Intraspecific virulence of entomopathogenic nematodes against the pests Frankliniella occidentalis (Thysanoptera: Thripidae) and Tuta absoluta (Lepidoptera: Gelechiidae)

Both insects, F. occidentalis and T. absoluta were provided by the R&D Department of Koppert (Spain) in the appropriate developmental instar, in scheduled and coordinated shipments. In all the cases, we ensured that in no case more than 2 days passed from their arrival until their experimentation, storing them under controlled conditions (60% Relative Humidity, RH, 22°C and a photoperiod of 16 hr of light -L- and 8 hr of darkness -D-) in isolated mesh-cages to avoid escapes.

A total of 10 EPN populations from three species (S. feltiae, S. carpocapsae, and H. bacteriophora) were investigated, comprising seven native and three commercial ones (Table 1). All nematodes were reproduced in the insect host Galleria mellonella (Lepidoptera: Pyralidae) (Woodring and Kaya, 1988). The infective juveniles (IJs) were collected in containers with mineral water and stored at 14°C. In any case, the nematodes were used in the studies within 15 to 20 days of emergence.

For the experiments with T. absoluta, tomato plants Solanum lycopersicum var. moneymaker of 5 to 6 true leaves were maintained in sterilized greenhouse plant substrate in 1 L containers under controlled conditions (60% RH, 22°C and 16L: 8D). Finally, as substrate in the F. occidentalis experiments, we used pure mineral sand (Vale do Lobo, Loulé, Portugal). Before the experiments, the sand was washed several times with running water, autoclaved for 1  h (two times in two consecutive days as suggested by Elhady et al., 2018), oven-dried at 40°C with ventilation, and stored in laboratory conditions for at least a week before use (Chiriboga et al., 2017).

We followed the procedures described by Ebssa et al. (2001) slightly modified. Briefly, the experimental unit was a mini-container with a transparent lid with hermetic closure to avoid escapes (2.5 cm diam. X 1.5 cm height). Each container was filled with 1 g of sterile sand. The final volume per mini-container was 100 μL (10% moisture w/v). With the aid of an A000 brush, 10 late-stage F. occidentalis larvae were individually caught for each mini-container, the corresponding treatments applied, and the containers were stored in controlled environment chamber (60% RH 22°C and 16L: 8D). Pollen was added to each mini-container 24 h postinoculation. The treatments included control (100 mL distilled water), and application of 100 μL suspension of 500 IJs (160 IJs/cm²) or of 250 IJs (80  IJs/cm²). Experiments were conducted by species, including in each trial all the corresponding populations. Treatments were replicated 5 times. The presence of adults was checked every 2 to 3 days post-inoculation, determining the adults’ emergence after 12 days (Campos-Herrera and Gutierrez, 2009). The experiment was performed twice, with new insects, nematodes, and substrate preparation.

The experimental unit was a 5.5 cm diam. Petri dish with one Whatman filter paper no. 1, a piece of 1 cm³ cotton, and two tomato leaves. In each dish, 8 last stage larvae of T. absoluta were included and 500 μL of distilled water was added to soak the cotton and partially moisten the filter paper. When studying the impact of EPN on pupae, the dishes were closed with parafilm and observed after 24 to 48 hr to verify the pupal status and the formation of galleries. When studying EPN efficacy against the last instar, nematodes were applied 6 hr after larval disposal in the dishes to ensure gallery formation but no pupation. In both studies, nematodes were applied in a final volume of 400 μL, with distilled water for controls. All dishes were stored in a controlled environment chamber (60% RH 22°C and 16L: 8D). In both, the pupa and last instar larva studies, the design and replication of the treatments were as for F. occidentalis, but with the concentrations of 500 IJs (21 IJs/cm²) and concentration of 100 IJs (4 IJs/cm²) applied in 400 μL. In the study against the pupal stage, the presence of adults was checked every 2 to 3 days post-inoculation for 12 days (Campos-Herrera and Gutierrez, 2009). In the study of the last instar larva, the mortality was checked daily until 5 days post-exposure, considering larval mortality and the number of days to the death as variables. The experiments were performed twice, with new insects, nematodes, and plants preparation.

We calculated the percentage of adult emergence and larval mortality. Before statistical analysis, percentage values were arcsine transformed. After checking the statistical similarity of the results, data from different trials were combined (data not shown). For each of the experiments, we ran generalized linear models (GLM, P < 0.05) for the analysis of the percentage of the variable of adult emergence, percentage of larval mortality, and the number of days needed to die. We explore the intraspecific variability considering the factors “population” for each species (five levels, comprising control and the four populations for S. feltiae and H. bacteriophora, and three levels, control and two populations for S. carpocapsae), concentrations (two levels, high and low as described above), and their interactions. Besides, we performed an individual one-way ANOVA and Tukey test (P < 0.05) for each species (interspecific analysis) or population (interspecific analysis) and concentration to disentangle the control potential against both insect pests in the corresponding instar. Finally, the Student’s t test (P < 0.05) investigated the differences between the two concentrations in each of the species/populations for all the variables. We performed all the analyses with SPSS 25.0 (SPSS Statistics, SPSS Inc., Chicago, IL, USA). We used least-square means ± S. E. as descriptive statistics.

