Efficacy of Entomopathogenic Fungi and Nematodes , and Low Risk Insecticides Against Wheat Stem Sawfly , Cephus cinctus ( Hymenoptera : Cephidae )

Entomopathogenic nematodes, fungi, and low risk insecticides were evaluated for the management of the wheat stem sawfly, Cephus cinctus Norton, in winter wheat at two locations (Devon and Western Triangle Ag Research center) in the Golden Triangle area of Montana (USA) in 2013. Two fungi (Beauveria bassiana and Metarhizium brunneum), four nematodes species (Steinernema carpocapsae, Steinernema kraussei, Steinernema feltiae, and Heterorthabditis bacteriophora), an insect growth regulator (diflubenzuron/dimilin), and a botanical-based chemical (azadirachtin/Aza-direct) were used as foliar sprays. These control agents significantly reduced damage caused by C. cinctus larvae, compared to the untreated control or treatment with water alone. No yield differences were observed among entomopathogenic fungi, nematodes, and low risk insecticides. The effectiveness of azadirachtin, diflubenzuron, the entomopathogenic fungi, and the nematodes persisted at the 28 day post application, by which time the wheat had been harvested. Stubbles collected after harvest showed significantly fewer sawfly larvae in the plots treated with entomopathogenic fungi, nematodes, diflubenzuron, and azadirachtin compared to the untreated and water spray plots, indicating that these biorational pesticides have potential to be used as alternatives to conventional pesticides for controlling the wheat stem sawfly larvae.


Inroduction
The wheat stem sawfly, Cephus cinctus Norton (Hymenoptera: Cephidae), is one of the most important pests of wheat, Triticum aestivum L. (Cyperales: Poaceae), especially in the Northern Great Plains of the United States and Canada (Weiss & Morill, 1992;Morill et al., 1993).Annual losses from this pest exceed $600 million (Beres et al., 2011).Sawfly larvae overwinter underground in dead stubbles, and adults emerge in late spring (Ainslie, 1929;Weiss & Morill, 1992;Gress et al., 2013).Males are haploid with nine chromosomes and typically emerge before females (Holmes, 1979).Mating takes place soon after emergence except for periods of strong wind or rain, which interfere with mating behaviors (Wallace & McNeal, 1966).Females usually fly near wheat plants and lay one egg per stem per visit, mostly in the upper developing internode (Holmes & Peterson, 1960).However, there can be multiple eggs deposited in a stem by different females (Buteler et al., 2009).One female can lay 30 to 50 eggs (Wenda-Piesik et al., 2009).The larvae feed on parenchymous and vascular tissues inside wheat stems, causing damage to the wheat by reducing head weight.As the wheat plant matures, the larva moves down to the base of the stem where it cuts a notch at ground level, leading to lodging before harvest, further reducing yield (Beres et al., 2007).
Attempts to control the wheat stem sawfly with conventional pesticides have been either ineffective or cost more than the economic yield return (Knodel et al., 2009).Alternative approaches such as biological control have therefore been considered for the management of this pest.Wenda-Piesik et al. (2009) demonstrated that Fusarium isolates caused mortality in both diapausing wheat stem sawfly larvae in a topical bioassay and developing larvae feeding in infested stems in a greenhouse experiment.Several commercially available fungi and nematodes have been used for biological control of various insect pests.These include Beauveria bassiana (Bals.-Criv.)Vuill (McGuire et al., 2005;Bhadauria et al., 2013), Metarhizium anisopliae (Metschnikoff) Sorokin (Bhat et al., 2010;Larramendy et al., 2011), S. carpocapsae Weiser (Cossentine et al., 2002;Chambers et al., 2010), S. kraussei Steiner (Haukeland & Lola-Luz, 2010), S. feltiae Filipjev (Shapiro-Ilan et al., 2004;Navaneethan et al., 2010), and Heterorhabditis bacteriophora Poinar (Toledo et al., 2005;Koppenhöfer et al., 2006).Dimilin is an insect growth regulator which has been reported to be effective in mosquito control (Msangi et al., 2011).Neem (Aza-direct) has also been reported to be effective against some insects (Mamoon-ur-Rashid et al., 2013, Sivasakthi et al., 2013).This study was aimed to investigate the potential use of these biorational control agents for the management of C. cinctus.

