Laboratory Bioassay of Selected Entomopathogenic Nematodes as Mortality Factors of Oulema melanopus (Coleoptera: Chrysomelidae)

Cereal crops occupy about 70% of the total area of arable lands in Poland (Wenda-Piesik and Piesik 1998). These cereal monocultures are susceptible to economic infestations of pests, including two species of cereal leaf beetles (Coleoptera: Chrysomelidae): Oulema melanopus (L.) and Oulema gallaeciana (Heyden). Of the two, O. melanopus is more prevalent in central and northern Poland, while O. gallaeciana prevails in southeastern Poland. Oulema melanopus is also the most important pest of wheat in other countries (Dimitrijević et al. 1999, Karić 2003), occurs in central Siberia, Sweden, Great Britain, Spain, and western Africa (Olfert et al. 2004, Tanasković et al. 2012), and is reported in cereal crops in the United States and Canada (LeSage et al. 2007, Olfert et al. 2004). In Europe, apart from winter and spring wheat, cereal leaf beetles are economic pests of barley, oats, and triticale (Malschi 2009, Meindl et al. 2001, Ulrich et al. 2004). Damage caused by cereal leaf beetles may be manifested in foliar loss, tillering of young plants, reduction of nutrient assimilation by the host plant, and vectoring of viral diseases (Trzmiel et al. 2015).

Efforts to identify alternatives to chemical control of pests in cereal crop production include the assessment of biological control agents. Natural enemies of cereal leaf beetles include predatory arthropods, parasitoids, and predation by some bird species. Parasitoids thus far used in biological control of cereal leaf beetle larvae include Tetrastichus julis (Walker) (Hymenoptera: Eulophidae), Diaparsis carinifer (Thomson), and Lemophagus curtus (Townes) (Hymenoptera: Ichneumonidae), while Anaphes flavipes (Foerster) (Hymenoptera: Mymaridae) has been assessed as an egg parasite (Evans et al. 2006, LeSage et al. 2007). Entomopathogenic nematodes (EPNs) should also be considered as they may also provide an environmentally friendly management approach for cereal production (Laznik et al. 2010b). Members of the Rhabditidae, Steinernematidae, and Heterorhabditidae families are obligatory generalist insect parasites with a free-living infective stage, that is, infective juveniles (IJs). Although their use in biological control has been traditionally for the control of soil-inhabiting pests (Ishibashi and Choi 1991), some research indicates their potential against foliar pests under special conditions (e.g., high concentrations combined with favorable abiotic factors of high humidity and optimum temperatures) (Laznik et al. 2012). Arthurs et al. (2004), among others, have shown the importance of selection of appropriate EPN species or strains.

The aim of the study presented here was to assess the biological activity of native isolates of Steinernema feltiae (Filipjev) and Heterorhabditis megidis (Poinar, Jackson, and Klein) against O. melanopus adults and larvae in laboratory assays. Laboratory assays provide information necessary to estimate specificity of species/ strains of EPNs to crop pests (Tóth 2006). As yet, no studies have been conducted to compare the potential of controlling the cereal leaf beetle with native isolates of EPNs in Poland. This also represents the first attempt to compare susceptibility of cereal leaf beetle adults and larvae to EPNs.

Cereal leaf beetles. Oulema melanopus adults used in the laboratory assays were collected in May from winter wheat and winter barley growing on experimental plots at the Institute of Plant Protection, State Research Institute, in Winna Góra, near Poznań, central Poland (N52°12′21.5″; E17°26′51.2″ 83 m elevation). Beetles were captured every second day during a 2-week period until egg laying by females was observed on the host plants.

Larvae used in the assays were obtained from a laboratory colony established by placing 11 pairs of field-collected adult beetles captured during the period of mating and egg laying on each of nine pots of tillering wheat plants. These were maintained in environmental chambers at an illumination of 53,000 lux (lamp SON-T Agro 400 W) on an 18/6-h photo regime at a temperature of 20°C ± 1°C and a relative humidity of 50–60%. After an egg-laying period of 7–10 d, living beetles were removed from the pots. Egg hatching and larval development (1 mm, 2 mm, 4 mm, and >4 mm in length) was monitored daily.

Nematodes. Five native isolates of EPNs representing either S. feltiae or H. megidis were assayed (Table 1). These were isolated from soil samples in 2010 and 2011 using the Egner's cane sampling device. Each soil core was 2.5 cm diameter and 30 cm deep. Four cores were obtained at each site, mixed, and placed in a plastic bag for transport to the laboratory. EPNs were isolated from these samples using Galleria mellonella (L.) (Lepidoptera: Pyralidae) larvae as “nematode traps” (Bedding and Akhurst 1975). White (1927) traps were used to collect IJs of each nematode isolate by placing the traps in an incubator at 25°C for 2 weeks, after which IJs were pipetted into tissue culture bottles which were then maintained at 4°C. IJs were then used to infect additional G. mellonella larvae to maintain cultures of each isolate. Species identification was performed based on morphometric criteria (Adams and Nguyen 2002, Poinar 1990) and genetic methods conducted at the Institute of Biochemistry and Biophysics, Polish Academy of Sciences (Wroclaw, Poland). Nematode cultures were subsequently maintained on G. mellonella larvae as per Kaya and Stock (1997).

