To evaluate the potential of newly described Rwandan EPNs (Yan et al., 2016) for the development of biological control products against soil insect pests, bioassay-based laboratory screenings and small-scale field efficacy trials had been implemented and the results, as presented here, are promising.
Laboratory bioassays
The small arena bioassays revealed that all the four tested Rwandan EPN species/strains were able to infect and kill white grubs, although it took quite a high number of infective juveniles per grub larva. Nevertheless, this is considered a good basis for further research, as it is often not too easy to find EPNs being infectious to grubs as they are often able to defend themselves to EPNs or other ground-living natural enemies (Ansari et al., 2003; Guo et al., 2013; Laznik and Trdan, 2015). And other EPNs, such as S. scarabaei, are not easily mass-produced. Consequently, there are only few EPN products on the market against white grubs, globally. However, as the tested Rwandan EPNs originated from small-holder farming habitats of Rwanda (Yan et al., 2016), they are, potentially, more adapted than EPNs from other habitats, such as sea shores. Moreover, the tested Rwandan EPNs seem to be as good as international species/strains commonly used for soil pest control. This is a hint that the tested EPNs may have evolved in areas of white grub prevalence in Rwanda.
EPN infectiousness against grubs
The overall accumulated mortality of A. graueri white grubs due to EPNs, as verified by dissections, was 13 ± 15 SD % until day 4; 17 ± 17% until day 7; and 20 ± 18% until day 14 (n = 291). Comparable results were obtained when correcting the total accumulated mortality of grubs in the treatments to the natural mortality in the untreated controls. That is, 13 ± 15% more grubs died in the EPN treatments than in the control until day 4; 16 ± 18% until day 7; and 17 ± 19% until day 14. Without any treatment, 6 ± 7% of grubs naturally died until day 4; 10 ± 13% until day 7; and 14 ± 18% until day 14.
Infectiousness depending on EPN species/strain
Overall, tested EPNs appeared to have medium infectiousness against white grubs in the laboratory bioassays. Nevertheless, all tested EPNs, regardless if Rwandan or international, were able to infect and kill A. graueri grubs (Figs. 1 and 2). However, species/strains differed in their infectiousness (GLM: “species/strain” for 7 day corrected mortality: F 6;290 = 16, p < 0.0001; “species × concentration”: F 24;290 = 2.5, p < 0.0003, adjusted R2 = 0.33; Fig. 2). The Rwandan H. bacteriophora RW14-N-C4a and S. carpocapsae RW14-G-R3a-2 reached the efficacies of their corresponding international strains, indicating that local EPNs could be used as biocontrol agents against Anomala grubs.
The heterorhabdits slightly more consistently and usually more effectively killed the grubs than did most steinernematids (Fig. 2); except for the Rwandan and international S. carpocapsae which reached the infectiousness level of the heterorhabdits in some of the assays.
On average across concentrations, the Rwandan H. bacteriophora RW14-N-C4a killed 29 ± 18 SD % of grubs, the international H. bacteriophora H06 27 ± 9%, the Rwandan S. carpocapsae RW14-G-R3a-2 14 ± 11% and the international S. carpocapsae All 18 ± 9%. Steinernema sp. RW14-M-C2a3 was, overall, the least performing (4 ± 2% of grubs killed across concentrations, Figs. 1 and 2). The Rwandan Steinernema sp. R14-M-C2b1 (10 ± 3%) and the international S. longicaudum X7 (11 ± 3%) were both of comparable medium, but relatively constant infectiousness (5 to 18% mortality across different concentrations), as well as easily mass-reared (data not shown), and therefore suggested for field trials.
Field assessments are the logical next step, because the infectiousness data from EPNs in the laboratory can only partly be transferred to the field situation (Peters et al., 1996). This is, for example, because the foraging behaviour of EPNs is less important for the attack of host insects in small arenas of bioassays, than under field conditions. Field conditions also require a longer survival of EPNs without the host, i.e. before finding the host, than in small arena bioassays.
