NEMATOLOGY
G. Salpiggidis et al. (2008) Phytoparasitica 36(1):95-100
Entomopathogenic Nematodes (Nematoda: Steinernematidae, Heterorhabditidae) as Control Agents for Parahypopta caestrum, a Pest in the Culture of
Asparagus officinalis G. Salpiggidis, *'1 E. Navrozidis 2 and M. Copland I The entomopathogenic nematodes (EPNs) Steinernema feltiae (under the trade name Entonem) and Heterorhabditis bacteriophora (under the trade name Larvanem) were evaluated as potential control agents of Parahypopta caestrum, the major pest of the asparagus crop in Greece. In laboratory experiments the two nematodes provided equal insect suppression, but significant differences were found in the time it took them to kill the larvae. S. feltiae caused high levels of mortality within 24 h and the highest level at 48 h. In contrast, H. bacteriophora required 96 h to achieve the highest level of mortality. In field experiments, the nematodes provided equal insect suppression as compared with the insecticide cadusaphos and the mixture teflubenzuron + Bacillus thuringiensis var. kurstaki. No significant differences were found in the effectiveness of the insecticides used, but there were significant differences between the control and treatments. The findings showed that S. feltiae and H. bacteriophora could be used to control the insect P. caestrum in asparagus culture. KEY WORDS: Bioagents; Heterorhabditis bacteriophora; Steinernema feltiae. INTRODUCTION
Asparagus officinalis (L.) is one of the most important cultivated crops in Greece. Its local consumption is almost zero, and most of the Greek asparagus production is exported, making Greece one of the leaders in the fresh asparagus trade (14). Establishment as well as cultivation of the crop requires much hand labor at all phases (16). This factor, along with the fact that harvest may begin only after the third year of cultivation, makes asparagus cultivation a very expensive investment (15). Parahypopta caestrum (Hfibner) was first recorded as a pest of asparagus in Greece in 1992 (11). The insect has one generation per year and the larvae bore mines into the interior of shoots and roots. After 2 or 3 years, an infestation will cause total loss of the plantation (11). It is obvious that an effective control strategy is needed. The use of insect growth regulators (IGRs) and other insecticides, such as organophosphate and Bacillus thuringiensis (Bt) toxins, did not provide satisfactory control although they reduced damage significantly (11). However, entomopathogenic nematodes (EPNs) belonging to Steinernema spp. and Heterorhabditis spp. have proved effective against a wide range of soil-inhabiting insects Received Aug. 19, 2005; revised ms. received March 21, 2007; accepted Sept. 5, 2007; http://www.phytoparasitica.orgposting Jan. 10, 2007. 1Dept. of Agricultural Sciences, Imperial College, London SW7 2BZ, UK. *Corresponding author [e-mail:
[email protected]]. 2Technological Educational Institute of Thessaloniki, T.K. 57400 Thessaloniki, Greece.
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(3). Several lepidopteran species, such as Helicoverpa zea (Boddie), Spodopterafrugiperda (Smith) (13) and Pectinophora gossypiella (Saunders) (10), were controlled by these nematodes. In the mentioned studies, the larvae and pupal stages of each lepidopteran species were tested for susceptibility to EPNs. Their entomopathogenic action is associated with bacterial symbiont Xenorhabdus spp. and Photorhabdus spp., respectively (1). Only the infective third stage juveniles of EPNs are infectious. After the infection, the above bacteria are released - causing septicemia and death (10). The EPNs Steinernema feltiae (Filipjev) under the trade name Entonem and Heterorhabditis bacteriophora (Poinar) under the trade name Larvanem (both supplied by Koppert| Systems) were evaluated as potential control agents of P. caestrum. MATERIALS AND METHODS L a b o r a t o r y assays were conducted to determine the effectiveness of the EPNs in controlling different instars of P. caestrum. Nematodes were stored at 8~ (for no longer than 4 h before use). First and second instars of P. caestrum were obtained from a colony reared on artificial diet. In order to obtain diapausing larvae from the same generation, some larvae were fed further on the artificial diet and placed under diapause-inducing conditions. A mixture of 1st, 2nd and 5th larval instars was placed on the surface of sterile sandy soil (10% moisture