Two species-specific TaqMan-based quantitative polymerase chain reaction assays for the detection in soil of Paenibacillus polymyxa inocula.

AIMS: The increasingly widespread use of beneficial microbial inocula in agriculture gives rise to two primary needs: i) the assessment of the environmental risk, i.e. their impact on local soil microbiome and soil properties; ii) being able to track them and monitor their persistence and fate to both optimize their formulation and application method. In previous years, PCR-based methods have detected bacterial or fungal bioinoculant at the species or strain level. However, the selective detection, quantification, and monitoring of target microbial species in a complex ecosystem such as soil require that the tests possess high specificity and sensitivity. METHODS AND RESULTS: The work proposes a quantitative real-time PCR detection method using TaqMan chemistry, showing high specificity and sensitivity for the Paenibacillus polymyxa K16 strain. The primer and probe sets were designed using the polymyxin gene cluster targeting pmxC and pmxE sequences. Validation tests showed that these assays allowed a discriminant and specific detection of P. polymyxa K16 in soil. CONCLUSION: The TaqMan-assay developed could thus ensure the necessary level of discrimination required by commercial and regulatory purposes to detect and monitor the bioinoculant in soil.

Co-inoculum of Beauveria brongniartii and B. bassiana shows in vitro different metabolic behaviour in comparison to single inoculums.

The use of entomopathogenic fungi for biocontrol of plant pests is recently receiving an increased interest due to the need of reducing the impact of agricultural practices on the environment. Biocontrol efficacy could be improved by co-inoculation of different microorganisms. However, interactions between the fungal species can trigger or depress the biocontrol activity. Co-inoculation of two entomopathogenic fungi (Beauveria bassiana and B. brongniartii) was performed in vitro to evaluate the effects of their joint behaviour on a range of different carbon sources in comparison to single inoculation. The two species showed a very different metabolic profile by Phenotype MicroArrayTM. B. bassiana showed a broader metabolism than B. brongniartii on a range of substrates. B. brongniartii showed a greater specificity in substrate utilization. Several carbon sources (L-Asparagine, L-Aspartic Acid, L- Glutamic Acid, m- Erythritol, D-Melezitose, D-Sorbitol) triggered the fungal metabolism in the co-inoculum. SSR markers and Real Time qPCR analysis showed that different substrates promoted either the growth of one or the other species, suggesting a form of interaction between the two fungi, related to their different ecological niches. The methodological approach that combines Phenotype MicroArrayTM and SSR genotyping appeared useful to assess the performance and potential competition of co-inoculated entomopathogenic fungi.

Development of a method for detection and quantification of B. brongniartii and B. bassiana in soil.

A culture independent method based on qPCR was developed for the detection and quantification of two fungal inoculants in soil. The aim was to adapt a genotyping approach based on SSR (Simple Sequence Repeat) marker to a discriminating tracing of two different species of bioinoculants in soil, after their in-field release. Two entomopathogenic fungi, Beauveria bassiana and B. brongniartii, were traced and quantified in soil samples obtained from field trials. These two fungal species were used as biological agents in Poland to control Melolontha melolontha (European cockchafer), whose larvae live in soil menacing horticultural crops. Specificity of SSR markers was verified using controls consisting of: i) soil samples containing fungal spores of B. bassiana and B. brongniartii in known dilutions; ii) the DNA of the fungal microorganisms; iii) soil samples singly inoculated with each fungus species. An initial evaluation of the protocol was performed with analyses of soil DNA and mycelial DNA. Further, the simultaneous detection and quantification of B. bassiana and B. brongniartii in soil was achieved in field samples after application of the bio-inoculants. The protocol can be considered as a relatively low cost solution for the detection, identification and traceability of fungal bio-inoculants in soil.

Unexploited potential of some biotechnological techniques for biofertilizer production and formulation.

