Susceptibility of white grubs from forestry and sugarcane plantations in South Africa to entomopathogenic nematodes

The control of white grub (Coleoptera: Scarabaeidae) pests of sugarcane and forest plantations is difficult due to their cryptic nature and resistance to chemicals. This study evaluated the potential use of entomopathogenic nematodes (EPNs) as an alternative control method. Laboratory bioassays were performed with 12 locally isolated EPN species to determine the susceptibility of third instar larvae of the white grubs Schizonycha affinis Boheman, Pegylis sommeri Burmeister, Monochelus sp. and Maladera sp. 4. Concentration trials to determine lethal dosages for three of the white grub species were performed using Heterorhabditis zealandica Poinar MJ2C. Bioassays were performed to determine whether nematodes could develop inside the cadavers of S. affinis and P. sommeri as these had shown the lowest susceptibility to EPNs. The mortality percentage of the white grubs, although varying significantly, was found to be low for most of the EPN species, except H. zealandica. The highest percentage mortality of white grubs was observed at four weeks post inoculation with the rate of mortality being highest in the first week. Schizonycha affinis had the lowest LD50 of 38 Infective juveniles (IJs) per larva after 28 days, compared to Maladera sp. 4, with 284 IJs per larva, and P. sommeri, with 1035 IJs per larva. The dissection of insect cadavers revealed possible limiting factors for low susceptibility, due to the nematodes and their associated symbiotic bacteria’s inability to infect the insect haemocoel, with no EPNs being found inside some of the cadavers.

important establishment pests in plantation forestry (Echeverri-Molina and Govender 2016a, b;Sivparsad et al. 2018). Govender (2007Govender ( , 2014 reported that white grubs were responsible for 13% of the damage caused in black wattle plantations in the KwaZulu-Natal (KZN) province, while McArthur and Leslie (2004) reported an average of 23-55% reduction in sugarcane yield in the KZN Midlands North area. Forestry and sugarcane plantations are often in proximity and an overlap of white grub species affecting these two crops has been observed (Sivparsad et al. 2018).
The control of white grubs is difficult mainly because of their soil-dwelling nature, their resistance to chemical insecticides (Grewal et al. 2005) and the nocturnal lifestyle of the adults (Jackson and Klein 2006). While white grub control efforts have been dominated using chemical insecticides, the use of entomopathogenic nematodes (EPNs) offers a relatively safe and efficient control option (Grewal et al. 2005;Koppenhöfer et al. 2020). EPNs (Rhabditida: Steinernematidae and Heterorhabditidae) are known to infect various below-and above-ground insects (Grewal et al. 2005;Lacey and Georgis 2012). The two EPN families are used worldwide in the biological control of various insect pests (Shapiro-llan et al. 2010;Lacey and Georgis 2012). EPNs kill their insect hosts using symbiotic bacteria, which they carry in their digestive system and in specialised bacterial chambers (Nobuyoshi 2002). Death of the insect normally occurs within 48 h after infection (Kaya et al. 1993;Nobuyoshi 2002).
South Africa has a diversity of native EPNs, with a total of 17 Steinernema and seven Heterorhabditis species reported by 2017 (Malan and Ferreira 2017). A recent description of Steinernema bertusi Katumanyane, Tiedt, Malan & Hurley brought the total of Steinernema species described from South Africa to 12, constituting 12% of the described species of this genus in the world (Katumanyane et al. 2020). Despite the diversity of native EPNs in South Africa and their potential as biological control agents for white grubs, so far only one study has been undertaken to evaluate the potential use of EPNs to control white grubs in South Africa. Abate et al. (2019) evaluated the efficacy of both native and non-native EPNs to control the white grub, Heteronychus licas Klug in sugarcane plantations. There is need to evaluate the use of EPNs on more white grub pests in sugarcane and plantation forests in South Africa.
In the current study, we screened locally isolated EPNs for their biocontrol potential against native white grubs from forestry and sugarcane plantations in South Africa. More specifically, we (1) screened twelve locally isolated EPN species for their biocontrol potential against four most dominant white grub species, (2) determined whether (and how) mortality rate of white grub species varied with infected EPN species and over time, (3) tested different concentrations of EPN to determine the lethal doses needed, and (4) examined the potential for different EPNs to infect, kill and reproduce in two of the most resistant white grub species.

