Plant-parasitic nematodes of the genus Meloidogyne cause significant economic losses worldwide (^{Abd-Elgawad and Askary, 2015}). One hundred species of the genus Meloidogyne that parasitize vascular plants have been described (^{Moens et al., 2009}). M. incognita is one of the most widely distributed species, followed by M. javanica, M. arenaria, and M. hapla (^{Carrillo-Fasio et al., 2019}).

However, M. enterolobii (= M. mayaguensis) has become one of the most important species worldwide in recent decades because of its high aggressiveness (^{Castagnone-Sereno, 2012}), its reproductive potential, wide geographic distribution, large host range, including both herbaceous and woody plants, and its ability to establish on plants carrying resistance genes to other Meloidogyne spp., such as some genotypes of tomato (Mi-1), potato (Mh), soybean (Mir1), and chili bell pepper (N and Tabasco) (^{Castagnone-Sereno, 2012}). M. enterolobii has been reported in Africa, Europe, the United States of America, Central America, and South America (Castagnone-Sereno, 2012; ^{Moens et al., 2009}). Recently, in Mexico, it was reported parasitizing watermelon (Citrullus lanatus) in the state of Veracruz and chilli (Capsicum annuum) in Sinaloa (^{Ramirez-Suarez et al., 2014}; ^{Villar-Luna et al., 2016}). In the states of Sinaloa and Baja California Sur, M. enterolobii is more prevalent than M. incognita (^{Carrillo-Fasio et al., 2019}; ^{Romero et al., 2019}).

Management of Meloidogyne spp. has focused mainly on chemical control, soil biofumigation, amendments, and resistant varieties (^{Cid del Prado-Vera et al., 2018}). However, these control methods have not been sufficient for M. enterolobii given the intrinsic characteristics of its biology. Therefore, it is important to continue searching for other management alternatives for this nematode species. One of the alternatives is the use of nematocidal compounds found in the environment (^{Akhtar and Malik, 2000}). On the other hand, one of the sustainable alternatives is the use of endophytic bacteria such as Serratia ureilytica, which has been evaluated in vitro against Nacobbus aberrans (J2) and in infected chilli plants, obtaining encouraging results (^{Wong et al., 2021}).

These types of compounds with nematocidal activity have been reported in mushrooms of the genus Pleurotus (^{Barron and Thorn, 1987}), in addition to having the ability to colonize and degrade a great variety of lignocellulosic residues and high medicinal and nutritional value (^{Cohen et al., 2002}). A toxin was identified in this genus of fungi as trans-2-decenedioic acid, with a nematocidal effect against Panagrellus redivivus (^{Kwok et al., 1992}). There have also been studies with activity against phytoparasites (^{Castañeda-Ramírez et al., 2020}). Furthermore, ^{Heydari et al. (2006}) reported the presence of a toxin in five Pleurotus spp. in water agar with an effect against M. javanica (J2). Likewise, the proteolytic activity of P. ostreatus is reported on the free-living nematode Panagrellus sp. (^{Genier et al., 2015}).

Additionally, it has been attributed to antiparasitic properties, mainly cestocidal (^{Samsam-Shariat et al., 1994}) and nematocidal in the livestock area (^{Pineda-Alegría et al., 2017}); however, in the agricultural area, it is important to conduct studies that contribute to the development of natural bionematicides based on edible fungi. The PdR-2 fraction of the edible mushroom P. djamor has been previously reported with nematocidal activity on infective larvae (L3) of Haemonchus contortus in vitro studies (^{González-Cortazar et al., 2020}). For this reason, the nematocidal activity of the PdR-2 fraction of P. djamor against J2s of M. enterolobii was evaluated in the present study.

The present study was carried out at the Helminthology Laboratory of the National Center for Disciplinary Research in Animal Health and Safety (CENID-SAI) of the National Institute of Forestry, Agriculture, and Livestock Research (INIFAP), located in Jiutepec, Morelos, Mexico. J2 were obtained from the root-knot of tomato cv. ‘Rio Grande’ (monoxenic crop, population of Ahome, Sinaloa) at the Postgraduate College, Montecillo Campus, Texcoco. Eggs were extracted according to the methodology of ^{Vrain (1977}) and incubated at 28±1 °C in Petri dishes with sterile distilled water to obtain J2.

Obtaining the PdR-2 fraction of P. djamor. The PdR-2 fraction was obtained from the hydroalcoholic extract (ethanol-water 7:3) of P. djamor strain ECS-0127 basidiomata. The extract (30 g) was adsorbed on 40 g of normal phase silica gel (70-230 mesh, Merck) and placed on a pre-packed glass chromatographic column. The elution system was performed with dichloromethane:methanol with the following system changes: 100, 90:10, 70:30, 50:50, and 100%. Samples were collected and concentrated in a rotary evaporator. The fractions were analyzed by thin layer chromatography and pooled according to their retention factor and elution system. From this fractionation, 22 fractions were obtained, of which fractions 11 to 18 formed the PdR-2 fraction (^{González-Cortazar et al., 2020}).

