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Citation Information : Journal of Nematology. Volume 50, Issue 3, Pages 273-280, DOI: https://doi.org/10.21307/jofnem-2018-037
License : (PUBLISHER)
Published Online: 17-October-2018
An alternative control method to the use of chemical insecticides against soil dwelling insect pests is the application of entomopathogenic nematodes formulated in alginate beads for enhanced shelf life. The aim was to compare the benefit on nematode survival and infectivity of: (i) pre-conditioning of juveniles, and (ii) coating of alginate beads. The nematodes
Entomopathogenic nematodes (EPNs) (Nematoda: Heterorhabditidae and Steinernematidae) are effective biological control agents of insects pests due to their ease of adaptation, high lethality against major pests and safety (Grewal, 2002), are exempt from registration by the Environmental Protection Agency (EPA) in the U.S.A, are compatible with various chemical pesticides and are feasible for genetic selection (Kaya and Koppenhöfer, 2004). The applications of Steinernematidae have demonstrated efficacy for the control of insect pests from soil as white grubs (Subramanian and Muthulakshmi, 2016).
EPNs are commonly stored in an aqueous suspension and achieve a survival time of 6 to 9 mon for Steinernematids and 3 to 4 mon for Heterorhabditids at 4°C to 15°C (Koppenhöfer, 2007; Stock and Goodrich-Blair, 2014). However, the application of EPN aqueous suspension on soil surface with conventional liquid spray technology, such as hand-operated sprinklers and hydraulic or pneumatic actuators, can affect the post-application effectiveness (Shapiro-Ilan et al., 2006; Hiltpold, 2015). Also, the cooling system required to maintain its storage stability during the transportation and spray technology required for the application increase the cost of use of EPNs (Chen and Glazer, 2005).
To promote the expansion of the use of EPNs as biological control agents, current research focuses on the development of low-cost formulations with: greater storage stability at room temperature, increased resistance during the transportation to the application site, where it dissolves for application in aqueous medium or it is deposited directly on moist soil (Burges, 1998; Grewal, 2000; Grzywacz et al., 2014), effective control of target insect pest and greater persistence in the field (Grewal, 2002; Shapiro-Ilan et al., 2012). The EPN formulation in gelling materials partially satisfies these requirements since it has been designed to provide a hydrophilic environment that prevents or delays the desiccation to increase its survival, especially on foliar surfaces (Navon et al., 2002). Since alginate biomaterials are used for tissue regeneration due to biocompatibility (Szekalska et al., 2016), its use in EPN formulation benefit its shelf life, is cheaper, and easier to elaborate and apply (Vemmer and Patel, 2013).
The success of EPN infection depends on the storage stability of the formulated product, which is also important for marketing; therefore, there is still scope for increasing that parameter (Navon et al., 2002; Sharma et al., 2011), but keeping it cheap and attending to its ease of use (Grzywacz et al., 2014). Some suggested strategies to improve the alginate formulation are: harden the surface to retain EPNs over an extended period of time (Hiltpold et al., 2012), slow down EPN metabolism before the formulation, probably forcing them into a partial anhidrobiosis through slow desiccation in glycerol, but maintaining the EPN infectiousness, and enhance it once the state dormancy breaks (Hiltpold, 2015; Kary et al., 2016). However, the survival of EPNs formulated in alginate beads (ABs) is no longer than 3 to 4 mon when stored at room temperature (22–25°C) (Kagimu et al., 2017).
The natural pre-conditionings of EPNs, before the formulation and coating of Abs, are cheaper strategies to improve the shelf life and their benefits and have been investigated marginally. The pre-conditioning of infective juveniles (IJs) in plaster of Paris prior to formulation in sodium ABs may have a positive effect on their survival time and infectivity since Cortés-Martínez et al. (2017) found that the survival of Steinernema glaseri mechanically pelletized in diatomaceous earth was improved if the IJs used were pre-conditioned on wet plaster of Paris after host emergence, compared to those from white trap.
