41st Annual Meeting of the Society for Invertebrate Pathology
and 9th International Conference on Bacillus thuringiensis

Stored product mites represent a serious pest due to their contamination of human food and animal feed by allergens. There is a strong effort in eliminating stored product mite infestation to zero or sub-zero level. The public demand for elimination of pesticides from the food industry for their negative effects on human health and the environment limited chemical pesticides application. Microbial pathogens and viruses toxic to mites are suggested as an alternative to chemical pesticides. We summarize the results of testing Bacillus thuringiensis toxins on acaridid mites. In addition we report the presence of gram negative proteobacteria that act as entomopathogenic symbionts in the digestive tract of nematodes (Steinernematidae and Heterorhabditidae). The clones of 16S rRNA genes from stored product mite homogenates showed high similarity to Xenorhabdus cabanillasii and 91% similarity to Photorhabdus temperata. In midgut cells of stored product mites, the unidentified icosahedral viral particles were observed. Many viral particles were found in the postcolon inside the microvilli and formed chain-like structures. The potential of abovementioned bacteria and viruses in control of stored product mites are discussed. This work was supported by the projects COST OC08065 and NAZV 1B 53043.

The red palm weevil Rhynchophorus ferrugineus (RPW) is a serious pest of date palm (Phoenix dactylifera) and other Palmaceae in Spain and other Mediterranean countries. Recent outbreaks of RPW in Spain promted approaches to control the pest. Chemical control approaches of RPW in Spain have proven inefficient. As a consequence, RPW first detected in Granada (southern Spain) it has expanded throught the Spanish Mediterranean zone. We reported natural infection of RPW with the entomopathogenic fungus Beauveria bassiana in SE Spain. This suggested the development of a biocontrol strategy of RPW using the fungus. For this purpose, pathogenicity of 9 B. bassiana strains (including RPW isolates) was evaluated in laboratory bioassays with RPW larvae. LT50 values of the strains were between 3-10 days. The best B. bassiana isolates were tested in RPW adults and these were formulated for use against RPW. These results suggest that B. bassiana has the potential to be developed in a biological control strategy of RPW. Furthermore they also show that the choice of the adequate strain is a key step in the development of an efficient biocontrol agent for RPW.

In the Pacific Northwest of North America, western cherry fruit fly, Rhagoletis indifferens (Diptera: Tephritidae) drop from infested fruit as late instar larvae and overwinter as pupae in the orchard soil. Both the larvae and pupae are susceptible to infection by Beauveria bassiana-GHA and the isolate could potentially be used as a bioinsecticide versus this important cherry pest. A soil survey of organically and chemically managed cherry orchards in southern British Columbia, Canada was conducted to determine the incidence of indigenous B. bassiana isolates. When the same soils were tested for their ability to support introduced Bb-GHA over time, the persistence of conidia germination in non-sterile soils was variable for four weeks post-treatment. The efficacy and logic of using Bb-GHA to cause western cherry fruit fly mortality within orchard soils was evaluated.

The fall webworm, Hyphantria cunea (Lepidoptera: Arctiidae) is dangerous quarantine pest damaging the agriculture crops, forest and ornamental plants, it distributed through the territory of West Georgia and Black Sea Coast. The insect mainly inhabits in the populated area – home sites, communities suburban parks and places of mass restoring where by the viewpoint of sanitation-hygiene the using chemical pesticides are prohibited. Generally the urban horticulture is under the threat of fall webworm. In this connection it is necessary the use of environmentally safe means to plant protection from this dangerous pest. The biological control potential of different means: bacterial - XenTari DF, Dipel, fungal - BotaniGard ES (Project – GRDF-GEB2-3337-TB-04, USA) and entomopathogenic nematode (EPN) introduced from Israel (CDR-CAR Project CA CA22-007) – Steinernema feltiae were tested against 2nd, 3rd instars larvae and pupae of H.cunea in laboratory and fields. Infectivity of H.cunea by the suspensions (0.7%) XenTari DF and BotaniGard ES on 7 days have caused 96-100% mortality. The mix infection at the reduced concentrations of microbial means with entomopathogenic nematodae, S.feltiae (1.500 unit/ml) on 3 days has caused 100% mortality of larvae, which may serve for cultivation of nematodes. Biological control will take the important place in IPM.

Indigenous isolates of the entomopathogenic fungus Metarhizium anisopliae were screened for virulence against the wireworm species Agriotes obscurus, A. lineatus and A. sputator. In 2006, thirteen isolates were tested by dipping larvae into blastospore suspensions (1x107 spores/mL). For the most virulent isolates, a maximum of 38.3%, 30.0%, and 30.4% of the A. obscurus, A. lineatus and A. sputator larvae, respectively, were infected after nine weeks. In 2008, a similar bioassay was performed using conidial (1x107 spores/mL) instead of blastospore suspensions. For the most virulent isolates infection rates were 80.0 %, 33.3%, and 40.0% for A. obscurus, A. lineatus and A. sputator larvae, respectively, after nine weeks. The results suggest that the M. anisopliae isolates may be particularly useful to control A. obscurus, while control of the other two wireworm species may be less efficient.

