Heterorhabditis bacteriophora Poinar, 1976 (Rhabditida: Heterorhabditidae) HBH hibrit ırkının in vitro katı kültürde üretiminin optimizasyonu

Entomopatojen nematodlar (EPN), toprakta yaşayan biyolojik mücadele ajanlarıdır. EPN'ler yaşam döngüleri boyunca bir konukçu böceğe ihtiyaç duyarlar ve gelişimleri sırasında konukçusunu öldürürler. EPN'lerin en büyük dezavantajı, ticari ürünlerin yüksek maliyetidir. Bu nedenle kitle üretimin optimizasyonu ile üretim maliyetlerinin düşürülmesine odaklanan birçok çalışma bulunmaktadır. Daha önceki bir proje kapsamında iki yerel izolattan Heterorhabditis bacteriophora Poinar, 1976 (Rhabditida: Heterorhabditidae) HBH hibrit ırkı geliştirilmiştir. Bu hibrit ırk, üstün biyo-ekolojik özelliklerinden dolayı patentlidir. Bu çalışmada, hibrit HBH ırkının in vitro katı kültürde kitle üretiminin optimize edilmesi amaçlanmıştır. Tüm laboratuvar denemeleri 2017-2018 yılları arasında Bursa Uludağ Üniversitesi, Ziraat Fakültesi, Bitki Koruma Bölümü, Nematoloji Laboratuvarı'nda gerçekleştirilmiştir. Optimizasyonda standart ortama ek katkı maddeleri (soya lesitini ve yumurta sarısı), sıcaklık (24, 28 ve 32°C) ve ortam pH'ı (5, 7 ve 9) üretim parametreleri olarak seçilmiştir. Optimizasyon, hermafrodit yumurta sayıları, toplam infektif jüvenil (IJ) sayısı, IJ vücut uzunluğu ve IJ etkinliği ile değerlendirilmiştir. Sonuçlara göre, en iyi üretim kombinasyonu, soya lesitini içeren agar, 28°C ve pH 7 olarak bulunmuştur. Ayrıca pH 9'lu agarlar, üretim verimini önemli ölçüde azaltmıştır. Sonuç olarak, HBH hibrit ırkının bazı önemli in vitro katı üretim parametreleri için optimum değerler belirlenmiştir.

Anahtar Kelimeler:

Heterorhabditis bacteriophora, kitle üretimi, monoksenik kültür, optimizasyon

Optimization of in vitro solid culture of Heterorhabditis bacteriophora Poinar, 1976 (Rhabditida: Heterorhabditidae) HBH hybrid strain

Entomopathogenic nematodes are soil-dwelling biocontrol agents. EPNs need an insect host to complete their life cycle, and they kill their host during its development. The major disadvantage of EPNs is the high cost of commercial products. Thus, there are many studies focused on reducing production costs by optimization of mass production. In a previous project, Heterorhabditis bacteriophora Poinar, 1976 (Rhabditida: Heterorhabditidae) HBH hybrid strain was developed from local isolates. This hybrid strain was patented due to superior bioecological characteristics. This study aimed to optimize in vitro solid mass production of hybrid strain. All laboratory trials were performed between 2017 and 2018, in Nematology Laboratory of Bursa Uludağ University, Faculty of Agriculture, Department of Plant Protection. For optimization, additional supplementary ingredients (soy lecithin and egg yolk), temperature (24, 28 and 32°C) and medium pH (5, 7 and 9) were selected as production parameters. Optimization was evaluated based on hermaphrodite egg numbers, total infective juveniles (IJs), IJ body length and IJ virulence. Based on results, best production combination was found as agar containing soy lecithin, 28°C and pH 7. Also, agar media with pH 9 markedly reduced production yield. Consequently, optimum values for some important in vitro solid production parameters of HBH hybrid strain were determined.

