Naturally occurring entomopathogenic nematodes in

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Abstract. The presence of naturally occurring entomopathogenic nematodes (epns) was surveyed in the West-Flanders province in the north-west of Belgium.

Journal ojHelminthology (1996) 70, 319-327

319

Naturally occurring entomopathogenic nematodes in the province of West-Flanders, Belgium

1

J.S. Miduturil, M. Moensl, W.M. Horninick 2, B.R. Briscoé and A.P. Reid 2 ICLü-Rijksstation voor Nematologie en Entomologie,

Van Gansberghelaan 96, B-9820 Merelbeke, Belgium

2International Institute of Parasitology, 395A Hatfield Road,

St Albans, Herts, AL4 OXU, UK

Abstract The presence of naturally occurring entomopathogenic nematodes (epns) was surveyed in the West-Flanders province in the north-west of Belgium. In 21 sites of different agronomical situations, 130 soil samples were taken. Using theGalleria larva bait technique, 16 soil samples were found positive for epns. Fifteen samples were found to contain Steinernemaspp. (nine S.jeitiae, five S. affinis, one Steinernema species B3). The remaining positive sample contained Heterorhabditis sp. (North West European strain). The morphometric characters of the isolates were highly variabie and did not correspond precisely to the original descriptions; however, biochemical charaderization confirmed their identity. The epns were isolated from 50%,18.8% or 12.3% of the samples taken in sand dunes, grassland or woodlands, respectively. S. jeltiae and S. affinis were isolated in these three habitats; Heterorhabditis sp. was found in a grassland habitat. Steinernema jeitiae was prevalent in loamy sand soils with a wide range of organic matter content; S. affinis, Heterorhabditis sp. and Steinernema species B3 were isolated in sandy loam soils. All the positive sample sites were in the pH range of 4.0-8.1. This is the first report of naturally occurring entomopathogenic nematodes in Belgium.

Introduction The economie importance of entomopathogenic nematodes (epns) of the families Steinernematidae and Heterorhabditidae is increasing because of their potential use in biological control of diHerent insect species. The non-feeding infcctive juveniles carry the symbiotic bacteria Xenorhabdus/Photorlzabdus in their gut. The nematodes search for or ambush a suitabJe insect host, enter through natura] openings but also through the cuticle and release their symbiont into the haemolymph. Proliferation of the bacteria leads to death of the insect host within 24-48 h, followed by nematode development and reproduction (Kaya ct nl., 1993). The search for potential control agents has led to surveys of naturally occurring epns in many countries. Surveys using the Gal/erin bait technique (Bedding &Akhurst, 1975) have shown that these nematodes are distributed in Asia

I

(Zhang et al., 1992; Amarasinghe et al., 1994), North Africa (Shamseldean & Abd Elgawad, 1994), Australia & New Zealand (Akhurst & Bedding, 1986; Wouts, 1979), Canada (Mracek & Webster, 1994), Europe (Mracek, 1980; Deseo ct al., 1984; Burman et al., 1986; Blackshaw, 1988; Vanninen et al., 1989; Hominick & Briscoe, 1990a, b; Ehlers et al., 1991; Griffin ct al., 1991, 1994; Boag et al., 1992; Haukeland, 1993; Tallosiet al., 1995), Mediterranean region (Glazeret al, 1991; Ozer et al., 1995), South America (Doucet ,1986; Nguyen & Smart, 1988; Doucet & Doucet, 1990) and USA (Akhurst & Brooks, 1984; Hara et al., 1991; Rueda el al., 1(93). These surveys indicated that thc steinernematids öre distributcd widely, whereas heterorhabditids are concentrated closer to the sea (Griffin et al., 1991, 1994). Species or strains of epns show differcnces in survival and infectivity which means that some may be more suitable for particular biocontrol progrömmes (Bedding et al., 1983). The biotic and abiotic conditions of their

320

l.s. Miduturi et al.

isolation site play a major role in their adaptation. Hence, new species or strains of epns are always being sought. In Belgium, the commercial use of epns in glasshouse pest control was initiated by Coomans & De Grisse (1985) and Miduturi et al. (1994) but knowledge of naturally occurring epns is lacking. The present survey was conducted in the West-Flanders province (fig. 1) from January toApril1994 with the aim of isolating indigenous entomopathogenic nematodes and relating distribution to the soil type and habitat.