The incorporation of certain stilbene optical brighteners into virus-based formulations has been demonstrated to increase viral pathogenicity (as indicated by reduced LD/LC50 values) but their effect on B. thuringiensis activity has been scarcely investigated. We determined the effect of nine optical brighteners on the insecticidal activity of B. thuringiensis ser. kurstaki HD-1 strain (Bt HD-1) on Helicoverpa armigera and also compared the effect of two optical brighteners on the insecticidal activity of Bt HD-1 and H. armigera single nucleocapsid nucleopolyhedrovirus strain SP1 (HearNPV-SP1). Blankophor CLE, Blankophor DRS, Blankophor ER, and Leucophor SAC significantly increased the pathogenicity of Bt HD1. In contrast, Tinopal UNPA-GX, Tinopal CBS, Blankophor BA, Leucophor AP, and Leucophor UO had an adverse or no effect on its activity. Tinopal LPW or Leucophor UO to HearNPV-SP1 significantly enhanced viral pathogenicity by 31.4 and 11.4 fold, respectively, when used at 1% and by 11.4 and 6.3 fold, respectively at 0.1%. However, none of these brighteners increased Bt-HD1 activity. We hypothesize that the degradation of the peritrophic membrane by optical brighteners causes the enhancement of HearNPV pathogenicity and the null or antagonistic activity on Bt HD-1 against H. armigera. Inclusion of optical brighteners on Bt-based formulations is discussed.

Palms are an important crop in Mediterranean zone and in dry areas worldwide. The red palm weevil (RPW, Rhynchophorus ferrugineus) was introduced in Europe with N Africa importations of infested plants fifteen years ago. Ineffective control measures (repeatedly applied chemicals have been futile for RPW control) against RPW are causing both severe economic and environmental damages. Consequently, there is a need for a Sustainable Pest Control of RPW. Infection of RPW adults and larvae by strains of the entomopathogenic fungus Beauveria bassiana, recently isolated from naturally infected RPW in the field, was investigated using Scanning Electron Microscope (SEM). RPW adults were treated with dry conidia and diluted conidia in 0.02% Tween-20. More apresoria were observed in the treatment of adults with dry conidia than in diluted conidia. Apresoria were also observed in larvae treated with dry conidia. The obtainment of fungal inoculum became favoured by different structures of the insect. These results suggest several ways of inoculum acquisition by the insect. This will help to fully develop B. bassiana as a biological control agent of RPW.

Bacillus thuringiensis subsp. israelensis strain Bti-11 based biological mosquito larvicides produced in Vietnam in different slowly released solid formulations were laboratory evaluated activity against main vector mosquitoes, Anopheles minimus, Aedes aegypti, Culex quinquefasciatus. The formulations were made in the small round cake form with 3 cm diameter, 2-5 mm thickness. Raw materials used were cheap and available in Vietnam: corn cob, sugar cane bagasse, cork, popcorn. Results showed that the formulation made of corn cob (CT1), the formulation made of sugar cane bagasse and polyvinylalcohol adhesive (CT4) got the highest efficacy, 96.6 and 100 % respectively. In experiment for long-term effect of products, the formulations CT1, CT4 and CT7 had high efficiency maintenance, larvae mortality was 95% after 11 days. The formulation CT4 was remarkably degraded after 11 days, while the formulation CT1 was not noticeably degraded. The formulation CT1 was used for field trail in some ponds in urbanizing areas in Thanh Xuan district, Hanoi city. Mosquito larvae density in experimental ponds reduced from 90.8 to 100 % after 72 hours of treatment. These indicate that Bti preparations produced in Vietnam have high efficacy in the field condition and could be promising products for mosquito control programs.

A new Bacillus subtilis isolate showed high anti-fungal activities (more than 80% control efficacy) against several plant diseases such as rice blast (Magnaporthe grisea), tomato gray mold (Botrytis cinerea), tomato late blight (Phytophthora infestans) and wheat leaf rust (Puccinia recondita). We tried to confer an insecticidal activity to this B. subtilis isolate for constructing a recombinant strain which has dual functions, anti-fungal and insecticidal activity. The insecticidal cry1Ac gene of B. thuringiensis was constructed under its own promoter in a minimal E. coli-B. thuringiensis shuttle vector (pHT1K-1Ac). The plasmid, pHT1K-1Ac was introduced into B. subtilis isolate by electroporation and the transformant was confirmed by PCR with cry1Ac specific primers. B. subtilis transformant produced a parasporal inclusion in the cells as in B. thuringiensis and the size of that protein was appox. 130 kDa. The insecticidal activity of the transformant was checked against lepidopteran pest. This result suggests that this recombinant B. subtilis strain shows the possibility of controlling harmful insect pests as well as plant fungal diseases simultaneously at one crop or on industrial downstream, the culture broth and harvested cells can be used as individual biological control agents separately for integrated crop protection.

The insect cuticle is the first barrier against biological or chemical contact insecticides. A thin layer of lipids, mainly hydrocarbons, protects insects against lethal desiccation; they are proposed as a new target for triatomine control. We studied the effect of alkane-growth adaptation of the entomopathogenic fungus Beauveria bassiana on the ability to infect the Chagas disease vector Triatoma infestans. The bioinsecticide capacity of two fungal strains (Bb GHA and Bb 10) was compared in fungi grown in two different carbon sources (glucose and insect-like hydrocarbons) as the sole carbon source. Mortality and median lethal time were evaluated at different doses (107, 108, and 109conidia/ml). The alkane-grown fungi showed enhanced virulence parameters. An increased mortality percentage (>50%) and/or a significant reduction (>15%) in the time to kill T. infestans were observed, compared to controls grown in complete medium. These evidences suggest that the initial steps of infection might be favored by using an insect-host hydrocarbon mimic as the sole carbon source for fungal growth.