Nemato!' medit. (2003),31: 157-162 157

SUSCEPTIBILITY OF ROOT MAGGOT, RADICUM, TO ENTOMOPATHOGENIC NEMATODES (STEINERNEMATIDAE AND HETERORHABDITIDAE)

S. Chenl and M. Moensl ,2

1 Agricultural Research Centre, Department of Crop Protection, Burg. Van Gansberghelaan 96, 9820 Merelbeke, Belgium; 2 Unz:versity Gent, Laboratory for Agrozoology, Coupure 555, 9000 Gent, Belgium

Summary. Susceptibility of last ins tar maggots of to 13 isolates of five 5teinernema (5. feltiae, 5. carpocapsae, 5. affine, 5. arenarium and 5. glaseri) and four isolates of Heterorhabditis (H. megidis and H. bacteriophortl) was evaluated in the labo­ ratory. Each of the populations controlled the maggot to some extent but differed in efficiency. When exposing one maggot to ten infective juveniles (IJs), 5. feltitle isolates showed the highest efficiency: 12-32% mortality at two days after infection (dail and 26- 73 % mortality at four dai. 5. gltlseri had the lowest pathogenicity (5.6% mortality at four dai), whereas 5. CtlrpOCtlpstle,5. tlffine, 5. tlrentlrium, H. megidis, and H. btlcteriophora were intermediately active (8-20% mortality at four dai). Comparisons of 5. feltitle populations showed Lxm31 as the best performing one. Experiments in which last instar maggots were exposed during 48 hrs to 12,25,50, 100, or 200 lJs yielded a LDso of 5. feltitle Lxm31, 5. ctlrpoctlpstle All,S. tlrentlrium, H. megidis Sn5 and H. btlcteriophora NJ-hbr of 12, 102, 258, 196, and 344 1J s per , respectively. As the effect of 5. gltlseri on maggots was too weak, a linear rela­ tion between dose and mortality was not found. The LTso for 5. feltitle Lxm31, 5. ctlrpoctlpstle All,S. tlrentlrium, H. megidis Sn5 and H. btlcteriophorcz (NJ-hbr) was calculated at 17, 82,192,77, and 104 hours per larva, respectively.

The cabbage root maggot, Delitl mdicum (Diptera: An­ Knowing and understanding the differences in infec­ thomyiidae), is a destructive of cruciferous crops tivity between epn and isolates is necessary. Pre­ throughout Europe and North America (Finch, 1989). It is diction of the mortality by the establishment of a the most serious pest of and cabbage but also relation between the nematode dose and efficacy, and damages Brussels sprout, , and garden brassi­ the exposure time and efficacy, provides vital informa­ ca (McKinlay, 1992). The adult lays eggs mainly at the tion for making strategic decisions in biological control. base of the plant stem but also at other plant parts such as In this paper we report on a series of experiments to de­ the fold of the enveloping leaflets of the Brussels sprout termine the efficacy of different epn species and isolates buttons. The hatched maggots burrow into the roots or - mainly collected in Belgium - against D. mdicum. the sprouts and plants may be attacked at all growth stages. The most serious damage, however, is usually ob­ served on young plants, which can be killed if severely at­ MATERIALS AND METHODS tacked in the seedbed or shortly after transplanting. are frequently used to control the cab­ Nematodes. The nematode species and isolates used bage root fly. Increased awareness of environmental in this study were selected on a geographical basis and hazards and toxicity of insecticides as well as the resis­ are shown in Table 1. They were cultured in last-instal' tance of to insecticides has prompted the search larvae of Gtllleria mellonella L. (Woodring and Kaya, for alternative means of pest control. Biological control 1988) and stored in distilled water at 5°C for a period of using entomopathogenic nematodes (epn) is one possi­ no longer than two weeks. The nematodes were kept at bility. Species of the Steinernematidae and Heterorhab­ room temperature (18-22 DC) for 24 hours before using. ditidae are lethal insect parasites that have a mutualistic Insect. Delia radicum Bouche maggots were collected association with bacteria of the genera Xenorhtlbdus and from the edges of cauliflower fields in the province of Photorhabdus, respectively. They are successful in the West Flanders in Belgium and reared on (Bras­ control of many insects (Klein, 1990). sica napus napobrassica L. Rchb.) at 20°C according the When searching for effective nematodes, it is prefer­ method described by Harris and Svec (1966). able to screen species rather than isolates, because there Susceptibility of D. radicum to different species and is greater variation between species than between iso­ isolates. The susceptibility of last stage larvae of D. lates (Bedding, 1990). Besides the intrinsic qualities of radicum to epn was studied in 24-well plates (Falcon, Le the species, epn efficiency is highly affected by the dose Pont de Claix, France). Each well was filled with 2 g (Hominick and Reid, 1990). There is a positive correla­ sterilised sand (225-425 pm) and incubated at room tion between the insect mortality and the dose of nema­ temperature for 24 hours before use. In each well one todes applied (Forschler and Nordin, 1988; Glazer and last ins tar larva of D. radicum was exposed to ten infec­ Navon, 1990; Peters and Ehlers, 1994). tive juveniles (IJ s) of one of the isolates. The insect lar- 158