Materials and methods

random subsamples of approximately 200 mI each were collected to a depth of 10 cm over an area of 50 m2 . The five samples were pooled and transported to the laboratory. A representative sample of 250 mi was placed in a plastic box and baited with five last instar larvae of Gal/cria 1Jlcl/oncl/a. The boxes were stored at 20-25°C.After 5 days the dead Gal/eria larvae were collected and transferred onto a White trap (White, 1927) to extract the infective jliveniles.All the samples were baited three times with Cal/cria to obtain the maximum number of positive soi! samples. The infective juveniles collected 10 days later were then checked for their pathogenicity to Gal/cria larvae.

Col/ection ofsoil samples

Prcparation of infective juveniles for morphometric studies

A total of 130 soi! samples was collected from 21 different locations of varied habitat type (grassland, woodland, cultivated land, roadside verge, sand dunes and sea shore) from the West-Flanders province of Belgium. However, three locations were bordering on the adjacent province (fig. 1). At each sampling site five

Identification of nematodes to genus level was attempted by making temporary mounts of ten infective juveniles for each isolate. Infective jllveniles were killed and fixed in 4% hot formaldehyde. Fixed nematodes were transferred to anhydrous glycerine according to Seinhorst's rapid method (Seinhorst, 1959) as modified by De Grisse (1969). Permanent slides were prepared according to Cobb (1918). All measurements were made using a drawing tube attached to the light microscope.

Soil parameters

eb

-... -*

6c 9_

10-'" 11.

6

-*

pH and EC were measured on soi! sus pensions prepared in the laboratory (20 g soi! suspended in 100 mi distilled water and shaken for 3 h). The organic matter content of each soil sample was determined by use of the ignition proccss and calculated using the percentage by weight method (Andrews, 1973). The soit samples were processed with a COlilter LS 100 fluid module apparatlls for particle size analysis. It gave the relative presence of the clay fraction «4 pm), the silt fraction (4-63 llm) and the sand fraction (>63 llm).

5-6 Nernatodc charactcrization through RFLPs 12.

Fig. 1. Entomopathogenic nematodes in the province of WestFlandl'rs, Belgium, showing sampling locations (0), samples fol' Steinernema fel/iae (0), Steinernema affinis (0), Steinernema sp. B3 (0) and Heterorirabrtitis IIlegidis (NWE type) (0). Sampling [ocations (nwnber of samples): 1. Knokke Zwin (20), 2. Knokke Heist (6), 3. Woesten (6), 4. Oedelem (10), 5. Snellegem (10), 6. Thillegembos (10), 7. Maldegem (2), S. Duinbergen/Wenduinel Dchaan (4), 9. Klemskerke (2), lD. Palingpot (4), 11. SJijpe (2), 12. WiJnendaele (4), 13. Bulskampveld (7), ]4. Houthulst (S), ]5. Zonnebeke (7),16. Poperinge (5), 17. Rodeberg (5), lS. Kemmel (5), 19. Louise Marie (5), 20. Kluisbergen (5), 21. Sijsele (3).

DNA was exbracted from single infective juveniles and used for PCR amplification Ooyceet al., 1994b) of the rDNA lTS region. Primers used were those described by Vrain et al. (1992). Amplification products were stored at -20°C until used. Following PCR, each sample together with contrals were digested with either Alul, HinfI or RsaI (1 III 10 x enzyme buffer, 5 III PCR product, 3.5 pi dH2 0 and 0.5 III restriction enzyme). Digests were incubated at 37°C for 1-4 hand fragments separated by electraphoresis in 1.5% (w Iv) agarose gel in 0.5 x TBE at 100V for 3-4 h. Gels were viewed on a UV-transilluminator. Nematodes were identified by comparing their RFLPs to a RFLP database of known isolates (Reid, 1994).

