was caused by Mentha villosa at 30 hours.
Extracts varied on effectiveness and many of them kept the nematode population stable throughout the experiment, such as Garcinia gardneriana, Psychotria carthagenensis, Bauhinia forficata, P. fuchsiaefolia, Genipa americana, Ligustrum lucidum, Table 1. Common names, scientific names and botanical family of collected plants.
Surprisingly, among the plants randomly chosen, some ornamentals, such as C. variegate, A. angustifolia, E. milii, Z. zamiifolia, Dieffenbachia seguine, and Sansevieria trifasciata killed nematodes in a specific concentration, especially at 20%, although they were stimulated in others. E. milii kept population low and stable in concentrations lower than 20%. In the ornamental group, only B. suaveolens and S. arboricola did not control nematodes at any concentration (Table 3). P. carthagenensis controlled nematodes in all concentrations, but higher concentrations did not determine higher control percentages, revealing no linear relation between dose and its respective controlling percentage (Table 4).
In respect to regression analysis, ANOVA showed significance for time, dose and their interaction (data not presented). As a biological characteristic, extracts did not express linear behavior, fitting better at second degree polynomial equation (Table 4). Graphs allow a better individual interpretation of time, and dose influences contrasting extracts. There was an additional effect for M. villosa (Fig. 1A), where longer exposure time increased reproduction, as well as dose, although dose was not significant (Table 4). Doses of C. papaya influenced nematodes more as to death, whereas time only kept population stable (Fig. 1B). For Z. zamiifolia, nematodes were not stimulated for time or dose ( Fig. 2A); on the other hand, time caused nematode reproduction for C. pachystachya, whereas dose had no significant effects on it (Fig. 2B).
After evaluating the effect of six concentrations, the balance of alive nematodes was influenced by this combination five times more, and both Scott-Knott and regression tests masked some remarkable results, such as for C. papaya, that controlled 100% of nematodes after 24 hours at 10% w/v, and after 12 hours at 20% w/v.   All extracts showed significance to the combination between time and dose, although some of them presented no influence by dose or time variable by itself. For the extracts in which nematodes had no influence of the variables, population was kept stable throughout the experiment (Table 4).

DISCUSSION
The search for new plants with anthelmintic potential may be random or focused on no host species to phytonematodes, since these plants could contain nematicide compounds (MARTINS; SANTOS, 2016). Seen that, WIRATNO et al. (2009) tested nematicidal activity of 17 plant extracts against Meloidogyne incognita with an ethanolic extraction of chemical compounds, composing buds, leaves, flowers, roots, seeds, and stems. According to them, tobacco, clove and betelvine presented high toxicity levels, killing over 80% of nematodes at a 5 mg mL -1 dosage, followed by sweet flag, pyrethrum, and citronella, whose control potential ranged from 10 to 20%. In our study, no extract was more effective than 66%. One explanation lies on the fact that extraction method changes effectiveness of secondary metabolites against phytopathogens, also related to the influence of temperature (VENTUROSO et al., 2010). KLIMPEL et al. (2011) evaluated the effectiveness of 13 plant extracts submitted to different extraction procedures (aqueous, ethanolic, methanolic, or chloroform) against three nematodes species. According to them, each methodology eluted compounds in different grades, presenting a varied potential. Aqueous extracts performed better control in vitro, especially after 24 hours.
Extraction methods seem to vary among plant species, once methanolic extracts prepared from 24 plants were used to verify their nematicide potential against P. redivivus, revealing a reduction of over 94% by Leucaena leucocephala and Paspalum notatum (CUNHA et al., 2003), whereas other six plant extracts varied their efficiency from 11.054 to 36.33% after 48 hours. These control indexes surpass most of ours, possibly because authors applied the Tukey test, that compares all possible treatment medias among themselves, two-by-two, and the Scott-Knott test groups treatments minimize variation within groups and maximize variation among groups to compare them without ambiguity, leading to a lesser groups number formation.
It shall be said that statistical analysis also plays an important role. The Scott-Knott test is ideal to compare a large number of treatments for grouping related media without ambiguity (BHERING et al., 2008), considering that other tests may present data overlap (CANTERI et al., 2001). Nonetheless, results were hidden by multiple comparison of media (Tables 2  and 3), because statistical breakdown evaluates general dose at each time (vice-versa), and, evidently, any concentration in earlier evaluation periods had less effects. This is why, statistically, the highest control did not exceed 60% when it actually did happen, especially for C. papaya extract, that killed 100% of nematodes at 20% w/v after 12, 24 and 30 hours.
