4d.Progress report.
This report serves to document research conducted under a trust fund cooperative agreement between ARS and the Rural Development Administration (RDA) of South Korea. Under the auspices of this agreement, an RDA scientist (Hyun Gwan Goh) works with ARS cooperators in the Insect Biocontrol Laboratory, Beltsville, MD, to Develop insect-specific microbial toxins for use as biopesticides. During the reporting period, twenty-eight strains of Bacillus thuringiensis (Bt) were screened for toxicity against he diamondback moth (DBM), Plutella xylostella, which is a serious insect pest of cruciferous plants, e.g., cabbage, cauliflower, kale, turnips, brussel sprouts and mustard greens. Bt strains were cultured on agar plates, examined for the presence of crystals and then harvested in water. Samples of Bt whole culture preparations, those treated with base [to solublize the crystal protein (pH-treated Bt)], as well as isolated spores were applied to artificial diet prior to placing 2nd instar DBM larvae on the diet. The soluble protein concentration was 2 or 3 times higher in Bt preparations that had been treated with NaOH, centrifuged to remove spores, restored to neutrality with HCl and filtered (0.45 micrometer filter) than in supernatants of whole culture preparations. Of the 28 Bt strains tested, eight [IBL 24, 136, 156, 194, 425, 455 (Dipel, commercial strain), 465 and 745] caused high levels of larval mortality after two-three days of feeding. Whole culture Bt preparations were more toxic than samples that contained either spores alone or primarily dissolved crystal protein. With the addition of any one of the three preparations of Bt, DBM mortality increased in a dose-dependent manner. Based on LC 50 values (concentration expressed in plate equivalents required to kill 50% of the DBMs 3 or 6 days after feeding was initiated) for whole culture suspensions of IBL Bt strains, potency from greatest to least was as follows: IBL 465, 745, 455, 425, 136, 194, 24, and 156. Spores that were damaged (shaken with beads) were no longer insecticidal, suggesting that spores must be intact to cause mortality. Previously, it had been reported that spores complemented crystal activity and that crystal protein, once digested by enzymes in the gut produced the active Cry protein that compromised the insect midgut allowing spores to enter the normally sterile body cavity. Based on electrophoretic studies, it was discovered that washed spore preparations do not contain crystal protein so that the mechanism of action by which spores alone kill DBM remains to be investigated. Preparations of pH-treated Bt were stable for ten or more weeks of refrigeration, but were not stable to boiling (therefore, not a heat-stable exotoxin) or, with the possible exception of Strain IBL 425, to extraction with methanol. From experiments in which dissolved crystal preparations of IBL strains, 425, 24, 465 and 455 (Dipel) were subjected to gel electrophoresis, it was learned that all strains except IBL 24 produced bands that comigrated with the Cry 1 proteins. From these preliminary experiments, it was hypothesized that the toxic protein in IBL 24 either is not Cry 1 or is active in the form of a precursor or metabolite of Cry 1. When the various strains of Bt were tested on Dipel-resistant DBM, IBL 425 had the lowest LC50 (was the most toxic), followed by 24 and 194. Thus, IBL 425 was the most promising for development as a bioinsecticide against DBM. Experiments are underway to characterize the toxic Bt-produced factor(s) and to conduct field tests in the fall.