Results Sixteen isolates of entomopathogenic nematodes were recovered from different habitats. All the isolates were able to multiply on G. mcl/onclla larvae and so confirmed their entomopathogenic character. The overall average length and overall average width of 15 isolates out of 16 isolates were 684 llm (476-885 pm) and 25 pm (16-42 pm), respectively (tabIe 1 and 2). The

Table 1. Morphometrics of infective juveniles of isolates from West-Flanders, Belgium comparable to Steinernema feitine a. Character

BI

R53

R56

R519

R550

R551

R555

R564

R571

Z7

S. feltiae (after Poinar, 1990)

Total length

736 (655-826)

617 (538-738)

647 (476-785)

705 (618-856)

721 (566-824)

762 (580-885)

704 (571-857)

712 (666-785)

759 (642-857)

735 (566-833)

849 (736-950)

Greatest width

26 (22-30)

23 (16-30)

20 (17-23)

24 (26-29)

26 (24-31)

24 (19-28)

23 (19-26)

30 (27-33)

32 (27-42)

26 (23-30)

26 (22-29)

Distance head to excretory pore

41 (35-48)

52 (42-65)

50 (40-58)

52 (46-61)

53 (45-67)

49 (38-59)

53 (43-64)

48 (37-56)

50 (40-65)

50 (46-56)

62 (53-67)

Distance head to nerve ring

70 (56-82)

74 (65-91)

76 (57-86)

76 (66-86)

76 (66-88)

77 (60-92)

80 (65-88)

75 (64-88)

75 (67-98)

77 (68-90)

99 (88-112)

0 '"Cl

Distance head to pharynx base

107 (82-128)

112 (94-138)

123 (96-139)

119 (109-131)

116 (102-131)

122 (109-142)

126 (100-136)

111 (98-133)

120 (104-135)

123 (108-131)

136 (115-150)

OQ

Tail length

64 (51-74)

51 (43-68)

66 (51-73)

76 (65-87)

67 (50-78)

75 (60-85)

75 (64-87)

69 (50-83)

79 (60-92)

67 (47-83)

81 (70-92)

::l ro

Ratio Ab

27 (22-32)

28 (23-33)

32 (28-37)

29 (24-32)

27 (23-31)

31 (27-33)

31 (29-38)

24 (22-25)

23 (21-30)

28 (23-33)

31 (29-33)

'"Ö0-

Ratio Be

6.9 (6.0-8.4)

5.6 (4.5-6.9)

5.3 (4.4-5.7)

5.9 (51-7.3)

6.1 (5.0-6.8)

6.2 (5.1-7.5)

5.6 (4.9-6.7)

6.4 (5.6-7.0)

6.0 (5.4-7.6)

5.9 (5.2-6.7)

6.0 (5.3-6.4)

Ratio Cd

11.4 (9.5-13.7)

12.1 (9.7-16.3)

9.9 (8.5-12)

9.3 (7.8-9.8)

10.6 (9.6-11.7)

10.1 (8.6-11.7)

9.5 (8.2-11)

10.4 (8.21-5.2)

9.7 (8.4-16.4)

11.0 (9.91-3.7)

10.4 (9.2-12.6)

Ratio De

0.38 (0.32-0.48)

0.41 0.43 0.45 0.46 0.4 (0.41-0.51) (0.37-0.45) (0.38-0.52) (0.39-0.55) (0.31-051)

0.42 0.43 0.42 (0.38-0.52) (0.38-0.48) (0.37-0.5)

0.40 (0.37-0.44)

0.45 (0.42-0.51)

Ratio Er

0.65 (0.54-0.88)

0.76 1.02 (0.84-1.22) (0.7-0.85)

0.68 (0.6-0.89)

0.78 0.67 (0.68-0.92) (0.45-0.85)

0.71 0.71 (0.55-0.87) (0.52-1.0)

0.65 0.74 (0.55-0.78) (0.63-1.08)

0.78 (0.69-0.86)

N

17

19

16

26

24

16

25

19

18

11

19

tTJ ::l

Ö

:3

'"g:.

