Luca Ruiu

Brevibacillus laterosporus, a Pathogen of Invertebrates and a Broad-Spectrum Antimicrobial Species.

Brevibacillus laterosporus, a bacterium characterized by the production of a unique canoe-shaped lamellar body attached to one side of the spore, is a natural inhabitant of water, soil and insects. Its biopesticidal potential has been reported against insects in different orders including Coleoptera, Lepidoptera, Diptera and against nematodes and mollusks. In addition to its pathogenicity against invertebrates, different B. laterosporus strains show a broad-spectrum antimicrobial activity including activity against phytopathogenic bacteria and fungi. A wide variety of molecules, including proteins and antibiotics, have been associated with the observed pathogenicity and mode of action. Before being considered as a biological control agent against plant pathogens, the antifungal and antibacterial properties of certain B. laterosporus strains have found medical interest, associated with the production of antibiotics with therapeutic effects. The recent whole genome sequencing of this species revealed its potential to produce polyketides, nonribosomal peptides, and toxins. Another field of growing interest is the use of this bacterium for bioremediation of contaminated sites by exploiting its biodegradation properties. The aim of the present review is to gather and discuss all recent findings on this emerging entomopathogen, giving a wider picture of its complex and broad-spectrum biocontrol activity.

Lethal and sublethal effects of Brevibacillus laterosporus on the housefly ( Musca domestica )

Sporulated cultures of an isolate of the entomopathogenic bacterium Brevibacillus laterosporus (Laubach) from soil in Sardinia (Italy) were highly toxic by ingestion to both adults and juveniles of the housefly, Musca domestica L. (Diptera: Muscidae). Toxicity was associated with spores as the culture supernatant was not toxic and no parasporal inclusions were observed. The level of toxicity varied with the concentration of spores, the time of exposure to the treated diet, and the stage of insect development. Comparative LC50 values were 0.72 × 108 and 1.75 × 108 spores per gram of diet for 1st and 2nd instar larvae, respectively, and 3.84 × 108 spores per gram for adults. A significant increase in larval development time and reductions in pupal weight and emergence rate were observed when larvae were fed on diets containing sublethal concentrations of spores. Adults surviving after feeding on a sublethal diet showed a significant reduction in fecundity and longevity. Similarly, adults from treated larvae exhibited a lower fecundity, although their longevity was not influenced. A relationship between pupal weight and adult fecundity was found. Pupal stage duration and egg eclosion were not significantly affected.

Insect Pathogenic Bacteria in Integrated Pest Management

The scientific community working in the field of insect pathology is experiencing an increasing academic and industrial interest in the discovery and development of new bioinsecticides as environmentally friendly pest control tools to be integrated, in combination or rotation, with chemicals in pest management programs. In this scientific context, market data report a significant growth of the biopesticide segment. Acquisition of new technologies by multinational Ag-tech companies is the center of the present industrial environment. This trend is in line with the requirements of new regulations on Integrated Pest Management. After a few decades of research on microbial pest management dominated by Bacillus thuringiensis (Bt), novel bacterial species with innovative modes of action are being discovered and developed into new products. Significant cases include the entomopathogenic nematode symbionts Photorhabdus spp. and Xenorhabdus spp., Serratia species, Yersinia entomophaga, Pseudomonas entomophila, and the recently discovered Betaproteobacteria species Burkholderia spp. and Chromobacterium spp. Lastly, Actinobacteria species like Streptomyces spp. and Saccharopolyspora spp. have gained high commercial interest for the production of a variety of metabolites acting as potent insecticides. With the aim to give a timely picture of the cutting-edge advancements in this renewed research field, different representative cases are reported and discussed.

Combination of beehive matrices analysis and ant biodiversity to study heavy metal pollution impact in a post-mining area (Sardinia, Italy)

Mining activities represent a major source of environment contamination. The aim of this study was to evaluate the use of bees and ants as bioindicators to detect the heavy metal impact in post-mining areas. A biomonitoring programme involving a combination of honeybee hive matrices analysis and ant biodiversity survey was conducted over a 3-year period. The experimental design involved three monitoring stations where repeated sampling activities focused on chemical detection of cadmium (Cd), chrome (Cr) and lead (Pb) from different matrices, both from hosted beehives (foraging bees, honey and pollen) and from the surrounding environment (stream water and soil). At the same time, ant biodiversity (number and abundance of species) was determined through a monitoring programme based on the use of pitfall traps placed in different habitats inside each mining site. The heavy metal content detected in stream water from the control station was always below the analytical limit of quantification. In the case of soil, the content of Cd and Pb from the control was lower than that of mining sites. The mean heavy metal concentrations in beehive matrices from mining sites were mainly higher than the control, and as a result of regression and discriminant analysis, forager bee sampling was an efficient environmental pollution bioindicator. Ant collection and identification highlighted a wide species variety with differences among habitats mostly associated with vegetation features. A lower variability was observed in the polluted landfill characterised by lack of vegetation. Combined biomonitoring with forager bees and ants represents a reliable tool for heavy metal environmental impact studies.

Observations on house fly larvae midgut ultrastructure after Brevibacillus laterosporus ingestion.

The pathological and histopathological course caused by Brevibacillus laterosporus on house fly larvae has been investigated conducting observations on insect behavior and midgut ultrastructure. After dissection and fixation, gut tissues were analyzed under transmission electron microscopy (TEM) in order to compare in vivo-treated and non-treated (control) fly specimens. Treated larvae showed extensively reduced feeding and growth rate, then became sluggish and died within 72 h. A progressive midgut epithelium deterioration was observed in treated larvae, compared to the control. Ultrastructural changes consisted of microvilli disruption, cytoplasm vacuolization and general disorganization, endoplasmic reticulum deformation, mitochondria alteration. Deterioration became progressively more dramatic until the infected cells released their content into the gut lumen. Disruption was associated also with midgut muscular sheath and connective tissue. These ultrastructural changes are similar to those widely described for other entomopathogenic bacteria, such as Bacillus thuringiensis, against different insect species. The rapid disruption of cellular fine structure supports a hypothesis based on an interaction of toxins with the epithelial cell membranes reminiscent of the specific B. thuringiensis δ-endotoxins mechanism of action on other insect targets.

