Petri plate bioassay
There were significant differences in mortality of L3 of A. segetum across EPN isolates. H. indica caused the maximum larval mortality (93.3%) 96 h post treatment, at the concentration of 40 IJs/larva (Fig. 1a). Among the locally extracted EPN isolates, Heterorhabditis spp. (HKM) caused a maximum mortality (81.3%) of A. segetum in comparison to the other two isolates. The data revealed significant differences in the percent larval mortality of A. segetum for each nematode species, population species (S) wise F = 43.2, df = 3, p = < 0.001; exposure (E) period wise F = 421.4, df = 3, p = < 0.001; and population (P) number wise F = 228.52, df = 3, p = < 0.001.
The differences in potential of the different isolates of EPNs against L4 of A. segetum were insignificant but differences across the concentrations were significant. All EPN isolates caused equivalent amount rates of larval mortality 96 h post inoculation, at the concentration of 40 IJs/larva (Fig. 1b). The data revealed significant differences in percent larval mortality of A. segetum for each nematode species, population S wise F = 30.36, df = 3, p = < 0.001; E period wise F = 323.19, df = 3, p = < 0.001; and P number wise F = 183.31, df = 3, p = < 0.001.
Among local EPN isolates, H. bacteriophora (HRJ) caused the maximum larval mortality in L5 of A. segetum 96 h post inoculation, at the concentration of 40 IJs/larva (Fig. 1c), whereas the commercial isolate H. indica caused the maximum larval mortality (56.6%). The data revealed significant differences in larval mortality of A. segetum for each nematode species, population S wise F = 0.7, df = 3, p = < 0.001; E period wise F = 112.78, df = 3, p = < 0.001; and P number wise F = 70.07, df = 3, p = < 0.001. Comparative data pertaining to efficacy of Heterorhabditis sp. (HSG), Heterorhabditis sp. (HKM), H. bacteriophora (HRJ) and H. indica 96 h post inoculation, at the concentration of 40 IJs/larva against L3-L5 of turnip moth were illustrated in Fig. 2.
Soil inoculation bioassay
Heterorhabditis sp. (HSG) achieved 28.33% mortality of A. segetum L4 at 10,000 IJs/kg soil, whereas at 1000 IJs/kg of soil, only 3.33% mortality was recorded 3 days post treatment. After 5 days of treatment, 8.33, 30.10, and 53.33% mortality were recorded at 1000, 5000, and 10,000 IJs/kg soil, respectively. The highest mortality rate (78.33%) was obtained at 10,000 IJs/kg of soil 7 days post treatment (Fig. 3a). The data revealed significant differences in larval mortality rates of A. segetum for each nematode species, population S wise F = 16.67, df = 3, p = < 0.001; E period wise F = 462.68, df = 2, p = < 0.001; and P number wise F = 772.24, df = 2, p = < 0.001.
Heterorhabditis sp. (HKM) showed a slightly higher mortality against L4 of A. segetum, compared to Heterorhabditis sp. (HSG) at the tested concentrations. There was 31.67% mortality with this EPN isolate, at 10,000 IJs/kg of soil 3 days post treatment. After 5 and 7 days of treatment, the concentration of 10,000 IJs/kg soil caused 58.33 and 80.00% mortality rates, respectively. At the population level of 5000 IJs/kg soil, the mortality varied from 18.33 to 58.33%, 3 to 7 days post treatment. The minimum mortality rate (21.67%) was recorded 7 days post treatment, at 1000 IJs/kg of soil (Fig. 3b).
H. bacteriophora (HRJ) was comparatively more effective than Heterorhabditis spp. (HSG) and Heterorhabditis sp. (HKM). There achieved 20.00 and 33.33% mortality rates, at 5000 and 10,000 IJs/kg soil, 3 days post treatment. The lowest concentration of 1000 IJs/kg of soil recorded 3.33–26.67% mortality, 3 to 7 days post treatment. At a concentration of 5000 IJs/kg of soil, the mortality rate varied from 36.67 to 60.00%, 5 to 7 days post treatment. The maximum mortality rate (81.67%) was recorded at 10,000 IJs/kg soil 7 days post treatment (Fig. 3c).