0

ro ::J

:=i"

:3 ro

-'

Js. Miduturi et al.

322

Table 2. Morphometrics of infective juveniles of isolates from West-Flanders, Belgium comparable to Steinernema affinis ". Character

RS5

RS10

215

216

218

S. affillis (after Poinar, 1990)

Total length Greatest width Distance head to excretory pore Distance head to nerve ring Distance head to pharynx base Taillength Ratio Ab Ratio Be Ratio Cd Ratio De Ratio Ef N

655 (603-738) 21 (16-24) 50 (38-59)

590 (531-730) 24 (20-28) 55 (44-61)

613 (476-712) 23 (18~26) 54 (41-60)

635 (546-799) 24 (22-27) 51 (44-64)

665 (571-809) 27 (24-32) 53 (46-61)

693 (608-800) 30 (28-34) 62 (51-69)

76 (64-92)

71 (62-86)

77 (53-89)

73 (62-88)

82 (75-94)

95 (88-104)

115 (92-145)

110 (92-136)

118 (79-134)

121 (104-150)

123 (108-133)

126 (115-134)

57 (45-68) 31 (26-34) 5.7 (5.4-6.1) 11.6 (10.0-12.3) 0.44 (0.41-0.49) 0.89 (0.76-1.02) 6

57 (51-65) 25 (23-28) 5.3 (4.9-6.1) 10.3 (9.7-10.8) 0.45 (0.41-0.52) 0.86 (0.74-1.0) 19

53 (42-66) 26 (23-33) 5.7 (4.3-6.0) 11.6 (7.0-13.2) 0.5 (0.4-0.5) 1.0 (0.70-1.1) 20

54 (40-65) 26 (21-31) 5.2 (4.6-6.1) 11.7 (10.1~13.5) 042 (0.34-0.48) 0.94 (0.74-1.16) 18

57 (50-65) 24 (22-29) 5.4 (4.5-6.2) 11.6 (10.2-12.7) 0.43 (0.31-0.49) 093 (0.76-1.06) 16

66 (64-74) 23 (21-28) 5.5 (5.1-6.0) 10.5 (9.5-11.5) 0.49 (0.43-0.53) 0.94 (0.74-1.08) 25

A

1

B

1 Z

aAll the measurements are in micrometres, range is given in brackets and precedes the mean; bLength divided by width; eLength divided by distance from head to pharynx base; dLength divided by tail length; l'Distance from head to excretory pore divided by distance from head to pharynx base; f[)istance from head to excretory pore divided by tail length. Table 3. Infective juvenile body measurements and ratios of a Belgian isolate of Heterorhabditis spa Character Belgian isolate (Sn 5)

H. rl/cgidis (Poinar et al., 1987)

Tota I length Greatest width Distance head to excretory pore Distance head to nerve ring Distance head to Pharynx base Tail length Ratio Ab Ratio Be Ratio Cd Ratio De Ratio Ef Ratio F'

768 (736-800) 29 (27-32) 131 (123-142) 109 (104-115) 155 (147-160) 119 (112-128) 26 (23-28) 5.0 (4.6-5.9) 6.5 (6.1-6.9) 0.85 (0.81-0.91) 1.1 (1.03~ 1.2) 0.25 (0.23-0.28)

I

739 (603-788) 24 (22-28) 109 (98-120) 85 (76-96) 129 (122-140) 112 (104-130) 30 (21-33) 58 (4.4-6.3) 6.6 (5.5-7.2) 0.85 (0.75-0.96) 0.97 (0.81-1.07) 0.21 (0.18-0.25)

aAll measurements are in micrometres, range is given in brackets and precedes the mean; bLength divided by width; eLength divided by distance from head to pharynx base; dLength divided by taillength; eDistance from head to excretory pore divided by Distance from head to pharynx base; fDistance from head to excretory pore divided by tail length; gWidth divided by tail Jength.