The research questions we tried to answer was: can the functionality of the sodium ABs be improved to preserve the survival and infectivity of the EPNs by pre-conditioning the IJs prior to formulation and by slowing down the drying process through coating the ABs with an extra layer of sodium alginate? Therefore, the objective was to evaluate the survival and infectivity of IJs of three species of nematodes, with or without pre-conditioning in a modified trap of plaster of Paris, formulated in ABs with and without coating, under laboratory conditions at room temperature.
Infective juveniles of the nematodes Steinernema carpocapsae (Sc), Heterorhabditis bacteriophora (Hb), and Steinernema glaseri (Sg) collected within a week after their emergence from the host cadavers were used. The nematodes had been reared in last instar larvae of the greater wax moth, Galleria mellonella, at 23 ± 3°C and stored at 12°C.
Two capture methods of IJs were used, a modified White trap (WT) and a WT filled with a layer of plaster of Paris (PP) instead of the filter paper on small petri dish (Stock and Goodrich-Blair, 2014). The PP was used for the pre-conditioning of IJs upon emergence from infected hosts. A total of 30 Galleria mellonella larvae were inoculated with Sc or Hb and 60 with Sg IJs in petri dishes with a filter paper disc (90 mm diameter, Whatman No. 1). The inoculation rate was 100 IJs per insect. Three days after inoculation, the infected hosts were divided between the two IJs capture treatments and incubated at 23 ± 3°C. A few drops of bidistilled water were occasionally applied on the plaster to prevent desiccation of the host.
Infected cadavers placed in WT or PP were checked daily to inspect the emergence of IJs. Due to the faster movement of juveniles in PP, they were collected from the surrounding water three days after the onset of the massive emergence. The IJs from the water in the WTs were collected 7 d after the emergence. Then, the collected juveniles were separately concentrated in bidistilled water suspension at the rate of 25,000 IJs/ml into partially closed 250 ml tissue culture flasks and stored at 12°C for a week.
The ABs were made following the ionic gelation method (Vemmer and Patel, 2013). 20 ml of IJs aqueous suspension was deposited in 20 ml of a 1% solution of sodium alginate, 36% glycerol, and 0.05% green vegetable dye for 24 h at 12°C. Final composition was 0.5% of sodium alginate, 18% glycerol, and 0.025% green vegetable dye. Droplets of 83.33 µl of the alginate nematode suspension containing approximately 1,000 IJs were deposited in a 2% CaCl2 solution using a 10 ml Mohr pipette with an exit hole of 3 mm in diameter, the drops remained in the solution for 20 min. A total of 470 ABs were recovered by treatment with a sieve, rinsed three times with bidistilled water to remove residues from the CaCl2 solution and deposited in a 500 ml beaker which was covered with Parafilm® tape.
Each AB was individually immersed for 2 s in a sodium alginate solution containing 0.5% of sodium alginate, 18% glycerol, and 0.025% green vegetable dye, to be deposited afterwards in the CaCl2 solution for 20 min. The ABs were recovered with a sieve, rinsed three times with bidistilled water, and deposited in a 500 ml beaker which was covered with Parafilm® tape.
The beakers containing the ABs were stored at room temperature (23 ± 3°C) for 50 d. Monitoring of storage conditions was done with a temperature and relative humidity data logger (Control Company®, model 4040, accuracy of ±1°C and ±5% RH).
Every two days, a dissected AB was placed in a petri dish (60 mm diameter) and rehydrated with 7 ml of bidistilled water for a period of 24 h to soften them and facilitate the release of IJs (Kaya and Nelsen, 1985). Then, a 1 ml sample containing approximately 143 IJs was taken, divided in ten 100 µl-subsamples and the alive and dead juveniles were counted using a stereoscopic microscope at 30×; the living ones had mobility by themselves or moved when stimulated with a dissecting needle, while dead IJs had shriveled surface or gas bubbles inside body (Peters, 2016). The survival rate (SR) was calculated as the proportion of alive juveniles over the total juveniles (alive and dead).
