The use of predatory nematodes as biological control agents of phytoparasitic nematodes has been re-evaluated only recently. To be effective, predatory nematodes should be easy to grow, cheap, and available on a commercial scale. In addition, they must show a reproductive rate sufficient to sustain a high population density and a significant longevity and stability for storage. The predators are not phytopathogenic nematodes, and among the positive outcomes of their introduction are remarkable their environment compatibility and safety for other non-target organisms, as well as the ability to search for prey. These features are present in Diplogasteridae. Their adaptability enables them to withstand changing climatic conditions, as well as the temporal variability between predator and prey. It should also be emphasized the ability to disperse, persist and reproduce in the absence of prey, the spectrum of action, all factors ideal for the management of phytoparasitic species. Adverse factor is cannibalism, due the lack of preys, that can reduce their biological control potential. We herein present preliminary data on occurrence, reproduction and intestine bacterial flora of the predatory nematode, Koerneria sudhausi

Colagiero, Mariantonietta

Murga-Gutierrez, Santos Nelida

Rosso, Laura Cristina

CREA Journals (Consiglio per la ricerca in agricoltura e l’analisi dell’economia agraria)

REDIA, XCIV, 2011: 133-135(*) CNR, Istituto per la Protezione delle Piante, Via Amendola 122/D, 70126 Bari, Italy; e.mail: a.ciancio@ba.ipp.cnr.it(**) Universidad Nacional de Trujillo, Departamento de Microbiología y Parasitología, Trujillo, PeruColagiero M., Rosso L.C., Ciancio A., Murga-Gutierrez S.N.  – Observations on the biology of a predatory nematodebelonging to Diplogasteridae. The use of predatory nematodes as biological control agents of phytoparasitic nematodes has been re-evaluatedonly recently. To be effective, predatory nematodes should be easy to grow, cheap, and available on a commercial scale.In addition, they must show a reproductive rate sufficient to sustain a high population density and a significant longevityand stability for storage. The predators are not phytopathogenic nematodes, and among the positive outcomes of theirintroduction are remarkable their environment compatibility and safety for other non-target organisms, as well as theability to search for prey. These features are present in Diplogasteridae. Their adaptability enables them to withstandchanging climatic conditions, as well as the temporal variability between predator and prey. It should also beemphasized the ability to disperse, persist and reproduce in the absence of prey, the spectrum of action, all factors idealfor the management of phytoparasitic species. Adverse factor is cannibalism, due the lack of preys, that can reduce theirbiological control potential. We herein present preliminary data on occurrence, reproduction and intestine bacterialflora of the predatory nematode, Koerneria sudhausi.KEY WORDS: nematodes, predation, biocontrol.MARIANTONIETTA COLAGIERO (*) - LAURA CRISTINA ROSSO (*) - AURELIO CIANCIO (*)SANTOS NELIDA MURGA-GUTIERREZ (**)OBSERVATIONS ON THE BIOLOGY OF A PREDATORY NEMATODEBELONGING TO DIPLOGASTERIDAEINTRODUCTIONThe damages caused by phytoparasitic nematodes areconsiderable, and in mediterranean environments manyvegetable, pulse and fruit productions are under pressuredue to their attacks. In recent years the expansion oforganic croppings promoted the development of newagricultural inputs, including products and protocolsbased on biological control agents as possible alternativesto pesticides.The use of nematodes as biological control agents ofother phytoparasitic nematodes dates back severaldecades, since first proposal by COBB (1917), but onlyrecently it was re-evaluated by studies on the role of anumber of predators (BILGRAMI, 2009). To act as effectivebiological control agents, predators must meet a numberof specific requirements including ease of cultivation andlow cost commercial production, together with a highreproduction rate allowing high population densities.Furthermore, they must have significant longevity andstability, in order to be preserved. To achieve success intheir use also important are their safety and compatibilitywith