Roberto M. Pereira

Beauveria bassiana: Endophytic colonization and plant disease control

Seed application of Beauveria bassiana 11-98 resulted in endophytic colonization of tomato and cotton seedlings and protection against plant pathogenic Rhizoctonia solani and Pythium myriotylum. Both pathogens cause damping off of seedlings and root rot of older plants. The degree of disease control achieved depended upon the population density of B. bassiana conidia on seed. Using standard plating techniques onto selective medium, endophytic 11-98 was recovered from surface-sterilized roots, stems, and leaves of tomato, cotton, and snap bean seedlings grown from seed treated with B. bassiana 11-98. As the rate of conidia applied to seed increased, the proportion of plant tissues from which B. bassiana 11-98 was recovered increased. For rapid detection of B. bassiana 11-98 in cotton tissues, we developed new ITS primers that produce a PCR product for B. bassiana 11-98, but not for cotton. In cotton samples containing DNA from B. bassiana11-98, the fungus was detected at DNA ratios of 1:1000; B. bassiana 11-98 was detected also in seedlings grown from seed treated with B. bassiana 11-98. Using SEM, hyphae of B. bassiana11-98 were observed penetrating epithelial cells of cotton and ramifying through palisade parenchyma and mesophyll leaf tissues. B. bassiana11-98 induced systemic resistance in cotton against Xanthomonas axonopodis pv. malvacearum (bacterial blight). In parasitism assays, hyphae of B. bassiana 11-98 were observed coiling around hyphae of Pythium myriotylum.

Susceptibility of Aedes aegypti, Culex quinquefasciatus Say, and Anopheles quadrimaculatus Say to 19 pesticides with different modes of action.

Abstract To access the relative potency of pesticides to control adult mosquitoes, 19 pesticides with various modes of action were evaluated against Aedes aegypti, Culex quinquefasciatus Say, and Anopheles quadrimaculatus Say. On the basis of 24-h LD50 values after topical application, the only pesticide that had higher activity than permethrin was fipronil, with LD50 values lower than permethrin for 107-, 4,849-, and 2-fold against Ae. aegypti, Cx. quinquefasciatus Say, and An. quadrimaculatus Say, respectively. Abamectin, imidacloprid, spinosad, diazinon, and carbaryl showed slightly lower activity than permethrin (<20-fold). However, bifenazate showed very low activity against the three mosquito species tested, with LD50 values higher than permethrin for >1000-fold. On the basis of 24-h LD50 values, Cx. quinquefasciatus was the least susceptible species to nine pesticides tested (DNOC, azocyclotin, chlorfenapyr, carbaryl, spinosad, imidacloprid, diazinon, abamectin, and permethrin), whereas Ae. aegypti was the least susceptible species to six pesticides tested (dicofol, amitraz, propargite, hydramethylnon, cyhexatin, and diafenthiuron), and An. quadrimaculatus was the least susceptible species to four pesticides tested (bifenazate, pyridaben, indoxacarb, and fipronil). Our results revealed that different species of mosquitoes had different susceptibility to pesticides, showing the need to select the most efficacious compounds for the least susceptible mosquito species to achieve successful mosquito control.

Beauveria bassiana yeast phase on agar medium and its pathogenicity against Diatraea saccharalis (Lepidoptera: Crambidae) and Tetranychus urticae (Acari: Tetranychidae).