Percentage emergence of adults was significant for all the factors (populations and concentration) and their interactions except for S. feltiae (population and interaction) (Supplementary data Table S1, Fig. 1). We analyzed both factors individually to disentangle for concentration or population effects. Differences among populations were observed for both concentrations for each of the species (Supplementary data Table S2), while differences between concentrations for the same population were only observed in one population of each species (Fig. 1, Supplementary data Table S3). In detail, in the analysis of H. bacteriophora populations, differences with the emergence observed in the control were only observed for the native populations (Hb_RM-102, Hb_AM-203, and Hb_MG-618b), that reached 58 to 43% emergence (Fig. 1A). In the case of the S. feltiae populations, all the populations except Sf_RM-107 registered lower adult emergence than the control for both concentrations tested, with Sf_Koppert registering 46% adult emergence, the lowest value of all of the S. feltiae populations (Fig. 1B). Finally, for the S. carpocapsae intraspecific analysis, only Sca_MG-596a applied at the high concentration resulted significantly lower than the control emergence (Fig. 1C, Table S2).

Figure 1:

Percentage emergence of adults was significant for all the factors (populations and concentration) and their interactions except for S. feltiae (population and interaction), and the interactions in the intraspecific analysis for S. carpocapsae populations (Table S1, Fig. 2). We analyzed both factors individually to disentangle for concentration or population effects. Differences among populations were observed for the 500 IJs for all the species, but only among S. feltiae populations in the 100 IJs concentration (Table S2). In detail, in the analysis of H. bacteriophora populations, differences with the emergence observed in the control were only observed when the 500 IJs concentration was applied, registering values of 48-24% emergence (Fig. 2A). Differences between concentrations were observed for the populations Hb_AM-203 and Hb_MG-618b (Table S3, Fig. 2A), which showed values < 50% adult emergence even at the low concentration of 100 IJs. In the case of the S. feltiae populations, only Sf_Koppert resulted significantly lower than the control for both concentrations (Fig. 2B), and no population registered differences between concentrations (Table S3). Finally, for the S. carpocapsae intraspecific analysis, only the high concentration resulted significantly lower than the control emergence (Fig. 2C, Table S2), being the adult emergence of both populations significantly lower when applied at 500 IJs concentration (Fig. 2C, Table S3).

Figure 2:

Percentage larval mortality was significant for all the factors (populations and concentration), but not the interaction (Table S1, Fig. 3). When the number of days to kill the larva were considered, H. bacteriophora and S. carpocapase showed differences for both factors and the interactions (Table S1, Supplementary data Fig. S1). However, the intraspecific analysis of S. feltiae was not affected by concentration (Table S1, Fig. S1B). We analyzed both factors individually for each of the variables (larval mortality and time to kill) to disentangle for concentration or population effects. Differences among populations were observed in both concentrations for larval mortality percentage and number of days to kill the insect, except for the high concentration in S. carpocapsae (Table S2, Fig. 3, Fig S1). In detail, in the analysis of H. bacteriophora, all the populations reported significantly higher mortalities than the control for both concentrations (Fig. 3A), with differences between the two concentrations for Hb_Koppert and Hb_RM-102 (Table S3). Considering the time to kill the larva, only the population Hb_RM-102 killed faster than the mortality observed in the control treatments (Fig S1A). Differences between the concentrations were observed for Hb_Koppert and Hb_RM-102 (Table S3). In the case of the S. feltiae, all populations and both concentrations resulted different than the mortality observed in the control, ranging 95-100% (Fig. 3B, Table S2, Table S3). Differences between the concentrations were registered for Sf_RM-107 and Sf_RS-5. Considering the time to kill the larvae, all the populations were faster than control in the 500 IJs concentrations, while only Sf_Koppert and Sf_AM-25 did were at the low 100 IJs concentration (Fig. S1). All the populations were faster at the high concentration, except Sf_Koppert (Fig. S1, Table S3).Finally, for the S. carpocapsae intraspecific analysis, both populations and concentrations generated higher larval mortality than the control (Fig. 3C), but none resulted faster than control (Fig. S1). Higher IJs concentration resulted in higher mortality for both S. carpocapsae populations (Fig. 3C, Table S3), while only Sca_MG-596a resulted faster at the high concentration (Fig. S1, Table S3).

Figure 3:

Figure S1: