Data were analyzed using a 3-way ANOVA with density of seed , application of B. bassiana , and date as factors . The commercial blueberry test site selected was located north of Bakersfield in Delano, CA. The trial began in August of 2008 and was conducted post blueberry harvest. The V. corymbosum varieties contained within the test area were, ‘Santa Fe’, ‘Jewel’, and ‘Star’. The most susceptible variety of blueberry to citrus thrips damage grown at the test site was the ‘Star’ variety and ‘Star’ was used consistently for evaluation of thrips numbers for all aspects of the trial . Our cooperator was interested in alternatives to traditional pesticides as the farm regularly was dealing with extremely high citrus thrips populations. For example, in 2008 the grower sprayed 5-10 times per field , rotating with traditional chemicals to reduce thrips impact on the subsequent year’s fruit set. Irrigation in all fields took place via drip irrigation with one water delivery emitter per line at each plant base , but additionally, one portion of the blueberry field was equipped with 360° overhead sprinklers. This irrigation setup provided the ideal situation to test B. bassiana under two watering regimes. The commercially available GHA strain is formulated to be mixed with water and for application via chemigation or as a foliar spray.
The label states that no surfactant is needed to keep the spores in suspension. However,plastic plants pots agitation alone in the 1,892.7 L holding tank was not sufficient to keep the material from precipitating, therefore 312.3 ml of Silwet L-77 was added to the tank mix. Mycotrol O® was applied directly to the soil surface with a gas-powered sprayer with a hand spray gun equipped with an adjustable flow meter. The dimensions of the plots were used to calculate the amount of material needed for both B. bassiana formulations . Plants in the test field were spaced every 0.92 m down each row, 3.35 m between each row, and each row was about 165 meters in length. Our studies were conducted inan 18-row section of a 4.04 ha field. The overhead sprinklers were spaced every 7 meters in the row and were located every other row for 12 rows. We chose to investigate the effectiveness of the B. bassiana colonized millet seed versus a Mycotrol O® soil application under two watering regimes, drip-line alone versus drip-line with overhead sprinkler, because B. bassiana conidia are highly subject to desiccation. Comparing the soil drench in both irrigation types with the colonized millet elucidated the effectiveness of the treatments when compared to the control. The blocks were laid out in a 3 x 2 factorial design, with each block consisting of most of five rows of blueberries , each being 27.4 m long . The berm used to grow blueberries at the commercial farm was 1.21 meters wide and each plot was 27.4 meters long. The spacing between adjacent rows was 3.35 m, while the spacing between the plants down a row was approximately 0.92 m with 30 plants per treatment plot . These dimensions result in 0.157 ha treated with raw spores but because the top of the berm was where thrips activity was evident and would be sampled, only 36% of the soil surface area was treated.
The Mycotrol O® label states that the maximum field rate is 6.9 L/ha mixed in 935.3 L/ha water. We therefore chose to apply the entire 6.9 L of Mycotrol O® in 378.5 L of water per ha directly to the berm with no application between the rows, which resulted in 100% of the per ha rate of product being applied to 36% of the area and allowed the maximum amount of active ingredient to be applied to the area that would have almost all thrips activity . Our field trial was intended to determine the extent to which B. bassiana might fit into a program projected to both control citrus thrips effectively and provide rotation among available chemistries so as to reduce thrips resistance evolution. Thus, we felt it was important to operate under the best possible conditions for thrips infection by Mycotrol O® , regardless of financial considerations, i.e. application of product at the maximum label rate in the area where thrips were most likely to be active. The amount of millet seed used in the field trail was calculated based on the area of the berm to be treated and likewise with the Mycotrol O® treatment, only 36% of the total field area was treated. The amount of seed used was one colonized seed/ 2 cm2 over an area of 576 m2 ; the fact that 0.45 kg of seed was needed per 840 cm2 resulted in the application of 3.40 kg of colonized millet seed for the 8 treated plots . Every other plant within the middle ten plants of the middle row of each plot were sampled with pupation emergence cages . These cages were placed tight against the base of each set of canes on the east side . With 5 cages per block and 4 replicate blocks per treatment, a total of 20 cages sampled thrips pupation per treatment over two sample periods, i.e. for two consecutive 3-day periods after the Mycotrol O® soil drench. The treatments were: no B. bassiana with and without overhead sprinkler; colonized millet seed with and without overhead sprinkler; and a soil drench of Mycotrol O® with and without overhead sprinkler . In total, data were collected from 240 emergence cages over the duration of the trial .