2

Fig. 2. RFLPs products, amp Panel A RFLPs obtained by dit digestion withj 2, S.feltiae (Bel! S. affinis (Belgi1 RSSO); 6, S. fel

mouth and anus were closed, the pharynx and intestine collapsed, with the excretory pore anterior to the nerve ring, and symbiotic bacteria present at the base of the pharyngeal bulb. A short tail was also present (less than 79 f-Im). Infective juveniles of some populations had a small refractile spine at the tip of the tail. All these characters are consistent with descriptions of Steinernema sp. The remaining isolate differed. lts mean body leng th and body width were 739 f-Im and 24 f-Im, respectively. The third stage infective juvenile was inside the second stage cuticle, which showed a number of longitudinal ridges. The head possessed a small projection on the dorsal side. The excretory pore was posterior to the nerve ring.

Cells of symbiotic baderia were found in the lumen of the intestine. These characters indicated that this isolate was Hctcrorhabditis sp. Further investigation on morphometric characters was carried out on all the 15 isolates of Steincrncma and the results were compared with the original morphometrics of Steinernema spp. Poinar (1990) considers the total body length and the ratio B as major characteristics of S. feltiae. Within the populations belonging to the genusSteinernema, ten were found comparable to S. feltiae (Filipjev) (tabIe 1). For all these isolates, ratios Band C were consistent with the revised S.feltiae descriptions (Poinar, 1990). However, mean va lues were overlying the range of this description

Steinernema sp. 10, S. carpocap Marker lane (b pairs); 13 and (Belgiwn site ~ 211);16,H.meg

for following

RS19, RSSO, R RS71); distan, RS19, RSS1, R

ring was smal

Entomopathogenic nematodes in Belgiwn

BI2345bï

Fig. 2. RFLPs obtained by restriction digestion of lTS PCR products, amplified adult females isolated during the survey. Panel A RFLPs obtained by digestion with A/u 1, Panel B RFLPs obtained by digestion withHilif·[ and Panel C RFLPs obtained by digestion withRsa l. Lanel, Steinemerna jeltiae (BelgilUnsite RS3); 2, S.feltiae (Belgium site RS64); 3, S. affinis (Belgiwn site 215); 4, S. affinis (Belgiwn site 216); 5, Steinernema sp. B3 (Belgiwn site RSSO); 6, S. jeltiae (UK site 76); 7, S. affinis (UK site 464); 8, Steinernema sp. B3 (UK site 249); 9, S. kraussei (Czechoslovakia); 10, S. carpocapsae (UK strain); 11, S. intermeditlm (USA); 12, Marker lane (band sizes 2000, 1200, SOO, 400, 200 and 100 base pairs); 13 and 14, Heterorhabditis megidis, NW European type (Belgium site Sn5); 15, H. megidis NW European type (UK site 211);16, H. megidis (Ohio);17, Heterorhabditis sp. Irish type (Ml45) and 18, H. bacterioplwra (USA). for following characters and isolates: totallength (RS3, RS6, RS19, RS50, RS55 and RS64); greatest width (RS6, RS64 and RS71); distance from head to excretory pore (BI, RS3, RS6, RS19, RS51, RS64, R571 and 27); distance from head to nerve ring WilS smaller for all the ten isolates; distance from head to