Trial Design and Location
Two trials were conducted, one at the Montana State University Western Triangle Agricultural Research Center (WTARC) (N48º18′24.88′′W111º55′28.45′′) and the other at Devon, Montana (N48º33′14.94′′W111º23′42.96′′).The distance between the two locations was 58 miles.The experiments were carried out from May-September 2013.Winter wheat Yellowstone variety was used for these trials.The wheat was seeded at the rate of 194 live seeds per m 2 .In both trials, the wheat was planted in four rows, with 30 cm between rows.Glyphosate (Roundup Powermax) was applied at the rate of 2.5 L/ ha (active ingredient of 540 g/L of acid glyphosate) before the wheat was seeded to control weed growth.Fertilizer N, P, and K ratio at 224.2, 0, and 22.4 kg/ha was broadcasted while planting, and an additional application of 12.3, 25.2, and 0 kg/ha of these three nutrients were placed through seed plot drill.The treatment plots were arranged in a complete randomized design (CRD) with four replicates.
Treatment plots were 8 m × 4 m and there was 2 m-distance between adjacent treatment plots to avoid spray drift.Each plot consisted of four rows.Standing plants were counted after seed germination.There were approximately 142 standing plants per m 2 in each plot.In each plot, control agents (Table 1) were sprayed after stem elongation had begun.The spray of each treatment was conducted only once at this plant stage.Treatment materials were mixed in a Chapin Lawn & Garden Sprayer, and sprayed at 297 L water per hectare, pressure ranges 241 to 310 Kpa.  2).In the third week, the untreated and water sprayed plots had significantly more larvae than other treatments (F 9, 70 = 2.34, P < 0.05).There were no significant differences in the number of larvae found among treatments with S. carpocapsae, S. kraussei, H. bacteriophora, Dimilin and Aza-direct (F 4, 35 = 0.94, P > 0.05).Plots treated with B. bassiana, M. brunneum, and S. feltiae had significantly fewer larvae than other treatments (F 9, 70 = 2.23, P < 0.05).In the fourth week, only the untreated and water sprayed plots had significantly more larvae than other treatments (F 9, 70 = 4.39, P < 0.05); the water spray treatment did not have a significantly different effect from the untreated control (F 1, 14 = 0.00, P > 0.05).No larvae were found in plots treated with B. bassiana, M. brunneum, S. carpocapsae, S. kraussei, S. feltiae, H. bacteriophora, Dimilin and Aza-direct.

Effect on Yield
There were no significant differences in wheat yield between the control plots and plots treated with water (F 1, 14 = 0.15, P >0.05).There were no significant difference in yield among plots treated with B. bassiana, M. brunneum, S. carpocapsae, S. kraussei, S. feltiae, H. bacteriophora, Dimilin, and Aza-direct.However, plots treated with these agents produced significantly higher yield than either the untreated control or the water spray (F 9, 70 = 2.27, P < 0.05) (Figure 3).