other non-target soil organisms.The ability to seek prey is an important factor that apredator should also possess, and some groups, i.e.,Diplogasteridae, can efficiently perform this task (BIL -GRAMI & JAIRAJPURI, 1988). Diplogastreidae are bacterialfeeders which can also prey on nematodes. Due to theirbiology they can be multiplied in vitro, feeding onbacterial cells (BILGRAMI, 2009).In this study, specimens of a population of Koerneriasudhausi, a member of the Diplogasteridae (VON LIEVEN,2008), were studied in detail. This species is known to act asan efficient predator of root-knot nematodes (BAR-EYAL etal., 2008). Preliminary data on its occurrence, reproductionand intestine bacterial flora, useful for multiplication incontrolled conditions, are herein provided.MATERIALS AND METHODSFor collections, standard nematological techniques wereused. Soil and root samples were collected from farmsspecialized in Asparagus productions at Virú district (LaLibertad, Peru). A few g of soil were collected on filterpaper and inoculated on 1% water agar (WA) in Petriplates, to allow multiplication of bacteria and of bacteriafeeding nematodes. The free living and predatory specieswere identified in temporary water mounts with a LeitzOrthoplan light microscope at 200-400×. Eggs or adultswere hand-picked and placed on 1% water agar andstored at 26°C, for subsequent observations.Studies on the biology of K. sudhausi were carried out invitro. Since the diplogasterid can also feed on bacteria, westudied the intestine bacterial flora. For identification ofthe bacterial species from the intestine of K. sudhausi,nematodes were picked up from the WA plates andsurface sterilized by immersion for 3 min in 0.5% NaOCl,followed by incubation with antibiotic solution (0.1%ampicillin, streptomycin and chloramphenicol) for 5 min.The nematodes were later rinsed twice in sterile distilledwater (SDW), followed by the rupture of their bodies in10 µl of SDW on a glass slide, with a sterile blade. Thebacterial cells thus released were smeared on Luria-Bertani(LB) agar medium with a sterile loop. Subsequently, theplates were incubated at 26°C for 48 hours, and thebacterial colonies were isolated in pure cultures on LBmedium.– Received 25 July 2011     Accepted 5 September 2011     Published 1 December 2011For DNA extraction, the bacterial cells were recoveredfrom the plates with a sterile spatula. The cells weresuspended in 350 µl of STET buffer (8% sucrose; 20 mMTris HCl pH 8; 50 mM EDTA; 0.5% Triton.) and 300 µgof lisozyme. The cells were then crushed by incubation at95°C for 40 sec and immediately cooled on ice. Anamount of 175 µl of phenol and 175 µl chloroform/iso -propanol (24:1) was added to the suspension, vortexedand centrifuged at 12000 rpm for 10 min. DNA wasprecipitated from the aqueous layer by adding 1/10 v/v of5M ammonium acetate and 2 volume of cold ethanol.After centrifugation, pellets were washed with 70%ethanol, dried under vacuum, and re-suspended in 50 µlsterile-distilled water.For species identification, PCR amplification of the 16SrDNA regions was performed using the primers 27F and1492R (LANE et al., 1985), on a thermocycler (iCycler,Biorad) with the following cycles: a first cycle at 95°C for 7min; 35 denaturation cycles (30 sec at 94°C), annealing (30sec at 50°C) and extension (1 min at 72°C), and a singlefinal extension cycle at 72°C for 7 min. Amplifiedproducts were examined on 1.5% agarose gels, purifiedusing a commercial GenElute PCR Clean- up kit(SIGMA) and directly sequenced through an availablecommercial service (Eurofins MWG Synthesis GmbH,Germany). The sequences produced were then assigned totheir corresponding species by means of BLAST analyses(ALTSCHUL et al., 1997) in the sequence databases availableat NCBI (http://www.ncbi.nlm.nih.gov/).RESULTS AND DISCUSSIONAdults of K. sudhausi were observed on WA petri plates.The nematodes were identified according to the originalspecies description and were multiplied on WA with theiroriginal bacterial microflora for several weeks. Adultfemales produced 6-8 eggs per day on WA, at 26°C. Theeggs irregular external formations and swellings,characteristic of this species, persisted during the develo -pment of the embryo until hatching, and were graduallycovered by a dense layer of bacterial