Comparative data pertaining to efficacy of Heterorhabditis sp. (HSG), Heterorhabditis sp. (HKM), and H. bacteriophora (HRJ) were illustrated in Fig. 4. All the three local isolates were statistically at par with each other, at the concentration of 1000 IJs/kg soil. The mortality rate varied from 11.66 to 13.89% at this concentration. At 5000 IJs/kg of soil, H. bacteriophora (HRJ) and Heterorhabditis sp. (HKM) showed 38.89 and 37.22% mortality and both were statistically at par with each other. Heterorhabditis sp. (HSG) recorded 33.33% mortality rate, which differed significantly than Heterorhabditis sp. (HKM) and H. bacteriophora (HRJ). At the highest population level of 10,000 IJs/kg of soil, Heterorhabditis sp. (HKM) was statistically at par with Heterorhabditis sp. (HSG) and H. bacteriophora (HRJ), whereas Heterorhabditis sp. (HSG) differed significantly than H. bacteriophora (HRJ). The highest mortality rate (57.78%) was recorded by H. bacteriophora (HRJ) at this concentration. The mortality rate varied from 16.11–18.89%, 3 days post treatment, and the differences among isolates were insignificant (Fig. 4).
In order to study the reproduction of EPNs, the A. segetum larvae were exposed to 10, 20, 30, and 40 IJs/larva of each nematode species. The host mortality rate and the emerging IJs from host cadavers were collected and counted. The data revealed that all four test nematodes were successfully invaded and propagated in the insect larvae and produced IJs (Figs. 5a–d). It was also evident that all nematode species exhibited a linear relationship between the concentrations of IJs applied and the total number of IJs produced per infected larva. In this study, H. indica and H. bacteriophora (HRJ) produced significantly more number of IJs per insect larva than the other two nematode species (Fig. 2c, d). For H. bacteriophora (HRJ) and H. indica, the maximum production of IJs per larva (14.23 ± 1.34 × 103 IJs/larva and 11.09 ± 1.14 × 103 IJs/larva) was obtained at 40 IJs/larva concentration. Among the four EPNs studied, the least progeny production was recorded for Heterorhabditis sp. (HKM). It increased linearly with the increase of IJ concentration where it reached its maximum of 7.62 ± 1.04 × 103 IJs/larva, at the concentration of 40 IJs/larva.
There were significant differences in the efficacy of different isolates of EPNs against the L3 of A. segetum across concentrations and the observation periods. The efficiency of EPNs against a given host partly depends on the host-finding, ability, and penetration capability of the IJs (Peters & Ehlers, 1994). EPNs have been tested against a large number of insect pest species, with results varying from poor to excellent control (Laznik and Trdan 2015). Many factors can influence the successful use of nematodes as biological agents, but matching the biology and ecology of both the nematode and the target pest is a crucial step towards successful application. The H. bacteriophora (HRJ) resulted in a significantly greater mortality of A. segetum than the isolates from Sangla and Kamand across the isolates and observation periods, though the differences in larval mortality between the isolates were much smaller. These findings are in partial agreement to the findings of Chandel et al. (2009) who reported 100% mortality of L3 and L4 of A. segetum in a Petri plate bioassay, at 10–40 IJs/cm2, due to infection with H. bacteriophora. Fetoh et al. (2009) tested an Egyptian strain of H. bacteriophora against L4 of A. ipsilon under laboratory conditions and recorded 80 ± 4.0 to 100 ± 0.0% mortality rate, at 25–100 IJs/ml and reported that H. bacteriophora was highly virulent against A. ipsilon. Larval mortality may be presumed to be related to the number of viable nematodes ingested by the insect during feeding, or infection could take place by invasion of the nematodes through the natural openings/cuticle of the insect with an undetermined minimum number required for mortality to occur. The maximum mortality (93.3%) was caused by H. indica (commercially available isolate during studies) against L3 of A. segetum. Hussaini et al. (2005) studied the infectivity of H. indica PDBC EN6.71 along with other EPNs against the last-instar larvae of A. ipsilon and obtained absolute mortality after 48 and 72 h at 25 and 32 °C. Against A. segetum, there was a 66.70% mortality rate, with H. indica PDBC EN 6.71 72 h post inoculation (Hussaini et al. 2000). Yan et al. (2014) also reported that H. indica LN2 was the most virulent and promising species, causing 83.3% mortality to the L3 72 h post infection.