Assays. The two concentrations of EPNs assayed against O. melanopus adults were 100 and 500 IJs per beetle. The concentrations assayed against larvae were 50 and 100 IJs per larva. Each of these concentrations for each insect stage was assayed at two temperatures (15°C and 25°C). In the assays, five adult or larval (3-mm length) beetles were placed in individual 9-cm-diameter petri dishes lined with moist filter paper along with several pieces of wheat leaves. Appropriate concentrations of IJs of the nematode isolates were then applied to the filter paper in the petri dishes. Controls were treated with distilled water only. Treated dishes were placed in chambers held at either 15°C or 25°C. Each of the 60 treatment combinations (isolate × insect stage × nematode concentration × temperature) was replicated 11 times. Insect mortality in each of the treatments was determined 48, 72, 96, and 120 h after initial exposure. Parasitism by the EPNs was confirmed by microscopic examination of dead insects.

Statistical analyses. Mortality data were statistically analyzed using of Statistica, version 12, of StatSoft^®Polska (Kroków, Poland). A Pearson chi-square test was used to determine the relationship between two variables.

Adult mortality response. At a dose of 100 IJs/adult, temperature significantly affected adult mortality caused by all EPN isolates tested, with the exception of H. megidis Wipsowo, with higher mortality levels at 25°C (Table 2). At 500 IJs/adult, the higher temperature resulted in higher adult mortality caused by the S. feltiae ZWO4, ZAG 15, and K13 isolates after 48 and 72 h of exposure, and the higher temperature increased adult mortality caused by the S. feltiae ZAG15 and K13 isolates 96 and 120 h after exposure. Higher temperature did not increase adult mortality by the S. feltiae ZWO23 isolate, with the exception of mortality after 96 h, which was higher at 25°C (Table 2).

At 15°C, adult mortality of beetles was found to be dose-dependent with the S. feltiae ZWO4, ZAG15, and K13 isolates regardless of the length of time after exposure, while the dose-dependent mortality response to the S. feltiae ZWO23 isolate was observed only at 48 h after application but with no effect observed at 72, 96, and 120 h (Table 3).

Variable results were obtained at 25°C. With the S. feltiae ZWO23 isolate, the higher dose had no effect on adult mortality after 48 and 72 h, but caused significantly higher mortality after 96 and 120 h. No dose-mortality effect was found with the S. feltiae ZWO4 isolate. With the S. feltiae ZAG15 isolate, the higher dose caused significantly higher mortality 96 h after application, but had no effect on mortality at 120 h. Likewise, the higher dose of the S. feltiae K13 isolate resulted in higher mortality of insects at 48 h but had no effect at 72 to 120 h. The higher dose of the H. megidis Wipsowo isolate resulted in higher adult mortality regardless of length of time after initial exposure (Table 3).

Overall, of the EPN isolates tested, the most pathogenic appeared to be the S. feltiae ZAG 15 isolate, which caused 100% mortality of O. melanopus adults at a temperature of 25°C and a dose of 500 IJs/adult 48 h following initial exposure. A relatively high level of mortality (84.4%) was also caused by exposure to a lower dose of IJs. A similar level of mortality (81.3%), but at higher dose, occurred following exposure to the S. feltiae K13 isolate.

At 15°C, mortality caused by the S. feltiae ZWO4 was only 6.5% at a dose of 100 IJs/adult and 30% at a dose of 500 IJs/adult at 48 h after exposure. At 72 h after application at 15°C, the S. feltiae ZWO4 isolate remained as the lowest performer at 100 IJs/adult, causing only 16.1% mortality, while the highest mortality of 79.3% was achieved with the S. feltiae ZAG15 at a dose of 500 IJs/adult. At 96 h, the S. feltiae ZAG15 isolate caused 82.8% mortality at the higher dose, while the S. feltiae ZWO23 isolate caused only 33.3% at the lower dose. The highest mortality observed at 120 h after application was 86.2% caused by the S. feltiae ZAG15 isolate at the higher dose, while the S. feltiae ZWO23 and K13 isolates caused 32.3% mortality applied at lower doses.

At 25°C, the S. feltiae K13 isolate was most pathogenic, causing 84.8% mortality at the higher dose. The weakest performer was the H. megidis Wipsowo isolate, which caused only 33.3% mortality at a dose of 100 IJs/adult. Longer exposure of the adults to the EPNs at this temperature did not alter mortality levels observed.

