DOI: 10.2478/v10014-008-0015-4
Agrovoc descriptors: nematoda; insect nematodes; new species; fauna; population structure; identification; population distribution; biological control
Agris category code: H10
COBISS code 1.01
University of Ljubljana Biotechnical Faculty Department of Agronomy
Entomopathogenic nematode Steinernema carpocapsae (Weiser) (Rhabditida: Steinernematidae), a new member
of Slovenian fauna
Žiga LAZNIK1, Timea TÓTH2, Tamàs LAKATOS3, Stanislav
TRDAN4
Received July 24, 2008; accepted August 04, 2008. Delo je prispelo 24. julija 2008, sprejeto 4. avgusta 2008.
ABSTRACT
In April 2008, 120 soil samples from 24 locations were collected in Gorenjska, Notranjska and Primorska regions as well as in Ljubljansko barje. The presence of entomopathogenic nematodes was confirmed in 9 samples from 6 locations. Only the sample C101, which was taken in the village Svino in the area of Breginjski kot (western part of Slovenia, the vicinity of Italian border), was sent to genetic analysis. Molecular biological analysis was proved the identity of the sample with the species Steinernema carpocapsae (Weiser). This was the first record of Steinernema carpocapsae in Slovenia. In preceding researches on the fauna of entomopathogenic nematodes in Slovenia, which started in 2007, we already established the occurrence of Steinernema affine (Bovien) and Steinernema feltiae (Filipjev).
Key words: entomopathogenic nematodes, Slovenia, Steinernema carpocapsae, biological control
IZVLEČEK
ENTOMOPATOGENA OGORČICA Steinernema carpocapsae (Weiser) (Rhabditida: Steinernematidae), NOVI PREDSTAVNIK SLOVENSKE FAVNE
V aprilu 2008 smo na območju Gorenjske, Notranjske, Primorske in Ljubljanskega barja na 24 lokacijah nabrali 120 talnih vzorcev. Zastopanost entomopatogenih ogorčic smo ugotovili v 9
1	Young researcher, B. Sc., Jamnikarjeva 101, SI-1111 Ljubljana, email:ziga.laznik@bf.uni-lj.si
2	Ph. D student, M. Sc., Vadastag 2, H-4244 Ùjfehértó, Hungary
3	Ph. D, Vadastag 2, H-4244 Ùjfehértó, Hungary
4	Assist. Prof., Ph. D, Jamnikarjeva 101, SI-1111 Ljubljana
vzorcih s 6 lokacij. V nadaljnjo genetsko analizo smo poslali le vzorec C101. Ta je bil odvzet v vasi Svino na območju Breginjskega kota (skrajni zahod Slovenije na meji z Italijo). Z molekulsko analizo smo identificirali vrsto Steinernema carpocapsae (Weiser). Gre za prvo odkritje omenjene vrste entomopatogene ogorčice pri nas. V predhodnjih raziskavah preučevanja faune entomopatogenih ogorčic v Sloveniji, ki potekajo od leta 2007, smo ugotovili zastopanost vrst Steinernema affine (Bovien) in Steinernema feltiae (Filipjev).
Ključne besede: entomopatogene ogorčice, Slovenija, Steinernema carpocapsae, biotično varstvo
1 INTRODUCTION
It is well known that entomopathogenic nematodes, which are classified into Steinernematidae and Heterorhabditidae families, have great potential as biological control agents in plant protection (Klein, 1990). Their activity against different pest insects is already well studied (Kaya in Gaugler, 1993; Ebssa, 2005). Entomopathogenic nematodes are soil organisms, which live with bacteria in symbiotic-mutualistic relationship. For the first time their importance in biological control was discovered in the United States of America in the thirties of past century (Laznik in Trdan, 2008).
At the time of the first record of entomopathogenic nematodes, a hypothesis was raised that these agents by themselves cause death of attacked insects (Gaugler in Kaya, 1990). In 1937, Bovien first mentioned a possibility of symbiotic bacteria to live in mutualistic relationship with entomopathogenic nematodes. His hypothesis was confirmed in 1955 by Dutky and Weiser (Weiser, 1955). In 1982, Boemare gave a proof for production of toxic substances by the nematodes from genus Steinernema. These substances have negative influence on the immune system of infected insect and could also cause death of the host without the presence of symbiotic bacteria. Until now no evidence was given that nematodes from genus Heterorhabditis are capable for their own production of toxins, which would be able to influence a poor viability of invaded insects (Klein, 1990).