In vitro evaluation of the PdR-2 fraction of P. djamor against J2 of M. enterolobii. Different concentrations of the PdR-2 fraction and 100 J2 specimens of M. enterolobii were placed in a 96-well plate. The final concentrations of the fraction were 0.039, 0.078, 0.156, 0.132, 0.625, and 1.25 mg mL^-1 and controls, which were distilled water and Levamisole (commercial anthelmintic) 5 mg mL^-1 in a volume of 100 µL (n=4). The J2 were exposed for 24 h, and after the exposure time, 10 aliquots of 10 µL were taken from each repetition, and the number of dead and alive individuals were counted. The number of dead individuals was used to calculate the percentage of mortality using the following formula:

larval mortality=dead nematodesdead nematodes+  live nematodes×100

Data were analyzed by analysis of variance (ANOVA) with the general linear model and a comparison of means with Tukey’s test (*p ≤ 0.05). STATGRAPHICS Centurion XV, V. 15.2.06 software was used for statistical procedures.

The mortality of M. enterolobii J2 is presented in Table 1. No dead individuals were recorded when distilled water was used, and the J2 presented movement as shown in Figure 1A; however, with the anthelmintic (Levamisole), 100% mortality was recorded at 24 h of exposure (Figure 1B) with statistically significant differences (*p ≤ 0. 05) with respect to the lowest concentrations of the PdR-2 fraction (0.039, 0.078, and 0.156 mg mL^-1) and water. The mortality for the lowest concentrations were 40.3 to 65%. On the other hand, concentrations of 0.156, 0.132, 0.625, and 1.25 mg mL^-1 of the PdR-2 fraction showed higher mortality percentages (76.8-86.3%), and these were significantly different from the water control (*P≤ 0.05).

Figure 1 J2 of Meloidogyne enterolobii exposed to H₂O and levamisole for 24 h, observed under light microscopy (20X). A) J2 exposed to H₂O and B) J2 exposed to levamisole. 

Currently, research carried out for the control of M. enterolobii has focused on the search for crops resistant to this phytonematode, given that this species is able to reproduce in different crops that possess the resistance genes currently available for the main species of Meloidogyne. Some of these genes are Me1-Me6, Mech1 and Mech2; however, it has been reported that M. enterolobii can infect plants with these Meloidogyne-resistant genes (^{Carrillo-Fasio et al., 2020}; ^{Philbrick et al., 2020}). Therefore, another sustainable alternative for the control of this nematode could be the use of compounds isolated from edible mushrooms.

Several species of edible mushrooms belonging to the genus Pleurotus have been shown to possess nematocidal properties; such studies have been conducted mainly in the livestock area using gastrointestinal nematode models of small ruminants, reporting a new line of research to be addressed to demonstrate their effectiveness in the sustainable control of these parasites (^{Castañeda-Ramírez et al., 2020}). The nematocidal activity is attributed to secondary metabolites, such as fatty acids, isolated from P. djamor species (e.g., linoleic acid) (^{Pineda-Alegría et al., 2017}). Other secondary metabolites, such as polyols, trehalose, mannitol, squalene, stearic acid, and β-sitosterol, have been isolated from P. eryngii (^{Cruz-Arévalo et al., 2020}). Regarding the PdR-2 fraction obtained from P. djamor (ECS-0127) was reported in a study by ^{González-Cortázar et al. (2020)}, 100% lethal nematocidal activity was reported against eggs and infective larvae (L3) of H. contortus in in vitro experiments. Similarly, in vivo evaluation of the PdR-2 fraction reached a reduction of up to 80% of the H. contortus parasite load using as a study model the gerbil Meriones unguiculatus. Finally, the compound responsible for the nematocidal activity of the PdR-2 fraction was identified as allitol and a terpene in a 9:1 ratio by nuclear magnetic resonance (NMR) proton (1^H) and carbon (13^C) spectroscopic analysis.

Most of the research works where the use of Pleurotus mushroom metabolites has been explored are focused on Meloidogyne spp., such as M. javanica (^{Heydari et al., 2006}; ^{Oka, 2001}); however, for the recently reported species in Mexico, M. enterolobii, research for its management is required. Therefore, our research provides relevant information. At a concentration of 0.132 mg mL^-1, mortality percentages higher than 84% were obtained on J2s of M. enterolobii. Similar results were reported by ^{Li et al. (2007}), who isolated three metabolites from P. ferulae extracts with a nematocidal effect on Bursaphelenchus xylophilus and Panagrellus redivivus. Of these, only the compound cheimonophyllon E registered 84% mortality on B. xylophilus at 72 h, while the PdR-2 fraction evaluated in this work registered a similar percentage at 24 h. Therefore, The PdR2 fraction showed nematocidal effect against J2s larvae of M. enterolobii and could potentially be used for the sustainable control of nematodes of agricultural importance.