Soil texture, infective juvenile concentration, and soil organic matter influence the efficacy of Steinernema feltiae isolate Lican Ray

Optimal concentration of S. feltiae LR in three soil types

The matrices were placed in vertical position and through the upper opening IJs were pipetted in one of the following suspension 0, 440, 2,200 or 4,400 IJs/5 mL water (Sharma et al., 2011). The distance between the petri dishes and the inoculation point was 20 cm. Each type of soil in combination with each IJ concentration defined the treatments (12 treatments), with five repetitions each. The matrices were maintained vertically in darkness at 25 ± 2°C. Three, six and nine days after inoculation (DAI), the petri dishes were removed, and larval mortality assessed.

Horizontal and vertical dispersal capacity of S. feltiae LR in three soil types

For this assay, another tube (Fig. 1C) of variable length according to the treatment, was adapted in the upper opening of the matrix, resulting in tubes with soil columns (distance between inoculation point and petri dishes) of total length of 10, 20, 30 and 40 cm for evaluating horizontal movement and 30, 50 and 70 cm for evaluating vertical movement of EPNs. The matrices were placed either in vertical or horizontal position, and then inoculated, with 4,400 IJs in 5 mL of water (the best concentration in the previous experiment) through the upper opening, closed and incubated as previously described. Combining the factors (soil type × distance) resulted in 12 (3 × 4) and 9 (3 × 3) treatments in horizontal and vertical assays, respectively, with five repetitions each. Three, six and nine days after inoculation (DAI), the petri dishes were removed, and larval mortality assessed.

Effect of OM at different decomposition degree on the dispersal of S. feltiae LR

Two, 4 and 6% v/v OM at different decomposition degree was added to loam soil with a natural content of 2% OM (the soil with best results in Assay 2) obtaining final concentrations of 4, 6 and 8%. In addition, two control treatments were set, the first consisted on the same soil with no OM added (natural content 2%) and the second, the same soil but eliminating OM by calcination in a muffle furnace (LabTech^®, Korea) at 360°C for 16 hr. Finally, each matrix (containing a soil column of 20 cm) was inoculated with 5 mL of a suspension of 4,400 IJs. The combination of the factors (percentage of OM × decomposition degree) and the control treatments resulted in 11 treatments, repeated five times each. Two and four days after inoculation (DAI), the petri dishes were removed, and larval mortality assessed.

In all the three assays the movement capacity of IJs was evaluated through the mortality level of the insect larvae, as IJs were capable of reaching and infecting the host (G. mellonella) at certain distances. In all cases dead larvae were washed with distilled water and placed in white traps (Kaya and Stock, 1997) at 25°C until IJ emergence occurred. In all assays, sentinel treatments, consisting on five G. mellonella larvae inoculated in petri dish with 500 IJ, were left in the chamber at the same temperature, to verify EPN viability and correct handling of the assay.

Experimental design and statistic analysis

All assays were conducted under laboratory conditions and arranged in a randomized block design with factorial structure. Petri dishes with G. mellonella larvae were considered as the experimental unit. Percentage of mortality was angular transformed and analyzed through general linear model. Data in Assay 1 were corrected by the Schneider-Orelli’s formula (Püntener, 1981). When F values were significant, differences between means were evaluated using Tukey’s multiple range (p ≤ 0.05). Minitab^® V17 was used for data analysis. All assays were repeated twice.

Results

Optimal concentration of S. feltiae LR in three soil types

At three DAI the analysis showed no interaction between IJ concentration and soil types (F _(4,36) = 1.13; p = 0.357) therefore, factors were analyzed independently. Considering the factor soil texture, and independently from the IJ concentration, the highest G. mellonella mortality occurred in loam soil (96%), followed by clay soil (75%), and sandy soil (50.7%), and significant differences were observed only between loam and sandy soils (F _(2,36) = 9.59; p ≤ 0.0001; Fig. 2I, capital letters). However, considering the factor IJ concentration and regardless of soil texture, no significant differences were observed between treatments at this time (F _(2,36) = 3.23; p = 0.051) (therefore, no lower-case letters on bars, Fig. 2I).