In the soil bioassay carried out against L4 of A. segetum, H. bacteriophora (HRJ) was found highly effective, followed by Heterorhabditis sp. (HKM) and then Heterorhabditis sp. (HSG) at10,000 IJs/kg of soil. The mortality rate varied from 78.33 to 81.67% 7 days post treatment, at local isolates. Chandel et al. (2009) found that the concentration of 1000 IJs/kg of soil of H. bacteriophora was sufficient to initiate infection in the larvae of A. segetum. They reported 61.3–91.6% mortality rate in L4 at 1000–10,000 IJs/kg of soil. Obtained data support the findings of Hussaini et al. (2001) who recorded that Heterorhabditis was virulent against A. ipsilon larvae in sand column assay. Gupta (2003) studied the efficacy of EPNs against A. ipsilon and found that the nematodes applied as foliar spray (50–100 IJs/larva), paper wrapping method (25–75 IJs/larva), and food dip (25–75 IJs/larva) caused 40–80, 100, and 40–60% pest mortality rates, respectively.
When A. segetum were exposed to Heterorhabditis spp. in sand, higher inoculation concentrations were required as compared to inoculation concentrations on filter paper. The differences in the two inoculation methods may be attributed to the differences in the inoculation substrates and bioassay arenas. In the sand bioassay technique, A. segetum was exposed to Heterorhabditis spp. in three-dimensional substrates, where the nematode has to search the insect host. In the Petri plate bioassay method, A. segetum was exposed to Heterorhabditis spp. in two-dimensional substrates, where the nematode and the host were in direct and close contact with each other. Obtained results are in agreement with that of Grewal et al. (1994) who reported that the substrate had a profound effect on host finding by EPNs. A major factor that restricts the EPNs’ host range was the foraging behavior of the IJs. These nematodes employed different foraging strategies to locate and infect hosts that range from one extreme of sit-and-wait (ambush) to the other of widely foraging strategy (cruise) (Lewis 2002; Laznik and Trdan 2013).
Timing of nematode applications is also an important consideration. Different turnip moths may arrive to the root feeding zone near the soil surface at varying times during the growing season. Nematodes applied too early may provide poor insect control and may not reach deep in the soil before their upward seasonal migration. To overcome the dispersal behavior of the EPNs, Heterorhabditis spp., so that it can infect its host, the application of high dosages to the soil surface may increase the infection rate. However, it is also important to note that results from the laboratory tests are not always comparable to field testing (Cantelo and Nickle 1992) as the functioning of EPNs in the open is influenced by an extensive list of factors. In one relevant study, the 100% efficacy rate of S. carpocapsae in controlling Colorado potato beetle adults, pupae, and larvae in the laboratory manifested as only a 31% reduction rate in this pest population when the test was repeated outdoors (Stewart et al. 1998).
Reproducing and recycling of EPNs in a host play an important role in their persistence in the soil and also in their overall effectiveness in pest control (Georgis and Hague 1991). A prior knowledge about reproducing and recycling nematodes is considered important in determining the time and concentration of subsequent EPN application, which may be useful in reducing the cost of application. The data in the present study suggested that following application, all the tested species of nematodes were able to infect and propagate within the insect host and produce IJs. The evidence obtained in this study suggests that all three tested indigenous species of EPNs were virulent enough to produce 100% mortality to the larvae of A. segetum. Furthermore, all EPNs could also propagate in the infected larva and produce F1 generation IJs.