Introduction

Mexico is one of the world's leading vegetable producers, ranking ninth in tomato (Solanum lycopersicum) production with 4,271,914 tons in 2022, and fifth in cucumber (Cucumis sativus) production with 1,038,999 tons. The state of Sinaloa is the main producer of both tomato and cucumber, with 677,612 and 314,150 tons, respectively (SIAP, 2023).

Rotylenchulus reniformis and Meloidogyne enterolobii are highly significant species and are among the top 10 most economically impactful and scientifically important plant- parasitic nematodes worldwide (^{Jones et al., 2013}). These nematodes have been reported to severely affect vegetable crops in the state of Sinaloa (^{Martínez-Gallardo et al., 2019}; ^{Salazar-Mesta et al., 2023}a, 2023b; ^{Valdez-Morales et al., 2024}).

Both Rotylenchulus spp. and Meloidogyne spp. form feeding sites in the roots of plants. Meloidogyne enterolobii is a sedentary endoparasitic nematode, and its parasitism involves the induction of giant cells in the parenchyma (^{Starr et al., 2002}; ^{Perry and Moens, 2013}). Meanwhile, R. reniformis is a semi-endoparasitic nematode and induces the formation of syncytia, primarily in pericycle cells (Starr et al., 2002). Although the infection sites of ectoparasitic and endoparasitic nematodes differ, interactions between species can occur, and these interactions may be antagonistic or suppressive to one of the species involved (^{Khan et al., 1985}; ^{Robinson et al., 2007}; ^{Gomes et al., 2014}).

Studies on interactions between plant-parasitic nematodes have shown that such relationships can be antagonistic for either nematode when feeding on the same plant (^{Diez et al., 2003}; ^{Melakeberhan and Dey, 2003}; ^{Manzanilla-Lopez and Starr, 2009}; ^{Ferreira et al., 2015}; ^{Fontana et al., 2018}). Moreover, the effects of nematode species interactions are related to the nature of parasitism and competition, as the competitive relationship increases with the complexity of the host-parasite interaction (^{Kesba et al., 2005}; ^{Moens et al., 2006}; ^{Robinson et al., 2007}; ^{Bhat and Parveen, 2012}).

In the case of plant-parasitic nematodes from the genera Helicotylenchus, Pratylenchus, and Rotylenchulus, it has been demonstrated that interactions occur due to competition for space and nutrients (^{Bhat and Parveen, 2012}; ^{Ferreira et al., 2015}a, 2015b; ^{Gomes et al., 2014}; ^{Fontana et al., 2018}). Additionally, several studies have determined the interaction between R. reniformis and Meloidogyne spp., including M. javanica and M. incognita, in various host plants such as pineapple (Ananas comosus), cotton (Gossypium hirsutum), tomato, and castor bean (Ricinus communis).

These studies recorded that R. reniformis has a competitive advantage over Meloidogyne species in joint inoculations, as a reduction in root galling was observed (^{Diez et al., 2003}; ^{Faske and Hurd, 2015}; ^{Ferreira et al., 2015}a, 2015b). The interaction of M. enterolobii with lesion nematodes, such as Helicotylenchus dihysteroides, has also been evaluated in guava (Psidium guajava) seedlings; however, symptoms caused by M. enterolobii were observed in both separate and combined inoculation treatments (^{Gomes et al., 2014}).

The interactions between M. enterolobii and R. reniformis in tomato and cucumber plants have not been studied, but it is suggested that R. reniformis may have a competitive advantage over the root-knot nematode due to its high degree of parasitism (^{Diez et al., 2003}; ^{Robinson, 2007}; ^{Aryal et al., 2011}). Therefore, the objective of this study was to determine the competitive interaction between R. reniformis and M. enterolobii in tomato and cucumber plants under greenhouse conditions.

