Plants are an important source of bioactive compounds against agricultural pests. Particularly plants with a nematicide activity and their by-products have the potential of being used on the field and of covering part of the demand for chemical nematicides. Chemical compounds have been isolated from various plant families, particularly Asteraceae, in vitro (α-Terthienyl) (^{Chitwood, 2002}; ^{Oka, 2010}, 2012). However, these compounds have not yet proven to be effective on the field and have not been commercially developed (^{Gommers and Bakker, 1988}). Inula viscosa, a common Asteraceae in Mediterranean countries, has proven to have nematocidal activity, due to the production in its sprouts of cosic and isocosic acids, which were isolated and applied against Meloidogyne javanica (^{Oka, 2001}). The extracts of the Verbesina encelioides leaves and flowers were also evaluated against the nematode M. javanica, with favorable results (^{Oka, 2012}). In addition to the Asteraceae, essential oils and the monoterpene components of other herbaceous plants such as Ocimum sanctum, Xylopia aethiopica, Thymus vulgaris and others have displayed nematocidal effects (^{Eloh et al., 2019}).

Meloidogyne incognita is one of the most harmful nematodes in agriculture worldwide, with damages in diverse annual and perennial crops. The feeding and development of the females cause root thickening and produce important lesions when releasing their eggs onto the soil. When the population density is high, damages to the roots are such that it can cause a loss in yield and quality of the production (^{Aissani et al., 2013}; ^{Pavaraj et al., 2012}). The most widely used control strategy for nematodes is synthetic pesticides. However, these pesticides increase production costs and affect the dynamics of the soil due to the molecules from the group of organophosphates, carbamates and some high-residue products that lead to important environmental pollution problems (^{Murcia and Stashenko, 2008}; ^{Álvarez et al., 2015}).

In the light of this problem, it is increasingly important to evaluate alternatives with lower impacts, such as the use of bio-rational strategies like the use of Verbesina sphaerocephala. This Asteraceae is endemic to western Mexico (Jalisco, Michoacán, Nayarit, Guanajuato, Guerrero) (^{Rzedowski et al., 2011}) and it is commonly known as capitaneja, capitana, árnica capitaneja, vara blanca or palo espino. The use of this species is not as extensive and only taxonomic and ethnobotanical information is available, the latter provided by the town of San Martin de las Flores, Jalisco, where the population uses it for its ethnopharmacological properties and agricultural potential (^{Velasco-Ramírez et al., 2019}). It is therefore proposed as a botanical nematocidal control (aqueous extract), since this type of extracts can be produced by the farmers themselves to be used as an alternative to minimize the incidence and damages caused by nematodes in crops of commercial interest, such as cucumbers (Cucumis sativus).

The cucumber is the fourth most widely produced crop in the world, with a production of 1,7 million tons in 2016 (^{Sayedain et al., 2021}), and Mexico is the fifth largest producing country in the world, with a production of 826,485 tons. The main cucumber producing states in Mexico are Sinaloa (268,878 t ha^-1), Sonora (152,457 t ha^-1), Michoacán (67,653 t ha^-1) and Jalisco is the tenth state (20,454 t ha^-1) (^{SIAP, 2020}). Despite its importance, the yields of this crop have considerable reductions due to pests and diseases. For example, diseases have included Fusarium spp, Rhizoctonia spp, Damping off (Phytophthora spp. andPythium spp.), angular leaf spot (Pseudomonas syringae) and the root-knotting nematode (Meloidogyne spp.) (^{Satyendra and Rekha, 2021}). Due to the above, the aim of this investigation was to test whether aqueous extracts taken from the leaves of V. sphaerocephala promote the development and reduce damages caused by M. incognita in cucumber plants (Cucumis sativus).

Several healthy (assymptomatic) young leaves (vegetative phase) of the wild species Verbesina sphaerocephala were gathered from the hills surrounding San Martin de las Flores in the municipal area of San Pedro Tlaquepaque, Jalisco (longitude: -103.282778 latitude: 20.585278 at 1540 masl). The leaves were dried at room temperature in the laboratory (~26 °C), pulverized using a blade mill (Hamilton Beach® 80335) and placed under a proximal chemistry test and a phytochemical analysis to determine the main biomolecules contained in the leaves. Next, 100 g of dry weight were placed in 1 L of distilled water, stirring constantly for 15 min, followed by an autoclave at 121 °C for 1 h at 1.2 kg cm². The hot extracts were poured through Whatman No. 40 filter paper and stored in glass jars at 4 °C. The liquid V. sphaerocephala extracts were designated as a stock solution; the pH and electric conductivity (CE, dS m^-1) were then measured. Finally, the color of the extracts was determined visually. All parameters were evaluated in triplicate.