Infectiousness depending on EPN concentration
The concentration of applied EPN species/strains influenced their infectiousness on A. graueri grubs (GLM “concentration” for 7 day corrected mortality: F 4;290 = 5.5, p < 0.0003; “concentration × species/strain” F 24;290 = 2.5, p < 0.0003, adjusted R2 = 0.33) (Fig. 2). This effect was due to an increasing infectiousness at high concentration of 1000 IJs per larva, whereas concentration between 100 and 400 IJs did not matter much (GLM F 3;236 = 0.8, p = 0.49). When analysing concentration effects separately per EPN species, often no dose efficacy-response was detected, that is, for S. carpocapsae All, Steinernema sp. RW14-M-C2a-3, Steinernema sp. RW14-M-C2b-1 and S. longicaudum X7 (GLM F 3;44 to 65) < 3, p > 0.05). Only the following species/strains showed a positive dose-infectiousness response, i.e. H. bacteriophora RW14-N-C4a and H06 and S. carpocapsae RW14-G-R3a-2. In other words, at low concentrations of 100 to 200 IJs per larva, both the Rwandan and international H. bacteriophora strains as well as the international S. carpocapsae All performed best (12 to 30% additional mortality compared to untreated control) (GLM in Fig. 1). At high concentrations of 1000 IJs per larvae, again both H. bacteriophora strains were the best as well as the Rwandan S. carpocapsae RW14-G-R3a-2 (34 to 58% mortality compared to control).
In many cases, results were variable, and therefore a clear dose-response can hardly be concluded. This is typical for natural strains of EPNs that have not yet gone through many cycles of mass production and therefore through a selection for constant traits. It also shows that higher concentrations than 1000 IJs per grub should probably have been included in the experiments to get better dose-response trendlines. Nevertheless, we conclude that all Rwandan EPNs are worth to be further investigated on a number of target soil insect pests of Rwanda.
Infectiousness depending on exposure period to EPNs
Overall, the 7-day assessment appeared to be promising to detect differences in the infectiousness of the different EPN species/strains as well as concentrations. Differences among EPN species/strains in their infectiousness on A. graueri grubs over time of exposure were more obvious at low EPN concentrations than at medium or high concentrations (Fig. 1a). Variability of data depended more on the EPN species/strain or concentration (see large SEMs for 1000 IJs per grub in Fig. 1b), than on exposure period. In other words, increasing exposure period from 4 up to 14 days did not consistently increase or decrease variability of data, except at low concentrations (see below).
As for low EPN concentrations of 100 to 200 IJs per larva, differences between EPN species/strains seemed most obvious for the 7-day assessment as data later became disturbed by increasing natural mortality. Infectiousness usually only little increased with time (Fig. 1a), except from day 4 to day 7 for S. carpocapsae All and Steinernema sp. RW14M-C2b-1, and up to day 14 for Steinernema sp. RW14M-C2a-3 and Steinernema sp. RW14M-C2b-1 (Kruskal-Wallis H tests at p < 0.05, d.f. 2; 40 to 60). For all tested EPNs, the variability of relative infectiousness increased over time due to an increase in natural mortality (Fig. 1b). In conclusion, the 7-day assessment should provide reliable information from the bioassay on differences between EPNs at an acceptable variability.
As for medium EPN concentrations of 300 to 400 IJs per larva, infectiousness did not or only little increase with time (Fig. 1). The 14-day assessment added little additional information and/or was disturbed by natural mortality, as seen in the decreasing values of H. bacteriophora RW14-N-C4a with time. The variability of relative infectiousness data was comparable among time periods, but increased in few cases for the 14-day assessment (Fig. 1). In conclusion, the 4- and 7-day assessment should provide reliable information from the bioassays.
As for high EPN concentrations of 1000 IJs per larva, infectiousness increased particularly after day 4 up to day 7, but only in few cases or not at all after day 7 (Fig. 1). This indicates that the high concentration led to maximum infection already at day 7 and no additional information could be obtained running the assays until day 14.
Field efficacy trials
White grub infestation
The natural population levels of grubs ranged from 0.1 to 0.25 larvae per plant in field M and from 0.3 to 0.9 larvae per plant in field N in 2015 (average across fields and cropping season 0.3 ± 0.29 SD larvae per plant). This equals about 4000 to 10,000 white grubs per hectare at site M and 4000 to 35,000 at site N, a pest level that can lead to significant yield loss as a single grub usually destroys at least one planted tuber.