Table I. Species and isolates of entomopathogenic nematodes used in the study.

Species Isolate Origin

Steinernema/eltiae (Filipjev, 1934) Wouts, Lxm31, Rs51, VBM5, Lxm16, Rs50, Nm30 and Belgium (Miduturi et al., 1997) Mracek, Gerdin and Bedding, 1982 Ma41 D-hbr R-U. Ehlers NeS Nemasys

S. affine (Bovien, 1937) Wouts, Mracek, Hm13 and Z15 Belgium (Miduturi et at., 1997) Gerdin and Bedding, 1982

S. carpocapsae (Weiser, 1955) Wouts, All R-U. Ehlers Mracek, Gerdin and Bedding, 1982

5 arenarium (Artyukbovsky, 1967) Wouts, S.E. Spiridonov Mracek, Gerdin and Bedding, 1982

5 glaseri (Steiner, 1929) Wouts, Mracek, S.E. Spiridonov Gerdin and Bedding, 1982

Heterorhabditis bacteriophora Poinar, 1976 NJ-hbr R-U. Ehlers

H. megidis Poinar,]ackson & Klein, 1987 SuS and VBM30 Belgium (Miduturi et al., 1997) NeH Nemasys

vae that were used had been collected close to pupation; Insect mortality was recorded after that period and consequently there was no or very low natural larval dead insects were dissected in Ringer solution. The mortality. If in a separated control treated with distilled number of nematodes that penetrated into the insect water, insect mortality exceeded 5 %, the experiment was counted. Each combination of epn species/isolate was repeated. One hundred D. radicum larvae were ex­ and exposure time was examined on 24 larvae. amined for each epn isolate. The plates were incubated Data analysis. NCSS software package was used for at 20°C and insect mortality was checked at two and all data analysis. X2 test by making a contingency table four days after infection (dai). Dead larvae were dissect­ was used to check significant differences (P<0.05) in ed to ascertain that their death was caused by the epn. susceptibility of D. radicum to the epn species. LD50 and Relation between nematode dose, infectivity and D. LT50 values were calculated by probit analysis. Invasion radicum mortality. Steinernema /eltiae Lxm31, S. car­ rate of nematode species at different doses and expo­ pocapsae All, S. glaseri, S. arenarium, Heterorhabditis sure time was compared with the X2 test. megidis Sn5, and H. bacteriophora NJ-hbr (Table I) were selected for the experiment conducted at 20°C in 24- well plates (preparation as above). Each last instar larva RESULTS of D. radicum was exposed to 12,25,50, 100 or 200 IJs. For each combination of nematode species and dose, 24 Susceptibility of D. radicum to different nematodes insect larvae were examined. Two dai, the insect larvae species and isolates. All S. /eltiae isolates killed D. were washed free of nematodes and incubated on dry fil­ radicum larvae (Fig. 1). The mortality obtained with this ter paper in a Petri dish for another two days at 20°C. At species depended on the isolate and varied between 12 the end of this period, dead insects were recorded and and 32 % at two dai and between 26 and 73 % at four dissected in Ringer solution (Woodring and Kaya, 1988) dai. At both observations, insect mortality differed sig­ to count the number of nematodes that had penetrated. nificantly between populations (P