It is discussed upon symbiotic-mutualistic relationship because nematodes provide shelter and protection for bacteria in an exchange for killing the attacked insects. Latter, bacteria also produce antibiotics, which prevent the development of intra-and interspecific competitors, which would also feed on cadavers. Bacteria transform the content of the host into feed, suitable for nematodes and also bacteria themselves are feed for nematodes (Kaya in Koppenhöfer, 1999). Nematodes from the family Steinernematidae live in symbiosis with bacteria from genus Xenorhabdus, meanwhile it is well know for the ones from the family Heterorhabditidae that they have the same association with bacteria from genus Photorhabdus (Forst et al, 1997).
In Slovenia, momentarily only entomopathogenic nematode Steinernema feltiae has a status of indigenous species (MAFF, 2008); therefore only this species can be applied in the field. With the researches, which results we also present in this paper, we want to enlist as more species of entomopathogenic nematodes as it is possible,
while in foreign countries they worth as alternatives to insecticides in controling pest insects. The strain C101, which we present in a current paper, we plan to use in extensive experiments in the future; first in the laboratory and afterward, when its status will be administratively entrenched, also in the field.
2	MATERIALS AND METHODS
In April 2008, we examined 120 soil samples from 24 different locations on the occurrence of EPNs in Slovenia. The soil samples, four from each region, were taken in Gorenjska, Notranjska and Primorska region as well as in Ljubljansko barje. We used »Galleria bait method«, which is the most frequently used method of EPNs detection from soil. After the death of greater wax moth (Galleria mellonella [Linnaeus]) larvae, we dried cadavers for 12 days and put them in so called »White trap« (Bedding and Akhurst, 1975) to separate the nematodes from death larvae. The suspension, which was acquired in this way, was used for artificial infection of the larvae of greater wax moth. Following procedure contained the use of centrifuge and 5 % concentration of sodium hypochlorate. The aim of this process was to acquire infective juveniles from the suspension. We confirmed the presence of nematodes in 9 soil samples of 6 locations. Only 1 positive sample, C101 (taken on the arable field near village Svino in Breginjski kot (NW Slovenia, 46°14'N, 13°33'E, 285 m alt.) was identified to this time.
3	RESULTS
To confirm the identification of isolated nematodes from larvae of wax moth, a selected sample was analysed by molecular biological approach. Genomic DNA was extracted from individual nematodes and PCR was performed to multiply ITS region using primers TW81 and AB28 after Hominick et al. (1997). PCR product were reisolated from 1 % TAE-buffered agarose gel using QIAquick Gel Extraction Kit (Qiagen, USA). Reisolated sample was sequenced in the laboratory of Biological Research Centre in Szeged, Hungary. The sequence was submitted into GenBank public database (Accession Number: EU914854). Sample DNA sequence was compared to sequences of species Steinernema using BLAST search in National Centre for Biotechnology Information (NCBI) web site (www.ncbi.nlm.nih.gov). The sequences producing significant alignments and at least 99 % identity were derived from Steinernema carpocapsae: GenBank Accession No. AY171282 and EU122951 (Spiridonov et al, 2004) (Fig. 1).
9833	1	GGAA-GA-CATTATTGAGCTAATATT-TTCC-TTTTCT-ATCAAGT
TTTCGCTGCTCGT		54
AY171282	1	.............-....-....-.A.......
........T.		43
EU122 951	103	....G..T..................-....-....-.A.......