Source of white grubs
Four white grub species, namely Schizonycha affinis Boheman, Pegylis sommeri Burmeister, Maladera sp. 4 and a Monochelus sp. were used in this study. Insect identification was done by means of a LUCID key, previously developed for white grubs in sugarcane plantations in southern Africa:https:// keys. lucid centr al. org/ keys/ v3/ sugar cane_ white_ grubs/ sugar cane_ white_ grubs. html. The white grubs were collected from wattle and sugarcane plantations in the KwaZulu-Natal province of South Africa. Each white grub was placed in a 30 ml plastic vial, filled with moist autoclaved peat moss (Hygrotech sustainable solutions) and provided with a fresh carrot disc to feed. The vials containing white grubs were placed in cooler boxes for transport and later rearing at the Forestry and Agricultural Biotechnology Institute (FABI) Biocontrol Centre at the University of Pretoria, South Africa. Rearing was done at 23 °C and 60-70% RH. The autoclaved peat and carrot discs were replaced weekly. Only third instar larvae were used in the experiments. The grubs were only used three weeks onwards after collection to eliminate those that could have come infected from the field. All nematodes used were reared using the third instar larvae of the greater wax moth, Galleria mellonella L. (Lepidoptera: Pyralidae). Infective juveniles (IJs) were harvested on modified White's traps and stored in distilled water in horizontally placed culture flasks at 12 °C. The EPNs were shaken periodically for ventilation and used within three weeks after harvesting. A fresh batch of nematodes was used for each repetition of the experiment.

Soil bioassays
The methods used by Koppenhöfer and Fuzy (2003), An et al. (2012) and Wu et al. (2014) were modified and used in the present experiment. The EPNs H. bacteriophora, H. zealandica-MJ2C, S. fabii, S. jeffreyense and S. yirgalemense were separately tested for their potential to kill the third instar larvae of P. sommeri, Monochelus sp., Maladera sp. 4. Two additional species, namely S. sacchari and a strain of S. fabii-SCH10, were further tested against the larvae of S. affinis.
For the setup, 30 ml plastic cups were filled with 30 g autoclaved sandy loam soil. A white grub was then placed on top and allowed to dig itself into the soil, and the soil was inoculated with the EPNs. From a nematode suspension, 400 IJs were sprayed onto the surface of the soil in each cup and the soil moisture was adjusted to 18% v/w. A carrot disc was provided for white grub feeding. The vials were then placed in a plastic container lined with moist tissue paper (100% moisture) and stored at 25 °C in a dark room. The experiment included five treatments (EPN species), and each was tested on ten white grubs. This was replicated three times. Each replicate was undertaken on a different test date using a different batch of nematodes. The cumulative percentage mortality for each treatment was recorded over a period of one month, at weekly intervals. For S. affinis, two additional EPN species were tested.