A Meloidogyne incognita population, previously identified with morphometric and pictorial keys (^{Eisenback et al., 1983}) and obtained from the tomato plantation located in the school greenhouse of the Centro Universitario de Ciencias Biológicas y Agropecuarias (CUCBA) of the Universidad de Guadalajara, was isolated for its growth and reproduction in vivo on tomato plants (Solanum lycopersicum) in 3 kg (20 x 15 cm) polyethylene bags, under greenhouse conditions in the Phytopathology area of the CUCBA.

To obtain the inoculant, the roots were washed using tap water, cut into pieces of approximately 1 cm in length, 10 g were dried and placed in a blender. Next, 20 mL of sodium hypochlorite solution (NaOCl) at 0.5% were added, along with 180 mL of distilled water, mixing for 3 min. It was then filtered through a 25 µm sieve, where the eggs remained and were later stored in a cup containing a precipitate; the suspension with eggs was diluted to 200 mL. A 1 mL aliquot was taken from the egg suspension from the precipitates cup, and with the help of the grid of a dissection counting box, counting began under the compound microscope (^{Hussey and Barker, 1973}). Twenty days after planting, using 24 polyethylene bags, the nematode was inoculated by emptying 105 mL of the egg suspension into every pot, distributed into three orifices, each one 5 cm deep. In this way, 3,030 eggs were inoculated in each pot.

The experiment was carried out in September 2019 in the CUCBA greenhouse using paraíso Enza Zaden cucumber seeds (100 seeds), previously sterilized in a 10% sodium hypochlorite solution for 30 min and then rinsed with distilled water. The seeds were planted in a germination tray with a mixture of peat substrate and vermiculite, 50:50 (sterilized in an autoclave at 121 °C for 20 min at 1.21 kg cm²), and irrigated on a daily basis at field capacity. They were left to grow in the greenhouse for 30 days and transplanted in plastic pots with a homogenized mixture of sand substrate and leaf soil, previously sterilized in an autoclave in a 70:30 proportion. The plants were kept in the greenhouse at 27-32 °C under sunlight, irrigated every 2 days and fertilized every 72 h using the fertilizer Poly-Feed® (19-19-19) with a dose of 1g L^-1 until the end of the experiment.

Seven different treatments were set up: 1: Control, plants without nematodes (P), 2: plants with nematodes (P+N), 3: plants with nematodes and carbofuran (Furadan®) 350 g L^-1 (P+N+C), 4: plants with nematodes and Trichoderma sp. 200 mL L^-1 (P+N+T), 5: plants with nematodes and a 25% V. sphaerocephala extract (P+N+V 25%), 6: plants with nematodes and 15% V. sphaerocephala extract (P+N+V 15%) and 7: plants with nematodes and a 10% V. sphaerocephala extract (P+N+V 10%). We applied 50 mL of extract for every pot. All treatments were applied two days after transplanting and directly after inoculation. The experiment was organized in a random block design with five repetitions (pots) and was carried out twice.

Three evaluations were carried out (every 10 days) during the initial growth of the crop until the end of the end of the experiment and the variables considered were plant height (cm, from the apical meristem to the main root), stem diameter (mm), concentration of chlorophyll [SPAD (Soil Plant Analysis Development)- 502], number of leaves and of flowers. By the end of the experiment, 40 days later, plants and roots were removed, washed with drinkable tap water and the root length was recorded (cm), along with the index of root knotting and the number of eggs in 10 g of roots. On the other hand, larvae were extracted and counted out of 20 g of roots, following ^{Bridge and Page (1980}) and a record was made of the dry weight of the tissue, which was kept at 60 °C for 48 h in a Felisa® brand drying oven. Finally, the number of larvae in 100 g of soil per treatment were counted, based on Baermann’s funnel technique from moist substrate (^{Barker, 1985}).

The data gathered from the experiment were analyzed with a one-way ANOVA for each variable per treatment and evaluation times and Tukey’s mean comparison test (p <0.05) using the statistical software Statgraphics® Centurion XV (New Jersey, United States) for Windows.

