Each cell was carefully removed daily and the filter paper doused with water to prevent leaf desiccation

Currently, there are no integrated pest management plans available for control of citrus thrips in blueberry. This is primarily due to the recent nature of this crop-pest association. Avocado thrips, Scirtothrips perseae Nakahara, is a relatively new pest of avocados in California. It appeared in the state in 1996, and, at the time, was a species new to science . By 1998, crop damage reduced industry revenues by 12% . Avocado thrips adults can feed on over 11 plant species’, however, larvae have been found only on avocados in the field in both California and Mexico, suggesting that S. perseae has a highly restricted host range . Although it has little effect on tree health, avocado thrips feed directly on immature fruit , and obvious feeding scars cause severe downgrading and culling of damaged fruit . With a limited number of pesticides available for thrips control and the propensity with which economically important thrips develop insecticide resistance, it is wise to monitor population levels carefully, limit treatments to population levels of economic concern and time treatments optimally . Appropriate cultural practices and conservation of natural enemies should be practiced in concert with the use of pesticides only on an as-needed basis. Thus, continuing the search for effective biological and chemical controls useful in citrus and avocado thrips management is important. For both species of thrips, some pupation occurs on the tree in cracks and in crevices’, however, the majority of both species drop as late second instars from trees to pupate in the upper layer of the leaf litter under trees . Propupae and pupae are rarely seen, move only if disturbed, and do not feed. Thus, pupation in the upper layers of the soil surface may create the ideal interface for control using the entomopathogenic fungi Beauveria bassiana . Coarse organic mulch beneath trees and the maintenance of a mulch layer,plastic flower bucket a common practice by many growers as a method of Phytophthora spp. management in avocados , may reduce survival of thrips that drop from trees to pupate below the tree.

The effectiveness of mulching to control thrips is uncertain and labor costs are required to add mulch may not be justified solely for thrips control. There is increasing pressure in the U.S. to move away from broad-spectrum insecticides and focus on alternative methods of control, e.g., genetically modified crop plants expressing Bacillus thuringiensis toxins , use of entomopathogens, and similar approaches. Applications of B. bassiana have been reported to decrease populations of thrips in greenhouse cucumbers, chrysanthemums, gerbera daisies, roses, and carnations . Microbial insecticides containing δ-endotoxins from Bt have been used as alternatives to conventional chemical insecticides for almost 70 years . Bt produces insecticidal proteins during the sporulation phase as parasporal crystals. These crystals are primarily comprised of one or more proteins, i.e. Crystal and Cytolitic toxins, also called δ-endotoxins. From a practical perspective, Cry proteins are parasporal inclusion proteins from Bt that exhibit experimentally verifiable toxic effects to a target organism or have significant sequence similarity to a known Cry protein . Similarly, Cyt proteins are parasporal inclusion proteins from Bt that exhibit hemolytic activity or has obvious sequence similarity to a known Cyt protein. These toxins are highly specific to their target insect, are innocuous to humans, vertebrates and plants, are regarded as environmentally friendly, are completely biodegradable, and show little adverse effect on non-target species . The Cyt proteins are significantly different both in their structure and their biological activities from the Cryproteins. However, Cyt proteins have shown toxicity to non-dipterous insects . In fact, Cyt proteins in some cases can extend activity to other Bacillus spp. for mosquitoes that lack the proper receptor . Many studies with thrips involving Bt proteins have typically evaluated Cry toxins in transgenic crops targeted mainly toward lepidopterous pests and there are no published studies we know of representing the impact of Cyt proteins on thrips. Due to the synergism seen between these two Bt proteins and the method of thrips feeding, commonly described as ‘punch and suck’ , whereby leaf tissue is macerated prior to ingestion, we hypothesized that Cry or Cyt proteins could potentially be useful against thrips pests.

The goal of this investigation was to determine if Cry or Cyt proteins or B. bassiana could be used effectively to manage citrus and avocado thrips. Field management of both thrips species is the ultimate goal with these biopesticides but field studies are laborious and expensive. Thus, we evaluated these materials in the laboratory to determine which were sufficiently efficacious to warrant follow-up field studies. Leaves of both avocado and citrus for all bioassays were chosen in observably identical states; young and soft but fully expanded leaves were used as these are the type on which both species of thrips prefer to feed and large leaves were needed to fit in the Munger cell bioassay units that confined the thrips on treated leaves . Briefly, Munger cells were constructed by using a Plexiglas sandwich; the middle cell layer was drilled with 3.2-cm diameter bit to provide a circular test arena . The upper and lower parts of the Plexiglas sandwich were solid and between the lower base and test arena a piece a piece of filter paper was placed to allow moisture exchange and to extend the life of the leaf during the bioassay. Airflow through the test arena was provided through two holes drilled through the center cell layer directly opposite one another, with fine-mesh screening melted onto the interior of the test arena to prevent escape. The Plexiglas sandwich was held together with four binder clips positioned such that the airflow was not covered. Once dry, the leaves were placed on the filter paper in Munger cells and the respective thrips species was added. The lid was placed on the cell but leaving the cell arena exposed, so that once the thrips were added, the cells could be closed quickly. Female and late second-instar avocado thrips and citrus thrips were then placed on treated leaves of their respective host plants inside the Munger cell. Control leaves for both species were treated with a mixture of the same suspension ingredients minus the protein. Bioassays were conducted concurrently in the following manner for both species: adult female thrips were placed on leaves coated with activated or inactivated forms of both Cyt1A and Cry11A, immature thrips were also placed on leaves coated with activated or inactivated forms of both Cyt1A and Cry11A, and all combinations for adults and immature thrips were carried out along with the corresponding control cells.