12-well laboratory bioassays
Based on the preliminary results of the soil bioassays, the most resistant white grubs, namely S. affinis and P. sommeri, were subjected to further tests in 12-well bioassay plates. The aim was to investigate any changes that occurred in the susceptibility of the white grub species, when they were directly exposed to the EPNs. The susceptibility of the two white grub species to nine EPNs, namely H. bacteriophora,H. baujardi,H. indica H. noenieputensis,H. safricana,O myriophila and S. fabii-SCH10, was tested. Additionally, the susceptibility of S. affinis to S. bertusi was also tested.
For the experimental setup, a single white grub was placed in a cell of a 12-well bioassay plate lined with a filter paper disc. It was inoculated with 200 IJs suspended in 50 µl distilled water. Ten grubs of each white grub species were used for each of the treatments plus the control. The controls received water only. The plates were placed in a plastic container lined with wet paper towels (at 100% moisture level), closed with the lid, and kept in a growth chamber, adjusted at 25 °C. Mortality reading was taken daily for up to seven days for S. affinis and for up to ten days for P. sommeri, as the mortality for the latter was observed to take longer than for the former. Any dead larvae were removed, rinsed with distilled water and placed in a new 12-well plate lined with moist filter paper. Dead larvae were returned to the growth chamber to allow any EPNs, if present, to develop. All dissections of the cadavers, aimed at confirming the presence of EPNs, were performed on the 10 th day post-inoculation (PI). Only those cadavers that were observed to have EPNs present were counted as part of the mortality (equal infection) data. The experiment was replicated three times, on a different test date, for each nematode species, and using a fresh batch of nematodes.

Concentration trial
Based on the preliminary results obtained with both the soil and the 12-well bioassay disk (Figs. 1, 2, 3), H. zealandica MJ2C was selected for the probit test to determine its lethal dosages (LD 50 ) on the third instar larvae of P. sommeri, S. affinis and Maladera sp. 4. Monochelus sp. was not included in the experiment, as its larvae were not available in sufficient numbers. Similar methods to those that were used in the soil bioassay were employed in this experiment. However, the treatments included different concentrations of H. zealandica MJ2C, namely 0, 100, 200, 400, 800 and 1600 IJs per vial. Ten white grubs were used in each treatment (EPN concentration × ten white grub species), with the experiment being replicated three times, and with each replicate being undertaken on a different test date, using a different batch of nematodes. Cumulative percentage mortality was recorded at weekly intervals over a period of one month.

Examining EPN survival and development inside insect cadavers
The ability of EPNs to develop in the insect cadavers of S. affinis and P. sommeri was tested using H. safricana,H. noenieputensis,H. indica,H. baujardi,H. bacteriophora,O. myriophila and S. bertusi. The aim was to test the survival and development Fig. 1 Effects of different entomopathogenic nematode treatments and time (7-28 days) on the mortality rate of the white grub, Schizonycha affinis, placed in vials containing soil (a) and 12-well bioassay disks mortality recorded after seven days (b). Error bars on the graph represent SE. The probability values presented are derived from Kruskal-Wallis statistic testing the difference in the treatments for each period of the nematodes to gain improved understanding of the mechanisms of resistance employed by the two most resistant grub species, namely S. affinis and P. sommeri, as observed in the soil and 12-well bioassays. Similar methods to those used in the 12-well bioassays were employed in the experiment. Grubs were individually placed in 90 mm diam. Petri dishes and inoculated with 200 IJs of the nematodes. Ten grubs each of S. affinis and P. sommeri were used per EPN treatment. The inoculated insects were incubated at 25 °C, in containers lined with moist tissue paper. Insect mortality was recorded daily for a period of ten days. Irrespective of the day of insect death, all dissections of dead insects were performed on the 10 th day PI to be able to observe and record the status of the EPNs developing inside the haemocoel. The presence or absence of the EPNs and the symbiotic bacteria, the development stage of the EPNs, and the general state of the cadaver were recorded. The EPN symbiotic bacteria colonisation was visually observed in the haemolymph for characteristics typical to a successful entomopathogenic symbiotic bacteria (ESB) colonisation which included gumminess of the bacterial body mass for Photorhabdus bacteria, colour changes, absence of a putrid smell. The limitation with visual observation for ESB could lie in the inability to observe these characters in the presence of a supressed ESB colonization.