Materials and methods

The experiments were conducted under greenhouse conditions from December 2022 to May 2023. Tomato (Solanum lycopersicum cv. Imperial) and cucumber (Cucumis sativus cv. Zeus) seeds were germinated in 60-cell trays filled with sterile substrate (Peat Moss). Twenty-one days after germination, seedlings were directly inoculated into the germination trays with 2000 juveniles (J2) of each nematode (R. reniformis and M. enterolobii). The experimental unit was one plant in a cell. Treatments were as follows: T1 = R. reniformis; T2 = M. enterolobii; T3 = R. reniformis was inoculated, followed by

M. enterolobii 15 days later; T4 = M. enterolobii was inoculated, followed by R. reniformis 15 days later; T5 = both species were inoculated on the same day; T6 = uninoculated control. The populations of R. reniformis and M. enterolobii used in this experiment were previously characterized and identified through a combination of morphological and molecular analyses by ^{Valdez-Morales et al. (2024}) and ^{Salazar Mesta et al. (2023}a), respectively.

Evaluations were conducted at 30 and 50 days after inoculation (DAI). The variables measured were the reproduction factor (RF) of each nematode, the galling index for M. enterolobii, and root necrosis percentage for R. reniformis. To evaluate the galling index, roots were extracted, washed with tap water, and the number of galls per root was counted for each treatment.

For the reproduction factor assessment, the final population of each nematode species was determined using the formula RF = Pf/Pi, where Pi represents the initial nematode population at the time of inoculation, and Pf is the final nematode count at root extraction (^{Ferris et al., 1993}). Roots were stained with 1% acid fuchsin (^{Byrd et al., 1983}), females and egg masses of each species were observed and counted using a stereoscopic microscope. In addition, juvenile and male nematodes were extracted from the substrate of each root using the Baermann funnel and Cobb's sieving methods (^{Christie and Perry, 1951}).

Statistical analysis. The experiment followed a completely randomized three-factor design with six treatments and three replicates. The response variables were the reproduction factor of both nematodes and disease severity, which included the galling index of M. enterolobii, and the percentage of root necrosis caused by R. reniformis. Root necrosis was evaluated on a visual scale of 1 to 100% based on root damage (^{Speijer and De Waele, 1997}; ^{Nega and Fetena, 2015}). The galling index (GI) was evaluated using the ^{Taylor and Sasser (1978}) scale, where: 0 = 0 galls, 1 = 1-2 galls, 2 = 3-10 galls, 3 = 11- 30 galls, 4 = 31-100 galls, and 5 = >100 galls. The data obtained were transformed to log (x + 1) to standardize variance. The transformed means were compared using Fisher's LSD test (P < 0.05) with SAS software version 9.3 (SAS Institute, Cary, North Carolina, USA).

Results

Reproduction and pathogenicity of R. reniformis and M. enterolobii. The initial inoculum levels of both nematodes increased during the trial, and the species displayed variable reproduction factors depending on the host. However, M. enterolobii exhibited a higher reproduction factor (RF) (41.9 and 20.9 in cucumber and tomato, respectively) compared to R. reniformis (7 and 1.8 in cucumber and tomato, respectively) on both hosts (Figures 1 A, C and 2 A, C).

Figure 1 Reproduction and severity of R. reniformis and M. enterolobii in cucumber plants at 30 and 50 days after inoculation (DAI). A) Reproduction factor of M. enterolobii. B) Number of galls caused by M. enterolobii. C) Reproduction factor of R. reniformis. D) Percentage of root necrosis caused by R. reniformis. The comparison of means was conducted between bars of the same color; means that do not share a letter are significantly different. 

Figure 2 Reproduction and severity of R. reniformis and M. enterolobii in tomato plants at 30 and 50 days after inoculation (DAI). A) Reproduction factor of M. enterolobii. B) Number of galls caused by M. enterolobii. C) Reproduction factor of R. reniformis. D) Percentage of root necrosis caused by R. reniformis. The comparison of means was conducted between bars of the same color; means that do not share a letter are significantly different. 

In cucumber plants, mature females and egg masses of both nematodes were observed in all inoculated treatments at 30 days after inoculation (DAI) (Figure 3A, C, E). In contrast, in tomato plants, mature females and egg masses of M. enterolobii were seen at 30 DAI, but R. reniformis only showed mature females without egg masses. The highest reproduction of both nematodes occurred at 50 DAI (mature females and egg masses) in both plant species (Figures 3B, D, F).