The proximal chemistry evaluations performed on V. sphaerocephala displayed 17.7% proteins, 10.4% raw fiber, 15.2% total ashes, 4.6% ethereal extract, 2.9% nitrogen, 45.2% carbohydrates and 6.8% moisture, which contains a large amount of carbohydrates, thus explaining the high capacity to absorb water. The phytochemical analysis (Table 1) gave results that show the presence of phenolic and alkaloid secondary metabolites in aqueous extracts obtained from leaves, most of which may be present in the plant-pathogen interaction and the defensive function against parasites or insects due to their toxicity, respectively. The pH values for the V. sphaerocephala extracts were alkaline (8.33), the CE was 1 and 1.3 dS m^-1 and the color was dark green.

The effect of the V. sphaerocephala extracts on the development of C. sativus plants for the variable of height was significantly (P= 0.0103) higher in treatment P+N during the first evaluation with an average of 24.6 cm; in the second evaluation, the treatment of P+N+V10% obtained a higher growth, with 26 cm, and in the third evaluation, the treatment of P+N+V25% obtained an average of 27.2 cm (Figure 1A). The stem diameter was significantly (P= 0.0514) greater in treatments P+N+T and P+N+V15% during the first evaluation with averages of 0.48 y 0.50 mm respectively. In the second evaluation, the diameter of the stems of the plants in treatment P+N+V25% was significantly (P= 0.06521) greater, with an average of 0.50 mm. At the end of the experiment, the diameter of the stems of the plants in treatments P+N+V10% and P+N+V15% was significantly (P= 0.09524) greater, with values of 0.56 and 0.51 mm respectively (Figure 1B). Regarding the number of leaves, in the first evaluation, treatments P+N, P+N+T and P+N+C were greater (P= 0.0605), with an average of eight leaves, in the second evaluation, treatment P+N+V10% obtained the highest (P= 0.0001) number of leaves (12), and in the third evaluation, all plants treated with V. sphaerocephala displayed a higher (P= 0.0009) average number of leaves (10) in comparison with the other treatments (Figure 1C). The number of flowers was higher (P= 0.0517) in treatment P+N with six flowers per plant on average in the first evaluation. In the second evaluation, the highest number of flowers was found in treatment P+N+V10% (P= 0.0001), with an average of nine flowers. In the third evaluation, the highest number of flowers was found in treatments P, P+N and P+N+C, since all plants treated with V. sphaerocephala were already in the stage of fruition (Figure 1D). The index of chlorophyll recorded in the first evaluation was similar in all treatments (P= 0.0565). In the second evaluation, treatments P+N+T, P+N+C, P+N+V15% and P+N+V25% displayed a higher chlorophyll index than the rest of the treatments. In the third evaluation, there was a significant decrease in all treatments, due to the number of fruits and flowers in the plants (Figure 1E).

Figure 1 Effect of the extracts of V. sphaerocephala on C. sativus plant development during the vegetative state (E1), flowering (E2) and maturation (E3). A) Plant height; B) Stem diameter; C) Number of leaves; D) Number of flowers; E) Chlorophyll index; F) Root length, and G) Dry weight of root. Means with different letters represent statistical differences according to Tukey’s test (P≤ 0.05). P (without nematodes), P+N (with nematodes), P+N+T (nematodes + Trichoderma ), P+N+C (nematodes + carbofuran), P+N+V at 10, 15 and 25% (nematodes + Verbesina extracts in different concentrations). 

The root length in all V. sphaerocephala treatments were significant (P= 0.0968), with an average of 25 cm (Figure 1F). The dry weight of the roots was significantly (P= 0.0650) higher for treatment P+N+V10% with an average of 1.7 g (Figure 1G).

The effect of the V. sphaerocephala extracts on the damage of M. incognita on C. sativus plants 50 days after transplanting, presented a lower rate of root-knotting (P= 0.05124) in comparison with the treatment P+N. The roots of treatments P+N+V10% and P+N+T presented the lowest index of root-knotting (14.3%), followed by the treatments with extracts at 15%, Carbofuran and extracts at 25%. Plants with treatments V. sphaerocephala al 10% (P+N+V10%) presented no root-knots or egg masses, and these extracts were found to reduce the reproduction of females and the hatching of eggs (Figure 2A). Significant differences (P= 0.0001) were found for the number of eggs in 10 g of roots in treatment P+N and the lowest values were found in treatments P+N+V10%, P+N+V15% and Trichoderma, which caused reductions of 20, 23.6 and 24.8% in the number of eggs in comparison with the control treatment (Figure 2B).

Regarding the number of larvae in the substrate, the treatments with carbofuran and with V. sphaerocephala extracts at 15% presented the lowest number, with 100 and 200 larvae, respectively. The larva population density in these treatments was significantly lower than the larva population densities presented in the control treatment. On the contrary, the treatment with Trichoderma displayed the highest number of larvae (380) for every 100 g of substrate. The V. sphaerocephala extracts did not entirely stop larvae from developing, probably since these products only paralyze the nematodes instead of causing their death (Figure 2C).