The Munger cells were closed and placed in an environmental chamber at 28ºC, 55% RH, and long daylight conditions . The bioassay was replicated on two separate dates . A minimum of 10 individuals was placed into each Munger cell and thrips were checked daily for eight days to assess mortality. Post seven days,flower buckets wholesale the integrity of the leaves was questionable and in all but one bioassay, mortality was observed before seven days; thus data were analyzed using day 7 mortality. Mortality was determined by lack of movement after gently probing each thrips with a small brush. Six strains of B. bassiana were obtained from the USDA-ARS Western Integrated Cropping Systems Research Unit located in Shafter, CA. GHA is the commercially available strain found in the field formulation of B. bassiana, Mycotrol O and the greenhouse formulation BotaniGard ES, and each of the other five strains were obtained via isolation from soils in Kern County by USDA-ARS collaborators in 2000. They were stored at – 80ºC. Culture methods for the thrips experiments were similar to those described previously for Lygus hesperus Knight bioassays and were conducted by collaborators from USDA-ARS, Shafter, CA. Briefly, isolates were grown on SDAY media, or Sabouraud’s dextrose agar plus yeast extract . The conidia were harvested from culture plates after 10–14 days incubation by scraping with a sterile rubber policeman into a 0.01% solution of Silwet L- 77 . The conidia were then enumerated with a hemocytometer. For preservation and storage, glycerol was added to the conidial suspension and stored in aliquots of 2 × 108 in a 2 ml solution at −80°C until needed for bioassays. Conidial viability was assessed following incubation for 16 h in potato dextrose broth just prior to use in experiments. Viability was determined by adding a sample of approximately 107 conidia to 20 ml potato dextrose broth and incubating ca. 16 h in a rotary shaker at 28°C. Conidia germination was examined under a compound microscope at 400× and scored as viable if the germ tube was at least twice the length of the conidium. Percentage viability was measured on 250 conidia of each isolate. All bioassays were conducted on the basis of the number of viable conidia measured after thawing and the desired concentrations were formulated by serial dilution. The strain from Mycotrol was isolated and cultured exactly as above to eliminate possible effects of production methods and formulation ingredients on insecticidal activity. Glycerol was not removed prior to using the conidia in bioassays. All six B. bassiana strains were suspended in 0.01% Silwet in a de-ionized water solution and evaluated on the same date at four concentrations for each thrips species. The control consisted of 0.01% Silwet in de-ionized water solution. Each of the 25 treatments was evaluated using five Munger cells , which contained a minimum of ten adult female thrips. These bioassays were repeated on 10 dates with both species tested simultaneously on each date .

Groups of thrips were anesthetized by exposure to CO2 for 15-30 sec, and each strain was administered to the dorsum of the abdomen of each knocked out thrips quickly and carefully in a 1µl drop with a Burkard Hand Microapplicator over filter paper. The droplet spread the length of the thrips immediately and the thrips was then deposited, still knocked out, onto the leaf tissue in the Munger cell. Once a minimum of 10 treated thrips were added, Munger cells were closed and sealed with binder clips and placed in an environmental chamber at 28ºC, 55% RH, and long daylight conditions . Each cell was checked daily for seven days to observe infection by the fungus. Each cell was carefully removed daily and the filter paper doused with water to prevent leaf desiccation. Individuals infected with B. bassiana were defined as those whose natural activity was retarded and/or showed arrestment and subsequently produced mycelia, which was confirmed post bioassay. Mortality caused by mycosis was confirmed on the basis of visual observation and then crushing individuals to reveal the presence of mycelial growth. When mycelial growth was not apparent, crushed individual thrips were placed on potato-dextrose agar plates for 5 days and then re-examined for the presence of mycelial growth. Data were analyzed after Abbott’s correction for control mortality using log-probit analysis with PROC PROBIT on SAS 9.2 and using the Raymond Statistics package . The purpose of the probit analysis was strictly for gross strain comparison. Probit analysis was used to estimate the LC50 and LC95 levels, confidence intervals, and χ 2 values for each strains. Lethal concentrations with overlapping 95% confidence intervals were not considered significantly different. The daily check data were analyzed as non-cumulative counts per day via the Survival Distribution Function on SAS 9.2 , where observation time represented the probability that the experimental unit from the population would have a lifetime exceeding that time with the variables strain and concentration. Assessments for each variable by species were done with Log-rank and Wilcoxon tests and multiple comparisons for the log-rank test were adjusted by using Tukey-Kramer method. Data were then plotted as estimates of the survivor function for the different strains separately for each species. Bacillus thuringiensis israelensis produces two groups of toxic proteins, the Cry and Cyt toxins that have different modes of action.In this investigation, results with Cyt1A and Cry11A were disappointing as both activated and inactivated forms of both proteins showed little effect against adult and second instar citrus thrips and avocado thrips. To our knowledge, there have been no reports of Bt endotoxins with activity against Thysanoptera, although Cyt1Aa was found to be toxic to the non-target species Chrysomela scripta Fabricius .