Honeys of Sardinia (Italy)

Summary Honey is a product closely related to its area of production and whose composition and flavour depend on the types of flowers foraged by bees. On the other hand, the main features of final products are also influenced by soil and climatic conditions, as well as human activities. Sardinia (Italy) is an island located in the centre of the Mediterranean basin and covers about 24,000 Km2. The most part of the surface is characterized by the presence of natural vegetation and extensive agro-pastoral agriculture. There are limited areas with intensive agriculture based on the use of chemicals (pesticides or fertilizers), and also limited industrial districts. Therefore, the island is for the most part natural, which represents the ideal status for high quality honey production. The melliferous plants of Sardinia include more than 200 species, which allow the production of different monofloral and polyfloral honeys, including typical productions from Asphodel, Thistle and Strawberry-tree (bitter honey) and other typical Mediterranean or worldwide spread products such as Eucalyptus and Citrus. This paper reports a descriptive catalogue of the monofloral and polyfloral Sardinian honeys, and, for each type, gives information on botanical origins (nectar sources), melissopalynological, organoleptic and chemical-physical characteristics.

Pathogenicity and characterization of a novel Bacillus cereus sensu lato isolate toxic to the Mediterranean fruit fly Ceratitis capitata Wied.

The lethal and sub-lethal effects of sporulated cultures of a novel Bacillus cereus sensu lato strain lacking detectable cry genes and identified through morphological and genetic analyses, have been studied on the Mediterranean fruit fly Ceratitis capitata. The lethal effects on young larvae were concentration dependent, with a median lethal concentration (LC50) of 4.48 × 10(8)spores/g of diet. Sporulated cultures of this strain significantly extended development time and reduced immature survival, and the size of emerging fly adults. Besides spores, the toxicity has been associated to the insoluble extra-spore fraction characterized through a proteomic approach. The profile of the extra-spore protein fraction (ES) showed major protein bands within the 35-65 kDa range. The results of mass spectrometry analysis highlighted the presence of putative virulence factors, including members of protein families previously associated to the insecticidal action of other microbial entomopathogens. These proteins include metalloproteases, peptidases and other enzymes.

Administration of Brevibacillus laterosporus spores as a poultry feed additive to inhibit house fly development in feces: A new eco-sustainable concept

The success of a microbial pesticide application against house flies developing in manure should accomplish the uniform mixing of active ingredients with this breeding medium, thus enhancing residual effects. The oral administration of the entomopathogenic bacterium Brevibacillus laterosporus to caged poultry species allows the homogeneous incorporation of its active ingredients with fly breeding media. Feces from treated broilers or hens show toxicity against exposed fly adults and larvae. Insecticidal effects are concentration-dependent with a lethal median concentration (LC50) value of 1.34 × 10(8) and 0.61 × 10(8) spores/g of feces for adults and larvae, respectively. Manure toxicity against flies was maintained as long as chickens were fed a diet containing adequate concentrations of B. laterosporus spores. Toxicity significantly decreased after spore administration to birds was interrupted. When poultry diet contained 10(10) spores/g, mortality of flies reared on feces exceeded 80%. The use of B. lateroporus spores as a feed additive in poultry production systems fostering a more integrated approach to farming is discussed.

Comparative Applications of Azadirachtin- and Brevibacillus laterosporus-Based Formulations for House Fly Management Experiments in Dairy Farms

ABSTRACT The potential of two bioinsecticidal formulations containing Brevibacillus laterosporus spores and azadirachtin, respectively, was assayed in laboratory and in comparative field treatments for the management of immature house flies on dairy farms. As already known for B. laterosporus, preliminary laboratory experiments with azadirachtin evidenced a concentration-dependent effect. Azadirachtin median lethal concentration (LC50) value determined for second instar larvae was 24.5 µg/g diet. Applications in dairy farms were performed at dosages and concentrations predetermined in laboratory experiments, to employ the two formulations at an equal insecticidal potential. Repeated applications on the cow pen caused a significant fly development depression in areas treated with azadirachtin (63%) and B. laterosporus (46%), compared with the control. Formulations were applied at a dosage of 3 liters/m2, and concentrations of 2 × 108 B. laterosporus spores/ml and 25 µg azadirachtin/ml, respectively.

Immature House Fly (Musca domestica) Control in Breeding Sites With a New Brevibacillus laterosporus Formulation

Abstract A bacterial formulation containing spores of a Brevibacillus laterosporus strain from Sardinia, known to be toxic by ingestion to the house fly (Musca domestica), was assayed in laboratory, outdoor, and field conditions for the control of immature stages of this pest. In all laboratory assays, the bacterial formulation exhibited toxicity against house fly larvae. A concentration of 1 × 108 spores of B. laterosporus/g of diet caused 100% mortality of house fly immature stages. The same formulation, applied at a concentration of 1 × 108 spores/ml, equivalent to a dose of 2 liters/m2, caused a reduction in adult emergence from laboratory and natural breeding substrates (outdoor cage experiments) up to 80.3 and 57.8%, respectively. Similarly, this formulation applied in the cow pen of a diary farm at a dose of 2 liters/m2 produced a significant reduction (30%) in immature fly development. Therefore, the use of this bacterial preparation in microbiological control strategies for the integrated pest management of this species is promising.