........T.		158
AY171280	1	..........-..C....A....-A-
....G..C..	.-..	..T____ 43
9833 55 TTCTAAGCTTTAACTTGACCTC-TAACGGCTTTGAAAGGTTTCTACAGATGTTTGGAGCA 113 AY171282 44 ..................T........................................ 102
EU122 951 159 .................-G........................................ 216
AY17128 0 44 ........A..G...C.. — ..-.---
T........T........T...C.......... 97
9833	114 GTTG-TAT-GAGCGTGACTGTGCTGATGAA-
CATTGTACATTGTTATCTAAGC-GTTTCG 169
AY171282 103 ____-...-.....................-
......................-...... 158
EU122 951 217 ____-...-.....................-
......................-...... 272
AY17128 0 98 ..CA-.-.CA............G....-..G.G..T...T...C.-
.-.--..A.-____ 149
9833	170 AT-
GTTTCTAGAATGCTTAGTGATGAGAATTAAAGAGGTCTGCTGACTCGCCATTCTTTG 22 8 AY171282 159 ..-
......................................................... 217
EU122 951 273 ..-
......................................................... 331
AY17128 0 150 -
.C......T..........C...............A..................-....
207
9833	229
ATTGCTAACAAAAACGTTTTGTTTCGATAATTGTGTCACTCGTTGATGCATTTTTTAAA-287
AY171282 218
..........................................................T-
276
EU122 951 332 ..............-......G..-.......-.....-
...........-.....-..- 384
AY17128 0 208
T...TT..........T......A....A..C..T-
266
9833	288 NATC-
AAGTCTTATCGGTGGATCACTCGGTTCGTAGGTCGATGAAAAACGGGGCAAAAAC 34 6 AY171282 277 T.......................................................... 335
EU122 951 385 TT..-......A...............................-
..........-..... 441
AY17128 0 2 67 T.......................................................... 325
9833	347
CGTTATTTGGCGTGAATTGCAGACATATTGAGCGCTAAAATTTTGAACGCAAATGGCACT 406
AY171282 336
395
EU122951 442
499
AY171280 326
385
9833	407
AACAGGTTTTTATCTGTTAGTATGTTCAATTGAGGGTCTTTTGACTAGAATCTGGCAATC 466
AY171282 396
455
EU122 951 500 ......-
..................................................... 558
AY17128 0 386 ......G..-................................................. 443
9833	4 67	CGCTGTGATTGCTTTTTCGGTAA-GCTACTTTGCT-T-T—T-----
--G---T-G--AA	508
AY171282 456	G......................-...........-.-.--.-----
--.---.-.--..	497
EU122 951 559	G......................-...........-.-.--.-----
--.---.-.--..	600
AY17128 0 444	G.............C.....A..-...........-.-.AG.-----
—.---.A.TG..	490
9833	509
GTACCTTTTCNGTATGGCTATTTGATTGTCTAACGGATGTCTGGCTAGCTGCTTCTTTGC 568
AY171282 498
G......................T................. T
557 EU122 951	601 .G..........		.T.....		...T........
660					
AY17128 0	491				
	.G..........	........A..	.T.....	.....T...	...T........
550
9833	5 69 TAGACGTCTGCAATCATTCGGCATTGCGTAGTGTTTGATTAAT-
GGTTTAGCGCGTTTCT 627
AY171282 558 ..................T........................-
................ 616
EU122 951 661 ..................T........................-
................ 719
AY17128 0 551
..................T...T...............AC...A....-...........
609
9833	628
TGCTAACTGACTTTTACACAAGCAAGTGTAATACGTTTCTTAAAGTCAGCTCATTAATCA 687
AY171282 617
676
356 Acta agriculturae Slovenica, 91 - 2, september 2008 EU122 951 720
779
AY17128 0 610
...............G................T........G.........-....T
668
9833	688 ATGTGGTTTTCTGACTTGATTTGTC-
GGTCAATTGTGCTATGCTCTG-CTAATCTTTTCG 745
AY171282 677 .........................-
.....................-............ 734
EU122 951 780 .........................-
.....................-............ 837
AY17128 0 669 ..T.............C.C......-
...TT.C..............T.-.......... 726
9833	746	AACT-AGACCTCAATT-GAGC	764
AY171282	735	____-..............745
EU122 951	838	____-...........T________857
AY17128 0	727	____-..................739
Figure 1 : Multiple sequence alignment of the ITS rDNA region (including partial fragments of the 18S and 28S rRNA genes) of 4 Steinernema species. Code 9833 correspond to the Slovenian isolate of Steinernema carpocapsae (C101). Codes AY171282 and EU122951 are Steinernema carpocapsae strains from Russia and Iran, respectively. Code AY171280 correspond to Steinernema tami strain from Vietnam.