The massive application of chemical fertilizers to support crop production has resulted in soil, water, and air pollution at a global scale. In the same time, this situation escalated consumers' concerns regarding quality and safety of food production which, due to increase of fertilizer prices, have provoked corresponding price increase of food products. It is widely accepted that the only solution is to boost exploitation of plant-beneficial microorganisms which in conditions of undisturbed soils play a key role in increasing the availability of minerals that otherwise are inaccessible to plants. This review paper is focused on the employment of microbial inoculants and their production and formulation. Special attention is given to biotechniques that are not fully exploited as tools for biofertilizer manufacturing such as microbial co-cultivation and co-immobilization. Another emerging area includes biotechnological production and combined usage of microorganisms/active natural compounds (biostimulants) such as plant extracts and exudates, compost extracts, and products like strigolactones, which improve not only plant growth and development but also plant-microbial interactions. The most important potential and novel strategies in this field are presented as well as the tendencies that will be developed in the near future.

A contribution to set a legal framework for biofertilisers.

The extensive research, production and use of microorganisms to improve plant nutrition have resulted in an inconsistent definition of the term "biofertiliser" which, in some cases, is due to the different microbial mechanisms involved. The rationale for adopting the term biofertiliser is that it derives from "biological fertiliser", that, in turn, implies the use of living microorganisms. Here, we propose a definition for this kind of products which is distinguishing them from biostimulants or other inorganic and organic fertilisers. Special emphasis is given to microorganism(s) with multifunctional properties and biofertilisers containing more than one microorganism. This definition could be included in legal provisions regulating registration and marketing requirements. A set of rules is also proposed which could guarantee the quality of biofertilisers present on the market and thus foster their use by farmers.

Technologies for beneficial microorganisms inocula used as biofertilizers.

The increasing need for environmentaly friendly agricultural practices is driving the use of fertilizers based on beneficial microorganisms. The latter belong to a wide array of genera, classes, and phyla, ranging from bacteria to yeasts and fungi, which can support plant nutrition with different mechanisms. Moreover, studies on the interactions between plant, soil, and the different microorganisms are shedding light on their interrelationships thus providing new possible ways to exploit them for agricultural purposes. However, even though the inoculation of plants with these microorganisms is a well-known practice, the formulation of inocula with a reliable and consistent effect under field conditions is still a bottleneck for their wider use. The choice of the technology for inocula production and of the carrier for the formulation is key to their successful application. This paper focuses on how inoculation issues can be approached to improve the performance of beneficial microorganisms used as a tool for enhancing plant growth and yield.

Changes in microorganisms populations in the soil after fumigation.

Soil fumigation with dazomet, metam sodium, chloropicrin and chloropicrin + 1.3 D resulted in significant decrease of fungi and increase of bacteria populations in trials carried out in four farms located in different areas. Depending on the farm and the active substance applied, the fungi population was decreased by 1.4- to 3500-fold in comparison to control. Metam sodium and chloropicrin showed the best efficacy, both of them almost totally eliminated the fungi from the soil environment.The total number of bacteria was increased by the chemical fumigation with all tested products. While the population of fluorescent Pseudomonads in all treated plots increased from 2- to 100-fold, depending on the farm, the number of Bacillus spp. was not changed or decreased compared with non fumigated soil.The nematology analysis of the soil indicated that any chemical fumigant significantly limited the population of plant parasitic nematodes, which number was, anyway, below the damage threshold. However, in most cases dazomet and metam sodium reduced the total number of all nematodes present in the soil.The fumigation with chloropicrin and 1,3 D at dose of 30 g/m2 resulted in an increase of the total number of all nematodes in soil.

Phosphorous acid residues in apples after foliar fertilization: results of field trials.