Figure 2:

At six DAI the analysis showed significant interactions between IJ concentration and soil type (F _(4,36) = 4.59; p = 0.004), with significantly lowest G. mellonella mortality in sandy soils at the concentration of 440 IJs (30%) compared to all other treatments, where mortality varied between 70.8 and 100% (Fig. 2II). A similar situation occurred at nine DAI (F _(4,36) = 4.91; p = 0.003), where mortality varied between 71.4 and 100%, with significantly lowest mortality in sandy soils and 440 IJ (Fig. 2III).

At the end of the experimental period (nine DAI), 4,400 was the only IJ concentration that achieved 100% of larval mortality in all soil types, therefore, this concentration was used in further assays.

Horizontal and vertical dispersal capacity of S. feltiae LR in three soil types

In all evaluation times the interaction between the factors soil texture and distance was significant, affecting IJ capacity to reach the host and infect it. The general trend was that an increase in the distance resulted in lower mortality; however, particular variations were observed according to soil type and matrix position.

Horizontal movement

At three DAI (F _{(6, 48)} = 4.21; p = 0.002), when larvae were 10 and 20 cm from the IJ inoculation point, in all soil textures, and 30 cm in loamy soil, mortality reached significantly highest values (80–100%). The lowest mortality levels were observed at 30 and 40 cm in most soils (approximately 0–20%) (Fig. 3).

Figure 3:

At six DAI (F _{(6, 48)} = 3.07; p = 0.013) and nine DAI (F _{(6, 48)} = 2.84; p = 0.019), significantly highest mortality was recorded at 10 and 20 in all soil textures, at 30 cm in loam soil (100%) and 30 cm in sandy soil (75%), followed by clay and sandy at 30 cm, and loam soil at 40 (35–75%). The lowest larval mortality occurred in clay and sandy soil at 40 cm (Fig. 3).

Vertical movement

At three DAI (F _{(4, 36)} = 4.31; p = 0.006), highest mortality was recorded in loam soil at 30 cm (100%) and 50 cm (60%), and in sandy loam soil at 30 cm (64%). The lowest mortality level was observed at 50 and 70 cm in most soils (approximately 0–12%) (Fig. 4).

At six DAI (F _{(4, 36)} = 5.54; p = 0.001) and nine DAI (F _{(4, 36)} = 7.78; p < 0.001), significantly highest mortality was recorded in loam soil at 30 and 50 cm and in sandy soil at 30 cm (100%), followed by clay at 30 cm, sandy at 50 cm and loam soil at 70 cm. In addition, at six DAI there was no larval mortality at 50 and 70 cm in clay, and at 70 cm in sandy soils. Finally, in clay soils, at nine DAI, mortality tended to increase at 30 and 50 cm compared to six DAI (Fig. 4).

Figure 4:

Effect of OM at different decomposition degree on the dispersal of S. feltiae LR

At two DAI (F _{(4, 40)} = 0.14; p = 0.965) and four DAI (F _{(4, 40)} = 0.53; p = 0.718), the interaction between the factors OM content and OM decomposition degree was no significant, therefore, these factors were analyzed independently in relation to their effect on the IJ capacity to reach the host and infect it. On one hand, considering the factor OM content, at two DAI (Fig. 5, lower-case letters) the only treatment where no mortality was observed was the soil with no OM. In general, in soils with OM (2–8%) larval mortality increased with OM content, however, these differences were not significant among all treatments. At four DAI (Fig. 5, capital letters) larval mortality was significantly higher in all treatments with OM (88–96%) compared to the calcined soil (16%). On the other hand, considering the factor OM decomposition degree, the independent analysis resulted in the comparison of five treatments: original soil with unknown OM decomposition degree; calcined soil without OM; soils with initial; medium; and advanced degree of OM decomposition. At two DAI, except for the soil with no OM, all treatments had a similar larval mortality level of approximately 34%, however these differences were not significant. Similar results were obtained at four DAI, with mortality near 93% for all treatments with OM, which was higher than the treatment with no OM, but this difference was no significant (Fig. 6).

Figure 5:

Figure 6:

Discussion

The S. feltiae isolate LR evaluated in this work displayed the expected dispersal behavior in the soil, according to S. feltiae foraging strategy (Alekseev et al., 2006; Campos-Herrera and Gutiérrez, 2014). The geographic diversity of Chile (from 18 °S latitude to 56 °S latitude) results in a myriad of soils under different management strategies, and biotic and abiotic characteristics. In this work, we verified the potential of this native isolate to be used under different conditions for the control of soils pests.