Beauveria bassiana colonizes insect hosts initially through a yeast phase, which is common in some artificial liquid cultures, but not reported on artificial solid media. We describe a yeast-like phase for B. bassiana isolate 447 (ATCC 20872) on MacConkey agar and its virulence toward Diatraea saccharalis and Tetranychus urticae. The yeast-like cells of B. bassiana developed by budding from germinating conidia after 24-h incubation. Cells were typically 5-10 microm and fungal colonies were initially circular and mucoid, but later were covered with mycelia and conidia. Ability to produce yeast-like cells on MacConkey medium was relatively common among different B. bassiana isolates, but growth rate and timing of yeast-like cell production also varied. Metarhizium anisopliae and Paecilomyces spp. isolates did not grow as yeast-like cells on MacConkey medium. Yeast-like cells of B. bassiana 447 were more virulent against D. saccharalis than conidia when 10(7)cells/ml were used. At 10(8)cells/ml, the estimated mean survival time was 5.4 days for the yeast suspension and 7.7 days for the conidial suspension, perhaps due to faster germination. The LC(50) was also lower for yeast than conidial suspensions. Yeast-like cells and conidia had similar virulence against T. urticae; the average mortalities with yeast-like cells and conidia were, respectively, 42.8 and 45.0%, with 10(7)cells/ml, and 77.8 and 74.4%, with 10(8)cells/ml. The estimated mean survival times were 3.6 and 3.9 for yeast and conidial suspensions, respectively. The bioassay results demonstrate the yeast-like structures produced on MacConkey agar are effective as inoculum for B. bassiana applications against arthropod pests, and possibly superior to conidia against some species. Obtaining well-defined yeast phase cultures of entomopathogenic hyphomycetes may be an important step in studies of the biology and nutrition, pathogenesis, and the genetic manipulation of these fungi.

ANT BEHAVIOR AND MICROBIAL PATHOGENS (HYMENOPTERA: FORMICIDAE)

The effectiveness of microbial controls for pest ants can be reduced by ant behaviors. Introductions of pathogens, including nematodes, into ant nests result in behavioral responses by ants that affect infection rates to ants exposed to inocula, affect the dissemination of inocula among nestmates, and affect the dispersal of inocula outside the nest. These behaviors include grooming, secretion of antibiotics, nest hygiene, avoidance, and dispersal. Ant behaviors must be considered in developing microbial control agents. Approaches to overcoming the behavioral responses of the red imported fire ant to the entomopathogen Beauveria bassiana (Balsamo) Vuillemin are discussed.

Ability of Bed Bug-Detecting Canines to Locate Live Bed Bugs and Viable Bed Bug Eggs

Abstract The bed bug, Cimex lectularius L., like other bed bug species, is difficult to visually locate because it is cryptic. Detector dogs are useful for locating bed bugs because they use olfaction rather than vision. Dogs were trained to detect the bed bug (as few as one adult male or female) and viable bed bug eggs (five, collected 5–6 d after feeding) by using a modified food and verbal reward system. Their efficacy was tested with bed bugs and viable bed bug eggs placed in vented polyvinyl chloride containers. Dogs were able to discriminate bed bugs from Camponotus floridanus Buckley, Blattella germanica (L.), and Reticulitermes flavipes (Kollar), with a 97.5% positive indication rate (correct indication of bed bugs when present) and 0% false positives (incorrect indication of bed bugs when not present). Dogs also were able to discriminate live bed bugs and viable bed bug eggs from dead bed bugs, cast skins, and feces, with a 95% positive indication rate and a 3% false positive rate on bed bug feces. In a controlled experiment in hotel rooms, dogs were 98% accurate in locating live bed bugs. A pseudoscent prepared from pentane extraction of bed bugs was recognized by trained dogs as bed bug scent (100% indication). The pseudoscent could be used to facilitate detector dog training and quality assurance programs. If trained properly, dogs can be used effectively to locate live bed bugs and viable bed bug eggs.

Efficacy of Beauveria bassiana (Balsamo) Vuillemin isolates for control of stored-grain pests

Abstract: Seventy two isolates of Beauveria bassiana or Metarhizium anisopliae were screened for control of Sitophilus oryzae, S. zeamais (Col., Curculionidae), and Rhyzopertha dominica (Col., Bostrychidae). Beauveria bassiana isolates produced highest mortalities against all three hosts. Ten isolates (all B. bassiana) were selected for further experimentation in the second phase of selection process. Bioassays were conducted by inoculating 50 adult insects of each species, maintained at 26 ± 0.5°C, 70 ± 10% RH, 12 h photophase, for 10 days. The cumulative mortalities after 10 days exposure and lethal time for 50% of the population (LT50) were calculated and compared. S. oryzae and S. zeamais were less susceptible to B. bassiana isolates than R. dominica, which was completely killed by several isolates. Isolate 476 was the most virulent to S. oryzae, and isolate 604 was the most virulent to S. zeamais. Isolate 604 was best overall, but isolates 476 and 643 should also be considered as control agents for stored‐grain pests. A combination of isolates may be advantageous if mixed pest infestations occur.