Keywords:

Heterorhabditis bacteriophora, mass production, monoxenic culture, optimization,

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Addis, T., A. Teshome, O. Strauch & R. Ehlers, 2016. Life history trait analysis of the entomopathogenic nematode Steinernema feltiae provides the basis for prediction of dauer juvenile yields in monoxenic liquid culture. Applied Microbiology and Biotechnology, 100 (10): 4357-4366.
Alsaidi, A., J. Valencia, D. Upadhyay, S. Mandjiny, R. Bullard-Dillard, J. Frederick & L. Holmes, 2018. Mass production of the beneficial nematode Steinernema carpocapsae using solid state fermentation. Journal of Advanced Agricultural Technologies, 5 (4): 276-280.
Anbesse, S., N. H. Sumaya, A. V. Dörfler, O. Strauch & R. U. Ehlers, 2013. Stabilization of heat tolerance traits in Heterorhabditis bacteriophora through selective breeding and creation of inbred lines in liquid culture. BioControl, 58 (1): 85-93.
Atwa, A. A., M. M. Shamseldean & F. A. Yonis, 2013. The effect of different pesticides on reproduction of entomopathogenic nematodes. Turkish Journal of Entomology, 37 (4): 493-502.
Blackburn, D., D. Shapiro-Ilan & B. J. Adams, 2016. Biological control and nutrition: Food for thought. Biological Control, 97: 131-138.
Boemare, N., C. Laumond & H. Mauleon, 1996. The entomopathogenic nematode-bacterium complex: Biology, life cycle and vertebrate safety. Biocontrol Science and Technology, 6 (3): 333-346.
Cabral, C. M., A. Cherqui, A. Pereira & N. Simões 2004. Purification and characterization of two distinct metalloproteases secreted by the entomopathogenic bacterium Photorhabdus sp. strain Az29. Applied and Environmental Microbiology, 70 (7): 3831-3838.
Chavarría-Hernández, N., G. Maciel-Vergara, J. C. Chavarría-Hernández & A. I. Rodriguez-Hernández, 2011. Mass production of the entomopathogenic nematode, Steinernema carpocapsae CABA01, through the submerged monoxenic culture in two internal-loop airlift bioreactors with some geometric differences. Biochemical Engineering Journal, 55 (3): 145-153.
Ciche, T. A., K. S. Kim, B. Kaufmann-Daszczuk, K. C. Q. Nguyen & D. H. Hall, 2008. Cell invasion and matricide during Photorhabdus luminescens transmission by Heterorhabditis bacteriophora nematodes. Applied and Environmental Microbiology, 74 (8): 2275-2287.
Clarke, D. J., 2008. Photorhabdus: a model for the analysis of pathogenicity and mutualism. Cellular Microbiology, 10 (11): 2159-2167.
Ehlers, R., 2001. Mass production of entomopathogenic nematodes for plant protection. Applied Microbiology and Biotechnology, 56 (5-6): 623-633.
Ehlers, R., I. Niemann, S. Hollmer, O. Strauch, D. Jende, M. Shanmugasundaram, U. Mehta, S. Easwaramoorthy & A. Burnell, 2000. Mass production potential of the bacto-helminthic biocontrol complex Heterorhabditis indica-Photorhabdus luminescens. Biocontrol Science and Technology, 10 (5): 607-616.
El-Sadawy, H. A., 2011. Mass production of Steinernema spp. on in-vitro developed solid medium. World Applied Sciences Journal, 14 (6): 803-813.
Ferreira, T., M. Addison & A. Malan, 2014. In vitro liquid culture of a South African isolate of Heterorhabditis zealandica for the control of insect pests. African Entomology, 22 (1): 80-92.
García del Pino, F. & M. Jové, 2005. Compatibility of entomopathogenic nematodes with fipronil. Journal of Helminthology, 79 (4): 333-337.
Gaugler, R., I. Brown, D., Shapiro-Ilan & A. Atwa, 2002. Automated technology for in vivo mass production of entomopathogenic nematodes. Biological Control, 24 (2): 199-206.