Although IJ concentration for the control of different pests have been widely studied (Gazit et al., 2000; Batista et al., 2014; Guo et al., 2017), the influence of soil texture at different IJ concentrations on pest control is poorly understood. For instance, Koppenhöfer and Fuzy (2006) found that the highest concentration of Steinernema scarabaei caused the highest mortality levels on Anomala orientalis (Coleoptera: Scarabeidae) in all substrates studied (loamy sand, sandy loam, loam, silt loam, acidic sand and potting mix). In contrast, in a study by Campos-Herrera and Gutiérrez (2009), the infectivity of S. feltiae isolate the Rioja against S. littoralis was affected by both soil type and IJ dosage, with higher mortality achieved with higher IJ doses in heavier soils. However, in these soils, the number of days to larval death was highest. Similarly, we observed that IJ dosage and soil texture affect IJ movement and its capacity to reach and kill the insect host. In our study, initially, the highest levels of mortality were achieved in loam soil, regardless of the IJ concentration, achieving the highest levels of mortality at six DAI. However, as time passed, both texture and IJ concentration influenced larval mortality, particularly in light-textured soil (sandy loam), where there was a clear tendency to higher mortality as the concentration increased.

These results suggest that in field conditions, higher IJ concentrations of S. feltiae should be utilized in applications for light soils. In heavy soils (clay), even though no significant differences were observed, the highest mortality was achieved with the highest IJ concentration, but it is likely that the percentage of clay in the soil will define the feasibility for EPN to disperse. According to Wallace (1968), the nematodes are confined to the existing soil geometry, and pore size correlates with movement; nematodes cannot pass through spaces much narrower than their body diameter. Therefore, the isolate selected should consider its body size and soil clay content.

Several studies have shown that soil texture affects the capacity of EPN to disperse and infect the host, such as Steinernema riobrave and H. bacteriophora on Diaprepes abbreviatus (Coleoptera: Curculionidae) (Shapiro et al., 2000), H. bacteriophora, H. megidis and S. feltiae on Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae) (Toepfer et al., 2010), and S. carpocapsae and H. bacteriophora on potato tuber moth, P. operculella (Hassani-Kakhki et al., 2013). In some cases, light textures were the most beneficial for various EPNs species (Hassani-Kakhki et al., 2013).

However, in our study, the native EPN, S. feltiae LR, was capable to disperse more in intermediate (loam), both vertically and horizontally. This isolate moved through the soil and reached the host at a distance of 30 cm horizontally in all soil types evaluated, except in loam, where they were able to move up to 40 cm. Furthermore, they achieved vertical distances of 70, 50 and 30 cm in loam, sandy loam and clay soil, respectively. Other studies found similar results with vertical movement of S. feltiae on the host G. mellonella (Neumann and Shields, 2006), also of S. carpocapsae on Anastrepha obliqua (Diptera: Tephritidae) (Toledo et al., 2009), and vertical and horizontal of Heterorhabdits amazonensis and Steinernema arenarium on Spodoptera frugiperda (Lepidopera: Noctuidae) (Andaló et al., 2012). In addition, in our study, mortality of G. mellonella decreased with the extreme soil textures evaluated. Horizontally at 30 cm, while in loam soil mortality reached 100% at the end of the experimental period, in sandy and clay soil it reached 80 and 45%, respectively. Similarly, at 40 cm, while in loam soil mortality reached 50%, in sandy and clay soil it reached only 20 and 0%, respectively.