Lethal Effects of Heat and Use of Localized Heat Treatment for Control of Bed Bug Infestations

ABSTRACT Bed bugs, Cimex lectularius L., hide in cracks and crevices in furniture and are difficult to control. The bed bug thermal death kinetics were examined to develop a heat treatment method to eliminate bed bug infestations in room contents. High temperatures caused temporary immobilization (knockdown) of bed bugs even with exposures that did not have lethal effects. Exposure of bed bug adults to 39°C for 240 min caused no mortality; however, as temperatures increased from 41 to 49°C, exposure times that caused 100% mortality decreased. The temperature difference to provide a 10-fold change in the mortality was estimated at 4°C, and the estimated activation energy (EA) was between 484 and 488.3 kJ/mol. This demonstrates that bed bugs are not more resistant or susceptible to changes in temperature than other tested insects and that the temperatures needed to kill bed bugs are relatively low. In room treatment tests, heat treatment times varied from 2 to 7 h with complete mortality of exposed bed bugs within the treatment envelope created by surrounding the treated furniture with polystyrene sheathing boards. Containment and circulation of heat around the treated material were crucial factors in an efficient heat treatment for bed bug control. The room floor material greatly affected containment of the heat. The tested method for limited heat treatment of furniture and other room contents required equipment costing less than US

FIELD RELEASES OF THE DECAPITATING FLY PSEUDACTEON CURVATUS (DIPTERA: PHORIDAE) FOR CONTROL OF IMPORTED FIRE ANTS (HYMENOPTERA: FORMICIDAE) IN ALABAMA, FLORIDA, AND TENNESSEE

400 and provided opportunity for residual pesticide application around the room with minimal disruption in use of treated room.

Structure-Activity Relationships of 33 Piperidines as Toxicants Against Female Adults of Aedes aegypti (Diptera: Culicidae)

Abstract The little decapitating fly, Pseudacteon curvatus Borgmeier, was released at 11 sites in Alabama, Florida, and Tennessee as a potential self-sustaining biocontrol agent of imported fire ants. We used a biotype from Buenos Aires Province, Argentina that parasitizes black fire ants (Solenopsis richteri Forel). Generally, several thousand flies were released as larvae in parasitized ant workers over a 1-2 week period. Pseudacteon curvatus flies were successfully established on hybrid fire ants (Solenopsis invicta × Solenopsis richteri) at a site near Talladega, Alabama where they have persisted more than two years and expanded out 5-20 km from the original release site. Flies failed to establish in Florida and Tennessee although a few 1st-generation field-reared flies were recovered at four sites in Florida. This fly is only the second parasitoid species to be successfully released against imported fire ants or any other pest ant species. Possible reasons for failures at the other sites include insufficient vegetation cover, competition with another Pseudacteon species in Florida, severe winter kill of ants at a site in Tennessee, and the possibility that the biotype of P. curvatus released was not a viable parasitoid of red imported fire ants.

The nonsusceptibility of soil Collembola to insect pathogens and their potential as scavengers of microbial pesticides

Abstract Aedes aegypti (L.) (Diptera: Culicidae) is the primary vector of both dengue and yellow fever. Use of insecticides is one of the primary ways to control this medically important insect pest. However, few new insecticides have been developed for mosquito control in recent years. As a part of our collaborative effort to search for new insecticides to control mosquitoes, piperidine was used as base compound for further optimization. Herein, we report the structure–activity relationships of 33 piperidines against adult female Ae. aegypti. On the basis of 24-h LD50 values after topical application, the most toxic compound was 2-ethyl-piperidine, with an LD50 as low as 0.8 &mgr;g per mosquito. The toxicities of piperidine derivatives were significantly decreased when a benzyl moiety was attached to the carbon of the piperidine ring, with an LD50 value as high as 29.2 &mgr;g per mosquito. The toxicity order of three moieties attached to the carbon of the piperidine ring was ethyl- > methyl- > benzyl-derivatives. When the same moiety was attached to the piperidine ring, the carbon position to which the moiety was attached conferred different toxicity and the toxicity order was second carbon > third carbon > fourth carbon. Together, these preliminary results may be useful in guiding further piperidine ring modifications in the development of potential new insecticides.