In-vitro mass production of the beneficial nematode Heterorhabditis bacteriophora using liquid culture fermentation technology Len Holmes, Michael Menefee and Floyd Inman III Biotechnology Research and Training Center University of North Carolina at Pembroke Pembroke, NC 28372

Agricultural Biological Control Products Biocontrol: Reduction of pest populations by natural enemies; involves active human role. Natural enemies include: Parasitoids, predators, microbes & beneficial nematodes. Targets: animals, weeds and disease. During last century hundreds of exotic control agents have been used. (North Carolina State University)

How Biocontrol Directly Benefits Agriculture Controls insect or weed pests to manageable levels (potentially permanent). Reduces hazard of chemical use and acute or long-term impact on humans and non-target organisms. No potential resistance build-up as with chemical inputs. No delay between application and harvesting. Residue-free products from farms. No phytotoxic effects on young plants (abortion of flowers). Public is more accepting of biological control than chemical agents.

Barriers to Adoption of Biocontrol Approaches I. Necessity of educated management and planning. II. More complete and accessible information on risks to environment and non-target organisms is needed. III. Knowledge on insect pest range and biology of insect host(s). IV. Abiotic and biotic factor interactions with biocontrol agent. V. Knowledge of synchronization of host and it natural enemies. VI. Knowledge of dispersal technology

Beneficial Nematodes How they are beneficial? Environmentally-friendly substitute for chemical insecticide Photorhabdus luminescens bacteria are symbiotically associated with the beneficial nematode Heterorhabditis bacteriophora Nematode recovery in liquid culture can vary considerably based upon many factors such as: bacterial phase variant, media formulation, bacterial density, etc.

Life-cycle complexes for beneficial nematodes Bacteria colonizes inside the infective juveniles (IJs) IJs live in the soil until they invade a susceptible insect host, seeking the hemolymph IJs release the bacteria into the insect’s hemolymph The bacteria participate in overcoming the insect’s defense system and killing the host Nematode growth and reproduction take place Nutrient limitation in the host insect cadaver Non-feeding IJ stage emerges into the soil to forage for a new host Tripartite interaction Ref: Ehlers RU (2001) Mass production of entomopathogenic nematodes for plant protection. Appl Microbiol Biotechnol (2001) 56:623–633

Development stages of Nematodes IJ3 Young hermaphrodite Adult Egg J1 Endotokia

Objective Mass production of EPN Heterorhabditis bacteriophora by In-vitro liquid culture fermentation technology using three different bioreactor working volumes of 5, 10 and 30 liters and scale-up. In vivo culture In vitro: solid culture In vitro: liquid culture

Outline of beneficial nematode culturing protocol

Challenges of the microbiology Phase variation of bacterial symbiont Fecundity of nematode inoculum Optimization of media composition Agitation, dissolved oxygen and pH Contamination (t = 0) (T = 24 hr)

Scale-up for ProDUCtion 5 Liter 10 Liter 30 Liter

Challenges in Production and Commercialization Consistency of mass production Production capacity Production costs Pricing and shipping costs Marketing UNCP Thomas Family Center for Entrepreneurship Website (http://sites.google.com/site/braveguardnematodes Two kinds of customers: Internet & regional Regional Field Testing

References Ehlers, R., Lunau, S., Krasomil K. and Osterfeld, K. 1998. Liquid culture of the entomopathogenic nematodebacterium-complex Heterorhabditis megidis/Photorhabdus luminescens. BioControl 43: 77–86 Ehlers, R.U. 2001. Mass production of entomopathogenic nematodes for plant protection. Appl. Microbiol. Biotechnol. 56: 623–633. Inman, F.L. III and L.D. Holmes. 2012a. Mass production of the beneficial nematode Heterorhabditis bacteriophora and its bacterial symbiont Photorhabdus luminescens. Indian J. Microbiol. 52(3): 316-324. doi:10.1007/s12088-012-0270-2 Shapiro-Ilan, D. and Gaugler, R. 2002. Production technology for entomopathogenic nematodes and their bacterial symbionts. Journal of Industrial Microbiology & Biotechnology. 28: 137 –146 Upadhyay, D., Kooliyottil, R., Mandjiny, S., Inman III, F. and Holmes, L. 2013. Mass production of the beneficial nematode Steinernema carpocapsae utilizing a fed-batch culturing process. ESci J. Plant Pathol. 02 (01): 52-58 Yoo, S., Brown, I. and Gaugler, R. 2000. Liquid media development for Heterorhabditis bacteriophora: lipid source and concentration. Appl microbial Biotechnol. 54: 759-763

THANK YOU!