323

pharynx base (BI, RS3 and RS64); taillength (BI, RS3, RS6, RSSO, RS64 and 27); ratio A (BI, RS6, RS50, RS64, RS71 and 27); ratio D (BI, RS6, RS51 and 27) and ratio E (BI, RS19, RS51 and RS71). The mean values of the remaining five isolates fit the descriptions ofS. affinis by Poinar (1990) for the following characteristics: totallength, ratio A, ratio Band ratio E (tabie 2). Differences were found for: distance from head to excretory pare (RS5); distance from head to pharynx base (RSlO) ratio C (RS5, 215, 216 and 218); ratio D (216). Mean values of greatest width, distance from head to nerve ring and taillength of all five isolates differed from Poinar (1990). The Heterorhabditis isolate (Sn5) had an ave rage length of 739 11111 (603-788 flm) and taillength of112 flm (104-130 pm), which are close to the described measurements ofH. megidis; the ratias were overlapping (tabIe 3). Same of the remaining characters (greatest width, distanc.e from head to excretory pare, to nerve ring and to pharynxbase), however, are smaller than in the original descriptions. The variability of morphometric characters and the lack of agreement with the descriptions of Poinar (1990) necessitated further examination of these isolates using PCR based methods. All the isolates yielded a c. 1.lkb fragment upon PCR amplification with ITS primers. The resulting PCR products were digested with either AluI, HinfI or RsaI and run against standard species found in northem Europe. A selection of the RFLPs obtained are shown in fig. 2. The patterns yieldedby the Belgian isolates clearly show three steinerr,ematid species: SfeUiae (lanes 1 and 2),S. affinis (lanes 3 and 4) and Steinernema sp. B3 (lane 5) all of which yield the same patterns as their British counterparts (lanes 6,7 and 8 respectively). The heterorhabditid isolate (lanes 13 and 14) yielded the same pattem as the North West European type of H. megidis found in the United Kingdom (lane 15). The original isolate ofH. megidis from Ohio (lane 16) and the Irish type (lane 17) bath show polymorphic bands in the case of digestion with RsaI but onIy in the case of the Irish type with Hinj!. In contrastH. bacterioplwra (lane 18) which is also found in Europe shows clear differences with all truee enzymes. The restrichon digests of the amplified lTS products identified nine populations as s. jeltiae Al, five as S. affmis, one as Steinernenw species B3 and one as H. megidis NWE type. Entomopathogenic nematodes were recovered from 16 (12.3%) of the 130 soil samples collected. Heterorhabditis was recovered from onIy one site, the remaining isolates were Steinemema spp. Entomopathogenic nematodes were isolated from 50%,18.8% or 12.3% of the samples taken in, respectively, sand dunes, grassland or woodland. Steinernema jeltiae and S. affinis were isolated in these three habitats. Species B3 was isolated from woodland. TheHeterorhabditis sp. was isolated from a grassland site. Roadside verges, cultivated land and sea share sites did not yield any epns (tabIe 4). Steinernema jeUiae Al was prevalent in loamy sand soils with a wide range of organic matter content (1.2-8.4%). Steinernema affinis was isolated in loamy sand to sandy loams with 4.2-5.3% organic matter content. Hetero1"habditis sp. (NWE type) and species B3 were also isolated in sandy loam, loam soils with organic matter content of 5.6% and 7.5%, respectively (table 5). The soils of positive sampling sites were in the pH range of 4.Q-8.1. Steinernemajeltiae and species B3 were isolated from acidic to neutral soils (pH 4.2-7.1), Steinernema affinis was found in neutral to alkaline soils (pH 6.5-8.1). Hetemrhabditis was present in an almost neutral soil (pH 6.8).

J.5. Miduturi et al.

324

Table 4. Distribution of stcinernematid and heterorhabditid nematodes in different habitats in the Province of West-Flanders, Belgium. Habitat

Grassland Woodland Roadside verge Sand dunes Sea share Cultivated land

Col.lected

posihve for S. feltine

Number of samples positive for positive for S. nffinis species B3

16 81 4 6 6 17

1 6 0 2 0 0

1 3 0 1 0 0

0 1 0 0 0 0

positive for Heterorhnbditis sp. 1 0 0 0 ()

0

Table 5. Habitat, soit characteristics and epn identity at positive sampling sites. Sample number