by weight) in 10-cm-diam petri dishes. Nematodes were pipetted onto the sand surface in 1 ml of distilled water at concentrations of 1000, 5000 or 25,000 infective juveniles (IJs) per petri dish. Only distilled water (1 ml) was added to controls. Each treatment was replicated five times. The petri dishes of each replication were subsequently placed in a double plastic bag and incubated in the dark at 24•176 to avoid desiccation. Larval mortality was recorded every 24 h for 120 h. The same procedure and the same concentration were used for the diapausing larvae tests but larvae were placed in larger plastic boxes (29 x 19 x 15 cm). Field tests were conducted to evaluate the effectiveness of the above EPNs under field conditions in comparison with the insecticide cadusaphos (Rugby 10 G; FMC, Hellas, Greece) and the mixture tefiubenzuron (Nomolt 15 SC; BASF Agro, Hellas) + Bacillus thuringiensis var. kurstaki (Bt) (Agree 3.8 WP; Sygenta, Hellas). Preliminary studies showed that both cadusaphos and the mixture teflubenzuron + Bt were effective against P. caestrum (unpublished data). The experimental field was a 7-year-old plantation, where the cultivar 'Stelina' was established with inter-row spacing of 2.1 m, and intra-row spacing of 30 cm; plot length was 60 m. In order to estimate the correct timing for application, light traps were placed along the side of the experimental field in mid May. Once the first moths were trapped, the first application date was calculated using data from the insect's life cycle (10). Three sprays, targeting young larvae at 12-15-day intervals, were applied onto the collar of the asparagus shoots and covering an area of 15-20 cm on both sides of the asparagus rows. The rates and volumes of each insecticide used are presented in Table 1. Each treatment was replicated four times. Cadusaphos and teflubenzuron were applied by a mechanical sprayer mounted on a tractor. Since the insecticide cadusaphos has nematicidal action (6), a new tank (plastic barrel) was fitted to the mechanical sprayer and a new rubber pipeline and hose were used. In addition, the screens were removed to avoid blockage of or damage to the nematodes. The suspension of nematodes was prepared by transferring the commercial materials into a bucket containing 5 l of water and stirred. The entire suspension was left to stand 96
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for 5 min, stirred again and then transferred to the tank which was filled with water until the required amount of spray volume was reached. Continuous mixing prevented the nematodes from sinking to the bottom. Only water was sprayed on the control plots. No further pesticides were used and weeding was done manually. The evaluation was conducted the following spring by recording the number of cocoons in a standard soil volume of 7.5 m 3 (60 m length • 0,5 m width • 0.25 m depth). Data were analyzed by one-way analysis of variance (Minitab 13,31) and Least Significant Difference (P=0.05) was used to separate treatment means. RESULTS Laboratory assays
There was very low mortality in control treatments (Table 2), S.
feltiae infected and killed 1st and 2nd instars at the same rate, with no significant differences between treatments (Table 2). However, when diapausing larvae were examined for susceptibility to nematodes, there were differences in the mean mortality, depending on concentration. Only the highest concentration of 25,000 IJs per dish gave satisfactory control. The same results were obtained when 14. bacteriophora was used as bioagent (Table 3). TABLE 1. Data on insecticides used in the field experiment for the control of Parahypopta caestrum (each applied in a volume of 100 1 ha -1) Commercial name and formulation Rugby 10G Mixture of Nomolt 15SC + Agree 3.8 WP Larvanem Entonem I.J., infectivejuveniles.
Common name
a.i. ha- ~
Cadusaphos Teflubenzuron + Bt var.
20 g 25.5 ml + 4.94 g
kurstaki Steinernemafeltiae Heterorhabditis bacteriophora
5x 108 l.J. 6:< 108 I.J.
TABLE 2. Mean (• mortality of 1st and 2nd instars and diapausing larvae of Parahypopta caestrum caused by Steinernema feltiae after 120 h at 24•176 Infectivejuveniles
Number of dead Number of dead 1st and 2rid instars diapausing larvae 25,000 23.64-0.4a 21.8:]:0.9a 5,000 23.4• 17.84-1.2b 1,000 23.44-0.7a 3.04-0.70c Control 2.84-1.0b 2.04-0.8c LSD 2.3796 2.7551 F 169.5 121.4 zWithin columns, means followed by the same letter do not differ significantly,with LSD at P_