At day of planting Irish potatoes, 0.1 ± 0.03 grubs were found per plant in field M and 0.7 ± 0.17 in field N. Before EPN treatment about 30 to 55 days after planting, 0.1 ± 0.1 grubs were found per plant in field M and 0.9 ± 0.52 in field N. Subsequently, the population remained relatively stable until harvest in field M (see untreated controls), but it decreased within 30 days after planting in field N until it remained relatively stable until harvest M (Fig. 3). This means, during the period of agents’ efficacy assessments, a relatively constant natural pest population was present. The overall average pest populations across time periods was not different between the two fields (independent samples t test, t 78 = −1.2, p = 0.26).
EPN efficacy at reducing grubs
Overall, treatments reduced Anomala and Hoplochelus white grub numbers (GLM, averages pooled over season; F 7;79 = 2.4; p = 0.03; adjusted R2 = 0.1; Fig. 4). Variability of grub data between plots was high as well as between time steps (Fig. 4). Therefore, it was difficult to come to quantitative conclusions particularly when separately looking at grub values of each field and time step.
When standardising grub data to their respective controls and combining data of both fields, the control effects of treatments became obvious (Fig. 5). Biological and insecticidal treatments were effective in reducing grubs, sometimes at highly efficient rates. Biological and insecticidal treatments were comparably effective when considering overall effects along the entire season (multiple Tukey post hoc comparisons, all p > 0.05). Control effects (compared to untreated controls) were highest about 60 to 90 days after planting, but less at earlier stages as well as towards harvest time. Only the handpicking method did not significantly reduce white grubs (Fig. 5).
As for EPNs, effects were highest about 30 to 60 days after their application when they reached up to 87 and 96% control on average across fields (Fig. 4). In contrast, 7 days after treatment, hardly any effects of EPNs were observed. This is because EPNs needed time to find and kill the hosts. However, the 7-day assessment as a first sample time is a common practice to evaluate dynamics of EPN efficacy after application (Yan et al., 2013). But, it is not surprising that shortly after the EPN treatment, still no effects were found.
The Rwandan isolate Steinernema sp. RW14-M-C2b-1 reached 29 ± 33% efficacies after 30 days and 96 ± 3% after 60 days, an increase likely due to EPN propagation in the soil. The Rwandan isolate was at least as good as the international strain S. longicaudum X7 or even better (e.g. see at concentration of 0.75 × 109 IJs/ha in Fig. 4).
The international S. longicaudum X7 reached, at least at medium and high application concentrations, high control efficacies (77 ± 16 up to 85 ± 9% respectively about 30 days after application, and 95 ± 3% and 82 ± 17% after about 60 days). Also, the low concentration reduced grubs, but it took longer to reach effects (Fig. 5). As for other studies, S. longicaudum X7 is known to attack white grubs, such as Holotrichia parallela or H. oblita in peanut fields in China, and is used by growers for biological control purposes (Guo et al., 2013, 2015).
However, more field trials with more Rwandan EPN species and strains will be needed once the EPN-based biocontrol factory at RAB Rubona will run at full capacity. Particularly, both heterorhabditids, i.e. the Rwandan H. bacteriophora RW14-N-C4a and the international H. bacteriophora H06, need further investigation under field conditions. Both revealed promising infectiousness levels in the laboratory screenings. Moreover, H. bacteriophora is known to attack white grubs, such as Popillia japonica in turf grass (Klein and Georgis, 1992; Selven et al., 1993; Downing, 1994), and H. parallela and H. oblita in peanut fields and grasslands in China (Guo et al., 2013, 2015).
As for the insecticides, both were comparably effective on average over the season, this is 39 ± 35% for Avermectin + Chlorpyrifos and 27 ± 40% for Fipronil + Chlorpyrifos (independent samples t test, t 18 = 0.7, p = 0.49). However, 37 to 60 days after transplanting, Avermectin + Chlorpyrifos reached efficacies of 58 up to 87%, whereas Fipronil + Chlorpyrifos only reached 4 to 44%. However, the pesticides are difficult to be compared to the EPN treatments due to different application types (coating versus into-soil stream spray), application periods and water amounts. Thus, it is likely that the insecticides have caused effects earlier in the season, i.e. soon after they had been applied at the moment of planting. It needs to be, however, mentioned that Rwandan small-holder farmers do not have or have only limited personal protective clothing. Therefore, there is an acute risk that farmers are intoxicated (WHO, 2009) when coating tubers with pesticides, and therefore this is not advised. Chlorpyrifos is of WHO acute toxicity class II, thus moderately hazardous and Fipronil and Avermectin are slightly hazardous (WHO, 2009).