cells (fig. I). Althoughthe function of the surface eggs formations is not known,freshly hatched juveniles were often observed movingaround the eggs and touching the shell irregularities withtheir lips, during feeding on bacteria. One generation wascompleted within 4-7 days, at 26°C.A total of 7 bacterial culture isolates were obtained fromthe K. sudhausi intestine content. The PCR amplificationof the 16S gene fragments yielded several amplicons of theexpected range size. BLAST analyses showed a 99%homology with the following species: Pseudomonas putida(fragment length: 780 bp, accession n. AM942549),Pigmentiphaga kullae (860 bp, NR_025112), Acidovoraxavenae (954 bp, AY512827), Bosea thioxidans (804 bp,FJ581444), Xenophilus sp. (950 bp, GQ246689) andBradyrhizobium sp. (900 bp, DQ520809).Predatory nematodes like K. sudhausi show a potentialin biocontrol, as their biology allows the development anduse as possible, innovative bio-nematocides. Theirmultiplication in vitro on bacteria allows production inlarge numbers, facilitating applications as massive inoculathat may have a significant impact on phytoparasiticnematodes (YEATES, 1969). Diplogasteridae showselectivity in predation (CHITAMBAR & NOFFSINGER, 1989).A prey preference, including endoparasitic nematodes,was reported for Mononchoides fortidens, M. longicaudatusand M. gaugleri (BILGRAMI & JAIRAJPURI, 1988; 1989;BILGRAMI et al., 2005). Studies on predation also showed achemoattraction towards prey (BILGRAMI, 2009). Theseproperties of Diplogasteridae are useful in biologicalcontrol, increasing their regulation efficiency. Treatmentswith Diplogasteridae reduced densities of the root-knotnematodes Meloidogyne javanica and M. arenaria or thecitrus nematode Tylenchulus semipenetrans in pots(BILGRAMI, 2009). Assays carried out in controlledconditions with K. sudhausi confirmed the assumptionsabout its potentials, and showed promising results againstM. javanica (BAR-EYAL et al., 2008).As candidate biological control agents of phytoparasitic134 M. COLAGIERO ET AL. REDIA, Vol. XCIV, 2011Figure I – Developmental stages of Koerneria sudhausi eggs on WA, showing the typical surface irregularswellings and the increasing density of surrounding bacterial cells, from early (1, 2) to the final pre-hatchingstages (3, 4).1 23 4nematodes, the adaptability of Diplogasteridae allows themto tolerate changing climatic conditions, as well as todisplay a stable, temporal compatibility with preys(BILGRAMI, 2009). The capacities to disperse, persist, sur -vive and reproduce, even in absence of prey (GROOTAERT etal., 1977), and the presence of a broad spectrum of action,are ideal traits for the biological management ofphytoparasitic nematodes with such predators. The onlydrawbacks are represented by the occurrence of pathogensor parasites, as well as by hyperparasitism that, in absenceof prey can lead to cannibalism.The possibility to introduce useful bacteria (i.e. Brady -rhi zobium), represents a favourable trait of K. sudhausi andneeds to be further checked also for other Diplogasteridaespecies. The range of Diplogasteridae interactions withbacteria appears wide, as i.e. Pristionchus pacificus showeddissemination, reduction in brood size, chemoattraction toparticular species and also avoidance and mortality. Incontrolled assays this species avoided Serratia marcescens,Bacillus thuringiensis and other insect pathogenic bacteria,because of their pathogenicity (RAE et al., 2008).For practical applications, i.e. introduction in soil orinundative treatments, parameters on conservation,transport, formulation as well as doses of application ofDiplogasteridae appear crucial. Practical beneficial poten -tialities for exploitation of predatory nematodes alsoinclude the absence of a registration procedure and theirvery low environmental impact. Finally, further benefits ofa management  approach based on Diplogasteridaeinclude the possibility to maintain and apply the inocula insitu, in simple organic compost or production systems, aswell as the possibility of inundative applications, in aclassical biocontrol approach.ACKNOWLEDGEMENTSResearch partially funded by Bilateral AgreementCNR/CONCYTEC and MIPAF, Project BIOMED. MrNicola Salatino is gratefully acknowledged for thetechnical assistance provided during this