Georgis, R., 1990. “Formulation and Application Technology, 173-191”. In: Entomopathogenic Nematodes in Biological Control (Eds. R. Gaugler & H. K. Kaya). CRC Press, CRC Press, Boca Raton, USA, 365 pp.
Glare, T., J. Caradus, W. Gelernter, T. Jackson, N. Keyhani, J. Köhl, P. Marrone, L. Morin & A. Stewart, 2012. Have biopesticides come of age? Trends in Biotechnology, 30 (5): 250-258.
Grewal, P., 2002. “Formulation and Application Technology, 265-287”. In: Entomopathogenic Nematology (Ed. R. Gaugler). CABI, Wallingford, UK, 357 pp.
Griffin, C. T., M. J. Downes & W. Block, 1990. Tests of Antarctic soils for insect parasitic nematodes. Antarctic Science, 2 (3): 221-222.
Hirao, A. & R. U. Ehlers, 2009. Effect of temperature on the development of Steinernema carpocapsae and Steinernema feltiae (Nematoda: Rhabditida) in liquid culture. Applied Microbiology and Biotechnology, 84 (6): 1061-1067.
Hirao, A. & R. U. Ehlers, 2010. Influence of inoculum density on population dynamics and dauer juvenile yields in liquid culture of biocontrol nematodes Steinernema carpocapsae and S. feltiae (Nematoda: Rhabditida). Applied Microbiology and Biotechnology, 85 (3): 507-515.
Hirao, A., R. U. Ehlers & O. Strauch, 2010. Life cycle and population development of the entomopathogenic nematodes Steinernema carpocapsae and S. feltiae (Nematoda, Rhabditida) in monoxenic liquid culture. Nematology, 12 (2): 201-210.
Hominick, W. M., 2002. “Biogeography, 115-143”. In: Entomopathogenic Nematology (Ed. R. Gaugler). CABI, Wallingford, UK, 357 pp.
Johnigk, S. & R. Ehlers, 1999. Endotokia matricida in hermaphrodites of Heterorhabditis spp. and the effect of the food supply. Nematology, 1 (7-8): 717-726.
Kaya, H. K. & P. Stock, 1997. “Techniques in Insect Nematology, 281-324”. In: Manual of Techniques in Insect Pathology (Ed. A. Lawrence). Academic Press, Elsevier, USA, 409 pp.
Lacey, L. A., D. Grzywacz, D. I. Shapiro-Ilan, R. Frutos, M. Brownbridge & M. S. Goettel, 2015. Insect pathogens as biological control agents: Back to the future. Journal of Invertebrate Pathology, 132: 1-41.
Leite, L. G., D. Shapiro-Ilan, S. Hazir & M. A. Jackson, 2017. Effect of inoculum age and physical parameters on in vitro culture of the entomopathogenic nematode Steinernema feltiae. Journal of Helminthology, 91 (6): 686-695.
Lewis, E. E., R. Gaugler & R. Harrison, 1992. Entomopathogenic nematode host finding: Response to host contact cues by cruise and ambush foragers. Parasitology, 105 (2): 309-315.
Lunau, S., S. Stoessel, A. J. Schmidt-Peisker & R. Ehlers, 1993. Establishment of monoxenic inocula for scaling up in vitro cultures of the entomopathogenic nematodes Steinernema spp. and Heterorhabditis spp. Nematologica, 39 (1-4): 385-399.
Olson, S., 2015. An analysis of the biopesticide market now and where it is going. Outlooks on Pest Management, 26 (5): 203-206.
Peters, A., 1996. The natural host range of Steinernema and Heterorhabditis spp. and their impact on insect populations. Biocontrol Science and Technology, 6 (3): 389-402.
Qiu, L. & R. A. Bedding, 2002. Characteristics of protectant synthesis of infective juveniles of Steinernema carpocapsae and importance of glycerol as a protectant for survival of the nematodes during osmotic dehydration. Comparative Biochemistry and Physiology Part B, 131 (4): 757-765.
Ramakuwela, T., J. Hatting, M. D. Laing, S. Hazir & N. Thiebaut, 2016. In vitro solid-state production of Steinernema innovationi with cost analysis. Biocontrol Science and Technology, 26 (6): 792-808.