As mentioned above, the local common practice of handpicking did not significantly reduce white grub populations. This might be due to the fact that the grubs were collected only from a small proportional area of the field, such as from the opened furrow for planting. At that time period, grubs are still not aggregated around crop roots or tubers as there is still no crop. And later, during mechanical weeding and during earthing up of potatoes, only between-row soil was searched. But at this time, grubs are expected feeding on the tubers and roots. Consequently, handpicking likely misses a large part of the pest population; thus, the effect of this method is limited.
Considering factor “time” after planting and/or after EPN treatment the following effects on white grubs were found. Around 30 days after planting, the insecticide and handpicking treatment had no detectable effect on grub numbers (Kruskal-Wallis H test, chi square = 2, p = 0.37; Fig. 4). At 37 days after planting (7 days after EPN treatment), the factor “treatment” had no detectable effect on grub numbers (sqrt-transformed data, Univariate GLM, F7;16 = 0.8, p = 0.6; Fig. 4).
Around 60 days after planting (30 days after EPN treatment), treatments had reduced grub numbers (sqrt-transformed, Univariate GLM, F 7;16 = 3.6, p = 0.045) as well as around 90 days after planting at day 60 after EPN treatment; GLM, F 7;16 = 4, p = 0.034; Fig. 4).
At harvest, 128 days after planting (day 71 to 108 after EPN treatment), no effects of EPNs on grub numbers were detected anymore (sqrt-transformed, Univariate GLM, F 7;16 = 0.7, p = 0.67) (Fig. 4).
EPN efficacy at preventing yield loss
The average yield was 0.3 ± 0.14 kg Irish potato tubers per plant, equalling about 9 to 10 tons per hectare. The yield was double in field M as in field N, that is, 0.4 ± 0.03 kg versus 0.2 ± 0.02 kg per plant, respectively (independent sample t test, t 62 = 11.8, p < 0.0001).
However, treatments had no detectable effect towards an increased yield (GLM of yields compared to control, F 7; 63, = 1.6; p = 0.15).
EPN persistence
The Rwandan or international EPNs were recovered from the soil of the treated experimental plots, but no natural EPN population was detected in the untreated plots.
Both applied EPN species well-established in the soil, because soil samples from the treated plots were all found EPN-positive 7 days later (Fig. 5). Later on, EPN persistence decreased with time (Pearson correlation r = −0.67 with time, p < 0.001, n = 40). Multiple comparison tests at each time step did not reveal differences between the persistence of the two EPNs and their concentrations (too few data), but a slight overall treatment effect remained (e.g. GLM at 30 days, F 4;9 = 5.8, p = 0.041; Fig. 5). S. longicaudum X7 persisted in the soil for at least 60 days after treatment in both fields. The Rwandan Steinernema sp. RW14-M-C2b1 persisted in the soil for at least 60 days in field N, whereas it was only detected until day 30 in field M. No EPNs were anymore detected towards time of harvest in both fields.
In summary, baited soil samples in our study proved that both the Rwandan and international EPN can establish in the soil after treatment. This indicates that the applied EPNs were of good quality as they need energy to survive some time in the soil until finding and propagating in the host insects. It moreover showed that the application method of EPNs into the moist just-opened soil furrow is a method appropriate for EPNs. Applications into the soil are known to be a good practice for EPN use as they prefer permanent moist conditions (Dutky, 1969). Also against other soil pests at a field scale, such as against the chrysomelid larvae of Diabrotica rootworms in maize, the fluid application into the soil had proven most suitable for EPNs compared with onto-soil fluid row-applications or into-soil granule applications (Toepfer et al., 2010). Our results also showed that the applied EPNs persisted in the fields of Rwanda for at least 2 months, which indicates that they were able to propagate. After more than 3 months, i.e. during harvest time, no EPNs were detected anymore. This is a typical situation for crops with large surfaces of bare soil such as maize (Kurtz et al., 2007), or, as here, in wide-spaced senescent Irish potatoes. One season persistence is advantageous in case of commercialisation of the EPNs, as they would need to be more frequently applied. Short persistence is however less typical for vegetables or berries where EPNs persist longer (Burlando and Kaya, 1992), or grasslands or orchards where they can persist for years (Belair et al., 1994).