Background

Alfalfa [Medicago sativa L. (Fabaceae)] plant is a forage plant that provides a high-value feed and has a very dense production area in Turkey (Karakurt and Fırıncıoğlu 2003). The alfalfa weevil [Hypera postica (Gyllenhal, 1813) (Coleoptera: Curculionidae)] and the lucerne beetle [Gonioctena fornicata (Brüggemann, 1873) (Coleoptera: Chrysomelidae)] are among the alfalfa pests, spread to almost all grown areas of alfalfa in Turkey and cause significant crop losses if no control is made (Efe and Özgökçe 2014; Efil 2018). Larvae and adults of both species feed on leaves, young shoots, and the tips of the stems. As a result of feeding, the plant turns yellow and withers, and then the leaves dry completely (Atanasova and Semerdjieva 2009; Majić et al. 2013).

Frequent cuttings are the most effective cultural method of the pest in the alfalfa fields. Gözüaçık and İreç (2019) reported that cutting or grazing in the fall will reduce the spring larval population of alfalfa weevil. Chemical control against H. postica is recommended in Turkey, but no chemical pesticides have been registered for the control of G. fornicata. Therefore, pest control activities that are environmentally friendly are needed.

Entomopathogens nematodes (EPNs) are highly virulent. They have recently become one of the most emphasized groups due to these features. Studies have indicated that more than 30 nematode families are in contact with insects (Kaya and Stock 1997). EPNs have a variety of distinct and advantageous characteristics, such as high virulence and the ability to actively seek out hosts, making them promising chemical alternatives (Gulcu et al. 2017). EPNs are now used in many countries to control a wide range of economically important insect pests (Odendaal et al. 2015; Belien 2018). EPNs belong to the Steinernematidae and Heterorhabditidae families and live in the soil and are obligatory insect pathogens. They are one of the most frequently used groups in microbial control of pests (Glazer and Lewis 2000).

Studies using EPNs against G. fornicata and H. postica are limited (Falahi et al. 2011; Roodaki et al. 2011). Therefore, the aim of this study was to investigate the virulence of EPNs against G. fornicata and H. postica adults under laboratory conditions.

Methods

Nematode sources

Native Turkish EPNs, Steinernema feltiae (isolate 09-31) from a vegetable garden in Aydin, S. carpocapsae (Black sea isolate) from grassland in Rize, and Heterorhabditis bacteriophora (isolate 09-43) from peach orchard in Aydın, Turkey, were obtained and supplied by Prof. Dr. Selçuk HAZIR (Aydın Adnan Menderes University, Faculty of Arts and Sciences, Department of Biology, Aydın, Turkey). S. carpocapsae Tokat-Ulas and H. bacteriophora Tokat-Songut were isolated from alfalfa cultivated areas of Tokat province (Turkey) (Çağlayan et al. 2020). The nematodes were cultured in last instar larvae of wax moth, Galleria mellonella (Linnaeus, 1758) (Lepidoptera: Pyralidae) at room temperature (23–24 °C) using methods described by Kaya and Stock (1997). The harvested infective juveniles (IJs) were used within 2 weeks after emergence for the experiments.

Insect sources

The lucerne beetle and alfalfa weevil adults were obtained from alfalfa fields in Emirseyit town of Tokat, Turkey (40° 2′ 13″ N, 36° 23′ 59″ E, 554 m).

Bioassays

Experiments were carried out in Petri-dishes (9 cm diameter). In the bottom of each Petri dish, filter paper was placed and 1 ml of prepared EPNs concentrations was poured onto the filter paper. For the control group, 1 ml of no-nematode water was used (Shahina and Salma 2010). In each Petri dish, 5 adult insects were released using the camel’s hair brush. Each EPN species was applied at 3 concentrations 500, 1000, and 2000 IJs ml−1. A micropipette was used to make the applications. After each treatment, the pipette tips were replaced. Alfalfa leaves were placed in Petri dishes to feed the insects. Experiments were carried at 4 replicates and 2 different times for each concentration. The data for mortality were recorded at 8-h intervals and the counts continued until to 120th hour. Dead insects were either dissected under a stereomicroscope or placed into White traps for nematode emergence to ensure that they were killed with EPNs (White 1927).

Statistical analysis

Data was analyzed using analysis of variance (ANOVA), and the means were compared by Tukey’s multiple comparison test. All statistical analyses were carried out using the MINITAB Release 16 packet program. LT30, LT50, and LT90 values were calculated by using the Probit analysis.