Sarwar, M., 2015. The killer chemicals as controller of agriculture insect pests: The conventional insecticides. International Journal of Chemical and Biomolecular Science, 1 (3): 141-147.
Schumann, M., A. Patel, M. Vemmer & S. Vidal, 2014. The role of carbon dioxide as an orientation cue for western corn rootworm larvae within the maize root system: Implications for an attract-and-kill approach. Pest Management Science, 70 (4): 642-650.
Serwe-Rodriguez, J., K. Sonnenberg, B. Appleman & S. Bornstein-Forst, 2004. Effects of host desiccation on development, survival, and infectivity of entomopathogenic nematode Steinernema carpocapsae. Journal of Invertebrate Pathology, 85 (3): 175-181.
Shapiro-Ilan, D., R. Gaugler, W. L. Tedders, I. Brown & E. E. Lewis, 2002. Optimization of inoculation for in vivo production of entomopathogenic nematodes. Journal of Nematology, 34 (4): 343-350.
Shapiro-Ilan, D. I., R. Han & C. Dolinksi, 2012. Entomopathogenic nematode production and application technology. Journal of Nematology, 44 (2): 206-217.
Shapiro-Ilan, D., E. Lewis, J. Campbell & D. Kim-Shapiro, 2012. Directional movement of entomopathogenic nematodes in response to electrical field: Effects of species, magnitude of voltage, and infective juvenile age. Journal of Invertebrate Pathology, 109 (1): 34-40.
Singh, A. & V. Upadhyay, 2018. A Review on entomopathogenic nematodes: Heterorhabditis and Steinernema. Advances in Bioresearch, 9 (2): 214-222.
Smart, G. C., 1995. Entomopathogenic nematodes for the biological control of insects. Journal of Nematology, 27 (4S): 529-534.
Strauch, O. & R. Ehlers, 2000. Influence of the aeration rate on the yields of the biocontrol nematode Heterorhabditis megidis in monoxenic liquid cultures. Applied Microbiology and Biotechnology, 54 (1): 9-13.
Susurluk, A. I., Y. Kongu & T. C. Ulu, 2013. Quality control of in vitro produced Heterorhabditis bacteriophora (Rhabditida: Heterorhabditidae) strains isolated from Turkey. Turkish Journal of Entomology, 37 (3): 283-291.
Tabassum, K. A. & F. Shahina, 2004. In vitro mass rearing of different species of entomopathogenic nematodes in monoxenic solid culture. Pakistan Journal of Nematology, 22 (2): 167-175.
Testa, A. M. & E. J. Shields, 2017. Low labor “in vivo” mass rearing method for entomopathogenic nematodes. Biological Control, 106: 77-82.
Ulu, T. & I. A. Susurluk, 2014. Heat and desiccation tolerances of Heterorhabditis bacteriophora strains and relationships between their tolerances and some bioecological characteristics. Invertebrate Survival Journal, 11 (1): 4-10.
Wouts, W. M., 1981. Mass production of the entomogenous nematode Heterorhabditis heliothidis (Nematoda: Heterorhabditidae) on artificial media. Journal of Nematology, 13 (4): 467-469.
Wright, D. J., A. Peters, S. Schroer & J. Fife, 2005. “Application Technology, 91-106”. In: Nematodes as Biocontrol Agents (Eds. P. S. Grewal, R. U. Ehlers & D. I. Shapiro-Ilan). CABI, Wallingford, UK, 528 pp.
Yoo, S. K., I. Brown & R. Gaugler, 2000. Liquid media development for Heterorhabditis bacteriophora: Lipid source and concentration. Applied Microbiology and Biotechnology, 54 (6): 759-763.
Yoo, S. K., R. Gaugler & C. W. Brey, 2001. Growth optimization of Photorhabdus luminescens isolated from entomopathogenic nematode Heterorhabditis bacteriophora. Korean Journal of Applied Microbiology and Biotechnology, 29 (2): 104-109.
Zioni, S., I. Glazer & D. Segal, 1992. Life cycle and reproductive potential of the nematode Heterorhabditis bacteriophora strain HP88. Journal of Nematology, 24 (3): 352-358.