Figure 3 Micrographs of Rotylenchulus reniformis and Meloidogyne enterolobii parasitizing cucumber and tomato roots at 50 days after inoculation (DAI). A) Female of M. enterolobii in cucumber root. B) Female of M. enterolobii causing galls in tomato root. C) Female of R. reniformis affecting cucumber roots. D) Female of R. reniformis in tomato root. E) Cucumber root with a gall caused by M. enterolobii and three mature females of R. reniformis around it. F) Tomato root with a gall from M. enterolobii and necrosis caused by R. reniformis. Scale Bars: A= 300 μm; B, E, F= 500; C, D= 100 μm. 

Both R. reniformis and M. enterolobii showed significant differences between treatments across both plant species. Notably, both nematodes had higher RF when inoculated separately (T1 and T2) with values of 49.1 and 20.9, respectively, in cucumber plants, and 7 and 1.8, respectively, in tomato plants, compared to the combined inoculation treatments (T3, T4, and T5). In treatments where one species was inoculated first and the other 15 DAI (T3 and T4), the species inoculated first exhibited a higher number of eggs, females, juveniles, and consequently a higher RF (Table 1), and caused greater symptom severity compared to the species inoculated later (Figures 1 and 2).

Rotylenchulus reniformis displayed higher RF in individual inoculations for both cucumber and tomato plants; however, its RF decreased when co-inoculated with M. enterolobii in the same root. Specifically, R. reniformis showed an RF reduction of up to 83% in cucumber and 88% in tomato compared to the treatment where only R. reniformis was inoculated. Moreover, when R. reniformis was inoculated first and M. enterolobii 15 DAI, the reproduction of R. reniformis decreased by 58 and 44% in cucumber and tomato plants, respectively, in the presence of M. enterolobii (Figures 2A, C).

Meloidogyne enterolobii also exhibited a higher RF in individual inoculations, but its RF decreased by 76 and 66% in cucumber and tomato plants, respectively, when co- inoculated with R. reniformis (Figures 1A, C).

Symptom severity caused by R. reniformis and M. enterolobii. All plants inoculated with R. reniformis showed root necrosis symptoms, and all treatments inoculated with M. enterolobii exhibited gall symptoms. Both nematodes caused symptoms in roots at 30 DAI, with greater severity observed at 50 DAI (Figures 4 and 5). In both crops, all R. reniformis and M. enterolobii treatments showed significant differences (P < 0.05) compared to the uninoculated control.

Figure 4 Tomato roots at 50 days after inoculation (DAI). A) Control without inoculation. B) Root with galls caused by M. enterolobii. C) Root with stunting and necrosis caused by R. reniformis. D) Root with galls, necrosis, and stunting symptoms, inoculated with R. reniformis and M. enterolobii simultaneously. 

Figure 5 Cucumber roots at 50 days after inoculation (DAI). A) Control without inoculation. B) Root with galls caused by M. enterolobii. C) Root with symptoms of stunting and necrosis caused by R. reniformis. D) Root with galls, necrosis, and stunting symptoms, inoculated with R. reniformis and M. enterolobii simultaneously. 

At 50 DAI, R. reniformis caused 93.3 and 58.3% necrosis in tomato and cucumber roots, respectively. However, when co-inoculated with M. enterolobii, the necrosis percentage decreased by 82% in cucumber roots and 85% in tomato roots. In other words, the presence of M. enterolobii in the same root reduced the severity of symptoms caused by R. reniformis in both cucumber and tomato plants (Figures 1D and 2D).

Meloidogyne enterolobii exhibited a similar pattern to R. reniformis in those individual inoculations resulted in more severe symptoms. However, when co-inoculated with R. reniformis, the galling index (GI) decreased, with T3 (where R. reniformis was inoculated first and M. enterolobii 15 DAI) showing the lowest symptom severity, with GI reductions of 76 and 60% in cucumber and tomato plants, respectively (Figures 1B and 2B). Overall, the presence of R. reniformis in the same root reduced the severity of symptoms caused by M. enterolobii in both cucumber and tomato plants (Figures 4 and 5).

It is worth mentioning that the highest severity in both cucumber and tomato roots was observed in those where both nematodes were co-inoculated, as these roots showed a combination of symptoms such as necrosis, galls, stunting, etc., caused by both nematode species (Figures 4D and 5D).