study. RIASSUNTOOSSERVAZIONI SULLA BIOLOGIADI UN NEMATODE PREDATOREAPPERTENENTE AI DIPLOGASTERIDAEL’impiego di nematodi predatori come agenti di controllobiologico contro nematodi fitoparassiti è stato rivalutato solorecentemente. Per essere efficaci, i nematodi predatori devonoessere facili da allevare e a buon mercato per essere disponi-bili su scala commerciale. Inoltre, essi devono mostrare untasso di riproduzione sufficiente a sostenere un’elevata den-sità di popolazione e una notevole longevità e stabilità per laloro conservazione. I predatori non sono nematodi fitopa-togeni e, tra i risultati positivi della loro introduzione, sono lacompatibilità ambientale e la sicurezza per gli altri organisminon bersaglio così come la capacità di ricerca delle prede.Queste caratteristiche sono presenti nei Diplogasteridae. Lacapacità di adattamento consente loro di resistere a condi-zioni climatiche variabili così come alla variabilità tempo-rale tra predatore e preda. Si sottolineano anche la capacitàdi disperdersi, persistere e riprodursi in assenza di prede, lospettro d’azione, fattori ideali per la gestione delle specie difitoparassiti. Fattore negativo è il cannibalismo, a causa dellamancanza di prede, che può ridurre il potenziale biologico dicontrollo. Vengono presentati dati preliminari sulla presenza,riproduzione e flora batterica dell’intestino di un nematodepredatore, Koerneria sudhausi.REFERENCESALTSCHUL S.F., MADDEN T.L., SCHAFFER A.A., ZHANG J.,ZHANG Z., MILLER W., LIPMAN D.J., 1997 – GappedBLAST and PSIBLAST: a new generation of proteindatabase search programs. - Nuc. Ac. Res., 25: 3389-3402.BAR-EYAL M., SHARON E., SPIEGEL Y., OKA Y., 2008 –Laboratory studies on the biocontrol potential of thepredatory nematode Koerneria sudhausi (Nematoda:Diplogastridae). - Nematology, 10: 633-637.BILGRAMI A.L., GAUGLER R., BREY C., 2005 – Preypreference and feeding behaviour of the diplogastridpredator Mononchoides gaugleri (Nematoda:Diplogastrida). - Nematology, 7: 333-342.BILGRAMI A. L., 2009 – Biological control potentials ofpredatory nematodes. In: A. Ciancio & K. G. Mukerji(eds.), Integrated management and biocontrol ofvegetable and grain crops nematodes; Springer,Dordrecht, NL, pp. 3-28.BILGRAMI A.L., JAIRAJPURI M.S., l988 – Attraction ofMononchoides longicaudatus and M. fortidens(Nematoda: Diplogasteridae) towards prey and factorsinfluencing attraction.- Rev. Nematol., 11: 195-202.BILGRAMI A.L., JAIRAJPURI M.S., 1989 – Resistance of preyto predation and strike rate of the predatorsMononchoides longicaudatus and M. fortidens(Nematoda: Diplogasteridae).- Rev. Nematol., 12: 45-49.BILGRAMI A.L., GAUGLER R., BREY C., 2005 – Preypreference and feeding behaviour of the diplogastridpredator Mononchoides gaugleri (Nematoda:Diplogastridae). - Nematology 7: 333-342.CHITAMBAR J.J., NOFFSINGER M., 1989 – Predaceousbehaviour and life history of Odontopharynxlongicaudata (Diplogasteridae). - J. Nematol., 21: 284-291.COBB N., 1917 – The Mononchus: a genus of free livingpredatory nematodes.- Soil Sc., 3: 431-86.GROOTAERT P., JAQUES A., SMALL R.W., 1977 – Preyselection in Butlerius sp. (Rhabditida: Diplogasteridae).-Med. van de Fac. Landbouw. Rijksuniv. Gent, 42: 1559-1563.LANE D.J., PACE B., OLSEN G.J., STAHL D.A., SOGIN M.L,PACE N.R., 1985 – Rapid determination of 16S ribosomalRNA sequences for phylogenetic analyses.- Proc. Natl.Acad. Sci. USA, 82: 6955–6959.RAE R., RIEBESELL M., DINKELACKER I., WANG Q.,HERRMANN M., WELLER A. M., DIETERICH C., SOMMERR. J., 2008 – Isolation of naturally associated bacteria ofnecromenic Pristionchus nematodes and fitnessconsequences.- Journal of Experimental Biology, 211:1927-1936.VON LIEVEN, A.F., 2008 – Koerneria sudhausi n. sp.(Nematoda: Diplogastridae) a hermaphroditic diplogastridwith an egg shell formed by zygote and uterinecomponents. - Nematology, 10: 27-45.YEATES G.W., 1969 – Predation by Mononchoidespotohikus in laboratory culture.- Nematologica, 15: 1-9.OBSERVATIONS ON THE BIOLOGY OF A PREDATORY NEMATODE BELONGING TO DIPLOGASTERIDAE 135

Observations on the biology of a predatory nematode belonging to Diplogasteridae

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Observations on the biology of a predatory nematode belonging to Diplogasteridae

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