Larval mortality response. Of the EPN isolates tested, the H. megidis Wipsowo isolate caused the greatest larval mortality (89.3–100%) after 48 h at 25°C. The H. megidis Wipsowo isolate also caused the lowest mortality response (34.4%) at a temperature of 15°C and a dose of 50 IJs/larva after 48 h of exposure, but the same isolate caused 96.7% larval mortality at a dose of 100 IJs/larva after 72 h of exposure at the same temperature.

At a dose of 50 IJs/larva, temperature had no effect on the mortality of larvae 48 h after application irrespective of the EPN isolate. At a dose of 100 IJs/larva, the higher temperature resulted in higher mortality of larvae only with the S. feltiae ZAG 15 and K13 isolates after 48 h (Table 4). No effect of dose was observed with larval mortality at both temperatures tested. The sole exception was the mortality of larvae following exposure to the S. feltiae Wipsowo isolate at 15°C at 48 h (Table 5).

No adult or larval mortality was observed in the control treatments regardless of temperature or EPN dose.

Our previous studies of the susceptibility of Pieris spp., Agrotis exlamationis (L.), and Hylobius abietis (L.) to native EPN isolates demonstrated that hosts differ in their susceptibility to isolates (Mazurkiewicz et al. 2016, 2017; Tumialis et al. 2013). Thus, we recognize the need to assay selected EPNs against each target pest. In the study reported herein, the most effective EPN isolate tested against O. melanopus was S. feltiae ZAG 15, while the H. megidis Wipsowo isolate was the least effective, causing mortality levels that did not exceed 58%.

Laznik et al. (2010a, b) also reported that the native Steinernema carpocapsae C101 isolate caused 100% mortality of cereal leaf beetle adults within 48 h of exposure to a dose of 500 IJs/adult. The Heterorhabditis bacteriophora D54 isolate was the least effective at temperatures of 15°C and 20°C. Laznik et al. (2010a, b) also found similar patterns of mortality response to EPN dose and duration of exposure, as we noted in our study.

Laznik et al. (2010c) and Trdan et al. (2009) indicate that 25°C is the optimum temperature for S. feltiae strains used to manage the rice weevil, Sitophilus oryzae (L.). Similarly, Trdan et al. (2008) found that both steinernematids and heterorhabditids were more effective at 25°C than at 15°C or 20°C, especially when applied at higher doses, against Phyllotreta spp. adults. Our results corroborate those findings as we also found that the EPN isolates we tested were more effective at the higher temperatures tested.

Our results and those of Laznik et al. (2010b) demonstrate the potential for using EPNs for management of the cereal leaf beetle. Other studies with other beetle species support this possibility. Campos-Herrera and Gutiérrez (2009) and Wright et al. (1987) obtained less than desirable levels of mortality (30–40%) of Colorado potato beetle, Leptinotarsa decemlineata (Say), larvae treated with S. feltiae. Tumialis et al. (2013), however, reported larval mortality exceeding 80% after challenging large pine weevils, H. abietis, with isolates of S. feltiae and Steinernema kraussei Steiner. Mortality ranging from 78 to 94% were observed by Santos et al. (2011) following exposure of Diabrotica speciosa Germar larvae to various isolates of Heterorhabditis sp. and Steinernema glaseri Steiner. And, Yadav and Lalramliana (2012) reported 100% mortality of Aplosonyx chalybaeus (Hope) larvae treated with isolates of Heterorhabditis indica Poinar, Karunakar and David, Steinernema thermophilum Ganguly and Singh, and S. glaseri.

Beetle larvae, in general, appear to be more susceptible to EPNs than adults (Toepfer et al. 2005, Trdan et al. 2009). Determination of appropriate EPN dose might be affected by the presence of coat of fecal wastes on larvae (Kher et al. 2011), which might be a barrier to penetration of EPN IJs.

In our opinion, an important objective of laboratory bioassays as reported herein is the selection of EPN isolates as potential biological control agents. The study by Santos et al. (2011) demonstrated that mortality obtained in laboratory bioassays may translate to effective (e.g., 70% mortality) levels of biological control in crops. Similarly, Laznik et al. (2010d) proved that an S. feltiae isolate identified in laboratory studies was effective in field trials against Colorado potato beetle larvae. Those results also showed efficacy of S. feltiae against the foliage-feeding pest (Laznik et al. 2010d). Therefore, our laboratory assays indicate that native isolates of EPNs have potential as biological control agents of O. melanopus in crops.

Additional research is required to further develop these EPN isolates for these uses.

The authors thank Radosław Jabłoński and Katarzyna Góral-Radziszewska (Warsaw University of Life Sciences–SGGW, Faculty of Animal Science, Department of Genetics and Animal Breeding) for statistical analysis consultation and advice.