Location

Habitat

Soil type

Organic matter(%)

pH

Nematode species/ RFLP type

Z7 Z 15 Z 16 Z 18 Sn 5

Knokke Zwin Knokke Zwin Knokke Zwin Knokke Zwin Snellegem

woodland woodland woodland woodland grassland

loamy sand loam sand sandy loam sandy loam sandy loam

8.4 4.3 4.2 5.3 5.6

7.1 6.5 8.1 7.4 6.8

RS 3 RS 5 RS 6 B1 RS 10 RS 19 RS 50 RS 51 RS 55 RS 64 RS 71

Duinbergen Duinbergen Wendlline 1illegembos Palingpot BlIlskamveld Rodeberg Kemmel Kemmel Louise Marie Sijsele

sand dunes sand dllnes sand dllnes wood land grassland wood land woodland wood land woodland wood land grassland

loamy sand loam sand loam sand loamy sand 10am loamy sand loamy sand silt loam 10am loamy sand loamy sand

4.9 4.1 1.2 4.2 4.6 7.3 7.5 7.4 5.2 2.5 4.4

7.0 7.3 5.4 5.5 7.6 4.2 4.0 4.7 4.3 4.4 7.0

fe/tinr Al affinis nffinis nffinis Heferorhnbdilis sp. (NWE type) S. fe/tine Al S. affinis S. feitiae Al S. feitine Al S. affinis S. fclfiae Al Species B3 S.fe/tiar Al S. feltine Al S. fe/tine Al S. fe/tiae Al

Discussion This survey provides the relative importance of the occurrence of steinernematids and heterorhabditids in the province of West-Flanders, Belgium. It yielded 15 Steinernema isolates and one Heterorhabditis sp. Variations in morphometrics were observed within the isolates comparable to S. feltiae, S. affil1is and Heierorhabditis sp. Gwynn (1993) also observed inconsistency in morphometric measurements of UK isolates of steinernematids. Nguyen & Smart (1995), aftel' a study of the morphometrics of H. bacteriophora and seven species of Steincrncma, noticed that the measurements of infective juveniles vary considerably depending upon the time of harvest in the il1 vivo culture. They suggested that for morphometric studies, only infective juveniles collected over a one week period aftel' first emergence from the insect host should be used. Presumably the reduction in size of infective juveniles is related to diminishing nutrient supplies in the insect cadaver. The polymerase chain reaction based RFLP method used in our study aUows quick and more precise discrimination of species. Amplified products of the lTS reg ion have, upon restrietion enzyme digestion, yielded

S. S. S. S.

many RFLPs that can be used for species identification of Steinernema isolates (Reid, 1994). The present study with PCR-RFLP based identification yielded nine S. feltiae Al, five S. affinis and one Species B3 which confirmed the morphometric characters. Joyce et al. (1994a) identified six RFLP profiles for Hetcrorhabditis mtDNA and rDNA amplification produets which species groups included Irish, North West European, tropical and the H. bacteriophora complex. The RFLP pattem of our Heterorhabditis isolate fits into the North West European type. Entomopathogenic nematodes have been found with various frequencies in most terrestrial habitats. The present survey yielded 12.3% positive samples with a predominance of steinernematids (11.5%) and only one sample of heterorhabditids (0.8%). The recovery ra te of Stcil1emema sp. from the present survey was higher than in surveys undertaken in Scotland (2.2%) (Boag et al., 1992), Northern lreland (3.8%) (Blackshaw, 1988) and Ireland (10.5%) (Griffin et al., 1991). It was almost four times less than in England, Wales and southern Scotland (48.6%) (Hominick & Briscoe, 1990a). Steil1crnema feitiae was the only species recovered during a survey in Northern lreland (Blackshaw, 1988) while in lreland S. feltiae and S. affinis were found in

respectively J 1991). Steincl species in Bril recovered fm Briscoe, 1990, was more wie 1995). Our rE commonspec (3.0%). Uniik, examples of species B3 ani Steinerner particularlyat and Israel (B\ et al, 1991) a northern Irela found associal 1990b). Stei Czechoslovak found in meac (1986) attribul in the distribu of nematodE steinernemati sandy la,llTISO of steinernem; habitats (tabiE

Hetcrorhabditi..

of coastal site: any of the 40

Heterorl1G1Jditis

metres from t matter conten noted a similar islands. Durin isolate belang detected.ltwa soil (73.12'X, sa site is situatee pesticides nor This may havi hasts on whic The soil p were betl·vee 4-8 did not pathogenicity were adversE However, as I soils does not the reasons Fe in different a . DifferencE occurred bel elsewhere. TI1 unknown du importance of seasons, habil The prese: presence of habitats in W~ an investigati entomopatho in Belgillm.