4 DISCUSSION
Genetic studies proved that the nematode species is Steinernema carpocapsae (Weiser, 1955) (Fig. 1). The ITS1-5.8S-ITS2 region, including the partial 18S and 28S rRNA genes (flanked by above primers) of Slovenian isolate C101 is 746 bp long. BLAST searches (Altschul et al, 1990) in GenBank showed that Slovenian isolate C101 (Fig. 2) has a high similarity (99 %) with those sequences available for S. carpocapsae populations (e.g. accession numbers AY171282 and EU122951). Sequence of other species from carpocapsae group, namely S. tami was obtained from GenBank searches that exhibited a lesser degree of similarity with the Slovenian isolate and other S. carpocapsae populations (e.g. accesion number AY171280) (Fig. 1). The present study constitutes the first report of S. carpocapsae in Slovenia. S. carpocapsae has a wide distribution in temperate regions, being one of the most common species found in Europe (overall in 15 countries), and in many other parts of the world (for a detailed EPN species distribution see Hominick, 2002).
We can place mentioned species into »carpocapsae group« of nematodes from genus Steinernema (Nguyen, 2006); for infective juveniles it is known that they are less than 600 ^m long (Fig. 2). This nematode lives in symbiosis with bacterium Xenorhabdus nematophila (Akhurst, 1980). The nematode was first recorded in
1955. Steinernema carpocapsae is the most studied, available, and versatile of all entomopathogenic nematodes (Gaugler, 2002).
Figure 2: Entomopathogenic nematode Steinernema carpocapsae (strain C101).
Important attributes include ease of mass production and ability to formulate in a partially desiccated state that provides several months of room-temperature shelf-life (Gaugler et al., 2002). Particularly effective against lepidopterous larvae, including various webworms, cutworms, armyworms, girdlers, and wood-borers (Georgis et al., 1991). This species is a classic sit-and-wait or "ambush" forager, standing on its tail in an upright position near the soil surface and attaching to passing hosts (Campbell et al., 2003). Consequently, S. carpocapsae tends to be most effective when applied against highly mobile surface-adapted insects. Highly responsive to carbon dioxide once a host has been contacted, the spiracles are a key portal of host entry. It is most effective at temperatures ranging from 22 to 28 °C (Georgis et al, 1991).
In Europe, the occurrence of S. carpocapsae was up to now confirmed in Austria, Bulgaria, Czech Republic, France, Germany, Great Britain, Italy, Norway, Poland, Portugal, Slovakia, Spain, Sweden and Switzerland (Hominick, 2002). Among entomopathogenic nematodes only Steinernema feltiae has a status of indigenous species for the time being (Laznik et al., 2008; MAFF, 2008). While in Slovenia, the effectiveness of this species was already tested in the field experiment (Laznik, 2008, unpubl.), this was yet not the case for S. carpocapsae. When also the latter species will shift from exotic agents list, we will test its activity against the pest insects in the open too. The most intensive experiments will be done against these insect pests against which we do not have registered insecticides, their number is limited, and specially against the insects, which already developed the resistance to insecticides.
5 ACKNOWLEDGEMENTS
This work is a part of program Horticulture No P4-0013-0481 granted by Slovenian Ministry of Higher Education, Science and Technology.
6 REFERENCES
Akhurst, R. J. 1980. Morphological and functional dimorphism inXenorhabdus spp., bacteria symbiotically associated with the insect pathogenic nematodes Neoaplectana and Heterorhabditis. J. General Microbiol. 121: 303-309.
Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. 1990. Basic local alignment search tool. J. Mol. Biol. 215: 403-410.
Bedding R. A., Akhurst R. J. 1975. Simple technique for the detection of insect parasitic rhabditid nematodes in soil. Nematologica 21: 109-110.
Boemare, N. E., Laumond, C., Luciani, J. 1982. Mise en evidence d'une toxicogenese provoquee par le nematode entomophage Neoplectana carpocapsae Weiser chez l'insecte Galleria mellonella L. Compets Rendus des séances de l'Academie des Scences, Paris, Ser. III. 295: 543-546.
Bovien, P. 1937. Some type of association between nematodes and insects. Videnk. Meddr. Naturh. Foren. 101: 1-114.