Discussion

In this study, both M. enterolobii and R. reniformis reproduced and caused symptoms in cucumber and tomato plants. However, both nematodes exhibited higher reproduction factors (RF) and caused more severe symptoms in cucumber, making it the more susceptible host. This observation aligns with findings by ^{Salazar-Mesta et al. (2023}b), who determined that cucumber is more susceptible to M. enterolobii compared to tomato and pepper (Capsicum annuum). This increased susceptibility may be due to the larger size and branching of cucumber roots compared to tomato roots, leading to greater root exudation, which nematodes detect through their sensory systems. Additionally, cucumber root exudates are primarily composed of sugars and compounds preferred by nematodes, such as stigmasterol, while tomato roots mainly release phenolics, sterols, terpenoids, glycosides, and other substances that are less favorable to nematodes (^{Mateos and Leal, 2003}; Lagunes and Zavaleta, 2016).

Both R. reniformis and M. enterolobii exhibited their highest RFs at 50 days after inoculation (DAI), which was expected since both nematodes typically complete their reproductive cycle (egg mass formation) between 18 and 30 days after inoculation in host plants such as tomato, eggplant (Solanum melongena), and cucumber (^{Salazar-Mesta et al., 2023}a, 2023b; ^{Valdez-Morales et al., 2024}). The results indicated that reducing the RF of both nematodes also decreased symptom severity. It has been observed that RF is closely related to the severity of root symptoms. Previous studies have reported that an increase in M. enterolobii RF elevates galling in tomato and pepper plants (^{Salazar-Antón et al., 2014}; Salazar-Mesta et al., 2023b; Salazar-Mesta et al., 2024). Similarly, an increase in R. reniformis RF has been associated with higher necrosis percentages in cowpea (Vigna unguiculata), cotton, and tomato plants (^{Aryal, 2011}; ^{Karthika et al., 2020}).

In tomato plants, M. enterolobii reproduction was reduced in the presence of R. reniformis, regardless of the order of inoculation. Treatments T3, T4, and T5 were statistically similar (Figure 5). This may be due to the high reproductive rate of M. enterolobii compared to R. reniformis. Under favorable conditions, the life cycle of most Meloidogyne species, including M. enterolobii, is completed in 25-30 days, with each female producing 500-1000 eggs per mass (Da Silva et al., 2019; ^{Philbrick et al., 2020}). In contrast, R. reniformis completes its life cycle in 18-25 days, with each female producing 30-200 eggs under favorable conditions (^{Robinson, 2002}).

The life cycle duration of plant-parasitic nematodes largely depends on soil temperature (^{Bridge and Starr, 2007}). The optimal temperature for M. enterolobii development ranges between 20 and 30 °C (^{Greco and Di Vito, 2009}; ^{Tomaz et al., 2021}), while for R. reniformis, it fluctuates between 23 and 30 °C (Starr et al., 2002). Our experiments were conducted under temperatures close to the optimal ranges for both nematodes, with the first experiment held at 18 to 30 °C and the second at 20 to 35 °C.

In cucumber plants, R. reniformis exhibited a similar behavior to M. enterolobii in terms of RF and symptom severity in treatments where both nematodes were inoculated simultaneously. This indicates that nematode reproduction depends significantly on host susceptibility. According to other authors, R. reniformis has a broad host range, with cucumber and eggplant being among its preferred hosts (^{Starr et al., 1991}; ^{Robinson et al., 1997}; Vhadera et al., 2001; ^{Valdez-Morales et al., 2024}). It has been reported that R. reniformis population densities are dependent on host susceptibility (^{Davis et al., 2003}; ^{Stetina et al., 2007}; ^{Moore and Lawrence, 2013}).

In the combined inoculations, the nematode species inoculated first-whether M. enterolobii or R. reniformis-showed a higher RF in both tomato and cucumber plants. This result could be due to the clustering of infective units of the first species in the root, leading to competition for space with the second species. This reduced penetration influenced the competition between both nematodes. A high initial population may deteriorate infection sites and lead to an accumulation of metabolic waste, affecting nematode development. Therefore, RF decreases when nematode populations are excessive due to limited food and space (^{Salazar-Antón et al., 2013}). These findings align with other studies that have evaluated the interaction of R. reniformis with various Meloidogyne species in cotton, pineapple, and castor plants (^{Diez et al., 2003}; ^{Aryal et al., 2011}). However, these studies propose space and nutrient competition as the mechanism for the antagonistic interaction between the nematodes. Recent studies, however, have concluded that prior infection of the host with Rotylenchulus or Meloidogyne can induce greater defense against the other species through the induction of systemic resistance (Aryal et al., 2011; Aryal et al., 2012; ^{Osman et al., 2012}). This resistance, mediated by the salicylic acid pathway (^{Van Loon et al., 2006}), enhances the plant natural defense systems against subsequent infections and provides broad-spectrum resistance to various pathogens, including plant-parasitic nematodes (Osman et al., 2012).