Infectivity was evaluated every 7 d according to the mortality of Galleria mellonella (Ricci et al., 1996). A filter paper disc (15 mm diameter, Whatman no. 413) was placed in each cell of the 24-well plate, where 100 μl of bidistilled water and a dissected AB were placed afterwards. The plates were maintained in an incubator at 25 ± 2°C for 24 h for reactivation of the EPNs. Subsequently, the Galleria mellonella larvae were individually placed in each well plate, covered with Parafilm® tape, and returned to the incubator. Insect mortality was checked 72 h later. To verify the mortality of Galleria mellonella larvae by EPNs, each host with signs of infection was placed in a petri dish and incubated at 25 ± 2°C until the IJs emerged.
The experiment was performed by duplication and each bioassay was repeated five times. The results of the duplicate tests were combined for the final analysis. Survival analysis was performed using Kaplan-Meier plots and the Log-Rank test (P ⩽ 0.001). The analysis of variance (ANOVA on ranks) and the SNK test (P < 0.05) were performed between the species of EPNs, pre-conditioning, coating of Abs and % infectivity on Galleria mellonella. All analyzes were performed using SigmaPlot® 12 software (Systat Software, Inc., San Jose, CA, USA).
The survival rates per week of EPNs in ABs are presented in Table 1, which shows that Sc had the best viability, maintaining a SR value of 58.8% for 28 d, followed by Hb with a SR of 51.4% at 21 d and Sg with the shortest survival time with 7 d and 15% of SR. The differences between the survival curves by nematode species were highly significant (χ 2 = 2,507, Log-Rank < 0.001).
The SR of pre-conditioned EPNs in PP and formulated in ABs remained 39.6% and 53.3% in 7 d for Sg and Sc, respectively, while SR of Hb was above 9.8% in 21 d (Table 1). The maximum extension of the survival time and the highest SR were, using Sg, 21 d and 22.5%, respectively. The differences between the survival curves of the treatments with pre-conditioning IJs were statistically significant (χ 2 = 3,207, Log-Rank < 0.001).
Amongst the three EPNs formulated in ABs coated with a layer of sodium alginate, Sc was maintained with an SR of 41.6% for 28 d, Hb with 21.7% for 28 d, and Sg with 20% for 7 d (Table 1). The differences between the survival curves of the EPNs in coated ABs were statistically significant (χ 2 = 5,086, Log-Rank < 0.001). The SR trends for Sg and Sc are similar to those observed in the corresponding treatments by EPN factor; therefore, no benefit on survival by coating of ABs was observed on EPNs.
The maximum extent of the survival time of Sg and Hb was just 7 d when pre-conditioned IJs were formulated in coated ABs. The SR of Sg decreased rapidly to 40% in the seventh day, whereas SR of Hb was maintained above 92.4% in the seventh day (Table 1). The SR of Sc remained above 67% in 14 d and its survival time was extended to 35 d, albeit with about 10% SR. The differences between the survival curves of the preconditioned EPNs in coated ABs were statistically significant (χ 2 = 4,288, Log-Rank < 0.001).
All three treatments within the four factors tested: nematode species (EPN), pre-conditioning (Prec), coating (C), and the Prec-C combination, were statistically significant different, reaching the lower P-values the first two (Table 2). For example, according to ANOVA, where the F-test applies to the means by factor, the infectivity (IN) of the EPNs in sodium ABs against Galleria mellonella was significantly different for each species applied (F = 6.289, P = 0.023) and the difference in the mean IN between Sc and Sg was greater (P < 0.05) than would be expected by chance after allowing for effects of differences in time after formulation. Sc was the most infectious, killing all larvae in a period of 28 d (100%), Hb killed almost all of them in a period of 14 d (~100%), but Sg killed a few larvae in the first 7 d (15%), which lowered its mean IN to 3.0% for a period of 35 d.
The IN of preconditioned EPNs formulated in sodium ABs against Galleria mellonella over time ranged from 55% to 100% (Table 2). The difference in the mean IN among the different EPN species is greater (F = 5.795, P = 0.006) than would be expected by chance after allowing for effects of differences in time after formulation, and the statistical difference was significant only between Sg and Sc nematodes (P < 0.05).
