Temperature: a driving factor for Meloidogyne floridensis migration toward different hosts

Less than 8% of all injected J2 (96,000) were recovered throughout the experiment, with an average of 1.86% recovery rate per column. The presence of tomato or French marigold plants did not improve the migration rate of J2 when compared with the control (p ≥ 0.05). The distribution of recovered J2 along the columns was significantly influence by temperature and time (p < 0.05), while the migration of active J2 differed between temperatures (p < 0.05) and rings (p < 0.0001, Table 1) over time. No J2 were found trapped in either nylon meshes.

At 3 DAI, more than 50% (775) of recovered J2 were found at the injection ring (< 1 cm) regardless of temperature; however, none were observed above 9 cm at 26°C, whereas less than 1% (11) migrated > 13 cm at 20°C (Fig. 2A). At 6 DAI, although most J2 still remained at the injection ring, 8% (75) were able to migrate up to 13 cm at 26°C; the percentage of recovered J2 at 20°C decreased at the injection ring and became evenly distributed from 1 to 13 cm (Fig. 2B). There was a significant increase (10%) on the percentage of J2 migrating > 13 cm at 20°C at 9 DAI, whereas J2 concentrated within the first 9 cm of the columns at 26°C (Fig. 2C). By the end of the experiment, recovered J2 were uniformly distributed along the columns, with similar recovery percentages at distances of up to 13 and > 13 cm at both temperatures (Fig. 2D).

Figure 2:

At 20°C, the percentage of active J2 decreased from 27% (953) to 21% (726) at 3 and 12 DAI, respectively, whereas there was a 30% (from 732 J2 at 3 DAI to 360 J2 at 12 DAI) reduction on the percentage of active J2 at 26°C. At 3 and 6 DAI greater percentages of J2 were recovered from columns at 26°C, however this behavior changed from 9 DAI onward, with 21% (726) and 8% (360) of active J2 recovered at 20 and 26°C, respectively, at 12 DAI (Fig. 3).

Figure 3:

There was no significant interaction between temperature, nematode travel distance and time (p ≥ 0.05, Table 1), thus data of 20 and 26°C were combined (Fig. 4). The distribution pattern of active J2 along the soil columns changed over time. At 3 DAI, more than 50% (973) active J2 was recovered from the injection ring, while the remaining were concentrated between 1 and 9 cm; only 1% (19) of active J2 were able to migrate more than 9 cm. At 6 DAI, 26 to 28% (465–490) of active J2 were extracted from the first three rings, indicating a progressive and even vertical migration within the first 9 cm; even so, only 1.4% (24) were able to reach the top of the columns (>13 cm). A turning point occurred at 9 DAI, where about 40% (539) of active J2 migrated up to 5 cm and 7% (96) were recovered from the soil inside the Styrofoam cup, which means a migration of more than 13 cm. A similar behavior was observed at 12 DAI, with almost 9% (82) of J2 extracted from distances greater than 13 cm (Fig. 4).

Figure 4:

There was no effect of temperature on plant growth variables (data not shown) or penetration of J2 (p ≥ 0.05), nor was there a significant interaction between any of the factors (p ≥ 0.05, Table 2). Penetration of J2 was greater on tomato than on French marigold plants (p < 0.001) with increasing number of J2 inside roots over time (p < 0.0001, Table 2).

Juveniles were able to parasitize both plant species, however a greater number of J2 were observed inside tomato roots (mean of 2.5 J2 per 2 g of fresh root), whereas less than 1 J2, on average, were counted inside French marigold roots (Fig. 5A). Regarding penetration over time, less than 0.1% of J2 were able to migrate more than 13 cm through the column and parasitize roots regardless of the presence of plants or temperature at 3 DAI. A peak of 3 J2 per 2 g of fresh roots were observed at 9 DAI (Fig. 5B).

Figure 5:

The presence of tomato or French marigold plants on the top of the columns did not contribute significantly to the vertical migration of M. floridensis J2 compared to plant-free treatments in our experiment. Similarly, the migration rate of M. chitwoodi under host-free conditions was found to not differ from that under tomato plants (Pinkerton et al., 1987). However, preferential migration of M. incognita and M. javanica in soil columns toward suitable hosts was previously reported (Dalzell et al., 2011; Prot, 1976). Feltham and Chaplain (2000) described a migration model for nematodes in restricted domains and pointed out that nematodes may present a biased-random motion strategy, that is, J2 would migrate with a bias toward a host root while simultaneously searching for better food sources. This assumption might explain the results obtained in our experiment.

Although the great majority of J2 were recovered near or at the injection ring, the percentage of J2 at distances greater than 13 cm significantly increased over time, changing from less than 1% at 3 DAI to 9% of active J2 at 12 DAI (Fig. 4). Some studies have reported greater recovery rates of J2 near the plant roots. Prot (1976) observed that 20% of injected J2 of M. javanica were able to migrate 50 cm in soil columns with 1.2-cm d rings. Dalzell et al. (2011) reported a recovery rate of nearly 80% of M. incognita near tomato rhizosphere in a pipette-bulb assay. On the other hand, Pinkerton et al. (1987) mentioned that less than 0.1% of injected J2 of M. chitwoodi and M. hapla were able to move 45 cm in soil columns with 8.5-cm d rings. Such different findings might be related to the apparatus dimensions and/or soil physical attributes.