The levels of phosphorous acid residues in apples after foliar fertilization with P fertilizers and after treatment with a phosphonate fungicide (Fosetyl-Al) were determined and compared. Two field trials and a glasshouse experiment, using different genotypes and plants of different age, were carried out and monitored over a three-year period. Phosphorous acid residues were found in apples after application of foliar P fertilizers. Concentrations of the residues ranged between 0.02 and 14 mg kg(-1) depending on the phosphorous acid content in the fertilizer used and the plant size and yield. The treatments induced an accumulation of the residue in the course of the experiments, which in some cases reached a level exceeding the maximum limit set by EU legislation. Residues were also detected in other plant organs, i.e., roots and buds. Plants treated with Fosetyl-Al contained phosphorous acid residues in their fruits and buds two years after the suspension of the treatment, suggesting a long-term persistence of the substance in plant storage organs. A second experiment, involving treatment of trees with seven foliar fertilizers of different composition, also induced accumulation of phosphorous acid residues in fruits. It is concluded that a wide array of foliar products containing phosphorous acid, even as a minor component, could mimic the residue effect of phosphonate fungicide treatments.

Work and chronic health effects among fishermen in Chioggia, Italy.

OBJECTIVES: To evaluate whether there is an association between deep-sea fishing and common chronic disease. METHODS: The study was cross-sectional, simultaneously considering groups of fishermen and non-fishermen. Information on life-style and work was collected by means of questionnaires, and clinical data were collected by specialists in: Internal Medicine (general clinical examination), Cardiology (ECG, measurement of arterial pressure), Pneumology (measurement of spirometric volumes), ENT (clinical examination of the ear, nose and throat, including audiometry), Ophthalmology (examination of lens). Beside the common statistical methods, the logistic stepwise regression analysis was used in order to find the risk factors of the diseases, and to correct the risk estimates for the confounding variables. RESULTS: Fisherman had prolonged hours of continuous work, which were found to be correlated with high cigarette and alcohol consumption. Significant associations were found between, on the one hand, work accidents, noise-induced hearing loss, solar keratosis, cataracts, obstructive bronchitis, rhino-sinusitis, otitis media with tympanic perforation, ECG alterations, and, on the other hand, various aspects of fisherman occupation, mainly fishing in high sea and work duration as fisherman. CONCLUSION: Deep-sea fishing is a stressful and risky work; a reduction in the number of years at sea with reduced exposure to noise, poor weather conditions and sun, and a lower consumption of cigarettes and alcohol might result in fewer skin, eye respiratory and cardiovascular diseases, and injuries.

Mortality from lung cancer and other diseases related to smoking among fishermen in north east Italy.

OBJECTIVES: To study the mortality of fishermen. This was suggested by a cluster of cases of lung cancer in Chioggia, a large fishing harbour in the Veneto. The aim was to weight occupation against smoking with respect to risk of lung cancer. METHODS: 7530 fishermen registered in the 1971-86 port authority registers of Chioggia and Venice were followed up for mortality from 1971 to 1989. Of 475 causes of death, 460 were traced. Standardised mortality ratio (SMR) was calculated with the regional population as a reference. A nested case-control study was carried out in Chioggia decedents only by interviewing next of kin. Complete data were obtained in 172 (70% response). Cases (lung cancer deaths) and controls (other causes of death) were compared for smoking and occupation, as a group of non-fishermen was available in the cohort. Logistic regression analysis was used to adjust for confounding factors. RESULTS: SMRs were high for lung cancer but low for other diseases related to smoking: circulatory and respiratory disease, tumours of the mouth, pharynx, oesophagus, and bladder. Likewise, SMRs were high for liver cancer but low for other diseases related to alcohol: digestive and circulatory disease, buccal, pharyngeal, and oesophageal tumours. In the case-control study, the risk of lung cancer was found to be related to smoking, and there was no interaction between occupation and smoking. When adjusted for age and smoking, the occupational indicators had no influence on the risk of mortality from lung cancer. CONCLUSIONS: Among fishermen in north east Italy the greatest health hazard is lung cancer, and the factor involved in this risk is smoking, not occupation. Smoking, however, was linked with long hours at sea in another study.