Statistical analysis
All statistical analyses were performed using the R statistical software, version 4.00 (R Development Core Team 2020). To determine whether mortality rates of white grub species (percentage of white grubs that died during the experiment) varied with infected EPN species and over time, we used generalised linear models (GLM) with a binomial distribution and a logit link function (Zuur et al. 2009), which assessed the main and interaction effects of time and EPN species. In order to better depict the trends in these effects, the variation in mortality among treatments (EPN species) was analysed and represented using bar plot (with error bars representing SE) for Fig. 2 Individual effects of different EPN treatments and time (7-28 days) on the mortality rate of the white grub, Pegylis sommeri, placed in vials containing soil (a) and 12-well bioassay disks mortality recorded after ten days (b). Error bars on the graph represent SE. The probability values presented are derived from Kruskal-Wallis statistic testing the difference in the treatments for each period each time interval (7, 14, 21 and 28 days). Due to the limited number of observations for each time interval, the Kruskal-Wallis non-parametric tests were performed to compare treatments (EPN species) for each time interval.
To test for the effect of time and different concentrations of EPN on mortality of white grubs, and determine the lethal doses needed, the log-logistic dose-response model (Eq. 1) was fitted with the 'drm' function of the package drc (Ritz et al. 2015) in R: where M(t) is the expected mortality rate at time t (number of weeks); b is proportional to the slope of M, at time t; and LD 50 is the concentration dose by which 50% of the white grubs would have died. (1)

Schizonycha affinis
The results of the soil bioassays showed that the mortality rate of S. affinis varied significantly among different EPN species (χ 2 = 167.48; df = 7; p < 0.001) and over time (χ 2 = 29.42; df = 3; p < 0.001). However, we found no interaction effects between EPN species and time (χ 2 = 8.73; df = 21; p = 0.991). Schizonycha affinis mortality increased over time for all treatments (Fig. 1a). Its highest mortality rate was constantly observed with H. zealandica MJ2C (Fig. 1a). The maximum percentage mortality of 70.0% ± 1.2%, which was obtained after 21 days, was significantly different from the next closest efficient species, S. jeffreyense, at 43.0% ± 1.2%, and S. yirgalemense, at 30.0% ± 1.2%. Overall, H. bacteriophora, S. fabii (BA), S. sacchari and an isolate of S. fabii (SCH10) showed very low mortality rates of less than 20% Fig. 3 Individual effects of treatments (EPN species) and time (7-28 days) on the mortality rate of the white grub a Maladera sp. 4 and b Monochelus sp. Error bars on the graph represent SE. The probability values presented are derived from Kruskal-Wallis statistic testing the difference in the treatments for each period after 28 days. For all the tested nematodes, the mortality graph levelled off after 21 days (Fig. 1a). In keeping with the results obtained in the soil bioassays, the highest percentage mortality for the 12-well laboratory bioassays was obtained from H. zealandica MJ2C at 63.0% ± 2.2%, seven days PI. Heterorhabditis zealandica SF41 gave the second highest mean mortality at 46.7% ± 3.3%, followed by Oscheius myriophila Poinar, at 40.0% ± 1.5%. The rest of the tested nematodes resulted in less than 30% mortality, seven days PI (Fig. 1b).

Concentration trial
Heterorhabditis zealandica MJ2C was used in the trial because it was the most efficient EPN for three of the white grub species, namely S. affinis, P. sommeri and Monochelus sp., in the screening bioassays. According to the results of the study, the LD 50 was found to be generally lowest for S. affinis, at 38 IJs per grub, intermediate for Maladera sp. 4, at 284 IJs per grub, and highest for Pegylis sommeri, at 1035 IJs per grub, at 28 days PI (Table 1).

