Isolation and characterization of soil bacteria able to rapidly degrade the organophosphorus nematicide fosthiazate.

Foshtiazate is an organophosphorus nematicide commonly used in protected crops and potato plantations. It is toxic to mammals, birds and honeybees, it is persistent in certain soils and can be transported to water resources. Recent studies by our group demonstrated, for the first time, the development of enhanced biodegradation of fosthiazate in agricultural soils. However, the micro-organisms driving this process are still unknown. We aimed to isolate soil bacteria responsible for the enhanced biodegradation of fosthiazate and assess their degradation potential against high concentrations of the nematicide. Enrichment cultures led to the isolation of two bacterial cultures actively degrading fosthiazate. Denaturating Gradient Gel Electrophoresis analysis revealed that they were composed of a single phylotype, identified via 16S rRNA cloning and phylogenetic analysis as Variovorax boronicumulans. This strain showed high degradation potential against fosthiazate. It degraded up to 100 mg l-1 in liquid cultures (DT50  = 11·2 days), whereas its degrading capacity was reduced at higher concentration levels (500 mg l-1 , DT50  = 20 days). This is the first report for the isolation of a fosthiazate-degrading bacterium, which showed high potential for use in future biodepuration and bioremediation applications. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reported for the first time the isolation and molecular identification of bacteria able to rapidly degrade the organophosphorus nematicide fosthiazate; one of the few synthetic nematicides still available on the global market. Further tests demonstrated the high capacity of the isolated strain to degrade high concentrations of fosthiazate suggesting its high potential for future bioremediation applications in contaminated environmental sites, considering high acute toxicity and high persistence and mobility of fosthiazate in acidic and low in organic matter content soils.

Are leaf glandular trichomes of oregano hospitable habitats for bacterial growth?

Phyllospheric bacteria were isolated from microsites around essential-oil-containing glands of two oregano (Origanum vulgare subsp. hirtum) lines. These bacteria, 20 isolates in total, were subjected to bioassays to examine their growth potential in the presence of essential oils at different concentrations. Although there were qualitative and quantitative differences in the essential oil composition between the two oregano lines, no differences were recorded in their antibacterial activity. In disk diffusion bioassays, four of the isolated strains could grow almost unrestrained in the presence of oregano oil, another five proved very sensitive, and the remaining 11 showed intermediate sensitivity. The strain least inhibited by oregano essential oil was further identified by complete16s rRNA gene sequencing as Pseudomonas putida. It was capable of forming biofilms even in the presence of oregano oil at high concentrations. Resistance of P. putida to oregano oil was further elaborated by microwell dilution bioassays, and its topology on oregano leaves was studied by electron microscopy. When inoculated on intact oregano plants, P. putida was able not only to colonize sites adjacent to essential oil-containing glands, but even to grow intracellularly. This is the first time that such prolific bacterial growth inside the glands has been visually observed. Results of this study further revealed that several bacteria can be established on oregano leaves, suggesting that these bacteria have attributes that allow them to tolerate or benefit from oregano secondary metabolites.

The effect of initial concentration of carbofuran on the development and stability of its enhanced biodegradation in top-soil and sub-soil.

Carbofuran was incubated in top-soil and sub-soil samples from a pesticide-free site at a range of initial concentrations from 0.1 to 10 mg kg-1. Amounts of the incubated soils were removed at intervals over the subsequent 12 months, and the rate of degradation of a second carbofuran dose at 10 mg kg-1 was assessed. An applied concentration as low as 0.1 mg kg-1 to top-soil resulted in more rapid degradation of the fresh addition of carbofuran for at least 12 months. The degree of enhancement was generally more pronounced with the higher initial concentrations. When the same study was conducted in sub-soil samples from the same site, an initial dose of carbofuran at 0.1 mg kg-1 resulted in only small increases in rates of degradation of a second carbofuran dose. However, degradation rates in the sub-soil samples were, in many instances, considerably greater than in the corresponding top-soil samples, irrespective of pre-treatment concentration or pre-incubated period. Initial doses of 0.5 mg kg-1 and higher applied to sub-soil successfully activated the sub-soil microflora. Application of the VARLEACH model to simulate carbofuran movement through the soil profile indicated that approximately 0.01 mg kg-1 of carbofuran may reach a depth of 70 cm 400 days after a standard field application. The results therefore imply that adaptation of the sub-soil microflora (c 1 m depth) by normal field rate applications of carbofuran is unlikely to occur. In experiments to investigate this in soils exposed to carbofuran in the field, there was no apparent relationship between top-soil exposure and degradation rates in the corresponding sub-soils. The results further confirmed that same sub-soil samples have an inherent capacity for rapid biodegradation of carbofuran. The high levels of variability observed between replicates in some of the sub-soil samples were attributed to the uneven distribution of a low population of carbofuran-degrading micro-organisms in sub-surface soil. There was no apparent relationship between soil microbial biomass and degradation rates within or between top-soil and sub-soil samples.