Under natural conditions, the movement of nematodes takes place within an infinite domain (Feltham et al., 2002). However, the evaluation of nematode migratory ability under laboratory conditions occurs in bounded domains and without root growth into the system. Although soil columns have introduced a 3D aspect to migration assays, the dimensional restraint imposed by such columns favors vertical rather than horizontal migration (Spence et al., 2008); therefore, the smaller the internal diameter of the apparatus, the greater the upward migration of J2. According to Hunt et al. (2001), the differences in experimental apparatus and conditions for nematode migration assays make it difficult to create a general model to describe nematode migration within the soil.

While moving within the soil, plant-parasitic nematodes are subjected to environmental changes, including temperature oscillations. In our experiment, J2 migration within soil was best at 20°C for 3, 6, and 9 DAI, where the percentage of recovered J2 was always higher at distances greater than 9 cm when compared to 26°C; by the end of the experiment (12 DAI) J2 distribution was similar between both temperatures (Fig. 1). Several studies have reported that temperature highly influences the migration distance of J2 of plant-parasitic nematodes, and when a gradient is present, they show thermotaxis (Dusenbery, 1988) and tend to migrate toward the warmer end (Robinson, 1994). Prot and van Gundy (1981) have observed maximum vertical migration rates of M. incognita at 20°C, while M. chitwoodi and M. hapla migrated furthest at 18°C (Pinkerton et al., 1987) and M. javanica at 25°C (Wallace, 1966). Migratory endoparasitic nematodes have shown similar behavior, i.e. Pratylenchus penetrans migration was best at 21°C when compared to 11 and 16°C (Pudasaini et al., 2007).

In our experiment, temperature did not influence the penetration of M. floridensis J2 since they are protected from environmental stress when inside healthy host roots (Adam et al., 2014; McSorley, 2003). However, the recovery rate of active J2 from the soil decreased faster at 26°C than at 20°C, suggesting penetration of J2 into tomato or French marigold roots or a greater mortality/inactivity rate of M. floridensis J2. In fact, high temperature may hinder root-knot nematode migratory ability (Wallace, 1966). As nematodes are poikilothermic animals (Pudasaini et al., 2007), J2 metabolism increases concomitantly with temperature and their consumption of lipids reserves also increases (Das et al., 2011; Rocha et al., 2016), thus higher inactivity rates might occur.

Tomato is considered a host for several Meloidogyne spp. (Seid et al., 2015), including M. floridensis (Brito et al., 2015). However, Cetintas et al. (2007) reported that M. floridensis J2 showed significant infectivity levels on tomato ‘Florida 47’, whereas they were not as pathogenic on ‘Solar Set’. The authors ruled out low inoculum viability because J2 produced high galling indices on tomato ‘Solar Set’ grown in clay pots. An average of 2.5 J2 of M. floridensis per 2 g of fresh roots were found inside tomato ‘Cobra’ in our experiment (Fig. 5), which is comparable to the results observed in microplots experiments using tomato ‘Solar Set’ (Cetintas et al., 2007). Other tomato cultivars have been reported as susceptible (‘Rutgers’ and ‘Talladega’) or resistant (‘Crista’) to M. floridensis (Stanley et al., 2009). The rationale behind the discrepancies on host status of different tomato cultivars to M. floridensis is still unknown and should be studied to improve peach root-knot nematode management at field scale.

Although we have hypothesized that J2 of M. floridensis would be repelled by French marigold exudates, a few J2 were able to penetrate their roots but did not show further development, suggesting French marigold as a nonhost (Kokalis-Burelle and Nyczepir, 2004). The chemotactic responses of active J2 of plant-parasitic nematodes lead them to the vicinity of host root tips or away from non-host plants (Wang et al., 2018). Nematode’s host-finding ability is a complex mechanism that rely on a blend of attractant and repellent compounds exuded by the same plant species (Chaisson and Hallem, 2012; Dutta et al., 2011). Different concentrations of extracts from tomato (Diez and Dusenbery, 1989) and cucumber (Castro et al., 1989) have shown both attractive and repellent properties to Meloidogyne spp.

The migratory behavior toward host roots may be species-specific. Meloidogyne graminicola, which is a species of root-knot nematode with narrow host range, responded differently to chemical cues from root exudates when compared to species that have a broader host range, such as M. incognita (Reynolds et al., 2011). This behavior was also reported for migratory endoparasitic nematodes. The vertical migration of individuals of P. penetrans was evaluated under different hosts and column lengths, where a preferential movement under good hosts (maize and bean) was observed regardless of the distance between host and injection point (Pudasaini et al., 2007). Further studies need to focus on understanding how plant-parasitic nematodes perceive exudate compounds as repellent or attractive from a given host (Dutta et al., 2012).

In summary, temperature directly affects the migration of M. floridensis J2 within the soil but shows no influence on J2 after they penetrate host tissue. Migration was best at 20°C, but J2 showed similar distribution along the columns over time (i.e. 12 DAI). J2 of M. floridensis can migrate great distances (>13 cm) regardless of the presence of plants due to random movement during their host-finding phase. French marigold plants did not repel J2 of M. floridensis nor hinder their migration, but fewer J2 were able to penetrate their roots.