Examining EPN development inside the insect cadaver
Heterorhabditis zealandica MJ2C showed a high mortality potential for S. affinis, causing the highest number of infections (19 out of the 30 white grubs tested), and was able to complete its life cycle inside the haemocoel of the grubs (Table 2). It was also observed to be the only nematode species able to protect its cadavers from attack by unidentified mite species. The cadavers were observed to remain intact, not smelly, or semi-decomposed, typical of EPN infected cadavers. Oscheius myriophila showed a comparatively high mortality for S. affinis, killing, on average, four out of ten individuals and being able to complete its life cycle within a week (Table 2). However, O. myriophila was unable to protect any of its cadavers, resulting in most of them being putrid and full of mites. Whenever H. indica colonised and killed S. affinis, the EPNs were unable to develop inside the cadavers, with only bacteria being observable at the end of the experiment (Table 2). For H. bacteriophora and H. zealandica SF41, mostly entomopathogenic nematode symbiotic bacterial (ESB) colonisation was observed, with no surviving EPNs appearing to remain in the cadavers. Steinernema bertusi showed a low percentage mortality towards S. affinis, with all the infections recorded being successful but was unable to keep the cadavers clean. The rest of the EPN species tested showed very low mortality rates. For H. safricana, H. noenieputensis and S. fabii SCH10, no ESBs/EPNs were observed in the dead cadavers (Table 2).
Heterorhabditis bacteriophora were found to have killed 20% of the P. sommeri grubs (Table 3) and were observed to have kept the cadavers clean and free of mites. In the cadavers, the red colour of the symbiotic bacteria was observed, although no EPN development of H. bacteriophora was observed. In two of the grubs, the symbiotic bacteria of H. bacteriophora were observed to have colonised only the upper body of P. sommeri. However, the rest of the EPNs used were found mostly to be unable to infect P. sommeri. Heterorhabditis zealandica SF41 was also found to have infected about 20% of the grubs involved (Table 3), but the EPNs were unable to complete their life cycle. Instead, stunted and dead nematodes were observed.
Most of the EPN-associated bacteria were generally observed to be unable to protect the P. sommeri and S. affinis cadavers from colonisation by other bacteria, possibly due to interference from the insects' own gut bacteria. The above could be seen in terms of the colour changes occurring in the cadaver, from colours typical of the symbiotic bacteria concerned to darkened colours only hours after infection, coupled with the emission of a foul odour after a few days had elapsed.