Studies have pointed out the presence of terpenoid and flavonoid compounds and of free aromatic acids (^{Bohlmann et al., 1980}) in species such as V. virgata (^{Martínez et al., 1983}), V. sordescens (^{Bohlmann et al., 1982}), V. glabrata and V. luetzelgurgii (^{Bohlmann et al., 1980}). This investigation presents the first phytochemical study of V. sphaerocephala (Table 1), in which the presence of phenols (phenolic and/or polyphenols, mainly) that function as antioxidants and may have preventive properties for some diseases and are involved as regulators in defensive processes of plants, and even have allelopathic effects (^{Willians et al., 2004}), became evident. Also clear was the presence of alkaloids, which have properties that have been documented as protective of plants against the attack of bacteria, viruses, fungi and herbivores (^{Bruneton, 2001}). Based on these results, there is an intention for future investigations of the antioxidant and inhibiting activities of radicals, as well as the type of alkaloids found in V. sphaerocephala to determine the way in which they react.

Figure 2 Effect of the V. sphaerocephala extracts on the damage caused by M. incognita on C. sativus plants. A. Root-knotting index. B. Eggs in 10 g of roots. C. Larvae in 100 g of soil. Means with different letters represent statistical differences according to Tukey’s test (P≤ 0.05). P (without nematodes), P+N (with nematodes), P+N+T (nematodes + Trichoderma ), P+N+C (nematodes + carbofuran), P+N+V at 10, 15 y 25% (nematodes + Verbesina extracts in different concentrations). 

During the evaluations throughout the experiment, the 10 and 25% V. sphaerocephala extracts in all variables showed that they stimulate plant development, even in pots and in the presence of M. incognita, as mentioned by ^{Mora et al. (2013}), who point out that several Verbesina species contain active organic compounds as growth regulators.

According to the results obtained in each of the cucumber growth variables in pots, the 10% Verbesina extracts were efficient for plant height, number of leaves, flowers, chlorophyll index and dry weight of roots, as indicated by ^{Oka (2012}) in an investigation carried out on V. encelioides aqueous extracts, in which the nematocidal activity of M. javanica and the effect on the growth of Inula viscosa. The investigation coincides with the results of this investigation, in which Verbesina was found to be an efficient bio-stimulant, since it acted on the physiology of cucumber plants in different ways, and vigor and crop yield were not affected by the infestation of M. incognita.

Regarding the damage of M. incognita on cucumber plants, Trichoderma improves the resistance of the roots to the. Because it is considered an inoculant that promotes the growth and provides benefits and protection to the plants, it may have stimulated root development, helping reduce damage caused by nematodes (^{Hernández-Melchor et al., 2019}). ^{Baños et al. (2010}) recommend the use of Trichoderma spp. in the management of Meloidogyne spp. in vegetables, since they proved to be an effective bioregulator against nematodes of this genus due to its toxins and hyphae. On the other hand, applying Trichoderma showed no nematocidal activity, since a greater number of nematodes were found on the soil. Stimulating the development of roots may have allowed a greater number of females to establish their feeding site, leading to a higher reproduction rate. It must be pointed out that the cucumber crop is susceptible to the attack of the M. incognita nematode, as expressed by ^{Julca et al. (2001}).Similar results were reported by ^{Oka (2012}) who, using V. encelioides, evaluated aqueous leaf extracts against M. javanica. ^{Vázquez-Sánchez et al. (2018}) determined the effectiveness of aqueous extracts of V. sphaerocephala leaves against Naccobus immobilizing the nematode, hindering their ability to feed and invade host leaves. Extracts of garlic (Allium satium), marigold (Tagetes erecta), papaya (Carica papaya) and Bermuda grass (Cynodon dactylon) have also been used to determine the nematocidal effects on Meloidogyne spp. in bean plants (Phaseolus vulgaris) (^{Parada and Guzmán, 1997}). The use of Tagetes zypaquirensis essential oils has also been useful in the management of the nematode Meloidogyne spp. as published by ^{Álvarez et al. (2015}) and recommended by ^{Eloh et al. (2019}) in an evaluation carried out with the selection of 10 plants grown in West Africa against M. incognita.

This investigation determined that the use of aqueous extracts of V. sphaerocephala at 10 and 15% may have the biological potential to minimize damages on roots caused by M. incognita. It is therefore necessary to out carry out more rigorous studies to determine the effect of the extracts on the reproduction rate of M. incognita.