Effects of temperature for optimizing production and storage of Steinernema rarum in a novel biphasic process, and efficacy of the nematode against Sphenophorus levis

Authors: LEITE, LUIS - Instituto Biologicio - Brazil; CHACON-OROZCO, JULIE - Instituto Biologicio - Brazil; Shapiro Ilan, David; BALDO, FERNADO - Instituto Biologicio - Brazil; CARDOSO, JORGE - Instituto Biologicio - Brazil

Submitted to: Biological Control
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/21/2023
Publication Date: 10/27/2023
Citation: Leite, L.G., Chacon-Orozco, J.G., Shapiro Ilan, D.I., Baldo, F.B., Cardoso, J.M. 2023. Effects of temperature for optimizing production and storage of Steinernema rarum in a novel biphasic process, and efficacy of the nematode against Sphenophorus levis. Biological Control. 187/105381. https://doi.org/10.1016/j.biocontrol.2023.105381.
DOI: https://doi.org/10.1016/j.biocontrol.2023.105381

Interpretive Summary: Beneficial nematodes (also known as entomopathogenic nematodes) are small round worms that are used as natural biopesticides. Unlike many chemical insecticides that are toxic to humans, other nontarget organisms, and the environment, beneficial nematodes only kill insects and are considered safe to the environment. Beneficial nematodes, however, can be expensive due to their production costs. In this paper, we describe a new improved mass production approach to grow the nematodes. The method involves growing the nematodes in a a nutritive broth followed by growth in a specialized sponge (phenolic sponge). The advantage of this sponge is it can be used to store the nematodes after production, and also can be applied directly to the crop site; the sponge is simply crumbled so that it can be applied through conventional agricultural sprayers. The new method promises to be highly economical. In a field test, nematodes produced using the new system caused high levels of control against an important sugarcane pest (sugarcane billbug). The new production system has great promise for reducing the costs, and increasing the efficacy of beneficial nematodes as natural biopesticides.

Technical Abstract: Entomopathogenic nematodes have been mass produced in vitro in a monoxenic system using two different approaches: the sponge (polyurethane) process soaked in symbiotic bacterial culture, and by liquid fermentation conducted in submerged bacterial culture. Another approach to produce nematodes under in vitro conditions is the biphasic process, which starts with liquid culture and ends with solid culture. This study was aimed at testing and optimizing a biphasic process for production of Steinernema rarum, as well as testing the efficacy of the produced nematode against the sugarcane billbug Sphenophorus levis. The process was accomplished by the initial growth of nematodes in liquid culture followed by inoculation on flaked phenolic sponges soaked with liquid medium and pre-inoculated with the symbiotic bacteria. The study consisted of three treatments represented by the three temperatures (15°C, 25°C, 35°C) and evaluation at 0, 15, 30, 60, 90 and 120 days after storage at the specified temperatures. The experimental unit consisted of a plastic bag with a porous air filter containing 5.5 g of flocked phenolic sponge soaked in 250 ml of rich medium. Two tests in sugarcane fields were conducted with the nematode produced by the optimized biphasic process, the first experiment to assess the nematode application rates of 1x108 and 1x109 IJs/ha, and the second to confirm the lower dosage of 1x108 IJs/ha. The nematode inoculated in the sponge flakes continued to reproduce after the 21 days of incubation at 21°C, reaching a peak of infective juvenile (IJ) yield by 30 days after the bags were transferred to storage conditions, regardless of the temperature at which they were stored. The nematode reached yields above 200,000 IJs /ml of culture in the treatments of 15°C and 25°C, which did not significantly differ from each other nor from the temperature of 35°C that generated 180,000 IJs/ml. The temperature of 15°C maintained the nematode viability above 80% for up to 60 days after storage at this temperature, differing significantly from the temperatures of 25°C and 35°C, which also differed among themselves and showed a drop in nematode viability to less than 22% after 30 days of storage. Steinernema rarum produced by the biphasic process provided >60% and >80% control of S. levis at 29 and 37 days post application in sugarcane fields, demonstrating that the rate of 1x108 IJs/ha should be recommended since it did not differed from the higher rate (1x109 IJs/ha) tested. We conclude that the phenolic sponge bi-phasic system is a highly efficient approach for producing entomopathogenic nematodes; moreover, the process is advantageous relative to other systems because the sponge acts as a medium for both production and storage, and the sponge-nematode mixture can be directly applied to the field without any extraction or harvest steps.