8.3 %, 8.3 % and 5.6 %, respectively. Dissection of dead ~ 50 • m\) days afiErinoculation (l) 45 insects showed that all species, except S. affine, pro­ D tOOf days afierinorulation '" 40 duced offspring (data not shown). E 35 Relation between nematode dose, infectivity and D. =: 30 e 25 radicum mortality. The median lethal dosage (LD50) of '6 20 ~ S. /eltiae Lxm31, S. carpocapsae All, S. arenarium, H. ~ 15 megidis Sn5 and H. bacteriophora NJ-hbr was calculated ~ 10 Q 4-< at 12, 102,258, 196 and 344 IJs per larva, respectively o 0 (Table II). For the same series of species, the LD95 was .~' '"@ . ~~ 119, 1709, 14414,5881 and 16583 Us per larva. The ~ ,t;."'" 95% fiducial limit of both the LD50 and LD95 demon­ ~ ,?' strated significant differences between S. /eltiae Lxm31 and the other species. The LD50 and LD95 of S. car­ Nematode species pocapsae All, S. arenarium. H. bacteriophora NJ-hbr and H. megidis Sn5 were not significantly different. Fig. 2. Mortality of D. radicum last instar larvae after exposure For all epn species, the mean number of IJs that es­ to 10 infective juveniles of different species of entomopatho­ tablished in D. radicum larvae increased with increasing genic nematodes at 20 DC for two (black columns) or four inoculation dose. The linear relationship between the days (white columns). The result for S. feltiae, S. affine and Heterorhabditis megidis is the average of 9,2 and 3 isolates, re­ number of nematodes established in D. radicum larvae spectively. and the number of nematode applied is shown in Table III. The x2-test showed no significant differences be­ 30 tween the mean percentages of nematodes obtained af­ ~ 00 ter different inoculation doses (P>0.05). 25 "D'" -+--Sfeltiae Relation between the nematode exposure time, in­ 0 ~ 20 ---S. carpocapsae 8 ~S.arenarium fectivity and D. radicum mortality. The number of IJs GO 0:: IS -e--Ii meg,,/is extracted from D. radicum larvae increased with increas­ 4-< 0 ~ H bacteriophora ing exposure time (Fig. 3). The majority of IJ s, however, D 10 '[; penetrated insect larvae during the first 48 hours. The './3 u 5 GO number of Us dissected from the insects was significant­ 4-< ly influenced by the epn species (P

Table II. The LD50 and LD95 and their fiducial limit of entomopathogenic nematode species to Delia radicum last stage larvae.

Nematode species Isolate LD50 (95 % fiducial limit) LD95 (95% fiducial limit) 5teinernema /eltiae Lxm31 12 (6-21) 119 (57-245) 5. carpocapsae All 102 (63-167) 1709 (358-8142) 5. arenarium 258 (57 -529) 14414 (415-499997) Heterorhabditis megidis Sn5 196 (74-384) 5881 (456-75864) H. bacteriophora NJ-hbr 344 (98-1201) 16583 (433-634453)

Table III. Relationship between the number of infective juveniles (yl infecting D. radicum last instar stage larvae and the nema­ tode doses (xl applied for five species of entomopathogenic nematodes.

Nematode species Isolate Regression R2

5teinernema /eltiae Lxm31 y = 0.2914x + 1.6141 0.9913 5. carpocapsae All y = 0.0735x - 0.1519 0.9968 5. arenarium y = 0.0487x - 0.1686 0.9967 Heterorhabditis megidis Sn5 y = 0.0543x - 0.0391 0.9971 H. bacteriophora NJ-hbr y = 0.0385x + 0.0859 0.9938

Table IV. The LT50 and LT95 and their fiducial limit of entomopathogenic nematode species to Delia radicum last instar stage larvae.

Nematode species Isolate LT50 (95% fiducial limit) LT95 (95% fiducial limit)

5teinernema /eltiae Lxm31 17 (11-25) 295 (89-978) 5. carpocapsae All 82 (60-113) 688 (190-2490) 5. arellarium 192 (96-385) 2286 (197-26591) Heterorhabditis megidis Sn5 77 (56-105) 702 (190-2602) H. bacteriophora NJ-hbr 104 (70-155) 1020 (205-5066) showed that for the same nematode species D. radicum ences in virulence among different species or isolates mortality was significantly different between different (Dunphy and Webster, 1986). exposure times (P