The IN of EPNs formulated in coated ABs on Galleria mellonella was between 35% and 100% and mean IN was significantly different between each specie applied (P < 0.001). Sg was the less infectious and therefore statistically significant different if compared with Sc or Hb (P < 0.001). As it can be seen in Table 2, this trend in statistical difference was maintained during all times after formulation.
The formulation of preconditioned EPNs in coated ABs results in statistically significant difference on mean IN for each species applied on Galleria mellonella larvae (F = 160.548, P < 0.001). The combined treatment Prec-C improves the mean IN of Sc much more than on Sg or Hb (P < 0.05). Table 2 shows that the mean IN of Sc had increased by 45% when compared with Prec and by 20% when compared with C and, the combination Prec-C produced negative results in Sg with respect to Prec only, while Hb-Prec-C had lower IN with respect to Hb-C only.
In Table 1, the mean survival time (MSt) of EPNs in ABs by each factor is shown for a time of 35 dpf. Non pre-conditioned Sc in uncoated ABs had the highest MSt (26.07 ± 0.389 d), but pre-conditioning of IJs before the formulation shows a negative effect on MSt (13.02 ± 0.218 d), and the same trend in MSt was observed using pre-conditioned Hb. The coating of ABs did not show a significant difference on MSt of Sg, Sc or Hb, in relation to ABs uncoated. Only in the case of Sc, the coating of ABs reversed the negative effect of pre-conditioning of IJs, when both factors were combined, increasing MSt from 13.02 ± 0.218 d to 22.06 ± 0.372 d. Sg had the lowest performance formulated in Abs, but pre-conditioning alone increased the MSt significantly from 8.05 ± 0.079 d to 13.21 ± 0.262 d.
The mean infectivity (mean IN) increased significantly with the pre-conditioning of Sg, since it went from 3 ± 2.19% to 29 ± 9.5%, while for Sc a significant decrease occurred from 80 ± 9.18% to 51 ± 11.3% (Table 2). Coating of ABs had its greatest effect on the mean IN of Hb, with a value of 77 ± 9.0%, while the pre-conditioning combined with the ABs coating increase the mean IN of Sc to 96 ± 2.7%. Based on these mean results, it could be recommended doing more research to validate the use both pre-conditioning and coating treatments for the entire formulation process of Sc in ABs.
The nematode Sc had the longest survival time (35 d), followed by Hb (28 d), and Sg (14 d); all of them were formulated in ABs stored at room temperature (23 ± 3°C). The differences in the maximum survival time were highly significant with the other EPN. The survival and infective fitness of EPNs are usually well maintained in sodium ABs, but the results of this study show marginal improvements, not enough to compare the values observed by other studies on formulations with the same species used.
A difference in the experiments reported is that the storage conditions were usually maintained at constant temperature. For example, after six months of storage in sodium ABs at 23°C and high relative humidity (100%), the SR of the S. feltiae IJs (IS-6) was almost 100% and its infectivity on Galleria mellonella was maintained similar to that of newly harvested IJs (Chen and Glazer, 2005). After three months of storage at 8°C and 25°C, the survival rate of 1,500 IJs glycerol dehydrated Steinernema carpocapsae (SH1) formulated in alginate granules was approximately 30% (Kary et al., 2016). Hiltpold et al. (2012) reported that Hb Poinar nematodes escape from alginate capsules stored at room temperature (25°C) and are able to kill Galleria mellonella larvae in 1 to 6 d. Recently, Kary et al. (2018) reported the survival of glycerol dehydrated Heterorhabditis bacteriophora (NIR1) formulated in sodium ABs stored at 25°C; in two months, their SR was 40% and minor to 10% in the third month. Non-published data on the formulation of Steinernema glaseri IJs in ABs to compare were found in the databases of Web of Science®.
The reported experiments of nematode formulation under variable storage conditions are limited. Only the survival of Egyptian strains of two EPNs contained in plastic bags with calcium alginate as carrier reported by Hussein and Abdel-Aty (2012) is similar to the survival of EPNs formulated in ABs, because they found that their nematodes show a decreasing trend in SR after 50 d of storage at 25 ± 2°C, resulting in an SR of approximately 25% and 50% for Hb (BA1) and Sc (BA2), respectively.