Results

Mortality rates caused by EPNs isolates varied according to the test insect, time, isolates, and concentration. There was a positive correlation between nematode concentration and exposure time and mortality. S. carpocapsae (Black sea isolate) started to show a significant effect at all doses against G. fornicata from the 32nd hour (F:21.67, DF: 3.28, P < 0.05), and this effect increased over 90% at the 64th hour (F:103.29, DF: 3.28, P < 0.05) (Table 1). S. feltiae (isolate 09-31) was 92% effective in 64 h at a dose of 2000 IJs ml−1. Other doses caused a mortality rate of over 50% at the same time period (F:30.51, DF: 3.27, P < 0.05). By the 112th hour, all doses produced over 90% death (F:68.59, DF: 3.27, P < 0.05) (Table 1). Similar to the S. carpocapsae (Black sea isolate) isolate, S. carpocapsae Tokat-Ulas showed a significant efficacy after 48 h and caused more than 90% mortality at all doses (F:148.74, DF: 3.26, P < 0.05) (Table 1). H. bacteriophora (isolate 09-43) caused an effect of 98% at the 120th hour at a dose of 2000 IJs ml−1, while this effect did not exceed 70% at other doses (F:34.25, DF: 3.21, P < 0.05) (Table 2). H. bacteriophora Tokat-Songut revealed a mortality rate of over 90% at doses of 1000 and 2000 IJs ml−1 at 72 h (F:54.81, DF: 3.28, P < 0.05). At the 112th hour, the effect of all doses was over 90% (F:91.73, DF: 3.28, P < 0.05) (Table 2).

Table 1 Mortality rates of Gonioctena fornicata adults exposed to Steinernema carpocapsae (Black sea isolate), S. feltiae (isolate 09-31), and S. carpocapsae Tokat-Ulas and controls over 120 h from treatment
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Table 2 Mortality rates of Gonioctena fornicata adults exposed to Heterorhabditis bacteriophora (isolate 09-43) and H. bacteriophora Tokat-Songut and controls over 120 h from treatment
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When the LT30 rates of the isolates against G. fornicata were examined, the most effective isolate was S. carpocapsae Tokat-Ulas. This was followed by S. carpocapsae (Black sea isolate), H. bacteriophora Tokat-Songut, S. feltiae (isolate 09-31), and H. bacteriophora (isolate 09-43). The order formed by the isolates for LT30 value is valid for LT50 and LT90 values (Table 3).

Table 3 Lethal time (LT30, LT50, and LT90) values of treated isolates against adults of Gonioctena fornicata (hour)
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Steinernema carpocapsae (Black sea isolate) showed a mortality rate of over 90% against H. postica from 72 h onwards at all doses (F:57.55, DF: 3.26, P < 0.05) (Table 4). Similarly, S. carpocapsae Tokat-Ulas caused a significant mortality rate against H. postica by more than 85% from the 64th hour at all doses (F:31.87, DF: 3.27, P < 0.05) (Table 4). S. feltiae (isolate 09-31) reached 90% effect only at 2000 IJs ml−1 doses and at the end of 112th hour (F:27.80, DF: 3.26, P < 0.05) (Table 4). The rate of effect of H. bacteriophora (isolate 09-43) remained quite low than other isolates and did not exceed 26% at all doses at the end of 120th hour (F:8.41, DF: 3.24, P < 0.05) (Table 5). At 120 h, the effect of H. bacteriophora Tokat-Songut was only 37% at the highest dose and its effect remained quite low, similar to that of H. bacteriophora (isolate 09-43) (F:9.28, DF: 3.25, P < 0.05) (Table 5).

Table 4 Mortality rates of Hypera postica adults exposed to Steinernema carpocapsae (Black sea isolate), S. feltiae (isolate 09-31), and S. carpocapsae Tokat-Ulas and controls over 120 h from treatment
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Table 5 Mortality rates of Hypera postica adults exposed to Heterorhabditis bacteriophora (isolate 09-43) and H. bacteriophora Tokat-Songut and controls over 120 h from treatment
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When the LT rates of the isolates against H. postica were examined, the order formed by LT30 and LT50 values was S. carpocapsae (Black sea isolate), S. carpocapsae Tokat-Ulas, S. feltiae (isolate 09-31), H. bacteriophora Tokat-Songut, and H. bacteriophora (isolate 09-43). This ranking was S. carpocapsae Tokat-Ulas, S. carpocapsae (Black sea isolate), S. feltiae (isolate 09-31), H. bacteriophora Tokat-Songut, and H. bacteriophora (isolate 09-43) at LT90 value (Table 6).