1980) or CO2 (Gaugler et al., 1980). Once they enter the host's body, epn are exposed to the immune reaction DISCUSSION of the host. If the nematode can overcome this reaction and release its symbiotic bacteria, the host will die with­ Screening of biological pesticides in the laboratory is in 48 hours. In the 24-well plates used in our study, the a standard procedure before a new antagonist is devel­ nematodes had no distance to overcome. Differences in oped and used in the field. Bedding (1990) underlined attraction to the host may thus be excluded. We never the importance of testing a number of epn species observed any IJ encapsulated in insect larvae. A single against any particular insect before initiating field evalu­ nematode present in the haemocoel always caused the ation for biological control. Laboratory studies are es­ death of the insect (S. Chen, unpublished). As these cri­ sential steps for maximising the chances for success and teria do not differentiate between the isolates, differ­ labour saving in the field experiments. ences in pathogenicity might be explained by differ­ Differences in infectivity between nematode isolates ences in ability to penetrate the insect body. We ob­ have been documented for many insect hosts (Forschler served that the cuticle of D. radicum was difficult to and Nordin, 1988; Griffin et al., 1989). Our study con­ penetrate directly by Heterorhabditis spp. On G. mel­ firms results obtained in previous studies (Bracken, Ion ella most IJs of these species accumulated at the in­ 1990; Sulistyanto et al., 1994) by demonstrating that 5. ter-segments and there caused many black spots. On D. /eltiae is much more virulent against D. radicum than 'radicum, however, we observed only a few black spots. other epn species. Isolates Lxm31 and D-hbr were the Compared to the small size of the insect host, IJs of 5. most virulent. Behavioural differences or differences in glaseri and 5. arenarium may be too large, which may the number of bacteria per TJs may account for differ- partially explain their low infectivity. 5. carpocapsae got Chen and Moens 161 easily in contact with the insect, but only a few Us pene­ Burman M. and Pye AE., 1980. Neoaplectana carpocapsae: trated the insect (S. Chen, unpublished). Movements of the nematode populations on a thermal gra­ The results of the dose-response experiments demon­ dient. Experimental Parasitology, 49: 258-265. strated a positive correlation between the dosage of epn Dunphy G. B. and Webster J. M., 1986. Temperature effects and the level of control of D. radicum. Similar results on the growth and virulence of Steinemema /eltiae strains have been found for other combinations of the epns and and Heterorhabditis heliothidis. Journal 0/ Nematology, 18: insects (Kaya, 1978; Simons, 1981; Molyneux et al., 270-272. 1983). As the slope of the regression line is an indica­ Fan X. and Hominick W.M., 1991. Efficiency of the Galleria tion of the virulence of the nematode species - because (Wax moth) baiting technique for recovering infective it measures the infection by all individuals capable of in­ stages of entomopathogenic rhabditids (Steinernematidae and Heterorhabditidae) from sand and . Revue de Ne­ vading and establishing within insects (Hominick and matologie, 14: 381-387. Reid, 1990) - our study proved S. /eltiae to be the most virulent species against D. radicum. This agrees with Finch S., 1989. Ecological considerations in the management of Delia pest species in vegetable crops. Annual Review 0/ most of the previous studies (Sulistyanto et al., 1994; Entomology, 34: 117-137. . Schroeder et al., 1996). The number of nematodes that established in the Forschler B.T. and Nordin G.L., 1988. Comparative patho­ genicity of selected entomogenous nematodes to the hard­ host was in proportion to the dose applied. Mracek wood borers, Prionoxystus robiniae (Lepidoptera: Cossi­ (1982) estimated that 10-20% of a dose of S. cmpocap­ dae) and Megacyllene robiniae (Coleoptera: Cerambyci­ 3 sae applied to OJ dm of soil was recovered from twen­ dae). Journal o/Invertebrate Pathology, 52: 343-347. ty G. mellon ella larvae. Fan and Hominick (1991) re­ Gaugler R, Lebeck L.M. and Boush G.M., 1980. Orientation ported that 36% of Steinernema spp. (Nash isolate) of the entomopathogenic nematode Neoaplectana carpocap­ could penetrate and establish in G. mellon ella; this per­ sae to carbon dioxide. Environmental Entomology, 9: 649- centage was independent of the dose. In our study, 29% 652. of the IJ s of S. /eltiae were recovered after 48 hours. Glazer 1. and Navon A, 1990. Activity and persistence of en­ However, only 7.4% and 5.4% of the initial dose ap­ tomoparasitic nematodes tested against Heliothis armigera plied were recovered for S. carpocapsae and H. megzdis, (Lepidoptera: Noctuidae). Journal 0/ Economic Entomolo­ respectively (Table II). Obviously, the percentage of ne­ gy, 83: 1795-1800. matodes recovered vary with both the epn and the in­ Griffin c.T., Simons W.R and Smits P.H., 1989. Activity and sect species. infectivity of four isolates of Heterorhabditis spp. Journal 0/ The LT50 is another parameter for measuring the per­ Invertebrate Pathology, 53: 107-112. formance of epn. A good isolate should have a short Harris C.R and Svec H,J., 1966. Mass rearing of the cabbage LT50 as after their release epn are immediately exposed maggot under controlled environmental condition, with ob­ to unfavourable environmental factors. 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Accepted for publicationon 12 June 2003.