Although pre-conditioned Sg and Hb IJs survived in low percentages when are formulated in sodium ABs after 14 d, a high percentage of infectivity on Galleria mellonella larvae was registered at this time. The sodium alginate is a material that usually has good biocompatibility because it does not react to contact with the biological materials with which it interacts (Orive et al., 2002), so it has been acknowledged that this property has a positive effect on the maintenance of the infectivity of EPNs, as confirmed by our study.
The capture of IJs after the migration over plaster of Paris was a useful treatment for pre-conditioning Sg prior to formulation in ABs as it increased SU. Therefore, we confirmed that the formulation of pre-conditioned IJs of Sg (NJ-43) on PP significantly improved their survivorship as Perez et al. (2003) and Cortés-Martínez et al. (2017) suggested. However, we cannot offer a conclusion about the benefit of pre-conditioning IJs on infectivity of Sg, because the insect pests and results were different.
The coating of ABs produced a very high increase in the infectivity of Sc or Hb on Galleria mellonella with respect to Sg. As suggested by Ruiz-Vega et al. (2011), it is possible that the first two have a lower tolerance to desiccation, while Sg showed the highest drying tolerance, at the beginning of a selection process for tolerance to low Aw. As a result, the coating reduced the drying rate of ABs, giving additional moisture to the Sc and Hb nematodes which allowed them to remain viable for a longer time under the evaporative and osmotic conditions created by gelling materials such as sodium alginate (Block, 2002).
The combination of pre-conditioning and coating produced similar results to those observed in Sc or Hb when coating was applied alone; but on average, Sc significantly exceeded Hb as its infectivity had a significant increase associated with Prec. This behavior could also be associated with the higher response of Sc to Prec due to the greater tolerance of the genus Steinernema to low relative humidity. In the study by Ruiz-Vega et al. (2011), the nematode Steinernema glaseri showed the highest percentage of survival at low Aw, which agrees with the results obtained by Brown and Gaugler (1997), who found that the emergence of nematodes of the genus Steinernema was less affected than that of Heterorhabditis bacteriophora under low relative humidity conditions.
Compared with the shelf life of available formulations as EPN infected insect cadavers or water dispersible granules (Kagimu et al., 2017), the proposed ABs are still not better because the two strategies tested marginally improve the survival and infectivity of nematodes, mainly depending on the fitness of each EPN species tested. Pre-conditioning benefits to Sg, coating of ABs benefits to Hb and Prec-C benefits slightly to Sc. However, the advantages of ease of IJs pre-conditioning and ABs preparation are maintained, which is important to develop low cost and simplest biopesticide technology for small farmers in less industrialized countries, where resources and equipment as liquid spray technology or air-conditioning are limited (Grzywacz et al., 2014).
Under a particular combination of formulation characteristics as EPN species, alginate coating and response of the EPNs to the pre-conditioning in plaster of Paris, the functionality of the sodium ABs to maintain viable and pathogenic IJs was favored. In relation to the pre-conditioning, only Steinernema glaseri IJs marginally favored their survival, as well as their infectivity on Galleria mellonella. The preconditioned IJs of Steinernema glaseri and Heterorhabditis bacteriophora showed a low percentage of survival to the formulation in sodium ABs; however, in the bioassays, a high percentage of infectivity was observed on 3rd instar larvae of Galleria mellonella. The coating of the sodium ABs resulted in the maintenance of a high infectivity in Sc and Hb up to 28 d, while the combination of pre-conditioning and coating slightly favored Sc followed by Sg.
The most promising combination to maintain nematode survival and infectivity at room temperature (23 ± 3°C) was using Steinernema carpocapsae IJs with or without pre-conditioning in coated or uncoated sodium ABs, which extended its survival time to 35 d with a minimal survival rate of 10% and preserved its infectivity (100%) against Galleria mellonella larvae.