However, the results have been diverse regarding which soil texture benefits EPNs activity, as the soil pore size can also affect IJ activity in the soil, because energy reserves are depleted with more movement. For example, smaller pores result in higher opposition to IJ movement and/or less oxygen available and consequently, IJs may lose efficacy finding, reaching and infecting their hosts (Wallace, 1968). Native isolates, even from the same species, may show better performance in specific soil textures. For example, in a study by Toepfer et al. (2010), the efficacy of three EPNs species, including S. feltiae, on D. v. virgifera larvae was higher in maize fields with heavy clay or silty clay soils than in sandy soils. In addition, S. carpocapsae and S. riobrave moved more easily in sandy loam and marine sand soils (Alekseev et al., 2006), and S. riobrave and H. bacteriophora dispersed more easily in sandy soils (Shapiro et al., 2000). Our study showed greater infective capacity efficacy of IJ in soils with medium pore size, however, other species, such as S. riobrave and H. bacteriophora, showed greater infection capacity on D. abbreviatus and longer persistence in Marl soil (80% silt and 15% clay) compared to sandy soil (Shapiro et al., 2000). Likewise, S. carpocapsae showed the highest percentage of infection on A. obliqua larvae in sand-clay mixture (60–82% depending on depth), compared to 45–64% in sand, and 23–30% in loamy-sand soil (Toledo et al., 2009).

Therefore, light soils (containing more sand and less clay) do not necessarily guarantee IJs efficacy, because there may be a strong relationship between soil pores and IJ size, which can benefit IJ dispersal. The dispersal rate in our study was higher in loam soil compared to the other textures, where IJs of S. feltiae LR reached G. mellonella (a non-mobile host), located 30 and 50 cm horizontally and vertically, respectively, in only three days. Similar studies were conducted by Bal and Grewal (2015) where S. carpocapsae and H. bacteriophora, moved horizontally approximately 15 and 10 cm in the same period, reaching a non-mobile host and 24 and 15 cm with a mobile host, respectively, in silt-loamy soil. However, further studies by Bal et al. (2017) showed that the same species moved an estimate of 150 cm in 3 days, under field conditions. Therefore, it is crucial to define the adequate EPN isolate for each soil type, which optimizes dispersal and time to reach the host, factors that should be considered together with IJ concentration in further field studies.

Although soil structure may be improved with the addition of OM, in this work, structure was not considered, because it was altered in the process of mixing the soil with OM and filling the tubes. Only the content of OM and the decomposition degree were considered. While EPN movement in organic substrate has received little attention, Griffin (2015), Ansari and Butt (2011) and Kapranas et al. (2017) point out that EPN behavior is influenced by the species and soil quality. For instance, both compaction and increasing peat content in sand, decreased IJ success in S. carpocapsae, Heterorhabditis downesi and S. feltiae (Kapranas et al., 2017). Also, according to Williams et al. (2013), it is likely that organic soils facilitate the active or passive movement of nematodes through the medium or promote their post-application survival. For example, Kruitbos et al. (2010) observed that while S. carpocapsae moved significantly less in sand than in peat, H. megidis dispersed well in both media, but only showed taxis toward hosts in sand. In addition, MacMillan et al. (2009) observed a more active dispersal of two Phasmarhabditis hermaphrodita strains in organic media (bark chips and leaf litter, and to a lesser extent peat) than in mineral soils. Similarly, our study evidenced, first, that the OM content influences IJ movement of S. feltiae LR, especially those treatments with OM content different from zero. Even though no significant differences in G. mellonella mortality were observed in the treatments that contained OM (2–8%), our results suggest that 4% would be the optimal content for this EPN and values above or below this, affect its capacity to reach and kill the host. Second, we observed that the OM decomposition degree (particle size), does not influence the capacity of S. feltiae LR to reach and kill the host. Regarding survival of the EPN, Herren et al. (2018) observed higher survival of S. feltiae in mature compost that in soil with no compost added; however, this was also affected by the application mode of the EPN. In their work, the survival ranged from only 3.2%, when EPNs were applied in aqueous suspension to soils with no compost added, to 62% when they were applied in clay-coated G. mellonella cadavers on soil with mature compost added.

Even though S. feltiae is a well-studied species, and several companies around the world have used it to formulate products commercially available, EPN intra-species variability concerning phenotypical and biological behavior has been demonstrated (Gaugler et al., 1989; Campos-Herrera and Gutiérrez, 2014). Therefore, when developing a product based on native EPN, it is important to study specific characteristics of these ecotypes to optimize its use under local conditions. Further studies under field conditions, using a wider range of soil OM content and including those textures that affected negatively the movement of S. feltiae LR (extreme textures), which could be improved adding OM, should be also considered.