Campbell, J. F., Lewis, E. E., Stock, S. P., Nadler, S., Kaya, H. K. 2003. Evolution of host search strategies in entomopathogenic nematodes (Nematoda: Steinernematidae). J. Nematol. 35: 142-145.
Ebssa, L. 2005. Efficacy of entomopathogenic nematodes for the control of the western
flower thrips Frankliniella occidentalis. Ph. D. Thesis, Hannover University: 141 pp.
Forst, S., Dowds, B., Boemare, N. E., Stackebrandt, E. 1997. Xenorhabdus spp. and Photorhabdus spp.: bugs that kill bugs. Annu. Rev. Microbiol. 51, 47-72.
Gaugler R. 2002. Entomopathogenic Nematology. New Jersey, CABI Publishing: 372 str.
Gaugler R., Kaya H. K. 1990. Entomopathogenic Nematodes in Biological Control. Florida, Boca Raton, CRC Press: 365 pp.
Georgis, R., Kaya, H., Gaugler, R. 1991. Effect of steinernematid and heterorhabditid nematodes on nontarget arthropods. Environ. Entomol. 20: 815-22.
Hominick W. M. 2002. Biogeography. In: Gaugler R (ed.) Entomopathogenic Nematology. CABI Publishing, Wallingford: 115-143.
Hominick W. M., Briscoe B. R., del Pino F. G., Heng J., Hunt D. J., Kozodoy E., Mracek Z., Nguyen K. B., Reid A. P., Spiridonov S., Stock P., Sturhan D., Waturu C., Yoshida M. 1997. Biosystematics of entomopathogenic nematodes: current status, protocols and definitions. J. Helminthol. 71: 271-298.
Kaya K.H., Koppenhöfer A.M. 1999. Biology and ecology of insectidal nematodes. In: Optimal use of insecticidal nematodes in pest management. Poravarapu S. (ed.). New Jersey, Bluberry Cranberry Research and Extension Center: 1-8.
Kaya, H. K., Gaugler, R. 1993. Entomopathogenic nematodes. Annu. Rev. Entomol. 38: 181206.
Klein, M. G. 1990. Efficacy against soil-inhabiting insect pests. Entomopathogenic Nematodes in Biological Control (Gaugler, R., Kaya, H. K., eds.). CRC Press, Boca Raton, FL, pp. 195-214.
Laznik, Ž., Tóth, T., Lakatos, T., Trdan, S. 2008. Entomopathogenic nematode Steinernema feltiae (Filipjev) (Rhabditida: Steinernematidae) recorded for the first time in Slovenia. Acta Agric. Slov., 91: 37-45.
Laznik, Ž., Trdan, S. 2007. After the first record of entomopathogenic nematodes in Slovenia.
Lect. pap. present. 8th Slov. Conf. Plant Prot. Radenci, March 6-7 2007. Ljubljana, Plant Prot. Soc. Slov.: 99-106 [Slovenian].
Laznik, Ž., Trdan, S. 2008. Entomopatogene ogorčice, naravni sovražniki nadzemskih škodljivcev kapusnic. Acta agriculturae Slovenica, 91: 227-237.
Ministry of Agriculture, Food, and Forestry of Republic Slovenia [MAFF]. 2008: Decision on the change of the status of the exotic agent for biological control (no. 34309/2008/5): 2 p. [Slovenian]
Nguyen, K. B. 2006. Entomopathogenic Nematodes. Entomology and Nematology Department (31 Jan 2006) http://kbn.ifas.ufl.edu/kbnstein.htm (21.11.2007)
Spiridonov, S. E., Reid, A. P., Podrucka, K., Subbotin, S. A., Moens, M. 2004. Phylogenetic relationship within the genus Steinernema (Nematoda: Rhabditida) as inferred from analyses of sequences of ITS1-5.8S-ITS2 region of the rDNA and morphological features. Nematology 6: 547-566.
Weiser, J. 1955. Neoaplectana carpocapsae n. sp. (Anguillulata, Steinernematidae) novy cizopasnic housenik obalece jablecneho, Carpocapsa pomonella L. Vestnik Cesk. Zool. Spolecnosti 19: 44-52.