Discussion
The results of the current study showed varying degrees of susceptibility of the white grubs S. affinis, P. sommeri, Maladera sp. 4 and Monochelus sp. to South African EPNs. Generally, Maladera sp. 4 was found to be the most susceptible, while P. sommeri was found to be the least susceptible. Schizonycha affinis and Monochelus sp. were found to be moderately susceptible. Other than P. sommeri, all the white grubs tested were found to be moderately susceptible to H. zealandica MJ2C. From the probit analysis, the LD 50 values obtained using H. zealandica MJ2C were found to be relatively high for P. sommeri (1035 IJs per larva), followed by the values obtained for Maladera sp. 4 (284 IJs per larva) and, lastly, the values obtained for S. affinis (38 IJs per larva), at 28 days PI.
The varying degrees of susceptibility of white grubs for the different EPN species or isolates in the current study aligns well with what has already been shown for white grub susceptibility to EPNs elsewhere (Grewal et al. 2005;An et al. 2012;Abate et al. 2019). Consensus exists that the efficacy of EPNs against white grubs can be difficult to predict, due to the control efficacy recorded from previous experiments being inconsistent (Grewal et al. 2005). Such inconsistency can be explained by the presence of various biotic differences, including differences in the attractiveness of hosts, EPN species, their dispersal rates and ability to penetrate the grubs involved through their cuticle/gut wall (Georgis and Gaugler 1991;Koppenhöfer et al. 2007). In addition, environmental factors, like moisture, temperature and the physical properties of soil (Kaya et al. 1993), can also be responsible for the inconsistencies. In our experiments, only H. zealandica MJ2C was able to provide The table shows the cumulative number of dead white grubs out of 30 white grubs inoculated per EPN species, the status of the entomopathogenic nematodes (EPNs) and of the symbiotic bacteria (ESB) inside the insect cadaver, as well as the status of the insect cadaver over a period of ten days post-infection, PI. No new mortalities recorded after five days PI. The nematode species are listed alphabetically
Observing the EPN development occurring in the haemocoel of S. affinis and P. sommeri revealed several factors that might cause the low susceptibility of the two white grubs to EPNs. Such factors might include the inability of the EPNs to penetrate the insect's cuticle and the gut cavity, as the haemocoel of some of the grubs dissected was found to contain no EPNs. Additionally, the inability of the EPN symbiotic bacteria to multiply and protect the EPNs and the cadaver concerned might have hindered the spreading of otherwise successful infection. Stokwe and Malan (2017) observed that ESB were unable to multiply on the haemolymph of the woolly apple aphid, Eriosoma lanigerum, and that the nematodes involved were unable to effect successful infection. For white grubs, possible interference with symbiotic bacteria multiplication can include antagonistic effects brought about through the action of the white grubs' gut bacteria (Skowronek et al. 2020). For example, H. zealandica MJ2C was observed to be able to protect its cadaver from the inroads of mites, with the cadavers not developing a foul smell. The fact that H. zealandica SF41 did not yield similar results suggests that H. zealandica MJ2C's unique characteristic may be attributed to its different symbiotic bacteria. Booyzen (2022) reported the existence of three different symbiotic bacteria associated with South African H. zealandica, turning early infected Galleria mellonella larvae 'blue', 'green' and 'red'. The H. zealandica MJ2C (green) and isolate SF41 (blue) used in the current study are associated with P. thracensis and P. heterorhabditis subsp. Heterorhabditis, respectively. Of key importance is the fact that the H. zealandica (MJ2C) associated with P. thracensis was found to be the most pathogenic EPN, which can probably be ascribed to the nature of the associated bacteria. The observation of the red colour in the H. bacteriophora infected cadaver and yet no EPNs in the dissected cadavers indicate that the bacteria of H. bacteriophora is able to multiply in the haemolymph of the white grubs.
Previously, Skowronek et al. (2020) found that bacteria from the midgut of the common white grub, Melolantha melolantha L. larvae, exhibited antagonistic activity against ESB. The possibility that the symbiotic bacteria of H. zealandica MJ2C can counteract the white grubs' antagonistic bacteria requires further investigation. Similar to the observations made in the current study are the observations made in the study conducted by Karagoz et al. (2007). These authors observed that when the white grubs investigated were killed by EPNs the associated Sancassania sp. (Acari: Acaridae) mites moulted to the adult stage and began feeding on the host tissues and/ or microbes associated with the cadavers, as well as on the IJs. Such a finding could serve to emphasise still further H. zealandica MJ2C's use of its symbiotic bacteria to protect its cadaver.
The results of the current study confirm the results obtained in previous studies, showing that white grubs have varying degrees of susceptibility to EPNs. Variations in susceptibility cut across different EPN species and strains of the same EPN species. The implication of the occurrence of such a pattern for the biological control of white grubs, through the effective utilisation of EPNs, is that EPN screening is required for each white grub species. Furthermore, different strains of the same EPN species might need to be tested for their utilisation in different geographical regions, as inconsistency in efficacy has been shown in such regard.
For South African white grub species, the current study shows that high mortality can be achieved using H. zealandica MJ2C. However, H. zealandica MJ2C showed limited capacity to control P. sommeri. Further investigation is therefore required to test for the resistance mechanisms of P. sommeri. Most of the EPNs tested were found to lack the ability to develop within the haemocoel of the two most resistant grubs investigated, namely P. sommeri and S. affinis. This finding implies that such EPNs, even when they prove capable of penetrating the white grubs involved, tend to lack the biological compatibility that they require to kill the white grubs. Such incompatibility might be present because of the immunological defences possessed by the white grubs. Future studies should focus on identifying the specific mechanisms used by the white grubs to defend themselves against the EPNs investigated, while further screening studies can be conducted to identify the comparatively efficient EPN species.