Mortality rates increased during exposure time (ELBADRI et al., 2008). However, only few extracts fit in such pattern, especially C. papaya, P. fuchsiaefolia, and P. carthagenensis (Table 2). As to ornamental plants, CUNHA et al. (2003) did not notice significant effects on P. redivivus control after 48 hours by methanolic extracts of Asparagus densiflorus, Pelargonium hortorum, Zinnia elegans, Tagetes erecta, and Euphorbia pulcherrima, which belongs to the Euphorbiaceae family, just like E. milii -that kept population low and stable until 12 hours (Table 2). However, the authors reported control set by Dendranthema grandiflorum (36.33%), and Bougainvillea glabra (23.39 %). In addition, KLIMPEL et al. (2011) did not observe any immediate effect (0 hours) of aqueous extracts on nematodes movement, exactly like what we found.
Most authors do not consider nematodes reproduction on research, solely counting the killed ones. Despite that, we considered this an important factor, because some plant extracts may stimulate nematodes, as noticed by many species in different concentrations and times (Tables 1 and 2). Some of these extracts had killed some individuals, but stimulated them more, and the final balance of alive nematodes masked those that were dead. ELBADRI et al. (2008) tested methanol and hexane extracts (0.05% concentration) of 21 medicinal plants for their toxicity against M. incognita. Nematodes were sensitive to all of them at some level, mainly after 72 hours. In this study, all concentrations of Baccharis trimera, M. villosa, and A. vera led to an increase of over than 30% in nematode reproduction (Table 3). Petiveria alliaceae had controlled over 10% of nematodes at 5 and 20% w/v concentrations, whereas A. absinthium did not control only at 5% w/v, and M. sect. pulegium had irrelevant control (0.5%) at 2.25% w/v. MARTINS; SANTOS (2016) prepared aqueous extracts of 10 medicinal plants to confront with M. incognita race 2 for 48 hours, counting inactive nematodes. Juveniles were put into water for 24 hours to analyze any eventual recovery. According to them, Eclipta alba, Ocimum basilicum, Artemisia vulgaris, Justicia pectoralis var. stenophylla, Spigelia   anthelmia, and Chenopodium ambrosioides killed over 75% of nematodes at the concentration of 10%, prepared by either infusion or maceration. Nonetheless, M. x villosa was the less effective extract against M. incognita race 2, and a stimulator to P. redivivus at any concentration (Table 3). Panagrellus redivivus as a free-living nematode moves freely in water; the same behavior was seen when nematodes were exposed to many extracts, especially the stimulator ones. This way, it was easy to check dead individuals, because their body got very strict or even end-curled, without any physical moves. WIRATNO et al. (2009) reported shape changings, depending on the plant extract. An excessive movement for stimulator extracts was observed, to which the presence of many juveniles was very common, but it did not provoke death, unlikely noticed by WIRATNO et al. (2009). MARTINS; SANTOS (2016) demonstrated differences on nematicide and static effects. ELBADRI et al. (2008) assumed that all extracts from seed had a higher nematicidal activity, matching with those we found (C. papaya). Seeds present higher chemical concentration of many soluble substances, such as alkaloids, tannin, phenolic compounds, and others (MARCOS FILHO, 2005).
In vitro evaluations aim at ranking extract plants to screen the best ones for further tests. MATEUS et al. (2014) evaluated the effect of biweekly application of Erythrina mulungu Mart. ex Benth aqueous extract, observing a reduction of 40% in M. incognita infectiveness in tomato roots, and 97.1% less eggs. This activity against nematodes is believed to result from secondary metabolites. Our in vitro results revealed the time factor, increasing P. redivivus population when treated with Erythrina verna (Table 4). FERREIRA et al. (2013) tried aqueous extracts of Sphagneticola trilobata, Tagetes patula, Tithonia diversifolia, Tridax procumbens, Unxia suffruticosa, and Zinnia peruviana on M. incognita, recording eggs outbreak over 85% in vitro; when they were tested in tomato plants, no statistical difference for root weight and nematode reproduction index was noticed, compared to treatment with water (control). Therefore, our results are promising, although they may not display the same reaction in in vivo situations (KLIMPEL et al., 2011), demanding future assays.
The hypothesis was confirmed. because some ornamental plants randomly chosen showed a control effect on P. redivivus, indicating the existence of new nematicides compounds. We first reported the action of C. variegate, Z. zamiifolia, and P. carthagenensis on P. redivivus.
For some extracts, dose and time had a concomitant influence on nematodes control, whereas for others, only one of such parameters was significant.