This study demonstrated that in co-inoculations, R. reniformis was more affected in terms of RF and symptom severity compared to M. enterolobii in tomato and cucumber plants. This contrasts with results from other authors, who determined that R. reniformis has a competitive advantage over Meloidogyne species (^{Diez et al., 2003}; ^{Faske and Hurd, 2015}; ^{Ferreira et al., 2015}). The effects of nematode species interactions are related to the nature of parasitism and competition, as the competitive relationship intensifies with increased host-parasite complexity (^{Kesba et al., 2005}; ^{Robinson et al., 2007}).

The parasitism of M. enterolobii involves the induction of giant cells in the parenchyma (^{Starr et al., 2002}; Perry et al., 2009; Perry and Moens, 2013). Through its stylet, this nematode introduces effector molecules (pectolytic and cellulolytic enzymes) into the host, reprogramming gene expression to modify host cell metabolism, physiology, and structure, forming specialized feeding sites. The plant responds by activating defense mechanisms, such as increased activity of key enzymes in the phenylpropanoid pathway, which synthesizes secondary metabolites with antimicrobial properties and lignin monomers that play a structural and defensive role in plants (^{Lagunes-Fortiz and Zavaleta-Mejía, 2016}).

On the other hand, R. reniformis is a semi-endoparasite, and its parasitism induces syncytium formation, primarily in pericycle cells (^{Starr et al., 2002}). Females insert their stylets into the host’s endodermal cells and release a mix of effectors, including CLE and CEP peptide mimics (^{Eves-Van den Akker et al., 2016}; ^{Wubben et al., 2015}), causing cell wall lysis in the initial cell and adjacent pericycle cells, allowing multiple cytoplasms to merge into a single syncytium (Wubben et al., 2015). Effector cells are regulated by parasitism genes, such as glutathione peroxidase, pectin lyase, polygalacturonase, β-1,4- endoglucanase, β-1,4-endoxylanase, and chorismate mutase (Wubben et al., 2010).

In addition to the parasitism nature of each nematode, biochemical and molecular aspects may influence their competitive interaction. Both species modify host plant metabolism and gene expression. Some studies have shown that root-knot nematodes alter host gene expression related to cell cycle control, cell wall modification, and hormone regulation to induce giant cell formation in the roots (^{Cabrera et al., 2015}; ^{Wieczorek, 2015}). Both nematodes (reniform and root-knot) suppress defense-related signaling pathways throughout the lifespan of their feeding structures (^{Jaouannet et al., 2013}; ^{Quentin et al., 2013}). Previously, several genes involved in nematode parasitism and gall formation have been reported (^{Mathesius, 2003}). For example, nodulin N-26, which encodes an aquaglyceroporin, is induced during both root-knot nematode gall formation and reniform nematode parasitism (^{Favery et al., 2002}). Similarly, the leucine-rich repeat (LRR) receptor kinase SYMRK/NORK, part of a receptor complex involved in root-knot nematode perception, has been reported to influence reniform nematode parasitism (^{Endre et al., 2002}; ^{Stracke et al., 2002}; ^{Weerasinghe et al., 2005}; ^{Redding et al., 2018}). Further physiological, biochemical, and molecular studies are recommended to understand in detail the key aspects of the competitive interaction between M. enterolobiiand R. reniformis in tomato and cucumber plants.

Conclusions

In individual inoculations, Meloidogyne enterolobii and Rotylenchulus reniformis exhibited the highest reproduction factor (RF) and disease severity in tomato and cucumber plants. In treatments where one species was inoculated before the other, the species inoculated first showed a higher RF and caused greater disease severity. In simultaneous co-inoculations, both nematodes reduced their RF and caused lower symptom severity in tomato and cucumber plants. However, in tomato plants, the reproduction of R. reniformis was more affected compared to that of M. enterolobii.