Discussion
Currently, the management of C. cinctus is often limited to the use of solid-stemmed resistant cultivars.Chemical pesticides are either ineffective or more costly than the economic yield return (Knodel et al., 2009).The development of biological control agents against this pest is an important alternative approach for the management of C. cinctus.In the current study, treatments with the entomopathogenic fungi and nematodes resulted in higher yield production and better control of C. cinctus compared to the untreated control and water spray.
Results of our study showed that the fungi B. bassiana (Mycotrol) and M. brunneum (Met 52) effectively reduced wheat stem damage caused by C. cinctus larvae compared to the untreated plots or water spray only (Figure 1).Beauveria bassiana has been found to control many insects (Bhadauria et al., 2013).Also, M. brunneum has been documented to infect more than 200 pest species (Robert & Leger, 2004).However, to our knowledge, no work has been done to evaluate the effectiveness of these entomopathogenic fungi against C. cinctus.Both B. bassiana and M. brunneum kill insects via infection following spore contact with spray droplets or treated surfaces, or by consuming plant tissue treated with the fungus.After contact, spores germinate and hyphae infect the insect (EPA, 2011).Infected insects die in 3-5 days, and the cadavers may serve as source of spores for secondary spread of the fungal entomopathogen.In addition, adult insects may spread the fungus through mating (Long et al., 2000).In our study, we found that the effectiveness of the two fungal entomopathogens started to take effect 21 to 28 days after application, in terms of reducing number of cut stems, which occurred at the third week for M. brunneum (Met52) and the fourth week for B. bassiana (Mycotrol) (Figure 2).Being consistent with stem cuts, the plots treated with the two fungi showed significantly lower number of C. cinctus larvae by the third and fourth weeks after wheat harvest (Figure 2).Entomopathogenic nematodes have many attributes of effective biological control agents (Kaya & Gaugler, 1993;Grewal et al., 2005;Koppenhöfer, 2007).In our current study, S. carpocapsae (Millenium ® ), S. kraussei (Nemasys ® L), S. feltiae (Nemasys ® ), and H. bacteriophora (Nemasys ® G) appeared to be effective in reducing the damage caused by C. cinctus larvae and increased the yields compared to the untreated control and treatment with water spray alone (Figure 3).Georgis et al. (1991) also reported that Steinernematid and Heterorhabditid nematodes significantly suppressed pest populations such as Popilla japonica Newman (Coleoptera: Scarabaeidae), Scapteriscus vicinus Scudder (Orthoptera: Gryllotalpidae), Otiorhynchus sulcatus Fabricius (Coleoptera: Curculiondae), Delia radicum Linnaaeus (Diptera: Anthomylidae), and Diabrotica virgifera viergifera LeConte (Coleoptera: Chrysomelidae) in the field.These entomopathogenic nematodes were also proven to have no adverse effect to nontarget arthropods especially when used for short-term control of insect pests (Georgis et al., 1991).All entomopathogenic nematodes used in our experiment were equally effective in reducing the stem damage within three weeks after spray (Figure 1).However, by 28 days after application, H. bacteriophora (Nemasys ® G) showed significantly higher efficacy in reducing stem cut damage compared to other nematode species (Figure 1).Different from our study, Kamali et al. (2013) reported that S. carpocapsae had higher virulence and better ability to locate larvae of Dacus ciliatus Loew (Diptera: Tephritidae) within infected fruits.Temperature thresholds for survival and infectivity vary with nematode species, their native habitat, and center of origin (Kaya, 1990).The average temperatures in the first 2 and 3 weeks after treatment were 29.3°C and 21.7 °C, respectively.The optimal temperature for S. feltiae to be infective ranges from 20 to 30°C, whereas some heterorhabiditids can infect host from 7 to 35 °C, and S. carpocapsae fails to cause infection when temperature drops below 10 °C (Kaya, 1990;Georgis et al., 2006;Lacey et al., 2006).However, this may also vary with host species.Different host insects might have different cues and characteristics under different temperatures which means the temperature can affect host recognition by nematodes and also the host itself can produce less cues depending on temperatures (Chen et al., 2003).By the fourth week after harvest, we found no sawfly larvae in wheat stubbles in any nematode treatment, indicating that these nematodes effectively controlled the insect within 28 days after harvest.
In addition, treatment with neem extracts showed significantly lower stem damage and fewer C. cinctus larvae compared to the untreated control and treatment with water spray alone (Figures 1, 2).Neem extracts are reported to affect over six hundred species of pests (Sivasakthi et al., 2013).Reddy and Guerrero (2000) suggested that neem had the potential to be used as a good alternative to conventional insecticides in IPM programs.In the current study, neem effectively suppressed C. cinctus populations (Figure 2).
Overall, all bio-rational agents used in the current study demonstrated good potential for controlling the C. cinctus.Among them, B. bassiana (Mycotrol), M. brunneum (Met 52), and S. feltiae (Numasys) were effective in killing the larvae within a shorter period of time compared to other agents.Our study indicated that these biopesticides may serve as alternative methods for controlling C. cinctus and may be incorporated into IPM programs, to mitigate the environmental pressure from the use of conventional insecticides.