The colonized millet seed was set to imbibe water and allowed to sporulate for three days before application and was applied using a hand fertilizer applicator . Four days post application of the millet seed, the soil drench of Mycotrol O® was applied and pupation emergence cages were placed in the field and left out for 3 days . After three days, the sticky cards from each emergence cage were collected and replaced with new cards and the traps were switched to the next plant on the east side. These traps were left in the field to sample thrips for another 3 days . Because the traps were placed out every other plant, this ensured that all of the middle ten plants were sampled over the two, 3-day sampling periods . For two weeks before through two weeks after the applications of B. bassiana , counts were taken of thrips levels on plants twice per week. Beat samples were taken by beating random canes of flush foliage such that the thrips would fall onto a 12 x 12 cm black acrylic beat tray. The numbers of thrips on the beat trays were counted quickly in the field. The counts were taken twice per week from each of the 10 central “data plants” from the ‘Star’ variety of each of the 24 test plots. The new green flush growth was measured on three dates to record the amount of growth since the beginning of the fungal treatment applications to determine if there were differences based on the treatments and amount of water applied to the different plots. Measurements were made of the average cm of new shoot growth over the 6-week trial period. Due to the complex nature of the experimental design, i.e. treatments nested in a 5-way ANOVA , beat count data were analyzed using PROC MIXED and means were separated using Tukey’s test . Figure 3-2 shows the location of late second instar citrus thrips at death in the greenhouse study as well as those that located pupal refuges on the plant. Based on where they dropped off the plant,blueberry pot data indicated that more than 92% of the thrips would have pupated off the plant, likely in the soil near the base of the plant. Numbers did not vary significantly by location over the seven sample dates of this study; therefore data were pooled . A key result was that the proportion of second instar thrips crawling down the base of the plant was higher than the proportion dropping off the plant at distances measured past the base the plant . The four emergence cages placed under the field blueberry plants in each cardinal direction provided a means of sampling late second instar thrips moving towards the soil to pupae versus adults emerging out of the soil following pupation . Total numbers of thrips collected were pooled for the four traps in each direction at each respective location to determine which cardinal direction showed the most activity, and therefore was the most appropriate location to sample for citrus thrips in the field trial. Emergence cage data were summarized in two ways; the number of thrips moving off the plant to pupate in the leaf litter and the number of thrips emerging from the leaf . Data from the nested ANOVA generated p-values for direction , as well as distance grouping from the base of the plant . The cage closest to the base of the plant had significantly higher numbers of thrips emerging from the soil . Numbers of thrips trapped from the eastern cardinal direction were significantly higher for both mean numbers of thrips moving to and from the soil , indicating that for the field trial, emergence cages should be placed directly next to the base of the plant on the eastern side to sample the location that would have the most thrips activity.
Of the proportion of thrips not finding pupal refuge on the plants with colonized seed , 100% infection was seen with each of the different quantities of seed, i.e. each of 0.5, 1, or 2 seeds/cm2 was a sufficient density to infect and kill all late second instar thrips in the greenhouse study. No thrips were infected in the control treatment. There were insufficient data to conduct a 3-way ANOVA because all recovered thrips were infected with the fungus. Because all three densities tested were effective, we chose to utilize the most economical density in the field trial, i.e. 0.5 seeds/cm2 . In the split-plot design model, the whole plot factor was water and the split-plot factor was fungus treatment in a type three analysis of variance . Water, time and treatment were the main effects in the full model. Thrips levels measured on pupation traps at 3 days after treatment were lowest with colonized millet seed, intermediate with Mycotrol O ® , and highest in the untreated control . Additionally, there was fewer thrips counted in the colonized millet seed treatment than in Mycotrol O® treated plots . However, at time two , thrips levels with Mycotrol O® were no longer significantly reduced in relation to the control . While thrips levels measured using pupation traps were significantly less than observed in the control in all plots , thrips levels on plants measured using beat samples did not show a significant decrease , although comparing data with no overhead water that with overhead water, there appeared to be fewer numbers of thrips in the overhead sprinkler plots . The measurements from the new green flush growth in the overhead sprinkler treatments showed that those plants had longer growth than those without overhead sprinklers , but thrips numbers were not significantly lower on those plants. At none of the times when foliar beat counts were taken were there significant differences in thrips numbers across any of the three treatments . The ultimate goal of this work was to determine if the GHA strain of Beauveria bassiana could be used effectively as an alternative to traditional insecticides in commercial blueberries in California. Laboratory and greenhouse trials with Beauveria bassiana have shown variable success in controlling thrips and several other insect species , whereas field trials have shown limited overall success, but very few field trials included Thysanoptera . This is mainly due to the fact that climatic conditions in the laboratory and greenhouse situations are stable and often more humid than the ambient field environment in arid areas like most of California.