Table 6 Lethal time (LT30, LT50, and LT90) values of treated isolates against adults of Hypera postica (hour)
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Discussion

The results showed that S. carpocapsae (Black sea isolate) and S. carpocapsae Tokat-Ulas were more effective against both H. postica and G. fornicata adults than other isolates. Similarly, Atay and Kepenekci (2016) reported that the S. carpocapsae (Black sea isolate) had an effect of 80, 83, and 82% on another alfalfa pest, Holotrichapion pullum (Gyllenhal, 1833) (Coleoptera: Apionidae) adults, at 20 degrees and at 3 different concentrations (500, 1000, 5000 IJs ml−1), respectively. In addition, Kepenekci et al. (2018) stated that S. carpocapsae (Black sea isolate) showed 99% efficacy in adults of Xylosandrus germanus (Blandford) (Coleoptera: Curculionidae) at a dose of 1000 IJs ml−1. Brivio and Mastore (2018) emphasized that symbiotic bacteria within the nematodes affect its virulence. The other possibility of variation in pathogenicity of EPN could be the host finding and penetration capability of nematodes. H. bacteriophora (isolate 09-43) and H. bacteriophora Tokat-Songut showed 98 and 100% effect on G. fornicata at 2000 IJs ml−1 doses in 120 h, respectively, while this effect was determined as 26 and 37% for H. postica at the same time and dose. Difference in the susceptibility of host insect species may be related with their immune system and the physical structure.

Kim et al. (2007) investigated the efficiency of 4 Korean EPNs against late larvae of alfalfa weevil and reported that S. glaseri (Steiner, 1929) Dongrae strain and Heterorhabditis sp. Gyeongsan strain were more effective against H. postica larva than S. carpocapsae GSN1 strain and H. bacteriophora Hamyang strain. Shah et al. (2011) stated that application of H. indica Poinar, Karunakar & David, 1992 and S. carpocapase at the rate 1 billion IJs/acre can decrease 72 and 50% population of alfalfa weevil, H. postica grubs, respectively. Falahi et al. (2011) tested S. carpocapsae against H. postica adults under laboratory conditions at doses of 250, 500, 1000, and 2000 IJs ml−1 and determined the highest effect as 97% at the dose of 2000 IJs ml−1 at the 72nd hour as our study.

Majić et al. (2013) showed that H. bacteriophora application in rate of 1000 IJs per adult of G. fornicata caused 100% mortality rate on 3 day post-treatment in the laboratory. Similarly, in the present study, H. bacteriophora isolates had a significant effect on adult of G. fornicata. On other chrysomelids, mortality studies have been carried out with success using a variety of steinernematid and heterorhabditid. Trdan et al. (2008) assessed virulence of EPNs S. feltiae, S. carpocapsae, H. bacteriophora, and H. megidis against adult flea beetles, Phyllotreta spp. (Coleoptera: Chrysomelidae) under laboratory conditions and revealed that S. feltiae was the most effective nematode. Also, Trdan et al. (2009) stated that S. feltiae showed the highest efficacy in controlling adults of Leptinotarsa decemlineata (Say, 1824) (Coleoptera: Chrysomelidae) at 15 °C. Similarly, in the present study, S. feltiae (isolate 09-31) was more effective on G. fornicata than H. postica. Unlike these studies, Laznik et al. (2010) determined that S. carpocapsae C101 caused more mortality than S. feltiae B30 and H. bacteriophora D54 against Oulema melanopus (Linnaeus, 1758) (Coleoptera: Chrysomelidae) adults.

Conclusions

This study highlighted the first analysis of EPNs’ efficacy against H. postica and G. fornicata in Turkey. G. fornicata adults were susceptible to all isolates tested, while H. postica adults were susceptible to S. carpocapsae (Black Sea isolate), S. feltiae (isolate 09-31), and S. carpocapsae Tokat-Ulas isolates. Therefore, these native EPNs can be used as bio-control agents against the adults of these 2 important alfalfa pests. Further studies should be conducted under field conditions.