Entomopatbogenic nematodes in Belgium

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respectively 7.1 % and 3.3% of the samples (Griffin et al., 1991). Steincrnema feltiae was also the most prevalent species in British soils. Heterorhabditids were rare, being recovered from only one site on each island (Hominick & Briscoe, 1990a). Later reports showed that Heterorhabditis was more widespread (Griffin et al., 1994; Hominick et al., 1995). Our results also show that S. feltiae was the most common species (6.9%) in Belgian soils followed by S. affinis (3.0%). Unlike other surveys (Hominick et al., 1995) no examples of S. kraussei were isolated. One Steinernema species 83 and one HeterorlJabditis sp. were isojated. Steinernematids are found in all types of soils but are particuJariy associated with sandy sods in Sweden, Hawaii and Israel (Burman et al., 1986; Hara et al., 1991; GJazer et al., 1991) and with sandy loam and loamy soils in northern Ireland (Blackshaw, 1988). In England they were found associa ted with calcareous soi! (Hominick & Briscoe, 1990b). Steinernematids were more common in Czechoslovakian forests than in erop land and were not found in meadows (Mraceket al., 1982). Akhurst & Bedding (1986) attributed differences in distribution to differences in the distribution of suitable insect hosts and to the species of nematodes involved. In the present survey, the steinernematids were isolated either in loamy sand or sandy loam sods except in one case, in silt loam; the majority of steinernematid isolates were found in woodland / forest habitats (tabks 4 and 5). Griffin et al. (1994) reported that Heterorhabditis sp.(Irish group) was recovered from 14.5% of coastal sites in Ireland and Britain and not detected at anY of the 40 inland sampling sites. The sites at which Heteror/mbdifis sp. was detected were within a few hundred metres from the sea in sandy soils with a 3-7% organic matter content and a pH range of 4.6-8.1. Hara et al. (1991) noted a similar distribution of heterorhabditids in Hawaiian islands. During the present survey only one Heterorhabditis isolate belonging to the North West European group was detected. It was recovered from grassland on a sandy loam soil (73.12% sand, 24.47% clay and 2.41 % silt). The sampling site is situated c. 18 km away from the sea and neither pesticides nor mineral fertilizers were used on this site. This may have encouraged the natural presence of insect hosts on which these epns multiply and survive. The soil pH measurements of present sampling sites were between 4.0-8.1. Tt was reported that pH values of 4-8 did not significantly affect the survival and pathogenicity of entomopathogenic nematodes but they were adversely affected at pH 10 (Kung et al., 1990). However, as Kaya (1990) pointed out, the pH of normal soils does not act as a limiting factor. This may be one of the reasons for the wide distribution of these nematodes in different agronomical situations. Differences in the frequency of nematodes detected occurred between the surveys conducted here and elsewhere. The reasons for these differences are at present unknown due to a lack of our understanding of the importance of different sampling strategics, differcnces in seasons, habitat, soil type and baiting technique. The present survey has shown for the first time the presence of entomopathogenic nematodes in different habitats in West-Flanders, Belgium. Future work inc1udes an investigation inta the possibility of the use of these entomopathogenic isolates for pest control programmes in Belgium.

325

Acknowledgements We thank Ing. G. Hendrickx & MI's M. Vandeputte for their technical assistance during the survey and also to the CLO-Rijksstation voor Nematologie en Entomologie, Merelbeke for financial assistance. Thanks also ta the COST 819 'Entomopathogenic nematodes' project for granting financiaJ assistance for a short-term scientific mission.

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