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Selection of a Bacterium for the Mass Production of Phasmarhabditis Hermaphrodita (Nematoda : Rhabditidae) As a Biocontrol Agent for Slugs

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Selection of a Bacterium for the Mass Production of Phasmarhabditis Hermaphrodita (Nematoda : Rhabditidae) As a Biocontrol Agent for Slugs Fundam. appl. NemalOl., 1995,18 (5),419-425 Selection of a bacterium for the mass production of Phasmarhabditis hermaphrodita (Nematoda : Rhabditidae) as a biocontrol agent for slugs Michael J. WILSON *, David M. GLEN *, Susan K GEORGE * and Jeremy D. PEARCE ** * Department ofAgricullural Sciences, University ofBristol, !nstitute ofArable Crops Research, Long Ashton Research Station, Bristol BSIB 9AF, u.K. and ** MiCToBio Ltd., Church Street, Thriplow, Royston, Herts. SGB 7RE, u.K. Accepted for publication 16 September 1994. Summary - Dauer larvae of the slug-parasitic nematode, Phasmarhabditis hermaphrodir.a, were found to carry a mean of ca . 70 bacterial colony forming units with up to five different bacterial types present in individual nematodes. Nine bacterial isolates found associated with P. hermaphrodir.a, were injected into the body cavity of the slug, DerOGeras reliculalum. Only two isolates (Aeromonas hydrophila and Pseudomonasfluorescens, isolate 140), were found to be pathogenic. Phasmarhabditis hermaphrodila grown in monoxenic culture with five bacterial isolates (Moraxella osloensis, Providencia rellgeri, Serralia proteamaculans, P. fluorescens, isolate 140 and P. fluorescens, isolate 141) which supported strong growth were bioassayed against D. reliculalum. Nematodes grown with M. osloensis or P. fluorescens (isolate 141) were pathogenic, those grown with P. reugeri gave inconsistent results and those grown with the other two bacteria were non-pathogenic. These results demonstrate that differences in pathogenicity between nematode/ bacterium combinations did not result from differences in bacteriaJ pathogenicity a1one. M. osloensis was selected as the bacterium for rearing P. hermaphrodita as a biocontrol agent for slugs. Résumé - Sélection d'une bactérie pour la production en masse ck Phasmarhabditis hennaphrodita (Nematoda : Rhabditidae), agent de contrôle biologique cks limaces -Il a été observé que le nématode parasite des limaces Phasmarhab­ ditis hermaphrodita était porteur d'environ 70 « unités formant une colonie » (CFU) appartenant jusqu'à cinq types différents de bactéries par individu. Neuf isolats bactériens associés à P. hermaphrodir.a sont injectés dans la cavité du corps de la limace Deroceras reliculalUm. Seuls deux isolats - Aeromonas hydrophila et Pseudomonas fluorescens isolat 140 - se sont révélés pathogènes. Phasma­ rhabditis hermaphrodir.a en élevage monoxénique avec cinq isolats bactériens (Moraxella osloensis, Providencia reugeri, Serralia proteomaculans, P. fluoresens isolat 140 et P. fluorescens isolat 141) permettant un taux élevé de croissance a été testé envers D. reliculalUm. Les nématodes élevés avec M. osloensis et P. fluorescens isolat 141 se sont montrés pathogènes, ceux élevés avec P. rellgeli donnent des résultats inconstants et ceux élevés avec les autres bactéries ne sont pas pathogènes. Ces résultats prouvent que les différences dans la nocuité des combinaisons nématode/bactérie ne provielU1ent pas seulement de la nocuité de la bactérie. M. osloensis a été sélectiolU1é en vue de l'élevage de P. hermaphrodir.a comme agent de contrôle biologique envers les limaces. Key-words : Phasmarhabditis hermaphrodir.a, rhabditid nematodes, bacteria, parasite, slug, Deroceras reliculalUm, monoxeny, pa­ thogenicity, biologicaJ control. The rhabditid nematade, Phasmarhabdùis herma­ & Poinar, 1979), respectively, which are carried monox­ phrodita (Schneider), is a parasite capable of killing sev­ enically in the anterior portion of the nematode gut. eral species of pest slugs and has the potential to be used These murualistic bacteria are thought to play an impor­ as a biocontrol agent (Wilson el al., 1993 a). It is a tant raIe in the pathogeniciry of the nematode/bacterium bacterial feeder and has been mass-produced in xenic complex and when these nematades are mass-produced culrure on nutrient media supporting a mixed bacterial for commercial use, they are grown in monoxenic cul­ flora (Wilson el al., 1993 b) using similar techniques to rure with their preferred species of Xenorhabdus or Pho­ those developed to mass-praduce entomopathogenic nematodes of the families Heterarhabditidae and Stei­ lorhabdus. This generally resuJts in consistent and pre­ nernematidae, which have been used successfully ta dictable yields of reliably virulent nematodes. These control several insect pest species. bacteria, however, can spontaneously alternate between Heterorhabditid and Steinernematid nematodes are two phases, and phase two provides a Jess suitable envi­ symbiotically associated with bacteria of the genera Pho­ ronment for