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

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 degrees C, 25 degrees C, 35 degrees C) and evaluation at 0, 15, 30, 60, 90 and 120 days after storage at the specified temperatures. Two tests in sugarcane fields were conducted to assess the efficiency of S. rarum against S. levis. The nematode inoculated in the sponge flakes continued to reproduce after the 21 days of incubation at 21 degrees 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 degrees C and 25 degrees C, which did not significantly differ from each other nor from the temperature of 35 degrees C that generated 180,000 IJs/ml. The temperature of 15 degrees C maintained nematode viability above 80% for up to 60 days after storage, differing significantly from the temperatures of 25 degrees C and 35 degrees C, which also differed among themselves and showed a drop in nematode viability to <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 10(8) IJs/ha should be recom-mended since it did not differed from the higher rate (10(9) IJs/ha) tested. We conclude that the phenolic sponge biphasic system is a highly efficient approach for producing entomopathogenic nematodes; moreover, the pro-cess 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.