Canadian Forest Service Publications
Effect of rainfall sunlight radiation and droplet size on the persistence of Bacillus thuringiensis subspp. Kurstaki formulation Dipel 76F, after application onto conifers. 1996. Sundaram, A.; Sundaram, K.M.S. Report to Abbott Laboratories. 44p.
Issued by: Great Lakes Forestry Centre
Catalog ID: 34052
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The effect of rainfall, sunlight radiation, and droplet size spectra of sprays on the persistence of Bacillus thuringiensis subspp. kurstaki (Btk) formulation, DiPel® 76AF, was investigated after application onto spruce foliage. The investigation consisted of three different studies: (i) Study I: a laboratory microcosm study to examine the photostability of Btk deposits on spruce foliage, after different periods of exposure to two radiation intensities, (ii) Study II: a laboratory microcosm study to examine the rainfastness of foliar deposits of DiPel 76AF after exposure to different amounts of cumulative rainfall consisting of two separate droplet size spectra, and (iii) Study III: a field microcosm study to investigate the influence of two different droplet spectra of DiPel 76AF sprays on foliar persistence of Btk under natural weathering conditions. In Study Iand Study II, the formulation was applied over spruce branch tips using mono-sized droplets generated from a non-rotary type of monodispersed droplet generator. In Study III, however, two different pressure-atomizers that can produce two distinctly different droplet size spectra were used, and spray was applied onto single trees surrounded by a portable enclosure. In all the three studies, the formulation was applied in such a manner as to provide a deposit that would be just enough to cause 100% mortality to spruce budworm in bioassay procedures, and persistence of Btk was investigated using both bioassay and total protein quantification methods. In Study I, uniform droplets (130 ± 7 \im in diameter) were applied over fully developed current-year spruce branch tips, and the branch tips were exposed to simulated sunlight with radiation intensities of 437 and 618 W/m2, for 0, 2, 4, and 6 d. After each exposure period, the branches were fed to laboratory-reared 6th instar spruce budworm larvae for bioassay. Mortalities were noted for 6 d and corrected for the control. Using the mortality data and the cumulative amount of needles consumed by the larvae, the average Btk concentration required to cause the observed mortality was calculated in IU per larva. From this value, the number of Ill's present in one gfoliage and per cm2 area of foliage were j computed. The data (expressed in lU/cnrr) indicated that bioactivity of foliar deposits of DiPel 76AF decreased gradually with increasing duration of exposure to radiation, and with increasing radiation intensity. The half-life (DT50, the exposure period required for 50% of the 0) f initial bioactivity to disappear) was 5.1 dfor the low intensity (437 W/m2), and 3.9 dfor the higher intensity (618 W/m2). In contrast with the bioassay results, the total protein levels I [determined by using the bicinchoninic acid (BCA) method] showed no decrease with increasing duration of exposure, or with increasing radiation intensity, thus indicating the unsuitability of the BCA method to detect photoinactivation of the protein fractions. In Study II, uniform droplets (150 ±9 ^im in diameter) were applied over fully developed current-year spruce branch tips, and the branch tips were exposed to simulated rainfall of two different intensities, to provide a cumulative rain of 0, 1, 2, 4, and 6 mm. The low intensity rainfall consisted of a rain droplet size spectrum with a volume median diameter (VMD) of 315 urn, whereas the high intensity rainfall provided a droplet spectrum with a VMD of 780 |im. After each exposure, the branches were fed to laboratory-reared 6th instar spruce^ budworm larvae for bioassay. The data (expressed in IU/cm2) indicated that bioactivity of foliar deposits decreased gradually with increasing cumulative rainfall. A new term, RF50 [the P amount of rain (in mm) required to wash off 50% of the initial deposit], was introduced to understand the relationship between the rainfall intensity and reduction in bioactivity. When the same amount of rain was applied in different droplet sizes, the RF50 value was high (5.2 mm) for small rain droplet sizes, compared to the small RF50 value (2.9) observed for the large rain droplet sizes, thus indicating an inverse relationship between rainfall intensity and RF50. Similar to the bioassay results, there was a gradual decrease in the total protein concentrations (as determined by the BCA method) with increasing amount of rain and with increasing rainfall intensity, thus indicating the suitability of the BCA method to detect rainwashing of the different protein fractions. Thus, both the bioassay and protein quantification methods were quite suitable to detect rainfastness of the total protein deposits on foliage. The RF50 value obtained using the total protein concentrations (expressed in ng/cm2) was 5.4 mm for the small rain droplets, as compared to only 3.4 mm for the large rain droplets. It appears that the high kinetic energy of the large rain droplet sizes physically dislodged the Btk particles from foliar surface, thus contributing to a low RF50 value. The field microcosm study, conducted for a 14-d period after Btk application, indicated that when DiPel 76AF was applied in small droplets (VMD of 65 urn), the duration of persistence (as determined by bioassay procedures) was shorter (ca 8.0 d) than when it was 00 ws^j P applied in large droplets (VMD of 130 urn) (ca 11 d). The data indicated an exponential relationship between Btk concentration (in IU/cm2) and time after spray, and the DT50 value (i.e., the time in days required for 50% of the initial deposit to disappear) was 1.98 d for the spray of small droplets, and 2.87 d for that of large droplets. The slower rate of inactivation of Btk when present in an agglomerated form in a few large droplets as compared to its presence in a disintegrated form in several small droplets, could be attributed to the surface area of exposure to radiation. The amount of Btk particles present in one 130 |im droplet would be eight times higher than the amount in a 65 urn droplet. Thus, the probability of exposure to sunlight would be increased markedly whenthe toxic protein fraction was present r in several small droplets than when it was present in a single large droplet. ' Similar to the reduction in bioactivity over the 14-d study period, the total protein concentrations also decreased gradually with time after spray. This reduction was probably caused by physical dislodgement of the droplets from foliar surface, either due to wind erosion, or by rubbing of branches one against the other during wind flow. The protein concentrations were fitted into the exponential equation, and the DT50 value for the small P droplet spectra was found to be 3.45 d, whereas for the large droplet spectra it was 6.07 d, thus confirming the direct relationship between droplet size spectra of sprays and DT50. T The present study suggests that partly cloudy and non-rainy weather conditions would be more appropriate for field application of the DiPel 76AF formulation than sunny and rainy j conditions, in order to increase the duration of field persistence of Btk. Regarding the role of droplet size spectra of sprays on persistence, the benefits of using large droplets (VMD of 130 ^im) were indicated in the present study. However, in actual field trials we use a constant application rate, and the number of droplets we can generate from a fixed volume rate is controlled by the droplet sizes of sprays. By applying small droplets, we can obtain a high number of droplets per unit area of the target (i.e., good coverage), whereas if we apply large droplets, the number of droplets per unit area would be severely reduced (i.e., poor coverage). Coverage may influence the manner in which the insect consumes the Btk toxin and could affect the bioassay results. This in turn could alter the persistence data. Thus, we may need to come to a compromise while choosing the droplet sizes of sprays for field application of the DiPel 76AF formulation.