nematode growth than phase one (Akhurst lorhabdus (Boemare, 1993) and Xenorhabdus (Thomas & Boemare, 1990). ISSN 1164-5571/95/05 S 4.00/ © Gauthier-Villars - ORSTOM 419 M. J. Wilson et al. There are many ways in which nematodes can inter­ To obtain monoxenic cultures of P. hennaphrodita, act with bacteria (Poinar & Hansen, 1986)", but Iittle is xenically-cultured individuais were first freed ofbacteria known about such interactions for P. hennaphrodita. then grown with the selected bacterium on a kidney­ Wilson et al. (1995) found that many species of bacteria based medium on 3 cm diameter Petri dishes, as de­ supported growth ofP. hennaphrodita in monoxenic cul­ scribed by Wilson et al. (1995 a). Gravid adult P. her­ tures, but that different bacterial isolates caused differ­ maphrodita were transferred, through n'lO changes of ences in the total numbers of nematodes produced in sterile water, to a sterile watch glass containing water culture and influenced the proportion of the population with 0.02 % sodium ethyl mercurithiosalicylate (" Thi­ which formed dauer larvae after three weeks. As dauer merosal ", Sigma) where they were left for 16 h at 10°C. larvae are the stage of P. hemzaphrodita which infect Nter this period, the larvae (L11L2) produced by the slugs, this has important implications for production of aduJts were transferred to centrifuge tubes containing P. hennaphrodita as a biocontrol agent. Wilson et al. la rrù quarter strength Ringer's solution (Poinar & Tho­ (1995) did not investigate differences in the quality ofP. mas, 1984) with 500 units ml -1 ofboth benzyl penicillin hennaphrodita grown in monoxenic culture with differ­ and streptomycin sulphate (Sigma). The larvae were ent bacteria, in particular, any effect that the bacterial kept in this solution at la oC for 24 h then concentrated food-source might have on the ability of dauer larvae to by centrifugation at 90 g for la min, re-suspended in infect and kill slugs. sterile quarter strength Ringer's solution and centri­ The investigations described herein into the relation­ fuged again. Re-suspension and centrifugation were re­ ships between P. hennaphrodita and bacteria had two peated once more to remove any traces of antibiotics, related aims. First to determine the direct effects of bac­ then sterile (axenic) larvae were transferred to a sterile teria associated with P. hermaphrodita on the field slug, watch glass, where they could be handled individually Deroeeras retieulatum (Müller). And, second, to deter­ using sterile micro-pipettes. For larger scale production mine whether the pathogenicity of P. hennaphrodita to of monoxenic nematodes for use in bioassays, mono­ D. retieulatum is influenced by growth with different xenic P. hennaphrodita from Petri dish-cultures were bacteria. Bacteria were isolated from dauer larvae of P. introduced into 250 ml flasks by pippeting 1 ml of sterile hennaphrodita, from xenic cultures of P. hennaphrodita broth over the surface of the agar, then re-pippeting the which were producing rughly pathogenic nematodes liquid containing a suspension of bacteria and 50 000­ and from living or dead individuals of D. reticulatum 100 000 nematodes into each flask, containing either a infested by P. hennaphrodita. The effects of nine isolates solid or liquid medium. For solid phase cultures, flasks on slugs were tested by injecting cultures of bacteria were filled with foam chips impregnated with kidney directly into the haemocoel of D. retieulatum. Five of medium (Wilson et al., 1993 b) and incubated at 15 oC these bacteria which had been found to support good for 3 weeks; dauer larvae were then harvested by placing monoxenic growth of P. hennaphrodita (Wilson et al., the foam on sieves in water (Wilson et al., 1993 b). For 1995) were used to grow monoxenic cultures of P. her­ liquid cultures, nematodes were introduced into 250 rrù maphrodita wruch were then bioassayed for their effects flasks containing 50 rrù of liquid kidney medium (KYO) on D. retieulatum. (Wilson et al., 1995) which were incubated at 15 oC in a gyrotatory flask shakerlincubator at 200 rpm for 3 weeks. Dauer larvae were harvested by aUowing the Materials and methods nematodes to settle out in tap water. COLLECTION AND MAINTENAt"lCE OF SLUGS ISOLATION AND MAINTENANCE OF BACTERlAL CUL­ Slugs (Deroceras retieulatum) were collected from TURES traps consisting of inverted plastic flowerpot saucers Bacteria were isolated from within dauer larvae of P. baited with bran in rough grassland at Long Ashton hennaphrodita, from living and dead D. retieulatum in­ Research Station (LARS). They were kept at 10°C with fected with P. hermaphrodita, and from xenic foam chip a 12 h night and day cycle in non-airtight plastic boxes cultures of P. hennaphrodita, as described below. lined with moistened absorbent cotton wool. Dauer larvae were first surface-sterilised by immer­ sion in 0.1 % (w/v) sodium ethyl mercurithiosalicylate
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